systems-adme/tox: resources and network approacheserdi/ekins2.pdf · development of ht methods,...
TRANSCRIPT
wwwelseviercomlocatejpharmtox
Journal of Pharmacological and Toxicol
Appraisal of state-of-the art
Systems-ADMETox Resources and network approaches
Sean Ekins
GeneGo 500 Renaissance Drive Suite 106 St Joseph MI 49085 USA
School of Pharmacy Department of Pharmaceutical Sciences University of Maryland USA
Received 23 May 2005 accepted 23 May 2005
Abstract
The increasing cost of drug development is partially due to our failure to identify undesirable compounds at an early enough stage of
development The application of higher throughput screening methods have resulted in the generation of very large datasets from cells in
vitro or from in vivo experiments following the treatment with drugs or known toxins In recent years the development of systems biology
databases and pathway software has enabled the analysis of the high-throughput data in the context of the whole cell One of the latest
technology paradigms to be applied alongside the existing in vitro and computational models for absorption distribution metabolism
excretion and toxicology (ADMETox) involves the integration of complex multidimensional datasets termed toxicogenomics The goal is to
provide a more complete understanding of the effects a molecule might have on the entire biological system However due to the sheer
complexity of this data it may be necessary to apply one or more different types of computational approaches that have as yet not been fully
utilized in this field
The present review describes the data generated currently and introduces computational approaches as a component of ADMETox These
methods include network algorithms and manually curated databases of interactions that have been separately classified under systems
biology methods The integration of these disparate tools will result in systems-ADMETox and it is important to understand exactly what
data resources and technologies are available and applicable Examples of networks derived with important drug transporters and drug
metabolizing enzymes are provided to demonstrate the network technologies
D 2005 Elsevier Inc All rights reserved
Keywords Algorithms Human Microarray Mouse Networks Rat Software Toxicogenomics Toxicoproteomics
1 Introduction
Metabolism and safety assessment have witnessed some
growth in the number of new technologies and methods
that have been introduced within the last decade However
according to a recent FDA white paper there is still
considerable scope for additional new methods (FDA
2004) For example recently various reports have
described new software and methods for metabolism
prediction (Balakin et al 2004a 2004b Borodina et al
2003 Borodina et al 2004 Boyer amp Zamora 2002
Korolev et al 2003) Simultaneously the use of high
throughput (HT) methods for genomics proteomics and
metabonomics have taken off in terms of their acceptance
1056-8719$ - see front matter D 2005 Elsevier Inc All rights reserved
doi101016jvascn200505005
Corresponding author Tel +1 269 930 0974 fax +1 269 983 7654
E-mail addresses seangenegocom ekinsseanyahoocom
in the industry as they have been evaluated primarily for
toxicology and metabolism assessment (Waring et al
2003 2002 2001) with some considerable focus on
hepatotoxicity (Harris Dial amp Casciano 2004 Hartley
et al 2004 Heijne et al 2004 Huang et al 2004
Liguori et al 2005 Ulrich Rockett Gibson amp Pettit
2004) For example searching PubMed for publications in
the last 5 years with the keywords Fmicroarray and
toxicology_ or Ftoxicogenomics_ indicates that the accu-
mulation of papers describing the latter is doubling every
year (Fig 1) which perhaps is mirrored by the application
in the pharmaceutical industry for predictive toxicology
(Suter Babiss amp Wheeldon 2004)
To date toxicogenomics experiments have been carried
out under non-standardized conditions Most of the studies
have been conducted with rats less often mice using
multiple different microarray formats and statistical proce-
ogical Methods 53 (2006) 38 ndash 66
0
20
40
60
80
100
2000 2001 2002 2003 2004 2005Year
Pu
bM
edN
um
ber
of
pu
blic
atio
ns
in
Fig 1 Annual frequency of articles appearing with the words lsquolsquotoxicoge-
nomicsrsquorsquo (squares) or lsquolsquomicroarray and toxicologyrsquorsquo (diamonds)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 39
dures (Tables 1-4) There have been relatively few cross-
platform toxicogenomics studies under controlled condi-
tions (Thompson et al 2004) Despite the suggested poor
compatibility between the different array types this latter
study demonstrated a high (90) consistency between the
expression of the genes that were shared between the
platforms The development of methods to visualize such
complex expression data has also expanded beyond the
widely used clustering methods (Eisen Spellman Brown amp
Botstein 1998) With the outcome of microarray analysis
being dependent on the widely used statistical procedures
applied to derive those genes that are significantly differ-
entially expressed (Butte 2002) newer approaches that do
not necessarily require data clustering may be an advantage
As rat and mouse are the most widely used in vivo toxicity
models it is assumed that acute and chronic toxicity shown
in animals largely coincides with human toxicity Therefore
differential expression patterns in animal models are also
assumed to be predictive of the end point toxic response in
human This is not always the case due to differences
between human and rodent physiology genetics metabo-
lism and signaling pathways For example the mechanism
of toxicity for pyrazinamide has been reconstructed
(Bugrim Nikolskaya amp Nikolsky 2004) to illustrate that
the accumulation of uric acid occurs in human but not in
mice and this results in toxicity in the former The relatively
poor understanding of such species differences may be
reflected in the relatively large number of late stage
molecules that have undergone in vivo toxicity assessment
yet have been later withdrawn due to adverse events in
humans
In recent years the appearance of systems biology which
uses the relationships of all elements of biology rather than
approaching them separately has been evident and will
likely reunite biological fields (Harrison 2004 Hood amp
Galas 2003) These systems approaches are the latest
incarnation of the importance of the Fparts vs wholes_debate (Ekins amp McGowan 2001) and interpreting ADME
Tox in this context may improve our understanding and
ultimate predictions (Bugrim et al 2004 Ekins Boulanger
Swaan amp Hupcey 2002a Kitano 2002a Werner 2003)
The perturbing effect of a molecule on the complete
biological system can be observed across all metabolic
and signaling pathways or networks and can provide some
limited insight into the binding to multiple proteins or
effects on gene expression simultaneously This requires the
collection of high-throughput data including global gene
expression protein content metabolic profiles for the same
samples as well as individual genetic clinical and pheno-
typic data However there are difficulties with such an
approach as there are likely to be differences between the
Fstimulus to effect_ durations for all the genendashprotein
relationships (Nicholson Holmes Lindon amp Wilson
2004)
We can now use either the growing number of academic
or commercially available pathway database and network
building tools with expression data These enable the
connection of interacting differentially expressed genes as
networks (Barabasi amp Oltvai 2004 Hanisch Zien Zimmer
amp Lengauer 2002 Ideker Ozier Schwikowski amp Siegal
2002 Ideker et al 2001 Segal et al 2003a Segal Wang amp
Koller 2003b Spirin amp Mirny 2003 Tornow amp Mewes
2003) as well as allowing the reverse engineering of
functional connections (Somogyi Fuhrman amp Wen 2001)
The use of such network visualizations suggests an
organized modularity in complex systems (Han et al
2004) which has also been applied to interpret the
connectivity of small molecules and their interaction with
proteins in the subfield of chemogenomics (Bredel amp Jacoby
2004 Csermely Agoston amp Pongor 2005 Parsons et al
2004 Sharom Bellows amp Tyers 2004) The parallel
development of HT methods databases ADMETox model-
ing and systems modeling is ongoing (Ekins Nikolsky amp
Nikolskaya 2005e) The present review is therefore timely
as it discusses some of the data resources limitations and
technologies that are available for Systems-ADMETox
(Fig 2) along with some illustration of their applications to
drug metabolism and drug transport which are key com-
ponents of the ADMETox process The ultimate aim of
this discussion is to provide awareness of an integrated
approach rather than a technology silo mentality represent-
ing the latest proposed research model in the field (Fig 2)
2 Data available
A recently published book provides an excellent over-
view of toxicogenomics and the reader is referred to this to
gain more insight into the applications and limitations
(Hamadeh amp Afshari 2004) The growing number of
toxicogenomic datasets derived from in vivo studies with
rat (Table 1) and mouse (Table 2) as well as in vitro cell
derived data (Table 3) highlights the different strains
microarray types and compounds that are routinely assessed
Also there are numerous instances of multiple groups testing
the same compound at similar or different doses eg well
known hepatotoxicants or nephrotoxicants such as clofibrate
Table 1
Literature toxicogenomics data derived from rat in vivo studies
Compounds Rat strain Microarray type Compound dose Microarray data
availability
Reference
Acetaminophen furan
methotrexate
methapyrilene
phenytoin
Male Spraguendash
Dawley VAF+
albino
Phase 1 Molecular
toxicology array
Acetaminophen
(4500 mgkgday)
methotrexate (1 mg
kg day)
methapyrilene (100
mgkgday) furan (40
mgkgday) or
phenytoin (300 mg
kgday)
Gene name accession
number and fold changes
are provided in a
manuscript table
(Huang et al
2004)
A-277249 3MC
Aroclor
SpraguendashDawley Affymetrix rat
toxicology U34 array
10 or 100 mgkg Gene name and
Affymetrix ID and fold
change in a manuscript
table
(Waring et al
2002)
Clofibrate paracetamol
benzoapyrene
Male Spraguendash
Dawley
Custom chip CLO at 250 mgkg
BP at 10 mgkg bw
given 3 times per
week for 2 weeks
APAP at 1000 mgkg
Gene name and fold
change in a manuscript
table
(Cunningham
Liang Fuhrman
Seilhamer amp
Somogyi 2000)
L-742694
Dexamethasone
Female
SpraguendashDawley
25K rat microarray L-742694 (50 mgkg
day) DEX (50 mg
kgday)
Tables of accession
number and gene names
only in a manuscript
table for liver and
intestine
(Hartley et al
2004)
Bromobenzene Male Wistar Custom 3000 rat gene
array
05 2 5 mmolkg Gene name accession
number and log fold
change for 3 dose levels
in a manuscript table
Supplemental data also
available
(Heijne et al
2004)
125-Dihydroxyvitamin
D3
Male Spraguendash
Dawley (small
intestine)
Affymetrix high-density
rat oligonucleotide
arrays (GeneChips
RG-U34A)
730 ngkg Gene name Genbank
accession number and
fold change in a
manuscript table
(Kutuzova amp
Deluca 2004)
Fenofibrate clofibrate
bezafibrate
gemfibrozi
ciprofibrate
beclofibrate etofibrate
Male CD Agilent arrays Dose ranging for up
to 14 days
Gene name and genbank
accession number in a
manuscript table
(Cornwell Souza
amp Ulrich 2004)
Furan Male Spraguendash
Dawley
NIEHS rat chip uml7000
clones
Exposed to 4 or 40
mgkg furan for up
to 14 days
Gene ID and gene
namemdashbinary data in a
manuscript table Dataset
also available on NIEHS
website
(Hamadeh et al
2004)
Paracetamol Male F344N Rat NIEHS tox chip
(httpdirniehsnihgov
microarraychipshtm)
0 50 150
1500 mgkg
Unigene accession
number gene name fold
change in a manuscript
table
(Heinloth et al
2004)
Carbon tetrachloride Male Spraguendash
Dawley
ADME Rat expression
bioarray (Motorola Life
Sciences) consists of
1040 single-stranded
oligonucleotide probes
Up to 14 day
treatment
Accession numbers and
binary data in a
manuscript table
(Young et al
2003)
Carbon tetrachloride and
chloroform
Male Spraguendash
Dawley
The rat CT arrays
contain sequences from
almost 700 rat genes
with known or
discovered
responsiveness to toxic
treatments
6 24 72 h high and
low doses
Gene names and fold
changes at multiple time
points in a manuscript
table
(Kier et al 2004)
(continued on next page)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6640
Table 1 (continued)
Compounds Rat strain Microarray type Compound dose Microarray data
availability
Reference
Dimethylarsinic acid Female F344
(bladder
epithelium)
Rat 10K chip (MWG
Biotech Inc) containing
10000 genes
100 ppm Genbank accession
number gene name in a
manuscript table
(Wei Arnold
Cano amp Cohen
2005)
Dexamethasone
troleandomycin
miconazole
clotrimazole
isoniazid
methylclofanapate
Female Spraguendash
Dawley
Rat HepatoChip DEX (50 mgkgday)
TAO (500 mgkg
day) MIC (100 mg
kgday) CLOT (100
mgkg per day) ISN
(100 mgkgday)
MCP (75 mgkgday)
Gene name and Genbank
accession numbermdashdata
displayed as a heatmap
not readily extracted
from publication
(Meneses-Lorente
et al 2003)
TCDD PeCDF PCB126
PCB153
Female Harlan
SpraguendashDawley
Affymetrix GeneChip
Test3 arrays
Exposed for 13 weeks
to toxicologically
equivalent doses
Accession number gene
name and fold change in
a manuscript table
(Vezina Walker
amp Olson 2004)
Paclitaxel Male and female
SpraguendashDawley
Genocheck 48K cDNA 4 mgkgday male
7 mgkgday female
Accession numbers and
gene names in a
manuscript table
(Lee et al 2004)
Clofibrate gemfibrozil
phenytoin
Male Spraguendash
Dawley VAF(+)
albino
48 K cDNA microarray
in house
Treated with each
compound for 24 h
and 2 weeks
Gene name and fold
change in a manuscript
table
(Jung et al 2004)
Clofibrate
dexamethasone
phenobarbital
3-methylcholanthrene
SpraguendashDawley Merck Drug Safety Chip
1443 genes (rat human
and mouse)
30 mgkgday Gene name GenBank
accession number and
relative fluorescence
levels in a manuscript
table
(Gerhold et al
2001)
Bemitradine clofibrate
doxylamine
methapyrilene
phenobarbital
tamoxifen
2-acetylaminofluorene
4-acetylaminofluorene
isoniazid
Male CD IGS Incyte RatGEM10
uml7800 rat cDNAs
Low mid and high
doses
A bar chart of 2 genes
that are affected by
compounds
(Kramer et al
2004)
PhIP Female Spraguendash
Dawley
Mouse cDNA
microarray containing
9984 cDNA clones
(National Cancer
Institute
75 mgkgday Gene names in a
manuscript table
(Shan Yu Schut
amp Snyderwine
2004)
Ethinylestradiol Male and female
SpraguendashDawley
Custom chip 3776 genes 0 001 01 and
10 ppm
Accession number gene
name and fold change at
different exposure levels
in a manuscript table
(Kato et al 2004)
Paraquat Male Wistar 1090 genes 7 mgkgday Gene name and
expression ratio in a
manuscript table
(Satomi et al
2004)
Hexachlorobenzene Female Brown
Norway
Affymetrix rat
RGU-34A GeneChip
microarray
0 150 or 450 mgkg Accession number gene
name and fold change
data for multiple organs
in a manuscript table
(Ezendam et al
2004)
N-methyl-NV-nitro-N-nitrosoguanidine
(MNNG)
Rat pyloric muco-
sae male congenic
rat strain that has a
homozygous_LIZ
transgene of
BigBlue rat
AFFYMETRIX Rat
Genome U34A
83 mgl AFFY ID gene name
gene symbol and fold
change in a manuscript
table
(Yamashita et al
2004)
N-methyl-NV-nitro-N-nitrosoguanidine
Male ACINJcI
(ACI)
Affymetrix GeneChip
Rat genome U34A
arrays
83 mgl from the age
of 8 weeks through to
40 weeks
Accession number gene
name symbol fold change
in a manuscript table also
rat vs human stomach
cancer comparison
(Abe et al 2003)
Cisplatin SpraguendashDawley Different arrays tox
chip incyte phase 1 etc
03ndash5 mgkg over a 4 to
144 h
Unigene gene ID gene
name NIEHS ID and
data for 5 platforms in a
manuscript table
(Thompson et al
2004)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 41
Table 1 (continued)
Compounds Rat strain Microarray type Compound dose Microarray data
availability
Reference
Clofibrate Male Spraguendash
Dawley
Atlas Rat Toxicology II
arrays (Clontech Palo
Alto CA USA)
containing 465 genes
High (250 mgkgday) or
low (25 mgkgday)
Genbank accession
number and data for 3
platforms in a
manuscript table
(Baker et al
2004)
Di(2-ethylhexyl)
phthalate
Male Spraguendash
Dawleymdash(testes)
An in-house cDNA
microarray
20 or 2000 mgkg Gene names Genbank
ID and fold change in a
manuscript table
(Kijima et al
2004)
Ciprofibrate Female Fischer An in-house cDNA
microarray
50 mgkg body weight Gene names symbol
accession number mean
ratio and SD
(Yadetie et al
2003)
Methapyrilene Male Spraguendash
Dawley
Rat Tox Chip 10 10 or 100 mgkgday Gene Name accession
number and indication of
up or down regulation
original data available on
NIEHS website
(Hamadeh et al
2002b)
Ecteinascidin-743
(ET-743)
Female Wistar Custom chip cDNA
microarrays containing
approximately 4700
hybridizable mouse
expressed sequence tags
derived from IMAGE
clones obtained from
Research Genetics
(Huntsville AL) or from
the MRC Human Gene
Mapping Project
40 ugkg Data available on
laboratory websitemdashnot
available at present
(Donald et al
2002)
Clofibrate Wyeth
14643 Gemfibrozil
phenobarbital
Male Spraguendash
Dawley VAF+
NIEHS rat chip v10 Clofibrate (250
mgkgday Wyeth
14643 (250 mgkg
day) Gemfibrozil
(100 mgkgday)
Phenobarbital
(120 mgkgday)
Gene names and fold
changes in a manuscript
table
(Hamadeh et al
2002a)
Vinclozin procymidone Male Spraguendash
Dawleymdashprostate
Clontech Atlas Rat 12
Toxicology array
200 mgkg Gene name accession
number average fold
change
(Rosen Wilson
Schmid amp Gray
2005)
Cisplatin Male Spraguendash
Dawley VAF1
albino (CRL
CD(SD) BR
Rat Tox Microarrays
were purchased from
Phase-1 Molecular
Toxicology
05 or 1 mgkgday Gene name accession
number fold change in
kidney
(Huang et al
2001)
Allyl alcohol
miodarone Aroclor
1254 arsenic
carbamazepine
carbon tetrachloride
diethylnitrosamine
dimethylformamide
diquat etoposide
indomethacin
methapyrilene
methotrexate
monocrotaline
3-methylcholanthrene
Male Spraguendash
Dawley
Affymetrix GeneChip
Test 2 Array
Allyl alcohol (40 mgkg
day) miodarone (100 mg
kgday) Aroclor 1254
(400 mgkgday) arsenic
(20 mgkgday)
carbamazepine (250 mg
kgday) carbon
tetrachloride (1000mgkg
day) diethylnitrosamine
(100 mgkgday)
dimethylformamide (1000
mgkgday) diquat (172
mgkgday) etoposide (50
mgkgday) indomethacin
(20 mgkgday)
methapyrilene (250 mg
kgday) methotrexate
(250 mgkgday)
monocrotaline (50 mgkg
day)
3-methylcholanthrene
(100 mgkgday)
Heatmap figures and a
table of Affymetrix
names for genes
correlated with clinical
chemistry changes in a
manuscript table
(Waring et al
2001)
(continued on next page)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6642
Table 1 (continued)
Compounds Rat strain Microarray type Compound dose Microarray data
availability
Reference
Microcystin-LR (MLR)
phenobarbital (PB)
lipopolysaccharide
(LPS) carbon
tetrachloride (CT)
thioacetamide (THA)
and cyproterone
acetate (CPA)
Male Wistar Purpose-made rat DNA
microarray (Affymetrix
Santa Clara CA)
containing 1600 rat
DNA sequences
Various Accession number gene
name fold changes
shown as a heat map
table in publicationmdash
data extraction would be
laborious
(Bulera et al
2001)
Acetamidofluorene
aniline bromobenzene
butyl hydroxytol
dieldrin disulfiram
ethinyl estradiol
hexachlorocyclohexane
g 4-methylthiazole
nimesulide piperonyl
butoxide precocene I
pulegone tannic acid
trans-anethole
Male Spraguendash
Dawley
Custom Rat MegaA
cDNA chip 3434-gene
Acetamidofluorene (200
mgkg) aniline (200 mg
kg) bromobenzene (900
mgkg) butyl hydroxytol
(1000 mgkg) dieldrin
(30 and 45 mgkg)
disulfiram (2000 mgkg)
ethinyl estradiol (500
mgkg)
hexachlorocyclohexane
gamma (40 65 80 mg
kg) 4-methylthiazole
(120 mgkg) nimesulide
(500 mgkg) piperonyl
butoxide (4000 mgkg)
precocene I (500 mgkg)
pulegone (400 mgkg)
tannic acid (3000 mg
kg) trans-anethole
(600 mgkg)
Gene name accession
number in a manuscript
table
(McMillian et al
2004)
NIEHS website at httpdirniehsnihgovmicroarraydatasetshome-pubhtm
EDGE website at httpedgeoncologywiscedu
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 43
and cisplatin (Table 1) In the majority of cases the resulting
number of differentially expressed genes is a very small
subset of the starting number on the microarray following
clustering or other types of analysis Upon closer examina-
tion of these publications the majority of them either
provide images of a heat map andor a table listing a gene
name accession number and expression change Very few of
the published studies (Tables 1ndash3) provide the original raw
microarray data file at a freely accessible website hence
restricting further analysis by scientists using other software
Although in some cases it is possible to cut and paste the
gene expression data tables from the publication pdf files
this is not always the case In the worst case scenario one
would have to manually retype gene lists or extract them
from heatmaps as binary type data As not all computational
researchers will have a laboratory available to them to
generate such quantities of toxicogenomics data the latter
points are important if we are going to continue to see
innovation in software development for this data This will
require free unrestricted access to data published Similarly
if we are to discern lsquolsquofingerprintsrsquorsquo for molecules acting with
a similar or identical mechanism we will need databases of
many diverse chemical structures that have been tested in a
similar manner There is therefore considerable interest in
the current databases being developed by the NIEHS FDA
and other groups which should help to improve the situation
by providing freely accessible microarray and other toxicity
related data Two of these databases being developed in the
public domain are Chemical Effects in Biological Systems
(CEBS) (httpwwwniehsnihgovnctcebshtm) (Mattes
Pettit Sansone Bushel amp Waters 2004 Waters et al
2003) which will accommodate gene expression profiles
proteomics and metabolomics data and allow complex
queries (Hood 2003a Mattes et al 2004) Similar goals
are being pursued in the development of the ArrayTrack
database at the FDA (Tong et al 2003) The EDGE
database (httpedgeoncologywisceduedgephp) an
expanding public effort at The University of Wisconsin
contains mouse gene expression profiles following treat-
ment with different toxic molecules (Hayes et al 2005
Thomas et al 2001) These separate efforts if widely
adopted should make published studies describing HT data
more readily accessible although it might have been more
efficient to evolve these into a single global database instead
of fragmented repositories
Proteomics data has also been generated in a limited
number of toxicology studies (Table 4) once again this has
been produced with different strains of rats and mice using
different protein chips 2-D gel electrophoresis (2-DIGE)
and mass spectroscopy methods (eg MALDI-MS) The
proteomic data is very rarely accessible to the reader for
their own computational analysis Subsequently there have
Table 2
Literature toxicogenomics data derived from mouse in vivo studies
Compounds Mouse strain Microarray type Compound dose Microarray data
availability
Reference
Benzene Male and female P53
KO mice and
C57BL6
Affymetrix and Incyte
GEM system
300 ppm 6 h per day 5
days a week for 2 weeks
Gene name accession
number and fold change
data in a manuscript
table
(Yoon et al 2003)
Benzene Male 129SvJ Affymetrix MG-U74Av2 100 ppm 6 h per day 5
days a week for 2 weeks
Gene name accession
number and fold change
data in a manuscript
table
(Faiola Fuller
Wong amp Recio
2004)
Phenobarbital CAR and wild type NIEHS Mouse tox chip
8736 genes
100 mgkg for 12 h Accession number gene
name and fold change
for wild type and knock
out mice in a manuscript
table
(Ueda et al 2002)
Aroclor BNF
ciprofloxacin cobalt
chloride TCDD IL-6
LPS PCB-153
phenobarbital
phenylhyrzn TNFa
WY-16463
C57BL6J Custom array with 1200
cDNAs
Aroclor (200 mgkg)
BNF (5 mgkg)
ciprofloxacin (250 mg
kg) cobalt chloride (60
mgkg) TCDD (10 ug
kg) IL-6 (25 ugkg)
LPS (1 mgkg)
PCB-153 (80 mgkg)
phenobarbital (100 mg
kgday) 3 days
phenylhyrzn (100 mg
kg) TNFa (50 ugkg)
WY-16463 (0125wv)
Gene names and fold
change as a heat map
Data is also available in
the EDGE database
(Thomas et al
2001)
TCPOBOP CD-1 female Custom 9000 cDNA
mouse array
1ndash3 h treatment 3 mg
kg body wt
Accession numbers and
fold change data in a
manuscript table
(Locker et al
2003)
3H-12-dithiole-3-thione
(D3T)
Male wild-type and
nrf2-disrupted
Affymetrix murine
genome U74Av2
GeneChip
05 mmolkg Accession number gene
name fold in a
manuscript table
(Kwak et al
2003)
MDMA Male albino Swissndash
Webster (neurons)
15 K mouse cDNA clone
set
47 mgkg Gene name and fold
change in a manuscript
table
(Xie et al 2004)
Phenytoin Female C57BL6 and
LDLRMurine genome-U74Av2 300 mgl Gene name accession
number and fold change
in a manuscript table
(Trocho et al
2004)
Genistein (1000 Agmouseday) or
diethylstilbestrol
(DES) (50 Agmouse
day)
ICR (testes) Custom cDNA
microarray containing
1754 cDNA probes
Genistein (1000 Agmouseday)
diethylstilbestrol (50 Agmouseday)
Accession number gene
name and fold change in
a manuscript tablemdashnote
very few genes
(Adachi et al
2004)
Cocaine and
buprenorphine
Male ICR Mouse DiscoveryArrayi
type I array containing
2688brain-derivedprobes
(Display Systems Biotech
Inc Copenhagen
Denmark)
40 mg kgmdash1 cocaine
once a day for 4 days 40
mg kgmdash1 cocaine plus
025 mg kgmdash1 BUP for
4 days
Data apparently not
available
(Hayase
Yamamoto
Yamamoto Muso
amp Shiota 2004)
Methamphetamine Male C57BLJ6
(striatum)
Affymetrix mouse
genechip mg-U74Av2
oligonucleotides arrays
12 488 genes
40 mgkg Gene accession number
gene ID and name and
signal log ratio in a
manuscript table
(Thomas
Francescutti-
Verbeem Liu amp
Kuhn 2004)
Di(2-ethylhexyl)
phthalate
Male C57BL6 Murine genome U74Av2
Arrays (MGU74Av2)
10 dietary DEHP for
13 weeks
Genbank accession
numbers gene name log
ratio data present as a bar
chart in the publication
data extraction would
take some effort
(Wong amp Gill
2002)
Diethylhexylphthalate Male PPARa null and
wild type
Custom made containing
600 tox genes
1150 mgkgday Gene names available on
a bar chartmdashquantitative
data not easily accessible
(Hasmall et al
2002)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6644
Table 2 (continued)
Compounds Mouse strain Microarray type Compound dose Microarray data
availability
Reference
Acetaminophen C57B16 3 129Ola
hybrid
Test-2 Chips (Affymetrix)
then individual
oligonucleotide
microarrays (Mul1K sub
A and sub B Affymetrix)
that can detect the
expression of 11000
known genes and
expressed sequence tags
(ESTs)
300 mgkg Genbank or SwissProt
ID and fold change data
in a manuscript table
(Reilly et al
2001)
Cadmium chloride
benzo(a)pyrene (BaP)
and trichloroethylene
(TCE)
Male Swiss Webster Custom Chips
containing 148 unique
genes
Various Data in table form few
genes affected for BAP
and TCE
(Bartosiewicz
Penn amp Buckpitt
2001)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 45
been only a very small number of studies that have
combined both transcriptomic and proteomic methods with
a single animal strain after treatment with a drug Hopefully
we will see this change in the future but this will in turn
present considerable challenges as huge amounts of
proteomic data are combined with the equally large
transcript data files
3 Network analysis and databases
From some of the early reviews of systems biology there
has been discussion of its application to drug discovery
(Kitano 2002ab) as well as the utility for ADMETox
(Ekins et al 2002a 2000b) More recently several other
journals have dedicated whole issues to the field of systems
biology However one could consider this quite a broad field
from network or pathway analysis to quantitative simulation
of organelles (Vo Greenberg amp Palsson 2004) whole cells
and organs It is apparent that we are now understanding
organisms from the perspective of computationally gener-
ated networks of protein and ligand interactions (Barabasi amp
Oltvai 2004) Network and pathway tools enable the
analysis of HT data in the context of all known interactions
when using a database as the source Individual reviews
have in some cases indicated that networks will be valuable
for understanding adverse events (Hood amp Perlmutter
2004) drug target identification or validation (Butcher
Berg amp Kunkel 2004) and complex metabolic interactions
(Nicholson et al 2004) A general schematic has been
generated in order to provide a description of the utilization
of such pathway databases and network building algorithms
from the initial parsing of high throughput data to network
comparisons and visualization (Fig 3) High throughput
data can be superimposed and visualized on the various
protein interaction databases available This is accomplished
by using either preset maps that capture current biological
knowledge or by building custom interaction networks
using many different algorithms which can be compared and
statistically evaluated as demonstrated in a very large
number of published examples (Dobrin Beg Karabasi amp
Oltvai 2004 Fiehn 2001 Han et al 2004 Hanisch et al
2002 Ideker et al 2002 Jeong Mason Barabasi amp Oltvai
2001 Jeong Tombor Albert Oltvai amp Barabasi 2000 Li
et al 2004 Milo et al 2002 Nikitin Egorov Daraselia amp
Mazo 2003 Pereira-Leal Enright amp Ouzounis 2004
Rives amp Galitski 2003 Segal et al 2003a Somogyi et al
2001 Spirin amp Mirny 2003 Tornow amp Mewes 2003
Vasquez Flammini Maritan amp Vespignani 2003 Yeger-
Lotem amp Margalit 2003 Yu Zhu Greenbaum Karro amp
Gerstein 2004)
31 Network applications
For example one group has used as an inference the
Bayesian network method for analysis of tissue toxicity from
microarray data as well as a mechanistic simulation for a
different pharmaceutically relevant molecule (Aksenov et
al 2005) Pathway tools and various resources have also
been applied to modeling the networks of nuclear hormone
receptors and their connections with other genes and small
molecules using a manually curated database MetaDrug
(Ekins Kirillov Rakmatulin amp Nikolskaya 2005d) or
MetaCore (Ekins Bugrim Nikolsky amp Nikolskaya 2005)
Transcriptional regulation of many transporters CYPs and
phase II enzymes are regulated by these receptors affecting
endogenous molecule transport metabolism cell growth
proliferation and oxidative stress (Ulrich 2003 Ulrich et al
2004) When the signaling networks and interacting ligands
for the transcriptional factors PPAR FXRRXRA ESR1
AHR HNF4A GCR-h MCR CAR-beta GCR-a LXR-a
CARRXR HNF4 FXR PXRRXR heterodimer PXR
AHRARNT heterodimer PPARaLXRa VDR PPAR-a
are visualized a very complex picture of interactions can be
created (Ekins et al 2005d) This suggests that when we
consider a molecule binding with only one nuclear receptor
we are observing only a fraction of the likely possible
feasible interactions based on the data gathered to date
Table 3
Literature toxicogenomics data derived from in vitro cell studies
Compounds Cell type Microarray type Compound dose Microarray data
availability
Reference
4-Hydroxytamoxifen
estrogen
MCF-7 breast cancer NIEHS ToxChip
microarray consisting of
1901 genes
1 uM hydroxytamoxifen
for a year 10 nM
17b-estradiol
Data available at NIEHS
website
(Hodges et al
2003)
Trovafloxacin Human hepatocytes Affymetrix U133A array 30ndash800 uM 142 genes available in
supplemental tablemdashnot
easily extracted
(Liguori et al
2005)
Estrogen MCF-7 breast cancer NIEHS ToxChip
microarray consisting of
1901 genes
10ndash10 M 17b-estradiol Data available at NIEHS
website
(Lobenhofer et al
2002)
Valproic acid NMRI mice embryo
and P19 mouse
embryocarcinoma
Custom chip including
15K mouse cDNA clone
set
600 mgkg body weight Gene symbol gene
name NIA EST log fold
change in a manuscript
table
(Kultima et al
2004)
Sulindac sulfide Human colorectal
carcinoma SW-480
and HCT-116
NIEHS human 12K chip 10 uM Genbank accession
number gene name and
fold change at various
time points in a
manuscript table data
also available on NIEHS
website
(Bottone
Martinez Collins
Afshari amp Eling
2003)
17Beta-estradiol estriol
estrone genistein
diethylstilbestrol
bisphenol A
nonylphenol
methoxychlor
MCF-7 U95A oligonucleotide
probe arrays (Affymetrix
10 nM (E2 estriol
estrone DES) 10 AM(genistein bisphenol A
nonylphenol and
methoxychlor)
Unigene name gene
name and fold change
for estrogen responsive
and nonresponsive in a
manuscript table
(Terasaka et al
2004)
Ouabain lauryl sulfate
dimethylsulfoxide
cycloheximide
tolbutamide sodium
fluoride diethyl
maleate buthionine
sulfoxamine
potassium bromate
sodium selenite
alloxan adriamycin
hydrogen peroxide
HepG2 Clontech Atlas Human
Stress Toxicology cDNA
arrays (234 genes)
Ouabain (43 uM) lauryl
sulfate (260 uM)
dimethylsulfoxide (128
M) cycloheximide (625
uM) tolbutamide (128
mM) sodium fluoride (3
mM) diethyl maleate
(125 mM) buthionine
sulfoxamine (30 mM)
potassium bromate (25
mM) sodium selenite (30
uM) alloxan (130 mM)
adriamycin (40 uM)
hydrogen peroxide
(4 mM)
Gene name ratio p -
value in downloadable
tables at journal website
(Morgan et a l
2002)
Aflatoxin B(1) (AFB(1))
2-acetylaminofluorene
(2AAF)
dimethylnitrosamine
(DMN)
acetaminophen (APAP)
HepG2 and primary
hepatocytes
Gene filter arrays
containing 31000 genes
10 uM aflatoxin B1
40 mM acetaminophen
100 uM
dimethylnitrosamine
10 uM
2-acetylaminofluorene
None (Harris et al
2004)
Mitomycin C (MMC)
and cisplatin (CIS)
and an alkylating
agent methyl
methanesulfonate
(MMS)
indirect-acting
genotoxins included
hydroxyurea (HU) a
ribonucleotide
reductase inhibitor
taxol (TXL) a
microtubule inhibitor
and etoposide
(ETOP)
L5178Y TK(+-)
mouse lymphoma
Affymetrix mouse
MG-U74A for MMC
and MG-U745Av2
(Affymetrix Inc Santa
Clara CA) for all the
other chemicals a total
of 9977 probe sets
(genes or ESTs) common
to these two array
models
Low mid and high doses Accession number gene
name gene ID statistical
significance at each time
point in a manuscript
table
(Hu et al 2004)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6646
Table 3 (continued)
Compounds Cell type Microarray type Compound dose Microarray data
availability
Reference
Hydroxyurea
(a carcinogen)
p-anisidine
(a noncarcinogen)
and paclitaxel
L5178Y Tk_-mouse
lymphoma
The Twin - Chip Mouse-
74K Digital Genomics
cDNA microarray
10 ngml paclitaxel 313
ugml hydroxyurea 32
ugml p-anisidine
Gene symbol and fold
change in a manuscript
table
(Lee et al 2003)
Acetaminophen
amiodarone clofibrate
erythromycin estolate
isoniazid alpha-
naphtylylisothiocyanate
beta-naphtoflavone
4-pentenoic acid
phenobarbital
tetracycline and
zileuton
Wistar Rat
hepatocytes
DualChip rat hepato
(Eppendorf Hamburg
Germany)
A single concentration
which varied for each
compound
Accession numbers and
gene namemdashfold
changes shown as
colored heat mapmdashnote
easily extracted from
publication
(de Longueville
et al 2003)
Bleomycin and hydrogen
peroxide
Mouse lymphoma
L5178Y TK(+ -)
Clontech Mouse 12K
cDNA microarray (1185
genes)
Bleomycin (25 and 20
ugml) hydrogen peroxide
(5 and 10 ugml)
Gene names and fold
change presented as bar
charts in publication
(Seidel Kan
Stott Schisler amp
Gollapudi 2003)
Bupivicaine
camptothecin
HL-60 Agilent human cDNA
microarray
1 mM Gene name GenBank
accession number
unigene and ratio in a
manuscript table
(Unami
Shinohara
Ichikawa amp Baba
2003)
Bisphenol A Mouse Sertoli TTE3 IntelliGene mouse
expression glass
microarrays (Version
10 Takara Shuzo)
which were spotted with
564 cDNA fragments of
mouse known genes and
approximately 301
expressed sequence tags
(ESTs)
0ndash400 uM Gene name GenBank
accession number and
fold change at time
points in a manuscript
table
(Tabuchi amp
Kondo 2003)
Mitomycin C or
doxorubicin
Hep G2 85 human gene custom
array
10 um mitomycin C
2 um doxorubicin or
2 ethanol
Bar charts with fold
changes in publication
very few genes
(Hong Muller amp
Lai 2003)
Amphotericin B Human peripheral
blood mononuclear
and THP-1
GF211 FKnown Genes_
Genefilter cDNA array
(ResGen) this array
consists of gt4000
individual elements
each representing a
known human gene
5 ugml Accession numbers and
fold expression in a
manuscript table
(Cleary Rogers amp
Chapman 2001
Rogers Pearson
Cleary Sullivan
amp Chapman
2002)
Benzo(a)pyrene diol
epoxide
TK6 human
lymphoblastoid
Human-350 microarray
a glass slide with 350
spotted human cDNA
probes (Phase-1
Molecular Toxicology
0 001 010 or
10 ugml)
Gene names and fold
change at doses in a
manuscript table
(Akerman et al
2004)
Etomoxir HepG2 Clontech AtlasiHuman Stress
Toxicology cDNA arrays
(234 genes)
1 mM etomoxir Gene names and fold
change in a manuscript
table
(Merrill et al
2002)
Tetrodotoxin Human glioma cell
line HTB-138
Using Affymetrix
GeneChip (HG-U133A
10 and 20 mM Affymetrix ID Genbank
ID gene name gene
symbol and fold change
in a manuscript table
(Raghavendra
Prasad Qi
Srinivasan amp
Gopalakrishnakone
2004)
Methotrexate
mercaptopurine
Human acute
lymphoblastic
leukemia
Affymetrix U133A chip Low and high dose and
combination
Data available as
supplemental data online
(Cheok et al 2003)
Prednisolone
vincristine
asparaginase
daunorubicin
Human acute
lymphoblastic
leukemia
Affymetrix U133A chip Various Data available as
supplemental data online
(Holleman et al
2004)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 47
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6648
A second study has indicated how a natural language
processing method CCNet was used to show the genes
regulated by the nuclear hormone receptor FXR (Apic
Ignjatovic Boyer amp Russell 2005) These automated
methods enable a more complete understanding of the
complexity of the transcriptional factors (Ekins Mirny amp
Schueltz 2002b Plant 2004 Ulrich 2003) but ultimately
rely on the quality of the content of the underlying database
of literature interactions This is a key consideration that is
often overlooked For example the gold standard database is
one that is manually curated to ensure the fidelity of the
direct interaction and is preferable to one generated
computationally by algorithms like natural language pro-
cessing (Nikolsky Nikolskaya amp Bugrim 2005) The
advantage of interaction networks over clustering has been
demonstrated in one study using MetaCore (Nikolsky
Ekins Nikolskaya amp Bugrim 2005) by reanalysis of a
published microarray study of G0-arrested MCF-7 breast
cancer cells treated with estrogen and 4-hydroxytamoxifen
(Hodges et al 2003) After producing integrated gene
networks for each treatment strikingly different patterns
were displayed although both contained early transcriptional
factors myc jun and fos Only the estrogen network featured
induced genes essential for all cell cycle phases (Nikolsky et
al 2005) Similarly microarray data for benzene toxicity
(Yoon et al 2003) has been re-analyzed by focusing on the
genes assessed on the p53 pathway (Ekins et al 2005e) We
envisage that a database of such networks for toxic
compounds will be used for comparing between different
molecules and used in the development of predictive
algorithms for Systems-ADMETox modeling in future
Another approach to using such pathway approaches is to
visualize the results of quantitative structure activity models
for predicting molecules binding to enzymes transporters
receptors and ion channels (Ekins Andreyev et al in press
2005e)
It is also possible to simultaneously interpret high
throughput data and predictions on interaction networks
providing a novel approach to predicting and understanding
potential undesirable drugndashdrug or off target effects in the
area of systems pharmacology An example data set uses
percent inhibition data for clotrimazole and ticonazole
which were screened against many different assays at a
single concentration in a commercially available database
BioPrint (Cerep Redmond WA) as published recently
(Fliri Loging Thadejo amp VOlkman 2005) The data for
10 assays has been arbitrarily encoded as inhibitors (gt50
inhibition) or non-inhibitors (lt50 inhibition) in a text file
which was then loaded into MetaCore The analyzed
network algorithm was then used which generates a large
network and fragments it into sub-networks each with a Z-
score and p-values for ranking according to saturation with
objects from the initial gene list The Gene-ontology
processes are also mapped to the gene list and individual
networks In this example a statistically significant network
was generated for the different proteins (Fig 4
p =2838e31) This network also maps the Gene Ontology
processes for the activation of MAPK (118 of genes p
value 9143e07) signal transduction (333 1600e05)
regulation of transcription DNA-dependent (294
2786e04) regulation of inflammatory response (39
3746e04) and the regulation of blood pressure (78
4230e04) This example network indicates how molecules
of the same or different therapeutic classes could be
evaluated for their effects as a graph either together as in
this case or individually This would be useful to indicate
potential off target effects and identify structurally dissimilar
molecules with similar network patterns Such networks
could then be compared to assess network overlap or
differences between molecules and their inhibition of
multiple proteins This type of unique visualization of high
throughput screening data illustrates how the target proteins
may be connected as a network to infer the possible
downstream effects of inhibition
With the burgeoning number of freely available online
and commercial databases that can be used for pathway
construction numbering in the hundreds there have been
suggestions to impose standards for model exchange
querying and visualization (Cary Bader amp Sander 2005)
To date there has been little discussion with regards to
standardization of ADMETox related databases although
there has been considerable discussion relating to drug
metabolism database generation (Erhardt 2003) This is
certainly an important area to address in the future There is
already a growing literature related to ADMETox that is
partially captured in the several commercially available
databases (Ekins et al 2005e) but to date there have been
limited academic efforts to capture data for transporters
with the human membrane transporter database (Yan amp
Sadee 2000) TP-search transporter database (httpwww
ilabrisewasedaacjptp-search) drug interaction database
(httpwwwdruginteractioninfoorgDatabaseinfoaspx)
nuclear hormone receptors (Nakata Yukawa Komiyama
Nakano amp Kaminuma 2002) the ADME-AP database
(Sun Ji Chen Wang amp Chen 2002) and PharmaGKB
(Oliver et al 2002) DSStox (httpwwwepagovnheert
dsstox) TOXNET (httptoxnetnimnihgov) that are
readily accessible
In order to generate accessible pathways using any of
the available software a large enough set of object
identifiers are required to map onto the underlying data-
base To demonstrate this datasets from toxicogenomics
studies have been evaluated with both the KEGG pathway
database and a commercially available product MetaCore
(wwwgenegocom) These gene or protein lists range in
size from 21 to 1853 objects In virtually all cases more
identifiers are mapped to networks in MetaCore and this
also seems independent of the identifier type used
(Unigene Affymetrix Genbank or Locuslink Table 5)
These numbers will obviously change as more information
is added to the respective databases hence the more
objects that are mapped from a dataset The more extensive
Table 4
Literature derived toxicoproteomics data
Compounds Data source Microarray type Compound dose Microarray data
availability
Reference
Carbon
tetrachloride
Male Wistar rats Affymetrix rat chip 8799
probes+proteomics
study
6ndash24 h exposure Genbank SwissProt and
binary data in a
manuscript table
(Fountoulakis
2004)
Paracetamol CD-1 male mice Custom mouse tox blots
with 450 genes
RTQ-PCR+proteomics
study 2-DIGE+
MALDI-MS
150 or 500 mgkg Gene name IMAGE ID
GenBank accession
number fold induction
SwissProt identifier and
protein abundance
change in manuscript
tables
(Ruepp Tonge
Shaw Wallis amp
Pognan 2002)
Paracetamol AP-1 male mice Affymetrix murine 11K
set+proteomics study
Up to 500 mgkg Gene names and fold
changes at multiple
doses and time points in
a manuscript tablemdash
proteomics data not
accessible
(Coen et al 2004)
Oxazepam or
Wy-14643
Male B6C3F1 mice NIEHS Mouse Chip
(8700 genes) 2-DIGE
and MS
Oxazepam (2500 ppm)
Wyeth (Wy)-14643
(500 ppm)
Proteomics data in a
manuscript table
Genbank accession
number gene name and
fold change in a
manuscript table data
also available to
download from NIEHS
website
(Iida et al 2003)
Compound A
(PPAR gamma
ligand)
Female rats CrlCD
(SD)IGS BR
Proteomics 2-DIGE and
MS
250 mgkgday up to
5 days
Accession number and
protein name and
average ratio in a
manuscript table
(Meneses-Lorente
et al 2004)
Bromobenzene Male Wistar rats Proteomics 2-DIGE and
MS custom 3000 cDNA
rat chip
5 mmolkg Accession number gene
name fold change in a
manuscript table protein
names along with bar
charts
(Heijne Stierum
Slijper van
Bladeren amp van
Ommen 2003)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 49
the network that can be generated (as it will consist of more
nodes) then a more comprehensive understanding of the
networks is possible The data available currently in the
literature can be used to evaluate such pathway and
network generation tools Recently we have used several
of the published studies (Tables 1ndash3) with MetaCore to
visualize networks for acetaminophen furan carbon tetra-
chloride benzene and cisplatin showing genes involved in
In vivo
In vitro
In Silico
1970s 1980s 1990s 2000s
Systems Biology ()
OMICS
Fig 2 The timeline for major paradigms in ADMETox
oxidative stress (Ekins Giroux Nikolsky Bugrim amp
Nikolskaya 2005c)
Other important pathwaynetwork building tools that could
potentially be applied to toxicogenomics data include
Ingenuity pathways analysis (httpwwwingenuitycom)
PathArt (httpwwwjubilantbiosyscompdhtm) Pathway
Assist (httpwwwariadnegenomicscomproctspathway
html) (Nikitin et al 2003) and several other databases
deposited at the Pathway Resource List (httpcbiiomskcc
orgprl) All of these products have unique underlying
proprietary pathway databases which are compiled manually
or automatically with text mining tools or a combination of
both We are still waiting for studies that provide a comparison
of different database tools or network building algorithms
Until then theremay be someoverlap but also some differences
between the results obtained from more than one network
method due to the database content and the algorithms used
The reader is recommended to evaluate for themselves several
technologies and select those with the most appropriate fit to
their own specialized needs
In the next Sections the further application of some
available network and database tools will be described with
Microarray or other high throughput data
Upload list of geneprotein identifiers and fold change significance etc (raw data or after selection of the differentially expressed
Genes using other statistical methods)
Parse database and generate interaction network with different algorithms or visualize on maps
Filter Networks
Access significance of the interaction networks
Determine GO processes
Compare 2 or more networks intersection overlap etc
Export gene list
Export gene list
generate interaction network with different algorithms or visualize on maps
Visualization
Fig 3 Schematic for the utilization of pathway tools for assessing high throughput data
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6650
specific detailed reference to transporters and enzymes but
these technologies can also be applied elsewhere as
described above
Fig 4 Network for high throughput screening data for clotrimazole and ticona
cholecystokinin CCR1 Choline transporter Chloride channel catechol-O-methyl
and co workers (Fliri et al 2005) Nodes highlighted with large blue circles rep
molecules showing gt50 inhibition in the assay blue circles represents both mol
inhibits gt50 and one inhibits lt50 Ligands (large) linked to transcriptional f
(wwgenegocom) (For interpretation of the references to colour in this figure leg
311 The role of transporters
A diverse array of organic solutes such as nutrients
neurotransmitters and drugs are transported by specialized
zole screened against 10 in vitro assays (Human Cannabinoid 1 Human
transferase COX2 CYP1A2 CYP2B6 CYP2C19) data published by Fliri
resent assays used in the study Red circles on these nodes represent both
ecules showing lt50 inhibition chequered circles represent one molecule
actors enzymes and transporters via edges using the MetaCorei database
end the reader is referred to the web version of this article)
Table 5
Datasets acquired from publications and other sources and used to evaluate two databasepathway tools KEGG and metaCore
Dataset Number of objects
genes proteins in list
Number of objects
mapped with KEGG
Number of objects mapped
with MetaCore on networks
Reference for microarray
data gene list
Tox Chip 10 (NIEHS) 1853 405 1498 httpsdir-appsniehsnihgov
mapsguestclonesrchcfm
4-Hydroxytamoxifen and
estrogen
1617 434 1343 (Hodges et al 2003)
Mitochondrial proteins 722 156 388 (Gaucher et al 2004
Taylor et al 2003)
Bromobenzene
(24 and 48 h)
130 41 89 (Heijne et al 2004)
Acetaminophen 30 19 23 (Heinloth et al 2004)
Acetaminophen 84 29 64 (Huang et al 2004)
Furan 185 64 139 (Huang et al 2004)
Tetrodotoxin 116 31 86 (Raghavendra Prasad et al
2004)
Benzene 73 16 62 (Yoon et al 2003)
Benzene 76 5 53 (Faiola et al 2004)
Carbon tetrachloride 37 8 26 (Young et al 2003)
Estrogen 94 33 90 (Lobenhofer et al 2002)
Trovafloxacin 142 20 82 (Liguori et al 2005)
Phenobarbital 37 13 28 (Ueda et al 2002)
L-742694 (liver)+ 45 17 19 (Hartley et al 2004)
L-742694 (intestine)+ 23 11 10 (Hartley et al 2004)
A-277249 21 7 9 (Waring et al 2002)
All files were converted to Locuslink identifiers except as noted () unigene () Affymetrix (+) Genbank identifiers were used
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 51
proteins across cellular membranes These may function as
passive processes or active processes energized by the
hydrolysis of ATP or coupling to the co-transport of counter
ions down an electrochemical gradient such as Na+ H+ and
Cl There are many thousands of transporters which can be
classified into distinct superfamilies One of these the solute
carrier class (SLC) is rapidly expanding and contains over
30 families and 200 members The ATP-binding cassette
(ABC) contains 7 families and over 48 members including
P-glycoprotein (P-gp) and MRP subfamilies (Zhang Knipp
Ekins amp Swaan 2002a Zhang Phelps Cheng Ekins amp
Swaan 2002b) Transporters have a key role in clinical
pharmacology with many drugs specifically targeting them
Numerous drugs share transport pathways with nutrients
and transporters have a role in oral absorption drug
bioavailability drug resistance excretion and ultimately
pharmacokinetics and pharmacodynamics
Polymorphism of drug transporters may be a key factor
in drug interactions and lack of effectiveness This field has
become known as pharmacogenomics and is focused on
understanding of inherited DNA sequence variations (poly-
morphisms and mutations) revealed by xenobiotics (Evans
amp McLeod 2003 Weinshilboum 2003) Over the last few
decades many genes have been directly linked to the
mechanisms of response (Evans amp McLeod 2003 Wein-
shilboum 2003) such that 20ndash95 of variability to drug
response is inheritable (Evans amp McLeod 2003) This
phenotypical variability is mainly caused by single nucleo-
tide polymorphisms (SNPs) present in anywhere from 1 to
50 of the population resulting in either lower protein
activity incorrect folding or rapid degradation via proteo-
somes (Weinshilboum amp Wang 2004) A number of
structurally diverse molecules bind to P-gp which is
expressed in many tissues and has numerous SNPs one of
which (C3435T) affects the expression level in the
duodenum and therefore can impact the absorption of
molecules which would be substrates for this transporter
(Sakaeda Nakamura amp Okumura 2002) The human
proton-dependent dipeptide transporter (hPEPT1) can also
affect the absorption of molecules in the intestine and
recently 9 SNPs were found with only one displaying a
reduced transport capacity (Zhang et al 2004) The sodium-
dependent carnitine cotransporter OCTN2 can possess
mutations and these result in primary carnitine deficiency
which impacts fatty acid oxidation and is characterized by
many clinical manifestations (Lahjouji Mitchel amp Qureshi
2001) The organic cation transporter 1 (OCT1) is also
important in the transport of numerous xenobiotics and
endobiotics Recently 4 SNPs were identified in the
Japanese population and when functionally characterized
in vitro the uptake of cations was reduced significantly for
some of these mutations indicating that this would likely
contribute to inter-individual variations in metabolism of
drugs which were transported via OCT1 (Sakata et al
2004)
3111 Clinical relevance of transporters The pregnane
X-receptor (PXR) is a transcriptional regulator of the
enzyme human MDR1 (P-gp) MRPs and OATP (Wang amp
LeCluyse 2003) CYP3A (Bertilsson et al 1998 Blumberg
et al 1998 Kliewer et al 1998) and CYP2C89 (Synold
Dussault amp Forman 2001) as well as many other genes
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6652
involved in the transport metabolism and biosynthesis of
bile acids (Staudinger Liu Madan Habeebu amp Klaassen
2001) However the additional receptors CAR FXR LXR
and other nuclear receptors take part in a complex network
of interactions to control these and other proteins Thus
elucidation of the regulatory networks which control the
expression of these transporters is also important To date
most of the research has centered on efflux transporters but
there has been considerable interest in uptake transporters
such as the organic anion transporter polypeptide (OATP
((Kim 2003) see also later Section on OATP)
There are several specific examples of the importance of
drug transporters to the clinical development of drugs One
example is the insulin sensitizer troglitazone which was
withdrawn due to hepatotoxicity although the precise
mechanism appears to have been unclear until recently
The major metabolite is a sulfated species and is suspected
of being responsible for the observed toxicity The recent
assessment of the organic anion transporting polypeptides
OATP-C and OATP8 expressed on the hepatocyte baso-
lateral membrane indicated that sulphated troglitazone has a
high affinity for the former and possibly lower affinity for
the latter (Nozawa et al 2004) This metabolite would
therefore be expected to accumulate in hepatocytes and
inhibit the bile salt export pump and Mrp2 Because
polymorphisms have been shown for OATP-C (Tirona
Leake Merino amp Kim 2001) it is also possible that these
may result in the accumulation of the metabolite and in turn
elicit idiosyncratic toxicity A second relevant example of
the impact of transporters is the clinically significant drugndash
drug interaction between cerivastatin and cyclosporine A
which occurs via the OATP-C transporter (Shitara Itoh
Sato Li amp Sugiyama 2003) A third example are the HIV
protease inhibitors saquinavir ritonavir and indinavir which
are transported by MRP2 in vitro and other drugs such as
probenecid and sulfinpyrazone are able to enhance this
transport Transport by MRP2 suggests that these com-
pounds will have decreased bioavailability due to increased
clearance and other drugs could aggravate this situation by
further enhancing transport (Huisman et al 2002) Sim-
ilarly the rifampicin mediated induction via PXR of MRP2
and P-gp in healthy subjects was found to significantly
decrease the AUC and also correlated with intestinal
expression of these transporters This transporter is also
inducible by cisplatin 2-AAF and phenobarbital (Schrenk
et al 2001) indicating multiple mechanisms may be
involved In other species such as rat commonly used as a
toxicity model orthologs of the transporters such as OATP2
are expressed and can be induced with ligands for PXR like
PCN (Guo Choudhuri amp Klaassen 2002) This is useful
knowledge because the advent of microarray technology
allows one to dose a rat with a xenobiotic and assess
thousands of genes simultaneously in a particular tissue For
instance animals dosed with known nephrotoxins have
shown some upregulation of the NandashKndashCl transporter
however the authors suggested genomic responses are
stronger soon after exposure before declining (Fleck et
al 2003) Some transporters may therefore be specifically
targeted by drugs in one tissue such as the CNS but these
same transporters may also be expressed elsewhere in the
body hence off-target effects may result in toxicity The
serotonin transporter is one such example which is
expressed in the lungs and brain Some substrates for this
channel like fenfluramine can result in primary pulmonary
hypertension as they accumulate in pulmonary cells (Roth-
man Ayestas Dersch amp Baumann 1999) Similarly P-gp is
expressed at the blood brain barrier and intestine impacting
the efficacy and bioavailability of drugs
3112 Transporter network examples ABCA1 The
ABCA1 transporter mediates the first step of cholesterol
transport Mutations in this gene cause Tangier disease
which results in severe HDL deficiency cholesterol
accumulation in macrophages and attendant atherosclerosis
This transporter represents a drug target for upregulation
modulating cholesterol metabolism and prevention of
cardiovascular disease (Oram amp Lawn 2001) In vitro
ABCA1 can be inhibited by the sulfonylurea glybenclamide
(Field Burn amp Mathur 2004 Wang Silver Thiele amp Tall
2001) In order to illustrate the advantages of mapping drug
transporters as networks onto functional models alongside
other proteins one can consider the example of ABCA1
The query of the MetaCorei database shows that this
transporter appears on three manually curated pathway maps
representing the Fstate of the art_ knowledge derived from
reliable high quality literature sources One can also use the
individual maps as an interface to access the underlying
layers of information about the transporter including the list
of encoding genessplice variants with known SNPs In
addition to browsing MetaCorei a user can also build
custom networks around ABCA1 using the network-
construction tool (Fig 5) Such a visualization utility may
be very helpful for identification of all putative pathways
around a particular transporter or compound of interest The
ABCA1 network created by this tool shows that this
transporter at the time of writing is linked directly to
twenty-five other objects such as APOE1 and LXR Many
of its neighbors have their own SNPs that could be
important in determining interactions between transport of
a drug and normal human transport of endogenous ligands
in health or disease
3113 Transporter network examples OATP The
OATPs are key membrane bound transporters expressed in
many organs including intestine liver lung choroid plexus
blood brain barrier and other organs (Tamai et al 2000)
This family of transporters is capable of mediating the
sodium-independent transport of a diverse array of mole-
cules such as steroid conjugates organic anions and
xenobiotics by coupling uptake with efflux of bicarbonate
(Satlin Amin amp Wolkoff 1997) glutathione or its
conjugates (Hagenbuch amp Meier 2004) The inhibition of
Fig 5 The network of direct gene interactions around the ABCA1 transporter gene (centre) generated with MetaCorei The interation types between nodes are shown as small colored hexagons eg unspecified
allosteric regulation binding cleavage competition covalent modification dephosphorylation phosphorylation transcription regulation transformation When applicable interactions also have a positive or
negative effect and direction Ligands (purple) linked to other proteins (blue blobs) transfactors (red) enzymes (orange) (For interpretation of the references to colour in this figure legend the reader is referred to
the web version of this article)
SEkin
sJournalofPharm
acologica
landToxico
logica
lMeth
ods53(2006)38ndash66
53
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6654
this transporterrsquos hepatic uptake of other compounds may be
important for reported drugndashdrug interactions (Kim 2003)
described earlier (Shitara et al 2003) as well as cerivastatin
with gemfibrozil (Shitara Hirano Sato amp Sugiyama 2004)
OATP1B1 (previous names OATP-C LST-1 OATP2
SLC21A6) represents the most studied human OATP to
date (Meier amp Stieger 2000) and is expressed on the
basolateral plasma membrane of hepatocytes Several single
nucleotide polymorphisms have been identified in the
OATP1B1 gene in European-Americans African-Americans
(Tirona et al 2001) and Japanese (Nozawa et al 2002)
dramatically impacting the transport of ligands such as
pravastatin (Mwinyi Johne Bauer Roots amp Gerloff 2004
Nishizato et al 2003) estrone-3-sulfate (Nozawa et al
2002 Tirona et al 2001) Rifampin (Tirona Leake
Wolkoff amp Kim 2003) and estradiol 17h-d-glucuronide(Tirona et al 2001)
The regulation of SLCO may be affected during
extrahepatic cholestasis bile duct ligation bile salt induced
Fig 6 Networks generated with the autoexpand algorithm in MetaCorei to illustr
be interconnected with other protein regulatory signaling information Informa
unspecified allosteric regulation binding cleavage competition covalent m
transformation When applicable interactions also have a positive or negative e
transfactors (red) enzymes (orange) (For interpretation of the references to colour
cholestatic hepatitis (Dumont et al 1997 Meier amp Stieger
2000 Rost et al 2003) and primary sclerosing cholangitis
(Oswald Kullack-Ublick Paumgarter amp Beuer 2001) In
particular OATP1B1 appears to be regulated by the liver-
enriched transcription factor hepatocyte nuclear factor 1a
(HNF1a) which binds to the promoter region of this
transporter (Jung et al 2001) Site directed mutagenesis
of this binding site resulted in inactivation suggesting the
critical nature of the interaction with HNF1a Bile acids
such as CDCA have been shown to transcriptionally repress
HNF1a in vitro via inhibition of the transactivating effect of
HNF4a on HNF1a (Jung amp Kullak-Ublick 2003) After
screening many rat and human uptake transporters in vitro
OATP1B1 was also shown to modulate the PXR response
by controlling rifampin retention in the cell and therefore
affecting the induction of CYP3A4 and other gene products
such as P-gp (Tirona et al 2003)
Some of the literature for OATP1B1 human substrate
data has been annotated into MetaCorei to illustrate the
ate how ligands for the human OATP-C (OATP1B1) transporter (centre) can
tion on the type of interaction between objects is hidden for clarity eg
odification dephosphorylation phosphorylation transcription regulation
ffect and direction Ligands (purple) linked to other proteins (blue blobs)
in this figure legend the reader is referred to the web version of this article)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 55
visualization of the complex interconnections between this
transporter its ligands regulatory factors and signaling
molecules already in the database (Fig 6) The network was
generated with the autoexpand algorithm in the software
representing one of multiple available algorithms for
connecting genes ligands and other objects in the database
Clearly if more ligands and their connections are added to
the database the complexity of the network will increase
considerably The OATP-C (OATP1B1) gene details can be
viewed upon querying the database and links are provided
to other public databases This page can be used to highlight
the multiple synonyms for this gene as well as links to the
multiple SNPs identified to date
3114 Transporter microarray data Microarrays have
generally been limited in the number of transporters present
on them (Annereau et al 2004) however they have been used
in an attempt to correlate pharmacokinetic properties with
gene expression for valacyclovir (Landowski et al 2003) as
well as understand the expression profile in different tissues
or cell lines upon food component or xenobiotic treatment
(Anderle Huang amp Sadee 2004) This lack of transporters
on microarrays has prompted some groups to produce their
own arrays with a heavier emphasis on transporters These
arrays have for example then been used to demonstrate the
upregulation of ABC transporters and down-regulation of
GST-Pi in cell lines resistant to colchicines or 9-nitro-
camptothecin (Annereau et al 2004) The genes that were
significantly up or down-regulated in this particular study
were used to build networks with MetaCore (Fig 7A B) and
the similarities between them were assessed (Fig 7C)
Although there were only a small number of significantly
changed genes in common (IL-8 Fos GST-Pi Calpactin and
Ubiquitin hydrolase) it is perhaps likely that there is a much
larger common gene network that is important for drug
resistance although a much larger number of cell lines and
drug treatments need to be evaluated to produce a definitive
drug resistance signature involving transporters enzymes
and transcriptional regulators
312 Applications to enzymes
As we have already described it may be particularly
valuable to visualize enzymes as networks to show
interactions with transcriptional regulators and ligands
For example a key enzyme is CYP3A4 which metabolizes
40ndash45 of all drugs and has relatively few SNPs
(Ingelman-Sundberg 2004) Using a second software suite
MetaDrugi (wwwgenegocom) it is possible to construct
a custom network around this or other drug metabolizing
enzymes (Fig 8) In this case the gene network for
CYP3A4 highlights all of the major transcriptional
regulators and several more distant linked proteins and
ligands connected on the network that may be useful for
further study (Fig 8) Substrates inhibitors as well as
regulatory factors and other enzymes can be observed
connected on this network Due to the many hundreds of
known inhibitors and substrates only a small number are
shown here for clarity We assume that if a perturbation in
a pathway (eg due to a nonfunctional enzyme) is linked
to a certain pathologic condition a similar perturbation
caused by the interference from xenobiotic metabolism
(eg competitive inhibition of the same enzyme) may
result in identical effects As microarray gene expression
data is increasingly generated the role of enzyme
regulation in toxicity of certain xenobiotics will become
more apparent from either in vivo or in vitro studies The
visualization of such signature gene networks involving
transporters and enzymes their ligands and regulatory
factors will also be important for future toxicity prediction
methods We have recently generated visualizations of
microarray data from MCF-7 cells treated with 4-hydrox-
ytamoxifen to show that some of the key genes involved in
metabolism and transport are upregulated (Ekins et al
2005d) In addition we have made predictions with various
QSAR models in MetaDrug to indicate the involvement of
PXR CYP3A4 and P-gp (Ekins et al 2005e) Therefore it
appears likely that 4-hydroxytamoxifen could increase its
own metabolism as well as efflux from cells via P-gp
which can be visualized on networks Any decrease of
function of these enzymes or transporters in a population
would likely result in changes in the metabolism and
transport of this active metabolite potentially impacting
the clinical effect This represents one example of how
both pathway tools QSAR models and network building
algorithms can be used with different types of predicted
and experimental data to allow visualization of potential
compound interactions or toxicity
313 Future network applications
As the population ages an increasing incidence and
prevalence of systemic diseases especially chronic diseases
have occurred among older adults This has resulted in an
increase in medications used concomitantly by this
population which presents challenges for drugndashdrug
interactions Physiologically elderly patients may behave
differently to the younger patients for which the drugs are
initially developed for and tested on Many pharmacoki-
netic investigations in the elderly population reveal
decreased clearance of lipophilic drugs metabolized by
the cytochrome P450 enzymes however few studies have
evaluated aging-dependent or gender-related changes in
specific P450 enzymes (Hunt Westerkam amp Slave 1992)
Age-related physiological changes such as a reduction in
liver mass hepatic metabolizing enzyme activity liver
blood flow and alterations in plasma drug binding may
account for the decreased elimination of some metabolized
drugs in the elderly It is particularly difficult to separate an
effect of aging from the variation in the rate of metabolism
due to factors such as individual metabolic phenotype
(slow or fast metabolizer due to SNPs) environmental
influences concomitant disease states and drug intake
(drugndashdrug interactions) The available data suggest that
Fig 7 Networks for drug resistant cell lines generated with the Auto expand algorithm in MetaCore A KB-3-1 vs KB-8-5 B ATC-DU-145 vs RCO1 C node overlap of A and B with initial nodes highlighted
with blue or red solid circles (Initial gene lists derived from (Annereau et al 2004)) Information on the type of interaction between objects is hidden for clarity eg unspecified allosteric regulation binding
cleavage competition covalent modification dephosphorylation phosphorylation transcription regulation transformation When applicable interactions also have a positive or negative effect and direction
Ligands (purple) linked to other proteins (blue blobs) transfactors (red) enzymes (orange) (For interpretation of the references to colour in this figure legend the reader is referred to the web version of this article)
SEkin
sJournalofPharm
acologica
landToxico
logica
lMeth
ods53(2006)38ndash66
56
Fig7(continued)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 57
Fig7(continued)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6658
Fig 8 The network of gene interactions around the CYP3A4 enzyme (centre) from MetaCorei Information on the type of interaction between objects is
hidden for clarity eg unspecified allosteric regulation binding cleavage competition covalent modification dephosphorylation phosphorylation
transcription regulation transformation When applicable interactions also have a positive or negative effect and direction Ligands (purple) linked to other
proteins (blue blobs) transfactors (red) enzymes (orange) (For interpretation of the references to colour in this figure legend the reader is referred to the web
version of this article)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 59
the initial doses of drugs metabolized by these enzymes
should be reduced in older patients according to the clinical
response In most published studies the elderly appear at
least as responsive as the young to inducers or inhibitors of
P450s (Durnas Loi amp Cusack 1990) More recently there
has been some suggestion that there are age related
reductions in function of some specific P450s such as
CYP3A4 (Patki et al 2004) and this could occur at the
level of regulation However earlier studies with the same
enzyme showed no change in clearance with age (Hunt et
al 1992) To date there has been even less examination of
the transporter functions and any changes with age
(Kinirons amp OrsquoMahony 2004) so the current understand-
ing of the effects of aging on metabolism and transport is
anything but transparent This represents an extreme
challenge for the pharmaceutical industry how to predict
whether a drug has an affinity for an enzyme or transporter
and whether this may be clinically important if it shows a
decline in expression or function with aging Any decrease
of function of these enzymes or transporters in an elderly
population would result in changes in the metabolism and
transport of metabolites potentially impacting the clinical
effect This represents another example of how computa-
tional approaches may perhaps be used with different types
of predicted and experimental data to allow visualization of
potential compound interactions or toxicity in elderly
populations We may see different gene networks high-
lighted as humans age and these may be modified by drug
treatment and coadministration This represents an area
were network analysis could be applied in the future and is
in need of considerable further research
The collection of microarray data in databases such as
CEBS ArrayTrack and EDGE represents a future resource
for computational gene network analysis One could
envisage that ultimately in each case such data is converted
to one or more networks that are also displayed for the user
and can be used to compare treatments from in vivo and in
vitro experiments This would represent a different approach
to clustering the data as currently implemented in one of
these efforts (Hayes et al 2005) and may condense large
amounts of experimentally derived data into a readily
interpreted network
4 Discussion
Previously in this journal the progress of many
research groups in predicting human ADME parameters
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6660
in silico (Ekins et al 2000b) and approaches for drug
metabolism (Ekins Ring Grace McRobie-Belle amp
Wrighton 2000a) have been described Both of these
reviews commented on moving HT assays for ADME
Tox much earlier in drug discovery which had also been
indicated by other groups The initial reviews also
highlighted the likely wealth of data that would become
available and how this could be used for structure
activity relationships alongside the bioactivity data in
computational models It was noted that there was a
paucity of predictive metabolism tools at that time In
addition a growing number of efforts to model whole
cells and organs now a field called systems biology
were recognized as models that could be integrated with
the in silico ADME approaches In summary since these
past reviews virtually all pharmaceutical companies have
attempted earlier high throughput screening for ADME
Tox properties and to some extent the wider application
of computational approaches for physicochemical proper-
ties Systems biology is being quite widely acknowledged
as the new paradigm for understanding complex bio-
logical datasets derived from high throughput technolo-
gies and the accumulated knowledge on human protein
interactions (Hartwell Hopfield Leibler amp Murray 1999
Hood 2003b) Therefore systems biology can be defined
as the integration of genetic proteomic transcriptomic
and metabonomic data using computational methods
(Nicholson amp Wilson 2003) When taken together
information on molecular processes derived from different
sources represents a lsquolsquouniversersquorsquo of putative biological
functionality of which only a small fraction of it will be
realized in a cell at any given time To date systems
biology has been driven by academia and funding bodies
such as the NIH rather than the pharmaceutical compa-
nies Presently there is a great deal of interest from
scientists of all backgrounds in identifying the networks
of cellular pathways and the corresponding physically
interacting proteins
The network building software for systems biology
described in this current review will be valuable to query
high throughput data and known literature interactions in
order to predict potential toxicity in different species In the
future the compilation of published toxicogenomics data-
sets characteristic of different types of toxicity will likely
be available in these software systems to act as a reference
database It is also feasible that we will be able to generate
the annotated datasets which specifically address the
differences between human and rat networks implicated
in toxicity The identification of sub-network modules
conserved between human and rat distinct for toxicity
types or predictive for toxic end-points in human will be
possible Such signature gene networks (Nikolsky et al
2005) can then be verified with other experimentally
derived data prospectively or from preexisting databases
The combined Fhigh throughput datandashin silico network_approaches therefore represents a novel method for access-
ing complex tissue-level processes as networks integrating
all data types based on functional interactions The data-
bases developed and used in this approach will certainly
benefit from further annotation around the drug metaboliz-
ing enzymes and transporters as described herein in terms
of transcriptional regulation and the ligands associated with
them which frequently appear in the literature It will be
important to capture disparities in the assignment of ligands
to enzymes transporters and other proteins as well as
negative data
In summary although there have been numerous
toxicogenomics studies published there is presently a
relatively small number of datasets that are freely available
to perform network analysis of microarray data The
number of studies identifying large numbers of proteins
which are affected by molecule treatment are even scarcer
still while there are several examples of some published
studies that combine such data It is hoped that the
numerous database initiatives for high content and toxicol-
ogy data that are being undertaken will improve the
situation for other researchers that are not currently
equipped to do such microarray studies themselves The
addition of requirements by journals to deposit such raw
data in a freely accessible resource will aid these initiatives
ADMETox groups have seen new technologies and
approaches developed over the last decade that have all
been applied to identify poor compounds earlier (Fig 2)
The latest technologies integrate network building tools
with high content data and databases The current review
described the limited number of networks generated for
ADMETox at present and one hopes that the impact of
such analyses will be commonplace in the future Systems
biology is however more than just applying a network
approach and hence systems-ADMETox will have to
evolve due to the continual pressure to develop newer
technologies This current paradigm combining empirical
data and computational methods should integrate the
complex data already generated making it readily inter-
pretable and valuable for identifying the most promising
compounds in the future
Acknowledgements
Dr Maggie AZ Hupcey is gratefully acknowledged for
editorial assistance Dr Peter W Swaan (University of
Maryland) and Dr Cheng Chang (Ohio State University)
Dr Steve Wright (University of Arizona) Dr K Sandy
Pang (University of Toronto) and Dr Craig Giroux (Wayne
State University) are kindly acknowledged for their support
and discussions My colleagues at GeneGo Sergey
Andreyev Andy Ryabov Eugene Kirillov Eugene A
Rakhmatulin Svetlana Sorokina Andrej Bugrim Tatiana
Nikolskaya Yuri Nikolsky John Metz and Julie Bryant are
all thanked for their considerable contributions to software
development and data annotation
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 61
References
Abe M Yamashita S Kuramoto T Hirayama Y Tsukamoto T Ohta
T et al (2003) Global expression analysis of N-methyl-NV-nitro-N-nitrosoguanidine-induced rat stomach carcinomas using oligonucleotide
microarrays Carcinogenesis 24 861ndash867
Adachi T Ono Y Koh K B Takashima K Tainaka H Matsuno Y
et al (2004) Long-term alteration of gene expression without
morphological change in testis after neonatal exposure to genistein in
mice Toxicogenomic analysis using cDNA microarray Food and
Chemical Toxicology 42 445ndash452
Akerman G S Rosenzweig B A Domon O E McGarrity L J
Blankenship L R Tsai C A et al (2004) Gene expression profiles
and genetic damage in benzo(a)pyrene diol epoxide-exposed TK6 cells
Mutation Research 549 43ndash64
Aksenov S V Church B Dhiman A Georgieva A Sarangapani R
Helmlinger G et al (2005) An integrated approach for inference and
mechanistic modeling for advancing drug development FEBS Letters
579 1878ndash1883
Anderle P Huang Y amp Sadee W (2004) Intestinal membrane transport
of drugs and nutrients Genomics of membrane transporters using
expression microarrays European Journal of Pharmaceutical Sciences
21 17ndash24
Annereau J P Szakacs G Tucker C J Arciello A Cardarelli C
Collins J et al (2004) Analysis of ATP-binding cassette transporter
expression in drug-selected cell lines by a microarray dedicated to
multidrug resistance Molecular Pharmacology 66 1397ndash1405
Apic G Ignjatovic T Boyer S amp Russell R B (2005) Illuminating drug
discovery with biological pathways FEBS Letters 579 1872ndash1877
Baker V A Harries H M Waring J F Duggan C M Ni H A Jolly
R A et al (2004) Clofibrate-induced gene expression changes in rat
liver A cross-laboratory analysis using membrane cDNA arrays
Environmental Health Perspectives 112 428ndash438
Balakin K V Ekins S Bugrim A Ivanenkov Y A Korolev D amp
Nikolsky Y et al (2004a) Quantitative structurendashmetabolism relation-
ship modeling of the metabolic N-dealkylation rates Drug Metabolism
and Disposition 32 1111ndash1120
Balakin K V Ekins S Bugrim A Ivanenkov Y A Korolev D amp
Nikolsky Y et al (2004b) Kohonen maps for prediction of binding to
human cytochrome P450 3A4 Drug Metabolism and Disposition 32
1183ndash1189
Barabasi A -L amp Oltvai Z N (2004) Network biology Under-
standing the cellrsquos functional organization Nature Reviews Genetics
5 101ndash113
Bartosiewicz M Penn S G amp Buckpitt A (2001) Applications of gene
arrays in environmental toxicology Fingerprints of gene regulation
associated with cadmium chloride benzo(a)pyrene and trichloro-
ethylene Environmental Health Perspectives 109 71ndash74
Bertilsson G Heidrich J Svensson K Asman M Jendeberg L
Sydow-Backman M et al (1998) Identification of a human nuclear
receptor defines a new signaling pathway for CYP3A induction
Proceedings of the National Academy of Sciences of the United States
of America 95 12208ndash12213
Blumberg B Sabbagh Jr W Juguilon H Bolado Jr J van Meter
C M Ong E S et al (1998) SXR a novel steroid and xenobiotic-
sensing nuclear receptor Genes and Development 12 3195ndash3205
Borodina Y Rudik A Filimonov D Kharchevnikova N Dmitriev A
Blinova V et al (2004) A new statistical approach to predicting
aromatic hydroxylation sites Comparison with model-based
approaches Journal of Chemical Information and Computer Sciences
44 1998ndash2009
Borodina Y Sadym A Filimonov D Blinova V Dmitriev A amp
Poroikov V (2003) Predicting biotransformation potential from
molecular structure Journal of Chemical Information and Computer
Sciences 43 1636ndash1646
Bottone Jr F G Martinez J M Collins J B Afshari C A amp Eling
T E (2003) Gene modulation by the cyclooxygenase inhibitor
sulindac sulfide in human colorectal carcinoma cells Possible link to
apoptosis Journal of Biological Chemistry 278 25790ndash25801
Boyer S amp Zamora I (2002) New methods in predictive metabolism
Journal of Computer-Aided Molecular Design 16 403ndash413
Bredel M amp Jacoby E (2004) Chemogenomics An emerging strategy
for rapid target and drug discovery Nature Reviews Genetics 5
262ndash275
Bugrim A Nikolskaya T amp Nikolsky Y (2004) Early prediction of drug
metabolism and toxicity Systems biology approach and modeling
Drug Discovery Today 9 127ndash135
Bulera S J Eddy S M Ferguson E Jatkoe T A Reindel J F
Bleavins M R et al (2001) RNA expression in the early character-
ization of hepatotoxicants in Wistar rats by high-density DNA micro-
arrays Hepatology 33 1239ndash1258
Butcher E C Berg E L amp Kunkel E J (2004) Systems biology in drug
discovery Nature Biotechnology 22 1253ndash1259
Butte A (2002) The use and analysis of microarray data Naturalist
Review of Drug Discovery 1 951ndash960
Cary M P Bader G D amp Sander C (2005) Pathway information for
systems biology FEBS Letters 579 1815ndash1820
Cheok M H Yang W Pui C H Downing J R Cheng C Naeve
C W et al (2003) Treatment-specific changes in gene expression
discriminate in vivo drug response in human leukemia cells Nature
Genetics 34 85ndash90
Cleary J D Rogers P D amp Chapman S W (2001) Differential
transcription factor expression in human mononuclear cells in response
to amphotericin B Identification with complementary DNA microarray
technology Pharmacotherapy 21 1046ndash1054
Coen M Ruepp S U Lindon J C Nicholson J K Pognan F Lenz
E M et al (2004) Integrated application of transcriptomics and
metabonomics yields new insight into the toxicity due to paracetamol in
the mouse Journal of Pharmaceutical and Biomedical Analysis 35
93ndash105
Cornwell P D De Souza A T amp Ulrich R G (2004) Profiling of
hepatic gene expression in rats treated with fibric acid analogs
Mutation Research 549 131ndash145
Csermely P Agoston V amp Pongor S (2005) The efficiency of multi-
target drugs The network approach might help drug design Trends in
Pharmacological Sciences 26 178ndash182
Cunningham M J Liang S Fuhrman S Seilhamer J J amp
Somogyi R (2000) Gene expression microarray data analysis for
toxicology profiling Annals of the New York Academy of Sciences 919
52ndash67
de Longueville F Atienzar F A Marcq L Dufrane S Evrard S
Wouters L et al (2003) Use of a low density microarray for studying
gene expression patterns induced by hepatotoxicants on primary
cultures of rat hepatocytes Toxicological Sciences 75 378ndash392
Dobrin R Beg Q K Barabasi A L amp Oltvai Z N (2004)
Aggregation of topological motifs in the Escherichia coli transcriptional
regulatory network BMC Bioinformatics 5 10
Donald S Verschoyle R D Edwards R Judah D J Davies R
Riley J et al (2002) Hepatobiliary damage and changes in hepatic
gene expression caused by the antitumor drug ecteinascidin-743 (ET-
743) in the female rat Cancer Research 62 4256ndash4262
Dumont M Jacquemin E DrsquoHont C Descout C Cresteil D Haouzi
D et al (1997) Expression of the liver Na+-independent organic anion
transporting polypeptide (oatp-1) in rats with bile duct ligation Journal
of Hepatology 27 1051ndash1056
Durnas C Loi C M amp Cusack B J (1990) Hepatic drug metabolism
and aging Clinical Pharmacokinetics 19 359ndash389
Eisen M B Spellman P T Brown P O amp Botstein D (1998) Cluster
analysis and display of genome-wide expression patterns Proceedings
of the National Academy of Sciences of the United States of America
95 14863ndash14868
Ekins S Andreyev S Ryabov A Kirilov E Rakhmatulin E A
Bugrim A et al (in press) Computational Prediction of Human Drug
Metabolism Exp Opin Drug Metab Toxicol
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6662
Ekins S Boulanger B Swaan P W amp Hupcey M A Z (2002)
Towards a new age of virtual ADMETOX and multidimensional drug
discovery Journal of Computer-Aided Molecular Design 16 381ndash401
Ekins S Bugrim A Nikolsky Y amp Nikolskaya T (2005) Systems
biology Applications in drug discovery In S Gad (Ed) Drug
Discovery Handbook (pp 123ndash183) Hoboken NJrsquo Wiley and Sons
Ekins S Giroux C N Nikolsky Y Bugrim A amp Nikolskaya T
(2005) A signature gene network approach to toxicity The Toxoco-
logists 84
Ekins S Kirillov E Rakhmatulin E amp Nikolskaya T (2005) A novel
method for visualizing nuclear hormone receptor networks relevant to
drug metabolism Drug Metabolism and Disposition 33 474ndash481
Ekins S amp McGowan R J (2001) The limits of reductionism The
shifting genomic paradigmrsquos impact on industry and academia
Philsophy in Science 9 1ndash23
Ekins S Mirny L amp Schuetz E G (2002) A ligand-based approach to
understanding selectivity of nuclear hormone receptors PXR CAR
FXR LXRa and LXRb Pharmaceutical Research 19 1788ndash1800
Ekins S Nikolsky Y amp Nikolskaya T (2005) Techniques Application
of systems biology to absorption distribution metabolism excretion
and toxicity Trends in Pharmacological Sciences 26 202ndash209
Ekins S Ring B J Grace J McRobie-Belle D J amp Wrighton S
A (2000) Present and future in vitro approaches for drug
metabolism Journal of Pharmacological and Toxicological Methods
44 313ndash324
Ekins S Waller C L Swaan P W Cruciani G Wrighton S A amp
Wikel J H (2000) Progress in predicting human ADME parameters in
silico Journal of Pharmacological and Toxicological Methods 44
251ndash272
Erhardt P W (2003) A human drug metabolism database Potential roles
in the quantitative predictions of drug metabolism and metabolism-
related drugndashdrug interactions Current Drug Metabolism 4 411ndash422
Evans W E amp McLeod H L (2003) Pharmacogenomicsmdashdrug
disposition drug targets and side effects New England Journal of
Medicine 348 538ndash549
Ezendam J Staedtler F Pennings J Vandebriel R J Pieters R
Boffetta P et al (2004) Toxicogenomics of subchronic hexachlor-
obenzene exposure in Brown Norway rats Environmental Health
Perspectives 112 782ndash791
Faiola B Fuller E S Wong V A amp Recio L (2004) Gene expression
profile in bone marrow and hematopoietic stem cells in mice exposed to
inhaled benzene Mutation Research 549 195ndash212
FDA (2004) Innovation stagnation Challenge and opportunity on the
critical path to new medicinal products
Fiehn O (2001) Combining genomics metabolome analysis and bio-
chemical modelling to understand metabolic networks Comparative
Functional Genomics 2 155ndash168
Field F J Born E amp Mathur S N (2004) LXRRXR ligand activation
enhances basolateral efflux of beta-sitosterol in CaCo-2 cells Journal of
Lipid Research 45 905ndash913
Fleck C Sutter L Appenroth D Koch B Meinhold T Pitack M
et al (2003) Use of gene chip technology for the characterisation of
the regulation of renal transport processes and of nephrotoxicity in
rats Experimental and Toxicologic Pathology 54 401ndash410
Fliri A F Loging W T Thadeio P F amp Volkmann R A (2005)
Biological spectra analysis Linking biological activity profiles to
molecular structure Proceedings of the National Academy of Sciences
of the United States of America 102 261ndash266
Fountoulakis M (2004) Application of proteomics technologies in the
investigation of the brain Mass Spectrometry Reviews 23 231ndash258
Gaucher S P Taylor S W Fahy E Zhang B Warnock D E Ghosh
S S et al (2004) Expanded coverage of the human heart
mitochondrial proteome using multidimensional liquid chromatography
coupled with tandem mass spectrometry Journal of Proteome
Research 3 495ndash505
Gerhold D Lu M Xu J Austin C Caskey C T amp Rushmore T
(2001) Monitoring expression of genes involved in drug metabolism
and toxicology using DNA microarrays Physiological Genomics 5
161ndash170
Guo G L Choudhuri S amp Klaassen C D (2002) Induction profile of
rat organic anion transporting polypeptide 2 (oatp2) by prototypical
drug-metabolizing enzyme inducers that activate gene expression
through ligand-activated transcription factor pathways Journal of
Pharmacology and Experimental Therapeutics 300 206ndash212
Hagenbuch B amp Meier P J (2004) Organic anion transporting
polypeptides of the OATP SLC21 family Phylogenetic classification
as OATP SLCO superfamily new nomenclature and molecularfunc-
tional properties Pflugers Archiv 447 653ndash665
Hamadeh H K amp Afshari C A (2004) Toxicogenomics principles and
applications Hobokenrsquo John Wiley and Sons
Hamadeh H K Bushel P R Jayadev S Martin K DiSorbo O Sieber
S et al (2002) Gene expression analysis reveals chemical-specific
profiles Toxicological Sciences 67 219ndash231
Hamadeh H K Jayadev S Gaillard E T Huang Q Stoll R
Blanchard K et al (2004) Integration of clinical and gene expression
endpoints to explore furan-mediated hepatotoxicity Mutation Research
549 169ndash183
Hamadeh H K Knight B L Haugen A C Sieber S Amin R P
Bushel P R et al (2002) Methapyrilene toxicity Anchorage of
pathologic observations to gene expression alterations Toxicologic
Pathology 30 470ndash482
Han J D Bertin N Hao T Goldberg D S Berriz G F Zhang L V
et al (2004) Evidence for dynamically organized modularity in the
yeast proteinndashprotein interaction network Nature 430 88ndash93
Hanisch D Zien A Zimmer R amp Lengauer T (2002) Co-clustering of
biological networks and gene expression data Bioinformatics 18
S145ndashS154
Harris A J Dial S L amp Casciano D A (2004) Comparison of basal
gene expression profiles and effects of hepatocarcinogens on gene
expression in cultured primary human hepatocytes and HepG2 cells
Mutation Research 549 79ndash99
Harrison S C (2004) Whither structural biology Nature Structural
Molecular Biology 11 12ndash15
Hartley D P Dai X He Y D Carlini E J Wang B Huskey S E
et al (2004) Activators of the rat pregnane X receptor differentially
modulate hepatic and intestinal gene expression Molecular Pharma-
cology 65 1159ndash1171
Hartwell L H Hopfield J J Leibler S amp Murray A W (1999) From
molecular to modular cell biology Nature 402 C47ndashC52
Hasmall S Orphanides G James N Pennie W Hedley K Soames
A et al (2002) Downregulation of lactoferrin by PPARalpha ligands
Role in perturbation of hepatocyte proliferation and apoptosis
Toxicology Sciences 68 304ndash313
Hayase T Yamamoto Y Yamamoto K Muso E amp Shiota K (2004)
Microarray profile analysis of toxic cocaine-induced alterations in the
expression of mouse brain gene sequences A possible rsquoprotectiversquo
effect of buprenorphine Journal of Applied Toxicology 24 15ndash20
Hayes K R Vollrath A L Zastrow G M McMillan B J Craven M
Jovanovich S et al (2005) EDGE A centralized resource for the
comparison analysis and distribution of toxicogenomic information
Molecular Pharmacology 67 1360ndash1368
Heijne W H Slitt A L van Bladeren P J Groten J P Klaassen C D
Stierum R H et al (2004) Bromobenzene-induced hepatotoxicity at
the transcriptome level Toxicology Sciences 79 411ndash422
Heijne W H Stierum R H Slijper M van Bladeren P J amp van
Ommen B (2003) Toxicogenomics of bromobenzene hepatotoxicity
A combined transcriptomics and proteomics approach Biochemical
Pharmacology 65 857ndash875
Heinloth A N Irwin R D Boorman G A Nettesheim P Fannin
R D Sieber S O et al (2004) Gene expression profiling of rat
livers reveals indicators of potential adverse effects Toxicology
Sciences 80 193ndash202
Hodges L C Cook J D Lobenhofer E K Li L Bennett L Bushel
P R et al (2003) Tamoxifen functions as a molecular agonist inducing
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 63
cell cycle-associated genes in breast cancer cells Molecular Cancer
Research 1 300ndash311
Holleman A Cheok M H den Boer M L Yang W Veerman KM
A J Kazemier et al (2004) Gene-expression patterns in drug-resistant
acute lymphoblastic leukemia cells and response to treatment New
England Journal of Medicine 351 533ndash542
Hong Y Muller U R amp Lai F (2003) Discriminating two classes of
toxicants through expression analysis of HepG2 cells with DNA arrays
Toxicology in Vitro 17 85ndash92
Hood E (2003) Proteomics Characterizing the cogs in the machinery of
life Environmental Health Perspectives 111 A816ndashA825
Hood L (2003) Systems biology Integrating technology biology and
computation Mechanism of Ageing and Development 124 9ndash16
Hood L amp Galas D (2003) The digital code of DNA Nature 421
444ndash448
Hood L amp Perlmutter R M (2004) The impact of systems approaches on
biological problems in drug discovery Nature Biotechnology 22
1215ndash1217
Hu T Gibson D P Carr G J Torontali S M Tiesman J P Chaney
J G et al (2004) Identification of a gene expression profile that
discriminates indirect-acting genotoxins from direct-acting genotoxins
Mutation Research 549 5ndash27
Huang Q Dunn II R T Jayadev S DiSorbo O Pack F D Farr S B
et al (2001) Assessment of cisplatin-induced nephrotoxicity by
microarray technology Toxicology Sciences 63 196ndash207
Huang Q Jin X Gaillard E T Knight B L Pack F D Stoltz J H et
al (2004) Gene expression profiling reveals multiple toxicity endpoints
induced by hepatotoxicants Mutation Research 549 147ndash167
Huisman M T Smit J W Crommentuyn K M Zelcer N Wiltshire
H R Beijnen J H et al (2002) Multidrug resistance protein 2
(MRP2) transports HIV protease inhibitors and transport can be
enhanced by other drugs Aids 16 2295ndash2301
Hunt C M Westerkam W R amp Stave G M (1992) Effect of age and
gender on the activity of human hepatic CYP3A Biochemical
Pharmacology 44 275ndash283
Ideker T Ozier O Schwikowski B amp Siegal A F (2002) Discovering
regulatory and signalling circuits in molecular interaction networks
Bioinformatics 18 S233ndashS240
Ideker T Thorsson V Ranish J A Christmas R Buhler J Eng J K
et al (2001) Integrated genomic and proteomic analyses of a system-
atically perturbed network Science 292 929ndash934
Iida M Anna C H Hartis J Bruno M Wetmore B Dubin J R et al
(2003) Changes in global gene and protein expression during early
mouse liver carcinogenesis induced by non-genotoxic model carcino-
gens oxazepam and Wyeth-14643 Carcinogenesis 24 757ndash770
Ingelman-Sundberg M (2004) Pharmacogenetics of cytochrome P450 and
its applications in drug therapy The past present and future Trends in
Pharmacological Sciences 25 193ndash200
Jeong H Mason S P Barabasi A L amp Oltvai Z N (2001) Lethality
and centrality in protein networks Nature 411 41ndash42
Jeong H Tombor B Albert R Oltvai Z N amp Barabasi A L
(2000) The large-scale organization of metabolic networks Nature
407 651ndash654
Jung D Hagenbuch B Gresh L Pontoglio M Meier P J amp Kullak-
Ublick G A (2001) Characterization of the human OATP-C
(SLC21A6) gene promoter and regulation of liver-specific OATP genes
by hepatocyte nuclear factor 1 alpha Journal of Biological Chemistry
276 37206ndash37214
Jung D amp Kullak-Ublick G A (2003) Hepatocyte nuclear factor 1
alpha A key mediator of the effect of bile acids on gene expression
Hepatology 37 622ndash631
Jung J -W Park J -S Hwang J -W Kang K -S YS L Song B
-S et al (2004) Gene expression analysis of peroxisome prolifer-
ators- and phenytoin-induced hepatotoxicity using cDNA microarray
Journal of Veterinary Medical Science 66 1329ndash1333
Kato N Shibutani M Takagi H Uneyama C Lee K -Y Takiami S
et al (2004) Gene expression profile in the livers of rats orally
administered ethinylestradiol for 28 days using a microarray technique
Toxicology 200 179ndash192
Kier L D Neft R Tang L Suizu R Cook T Onsurez K et al
(2004) Applications of microarrays with toxicologically relevant genes
(tox genes) for the evaluation of chemical toxicants in Sprague Dawley
rats in vivo and human hepatocytes in vitro Mutation Research 549
101ndash113
Kijima K Toyosawa K Yasuba M Matsuoka N Adachi T
Komiyama M et al (2004) Gene expression analysis of the rat testis
after treatment with di(2-ethylhexyl) phthalate using cDNA microarray
and real-time RT-PCR Toxicology and Applied Pharmacology 200
103ndash110
Kim R B (2003) Organic anion-transporting polypeptide (OATP) trans-
porter family and drug disposition European Journal of Clinical
Investigation 33 1ndash5
Kinirons M T amp OrsquoMahony M S (2004) Drug metabolism and ageing
British Journal of Clinical Pharmacology 57 540ndash544
Kitano H (2002a) Computational systems biology Nature 420 206ndash210
Kitano H (2002b) Systems biology A brief overview Science 295
1662ndash1664
Kliewer S A Moore J T Wade L Staudinger J L Watson M
A Jones S A et al (1998) An orphan nuclear receptor activated
by pregnanes defines a novel steroid signalling pathway Cell 92
73ndash82
Korolev D Balakin K V Nikolsky Y Kirillov E Ivanenkov Y A
Savchuk N P et al (2003) Modeling of human cytochrome p450-
mediated drug metabolism using unsupervised machine learning
approach Journal of Medicinal Chemistry 46 3631ndash3643
Kramer J A Pettit S D Amin R P Bertram T A Car B
Cunningham M et al (2004) Overview on the application of
transcription profiling using selected nephrotoxicants for toxicology
assessment Environmental Health Perspectives 112 460ndash464
Kultima K Nystrom A -M Scholz B Gustafson A -L Dencker L
amp Stigson M (2004) Valproic acid teratogenicity A toxicogenomics
approach Environmental Health Perspectives 112 1225ndash1235
Kutuzova G D amp Deluca H F (2004) Gene expression profiles in rat
intestine identify pathways for 125-dihydroxyvitamin D(3) stimulated
calcium absorption and clarify its immunomodulatory properties
Archives of Biochemistry and Biophysics 432 152ndash166
Kwak M K Wakabayashi N Itoh K Motohashi H Yamamoto M amp
Kensler T W (2003) Modulation of gene expression by cancer
chemopreventive dithiolethiones through the Keap1-Nrf2 pathway
Identification of novel gene clusters for cell survival Journal of
Biological Chemistry 278 8135ndash8145
Lahjouji K Mitchell G A amp Qureshi I A (2001) Carnitine transport
by organic cation transporters and systemic carnitine deficiency
Molecular Genetics and Metabolism 73 287ndash297
Landowski C P Sun D Foster D R Menon S S Barnett J L
Welage L S et al (2003) Gene expression in the human intestine and
correlation with oral valacyclovir pharmacokinetic parameters Journal
of Pharmacology and Experimental Therapeutics 306 778ndash786
Lee G -J Lee W -S Jeon K -S Um C -H Kim S -J Lee C -H
et al (2004) cDNA microarray gene expression analysis and
toxicological phenotype for anticancer drug Journal of Veterinary
Medical Science 66 1339ndash1345
Lee M Kwon J Kim S N Kim J E Koh W S Kim E J et al
(2003) cDNA microarray gene expression profiling of hydroxyurea
paclitaxel and p-anisidine genotoxic compounds with differing
tumorigenicity results Environmental and Molecular Mutagenesis
42 91ndash97
Li S Armstrong C M Bertin N Ge H Milstein S Boxem M et al
(2004) A map of the interactome network of the metazoan C elegans
Science 303 540ndash543
Liguori M J Anderson L M Bukofzer S McKim J Pregenzer J F
Retief J et al (2005) Microarray analysis in human hepatocytes
suggests a mechanism for hepatotoxicity induced by trovafloxacin
Hepatology 41 177ndash186
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6664
Lobenhofer E K Bennett L Cable P L Li L Bushel P R amp Afshari
C A (2002) Regulation of DNA replication fork genes by 17beta-
estradiol Molecular Endocrinology 16 1215ndash1229
Locker J Tian J Carver R Concas D Cossu C Ledda-Columbano
G M et al (2003) A common set of immediate-early response genes
in liver regeneration and hyperplasia Hepatology 38 314ndash325
Mattes W B Pettit S D Sansone S A Bushel P R amp Waters M D
(2004) Database development in toxicogenomics Issues and efforts
Environmental Health Perspectives 112 495ndash505
McMillian M Nie A Y Parker J B Leone A Bryant S Kemmerer
M et al (2004) A gene expression signature for oxidant stressreactive
metabolites in rat liver Biochemical Pharmacology 68 2249ndash2261
Meier P J amp Stieger B (2000) Molecular mechanisms in bile formation
News in Physiological Sciences 15 89ndash93
Meneses-Lorente G de Longueville F Dos Santos-Mendes S Bonnert
T P Jack A Evrard S et al (2003) An evaluation of a low-density
DNA microarray using cytochrome P450 inducers Chemical Research
in Toxicology 16 1070ndash1077
Meneses-Lorente G Guest P C Lawrence J Muniappa N Knowles
M R Skynner H A et al (2004) A proteomic investigation of drug-
induced steatosis in rat liver Chemical Research in Toxicology 17
605ndash612
Merrill C L Ni H Yoon L W Tirmenstein M A Narayanan P
Benavides G R et al (2002) Etomoxir-induced oxidative stress in
HepG2 cells detected by differential gene expression is confirmed
biochemically Toxicology Sciences 68 93ndash101
Milo R Shen-Orr S Itzkovitz S Kashtan N Chklovskii D amp Alon
U (2002) Network motifs Simple building blocks of complex
networks Science 298 824ndash827
Morgan K T Ni H Brown H R Yoon L Qualls C W Crosby L
M et al (2002) Application of cDNA microarray technology to in
vitro toxicology and the selection of genes for a real-time RT-PCR-
based screen for oxidative stress in Hep-G2 cells Toxicologic
Pathology 30 435ndash451
Mwinyi J Johne A Bauer S Roots I amp Gerloff T (2004) Evidence
for inverse effects of OATP-C (SLC21A6) 5 and 1b haplotypes on
pravastatin kinetics Clinical Pharmacology and Therapeutics 75
415ndash421
Nakata K Yukawa M Komiyama N Nakano T amp Kaminuma T
(2002) A nuclear receptor database that maps pathways to diseases
Genome Infomatics 13 515ndash516
Nicholson J K Holmes E Lindon J C amp Wilson I D (2004) The
challenges of modeling mammalian biocomplexity Nature Biotechnol-
ogy 22 1268ndash1274
Nicholson J K amp Wilson I D (2003) Understanding rsquoglobal systems
biology Metabonomics and the continuum of metabolism Nature
Reviews on Drug Discovery 2 668ndash676
Nikitin A Egorov S Daraselia N amp Mazo I (2003) Pathway studiomdash
the analysis and navigation of molecular networks Bioinformatics 19
2155ndash2157
Nikolsky Y Ekins S Nikolskaya T amp Bugrim A (2005) A novel
method for generation of signature networks as biomarkers from
complex high throughput data Toxicology Letters 158 20ndash29
Nikolsky Y Nikolskaya T amp Bugrim A (2005) Biological networks
and analysis of experimental data in drug discovery Drug Discovery
Today 10 653ndash662
Nishizato Y Ieiri I Suzuki H Kimura M Kawabata K Hirota T
et al (2003) Polymorphisms of OATP-C (SLC21A6) and OAT3
(SLC22A8) genes Consequences for pravastatin pharmacokinetics
Clinical Pharmacology and Therapeutics 73 554ndash565
Nozawa T Nakajima M Tamai I Noda K Nezu J Sai Y et al
(2002) Genetic polymorphisms of human organic anion transporters
OATP-C (SLC21A6) and OATP-B (SLC21A9) Allele frequencies in
the Japanese population and functional analysis Journal of Pharmacol-
ogy and Experimental Therapeutics 302 804ndash813
Nozawa T Sugiura S Nakajima M Goto A Yokoi T Nezu J et al
(2004) Involvement of organic anion transporting polypeptides in the
transport of troglitazone sulfate Implications for understanding trogli-
tazone hepatotoxicity Drug Metabolism and Disposition 32 291ndash294
Oliver D E Rubin D L Stuart J M Hewett M Klein T E amp
Altman R B (2002) Ontology development for a pharmacogenetics
knowledge base Package Symposion 88ndash99
Oram J F amp Lawn R M (2001) ABCA1 The gatekeeper for eliminating
excess tissue cholesterol Journal of Lipid Research 42 1173ndash1179
Oswald M Kullak-Ublick G A Paumgartner G amp Beuers U (2001)
Expression of hepatic transporters OATP-C and MRP2 in primary
sclerosing cholangitis Liver 21 247ndash253
Parsons A B Brost R I Ding H Li Z Zhang C Sheikh B et al
(2004) Integration of chemical-genetic and genetic interaction data
links bioactive compounds to cellular target pathways Nature Bio-
technology 22 62ndash69
Patki K C von Moltke L L Harmatz J S Hesse L M Court M H
amp Greenblatt D J (2004) Effect of age on in vitro triazolam
biotransformation in male human liver microsomes Journal of
Pharmacology and Experimental Therapeutics 308 874ndash879
Pereira-Leal J B Enright A J amp Ouzounis C A (2004) Detection of
functional modules from protein interaction networks Proteins 54
49ndash57
Plant N (2004) Interaction networks Coordinating responses to xeno-
biotic exposure Toxicology 202 21ndash32
Raghavendra Prasad H S Qi Z Srinivasan K N amp Gopalakrish-
nakone P (2004) Potential effects of tetrodotoxin exposure to
human glial cells postulated using microarray approach Toxicon 44
597ndash608
Reilly T P Bourdi M Brady J N Pise-Masison C A Radonovich M
F George J W et al (2001) Expression profiling of acetaminophen
liver toxicity in mice using microarray technology Biochemical and
Biophysical Research Communications 282 321ndash328
Rives A W amp Galitski T (2003) Modular organization of cellular
networks Proceedings of the National Academy of Sciences of the
United States of America 100 1128ndash1133
Rogers P D Pearson M M Cleary J D Sullivan D C amp Chapman
S W (2002) Differential expression of genes encoding immunomodu-
latory proteins in response to amphotericin B in human mononuclear
cells identified by cDNA microarray analysis Journal of Antimicrobial
Chemotherapy 50 811ndash817
Rosen M B Wilson V S Schmid J E amp Gray L E (2005) Gene
expression analysis in the ventral prostate of rats exposed to vinclozolin
or procymidone Reproductive Toxicology 19 367ndash379
Rost D Herrmann T Sauer P Schmidts H L Stieger B Meier P J
et al (2003) Regulation of rat organic anion transporters in bile salt-
induced cholestatic hepatitis Effect of ursodeoxycholate Hepatology
38 187ndash195
Rothman R B Ayestas M A Dersch C M amp Baumann M H (1999)
Aminorex fenfluramine and chlorphentermine are serotonin transporter
substrates Implications for primary pulmonary hypertension Circula-
tion 100 869ndash875
Ruepp S U Tonge R P Shaw J Wallis N amp Pognan F (2002)
Genomics and proteomics analysis of acetaminophen toxicity in mouse
liver Toxicology Sciences 65 135ndash150
Sakaeda T Nakamura T amp Okumura K (2002) MDR1 genotype-
related pharmacokinetics and pharmacodynamics Biological amp Phar-
maceutical Bulletin 25 1391ndash1400
Sakata T Anzai N Shin H J Noshiro R Hirata T Yokoyama H
et al (2004) Novel single nucleotide polymorphisms of organic cation
transporter 1 (SLC22A1) affecting transport functions Biochemical
and Biophysical Research Communications 313 789ndash793
Satlin L M Amin V amp Wolkoff A W (1997) Organic anion
transporting polypeptide mediates organic anionHCO3-exchange
Journal of Biological Chemistry 272 26340ndash26345
Satomi Y Tsuchiya W Mihara K Ota M Kasahara Y amp Akahori F
(2004) Gene expression analysis of the lung following paraquat
administration in rats using DNA microarray Journal of Toxicological
Sciences 29 91ndash100
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 65
Schrenk D Baus P R Ermel N Klein C Vorderstemann B amp
Kauffmann H M (2001) Up-regulation of transporters of the MRP
family by drugs and toxins Toxicology Letters 120 51ndash57
Segal E Shapira M Regev A Persquoer D Botstein D Koller D et al
(2003) Module networks Identifying regulatory modules and their
condition-specific regulators from gene expression data Nature
Genetics 34 166ndash176
Segal E Wang H amp Koller D (2003) Discovering molecular pathways
from protein interaction and gene expression Bioinformatics 19
i264ndash i272
Seidel S D Kan H L Stott W T Schisler M R amp Gollapudi B B
(2003) Identification of transcriptome profiles for the DNA-damaging
agents bleomycin and hydrogen peroxide in L5178Y mouse lymphoma
cells Environmental and Molecular Mutagenesis 42 19ndash25
Shan L Yu Y Schut H A J amp Snyderwine E G (2004) Susceptibility
of rats to mammary gland carcinogenesis by the food-derived
carcinogen 2-Amino-1-Methyl-6-Phenylimidazo[45-b]Pyridine (PhIP)
varies with age and is associated with the induction of differential gene
expression American Journal of Pathology 165 191ndash202
Sharom J R Bellows D S amp Tyers M (2004) From large networks to
small molecules Current Opinion in Chemical Biology 8 81ndash90
Shitara Y Hirano M Sato H amp Sugiyama Y (2004) Gemfibrozil and
its glucuronide inhibit the OATP2(OATP1B1 SLC21A6)-mediated
hepatic uptake and CYP2C8-mediated metabolism of cerivastatinmdash
Analysis of the mechanism of the clinically relevant drugndashdrug
interaction between cerivastatin and gemfibrozil Journal of Pharma-
cology and Experimental Therapeutics
Shitara Y Itoh T Sato H Li A P amp Sugiyama Y (2003) Inhibition of
transporter-mediated hepatic uptake as a mechanism for drugndashdrug
interaction between cerivastatin and cyclosporin A Journal of
Pharmacology and Experimental Therapeutics 304 610ndash616
Somogyi R Fuhrman S amp Wen X (2001) Genetic network inference in
computational models and applications to large-scale gene expression
data In M Bower amp H Bolouri (Eds) Computational modeling of
genetic and biochemical networks (pp 119ndash157) Cambridge MArsquoThe
MIT Press
Spirin V amp Mirny L A (2003) Protein complexes and functional
modules in molecular networks Proceedings of the National Academy
of Sciences of the United States of America 100 12123ndash12128
Staudinger J Liu Y Madan A Habeebu S amp Klaassen C D
(2001) Coordinate regulation of xenobiotic and bile acid homeo-
stasis by pregnane X receptor Drug Metabolism and Disposition
29 1467ndash1472
Sun L Z Ji Z L Chen X Wang J F amp Chen Y Z (2002) ADME-
AP A database of ADME associated proteins Bioinformatics 18
1699ndash1700
Suter L Babiss L E amp Wheeldon E B (2004) Toxicogenomics in
predictive toxicology in drug development Chemistry and Biology 11
161ndash171
Synold T W Dussault I amp Forman B M (2001) The orphan nuclear
receptor SXR coordinately regulates drug metabolism and efflux
Nature Medicine 7 584ndash590
Tabuchi Y amp Kondo T (2003) cDNA microarray analysis reveals chop-
10 plays a key role in Sertoli cell injury induced by bisphenol A
Biochemical and Biophysical Research Communications 305 54ndash61
Tamai I Nezu J Uchino H Sai Y Oku A Shimane M et al (2000)
Molecular identification and characterization of novel members of the
human organic anion transporter (OATP) family Biochemical and
Biophysical Research Communications 273 251ndash260
Taylor S W Fahy E Zhang B Glenn G M Warnock D E Wiley S
et al (2003) Characterization of the human heart mitochondrial
proteome Nature Biotechnology 21 281ndash286
Terasaka S Aita Y Inoue A Hayashi S Nishigaki M Aoyagi K
et al (2004) Using a customized DNA microarray for expression
profiling of the estrogen-responsive genes to evaluate estrogen activity
among natural estrogens and industrial chemicals Environmental
Health Perspectives 112 773ndash781
Thomas D M Francescutti-Verbeem D M Liu X amp Kuhn D M
(2004) Identification of differentially regulated transcripts in mouse
striatum following methamphetamine treatmentmdashan oligonucleotide
microarray approach Journal of Neurochemistry 88 380ndash393
Thomas R S Rank D R Penn S G Zastrow G M Hayes K R
Pande K et al (2001) Identification of toxicologically predictive
gene sets using cDNA microarrays Molecular Pharmacology 60
1189ndash1194
Thompson K L Afshari C A Amin R P Bertram T A Car B
Cunningham M et al (2004) Identification of platform-independent
gene expression markers of cisplatin nephrotoxicity Environmental
Health Perspectives 112 488ndash494
Tirona R G Leake B F Merino G amp Kim R B (2001)
Polymorphisms in OATP-C Identification of multiple allelic
variants associated with altered transport activity among European-
and African-Americans Journal of Biological Chemistry 276
35669ndash35675
Tirona R G Leake B F Wolkoff A W amp Kim R B (2003)
Human organic anion transporting polypeptide-C (SLC21A6) is a
major determinant of the rifampin-mediated pregnane X receptor
activation Journal of Pharmacology and Experimental Therapeutics
304 223ndash228
Tong W Cao X Harris S Sun H Fang H Fuscoe J et al (2003)
ArrayTrackmdashsupporting toxicogenomic research at the US Food and
Drug Administration National Center for Toxicological Research
Environmental Health Perspectives 111 1819ndash1826
Tornow S amp Mewes H W (2003) Functional modules by relating
protein interaction networks and gene expression Nucleic Acids
Research 31 6283ndash6289
Trocho C Escola-Gil J C Ribas V Benitez S Martin-Campos J M
Rotllan N et al (2004) Phenytoin treatment reduces atherosclerosis in
mice through mechanisms independent of plasma HDL-cholesterol
concentration Atherosclerosis 174 275ndash285
Ueda A Hamadeh H K Webb H K Yamamoto Y Sueyoshi T
Afshari C A et al (2002) Diverse roles of the nuclear orphan
receptor CAR in regulating hepatic genes in response to phenobarbital
Molecular Pharmacology 61 1ndash6
Ulrich R G (2003) The toxicogenomics of nuclear receptor agonists
Current Opinion in Chemical Biology 7 505ndash510
Ulrich R G Rockett J C Gibson G G amp Pettit S D (2004)
Overview of an interlaboratory collaboration on evaluating the effects
of model hepatotoxicants on hepatic gene expression Environmental
Health Perspectives 112 423ndash427
Unami A Shinohara Y Ichikawa T amp Baba Y (2003) Biochemical
and microarray analyses of bupivacaine-induced apoptosis Journal of
Toxicological Sciences 28 77ndash94
Vasquez A Flammini A Maritan A amp Vespignani A (2003) Global
protein function prediction from proteinndashprotein interaction networks
Nature Biotechnology 21 697ndash700
Vezina C M Walker N J amp Olson J R (2004) Subchronic Exposure to
TCDD PeCDF PCB126 and PCB153 Effect on hepatic gene
expression Environmental Health Perspectives 112 1636ndash1644
Vo T D Greenberg H J amp Palsson B O (2004) Reconstruction and
functional characterization of the human mitochondrial metabolic
network based on proteomic and biochemical data Journal of Bio-
logical Chemistry 279 39532ndash39540
Wang H amp LeCluyse E L (2003) Role of orphan nuclear receptors in
the regulation of drug-metabolising enzymes Clinical Pharmacoki-
netics 42 1331ndash1357
Wang N Silver D L Thiele C amp Tall A R (2001) ATP-
binding cassette transporter A1 (ABCA1) functions as a choles-
terol efflux regulatory protein Journal of Biological Chemistry 276
23742ndash23747
Waring J F Cavet G Jolly R A McDowell J Dai H Ciurlionis R
et al (2003) Development of a DNA Microarray for toxicology based
on hepatoxin-regulated sequences Environmental Health Perspectives
111 863ndash870
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6666
Waring J F Gum R Morfitt D Jolly R A Ciurlionis R Heindel M
et al (2002) Identifying toxic mechanisms using DNA microarrays
Evidence that an experimental inhibitor of cell adhesion molecule
expression signals through the aryl hydrocarbon nuclear receptor
Toxicology 181ndash182 537ndash550
Waring J F Jolly R A Ciurlionis R Lum P Y Praestgaard J T
Morfitt D C et al (2001) Clustering of hepatotoxins based on
mechanism of toxicity using gene expression profiles Toxicology and
Applied Pharmacology 175 28ndash42
Waters M Boorman G Bushel P Cunningham M Irwin R Merrick
A et al (2003) Systems toxicology and the Chemical Effects in
Biological Systems (CEBS) knowledge base EHP Toxicogenomics
111 15ndash28
Wei M Arnold L Cano M amp Cohen S M (2005) Effects of co-
administration of antioxidants and arsenicals on the rat urinary bladder
epithelium Toxicology Sciences 83 237ndash245
Weinshilboum R (2003) Inheritance and drug response New England
Journal of Medicine 348 529ndash537
Weinshilboum R amp Wang L (2004) Pharmacogenetics Inherited
variation in amino acid sequence and altered protein quantity Clinical
Pharmacology and Therapeutics 75 253ndash258
Werner E (2003) In silico multicellular systems biology and minimal
genomes Drug Discovery Today 8 1121ndash1127
Wong J S amp Gill S S (2002) Gene expression changes induced in
mouse liver by di(2-ethylhexyl) phthalate Toxicology and Applied
Pharmacology 185 180ndash196
Xie T Tong L McCann U D Yuan J Becker K G Mechan A O
et al (2004) Identification and characterization of metallothionein-1
and -2 gene expression in the context of ()34-methylenedioxymetham-
phetamine-induced toxicity to brain dopaminergic neurons Journal of
Neuroscience 24 7043ndash7050
Yadetie F Laegreid A Bakke I Kusnierczyk W Komorowski J
Waldum H L et al (2003) Liver gene expression in rats in response
to the peroxisome proliferator-activated receptor-alpha agonist ciprofi-
brate Physiological Genomics 15 9ndash19
Yamashita S Nomoto T Abe M Tatematsu M Sugimura T amp
Ushijima T (2004) Persistence of gene expression changes in stomach
mucosae induced by short-term N-methyl-NV-nitro-N-nitrosoguanidinetreatment and their presence in stomach cancers Mutation Research
549 185ndash193
Yan Q amp Sadee W (2000) Human membrane transporter database A
web-accessible relational database for drug transport studies and
pharmacogenomics AAPS Pharmsci 2 E20
Yeger-Lotem E amp Margalit H (2003) Detection of regulatory circuits by
integrating the cellular networks of proteinndashprotein interactions and
transcription regulation Nucleic Acids Research 31 6053ndash6061
Yoon B I Li G -X Kitada K Kawasaki Y Igarashi K amp Kodama
Y et al (2003) Mechanisms of benzene-induced hematotoxicity and
leukemogenicity cDNA microarray analyses using mouse bone marrow
tissue Environmental Health Perspectives 111 1411ndash1420
Young M B DiSilvestro M R Sendera T J Freund J Kriete A amp
Magnuson S R (2003) Analysis of gene expression in carbon
tetrachloride-treated rat livers using a novel bioarray technology
Pharmacogenomics Journal 3 41ndash52
Yu H Zhu X Greenbaum D Karro J amp Gerstein M (2004) TopNet
A tool for comparing biological sub-networks correlating protein
properties with topological statistics Nucleic Acids Research 32
328ndash337
Zhang E Y Fu D J Pak Y A Stewart T Mukhopadhyay N
Wrighton S A et al (2004) Genetic polymorphisms in human proton-
dependent dipeptide transporter PEPT1 Implications for the functional
role of Pro586 Journal of Pharmacology and Experimental Therapeu-
tics 310 437ndash445
Zhang E Y Knipp G T Ekins S amp Swaan P W (2002)
Structural biology and function of solute transporters Implications
for identifying and designing substrates Drug Metabolism Reviews
34 709ndash750
Zhang E Y Phelps M A Cheng C Ekins S amp Swaan P W (2002)
Modeling of active transport systems Advanced Drug Delivery
Reviews 54 329ndash354
- Systems-ADMETox Resources and network approaches
-
- Introduction
- Data available
- Network analysis and databases
-
- Network applications
-
- The role of transporters
-
- Clinical relevance of transporters
- Transporter network examples ABCA1
- Transporter network examples OATP
- Transporter microarray data
-
- Applications to enzymes
- Future network applications
-
- Discussion
- Acknowledgements
- References
-
0
20
40
60
80
100
2000 2001 2002 2003 2004 2005Year
Pu
bM
edN
um
ber
of
pu
blic
atio
ns
in
Fig 1 Annual frequency of articles appearing with the words lsquolsquotoxicoge-
nomicsrsquorsquo (squares) or lsquolsquomicroarray and toxicologyrsquorsquo (diamonds)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 39
dures (Tables 1-4) There have been relatively few cross-
platform toxicogenomics studies under controlled condi-
tions (Thompson et al 2004) Despite the suggested poor
compatibility between the different array types this latter
study demonstrated a high (90) consistency between the
expression of the genes that were shared between the
platforms The development of methods to visualize such
complex expression data has also expanded beyond the
widely used clustering methods (Eisen Spellman Brown amp
Botstein 1998) With the outcome of microarray analysis
being dependent on the widely used statistical procedures
applied to derive those genes that are significantly differ-
entially expressed (Butte 2002) newer approaches that do
not necessarily require data clustering may be an advantage
As rat and mouse are the most widely used in vivo toxicity
models it is assumed that acute and chronic toxicity shown
in animals largely coincides with human toxicity Therefore
differential expression patterns in animal models are also
assumed to be predictive of the end point toxic response in
human This is not always the case due to differences
between human and rodent physiology genetics metabo-
lism and signaling pathways For example the mechanism
of toxicity for pyrazinamide has been reconstructed
(Bugrim Nikolskaya amp Nikolsky 2004) to illustrate that
the accumulation of uric acid occurs in human but not in
mice and this results in toxicity in the former The relatively
poor understanding of such species differences may be
reflected in the relatively large number of late stage
molecules that have undergone in vivo toxicity assessment
yet have been later withdrawn due to adverse events in
humans
In recent years the appearance of systems biology which
uses the relationships of all elements of biology rather than
approaching them separately has been evident and will
likely reunite biological fields (Harrison 2004 Hood amp
Galas 2003) These systems approaches are the latest
incarnation of the importance of the Fparts vs wholes_debate (Ekins amp McGowan 2001) and interpreting ADME
Tox in this context may improve our understanding and
ultimate predictions (Bugrim et al 2004 Ekins Boulanger
Swaan amp Hupcey 2002a Kitano 2002a Werner 2003)
The perturbing effect of a molecule on the complete
biological system can be observed across all metabolic
and signaling pathways or networks and can provide some
limited insight into the binding to multiple proteins or
effects on gene expression simultaneously This requires the
collection of high-throughput data including global gene
expression protein content metabolic profiles for the same
samples as well as individual genetic clinical and pheno-
typic data However there are difficulties with such an
approach as there are likely to be differences between the
Fstimulus to effect_ durations for all the genendashprotein
relationships (Nicholson Holmes Lindon amp Wilson
2004)
We can now use either the growing number of academic
or commercially available pathway database and network
building tools with expression data These enable the
connection of interacting differentially expressed genes as
networks (Barabasi amp Oltvai 2004 Hanisch Zien Zimmer
amp Lengauer 2002 Ideker Ozier Schwikowski amp Siegal
2002 Ideker et al 2001 Segal et al 2003a Segal Wang amp
Koller 2003b Spirin amp Mirny 2003 Tornow amp Mewes
2003) as well as allowing the reverse engineering of
functional connections (Somogyi Fuhrman amp Wen 2001)
The use of such network visualizations suggests an
organized modularity in complex systems (Han et al
2004) which has also been applied to interpret the
connectivity of small molecules and their interaction with
proteins in the subfield of chemogenomics (Bredel amp Jacoby
2004 Csermely Agoston amp Pongor 2005 Parsons et al
2004 Sharom Bellows amp Tyers 2004) The parallel
development of HT methods databases ADMETox model-
ing and systems modeling is ongoing (Ekins Nikolsky amp
Nikolskaya 2005e) The present review is therefore timely
as it discusses some of the data resources limitations and
technologies that are available for Systems-ADMETox
(Fig 2) along with some illustration of their applications to
drug metabolism and drug transport which are key com-
ponents of the ADMETox process The ultimate aim of
this discussion is to provide awareness of an integrated
approach rather than a technology silo mentality represent-
ing the latest proposed research model in the field (Fig 2)
2 Data available
A recently published book provides an excellent over-
view of toxicogenomics and the reader is referred to this to
gain more insight into the applications and limitations
(Hamadeh amp Afshari 2004) The growing number of
toxicogenomic datasets derived from in vivo studies with
rat (Table 1) and mouse (Table 2) as well as in vitro cell
derived data (Table 3) highlights the different strains
microarray types and compounds that are routinely assessed
Also there are numerous instances of multiple groups testing
the same compound at similar or different doses eg well
known hepatotoxicants or nephrotoxicants such as clofibrate
Table 1
Literature toxicogenomics data derived from rat in vivo studies
Compounds Rat strain Microarray type Compound dose Microarray data
availability
Reference
Acetaminophen furan
methotrexate
methapyrilene
phenytoin
Male Spraguendash
Dawley VAF+
albino
Phase 1 Molecular
toxicology array
Acetaminophen
(4500 mgkgday)
methotrexate (1 mg
kg day)
methapyrilene (100
mgkgday) furan (40
mgkgday) or
phenytoin (300 mg
kgday)
Gene name accession
number and fold changes
are provided in a
manuscript table
(Huang et al
2004)
A-277249 3MC
Aroclor
SpraguendashDawley Affymetrix rat
toxicology U34 array
10 or 100 mgkg Gene name and
Affymetrix ID and fold
change in a manuscript
table
(Waring et al
2002)
Clofibrate paracetamol
benzoapyrene
Male Spraguendash
Dawley
Custom chip CLO at 250 mgkg
BP at 10 mgkg bw
given 3 times per
week for 2 weeks
APAP at 1000 mgkg
Gene name and fold
change in a manuscript
table
(Cunningham
Liang Fuhrman
Seilhamer amp
Somogyi 2000)
L-742694
Dexamethasone
Female
SpraguendashDawley
25K rat microarray L-742694 (50 mgkg
day) DEX (50 mg
kgday)
Tables of accession
number and gene names
only in a manuscript
table for liver and
intestine
(Hartley et al
2004)
Bromobenzene Male Wistar Custom 3000 rat gene
array
05 2 5 mmolkg Gene name accession
number and log fold
change for 3 dose levels
in a manuscript table
Supplemental data also
available
(Heijne et al
2004)
125-Dihydroxyvitamin
D3
Male Spraguendash
Dawley (small
intestine)
Affymetrix high-density
rat oligonucleotide
arrays (GeneChips
RG-U34A)
730 ngkg Gene name Genbank
accession number and
fold change in a
manuscript table
(Kutuzova amp
Deluca 2004)
Fenofibrate clofibrate
bezafibrate
gemfibrozi
ciprofibrate
beclofibrate etofibrate
Male CD Agilent arrays Dose ranging for up
to 14 days
Gene name and genbank
accession number in a
manuscript table
(Cornwell Souza
amp Ulrich 2004)
Furan Male Spraguendash
Dawley
NIEHS rat chip uml7000
clones
Exposed to 4 or 40
mgkg furan for up
to 14 days
Gene ID and gene
namemdashbinary data in a
manuscript table Dataset
also available on NIEHS
website
(Hamadeh et al
2004)
Paracetamol Male F344N Rat NIEHS tox chip
(httpdirniehsnihgov
microarraychipshtm)
0 50 150
1500 mgkg
Unigene accession
number gene name fold
change in a manuscript
table
(Heinloth et al
2004)
Carbon tetrachloride Male Spraguendash
Dawley
ADME Rat expression
bioarray (Motorola Life
Sciences) consists of
1040 single-stranded
oligonucleotide probes
Up to 14 day
treatment
Accession numbers and
binary data in a
manuscript table
(Young et al
2003)
Carbon tetrachloride and
chloroform
Male Spraguendash
Dawley
The rat CT arrays
contain sequences from
almost 700 rat genes
with known or
discovered
responsiveness to toxic
treatments
6 24 72 h high and
low doses
Gene names and fold
changes at multiple time
points in a manuscript
table
(Kier et al 2004)
(continued on next page)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6640
Table 1 (continued)
Compounds Rat strain Microarray type Compound dose Microarray data
availability
Reference
Dimethylarsinic acid Female F344
(bladder
epithelium)
Rat 10K chip (MWG
Biotech Inc) containing
10000 genes
100 ppm Genbank accession
number gene name in a
manuscript table
(Wei Arnold
Cano amp Cohen
2005)
Dexamethasone
troleandomycin
miconazole
clotrimazole
isoniazid
methylclofanapate
Female Spraguendash
Dawley
Rat HepatoChip DEX (50 mgkgday)
TAO (500 mgkg
day) MIC (100 mg
kgday) CLOT (100
mgkg per day) ISN
(100 mgkgday)
MCP (75 mgkgday)
Gene name and Genbank
accession numbermdashdata
displayed as a heatmap
not readily extracted
from publication
(Meneses-Lorente
et al 2003)
TCDD PeCDF PCB126
PCB153
Female Harlan
SpraguendashDawley
Affymetrix GeneChip
Test3 arrays
Exposed for 13 weeks
to toxicologically
equivalent doses
Accession number gene
name and fold change in
a manuscript table
(Vezina Walker
amp Olson 2004)
Paclitaxel Male and female
SpraguendashDawley
Genocheck 48K cDNA 4 mgkgday male
7 mgkgday female
Accession numbers and
gene names in a
manuscript table
(Lee et al 2004)
Clofibrate gemfibrozil
phenytoin
Male Spraguendash
Dawley VAF(+)
albino
48 K cDNA microarray
in house
Treated with each
compound for 24 h
and 2 weeks
Gene name and fold
change in a manuscript
table
(Jung et al 2004)
Clofibrate
dexamethasone
phenobarbital
3-methylcholanthrene
SpraguendashDawley Merck Drug Safety Chip
1443 genes (rat human
and mouse)
30 mgkgday Gene name GenBank
accession number and
relative fluorescence
levels in a manuscript
table
(Gerhold et al
2001)
Bemitradine clofibrate
doxylamine
methapyrilene
phenobarbital
tamoxifen
2-acetylaminofluorene
4-acetylaminofluorene
isoniazid
Male CD IGS Incyte RatGEM10
uml7800 rat cDNAs
Low mid and high
doses
A bar chart of 2 genes
that are affected by
compounds
(Kramer et al
2004)
PhIP Female Spraguendash
Dawley
Mouse cDNA
microarray containing
9984 cDNA clones
(National Cancer
Institute
75 mgkgday Gene names in a
manuscript table
(Shan Yu Schut
amp Snyderwine
2004)
Ethinylestradiol Male and female
SpraguendashDawley
Custom chip 3776 genes 0 001 01 and
10 ppm
Accession number gene
name and fold change at
different exposure levels
in a manuscript table
(Kato et al 2004)
Paraquat Male Wistar 1090 genes 7 mgkgday Gene name and
expression ratio in a
manuscript table
(Satomi et al
2004)
Hexachlorobenzene Female Brown
Norway
Affymetrix rat
RGU-34A GeneChip
microarray
0 150 or 450 mgkg Accession number gene
name and fold change
data for multiple organs
in a manuscript table
(Ezendam et al
2004)
N-methyl-NV-nitro-N-nitrosoguanidine
(MNNG)
Rat pyloric muco-
sae male congenic
rat strain that has a
homozygous_LIZ
transgene of
BigBlue rat
AFFYMETRIX Rat
Genome U34A
83 mgl AFFY ID gene name
gene symbol and fold
change in a manuscript
table
(Yamashita et al
2004)
N-methyl-NV-nitro-N-nitrosoguanidine
Male ACINJcI
(ACI)
Affymetrix GeneChip
Rat genome U34A
arrays
83 mgl from the age
of 8 weeks through to
40 weeks
Accession number gene
name symbol fold change
in a manuscript table also
rat vs human stomach
cancer comparison
(Abe et al 2003)
Cisplatin SpraguendashDawley Different arrays tox
chip incyte phase 1 etc
03ndash5 mgkg over a 4 to
144 h
Unigene gene ID gene
name NIEHS ID and
data for 5 platforms in a
manuscript table
(Thompson et al
2004)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 41
Table 1 (continued)
Compounds Rat strain Microarray type Compound dose Microarray data
availability
Reference
Clofibrate Male Spraguendash
Dawley
Atlas Rat Toxicology II
arrays (Clontech Palo
Alto CA USA)
containing 465 genes
High (250 mgkgday) or
low (25 mgkgday)
Genbank accession
number and data for 3
platforms in a
manuscript table
(Baker et al
2004)
Di(2-ethylhexyl)
phthalate
Male Spraguendash
Dawleymdash(testes)
An in-house cDNA
microarray
20 or 2000 mgkg Gene names Genbank
ID and fold change in a
manuscript table
(Kijima et al
2004)
Ciprofibrate Female Fischer An in-house cDNA
microarray
50 mgkg body weight Gene names symbol
accession number mean
ratio and SD
(Yadetie et al
2003)
Methapyrilene Male Spraguendash
Dawley
Rat Tox Chip 10 10 or 100 mgkgday Gene Name accession
number and indication of
up or down regulation
original data available on
NIEHS website
(Hamadeh et al
2002b)
Ecteinascidin-743
(ET-743)
Female Wistar Custom chip cDNA
microarrays containing
approximately 4700
hybridizable mouse
expressed sequence tags
derived from IMAGE
clones obtained from
Research Genetics
(Huntsville AL) or from
the MRC Human Gene
Mapping Project
40 ugkg Data available on
laboratory websitemdashnot
available at present
(Donald et al
2002)
Clofibrate Wyeth
14643 Gemfibrozil
phenobarbital
Male Spraguendash
Dawley VAF+
NIEHS rat chip v10 Clofibrate (250
mgkgday Wyeth
14643 (250 mgkg
day) Gemfibrozil
(100 mgkgday)
Phenobarbital
(120 mgkgday)
Gene names and fold
changes in a manuscript
table
(Hamadeh et al
2002a)
Vinclozin procymidone Male Spraguendash
Dawleymdashprostate
Clontech Atlas Rat 12
Toxicology array
200 mgkg Gene name accession
number average fold
change
(Rosen Wilson
Schmid amp Gray
2005)
Cisplatin Male Spraguendash
Dawley VAF1
albino (CRL
CD(SD) BR
Rat Tox Microarrays
were purchased from
Phase-1 Molecular
Toxicology
05 or 1 mgkgday Gene name accession
number fold change in
kidney
(Huang et al
2001)
Allyl alcohol
miodarone Aroclor
1254 arsenic
carbamazepine
carbon tetrachloride
diethylnitrosamine
dimethylformamide
diquat etoposide
indomethacin
methapyrilene
methotrexate
monocrotaline
3-methylcholanthrene
Male Spraguendash
Dawley
Affymetrix GeneChip
Test 2 Array
Allyl alcohol (40 mgkg
day) miodarone (100 mg
kgday) Aroclor 1254
(400 mgkgday) arsenic
(20 mgkgday)
carbamazepine (250 mg
kgday) carbon
tetrachloride (1000mgkg
day) diethylnitrosamine
(100 mgkgday)
dimethylformamide (1000
mgkgday) diquat (172
mgkgday) etoposide (50
mgkgday) indomethacin
(20 mgkgday)
methapyrilene (250 mg
kgday) methotrexate
(250 mgkgday)
monocrotaline (50 mgkg
day)
3-methylcholanthrene
(100 mgkgday)
Heatmap figures and a
table of Affymetrix
names for genes
correlated with clinical
chemistry changes in a
manuscript table
(Waring et al
2001)
(continued on next page)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6642
Table 1 (continued)
Compounds Rat strain Microarray type Compound dose Microarray data
availability
Reference
Microcystin-LR (MLR)
phenobarbital (PB)
lipopolysaccharide
(LPS) carbon
tetrachloride (CT)
thioacetamide (THA)
and cyproterone
acetate (CPA)
Male Wistar Purpose-made rat DNA
microarray (Affymetrix
Santa Clara CA)
containing 1600 rat
DNA sequences
Various Accession number gene
name fold changes
shown as a heat map
table in publicationmdash
data extraction would be
laborious
(Bulera et al
2001)
Acetamidofluorene
aniline bromobenzene
butyl hydroxytol
dieldrin disulfiram
ethinyl estradiol
hexachlorocyclohexane
g 4-methylthiazole
nimesulide piperonyl
butoxide precocene I
pulegone tannic acid
trans-anethole
Male Spraguendash
Dawley
Custom Rat MegaA
cDNA chip 3434-gene
Acetamidofluorene (200
mgkg) aniline (200 mg
kg) bromobenzene (900
mgkg) butyl hydroxytol
(1000 mgkg) dieldrin
(30 and 45 mgkg)
disulfiram (2000 mgkg)
ethinyl estradiol (500
mgkg)
hexachlorocyclohexane
gamma (40 65 80 mg
kg) 4-methylthiazole
(120 mgkg) nimesulide
(500 mgkg) piperonyl
butoxide (4000 mgkg)
precocene I (500 mgkg)
pulegone (400 mgkg)
tannic acid (3000 mg
kg) trans-anethole
(600 mgkg)
Gene name accession
number in a manuscript
table
(McMillian et al
2004)
NIEHS website at httpdirniehsnihgovmicroarraydatasetshome-pubhtm
EDGE website at httpedgeoncologywiscedu
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 43
and cisplatin (Table 1) In the majority of cases the resulting
number of differentially expressed genes is a very small
subset of the starting number on the microarray following
clustering or other types of analysis Upon closer examina-
tion of these publications the majority of them either
provide images of a heat map andor a table listing a gene
name accession number and expression change Very few of
the published studies (Tables 1ndash3) provide the original raw
microarray data file at a freely accessible website hence
restricting further analysis by scientists using other software
Although in some cases it is possible to cut and paste the
gene expression data tables from the publication pdf files
this is not always the case In the worst case scenario one
would have to manually retype gene lists or extract them
from heatmaps as binary type data As not all computational
researchers will have a laboratory available to them to
generate such quantities of toxicogenomics data the latter
points are important if we are going to continue to see
innovation in software development for this data This will
require free unrestricted access to data published Similarly
if we are to discern lsquolsquofingerprintsrsquorsquo for molecules acting with
a similar or identical mechanism we will need databases of
many diverse chemical structures that have been tested in a
similar manner There is therefore considerable interest in
the current databases being developed by the NIEHS FDA
and other groups which should help to improve the situation
by providing freely accessible microarray and other toxicity
related data Two of these databases being developed in the
public domain are Chemical Effects in Biological Systems
(CEBS) (httpwwwniehsnihgovnctcebshtm) (Mattes
Pettit Sansone Bushel amp Waters 2004 Waters et al
2003) which will accommodate gene expression profiles
proteomics and metabolomics data and allow complex
queries (Hood 2003a Mattes et al 2004) Similar goals
are being pursued in the development of the ArrayTrack
database at the FDA (Tong et al 2003) The EDGE
database (httpedgeoncologywisceduedgephp) an
expanding public effort at The University of Wisconsin
contains mouse gene expression profiles following treat-
ment with different toxic molecules (Hayes et al 2005
Thomas et al 2001) These separate efforts if widely
adopted should make published studies describing HT data
more readily accessible although it might have been more
efficient to evolve these into a single global database instead
of fragmented repositories
Proteomics data has also been generated in a limited
number of toxicology studies (Table 4) once again this has
been produced with different strains of rats and mice using
different protein chips 2-D gel electrophoresis (2-DIGE)
and mass spectroscopy methods (eg MALDI-MS) The
proteomic data is very rarely accessible to the reader for
their own computational analysis Subsequently there have
Table 2
Literature toxicogenomics data derived from mouse in vivo studies
Compounds Mouse strain Microarray type Compound dose Microarray data
availability
Reference
Benzene Male and female P53
KO mice and
C57BL6
Affymetrix and Incyte
GEM system
300 ppm 6 h per day 5
days a week for 2 weeks
Gene name accession
number and fold change
data in a manuscript
table
(Yoon et al 2003)
Benzene Male 129SvJ Affymetrix MG-U74Av2 100 ppm 6 h per day 5
days a week for 2 weeks
Gene name accession
number and fold change
data in a manuscript
table
(Faiola Fuller
Wong amp Recio
2004)
Phenobarbital CAR and wild type NIEHS Mouse tox chip
8736 genes
100 mgkg for 12 h Accession number gene
name and fold change
for wild type and knock
out mice in a manuscript
table
(Ueda et al 2002)
Aroclor BNF
ciprofloxacin cobalt
chloride TCDD IL-6
LPS PCB-153
phenobarbital
phenylhyrzn TNFa
WY-16463
C57BL6J Custom array with 1200
cDNAs
Aroclor (200 mgkg)
BNF (5 mgkg)
ciprofloxacin (250 mg
kg) cobalt chloride (60
mgkg) TCDD (10 ug
kg) IL-6 (25 ugkg)
LPS (1 mgkg)
PCB-153 (80 mgkg)
phenobarbital (100 mg
kgday) 3 days
phenylhyrzn (100 mg
kg) TNFa (50 ugkg)
WY-16463 (0125wv)
Gene names and fold
change as a heat map
Data is also available in
the EDGE database
(Thomas et al
2001)
TCPOBOP CD-1 female Custom 9000 cDNA
mouse array
1ndash3 h treatment 3 mg
kg body wt
Accession numbers and
fold change data in a
manuscript table
(Locker et al
2003)
3H-12-dithiole-3-thione
(D3T)
Male wild-type and
nrf2-disrupted
Affymetrix murine
genome U74Av2
GeneChip
05 mmolkg Accession number gene
name fold in a
manuscript table
(Kwak et al
2003)
MDMA Male albino Swissndash
Webster (neurons)
15 K mouse cDNA clone
set
47 mgkg Gene name and fold
change in a manuscript
table
(Xie et al 2004)
Phenytoin Female C57BL6 and
LDLRMurine genome-U74Av2 300 mgl Gene name accession
number and fold change
in a manuscript table
(Trocho et al
2004)
Genistein (1000 Agmouseday) or
diethylstilbestrol
(DES) (50 Agmouse
day)
ICR (testes) Custom cDNA
microarray containing
1754 cDNA probes
Genistein (1000 Agmouseday)
diethylstilbestrol (50 Agmouseday)
Accession number gene
name and fold change in
a manuscript tablemdashnote
very few genes
(Adachi et al
2004)
Cocaine and
buprenorphine
Male ICR Mouse DiscoveryArrayi
type I array containing
2688brain-derivedprobes
(Display Systems Biotech
Inc Copenhagen
Denmark)
40 mg kgmdash1 cocaine
once a day for 4 days 40
mg kgmdash1 cocaine plus
025 mg kgmdash1 BUP for
4 days
Data apparently not
available
(Hayase
Yamamoto
Yamamoto Muso
amp Shiota 2004)
Methamphetamine Male C57BLJ6
(striatum)
Affymetrix mouse
genechip mg-U74Av2
oligonucleotides arrays
12 488 genes
40 mgkg Gene accession number
gene ID and name and
signal log ratio in a
manuscript table
(Thomas
Francescutti-
Verbeem Liu amp
Kuhn 2004)
Di(2-ethylhexyl)
phthalate
Male C57BL6 Murine genome U74Av2
Arrays (MGU74Av2)
10 dietary DEHP for
13 weeks
Genbank accession
numbers gene name log
ratio data present as a bar
chart in the publication
data extraction would
take some effort
(Wong amp Gill
2002)
Diethylhexylphthalate Male PPARa null and
wild type
Custom made containing
600 tox genes
1150 mgkgday Gene names available on
a bar chartmdashquantitative
data not easily accessible
(Hasmall et al
2002)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6644
Table 2 (continued)
Compounds Mouse strain Microarray type Compound dose Microarray data
availability
Reference
Acetaminophen C57B16 3 129Ola
hybrid
Test-2 Chips (Affymetrix)
then individual
oligonucleotide
microarrays (Mul1K sub
A and sub B Affymetrix)
that can detect the
expression of 11000
known genes and
expressed sequence tags
(ESTs)
300 mgkg Genbank or SwissProt
ID and fold change data
in a manuscript table
(Reilly et al
2001)
Cadmium chloride
benzo(a)pyrene (BaP)
and trichloroethylene
(TCE)
Male Swiss Webster Custom Chips
containing 148 unique
genes
Various Data in table form few
genes affected for BAP
and TCE
(Bartosiewicz
Penn amp Buckpitt
2001)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 45
been only a very small number of studies that have
combined both transcriptomic and proteomic methods with
a single animal strain after treatment with a drug Hopefully
we will see this change in the future but this will in turn
present considerable challenges as huge amounts of
proteomic data are combined with the equally large
transcript data files
3 Network analysis and databases
From some of the early reviews of systems biology there
has been discussion of its application to drug discovery
(Kitano 2002ab) as well as the utility for ADMETox
(Ekins et al 2002a 2000b) More recently several other
journals have dedicated whole issues to the field of systems
biology However one could consider this quite a broad field
from network or pathway analysis to quantitative simulation
of organelles (Vo Greenberg amp Palsson 2004) whole cells
and organs It is apparent that we are now understanding
organisms from the perspective of computationally gener-
ated networks of protein and ligand interactions (Barabasi amp
Oltvai 2004) Network and pathway tools enable the
analysis of HT data in the context of all known interactions
when using a database as the source Individual reviews
have in some cases indicated that networks will be valuable
for understanding adverse events (Hood amp Perlmutter
2004) drug target identification or validation (Butcher
Berg amp Kunkel 2004) and complex metabolic interactions
(Nicholson et al 2004) A general schematic has been
generated in order to provide a description of the utilization
of such pathway databases and network building algorithms
from the initial parsing of high throughput data to network
comparisons and visualization (Fig 3) High throughput
data can be superimposed and visualized on the various
protein interaction databases available This is accomplished
by using either preset maps that capture current biological
knowledge or by building custom interaction networks
using many different algorithms which can be compared and
statistically evaluated as demonstrated in a very large
number of published examples (Dobrin Beg Karabasi amp
Oltvai 2004 Fiehn 2001 Han et al 2004 Hanisch et al
2002 Ideker et al 2002 Jeong Mason Barabasi amp Oltvai
2001 Jeong Tombor Albert Oltvai amp Barabasi 2000 Li
et al 2004 Milo et al 2002 Nikitin Egorov Daraselia amp
Mazo 2003 Pereira-Leal Enright amp Ouzounis 2004
Rives amp Galitski 2003 Segal et al 2003a Somogyi et al
2001 Spirin amp Mirny 2003 Tornow amp Mewes 2003
Vasquez Flammini Maritan amp Vespignani 2003 Yeger-
Lotem amp Margalit 2003 Yu Zhu Greenbaum Karro amp
Gerstein 2004)
31 Network applications
For example one group has used as an inference the
Bayesian network method for analysis of tissue toxicity from
microarray data as well as a mechanistic simulation for a
different pharmaceutically relevant molecule (Aksenov et
al 2005) Pathway tools and various resources have also
been applied to modeling the networks of nuclear hormone
receptors and their connections with other genes and small
molecules using a manually curated database MetaDrug
(Ekins Kirillov Rakmatulin amp Nikolskaya 2005d) or
MetaCore (Ekins Bugrim Nikolsky amp Nikolskaya 2005)
Transcriptional regulation of many transporters CYPs and
phase II enzymes are regulated by these receptors affecting
endogenous molecule transport metabolism cell growth
proliferation and oxidative stress (Ulrich 2003 Ulrich et al
2004) When the signaling networks and interacting ligands
for the transcriptional factors PPAR FXRRXRA ESR1
AHR HNF4A GCR-h MCR CAR-beta GCR-a LXR-a
CARRXR HNF4 FXR PXRRXR heterodimer PXR
AHRARNT heterodimer PPARaLXRa VDR PPAR-a
are visualized a very complex picture of interactions can be
created (Ekins et al 2005d) This suggests that when we
consider a molecule binding with only one nuclear receptor
we are observing only a fraction of the likely possible
feasible interactions based on the data gathered to date
Table 3
Literature toxicogenomics data derived from in vitro cell studies
Compounds Cell type Microarray type Compound dose Microarray data
availability
Reference
4-Hydroxytamoxifen
estrogen
MCF-7 breast cancer NIEHS ToxChip
microarray consisting of
1901 genes
1 uM hydroxytamoxifen
for a year 10 nM
17b-estradiol
Data available at NIEHS
website
(Hodges et al
2003)
Trovafloxacin Human hepatocytes Affymetrix U133A array 30ndash800 uM 142 genes available in
supplemental tablemdashnot
easily extracted
(Liguori et al
2005)
Estrogen MCF-7 breast cancer NIEHS ToxChip
microarray consisting of
1901 genes
10ndash10 M 17b-estradiol Data available at NIEHS
website
(Lobenhofer et al
2002)
Valproic acid NMRI mice embryo
and P19 mouse
embryocarcinoma
Custom chip including
15K mouse cDNA clone
set
600 mgkg body weight Gene symbol gene
name NIA EST log fold
change in a manuscript
table
(Kultima et al
2004)
Sulindac sulfide Human colorectal
carcinoma SW-480
and HCT-116
NIEHS human 12K chip 10 uM Genbank accession
number gene name and
fold change at various
time points in a
manuscript table data
also available on NIEHS
website
(Bottone
Martinez Collins
Afshari amp Eling
2003)
17Beta-estradiol estriol
estrone genistein
diethylstilbestrol
bisphenol A
nonylphenol
methoxychlor
MCF-7 U95A oligonucleotide
probe arrays (Affymetrix
10 nM (E2 estriol
estrone DES) 10 AM(genistein bisphenol A
nonylphenol and
methoxychlor)
Unigene name gene
name and fold change
for estrogen responsive
and nonresponsive in a
manuscript table
(Terasaka et al
2004)
Ouabain lauryl sulfate
dimethylsulfoxide
cycloheximide
tolbutamide sodium
fluoride diethyl
maleate buthionine
sulfoxamine
potassium bromate
sodium selenite
alloxan adriamycin
hydrogen peroxide
HepG2 Clontech Atlas Human
Stress Toxicology cDNA
arrays (234 genes)
Ouabain (43 uM) lauryl
sulfate (260 uM)
dimethylsulfoxide (128
M) cycloheximide (625
uM) tolbutamide (128
mM) sodium fluoride (3
mM) diethyl maleate
(125 mM) buthionine
sulfoxamine (30 mM)
potassium bromate (25
mM) sodium selenite (30
uM) alloxan (130 mM)
adriamycin (40 uM)
hydrogen peroxide
(4 mM)
Gene name ratio p -
value in downloadable
tables at journal website
(Morgan et a l
2002)
Aflatoxin B(1) (AFB(1))
2-acetylaminofluorene
(2AAF)
dimethylnitrosamine
(DMN)
acetaminophen (APAP)
HepG2 and primary
hepatocytes
Gene filter arrays
containing 31000 genes
10 uM aflatoxin B1
40 mM acetaminophen
100 uM
dimethylnitrosamine
10 uM
2-acetylaminofluorene
None (Harris et al
2004)
Mitomycin C (MMC)
and cisplatin (CIS)
and an alkylating
agent methyl
methanesulfonate
(MMS)
indirect-acting
genotoxins included
hydroxyurea (HU) a
ribonucleotide
reductase inhibitor
taxol (TXL) a
microtubule inhibitor
and etoposide
(ETOP)
L5178Y TK(+-)
mouse lymphoma
Affymetrix mouse
MG-U74A for MMC
and MG-U745Av2
(Affymetrix Inc Santa
Clara CA) for all the
other chemicals a total
of 9977 probe sets
(genes or ESTs) common
to these two array
models
Low mid and high doses Accession number gene
name gene ID statistical
significance at each time
point in a manuscript
table
(Hu et al 2004)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6646
Table 3 (continued)
Compounds Cell type Microarray type Compound dose Microarray data
availability
Reference
Hydroxyurea
(a carcinogen)
p-anisidine
(a noncarcinogen)
and paclitaxel
L5178Y Tk_-mouse
lymphoma
The Twin - Chip Mouse-
74K Digital Genomics
cDNA microarray
10 ngml paclitaxel 313
ugml hydroxyurea 32
ugml p-anisidine
Gene symbol and fold
change in a manuscript
table
(Lee et al 2003)
Acetaminophen
amiodarone clofibrate
erythromycin estolate
isoniazid alpha-
naphtylylisothiocyanate
beta-naphtoflavone
4-pentenoic acid
phenobarbital
tetracycline and
zileuton
Wistar Rat
hepatocytes
DualChip rat hepato
(Eppendorf Hamburg
Germany)
A single concentration
which varied for each
compound
Accession numbers and
gene namemdashfold
changes shown as
colored heat mapmdashnote
easily extracted from
publication
(de Longueville
et al 2003)
Bleomycin and hydrogen
peroxide
Mouse lymphoma
L5178Y TK(+ -)
Clontech Mouse 12K
cDNA microarray (1185
genes)
Bleomycin (25 and 20
ugml) hydrogen peroxide
(5 and 10 ugml)
Gene names and fold
change presented as bar
charts in publication
(Seidel Kan
Stott Schisler amp
Gollapudi 2003)
Bupivicaine
camptothecin
HL-60 Agilent human cDNA
microarray
1 mM Gene name GenBank
accession number
unigene and ratio in a
manuscript table
(Unami
Shinohara
Ichikawa amp Baba
2003)
Bisphenol A Mouse Sertoli TTE3 IntelliGene mouse
expression glass
microarrays (Version
10 Takara Shuzo)
which were spotted with
564 cDNA fragments of
mouse known genes and
approximately 301
expressed sequence tags
(ESTs)
0ndash400 uM Gene name GenBank
accession number and
fold change at time
points in a manuscript
table
(Tabuchi amp
Kondo 2003)
Mitomycin C or
doxorubicin
Hep G2 85 human gene custom
array
10 um mitomycin C
2 um doxorubicin or
2 ethanol
Bar charts with fold
changes in publication
very few genes
(Hong Muller amp
Lai 2003)
Amphotericin B Human peripheral
blood mononuclear
and THP-1
GF211 FKnown Genes_
Genefilter cDNA array
(ResGen) this array
consists of gt4000
individual elements
each representing a
known human gene
5 ugml Accession numbers and
fold expression in a
manuscript table
(Cleary Rogers amp
Chapman 2001
Rogers Pearson
Cleary Sullivan
amp Chapman
2002)
Benzo(a)pyrene diol
epoxide
TK6 human
lymphoblastoid
Human-350 microarray
a glass slide with 350
spotted human cDNA
probes (Phase-1
Molecular Toxicology
0 001 010 or
10 ugml)
Gene names and fold
change at doses in a
manuscript table
(Akerman et al
2004)
Etomoxir HepG2 Clontech AtlasiHuman Stress
Toxicology cDNA arrays
(234 genes)
1 mM etomoxir Gene names and fold
change in a manuscript
table
(Merrill et al
2002)
Tetrodotoxin Human glioma cell
line HTB-138
Using Affymetrix
GeneChip (HG-U133A
10 and 20 mM Affymetrix ID Genbank
ID gene name gene
symbol and fold change
in a manuscript table
(Raghavendra
Prasad Qi
Srinivasan amp
Gopalakrishnakone
2004)
Methotrexate
mercaptopurine
Human acute
lymphoblastic
leukemia
Affymetrix U133A chip Low and high dose and
combination
Data available as
supplemental data online
(Cheok et al 2003)
Prednisolone
vincristine
asparaginase
daunorubicin
Human acute
lymphoblastic
leukemia
Affymetrix U133A chip Various Data available as
supplemental data online
(Holleman et al
2004)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 47
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6648
A second study has indicated how a natural language
processing method CCNet was used to show the genes
regulated by the nuclear hormone receptor FXR (Apic
Ignjatovic Boyer amp Russell 2005) These automated
methods enable a more complete understanding of the
complexity of the transcriptional factors (Ekins Mirny amp
Schueltz 2002b Plant 2004 Ulrich 2003) but ultimately
rely on the quality of the content of the underlying database
of literature interactions This is a key consideration that is
often overlooked For example the gold standard database is
one that is manually curated to ensure the fidelity of the
direct interaction and is preferable to one generated
computationally by algorithms like natural language pro-
cessing (Nikolsky Nikolskaya amp Bugrim 2005) The
advantage of interaction networks over clustering has been
demonstrated in one study using MetaCore (Nikolsky
Ekins Nikolskaya amp Bugrim 2005) by reanalysis of a
published microarray study of G0-arrested MCF-7 breast
cancer cells treated with estrogen and 4-hydroxytamoxifen
(Hodges et al 2003) After producing integrated gene
networks for each treatment strikingly different patterns
were displayed although both contained early transcriptional
factors myc jun and fos Only the estrogen network featured
induced genes essential for all cell cycle phases (Nikolsky et
al 2005) Similarly microarray data for benzene toxicity
(Yoon et al 2003) has been re-analyzed by focusing on the
genes assessed on the p53 pathway (Ekins et al 2005e) We
envisage that a database of such networks for toxic
compounds will be used for comparing between different
molecules and used in the development of predictive
algorithms for Systems-ADMETox modeling in future
Another approach to using such pathway approaches is to
visualize the results of quantitative structure activity models
for predicting molecules binding to enzymes transporters
receptors and ion channels (Ekins Andreyev et al in press
2005e)
It is also possible to simultaneously interpret high
throughput data and predictions on interaction networks
providing a novel approach to predicting and understanding
potential undesirable drugndashdrug or off target effects in the
area of systems pharmacology An example data set uses
percent inhibition data for clotrimazole and ticonazole
which were screened against many different assays at a
single concentration in a commercially available database
BioPrint (Cerep Redmond WA) as published recently
(Fliri Loging Thadejo amp VOlkman 2005) The data for
10 assays has been arbitrarily encoded as inhibitors (gt50
inhibition) or non-inhibitors (lt50 inhibition) in a text file
which was then loaded into MetaCore The analyzed
network algorithm was then used which generates a large
network and fragments it into sub-networks each with a Z-
score and p-values for ranking according to saturation with
objects from the initial gene list The Gene-ontology
processes are also mapped to the gene list and individual
networks In this example a statistically significant network
was generated for the different proteins (Fig 4
p =2838e31) This network also maps the Gene Ontology
processes for the activation of MAPK (118 of genes p
value 9143e07) signal transduction (333 1600e05)
regulation of transcription DNA-dependent (294
2786e04) regulation of inflammatory response (39
3746e04) and the regulation of blood pressure (78
4230e04) This example network indicates how molecules
of the same or different therapeutic classes could be
evaluated for their effects as a graph either together as in
this case or individually This would be useful to indicate
potential off target effects and identify structurally dissimilar
molecules with similar network patterns Such networks
could then be compared to assess network overlap or
differences between molecules and their inhibition of
multiple proteins This type of unique visualization of high
throughput screening data illustrates how the target proteins
may be connected as a network to infer the possible
downstream effects of inhibition
With the burgeoning number of freely available online
and commercial databases that can be used for pathway
construction numbering in the hundreds there have been
suggestions to impose standards for model exchange
querying and visualization (Cary Bader amp Sander 2005)
To date there has been little discussion with regards to
standardization of ADMETox related databases although
there has been considerable discussion relating to drug
metabolism database generation (Erhardt 2003) This is
certainly an important area to address in the future There is
already a growing literature related to ADMETox that is
partially captured in the several commercially available
databases (Ekins et al 2005e) but to date there have been
limited academic efforts to capture data for transporters
with the human membrane transporter database (Yan amp
Sadee 2000) TP-search transporter database (httpwww
ilabrisewasedaacjptp-search) drug interaction database
(httpwwwdruginteractioninfoorgDatabaseinfoaspx)
nuclear hormone receptors (Nakata Yukawa Komiyama
Nakano amp Kaminuma 2002) the ADME-AP database
(Sun Ji Chen Wang amp Chen 2002) and PharmaGKB
(Oliver et al 2002) DSStox (httpwwwepagovnheert
dsstox) TOXNET (httptoxnetnimnihgov) that are
readily accessible
In order to generate accessible pathways using any of
the available software a large enough set of object
identifiers are required to map onto the underlying data-
base To demonstrate this datasets from toxicogenomics
studies have been evaluated with both the KEGG pathway
database and a commercially available product MetaCore
(wwwgenegocom) These gene or protein lists range in
size from 21 to 1853 objects In virtually all cases more
identifiers are mapped to networks in MetaCore and this
also seems independent of the identifier type used
(Unigene Affymetrix Genbank or Locuslink Table 5)
These numbers will obviously change as more information
is added to the respective databases hence the more
objects that are mapped from a dataset The more extensive
Table 4
Literature derived toxicoproteomics data
Compounds Data source Microarray type Compound dose Microarray data
availability
Reference
Carbon
tetrachloride
Male Wistar rats Affymetrix rat chip 8799
probes+proteomics
study
6ndash24 h exposure Genbank SwissProt and
binary data in a
manuscript table
(Fountoulakis
2004)
Paracetamol CD-1 male mice Custom mouse tox blots
with 450 genes
RTQ-PCR+proteomics
study 2-DIGE+
MALDI-MS
150 or 500 mgkg Gene name IMAGE ID
GenBank accession
number fold induction
SwissProt identifier and
protein abundance
change in manuscript
tables
(Ruepp Tonge
Shaw Wallis amp
Pognan 2002)
Paracetamol AP-1 male mice Affymetrix murine 11K
set+proteomics study
Up to 500 mgkg Gene names and fold
changes at multiple
doses and time points in
a manuscript tablemdash
proteomics data not
accessible
(Coen et al 2004)
Oxazepam or
Wy-14643
Male B6C3F1 mice NIEHS Mouse Chip
(8700 genes) 2-DIGE
and MS
Oxazepam (2500 ppm)
Wyeth (Wy)-14643
(500 ppm)
Proteomics data in a
manuscript table
Genbank accession
number gene name and
fold change in a
manuscript table data
also available to
download from NIEHS
website
(Iida et al 2003)
Compound A
(PPAR gamma
ligand)
Female rats CrlCD
(SD)IGS BR
Proteomics 2-DIGE and
MS
250 mgkgday up to
5 days
Accession number and
protein name and
average ratio in a
manuscript table
(Meneses-Lorente
et al 2004)
Bromobenzene Male Wistar rats Proteomics 2-DIGE and
MS custom 3000 cDNA
rat chip
5 mmolkg Accession number gene
name fold change in a
manuscript table protein
names along with bar
charts
(Heijne Stierum
Slijper van
Bladeren amp van
Ommen 2003)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 49
the network that can be generated (as it will consist of more
nodes) then a more comprehensive understanding of the
networks is possible The data available currently in the
literature can be used to evaluate such pathway and
network generation tools Recently we have used several
of the published studies (Tables 1ndash3) with MetaCore to
visualize networks for acetaminophen furan carbon tetra-
chloride benzene and cisplatin showing genes involved in
In vivo
In vitro
In Silico
1970s 1980s 1990s 2000s
Systems Biology ()
OMICS
Fig 2 The timeline for major paradigms in ADMETox
oxidative stress (Ekins Giroux Nikolsky Bugrim amp
Nikolskaya 2005c)
Other important pathwaynetwork building tools that could
potentially be applied to toxicogenomics data include
Ingenuity pathways analysis (httpwwwingenuitycom)
PathArt (httpwwwjubilantbiosyscompdhtm) Pathway
Assist (httpwwwariadnegenomicscomproctspathway
html) (Nikitin et al 2003) and several other databases
deposited at the Pathway Resource List (httpcbiiomskcc
orgprl) All of these products have unique underlying
proprietary pathway databases which are compiled manually
or automatically with text mining tools or a combination of
both We are still waiting for studies that provide a comparison
of different database tools or network building algorithms
Until then theremay be someoverlap but also some differences
between the results obtained from more than one network
method due to the database content and the algorithms used
The reader is recommended to evaluate for themselves several
technologies and select those with the most appropriate fit to
their own specialized needs
In the next Sections the further application of some
available network and database tools will be described with
Microarray or other high throughput data
Upload list of geneprotein identifiers and fold change significance etc (raw data or after selection of the differentially expressed
Genes using other statistical methods)
Parse database and generate interaction network with different algorithms or visualize on maps
Filter Networks
Access significance of the interaction networks
Determine GO processes
Compare 2 or more networks intersection overlap etc
Export gene list
Export gene list
generate interaction network with different algorithms or visualize on maps
Visualization
Fig 3 Schematic for the utilization of pathway tools for assessing high throughput data
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6650
specific detailed reference to transporters and enzymes but
these technologies can also be applied elsewhere as
described above
Fig 4 Network for high throughput screening data for clotrimazole and ticona
cholecystokinin CCR1 Choline transporter Chloride channel catechol-O-methyl
and co workers (Fliri et al 2005) Nodes highlighted with large blue circles rep
molecules showing gt50 inhibition in the assay blue circles represents both mol
inhibits gt50 and one inhibits lt50 Ligands (large) linked to transcriptional f
(wwgenegocom) (For interpretation of the references to colour in this figure leg
311 The role of transporters
A diverse array of organic solutes such as nutrients
neurotransmitters and drugs are transported by specialized
zole screened against 10 in vitro assays (Human Cannabinoid 1 Human
transferase COX2 CYP1A2 CYP2B6 CYP2C19) data published by Fliri
resent assays used in the study Red circles on these nodes represent both
ecules showing lt50 inhibition chequered circles represent one molecule
actors enzymes and transporters via edges using the MetaCorei database
end the reader is referred to the web version of this article)
Table 5
Datasets acquired from publications and other sources and used to evaluate two databasepathway tools KEGG and metaCore
Dataset Number of objects
genes proteins in list
Number of objects
mapped with KEGG
Number of objects mapped
with MetaCore on networks
Reference for microarray
data gene list
Tox Chip 10 (NIEHS) 1853 405 1498 httpsdir-appsniehsnihgov
mapsguestclonesrchcfm
4-Hydroxytamoxifen and
estrogen
1617 434 1343 (Hodges et al 2003)
Mitochondrial proteins 722 156 388 (Gaucher et al 2004
Taylor et al 2003)
Bromobenzene
(24 and 48 h)
130 41 89 (Heijne et al 2004)
Acetaminophen 30 19 23 (Heinloth et al 2004)
Acetaminophen 84 29 64 (Huang et al 2004)
Furan 185 64 139 (Huang et al 2004)
Tetrodotoxin 116 31 86 (Raghavendra Prasad et al
2004)
Benzene 73 16 62 (Yoon et al 2003)
Benzene 76 5 53 (Faiola et al 2004)
Carbon tetrachloride 37 8 26 (Young et al 2003)
Estrogen 94 33 90 (Lobenhofer et al 2002)
Trovafloxacin 142 20 82 (Liguori et al 2005)
Phenobarbital 37 13 28 (Ueda et al 2002)
L-742694 (liver)+ 45 17 19 (Hartley et al 2004)
L-742694 (intestine)+ 23 11 10 (Hartley et al 2004)
A-277249 21 7 9 (Waring et al 2002)
All files were converted to Locuslink identifiers except as noted () unigene () Affymetrix (+) Genbank identifiers were used
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 51
proteins across cellular membranes These may function as
passive processes or active processes energized by the
hydrolysis of ATP or coupling to the co-transport of counter
ions down an electrochemical gradient such as Na+ H+ and
Cl There are many thousands of transporters which can be
classified into distinct superfamilies One of these the solute
carrier class (SLC) is rapidly expanding and contains over
30 families and 200 members The ATP-binding cassette
(ABC) contains 7 families and over 48 members including
P-glycoprotein (P-gp) and MRP subfamilies (Zhang Knipp
Ekins amp Swaan 2002a Zhang Phelps Cheng Ekins amp
Swaan 2002b) Transporters have a key role in clinical
pharmacology with many drugs specifically targeting them
Numerous drugs share transport pathways with nutrients
and transporters have a role in oral absorption drug
bioavailability drug resistance excretion and ultimately
pharmacokinetics and pharmacodynamics
Polymorphism of drug transporters may be a key factor
in drug interactions and lack of effectiveness This field has
become known as pharmacogenomics and is focused on
understanding of inherited DNA sequence variations (poly-
morphisms and mutations) revealed by xenobiotics (Evans
amp McLeod 2003 Weinshilboum 2003) Over the last few
decades many genes have been directly linked to the
mechanisms of response (Evans amp McLeod 2003 Wein-
shilboum 2003) such that 20ndash95 of variability to drug
response is inheritable (Evans amp McLeod 2003) This
phenotypical variability is mainly caused by single nucleo-
tide polymorphisms (SNPs) present in anywhere from 1 to
50 of the population resulting in either lower protein
activity incorrect folding or rapid degradation via proteo-
somes (Weinshilboum amp Wang 2004) A number of
structurally diverse molecules bind to P-gp which is
expressed in many tissues and has numerous SNPs one of
which (C3435T) affects the expression level in the
duodenum and therefore can impact the absorption of
molecules which would be substrates for this transporter
(Sakaeda Nakamura amp Okumura 2002) The human
proton-dependent dipeptide transporter (hPEPT1) can also
affect the absorption of molecules in the intestine and
recently 9 SNPs were found with only one displaying a
reduced transport capacity (Zhang et al 2004) The sodium-
dependent carnitine cotransporter OCTN2 can possess
mutations and these result in primary carnitine deficiency
which impacts fatty acid oxidation and is characterized by
many clinical manifestations (Lahjouji Mitchel amp Qureshi
2001) The organic cation transporter 1 (OCT1) is also
important in the transport of numerous xenobiotics and
endobiotics Recently 4 SNPs were identified in the
Japanese population and when functionally characterized
in vitro the uptake of cations was reduced significantly for
some of these mutations indicating that this would likely
contribute to inter-individual variations in metabolism of
drugs which were transported via OCT1 (Sakata et al
2004)
3111 Clinical relevance of transporters The pregnane
X-receptor (PXR) is a transcriptional regulator of the
enzyme human MDR1 (P-gp) MRPs and OATP (Wang amp
LeCluyse 2003) CYP3A (Bertilsson et al 1998 Blumberg
et al 1998 Kliewer et al 1998) and CYP2C89 (Synold
Dussault amp Forman 2001) as well as many other genes
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6652
involved in the transport metabolism and biosynthesis of
bile acids (Staudinger Liu Madan Habeebu amp Klaassen
2001) However the additional receptors CAR FXR LXR
and other nuclear receptors take part in a complex network
of interactions to control these and other proteins Thus
elucidation of the regulatory networks which control the
expression of these transporters is also important To date
most of the research has centered on efflux transporters but
there has been considerable interest in uptake transporters
such as the organic anion transporter polypeptide (OATP
((Kim 2003) see also later Section on OATP)
There are several specific examples of the importance of
drug transporters to the clinical development of drugs One
example is the insulin sensitizer troglitazone which was
withdrawn due to hepatotoxicity although the precise
mechanism appears to have been unclear until recently
The major metabolite is a sulfated species and is suspected
of being responsible for the observed toxicity The recent
assessment of the organic anion transporting polypeptides
OATP-C and OATP8 expressed on the hepatocyte baso-
lateral membrane indicated that sulphated troglitazone has a
high affinity for the former and possibly lower affinity for
the latter (Nozawa et al 2004) This metabolite would
therefore be expected to accumulate in hepatocytes and
inhibit the bile salt export pump and Mrp2 Because
polymorphisms have been shown for OATP-C (Tirona
Leake Merino amp Kim 2001) it is also possible that these
may result in the accumulation of the metabolite and in turn
elicit idiosyncratic toxicity A second relevant example of
the impact of transporters is the clinically significant drugndash
drug interaction between cerivastatin and cyclosporine A
which occurs via the OATP-C transporter (Shitara Itoh
Sato Li amp Sugiyama 2003) A third example are the HIV
protease inhibitors saquinavir ritonavir and indinavir which
are transported by MRP2 in vitro and other drugs such as
probenecid and sulfinpyrazone are able to enhance this
transport Transport by MRP2 suggests that these com-
pounds will have decreased bioavailability due to increased
clearance and other drugs could aggravate this situation by
further enhancing transport (Huisman et al 2002) Sim-
ilarly the rifampicin mediated induction via PXR of MRP2
and P-gp in healthy subjects was found to significantly
decrease the AUC and also correlated with intestinal
expression of these transporters This transporter is also
inducible by cisplatin 2-AAF and phenobarbital (Schrenk
et al 2001) indicating multiple mechanisms may be
involved In other species such as rat commonly used as a
toxicity model orthologs of the transporters such as OATP2
are expressed and can be induced with ligands for PXR like
PCN (Guo Choudhuri amp Klaassen 2002) This is useful
knowledge because the advent of microarray technology
allows one to dose a rat with a xenobiotic and assess
thousands of genes simultaneously in a particular tissue For
instance animals dosed with known nephrotoxins have
shown some upregulation of the NandashKndashCl transporter
however the authors suggested genomic responses are
stronger soon after exposure before declining (Fleck et
al 2003) Some transporters may therefore be specifically
targeted by drugs in one tissue such as the CNS but these
same transporters may also be expressed elsewhere in the
body hence off-target effects may result in toxicity The
serotonin transporter is one such example which is
expressed in the lungs and brain Some substrates for this
channel like fenfluramine can result in primary pulmonary
hypertension as they accumulate in pulmonary cells (Roth-
man Ayestas Dersch amp Baumann 1999) Similarly P-gp is
expressed at the blood brain barrier and intestine impacting
the efficacy and bioavailability of drugs
3112 Transporter network examples ABCA1 The
ABCA1 transporter mediates the first step of cholesterol
transport Mutations in this gene cause Tangier disease
which results in severe HDL deficiency cholesterol
accumulation in macrophages and attendant atherosclerosis
This transporter represents a drug target for upregulation
modulating cholesterol metabolism and prevention of
cardiovascular disease (Oram amp Lawn 2001) In vitro
ABCA1 can be inhibited by the sulfonylurea glybenclamide
(Field Burn amp Mathur 2004 Wang Silver Thiele amp Tall
2001) In order to illustrate the advantages of mapping drug
transporters as networks onto functional models alongside
other proteins one can consider the example of ABCA1
The query of the MetaCorei database shows that this
transporter appears on three manually curated pathway maps
representing the Fstate of the art_ knowledge derived from
reliable high quality literature sources One can also use the
individual maps as an interface to access the underlying
layers of information about the transporter including the list
of encoding genessplice variants with known SNPs In
addition to browsing MetaCorei a user can also build
custom networks around ABCA1 using the network-
construction tool (Fig 5) Such a visualization utility may
be very helpful for identification of all putative pathways
around a particular transporter or compound of interest The
ABCA1 network created by this tool shows that this
transporter at the time of writing is linked directly to
twenty-five other objects such as APOE1 and LXR Many
of its neighbors have their own SNPs that could be
important in determining interactions between transport of
a drug and normal human transport of endogenous ligands
in health or disease
3113 Transporter network examples OATP The
OATPs are key membrane bound transporters expressed in
many organs including intestine liver lung choroid plexus
blood brain barrier and other organs (Tamai et al 2000)
This family of transporters is capable of mediating the
sodium-independent transport of a diverse array of mole-
cules such as steroid conjugates organic anions and
xenobiotics by coupling uptake with efflux of bicarbonate
(Satlin Amin amp Wolkoff 1997) glutathione or its
conjugates (Hagenbuch amp Meier 2004) The inhibition of
Fig 5 The network of direct gene interactions around the ABCA1 transporter gene (centre) generated with MetaCorei The interation types between nodes are shown as small colored hexagons eg unspecified
allosteric regulation binding cleavage competition covalent modification dephosphorylation phosphorylation transcription regulation transformation When applicable interactions also have a positive or
negative effect and direction Ligands (purple) linked to other proteins (blue blobs) transfactors (red) enzymes (orange) (For interpretation of the references to colour in this figure legend the reader is referred to
the web version of this article)
SEkin
sJournalofPharm
acologica
landToxico
logica
lMeth
ods53(2006)38ndash66
53
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6654
this transporterrsquos hepatic uptake of other compounds may be
important for reported drugndashdrug interactions (Kim 2003)
described earlier (Shitara et al 2003) as well as cerivastatin
with gemfibrozil (Shitara Hirano Sato amp Sugiyama 2004)
OATP1B1 (previous names OATP-C LST-1 OATP2
SLC21A6) represents the most studied human OATP to
date (Meier amp Stieger 2000) and is expressed on the
basolateral plasma membrane of hepatocytes Several single
nucleotide polymorphisms have been identified in the
OATP1B1 gene in European-Americans African-Americans
(Tirona et al 2001) and Japanese (Nozawa et al 2002)
dramatically impacting the transport of ligands such as
pravastatin (Mwinyi Johne Bauer Roots amp Gerloff 2004
Nishizato et al 2003) estrone-3-sulfate (Nozawa et al
2002 Tirona et al 2001) Rifampin (Tirona Leake
Wolkoff amp Kim 2003) and estradiol 17h-d-glucuronide(Tirona et al 2001)
The regulation of SLCO may be affected during
extrahepatic cholestasis bile duct ligation bile salt induced
Fig 6 Networks generated with the autoexpand algorithm in MetaCorei to illustr
be interconnected with other protein regulatory signaling information Informa
unspecified allosteric regulation binding cleavage competition covalent m
transformation When applicable interactions also have a positive or negative e
transfactors (red) enzymes (orange) (For interpretation of the references to colour
cholestatic hepatitis (Dumont et al 1997 Meier amp Stieger
2000 Rost et al 2003) and primary sclerosing cholangitis
(Oswald Kullack-Ublick Paumgarter amp Beuer 2001) In
particular OATP1B1 appears to be regulated by the liver-
enriched transcription factor hepatocyte nuclear factor 1a
(HNF1a) which binds to the promoter region of this
transporter (Jung et al 2001) Site directed mutagenesis
of this binding site resulted in inactivation suggesting the
critical nature of the interaction with HNF1a Bile acids
such as CDCA have been shown to transcriptionally repress
HNF1a in vitro via inhibition of the transactivating effect of
HNF4a on HNF1a (Jung amp Kullak-Ublick 2003) After
screening many rat and human uptake transporters in vitro
OATP1B1 was also shown to modulate the PXR response
by controlling rifampin retention in the cell and therefore
affecting the induction of CYP3A4 and other gene products
such as P-gp (Tirona et al 2003)
Some of the literature for OATP1B1 human substrate
data has been annotated into MetaCorei to illustrate the
ate how ligands for the human OATP-C (OATP1B1) transporter (centre) can
tion on the type of interaction between objects is hidden for clarity eg
odification dephosphorylation phosphorylation transcription regulation
ffect and direction Ligands (purple) linked to other proteins (blue blobs)
in this figure legend the reader is referred to the web version of this article)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 55
visualization of the complex interconnections between this
transporter its ligands regulatory factors and signaling
molecules already in the database (Fig 6) The network was
generated with the autoexpand algorithm in the software
representing one of multiple available algorithms for
connecting genes ligands and other objects in the database
Clearly if more ligands and their connections are added to
the database the complexity of the network will increase
considerably The OATP-C (OATP1B1) gene details can be
viewed upon querying the database and links are provided
to other public databases This page can be used to highlight
the multiple synonyms for this gene as well as links to the
multiple SNPs identified to date
3114 Transporter microarray data Microarrays have
generally been limited in the number of transporters present
on them (Annereau et al 2004) however they have been used
in an attempt to correlate pharmacokinetic properties with
gene expression for valacyclovir (Landowski et al 2003) as
well as understand the expression profile in different tissues
or cell lines upon food component or xenobiotic treatment
(Anderle Huang amp Sadee 2004) This lack of transporters
on microarrays has prompted some groups to produce their
own arrays with a heavier emphasis on transporters These
arrays have for example then been used to demonstrate the
upregulation of ABC transporters and down-regulation of
GST-Pi in cell lines resistant to colchicines or 9-nitro-
camptothecin (Annereau et al 2004) The genes that were
significantly up or down-regulated in this particular study
were used to build networks with MetaCore (Fig 7A B) and
the similarities between them were assessed (Fig 7C)
Although there were only a small number of significantly
changed genes in common (IL-8 Fos GST-Pi Calpactin and
Ubiquitin hydrolase) it is perhaps likely that there is a much
larger common gene network that is important for drug
resistance although a much larger number of cell lines and
drug treatments need to be evaluated to produce a definitive
drug resistance signature involving transporters enzymes
and transcriptional regulators
312 Applications to enzymes
As we have already described it may be particularly
valuable to visualize enzymes as networks to show
interactions with transcriptional regulators and ligands
For example a key enzyme is CYP3A4 which metabolizes
40ndash45 of all drugs and has relatively few SNPs
(Ingelman-Sundberg 2004) Using a second software suite
MetaDrugi (wwwgenegocom) it is possible to construct
a custom network around this or other drug metabolizing
enzymes (Fig 8) In this case the gene network for
CYP3A4 highlights all of the major transcriptional
regulators and several more distant linked proteins and
ligands connected on the network that may be useful for
further study (Fig 8) Substrates inhibitors as well as
regulatory factors and other enzymes can be observed
connected on this network Due to the many hundreds of
known inhibitors and substrates only a small number are
shown here for clarity We assume that if a perturbation in
a pathway (eg due to a nonfunctional enzyme) is linked
to a certain pathologic condition a similar perturbation
caused by the interference from xenobiotic metabolism
(eg competitive inhibition of the same enzyme) may
result in identical effects As microarray gene expression
data is increasingly generated the role of enzyme
regulation in toxicity of certain xenobiotics will become
more apparent from either in vivo or in vitro studies The
visualization of such signature gene networks involving
transporters and enzymes their ligands and regulatory
factors will also be important for future toxicity prediction
methods We have recently generated visualizations of
microarray data from MCF-7 cells treated with 4-hydrox-
ytamoxifen to show that some of the key genes involved in
metabolism and transport are upregulated (Ekins et al
2005d) In addition we have made predictions with various
QSAR models in MetaDrug to indicate the involvement of
PXR CYP3A4 and P-gp (Ekins et al 2005e) Therefore it
appears likely that 4-hydroxytamoxifen could increase its
own metabolism as well as efflux from cells via P-gp
which can be visualized on networks Any decrease of
function of these enzymes or transporters in a population
would likely result in changes in the metabolism and
transport of this active metabolite potentially impacting
the clinical effect This represents one example of how
both pathway tools QSAR models and network building
algorithms can be used with different types of predicted
and experimental data to allow visualization of potential
compound interactions or toxicity
313 Future network applications
As the population ages an increasing incidence and
prevalence of systemic diseases especially chronic diseases
have occurred among older adults This has resulted in an
increase in medications used concomitantly by this
population which presents challenges for drugndashdrug
interactions Physiologically elderly patients may behave
differently to the younger patients for which the drugs are
initially developed for and tested on Many pharmacoki-
netic investigations in the elderly population reveal
decreased clearance of lipophilic drugs metabolized by
the cytochrome P450 enzymes however few studies have
evaluated aging-dependent or gender-related changes in
specific P450 enzymes (Hunt Westerkam amp Slave 1992)
Age-related physiological changes such as a reduction in
liver mass hepatic metabolizing enzyme activity liver
blood flow and alterations in plasma drug binding may
account for the decreased elimination of some metabolized
drugs in the elderly It is particularly difficult to separate an
effect of aging from the variation in the rate of metabolism
due to factors such as individual metabolic phenotype
(slow or fast metabolizer due to SNPs) environmental
influences concomitant disease states and drug intake
(drugndashdrug interactions) The available data suggest that
Fig 7 Networks for drug resistant cell lines generated with the Auto expand algorithm in MetaCore A KB-3-1 vs KB-8-5 B ATC-DU-145 vs RCO1 C node overlap of A and B with initial nodes highlighted
with blue or red solid circles (Initial gene lists derived from (Annereau et al 2004)) Information on the type of interaction between objects is hidden for clarity eg unspecified allosteric regulation binding
cleavage competition covalent modification dephosphorylation phosphorylation transcription regulation transformation When applicable interactions also have a positive or negative effect and direction
Ligands (purple) linked to other proteins (blue blobs) transfactors (red) enzymes (orange) (For interpretation of the references to colour in this figure legend the reader is referred to the web version of this article)
SEkin
sJournalofPharm
acologica
landToxico
logica
lMeth
ods53(2006)38ndash66
56
Fig7(continued)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 57
Fig7(continued)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6658
Fig 8 The network of gene interactions around the CYP3A4 enzyme (centre) from MetaCorei Information on the type of interaction between objects is
hidden for clarity eg unspecified allosteric regulation binding cleavage competition covalent modification dephosphorylation phosphorylation
transcription regulation transformation When applicable interactions also have a positive or negative effect and direction Ligands (purple) linked to other
proteins (blue blobs) transfactors (red) enzymes (orange) (For interpretation of the references to colour in this figure legend the reader is referred to the web
version of this article)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 59
the initial doses of drugs metabolized by these enzymes
should be reduced in older patients according to the clinical
response In most published studies the elderly appear at
least as responsive as the young to inducers or inhibitors of
P450s (Durnas Loi amp Cusack 1990) More recently there
has been some suggestion that there are age related
reductions in function of some specific P450s such as
CYP3A4 (Patki et al 2004) and this could occur at the
level of regulation However earlier studies with the same
enzyme showed no change in clearance with age (Hunt et
al 1992) To date there has been even less examination of
the transporter functions and any changes with age
(Kinirons amp OrsquoMahony 2004) so the current understand-
ing of the effects of aging on metabolism and transport is
anything but transparent This represents an extreme
challenge for the pharmaceutical industry how to predict
whether a drug has an affinity for an enzyme or transporter
and whether this may be clinically important if it shows a
decline in expression or function with aging Any decrease
of function of these enzymes or transporters in an elderly
population would result in changes in the metabolism and
transport of metabolites potentially impacting the clinical
effect This represents another example of how computa-
tional approaches may perhaps be used with different types
of predicted and experimental data to allow visualization of
potential compound interactions or toxicity in elderly
populations We may see different gene networks high-
lighted as humans age and these may be modified by drug
treatment and coadministration This represents an area
were network analysis could be applied in the future and is
in need of considerable further research
The collection of microarray data in databases such as
CEBS ArrayTrack and EDGE represents a future resource
for computational gene network analysis One could
envisage that ultimately in each case such data is converted
to one or more networks that are also displayed for the user
and can be used to compare treatments from in vivo and in
vitro experiments This would represent a different approach
to clustering the data as currently implemented in one of
these efforts (Hayes et al 2005) and may condense large
amounts of experimentally derived data into a readily
interpreted network
4 Discussion
Previously in this journal the progress of many
research groups in predicting human ADME parameters
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6660
in silico (Ekins et al 2000b) and approaches for drug
metabolism (Ekins Ring Grace McRobie-Belle amp
Wrighton 2000a) have been described Both of these
reviews commented on moving HT assays for ADME
Tox much earlier in drug discovery which had also been
indicated by other groups The initial reviews also
highlighted the likely wealth of data that would become
available and how this could be used for structure
activity relationships alongside the bioactivity data in
computational models It was noted that there was a
paucity of predictive metabolism tools at that time In
addition a growing number of efforts to model whole
cells and organs now a field called systems biology
were recognized as models that could be integrated with
the in silico ADME approaches In summary since these
past reviews virtually all pharmaceutical companies have
attempted earlier high throughput screening for ADME
Tox properties and to some extent the wider application
of computational approaches for physicochemical proper-
ties Systems biology is being quite widely acknowledged
as the new paradigm for understanding complex bio-
logical datasets derived from high throughput technolo-
gies and the accumulated knowledge on human protein
interactions (Hartwell Hopfield Leibler amp Murray 1999
Hood 2003b) Therefore systems biology can be defined
as the integration of genetic proteomic transcriptomic
and metabonomic data using computational methods
(Nicholson amp Wilson 2003) When taken together
information on molecular processes derived from different
sources represents a lsquolsquouniversersquorsquo of putative biological
functionality of which only a small fraction of it will be
realized in a cell at any given time To date systems
biology has been driven by academia and funding bodies
such as the NIH rather than the pharmaceutical compa-
nies Presently there is a great deal of interest from
scientists of all backgrounds in identifying the networks
of cellular pathways and the corresponding physically
interacting proteins
The network building software for systems biology
described in this current review will be valuable to query
high throughput data and known literature interactions in
order to predict potential toxicity in different species In the
future the compilation of published toxicogenomics data-
sets characteristic of different types of toxicity will likely
be available in these software systems to act as a reference
database It is also feasible that we will be able to generate
the annotated datasets which specifically address the
differences between human and rat networks implicated
in toxicity The identification of sub-network modules
conserved between human and rat distinct for toxicity
types or predictive for toxic end-points in human will be
possible Such signature gene networks (Nikolsky et al
2005) can then be verified with other experimentally
derived data prospectively or from preexisting databases
The combined Fhigh throughput datandashin silico network_approaches therefore represents a novel method for access-
ing complex tissue-level processes as networks integrating
all data types based on functional interactions The data-
bases developed and used in this approach will certainly
benefit from further annotation around the drug metaboliz-
ing enzymes and transporters as described herein in terms
of transcriptional regulation and the ligands associated with
them which frequently appear in the literature It will be
important to capture disparities in the assignment of ligands
to enzymes transporters and other proteins as well as
negative data
In summary although there have been numerous
toxicogenomics studies published there is presently a
relatively small number of datasets that are freely available
to perform network analysis of microarray data The
number of studies identifying large numbers of proteins
which are affected by molecule treatment are even scarcer
still while there are several examples of some published
studies that combine such data It is hoped that the
numerous database initiatives for high content and toxicol-
ogy data that are being undertaken will improve the
situation for other researchers that are not currently
equipped to do such microarray studies themselves The
addition of requirements by journals to deposit such raw
data in a freely accessible resource will aid these initiatives
ADMETox groups have seen new technologies and
approaches developed over the last decade that have all
been applied to identify poor compounds earlier (Fig 2)
The latest technologies integrate network building tools
with high content data and databases The current review
described the limited number of networks generated for
ADMETox at present and one hopes that the impact of
such analyses will be commonplace in the future Systems
biology is however more than just applying a network
approach and hence systems-ADMETox will have to
evolve due to the continual pressure to develop newer
technologies This current paradigm combining empirical
data and computational methods should integrate the
complex data already generated making it readily inter-
pretable and valuable for identifying the most promising
compounds in the future
Acknowledgements
Dr Maggie AZ Hupcey is gratefully acknowledged for
editorial assistance Dr Peter W Swaan (University of
Maryland) and Dr Cheng Chang (Ohio State University)
Dr Steve Wright (University of Arizona) Dr K Sandy
Pang (University of Toronto) and Dr Craig Giroux (Wayne
State University) are kindly acknowledged for their support
and discussions My colleagues at GeneGo Sergey
Andreyev Andy Ryabov Eugene Kirillov Eugene A
Rakhmatulin Svetlana Sorokina Andrej Bugrim Tatiana
Nikolskaya Yuri Nikolsky John Metz and Julie Bryant are
all thanked for their considerable contributions to software
development and data annotation
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 61
References
Abe M Yamashita S Kuramoto T Hirayama Y Tsukamoto T Ohta
T et al (2003) Global expression analysis of N-methyl-NV-nitro-N-nitrosoguanidine-induced rat stomach carcinomas using oligonucleotide
microarrays Carcinogenesis 24 861ndash867
Adachi T Ono Y Koh K B Takashima K Tainaka H Matsuno Y
et al (2004) Long-term alteration of gene expression without
morphological change in testis after neonatal exposure to genistein in
mice Toxicogenomic analysis using cDNA microarray Food and
Chemical Toxicology 42 445ndash452
Akerman G S Rosenzweig B A Domon O E McGarrity L J
Blankenship L R Tsai C A et al (2004) Gene expression profiles
and genetic damage in benzo(a)pyrene diol epoxide-exposed TK6 cells
Mutation Research 549 43ndash64
Aksenov S V Church B Dhiman A Georgieva A Sarangapani R
Helmlinger G et al (2005) An integrated approach for inference and
mechanistic modeling for advancing drug development FEBS Letters
579 1878ndash1883
Anderle P Huang Y amp Sadee W (2004) Intestinal membrane transport
of drugs and nutrients Genomics of membrane transporters using
expression microarrays European Journal of Pharmaceutical Sciences
21 17ndash24
Annereau J P Szakacs G Tucker C J Arciello A Cardarelli C
Collins J et al (2004) Analysis of ATP-binding cassette transporter
expression in drug-selected cell lines by a microarray dedicated to
multidrug resistance Molecular Pharmacology 66 1397ndash1405
Apic G Ignjatovic T Boyer S amp Russell R B (2005) Illuminating drug
discovery with biological pathways FEBS Letters 579 1872ndash1877
Baker V A Harries H M Waring J F Duggan C M Ni H A Jolly
R A et al (2004) Clofibrate-induced gene expression changes in rat
liver A cross-laboratory analysis using membrane cDNA arrays
Environmental Health Perspectives 112 428ndash438
Balakin K V Ekins S Bugrim A Ivanenkov Y A Korolev D amp
Nikolsky Y et al (2004a) Quantitative structurendashmetabolism relation-
ship modeling of the metabolic N-dealkylation rates Drug Metabolism
and Disposition 32 1111ndash1120
Balakin K V Ekins S Bugrim A Ivanenkov Y A Korolev D amp
Nikolsky Y et al (2004b) Kohonen maps for prediction of binding to
human cytochrome P450 3A4 Drug Metabolism and Disposition 32
1183ndash1189
Barabasi A -L amp Oltvai Z N (2004) Network biology Under-
standing the cellrsquos functional organization Nature Reviews Genetics
5 101ndash113
Bartosiewicz M Penn S G amp Buckpitt A (2001) Applications of gene
arrays in environmental toxicology Fingerprints of gene regulation
associated with cadmium chloride benzo(a)pyrene and trichloro-
ethylene Environmental Health Perspectives 109 71ndash74
Bertilsson G Heidrich J Svensson K Asman M Jendeberg L
Sydow-Backman M et al (1998) Identification of a human nuclear
receptor defines a new signaling pathway for CYP3A induction
Proceedings of the National Academy of Sciences of the United States
of America 95 12208ndash12213
Blumberg B Sabbagh Jr W Juguilon H Bolado Jr J van Meter
C M Ong E S et al (1998) SXR a novel steroid and xenobiotic-
sensing nuclear receptor Genes and Development 12 3195ndash3205
Borodina Y Rudik A Filimonov D Kharchevnikova N Dmitriev A
Blinova V et al (2004) A new statistical approach to predicting
aromatic hydroxylation sites Comparison with model-based
approaches Journal of Chemical Information and Computer Sciences
44 1998ndash2009
Borodina Y Sadym A Filimonov D Blinova V Dmitriev A amp
Poroikov V (2003) Predicting biotransformation potential from
molecular structure Journal of Chemical Information and Computer
Sciences 43 1636ndash1646
Bottone Jr F G Martinez J M Collins J B Afshari C A amp Eling
T E (2003) Gene modulation by the cyclooxygenase inhibitor
sulindac sulfide in human colorectal carcinoma cells Possible link to
apoptosis Journal of Biological Chemistry 278 25790ndash25801
Boyer S amp Zamora I (2002) New methods in predictive metabolism
Journal of Computer-Aided Molecular Design 16 403ndash413
Bredel M amp Jacoby E (2004) Chemogenomics An emerging strategy
for rapid target and drug discovery Nature Reviews Genetics 5
262ndash275
Bugrim A Nikolskaya T amp Nikolsky Y (2004) Early prediction of drug
metabolism and toxicity Systems biology approach and modeling
Drug Discovery Today 9 127ndash135
Bulera S J Eddy S M Ferguson E Jatkoe T A Reindel J F
Bleavins M R et al (2001) RNA expression in the early character-
ization of hepatotoxicants in Wistar rats by high-density DNA micro-
arrays Hepatology 33 1239ndash1258
Butcher E C Berg E L amp Kunkel E J (2004) Systems biology in drug
discovery Nature Biotechnology 22 1253ndash1259
Butte A (2002) The use and analysis of microarray data Naturalist
Review of Drug Discovery 1 951ndash960
Cary M P Bader G D amp Sander C (2005) Pathway information for
systems biology FEBS Letters 579 1815ndash1820
Cheok M H Yang W Pui C H Downing J R Cheng C Naeve
C W et al (2003) Treatment-specific changes in gene expression
discriminate in vivo drug response in human leukemia cells Nature
Genetics 34 85ndash90
Cleary J D Rogers P D amp Chapman S W (2001) Differential
transcription factor expression in human mononuclear cells in response
to amphotericin B Identification with complementary DNA microarray
technology Pharmacotherapy 21 1046ndash1054
Coen M Ruepp S U Lindon J C Nicholson J K Pognan F Lenz
E M et al (2004) Integrated application of transcriptomics and
metabonomics yields new insight into the toxicity due to paracetamol in
the mouse Journal of Pharmaceutical and Biomedical Analysis 35
93ndash105
Cornwell P D De Souza A T amp Ulrich R G (2004) Profiling of
hepatic gene expression in rats treated with fibric acid analogs
Mutation Research 549 131ndash145
Csermely P Agoston V amp Pongor S (2005) The efficiency of multi-
target drugs The network approach might help drug design Trends in
Pharmacological Sciences 26 178ndash182
Cunningham M J Liang S Fuhrman S Seilhamer J J amp
Somogyi R (2000) Gene expression microarray data analysis for
toxicology profiling Annals of the New York Academy of Sciences 919
52ndash67
de Longueville F Atienzar F A Marcq L Dufrane S Evrard S
Wouters L et al (2003) Use of a low density microarray for studying
gene expression patterns induced by hepatotoxicants on primary
cultures of rat hepatocytes Toxicological Sciences 75 378ndash392
Dobrin R Beg Q K Barabasi A L amp Oltvai Z N (2004)
Aggregation of topological motifs in the Escherichia coli transcriptional
regulatory network BMC Bioinformatics 5 10
Donald S Verschoyle R D Edwards R Judah D J Davies R
Riley J et al (2002) Hepatobiliary damage and changes in hepatic
gene expression caused by the antitumor drug ecteinascidin-743 (ET-
743) in the female rat Cancer Research 62 4256ndash4262
Dumont M Jacquemin E DrsquoHont C Descout C Cresteil D Haouzi
D et al (1997) Expression of the liver Na+-independent organic anion
transporting polypeptide (oatp-1) in rats with bile duct ligation Journal
of Hepatology 27 1051ndash1056
Durnas C Loi C M amp Cusack B J (1990) Hepatic drug metabolism
and aging Clinical Pharmacokinetics 19 359ndash389
Eisen M B Spellman P T Brown P O amp Botstein D (1998) Cluster
analysis and display of genome-wide expression patterns Proceedings
of the National Academy of Sciences of the United States of America
95 14863ndash14868
Ekins S Andreyev S Ryabov A Kirilov E Rakhmatulin E A
Bugrim A et al (in press) Computational Prediction of Human Drug
Metabolism Exp Opin Drug Metab Toxicol
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6662
Ekins S Boulanger B Swaan P W amp Hupcey M A Z (2002)
Towards a new age of virtual ADMETOX and multidimensional drug
discovery Journal of Computer-Aided Molecular Design 16 381ndash401
Ekins S Bugrim A Nikolsky Y amp Nikolskaya T (2005) Systems
biology Applications in drug discovery In S Gad (Ed) Drug
Discovery Handbook (pp 123ndash183) Hoboken NJrsquo Wiley and Sons
Ekins S Giroux C N Nikolsky Y Bugrim A amp Nikolskaya T
(2005) A signature gene network approach to toxicity The Toxoco-
logists 84
Ekins S Kirillov E Rakhmatulin E amp Nikolskaya T (2005) A novel
method for visualizing nuclear hormone receptor networks relevant to
drug metabolism Drug Metabolism and Disposition 33 474ndash481
Ekins S amp McGowan R J (2001) The limits of reductionism The
shifting genomic paradigmrsquos impact on industry and academia
Philsophy in Science 9 1ndash23
Ekins S Mirny L amp Schuetz E G (2002) A ligand-based approach to
understanding selectivity of nuclear hormone receptors PXR CAR
FXR LXRa and LXRb Pharmaceutical Research 19 1788ndash1800
Ekins S Nikolsky Y amp Nikolskaya T (2005) Techniques Application
of systems biology to absorption distribution metabolism excretion
and toxicity Trends in Pharmacological Sciences 26 202ndash209
Ekins S Ring B J Grace J McRobie-Belle D J amp Wrighton S
A (2000) Present and future in vitro approaches for drug
metabolism Journal of Pharmacological and Toxicological Methods
44 313ndash324
Ekins S Waller C L Swaan P W Cruciani G Wrighton S A amp
Wikel J H (2000) Progress in predicting human ADME parameters in
silico Journal of Pharmacological and Toxicological Methods 44
251ndash272
Erhardt P W (2003) A human drug metabolism database Potential roles
in the quantitative predictions of drug metabolism and metabolism-
related drugndashdrug interactions Current Drug Metabolism 4 411ndash422
Evans W E amp McLeod H L (2003) Pharmacogenomicsmdashdrug
disposition drug targets and side effects New England Journal of
Medicine 348 538ndash549
Ezendam J Staedtler F Pennings J Vandebriel R J Pieters R
Boffetta P et al (2004) Toxicogenomics of subchronic hexachlor-
obenzene exposure in Brown Norway rats Environmental Health
Perspectives 112 782ndash791
Faiola B Fuller E S Wong V A amp Recio L (2004) Gene expression
profile in bone marrow and hematopoietic stem cells in mice exposed to
inhaled benzene Mutation Research 549 195ndash212
FDA (2004) Innovation stagnation Challenge and opportunity on the
critical path to new medicinal products
Fiehn O (2001) Combining genomics metabolome analysis and bio-
chemical modelling to understand metabolic networks Comparative
Functional Genomics 2 155ndash168
Field F J Born E amp Mathur S N (2004) LXRRXR ligand activation
enhances basolateral efflux of beta-sitosterol in CaCo-2 cells Journal of
Lipid Research 45 905ndash913
Fleck C Sutter L Appenroth D Koch B Meinhold T Pitack M
et al (2003) Use of gene chip technology for the characterisation of
the regulation of renal transport processes and of nephrotoxicity in
rats Experimental and Toxicologic Pathology 54 401ndash410
Fliri A F Loging W T Thadeio P F amp Volkmann R A (2005)
Biological spectra analysis Linking biological activity profiles to
molecular structure Proceedings of the National Academy of Sciences
of the United States of America 102 261ndash266
Fountoulakis M (2004) Application of proteomics technologies in the
investigation of the brain Mass Spectrometry Reviews 23 231ndash258
Gaucher S P Taylor S W Fahy E Zhang B Warnock D E Ghosh
S S et al (2004) Expanded coverage of the human heart
mitochondrial proteome using multidimensional liquid chromatography
coupled with tandem mass spectrometry Journal of Proteome
Research 3 495ndash505
Gerhold D Lu M Xu J Austin C Caskey C T amp Rushmore T
(2001) Monitoring expression of genes involved in drug metabolism
and toxicology using DNA microarrays Physiological Genomics 5
161ndash170
Guo G L Choudhuri S amp Klaassen C D (2002) Induction profile of
rat organic anion transporting polypeptide 2 (oatp2) by prototypical
drug-metabolizing enzyme inducers that activate gene expression
through ligand-activated transcription factor pathways Journal of
Pharmacology and Experimental Therapeutics 300 206ndash212
Hagenbuch B amp Meier P J (2004) Organic anion transporting
polypeptides of the OATP SLC21 family Phylogenetic classification
as OATP SLCO superfamily new nomenclature and molecularfunc-
tional properties Pflugers Archiv 447 653ndash665
Hamadeh H K amp Afshari C A (2004) Toxicogenomics principles and
applications Hobokenrsquo John Wiley and Sons
Hamadeh H K Bushel P R Jayadev S Martin K DiSorbo O Sieber
S et al (2002) Gene expression analysis reveals chemical-specific
profiles Toxicological Sciences 67 219ndash231
Hamadeh H K Jayadev S Gaillard E T Huang Q Stoll R
Blanchard K et al (2004) Integration of clinical and gene expression
endpoints to explore furan-mediated hepatotoxicity Mutation Research
549 169ndash183
Hamadeh H K Knight B L Haugen A C Sieber S Amin R P
Bushel P R et al (2002) Methapyrilene toxicity Anchorage of
pathologic observations to gene expression alterations Toxicologic
Pathology 30 470ndash482
Han J D Bertin N Hao T Goldberg D S Berriz G F Zhang L V
et al (2004) Evidence for dynamically organized modularity in the
yeast proteinndashprotein interaction network Nature 430 88ndash93
Hanisch D Zien A Zimmer R amp Lengauer T (2002) Co-clustering of
biological networks and gene expression data Bioinformatics 18
S145ndashS154
Harris A J Dial S L amp Casciano D A (2004) Comparison of basal
gene expression profiles and effects of hepatocarcinogens on gene
expression in cultured primary human hepatocytes and HepG2 cells
Mutation Research 549 79ndash99
Harrison S C (2004) Whither structural biology Nature Structural
Molecular Biology 11 12ndash15
Hartley D P Dai X He Y D Carlini E J Wang B Huskey S E
et al (2004) Activators of the rat pregnane X receptor differentially
modulate hepatic and intestinal gene expression Molecular Pharma-
cology 65 1159ndash1171
Hartwell L H Hopfield J J Leibler S amp Murray A W (1999) From
molecular to modular cell biology Nature 402 C47ndashC52
Hasmall S Orphanides G James N Pennie W Hedley K Soames
A et al (2002) Downregulation of lactoferrin by PPARalpha ligands
Role in perturbation of hepatocyte proliferation and apoptosis
Toxicology Sciences 68 304ndash313
Hayase T Yamamoto Y Yamamoto K Muso E amp Shiota K (2004)
Microarray profile analysis of toxic cocaine-induced alterations in the
expression of mouse brain gene sequences A possible rsquoprotectiversquo
effect of buprenorphine Journal of Applied Toxicology 24 15ndash20
Hayes K R Vollrath A L Zastrow G M McMillan B J Craven M
Jovanovich S et al (2005) EDGE A centralized resource for the
comparison analysis and distribution of toxicogenomic information
Molecular Pharmacology 67 1360ndash1368
Heijne W H Slitt A L van Bladeren P J Groten J P Klaassen C D
Stierum R H et al (2004) Bromobenzene-induced hepatotoxicity at
the transcriptome level Toxicology Sciences 79 411ndash422
Heijne W H Stierum R H Slijper M van Bladeren P J amp van
Ommen B (2003) Toxicogenomics of bromobenzene hepatotoxicity
A combined transcriptomics and proteomics approach Biochemical
Pharmacology 65 857ndash875
Heinloth A N Irwin R D Boorman G A Nettesheim P Fannin
R D Sieber S O et al (2004) Gene expression profiling of rat
livers reveals indicators of potential adverse effects Toxicology
Sciences 80 193ndash202
Hodges L C Cook J D Lobenhofer E K Li L Bennett L Bushel
P R et al (2003) Tamoxifen functions as a molecular agonist inducing
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 63
cell cycle-associated genes in breast cancer cells Molecular Cancer
Research 1 300ndash311
Holleman A Cheok M H den Boer M L Yang W Veerman KM
A J Kazemier et al (2004) Gene-expression patterns in drug-resistant
acute lymphoblastic leukemia cells and response to treatment New
England Journal of Medicine 351 533ndash542
Hong Y Muller U R amp Lai F (2003) Discriminating two classes of
toxicants through expression analysis of HepG2 cells with DNA arrays
Toxicology in Vitro 17 85ndash92
Hood E (2003) Proteomics Characterizing the cogs in the machinery of
life Environmental Health Perspectives 111 A816ndashA825
Hood L (2003) Systems biology Integrating technology biology and
computation Mechanism of Ageing and Development 124 9ndash16
Hood L amp Galas D (2003) The digital code of DNA Nature 421
444ndash448
Hood L amp Perlmutter R M (2004) The impact of systems approaches on
biological problems in drug discovery Nature Biotechnology 22
1215ndash1217
Hu T Gibson D P Carr G J Torontali S M Tiesman J P Chaney
J G et al (2004) Identification of a gene expression profile that
discriminates indirect-acting genotoxins from direct-acting genotoxins
Mutation Research 549 5ndash27
Huang Q Dunn II R T Jayadev S DiSorbo O Pack F D Farr S B
et al (2001) Assessment of cisplatin-induced nephrotoxicity by
microarray technology Toxicology Sciences 63 196ndash207
Huang Q Jin X Gaillard E T Knight B L Pack F D Stoltz J H et
al (2004) Gene expression profiling reveals multiple toxicity endpoints
induced by hepatotoxicants Mutation Research 549 147ndash167
Huisman M T Smit J W Crommentuyn K M Zelcer N Wiltshire
H R Beijnen J H et al (2002) Multidrug resistance protein 2
(MRP2) transports HIV protease inhibitors and transport can be
enhanced by other drugs Aids 16 2295ndash2301
Hunt C M Westerkam W R amp Stave G M (1992) Effect of age and
gender on the activity of human hepatic CYP3A Biochemical
Pharmacology 44 275ndash283
Ideker T Ozier O Schwikowski B amp Siegal A F (2002) Discovering
regulatory and signalling circuits in molecular interaction networks
Bioinformatics 18 S233ndashS240
Ideker T Thorsson V Ranish J A Christmas R Buhler J Eng J K
et al (2001) Integrated genomic and proteomic analyses of a system-
atically perturbed network Science 292 929ndash934
Iida M Anna C H Hartis J Bruno M Wetmore B Dubin J R et al
(2003) Changes in global gene and protein expression during early
mouse liver carcinogenesis induced by non-genotoxic model carcino-
gens oxazepam and Wyeth-14643 Carcinogenesis 24 757ndash770
Ingelman-Sundberg M (2004) Pharmacogenetics of cytochrome P450 and
its applications in drug therapy The past present and future Trends in
Pharmacological Sciences 25 193ndash200
Jeong H Mason S P Barabasi A L amp Oltvai Z N (2001) Lethality
and centrality in protein networks Nature 411 41ndash42
Jeong H Tombor B Albert R Oltvai Z N amp Barabasi A L
(2000) The large-scale organization of metabolic networks Nature
407 651ndash654
Jung D Hagenbuch B Gresh L Pontoglio M Meier P J amp Kullak-
Ublick G A (2001) Characterization of the human OATP-C
(SLC21A6) gene promoter and regulation of liver-specific OATP genes
by hepatocyte nuclear factor 1 alpha Journal of Biological Chemistry
276 37206ndash37214
Jung D amp Kullak-Ublick G A (2003) Hepatocyte nuclear factor 1
alpha A key mediator of the effect of bile acids on gene expression
Hepatology 37 622ndash631
Jung J -W Park J -S Hwang J -W Kang K -S YS L Song B
-S et al (2004) Gene expression analysis of peroxisome prolifer-
ators- and phenytoin-induced hepatotoxicity using cDNA microarray
Journal of Veterinary Medical Science 66 1329ndash1333
Kato N Shibutani M Takagi H Uneyama C Lee K -Y Takiami S
et al (2004) Gene expression profile in the livers of rats orally
administered ethinylestradiol for 28 days using a microarray technique
Toxicology 200 179ndash192
Kier L D Neft R Tang L Suizu R Cook T Onsurez K et al
(2004) Applications of microarrays with toxicologically relevant genes
(tox genes) for the evaluation of chemical toxicants in Sprague Dawley
rats in vivo and human hepatocytes in vitro Mutation Research 549
101ndash113
Kijima K Toyosawa K Yasuba M Matsuoka N Adachi T
Komiyama M et al (2004) Gene expression analysis of the rat testis
after treatment with di(2-ethylhexyl) phthalate using cDNA microarray
and real-time RT-PCR Toxicology and Applied Pharmacology 200
103ndash110
Kim R B (2003) Organic anion-transporting polypeptide (OATP) trans-
porter family and drug disposition European Journal of Clinical
Investigation 33 1ndash5
Kinirons M T amp OrsquoMahony M S (2004) Drug metabolism and ageing
British Journal of Clinical Pharmacology 57 540ndash544
Kitano H (2002a) Computational systems biology Nature 420 206ndash210
Kitano H (2002b) Systems biology A brief overview Science 295
1662ndash1664
Kliewer S A Moore J T Wade L Staudinger J L Watson M
A Jones S A et al (1998) An orphan nuclear receptor activated
by pregnanes defines a novel steroid signalling pathway Cell 92
73ndash82
Korolev D Balakin K V Nikolsky Y Kirillov E Ivanenkov Y A
Savchuk N P et al (2003) Modeling of human cytochrome p450-
mediated drug metabolism using unsupervised machine learning
approach Journal of Medicinal Chemistry 46 3631ndash3643
Kramer J A Pettit S D Amin R P Bertram T A Car B
Cunningham M et al (2004) Overview on the application of
transcription profiling using selected nephrotoxicants for toxicology
assessment Environmental Health Perspectives 112 460ndash464
Kultima K Nystrom A -M Scholz B Gustafson A -L Dencker L
amp Stigson M (2004) Valproic acid teratogenicity A toxicogenomics
approach Environmental Health Perspectives 112 1225ndash1235
Kutuzova G D amp Deluca H F (2004) Gene expression profiles in rat
intestine identify pathways for 125-dihydroxyvitamin D(3) stimulated
calcium absorption and clarify its immunomodulatory properties
Archives of Biochemistry and Biophysics 432 152ndash166
Kwak M K Wakabayashi N Itoh K Motohashi H Yamamoto M amp
Kensler T W (2003) Modulation of gene expression by cancer
chemopreventive dithiolethiones through the Keap1-Nrf2 pathway
Identification of novel gene clusters for cell survival Journal of
Biological Chemistry 278 8135ndash8145
Lahjouji K Mitchell G A amp Qureshi I A (2001) Carnitine transport
by organic cation transporters and systemic carnitine deficiency
Molecular Genetics and Metabolism 73 287ndash297
Landowski C P Sun D Foster D R Menon S S Barnett J L
Welage L S et al (2003) Gene expression in the human intestine and
correlation with oral valacyclovir pharmacokinetic parameters Journal
of Pharmacology and Experimental Therapeutics 306 778ndash786
Lee G -J Lee W -S Jeon K -S Um C -H Kim S -J Lee C -H
et al (2004) cDNA microarray gene expression analysis and
toxicological phenotype for anticancer drug Journal of Veterinary
Medical Science 66 1339ndash1345
Lee M Kwon J Kim S N Kim J E Koh W S Kim E J et al
(2003) cDNA microarray gene expression profiling of hydroxyurea
paclitaxel and p-anisidine genotoxic compounds with differing
tumorigenicity results Environmental and Molecular Mutagenesis
42 91ndash97
Li S Armstrong C M Bertin N Ge H Milstein S Boxem M et al
(2004) A map of the interactome network of the metazoan C elegans
Science 303 540ndash543
Liguori M J Anderson L M Bukofzer S McKim J Pregenzer J F
Retief J et al (2005) Microarray analysis in human hepatocytes
suggests a mechanism for hepatotoxicity induced by trovafloxacin
Hepatology 41 177ndash186
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6664
Lobenhofer E K Bennett L Cable P L Li L Bushel P R amp Afshari
C A (2002) Regulation of DNA replication fork genes by 17beta-
estradiol Molecular Endocrinology 16 1215ndash1229
Locker J Tian J Carver R Concas D Cossu C Ledda-Columbano
G M et al (2003) A common set of immediate-early response genes
in liver regeneration and hyperplasia Hepatology 38 314ndash325
Mattes W B Pettit S D Sansone S A Bushel P R amp Waters M D
(2004) Database development in toxicogenomics Issues and efforts
Environmental Health Perspectives 112 495ndash505
McMillian M Nie A Y Parker J B Leone A Bryant S Kemmerer
M et al (2004) A gene expression signature for oxidant stressreactive
metabolites in rat liver Biochemical Pharmacology 68 2249ndash2261
Meier P J amp Stieger B (2000) Molecular mechanisms in bile formation
News in Physiological Sciences 15 89ndash93
Meneses-Lorente G de Longueville F Dos Santos-Mendes S Bonnert
T P Jack A Evrard S et al (2003) An evaluation of a low-density
DNA microarray using cytochrome P450 inducers Chemical Research
in Toxicology 16 1070ndash1077
Meneses-Lorente G Guest P C Lawrence J Muniappa N Knowles
M R Skynner H A et al (2004) A proteomic investigation of drug-
induced steatosis in rat liver Chemical Research in Toxicology 17
605ndash612
Merrill C L Ni H Yoon L W Tirmenstein M A Narayanan P
Benavides G R et al (2002) Etomoxir-induced oxidative stress in
HepG2 cells detected by differential gene expression is confirmed
biochemically Toxicology Sciences 68 93ndash101
Milo R Shen-Orr S Itzkovitz S Kashtan N Chklovskii D amp Alon
U (2002) Network motifs Simple building blocks of complex
networks Science 298 824ndash827
Morgan K T Ni H Brown H R Yoon L Qualls C W Crosby L
M et al (2002) Application of cDNA microarray technology to in
vitro toxicology and the selection of genes for a real-time RT-PCR-
based screen for oxidative stress in Hep-G2 cells Toxicologic
Pathology 30 435ndash451
Mwinyi J Johne A Bauer S Roots I amp Gerloff T (2004) Evidence
for inverse effects of OATP-C (SLC21A6) 5 and 1b haplotypes on
pravastatin kinetics Clinical Pharmacology and Therapeutics 75
415ndash421
Nakata K Yukawa M Komiyama N Nakano T amp Kaminuma T
(2002) A nuclear receptor database that maps pathways to diseases
Genome Infomatics 13 515ndash516
Nicholson J K Holmes E Lindon J C amp Wilson I D (2004) The
challenges of modeling mammalian biocomplexity Nature Biotechnol-
ogy 22 1268ndash1274
Nicholson J K amp Wilson I D (2003) Understanding rsquoglobal systems
biology Metabonomics and the continuum of metabolism Nature
Reviews on Drug Discovery 2 668ndash676
Nikitin A Egorov S Daraselia N amp Mazo I (2003) Pathway studiomdash
the analysis and navigation of molecular networks Bioinformatics 19
2155ndash2157
Nikolsky Y Ekins S Nikolskaya T amp Bugrim A (2005) A novel
method for generation of signature networks as biomarkers from
complex high throughput data Toxicology Letters 158 20ndash29
Nikolsky Y Nikolskaya T amp Bugrim A (2005) Biological networks
and analysis of experimental data in drug discovery Drug Discovery
Today 10 653ndash662
Nishizato Y Ieiri I Suzuki H Kimura M Kawabata K Hirota T
et al (2003) Polymorphisms of OATP-C (SLC21A6) and OAT3
(SLC22A8) genes Consequences for pravastatin pharmacokinetics
Clinical Pharmacology and Therapeutics 73 554ndash565
Nozawa T Nakajima M Tamai I Noda K Nezu J Sai Y et al
(2002) Genetic polymorphisms of human organic anion transporters
OATP-C (SLC21A6) and OATP-B (SLC21A9) Allele frequencies in
the Japanese population and functional analysis Journal of Pharmacol-
ogy and Experimental Therapeutics 302 804ndash813
Nozawa T Sugiura S Nakajima M Goto A Yokoi T Nezu J et al
(2004) Involvement of organic anion transporting polypeptides in the
transport of troglitazone sulfate Implications for understanding trogli-
tazone hepatotoxicity Drug Metabolism and Disposition 32 291ndash294
Oliver D E Rubin D L Stuart J M Hewett M Klein T E amp
Altman R B (2002) Ontology development for a pharmacogenetics
knowledge base Package Symposion 88ndash99
Oram J F amp Lawn R M (2001) ABCA1 The gatekeeper for eliminating
excess tissue cholesterol Journal of Lipid Research 42 1173ndash1179
Oswald M Kullak-Ublick G A Paumgartner G amp Beuers U (2001)
Expression of hepatic transporters OATP-C and MRP2 in primary
sclerosing cholangitis Liver 21 247ndash253
Parsons A B Brost R I Ding H Li Z Zhang C Sheikh B et al
(2004) Integration of chemical-genetic and genetic interaction data
links bioactive compounds to cellular target pathways Nature Bio-
technology 22 62ndash69
Patki K C von Moltke L L Harmatz J S Hesse L M Court M H
amp Greenblatt D J (2004) Effect of age on in vitro triazolam
biotransformation in male human liver microsomes Journal of
Pharmacology and Experimental Therapeutics 308 874ndash879
Pereira-Leal J B Enright A J amp Ouzounis C A (2004) Detection of
functional modules from protein interaction networks Proteins 54
49ndash57
Plant N (2004) Interaction networks Coordinating responses to xeno-
biotic exposure Toxicology 202 21ndash32
Raghavendra Prasad H S Qi Z Srinivasan K N amp Gopalakrish-
nakone P (2004) Potential effects of tetrodotoxin exposure to
human glial cells postulated using microarray approach Toxicon 44
597ndash608
Reilly T P Bourdi M Brady J N Pise-Masison C A Radonovich M
F George J W et al (2001) Expression profiling of acetaminophen
liver toxicity in mice using microarray technology Biochemical and
Biophysical Research Communications 282 321ndash328
Rives A W amp Galitski T (2003) Modular organization of cellular
networks Proceedings of the National Academy of Sciences of the
United States of America 100 1128ndash1133
Rogers P D Pearson M M Cleary J D Sullivan D C amp Chapman
S W (2002) Differential expression of genes encoding immunomodu-
latory proteins in response to amphotericin B in human mononuclear
cells identified by cDNA microarray analysis Journal of Antimicrobial
Chemotherapy 50 811ndash817
Rosen M B Wilson V S Schmid J E amp Gray L E (2005) Gene
expression analysis in the ventral prostate of rats exposed to vinclozolin
or procymidone Reproductive Toxicology 19 367ndash379
Rost D Herrmann T Sauer P Schmidts H L Stieger B Meier P J
et al (2003) Regulation of rat organic anion transporters in bile salt-
induced cholestatic hepatitis Effect of ursodeoxycholate Hepatology
38 187ndash195
Rothman R B Ayestas M A Dersch C M amp Baumann M H (1999)
Aminorex fenfluramine and chlorphentermine are serotonin transporter
substrates Implications for primary pulmonary hypertension Circula-
tion 100 869ndash875
Ruepp S U Tonge R P Shaw J Wallis N amp Pognan F (2002)
Genomics and proteomics analysis of acetaminophen toxicity in mouse
liver Toxicology Sciences 65 135ndash150
Sakaeda T Nakamura T amp Okumura K (2002) MDR1 genotype-
related pharmacokinetics and pharmacodynamics Biological amp Phar-
maceutical Bulletin 25 1391ndash1400
Sakata T Anzai N Shin H J Noshiro R Hirata T Yokoyama H
et al (2004) Novel single nucleotide polymorphisms of organic cation
transporter 1 (SLC22A1) affecting transport functions Biochemical
and Biophysical Research Communications 313 789ndash793
Satlin L M Amin V amp Wolkoff A W (1997) Organic anion
transporting polypeptide mediates organic anionHCO3-exchange
Journal of Biological Chemistry 272 26340ndash26345
Satomi Y Tsuchiya W Mihara K Ota M Kasahara Y amp Akahori F
(2004) Gene expression analysis of the lung following paraquat
administration in rats using DNA microarray Journal of Toxicological
Sciences 29 91ndash100
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 65
Schrenk D Baus P R Ermel N Klein C Vorderstemann B amp
Kauffmann H M (2001) Up-regulation of transporters of the MRP
family by drugs and toxins Toxicology Letters 120 51ndash57
Segal E Shapira M Regev A Persquoer D Botstein D Koller D et al
(2003) Module networks Identifying regulatory modules and their
condition-specific regulators from gene expression data Nature
Genetics 34 166ndash176
Segal E Wang H amp Koller D (2003) Discovering molecular pathways
from protein interaction and gene expression Bioinformatics 19
i264ndash i272
Seidel S D Kan H L Stott W T Schisler M R amp Gollapudi B B
(2003) Identification of transcriptome profiles for the DNA-damaging
agents bleomycin and hydrogen peroxide in L5178Y mouse lymphoma
cells Environmental and Molecular Mutagenesis 42 19ndash25
Shan L Yu Y Schut H A J amp Snyderwine E G (2004) Susceptibility
of rats to mammary gland carcinogenesis by the food-derived
carcinogen 2-Amino-1-Methyl-6-Phenylimidazo[45-b]Pyridine (PhIP)
varies with age and is associated with the induction of differential gene
expression American Journal of Pathology 165 191ndash202
Sharom J R Bellows D S amp Tyers M (2004) From large networks to
small molecules Current Opinion in Chemical Biology 8 81ndash90
Shitara Y Hirano M Sato H amp Sugiyama Y (2004) Gemfibrozil and
its glucuronide inhibit the OATP2(OATP1B1 SLC21A6)-mediated
hepatic uptake and CYP2C8-mediated metabolism of cerivastatinmdash
Analysis of the mechanism of the clinically relevant drugndashdrug
interaction between cerivastatin and gemfibrozil Journal of Pharma-
cology and Experimental Therapeutics
Shitara Y Itoh T Sato H Li A P amp Sugiyama Y (2003) Inhibition of
transporter-mediated hepatic uptake as a mechanism for drugndashdrug
interaction between cerivastatin and cyclosporin A Journal of
Pharmacology and Experimental Therapeutics 304 610ndash616
Somogyi R Fuhrman S amp Wen X (2001) Genetic network inference in
computational models and applications to large-scale gene expression
data In M Bower amp H Bolouri (Eds) Computational modeling of
genetic and biochemical networks (pp 119ndash157) Cambridge MArsquoThe
MIT Press
Spirin V amp Mirny L A (2003) Protein complexes and functional
modules in molecular networks Proceedings of the National Academy
of Sciences of the United States of America 100 12123ndash12128
Staudinger J Liu Y Madan A Habeebu S amp Klaassen C D
(2001) Coordinate regulation of xenobiotic and bile acid homeo-
stasis by pregnane X receptor Drug Metabolism and Disposition
29 1467ndash1472
Sun L Z Ji Z L Chen X Wang J F amp Chen Y Z (2002) ADME-
AP A database of ADME associated proteins Bioinformatics 18
1699ndash1700
Suter L Babiss L E amp Wheeldon E B (2004) Toxicogenomics in
predictive toxicology in drug development Chemistry and Biology 11
161ndash171
Synold T W Dussault I amp Forman B M (2001) The orphan nuclear
receptor SXR coordinately regulates drug metabolism and efflux
Nature Medicine 7 584ndash590
Tabuchi Y amp Kondo T (2003) cDNA microarray analysis reveals chop-
10 plays a key role in Sertoli cell injury induced by bisphenol A
Biochemical and Biophysical Research Communications 305 54ndash61
Tamai I Nezu J Uchino H Sai Y Oku A Shimane M et al (2000)
Molecular identification and characterization of novel members of the
human organic anion transporter (OATP) family Biochemical and
Biophysical Research Communications 273 251ndash260
Taylor S W Fahy E Zhang B Glenn G M Warnock D E Wiley S
et al (2003) Characterization of the human heart mitochondrial
proteome Nature Biotechnology 21 281ndash286
Terasaka S Aita Y Inoue A Hayashi S Nishigaki M Aoyagi K
et al (2004) Using a customized DNA microarray for expression
profiling of the estrogen-responsive genes to evaluate estrogen activity
among natural estrogens and industrial chemicals Environmental
Health Perspectives 112 773ndash781
Thomas D M Francescutti-Verbeem D M Liu X amp Kuhn D M
(2004) Identification of differentially regulated transcripts in mouse
striatum following methamphetamine treatmentmdashan oligonucleotide
microarray approach Journal of Neurochemistry 88 380ndash393
Thomas R S Rank D R Penn S G Zastrow G M Hayes K R
Pande K et al (2001) Identification of toxicologically predictive
gene sets using cDNA microarrays Molecular Pharmacology 60
1189ndash1194
Thompson K L Afshari C A Amin R P Bertram T A Car B
Cunningham M et al (2004) Identification of platform-independent
gene expression markers of cisplatin nephrotoxicity Environmental
Health Perspectives 112 488ndash494
Tirona R G Leake B F Merino G amp Kim R B (2001)
Polymorphisms in OATP-C Identification of multiple allelic
variants associated with altered transport activity among European-
and African-Americans Journal of Biological Chemistry 276
35669ndash35675
Tirona R G Leake B F Wolkoff A W amp Kim R B (2003)
Human organic anion transporting polypeptide-C (SLC21A6) is a
major determinant of the rifampin-mediated pregnane X receptor
activation Journal of Pharmacology and Experimental Therapeutics
304 223ndash228
Tong W Cao X Harris S Sun H Fang H Fuscoe J et al (2003)
ArrayTrackmdashsupporting toxicogenomic research at the US Food and
Drug Administration National Center for Toxicological Research
Environmental Health Perspectives 111 1819ndash1826
Tornow S amp Mewes H W (2003) Functional modules by relating
protein interaction networks and gene expression Nucleic Acids
Research 31 6283ndash6289
Trocho C Escola-Gil J C Ribas V Benitez S Martin-Campos J M
Rotllan N et al (2004) Phenytoin treatment reduces atherosclerosis in
mice through mechanisms independent of plasma HDL-cholesterol
concentration Atherosclerosis 174 275ndash285
Ueda A Hamadeh H K Webb H K Yamamoto Y Sueyoshi T
Afshari C A et al (2002) Diverse roles of the nuclear orphan
receptor CAR in regulating hepatic genes in response to phenobarbital
Molecular Pharmacology 61 1ndash6
Ulrich R G (2003) The toxicogenomics of nuclear receptor agonists
Current Opinion in Chemical Biology 7 505ndash510
Ulrich R G Rockett J C Gibson G G amp Pettit S D (2004)
Overview of an interlaboratory collaboration on evaluating the effects
of model hepatotoxicants on hepatic gene expression Environmental
Health Perspectives 112 423ndash427
Unami A Shinohara Y Ichikawa T amp Baba Y (2003) Biochemical
and microarray analyses of bupivacaine-induced apoptosis Journal of
Toxicological Sciences 28 77ndash94
Vasquez A Flammini A Maritan A amp Vespignani A (2003) Global
protein function prediction from proteinndashprotein interaction networks
Nature Biotechnology 21 697ndash700
Vezina C M Walker N J amp Olson J R (2004) Subchronic Exposure to
TCDD PeCDF PCB126 and PCB153 Effect on hepatic gene
expression Environmental Health Perspectives 112 1636ndash1644
Vo T D Greenberg H J amp Palsson B O (2004) Reconstruction and
functional characterization of the human mitochondrial metabolic
network based on proteomic and biochemical data Journal of Bio-
logical Chemistry 279 39532ndash39540
Wang H amp LeCluyse E L (2003) Role of orphan nuclear receptors in
the regulation of drug-metabolising enzymes Clinical Pharmacoki-
netics 42 1331ndash1357
Wang N Silver D L Thiele C amp Tall A R (2001) ATP-
binding cassette transporter A1 (ABCA1) functions as a choles-
terol efflux regulatory protein Journal of Biological Chemistry 276
23742ndash23747
Waring J F Cavet G Jolly R A McDowell J Dai H Ciurlionis R
et al (2003) Development of a DNA Microarray for toxicology based
on hepatoxin-regulated sequences Environmental Health Perspectives
111 863ndash870
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6666
Waring J F Gum R Morfitt D Jolly R A Ciurlionis R Heindel M
et al (2002) Identifying toxic mechanisms using DNA microarrays
Evidence that an experimental inhibitor of cell adhesion molecule
expression signals through the aryl hydrocarbon nuclear receptor
Toxicology 181ndash182 537ndash550
Waring J F Jolly R A Ciurlionis R Lum P Y Praestgaard J T
Morfitt D C et al (2001) Clustering of hepatotoxins based on
mechanism of toxicity using gene expression profiles Toxicology and
Applied Pharmacology 175 28ndash42
Waters M Boorman G Bushel P Cunningham M Irwin R Merrick
A et al (2003) Systems toxicology and the Chemical Effects in
Biological Systems (CEBS) knowledge base EHP Toxicogenomics
111 15ndash28
Wei M Arnold L Cano M amp Cohen S M (2005) Effects of co-
administration of antioxidants and arsenicals on the rat urinary bladder
epithelium Toxicology Sciences 83 237ndash245
Weinshilboum R (2003) Inheritance and drug response New England
Journal of Medicine 348 529ndash537
Weinshilboum R amp Wang L (2004) Pharmacogenetics Inherited
variation in amino acid sequence and altered protein quantity Clinical
Pharmacology and Therapeutics 75 253ndash258
Werner E (2003) In silico multicellular systems biology and minimal
genomes Drug Discovery Today 8 1121ndash1127
Wong J S amp Gill S S (2002) Gene expression changes induced in
mouse liver by di(2-ethylhexyl) phthalate Toxicology and Applied
Pharmacology 185 180ndash196
Xie T Tong L McCann U D Yuan J Becker K G Mechan A O
et al (2004) Identification and characterization of metallothionein-1
and -2 gene expression in the context of ()34-methylenedioxymetham-
phetamine-induced toxicity to brain dopaminergic neurons Journal of
Neuroscience 24 7043ndash7050
Yadetie F Laegreid A Bakke I Kusnierczyk W Komorowski J
Waldum H L et al (2003) Liver gene expression in rats in response
to the peroxisome proliferator-activated receptor-alpha agonist ciprofi-
brate Physiological Genomics 15 9ndash19
Yamashita S Nomoto T Abe M Tatematsu M Sugimura T amp
Ushijima T (2004) Persistence of gene expression changes in stomach
mucosae induced by short-term N-methyl-NV-nitro-N-nitrosoguanidinetreatment and their presence in stomach cancers Mutation Research
549 185ndash193
Yan Q amp Sadee W (2000) Human membrane transporter database A
web-accessible relational database for drug transport studies and
pharmacogenomics AAPS Pharmsci 2 E20
Yeger-Lotem E amp Margalit H (2003) Detection of regulatory circuits by
integrating the cellular networks of proteinndashprotein interactions and
transcription regulation Nucleic Acids Research 31 6053ndash6061
Yoon B I Li G -X Kitada K Kawasaki Y Igarashi K amp Kodama
Y et al (2003) Mechanisms of benzene-induced hematotoxicity and
leukemogenicity cDNA microarray analyses using mouse bone marrow
tissue Environmental Health Perspectives 111 1411ndash1420
Young M B DiSilvestro M R Sendera T J Freund J Kriete A amp
Magnuson S R (2003) Analysis of gene expression in carbon
tetrachloride-treated rat livers using a novel bioarray technology
Pharmacogenomics Journal 3 41ndash52
Yu H Zhu X Greenbaum D Karro J amp Gerstein M (2004) TopNet
A tool for comparing biological sub-networks correlating protein
properties with topological statistics Nucleic Acids Research 32
328ndash337
Zhang E Y Fu D J Pak Y A Stewart T Mukhopadhyay N
Wrighton S A et al (2004) Genetic polymorphisms in human proton-
dependent dipeptide transporter PEPT1 Implications for the functional
role of Pro586 Journal of Pharmacology and Experimental Therapeu-
tics 310 437ndash445
Zhang E Y Knipp G T Ekins S amp Swaan P W (2002)
Structural biology and function of solute transporters Implications
for identifying and designing substrates Drug Metabolism Reviews
34 709ndash750
Zhang E Y Phelps M A Cheng C Ekins S amp Swaan P W (2002)
Modeling of active transport systems Advanced Drug Delivery
Reviews 54 329ndash354
- Systems-ADMETox Resources and network approaches
-
- Introduction
- Data available
- Network analysis and databases
-
- Network applications
-
- The role of transporters
-
- Clinical relevance of transporters
- Transporter network examples ABCA1
- Transporter network examples OATP
- Transporter microarray data
-
- Applications to enzymes
- Future network applications
-
- Discussion
- Acknowledgements
- References
-
Table 1
Literature toxicogenomics data derived from rat in vivo studies
Compounds Rat strain Microarray type Compound dose Microarray data
availability
Reference
Acetaminophen furan
methotrexate
methapyrilene
phenytoin
Male Spraguendash
Dawley VAF+
albino
Phase 1 Molecular
toxicology array
Acetaminophen
(4500 mgkgday)
methotrexate (1 mg
kg day)
methapyrilene (100
mgkgday) furan (40
mgkgday) or
phenytoin (300 mg
kgday)
Gene name accession
number and fold changes
are provided in a
manuscript table
(Huang et al
2004)
A-277249 3MC
Aroclor
SpraguendashDawley Affymetrix rat
toxicology U34 array
10 or 100 mgkg Gene name and
Affymetrix ID and fold
change in a manuscript
table
(Waring et al
2002)
Clofibrate paracetamol
benzoapyrene
Male Spraguendash
Dawley
Custom chip CLO at 250 mgkg
BP at 10 mgkg bw
given 3 times per
week for 2 weeks
APAP at 1000 mgkg
Gene name and fold
change in a manuscript
table
(Cunningham
Liang Fuhrman
Seilhamer amp
Somogyi 2000)
L-742694
Dexamethasone
Female
SpraguendashDawley
25K rat microarray L-742694 (50 mgkg
day) DEX (50 mg
kgday)
Tables of accession
number and gene names
only in a manuscript
table for liver and
intestine
(Hartley et al
2004)
Bromobenzene Male Wistar Custom 3000 rat gene
array
05 2 5 mmolkg Gene name accession
number and log fold
change for 3 dose levels
in a manuscript table
Supplemental data also
available
(Heijne et al
2004)
125-Dihydroxyvitamin
D3
Male Spraguendash
Dawley (small
intestine)
Affymetrix high-density
rat oligonucleotide
arrays (GeneChips
RG-U34A)
730 ngkg Gene name Genbank
accession number and
fold change in a
manuscript table
(Kutuzova amp
Deluca 2004)
Fenofibrate clofibrate
bezafibrate
gemfibrozi
ciprofibrate
beclofibrate etofibrate
Male CD Agilent arrays Dose ranging for up
to 14 days
Gene name and genbank
accession number in a
manuscript table
(Cornwell Souza
amp Ulrich 2004)
Furan Male Spraguendash
Dawley
NIEHS rat chip uml7000
clones
Exposed to 4 or 40
mgkg furan for up
to 14 days
Gene ID and gene
namemdashbinary data in a
manuscript table Dataset
also available on NIEHS
website
(Hamadeh et al
2004)
Paracetamol Male F344N Rat NIEHS tox chip
(httpdirniehsnihgov
microarraychipshtm)
0 50 150
1500 mgkg
Unigene accession
number gene name fold
change in a manuscript
table
(Heinloth et al
2004)
Carbon tetrachloride Male Spraguendash
Dawley
ADME Rat expression
bioarray (Motorola Life
Sciences) consists of
1040 single-stranded
oligonucleotide probes
Up to 14 day
treatment
Accession numbers and
binary data in a
manuscript table
(Young et al
2003)
Carbon tetrachloride and
chloroform
Male Spraguendash
Dawley
The rat CT arrays
contain sequences from
almost 700 rat genes
with known or
discovered
responsiveness to toxic
treatments
6 24 72 h high and
low doses
Gene names and fold
changes at multiple time
points in a manuscript
table
(Kier et al 2004)
(continued on next page)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6640
Table 1 (continued)
Compounds Rat strain Microarray type Compound dose Microarray data
availability
Reference
Dimethylarsinic acid Female F344
(bladder
epithelium)
Rat 10K chip (MWG
Biotech Inc) containing
10000 genes
100 ppm Genbank accession
number gene name in a
manuscript table
(Wei Arnold
Cano amp Cohen
2005)
Dexamethasone
troleandomycin
miconazole
clotrimazole
isoniazid
methylclofanapate
Female Spraguendash
Dawley
Rat HepatoChip DEX (50 mgkgday)
TAO (500 mgkg
day) MIC (100 mg
kgday) CLOT (100
mgkg per day) ISN
(100 mgkgday)
MCP (75 mgkgday)
Gene name and Genbank
accession numbermdashdata
displayed as a heatmap
not readily extracted
from publication
(Meneses-Lorente
et al 2003)
TCDD PeCDF PCB126
PCB153
Female Harlan
SpraguendashDawley
Affymetrix GeneChip
Test3 arrays
Exposed for 13 weeks
to toxicologically
equivalent doses
Accession number gene
name and fold change in
a manuscript table
(Vezina Walker
amp Olson 2004)
Paclitaxel Male and female
SpraguendashDawley
Genocheck 48K cDNA 4 mgkgday male
7 mgkgday female
Accession numbers and
gene names in a
manuscript table
(Lee et al 2004)
Clofibrate gemfibrozil
phenytoin
Male Spraguendash
Dawley VAF(+)
albino
48 K cDNA microarray
in house
Treated with each
compound for 24 h
and 2 weeks
Gene name and fold
change in a manuscript
table
(Jung et al 2004)
Clofibrate
dexamethasone
phenobarbital
3-methylcholanthrene
SpraguendashDawley Merck Drug Safety Chip
1443 genes (rat human
and mouse)
30 mgkgday Gene name GenBank
accession number and
relative fluorescence
levels in a manuscript
table
(Gerhold et al
2001)
Bemitradine clofibrate
doxylamine
methapyrilene
phenobarbital
tamoxifen
2-acetylaminofluorene
4-acetylaminofluorene
isoniazid
Male CD IGS Incyte RatGEM10
uml7800 rat cDNAs
Low mid and high
doses
A bar chart of 2 genes
that are affected by
compounds
(Kramer et al
2004)
PhIP Female Spraguendash
Dawley
Mouse cDNA
microarray containing
9984 cDNA clones
(National Cancer
Institute
75 mgkgday Gene names in a
manuscript table
(Shan Yu Schut
amp Snyderwine
2004)
Ethinylestradiol Male and female
SpraguendashDawley
Custom chip 3776 genes 0 001 01 and
10 ppm
Accession number gene
name and fold change at
different exposure levels
in a manuscript table
(Kato et al 2004)
Paraquat Male Wistar 1090 genes 7 mgkgday Gene name and
expression ratio in a
manuscript table
(Satomi et al
2004)
Hexachlorobenzene Female Brown
Norway
Affymetrix rat
RGU-34A GeneChip
microarray
0 150 or 450 mgkg Accession number gene
name and fold change
data for multiple organs
in a manuscript table
(Ezendam et al
2004)
N-methyl-NV-nitro-N-nitrosoguanidine
(MNNG)
Rat pyloric muco-
sae male congenic
rat strain that has a
homozygous_LIZ
transgene of
BigBlue rat
AFFYMETRIX Rat
Genome U34A
83 mgl AFFY ID gene name
gene symbol and fold
change in a manuscript
table
(Yamashita et al
2004)
N-methyl-NV-nitro-N-nitrosoguanidine
Male ACINJcI
(ACI)
Affymetrix GeneChip
Rat genome U34A
arrays
83 mgl from the age
of 8 weeks through to
40 weeks
Accession number gene
name symbol fold change
in a manuscript table also
rat vs human stomach
cancer comparison
(Abe et al 2003)
Cisplatin SpraguendashDawley Different arrays tox
chip incyte phase 1 etc
03ndash5 mgkg over a 4 to
144 h
Unigene gene ID gene
name NIEHS ID and
data for 5 platforms in a
manuscript table
(Thompson et al
2004)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 41
Table 1 (continued)
Compounds Rat strain Microarray type Compound dose Microarray data
availability
Reference
Clofibrate Male Spraguendash
Dawley
Atlas Rat Toxicology II
arrays (Clontech Palo
Alto CA USA)
containing 465 genes
High (250 mgkgday) or
low (25 mgkgday)
Genbank accession
number and data for 3
platforms in a
manuscript table
(Baker et al
2004)
Di(2-ethylhexyl)
phthalate
Male Spraguendash
Dawleymdash(testes)
An in-house cDNA
microarray
20 or 2000 mgkg Gene names Genbank
ID and fold change in a
manuscript table
(Kijima et al
2004)
Ciprofibrate Female Fischer An in-house cDNA
microarray
50 mgkg body weight Gene names symbol
accession number mean
ratio and SD
(Yadetie et al
2003)
Methapyrilene Male Spraguendash
Dawley
Rat Tox Chip 10 10 or 100 mgkgday Gene Name accession
number and indication of
up or down regulation
original data available on
NIEHS website
(Hamadeh et al
2002b)
Ecteinascidin-743
(ET-743)
Female Wistar Custom chip cDNA
microarrays containing
approximately 4700
hybridizable mouse
expressed sequence tags
derived from IMAGE
clones obtained from
Research Genetics
(Huntsville AL) or from
the MRC Human Gene
Mapping Project
40 ugkg Data available on
laboratory websitemdashnot
available at present
(Donald et al
2002)
Clofibrate Wyeth
14643 Gemfibrozil
phenobarbital
Male Spraguendash
Dawley VAF+
NIEHS rat chip v10 Clofibrate (250
mgkgday Wyeth
14643 (250 mgkg
day) Gemfibrozil
(100 mgkgday)
Phenobarbital
(120 mgkgday)
Gene names and fold
changes in a manuscript
table
(Hamadeh et al
2002a)
Vinclozin procymidone Male Spraguendash
Dawleymdashprostate
Clontech Atlas Rat 12
Toxicology array
200 mgkg Gene name accession
number average fold
change
(Rosen Wilson
Schmid amp Gray
2005)
Cisplatin Male Spraguendash
Dawley VAF1
albino (CRL
CD(SD) BR
Rat Tox Microarrays
were purchased from
Phase-1 Molecular
Toxicology
05 or 1 mgkgday Gene name accession
number fold change in
kidney
(Huang et al
2001)
Allyl alcohol
miodarone Aroclor
1254 arsenic
carbamazepine
carbon tetrachloride
diethylnitrosamine
dimethylformamide
diquat etoposide
indomethacin
methapyrilene
methotrexate
monocrotaline
3-methylcholanthrene
Male Spraguendash
Dawley
Affymetrix GeneChip
Test 2 Array
Allyl alcohol (40 mgkg
day) miodarone (100 mg
kgday) Aroclor 1254
(400 mgkgday) arsenic
(20 mgkgday)
carbamazepine (250 mg
kgday) carbon
tetrachloride (1000mgkg
day) diethylnitrosamine
(100 mgkgday)
dimethylformamide (1000
mgkgday) diquat (172
mgkgday) etoposide (50
mgkgday) indomethacin
(20 mgkgday)
methapyrilene (250 mg
kgday) methotrexate
(250 mgkgday)
monocrotaline (50 mgkg
day)
3-methylcholanthrene
(100 mgkgday)
Heatmap figures and a
table of Affymetrix
names for genes
correlated with clinical
chemistry changes in a
manuscript table
(Waring et al
2001)
(continued on next page)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6642
Table 1 (continued)
Compounds Rat strain Microarray type Compound dose Microarray data
availability
Reference
Microcystin-LR (MLR)
phenobarbital (PB)
lipopolysaccharide
(LPS) carbon
tetrachloride (CT)
thioacetamide (THA)
and cyproterone
acetate (CPA)
Male Wistar Purpose-made rat DNA
microarray (Affymetrix
Santa Clara CA)
containing 1600 rat
DNA sequences
Various Accession number gene
name fold changes
shown as a heat map
table in publicationmdash
data extraction would be
laborious
(Bulera et al
2001)
Acetamidofluorene
aniline bromobenzene
butyl hydroxytol
dieldrin disulfiram
ethinyl estradiol
hexachlorocyclohexane
g 4-methylthiazole
nimesulide piperonyl
butoxide precocene I
pulegone tannic acid
trans-anethole
Male Spraguendash
Dawley
Custom Rat MegaA
cDNA chip 3434-gene
Acetamidofluorene (200
mgkg) aniline (200 mg
kg) bromobenzene (900
mgkg) butyl hydroxytol
(1000 mgkg) dieldrin
(30 and 45 mgkg)
disulfiram (2000 mgkg)
ethinyl estradiol (500
mgkg)
hexachlorocyclohexane
gamma (40 65 80 mg
kg) 4-methylthiazole
(120 mgkg) nimesulide
(500 mgkg) piperonyl
butoxide (4000 mgkg)
precocene I (500 mgkg)
pulegone (400 mgkg)
tannic acid (3000 mg
kg) trans-anethole
(600 mgkg)
Gene name accession
number in a manuscript
table
(McMillian et al
2004)
NIEHS website at httpdirniehsnihgovmicroarraydatasetshome-pubhtm
EDGE website at httpedgeoncologywiscedu
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 43
and cisplatin (Table 1) In the majority of cases the resulting
number of differentially expressed genes is a very small
subset of the starting number on the microarray following
clustering or other types of analysis Upon closer examina-
tion of these publications the majority of them either
provide images of a heat map andor a table listing a gene
name accession number and expression change Very few of
the published studies (Tables 1ndash3) provide the original raw
microarray data file at a freely accessible website hence
restricting further analysis by scientists using other software
Although in some cases it is possible to cut and paste the
gene expression data tables from the publication pdf files
this is not always the case In the worst case scenario one
would have to manually retype gene lists or extract them
from heatmaps as binary type data As not all computational
researchers will have a laboratory available to them to
generate such quantities of toxicogenomics data the latter
points are important if we are going to continue to see
innovation in software development for this data This will
require free unrestricted access to data published Similarly
if we are to discern lsquolsquofingerprintsrsquorsquo for molecules acting with
a similar or identical mechanism we will need databases of
many diverse chemical structures that have been tested in a
similar manner There is therefore considerable interest in
the current databases being developed by the NIEHS FDA
and other groups which should help to improve the situation
by providing freely accessible microarray and other toxicity
related data Two of these databases being developed in the
public domain are Chemical Effects in Biological Systems
(CEBS) (httpwwwniehsnihgovnctcebshtm) (Mattes
Pettit Sansone Bushel amp Waters 2004 Waters et al
2003) which will accommodate gene expression profiles
proteomics and metabolomics data and allow complex
queries (Hood 2003a Mattes et al 2004) Similar goals
are being pursued in the development of the ArrayTrack
database at the FDA (Tong et al 2003) The EDGE
database (httpedgeoncologywisceduedgephp) an
expanding public effort at The University of Wisconsin
contains mouse gene expression profiles following treat-
ment with different toxic molecules (Hayes et al 2005
Thomas et al 2001) These separate efforts if widely
adopted should make published studies describing HT data
more readily accessible although it might have been more
efficient to evolve these into a single global database instead
of fragmented repositories
Proteomics data has also been generated in a limited
number of toxicology studies (Table 4) once again this has
been produced with different strains of rats and mice using
different protein chips 2-D gel electrophoresis (2-DIGE)
and mass spectroscopy methods (eg MALDI-MS) The
proteomic data is very rarely accessible to the reader for
their own computational analysis Subsequently there have
Table 2
Literature toxicogenomics data derived from mouse in vivo studies
Compounds Mouse strain Microarray type Compound dose Microarray data
availability
Reference
Benzene Male and female P53
KO mice and
C57BL6
Affymetrix and Incyte
GEM system
300 ppm 6 h per day 5
days a week for 2 weeks
Gene name accession
number and fold change
data in a manuscript
table
(Yoon et al 2003)
Benzene Male 129SvJ Affymetrix MG-U74Av2 100 ppm 6 h per day 5
days a week for 2 weeks
Gene name accession
number and fold change
data in a manuscript
table
(Faiola Fuller
Wong amp Recio
2004)
Phenobarbital CAR and wild type NIEHS Mouse tox chip
8736 genes
100 mgkg for 12 h Accession number gene
name and fold change
for wild type and knock
out mice in a manuscript
table
(Ueda et al 2002)
Aroclor BNF
ciprofloxacin cobalt
chloride TCDD IL-6
LPS PCB-153
phenobarbital
phenylhyrzn TNFa
WY-16463
C57BL6J Custom array with 1200
cDNAs
Aroclor (200 mgkg)
BNF (5 mgkg)
ciprofloxacin (250 mg
kg) cobalt chloride (60
mgkg) TCDD (10 ug
kg) IL-6 (25 ugkg)
LPS (1 mgkg)
PCB-153 (80 mgkg)
phenobarbital (100 mg
kgday) 3 days
phenylhyrzn (100 mg
kg) TNFa (50 ugkg)
WY-16463 (0125wv)
Gene names and fold
change as a heat map
Data is also available in
the EDGE database
(Thomas et al
2001)
TCPOBOP CD-1 female Custom 9000 cDNA
mouse array
1ndash3 h treatment 3 mg
kg body wt
Accession numbers and
fold change data in a
manuscript table
(Locker et al
2003)
3H-12-dithiole-3-thione
(D3T)
Male wild-type and
nrf2-disrupted
Affymetrix murine
genome U74Av2
GeneChip
05 mmolkg Accession number gene
name fold in a
manuscript table
(Kwak et al
2003)
MDMA Male albino Swissndash
Webster (neurons)
15 K mouse cDNA clone
set
47 mgkg Gene name and fold
change in a manuscript
table
(Xie et al 2004)
Phenytoin Female C57BL6 and
LDLRMurine genome-U74Av2 300 mgl Gene name accession
number and fold change
in a manuscript table
(Trocho et al
2004)
Genistein (1000 Agmouseday) or
diethylstilbestrol
(DES) (50 Agmouse
day)
ICR (testes) Custom cDNA
microarray containing
1754 cDNA probes
Genistein (1000 Agmouseday)
diethylstilbestrol (50 Agmouseday)
Accession number gene
name and fold change in
a manuscript tablemdashnote
very few genes
(Adachi et al
2004)
Cocaine and
buprenorphine
Male ICR Mouse DiscoveryArrayi
type I array containing
2688brain-derivedprobes
(Display Systems Biotech
Inc Copenhagen
Denmark)
40 mg kgmdash1 cocaine
once a day for 4 days 40
mg kgmdash1 cocaine plus
025 mg kgmdash1 BUP for
4 days
Data apparently not
available
(Hayase
Yamamoto
Yamamoto Muso
amp Shiota 2004)
Methamphetamine Male C57BLJ6
(striatum)
Affymetrix mouse
genechip mg-U74Av2
oligonucleotides arrays
12 488 genes
40 mgkg Gene accession number
gene ID and name and
signal log ratio in a
manuscript table
(Thomas
Francescutti-
Verbeem Liu amp
Kuhn 2004)
Di(2-ethylhexyl)
phthalate
Male C57BL6 Murine genome U74Av2
Arrays (MGU74Av2)
10 dietary DEHP for
13 weeks
Genbank accession
numbers gene name log
ratio data present as a bar
chart in the publication
data extraction would
take some effort
(Wong amp Gill
2002)
Diethylhexylphthalate Male PPARa null and
wild type
Custom made containing
600 tox genes
1150 mgkgday Gene names available on
a bar chartmdashquantitative
data not easily accessible
(Hasmall et al
2002)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6644
Table 2 (continued)
Compounds Mouse strain Microarray type Compound dose Microarray data
availability
Reference
Acetaminophen C57B16 3 129Ola
hybrid
Test-2 Chips (Affymetrix)
then individual
oligonucleotide
microarrays (Mul1K sub
A and sub B Affymetrix)
that can detect the
expression of 11000
known genes and
expressed sequence tags
(ESTs)
300 mgkg Genbank or SwissProt
ID and fold change data
in a manuscript table
(Reilly et al
2001)
Cadmium chloride
benzo(a)pyrene (BaP)
and trichloroethylene
(TCE)
Male Swiss Webster Custom Chips
containing 148 unique
genes
Various Data in table form few
genes affected for BAP
and TCE
(Bartosiewicz
Penn amp Buckpitt
2001)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 45
been only a very small number of studies that have
combined both transcriptomic and proteomic methods with
a single animal strain after treatment with a drug Hopefully
we will see this change in the future but this will in turn
present considerable challenges as huge amounts of
proteomic data are combined with the equally large
transcript data files
3 Network analysis and databases
From some of the early reviews of systems biology there
has been discussion of its application to drug discovery
(Kitano 2002ab) as well as the utility for ADMETox
(Ekins et al 2002a 2000b) More recently several other
journals have dedicated whole issues to the field of systems
biology However one could consider this quite a broad field
from network or pathway analysis to quantitative simulation
of organelles (Vo Greenberg amp Palsson 2004) whole cells
and organs It is apparent that we are now understanding
organisms from the perspective of computationally gener-
ated networks of protein and ligand interactions (Barabasi amp
Oltvai 2004) Network and pathway tools enable the
analysis of HT data in the context of all known interactions
when using a database as the source Individual reviews
have in some cases indicated that networks will be valuable
for understanding adverse events (Hood amp Perlmutter
2004) drug target identification or validation (Butcher
Berg amp Kunkel 2004) and complex metabolic interactions
(Nicholson et al 2004) A general schematic has been
generated in order to provide a description of the utilization
of such pathway databases and network building algorithms
from the initial parsing of high throughput data to network
comparisons and visualization (Fig 3) High throughput
data can be superimposed and visualized on the various
protein interaction databases available This is accomplished
by using either preset maps that capture current biological
knowledge or by building custom interaction networks
using many different algorithms which can be compared and
statistically evaluated as demonstrated in a very large
number of published examples (Dobrin Beg Karabasi amp
Oltvai 2004 Fiehn 2001 Han et al 2004 Hanisch et al
2002 Ideker et al 2002 Jeong Mason Barabasi amp Oltvai
2001 Jeong Tombor Albert Oltvai amp Barabasi 2000 Li
et al 2004 Milo et al 2002 Nikitin Egorov Daraselia amp
Mazo 2003 Pereira-Leal Enright amp Ouzounis 2004
Rives amp Galitski 2003 Segal et al 2003a Somogyi et al
2001 Spirin amp Mirny 2003 Tornow amp Mewes 2003
Vasquez Flammini Maritan amp Vespignani 2003 Yeger-
Lotem amp Margalit 2003 Yu Zhu Greenbaum Karro amp
Gerstein 2004)
31 Network applications
For example one group has used as an inference the
Bayesian network method for analysis of tissue toxicity from
microarray data as well as a mechanistic simulation for a
different pharmaceutically relevant molecule (Aksenov et
al 2005) Pathway tools and various resources have also
been applied to modeling the networks of nuclear hormone
receptors and their connections with other genes and small
molecules using a manually curated database MetaDrug
(Ekins Kirillov Rakmatulin amp Nikolskaya 2005d) or
MetaCore (Ekins Bugrim Nikolsky amp Nikolskaya 2005)
Transcriptional regulation of many transporters CYPs and
phase II enzymes are regulated by these receptors affecting
endogenous molecule transport metabolism cell growth
proliferation and oxidative stress (Ulrich 2003 Ulrich et al
2004) When the signaling networks and interacting ligands
for the transcriptional factors PPAR FXRRXRA ESR1
AHR HNF4A GCR-h MCR CAR-beta GCR-a LXR-a
CARRXR HNF4 FXR PXRRXR heterodimer PXR
AHRARNT heterodimer PPARaLXRa VDR PPAR-a
are visualized a very complex picture of interactions can be
created (Ekins et al 2005d) This suggests that when we
consider a molecule binding with only one nuclear receptor
we are observing only a fraction of the likely possible
feasible interactions based on the data gathered to date
Table 3
Literature toxicogenomics data derived from in vitro cell studies
Compounds Cell type Microarray type Compound dose Microarray data
availability
Reference
4-Hydroxytamoxifen
estrogen
MCF-7 breast cancer NIEHS ToxChip
microarray consisting of
1901 genes
1 uM hydroxytamoxifen
for a year 10 nM
17b-estradiol
Data available at NIEHS
website
(Hodges et al
2003)
Trovafloxacin Human hepatocytes Affymetrix U133A array 30ndash800 uM 142 genes available in
supplemental tablemdashnot
easily extracted
(Liguori et al
2005)
Estrogen MCF-7 breast cancer NIEHS ToxChip
microarray consisting of
1901 genes
10ndash10 M 17b-estradiol Data available at NIEHS
website
(Lobenhofer et al
2002)
Valproic acid NMRI mice embryo
and P19 mouse
embryocarcinoma
Custom chip including
15K mouse cDNA clone
set
600 mgkg body weight Gene symbol gene
name NIA EST log fold
change in a manuscript
table
(Kultima et al
2004)
Sulindac sulfide Human colorectal
carcinoma SW-480
and HCT-116
NIEHS human 12K chip 10 uM Genbank accession
number gene name and
fold change at various
time points in a
manuscript table data
also available on NIEHS
website
(Bottone
Martinez Collins
Afshari amp Eling
2003)
17Beta-estradiol estriol
estrone genistein
diethylstilbestrol
bisphenol A
nonylphenol
methoxychlor
MCF-7 U95A oligonucleotide
probe arrays (Affymetrix
10 nM (E2 estriol
estrone DES) 10 AM(genistein bisphenol A
nonylphenol and
methoxychlor)
Unigene name gene
name and fold change
for estrogen responsive
and nonresponsive in a
manuscript table
(Terasaka et al
2004)
Ouabain lauryl sulfate
dimethylsulfoxide
cycloheximide
tolbutamide sodium
fluoride diethyl
maleate buthionine
sulfoxamine
potassium bromate
sodium selenite
alloxan adriamycin
hydrogen peroxide
HepG2 Clontech Atlas Human
Stress Toxicology cDNA
arrays (234 genes)
Ouabain (43 uM) lauryl
sulfate (260 uM)
dimethylsulfoxide (128
M) cycloheximide (625
uM) tolbutamide (128
mM) sodium fluoride (3
mM) diethyl maleate
(125 mM) buthionine
sulfoxamine (30 mM)
potassium bromate (25
mM) sodium selenite (30
uM) alloxan (130 mM)
adriamycin (40 uM)
hydrogen peroxide
(4 mM)
Gene name ratio p -
value in downloadable
tables at journal website
(Morgan et a l
2002)
Aflatoxin B(1) (AFB(1))
2-acetylaminofluorene
(2AAF)
dimethylnitrosamine
(DMN)
acetaminophen (APAP)
HepG2 and primary
hepatocytes
Gene filter arrays
containing 31000 genes
10 uM aflatoxin B1
40 mM acetaminophen
100 uM
dimethylnitrosamine
10 uM
2-acetylaminofluorene
None (Harris et al
2004)
Mitomycin C (MMC)
and cisplatin (CIS)
and an alkylating
agent methyl
methanesulfonate
(MMS)
indirect-acting
genotoxins included
hydroxyurea (HU) a
ribonucleotide
reductase inhibitor
taxol (TXL) a
microtubule inhibitor
and etoposide
(ETOP)
L5178Y TK(+-)
mouse lymphoma
Affymetrix mouse
MG-U74A for MMC
and MG-U745Av2
(Affymetrix Inc Santa
Clara CA) for all the
other chemicals a total
of 9977 probe sets
(genes or ESTs) common
to these two array
models
Low mid and high doses Accession number gene
name gene ID statistical
significance at each time
point in a manuscript
table
(Hu et al 2004)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6646
Table 3 (continued)
Compounds Cell type Microarray type Compound dose Microarray data
availability
Reference
Hydroxyurea
(a carcinogen)
p-anisidine
(a noncarcinogen)
and paclitaxel
L5178Y Tk_-mouse
lymphoma
The Twin - Chip Mouse-
74K Digital Genomics
cDNA microarray
10 ngml paclitaxel 313
ugml hydroxyurea 32
ugml p-anisidine
Gene symbol and fold
change in a manuscript
table
(Lee et al 2003)
Acetaminophen
amiodarone clofibrate
erythromycin estolate
isoniazid alpha-
naphtylylisothiocyanate
beta-naphtoflavone
4-pentenoic acid
phenobarbital
tetracycline and
zileuton
Wistar Rat
hepatocytes
DualChip rat hepato
(Eppendorf Hamburg
Germany)
A single concentration
which varied for each
compound
Accession numbers and
gene namemdashfold
changes shown as
colored heat mapmdashnote
easily extracted from
publication
(de Longueville
et al 2003)
Bleomycin and hydrogen
peroxide
Mouse lymphoma
L5178Y TK(+ -)
Clontech Mouse 12K
cDNA microarray (1185
genes)
Bleomycin (25 and 20
ugml) hydrogen peroxide
(5 and 10 ugml)
Gene names and fold
change presented as bar
charts in publication
(Seidel Kan
Stott Schisler amp
Gollapudi 2003)
Bupivicaine
camptothecin
HL-60 Agilent human cDNA
microarray
1 mM Gene name GenBank
accession number
unigene and ratio in a
manuscript table
(Unami
Shinohara
Ichikawa amp Baba
2003)
Bisphenol A Mouse Sertoli TTE3 IntelliGene mouse
expression glass
microarrays (Version
10 Takara Shuzo)
which were spotted with
564 cDNA fragments of
mouse known genes and
approximately 301
expressed sequence tags
(ESTs)
0ndash400 uM Gene name GenBank
accession number and
fold change at time
points in a manuscript
table
(Tabuchi amp
Kondo 2003)
Mitomycin C or
doxorubicin
Hep G2 85 human gene custom
array
10 um mitomycin C
2 um doxorubicin or
2 ethanol
Bar charts with fold
changes in publication
very few genes
(Hong Muller amp
Lai 2003)
Amphotericin B Human peripheral
blood mononuclear
and THP-1
GF211 FKnown Genes_
Genefilter cDNA array
(ResGen) this array
consists of gt4000
individual elements
each representing a
known human gene
5 ugml Accession numbers and
fold expression in a
manuscript table
(Cleary Rogers amp
Chapman 2001
Rogers Pearson
Cleary Sullivan
amp Chapman
2002)
Benzo(a)pyrene diol
epoxide
TK6 human
lymphoblastoid
Human-350 microarray
a glass slide with 350
spotted human cDNA
probes (Phase-1
Molecular Toxicology
0 001 010 or
10 ugml)
Gene names and fold
change at doses in a
manuscript table
(Akerman et al
2004)
Etomoxir HepG2 Clontech AtlasiHuman Stress
Toxicology cDNA arrays
(234 genes)
1 mM etomoxir Gene names and fold
change in a manuscript
table
(Merrill et al
2002)
Tetrodotoxin Human glioma cell
line HTB-138
Using Affymetrix
GeneChip (HG-U133A
10 and 20 mM Affymetrix ID Genbank
ID gene name gene
symbol and fold change
in a manuscript table
(Raghavendra
Prasad Qi
Srinivasan amp
Gopalakrishnakone
2004)
Methotrexate
mercaptopurine
Human acute
lymphoblastic
leukemia
Affymetrix U133A chip Low and high dose and
combination
Data available as
supplemental data online
(Cheok et al 2003)
Prednisolone
vincristine
asparaginase
daunorubicin
Human acute
lymphoblastic
leukemia
Affymetrix U133A chip Various Data available as
supplemental data online
(Holleman et al
2004)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 47
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6648
A second study has indicated how a natural language
processing method CCNet was used to show the genes
regulated by the nuclear hormone receptor FXR (Apic
Ignjatovic Boyer amp Russell 2005) These automated
methods enable a more complete understanding of the
complexity of the transcriptional factors (Ekins Mirny amp
Schueltz 2002b Plant 2004 Ulrich 2003) but ultimately
rely on the quality of the content of the underlying database
of literature interactions This is a key consideration that is
often overlooked For example the gold standard database is
one that is manually curated to ensure the fidelity of the
direct interaction and is preferable to one generated
computationally by algorithms like natural language pro-
cessing (Nikolsky Nikolskaya amp Bugrim 2005) The
advantage of interaction networks over clustering has been
demonstrated in one study using MetaCore (Nikolsky
Ekins Nikolskaya amp Bugrim 2005) by reanalysis of a
published microarray study of G0-arrested MCF-7 breast
cancer cells treated with estrogen and 4-hydroxytamoxifen
(Hodges et al 2003) After producing integrated gene
networks for each treatment strikingly different patterns
were displayed although both contained early transcriptional
factors myc jun and fos Only the estrogen network featured
induced genes essential for all cell cycle phases (Nikolsky et
al 2005) Similarly microarray data for benzene toxicity
(Yoon et al 2003) has been re-analyzed by focusing on the
genes assessed on the p53 pathway (Ekins et al 2005e) We
envisage that a database of such networks for toxic
compounds will be used for comparing between different
molecules and used in the development of predictive
algorithms for Systems-ADMETox modeling in future
Another approach to using such pathway approaches is to
visualize the results of quantitative structure activity models
for predicting molecules binding to enzymes transporters
receptors and ion channels (Ekins Andreyev et al in press
2005e)
It is also possible to simultaneously interpret high
throughput data and predictions on interaction networks
providing a novel approach to predicting and understanding
potential undesirable drugndashdrug or off target effects in the
area of systems pharmacology An example data set uses
percent inhibition data for clotrimazole and ticonazole
which were screened against many different assays at a
single concentration in a commercially available database
BioPrint (Cerep Redmond WA) as published recently
(Fliri Loging Thadejo amp VOlkman 2005) The data for
10 assays has been arbitrarily encoded as inhibitors (gt50
inhibition) or non-inhibitors (lt50 inhibition) in a text file
which was then loaded into MetaCore The analyzed
network algorithm was then used which generates a large
network and fragments it into sub-networks each with a Z-
score and p-values for ranking according to saturation with
objects from the initial gene list The Gene-ontology
processes are also mapped to the gene list and individual
networks In this example a statistically significant network
was generated for the different proteins (Fig 4
p =2838e31) This network also maps the Gene Ontology
processes for the activation of MAPK (118 of genes p
value 9143e07) signal transduction (333 1600e05)
regulation of transcription DNA-dependent (294
2786e04) regulation of inflammatory response (39
3746e04) and the regulation of blood pressure (78
4230e04) This example network indicates how molecules
of the same or different therapeutic classes could be
evaluated for their effects as a graph either together as in
this case or individually This would be useful to indicate
potential off target effects and identify structurally dissimilar
molecules with similar network patterns Such networks
could then be compared to assess network overlap or
differences between molecules and their inhibition of
multiple proteins This type of unique visualization of high
throughput screening data illustrates how the target proteins
may be connected as a network to infer the possible
downstream effects of inhibition
With the burgeoning number of freely available online
and commercial databases that can be used for pathway
construction numbering in the hundreds there have been
suggestions to impose standards for model exchange
querying and visualization (Cary Bader amp Sander 2005)
To date there has been little discussion with regards to
standardization of ADMETox related databases although
there has been considerable discussion relating to drug
metabolism database generation (Erhardt 2003) This is
certainly an important area to address in the future There is
already a growing literature related to ADMETox that is
partially captured in the several commercially available
databases (Ekins et al 2005e) but to date there have been
limited academic efforts to capture data for transporters
with the human membrane transporter database (Yan amp
Sadee 2000) TP-search transporter database (httpwww
ilabrisewasedaacjptp-search) drug interaction database
(httpwwwdruginteractioninfoorgDatabaseinfoaspx)
nuclear hormone receptors (Nakata Yukawa Komiyama
Nakano amp Kaminuma 2002) the ADME-AP database
(Sun Ji Chen Wang amp Chen 2002) and PharmaGKB
(Oliver et al 2002) DSStox (httpwwwepagovnheert
dsstox) TOXNET (httptoxnetnimnihgov) that are
readily accessible
In order to generate accessible pathways using any of
the available software a large enough set of object
identifiers are required to map onto the underlying data-
base To demonstrate this datasets from toxicogenomics
studies have been evaluated with both the KEGG pathway
database and a commercially available product MetaCore
(wwwgenegocom) These gene or protein lists range in
size from 21 to 1853 objects In virtually all cases more
identifiers are mapped to networks in MetaCore and this
also seems independent of the identifier type used
(Unigene Affymetrix Genbank or Locuslink Table 5)
These numbers will obviously change as more information
is added to the respective databases hence the more
objects that are mapped from a dataset The more extensive
Table 4
Literature derived toxicoproteomics data
Compounds Data source Microarray type Compound dose Microarray data
availability
Reference
Carbon
tetrachloride
Male Wistar rats Affymetrix rat chip 8799
probes+proteomics
study
6ndash24 h exposure Genbank SwissProt and
binary data in a
manuscript table
(Fountoulakis
2004)
Paracetamol CD-1 male mice Custom mouse tox blots
with 450 genes
RTQ-PCR+proteomics
study 2-DIGE+
MALDI-MS
150 or 500 mgkg Gene name IMAGE ID
GenBank accession
number fold induction
SwissProt identifier and
protein abundance
change in manuscript
tables
(Ruepp Tonge
Shaw Wallis amp
Pognan 2002)
Paracetamol AP-1 male mice Affymetrix murine 11K
set+proteomics study
Up to 500 mgkg Gene names and fold
changes at multiple
doses and time points in
a manuscript tablemdash
proteomics data not
accessible
(Coen et al 2004)
Oxazepam or
Wy-14643
Male B6C3F1 mice NIEHS Mouse Chip
(8700 genes) 2-DIGE
and MS
Oxazepam (2500 ppm)
Wyeth (Wy)-14643
(500 ppm)
Proteomics data in a
manuscript table
Genbank accession
number gene name and
fold change in a
manuscript table data
also available to
download from NIEHS
website
(Iida et al 2003)
Compound A
(PPAR gamma
ligand)
Female rats CrlCD
(SD)IGS BR
Proteomics 2-DIGE and
MS
250 mgkgday up to
5 days
Accession number and
protein name and
average ratio in a
manuscript table
(Meneses-Lorente
et al 2004)
Bromobenzene Male Wistar rats Proteomics 2-DIGE and
MS custom 3000 cDNA
rat chip
5 mmolkg Accession number gene
name fold change in a
manuscript table protein
names along with bar
charts
(Heijne Stierum
Slijper van
Bladeren amp van
Ommen 2003)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 49
the network that can be generated (as it will consist of more
nodes) then a more comprehensive understanding of the
networks is possible The data available currently in the
literature can be used to evaluate such pathway and
network generation tools Recently we have used several
of the published studies (Tables 1ndash3) with MetaCore to
visualize networks for acetaminophen furan carbon tetra-
chloride benzene and cisplatin showing genes involved in
In vivo
In vitro
In Silico
1970s 1980s 1990s 2000s
Systems Biology ()
OMICS
Fig 2 The timeline for major paradigms in ADMETox
oxidative stress (Ekins Giroux Nikolsky Bugrim amp
Nikolskaya 2005c)
Other important pathwaynetwork building tools that could
potentially be applied to toxicogenomics data include
Ingenuity pathways analysis (httpwwwingenuitycom)
PathArt (httpwwwjubilantbiosyscompdhtm) Pathway
Assist (httpwwwariadnegenomicscomproctspathway
html) (Nikitin et al 2003) and several other databases
deposited at the Pathway Resource List (httpcbiiomskcc
orgprl) All of these products have unique underlying
proprietary pathway databases which are compiled manually
or automatically with text mining tools or a combination of
both We are still waiting for studies that provide a comparison
of different database tools or network building algorithms
Until then theremay be someoverlap but also some differences
between the results obtained from more than one network
method due to the database content and the algorithms used
The reader is recommended to evaluate for themselves several
technologies and select those with the most appropriate fit to
their own specialized needs
In the next Sections the further application of some
available network and database tools will be described with
Microarray or other high throughput data
Upload list of geneprotein identifiers and fold change significance etc (raw data or after selection of the differentially expressed
Genes using other statistical methods)
Parse database and generate interaction network with different algorithms or visualize on maps
Filter Networks
Access significance of the interaction networks
Determine GO processes
Compare 2 or more networks intersection overlap etc
Export gene list
Export gene list
generate interaction network with different algorithms or visualize on maps
Visualization
Fig 3 Schematic for the utilization of pathway tools for assessing high throughput data
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6650
specific detailed reference to transporters and enzymes but
these technologies can also be applied elsewhere as
described above
Fig 4 Network for high throughput screening data for clotrimazole and ticona
cholecystokinin CCR1 Choline transporter Chloride channel catechol-O-methyl
and co workers (Fliri et al 2005) Nodes highlighted with large blue circles rep
molecules showing gt50 inhibition in the assay blue circles represents both mol
inhibits gt50 and one inhibits lt50 Ligands (large) linked to transcriptional f
(wwgenegocom) (For interpretation of the references to colour in this figure leg
311 The role of transporters
A diverse array of organic solutes such as nutrients
neurotransmitters and drugs are transported by specialized
zole screened against 10 in vitro assays (Human Cannabinoid 1 Human
transferase COX2 CYP1A2 CYP2B6 CYP2C19) data published by Fliri
resent assays used in the study Red circles on these nodes represent both
ecules showing lt50 inhibition chequered circles represent one molecule
actors enzymes and transporters via edges using the MetaCorei database
end the reader is referred to the web version of this article)
Table 5
Datasets acquired from publications and other sources and used to evaluate two databasepathway tools KEGG and metaCore
Dataset Number of objects
genes proteins in list
Number of objects
mapped with KEGG
Number of objects mapped
with MetaCore on networks
Reference for microarray
data gene list
Tox Chip 10 (NIEHS) 1853 405 1498 httpsdir-appsniehsnihgov
mapsguestclonesrchcfm
4-Hydroxytamoxifen and
estrogen
1617 434 1343 (Hodges et al 2003)
Mitochondrial proteins 722 156 388 (Gaucher et al 2004
Taylor et al 2003)
Bromobenzene
(24 and 48 h)
130 41 89 (Heijne et al 2004)
Acetaminophen 30 19 23 (Heinloth et al 2004)
Acetaminophen 84 29 64 (Huang et al 2004)
Furan 185 64 139 (Huang et al 2004)
Tetrodotoxin 116 31 86 (Raghavendra Prasad et al
2004)
Benzene 73 16 62 (Yoon et al 2003)
Benzene 76 5 53 (Faiola et al 2004)
Carbon tetrachloride 37 8 26 (Young et al 2003)
Estrogen 94 33 90 (Lobenhofer et al 2002)
Trovafloxacin 142 20 82 (Liguori et al 2005)
Phenobarbital 37 13 28 (Ueda et al 2002)
L-742694 (liver)+ 45 17 19 (Hartley et al 2004)
L-742694 (intestine)+ 23 11 10 (Hartley et al 2004)
A-277249 21 7 9 (Waring et al 2002)
All files were converted to Locuslink identifiers except as noted () unigene () Affymetrix (+) Genbank identifiers were used
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 51
proteins across cellular membranes These may function as
passive processes or active processes energized by the
hydrolysis of ATP or coupling to the co-transport of counter
ions down an electrochemical gradient such as Na+ H+ and
Cl There are many thousands of transporters which can be
classified into distinct superfamilies One of these the solute
carrier class (SLC) is rapidly expanding and contains over
30 families and 200 members The ATP-binding cassette
(ABC) contains 7 families and over 48 members including
P-glycoprotein (P-gp) and MRP subfamilies (Zhang Knipp
Ekins amp Swaan 2002a Zhang Phelps Cheng Ekins amp
Swaan 2002b) Transporters have a key role in clinical
pharmacology with many drugs specifically targeting them
Numerous drugs share transport pathways with nutrients
and transporters have a role in oral absorption drug
bioavailability drug resistance excretion and ultimately
pharmacokinetics and pharmacodynamics
Polymorphism of drug transporters may be a key factor
in drug interactions and lack of effectiveness This field has
become known as pharmacogenomics and is focused on
understanding of inherited DNA sequence variations (poly-
morphisms and mutations) revealed by xenobiotics (Evans
amp McLeod 2003 Weinshilboum 2003) Over the last few
decades many genes have been directly linked to the
mechanisms of response (Evans amp McLeod 2003 Wein-
shilboum 2003) such that 20ndash95 of variability to drug
response is inheritable (Evans amp McLeod 2003) This
phenotypical variability is mainly caused by single nucleo-
tide polymorphisms (SNPs) present in anywhere from 1 to
50 of the population resulting in either lower protein
activity incorrect folding or rapid degradation via proteo-
somes (Weinshilboum amp Wang 2004) A number of
structurally diverse molecules bind to P-gp which is
expressed in many tissues and has numerous SNPs one of
which (C3435T) affects the expression level in the
duodenum and therefore can impact the absorption of
molecules which would be substrates for this transporter
(Sakaeda Nakamura amp Okumura 2002) The human
proton-dependent dipeptide transporter (hPEPT1) can also
affect the absorption of molecules in the intestine and
recently 9 SNPs were found with only one displaying a
reduced transport capacity (Zhang et al 2004) The sodium-
dependent carnitine cotransporter OCTN2 can possess
mutations and these result in primary carnitine deficiency
which impacts fatty acid oxidation and is characterized by
many clinical manifestations (Lahjouji Mitchel amp Qureshi
2001) The organic cation transporter 1 (OCT1) is also
important in the transport of numerous xenobiotics and
endobiotics Recently 4 SNPs were identified in the
Japanese population and when functionally characterized
in vitro the uptake of cations was reduced significantly for
some of these mutations indicating that this would likely
contribute to inter-individual variations in metabolism of
drugs which were transported via OCT1 (Sakata et al
2004)
3111 Clinical relevance of transporters The pregnane
X-receptor (PXR) is a transcriptional regulator of the
enzyme human MDR1 (P-gp) MRPs and OATP (Wang amp
LeCluyse 2003) CYP3A (Bertilsson et al 1998 Blumberg
et al 1998 Kliewer et al 1998) and CYP2C89 (Synold
Dussault amp Forman 2001) as well as many other genes
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6652
involved in the transport metabolism and biosynthesis of
bile acids (Staudinger Liu Madan Habeebu amp Klaassen
2001) However the additional receptors CAR FXR LXR
and other nuclear receptors take part in a complex network
of interactions to control these and other proteins Thus
elucidation of the regulatory networks which control the
expression of these transporters is also important To date
most of the research has centered on efflux transporters but
there has been considerable interest in uptake transporters
such as the organic anion transporter polypeptide (OATP
((Kim 2003) see also later Section on OATP)
There are several specific examples of the importance of
drug transporters to the clinical development of drugs One
example is the insulin sensitizer troglitazone which was
withdrawn due to hepatotoxicity although the precise
mechanism appears to have been unclear until recently
The major metabolite is a sulfated species and is suspected
of being responsible for the observed toxicity The recent
assessment of the organic anion transporting polypeptides
OATP-C and OATP8 expressed on the hepatocyte baso-
lateral membrane indicated that sulphated troglitazone has a
high affinity for the former and possibly lower affinity for
the latter (Nozawa et al 2004) This metabolite would
therefore be expected to accumulate in hepatocytes and
inhibit the bile salt export pump and Mrp2 Because
polymorphisms have been shown for OATP-C (Tirona
Leake Merino amp Kim 2001) it is also possible that these
may result in the accumulation of the metabolite and in turn
elicit idiosyncratic toxicity A second relevant example of
the impact of transporters is the clinically significant drugndash
drug interaction between cerivastatin and cyclosporine A
which occurs via the OATP-C transporter (Shitara Itoh
Sato Li amp Sugiyama 2003) A third example are the HIV
protease inhibitors saquinavir ritonavir and indinavir which
are transported by MRP2 in vitro and other drugs such as
probenecid and sulfinpyrazone are able to enhance this
transport Transport by MRP2 suggests that these com-
pounds will have decreased bioavailability due to increased
clearance and other drugs could aggravate this situation by
further enhancing transport (Huisman et al 2002) Sim-
ilarly the rifampicin mediated induction via PXR of MRP2
and P-gp in healthy subjects was found to significantly
decrease the AUC and also correlated with intestinal
expression of these transporters This transporter is also
inducible by cisplatin 2-AAF and phenobarbital (Schrenk
et al 2001) indicating multiple mechanisms may be
involved In other species such as rat commonly used as a
toxicity model orthologs of the transporters such as OATP2
are expressed and can be induced with ligands for PXR like
PCN (Guo Choudhuri amp Klaassen 2002) This is useful
knowledge because the advent of microarray technology
allows one to dose a rat with a xenobiotic and assess
thousands of genes simultaneously in a particular tissue For
instance animals dosed with known nephrotoxins have
shown some upregulation of the NandashKndashCl transporter
however the authors suggested genomic responses are
stronger soon after exposure before declining (Fleck et
al 2003) Some transporters may therefore be specifically
targeted by drugs in one tissue such as the CNS but these
same transporters may also be expressed elsewhere in the
body hence off-target effects may result in toxicity The
serotonin transporter is one such example which is
expressed in the lungs and brain Some substrates for this
channel like fenfluramine can result in primary pulmonary
hypertension as they accumulate in pulmonary cells (Roth-
man Ayestas Dersch amp Baumann 1999) Similarly P-gp is
expressed at the blood brain barrier and intestine impacting
the efficacy and bioavailability of drugs
3112 Transporter network examples ABCA1 The
ABCA1 transporter mediates the first step of cholesterol
transport Mutations in this gene cause Tangier disease
which results in severe HDL deficiency cholesterol
accumulation in macrophages and attendant atherosclerosis
This transporter represents a drug target for upregulation
modulating cholesterol metabolism and prevention of
cardiovascular disease (Oram amp Lawn 2001) In vitro
ABCA1 can be inhibited by the sulfonylurea glybenclamide
(Field Burn amp Mathur 2004 Wang Silver Thiele amp Tall
2001) In order to illustrate the advantages of mapping drug
transporters as networks onto functional models alongside
other proteins one can consider the example of ABCA1
The query of the MetaCorei database shows that this
transporter appears on three manually curated pathway maps
representing the Fstate of the art_ knowledge derived from
reliable high quality literature sources One can also use the
individual maps as an interface to access the underlying
layers of information about the transporter including the list
of encoding genessplice variants with known SNPs In
addition to browsing MetaCorei a user can also build
custom networks around ABCA1 using the network-
construction tool (Fig 5) Such a visualization utility may
be very helpful for identification of all putative pathways
around a particular transporter or compound of interest The
ABCA1 network created by this tool shows that this
transporter at the time of writing is linked directly to
twenty-five other objects such as APOE1 and LXR Many
of its neighbors have their own SNPs that could be
important in determining interactions between transport of
a drug and normal human transport of endogenous ligands
in health or disease
3113 Transporter network examples OATP The
OATPs are key membrane bound transporters expressed in
many organs including intestine liver lung choroid plexus
blood brain barrier and other organs (Tamai et al 2000)
This family of transporters is capable of mediating the
sodium-independent transport of a diverse array of mole-
cules such as steroid conjugates organic anions and
xenobiotics by coupling uptake with efflux of bicarbonate
(Satlin Amin amp Wolkoff 1997) glutathione or its
conjugates (Hagenbuch amp Meier 2004) The inhibition of
Fig 5 The network of direct gene interactions around the ABCA1 transporter gene (centre) generated with MetaCorei The interation types between nodes are shown as small colored hexagons eg unspecified
allosteric regulation binding cleavage competition covalent modification dephosphorylation phosphorylation transcription regulation transformation When applicable interactions also have a positive or
negative effect and direction Ligands (purple) linked to other proteins (blue blobs) transfactors (red) enzymes (orange) (For interpretation of the references to colour in this figure legend the reader is referred to
the web version of this article)
SEkin
sJournalofPharm
acologica
landToxico
logica
lMeth
ods53(2006)38ndash66
53
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6654
this transporterrsquos hepatic uptake of other compounds may be
important for reported drugndashdrug interactions (Kim 2003)
described earlier (Shitara et al 2003) as well as cerivastatin
with gemfibrozil (Shitara Hirano Sato amp Sugiyama 2004)
OATP1B1 (previous names OATP-C LST-1 OATP2
SLC21A6) represents the most studied human OATP to
date (Meier amp Stieger 2000) and is expressed on the
basolateral plasma membrane of hepatocytes Several single
nucleotide polymorphisms have been identified in the
OATP1B1 gene in European-Americans African-Americans
(Tirona et al 2001) and Japanese (Nozawa et al 2002)
dramatically impacting the transport of ligands such as
pravastatin (Mwinyi Johne Bauer Roots amp Gerloff 2004
Nishizato et al 2003) estrone-3-sulfate (Nozawa et al
2002 Tirona et al 2001) Rifampin (Tirona Leake
Wolkoff amp Kim 2003) and estradiol 17h-d-glucuronide(Tirona et al 2001)
The regulation of SLCO may be affected during
extrahepatic cholestasis bile duct ligation bile salt induced
Fig 6 Networks generated with the autoexpand algorithm in MetaCorei to illustr
be interconnected with other protein regulatory signaling information Informa
unspecified allosteric regulation binding cleavage competition covalent m
transformation When applicable interactions also have a positive or negative e
transfactors (red) enzymes (orange) (For interpretation of the references to colour
cholestatic hepatitis (Dumont et al 1997 Meier amp Stieger
2000 Rost et al 2003) and primary sclerosing cholangitis
(Oswald Kullack-Ublick Paumgarter amp Beuer 2001) In
particular OATP1B1 appears to be regulated by the liver-
enriched transcription factor hepatocyte nuclear factor 1a
(HNF1a) which binds to the promoter region of this
transporter (Jung et al 2001) Site directed mutagenesis
of this binding site resulted in inactivation suggesting the
critical nature of the interaction with HNF1a Bile acids
such as CDCA have been shown to transcriptionally repress
HNF1a in vitro via inhibition of the transactivating effect of
HNF4a on HNF1a (Jung amp Kullak-Ublick 2003) After
screening many rat and human uptake transporters in vitro
OATP1B1 was also shown to modulate the PXR response
by controlling rifampin retention in the cell and therefore
affecting the induction of CYP3A4 and other gene products
such as P-gp (Tirona et al 2003)
Some of the literature for OATP1B1 human substrate
data has been annotated into MetaCorei to illustrate the
ate how ligands for the human OATP-C (OATP1B1) transporter (centre) can
tion on the type of interaction between objects is hidden for clarity eg
odification dephosphorylation phosphorylation transcription regulation
ffect and direction Ligands (purple) linked to other proteins (blue blobs)
in this figure legend the reader is referred to the web version of this article)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 55
visualization of the complex interconnections between this
transporter its ligands regulatory factors and signaling
molecules already in the database (Fig 6) The network was
generated with the autoexpand algorithm in the software
representing one of multiple available algorithms for
connecting genes ligands and other objects in the database
Clearly if more ligands and their connections are added to
the database the complexity of the network will increase
considerably The OATP-C (OATP1B1) gene details can be
viewed upon querying the database and links are provided
to other public databases This page can be used to highlight
the multiple synonyms for this gene as well as links to the
multiple SNPs identified to date
3114 Transporter microarray data Microarrays have
generally been limited in the number of transporters present
on them (Annereau et al 2004) however they have been used
in an attempt to correlate pharmacokinetic properties with
gene expression for valacyclovir (Landowski et al 2003) as
well as understand the expression profile in different tissues
or cell lines upon food component or xenobiotic treatment
(Anderle Huang amp Sadee 2004) This lack of transporters
on microarrays has prompted some groups to produce their
own arrays with a heavier emphasis on transporters These
arrays have for example then been used to demonstrate the
upregulation of ABC transporters and down-regulation of
GST-Pi in cell lines resistant to colchicines or 9-nitro-
camptothecin (Annereau et al 2004) The genes that were
significantly up or down-regulated in this particular study
were used to build networks with MetaCore (Fig 7A B) and
the similarities between them were assessed (Fig 7C)
Although there were only a small number of significantly
changed genes in common (IL-8 Fos GST-Pi Calpactin and
Ubiquitin hydrolase) it is perhaps likely that there is a much
larger common gene network that is important for drug
resistance although a much larger number of cell lines and
drug treatments need to be evaluated to produce a definitive
drug resistance signature involving transporters enzymes
and transcriptional regulators
312 Applications to enzymes
As we have already described it may be particularly
valuable to visualize enzymes as networks to show
interactions with transcriptional regulators and ligands
For example a key enzyme is CYP3A4 which metabolizes
40ndash45 of all drugs and has relatively few SNPs
(Ingelman-Sundberg 2004) Using a second software suite
MetaDrugi (wwwgenegocom) it is possible to construct
a custom network around this or other drug metabolizing
enzymes (Fig 8) In this case the gene network for
CYP3A4 highlights all of the major transcriptional
regulators and several more distant linked proteins and
ligands connected on the network that may be useful for
further study (Fig 8) Substrates inhibitors as well as
regulatory factors and other enzymes can be observed
connected on this network Due to the many hundreds of
known inhibitors and substrates only a small number are
shown here for clarity We assume that if a perturbation in
a pathway (eg due to a nonfunctional enzyme) is linked
to a certain pathologic condition a similar perturbation
caused by the interference from xenobiotic metabolism
(eg competitive inhibition of the same enzyme) may
result in identical effects As microarray gene expression
data is increasingly generated the role of enzyme
regulation in toxicity of certain xenobiotics will become
more apparent from either in vivo or in vitro studies The
visualization of such signature gene networks involving
transporters and enzymes their ligands and regulatory
factors will also be important for future toxicity prediction
methods We have recently generated visualizations of
microarray data from MCF-7 cells treated with 4-hydrox-
ytamoxifen to show that some of the key genes involved in
metabolism and transport are upregulated (Ekins et al
2005d) In addition we have made predictions with various
QSAR models in MetaDrug to indicate the involvement of
PXR CYP3A4 and P-gp (Ekins et al 2005e) Therefore it
appears likely that 4-hydroxytamoxifen could increase its
own metabolism as well as efflux from cells via P-gp
which can be visualized on networks Any decrease of
function of these enzymes or transporters in a population
would likely result in changes in the metabolism and
transport of this active metabolite potentially impacting
the clinical effect This represents one example of how
both pathway tools QSAR models and network building
algorithms can be used with different types of predicted
and experimental data to allow visualization of potential
compound interactions or toxicity
313 Future network applications
As the population ages an increasing incidence and
prevalence of systemic diseases especially chronic diseases
have occurred among older adults This has resulted in an
increase in medications used concomitantly by this
population which presents challenges for drugndashdrug
interactions Physiologically elderly patients may behave
differently to the younger patients for which the drugs are
initially developed for and tested on Many pharmacoki-
netic investigations in the elderly population reveal
decreased clearance of lipophilic drugs metabolized by
the cytochrome P450 enzymes however few studies have
evaluated aging-dependent or gender-related changes in
specific P450 enzymes (Hunt Westerkam amp Slave 1992)
Age-related physiological changes such as a reduction in
liver mass hepatic metabolizing enzyme activity liver
blood flow and alterations in plasma drug binding may
account for the decreased elimination of some metabolized
drugs in the elderly It is particularly difficult to separate an
effect of aging from the variation in the rate of metabolism
due to factors such as individual metabolic phenotype
(slow or fast metabolizer due to SNPs) environmental
influences concomitant disease states and drug intake
(drugndashdrug interactions) The available data suggest that
Fig 7 Networks for drug resistant cell lines generated with the Auto expand algorithm in MetaCore A KB-3-1 vs KB-8-5 B ATC-DU-145 vs RCO1 C node overlap of A and B with initial nodes highlighted
with blue or red solid circles (Initial gene lists derived from (Annereau et al 2004)) Information on the type of interaction between objects is hidden for clarity eg unspecified allosteric regulation binding
cleavage competition covalent modification dephosphorylation phosphorylation transcription regulation transformation When applicable interactions also have a positive or negative effect and direction
Ligands (purple) linked to other proteins (blue blobs) transfactors (red) enzymes (orange) (For interpretation of the references to colour in this figure legend the reader is referred to the web version of this article)
SEkin
sJournalofPharm
acologica
landToxico
logica
lMeth
ods53(2006)38ndash66
56
Fig7(continued)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 57
Fig7(continued)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6658
Fig 8 The network of gene interactions around the CYP3A4 enzyme (centre) from MetaCorei Information on the type of interaction between objects is
hidden for clarity eg unspecified allosteric regulation binding cleavage competition covalent modification dephosphorylation phosphorylation
transcription regulation transformation When applicable interactions also have a positive or negative effect and direction Ligands (purple) linked to other
proteins (blue blobs) transfactors (red) enzymes (orange) (For interpretation of the references to colour in this figure legend the reader is referred to the web
version of this article)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 59
the initial doses of drugs metabolized by these enzymes
should be reduced in older patients according to the clinical
response In most published studies the elderly appear at
least as responsive as the young to inducers or inhibitors of
P450s (Durnas Loi amp Cusack 1990) More recently there
has been some suggestion that there are age related
reductions in function of some specific P450s such as
CYP3A4 (Patki et al 2004) and this could occur at the
level of regulation However earlier studies with the same
enzyme showed no change in clearance with age (Hunt et
al 1992) To date there has been even less examination of
the transporter functions and any changes with age
(Kinirons amp OrsquoMahony 2004) so the current understand-
ing of the effects of aging on metabolism and transport is
anything but transparent This represents an extreme
challenge for the pharmaceutical industry how to predict
whether a drug has an affinity for an enzyme or transporter
and whether this may be clinically important if it shows a
decline in expression or function with aging Any decrease
of function of these enzymes or transporters in an elderly
population would result in changes in the metabolism and
transport of metabolites potentially impacting the clinical
effect This represents another example of how computa-
tional approaches may perhaps be used with different types
of predicted and experimental data to allow visualization of
potential compound interactions or toxicity in elderly
populations We may see different gene networks high-
lighted as humans age and these may be modified by drug
treatment and coadministration This represents an area
were network analysis could be applied in the future and is
in need of considerable further research
The collection of microarray data in databases such as
CEBS ArrayTrack and EDGE represents a future resource
for computational gene network analysis One could
envisage that ultimately in each case such data is converted
to one or more networks that are also displayed for the user
and can be used to compare treatments from in vivo and in
vitro experiments This would represent a different approach
to clustering the data as currently implemented in one of
these efforts (Hayes et al 2005) and may condense large
amounts of experimentally derived data into a readily
interpreted network
4 Discussion
Previously in this journal the progress of many
research groups in predicting human ADME parameters
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6660
in silico (Ekins et al 2000b) and approaches for drug
metabolism (Ekins Ring Grace McRobie-Belle amp
Wrighton 2000a) have been described Both of these
reviews commented on moving HT assays for ADME
Tox much earlier in drug discovery which had also been
indicated by other groups The initial reviews also
highlighted the likely wealth of data that would become
available and how this could be used for structure
activity relationships alongside the bioactivity data in
computational models It was noted that there was a
paucity of predictive metabolism tools at that time In
addition a growing number of efforts to model whole
cells and organs now a field called systems biology
were recognized as models that could be integrated with
the in silico ADME approaches In summary since these
past reviews virtually all pharmaceutical companies have
attempted earlier high throughput screening for ADME
Tox properties and to some extent the wider application
of computational approaches for physicochemical proper-
ties Systems biology is being quite widely acknowledged
as the new paradigm for understanding complex bio-
logical datasets derived from high throughput technolo-
gies and the accumulated knowledge on human protein
interactions (Hartwell Hopfield Leibler amp Murray 1999
Hood 2003b) Therefore systems biology can be defined
as the integration of genetic proteomic transcriptomic
and metabonomic data using computational methods
(Nicholson amp Wilson 2003) When taken together
information on molecular processes derived from different
sources represents a lsquolsquouniversersquorsquo of putative biological
functionality of which only a small fraction of it will be
realized in a cell at any given time To date systems
biology has been driven by academia and funding bodies
such as the NIH rather than the pharmaceutical compa-
nies Presently there is a great deal of interest from
scientists of all backgrounds in identifying the networks
of cellular pathways and the corresponding physically
interacting proteins
The network building software for systems biology
described in this current review will be valuable to query
high throughput data and known literature interactions in
order to predict potential toxicity in different species In the
future the compilation of published toxicogenomics data-
sets characteristic of different types of toxicity will likely
be available in these software systems to act as a reference
database It is also feasible that we will be able to generate
the annotated datasets which specifically address the
differences between human and rat networks implicated
in toxicity The identification of sub-network modules
conserved between human and rat distinct for toxicity
types or predictive for toxic end-points in human will be
possible Such signature gene networks (Nikolsky et al
2005) can then be verified with other experimentally
derived data prospectively or from preexisting databases
The combined Fhigh throughput datandashin silico network_approaches therefore represents a novel method for access-
ing complex tissue-level processes as networks integrating
all data types based on functional interactions The data-
bases developed and used in this approach will certainly
benefit from further annotation around the drug metaboliz-
ing enzymes and transporters as described herein in terms
of transcriptional regulation and the ligands associated with
them which frequently appear in the literature It will be
important to capture disparities in the assignment of ligands
to enzymes transporters and other proteins as well as
negative data
In summary although there have been numerous
toxicogenomics studies published there is presently a
relatively small number of datasets that are freely available
to perform network analysis of microarray data The
number of studies identifying large numbers of proteins
which are affected by molecule treatment are even scarcer
still while there are several examples of some published
studies that combine such data It is hoped that the
numerous database initiatives for high content and toxicol-
ogy data that are being undertaken will improve the
situation for other researchers that are not currently
equipped to do such microarray studies themselves The
addition of requirements by journals to deposit such raw
data in a freely accessible resource will aid these initiatives
ADMETox groups have seen new technologies and
approaches developed over the last decade that have all
been applied to identify poor compounds earlier (Fig 2)
The latest technologies integrate network building tools
with high content data and databases The current review
described the limited number of networks generated for
ADMETox at present and one hopes that the impact of
such analyses will be commonplace in the future Systems
biology is however more than just applying a network
approach and hence systems-ADMETox will have to
evolve due to the continual pressure to develop newer
technologies This current paradigm combining empirical
data and computational methods should integrate the
complex data already generated making it readily inter-
pretable and valuable for identifying the most promising
compounds in the future
Acknowledgements
Dr Maggie AZ Hupcey is gratefully acknowledged for
editorial assistance Dr Peter W Swaan (University of
Maryland) and Dr Cheng Chang (Ohio State University)
Dr Steve Wright (University of Arizona) Dr K Sandy
Pang (University of Toronto) and Dr Craig Giroux (Wayne
State University) are kindly acknowledged for their support
and discussions My colleagues at GeneGo Sergey
Andreyev Andy Ryabov Eugene Kirillov Eugene A
Rakhmatulin Svetlana Sorokina Andrej Bugrim Tatiana
Nikolskaya Yuri Nikolsky John Metz and Julie Bryant are
all thanked for their considerable contributions to software
development and data annotation
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 61
References
Abe M Yamashita S Kuramoto T Hirayama Y Tsukamoto T Ohta
T et al (2003) Global expression analysis of N-methyl-NV-nitro-N-nitrosoguanidine-induced rat stomach carcinomas using oligonucleotide
microarrays Carcinogenesis 24 861ndash867
Adachi T Ono Y Koh K B Takashima K Tainaka H Matsuno Y
et al (2004) Long-term alteration of gene expression without
morphological change in testis after neonatal exposure to genistein in
mice Toxicogenomic analysis using cDNA microarray Food and
Chemical Toxicology 42 445ndash452
Akerman G S Rosenzweig B A Domon O E McGarrity L J
Blankenship L R Tsai C A et al (2004) Gene expression profiles
and genetic damage in benzo(a)pyrene diol epoxide-exposed TK6 cells
Mutation Research 549 43ndash64
Aksenov S V Church B Dhiman A Georgieva A Sarangapani R
Helmlinger G et al (2005) An integrated approach for inference and
mechanistic modeling for advancing drug development FEBS Letters
579 1878ndash1883
Anderle P Huang Y amp Sadee W (2004) Intestinal membrane transport
of drugs and nutrients Genomics of membrane transporters using
expression microarrays European Journal of Pharmaceutical Sciences
21 17ndash24
Annereau J P Szakacs G Tucker C J Arciello A Cardarelli C
Collins J et al (2004) Analysis of ATP-binding cassette transporter
expression in drug-selected cell lines by a microarray dedicated to
multidrug resistance Molecular Pharmacology 66 1397ndash1405
Apic G Ignjatovic T Boyer S amp Russell R B (2005) Illuminating drug
discovery with biological pathways FEBS Letters 579 1872ndash1877
Baker V A Harries H M Waring J F Duggan C M Ni H A Jolly
R A et al (2004) Clofibrate-induced gene expression changes in rat
liver A cross-laboratory analysis using membrane cDNA arrays
Environmental Health Perspectives 112 428ndash438
Balakin K V Ekins S Bugrim A Ivanenkov Y A Korolev D amp
Nikolsky Y et al (2004a) Quantitative structurendashmetabolism relation-
ship modeling of the metabolic N-dealkylation rates Drug Metabolism
and Disposition 32 1111ndash1120
Balakin K V Ekins S Bugrim A Ivanenkov Y A Korolev D amp
Nikolsky Y et al (2004b) Kohonen maps for prediction of binding to
human cytochrome P450 3A4 Drug Metabolism and Disposition 32
1183ndash1189
Barabasi A -L amp Oltvai Z N (2004) Network biology Under-
standing the cellrsquos functional organization Nature Reviews Genetics
5 101ndash113
Bartosiewicz M Penn S G amp Buckpitt A (2001) Applications of gene
arrays in environmental toxicology Fingerprints of gene regulation
associated with cadmium chloride benzo(a)pyrene and trichloro-
ethylene Environmental Health Perspectives 109 71ndash74
Bertilsson G Heidrich J Svensson K Asman M Jendeberg L
Sydow-Backman M et al (1998) Identification of a human nuclear
receptor defines a new signaling pathway for CYP3A induction
Proceedings of the National Academy of Sciences of the United States
of America 95 12208ndash12213
Blumberg B Sabbagh Jr W Juguilon H Bolado Jr J van Meter
C M Ong E S et al (1998) SXR a novel steroid and xenobiotic-
sensing nuclear receptor Genes and Development 12 3195ndash3205
Borodina Y Rudik A Filimonov D Kharchevnikova N Dmitriev A
Blinova V et al (2004) A new statistical approach to predicting
aromatic hydroxylation sites Comparison with model-based
approaches Journal of Chemical Information and Computer Sciences
44 1998ndash2009
Borodina Y Sadym A Filimonov D Blinova V Dmitriev A amp
Poroikov V (2003) Predicting biotransformation potential from
molecular structure Journal of Chemical Information and Computer
Sciences 43 1636ndash1646
Bottone Jr F G Martinez J M Collins J B Afshari C A amp Eling
T E (2003) Gene modulation by the cyclooxygenase inhibitor
sulindac sulfide in human colorectal carcinoma cells Possible link to
apoptosis Journal of Biological Chemistry 278 25790ndash25801
Boyer S amp Zamora I (2002) New methods in predictive metabolism
Journal of Computer-Aided Molecular Design 16 403ndash413
Bredel M amp Jacoby E (2004) Chemogenomics An emerging strategy
for rapid target and drug discovery Nature Reviews Genetics 5
262ndash275
Bugrim A Nikolskaya T amp Nikolsky Y (2004) Early prediction of drug
metabolism and toxicity Systems biology approach and modeling
Drug Discovery Today 9 127ndash135
Bulera S J Eddy S M Ferguson E Jatkoe T A Reindel J F
Bleavins M R et al (2001) RNA expression in the early character-
ization of hepatotoxicants in Wistar rats by high-density DNA micro-
arrays Hepatology 33 1239ndash1258
Butcher E C Berg E L amp Kunkel E J (2004) Systems biology in drug
discovery Nature Biotechnology 22 1253ndash1259
Butte A (2002) The use and analysis of microarray data Naturalist
Review of Drug Discovery 1 951ndash960
Cary M P Bader G D amp Sander C (2005) Pathway information for
systems biology FEBS Letters 579 1815ndash1820
Cheok M H Yang W Pui C H Downing J R Cheng C Naeve
C W et al (2003) Treatment-specific changes in gene expression
discriminate in vivo drug response in human leukemia cells Nature
Genetics 34 85ndash90
Cleary J D Rogers P D amp Chapman S W (2001) Differential
transcription factor expression in human mononuclear cells in response
to amphotericin B Identification with complementary DNA microarray
technology Pharmacotherapy 21 1046ndash1054
Coen M Ruepp S U Lindon J C Nicholson J K Pognan F Lenz
E M et al (2004) Integrated application of transcriptomics and
metabonomics yields new insight into the toxicity due to paracetamol in
the mouse Journal of Pharmaceutical and Biomedical Analysis 35
93ndash105
Cornwell P D De Souza A T amp Ulrich R G (2004) Profiling of
hepatic gene expression in rats treated with fibric acid analogs
Mutation Research 549 131ndash145
Csermely P Agoston V amp Pongor S (2005) The efficiency of multi-
target drugs The network approach might help drug design Trends in
Pharmacological Sciences 26 178ndash182
Cunningham M J Liang S Fuhrman S Seilhamer J J amp
Somogyi R (2000) Gene expression microarray data analysis for
toxicology profiling Annals of the New York Academy of Sciences 919
52ndash67
de Longueville F Atienzar F A Marcq L Dufrane S Evrard S
Wouters L et al (2003) Use of a low density microarray for studying
gene expression patterns induced by hepatotoxicants on primary
cultures of rat hepatocytes Toxicological Sciences 75 378ndash392
Dobrin R Beg Q K Barabasi A L amp Oltvai Z N (2004)
Aggregation of topological motifs in the Escherichia coli transcriptional
regulatory network BMC Bioinformatics 5 10
Donald S Verschoyle R D Edwards R Judah D J Davies R
Riley J et al (2002) Hepatobiliary damage and changes in hepatic
gene expression caused by the antitumor drug ecteinascidin-743 (ET-
743) in the female rat Cancer Research 62 4256ndash4262
Dumont M Jacquemin E DrsquoHont C Descout C Cresteil D Haouzi
D et al (1997) Expression of the liver Na+-independent organic anion
transporting polypeptide (oatp-1) in rats with bile duct ligation Journal
of Hepatology 27 1051ndash1056
Durnas C Loi C M amp Cusack B J (1990) Hepatic drug metabolism
and aging Clinical Pharmacokinetics 19 359ndash389
Eisen M B Spellman P T Brown P O amp Botstein D (1998) Cluster
analysis and display of genome-wide expression patterns Proceedings
of the National Academy of Sciences of the United States of America
95 14863ndash14868
Ekins S Andreyev S Ryabov A Kirilov E Rakhmatulin E A
Bugrim A et al (in press) Computational Prediction of Human Drug
Metabolism Exp Opin Drug Metab Toxicol
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6662
Ekins S Boulanger B Swaan P W amp Hupcey M A Z (2002)
Towards a new age of virtual ADMETOX and multidimensional drug
discovery Journal of Computer-Aided Molecular Design 16 381ndash401
Ekins S Bugrim A Nikolsky Y amp Nikolskaya T (2005) Systems
biology Applications in drug discovery In S Gad (Ed) Drug
Discovery Handbook (pp 123ndash183) Hoboken NJrsquo Wiley and Sons
Ekins S Giroux C N Nikolsky Y Bugrim A amp Nikolskaya T
(2005) A signature gene network approach to toxicity The Toxoco-
logists 84
Ekins S Kirillov E Rakhmatulin E amp Nikolskaya T (2005) A novel
method for visualizing nuclear hormone receptor networks relevant to
drug metabolism Drug Metabolism and Disposition 33 474ndash481
Ekins S amp McGowan R J (2001) The limits of reductionism The
shifting genomic paradigmrsquos impact on industry and academia
Philsophy in Science 9 1ndash23
Ekins S Mirny L amp Schuetz E G (2002) A ligand-based approach to
understanding selectivity of nuclear hormone receptors PXR CAR
FXR LXRa and LXRb Pharmaceutical Research 19 1788ndash1800
Ekins S Nikolsky Y amp Nikolskaya T (2005) Techniques Application
of systems biology to absorption distribution metabolism excretion
and toxicity Trends in Pharmacological Sciences 26 202ndash209
Ekins S Ring B J Grace J McRobie-Belle D J amp Wrighton S
A (2000) Present and future in vitro approaches for drug
metabolism Journal of Pharmacological and Toxicological Methods
44 313ndash324
Ekins S Waller C L Swaan P W Cruciani G Wrighton S A amp
Wikel J H (2000) Progress in predicting human ADME parameters in
silico Journal of Pharmacological and Toxicological Methods 44
251ndash272
Erhardt P W (2003) A human drug metabolism database Potential roles
in the quantitative predictions of drug metabolism and metabolism-
related drugndashdrug interactions Current Drug Metabolism 4 411ndash422
Evans W E amp McLeod H L (2003) Pharmacogenomicsmdashdrug
disposition drug targets and side effects New England Journal of
Medicine 348 538ndash549
Ezendam J Staedtler F Pennings J Vandebriel R J Pieters R
Boffetta P et al (2004) Toxicogenomics of subchronic hexachlor-
obenzene exposure in Brown Norway rats Environmental Health
Perspectives 112 782ndash791
Faiola B Fuller E S Wong V A amp Recio L (2004) Gene expression
profile in bone marrow and hematopoietic stem cells in mice exposed to
inhaled benzene Mutation Research 549 195ndash212
FDA (2004) Innovation stagnation Challenge and opportunity on the
critical path to new medicinal products
Fiehn O (2001) Combining genomics metabolome analysis and bio-
chemical modelling to understand metabolic networks Comparative
Functional Genomics 2 155ndash168
Field F J Born E amp Mathur S N (2004) LXRRXR ligand activation
enhances basolateral efflux of beta-sitosterol in CaCo-2 cells Journal of
Lipid Research 45 905ndash913
Fleck C Sutter L Appenroth D Koch B Meinhold T Pitack M
et al (2003) Use of gene chip technology for the characterisation of
the regulation of renal transport processes and of nephrotoxicity in
rats Experimental and Toxicologic Pathology 54 401ndash410
Fliri A F Loging W T Thadeio P F amp Volkmann R A (2005)
Biological spectra analysis Linking biological activity profiles to
molecular structure Proceedings of the National Academy of Sciences
of the United States of America 102 261ndash266
Fountoulakis M (2004) Application of proteomics technologies in the
investigation of the brain Mass Spectrometry Reviews 23 231ndash258
Gaucher S P Taylor S W Fahy E Zhang B Warnock D E Ghosh
S S et al (2004) Expanded coverage of the human heart
mitochondrial proteome using multidimensional liquid chromatography
coupled with tandem mass spectrometry Journal of Proteome
Research 3 495ndash505
Gerhold D Lu M Xu J Austin C Caskey C T amp Rushmore T
(2001) Monitoring expression of genes involved in drug metabolism
and toxicology using DNA microarrays Physiological Genomics 5
161ndash170
Guo G L Choudhuri S amp Klaassen C D (2002) Induction profile of
rat organic anion transporting polypeptide 2 (oatp2) by prototypical
drug-metabolizing enzyme inducers that activate gene expression
through ligand-activated transcription factor pathways Journal of
Pharmacology and Experimental Therapeutics 300 206ndash212
Hagenbuch B amp Meier P J (2004) Organic anion transporting
polypeptides of the OATP SLC21 family Phylogenetic classification
as OATP SLCO superfamily new nomenclature and molecularfunc-
tional properties Pflugers Archiv 447 653ndash665
Hamadeh H K amp Afshari C A (2004) Toxicogenomics principles and
applications Hobokenrsquo John Wiley and Sons
Hamadeh H K Bushel P R Jayadev S Martin K DiSorbo O Sieber
S et al (2002) Gene expression analysis reveals chemical-specific
profiles Toxicological Sciences 67 219ndash231
Hamadeh H K Jayadev S Gaillard E T Huang Q Stoll R
Blanchard K et al (2004) Integration of clinical and gene expression
endpoints to explore furan-mediated hepatotoxicity Mutation Research
549 169ndash183
Hamadeh H K Knight B L Haugen A C Sieber S Amin R P
Bushel P R et al (2002) Methapyrilene toxicity Anchorage of
pathologic observations to gene expression alterations Toxicologic
Pathology 30 470ndash482
Han J D Bertin N Hao T Goldberg D S Berriz G F Zhang L V
et al (2004) Evidence for dynamically organized modularity in the
yeast proteinndashprotein interaction network Nature 430 88ndash93
Hanisch D Zien A Zimmer R amp Lengauer T (2002) Co-clustering of
biological networks and gene expression data Bioinformatics 18
S145ndashS154
Harris A J Dial S L amp Casciano D A (2004) Comparison of basal
gene expression profiles and effects of hepatocarcinogens on gene
expression in cultured primary human hepatocytes and HepG2 cells
Mutation Research 549 79ndash99
Harrison S C (2004) Whither structural biology Nature Structural
Molecular Biology 11 12ndash15
Hartley D P Dai X He Y D Carlini E J Wang B Huskey S E
et al (2004) Activators of the rat pregnane X receptor differentially
modulate hepatic and intestinal gene expression Molecular Pharma-
cology 65 1159ndash1171
Hartwell L H Hopfield J J Leibler S amp Murray A W (1999) From
molecular to modular cell biology Nature 402 C47ndashC52
Hasmall S Orphanides G James N Pennie W Hedley K Soames
A et al (2002) Downregulation of lactoferrin by PPARalpha ligands
Role in perturbation of hepatocyte proliferation and apoptosis
Toxicology Sciences 68 304ndash313
Hayase T Yamamoto Y Yamamoto K Muso E amp Shiota K (2004)
Microarray profile analysis of toxic cocaine-induced alterations in the
expression of mouse brain gene sequences A possible rsquoprotectiversquo
effect of buprenorphine Journal of Applied Toxicology 24 15ndash20
Hayes K R Vollrath A L Zastrow G M McMillan B J Craven M
Jovanovich S et al (2005) EDGE A centralized resource for the
comparison analysis and distribution of toxicogenomic information
Molecular Pharmacology 67 1360ndash1368
Heijne W H Slitt A L van Bladeren P J Groten J P Klaassen C D
Stierum R H et al (2004) Bromobenzene-induced hepatotoxicity at
the transcriptome level Toxicology Sciences 79 411ndash422
Heijne W H Stierum R H Slijper M van Bladeren P J amp van
Ommen B (2003) Toxicogenomics of bromobenzene hepatotoxicity
A combined transcriptomics and proteomics approach Biochemical
Pharmacology 65 857ndash875
Heinloth A N Irwin R D Boorman G A Nettesheim P Fannin
R D Sieber S O et al (2004) Gene expression profiling of rat
livers reveals indicators of potential adverse effects Toxicology
Sciences 80 193ndash202
Hodges L C Cook J D Lobenhofer E K Li L Bennett L Bushel
P R et al (2003) Tamoxifen functions as a molecular agonist inducing
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 63
cell cycle-associated genes in breast cancer cells Molecular Cancer
Research 1 300ndash311
Holleman A Cheok M H den Boer M L Yang W Veerman KM
A J Kazemier et al (2004) Gene-expression patterns in drug-resistant
acute lymphoblastic leukemia cells and response to treatment New
England Journal of Medicine 351 533ndash542
Hong Y Muller U R amp Lai F (2003) Discriminating two classes of
toxicants through expression analysis of HepG2 cells with DNA arrays
Toxicology in Vitro 17 85ndash92
Hood E (2003) Proteomics Characterizing the cogs in the machinery of
life Environmental Health Perspectives 111 A816ndashA825
Hood L (2003) Systems biology Integrating technology biology and
computation Mechanism of Ageing and Development 124 9ndash16
Hood L amp Galas D (2003) The digital code of DNA Nature 421
444ndash448
Hood L amp Perlmutter R M (2004) The impact of systems approaches on
biological problems in drug discovery Nature Biotechnology 22
1215ndash1217
Hu T Gibson D P Carr G J Torontali S M Tiesman J P Chaney
J G et al (2004) Identification of a gene expression profile that
discriminates indirect-acting genotoxins from direct-acting genotoxins
Mutation Research 549 5ndash27
Huang Q Dunn II R T Jayadev S DiSorbo O Pack F D Farr S B
et al (2001) Assessment of cisplatin-induced nephrotoxicity by
microarray technology Toxicology Sciences 63 196ndash207
Huang Q Jin X Gaillard E T Knight B L Pack F D Stoltz J H et
al (2004) Gene expression profiling reveals multiple toxicity endpoints
induced by hepatotoxicants Mutation Research 549 147ndash167
Huisman M T Smit J W Crommentuyn K M Zelcer N Wiltshire
H R Beijnen J H et al (2002) Multidrug resistance protein 2
(MRP2) transports HIV protease inhibitors and transport can be
enhanced by other drugs Aids 16 2295ndash2301
Hunt C M Westerkam W R amp Stave G M (1992) Effect of age and
gender on the activity of human hepatic CYP3A Biochemical
Pharmacology 44 275ndash283
Ideker T Ozier O Schwikowski B amp Siegal A F (2002) Discovering
regulatory and signalling circuits in molecular interaction networks
Bioinformatics 18 S233ndashS240
Ideker T Thorsson V Ranish J A Christmas R Buhler J Eng J K
et al (2001) Integrated genomic and proteomic analyses of a system-
atically perturbed network Science 292 929ndash934
Iida M Anna C H Hartis J Bruno M Wetmore B Dubin J R et al
(2003) Changes in global gene and protein expression during early
mouse liver carcinogenesis induced by non-genotoxic model carcino-
gens oxazepam and Wyeth-14643 Carcinogenesis 24 757ndash770
Ingelman-Sundberg M (2004) Pharmacogenetics of cytochrome P450 and
its applications in drug therapy The past present and future Trends in
Pharmacological Sciences 25 193ndash200
Jeong H Mason S P Barabasi A L amp Oltvai Z N (2001) Lethality
and centrality in protein networks Nature 411 41ndash42
Jeong H Tombor B Albert R Oltvai Z N amp Barabasi A L
(2000) The large-scale organization of metabolic networks Nature
407 651ndash654
Jung D Hagenbuch B Gresh L Pontoglio M Meier P J amp Kullak-
Ublick G A (2001) Characterization of the human OATP-C
(SLC21A6) gene promoter and regulation of liver-specific OATP genes
by hepatocyte nuclear factor 1 alpha Journal of Biological Chemistry
276 37206ndash37214
Jung D amp Kullak-Ublick G A (2003) Hepatocyte nuclear factor 1
alpha A key mediator of the effect of bile acids on gene expression
Hepatology 37 622ndash631
Jung J -W Park J -S Hwang J -W Kang K -S YS L Song B
-S et al (2004) Gene expression analysis of peroxisome prolifer-
ators- and phenytoin-induced hepatotoxicity using cDNA microarray
Journal of Veterinary Medical Science 66 1329ndash1333
Kato N Shibutani M Takagi H Uneyama C Lee K -Y Takiami S
et al (2004) Gene expression profile in the livers of rats orally
administered ethinylestradiol for 28 days using a microarray technique
Toxicology 200 179ndash192
Kier L D Neft R Tang L Suizu R Cook T Onsurez K et al
(2004) Applications of microarrays with toxicologically relevant genes
(tox genes) for the evaluation of chemical toxicants in Sprague Dawley
rats in vivo and human hepatocytes in vitro Mutation Research 549
101ndash113
Kijima K Toyosawa K Yasuba M Matsuoka N Adachi T
Komiyama M et al (2004) Gene expression analysis of the rat testis
after treatment with di(2-ethylhexyl) phthalate using cDNA microarray
and real-time RT-PCR Toxicology and Applied Pharmacology 200
103ndash110
Kim R B (2003) Organic anion-transporting polypeptide (OATP) trans-
porter family and drug disposition European Journal of Clinical
Investigation 33 1ndash5
Kinirons M T amp OrsquoMahony M S (2004) Drug metabolism and ageing
British Journal of Clinical Pharmacology 57 540ndash544
Kitano H (2002a) Computational systems biology Nature 420 206ndash210
Kitano H (2002b) Systems biology A brief overview Science 295
1662ndash1664
Kliewer S A Moore J T Wade L Staudinger J L Watson M
A Jones S A et al (1998) An orphan nuclear receptor activated
by pregnanes defines a novel steroid signalling pathway Cell 92
73ndash82
Korolev D Balakin K V Nikolsky Y Kirillov E Ivanenkov Y A
Savchuk N P et al (2003) Modeling of human cytochrome p450-
mediated drug metabolism using unsupervised machine learning
approach Journal of Medicinal Chemistry 46 3631ndash3643
Kramer J A Pettit S D Amin R P Bertram T A Car B
Cunningham M et al (2004) Overview on the application of
transcription profiling using selected nephrotoxicants for toxicology
assessment Environmental Health Perspectives 112 460ndash464
Kultima K Nystrom A -M Scholz B Gustafson A -L Dencker L
amp Stigson M (2004) Valproic acid teratogenicity A toxicogenomics
approach Environmental Health Perspectives 112 1225ndash1235
Kutuzova G D amp Deluca H F (2004) Gene expression profiles in rat
intestine identify pathways for 125-dihydroxyvitamin D(3) stimulated
calcium absorption and clarify its immunomodulatory properties
Archives of Biochemistry and Biophysics 432 152ndash166
Kwak M K Wakabayashi N Itoh K Motohashi H Yamamoto M amp
Kensler T W (2003) Modulation of gene expression by cancer
chemopreventive dithiolethiones through the Keap1-Nrf2 pathway
Identification of novel gene clusters for cell survival Journal of
Biological Chemistry 278 8135ndash8145
Lahjouji K Mitchell G A amp Qureshi I A (2001) Carnitine transport
by organic cation transporters and systemic carnitine deficiency
Molecular Genetics and Metabolism 73 287ndash297
Landowski C P Sun D Foster D R Menon S S Barnett J L
Welage L S et al (2003) Gene expression in the human intestine and
correlation with oral valacyclovir pharmacokinetic parameters Journal
of Pharmacology and Experimental Therapeutics 306 778ndash786
Lee G -J Lee W -S Jeon K -S Um C -H Kim S -J Lee C -H
et al (2004) cDNA microarray gene expression analysis and
toxicological phenotype for anticancer drug Journal of Veterinary
Medical Science 66 1339ndash1345
Lee M Kwon J Kim S N Kim J E Koh W S Kim E J et al
(2003) cDNA microarray gene expression profiling of hydroxyurea
paclitaxel and p-anisidine genotoxic compounds with differing
tumorigenicity results Environmental and Molecular Mutagenesis
42 91ndash97
Li S Armstrong C M Bertin N Ge H Milstein S Boxem M et al
(2004) A map of the interactome network of the metazoan C elegans
Science 303 540ndash543
Liguori M J Anderson L M Bukofzer S McKim J Pregenzer J F
Retief J et al (2005) Microarray analysis in human hepatocytes
suggests a mechanism for hepatotoxicity induced by trovafloxacin
Hepatology 41 177ndash186
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6664
Lobenhofer E K Bennett L Cable P L Li L Bushel P R amp Afshari
C A (2002) Regulation of DNA replication fork genes by 17beta-
estradiol Molecular Endocrinology 16 1215ndash1229
Locker J Tian J Carver R Concas D Cossu C Ledda-Columbano
G M et al (2003) A common set of immediate-early response genes
in liver regeneration and hyperplasia Hepatology 38 314ndash325
Mattes W B Pettit S D Sansone S A Bushel P R amp Waters M D
(2004) Database development in toxicogenomics Issues and efforts
Environmental Health Perspectives 112 495ndash505
McMillian M Nie A Y Parker J B Leone A Bryant S Kemmerer
M et al (2004) A gene expression signature for oxidant stressreactive
metabolites in rat liver Biochemical Pharmacology 68 2249ndash2261
Meier P J amp Stieger B (2000) Molecular mechanisms in bile formation
News in Physiological Sciences 15 89ndash93
Meneses-Lorente G de Longueville F Dos Santos-Mendes S Bonnert
T P Jack A Evrard S et al (2003) An evaluation of a low-density
DNA microarray using cytochrome P450 inducers Chemical Research
in Toxicology 16 1070ndash1077
Meneses-Lorente G Guest P C Lawrence J Muniappa N Knowles
M R Skynner H A et al (2004) A proteomic investigation of drug-
induced steatosis in rat liver Chemical Research in Toxicology 17
605ndash612
Merrill C L Ni H Yoon L W Tirmenstein M A Narayanan P
Benavides G R et al (2002) Etomoxir-induced oxidative stress in
HepG2 cells detected by differential gene expression is confirmed
biochemically Toxicology Sciences 68 93ndash101
Milo R Shen-Orr S Itzkovitz S Kashtan N Chklovskii D amp Alon
U (2002) Network motifs Simple building blocks of complex
networks Science 298 824ndash827
Morgan K T Ni H Brown H R Yoon L Qualls C W Crosby L
M et al (2002) Application of cDNA microarray technology to in
vitro toxicology and the selection of genes for a real-time RT-PCR-
based screen for oxidative stress in Hep-G2 cells Toxicologic
Pathology 30 435ndash451
Mwinyi J Johne A Bauer S Roots I amp Gerloff T (2004) Evidence
for inverse effects of OATP-C (SLC21A6) 5 and 1b haplotypes on
pravastatin kinetics Clinical Pharmacology and Therapeutics 75
415ndash421
Nakata K Yukawa M Komiyama N Nakano T amp Kaminuma T
(2002) A nuclear receptor database that maps pathways to diseases
Genome Infomatics 13 515ndash516
Nicholson J K Holmes E Lindon J C amp Wilson I D (2004) The
challenges of modeling mammalian biocomplexity Nature Biotechnol-
ogy 22 1268ndash1274
Nicholson J K amp Wilson I D (2003) Understanding rsquoglobal systems
biology Metabonomics and the continuum of metabolism Nature
Reviews on Drug Discovery 2 668ndash676
Nikitin A Egorov S Daraselia N amp Mazo I (2003) Pathway studiomdash
the analysis and navigation of molecular networks Bioinformatics 19
2155ndash2157
Nikolsky Y Ekins S Nikolskaya T amp Bugrim A (2005) A novel
method for generation of signature networks as biomarkers from
complex high throughput data Toxicology Letters 158 20ndash29
Nikolsky Y Nikolskaya T amp Bugrim A (2005) Biological networks
and analysis of experimental data in drug discovery Drug Discovery
Today 10 653ndash662
Nishizato Y Ieiri I Suzuki H Kimura M Kawabata K Hirota T
et al (2003) Polymorphisms of OATP-C (SLC21A6) and OAT3
(SLC22A8) genes Consequences for pravastatin pharmacokinetics
Clinical Pharmacology and Therapeutics 73 554ndash565
Nozawa T Nakajima M Tamai I Noda K Nezu J Sai Y et al
(2002) Genetic polymorphisms of human organic anion transporters
OATP-C (SLC21A6) and OATP-B (SLC21A9) Allele frequencies in
the Japanese population and functional analysis Journal of Pharmacol-
ogy and Experimental Therapeutics 302 804ndash813
Nozawa T Sugiura S Nakajima M Goto A Yokoi T Nezu J et al
(2004) Involvement of organic anion transporting polypeptides in the
transport of troglitazone sulfate Implications for understanding trogli-
tazone hepatotoxicity Drug Metabolism and Disposition 32 291ndash294
Oliver D E Rubin D L Stuart J M Hewett M Klein T E amp
Altman R B (2002) Ontology development for a pharmacogenetics
knowledge base Package Symposion 88ndash99
Oram J F amp Lawn R M (2001) ABCA1 The gatekeeper for eliminating
excess tissue cholesterol Journal of Lipid Research 42 1173ndash1179
Oswald M Kullak-Ublick G A Paumgartner G amp Beuers U (2001)
Expression of hepatic transporters OATP-C and MRP2 in primary
sclerosing cholangitis Liver 21 247ndash253
Parsons A B Brost R I Ding H Li Z Zhang C Sheikh B et al
(2004) Integration of chemical-genetic and genetic interaction data
links bioactive compounds to cellular target pathways Nature Bio-
technology 22 62ndash69
Patki K C von Moltke L L Harmatz J S Hesse L M Court M H
amp Greenblatt D J (2004) Effect of age on in vitro triazolam
biotransformation in male human liver microsomes Journal of
Pharmacology and Experimental Therapeutics 308 874ndash879
Pereira-Leal J B Enright A J amp Ouzounis C A (2004) Detection of
functional modules from protein interaction networks Proteins 54
49ndash57
Plant N (2004) Interaction networks Coordinating responses to xeno-
biotic exposure Toxicology 202 21ndash32
Raghavendra Prasad H S Qi Z Srinivasan K N amp Gopalakrish-
nakone P (2004) Potential effects of tetrodotoxin exposure to
human glial cells postulated using microarray approach Toxicon 44
597ndash608
Reilly T P Bourdi M Brady J N Pise-Masison C A Radonovich M
F George J W et al (2001) Expression profiling of acetaminophen
liver toxicity in mice using microarray technology Biochemical and
Biophysical Research Communications 282 321ndash328
Rives A W amp Galitski T (2003) Modular organization of cellular
networks Proceedings of the National Academy of Sciences of the
United States of America 100 1128ndash1133
Rogers P D Pearson M M Cleary J D Sullivan D C amp Chapman
S W (2002) Differential expression of genes encoding immunomodu-
latory proteins in response to amphotericin B in human mononuclear
cells identified by cDNA microarray analysis Journal of Antimicrobial
Chemotherapy 50 811ndash817
Rosen M B Wilson V S Schmid J E amp Gray L E (2005) Gene
expression analysis in the ventral prostate of rats exposed to vinclozolin
or procymidone Reproductive Toxicology 19 367ndash379
Rost D Herrmann T Sauer P Schmidts H L Stieger B Meier P J
et al (2003) Regulation of rat organic anion transporters in bile salt-
induced cholestatic hepatitis Effect of ursodeoxycholate Hepatology
38 187ndash195
Rothman R B Ayestas M A Dersch C M amp Baumann M H (1999)
Aminorex fenfluramine and chlorphentermine are serotonin transporter
substrates Implications for primary pulmonary hypertension Circula-
tion 100 869ndash875
Ruepp S U Tonge R P Shaw J Wallis N amp Pognan F (2002)
Genomics and proteomics analysis of acetaminophen toxicity in mouse
liver Toxicology Sciences 65 135ndash150
Sakaeda T Nakamura T amp Okumura K (2002) MDR1 genotype-
related pharmacokinetics and pharmacodynamics Biological amp Phar-
maceutical Bulletin 25 1391ndash1400
Sakata T Anzai N Shin H J Noshiro R Hirata T Yokoyama H
et al (2004) Novel single nucleotide polymorphisms of organic cation
transporter 1 (SLC22A1) affecting transport functions Biochemical
and Biophysical Research Communications 313 789ndash793
Satlin L M Amin V amp Wolkoff A W (1997) Organic anion
transporting polypeptide mediates organic anionHCO3-exchange
Journal of Biological Chemistry 272 26340ndash26345
Satomi Y Tsuchiya W Mihara K Ota M Kasahara Y amp Akahori F
(2004) Gene expression analysis of the lung following paraquat
administration in rats using DNA microarray Journal of Toxicological
Sciences 29 91ndash100
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 65
Schrenk D Baus P R Ermel N Klein C Vorderstemann B amp
Kauffmann H M (2001) Up-regulation of transporters of the MRP
family by drugs and toxins Toxicology Letters 120 51ndash57
Segal E Shapira M Regev A Persquoer D Botstein D Koller D et al
(2003) Module networks Identifying regulatory modules and their
condition-specific regulators from gene expression data Nature
Genetics 34 166ndash176
Segal E Wang H amp Koller D (2003) Discovering molecular pathways
from protein interaction and gene expression Bioinformatics 19
i264ndash i272
Seidel S D Kan H L Stott W T Schisler M R amp Gollapudi B B
(2003) Identification of transcriptome profiles for the DNA-damaging
agents bleomycin and hydrogen peroxide in L5178Y mouse lymphoma
cells Environmental and Molecular Mutagenesis 42 19ndash25
Shan L Yu Y Schut H A J amp Snyderwine E G (2004) Susceptibility
of rats to mammary gland carcinogenesis by the food-derived
carcinogen 2-Amino-1-Methyl-6-Phenylimidazo[45-b]Pyridine (PhIP)
varies with age and is associated with the induction of differential gene
expression American Journal of Pathology 165 191ndash202
Sharom J R Bellows D S amp Tyers M (2004) From large networks to
small molecules Current Opinion in Chemical Biology 8 81ndash90
Shitara Y Hirano M Sato H amp Sugiyama Y (2004) Gemfibrozil and
its glucuronide inhibit the OATP2(OATP1B1 SLC21A6)-mediated
hepatic uptake and CYP2C8-mediated metabolism of cerivastatinmdash
Analysis of the mechanism of the clinically relevant drugndashdrug
interaction between cerivastatin and gemfibrozil Journal of Pharma-
cology and Experimental Therapeutics
Shitara Y Itoh T Sato H Li A P amp Sugiyama Y (2003) Inhibition of
transporter-mediated hepatic uptake as a mechanism for drugndashdrug
interaction between cerivastatin and cyclosporin A Journal of
Pharmacology and Experimental Therapeutics 304 610ndash616
Somogyi R Fuhrman S amp Wen X (2001) Genetic network inference in
computational models and applications to large-scale gene expression
data In M Bower amp H Bolouri (Eds) Computational modeling of
genetic and biochemical networks (pp 119ndash157) Cambridge MArsquoThe
MIT Press
Spirin V amp Mirny L A (2003) Protein complexes and functional
modules in molecular networks Proceedings of the National Academy
of Sciences of the United States of America 100 12123ndash12128
Staudinger J Liu Y Madan A Habeebu S amp Klaassen C D
(2001) Coordinate regulation of xenobiotic and bile acid homeo-
stasis by pregnane X receptor Drug Metabolism and Disposition
29 1467ndash1472
Sun L Z Ji Z L Chen X Wang J F amp Chen Y Z (2002) ADME-
AP A database of ADME associated proteins Bioinformatics 18
1699ndash1700
Suter L Babiss L E amp Wheeldon E B (2004) Toxicogenomics in
predictive toxicology in drug development Chemistry and Biology 11
161ndash171
Synold T W Dussault I amp Forman B M (2001) The orphan nuclear
receptor SXR coordinately regulates drug metabolism and efflux
Nature Medicine 7 584ndash590
Tabuchi Y amp Kondo T (2003) cDNA microarray analysis reveals chop-
10 plays a key role in Sertoli cell injury induced by bisphenol A
Biochemical and Biophysical Research Communications 305 54ndash61
Tamai I Nezu J Uchino H Sai Y Oku A Shimane M et al (2000)
Molecular identification and characterization of novel members of the
human organic anion transporter (OATP) family Biochemical and
Biophysical Research Communications 273 251ndash260
Taylor S W Fahy E Zhang B Glenn G M Warnock D E Wiley S
et al (2003) Characterization of the human heart mitochondrial
proteome Nature Biotechnology 21 281ndash286
Terasaka S Aita Y Inoue A Hayashi S Nishigaki M Aoyagi K
et al (2004) Using a customized DNA microarray for expression
profiling of the estrogen-responsive genes to evaluate estrogen activity
among natural estrogens and industrial chemicals Environmental
Health Perspectives 112 773ndash781
Thomas D M Francescutti-Verbeem D M Liu X amp Kuhn D M
(2004) Identification of differentially regulated transcripts in mouse
striatum following methamphetamine treatmentmdashan oligonucleotide
microarray approach Journal of Neurochemistry 88 380ndash393
Thomas R S Rank D R Penn S G Zastrow G M Hayes K R
Pande K et al (2001) Identification of toxicologically predictive
gene sets using cDNA microarrays Molecular Pharmacology 60
1189ndash1194
Thompson K L Afshari C A Amin R P Bertram T A Car B
Cunningham M et al (2004) Identification of platform-independent
gene expression markers of cisplatin nephrotoxicity Environmental
Health Perspectives 112 488ndash494
Tirona R G Leake B F Merino G amp Kim R B (2001)
Polymorphisms in OATP-C Identification of multiple allelic
variants associated with altered transport activity among European-
and African-Americans Journal of Biological Chemistry 276
35669ndash35675
Tirona R G Leake B F Wolkoff A W amp Kim R B (2003)
Human organic anion transporting polypeptide-C (SLC21A6) is a
major determinant of the rifampin-mediated pregnane X receptor
activation Journal of Pharmacology and Experimental Therapeutics
304 223ndash228
Tong W Cao X Harris S Sun H Fang H Fuscoe J et al (2003)
ArrayTrackmdashsupporting toxicogenomic research at the US Food and
Drug Administration National Center for Toxicological Research
Environmental Health Perspectives 111 1819ndash1826
Tornow S amp Mewes H W (2003) Functional modules by relating
protein interaction networks and gene expression Nucleic Acids
Research 31 6283ndash6289
Trocho C Escola-Gil J C Ribas V Benitez S Martin-Campos J M
Rotllan N et al (2004) Phenytoin treatment reduces atherosclerosis in
mice through mechanisms independent of plasma HDL-cholesterol
concentration Atherosclerosis 174 275ndash285
Ueda A Hamadeh H K Webb H K Yamamoto Y Sueyoshi T
Afshari C A et al (2002) Diverse roles of the nuclear orphan
receptor CAR in regulating hepatic genes in response to phenobarbital
Molecular Pharmacology 61 1ndash6
Ulrich R G (2003) The toxicogenomics of nuclear receptor agonists
Current Opinion in Chemical Biology 7 505ndash510
Ulrich R G Rockett J C Gibson G G amp Pettit S D (2004)
Overview of an interlaboratory collaboration on evaluating the effects
of model hepatotoxicants on hepatic gene expression Environmental
Health Perspectives 112 423ndash427
Unami A Shinohara Y Ichikawa T amp Baba Y (2003) Biochemical
and microarray analyses of bupivacaine-induced apoptosis Journal of
Toxicological Sciences 28 77ndash94
Vasquez A Flammini A Maritan A amp Vespignani A (2003) Global
protein function prediction from proteinndashprotein interaction networks
Nature Biotechnology 21 697ndash700
Vezina C M Walker N J amp Olson J R (2004) Subchronic Exposure to
TCDD PeCDF PCB126 and PCB153 Effect on hepatic gene
expression Environmental Health Perspectives 112 1636ndash1644
Vo T D Greenberg H J amp Palsson B O (2004) Reconstruction and
functional characterization of the human mitochondrial metabolic
network based on proteomic and biochemical data Journal of Bio-
logical Chemistry 279 39532ndash39540
Wang H amp LeCluyse E L (2003) Role of orphan nuclear receptors in
the regulation of drug-metabolising enzymes Clinical Pharmacoki-
netics 42 1331ndash1357
Wang N Silver D L Thiele C amp Tall A R (2001) ATP-
binding cassette transporter A1 (ABCA1) functions as a choles-
terol efflux regulatory protein Journal of Biological Chemistry 276
23742ndash23747
Waring J F Cavet G Jolly R A McDowell J Dai H Ciurlionis R
et al (2003) Development of a DNA Microarray for toxicology based
on hepatoxin-regulated sequences Environmental Health Perspectives
111 863ndash870
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6666
Waring J F Gum R Morfitt D Jolly R A Ciurlionis R Heindel M
et al (2002) Identifying toxic mechanisms using DNA microarrays
Evidence that an experimental inhibitor of cell adhesion molecule
expression signals through the aryl hydrocarbon nuclear receptor
Toxicology 181ndash182 537ndash550
Waring J F Jolly R A Ciurlionis R Lum P Y Praestgaard J T
Morfitt D C et al (2001) Clustering of hepatotoxins based on
mechanism of toxicity using gene expression profiles Toxicology and
Applied Pharmacology 175 28ndash42
Waters M Boorman G Bushel P Cunningham M Irwin R Merrick
A et al (2003) Systems toxicology and the Chemical Effects in
Biological Systems (CEBS) knowledge base EHP Toxicogenomics
111 15ndash28
Wei M Arnold L Cano M amp Cohen S M (2005) Effects of co-
administration of antioxidants and arsenicals on the rat urinary bladder
epithelium Toxicology Sciences 83 237ndash245
Weinshilboum R (2003) Inheritance and drug response New England
Journal of Medicine 348 529ndash537
Weinshilboum R amp Wang L (2004) Pharmacogenetics Inherited
variation in amino acid sequence and altered protein quantity Clinical
Pharmacology and Therapeutics 75 253ndash258
Werner E (2003) In silico multicellular systems biology and minimal
genomes Drug Discovery Today 8 1121ndash1127
Wong J S amp Gill S S (2002) Gene expression changes induced in
mouse liver by di(2-ethylhexyl) phthalate Toxicology and Applied
Pharmacology 185 180ndash196
Xie T Tong L McCann U D Yuan J Becker K G Mechan A O
et al (2004) Identification and characterization of metallothionein-1
and -2 gene expression in the context of ()34-methylenedioxymetham-
phetamine-induced toxicity to brain dopaminergic neurons Journal of
Neuroscience 24 7043ndash7050
Yadetie F Laegreid A Bakke I Kusnierczyk W Komorowski J
Waldum H L et al (2003) Liver gene expression in rats in response
to the peroxisome proliferator-activated receptor-alpha agonist ciprofi-
brate Physiological Genomics 15 9ndash19
Yamashita S Nomoto T Abe M Tatematsu M Sugimura T amp
Ushijima T (2004) Persistence of gene expression changes in stomach
mucosae induced by short-term N-methyl-NV-nitro-N-nitrosoguanidinetreatment and their presence in stomach cancers Mutation Research
549 185ndash193
Yan Q amp Sadee W (2000) Human membrane transporter database A
web-accessible relational database for drug transport studies and
pharmacogenomics AAPS Pharmsci 2 E20
Yeger-Lotem E amp Margalit H (2003) Detection of regulatory circuits by
integrating the cellular networks of proteinndashprotein interactions and
transcription regulation Nucleic Acids Research 31 6053ndash6061
Yoon B I Li G -X Kitada K Kawasaki Y Igarashi K amp Kodama
Y et al (2003) Mechanisms of benzene-induced hematotoxicity and
leukemogenicity cDNA microarray analyses using mouse bone marrow
tissue Environmental Health Perspectives 111 1411ndash1420
Young M B DiSilvestro M R Sendera T J Freund J Kriete A amp
Magnuson S R (2003) Analysis of gene expression in carbon
tetrachloride-treated rat livers using a novel bioarray technology
Pharmacogenomics Journal 3 41ndash52
Yu H Zhu X Greenbaum D Karro J amp Gerstein M (2004) TopNet
A tool for comparing biological sub-networks correlating protein
properties with topological statistics Nucleic Acids Research 32
328ndash337
Zhang E Y Fu D J Pak Y A Stewart T Mukhopadhyay N
Wrighton S A et al (2004) Genetic polymorphisms in human proton-
dependent dipeptide transporter PEPT1 Implications for the functional
role of Pro586 Journal of Pharmacology and Experimental Therapeu-
tics 310 437ndash445
Zhang E Y Knipp G T Ekins S amp Swaan P W (2002)
Structural biology and function of solute transporters Implications
for identifying and designing substrates Drug Metabolism Reviews
34 709ndash750
Zhang E Y Phelps M A Cheng C Ekins S amp Swaan P W (2002)
Modeling of active transport systems Advanced Drug Delivery
Reviews 54 329ndash354
- Systems-ADMETox Resources and network approaches
-
- Introduction
- Data available
- Network analysis and databases
-
- Network applications
-
- The role of transporters
-
- Clinical relevance of transporters
- Transporter network examples ABCA1
- Transporter network examples OATP
- Transporter microarray data
-
- Applications to enzymes
- Future network applications
-
- Discussion
- Acknowledgements
- References
-
Table 1 (continued)
Compounds Rat strain Microarray type Compound dose Microarray data
availability
Reference
Dimethylarsinic acid Female F344
(bladder
epithelium)
Rat 10K chip (MWG
Biotech Inc) containing
10000 genes
100 ppm Genbank accession
number gene name in a
manuscript table
(Wei Arnold
Cano amp Cohen
2005)
Dexamethasone
troleandomycin
miconazole
clotrimazole
isoniazid
methylclofanapate
Female Spraguendash
Dawley
Rat HepatoChip DEX (50 mgkgday)
TAO (500 mgkg
day) MIC (100 mg
kgday) CLOT (100
mgkg per day) ISN
(100 mgkgday)
MCP (75 mgkgday)
Gene name and Genbank
accession numbermdashdata
displayed as a heatmap
not readily extracted
from publication
(Meneses-Lorente
et al 2003)
TCDD PeCDF PCB126
PCB153
Female Harlan
SpraguendashDawley
Affymetrix GeneChip
Test3 arrays
Exposed for 13 weeks
to toxicologically
equivalent doses
Accession number gene
name and fold change in
a manuscript table
(Vezina Walker
amp Olson 2004)
Paclitaxel Male and female
SpraguendashDawley
Genocheck 48K cDNA 4 mgkgday male
7 mgkgday female
Accession numbers and
gene names in a
manuscript table
(Lee et al 2004)
Clofibrate gemfibrozil
phenytoin
Male Spraguendash
Dawley VAF(+)
albino
48 K cDNA microarray
in house
Treated with each
compound for 24 h
and 2 weeks
Gene name and fold
change in a manuscript
table
(Jung et al 2004)
Clofibrate
dexamethasone
phenobarbital
3-methylcholanthrene
SpraguendashDawley Merck Drug Safety Chip
1443 genes (rat human
and mouse)
30 mgkgday Gene name GenBank
accession number and
relative fluorescence
levels in a manuscript
table
(Gerhold et al
2001)
Bemitradine clofibrate
doxylamine
methapyrilene
phenobarbital
tamoxifen
2-acetylaminofluorene
4-acetylaminofluorene
isoniazid
Male CD IGS Incyte RatGEM10
uml7800 rat cDNAs
Low mid and high
doses
A bar chart of 2 genes
that are affected by
compounds
(Kramer et al
2004)
PhIP Female Spraguendash
Dawley
Mouse cDNA
microarray containing
9984 cDNA clones
(National Cancer
Institute
75 mgkgday Gene names in a
manuscript table
(Shan Yu Schut
amp Snyderwine
2004)
Ethinylestradiol Male and female
SpraguendashDawley
Custom chip 3776 genes 0 001 01 and
10 ppm
Accession number gene
name and fold change at
different exposure levels
in a manuscript table
(Kato et al 2004)
Paraquat Male Wistar 1090 genes 7 mgkgday Gene name and
expression ratio in a
manuscript table
(Satomi et al
2004)
Hexachlorobenzene Female Brown
Norway
Affymetrix rat
RGU-34A GeneChip
microarray
0 150 or 450 mgkg Accession number gene
name and fold change
data for multiple organs
in a manuscript table
(Ezendam et al
2004)
N-methyl-NV-nitro-N-nitrosoguanidine
(MNNG)
Rat pyloric muco-
sae male congenic
rat strain that has a
homozygous_LIZ
transgene of
BigBlue rat
AFFYMETRIX Rat
Genome U34A
83 mgl AFFY ID gene name
gene symbol and fold
change in a manuscript
table
(Yamashita et al
2004)
N-methyl-NV-nitro-N-nitrosoguanidine
Male ACINJcI
(ACI)
Affymetrix GeneChip
Rat genome U34A
arrays
83 mgl from the age
of 8 weeks through to
40 weeks
Accession number gene
name symbol fold change
in a manuscript table also
rat vs human stomach
cancer comparison
(Abe et al 2003)
Cisplatin SpraguendashDawley Different arrays tox
chip incyte phase 1 etc
03ndash5 mgkg over a 4 to
144 h
Unigene gene ID gene
name NIEHS ID and
data for 5 platforms in a
manuscript table
(Thompson et al
2004)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 41
Table 1 (continued)
Compounds Rat strain Microarray type Compound dose Microarray data
availability
Reference
Clofibrate Male Spraguendash
Dawley
Atlas Rat Toxicology II
arrays (Clontech Palo
Alto CA USA)
containing 465 genes
High (250 mgkgday) or
low (25 mgkgday)
Genbank accession
number and data for 3
platforms in a
manuscript table
(Baker et al
2004)
Di(2-ethylhexyl)
phthalate
Male Spraguendash
Dawleymdash(testes)
An in-house cDNA
microarray
20 or 2000 mgkg Gene names Genbank
ID and fold change in a
manuscript table
(Kijima et al
2004)
Ciprofibrate Female Fischer An in-house cDNA
microarray
50 mgkg body weight Gene names symbol
accession number mean
ratio and SD
(Yadetie et al
2003)
Methapyrilene Male Spraguendash
Dawley
Rat Tox Chip 10 10 or 100 mgkgday Gene Name accession
number and indication of
up or down regulation
original data available on
NIEHS website
(Hamadeh et al
2002b)
Ecteinascidin-743
(ET-743)
Female Wistar Custom chip cDNA
microarrays containing
approximately 4700
hybridizable mouse
expressed sequence tags
derived from IMAGE
clones obtained from
Research Genetics
(Huntsville AL) or from
the MRC Human Gene
Mapping Project
40 ugkg Data available on
laboratory websitemdashnot
available at present
(Donald et al
2002)
Clofibrate Wyeth
14643 Gemfibrozil
phenobarbital
Male Spraguendash
Dawley VAF+
NIEHS rat chip v10 Clofibrate (250
mgkgday Wyeth
14643 (250 mgkg
day) Gemfibrozil
(100 mgkgday)
Phenobarbital
(120 mgkgday)
Gene names and fold
changes in a manuscript
table
(Hamadeh et al
2002a)
Vinclozin procymidone Male Spraguendash
Dawleymdashprostate
Clontech Atlas Rat 12
Toxicology array
200 mgkg Gene name accession
number average fold
change
(Rosen Wilson
Schmid amp Gray
2005)
Cisplatin Male Spraguendash
Dawley VAF1
albino (CRL
CD(SD) BR
Rat Tox Microarrays
were purchased from
Phase-1 Molecular
Toxicology
05 or 1 mgkgday Gene name accession
number fold change in
kidney
(Huang et al
2001)
Allyl alcohol
miodarone Aroclor
1254 arsenic
carbamazepine
carbon tetrachloride
diethylnitrosamine
dimethylformamide
diquat etoposide
indomethacin
methapyrilene
methotrexate
monocrotaline
3-methylcholanthrene
Male Spraguendash
Dawley
Affymetrix GeneChip
Test 2 Array
Allyl alcohol (40 mgkg
day) miodarone (100 mg
kgday) Aroclor 1254
(400 mgkgday) arsenic
(20 mgkgday)
carbamazepine (250 mg
kgday) carbon
tetrachloride (1000mgkg
day) diethylnitrosamine
(100 mgkgday)
dimethylformamide (1000
mgkgday) diquat (172
mgkgday) etoposide (50
mgkgday) indomethacin
(20 mgkgday)
methapyrilene (250 mg
kgday) methotrexate
(250 mgkgday)
monocrotaline (50 mgkg
day)
3-methylcholanthrene
(100 mgkgday)
Heatmap figures and a
table of Affymetrix
names for genes
correlated with clinical
chemistry changes in a
manuscript table
(Waring et al
2001)
(continued on next page)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6642
Table 1 (continued)
Compounds Rat strain Microarray type Compound dose Microarray data
availability
Reference
Microcystin-LR (MLR)
phenobarbital (PB)
lipopolysaccharide
(LPS) carbon
tetrachloride (CT)
thioacetamide (THA)
and cyproterone
acetate (CPA)
Male Wistar Purpose-made rat DNA
microarray (Affymetrix
Santa Clara CA)
containing 1600 rat
DNA sequences
Various Accession number gene
name fold changes
shown as a heat map
table in publicationmdash
data extraction would be
laborious
(Bulera et al
2001)
Acetamidofluorene
aniline bromobenzene
butyl hydroxytol
dieldrin disulfiram
ethinyl estradiol
hexachlorocyclohexane
g 4-methylthiazole
nimesulide piperonyl
butoxide precocene I
pulegone tannic acid
trans-anethole
Male Spraguendash
Dawley
Custom Rat MegaA
cDNA chip 3434-gene
Acetamidofluorene (200
mgkg) aniline (200 mg
kg) bromobenzene (900
mgkg) butyl hydroxytol
(1000 mgkg) dieldrin
(30 and 45 mgkg)
disulfiram (2000 mgkg)
ethinyl estradiol (500
mgkg)
hexachlorocyclohexane
gamma (40 65 80 mg
kg) 4-methylthiazole
(120 mgkg) nimesulide
(500 mgkg) piperonyl
butoxide (4000 mgkg)
precocene I (500 mgkg)
pulegone (400 mgkg)
tannic acid (3000 mg
kg) trans-anethole
(600 mgkg)
Gene name accession
number in a manuscript
table
(McMillian et al
2004)
NIEHS website at httpdirniehsnihgovmicroarraydatasetshome-pubhtm
EDGE website at httpedgeoncologywiscedu
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 43
and cisplatin (Table 1) In the majority of cases the resulting
number of differentially expressed genes is a very small
subset of the starting number on the microarray following
clustering or other types of analysis Upon closer examina-
tion of these publications the majority of them either
provide images of a heat map andor a table listing a gene
name accession number and expression change Very few of
the published studies (Tables 1ndash3) provide the original raw
microarray data file at a freely accessible website hence
restricting further analysis by scientists using other software
Although in some cases it is possible to cut and paste the
gene expression data tables from the publication pdf files
this is not always the case In the worst case scenario one
would have to manually retype gene lists or extract them
from heatmaps as binary type data As not all computational
researchers will have a laboratory available to them to
generate such quantities of toxicogenomics data the latter
points are important if we are going to continue to see
innovation in software development for this data This will
require free unrestricted access to data published Similarly
if we are to discern lsquolsquofingerprintsrsquorsquo for molecules acting with
a similar or identical mechanism we will need databases of
many diverse chemical structures that have been tested in a
similar manner There is therefore considerable interest in
the current databases being developed by the NIEHS FDA
and other groups which should help to improve the situation
by providing freely accessible microarray and other toxicity
related data Two of these databases being developed in the
public domain are Chemical Effects in Biological Systems
(CEBS) (httpwwwniehsnihgovnctcebshtm) (Mattes
Pettit Sansone Bushel amp Waters 2004 Waters et al
2003) which will accommodate gene expression profiles
proteomics and metabolomics data and allow complex
queries (Hood 2003a Mattes et al 2004) Similar goals
are being pursued in the development of the ArrayTrack
database at the FDA (Tong et al 2003) The EDGE
database (httpedgeoncologywisceduedgephp) an
expanding public effort at The University of Wisconsin
contains mouse gene expression profiles following treat-
ment with different toxic molecules (Hayes et al 2005
Thomas et al 2001) These separate efforts if widely
adopted should make published studies describing HT data
more readily accessible although it might have been more
efficient to evolve these into a single global database instead
of fragmented repositories
Proteomics data has also been generated in a limited
number of toxicology studies (Table 4) once again this has
been produced with different strains of rats and mice using
different protein chips 2-D gel electrophoresis (2-DIGE)
and mass spectroscopy methods (eg MALDI-MS) The
proteomic data is very rarely accessible to the reader for
their own computational analysis Subsequently there have
Table 2
Literature toxicogenomics data derived from mouse in vivo studies
Compounds Mouse strain Microarray type Compound dose Microarray data
availability
Reference
Benzene Male and female P53
KO mice and
C57BL6
Affymetrix and Incyte
GEM system
300 ppm 6 h per day 5
days a week for 2 weeks
Gene name accession
number and fold change
data in a manuscript
table
(Yoon et al 2003)
Benzene Male 129SvJ Affymetrix MG-U74Av2 100 ppm 6 h per day 5
days a week for 2 weeks
Gene name accession
number and fold change
data in a manuscript
table
(Faiola Fuller
Wong amp Recio
2004)
Phenobarbital CAR and wild type NIEHS Mouse tox chip
8736 genes
100 mgkg for 12 h Accession number gene
name and fold change
for wild type and knock
out mice in a manuscript
table
(Ueda et al 2002)
Aroclor BNF
ciprofloxacin cobalt
chloride TCDD IL-6
LPS PCB-153
phenobarbital
phenylhyrzn TNFa
WY-16463
C57BL6J Custom array with 1200
cDNAs
Aroclor (200 mgkg)
BNF (5 mgkg)
ciprofloxacin (250 mg
kg) cobalt chloride (60
mgkg) TCDD (10 ug
kg) IL-6 (25 ugkg)
LPS (1 mgkg)
PCB-153 (80 mgkg)
phenobarbital (100 mg
kgday) 3 days
phenylhyrzn (100 mg
kg) TNFa (50 ugkg)
WY-16463 (0125wv)
Gene names and fold
change as a heat map
Data is also available in
the EDGE database
(Thomas et al
2001)
TCPOBOP CD-1 female Custom 9000 cDNA
mouse array
1ndash3 h treatment 3 mg
kg body wt
Accession numbers and
fold change data in a
manuscript table
(Locker et al
2003)
3H-12-dithiole-3-thione
(D3T)
Male wild-type and
nrf2-disrupted
Affymetrix murine
genome U74Av2
GeneChip
05 mmolkg Accession number gene
name fold in a
manuscript table
(Kwak et al
2003)
MDMA Male albino Swissndash
Webster (neurons)
15 K mouse cDNA clone
set
47 mgkg Gene name and fold
change in a manuscript
table
(Xie et al 2004)
Phenytoin Female C57BL6 and
LDLRMurine genome-U74Av2 300 mgl Gene name accession
number and fold change
in a manuscript table
(Trocho et al
2004)
Genistein (1000 Agmouseday) or
diethylstilbestrol
(DES) (50 Agmouse
day)
ICR (testes) Custom cDNA
microarray containing
1754 cDNA probes
Genistein (1000 Agmouseday)
diethylstilbestrol (50 Agmouseday)
Accession number gene
name and fold change in
a manuscript tablemdashnote
very few genes
(Adachi et al
2004)
Cocaine and
buprenorphine
Male ICR Mouse DiscoveryArrayi
type I array containing
2688brain-derivedprobes
(Display Systems Biotech
Inc Copenhagen
Denmark)
40 mg kgmdash1 cocaine
once a day for 4 days 40
mg kgmdash1 cocaine plus
025 mg kgmdash1 BUP for
4 days
Data apparently not
available
(Hayase
Yamamoto
Yamamoto Muso
amp Shiota 2004)
Methamphetamine Male C57BLJ6
(striatum)
Affymetrix mouse
genechip mg-U74Av2
oligonucleotides arrays
12 488 genes
40 mgkg Gene accession number
gene ID and name and
signal log ratio in a
manuscript table
(Thomas
Francescutti-
Verbeem Liu amp
Kuhn 2004)
Di(2-ethylhexyl)
phthalate
Male C57BL6 Murine genome U74Av2
Arrays (MGU74Av2)
10 dietary DEHP for
13 weeks
Genbank accession
numbers gene name log
ratio data present as a bar
chart in the publication
data extraction would
take some effort
(Wong amp Gill
2002)
Diethylhexylphthalate Male PPARa null and
wild type
Custom made containing
600 tox genes
1150 mgkgday Gene names available on
a bar chartmdashquantitative
data not easily accessible
(Hasmall et al
2002)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6644
Table 2 (continued)
Compounds Mouse strain Microarray type Compound dose Microarray data
availability
Reference
Acetaminophen C57B16 3 129Ola
hybrid
Test-2 Chips (Affymetrix)
then individual
oligonucleotide
microarrays (Mul1K sub
A and sub B Affymetrix)
that can detect the
expression of 11000
known genes and
expressed sequence tags
(ESTs)
300 mgkg Genbank or SwissProt
ID and fold change data
in a manuscript table
(Reilly et al
2001)
Cadmium chloride
benzo(a)pyrene (BaP)
and trichloroethylene
(TCE)
Male Swiss Webster Custom Chips
containing 148 unique
genes
Various Data in table form few
genes affected for BAP
and TCE
(Bartosiewicz
Penn amp Buckpitt
2001)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 45
been only a very small number of studies that have
combined both transcriptomic and proteomic methods with
a single animal strain after treatment with a drug Hopefully
we will see this change in the future but this will in turn
present considerable challenges as huge amounts of
proteomic data are combined with the equally large
transcript data files
3 Network analysis and databases
From some of the early reviews of systems biology there
has been discussion of its application to drug discovery
(Kitano 2002ab) as well as the utility for ADMETox
(Ekins et al 2002a 2000b) More recently several other
journals have dedicated whole issues to the field of systems
biology However one could consider this quite a broad field
from network or pathway analysis to quantitative simulation
of organelles (Vo Greenberg amp Palsson 2004) whole cells
and organs It is apparent that we are now understanding
organisms from the perspective of computationally gener-
ated networks of protein and ligand interactions (Barabasi amp
Oltvai 2004) Network and pathway tools enable the
analysis of HT data in the context of all known interactions
when using a database as the source Individual reviews
have in some cases indicated that networks will be valuable
for understanding adverse events (Hood amp Perlmutter
2004) drug target identification or validation (Butcher
Berg amp Kunkel 2004) and complex metabolic interactions
(Nicholson et al 2004) A general schematic has been
generated in order to provide a description of the utilization
of such pathway databases and network building algorithms
from the initial parsing of high throughput data to network
comparisons and visualization (Fig 3) High throughput
data can be superimposed and visualized on the various
protein interaction databases available This is accomplished
by using either preset maps that capture current biological
knowledge or by building custom interaction networks
using many different algorithms which can be compared and
statistically evaluated as demonstrated in a very large
number of published examples (Dobrin Beg Karabasi amp
Oltvai 2004 Fiehn 2001 Han et al 2004 Hanisch et al
2002 Ideker et al 2002 Jeong Mason Barabasi amp Oltvai
2001 Jeong Tombor Albert Oltvai amp Barabasi 2000 Li
et al 2004 Milo et al 2002 Nikitin Egorov Daraselia amp
Mazo 2003 Pereira-Leal Enright amp Ouzounis 2004
Rives amp Galitski 2003 Segal et al 2003a Somogyi et al
2001 Spirin amp Mirny 2003 Tornow amp Mewes 2003
Vasquez Flammini Maritan amp Vespignani 2003 Yeger-
Lotem amp Margalit 2003 Yu Zhu Greenbaum Karro amp
Gerstein 2004)
31 Network applications
For example one group has used as an inference the
Bayesian network method for analysis of tissue toxicity from
microarray data as well as a mechanistic simulation for a
different pharmaceutically relevant molecule (Aksenov et
al 2005) Pathway tools and various resources have also
been applied to modeling the networks of nuclear hormone
receptors and their connections with other genes and small
molecules using a manually curated database MetaDrug
(Ekins Kirillov Rakmatulin amp Nikolskaya 2005d) or
MetaCore (Ekins Bugrim Nikolsky amp Nikolskaya 2005)
Transcriptional regulation of many transporters CYPs and
phase II enzymes are regulated by these receptors affecting
endogenous molecule transport metabolism cell growth
proliferation and oxidative stress (Ulrich 2003 Ulrich et al
2004) When the signaling networks and interacting ligands
for the transcriptional factors PPAR FXRRXRA ESR1
AHR HNF4A GCR-h MCR CAR-beta GCR-a LXR-a
CARRXR HNF4 FXR PXRRXR heterodimer PXR
AHRARNT heterodimer PPARaLXRa VDR PPAR-a
are visualized a very complex picture of interactions can be
created (Ekins et al 2005d) This suggests that when we
consider a molecule binding with only one nuclear receptor
we are observing only a fraction of the likely possible
feasible interactions based on the data gathered to date
Table 3
Literature toxicogenomics data derived from in vitro cell studies
Compounds Cell type Microarray type Compound dose Microarray data
availability
Reference
4-Hydroxytamoxifen
estrogen
MCF-7 breast cancer NIEHS ToxChip
microarray consisting of
1901 genes
1 uM hydroxytamoxifen
for a year 10 nM
17b-estradiol
Data available at NIEHS
website
(Hodges et al
2003)
Trovafloxacin Human hepatocytes Affymetrix U133A array 30ndash800 uM 142 genes available in
supplemental tablemdashnot
easily extracted
(Liguori et al
2005)
Estrogen MCF-7 breast cancer NIEHS ToxChip
microarray consisting of
1901 genes
10ndash10 M 17b-estradiol Data available at NIEHS
website
(Lobenhofer et al
2002)
Valproic acid NMRI mice embryo
and P19 mouse
embryocarcinoma
Custom chip including
15K mouse cDNA clone
set
600 mgkg body weight Gene symbol gene
name NIA EST log fold
change in a manuscript
table
(Kultima et al
2004)
Sulindac sulfide Human colorectal
carcinoma SW-480
and HCT-116
NIEHS human 12K chip 10 uM Genbank accession
number gene name and
fold change at various
time points in a
manuscript table data
also available on NIEHS
website
(Bottone
Martinez Collins
Afshari amp Eling
2003)
17Beta-estradiol estriol
estrone genistein
diethylstilbestrol
bisphenol A
nonylphenol
methoxychlor
MCF-7 U95A oligonucleotide
probe arrays (Affymetrix
10 nM (E2 estriol
estrone DES) 10 AM(genistein bisphenol A
nonylphenol and
methoxychlor)
Unigene name gene
name and fold change
for estrogen responsive
and nonresponsive in a
manuscript table
(Terasaka et al
2004)
Ouabain lauryl sulfate
dimethylsulfoxide
cycloheximide
tolbutamide sodium
fluoride diethyl
maleate buthionine
sulfoxamine
potassium bromate
sodium selenite
alloxan adriamycin
hydrogen peroxide
HepG2 Clontech Atlas Human
Stress Toxicology cDNA
arrays (234 genes)
Ouabain (43 uM) lauryl
sulfate (260 uM)
dimethylsulfoxide (128
M) cycloheximide (625
uM) tolbutamide (128
mM) sodium fluoride (3
mM) diethyl maleate
(125 mM) buthionine
sulfoxamine (30 mM)
potassium bromate (25
mM) sodium selenite (30
uM) alloxan (130 mM)
adriamycin (40 uM)
hydrogen peroxide
(4 mM)
Gene name ratio p -
value in downloadable
tables at journal website
(Morgan et a l
2002)
Aflatoxin B(1) (AFB(1))
2-acetylaminofluorene
(2AAF)
dimethylnitrosamine
(DMN)
acetaminophen (APAP)
HepG2 and primary
hepatocytes
Gene filter arrays
containing 31000 genes
10 uM aflatoxin B1
40 mM acetaminophen
100 uM
dimethylnitrosamine
10 uM
2-acetylaminofluorene
None (Harris et al
2004)
Mitomycin C (MMC)
and cisplatin (CIS)
and an alkylating
agent methyl
methanesulfonate
(MMS)
indirect-acting
genotoxins included
hydroxyurea (HU) a
ribonucleotide
reductase inhibitor
taxol (TXL) a
microtubule inhibitor
and etoposide
(ETOP)
L5178Y TK(+-)
mouse lymphoma
Affymetrix mouse
MG-U74A for MMC
and MG-U745Av2
(Affymetrix Inc Santa
Clara CA) for all the
other chemicals a total
of 9977 probe sets
(genes or ESTs) common
to these two array
models
Low mid and high doses Accession number gene
name gene ID statistical
significance at each time
point in a manuscript
table
(Hu et al 2004)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6646
Table 3 (continued)
Compounds Cell type Microarray type Compound dose Microarray data
availability
Reference
Hydroxyurea
(a carcinogen)
p-anisidine
(a noncarcinogen)
and paclitaxel
L5178Y Tk_-mouse
lymphoma
The Twin - Chip Mouse-
74K Digital Genomics
cDNA microarray
10 ngml paclitaxel 313
ugml hydroxyurea 32
ugml p-anisidine
Gene symbol and fold
change in a manuscript
table
(Lee et al 2003)
Acetaminophen
amiodarone clofibrate
erythromycin estolate
isoniazid alpha-
naphtylylisothiocyanate
beta-naphtoflavone
4-pentenoic acid
phenobarbital
tetracycline and
zileuton
Wistar Rat
hepatocytes
DualChip rat hepato
(Eppendorf Hamburg
Germany)
A single concentration
which varied for each
compound
Accession numbers and
gene namemdashfold
changes shown as
colored heat mapmdashnote
easily extracted from
publication
(de Longueville
et al 2003)
Bleomycin and hydrogen
peroxide
Mouse lymphoma
L5178Y TK(+ -)
Clontech Mouse 12K
cDNA microarray (1185
genes)
Bleomycin (25 and 20
ugml) hydrogen peroxide
(5 and 10 ugml)
Gene names and fold
change presented as bar
charts in publication
(Seidel Kan
Stott Schisler amp
Gollapudi 2003)
Bupivicaine
camptothecin
HL-60 Agilent human cDNA
microarray
1 mM Gene name GenBank
accession number
unigene and ratio in a
manuscript table
(Unami
Shinohara
Ichikawa amp Baba
2003)
Bisphenol A Mouse Sertoli TTE3 IntelliGene mouse
expression glass
microarrays (Version
10 Takara Shuzo)
which were spotted with
564 cDNA fragments of
mouse known genes and
approximately 301
expressed sequence tags
(ESTs)
0ndash400 uM Gene name GenBank
accession number and
fold change at time
points in a manuscript
table
(Tabuchi amp
Kondo 2003)
Mitomycin C or
doxorubicin
Hep G2 85 human gene custom
array
10 um mitomycin C
2 um doxorubicin or
2 ethanol
Bar charts with fold
changes in publication
very few genes
(Hong Muller amp
Lai 2003)
Amphotericin B Human peripheral
blood mononuclear
and THP-1
GF211 FKnown Genes_
Genefilter cDNA array
(ResGen) this array
consists of gt4000
individual elements
each representing a
known human gene
5 ugml Accession numbers and
fold expression in a
manuscript table
(Cleary Rogers amp
Chapman 2001
Rogers Pearson
Cleary Sullivan
amp Chapman
2002)
Benzo(a)pyrene diol
epoxide
TK6 human
lymphoblastoid
Human-350 microarray
a glass slide with 350
spotted human cDNA
probes (Phase-1
Molecular Toxicology
0 001 010 or
10 ugml)
Gene names and fold
change at doses in a
manuscript table
(Akerman et al
2004)
Etomoxir HepG2 Clontech AtlasiHuman Stress
Toxicology cDNA arrays
(234 genes)
1 mM etomoxir Gene names and fold
change in a manuscript
table
(Merrill et al
2002)
Tetrodotoxin Human glioma cell
line HTB-138
Using Affymetrix
GeneChip (HG-U133A
10 and 20 mM Affymetrix ID Genbank
ID gene name gene
symbol and fold change
in a manuscript table
(Raghavendra
Prasad Qi
Srinivasan amp
Gopalakrishnakone
2004)
Methotrexate
mercaptopurine
Human acute
lymphoblastic
leukemia
Affymetrix U133A chip Low and high dose and
combination
Data available as
supplemental data online
(Cheok et al 2003)
Prednisolone
vincristine
asparaginase
daunorubicin
Human acute
lymphoblastic
leukemia
Affymetrix U133A chip Various Data available as
supplemental data online
(Holleman et al
2004)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 47
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6648
A second study has indicated how a natural language
processing method CCNet was used to show the genes
regulated by the nuclear hormone receptor FXR (Apic
Ignjatovic Boyer amp Russell 2005) These automated
methods enable a more complete understanding of the
complexity of the transcriptional factors (Ekins Mirny amp
Schueltz 2002b Plant 2004 Ulrich 2003) but ultimately
rely on the quality of the content of the underlying database
of literature interactions This is a key consideration that is
often overlooked For example the gold standard database is
one that is manually curated to ensure the fidelity of the
direct interaction and is preferable to one generated
computationally by algorithms like natural language pro-
cessing (Nikolsky Nikolskaya amp Bugrim 2005) The
advantage of interaction networks over clustering has been
demonstrated in one study using MetaCore (Nikolsky
Ekins Nikolskaya amp Bugrim 2005) by reanalysis of a
published microarray study of G0-arrested MCF-7 breast
cancer cells treated with estrogen and 4-hydroxytamoxifen
(Hodges et al 2003) After producing integrated gene
networks for each treatment strikingly different patterns
were displayed although both contained early transcriptional
factors myc jun and fos Only the estrogen network featured
induced genes essential for all cell cycle phases (Nikolsky et
al 2005) Similarly microarray data for benzene toxicity
(Yoon et al 2003) has been re-analyzed by focusing on the
genes assessed on the p53 pathway (Ekins et al 2005e) We
envisage that a database of such networks for toxic
compounds will be used for comparing between different
molecules and used in the development of predictive
algorithms for Systems-ADMETox modeling in future
Another approach to using such pathway approaches is to
visualize the results of quantitative structure activity models
for predicting molecules binding to enzymes transporters
receptors and ion channels (Ekins Andreyev et al in press
2005e)
It is also possible to simultaneously interpret high
throughput data and predictions on interaction networks
providing a novel approach to predicting and understanding
potential undesirable drugndashdrug or off target effects in the
area of systems pharmacology An example data set uses
percent inhibition data for clotrimazole and ticonazole
which were screened against many different assays at a
single concentration in a commercially available database
BioPrint (Cerep Redmond WA) as published recently
(Fliri Loging Thadejo amp VOlkman 2005) The data for
10 assays has been arbitrarily encoded as inhibitors (gt50
inhibition) or non-inhibitors (lt50 inhibition) in a text file
which was then loaded into MetaCore The analyzed
network algorithm was then used which generates a large
network and fragments it into sub-networks each with a Z-
score and p-values for ranking according to saturation with
objects from the initial gene list The Gene-ontology
processes are also mapped to the gene list and individual
networks In this example a statistically significant network
was generated for the different proteins (Fig 4
p =2838e31) This network also maps the Gene Ontology
processes for the activation of MAPK (118 of genes p
value 9143e07) signal transduction (333 1600e05)
regulation of transcription DNA-dependent (294
2786e04) regulation of inflammatory response (39
3746e04) and the regulation of blood pressure (78
4230e04) This example network indicates how molecules
of the same or different therapeutic classes could be
evaluated for their effects as a graph either together as in
this case or individually This would be useful to indicate
potential off target effects and identify structurally dissimilar
molecules with similar network patterns Such networks
could then be compared to assess network overlap or
differences between molecules and their inhibition of
multiple proteins This type of unique visualization of high
throughput screening data illustrates how the target proteins
may be connected as a network to infer the possible
downstream effects of inhibition
With the burgeoning number of freely available online
and commercial databases that can be used for pathway
construction numbering in the hundreds there have been
suggestions to impose standards for model exchange
querying and visualization (Cary Bader amp Sander 2005)
To date there has been little discussion with regards to
standardization of ADMETox related databases although
there has been considerable discussion relating to drug
metabolism database generation (Erhardt 2003) This is
certainly an important area to address in the future There is
already a growing literature related to ADMETox that is
partially captured in the several commercially available
databases (Ekins et al 2005e) but to date there have been
limited academic efforts to capture data for transporters
with the human membrane transporter database (Yan amp
Sadee 2000) TP-search transporter database (httpwww
ilabrisewasedaacjptp-search) drug interaction database
(httpwwwdruginteractioninfoorgDatabaseinfoaspx)
nuclear hormone receptors (Nakata Yukawa Komiyama
Nakano amp Kaminuma 2002) the ADME-AP database
(Sun Ji Chen Wang amp Chen 2002) and PharmaGKB
(Oliver et al 2002) DSStox (httpwwwepagovnheert
dsstox) TOXNET (httptoxnetnimnihgov) that are
readily accessible
In order to generate accessible pathways using any of
the available software a large enough set of object
identifiers are required to map onto the underlying data-
base To demonstrate this datasets from toxicogenomics
studies have been evaluated with both the KEGG pathway
database and a commercially available product MetaCore
(wwwgenegocom) These gene or protein lists range in
size from 21 to 1853 objects In virtually all cases more
identifiers are mapped to networks in MetaCore and this
also seems independent of the identifier type used
(Unigene Affymetrix Genbank or Locuslink Table 5)
These numbers will obviously change as more information
is added to the respective databases hence the more
objects that are mapped from a dataset The more extensive
Table 4
Literature derived toxicoproteomics data
Compounds Data source Microarray type Compound dose Microarray data
availability
Reference
Carbon
tetrachloride
Male Wistar rats Affymetrix rat chip 8799
probes+proteomics
study
6ndash24 h exposure Genbank SwissProt and
binary data in a
manuscript table
(Fountoulakis
2004)
Paracetamol CD-1 male mice Custom mouse tox blots
with 450 genes
RTQ-PCR+proteomics
study 2-DIGE+
MALDI-MS
150 or 500 mgkg Gene name IMAGE ID
GenBank accession
number fold induction
SwissProt identifier and
protein abundance
change in manuscript
tables
(Ruepp Tonge
Shaw Wallis amp
Pognan 2002)
Paracetamol AP-1 male mice Affymetrix murine 11K
set+proteomics study
Up to 500 mgkg Gene names and fold
changes at multiple
doses and time points in
a manuscript tablemdash
proteomics data not
accessible
(Coen et al 2004)
Oxazepam or
Wy-14643
Male B6C3F1 mice NIEHS Mouse Chip
(8700 genes) 2-DIGE
and MS
Oxazepam (2500 ppm)
Wyeth (Wy)-14643
(500 ppm)
Proteomics data in a
manuscript table
Genbank accession
number gene name and
fold change in a
manuscript table data
also available to
download from NIEHS
website
(Iida et al 2003)
Compound A
(PPAR gamma
ligand)
Female rats CrlCD
(SD)IGS BR
Proteomics 2-DIGE and
MS
250 mgkgday up to
5 days
Accession number and
protein name and
average ratio in a
manuscript table
(Meneses-Lorente
et al 2004)
Bromobenzene Male Wistar rats Proteomics 2-DIGE and
MS custom 3000 cDNA
rat chip
5 mmolkg Accession number gene
name fold change in a
manuscript table protein
names along with bar
charts
(Heijne Stierum
Slijper van
Bladeren amp van
Ommen 2003)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 49
the network that can be generated (as it will consist of more
nodes) then a more comprehensive understanding of the
networks is possible The data available currently in the
literature can be used to evaluate such pathway and
network generation tools Recently we have used several
of the published studies (Tables 1ndash3) with MetaCore to
visualize networks for acetaminophen furan carbon tetra-
chloride benzene and cisplatin showing genes involved in
In vivo
In vitro
In Silico
1970s 1980s 1990s 2000s
Systems Biology ()
OMICS
Fig 2 The timeline for major paradigms in ADMETox
oxidative stress (Ekins Giroux Nikolsky Bugrim amp
Nikolskaya 2005c)
Other important pathwaynetwork building tools that could
potentially be applied to toxicogenomics data include
Ingenuity pathways analysis (httpwwwingenuitycom)
PathArt (httpwwwjubilantbiosyscompdhtm) Pathway
Assist (httpwwwariadnegenomicscomproctspathway
html) (Nikitin et al 2003) and several other databases
deposited at the Pathway Resource List (httpcbiiomskcc
orgprl) All of these products have unique underlying
proprietary pathway databases which are compiled manually
or automatically with text mining tools or a combination of
both We are still waiting for studies that provide a comparison
of different database tools or network building algorithms
Until then theremay be someoverlap but also some differences
between the results obtained from more than one network
method due to the database content and the algorithms used
The reader is recommended to evaluate for themselves several
technologies and select those with the most appropriate fit to
their own specialized needs
In the next Sections the further application of some
available network and database tools will be described with
Microarray or other high throughput data
Upload list of geneprotein identifiers and fold change significance etc (raw data or after selection of the differentially expressed
Genes using other statistical methods)
Parse database and generate interaction network with different algorithms or visualize on maps
Filter Networks
Access significance of the interaction networks
Determine GO processes
Compare 2 or more networks intersection overlap etc
Export gene list
Export gene list
generate interaction network with different algorithms or visualize on maps
Visualization
Fig 3 Schematic for the utilization of pathway tools for assessing high throughput data
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6650
specific detailed reference to transporters and enzymes but
these technologies can also be applied elsewhere as
described above
Fig 4 Network for high throughput screening data for clotrimazole and ticona
cholecystokinin CCR1 Choline transporter Chloride channel catechol-O-methyl
and co workers (Fliri et al 2005) Nodes highlighted with large blue circles rep
molecules showing gt50 inhibition in the assay blue circles represents both mol
inhibits gt50 and one inhibits lt50 Ligands (large) linked to transcriptional f
(wwgenegocom) (For interpretation of the references to colour in this figure leg
311 The role of transporters
A diverse array of organic solutes such as nutrients
neurotransmitters and drugs are transported by specialized
zole screened against 10 in vitro assays (Human Cannabinoid 1 Human
transferase COX2 CYP1A2 CYP2B6 CYP2C19) data published by Fliri
resent assays used in the study Red circles on these nodes represent both
ecules showing lt50 inhibition chequered circles represent one molecule
actors enzymes and transporters via edges using the MetaCorei database
end the reader is referred to the web version of this article)
Table 5
Datasets acquired from publications and other sources and used to evaluate two databasepathway tools KEGG and metaCore
Dataset Number of objects
genes proteins in list
Number of objects
mapped with KEGG
Number of objects mapped
with MetaCore on networks
Reference for microarray
data gene list
Tox Chip 10 (NIEHS) 1853 405 1498 httpsdir-appsniehsnihgov
mapsguestclonesrchcfm
4-Hydroxytamoxifen and
estrogen
1617 434 1343 (Hodges et al 2003)
Mitochondrial proteins 722 156 388 (Gaucher et al 2004
Taylor et al 2003)
Bromobenzene
(24 and 48 h)
130 41 89 (Heijne et al 2004)
Acetaminophen 30 19 23 (Heinloth et al 2004)
Acetaminophen 84 29 64 (Huang et al 2004)
Furan 185 64 139 (Huang et al 2004)
Tetrodotoxin 116 31 86 (Raghavendra Prasad et al
2004)
Benzene 73 16 62 (Yoon et al 2003)
Benzene 76 5 53 (Faiola et al 2004)
Carbon tetrachloride 37 8 26 (Young et al 2003)
Estrogen 94 33 90 (Lobenhofer et al 2002)
Trovafloxacin 142 20 82 (Liguori et al 2005)
Phenobarbital 37 13 28 (Ueda et al 2002)
L-742694 (liver)+ 45 17 19 (Hartley et al 2004)
L-742694 (intestine)+ 23 11 10 (Hartley et al 2004)
A-277249 21 7 9 (Waring et al 2002)
All files were converted to Locuslink identifiers except as noted () unigene () Affymetrix (+) Genbank identifiers were used
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 51
proteins across cellular membranes These may function as
passive processes or active processes energized by the
hydrolysis of ATP or coupling to the co-transport of counter
ions down an electrochemical gradient such as Na+ H+ and
Cl There are many thousands of transporters which can be
classified into distinct superfamilies One of these the solute
carrier class (SLC) is rapidly expanding and contains over
30 families and 200 members The ATP-binding cassette
(ABC) contains 7 families and over 48 members including
P-glycoprotein (P-gp) and MRP subfamilies (Zhang Knipp
Ekins amp Swaan 2002a Zhang Phelps Cheng Ekins amp
Swaan 2002b) Transporters have a key role in clinical
pharmacology with many drugs specifically targeting them
Numerous drugs share transport pathways with nutrients
and transporters have a role in oral absorption drug
bioavailability drug resistance excretion and ultimately
pharmacokinetics and pharmacodynamics
Polymorphism of drug transporters may be a key factor
in drug interactions and lack of effectiveness This field has
become known as pharmacogenomics and is focused on
understanding of inherited DNA sequence variations (poly-
morphisms and mutations) revealed by xenobiotics (Evans
amp McLeod 2003 Weinshilboum 2003) Over the last few
decades many genes have been directly linked to the
mechanisms of response (Evans amp McLeod 2003 Wein-
shilboum 2003) such that 20ndash95 of variability to drug
response is inheritable (Evans amp McLeod 2003) This
phenotypical variability is mainly caused by single nucleo-
tide polymorphisms (SNPs) present in anywhere from 1 to
50 of the population resulting in either lower protein
activity incorrect folding or rapid degradation via proteo-
somes (Weinshilboum amp Wang 2004) A number of
structurally diverse molecules bind to P-gp which is
expressed in many tissues and has numerous SNPs one of
which (C3435T) affects the expression level in the
duodenum and therefore can impact the absorption of
molecules which would be substrates for this transporter
(Sakaeda Nakamura amp Okumura 2002) The human
proton-dependent dipeptide transporter (hPEPT1) can also
affect the absorption of molecules in the intestine and
recently 9 SNPs were found with only one displaying a
reduced transport capacity (Zhang et al 2004) The sodium-
dependent carnitine cotransporter OCTN2 can possess
mutations and these result in primary carnitine deficiency
which impacts fatty acid oxidation and is characterized by
many clinical manifestations (Lahjouji Mitchel amp Qureshi
2001) The organic cation transporter 1 (OCT1) is also
important in the transport of numerous xenobiotics and
endobiotics Recently 4 SNPs were identified in the
Japanese population and when functionally characterized
in vitro the uptake of cations was reduced significantly for
some of these mutations indicating that this would likely
contribute to inter-individual variations in metabolism of
drugs which were transported via OCT1 (Sakata et al
2004)
3111 Clinical relevance of transporters The pregnane
X-receptor (PXR) is a transcriptional regulator of the
enzyme human MDR1 (P-gp) MRPs and OATP (Wang amp
LeCluyse 2003) CYP3A (Bertilsson et al 1998 Blumberg
et al 1998 Kliewer et al 1998) and CYP2C89 (Synold
Dussault amp Forman 2001) as well as many other genes
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6652
involved in the transport metabolism and biosynthesis of
bile acids (Staudinger Liu Madan Habeebu amp Klaassen
2001) However the additional receptors CAR FXR LXR
and other nuclear receptors take part in a complex network
of interactions to control these and other proteins Thus
elucidation of the regulatory networks which control the
expression of these transporters is also important To date
most of the research has centered on efflux transporters but
there has been considerable interest in uptake transporters
such as the organic anion transporter polypeptide (OATP
((Kim 2003) see also later Section on OATP)
There are several specific examples of the importance of
drug transporters to the clinical development of drugs One
example is the insulin sensitizer troglitazone which was
withdrawn due to hepatotoxicity although the precise
mechanism appears to have been unclear until recently
The major metabolite is a sulfated species and is suspected
of being responsible for the observed toxicity The recent
assessment of the organic anion transporting polypeptides
OATP-C and OATP8 expressed on the hepatocyte baso-
lateral membrane indicated that sulphated troglitazone has a
high affinity for the former and possibly lower affinity for
the latter (Nozawa et al 2004) This metabolite would
therefore be expected to accumulate in hepatocytes and
inhibit the bile salt export pump and Mrp2 Because
polymorphisms have been shown for OATP-C (Tirona
Leake Merino amp Kim 2001) it is also possible that these
may result in the accumulation of the metabolite and in turn
elicit idiosyncratic toxicity A second relevant example of
the impact of transporters is the clinically significant drugndash
drug interaction between cerivastatin and cyclosporine A
which occurs via the OATP-C transporter (Shitara Itoh
Sato Li amp Sugiyama 2003) A third example are the HIV
protease inhibitors saquinavir ritonavir and indinavir which
are transported by MRP2 in vitro and other drugs such as
probenecid and sulfinpyrazone are able to enhance this
transport Transport by MRP2 suggests that these com-
pounds will have decreased bioavailability due to increased
clearance and other drugs could aggravate this situation by
further enhancing transport (Huisman et al 2002) Sim-
ilarly the rifampicin mediated induction via PXR of MRP2
and P-gp in healthy subjects was found to significantly
decrease the AUC and also correlated with intestinal
expression of these transporters This transporter is also
inducible by cisplatin 2-AAF and phenobarbital (Schrenk
et al 2001) indicating multiple mechanisms may be
involved In other species such as rat commonly used as a
toxicity model orthologs of the transporters such as OATP2
are expressed and can be induced with ligands for PXR like
PCN (Guo Choudhuri amp Klaassen 2002) This is useful
knowledge because the advent of microarray technology
allows one to dose a rat with a xenobiotic and assess
thousands of genes simultaneously in a particular tissue For
instance animals dosed with known nephrotoxins have
shown some upregulation of the NandashKndashCl transporter
however the authors suggested genomic responses are
stronger soon after exposure before declining (Fleck et
al 2003) Some transporters may therefore be specifically
targeted by drugs in one tissue such as the CNS but these
same transporters may also be expressed elsewhere in the
body hence off-target effects may result in toxicity The
serotonin transporter is one such example which is
expressed in the lungs and brain Some substrates for this
channel like fenfluramine can result in primary pulmonary
hypertension as they accumulate in pulmonary cells (Roth-
man Ayestas Dersch amp Baumann 1999) Similarly P-gp is
expressed at the blood brain barrier and intestine impacting
the efficacy and bioavailability of drugs
3112 Transporter network examples ABCA1 The
ABCA1 transporter mediates the first step of cholesterol
transport Mutations in this gene cause Tangier disease
which results in severe HDL deficiency cholesterol
accumulation in macrophages and attendant atherosclerosis
This transporter represents a drug target for upregulation
modulating cholesterol metabolism and prevention of
cardiovascular disease (Oram amp Lawn 2001) In vitro
ABCA1 can be inhibited by the sulfonylurea glybenclamide
(Field Burn amp Mathur 2004 Wang Silver Thiele amp Tall
2001) In order to illustrate the advantages of mapping drug
transporters as networks onto functional models alongside
other proteins one can consider the example of ABCA1
The query of the MetaCorei database shows that this
transporter appears on three manually curated pathway maps
representing the Fstate of the art_ knowledge derived from
reliable high quality literature sources One can also use the
individual maps as an interface to access the underlying
layers of information about the transporter including the list
of encoding genessplice variants with known SNPs In
addition to browsing MetaCorei a user can also build
custom networks around ABCA1 using the network-
construction tool (Fig 5) Such a visualization utility may
be very helpful for identification of all putative pathways
around a particular transporter or compound of interest The
ABCA1 network created by this tool shows that this
transporter at the time of writing is linked directly to
twenty-five other objects such as APOE1 and LXR Many
of its neighbors have their own SNPs that could be
important in determining interactions between transport of
a drug and normal human transport of endogenous ligands
in health or disease
3113 Transporter network examples OATP The
OATPs are key membrane bound transporters expressed in
many organs including intestine liver lung choroid plexus
blood brain barrier and other organs (Tamai et al 2000)
This family of transporters is capable of mediating the
sodium-independent transport of a diverse array of mole-
cules such as steroid conjugates organic anions and
xenobiotics by coupling uptake with efflux of bicarbonate
(Satlin Amin amp Wolkoff 1997) glutathione or its
conjugates (Hagenbuch amp Meier 2004) The inhibition of
Fig 5 The network of direct gene interactions around the ABCA1 transporter gene (centre) generated with MetaCorei The interation types between nodes are shown as small colored hexagons eg unspecified
allosteric regulation binding cleavage competition covalent modification dephosphorylation phosphorylation transcription regulation transformation When applicable interactions also have a positive or
negative effect and direction Ligands (purple) linked to other proteins (blue blobs) transfactors (red) enzymes (orange) (For interpretation of the references to colour in this figure legend the reader is referred to
the web version of this article)
SEkin
sJournalofPharm
acologica
landToxico
logica
lMeth
ods53(2006)38ndash66
53
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6654
this transporterrsquos hepatic uptake of other compounds may be
important for reported drugndashdrug interactions (Kim 2003)
described earlier (Shitara et al 2003) as well as cerivastatin
with gemfibrozil (Shitara Hirano Sato amp Sugiyama 2004)
OATP1B1 (previous names OATP-C LST-1 OATP2
SLC21A6) represents the most studied human OATP to
date (Meier amp Stieger 2000) and is expressed on the
basolateral plasma membrane of hepatocytes Several single
nucleotide polymorphisms have been identified in the
OATP1B1 gene in European-Americans African-Americans
(Tirona et al 2001) and Japanese (Nozawa et al 2002)
dramatically impacting the transport of ligands such as
pravastatin (Mwinyi Johne Bauer Roots amp Gerloff 2004
Nishizato et al 2003) estrone-3-sulfate (Nozawa et al
2002 Tirona et al 2001) Rifampin (Tirona Leake
Wolkoff amp Kim 2003) and estradiol 17h-d-glucuronide(Tirona et al 2001)
The regulation of SLCO may be affected during
extrahepatic cholestasis bile duct ligation bile salt induced
Fig 6 Networks generated with the autoexpand algorithm in MetaCorei to illustr
be interconnected with other protein regulatory signaling information Informa
unspecified allosteric regulation binding cleavage competition covalent m
transformation When applicable interactions also have a positive or negative e
transfactors (red) enzymes (orange) (For interpretation of the references to colour
cholestatic hepatitis (Dumont et al 1997 Meier amp Stieger
2000 Rost et al 2003) and primary sclerosing cholangitis
(Oswald Kullack-Ublick Paumgarter amp Beuer 2001) In
particular OATP1B1 appears to be regulated by the liver-
enriched transcription factor hepatocyte nuclear factor 1a
(HNF1a) which binds to the promoter region of this
transporter (Jung et al 2001) Site directed mutagenesis
of this binding site resulted in inactivation suggesting the
critical nature of the interaction with HNF1a Bile acids
such as CDCA have been shown to transcriptionally repress
HNF1a in vitro via inhibition of the transactivating effect of
HNF4a on HNF1a (Jung amp Kullak-Ublick 2003) After
screening many rat and human uptake transporters in vitro
OATP1B1 was also shown to modulate the PXR response
by controlling rifampin retention in the cell and therefore
affecting the induction of CYP3A4 and other gene products
such as P-gp (Tirona et al 2003)
Some of the literature for OATP1B1 human substrate
data has been annotated into MetaCorei to illustrate the
ate how ligands for the human OATP-C (OATP1B1) transporter (centre) can
tion on the type of interaction between objects is hidden for clarity eg
odification dephosphorylation phosphorylation transcription regulation
ffect and direction Ligands (purple) linked to other proteins (blue blobs)
in this figure legend the reader is referred to the web version of this article)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 55
visualization of the complex interconnections between this
transporter its ligands regulatory factors and signaling
molecules already in the database (Fig 6) The network was
generated with the autoexpand algorithm in the software
representing one of multiple available algorithms for
connecting genes ligands and other objects in the database
Clearly if more ligands and their connections are added to
the database the complexity of the network will increase
considerably The OATP-C (OATP1B1) gene details can be
viewed upon querying the database and links are provided
to other public databases This page can be used to highlight
the multiple synonyms for this gene as well as links to the
multiple SNPs identified to date
3114 Transporter microarray data Microarrays have
generally been limited in the number of transporters present
on them (Annereau et al 2004) however they have been used
in an attempt to correlate pharmacokinetic properties with
gene expression for valacyclovir (Landowski et al 2003) as
well as understand the expression profile in different tissues
or cell lines upon food component or xenobiotic treatment
(Anderle Huang amp Sadee 2004) This lack of transporters
on microarrays has prompted some groups to produce their
own arrays with a heavier emphasis on transporters These
arrays have for example then been used to demonstrate the
upregulation of ABC transporters and down-regulation of
GST-Pi in cell lines resistant to colchicines or 9-nitro-
camptothecin (Annereau et al 2004) The genes that were
significantly up or down-regulated in this particular study
were used to build networks with MetaCore (Fig 7A B) and
the similarities between them were assessed (Fig 7C)
Although there were only a small number of significantly
changed genes in common (IL-8 Fos GST-Pi Calpactin and
Ubiquitin hydrolase) it is perhaps likely that there is a much
larger common gene network that is important for drug
resistance although a much larger number of cell lines and
drug treatments need to be evaluated to produce a definitive
drug resistance signature involving transporters enzymes
and transcriptional regulators
312 Applications to enzymes
As we have already described it may be particularly
valuable to visualize enzymes as networks to show
interactions with transcriptional regulators and ligands
For example a key enzyme is CYP3A4 which metabolizes
40ndash45 of all drugs and has relatively few SNPs
(Ingelman-Sundberg 2004) Using a second software suite
MetaDrugi (wwwgenegocom) it is possible to construct
a custom network around this or other drug metabolizing
enzymes (Fig 8) In this case the gene network for
CYP3A4 highlights all of the major transcriptional
regulators and several more distant linked proteins and
ligands connected on the network that may be useful for
further study (Fig 8) Substrates inhibitors as well as
regulatory factors and other enzymes can be observed
connected on this network Due to the many hundreds of
known inhibitors and substrates only a small number are
shown here for clarity We assume that if a perturbation in
a pathway (eg due to a nonfunctional enzyme) is linked
to a certain pathologic condition a similar perturbation
caused by the interference from xenobiotic metabolism
(eg competitive inhibition of the same enzyme) may
result in identical effects As microarray gene expression
data is increasingly generated the role of enzyme
regulation in toxicity of certain xenobiotics will become
more apparent from either in vivo or in vitro studies The
visualization of such signature gene networks involving
transporters and enzymes their ligands and regulatory
factors will also be important for future toxicity prediction
methods We have recently generated visualizations of
microarray data from MCF-7 cells treated with 4-hydrox-
ytamoxifen to show that some of the key genes involved in
metabolism and transport are upregulated (Ekins et al
2005d) In addition we have made predictions with various
QSAR models in MetaDrug to indicate the involvement of
PXR CYP3A4 and P-gp (Ekins et al 2005e) Therefore it
appears likely that 4-hydroxytamoxifen could increase its
own metabolism as well as efflux from cells via P-gp
which can be visualized on networks Any decrease of
function of these enzymes or transporters in a population
would likely result in changes in the metabolism and
transport of this active metabolite potentially impacting
the clinical effect This represents one example of how
both pathway tools QSAR models and network building
algorithms can be used with different types of predicted
and experimental data to allow visualization of potential
compound interactions or toxicity
313 Future network applications
As the population ages an increasing incidence and
prevalence of systemic diseases especially chronic diseases
have occurred among older adults This has resulted in an
increase in medications used concomitantly by this
population which presents challenges for drugndashdrug
interactions Physiologically elderly patients may behave
differently to the younger patients for which the drugs are
initially developed for and tested on Many pharmacoki-
netic investigations in the elderly population reveal
decreased clearance of lipophilic drugs metabolized by
the cytochrome P450 enzymes however few studies have
evaluated aging-dependent or gender-related changes in
specific P450 enzymes (Hunt Westerkam amp Slave 1992)
Age-related physiological changes such as a reduction in
liver mass hepatic metabolizing enzyme activity liver
blood flow and alterations in plasma drug binding may
account for the decreased elimination of some metabolized
drugs in the elderly It is particularly difficult to separate an
effect of aging from the variation in the rate of metabolism
due to factors such as individual metabolic phenotype
(slow or fast metabolizer due to SNPs) environmental
influences concomitant disease states and drug intake
(drugndashdrug interactions) The available data suggest that
Fig 7 Networks for drug resistant cell lines generated with the Auto expand algorithm in MetaCore A KB-3-1 vs KB-8-5 B ATC-DU-145 vs RCO1 C node overlap of A and B with initial nodes highlighted
with blue or red solid circles (Initial gene lists derived from (Annereau et al 2004)) Information on the type of interaction between objects is hidden for clarity eg unspecified allosteric regulation binding
cleavage competition covalent modification dephosphorylation phosphorylation transcription regulation transformation When applicable interactions also have a positive or negative effect and direction
Ligands (purple) linked to other proteins (blue blobs) transfactors (red) enzymes (orange) (For interpretation of the references to colour in this figure legend the reader is referred to the web version of this article)
SEkin
sJournalofPharm
acologica
landToxico
logica
lMeth
ods53(2006)38ndash66
56
Fig7(continued)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 57
Fig7(continued)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6658
Fig 8 The network of gene interactions around the CYP3A4 enzyme (centre) from MetaCorei Information on the type of interaction between objects is
hidden for clarity eg unspecified allosteric regulation binding cleavage competition covalent modification dephosphorylation phosphorylation
transcription regulation transformation When applicable interactions also have a positive or negative effect and direction Ligands (purple) linked to other
proteins (blue blobs) transfactors (red) enzymes (orange) (For interpretation of the references to colour in this figure legend the reader is referred to the web
version of this article)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 59
the initial doses of drugs metabolized by these enzymes
should be reduced in older patients according to the clinical
response In most published studies the elderly appear at
least as responsive as the young to inducers or inhibitors of
P450s (Durnas Loi amp Cusack 1990) More recently there
has been some suggestion that there are age related
reductions in function of some specific P450s such as
CYP3A4 (Patki et al 2004) and this could occur at the
level of regulation However earlier studies with the same
enzyme showed no change in clearance with age (Hunt et
al 1992) To date there has been even less examination of
the transporter functions and any changes with age
(Kinirons amp OrsquoMahony 2004) so the current understand-
ing of the effects of aging on metabolism and transport is
anything but transparent This represents an extreme
challenge for the pharmaceutical industry how to predict
whether a drug has an affinity for an enzyme or transporter
and whether this may be clinically important if it shows a
decline in expression or function with aging Any decrease
of function of these enzymes or transporters in an elderly
population would result in changes in the metabolism and
transport of metabolites potentially impacting the clinical
effect This represents another example of how computa-
tional approaches may perhaps be used with different types
of predicted and experimental data to allow visualization of
potential compound interactions or toxicity in elderly
populations We may see different gene networks high-
lighted as humans age and these may be modified by drug
treatment and coadministration This represents an area
were network analysis could be applied in the future and is
in need of considerable further research
The collection of microarray data in databases such as
CEBS ArrayTrack and EDGE represents a future resource
for computational gene network analysis One could
envisage that ultimately in each case such data is converted
to one or more networks that are also displayed for the user
and can be used to compare treatments from in vivo and in
vitro experiments This would represent a different approach
to clustering the data as currently implemented in one of
these efforts (Hayes et al 2005) and may condense large
amounts of experimentally derived data into a readily
interpreted network
4 Discussion
Previously in this journal the progress of many
research groups in predicting human ADME parameters
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6660
in silico (Ekins et al 2000b) and approaches for drug
metabolism (Ekins Ring Grace McRobie-Belle amp
Wrighton 2000a) have been described Both of these
reviews commented on moving HT assays for ADME
Tox much earlier in drug discovery which had also been
indicated by other groups The initial reviews also
highlighted the likely wealth of data that would become
available and how this could be used for structure
activity relationships alongside the bioactivity data in
computational models It was noted that there was a
paucity of predictive metabolism tools at that time In
addition a growing number of efforts to model whole
cells and organs now a field called systems biology
were recognized as models that could be integrated with
the in silico ADME approaches In summary since these
past reviews virtually all pharmaceutical companies have
attempted earlier high throughput screening for ADME
Tox properties and to some extent the wider application
of computational approaches for physicochemical proper-
ties Systems biology is being quite widely acknowledged
as the new paradigm for understanding complex bio-
logical datasets derived from high throughput technolo-
gies and the accumulated knowledge on human protein
interactions (Hartwell Hopfield Leibler amp Murray 1999
Hood 2003b) Therefore systems biology can be defined
as the integration of genetic proteomic transcriptomic
and metabonomic data using computational methods
(Nicholson amp Wilson 2003) When taken together
information on molecular processes derived from different
sources represents a lsquolsquouniversersquorsquo of putative biological
functionality of which only a small fraction of it will be
realized in a cell at any given time To date systems
biology has been driven by academia and funding bodies
such as the NIH rather than the pharmaceutical compa-
nies Presently there is a great deal of interest from
scientists of all backgrounds in identifying the networks
of cellular pathways and the corresponding physically
interacting proteins
The network building software for systems biology
described in this current review will be valuable to query
high throughput data and known literature interactions in
order to predict potential toxicity in different species In the
future the compilation of published toxicogenomics data-
sets characteristic of different types of toxicity will likely
be available in these software systems to act as a reference
database It is also feasible that we will be able to generate
the annotated datasets which specifically address the
differences between human and rat networks implicated
in toxicity The identification of sub-network modules
conserved between human and rat distinct for toxicity
types or predictive for toxic end-points in human will be
possible Such signature gene networks (Nikolsky et al
2005) can then be verified with other experimentally
derived data prospectively or from preexisting databases
The combined Fhigh throughput datandashin silico network_approaches therefore represents a novel method for access-
ing complex tissue-level processes as networks integrating
all data types based on functional interactions The data-
bases developed and used in this approach will certainly
benefit from further annotation around the drug metaboliz-
ing enzymes and transporters as described herein in terms
of transcriptional regulation and the ligands associated with
them which frequently appear in the literature It will be
important to capture disparities in the assignment of ligands
to enzymes transporters and other proteins as well as
negative data
In summary although there have been numerous
toxicogenomics studies published there is presently a
relatively small number of datasets that are freely available
to perform network analysis of microarray data The
number of studies identifying large numbers of proteins
which are affected by molecule treatment are even scarcer
still while there are several examples of some published
studies that combine such data It is hoped that the
numerous database initiatives for high content and toxicol-
ogy data that are being undertaken will improve the
situation for other researchers that are not currently
equipped to do such microarray studies themselves The
addition of requirements by journals to deposit such raw
data in a freely accessible resource will aid these initiatives
ADMETox groups have seen new technologies and
approaches developed over the last decade that have all
been applied to identify poor compounds earlier (Fig 2)
The latest technologies integrate network building tools
with high content data and databases The current review
described the limited number of networks generated for
ADMETox at present and one hopes that the impact of
such analyses will be commonplace in the future Systems
biology is however more than just applying a network
approach and hence systems-ADMETox will have to
evolve due to the continual pressure to develop newer
technologies This current paradigm combining empirical
data and computational methods should integrate the
complex data already generated making it readily inter-
pretable and valuable for identifying the most promising
compounds in the future
Acknowledgements
Dr Maggie AZ Hupcey is gratefully acknowledged for
editorial assistance Dr Peter W Swaan (University of
Maryland) and Dr Cheng Chang (Ohio State University)
Dr Steve Wright (University of Arizona) Dr K Sandy
Pang (University of Toronto) and Dr Craig Giroux (Wayne
State University) are kindly acknowledged for their support
and discussions My colleagues at GeneGo Sergey
Andreyev Andy Ryabov Eugene Kirillov Eugene A
Rakhmatulin Svetlana Sorokina Andrej Bugrim Tatiana
Nikolskaya Yuri Nikolsky John Metz and Julie Bryant are
all thanked for their considerable contributions to software
development and data annotation
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 61
References
Abe M Yamashita S Kuramoto T Hirayama Y Tsukamoto T Ohta
T et al (2003) Global expression analysis of N-methyl-NV-nitro-N-nitrosoguanidine-induced rat stomach carcinomas using oligonucleotide
microarrays Carcinogenesis 24 861ndash867
Adachi T Ono Y Koh K B Takashima K Tainaka H Matsuno Y
et al (2004) Long-term alteration of gene expression without
morphological change in testis after neonatal exposure to genistein in
mice Toxicogenomic analysis using cDNA microarray Food and
Chemical Toxicology 42 445ndash452
Akerman G S Rosenzweig B A Domon O E McGarrity L J
Blankenship L R Tsai C A et al (2004) Gene expression profiles
and genetic damage in benzo(a)pyrene diol epoxide-exposed TK6 cells
Mutation Research 549 43ndash64
Aksenov S V Church B Dhiman A Georgieva A Sarangapani R
Helmlinger G et al (2005) An integrated approach for inference and
mechanistic modeling for advancing drug development FEBS Letters
579 1878ndash1883
Anderle P Huang Y amp Sadee W (2004) Intestinal membrane transport
of drugs and nutrients Genomics of membrane transporters using
expression microarrays European Journal of Pharmaceutical Sciences
21 17ndash24
Annereau J P Szakacs G Tucker C J Arciello A Cardarelli C
Collins J et al (2004) Analysis of ATP-binding cassette transporter
expression in drug-selected cell lines by a microarray dedicated to
multidrug resistance Molecular Pharmacology 66 1397ndash1405
Apic G Ignjatovic T Boyer S amp Russell R B (2005) Illuminating drug
discovery with biological pathways FEBS Letters 579 1872ndash1877
Baker V A Harries H M Waring J F Duggan C M Ni H A Jolly
R A et al (2004) Clofibrate-induced gene expression changes in rat
liver A cross-laboratory analysis using membrane cDNA arrays
Environmental Health Perspectives 112 428ndash438
Balakin K V Ekins S Bugrim A Ivanenkov Y A Korolev D amp
Nikolsky Y et al (2004a) Quantitative structurendashmetabolism relation-
ship modeling of the metabolic N-dealkylation rates Drug Metabolism
and Disposition 32 1111ndash1120
Balakin K V Ekins S Bugrim A Ivanenkov Y A Korolev D amp
Nikolsky Y et al (2004b) Kohonen maps for prediction of binding to
human cytochrome P450 3A4 Drug Metabolism and Disposition 32
1183ndash1189
Barabasi A -L amp Oltvai Z N (2004) Network biology Under-
standing the cellrsquos functional organization Nature Reviews Genetics
5 101ndash113
Bartosiewicz M Penn S G amp Buckpitt A (2001) Applications of gene
arrays in environmental toxicology Fingerprints of gene regulation
associated with cadmium chloride benzo(a)pyrene and trichloro-
ethylene Environmental Health Perspectives 109 71ndash74
Bertilsson G Heidrich J Svensson K Asman M Jendeberg L
Sydow-Backman M et al (1998) Identification of a human nuclear
receptor defines a new signaling pathway for CYP3A induction
Proceedings of the National Academy of Sciences of the United States
of America 95 12208ndash12213
Blumberg B Sabbagh Jr W Juguilon H Bolado Jr J van Meter
C M Ong E S et al (1998) SXR a novel steroid and xenobiotic-
sensing nuclear receptor Genes and Development 12 3195ndash3205
Borodina Y Rudik A Filimonov D Kharchevnikova N Dmitriev A
Blinova V et al (2004) A new statistical approach to predicting
aromatic hydroxylation sites Comparison with model-based
approaches Journal of Chemical Information and Computer Sciences
44 1998ndash2009
Borodina Y Sadym A Filimonov D Blinova V Dmitriev A amp
Poroikov V (2003) Predicting biotransformation potential from
molecular structure Journal of Chemical Information and Computer
Sciences 43 1636ndash1646
Bottone Jr F G Martinez J M Collins J B Afshari C A amp Eling
T E (2003) Gene modulation by the cyclooxygenase inhibitor
sulindac sulfide in human colorectal carcinoma cells Possible link to
apoptosis Journal of Biological Chemistry 278 25790ndash25801
Boyer S amp Zamora I (2002) New methods in predictive metabolism
Journal of Computer-Aided Molecular Design 16 403ndash413
Bredel M amp Jacoby E (2004) Chemogenomics An emerging strategy
for rapid target and drug discovery Nature Reviews Genetics 5
262ndash275
Bugrim A Nikolskaya T amp Nikolsky Y (2004) Early prediction of drug
metabolism and toxicity Systems biology approach and modeling
Drug Discovery Today 9 127ndash135
Bulera S J Eddy S M Ferguson E Jatkoe T A Reindel J F
Bleavins M R et al (2001) RNA expression in the early character-
ization of hepatotoxicants in Wistar rats by high-density DNA micro-
arrays Hepatology 33 1239ndash1258
Butcher E C Berg E L amp Kunkel E J (2004) Systems biology in drug
discovery Nature Biotechnology 22 1253ndash1259
Butte A (2002) The use and analysis of microarray data Naturalist
Review of Drug Discovery 1 951ndash960
Cary M P Bader G D amp Sander C (2005) Pathway information for
systems biology FEBS Letters 579 1815ndash1820
Cheok M H Yang W Pui C H Downing J R Cheng C Naeve
C W et al (2003) Treatment-specific changes in gene expression
discriminate in vivo drug response in human leukemia cells Nature
Genetics 34 85ndash90
Cleary J D Rogers P D amp Chapman S W (2001) Differential
transcription factor expression in human mononuclear cells in response
to amphotericin B Identification with complementary DNA microarray
technology Pharmacotherapy 21 1046ndash1054
Coen M Ruepp S U Lindon J C Nicholson J K Pognan F Lenz
E M et al (2004) Integrated application of transcriptomics and
metabonomics yields new insight into the toxicity due to paracetamol in
the mouse Journal of Pharmaceutical and Biomedical Analysis 35
93ndash105
Cornwell P D De Souza A T amp Ulrich R G (2004) Profiling of
hepatic gene expression in rats treated with fibric acid analogs
Mutation Research 549 131ndash145
Csermely P Agoston V amp Pongor S (2005) The efficiency of multi-
target drugs The network approach might help drug design Trends in
Pharmacological Sciences 26 178ndash182
Cunningham M J Liang S Fuhrman S Seilhamer J J amp
Somogyi R (2000) Gene expression microarray data analysis for
toxicology profiling Annals of the New York Academy of Sciences 919
52ndash67
de Longueville F Atienzar F A Marcq L Dufrane S Evrard S
Wouters L et al (2003) Use of a low density microarray for studying
gene expression patterns induced by hepatotoxicants on primary
cultures of rat hepatocytes Toxicological Sciences 75 378ndash392
Dobrin R Beg Q K Barabasi A L amp Oltvai Z N (2004)
Aggregation of topological motifs in the Escherichia coli transcriptional
regulatory network BMC Bioinformatics 5 10
Donald S Verschoyle R D Edwards R Judah D J Davies R
Riley J et al (2002) Hepatobiliary damage and changes in hepatic
gene expression caused by the antitumor drug ecteinascidin-743 (ET-
743) in the female rat Cancer Research 62 4256ndash4262
Dumont M Jacquemin E DrsquoHont C Descout C Cresteil D Haouzi
D et al (1997) Expression of the liver Na+-independent organic anion
transporting polypeptide (oatp-1) in rats with bile duct ligation Journal
of Hepatology 27 1051ndash1056
Durnas C Loi C M amp Cusack B J (1990) Hepatic drug metabolism
and aging Clinical Pharmacokinetics 19 359ndash389
Eisen M B Spellman P T Brown P O amp Botstein D (1998) Cluster
analysis and display of genome-wide expression patterns Proceedings
of the National Academy of Sciences of the United States of America
95 14863ndash14868
Ekins S Andreyev S Ryabov A Kirilov E Rakhmatulin E A
Bugrim A et al (in press) Computational Prediction of Human Drug
Metabolism Exp Opin Drug Metab Toxicol
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6662
Ekins S Boulanger B Swaan P W amp Hupcey M A Z (2002)
Towards a new age of virtual ADMETOX and multidimensional drug
discovery Journal of Computer-Aided Molecular Design 16 381ndash401
Ekins S Bugrim A Nikolsky Y amp Nikolskaya T (2005) Systems
biology Applications in drug discovery In S Gad (Ed) Drug
Discovery Handbook (pp 123ndash183) Hoboken NJrsquo Wiley and Sons
Ekins S Giroux C N Nikolsky Y Bugrim A amp Nikolskaya T
(2005) A signature gene network approach to toxicity The Toxoco-
logists 84
Ekins S Kirillov E Rakhmatulin E amp Nikolskaya T (2005) A novel
method for visualizing nuclear hormone receptor networks relevant to
drug metabolism Drug Metabolism and Disposition 33 474ndash481
Ekins S amp McGowan R J (2001) The limits of reductionism The
shifting genomic paradigmrsquos impact on industry and academia
Philsophy in Science 9 1ndash23
Ekins S Mirny L amp Schuetz E G (2002) A ligand-based approach to
understanding selectivity of nuclear hormone receptors PXR CAR
FXR LXRa and LXRb Pharmaceutical Research 19 1788ndash1800
Ekins S Nikolsky Y amp Nikolskaya T (2005) Techniques Application
of systems biology to absorption distribution metabolism excretion
and toxicity Trends in Pharmacological Sciences 26 202ndash209
Ekins S Ring B J Grace J McRobie-Belle D J amp Wrighton S
A (2000) Present and future in vitro approaches for drug
metabolism Journal of Pharmacological and Toxicological Methods
44 313ndash324
Ekins S Waller C L Swaan P W Cruciani G Wrighton S A amp
Wikel J H (2000) Progress in predicting human ADME parameters in
silico Journal of Pharmacological and Toxicological Methods 44
251ndash272
Erhardt P W (2003) A human drug metabolism database Potential roles
in the quantitative predictions of drug metabolism and metabolism-
related drugndashdrug interactions Current Drug Metabolism 4 411ndash422
Evans W E amp McLeod H L (2003) Pharmacogenomicsmdashdrug
disposition drug targets and side effects New England Journal of
Medicine 348 538ndash549
Ezendam J Staedtler F Pennings J Vandebriel R J Pieters R
Boffetta P et al (2004) Toxicogenomics of subchronic hexachlor-
obenzene exposure in Brown Norway rats Environmental Health
Perspectives 112 782ndash791
Faiola B Fuller E S Wong V A amp Recio L (2004) Gene expression
profile in bone marrow and hematopoietic stem cells in mice exposed to
inhaled benzene Mutation Research 549 195ndash212
FDA (2004) Innovation stagnation Challenge and opportunity on the
critical path to new medicinal products
Fiehn O (2001) Combining genomics metabolome analysis and bio-
chemical modelling to understand metabolic networks Comparative
Functional Genomics 2 155ndash168
Field F J Born E amp Mathur S N (2004) LXRRXR ligand activation
enhances basolateral efflux of beta-sitosterol in CaCo-2 cells Journal of
Lipid Research 45 905ndash913
Fleck C Sutter L Appenroth D Koch B Meinhold T Pitack M
et al (2003) Use of gene chip technology for the characterisation of
the regulation of renal transport processes and of nephrotoxicity in
rats Experimental and Toxicologic Pathology 54 401ndash410
Fliri A F Loging W T Thadeio P F amp Volkmann R A (2005)
Biological spectra analysis Linking biological activity profiles to
molecular structure Proceedings of the National Academy of Sciences
of the United States of America 102 261ndash266
Fountoulakis M (2004) Application of proteomics technologies in the
investigation of the brain Mass Spectrometry Reviews 23 231ndash258
Gaucher S P Taylor S W Fahy E Zhang B Warnock D E Ghosh
S S et al (2004) Expanded coverage of the human heart
mitochondrial proteome using multidimensional liquid chromatography
coupled with tandem mass spectrometry Journal of Proteome
Research 3 495ndash505
Gerhold D Lu M Xu J Austin C Caskey C T amp Rushmore T
(2001) Monitoring expression of genes involved in drug metabolism
and toxicology using DNA microarrays Physiological Genomics 5
161ndash170
Guo G L Choudhuri S amp Klaassen C D (2002) Induction profile of
rat organic anion transporting polypeptide 2 (oatp2) by prototypical
drug-metabolizing enzyme inducers that activate gene expression
through ligand-activated transcription factor pathways Journal of
Pharmacology and Experimental Therapeutics 300 206ndash212
Hagenbuch B amp Meier P J (2004) Organic anion transporting
polypeptides of the OATP SLC21 family Phylogenetic classification
as OATP SLCO superfamily new nomenclature and molecularfunc-
tional properties Pflugers Archiv 447 653ndash665
Hamadeh H K amp Afshari C A (2004) Toxicogenomics principles and
applications Hobokenrsquo John Wiley and Sons
Hamadeh H K Bushel P R Jayadev S Martin K DiSorbo O Sieber
S et al (2002) Gene expression analysis reveals chemical-specific
profiles Toxicological Sciences 67 219ndash231
Hamadeh H K Jayadev S Gaillard E T Huang Q Stoll R
Blanchard K et al (2004) Integration of clinical and gene expression
endpoints to explore furan-mediated hepatotoxicity Mutation Research
549 169ndash183
Hamadeh H K Knight B L Haugen A C Sieber S Amin R P
Bushel P R et al (2002) Methapyrilene toxicity Anchorage of
pathologic observations to gene expression alterations Toxicologic
Pathology 30 470ndash482
Han J D Bertin N Hao T Goldberg D S Berriz G F Zhang L V
et al (2004) Evidence for dynamically organized modularity in the
yeast proteinndashprotein interaction network Nature 430 88ndash93
Hanisch D Zien A Zimmer R amp Lengauer T (2002) Co-clustering of
biological networks and gene expression data Bioinformatics 18
S145ndashS154
Harris A J Dial S L amp Casciano D A (2004) Comparison of basal
gene expression profiles and effects of hepatocarcinogens on gene
expression in cultured primary human hepatocytes and HepG2 cells
Mutation Research 549 79ndash99
Harrison S C (2004) Whither structural biology Nature Structural
Molecular Biology 11 12ndash15
Hartley D P Dai X He Y D Carlini E J Wang B Huskey S E
et al (2004) Activators of the rat pregnane X receptor differentially
modulate hepatic and intestinal gene expression Molecular Pharma-
cology 65 1159ndash1171
Hartwell L H Hopfield J J Leibler S amp Murray A W (1999) From
molecular to modular cell biology Nature 402 C47ndashC52
Hasmall S Orphanides G James N Pennie W Hedley K Soames
A et al (2002) Downregulation of lactoferrin by PPARalpha ligands
Role in perturbation of hepatocyte proliferation and apoptosis
Toxicology Sciences 68 304ndash313
Hayase T Yamamoto Y Yamamoto K Muso E amp Shiota K (2004)
Microarray profile analysis of toxic cocaine-induced alterations in the
expression of mouse brain gene sequences A possible rsquoprotectiversquo
effect of buprenorphine Journal of Applied Toxicology 24 15ndash20
Hayes K R Vollrath A L Zastrow G M McMillan B J Craven M
Jovanovich S et al (2005) EDGE A centralized resource for the
comparison analysis and distribution of toxicogenomic information
Molecular Pharmacology 67 1360ndash1368
Heijne W H Slitt A L van Bladeren P J Groten J P Klaassen C D
Stierum R H et al (2004) Bromobenzene-induced hepatotoxicity at
the transcriptome level Toxicology Sciences 79 411ndash422
Heijne W H Stierum R H Slijper M van Bladeren P J amp van
Ommen B (2003) Toxicogenomics of bromobenzene hepatotoxicity
A combined transcriptomics and proteomics approach Biochemical
Pharmacology 65 857ndash875
Heinloth A N Irwin R D Boorman G A Nettesheim P Fannin
R D Sieber S O et al (2004) Gene expression profiling of rat
livers reveals indicators of potential adverse effects Toxicology
Sciences 80 193ndash202
Hodges L C Cook J D Lobenhofer E K Li L Bennett L Bushel
P R et al (2003) Tamoxifen functions as a molecular agonist inducing
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 63
cell cycle-associated genes in breast cancer cells Molecular Cancer
Research 1 300ndash311
Holleman A Cheok M H den Boer M L Yang W Veerman KM
A J Kazemier et al (2004) Gene-expression patterns in drug-resistant
acute lymphoblastic leukemia cells and response to treatment New
England Journal of Medicine 351 533ndash542
Hong Y Muller U R amp Lai F (2003) Discriminating two classes of
toxicants through expression analysis of HepG2 cells with DNA arrays
Toxicology in Vitro 17 85ndash92
Hood E (2003) Proteomics Characterizing the cogs in the machinery of
life Environmental Health Perspectives 111 A816ndashA825
Hood L (2003) Systems biology Integrating technology biology and
computation Mechanism of Ageing and Development 124 9ndash16
Hood L amp Galas D (2003) The digital code of DNA Nature 421
444ndash448
Hood L amp Perlmutter R M (2004) The impact of systems approaches on
biological problems in drug discovery Nature Biotechnology 22
1215ndash1217
Hu T Gibson D P Carr G J Torontali S M Tiesman J P Chaney
J G et al (2004) Identification of a gene expression profile that
discriminates indirect-acting genotoxins from direct-acting genotoxins
Mutation Research 549 5ndash27
Huang Q Dunn II R T Jayadev S DiSorbo O Pack F D Farr S B
et al (2001) Assessment of cisplatin-induced nephrotoxicity by
microarray technology Toxicology Sciences 63 196ndash207
Huang Q Jin X Gaillard E T Knight B L Pack F D Stoltz J H et
al (2004) Gene expression profiling reveals multiple toxicity endpoints
induced by hepatotoxicants Mutation Research 549 147ndash167
Huisman M T Smit J W Crommentuyn K M Zelcer N Wiltshire
H R Beijnen J H et al (2002) Multidrug resistance protein 2
(MRP2) transports HIV protease inhibitors and transport can be
enhanced by other drugs Aids 16 2295ndash2301
Hunt C M Westerkam W R amp Stave G M (1992) Effect of age and
gender on the activity of human hepatic CYP3A Biochemical
Pharmacology 44 275ndash283
Ideker T Ozier O Schwikowski B amp Siegal A F (2002) Discovering
regulatory and signalling circuits in molecular interaction networks
Bioinformatics 18 S233ndashS240
Ideker T Thorsson V Ranish J A Christmas R Buhler J Eng J K
et al (2001) Integrated genomic and proteomic analyses of a system-
atically perturbed network Science 292 929ndash934
Iida M Anna C H Hartis J Bruno M Wetmore B Dubin J R et al
(2003) Changes in global gene and protein expression during early
mouse liver carcinogenesis induced by non-genotoxic model carcino-
gens oxazepam and Wyeth-14643 Carcinogenesis 24 757ndash770
Ingelman-Sundberg M (2004) Pharmacogenetics of cytochrome P450 and
its applications in drug therapy The past present and future Trends in
Pharmacological Sciences 25 193ndash200
Jeong H Mason S P Barabasi A L amp Oltvai Z N (2001) Lethality
and centrality in protein networks Nature 411 41ndash42
Jeong H Tombor B Albert R Oltvai Z N amp Barabasi A L
(2000) The large-scale organization of metabolic networks Nature
407 651ndash654
Jung D Hagenbuch B Gresh L Pontoglio M Meier P J amp Kullak-
Ublick G A (2001) Characterization of the human OATP-C
(SLC21A6) gene promoter and regulation of liver-specific OATP genes
by hepatocyte nuclear factor 1 alpha Journal of Biological Chemistry
276 37206ndash37214
Jung D amp Kullak-Ublick G A (2003) Hepatocyte nuclear factor 1
alpha A key mediator of the effect of bile acids on gene expression
Hepatology 37 622ndash631
Jung J -W Park J -S Hwang J -W Kang K -S YS L Song B
-S et al (2004) Gene expression analysis of peroxisome prolifer-
ators- and phenytoin-induced hepatotoxicity using cDNA microarray
Journal of Veterinary Medical Science 66 1329ndash1333
Kato N Shibutani M Takagi H Uneyama C Lee K -Y Takiami S
et al (2004) Gene expression profile in the livers of rats orally
administered ethinylestradiol for 28 days using a microarray technique
Toxicology 200 179ndash192
Kier L D Neft R Tang L Suizu R Cook T Onsurez K et al
(2004) Applications of microarrays with toxicologically relevant genes
(tox genes) for the evaluation of chemical toxicants in Sprague Dawley
rats in vivo and human hepatocytes in vitro Mutation Research 549
101ndash113
Kijima K Toyosawa K Yasuba M Matsuoka N Adachi T
Komiyama M et al (2004) Gene expression analysis of the rat testis
after treatment with di(2-ethylhexyl) phthalate using cDNA microarray
and real-time RT-PCR Toxicology and Applied Pharmacology 200
103ndash110
Kim R B (2003) Organic anion-transporting polypeptide (OATP) trans-
porter family and drug disposition European Journal of Clinical
Investigation 33 1ndash5
Kinirons M T amp OrsquoMahony M S (2004) Drug metabolism and ageing
British Journal of Clinical Pharmacology 57 540ndash544
Kitano H (2002a) Computational systems biology Nature 420 206ndash210
Kitano H (2002b) Systems biology A brief overview Science 295
1662ndash1664
Kliewer S A Moore J T Wade L Staudinger J L Watson M
A Jones S A et al (1998) An orphan nuclear receptor activated
by pregnanes defines a novel steroid signalling pathway Cell 92
73ndash82
Korolev D Balakin K V Nikolsky Y Kirillov E Ivanenkov Y A
Savchuk N P et al (2003) Modeling of human cytochrome p450-
mediated drug metabolism using unsupervised machine learning
approach Journal of Medicinal Chemistry 46 3631ndash3643
Kramer J A Pettit S D Amin R P Bertram T A Car B
Cunningham M et al (2004) Overview on the application of
transcription profiling using selected nephrotoxicants for toxicology
assessment Environmental Health Perspectives 112 460ndash464
Kultima K Nystrom A -M Scholz B Gustafson A -L Dencker L
amp Stigson M (2004) Valproic acid teratogenicity A toxicogenomics
approach Environmental Health Perspectives 112 1225ndash1235
Kutuzova G D amp Deluca H F (2004) Gene expression profiles in rat
intestine identify pathways for 125-dihydroxyvitamin D(3) stimulated
calcium absorption and clarify its immunomodulatory properties
Archives of Biochemistry and Biophysics 432 152ndash166
Kwak M K Wakabayashi N Itoh K Motohashi H Yamamoto M amp
Kensler T W (2003) Modulation of gene expression by cancer
chemopreventive dithiolethiones through the Keap1-Nrf2 pathway
Identification of novel gene clusters for cell survival Journal of
Biological Chemistry 278 8135ndash8145
Lahjouji K Mitchell G A amp Qureshi I A (2001) Carnitine transport
by organic cation transporters and systemic carnitine deficiency
Molecular Genetics and Metabolism 73 287ndash297
Landowski C P Sun D Foster D R Menon S S Barnett J L
Welage L S et al (2003) Gene expression in the human intestine and
correlation with oral valacyclovir pharmacokinetic parameters Journal
of Pharmacology and Experimental Therapeutics 306 778ndash786
Lee G -J Lee W -S Jeon K -S Um C -H Kim S -J Lee C -H
et al (2004) cDNA microarray gene expression analysis and
toxicological phenotype for anticancer drug Journal of Veterinary
Medical Science 66 1339ndash1345
Lee M Kwon J Kim S N Kim J E Koh W S Kim E J et al
(2003) cDNA microarray gene expression profiling of hydroxyurea
paclitaxel and p-anisidine genotoxic compounds with differing
tumorigenicity results Environmental and Molecular Mutagenesis
42 91ndash97
Li S Armstrong C M Bertin N Ge H Milstein S Boxem M et al
(2004) A map of the interactome network of the metazoan C elegans
Science 303 540ndash543
Liguori M J Anderson L M Bukofzer S McKim J Pregenzer J F
Retief J et al (2005) Microarray analysis in human hepatocytes
suggests a mechanism for hepatotoxicity induced by trovafloxacin
Hepatology 41 177ndash186
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6664
Lobenhofer E K Bennett L Cable P L Li L Bushel P R amp Afshari
C A (2002) Regulation of DNA replication fork genes by 17beta-
estradiol Molecular Endocrinology 16 1215ndash1229
Locker J Tian J Carver R Concas D Cossu C Ledda-Columbano
G M et al (2003) A common set of immediate-early response genes
in liver regeneration and hyperplasia Hepatology 38 314ndash325
Mattes W B Pettit S D Sansone S A Bushel P R amp Waters M D
(2004) Database development in toxicogenomics Issues and efforts
Environmental Health Perspectives 112 495ndash505
McMillian M Nie A Y Parker J B Leone A Bryant S Kemmerer
M et al (2004) A gene expression signature for oxidant stressreactive
metabolites in rat liver Biochemical Pharmacology 68 2249ndash2261
Meier P J amp Stieger B (2000) Molecular mechanisms in bile formation
News in Physiological Sciences 15 89ndash93
Meneses-Lorente G de Longueville F Dos Santos-Mendes S Bonnert
T P Jack A Evrard S et al (2003) An evaluation of a low-density
DNA microarray using cytochrome P450 inducers Chemical Research
in Toxicology 16 1070ndash1077
Meneses-Lorente G Guest P C Lawrence J Muniappa N Knowles
M R Skynner H A et al (2004) A proteomic investigation of drug-
induced steatosis in rat liver Chemical Research in Toxicology 17
605ndash612
Merrill C L Ni H Yoon L W Tirmenstein M A Narayanan P
Benavides G R et al (2002) Etomoxir-induced oxidative stress in
HepG2 cells detected by differential gene expression is confirmed
biochemically Toxicology Sciences 68 93ndash101
Milo R Shen-Orr S Itzkovitz S Kashtan N Chklovskii D amp Alon
U (2002) Network motifs Simple building blocks of complex
networks Science 298 824ndash827
Morgan K T Ni H Brown H R Yoon L Qualls C W Crosby L
M et al (2002) Application of cDNA microarray technology to in
vitro toxicology and the selection of genes for a real-time RT-PCR-
based screen for oxidative stress in Hep-G2 cells Toxicologic
Pathology 30 435ndash451
Mwinyi J Johne A Bauer S Roots I amp Gerloff T (2004) Evidence
for inverse effects of OATP-C (SLC21A6) 5 and 1b haplotypes on
pravastatin kinetics Clinical Pharmacology and Therapeutics 75
415ndash421
Nakata K Yukawa M Komiyama N Nakano T amp Kaminuma T
(2002) A nuclear receptor database that maps pathways to diseases
Genome Infomatics 13 515ndash516
Nicholson J K Holmes E Lindon J C amp Wilson I D (2004) The
challenges of modeling mammalian biocomplexity Nature Biotechnol-
ogy 22 1268ndash1274
Nicholson J K amp Wilson I D (2003) Understanding rsquoglobal systems
biology Metabonomics and the continuum of metabolism Nature
Reviews on Drug Discovery 2 668ndash676
Nikitin A Egorov S Daraselia N amp Mazo I (2003) Pathway studiomdash
the analysis and navigation of molecular networks Bioinformatics 19
2155ndash2157
Nikolsky Y Ekins S Nikolskaya T amp Bugrim A (2005) A novel
method for generation of signature networks as biomarkers from
complex high throughput data Toxicology Letters 158 20ndash29
Nikolsky Y Nikolskaya T amp Bugrim A (2005) Biological networks
and analysis of experimental data in drug discovery Drug Discovery
Today 10 653ndash662
Nishizato Y Ieiri I Suzuki H Kimura M Kawabata K Hirota T
et al (2003) Polymorphisms of OATP-C (SLC21A6) and OAT3
(SLC22A8) genes Consequences for pravastatin pharmacokinetics
Clinical Pharmacology and Therapeutics 73 554ndash565
Nozawa T Nakajima M Tamai I Noda K Nezu J Sai Y et al
(2002) Genetic polymorphisms of human organic anion transporters
OATP-C (SLC21A6) and OATP-B (SLC21A9) Allele frequencies in
the Japanese population and functional analysis Journal of Pharmacol-
ogy and Experimental Therapeutics 302 804ndash813
Nozawa T Sugiura S Nakajima M Goto A Yokoi T Nezu J et al
(2004) Involvement of organic anion transporting polypeptides in the
transport of troglitazone sulfate Implications for understanding trogli-
tazone hepatotoxicity Drug Metabolism and Disposition 32 291ndash294
Oliver D E Rubin D L Stuart J M Hewett M Klein T E amp
Altman R B (2002) Ontology development for a pharmacogenetics
knowledge base Package Symposion 88ndash99
Oram J F amp Lawn R M (2001) ABCA1 The gatekeeper for eliminating
excess tissue cholesterol Journal of Lipid Research 42 1173ndash1179
Oswald M Kullak-Ublick G A Paumgartner G amp Beuers U (2001)
Expression of hepatic transporters OATP-C and MRP2 in primary
sclerosing cholangitis Liver 21 247ndash253
Parsons A B Brost R I Ding H Li Z Zhang C Sheikh B et al
(2004) Integration of chemical-genetic and genetic interaction data
links bioactive compounds to cellular target pathways Nature Bio-
technology 22 62ndash69
Patki K C von Moltke L L Harmatz J S Hesse L M Court M H
amp Greenblatt D J (2004) Effect of age on in vitro triazolam
biotransformation in male human liver microsomes Journal of
Pharmacology and Experimental Therapeutics 308 874ndash879
Pereira-Leal J B Enright A J amp Ouzounis C A (2004) Detection of
functional modules from protein interaction networks Proteins 54
49ndash57
Plant N (2004) Interaction networks Coordinating responses to xeno-
biotic exposure Toxicology 202 21ndash32
Raghavendra Prasad H S Qi Z Srinivasan K N amp Gopalakrish-
nakone P (2004) Potential effects of tetrodotoxin exposure to
human glial cells postulated using microarray approach Toxicon 44
597ndash608
Reilly T P Bourdi M Brady J N Pise-Masison C A Radonovich M
F George J W et al (2001) Expression profiling of acetaminophen
liver toxicity in mice using microarray technology Biochemical and
Biophysical Research Communications 282 321ndash328
Rives A W amp Galitski T (2003) Modular organization of cellular
networks Proceedings of the National Academy of Sciences of the
United States of America 100 1128ndash1133
Rogers P D Pearson M M Cleary J D Sullivan D C amp Chapman
S W (2002) Differential expression of genes encoding immunomodu-
latory proteins in response to amphotericin B in human mononuclear
cells identified by cDNA microarray analysis Journal of Antimicrobial
Chemotherapy 50 811ndash817
Rosen M B Wilson V S Schmid J E amp Gray L E (2005) Gene
expression analysis in the ventral prostate of rats exposed to vinclozolin
or procymidone Reproductive Toxicology 19 367ndash379
Rost D Herrmann T Sauer P Schmidts H L Stieger B Meier P J
et al (2003) Regulation of rat organic anion transporters in bile salt-
induced cholestatic hepatitis Effect of ursodeoxycholate Hepatology
38 187ndash195
Rothman R B Ayestas M A Dersch C M amp Baumann M H (1999)
Aminorex fenfluramine and chlorphentermine are serotonin transporter
substrates Implications for primary pulmonary hypertension Circula-
tion 100 869ndash875
Ruepp S U Tonge R P Shaw J Wallis N amp Pognan F (2002)
Genomics and proteomics analysis of acetaminophen toxicity in mouse
liver Toxicology Sciences 65 135ndash150
Sakaeda T Nakamura T amp Okumura K (2002) MDR1 genotype-
related pharmacokinetics and pharmacodynamics Biological amp Phar-
maceutical Bulletin 25 1391ndash1400
Sakata T Anzai N Shin H J Noshiro R Hirata T Yokoyama H
et al (2004) Novel single nucleotide polymorphisms of organic cation
transporter 1 (SLC22A1) affecting transport functions Biochemical
and Biophysical Research Communications 313 789ndash793
Satlin L M Amin V amp Wolkoff A W (1997) Organic anion
transporting polypeptide mediates organic anionHCO3-exchange
Journal of Biological Chemistry 272 26340ndash26345
Satomi Y Tsuchiya W Mihara K Ota M Kasahara Y amp Akahori F
(2004) Gene expression analysis of the lung following paraquat
administration in rats using DNA microarray Journal of Toxicological
Sciences 29 91ndash100
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 65
Schrenk D Baus P R Ermel N Klein C Vorderstemann B amp
Kauffmann H M (2001) Up-regulation of transporters of the MRP
family by drugs and toxins Toxicology Letters 120 51ndash57
Segal E Shapira M Regev A Persquoer D Botstein D Koller D et al
(2003) Module networks Identifying regulatory modules and their
condition-specific regulators from gene expression data Nature
Genetics 34 166ndash176
Segal E Wang H amp Koller D (2003) Discovering molecular pathways
from protein interaction and gene expression Bioinformatics 19
i264ndash i272
Seidel S D Kan H L Stott W T Schisler M R amp Gollapudi B B
(2003) Identification of transcriptome profiles for the DNA-damaging
agents bleomycin and hydrogen peroxide in L5178Y mouse lymphoma
cells Environmental and Molecular Mutagenesis 42 19ndash25
Shan L Yu Y Schut H A J amp Snyderwine E G (2004) Susceptibility
of rats to mammary gland carcinogenesis by the food-derived
carcinogen 2-Amino-1-Methyl-6-Phenylimidazo[45-b]Pyridine (PhIP)
varies with age and is associated with the induction of differential gene
expression American Journal of Pathology 165 191ndash202
Sharom J R Bellows D S amp Tyers M (2004) From large networks to
small molecules Current Opinion in Chemical Biology 8 81ndash90
Shitara Y Hirano M Sato H amp Sugiyama Y (2004) Gemfibrozil and
its glucuronide inhibit the OATP2(OATP1B1 SLC21A6)-mediated
hepatic uptake and CYP2C8-mediated metabolism of cerivastatinmdash
Analysis of the mechanism of the clinically relevant drugndashdrug
interaction between cerivastatin and gemfibrozil Journal of Pharma-
cology and Experimental Therapeutics
Shitara Y Itoh T Sato H Li A P amp Sugiyama Y (2003) Inhibition of
transporter-mediated hepatic uptake as a mechanism for drugndashdrug
interaction between cerivastatin and cyclosporin A Journal of
Pharmacology and Experimental Therapeutics 304 610ndash616
Somogyi R Fuhrman S amp Wen X (2001) Genetic network inference in
computational models and applications to large-scale gene expression
data In M Bower amp H Bolouri (Eds) Computational modeling of
genetic and biochemical networks (pp 119ndash157) Cambridge MArsquoThe
MIT Press
Spirin V amp Mirny L A (2003) Protein complexes and functional
modules in molecular networks Proceedings of the National Academy
of Sciences of the United States of America 100 12123ndash12128
Staudinger J Liu Y Madan A Habeebu S amp Klaassen C D
(2001) Coordinate regulation of xenobiotic and bile acid homeo-
stasis by pregnane X receptor Drug Metabolism and Disposition
29 1467ndash1472
Sun L Z Ji Z L Chen X Wang J F amp Chen Y Z (2002) ADME-
AP A database of ADME associated proteins Bioinformatics 18
1699ndash1700
Suter L Babiss L E amp Wheeldon E B (2004) Toxicogenomics in
predictive toxicology in drug development Chemistry and Biology 11
161ndash171
Synold T W Dussault I amp Forman B M (2001) The orphan nuclear
receptor SXR coordinately regulates drug metabolism and efflux
Nature Medicine 7 584ndash590
Tabuchi Y amp Kondo T (2003) cDNA microarray analysis reveals chop-
10 plays a key role in Sertoli cell injury induced by bisphenol A
Biochemical and Biophysical Research Communications 305 54ndash61
Tamai I Nezu J Uchino H Sai Y Oku A Shimane M et al (2000)
Molecular identification and characterization of novel members of the
human organic anion transporter (OATP) family Biochemical and
Biophysical Research Communications 273 251ndash260
Taylor S W Fahy E Zhang B Glenn G M Warnock D E Wiley S
et al (2003) Characterization of the human heart mitochondrial
proteome Nature Biotechnology 21 281ndash286
Terasaka S Aita Y Inoue A Hayashi S Nishigaki M Aoyagi K
et al (2004) Using a customized DNA microarray for expression
profiling of the estrogen-responsive genes to evaluate estrogen activity
among natural estrogens and industrial chemicals Environmental
Health Perspectives 112 773ndash781
Thomas D M Francescutti-Verbeem D M Liu X amp Kuhn D M
(2004) Identification of differentially regulated transcripts in mouse
striatum following methamphetamine treatmentmdashan oligonucleotide
microarray approach Journal of Neurochemistry 88 380ndash393
Thomas R S Rank D R Penn S G Zastrow G M Hayes K R
Pande K et al (2001) Identification of toxicologically predictive
gene sets using cDNA microarrays Molecular Pharmacology 60
1189ndash1194
Thompson K L Afshari C A Amin R P Bertram T A Car B
Cunningham M et al (2004) Identification of platform-independent
gene expression markers of cisplatin nephrotoxicity Environmental
Health Perspectives 112 488ndash494
Tirona R G Leake B F Merino G amp Kim R B (2001)
Polymorphisms in OATP-C Identification of multiple allelic
variants associated with altered transport activity among European-
and African-Americans Journal of Biological Chemistry 276
35669ndash35675
Tirona R G Leake B F Wolkoff A W amp Kim R B (2003)
Human organic anion transporting polypeptide-C (SLC21A6) is a
major determinant of the rifampin-mediated pregnane X receptor
activation Journal of Pharmacology and Experimental Therapeutics
304 223ndash228
Tong W Cao X Harris S Sun H Fang H Fuscoe J et al (2003)
ArrayTrackmdashsupporting toxicogenomic research at the US Food and
Drug Administration National Center for Toxicological Research
Environmental Health Perspectives 111 1819ndash1826
Tornow S amp Mewes H W (2003) Functional modules by relating
protein interaction networks and gene expression Nucleic Acids
Research 31 6283ndash6289
Trocho C Escola-Gil J C Ribas V Benitez S Martin-Campos J M
Rotllan N et al (2004) Phenytoin treatment reduces atherosclerosis in
mice through mechanisms independent of plasma HDL-cholesterol
concentration Atherosclerosis 174 275ndash285
Ueda A Hamadeh H K Webb H K Yamamoto Y Sueyoshi T
Afshari C A et al (2002) Diverse roles of the nuclear orphan
receptor CAR in regulating hepatic genes in response to phenobarbital
Molecular Pharmacology 61 1ndash6
Ulrich R G (2003) The toxicogenomics of nuclear receptor agonists
Current Opinion in Chemical Biology 7 505ndash510
Ulrich R G Rockett J C Gibson G G amp Pettit S D (2004)
Overview of an interlaboratory collaboration on evaluating the effects
of model hepatotoxicants on hepatic gene expression Environmental
Health Perspectives 112 423ndash427
Unami A Shinohara Y Ichikawa T amp Baba Y (2003) Biochemical
and microarray analyses of bupivacaine-induced apoptosis Journal of
Toxicological Sciences 28 77ndash94
Vasquez A Flammini A Maritan A amp Vespignani A (2003) Global
protein function prediction from proteinndashprotein interaction networks
Nature Biotechnology 21 697ndash700
Vezina C M Walker N J amp Olson J R (2004) Subchronic Exposure to
TCDD PeCDF PCB126 and PCB153 Effect on hepatic gene
expression Environmental Health Perspectives 112 1636ndash1644
Vo T D Greenberg H J amp Palsson B O (2004) Reconstruction and
functional characterization of the human mitochondrial metabolic
network based on proteomic and biochemical data Journal of Bio-
logical Chemistry 279 39532ndash39540
Wang H amp LeCluyse E L (2003) Role of orphan nuclear receptors in
the regulation of drug-metabolising enzymes Clinical Pharmacoki-
netics 42 1331ndash1357
Wang N Silver D L Thiele C amp Tall A R (2001) ATP-
binding cassette transporter A1 (ABCA1) functions as a choles-
terol efflux regulatory protein Journal of Biological Chemistry 276
23742ndash23747
Waring J F Cavet G Jolly R A McDowell J Dai H Ciurlionis R
et al (2003) Development of a DNA Microarray for toxicology based
on hepatoxin-regulated sequences Environmental Health Perspectives
111 863ndash870
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6666
Waring J F Gum R Morfitt D Jolly R A Ciurlionis R Heindel M
et al (2002) Identifying toxic mechanisms using DNA microarrays
Evidence that an experimental inhibitor of cell adhesion molecule
expression signals through the aryl hydrocarbon nuclear receptor
Toxicology 181ndash182 537ndash550
Waring J F Jolly R A Ciurlionis R Lum P Y Praestgaard J T
Morfitt D C et al (2001) Clustering of hepatotoxins based on
mechanism of toxicity using gene expression profiles Toxicology and
Applied Pharmacology 175 28ndash42
Waters M Boorman G Bushel P Cunningham M Irwin R Merrick
A et al (2003) Systems toxicology and the Chemical Effects in
Biological Systems (CEBS) knowledge base EHP Toxicogenomics
111 15ndash28
Wei M Arnold L Cano M amp Cohen S M (2005) Effects of co-
administration of antioxidants and arsenicals on the rat urinary bladder
epithelium Toxicology Sciences 83 237ndash245
Weinshilboum R (2003) Inheritance and drug response New England
Journal of Medicine 348 529ndash537
Weinshilboum R amp Wang L (2004) Pharmacogenetics Inherited
variation in amino acid sequence and altered protein quantity Clinical
Pharmacology and Therapeutics 75 253ndash258
Werner E (2003) In silico multicellular systems biology and minimal
genomes Drug Discovery Today 8 1121ndash1127
Wong J S amp Gill S S (2002) Gene expression changes induced in
mouse liver by di(2-ethylhexyl) phthalate Toxicology and Applied
Pharmacology 185 180ndash196
Xie T Tong L McCann U D Yuan J Becker K G Mechan A O
et al (2004) Identification and characterization of metallothionein-1
and -2 gene expression in the context of ()34-methylenedioxymetham-
phetamine-induced toxicity to brain dopaminergic neurons Journal of
Neuroscience 24 7043ndash7050
Yadetie F Laegreid A Bakke I Kusnierczyk W Komorowski J
Waldum H L et al (2003) Liver gene expression in rats in response
to the peroxisome proliferator-activated receptor-alpha agonist ciprofi-
brate Physiological Genomics 15 9ndash19
Yamashita S Nomoto T Abe M Tatematsu M Sugimura T amp
Ushijima T (2004) Persistence of gene expression changes in stomach
mucosae induced by short-term N-methyl-NV-nitro-N-nitrosoguanidinetreatment and their presence in stomach cancers Mutation Research
549 185ndash193
Yan Q amp Sadee W (2000) Human membrane transporter database A
web-accessible relational database for drug transport studies and
pharmacogenomics AAPS Pharmsci 2 E20
Yeger-Lotem E amp Margalit H (2003) Detection of regulatory circuits by
integrating the cellular networks of proteinndashprotein interactions and
transcription regulation Nucleic Acids Research 31 6053ndash6061
Yoon B I Li G -X Kitada K Kawasaki Y Igarashi K amp Kodama
Y et al (2003) Mechanisms of benzene-induced hematotoxicity and
leukemogenicity cDNA microarray analyses using mouse bone marrow
tissue Environmental Health Perspectives 111 1411ndash1420
Young M B DiSilvestro M R Sendera T J Freund J Kriete A amp
Magnuson S R (2003) Analysis of gene expression in carbon
tetrachloride-treated rat livers using a novel bioarray technology
Pharmacogenomics Journal 3 41ndash52
Yu H Zhu X Greenbaum D Karro J amp Gerstein M (2004) TopNet
A tool for comparing biological sub-networks correlating protein
properties with topological statistics Nucleic Acids Research 32
328ndash337
Zhang E Y Fu D J Pak Y A Stewart T Mukhopadhyay N
Wrighton S A et al (2004) Genetic polymorphisms in human proton-
dependent dipeptide transporter PEPT1 Implications for the functional
role of Pro586 Journal of Pharmacology and Experimental Therapeu-
tics 310 437ndash445
Zhang E Y Knipp G T Ekins S amp Swaan P W (2002)
Structural biology and function of solute transporters Implications
for identifying and designing substrates Drug Metabolism Reviews
34 709ndash750
Zhang E Y Phelps M A Cheng C Ekins S amp Swaan P W (2002)
Modeling of active transport systems Advanced Drug Delivery
Reviews 54 329ndash354
- Systems-ADMETox Resources and network approaches
-
- Introduction
- Data available
- Network analysis and databases
-
- Network applications
-
- The role of transporters
-
- Clinical relevance of transporters
- Transporter network examples ABCA1
- Transporter network examples OATP
- Transporter microarray data
-
- Applications to enzymes
- Future network applications
-
- Discussion
- Acknowledgements
- References
-
Table 1 (continued)
Compounds Rat strain Microarray type Compound dose Microarray data
availability
Reference
Clofibrate Male Spraguendash
Dawley
Atlas Rat Toxicology II
arrays (Clontech Palo
Alto CA USA)
containing 465 genes
High (250 mgkgday) or
low (25 mgkgday)
Genbank accession
number and data for 3
platforms in a
manuscript table
(Baker et al
2004)
Di(2-ethylhexyl)
phthalate
Male Spraguendash
Dawleymdash(testes)
An in-house cDNA
microarray
20 or 2000 mgkg Gene names Genbank
ID and fold change in a
manuscript table
(Kijima et al
2004)
Ciprofibrate Female Fischer An in-house cDNA
microarray
50 mgkg body weight Gene names symbol
accession number mean
ratio and SD
(Yadetie et al
2003)
Methapyrilene Male Spraguendash
Dawley
Rat Tox Chip 10 10 or 100 mgkgday Gene Name accession
number and indication of
up or down regulation
original data available on
NIEHS website
(Hamadeh et al
2002b)
Ecteinascidin-743
(ET-743)
Female Wistar Custom chip cDNA
microarrays containing
approximately 4700
hybridizable mouse
expressed sequence tags
derived from IMAGE
clones obtained from
Research Genetics
(Huntsville AL) or from
the MRC Human Gene
Mapping Project
40 ugkg Data available on
laboratory websitemdashnot
available at present
(Donald et al
2002)
Clofibrate Wyeth
14643 Gemfibrozil
phenobarbital
Male Spraguendash
Dawley VAF+
NIEHS rat chip v10 Clofibrate (250
mgkgday Wyeth
14643 (250 mgkg
day) Gemfibrozil
(100 mgkgday)
Phenobarbital
(120 mgkgday)
Gene names and fold
changes in a manuscript
table
(Hamadeh et al
2002a)
Vinclozin procymidone Male Spraguendash
Dawleymdashprostate
Clontech Atlas Rat 12
Toxicology array
200 mgkg Gene name accession
number average fold
change
(Rosen Wilson
Schmid amp Gray
2005)
Cisplatin Male Spraguendash
Dawley VAF1
albino (CRL
CD(SD) BR
Rat Tox Microarrays
were purchased from
Phase-1 Molecular
Toxicology
05 or 1 mgkgday Gene name accession
number fold change in
kidney
(Huang et al
2001)
Allyl alcohol
miodarone Aroclor
1254 arsenic
carbamazepine
carbon tetrachloride
diethylnitrosamine
dimethylformamide
diquat etoposide
indomethacin
methapyrilene
methotrexate
monocrotaline
3-methylcholanthrene
Male Spraguendash
Dawley
Affymetrix GeneChip
Test 2 Array
Allyl alcohol (40 mgkg
day) miodarone (100 mg
kgday) Aroclor 1254
(400 mgkgday) arsenic
(20 mgkgday)
carbamazepine (250 mg
kgday) carbon
tetrachloride (1000mgkg
day) diethylnitrosamine
(100 mgkgday)
dimethylformamide (1000
mgkgday) diquat (172
mgkgday) etoposide (50
mgkgday) indomethacin
(20 mgkgday)
methapyrilene (250 mg
kgday) methotrexate
(250 mgkgday)
monocrotaline (50 mgkg
day)
3-methylcholanthrene
(100 mgkgday)
Heatmap figures and a
table of Affymetrix
names for genes
correlated with clinical
chemistry changes in a
manuscript table
(Waring et al
2001)
(continued on next page)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6642
Table 1 (continued)
Compounds Rat strain Microarray type Compound dose Microarray data
availability
Reference
Microcystin-LR (MLR)
phenobarbital (PB)
lipopolysaccharide
(LPS) carbon
tetrachloride (CT)
thioacetamide (THA)
and cyproterone
acetate (CPA)
Male Wistar Purpose-made rat DNA
microarray (Affymetrix
Santa Clara CA)
containing 1600 rat
DNA sequences
Various Accession number gene
name fold changes
shown as a heat map
table in publicationmdash
data extraction would be
laborious
(Bulera et al
2001)
Acetamidofluorene
aniline bromobenzene
butyl hydroxytol
dieldrin disulfiram
ethinyl estradiol
hexachlorocyclohexane
g 4-methylthiazole
nimesulide piperonyl
butoxide precocene I
pulegone tannic acid
trans-anethole
Male Spraguendash
Dawley
Custom Rat MegaA
cDNA chip 3434-gene
Acetamidofluorene (200
mgkg) aniline (200 mg
kg) bromobenzene (900
mgkg) butyl hydroxytol
(1000 mgkg) dieldrin
(30 and 45 mgkg)
disulfiram (2000 mgkg)
ethinyl estradiol (500
mgkg)
hexachlorocyclohexane
gamma (40 65 80 mg
kg) 4-methylthiazole
(120 mgkg) nimesulide
(500 mgkg) piperonyl
butoxide (4000 mgkg)
precocene I (500 mgkg)
pulegone (400 mgkg)
tannic acid (3000 mg
kg) trans-anethole
(600 mgkg)
Gene name accession
number in a manuscript
table
(McMillian et al
2004)
NIEHS website at httpdirniehsnihgovmicroarraydatasetshome-pubhtm
EDGE website at httpedgeoncologywiscedu
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 43
and cisplatin (Table 1) In the majority of cases the resulting
number of differentially expressed genes is a very small
subset of the starting number on the microarray following
clustering or other types of analysis Upon closer examina-
tion of these publications the majority of them either
provide images of a heat map andor a table listing a gene
name accession number and expression change Very few of
the published studies (Tables 1ndash3) provide the original raw
microarray data file at a freely accessible website hence
restricting further analysis by scientists using other software
Although in some cases it is possible to cut and paste the
gene expression data tables from the publication pdf files
this is not always the case In the worst case scenario one
would have to manually retype gene lists or extract them
from heatmaps as binary type data As not all computational
researchers will have a laboratory available to them to
generate such quantities of toxicogenomics data the latter
points are important if we are going to continue to see
innovation in software development for this data This will
require free unrestricted access to data published Similarly
if we are to discern lsquolsquofingerprintsrsquorsquo for molecules acting with
a similar or identical mechanism we will need databases of
many diverse chemical structures that have been tested in a
similar manner There is therefore considerable interest in
the current databases being developed by the NIEHS FDA
and other groups which should help to improve the situation
by providing freely accessible microarray and other toxicity
related data Two of these databases being developed in the
public domain are Chemical Effects in Biological Systems
(CEBS) (httpwwwniehsnihgovnctcebshtm) (Mattes
Pettit Sansone Bushel amp Waters 2004 Waters et al
2003) which will accommodate gene expression profiles
proteomics and metabolomics data and allow complex
queries (Hood 2003a Mattes et al 2004) Similar goals
are being pursued in the development of the ArrayTrack
database at the FDA (Tong et al 2003) The EDGE
database (httpedgeoncologywisceduedgephp) an
expanding public effort at The University of Wisconsin
contains mouse gene expression profiles following treat-
ment with different toxic molecules (Hayes et al 2005
Thomas et al 2001) These separate efforts if widely
adopted should make published studies describing HT data
more readily accessible although it might have been more
efficient to evolve these into a single global database instead
of fragmented repositories
Proteomics data has also been generated in a limited
number of toxicology studies (Table 4) once again this has
been produced with different strains of rats and mice using
different protein chips 2-D gel electrophoresis (2-DIGE)
and mass spectroscopy methods (eg MALDI-MS) The
proteomic data is very rarely accessible to the reader for
their own computational analysis Subsequently there have
Table 2
Literature toxicogenomics data derived from mouse in vivo studies
Compounds Mouse strain Microarray type Compound dose Microarray data
availability
Reference
Benzene Male and female P53
KO mice and
C57BL6
Affymetrix and Incyte
GEM system
300 ppm 6 h per day 5
days a week for 2 weeks
Gene name accession
number and fold change
data in a manuscript
table
(Yoon et al 2003)
Benzene Male 129SvJ Affymetrix MG-U74Av2 100 ppm 6 h per day 5
days a week for 2 weeks
Gene name accession
number and fold change
data in a manuscript
table
(Faiola Fuller
Wong amp Recio
2004)
Phenobarbital CAR and wild type NIEHS Mouse tox chip
8736 genes
100 mgkg for 12 h Accession number gene
name and fold change
for wild type and knock
out mice in a manuscript
table
(Ueda et al 2002)
Aroclor BNF
ciprofloxacin cobalt
chloride TCDD IL-6
LPS PCB-153
phenobarbital
phenylhyrzn TNFa
WY-16463
C57BL6J Custom array with 1200
cDNAs
Aroclor (200 mgkg)
BNF (5 mgkg)
ciprofloxacin (250 mg
kg) cobalt chloride (60
mgkg) TCDD (10 ug
kg) IL-6 (25 ugkg)
LPS (1 mgkg)
PCB-153 (80 mgkg)
phenobarbital (100 mg
kgday) 3 days
phenylhyrzn (100 mg
kg) TNFa (50 ugkg)
WY-16463 (0125wv)
Gene names and fold
change as a heat map
Data is also available in
the EDGE database
(Thomas et al
2001)
TCPOBOP CD-1 female Custom 9000 cDNA
mouse array
1ndash3 h treatment 3 mg
kg body wt
Accession numbers and
fold change data in a
manuscript table
(Locker et al
2003)
3H-12-dithiole-3-thione
(D3T)
Male wild-type and
nrf2-disrupted
Affymetrix murine
genome U74Av2
GeneChip
05 mmolkg Accession number gene
name fold in a
manuscript table
(Kwak et al
2003)
MDMA Male albino Swissndash
Webster (neurons)
15 K mouse cDNA clone
set
47 mgkg Gene name and fold
change in a manuscript
table
(Xie et al 2004)
Phenytoin Female C57BL6 and
LDLRMurine genome-U74Av2 300 mgl Gene name accession
number and fold change
in a manuscript table
(Trocho et al
2004)
Genistein (1000 Agmouseday) or
diethylstilbestrol
(DES) (50 Agmouse
day)
ICR (testes) Custom cDNA
microarray containing
1754 cDNA probes
Genistein (1000 Agmouseday)
diethylstilbestrol (50 Agmouseday)
Accession number gene
name and fold change in
a manuscript tablemdashnote
very few genes
(Adachi et al
2004)
Cocaine and
buprenorphine
Male ICR Mouse DiscoveryArrayi
type I array containing
2688brain-derivedprobes
(Display Systems Biotech
Inc Copenhagen
Denmark)
40 mg kgmdash1 cocaine
once a day for 4 days 40
mg kgmdash1 cocaine plus
025 mg kgmdash1 BUP for
4 days
Data apparently not
available
(Hayase
Yamamoto
Yamamoto Muso
amp Shiota 2004)
Methamphetamine Male C57BLJ6
(striatum)
Affymetrix mouse
genechip mg-U74Av2
oligonucleotides arrays
12 488 genes
40 mgkg Gene accession number
gene ID and name and
signal log ratio in a
manuscript table
(Thomas
Francescutti-
Verbeem Liu amp
Kuhn 2004)
Di(2-ethylhexyl)
phthalate
Male C57BL6 Murine genome U74Av2
Arrays (MGU74Av2)
10 dietary DEHP for
13 weeks
Genbank accession
numbers gene name log
ratio data present as a bar
chart in the publication
data extraction would
take some effort
(Wong amp Gill
2002)
Diethylhexylphthalate Male PPARa null and
wild type
Custom made containing
600 tox genes
1150 mgkgday Gene names available on
a bar chartmdashquantitative
data not easily accessible
(Hasmall et al
2002)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6644
Table 2 (continued)
Compounds Mouse strain Microarray type Compound dose Microarray data
availability
Reference
Acetaminophen C57B16 3 129Ola
hybrid
Test-2 Chips (Affymetrix)
then individual
oligonucleotide
microarrays (Mul1K sub
A and sub B Affymetrix)
that can detect the
expression of 11000
known genes and
expressed sequence tags
(ESTs)
300 mgkg Genbank or SwissProt
ID and fold change data
in a manuscript table
(Reilly et al
2001)
Cadmium chloride
benzo(a)pyrene (BaP)
and trichloroethylene
(TCE)
Male Swiss Webster Custom Chips
containing 148 unique
genes
Various Data in table form few
genes affected for BAP
and TCE
(Bartosiewicz
Penn amp Buckpitt
2001)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 45
been only a very small number of studies that have
combined both transcriptomic and proteomic methods with
a single animal strain after treatment with a drug Hopefully
we will see this change in the future but this will in turn
present considerable challenges as huge amounts of
proteomic data are combined with the equally large
transcript data files
3 Network analysis and databases
From some of the early reviews of systems biology there
has been discussion of its application to drug discovery
(Kitano 2002ab) as well as the utility for ADMETox
(Ekins et al 2002a 2000b) More recently several other
journals have dedicated whole issues to the field of systems
biology However one could consider this quite a broad field
from network or pathway analysis to quantitative simulation
of organelles (Vo Greenberg amp Palsson 2004) whole cells
and organs It is apparent that we are now understanding
organisms from the perspective of computationally gener-
ated networks of protein and ligand interactions (Barabasi amp
Oltvai 2004) Network and pathway tools enable the
analysis of HT data in the context of all known interactions
when using a database as the source Individual reviews
have in some cases indicated that networks will be valuable
for understanding adverse events (Hood amp Perlmutter
2004) drug target identification or validation (Butcher
Berg amp Kunkel 2004) and complex metabolic interactions
(Nicholson et al 2004) A general schematic has been
generated in order to provide a description of the utilization
of such pathway databases and network building algorithms
from the initial parsing of high throughput data to network
comparisons and visualization (Fig 3) High throughput
data can be superimposed and visualized on the various
protein interaction databases available This is accomplished
by using either preset maps that capture current biological
knowledge or by building custom interaction networks
using many different algorithms which can be compared and
statistically evaluated as demonstrated in a very large
number of published examples (Dobrin Beg Karabasi amp
Oltvai 2004 Fiehn 2001 Han et al 2004 Hanisch et al
2002 Ideker et al 2002 Jeong Mason Barabasi amp Oltvai
2001 Jeong Tombor Albert Oltvai amp Barabasi 2000 Li
et al 2004 Milo et al 2002 Nikitin Egorov Daraselia amp
Mazo 2003 Pereira-Leal Enright amp Ouzounis 2004
Rives amp Galitski 2003 Segal et al 2003a Somogyi et al
2001 Spirin amp Mirny 2003 Tornow amp Mewes 2003
Vasquez Flammini Maritan amp Vespignani 2003 Yeger-
Lotem amp Margalit 2003 Yu Zhu Greenbaum Karro amp
Gerstein 2004)
31 Network applications
For example one group has used as an inference the
Bayesian network method for analysis of tissue toxicity from
microarray data as well as a mechanistic simulation for a
different pharmaceutically relevant molecule (Aksenov et
al 2005) Pathway tools and various resources have also
been applied to modeling the networks of nuclear hormone
receptors and their connections with other genes and small
molecules using a manually curated database MetaDrug
(Ekins Kirillov Rakmatulin amp Nikolskaya 2005d) or
MetaCore (Ekins Bugrim Nikolsky amp Nikolskaya 2005)
Transcriptional regulation of many transporters CYPs and
phase II enzymes are regulated by these receptors affecting
endogenous molecule transport metabolism cell growth
proliferation and oxidative stress (Ulrich 2003 Ulrich et al
2004) When the signaling networks and interacting ligands
for the transcriptional factors PPAR FXRRXRA ESR1
AHR HNF4A GCR-h MCR CAR-beta GCR-a LXR-a
CARRXR HNF4 FXR PXRRXR heterodimer PXR
AHRARNT heterodimer PPARaLXRa VDR PPAR-a
are visualized a very complex picture of interactions can be
created (Ekins et al 2005d) This suggests that when we
consider a molecule binding with only one nuclear receptor
we are observing only a fraction of the likely possible
feasible interactions based on the data gathered to date
Table 3
Literature toxicogenomics data derived from in vitro cell studies
Compounds Cell type Microarray type Compound dose Microarray data
availability
Reference
4-Hydroxytamoxifen
estrogen
MCF-7 breast cancer NIEHS ToxChip
microarray consisting of
1901 genes
1 uM hydroxytamoxifen
for a year 10 nM
17b-estradiol
Data available at NIEHS
website
(Hodges et al
2003)
Trovafloxacin Human hepatocytes Affymetrix U133A array 30ndash800 uM 142 genes available in
supplemental tablemdashnot
easily extracted
(Liguori et al
2005)
Estrogen MCF-7 breast cancer NIEHS ToxChip
microarray consisting of
1901 genes
10ndash10 M 17b-estradiol Data available at NIEHS
website
(Lobenhofer et al
2002)
Valproic acid NMRI mice embryo
and P19 mouse
embryocarcinoma
Custom chip including
15K mouse cDNA clone
set
600 mgkg body weight Gene symbol gene
name NIA EST log fold
change in a manuscript
table
(Kultima et al
2004)
Sulindac sulfide Human colorectal
carcinoma SW-480
and HCT-116
NIEHS human 12K chip 10 uM Genbank accession
number gene name and
fold change at various
time points in a
manuscript table data
also available on NIEHS
website
(Bottone
Martinez Collins
Afshari amp Eling
2003)
17Beta-estradiol estriol
estrone genistein
diethylstilbestrol
bisphenol A
nonylphenol
methoxychlor
MCF-7 U95A oligonucleotide
probe arrays (Affymetrix
10 nM (E2 estriol
estrone DES) 10 AM(genistein bisphenol A
nonylphenol and
methoxychlor)
Unigene name gene
name and fold change
for estrogen responsive
and nonresponsive in a
manuscript table
(Terasaka et al
2004)
Ouabain lauryl sulfate
dimethylsulfoxide
cycloheximide
tolbutamide sodium
fluoride diethyl
maleate buthionine
sulfoxamine
potassium bromate
sodium selenite
alloxan adriamycin
hydrogen peroxide
HepG2 Clontech Atlas Human
Stress Toxicology cDNA
arrays (234 genes)
Ouabain (43 uM) lauryl
sulfate (260 uM)
dimethylsulfoxide (128
M) cycloheximide (625
uM) tolbutamide (128
mM) sodium fluoride (3
mM) diethyl maleate
(125 mM) buthionine
sulfoxamine (30 mM)
potassium bromate (25
mM) sodium selenite (30
uM) alloxan (130 mM)
adriamycin (40 uM)
hydrogen peroxide
(4 mM)
Gene name ratio p -
value in downloadable
tables at journal website
(Morgan et a l
2002)
Aflatoxin B(1) (AFB(1))
2-acetylaminofluorene
(2AAF)
dimethylnitrosamine
(DMN)
acetaminophen (APAP)
HepG2 and primary
hepatocytes
Gene filter arrays
containing 31000 genes
10 uM aflatoxin B1
40 mM acetaminophen
100 uM
dimethylnitrosamine
10 uM
2-acetylaminofluorene
None (Harris et al
2004)
Mitomycin C (MMC)
and cisplatin (CIS)
and an alkylating
agent methyl
methanesulfonate
(MMS)
indirect-acting
genotoxins included
hydroxyurea (HU) a
ribonucleotide
reductase inhibitor
taxol (TXL) a
microtubule inhibitor
and etoposide
(ETOP)
L5178Y TK(+-)
mouse lymphoma
Affymetrix mouse
MG-U74A for MMC
and MG-U745Av2
(Affymetrix Inc Santa
Clara CA) for all the
other chemicals a total
of 9977 probe sets
(genes or ESTs) common
to these two array
models
Low mid and high doses Accession number gene
name gene ID statistical
significance at each time
point in a manuscript
table
(Hu et al 2004)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6646
Table 3 (continued)
Compounds Cell type Microarray type Compound dose Microarray data
availability
Reference
Hydroxyurea
(a carcinogen)
p-anisidine
(a noncarcinogen)
and paclitaxel
L5178Y Tk_-mouse
lymphoma
The Twin - Chip Mouse-
74K Digital Genomics
cDNA microarray
10 ngml paclitaxel 313
ugml hydroxyurea 32
ugml p-anisidine
Gene symbol and fold
change in a manuscript
table
(Lee et al 2003)
Acetaminophen
amiodarone clofibrate
erythromycin estolate
isoniazid alpha-
naphtylylisothiocyanate
beta-naphtoflavone
4-pentenoic acid
phenobarbital
tetracycline and
zileuton
Wistar Rat
hepatocytes
DualChip rat hepato
(Eppendorf Hamburg
Germany)
A single concentration
which varied for each
compound
Accession numbers and
gene namemdashfold
changes shown as
colored heat mapmdashnote
easily extracted from
publication
(de Longueville
et al 2003)
Bleomycin and hydrogen
peroxide
Mouse lymphoma
L5178Y TK(+ -)
Clontech Mouse 12K
cDNA microarray (1185
genes)
Bleomycin (25 and 20
ugml) hydrogen peroxide
(5 and 10 ugml)
Gene names and fold
change presented as bar
charts in publication
(Seidel Kan
Stott Schisler amp
Gollapudi 2003)
Bupivicaine
camptothecin
HL-60 Agilent human cDNA
microarray
1 mM Gene name GenBank
accession number
unigene and ratio in a
manuscript table
(Unami
Shinohara
Ichikawa amp Baba
2003)
Bisphenol A Mouse Sertoli TTE3 IntelliGene mouse
expression glass
microarrays (Version
10 Takara Shuzo)
which were spotted with
564 cDNA fragments of
mouse known genes and
approximately 301
expressed sequence tags
(ESTs)
0ndash400 uM Gene name GenBank
accession number and
fold change at time
points in a manuscript
table
(Tabuchi amp
Kondo 2003)
Mitomycin C or
doxorubicin
Hep G2 85 human gene custom
array
10 um mitomycin C
2 um doxorubicin or
2 ethanol
Bar charts with fold
changes in publication
very few genes
(Hong Muller amp
Lai 2003)
Amphotericin B Human peripheral
blood mononuclear
and THP-1
GF211 FKnown Genes_
Genefilter cDNA array
(ResGen) this array
consists of gt4000
individual elements
each representing a
known human gene
5 ugml Accession numbers and
fold expression in a
manuscript table
(Cleary Rogers amp
Chapman 2001
Rogers Pearson
Cleary Sullivan
amp Chapman
2002)
Benzo(a)pyrene diol
epoxide
TK6 human
lymphoblastoid
Human-350 microarray
a glass slide with 350
spotted human cDNA
probes (Phase-1
Molecular Toxicology
0 001 010 or
10 ugml)
Gene names and fold
change at doses in a
manuscript table
(Akerman et al
2004)
Etomoxir HepG2 Clontech AtlasiHuman Stress
Toxicology cDNA arrays
(234 genes)
1 mM etomoxir Gene names and fold
change in a manuscript
table
(Merrill et al
2002)
Tetrodotoxin Human glioma cell
line HTB-138
Using Affymetrix
GeneChip (HG-U133A
10 and 20 mM Affymetrix ID Genbank
ID gene name gene
symbol and fold change
in a manuscript table
(Raghavendra
Prasad Qi
Srinivasan amp
Gopalakrishnakone
2004)
Methotrexate
mercaptopurine
Human acute
lymphoblastic
leukemia
Affymetrix U133A chip Low and high dose and
combination
Data available as
supplemental data online
(Cheok et al 2003)
Prednisolone
vincristine
asparaginase
daunorubicin
Human acute
lymphoblastic
leukemia
Affymetrix U133A chip Various Data available as
supplemental data online
(Holleman et al
2004)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 47
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6648
A second study has indicated how a natural language
processing method CCNet was used to show the genes
regulated by the nuclear hormone receptor FXR (Apic
Ignjatovic Boyer amp Russell 2005) These automated
methods enable a more complete understanding of the
complexity of the transcriptional factors (Ekins Mirny amp
Schueltz 2002b Plant 2004 Ulrich 2003) but ultimately
rely on the quality of the content of the underlying database
of literature interactions This is a key consideration that is
often overlooked For example the gold standard database is
one that is manually curated to ensure the fidelity of the
direct interaction and is preferable to one generated
computationally by algorithms like natural language pro-
cessing (Nikolsky Nikolskaya amp Bugrim 2005) The
advantage of interaction networks over clustering has been
demonstrated in one study using MetaCore (Nikolsky
Ekins Nikolskaya amp Bugrim 2005) by reanalysis of a
published microarray study of G0-arrested MCF-7 breast
cancer cells treated with estrogen and 4-hydroxytamoxifen
(Hodges et al 2003) After producing integrated gene
networks for each treatment strikingly different patterns
were displayed although both contained early transcriptional
factors myc jun and fos Only the estrogen network featured
induced genes essential for all cell cycle phases (Nikolsky et
al 2005) Similarly microarray data for benzene toxicity
(Yoon et al 2003) has been re-analyzed by focusing on the
genes assessed on the p53 pathway (Ekins et al 2005e) We
envisage that a database of such networks for toxic
compounds will be used for comparing between different
molecules and used in the development of predictive
algorithms for Systems-ADMETox modeling in future
Another approach to using such pathway approaches is to
visualize the results of quantitative structure activity models
for predicting molecules binding to enzymes transporters
receptors and ion channels (Ekins Andreyev et al in press
2005e)
It is also possible to simultaneously interpret high
throughput data and predictions on interaction networks
providing a novel approach to predicting and understanding
potential undesirable drugndashdrug or off target effects in the
area of systems pharmacology An example data set uses
percent inhibition data for clotrimazole and ticonazole
which were screened against many different assays at a
single concentration in a commercially available database
BioPrint (Cerep Redmond WA) as published recently
(Fliri Loging Thadejo amp VOlkman 2005) The data for
10 assays has been arbitrarily encoded as inhibitors (gt50
inhibition) or non-inhibitors (lt50 inhibition) in a text file
which was then loaded into MetaCore The analyzed
network algorithm was then used which generates a large
network and fragments it into sub-networks each with a Z-
score and p-values for ranking according to saturation with
objects from the initial gene list The Gene-ontology
processes are also mapped to the gene list and individual
networks In this example a statistically significant network
was generated for the different proteins (Fig 4
p =2838e31) This network also maps the Gene Ontology
processes for the activation of MAPK (118 of genes p
value 9143e07) signal transduction (333 1600e05)
regulation of transcription DNA-dependent (294
2786e04) regulation of inflammatory response (39
3746e04) and the regulation of blood pressure (78
4230e04) This example network indicates how molecules
of the same or different therapeutic classes could be
evaluated for their effects as a graph either together as in
this case or individually This would be useful to indicate
potential off target effects and identify structurally dissimilar
molecules with similar network patterns Such networks
could then be compared to assess network overlap or
differences between molecules and their inhibition of
multiple proteins This type of unique visualization of high
throughput screening data illustrates how the target proteins
may be connected as a network to infer the possible
downstream effects of inhibition
With the burgeoning number of freely available online
and commercial databases that can be used for pathway
construction numbering in the hundreds there have been
suggestions to impose standards for model exchange
querying and visualization (Cary Bader amp Sander 2005)
To date there has been little discussion with regards to
standardization of ADMETox related databases although
there has been considerable discussion relating to drug
metabolism database generation (Erhardt 2003) This is
certainly an important area to address in the future There is
already a growing literature related to ADMETox that is
partially captured in the several commercially available
databases (Ekins et al 2005e) but to date there have been
limited academic efforts to capture data for transporters
with the human membrane transporter database (Yan amp
Sadee 2000) TP-search transporter database (httpwww
ilabrisewasedaacjptp-search) drug interaction database
(httpwwwdruginteractioninfoorgDatabaseinfoaspx)
nuclear hormone receptors (Nakata Yukawa Komiyama
Nakano amp Kaminuma 2002) the ADME-AP database
(Sun Ji Chen Wang amp Chen 2002) and PharmaGKB
(Oliver et al 2002) DSStox (httpwwwepagovnheert
dsstox) TOXNET (httptoxnetnimnihgov) that are
readily accessible
In order to generate accessible pathways using any of
the available software a large enough set of object
identifiers are required to map onto the underlying data-
base To demonstrate this datasets from toxicogenomics
studies have been evaluated with both the KEGG pathway
database and a commercially available product MetaCore
(wwwgenegocom) These gene or protein lists range in
size from 21 to 1853 objects In virtually all cases more
identifiers are mapped to networks in MetaCore and this
also seems independent of the identifier type used
(Unigene Affymetrix Genbank or Locuslink Table 5)
These numbers will obviously change as more information
is added to the respective databases hence the more
objects that are mapped from a dataset The more extensive
Table 4
Literature derived toxicoproteomics data
Compounds Data source Microarray type Compound dose Microarray data
availability
Reference
Carbon
tetrachloride
Male Wistar rats Affymetrix rat chip 8799
probes+proteomics
study
6ndash24 h exposure Genbank SwissProt and
binary data in a
manuscript table
(Fountoulakis
2004)
Paracetamol CD-1 male mice Custom mouse tox blots
with 450 genes
RTQ-PCR+proteomics
study 2-DIGE+
MALDI-MS
150 or 500 mgkg Gene name IMAGE ID
GenBank accession
number fold induction
SwissProt identifier and
protein abundance
change in manuscript
tables
(Ruepp Tonge
Shaw Wallis amp
Pognan 2002)
Paracetamol AP-1 male mice Affymetrix murine 11K
set+proteomics study
Up to 500 mgkg Gene names and fold
changes at multiple
doses and time points in
a manuscript tablemdash
proteomics data not
accessible
(Coen et al 2004)
Oxazepam or
Wy-14643
Male B6C3F1 mice NIEHS Mouse Chip
(8700 genes) 2-DIGE
and MS
Oxazepam (2500 ppm)
Wyeth (Wy)-14643
(500 ppm)
Proteomics data in a
manuscript table
Genbank accession
number gene name and
fold change in a
manuscript table data
also available to
download from NIEHS
website
(Iida et al 2003)
Compound A
(PPAR gamma
ligand)
Female rats CrlCD
(SD)IGS BR
Proteomics 2-DIGE and
MS
250 mgkgday up to
5 days
Accession number and
protein name and
average ratio in a
manuscript table
(Meneses-Lorente
et al 2004)
Bromobenzene Male Wistar rats Proteomics 2-DIGE and
MS custom 3000 cDNA
rat chip
5 mmolkg Accession number gene
name fold change in a
manuscript table protein
names along with bar
charts
(Heijne Stierum
Slijper van
Bladeren amp van
Ommen 2003)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 49
the network that can be generated (as it will consist of more
nodes) then a more comprehensive understanding of the
networks is possible The data available currently in the
literature can be used to evaluate such pathway and
network generation tools Recently we have used several
of the published studies (Tables 1ndash3) with MetaCore to
visualize networks for acetaminophen furan carbon tetra-
chloride benzene and cisplatin showing genes involved in
In vivo
In vitro
In Silico
1970s 1980s 1990s 2000s
Systems Biology ()
OMICS
Fig 2 The timeline for major paradigms in ADMETox
oxidative stress (Ekins Giroux Nikolsky Bugrim amp
Nikolskaya 2005c)
Other important pathwaynetwork building tools that could
potentially be applied to toxicogenomics data include
Ingenuity pathways analysis (httpwwwingenuitycom)
PathArt (httpwwwjubilantbiosyscompdhtm) Pathway
Assist (httpwwwariadnegenomicscomproctspathway
html) (Nikitin et al 2003) and several other databases
deposited at the Pathway Resource List (httpcbiiomskcc
orgprl) All of these products have unique underlying
proprietary pathway databases which are compiled manually
or automatically with text mining tools or a combination of
both We are still waiting for studies that provide a comparison
of different database tools or network building algorithms
Until then theremay be someoverlap but also some differences
between the results obtained from more than one network
method due to the database content and the algorithms used
The reader is recommended to evaluate for themselves several
technologies and select those with the most appropriate fit to
their own specialized needs
In the next Sections the further application of some
available network and database tools will be described with
Microarray or other high throughput data
Upload list of geneprotein identifiers and fold change significance etc (raw data or after selection of the differentially expressed
Genes using other statistical methods)
Parse database and generate interaction network with different algorithms or visualize on maps
Filter Networks
Access significance of the interaction networks
Determine GO processes
Compare 2 or more networks intersection overlap etc
Export gene list
Export gene list
generate interaction network with different algorithms or visualize on maps
Visualization
Fig 3 Schematic for the utilization of pathway tools for assessing high throughput data
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6650
specific detailed reference to transporters and enzymes but
these technologies can also be applied elsewhere as
described above
Fig 4 Network for high throughput screening data for clotrimazole and ticona
cholecystokinin CCR1 Choline transporter Chloride channel catechol-O-methyl
and co workers (Fliri et al 2005) Nodes highlighted with large blue circles rep
molecules showing gt50 inhibition in the assay blue circles represents both mol
inhibits gt50 and one inhibits lt50 Ligands (large) linked to transcriptional f
(wwgenegocom) (For interpretation of the references to colour in this figure leg
311 The role of transporters
A diverse array of organic solutes such as nutrients
neurotransmitters and drugs are transported by specialized
zole screened against 10 in vitro assays (Human Cannabinoid 1 Human
transferase COX2 CYP1A2 CYP2B6 CYP2C19) data published by Fliri
resent assays used in the study Red circles on these nodes represent both
ecules showing lt50 inhibition chequered circles represent one molecule
actors enzymes and transporters via edges using the MetaCorei database
end the reader is referred to the web version of this article)
Table 5
Datasets acquired from publications and other sources and used to evaluate two databasepathway tools KEGG and metaCore
Dataset Number of objects
genes proteins in list
Number of objects
mapped with KEGG
Number of objects mapped
with MetaCore on networks
Reference for microarray
data gene list
Tox Chip 10 (NIEHS) 1853 405 1498 httpsdir-appsniehsnihgov
mapsguestclonesrchcfm
4-Hydroxytamoxifen and
estrogen
1617 434 1343 (Hodges et al 2003)
Mitochondrial proteins 722 156 388 (Gaucher et al 2004
Taylor et al 2003)
Bromobenzene
(24 and 48 h)
130 41 89 (Heijne et al 2004)
Acetaminophen 30 19 23 (Heinloth et al 2004)
Acetaminophen 84 29 64 (Huang et al 2004)
Furan 185 64 139 (Huang et al 2004)
Tetrodotoxin 116 31 86 (Raghavendra Prasad et al
2004)
Benzene 73 16 62 (Yoon et al 2003)
Benzene 76 5 53 (Faiola et al 2004)
Carbon tetrachloride 37 8 26 (Young et al 2003)
Estrogen 94 33 90 (Lobenhofer et al 2002)
Trovafloxacin 142 20 82 (Liguori et al 2005)
Phenobarbital 37 13 28 (Ueda et al 2002)
L-742694 (liver)+ 45 17 19 (Hartley et al 2004)
L-742694 (intestine)+ 23 11 10 (Hartley et al 2004)
A-277249 21 7 9 (Waring et al 2002)
All files were converted to Locuslink identifiers except as noted () unigene () Affymetrix (+) Genbank identifiers were used
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 51
proteins across cellular membranes These may function as
passive processes or active processes energized by the
hydrolysis of ATP or coupling to the co-transport of counter
ions down an electrochemical gradient such as Na+ H+ and
Cl There are many thousands of transporters which can be
classified into distinct superfamilies One of these the solute
carrier class (SLC) is rapidly expanding and contains over
30 families and 200 members The ATP-binding cassette
(ABC) contains 7 families and over 48 members including
P-glycoprotein (P-gp) and MRP subfamilies (Zhang Knipp
Ekins amp Swaan 2002a Zhang Phelps Cheng Ekins amp
Swaan 2002b) Transporters have a key role in clinical
pharmacology with many drugs specifically targeting them
Numerous drugs share transport pathways with nutrients
and transporters have a role in oral absorption drug
bioavailability drug resistance excretion and ultimately
pharmacokinetics and pharmacodynamics
Polymorphism of drug transporters may be a key factor
in drug interactions and lack of effectiveness This field has
become known as pharmacogenomics and is focused on
understanding of inherited DNA sequence variations (poly-
morphisms and mutations) revealed by xenobiotics (Evans
amp McLeod 2003 Weinshilboum 2003) Over the last few
decades many genes have been directly linked to the
mechanisms of response (Evans amp McLeod 2003 Wein-
shilboum 2003) such that 20ndash95 of variability to drug
response is inheritable (Evans amp McLeod 2003) This
phenotypical variability is mainly caused by single nucleo-
tide polymorphisms (SNPs) present in anywhere from 1 to
50 of the population resulting in either lower protein
activity incorrect folding or rapid degradation via proteo-
somes (Weinshilboum amp Wang 2004) A number of
structurally diverse molecules bind to P-gp which is
expressed in many tissues and has numerous SNPs one of
which (C3435T) affects the expression level in the
duodenum and therefore can impact the absorption of
molecules which would be substrates for this transporter
(Sakaeda Nakamura amp Okumura 2002) The human
proton-dependent dipeptide transporter (hPEPT1) can also
affect the absorption of molecules in the intestine and
recently 9 SNPs were found with only one displaying a
reduced transport capacity (Zhang et al 2004) The sodium-
dependent carnitine cotransporter OCTN2 can possess
mutations and these result in primary carnitine deficiency
which impacts fatty acid oxidation and is characterized by
many clinical manifestations (Lahjouji Mitchel amp Qureshi
2001) The organic cation transporter 1 (OCT1) is also
important in the transport of numerous xenobiotics and
endobiotics Recently 4 SNPs were identified in the
Japanese population and when functionally characterized
in vitro the uptake of cations was reduced significantly for
some of these mutations indicating that this would likely
contribute to inter-individual variations in metabolism of
drugs which were transported via OCT1 (Sakata et al
2004)
3111 Clinical relevance of transporters The pregnane
X-receptor (PXR) is a transcriptional regulator of the
enzyme human MDR1 (P-gp) MRPs and OATP (Wang amp
LeCluyse 2003) CYP3A (Bertilsson et al 1998 Blumberg
et al 1998 Kliewer et al 1998) and CYP2C89 (Synold
Dussault amp Forman 2001) as well as many other genes
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6652
involved in the transport metabolism and biosynthesis of
bile acids (Staudinger Liu Madan Habeebu amp Klaassen
2001) However the additional receptors CAR FXR LXR
and other nuclear receptors take part in a complex network
of interactions to control these and other proteins Thus
elucidation of the regulatory networks which control the
expression of these transporters is also important To date
most of the research has centered on efflux transporters but
there has been considerable interest in uptake transporters
such as the organic anion transporter polypeptide (OATP
((Kim 2003) see also later Section on OATP)
There are several specific examples of the importance of
drug transporters to the clinical development of drugs One
example is the insulin sensitizer troglitazone which was
withdrawn due to hepatotoxicity although the precise
mechanism appears to have been unclear until recently
The major metabolite is a sulfated species and is suspected
of being responsible for the observed toxicity The recent
assessment of the organic anion transporting polypeptides
OATP-C and OATP8 expressed on the hepatocyte baso-
lateral membrane indicated that sulphated troglitazone has a
high affinity for the former and possibly lower affinity for
the latter (Nozawa et al 2004) This metabolite would
therefore be expected to accumulate in hepatocytes and
inhibit the bile salt export pump and Mrp2 Because
polymorphisms have been shown for OATP-C (Tirona
Leake Merino amp Kim 2001) it is also possible that these
may result in the accumulation of the metabolite and in turn
elicit idiosyncratic toxicity A second relevant example of
the impact of transporters is the clinically significant drugndash
drug interaction between cerivastatin and cyclosporine A
which occurs via the OATP-C transporter (Shitara Itoh
Sato Li amp Sugiyama 2003) A third example are the HIV
protease inhibitors saquinavir ritonavir and indinavir which
are transported by MRP2 in vitro and other drugs such as
probenecid and sulfinpyrazone are able to enhance this
transport Transport by MRP2 suggests that these com-
pounds will have decreased bioavailability due to increased
clearance and other drugs could aggravate this situation by
further enhancing transport (Huisman et al 2002) Sim-
ilarly the rifampicin mediated induction via PXR of MRP2
and P-gp in healthy subjects was found to significantly
decrease the AUC and also correlated with intestinal
expression of these transporters This transporter is also
inducible by cisplatin 2-AAF and phenobarbital (Schrenk
et al 2001) indicating multiple mechanisms may be
involved In other species such as rat commonly used as a
toxicity model orthologs of the transporters such as OATP2
are expressed and can be induced with ligands for PXR like
PCN (Guo Choudhuri amp Klaassen 2002) This is useful
knowledge because the advent of microarray technology
allows one to dose a rat with a xenobiotic and assess
thousands of genes simultaneously in a particular tissue For
instance animals dosed with known nephrotoxins have
shown some upregulation of the NandashKndashCl transporter
however the authors suggested genomic responses are
stronger soon after exposure before declining (Fleck et
al 2003) Some transporters may therefore be specifically
targeted by drugs in one tissue such as the CNS but these
same transporters may also be expressed elsewhere in the
body hence off-target effects may result in toxicity The
serotonin transporter is one such example which is
expressed in the lungs and brain Some substrates for this
channel like fenfluramine can result in primary pulmonary
hypertension as they accumulate in pulmonary cells (Roth-
man Ayestas Dersch amp Baumann 1999) Similarly P-gp is
expressed at the blood brain barrier and intestine impacting
the efficacy and bioavailability of drugs
3112 Transporter network examples ABCA1 The
ABCA1 transporter mediates the first step of cholesterol
transport Mutations in this gene cause Tangier disease
which results in severe HDL deficiency cholesterol
accumulation in macrophages and attendant atherosclerosis
This transporter represents a drug target for upregulation
modulating cholesterol metabolism and prevention of
cardiovascular disease (Oram amp Lawn 2001) In vitro
ABCA1 can be inhibited by the sulfonylurea glybenclamide
(Field Burn amp Mathur 2004 Wang Silver Thiele amp Tall
2001) In order to illustrate the advantages of mapping drug
transporters as networks onto functional models alongside
other proteins one can consider the example of ABCA1
The query of the MetaCorei database shows that this
transporter appears on three manually curated pathway maps
representing the Fstate of the art_ knowledge derived from
reliable high quality literature sources One can also use the
individual maps as an interface to access the underlying
layers of information about the transporter including the list
of encoding genessplice variants with known SNPs In
addition to browsing MetaCorei a user can also build
custom networks around ABCA1 using the network-
construction tool (Fig 5) Such a visualization utility may
be very helpful for identification of all putative pathways
around a particular transporter or compound of interest The
ABCA1 network created by this tool shows that this
transporter at the time of writing is linked directly to
twenty-five other objects such as APOE1 and LXR Many
of its neighbors have their own SNPs that could be
important in determining interactions between transport of
a drug and normal human transport of endogenous ligands
in health or disease
3113 Transporter network examples OATP The
OATPs are key membrane bound transporters expressed in
many organs including intestine liver lung choroid plexus
blood brain barrier and other organs (Tamai et al 2000)
This family of transporters is capable of mediating the
sodium-independent transport of a diverse array of mole-
cules such as steroid conjugates organic anions and
xenobiotics by coupling uptake with efflux of bicarbonate
(Satlin Amin amp Wolkoff 1997) glutathione or its
conjugates (Hagenbuch amp Meier 2004) The inhibition of
Fig 5 The network of direct gene interactions around the ABCA1 transporter gene (centre) generated with MetaCorei The interation types between nodes are shown as small colored hexagons eg unspecified
allosteric regulation binding cleavage competition covalent modification dephosphorylation phosphorylation transcription regulation transformation When applicable interactions also have a positive or
negative effect and direction Ligands (purple) linked to other proteins (blue blobs) transfactors (red) enzymes (orange) (For interpretation of the references to colour in this figure legend the reader is referred to
the web version of this article)
SEkin
sJournalofPharm
acologica
landToxico
logica
lMeth
ods53(2006)38ndash66
53
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6654
this transporterrsquos hepatic uptake of other compounds may be
important for reported drugndashdrug interactions (Kim 2003)
described earlier (Shitara et al 2003) as well as cerivastatin
with gemfibrozil (Shitara Hirano Sato amp Sugiyama 2004)
OATP1B1 (previous names OATP-C LST-1 OATP2
SLC21A6) represents the most studied human OATP to
date (Meier amp Stieger 2000) and is expressed on the
basolateral plasma membrane of hepatocytes Several single
nucleotide polymorphisms have been identified in the
OATP1B1 gene in European-Americans African-Americans
(Tirona et al 2001) and Japanese (Nozawa et al 2002)
dramatically impacting the transport of ligands such as
pravastatin (Mwinyi Johne Bauer Roots amp Gerloff 2004
Nishizato et al 2003) estrone-3-sulfate (Nozawa et al
2002 Tirona et al 2001) Rifampin (Tirona Leake
Wolkoff amp Kim 2003) and estradiol 17h-d-glucuronide(Tirona et al 2001)
The regulation of SLCO may be affected during
extrahepatic cholestasis bile duct ligation bile salt induced
Fig 6 Networks generated with the autoexpand algorithm in MetaCorei to illustr
be interconnected with other protein regulatory signaling information Informa
unspecified allosteric regulation binding cleavage competition covalent m
transformation When applicable interactions also have a positive or negative e
transfactors (red) enzymes (orange) (For interpretation of the references to colour
cholestatic hepatitis (Dumont et al 1997 Meier amp Stieger
2000 Rost et al 2003) and primary sclerosing cholangitis
(Oswald Kullack-Ublick Paumgarter amp Beuer 2001) In
particular OATP1B1 appears to be regulated by the liver-
enriched transcription factor hepatocyte nuclear factor 1a
(HNF1a) which binds to the promoter region of this
transporter (Jung et al 2001) Site directed mutagenesis
of this binding site resulted in inactivation suggesting the
critical nature of the interaction with HNF1a Bile acids
such as CDCA have been shown to transcriptionally repress
HNF1a in vitro via inhibition of the transactivating effect of
HNF4a on HNF1a (Jung amp Kullak-Ublick 2003) After
screening many rat and human uptake transporters in vitro
OATP1B1 was also shown to modulate the PXR response
by controlling rifampin retention in the cell and therefore
affecting the induction of CYP3A4 and other gene products
such as P-gp (Tirona et al 2003)
Some of the literature for OATP1B1 human substrate
data has been annotated into MetaCorei to illustrate the
ate how ligands for the human OATP-C (OATP1B1) transporter (centre) can
tion on the type of interaction between objects is hidden for clarity eg
odification dephosphorylation phosphorylation transcription regulation
ffect and direction Ligands (purple) linked to other proteins (blue blobs)
in this figure legend the reader is referred to the web version of this article)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 55
visualization of the complex interconnections between this
transporter its ligands regulatory factors and signaling
molecules already in the database (Fig 6) The network was
generated with the autoexpand algorithm in the software
representing one of multiple available algorithms for
connecting genes ligands and other objects in the database
Clearly if more ligands and their connections are added to
the database the complexity of the network will increase
considerably The OATP-C (OATP1B1) gene details can be
viewed upon querying the database and links are provided
to other public databases This page can be used to highlight
the multiple synonyms for this gene as well as links to the
multiple SNPs identified to date
3114 Transporter microarray data Microarrays have
generally been limited in the number of transporters present
on them (Annereau et al 2004) however they have been used
in an attempt to correlate pharmacokinetic properties with
gene expression for valacyclovir (Landowski et al 2003) as
well as understand the expression profile in different tissues
or cell lines upon food component or xenobiotic treatment
(Anderle Huang amp Sadee 2004) This lack of transporters
on microarrays has prompted some groups to produce their
own arrays with a heavier emphasis on transporters These
arrays have for example then been used to demonstrate the
upregulation of ABC transporters and down-regulation of
GST-Pi in cell lines resistant to colchicines or 9-nitro-
camptothecin (Annereau et al 2004) The genes that were
significantly up or down-regulated in this particular study
were used to build networks with MetaCore (Fig 7A B) and
the similarities between them were assessed (Fig 7C)
Although there were only a small number of significantly
changed genes in common (IL-8 Fos GST-Pi Calpactin and
Ubiquitin hydrolase) it is perhaps likely that there is a much
larger common gene network that is important for drug
resistance although a much larger number of cell lines and
drug treatments need to be evaluated to produce a definitive
drug resistance signature involving transporters enzymes
and transcriptional regulators
312 Applications to enzymes
As we have already described it may be particularly
valuable to visualize enzymes as networks to show
interactions with transcriptional regulators and ligands
For example a key enzyme is CYP3A4 which metabolizes
40ndash45 of all drugs and has relatively few SNPs
(Ingelman-Sundberg 2004) Using a second software suite
MetaDrugi (wwwgenegocom) it is possible to construct
a custom network around this or other drug metabolizing
enzymes (Fig 8) In this case the gene network for
CYP3A4 highlights all of the major transcriptional
regulators and several more distant linked proteins and
ligands connected on the network that may be useful for
further study (Fig 8) Substrates inhibitors as well as
regulatory factors and other enzymes can be observed
connected on this network Due to the many hundreds of
known inhibitors and substrates only a small number are
shown here for clarity We assume that if a perturbation in
a pathway (eg due to a nonfunctional enzyme) is linked
to a certain pathologic condition a similar perturbation
caused by the interference from xenobiotic metabolism
(eg competitive inhibition of the same enzyme) may
result in identical effects As microarray gene expression
data is increasingly generated the role of enzyme
regulation in toxicity of certain xenobiotics will become
more apparent from either in vivo or in vitro studies The
visualization of such signature gene networks involving
transporters and enzymes their ligands and regulatory
factors will also be important for future toxicity prediction
methods We have recently generated visualizations of
microarray data from MCF-7 cells treated with 4-hydrox-
ytamoxifen to show that some of the key genes involved in
metabolism and transport are upregulated (Ekins et al
2005d) In addition we have made predictions with various
QSAR models in MetaDrug to indicate the involvement of
PXR CYP3A4 and P-gp (Ekins et al 2005e) Therefore it
appears likely that 4-hydroxytamoxifen could increase its
own metabolism as well as efflux from cells via P-gp
which can be visualized on networks Any decrease of
function of these enzymes or transporters in a population
would likely result in changes in the metabolism and
transport of this active metabolite potentially impacting
the clinical effect This represents one example of how
both pathway tools QSAR models and network building
algorithms can be used with different types of predicted
and experimental data to allow visualization of potential
compound interactions or toxicity
313 Future network applications
As the population ages an increasing incidence and
prevalence of systemic diseases especially chronic diseases
have occurred among older adults This has resulted in an
increase in medications used concomitantly by this
population which presents challenges for drugndashdrug
interactions Physiologically elderly patients may behave
differently to the younger patients for which the drugs are
initially developed for and tested on Many pharmacoki-
netic investigations in the elderly population reveal
decreased clearance of lipophilic drugs metabolized by
the cytochrome P450 enzymes however few studies have
evaluated aging-dependent or gender-related changes in
specific P450 enzymes (Hunt Westerkam amp Slave 1992)
Age-related physiological changes such as a reduction in
liver mass hepatic metabolizing enzyme activity liver
blood flow and alterations in plasma drug binding may
account for the decreased elimination of some metabolized
drugs in the elderly It is particularly difficult to separate an
effect of aging from the variation in the rate of metabolism
due to factors such as individual metabolic phenotype
(slow or fast metabolizer due to SNPs) environmental
influences concomitant disease states and drug intake
(drugndashdrug interactions) The available data suggest that
Fig 7 Networks for drug resistant cell lines generated with the Auto expand algorithm in MetaCore A KB-3-1 vs KB-8-5 B ATC-DU-145 vs RCO1 C node overlap of A and B with initial nodes highlighted
with blue or red solid circles (Initial gene lists derived from (Annereau et al 2004)) Information on the type of interaction between objects is hidden for clarity eg unspecified allosteric regulation binding
cleavage competition covalent modification dephosphorylation phosphorylation transcription regulation transformation When applicable interactions also have a positive or negative effect and direction
Ligands (purple) linked to other proteins (blue blobs) transfactors (red) enzymes (orange) (For interpretation of the references to colour in this figure legend the reader is referred to the web version of this article)
SEkin
sJournalofPharm
acologica
landToxico
logica
lMeth
ods53(2006)38ndash66
56
Fig7(continued)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 57
Fig7(continued)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6658
Fig 8 The network of gene interactions around the CYP3A4 enzyme (centre) from MetaCorei Information on the type of interaction between objects is
hidden for clarity eg unspecified allosteric regulation binding cleavage competition covalent modification dephosphorylation phosphorylation
transcription regulation transformation When applicable interactions also have a positive or negative effect and direction Ligands (purple) linked to other
proteins (blue blobs) transfactors (red) enzymes (orange) (For interpretation of the references to colour in this figure legend the reader is referred to the web
version of this article)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 59
the initial doses of drugs metabolized by these enzymes
should be reduced in older patients according to the clinical
response In most published studies the elderly appear at
least as responsive as the young to inducers or inhibitors of
P450s (Durnas Loi amp Cusack 1990) More recently there
has been some suggestion that there are age related
reductions in function of some specific P450s such as
CYP3A4 (Patki et al 2004) and this could occur at the
level of regulation However earlier studies with the same
enzyme showed no change in clearance with age (Hunt et
al 1992) To date there has been even less examination of
the transporter functions and any changes with age
(Kinirons amp OrsquoMahony 2004) so the current understand-
ing of the effects of aging on metabolism and transport is
anything but transparent This represents an extreme
challenge for the pharmaceutical industry how to predict
whether a drug has an affinity for an enzyme or transporter
and whether this may be clinically important if it shows a
decline in expression or function with aging Any decrease
of function of these enzymes or transporters in an elderly
population would result in changes in the metabolism and
transport of metabolites potentially impacting the clinical
effect This represents another example of how computa-
tional approaches may perhaps be used with different types
of predicted and experimental data to allow visualization of
potential compound interactions or toxicity in elderly
populations We may see different gene networks high-
lighted as humans age and these may be modified by drug
treatment and coadministration This represents an area
were network analysis could be applied in the future and is
in need of considerable further research
The collection of microarray data in databases such as
CEBS ArrayTrack and EDGE represents a future resource
for computational gene network analysis One could
envisage that ultimately in each case such data is converted
to one or more networks that are also displayed for the user
and can be used to compare treatments from in vivo and in
vitro experiments This would represent a different approach
to clustering the data as currently implemented in one of
these efforts (Hayes et al 2005) and may condense large
amounts of experimentally derived data into a readily
interpreted network
4 Discussion
Previously in this journal the progress of many
research groups in predicting human ADME parameters
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6660
in silico (Ekins et al 2000b) and approaches for drug
metabolism (Ekins Ring Grace McRobie-Belle amp
Wrighton 2000a) have been described Both of these
reviews commented on moving HT assays for ADME
Tox much earlier in drug discovery which had also been
indicated by other groups The initial reviews also
highlighted the likely wealth of data that would become
available and how this could be used for structure
activity relationships alongside the bioactivity data in
computational models It was noted that there was a
paucity of predictive metabolism tools at that time In
addition a growing number of efforts to model whole
cells and organs now a field called systems biology
were recognized as models that could be integrated with
the in silico ADME approaches In summary since these
past reviews virtually all pharmaceutical companies have
attempted earlier high throughput screening for ADME
Tox properties and to some extent the wider application
of computational approaches for physicochemical proper-
ties Systems biology is being quite widely acknowledged
as the new paradigm for understanding complex bio-
logical datasets derived from high throughput technolo-
gies and the accumulated knowledge on human protein
interactions (Hartwell Hopfield Leibler amp Murray 1999
Hood 2003b) Therefore systems biology can be defined
as the integration of genetic proteomic transcriptomic
and metabonomic data using computational methods
(Nicholson amp Wilson 2003) When taken together
information on molecular processes derived from different
sources represents a lsquolsquouniversersquorsquo of putative biological
functionality of which only a small fraction of it will be
realized in a cell at any given time To date systems
biology has been driven by academia and funding bodies
such as the NIH rather than the pharmaceutical compa-
nies Presently there is a great deal of interest from
scientists of all backgrounds in identifying the networks
of cellular pathways and the corresponding physically
interacting proteins
The network building software for systems biology
described in this current review will be valuable to query
high throughput data and known literature interactions in
order to predict potential toxicity in different species In the
future the compilation of published toxicogenomics data-
sets characteristic of different types of toxicity will likely
be available in these software systems to act as a reference
database It is also feasible that we will be able to generate
the annotated datasets which specifically address the
differences between human and rat networks implicated
in toxicity The identification of sub-network modules
conserved between human and rat distinct for toxicity
types or predictive for toxic end-points in human will be
possible Such signature gene networks (Nikolsky et al
2005) can then be verified with other experimentally
derived data prospectively or from preexisting databases
The combined Fhigh throughput datandashin silico network_approaches therefore represents a novel method for access-
ing complex tissue-level processes as networks integrating
all data types based on functional interactions The data-
bases developed and used in this approach will certainly
benefit from further annotation around the drug metaboliz-
ing enzymes and transporters as described herein in terms
of transcriptional regulation and the ligands associated with
them which frequently appear in the literature It will be
important to capture disparities in the assignment of ligands
to enzymes transporters and other proteins as well as
negative data
In summary although there have been numerous
toxicogenomics studies published there is presently a
relatively small number of datasets that are freely available
to perform network analysis of microarray data The
number of studies identifying large numbers of proteins
which are affected by molecule treatment are even scarcer
still while there are several examples of some published
studies that combine such data It is hoped that the
numerous database initiatives for high content and toxicol-
ogy data that are being undertaken will improve the
situation for other researchers that are not currently
equipped to do such microarray studies themselves The
addition of requirements by journals to deposit such raw
data in a freely accessible resource will aid these initiatives
ADMETox groups have seen new technologies and
approaches developed over the last decade that have all
been applied to identify poor compounds earlier (Fig 2)
The latest technologies integrate network building tools
with high content data and databases The current review
described the limited number of networks generated for
ADMETox at present and one hopes that the impact of
such analyses will be commonplace in the future Systems
biology is however more than just applying a network
approach and hence systems-ADMETox will have to
evolve due to the continual pressure to develop newer
technologies This current paradigm combining empirical
data and computational methods should integrate the
complex data already generated making it readily inter-
pretable and valuable for identifying the most promising
compounds in the future
Acknowledgements
Dr Maggie AZ Hupcey is gratefully acknowledged for
editorial assistance Dr Peter W Swaan (University of
Maryland) and Dr Cheng Chang (Ohio State University)
Dr Steve Wright (University of Arizona) Dr K Sandy
Pang (University of Toronto) and Dr Craig Giroux (Wayne
State University) are kindly acknowledged for their support
and discussions My colleagues at GeneGo Sergey
Andreyev Andy Ryabov Eugene Kirillov Eugene A
Rakhmatulin Svetlana Sorokina Andrej Bugrim Tatiana
Nikolskaya Yuri Nikolsky John Metz and Julie Bryant are
all thanked for their considerable contributions to software
development and data annotation
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 61
References
Abe M Yamashita S Kuramoto T Hirayama Y Tsukamoto T Ohta
T et al (2003) Global expression analysis of N-methyl-NV-nitro-N-nitrosoguanidine-induced rat stomach carcinomas using oligonucleotide
microarrays Carcinogenesis 24 861ndash867
Adachi T Ono Y Koh K B Takashima K Tainaka H Matsuno Y
et al (2004) Long-term alteration of gene expression without
morphological change in testis after neonatal exposure to genistein in
mice Toxicogenomic analysis using cDNA microarray Food and
Chemical Toxicology 42 445ndash452
Akerman G S Rosenzweig B A Domon O E McGarrity L J
Blankenship L R Tsai C A et al (2004) Gene expression profiles
and genetic damage in benzo(a)pyrene diol epoxide-exposed TK6 cells
Mutation Research 549 43ndash64
Aksenov S V Church B Dhiman A Georgieva A Sarangapani R
Helmlinger G et al (2005) An integrated approach for inference and
mechanistic modeling for advancing drug development FEBS Letters
579 1878ndash1883
Anderle P Huang Y amp Sadee W (2004) Intestinal membrane transport
of drugs and nutrients Genomics of membrane transporters using
expression microarrays European Journal of Pharmaceutical Sciences
21 17ndash24
Annereau J P Szakacs G Tucker C J Arciello A Cardarelli C
Collins J et al (2004) Analysis of ATP-binding cassette transporter
expression in drug-selected cell lines by a microarray dedicated to
multidrug resistance Molecular Pharmacology 66 1397ndash1405
Apic G Ignjatovic T Boyer S amp Russell R B (2005) Illuminating drug
discovery with biological pathways FEBS Letters 579 1872ndash1877
Baker V A Harries H M Waring J F Duggan C M Ni H A Jolly
R A et al (2004) Clofibrate-induced gene expression changes in rat
liver A cross-laboratory analysis using membrane cDNA arrays
Environmental Health Perspectives 112 428ndash438
Balakin K V Ekins S Bugrim A Ivanenkov Y A Korolev D amp
Nikolsky Y et al (2004a) Quantitative structurendashmetabolism relation-
ship modeling of the metabolic N-dealkylation rates Drug Metabolism
and Disposition 32 1111ndash1120
Balakin K V Ekins S Bugrim A Ivanenkov Y A Korolev D amp
Nikolsky Y et al (2004b) Kohonen maps for prediction of binding to
human cytochrome P450 3A4 Drug Metabolism and Disposition 32
1183ndash1189
Barabasi A -L amp Oltvai Z N (2004) Network biology Under-
standing the cellrsquos functional organization Nature Reviews Genetics
5 101ndash113
Bartosiewicz M Penn S G amp Buckpitt A (2001) Applications of gene
arrays in environmental toxicology Fingerprints of gene regulation
associated with cadmium chloride benzo(a)pyrene and trichloro-
ethylene Environmental Health Perspectives 109 71ndash74
Bertilsson G Heidrich J Svensson K Asman M Jendeberg L
Sydow-Backman M et al (1998) Identification of a human nuclear
receptor defines a new signaling pathway for CYP3A induction
Proceedings of the National Academy of Sciences of the United States
of America 95 12208ndash12213
Blumberg B Sabbagh Jr W Juguilon H Bolado Jr J van Meter
C M Ong E S et al (1998) SXR a novel steroid and xenobiotic-
sensing nuclear receptor Genes and Development 12 3195ndash3205
Borodina Y Rudik A Filimonov D Kharchevnikova N Dmitriev A
Blinova V et al (2004) A new statistical approach to predicting
aromatic hydroxylation sites Comparison with model-based
approaches Journal of Chemical Information and Computer Sciences
44 1998ndash2009
Borodina Y Sadym A Filimonov D Blinova V Dmitriev A amp
Poroikov V (2003) Predicting biotransformation potential from
molecular structure Journal of Chemical Information and Computer
Sciences 43 1636ndash1646
Bottone Jr F G Martinez J M Collins J B Afshari C A amp Eling
T E (2003) Gene modulation by the cyclooxygenase inhibitor
sulindac sulfide in human colorectal carcinoma cells Possible link to
apoptosis Journal of Biological Chemistry 278 25790ndash25801
Boyer S amp Zamora I (2002) New methods in predictive metabolism
Journal of Computer-Aided Molecular Design 16 403ndash413
Bredel M amp Jacoby E (2004) Chemogenomics An emerging strategy
for rapid target and drug discovery Nature Reviews Genetics 5
262ndash275
Bugrim A Nikolskaya T amp Nikolsky Y (2004) Early prediction of drug
metabolism and toxicity Systems biology approach and modeling
Drug Discovery Today 9 127ndash135
Bulera S J Eddy S M Ferguson E Jatkoe T A Reindel J F
Bleavins M R et al (2001) RNA expression in the early character-
ization of hepatotoxicants in Wistar rats by high-density DNA micro-
arrays Hepatology 33 1239ndash1258
Butcher E C Berg E L amp Kunkel E J (2004) Systems biology in drug
discovery Nature Biotechnology 22 1253ndash1259
Butte A (2002) The use and analysis of microarray data Naturalist
Review of Drug Discovery 1 951ndash960
Cary M P Bader G D amp Sander C (2005) Pathway information for
systems biology FEBS Letters 579 1815ndash1820
Cheok M H Yang W Pui C H Downing J R Cheng C Naeve
C W et al (2003) Treatment-specific changes in gene expression
discriminate in vivo drug response in human leukemia cells Nature
Genetics 34 85ndash90
Cleary J D Rogers P D amp Chapman S W (2001) Differential
transcription factor expression in human mononuclear cells in response
to amphotericin B Identification with complementary DNA microarray
technology Pharmacotherapy 21 1046ndash1054
Coen M Ruepp S U Lindon J C Nicholson J K Pognan F Lenz
E M et al (2004) Integrated application of transcriptomics and
metabonomics yields new insight into the toxicity due to paracetamol in
the mouse Journal of Pharmaceutical and Biomedical Analysis 35
93ndash105
Cornwell P D De Souza A T amp Ulrich R G (2004) Profiling of
hepatic gene expression in rats treated with fibric acid analogs
Mutation Research 549 131ndash145
Csermely P Agoston V amp Pongor S (2005) The efficiency of multi-
target drugs The network approach might help drug design Trends in
Pharmacological Sciences 26 178ndash182
Cunningham M J Liang S Fuhrman S Seilhamer J J amp
Somogyi R (2000) Gene expression microarray data analysis for
toxicology profiling Annals of the New York Academy of Sciences 919
52ndash67
de Longueville F Atienzar F A Marcq L Dufrane S Evrard S
Wouters L et al (2003) Use of a low density microarray for studying
gene expression patterns induced by hepatotoxicants on primary
cultures of rat hepatocytes Toxicological Sciences 75 378ndash392
Dobrin R Beg Q K Barabasi A L amp Oltvai Z N (2004)
Aggregation of topological motifs in the Escherichia coli transcriptional
regulatory network BMC Bioinformatics 5 10
Donald S Verschoyle R D Edwards R Judah D J Davies R
Riley J et al (2002) Hepatobiliary damage and changes in hepatic
gene expression caused by the antitumor drug ecteinascidin-743 (ET-
743) in the female rat Cancer Research 62 4256ndash4262
Dumont M Jacquemin E DrsquoHont C Descout C Cresteil D Haouzi
D et al (1997) Expression of the liver Na+-independent organic anion
transporting polypeptide (oatp-1) in rats with bile duct ligation Journal
of Hepatology 27 1051ndash1056
Durnas C Loi C M amp Cusack B J (1990) Hepatic drug metabolism
and aging Clinical Pharmacokinetics 19 359ndash389
Eisen M B Spellman P T Brown P O amp Botstein D (1998) Cluster
analysis and display of genome-wide expression patterns Proceedings
of the National Academy of Sciences of the United States of America
95 14863ndash14868
Ekins S Andreyev S Ryabov A Kirilov E Rakhmatulin E A
Bugrim A et al (in press) Computational Prediction of Human Drug
Metabolism Exp Opin Drug Metab Toxicol
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6662
Ekins S Boulanger B Swaan P W amp Hupcey M A Z (2002)
Towards a new age of virtual ADMETOX and multidimensional drug
discovery Journal of Computer-Aided Molecular Design 16 381ndash401
Ekins S Bugrim A Nikolsky Y amp Nikolskaya T (2005) Systems
biology Applications in drug discovery In S Gad (Ed) Drug
Discovery Handbook (pp 123ndash183) Hoboken NJrsquo Wiley and Sons
Ekins S Giroux C N Nikolsky Y Bugrim A amp Nikolskaya T
(2005) A signature gene network approach to toxicity The Toxoco-
logists 84
Ekins S Kirillov E Rakhmatulin E amp Nikolskaya T (2005) A novel
method for visualizing nuclear hormone receptor networks relevant to
drug metabolism Drug Metabolism and Disposition 33 474ndash481
Ekins S amp McGowan R J (2001) The limits of reductionism The
shifting genomic paradigmrsquos impact on industry and academia
Philsophy in Science 9 1ndash23
Ekins S Mirny L amp Schuetz E G (2002) A ligand-based approach to
understanding selectivity of nuclear hormone receptors PXR CAR
FXR LXRa and LXRb Pharmaceutical Research 19 1788ndash1800
Ekins S Nikolsky Y amp Nikolskaya T (2005) Techniques Application
of systems biology to absorption distribution metabolism excretion
and toxicity Trends in Pharmacological Sciences 26 202ndash209
Ekins S Ring B J Grace J McRobie-Belle D J amp Wrighton S
A (2000) Present and future in vitro approaches for drug
metabolism Journal of Pharmacological and Toxicological Methods
44 313ndash324
Ekins S Waller C L Swaan P W Cruciani G Wrighton S A amp
Wikel J H (2000) Progress in predicting human ADME parameters in
silico Journal of Pharmacological and Toxicological Methods 44
251ndash272
Erhardt P W (2003) A human drug metabolism database Potential roles
in the quantitative predictions of drug metabolism and metabolism-
related drugndashdrug interactions Current Drug Metabolism 4 411ndash422
Evans W E amp McLeod H L (2003) Pharmacogenomicsmdashdrug
disposition drug targets and side effects New England Journal of
Medicine 348 538ndash549
Ezendam J Staedtler F Pennings J Vandebriel R J Pieters R
Boffetta P et al (2004) Toxicogenomics of subchronic hexachlor-
obenzene exposure in Brown Norway rats Environmental Health
Perspectives 112 782ndash791
Faiola B Fuller E S Wong V A amp Recio L (2004) Gene expression
profile in bone marrow and hematopoietic stem cells in mice exposed to
inhaled benzene Mutation Research 549 195ndash212
FDA (2004) Innovation stagnation Challenge and opportunity on the
critical path to new medicinal products
Fiehn O (2001) Combining genomics metabolome analysis and bio-
chemical modelling to understand metabolic networks Comparative
Functional Genomics 2 155ndash168
Field F J Born E amp Mathur S N (2004) LXRRXR ligand activation
enhances basolateral efflux of beta-sitosterol in CaCo-2 cells Journal of
Lipid Research 45 905ndash913
Fleck C Sutter L Appenroth D Koch B Meinhold T Pitack M
et al (2003) Use of gene chip technology for the characterisation of
the regulation of renal transport processes and of nephrotoxicity in
rats Experimental and Toxicologic Pathology 54 401ndash410
Fliri A F Loging W T Thadeio P F amp Volkmann R A (2005)
Biological spectra analysis Linking biological activity profiles to
molecular structure Proceedings of the National Academy of Sciences
of the United States of America 102 261ndash266
Fountoulakis M (2004) Application of proteomics technologies in the
investigation of the brain Mass Spectrometry Reviews 23 231ndash258
Gaucher S P Taylor S W Fahy E Zhang B Warnock D E Ghosh
S S et al (2004) Expanded coverage of the human heart
mitochondrial proteome using multidimensional liquid chromatography
coupled with tandem mass spectrometry Journal of Proteome
Research 3 495ndash505
Gerhold D Lu M Xu J Austin C Caskey C T amp Rushmore T
(2001) Monitoring expression of genes involved in drug metabolism
and toxicology using DNA microarrays Physiological Genomics 5
161ndash170
Guo G L Choudhuri S amp Klaassen C D (2002) Induction profile of
rat organic anion transporting polypeptide 2 (oatp2) by prototypical
drug-metabolizing enzyme inducers that activate gene expression
through ligand-activated transcription factor pathways Journal of
Pharmacology and Experimental Therapeutics 300 206ndash212
Hagenbuch B amp Meier P J (2004) Organic anion transporting
polypeptides of the OATP SLC21 family Phylogenetic classification
as OATP SLCO superfamily new nomenclature and molecularfunc-
tional properties Pflugers Archiv 447 653ndash665
Hamadeh H K amp Afshari C A (2004) Toxicogenomics principles and
applications Hobokenrsquo John Wiley and Sons
Hamadeh H K Bushel P R Jayadev S Martin K DiSorbo O Sieber
S et al (2002) Gene expression analysis reveals chemical-specific
profiles Toxicological Sciences 67 219ndash231
Hamadeh H K Jayadev S Gaillard E T Huang Q Stoll R
Blanchard K et al (2004) Integration of clinical and gene expression
endpoints to explore furan-mediated hepatotoxicity Mutation Research
549 169ndash183
Hamadeh H K Knight B L Haugen A C Sieber S Amin R P
Bushel P R et al (2002) Methapyrilene toxicity Anchorage of
pathologic observations to gene expression alterations Toxicologic
Pathology 30 470ndash482
Han J D Bertin N Hao T Goldberg D S Berriz G F Zhang L V
et al (2004) Evidence for dynamically organized modularity in the
yeast proteinndashprotein interaction network Nature 430 88ndash93
Hanisch D Zien A Zimmer R amp Lengauer T (2002) Co-clustering of
biological networks and gene expression data Bioinformatics 18
S145ndashS154
Harris A J Dial S L amp Casciano D A (2004) Comparison of basal
gene expression profiles and effects of hepatocarcinogens on gene
expression in cultured primary human hepatocytes and HepG2 cells
Mutation Research 549 79ndash99
Harrison S C (2004) Whither structural biology Nature Structural
Molecular Biology 11 12ndash15
Hartley D P Dai X He Y D Carlini E J Wang B Huskey S E
et al (2004) Activators of the rat pregnane X receptor differentially
modulate hepatic and intestinal gene expression Molecular Pharma-
cology 65 1159ndash1171
Hartwell L H Hopfield J J Leibler S amp Murray A W (1999) From
molecular to modular cell biology Nature 402 C47ndashC52
Hasmall S Orphanides G James N Pennie W Hedley K Soames
A et al (2002) Downregulation of lactoferrin by PPARalpha ligands
Role in perturbation of hepatocyte proliferation and apoptosis
Toxicology Sciences 68 304ndash313
Hayase T Yamamoto Y Yamamoto K Muso E amp Shiota K (2004)
Microarray profile analysis of toxic cocaine-induced alterations in the
expression of mouse brain gene sequences A possible rsquoprotectiversquo
effect of buprenorphine Journal of Applied Toxicology 24 15ndash20
Hayes K R Vollrath A L Zastrow G M McMillan B J Craven M
Jovanovich S et al (2005) EDGE A centralized resource for the
comparison analysis and distribution of toxicogenomic information
Molecular Pharmacology 67 1360ndash1368
Heijne W H Slitt A L van Bladeren P J Groten J P Klaassen C D
Stierum R H et al (2004) Bromobenzene-induced hepatotoxicity at
the transcriptome level Toxicology Sciences 79 411ndash422
Heijne W H Stierum R H Slijper M van Bladeren P J amp van
Ommen B (2003) Toxicogenomics of bromobenzene hepatotoxicity
A combined transcriptomics and proteomics approach Biochemical
Pharmacology 65 857ndash875
Heinloth A N Irwin R D Boorman G A Nettesheim P Fannin
R D Sieber S O et al (2004) Gene expression profiling of rat
livers reveals indicators of potential adverse effects Toxicology
Sciences 80 193ndash202
Hodges L C Cook J D Lobenhofer E K Li L Bennett L Bushel
P R et al (2003) Tamoxifen functions as a molecular agonist inducing
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 63
cell cycle-associated genes in breast cancer cells Molecular Cancer
Research 1 300ndash311
Holleman A Cheok M H den Boer M L Yang W Veerman KM
A J Kazemier et al (2004) Gene-expression patterns in drug-resistant
acute lymphoblastic leukemia cells and response to treatment New
England Journal of Medicine 351 533ndash542
Hong Y Muller U R amp Lai F (2003) Discriminating two classes of
toxicants through expression analysis of HepG2 cells with DNA arrays
Toxicology in Vitro 17 85ndash92
Hood E (2003) Proteomics Characterizing the cogs in the machinery of
life Environmental Health Perspectives 111 A816ndashA825
Hood L (2003) Systems biology Integrating technology biology and
computation Mechanism of Ageing and Development 124 9ndash16
Hood L amp Galas D (2003) The digital code of DNA Nature 421
444ndash448
Hood L amp Perlmutter R M (2004) The impact of systems approaches on
biological problems in drug discovery Nature Biotechnology 22
1215ndash1217
Hu T Gibson D P Carr G J Torontali S M Tiesman J P Chaney
J G et al (2004) Identification of a gene expression profile that
discriminates indirect-acting genotoxins from direct-acting genotoxins
Mutation Research 549 5ndash27
Huang Q Dunn II R T Jayadev S DiSorbo O Pack F D Farr S B
et al (2001) Assessment of cisplatin-induced nephrotoxicity by
microarray technology Toxicology Sciences 63 196ndash207
Huang Q Jin X Gaillard E T Knight B L Pack F D Stoltz J H et
al (2004) Gene expression profiling reveals multiple toxicity endpoints
induced by hepatotoxicants Mutation Research 549 147ndash167
Huisman M T Smit J W Crommentuyn K M Zelcer N Wiltshire
H R Beijnen J H et al (2002) Multidrug resistance protein 2
(MRP2) transports HIV protease inhibitors and transport can be
enhanced by other drugs Aids 16 2295ndash2301
Hunt C M Westerkam W R amp Stave G M (1992) Effect of age and
gender on the activity of human hepatic CYP3A Biochemical
Pharmacology 44 275ndash283
Ideker T Ozier O Schwikowski B amp Siegal A F (2002) Discovering
regulatory and signalling circuits in molecular interaction networks
Bioinformatics 18 S233ndashS240
Ideker T Thorsson V Ranish J A Christmas R Buhler J Eng J K
et al (2001) Integrated genomic and proteomic analyses of a system-
atically perturbed network Science 292 929ndash934
Iida M Anna C H Hartis J Bruno M Wetmore B Dubin J R et al
(2003) Changes in global gene and protein expression during early
mouse liver carcinogenesis induced by non-genotoxic model carcino-
gens oxazepam and Wyeth-14643 Carcinogenesis 24 757ndash770
Ingelman-Sundberg M (2004) Pharmacogenetics of cytochrome P450 and
its applications in drug therapy The past present and future Trends in
Pharmacological Sciences 25 193ndash200
Jeong H Mason S P Barabasi A L amp Oltvai Z N (2001) Lethality
and centrality in protein networks Nature 411 41ndash42
Jeong H Tombor B Albert R Oltvai Z N amp Barabasi A L
(2000) The large-scale organization of metabolic networks Nature
407 651ndash654
Jung D Hagenbuch B Gresh L Pontoglio M Meier P J amp Kullak-
Ublick G A (2001) Characterization of the human OATP-C
(SLC21A6) gene promoter and regulation of liver-specific OATP genes
by hepatocyte nuclear factor 1 alpha Journal of Biological Chemistry
276 37206ndash37214
Jung D amp Kullak-Ublick G A (2003) Hepatocyte nuclear factor 1
alpha A key mediator of the effect of bile acids on gene expression
Hepatology 37 622ndash631
Jung J -W Park J -S Hwang J -W Kang K -S YS L Song B
-S et al (2004) Gene expression analysis of peroxisome prolifer-
ators- and phenytoin-induced hepatotoxicity using cDNA microarray
Journal of Veterinary Medical Science 66 1329ndash1333
Kato N Shibutani M Takagi H Uneyama C Lee K -Y Takiami S
et al (2004) Gene expression profile in the livers of rats orally
administered ethinylestradiol for 28 days using a microarray technique
Toxicology 200 179ndash192
Kier L D Neft R Tang L Suizu R Cook T Onsurez K et al
(2004) Applications of microarrays with toxicologically relevant genes
(tox genes) for the evaluation of chemical toxicants in Sprague Dawley
rats in vivo and human hepatocytes in vitro Mutation Research 549
101ndash113
Kijima K Toyosawa K Yasuba M Matsuoka N Adachi T
Komiyama M et al (2004) Gene expression analysis of the rat testis
after treatment with di(2-ethylhexyl) phthalate using cDNA microarray
and real-time RT-PCR Toxicology and Applied Pharmacology 200
103ndash110
Kim R B (2003) Organic anion-transporting polypeptide (OATP) trans-
porter family and drug disposition European Journal of Clinical
Investigation 33 1ndash5
Kinirons M T amp OrsquoMahony M S (2004) Drug metabolism and ageing
British Journal of Clinical Pharmacology 57 540ndash544
Kitano H (2002a) Computational systems biology Nature 420 206ndash210
Kitano H (2002b) Systems biology A brief overview Science 295
1662ndash1664
Kliewer S A Moore J T Wade L Staudinger J L Watson M
A Jones S A et al (1998) An orphan nuclear receptor activated
by pregnanes defines a novel steroid signalling pathway Cell 92
73ndash82
Korolev D Balakin K V Nikolsky Y Kirillov E Ivanenkov Y A
Savchuk N P et al (2003) Modeling of human cytochrome p450-
mediated drug metabolism using unsupervised machine learning
approach Journal of Medicinal Chemistry 46 3631ndash3643
Kramer J A Pettit S D Amin R P Bertram T A Car B
Cunningham M et al (2004) Overview on the application of
transcription profiling using selected nephrotoxicants for toxicology
assessment Environmental Health Perspectives 112 460ndash464
Kultima K Nystrom A -M Scholz B Gustafson A -L Dencker L
amp Stigson M (2004) Valproic acid teratogenicity A toxicogenomics
approach Environmental Health Perspectives 112 1225ndash1235
Kutuzova G D amp Deluca H F (2004) Gene expression profiles in rat
intestine identify pathways for 125-dihydroxyvitamin D(3) stimulated
calcium absorption and clarify its immunomodulatory properties
Archives of Biochemistry and Biophysics 432 152ndash166
Kwak M K Wakabayashi N Itoh K Motohashi H Yamamoto M amp
Kensler T W (2003) Modulation of gene expression by cancer
chemopreventive dithiolethiones through the Keap1-Nrf2 pathway
Identification of novel gene clusters for cell survival Journal of
Biological Chemistry 278 8135ndash8145
Lahjouji K Mitchell G A amp Qureshi I A (2001) Carnitine transport
by organic cation transporters and systemic carnitine deficiency
Molecular Genetics and Metabolism 73 287ndash297
Landowski C P Sun D Foster D R Menon S S Barnett J L
Welage L S et al (2003) Gene expression in the human intestine and
correlation with oral valacyclovir pharmacokinetic parameters Journal
of Pharmacology and Experimental Therapeutics 306 778ndash786
Lee G -J Lee W -S Jeon K -S Um C -H Kim S -J Lee C -H
et al (2004) cDNA microarray gene expression analysis and
toxicological phenotype for anticancer drug Journal of Veterinary
Medical Science 66 1339ndash1345
Lee M Kwon J Kim S N Kim J E Koh W S Kim E J et al
(2003) cDNA microarray gene expression profiling of hydroxyurea
paclitaxel and p-anisidine genotoxic compounds with differing
tumorigenicity results Environmental and Molecular Mutagenesis
42 91ndash97
Li S Armstrong C M Bertin N Ge H Milstein S Boxem M et al
(2004) A map of the interactome network of the metazoan C elegans
Science 303 540ndash543
Liguori M J Anderson L M Bukofzer S McKim J Pregenzer J F
Retief J et al (2005) Microarray analysis in human hepatocytes
suggests a mechanism for hepatotoxicity induced by trovafloxacin
Hepatology 41 177ndash186
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6664
Lobenhofer E K Bennett L Cable P L Li L Bushel P R amp Afshari
C A (2002) Regulation of DNA replication fork genes by 17beta-
estradiol Molecular Endocrinology 16 1215ndash1229
Locker J Tian J Carver R Concas D Cossu C Ledda-Columbano
G M et al (2003) A common set of immediate-early response genes
in liver regeneration and hyperplasia Hepatology 38 314ndash325
Mattes W B Pettit S D Sansone S A Bushel P R amp Waters M D
(2004) Database development in toxicogenomics Issues and efforts
Environmental Health Perspectives 112 495ndash505
McMillian M Nie A Y Parker J B Leone A Bryant S Kemmerer
M et al (2004) A gene expression signature for oxidant stressreactive
metabolites in rat liver Biochemical Pharmacology 68 2249ndash2261
Meier P J amp Stieger B (2000) Molecular mechanisms in bile formation
News in Physiological Sciences 15 89ndash93
Meneses-Lorente G de Longueville F Dos Santos-Mendes S Bonnert
T P Jack A Evrard S et al (2003) An evaluation of a low-density
DNA microarray using cytochrome P450 inducers Chemical Research
in Toxicology 16 1070ndash1077
Meneses-Lorente G Guest P C Lawrence J Muniappa N Knowles
M R Skynner H A et al (2004) A proteomic investigation of drug-
induced steatosis in rat liver Chemical Research in Toxicology 17
605ndash612
Merrill C L Ni H Yoon L W Tirmenstein M A Narayanan P
Benavides G R et al (2002) Etomoxir-induced oxidative stress in
HepG2 cells detected by differential gene expression is confirmed
biochemically Toxicology Sciences 68 93ndash101
Milo R Shen-Orr S Itzkovitz S Kashtan N Chklovskii D amp Alon
U (2002) Network motifs Simple building blocks of complex
networks Science 298 824ndash827
Morgan K T Ni H Brown H R Yoon L Qualls C W Crosby L
M et al (2002) Application of cDNA microarray technology to in
vitro toxicology and the selection of genes for a real-time RT-PCR-
based screen for oxidative stress in Hep-G2 cells Toxicologic
Pathology 30 435ndash451
Mwinyi J Johne A Bauer S Roots I amp Gerloff T (2004) Evidence
for inverse effects of OATP-C (SLC21A6) 5 and 1b haplotypes on
pravastatin kinetics Clinical Pharmacology and Therapeutics 75
415ndash421
Nakata K Yukawa M Komiyama N Nakano T amp Kaminuma T
(2002) A nuclear receptor database that maps pathways to diseases
Genome Infomatics 13 515ndash516
Nicholson J K Holmes E Lindon J C amp Wilson I D (2004) The
challenges of modeling mammalian biocomplexity Nature Biotechnol-
ogy 22 1268ndash1274
Nicholson J K amp Wilson I D (2003) Understanding rsquoglobal systems
biology Metabonomics and the continuum of metabolism Nature
Reviews on Drug Discovery 2 668ndash676
Nikitin A Egorov S Daraselia N amp Mazo I (2003) Pathway studiomdash
the analysis and navigation of molecular networks Bioinformatics 19
2155ndash2157
Nikolsky Y Ekins S Nikolskaya T amp Bugrim A (2005) A novel
method for generation of signature networks as biomarkers from
complex high throughput data Toxicology Letters 158 20ndash29
Nikolsky Y Nikolskaya T amp Bugrim A (2005) Biological networks
and analysis of experimental data in drug discovery Drug Discovery
Today 10 653ndash662
Nishizato Y Ieiri I Suzuki H Kimura M Kawabata K Hirota T
et al (2003) Polymorphisms of OATP-C (SLC21A6) and OAT3
(SLC22A8) genes Consequences for pravastatin pharmacokinetics
Clinical Pharmacology and Therapeutics 73 554ndash565
Nozawa T Nakajima M Tamai I Noda K Nezu J Sai Y et al
(2002) Genetic polymorphisms of human organic anion transporters
OATP-C (SLC21A6) and OATP-B (SLC21A9) Allele frequencies in
the Japanese population and functional analysis Journal of Pharmacol-
ogy and Experimental Therapeutics 302 804ndash813
Nozawa T Sugiura S Nakajima M Goto A Yokoi T Nezu J et al
(2004) Involvement of organic anion transporting polypeptides in the
transport of troglitazone sulfate Implications for understanding trogli-
tazone hepatotoxicity Drug Metabolism and Disposition 32 291ndash294
Oliver D E Rubin D L Stuart J M Hewett M Klein T E amp
Altman R B (2002) Ontology development for a pharmacogenetics
knowledge base Package Symposion 88ndash99
Oram J F amp Lawn R M (2001) ABCA1 The gatekeeper for eliminating
excess tissue cholesterol Journal of Lipid Research 42 1173ndash1179
Oswald M Kullak-Ublick G A Paumgartner G amp Beuers U (2001)
Expression of hepatic transporters OATP-C and MRP2 in primary
sclerosing cholangitis Liver 21 247ndash253
Parsons A B Brost R I Ding H Li Z Zhang C Sheikh B et al
(2004) Integration of chemical-genetic and genetic interaction data
links bioactive compounds to cellular target pathways Nature Bio-
technology 22 62ndash69
Patki K C von Moltke L L Harmatz J S Hesse L M Court M H
amp Greenblatt D J (2004) Effect of age on in vitro triazolam
biotransformation in male human liver microsomes Journal of
Pharmacology and Experimental Therapeutics 308 874ndash879
Pereira-Leal J B Enright A J amp Ouzounis C A (2004) Detection of
functional modules from protein interaction networks Proteins 54
49ndash57
Plant N (2004) Interaction networks Coordinating responses to xeno-
biotic exposure Toxicology 202 21ndash32
Raghavendra Prasad H S Qi Z Srinivasan K N amp Gopalakrish-
nakone P (2004) Potential effects of tetrodotoxin exposure to
human glial cells postulated using microarray approach Toxicon 44
597ndash608
Reilly T P Bourdi M Brady J N Pise-Masison C A Radonovich M
F George J W et al (2001) Expression profiling of acetaminophen
liver toxicity in mice using microarray technology Biochemical and
Biophysical Research Communications 282 321ndash328
Rives A W amp Galitski T (2003) Modular organization of cellular
networks Proceedings of the National Academy of Sciences of the
United States of America 100 1128ndash1133
Rogers P D Pearson M M Cleary J D Sullivan D C amp Chapman
S W (2002) Differential expression of genes encoding immunomodu-
latory proteins in response to amphotericin B in human mononuclear
cells identified by cDNA microarray analysis Journal of Antimicrobial
Chemotherapy 50 811ndash817
Rosen M B Wilson V S Schmid J E amp Gray L E (2005) Gene
expression analysis in the ventral prostate of rats exposed to vinclozolin
or procymidone Reproductive Toxicology 19 367ndash379
Rost D Herrmann T Sauer P Schmidts H L Stieger B Meier P J
et al (2003) Regulation of rat organic anion transporters in bile salt-
induced cholestatic hepatitis Effect of ursodeoxycholate Hepatology
38 187ndash195
Rothman R B Ayestas M A Dersch C M amp Baumann M H (1999)
Aminorex fenfluramine and chlorphentermine are serotonin transporter
substrates Implications for primary pulmonary hypertension Circula-
tion 100 869ndash875
Ruepp S U Tonge R P Shaw J Wallis N amp Pognan F (2002)
Genomics and proteomics analysis of acetaminophen toxicity in mouse
liver Toxicology Sciences 65 135ndash150
Sakaeda T Nakamura T amp Okumura K (2002) MDR1 genotype-
related pharmacokinetics and pharmacodynamics Biological amp Phar-
maceutical Bulletin 25 1391ndash1400
Sakata T Anzai N Shin H J Noshiro R Hirata T Yokoyama H
et al (2004) Novel single nucleotide polymorphisms of organic cation
transporter 1 (SLC22A1) affecting transport functions Biochemical
and Biophysical Research Communications 313 789ndash793
Satlin L M Amin V amp Wolkoff A W (1997) Organic anion
transporting polypeptide mediates organic anionHCO3-exchange
Journal of Biological Chemistry 272 26340ndash26345
Satomi Y Tsuchiya W Mihara K Ota M Kasahara Y amp Akahori F
(2004) Gene expression analysis of the lung following paraquat
administration in rats using DNA microarray Journal of Toxicological
Sciences 29 91ndash100
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 65
Schrenk D Baus P R Ermel N Klein C Vorderstemann B amp
Kauffmann H M (2001) Up-regulation of transporters of the MRP
family by drugs and toxins Toxicology Letters 120 51ndash57
Segal E Shapira M Regev A Persquoer D Botstein D Koller D et al
(2003) Module networks Identifying regulatory modules and their
condition-specific regulators from gene expression data Nature
Genetics 34 166ndash176
Segal E Wang H amp Koller D (2003) Discovering molecular pathways
from protein interaction and gene expression Bioinformatics 19
i264ndash i272
Seidel S D Kan H L Stott W T Schisler M R amp Gollapudi B B
(2003) Identification of transcriptome profiles for the DNA-damaging
agents bleomycin and hydrogen peroxide in L5178Y mouse lymphoma
cells Environmental and Molecular Mutagenesis 42 19ndash25
Shan L Yu Y Schut H A J amp Snyderwine E G (2004) Susceptibility
of rats to mammary gland carcinogenesis by the food-derived
carcinogen 2-Amino-1-Methyl-6-Phenylimidazo[45-b]Pyridine (PhIP)
varies with age and is associated with the induction of differential gene
expression American Journal of Pathology 165 191ndash202
Sharom J R Bellows D S amp Tyers M (2004) From large networks to
small molecules Current Opinion in Chemical Biology 8 81ndash90
Shitara Y Hirano M Sato H amp Sugiyama Y (2004) Gemfibrozil and
its glucuronide inhibit the OATP2(OATP1B1 SLC21A6)-mediated
hepatic uptake and CYP2C8-mediated metabolism of cerivastatinmdash
Analysis of the mechanism of the clinically relevant drugndashdrug
interaction between cerivastatin and gemfibrozil Journal of Pharma-
cology and Experimental Therapeutics
Shitara Y Itoh T Sato H Li A P amp Sugiyama Y (2003) Inhibition of
transporter-mediated hepatic uptake as a mechanism for drugndashdrug
interaction between cerivastatin and cyclosporin A Journal of
Pharmacology and Experimental Therapeutics 304 610ndash616
Somogyi R Fuhrman S amp Wen X (2001) Genetic network inference in
computational models and applications to large-scale gene expression
data In M Bower amp H Bolouri (Eds) Computational modeling of
genetic and biochemical networks (pp 119ndash157) Cambridge MArsquoThe
MIT Press
Spirin V amp Mirny L A (2003) Protein complexes and functional
modules in molecular networks Proceedings of the National Academy
of Sciences of the United States of America 100 12123ndash12128
Staudinger J Liu Y Madan A Habeebu S amp Klaassen C D
(2001) Coordinate regulation of xenobiotic and bile acid homeo-
stasis by pregnane X receptor Drug Metabolism and Disposition
29 1467ndash1472
Sun L Z Ji Z L Chen X Wang J F amp Chen Y Z (2002) ADME-
AP A database of ADME associated proteins Bioinformatics 18
1699ndash1700
Suter L Babiss L E amp Wheeldon E B (2004) Toxicogenomics in
predictive toxicology in drug development Chemistry and Biology 11
161ndash171
Synold T W Dussault I amp Forman B M (2001) The orphan nuclear
receptor SXR coordinately regulates drug metabolism and efflux
Nature Medicine 7 584ndash590
Tabuchi Y amp Kondo T (2003) cDNA microarray analysis reveals chop-
10 plays a key role in Sertoli cell injury induced by bisphenol A
Biochemical and Biophysical Research Communications 305 54ndash61
Tamai I Nezu J Uchino H Sai Y Oku A Shimane M et al (2000)
Molecular identification and characterization of novel members of the
human organic anion transporter (OATP) family Biochemical and
Biophysical Research Communications 273 251ndash260
Taylor S W Fahy E Zhang B Glenn G M Warnock D E Wiley S
et al (2003) Characterization of the human heart mitochondrial
proteome Nature Biotechnology 21 281ndash286
Terasaka S Aita Y Inoue A Hayashi S Nishigaki M Aoyagi K
et al (2004) Using a customized DNA microarray for expression
profiling of the estrogen-responsive genes to evaluate estrogen activity
among natural estrogens and industrial chemicals Environmental
Health Perspectives 112 773ndash781
Thomas D M Francescutti-Verbeem D M Liu X amp Kuhn D M
(2004) Identification of differentially regulated transcripts in mouse
striatum following methamphetamine treatmentmdashan oligonucleotide
microarray approach Journal of Neurochemistry 88 380ndash393
Thomas R S Rank D R Penn S G Zastrow G M Hayes K R
Pande K et al (2001) Identification of toxicologically predictive
gene sets using cDNA microarrays Molecular Pharmacology 60
1189ndash1194
Thompson K L Afshari C A Amin R P Bertram T A Car B
Cunningham M et al (2004) Identification of platform-independent
gene expression markers of cisplatin nephrotoxicity Environmental
Health Perspectives 112 488ndash494
Tirona R G Leake B F Merino G amp Kim R B (2001)
Polymorphisms in OATP-C Identification of multiple allelic
variants associated with altered transport activity among European-
and African-Americans Journal of Biological Chemistry 276
35669ndash35675
Tirona R G Leake B F Wolkoff A W amp Kim R B (2003)
Human organic anion transporting polypeptide-C (SLC21A6) is a
major determinant of the rifampin-mediated pregnane X receptor
activation Journal of Pharmacology and Experimental Therapeutics
304 223ndash228
Tong W Cao X Harris S Sun H Fang H Fuscoe J et al (2003)
ArrayTrackmdashsupporting toxicogenomic research at the US Food and
Drug Administration National Center for Toxicological Research
Environmental Health Perspectives 111 1819ndash1826
Tornow S amp Mewes H W (2003) Functional modules by relating
protein interaction networks and gene expression Nucleic Acids
Research 31 6283ndash6289
Trocho C Escola-Gil J C Ribas V Benitez S Martin-Campos J M
Rotllan N et al (2004) Phenytoin treatment reduces atherosclerosis in
mice through mechanisms independent of plasma HDL-cholesterol
concentration Atherosclerosis 174 275ndash285
Ueda A Hamadeh H K Webb H K Yamamoto Y Sueyoshi T
Afshari C A et al (2002) Diverse roles of the nuclear orphan
receptor CAR in regulating hepatic genes in response to phenobarbital
Molecular Pharmacology 61 1ndash6
Ulrich R G (2003) The toxicogenomics of nuclear receptor agonists
Current Opinion in Chemical Biology 7 505ndash510
Ulrich R G Rockett J C Gibson G G amp Pettit S D (2004)
Overview of an interlaboratory collaboration on evaluating the effects
of model hepatotoxicants on hepatic gene expression Environmental
Health Perspectives 112 423ndash427
Unami A Shinohara Y Ichikawa T amp Baba Y (2003) Biochemical
and microarray analyses of bupivacaine-induced apoptosis Journal of
Toxicological Sciences 28 77ndash94
Vasquez A Flammini A Maritan A amp Vespignani A (2003) Global
protein function prediction from proteinndashprotein interaction networks
Nature Biotechnology 21 697ndash700
Vezina C M Walker N J amp Olson J R (2004) Subchronic Exposure to
TCDD PeCDF PCB126 and PCB153 Effect on hepatic gene
expression Environmental Health Perspectives 112 1636ndash1644
Vo T D Greenberg H J amp Palsson B O (2004) Reconstruction and
functional characterization of the human mitochondrial metabolic
network based on proteomic and biochemical data Journal of Bio-
logical Chemistry 279 39532ndash39540
Wang H amp LeCluyse E L (2003) Role of orphan nuclear receptors in
the regulation of drug-metabolising enzymes Clinical Pharmacoki-
netics 42 1331ndash1357
Wang N Silver D L Thiele C amp Tall A R (2001) ATP-
binding cassette transporter A1 (ABCA1) functions as a choles-
terol efflux regulatory protein Journal of Biological Chemistry 276
23742ndash23747
Waring J F Cavet G Jolly R A McDowell J Dai H Ciurlionis R
et al (2003) Development of a DNA Microarray for toxicology based
on hepatoxin-regulated sequences Environmental Health Perspectives
111 863ndash870
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6666
Waring J F Gum R Morfitt D Jolly R A Ciurlionis R Heindel M
et al (2002) Identifying toxic mechanisms using DNA microarrays
Evidence that an experimental inhibitor of cell adhesion molecule
expression signals through the aryl hydrocarbon nuclear receptor
Toxicology 181ndash182 537ndash550
Waring J F Jolly R A Ciurlionis R Lum P Y Praestgaard J T
Morfitt D C et al (2001) Clustering of hepatotoxins based on
mechanism of toxicity using gene expression profiles Toxicology and
Applied Pharmacology 175 28ndash42
Waters M Boorman G Bushel P Cunningham M Irwin R Merrick
A et al (2003) Systems toxicology and the Chemical Effects in
Biological Systems (CEBS) knowledge base EHP Toxicogenomics
111 15ndash28
Wei M Arnold L Cano M amp Cohen S M (2005) Effects of co-
administration of antioxidants and arsenicals on the rat urinary bladder
epithelium Toxicology Sciences 83 237ndash245
Weinshilboum R (2003) Inheritance and drug response New England
Journal of Medicine 348 529ndash537
Weinshilboum R amp Wang L (2004) Pharmacogenetics Inherited
variation in amino acid sequence and altered protein quantity Clinical
Pharmacology and Therapeutics 75 253ndash258
Werner E (2003) In silico multicellular systems biology and minimal
genomes Drug Discovery Today 8 1121ndash1127
Wong J S amp Gill S S (2002) Gene expression changes induced in
mouse liver by di(2-ethylhexyl) phthalate Toxicology and Applied
Pharmacology 185 180ndash196
Xie T Tong L McCann U D Yuan J Becker K G Mechan A O
et al (2004) Identification and characterization of metallothionein-1
and -2 gene expression in the context of ()34-methylenedioxymetham-
phetamine-induced toxicity to brain dopaminergic neurons Journal of
Neuroscience 24 7043ndash7050
Yadetie F Laegreid A Bakke I Kusnierczyk W Komorowski J
Waldum H L et al (2003) Liver gene expression in rats in response
to the peroxisome proliferator-activated receptor-alpha agonist ciprofi-
brate Physiological Genomics 15 9ndash19
Yamashita S Nomoto T Abe M Tatematsu M Sugimura T amp
Ushijima T (2004) Persistence of gene expression changes in stomach
mucosae induced by short-term N-methyl-NV-nitro-N-nitrosoguanidinetreatment and their presence in stomach cancers Mutation Research
549 185ndash193
Yan Q amp Sadee W (2000) Human membrane transporter database A
web-accessible relational database for drug transport studies and
pharmacogenomics AAPS Pharmsci 2 E20
Yeger-Lotem E amp Margalit H (2003) Detection of regulatory circuits by
integrating the cellular networks of proteinndashprotein interactions and
transcription regulation Nucleic Acids Research 31 6053ndash6061
Yoon B I Li G -X Kitada K Kawasaki Y Igarashi K amp Kodama
Y et al (2003) Mechanisms of benzene-induced hematotoxicity and
leukemogenicity cDNA microarray analyses using mouse bone marrow
tissue Environmental Health Perspectives 111 1411ndash1420
Young M B DiSilvestro M R Sendera T J Freund J Kriete A amp
Magnuson S R (2003) Analysis of gene expression in carbon
tetrachloride-treated rat livers using a novel bioarray technology
Pharmacogenomics Journal 3 41ndash52
Yu H Zhu X Greenbaum D Karro J amp Gerstein M (2004) TopNet
A tool for comparing biological sub-networks correlating protein
properties with topological statistics Nucleic Acids Research 32
328ndash337
Zhang E Y Fu D J Pak Y A Stewart T Mukhopadhyay N
Wrighton S A et al (2004) Genetic polymorphisms in human proton-
dependent dipeptide transporter PEPT1 Implications for the functional
role of Pro586 Journal of Pharmacology and Experimental Therapeu-
tics 310 437ndash445
Zhang E Y Knipp G T Ekins S amp Swaan P W (2002)
Structural biology and function of solute transporters Implications
for identifying and designing substrates Drug Metabolism Reviews
34 709ndash750
Zhang E Y Phelps M A Cheng C Ekins S amp Swaan P W (2002)
Modeling of active transport systems Advanced Drug Delivery
Reviews 54 329ndash354
- Systems-ADMETox Resources and network approaches
-
- Introduction
- Data available
- Network analysis and databases
-
- Network applications
-
- The role of transporters
-
- Clinical relevance of transporters
- Transporter network examples ABCA1
- Transporter network examples OATP
- Transporter microarray data
-
- Applications to enzymes
- Future network applications
-
- Discussion
- Acknowledgements
- References
-
Table 1 (continued)
Compounds Rat strain Microarray type Compound dose Microarray data
availability
Reference
Microcystin-LR (MLR)
phenobarbital (PB)
lipopolysaccharide
(LPS) carbon
tetrachloride (CT)
thioacetamide (THA)
and cyproterone
acetate (CPA)
Male Wistar Purpose-made rat DNA
microarray (Affymetrix
Santa Clara CA)
containing 1600 rat
DNA sequences
Various Accession number gene
name fold changes
shown as a heat map
table in publicationmdash
data extraction would be
laborious
(Bulera et al
2001)
Acetamidofluorene
aniline bromobenzene
butyl hydroxytol
dieldrin disulfiram
ethinyl estradiol
hexachlorocyclohexane
g 4-methylthiazole
nimesulide piperonyl
butoxide precocene I
pulegone tannic acid
trans-anethole
Male Spraguendash
Dawley
Custom Rat MegaA
cDNA chip 3434-gene
Acetamidofluorene (200
mgkg) aniline (200 mg
kg) bromobenzene (900
mgkg) butyl hydroxytol
(1000 mgkg) dieldrin
(30 and 45 mgkg)
disulfiram (2000 mgkg)
ethinyl estradiol (500
mgkg)
hexachlorocyclohexane
gamma (40 65 80 mg
kg) 4-methylthiazole
(120 mgkg) nimesulide
(500 mgkg) piperonyl
butoxide (4000 mgkg)
precocene I (500 mgkg)
pulegone (400 mgkg)
tannic acid (3000 mg
kg) trans-anethole
(600 mgkg)
Gene name accession
number in a manuscript
table
(McMillian et al
2004)
NIEHS website at httpdirniehsnihgovmicroarraydatasetshome-pubhtm
EDGE website at httpedgeoncologywiscedu
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 43
and cisplatin (Table 1) In the majority of cases the resulting
number of differentially expressed genes is a very small
subset of the starting number on the microarray following
clustering or other types of analysis Upon closer examina-
tion of these publications the majority of them either
provide images of a heat map andor a table listing a gene
name accession number and expression change Very few of
the published studies (Tables 1ndash3) provide the original raw
microarray data file at a freely accessible website hence
restricting further analysis by scientists using other software
Although in some cases it is possible to cut and paste the
gene expression data tables from the publication pdf files
this is not always the case In the worst case scenario one
would have to manually retype gene lists or extract them
from heatmaps as binary type data As not all computational
researchers will have a laboratory available to them to
generate such quantities of toxicogenomics data the latter
points are important if we are going to continue to see
innovation in software development for this data This will
require free unrestricted access to data published Similarly
if we are to discern lsquolsquofingerprintsrsquorsquo for molecules acting with
a similar or identical mechanism we will need databases of
many diverse chemical structures that have been tested in a
similar manner There is therefore considerable interest in
the current databases being developed by the NIEHS FDA
and other groups which should help to improve the situation
by providing freely accessible microarray and other toxicity
related data Two of these databases being developed in the
public domain are Chemical Effects in Biological Systems
(CEBS) (httpwwwniehsnihgovnctcebshtm) (Mattes
Pettit Sansone Bushel amp Waters 2004 Waters et al
2003) which will accommodate gene expression profiles
proteomics and metabolomics data and allow complex
queries (Hood 2003a Mattes et al 2004) Similar goals
are being pursued in the development of the ArrayTrack
database at the FDA (Tong et al 2003) The EDGE
database (httpedgeoncologywisceduedgephp) an
expanding public effort at The University of Wisconsin
contains mouse gene expression profiles following treat-
ment with different toxic molecules (Hayes et al 2005
Thomas et al 2001) These separate efforts if widely
adopted should make published studies describing HT data
more readily accessible although it might have been more
efficient to evolve these into a single global database instead
of fragmented repositories
Proteomics data has also been generated in a limited
number of toxicology studies (Table 4) once again this has
been produced with different strains of rats and mice using
different protein chips 2-D gel electrophoresis (2-DIGE)
and mass spectroscopy methods (eg MALDI-MS) The
proteomic data is very rarely accessible to the reader for
their own computational analysis Subsequently there have
Table 2
Literature toxicogenomics data derived from mouse in vivo studies
Compounds Mouse strain Microarray type Compound dose Microarray data
availability
Reference
Benzene Male and female P53
KO mice and
C57BL6
Affymetrix and Incyte
GEM system
300 ppm 6 h per day 5
days a week for 2 weeks
Gene name accession
number and fold change
data in a manuscript
table
(Yoon et al 2003)
Benzene Male 129SvJ Affymetrix MG-U74Av2 100 ppm 6 h per day 5
days a week for 2 weeks
Gene name accession
number and fold change
data in a manuscript
table
(Faiola Fuller
Wong amp Recio
2004)
Phenobarbital CAR and wild type NIEHS Mouse tox chip
8736 genes
100 mgkg for 12 h Accession number gene
name and fold change
for wild type and knock
out mice in a manuscript
table
(Ueda et al 2002)
Aroclor BNF
ciprofloxacin cobalt
chloride TCDD IL-6
LPS PCB-153
phenobarbital
phenylhyrzn TNFa
WY-16463
C57BL6J Custom array with 1200
cDNAs
Aroclor (200 mgkg)
BNF (5 mgkg)
ciprofloxacin (250 mg
kg) cobalt chloride (60
mgkg) TCDD (10 ug
kg) IL-6 (25 ugkg)
LPS (1 mgkg)
PCB-153 (80 mgkg)
phenobarbital (100 mg
kgday) 3 days
phenylhyrzn (100 mg
kg) TNFa (50 ugkg)
WY-16463 (0125wv)
Gene names and fold
change as a heat map
Data is also available in
the EDGE database
(Thomas et al
2001)
TCPOBOP CD-1 female Custom 9000 cDNA
mouse array
1ndash3 h treatment 3 mg
kg body wt
Accession numbers and
fold change data in a
manuscript table
(Locker et al
2003)
3H-12-dithiole-3-thione
(D3T)
Male wild-type and
nrf2-disrupted
Affymetrix murine
genome U74Av2
GeneChip
05 mmolkg Accession number gene
name fold in a
manuscript table
(Kwak et al
2003)
MDMA Male albino Swissndash
Webster (neurons)
15 K mouse cDNA clone
set
47 mgkg Gene name and fold
change in a manuscript
table
(Xie et al 2004)
Phenytoin Female C57BL6 and
LDLRMurine genome-U74Av2 300 mgl Gene name accession
number and fold change
in a manuscript table
(Trocho et al
2004)
Genistein (1000 Agmouseday) or
diethylstilbestrol
(DES) (50 Agmouse
day)
ICR (testes) Custom cDNA
microarray containing
1754 cDNA probes
Genistein (1000 Agmouseday)
diethylstilbestrol (50 Agmouseday)
Accession number gene
name and fold change in
a manuscript tablemdashnote
very few genes
(Adachi et al
2004)
Cocaine and
buprenorphine
Male ICR Mouse DiscoveryArrayi
type I array containing
2688brain-derivedprobes
(Display Systems Biotech
Inc Copenhagen
Denmark)
40 mg kgmdash1 cocaine
once a day for 4 days 40
mg kgmdash1 cocaine plus
025 mg kgmdash1 BUP for
4 days
Data apparently not
available
(Hayase
Yamamoto
Yamamoto Muso
amp Shiota 2004)
Methamphetamine Male C57BLJ6
(striatum)
Affymetrix mouse
genechip mg-U74Av2
oligonucleotides arrays
12 488 genes
40 mgkg Gene accession number
gene ID and name and
signal log ratio in a
manuscript table
(Thomas
Francescutti-
Verbeem Liu amp
Kuhn 2004)
Di(2-ethylhexyl)
phthalate
Male C57BL6 Murine genome U74Av2
Arrays (MGU74Av2)
10 dietary DEHP for
13 weeks
Genbank accession
numbers gene name log
ratio data present as a bar
chart in the publication
data extraction would
take some effort
(Wong amp Gill
2002)
Diethylhexylphthalate Male PPARa null and
wild type
Custom made containing
600 tox genes
1150 mgkgday Gene names available on
a bar chartmdashquantitative
data not easily accessible
(Hasmall et al
2002)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6644
Table 2 (continued)
Compounds Mouse strain Microarray type Compound dose Microarray data
availability
Reference
Acetaminophen C57B16 3 129Ola
hybrid
Test-2 Chips (Affymetrix)
then individual
oligonucleotide
microarrays (Mul1K sub
A and sub B Affymetrix)
that can detect the
expression of 11000
known genes and
expressed sequence tags
(ESTs)
300 mgkg Genbank or SwissProt
ID and fold change data
in a manuscript table
(Reilly et al
2001)
Cadmium chloride
benzo(a)pyrene (BaP)
and trichloroethylene
(TCE)
Male Swiss Webster Custom Chips
containing 148 unique
genes
Various Data in table form few
genes affected for BAP
and TCE
(Bartosiewicz
Penn amp Buckpitt
2001)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 45
been only a very small number of studies that have
combined both transcriptomic and proteomic methods with
a single animal strain after treatment with a drug Hopefully
we will see this change in the future but this will in turn
present considerable challenges as huge amounts of
proteomic data are combined with the equally large
transcript data files
3 Network analysis and databases
From some of the early reviews of systems biology there
has been discussion of its application to drug discovery
(Kitano 2002ab) as well as the utility for ADMETox
(Ekins et al 2002a 2000b) More recently several other
journals have dedicated whole issues to the field of systems
biology However one could consider this quite a broad field
from network or pathway analysis to quantitative simulation
of organelles (Vo Greenberg amp Palsson 2004) whole cells
and organs It is apparent that we are now understanding
organisms from the perspective of computationally gener-
ated networks of protein and ligand interactions (Barabasi amp
Oltvai 2004) Network and pathway tools enable the
analysis of HT data in the context of all known interactions
when using a database as the source Individual reviews
have in some cases indicated that networks will be valuable
for understanding adverse events (Hood amp Perlmutter
2004) drug target identification or validation (Butcher
Berg amp Kunkel 2004) and complex metabolic interactions
(Nicholson et al 2004) A general schematic has been
generated in order to provide a description of the utilization
of such pathway databases and network building algorithms
from the initial parsing of high throughput data to network
comparisons and visualization (Fig 3) High throughput
data can be superimposed and visualized on the various
protein interaction databases available This is accomplished
by using either preset maps that capture current biological
knowledge or by building custom interaction networks
using many different algorithms which can be compared and
statistically evaluated as demonstrated in a very large
number of published examples (Dobrin Beg Karabasi amp
Oltvai 2004 Fiehn 2001 Han et al 2004 Hanisch et al
2002 Ideker et al 2002 Jeong Mason Barabasi amp Oltvai
2001 Jeong Tombor Albert Oltvai amp Barabasi 2000 Li
et al 2004 Milo et al 2002 Nikitin Egorov Daraselia amp
Mazo 2003 Pereira-Leal Enright amp Ouzounis 2004
Rives amp Galitski 2003 Segal et al 2003a Somogyi et al
2001 Spirin amp Mirny 2003 Tornow amp Mewes 2003
Vasquez Flammini Maritan amp Vespignani 2003 Yeger-
Lotem amp Margalit 2003 Yu Zhu Greenbaum Karro amp
Gerstein 2004)
31 Network applications
For example one group has used as an inference the
Bayesian network method for analysis of tissue toxicity from
microarray data as well as a mechanistic simulation for a
different pharmaceutically relevant molecule (Aksenov et
al 2005) Pathway tools and various resources have also
been applied to modeling the networks of nuclear hormone
receptors and their connections with other genes and small
molecules using a manually curated database MetaDrug
(Ekins Kirillov Rakmatulin amp Nikolskaya 2005d) or
MetaCore (Ekins Bugrim Nikolsky amp Nikolskaya 2005)
Transcriptional regulation of many transporters CYPs and
phase II enzymes are regulated by these receptors affecting
endogenous molecule transport metabolism cell growth
proliferation and oxidative stress (Ulrich 2003 Ulrich et al
2004) When the signaling networks and interacting ligands
for the transcriptional factors PPAR FXRRXRA ESR1
AHR HNF4A GCR-h MCR CAR-beta GCR-a LXR-a
CARRXR HNF4 FXR PXRRXR heterodimer PXR
AHRARNT heterodimer PPARaLXRa VDR PPAR-a
are visualized a very complex picture of interactions can be
created (Ekins et al 2005d) This suggests that when we
consider a molecule binding with only one nuclear receptor
we are observing only a fraction of the likely possible
feasible interactions based on the data gathered to date
Table 3
Literature toxicogenomics data derived from in vitro cell studies
Compounds Cell type Microarray type Compound dose Microarray data
availability
Reference
4-Hydroxytamoxifen
estrogen
MCF-7 breast cancer NIEHS ToxChip
microarray consisting of
1901 genes
1 uM hydroxytamoxifen
for a year 10 nM
17b-estradiol
Data available at NIEHS
website
(Hodges et al
2003)
Trovafloxacin Human hepatocytes Affymetrix U133A array 30ndash800 uM 142 genes available in
supplemental tablemdashnot
easily extracted
(Liguori et al
2005)
Estrogen MCF-7 breast cancer NIEHS ToxChip
microarray consisting of
1901 genes
10ndash10 M 17b-estradiol Data available at NIEHS
website
(Lobenhofer et al
2002)
Valproic acid NMRI mice embryo
and P19 mouse
embryocarcinoma
Custom chip including
15K mouse cDNA clone
set
600 mgkg body weight Gene symbol gene
name NIA EST log fold
change in a manuscript
table
(Kultima et al
2004)
Sulindac sulfide Human colorectal
carcinoma SW-480
and HCT-116
NIEHS human 12K chip 10 uM Genbank accession
number gene name and
fold change at various
time points in a
manuscript table data
also available on NIEHS
website
(Bottone
Martinez Collins
Afshari amp Eling
2003)
17Beta-estradiol estriol
estrone genistein
diethylstilbestrol
bisphenol A
nonylphenol
methoxychlor
MCF-7 U95A oligonucleotide
probe arrays (Affymetrix
10 nM (E2 estriol
estrone DES) 10 AM(genistein bisphenol A
nonylphenol and
methoxychlor)
Unigene name gene
name and fold change
for estrogen responsive
and nonresponsive in a
manuscript table
(Terasaka et al
2004)
Ouabain lauryl sulfate
dimethylsulfoxide
cycloheximide
tolbutamide sodium
fluoride diethyl
maleate buthionine
sulfoxamine
potassium bromate
sodium selenite
alloxan adriamycin
hydrogen peroxide
HepG2 Clontech Atlas Human
Stress Toxicology cDNA
arrays (234 genes)
Ouabain (43 uM) lauryl
sulfate (260 uM)
dimethylsulfoxide (128
M) cycloheximide (625
uM) tolbutamide (128
mM) sodium fluoride (3
mM) diethyl maleate
(125 mM) buthionine
sulfoxamine (30 mM)
potassium bromate (25
mM) sodium selenite (30
uM) alloxan (130 mM)
adriamycin (40 uM)
hydrogen peroxide
(4 mM)
Gene name ratio p -
value in downloadable
tables at journal website
(Morgan et a l
2002)
Aflatoxin B(1) (AFB(1))
2-acetylaminofluorene
(2AAF)
dimethylnitrosamine
(DMN)
acetaminophen (APAP)
HepG2 and primary
hepatocytes
Gene filter arrays
containing 31000 genes
10 uM aflatoxin B1
40 mM acetaminophen
100 uM
dimethylnitrosamine
10 uM
2-acetylaminofluorene
None (Harris et al
2004)
Mitomycin C (MMC)
and cisplatin (CIS)
and an alkylating
agent methyl
methanesulfonate
(MMS)
indirect-acting
genotoxins included
hydroxyurea (HU) a
ribonucleotide
reductase inhibitor
taxol (TXL) a
microtubule inhibitor
and etoposide
(ETOP)
L5178Y TK(+-)
mouse lymphoma
Affymetrix mouse
MG-U74A for MMC
and MG-U745Av2
(Affymetrix Inc Santa
Clara CA) for all the
other chemicals a total
of 9977 probe sets
(genes or ESTs) common
to these two array
models
Low mid and high doses Accession number gene
name gene ID statistical
significance at each time
point in a manuscript
table
(Hu et al 2004)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6646
Table 3 (continued)
Compounds Cell type Microarray type Compound dose Microarray data
availability
Reference
Hydroxyurea
(a carcinogen)
p-anisidine
(a noncarcinogen)
and paclitaxel
L5178Y Tk_-mouse
lymphoma
The Twin - Chip Mouse-
74K Digital Genomics
cDNA microarray
10 ngml paclitaxel 313
ugml hydroxyurea 32
ugml p-anisidine
Gene symbol and fold
change in a manuscript
table
(Lee et al 2003)
Acetaminophen
amiodarone clofibrate
erythromycin estolate
isoniazid alpha-
naphtylylisothiocyanate
beta-naphtoflavone
4-pentenoic acid
phenobarbital
tetracycline and
zileuton
Wistar Rat
hepatocytes
DualChip rat hepato
(Eppendorf Hamburg
Germany)
A single concentration
which varied for each
compound
Accession numbers and
gene namemdashfold
changes shown as
colored heat mapmdashnote
easily extracted from
publication
(de Longueville
et al 2003)
Bleomycin and hydrogen
peroxide
Mouse lymphoma
L5178Y TK(+ -)
Clontech Mouse 12K
cDNA microarray (1185
genes)
Bleomycin (25 and 20
ugml) hydrogen peroxide
(5 and 10 ugml)
Gene names and fold
change presented as bar
charts in publication
(Seidel Kan
Stott Schisler amp
Gollapudi 2003)
Bupivicaine
camptothecin
HL-60 Agilent human cDNA
microarray
1 mM Gene name GenBank
accession number
unigene and ratio in a
manuscript table
(Unami
Shinohara
Ichikawa amp Baba
2003)
Bisphenol A Mouse Sertoli TTE3 IntelliGene mouse
expression glass
microarrays (Version
10 Takara Shuzo)
which were spotted with
564 cDNA fragments of
mouse known genes and
approximately 301
expressed sequence tags
(ESTs)
0ndash400 uM Gene name GenBank
accession number and
fold change at time
points in a manuscript
table
(Tabuchi amp
Kondo 2003)
Mitomycin C or
doxorubicin
Hep G2 85 human gene custom
array
10 um mitomycin C
2 um doxorubicin or
2 ethanol
Bar charts with fold
changes in publication
very few genes
(Hong Muller amp
Lai 2003)
Amphotericin B Human peripheral
blood mononuclear
and THP-1
GF211 FKnown Genes_
Genefilter cDNA array
(ResGen) this array
consists of gt4000
individual elements
each representing a
known human gene
5 ugml Accession numbers and
fold expression in a
manuscript table
(Cleary Rogers amp
Chapman 2001
Rogers Pearson
Cleary Sullivan
amp Chapman
2002)
Benzo(a)pyrene diol
epoxide
TK6 human
lymphoblastoid
Human-350 microarray
a glass slide with 350
spotted human cDNA
probes (Phase-1
Molecular Toxicology
0 001 010 or
10 ugml)
Gene names and fold
change at doses in a
manuscript table
(Akerman et al
2004)
Etomoxir HepG2 Clontech AtlasiHuman Stress
Toxicology cDNA arrays
(234 genes)
1 mM etomoxir Gene names and fold
change in a manuscript
table
(Merrill et al
2002)
Tetrodotoxin Human glioma cell
line HTB-138
Using Affymetrix
GeneChip (HG-U133A
10 and 20 mM Affymetrix ID Genbank
ID gene name gene
symbol and fold change
in a manuscript table
(Raghavendra
Prasad Qi
Srinivasan amp
Gopalakrishnakone
2004)
Methotrexate
mercaptopurine
Human acute
lymphoblastic
leukemia
Affymetrix U133A chip Low and high dose and
combination
Data available as
supplemental data online
(Cheok et al 2003)
Prednisolone
vincristine
asparaginase
daunorubicin
Human acute
lymphoblastic
leukemia
Affymetrix U133A chip Various Data available as
supplemental data online
(Holleman et al
2004)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 47
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6648
A second study has indicated how a natural language
processing method CCNet was used to show the genes
regulated by the nuclear hormone receptor FXR (Apic
Ignjatovic Boyer amp Russell 2005) These automated
methods enable a more complete understanding of the
complexity of the transcriptional factors (Ekins Mirny amp
Schueltz 2002b Plant 2004 Ulrich 2003) but ultimately
rely on the quality of the content of the underlying database
of literature interactions This is a key consideration that is
often overlooked For example the gold standard database is
one that is manually curated to ensure the fidelity of the
direct interaction and is preferable to one generated
computationally by algorithms like natural language pro-
cessing (Nikolsky Nikolskaya amp Bugrim 2005) The
advantage of interaction networks over clustering has been
demonstrated in one study using MetaCore (Nikolsky
Ekins Nikolskaya amp Bugrim 2005) by reanalysis of a
published microarray study of G0-arrested MCF-7 breast
cancer cells treated with estrogen and 4-hydroxytamoxifen
(Hodges et al 2003) After producing integrated gene
networks for each treatment strikingly different patterns
were displayed although both contained early transcriptional
factors myc jun and fos Only the estrogen network featured
induced genes essential for all cell cycle phases (Nikolsky et
al 2005) Similarly microarray data for benzene toxicity
(Yoon et al 2003) has been re-analyzed by focusing on the
genes assessed on the p53 pathway (Ekins et al 2005e) We
envisage that a database of such networks for toxic
compounds will be used for comparing between different
molecules and used in the development of predictive
algorithms for Systems-ADMETox modeling in future
Another approach to using such pathway approaches is to
visualize the results of quantitative structure activity models
for predicting molecules binding to enzymes transporters
receptors and ion channels (Ekins Andreyev et al in press
2005e)
It is also possible to simultaneously interpret high
throughput data and predictions on interaction networks
providing a novel approach to predicting and understanding
potential undesirable drugndashdrug or off target effects in the
area of systems pharmacology An example data set uses
percent inhibition data for clotrimazole and ticonazole
which were screened against many different assays at a
single concentration in a commercially available database
BioPrint (Cerep Redmond WA) as published recently
(Fliri Loging Thadejo amp VOlkman 2005) The data for
10 assays has been arbitrarily encoded as inhibitors (gt50
inhibition) or non-inhibitors (lt50 inhibition) in a text file
which was then loaded into MetaCore The analyzed
network algorithm was then used which generates a large
network and fragments it into sub-networks each with a Z-
score and p-values for ranking according to saturation with
objects from the initial gene list The Gene-ontology
processes are also mapped to the gene list and individual
networks In this example a statistically significant network
was generated for the different proteins (Fig 4
p =2838e31) This network also maps the Gene Ontology
processes for the activation of MAPK (118 of genes p
value 9143e07) signal transduction (333 1600e05)
regulation of transcription DNA-dependent (294
2786e04) regulation of inflammatory response (39
3746e04) and the regulation of blood pressure (78
4230e04) This example network indicates how molecules
of the same or different therapeutic classes could be
evaluated for their effects as a graph either together as in
this case or individually This would be useful to indicate
potential off target effects and identify structurally dissimilar
molecules with similar network patterns Such networks
could then be compared to assess network overlap or
differences between molecules and their inhibition of
multiple proteins This type of unique visualization of high
throughput screening data illustrates how the target proteins
may be connected as a network to infer the possible
downstream effects of inhibition
With the burgeoning number of freely available online
and commercial databases that can be used for pathway
construction numbering in the hundreds there have been
suggestions to impose standards for model exchange
querying and visualization (Cary Bader amp Sander 2005)
To date there has been little discussion with regards to
standardization of ADMETox related databases although
there has been considerable discussion relating to drug
metabolism database generation (Erhardt 2003) This is
certainly an important area to address in the future There is
already a growing literature related to ADMETox that is
partially captured in the several commercially available
databases (Ekins et al 2005e) but to date there have been
limited academic efforts to capture data for transporters
with the human membrane transporter database (Yan amp
Sadee 2000) TP-search transporter database (httpwww
ilabrisewasedaacjptp-search) drug interaction database
(httpwwwdruginteractioninfoorgDatabaseinfoaspx)
nuclear hormone receptors (Nakata Yukawa Komiyama
Nakano amp Kaminuma 2002) the ADME-AP database
(Sun Ji Chen Wang amp Chen 2002) and PharmaGKB
(Oliver et al 2002) DSStox (httpwwwepagovnheert
dsstox) TOXNET (httptoxnetnimnihgov) that are
readily accessible
In order to generate accessible pathways using any of
the available software a large enough set of object
identifiers are required to map onto the underlying data-
base To demonstrate this datasets from toxicogenomics
studies have been evaluated with both the KEGG pathway
database and a commercially available product MetaCore
(wwwgenegocom) These gene or protein lists range in
size from 21 to 1853 objects In virtually all cases more
identifiers are mapped to networks in MetaCore and this
also seems independent of the identifier type used
(Unigene Affymetrix Genbank or Locuslink Table 5)
These numbers will obviously change as more information
is added to the respective databases hence the more
objects that are mapped from a dataset The more extensive
Table 4
Literature derived toxicoproteomics data
Compounds Data source Microarray type Compound dose Microarray data
availability
Reference
Carbon
tetrachloride
Male Wistar rats Affymetrix rat chip 8799
probes+proteomics
study
6ndash24 h exposure Genbank SwissProt and
binary data in a
manuscript table
(Fountoulakis
2004)
Paracetamol CD-1 male mice Custom mouse tox blots
with 450 genes
RTQ-PCR+proteomics
study 2-DIGE+
MALDI-MS
150 or 500 mgkg Gene name IMAGE ID
GenBank accession
number fold induction
SwissProt identifier and
protein abundance
change in manuscript
tables
(Ruepp Tonge
Shaw Wallis amp
Pognan 2002)
Paracetamol AP-1 male mice Affymetrix murine 11K
set+proteomics study
Up to 500 mgkg Gene names and fold
changes at multiple
doses and time points in
a manuscript tablemdash
proteomics data not
accessible
(Coen et al 2004)
Oxazepam or
Wy-14643
Male B6C3F1 mice NIEHS Mouse Chip
(8700 genes) 2-DIGE
and MS
Oxazepam (2500 ppm)
Wyeth (Wy)-14643
(500 ppm)
Proteomics data in a
manuscript table
Genbank accession
number gene name and
fold change in a
manuscript table data
also available to
download from NIEHS
website
(Iida et al 2003)
Compound A
(PPAR gamma
ligand)
Female rats CrlCD
(SD)IGS BR
Proteomics 2-DIGE and
MS
250 mgkgday up to
5 days
Accession number and
protein name and
average ratio in a
manuscript table
(Meneses-Lorente
et al 2004)
Bromobenzene Male Wistar rats Proteomics 2-DIGE and
MS custom 3000 cDNA
rat chip
5 mmolkg Accession number gene
name fold change in a
manuscript table protein
names along with bar
charts
(Heijne Stierum
Slijper van
Bladeren amp van
Ommen 2003)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 49
the network that can be generated (as it will consist of more
nodes) then a more comprehensive understanding of the
networks is possible The data available currently in the
literature can be used to evaluate such pathway and
network generation tools Recently we have used several
of the published studies (Tables 1ndash3) with MetaCore to
visualize networks for acetaminophen furan carbon tetra-
chloride benzene and cisplatin showing genes involved in
In vivo
In vitro
In Silico
1970s 1980s 1990s 2000s
Systems Biology ()
OMICS
Fig 2 The timeline for major paradigms in ADMETox
oxidative stress (Ekins Giroux Nikolsky Bugrim amp
Nikolskaya 2005c)
Other important pathwaynetwork building tools that could
potentially be applied to toxicogenomics data include
Ingenuity pathways analysis (httpwwwingenuitycom)
PathArt (httpwwwjubilantbiosyscompdhtm) Pathway
Assist (httpwwwariadnegenomicscomproctspathway
html) (Nikitin et al 2003) and several other databases
deposited at the Pathway Resource List (httpcbiiomskcc
orgprl) All of these products have unique underlying
proprietary pathway databases which are compiled manually
or automatically with text mining tools or a combination of
both We are still waiting for studies that provide a comparison
of different database tools or network building algorithms
Until then theremay be someoverlap but also some differences
between the results obtained from more than one network
method due to the database content and the algorithms used
The reader is recommended to evaluate for themselves several
technologies and select those with the most appropriate fit to
their own specialized needs
In the next Sections the further application of some
available network and database tools will be described with
Microarray or other high throughput data
Upload list of geneprotein identifiers and fold change significance etc (raw data or after selection of the differentially expressed
Genes using other statistical methods)
Parse database and generate interaction network with different algorithms or visualize on maps
Filter Networks
Access significance of the interaction networks
Determine GO processes
Compare 2 or more networks intersection overlap etc
Export gene list
Export gene list
generate interaction network with different algorithms or visualize on maps
Visualization
Fig 3 Schematic for the utilization of pathway tools for assessing high throughput data
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6650
specific detailed reference to transporters and enzymes but
these technologies can also be applied elsewhere as
described above
Fig 4 Network for high throughput screening data for clotrimazole and ticona
cholecystokinin CCR1 Choline transporter Chloride channel catechol-O-methyl
and co workers (Fliri et al 2005) Nodes highlighted with large blue circles rep
molecules showing gt50 inhibition in the assay blue circles represents both mol
inhibits gt50 and one inhibits lt50 Ligands (large) linked to transcriptional f
(wwgenegocom) (For interpretation of the references to colour in this figure leg
311 The role of transporters
A diverse array of organic solutes such as nutrients
neurotransmitters and drugs are transported by specialized
zole screened against 10 in vitro assays (Human Cannabinoid 1 Human
transferase COX2 CYP1A2 CYP2B6 CYP2C19) data published by Fliri
resent assays used in the study Red circles on these nodes represent both
ecules showing lt50 inhibition chequered circles represent one molecule
actors enzymes and transporters via edges using the MetaCorei database
end the reader is referred to the web version of this article)
Table 5
Datasets acquired from publications and other sources and used to evaluate two databasepathway tools KEGG and metaCore
Dataset Number of objects
genes proteins in list
Number of objects
mapped with KEGG
Number of objects mapped
with MetaCore on networks
Reference for microarray
data gene list
Tox Chip 10 (NIEHS) 1853 405 1498 httpsdir-appsniehsnihgov
mapsguestclonesrchcfm
4-Hydroxytamoxifen and
estrogen
1617 434 1343 (Hodges et al 2003)
Mitochondrial proteins 722 156 388 (Gaucher et al 2004
Taylor et al 2003)
Bromobenzene
(24 and 48 h)
130 41 89 (Heijne et al 2004)
Acetaminophen 30 19 23 (Heinloth et al 2004)
Acetaminophen 84 29 64 (Huang et al 2004)
Furan 185 64 139 (Huang et al 2004)
Tetrodotoxin 116 31 86 (Raghavendra Prasad et al
2004)
Benzene 73 16 62 (Yoon et al 2003)
Benzene 76 5 53 (Faiola et al 2004)
Carbon tetrachloride 37 8 26 (Young et al 2003)
Estrogen 94 33 90 (Lobenhofer et al 2002)
Trovafloxacin 142 20 82 (Liguori et al 2005)
Phenobarbital 37 13 28 (Ueda et al 2002)
L-742694 (liver)+ 45 17 19 (Hartley et al 2004)
L-742694 (intestine)+ 23 11 10 (Hartley et al 2004)
A-277249 21 7 9 (Waring et al 2002)
All files were converted to Locuslink identifiers except as noted () unigene () Affymetrix (+) Genbank identifiers were used
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 51
proteins across cellular membranes These may function as
passive processes or active processes energized by the
hydrolysis of ATP or coupling to the co-transport of counter
ions down an electrochemical gradient such as Na+ H+ and
Cl There are many thousands of transporters which can be
classified into distinct superfamilies One of these the solute
carrier class (SLC) is rapidly expanding and contains over
30 families and 200 members The ATP-binding cassette
(ABC) contains 7 families and over 48 members including
P-glycoprotein (P-gp) and MRP subfamilies (Zhang Knipp
Ekins amp Swaan 2002a Zhang Phelps Cheng Ekins amp
Swaan 2002b) Transporters have a key role in clinical
pharmacology with many drugs specifically targeting them
Numerous drugs share transport pathways with nutrients
and transporters have a role in oral absorption drug
bioavailability drug resistance excretion and ultimately
pharmacokinetics and pharmacodynamics
Polymorphism of drug transporters may be a key factor
in drug interactions and lack of effectiveness This field has
become known as pharmacogenomics and is focused on
understanding of inherited DNA sequence variations (poly-
morphisms and mutations) revealed by xenobiotics (Evans
amp McLeod 2003 Weinshilboum 2003) Over the last few
decades many genes have been directly linked to the
mechanisms of response (Evans amp McLeod 2003 Wein-
shilboum 2003) such that 20ndash95 of variability to drug
response is inheritable (Evans amp McLeod 2003) This
phenotypical variability is mainly caused by single nucleo-
tide polymorphisms (SNPs) present in anywhere from 1 to
50 of the population resulting in either lower protein
activity incorrect folding or rapid degradation via proteo-
somes (Weinshilboum amp Wang 2004) A number of
structurally diverse molecules bind to P-gp which is
expressed in many tissues and has numerous SNPs one of
which (C3435T) affects the expression level in the
duodenum and therefore can impact the absorption of
molecules which would be substrates for this transporter
(Sakaeda Nakamura amp Okumura 2002) The human
proton-dependent dipeptide transporter (hPEPT1) can also
affect the absorption of molecules in the intestine and
recently 9 SNPs were found with only one displaying a
reduced transport capacity (Zhang et al 2004) The sodium-
dependent carnitine cotransporter OCTN2 can possess
mutations and these result in primary carnitine deficiency
which impacts fatty acid oxidation and is characterized by
many clinical manifestations (Lahjouji Mitchel amp Qureshi
2001) The organic cation transporter 1 (OCT1) is also
important in the transport of numerous xenobiotics and
endobiotics Recently 4 SNPs were identified in the
Japanese population and when functionally characterized
in vitro the uptake of cations was reduced significantly for
some of these mutations indicating that this would likely
contribute to inter-individual variations in metabolism of
drugs which were transported via OCT1 (Sakata et al
2004)
3111 Clinical relevance of transporters The pregnane
X-receptor (PXR) is a transcriptional regulator of the
enzyme human MDR1 (P-gp) MRPs and OATP (Wang amp
LeCluyse 2003) CYP3A (Bertilsson et al 1998 Blumberg
et al 1998 Kliewer et al 1998) and CYP2C89 (Synold
Dussault amp Forman 2001) as well as many other genes
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6652
involved in the transport metabolism and biosynthesis of
bile acids (Staudinger Liu Madan Habeebu amp Klaassen
2001) However the additional receptors CAR FXR LXR
and other nuclear receptors take part in a complex network
of interactions to control these and other proteins Thus
elucidation of the regulatory networks which control the
expression of these transporters is also important To date
most of the research has centered on efflux transporters but
there has been considerable interest in uptake transporters
such as the organic anion transporter polypeptide (OATP
((Kim 2003) see also later Section on OATP)
There are several specific examples of the importance of
drug transporters to the clinical development of drugs One
example is the insulin sensitizer troglitazone which was
withdrawn due to hepatotoxicity although the precise
mechanism appears to have been unclear until recently
The major metabolite is a sulfated species and is suspected
of being responsible for the observed toxicity The recent
assessment of the organic anion transporting polypeptides
OATP-C and OATP8 expressed on the hepatocyte baso-
lateral membrane indicated that sulphated troglitazone has a
high affinity for the former and possibly lower affinity for
the latter (Nozawa et al 2004) This metabolite would
therefore be expected to accumulate in hepatocytes and
inhibit the bile salt export pump and Mrp2 Because
polymorphisms have been shown for OATP-C (Tirona
Leake Merino amp Kim 2001) it is also possible that these
may result in the accumulation of the metabolite and in turn
elicit idiosyncratic toxicity A second relevant example of
the impact of transporters is the clinically significant drugndash
drug interaction between cerivastatin and cyclosporine A
which occurs via the OATP-C transporter (Shitara Itoh
Sato Li amp Sugiyama 2003) A third example are the HIV
protease inhibitors saquinavir ritonavir and indinavir which
are transported by MRP2 in vitro and other drugs such as
probenecid and sulfinpyrazone are able to enhance this
transport Transport by MRP2 suggests that these com-
pounds will have decreased bioavailability due to increased
clearance and other drugs could aggravate this situation by
further enhancing transport (Huisman et al 2002) Sim-
ilarly the rifampicin mediated induction via PXR of MRP2
and P-gp in healthy subjects was found to significantly
decrease the AUC and also correlated with intestinal
expression of these transporters This transporter is also
inducible by cisplatin 2-AAF and phenobarbital (Schrenk
et al 2001) indicating multiple mechanisms may be
involved In other species such as rat commonly used as a
toxicity model orthologs of the transporters such as OATP2
are expressed and can be induced with ligands for PXR like
PCN (Guo Choudhuri amp Klaassen 2002) This is useful
knowledge because the advent of microarray technology
allows one to dose a rat with a xenobiotic and assess
thousands of genes simultaneously in a particular tissue For
instance animals dosed with known nephrotoxins have
shown some upregulation of the NandashKndashCl transporter
however the authors suggested genomic responses are
stronger soon after exposure before declining (Fleck et
al 2003) Some transporters may therefore be specifically
targeted by drugs in one tissue such as the CNS but these
same transporters may also be expressed elsewhere in the
body hence off-target effects may result in toxicity The
serotonin transporter is one such example which is
expressed in the lungs and brain Some substrates for this
channel like fenfluramine can result in primary pulmonary
hypertension as they accumulate in pulmonary cells (Roth-
man Ayestas Dersch amp Baumann 1999) Similarly P-gp is
expressed at the blood brain barrier and intestine impacting
the efficacy and bioavailability of drugs
3112 Transporter network examples ABCA1 The
ABCA1 transporter mediates the first step of cholesterol
transport Mutations in this gene cause Tangier disease
which results in severe HDL deficiency cholesterol
accumulation in macrophages and attendant atherosclerosis
This transporter represents a drug target for upregulation
modulating cholesterol metabolism and prevention of
cardiovascular disease (Oram amp Lawn 2001) In vitro
ABCA1 can be inhibited by the sulfonylurea glybenclamide
(Field Burn amp Mathur 2004 Wang Silver Thiele amp Tall
2001) In order to illustrate the advantages of mapping drug
transporters as networks onto functional models alongside
other proteins one can consider the example of ABCA1
The query of the MetaCorei database shows that this
transporter appears on three manually curated pathway maps
representing the Fstate of the art_ knowledge derived from
reliable high quality literature sources One can also use the
individual maps as an interface to access the underlying
layers of information about the transporter including the list
of encoding genessplice variants with known SNPs In
addition to browsing MetaCorei a user can also build
custom networks around ABCA1 using the network-
construction tool (Fig 5) Such a visualization utility may
be very helpful for identification of all putative pathways
around a particular transporter or compound of interest The
ABCA1 network created by this tool shows that this
transporter at the time of writing is linked directly to
twenty-five other objects such as APOE1 and LXR Many
of its neighbors have their own SNPs that could be
important in determining interactions between transport of
a drug and normal human transport of endogenous ligands
in health or disease
3113 Transporter network examples OATP The
OATPs are key membrane bound transporters expressed in
many organs including intestine liver lung choroid plexus
blood brain barrier and other organs (Tamai et al 2000)
This family of transporters is capable of mediating the
sodium-independent transport of a diverse array of mole-
cules such as steroid conjugates organic anions and
xenobiotics by coupling uptake with efflux of bicarbonate
(Satlin Amin amp Wolkoff 1997) glutathione or its
conjugates (Hagenbuch amp Meier 2004) The inhibition of
Fig 5 The network of direct gene interactions around the ABCA1 transporter gene (centre) generated with MetaCorei The interation types between nodes are shown as small colored hexagons eg unspecified
allosteric regulation binding cleavage competition covalent modification dephosphorylation phosphorylation transcription regulation transformation When applicable interactions also have a positive or
negative effect and direction Ligands (purple) linked to other proteins (blue blobs) transfactors (red) enzymes (orange) (For interpretation of the references to colour in this figure legend the reader is referred to
the web version of this article)
SEkin
sJournalofPharm
acologica
landToxico
logica
lMeth
ods53(2006)38ndash66
53
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6654
this transporterrsquos hepatic uptake of other compounds may be
important for reported drugndashdrug interactions (Kim 2003)
described earlier (Shitara et al 2003) as well as cerivastatin
with gemfibrozil (Shitara Hirano Sato amp Sugiyama 2004)
OATP1B1 (previous names OATP-C LST-1 OATP2
SLC21A6) represents the most studied human OATP to
date (Meier amp Stieger 2000) and is expressed on the
basolateral plasma membrane of hepatocytes Several single
nucleotide polymorphisms have been identified in the
OATP1B1 gene in European-Americans African-Americans
(Tirona et al 2001) and Japanese (Nozawa et al 2002)
dramatically impacting the transport of ligands such as
pravastatin (Mwinyi Johne Bauer Roots amp Gerloff 2004
Nishizato et al 2003) estrone-3-sulfate (Nozawa et al
2002 Tirona et al 2001) Rifampin (Tirona Leake
Wolkoff amp Kim 2003) and estradiol 17h-d-glucuronide(Tirona et al 2001)
The regulation of SLCO may be affected during
extrahepatic cholestasis bile duct ligation bile salt induced
Fig 6 Networks generated with the autoexpand algorithm in MetaCorei to illustr
be interconnected with other protein regulatory signaling information Informa
unspecified allosteric regulation binding cleavage competition covalent m
transformation When applicable interactions also have a positive or negative e
transfactors (red) enzymes (orange) (For interpretation of the references to colour
cholestatic hepatitis (Dumont et al 1997 Meier amp Stieger
2000 Rost et al 2003) and primary sclerosing cholangitis
(Oswald Kullack-Ublick Paumgarter amp Beuer 2001) In
particular OATP1B1 appears to be regulated by the liver-
enriched transcription factor hepatocyte nuclear factor 1a
(HNF1a) which binds to the promoter region of this
transporter (Jung et al 2001) Site directed mutagenesis
of this binding site resulted in inactivation suggesting the
critical nature of the interaction with HNF1a Bile acids
such as CDCA have been shown to transcriptionally repress
HNF1a in vitro via inhibition of the transactivating effect of
HNF4a on HNF1a (Jung amp Kullak-Ublick 2003) After
screening many rat and human uptake transporters in vitro
OATP1B1 was also shown to modulate the PXR response
by controlling rifampin retention in the cell and therefore
affecting the induction of CYP3A4 and other gene products
such as P-gp (Tirona et al 2003)
Some of the literature for OATP1B1 human substrate
data has been annotated into MetaCorei to illustrate the
ate how ligands for the human OATP-C (OATP1B1) transporter (centre) can
tion on the type of interaction between objects is hidden for clarity eg
odification dephosphorylation phosphorylation transcription regulation
ffect and direction Ligands (purple) linked to other proteins (blue blobs)
in this figure legend the reader is referred to the web version of this article)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 55
visualization of the complex interconnections between this
transporter its ligands regulatory factors and signaling
molecules already in the database (Fig 6) The network was
generated with the autoexpand algorithm in the software
representing one of multiple available algorithms for
connecting genes ligands and other objects in the database
Clearly if more ligands and their connections are added to
the database the complexity of the network will increase
considerably The OATP-C (OATP1B1) gene details can be
viewed upon querying the database and links are provided
to other public databases This page can be used to highlight
the multiple synonyms for this gene as well as links to the
multiple SNPs identified to date
3114 Transporter microarray data Microarrays have
generally been limited in the number of transporters present
on them (Annereau et al 2004) however they have been used
in an attempt to correlate pharmacokinetic properties with
gene expression for valacyclovir (Landowski et al 2003) as
well as understand the expression profile in different tissues
or cell lines upon food component or xenobiotic treatment
(Anderle Huang amp Sadee 2004) This lack of transporters
on microarrays has prompted some groups to produce their
own arrays with a heavier emphasis on transporters These
arrays have for example then been used to demonstrate the
upregulation of ABC transporters and down-regulation of
GST-Pi in cell lines resistant to colchicines or 9-nitro-
camptothecin (Annereau et al 2004) The genes that were
significantly up or down-regulated in this particular study
were used to build networks with MetaCore (Fig 7A B) and
the similarities between them were assessed (Fig 7C)
Although there were only a small number of significantly
changed genes in common (IL-8 Fos GST-Pi Calpactin and
Ubiquitin hydrolase) it is perhaps likely that there is a much
larger common gene network that is important for drug
resistance although a much larger number of cell lines and
drug treatments need to be evaluated to produce a definitive
drug resistance signature involving transporters enzymes
and transcriptional regulators
312 Applications to enzymes
As we have already described it may be particularly
valuable to visualize enzymes as networks to show
interactions with transcriptional regulators and ligands
For example a key enzyme is CYP3A4 which metabolizes
40ndash45 of all drugs and has relatively few SNPs
(Ingelman-Sundberg 2004) Using a second software suite
MetaDrugi (wwwgenegocom) it is possible to construct
a custom network around this or other drug metabolizing
enzymes (Fig 8) In this case the gene network for
CYP3A4 highlights all of the major transcriptional
regulators and several more distant linked proteins and
ligands connected on the network that may be useful for
further study (Fig 8) Substrates inhibitors as well as
regulatory factors and other enzymes can be observed
connected on this network Due to the many hundreds of
known inhibitors and substrates only a small number are
shown here for clarity We assume that if a perturbation in
a pathway (eg due to a nonfunctional enzyme) is linked
to a certain pathologic condition a similar perturbation
caused by the interference from xenobiotic metabolism
(eg competitive inhibition of the same enzyme) may
result in identical effects As microarray gene expression
data is increasingly generated the role of enzyme
regulation in toxicity of certain xenobiotics will become
more apparent from either in vivo or in vitro studies The
visualization of such signature gene networks involving
transporters and enzymes their ligands and regulatory
factors will also be important for future toxicity prediction
methods We have recently generated visualizations of
microarray data from MCF-7 cells treated with 4-hydrox-
ytamoxifen to show that some of the key genes involved in
metabolism and transport are upregulated (Ekins et al
2005d) In addition we have made predictions with various
QSAR models in MetaDrug to indicate the involvement of
PXR CYP3A4 and P-gp (Ekins et al 2005e) Therefore it
appears likely that 4-hydroxytamoxifen could increase its
own metabolism as well as efflux from cells via P-gp
which can be visualized on networks Any decrease of
function of these enzymes or transporters in a population
would likely result in changes in the metabolism and
transport of this active metabolite potentially impacting
the clinical effect This represents one example of how
both pathway tools QSAR models and network building
algorithms can be used with different types of predicted
and experimental data to allow visualization of potential
compound interactions or toxicity
313 Future network applications
As the population ages an increasing incidence and
prevalence of systemic diseases especially chronic diseases
have occurred among older adults This has resulted in an
increase in medications used concomitantly by this
population which presents challenges for drugndashdrug
interactions Physiologically elderly patients may behave
differently to the younger patients for which the drugs are
initially developed for and tested on Many pharmacoki-
netic investigations in the elderly population reveal
decreased clearance of lipophilic drugs metabolized by
the cytochrome P450 enzymes however few studies have
evaluated aging-dependent or gender-related changes in
specific P450 enzymes (Hunt Westerkam amp Slave 1992)
Age-related physiological changes such as a reduction in
liver mass hepatic metabolizing enzyme activity liver
blood flow and alterations in plasma drug binding may
account for the decreased elimination of some metabolized
drugs in the elderly It is particularly difficult to separate an
effect of aging from the variation in the rate of metabolism
due to factors such as individual metabolic phenotype
(slow or fast metabolizer due to SNPs) environmental
influences concomitant disease states and drug intake
(drugndashdrug interactions) The available data suggest that
Fig 7 Networks for drug resistant cell lines generated with the Auto expand algorithm in MetaCore A KB-3-1 vs KB-8-5 B ATC-DU-145 vs RCO1 C node overlap of A and B with initial nodes highlighted
with blue or red solid circles (Initial gene lists derived from (Annereau et al 2004)) Information on the type of interaction between objects is hidden for clarity eg unspecified allosteric regulation binding
cleavage competition covalent modification dephosphorylation phosphorylation transcription regulation transformation When applicable interactions also have a positive or negative effect and direction
Ligands (purple) linked to other proteins (blue blobs) transfactors (red) enzymes (orange) (For interpretation of the references to colour in this figure legend the reader is referred to the web version of this article)
SEkin
sJournalofPharm
acologica
landToxico
logica
lMeth
ods53(2006)38ndash66
56
Fig7(continued)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 57
Fig7(continued)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6658
Fig 8 The network of gene interactions around the CYP3A4 enzyme (centre) from MetaCorei Information on the type of interaction between objects is
hidden for clarity eg unspecified allosteric regulation binding cleavage competition covalent modification dephosphorylation phosphorylation
transcription regulation transformation When applicable interactions also have a positive or negative effect and direction Ligands (purple) linked to other
proteins (blue blobs) transfactors (red) enzymes (orange) (For interpretation of the references to colour in this figure legend the reader is referred to the web
version of this article)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 59
the initial doses of drugs metabolized by these enzymes
should be reduced in older patients according to the clinical
response In most published studies the elderly appear at
least as responsive as the young to inducers or inhibitors of
P450s (Durnas Loi amp Cusack 1990) More recently there
has been some suggestion that there are age related
reductions in function of some specific P450s such as
CYP3A4 (Patki et al 2004) and this could occur at the
level of regulation However earlier studies with the same
enzyme showed no change in clearance with age (Hunt et
al 1992) To date there has been even less examination of
the transporter functions and any changes with age
(Kinirons amp OrsquoMahony 2004) so the current understand-
ing of the effects of aging on metabolism and transport is
anything but transparent This represents an extreme
challenge for the pharmaceutical industry how to predict
whether a drug has an affinity for an enzyme or transporter
and whether this may be clinically important if it shows a
decline in expression or function with aging Any decrease
of function of these enzymes or transporters in an elderly
population would result in changes in the metabolism and
transport of metabolites potentially impacting the clinical
effect This represents another example of how computa-
tional approaches may perhaps be used with different types
of predicted and experimental data to allow visualization of
potential compound interactions or toxicity in elderly
populations We may see different gene networks high-
lighted as humans age and these may be modified by drug
treatment and coadministration This represents an area
were network analysis could be applied in the future and is
in need of considerable further research
The collection of microarray data in databases such as
CEBS ArrayTrack and EDGE represents a future resource
for computational gene network analysis One could
envisage that ultimately in each case such data is converted
to one or more networks that are also displayed for the user
and can be used to compare treatments from in vivo and in
vitro experiments This would represent a different approach
to clustering the data as currently implemented in one of
these efforts (Hayes et al 2005) and may condense large
amounts of experimentally derived data into a readily
interpreted network
4 Discussion
Previously in this journal the progress of many
research groups in predicting human ADME parameters
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6660
in silico (Ekins et al 2000b) and approaches for drug
metabolism (Ekins Ring Grace McRobie-Belle amp
Wrighton 2000a) have been described Both of these
reviews commented on moving HT assays for ADME
Tox much earlier in drug discovery which had also been
indicated by other groups The initial reviews also
highlighted the likely wealth of data that would become
available and how this could be used for structure
activity relationships alongside the bioactivity data in
computational models It was noted that there was a
paucity of predictive metabolism tools at that time In
addition a growing number of efforts to model whole
cells and organs now a field called systems biology
were recognized as models that could be integrated with
the in silico ADME approaches In summary since these
past reviews virtually all pharmaceutical companies have
attempted earlier high throughput screening for ADME
Tox properties and to some extent the wider application
of computational approaches for physicochemical proper-
ties Systems biology is being quite widely acknowledged
as the new paradigm for understanding complex bio-
logical datasets derived from high throughput technolo-
gies and the accumulated knowledge on human protein
interactions (Hartwell Hopfield Leibler amp Murray 1999
Hood 2003b) Therefore systems biology can be defined
as the integration of genetic proteomic transcriptomic
and metabonomic data using computational methods
(Nicholson amp Wilson 2003) When taken together
information on molecular processes derived from different
sources represents a lsquolsquouniversersquorsquo of putative biological
functionality of which only a small fraction of it will be
realized in a cell at any given time To date systems
biology has been driven by academia and funding bodies
such as the NIH rather than the pharmaceutical compa-
nies Presently there is a great deal of interest from
scientists of all backgrounds in identifying the networks
of cellular pathways and the corresponding physically
interacting proteins
The network building software for systems biology
described in this current review will be valuable to query
high throughput data and known literature interactions in
order to predict potential toxicity in different species In the
future the compilation of published toxicogenomics data-
sets characteristic of different types of toxicity will likely
be available in these software systems to act as a reference
database It is also feasible that we will be able to generate
the annotated datasets which specifically address the
differences between human and rat networks implicated
in toxicity The identification of sub-network modules
conserved between human and rat distinct for toxicity
types or predictive for toxic end-points in human will be
possible Such signature gene networks (Nikolsky et al
2005) can then be verified with other experimentally
derived data prospectively or from preexisting databases
The combined Fhigh throughput datandashin silico network_approaches therefore represents a novel method for access-
ing complex tissue-level processes as networks integrating
all data types based on functional interactions The data-
bases developed and used in this approach will certainly
benefit from further annotation around the drug metaboliz-
ing enzymes and transporters as described herein in terms
of transcriptional regulation and the ligands associated with
them which frequently appear in the literature It will be
important to capture disparities in the assignment of ligands
to enzymes transporters and other proteins as well as
negative data
In summary although there have been numerous
toxicogenomics studies published there is presently a
relatively small number of datasets that are freely available
to perform network analysis of microarray data The
number of studies identifying large numbers of proteins
which are affected by molecule treatment are even scarcer
still while there are several examples of some published
studies that combine such data It is hoped that the
numerous database initiatives for high content and toxicol-
ogy data that are being undertaken will improve the
situation for other researchers that are not currently
equipped to do such microarray studies themselves The
addition of requirements by journals to deposit such raw
data in a freely accessible resource will aid these initiatives
ADMETox groups have seen new technologies and
approaches developed over the last decade that have all
been applied to identify poor compounds earlier (Fig 2)
The latest technologies integrate network building tools
with high content data and databases The current review
described the limited number of networks generated for
ADMETox at present and one hopes that the impact of
such analyses will be commonplace in the future Systems
biology is however more than just applying a network
approach and hence systems-ADMETox will have to
evolve due to the continual pressure to develop newer
technologies This current paradigm combining empirical
data and computational methods should integrate the
complex data already generated making it readily inter-
pretable and valuable for identifying the most promising
compounds in the future
Acknowledgements
Dr Maggie AZ Hupcey is gratefully acknowledged for
editorial assistance Dr Peter W Swaan (University of
Maryland) and Dr Cheng Chang (Ohio State University)
Dr Steve Wright (University of Arizona) Dr K Sandy
Pang (University of Toronto) and Dr Craig Giroux (Wayne
State University) are kindly acknowledged for their support
and discussions My colleagues at GeneGo Sergey
Andreyev Andy Ryabov Eugene Kirillov Eugene A
Rakhmatulin Svetlana Sorokina Andrej Bugrim Tatiana
Nikolskaya Yuri Nikolsky John Metz and Julie Bryant are
all thanked for their considerable contributions to software
development and data annotation
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 61
References
Abe M Yamashita S Kuramoto T Hirayama Y Tsukamoto T Ohta
T et al (2003) Global expression analysis of N-methyl-NV-nitro-N-nitrosoguanidine-induced rat stomach carcinomas using oligonucleotide
microarrays Carcinogenesis 24 861ndash867
Adachi T Ono Y Koh K B Takashima K Tainaka H Matsuno Y
et al (2004) Long-term alteration of gene expression without
morphological change in testis after neonatal exposure to genistein in
mice Toxicogenomic analysis using cDNA microarray Food and
Chemical Toxicology 42 445ndash452
Akerman G S Rosenzweig B A Domon O E McGarrity L J
Blankenship L R Tsai C A et al (2004) Gene expression profiles
and genetic damage in benzo(a)pyrene diol epoxide-exposed TK6 cells
Mutation Research 549 43ndash64
Aksenov S V Church B Dhiman A Georgieva A Sarangapani R
Helmlinger G et al (2005) An integrated approach for inference and
mechanistic modeling for advancing drug development FEBS Letters
579 1878ndash1883
Anderle P Huang Y amp Sadee W (2004) Intestinal membrane transport
of drugs and nutrients Genomics of membrane transporters using
expression microarrays European Journal of Pharmaceutical Sciences
21 17ndash24
Annereau J P Szakacs G Tucker C J Arciello A Cardarelli C
Collins J et al (2004) Analysis of ATP-binding cassette transporter
expression in drug-selected cell lines by a microarray dedicated to
multidrug resistance Molecular Pharmacology 66 1397ndash1405
Apic G Ignjatovic T Boyer S amp Russell R B (2005) Illuminating drug
discovery with biological pathways FEBS Letters 579 1872ndash1877
Baker V A Harries H M Waring J F Duggan C M Ni H A Jolly
R A et al (2004) Clofibrate-induced gene expression changes in rat
liver A cross-laboratory analysis using membrane cDNA arrays
Environmental Health Perspectives 112 428ndash438
Balakin K V Ekins S Bugrim A Ivanenkov Y A Korolev D amp
Nikolsky Y et al (2004a) Quantitative structurendashmetabolism relation-
ship modeling of the metabolic N-dealkylation rates Drug Metabolism
and Disposition 32 1111ndash1120
Balakin K V Ekins S Bugrim A Ivanenkov Y A Korolev D amp
Nikolsky Y et al (2004b) Kohonen maps for prediction of binding to
human cytochrome P450 3A4 Drug Metabolism and Disposition 32
1183ndash1189
Barabasi A -L amp Oltvai Z N (2004) Network biology Under-
standing the cellrsquos functional organization Nature Reviews Genetics
5 101ndash113
Bartosiewicz M Penn S G amp Buckpitt A (2001) Applications of gene
arrays in environmental toxicology Fingerprints of gene regulation
associated with cadmium chloride benzo(a)pyrene and trichloro-
ethylene Environmental Health Perspectives 109 71ndash74
Bertilsson G Heidrich J Svensson K Asman M Jendeberg L
Sydow-Backman M et al (1998) Identification of a human nuclear
receptor defines a new signaling pathway for CYP3A induction
Proceedings of the National Academy of Sciences of the United States
of America 95 12208ndash12213
Blumberg B Sabbagh Jr W Juguilon H Bolado Jr J van Meter
C M Ong E S et al (1998) SXR a novel steroid and xenobiotic-
sensing nuclear receptor Genes and Development 12 3195ndash3205
Borodina Y Rudik A Filimonov D Kharchevnikova N Dmitriev A
Blinova V et al (2004) A new statistical approach to predicting
aromatic hydroxylation sites Comparison with model-based
approaches Journal of Chemical Information and Computer Sciences
44 1998ndash2009
Borodina Y Sadym A Filimonov D Blinova V Dmitriev A amp
Poroikov V (2003) Predicting biotransformation potential from
molecular structure Journal of Chemical Information and Computer
Sciences 43 1636ndash1646
Bottone Jr F G Martinez J M Collins J B Afshari C A amp Eling
T E (2003) Gene modulation by the cyclooxygenase inhibitor
sulindac sulfide in human colorectal carcinoma cells Possible link to
apoptosis Journal of Biological Chemistry 278 25790ndash25801
Boyer S amp Zamora I (2002) New methods in predictive metabolism
Journal of Computer-Aided Molecular Design 16 403ndash413
Bredel M amp Jacoby E (2004) Chemogenomics An emerging strategy
for rapid target and drug discovery Nature Reviews Genetics 5
262ndash275
Bugrim A Nikolskaya T amp Nikolsky Y (2004) Early prediction of drug
metabolism and toxicity Systems biology approach and modeling
Drug Discovery Today 9 127ndash135
Bulera S J Eddy S M Ferguson E Jatkoe T A Reindel J F
Bleavins M R et al (2001) RNA expression in the early character-
ization of hepatotoxicants in Wistar rats by high-density DNA micro-
arrays Hepatology 33 1239ndash1258
Butcher E C Berg E L amp Kunkel E J (2004) Systems biology in drug
discovery Nature Biotechnology 22 1253ndash1259
Butte A (2002) The use and analysis of microarray data Naturalist
Review of Drug Discovery 1 951ndash960
Cary M P Bader G D amp Sander C (2005) Pathway information for
systems biology FEBS Letters 579 1815ndash1820
Cheok M H Yang W Pui C H Downing J R Cheng C Naeve
C W et al (2003) Treatment-specific changes in gene expression
discriminate in vivo drug response in human leukemia cells Nature
Genetics 34 85ndash90
Cleary J D Rogers P D amp Chapman S W (2001) Differential
transcription factor expression in human mononuclear cells in response
to amphotericin B Identification with complementary DNA microarray
technology Pharmacotherapy 21 1046ndash1054
Coen M Ruepp S U Lindon J C Nicholson J K Pognan F Lenz
E M et al (2004) Integrated application of transcriptomics and
metabonomics yields new insight into the toxicity due to paracetamol in
the mouse Journal of Pharmaceutical and Biomedical Analysis 35
93ndash105
Cornwell P D De Souza A T amp Ulrich R G (2004) Profiling of
hepatic gene expression in rats treated with fibric acid analogs
Mutation Research 549 131ndash145
Csermely P Agoston V amp Pongor S (2005) The efficiency of multi-
target drugs The network approach might help drug design Trends in
Pharmacological Sciences 26 178ndash182
Cunningham M J Liang S Fuhrman S Seilhamer J J amp
Somogyi R (2000) Gene expression microarray data analysis for
toxicology profiling Annals of the New York Academy of Sciences 919
52ndash67
de Longueville F Atienzar F A Marcq L Dufrane S Evrard S
Wouters L et al (2003) Use of a low density microarray for studying
gene expression patterns induced by hepatotoxicants on primary
cultures of rat hepatocytes Toxicological Sciences 75 378ndash392
Dobrin R Beg Q K Barabasi A L amp Oltvai Z N (2004)
Aggregation of topological motifs in the Escherichia coli transcriptional
regulatory network BMC Bioinformatics 5 10
Donald S Verschoyle R D Edwards R Judah D J Davies R
Riley J et al (2002) Hepatobiliary damage and changes in hepatic
gene expression caused by the antitumor drug ecteinascidin-743 (ET-
743) in the female rat Cancer Research 62 4256ndash4262
Dumont M Jacquemin E DrsquoHont C Descout C Cresteil D Haouzi
D et al (1997) Expression of the liver Na+-independent organic anion
transporting polypeptide (oatp-1) in rats with bile duct ligation Journal
of Hepatology 27 1051ndash1056
Durnas C Loi C M amp Cusack B J (1990) Hepatic drug metabolism
and aging Clinical Pharmacokinetics 19 359ndash389
Eisen M B Spellman P T Brown P O amp Botstein D (1998) Cluster
analysis and display of genome-wide expression patterns Proceedings
of the National Academy of Sciences of the United States of America
95 14863ndash14868
Ekins S Andreyev S Ryabov A Kirilov E Rakhmatulin E A
Bugrim A et al (in press) Computational Prediction of Human Drug
Metabolism Exp Opin Drug Metab Toxicol
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6662
Ekins S Boulanger B Swaan P W amp Hupcey M A Z (2002)
Towards a new age of virtual ADMETOX and multidimensional drug
discovery Journal of Computer-Aided Molecular Design 16 381ndash401
Ekins S Bugrim A Nikolsky Y amp Nikolskaya T (2005) Systems
biology Applications in drug discovery In S Gad (Ed) Drug
Discovery Handbook (pp 123ndash183) Hoboken NJrsquo Wiley and Sons
Ekins S Giroux C N Nikolsky Y Bugrim A amp Nikolskaya T
(2005) A signature gene network approach to toxicity The Toxoco-
logists 84
Ekins S Kirillov E Rakhmatulin E amp Nikolskaya T (2005) A novel
method for visualizing nuclear hormone receptor networks relevant to
drug metabolism Drug Metabolism and Disposition 33 474ndash481
Ekins S amp McGowan R J (2001) The limits of reductionism The
shifting genomic paradigmrsquos impact on industry and academia
Philsophy in Science 9 1ndash23
Ekins S Mirny L amp Schuetz E G (2002) A ligand-based approach to
understanding selectivity of nuclear hormone receptors PXR CAR
FXR LXRa and LXRb Pharmaceutical Research 19 1788ndash1800
Ekins S Nikolsky Y amp Nikolskaya T (2005) Techniques Application
of systems biology to absorption distribution metabolism excretion
and toxicity Trends in Pharmacological Sciences 26 202ndash209
Ekins S Ring B J Grace J McRobie-Belle D J amp Wrighton S
A (2000) Present and future in vitro approaches for drug
metabolism Journal of Pharmacological and Toxicological Methods
44 313ndash324
Ekins S Waller C L Swaan P W Cruciani G Wrighton S A amp
Wikel J H (2000) Progress in predicting human ADME parameters in
silico Journal of Pharmacological and Toxicological Methods 44
251ndash272
Erhardt P W (2003) A human drug metabolism database Potential roles
in the quantitative predictions of drug metabolism and metabolism-
related drugndashdrug interactions Current Drug Metabolism 4 411ndash422
Evans W E amp McLeod H L (2003) Pharmacogenomicsmdashdrug
disposition drug targets and side effects New England Journal of
Medicine 348 538ndash549
Ezendam J Staedtler F Pennings J Vandebriel R J Pieters R
Boffetta P et al (2004) Toxicogenomics of subchronic hexachlor-
obenzene exposure in Brown Norway rats Environmental Health
Perspectives 112 782ndash791
Faiola B Fuller E S Wong V A amp Recio L (2004) Gene expression
profile in bone marrow and hematopoietic stem cells in mice exposed to
inhaled benzene Mutation Research 549 195ndash212
FDA (2004) Innovation stagnation Challenge and opportunity on the
critical path to new medicinal products
Fiehn O (2001) Combining genomics metabolome analysis and bio-
chemical modelling to understand metabolic networks Comparative
Functional Genomics 2 155ndash168
Field F J Born E amp Mathur S N (2004) LXRRXR ligand activation
enhances basolateral efflux of beta-sitosterol in CaCo-2 cells Journal of
Lipid Research 45 905ndash913
Fleck C Sutter L Appenroth D Koch B Meinhold T Pitack M
et al (2003) Use of gene chip technology for the characterisation of
the regulation of renal transport processes and of nephrotoxicity in
rats Experimental and Toxicologic Pathology 54 401ndash410
Fliri A F Loging W T Thadeio P F amp Volkmann R A (2005)
Biological spectra analysis Linking biological activity profiles to
molecular structure Proceedings of the National Academy of Sciences
of the United States of America 102 261ndash266
Fountoulakis M (2004) Application of proteomics technologies in the
investigation of the brain Mass Spectrometry Reviews 23 231ndash258
Gaucher S P Taylor S W Fahy E Zhang B Warnock D E Ghosh
S S et al (2004) Expanded coverage of the human heart
mitochondrial proteome using multidimensional liquid chromatography
coupled with tandem mass spectrometry Journal of Proteome
Research 3 495ndash505
Gerhold D Lu M Xu J Austin C Caskey C T amp Rushmore T
(2001) Monitoring expression of genes involved in drug metabolism
and toxicology using DNA microarrays Physiological Genomics 5
161ndash170
Guo G L Choudhuri S amp Klaassen C D (2002) Induction profile of
rat organic anion transporting polypeptide 2 (oatp2) by prototypical
drug-metabolizing enzyme inducers that activate gene expression
through ligand-activated transcription factor pathways Journal of
Pharmacology and Experimental Therapeutics 300 206ndash212
Hagenbuch B amp Meier P J (2004) Organic anion transporting
polypeptides of the OATP SLC21 family Phylogenetic classification
as OATP SLCO superfamily new nomenclature and molecularfunc-
tional properties Pflugers Archiv 447 653ndash665
Hamadeh H K amp Afshari C A (2004) Toxicogenomics principles and
applications Hobokenrsquo John Wiley and Sons
Hamadeh H K Bushel P R Jayadev S Martin K DiSorbo O Sieber
S et al (2002) Gene expression analysis reveals chemical-specific
profiles Toxicological Sciences 67 219ndash231
Hamadeh H K Jayadev S Gaillard E T Huang Q Stoll R
Blanchard K et al (2004) Integration of clinical and gene expression
endpoints to explore furan-mediated hepatotoxicity Mutation Research
549 169ndash183
Hamadeh H K Knight B L Haugen A C Sieber S Amin R P
Bushel P R et al (2002) Methapyrilene toxicity Anchorage of
pathologic observations to gene expression alterations Toxicologic
Pathology 30 470ndash482
Han J D Bertin N Hao T Goldberg D S Berriz G F Zhang L V
et al (2004) Evidence for dynamically organized modularity in the
yeast proteinndashprotein interaction network Nature 430 88ndash93
Hanisch D Zien A Zimmer R amp Lengauer T (2002) Co-clustering of
biological networks and gene expression data Bioinformatics 18
S145ndashS154
Harris A J Dial S L amp Casciano D A (2004) Comparison of basal
gene expression profiles and effects of hepatocarcinogens on gene
expression in cultured primary human hepatocytes and HepG2 cells
Mutation Research 549 79ndash99
Harrison S C (2004) Whither structural biology Nature Structural
Molecular Biology 11 12ndash15
Hartley D P Dai X He Y D Carlini E J Wang B Huskey S E
et al (2004) Activators of the rat pregnane X receptor differentially
modulate hepatic and intestinal gene expression Molecular Pharma-
cology 65 1159ndash1171
Hartwell L H Hopfield J J Leibler S amp Murray A W (1999) From
molecular to modular cell biology Nature 402 C47ndashC52
Hasmall S Orphanides G James N Pennie W Hedley K Soames
A et al (2002) Downregulation of lactoferrin by PPARalpha ligands
Role in perturbation of hepatocyte proliferation and apoptosis
Toxicology Sciences 68 304ndash313
Hayase T Yamamoto Y Yamamoto K Muso E amp Shiota K (2004)
Microarray profile analysis of toxic cocaine-induced alterations in the
expression of mouse brain gene sequences A possible rsquoprotectiversquo
effect of buprenorphine Journal of Applied Toxicology 24 15ndash20
Hayes K R Vollrath A L Zastrow G M McMillan B J Craven M
Jovanovich S et al (2005) EDGE A centralized resource for the
comparison analysis and distribution of toxicogenomic information
Molecular Pharmacology 67 1360ndash1368
Heijne W H Slitt A L van Bladeren P J Groten J P Klaassen C D
Stierum R H et al (2004) Bromobenzene-induced hepatotoxicity at
the transcriptome level Toxicology Sciences 79 411ndash422
Heijne W H Stierum R H Slijper M van Bladeren P J amp van
Ommen B (2003) Toxicogenomics of bromobenzene hepatotoxicity
A combined transcriptomics and proteomics approach Biochemical
Pharmacology 65 857ndash875
Heinloth A N Irwin R D Boorman G A Nettesheim P Fannin
R D Sieber S O et al (2004) Gene expression profiling of rat
livers reveals indicators of potential adverse effects Toxicology
Sciences 80 193ndash202
Hodges L C Cook J D Lobenhofer E K Li L Bennett L Bushel
P R et al (2003) Tamoxifen functions as a molecular agonist inducing
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 63
cell cycle-associated genes in breast cancer cells Molecular Cancer
Research 1 300ndash311
Holleman A Cheok M H den Boer M L Yang W Veerman KM
A J Kazemier et al (2004) Gene-expression patterns in drug-resistant
acute lymphoblastic leukemia cells and response to treatment New
England Journal of Medicine 351 533ndash542
Hong Y Muller U R amp Lai F (2003) Discriminating two classes of
toxicants through expression analysis of HepG2 cells with DNA arrays
Toxicology in Vitro 17 85ndash92
Hood E (2003) Proteomics Characterizing the cogs in the machinery of
life Environmental Health Perspectives 111 A816ndashA825
Hood L (2003) Systems biology Integrating technology biology and
computation Mechanism of Ageing and Development 124 9ndash16
Hood L amp Galas D (2003) The digital code of DNA Nature 421
444ndash448
Hood L amp Perlmutter R M (2004) The impact of systems approaches on
biological problems in drug discovery Nature Biotechnology 22
1215ndash1217
Hu T Gibson D P Carr G J Torontali S M Tiesman J P Chaney
J G et al (2004) Identification of a gene expression profile that
discriminates indirect-acting genotoxins from direct-acting genotoxins
Mutation Research 549 5ndash27
Huang Q Dunn II R T Jayadev S DiSorbo O Pack F D Farr S B
et al (2001) Assessment of cisplatin-induced nephrotoxicity by
microarray technology Toxicology Sciences 63 196ndash207
Huang Q Jin X Gaillard E T Knight B L Pack F D Stoltz J H et
al (2004) Gene expression profiling reveals multiple toxicity endpoints
induced by hepatotoxicants Mutation Research 549 147ndash167
Huisman M T Smit J W Crommentuyn K M Zelcer N Wiltshire
H R Beijnen J H et al (2002) Multidrug resistance protein 2
(MRP2) transports HIV protease inhibitors and transport can be
enhanced by other drugs Aids 16 2295ndash2301
Hunt C M Westerkam W R amp Stave G M (1992) Effect of age and
gender on the activity of human hepatic CYP3A Biochemical
Pharmacology 44 275ndash283
Ideker T Ozier O Schwikowski B amp Siegal A F (2002) Discovering
regulatory and signalling circuits in molecular interaction networks
Bioinformatics 18 S233ndashS240
Ideker T Thorsson V Ranish J A Christmas R Buhler J Eng J K
et al (2001) Integrated genomic and proteomic analyses of a system-
atically perturbed network Science 292 929ndash934
Iida M Anna C H Hartis J Bruno M Wetmore B Dubin J R et al
(2003) Changes in global gene and protein expression during early
mouse liver carcinogenesis induced by non-genotoxic model carcino-
gens oxazepam and Wyeth-14643 Carcinogenesis 24 757ndash770
Ingelman-Sundberg M (2004) Pharmacogenetics of cytochrome P450 and
its applications in drug therapy The past present and future Trends in
Pharmacological Sciences 25 193ndash200
Jeong H Mason S P Barabasi A L amp Oltvai Z N (2001) Lethality
and centrality in protein networks Nature 411 41ndash42
Jeong H Tombor B Albert R Oltvai Z N amp Barabasi A L
(2000) The large-scale organization of metabolic networks Nature
407 651ndash654
Jung D Hagenbuch B Gresh L Pontoglio M Meier P J amp Kullak-
Ublick G A (2001) Characterization of the human OATP-C
(SLC21A6) gene promoter and regulation of liver-specific OATP genes
by hepatocyte nuclear factor 1 alpha Journal of Biological Chemistry
276 37206ndash37214
Jung D amp Kullak-Ublick G A (2003) Hepatocyte nuclear factor 1
alpha A key mediator of the effect of bile acids on gene expression
Hepatology 37 622ndash631
Jung J -W Park J -S Hwang J -W Kang K -S YS L Song B
-S et al (2004) Gene expression analysis of peroxisome prolifer-
ators- and phenytoin-induced hepatotoxicity using cDNA microarray
Journal of Veterinary Medical Science 66 1329ndash1333
Kato N Shibutani M Takagi H Uneyama C Lee K -Y Takiami S
et al (2004) Gene expression profile in the livers of rats orally
administered ethinylestradiol for 28 days using a microarray technique
Toxicology 200 179ndash192
Kier L D Neft R Tang L Suizu R Cook T Onsurez K et al
(2004) Applications of microarrays with toxicologically relevant genes
(tox genes) for the evaluation of chemical toxicants in Sprague Dawley
rats in vivo and human hepatocytes in vitro Mutation Research 549
101ndash113
Kijima K Toyosawa K Yasuba M Matsuoka N Adachi T
Komiyama M et al (2004) Gene expression analysis of the rat testis
after treatment with di(2-ethylhexyl) phthalate using cDNA microarray
and real-time RT-PCR Toxicology and Applied Pharmacology 200
103ndash110
Kim R B (2003) Organic anion-transporting polypeptide (OATP) trans-
porter family and drug disposition European Journal of Clinical
Investigation 33 1ndash5
Kinirons M T amp OrsquoMahony M S (2004) Drug metabolism and ageing
British Journal of Clinical Pharmacology 57 540ndash544
Kitano H (2002a) Computational systems biology Nature 420 206ndash210
Kitano H (2002b) Systems biology A brief overview Science 295
1662ndash1664
Kliewer S A Moore J T Wade L Staudinger J L Watson M
A Jones S A et al (1998) An orphan nuclear receptor activated
by pregnanes defines a novel steroid signalling pathway Cell 92
73ndash82
Korolev D Balakin K V Nikolsky Y Kirillov E Ivanenkov Y A
Savchuk N P et al (2003) Modeling of human cytochrome p450-
mediated drug metabolism using unsupervised machine learning
approach Journal of Medicinal Chemistry 46 3631ndash3643
Kramer J A Pettit S D Amin R P Bertram T A Car B
Cunningham M et al (2004) Overview on the application of
transcription profiling using selected nephrotoxicants for toxicology
assessment Environmental Health Perspectives 112 460ndash464
Kultima K Nystrom A -M Scholz B Gustafson A -L Dencker L
amp Stigson M (2004) Valproic acid teratogenicity A toxicogenomics
approach Environmental Health Perspectives 112 1225ndash1235
Kutuzova G D amp Deluca H F (2004) Gene expression profiles in rat
intestine identify pathways for 125-dihydroxyvitamin D(3) stimulated
calcium absorption and clarify its immunomodulatory properties
Archives of Biochemistry and Biophysics 432 152ndash166
Kwak M K Wakabayashi N Itoh K Motohashi H Yamamoto M amp
Kensler T W (2003) Modulation of gene expression by cancer
chemopreventive dithiolethiones through the Keap1-Nrf2 pathway
Identification of novel gene clusters for cell survival Journal of
Biological Chemistry 278 8135ndash8145
Lahjouji K Mitchell G A amp Qureshi I A (2001) Carnitine transport
by organic cation transporters and systemic carnitine deficiency
Molecular Genetics and Metabolism 73 287ndash297
Landowski C P Sun D Foster D R Menon S S Barnett J L
Welage L S et al (2003) Gene expression in the human intestine and
correlation with oral valacyclovir pharmacokinetic parameters Journal
of Pharmacology and Experimental Therapeutics 306 778ndash786
Lee G -J Lee W -S Jeon K -S Um C -H Kim S -J Lee C -H
et al (2004) cDNA microarray gene expression analysis and
toxicological phenotype for anticancer drug Journal of Veterinary
Medical Science 66 1339ndash1345
Lee M Kwon J Kim S N Kim J E Koh W S Kim E J et al
(2003) cDNA microarray gene expression profiling of hydroxyurea
paclitaxel and p-anisidine genotoxic compounds with differing
tumorigenicity results Environmental and Molecular Mutagenesis
42 91ndash97
Li S Armstrong C M Bertin N Ge H Milstein S Boxem M et al
(2004) A map of the interactome network of the metazoan C elegans
Science 303 540ndash543
Liguori M J Anderson L M Bukofzer S McKim J Pregenzer J F
Retief J et al (2005) Microarray analysis in human hepatocytes
suggests a mechanism for hepatotoxicity induced by trovafloxacin
Hepatology 41 177ndash186
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6664
Lobenhofer E K Bennett L Cable P L Li L Bushel P R amp Afshari
C A (2002) Regulation of DNA replication fork genes by 17beta-
estradiol Molecular Endocrinology 16 1215ndash1229
Locker J Tian J Carver R Concas D Cossu C Ledda-Columbano
G M et al (2003) A common set of immediate-early response genes
in liver regeneration and hyperplasia Hepatology 38 314ndash325
Mattes W B Pettit S D Sansone S A Bushel P R amp Waters M D
(2004) Database development in toxicogenomics Issues and efforts
Environmental Health Perspectives 112 495ndash505
McMillian M Nie A Y Parker J B Leone A Bryant S Kemmerer
M et al (2004) A gene expression signature for oxidant stressreactive
metabolites in rat liver Biochemical Pharmacology 68 2249ndash2261
Meier P J amp Stieger B (2000) Molecular mechanisms in bile formation
News in Physiological Sciences 15 89ndash93
Meneses-Lorente G de Longueville F Dos Santos-Mendes S Bonnert
T P Jack A Evrard S et al (2003) An evaluation of a low-density
DNA microarray using cytochrome P450 inducers Chemical Research
in Toxicology 16 1070ndash1077
Meneses-Lorente G Guest P C Lawrence J Muniappa N Knowles
M R Skynner H A et al (2004) A proteomic investigation of drug-
induced steatosis in rat liver Chemical Research in Toxicology 17
605ndash612
Merrill C L Ni H Yoon L W Tirmenstein M A Narayanan P
Benavides G R et al (2002) Etomoxir-induced oxidative stress in
HepG2 cells detected by differential gene expression is confirmed
biochemically Toxicology Sciences 68 93ndash101
Milo R Shen-Orr S Itzkovitz S Kashtan N Chklovskii D amp Alon
U (2002) Network motifs Simple building blocks of complex
networks Science 298 824ndash827
Morgan K T Ni H Brown H R Yoon L Qualls C W Crosby L
M et al (2002) Application of cDNA microarray technology to in
vitro toxicology and the selection of genes for a real-time RT-PCR-
based screen for oxidative stress in Hep-G2 cells Toxicologic
Pathology 30 435ndash451
Mwinyi J Johne A Bauer S Roots I amp Gerloff T (2004) Evidence
for inverse effects of OATP-C (SLC21A6) 5 and 1b haplotypes on
pravastatin kinetics Clinical Pharmacology and Therapeutics 75
415ndash421
Nakata K Yukawa M Komiyama N Nakano T amp Kaminuma T
(2002) A nuclear receptor database that maps pathways to diseases
Genome Infomatics 13 515ndash516
Nicholson J K Holmes E Lindon J C amp Wilson I D (2004) The
challenges of modeling mammalian biocomplexity Nature Biotechnol-
ogy 22 1268ndash1274
Nicholson J K amp Wilson I D (2003) Understanding rsquoglobal systems
biology Metabonomics and the continuum of metabolism Nature
Reviews on Drug Discovery 2 668ndash676
Nikitin A Egorov S Daraselia N amp Mazo I (2003) Pathway studiomdash
the analysis and navigation of molecular networks Bioinformatics 19
2155ndash2157
Nikolsky Y Ekins S Nikolskaya T amp Bugrim A (2005) A novel
method for generation of signature networks as biomarkers from
complex high throughput data Toxicology Letters 158 20ndash29
Nikolsky Y Nikolskaya T amp Bugrim A (2005) Biological networks
and analysis of experimental data in drug discovery Drug Discovery
Today 10 653ndash662
Nishizato Y Ieiri I Suzuki H Kimura M Kawabata K Hirota T
et al (2003) Polymorphisms of OATP-C (SLC21A6) and OAT3
(SLC22A8) genes Consequences for pravastatin pharmacokinetics
Clinical Pharmacology and Therapeutics 73 554ndash565
Nozawa T Nakajima M Tamai I Noda K Nezu J Sai Y et al
(2002) Genetic polymorphisms of human organic anion transporters
OATP-C (SLC21A6) and OATP-B (SLC21A9) Allele frequencies in
the Japanese population and functional analysis Journal of Pharmacol-
ogy and Experimental Therapeutics 302 804ndash813
Nozawa T Sugiura S Nakajima M Goto A Yokoi T Nezu J et al
(2004) Involvement of organic anion transporting polypeptides in the
transport of troglitazone sulfate Implications for understanding trogli-
tazone hepatotoxicity Drug Metabolism and Disposition 32 291ndash294
Oliver D E Rubin D L Stuart J M Hewett M Klein T E amp
Altman R B (2002) Ontology development for a pharmacogenetics
knowledge base Package Symposion 88ndash99
Oram J F amp Lawn R M (2001) ABCA1 The gatekeeper for eliminating
excess tissue cholesterol Journal of Lipid Research 42 1173ndash1179
Oswald M Kullak-Ublick G A Paumgartner G amp Beuers U (2001)
Expression of hepatic transporters OATP-C and MRP2 in primary
sclerosing cholangitis Liver 21 247ndash253
Parsons A B Brost R I Ding H Li Z Zhang C Sheikh B et al
(2004) Integration of chemical-genetic and genetic interaction data
links bioactive compounds to cellular target pathways Nature Bio-
technology 22 62ndash69
Patki K C von Moltke L L Harmatz J S Hesse L M Court M H
amp Greenblatt D J (2004) Effect of age on in vitro triazolam
biotransformation in male human liver microsomes Journal of
Pharmacology and Experimental Therapeutics 308 874ndash879
Pereira-Leal J B Enright A J amp Ouzounis C A (2004) Detection of
functional modules from protein interaction networks Proteins 54
49ndash57
Plant N (2004) Interaction networks Coordinating responses to xeno-
biotic exposure Toxicology 202 21ndash32
Raghavendra Prasad H S Qi Z Srinivasan K N amp Gopalakrish-
nakone P (2004) Potential effects of tetrodotoxin exposure to
human glial cells postulated using microarray approach Toxicon 44
597ndash608
Reilly T P Bourdi M Brady J N Pise-Masison C A Radonovich M
F George J W et al (2001) Expression profiling of acetaminophen
liver toxicity in mice using microarray technology Biochemical and
Biophysical Research Communications 282 321ndash328
Rives A W amp Galitski T (2003) Modular organization of cellular
networks Proceedings of the National Academy of Sciences of the
United States of America 100 1128ndash1133
Rogers P D Pearson M M Cleary J D Sullivan D C amp Chapman
S W (2002) Differential expression of genes encoding immunomodu-
latory proteins in response to amphotericin B in human mononuclear
cells identified by cDNA microarray analysis Journal of Antimicrobial
Chemotherapy 50 811ndash817
Rosen M B Wilson V S Schmid J E amp Gray L E (2005) Gene
expression analysis in the ventral prostate of rats exposed to vinclozolin
or procymidone Reproductive Toxicology 19 367ndash379
Rost D Herrmann T Sauer P Schmidts H L Stieger B Meier P J
et al (2003) Regulation of rat organic anion transporters in bile salt-
induced cholestatic hepatitis Effect of ursodeoxycholate Hepatology
38 187ndash195
Rothman R B Ayestas M A Dersch C M amp Baumann M H (1999)
Aminorex fenfluramine and chlorphentermine are serotonin transporter
substrates Implications for primary pulmonary hypertension Circula-
tion 100 869ndash875
Ruepp S U Tonge R P Shaw J Wallis N amp Pognan F (2002)
Genomics and proteomics analysis of acetaminophen toxicity in mouse
liver Toxicology Sciences 65 135ndash150
Sakaeda T Nakamura T amp Okumura K (2002) MDR1 genotype-
related pharmacokinetics and pharmacodynamics Biological amp Phar-
maceutical Bulletin 25 1391ndash1400
Sakata T Anzai N Shin H J Noshiro R Hirata T Yokoyama H
et al (2004) Novel single nucleotide polymorphisms of organic cation
transporter 1 (SLC22A1) affecting transport functions Biochemical
and Biophysical Research Communications 313 789ndash793
Satlin L M Amin V amp Wolkoff A W (1997) Organic anion
transporting polypeptide mediates organic anionHCO3-exchange
Journal of Biological Chemistry 272 26340ndash26345
Satomi Y Tsuchiya W Mihara K Ota M Kasahara Y amp Akahori F
(2004) Gene expression analysis of the lung following paraquat
administration in rats using DNA microarray Journal of Toxicological
Sciences 29 91ndash100
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 65
Schrenk D Baus P R Ermel N Klein C Vorderstemann B amp
Kauffmann H M (2001) Up-regulation of transporters of the MRP
family by drugs and toxins Toxicology Letters 120 51ndash57
Segal E Shapira M Regev A Persquoer D Botstein D Koller D et al
(2003) Module networks Identifying regulatory modules and their
condition-specific regulators from gene expression data Nature
Genetics 34 166ndash176
Segal E Wang H amp Koller D (2003) Discovering molecular pathways
from protein interaction and gene expression Bioinformatics 19
i264ndash i272
Seidel S D Kan H L Stott W T Schisler M R amp Gollapudi B B
(2003) Identification of transcriptome profiles for the DNA-damaging
agents bleomycin and hydrogen peroxide in L5178Y mouse lymphoma
cells Environmental and Molecular Mutagenesis 42 19ndash25
Shan L Yu Y Schut H A J amp Snyderwine E G (2004) Susceptibility
of rats to mammary gland carcinogenesis by the food-derived
carcinogen 2-Amino-1-Methyl-6-Phenylimidazo[45-b]Pyridine (PhIP)
varies with age and is associated with the induction of differential gene
expression American Journal of Pathology 165 191ndash202
Sharom J R Bellows D S amp Tyers M (2004) From large networks to
small molecules Current Opinion in Chemical Biology 8 81ndash90
Shitara Y Hirano M Sato H amp Sugiyama Y (2004) Gemfibrozil and
its glucuronide inhibit the OATP2(OATP1B1 SLC21A6)-mediated
hepatic uptake and CYP2C8-mediated metabolism of cerivastatinmdash
Analysis of the mechanism of the clinically relevant drugndashdrug
interaction between cerivastatin and gemfibrozil Journal of Pharma-
cology and Experimental Therapeutics
Shitara Y Itoh T Sato H Li A P amp Sugiyama Y (2003) Inhibition of
transporter-mediated hepatic uptake as a mechanism for drugndashdrug
interaction between cerivastatin and cyclosporin A Journal of
Pharmacology and Experimental Therapeutics 304 610ndash616
Somogyi R Fuhrman S amp Wen X (2001) Genetic network inference in
computational models and applications to large-scale gene expression
data In M Bower amp H Bolouri (Eds) Computational modeling of
genetic and biochemical networks (pp 119ndash157) Cambridge MArsquoThe
MIT Press
Spirin V amp Mirny L A (2003) Protein complexes and functional
modules in molecular networks Proceedings of the National Academy
of Sciences of the United States of America 100 12123ndash12128
Staudinger J Liu Y Madan A Habeebu S amp Klaassen C D
(2001) Coordinate regulation of xenobiotic and bile acid homeo-
stasis by pregnane X receptor Drug Metabolism and Disposition
29 1467ndash1472
Sun L Z Ji Z L Chen X Wang J F amp Chen Y Z (2002) ADME-
AP A database of ADME associated proteins Bioinformatics 18
1699ndash1700
Suter L Babiss L E amp Wheeldon E B (2004) Toxicogenomics in
predictive toxicology in drug development Chemistry and Biology 11
161ndash171
Synold T W Dussault I amp Forman B M (2001) The orphan nuclear
receptor SXR coordinately regulates drug metabolism and efflux
Nature Medicine 7 584ndash590
Tabuchi Y amp Kondo T (2003) cDNA microarray analysis reveals chop-
10 plays a key role in Sertoli cell injury induced by bisphenol A
Biochemical and Biophysical Research Communications 305 54ndash61
Tamai I Nezu J Uchino H Sai Y Oku A Shimane M et al (2000)
Molecular identification and characterization of novel members of the
human organic anion transporter (OATP) family Biochemical and
Biophysical Research Communications 273 251ndash260
Taylor S W Fahy E Zhang B Glenn G M Warnock D E Wiley S
et al (2003) Characterization of the human heart mitochondrial
proteome Nature Biotechnology 21 281ndash286
Terasaka S Aita Y Inoue A Hayashi S Nishigaki M Aoyagi K
et al (2004) Using a customized DNA microarray for expression
profiling of the estrogen-responsive genes to evaluate estrogen activity
among natural estrogens and industrial chemicals Environmental
Health Perspectives 112 773ndash781
Thomas D M Francescutti-Verbeem D M Liu X amp Kuhn D M
(2004) Identification of differentially regulated transcripts in mouse
striatum following methamphetamine treatmentmdashan oligonucleotide
microarray approach Journal of Neurochemistry 88 380ndash393
Thomas R S Rank D R Penn S G Zastrow G M Hayes K R
Pande K et al (2001) Identification of toxicologically predictive
gene sets using cDNA microarrays Molecular Pharmacology 60
1189ndash1194
Thompson K L Afshari C A Amin R P Bertram T A Car B
Cunningham M et al (2004) Identification of platform-independent
gene expression markers of cisplatin nephrotoxicity Environmental
Health Perspectives 112 488ndash494
Tirona R G Leake B F Merino G amp Kim R B (2001)
Polymorphisms in OATP-C Identification of multiple allelic
variants associated with altered transport activity among European-
and African-Americans Journal of Biological Chemistry 276
35669ndash35675
Tirona R G Leake B F Wolkoff A W amp Kim R B (2003)
Human organic anion transporting polypeptide-C (SLC21A6) is a
major determinant of the rifampin-mediated pregnane X receptor
activation Journal of Pharmacology and Experimental Therapeutics
304 223ndash228
Tong W Cao X Harris S Sun H Fang H Fuscoe J et al (2003)
ArrayTrackmdashsupporting toxicogenomic research at the US Food and
Drug Administration National Center for Toxicological Research
Environmental Health Perspectives 111 1819ndash1826
Tornow S amp Mewes H W (2003) Functional modules by relating
protein interaction networks and gene expression Nucleic Acids
Research 31 6283ndash6289
Trocho C Escola-Gil J C Ribas V Benitez S Martin-Campos J M
Rotllan N et al (2004) Phenytoin treatment reduces atherosclerosis in
mice through mechanisms independent of plasma HDL-cholesterol
concentration Atherosclerosis 174 275ndash285
Ueda A Hamadeh H K Webb H K Yamamoto Y Sueyoshi T
Afshari C A et al (2002) Diverse roles of the nuclear orphan
receptor CAR in regulating hepatic genes in response to phenobarbital
Molecular Pharmacology 61 1ndash6
Ulrich R G (2003) The toxicogenomics of nuclear receptor agonists
Current Opinion in Chemical Biology 7 505ndash510
Ulrich R G Rockett J C Gibson G G amp Pettit S D (2004)
Overview of an interlaboratory collaboration on evaluating the effects
of model hepatotoxicants on hepatic gene expression Environmental
Health Perspectives 112 423ndash427
Unami A Shinohara Y Ichikawa T amp Baba Y (2003) Biochemical
and microarray analyses of bupivacaine-induced apoptosis Journal of
Toxicological Sciences 28 77ndash94
Vasquez A Flammini A Maritan A amp Vespignani A (2003) Global
protein function prediction from proteinndashprotein interaction networks
Nature Biotechnology 21 697ndash700
Vezina C M Walker N J amp Olson J R (2004) Subchronic Exposure to
TCDD PeCDF PCB126 and PCB153 Effect on hepatic gene
expression Environmental Health Perspectives 112 1636ndash1644
Vo T D Greenberg H J amp Palsson B O (2004) Reconstruction and
functional characterization of the human mitochondrial metabolic
network based on proteomic and biochemical data Journal of Bio-
logical Chemistry 279 39532ndash39540
Wang H amp LeCluyse E L (2003) Role of orphan nuclear receptors in
the regulation of drug-metabolising enzymes Clinical Pharmacoki-
netics 42 1331ndash1357
Wang N Silver D L Thiele C amp Tall A R (2001) ATP-
binding cassette transporter A1 (ABCA1) functions as a choles-
terol efflux regulatory protein Journal of Biological Chemistry 276
23742ndash23747
Waring J F Cavet G Jolly R A McDowell J Dai H Ciurlionis R
et al (2003) Development of a DNA Microarray for toxicology based
on hepatoxin-regulated sequences Environmental Health Perspectives
111 863ndash870
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6666
Waring J F Gum R Morfitt D Jolly R A Ciurlionis R Heindel M
et al (2002) Identifying toxic mechanisms using DNA microarrays
Evidence that an experimental inhibitor of cell adhesion molecule
expression signals through the aryl hydrocarbon nuclear receptor
Toxicology 181ndash182 537ndash550
Waring J F Jolly R A Ciurlionis R Lum P Y Praestgaard J T
Morfitt D C et al (2001) Clustering of hepatotoxins based on
mechanism of toxicity using gene expression profiles Toxicology and
Applied Pharmacology 175 28ndash42
Waters M Boorman G Bushel P Cunningham M Irwin R Merrick
A et al (2003) Systems toxicology and the Chemical Effects in
Biological Systems (CEBS) knowledge base EHP Toxicogenomics
111 15ndash28
Wei M Arnold L Cano M amp Cohen S M (2005) Effects of co-
administration of antioxidants and arsenicals on the rat urinary bladder
epithelium Toxicology Sciences 83 237ndash245
Weinshilboum R (2003) Inheritance and drug response New England
Journal of Medicine 348 529ndash537
Weinshilboum R amp Wang L (2004) Pharmacogenetics Inherited
variation in amino acid sequence and altered protein quantity Clinical
Pharmacology and Therapeutics 75 253ndash258
Werner E (2003) In silico multicellular systems biology and minimal
genomes Drug Discovery Today 8 1121ndash1127
Wong J S amp Gill S S (2002) Gene expression changes induced in
mouse liver by di(2-ethylhexyl) phthalate Toxicology and Applied
Pharmacology 185 180ndash196
Xie T Tong L McCann U D Yuan J Becker K G Mechan A O
et al (2004) Identification and characterization of metallothionein-1
and -2 gene expression in the context of ()34-methylenedioxymetham-
phetamine-induced toxicity to brain dopaminergic neurons Journal of
Neuroscience 24 7043ndash7050
Yadetie F Laegreid A Bakke I Kusnierczyk W Komorowski J
Waldum H L et al (2003) Liver gene expression in rats in response
to the peroxisome proliferator-activated receptor-alpha agonist ciprofi-
brate Physiological Genomics 15 9ndash19
Yamashita S Nomoto T Abe M Tatematsu M Sugimura T amp
Ushijima T (2004) Persistence of gene expression changes in stomach
mucosae induced by short-term N-methyl-NV-nitro-N-nitrosoguanidinetreatment and their presence in stomach cancers Mutation Research
549 185ndash193
Yan Q amp Sadee W (2000) Human membrane transporter database A
web-accessible relational database for drug transport studies and
pharmacogenomics AAPS Pharmsci 2 E20
Yeger-Lotem E amp Margalit H (2003) Detection of regulatory circuits by
integrating the cellular networks of proteinndashprotein interactions and
transcription regulation Nucleic Acids Research 31 6053ndash6061
Yoon B I Li G -X Kitada K Kawasaki Y Igarashi K amp Kodama
Y et al (2003) Mechanisms of benzene-induced hematotoxicity and
leukemogenicity cDNA microarray analyses using mouse bone marrow
tissue Environmental Health Perspectives 111 1411ndash1420
Young M B DiSilvestro M R Sendera T J Freund J Kriete A amp
Magnuson S R (2003) Analysis of gene expression in carbon
tetrachloride-treated rat livers using a novel bioarray technology
Pharmacogenomics Journal 3 41ndash52
Yu H Zhu X Greenbaum D Karro J amp Gerstein M (2004) TopNet
A tool for comparing biological sub-networks correlating protein
properties with topological statistics Nucleic Acids Research 32
328ndash337
Zhang E Y Fu D J Pak Y A Stewart T Mukhopadhyay N
Wrighton S A et al (2004) Genetic polymorphisms in human proton-
dependent dipeptide transporter PEPT1 Implications for the functional
role of Pro586 Journal of Pharmacology and Experimental Therapeu-
tics 310 437ndash445
Zhang E Y Knipp G T Ekins S amp Swaan P W (2002)
Structural biology and function of solute transporters Implications
for identifying and designing substrates Drug Metabolism Reviews
34 709ndash750
Zhang E Y Phelps M A Cheng C Ekins S amp Swaan P W (2002)
Modeling of active transport systems Advanced Drug Delivery
Reviews 54 329ndash354
- Systems-ADMETox Resources and network approaches
-
- Introduction
- Data available
- Network analysis and databases
-
- Network applications
-
- The role of transporters
-
- Clinical relevance of transporters
- Transporter network examples ABCA1
- Transporter network examples OATP
- Transporter microarray data
-
- Applications to enzymes
- Future network applications
-
- Discussion
- Acknowledgements
- References
-
Table 2
Literature toxicogenomics data derived from mouse in vivo studies
Compounds Mouse strain Microarray type Compound dose Microarray data
availability
Reference
Benzene Male and female P53
KO mice and
C57BL6
Affymetrix and Incyte
GEM system
300 ppm 6 h per day 5
days a week for 2 weeks
Gene name accession
number and fold change
data in a manuscript
table
(Yoon et al 2003)
Benzene Male 129SvJ Affymetrix MG-U74Av2 100 ppm 6 h per day 5
days a week for 2 weeks
Gene name accession
number and fold change
data in a manuscript
table
(Faiola Fuller
Wong amp Recio
2004)
Phenobarbital CAR and wild type NIEHS Mouse tox chip
8736 genes
100 mgkg for 12 h Accession number gene
name and fold change
for wild type and knock
out mice in a manuscript
table
(Ueda et al 2002)
Aroclor BNF
ciprofloxacin cobalt
chloride TCDD IL-6
LPS PCB-153
phenobarbital
phenylhyrzn TNFa
WY-16463
C57BL6J Custom array with 1200
cDNAs
Aroclor (200 mgkg)
BNF (5 mgkg)
ciprofloxacin (250 mg
kg) cobalt chloride (60
mgkg) TCDD (10 ug
kg) IL-6 (25 ugkg)
LPS (1 mgkg)
PCB-153 (80 mgkg)
phenobarbital (100 mg
kgday) 3 days
phenylhyrzn (100 mg
kg) TNFa (50 ugkg)
WY-16463 (0125wv)
Gene names and fold
change as a heat map
Data is also available in
the EDGE database
(Thomas et al
2001)
TCPOBOP CD-1 female Custom 9000 cDNA
mouse array
1ndash3 h treatment 3 mg
kg body wt
Accession numbers and
fold change data in a
manuscript table
(Locker et al
2003)
3H-12-dithiole-3-thione
(D3T)
Male wild-type and
nrf2-disrupted
Affymetrix murine
genome U74Av2
GeneChip
05 mmolkg Accession number gene
name fold in a
manuscript table
(Kwak et al
2003)
MDMA Male albino Swissndash
Webster (neurons)
15 K mouse cDNA clone
set
47 mgkg Gene name and fold
change in a manuscript
table
(Xie et al 2004)
Phenytoin Female C57BL6 and
LDLRMurine genome-U74Av2 300 mgl Gene name accession
number and fold change
in a manuscript table
(Trocho et al
2004)
Genistein (1000 Agmouseday) or
diethylstilbestrol
(DES) (50 Agmouse
day)
ICR (testes) Custom cDNA
microarray containing
1754 cDNA probes
Genistein (1000 Agmouseday)
diethylstilbestrol (50 Agmouseday)
Accession number gene
name and fold change in
a manuscript tablemdashnote
very few genes
(Adachi et al
2004)
Cocaine and
buprenorphine
Male ICR Mouse DiscoveryArrayi
type I array containing
2688brain-derivedprobes
(Display Systems Biotech
Inc Copenhagen
Denmark)
40 mg kgmdash1 cocaine
once a day for 4 days 40
mg kgmdash1 cocaine plus
025 mg kgmdash1 BUP for
4 days
Data apparently not
available
(Hayase
Yamamoto
Yamamoto Muso
amp Shiota 2004)
Methamphetamine Male C57BLJ6
(striatum)
Affymetrix mouse
genechip mg-U74Av2
oligonucleotides arrays
12 488 genes
40 mgkg Gene accession number
gene ID and name and
signal log ratio in a
manuscript table
(Thomas
Francescutti-
Verbeem Liu amp
Kuhn 2004)
Di(2-ethylhexyl)
phthalate
Male C57BL6 Murine genome U74Av2
Arrays (MGU74Av2)
10 dietary DEHP for
13 weeks
Genbank accession
numbers gene name log
ratio data present as a bar
chart in the publication
data extraction would
take some effort
(Wong amp Gill
2002)
Diethylhexylphthalate Male PPARa null and
wild type
Custom made containing
600 tox genes
1150 mgkgday Gene names available on
a bar chartmdashquantitative
data not easily accessible
(Hasmall et al
2002)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6644
Table 2 (continued)
Compounds Mouse strain Microarray type Compound dose Microarray data
availability
Reference
Acetaminophen C57B16 3 129Ola
hybrid
Test-2 Chips (Affymetrix)
then individual
oligonucleotide
microarrays (Mul1K sub
A and sub B Affymetrix)
that can detect the
expression of 11000
known genes and
expressed sequence tags
(ESTs)
300 mgkg Genbank or SwissProt
ID and fold change data
in a manuscript table
(Reilly et al
2001)
Cadmium chloride
benzo(a)pyrene (BaP)
and trichloroethylene
(TCE)
Male Swiss Webster Custom Chips
containing 148 unique
genes
Various Data in table form few
genes affected for BAP
and TCE
(Bartosiewicz
Penn amp Buckpitt
2001)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 45
been only a very small number of studies that have
combined both transcriptomic and proteomic methods with
a single animal strain after treatment with a drug Hopefully
we will see this change in the future but this will in turn
present considerable challenges as huge amounts of
proteomic data are combined with the equally large
transcript data files
3 Network analysis and databases
From some of the early reviews of systems biology there
has been discussion of its application to drug discovery
(Kitano 2002ab) as well as the utility for ADMETox
(Ekins et al 2002a 2000b) More recently several other
journals have dedicated whole issues to the field of systems
biology However one could consider this quite a broad field
from network or pathway analysis to quantitative simulation
of organelles (Vo Greenberg amp Palsson 2004) whole cells
and organs It is apparent that we are now understanding
organisms from the perspective of computationally gener-
ated networks of protein and ligand interactions (Barabasi amp
Oltvai 2004) Network and pathway tools enable the
analysis of HT data in the context of all known interactions
when using a database as the source Individual reviews
have in some cases indicated that networks will be valuable
for understanding adverse events (Hood amp Perlmutter
2004) drug target identification or validation (Butcher
Berg amp Kunkel 2004) and complex metabolic interactions
(Nicholson et al 2004) A general schematic has been
generated in order to provide a description of the utilization
of such pathway databases and network building algorithms
from the initial parsing of high throughput data to network
comparisons and visualization (Fig 3) High throughput
data can be superimposed and visualized on the various
protein interaction databases available This is accomplished
by using either preset maps that capture current biological
knowledge or by building custom interaction networks
using many different algorithms which can be compared and
statistically evaluated as demonstrated in a very large
number of published examples (Dobrin Beg Karabasi amp
Oltvai 2004 Fiehn 2001 Han et al 2004 Hanisch et al
2002 Ideker et al 2002 Jeong Mason Barabasi amp Oltvai
2001 Jeong Tombor Albert Oltvai amp Barabasi 2000 Li
et al 2004 Milo et al 2002 Nikitin Egorov Daraselia amp
Mazo 2003 Pereira-Leal Enright amp Ouzounis 2004
Rives amp Galitski 2003 Segal et al 2003a Somogyi et al
2001 Spirin amp Mirny 2003 Tornow amp Mewes 2003
Vasquez Flammini Maritan amp Vespignani 2003 Yeger-
Lotem amp Margalit 2003 Yu Zhu Greenbaum Karro amp
Gerstein 2004)
31 Network applications
For example one group has used as an inference the
Bayesian network method for analysis of tissue toxicity from
microarray data as well as a mechanistic simulation for a
different pharmaceutically relevant molecule (Aksenov et
al 2005) Pathway tools and various resources have also
been applied to modeling the networks of nuclear hormone
receptors and their connections with other genes and small
molecules using a manually curated database MetaDrug
(Ekins Kirillov Rakmatulin amp Nikolskaya 2005d) or
MetaCore (Ekins Bugrim Nikolsky amp Nikolskaya 2005)
Transcriptional regulation of many transporters CYPs and
phase II enzymes are regulated by these receptors affecting
endogenous molecule transport metabolism cell growth
proliferation and oxidative stress (Ulrich 2003 Ulrich et al
2004) When the signaling networks and interacting ligands
for the transcriptional factors PPAR FXRRXRA ESR1
AHR HNF4A GCR-h MCR CAR-beta GCR-a LXR-a
CARRXR HNF4 FXR PXRRXR heterodimer PXR
AHRARNT heterodimer PPARaLXRa VDR PPAR-a
are visualized a very complex picture of interactions can be
created (Ekins et al 2005d) This suggests that when we
consider a molecule binding with only one nuclear receptor
we are observing only a fraction of the likely possible
feasible interactions based on the data gathered to date
Table 3
Literature toxicogenomics data derived from in vitro cell studies
Compounds Cell type Microarray type Compound dose Microarray data
availability
Reference
4-Hydroxytamoxifen
estrogen
MCF-7 breast cancer NIEHS ToxChip
microarray consisting of
1901 genes
1 uM hydroxytamoxifen
for a year 10 nM
17b-estradiol
Data available at NIEHS
website
(Hodges et al
2003)
Trovafloxacin Human hepatocytes Affymetrix U133A array 30ndash800 uM 142 genes available in
supplemental tablemdashnot
easily extracted
(Liguori et al
2005)
Estrogen MCF-7 breast cancer NIEHS ToxChip
microarray consisting of
1901 genes
10ndash10 M 17b-estradiol Data available at NIEHS
website
(Lobenhofer et al
2002)
Valproic acid NMRI mice embryo
and P19 mouse
embryocarcinoma
Custom chip including
15K mouse cDNA clone
set
600 mgkg body weight Gene symbol gene
name NIA EST log fold
change in a manuscript
table
(Kultima et al
2004)
Sulindac sulfide Human colorectal
carcinoma SW-480
and HCT-116
NIEHS human 12K chip 10 uM Genbank accession
number gene name and
fold change at various
time points in a
manuscript table data
also available on NIEHS
website
(Bottone
Martinez Collins
Afshari amp Eling
2003)
17Beta-estradiol estriol
estrone genistein
diethylstilbestrol
bisphenol A
nonylphenol
methoxychlor
MCF-7 U95A oligonucleotide
probe arrays (Affymetrix
10 nM (E2 estriol
estrone DES) 10 AM(genistein bisphenol A
nonylphenol and
methoxychlor)
Unigene name gene
name and fold change
for estrogen responsive
and nonresponsive in a
manuscript table
(Terasaka et al
2004)
Ouabain lauryl sulfate
dimethylsulfoxide
cycloheximide
tolbutamide sodium
fluoride diethyl
maleate buthionine
sulfoxamine
potassium bromate
sodium selenite
alloxan adriamycin
hydrogen peroxide
HepG2 Clontech Atlas Human
Stress Toxicology cDNA
arrays (234 genes)
Ouabain (43 uM) lauryl
sulfate (260 uM)
dimethylsulfoxide (128
M) cycloheximide (625
uM) tolbutamide (128
mM) sodium fluoride (3
mM) diethyl maleate
(125 mM) buthionine
sulfoxamine (30 mM)
potassium bromate (25
mM) sodium selenite (30
uM) alloxan (130 mM)
adriamycin (40 uM)
hydrogen peroxide
(4 mM)
Gene name ratio p -
value in downloadable
tables at journal website
(Morgan et a l
2002)
Aflatoxin B(1) (AFB(1))
2-acetylaminofluorene
(2AAF)
dimethylnitrosamine
(DMN)
acetaminophen (APAP)
HepG2 and primary
hepatocytes
Gene filter arrays
containing 31000 genes
10 uM aflatoxin B1
40 mM acetaminophen
100 uM
dimethylnitrosamine
10 uM
2-acetylaminofluorene
None (Harris et al
2004)
Mitomycin C (MMC)
and cisplatin (CIS)
and an alkylating
agent methyl
methanesulfonate
(MMS)
indirect-acting
genotoxins included
hydroxyurea (HU) a
ribonucleotide
reductase inhibitor
taxol (TXL) a
microtubule inhibitor
and etoposide
(ETOP)
L5178Y TK(+-)
mouse lymphoma
Affymetrix mouse
MG-U74A for MMC
and MG-U745Av2
(Affymetrix Inc Santa
Clara CA) for all the
other chemicals a total
of 9977 probe sets
(genes or ESTs) common
to these two array
models
Low mid and high doses Accession number gene
name gene ID statistical
significance at each time
point in a manuscript
table
(Hu et al 2004)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6646
Table 3 (continued)
Compounds Cell type Microarray type Compound dose Microarray data
availability
Reference
Hydroxyurea
(a carcinogen)
p-anisidine
(a noncarcinogen)
and paclitaxel
L5178Y Tk_-mouse
lymphoma
The Twin - Chip Mouse-
74K Digital Genomics
cDNA microarray
10 ngml paclitaxel 313
ugml hydroxyurea 32
ugml p-anisidine
Gene symbol and fold
change in a manuscript
table
(Lee et al 2003)
Acetaminophen
amiodarone clofibrate
erythromycin estolate
isoniazid alpha-
naphtylylisothiocyanate
beta-naphtoflavone
4-pentenoic acid
phenobarbital
tetracycline and
zileuton
Wistar Rat
hepatocytes
DualChip rat hepato
(Eppendorf Hamburg
Germany)
A single concentration
which varied for each
compound
Accession numbers and
gene namemdashfold
changes shown as
colored heat mapmdashnote
easily extracted from
publication
(de Longueville
et al 2003)
Bleomycin and hydrogen
peroxide
Mouse lymphoma
L5178Y TK(+ -)
Clontech Mouse 12K
cDNA microarray (1185
genes)
Bleomycin (25 and 20
ugml) hydrogen peroxide
(5 and 10 ugml)
Gene names and fold
change presented as bar
charts in publication
(Seidel Kan
Stott Schisler amp
Gollapudi 2003)
Bupivicaine
camptothecin
HL-60 Agilent human cDNA
microarray
1 mM Gene name GenBank
accession number
unigene and ratio in a
manuscript table
(Unami
Shinohara
Ichikawa amp Baba
2003)
Bisphenol A Mouse Sertoli TTE3 IntelliGene mouse
expression glass
microarrays (Version
10 Takara Shuzo)
which were spotted with
564 cDNA fragments of
mouse known genes and
approximately 301
expressed sequence tags
(ESTs)
0ndash400 uM Gene name GenBank
accession number and
fold change at time
points in a manuscript
table
(Tabuchi amp
Kondo 2003)
Mitomycin C or
doxorubicin
Hep G2 85 human gene custom
array
10 um mitomycin C
2 um doxorubicin or
2 ethanol
Bar charts with fold
changes in publication
very few genes
(Hong Muller amp
Lai 2003)
Amphotericin B Human peripheral
blood mononuclear
and THP-1
GF211 FKnown Genes_
Genefilter cDNA array
(ResGen) this array
consists of gt4000
individual elements
each representing a
known human gene
5 ugml Accession numbers and
fold expression in a
manuscript table
(Cleary Rogers amp
Chapman 2001
Rogers Pearson
Cleary Sullivan
amp Chapman
2002)
Benzo(a)pyrene diol
epoxide
TK6 human
lymphoblastoid
Human-350 microarray
a glass slide with 350
spotted human cDNA
probes (Phase-1
Molecular Toxicology
0 001 010 or
10 ugml)
Gene names and fold
change at doses in a
manuscript table
(Akerman et al
2004)
Etomoxir HepG2 Clontech AtlasiHuman Stress
Toxicology cDNA arrays
(234 genes)
1 mM etomoxir Gene names and fold
change in a manuscript
table
(Merrill et al
2002)
Tetrodotoxin Human glioma cell
line HTB-138
Using Affymetrix
GeneChip (HG-U133A
10 and 20 mM Affymetrix ID Genbank
ID gene name gene
symbol and fold change
in a manuscript table
(Raghavendra
Prasad Qi
Srinivasan amp
Gopalakrishnakone
2004)
Methotrexate
mercaptopurine
Human acute
lymphoblastic
leukemia
Affymetrix U133A chip Low and high dose and
combination
Data available as
supplemental data online
(Cheok et al 2003)
Prednisolone
vincristine
asparaginase
daunorubicin
Human acute
lymphoblastic
leukemia
Affymetrix U133A chip Various Data available as
supplemental data online
(Holleman et al
2004)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 47
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6648
A second study has indicated how a natural language
processing method CCNet was used to show the genes
regulated by the nuclear hormone receptor FXR (Apic
Ignjatovic Boyer amp Russell 2005) These automated
methods enable a more complete understanding of the
complexity of the transcriptional factors (Ekins Mirny amp
Schueltz 2002b Plant 2004 Ulrich 2003) but ultimately
rely on the quality of the content of the underlying database
of literature interactions This is a key consideration that is
often overlooked For example the gold standard database is
one that is manually curated to ensure the fidelity of the
direct interaction and is preferable to one generated
computationally by algorithms like natural language pro-
cessing (Nikolsky Nikolskaya amp Bugrim 2005) The
advantage of interaction networks over clustering has been
demonstrated in one study using MetaCore (Nikolsky
Ekins Nikolskaya amp Bugrim 2005) by reanalysis of a
published microarray study of G0-arrested MCF-7 breast
cancer cells treated with estrogen and 4-hydroxytamoxifen
(Hodges et al 2003) After producing integrated gene
networks for each treatment strikingly different patterns
were displayed although both contained early transcriptional
factors myc jun and fos Only the estrogen network featured
induced genes essential for all cell cycle phases (Nikolsky et
al 2005) Similarly microarray data for benzene toxicity
(Yoon et al 2003) has been re-analyzed by focusing on the
genes assessed on the p53 pathway (Ekins et al 2005e) We
envisage that a database of such networks for toxic
compounds will be used for comparing between different
molecules and used in the development of predictive
algorithms for Systems-ADMETox modeling in future
Another approach to using such pathway approaches is to
visualize the results of quantitative structure activity models
for predicting molecules binding to enzymes transporters
receptors and ion channels (Ekins Andreyev et al in press
2005e)
It is also possible to simultaneously interpret high
throughput data and predictions on interaction networks
providing a novel approach to predicting and understanding
potential undesirable drugndashdrug or off target effects in the
area of systems pharmacology An example data set uses
percent inhibition data for clotrimazole and ticonazole
which were screened against many different assays at a
single concentration in a commercially available database
BioPrint (Cerep Redmond WA) as published recently
(Fliri Loging Thadejo amp VOlkman 2005) The data for
10 assays has been arbitrarily encoded as inhibitors (gt50
inhibition) or non-inhibitors (lt50 inhibition) in a text file
which was then loaded into MetaCore The analyzed
network algorithm was then used which generates a large
network and fragments it into sub-networks each with a Z-
score and p-values for ranking according to saturation with
objects from the initial gene list The Gene-ontology
processes are also mapped to the gene list and individual
networks In this example a statistically significant network
was generated for the different proteins (Fig 4
p =2838e31) This network also maps the Gene Ontology
processes for the activation of MAPK (118 of genes p
value 9143e07) signal transduction (333 1600e05)
regulation of transcription DNA-dependent (294
2786e04) regulation of inflammatory response (39
3746e04) and the regulation of blood pressure (78
4230e04) This example network indicates how molecules
of the same or different therapeutic classes could be
evaluated for their effects as a graph either together as in
this case or individually This would be useful to indicate
potential off target effects and identify structurally dissimilar
molecules with similar network patterns Such networks
could then be compared to assess network overlap or
differences between molecules and their inhibition of
multiple proteins This type of unique visualization of high
throughput screening data illustrates how the target proteins
may be connected as a network to infer the possible
downstream effects of inhibition
With the burgeoning number of freely available online
and commercial databases that can be used for pathway
construction numbering in the hundreds there have been
suggestions to impose standards for model exchange
querying and visualization (Cary Bader amp Sander 2005)
To date there has been little discussion with regards to
standardization of ADMETox related databases although
there has been considerable discussion relating to drug
metabolism database generation (Erhardt 2003) This is
certainly an important area to address in the future There is
already a growing literature related to ADMETox that is
partially captured in the several commercially available
databases (Ekins et al 2005e) but to date there have been
limited academic efforts to capture data for transporters
with the human membrane transporter database (Yan amp
Sadee 2000) TP-search transporter database (httpwww
ilabrisewasedaacjptp-search) drug interaction database
(httpwwwdruginteractioninfoorgDatabaseinfoaspx)
nuclear hormone receptors (Nakata Yukawa Komiyama
Nakano amp Kaminuma 2002) the ADME-AP database
(Sun Ji Chen Wang amp Chen 2002) and PharmaGKB
(Oliver et al 2002) DSStox (httpwwwepagovnheert
dsstox) TOXNET (httptoxnetnimnihgov) that are
readily accessible
In order to generate accessible pathways using any of
the available software a large enough set of object
identifiers are required to map onto the underlying data-
base To demonstrate this datasets from toxicogenomics
studies have been evaluated with both the KEGG pathway
database and a commercially available product MetaCore
(wwwgenegocom) These gene or protein lists range in
size from 21 to 1853 objects In virtually all cases more
identifiers are mapped to networks in MetaCore and this
also seems independent of the identifier type used
(Unigene Affymetrix Genbank or Locuslink Table 5)
These numbers will obviously change as more information
is added to the respective databases hence the more
objects that are mapped from a dataset The more extensive
Table 4
Literature derived toxicoproteomics data
Compounds Data source Microarray type Compound dose Microarray data
availability
Reference
Carbon
tetrachloride
Male Wistar rats Affymetrix rat chip 8799
probes+proteomics
study
6ndash24 h exposure Genbank SwissProt and
binary data in a
manuscript table
(Fountoulakis
2004)
Paracetamol CD-1 male mice Custom mouse tox blots
with 450 genes
RTQ-PCR+proteomics
study 2-DIGE+
MALDI-MS
150 or 500 mgkg Gene name IMAGE ID
GenBank accession
number fold induction
SwissProt identifier and
protein abundance
change in manuscript
tables
(Ruepp Tonge
Shaw Wallis amp
Pognan 2002)
Paracetamol AP-1 male mice Affymetrix murine 11K
set+proteomics study
Up to 500 mgkg Gene names and fold
changes at multiple
doses and time points in
a manuscript tablemdash
proteomics data not
accessible
(Coen et al 2004)
Oxazepam or
Wy-14643
Male B6C3F1 mice NIEHS Mouse Chip
(8700 genes) 2-DIGE
and MS
Oxazepam (2500 ppm)
Wyeth (Wy)-14643
(500 ppm)
Proteomics data in a
manuscript table
Genbank accession
number gene name and
fold change in a
manuscript table data
also available to
download from NIEHS
website
(Iida et al 2003)
Compound A
(PPAR gamma
ligand)
Female rats CrlCD
(SD)IGS BR
Proteomics 2-DIGE and
MS
250 mgkgday up to
5 days
Accession number and
protein name and
average ratio in a
manuscript table
(Meneses-Lorente
et al 2004)
Bromobenzene Male Wistar rats Proteomics 2-DIGE and
MS custom 3000 cDNA
rat chip
5 mmolkg Accession number gene
name fold change in a
manuscript table protein
names along with bar
charts
(Heijne Stierum
Slijper van
Bladeren amp van
Ommen 2003)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 49
the network that can be generated (as it will consist of more
nodes) then a more comprehensive understanding of the
networks is possible The data available currently in the
literature can be used to evaluate such pathway and
network generation tools Recently we have used several
of the published studies (Tables 1ndash3) with MetaCore to
visualize networks for acetaminophen furan carbon tetra-
chloride benzene and cisplatin showing genes involved in
In vivo
In vitro
In Silico
1970s 1980s 1990s 2000s
Systems Biology ()
OMICS
Fig 2 The timeline for major paradigms in ADMETox
oxidative stress (Ekins Giroux Nikolsky Bugrim amp
Nikolskaya 2005c)
Other important pathwaynetwork building tools that could
potentially be applied to toxicogenomics data include
Ingenuity pathways analysis (httpwwwingenuitycom)
PathArt (httpwwwjubilantbiosyscompdhtm) Pathway
Assist (httpwwwariadnegenomicscomproctspathway
html) (Nikitin et al 2003) and several other databases
deposited at the Pathway Resource List (httpcbiiomskcc
orgprl) All of these products have unique underlying
proprietary pathway databases which are compiled manually
or automatically with text mining tools or a combination of
both We are still waiting for studies that provide a comparison
of different database tools or network building algorithms
Until then theremay be someoverlap but also some differences
between the results obtained from more than one network
method due to the database content and the algorithms used
The reader is recommended to evaluate for themselves several
technologies and select those with the most appropriate fit to
their own specialized needs
In the next Sections the further application of some
available network and database tools will be described with
Microarray or other high throughput data
Upload list of geneprotein identifiers and fold change significance etc (raw data or after selection of the differentially expressed
Genes using other statistical methods)
Parse database and generate interaction network with different algorithms or visualize on maps
Filter Networks
Access significance of the interaction networks
Determine GO processes
Compare 2 or more networks intersection overlap etc
Export gene list
Export gene list
generate interaction network with different algorithms or visualize on maps
Visualization
Fig 3 Schematic for the utilization of pathway tools for assessing high throughput data
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6650
specific detailed reference to transporters and enzymes but
these technologies can also be applied elsewhere as
described above
Fig 4 Network for high throughput screening data for clotrimazole and ticona
cholecystokinin CCR1 Choline transporter Chloride channel catechol-O-methyl
and co workers (Fliri et al 2005) Nodes highlighted with large blue circles rep
molecules showing gt50 inhibition in the assay blue circles represents both mol
inhibits gt50 and one inhibits lt50 Ligands (large) linked to transcriptional f
(wwgenegocom) (For interpretation of the references to colour in this figure leg
311 The role of transporters
A diverse array of organic solutes such as nutrients
neurotransmitters and drugs are transported by specialized
zole screened against 10 in vitro assays (Human Cannabinoid 1 Human
transferase COX2 CYP1A2 CYP2B6 CYP2C19) data published by Fliri
resent assays used in the study Red circles on these nodes represent both
ecules showing lt50 inhibition chequered circles represent one molecule
actors enzymes and transporters via edges using the MetaCorei database
end the reader is referred to the web version of this article)
Table 5
Datasets acquired from publications and other sources and used to evaluate two databasepathway tools KEGG and metaCore
Dataset Number of objects
genes proteins in list
Number of objects
mapped with KEGG
Number of objects mapped
with MetaCore on networks
Reference for microarray
data gene list
Tox Chip 10 (NIEHS) 1853 405 1498 httpsdir-appsniehsnihgov
mapsguestclonesrchcfm
4-Hydroxytamoxifen and
estrogen
1617 434 1343 (Hodges et al 2003)
Mitochondrial proteins 722 156 388 (Gaucher et al 2004
Taylor et al 2003)
Bromobenzene
(24 and 48 h)
130 41 89 (Heijne et al 2004)
Acetaminophen 30 19 23 (Heinloth et al 2004)
Acetaminophen 84 29 64 (Huang et al 2004)
Furan 185 64 139 (Huang et al 2004)
Tetrodotoxin 116 31 86 (Raghavendra Prasad et al
2004)
Benzene 73 16 62 (Yoon et al 2003)
Benzene 76 5 53 (Faiola et al 2004)
Carbon tetrachloride 37 8 26 (Young et al 2003)
Estrogen 94 33 90 (Lobenhofer et al 2002)
Trovafloxacin 142 20 82 (Liguori et al 2005)
Phenobarbital 37 13 28 (Ueda et al 2002)
L-742694 (liver)+ 45 17 19 (Hartley et al 2004)
L-742694 (intestine)+ 23 11 10 (Hartley et al 2004)
A-277249 21 7 9 (Waring et al 2002)
All files were converted to Locuslink identifiers except as noted () unigene () Affymetrix (+) Genbank identifiers were used
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 51
proteins across cellular membranes These may function as
passive processes or active processes energized by the
hydrolysis of ATP or coupling to the co-transport of counter
ions down an electrochemical gradient such as Na+ H+ and
Cl There are many thousands of transporters which can be
classified into distinct superfamilies One of these the solute
carrier class (SLC) is rapidly expanding and contains over
30 families and 200 members The ATP-binding cassette
(ABC) contains 7 families and over 48 members including
P-glycoprotein (P-gp) and MRP subfamilies (Zhang Knipp
Ekins amp Swaan 2002a Zhang Phelps Cheng Ekins amp
Swaan 2002b) Transporters have a key role in clinical
pharmacology with many drugs specifically targeting them
Numerous drugs share transport pathways with nutrients
and transporters have a role in oral absorption drug
bioavailability drug resistance excretion and ultimately
pharmacokinetics and pharmacodynamics
Polymorphism of drug transporters may be a key factor
in drug interactions and lack of effectiveness This field has
become known as pharmacogenomics and is focused on
understanding of inherited DNA sequence variations (poly-
morphisms and mutations) revealed by xenobiotics (Evans
amp McLeod 2003 Weinshilboum 2003) Over the last few
decades many genes have been directly linked to the
mechanisms of response (Evans amp McLeod 2003 Wein-
shilboum 2003) such that 20ndash95 of variability to drug
response is inheritable (Evans amp McLeod 2003) This
phenotypical variability is mainly caused by single nucleo-
tide polymorphisms (SNPs) present in anywhere from 1 to
50 of the population resulting in either lower protein
activity incorrect folding or rapid degradation via proteo-
somes (Weinshilboum amp Wang 2004) A number of
structurally diverse molecules bind to P-gp which is
expressed in many tissues and has numerous SNPs one of
which (C3435T) affects the expression level in the
duodenum and therefore can impact the absorption of
molecules which would be substrates for this transporter
(Sakaeda Nakamura amp Okumura 2002) The human
proton-dependent dipeptide transporter (hPEPT1) can also
affect the absorption of molecules in the intestine and
recently 9 SNPs were found with only one displaying a
reduced transport capacity (Zhang et al 2004) The sodium-
dependent carnitine cotransporter OCTN2 can possess
mutations and these result in primary carnitine deficiency
which impacts fatty acid oxidation and is characterized by
many clinical manifestations (Lahjouji Mitchel amp Qureshi
2001) The organic cation transporter 1 (OCT1) is also
important in the transport of numerous xenobiotics and
endobiotics Recently 4 SNPs were identified in the
Japanese population and when functionally characterized
in vitro the uptake of cations was reduced significantly for
some of these mutations indicating that this would likely
contribute to inter-individual variations in metabolism of
drugs which were transported via OCT1 (Sakata et al
2004)
3111 Clinical relevance of transporters The pregnane
X-receptor (PXR) is a transcriptional regulator of the
enzyme human MDR1 (P-gp) MRPs and OATP (Wang amp
LeCluyse 2003) CYP3A (Bertilsson et al 1998 Blumberg
et al 1998 Kliewer et al 1998) and CYP2C89 (Synold
Dussault amp Forman 2001) as well as many other genes
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6652
involved in the transport metabolism and biosynthesis of
bile acids (Staudinger Liu Madan Habeebu amp Klaassen
2001) However the additional receptors CAR FXR LXR
and other nuclear receptors take part in a complex network
of interactions to control these and other proteins Thus
elucidation of the regulatory networks which control the
expression of these transporters is also important To date
most of the research has centered on efflux transporters but
there has been considerable interest in uptake transporters
such as the organic anion transporter polypeptide (OATP
((Kim 2003) see also later Section on OATP)
There are several specific examples of the importance of
drug transporters to the clinical development of drugs One
example is the insulin sensitizer troglitazone which was
withdrawn due to hepatotoxicity although the precise
mechanism appears to have been unclear until recently
The major metabolite is a sulfated species and is suspected
of being responsible for the observed toxicity The recent
assessment of the organic anion transporting polypeptides
OATP-C and OATP8 expressed on the hepatocyte baso-
lateral membrane indicated that sulphated troglitazone has a
high affinity for the former and possibly lower affinity for
the latter (Nozawa et al 2004) This metabolite would
therefore be expected to accumulate in hepatocytes and
inhibit the bile salt export pump and Mrp2 Because
polymorphisms have been shown for OATP-C (Tirona
Leake Merino amp Kim 2001) it is also possible that these
may result in the accumulation of the metabolite and in turn
elicit idiosyncratic toxicity A second relevant example of
the impact of transporters is the clinically significant drugndash
drug interaction between cerivastatin and cyclosporine A
which occurs via the OATP-C transporter (Shitara Itoh
Sato Li amp Sugiyama 2003) A third example are the HIV
protease inhibitors saquinavir ritonavir and indinavir which
are transported by MRP2 in vitro and other drugs such as
probenecid and sulfinpyrazone are able to enhance this
transport Transport by MRP2 suggests that these com-
pounds will have decreased bioavailability due to increased
clearance and other drugs could aggravate this situation by
further enhancing transport (Huisman et al 2002) Sim-
ilarly the rifampicin mediated induction via PXR of MRP2
and P-gp in healthy subjects was found to significantly
decrease the AUC and also correlated with intestinal
expression of these transporters This transporter is also
inducible by cisplatin 2-AAF and phenobarbital (Schrenk
et al 2001) indicating multiple mechanisms may be
involved In other species such as rat commonly used as a
toxicity model orthologs of the transporters such as OATP2
are expressed and can be induced with ligands for PXR like
PCN (Guo Choudhuri amp Klaassen 2002) This is useful
knowledge because the advent of microarray technology
allows one to dose a rat with a xenobiotic and assess
thousands of genes simultaneously in a particular tissue For
instance animals dosed with known nephrotoxins have
shown some upregulation of the NandashKndashCl transporter
however the authors suggested genomic responses are
stronger soon after exposure before declining (Fleck et
al 2003) Some transporters may therefore be specifically
targeted by drugs in one tissue such as the CNS but these
same transporters may also be expressed elsewhere in the
body hence off-target effects may result in toxicity The
serotonin transporter is one such example which is
expressed in the lungs and brain Some substrates for this
channel like fenfluramine can result in primary pulmonary
hypertension as they accumulate in pulmonary cells (Roth-
man Ayestas Dersch amp Baumann 1999) Similarly P-gp is
expressed at the blood brain barrier and intestine impacting
the efficacy and bioavailability of drugs
3112 Transporter network examples ABCA1 The
ABCA1 transporter mediates the first step of cholesterol
transport Mutations in this gene cause Tangier disease
which results in severe HDL deficiency cholesterol
accumulation in macrophages and attendant atherosclerosis
This transporter represents a drug target for upregulation
modulating cholesterol metabolism and prevention of
cardiovascular disease (Oram amp Lawn 2001) In vitro
ABCA1 can be inhibited by the sulfonylurea glybenclamide
(Field Burn amp Mathur 2004 Wang Silver Thiele amp Tall
2001) In order to illustrate the advantages of mapping drug
transporters as networks onto functional models alongside
other proteins one can consider the example of ABCA1
The query of the MetaCorei database shows that this
transporter appears on three manually curated pathway maps
representing the Fstate of the art_ knowledge derived from
reliable high quality literature sources One can also use the
individual maps as an interface to access the underlying
layers of information about the transporter including the list
of encoding genessplice variants with known SNPs In
addition to browsing MetaCorei a user can also build
custom networks around ABCA1 using the network-
construction tool (Fig 5) Such a visualization utility may
be very helpful for identification of all putative pathways
around a particular transporter or compound of interest The
ABCA1 network created by this tool shows that this
transporter at the time of writing is linked directly to
twenty-five other objects such as APOE1 and LXR Many
of its neighbors have their own SNPs that could be
important in determining interactions between transport of
a drug and normal human transport of endogenous ligands
in health or disease
3113 Transporter network examples OATP The
OATPs are key membrane bound transporters expressed in
many organs including intestine liver lung choroid plexus
blood brain barrier and other organs (Tamai et al 2000)
This family of transporters is capable of mediating the
sodium-independent transport of a diverse array of mole-
cules such as steroid conjugates organic anions and
xenobiotics by coupling uptake with efflux of bicarbonate
(Satlin Amin amp Wolkoff 1997) glutathione or its
conjugates (Hagenbuch amp Meier 2004) The inhibition of
Fig 5 The network of direct gene interactions around the ABCA1 transporter gene (centre) generated with MetaCorei The interation types between nodes are shown as small colored hexagons eg unspecified
allosteric regulation binding cleavage competition covalent modification dephosphorylation phosphorylation transcription regulation transformation When applicable interactions also have a positive or
negative effect and direction Ligands (purple) linked to other proteins (blue blobs) transfactors (red) enzymes (orange) (For interpretation of the references to colour in this figure legend the reader is referred to
the web version of this article)
SEkin
sJournalofPharm
acologica
landToxico
logica
lMeth
ods53(2006)38ndash66
53
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6654
this transporterrsquos hepatic uptake of other compounds may be
important for reported drugndashdrug interactions (Kim 2003)
described earlier (Shitara et al 2003) as well as cerivastatin
with gemfibrozil (Shitara Hirano Sato amp Sugiyama 2004)
OATP1B1 (previous names OATP-C LST-1 OATP2
SLC21A6) represents the most studied human OATP to
date (Meier amp Stieger 2000) and is expressed on the
basolateral plasma membrane of hepatocytes Several single
nucleotide polymorphisms have been identified in the
OATP1B1 gene in European-Americans African-Americans
(Tirona et al 2001) and Japanese (Nozawa et al 2002)
dramatically impacting the transport of ligands such as
pravastatin (Mwinyi Johne Bauer Roots amp Gerloff 2004
Nishizato et al 2003) estrone-3-sulfate (Nozawa et al
2002 Tirona et al 2001) Rifampin (Tirona Leake
Wolkoff amp Kim 2003) and estradiol 17h-d-glucuronide(Tirona et al 2001)
The regulation of SLCO may be affected during
extrahepatic cholestasis bile duct ligation bile salt induced
Fig 6 Networks generated with the autoexpand algorithm in MetaCorei to illustr
be interconnected with other protein regulatory signaling information Informa
unspecified allosteric regulation binding cleavage competition covalent m
transformation When applicable interactions also have a positive or negative e
transfactors (red) enzymes (orange) (For interpretation of the references to colour
cholestatic hepatitis (Dumont et al 1997 Meier amp Stieger
2000 Rost et al 2003) and primary sclerosing cholangitis
(Oswald Kullack-Ublick Paumgarter amp Beuer 2001) In
particular OATP1B1 appears to be regulated by the liver-
enriched transcription factor hepatocyte nuclear factor 1a
(HNF1a) which binds to the promoter region of this
transporter (Jung et al 2001) Site directed mutagenesis
of this binding site resulted in inactivation suggesting the
critical nature of the interaction with HNF1a Bile acids
such as CDCA have been shown to transcriptionally repress
HNF1a in vitro via inhibition of the transactivating effect of
HNF4a on HNF1a (Jung amp Kullak-Ublick 2003) After
screening many rat and human uptake transporters in vitro
OATP1B1 was also shown to modulate the PXR response
by controlling rifampin retention in the cell and therefore
affecting the induction of CYP3A4 and other gene products
such as P-gp (Tirona et al 2003)
Some of the literature for OATP1B1 human substrate
data has been annotated into MetaCorei to illustrate the
ate how ligands for the human OATP-C (OATP1B1) transporter (centre) can
tion on the type of interaction between objects is hidden for clarity eg
odification dephosphorylation phosphorylation transcription regulation
ffect and direction Ligands (purple) linked to other proteins (blue blobs)
in this figure legend the reader is referred to the web version of this article)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 55
visualization of the complex interconnections between this
transporter its ligands regulatory factors and signaling
molecules already in the database (Fig 6) The network was
generated with the autoexpand algorithm in the software
representing one of multiple available algorithms for
connecting genes ligands and other objects in the database
Clearly if more ligands and their connections are added to
the database the complexity of the network will increase
considerably The OATP-C (OATP1B1) gene details can be
viewed upon querying the database and links are provided
to other public databases This page can be used to highlight
the multiple synonyms for this gene as well as links to the
multiple SNPs identified to date
3114 Transporter microarray data Microarrays have
generally been limited in the number of transporters present
on them (Annereau et al 2004) however they have been used
in an attempt to correlate pharmacokinetic properties with
gene expression for valacyclovir (Landowski et al 2003) as
well as understand the expression profile in different tissues
or cell lines upon food component or xenobiotic treatment
(Anderle Huang amp Sadee 2004) This lack of transporters
on microarrays has prompted some groups to produce their
own arrays with a heavier emphasis on transporters These
arrays have for example then been used to demonstrate the
upregulation of ABC transporters and down-regulation of
GST-Pi in cell lines resistant to colchicines or 9-nitro-
camptothecin (Annereau et al 2004) The genes that were
significantly up or down-regulated in this particular study
were used to build networks with MetaCore (Fig 7A B) and
the similarities between them were assessed (Fig 7C)
Although there were only a small number of significantly
changed genes in common (IL-8 Fos GST-Pi Calpactin and
Ubiquitin hydrolase) it is perhaps likely that there is a much
larger common gene network that is important for drug
resistance although a much larger number of cell lines and
drug treatments need to be evaluated to produce a definitive
drug resistance signature involving transporters enzymes
and transcriptional regulators
312 Applications to enzymes
As we have already described it may be particularly
valuable to visualize enzymes as networks to show
interactions with transcriptional regulators and ligands
For example a key enzyme is CYP3A4 which metabolizes
40ndash45 of all drugs and has relatively few SNPs
(Ingelman-Sundberg 2004) Using a second software suite
MetaDrugi (wwwgenegocom) it is possible to construct
a custom network around this or other drug metabolizing
enzymes (Fig 8) In this case the gene network for
CYP3A4 highlights all of the major transcriptional
regulators and several more distant linked proteins and
ligands connected on the network that may be useful for
further study (Fig 8) Substrates inhibitors as well as
regulatory factors and other enzymes can be observed
connected on this network Due to the many hundreds of
known inhibitors and substrates only a small number are
shown here for clarity We assume that if a perturbation in
a pathway (eg due to a nonfunctional enzyme) is linked
to a certain pathologic condition a similar perturbation
caused by the interference from xenobiotic metabolism
(eg competitive inhibition of the same enzyme) may
result in identical effects As microarray gene expression
data is increasingly generated the role of enzyme
regulation in toxicity of certain xenobiotics will become
more apparent from either in vivo or in vitro studies The
visualization of such signature gene networks involving
transporters and enzymes their ligands and regulatory
factors will also be important for future toxicity prediction
methods We have recently generated visualizations of
microarray data from MCF-7 cells treated with 4-hydrox-
ytamoxifen to show that some of the key genes involved in
metabolism and transport are upregulated (Ekins et al
2005d) In addition we have made predictions with various
QSAR models in MetaDrug to indicate the involvement of
PXR CYP3A4 and P-gp (Ekins et al 2005e) Therefore it
appears likely that 4-hydroxytamoxifen could increase its
own metabolism as well as efflux from cells via P-gp
which can be visualized on networks Any decrease of
function of these enzymes or transporters in a population
would likely result in changes in the metabolism and
transport of this active metabolite potentially impacting
the clinical effect This represents one example of how
both pathway tools QSAR models and network building
algorithms can be used with different types of predicted
and experimental data to allow visualization of potential
compound interactions or toxicity
313 Future network applications
As the population ages an increasing incidence and
prevalence of systemic diseases especially chronic diseases
have occurred among older adults This has resulted in an
increase in medications used concomitantly by this
population which presents challenges for drugndashdrug
interactions Physiologically elderly patients may behave
differently to the younger patients for which the drugs are
initially developed for and tested on Many pharmacoki-
netic investigations in the elderly population reveal
decreased clearance of lipophilic drugs metabolized by
the cytochrome P450 enzymes however few studies have
evaluated aging-dependent or gender-related changes in
specific P450 enzymes (Hunt Westerkam amp Slave 1992)
Age-related physiological changes such as a reduction in
liver mass hepatic metabolizing enzyme activity liver
blood flow and alterations in plasma drug binding may
account for the decreased elimination of some metabolized
drugs in the elderly It is particularly difficult to separate an
effect of aging from the variation in the rate of metabolism
due to factors such as individual metabolic phenotype
(slow or fast metabolizer due to SNPs) environmental
influences concomitant disease states and drug intake
(drugndashdrug interactions) The available data suggest that
Fig 7 Networks for drug resistant cell lines generated with the Auto expand algorithm in MetaCore A KB-3-1 vs KB-8-5 B ATC-DU-145 vs RCO1 C node overlap of A and B with initial nodes highlighted
with blue or red solid circles (Initial gene lists derived from (Annereau et al 2004)) Information on the type of interaction between objects is hidden for clarity eg unspecified allosteric regulation binding
cleavage competition covalent modification dephosphorylation phosphorylation transcription regulation transformation When applicable interactions also have a positive or negative effect and direction
Ligands (purple) linked to other proteins (blue blobs) transfactors (red) enzymes (orange) (For interpretation of the references to colour in this figure legend the reader is referred to the web version of this article)
SEkin
sJournalofPharm
acologica
landToxico
logica
lMeth
ods53(2006)38ndash66
56
Fig7(continued)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 57
Fig7(continued)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6658
Fig 8 The network of gene interactions around the CYP3A4 enzyme (centre) from MetaCorei Information on the type of interaction between objects is
hidden for clarity eg unspecified allosteric regulation binding cleavage competition covalent modification dephosphorylation phosphorylation
transcription regulation transformation When applicable interactions also have a positive or negative effect and direction Ligands (purple) linked to other
proteins (blue blobs) transfactors (red) enzymes (orange) (For interpretation of the references to colour in this figure legend the reader is referred to the web
version of this article)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 59
the initial doses of drugs metabolized by these enzymes
should be reduced in older patients according to the clinical
response In most published studies the elderly appear at
least as responsive as the young to inducers or inhibitors of
P450s (Durnas Loi amp Cusack 1990) More recently there
has been some suggestion that there are age related
reductions in function of some specific P450s such as
CYP3A4 (Patki et al 2004) and this could occur at the
level of regulation However earlier studies with the same
enzyme showed no change in clearance with age (Hunt et
al 1992) To date there has been even less examination of
the transporter functions and any changes with age
(Kinirons amp OrsquoMahony 2004) so the current understand-
ing of the effects of aging on metabolism and transport is
anything but transparent This represents an extreme
challenge for the pharmaceutical industry how to predict
whether a drug has an affinity for an enzyme or transporter
and whether this may be clinically important if it shows a
decline in expression or function with aging Any decrease
of function of these enzymes or transporters in an elderly
population would result in changes in the metabolism and
transport of metabolites potentially impacting the clinical
effect This represents another example of how computa-
tional approaches may perhaps be used with different types
of predicted and experimental data to allow visualization of
potential compound interactions or toxicity in elderly
populations We may see different gene networks high-
lighted as humans age and these may be modified by drug
treatment and coadministration This represents an area
were network analysis could be applied in the future and is
in need of considerable further research
The collection of microarray data in databases such as
CEBS ArrayTrack and EDGE represents a future resource
for computational gene network analysis One could
envisage that ultimately in each case such data is converted
to one or more networks that are also displayed for the user
and can be used to compare treatments from in vivo and in
vitro experiments This would represent a different approach
to clustering the data as currently implemented in one of
these efforts (Hayes et al 2005) and may condense large
amounts of experimentally derived data into a readily
interpreted network
4 Discussion
Previously in this journal the progress of many
research groups in predicting human ADME parameters
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6660
in silico (Ekins et al 2000b) and approaches for drug
metabolism (Ekins Ring Grace McRobie-Belle amp
Wrighton 2000a) have been described Both of these
reviews commented on moving HT assays for ADME
Tox much earlier in drug discovery which had also been
indicated by other groups The initial reviews also
highlighted the likely wealth of data that would become
available and how this could be used for structure
activity relationships alongside the bioactivity data in
computational models It was noted that there was a
paucity of predictive metabolism tools at that time In
addition a growing number of efforts to model whole
cells and organs now a field called systems biology
were recognized as models that could be integrated with
the in silico ADME approaches In summary since these
past reviews virtually all pharmaceutical companies have
attempted earlier high throughput screening for ADME
Tox properties and to some extent the wider application
of computational approaches for physicochemical proper-
ties Systems biology is being quite widely acknowledged
as the new paradigm for understanding complex bio-
logical datasets derived from high throughput technolo-
gies and the accumulated knowledge on human protein
interactions (Hartwell Hopfield Leibler amp Murray 1999
Hood 2003b) Therefore systems biology can be defined
as the integration of genetic proteomic transcriptomic
and metabonomic data using computational methods
(Nicholson amp Wilson 2003) When taken together
information on molecular processes derived from different
sources represents a lsquolsquouniversersquorsquo of putative biological
functionality of which only a small fraction of it will be
realized in a cell at any given time To date systems
biology has been driven by academia and funding bodies
such as the NIH rather than the pharmaceutical compa-
nies Presently there is a great deal of interest from
scientists of all backgrounds in identifying the networks
of cellular pathways and the corresponding physically
interacting proteins
The network building software for systems biology
described in this current review will be valuable to query
high throughput data and known literature interactions in
order to predict potential toxicity in different species In the
future the compilation of published toxicogenomics data-
sets characteristic of different types of toxicity will likely
be available in these software systems to act as a reference
database It is also feasible that we will be able to generate
the annotated datasets which specifically address the
differences between human and rat networks implicated
in toxicity The identification of sub-network modules
conserved between human and rat distinct for toxicity
types or predictive for toxic end-points in human will be
possible Such signature gene networks (Nikolsky et al
2005) can then be verified with other experimentally
derived data prospectively or from preexisting databases
The combined Fhigh throughput datandashin silico network_approaches therefore represents a novel method for access-
ing complex tissue-level processes as networks integrating
all data types based on functional interactions The data-
bases developed and used in this approach will certainly
benefit from further annotation around the drug metaboliz-
ing enzymes and transporters as described herein in terms
of transcriptional regulation and the ligands associated with
them which frequently appear in the literature It will be
important to capture disparities in the assignment of ligands
to enzymes transporters and other proteins as well as
negative data
In summary although there have been numerous
toxicogenomics studies published there is presently a
relatively small number of datasets that are freely available
to perform network analysis of microarray data The
number of studies identifying large numbers of proteins
which are affected by molecule treatment are even scarcer
still while there are several examples of some published
studies that combine such data It is hoped that the
numerous database initiatives for high content and toxicol-
ogy data that are being undertaken will improve the
situation for other researchers that are not currently
equipped to do such microarray studies themselves The
addition of requirements by journals to deposit such raw
data in a freely accessible resource will aid these initiatives
ADMETox groups have seen new technologies and
approaches developed over the last decade that have all
been applied to identify poor compounds earlier (Fig 2)
The latest technologies integrate network building tools
with high content data and databases The current review
described the limited number of networks generated for
ADMETox at present and one hopes that the impact of
such analyses will be commonplace in the future Systems
biology is however more than just applying a network
approach and hence systems-ADMETox will have to
evolve due to the continual pressure to develop newer
technologies This current paradigm combining empirical
data and computational methods should integrate the
complex data already generated making it readily inter-
pretable and valuable for identifying the most promising
compounds in the future
Acknowledgements
Dr Maggie AZ Hupcey is gratefully acknowledged for
editorial assistance Dr Peter W Swaan (University of
Maryland) and Dr Cheng Chang (Ohio State University)
Dr Steve Wright (University of Arizona) Dr K Sandy
Pang (University of Toronto) and Dr Craig Giroux (Wayne
State University) are kindly acknowledged for their support
and discussions My colleagues at GeneGo Sergey
Andreyev Andy Ryabov Eugene Kirillov Eugene A
Rakhmatulin Svetlana Sorokina Andrej Bugrim Tatiana
Nikolskaya Yuri Nikolsky John Metz and Julie Bryant are
all thanked for their considerable contributions to software
development and data annotation
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 61
References
Abe M Yamashita S Kuramoto T Hirayama Y Tsukamoto T Ohta
T et al (2003) Global expression analysis of N-methyl-NV-nitro-N-nitrosoguanidine-induced rat stomach carcinomas using oligonucleotide
microarrays Carcinogenesis 24 861ndash867
Adachi T Ono Y Koh K B Takashima K Tainaka H Matsuno Y
et al (2004) Long-term alteration of gene expression without
morphological change in testis after neonatal exposure to genistein in
mice Toxicogenomic analysis using cDNA microarray Food and
Chemical Toxicology 42 445ndash452
Akerman G S Rosenzweig B A Domon O E McGarrity L J
Blankenship L R Tsai C A et al (2004) Gene expression profiles
and genetic damage in benzo(a)pyrene diol epoxide-exposed TK6 cells
Mutation Research 549 43ndash64
Aksenov S V Church B Dhiman A Georgieva A Sarangapani R
Helmlinger G et al (2005) An integrated approach for inference and
mechanistic modeling for advancing drug development FEBS Letters
579 1878ndash1883
Anderle P Huang Y amp Sadee W (2004) Intestinal membrane transport
of drugs and nutrients Genomics of membrane transporters using
expression microarrays European Journal of Pharmaceutical Sciences
21 17ndash24
Annereau J P Szakacs G Tucker C J Arciello A Cardarelli C
Collins J et al (2004) Analysis of ATP-binding cassette transporter
expression in drug-selected cell lines by a microarray dedicated to
multidrug resistance Molecular Pharmacology 66 1397ndash1405
Apic G Ignjatovic T Boyer S amp Russell R B (2005) Illuminating drug
discovery with biological pathways FEBS Letters 579 1872ndash1877
Baker V A Harries H M Waring J F Duggan C M Ni H A Jolly
R A et al (2004) Clofibrate-induced gene expression changes in rat
liver A cross-laboratory analysis using membrane cDNA arrays
Environmental Health Perspectives 112 428ndash438
Balakin K V Ekins S Bugrim A Ivanenkov Y A Korolev D amp
Nikolsky Y et al (2004a) Quantitative structurendashmetabolism relation-
ship modeling of the metabolic N-dealkylation rates Drug Metabolism
and Disposition 32 1111ndash1120
Balakin K V Ekins S Bugrim A Ivanenkov Y A Korolev D amp
Nikolsky Y et al (2004b) Kohonen maps for prediction of binding to
human cytochrome P450 3A4 Drug Metabolism and Disposition 32
1183ndash1189
Barabasi A -L amp Oltvai Z N (2004) Network biology Under-
standing the cellrsquos functional organization Nature Reviews Genetics
5 101ndash113
Bartosiewicz M Penn S G amp Buckpitt A (2001) Applications of gene
arrays in environmental toxicology Fingerprints of gene regulation
associated with cadmium chloride benzo(a)pyrene and trichloro-
ethylene Environmental Health Perspectives 109 71ndash74
Bertilsson G Heidrich J Svensson K Asman M Jendeberg L
Sydow-Backman M et al (1998) Identification of a human nuclear
receptor defines a new signaling pathway for CYP3A induction
Proceedings of the National Academy of Sciences of the United States
of America 95 12208ndash12213
Blumberg B Sabbagh Jr W Juguilon H Bolado Jr J van Meter
C M Ong E S et al (1998) SXR a novel steroid and xenobiotic-
sensing nuclear receptor Genes and Development 12 3195ndash3205
Borodina Y Rudik A Filimonov D Kharchevnikova N Dmitriev A
Blinova V et al (2004) A new statistical approach to predicting
aromatic hydroxylation sites Comparison with model-based
approaches Journal of Chemical Information and Computer Sciences
44 1998ndash2009
Borodina Y Sadym A Filimonov D Blinova V Dmitriev A amp
Poroikov V (2003) Predicting biotransformation potential from
molecular structure Journal of Chemical Information and Computer
Sciences 43 1636ndash1646
Bottone Jr F G Martinez J M Collins J B Afshari C A amp Eling
T E (2003) Gene modulation by the cyclooxygenase inhibitor
sulindac sulfide in human colorectal carcinoma cells Possible link to
apoptosis Journal of Biological Chemistry 278 25790ndash25801
Boyer S amp Zamora I (2002) New methods in predictive metabolism
Journal of Computer-Aided Molecular Design 16 403ndash413
Bredel M amp Jacoby E (2004) Chemogenomics An emerging strategy
for rapid target and drug discovery Nature Reviews Genetics 5
262ndash275
Bugrim A Nikolskaya T amp Nikolsky Y (2004) Early prediction of drug
metabolism and toxicity Systems biology approach and modeling
Drug Discovery Today 9 127ndash135
Bulera S J Eddy S M Ferguson E Jatkoe T A Reindel J F
Bleavins M R et al (2001) RNA expression in the early character-
ization of hepatotoxicants in Wistar rats by high-density DNA micro-
arrays Hepatology 33 1239ndash1258
Butcher E C Berg E L amp Kunkel E J (2004) Systems biology in drug
discovery Nature Biotechnology 22 1253ndash1259
Butte A (2002) The use and analysis of microarray data Naturalist
Review of Drug Discovery 1 951ndash960
Cary M P Bader G D amp Sander C (2005) Pathway information for
systems biology FEBS Letters 579 1815ndash1820
Cheok M H Yang W Pui C H Downing J R Cheng C Naeve
C W et al (2003) Treatment-specific changes in gene expression
discriminate in vivo drug response in human leukemia cells Nature
Genetics 34 85ndash90
Cleary J D Rogers P D amp Chapman S W (2001) Differential
transcription factor expression in human mononuclear cells in response
to amphotericin B Identification with complementary DNA microarray
technology Pharmacotherapy 21 1046ndash1054
Coen M Ruepp S U Lindon J C Nicholson J K Pognan F Lenz
E M et al (2004) Integrated application of transcriptomics and
metabonomics yields new insight into the toxicity due to paracetamol in
the mouse Journal of Pharmaceutical and Biomedical Analysis 35
93ndash105
Cornwell P D De Souza A T amp Ulrich R G (2004) Profiling of
hepatic gene expression in rats treated with fibric acid analogs
Mutation Research 549 131ndash145
Csermely P Agoston V amp Pongor S (2005) The efficiency of multi-
target drugs The network approach might help drug design Trends in
Pharmacological Sciences 26 178ndash182
Cunningham M J Liang S Fuhrman S Seilhamer J J amp
Somogyi R (2000) Gene expression microarray data analysis for
toxicology profiling Annals of the New York Academy of Sciences 919
52ndash67
de Longueville F Atienzar F A Marcq L Dufrane S Evrard S
Wouters L et al (2003) Use of a low density microarray for studying
gene expression patterns induced by hepatotoxicants on primary
cultures of rat hepatocytes Toxicological Sciences 75 378ndash392
Dobrin R Beg Q K Barabasi A L amp Oltvai Z N (2004)
Aggregation of topological motifs in the Escherichia coli transcriptional
regulatory network BMC Bioinformatics 5 10
Donald S Verschoyle R D Edwards R Judah D J Davies R
Riley J et al (2002) Hepatobiliary damage and changes in hepatic
gene expression caused by the antitumor drug ecteinascidin-743 (ET-
743) in the female rat Cancer Research 62 4256ndash4262
Dumont M Jacquemin E DrsquoHont C Descout C Cresteil D Haouzi
D et al (1997) Expression of the liver Na+-independent organic anion
transporting polypeptide (oatp-1) in rats with bile duct ligation Journal
of Hepatology 27 1051ndash1056
Durnas C Loi C M amp Cusack B J (1990) Hepatic drug metabolism
and aging Clinical Pharmacokinetics 19 359ndash389
Eisen M B Spellman P T Brown P O amp Botstein D (1998) Cluster
analysis and display of genome-wide expression patterns Proceedings
of the National Academy of Sciences of the United States of America
95 14863ndash14868
Ekins S Andreyev S Ryabov A Kirilov E Rakhmatulin E A
Bugrim A et al (in press) Computational Prediction of Human Drug
Metabolism Exp Opin Drug Metab Toxicol
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6662
Ekins S Boulanger B Swaan P W amp Hupcey M A Z (2002)
Towards a new age of virtual ADMETOX and multidimensional drug
discovery Journal of Computer-Aided Molecular Design 16 381ndash401
Ekins S Bugrim A Nikolsky Y amp Nikolskaya T (2005) Systems
biology Applications in drug discovery In S Gad (Ed) Drug
Discovery Handbook (pp 123ndash183) Hoboken NJrsquo Wiley and Sons
Ekins S Giroux C N Nikolsky Y Bugrim A amp Nikolskaya T
(2005) A signature gene network approach to toxicity The Toxoco-
logists 84
Ekins S Kirillov E Rakhmatulin E amp Nikolskaya T (2005) A novel
method for visualizing nuclear hormone receptor networks relevant to
drug metabolism Drug Metabolism and Disposition 33 474ndash481
Ekins S amp McGowan R J (2001) The limits of reductionism The
shifting genomic paradigmrsquos impact on industry and academia
Philsophy in Science 9 1ndash23
Ekins S Mirny L amp Schuetz E G (2002) A ligand-based approach to
understanding selectivity of nuclear hormone receptors PXR CAR
FXR LXRa and LXRb Pharmaceutical Research 19 1788ndash1800
Ekins S Nikolsky Y amp Nikolskaya T (2005) Techniques Application
of systems biology to absorption distribution metabolism excretion
and toxicity Trends in Pharmacological Sciences 26 202ndash209
Ekins S Ring B J Grace J McRobie-Belle D J amp Wrighton S
A (2000) Present and future in vitro approaches for drug
metabolism Journal of Pharmacological and Toxicological Methods
44 313ndash324
Ekins S Waller C L Swaan P W Cruciani G Wrighton S A amp
Wikel J H (2000) Progress in predicting human ADME parameters in
silico Journal of Pharmacological and Toxicological Methods 44
251ndash272
Erhardt P W (2003) A human drug metabolism database Potential roles
in the quantitative predictions of drug metabolism and metabolism-
related drugndashdrug interactions Current Drug Metabolism 4 411ndash422
Evans W E amp McLeod H L (2003) Pharmacogenomicsmdashdrug
disposition drug targets and side effects New England Journal of
Medicine 348 538ndash549
Ezendam J Staedtler F Pennings J Vandebriel R J Pieters R
Boffetta P et al (2004) Toxicogenomics of subchronic hexachlor-
obenzene exposure in Brown Norway rats Environmental Health
Perspectives 112 782ndash791
Faiola B Fuller E S Wong V A amp Recio L (2004) Gene expression
profile in bone marrow and hematopoietic stem cells in mice exposed to
inhaled benzene Mutation Research 549 195ndash212
FDA (2004) Innovation stagnation Challenge and opportunity on the
critical path to new medicinal products
Fiehn O (2001) Combining genomics metabolome analysis and bio-
chemical modelling to understand metabolic networks Comparative
Functional Genomics 2 155ndash168
Field F J Born E amp Mathur S N (2004) LXRRXR ligand activation
enhances basolateral efflux of beta-sitosterol in CaCo-2 cells Journal of
Lipid Research 45 905ndash913
Fleck C Sutter L Appenroth D Koch B Meinhold T Pitack M
et al (2003) Use of gene chip technology for the characterisation of
the regulation of renal transport processes and of nephrotoxicity in
rats Experimental and Toxicologic Pathology 54 401ndash410
Fliri A F Loging W T Thadeio P F amp Volkmann R A (2005)
Biological spectra analysis Linking biological activity profiles to
molecular structure Proceedings of the National Academy of Sciences
of the United States of America 102 261ndash266
Fountoulakis M (2004) Application of proteomics technologies in the
investigation of the brain Mass Spectrometry Reviews 23 231ndash258
Gaucher S P Taylor S W Fahy E Zhang B Warnock D E Ghosh
S S et al (2004) Expanded coverage of the human heart
mitochondrial proteome using multidimensional liquid chromatography
coupled with tandem mass spectrometry Journal of Proteome
Research 3 495ndash505
Gerhold D Lu M Xu J Austin C Caskey C T amp Rushmore T
(2001) Monitoring expression of genes involved in drug metabolism
and toxicology using DNA microarrays Physiological Genomics 5
161ndash170
Guo G L Choudhuri S amp Klaassen C D (2002) Induction profile of
rat organic anion transporting polypeptide 2 (oatp2) by prototypical
drug-metabolizing enzyme inducers that activate gene expression
through ligand-activated transcription factor pathways Journal of
Pharmacology and Experimental Therapeutics 300 206ndash212
Hagenbuch B amp Meier P J (2004) Organic anion transporting
polypeptides of the OATP SLC21 family Phylogenetic classification
as OATP SLCO superfamily new nomenclature and molecularfunc-
tional properties Pflugers Archiv 447 653ndash665
Hamadeh H K amp Afshari C A (2004) Toxicogenomics principles and
applications Hobokenrsquo John Wiley and Sons
Hamadeh H K Bushel P R Jayadev S Martin K DiSorbo O Sieber
S et al (2002) Gene expression analysis reveals chemical-specific
profiles Toxicological Sciences 67 219ndash231
Hamadeh H K Jayadev S Gaillard E T Huang Q Stoll R
Blanchard K et al (2004) Integration of clinical and gene expression
endpoints to explore furan-mediated hepatotoxicity Mutation Research
549 169ndash183
Hamadeh H K Knight B L Haugen A C Sieber S Amin R P
Bushel P R et al (2002) Methapyrilene toxicity Anchorage of
pathologic observations to gene expression alterations Toxicologic
Pathology 30 470ndash482
Han J D Bertin N Hao T Goldberg D S Berriz G F Zhang L V
et al (2004) Evidence for dynamically organized modularity in the
yeast proteinndashprotein interaction network Nature 430 88ndash93
Hanisch D Zien A Zimmer R amp Lengauer T (2002) Co-clustering of
biological networks and gene expression data Bioinformatics 18
S145ndashS154
Harris A J Dial S L amp Casciano D A (2004) Comparison of basal
gene expression profiles and effects of hepatocarcinogens on gene
expression in cultured primary human hepatocytes and HepG2 cells
Mutation Research 549 79ndash99
Harrison S C (2004) Whither structural biology Nature Structural
Molecular Biology 11 12ndash15
Hartley D P Dai X He Y D Carlini E J Wang B Huskey S E
et al (2004) Activators of the rat pregnane X receptor differentially
modulate hepatic and intestinal gene expression Molecular Pharma-
cology 65 1159ndash1171
Hartwell L H Hopfield J J Leibler S amp Murray A W (1999) From
molecular to modular cell biology Nature 402 C47ndashC52
Hasmall S Orphanides G James N Pennie W Hedley K Soames
A et al (2002) Downregulation of lactoferrin by PPARalpha ligands
Role in perturbation of hepatocyte proliferation and apoptosis
Toxicology Sciences 68 304ndash313
Hayase T Yamamoto Y Yamamoto K Muso E amp Shiota K (2004)
Microarray profile analysis of toxic cocaine-induced alterations in the
expression of mouse brain gene sequences A possible rsquoprotectiversquo
effect of buprenorphine Journal of Applied Toxicology 24 15ndash20
Hayes K R Vollrath A L Zastrow G M McMillan B J Craven M
Jovanovich S et al (2005) EDGE A centralized resource for the
comparison analysis and distribution of toxicogenomic information
Molecular Pharmacology 67 1360ndash1368
Heijne W H Slitt A L van Bladeren P J Groten J P Klaassen C D
Stierum R H et al (2004) Bromobenzene-induced hepatotoxicity at
the transcriptome level Toxicology Sciences 79 411ndash422
Heijne W H Stierum R H Slijper M van Bladeren P J amp van
Ommen B (2003) Toxicogenomics of bromobenzene hepatotoxicity
A combined transcriptomics and proteomics approach Biochemical
Pharmacology 65 857ndash875
Heinloth A N Irwin R D Boorman G A Nettesheim P Fannin
R D Sieber S O et al (2004) Gene expression profiling of rat
livers reveals indicators of potential adverse effects Toxicology
Sciences 80 193ndash202
Hodges L C Cook J D Lobenhofer E K Li L Bennett L Bushel
P R et al (2003) Tamoxifen functions as a molecular agonist inducing
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 63
cell cycle-associated genes in breast cancer cells Molecular Cancer
Research 1 300ndash311
Holleman A Cheok M H den Boer M L Yang W Veerman KM
A J Kazemier et al (2004) Gene-expression patterns in drug-resistant
acute lymphoblastic leukemia cells and response to treatment New
England Journal of Medicine 351 533ndash542
Hong Y Muller U R amp Lai F (2003) Discriminating two classes of
toxicants through expression analysis of HepG2 cells with DNA arrays
Toxicology in Vitro 17 85ndash92
Hood E (2003) Proteomics Characterizing the cogs in the machinery of
life Environmental Health Perspectives 111 A816ndashA825
Hood L (2003) Systems biology Integrating technology biology and
computation Mechanism of Ageing and Development 124 9ndash16
Hood L amp Galas D (2003) The digital code of DNA Nature 421
444ndash448
Hood L amp Perlmutter R M (2004) The impact of systems approaches on
biological problems in drug discovery Nature Biotechnology 22
1215ndash1217
Hu T Gibson D P Carr G J Torontali S M Tiesman J P Chaney
J G et al (2004) Identification of a gene expression profile that
discriminates indirect-acting genotoxins from direct-acting genotoxins
Mutation Research 549 5ndash27
Huang Q Dunn II R T Jayadev S DiSorbo O Pack F D Farr S B
et al (2001) Assessment of cisplatin-induced nephrotoxicity by
microarray technology Toxicology Sciences 63 196ndash207
Huang Q Jin X Gaillard E T Knight B L Pack F D Stoltz J H et
al (2004) Gene expression profiling reveals multiple toxicity endpoints
induced by hepatotoxicants Mutation Research 549 147ndash167
Huisman M T Smit J W Crommentuyn K M Zelcer N Wiltshire
H R Beijnen J H et al (2002) Multidrug resistance protein 2
(MRP2) transports HIV protease inhibitors and transport can be
enhanced by other drugs Aids 16 2295ndash2301
Hunt C M Westerkam W R amp Stave G M (1992) Effect of age and
gender on the activity of human hepatic CYP3A Biochemical
Pharmacology 44 275ndash283
Ideker T Ozier O Schwikowski B amp Siegal A F (2002) Discovering
regulatory and signalling circuits in molecular interaction networks
Bioinformatics 18 S233ndashS240
Ideker T Thorsson V Ranish J A Christmas R Buhler J Eng J K
et al (2001) Integrated genomic and proteomic analyses of a system-
atically perturbed network Science 292 929ndash934
Iida M Anna C H Hartis J Bruno M Wetmore B Dubin J R et al
(2003) Changes in global gene and protein expression during early
mouse liver carcinogenesis induced by non-genotoxic model carcino-
gens oxazepam and Wyeth-14643 Carcinogenesis 24 757ndash770
Ingelman-Sundberg M (2004) Pharmacogenetics of cytochrome P450 and
its applications in drug therapy The past present and future Trends in
Pharmacological Sciences 25 193ndash200
Jeong H Mason S P Barabasi A L amp Oltvai Z N (2001) Lethality
and centrality in protein networks Nature 411 41ndash42
Jeong H Tombor B Albert R Oltvai Z N amp Barabasi A L
(2000) The large-scale organization of metabolic networks Nature
407 651ndash654
Jung D Hagenbuch B Gresh L Pontoglio M Meier P J amp Kullak-
Ublick G A (2001) Characterization of the human OATP-C
(SLC21A6) gene promoter and regulation of liver-specific OATP genes
by hepatocyte nuclear factor 1 alpha Journal of Biological Chemistry
276 37206ndash37214
Jung D amp Kullak-Ublick G A (2003) Hepatocyte nuclear factor 1
alpha A key mediator of the effect of bile acids on gene expression
Hepatology 37 622ndash631
Jung J -W Park J -S Hwang J -W Kang K -S YS L Song B
-S et al (2004) Gene expression analysis of peroxisome prolifer-
ators- and phenytoin-induced hepatotoxicity using cDNA microarray
Journal of Veterinary Medical Science 66 1329ndash1333
Kato N Shibutani M Takagi H Uneyama C Lee K -Y Takiami S
et al (2004) Gene expression profile in the livers of rats orally
administered ethinylestradiol for 28 days using a microarray technique
Toxicology 200 179ndash192
Kier L D Neft R Tang L Suizu R Cook T Onsurez K et al
(2004) Applications of microarrays with toxicologically relevant genes
(tox genes) for the evaluation of chemical toxicants in Sprague Dawley
rats in vivo and human hepatocytes in vitro Mutation Research 549
101ndash113
Kijima K Toyosawa K Yasuba M Matsuoka N Adachi T
Komiyama M et al (2004) Gene expression analysis of the rat testis
after treatment with di(2-ethylhexyl) phthalate using cDNA microarray
and real-time RT-PCR Toxicology and Applied Pharmacology 200
103ndash110
Kim R B (2003) Organic anion-transporting polypeptide (OATP) trans-
porter family and drug disposition European Journal of Clinical
Investigation 33 1ndash5
Kinirons M T amp OrsquoMahony M S (2004) Drug metabolism and ageing
British Journal of Clinical Pharmacology 57 540ndash544
Kitano H (2002a) Computational systems biology Nature 420 206ndash210
Kitano H (2002b) Systems biology A brief overview Science 295
1662ndash1664
Kliewer S A Moore J T Wade L Staudinger J L Watson M
A Jones S A et al (1998) An orphan nuclear receptor activated
by pregnanes defines a novel steroid signalling pathway Cell 92
73ndash82
Korolev D Balakin K V Nikolsky Y Kirillov E Ivanenkov Y A
Savchuk N P et al (2003) Modeling of human cytochrome p450-
mediated drug metabolism using unsupervised machine learning
approach Journal of Medicinal Chemistry 46 3631ndash3643
Kramer J A Pettit S D Amin R P Bertram T A Car B
Cunningham M et al (2004) Overview on the application of
transcription profiling using selected nephrotoxicants for toxicology
assessment Environmental Health Perspectives 112 460ndash464
Kultima K Nystrom A -M Scholz B Gustafson A -L Dencker L
amp Stigson M (2004) Valproic acid teratogenicity A toxicogenomics
approach Environmental Health Perspectives 112 1225ndash1235
Kutuzova G D amp Deluca H F (2004) Gene expression profiles in rat
intestine identify pathways for 125-dihydroxyvitamin D(3) stimulated
calcium absorption and clarify its immunomodulatory properties
Archives of Biochemistry and Biophysics 432 152ndash166
Kwak M K Wakabayashi N Itoh K Motohashi H Yamamoto M amp
Kensler T W (2003) Modulation of gene expression by cancer
chemopreventive dithiolethiones through the Keap1-Nrf2 pathway
Identification of novel gene clusters for cell survival Journal of
Biological Chemistry 278 8135ndash8145
Lahjouji K Mitchell G A amp Qureshi I A (2001) Carnitine transport
by organic cation transporters and systemic carnitine deficiency
Molecular Genetics and Metabolism 73 287ndash297
Landowski C P Sun D Foster D R Menon S S Barnett J L
Welage L S et al (2003) Gene expression in the human intestine and
correlation with oral valacyclovir pharmacokinetic parameters Journal
of Pharmacology and Experimental Therapeutics 306 778ndash786
Lee G -J Lee W -S Jeon K -S Um C -H Kim S -J Lee C -H
et al (2004) cDNA microarray gene expression analysis and
toxicological phenotype for anticancer drug Journal of Veterinary
Medical Science 66 1339ndash1345
Lee M Kwon J Kim S N Kim J E Koh W S Kim E J et al
(2003) cDNA microarray gene expression profiling of hydroxyurea
paclitaxel and p-anisidine genotoxic compounds with differing
tumorigenicity results Environmental and Molecular Mutagenesis
42 91ndash97
Li S Armstrong C M Bertin N Ge H Milstein S Boxem M et al
(2004) A map of the interactome network of the metazoan C elegans
Science 303 540ndash543
Liguori M J Anderson L M Bukofzer S McKim J Pregenzer J F
Retief J et al (2005) Microarray analysis in human hepatocytes
suggests a mechanism for hepatotoxicity induced by trovafloxacin
Hepatology 41 177ndash186
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6664
Lobenhofer E K Bennett L Cable P L Li L Bushel P R amp Afshari
C A (2002) Regulation of DNA replication fork genes by 17beta-
estradiol Molecular Endocrinology 16 1215ndash1229
Locker J Tian J Carver R Concas D Cossu C Ledda-Columbano
G M et al (2003) A common set of immediate-early response genes
in liver regeneration and hyperplasia Hepatology 38 314ndash325
Mattes W B Pettit S D Sansone S A Bushel P R amp Waters M D
(2004) Database development in toxicogenomics Issues and efforts
Environmental Health Perspectives 112 495ndash505
McMillian M Nie A Y Parker J B Leone A Bryant S Kemmerer
M et al (2004) A gene expression signature for oxidant stressreactive
metabolites in rat liver Biochemical Pharmacology 68 2249ndash2261
Meier P J amp Stieger B (2000) Molecular mechanisms in bile formation
News in Physiological Sciences 15 89ndash93
Meneses-Lorente G de Longueville F Dos Santos-Mendes S Bonnert
T P Jack A Evrard S et al (2003) An evaluation of a low-density
DNA microarray using cytochrome P450 inducers Chemical Research
in Toxicology 16 1070ndash1077
Meneses-Lorente G Guest P C Lawrence J Muniappa N Knowles
M R Skynner H A et al (2004) A proteomic investigation of drug-
induced steatosis in rat liver Chemical Research in Toxicology 17
605ndash612
Merrill C L Ni H Yoon L W Tirmenstein M A Narayanan P
Benavides G R et al (2002) Etomoxir-induced oxidative stress in
HepG2 cells detected by differential gene expression is confirmed
biochemically Toxicology Sciences 68 93ndash101
Milo R Shen-Orr S Itzkovitz S Kashtan N Chklovskii D amp Alon
U (2002) Network motifs Simple building blocks of complex
networks Science 298 824ndash827
Morgan K T Ni H Brown H R Yoon L Qualls C W Crosby L
M et al (2002) Application of cDNA microarray technology to in
vitro toxicology and the selection of genes for a real-time RT-PCR-
based screen for oxidative stress in Hep-G2 cells Toxicologic
Pathology 30 435ndash451
Mwinyi J Johne A Bauer S Roots I amp Gerloff T (2004) Evidence
for inverse effects of OATP-C (SLC21A6) 5 and 1b haplotypes on
pravastatin kinetics Clinical Pharmacology and Therapeutics 75
415ndash421
Nakata K Yukawa M Komiyama N Nakano T amp Kaminuma T
(2002) A nuclear receptor database that maps pathways to diseases
Genome Infomatics 13 515ndash516
Nicholson J K Holmes E Lindon J C amp Wilson I D (2004) The
challenges of modeling mammalian biocomplexity Nature Biotechnol-
ogy 22 1268ndash1274
Nicholson J K amp Wilson I D (2003) Understanding rsquoglobal systems
biology Metabonomics and the continuum of metabolism Nature
Reviews on Drug Discovery 2 668ndash676
Nikitin A Egorov S Daraselia N amp Mazo I (2003) Pathway studiomdash
the analysis and navigation of molecular networks Bioinformatics 19
2155ndash2157
Nikolsky Y Ekins S Nikolskaya T amp Bugrim A (2005) A novel
method for generation of signature networks as biomarkers from
complex high throughput data Toxicology Letters 158 20ndash29
Nikolsky Y Nikolskaya T amp Bugrim A (2005) Biological networks
and analysis of experimental data in drug discovery Drug Discovery
Today 10 653ndash662
Nishizato Y Ieiri I Suzuki H Kimura M Kawabata K Hirota T
et al (2003) Polymorphisms of OATP-C (SLC21A6) and OAT3
(SLC22A8) genes Consequences for pravastatin pharmacokinetics
Clinical Pharmacology and Therapeutics 73 554ndash565
Nozawa T Nakajima M Tamai I Noda K Nezu J Sai Y et al
(2002) Genetic polymorphisms of human organic anion transporters
OATP-C (SLC21A6) and OATP-B (SLC21A9) Allele frequencies in
the Japanese population and functional analysis Journal of Pharmacol-
ogy and Experimental Therapeutics 302 804ndash813
Nozawa T Sugiura S Nakajima M Goto A Yokoi T Nezu J et al
(2004) Involvement of organic anion transporting polypeptides in the
transport of troglitazone sulfate Implications for understanding trogli-
tazone hepatotoxicity Drug Metabolism and Disposition 32 291ndash294
Oliver D E Rubin D L Stuart J M Hewett M Klein T E amp
Altman R B (2002) Ontology development for a pharmacogenetics
knowledge base Package Symposion 88ndash99
Oram J F amp Lawn R M (2001) ABCA1 The gatekeeper for eliminating
excess tissue cholesterol Journal of Lipid Research 42 1173ndash1179
Oswald M Kullak-Ublick G A Paumgartner G amp Beuers U (2001)
Expression of hepatic transporters OATP-C and MRP2 in primary
sclerosing cholangitis Liver 21 247ndash253
Parsons A B Brost R I Ding H Li Z Zhang C Sheikh B et al
(2004) Integration of chemical-genetic and genetic interaction data
links bioactive compounds to cellular target pathways Nature Bio-
technology 22 62ndash69
Patki K C von Moltke L L Harmatz J S Hesse L M Court M H
amp Greenblatt D J (2004) Effect of age on in vitro triazolam
biotransformation in male human liver microsomes Journal of
Pharmacology and Experimental Therapeutics 308 874ndash879
Pereira-Leal J B Enright A J amp Ouzounis C A (2004) Detection of
functional modules from protein interaction networks Proteins 54
49ndash57
Plant N (2004) Interaction networks Coordinating responses to xeno-
biotic exposure Toxicology 202 21ndash32
Raghavendra Prasad H S Qi Z Srinivasan K N amp Gopalakrish-
nakone P (2004) Potential effects of tetrodotoxin exposure to
human glial cells postulated using microarray approach Toxicon 44
597ndash608
Reilly T P Bourdi M Brady J N Pise-Masison C A Radonovich M
F George J W et al (2001) Expression profiling of acetaminophen
liver toxicity in mice using microarray technology Biochemical and
Biophysical Research Communications 282 321ndash328
Rives A W amp Galitski T (2003) Modular organization of cellular
networks Proceedings of the National Academy of Sciences of the
United States of America 100 1128ndash1133
Rogers P D Pearson M M Cleary J D Sullivan D C amp Chapman
S W (2002) Differential expression of genes encoding immunomodu-
latory proteins in response to amphotericin B in human mononuclear
cells identified by cDNA microarray analysis Journal of Antimicrobial
Chemotherapy 50 811ndash817
Rosen M B Wilson V S Schmid J E amp Gray L E (2005) Gene
expression analysis in the ventral prostate of rats exposed to vinclozolin
or procymidone Reproductive Toxicology 19 367ndash379
Rost D Herrmann T Sauer P Schmidts H L Stieger B Meier P J
et al (2003) Regulation of rat organic anion transporters in bile salt-
induced cholestatic hepatitis Effect of ursodeoxycholate Hepatology
38 187ndash195
Rothman R B Ayestas M A Dersch C M amp Baumann M H (1999)
Aminorex fenfluramine and chlorphentermine are serotonin transporter
substrates Implications for primary pulmonary hypertension Circula-
tion 100 869ndash875
Ruepp S U Tonge R P Shaw J Wallis N amp Pognan F (2002)
Genomics and proteomics analysis of acetaminophen toxicity in mouse
liver Toxicology Sciences 65 135ndash150
Sakaeda T Nakamura T amp Okumura K (2002) MDR1 genotype-
related pharmacokinetics and pharmacodynamics Biological amp Phar-
maceutical Bulletin 25 1391ndash1400
Sakata T Anzai N Shin H J Noshiro R Hirata T Yokoyama H
et al (2004) Novel single nucleotide polymorphisms of organic cation
transporter 1 (SLC22A1) affecting transport functions Biochemical
and Biophysical Research Communications 313 789ndash793
Satlin L M Amin V amp Wolkoff A W (1997) Organic anion
transporting polypeptide mediates organic anionHCO3-exchange
Journal of Biological Chemistry 272 26340ndash26345
Satomi Y Tsuchiya W Mihara K Ota M Kasahara Y amp Akahori F
(2004) Gene expression analysis of the lung following paraquat
administration in rats using DNA microarray Journal of Toxicological
Sciences 29 91ndash100
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 65
Schrenk D Baus P R Ermel N Klein C Vorderstemann B amp
Kauffmann H M (2001) Up-regulation of transporters of the MRP
family by drugs and toxins Toxicology Letters 120 51ndash57
Segal E Shapira M Regev A Persquoer D Botstein D Koller D et al
(2003) Module networks Identifying regulatory modules and their
condition-specific regulators from gene expression data Nature
Genetics 34 166ndash176
Segal E Wang H amp Koller D (2003) Discovering molecular pathways
from protein interaction and gene expression Bioinformatics 19
i264ndash i272
Seidel S D Kan H L Stott W T Schisler M R amp Gollapudi B B
(2003) Identification of transcriptome profiles for the DNA-damaging
agents bleomycin and hydrogen peroxide in L5178Y mouse lymphoma
cells Environmental and Molecular Mutagenesis 42 19ndash25
Shan L Yu Y Schut H A J amp Snyderwine E G (2004) Susceptibility
of rats to mammary gland carcinogenesis by the food-derived
carcinogen 2-Amino-1-Methyl-6-Phenylimidazo[45-b]Pyridine (PhIP)
varies with age and is associated with the induction of differential gene
expression American Journal of Pathology 165 191ndash202
Sharom J R Bellows D S amp Tyers M (2004) From large networks to
small molecules Current Opinion in Chemical Biology 8 81ndash90
Shitara Y Hirano M Sato H amp Sugiyama Y (2004) Gemfibrozil and
its glucuronide inhibit the OATP2(OATP1B1 SLC21A6)-mediated
hepatic uptake and CYP2C8-mediated metabolism of cerivastatinmdash
Analysis of the mechanism of the clinically relevant drugndashdrug
interaction between cerivastatin and gemfibrozil Journal of Pharma-
cology and Experimental Therapeutics
Shitara Y Itoh T Sato H Li A P amp Sugiyama Y (2003) Inhibition of
transporter-mediated hepatic uptake as a mechanism for drugndashdrug
interaction between cerivastatin and cyclosporin A Journal of
Pharmacology and Experimental Therapeutics 304 610ndash616
Somogyi R Fuhrman S amp Wen X (2001) Genetic network inference in
computational models and applications to large-scale gene expression
data In M Bower amp H Bolouri (Eds) Computational modeling of
genetic and biochemical networks (pp 119ndash157) Cambridge MArsquoThe
MIT Press
Spirin V amp Mirny L A (2003) Protein complexes and functional
modules in molecular networks Proceedings of the National Academy
of Sciences of the United States of America 100 12123ndash12128
Staudinger J Liu Y Madan A Habeebu S amp Klaassen C D
(2001) Coordinate regulation of xenobiotic and bile acid homeo-
stasis by pregnane X receptor Drug Metabolism and Disposition
29 1467ndash1472
Sun L Z Ji Z L Chen X Wang J F amp Chen Y Z (2002) ADME-
AP A database of ADME associated proteins Bioinformatics 18
1699ndash1700
Suter L Babiss L E amp Wheeldon E B (2004) Toxicogenomics in
predictive toxicology in drug development Chemistry and Biology 11
161ndash171
Synold T W Dussault I amp Forman B M (2001) The orphan nuclear
receptor SXR coordinately regulates drug metabolism and efflux
Nature Medicine 7 584ndash590
Tabuchi Y amp Kondo T (2003) cDNA microarray analysis reveals chop-
10 plays a key role in Sertoli cell injury induced by bisphenol A
Biochemical and Biophysical Research Communications 305 54ndash61
Tamai I Nezu J Uchino H Sai Y Oku A Shimane M et al (2000)
Molecular identification and characterization of novel members of the
human organic anion transporter (OATP) family Biochemical and
Biophysical Research Communications 273 251ndash260
Taylor S W Fahy E Zhang B Glenn G M Warnock D E Wiley S
et al (2003) Characterization of the human heart mitochondrial
proteome Nature Biotechnology 21 281ndash286
Terasaka S Aita Y Inoue A Hayashi S Nishigaki M Aoyagi K
et al (2004) Using a customized DNA microarray for expression
profiling of the estrogen-responsive genes to evaluate estrogen activity
among natural estrogens and industrial chemicals Environmental
Health Perspectives 112 773ndash781
Thomas D M Francescutti-Verbeem D M Liu X amp Kuhn D M
(2004) Identification of differentially regulated transcripts in mouse
striatum following methamphetamine treatmentmdashan oligonucleotide
microarray approach Journal of Neurochemistry 88 380ndash393
Thomas R S Rank D R Penn S G Zastrow G M Hayes K R
Pande K et al (2001) Identification of toxicologically predictive
gene sets using cDNA microarrays Molecular Pharmacology 60
1189ndash1194
Thompson K L Afshari C A Amin R P Bertram T A Car B
Cunningham M et al (2004) Identification of platform-independent
gene expression markers of cisplatin nephrotoxicity Environmental
Health Perspectives 112 488ndash494
Tirona R G Leake B F Merino G amp Kim R B (2001)
Polymorphisms in OATP-C Identification of multiple allelic
variants associated with altered transport activity among European-
and African-Americans Journal of Biological Chemistry 276
35669ndash35675
Tirona R G Leake B F Wolkoff A W amp Kim R B (2003)
Human organic anion transporting polypeptide-C (SLC21A6) is a
major determinant of the rifampin-mediated pregnane X receptor
activation Journal of Pharmacology and Experimental Therapeutics
304 223ndash228
Tong W Cao X Harris S Sun H Fang H Fuscoe J et al (2003)
ArrayTrackmdashsupporting toxicogenomic research at the US Food and
Drug Administration National Center for Toxicological Research
Environmental Health Perspectives 111 1819ndash1826
Tornow S amp Mewes H W (2003) Functional modules by relating
protein interaction networks and gene expression Nucleic Acids
Research 31 6283ndash6289
Trocho C Escola-Gil J C Ribas V Benitez S Martin-Campos J M
Rotllan N et al (2004) Phenytoin treatment reduces atherosclerosis in
mice through mechanisms independent of plasma HDL-cholesterol
concentration Atherosclerosis 174 275ndash285
Ueda A Hamadeh H K Webb H K Yamamoto Y Sueyoshi T
Afshari C A et al (2002) Diverse roles of the nuclear orphan
receptor CAR in regulating hepatic genes in response to phenobarbital
Molecular Pharmacology 61 1ndash6
Ulrich R G (2003) The toxicogenomics of nuclear receptor agonists
Current Opinion in Chemical Biology 7 505ndash510
Ulrich R G Rockett J C Gibson G G amp Pettit S D (2004)
Overview of an interlaboratory collaboration on evaluating the effects
of model hepatotoxicants on hepatic gene expression Environmental
Health Perspectives 112 423ndash427
Unami A Shinohara Y Ichikawa T amp Baba Y (2003) Biochemical
and microarray analyses of bupivacaine-induced apoptosis Journal of
Toxicological Sciences 28 77ndash94
Vasquez A Flammini A Maritan A amp Vespignani A (2003) Global
protein function prediction from proteinndashprotein interaction networks
Nature Biotechnology 21 697ndash700
Vezina C M Walker N J amp Olson J R (2004) Subchronic Exposure to
TCDD PeCDF PCB126 and PCB153 Effect on hepatic gene
expression Environmental Health Perspectives 112 1636ndash1644
Vo T D Greenberg H J amp Palsson B O (2004) Reconstruction and
functional characterization of the human mitochondrial metabolic
network based on proteomic and biochemical data Journal of Bio-
logical Chemistry 279 39532ndash39540
Wang H amp LeCluyse E L (2003) Role of orphan nuclear receptors in
the regulation of drug-metabolising enzymes Clinical Pharmacoki-
netics 42 1331ndash1357
Wang N Silver D L Thiele C amp Tall A R (2001) ATP-
binding cassette transporter A1 (ABCA1) functions as a choles-
terol efflux regulatory protein Journal of Biological Chemistry 276
23742ndash23747
Waring J F Cavet G Jolly R A McDowell J Dai H Ciurlionis R
et al (2003) Development of a DNA Microarray for toxicology based
on hepatoxin-regulated sequences Environmental Health Perspectives
111 863ndash870
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6666
Waring J F Gum R Morfitt D Jolly R A Ciurlionis R Heindel M
et al (2002) Identifying toxic mechanisms using DNA microarrays
Evidence that an experimental inhibitor of cell adhesion molecule
expression signals through the aryl hydrocarbon nuclear receptor
Toxicology 181ndash182 537ndash550
Waring J F Jolly R A Ciurlionis R Lum P Y Praestgaard J T
Morfitt D C et al (2001) Clustering of hepatotoxins based on
mechanism of toxicity using gene expression profiles Toxicology and
Applied Pharmacology 175 28ndash42
Waters M Boorman G Bushel P Cunningham M Irwin R Merrick
A et al (2003) Systems toxicology and the Chemical Effects in
Biological Systems (CEBS) knowledge base EHP Toxicogenomics
111 15ndash28
Wei M Arnold L Cano M amp Cohen S M (2005) Effects of co-
administration of antioxidants and arsenicals on the rat urinary bladder
epithelium Toxicology Sciences 83 237ndash245
Weinshilboum R (2003) Inheritance and drug response New England
Journal of Medicine 348 529ndash537
Weinshilboum R amp Wang L (2004) Pharmacogenetics Inherited
variation in amino acid sequence and altered protein quantity Clinical
Pharmacology and Therapeutics 75 253ndash258
Werner E (2003) In silico multicellular systems biology and minimal
genomes Drug Discovery Today 8 1121ndash1127
Wong J S amp Gill S S (2002) Gene expression changes induced in
mouse liver by di(2-ethylhexyl) phthalate Toxicology and Applied
Pharmacology 185 180ndash196
Xie T Tong L McCann U D Yuan J Becker K G Mechan A O
et al (2004) Identification and characterization of metallothionein-1
and -2 gene expression in the context of ()34-methylenedioxymetham-
phetamine-induced toxicity to brain dopaminergic neurons Journal of
Neuroscience 24 7043ndash7050
Yadetie F Laegreid A Bakke I Kusnierczyk W Komorowski J
Waldum H L et al (2003) Liver gene expression in rats in response
to the peroxisome proliferator-activated receptor-alpha agonist ciprofi-
brate Physiological Genomics 15 9ndash19
Yamashita S Nomoto T Abe M Tatematsu M Sugimura T amp
Ushijima T (2004) Persistence of gene expression changes in stomach
mucosae induced by short-term N-methyl-NV-nitro-N-nitrosoguanidinetreatment and their presence in stomach cancers Mutation Research
549 185ndash193
Yan Q amp Sadee W (2000) Human membrane transporter database A
web-accessible relational database for drug transport studies and
pharmacogenomics AAPS Pharmsci 2 E20
Yeger-Lotem E amp Margalit H (2003) Detection of regulatory circuits by
integrating the cellular networks of proteinndashprotein interactions and
transcription regulation Nucleic Acids Research 31 6053ndash6061
Yoon B I Li G -X Kitada K Kawasaki Y Igarashi K amp Kodama
Y et al (2003) Mechanisms of benzene-induced hematotoxicity and
leukemogenicity cDNA microarray analyses using mouse bone marrow
tissue Environmental Health Perspectives 111 1411ndash1420
Young M B DiSilvestro M R Sendera T J Freund J Kriete A amp
Magnuson S R (2003) Analysis of gene expression in carbon
tetrachloride-treated rat livers using a novel bioarray technology
Pharmacogenomics Journal 3 41ndash52
Yu H Zhu X Greenbaum D Karro J amp Gerstein M (2004) TopNet
A tool for comparing biological sub-networks correlating protein
properties with topological statistics Nucleic Acids Research 32
328ndash337
Zhang E Y Fu D J Pak Y A Stewart T Mukhopadhyay N
Wrighton S A et al (2004) Genetic polymorphisms in human proton-
dependent dipeptide transporter PEPT1 Implications for the functional
role of Pro586 Journal of Pharmacology and Experimental Therapeu-
tics 310 437ndash445
Zhang E Y Knipp G T Ekins S amp Swaan P W (2002)
Structural biology and function of solute transporters Implications
for identifying and designing substrates Drug Metabolism Reviews
34 709ndash750
Zhang E Y Phelps M A Cheng C Ekins S amp Swaan P W (2002)
Modeling of active transport systems Advanced Drug Delivery
Reviews 54 329ndash354
- Systems-ADMETox Resources and network approaches
-
- Introduction
- Data available
- Network analysis and databases
-
- Network applications
-
- The role of transporters
-
- Clinical relevance of transporters
- Transporter network examples ABCA1
- Transporter network examples OATP
- Transporter microarray data
-
- Applications to enzymes
- Future network applications
-
- Discussion
- Acknowledgements
- References
-
Table 2 (continued)
Compounds Mouse strain Microarray type Compound dose Microarray data
availability
Reference
Acetaminophen C57B16 3 129Ola
hybrid
Test-2 Chips (Affymetrix)
then individual
oligonucleotide
microarrays (Mul1K sub
A and sub B Affymetrix)
that can detect the
expression of 11000
known genes and
expressed sequence tags
(ESTs)
300 mgkg Genbank or SwissProt
ID and fold change data
in a manuscript table
(Reilly et al
2001)
Cadmium chloride
benzo(a)pyrene (BaP)
and trichloroethylene
(TCE)
Male Swiss Webster Custom Chips
containing 148 unique
genes
Various Data in table form few
genes affected for BAP
and TCE
(Bartosiewicz
Penn amp Buckpitt
2001)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 45
been only a very small number of studies that have
combined both transcriptomic and proteomic methods with
a single animal strain after treatment with a drug Hopefully
we will see this change in the future but this will in turn
present considerable challenges as huge amounts of
proteomic data are combined with the equally large
transcript data files
3 Network analysis and databases
From some of the early reviews of systems biology there
has been discussion of its application to drug discovery
(Kitano 2002ab) as well as the utility for ADMETox
(Ekins et al 2002a 2000b) More recently several other
journals have dedicated whole issues to the field of systems
biology However one could consider this quite a broad field
from network or pathway analysis to quantitative simulation
of organelles (Vo Greenberg amp Palsson 2004) whole cells
and organs It is apparent that we are now understanding
organisms from the perspective of computationally gener-
ated networks of protein and ligand interactions (Barabasi amp
Oltvai 2004) Network and pathway tools enable the
analysis of HT data in the context of all known interactions
when using a database as the source Individual reviews
have in some cases indicated that networks will be valuable
for understanding adverse events (Hood amp Perlmutter
2004) drug target identification or validation (Butcher
Berg amp Kunkel 2004) and complex metabolic interactions
(Nicholson et al 2004) A general schematic has been
generated in order to provide a description of the utilization
of such pathway databases and network building algorithms
from the initial parsing of high throughput data to network
comparisons and visualization (Fig 3) High throughput
data can be superimposed and visualized on the various
protein interaction databases available This is accomplished
by using either preset maps that capture current biological
knowledge or by building custom interaction networks
using many different algorithms which can be compared and
statistically evaluated as demonstrated in a very large
number of published examples (Dobrin Beg Karabasi amp
Oltvai 2004 Fiehn 2001 Han et al 2004 Hanisch et al
2002 Ideker et al 2002 Jeong Mason Barabasi amp Oltvai
2001 Jeong Tombor Albert Oltvai amp Barabasi 2000 Li
et al 2004 Milo et al 2002 Nikitin Egorov Daraselia amp
Mazo 2003 Pereira-Leal Enright amp Ouzounis 2004
Rives amp Galitski 2003 Segal et al 2003a Somogyi et al
2001 Spirin amp Mirny 2003 Tornow amp Mewes 2003
Vasquez Flammini Maritan amp Vespignani 2003 Yeger-
Lotem amp Margalit 2003 Yu Zhu Greenbaum Karro amp
Gerstein 2004)
31 Network applications
For example one group has used as an inference the
Bayesian network method for analysis of tissue toxicity from
microarray data as well as a mechanistic simulation for a
different pharmaceutically relevant molecule (Aksenov et
al 2005) Pathway tools and various resources have also
been applied to modeling the networks of nuclear hormone
receptors and their connections with other genes and small
molecules using a manually curated database MetaDrug
(Ekins Kirillov Rakmatulin amp Nikolskaya 2005d) or
MetaCore (Ekins Bugrim Nikolsky amp Nikolskaya 2005)
Transcriptional regulation of many transporters CYPs and
phase II enzymes are regulated by these receptors affecting
endogenous molecule transport metabolism cell growth
proliferation and oxidative stress (Ulrich 2003 Ulrich et al
2004) When the signaling networks and interacting ligands
for the transcriptional factors PPAR FXRRXRA ESR1
AHR HNF4A GCR-h MCR CAR-beta GCR-a LXR-a
CARRXR HNF4 FXR PXRRXR heterodimer PXR
AHRARNT heterodimer PPARaLXRa VDR PPAR-a
are visualized a very complex picture of interactions can be
created (Ekins et al 2005d) This suggests that when we
consider a molecule binding with only one nuclear receptor
we are observing only a fraction of the likely possible
feasible interactions based on the data gathered to date
Table 3
Literature toxicogenomics data derived from in vitro cell studies
Compounds Cell type Microarray type Compound dose Microarray data
availability
Reference
4-Hydroxytamoxifen
estrogen
MCF-7 breast cancer NIEHS ToxChip
microarray consisting of
1901 genes
1 uM hydroxytamoxifen
for a year 10 nM
17b-estradiol
Data available at NIEHS
website
(Hodges et al
2003)
Trovafloxacin Human hepatocytes Affymetrix U133A array 30ndash800 uM 142 genes available in
supplemental tablemdashnot
easily extracted
(Liguori et al
2005)
Estrogen MCF-7 breast cancer NIEHS ToxChip
microarray consisting of
1901 genes
10ndash10 M 17b-estradiol Data available at NIEHS
website
(Lobenhofer et al
2002)
Valproic acid NMRI mice embryo
and P19 mouse
embryocarcinoma
Custom chip including
15K mouse cDNA clone
set
600 mgkg body weight Gene symbol gene
name NIA EST log fold
change in a manuscript
table
(Kultima et al
2004)
Sulindac sulfide Human colorectal
carcinoma SW-480
and HCT-116
NIEHS human 12K chip 10 uM Genbank accession
number gene name and
fold change at various
time points in a
manuscript table data
also available on NIEHS
website
(Bottone
Martinez Collins
Afshari amp Eling
2003)
17Beta-estradiol estriol
estrone genistein
diethylstilbestrol
bisphenol A
nonylphenol
methoxychlor
MCF-7 U95A oligonucleotide
probe arrays (Affymetrix
10 nM (E2 estriol
estrone DES) 10 AM(genistein bisphenol A
nonylphenol and
methoxychlor)
Unigene name gene
name and fold change
for estrogen responsive
and nonresponsive in a
manuscript table
(Terasaka et al
2004)
Ouabain lauryl sulfate
dimethylsulfoxide
cycloheximide
tolbutamide sodium
fluoride diethyl
maleate buthionine
sulfoxamine
potassium bromate
sodium selenite
alloxan adriamycin
hydrogen peroxide
HepG2 Clontech Atlas Human
Stress Toxicology cDNA
arrays (234 genes)
Ouabain (43 uM) lauryl
sulfate (260 uM)
dimethylsulfoxide (128
M) cycloheximide (625
uM) tolbutamide (128
mM) sodium fluoride (3
mM) diethyl maleate
(125 mM) buthionine
sulfoxamine (30 mM)
potassium bromate (25
mM) sodium selenite (30
uM) alloxan (130 mM)
adriamycin (40 uM)
hydrogen peroxide
(4 mM)
Gene name ratio p -
value in downloadable
tables at journal website
(Morgan et a l
2002)
Aflatoxin B(1) (AFB(1))
2-acetylaminofluorene
(2AAF)
dimethylnitrosamine
(DMN)
acetaminophen (APAP)
HepG2 and primary
hepatocytes
Gene filter arrays
containing 31000 genes
10 uM aflatoxin B1
40 mM acetaminophen
100 uM
dimethylnitrosamine
10 uM
2-acetylaminofluorene
None (Harris et al
2004)
Mitomycin C (MMC)
and cisplatin (CIS)
and an alkylating
agent methyl
methanesulfonate
(MMS)
indirect-acting
genotoxins included
hydroxyurea (HU) a
ribonucleotide
reductase inhibitor
taxol (TXL) a
microtubule inhibitor
and etoposide
(ETOP)
L5178Y TK(+-)
mouse lymphoma
Affymetrix mouse
MG-U74A for MMC
and MG-U745Av2
(Affymetrix Inc Santa
Clara CA) for all the
other chemicals a total
of 9977 probe sets
(genes or ESTs) common
to these two array
models
Low mid and high doses Accession number gene
name gene ID statistical
significance at each time
point in a manuscript
table
(Hu et al 2004)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6646
Table 3 (continued)
Compounds Cell type Microarray type Compound dose Microarray data
availability
Reference
Hydroxyurea
(a carcinogen)
p-anisidine
(a noncarcinogen)
and paclitaxel
L5178Y Tk_-mouse
lymphoma
The Twin - Chip Mouse-
74K Digital Genomics
cDNA microarray
10 ngml paclitaxel 313
ugml hydroxyurea 32
ugml p-anisidine
Gene symbol and fold
change in a manuscript
table
(Lee et al 2003)
Acetaminophen
amiodarone clofibrate
erythromycin estolate
isoniazid alpha-
naphtylylisothiocyanate
beta-naphtoflavone
4-pentenoic acid
phenobarbital
tetracycline and
zileuton
Wistar Rat
hepatocytes
DualChip rat hepato
(Eppendorf Hamburg
Germany)
A single concentration
which varied for each
compound
Accession numbers and
gene namemdashfold
changes shown as
colored heat mapmdashnote
easily extracted from
publication
(de Longueville
et al 2003)
Bleomycin and hydrogen
peroxide
Mouse lymphoma
L5178Y TK(+ -)
Clontech Mouse 12K
cDNA microarray (1185
genes)
Bleomycin (25 and 20
ugml) hydrogen peroxide
(5 and 10 ugml)
Gene names and fold
change presented as bar
charts in publication
(Seidel Kan
Stott Schisler amp
Gollapudi 2003)
Bupivicaine
camptothecin
HL-60 Agilent human cDNA
microarray
1 mM Gene name GenBank
accession number
unigene and ratio in a
manuscript table
(Unami
Shinohara
Ichikawa amp Baba
2003)
Bisphenol A Mouse Sertoli TTE3 IntelliGene mouse
expression glass
microarrays (Version
10 Takara Shuzo)
which were spotted with
564 cDNA fragments of
mouse known genes and
approximately 301
expressed sequence tags
(ESTs)
0ndash400 uM Gene name GenBank
accession number and
fold change at time
points in a manuscript
table
(Tabuchi amp
Kondo 2003)
Mitomycin C or
doxorubicin
Hep G2 85 human gene custom
array
10 um mitomycin C
2 um doxorubicin or
2 ethanol
Bar charts with fold
changes in publication
very few genes
(Hong Muller amp
Lai 2003)
Amphotericin B Human peripheral
blood mononuclear
and THP-1
GF211 FKnown Genes_
Genefilter cDNA array
(ResGen) this array
consists of gt4000
individual elements
each representing a
known human gene
5 ugml Accession numbers and
fold expression in a
manuscript table
(Cleary Rogers amp
Chapman 2001
Rogers Pearson
Cleary Sullivan
amp Chapman
2002)
Benzo(a)pyrene diol
epoxide
TK6 human
lymphoblastoid
Human-350 microarray
a glass slide with 350
spotted human cDNA
probes (Phase-1
Molecular Toxicology
0 001 010 or
10 ugml)
Gene names and fold
change at doses in a
manuscript table
(Akerman et al
2004)
Etomoxir HepG2 Clontech AtlasiHuman Stress
Toxicology cDNA arrays
(234 genes)
1 mM etomoxir Gene names and fold
change in a manuscript
table
(Merrill et al
2002)
Tetrodotoxin Human glioma cell
line HTB-138
Using Affymetrix
GeneChip (HG-U133A
10 and 20 mM Affymetrix ID Genbank
ID gene name gene
symbol and fold change
in a manuscript table
(Raghavendra
Prasad Qi
Srinivasan amp
Gopalakrishnakone
2004)
Methotrexate
mercaptopurine
Human acute
lymphoblastic
leukemia
Affymetrix U133A chip Low and high dose and
combination
Data available as
supplemental data online
(Cheok et al 2003)
Prednisolone
vincristine
asparaginase
daunorubicin
Human acute
lymphoblastic
leukemia
Affymetrix U133A chip Various Data available as
supplemental data online
(Holleman et al
2004)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 47
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6648
A second study has indicated how a natural language
processing method CCNet was used to show the genes
regulated by the nuclear hormone receptor FXR (Apic
Ignjatovic Boyer amp Russell 2005) These automated
methods enable a more complete understanding of the
complexity of the transcriptional factors (Ekins Mirny amp
Schueltz 2002b Plant 2004 Ulrich 2003) but ultimately
rely on the quality of the content of the underlying database
of literature interactions This is a key consideration that is
often overlooked For example the gold standard database is
one that is manually curated to ensure the fidelity of the
direct interaction and is preferable to one generated
computationally by algorithms like natural language pro-
cessing (Nikolsky Nikolskaya amp Bugrim 2005) The
advantage of interaction networks over clustering has been
demonstrated in one study using MetaCore (Nikolsky
Ekins Nikolskaya amp Bugrim 2005) by reanalysis of a
published microarray study of G0-arrested MCF-7 breast
cancer cells treated with estrogen and 4-hydroxytamoxifen
(Hodges et al 2003) After producing integrated gene
networks for each treatment strikingly different patterns
were displayed although both contained early transcriptional
factors myc jun and fos Only the estrogen network featured
induced genes essential for all cell cycle phases (Nikolsky et
al 2005) Similarly microarray data for benzene toxicity
(Yoon et al 2003) has been re-analyzed by focusing on the
genes assessed on the p53 pathway (Ekins et al 2005e) We
envisage that a database of such networks for toxic
compounds will be used for comparing between different
molecules and used in the development of predictive
algorithms for Systems-ADMETox modeling in future
Another approach to using such pathway approaches is to
visualize the results of quantitative structure activity models
for predicting molecules binding to enzymes transporters
receptors and ion channels (Ekins Andreyev et al in press
2005e)
It is also possible to simultaneously interpret high
throughput data and predictions on interaction networks
providing a novel approach to predicting and understanding
potential undesirable drugndashdrug or off target effects in the
area of systems pharmacology An example data set uses
percent inhibition data for clotrimazole and ticonazole
which were screened against many different assays at a
single concentration in a commercially available database
BioPrint (Cerep Redmond WA) as published recently
(Fliri Loging Thadejo amp VOlkman 2005) The data for
10 assays has been arbitrarily encoded as inhibitors (gt50
inhibition) or non-inhibitors (lt50 inhibition) in a text file
which was then loaded into MetaCore The analyzed
network algorithm was then used which generates a large
network and fragments it into sub-networks each with a Z-
score and p-values for ranking according to saturation with
objects from the initial gene list The Gene-ontology
processes are also mapped to the gene list and individual
networks In this example a statistically significant network
was generated for the different proteins (Fig 4
p =2838e31) This network also maps the Gene Ontology
processes for the activation of MAPK (118 of genes p
value 9143e07) signal transduction (333 1600e05)
regulation of transcription DNA-dependent (294
2786e04) regulation of inflammatory response (39
3746e04) and the regulation of blood pressure (78
4230e04) This example network indicates how molecules
of the same or different therapeutic classes could be
evaluated for their effects as a graph either together as in
this case or individually This would be useful to indicate
potential off target effects and identify structurally dissimilar
molecules with similar network patterns Such networks
could then be compared to assess network overlap or
differences between molecules and their inhibition of
multiple proteins This type of unique visualization of high
throughput screening data illustrates how the target proteins
may be connected as a network to infer the possible
downstream effects of inhibition
With the burgeoning number of freely available online
and commercial databases that can be used for pathway
construction numbering in the hundreds there have been
suggestions to impose standards for model exchange
querying and visualization (Cary Bader amp Sander 2005)
To date there has been little discussion with regards to
standardization of ADMETox related databases although
there has been considerable discussion relating to drug
metabolism database generation (Erhardt 2003) This is
certainly an important area to address in the future There is
already a growing literature related to ADMETox that is
partially captured in the several commercially available
databases (Ekins et al 2005e) but to date there have been
limited academic efforts to capture data for transporters
with the human membrane transporter database (Yan amp
Sadee 2000) TP-search transporter database (httpwww
ilabrisewasedaacjptp-search) drug interaction database
(httpwwwdruginteractioninfoorgDatabaseinfoaspx)
nuclear hormone receptors (Nakata Yukawa Komiyama
Nakano amp Kaminuma 2002) the ADME-AP database
(Sun Ji Chen Wang amp Chen 2002) and PharmaGKB
(Oliver et al 2002) DSStox (httpwwwepagovnheert
dsstox) TOXNET (httptoxnetnimnihgov) that are
readily accessible
In order to generate accessible pathways using any of
the available software a large enough set of object
identifiers are required to map onto the underlying data-
base To demonstrate this datasets from toxicogenomics
studies have been evaluated with both the KEGG pathway
database and a commercially available product MetaCore
(wwwgenegocom) These gene or protein lists range in
size from 21 to 1853 objects In virtually all cases more
identifiers are mapped to networks in MetaCore and this
also seems independent of the identifier type used
(Unigene Affymetrix Genbank or Locuslink Table 5)
These numbers will obviously change as more information
is added to the respective databases hence the more
objects that are mapped from a dataset The more extensive
Table 4
Literature derived toxicoproteomics data
Compounds Data source Microarray type Compound dose Microarray data
availability
Reference
Carbon
tetrachloride
Male Wistar rats Affymetrix rat chip 8799
probes+proteomics
study
6ndash24 h exposure Genbank SwissProt and
binary data in a
manuscript table
(Fountoulakis
2004)
Paracetamol CD-1 male mice Custom mouse tox blots
with 450 genes
RTQ-PCR+proteomics
study 2-DIGE+
MALDI-MS
150 or 500 mgkg Gene name IMAGE ID
GenBank accession
number fold induction
SwissProt identifier and
protein abundance
change in manuscript
tables
(Ruepp Tonge
Shaw Wallis amp
Pognan 2002)
Paracetamol AP-1 male mice Affymetrix murine 11K
set+proteomics study
Up to 500 mgkg Gene names and fold
changes at multiple
doses and time points in
a manuscript tablemdash
proteomics data not
accessible
(Coen et al 2004)
Oxazepam or
Wy-14643
Male B6C3F1 mice NIEHS Mouse Chip
(8700 genes) 2-DIGE
and MS
Oxazepam (2500 ppm)
Wyeth (Wy)-14643
(500 ppm)
Proteomics data in a
manuscript table
Genbank accession
number gene name and
fold change in a
manuscript table data
also available to
download from NIEHS
website
(Iida et al 2003)
Compound A
(PPAR gamma
ligand)
Female rats CrlCD
(SD)IGS BR
Proteomics 2-DIGE and
MS
250 mgkgday up to
5 days
Accession number and
protein name and
average ratio in a
manuscript table
(Meneses-Lorente
et al 2004)
Bromobenzene Male Wistar rats Proteomics 2-DIGE and
MS custom 3000 cDNA
rat chip
5 mmolkg Accession number gene
name fold change in a
manuscript table protein
names along with bar
charts
(Heijne Stierum
Slijper van
Bladeren amp van
Ommen 2003)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 49
the network that can be generated (as it will consist of more
nodes) then a more comprehensive understanding of the
networks is possible The data available currently in the
literature can be used to evaluate such pathway and
network generation tools Recently we have used several
of the published studies (Tables 1ndash3) with MetaCore to
visualize networks for acetaminophen furan carbon tetra-
chloride benzene and cisplatin showing genes involved in
In vivo
In vitro
In Silico
1970s 1980s 1990s 2000s
Systems Biology ()
OMICS
Fig 2 The timeline for major paradigms in ADMETox
oxidative stress (Ekins Giroux Nikolsky Bugrim amp
Nikolskaya 2005c)
Other important pathwaynetwork building tools that could
potentially be applied to toxicogenomics data include
Ingenuity pathways analysis (httpwwwingenuitycom)
PathArt (httpwwwjubilantbiosyscompdhtm) Pathway
Assist (httpwwwariadnegenomicscomproctspathway
html) (Nikitin et al 2003) and several other databases
deposited at the Pathway Resource List (httpcbiiomskcc
orgprl) All of these products have unique underlying
proprietary pathway databases which are compiled manually
or automatically with text mining tools or a combination of
both We are still waiting for studies that provide a comparison
of different database tools or network building algorithms
Until then theremay be someoverlap but also some differences
between the results obtained from more than one network
method due to the database content and the algorithms used
The reader is recommended to evaluate for themselves several
technologies and select those with the most appropriate fit to
their own specialized needs
In the next Sections the further application of some
available network and database tools will be described with
Microarray or other high throughput data
Upload list of geneprotein identifiers and fold change significance etc (raw data or after selection of the differentially expressed
Genes using other statistical methods)
Parse database and generate interaction network with different algorithms or visualize on maps
Filter Networks
Access significance of the interaction networks
Determine GO processes
Compare 2 or more networks intersection overlap etc
Export gene list
Export gene list
generate interaction network with different algorithms or visualize on maps
Visualization
Fig 3 Schematic for the utilization of pathway tools for assessing high throughput data
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6650
specific detailed reference to transporters and enzymes but
these technologies can also be applied elsewhere as
described above
Fig 4 Network for high throughput screening data for clotrimazole and ticona
cholecystokinin CCR1 Choline transporter Chloride channel catechol-O-methyl
and co workers (Fliri et al 2005) Nodes highlighted with large blue circles rep
molecules showing gt50 inhibition in the assay blue circles represents both mol
inhibits gt50 and one inhibits lt50 Ligands (large) linked to transcriptional f
(wwgenegocom) (For interpretation of the references to colour in this figure leg
311 The role of transporters
A diverse array of organic solutes such as nutrients
neurotransmitters and drugs are transported by specialized
zole screened against 10 in vitro assays (Human Cannabinoid 1 Human
transferase COX2 CYP1A2 CYP2B6 CYP2C19) data published by Fliri
resent assays used in the study Red circles on these nodes represent both
ecules showing lt50 inhibition chequered circles represent one molecule
actors enzymes and transporters via edges using the MetaCorei database
end the reader is referred to the web version of this article)
Table 5
Datasets acquired from publications and other sources and used to evaluate two databasepathway tools KEGG and metaCore
Dataset Number of objects
genes proteins in list
Number of objects
mapped with KEGG
Number of objects mapped
with MetaCore on networks
Reference for microarray
data gene list
Tox Chip 10 (NIEHS) 1853 405 1498 httpsdir-appsniehsnihgov
mapsguestclonesrchcfm
4-Hydroxytamoxifen and
estrogen
1617 434 1343 (Hodges et al 2003)
Mitochondrial proteins 722 156 388 (Gaucher et al 2004
Taylor et al 2003)
Bromobenzene
(24 and 48 h)
130 41 89 (Heijne et al 2004)
Acetaminophen 30 19 23 (Heinloth et al 2004)
Acetaminophen 84 29 64 (Huang et al 2004)
Furan 185 64 139 (Huang et al 2004)
Tetrodotoxin 116 31 86 (Raghavendra Prasad et al
2004)
Benzene 73 16 62 (Yoon et al 2003)
Benzene 76 5 53 (Faiola et al 2004)
Carbon tetrachloride 37 8 26 (Young et al 2003)
Estrogen 94 33 90 (Lobenhofer et al 2002)
Trovafloxacin 142 20 82 (Liguori et al 2005)
Phenobarbital 37 13 28 (Ueda et al 2002)
L-742694 (liver)+ 45 17 19 (Hartley et al 2004)
L-742694 (intestine)+ 23 11 10 (Hartley et al 2004)
A-277249 21 7 9 (Waring et al 2002)
All files were converted to Locuslink identifiers except as noted () unigene () Affymetrix (+) Genbank identifiers were used
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 51
proteins across cellular membranes These may function as
passive processes or active processes energized by the
hydrolysis of ATP or coupling to the co-transport of counter
ions down an electrochemical gradient such as Na+ H+ and
Cl There are many thousands of transporters which can be
classified into distinct superfamilies One of these the solute
carrier class (SLC) is rapidly expanding and contains over
30 families and 200 members The ATP-binding cassette
(ABC) contains 7 families and over 48 members including
P-glycoprotein (P-gp) and MRP subfamilies (Zhang Knipp
Ekins amp Swaan 2002a Zhang Phelps Cheng Ekins amp
Swaan 2002b) Transporters have a key role in clinical
pharmacology with many drugs specifically targeting them
Numerous drugs share transport pathways with nutrients
and transporters have a role in oral absorption drug
bioavailability drug resistance excretion and ultimately
pharmacokinetics and pharmacodynamics
Polymorphism of drug transporters may be a key factor
in drug interactions and lack of effectiveness This field has
become known as pharmacogenomics and is focused on
understanding of inherited DNA sequence variations (poly-
morphisms and mutations) revealed by xenobiotics (Evans
amp McLeod 2003 Weinshilboum 2003) Over the last few
decades many genes have been directly linked to the
mechanisms of response (Evans amp McLeod 2003 Wein-
shilboum 2003) such that 20ndash95 of variability to drug
response is inheritable (Evans amp McLeod 2003) This
phenotypical variability is mainly caused by single nucleo-
tide polymorphisms (SNPs) present in anywhere from 1 to
50 of the population resulting in either lower protein
activity incorrect folding or rapid degradation via proteo-
somes (Weinshilboum amp Wang 2004) A number of
structurally diverse molecules bind to P-gp which is
expressed in many tissues and has numerous SNPs one of
which (C3435T) affects the expression level in the
duodenum and therefore can impact the absorption of
molecules which would be substrates for this transporter
(Sakaeda Nakamura amp Okumura 2002) The human
proton-dependent dipeptide transporter (hPEPT1) can also
affect the absorption of molecules in the intestine and
recently 9 SNPs were found with only one displaying a
reduced transport capacity (Zhang et al 2004) The sodium-
dependent carnitine cotransporter OCTN2 can possess
mutations and these result in primary carnitine deficiency
which impacts fatty acid oxidation and is characterized by
many clinical manifestations (Lahjouji Mitchel amp Qureshi
2001) The organic cation transporter 1 (OCT1) is also
important in the transport of numerous xenobiotics and
endobiotics Recently 4 SNPs were identified in the
Japanese population and when functionally characterized
in vitro the uptake of cations was reduced significantly for
some of these mutations indicating that this would likely
contribute to inter-individual variations in metabolism of
drugs which were transported via OCT1 (Sakata et al
2004)
3111 Clinical relevance of transporters The pregnane
X-receptor (PXR) is a transcriptional regulator of the
enzyme human MDR1 (P-gp) MRPs and OATP (Wang amp
LeCluyse 2003) CYP3A (Bertilsson et al 1998 Blumberg
et al 1998 Kliewer et al 1998) and CYP2C89 (Synold
Dussault amp Forman 2001) as well as many other genes
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6652
involved in the transport metabolism and biosynthesis of
bile acids (Staudinger Liu Madan Habeebu amp Klaassen
2001) However the additional receptors CAR FXR LXR
and other nuclear receptors take part in a complex network
of interactions to control these and other proteins Thus
elucidation of the regulatory networks which control the
expression of these transporters is also important To date
most of the research has centered on efflux transporters but
there has been considerable interest in uptake transporters
such as the organic anion transporter polypeptide (OATP
((Kim 2003) see also later Section on OATP)
There are several specific examples of the importance of
drug transporters to the clinical development of drugs One
example is the insulin sensitizer troglitazone which was
withdrawn due to hepatotoxicity although the precise
mechanism appears to have been unclear until recently
The major metabolite is a sulfated species and is suspected
of being responsible for the observed toxicity The recent
assessment of the organic anion transporting polypeptides
OATP-C and OATP8 expressed on the hepatocyte baso-
lateral membrane indicated that sulphated troglitazone has a
high affinity for the former and possibly lower affinity for
the latter (Nozawa et al 2004) This metabolite would
therefore be expected to accumulate in hepatocytes and
inhibit the bile salt export pump and Mrp2 Because
polymorphisms have been shown for OATP-C (Tirona
Leake Merino amp Kim 2001) it is also possible that these
may result in the accumulation of the metabolite and in turn
elicit idiosyncratic toxicity A second relevant example of
the impact of transporters is the clinically significant drugndash
drug interaction between cerivastatin and cyclosporine A
which occurs via the OATP-C transporter (Shitara Itoh
Sato Li amp Sugiyama 2003) A third example are the HIV
protease inhibitors saquinavir ritonavir and indinavir which
are transported by MRP2 in vitro and other drugs such as
probenecid and sulfinpyrazone are able to enhance this
transport Transport by MRP2 suggests that these com-
pounds will have decreased bioavailability due to increased
clearance and other drugs could aggravate this situation by
further enhancing transport (Huisman et al 2002) Sim-
ilarly the rifampicin mediated induction via PXR of MRP2
and P-gp in healthy subjects was found to significantly
decrease the AUC and also correlated with intestinal
expression of these transporters This transporter is also
inducible by cisplatin 2-AAF and phenobarbital (Schrenk
et al 2001) indicating multiple mechanisms may be
involved In other species such as rat commonly used as a
toxicity model orthologs of the transporters such as OATP2
are expressed and can be induced with ligands for PXR like
PCN (Guo Choudhuri amp Klaassen 2002) This is useful
knowledge because the advent of microarray technology
allows one to dose a rat with a xenobiotic and assess
thousands of genes simultaneously in a particular tissue For
instance animals dosed with known nephrotoxins have
shown some upregulation of the NandashKndashCl transporter
however the authors suggested genomic responses are
stronger soon after exposure before declining (Fleck et
al 2003) Some transporters may therefore be specifically
targeted by drugs in one tissue such as the CNS but these
same transporters may also be expressed elsewhere in the
body hence off-target effects may result in toxicity The
serotonin transporter is one such example which is
expressed in the lungs and brain Some substrates for this
channel like fenfluramine can result in primary pulmonary
hypertension as they accumulate in pulmonary cells (Roth-
man Ayestas Dersch amp Baumann 1999) Similarly P-gp is
expressed at the blood brain barrier and intestine impacting
the efficacy and bioavailability of drugs
3112 Transporter network examples ABCA1 The
ABCA1 transporter mediates the first step of cholesterol
transport Mutations in this gene cause Tangier disease
which results in severe HDL deficiency cholesterol
accumulation in macrophages and attendant atherosclerosis
This transporter represents a drug target for upregulation
modulating cholesterol metabolism and prevention of
cardiovascular disease (Oram amp Lawn 2001) In vitro
ABCA1 can be inhibited by the sulfonylurea glybenclamide
(Field Burn amp Mathur 2004 Wang Silver Thiele amp Tall
2001) In order to illustrate the advantages of mapping drug
transporters as networks onto functional models alongside
other proteins one can consider the example of ABCA1
The query of the MetaCorei database shows that this
transporter appears on three manually curated pathway maps
representing the Fstate of the art_ knowledge derived from
reliable high quality literature sources One can also use the
individual maps as an interface to access the underlying
layers of information about the transporter including the list
of encoding genessplice variants with known SNPs In
addition to browsing MetaCorei a user can also build
custom networks around ABCA1 using the network-
construction tool (Fig 5) Such a visualization utility may
be very helpful for identification of all putative pathways
around a particular transporter or compound of interest The
ABCA1 network created by this tool shows that this
transporter at the time of writing is linked directly to
twenty-five other objects such as APOE1 and LXR Many
of its neighbors have their own SNPs that could be
important in determining interactions between transport of
a drug and normal human transport of endogenous ligands
in health or disease
3113 Transporter network examples OATP The
OATPs are key membrane bound transporters expressed in
many organs including intestine liver lung choroid plexus
blood brain barrier and other organs (Tamai et al 2000)
This family of transporters is capable of mediating the
sodium-independent transport of a diverse array of mole-
cules such as steroid conjugates organic anions and
xenobiotics by coupling uptake with efflux of bicarbonate
(Satlin Amin amp Wolkoff 1997) glutathione or its
conjugates (Hagenbuch amp Meier 2004) The inhibition of
Fig 5 The network of direct gene interactions around the ABCA1 transporter gene (centre) generated with MetaCorei The interation types between nodes are shown as small colored hexagons eg unspecified
allosteric regulation binding cleavage competition covalent modification dephosphorylation phosphorylation transcription regulation transformation When applicable interactions also have a positive or
negative effect and direction Ligands (purple) linked to other proteins (blue blobs) transfactors (red) enzymes (orange) (For interpretation of the references to colour in this figure legend the reader is referred to
the web version of this article)
SEkin
sJournalofPharm
acologica
landToxico
logica
lMeth
ods53(2006)38ndash66
53
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6654
this transporterrsquos hepatic uptake of other compounds may be
important for reported drugndashdrug interactions (Kim 2003)
described earlier (Shitara et al 2003) as well as cerivastatin
with gemfibrozil (Shitara Hirano Sato amp Sugiyama 2004)
OATP1B1 (previous names OATP-C LST-1 OATP2
SLC21A6) represents the most studied human OATP to
date (Meier amp Stieger 2000) and is expressed on the
basolateral plasma membrane of hepatocytes Several single
nucleotide polymorphisms have been identified in the
OATP1B1 gene in European-Americans African-Americans
(Tirona et al 2001) and Japanese (Nozawa et al 2002)
dramatically impacting the transport of ligands such as
pravastatin (Mwinyi Johne Bauer Roots amp Gerloff 2004
Nishizato et al 2003) estrone-3-sulfate (Nozawa et al
2002 Tirona et al 2001) Rifampin (Tirona Leake
Wolkoff amp Kim 2003) and estradiol 17h-d-glucuronide(Tirona et al 2001)
The regulation of SLCO may be affected during
extrahepatic cholestasis bile duct ligation bile salt induced
Fig 6 Networks generated with the autoexpand algorithm in MetaCorei to illustr
be interconnected with other protein regulatory signaling information Informa
unspecified allosteric regulation binding cleavage competition covalent m
transformation When applicable interactions also have a positive or negative e
transfactors (red) enzymes (orange) (For interpretation of the references to colour
cholestatic hepatitis (Dumont et al 1997 Meier amp Stieger
2000 Rost et al 2003) and primary sclerosing cholangitis
(Oswald Kullack-Ublick Paumgarter amp Beuer 2001) In
particular OATP1B1 appears to be regulated by the liver-
enriched transcription factor hepatocyte nuclear factor 1a
(HNF1a) which binds to the promoter region of this
transporter (Jung et al 2001) Site directed mutagenesis
of this binding site resulted in inactivation suggesting the
critical nature of the interaction with HNF1a Bile acids
such as CDCA have been shown to transcriptionally repress
HNF1a in vitro via inhibition of the transactivating effect of
HNF4a on HNF1a (Jung amp Kullak-Ublick 2003) After
screening many rat and human uptake transporters in vitro
OATP1B1 was also shown to modulate the PXR response
by controlling rifampin retention in the cell and therefore
affecting the induction of CYP3A4 and other gene products
such as P-gp (Tirona et al 2003)
Some of the literature for OATP1B1 human substrate
data has been annotated into MetaCorei to illustrate the
ate how ligands for the human OATP-C (OATP1B1) transporter (centre) can
tion on the type of interaction between objects is hidden for clarity eg
odification dephosphorylation phosphorylation transcription regulation
ffect and direction Ligands (purple) linked to other proteins (blue blobs)
in this figure legend the reader is referred to the web version of this article)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 55
visualization of the complex interconnections between this
transporter its ligands regulatory factors and signaling
molecules already in the database (Fig 6) The network was
generated with the autoexpand algorithm in the software
representing one of multiple available algorithms for
connecting genes ligands and other objects in the database
Clearly if more ligands and their connections are added to
the database the complexity of the network will increase
considerably The OATP-C (OATP1B1) gene details can be
viewed upon querying the database and links are provided
to other public databases This page can be used to highlight
the multiple synonyms for this gene as well as links to the
multiple SNPs identified to date
3114 Transporter microarray data Microarrays have
generally been limited in the number of transporters present
on them (Annereau et al 2004) however they have been used
in an attempt to correlate pharmacokinetic properties with
gene expression for valacyclovir (Landowski et al 2003) as
well as understand the expression profile in different tissues
or cell lines upon food component or xenobiotic treatment
(Anderle Huang amp Sadee 2004) This lack of transporters
on microarrays has prompted some groups to produce their
own arrays with a heavier emphasis on transporters These
arrays have for example then been used to demonstrate the
upregulation of ABC transporters and down-regulation of
GST-Pi in cell lines resistant to colchicines or 9-nitro-
camptothecin (Annereau et al 2004) The genes that were
significantly up or down-regulated in this particular study
were used to build networks with MetaCore (Fig 7A B) and
the similarities between them were assessed (Fig 7C)
Although there were only a small number of significantly
changed genes in common (IL-8 Fos GST-Pi Calpactin and
Ubiquitin hydrolase) it is perhaps likely that there is a much
larger common gene network that is important for drug
resistance although a much larger number of cell lines and
drug treatments need to be evaluated to produce a definitive
drug resistance signature involving transporters enzymes
and transcriptional regulators
312 Applications to enzymes
As we have already described it may be particularly
valuable to visualize enzymes as networks to show
interactions with transcriptional regulators and ligands
For example a key enzyme is CYP3A4 which metabolizes
40ndash45 of all drugs and has relatively few SNPs
(Ingelman-Sundberg 2004) Using a second software suite
MetaDrugi (wwwgenegocom) it is possible to construct
a custom network around this or other drug metabolizing
enzymes (Fig 8) In this case the gene network for
CYP3A4 highlights all of the major transcriptional
regulators and several more distant linked proteins and
ligands connected on the network that may be useful for
further study (Fig 8) Substrates inhibitors as well as
regulatory factors and other enzymes can be observed
connected on this network Due to the many hundreds of
known inhibitors and substrates only a small number are
shown here for clarity We assume that if a perturbation in
a pathway (eg due to a nonfunctional enzyme) is linked
to a certain pathologic condition a similar perturbation
caused by the interference from xenobiotic metabolism
(eg competitive inhibition of the same enzyme) may
result in identical effects As microarray gene expression
data is increasingly generated the role of enzyme
regulation in toxicity of certain xenobiotics will become
more apparent from either in vivo or in vitro studies The
visualization of such signature gene networks involving
transporters and enzymes their ligands and regulatory
factors will also be important for future toxicity prediction
methods We have recently generated visualizations of
microarray data from MCF-7 cells treated with 4-hydrox-
ytamoxifen to show that some of the key genes involved in
metabolism and transport are upregulated (Ekins et al
2005d) In addition we have made predictions with various
QSAR models in MetaDrug to indicate the involvement of
PXR CYP3A4 and P-gp (Ekins et al 2005e) Therefore it
appears likely that 4-hydroxytamoxifen could increase its
own metabolism as well as efflux from cells via P-gp
which can be visualized on networks Any decrease of
function of these enzymes or transporters in a population
would likely result in changes in the metabolism and
transport of this active metabolite potentially impacting
the clinical effect This represents one example of how
both pathway tools QSAR models and network building
algorithms can be used with different types of predicted
and experimental data to allow visualization of potential
compound interactions or toxicity
313 Future network applications
As the population ages an increasing incidence and
prevalence of systemic diseases especially chronic diseases
have occurred among older adults This has resulted in an
increase in medications used concomitantly by this
population which presents challenges for drugndashdrug
interactions Physiologically elderly patients may behave
differently to the younger patients for which the drugs are
initially developed for and tested on Many pharmacoki-
netic investigations in the elderly population reveal
decreased clearance of lipophilic drugs metabolized by
the cytochrome P450 enzymes however few studies have
evaluated aging-dependent or gender-related changes in
specific P450 enzymes (Hunt Westerkam amp Slave 1992)
Age-related physiological changes such as a reduction in
liver mass hepatic metabolizing enzyme activity liver
blood flow and alterations in plasma drug binding may
account for the decreased elimination of some metabolized
drugs in the elderly It is particularly difficult to separate an
effect of aging from the variation in the rate of metabolism
due to factors such as individual metabolic phenotype
(slow or fast metabolizer due to SNPs) environmental
influences concomitant disease states and drug intake
(drugndashdrug interactions) The available data suggest that
Fig 7 Networks for drug resistant cell lines generated with the Auto expand algorithm in MetaCore A KB-3-1 vs KB-8-5 B ATC-DU-145 vs RCO1 C node overlap of A and B with initial nodes highlighted
with blue or red solid circles (Initial gene lists derived from (Annereau et al 2004)) Information on the type of interaction between objects is hidden for clarity eg unspecified allosteric regulation binding
cleavage competition covalent modification dephosphorylation phosphorylation transcription regulation transformation When applicable interactions also have a positive or negative effect and direction
Ligands (purple) linked to other proteins (blue blobs) transfactors (red) enzymes (orange) (For interpretation of the references to colour in this figure legend the reader is referred to the web version of this article)
SEkin
sJournalofPharm
acologica
landToxico
logica
lMeth
ods53(2006)38ndash66
56
Fig7(continued)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 57
Fig7(continued)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6658
Fig 8 The network of gene interactions around the CYP3A4 enzyme (centre) from MetaCorei Information on the type of interaction between objects is
hidden for clarity eg unspecified allosteric regulation binding cleavage competition covalent modification dephosphorylation phosphorylation
transcription regulation transformation When applicable interactions also have a positive or negative effect and direction Ligands (purple) linked to other
proteins (blue blobs) transfactors (red) enzymes (orange) (For interpretation of the references to colour in this figure legend the reader is referred to the web
version of this article)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 59
the initial doses of drugs metabolized by these enzymes
should be reduced in older patients according to the clinical
response In most published studies the elderly appear at
least as responsive as the young to inducers or inhibitors of
P450s (Durnas Loi amp Cusack 1990) More recently there
has been some suggestion that there are age related
reductions in function of some specific P450s such as
CYP3A4 (Patki et al 2004) and this could occur at the
level of regulation However earlier studies with the same
enzyme showed no change in clearance with age (Hunt et
al 1992) To date there has been even less examination of
the transporter functions and any changes with age
(Kinirons amp OrsquoMahony 2004) so the current understand-
ing of the effects of aging on metabolism and transport is
anything but transparent This represents an extreme
challenge for the pharmaceutical industry how to predict
whether a drug has an affinity for an enzyme or transporter
and whether this may be clinically important if it shows a
decline in expression or function with aging Any decrease
of function of these enzymes or transporters in an elderly
population would result in changes in the metabolism and
transport of metabolites potentially impacting the clinical
effect This represents another example of how computa-
tional approaches may perhaps be used with different types
of predicted and experimental data to allow visualization of
potential compound interactions or toxicity in elderly
populations We may see different gene networks high-
lighted as humans age and these may be modified by drug
treatment and coadministration This represents an area
were network analysis could be applied in the future and is
in need of considerable further research
The collection of microarray data in databases such as
CEBS ArrayTrack and EDGE represents a future resource
for computational gene network analysis One could
envisage that ultimately in each case such data is converted
to one or more networks that are also displayed for the user
and can be used to compare treatments from in vivo and in
vitro experiments This would represent a different approach
to clustering the data as currently implemented in one of
these efforts (Hayes et al 2005) and may condense large
amounts of experimentally derived data into a readily
interpreted network
4 Discussion
Previously in this journal the progress of many
research groups in predicting human ADME parameters
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6660
in silico (Ekins et al 2000b) and approaches for drug
metabolism (Ekins Ring Grace McRobie-Belle amp
Wrighton 2000a) have been described Both of these
reviews commented on moving HT assays for ADME
Tox much earlier in drug discovery which had also been
indicated by other groups The initial reviews also
highlighted the likely wealth of data that would become
available and how this could be used for structure
activity relationships alongside the bioactivity data in
computational models It was noted that there was a
paucity of predictive metabolism tools at that time In
addition a growing number of efforts to model whole
cells and organs now a field called systems biology
were recognized as models that could be integrated with
the in silico ADME approaches In summary since these
past reviews virtually all pharmaceutical companies have
attempted earlier high throughput screening for ADME
Tox properties and to some extent the wider application
of computational approaches for physicochemical proper-
ties Systems biology is being quite widely acknowledged
as the new paradigm for understanding complex bio-
logical datasets derived from high throughput technolo-
gies and the accumulated knowledge on human protein
interactions (Hartwell Hopfield Leibler amp Murray 1999
Hood 2003b) Therefore systems biology can be defined
as the integration of genetic proteomic transcriptomic
and metabonomic data using computational methods
(Nicholson amp Wilson 2003) When taken together
information on molecular processes derived from different
sources represents a lsquolsquouniversersquorsquo of putative biological
functionality of which only a small fraction of it will be
realized in a cell at any given time To date systems
biology has been driven by academia and funding bodies
such as the NIH rather than the pharmaceutical compa-
nies Presently there is a great deal of interest from
scientists of all backgrounds in identifying the networks
of cellular pathways and the corresponding physically
interacting proteins
The network building software for systems biology
described in this current review will be valuable to query
high throughput data and known literature interactions in
order to predict potential toxicity in different species In the
future the compilation of published toxicogenomics data-
sets characteristic of different types of toxicity will likely
be available in these software systems to act as a reference
database It is also feasible that we will be able to generate
the annotated datasets which specifically address the
differences between human and rat networks implicated
in toxicity The identification of sub-network modules
conserved between human and rat distinct for toxicity
types or predictive for toxic end-points in human will be
possible Such signature gene networks (Nikolsky et al
2005) can then be verified with other experimentally
derived data prospectively or from preexisting databases
The combined Fhigh throughput datandashin silico network_approaches therefore represents a novel method for access-
ing complex tissue-level processes as networks integrating
all data types based on functional interactions The data-
bases developed and used in this approach will certainly
benefit from further annotation around the drug metaboliz-
ing enzymes and transporters as described herein in terms
of transcriptional regulation and the ligands associated with
them which frequently appear in the literature It will be
important to capture disparities in the assignment of ligands
to enzymes transporters and other proteins as well as
negative data
In summary although there have been numerous
toxicogenomics studies published there is presently a
relatively small number of datasets that are freely available
to perform network analysis of microarray data The
number of studies identifying large numbers of proteins
which are affected by molecule treatment are even scarcer
still while there are several examples of some published
studies that combine such data It is hoped that the
numerous database initiatives for high content and toxicol-
ogy data that are being undertaken will improve the
situation for other researchers that are not currently
equipped to do such microarray studies themselves The
addition of requirements by journals to deposit such raw
data in a freely accessible resource will aid these initiatives
ADMETox groups have seen new technologies and
approaches developed over the last decade that have all
been applied to identify poor compounds earlier (Fig 2)
The latest technologies integrate network building tools
with high content data and databases The current review
described the limited number of networks generated for
ADMETox at present and one hopes that the impact of
such analyses will be commonplace in the future Systems
biology is however more than just applying a network
approach and hence systems-ADMETox will have to
evolve due to the continual pressure to develop newer
technologies This current paradigm combining empirical
data and computational methods should integrate the
complex data already generated making it readily inter-
pretable and valuable for identifying the most promising
compounds in the future
Acknowledgements
Dr Maggie AZ Hupcey is gratefully acknowledged for
editorial assistance Dr Peter W Swaan (University of
Maryland) and Dr Cheng Chang (Ohio State University)
Dr Steve Wright (University of Arizona) Dr K Sandy
Pang (University of Toronto) and Dr Craig Giroux (Wayne
State University) are kindly acknowledged for their support
and discussions My colleagues at GeneGo Sergey
Andreyev Andy Ryabov Eugene Kirillov Eugene A
Rakhmatulin Svetlana Sorokina Andrej Bugrim Tatiana
Nikolskaya Yuri Nikolsky John Metz and Julie Bryant are
all thanked for their considerable contributions to software
development and data annotation
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 61
References
Abe M Yamashita S Kuramoto T Hirayama Y Tsukamoto T Ohta
T et al (2003) Global expression analysis of N-methyl-NV-nitro-N-nitrosoguanidine-induced rat stomach carcinomas using oligonucleotide
microarrays Carcinogenesis 24 861ndash867
Adachi T Ono Y Koh K B Takashima K Tainaka H Matsuno Y
et al (2004) Long-term alteration of gene expression without
morphological change in testis after neonatal exposure to genistein in
mice Toxicogenomic analysis using cDNA microarray Food and
Chemical Toxicology 42 445ndash452
Akerman G S Rosenzweig B A Domon O E McGarrity L J
Blankenship L R Tsai C A et al (2004) Gene expression profiles
and genetic damage in benzo(a)pyrene diol epoxide-exposed TK6 cells
Mutation Research 549 43ndash64
Aksenov S V Church B Dhiman A Georgieva A Sarangapani R
Helmlinger G et al (2005) An integrated approach for inference and
mechanistic modeling for advancing drug development FEBS Letters
579 1878ndash1883
Anderle P Huang Y amp Sadee W (2004) Intestinal membrane transport
of drugs and nutrients Genomics of membrane transporters using
expression microarrays European Journal of Pharmaceutical Sciences
21 17ndash24
Annereau J P Szakacs G Tucker C J Arciello A Cardarelli C
Collins J et al (2004) Analysis of ATP-binding cassette transporter
expression in drug-selected cell lines by a microarray dedicated to
multidrug resistance Molecular Pharmacology 66 1397ndash1405
Apic G Ignjatovic T Boyer S amp Russell R B (2005) Illuminating drug
discovery with biological pathways FEBS Letters 579 1872ndash1877
Baker V A Harries H M Waring J F Duggan C M Ni H A Jolly
R A et al (2004) Clofibrate-induced gene expression changes in rat
liver A cross-laboratory analysis using membrane cDNA arrays
Environmental Health Perspectives 112 428ndash438
Balakin K V Ekins S Bugrim A Ivanenkov Y A Korolev D amp
Nikolsky Y et al (2004a) Quantitative structurendashmetabolism relation-
ship modeling of the metabolic N-dealkylation rates Drug Metabolism
and Disposition 32 1111ndash1120
Balakin K V Ekins S Bugrim A Ivanenkov Y A Korolev D amp
Nikolsky Y et al (2004b) Kohonen maps for prediction of binding to
human cytochrome P450 3A4 Drug Metabolism and Disposition 32
1183ndash1189
Barabasi A -L amp Oltvai Z N (2004) Network biology Under-
standing the cellrsquos functional organization Nature Reviews Genetics
5 101ndash113
Bartosiewicz M Penn S G amp Buckpitt A (2001) Applications of gene
arrays in environmental toxicology Fingerprints of gene regulation
associated with cadmium chloride benzo(a)pyrene and trichloro-
ethylene Environmental Health Perspectives 109 71ndash74
Bertilsson G Heidrich J Svensson K Asman M Jendeberg L
Sydow-Backman M et al (1998) Identification of a human nuclear
receptor defines a new signaling pathway for CYP3A induction
Proceedings of the National Academy of Sciences of the United States
of America 95 12208ndash12213
Blumberg B Sabbagh Jr W Juguilon H Bolado Jr J van Meter
C M Ong E S et al (1998) SXR a novel steroid and xenobiotic-
sensing nuclear receptor Genes and Development 12 3195ndash3205
Borodina Y Rudik A Filimonov D Kharchevnikova N Dmitriev A
Blinova V et al (2004) A new statistical approach to predicting
aromatic hydroxylation sites Comparison with model-based
approaches Journal of Chemical Information and Computer Sciences
44 1998ndash2009
Borodina Y Sadym A Filimonov D Blinova V Dmitriev A amp
Poroikov V (2003) Predicting biotransformation potential from
molecular structure Journal of Chemical Information and Computer
Sciences 43 1636ndash1646
Bottone Jr F G Martinez J M Collins J B Afshari C A amp Eling
T E (2003) Gene modulation by the cyclooxygenase inhibitor
sulindac sulfide in human colorectal carcinoma cells Possible link to
apoptosis Journal of Biological Chemistry 278 25790ndash25801
Boyer S amp Zamora I (2002) New methods in predictive metabolism
Journal of Computer-Aided Molecular Design 16 403ndash413
Bredel M amp Jacoby E (2004) Chemogenomics An emerging strategy
for rapid target and drug discovery Nature Reviews Genetics 5
262ndash275
Bugrim A Nikolskaya T amp Nikolsky Y (2004) Early prediction of drug
metabolism and toxicity Systems biology approach and modeling
Drug Discovery Today 9 127ndash135
Bulera S J Eddy S M Ferguson E Jatkoe T A Reindel J F
Bleavins M R et al (2001) RNA expression in the early character-
ization of hepatotoxicants in Wistar rats by high-density DNA micro-
arrays Hepatology 33 1239ndash1258
Butcher E C Berg E L amp Kunkel E J (2004) Systems biology in drug
discovery Nature Biotechnology 22 1253ndash1259
Butte A (2002) The use and analysis of microarray data Naturalist
Review of Drug Discovery 1 951ndash960
Cary M P Bader G D amp Sander C (2005) Pathway information for
systems biology FEBS Letters 579 1815ndash1820
Cheok M H Yang W Pui C H Downing J R Cheng C Naeve
C W et al (2003) Treatment-specific changes in gene expression
discriminate in vivo drug response in human leukemia cells Nature
Genetics 34 85ndash90
Cleary J D Rogers P D amp Chapman S W (2001) Differential
transcription factor expression in human mononuclear cells in response
to amphotericin B Identification with complementary DNA microarray
technology Pharmacotherapy 21 1046ndash1054
Coen M Ruepp S U Lindon J C Nicholson J K Pognan F Lenz
E M et al (2004) Integrated application of transcriptomics and
metabonomics yields new insight into the toxicity due to paracetamol in
the mouse Journal of Pharmaceutical and Biomedical Analysis 35
93ndash105
Cornwell P D De Souza A T amp Ulrich R G (2004) Profiling of
hepatic gene expression in rats treated with fibric acid analogs
Mutation Research 549 131ndash145
Csermely P Agoston V amp Pongor S (2005) The efficiency of multi-
target drugs The network approach might help drug design Trends in
Pharmacological Sciences 26 178ndash182
Cunningham M J Liang S Fuhrman S Seilhamer J J amp
Somogyi R (2000) Gene expression microarray data analysis for
toxicology profiling Annals of the New York Academy of Sciences 919
52ndash67
de Longueville F Atienzar F A Marcq L Dufrane S Evrard S
Wouters L et al (2003) Use of a low density microarray for studying
gene expression patterns induced by hepatotoxicants on primary
cultures of rat hepatocytes Toxicological Sciences 75 378ndash392
Dobrin R Beg Q K Barabasi A L amp Oltvai Z N (2004)
Aggregation of topological motifs in the Escherichia coli transcriptional
regulatory network BMC Bioinformatics 5 10
Donald S Verschoyle R D Edwards R Judah D J Davies R
Riley J et al (2002) Hepatobiliary damage and changes in hepatic
gene expression caused by the antitumor drug ecteinascidin-743 (ET-
743) in the female rat Cancer Research 62 4256ndash4262
Dumont M Jacquemin E DrsquoHont C Descout C Cresteil D Haouzi
D et al (1997) Expression of the liver Na+-independent organic anion
transporting polypeptide (oatp-1) in rats with bile duct ligation Journal
of Hepatology 27 1051ndash1056
Durnas C Loi C M amp Cusack B J (1990) Hepatic drug metabolism
and aging Clinical Pharmacokinetics 19 359ndash389
Eisen M B Spellman P T Brown P O amp Botstein D (1998) Cluster
analysis and display of genome-wide expression patterns Proceedings
of the National Academy of Sciences of the United States of America
95 14863ndash14868
Ekins S Andreyev S Ryabov A Kirilov E Rakhmatulin E A
Bugrim A et al (in press) Computational Prediction of Human Drug
Metabolism Exp Opin Drug Metab Toxicol
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6662
Ekins S Boulanger B Swaan P W amp Hupcey M A Z (2002)
Towards a new age of virtual ADMETOX and multidimensional drug
discovery Journal of Computer-Aided Molecular Design 16 381ndash401
Ekins S Bugrim A Nikolsky Y amp Nikolskaya T (2005) Systems
biology Applications in drug discovery In S Gad (Ed) Drug
Discovery Handbook (pp 123ndash183) Hoboken NJrsquo Wiley and Sons
Ekins S Giroux C N Nikolsky Y Bugrim A amp Nikolskaya T
(2005) A signature gene network approach to toxicity The Toxoco-
logists 84
Ekins S Kirillov E Rakhmatulin E amp Nikolskaya T (2005) A novel
method for visualizing nuclear hormone receptor networks relevant to
drug metabolism Drug Metabolism and Disposition 33 474ndash481
Ekins S amp McGowan R J (2001) The limits of reductionism The
shifting genomic paradigmrsquos impact on industry and academia
Philsophy in Science 9 1ndash23
Ekins S Mirny L amp Schuetz E G (2002) A ligand-based approach to
understanding selectivity of nuclear hormone receptors PXR CAR
FXR LXRa and LXRb Pharmaceutical Research 19 1788ndash1800
Ekins S Nikolsky Y amp Nikolskaya T (2005) Techniques Application
of systems biology to absorption distribution metabolism excretion
and toxicity Trends in Pharmacological Sciences 26 202ndash209
Ekins S Ring B J Grace J McRobie-Belle D J amp Wrighton S
A (2000) Present and future in vitro approaches for drug
metabolism Journal of Pharmacological and Toxicological Methods
44 313ndash324
Ekins S Waller C L Swaan P W Cruciani G Wrighton S A amp
Wikel J H (2000) Progress in predicting human ADME parameters in
silico Journal of Pharmacological and Toxicological Methods 44
251ndash272
Erhardt P W (2003) A human drug metabolism database Potential roles
in the quantitative predictions of drug metabolism and metabolism-
related drugndashdrug interactions Current Drug Metabolism 4 411ndash422
Evans W E amp McLeod H L (2003) Pharmacogenomicsmdashdrug
disposition drug targets and side effects New England Journal of
Medicine 348 538ndash549
Ezendam J Staedtler F Pennings J Vandebriel R J Pieters R
Boffetta P et al (2004) Toxicogenomics of subchronic hexachlor-
obenzene exposure in Brown Norway rats Environmental Health
Perspectives 112 782ndash791
Faiola B Fuller E S Wong V A amp Recio L (2004) Gene expression
profile in bone marrow and hematopoietic stem cells in mice exposed to
inhaled benzene Mutation Research 549 195ndash212
FDA (2004) Innovation stagnation Challenge and opportunity on the
critical path to new medicinal products
Fiehn O (2001) Combining genomics metabolome analysis and bio-
chemical modelling to understand metabolic networks Comparative
Functional Genomics 2 155ndash168
Field F J Born E amp Mathur S N (2004) LXRRXR ligand activation
enhances basolateral efflux of beta-sitosterol in CaCo-2 cells Journal of
Lipid Research 45 905ndash913
Fleck C Sutter L Appenroth D Koch B Meinhold T Pitack M
et al (2003) Use of gene chip technology for the characterisation of
the regulation of renal transport processes and of nephrotoxicity in
rats Experimental and Toxicologic Pathology 54 401ndash410
Fliri A F Loging W T Thadeio P F amp Volkmann R A (2005)
Biological spectra analysis Linking biological activity profiles to
molecular structure Proceedings of the National Academy of Sciences
of the United States of America 102 261ndash266
Fountoulakis M (2004) Application of proteomics technologies in the
investigation of the brain Mass Spectrometry Reviews 23 231ndash258
Gaucher S P Taylor S W Fahy E Zhang B Warnock D E Ghosh
S S et al (2004) Expanded coverage of the human heart
mitochondrial proteome using multidimensional liquid chromatography
coupled with tandem mass spectrometry Journal of Proteome
Research 3 495ndash505
Gerhold D Lu M Xu J Austin C Caskey C T amp Rushmore T
(2001) Monitoring expression of genes involved in drug metabolism
and toxicology using DNA microarrays Physiological Genomics 5
161ndash170
Guo G L Choudhuri S amp Klaassen C D (2002) Induction profile of
rat organic anion transporting polypeptide 2 (oatp2) by prototypical
drug-metabolizing enzyme inducers that activate gene expression
through ligand-activated transcription factor pathways Journal of
Pharmacology and Experimental Therapeutics 300 206ndash212
Hagenbuch B amp Meier P J (2004) Organic anion transporting
polypeptides of the OATP SLC21 family Phylogenetic classification
as OATP SLCO superfamily new nomenclature and molecularfunc-
tional properties Pflugers Archiv 447 653ndash665
Hamadeh H K amp Afshari C A (2004) Toxicogenomics principles and
applications Hobokenrsquo John Wiley and Sons
Hamadeh H K Bushel P R Jayadev S Martin K DiSorbo O Sieber
S et al (2002) Gene expression analysis reveals chemical-specific
profiles Toxicological Sciences 67 219ndash231
Hamadeh H K Jayadev S Gaillard E T Huang Q Stoll R
Blanchard K et al (2004) Integration of clinical and gene expression
endpoints to explore furan-mediated hepatotoxicity Mutation Research
549 169ndash183
Hamadeh H K Knight B L Haugen A C Sieber S Amin R P
Bushel P R et al (2002) Methapyrilene toxicity Anchorage of
pathologic observations to gene expression alterations Toxicologic
Pathology 30 470ndash482
Han J D Bertin N Hao T Goldberg D S Berriz G F Zhang L V
et al (2004) Evidence for dynamically organized modularity in the
yeast proteinndashprotein interaction network Nature 430 88ndash93
Hanisch D Zien A Zimmer R amp Lengauer T (2002) Co-clustering of
biological networks and gene expression data Bioinformatics 18
S145ndashS154
Harris A J Dial S L amp Casciano D A (2004) Comparison of basal
gene expression profiles and effects of hepatocarcinogens on gene
expression in cultured primary human hepatocytes and HepG2 cells
Mutation Research 549 79ndash99
Harrison S C (2004) Whither structural biology Nature Structural
Molecular Biology 11 12ndash15
Hartley D P Dai X He Y D Carlini E J Wang B Huskey S E
et al (2004) Activators of the rat pregnane X receptor differentially
modulate hepatic and intestinal gene expression Molecular Pharma-
cology 65 1159ndash1171
Hartwell L H Hopfield J J Leibler S amp Murray A W (1999) From
molecular to modular cell biology Nature 402 C47ndashC52
Hasmall S Orphanides G James N Pennie W Hedley K Soames
A et al (2002) Downregulation of lactoferrin by PPARalpha ligands
Role in perturbation of hepatocyte proliferation and apoptosis
Toxicology Sciences 68 304ndash313
Hayase T Yamamoto Y Yamamoto K Muso E amp Shiota K (2004)
Microarray profile analysis of toxic cocaine-induced alterations in the
expression of mouse brain gene sequences A possible rsquoprotectiversquo
effect of buprenorphine Journal of Applied Toxicology 24 15ndash20
Hayes K R Vollrath A L Zastrow G M McMillan B J Craven M
Jovanovich S et al (2005) EDGE A centralized resource for the
comparison analysis and distribution of toxicogenomic information
Molecular Pharmacology 67 1360ndash1368
Heijne W H Slitt A L van Bladeren P J Groten J P Klaassen C D
Stierum R H et al (2004) Bromobenzene-induced hepatotoxicity at
the transcriptome level Toxicology Sciences 79 411ndash422
Heijne W H Stierum R H Slijper M van Bladeren P J amp van
Ommen B (2003) Toxicogenomics of bromobenzene hepatotoxicity
A combined transcriptomics and proteomics approach Biochemical
Pharmacology 65 857ndash875
Heinloth A N Irwin R D Boorman G A Nettesheim P Fannin
R D Sieber S O et al (2004) Gene expression profiling of rat
livers reveals indicators of potential adverse effects Toxicology
Sciences 80 193ndash202
Hodges L C Cook J D Lobenhofer E K Li L Bennett L Bushel
P R et al (2003) Tamoxifen functions as a molecular agonist inducing
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 63
cell cycle-associated genes in breast cancer cells Molecular Cancer
Research 1 300ndash311
Holleman A Cheok M H den Boer M L Yang W Veerman KM
A J Kazemier et al (2004) Gene-expression patterns in drug-resistant
acute lymphoblastic leukemia cells and response to treatment New
England Journal of Medicine 351 533ndash542
Hong Y Muller U R amp Lai F (2003) Discriminating two classes of
toxicants through expression analysis of HepG2 cells with DNA arrays
Toxicology in Vitro 17 85ndash92
Hood E (2003) Proteomics Characterizing the cogs in the machinery of
life Environmental Health Perspectives 111 A816ndashA825
Hood L (2003) Systems biology Integrating technology biology and
computation Mechanism of Ageing and Development 124 9ndash16
Hood L amp Galas D (2003) The digital code of DNA Nature 421
444ndash448
Hood L amp Perlmutter R M (2004) The impact of systems approaches on
biological problems in drug discovery Nature Biotechnology 22
1215ndash1217
Hu T Gibson D P Carr G J Torontali S M Tiesman J P Chaney
J G et al (2004) Identification of a gene expression profile that
discriminates indirect-acting genotoxins from direct-acting genotoxins
Mutation Research 549 5ndash27
Huang Q Dunn II R T Jayadev S DiSorbo O Pack F D Farr S B
et al (2001) Assessment of cisplatin-induced nephrotoxicity by
microarray technology Toxicology Sciences 63 196ndash207
Huang Q Jin X Gaillard E T Knight B L Pack F D Stoltz J H et
al (2004) Gene expression profiling reveals multiple toxicity endpoints
induced by hepatotoxicants Mutation Research 549 147ndash167
Huisman M T Smit J W Crommentuyn K M Zelcer N Wiltshire
H R Beijnen J H et al (2002) Multidrug resistance protein 2
(MRP2) transports HIV protease inhibitors and transport can be
enhanced by other drugs Aids 16 2295ndash2301
Hunt C M Westerkam W R amp Stave G M (1992) Effect of age and
gender on the activity of human hepatic CYP3A Biochemical
Pharmacology 44 275ndash283
Ideker T Ozier O Schwikowski B amp Siegal A F (2002) Discovering
regulatory and signalling circuits in molecular interaction networks
Bioinformatics 18 S233ndashS240
Ideker T Thorsson V Ranish J A Christmas R Buhler J Eng J K
et al (2001) Integrated genomic and proteomic analyses of a system-
atically perturbed network Science 292 929ndash934
Iida M Anna C H Hartis J Bruno M Wetmore B Dubin J R et al
(2003) Changes in global gene and protein expression during early
mouse liver carcinogenesis induced by non-genotoxic model carcino-
gens oxazepam and Wyeth-14643 Carcinogenesis 24 757ndash770
Ingelman-Sundberg M (2004) Pharmacogenetics of cytochrome P450 and
its applications in drug therapy The past present and future Trends in
Pharmacological Sciences 25 193ndash200
Jeong H Mason S P Barabasi A L amp Oltvai Z N (2001) Lethality
and centrality in protein networks Nature 411 41ndash42
Jeong H Tombor B Albert R Oltvai Z N amp Barabasi A L
(2000) The large-scale organization of metabolic networks Nature
407 651ndash654
Jung D Hagenbuch B Gresh L Pontoglio M Meier P J amp Kullak-
Ublick G A (2001) Characterization of the human OATP-C
(SLC21A6) gene promoter and regulation of liver-specific OATP genes
by hepatocyte nuclear factor 1 alpha Journal of Biological Chemistry
276 37206ndash37214
Jung D amp Kullak-Ublick G A (2003) Hepatocyte nuclear factor 1
alpha A key mediator of the effect of bile acids on gene expression
Hepatology 37 622ndash631
Jung J -W Park J -S Hwang J -W Kang K -S YS L Song B
-S et al (2004) Gene expression analysis of peroxisome prolifer-
ators- and phenytoin-induced hepatotoxicity using cDNA microarray
Journal of Veterinary Medical Science 66 1329ndash1333
Kato N Shibutani M Takagi H Uneyama C Lee K -Y Takiami S
et al (2004) Gene expression profile in the livers of rats orally
administered ethinylestradiol for 28 days using a microarray technique
Toxicology 200 179ndash192
Kier L D Neft R Tang L Suizu R Cook T Onsurez K et al
(2004) Applications of microarrays with toxicologically relevant genes
(tox genes) for the evaluation of chemical toxicants in Sprague Dawley
rats in vivo and human hepatocytes in vitro Mutation Research 549
101ndash113
Kijima K Toyosawa K Yasuba M Matsuoka N Adachi T
Komiyama M et al (2004) Gene expression analysis of the rat testis
after treatment with di(2-ethylhexyl) phthalate using cDNA microarray
and real-time RT-PCR Toxicology and Applied Pharmacology 200
103ndash110
Kim R B (2003) Organic anion-transporting polypeptide (OATP) trans-
porter family and drug disposition European Journal of Clinical
Investigation 33 1ndash5
Kinirons M T amp OrsquoMahony M S (2004) Drug metabolism and ageing
British Journal of Clinical Pharmacology 57 540ndash544
Kitano H (2002a) Computational systems biology Nature 420 206ndash210
Kitano H (2002b) Systems biology A brief overview Science 295
1662ndash1664
Kliewer S A Moore J T Wade L Staudinger J L Watson M
A Jones S A et al (1998) An orphan nuclear receptor activated
by pregnanes defines a novel steroid signalling pathway Cell 92
73ndash82
Korolev D Balakin K V Nikolsky Y Kirillov E Ivanenkov Y A
Savchuk N P et al (2003) Modeling of human cytochrome p450-
mediated drug metabolism using unsupervised machine learning
approach Journal of Medicinal Chemistry 46 3631ndash3643
Kramer J A Pettit S D Amin R P Bertram T A Car B
Cunningham M et al (2004) Overview on the application of
transcription profiling using selected nephrotoxicants for toxicology
assessment Environmental Health Perspectives 112 460ndash464
Kultima K Nystrom A -M Scholz B Gustafson A -L Dencker L
amp Stigson M (2004) Valproic acid teratogenicity A toxicogenomics
approach Environmental Health Perspectives 112 1225ndash1235
Kutuzova G D amp Deluca H F (2004) Gene expression profiles in rat
intestine identify pathways for 125-dihydroxyvitamin D(3) stimulated
calcium absorption and clarify its immunomodulatory properties
Archives of Biochemistry and Biophysics 432 152ndash166
Kwak M K Wakabayashi N Itoh K Motohashi H Yamamoto M amp
Kensler T W (2003) Modulation of gene expression by cancer
chemopreventive dithiolethiones through the Keap1-Nrf2 pathway
Identification of novel gene clusters for cell survival Journal of
Biological Chemistry 278 8135ndash8145
Lahjouji K Mitchell G A amp Qureshi I A (2001) Carnitine transport
by organic cation transporters and systemic carnitine deficiency
Molecular Genetics and Metabolism 73 287ndash297
Landowski C P Sun D Foster D R Menon S S Barnett J L
Welage L S et al (2003) Gene expression in the human intestine and
correlation with oral valacyclovir pharmacokinetic parameters Journal
of Pharmacology and Experimental Therapeutics 306 778ndash786
Lee G -J Lee W -S Jeon K -S Um C -H Kim S -J Lee C -H
et al (2004) cDNA microarray gene expression analysis and
toxicological phenotype for anticancer drug Journal of Veterinary
Medical Science 66 1339ndash1345
Lee M Kwon J Kim S N Kim J E Koh W S Kim E J et al
(2003) cDNA microarray gene expression profiling of hydroxyurea
paclitaxel and p-anisidine genotoxic compounds with differing
tumorigenicity results Environmental and Molecular Mutagenesis
42 91ndash97
Li S Armstrong C M Bertin N Ge H Milstein S Boxem M et al
(2004) A map of the interactome network of the metazoan C elegans
Science 303 540ndash543
Liguori M J Anderson L M Bukofzer S McKim J Pregenzer J F
Retief J et al (2005) Microarray analysis in human hepatocytes
suggests a mechanism for hepatotoxicity induced by trovafloxacin
Hepatology 41 177ndash186
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6664
Lobenhofer E K Bennett L Cable P L Li L Bushel P R amp Afshari
C A (2002) Regulation of DNA replication fork genes by 17beta-
estradiol Molecular Endocrinology 16 1215ndash1229
Locker J Tian J Carver R Concas D Cossu C Ledda-Columbano
G M et al (2003) A common set of immediate-early response genes
in liver regeneration and hyperplasia Hepatology 38 314ndash325
Mattes W B Pettit S D Sansone S A Bushel P R amp Waters M D
(2004) Database development in toxicogenomics Issues and efforts
Environmental Health Perspectives 112 495ndash505
McMillian M Nie A Y Parker J B Leone A Bryant S Kemmerer
M et al (2004) A gene expression signature for oxidant stressreactive
metabolites in rat liver Biochemical Pharmacology 68 2249ndash2261
Meier P J amp Stieger B (2000) Molecular mechanisms in bile formation
News in Physiological Sciences 15 89ndash93
Meneses-Lorente G de Longueville F Dos Santos-Mendes S Bonnert
T P Jack A Evrard S et al (2003) An evaluation of a low-density
DNA microarray using cytochrome P450 inducers Chemical Research
in Toxicology 16 1070ndash1077
Meneses-Lorente G Guest P C Lawrence J Muniappa N Knowles
M R Skynner H A et al (2004) A proteomic investigation of drug-
induced steatosis in rat liver Chemical Research in Toxicology 17
605ndash612
Merrill C L Ni H Yoon L W Tirmenstein M A Narayanan P
Benavides G R et al (2002) Etomoxir-induced oxidative stress in
HepG2 cells detected by differential gene expression is confirmed
biochemically Toxicology Sciences 68 93ndash101
Milo R Shen-Orr S Itzkovitz S Kashtan N Chklovskii D amp Alon
U (2002) Network motifs Simple building blocks of complex
networks Science 298 824ndash827
Morgan K T Ni H Brown H R Yoon L Qualls C W Crosby L
M et al (2002) Application of cDNA microarray technology to in
vitro toxicology and the selection of genes for a real-time RT-PCR-
based screen for oxidative stress in Hep-G2 cells Toxicologic
Pathology 30 435ndash451
Mwinyi J Johne A Bauer S Roots I amp Gerloff T (2004) Evidence
for inverse effects of OATP-C (SLC21A6) 5 and 1b haplotypes on
pravastatin kinetics Clinical Pharmacology and Therapeutics 75
415ndash421
Nakata K Yukawa M Komiyama N Nakano T amp Kaminuma T
(2002) A nuclear receptor database that maps pathways to diseases
Genome Infomatics 13 515ndash516
Nicholson J K Holmes E Lindon J C amp Wilson I D (2004) The
challenges of modeling mammalian biocomplexity Nature Biotechnol-
ogy 22 1268ndash1274
Nicholson J K amp Wilson I D (2003) Understanding rsquoglobal systems
biology Metabonomics and the continuum of metabolism Nature
Reviews on Drug Discovery 2 668ndash676
Nikitin A Egorov S Daraselia N amp Mazo I (2003) Pathway studiomdash
the analysis and navigation of molecular networks Bioinformatics 19
2155ndash2157
Nikolsky Y Ekins S Nikolskaya T amp Bugrim A (2005) A novel
method for generation of signature networks as biomarkers from
complex high throughput data Toxicology Letters 158 20ndash29
Nikolsky Y Nikolskaya T amp Bugrim A (2005) Biological networks
and analysis of experimental data in drug discovery Drug Discovery
Today 10 653ndash662
Nishizato Y Ieiri I Suzuki H Kimura M Kawabata K Hirota T
et al (2003) Polymorphisms of OATP-C (SLC21A6) and OAT3
(SLC22A8) genes Consequences for pravastatin pharmacokinetics
Clinical Pharmacology and Therapeutics 73 554ndash565
Nozawa T Nakajima M Tamai I Noda K Nezu J Sai Y et al
(2002) Genetic polymorphisms of human organic anion transporters
OATP-C (SLC21A6) and OATP-B (SLC21A9) Allele frequencies in
the Japanese population and functional analysis Journal of Pharmacol-
ogy and Experimental Therapeutics 302 804ndash813
Nozawa T Sugiura S Nakajima M Goto A Yokoi T Nezu J et al
(2004) Involvement of organic anion transporting polypeptides in the
transport of troglitazone sulfate Implications for understanding trogli-
tazone hepatotoxicity Drug Metabolism and Disposition 32 291ndash294
Oliver D E Rubin D L Stuart J M Hewett M Klein T E amp
Altman R B (2002) Ontology development for a pharmacogenetics
knowledge base Package Symposion 88ndash99
Oram J F amp Lawn R M (2001) ABCA1 The gatekeeper for eliminating
excess tissue cholesterol Journal of Lipid Research 42 1173ndash1179
Oswald M Kullak-Ublick G A Paumgartner G amp Beuers U (2001)
Expression of hepatic transporters OATP-C and MRP2 in primary
sclerosing cholangitis Liver 21 247ndash253
Parsons A B Brost R I Ding H Li Z Zhang C Sheikh B et al
(2004) Integration of chemical-genetic and genetic interaction data
links bioactive compounds to cellular target pathways Nature Bio-
technology 22 62ndash69
Patki K C von Moltke L L Harmatz J S Hesse L M Court M H
amp Greenblatt D J (2004) Effect of age on in vitro triazolam
biotransformation in male human liver microsomes Journal of
Pharmacology and Experimental Therapeutics 308 874ndash879
Pereira-Leal J B Enright A J amp Ouzounis C A (2004) Detection of
functional modules from protein interaction networks Proteins 54
49ndash57
Plant N (2004) Interaction networks Coordinating responses to xeno-
biotic exposure Toxicology 202 21ndash32
Raghavendra Prasad H S Qi Z Srinivasan K N amp Gopalakrish-
nakone P (2004) Potential effects of tetrodotoxin exposure to
human glial cells postulated using microarray approach Toxicon 44
597ndash608
Reilly T P Bourdi M Brady J N Pise-Masison C A Radonovich M
F George J W et al (2001) Expression profiling of acetaminophen
liver toxicity in mice using microarray technology Biochemical and
Biophysical Research Communications 282 321ndash328
Rives A W amp Galitski T (2003) Modular organization of cellular
networks Proceedings of the National Academy of Sciences of the
United States of America 100 1128ndash1133
Rogers P D Pearson M M Cleary J D Sullivan D C amp Chapman
S W (2002) Differential expression of genes encoding immunomodu-
latory proteins in response to amphotericin B in human mononuclear
cells identified by cDNA microarray analysis Journal of Antimicrobial
Chemotherapy 50 811ndash817
Rosen M B Wilson V S Schmid J E amp Gray L E (2005) Gene
expression analysis in the ventral prostate of rats exposed to vinclozolin
or procymidone Reproductive Toxicology 19 367ndash379
Rost D Herrmann T Sauer P Schmidts H L Stieger B Meier P J
et al (2003) Regulation of rat organic anion transporters in bile salt-
induced cholestatic hepatitis Effect of ursodeoxycholate Hepatology
38 187ndash195
Rothman R B Ayestas M A Dersch C M amp Baumann M H (1999)
Aminorex fenfluramine and chlorphentermine are serotonin transporter
substrates Implications for primary pulmonary hypertension Circula-
tion 100 869ndash875
Ruepp S U Tonge R P Shaw J Wallis N amp Pognan F (2002)
Genomics and proteomics analysis of acetaminophen toxicity in mouse
liver Toxicology Sciences 65 135ndash150
Sakaeda T Nakamura T amp Okumura K (2002) MDR1 genotype-
related pharmacokinetics and pharmacodynamics Biological amp Phar-
maceutical Bulletin 25 1391ndash1400
Sakata T Anzai N Shin H J Noshiro R Hirata T Yokoyama H
et al (2004) Novel single nucleotide polymorphisms of organic cation
transporter 1 (SLC22A1) affecting transport functions Biochemical
and Biophysical Research Communications 313 789ndash793
Satlin L M Amin V amp Wolkoff A W (1997) Organic anion
transporting polypeptide mediates organic anionHCO3-exchange
Journal of Biological Chemistry 272 26340ndash26345
Satomi Y Tsuchiya W Mihara K Ota M Kasahara Y amp Akahori F
(2004) Gene expression analysis of the lung following paraquat
administration in rats using DNA microarray Journal of Toxicological
Sciences 29 91ndash100
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 65
Schrenk D Baus P R Ermel N Klein C Vorderstemann B amp
Kauffmann H M (2001) Up-regulation of transporters of the MRP
family by drugs and toxins Toxicology Letters 120 51ndash57
Segal E Shapira M Regev A Persquoer D Botstein D Koller D et al
(2003) Module networks Identifying regulatory modules and their
condition-specific regulators from gene expression data Nature
Genetics 34 166ndash176
Segal E Wang H amp Koller D (2003) Discovering molecular pathways
from protein interaction and gene expression Bioinformatics 19
i264ndash i272
Seidel S D Kan H L Stott W T Schisler M R amp Gollapudi B B
(2003) Identification of transcriptome profiles for the DNA-damaging
agents bleomycin and hydrogen peroxide in L5178Y mouse lymphoma
cells Environmental and Molecular Mutagenesis 42 19ndash25
Shan L Yu Y Schut H A J amp Snyderwine E G (2004) Susceptibility
of rats to mammary gland carcinogenesis by the food-derived
carcinogen 2-Amino-1-Methyl-6-Phenylimidazo[45-b]Pyridine (PhIP)
varies with age and is associated with the induction of differential gene
expression American Journal of Pathology 165 191ndash202
Sharom J R Bellows D S amp Tyers M (2004) From large networks to
small molecules Current Opinion in Chemical Biology 8 81ndash90
Shitara Y Hirano M Sato H amp Sugiyama Y (2004) Gemfibrozil and
its glucuronide inhibit the OATP2(OATP1B1 SLC21A6)-mediated
hepatic uptake and CYP2C8-mediated metabolism of cerivastatinmdash
Analysis of the mechanism of the clinically relevant drugndashdrug
interaction between cerivastatin and gemfibrozil Journal of Pharma-
cology and Experimental Therapeutics
Shitara Y Itoh T Sato H Li A P amp Sugiyama Y (2003) Inhibition of
transporter-mediated hepatic uptake as a mechanism for drugndashdrug
interaction between cerivastatin and cyclosporin A Journal of
Pharmacology and Experimental Therapeutics 304 610ndash616
Somogyi R Fuhrman S amp Wen X (2001) Genetic network inference in
computational models and applications to large-scale gene expression
data In M Bower amp H Bolouri (Eds) Computational modeling of
genetic and biochemical networks (pp 119ndash157) Cambridge MArsquoThe
MIT Press
Spirin V amp Mirny L A (2003) Protein complexes and functional
modules in molecular networks Proceedings of the National Academy
of Sciences of the United States of America 100 12123ndash12128
Staudinger J Liu Y Madan A Habeebu S amp Klaassen C D
(2001) Coordinate regulation of xenobiotic and bile acid homeo-
stasis by pregnane X receptor Drug Metabolism and Disposition
29 1467ndash1472
Sun L Z Ji Z L Chen X Wang J F amp Chen Y Z (2002) ADME-
AP A database of ADME associated proteins Bioinformatics 18
1699ndash1700
Suter L Babiss L E amp Wheeldon E B (2004) Toxicogenomics in
predictive toxicology in drug development Chemistry and Biology 11
161ndash171
Synold T W Dussault I amp Forman B M (2001) The orphan nuclear
receptor SXR coordinately regulates drug metabolism and efflux
Nature Medicine 7 584ndash590
Tabuchi Y amp Kondo T (2003) cDNA microarray analysis reveals chop-
10 plays a key role in Sertoli cell injury induced by bisphenol A
Biochemical and Biophysical Research Communications 305 54ndash61
Tamai I Nezu J Uchino H Sai Y Oku A Shimane M et al (2000)
Molecular identification and characterization of novel members of the
human organic anion transporter (OATP) family Biochemical and
Biophysical Research Communications 273 251ndash260
Taylor S W Fahy E Zhang B Glenn G M Warnock D E Wiley S
et al (2003) Characterization of the human heart mitochondrial
proteome Nature Biotechnology 21 281ndash286
Terasaka S Aita Y Inoue A Hayashi S Nishigaki M Aoyagi K
et al (2004) Using a customized DNA microarray for expression
profiling of the estrogen-responsive genes to evaluate estrogen activity
among natural estrogens and industrial chemicals Environmental
Health Perspectives 112 773ndash781
Thomas D M Francescutti-Verbeem D M Liu X amp Kuhn D M
(2004) Identification of differentially regulated transcripts in mouse
striatum following methamphetamine treatmentmdashan oligonucleotide
microarray approach Journal of Neurochemistry 88 380ndash393
Thomas R S Rank D R Penn S G Zastrow G M Hayes K R
Pande K et al (2001) Identification of toxicologically predictive
gene sets using cDNA microarrays Molecular Pharmacology 60
1189ndash1194
Thompson K L Afshari C A Amin R P Bertram T A Car B
Cunningham M et al (2004) Identification of platform-independent
gene expression markers of cisplatin nephrotoxicity Environmental
Health Perspectives 112 488ndash494
Tirona R G Leake B F Merino G amp Kim R B (2001)
Polymorphisms in OATP-C Identification of multiple allelic
variants associated with altered transport activity among European-
and African-Americans Journal of Biological Chemistry 276
35669ndash35675
Tirona R G Leake B F Wolkoff A W amp Kim R B (2003)
Human organic anion transporting polypeptide-C (SLC21A6) is a
major determinant of the rifampin-mediated pregnane X receptor
activation Journal of Pharmacology and Experimental Therapeutics
304 223ndash228
Tong W Cao X Harris S Sun H Fang H Fuscoe J et al (2003)
ArrayTrackmdashsupporting toxicogenomic research at the US Food and
Drug Administration National Center for Toxicological Research
Environmental Health Perspectives 111 1819ndash1826
Tornow S amp Mewes H W (2003) Functional modules by relating
protein interaction networks and gene expression Nucleic Acids
Research 31 6283ndash6289
Trocho C Escola-Gil J C Ribas V Benitez S Martin-Campos J M
Rotllan N et al (2004) Phenytoin treatment reduces atherosclerosis in
mice through mechanisms independent of plasma HDL-cholesterol
concentration Atherosclerosis 174 275ndash285
Ueda A Hamadeh H K Webb H K Yamamoto Y Sueyoshi T
Afshari C A et al (2002) Diverse roles of the nuclear orphan
receptor CAR in regulating hepatic genes in response to phenobarbital
Molecular Pharmacology 61 1ndash6
Ulrich R G (2003) The toxicogenomics of nuclear receptor agonists
Current Opinion in Chemical Biology 7 505ndash510
Ulrich R G Rockett J C Gibson G G amp Pettit S D (2004)
Overview of an interlaboratory collaboration on evaluating the effects
of model hepatotoxicants on hepatic gene expression Environmental
Health Perspectives 112 423ndash427
Unami A Shinohara Y Ichikawa T amp Baba Y (2003) Biochemical
and microarray analyses of bupivacaine-induced apoptosis Journal of
Toxicological Sciences 28 77ndash94
Vasquez A Flammini A Maritan A amp Vespignani A (2003) Global
protein function prediction from proteinndashprotein interaction networks
Nature Biotechnology 21 697ndash700
Vezina C M Walker N J amp Olson J R (2004) Subchronic Exposure to
TCDD PeCDF PCB126 and PCB153 Effect on hepatic gene
expression Environmental Health Perspectives 112 1636ndash1644
Vo T D Greenberg H J amp Palsson B O (2004) Reconstruction and
functional characterization of the human mitochondrial metabolic
network based on proteomic and biochemical data Journal of Bio-
logical Chemistry 279 39532ndash39540
Wang H amp LeCluyse E L (2003) Role of orphan nuclear receptors in
the regulation of drug-metabolising enzymes Clinical Pharmacoki-
netics 42 1331ndash1357
Wang N Silver D L Thiele C amp Tall A R (2001) ATP-
binding cassette transporter A1 (ABCA1) functions as a choles-
terol efflux regulatory protein Journal of Biological Chemistry 276
23742ndash23747
Waring J F Cavet G Jolly R A McDowell J Dai H Ciurlionis R
et al (2003) Development of a DNA Microarray for toxicology based
on hepatoxin-regulated sequences Environmental Health Perspectives
111 863ndash870
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6666
Waring J F Gum R Morfitt D Jolly R A Ciurlionis R Heindel M
et al (2002) Identifying toxic mechanisms using DNA microarrays
Evidence that an experimental inhibitor of cell adhesion molecule
expression signals through the aryl hydrocarbon nuclear receptor
Toxicology 181ndash182 537ndash550
Waring J F Jolly R A Ciurlionis R Lum P Y Praestgaard J T
Morfitt D C et al (2001) Clustering of hepatotoxins based on
mechanism of toxicity using gene expression profiles Toxicology and
Applied Pharmacology 175 28ndash42
Waters M Boorman G Bushel P Cunningham M Irwin R Merrick
A et al (2003) Systems toxicology and the Chemical Effects in
Biological Systems (CEBS) knowledge base EHP Toxicogenomics
111 15ndash28
Wei M Arnold L Cano M amp Cohen S M (2005) Effects of co-
administration of antioxidants and arsenicals on the rat urinary bladder
epithelium Toxicology Sciences 83 237ndash245
Weinshilboum R (2003) Inheritance and drug response New England
Journal of Medicine 348 529ndash537
Weinshilboum R amp Wang L (2004) Pharmacogenetics Inherited
variation in amino acid sequence and altered protein quantity Clinical
Pharmacology and Therapeutics 75 253ndash258
Werner E (2003) In silico multicellular systems biology and minimal
genomes Drug Discovery Today 8 1121ndash1127
Wong J S amp Gill S S (2002) Gene expression changes induced in
mouse liver by di(2-ethylhexyl) phthalate Toxicology and Applied
Pharmacology 185 180ndash196
Xie T Tong L McCann U D Yuan J Becker K G Mechan A O
et al (2004) Identification and characterization of metallothionein-1
and -2 gene expression in the context of ()34-methylenedioxymetham-
phetamine-induced toxicity to brain dopaminergic neurons Journal of
Neuroscience 24 7043ndash7050
Yadetie F Laegreid A Bakke I Kusnierczyk W Komorowski J
Waldum H L et al (2003) Liver gene expression in rats in response
to the peroxisome proliferator-activated receptor-alpha agonist ciprofi-
brate Physiological Genomics 15 9ndash19
Yamashita S Nomoto T Abe M Tatematsu M Sugimura T amp
Ushijima T (2004) Persistence of gene expression changes in stomach
mucosae induced by short-term N-methyl-NV-nitro-N-nitrosoguanidinetreatment and their presence in stomach cancers Mutation Research
549 185ndash193
Yan Q amp Sadee W (2000) Human membrane transporter database A
web-accessible relational database for drug transport studies and
pharmacogenomics AAPS Pharmsci 2 E20
Yeger-Lotem E amp Margalit H (2003) Detection of regulatory circuits by
integrating the cellular networks of proteinndashprotein interactions and
transcription regulation Nucleic Acids Research 31 6053ndash6061
Yoon B I Li G -X Kitada K Kawasaki Y Igarashi K amp Kodama
Y et al (2003) Mechanisms of benzene-induced hematotoxicity and
leukemogenicity cDNA microarray analyses using mouse bone marrow
tissue Environmental Health Perspectives 111 1411ndash1420
Young M B DiSilvestro M R Sendera T J Freund J Kriete A amp
Magnuson S R (2003) Analysis of gene expression in carbon
tetrachloride-treated rat livers using a novel bioarray technology
Pharmacogenomics Journal 3 41ndash52
Yu H Zhu X Greenbaum D Karro J amp Gerstein M (2004) TopNet
A tool for comparing biological sub-networks correlating protein
properties with topological statistics Nucleic Acids Research 32
328ndash337
Zhang E Y Fu D J Pak Y A Stewart T Mukhopadhyay N
Wrighton S A et al (2004) Genetic polymorphisms in human proton-
dependent dipeptide transporter PEPT1 Implications for the functional
role of Pro586 Journal of Pharmacology and Experimental Therapeu-
tics 310 437ndash445
Zhang E Y Knipp G T Ekins S amp Swaan P W (2002)
Structural biology and function of solute transporters Implications
for identifying and designing substrates Drug Metabolism Reviews
34 709ndash750
Zhang E Y Phelps M A Cheng C Ekins S amp Swaan P W (2002)
Modeling of active transport systems Advanced Drug Delivery
Reviews 54 329ndash354
- Systems-ADMETox Resources and network approaches
-
- Introduction
- Data available
- Network analysis and databases
-
- Network applications
-
- The role of transporters
-
- Clinical relevance of transporters
- Transporter network examples ABCA1
- Transporter network examples OATP
- Transporter microarray data
-
- Applications to enzymes
- Future network applications
-
- Discussion
- Acknowledgements
- References
-
Table 3
Literature toxicogenomics data derived from in vitro cell studies
Compounds Cell type Microarray type Compound dose Microarray data
availability
Reference
4-Hydroxytamoxifen
estrogen
MCF-7 breast cancer NIEHS ToxChip
microarray consisting of
1901 genes
1 uM hydroxytamoxifen
for a year 10 nM
17b-estradiol
Data available at NIEHS
website
(Hodges et al
2003)
Trovafloxacin Human hepatocytes Affymetrix U133A array 30ndash800 uM 142 genes available in
supplemental tablemdashnot
easily extracted
(Liguori et al
2005)
Estrogen MCF-7 breast cancer NIEHS ToxChip
microarray consisting of
1901 genes
10ndash10 M 17b-estradiol Data available at NIEHS
website
(Lobenhofer et al
2002)
Valproic acid NMRI mice embryo
and P19 mouse
embryocarcinoma
Custom chip including
15K mouse cDNA clone
set
600 mgkg body weight Gene symbol gene
name NIA EST log fold
change in a manuscript
table
(Kultima et al
2004)
Sulindac sulfide Human colorectal
carcinoma SW-480
and HCT-116
NIEHS human 12K chip 10 uM Genbank accession
number gene name and
fold change at various
time points in a
manuscript table data
also available on NIEHS
website
(Bottone
Martinez Collins
Afshari amp Eling
2003)
17Beta-estradiol estriol
estrone genistein
diethylstilbestrol
bisphenol A
nonylphenol
methoxychlor
MCF-7 U95A oligonucleotide
probe arrays (Affymetrix
10 nM (E2 estriol
estrone DES) 10 AM(genistein bisphenol A
nonylphenol and
methoxychlor)
Unigene name gene
name and fold change
for estrogen responsive
and nonresponsive in a
manuscript table
(Terasaka et al
2004)
Ouabain lauryl sulfate
dimethylsulfoxide
cycloheximide
tolbutamide sodium
fluoride diethyl
maleate buthionine
sulfoxamine
potassium bromate
sodium selenite
alloxan adriamycin
hydrogen peroxide
HepG2 Clontech Atlas Human
Stress Toxicology cDNA
arrays (234 genes)
Ouabain (43 uM) lauryl
sulfate (260 uM)
dimethylsulfoxide (128
M) cycloheximide (625
uM) tolbutamide (128
mM) sodium fluoride (3
mM) diethyl maleate
(125 mM) buthionine
sulfoxamine (30 mM)
potassium bromate (25
mM) sodium selenite (30
uM) alloxan (130 mM)
adriamycin (40 uM)
hydrogen peroxide
(4 mM)
Gene name ratio p -
value in downloadable
tables at journal website
(Morgan et a l
2002)
Aflatoxin B(1) (AFB(1))
2-acetylaminofluorene
(2AAF)
dimethylnitrosamine
(DMN)
acetaminophen (APAP)
HepG2 and primary
hepatocytes
Gene filter arrays
containing 31000 genes
10 uM aflatoxin B1
40 mM acetaminophen
100 uM
dimethylnitrosamine
10 uM
2-acetylaminofluorene
None (Harris et al
2004)
Mitomycin C (MMC)
and cisplatin (CIS)
and an alkylating
agent methyl
methanesulfonate
(MMS)
indirect-acting
genotoxins included
hydroxyurea (HU) a
ribonucleotide
reductase inhibitor
taxol (TXL) a
microtubule inhibitor
and etoposide
(ETOP)
L5178Y TK(+-)
mouse lymphoma
Affymetrix mouse
MG-U74A for MMC
and MG-U745Av2
(Affymetrix Inc Santa
Clara CA) for all the
other chemicals a total
of 9977 probe sets
(genes or ESTs) common
to these two array
models
Low mid and high doses Accession number gene
name gene ID statistical
significance at each time
point in a manuscript
table
(Hu et al 2004)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6646
Table 3 (continued)
Compounds Cell type Microarray type Compound dose Microarray data
availability
Reference
Hydroxyurea
(a carcinogen)
p-anisidine
(a noncarcinogen)
and paclitaxel
L5178Y Tk_-mouse
lymphoma
The Twin - Chip Mouse-
74K Digital Genomics
cDNA microarray
10 ngml paclitaxel 313
ugml hydroxyurea 32
ugml p-anisidine
Gene symbol and fold
change in a manuscript
table
(Lee et al 2003)
Acetaminophen
amiodarone clofibrate
erythromycin estolate
isoniazid alpha-
naphtylylisothiocyanate
beta-naphtoflavone
4-pentenoic acid
phenobarbital
tetracycline and
zileuton
Wistar Rat
hepatocytes
DualChip rat hepato
(Eppendorf Hamburg
Germany)
A single concentration
which varied for each
compound
Accession numbers and
gene namemdashfold
changes shown as
colored heat mapmdashnote
easily extracted from
publication
(de Longueville
et al 2003)
Bleomycin and hydrogen
peroxide
Mouse lymphoma
L5178Y TK(+ -)
Clontech Mouse 12K
cDNA microarray (1185
genes)
Bleomycin (25 and 20
ugml) hydrogen peroxide
(5 and 10 ugml)
Gene names and fold
change presented as bar
charts in publication
(Seidel Kan
Stott Schisler amp
Gollapudi 2003)
Bupivicaine
camptothecin
HL-60 Agilent human cDNA
microarray
1 mM Gene name GenBank
accession number
unigene and ratio in a
manuscript table
(Unami
Shinohara
Ichikawa amp Baba
2003)
Bisphenol A Mouse Sertoli TTE3 IntelliGene mouse
expression glass
microarrays (Version
10 Takara Shuzo)
which were spotted with
564 cDNA fragments of
mouse known genes and
approximately 301
expressed sequence tags
(ESTs)
0ndash400 uM Gene name GenBank
accession number and
fold change at time
points in a manuscript
table
(Tabuchi amp
Kondo 2003)
Mitomycin C or
doxorubicin
Hep G2 85 human gene custom
array
10 um mitomycin C
2 um doxorubicin or
2 ethanol
Bar charts with fold
changes in publication
very few genes
(Hong Muller amp
Lai 2003)
Amphotericin B Human peripheral
blood mononuclear
and THP-1
GF211 FKnown Genes_
Genefilter cDNA array
(ResGen) this array
consists of gt4000
individual elements
each representing a
known human gene
5 ugml Accession numbers and
fold expression in a
manuscript table
(Cleary Rogers amp
Chapman 2001
Rogers Pearson
Cleary Sullivan
amp Chapman
2002)
Benzo(a)pyrene diol
epoxide
TK6 human
lymphoblastoid
Human-350 microarray
a glass slide with 350
spotted human cDNA
probes (Phase-1
Molecular Toxicology
0 001 010 or
10 ugml)
Gene names and fold
change at doses in a
manuscript table
(Akerman et al
2004)
Etomoxir HepG2 Clontech AtlasiHuman Stress
Toxicology cDNA arrays
(234 genes)
1 mM etomoxir Gene names and fold
change in a manuscript
table
(Merrill et al
2002)
Tetrodotoxin Human glioma cell
line HTB-138
Using Affymetrix
GeneChip (HG-U133A
10 and 20 mM Affymetrix ID Genbank
ID gene name gene
symbol and fold change
in a manuscript table
(Raghavendra
Prasad Qi
Srinivasan amp
Gopalakrishnakone
2004)
Methotrexate
mercaptopurine
Human acute
lymphoblastic
leukemia
Affymetrix U133A chip Low and high dose and
combination
Data available as
supplemental data online
(Cheok et al 2003)
Prednisolone
vincristine
asparaginase
daunorubicin
Human acute
lymphoblastic
leukemia
Affymetrix U133A chip Various Data available as
supplemental data online
(Holleman et al
2004)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 47
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6648
A second study has indicated how a natural language
processing method CCNet was used to show the genes
regulated by the nuclear hormone receptor FXR (Apic
Ignjatovic Boyer amp Russell 2005) These automated
methods enable a more complete understanding of the
complexity of the transcriptional factors (Ekins Mirny amp
Schueltz 2002b Plant 2004 Ulrich 2003) but ultimately
rely on the quality of the content of the underlying database
of literature interactions This is a key consideration that is
often overlooked For example the gold standard database is
one that is manually curated to ensure the fidelity of the
direct interaction and is preferable to one generated
computationally by algorithms like natural language pro-
cessing (Nikolsky Nikolskaya amp Bugrim 2005) The
advantage of interaction networks over clustering has been
demonstrated in one study using MetaCore (Nikolsky
Ekins Nikolskaya amp Bugrim 2005) by reanalysis of a
published microarray study of G0-arrested MCF-7 breast
cancer cells treated with estrogen and 4-hydroxytamoxifen
(Hodges et al 2003) After producing integrated gene
networks for each treatment strikingly different patterns
were displayed although both contained early transcriptional
factors myc jun and fos Only the estrogen network featured
induced genes essential for all cell cycle phases (Nikolsky et
al 2005) Similarly microarray data for benzene toxicity
(Yoon et al 2003) has been re-analyzed by focusing on the
genes assessed on the p53 pathway (Ekins et al 2005e) We
envisage that a database of such networks for toxic
compounds will be used for comparing between different
molecules and used in the development of predictive
algorithms for Systems-ADMETox modeling in future
Another approach to using such pathway approaches is to
visualize the results of quantitative structure activity models
for predicting molecules binding to enzymes transporters
receptors and ion channels (Ekins Andreyev et al in press
2005e)
It is also possible to simultaneously interpret high
throughput data and predictions on interaction networks
providing a novel approach to predicting and understanding
potential undesirable drugndashdrug or off target effects in the
area of systems pharmacology An example data set uses
percent inhibition data for clotrimazole and ticonazole
which were screened against many different assays at a
single concentration in a commercially available database
BioPrint (Cerep Redmond WA) as published recently
(Fliri Loging Thadejo amp VOlkman 2005) The data for
10 assays has been arbitrarily encoded as inhibitors (gt50
inhibition) or non-inhibitors (lt50 inhibition) in a text file
which was then loaded into MetaCore The analyzed
network algorithm was then used which generates a large
network and fragments it into sub-networks each with a Z-
score and p-values for ranking according to saturation with
objects from the initial gene list The Gene-ontology
processes are also mapped to the gene list and individual
networks In this example a statistically significant network
was generated for the different proteins (Fig 4
p =2838e31) This network also maps the Gene Ontology
processes for the activation of MAPK (118 of genes p
value 9143e07) signal transduction (333 1600e05)
regulation of transcription DNA-dependent (294
2786e04) regulation of inflammatory response (39
3746e04) and the regulation of blood pressure (78
4230e04) This example network indicates how molecules
of the same or different therapeutic classes could be
evaluated for their effects as a graph either together as in
this case or individually This would be useful to indicate
potential off target effects and identify structurally dissimilar
molecules with similar network patterns Such networks
could then be compared to assess network overlap or
differences between molecules and their inhibition of
multiple proteins This type of unique visualization of high
throughput screening data illustrates how the target proteins
may be connected as a network to infer the possible
downstream effects of inhibition
With the burgeoning number of freely available online
and commercial databases that can be used for pathway
construction numbering in the hundreds there have been
suggestions to impose standards for model exchange
querying and visualization (Cary Bader amp Sander 2005)
To date there has been little discussion with regards to
standardization of ADMETox related databases although
there has been considerable discussion relating to drug
metabolism database generation (Erhardt 2003) This is
certainly an important area to address in the future There is
already a growing literature related to ADMETox that is
partially captured in the several commercially available
databases (Ekins et al 2005e) but to date there have been
limited academic efforts to capture data for transporters
with the human membrane transporter database (Yan amp
Sadee 2000) TP-search transporter database (httpwww
ilabrisewasedaacjptp-search) drug interaction database
(httpwwwdruginteractioninfoorgDatabaseinfoaspx)
nuclear hormone receptors (Nakata Yukawa Komiyama
Nakano amp Kaminuma 2002) the ADME-AP database
(Sun Ji Chen Wang amp Chen 2002) and PharmaGKB
(Oliver et al 2002) DSStox (httpwwwepagovnheert
dsstox) TOXNET (httptoxnetnimnihgov) that are
readily accessible
In order to generate accessible pathways using any of
the available software a large enough set of object
identifiers are required to map onto the underlying data-
base To demonstrate this datasets from toxicogenomics
studies have been evaluated with both the KEGG pathway
database and a commercially available product MetaCore
(wwwgenegocom) These gene or protein lists range in
size from 21 to 1853 objects In virtually all cases more
identifiers are mapped to networks in MetaCore and this
also seems independent of the identifier type used
(Unigene Affymetrix Genbank or Locuslink Table 5)
These numbers will obviously change as more information
is added to the respective databases hence the more
objects that are mapped from a dataset The more extensive
Table 4
Literature derived toxicoproteomics data
Compounds Data source Microarray type Compound dose Microarray data
availability
Reference
Carbon
tetrachloride
Male Wistar rats Affymetrix rat chip 8799
probes+proteomics
study
6ndash24 h exposure Genbank SwissProt and
binary data in a
manuscript table
(Fountoulakis
2004)
Paracetamol CD-1 male mice Custom mouse tox blots
with 450 genes
RTQ-PCR+proteomics
study 2-DIGE+
MALDI-MS
150 or 500 mgkg Gene name IMAGE ID
GenBank accession
number fold induction
SwissProt identifier and
protein abundance
change in manuscript
tables
(Ruepp Tonge
Shaw Wallis amp
Pognan 2002)
Paracetamol AP-1 male mice Affymetrix murine 11K
set+proteomics study
Up to 500 mgkg Gene names and fold
changes at multiple
doses and time points in
a manuscript tablemdash
proteomics data not
accessible
(Coen et al 2004)
Oxazepam or
Wy-14643
Male B6C3F1 mice NIEHS Mouse Chip
(8700 genes) 2-DIGE
and MS
Oxazepam (2500 ppm)
Wyeth (Wy)-14643
(500 ppm)
Proteomics data in a
manuscript table
Genbank accession
number gene name and
fold change in a
manuscript table data
also available to
download from NIEHS
website
(Iida et al 2003)
Compound A
(PPAR gamma
ligand)
Female rats CrlCD
(SD)IGS BR
Proteomics 2-DIGE and
MS
250 mgkgday up to
5 days
Accession number and
protein name and
average ratio in a
manuscript table
(Meneses-Lorente
et al 2004)
Bromobenzene Male Wistar rats Proteomics 2-DIGE and
MS custom 3000 cDNA
rat chip
5 mmolkg Accession number gene
name fold change in a
manuscript table protein
names along with bar
charts
(Heijne Stierum
Slijper van
Bladeren amp van
Ommen 2003)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 49
the network that can be generated (as it will consist of more
nodes) then a more comprehensive understanding of the
networks is possible The data available currently in the
literature can be used to evaluate such pathway and
network generation tools Recently we have used several
of the published studies (Tables 1ndash3) with MetaCore to
visualize networks for acetaminophen furan carbon tetra-
chloride benzene and cisplatin showing genes involved in
In vivo
In vitro
In Silico
1970s 1980s 1990s 2000s
Systems Biology ()
OMICS
Fig 2 The timeline for major paradigms in ADMETox
oxidative stress (Ekins Giroux Nikolsky Bugrim amp
Nikolskaya 2005c)
Other important pathwaynetwork building tools that could
potentially be applied to toxicogenomics data include
Ingenuity pathways analysis (httpwwwingenuitycom)
PathArt (httpwwwjubilantbiosyscompdhtm) Pathway
Assist (httpwwwariadnegenomicscomproctspathway
html) (Nikitin et al 2003) and several other databases
deposited at the Pathway Resource List (httpcbiiomskcc
orgprl) All of these products have unique underlying
proprietary pathway databases which are compiled manually
or automatically with text mining tools or a combination of
both We are still waiting for studies that provide a comparison
of different database tools or network building algorithms
Until then theremay be someoverlap but also some differences
between the results obtained from more than one network
method due to the database content and the algorithms used
The reader is recommended to evaluate for themselves several
technologies and select those with the most appropriate fit to
their own specialized needs
In the next Sections the further application of some
available network and database tools will be described with
Microarray or other high throughput data
Upload list of geneprotein identifiers and fold change significance etc (raw data or after selection of the differentially expressed
Genes using other statistical methods)
Parse database and generate interaction network with different algorithms or visualize on maps
Filter Networks
Access significance of the interaction networks
Determine GO processes
Compare 2 or more networks intersection overlap etc
Export gene list
Export gene list
generate interaction network with different algorithms or visualize on maps
Visualization
Fig 3 Schematic for the utilization of pathway tools for assessing high throughput data
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6650
specific detailed reference to transporters and enzymes but
these technologies can also be applied elsewhere as
described above
Fig 4 Network for high throughput screening data for clotrimazole and ticona
cholecystokinin CCR1 Choline transporter Chloride channel catechol-O-methyl
and co workers (Fliri et al 2005) Nodes highlighted with large blue circles rep
molecules showing gt50 inhibition in the assay blue circles represents both mol
inhibits gt50 and one inhibits lt50 Ligands (large) linked to transcriptional f
(wwgenegocom) (For interpretation of the references to colour in this figure leg
311 The role of transporters
A diverse array of organic solutes such as nutrients
neurotransmitters and drugs are transported by specialized
zole screened against 10 in vitro assays (Human Cannabinoid 1 Human
transferase COX2 CYP1A2 CYP2B6 CYP2C19) data published by Fliri
resent assays used in the study Red circles on these nodes represent both
ecules showing lt50 inhibition chequered circles represent one molecule
actors enzymes and transporters via edges using the MetaCorei database
end the reader is referred to the web version of this article)
Table 5
Datasets acquired from publications and other sources and used to evaluate two databasepathway tools KEGG and metaCore
Dataset Number of objects
genes proteins in list
Number of objects
mapped with KEGG
Number of objects mapped
with MetaCore on networks
Reference for microarray
data gene list
Tox Chip 10 (NIEHS) 1853 405 1498 httpsdir-appsniehsnihgov
mapsguestclonesrchcfm
4-Hydroxytamoxifen and
estrogen
1617 434 1343 (Hodges et al 2003)
Mitochondrial proteins 722 156 388 (Gaucher et al 2004
Taylor et al 2003)
Bromobenzene
(24 and 48 h)
130 41 89 (Heijne et al 2004)
Acetaminophen 30 19 23 (Heinloth et al 2004)
Acetaminophen 84 29 64 (Huang et al 2004)
Furan 185 64 139 (Huang et al 2004)
Tetrodotoxin 116 31 86 (Raghavendra Prasad et al
2004)
Benzene 73 16 62 (Yoon et al 2003)
Benzene 76 5 53 (Faiola et al 2004)
Carbon tetrachloride 37 8 26 (Young et al 2003)
Estrogen 94 33 90 (Lobenhofer et al 2002)
Trovafloxacin 142 20 82 (Liguori et al 2005)
Phenobarbital 37 13 28 (Ueda et al 2002)
L-742694 (liver)+ 45 17 19 (Hartley et al 2004)
L-742694 (intestine)+ 23 11 10 (Hartley et al 2004)
A-277249 21 7 9 (Waring et al 2002)
All files were converted to Locuslink identifiers except as noted () unigene () Affymetrix (+) Genbank identifiers were used
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 51
proteins across cellular membranes These may function as
passive processes or active processes energized by the
hydrolysis of ATP or coupling to the co-transport of counter
ions down an electrochemical gradient such as Na+ H+ and
Cl There are many thousands of transporters which can be
classified into distinct superfamilies One of these the solute
carrier class (SLC) is rapidly expanding and contains over
30 families and 200 members The ATP-binding cassette
(ABC) contains 7 families and over 48 members including
P-glycoprotein (P-gp) and MRP subfamilies (Zhang Knipp
Ekins amp Swaan 2002a Zhang Phelps Cheng Ekins amp
Swaan 2002b) Transporters have a key role in clinical
pharmacology with many drugs specifically targeting them
Numerous drugs share transport pathways with nutrients
and transporters have a role in oral absorption drug
bioavailability drug resistance excretion and ultimately
pharmacokinetics and pharmacodynamics
Polymorphism of drug transporters may be a key factor
in drug interactions and lack of effectiveness This field has
become known as pharmacogenomics and is focused on
understanding of inherited DNA sequence variations (poly-
morphisms and mutations) revealed by xenobiotics (Evans
amp McLeod 2003 Weinshilboum 2003) Over the last few
decades many genes have been directly linked to the
mechanisms of response (Evans amp McLeod 2003 Wein-
shilboum 2003) such that 20ndash95 of variability to drug
response is inheritable (Evans amp McLeod 2003) This
phenotypical variability is mainly caused by single nucleo-
tide polymorphisms (SNPs) present in anywhere from 1 to
50 of the population resulting in either lower protein
activity incorrect folding or rapid degradation via proteo-
somes (Weinshilboum amp Wang 2004) A number of
structurally diverse molecules bind to P-gp which is
expressed in many tissues and has numerous SNPs one of
which (C3435T) affects the expression level in the
duodenum and therefore can impact the absorption of
molecules which would be substrates for this transporter
(Sakaeda Nakamura amp Okumura 2002) The human
proton-dependent dipeptide transporter (hPEPT1) can also
affect the absorption of molecules in the intestine and
recently 9 SNPs were found with only one displaying a
reduced transport capacity (Zhang et al 2004) The sodium-
dependent carnitine cotransporter OCTN2 can possess
mutations and these result in primary carnitine deficiency
which impacts fatty acid oxidation and is characterized by
many clinical manifestations (Lahjouji Mitchel amp Qureshi
2001) The organic cation transporter 1 (OCT1) is also
important in the transport of numerous xenobiotics and
endobiotics Recently 4 SNPs were identified in the
Japanese population and when functionally characterized
in vitro the uptake of cations was reduced significantly for
some of these mutations indicating that this would likely
contribute to inter-individual variations in metabolism of
drugs which were transported via OCT1 (Sakata et al
2004)
3111 Clinical relevance of transporters The pregnane
X-receptor (PXR) is a transcriptional regulator of the
enzyme human MDR1 (P-gp) MRPs and OATP (Wang amp
LeCluyse 2003) CYP3A (Bertilsson et al 1998 Blumberg
et al 1998 Kliewer et al 1998) and CYP2C89 (Synold
Dussault amp Forman 2001) as well as many other genes
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6652
involved in the transport metabolism and biosynthesis of
bile acids (Staudinger Liu Madan Habeebu amp Klaassen
2001) However the additional receptors CAR FXR LXR
and other nuclear receptors take part in a complex network
of interactions to control these and other proteins Thus
elucidation of the regulatory networks which control the
expression of these transporters is also important To date
most of the research has centered on efflux transporters but
there has been considerable interest in uptake transporters
such as the organic anion transporter polypeptide (OATP
((Kim 2003) see also later Section on OATP)
There are several specific examples of the importance of
drug transporters to the clinical development of drugs One
example is the insulin sensitizer troglitazone which was
withdrawn due to hepatotoxicity although the precise
mechanism appears to have been unclear until recently
The major metabolite is a sulfated species and is suspected
of being responsible for the observed toxicity The recent
assessment of the organic anion transporting polypeptides
OATP-C and OATP8 expressed on the hepatocyte baso-
lateral membrane indicated that sulphated troglitazone has a
high affinity for the former and possibly lower affinity for
the latter (Nozawa et al 2004) This metabolite would
therefore be expected to accumulate in hepatocytes and
inhibit the bile salt export pump and Mrp2 Because
polymorphisms have been shown for OATP-C (Tirona
Leake Merino amp Kim 2001) it is also possible that these
may result in the accumulation of the metabolite and in turn
elicit idiosyncratic toxicity A second relevant example of
the impact of transporters is the clinically significant drugndash
drug interaction between cerivastatin and cyclosporine A
which occurs via the OATP-C transporter (Shitara Itoh
Sato Li amp Sugiyama 2003) A third example are the HIV
protease inhibitors saquinavir ritonavir and indinavir which
are transported by MRP2 in vitro and other drugs such as
probenecid and sulfinpyrazone are able to enhance this
transport Transport by MRP2 suggests that these com-
pounds will have decreased bioavailability due to increased
clearance and other drugs could aggravate this situation by
further enhancing transport (Huisman et al 2002) Sim-
ilarly the rifampicin mediated induction via PXR of MRP2
and P-gp in healthy subjects was found to significantly
decrease the AUC and also correlated with intestinal
expression of these transporters This transporter is also
inducible by cisplatin 2-AAF and phenobarbital (Schrenk
et al 2001) indicating multiple mechanisms may be
involved In other species such as rat commonly used as a
toxicity model orthologs of the transporters such as OATP2
are expressed and can be induced with ligands for PXR like
PCN (Guo Choudhuri amp Klaassen 2002) This is useful
knowledge because the advent of microarray technology
allows one to dose a rat with a xenobiotic and assess
thousands of genes simultaneously in a particular tissue For
instance animals dosed with known nephrotoxins have
shown some upregulation of the NandashKndashCl transporter
however the authors suggested genomic responses are
stronger soon after exposure before declining (Fleck et
al 2003) Some transporters may therefore be specifically
targeted by drugs in one tissue such as the CNS but these
same transporters may also be expressed elsewhere in the
body hence off-target effects may result in toxicity The
serotonin transporter is one such example which is
expressed in the lungs and brain Some substrates for this
channel like fenfluramine can result in primary pulmonary
hypertension as they accumulate in pulmonary cells (Roth-
man Ayestas Dersch amp Baumann 1999) Similarly P-gp is
expressed at the blood brain barrier and intestine impacting
the efficacy and bioavailability of drugs
3112 Transporter network examples ABCA1 The
ABCA1 transporter mediates the first step of cholesterol
transport Mutations in this gene cause Tangier disease
which results in severe HDL deficiency cholesterol
accumulation in macrophages and attendant atherosclerosis
This transporter represents a drug target for upregulation
modulating cholesterol metabolism and prevention of
cardiovascular disease (Oram amp Lawn 2001) In vitro
ABCA1 can be inhibited by the sulfonylurea glybenclamide
(Field Burn amp Mathur 2004 Wang Silver Thiele amp Tall
2001) In order to illustrate the advantages of mapping drug
transporters as networks onto functional models alongside
other proteins one can consider the example of ABCA1
The query of the MetaCorei database shows that this
transporter appears on three manually curated pathway maps
representing the Fstate of the art_ knowledge derived from
reliable high quality literature sources One can also use the
individual maps as an interface to access the underlying
layers of information about the transporter including the list
of encoding genessplice variants with known SNPs In
addition to browsing MetaCorei a user can also build
custom networks around ABCA1 using the network-
construction tool (Fig 5) Such a visualization utility may
be very helpful for identification of all putative pathways
around a particular transporter or compound of interest The
ABCA1 network created by this tool shows that this
transporter at the time of writing is linked directly to
twenty-five other objects such as APOE1 and LXR Many
of its neighbors have their own SNPs that could be
important in determining interactions between transport of
a drug and normal human transport of endogenous ligands
in health or disease
3113 Transporter network examples OATP The
OATPs are key membrane bound transporters expressed in
many organs including intestine liver lung choroid plexus
blood brain barrier and other organs (Tamai et al 2000)
This family of transporters is capable of mediating the
sodium-independent transport of a diverse array of mole-
cules such as steroid conjugates organic anions and
xenobiotics by coupling uptake with efflux of bicarbonate
(Satlin Amin amp Wolkoff 1997) glutathione or its
conjugates (Hagenbuch amp Meier 2004) The inhibition of
Fig 5 The network of direct gene interactions around the ABCA1 transporter gene (centre) generated with MetaCorei The interation types between nodes are shown as small colored hexagons eg unspecified
allosteric regulation binding cleavage competition covalent modification dephosphorylation phosphorylation transcription regulation transformation When applicable interactions also have a positive or
negative effect and direction Ligands (purple) linked to other proteins (blue blobs) transfactors (red) enzymes (orange) (For interpretation of the references to colour in this figure legend the reader is referred to
the web version of this article)
SEkin
sJournalofPharm
acologica
landToxico
logica
lMeth
ods53(2006)38ndash66
53
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6654
this transporterrsquos hepatic uptake of other compounds may be
important for reported drugndashdrug interactions (Kim 2003)
described earlier (Shitara et al 2003) as well as cerivastatin
with gemfibrozil (Shitara Hirano Sato amp Sugiyama 2004)
OATP1B1 (previous names OATP-C LST-1 OATP2
SLC21A6) represents the most studied human OATP to
date (Meier amp Stieger 2000) and is expressed on the
basolateral plasma membrane of hepatocytes Several single
nucleotide polymorphisms have been identified in the
OATP1B1 gene in European-Americans African-Americans
(Tirona et al 2001) and Japanese (Nozawa et al 2002)
dramatically impacting the transport of ligands such as
pravastatin (Mwinyi Johne Bauer Roots amp Gerloff 2004
Nishizato et al 2003) estrone-3-sulfate (Nozawa et al
2002 Tirona et al 2001) Rifampin (Tirona Leake
Wolkoff amp Kim 2003) and estradiol 17h-d-glucuronide(Tirona et al 2001)
The regulation of SLCO may be affected during
extrahepatic cholestasis bile duct ligation bile salt induced
Fig 6 Networks generated with the autoexpand algorithm in MetaCorei to illustr
be interconnected with other protein regulatory signaling information Informa
unspecified allosteric regulation binding cleavage competition covalent m
transformation When applicable interactions also have a positive or negative e
transfactors (red) enzymes (orange) (For interpretation of the references to colour
cholestatic hepatitis (Dumont et al 1997 Meier amp Stieger
2000 Rost et al 2003) and primary sclerosing cholangitis
(Oswald Kullack-Ublick Paumgarter amp Beuer 2001) In
particular OATP1B1 appears to be regulated by the liver-
enriched transcription factor hepatocyte nuclear factor 1a
(HNF1a) which binds to the promoter region of this
transporter (Jung et al 2001) Site directed mutagenesis
of this binding site resulted in inactivation suggesting the
critical nature of the interaction with HNF1a Bile acids
such as CDCA have been shown to transcriptionally repress
HNF1a in vitro via inhibition of the transactivating effect of
HNF4a on HNF1a (Jung amp Kullak-Ublick 2003) After
screening many rat and human uptake transporters in vitro
OATP1B1 was also shown to modulate the PXR response
by controlling rifampin retention in the cell and therefore
affecting the induction of CYP3A4 and other gene products
such as P-gp (Tirona et al 2003)
Some of the literature for OATP1B1 human substrate
data has been annotated into MetaCorei to illustrate the
ate how ligands for the human OATP-C (OATP1B1) transporter (centre) can
tion on the type of interaction between objects is hidden for clarity eg
odification dephosphorylation phosphorylation transcription regulation
ffect and direction Ligands (purple) linked to other proteins (blue blobs)
in this figure legend the reader is referred to the web version of this article)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 55
visualization of the complex interconnections between this
transporter its ligands regulatory factors and signaling
molecules already in the database (Fig 6) The network was
generated with the autoexpand algorithm in the software
representing one of multiple available algorithms for
connecting genes ligands and other objects in the database
Clearly if more ligands and their connections are added to
the database the complexity of the network will increase
considerably The OATP-C (OATP1B1) gene details can be
viewed upon querying the database and links are provided
to other public databases This page can be used to highlight
the multiple synonyms for this gene as well as links to the
multiple SNPs identified to date
3114 Transporter microarray data Microarrays have
generally been limited in the number of transporters present
on them (Annereau et al 2004) however they have been used
in an attempt to correlate pharmacokinetic properties with
gene expression for valacyclovir (Landowski et al 2003) as
well as understand the expression profile in different tissues
or cell lines upon food component or xenobiotic treatment
(Anderle Huang amp Sadee 2004) This lack of transporters
on microarrays has prompted some groups to produce their
own arrays with a heavier emphasis on transporters These
arrays have for example then been used to demonstrate the
upregulation of ABC transporters and down-regulation of
GST-Pi in cell lines resistant to colchicines or 9-nitro-
camptothecin (Annereau et al 2004) The genes that were
significantly up or down-regulated in this particular study
were used to build networks with MetaCore (Fig 7A B) and
the similarities between them were assessed (Fig 7C)
Although there were only a small number of significantly
changed genes in common (IL-8 Fos GST-Pi Calpactin and
Ubiquitin hydrolase) it is perhaps likely that there is a much
larger common gene network that is important for drug
resistance although a much larger number of cell lines and
drug treatments need to be evaluated to produce a definitive
drug resistance signature involving transporters enzymes
and transcriptional regulators
312 Applications to enzymes
As we have already described it may be particularly
valuable to visualize enzymes as networks to show
interactions with transcriptional regulators and ligands
For example a key enzyme is CYP3A4 which metabolizes
40ndash45 of all drugs and has relatively few SNPs
(Ingelman-Sundberg 2004) Using a second software suite
MetaDrugi (wwwgenegocom) it is possible to construct
a custom network around this or other drug metabolizing
enzymes (Fig 8) In this case the gene network for
CYP3A4 highlights all of the major transcriptional
regulators and several more distant linked proteins and
ligands connected on the network that may be useful for
further study (Fig 8) Substrates inhibitors as well as
regulatory factors and other enzymes can be observed
connected on this network Due to the many hundreds of
known inhibitors and substrates only a small number are
shown here for clarity We assume that if a perturbation in
a pathway (eg due to a nonfunctional enzyme) is linked
to a certain pathologic condition a similar perturbation
caused by the interference from xenobiotic metabolism
(eg competitive inhibition of the same enzyme) may
result in identical effects As microarray gene expression
data is increasingly generated the role of enzyme
regulation in toxicity of certain xenobiotics will become
more apparent from either in vivo or in vitro studies The
visualization of such signature gene networks involving
transporters and enzymes their ligands and regulatory
factors will also be important for future toxicity prediction
methods We have recently generated visualizations of
microarray data from MCF-7 cells treated with 4-hydrox-
ytamoxifen to show that some of the key genes involved in
metabolism and transport are upregulated (Ekins et al
2005d) In addition we have made predictions with various
QSAR models in MetaDrug to indicate the involvement of
PXR CYP3A4 and P-gp (Ekins et al 2005e) Therefore it
appears likely that 4-hydroxytamoxifen could increase its
own metabolism as well as efflux from cells via P-gp
which can be visualized on networks Any decrease of
function of these enzymes or transporters in a population
would likely result in changes in the metabolism and
transport of this active metabolite potentially impacting
the clinical effect This represents one example of how
both pathway tools QSAR models and network building
algorithms can be used with different types of predicted
and experimental data to allow visualization of potential
compound interactions or toxicity
313 Future network applications
As the population ages an increasing incidence and
prevalence of systemic diseases especially chronic diseases
have occurred among older adults This has resulted in an
increase in medications used concomitantly by this
population which presents challenges for drugndashdrug
interactions Physiologically elderly patients may behave
differently to the younger patients for which the drugs are
initially developed for and tested on Many pharmacoki-
netic investigations in the elderly population reveal
decreased clearance of lipophilic drugs metabolized by
the cytochrome P450 enzymes however few studies have
evaluated aging-dependent or gender-related changes in
specific P450 enzymes (Hunt Westerkam amp Slave 1992)
Age-related physiological changes such as a reduction in
liver mass hepatic metabolizing enzyme activity liver
blood flow and alterations in plasma drug binding may
account for the decreased elimination of some metabolized
drugs in the elderly It is particularly difficult to separate an
effect of aging from the variation in the rate of metabolism
due to factors such as individual metabolic phenotype
(slow or fast metabolizer due to SNPs) environmental
influences concomitant disease states and drug intake
(drugndashdrug interactions) The available data suggest that
Fig 7 Networks for drug resistant cell lines generated with the Auto expand algorithm in MetaCore A KB-3-1 vs KB-8-5 B ATC-DU-145 vs RCO1 C node overlap of A and B with initial nodes highlighted
with blue or red solid circles (Initial gene lists derived from (Annereau et al 2004)) Information on the type of interaction between objects is hidden for clarity eg unspecified allosteric regulation binding
cleavage competition covalent modification dephosphorylation phosphorylation transcription regulation transformation When applicable interactions also have a positive or negative effect and direction
Ligands (purple) linked to other proteins (blue blobs) transfactors (red) enzymes (orange) (For interpretation of the references to colour in this figure legend the reader is referred to the web version of this article)
SEkin
sJournalofPharm
acologica
landToxico
logica
lMeth
ods53(2006)38ndash66
56
Fig7(continued)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 57
Fig7(continued)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6658
Fig 8 The network of gene interactions around the CYP3A4 enzyme (centre) from MetaCorei Information on the type of interaction between objects is
hidden for clarity eg unspecified allosteric regulation binding cleavage competition covalent modification dephosphorylation phosphorylation
transcription regulation transformation When applicable interactions also have a positive or negative effect and direction Ligands (purple) linked to other
proteins (blue blobs) transfactors (red) enzymes (orange) (For interpretation of the references to colour in this figure legend the reader is referred to the web
version of this article)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 59
the initial doses of drugs metabolized by these enzymes
should be reduced in older patients according to the clinical
response In most published studies the elderly appear at
least as responsive as the young to inducers or inhibitors of
P450s (Durnas Loi amp Cusack 1990) More recently there
has been some suggestion that there are age related
reductions in function of some specific P450s such as
CYP3A4 (Patki et al 2004) and this could occur at the
level of regulation However earlier studies with the same
enzyme showed no change in clearance with age (Hunt et
al 1992) To date there has been even less examination of
the transporter functions and any changes with age
(Kinirons amp OrsquoMahony 2004) so the current understand-
ing of the effects of aging on metabolism and transport is
anything but transparent This represents an extreme
challenge for the pharmaceutical industry how to predict
whether a drug has an affinity for an enzyme or transporter
and whether this may be clinically important if it shows a
decline in expression or function with aging Any decrease
of function of these enzymes or transporters in an elderly
population would result in changes in the metabolism and
transport of metabolites potentially impacting the clinical
effect This represents another example of how computa-
tional approaches may perhaps be used with different types
of predicted and experimental data to allow visualization of
potential compound interactions or toxicity in elderly
populations We may see different gene networks high-
lighted as humans age and these may be modified by drug
treatment and coadministration This represents an area
were network analysis could be applied in the future and is
in need of considerable further research
The collection of microarray data in databases such as
CEBS ArrayTrack and EDGE represents a future resource
for computational gene network analysis One could
envisage that ultimately in each case such data is converted
to one or more networks that are also displayed for the user
and can be used to compare treatments from in vivo and in
vitro experiments This would represent a different approach
to clustering the data as currently implemented in one of
these efforts (Hayes et al 2005) and may condense large
amounts of experimentally derived data into a readily
interpreted network
4 Discussion
Previously in this journal the progress of many
research groups in predicting human ADME parameters
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6660
in silico (Ekins et al 2000b) and approaches for drug
metabolism (Ekins Ring Grace McRobie-Belle amp
Wrighton 2000a) have been described Both of these
reviews commented on moving HT assays for ADME
Tox much earlier in drug discovery which had also been
indicated by other groups The initial reviews also
highlighted the likely wealth of data that would become
available and how this could be used for structure
activity relationships alongside the bioactivity data in
computational models It was noted that there was a
paucity of predictive metabolism tools at that time In
addition a growing number of efforts to model whole
cells and organs now a field called systems biology
were recognized as models that could be integrated with
the in silico ADME approaches In summary since these
past reviews virtually all pharmaceutical companies have
attempted earlier high throughput screening for ADME
Tox properties and to some extent the wider application
of computational approaches for physicochemical proper-
ties Systems biology is being quite widely acknowledged
as the new paradigm for understanding complex bio-
logical datasets derived from high throughput technolo-
gies and the accumulated knowledge on human protein
interactions (Hartwell Hopfield Leibler amp Murray 1999
Hood 2003b) Therefore systems biology can be defined
as the integration of genetic proteomic transcriptomic
and metabonomic data using computational methods
(Nicholson amp Wilson 2003) When taken together
information on molecular processes derived from different
sources represents a lsquolsquouniversersquorsquo of putative biological
functionality of which only a small fraction of it will be
realized in a cell at any given time To date systems
biology has been driven by academia and funding bodies
such as the NIH rather than the pharmaceutical compa-
nies Presently there is a great deal of interest from
scientists of all backgrounds in identifying the networks
of cellular pathways and the corresponding physically
interacting proteins
The network building software for systems biology
described in this current review will be valuable to query
high throughput data and known literature interactions in
order to predict potential toxicity in different species In the
future the compilation of published toxicogenomics data-
sets characteristic of different types of toxicity will likely
be available in these software systems to act as a reference
database It is also feasible that we will be able to generate
the annotated datasets which specifically address the
differences between human and rat networks implicated
in toxicity The identification of sub-network modules
conserved between human and rat distinct for toxicity
types or predictive for toxic end-points in human will be
possible Such signature gene networks (Nikolsky et al
2005) can then be verified with other experimentally
derived data prospectively or from preexisting databases
The combined Fhigh throughput datandashin silico network_approaches therefore represents a novel method for access-
ing complex tissue-level processes as networks integrating
all data types based on functional interactions The data-
bases developed and used in this approach will certainly
benefit from further annotation around the drug metaboliz-
ing enzymes and transporters as described herein in terms
of transcriptional regulation and the ligands associated with
them which frequently appear in the literature It will be
important to capture disparities in the assignment of ligands
to enzymes transporters and other proteins as well as
negative data
In summary although there have been numerous
toxicogenomics studies published there is presently a
relatively small number of datasets that are freely available
to perform network analysis of microarray data The
number of studies identifying large numbers of proteins
which are affected by molecule treatment are even scarcer
still while there are several examples of some published
studies that combine such data It is hoped that the
numerous database initiatives for high content and toxicol-
ogy data that are being undertaken will improve the
situation for other researchers that are not currently
equipped to do such microarray studies themselves The
addition of requirements by journals to deposit such raw
data in a freely accessible resource will aid these initiatives
ADMETox groups have seen new technologies and
approaches developed over the last decade that have all
been applied to identify poor compounds earlier (Fig 2)
The latest technologies integrate network building tools
with high content data and databases The current review
described the limited number of networks generated for
ADMETox at present and one hopes that the impact of
such analyses will be commonplace in the future Systems
biology is however more than just applying a network
approach and hence systems-ADMETox will have to
evolve due to the continual pressure to develop newer
technologies This current paradigm combining empirical
data and computational methods should integrate the
complex data already generated making it readily inter-
pretable and valuable for identifying the most promising
compounds in the future
Acknowledgements
Dr Maggie AZ Hupcey is gratefully acknowledged for
editorial assistance Dr Peter W Swaan (University of
Maryland) and Dr Cheng Chang (Ohio State University)
Dr Steve Wright (University of Arizona) Dr K Sandy
Pang (University of Toronto) and Dr Craig Giroux (Wayne
State University) are kindly acknowledged for their support
and discussions My colleagues at GeneGo Sergey
Andreyev Andy Ryabov Eugene Kirillov Eugene A
Rakhmatulin Svetlana Sorokina Andrej Bugrim Tatiana
Nikolskaya Yuri Nikolsky John Metz and Julie Bryant are
all thanked for their considerable contributions to software
development and data annotation
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 61
References
Abe M Yamashita S Kuramoto T Hirayama Y Tsukamoto T Ohta
T et al (2003) Global expression analysis of N-methyl-NV-nitro-N-nitrosoguanidine-induced rat stomach carcinomas using oligonucleotide
microarrays Carcinogenesis 24 861ndash867
Adachi T Ono Y Koh K B Takashima K Tainaka H Matsuno Y
et al (2004) Long-term alteration of gene expression without
morphological change in testis after neonatal exposure to genistein in
mice Toxicogenomic analysis using cDNA microarray Food and
Chemical Toxicology 42 445ndash452
Akerman G S Rosenzweig B A Domon O E McGarrity L J
Blankenship L R Tsai C A et al (2004) Gene expression profiles
and genetic damage in benzo(a)pyrene diol epoxide-exposed TK6 cells
Mutation Research 549 43ndash64
Aksenov S V Church B Dhiman A Georgieva A Sarangapani R
Helmlinger G et al (2005) An integrated approach for inference and
mechanistic modeling for advancing drug development FEBS Letters
579 1878ndash1883
Anderle P Huang Y amp Sadee W (2004) Intestinal membrane transport
of drugs and nutrients Genomics of membrane transporters using
expression microarrays European Journal of Pharmaceutical Sciences
21 17ndash24
Annereau J P Szakacs G Tucker C J Arciello A Cardarelli C
Collins J et al (2004) Analysis of ATP-binding cassette transporter
expression in drug-selected cell lines by a microarray dedicated to
multidrug resistance Molecular Pharmacology 66 1397ndash1405
Apic G Ignjatovic T Boyer S amp Russell R B (2005) Illuminating drug
discovery with biological pathways FEBS Letters 579 1872ndash1877
Baker V A Harries H M Waring J F Duggan C M Ni H A Jolly
R A et al (2004) Clofibrate-induced gene expression changes in rat
liver A cross-laboratory analysis using membrane cDNA arrays
Environmental Health Perspectives 112 428ndash438
Balakin K V Ekins S Bugrim A Ivanenkov Y A Korolev D amp
Nikolsky Y et al (2004a) Quantitative structurendashmetabolism relation-
ship modeling of the metabolic N-dealkylation rates Drug Metabolism
and Disposition 32 1111ndash1120
Balakin K V Ekins S Bugrim A Ivanenkov Y A Korolev D amp
Nikolsky Y et al (2004b) Kohonen maps for prediction of binding to
human cytochrome P450 3A4 Drug Metabolism and Disposition 32
1183ndash1189
Barabasi A -L amp Oltvai Z N (2004) Network biology Under-
standing the cellrsquos functional organization Nature Reviews Genetics
5 101ndash113
Bartosiewicz M Penn S G amp Buckpitt A (2001) Applications of gene
arrays in environmental toxicology Fingerprints of gene regulation
associated with cadmium chloride benzo(a)pyrene and trichloro-
ethylene Environmental Health Perspectives 109 71ndash74
Bertilsson G Heidrich J Svensson K Asman M Jendeberg L
Sydow-Backman M et al (1998) Identification of a human nuclear
receptor defines a new signaling pathway for CYP3A induction
Proceedings of the National Academy of Sciences of the United States
of America 95 12208ndash12213
Blumberg B Sabbagh Jr W Juguilon H Bolado Jr J van Meter
C M Ong E S et al (1998) SXR a novel steroid and xenobiotic-
sensing nuclear receptor Genes and Development 12 3195ndash3205
Borodina Y Rudik A Filimonov D Kharchevnikova N Dmitriev A
Blinova V et al (2004) A new statistical approach to predicting
aromatic hydroxylation sites Comparison with model-based
approaches Journal of Chemical Information and Computer Sciences
44 1998ndash2009
Borodina Y Sadym A Filimonov D Blinova V Dmitriev A amp
Poroikov V (2003) Predicting biotransformation potential from
molecular structure Journal of Chemical Information and Computer
Sciences 43 1636ndash1646
Bottone Jr F G Martinez J M Collins J B Afshari C A amp Eling
T E (2003) Gene modulation by the cyclooxygenase inhibitor
sulindac sulfide in human colorectal carcinoma cells Possible link to
apoptosis Journal of Biological Chemistry 278 25790ndash25801
Boyer S amp Zamora I (2002) New methods in predictive metabolism
Journal of Computer-Aided Molecular Design 16 403ndash413
Bredel M amp Jacoby E (2004) Chemogenomics An emerging strategy
for rapid target and drug discovery Nature Reviews Genetics 5
262ndash275
Bugrim A Nikolskaya T amp Nikolsky Y (2004) Early prediction of drug
metabolism and toxicity Systems biology approach and modeling
Drug Discovery Today 9 127ndash135
Bulera S J Eddy S M Ferguson E Jatkoe T A Reindel J F
Bleavins M R et al (2001) RNA expression in the early character-
ization of hepatotoxicants in Wistar rats by high-density DNA micro-
arrays Hepatology 33 1239ndash1258
Butcher E C Berg E L amp Kunkel E J (2004) Systems biology in drug
discovery Nature Biotechnology 22 1253ndash1259
Butte A (2002) The use and analysis of microarray data Naturalist
Review of Drug Discovery 1 951ndash960
Cary M P Bader G D amp Sander C (2005) Pathway information for
systems biology FEBS Letters 579 1815ndash1820
Cheok M H Yang W Pui C H Downing J R Cheng C Naeve
C W et al (2003) Treatment-specific changes in gene expression
discriminate in vivo drug response in human leukemia cells Nature
Genetics 34 85ndash90
Cleary J D Rogers P D amp Chapman S W (2001) Differential
transcription factor expression in human mononuclear cells in response
to amphotericin B Identification with complementary DNA microarray
technology Pharmacotherapy 21 1046ndash1054
Coen M Ruepp S U Lindon J C Nicholson J K Pognan F Lenz
E M et al (2004) Integrated application of transcriptomics and
metabonomics yields new insight into the toxicity due to paracetamol in
the mouse Journal of Pharmaceutical and Biomedical Analysis 35
93ndash105
Cornwell P D De Souza A T amp Ulrich R G (2004) Profiling of
hepatic gene expression in rats treated with fibric acid analogs
Mutation Research 549 131ndash145
Csermely P Agoston V amp Pongor S (2005) The efficiency of multi-
target drugs The network approach might help drug design Trends in
Pharmacological Sciences 26 178ndash182
Cunningham M J Liang S Fuhrman S Seilhamer J J amp
Somogyi R (2000) Gene expression microarray data analysis for
toxicology profiling Annals of the New York Academy of Sciences 919
52ndash67
de Longueville F Atienzar F A Marcq L Dufrane S Evrard S
Wouters L et al (2003) Use of a low density microarray for studying
gene expression patterns induced by hepatotoxicants on primary
cultures of rat hepatocytes Toxicological Sciences 75 378ndash392
Dobrin R Beg Q K Barabasi A L amp Oltvai Z N (2004)
Aggregation of topological motifs in the Escherichia coli transcriptional
regulatory network BMC Bioinformatics 5 10
Donald S Verschoyle R D Edwards R Judah D J Davies R
Riley J et al (2002) Hepatobiliary damage and changes in hepatic
gene expression caused by the antitumor drug ecteinascidin-743 (ET-
743) in the female rat Cancer Research 62 4256ndash4262
Dumont M Jacquemin E DrsquoHont C Descout C Cresteil D Haouzi
D et al (1997) Expression of the liver Na+-independent organic anion
transporting polypeptide (oatp-1) in rats with bile duct ligation Journal
of Hepatology 27 1051ndash1056
Durnas C Loi C M amp Cusack B J (1990) Hepatic drug metabolism
and aging Clinical Pharmacokinetics 19 359ndash389
Eisen M B Spellman P T Brown P O amp Botstein D (1998) Cluster
analysis and display of genome-wide expression patterns Proceedings
of the National Academy of Sciences of the United States of America
95 14863ndash14868
Ekins S Andreyev S Ryabov A Kirilov E Rakhmatulin E A
Bugrim A et al (in press) Computational Prediction of Human Drug
Metabolism Exp Opin Drug Metab Toxicol
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6662
Ekins S Boulanger B Swaan P W amp Hupcey M A Z (2002)
Towards a new age of virtual ADMETOX and multidimensional drug
discovery Journal of Computer-Aided Molecular Design 16 381ndash401
Ekins S Bugrim A Nikolsky Y amp Nikolskaya T (2005) Systems
biology Applications in drug discovery In S Gad (Ed) Drug
Discovery Handbook (pp 123ndash183) Hoboken NJrsquo Wiley and Sons
Ekins S Giroux C N Nikolsky Y Bugrim A amp Nikolskaya T
(2005) A signature gene network approach to toxicity The Toxoco-
logists 84
Ekins S Kirillov E Rakhmatulin E amp Nikolskaya T (2005) A novel
method for visualizing nuclear hormone receptor networks relevant to
drug metabolism Drug Metabolism and Disposition 33 474ndash481
Ekins S amp McGowan R J (2001) The limits of reductionism The
shifting genomic paradigmrsquos impact on industry and academia
Philsophy in Science 9 1ndash23
Ekins S Mirny L amp Schuetz E G (2002) A ligand-based approach to
understanding selectivity of nuclear hormone receptors PXR CAR
FXR LXRa and LXRb Pharmaceutical Research 19 1788ndash1800
Ekins S Nikolsky Y amp Nikolskaya T (2005) Techniques Application
of systems biology to absorption distribution metabolism excretion
and toxicity Trends in Pharmacological Sciences 26 202ndash209
Ekins S Ring B J Grace J McRobie-Belle D J amp Wrighton S
A (2000) Present and future in vitro approaches for drug
metabolism Journal of Pharmacological and Toxicological Methods
44 313ndash324
Ekins S Waller C L Swaan P W Cruciani G Wrighton S A amp
Wikel J H (2000) Progress in predicting human ADME parameters in
silico Journal of Pharmacological and Toxicological Methods 44
251ndash272
Erhardt P W (2003) A human drug metabolism database Potential roles
in the quantitative predictions of drug metabolism and metabolism-
related drugndashdrug interactions Current Drug Metabolism 4 411ndash422
Evans W E amp McLeod H L (2003) Pharmacogenomicsmdashdrug
disposition drug targets and side effects New England Journal of
Medicine 348 538ndash549
Ezendam J Staedtler F Pennings J Vandebriel R J Pieters R
Boffetta P et al (2004) Toxicogenomics of subchronic hexachlor-
obenzene exposure in Brown Norway rats Environmental Health
Perspectives 112 782ndash791
Faiola B Fuller E S Wong V A amp Recio L (2004) Gene expression
profile in bone marrow and hematopoietic stem cells in mice exposed to
inhaled benzene Mutation Research 549 195ndash212
FDA (2004) Innovation stagnation Challenge and opportunity on the
critical path to new medicinal products
Fiehn O (2001) Combining genomics metabolome analysis and bio-
chemical modelling to understand metabolic networks Comparative
Functional Genomics 2 155ndash168
Field F J Born E amp Mathur S N (2004) LXRRXR ligand activation
enhances basolateral efflux of beta-sitosterol in CaCo-2 cells Journal of
Lipid Research 45 905ndash913
Fleck C Sutter L Appenroth D Koch B Meinhold T Pitack M
et al (2003) Use of gene chip technology for the characterisation of
the regulation of renal transport processes and of nephrotoxicity in
rats Experimental and Toxicologic Pathology 54 401ndash410
Fliri A F Loging W T Thadeio P F amp Volkmann R A (2005)
Biological spectra analysis Linking biological activity profiles to
molecular structure Proceedings of the National Academy of Sciences
of the United States of America 102 261ndash266
Fountoulakis M (2004) Application of proteomics technologies in the
investigation of the brain Mass Spectrometry Reviews 23 231ndash258
Gaucher S P Taylor S W Fahy E Zhang B Warnock D E Ghosh
S S et al (2004) Expanded coverage of the human heart
mitochondrial proteome using multidimensional liquid chromatography
coupled with tandem mass spectrometry Journal of Proteome
Research 3 495ndash505
Gerhold D Lu M Xu J Austin C Caskey C T amp Rushmore T
(2001) Monitoring expression of genes involved in drug metabolism
and toxicology using DNA microarrays Physiological Genomics 5
161ndash170
Guo G L Choudhuri S amp Klaassen C D (2002) Induction profile of
rat organic anion transporting polypeptide 2 (oatp2) by prototypical
drug-metabolizing enzyme inducers that activate gene expression
through ligand-activated transcription factor pathways Journal of
Pharmacology and Experimental Therapeutics 300 206ndash212
Hagenbuch B amp Meier P J (2004) Organic anion transporting
polypeptides of the OATP SLC21 family Phylogenetic classification
as OATP SLCO superfamily new nomenclature and molecularfunc-
tional properties Pflugers Archiv 447 653ndash665
Hamadeh H K amp Afshari C A (2004) Toxicogenomics principles and
applications Hobokenrsquo John Wiley and Sons
Hamadeh H K Bushel P R Jayadev S Martin K DiSorbo O Sieber
S et al (2002) Gene expression analysis reveals chemical-specific
profiles Toxicological Sciences 67 219ndash231
Hamadeh H K Jayadev S Gaillard E T Huang Q Stoll R
Blanchard K et al (2004) Integration of clinical and gene expression
endpoints to explore furan-mediated hepatotoxicity Mutation Research
549 169ndash183
Hamadeh H K Knight B L Haugen A C Sieber S Amin R P
Bushel P R et al (2002) Methapyrilene toxicity Anchorage of
pathologic observations to gene expression alterations Toxicologic
Pathology 30 470ndash482
Han J D Bertin N Hao T Goldberg D S Berriz G F Zhang L V
et al (2004) Evidence for dynamically organized modularity in the
yeast proteinndashprotein interaction network Nature 430 88ndash93
Hanisch D Zien A Zimmer R amp Lengauer T (2002) Co-clustering of
biological networks and gene expression data Bioinformatics 18
S145ndashS154
Harris A J Dial S L amp Casciano D A (2004) Comparison of basal
gene expression profiles and effects of hepatocarcinogens on gene
expression in cultured primary human hepatocytes and HepG2 cells
Mutation Research 549 79ndash99
Harrison S C (2004) Whither structural biology Nature Structural
Molecular Biology 11 12ndash15
Hartley D P Dai X He Y D Carlini E J Wang B Huskey S E
et al (2004) Activators of the rat pregnane X receptor differentially
modulate hepatic and intestinal gene expression Molecular Pharma-
cology 65 1159ndash1171
Hartwell L H Hopfield J J Leibler S amp Murray A W (1999) From
molecular to modular cell biology Nature 402 C47ndashC52
Hasmall S Orphanides G James N Pennie W Hedley K Soames
A et al (2002) Downregulation of lactoferrin by PPARalpha ligands
Role in perturbation of hepatocyte proliferation and apoptosis
Toxicology Sciences 68 304ndash313
Hayase T Yamamoto Y Yamamoto K Muso E amp Shiota K (2004)
Microarray profile analysis of toxic cocaine-induced alterations in the
expression of mouse brain gene sequences A possible rsquoprotectiversquo
effect of buprenorphine Journal of Applied Toxicology 24 15ndash20
Hayes K R Vollrath A L Zastrow G M McMillan B J Craven M
Jovanovich S et al (2005) EDGE A centralized resource for the
comparison analysis and distribution of toxicogenomic information
Molecular Pharmacology 67 1360ndash1368
Heijne W H Slitt A L van Bladeren P J Groten J P Klaassen C D
Stierum R H et al (2004) Bromobenzene-induced hepatotoxicity at
the transcriptome level Toxicology Sciences 79 411ndash422
Heijne W H Stierum R H Slijper M van Bladeren P J amp van
Ommen B (2003) Toxicogenomics of bromobenzene hepatotoxicity
A combined transcriptomics and proteomics approach Biochemical
Pharmacology 65 857ndash875
Heinloth A N Irwin R D Boorman G A Nettesheim P Fannin
R D Sieber S O et al (2004) Gene expression profiling of rat
livers reveals indicators of potential adverse effects Toxicology
Sciences 80 193ndash202
Hodges L C Cook J D Lobenhofer E K Li L Bennett L Bushel
P R et al (2003) Tamoxifen functions as a molecular agonist inducing
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 63
cell cycle-associated genes in breast cancer cells Molecular Cancer
Research 1 300ndash311
Holleman A Cheok M H den Boer M L Yang W Veerman KM
A J Kazemier et al (2004) Gene-expression patterns in drug-resistant
acute lymphoblastic leukemia cells and response to treatment New
England Journal of Medicine 351 533ndash542
Hong Y Muller U R amp Lai F (2003) Discriminating two classes of
toxicants through expression analysis of HepG2 cells with DNA arrays
Toxicology in Vitro 17 85ndash92
Hood E (2003) Proteomics Characterizing the cogs in the machinery of
life Environmental Health Perspectives 111 A816ndashA825
Hood L (2003) Systems biology Integrating technology biology and
computation Mechanism of Ageing and Development 124 9ndash16
Hood L amp Galas D (2003) The digital code of DNA Nature 421
444ndash448
Hood L amp Perlmutter R M (2004) The impact of systems approaches on
biological problems in drug discovery Nature Biotechnology 22
1215ndash1217
Hu T Gibson D P Carr G J Torontali S M Tiesman J P Chaney
J G et al (2004) Identification of a gene expression profile that
discriminates indirect-acting genotoxins from direct-acting genotoxins
Mutation Research 549 5ndash27
Huang Q Dunn II R T Jayadev S DiSorbo O Pack F D Farr S B
et al (2001) Assessment of cisplatin-induced nephrotoxicity by
microarray technology Toxicology Sciences 63 196ndash207
Huang Q Jin X Gaillard E T Knight B L Pack F D Stoltz J H et
al (2004) Gene expression profiling reveals multiple toxicity endpoints
induced by hepatotoxicants Mutation Research 549 147ndash167
Huisman M T Smit J W Crommentuyn K M Zelcer N Wiltshire
H R Beijnen J H et al (2002) Multidrug resistance protein 2
(MRP2) transports HIV protease inhibitors and transport can be
enhanced by other drugs Aids 16 2295ndash2301
Hunt C M Westerkam W R amp Stave G M (1992) Effect of age and
gender on the activity of human hepatic CYP3A Biochemical
Pharmacology 44 275ndash283
Ideker T Ozier O Schwikowski B amp Siegal A F (2002) Discovering
regulatory and signalling circuits in molecular interaction networks
Bioinformatics 18 S233ndashS240
Ideker T Thorsson V Ranish J A Christmas R Buhler J Eng J K
et al (2001) Integrated genomic and proteomic analyses of a system-
atically perturbed network Science 292 929ndash934
Iida M Anna C H Hartis J Bruno M Wetmore B Dubin J R et al
(2003) Changes in global gene and protein expression during early
mouse liver carcinogenesis induced by non-genotoxic model carcino-
gens oxazepam and Wyeth-14643 Carcinogenesis 24 757ndash770
Ingelman-Sundberg M (2004) Pharmacogenetics of cytochrome P450 and
its applications in drug therapy The past present and future Trends in
Pharmacological Sciences 25 193ndash200
Jeong H Mason S P Barabasi A L amp Oltvai Z N (2001) Lethality
and centrality in protein networks Nature 411 41ndash42
Jeong H Tombor B Albert R Oltvai Z N amp Barabasi A L
(2000) The large-scale organization of metabolic networks Nature
407 651ndash654
Jung D Hagenbuch B Gresh L Pontoglio M Meier P J amp Kullak-
Ublick G A (2001) Characterization of the human OATP-C
(SLC21A6) gene promoter and regulation of liver-specific OATP genes
by hepatocyte nuclear factor 1 alpha Journal of Biological Chemistry
276 37206ndash37214
Jung D amp Kullak-Ublick G A (2003) Hepatocyte nuclear factor 1
alpha A key mediator of the effect of bile acids on gene expression
Hepatology 37 622ndash631
Jung J -W Park J -S Hwang J -W Kang K -S YS L Song B
-S et al (2004) Gene expression analysis of peroxisome prolifer-
ators- and phenytoin-induced hepatotoxicity using cDNA microarray
Journal of Veterinary Medical Science 66 1329ndash1333
Kato N Shibutani M Takagi H Uneyama C Lee K -Y Takiami S
et al (2004) Gene expression profile in the livers of rats orally
administered ethinylestradiol for 28 days using a microarray technique
Toxicology 200 179ndash192
Kier L D Neft R Tang L Suizu R Cook T Onsurez K et al
(2004) Applications of microarrays with toxicologically relevant genes
(tox genes) for the evaluation of chemical toxicants in Sprague Dawley
rats in vivo and human hepatocytes in vitro Mutation Research 549
101ndash113
Kijima K Toyosawa K Yasuba M Matsuoka N Adachi T
Komiyama M et al (2004) Gene expression analysis of the rat testis
after treatment with di(2-ethylhexyl) phthalate using cDNA microarray
and real-time RT-PCR Toxicology and Applied Pharmacology 200
103ndash110
Kim R B (2003) Organic anion-transporting polypeptide (OATP) trans-
porter family and drug disposition European Journal of Clinical
Investigation 33 1ndash5
Kinirons M T amp OrsquoMahony M S (2004) Drug metabolism and ageing
British Journal of Clinical Pharmacology 57 540ndash544
Kitano H (2002a) Computational systems biology Nature 420 206ndash210
Kitano H (2002b) Systems biology A brief overview Science 295
1662ndash1664
Kliewer S A Moore J T Wade L Staudinger J L Watson M
A Jones S A et al (1998) An orphan nuclear receptor activated
by pregnanes defines a novel steroid signalling pathway Cell 92
73ndash82
Korolev D Balakin K V Nikolsky Y Kirillov E Ivanenkov Y A
Savchuk N P et al (2003) Modeling of human cytochrome p450-
mediated drug metabolism using unsupervised machine learning
approach Journal of Medicinal Chemistry 46 3631ndash3643
Kramer J A Pettit S D Amin R P Bertram T A Car B
Cunningham M et al (2004) Overview on the application of
transcription profiling using selected nephrotoxicants for toxicology
assessment Environmental Health Perspectives 112 460ndash464
Kultima K Nystrom A -M Scholz B Gustafson A -L Dencker L
amp Stigson M (2004) Valproic acid teratogenicity A toxicogenomics
approach Environmental Health Perspectives 112 1225ndash1235
Kutuzova G D amp Deluca H F (2004) Gene expression profiles in rat
intestine identify pathways for 125-dihydroxyvitamin D(3) stimulated
calcium absorption and clarify its immunomodulatory properties
Archives of Biochemistry and Biophysics 432 152ndash166
Kwak M K Wakabayashi N Itoh K Motohashi H Yamamoto M amp
Kensler T W (2003) Modulation of gene expression by cancer
chemopreventive dithiolethiones through the Keap1-Nrf2 pathway
Identification of novel gene clusters for cell survival Journal of
Biological Chemistry 278 8135ndash8145
Lahjouji K Mitchell G A amp Qureshi I A (2001) Carnitine transport
by organic cation transporters and systemic carnitine deficiency
Molecular Genetics and Metabolism 73 287ndash297
Landowski C P Sun D Foster D R Menon S S Barnett J L
Welage L S et al (2003) Gene expression in the human intestine and
correlation with oral valacyclovir pharmacokinetic parameters Journal
of Pharmacology and Experimental Therapeutics 306 778ndash786
Lee G -J Lee W -S Jeon K -S Um C -H Kim S -J Lee C -H
et al (2004) cDNA microarray gene expression analysis and
toxicological phenotype for anticancer drug Journal of Veterinary
Medical Science 66 1339ndash1345
Lee M Kwon J Kim S N Kim J E Koh W S Kim E J et al
(2003) cDNA microarray gene expression profiling of hydroxyurea
paclitaxel and p-anisidine genotoxic compounds with differing
tumorigenicity results Environmental and Molecular Mutagenesis
42 91ndash97
Li S Armstrong C M Bertin N Ge H Milstein S Boxem M et al
(2004) A map of the interactome network of the metazoan C elegans
Science 303 540ndash543
Liguori M J Anderson L M Bukofzer S McKim J Pregenzer J F
Retief J et al (2005) Microarray analysis in human hepatocytes
suggests a mechanism for hepatotoxicity induced by trovafloxacin
Hepatology 41 177ndash186
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6664
Lobenhofer E K Bennett L Cable P L Li L Bushel P R amp Afshari
C A (2002) Regulation of DNA replication fork genes by 17beta-
estradiol Molecular Endocrinology 16 1215ndash1229
Locker J Tian J Carver R Concas D Cossu C Ledda-Columbano
G M et al (2003) A common set of immediate-early response genes
in liver regeneration and hyperplasia Hepatology 38 314ndash325
Mattes W B Pettit S D Sansone S A Bushel P R amp Waters M D
(2004) Database development in toxicogenomics Issues and efforts
Environmental Health Perspectives 112 495ndash505
McMillian M Nie A Y Parker J B Leone A Bryant S Kemmerer
M et al (2004) A gene expression signature for oxidant stressreactive
metabolites in rat liver Biochemical Pharmacology 68 2249ndash2261
Meier P J amp Stieger B (2000) Molecular mechanisms in bile formation
News in Physiological Sciences 15 89ndash93
Meneses-Lorente G de Longueville F Dos Santos-Mendes S Bonnert
T P Jack A Evrard S et al (2003) An evaluation of a low-density
DNA microarray using cytochrome P450 inducers Chemical Research
in Toxicology 16 1070ndash1077
Meneses-Lorente G Guest P C Lawrence J Muniappa N Knowles
M R Skynner H A et al (2004) A proteomic investigation of drug-
induced steatosis in rat liver Chemical Research in Toxicology 17
605ndash612
Merrill C L Ni H Yoon L W Tirmenstein M A Narayanan P
Benavides G R et al (2002) Etomoxir-induced oxidative stress in
HepG2 cells detected by differential gene expression is confirmed
biochemically Toxicology Sciences 68 93ndash101
Milo R Shen-Orr S Itzkovitz S Kashtan N Chklovskii D amp Alon
U (2002) Network motifs Simple building blocks of complex
networks Science 298 824ndash827
Morgan K T Ni H Brown H R Yoon L Qualls C W Crosby L
M et al (2002) Application of cDNA microarray technology to in
vitro toxicology and the selection of genes for a real-time RT-PCR-
based screen for oxidative stress in Hep-G2 cells Toxicologic
Pathology 30 435ndash451
Mwinyi J Johne A Bauer S Roots I amp Gerloff T (2004) Evidence
for inverse effects of OATP-C (SLC21A6) 5 and 1b haplotypes on
pravastatin kinetics Clinical Pharmacology and Therapeutics 75
415ndash421
Nakata K Yukawa M Komiyama N Nakano T amp Kaminuma T
(2002) A nuclear receptor database that maps pathways to diseases
Genome Infomatics 13 515ndash516
Nicholson J K Holmes E Lindon J C amp Wilson I D (2004) The
challenges of modeling mammalian biocomplexity Nature Biotechnol-
ogy 22 1268ndash1274
Nicholson J K amp Wilson I D (2003) Understanding rsquoglobal systems
biology Metabonomics and the continuum of metabolism Nature
Reviews on Drug Discovery 2 668ndash676
Nikitin A Egorov S Daraselia N amp Mazo I (2003) Pathway studiomdash
the analysis and navigation of molecular networks Bioinformatics 19
2155ndash2157
Nikolsky Y Ekins S Nikolskaya T amp Bugrim A (2005) A novel
method for generation of signature networks as biomarkers from
complex high throughput data Toxicology Letters 158 20ndash29
Nikolsky Y Nikolskaya T amp Bugrim A (2005) Biological networks
and analysis of experimental data in drug discovery Drug Discovery
Today 10 653ndash662
Nishizato Y Ieiri I Suzuki H Kimura M Kawabata K Hirota T
et al (2003) Polymorphisms of OATP-C (SLC21A6) and OAT3
(SLC22A8) genes Consequences for pravastatin pharmacokinetics
Clinical Pharmacology and Therapeutics 73 554ndash565
Nozawa T Nakajima M Tamai I Noda K Nezu J Sai Y et al
(2002) Genetic polymorphisms of human organic anion transporters
OATP-C (SLC21A6) and OATP-B (SLC21A9) Allele frequencies in
the Japanese population and functional analysis Journal of Pharmacol-
ogy and Experimental Therapeutics 302 804ndash813
Nozawa T Sugiura S Nakajima M Goto A Yokoi T Nezu J et al
(2004) Involvement of organic anion transporting polypeptides in the
transport of troglitazone sulfate Implications for understanding trogli-
tazone hepatotoxicity Drug Metabolism and Disposition 32 291ndash294
Oliver D E Rubin D L Stuart J M Hewett M Klein T E amp
Altman R B (2002) Ontology development for a pharmacogenetics
knowledge base Package Symposion 88ndash99
Oram J F amp Lawn R M (2001) ABCA1 The gatekeeper for eliminating
excess tissue cholesterol Journal of Lipid Research 42 1173ndash1179
Oswald M Kullak-Ublick G A Paumgartner G amp Beuers U (2001)
Expression of hepatic transporters OATP-C and MRP2 in primary
sclerosing cholangitis Liver 21 247ndash253
Parsons A B Brost R I Ding H Li Z Zhang C Sheikh B et al
(2004) Integration of chemical-genetic and genetic interaction data
links bioactive compounds to cellular target pathways Nature Bio-
technology 22 62ndash69
Patki K C von Moltke L L Harmatz J S Hesse L M Court M H
amp Greenblatt D J (2004) Effect of age on in vitro triazolam
biotransformation in male human liver microsomes Journal of
Pharmacology and Experimental Therapeutics 308 874ndash879
Pereira-Leal J B Enright A J amp Ouzounis C A (2004) Detection of
functional modules from protein interaction networks Proteins 54
49ndash57
Plant N (2004) Interaction networks Coordinating responses to xeno-
biotic exposure Toxicology 202 21ndash32
Raghavendra Prasad H S Qi Z Srinivasan K N amp Gopalakrish-
nakone P (2004) Potential effects of tetrodotoxin exposure to
human glial cells postulated using microarray approach Toxicon 44
597ndash608
Reilly T P Bourdi M Brady J N Pise-Masison C A Radonovich M
F George J W et al (2001) Expression profiling of acetaminophen
liver toxicity in mice using microarray technology Biochemical and
Biophysical Research Communications 282 321ndash328
Rives A W amp Galitski T (2003) Modular organization of cellular
networks Proceedings of the National Academy of Sciences of the
United States of America 100 1128ndash1133
Rogers P D Pearson M M Cleary J D Sullivan D C amp Chapman
S W (2002) Differential expression of genes encoding immunomodu-
latory proteins in response to amphotericin B in human mononuclear
cells identified by cDNA microarray analysis Journal of Antimicrobial
Chemotherapy 50 811ndash817
Rosen M B Wilson V S Schmid J E amp Gray L E (2005) Gene
expression analysis in the ventral prostate of rats exposed to vinclozolin
or procymidone Reproductive Toxicology 19 367ndash379
Rost D Herrmann T Sauer P Schmidts H L Stieger B Meier P J
et al (2003) Regulation of rat organic anion transporters in bile salt-
induced cholestatic hepatitis Effect of ursodeoxycholate Hepatology
38 187ndash195
Rothman R B Ayestas M A Dersch C M amp Baumann M H (1999)
Aminorex fenfluramine and chlorphentermine are serotonin transporter
substrates Implications for primary pulmonary hypertension Circula-
tion 100 869ndash875
Ruepp S U Tonge R P Shaw J Wallis N amp Pognan F (2002)
Genomics and proteomics analysis of acetaminophen toxicity in mouse
liver Toxicology Sciences 65 135ndash150
Sakaeda T Nakamura T amp Okumura K (2002) MDR1 genotype-
related pharmacokinetics and pharmacodynamics Biological amp Phar-
maceutical Bulletin 25 1391ndash1400
Sakata T Anzai N Shin H J Noshiro R Hirata T Yokoyama H
et al (2004) Novel single nucleotide polymorphisms of organic cation
transporter 1 (SLC22A1) affecting transport functions Biochemical
and Biophysical Research Communications 313 789ndash793
Satlin L M Amin V amp Wolkoff A W (1997) Organic anion
transporting polypeptide mediates organic anionHCO3-exchange
Journal of Biological Chemistry 272 26340ndash26345
Satomi Y Tsuchiya W Mihara K Ota M Kasahara Y amp Akahori F
(2004) Gene expression analysis of the lung following paraquat
administration in rats using DNA microarray Journal of Toxicological
Sciences 29 91ndash100
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 65
Schrenk D Baus P R Ermel N Klein C Vorderstemann B amp
Kauffmann H M (2001) Up-regulation of transporters of the MRP
family by drugs and toxins Toxicology Letters 120 51ndash57
Segal E Shapira M Regev A Persquoer D Botstein D Koller D et al
(2003) Module networks Identifying regulatory modules and their
condition-specific regulators from gene expression data Nature
Genetics 34 166ndash176
Segal E Wang H amp Koller D (2003) Discovering molecular pathways
from protein interaction and gene expression Bioinformatics 19
i264ndash i272
Seidel S D Kan H L Stott W T Schisler M R amp Gollapudi B B
(2003) Identification of transcriptome profiles for the DNA-damaging
agents bleomycin and hydrogen peroxide in L5178Y mouse lymphoma
cells Environmental and Molecular Mutagenesis 42 19ndash25
Shan L Yu Y Schut H A J amp Snyderwine E G (2004) Susceptibility
of rats to mammary gland carcinogenesis by the food-derived
carcinogen 2-Amino-1-Methyl-6-Phenylimidazo[45-b]Pyridine (PhIP)
varies with age and is associated with the induction of differential gene
expression American Journal of Pathology 165 191ndash202
Sharom J R Bellows D S amp Tyers M (2004) From large networks to
small molecules Current Opinion in Chemical Biology 8 81ndash90
Shitara Y Hirano M Sato H amp Sugiyama Y (2004) Gemfibrozil and
its glucuronide inhibit the OATP2(OATP1B1 SLC21A6)-mediated
hepatic uptake and CYP2C8-mediated metabolism of cerivastatinmdash
Analysis of the mechanism of the clinically relevant drugndashdrug
interaction between cerivastatin and gemfibrozil Journal of Pharma-
cology and Experimental Therapeutics
Shitara Y Itoh T Sato H Li A P amp Sugiyama Y (2003) Inhibition of
transporter-mediated hepatic uptake as a mechanism for drugndashdrug
interaction between cerivastatin and cyclosporin A Journal of
Pharmacology and Experimental Therapeutics 304 610ndash616
Somogyi R Fuhrman S amp Wen X (2001) Genetic network inference in
computational models and applications to large-scale gene expression
data In M Bower amp H Bolouri (Eds) Computational modeling of
genetic and biochemical networks (pp 119ndash157) Cambridge MArsquoThe
MIT Press
Spirin V amp Mirny L A (2003) Protein complexes and functional
modules in molecular networks Proceedings of the National Academy
of Sciences of the United States of America 100 12123ndash12128
Staudinger J Liu Y Madan A Habeebu S amp Klaassen C D
(2001) Coordinate regulation of xenobiotic and bile acid homeo-
stasis by pregnane X receptor Drug Metabolism and Disposition
29 1467ndash1472
Sun L Z Ji Z L Chen X Wang J F amp Chen Y Z (2002) ADME-
AP A database of ADME associated proteins Bioinformatics 18
1699ndash1700
Suter L Babiss L E amp Wheeldon E B (2004) Toxicogenomics in
predictive toxicology in drug development Chemistry and Biology 11
161ndash171
Synold T W Dussault I amp Forman B M (2001) The orphan nuclear
receptor SXR coordinately regulates drug metabolism and efflux
Nature Medicine 7 584ndash590
Tabuchi Y amp Kondo T (2003) cDNA microarray analysis reveals chop-
10 plays a key role in Sertoli cell injury induced by bisphenol A
Biochemical and Biophysical Research Communications 305 54ndash61
Tamai I Nezu J Uchino H Sai Y Oku A Shimane M et al (2000)
Molecular identification and characterization of novel members of the
human organic anion transporter (OATP) family Biochemical and
Biophysical Research Communications 273 251ndash260
Taylor S W Fahy E Zhang B Glenn G M Warnock D E Wiley S
et al (2003) Characterization of the human heart mitochondrial
proteome Nature Biotechnology 21 281ndash286
Terasaka S Aita Y Inoue A Hayashi S Nishigaki M Aoyagi K
et al (2004) Using a customized DNA microarray for expression
profiling of the estrogen-responsive genes to evaluate estrogen activity
among natural estrogens and industrial chemicals Environmental
Health Perspectives 112 773ndash781
Thomas D M Francescutti-Verbeem D M Liu X amp Kuhn D M
(2004) Identification of differentially regulated transcripts in mouse
striatum following methamphetamine treatmentmdashan oligonucleotide
microarray approach Journal of Neurochemistry 88 380ndash393
Thomas R S Rank D R Penn S G Zastrow G M Hayes K R
Pande K et al (2001) Identification of toxicologically predictive
gene sets using cDNA microarrays Molecular Pharmacology 60
1189ndash1194
Thompson K L Afshari C A Amin R P Bertram T A Car B
Cunningham M et al (2004) Identification of platform-independent
gene expression markers of cisplatin nephrotoxicity Environmental
Health Perspectives 112 488ndash494
Tirona R G Leake B F Merino G amp Kim R B (2001)
Polymorphisms in OATP-C Identification of multiple allelic
variants associated with altered transport activity among European-
and African-Americans Journal of Biological Chemistry 276
35669ndash35675
Tirona R G Leake B F Wolkoff A W amp Kim R B (2003)
Human organic anion transporting polypeptide-C (SLC21A6) is a
major determinant of the rifampin-mediated pregnane X receptor
activation Journal of Pharmacology and Experimental Therapeutics
304 223ndash228
Tong W Cao X Harris S Sun H Fang H Fuscoe J et al (2003)
ArrayTrackmdashsupporting toxicogenomic research at the US Food and
Drug Administration National Center for Toxicological Research
Environmental Health Perspectives 111 1819ndash1826
Tornow S amp Mewes H W (2003) Functional modules by relating
protein interaction networks and gene expression Nucleic Acids
Research 31 6283ndash6289
Trocho C Escola-Gil J C Ribas V Benitez S Martin-Campos J M
Rotllan N et al (2004) Phenytoin treatment reduces atherosclerosis in
mice through mechanisms independent of plasma HDL-cholesterol
concentration Atherosclerosis 174 275ndash285
Ueda A Hamadeh H K Webb H K Yamamoto Y Sueyoshi T
Afshari C A et al (2002) Diverse roles of the nuclear orphan
receptor CAR in regulating hepatic genes in response to phenobarbital
Molecular Pharmacology 61 1ndash6
Ulrich R G (2003) The toxicogenomics of nuclear receptor agonists
Current Opinion in Chemical Biology 7 505ndash510
Ulrich R G Rockett J C Gibson G G amp Pettit S D (2004)
Overview of an interlaboratory collaboration on evaluating the effects
of model hepatotoxicants on hepatic gene expression Environmental
Health Perspectives 112 423ndash427
Unami A Shinohara Y Ichikawa T amp Baba Y (2003) Biochemical
and microarray analyses of bupivacaine-induced apoptosis Journal of
Toxicological Sciences 28 77ndash94
Vasquez A Flammini A Maritan A amp Vespignani A (2003) Global
protein function prediction from proteinndashprotein interaction networks
Nature Biotechnology 21 697ndash700
Vezina C M Walker N J amp Olson J R (2004) Subchronic Exposure to
TCDD PeCDF PCB126 and PCB153 Effect on hepatic gene
expression Environmental Health Perspectives 112 1636ndash1644
Vo T D Greenberg H J amp Palsson B O (2004) Reconstruction and
functional characterization of the human mitochondrial metabolic
network based on proteomic and biochemical data Journal of Bio-
logical Chemistry 279 39532ndash39540
Wang H amp LeCluyse E L (2003) Role of orphan nuclear receptors in
the regulation of drug-metabolising enzymes Clinical Pharmacoki-
netics 42 1331ndash1357
Wang N Silver D L Thiele C amp Tall A R (2001) ATP-
binding cassette transporter A1 (ABCA1) functions as a choles-
terol efflux regulatory protein Journal of Biological Chemistry 276
23742ndash23747
Waring J F Cavet G Jolly R A McDowell J Dai H Ciurlionis R
et al (2003) Development of a DNA Microarray for toxicology based
on hepatoxin-regulated sequences Environmental Health Perspectives
111 863ndash870
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6666
Waring J F Gum R Morfitt D Jolly R A Ciurlionis R Heindel M
et al (2002) Identifying toxic mechanisms using DNA microarrays
Evidence that an experimental inhibitor of cell adhesion molecule
expression signals through the aryl hydrocarbon nuclear receptor
Toxicology 181ndash182 537ndash550
Waring J F Jolly R A Ciurlionis R Lum P Y Praestgaard J T
Morfitt D C et al (2001) Clustering of hepatotoxins based on
mechanism of toxicity using gene expression profiles Toxicology and
Applied Pharmacology 175 28ndash42
Waters M Boorman G Bushel P Cunningham M Irwin R Merrick
A et al (2003) Systems toxicology and the Chemical Effects in
Biological Systems (CEBS) knowledge base EHP Toxicogenomics
111 15ndash28
Wei M Arnold L Cano M amp Cohen S M (2005) Effects of co-
administration of antioxidants and arsenicals on the rat urinary bladder
epithelium Toxicology Sciences 83 237ndash245
Weinshilboum R (2003) Inheritance and drug response New England
Journal of Medicine 348 529ndash537
Weinshilboum R amp Wang L (2004) Pharmacogenetics Inherited
variation in amino acid sequence and altered protein quantity Clinical
Pharmacology and Therapeutics 75 253ndash258
Werner E (2003) In silico multicellular systems biology and minimal
genomes Drug Discovery Today 8 1121ndash1127
Wong J S amp Gill S S (2002) Gene expression changes induced in
mouse liver by di(2-ethylhexyl) phthalate Toxicology and Applied
Pharmacology 185 180ndash196
Xie T Tong L McCann U D Yuan J Becker K G Mechan A O
et al (2004) Identification and characterization of metallothionein-1
and -2 gene expression in the context of ()34-methylenedioxymetham-
phetamine-induced toxicity to brain dopaminergic neurons Journal of
Neuroscience 24 7043ndash7050
Yadetie F Laegreid A Bakke I Kusnierczyk W Komorowski J
Waldum H L et al (2003) Liver gene expression in rats in response
to the peroxisome proliferator-activated receptor-alpha agonist ciprofi-
brate Physiological Genomics 15 9ndash19
Yamashita S Nomoto T Abe M Tatematsu M Sugimura T amp
Ushijima T (2004) Persistence of gene expression changes in stomach
mucosae induced by short-term N-methyl-NV-nitro-N-nitrosoguanidinetreatment and their presence in stomach cancers Mutation Research
549 185ndash193
Yan Q amp Sadee W (2000) Human membrane transporter database A
web-accessible relational database for drug transport studies and
pharmacogenomics AAPS Pharmsci 2 E20
Yeger-Lotem E amp Margalit H (2003) Detection of regulatory circuits by
integrating the cellular networks of proteinndashprotein interactions and
transcription regulation Nucleic Acids Research 31 6053ndash6061
Yoon B I Li G -X Kitada K Kawasaki Y Igarashi K amp Kodama
Y et al (2003) Mechanisms of benzene-induced hematotoxicity and
leukemogenicity cDNA microarray analyses using mouse bone marrow
tissue Environmental Health Perspectives 111 1411ndash1420
Young M B DiSilvestro M R Sendera T J Freund J Kriete A amp
Magnuson S R (2003) Analysis of gene expression in carbon
tetrachloride-treated rat livers using a novel bioarray technology
Pharmacogenomics Journal 3 41ndash52
Yu H Zhu X Greenbaum D Karro J amp Gerstein M (2004) TopNet
A tool for comparing biological sub-networks correlating protein
properties with topological statistics Nucleic Acids Research 32
328ndash337
Zhang E Y Fu D J Pak Y A Stewart T Mukhopadhyay N
Wrighton S A et al (2004) Genetic polymorphisms in human proton-
dependent dipeptide transporter PEPT1 Implications for the functional
role of Pro586 Journal of Pharmacology and Experimental Therapeu-
tics 310 437ndash445
Zhang E Y Knipp G T Ekins S amp Swaan P W (2002)
Structural biology and function of solute transporters Implications
for identifying and designing substrates Drug Metabolism Reviews
34 709ndash750
Zhang E Y Phelps M A Cheng C Ekins S amp Swaan P W (2002)
Modeling of active transport systems Advanced Drug Delivery
Reviews 54 329ndash354
- Systems-ADMETox Resources and network approaches
-
- Introduction
- Data available
- Network analysis and databases
-
- Network applications
-
- The role of transporters
-
- Clinical relevance of transporters
- Transporter network examples ABCA1
- Transporter network examples OATP
- Transporter microarray data
-
- Applications to enzymes
- Future network applications
-
- Discussion
- Acknowledgements
- References
-
Table 3 (continued)
Compounds Cell type Microarray type Compound dose Microarray data
availability
Reference
Hydroxyurea
(a carcinogen)
p-anisidine
(a noncarcinogen)
and paclitaxel
L5178Y Tk_-mouse
lymphoma
The Twin - Chip Mouse-
74K Digital Genomics
cDNA microarray
10 ngml paclitaxel 313
ugml hydroxyurea 32
ugml p-anisidine
Gene symbol and fold
change in a manuscript
table
(Lee et al 2003)
Acetaminophen
amiodarone clofibrate
erythromycin estolate
isoniazid alpha-
naphtylylisothiocyanate
beta-naphtoflavone
4-pentenoic acid
phenobarbital
tetracycline and
zileuton
Wistar Rat
hepatocytes
DualChip rat hepato
(Eppendorf Hamburg
Germany)
A single concentration
which varied for each
compound
Accession numbers and
gene namemdashfold
changes shown as
colored heat mapmdashnote
easily extracted from
publication
(de Longueville
et al 2003)
Bleomycin and hydrogen
peroxide
Mouse lymphoma
L5178Y TK(+ -)
Clontech Mouse 12K
cDNA microarray (1185
genes)
Bleomycin (25 and 20
ugml) hydrogen peroxide
(5 and 10 ugml)
Gene names and fold
change presented as bar
charts in publication
(Seidel Kan
Stott Schisler amp
Gollapudi 2003)
Bupivicaine
camptothecin
HL-60 Agilent human cDNA
microarray
1 mM Gene name GenBank
accession number
unigene and ratio in a
manuscript table
(Unami
Shinohara
Ichikawa amp Baba
2003)
Bisphenol A Mouse Sertoli TTE3 IntelliGene mouse
expression glass
microarrays (Version
10 Takara Shuzo)
which were spotted with
564 cDNA fragments of
mouse known genes and
approximately 301
expressed sequence tags
(ESTs)
0ndash400 uM Gene name GenBank
accession number and
fold change at time
points in a manuscript
table
(Tabuchi amp
Kondo 2003)
Mitomycin C or
doxorubicin
Hep G2 85 human gene custom
array
10 um mitomycin C
2 um doxorubicin or
2 ethanol
Bar charts with fold
changes in publication
very few genes
(Hong Muller amp
Lai 2003)
Amphotericin B Human peripheral
blood mononuclear
and THP-1
GF211 FKnown Genes_
Genefilter cDNA array
(ResGen) this array
consists of gt4000
individual elements
each representing a
known human gene
5 ugml Accession numbers and
fold expression in a
manuscript table
(Cleary Rogers amp
Chapman 2001
Rogers Pearson
Cleary Sullivan
amp Chapman
2002)
Benzo(a)pyrene diol
epoxide
TK6 human
lymphoblastoid
Human-350 microarray
a glass slide with 350
spotted human cDNA
probes (Phase-1
Molecular Toxicology
0 001 010 or
10 ugml)
Gene names and fold
change at doses in a
manuscript table
(Akerman et al
2004)
Etomoxir HepG2 Clontech AtlasiHuman Stress
Toxicology cDNA arrays
(234 genes)
1 mM etomoxir Gene names and fold
change in a manuscript
table
(Merrill et al
2002)
Tetrodotoxin Human glioma cell
line HTB-138
Using Affymetrix
GeneChip (HG-U133A
10 and 20 mM Affymetrix ID Genbank
ID gene name gene
symbol and fold change
in a manuscript table
(Raghavendra
Prasad Qi
Srinivasan amp
Gopalakrishnakone
2004)
Methotrexate
mercaptopurine
Human acute
lymphoblastic
leukemia
Affymetrix U133A chip Low and high dose and
combination
Data available as
supplemental data online
(Cheok et al 2003)
Prednisolone
vincristine
asparaginase
daunorubicin
Human acute
lymphoblastic
leukemia
Affymetrix U133A chip Various Data available as
supplemental data online
(Holleman et al
2004)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 47
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6648
A second study has indicated how a natural language
processing method CCNet was used to show the genes
regulated by the nuclear hormone receptor FXR (Apic
Ignjatovic Boyer amp Russell 2005) These automated
methods enable a more complete understanding of the
complexity of the transcriptional factors (Ekins Mirny amp
Schueltz 2002b Plant 2004 Ulrich 2003) but ultimately
rely on the quality of the content of the underlying database
of literature interactions This is a key consideration that is
often overlooked For example the gold standard database is
one that is manually curated to ensure the fidelity of the
direct interaction and is preferable to one generated
computationally by algorithms like natural language pro-
cessing (Nikolsky Nikolskaya amp Bugrim 2005) The
advantage of interaction networks over clustering has been
demonstrated in one study using MetaCore (Nikolsky
Ekins Nikolskaya amp Bugrim 2005) by reanalysis of a
published microarray study of G0-arrested MCF-7 breast
cancer cells treated with estrogen and 4-hydroxytamoxifen
(Hodges et al 2003) After producing integrated gene
networks for each treatment strikingly different patterns
were displayed although both contained early transcriptional
factors myc jun and fos Only the estrogen network featured
induced genes essential for all cell cycle phases (Nikolsky et
al 2005) Similarly microarray data for benzene toxicity
(Yoon et al 2003) has been re-analyzed by focusing on the
genes assessed on the p53 pathway (Ekins et al 2005e) We
envisage that a database of such networks for toxic
compounds will be used for comparing between different
molecules and used in the development of predictive
algorithms for Systems-ADMETox modeling in future
Another approach to using such pathway approaches is to
visualize the results of quantitative structure activity models
for predicting molecules binding to enzymes transporters
receptors and ion channels (Ekins Andreyev et al in press
2005e)
It is also possible to simultaneously interpret high
throughput data and predictions on interaction networks
providing a novel approach to predicting and understanding
potential undesirable drugndashdrug or off target effects in the
area of systems pharmacology An example data set uses
percent inhibition data for clotrimazole and ticonazole
which were screened against many different assays at a
single concentration in a commercially available database
BioPrint (Cerep Redmond WA) as published recently
(Fliri Loging Thadejo amp VOlkman 2005) The data for
10 assays has been arbitrarily encoded as inhibitors (gt50
inhibition) or non-inhibitors (lt50 inhibition) in a text file
which was then loaded into MetaCore The analyzed
network algorithm was then used which generates a large
network and fragments it into sub-networks each with a Z-
score and p-values for ranking according to saturation with
objects from the initial gene list The Gene-ontology
processes are also mapped to the gene list and individual
networks In this example a statistically significant network
was generated for the different proteins (Fig 4
p =2838e31) This network also maps the Gene Ontology
processes for the activation of MAPK (118 of genes p
value 9143e07) signal transduction (333 1600e05)
regulation of transcription DNA-dependent (294
2786e04) regulation of inflammatory response (39
3746e04) and the regulation of blood pressure (78
4230e04) This example network indicates how molecules
of the same or different therapeutic classes could be
evaluated for their effects as a graph either together as in
this case or individually This would be useful to indicate
potential off target effects and identify structurally dissimilar
molecules with similar network patterns Such networks
could then be compared to assess network overlap or
differences between molecules and their inhibition of
multiple proteins This type of unique visualization of high
throughput screening data illustrates how the target proteins
may be connected as a network to infer the possible
downstream effects of inhibition
With the burgeoning number of freely available online
and commercial databases that can be used for pathway
construction numbering in the hundreds there have been
suggestions to impose standards for model exchange
querying and visualization (Cary Bader amp Sander 2005)
To date there has been little discussion with regards to
standardization of ADMETox related databases although
there has been considerable discussion relating to drug
metabolism database generation (Erhardt 2003) This is
certainly an important area to address in the future There is
already a growing literature related to ADMETox that is
partially captured in the several commercially available
databases (Ekins et al 2005e) but to date there have been
limited academic efforts to capture data for transporters
with the human membrane transporter database (Yan amp
Sadee 2000) TP-search transporter database (httpwww
ilabrisewasedaacjptp-search) drug interaction database
(httpwwwdruginteractioninfoorgDatabaseinfoaspx)
nuclear hormone receptors (Nakata Yukawa Komiyama
Nakano amp Kaminuma 2002) the ADME-AP database
(Sun Ji Chen Wang amp Chen 2002) and PharmaGKB
(Oliver et al 2002) DSStox (httpwwwepagovnheert
dsstox) TOXNET (httptoxnetnimnihgov) that are
readily accessible
In order to generate accessible pathways using any of
the available software a large enough set of object
identifiers are required to map onto the underlying data-
base To demonstrate this datasets from toxicogenomics
studies have been evaluated with both the KEGG pathway
database and a commercially available product MetaCore
(wwwgenegocom) These gene or protein lists range in
size from 21 to 1853 objects In virtually all cases more
identifiers are mapped to networks in MetaCore and this
also seems independent of the identifier type used
(Unigene Affymetrix Genbank or Locuslink Table 5)
These numbers will obviously change as more information
is added to the respective databases hence the more
objects that are mapped from a dataset The more extensive
Table 4
Literature derived toxicoproteomics data
Compounds Data source Microarray type Compound dose Microarray data
availability
Reference
Carbon
tetrachloride
Male Wistar rats Affymetrix rat chip 8799
probes+proteomics
study
6ndash24 h exposure Genbank SwissProt and
binary data in a
manuscript table
(Fountoulakis
2004)
Paracetamol CD-1 male mice Custom mouse tox blots
with 450 genes
RTQ-PCR+proteomics
study 2-DIGE+
MALDI-MS
150 or 500 mgkg Gene name IMAGE ID
GenBank accession
number fold induction
SwissProt identifier and
protein abundance
change in manuscript
tables
(Ruepp Tonge
Shaw Wallis amp
Pognan 2002)
Paracetamol AP-1 male mice Affymetrix murine 11K
set+proteomics study
Up to 500 mgkg Gene names and fold
changes at multiple
doses and time points in
a manuscript tablemdash
proteomics data not
accessible
(Coen et al 2004)
Oxazepam or
Wy-14643
Male B6C3F1 mice NIEHS Mouse Chip
(8700 genes) 2-DIGE
and MS
Oxazepam (2500 ppm)
Wyeth (Wy)-14643
(500 ppm)
Proteomics data in a
manuscript table
Genbank accession
number gene name and
fold change in a
manuscript table data
also available to
download from NIEHS
website
(Iida et al 2003)
Compound A
(PPAR gamma
ligand)
Female rats CrlCD
(SD)IGS BR
Proteomics 2-DIGE and
MS
250 mgkgday up to
5 days
Accession number and
protein name and
average ratio in a
manuscript table
(Meneses-Lorente
et al 2004)
Bromobenzene Male Wistar rats Proteomics 2-DIGE and
MS custom 3000 cDNA
rat chip
5 mmolkg Accession number gene
name fold change in a
manuscript table protein
names along with bar
charts
(Heijne Stierum
Slijper van
Bladeren amp van
Ommen 2003)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 49
the network that can be generated (as it will consist of more
nodes) then a more comprehensive understanding of the
networks is possible The data available currently in the
literature can be used to evaluate such pathway and
network generation tools Recently we have used several
of the published studies (Tables 1ndash3) with MetaCore to
visualize networks for acetaminophen furan carbon tetra-
chloride benzene and cisplatin showing genes involved in
In vivo
In vitro
In Silico
1970s 1980s 1990s 2000s
Systems Biology ()
OMICS
Fig 2 The timeline for major paradigms in ADMETox
oxidative stress (Ekins Giroux Nikolsky Bugrim amp
Nikolskaya 2005c)
Other important pathwaynetwork building tools that could
potentially be applied to toxicogenomics data include
Ingenuity pathways analysis (httpwwwingenuitycom)
PathArt (httpwwwjubilantbiosyscompdhtm) Pathway
Assist (httpwwwariadnegenomicscomproctspathway
html) (Nikitin et al 2003) and several other databases
deposited at the Pathway Resource List (httpcbiiomskcc
orgprl) All of these products have unique underlying
proprietary pathway databases which are compiled manually
or automatically with text mining tools or a combination of
both We are still waiting for studies that provide a comparison
of different database tools or network building algorithms
Until then theremay be someoverlap but also some differences
between the results obtained from more than one network
method due to the database content and the algorithms used
The reader is recommended to evaluate for themselves several
technologies and select those with the most appropriate fit to
their own specialized needs
In the next Sections the further application of some
available network and database tools will be described with
Microarray or other high throughput data
Upload list of geneprotein identifiers and fold change significance etc (raw data or after selection of the differentially expressed
Genes using other statistical methods)
Parse database and generate interaction network with different algorithms or visualize on maps
Filter Networks
Access significance of the interaction networks
Determine GO processes
Compare 2 or more networks intersection overlap etc
Export gene list
Export gene list
generate interaction network with different algorithms or visualize on maps
Visualization
Fig 3 Schematic for the utilization of pathway tools for assessing high throughput data
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6650
specific detailed reference to transporters and enzymes but
these technologies can also be applied elsewhere as
described above
Fig 4 Network for high throughput screening data for clotrimazole and ticona
cholecystokinin CCR1 Choline transporter Chloride channel catechol-O-methyl
and co workers (Fliri et al 2005) Nodes highlighted with large blue circles rep
molecules showing gt50 inhibition in the assay blue circles represents both mol
inhibits gt50 and one inhibits lt50 Ligands (large) linked to transcriptional f
(wwgenegocom) (For interpretation of the references to colour in this figure leg
311 The role of transporters
A diverse array of organic solutes such as nutrients
neurotransmitters and drugs are transported by specialized
zole screened against 10 in vitro assays (Human Cannabinoid 1 Human
transferase COX2 CYP1A2 CYP2B6 CYP2C19) data published by Fliri
resent assays used in the study Red circles on these nodes represent both
ecules showing lt50 inhibition chequered circles represent one molecule
actors enzymes and transporters via edges using the MetaCorei database
end the reader is referred to the web version of this article)
Table 5
Datasets acquired from publications and other sources and used to evaluate two databasepathway tools KEGG and metaCore
Dataset Number of objects
genes proteins in list
Number of objects
mapped with KEGG
Number of objects mapped
with MetaCore on networks
Reference for microarray
data gene list
Tox Chip 10 (NIEHS) 1853 405 1498 httpsdir-appsniehsnihgov
mapsguestclonesrchcfm
4-Hydroxytamoxifen and
estrogen
1617 434 1343 (Hodges et al 2003)
Mitochondrial proteins 722 156 388 (Gaucher et al 2004
Taylor et al 2003)
Bromobenzene
(24 and 48 h)
130 41 89 (Heijne et al 2004)
Acetaminophen 30 19 23 (Heinloth et al 2004)
Acetaminophen 84 29 64 (Huang et al 2004)
Furan 185 64 139 (Huang et al 2004)
Tetrodotoxin 116 31 86 (Raghavendra Prasad et al
2004)
Benzene 73 16 62 (Yoon et al 2003)
Benzene 76 5 53 (Faiola et al 2004)
Carbon tetrachloride 37 8 26 (Young et al 2003)
Estrogen 94 33 90 (Lobenhofer et al 2002)
Trovafloxacin 142 20 82 (Liguori et al 2005)
Phenobarbital 37 13 28 (Ueda et al 2002)
L-742694 (liver)+ 45 17 19 (Hartley et al 2004)
L-742694 (intestine)+ 23 11 10 (Hartley et al 2004)
A-277249 21 7 9 (Waring et al 2002)
All files were converted to Locuslink identifiers except as noted () unigene () Affymetrix (+) Genbank identifiers were used
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 51
proteins across cellular membranes These may function as
passive processes or active processes energized by the
hydrolysis of ATP or coupling to the co-transport of counter
ions down an electrochemical gradient such as Na+ H+ and
Cl There are many thousands of transporters which can be
classified into distinct superfamilies One of these the solute
carrier class (SLC) is rapidly expanding and contains over
30 families and 200 members The ATP-binding cassette
(ABC) contains 7 families and over 48 members including
P-glycoprotein (P-gp) and MRP subfamilies (Zhang Knipp
Ekins amp Swaan 2002a Zhang Phelps Cheng Ekins amp
Swaan 2002b) Transporters have a key role in clinical
pharmacology with many drugs specifically targeting them
Numerous drugs share transport pathways with nutrients
and transporters have a role in oral absorption drug
bioavailability drug resistance excretion and ultimately
pharmacokinetics and pharmacodynamics
Polymorphism of drug transporters may be a key factor
in drug interactions and lack of effectiveness This field has
become known as pharmacogenomics and is focused on
understanding of inherited DNA sequence variations (poly-
morphisms and mutations) revealed by xenobiotics (Evans
amp McLeod 2003 Weinshilboum 2003) Over the last few
decades many genes have been directly linked to the
mechanisms of response (Evans amp McLeod 2003 Wein-
shilboum 2003) such that 20ndash95 of variability to drug
response is inheritable (Evans amp McLeod 2003) This
phenotypical variability is mainly caused by single nucleo-
tide polymorphisms (SNPs) present in anywhere from 1 to
50 of the population resulting in either lower protein
activity incorrect folding or rapid degradation via proteo-
somes (Weinshilboum amp Wang 2004) A number of
structurally diverse molecules bind to P-gp which is
expressed in many tissues and has numerous SNPs one of
which (C3435T) affects the expression level in the
duodenum and therefore can impact the absorption of
molecules which would be substrates for this transporter
(Sakaeda Nakamura amp Okumura 2002) The human
proton-dependent dipeptide transporter (hPEPT1) can also
affect the absorption of molecules in the intestine and
recently 9 SNPs were found with only one displaying a
reduced transport capacity (Zhang et al 2004) The sodium-
dependent carnitine cotransporter OCTN2 can possess
mutations and these result in primary carnitine deficiency
which impacts fatty acid oxidation and is characterized by
many clinical manifestations (Lahjouji Mitchel amp Qureshi
2001) The organic cation transporter 1 (OCT1) is also
important in the transport of numerous xenobiotics and
endobiotics Recently 4 SNPs were identified in the
Japanese population and when functionally characterized
in vitro the uptake of cations was reduced significantly for
some of these mutations indicating that this would likely
contribute to inter-individual variations in metabolism of
drugs which were transported via OCT1 (Sakata et al
2004)
3111 Clinical relevance of transporters The pregnane
X-receptor (PXR) is a transcriptional regulator of the
enzyme human MDR1 (P-gp) MRPs and OATP (Wang amp
LeCluyse 2003) CYP3A (Bertilsson et al 1998 Blumberg
et al 1998 Kliewer et al 1998) and CYP2C89 (Synold
Dussault amp Forman 2001) as well as many other genes
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6652
involved in the transport metabolism and biosynthesis of
bile acids (Staudinger Liu Madan Habeebu amp Klaassen
2001) However the additional receptors CAR FXR LXR
and other nuclear receptors take part in a complex network
of interactions to control these and other proteins Thus
elucidation of the regulatory networks which control the
expression of these transporters is also important To date
most of the research has centered on efflux transporters but
there has been considerable interest in uptake transporters
such as the organic anion transporter polypeptide (OATP
((Kim 2003) see also later Section on OATP)
There are several specific examples of the importance of
drug transporters to the clinical development of drugs One
example is the insulin sensitizer troglitazone which was
withdrawn due to hepatotoxicity although the precise
mechanism appears to have been unclear until recently
The major metabolite is a sulfated species and is suspected
of being responsible for the observed toxicity The recent
assessment of the organic anion transporting polypeptides
OATP-C and OATP8 expressed on the hepatocyte baso-
lateral membrane indicated that sulphated troglitazone has a
high affinity for the former and possibly lower affinity for
the latter (Nozawa et al 2004) This metabolite would
therefore be expected to accumulate in hepatocytes and
inhibit the bile salt export pump and Mrp2 Because
polymorphisms have been shown for OATP-C (Tirona
Leake Merino amp Kim 2001) it is also possible that these
may result in the accumulation of the metabolite and in turn
elicit idiosyncratic toxicity A second relevant example of
the impact of transporters is the clinically significant drugndash
drug interaction between cerivastatin and cyclosporine A
which occurs via the OATP-C transporter (Shitara Itoh
Sato Li amp Sugiyama 2003) A third example are the HIV
protease inhibitors saquinavir ritonavir and indinavir which
are transported by MRP2 in vitro and other drugs such as
probenecid and sulfinpyrazone are able to enhance this
transport Transport by MRP2 suggests that these com-
pounds will have decreased bioavailability due to increased
clearance and other drugs could aggravate this situation by
further enhancing transport (Huisman et al 2002) Sim-
ilarly the rifampicin mediated induction via PXR of MRP2
and P-gp in healthy subjects was found to significantly
decrease the AUC and also correlated with intestinal
expression of these transporters This transporter is also
inducible by cisplatin 2-AAF and phenobarbital (Schrenk
et al 2001) indicating multiple mechanisms may be
involved In other species such as rat commonly used as a
toxicity model orthologs of the transporters such as OATP2
are expressed and can be induced with ligands for PXR like
PCN (Guo Choudhuri amp Klaassen 2002) This is useful
knowledge because the advent of microarray technology
allows one to dose a rat with a xenobiotic and assess
thousands of genes simultaneously in a particular tissue For
instance animals dosed with known nephrotoxins have
shown some upregulation of the NandashKndashCl transporter
however the authors suggested genomic responses are
stronger soon after exposure before declining (Fleck et
al 2003) Some transporters may therefore be specifically
targeted by drugs in one tissue such as the CNS but these
same transporters may also be expressed elsewhere in the
body hence off-target effects may result in toxicity The
serotonin transporter is one such example which is
expressed in the lungs and brain Some substrates for this
channel like fenfluramine can result in primary pulmonary
hypertension as they accumulate in pulmonary cells (Roth-
man Ayestas Dersch amp Baumann 1999) Similarly P-gp is
expressed at the blood brain barrier and intestine impacting
the efficacy and bioavailability of drugs
3112 Transporter network examples ABCA1 The
ABCA1 transporter mediates the first step of cholesterol
transport Mutations in this gene cause Tangier disease
which results in severe HDL deficiency cholesterol
accumulation in macrophages and attendant atherosclerosis
This transporter represents a drug target for upregulation
modulating cholesterol metabolism and prevention of
cardiovascular disease (Oram amp Lawn 2001) In vitro
ABCA1 can be inhibited by the sulfonylurea glybenclamide
(Field Burn amp Mathur 2004 Wang Silver Thiele amp Tall
2001) In order to illustrate the advantages of mapping drug
transporters as networks onto functional models alongside
other proteins one can consider the example of ABCA1
The query of the MetaCorei database shows that this
transporter appears on three manually curated pathway maps
representing the Fstate of the art_ knowledge derived from
reliable high quality literature sources One can also use the
individual maps as an interface to access the underlying
layers of information about the transporter including the list
of encoding genessplice variants with known SNPs In
addition to browsing MetaCorei a user can also build
custom networks around ABCA1 using the network-
construction tool (Fig 5) Such a visualization utility may
be very helpful for identification of all putative pathways
around a particular transporter or compound of interest The
ABCA1 network created by this tool shows that this
transporter at the time of writing is linked directly to
twenty-five other objects such as APOE1 and LXR Many
of its neighbors have their own SNPs that could be
important in determining interactions between transport of
a drug and normal human transport of endogenous ligands
in health or disease
3113 Transporter network examples OATP The
OATPs are key membrane bound transporters expressed in
many organs including intestine liver lung choroid plexus
blood brain barrier and other organs (Tamai et al 2000)
This family of transporters is capable of mediating the
sodium-independent transport of a diverse array of mole-
cules such as steroid conjugates organic anions and
xenobiotics by coupling uptake with efflux of bicarbonate
(Satlin Amin amp Wolkoff 1997) glutathione or its
conjugates (Hagenbuch amp Meier 2004) The inhibition of
Fig 5 The network of direct gene interactions around the ABCA1 transporter gene (centre) generated with MetaCorei The interation types between nodes are shown as small colored hexagons eg unspecified
allosteric regulation binding cleavage competition covalent modification dephosphorylation phosphorylation transcription regulation transformation When applicable interactions also have a positive or
negative effect and direction Ligands (purple) linked to other proteins (blue blobs) transfactors (red) enzymes (orange) (For interpretation of the references to colour in this figure legend the reader is referred to
the web version of this article)
SEkin
sJournalofPharm
acologica
landToxico
logica
lMeth
ods53(2006)38ndash66
53
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6654
this transporterrsquos hepatic uptake of other compounds may be
important for reported drugndashdrug interactions (Kim 2003)
described earlier (Shitara et al 2003) as well as cerivastatin
with gemfibrozil (Shitara Hirano Sato amp Sugiyama 2004)
OATP1B1 (previous names OATP-C LST-1 OATP2
SLC21A6) represents the most studied human OATP to
date (Meier amp Stieger 2000) and is expressed on the
basolateral plasma membrane of hepatocytes Several single
nucleotide polymorphisms have been identified in the
OATP1B1 gene in European-Americans African-Americans
(Tirona et al 2001) and Japanese (Nozawa et al 2002)
dramatically impacting the transport of ligands such as
pravastatin (Mwinyi Johne Bauer Roots amp Gerloff 2004
Nishizato et al 2003) estrone-3-sulfate (Nozawa et al
2002 Tirona et al 2001) Rifampin (Tirona Leake
Wolkoff amp Kim 2003) and estradiol 17h-d-glucuronide(Tirona et al 2001)
The regulation of SLCO may be affected during
extrahepatic cholestasis bile duct ligation bile salt induced
Fig 6 Networks generated with the autoexpand algorithm in MetaCorei to illustr
be interconnected with other protein regulatory signaling information Informa
unspecified allosteric regulation binding cleavage competition covalent m
transformation When applicable interactions also have a positive or negative e
transfactors (red) enzymes (orange) (For interpretation of the references to colour
cholestatic hepatitis (Dumont et al 1997 Meier amp Stieger
2000 Rost et al 2003) and primary sclerosing cholangitis
(Oswald Kullack-Ublick Paumgarter amp Beuer 2001) In
particular OATP1B1 appears to be regulated by the liver-
enriched transcription factor hepatocyte nuclear factor 1a
(HNF1a) which binds to the promoter region of this
transporter (Jung et al 2001) Site directed mutagenesis
of this binding site resulted in inactivation suggesting the
critical nature of the interaction with HNF1a Bile acids
such as CDCA have been shown to transcriptionally repress
HNF1a in vitro via inhibition of the transactivating effect of
HNF4a on HNF1a (Jung amp Kullak-Ublick 2003) After
screening many rat and human uptake transporters in vitro
OATP1B1 was also shown to modulate the PXR response
by controlling rifampin retention in the cell and therefore
affecting the induction of CYP3A4 and other gene products
such as P-gp (Tirona et al 2003)
Some of the literature for OATP1B1 human substrate
data has been annotated into MetaCorei to illustrate the
ate how ligands for the human OATP-C (OATP1B1) transporter (centre) can
tion on the type of interaction between objects is hidden for clarity eg
odification dephosphorylation phosphorylation transcription regulation
ffect and direction Ligands (purple) linked to other proteins (blue blobs)
in this figure legend the reader is referred to the web version of this article)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 55
visualization of the complex interconnections between this
transporter its ligands regulatory factors and signaling
molecules already in the database (Fig 6) The network was
generated with the autoexpand algorithm in the software
representing one of multiple available algorithms for
connecting genes ligands and other objects in the database
Clearly if more ligands and their connections are added to
the database the complexity of the network will increase
considerably The OATP-C (OATP1B1) gene details can be
viewed upon querying the database and links are provided
to other public databases This page can be used to highlight
the multiple synonyms for this gene as well as links to the
multiple SNPs identified to date
3114 Transporter microarray data Microarrays have
generally been limited in the number of transporters present
on them (Annereau et al 2004) however they have been used
in an attempt to correlate pharmacokinetic properties with
gene expression for valacyclovir (Landowski et al 2003) as
well as understand the expression profile in different tissues
or cell lines upon food component or xenobiotic treatment
(Anderle Huang amp Sadee 2004) This lack of transporters
on microarrays has prompted some groups to produce their
own arrays with a heavier emphasis on transporters These
arrays have for example then been used to demonstrate the
upregulation of ABC transporters and down-regulation of
GST-Pi in cell lines resistant to colchicines or 9-nitro-
camptothecin (Annereau et al 2004) The genes that were
significantly up or down-regulated in this particular study
were used to build networks with MetaCore (Fig 7A B) and
the similarities between them were assessed (Fig 7C)
Although there were only a small number of significantly
changed genes in common (IL-8 Fos GST-Pi Calpactin and
Ubiquitin hydrolase) it is perhaps likely that there is a much
larger common gene network that is important for drug
resistance although a much larger number of cell lines and
drug treatments need to be evaluated to produce a definitive
drug resistance signature involving transporters enzymes
and transcriptional regulators
312 Applications to enzymes
As we have already described it may be particularly
valuable to visualize enzymes as networks to show
interactions with transcriptional regulators and ligands
For example a key enzyme is CYP3A4 which metabolizes
40ndash45 of all drugs and has relatively few SNPs
(Ingelman-Sundberg 2004) Using a second software suite
MetaDrugi (wwwgenegocom) it is possible to construct
a custom network around this or other drug metabolizing
enzymes (Fig 8) In this case the gene network for
CYP3A4 highlights all of the major transcriptional
regulators and several more distant linked proteins and
ligands connected on the network that may be useful for
further study (Fig 8) Substrates inhibitors as well as
regulatory factors and other enzymes can be observed
connected on this network Due to the many hundreds of
known inhibitors and substrates only a small number are
shown here for clarity We assume that if a perturbation in
a pathway (eg due to a nonfunctional enzyme) is linked
to a certain pathologic condition a similar perturbation
caused by the interference from xenobiotic metabolism
(eg competitive inhibition of the same enzyme) may
result in identical effects As microarray gene expression
data is increasingly generated the role of enzyme
regulation in toxicity of certain xenobiotics will become
more apparent from either in vivo or in vitro studies The
visualization of such signature gene networks involving
transporters and enzymes their ligands and regulatory
factors will also be important for future toxicity prediction
methods We have recently generated visualizations of
microarray data from MCF-7 cells treated with 4-hydrox-
ytamoxifen to show that some of the key genes involved in
metabolism and transport are upregulated (Ekins et al
2005d) In addition we have made predictions with various
QSAR models in MetaDrug to indicate the involvement of
PXR CYP3A4 and P-gp (Ekins et al 2005e) Therefore it
appears likely that 4-hydroxytamoxifen could increase its
own metabolism as well as efflux from cells via P-gp
which can be visualized on networks Any decrease of
function of these enzymes or transporters in a population
would likely result in changes in the metabolism and
transport of this active metabolite potentially impacting
the clinical effect This represents one example of how
both pathway tools QSAR models and network building
algorithms can be used with different types of predicted
and experimental data to allow visualization of potential
compound interactions or toxicity
313 Future network applications
As the population ages an increasing incidence and
prevalence of systemic diseases especially chronic diseases
have occurred among older adults This has resulted in an
increase in medications used concomitantly by this
population which presents challenges for drugndashdrug
interactions Physiologically elderly patients may behave
differently to the younger patients for which the drugs are
initially developed for and tested on Many pharmacoki-
netic investigations in the elderly population reveal
decreased clearance of lipophilic drugs metabolized by
the cytochrome P450 enzymes however few studies have
evaluated aging-dependent or gender-related changes in
specific P450 enzymes (Hunt Westerkam amp Slave 1992)
Age-related physiological changes such as a reduction in
liver mass hepatic metabolizing enzyme activity liver
blood flow and alterations in plasma drug binding may
account for the decreased elimination of some metabolized
drugs in the elderly It is particularly difficult to separate an
effect of aging from the variation in the rate of metabolism
due to factors such as individual metabolic phenotype
(slow or fast metabolizer due to SNPs) environmental
influences concomitant disease states and drug intake
(drugndashdrug interactions) The available data suggest that
Fig 7 Networks for drug resistant cell lines generated with the Auto expand algorithm in MetaCore A KB-3-1 vs KB-8-5 B ATC-DU-145 vs RCO1 C node overlap of A and B with initial nodes highlighted
with blue or red solid circles (Initial gene lists derived from (Annereau et al 2004)) Information on the type of interaction between objects is hidden for clarity eg unspecified allosteric regulation binding
cleavage competition covalent modification dephosphorylation phosphorylation transcription regulation transformation When applicable interactions also have a positive or negative effect and direction
Ligands (purple) linked to other proteins (blue blobs) transfactors (red) enzymes (orange) (For interpretation of the references to colour in this figure legend the reader is referred to the web version of this article)
SEkin
sJournalofPharm
acologica
landToxico
logica
lMeth
ods53(2006)38ndash66
56
Fig7(continued)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 57
Fig7(continued)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6658
Fig 8 The network of gene interactions around the CYP3A4 enzyme (centre) from MetaCorei Information on the type of interaction between objects is
hidden for clarity eg unspecified allosteric regulation binding cleavage competition covalent modification dephosphorylation phosphorylation
transcription regulation transformation When applicable interactions also have a positive or negative effect and direction Ligands (purple) linked to other
proteins (blue blobs) transfactors (red) enzymes (orange) (For interpretation of the references to colour in this figure legend the reader is referred to the web
version of this article)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 59
the initial doses of drugs metabolized by these enzymes
should be reduced in older patients according to the clinical
response In most published studies the elderly appear at
least as responsive as the young to inducers or inhibitors of
P450s (Durnas Loi amp Cusack 1990) More recently there
has been some suggestion that there are age related
reductions in function of some specific P450s such as
CYP3A4 (Patki et al 2004) and this could occur at the
level of regulation However earlier studies with the same
enzyme showed no change in clearance with age (Hunt et
al 1992) To date there has been even less examination of
the transporter functions and any changes with age
(Kinirons amp OrsquoMahony 2004) so the current understand-
ing of the effects of aging on metabolism and transport is
anything but transparent This represents an extreme
challenge for the pharmaceutical industry how to predict
whether a drug has an affinity for an enzyme or transporter
and whether this may be clinically important if it shows a
decline in expression or function with aging Any decrease
of function of these enzymes or transporters in an elderly
population would result in changes in the metabolism and
transport of metabolites potentially impacting the clinical
effect This represents another example of how computa-
tional approaches may perhaps be used with different types
of predicted and experimental data to allow visualization of
potential compound interactions or toxicity in elderly
populations We may see different gene networks high-
lighted as humans age and these may be modified by drug
treatment and coadministration This represents an area
were network analysis could be applied in the future and is
in need of considerable further research
The collection of microarray data in databases such as
CEBS ArrayTrack and EDGE represents a future resource
for computational gene network analysis One could
envisage that ultimately in each case such data is converted
to one or more networks that are also displayed for the user
and can be used to compare treatments from in vivo and in
vitro experiments This would represent a different approach
to clustering the data as currently implemented in one of
these efforts (Hayes et al 2005) and may condense large
amounts of experimentally derived data into a readily
interpreted network
4 Discussion
Previously in this journal the progress of many
research groups in predicting human ADME parameters
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6660
in silico (Ekins et al 2000b) and approaches for drug
metabolism (Ekins Ring Grace McRobie-Belle amp
Wrighton 2000a) have been described Both of these
reviews commented on moving HT assays for ADME
Tox much earlier in drug discovery which had also been
indicated by other groups The initial reviews also
highlighted the likely wealth of data that would become
available and how this could be used for structure
activity relationships alongside the bioactivity data in
computational models It was noted that there was a
paucity of predictive metabolism tools at that time In
addition a growing number of efforts to model whole
cells and organs now a field called systems biology
were recognized as models that could be integrated with
the in silico ADME approaches In summary since these
past reviews virtually all pharmaceutical companies have
attempted earlier high throughput screening for ADME
Tox properties and to some extent the wider application
of computational approaches for physicochemical proper-
ties Systems biology is being quite widely acknowledged
as the new paradigm for understanding complex bio-
logical datasets derived from high throughput technolo-
gies and the accumulated knowledge on human protein
interactions (Hartwell Hopfield Leibler amp Murray 1999
Hood 2003b) Therefore systems biology can be defined
as the integration of genetic proteomic transcriptomic
and metabonomic data using computational methods
(Nicholson amp Wilson 2003) When taken together
information on molecular processes derived from different
sources represents a lsquolsquouniversersquorsquo of putative biological
functionality of which only a small fraction of it will be
realized in a cell at any given time To date systems
biology has been driven by academia and funding bodies
such as the NIH rather than the pharmaceutical compa-
nies Presently there is a great deal of interest from
scientists of all backgrounds in identifying the networks
of cellular pathways and the corresponding physically
interacting proteins
The network building software for systems biology
described in this current review will be valuable to query
high throughput data and known literature interactions in
order to predict potential toxicity in different species In the
future the compilation of published toxicogenomics data-
sets characteristic of different types of toxicity will likely
be available in these software systems to act as a reference
database It is also feasible that we will be able to generate
the annotated datasets which specifically address the
differences between human and rat networks implicated
in toxicity The identification of sub-network modules
conserved between human and rat distinct for toxicity
types or predictive for toxic end-points in human will be
possible Such signature gene networks (Nikolsky et al
2005) can then be verified with other experimentally
derived data prospectively or from preexisting databases
The combined Fhigh throughput datandashin silico network_approaches therefore represents a novel method for access-
ing complex tissue-level processes as networks integrating
all data types based on functional interactions The data-
bases developed and used in this approach will certainly
benefit from further annotation around the drug metaboliz-
ing enzymes and transporters as described herein in terms
of transcriptional regulation and the ligands associated with
them which frequently appear in the literature It will be
important to capture disparities in the assignment of ligands
to enzymes transporters and other proteins as well as
negative data
In summary although there have been numerous
toxicogenomics studies published there is presently a
relatively small number of datasets that are freely available
to perform network analysis of microarray data The
number of studies identifying large numbers of proteins
which are affected by molecule treatment are even scarcer
still while there are several examples of some published
studies that combine such data It is hoped that the
numerous database initiatives for high content and toxicol-
ogy data that are being undertaken will improve the
situation for other researchers that are not currently
equipped to do such microarray studies themselves The
addition of requirements by journals to deposit such raw
data in a freely accessible resource will aid these initiatives
ADMETox groups have seen new technologies and
approaches developed over the last decade that have all
been applied to identify poor compounds earlier (Fig 2)
The latest technologies integrate network building tools
with high content data and databases The current review
described the limited number of networks generated for
ADMETox at present and one hopes that the impact of
such analyses will be commonplace in the future Systems
biology is however more than just applying a network
approach and hence systems-ADMETox will have to
evolve due to the continual pressure to develop newer
technologies This current paradigm combining empirical
data and computational methods should integrate the
complex data already generated making it readily inter-
pretable and valuable for identifying the most promising
compounds in the future
Acknowledgements
Dr Maggie AZ Hupcey is gratefully acknowledged for
editorial assistance Dr Peter W Swaan (University of
Maryland) and Dr Cheng Chang (Ohio State University)
Dr Steve Wright (University of Arizona) Dr K Sandy
Pang (University of Toronto) and Dr Craig Giroux (Wayne
State University) are kindly acknowledged for their support
and discussions My colleagues at GeneGo Sergey
Andreyev Andy Ryabov Eugene Kirillov Eugene A
Rakhmatulin Svetlana Sorokina Andrej Bugrim Tatiana
Nikolskaya Yuri Nikolsky John Metz and Julie Bryant are
all thanked for their considerable contributions to software
development and data annotation
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 61
References
Abe M Yamashita S Kuramoto T Hirayama Y Tsukamoto T Ohta
T et al (2003) Global expression analysis of N-methyl-NV-nitro-N-nitrosoguanidine-induced rat stomach carcinomas using oligonucleotide
microarrays Carcinogenesis 24 861ndash867
Adachi T Ono Y Koh K B Takashima K Tainaka H Matsuno Y
et al (2004) Long-term alteration of gene expression without
morphological change in testis after neonatal exposure to genistein in
mice Toxicogenomic analysis using cDNA microarray Food and
Chemical Toxicology 42 445ndash452
Akerman G S Rosenzweig B A Domon O E McGarrity L J
Blankenship L R Tsai C A et al (2004) Gene expression profiles
and genetic damage in benzo(a)pyrene diol epoxide-exposed TK6 cells
Mutation Research 549 43ndash64
Aksenov S V Church B Dhiman A Georgieva A Sarangapani R
Helmlinger G et al (2005) An integrated approach for inference and
mechanistic modeling for advancing drug development FEBS Letters
579 1878ndash1883
Anderle P Huang Y amp Sadee W (2004) Intestinal membrane transport
of drugs and nutrients Genomics of membrane transporters using
expression microarrays European Journal of Pharmaceutical Sciences
21 17ndash24
Annereau J P Szakacs G Tucker C J Arciello A Cardarelli C
Collins J et al (2004) Analysis of ATP-binding cassette transporter
expression in drug-selected cell lines by a microarray dedicated to
multidrug resistance Molecular Pharmacology 66 1397ndash1405
Apic G Ignjatovic T Boyer S amp Russell R B (2005) Illuminating drug
discovery with biological pathways FEBS Letters 579 1872ndash1877
Baker V A Harries H M Waring J F Duggan C M Ni H A Jolly
R A et al (2004) Clofibrate-induced gene expression changes in rat
liver A cross-laboratory analysis using membrane cDNA arrays
Environmental Health Perspectives 112 428ndash438
Balakin K V Ekins S Bugrim A Ivanenkov Y A Korolev D amp
Nikolsky Y et al (2004a) Quantitative structurendashmetabolism relation-
ship modeling of the metabolic N-dealkylation rates Drug Metabolism
and Disposition 32 1111ndash1120
Balakin K V Ekins S Bugrim A Ivanenkov Y A Korolev D amp
Nikolsky Y et al (2004b) Kohonen maps for prediction of binding to
human cytochrome P450 3A4 Drug Metabolism and Disposition 32
1183ndash1189
Barabasi A -L amp Oltvai Z N (2004) Network biology Under-
standing the cellrsquos functional organization Nature Reviews Genetics
5 101ndash113
Bartosiewicz M Penn S G amp Buckpitt A (2001) Applications of gene
arrays in environmental toxicology Fingerprints of gene regulation
associated with cadmium chloride benzo(a)pyrene and trichloro-
ethylene Environmental Health Perspectives 109 71ndash74
Bertilsson G Heidrich J Svensson K Asman M Jendeberg L
Sydow-Backman M et al (1998) Identification of a human nuclear
receptor defines a new signaling pathway for CYP3A induction
Proceedings of the National Academy of Sciences of the United States
of America 95 12208ndash12213
Blumberg B Sabbagh Jr W Juguilon H Bolado Jr J van Meter
C M Ong E S et al (1998) SXR a novel steroid and xenobiotic-
sensing nuclear receptor Genes and Development 12 3195ndash3205
Borodina Y Rudik A Filimonov D Kharchevnikova N Dmitriev A
Blinova V et al (2004) A new statistical approach to predicting
aromatic hydroxylation sites Comparison with model-based
approaches Journal of Chemical Information and Computer Sciences
44 1998ndash2009
Borodina Y Sadym A Filimonov D Blinova V Dmitriev A amp
Poroikov V (2003) Predicting biotransformation potential from
molecular structure Journal of Chemical Information and Computer
Sciences 43 1636ndash1646
Bottone Jr F G Martinez J M Collins J B Afshari C A amp Eling
T E (2003) Gene modulation by the cyclooxygenase inhibitor
sulindac sulfide in human colorectal carcinoma cells Possible link to
apoptosis Journal of Biological Chemistry 278 25790ndash25801
Boyer S amp Zamora I (2002) New methods in predictive metabolism
Journal of Computer-Aided Molecular Design 16 403ndash413
Bredel M amp Jacoby E (2004) Chemogenomics An emerging strategy
for rapid target and drug discovery Nature Reviews Genetics 5
262ndash275
Bugrim A Nikolskaya T amp Nikolsky Y (2004) Early prediction of drug
metabolism and toxicity Systems biology approach and modeling
Drug Discovery Today 9 127ndash135
Bulera S J Eddy S M Ferguson E Jatkoe T A Reindel J F
Bleavins M R et al (2001) RNA expression in the early character-
ization of hepatotoxicants in Wistar rats by high-density DNA micro-
arrays Hepatology 33 1239ndash1258
Butcher E C Berg E L amp Kunkel E J (2004) Systems biology in drug
discovery Nature Biotechnology 22 1253ndash1259
Butte A (2002) The use and analysis of microarray data Naturalist
Review of Drug Discovery 1 951ndash960
Cary M P Bader G D amp Sander C (2005) Pathway information for
systems biology FEBS Letters 579 1815ndash1820
Cheok M H Yang W Pui C H Downing J R Cheng C Naeve
C W et al (2003) Treatment-specific changes in gene expression
discriminate in vivo drug response in human leukemia cells Nature
Genetics 34 85ndash90
Cleary J D Rogers P D amp Chapman S W (2001) Differential
transcription factor expression in human mononuclear cells in response
to amphotericin B Identification with complementary DNA microarray
technology Pharmacotherapy 21 1046ndash1054
Coen M Ruepp S U Lindon J C Nicholson J K Pognan F Lenz
E M et al (2004) Integrated application of transcriptomics and
metabonomics yields new insight into the toxicity due to paracetamol in
the mouse Journal of Pharmaceutical and Biomedical Analysis 35
93ndash105
Cornwell P D De Souza A T amp Ulrich R G (2004) Profiling of
hepatic gene expression in rats treated with fibric acid analogs
Mutation Research 549 131ndash145
Csermely P Agoston V amp Pongor S (2005) The efficiency of multi-
target drugs The network approach might help drug design Trends in
Pharmacological Sciences 26 178ndash182
Cunningham M J Liang S Fuhrman S Seilhamer J J amp
Somogyi R (2000) Gene expression microarray data analysis for
toxicology profiling Annals of the New York Academy of Sciences 919
52ndash67
de Longueville F Atienzar F A Marcq L Dufrane S Evrard S
Wouters L et al (2003) Use of a low density microarray for studying
gene expression patterns induced by hepatotoxicants on primary
cultures of rat hepatocytes Toxicological Sciences 75 378ndash392
Dobrin R Beg Q K Barabasi A L amp Oltvai Z N (2004)
Aggregation of topological motifs in the Escherichia coli transcriptional
regulatory network BMC Bioinformatics 5 10
Donald S Verschoyle R D Edwards R Judah D J Davies R
Riley J et al (2002) Hepatobiliary damage and changes in hepatic
gene expression caused by the antitumor drug ecteinascidin-743 (ET-
743) in the female rat Cancer Research 62 4256ndash4262
Dumont M Jacquemin E DrsquoHont C Descout C Cresteil D Haouzi
D et al (1997) Expression of the liver Na+-independent organic anion
transporting polypeptide (oatp-1) in rats with bile duct ligation Journal
of Hepatology 27 1051ndash1056
Durnas C Loi C M amp Cusack B J (1990) Hepatic drug metabolism
and aging Clinical Pharmacokinetics 19 359ndash389
Eisen M B Spellman P T Brown P O amp Botstein D (1998) Cluster
analysis and display of genome-wide expression patterns Proceedings
of the National Academy of Sciences of the United States of America
95 14863ndash14868
Ekins S Andreyev S Ryabov A Kirilov E Rakhmatulin E A
Bugrim A et al (in press) Computational Prediction of Human Drug
Metabolism Exp Opin Drug Metab Toxicol
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6662
Ekins S Boulanger B Swaan P W amp Hupcey M A Z (2002)
Towards a new age of virtual ADMETOX and multidimensional drug
discovery Journal of Computer-Aided Molecular Design 16 381ndash401
Ekins S Bugrim A Nikolsky Y amp Nikolskaya T (2005) Systems
biology Applications in drug discovery In S Gad (Ed) Drug
Discovery Handbook (pp 123ndash183) Hoboken NJrsquo Wiley and Sons
Ekins S Giroux C N Nikolsky Y Bugrim A amp Nikolskaya T
(2005) A signature gene network approach to toxicity The Toxoco-
logists 84
Ekins S Kirillov E Rakhmatulin E amp Nikolskaya T (2005) A novel
method for visualizing nuclear hormone receptor networks relevant to
drug metabolism Drug Metabolism and Disposition 33 474ndash481
Ekins S amp McGowan R J (2001) The limits of reductionism The
shifting genomic paradigmrsquos impact on industry and academia
Philsophy in Science 9 1ndash23
Ekins S Mirny L amp Schuetz E G (2002) A ligand-based approach to
understanding selectivity of nuclear hormone receptors PXR CAR
FXR LXRa and LXRb Pharmaceutical Research 19 1788ndash1800
Ekins S Nikolsky Y amp Nikolskaya T (2005) Techniques Application
of systems biology to absorption distribution metabolism excretion
and toxicity Trends in Pharmacological Sciences 26 202ndash209
Ekins S Ring B J Grace J McRobie-Belle D J amp Wrighton S
A (2000) Present and future in vitro approaches for drug
metabolism Journal of Pharmacological and Toxicological Methods
44 313ndash324
Ekins S Waller C L Swaan P W Cruciani G Wrighton S A amp
Wikel J H (2000) Progress in predicting human ADME parameters in
silico Journal of Pharmacological and Toxicological Methods 44
251ndash272
Erhardt P W (2003) A human drug metabolism database Potential roles
in the quantitative predictions of drug metabolism and metabolism-
related drugndashdrug interactions Current Drug Metabolism 4 411ndash422
Evans W E amp McLeod H L (2003) Pharmacogenomicsmdashdrug
disposition drug targets and side effects New England Journal of
Medicine 348 538ndash549
Ezendam J Staedtler F Pennings J Vandebriel R J Pieters R
Boffetta P et al (2004) Toxicogenomics of subchronic hexachlor-
obenzene exposure in Brown Norway rats Environmental Health
Perspectives 112 782ndash791
Faiola B Fuller E S Wong V A amp Recio L (2004) Gene expression
profile in bone marrow and hematopoietic stem cells in mice exposed to
inhaled benzene Mutation Research 549 195ndash212
FDA (2004) Innovation stagnation Challenge and opportunity on the
critical path to new medicinal products
Fiehn O (2001) Combining genomics metabolome analysis and bio-
chemical modelling to understand metabolic networks Comparative
Functional Genomics 2 155ndash168
Field F J Born E amp Mathur S N (2004) LXRRXR ligand activation
enhances basolateral efflux of beta-sitosterol in CaCo-2 cells Journal of
Lipid Research 45 905ndash913
Fleck C Sutter L Appenroth D Koch B Meinhold T Pitack M
et al (2003) Use of gene chip technology for the characterisation of
the regulation of renal transport processes and of nephrotoxicity in
rats Experimental and Toxicologic Pathology 54 401ndash410
Fliri A F Loging W T Thadeio P F amp Volkmann R A (2005)
Biological spectra analysis Linking biological activity profiles to
molecular structure Proceedings of the National Academy of Sciences
of the United States of America 102 261ndash266
Fountoulakis M (2004) Application of proteomics technologies in the
investigation of the brain Mass Spectrometry Reviews 23 231ndash258
Gaucher S P Taylor S W Fahy E Zhang B Warnock D E Ghosh
S S et al (2004) Expanded coverage of the human heart
mitochondrial proteome using multidimensional liquid chromatography
coupled with tandem mass spectrometry Journal of Proteome
Research 3 495ndash505
Gerhold D Lu M Xu J Austin C Caskey C T amp Rushmore T
(2001) Monitoring expression of genes involved in drug metabolism
and toxicology using DNA microarrays Physiological Genomics 5
161ndash170
Guo G L Choudhuri S amp Klaassen C D (2002) Induction profile of
rat organic anion transporting polypeptide 2 (oatp2) by prototypical
drug-metabolizing enzyme inducers that activate gene expression
through ligand-activated transcription factor pathways Journal of
Pharmacology and Experimental Therapeutics 300 206ndash212
Hagenbuch B amp Meier P J (2004) Organic anion transporting
polypeptides of the OATP SLC21 family Phylogenetic classification
as OATP SLCO superfamily new nomenclature and molecularfunc-
tional properties Pflugers Archiv 447 653ndash665
Hamadeh H K amp Afshari C A (2004) Toxicogenomics principles and
applications Hobokenrsquo John Wiley and Sons
Hamadeh H K Bushel P R Jayadev S Martin K DiSorbo O Sieber
S et al (2002) Gene expression analysis reveals chemical-specific
profiles Toxicological Sciences 67 219ndash231
Hamadeh H K Jayadev S Gaillard E T Huang Q Stoll R
Blanchard K et al (2004) Integration of clinical and gene expression
endpoints to explore furan-mediated hepatotoxicity Mutation Research
549 169ndash183
Hamadeh H K Knight B L Haugen A C Sieber S Amin R P
Bushel P R et al (2002) Methapyrilene toxicity Anchorage of
pathologic observations to gene expression alterations Toxicologic
Pathology 30 470ndash482
Han J D Bertin N Hao T Goldberg D S Berriz G F Zhang L V
et al (2004) Evidence for dynamically organized modularity in the
yeast proteinndashprotein interaction network Nature 430 88ndash93
Hanisch D Zien A Zimmer R amp Lengauer T (2002) Co-clustering of
biological networks and gene expression data Bioinformatics 18
S145ndashS154
Harris A J Dial S L amp Casciano D A (2004) Comparison of basal
gene expression profiles and effects of hepatocarcinogens on gene
expression in cultured primary human hepatocytes and HepG2 cells
Mutation Research 549 79ndash99
Harrison S C (2004) Whither structural biology Nature Structural
Molecular Biology 11 12ndash15
Hartley D P Dai X He Y D Carlini E J Wang B Huskey S E
et al (2004) Activators of the rat pregnane X receptor differentially
modulate hepatic and intestinal gene expression Molecular Pharma-
cology 65 1159ndash1171
Hartwell L H Hopfield J J Leibler S amp Murray A W (1999) From
molecular to modular cell biology Nature 402 C47ndashC52
Hasmall S Orphanides G James N Pennie W Hedley K Soames
A et al (2002) Downregulation of lactoferrin by PPARalpha ligands
Role in perturbation of hepatocyte proliferation and apoptosis
Toxicology Sciences 68 304ndash313
Hayase T Yamamoto Y Yamamoto K Muso E amp Shiota K (2004)
Microarray profile analysis of toxic cocaine-induced alterations in the
expression of mouse brain gene sequences A possible rsquoprotectiversquo
effect of buprenorphine Journal of Applied Toxicology 24 15ndash20
Hayes K R Vollrath A L Zastrow G M McMillan B J Craven M
Jovanovich S et al (2005) EDGE A centralized resource for the
comparison analysis and distribution of toxicogenomic information
Molecular Pharmacology 67 1360ndash1368
Heijne W H Slitt A L van Bladeren P J Groten J P Klaassen C D
Stierum R H et al (2004) Bromobenzene-induced hepatotoxicity at
the transcriptome level Toxicology Sciences 79 411ndash422
Heijne W H Stierum R H Slijper M van Bladeren P J amp van
Ommen B (2003) Toxicogenomics of bromobenzene hepatotoxicity
A combined transcriptomics and proteomics approach Biochemical
Pharmacology 65 857ndash875
Heinloth A N Irwin R D Boorman G A Nettesheim P Fannin
R D Sieber S O et al (2004) Gene expression profiling of rat
livers reveals indicators of potential adverse effects Toxicology
Sciences 80 193ndash202
Hodges L C Cook J D Lobenhofer E K Li L Bennett L Bushel
P R et al (2003) Tamoxifen functions as a molecular agonist inducing
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 63
cell cycle-associated genes in breast cancer cells Molecular Cancer
Research 1 300ndash311
Holleman A Cheok M H den Boer M L Yang W Veerman KM
A J Kazemier et al (2004) Gene-expression patterns in drug-resistant
acute lymphoblastic leukemia cells and response to treatment New
England Journal of Medicine 351 533ndash542
Hong Y Muller U R amp Lai F (2003) Discriminating two classes of
toxicants through expression analysis of HepG2 cells with DNA arrays
Toxicology in Vitro 17 85ndash92
Hood E (2003) Proteomics Characterizing the cogs in the machinery of
life Environmental Health Perspectives 111 A816ndashA825
Hood L (2003) Systems biology Integrating technology biology and
computation Mechanism of Ageing and Development 124 9ndash16
Hood L amp Galas D (2003) The digital code of DNA Nature 421
444ndash448
Hood L amp Perlmutter R M (2004) The impact of systems approaches on
biological problems in drug discovery Nature Biotechnology 22
1215ndash1217
Hu T Gibson D P Carr G J Torontali S M Tiesman J P Chaney
J G et al (2004) Identification of a gene expression profile that
discriminates indirect-acting genotoxins from direct-acting genotoxins
Mutation Research 549 5ndash27
Huang Q Dunn II R T Jayadev S DiSorbo O Pack F D Farr S B
et al (2001) Assessment of cisplatin-induced nephrotoxicity by
microarray technology Toxicology Sciences 63 196ndash207
Huang Q Jin X Gaillard E T Knight B L Pack F D Stoltz J H et
al (2004) Gene expression profiling reveals multiple toxicity endpoints
induced by hepatotoxicants Mutation Research 549 147ndash167
Huisman M T Smit J W Crommentuyn K M Zelcer N Wiltshire
H R Beijnen J H et al (2002) Multidrug resistance protein 2
(MRP2) transports HIV protease inhibitors and transport can be
enhanced by other drugs Aids 16 2295ndash2301
Hunt C M Westerkam W R amp Stave G M (1992) Effect of age and
gender on the activity of human hepatic CYP3A Biochemical
Pharmacology 44 275ndash283
Ideker T Ozier O Schwikowski B amp Siegal A F (2002) Discovering
regulatory and signalling circuits in molecular interaction networks
Bioinformatics 18 S233ndashS240
Ideker T Thorsson V Ranish J A Christmas R Buhler J Eng J K
et al (2001) Integrated genomic and proteomic analyses of a system-
atically perturbed network Science 292 929ndash934
Iida M Anna C H Hartis J Bruno M Wetmore B Dubin J R et al
(2003) Changes in global gene and protein expression during early
mouse liver carcinogenesis induced by non-genotoxic model carcino-
gens oxazepam and Wyeth-14643 Carcinogenesis 24 757ndash770
Ingelman-Sundberg M (2004) Pharmacogenetics of cytochrome P450 and
its applications in drug therapy The past present and future Trends in
Pharmacological Sciences 25 193ndash200
Jeong H Mason S P Barabasi A L amp Oltvai Z N (2001) Lethality
and centrality in protein networks Nature 411 41ndash42
Jeong H Tombor B Albert R Oltvai Z N amp Barabasi A L
(2000) The large-scale organization of metabolic networks Nature
407 651ndash654
Jung D Hagenbuch B Gresh L Pontoglio M Meier P J amp Kullak-
Ublick G A (2001) Characterization of the human OATP-C
(SLC21A6) gene promoter and regulation of liver-specific OATP genes
by hepatocyte nuclear factor 1 alpha Journal of Biological Chemistry
276 37206ndash37214
Jung D amp Kullak-Ublick G A (2003) Hepatocyte nuclear factor 1
alpha A key mediator of the effect of bile acids on gene expression
Hepatology 37 622ndash631
Jung J -W Park J -S Hwang J -W Kang K -S YS L Song B
-S et al (2004) Gene expression analysis of peroxisome prolifer-
ators- and phenytoin-induced hepatotoxicity using cDNA microarray
Journal of Veterinary Medical Science 66 1329ndash1333
Kato N Shibutani M Takagi H Uneyama C Lee K -Y Takiami S
et al (2004) Gene expression profile in the livers of rats orally
administered ethinylestradiol for 28 days using a microarray technique
Toxicology 200 179ndash192
Kier L D Neft R Tang L Suizu R Cook T Onsurez K et al
(2004) Applications of microarrays with toxicologically relevant genes
(tox genes) for the evaluation of chemical toxicants in Sprague Dawley
rats in vivo and human hepatocytes in vitro Mutation Research 549
101ndash113
Kijima K Toyosawa K Yasuba M Matsuoka N Adachi T
Komiyama M et al (2004) Gene expression analysis of the rat testis
after treatment with di(2-ethylhexyl) phthalate using cDNA microarray
and real-time RT-PCR Toxicology and Applied Pharmacology 200
103ndash110
Kim R B (2003) Organic anion-transporting polypeptide (OATP) trans-
porter family and drug disposition European Journal of Clinical
Investigation 33 1ndash5
Kinirons M T amp OrsquoMahony M S (2004) Drug metabolism and ageing
British Journal of Clinical Pharmacology 57 540ndash544
Kitano H (2002a) Computational systems biology Nature 420 206ndash210
Kitano H (2002b) Systems biology A brief overview Science 295
1662ndash1664
Kliewer S A Moore J T Wade L Staudinger J L Watson M
A Jones S A et al (1998) An orphan nuclear receptor activated
by pregnanes defines a novel steroid signalling pathway Cell 92
73ndash82
Korolev D Balakin K V Nikolsky Y Kirillov E Ivanenkov Y A
Savchuk N P et al (2003) Modeling of human cytochrome p450-
mediated drug metabolism using unsupervised machine learning
approach Journal of Medicinal Chemistry 46 3631ndash3643
Kramer J A Pettit S D Amin R P Bertram T A Car B
Cunningham M et al (2004) Overview on the application of
transcription profiling using selected nephrotoxicants for toxicology
assessment Environmental Health Perspectives 112 460ndash464
Kultima K Nystrom A -M Scholz B Gustafson A -L Dencker L
amp Stigson M (2004) Valproic acid teratogenicity A toxicogenomics
approach Environmental Health Perspectives 112 1225ndash1235
Kutuzova G D amp Deluca H F (2004) Gene expression profiles in rat
intestine identify pathways for 125-dihydroxyvitamin D(3) stimulated
calcium absorption and clarify its immunomodulatory properties
Archives of Biochemistry and Biophysics 432 152ndash166
Kwak M K Wakabayashi N Itoh K Motohashi H Yamamoto M amp
Kensler T W (2003) Modulation of gene expression by cancer
chemopreventive dithiolethiones through the Keap1-Nrf2 pathway
Identification of novel gene clusters for cell survival Journal of
Biological Chemistry 278 8135ndash8145
Lahjouji K Mitchell G A amp Qureshi I A (2001) Carnitine transport
by organic cation transporters and systemic carnitine deficiency
Molecular Genetics and Metabolism 73 287ndash297
Landowski C P Sun D Foster D R Menon S S Barnett J L
Welage L S et al (2003) Gene expression in the human intestine and
correlation with oral valacyclovir pharmacokinetic parameters Journal
of Pharmacology and Experimental Therapeutics 306 778ndash786
Lee G -J Lee W -S Jeon K -S Um C -H Kim S -J Lee C -H
et al (2004) cDNA microarray gene expression analysis and
toxicological phenotype for anticancer drug Journal of Veterinary
Medical Science 66 1339ndash1345
Lee M Kwon J Kim S N Kim J E Koh W S Kim E J et al
(2003) cDNA microarray gene expression profiling of hydroxyurea
paclitaxel and p-anisidine genotoxic compounds with differing
tumorigenicity results Environmental and Molecular Mutagenesis
42 91ndash97
Li S Armstrong C M Bertin N Ge H Milstein S Boxem M et al
(2004) A map of the interactome network of the metazoan C elegans
Science 303 540ndash543
Liguori M J Anderson L M Bukofzer S McKim J Pregenzer J F
Retief J et al (2005) Microarray analysis in human hepatocytes
suggests a mechanism for hepatotoxicity induced by trovafloxacin
Hepatology 41 177ndash186
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6664
Lobenhofer E K Bennett L Cable P L Li L Bushel P R amp Afshari
C A (2002) Regulation of DNA replication fork genes by 17beta-
estradiol Molecular Endocrinology 16 1215ndash1229
Locker J Tian J Carver R Concas D Cossu C Ledda-Columbano
G M et al (2003) A common set of immediate-early response genes
in liver regeneration and hyperplasia Hepatology 38 314ndash325
Mattes W B Pettit S D Sansone S A Bushel P R amp Waters M D
(2004) Database development in toxicogenomics Issues and efforts
Environmental Health Perspectives 112 495ndash505
McMillian M Nie A Y Parker J B Leone A Bryant S Kemmerer
M et al (2004) A gene expression signature for oxidant stressreactive
metabolites in rat liver Biochemical Pharmacology 68 2249ndash2261
Meier P J amp Stieger B (2000) Molecular mechanisms in bile formation
News in Physiological Sciences 15 89ndash93
Meneses-Lorente G de Longueville F Dos Santos-Mendes S Bonnert
T P Jack A Evrard S et al (2003) An evaluation of a low-density
DNA microarray using cytochrome P450 inducers Chemical Research
in Toxicology 16 1070ndash1077
Meneses-Lorente G Guest P C Lawrence J Muniappa N Knowles
M R Skynner H A et al (2004) A proteomic investigation of drug-
induced steatosis in rat liver Chemical Research in Toxicology 17
605ndash612
Merrill C L Ni H Yoon L W Tirmenstein M A Narayanan P
Benavides G R et al (2002) Etomoxir-induced oxidative stress in
HepG2 cells detected by differential gene expression is confirmed
biochemically Toxicology Sciences 68 93ndash101
Milo R Shen-Orr S Itzkovitz S Kashtan N Chklovskii D amp Alon
U (2002) Network motifs Simple building blocks of complex
networks Science 298 824ndash827
Morgan K T Ni H Brown H R Yoon L Qualls C W Crosby L
M et al (2002) Application of cDNA microarray technology to in
vitro toxicology and the selection of genes for a real-time RT-PCR-
based screen for oxidative stress in Hep-G2 cells Toxicologic
Pathology 30 435ndash451
Mwinyi J Johne A Bauer S Roots I amp Gerloff T (2004) Evidence
for inverse effects of OATP-C (SLC21A6) 5 and 1b haplotypes on
pravastatin kinetics Clinical Pharmacology and Therapeutics 75
415ndash421
Nakata K Yukawa M Komiyama N Nakano T amp Kaminuma T
(2002) A nuclear receptor database that maps pathways to diseases
Genome Infomatics 13 515ndash516
Nicholson J K Holmes E Lindon J C amp Wilson I D (2004) The
challenges of modeling mammalian biocomplexity Nature Biotechnol-
ogy 22 1268ndash1274
Nicholson J K amp Wilson I D (2003) Understanding rsquoglobal systems
biology Metabonomics and the continuum of metabolism Nature
Reviews on Drug Discovery 2 668ndash676
Nikitin A Egorov S Daraselia N amp Mazo I (2003) Pathway studiomdash
the analysis and navigation of molecular networks Bioinformatics 19
2155ndash2157
Nikolsky Y Ekins S Nikolskaya T amp Bugrim A (2005) A novel
method for generation of signature networks as biomarkers from
complex high throughput data Toxicology Letters 158 20ndash29
Nikolsky Y Nikolskaya T amp Bugrim A (2005) Biological networks
and analysis of experimental data in drug discovery Drug Discovery
Today 10 653ndash662
Nishizato Y Ieiri I Suzuki H Kimura M Kawabata K Hirota T
et al (2003) Polymorphisms of OATP-C (SLC21A6) and OAT3
(SLC22A8) genes Consequences for pravastatin pharmacokinetics
Clinical Pharmacology and Therapeutics 73 554ndash565
Nozawa T Nakajima M Tamai I Noda K Nezu J Sai Y et al
(2002) Genetic polymorphisms of human organic anion transporters
OATP-C (SLC21A6) and OATP-B (SLC21A9) Allele frequencies in
the Japanese population and functional analysis Journal of Pharmacol-
ogy and Experimental Therapeutics 302 804ndash813
Nozawa T Sugiura S Nakajima M Goto A Yokoi T Nezu J et al
(2004) Involvement of organic anion transporting polypeptides in the
transport of troglitazone sulfate Implications for understanding trogli-
tazone hepatotoxicity Drug Metabolism and Disposition 32 291ndash294
Oliver D E Rubin D L Stuart J M Hewett M Klein T E amp
Altman R B (2002) Ontology development for a pharmacogenetics
knowledge base Package Symposion 88ndash99
Oram J F amp Lawn R M (2001) ABCA1 The gatekeeper for eliminating
excess tissue cholesterol Journal of Lipid Research 42 1173ndash1179
Oswald M Kullak-Ublick G A Paumgartner G amp Beuers U (2001)
Expression of hepatic transporters OATP-C and MRP2 in primary
sclerosing cholangitis Liver 21 247ndash253
Parsons A B Brost R I Ding H Li Z Zhang C Sheikh B et al
(2004) Integration of chemical-genetic and genetic interaction data
links bioactive compounds to cellular target pathways Nature Bio-
technology 22 62ndash69
Patki K C von Moltke L L Harmatz J S Hesse L M Court M H
amp Greenblatt D J (2004) Effect of age on in vitro triazolam
biotransformation in male human liver microsomes Journal of
Pharmacology and Experimental Therapeutics 308 874ndash879
Pereira-Leal J B Enright A J amp Ouzounis C A (2004) Detection of
functional modules from protein interaction networks Proteins 54
49ndash57
Plant N (2004) Interaction networks Coordinating responses to xeno-
biotic exposure Toxicology 202 21ndash32
Raghavendra Prasad H S Qi Z Srinivasan K N amp Gopalakrish-
nakone P (2004) Potential effects of tetrodotoxin exposure to
human glial cells postulated using microarray approach Toxicon 44
597ndash608
Reilly T P Bourdi M Brady J N Pise-Masison C A Radonovich M
F George J W et al (2001) Expression profiling of acetaminophen
liver toxicity in mice using microarray technology Biochemical and
Biophysical Research Communications 282 321ndash328
Rives A W amp Galitski T (2003) Modular organization of cellular
networks Proceedings of the National Academy of Sciences of the
United States of America 100 1128ndash1133
Rogers P D Pearson M M Cleary J D Sullivan D C amp Chapman
S W (2002) Differential expression of genes encoding immunomodu-
latory proteins in response to amphotericin B in human mononuclear
cells identified by cDNA microarray analysis Journal of Antimicrobial
Chemotherapy 50 811ndash817
Rosen M B Wilson V S Schmid J E amp Gray L E (2005) Gene
expression analysis in the ventral prostate of rats exposed to vinclozolin
or procymidone Reproductive Toxicology 19 367ndash379
Rost D Herrmann T Sauer P Schmidts H L Stieger B Meier P J
et al (2003) Regulation of rat organic anion transporters in bile salt-
induced cholestatic hepatitis Effect of ursodeoxycholate Hepatology
38 187ndash195
Rothman R B Ayestas M A Dersch C M amp Baumann M H (1999)
Aminorex fenfluramine and chlorphentermine are serotonin transporter
substrates Implications for primary pulmonary hypertension Circula-
tion 100 869ndash875
Ruepp S U Tonge R P Shaw J Wallis N amp Pognan F (2002)
Genomics and proteomics analysis of acetaminophen toxicity in mouse
liver Toxicology Sciences 65 135ndash150
Sakaeda T Nakamura T amp Okumura K (2002) MDR1 genotype-
related pharmacokinetics and pharmacodynamics Biological amp Phar-
maceutical Bulletin 25 1391ndash1400
Sakata T Anzai N Shin H J Noshiro R Hirata T Yokoyama H
et al (2004) Novel single nucleotide polymorphisms of organic cation
transporter 1 (SLC22A1) affecting transport functions Biochemical
and Biophysical Research Communications 313 789ndash793
Satlin L M Amin V amp Wolkoff A W (1997) Organic anion
transporting polypeptide mediates organic anionHCO3-exchange
Journal of Biological Chemistry 272 26340ndash26345
Satomi Y Tsuchiya W Mihara K Ota M Kasahara Y amp Akahori F
(2004) Gene expression analysis of the lung following paraquat
administration in rats using DNA microarray Journal of Toxicological
Sciences 29 91ndash100
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 65
Schrenk D Baus P R Ermel N Klein C Vorderstemann B amp
Kauffmann H M (2001) Up-regulation of transporters of the MRP
family by drugs and toxins Toxicology Letters 120 51ndash57
Segal E Shapira M Regev A Persquoer D Botstein D Koller D et al
(2003) Module networks Identifying regulatory modules and their
condition-specific regulators from gene expression data Nature
Genetics 34 166ndash176
Segal E Wang H amp Koller D (2003) Discovering molecular pathways
from protein interaction and gene expression Bioinformatics 19
i264ndash i272
Seidel S D Kan H L Stott W T Schisler M R amp Gollapudi B B
(2003) Identification of transcriptome profiles for the DNA-damaging
agents bleomycin and hydrogen peroxide in L5178Y mouse lymphoma
cells Environmental and Molecular Mutagenesis 42 19ndash25
Shan L Yu Y Schut H A J amp Snyderwine E G (2004) Susceptibility
of rats to mammary gland carcinogenesis by the food-derived
carcinogen 2-Amino-1-Methyl-6-Phenylimidazo[45-b]Pyridine (PhIP)
varies with age and is associated with the induction of differential gene
expression American Journal of Pathology 165 191ndash202
Sharom J R Bellows D S amp Tyers M (2004) From large networks to
small molecules Current Opinion in Chemical Biology 8 81ndash90
Shitara Y Hirano M Sato H amp Sugiyama Y (2004) Gemfibrozil and
its glucuronide inhibit the OATP2(OATP1B1 SLC21A6)-mediated
hepatic uptake and CYP2C8-mediated metabolism of cerivastatinmdash
Analysis of the mechanism of the clinically relevant drugndashdrug
interaction between cerivastatin and gemfibrozil Journal of Pharma-
cology and Experimental Therapeutics
Shitara Y Itoh T Sato H Li A P amp Sugiyama Y (2003) Inhibition of
transporter-mediated hepatic uptake as a mechanism for drugndashdrug
interaction between cerivastatin and cyclosporin A Journal of
Pharmacology and Experimental Therapeutics 304 610ndash616
Somogyi R Fuhrman S amp Wen X (2001) Genetic network inference in
computational models and applications to large-scale gene expression
data In M Bower amp H Bolouri (Eds) Computational modeling of
genetic and biochemical networks (pp 119ndash157) Cambridge MArsquoThe
MIT Press
Spirin V amp Mirny L A (2003) Protein complexes and functional
modules in molecular networks Proceedings of the National Academy
of Sciences of the United States of America 100 12123ndash12128
Staudinger J Liu Y Madan A Habeebu S amp Klaassen C D
(2001) Coordinate regulation of xenobiotic and bile acid homeo-
stasis by pregnane X receptor Drug Metabolism and Disposition
29 1467ndash1472
Sun L Z Ji Z L Chen X Wang J F amp Chen Y Z (2002) ADME-
AP A database of ADME associated proteins Bioinformatics 18
1699ndash1700
Suter L Babiss L E amp Wheeldon E B (2004) Toxicogenomics in
predictive toxicology in drug development Chemistry and Biology 11
161ndash171
Synold T W Dussault I amp Forman B M (2001) The orphan nuclear
receptor SXR coordinately regulates drug metabolism and efflux
Nature Medicine 7 584ndash590
Tabuchi Y amp Kondo T (2003) cDNA microarray analysis reveals chop-
10 plays a key role in Sertoli cell injury induced by bisphenol A
Biochemical and Biophysical Research Communications 305 54ndash61
Tamai I Nezu J Uchino H Sai Y Oku A Shimane M et al (2000)
Molecular identification and characterization of novel members of the
human organic anion transporter (OATP) family Biochemical and
Biophysical Research Communications 273 251ndash260
Taylor S W Fahy E Zhang B Glenn G M Warnock D E Wiley S
et al (2003) Characterization of the human heart mitochondrial
proteome Nature Biotechnology 21 281ndash286
Terasaka S Aita Y Inoue A Hayashi S Nishigaki M Aoyagi K
et al (2004) Using a customized DNA microarray for expression
profiling of the estrogen-responsive genes to evaluate estrogen activity
among natural estrogens and industrial chemicals Environmental
Health Perspectives 112 773ndash781
Thomas D M Francescutti-Verbeem D M Liu X amp Kuhn D M
(2004) Identification of differentially regulated transcripts in mouse
striatum following methamphetamine treatmentmdashan oligonucleotide
microarray approach Journal of Neurochemistry 88 380ndash393
Thomas R S Rank D R Penn S G Zastrow G M Hayes K R
Pande K et al (2001) Identification of toxicologically predictive
gene sets using cDNA microarrays Molecular Pharmacology 60
1189ndash1194
Thompson K L Afshari C A Amin R P Bertram T A Car B
Cunningham M et al (2004) Identification of platform-independent
gene expression markers of cisplatin nephrotoxicity Environmental
Health Perspectives 112 488ndash494
Tirona R G Leake B F Merino G amp Kim R B (2001)
Polymorphisms in OATP-C Identification of multiple allelic
variants associated with altered transport activity among European-
and African-Americans Journal of Biological Chemistry 276
35669ndash35675
Tirona R G Leake B F Wolkoff A W amp Kim R B (2003)
Human organic anion transporting polypeptide-C (SLC21A6) is a
major determinant of the rifampin-mediated pregnane X receptor
activation Journal of Pharmacology and Experimental Therapeutics
304 223ndash228
Tong W Cao X Harris S Sun H Fang H Fuscoe J et al (2003)
ArrayTrackmdashsupporting toxicogenomic research at the US Food and
Drug Administration National Center for Toxicological Research
Environmental Health Perspectives 111 1819ndash1826
Tornow S amp Mewes H W (2003) Functional modules by relating
protein interaction networks and gene expression Nucleic Acids
Research 31 6283ndash6289
Trocho C Escola-Gil J C Ribas V Benitez S Martin-Campos J M
Rotllan N et al (2004) Phenytoin treatment reduces atherosclerosis in
mice through mechanisms independent of plasma HDL-cholesterol
concentration Atherosclerosis 174 275ndash285
Ueda A Hamadeh H K Webb H K Yamamoto Y Sueyoshi T
Afshari C A et al (2002) Diverse roles of the nuclear orphan
receptor CAR in regulating hepatic genes in response to phenobarbital
Molecular Pharmacology 61 1ndash6
Ulrich R G (2003) The toxicogenomics of nuclear receptor agonists
Current Opinion in Chemical Biology 7 505ndash510
Ulrich R G Rockett J C Gibson G G amp Pettit S D (2004)
Overview of an interlaboratory collaboration on evaluating the effects
of model hepatotoxicants on hepatic gene expression Environmental
Health Perspectives 112 423ndash427
Unami A Shinohara Y Ichikawa T amp Baba Y (2003) Biochemical
and microarray analyses of bupivacaine-induced apoptosis Journal of
Toxicological Sciences 28 77ndash94
Vasquez A Flammini A Maritan A amp Vespignani A (2003) Global
protein function prediction from proteinndashprotein interaction networks
Nature Biotechnology 21 697ndash700
Vezina C M Walker N J amp Olson J R (2004) Subchronic Exposure to
TCDD PeCDF PCB126 and PCB153 Effect on hepatic gene
expression Environmental Health Perspectives 112 1636ndash1644
Vo T D Greenberg H J amp Palsson B O (2004) Reconstruction and
functional characterization of the human mitochondrial metabolic
network based on proteomic and biochemical data Journal of Bio-
logical Chemistry 279 39532ndash39540
Wang H amp LeCluyse E L (2003) Role of orphan nuclear receptors in
the regulation of drug-metabolising enzymes Clinical Pharmacoki-
netics 42 1331ndash1357
Wang N Silver D L Thiele C amp Tall A R (2001) ATP-
binding cassette transporter A1 (ABCA1) functions as a choles-
terol efflux regulatory protein Journal of Biological Chemistry 276
23742ndash23747
Waring J F Cavet G Jolly R A McDowell J Dai H Ciurlionis R
et al (2003) Development of a DNA Microarray for toxicology based
on hepatoxin-regulated sequences Environmental Health Perspectives
111 863ndash870
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6666
Waring J F Gum R Morfitt D Jolly R A Ciurlionis R Heindel M
et al (2002) Identifying toxic mechanisms using DNA microarrays
Evidence that an experimental inhibitor of cell adhesion molecule
expression signals through the aryl hydrocarbon nuclear receptor
Toxicology 181ndash182 537ndash550
Waring J F Jolly R A Ciurlionis R Lum P Y Praestgaard J T
Morfitt D C et al (2001) Clustering of hepatotoxins based on
mechanism of toxicity using gene expression profiles Toxicology and
Applied Pharmacology 175 28ndash42
Waters M Boorman G Bushel P Cunningham M Irwin R Merrick
A et al (2003) Systems toxicology and the Chemical Effects in
Biological Systems (CEBS) knowledge base EHP Toxicogenomics
111 15ndash28
Wei M Arnold L Cano M amp Cohen S M (2005) Effects of co-
administration of antioxidants and arsenicals on the rat urinary bladder
epithelium Toxicology Sciences 83 237ndash245
Weinshilboum R (2003) Inheritance and drug response New England
Journal of Medicine 348 529ndash537
Weinshilboum R amp Wang L (2004) Pharmacogenetics Inherited
variation in amino acid sequence and altered protein quantity Clinical
Pharmacology and Therapeutics 75 253ndash258
Werner E (2003) In silico multicellular systems biology and minimal
genomes Drug Discovery Today 8 1121ndash1127
Wong J S amp Gill S S (2002) Gene expression changes induced in
mouse liver by di(2-ethylhexyl) phthalate Toxicology and Applied
Pharmacology 185 180ndash196
Xie T Tong L McCann U D Yuan J Becker K G Mechan A O
et al (2004) Identification and characterization of metallothionein-1
and -2 gene expression in the context of ()34-methylenedioxymetham-
phetamine-induced toxicity to brain dopaminergic neurons Journal of
Neuroscience 24 7043ndash7050
Yadetie F Laegreid A Bakke I Kusnierczyk W Komorowski J
Waldum H L et al (2003) Liver gene expression in rats in response
to the peroxisome proliferator-activated receptor-alpha agonist ciprofi-
brate Physiological Genomics 15 9ndash19
Yamashita S Nomoto T Abe M Tatematsu M Sugimura T amp
Ushijima T (2004) Persistence of gene expression changes in stomach
mucosae induced by short-term N-methyl-NV-nitro-N-nitrosoguanidinetreatment and their presence in stomach cancers Mutation Research
549 185ndash193
Yan Q amp Sadee W (2000) Human membrane transporter database A
web-accessible relational database for drug transport studies and
pharmacogenomics AAPS Pharmsci 2 E20
Yeger-Lotem E amp Margalit H (2003) Detection of regulatory circuits by
integrating the cellular networks of proteinndashprotein interactions and
transcription regulation Nucleic Acids Research 31 6053ndash6061
Yoon B I Li G -X Kitada K Kawasaki Y Igarashi K amp Kodama
Y et al (2003) Mechanisms of benzene-induced hematotoxicity and
leukemogenicity cDNA microarray analyses using mouse bone marrow
tissue Environmental Health Perspectives 111 1411ndash1420
Young M B DiSilvestro M R Sendera T J Freund J Kriete A amp
Magnuson S R (2003) Analysis of gene expression in carbon
tetrachloride-treated rat livers using a novel bioarray technology
Pharmacogenomics Journal 3 41ndash52
Yu H Zhu X Greenbaum D Karro J amp Gerstein M (2004) TopNet
A tool for comparing biological sub-networks correlating protein
properties with topological statistics Nucleic Acids Research 32
328ndash337
Zhang E Y Fu D J Pak Y A Stewart T Mukhopadhyay N
Wrighton S A et al (2004) Genetic polymorphisms in human proton-
dependent dipeptide transporter PEPT1 Implications for the functional
role of Pro586 Journal of Pharmacology and Experimental Therapeu-
tics 310 437ndash445
Zhang E Y Knipp G T Ekins S amp Swaan P W (2002)
Structural biology and function of solute transporters Implications
for identifying and designing substrates Drug Metabolism Reviews
34 709ndash750
Zhang E Y Phelps M A Cheng C Ekins S amp Swaan P W (2002)
Modeling of active transport systems Advanced Drug Delivery
Reviews 54 329ndash354
- Systems-ADMETox Resources and network approaches
-
- Introduction
- Data available
- Network analysis and databases
-
- Network applications
-
- The role of transporters
-
- Clinical relevance of transporters
- Transporter network examples ABCA1
- Transporter network examples OATP
- Transporter microarray data
-
- Applications to enzymes
- Future network applications
-
- Discussion
- Acknowledgements
- References
-
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6648
A second study has indicated how a natural language
processing method CCNet was used to show the genes
regulated by the nuclear hormone receptor FXR (Apic
Ignjatovic Boyer amp Russell 2005) These automated
methods enable a more complete understanding of the
complexity of the transcriptional factors (Ekins Mirny amp
Schueltz 2002b Plant 2004 Ulrich 2003) but ultimately
rely on the quality of the content of the underlying database
of literature interactions This is a key consideration that is
often overlooked For example the gold standard database is
one that is manually curated to ensure the fidelity of the
direct interaction and is preferable to one generated
computationally by algorithms like natural language pro-
cessing (Nikolsky Nikolskaya amp Bugrim 2005) The
advantage of interaction networks over clustering has been
demonstrated in one study using MetaCore (Nikolsky
Ekins Nikolskaya amp Bugrim 2005) by reanalysis of a
published microarray study of G0-arrested MCF-7 breast
cancer cells treated with estrogen and 4-hydroxytamoxifen
(Hodges et al 2003) After producing integrated gene
networks for each treatment strikingly different patterns
were displayed although both contained early transcriptional
factors myc jun and fos Only the estrogen network featured
induced genes essential for all cell cycle phases (Nikolsky et
al 2005) Similarly microarray data for benzene toxicity
(Yoon et al 2003) has been re-analyzed by focusing on the
genes assessed on the p53 pathway (Ekins et al 2005e) We
envisage that a database of such networks for toxic
compounds will be used for comparing between different
molecules and used in the development of predictive
algorithms for Systems-ADMETox modeling in future
Another approach to using such pathway approaches is to
visualize the results of quantitative structure activity models
for predicting molecules binding to enzymes transporters
receptors and ion channels (Ekins Andreyev et al in press
2005e)
It is also possible to simultaneously interpret high
throughput data and predictions on interaction networks
providing a novel approach to predicting and understanding
potential undesirable drugndashdrug or off target effects in the
area of systems pharmacology An example data set uses
percent inhibition data for clotrimazole and ticonazole
which were screened against many different assays at a
single concentration in a commercially available database
BioPrint (Cerep Redmond WA) as published recently
(Fliri Loging Thadejo amp VOlkman 2005) The data for
10 assays has been arbitrarily encoded as inhibitors (gt50
inhibition) or non-inhibitors (lt50 inhibition) in a text file
which was then loaded into MetaCore The analyzed
network algorithm was then used which generates a large
network and fragments it into sub-networks each with a Z-
score and p-values for ranking according to saturation with
objects from the initial gene list The Gene-ontology
processes are also mapped to the gene list and individual
networks In this example a statistically significant network
was generated for the different proteins (Fig 4
p =2838e31) This network also maps the Gene Ontology
processes for the activation of MAPK (118 of genes p
value 9143e07) signal transduction (333 1600e05)
regulation of transcription DNA-dependent (294
2786e04) regulation of inflammatory response (39
3746e04) and the regulation of blood pressure (78
4230e04) This example network indicates how molecules
of the same or different therapeutic classes could be
evaluated for their effects as a graph either together as in
this case or individually This would be useful to indicate
potential off target effects and identify structurally dissimilar
molecules with similar network patterns Such networks
could then be compared to assess network overlap or
differences between molecules and their inhibition of
multiple proteins This type of unique visualization of high
throughput screening data illustrates how the target proteins
may be connected as a network to infer the possible
downstream effects of inhibition
With the burgeoning number of freely available online
and commercial databases that can be used for pathway
construction numbering in the hundreds there have been
suggestions to impose standards for model exchange
querying and visualization (Cary Bader amp Sander 2005)
To date there has been little discussion with regards to
standardization of ADMETox related databases although
there has been considerable discussion relating to drug
metabolism database generation (Erhardt 2003) This is
certainly an important area to address in the future There is
already a growing literature related to ADMETox that is
partially captured in the several commercially available
databases (Ekins et al 2005e) but to date there have been
limited academic efforts to capture data for transporters
with the human membrane transporter database (Yan amp
Sadee 2000) TP-search transporter database (httpwww
ilabrisewasedaacjptp-search) drug interaction database
(httpwwwdruginteractioninfoorgDatabaseinfoaspx)
nuclear hormone receptors (Nakata Yukawa Komiyama
Nakano amp Kaminuma 2002) the ADME-AP database
(Sun Ji Chen Wang amp Chen 2002) and PharmaGKB
(Oliver et al 2002) DSStox (httpwwwepagovnheert
dsstox) TOXNET (httptoxnetnimnihgov) that are
readily accessible
In order to generate accessible pathways using any of
the available software a large enough set of object
identifiers are required to map onto the underlying data-
base To demonstrate this datasets from toxicogenomics
studies have been evaluated with both the KEGG pathway
database and a commercially available product MetaCore
(wwwgenegocom) These gene or protein lists range in
size from 21 to 1853 objects In virtually all cases more
identifiers are mapped to networks in MetaCore and this
also seems independent of the identifier type used
(Unigene Affymetrix Genbank or Locuslink Table 5)
These numbers will obviously change as more information
is added to the respective databases hence the more
objects that are mapped from a dataset The more extensive
Table 4
Literature derived toxicoproteomics data
Compounds Data source Microarray type Compound dose Microarray data
availability
Reference
Carbon
tetrachloride
Male Wistar rats Affymetrix rat chip 8799
probes+proteomics
study
6ndash24 h exposure Genbank SwissProt and
binary data in a
manuscript table
(Fountoulakis
2004)
Paracetamol CD-1 male mice Custom mouse tox blots
with 450 genes
RTQ-PCR+proteomics
study 2-DIGE+
MALDI-MS
150 or 500 mgkg Gene name IMAGE ID
GenBank accession
number fold induction
SwissProt identifier and
protein abundance
change in manuscript
tables
(Ruepp Tonge
Shaw Wallis amp
Pognan 2002)
Paracetamol AP-1 male mice Affymetrix murine 11K
set+proteomics study
Up to 500 mgkg Gene names and fold
changes at multiple
doses and time points in
a manuscript tablemdash
proteomics data not
accessible
(Coen et al 2004)
Oxazepam or
Wy-14643
Male B6C3F1 mice NIEHS Mouse Chip
(8700 genes) 2-DIGE
and MS
Oxazepam (2500 ppm)
Wyeth (Wy)-14643
(500 ppm)
Proteomics data in a
manuscript table
Genbank accession
number gene name and
fold change in a
manuscript table data
also available to
download from NIEHS
website
(Iida et al 2003)
Compound A
(PPAR gamma
ligand)
Female rats CrlCD
(SD)IGS BR
Proteomics 2-DIGE and
MS
250 mgkgday up to
5 days
Accession number and
protein name and
average ratio in a
manuscript table
(Meneses-Lorente
et al 2004)
Bromobenzene Male Wistar rats Proteomics 2-DIGE and
MS custom 3000 cDNA
rat chip
5 mmolkg Accession number gene
name fold change in a
manuscript table protein
names along with bar
charts
(Heijne Stierum
Slijper van
Bladeren amp van
Ommen 2003)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 49
the network that can be generated (as it will consist of more
nodes) then a more comprehensive understanding of the
networks is possible The data available currently in the
literature can be used to evaluate such pathway and
network generation tools Recently we have used several
of the published studies (Tables 1ndash3) with MetaCore to
visualize networks for acetaminophen furan carbon tetra-
chloride benzene and cisplatin showing genes involved in
In vivo
In vitro
In Silico
1970s 1980s 1990s 2000s
Systems Biology ()
OMICS
Fig 2 The timeline for major paradigms in ADMETox
oxidative stress (Ekins Giroux Nikolsky Bugrim amp
Nikolskaya 2005c)
Other important pathwaynetwork building tools that could
potentially be applied to toxicogenomics data include
Ingenuity pathways analysis (httpwwwingenuitycom)
PathArt (httpwwwjubilantbiosyscompdhtm) Pathway
Assist (httpwwwariadnegenomicscomproctspathway
html) (Nikitin et al 2003) and several other databases
deposited at the Pathway Resource List (httpcbiiomskcc
orgprl) All of these products have unique underlying
proprietary pathway databases which are compiled manually
or automatically with text mining tools or a combination of
both We are still waiting for studies that provide a comparison
of different database tools or network building algorithms
Until then theremay be someoverlap but also some differences
between the results obtained from more than one network
method due to the database content and the algorithms used
The reader is recommended to evaluate for themselves several
technologies and select those with the most appropriate fit to
their own specialized needs
In the next Sections the further application of some
available network and database tools will be described with
Microarray or other high throughput data
Upload list of geneprotein identifiers and fold change significance etc (raw data or after selection of the differentially expressed
Genes using other statistical methods)
Parse database and generate interaction network with different algorithms or visualize on maps
Filter Networks
Access significance of the interaction networks
Determine GO processes
Compare 2 or more networks intersection overlap etc
Export gene list
Export gene list
generate interaction network with different algorithms or visualize on maps
Visualization
Fig 3 Schematic for the utilization of pathway tools for assessing high throughput data
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6650
specific detailed reference to transporters and enzymes but
these technologies can also be applied elsewhere as
described above
Fig 4 Network for high throughput screening data for clotrimazole and ticona
cholecystokinin CCR1 Choline transporter Chloride channel catechol-O-methyl
and co workers (Fliri et al 2005) Nodes highlighted with large blue circles rep
molecules showing gt50 inhibition in the assay blue circles represents both mol
inhibits gt50 and one inhibits lt50 Ligands (large) linked to transcriptional f
(wwgenegocom) (For interpretation of the references to colour in this figure leg
311 The role of transporters
A diverse array of organic solutes such as nutrients
neurotransmitters and drugs are transported by specialized
zole screened against 10 in vitro assays (Human Cannabinoid 1 Human
transferase COX2 CYP1A2 CYP2B6 CYP2C19) data published by Fliri
resent assays used in the study Red circles on these nodes represent both
ecules showing lt50 inhibition chequered circles represent one molecule
actors enzymes and transporters via edges using the MetaCorei database
end the reader is referred to the web version of this article)
Table 5
Datasets acquired from publications and other sources and used to evaluate two databasepathway tools KEGG and metaCore
Dataset Number of objects
genes proteins in list
Number of objects
mapped with KEGG
Number of objects mapped
with MetaCore on networks
Reference for microarray
data gene list
Tox Chip 10 (NIEHS) 1853 405 1498 httpsdir-appsniehsnihgov
mapsguestclonesrchcfm
4-Hydroxytamoxifen and
estrogen
1617 434 1343 (Hodges et al 2003)
Mitochondrial proteins 722 156 388 (Gaucher et al 2004
Taylor et al 2003)
Bromobenzene
(24 and 48 h)
130 41 89 (Heijne et al 2004)
Acetaminophen 30 19 23 (Heinloth et al 2004)
Acetaminophen 84 29 64 (Huang et al 2004)
Furan 185 64 139 (Huang et al 2004)
Tetrodotoxin 116 31 86 (Raghavendra Prasad et al
2004)
Benzene 73 16 62 (Yoon et al 2003)
Benzene 76 5 53 (Faiola et al 2004)
Carbon tetrachloride 37 8 26 (Young et al 2003)
Estrogen 94 33 90 (Lobenhofer et al 2002)
Trovafloxacin 142 20 82 (Liguori et al 2005)
Phenobarbital 37 13 28 (Ueda et al 2002)
L-742694 (liver)+ 45 17 19 (Hartley et al 2004)
L-742694 (intestine)+ 23 11 10 (Hartley et al 2004)
A-277249 21 7 9 (Waring et al 2002)
All files were converted to Locuslink identifiers except as noted () unigene () Affymetrix (+) Genbank identifiers were used
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 51
proteins across cellular membranes These may function as
passive processes or active processes energized by the
hydrolysis of ATP or coupling to the co-transport of counter
ions down an electrochemical gradient such as Na+ H+ and
Cl There are many thousands of transporters which can be
classified into distinct superfamilies One of these the solute
carrier class (SLC) is rapidly expanding and contains over
30 families and 200 members The ATP-binding cassette
(ABC) contains 7 families and over 48 members including
P-glycoprotein (P-gp) and MRP subfamilies (Zhang Knipp
Ekins amp Swaan 2002a Zhang Phelps Cheng Ekins amp
Swaan 2002b) Transporters have a key role in clinical
pharmacology with many drugs specifically targeting them
Numerous drugs share transport pathways with nutrients
and transporters have a role in oral absorption drug
bioavailability drug resistance excretion and ultimately
pharmacokinetics and pharmacodynamics
Polymorphism of drug transporters may be a key factor
in drug interactions and lack of effectiveness This field has
become known as pharmacogenomics and is focused on
understanding of inherited DNA sequence variations (poly-
morphisms and mutations) revealed by xenobiotics (Evans
amp McLeod 2003 Weinshilboum 2003) Over the last few
decades many genes have been directly linked to the
mechanisms of response (Evans amp McLeod 2003 Wein-
shilboum 2003) such that 20ndash95 of variability to drug
response is inheritable (Evans amp McLeod 2003) This
phenotypical variability is mainly caused by single nucleo-
tide polymorphisms (SNPs) present in anywhere from 1 to
50 of the population resulting in either lower protein
activity incorrect folding or rapid degradation via proteo-
somes (Weinshilboum amp Wang 2004) A number of
structurally diverse molecules bind to P-gp which is
expressed in many tissues and has numerous SNPs one of
which (C3435T) affects the expression level in the
duodenum and therefore can impact the absorption of
molecules which would be substrates for this transporter
(Sakaeda Nakamura amp Okumura 2002) The human
proton-dependent dipeptide transporter (hPEPT1) can also
affect the absorption of molecules in the intestine and
recently 9 SNPs were found with only one displaying a
reduced transport capacity (Zhang et al 2004) The sodium-
dependent carnitine cotransporter OCTN2 can possess
mutations and these result in primary carnitine deficiency
which impacts fatty acid oxidation and is characterized by
many clinical manifestations (Lahjouji Mitchel amp Qureshi
2001) The organic cation transporter 1 (OCT1) is also
important in the transport of numerous xenobiotics and
endobiotics Recently 4 SNPs were identified in the
Japanese population and when functionally characterized
in vitro the uptake of cations was reduced significantly for
some of these mutations indicating that this would likely
contribute to inter-individual variations in metabolism of
drugs which were transported via OCT1 (Sakata et al
2004)
3111 Clinical relevance of transporters The pregnane
X-receptor (PXR) is a transcriptional regulator of the
enzyme human MDR1 (P-gp) MRPs and OATP (Wang amp
LeCluyse 2003) CYP3A (Bertilsson et al 1998 Blumberg
et al 1998 Kliewer et al 1998) and CYP2C89 (Synold
Dussault amp Forman 2001) as well as many other genes
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6652
involved in the transport metabolism and biosynthesis of
bile acids (Staudinger Liu Madan Habeebu amp Klaassen
2001) However the additional receptors CAR FXR LXR
and other nuclear receptors take part in a complex network
of interactions to control these and other proteins Thus
elucidation of the regulatory networks which control the
expression of these transporters is also important To date
most of the research has centered on efflux transporters but
there has been considerable interest in uptake transporters
such as the organic anion transporter polypeptide (OATP
((Kim 2003) see also later Section on OATP)
There are several specific examples of the importance of
drug transporters to the clinical development of drugs One
example is the insulin sensitizer troglitazone which was
withdrawn due to hepatotoxicity although the precise
mechanism appears to have been unclear until recently
The major metabolite is a sulfated species and is suspected
of being responsible for the observed toxicity The recent
assessment of the organic anion transporting polypeptides
OATP-C and OATP8 expressed on the hepatocyte baso-
lateral membrane indicated that sulphated troglitazone has a
high affinity for the former and possibly lower affinity for
the latter (Nozawa et al 2004) This metabolite would
therefore be expected to accumulate in hepatocytes and
inhibit the bile salt export pump and Mrp2 Because
polymorphisms have been shown for OATP-C (Tirona
Leake Merino amp Kim 2001) it is also possible that these
may result in the accumulation of the metabolite and in turn
elicit idiosyncratic toxicity A second relevant example of
the impact of transporters is the clinically significant drugndash
drug interaction between cerivastatin and cyclosporine A
which occurs via the OATP-C transporter (Shitara Itoh
Sato Li amp Sugiyama 2003) A third example are the HIV
protease inhibitors saquinavir ritonavir and indinavir which
are transported by MRP2 in vitro and other drugs such as
probenecid and sulfinpyrazone are able to enhance this
transport Transport by MRP2 suggests that these com-
pounds will have decreased bioavailability due to increased
clearance and other drugs could aggravate this situation by
further enhancing transport (Huisman et al 2002) Sim-
ilarly the rifampicin mediated induction via PXR of MRP2
and P-gp in healthy subjects was found to significantly
decrease the AUC and also correlated with intestinal
expression of these transporters This transporter is also
inducible by cisplatin 2-AAF and phenobarbital (Schrenk
et al 2001) indicating multiple mechanisms may be
involved In other species such as rat commonly used as a
toxicity model orthologs of the transporters such as OATP2
are expressed and can be induced with ligands for PXR like
PCN (Guo Choudhuri amp Klaassen 2002) This is useful
knowledge because the advent of microarray technology
allows one to dose a rat with a xenobiotic and assess
thousands of genes simultaneously in a particular tissue For
instance animals dosed with known nephrotoxins have
shown some upregulation of the NandashKndashCl transporter
however the authors suggested genomic responses are
stronger soon after exposure before declining (Fleck et
al 2003) Some transporters may therefore be specifically
targeted by drugs in one tissue such as the CNS but these
same transporters may also be expressed elsewhere in the
body hence off-target effects may result in toxicity The
serotonin transporter is one such example which is
expressed in the lungs and brain Some substrates for this
channel like fenfluramine can result in primary pulmonary
hypertension as they accumulate in pulmonary cells (Roth-
man Ayestas Dersch amp Baumann 1999) Similarly P-gp is
expressed at the blood brain barrier and intestine impacting
the efficacy and bioavailability of drugs
3112 Transporter network examples ABCA1 The
ABCA1 transporter mediates the first step of cholesterol
transport Mutations in this gene cause Tangier disease
which results in severe HDL deficiency cholesterol
accumulation in macrophages and attendant atherosclerosis
This transporter represents a drug target for upregulation
modulating cholesterol metabolism and prevention of
cardiovascular disease (Oram amp Lawn 2001) In vitro
ABCA1 can be inhibited by the sulfonylurea glybenclamide
(Field Burn amp Mathur 2004 Wang Silver Thiele amp Tall
2001) In order to illustrate the advantages of mapping drug
transporters as networks onto functional models alongside
other proteins one can consider the example of ABCA1
The query of the MetaCorei database shows that this
transporter appears on three manually curated pathway maps
representing the Fstate of the art_ knowledge derived from
reliable high quality literature sources One can also use the
individual maps as an interface to access the underlying
layers of information about the transporter including the list
of encoding genessplice variants with known SNPs In
addition to browsing MetaCorei a user can also build
custom networks around ABCA1 using the network-
construction tool (Fig 5) Such a visualization utility may
be very helpful for identification of all putative pathways
around a particular transporter or compound of interest The
ABCA1 network created by this tool shows that this
transporter at the time of writing is linked directly to
twenty-five other objects such as APOE1 and LXR Many
of its neighbors have their own SNPs that could be
important in determining interactions between transport of
a drug and normal human transport of endogenous ligands
in health or disease
3113 Transporter network examples OATP The
OATPs are key membrane bound transporters expressed in
many organs including intestine liver lung choroid plexus
blood brain barrier and other organs (Tamai et al 2000)
This family of transporters is capable of mediating the
sodium-independent transport of a diverse array of mole-
cules such as steroid conjugates organic anions and
xenobiotics by coupling uptake with efflux of bicarbonate
(Satlin Amin amp Wolkoff 1997) glutathione or its
conjugates (Hagenbuch amp Meier 2004) The inhibition of
Fig 5 The network of direct gene interactions around the ABCA1 transporter gene (centre) generated with MetaCorei The interation types between nodes are shown as small colored hexagons eg unspecified
allosteric regulation binding cleavage competition covalent modification dephosphorylation phosphorylation transcription regulation transformation When applicable interactions also have a positive or
negative effect and direction Ligands (purple) linked to other proteins (blue blobs) transfactors (red) enzymes (orange) (For interpretation of the references to colour in this figure legend the reader is referred to
the web version of this article)
SEkin
sJournalofPharm
acologica
landToxico
logica
lMeth
ods53(2006)38ndash66
53
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6654
this transporterrsquos hepatic uptake of other compounds may be
important for reported drugndashdrug interactions (Kim 2003)
described earlier (Shitara et al 2003) as well as cerivastatin
with gemfibrozil (Shitara Hirano Sato amp Sugiyama 2004)
OATP1B1 (previous names OATP-C LST-1 OATP2
SLC21A6) represents the most studied human OATP to
date (Meier amp Stieger 2000) and is expressed on the
basolateral plasma membrane of hepatocytes Several single
nucleotide polymorphisms have been identified in the
OATP1B1 gene in European-Americans African-Americans
(Tirona et al 2001) and Japanese (Nozawa et al 2002)
dramatically impacting the transport of ligands such as
pravastatin (Mwinyi Johne Bauer Roots amp Gerloff 2004
Nishizato et al 2003) estrone-3-sulfate (Nozawa et al
2002 Tirona et al 2001) Rifampin (Tirona Leake
Wolkoff amp Kim 2003) and estradiol 17h-d-glucuronide(Tirona et al 2001)
The regulation of SLCO may be affected during
extrahepatic cholestasis bile duct ligation bile salt induced
Fig 6 Networks generated with the autoexpand algorithm in MetaCorei to illustr
be interconnected with other protein regulatory signaling information Informa
unspecified allosteric regulation binding cleavage competition covalent m
transformation When applicable interactions also have a positive or negative e
transfactors (red) enzymes (orange) (For interpretation of the references to colour
cholestatic hepatitis (Dumont et al 1997 Meier amp Stieger
2000 Rost et al 2003) and primary sclerosing cholangitis
(Oswald Kullack-Ublick Paumgarter amp Beuer 2001) In
particular OATP1B1 appears to be regulated by the liver-
enriched transcription factor hepatocyte nuclear factor 1a
(HNF1a) which binds to the promoter region of this
transporter (Jung et al 2001) Site directed mutagenesis
of this binding site resulted in inactivation suggesting the
critical nature of the interaction with HNF1a Bile acids
such as CDCA have been shown to transcriptionally repress
HNF1a in vitro via inhibition of the transactivating effect of
HNF4a on HNF1a (Jung amp Kullak-Ublick 2003) After
screening many rat and human uptake transporters in vitro
OATP1B1 was also shown to modulate the PXR response
by controlling rifampin retention in the cell and therefore
affecting the induction of CYP3A4 and other gene products
such as P-gp (Tirona et al 2003)
Some of the literature for OATP1B1 human substrate
data has been annotated into MetaCorei to illustrate the
ate how ligands for the human OATP-C (OATP1B1) transporter (centre) can
tion on the type of interaction between objects is hidden for clarity eg
odification dephosphorylation phosphorylation transcription regulation
ffect and direction Ligands (purple) linked to other proteins (blue blobs)
in this figure legend the reader is referred to the web version of this article)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 55
visualization of the complex interconnections between this
transporter its ligands regulatory factors and signaling
molecules already in the database (Fig 6) The network was
generated with the autoexpand algorithm in the software
representing one of multiple available algorithms for
connecting genes ligands and other objects in the database
Clearly if more ligands and their connections are added to
the database the complexity of the network will increase
considerably The OATP-C (OATP1B1) gene details can be
viewed upon querying the database and links are provided
to other public databases This page can be used to highlight
the multiple synonyms for this gene as well as links to the
multiple SNPs identified to date
3114 Transporter microarray data Microarrays have
generally been limited in the number of transporters present
on them (Annereau et al 2004) however they have been used
in an attempt to correlate pharmacokinetic properties with
gene expression for valacyclovir (Landowski et al 2003) as
well as understand the expression profile in different tissues
or cell lines upon food component or xenobiotic treatment
(Anderle Huang amp Sadee 2004) This lack of transporters
on microarrays has prompted some groups to produce their
own arrays with a heavier emphasis on transporters These
arrays have for example then been used to demonstrate the
upregulation of ABC transporters and down-regulation of
GST-Pi in cell lines resistant to colchicines or 9-nitro-
camptothecin (Annereau et al 2004) The genes that were
significantly up or down-regulated in this particular study
were used to build networks with MetaCore (Fig 7A B) and
the similarities between them were assessed (Fig 7C)
Although there were only a small number of significantly
changed genes in common (IL-8 Fos GST-Pi Calpactin and
Ubiquitin hydrolase) it is perhaps likely that there is a much
larger common gene network that is important for drug
resistance although a much larger number of cell lines and
drug treatments need to be evaluated to produce a definitive
drug resistance signature involving transporters enzymes
and transcriptional regulators
312 Applications to enzymes
As we have already described it may be particularly
valuable to visualize enzymes as networks to show
interactions with transcriptional regulators and ligands
For example a key enzyme is CYP3A4 which metabolizes
40ndash45 of all drugs and has relatively few SNPs
(Ingelman-Sundberg 2004) Using a second software suite
MetaDrugi (wwwgenegocom) it is possible to construct
a custom network around this or other drug metabolizing
enzymes (Fig 8) In this case the gene network for
CYP3A4 highlights all of the major transcriptional
regulators and several more distant linked proteins and
ligands connected on the network that may be useful for
further study (Fig 8) Substrates inhibitors as well as
regulatory factors and other enzymes can be observed
connected on this network Due to the many hundreds of
known inhibitors and substrates only a small number are
shown here for clarity We assume that if a perturbation in
a pathway (eg due to a nonfunctional enzyme) is linked
to a certain pathologic condition a similar perturbation
caused by the interference from xenobiotic metabolism
(eg competitive inhibition of the same enzyme) may
result in identical effects As microarray gene expression
data is increasingly generated the role of enzyme
regulation in toxicity of certain xenobiotics will become
more apparent from either in vivo or in vitro studies The
visualization of such signature gene networks involving
transporters and enzymes their ligands and regulatory
factors will also be important for future toxicity prediction
methods We have recently generated visualizations of
microarray data from MCF-7 cells treated with 4-hydrox-
ytamoxifen to show that some of the key genes involved in
metabolism and transport are upregulated (Ekins et al
2005d) In addition we have made predictions with various
QSAR models in MetaDrug to indicate the involvement of
PXR CYP3A4 and P-gp (Ekins et al 2005e) Therefore it
appears likely that 4-hydroxytamoxifen could increase its
own metabolism as well as efflux from cells via P-gp
which can be visualized on networks Any decrease of
function of these enzymes or transporters in a population
would likely result in changes in the metabolism and
transport of this active metabolite potentially impacting
the clinical effect This represents one example of how
both pathway tools QSAR models and network building
algorithms can be used with different types of predicted
and experimental data to allow visualization of potential
compound interactions or toxicity
313 Future network applications
As the population ages an increasing incidence and
prevalence of systemic diseases especially chronic diseases
have occurred among older adults This has resulted in an
increase in medications used concomitantly by this
population which presents challenges for drugndashdrug
interactions Physiologically elderly patients may behave
differently to the younger patients for which the drugs are
initially developed for and tested on Many pharmacoki-
netic investigations in the elderly population reveal
decreased clearance of lipophilic drugs metabolized by
the cytochrome P450 enzymes however few studies have
evaluated aging-dependent or gender-related changes in
specific P450 enzymes (Hunt Westerkam amp Slave 1992)
Age-related physiological changes such as a reduction in
liver mass hepatic metabolizing enzyme activity liver
blood flow and alterations in plasma drug binding may
account for the decreased elimination of some metabolized
drugs in the elderly It is particularly difficult to separate an
effect of aging from the variation in the rate of metabolism
due to factors such as individual metabolic phenotype
(slow or fast metabolizer due to SNPs) environmental
influences concomitant disease states and drug intake
(drugndashdrug interactions) The available data suggest that
Fig 7 Networks for drug resistant cell lines generated with the Auto expand algorithm in MetaCore A KB-3-1 vs KB-8-5 B ATC-DU-145 vs RCO1 C node overlap of A and B with initial nodes highlighted
with blue or red solid circles (Initial gene lists derived from (Annereau et al 2004)) Information on the type of interaction between objects is hidden for clarity eg unspecified allosteric regulation binding
cleavage competition covalent modification dephosphorylation phosphorylation transcription regulation transformation When applicable interactions also have a positive or negative effect and direction
Ligands (purple) linked to other proteins (blue blobs) transfactors (red) enzymes (orange) (For interpretation of the references to colour in this figure legend the reader is referred to the web version of this article)
SEkin
sJournalofPharm
acologica
landToxico
logica
lMeth
ods53(2006)38ndash66
56
Fig7(continued)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 57
Fig7(continued)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6658
Fig 8 The network of gene interactions around the CYP3A4 enzyme (centre) from MetaCorei Information on the type of interaction between objects is
hidden for clarity eg unspecified allosteric regulation binding cleavage competition covalent modification dephosphorylation phosphorylation
transcription regulation transformation When applicable interactions also have a positive or negative effect and direction Ligands (purple) linked to other
proteins (blue blobs) transfactors (red) enzymes (orange) (For interpretation of the references to colour in this figure legend the reader is referred to the web
version of this article)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 59
the initial doses of drugs metabolized by these enzymes
should be reduced in older patients according to the clinical
response In most published studies the elderly appear at
least as responsive as the young to inducers or inhibitors of
P450s (Durnas Loi amp Cusack 1990) More recently there
has been some suggestion that there are age related
reductions in function of some specific P450s such as
CYP3A4 (Patki et al 2004) and this could occur at the
level of regulation However earlier studies with the same
enzyme showed no change in clearance with age (Hunt et
al 1992) To date there has been even less examination of
the transporter functions and any changes with age
(Kinirons amp OrsquoMahony 2004) so the current understand-
ing of the effects of aging on metabolism and transport is
anything but transparent This represents an extreme
challenge for the pharmaceutical industry how to predict
whether a drug has an affinity for an enzyme or transporter
and whether this may be clinically important if it shows a
decline in expression or function with aging Any decrease
of function of these enzymes or transporters in an elderly
population would result in changes in the metabolism and
transport of metabolites potentially impacting the clinical
effect This represents another example of how computa-
tional approaches may perhaps be used with different types
of predicted and experimental data to allow visualization of
potential compound interactions or toxicity in elderly
populations We may see different gene networks high-
lighted as humans age and these may be modified by drug
treatment and coadministration This represents an area
were network analysis could be applied in the future and is
in need of considerable further research
The collection of microarray data in databases such as
CEBS ArrayTrack and EDGE represents a future resource
for computational gene network analysis One could
envisage that ultimately in each case such data is converted
to one or more networks that are also displayed for the user
and can be used to compare treatments from in vivo and in
vitro experiments This would represent a different approach
to clustering the data as currently implemented in one of
these efforts (Hayes et al 2005) and may condense large
amounts of experimentally derived data into a readily
interpreted network
4 Discussion
Previously in this journal the progress of many
research groups in predicting human ADME parameters
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6660
in silico (Ekins et al 2000b) and approaches for drug
metabolism (Ekins Ring Grace McRobie-Belle amp
Wrighton 2000a) have been described Both of these
reviews commented on moving HT assays for ADME
Tox much earlier in drug discovery which had also been
indicated by other groups The initial reviews also
highlighted the likely wealth of data that would become
available and how this could be used for structure
activity relationships alongside the bioactivity data in
computational models It was noted that there was a
paucity of predictive metabolism tools at that time In
addition a growing number of efforts to model whole
cells and organs now a field called systems biology
were recognized as models that could be integrated with
the in silico ADME approaches In summary since these
past reviews virtually all pharmaceutical companies have
attempted earlier high throughput screening for ADME
Tox properties and to some extent the wider application
of computational approaches for physicochemical proper-
ties Systems biology is being quite widely acknowledged
as the new paradigm for understanding complex bio-
logical datasets derived from high throughput technolo-
gies and the accumulated knowledge on human protein
interactions (Hartwell Hopfield Leibler amp Murray 1999
Hood 2003b) Therefore systems biology can be defined
as the integration of genetic proteomic transcriptomic
and metabonomic data using computational methods
(Nicholson amp Wilson 2003) When taken together
information on molecular processes derived from different
sources represents a lsquolsquouniversersquorsquo of putative biological
functionality of which only a small fraction of it will be
realized in a cell at any given time To date systems
biology has been driven by academia and funding bodies
such as the NIH rather than the pharmaceutical compa-
nies Presently there is a great deal of interest from
scientists of all backgrounds in identifying the networks
of cellular pathways and the corresponding physically
interacting proteins
The network building software for systems biology
described in this current review will be valuable to query
high throughput data and known literature interactions in
order to predict potential toxicity in different species In the
future the compilation of published toxicogenomics data-
sets characteristic of different types of toxicity will likely
be available in these software systems to act as a reference
database It is also feasible that we will be able to generate
the annotated datasets which specifically address the
differences between human and rat networks implicated
in toxicity The identification of sub-network modules
conserved between human and rat distinct for toxicity
types or predictive for toxic end-points in human will be
possible Such signature gene networks (Nikolsky et al
2005) can then be verified with other experimentally
derived data prospectively or from preexisting databases
The combined Fhigh throughput datandashin silico network_approaches therefore represents a novel method for access-
ing complex tissue-level processes as networks integrating
all data types based on functional interactions The data-
bases developed and used in this approach will certainly
benefit from further annotation around the drug metaboliz-
ing enzymes and transporters as described herein in terms
of transcriptional regulation and the ligands associated with
them which frequently appear in the literature It will be
important to capture disparities in the assignment of ligands
to enzymes transporters and other proteins as well as
negative data
In summary although there have been numerous
toxicogenomics studies published there is presently a
relatively small number of datasets that are freely available
to perform network analysis of microarray data The
number of studies identifying large numbers of proteins
which are affected by molecule treatment are even scarcer
still while there are several examples of some published
studies that combine such data It is hoped that the
numerous database initiatives for high content and toxicol-
ogy data that are being undertaken will improve the
situation for other researchers that are not currently
equipped to do such microarray studies themselves The
addition of requirements by journals to deposit such raw
data in a freely accessible resource will aid these initiatives
ADMETox groups have seen new technologies and
approaches developed over the last decade that have all
been applied to identify poor compounds earlier (Fig 2)
The latest technologies integrate network building tools
with high content data and databases The current review
described the limited number of networks generated for
ADMETox at present and one hopes that the impact of
such analyses will be commonplace in the future Systems
biology is however more than just applying a network
approach and hence systems-ADMETox will have to
evolve due to the continual pressure to develop newer
technologies This current paradigm combining empirical
data and computational methods should integrate the
complex data already generated making it readily inter-
pretable and valuable for identifying the most promising
compounds in the future
Acknowledgements
Dr Maggie AZ Hupcey is gratefully acknowledged for
editorial assistance Dr Peter W Swaan (University of
Maryland) and Dr Cheng Chang (Ohio State University)
Dr Steve Wright (University of Arizona) Dr K Sandy
Pang (University of Toronto) and Dr Craig Giroux (Wayne
State University) are kindly acknowledged for their support
and discussions My colleagues at GeneGo Sergey
Andreyev Andy Ryabov Eugene Kirillov Eugene A
Rakhmatulin Svetlana Sorokina Andrej Bugrim Tatiana
Nikolskaya Yuri Nikolsky John Metz and Julie Bryant are
all thanked for their considerable contributions to software
development and data annotation
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 61
References
Abe M Yamashita S Kuramoto T Hirayama Y Tsukamoto T Ohta
T et al (2003) Global expression analysis of N-methyl-NV-nitro-N-nitrosoguanidine-induced rat stomach carcinomas using oligonucleotide
microarrays Carcinogenesis 24 861ndash867
Adachi T Ono Y Koh K B Takashima K Tainaka H Matsuno Y
et al (2004) Long-term alteration of gene expression without
morphological change in testis after neonatal exposure to genistein in
mice Toxicogenomic analysis using cDNA microarray Food and
Chemical Toxicology 42 445ndash452
Akerman G S Rosenzweig B A Domon O E McGarrity L J
Blankenship L R Tsai C A et al (2004) Gene expression profiles
and genetic damage in benzo(a)pyrene diol epoxide-exposed TK6 cells
Mutation Research 549 43ndash64
Aksenov S V Church B Dhiman A Georgieva A Sarangapani R
Helmlinger G et al (2005) An integrated approach for inference and
mechanistic modeling for advancing drug development FEBS Letters
579 1878ndash1883
Anderle P Huang Y amp Sadee W (2004) Intestinal membrane transport
of drugs and nutrients Genomics of membrane transporters using
expression microarrays European Journal of Pharmaceutical Sciences
21 17ndash24
Annereau J P Szakacs G Tucker C J Arciello A Cardarelli C
Collins J et al (2004) Analysis of ATP-binding cassette transporter
expression in drug-selected cell lines by a microarray dedicated to
multidrug resistance Molecular Pharmacology 66 1397ndash1405
Apic G Ignjatovic T Boyer S amp Russell R B (2005) Illuminating drug
discovery with biological pathways FEBS Letters 579 1872ndash1877
Baker V A Harries H M Waring J F Duggan C M Ni H A Jolly
R A et al (2004) Clofibrate-induced gene expression changes in rat
liver A cross-laboratory analysis using membrane cDNA arrays
Environmental Health Perspectives 112 428ndash438
Balakin K V Ekins S Bugrim A Ivanenkov Y A Korolev D amp
Nikolsky Y et al (2004a) Quantitative structurendashmetabolism relation-
ship modeling of the metabolic N-dealkylation rates Drug Metabolism
and Disposition 32 1111ndash1120
Balakin K V Ekins S Bugrim A Ivanenkov Y A Korolev D amp
Nikolsky Y et al (2004b) Kohonen maps for prediction of binding to
human cytochrome P450 3A4 Drug Metabolism and Disposition 32
1183ndash1189
Barabasi A -L amp Oltvai Z N (2004) Network biology Under-
standing the cellrsquos functional organization Nature Reviews Genetics
5 101ndash113
Bartosiewicz M Penn S G amp Buckpitt A (2001) Applications of gene
arrays in environmental toxicology Fingerprints of gene regulation
associated with cadmium chloride benzo(a)pyrene and trichloro-
ethylene Environmental Health Perspectives 109 71ndash74
Bertilsson G Heidrich J Svensson K Asman M Jendeberg L
Sydow-Backman M et al (1998) Identification of a human nuclear
receptor defines a new signaling pathway for CYP3A induction
Proceedings of the National Academy of Sciences of the United States
of America 95 12208ndash12213
Blumberg B Sabbagh Jr W Juguilon H Bolado Jr J van Meter
C M Ong E S et al (1998) SXR a novel steroid and xenobiotic-
sensing nuclear receptor Genes and Development 12 3195ndash3205
Borodina Y Rudik A Filimonov D Kharchevnikova N Dmitriev A
Blinova V et al (2004) A new statistical approach to predicting
aromatic hydroxylation sites Comparison with model-based
approaches Journal of Chemical Information and Computer Sciences
44 1998ndash2009
Borodina Y Sadym A Filimonov D Blinova V Dmitriev A amp
Poroikov V (2003) Predicting biotransformation potential from
molecular structure Journal of Chemical Information and Computer
Sciences 43 1636ndash1646
Bottone Jr F G Martinez J M Collins J B Afshari C A amp Eling
T E (2003) Gene modulation by the cyclooxygenase inhibitor
sulindac sulfide in human colorectal carcinoma cells Possible link to
apoptosis Journal of Biological Chemistry 278 25790ndash25801
Boyer S amp Zamora I (2002) New methods in predictive metabolism
Journal of Computer-Aided Molecular Design 16 403ndash413
Bredel M amp Jacoby E (2004) Chemogenomics An emerging strategy
for rapid target and drug discovery Nature Reviews Genetics 5
262ndash275
Bugrim A Nikolskaya T amp Nikolsky Y (2004) Early prediction of drug
metabolism and toxicity Systems biology approach and modeling
Drug Discovery Today 9 127ndash135
Bulera S J Eddy S M Ferguson E Jatkoe T A Reindel J F
Bleavins M R et al (2001) RNA expression in the early character-
ization of hepatotoxicants in Wistar rats by high-density DNA micro-
arrays Hepatology 33 1239ndash1258
Butcher E C Berg E L amp Kunkel E J (2004) Systems biology in drug
discovery Nature Biotechnology 22 1253ndash1259
Butte A (2002) The use and analysis of microarray data Naturalist
Review of Drug Discovery 1 951ndash960
Cary M P Bader G D amp Sander C (2005) Pathway information for
systems biology FEBS Letters 579 1815ndash1820
Cheok M H Yang W Pui C H Downing J R Cheng C Naeve
C W et al (2003) Treatment-specific changes in gene expression
discriminate in vivo drug response in human leukemia cells Nature
Genetics 34 85ndash90
Cleary J D Rogers P D amp Chapman S W (2001) Differential
transcription factor expression in human mononuclear cells in response
to amphotericin B Identification with complementary DNA microarray
technology Pharmacotherapy 21 1046ndash1054
Coen M Ruepp S U Lindon J C Nicholson J K Pognan F Lenz
E M et al (2004) Integrated application of transcriptomics and
metabonomics yields new insight into the toxicity due to paracetamol in
the mouse Journal of Pharmaceutical and Biomedical Analysis 35
93ndash105
Cornwell P D De Souza A T amp Ulrich R G (2004) Profiling of
hepatic gene expression in rats treated with fibric acid analogs
Mutation Research 549 131ndash145
Csermely P Agoston V amp Pongor S (2005) The efficiency of multi-
target drugs The network approach might help drug design Trends in
Pharmacological Sciences 26 178ndash182
Cunningham M J Liang S Fuhrman S Seilhamer J J amp
Somogyi R (2000) Gene expression microarray data analysis for
toxicology profiling Annals of the New York Academy of Sciences 919
52ndash67
de Longueville F Atienzar F A Marcq L Dufrane S Evrard S
Wouters L et al (2003) Use of a low density microarray for studying
gene expression patterns induced by hepatotoxicants on primary
cultures of rat hepatocytes Toxicological Sciences 75 378ndash392
Dobrin R Beg Q K Barabasi A L amp Oltvai Z N (2004)
Aggregation of topological motifs in the Escherichia coli transcriptional
regulatory network BMC Bioinformatics 5 10
Donald S Verschoyle R D Edwards R Judah D J Davies R
Riley J et al (2002) Hepatobiliary damage and changes in hepatic
gene expression caused by the antitumor drug ecteinascidin-743 (ET-
743) in the female rat Cancer Research 62 4256ndash4262
Dumont M Jacquemin E DrsquoHont C Descout C Cresteil D Haouzi
D et al (1997) Expression of the liver Na+-independent organic anion
transporting polypeptide (oatp-1) in rats with bile duct ligation Journal
of Hepatology 27 1051ndash1056
Durnas C Loi C M amp Cusack B J (1990) Hepatic drug metabolism
and aging Clinical Pharmacokinetics 19 359ndash389
Eisen M B Spellman P T Brown P O amp Botstein D (1998) Cluster
analysis and display of genome-wide expression patterns Proceedings
of the National Academy of Sciences of the United States of America
95 14863ndash14868
Ekins S Andreyev S Ryabov A Kirilov E Rakhmatulin E A
Bugrim A et al (in press) Computational Prediction of Human Drug
Metabolism Exp Opin Drug Metab Toxicol
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6662
Ekins S Boulanger B Swaan P W amp Hupcey M A Z (2002)
Towards a new age of virtual ADMETOX and multidimensional drug
discovery Journal of Computer-Aided Molecular Design 16 381ndash401
Ekins S Bugrim A Nikolsky Y amp Nikolskaya T (2005) Systems
biology Applications in drug discovery In S Gad (Ed) Drug
Discovery Handbook (pp 123ndash183) Hoboken NJrsquo Wiley and Sons
Ekins S Giroux C N Nikolsky Y Bugrim A amp Nikolskaya T
(2005) A signature gene network approach to toxicity The Toxoco-
logists 84
Ekins S Kirillov E Rakhmatulin E amp Nikolskaya T (2005) A novel
method for visualizing nuclear hormone receptor networks relevant to
drug metabolism Drug Metabolism and Disposition 33 474ndash481
Ekins S amp McGowan R J (2001) The limits of reductionism The
shifting genomic paradigmrsquos impact on industry and academia
Philsophy in Science 9 1ndash23
Ekins S Mirny L amp Schuetz E G (2002) A ligand-based approach to
understanding selectivity of nuclear hormone receptors PXR CAR
FXR LXRa and LXRb Pharmaceutical Research 19 1788ndash1800
Ekins S Nikolsky Y amp Nikolskaya T (2005) Techniques Application
of systems biology to absorption distribution metabolism excretion
and toxicity Trends in Pharmacological Sciences 26 202ndash209
Ekins S Ring B J Grace J McRobie-Belle D J amp Wrighton S
A (2000) Present and future in vitro approaches for drug
metabolism Journal of Pharmacological and Toxicological Methods
44 313ndash324
Ekins S Waller C L Swaan P W Cruciani G Wrighton S A amp
Wikel J H (2000) Progress in predicting human ADME parameters in
silico Journal of Pharmacological and Toxicological Methods 44
251ndash272
Erhardt P W (2003) A human drug metabolism database Potential roles
in the quantitative predictions of drug metabolism and metabolism-
related drugndashdrug interactions Current Drug Metabolism 4 411ndash422
Evans W E amp McLeod H L (2003) Pharmacogenomicsmdashdrug
disposition drug targets and side effects New England Journal of
Medicine 348 538ndash549
Ezendam J Staedtler F Pennings J Vandebriel R J Pieters R
Boffetta P et al (2004) Toxicogenomics of subchronic hexachlor-
obenzene exposure in Brown Norway rats Environmental Health
Perspectives 112 782ndash791
Faiola B Fuller E S Wong V A amp Recio L (2004) Gene expression
profile in bone marrow and hematopoietic stem cells in mice exposed to
inhaled benzene Mutation Research 549 195ndash212
FDA (2004) Innovation stagnation Challenge and opportunity on the
critical path to new medicinal products
Fiehn O (2001) Combining genomics metabolome analysis and bio-
chemical modelling to understand metabolic networks Comparative
Functional Genomics 2 155ndash168
Field F J Born E amp Mathur S N (2004) LXRRXR ligand activation
enhances basolateral efflux of beta-sitosterol in CaCo-2 cells Journal of
Lipid Research 45 905ndash913
Fleck C Sutter L Appenroth D Koch B Meinhold T Pitack M
et al (2003) Use of gene chip technology for the characterisation of
the regulation of renal transport processes and of nephrotoxicity in
rats Experimental and Toxicologic Pathology 54 401ndash410
Fliri A F Loging W T Thadeio P F amp Volkmann R A (2005)
Biological spectra analysis Linking biological activity profiles to
molecular structure Proceedings of the National Academy of Sciences
of the United States of America 102 261ndash266
Fountoulakis M (2004) Application of proteomics technologies in the
investigation of the brain Mass Spectrometry Reviews 23 231ndash258
Gaucher S P Taylor S W Fahy E Zhang B Warnock D E Ghosh
S S et al (2004) Expanded coverage of the human heart
mitochondrial proteome using multidimensional liquid chromatography
coupled with tandem mass spectrometry Journal of Proteome
Research 3 495ndash505
Gerhold D Lu M Xu J Austin C Caskey C T amp Rushmore T
(2001) Monitoring expression of genes involved in drug metabolism
and toxicology using DNA microarrays Physiological Genomics 5
161ndash170
Guo G L Choudhuri S amp Klaassen C D (2002) Induction profile of
rat organic anion transporting polypeptide 2 (oatp2) by prototypical
drug-metabolizing enzyme inducers that activate gene expression
through ligand-activated transcription factor pathways Journal of
Pharmacology and Experimental Therapeutics 300 206ndash212
Hagenbuch B amp Meier P J (2004) Organic anion transporting
polypeptides of the OATP SLC21 family Phylogenetic classification
as OATP SLCO superfamily new nomenclature and molecularfunc-
tional properties Pflugers Archiv 447 653ndash665
Hamadeh H K amp Afshari C A (2004) Toxicogenomics principles and
applications Hobokenrsquo John Wiley and Sons
Hamadeh H K Bushel P R Jayadev S Martin K DiSorbo O Sieber
S et al (2002) Gene expression analysis reveals chemical-specific
profiles Toxicological Sciences 67 219ndash231
Hamadeh H K Jayadev S Gaillard E T Huang Q Stoll R
Blanchard K et al (2004) Integration of clinical and gene expression
endpoints to explore furan-mediated hepatotoxicity Mutation Research
549 169ndash183
Hamadeh H K Knight B L Haugen A C Sieber S Amin R P
Bushel P R et al (2002) Methapyrilene toxicity Anchorage of
pathologic observations to gene expression alterations Toxicologic
Pathology 30 470ndash482
Han J D Bertin N Hao T Goldberg D S Berriz G F Zhang L V
et al (2004) Evidence for dynamically organized modularity in the
yeast proteinndashprotein interaction network Nature 430 88ndash93
Hanisch D Zien A Zimmer R amp Lengauer T (2002) Co-clustering of
biological networks and gene expression data Bioinformatics 18
S145ndashS154
Harris A J Dial S L amp Casciano D A (2004) Comparison of basal
gene expression profiles and effects of hepatocarcinogens on gene
expression in cultured primary human hepatocytes and HepG2 cells
Mutation Research 549 79ndash99
Harrison S C (2004) Whither structural biology Nature Structural
Molecular Biology 11 12ndash15
Hartley D P Dai X He Y D Carlini E J Wang B Huskey S E
et al (2004) Activators of the rat pregnane X receptor differentially
modulate hepatic and intestinal gene expression Molecular Pharma-
cology 65 1159ndash1171
Hartwell L H Hopfield J J Leibler S amp Murray A W (1999) From
molecular to modular cell biology Nature 402 C47ndashC52
Hasmall S Orphanides G James N Pennie W Hedley K Soames
A et al (2002) Downregulation of lactoferrin by PPARalpha ligands
Role in perturbation of hepatocyte proliferation and apoptosis
Toxicology Sciences 68 304ndash313
Hayase T Yamamoto Y Yamamoto K Muso E amp Shiota K (2004)
Microarray profile analysis of toxic cocaine-induced alterations in the
expression of mouse brain gene sequences A possible rsquoprotectiversquo
effect of buprenorphine Journal of Applied Toxicology 24 15ndash20
Hayes K R Vollrath A L Zastrow G M McMillan B J Craven M
Jovanovich S et al (2005) EDGE A centralized resource for the
comparison analysis and distribution of toxicogenomic information
Molecular Pharmacology 67 1360ndash1368
Heijne W H Slitt A L van Bladeren P J Groten J P Klaassen C D
Stierum R H et al (2004) Bromobenzene-induced hepatotoxicity at
the transcriptome level Toxicology Sciences 79 411ndash422
Heijne W H Stierum R H Slijper M van Bladeren P J amp van
Ommen B (2003) Toxicogenomics of bromobenzene hepatotoxicity
A combined transcriptomics and proteomics approach Biochemical
Pharmacology 65 857ndash875
Heinloth A N Irwin R D Boorman G A Nettesheim P Fannin
R D Sieber S O et al (2004) Gene expression profiling of rat
livers reveals indicators of potential adverse effects Toxicology
Sciences 80 193ndash202
Hodges L C Cook J D Lobenhofer E K Li L Bennett L Bushel
P R et al (2003) Tamoxifen functions as a molecular agonist inducing
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 63
cell cycle-associated genes in breast cancer cells Molecular Cancer
Research 1 300ndash311
Holleman A Cheok M H den Boer M L Yang W Veerman KM
A J Kazemier et al (2004) Gene-expression patterns in drug-resistant
acute lymphoblastic leukemia cells and response to treatment New
England Journal of Medicine 351 533ndash542
Hong Y Muller U R amp Lai F (2003) Discriminating two classes of
toxicants through expression analysis of HepG2 cells with DNA arrays
Toxicology in Vitro 17 85ndash92
Hood E (2003) Proteomics Characterizing the cogs in the machinery of
life Environmental Health Perspectives 111 A816ndashA825
Hood L (2003) Systems biology Integrating technology biology and
computation Mechanism of Ageing and Development 124 9ndash16
Hood L amp Galas D (2003) The digital code of DNA Nature 421
444ndash448
Hood L amp Perlmutter R M (2004) The impact of systems approaches on
biological problems in drug discovery Nature Biotechnology 22
1215ndash1217
Hu T Gibson D P Carr G J Torontali S M Tiesman J P Chaney
J G et al (2004) Identification of a gene expression profile that
discriminates indirect-acting genotoxins from direct-acting genotoxins
Mutation Research 549 5ndash27
Huang Q Dunn II R T Jayadev S DiSorbo O Pack F D Farr S B
et al (2001) Assessment of cisplatin-induced nephrotoxicity by
microarray technology Toxicology Sciences 63 196ndash207
Huang Q Jin X Gaillard E T Knight B L Pack F D Stoltz J H et
al (2004) Gene expression profiling reveals multiple toxicity endpoints
induced by hepatotoxicants Mutation Research 549 147ndash167
Huisman M T Smit J W Crommentuyn K M Zelcer N Wiltshire
H R Beijnen J H et al (2002) Multidrug resistance protein 2
(MRP2) transports HIV protease inhibitors and transport can be
enhanced by other drugs Aids 16 2295ndash2301
Hunt C M Westerkam W R amp Stave G M (1992) Effect of age and
gender on the activity of human hepatic CYP3A Biochemical
Pharmacology 44 275ndash283
Ideker T Ozier O Schwikowski B amp Siegal A F (2002) Discovering
regulatory and signalling circuits in molecular interaction networks
Bioinformatics 18 S233ndashS240
Ideker T Thorsson V Ranish J A Christmas R Buhler J Eng J K
et al (2001) Integrated genomic and proteomic analyses of a system-
atically perturbed network Science 292 929ndash934
Iida M Anna C H Hartis J Bruno M Wetmore B Dubin J R et al
(2003) Changes in global gene and protein expression during early
mouse liver carcinogenesis induced by non-genotoxic model carcino-
gens oxazepam and Wyeth-14643 Carcinogenesis 24 757ndash770
Ingelman-Sundberg M (2004) Pharmacogenetics of cytochrome P450 and
its applications in drug therapy The past present and future Trends in
Pharmacological Sciences 25 193ndash200
Jeong H Mason S P Barabasi A L amp Oltvai Z N (2001) Lethality
and centrality in protein networks Nature 411 41ndash42
Jeong H Tombor B Albert R Oltvai Z N amp Barabasi A L
(2000) The large-scale organization of metabolic networks Nature
407 651ndash654
Jung D Hagenbuch B Gresh L Pontoglio M Meier P J amp Kullak-
Ublick G A (2001) Characterization of the human OATP-C
(SLC21A6) gene promoter and regulation of liver-specific OATP genes
by hepatocyte nuclear factor 1 alpha Journal of Biological Chemistry
276 37206ndash37214
Jung D amp Kullak-Ublick G A (2003) Hepatocyte nuclear factor 1
alpha A key mediator of the effect of bile acids on gene expression
Hepatology 37 622ndash631
Jung J -W Park J -S Hwang J -W Kang K -S YS L Song B
-S et al (2004) Gene expression analysis of peroxisome prolifer-
ators- and phenytoin-induced hepatotoxicity using cDNA microarray
Journal of Veterinary Medical Science 66 1329ndash1333
Kato N Shibutani M Takagi H Uneyama C Lee K -Y Takiami S
et al (2004) Gene expression profile in the livers of rats orally
administered ethinylestradiol for 28 days using a microarray technique
Toxicology 200 179ndash192
Kier L D Neft R Tang L Suizu R Cook T Onsurez K et al
(2004) Applications of microarrays with toxicologically relevant genes
(tox genes) for the evaluation of chemical toxicants in Sprague Dawley
rats in vivo and human hepatocytes in vitro Mutation Research 549
101ndash113
Kijima K Toyosawa K Yasuba M Matsuoka N Adachi T
Komiyama M et al (2004) Gene expression analysis of the rat testis
after treatment with di(2-ethylhexyl) phthalate using cDNA microarray
and real-time RT-PCR Toxicology and Applied Pharmacology 200
103ndash110
Kim R B (2003) Organic anion-transporting polypeptide (OATP) trans-
porter family and drug disposition European Journal of Clinical
Investigation 33 1ndash5
Kinirons M T amp OrsquoMahony M S (2004) Drug metabolism and ageing
British Journal of Clinical Pharmacology 57 540ndash544
Kitano H (2002a) Computational systems biology Nature 420 206ndash210
Kitano H (2002b) Systems biology A brief overview Science 295
1662ndash1664
Kliewer S A Moore J T Wade L Staudinger J L Watson M
A Jones S A et al (1998) An orphan nuclear receptor activated
by pregnanes defines a novel steroid signalling pathway Cell 92
73ndash82
Korolev D Balakin K V Nikolsky Y Kirillov E Ivanenkov Y A
Savchuk N P et al (2003) Modeling of human cytochrome p450-
mediated drug metabolism using unsupervised machine learning
approach Journal of Medicinal Chemistry 46 3631ndash3643
Kramer J A Pettit S D Amin R P Bertram T A Car B
Cunningham M et al (2004) Overview on the application of
transcription profiling using selected nephrotoxicants for toxicology
assessment Environmental Health Perspectives 112 460ndash464
Kultima K Nystrom A -M Scholz B Gustafson A -L Dencker L
amp Stigson M (2004) Valproic acid teratogenicity A toxicogenomics
approach Environmental Health Perspectives 112 1225ndash1235
Kutuzova G D amp Deluca H F (2004) Gene expression profiles in rat
intestine identify pathways for 125-dihydroxyvitamin D(3) stimulated
calcium absorption and clarify its immunomodulatory properties
Archives of Biochemistry and Biophysics 432 152ndash166
Kwak M K Wakabayashi N Itoh K Motohashi H Yamamoto M amp
Kensler T W (2003) Modulation of gene expression by cancer
chemopreventive dithiolethiones through the Keap1-Nrf2 pathway
Identification of novel gene clusters for cell survival Journal of
Biological Chemistry 278 8135ndash8145
Lahjouji K Mitchell G A amp Qureshi I A (2001) Carnitine transport
by organic cation transporters and systemic carnitine deficiency
Molecular Genetics and Metabolism 73 287ndash297
Landowski C P Sun D Foster D R Menon S S Barnett J L
Welage L S et al (2003) Gene expression in the human intestine and
correlation with oral valacyclovir pharmacokinetic parameters Journal
of Pharmacology and Experimental Therapeutics 306 778ndash786
Lee G -J Lee W -S Jeon K -S Um C -H Kim S -J Lee C -H
et al (2004) cDNA microarray gene expression analysis and
toxicological phenotype for anticancer drug Journal of Veterinary
Medical Science 66 1339ndash1345
Lee M Kwon J Kim S N Kim J E Koh W S Kim E J et al
(2003) cDNA microarray gene expression profiling of hydroxyurea
paclitaxel and p-anisidine genotoxic compounds with differing
tumorigenicity results Environmental and Molecular Mutagenesis
42 91ndash97
Li S Armstrong C M Bertin N Ge H Milstein S Boxem M et al
(2004) A map of the interactome network of the metazoan C elegans
Science 303 540ndash543
Liguori M J Anderson L M Bukofzer S McKim J Pregenzer J F
Retief J et al (2005) Microarray analysis in human hepatocytes
suggests a mechanism for hepatotoxicity induced by trovafloxacin
Hepatology 41 177ndash186
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6664
Lobenhofer E K Bennett L Cable P L Li L Bushel P R amp Afshari
C A (2002) Regulation of DNA replication fork genes by 17beta-
estradiol Molecular Endocrinology 16 1215ndash1229
Locker J Tian J Carver R Concas D Cossu C Ledda-Columbano
G M et al (2003) A common set of immediate-early response genes
in liver regeneration and hyperplasia Hepatology 38 314ndash325
Mattes W B Pettit S D Sansone S A Bushel P R amp Waters M D
(2004) Database development in toxicogenomics Issues and efforts
Environmental Health Perspectives 112 495ndash505
McMillian M Nie A Y Parker J B Leone A Bryant S Kemmerer
M et al (2004) A gene expression signature for oxidant stressreactive
metabolites in rat liver Biochemical Pharmacology 68 2249ndash2261
Meier P J amp Stieger B (2000) Molecular mechanisms in bile formation
News in Physiological Sciences 15 89ndash93
Meneses-Lorente G de Longueville F Dos Santos-Mendes S Bonnert
T P Jack A Evrard S et al (2003) An evaluation of a low-density
DNA microarray using cytochrome P450 inducers Chemical Research
in Toxicology 16 1070ndash1077
Meneses-Lorente G Guest P C Lawrence J Muniappa N Knowles
M R Skynner H A et al (2004) A proteomic investigation of drug-
induced steatosis in rat liver Chemical Research in Toxicology 17
605ndash612
Merrill C L Ni H Yoon L W Tirmenstein M A Narayanan P
Benavides G R et al (2002) Etomoxir-induced oxidative stress in
HepG2 cells detected by differential gene expression is confirmed
biochemically Toxicology Sciences 68 93ndash101
Milo R Shen-Orr S Itzkovitz S Kashtan N Chklovskii D amp Alon
U (2002) Network motifs Simple building blocks of complex
networks Science 298 824ndash827
Morgan K T Ni H Brown H R Yoon L Qualls C W Crosby L
M et al (2002) Application of cDNA microarray technology to in
vitro toxicology and the selection of genes for a real-time RT-PCR-
based screen for oxidative stress in Hep-G2 cells Toxicologic
Pathology 30 435ndash451
Mwinyi J Johne A Bauer S Roots I amp Gerloff T (2004) Evidence
for inverse effects of OATP-C (SLC21A6) 5 and 1b haplotypes on
pravastatin kinetics Clinical Pharmacology and Therapeutics 75
415ndash421
Nakata K Yukawa M Komiyama N Nakano T amp Kaminuma T
(2002) A nuclear receptor database that maps pathways to diseases
Genome Infomatics 13 515ndash516
Nicholson J K Holmes E Lindon J C amp Wilson I D (2004) The
challenges of modeling mammalian biocomplexity Nature Biotechnol-
ogy 22 1268ndash1274
Nicholson J K amp Wilson I D (2003) Understanding rsquoglobal systems
biology Metabonomics and the continuum of metabolism Nature
Reviews on Drug Discovery 2 668ndash676
Nikitin A Egorov S Daraselia N amp Mazo I (2003) Pathway studiomdash
the analysis and navigation of molecular networks Bioinformatics 19
2155ndash2157
Nikolsky Y Ekins S Nikolskaya T amp Bugrim A (2005) A novel
method for generation of signature networks as biomarkers from
complex high throughput data Toxicology Letters 158 20ndash29
Nikolsky Y Nikolskaya T amp Bugrim A (2005) Biological networks
and analysis of experimental data in drug discovery Drug Discovery
Today 10 653ndash662
Nishizato Y Ieiri I Suzuki H Kimura M Kawabata K Hirota T
et al (2003) Polymorphisms of OATP-C (SLC21A6) and OAT3
(SLC22A8) genes Consequences for pravastatin pharmacokinetics
Clinical Pharmacology and Therapeutics 73 554ndash565
Nozawa T Nakajima M Tamai I Noda K Nezu J Sai Y et al
(2002) Genetic polymorphisms of human organic anion transporters
OATP-C (SLC21A6) and OATP-B (SLC21A9) Allele frequencies in
the Japanese population and functional analysis Journal of Pharmacol-
ogy and Experimental Therapeutics 302 804ndash813
Nozawa T Sugiura S Nakajima M Goto A Yokoi T Nezu J et al
(2004) Involvement of organic anion transporting polypeptides in the
transport of troglitazone sulfate Implications for understanding trogli-
tazone hepatotoxicity Drug Metabolism and Disposition 32 291ndash294
Oliver D E Rubin D L Stuart J M Hewett M Klein T E amp
Altman R B (2002) Ontology development for a pharmacogenetics
knowledge base Package Symposion 88ndash99
Oram J F amp Lawn R M (2001) ABCA1 The gatekeeper for eliminating
excess tissue cholesterol Journal of Lipid Research 42 1173ndash1179
Oswald M Kullak-Ublick G A Paumgartner G amp Beuers U (2001)
Expression of hepatic transporters OATP-C and MRP2 in primary
sclerosing cholangitis Liver 21 247ndash253
Parsons A B Brost R I Ding H Li Z Zhang C Sheikh B et al
(2004) Integration of chemical-genetic and genetic interaction data
links bioactive compounds to cellular target pathways Nature Bio-
technology 22 62ndash69
Patki K C von Moltke L L Harmatz J S Hesse L M Court M H
amp Greenblatt D J (2004) Effect of age on in vitro triazolam
biotransformation in male human liver microsomes Journal of
Pharmacology and Experimental Therapeutics 308 874ndash879
Pereira-Leal J B Enright A J amp Ouzounis C A (2004) Detection of
functional modules from protein interaction networks Proteins 54
49ndash57
Plant N (2004) Interaction networks Coordinating responses to xeno-
biotic exposure Toxicology 202 21ndash32
Raghavendra Prasad H S Qi Z Srinivasan K N amp Gopalakrish-
nakone P (2004) Potential effects of tetrodotoxin exposure to
human glial cells postulated using microarray approach Toxicon 44
597ndash608
Reilly T P Bourdi M Brady J N Pise-Masison C A Radonovich M
F George J W et al (2001) Expression profiling of acetaminophen
liver toxicity in mice using microarray technology Biochemical and
Biophysical Research Communications 282 321ndash328
Rives A W amp Galitski T (2003) Modular organization of cellular
networks Proceedings of the National Academy of Sciences of the
United States of America 100 1128ndash1133
Rogers P D Pearson M M Cleary J D Sullivan D C amp Chapman
S W (2002) Differential expression of genes encoding immunomodu-
latory proteins in response to amphotericin B in human mononuclear
cells identified by cDNA microarray analysis Journal of Antimicrobial
Chemotherapy 50 811ndash817
Rosen M B Wilson V S Schmid J E amp Gray L E (2005) Gene
expression analysis in the ventral prostate of rats exposed to vinclozolin
or procymidone Reproductive Toxicology 19 367ndash379
Rost D Herrmann T Sauer P Schmidts H L Stieger B Meier P J
et al (2003) Regulation of rat organic anion transporters in bile salt-
induced cholestatic hepatitis Effect of ursodeoxycholate Hepatology
38 187ndash195
Rothman R B Ayestas M A Dersch C M amp Baumann M H (1999)
Aminorex fenfluramine and chlorphentermine are serotonin transporter
substrates Implications for primary pulmonary hypertension Circula-
tion 100 869ndash875
Ruepp S U Tonge R P Shaw J Wallis N amp Pognan F (2002)
Genomics and proteomics analysis of acetaminophen toxicity in mouse
liver Toxicology Sciences 65 135ndash150
Sakaeda T Nakamura T amp Okumura K (2002) MDR1 genotype-
related pharmacokinetics and pharmacodynamics Biological amp Phar-
maceutical Bulletin 25 1391ndash1400
Sakata T Anzai N Shin H J Noshiro R Hirata T Yokoyama H
et al (2004) Novel single nucleotide polymorphisms of organic cation
transporter 1 (SLC22A1) affecting transport functions Biochemical
and Biophysical Research Communications 313 789ndash793
Satlin L M Amin V amp Wolkoff A W (1997) Organic anion
transporting polypeptide mediates organic anionHCO3-exchange
Journal of Biological Chemistry 272 26340ndash26345
Satomi Y Tsuchiya W Mihara K Ota M Kasahara Y amp Akahori F
(2004) Gene expression analysis of the lung following paraquat
administration in rats using DNA microarray Journal of Toxicological
Sciences 29 91ndash100
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 65
Schrenk D Baus P R Ermel N Klein C Vorderstemann B amp
Kauffmann H M (2001) Up-regulation of transporters of the MRP
family by drugs and toxins Toxicology Letters 120 51ndash57
Segal E Shapira M Regev A Persquoer D Botstein D Koller D et al
(2003) Module networks Identifying regulatory modules and their
condition-specific regulators from gene expression data Nature
Genetics 34 166ndash176
Segal E Wang H amp Koller D (2003) Discovering molecular pathways
from protein interaction and gene expression Bioinformatics 19
i264ndash i272
Seidel S D Kan H L Stott W T Schisler M R amp Gollapudi B B
(2003) Identification of transcriptome profiles for the DNA-damaging
agents bleomycin and hydrogen peroxide in L5178Y mouse lymphoma
cells Environmental and Molecular Mutagenesis 42 19ndash25
Shan L Yu Y Schut H A J amp Snyderwine E G (2004) Susceptibility
of rats to mammary gland carcinogenesis by the food-derived
carcinogen 2-Amino-1-Methyl-6-Phenylimidazo[45-b]Pyridine (PhIP)
varies with age and is associated with the induction of differential gene
expression American Journal of Pathology 165 191ndash202
Sharom J R Bellows D S amp Tyers M (2004) From large networks to
small molecules Current Opinion in Chemical Biology 8 81ndash90
Shitara Y Hirano M Sato H amp Sugiyama Y (2004) Gemfibrozil and
its glucuronide inhibit the OATP2(OATP1B1 SLC21A6)-mediated
hepatic uptake and CYP2C8-mediated metabolism of cerivastatinmdash
Analysis of the mechanism of the clinically relevant drugndashdrug
interaction between cerivastatin and gemfibrozil Journal of Pharma-
cology and Experimental Therapeutics
Shitara Y Itoh T Sato H Li A P amp Sugiyama Y (2003) Inhibition of
transporter-mediated hepatic uptake as a mechanism for drugndashdrug
interaction between cerivastatin and cyclosporin A Journal of
Pharmacology and Experimental Therapeutics 304 610ndash616
Somogyi R Fuhrman S amp Wen X (2001) Genetic network inference in
computational models and applications to large-scale gene expression
data In M Bower amp H Bolouri (Eds) Computational modeling of
genetic and biochemical networks (pp 119ndash157) Cambridge MArsquoThe
MIT Press
Spirin V amp Mirny L A (2003) Protein complexes and functional
modules in molecular networks Proceedings of the National Academy
of Sciences of the United States of America 100 12123ndash12128
Staudinger J Liu Y Madan A Habeebu S amp Klaassen C D
(2001) Coordinate regulation of xenobiotic and bile acid homeo-
stasis by pregnane X receptor Drug Metabolism and Disposition
29 1467ndash1472
Sun L Z Ji Z L Chen X Wang J F amp Chen Y Z (2002) ADME-
AP A database of ADME associated proteins Bioinformatics 18
1699ndash1700
Suter L Babiss L E amp Wheeldon E B (2004) Toxicogenomics in
predictive toxicology in drug development Chemistry and Biology 11
161ndash171
Synold T W Dussault I amp Forman B M (2001) The orphan nuclear
receptor SXR coordinately regulates drug metabolism and efflux
Nature Medicine 7 584ndash590
Tabuchi Y amp Kondo T (2003) cDNA microarray analysis reveals chop-
10 plays a key role in Sertoli cell injury induced by bisphenol A
Biochemical and Biophysical Research Communications 305 54ndash61
Tamai I Nezu J Uchino H Sai Y Oku A Shimane M et al (2000)
Molecular identification and characterization of novel members of the
human organic anion transporter (OATP) family Biochemical and
Biophysical Research Communications 273 251ndash260
Taylor S W Fahy E Zhang B Glenn G M Warnock D E Wiley S
et al (2003) Characterization of the human heart mitochondrial
proteome Nature Biotechnology 21 281ndash286
Terasaka S Aita Y Inoue A Hayashi S Nishigaki M Aoyagi K
et al (2004) Using a customized DNA microarray for expression
profiling of the estrogen-responsive genes to evaluate estrogen activity
among natural estrogens and industrial chemicals Environmental
Health Perspectives 112 773ndash781
Thomas D M Francescutti-Verbeem D M Liu X amp Kuhn D M
(2004) Identification of differentially regulated transcripts in mouse
striatum following methamphetamine treatmentmdashan oligonucleotide
microarray approach Journal of Neurochemistry 88 380ndash393
Thomas R S Rank D R Penn S G Zastrow G M Hayes K R
Pande K et al (2001) Identification of toxicologically predictive
gene sets using cDNA microarrays Molecular Pharmacology 60
1189ndash1194
Thompson K L Afshari C A Amin R P Bertram T A Car B
Cunningham M et al (2004) Identification of platform-independent
gene expression markers of cisplatin nephrotoxicity Environmental
Health Perspectives 112 488ndash494
Tirona R G Leake B F Merino G amp Kim R B (2001)
Polymorphisms in OATP-C Identification of multiple allelic
variants associated with altered transport activity among European-
and African-Americans Journal of Biological Chemistry 276
35669ndash35675
Tirona R G Leake B F Wolkoff A W amp Kim R B (2003)
Human organic anion transporting polypeptide-C (SLC21A6) is a
major determinant of the rifampin-mediated pregnane X receptor
activation Journal of Pharmacology and Experimental Therapeutics
304 223ndash228
Tong W Cao X Harris S Sun H Fang H Fuscoe J et al (2003)
ArrayTrackmdashsupporting toxicogenomic research at the US Food and
Drug Administration National Center for Toxicological Research
Environmental Health Perspectives 111 1819ndash1826
Tornow S amp Mewes H W (2003) Functional modules by relating
protein interaction networks and gene expression Nucleic Acids
Research 31 6283ndash6289
Trocho C Escola-Gil J C Ribas V Benitez S Martin-Campos J M
Rotllan N et al (2004) Phenytoin treatment reduces atherosclerosis in
mice through mechanisms independent of plasma HDL-cholesterol
concentration Atherosclerosis 174 275ndash285
Ueda A Hamadeh H K Webb H K Yamamoto Y Sueyoshi T
Afshari C A et al (2002) Diverse roles of the nuclear orphan
receptor CAR in regulating hepatic genes in response to phenobarbital
Molecular Pharmacology 61 1ndash6
Ulrich R G (2003) The toxicogenomics of nuclear receptor agonists
Current Opinion in Chemical Biology 7 505ndash510
Ulrich R G Rockett J C Gibson G G amp Pettit S D (2004)
Overview of an interlaboratory collaboration on evaluating the effects
of model hepatotoxicants on hepatic gene expression Environmental
Health Perspectives 112 423ndash427
Unami A Shinohara Y Ichikawa T amp Baba Y (2003) Biochemical
and microarray analyses of bupivacaine-induced apoptosis Journal of
Toxicological Sciences 28 77ndash94
Vasquez A Flammini A Maritan A amp Vespignani A (2003) Global
protein function prediction from proteinndashprotein interaction networks
Nature Biotechnology 21 697ndash700
Vezina C M Walker N J amp Olson J R (2004) Subchronic Exposure to
TCDD PeCDF PCB126 and PCB153 Effect on hepatic gene
expression Environmental Health Perspectives 112 1636ndash1644
Vo T D Greenberg H J amp Palsson B O (2004) Reconstruction and
functional characterization of the human mitochondrial metabolic
network based on proteomic and biochemical data Journal of Bio-
logical Chemistry 279 39532ndash39540
Wang H amp LeCluyse E L (2003) Role of orphan nuclear receptors in
the regulation of drug-metabolising enzymes Clinical Pharmacoki-
netics 42 1331ndash1357
Wang N Silver D L Thiele C amp Tall A R (2001) ATP-
binding cassette transporter A1 (ABCA1) functions as a choles-
terol efflux regulatory protein Journal of Biological Chemistry 276
23742ndash23747
Waring J F Cavet G Jolly R A McDowell J Dai H Ciurlionis R
et al (2003) Development of a DNA Microarray for toxicology based
on hepatoxin-regulated sequences Environmental Health Perspectives
111 863ndash870
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6666
Waring J F Gum R Morfitt D Jolly R A Ciurlionis R Heindel M
et al (2002) Identifying toxic mechanisms using DNA microarrays
Evidence that an experimental inhibitor of cell adhesion molecule
expression signals through the aryl hydrocarbon nuclear receptor
Toxicology 181ndash182 537ndash550
Waring J F Jolly R A Ciurlionis R Lum P Y Praestgaard J T
Morfitt D C et al (2001) Clustering of hepatotoxins based on
mechanism of toxicity using gene expression profiles Toxicology and
Applied Pharmacology 175 28ndash42
Waters M Boorman G Bushel P Cunningham M Irwin R Merrick
A et al (2003) Systems toxicology and the Chemical Effects in
Biological Systems (CEBS) knowledge base EHP Toxicogenomics
111 15ndash28
Wei M Arnold L Cano M amp Cohen S M (2005) Effects of co-
administration of antioxidants and arsenicals on the rat urinary bladder
epithelium Toxicology Sciences 83 237ndash245
Weinshilboum R (2003) Inheritance and drug response New England
Journal of Medicine 348 529ndash537
Weinshilboum R amp Wang L (2004) Pharmacogenetics Inherited
variation in amino acid sequence and altered protein quantity Clinical
Pharmacology and Therapeutics 75 253ndash258
Werner E (2003) In silico multicellular systems biology and minimal
genomes Drug Discovery Today 8 1121ndash1127
Wong J S amp Gill S S (2002) Gene expression changes induced in
mouse liver by di(2-ethylhexyl) phthalate Toxicology and Applied
Pharmacology 185 180ndash196
Xie T Tong L McCann U D Yuan J Becker K G Mechan A O
et al (2004) Identification and characterization of metallothionein-1
and -2 gene expression in the context of ()34-methylenedioxymetham-
phetamine-induced toxicity to brain dopaminergic neurons Journal of
Neuroscience 24 7043ndash7050
Yadetie F Laegreid A Bakke I Kusnierczyk W Komorowski J
Waldum H L et al (2003) Liver gene expression in rats in response
to the peroxisome proliferator-activated receptor-alpha agonist ciprofi-
brate Physiological Genomics 15 9ndash19
Yamashita S Nomoto T Abe M Tatematsu M Sugimura T amp
Ushijima T (2004) Persistence of gene expression changes in stomach
mucosae induced by short-term N-methyl-NV-nitro-N-nitrosoguanidinetreatment and their presence in stomach cancers Mutation Research
549 185ndash193
Yan Q amp Sadee W (2000) Human membrane transporter database A
web-accessible relational database for drug transport studies and
pharmacogenomics AAPS Pharmsci 2 E20
Yeger-Lotem E amp Margalit H (2003) Detection of regulatory circuits by
integrating the cellular networks of proteinndashprotein interactions and
transcription regulation Nucleic Acids Research 31 6053ndash6061
Yoon B I Li G -X Kitada K Kawasaki Y Igarashi K amp Kodama
Y et al (2003) Mechanisms of benzene-induced hematotoxicity and
leukemogenicity cDNA microarray analyses using mouse bone marrow
tissue Environmental Health Perspectives 111 1411ndash1420
Young M B DiSilvestro M R Sendera T J Freund J Kriete A amp
Magnuson S R (2003) Analysis of gene expression in carbon
tetrachloride-treated rat livers using a novel bioarray technology
Pharmacogenomics Journal 3 41ndash52
Yu H Zhu X Greenbaum D Karro J amp Gerstein M (2004) TopNet
A tool for comparing biological sub-networks correlating protein
properties with topological statistics Nucleic Acids Research 32
328ndash337
Zhang E Y Fu D J Pak Y A Stewart T Mukhopadhyay N
Wrighton S A et al (2004) Genetic polymorphisms in human proton-
dependent dipeptide transporter PEPT1 Implications for the functional
role of Pro586 Journal of Pharmacology and Experimental Therapeu-
tics 310 437ndash445
Zhang E Y Knipp G T Ekins S amp Swaan P W (2002)
Structural biology and function of solute transporters Implications
for identifying and designing substrates Drug Metabolism Reviews
34 709ndash750
Zhang E Y Phelps M A Cheng C Ekins S amp Swaan P W (2002)
Modeling of active transport systems Advanced Drug Delivery
Reviews 54 329ndash354
- Systems-ADMETox Resources and network approaches
-
- Introduction
- Data available
- Network analysis and databases
-
- Network applications
-
- The role of transporters
-
- Clinical relevance of transporters
- Transporter network examples ABCA1
- Transporter network examples OATP
- Transporter microarray data
-
- Applications to enzymes
- Future network applications
-
- Discussion
- Acknowledgements
- References
-
Table 4
Literature derived toxicoproteomics data
Compounds Data source Microarray type Compound dose Microarray data
availability
Reference
Carbon
tetrachloride
Male Wistar rats Affymetrix rat chip 8799
probes+proteomics
study
6ndash24 h exposure Genbank SwissProt and
binary data in a
manuscript table
(Fountoulakis
2004)
Paracetamol CD-1 male mice Custom mouse tox blots
with 450 genes
RTQ-PCR+proteomics
study 2-DIGE+
MALDI-MS
150 or 500 mgkg Gene name IMAGE ID
GenBank accession
number fold induction
SwissProt identifier and
protein abundance
change in manuscript
tables
(Ruepp Tonge
Shaw Wallis amp
Pognan 2002)
Paracetamol AP-1 male mice Affymetrix murine 11K
set+proteomics study
Up to 500 mgkg Gene names and fold
changes at multiple
doses and time points in
a manuscript tablemdash
proteomics data not
accessible
(Coen et al 2004)
Oxazepam or
Wy-14643
Male B6C3F1 mice NIEHS Mouse Chip
(8700 genes) 2-DIGE
and MS
Oxazepam (2500 ppm)
Wyeth (Wy)-14643
(500 ppm)
Proteomics data in a
manuscript table
Genbank accession
number gene name and
fold change in a
manuscript table data
also available to
download from NIEHS
website
(Iida et al 2003)
Compound A
(PPAR gamma
ligand)
Female rats CrlCD
(SD)IGS BR
Proteomics 2-DIGE and
MS
250 mgkgday up to
5 days
Accession number and
protein name and
average ratio in a
manuscript table
(Meneses-Lorente
et al 2004)
Bromobenzene Male Wistar rats Proteomics 2-DIGE and
MS custom 3000 cDNA
rat chip
5 mmolkg Accession number gene
name fold change in a
manuscript table protein
names along with bar
charts
(Heijne Stierum
Slijper van
Bladeren amp van
Ommen 2003)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 49
the network that can be generated (as it will consist of more
nodes) then a more comprehensive understanding of the
networks is possible The data available currently in the
literature can be used to evaluate such pathway and
network generation tools Recently we have used several
of the published studies (Tables 1ndash3) with MetaCore to
visualize networks for acetaminophen furan carbon tetra-
chloride benzene and cisplatin showing genes involved in
In vivo
In vitro
In Silico
1970s 1980s 1990s 2000s
Systems Biology ()
OMICS
Fig 2 The timeline for major paradigms in ADMETox
oxidative stress (Ekins Giroux Nikolsky Bugrim amp
Nikolskaya 2005c)
Other important pathwaynetwork building tools that could
potentially be applied to toxicogenomics data include
Ingenuity pathways analysis (httpwwwingenuitycom)
PathArt (httpwwwjubilantbiosyscompdhtm) Pathway
Assist (httpwwwariadnegenomicscomproctspathway
html) (Nikitin et al 2003) and several other databases
deposited at the Pathway Resource List (httpcbiiomskcc
orgprl) All of these products have unique underlying
proprietary pathway databases which are compiled manually
or automatically with text mining tools or a combination of
both We are still waiting for studies that provide a comparison
of different database tools or network building algorithms
Until then theremay be someoverlap but also some differences
between the results obtained from more than one network
method due to the database content and the algorithms used
The reader is recommended to evaluate for themselves several
technologies and select those with the most appropriate fit to
their own specialized needs
In the next Sections the further application of some
available network and database tools will be described with
Microarray or other high throughput data
Upload list of geneprotein identifiers and fold change significance etc (raw data or after selection of the differentially expressed
Genes using other statistical methods)
Parse database and generate interaction network with different algorithms or visualize on maps
Filter Networks
Access significance of the interaction networks
Determine GO processes
Compare 2 or more networks intersection overlap etc
Export gene list
Export gene list
generate interaction network with different algorithms or visualize on maps
Visualization
Fig 3 Schematic for the utilization of pathway tools for assessing high throughput data
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6650
specific detailed reference to transporters and enzymes but
these technologies can also be applied elsewhere as
described above
Fig 4 Network for high throughput screening data for clotrimazole and ticona
cholecystokinin CCR1 Choline transporter Chloride channel catechol-O-methyl
and co workers (Fliri et al 2005) Nodes highlighted with large blue circles rep
molecules showing gt50 inhibition in the assay blue circles represents both mol
inhibits gt50 and one inhibits lt50 Ligands (large) linked to transcriptional f
(wwgenegocom) (For interpretation of the references to colour in this figure leg
311 The role of transporters
A diverse array of organic solutes such as nutrients
neurotransmitters and drugs are transported by specialized
zole screened against 10 in vitro assays (Human Cannabinoid 1 Human
transferase COX2 CYP1A2 CYP2B6 CYP2C19) data published by Fliri
resent assays used in the study Red circles on these nodes represent both
ecules showing lt50 inhibition chequered circles represent one molecule
actors enzymes and transporters via edges using the MetaCorei database
end the reader is referred to the web version of this article)
Table 5
Datasets acquired from publications and other sources and used to evaluate two databasepathway tools KEGG and metaCore
Dataset Number of objects
genes proteins in list
Number of objects
mapped with KEGG
Number of objects mapped
with MetaCore on networks
Reference for microarray
data gene list
Tox Chip 10 (NIEHS) 1853 405 1498 httpsdir-appsniehsnihgov
mapsguestclonesrchcfm
4-Hydroxytamoxifen and
estrogen
1617 434 1343 (Hodges et al 2003)
Mitochondrial proteins 722 156 388 (Gaucher et al 2004
Taylor et al 2003)
Bromobenzene
(24 and 48 h)
130 41 89 (Heijne et al 2004)
Acetaminophen 30 19 23 (Heinloth et al 2004)
Acetaminophen 84 29 64 (Huang et al 2004)
Furan 185 64 139 (Huang et al 2004)
Tetrodotoxin 116 31 86 (Raghavendra Prasad et al
2004)
Benzene 73 16 62 (Yoon et al 2003)
Benzene 76 5 53 (Faiola et al 2004)
Carbon tetrachloride 37 8 26 (Young et al 2003)
Estrogen 94 33 90 (Lobenhofer et al 2002)
Trovafloxacin 142 20 82 (Liguori et al 2005)
Phenobarbital 37 13 28 (Ueda et al 2002)
L-742694 (liver)+ 45 17 19 (Hartley et al 2004)
L-742694 (intestine)+ 23 11 10 (Hartley et al 2004)
A-277249 21 7 9 (Waring et al 2002)
All files were converted to Locuslink identifiers except as noted () unigene () Affymetrix (+) Genbank identifiers were used
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 51
proteins across cellular membranes These may function as
passive processes or active processes energized by the
hydrolysis of ATP or coupling to the co-transport of counter
ions down an electrochemical gradient such as Na+ H+ and
Cl There are many thousands of transporters which can be
classified into distinct superfamilies One of these the solute
carrier class (SLC) is rapidly expanding and contains over
30 families and 200 members The ATP-binding cassette
(ABC) contains 7 families and over 48 members including
P-glycoprotein (P-gp) and MRP subfamilies (Zhang Knipp
Ekins amp Swaan 2002a Zhang Phelps Cheng Ekins amp
Swaan 2002b) Transporters have a key role in clinical
pharmacology with many drugs specifically targeting them
Numerous drugs share transport pathways with nutrients
and transporters have a role in oral absorption drug
bioavailability drug resistance excretion and ultimately
pharmacokinetics and pharmacodynamics
Polymorphism of drug transporters may be a key factor
in drug interactions and lack of effectiveness This field has
become known as pharmacogenomics and is focused on
understanding of inherited DNA sequence variations (poly-
morphisms and mutations) revealed by xenobiotics (Evans
amp McLeod 2003 Weinshilboum 2003) Over the last few
decades many genes have been directly linked to the
mechanisms of response (Evans amp McLeod 2003 Wein-
shilboum 2003) such that 20ndash95 of variability to drug
response is inheritable (Evans amp McLeod 2003) This
phenotypical variability is mainly caused by single nucleo-
tide polymorphisms (SNPs) present in anywhere from 1 to
50 of the population resulting in either lower protein
activity incorrect folding or rapid degradation via proteo-
somes (Weinshilboum amp Wang 2004) A number of
structurally diverse molecules bind to P-gp which is
expressed in many tissues and has numerous SNPs one of
which (C3435T) affects the expression level in the
duodenum and therefore can impact the absorption of
molecules which would be substrates for this transporter
(Sakaeda Nakamura amp Okumura 2002) The human
proton-dependent dipeptide transporter (hPEPT1) can also
affect the absorption of molecules in the intestine and
recently 9 SNPs were found with only one displaying a
reduced transport capacity (Zhang et al 2004) The sodium-
dependent carnitine cotransporter OCTN2 can possess
mutations and these result in primary carnitine deficiency
which impacts fatty acid oxidation and is characterized by
many clinical manifestations (Lahjouji Mitchel amp Qureshi
2001) The organic cation transporter 1 (OCT1) is also
important in the transport of numerous xenobiotics and
endobiotics Recently 4 SNPs were identified in the
Japanese population and when functionally characterized
in vitro the uptake of cations was reduced significantly for
some of these mutations indicating that this would likely
contribute to inter-individual variations in metabolism of
drugs which were transported via OCT1 (Sakata et al
2004)
3111 Clinical relevance of transporters The pregnane
X-receptor (PXR) is a transcriptional regulator of the
enzyme human MDR1 (P-gp) MRPs and OATP (Wang amp
LeCluyse 2003) CYP3A (Bertilsson et al 1998 Blumberg
et al 1998 Kliewer et al 1998) and CYP2C89 (Synold
Dussault amp Forman 2001) as well as many other genes
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6652
involved in the transport metabolism and biosynthesis of
bile acids (Staudinger Liu Madan Habeebu amp Klaassen
2001) However the additional receptors CAR FXR LXR
and other nuclear receptors take part in a complex network
of interactions to control these and other proteins Thus
elucidation of the regulatory networks which control the
expression of these transporters is also important To date
most of the research has centered on efflux transporters but
there has been considerable interest in uptake transporters
such as the organic anion transporter polypeptide (OATP
((Kim 2003) see also later Section on OATP)
There are several specific examples of the importance of
drug transporters to the clinical development of drugs One
example is the insulin sensitizer troglitazone which was
withdrawn due to hepatotoxicity although the precise
mechanism appears to have been unclear until recently
The major metabolite is a sulfated species and is suspected
of being responsible for the observed toxicity The recent
assessment of the organic anion transporting polypeptides
OATP-C and OATP8 expressed on the hepatocyte baso-
lateral membrane indicated that sulphated troglitazone has a
high affinity for the former and possibly lower affinity for
the latter (Nozawa et al 2004) This metabolite would
therefore be expected to accumulate in hepatocytes and
inhibit the bile salt export pump and Mrp2 Because
polymorphisms have been shown for OATP-C (Tirona
Leake Merino amp Kim 2001) it is also possible that these
may result in the accumulation of the metabolite and in turn
elicit idiosyncratic toxicity A second relevant example of
the impact of transporters is the clinically significant drugndash
drug interaction between cerivastatin and cyclosporine A
which occurs via the OATP-C transporter (Shitara Itoh
Sato Li amp Sugiyama 2003) A third example are the HIV
protease inhibitors saquinavir ritonavir and indinavir which
are transported by MRP2 in vitro and other drugs such as
probenecid and sulfinpyrazone are able to enhance this
transport Transport by MRP2 suggests that these com-
pounds will have decreased bioavailability due to increased
clearance and other drugs could aggravate this situation by
further enhancing transport (Huisman et al 2002) Sim-
ilarly the rifampicin mediated induction via PXR of MRP2
and P-gp in healthy subjects was found to significantly
decrease the AUC and also correlated with intestinal
expression of these transporters This transporter is also
inducible by cisplatin 2-AAF and phenobarbital (Schrenk
et al 2001) indicating multiple mechanisms may be
involved In other species such as rat commonly used as a
toxicity model orthologs of the transporters such as OATP2
are expressed and can be induced with ligands for PXR like
PCN (Guo Choudhuri amp Klaassen 2002) This is useful
knowledge because the advent of microarray technology
allows one to dose a rat with a xenobiotic and assess
thousands of genes simultaneously in a particular tissue For
instance animals dosed with known nephrotoxins have
shown some upregulation of the NandashKndashCl transporter
however the authors suggested genomic responses are
stronger soon after exposure before declining (Fleck et
al 2003) Some transporters may therefore be specifically
targeted by drugs in one tissue such as the CNS but these
same transporters may also be expressed elsewhere in the
body hence off-target effects may result in toxicity The
serotonin transporter is one such example which is
expressed in the lungs and brain Some substrates for this
channel like fenfluramine can result in primary pulmonary
hypertension as they accumulate in pulmonary cells (Roth-
man Ayestas Dersch amp Baumann 1999) Similarly P-gp is
expressed at the blood brain barrier and intestine impacting
the efficacy and bioavailability of drugs
3112 Transporter network examples ABCA1 The
ABCA1 transporter mediates the first step of cholesterol
transport Mutations in this gene cause Tangier disease
which results in severe HDL deficiency cholesterol
accumulation in macrophages and attendant atherosclerosis
This transporter represents a drug target for upregulation
modulating cholesterol metabolism and prevention of
cardiovascular disease (Oram amp Lawn 2001) In vitro
ABCA1 can be inhibited by the sulfonylurea glybenclamide
(Field Burn amp Mathur 2004 Wang Silver Thiele amp Tall
2001) In order to illustrate the advantages of mapping drug
transporters as networks onto functional models alongside
other proteins one can consider the example of ABCA1
The query of the MetaCorei database shows that this
transporter appears on three manually curated pathway maps
representing the Fstate of the art_ knowledge derived from
reliable high quality literature sources One can also use the
individual maps as an interface to access the underlying
layers of information about the transporter including the list
of encoding genessplice variants with known SNPs In
addition to browsing MetaCorei a user can also build
custom networks around ABCA1 using the network-
construction tool (Fig 5) Such a visualization utility may
be very helpful for identification of all putative pathways
around a particular transporter or compound of interest The
ABCA1 network created by this tool shows that this
transporter at the time of writing is linked directly to
twenty-five other objects such as APOE1 and LXR Many
of its neighbors have their own SNPs that could be
important in determining interactions between transport of
a drug and normal human transport of endogenous ligands
in health or disease
3113 Transporter network examples OATP The
OATPs are key membrane bound transporters expressed in
many organs including intestine liver lung choroid plexus
blood brain barrier and other organs (Tamai et al 2000)
This family of transporters is capable of mediating the
sodium-independent transport of a diverse array of mole-
cules such as steroid conjugates organic anions and
xenobiotics by coupling uptake with efflux of bicarbonate
(Satlin Amin amp Wolkoff 1997) glutathione or its
conjugates (Hagenbuch amp Meier 2004) The inhibition of
Fig 5 The network of direct gene interactions around the ABCA1 transporter gene (centre) generated with MetaCorei The interation types between nodes are shown as small colored hexagons eg unspecified
allosteric regulation binding cleavage competition covalent modification dephosphorylation phosphorylation transcription regulation transformation When applicable interactions also have a positive or
negative effect and direction Ligands (purple) linked to other proteins (blue blobs) transfactors (red) enzymes (orange) (For interpretation of the references to colour in this figure legend the reader is referred to
the web version of this article)
SEkin
sJournalofPharm
acologica
landToxico
logica
lMeth
ods53(2006)38ndash66
53
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6654
this transporterrsquos hepatic uptake of other compounds may be
important for reported drugndashdrug interactions (Kim 2003)
described earlier (Shitara et al 2003) as well as cerivastatin
with gemfibrozil (Shitara Hirano Sato amp Sugiyama 2004)
OATP1B1 (previous names OATP-C LST-1 OATP2
SLC21A6) represents the most studied human OATP to
date (Meier amp Stieger 2000) and is expressed on the
basolateral plasma membrane of hepatocytes Several single
nucleotide polymorphisms have been identified in the
OATP1B1 gene in European-Americans African-Americans
(Tirona et al 2001) and Japanese (Nozawa et al 2002)
dramatically impacting the transport of ligands such as
pravastatin (Mwinyi Johne Bauer Roots amp Gerloff 2004
Nishizato et al 2003) estrone-3-sulfate (Nozawa et al
2002 Tirona et al 2001) Rifampin (Tirona Leake
Wolkoff amp Kim 2003) and estradiol 17h-d-glucuronide(Tirona et al 2001)
The regulation of SLCO may be affected during
extrahepatic cholestasis bile duct ligation bile salt induced
Fig 6 Networks generated with the autoexpand algorithm in MetaCorei to illustr
be interconnected with other protein regulatory signaling information Informa
unspecified allosteric regulation binding cleavage competition covalent m
transformation When applicable interactions also have a positive or negative e
transfactors (red) enzymes (orange) (For interpretation of the references to colour
cholestatic hepatitis (Dumont et al 1997 Meier amp Stieger
2000 Rost et al 2003) and primary sclerosing cholangitis
(Oswald Kullack-Ublick Paumgarter amp Beuer 2001) In
particular OATP1B1 appears to be regulated by the liver-
enriched transcription factor hepatocyte nuclear factor 1a
(HNF1a) which binds to the promoter region of this
transporter (Jung et al 2001) Site directed mutagenesis
of this binding site resulted in inactivation suggesting the
critical nature of the interaction with HNF1a Bile acids
such as CDCA have been shown to transcriptionally repress
HNF1a in vitro via inhibition of the transactivating effect of
HNF4a on HNF1a (Jung amp Kullak-Ublick 2003) After
screening many rat and human uptake transporters in vitro
OATP1B1 was also shown to modulate the PXR response
by controlling rifampin retention in the cell and therefore
affecting the induction of CYP3A4 and other gene products
such as P-gp (Tirona et al 2003)
Some of the literature for OATP1B1 human substrate
data has been annotated into MetaCorei to illustrate the
ate how ligands for the human OATP-C (OATP1B1) transporter (centre) can
tion on the type of interaction between objects is hidden for clarity eg
odification dephosphorylation phosphorylation transcription regulation
ffect and direction Ligands (purple) linked to other proteins (blue blobs)
in this figure legend the reader is referred to the web version of this article)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 55
visualization of the complex interconnections between this
transporter its ligands regulatory factors and signaling
molecules already in the database (Fig 6) The network was
generated with the autoexpand algorithm in the software
representing one of multiple available algorithms for
connecting genes ligands and other objects in the database
Clearly if more ligands and their connections are added to
the database the complexity of the network will increase
considerably The OATP-C (OATP1B1) gene details can be
viewed upon querying the database and links are provided
to other public databases This page can be used to highlight
the multiple synonyms for this gene as well as links to the
multiple SNPs identified to date
3114 Transporter microarray data Microarrays have
generally been limited in the number of transporters present
on them (Annereau et al 2004) however they have been used
in an attempt to correlate pharmacokinetic properties with
gene expression for valacyclovir (Landowski et al 2003) as
well as understand the expression profile in different tissues
or cell lines upon food component or xenobiotic treatment
(Anderle Huang amp Sadee 2004) This lack of transporters
on microarrays has prompted some groups to produce their
own arrays with a heavier emphasis on transporters These
arrays have for example then been used to demonstrate the
upregulation of ABC transporters and down-regulation of
GST-Pi in cell lines resistant to colchicines or 9-nitro-
camptothecin (Annereau et al 2004) The genes that were
significantly up or down-regulated in this particular study
were used to build networks with MetaCore (Fig 7A B) and
the similarities between them were assessed (Fig 7C)
Although there were only a small number of significantly
changed genes in common (IL-8 Fos GST-Pi Calpactin and
Ubiquitin hydrolase) it is perhaps likely that there is a much
larger common gene network that is important for drug
resistance although a much larger number of cell lines and
drug treatments need to be evaluated to produce a definitive
drug resistance signature involving transporters enzymes
and transcriptional regulators
312 Applications to enzymes
As we have already described it may be particularly
valuable to visualize enzymes as networks to show
interactions with transcriptional regulators and ligands
For example a key enzyme is CYP3A4 which metabolizes
40ndash45 of all drugs and has relatively few SNPs
(Ingelman-Sundberg 2004) Using a second software suite
MetaDrugi (wwwgenegocom) it is possible to construct
a custom network around this or other drug metabolizing
enzymes (Fig 8) In this case the gene network for
CYP3A4 highlights all of the major transcriptional
regulators and several more distant linked proteins and
ligands connected on the network that may be useful for
further study (Fig 8) Substrates inhibitors as well as
regulatory factors and other enzymes can be observed
connected on this network Due to the many hundreds of
known inhibitors and substrates only a small number are
shown here for clarity We assume that if a perturbation in
a pathway (eg due to a nonfunctional enzyme) is linked
to a certain pathologic condition a similar perturbation
caused by the interference from xenobiotic metabolism
(eg competitive inhibition of the same enzyme) may
result in identical effects As microarray gene expression
data is increasingly generated the role of enzyme
regulation in toxicity of certain xenobiotics will become
more apparent from either in vivo or in vitro studies The
visualization of such signature gene networks involving
transporters and enzymes their ligands and regulatory
factors will also be important for future toxicity prediction
methods We have recently generated visualizations of
microarray data from MCF-7 cells treated with 4-hydrox-
ytamoxifen to show that some of the key genes involved in
metabolism and transport are upregulated (Ekins et al
2005d) In addition we have made predictions with various
QSAR models in MetaDrug to indicate the involvement of
PXR CYP3A4 and P-gp (Ekins et al 2005e) Therefore it
appears likely that 4-hydroxytamoxifen could increase its
own metabolism as well as efflux from cells via P-gp
which can be visualized on networks Any decrease of
function of these enzymes or transporters in a population
would likely result in changes in the metabolism and
transport of this active metabolite potentially impacting
the clinical effect This represents one example of how
both pathway tools QSAR models and network building
algorithms can be used with different types of predicted
and experimental data to allow visualization of potential
compound interactions or toxicity
313 Future network applications
As the population ages an increasing incidence and
prevalence of systemic diseases especially chronic diseases
have occurred among older adults This has resulted in an
increase in medications used concomitantly by this
population which presents challenges for drugndashdrug
interactions Physiologically elderly patients may behave
differently to the younger patients for which the drugs are
initially developed for and tested on Many pharmacoki-
netic investigations in the elderly population reveal
decreased clearance of lipophilic drugs metabolized by
the cytochrome P450 enzymes however few studies have
evaluated aging-dependent or gender-related changes in
specific P450 enzymes (Hunt Westerkam amp Slave 1992)
Age-related physiological changes such as a reduction in
liver mass hepatic metabolizing enzyme activity liver
blood flow and alterations in plasma drug binding may
account for the decreased elimination of some metabolized
drugs in the elderly It is particularly difficult to separate an
effect of aging from the variation in the rate of metabolism
due to factors such as individual metabolic phenotype
(slow or fast metabolizer due to SNPs) environmental
influences concomitant disease states and drug intake
(drugndashdrug interactions) The available data suggest that
Fig 7 Networks for drug resistant cell lines generated with the Auto expand algorithm in MetaCore A KB-3-1 vs KB-8-5 B ATC-DU-145 vs RCO1 C node overlap of A and B with initial nodes highlighted
with blue or red solid circles (Initial gene lists derived from (Annereau et al 2004)) Information on the type of interaction between objects is hidden for clarity eg unspecified allosteric regulation binding
cleavage competition covalent modification dephosphorylation phosphorylation transcription regulation transformation When applicable interactions also have a positive or negative effect and direction
Ligands (purple) linked to other proteins (blue blobs) transfactors (red) enzymes (orange) (For interpretation of the references to colour in this figure legend the reader is referred to the web version of this article)
SEkin
sJournalofPharm
acologica
landToxico
logica
lMeth
ods53(2006)38ndash66
56
Fig7(continued)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 57
Fig7(continued)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6658
Fig 8 The network of gene interactions around the CYP3A4 enzyme (centre) from MetaCorei Information on the type of interaction between objects is
hidden for clarity eg unspecified allosteric regulation binding cleavage competition covalent modification dephosphorylation phosphorylation
transcription regulation transformation When applicable interactions also have a positive or negative effect and direction Ligands (purple) linked to other
proteins (blue blobs) transfactors (red) enzymes (orange) (For interpretation of the references to colour in this figure legend the reader is referred to the web
version of this article)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 59
the initial doses of drugs metabolized by these enzymes
should be reduced in older patients according to the clinical
response In most published studies the elderly appear at
least as responsive as the young to inducers or inhibitors of
P450s (Durnas Loi amp Cusack 1990) More recently there
has been some suggestion that there are age related
reductions in function of some specific P450s such as
CYP3A4 (Patki et al 2004) and this could occur at the
level of regulation However earlier studies with the same
enzyme showed no change in clearance with age (Hunt et
al 1992) To date there has been even less examination of
the transporter functions and any changes with age
(Kinirons amp OrsquoMahony 2004) so the current understand-
ing of the effects of aging on metabolism and transport is
anything but transparent This represents an extreme
challenge for the pharmaceutical industry how to predict
whether a drug has an affinity for an enzyme or transporter
and whether this may be clinically important if it shows a
decline in expression or function with aging Any decrease
of function of these enzymes or transporters in an elderly
population would result in changes in the metabolism and
transport of metabolites potentially impacting the clinical
effect This represents another example of how computa-
tional approaches may perhaps be used with different types
of predicted and experimental data to allow visualization of
potential compound interactions or toxicity in elderly
populations We may see different gene networks high-
lighted as humans age and these may be modified by drug
treatment and coadministration This represents an area
were network analysis could be applied in the future and is
in need of considerable further research
The collection of microarray data in databases such as
CEBS ArrayTrack and EDGE represents a future resource
for computational gene network analysis One could
envisage that ultimately in each case such data is converted
to one or more networks that are also displayed for the user
and can be used to compare treatments from in vivo and in
vitro experiments This would represent a different approach
to clustering the data as currently implemented in one of
these efforts (Hayes et al 2005) and may condense large
amounts of experimentally derived data into a readily
interpreted network
4 Discussion
Previously in this journal the progress of many
research groups in predicting human ADME parameters
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6660
in silico (Ekins et al 2000b) and approaches for drug
metabolism (Ekins Ring Grace McRobie-Belle amp
Wrighton 2000a) have been described Both of these
reviews commented on moving HT assays for ADME
Tox much earlier in drug discovery which had also been
indicated by other groups The initial reviews also
highlighted the likely wealth of data that would become
available and how this could be used for structure
activity relationships alongside the bioactivity data in
computational models It was noted that there was a
paucity of predictive metabolism tools at that time In
addition a growing number of efforts to model whole
cells and organs now a field called systems biology
were recognized as models that could be integrated with
the in silico ADME approaches In summary since these
past reviews virtually all pharmaceutical companies have
attempted earlier high throughput screening for ADME
Tox properties and to some extent the wider application
of computational approaches for physicochemical proper-
ties Systems biology is being quite widely acknowledged
as the new paradigm for understanding complex bio-
logical datasets derived from high throughput technolo-
gies and the accumulated knowledge on human protein
interactions (Hartwell Hopfield Leibler amp Murray 1999
Hood 2003b) Therefore systems biology can be defined
as the integration of genetic proteomic transcriptomic
and metabonomic data using computational methods
(Nicholson amp Wilson 2003) When taken together
information on molecular processes derived from different
sources represents a lsquolsquouniversersquorsquo of putative biological
functionality of which only a small fraction of it will be
realized in a cell at any given time To date systems
biology has been driven by academia and funding bodies
such as the NIH rather than the pharmaceutical compa-
nies Presently there is a great deal of interest from
scientists of all backgrounds in identifying the networks
of cellular pathways and the corresponding physically
interacting proteins
The network building software for systems biology
described in this current review will be valuable to query
high throughput data and known literature interactions in
order to predict potential toxicity in different species In the
future the compilation of published toxicogenomics data-
sets characteristic of different types of toxicity will likely
be available in these software systems to act as a reference
database It is also feasible that we will be able to generate
the annotated datasets which specifically address the
differences between human and rat networks implicated
in toxicity The identification of sub-network modules
conserved between human and rat distinct for toxicity
types or predictive for toxic end-points in human will be
possible Such signature gene networks (Nikolsky et al
2005) can then be verified with other experimentally
derived data prospectively or from preexisting databases
The combined Fhigh throughput datandashin silico network_approaches therefore represents a novel method for access-
ing complex tissue-level processes as networks integrating
all data types based on functional interactions The data-
bases developed and used in this approach will certainly
benefit from further annotation around the drug metaboliz-
ing enzymes and transporters as described herein in terms
of transcriptional regulation and the ligands associated with
them which frequently appear in the literature It will be
important to capture disparities in the assignment of ligands
to enzymes transporters and other proteins as well as
negative data
In summary although there have been numerous
toxicogenomics studies published there is presently a
relatively small number of datasets that are freely available
to perform network analysis of microarray data The
number of studies identifying large numbers of proteins
which are affected by molecule treatment are even scarcer
still while there are several examples of some published
studies that combine such data It is hoped that the
numerous database initiatives for high content and toxicol-
ogy data that are being undertaken will improve the
situation for other researchers that are not currently
equipped to do such microarray studies themselves The
addition of requirements by journals to deposit such raw
data in a freely accessible resource will aid these initiatives
ADMETox groups have seen new technologies and
approaches developed over the last decade that have all
been applied to identify poor compounds earlier (Fig 2)
The latest technologies integrate network building tools
with high content data and databases The current review
described the limited number of networks generated for
ADMETox at present and one hopes that the impact of
such analyses will be commonplace in the future Systems
biology is however more than just applying a network
approach and hence systems-ADMETox will have to
evolve due to the continual pressure to develop newer
technologies This current paradigm combining empirical
data and computational methods should integrate the
complex data already generated making it readily inter-
pretable and valuable for identifying the most promising
compounds in the future
Acknowledgements
Dr Maggie AZ Hupcey is gratefully acknowledged for
editorial assistance Dr Peter W Swaan (University of
Maryland) and Dr Cheng Chang (Ohio State University)
Dr Steve Wright (University of Arizona) Dr K Sandy
Pang (University of Toronto) and Dr Craig Giroux (Wayne
State University) are kindly acknowledged for their support
and discussions My colleagues at GeneGo Sergey
Andreyev Andy Ryabov Eugene Kirillov Eugene A
Rakhmatulin Svetlana Sorokina Andrej Bugrim Tatiana
Nikolskaya Yuri Nikolsky John Metz and Julie Bryant are
all thanked for their considerable contributions to software
development and data annotation
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 61
References
Abe M Yamashita S Kuramoto T Hirayama Y Tsukamoto T Ohta
T et al (2003) Global expression analysis of N-methyl-NV-nitro-N-nitrosoguanidine-induced rat stomach carcinomas using oligonucleotide
microarrays Carcinogenesis 24 861ndash867
Adachi T Ono Y Koh K B Takashima K Tainaka H Matsuno Y
et al (2004) Long-term alteration of gene expression without
morphological change in testis after neonatal exposure to genistein in
mice Toxicogenomic analysis using cDNA microarray Food and
Chemical Toxicology 42 445ndash452
Akerman G S Rosenzweig B A Domon O E McGarrity L J
Blankenship L R Tsai C A et al (2004) Gene expression profiles
and genetic damage in benzo(a)pyrene diol epoxide-exposed TK6 cells
Mutation Research 549 43ndash64
Aksenov S V Church B Dhiman A Georgieva A Sarangapani R
Helmlinger G et al (2005) An integrated approach for inference and
mechanistic modeling for advancing drug development FEBS Letters
579 1878ndash1883
Anderle P Huang Y amp Sadee W (2004) Intestinal membrane transport
of drugs and nutrients Genomics of membrane transporters using
expression microarrays European Journal of Pharmaceutical Sciences
21 17ndash24
Annereau J P Szakacs G Tucker C J Arciello A Cardarelli C
Collins J et al (2004) Analysis of ATP-binding cassette transporter
expression in drug-selected cell lines by a microarray dedicated to
multidrug resistance Molecular Pharmacology 66 1397ndash1405
Apic G Ignjatovic T Boyer S amp Russell R B (2005) Illuminating drug
discovery with biological pathways FEBS Letters 579 1872ndash1877
Baker V A Harries H M Waring J F Duggan C M Ni H A Jolly
R A et al (2004) Clofibrate-induced gene expression changes in rat
liver A cross-laboratory analysis using membrane cDNA arrays
Environmental Health Perspectives 112 428ndash438
Balakin K V Ekins S Bugrim A Ivanenkov Y A Korolev D amp
Nikolsky Y et al (2004a) Quantitative structurendashmetabolism relation-
ship modeling of the metabolic N-dealkylation rates Drug Metabolism
and Disposition 32 1111ndash1120
Balakin K V Ekins S Bugrim A Ivanenkov Y A Korolev D amp
Nikolsky Y et al (2004b) Kohonen maps for prediction of binding to
human cytochrome P450 3A4 Drug Metabolism and Disposition 32
1183ndash1189
Barabasi A -L amp Oltvai Z N (2004) Network biology Under-
standing the cellrsquos functional organization Nature Reviews Genetics
5 101ndash113
Bartosiewicz M Penn S G amp Buckpitt A (2001) Applications of gene
arrays in environmental toxicology Fingerprints of gene regulation
associated with cadmium chloride benzo(a)pyrene and trichloro-
ethylene Environmental Health Perspectives 109 71ndash74
Bertilsson G Heidrich J Svensson K Asman M Jendeberg L
Sydow-Backman M et al (1998) Identification of a human nuclear
receptor defines a new signaling pathway for CYP3A induction
Proceedings of the National Academy of Sciences of the United States
of America 95 12208ndash12213
Blumberg B Sabbagh Jr W Juguilon H Bolado Jr J van Meter
C M Ong E S et al (1998) SXR a novel steroid and xenobiotic-
sensing nuclear receptor Genes and Development 12 3195ndash3205
Borodina Y Rudik A Filimonov D Kharchevnikova N Dmitriev A
Blinova V et al (2004) A new statistical approach to predicting
aromatic hydroxylation sites Comparison with model-based
approaches Journal of Chemical Information and Computer Sciences
44 1998ndash2009
Borodina Y Sadym A Filimonov D Blinova V Dmitriev A amp
Poroikov V (2003) Predicting biotransformation potential from
molecular structure Journal of Chemical Information and Computer
Sciences 43 1636ndash1646
Bottone Jr F G Martinez J M Collins J B Afshari C A amp Eling
T E (2003) Gene modulation by the cyclooxygenase inhibitor
sulindac sulfide in human colorectal carcinoma cells Possible link to
apoptosis Journal of Biological Chemistry 278 25790ndash25801
Boyer S amp Zamora I (2002) New methods in predictive metabolism
Journal of Computer-Aided Molecular Design 16 403ndash413
Bredel M amp Jacoby E (2004) Chemogenomics An emerging strategy
for rapid target and drug discovery Nature Reviews Genetics 5
262ndash275
Bugrim A Nikolskaya T amp Nikolsky Y (2004) Early prediction of drug
metabolism and toxicity Systems biology approach and modeling
Drug Discovery Today 9 127ndash135
Bulera S J Eddy S M Ferguson E Jatkoe T A Reindel J F
Bleavins M R et al (2001) RNA expression in the early character-
ization of hepatotoxicants in Wistar rats by high-density DNA micro-
arrays Hepatology 33 1239ndash1258
Butcher E C Berg E L amp Kunkel E J (2004) Systems biology in drug
discovery Nature Biotechnology 22 1253ndash1259
Butte A (2002) The use and analysis of microarray data Naturalist
Review of Drug Discovery 1 951ndash960
Cary M P Bader G D amp Sander C (2005) Pathway information for
systems biology FEBS Letters 579 1815ndash1820
Cheok M H Yang W Pui C H Downing J R Cheng C Naeve
C W et al (2003) Treatment-specific changes in gene expression
discriminate in vivo drug response in human leukemia cells Nature
Genetics 34 85ndash90
Cleary J D Rogers P D amp Chapman S W (2001) Differential
transcription factor expression in human mononuclear cells in response
to amphotericin B Identification with complementary DNA microarray
technology Pharmacotherapy 21 1046ndash1054
Coen M Ruepp S U Lindon J C Nicholson J K Pognan F Lenz
E M et al (2004) Integrated application of transcriptomics and
metabonomics yields new insight into the toxicity due to paracetamol in
the mouse Journal of Pharmaceutical and Biomedical Analysis 35
93ndash105
Cornwell P D De Souza A T amp Ulrich R G (2004) Profiling of
hepatic gene expression in rats treated with fibric acid analogs
Mutation Research 549 131ndash145
Csermely P Agoston V amp Pongor S (2005) The efficiency of multi-
target drugs The network approach might help drug design Trends in
Pharmacological Sciences 26 178ndash182
Cunningham M J Liang S Fuhrman S Seilhamer J J amp
Somogyi R (2000) Gene expression microarray data analysis for
toxicology profiling Annals of the New York Academy of Sciences 919
52ndash67
de Longueville F Atienzar F A Marcq L Dufrane S Evrard S
Wouters L et al (2003) Use of a low density microarray for studying
gene expression patterns induced by hepatotoxicants on primary
cultures of rat hepatocytes Toxicological Sciences 75 378ndash392
Dobrin R Beg Q K Barabasi A L amp Oltvai Z N (2004)
Aggregation of topological motifs in the Escherichia coli transcriptional
regulatory network BMC Bioinformatics 5 10
Donald S Verschoyle R D Edwards R Judah D J Davies R
Riley J et al (2002) Hepatobiliary damage and changes in hepatic
gene expression caused by the antitumor drug ecteinascidin-743 (ET-
743) in the female rat Cancer Research 62 4256ndash4262
Dumont M Jacquemin E DrsquoHont C Descout C Cresteil D Haouzi
D et al (1997) Expression of the liver Na+-independent organic anion
transporting polypeptide (oatp-1) in rats with bile duct ligation Journal
of Hepatology 27 1051ndash1056
Durnas C Loi C M amp Cusack B J (1990) Hepatic drug metabolism
and aging Clinical Pharmacokinetics 19 359ndash389
Eisen M B Spellman P T Brown P O amp Botstein D (1998) Cluster
analysis and display of genome-wide expression patterns Proceedings
of the National Academy of Sciences of the United States of America
95 14863ndash14868
Ekins S Andreyev S Ryabov A Kirilov E Rakhmatulin E A
Bugrim A et al (in press) Computational Prediction of Human Drug
Metabolism Exp Opin Drug Metab Toxicol
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6662
Ekins S Boulanger B Swaan P W amp Hupcey M A Z (2002)
Towards a new age of virtual ADMETOX and multidimensional drug
discovery Journal of Computer-Aided Molecular Design 16 381ndash401
Ekins S Bugrim A Nikolsky Y amp Nikolskaya T (2005) Systems
biology Applications in drug discovery In S Gad (Ed) Drug
Discovery Handbook (pp 123ndash183) Hoboken NJrsquo Wiley and Sons
Ekins S Giroux C N Nikolsky Y Bugrim A amp Nikolskaya T
(2005) A signature gene network approach to toxicity The Toxoco-
logists 84
Ekins S Kirillov E Rakhmatulin E amp Nikolskaya T (2005) A novel
method for visualizing nuclear hormone receptor networks relevant to
drug metabolism Drug Metabolism and Disposition 33 474ndash481
Ekins S amp McGowan R J (2001) The limits of reductionism The
shifting genomic paradigmrsquos impact on industry and academia
Philsophy in Science 9 1ndash23
Ekins S Mirny L amp Schuetz E G (2002) A ligand-based approach to
understanding selectivity of nuclear hormone receptors PXR CAR
FXR LXRa and LXRb Pharmaceutical Research 19 1788ndash1800
Ekins S Nikolsky Y amp Nikolskaya T (2005) Techniques Application
of systems biology to absorption distribution metabolism excretion
and toxicity Trends in Pharmacological Sciences 26 202ndash209
Ekins S Ring B J Grace J McRobie-Belle D J amp Wrighton S
A (2000) Present and future in vitro approaches for drug
metabolism Journal of Pharmacological and Toxicological Methods
44 313ndash324
Ekins S Waller C L Swaan P W Cruciani G Wrighton S A amp
Wikel J H (2000) Progress in predicting human ADME parameters in
silico Journal of Pharmacological and Toxicological Methods 44
251ndash272
Erhardt P W (2003) A human drug metabolism database Potential roles
in the quantitative predictions of drug metabolism and metabolism-
related drugndashdrug interactions Current Drug Metabolism 4 411ndash422
Evans W E amp McLeod H L (2003) Pharmacogenomicsmdashdrug
disposition drug targets and side effects New England Journal of
Medicine 348 538ndash549
Ezendam J Staedtler F Pennings J Vandebriel R J Pieters R
Boffetta P et al (2004) Toxicogenomics of subchronic hexachlor-
obenzene exposure in Brown Norway rats Environmental Health
Perspectives 112 782ndash791
Faiola B Fuller E S Wong V A amp Recio L (2004) Gene expression
profile in bone marrow and hematopoietic stem cells in mice exposed to
inhaled benzene Mutation Research 549 195ndash212
FDA (2004) Innovation stagnation Challenge and opportunity on the
critical path to new medicinal products
Fiehn O (2001) Combining genomics metabolome analysis and bio-
chemical modelling to understand metabolic networks Comparative
Functional Genomics 2 155ndash168
Field F J Born E amp Mathur S N (2004) LXRRXR ligand activation
enhances basolateral efflux of beta-sitosterol in CaCo-2 cells Journal of
Lipid Research 45 905ndash913
Fleck C Sutter L Appenroth D Koch B Meinhold T Pitack M
et al (2003) Use of gene chip technology for the characterisation of
the regulation of renal transport processes and of nephrotoxicity in
rats Experimental and Toxicologic Pathology 54 401ndash410
Fliri A F Loging W T Thadeio P F amp Volkmann R A (2005)
Biological spectra analysis Linking biological activity profiles to
molecular structure Proceedings of the National Academy of Sciences
of the United States of America 102 261ndash266
Fountoulakis M (2004) Application of proteomics technologies in the
investigation of the brain Mass Spectrometry Reviews 23 231ndash258
Gaucher S P Taylor S W Fahy E Zhang B Warnock D E Ghosh
S S et al (2004) Expanded coverage of the human heart
mitochondrial proteome using multidimensional liquid chromatography
coupled with tandem mass spectrometry Journal of Proteome
Research 3 495ndash505
Gerhold D Lu M Xu J Austin C Caskey C T amp Rushmore T
(2001) Monitoring expression of genes involved in drug metabolism
and toxicology using DNA microarrays Physiological Genomics 5
161ndash170
Guo G L Choudhuri S amp Klaassen C D (2002) Induction profile of
rat organic anion transporting polypeptide 2 (oatp2) by prototypical
drug-metabolizing enzyme inducers that activate gene expression
through ligand-activated transcription factor pathways Journal of
Pharmacology and Experimental Therapeutics 300 206ndash212
Hagenbuch B amp Meier P J (2004) Organic anion transporting
polypeptides of the OATP SLC21 family Phylogenetic classification
as OATP SLCO superfamily new nomenclature and molecularfunc-
tional properties Pflugers Archiv 447 653ndash665
Hamadeh H K amp Afshari C A (2004) Toxicogenomics principles and
applications Hobokenrsquo John Wiley and Sons
Hamadeh H K Bushel P R Jayadev S Martin K DiSorbo O Sieber
S et al (2002) Gene expression analysis reveals chemical-specific
profiles Toxicological Sciences 67 219ndash231
Hamadeh H K Jayadev S Gaillard E T Huang Q Stoll R
Blanchard K et al (2004) Integration of clinical and gene expression
endpoints to explore furan-mediated hepatotoxicity Mutation Research
549 169ndash183
Hamadeh H K Knight B L Haugen A C Sieber S Amin R P
Bushel P R et al (2002) Methapyrilene toxicity Anchorage of
pathologic observations to gene expression alterations Toxicologic
Pathology 30 470ndash482
Han J D Bertin N Hao T Goldberg D S Berriz G F Zhang L V
et al (2004) Evidence for dynamically organized modularity in the
yeast proteinndashprotein interaction network Nature 430 88ndash93
Hanisch D Zien A Zimmer R amp Lengauer T (2002) Co-clustering of
biological networks and gene expression data Bioinformatics 18
S145ndashS154
Harris A J Dial S L amp Casciano D A (2004) Comparison of basal
gene expression profiles and effects of hepatocarcinogens on gene
expression in cultured primary human hepatocytes and HepG2 cells
Mutation Research 549 79ndash99
Harrison S C (2004) Whither structural biology Nature Structural
Molecular Biology 11 12ndash15
Hartley D P Dai X He Y D Carlini E J Wang B Huskey S E
et al (2004) Activators of the rat pregnane X receptor differentially
modulate hepatic and intestinal gene expression Molecular Pharma-
cology 65 1159ndash1171
Hartwell L H Hopfield J J Leibler S amp Murray A W (1999) From
molecular to modular cell biology Nature 402 C47ndashC52
Hasmall S Orphanides G James N Pennie W Hedley K Soames
A et al (2002) Downregulation of lactoferrin by PPARalpha ligands
Role in perturbation of hepatocyte proliferation and apoptosis
Toxicology Sciences 68 304ndash313
Hayase T Yamamoto Y Yamamoto K Muso E amp Shiota K (2004)
Microarray profile analysis of toxic cocaine-induced alterations in the
expression of mouse brain gene sequences A possible rsquoprotectiversquo
effect of buprenorphine Journal of Applied Toxicology 24 15ndash20
Hayes K R Vollrath A L Zastrow G M McMillan B J Craven M
Jovanovich S et al (2005) EDGE A centralized resource for the
comparison analysis and distribution of toxicogenomic information
Molecular Pharmacology 67 1360ndash1368
Heijne W H Slitt A L van Bladeren P J Groten J P Klaassen C D
Stierum R H et al (2004) Bromobenzene-induced hepatotoxicity at
the transcriptome level Toxicology Sciences 79 411ndash422
Heijne W H Stierum R H Slijper M van Bladeren P J amp van
Ommen B (2003) Toxicogenomics of bromobenzene hepatotoxicity
A combined transcriptomics and proteomics approach Biochemical
Pharmacology 65 857ndash875
Heinloth A N Irwin R D Boorman G A Nettesheim P Fannin
R D Sieber S O et al (2004) Gene expression profiling of rat
livers reveals indicators of potential adverse effects Toxicology
Sciences 80 193ndash202
Hodges L C Cook J D Lobenhofer E K Li L Bennett L Bushel
P R et al (2003) Tamoxifen functions as a molecular agonist inducing
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 63
cell cycle-associated genes in breast cancer cells Molecular Cancer
Research 1 300ndash311
Holleman A Cheok M H den Boer M L Yang W Veerman KM
A J Kazemier et al (2004) Gene-expression patterns in drug-resistant
acute lymphoblastic leukemia cells and response to treatment New
England Journal of Medicine 351 533ndash542
Hong Y Muller U R amp Lai F (2003) Discriminating two classes of
toxicants through expression analysis of HepG2 cells with DNA arrays
Toxicology in Vitro 17 85ndash92
Hood E (2003) Proteomics Characterizing the cogs in the machinery of
life Environmental Health Perspectives 111 A816ndashA825
Hood L (2003) Systems biology Integrating technology biology and
computation Mechanism of Ageing and Development 124 9ndash16
Hood L amp Galas D (2003) The digital code of DNA Nature 421
444ndash448
Hood L amp Perlmutter R M (2004) The impact of systems approaches on
biological problems in drug discovery Nature Biotechnology 22
1215ndash1217
Hu T Gibson D P Carr G J Torontali S M Tiesman J P Chaney
J G et al (2004) Identification of a gene expression profile that
discriminates indirect-acting genotoxins from direct-acting genotoxins
Mutation Research 549 5ndash27
Huang Q Dunn II R T Jayadev S DiSorbo O Pack F D Farr S B
et al (2001) Assessment of cisplatin-induced nephrotoxicity by
microarray technology Toxicology Sciences 63 196ndash207
Huang Q Jin X Gaillard E T Knight B L Pack F D Stoltz J H et
al (2004) Gene expression profiling reveals multiple toxicity endpoints
induced by hepatotoxicants Mutation Research 549 147ndash167
Huisman M T Smit J W Crommentuyn K M Zelcer N Wiltshire
H R Beijnen J H et al (2002) Multidrug resistance protein 2
(MRP2) transports HIV protease inhibitors and transport can be
enhanced by other drugs Aids 16 2295ndash2301
Hunt C M Westerkam W R amp Stave G M (1992) Effect of age and
gender on the activity of human hepatic CYP3A Biochemical
Pharmacology 44 275ndash283
Ideker T Ozier O Schwikowski B amp Siegal A F (2002) Discovering
regulatory and signalling circuits in molecular interaction networks
Bioinformatics 18 S233ndashS240
Ideker T Thorsson V Ranish J A Christmas R Buhler J Eng J K
et al (2001) Integrated genomic and proteomic analyses of a system-
atically perturbed network Science 292 929ndash934
Iida M Anna C H Hartis J Bruno M Wetmore B Dubin J R et al
(2003) Changes in global gene and protein expression during early
mouse liver carcinogenesis induced by non-genotoxic model carcino-
gens oxazepam and Wyeth-14643 Carcinogenesis 24 757ndash770
Ingelman-Sundberg M (2004) Pharmacogenetics of cytochrome P450 and
its applications in drug therapy The past present and future Trends in
Pharmacological Sciences 25 193ndash200
Jeong H Mason S P Barabasi A L amp Oltvai Z N (2001) Lethality
and centrality in protein networks Nature 411 41ndash42
Jeong H Tombor B Albert R Oltvai Z N amp Barabasi A L
(2000) The large-scale organization of metabolic networks Nature
407 651ndash654
Jung D Hagenbuch B Gresh L Pontoglio M Meier P J amp Kullak-
Ublick G A (2001) Characterization of the human OATP-C
(SLC21A6) gene promoter and regulation of liver-specific OATP genes
by hepatocyte nuclear factor 1 alpha Journal of Biological Chemistry
276 37206ndash37214
Jung D amp Kullak-Ublick G A (2003) Hepatocyte nuclear factor 1
alpha A key mediator of the effect of bile acids on gene expression
Hepatology 37 622ndash631
Jung J -W Park J -S Hwang J -W Kang K -S YS L Song B
-S et al (2004) Gene expression analysis of peroxisome prolifer-
ators- and phenytoin-induced hepatotoxicity using cDNA microarray
Journal of Veterinary Medical Science 66 1329ndash1333
Kato N Shibutani M Takagi H Uneyama C Lee K -Y Takiami S
et al (2004) Gene expression profile in the livers of rats orally
administered ethinylestradiol for 28 days using a microarray technique
Toxicology 200 179ndash192
Kier L D Neft R Tang L Suizu R Cook T Onsurez K et al
(2004) Applications of microarrays with toxicologically relevant genes
(tox genes) for the evaluation of chemical toxicants in Sprague Dawley
rats in vivo and human hepatocytes in vitro Mutation Research 549
101ndash113
Kijima K Toyosawa K Yasuba M Matsuoka N Adachi T
Komiyama M et al (2004) Gene expression analysis of the rat testis
after treatment with di(2-ethylhexyl) phthalate using cDNA microarray
and real-time RT-PCR Toxicology and Applied Pharmacology 200
103ndash110
Kim R B (2003) Organic anion-transporting polypeptide (OATP) trans-
porter family and drug disposition European Journal of Clinical
Investigation 33 1ndash5
Kinirons M T amp OrsquoMahony M S (2004) Drug metabolism and ageing
British Journal of Clinical Pharmacology 57 540ndash544
Kitano H (2002a) Computational systems biology Nature 420 206ndash210
Kitano H (2002b) Systems biology A brief overview Science 295
1662ndash1664
Kliewer S A Moore J T Wade L Staudinger J L Watson M
A Jones S A et al (1998) An orphan nuclear receptor activated
by pregnanes defines a novel steroid signalling pathway Cell 92
73ndash82
Korolev D Balakin K V Nikolsky Y Kirillov E Ivanenkov Y A
Savchuk N P et al (2003) Modeling of human cytochrome p450-
mediated drug metabolism using unsupervised machine learning
approach Journal of Medicinal Chemistry 46 3631ndash3643
Kramer J A Pettit S D Amin R P Bertram T A Car B
Cunningham M et al (2004) Overview on the application of
transcription profiling using selected nephrotoxicants for toxicology
assessment Environmental Health Perspectives 112 460ndash464
Kultima K Nystrom A -M Scholz B Gustafson A -L Dencker L
amp Stigson M (2004) Valproic acid teratogenicity A toxicogenomics
approach Environmental Health Perspectives 112 1225ndash1235
Kutuzova G D amp Deluca H F (2004) Gene expression profiles in rat
intestine identify pathways for 125-dihydroxyvitamin D(3) stimulated
calcium absorption and clarify its immunomodulatory properties
Archives of Biochemistry and Biophysics 432 152ndash166
Kwak M K Wakabayashi N Itoh K Motohashi H Yamamoto M amp
Kensler T W (2003) Modulation of gene expression by cancer
chemopreventive dithiolethiones through the Keap1-Nrf2 pathway
Identification of novel gene clusters for cell survival Journal of
Biological Chemistry 278 8135ndash8145
Lahjouji K Mitchell G A amp Qureshi I A (2001) Carnitine transport
by organic cation transporters and systemic carnitine deficiency
Molecular Genetics and Metabolism 73 287ndash297
Landowski C P Sun D Foster D R Menon S S Barnett J L
Welage L S et al (2003) Gene expression in the human intestine and
correlation with oral valacyclovir pharmacokinetic parameters Journal
of Pharmacology and Experimental Therapeutics 306 778ndash786
Lee G -J Lee W -S Jeon K -S Um C -H Kim S -J Lee C -H
et al (2004) cDNA microarray gene expression analysis and
toxicological phenotype for anticancer drug Journal of Veterinary
Medical Science 66 1339ndash1345
Lee M Kwon J Kim S N Kim J E Koh W S Kim E J et al
(2003) cDNA microarray gene expression profiling of hydroxyurea
paclitaxel and p-anisidine genotoxic compounds with differing
tumorigenicity results Environmental and Molecular Mutagenesis
42 91ndash97
Li S Armstrong C M Bertin N Ge H Milstein S Boxem M et al
(2004) A map of the interactome network of the metazoan C elegans
Science 303 540ndash543
Liguori M J Anderson L M Bukofzer S McKim J Pregenzer J F
Retief J et al (2005) Microarray analysis in human hepatocytes
suggests a mechanism for hepatotoxicity induced by trovafloxacin
Hepatology 41 177ndash186
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6664
Lobenhofer E K Bennett L Cable P L Li L Bushel P R amp Afshari
C A (2002) Regulation of DNA replication fork genes by 17beta-
estradiol Molecular Endocrinology 16 1215ndash1229
Locker J Tian J Carver R Concas D Cossu C Ledda-Columbano
G M et al (2003) A common set of immediate-early response genes
in liver regeneration and hyperplasia Hepatology 38 314ndash325
Mattes W B Pettit S D Sansone S A Bushel P R amp Waters M D
(2004) Database development in toxicogenomics Issues and efforts
Environmental Health Perspectives 112 495ndash505
McMillian M Nie A Y Parker J B Leone A Bryant S Kemmerer
M et al (2004) A gene expression signature for oxidant stressreactive
metabolites in rat liver Biochemical Pharmacology 68 2249ndash2261
Meier P J amp Stieger B (2000) Molecular mechanisms in bile formation
News in Physiological Sciences 15 89ndash93
Meneses-Lorente G de Longueville F Dos Santos-Mendes S Bonnert
T P Jack A Evrard S et al (2003) An evaluation of a low-density
DNA microarray using cytochrome P450 inducers Chemical Research
in Toxicology 16 1070ndash1077
Meneses-Lorente G Guest P C Lawrence J Muniappa N Knowles
M R Skynner H A et al (2004) A proteomic investigation of drug-
induced steatosis in rat liver Chemical Research in Toxicology 17
605ndash612
Merrill C L Ni H Yoon L W Tirmenstein M A Narayanan P
Benavides G R et al (2002) Etomoxir-induced oxidative stress in
HepG2 cells detected by differential gene expression is confirmed
biochemically Toxicology Sciences 68 93ndash101
Milo R Shen-Orr S Itzkovitz S Kashtan N Chklovskii D amp Alon
U (2002) Network motifs Simple building blocks of complex
networks Science 298 824ndash827
Morgan K T Ni H Brown H R Yoon L Qualls C W Crosby L
M et al (2002) Application of cDNA microarray technology to in
vitro toxicology and the selection of genes for a real-time RT-PCR-
based screen for oxidative stress in Hep-G2 cells Toxicologic
Pathology 30 435ndash451
Mwinyi J Johne A Bauer S Roots I amp Gerloff T (2004) Evidence
for inverse effects of OATP-C (SLC21A6) 5 and 1b haplotypes on
pravastatin kinetics Clinical Pharmacology and Therapeutics 75
415ndash421
Nakata K Yukawa M Komiyama N Nakano T amp Kaminuma T
(2002) A nuclear receptor database that maps pathways to diseases
Genome Infomatics 13 515ndash516
Nicholson J K Holmes E Lindon J C amp Wilson I D (2004) The
challenges of modeling mammalian biocomplexity Nature Biotechnol-
ogy 22 1268ndash1274
Nicholson J K amp Wilson I D (2003) Understanding rsquoglobal systems
biology Metabonomics and the continuum of metabolism Nature
Reviews on Drug Discovery 2 668ndash676
Nikitin A Egorov S Daraselia N amp Mazo I (2003) Pathway studiomdash
the analysis and navigation of molecular networks Bioinformatics 19
2155ndash2157
Nikolsky Y Ekins S Nikolskaya T amp Bugrim A (2005) A novel
method for generation of signature networks as biomarkers from
complex high throughput data Toxicology Letters 158 20ndash29
Nikolsky Y Nikolskaya T amp Bugrim A (2005) Biological networks
and analysis of experimental data in drug discovery Drug Discovery
Today 10 653ndash662
Nishizato Y Ieiri I Suzuki H Kimura M Kawabata K Hirota T
et al (2003) Polymorphisms of OATP-C (SLC21A6) and OAT3
(SLC22A8) genes Consequences for pravastatin pharmacokinetics
Clinical Pharmacology and Therapeutics 73 554ndash565
Nozawa T Nakajima M Tamai I Noda K Nezu J Sai Y et al
(2002) Genetic polymorphisms of human organic anion transporters
OATP-C (SLC21A6) and OATP-B (SLC21A9) Allele frequencies in
the Japanese population and functional analysis Journal of Pharmacol-
ogy and Experimental Therapeutics 302 804ndash813
Nozawa T Sugiura S Nakajima M Goto A Yokoi T Nezu J et al
(2004) Involvement of organic anion transporting polypeptides in the
transport of troglitazone sulfate Implications for understanding trogli-
tazone hepatotoxicity Drug Metabolism and Disposition 32 291ndash294
Oliver D E Rubin D L Stuart J M Hewett M Klein T E amp
Altman R B (2002) Ontology development for a pharmacogenetics
knowledge base Package Symposion 88ndash99
Oram J F amp Lawn R M (2001) ABCA1 The gatekeeper for eliminating
excess tissue cholesterol Journal of Lipid Research 42 1173ndash1179
Oswald M Kullak-Ublick G A Paumgartner G amp Beuers U (2001)
Expression of hepatic transporters OATP-C and MRP2 in primary
sclerosing cholangitis Liver 21 247ndash253
Parsons A B Brost R I Ding H Li Z Zhang C Sheikh B et al
(2004) Integration of chemical-genetic and genetic interaction data
links bioactive compounds to cellular target pathways Nature Bio-
technology 22 62ndash69
Patki K C von Moltke L L Harmatz J S Hesse L M Court M H
amp Greenblatt D J (2004) Effect of age on in vitro triazolam
biotransformation in male human liver microsomes Journal of
Pharmacology and Experimental Therapeutics 308 874ndash879
Pereira-Leal J B Enright A J amp Ouzounis C A (2004) Detection of
functional modules from protein interaction networks Proteins 54
49ndash57
Plant N (2004) Interaction networks Coordinating responses to xeno-
biotic exposure Toxicology 202 21ndash32
Raghavendra Prasad H S Qi Z Srinivasan K N amp Gopalakrish-
nakone P (2004) Potential effects of tetrodotoxin exposure to
human glial cells postulated using microarray approach Toxicon 44
597ndash608
Reilly T P Bourdi M Brady J N Pise-Masison C A Radonovich M
F George J W et al (2001) Expression profiling of acetaminophen
liver toxicity in mice using microarray technology Biochemical and
Biophysical Research Communications 282 321ndash328
Rives A W amp Galitski T (2003) Modular organization of cellular
networks Proceedings of the National Academy of Sciences of the
United States of America 100 1128ndash1133
Rogers P D Pearson M M Cleary J D Sullivan D C amp Chapman
S W (2002) Differential expression of genes encoding immunomodu-
latory proteins in response to amphotericin B in human mononuclear
cells identified by cDNA microarray analysis Journal of Antimicrobial
Chemotherapy 50 811ndash817
Rosen M B Wilson V S Schmid J E amp Gray L E (2005) Gene
expression analysis in the ventral prostate of rats exposed to vinclozolin
or procymidone Reproductive Toxicology 19 367ndash379
Rost D Herrmann T Sauer P Schmidts H L Stieger B Meier P J
et al (2003) Regulation of rat organic anion transporters in bile salt-
induced cholestatic hepatitis Effect of ursodeoxycholate Hepatology
38 187ndash195
Rothman R B Ayestas M A Dersch C M amp Baumann M H (1999)
Aminorex fenfluramine and chlorphentermine are serotonin transporter
substrates Implications for primary pulmonary hypertension Circula-
tion 100 869ndash875
Ruepp S U Tonge R P Shaw J Wallis N amp Pognan F (2002)
Genomics and proteomics analysis of acetaminophen toxicity in mouse
liver Toxicology Sciences 65 135ndash150
Sakaeda T Nakamura T amp Okumura K (2002) MDR1 genotype-
related pharmacokinetics and pharmacodynamics Biological amp Phar-
maceutical Bulletin 25 1391ndash1400
Sakata T Anzai N Shin H J Noshiro R Hirata T Yokoyama H
et al (2004) Novel single nucleotide polymorphisms of organic cation
transporter 1 (SLC22A1) affecting transport functions Biochemical
and Biophysical Research Communications 313 789ndash793
Satlin L M Amin V amp Wolkoff A W (1997) Organic anion
transporting polypeptide mediates organic anionHCO3-exchange
Journal of Biological Chemistry 272 26340ndash26345
Satomi Y Tsuchiya W Mihara K Ota M Kasahara Y amp Akahori F
(2004) Gene expression analysis of the lung following paraquat
administration in rats using DNA microarray Journal of Toxicological
Sciences 29 91ndash100
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 65
Schrenk D Baus P R Ermel N Klein C Vorderstemann B amp
Kauffmann H M (2001) Up-regulation of transporters of the MRP
family by drugs and toxins Toxicology Letters 120 51ndash57
Segal E Shapira M Regev A Persquoer D Botstein D Koller D et al
(2003) Module networks Identifying regulatory modules and their
condition-specific regulators from gene expression data Nature
Genetics 34 166ndash176
Segal E Wang H amp Koller D (2003) Discovering molecular pathways
from protein interaction and gene expression Bioinformatics 19
i264ndash i272
Seidel S D Kan H L Stott W T Schisler M R amp Gollapudi B B
(2003) Identification of transcriptome profiles for the DNA-damaging
agents bleomycin and hydrogen peroxide in L5178Y mouse lymphoma
cells Environmental and Molecular Mutagenesis 42 19ndash25
Shan L Yu Y Schut H A J amp Snyderwine E G (2004) Susceptibility
of rats to mammary gland carcinogenesis by the food-derived
carcinogen 2-Amino-1-Methyl-6-Phenylimidazo[45-b]Pyridine (PhIP)
varies with age and is associated with the induction of differential gene
expression American Journal of Pathology 165 191ndash202
Sharom J R Bellows D S amp Tyers M (2004) From large networks to
small molecules Current Opinion in Chemical Biology 8 81ndash90
Shitara Y Hirano M Sato H amp Sugiyama Y (2004) Gemfibrozil and
its glucuronide inhibit the OATP2(OATP1B1 SLC21A6)-mediated
hepatic uptake and CYP2C8-mediated metabolism of cerivastatinmdash
Analysis of the mechanism of the clinically relevant drugndashdrug
interaction between cerivastatin and gemfibrozil Journal of Pharma-
cology and Experimental Therapeutics
Shitara Y Itoh T Sato H Li A P amp Sugiyama Y (2003) Inhibition of
transporter-mediated hepatic uptake as a mechanism for drugndashdrug
interaction between cerivastatin and cyclosporin A Journal of
Pharmacology and Experimental Therapeutics 304 610ndash616
Somogyi R Fuhrman S amp Wen X (2001) Genetic network inference in
computational models and applications to large-scale gene expression
data In M Bower amp H Bolouri (Eds) Computational modeling of
genetic and biochemical networks (pp 119ndash157) Cambridge MArsquoThe
MIT Press
Spirin V amp Mirny L A (2003) Protein complexes and functional
modules in molecular networks Proceedings of the National Academy
of Sciences of the United States of America 100 12123ndash12128
Staudinger J Liu Y Madan A Habeebu S amp Klaassen C D
(2001) Coordinate regulation of xenobiotic and bile acid homeo-
stasis by pregnane X receptor Drug Metabolism and Disposition
29 1467ndash1472
Sun L Z Ji Z L Chen X Wang J F amp Chen Y Z (2002) ADME-
AP A database of ADME associated proteins Bioinformatics 18
1699ndash1700
Suter L Babiss L E amp Wheeldon E B (2004) Toxicogenomics in
predictive toxicology in drug development Chemistry and Biology 11
161ndash171
Synold T W Dussault I amp Forman B M (2001) The orphan nuclear
receptor SXR coordinately regulates drug metabolism and efflux
Nature Medicine 7 584ndash590
Tabuchi Y amp Kondo T (2003) cDNA microarray analysis reveals chop-
10 plays a key role in Sertoli cell injury induced by bisphenol A
Biochemical and Biophysical Research Communications 305 54ndash61
Tamai I Nezu J Uchino H Sai Y Oku A Shimane M et al (2000)
Molecular identification and characterization of novel members of the
human organic anion transporter (OATP) family Biochemical and
Biophysical Research Communications 273 251ndash260
Taylor S W Fahy E Zhang B Glenn G M Warnock D E Wiley S
et al (2003) Characterization of the human heart mitochondrial
proteome Nature Biotechnology 21 281ndash286
Terasaka S Aita Y Inoue A Hayashi S Nishigaki M Aoyagi K
et al (2004) Using a customized DNA microarray for expression
profiling of the estrogen-responsive genes to evaluate estrogen activity
among natural estrogens and industrial chemicals Environmental
Health Perspectives 112 773ndash781
Thomas D M Francescutti-Verbeem D M Liu X amp Kuhn D M
(2004) Identification of differentially regulated transcripts in mouse
striatum following methamphetamine treatmentmdashan oligonucleotide
microarray approach Journal of Neurochemistry 88 380ndash393
Thomas R S Rank D R Penn S G Zastrow G M Hayes K R
Pande K et al (2001) Identification of toxicologically predictive
gene sets using cDNA microarrays Molecular Pharmacology 60
1189ndash1194
Thompson K L Afshari C A Amin R P Bertram T A Car B
Cunningham M et al (2004) Identification of platform-independent
gene expression markers of cisplatin nephrotoxicity Environmental
Health Perspectives 112 488ndash494
Tirona R G Leake B F Merino G amp Kim R B (2001)
Polymorphisms in OATP-C Identification of multiple allelic
variants associated with altered transport activity among European-
and African-Americans Journal of Biological Chemistry 276
35669ndash35675
Tirona R G Leake B F Wolkoff A W amp Kim R B (2003)
Human organic anion transporting polypeptide-C (SLC21A6) is a
major determinant of the rifampin-mediated pregnane X receptor
activation Journal of Pharmacology and Experimental Therapeutics
304 223ndash228
Tong W Cao X Harris S Sun H Fang H Fuscoe J et al (2003)
ArrayTrackmdashsupporting toxicogenomic research at the US Food and
Drug Administration National Center for Toxicological Research
Environmental Health Perspectives 111 1819ndash1826
Tornow S amp Mewes H W (2003) Functional modules by relating
protein interaction networks and gene expression Nucleic Acids
Research 31 6283ndash6289
Trocho C Escola-Gil J C Ribas V Benitez S Martin-Campos J M
Rotllan N et al (2004) Phenytoin treatment reduces atherosclerosis in
mice through mechanisms independent of plasma HDL-cholesterol
concentration Atherosclerosis 174 275ndash285
Ueda A Hamadeh H K Webb H K Yamamoto Y Sueyoshi T
Afshari C A et al (2002) Diverse roles of the nuclear orphan
receptor CAR in regulating hepatic genes in response to phenobarbital
Molecular Pharmacology 61 1ndash6
Ulrich R G (2003) The toxicogenomics of nuclear receptor agonists
Current Opinion in Chemical Biology 7 505ndash510
Ulrich R G Rockett J C Gibson G G amp Pettit S D (2004)
Overview of an interlaboratory collaboration on evaluating the effects
of model hepatotoxicants on hepatic gene expression Environmental
Health Perspectives 112 423ndash427
Unami A Shinohara Y Ichikawa T amp Baba Y (2003) Biochemical
and microarray analyses of bupivacaine-induced apoptosis Journal of
Toxicological Sciences 28 77ndash94
Vasquez A Flammini A Maritan A amp Vespignani A (2003) Global
protein function prediction from proteinndashprotein interaction networks
Nature Biotechnology 21 697ndash700
Vezina C M Walker N J amp Olson J R (2004) Subchronic Exposure to
TCDD PeCDF PCB126 and PCB153 Effect on hepatic gene
expression Environmental Health Perspectives 112 1636ndash1644
Vo T D Greenberg H J amp Palsson B O (2004) Reconstruction and
functional characterization of the human mitochondrial metabolic
network based on proteomic and biochemical data Journal of Bio-
logical Chemistry 279 39532ndash39540
Wang H amp LeCluyse E L (2003) Role of orphan nuclear receptors in
the regulation of drug-metabolising enzymes Clinical Pharmacoki-
netics 42 1331ndash1357
Wang N Silver D L Thiele C amp Tall A R (2001) ATP-
binding cassette transporter A1 (ABCA1) functions as a choles-
terol efflux regulatory protein Journal of Biological Chemistry 276
23742ndash23747
Waring J F Cavet G Jolly R A McDowell J Dai H Ciurlionis R
et al (2003) Development of a DNA Microarray for toxicology based
on hepatoxin-regulated sequences Environmental Health Perspectives
111 863ndash870
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6666
Waring J F Gum R Morfitt D Jolly R A Ciurlionis R Heindel M
et al (2002) Identifying toxic mechanisms using DNA microarrays
Evidence that an experimental inhibitor of cell adhesion molecule
expression signals through the aryl hydrocarbon nuclear receptor
Toxicology 181ndash182 537ndash550
Waring J F Jolly R A Ciurlionis R Lum P Y Praestgaard J T
Morfitt D C et al (2001) Clustering of hepatotoxins based on
mechanism of toxicity using gene expression profiles Toxicology and
Applied Pharmacology 175 28ndash42
Waters M Boorman G Bushel P Cunningham M Irwin R Merrick
A et al (2003) Systems toxicology and the Chemical Effects in
Biological Systems (CEBS) knowledge base EHP Toxicogenomics
111 15ndash28
Wei M Arnold L Cano M amp Cohen S M (2005) Effects of co-
administration of antioxidants and arsenicals on the rat urinary bladder
epithelium Toxicology Sciences 83 237ndash245
Weinshilboum R (2003) Inheritance and drug response New England
Journal of Medicine 348 529ndash537
Weinshilboum R amp Wang L (2004) Pharmacogenetics Inherited
variation in amino acid sequence and altered protein quantity Clinical
Pharmacology and Therapeutics 75 253ndash258
Werner E (2003) In silico multicellular systems biology and minimal
genomes Drug Discovery Today 8 1121ndash1127
Wong J S amp Gill S S (2002) Gene expression changes induced in
mouse liver by di(2-ethylhexyl) phthalate Toxicology and Applied
Pharmacology 185 180ndash196
Xie T Tong L McCann U D Yuan J Becker K G Mechan A O
et al (2004) Identification and characterization of metallothionein-1
and -2 gene expression in the context of ()34-methylenedioxymetham-
phetamine-induced toxicity to brain dopaminergic neurons Journal of
Neuroscience 24 7043ndash7050
Yadetie F Laegreid A Bakke I Kusnierczyk W Komorowski J
Waldum H L et al (2003) Liver gene expression in rats in response
to the peroxisome proliferator-activated receptor-alpha agonist ciprofi-
brate Physiological Genomics 15 9ndash19
Yamashita S Nomoto T Abe M Tatematsu M Sugimura T amp
Ushijima T (2004) Persistence of gene expression changes in stomach
mucosae induced by short-term N-methyl-NV-nitro-N-nitrosoguanidinetreatment and their presence in stomach cancers Mutation Research
549 185ndash193
Yan Q amp Sadee W (2000) Human membrane transporter database A
web-accessible relational database for drug transport studies and
pharmacogenomics AAPS Pharmsci 2 E20
Yeger-Lotem E amp Margalit H (2003) Detection of regulatory circuits by
integrating the cellular networks of proteinndashprotein interactions and
transcription regulation Nucleic Acids Research 31 6053ndash6061
Yoon B I Li G -X Kitada K Kawasaki Y Igarashi K amp Kodama
Y et al (2003) Mechanisms of benzene-induced hematotoxicity and
leukemogenicity cDNA microarray analyses using mouse bone marrow
tissue Environmental Health Perspectives 111 1411ndash1420
Young M B DiSilvestro M R Sendera T J Freund J Kriete A amp
Magnuson S R (2003) Analysis of gene expression in carbon
tetrachloride-treated rat livers using a novel bioarray technology
Pharmacogenomics Journal 3 41ndash52
Yu H Zhu X Greenbaum D Karro J amp Gerstein M (2004) TopNet
A tool for comparing biological sub-networks correlating protein
properties with topological statistics Nucleic Acids Research 32
328ndash337
Zhang E Y Fu D J Pak Y A Stewart T Mukhopadhyay N
Wrighton S A et al (2004) Genetic polymorphisms in human proton-
dependent dipeptide transporter PEPT1 Implications for the functional
role of Pro586 Journal of Pharmacology and Experimental Therapeu-
tics 310 437ndash445
Zhang E Y Knipp G T Ekins S amp Swaan P W (2002)
Structural biology and function of solute transporters Implications
for identifying and designing substrates Drug Metabolism Reviews
34 709ndash750
Zhang E Y Phelps M A Cheng C Ekins S amp Swaan P W (2002)
Modeling of active transport systems Advanced Drug Delivery
Reviews 54 329ndash354
- Systems-ADMETox Resources and network approaches
-
- Introduction
- Data available
- Network analysis and databases
-
- Network applications
-
- The role of transporters
-
- Clinical relevance of transporters
- Transporter network examples ABCA1
- Transporter network examples OATP
- Transporter microarray data
-
- Applications to enzymes
- Future network applications
-
- Discussion
- Acknowledgements
- References
-
Microarray or other high throughput data
Upload list of geneprotein identifiers and fold change significance etc (raw data or after selection of the differentially expressed
Genes using other statistical methods)
Parse database and generate interaction network with different algorithms or visualize on maps
Filter Networks
Access significance of the interaction networks
Determine GO processes
Compare 2 or more networks intersection overlap etc
Export gene list
Export gene list
generate interaction network with different algorithms or visualize on maps
Visualization
Fig 3 Schematic for the utilization of pathway tools for assessing high throughput data
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6650
specific detailed reference to transporters and enzymes but
these technologies can also be applied elsewhere as
described above
Fig 4 Network for high throughput screening data for clotrimazole and ticona
cholecystokinin CCR1 Choline transporter Chloride channel catechol-O-methyl
and co workers (Fliri et al 2005) Nodes highlighted with large blue circles rep
molecules showing gt50 inhibition in the assay blue circles represents both mol
inhibits gt50 and one inhibits lt50 Ligands (large) linked to transcriptional f
(wwgenegocom) (For interpretation of the references to colour in this figure leg
311 The role of transporters
A diverse array of organic solutes such as nutrients
neurotransmitters and drugs are transported by specialized
zole screened against 10 in vitro assays (Human Cannabinoid 1 Human
transferase COX2 CYP1A2 CYP2B6 CYP2C19) data published by Fliri
resent assays used in the study Red circles on these nodes represent both
ecules showing lt50 inhibition chequered circles represent one molecule
actors enzymes and transporters via edges using the MetaCorei database
end the reader is referred to the web version of this article)
Table 5
Datasets acquired from publications and other sources and used to evaluate two databasepathway tools KEGG and metaCore
Dataset Number of objects
genes proteins in list
Number of objects
mapped with KEGG
Number of objects mapped
with MetaCore on networks
Reference for microarray
data gene list
Tox Chip 10 (NIEHS) 1853 405 1498 httpsdir-appsniehsnihgov
mapsguestclonesrchcfm
4-Hydroxytamoxifen and
estrogen
1617 434 1343 (Hodges et al 2003)
Mitochondrial proteins 722 156 388 (Gaucher et al 2004
Taylor et al 2003)
Bromobenzene
(24 and 48 h)
130 41 89 (Heijne et al 2004)
Acetaminophen 30 19 23 (Heinloth et al 2004)
Acetaminophen 84 29 64 (Huang et al 2004)
Furan 185 64 139 (Huang et al 2004)
Tetrodotoxin 116 31 86 (Raghavendra Prasad et al
2004)
Benzene 73 16 62 (Yoon et al 2003)
Benzene 76 5 53 (Faiola et al 2004)
Carbon tetrachloride 37 8 26 (Young et al 2003)
Estrogen 94 33 90 (Lobenhofer et al 2002)
Trovafloxacin 142 20 82 (Liguori et al 2005)
Phenobarbital 37 13 28 (Ueda et al 2002)
L-742694 (liver)+ 45 17 19 (Hartley et al 2004)
L-742694 (intestine)+ 23 11 10 (Hartley et al 2004)
A-277249 21 7 9 (Waring et al 2002)
All files were converted to Locuslink identifiers except as noted () unigene () Affymetrix (+) Genbank identifiers were used
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 51
proteins across cellular membranes These may function as
passive processes or active processes energized by the
hydrolysis of ATP or coupling to the co-transport of counter
ions down an electrochemical gradient such as Na+ H+ and
Cl There are many thousands of transporters which can be
classified into distinct superfamilies One of these the solute
carrier class (SLC) is rapidly expanding and contains over
30 families and 200 members The ATP-binding cassette
(ABC) contains 7 families and over 48 members including
P-glycoprotein (P-gp) and MRP subfamilies (Zhang Knipp
Ekins amp Swaan 2002a Zhang Phelps Cheng Ekins amp
Swaan 2002b) Transporters have a key role in clinical
pharmacology with many drugs specifically targeting them
Numerous drugs share transport pathways with nutrients
and transporters have a role in oral absorption drug
bioavailability drug resistance excretion and ultimately
pharmacokinetics and pharmacodynamics
Polymorphism of drug transporters may be a key factor
in drug interactions and lack of effectiveness This field has
become known as pharmacogenomics and is focused on
understanding of inherited DNA sequence variations (poly-
morphisms and mutations) revealed by xenobiotics (Evans
amp McLeod 2003 Weinshilboum 2003) Over the last few
decades many genes have been directly linked to the
mechanisms of response (Evans amp McLeod 2003 Wein-
shilboum 2003) such that 20ndash95 of variability to drug
response is inheritable (Evans amp McLeod 2003) This
phenotypical variability is mainly caused by single nucleo-
tide polymorphisms (SNPs) present in anywhere from 1 to
50 of the population resulting in either lower protein
activity incorrect folding or rapid degradation via proteo-
somes (Weinshilboum amp Wang 2004) A number of
structurally diverse molecules bind to P-gp which is
expressed in many tissues and has numerous SNPs one of
which (C3435T) affects the expression level in the
duodenum and therefore can impact the absorption of
molecules which would be substrates for this transporter
(Sakaeda Nakamura amp Okumura 2002) The human
proton-dependent dipeptide transporter (hPEPT1) can also
affect the absorption of molecules in the intestine and
recently 9 SNPs were found with only one displaying a
reduced transport capacity (Zhang et al 2004) The sodium-
dependent carnitine cotransporter OCTN2 can possess
mutations and these result in primary carnitine deficiency
which impacts fatty acid oxidation and is characterized by
many clinical manifestations (Lahjouji Mitchel amp Qureshi
2001) The organic cation transporter 1 (OCT1) is also
important in the transport of numerous xenobiotics and
endobiotics Recently 4 SNPs were identified in the
Japanese population and when functionally characterized
in vitro the uptake of cations was reduced significantly for
some of these mutations indicating that this would likely
contribute to inter-individual variations in metabolism of
drugs which were transported via OCT1 (Sakata et al
2004)
3111 Clinical relevance of transporters The pregnane
X-receptor (PXR) is a transcriptional regulator of the
enzyme human MDR1 (P-gp) MRPs and OATP (Wang amp
LeCluyse 2003) CYP3A (Bertilsson et al 1998 Blumberg
et al 1998 Kliewer et al 1998) and CYP2C89 (Synold
Dussault amp Forman 2001) as well as many other genes
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6652
involved in the transport metabolism and biosynthesis of
bile acids (Staudinger Liu Madan Habeebu amp Klaassen
2001) However the additional receptors CAR FXR LXR
and other nuclear receptors take part in a complex network
of interactions to control these and other proteins Thus
elucidation of the regulatory networks which control the
expression of these transporters is also important To date
most of the research has centered on efflux transporters but
there has been considerable interest in uptake transporters
such as the organic anion transporter polypeptide (OATP
((Kim 2003) see also later Section on OATP)
There are several specific examples of the importance of
drug transporters to the clinical development of drugs One
example is the insulin sensitizer troglitazone which was
withdrawn due to hepatotoxicity although the precise
mechanism appears to have been unclear until recently
The major metabolite is a sulfated species and is suspected
of being responsible for the observed toxicity The recent
assessment of the organic anion transporting polypeptides
OATP-C and OATP8 expressed on the hepatocyte baso-
lateral membrane indicated that sulphated troglitazone has a
high affinity for the former and possibly lower affinity for
the latter (Nozawa et al 2004) This metabolite would
therefore be expected to accumulate in hepatocytes and
inhibit the bile salt export pump and Mrp2 Because
polymorphisms have been shown for OATP-C (Tirona
Leake Merino amp Kim 2001) it is also possible that these
may result in the accumulation of the metabolite and in turn
elicit idiosyncratic toxicity A second relevant example of
the impact of transporters is the clinically significant drugndash
drug interaction between cerivastatin and cyclosporine A
which occurs via the OATP-C transporter (Shitara Itoh
Sato Li amp Sugiyama 2003) A third example are the HIV
protease inhibitors saquinavir ritonavir and indinavir which
are transported by MRP2 in vitro and other drugs such as
probenecid and sulfinpyrazone are able to enhance this
transport Transport by MRP2 suggests that these com-
pounds will have decreased bioavailability due to increased
clearance and other drugs could aggravate this situation by
further enhancing transport (Huisman et al 2002) Sim-
ilarly the rifampicin mediated induction via PXR of MRP2
and P-gp in healthy subjects was found to significantly
decrease the AUC and also correlated with intestinal
expression of these transporters This transporter is also
inducible by cisplatin 2-AAF and phenobarbital (Schrenk
et al 2001) indicating multiple mechanisms may be
involved In other species such as rat commonly used as a
toxicity model orthologs of the transporters such as OATP2
are expressed and can be induced with ligands for PXR like
PCN (Guo Choudhuri amp Klaassen 2002) This is useful
knowledge because the advent of microarray technology
allows one to dose a rat with a xenobiotic and assess
thousands of genes simultaneously in a particular tissue For
instance animals dosed with known nephrotoxins have
shown some upregulation of the NandashKndashCl transporter
however the authors suggested genomic responses are
stronger soon after exposure before declining (Fleck et
al 2003) Some transporters may therefore be specifically
targeted by drugs in one tissue such as the CNS but these
same transporters may also be expressed elsewhere in the
body hence off-target effects may result in toxicity The
serotonin transporter is one such example which is
expressed in the lungs and brain Some substrates for this
channel like fenfluramine can result in primary pulmonary
hypertension as they accumulate in pulmonary cells (Roth-
man Ayestas Dersch amp Baumann 1999) Similarly P-gp is
expressed at the blood brain barrier and intestine impacting
the efficacy and bioavailability of drugs
3112 Transporter network examples ABCA1 The
ABCA1 transporter mediates the first step of cholesterol
transport Mutations in this gene cause Tangier disease
which results in severe HDL deficiency cholesterol
accumulation in macrophages and attendant atherosclerosis
This transporter represents a drug target for upregulation
modulating cholesterol metabolism and prevention of
cardiovascular disease (Oram amp Lawn 2001) In vitro
ABCA1 can be inhibited by the sulfonylurea glybenclamide
(Field Burn amp Mathur 2004 Wang Silver Thiele amp Tall
2001) In order to illustrate the advantages of mapping drug
transporters as networks onto functional models alongside
other proteins one can consider the example of ABCA1
The query of the MetaCorei database shows that this
transporter appears on three manually curated pathway maps
representing the Fstate of the art_ knowledge derived from
reliable high quality literature sources One can also use the
individual maps as an interface to access the underlying
layers of information about the transporter including the list
of encoding genessplice variants with known SNPs In
addition to browsing MetaCorei a user can also build
custom networks around ABCA1 using the network-
construction tool (Fig 5) Such a visualization utility may
be very helpful for identification of all putative pathways
around a particular transporter or compound of interest The
ABCA1 network created by this tool shows that this
transporter at the time of writing is linked directly to
twenty-five other objects such as APOE1 and LXR Many
of its neighbors have their own SNPs that could be
important in determining interactions between transport of
a drug and normal human transport of endogenous ligands
in health or disease
3113 Transporter network examples OATP The
OATPs are key membrane bound transporters expressed in
many organs including intestine liver lung choroid plexus
blood brain barrier and other organs (Tamai et al 2000)
This family of transporters is capable of mediating the
sodium-independent transport of a diverse array of mole-
cules such as steroid conjugates organic anions and
xenobiotics by coupling uptake with efflux of bicarbonate
(Satlin Amin amp Wolkoff 1997) glutathione or its
conjugates (Hagenbuch amp Meier 2004) The inhibition of
Fig 5 The network of direct gene interactions around the ABCA1 transporter gene (centre) generated with MetaCorei The interation types between nodes are shown as small colored hexagons eg unspecified
allosteric regulation binding cleavage competition covalent modification dephosphorylation phosphorylation transcription regulation transformation When applicable interactions also have a positive or
negative effect and direction Ligands (purple) linked to other proteins (blue blobs) transfactors (red) enzymes (orange) (For interpretation of the references to colour in this figure legend the reader is referred to
the web version of this article)
SEkin
sJournalofPharm
acologica
landToxico
logica
lMeth
ods53(2006)38ndash66
53
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6654
this transporterrsquos hepatic uptake of other compounds may be
important for reported drugndashdrug interactions (Kim 2003)
described earlier (Shitara et al 2003) as well as cerivastatin
with gemfibrozil (Shitara Hirano Sato amp Sugiyama 2004)
OATP1B1 (previous names OATP-C LST-1 OATP2
SLC21A6) represents the most studied human OATP to
date (Meier amp Stieger 2000) and is expressed on the
basolateral plasma membrane of hepatocytes Several single
nucleotide polymorphisms have been identified in the
OATP1B1 gene in European-Americans African-Americans
(Tirona et al 2001) and Japanese (Nozawa et al 2002)
dramatically impacting the transport of ligands such as
pravastatin (Mwinyi Johne Bauer Roots amp Gerloff 2004
Nishizato et al 2003) estrone-3-sulfate (Nozawa et al
2002 Tirona et al 2001) Rifampin (Tirona Leake
Wolkoff amp Kim 2003) and estradiol 17h-d-glucuronide(Tirona et al 2001)
The regulation of SLCO may be affected during
extrahepatic cholestasis bile duct ligation bile salt induced
Fig 6 Networks generated with the autoexpand algorithm in MetaCorei to illustr
be interconnected with other protein regulatory signaling information Informa
unspecified allosteric regulation binding cleavage competition covalent m
transformation When applicable interactions also have a positive or negative e
transfactors (red) enzymes (orange) (For interpretation of the references to colour
cholestatic hepatitis (Dumont et al 1997 Meier amp Stieger
2000 Rost et al 2003) and primary sclerosing cholangitis
(Oswald Kullack-Ublick Paumgarter amp Beuer 2001) In
particular OATP1B1 appears to be regulated by the liver-
enriched transcription factor hepatocyte nuclear factor 1a
(HNF1a) which binds to the promoter region of this
transporter (Jung et al 2001) Site directed mutagenesis
of this binding site resulted in inactivation suggesting the
critical nature of the interaction with HNF1a Bile acids
such as CDCA have been shown to transcriptionally repress
HNF1a in vitro via inhibition of the transactivating effect of
HNF4a on HNF1a (Jung amp Kullak-Ublick 2003) After
screening many rat and human uptake transporters in vitro
OATP1B1 was also shown to modulate the PXR response
by controlling rifampin retention in the cell and therefore
affecting the induction of CYP3A4 and other gene products
such as P-gp (Tirona et al 2003)
Some of the literature for OATP1B1 human substrate
data has been annotated into MetaCorei to illustrate the
ate how ligands for the human OATP-C (OATP1B1) transporter (centre) can
tion on the type of interaction between objects is hidden for clarity eg
odification dephosphorylation phosphorylation transcription regulation
ffect and direction Ligands (purple) linked to other proteins (blue blobs)
in this figure legend the reader is referred to the web version of this article)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 55
visualization of the complex interconnections between this
transporter its ligands regulatory factors and signaling
molecules already in the database (Fig 6) The network was
generated with the autoexpand algorithm in the software
representing one of multiple available algorithms for
connecting genes ligands and other objects in the database
Clearly if more ligands and their connections are added to
the database the complexity of the network will increase
considerably The OATP-C (OATP1B1) gene details can be
viewed upon querying the database and links are provided
to other public databases This page can be used to highlight
the multiple synonyms for this gene as well as links to the
multiple SNPs identified to date
3114 Transporter microarray data Microarrays have
generally been limited in the number of transporters present
on them (Annereau et al 2004) however they have been used
in an attempt to correlate pharmacokinetic properties with
gene expression for valacyclovir (Landowski et al 2003) as
well as understand the expression profile in different tissues
or cell lines upon food component or xenobiotic treatment
(Anderle Huang amp Sadee 2004) This lack of transporters
on microarrays has prompted some groups to produce their
own arrays with a heavier emphasis on transporters These
arrays have for example then been used to demonstrate the
upregulation of ABC transporters and down-regulation of
GST-Pi in cell lines resistant to colchicines or 9-nitro-
camptothecin (Annereau et al 2004) The genes that were
significantly up or down-regulated in this particular study
were used to build networks with MetaCore (Fig 7A B) and
the similarities between them were assessed (Fig 7C)
Although there were only a small number of significantly
changed genes in common (IL-8 Fos GST-Pi Calpactin and
Ubiquitin hydrolase) it is perhaps likely that there is a much
larger common gene network that is important for drug
resistance although a much larger number of cell lines and
drug treatments need to be evaluated to produce a definitive
drug resistance signature involving transporters enzymes
and transcriptional regulators
312 Applications to enzymes
As we have already described it may be particularly
valuable to visualize enzymes as networks to show
interactions with transcriptional regulators and ligands
For example a key enzyme is CYP3A4 which metabolizes
40ndash45 of all drugs and has relatively few SNPs
(Ingelman-Sundberg 2004) Using a second software suite
MetaDrugi (wwwgenegocom) it is possible to construct
a custom network around this or other drug metabolizing
enzymes (Fig 8) In this case the gene network for
CYP3A4 highlights all of the major transcriptional
regulators and several more distant linked proteins and
ligands connected on the network that may be useful for
further study (Fig 8) Substrates inhibitors as well as
regulatory factors and other enzymes can be observed
connected on this network Due to the many hundreds of
known inhibitors and substrates only a small number are
shown here for clarity We assume that if a perturbation in
a pathway (eg due to a nonfunctional enzyme) is linked
to a certain pathologic condition a similar perturbation
caused by the interference from xenobiotic metabolism
(eg competitive inhibition of the same enzyme) may
result in identical effects As microarray gene expression
data is increasingly generated the role of enzyme
regulation in toxicity of certain xenobiotics will become
more apparent from either in vivo or in vitro studies The
visualization of such signature gene networks involving
transporters and enzymes their ligands and regulatory
factors will also be important for future toxicity prediction
methods We have recently generated visualizations of
microarray data from MCF-7 cells treated with 4-hydrox-
ytamoxifen to show that some of the key genes involved in
metabolism and transport are upregulated (Ekins et al
2005d) In addition we have made predictions with various
QSAR models in MetaDrug to indicate the involvement of
PXR CYP3A4 and P-gp (Ekins et al 2005e) Therefore it
appears likely that 4-hydroxytamoxifen could increase its
own metabolism as well as efflux from cells via P-gp
which can be visualized on networks Any decrease of
function of these enzymes or transporters in a population
would likely result in changes in the metabolism and
transport of this active metabolite potentially impacting
the clinical effect This represents one example of how
both pathway tools QSAR models and network building
algorithms can be used with different types of predicted
and experimental data to allow visualization of potential
compound interactions or toxicity
313 Future network applications
As the population ages an increasing incidence and
prevalence of systemic diseases especially chronic diseases
have occurred among older adults This has resulted in an
increase in medications used concomitantly by this
population which presents challenges for drugndashdrug
interactions Physiologically elderly patients may behave
differently to the younger patients for which the drugs are
initially developed for and tested on Many pharmacoki-
netic investigations in the elderly population reveal
decreased clearance of lipophilic drugs metabolized by
the cytochrome P450 enzymes however few studies have
evaluated aging-dependent or gender-related changes in
specific P450 enzymes (Hunt Westerkam amp Slave 1992)
Age-related physiological changes such as a reduction in
liver mass hepatic metabolizing enzyme activity liver
blood flow and alterations in plasma drug binding may
account for the decreased elimination of some metabolized
drugs in the elderly It is particularly difficult to separate an
effect of aging from the variation in the rate of metabolism
due to factors such as individual metabolic phenotype
(slow or fast metabolizer due to SNPs) environmental
influences concomitant disease states and drug intake
(drugndashdrug interactions) The available data suggest that
Fig 7 Networks for drug resistant cell lines generated with the Auto expand algorithm in MetaCore A KB-3-1 vs KB-8-5 B ATC-DU-145 vs RCO1 C node overlap of A and B with initial nodes highlighted
with blue or red solid circles (Initial gene lists derived from (Annereau et al 2004)) Information on the type of interaction between objects is hidden for clarity eg unspecified allosteric regulation binding
cleavage competition covalent modification dephosphorylation phosphorylation transcription regulation transformation When applicable interactions also have a positive or negative effect and direction
Ligands (purple) linked to other proteins (blue blobs) transfactors (red) enzymes (orange) (For interpretation of the references to colour in this figure legend the reader is referred to the web version of this article)
SEkin
sJournalofPharm
acologica
landToxico
logica
lMeth
ods53(2006)38ndash66
56
Fig7(continued)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 57
Fig7(continued)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6658
Fig 8 The network of gene interactions around the CYP3A4 enzyme (centre) from MetaCorei Information on the type of interaction between objects is
hidden for clarity eg unspecified allosteric regulation binding cleavage competition covalent modification dephosphorylation phosphorylation
transcription regulation transformation When applicable interactions also have a positive or negative effect and direction Ligands (purple) linked to other
proteins (blue blobs) transfactors (red) enzymes (orange) (For interpretation of the references to colour in this figure legend the reader is referred to the web
version of this article)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 59
the initial doses of drugs metabolized by these enzymes
should be reduced in older patients according to the clinical
response In most published studies the elderly appear at
least as responsive as the young to inducers or inhibitors of
P450s (Durnas Loi amp Cusack 1990) More recently there
has been some suggestion that there are age related
reductions in function of some specific P450s such as
CYP3A4 (Patki et al 2004) and this could occur at the
level of regulation However earlier studies with the same
enzyme showed no change in clearance with age (Hunt et
al 1992) To date there has been even less examination of
the transporter functions and any changes with age
(Kinirons amp OrsquoMahony 2004) so the current understand-
ing of the effects of aging on metabolism and transport is
anything but transparent This represents an extreme
challenge for the pharmaceutical industry how to predict
whether a drug has an affinity for an enzyme or transporter
and whether this may be clinically important if it shows a
decline in expression or function with aging Any decrease
of function of these enzymes or transporters in an elderly
population would result in changes in the metabolism and
transport of metabolites potentially impacting the clinical
effect This represents another example of how computa-
tional approaches may perhaps be used with different types
of predicted and experimental data to allow visualization of
potential compound interactions or toxicity in elderly
populations We may see different gene networks high-
lighted as humans age and these may be modified by drug
treatment and coadministration This represents an area
were network analysis could be applied in the future and is
in need of considerable further research
The collection of microarray data in databases such as
CEBS ArrayTrack and EDGE represents a future resource
for computational gene network analysis One could
envisage that ultimately in each case such data is converted
to one or more networks that are also displayed for the user
and can be used to compare treatments from in vivo and in
vitro experiments This would represent a different approach
to clustering the data as currently implemented in one of
these efforts (Hayes et al 2005) and may condense large
amounts of experimentally derived data into a readily
interpreted network
4 Discussion
Previously in this journal the progress of many
research groups in predicting human ADME parameters
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6660
in silico (Ekins et al 2000b) and approaches for drug
metabolism (Ekins Ring Grace McRobie-Belle amp
Wrighton 2000a) have been described Both of these
reviews commented on moving HT assays for ADME
Tox much earlier in drug discovery which had also been
indicated by other groups The initial reviews also
highlighted the likely wealth of data that would become
available and how this could be used for structure
activity relationships alongside the bioactivity data in
computational models It was noted that there was a
paucity of predictive metabolism tools at that time In
addition a growing number of efforts to model whole
cells and organs now a field called systems biology
were recognized as models that could be integrated with
the in silico ADME approaches In summary since these
past reviews virtually all pharmaceutical companies have
attempted earlier high throughput screening for ADME
Tox properties and to some extent the wider application
of computational approaches for physicochemical proper-
ties Systems biology is being quite widely acknowledged
as the new paradigm for understanding complex bio-
logical datasets derived from high throughput technolo-
gies and the accumulated knowledge on human protein
interactions (Hartwell Hopfield Leibler amp Murray 1999
Hood 2003b) Therefore systems biology can be defined
as the integration of genetic proteomic transcriptomic
and metabonomic data using computational methods
(Nicholson amp Wilson 2003) When taken together
information on molecular processes derived from different
sources represents a lsquolsquouniversersquorsquo of putative biological
functionality of which only a small fraction of it will be
realized in a cell at any given time To date systems
biology has been driven by academia and funding bodies
such as the NIH rather than the pharmaceutical compa-
nies Presently there is a great deal of interest from
scientists of all backgrounds in identifying the networks
of cellular pathways and the corresponding physically
interacting proteins
The network building software for systems biology
described in this current review will be valuable to query
high throughput data and known literature interactions in
order to predict potential toxicity in different species In the
future the compilation of published toxicogenomics data-
sets characteristic of different types of toxicity will likely
be available in these software systems to act as a reference
database It is also feasible that we will be able to generate
the annotated datasets which specifically address the
differences between human and rat networks implicated
in toxicity The identification of sub-network modules
conserved between human and rat distinct for toxicity
types or predictive for toxic end-points in human will be
possible Such signature gene networks (Nikolsky et al
2005) can then be verified with other experimentally
derived data prospectively or from preexisting databases
The combined Fhigh throughput datandashin silico network_approaches therefore represents a novel method for access-
ing complex tissue-level processes as networks integrating
all data types based on functional interactions The data-
bases developed and used in this approach will certainly
benefit from further annotation around the drug metaboliz-
ing enzymes and transporters as described herein in terms
of transcriptional regulation and the ligands associated with
them which frequently appear in the literature It will be
important to capture disparities in the assignment of ligands
to enzymes transporters and other proteins as well as
negative data
In summary although there have been numerous
toxicogenomics studies published there is presently a
relatively small number of datasets that are freely available
to perform network analysis of microarray data The
number of studies identifying large numbers of proteins
which are affected by molecule treatment are even scarcer
still while there are several examples of some published
studies that combine such data It is hoped that the
numerous database initiatives for high content and toxicol-
ogy data that are being undertaken will improve the
situation for other researchers that are not currently
equipped to do such microarray studies themselves The
addition of requirements by journals to deposit such raw
data in a freely accessible resource will aid these initiatives
ADMETox groups have seen new technologies and
approaches developed over the last decade that have all
been applied to identify poor compounds earlier (Fig 2)
The latest technologies integrate network building tools
with high content data and databases The current review
described the limited number of networks generated for
ADMETox at present and one hopes that the impact of
such analyses will be commonplace in the future Systems
biology is however more than just applying a network
approach and hence systems-ADMETox will have to
evolve due to the continual pressure to develop newer
technologies This current paradigm combining empirical
data and computational methods should integrate the
complex data already generated making it readily inter-
pretable and valuable for identifying the most promising
compounds in the future
Acknowledgements
Dr Maggie AZ Hupcey is gratefully acknowledged for
editorial assistance Dr Peter W Swaan (University of
Maryland) and Dr Cheng Chang (Ohio State University)
Dr Steve Wright (University of Arizona) Dr K Sandy
Pang (University of Toronto) and Dr Craig Giroux (Wayne
State University) are kindly acknowledged for their support
and discussions My colleagues at GeneGo Sergey
Andreyev Andy Ryabov Eugene Kirillov Eugene A
Rakhmatulin Svetlana Sorokina Andrej Bugrim Tatiana
Nikolskaya Yuri Nikolsky John Metz and Julie Bryant are
all thanked for their considerable contributions to software
development and data annotation
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 61
References
Abe M Yamashita S Kuramoto T Hirayama Y Tsukamoto T Ohta
T et al (2003) Global expression analysis of N-methyl-NV-nitro-N-nitrosoguanidine-induced rat stomach carcinomas using oligonucleotide
microarrays Carcinogenesis 24 861ndash867
Adachi T Ono Y Koh K B Takashima K Tainaka H Matsuno Y
et al (2004) Long-term alteration of gene expression without
morphological change in testis after neonatal exposure to genistein in
mice Toxicogenomic analysis using cDNA microarray Food and
Chemical Toxicology 42 445ndash452
Akerman G S Rosenzweig B A Domon O E McGarrity L J
Blankenship L R Tsai C A et al (2004) Gene expression profiles
and genetic damage in benzo(a)pyrene diol epoxide-exposed TK6 cells
Mutation Research 549 43ndash64
Aksenov S V Church B Dhiman A Georgieva A Sarangapani R
Helmlinger G et al (2005) An integrated approach for inference and
mechanistic modeling for advancing drug development FEBS Letters
579 1878ndash1883
Anderle P Huang Y amp Sadee W (2004) Intestinal membrane transport
of drugs and nutrients Genomics of membrane transporters using
expression microarrays European Journal of Pharmaceutical Sciences
21 17ndash24
Annereau J P Szakacs G Tucker C J Arciello A Cardarelli C
Collins J et al (2004) Analysis of ATP-binding cassette transporter
expression in drug-selected cell lines by a microarray dedicated to
multidrug resistance Molecular Pharmacology 66 1397ndash1405
Apic G Ignjatovic T Boyer S amp Russell R B (2005) Illuminating drug
discovery with biological pathways FEBS Letters 579 1872ndash1877
Baker V A Harries H M Waring J F Duggan C M Ni H A Jolly
R A et al (2004) Clofibrate-induced gene expression changes in rat
liver A cross-laboratory analysis using membrane cDNA arrays
Environmental Health Perspectives 112 428ndash438
Balakin K V Ekins S Bugrim A Ivanenkov Y A Korolev D amp
Nikolsky Y et al (2004a) Quantitative structurendashmetabolism relation-
ship modeling of the metabolic N-dealkylation rates Drug Metabolism
and Disposition 32 1111ndash1120
Balakin K V Ekins S Bugrim A Ivanenkov Y A Korolev D amp
Nikolsky Y et al (2004b) Kohonen maps for prediction of binding to
human cytochrome P450 3A4 Drug Metabolism and Disposition 32
1183ndash1189
Barabasi A -L amp Oltvai Z N (2004) Network biology Under-
standing the cellrsquos functional organization Nature Reviews Genetics
5 101ndash113
Bartosiewicz M Penn S G amp Buckpitt A (2001) Applications of gene
arrays in environmental toxicology Fingerprints of gene regulation
associated with cadmium chloride benzo(a)pyrene and trichloro-
ethylene Environmental Health Perspectives 109 71ndash74
Bertilsson G Heidrich J Svensson K Asman M Jendeberg L
Sydow-Backman M et al (1998) Identification of a human nuclear
receptor defines a new signaling pathway for CYP3A induction
Proceedings of the National Academy of Sciences of the United States
of America 95 12208ndash12213
Blumberg B Sabbagh Jr W Juguilon H Bolado Jr J van Meter
C M Ong E S et al (1998) SXR a novel steroid and xenobiotic-
sensing nuclear receptor Genes and Development 12 3195ndash3205
Borodina Y Rudik A Filimonov D Kharchevnikova N Dmitriev A
Blinova V et al (2004) A new statistical approach to predicting
aromatic hydroxylation sites Comparison with model-based
approaches Journal of Chemical Information and Computer Sciences
44 1998ndash2009
Borodina Y Sadym A Filimonov D Blinova V Dmitriev A amp
Poroikov V (2003) Predicting biotransformation potential from
molecular structure Journal of Chemical Information and Computer
Sciences 43 1636ndash1646
Bottone Jr F G Martinez J M Collins J B Afshari C A amp Eling
T E (2003) Gene modulation by the cyclooxygenase inhibitor
sulindac sulfide in human colorectal carcinoma cells Possible link to
apoptosis Journal of Biological Chemistry 278 25790ndash25801
Boyer S amp Zamora I (2002) New methods in predictive metabolism
Journal of Computer-Aided Molecular Design 16 403ndash413
Bredel M amp Jacoby E (2004) Chemogenomics An emerging strategy
for rapid target and drug discovery Nature Reviews Genetics 5
262ndash275
Bugrim A Nikolskaya T amp Nikolsky Y (2004) Early prediction of drug
metabolism and toxicity Systems biology approach and modeling
Drug Discovery Today 9 127ndash135
Bulera S J Eddy S M Ferguson E Jatkoe T A Reindel J F
Bleavins M R et al (2001) RNA expression in the early character-
ization of hepatotoxicants in Wistar rats by high-density DNA micro-
arrays Hepatology 33 1239ndash1258
Butcher E C Berg E L amp Kunkel E J (2004) Systems biology in drug
discovery Nature Biotechnology 22 1253ndash1259
Butte A (2002) The use and analysis of microarray data Naturalist
Review of Drug Discovery 1 951ndash960
Cary M P Bader G D amp Sander C (2005) Pathway information for
systems biology FEBS Letters 579 1815ndash1820
Cheok M H Yang W Pui C H Downing J R Cheng C Naeve
C W et al (2003) Treatment-specific changes in gene expression
discriminate in vivo drug response in human leukemia cells Nature
Genetics 34 85ndash90
Cleary J D Rogers P D amp Chapman S W (2001) Differential
transcription factor expression in human mononuclear cells in response
to amphotericin B Identification with complementary DNA microarray
technology Pharmacotherapy 21 1046ndash1054
Coen M Ruepp S U Lindon J C Nicholson J K Pognan F Lenz
E M et al (2004) Integrated application of transcriptomics and
metabonomics yields new insight into the toxicity due to paracetamol in
the mouse Journal of Pharmaceutical and Biomedical Analysis 35
93ndash105
Cornwell P D De Souza A T amp Ulrich R G (2004) Profiling of
hepatic gene expression in rats treated with fibric acid analogs
Mutation Research 549 131ndash145
Csermely P Agoston V amp Pongor S (2005) The efficiency of multi-
target drugs The network approach might help drug design Trends in
Pharmacological Sciences 26 178ndash182
Cunningham M J Liang S Fuhrman S Seilhamer J J amp
Somogyi R (2000) Gene expression microarray data analysis for
toxicology profiling Annals of the New York Academy of Sciences 919
52ndash67
de Longueville F Atienzar F A Marcq L Dufrane S Evrard S
Wouters L et al (2003) Use of a low density microarray for studying
gene expression patterns induced by hepatotoxicants on primary
cultures of rat hepatocytes Toxicological Sciences 75 378ndash392
Dobrin R Beg Q K Barabasi A L amp Oltvai Z N (2004)
Aggregation of topological motifs in the Escherichia coli transcriptional
regulatory network BMC Bioinformatics 5 10
Donald S Verschoyle R D Edwards R Judah D J Davies R
Riley J et al (2002) Hepatobiliary damage and changes in hepatic
gene expression caused by the antitumor drug ecteinascidin-743 (ET-
743) in the female rat Cancer Research 62 4256ndash4262
Dumont M Jacquemin E DrsquoHont C Descout C Cresteil D Haouzi
D et al (1997) Expression of the liver Na+-independent organic anion
transporting polypeptide (oatp-1) in rats with bile duct ligation Journal
of Hepatology 27 1051ndash1056
Durnas C Loi C M amp Cusack B J (1990) Hepatic drug metabolism
and aging Clinical Pharmacokinetics 19 359ndash389
Eisen M B Spellman P T Brown P O amp Botstein D (1998) Cluster
analysis and display of genome-wide expression patterns Proceedings
of the National Academy of Sciences of the United States of America
95 14863ndash14868
Ekins S Andreyev S Ryabov A Kirilov E Rakhmatulin E A
Bugrim A et al (in press) Computational Prediction of Human Drug
Metabolism Exp Opin Drug Metab Toxicol
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6662
Ekins S Boulanger B Swaan P W amp Hupcey M A Z (2002)
Towards a new age of virtual ADMETOX and multidimensional drug
discovery Journal of Computer-Aided Molecular Design 16 381ndash401
Ekins S Bugrim A Nikolsky Y amp Nikolskaya T (2005) Systems
biology Applications in drug discovery In S Gad (Ed) Drug
Discovery Handbook (pp 123ndash183) Hoboken NJrsquo Wiley and Sons
Ekins S Giroux C N Nikolsky Y Bugrim A amp Nikolskaya T
(2005) A signature gene network approach to toxicity The Toxoco-
logists 84
Ekins S Kirillov E Rakhmatulin E amp Nikolskaya T (2005) A novel
method for visualizing nuclear hormone receptor networks relevant to
drug metabolism Drug Metabolism and Disposition 33 474ndash481
Ekins S amp McGowan R J (2001) The limits of reductionism The
shifting genomic paradigmrsquos impact on industry and academia
Philsophy in Science 9 1ndash23
Ekins S Mirny L amp Schuetz E G (2002) A ligand-based approach to
understanding selectivity of nuclear hormone receptors PXR CAR
FXR LXRa and LXRb Pharmaceutical Research 19 1788ndash1800
Ekins S Nikolsky Y amp Nikolskaya T (2005) Techniques Application
of systems biology to absorption distribution metabolism excretion
and toxicity Trends in Pharmacological Sciences 26 202ndash209
Ekins S Ring B J Grace J McRobie-Belle D J amp Wrighton S
A (2000) Present and future in vitro approaches for drug
metabolism Journal of Pharmacological and Toxicological Methods
44 313ndash324
Ekins S Waller C L Swaan P W Cruciani G Wrighton S A amp
Wikel J H (2000) Progress in predicting human ADME parameters in
silico Journal of Pharmacological and Toxicological Methods 44
251ndash272
Erhardt P W (2003) A human drug metabolism database Potential roles
in the quantitative predictions of drug metabolism and metabolism-
related drugndashdrug interactions Current Drug Metabolism 4 411ndash422
Evans W E amp McLeod H L (2003) Pharmacogenomicsmdashdrug
disposition drug targets and side effects New England Journal of
Medicine 348 538ndash549
Ezendam J Staedtler F Pennings J Vandebriel R J Pieters R
Boffetta P et al (2004) Toxicogenomics of subchronic hexachlor-
obenzene exposure in Brown Norway rats Environmental Health
Perspectives 112 782ndash791
Faiola B Fuller E S Wong V A amp Recio L (2004) Gene expression
profile in bone marrow and hematopoietic stem cells in mice exposed to
inhaled benzene Mutation Research 549 195ndash212
FDA (2004) Innovation stagnation Challenge and opportunity on the
critical path to new medicinal products
Fiehn O (2001) Combining genomics metabolome analysis and bio-
chemical modelling to understand metabolic networks Comparative
Functional Genomics 2 155ndash168
Field F J Born E amp Mathur S N (2004) LXRRXR ligand activation
enhances basolateral efflux of beta-sitosterol in CaCo-2 cells Journal of
Lipid Research 45 905ndash913
Fleck C Sutter L Appenroth D Koch B Meinhold T Pitack M
et al (2003) Use of gene chip technology for the characterisation of
the regulation of renal transport processes and of nephrotoxicity in
rats Experimental and Toxicologic Pathology 54 401ndash410
Fliri A F Loging W T Thadeio P F amp Volkmann R A (2005)
Biological spectra analysis Linking biological activity profiles to
molecular structure Proceedings of the National Academy of Sciences
of the United States of America 102 261ndash266
Fountoulakis M (2004) Application of proteomics technologies in the
investigation of the brain Mass Spectrometry Reviews 23 231ndash258
Gaucher S P Taylor S W Fahy E Zhang B Warnock D E Ghosh
S S et al (2004) Expanded coverage of the human heart
mitochondrial proteome using multidimensional liquid chromatography
coupled with tandem mass spectrometry Journal of Proteome
Research 3 495ndash505
Gerhold D Lu M Xu J Austin C Caskey C T amp Rushmore T
(2001) Monitoring expression of genes involved in drug metabolism
and toxicology using DNA microarrays Physiological Genomics 5
161ndash170
Guo G L Choudhuri S amp Klaassen C D (2002) Induction profile of
rat organic anion transporting polypeptide 2 (oatp2) by prototypical
drug-metabolizing enzyme inducers that activate gene expression
through ligand-activated transcription factor pathways Journal of
Pharmacology and Experimental Therapeutics 300 206ndash212
Hagenbuch B amp Meier P J (2004) Organic anion transporting
polypeptides of the OATP SLC21 family Phylogenetic classification
as OATP SLCO superfamily new nomenclature and molecularfunc-
tional properties Pflugers Archiv 447 653ndash665
Hamadeh H K amp Afshari C A (2004) Toxicogenomics principles and
applications Hobokenrsquo John Wiley and Sons
Hamadeh H K Bushel P R Jayadev S Martin K DiSorbo O Sieber
S et al (2002) Gene expression analysis reveals chemical-specific
profiles Toxicological Sciences 67 219ndash231
Hamadeh H K Jayadev S Gaillard E T Huang Q Stoll R
Blanchard K et al (2004) Integration of clinical and gene expression
endpoints to explore furan-mediated hepatotoxicity Mutation Research
549 169ndash183
Hamadeh H K Knight B L Haugen A C Sieber S Amin R P
Bushel P R et al (2002) Methapyrilene toxicity Anchorage of
pathologic observations to gene expression alterations Toxicologic
Pathology 30 470ndash482
Han J D Bertin N Hao T Goldberg D S Berriz G F Zhang L V
et al (2004) Evidence for dynamically organized modularity in the
yeast proteinndashprotein interaction network Nature 430 88ndash93
Hanisch D Zien A Zimmer R amp Lengauer T (2002) Co-clustering of
biological networks and gene expression data Bioinformatics 18
S145ndashS154
Harris A J Dial S L amp Casciano D A (2004) Comparison of basal
gene expression profiles and effects of hepatocarcinogens on gene
expression in cultured primary human hepatocytes and HepG2 cells
Mutation Research 549 79ndash99
Harrison S C (2004) Whither structural biology Nature Structural
Molecular Biology 11 12ndash15
Hartley D P Dai X He Y D Carlini E J Wang B Huskey S E
et al (2004) Activators of the rat pregnane X receptor differentially
modulate hepatic and intestinal gene expression Molecular Pharma-
cology 65 1159ndash1171
Hartwell L H Hopfield J J Leibler S amp Murray A W (1999) From
molecular to modular cell biology Nature 402 C47ndashC52
Hasmall S Orphanides G James N Pennie W Hedley K Soames
A et al (2002) Downregulation of lactoferrin by PPARalpha ligands
Role in perturbation of hepatocyte proliferation and apoptosis
Toxicology Sciences 68 304ndash313
Hayase T Yamamoto Y Yamamoto K Muso E amp Shiota K (2004)
Microarray profile analysis of toxic cocaine-induced alterations in the
expression of mouse brain gene sequences A possible rsquoprotectiversquo
effect of buprenorphine Journal of Applied Toxicology 24 15ndash20
Hayes K R Vollrath A L Zastrow G M McMillan B J Craven M
Jovanovich S et al (2005) EDGE A centralized resource for the
comparison analysis and distribution of toxicogenomic information
Molecular Pharmacology 67 1360ndash1368
Heijne W H Slitt A L van Bladeren P J Groten J P Klaassen C D
Stierum R H et al (2004) Bromobenzene-induced hepatotoxicity at
the transcriptome level Toxicology Sciences 79 411ndash422
Heijne W H Stierum R H Slijper M van Bladeren P J amp van
Ommen B (2003) Toxicogenomics of bromobenzene hepatotoxicity
A combined transcriptomics and proteomics approach Biochemical
Pharmacology 65 857ndash875
Heinloth A N Irwin R D Boorman G A Nettesheim P Fannin
R D Sieber S O et al (2004) Gene expression profiling of rat
livers reveals indicators of potential adverse effects Toxicology
Sciences 80 193ndash202
Hodges L C Cook J D Lobenhofer E K Li L Bennett L Bushel
P R et al (2003) Tamoxifen functions as a molecular agonist inducing
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 63
cell cycle-associated genes in breast cancer cells Molecular Cancer
Research 1 300ndash311
Holleman A Cheok M H den Boer M L Yang W Veerman KM
A J Kazemier et al (2004) Gene-expression patterns in drug-resistant
acute lymphoblastic leukemia cells and response to treatment New
England Journal of Medicine 351 533ndash542
Hong Y Muller U R amp Lai F (2003) Discriminating two classes of
toxicants through expression analysis of HepG2 cells with DNA arrays
Toxicology in Vitro 17 85ndash92
Hood E (2003) Proteomics Characterizing the cogs in the machinery of
life Environmental Health Perspectives 111 A816ndashA825
Hood L (2003) Systems biology Integrating technology biology and
computation Mechanism of Ageing and Development 124 9ndash16
Hood L amp Galas D (2003) The digital code of DNA Nature 421
444ndash448
Hood L amp Perlmutter R M (2004) The impact of systems approaches on
biological problems in drug discovery Nature Biotechnology 22
1215ndash1217
Hu T Gibson D P Carr G J Torontali S M Tiesman J P Chaney
J G et al (2004) Identification of a gene expression profile that
discriminates indirect-acting genotoxins from direct-acting genotoxins
Mutation Research 549 5ndash27
Huang Q Dunn II R T Jayadev S DiSorbo O Pack F D Farr S B
et al (2001) Assessment of cisplatin-induced nephrotoxicity by
microarray technology Toxicology Sciences 63 196ndash207
Huang Q Jin X Gaillard E T Knight B L Pack F D Stoltz J H et
al (2004) Gene expression profiling reveals multiple toxicity endpoints
induced by hepatotoxicants Mutation Research 549 147ndash167
Huisman M T Smit J W Crommentuyn K M Zelcer N Wiltshire
H R Beijnen J H et al (2002) Multidrug resistance protein 2
(MRP2) transports HIV protease inhibitors and transport can be
enhanced by other drugs Aids 16 2295ndash2301
Hunt C M Westerkam W R amp Stave G M (1992) Effect of age and
gender on the activity of human hepatic CYP3A Biochemical
Pharmacology 44 275ndash283
Ideker T Ozier O Schwikowski B amp Siegal A F (2002) Discovering
regulatory and signalling circuits in molecular interaction networks
Bioinformatics 18 S233ndashS240
Ideker T Thorsson V Ranish J A Christmas R Buhler J Eng J K
et al (2001) Integrated genomic and proteomic analyses of a system-
atically perturbed network Science 292 929ndash934
Iida M Anna C H Hartis J Bruno M Wetmore B Dubin J R et al
(2003) Changes in global gene and protein expression during early
mouse liver carcinogenesis induced by non-genotoxic model carcino-
gens oxazepam and Wyeth-14643 Carcinogenesis 24 757ndash770
Ingelman-Sundberg M (2004) Pharmacogenetics of cytochrome P450 and
its applications in drug therapy The past present and future Trends in
Pharmacological Sciences 25 193ndash200
Jeong H Mason S P Barabasi A L amp Oltvai Z N (2001) Lethality
and centrality in protein networks Nature 411 41ndash42
Jeong H Tombor B Albert R Oltvai Z N amp Barabasi A L
(2000) The large-scale organization of metabolic networks Nature
407 651ndash654
Jung D Hagenbuch B Gresh L Pontoglio M Meier P J amp Kullak-
Ublick G A (2001) Characterization of the human OATP-C
(SLC21A6) gene promoter and regulation of liver-specific OATP genes
by hepatocyte nuclear factor 1 alpha Journal of Biological Chemistry
276 37206ndash37214
Jung D amp Kullak-Ublick G A (2003) Hepatocyte nuclear factor 1
alpha A key mediator of the effect of bile acids on gene expression
Hepatology 37 622ndash631
Jung J -W Park J -S Hwang J -W Kang K -S YS L Song B
-S et al (2004) Gene expression analysis of peroxisome prolifer-
ators- and phenytoin-induced hepatotoxicity using cDNA microarray
Journal of Veterinary Medical Science 66 1329ndash1333
Kato N Shibutani M Takagi H Uneyama C Lee K -Y Takiami S
et al (2004) Gene expression profile in the livers of rats orally
administered ethinylestradiol for 28 days using a microarray technique
Toxicology 200 179ndash192
Kier L D Neft R Tang L Suizu R Cook T Onsurez K et al
(2004) Applications of microarrays with toxicologically relevant genes
(tox genes) for the evaluation of chemical toxicants in Sprague Dawley
rats in vivo and human hepatocytes in vitro Mutation Research 549
101ndash113
Kijima K Toyosawa K Yasuba M Matsuoka N Adachi T
Komiyama M et al (2004) Gene expression analysis of the rat testis
after treatment with di(2-ethylhexyl) phthalate using cDNA microarray
and real-time RT-PCR Toxicology and Applied Pharmacology 200
103ndash110
Kim R B (2003) Organic anion-transporting polypeptide (OATP) trans-
porter family and drug disposition European Journal of Clinical
Investigation 33 1ndash5
Kinirons M T amp OrsquoMahony M S (2004) Drug metabolism and ageing
British Journal of Clinical Pharmacology 57 540ndash544
Kitano H (2002a) Computational systems biology Nature 420 206ndash210
Kitano H (2002b) Systems biology A brief overview Science 295
1662ndash1664
Kliewer S A Moore J T Wade L Staudinger J L Watson M
A Jones S A et al (1998) An orphan nuclear receptor activated
by pregnanes defines a novel steroid signalling pathway Cell 92
73ndash82
Korolev D Balakin K V Nikolsky Y Kirillov E Ivanenkov Y A
Savchuk N P et al (2003) Modeling of human cytochrome p450-
mediated drug metabolism using unsupervised machine learning
approach Journal of Medicinal Chemistry 46 3631ndash3643
Kramer J A Pettit S D Amin R P Bertram T A Car B
Cunningham M et al (2004) Overview on the application of
transcription profiling using selected nephrotoxicants for toxicology
assessment Environmental Health Perspectives 112 460ndash464
Kultima K Nystrom A -M Scholz B Gustafson A -L Dencker L
amp Stigson M (2004) Valproic acid teratogenicity A toxicogenomics
approach Environmental Health Perspectives 112 1225ndash1235
Kutuzova G D amp Deluca H F (2004) Gene expression profiles in rat
intestine identify pathways for 125-dihydroxyvitamin D(3) stimulated
calcium absorption and clarify its immunomodulatory properties
Archives of Biochemistry and Biophysics 432 152ndash166
Kwak M K Wakabayashi N Itoh K Motohashi H Yamamoto M amp
Kensler T W (2003) Modulation of gene expression by cancer
chemopreventive dithiolethiones through the Keap1-Nrf2 pathway
Identification of novel gene clusters for cell survival Journal of
Biological Chemistry 278 8135ndash8145
Lahjouji K Mitchell G A amp Qureshi I A (2001) Carnitine transport
by organic cation transporters and systemic carnitine deficiency
Molecular Genetics and Metabolism 73 287ndash297
Landowski C P Sun D Foster D R Menon S S Barnett J L
Welage L S et al (2003) Gene expression in the human intestine and
correlation with oral valacyclovir pharmacokinetic parameters Journal
of Pharmacology and Experimental Therapeutics 306 778ndash786
Lee G -J Lee W -S Jeon K -S Um C -H Kim S -J Lee C -H
et al (2004) cDNA microarray gene expression analysis and
toxicological phenotype for anticancer drug Journal of Veterinary
Medical Science 66 1339ndash1345
Lee M Kwon J Kim S N Kim J E Koh W S Kim E J et al
(2003) cDNA microarray gene expression profiling of hydroxyurea
paclitaxel and p-anisidine genotoxic compounds with differing
tumorigenicity results Environmental and Molecular Mutagenesis
42 91ndash97
Li S Armstrong C M Bertin N Ge H Milstein S Boxem M et al
(2004) A map of the interactome network of the metazoan C elegans
Science 303 540ndash543
Liguori M J Anderson L M Bukofzer S McKim J Pregenzer J F
Retief J et al (2005) Microarray analysis in human hepatocytes
suggests a mechanism for hepatotoxicity induced by trovafloxacin
Hepatology 41 177ndash186
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6664
Lobenhofer E K Bennett L Cable P L Li L Bushel P R amp Afshari
C A (2002) Regulation of DNA replication fork genes by 17beta-
estradiol Molecular Endocrinology 16 1215ndash1229
Locker J Tian J Carver R Concas D Cossu C Ledda-Columbano
G M et al (2003) A common set of immediate-early response genes
in liver regeneration and hyperplasia Hepatology 38 314ndash325
Mattes W B Pettit S D Sansone S A Bushel P R amp Waters M D
(2004) Database development in toxicogenomics Issues and efforts
Environmental Health Perspectives 112 495ndash505
McMillian M Nie A Y Parker J B Leone A Bryant S Kemmerer
M et al (2004) A gene expression signature for oxidant stressreactive
metabolites in rat liver Biochemical Pharmacology 68 2249ndash2261
Meier P J amp Stieger B (2000) Molecular mechanisms in bile formation
News in Physiological Sciences 15 89ndash93
Meneses-Lorente G de Longueville F Dos Santos-Mendes S Bonnert
T P Jack A Evrard S et al (2003) An evaluation of a low-density
DNA microarray using cytochrome P450 inducers Chemical Research
in Toxicology 16 1070ndash1077
Meneses-Lorente G Guest P C Lawrence J Muniappa N Knowles
M R Skynner H A et al (2004) A proteomic investigation of drug-
induced steatosis in rat liver Chemical Research in Toxicology 17
605ndash612
Merrill C L Ni H Yoon L W Tirmenstein M A Narayanan P
Benavides G R et al (2002) Etomoxir-induced oxidative stress in
HepG2 cells detected by differential gene expression is confirmed
biochemically Toxicology Sciences 68 93ndash101
Milo R Shen-Orr S Itzkovitz S Kashtan N Chklovskii D amp Alon
U (2002) Network motifs Simple building blocks of complex
networks Science 298 824ndash827
Morgan K T Ni H Brown H R Yoon L Qualls C W Crosby L
M et al (2002) Application of cDNA microarray technology to in
vitro toxicology and the selection of genes for a real-time RT-PCR-
based screen for oxidative stress in Hep-G2 cells Toxicologic
Pathology 30 435ndash451
Mwinyi J Johne A Bauer S Roots I amp Gerloff T (2004) Evidence
for inverse effects of OATP-C (SLC21A6) 5 and 1b haplotypes on
pravastatin kinetics Clinical Pharmacology and Therapeutics 75
415ndash421
Nakata K Yukawa M Komiyama N Nakano T amp Kaminuma T
(2002) A nuclear receptor database that maps pathways to diseases
Genome Infomatics 13 515ndash516
Nicholson J K Holmes E Lindon J C amp Wilson I D (2004) The
challenges of modeling mammalian biocomplexity Nature Biotechnol-
ogy 22 1268ndash1274
Nicholson J K amp Wilson I D (2003) Understanding rsquoglobal systems
biology Metabonomics and the continuum of metabolism Nature
Reviews on Drug Discovery 2 668ndash676
Nikitin A Egorov S Daraselia N amp Mazo I (2003) Pathway studiomdash
the analysis and navigation of molecular networks Bioinformatics 19
2155ndash2157
Nikolsky Y Ekins S Nikolskaya T amp Bugrim A (2005) A novel
method for generation of signature networks as biomarkers from
complex high throughput data Toxicology Letters 158 20ndash29
Nikolsky Y Nikolskaya T amp Bugrim A (2005) Biological networks
and analysis of experimental data in drug discovery Drug Discovery
Today 10 653ndash662
Nishizato Y Ieiri I Suzuki H Kimura M Kawabata K Hirota T
et al (2003) Polymorphisms of OATP-C (SLC21A6) and OAT3
(SLC22A8) genes Consequences for pravastatin pharmacokinetics
Clinical Pharmacology and Therapeutics 73 554ndash565
Nozawa T Nakajima M Tamai I Noda K Nezu J Sai Y et al
(2002) Genetic polymorphisms of human organic anion transporters
OATP-C (SLC21A6) and OATP-B (SLC21A9) Allele frequencies in
the Japanese population and functional analysis Journal of Pharmacol-
ogy and Experimental Therapeutics 302 804ndash813
Nozawa T Sugiura S Nakajima M Goto A Yokoi T Nezu J et al
(2004) Involvement of organic anion transporting polypeptides in the
transport of troglitazone sulfate Implications for understanding trogli-
tazone hepatotoxicity Drug Metabolism and Disposition 32 291ndash294
Oliver D E Rubin D L Stuart J M Hewett M Klein T E amp
Altman R B (2002) Ontology development for a pharmacogenetics
knowledge base Package Symposion 88ndash99
Oram J F amp Lawn R M (2001) ABCA1 The gatekeeper for eliminating
excess tissue cholesterol Journal of Lipid Research 42 1173ndash1179
Oswald M Kullak-Ublick G A Paumgartner G amp Beuers U (2001)
Expression of hepatic transporters OATP-C and MRP2 in primary
sclerosing cholangitis Liver 21 247ndash253
Parsons A B Brost R I Ding H Li Z Zhang C Sheikh B et al
(2004) Integration of chemical-genetic and genetic interaction data
links bioactive compounds to cellular target pathways Nature Bio-
technology 22 62ndash69
Patki K C von Moltke L L Harmatz J S Hesse L M Court M H
amp Greenblatt D J (2004) Effect of age on in vitro triazolam
biotransformation in male human liver microsomes Journal of
Pharmacology and Experimental Therapeutics 308 874ndash879
Pereira-Leal J B Enright A J amp Ouzounis C A (2004) Detection of
functional modules from protein interaction networks Proteins 54
49ndash57
Plant N (2004) Interaction networks Coordinating responses to xeno-
biotic exposure Toxicology 202 21ndash32
Raghavendra Prasad H S Qi Z Srinivasan K N amp Gopalakrish-
nakone P (2004) Potential effects of tetrodotoxin exposure to
human glial cells postulated using microarray approach Toxicon 44
597ndash608
Reilly T P Bourdi M Brady J N Pise-Masison C A Radonovich M
F George J W et al (2001) Expression profiling of acetaminophen
liver toxicity in mice using microarray technology Biochemical and
Biophysical Research Communications 282 321ndash328
Rives A W amp Galitski T (2003) Modular organization of cellular
networks Proceedings of the National Academy of Sciences of the
United States of America 100 1128ndash1133
Rogers P D Pearson M M Cleary J D Sullivan D C amp Chapman
S W (2002) Differential expression of genes encoding immunomodu-
latory proteins in response to amphotericin B in human mononuclear
cells identified by cDNA microarray analysis Journal of Antimicrobial
Chemotherapy 50 811ndash817
Rosen M B Wilson V S Schmid J E amp Gray L E (2005) Gene
expression analysis in the ventral prostate of rats exposed to vinclozolin
or procymidone Reproductive Toxicology 19 367ndash379
Rost D Herrmann T Sauer P Schmidts H L Stieger B Meier P J
et al (2003) Regulation of rat organic anion transporters in bile salt-
induced cholestatic hepatitis Effect of ursodeoxycholate Hepatology
38 187ndash195
Rothman R B Ayestas M A Dersch C M amp Baumann M H (1999)
Aminorex fenfluramine and chlorphentermine are serotonin transporter
substrates Implications for primary pulmonary hypertension Circula-
tion 100 869ndash875
Ruepp S U Tonge R P Shaw J Wallis N amp Pognan F (2002)
Genomics and proteomics analysis of acetaminophen toxicity in mouse
liver Toxicology Sciences 65 135ndash150
Sakaeda T Nakamura T amp Okumura K (2002) MDR1 genotype-
related pharmacokinetics and pharmacodynamics Biological amp Phar-
maceutical Bulletin 25 1391ndash1400
Sakata T Anzai N Shin H J Noshiro R Hirata T Yokoyama H
et al (2004) Novel single nucleotide polymorphisms of organic cation
transporter 1 (SLC22A1) affecting transport functions Biochemical
and Biophysical Research Communications 313 789ndash793
Satlin L M Amin V amp Wolkoff A W (1997) Organic anion
transporting polypeptide mediates organic anionHCO3-exchange
Journal of Biological Chemistry 272 26340ndash26345
Satomi Y Tsuchiya W Mihara K Ota M Kasahara Y amp Akahori F
(2004) Gene expression analysis of the lung following paraquat
administration in rats using DNA microarray Journal of Toxicological
Sciences 29 91ndash100
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 65
Schrenk D Baus P R Ermel N Klein C Vorderstemann B amp
Kauffmann H M (2001) Up-regulation of transporters of the MRP
family by drugs and toxins Toxicology Letters 120 51ndash57
Segal E Shapira M Regev A Persquoer D Botstein D Koller D et al
(2003) Module networks Identifying regulatory modules and their
condition-specific regulators from gene expression data Nature
Genetics 34 166ndash176
Segal E Wang H amp Koller D (2003) Discovering molecular pathways
from protein interaction and gene expression Bioinformatics 19
i264ndash i272
Seidel S D Kan H L Stott W T Schisler M R amp Gollapudi B B
(2003) Identification of transcriptome profiles for the DNA-damaging
agents bleomycin and hydrogen peroxide in L5178Y mouse lymphoma
cells Environmental and Molecular Mutagenesis 42 19ndash25
Shan L Yu Y Schut H A J amp Snyderwine E G (2004) Susceptibility
of rats to mammary gland carcinogenesis by the food-derived
carcinogen 2-Amino-1-Methyl-6-Phenylimidazo[45-b]Pyridine (PhIP)
varies with age and is associated with the induction of differential gene
expression American Journal of Pathology 165 191ndash202
Sharom J R Bellows D S amp Tyers M (2004) From large networks to
small molecules Current Opinion in Chemical Biology 8 81ndash90
Shitara Y Hirano M Sato H amp Sugiyama Y (2004) Gemfibrozil and
its glucuronide inhibit the OATP2(OATP1B1 SLC21A6)-mediated
hepatic uptake and CYP2C8-mediated metabolism of cerivastatinmdash
Analysis of the mechanism of the clinically relevant drugndashdrug
interaction between cerivastatin and gemfibrozil Journal of Pharma-
cology and Experimental Therapeutics
Shitara Y Itoh T Sato H Li A P amp Sugiyama Y (2003) Inhibition of
transporter-mediated hepatic uptake as a mechanism for drugndashdrug
interaction between cerivastatin and cyclosporin A Journal of
Pharmacology and Experimental Therapeutics 304 610ndash616
Somogyi R Fuhrman S amp Wen X (2001) Genetic network inference in
computational models and applications to large-scale gene expression
data In M Bower amp H Bolouri (Eds) Computational modeling of
genetic and biochemical networks (pp 119ndash157) Cambridge MArsquoThe
MIT Press
Spirin V amp Mirny L A (2003) Protein complexes and functional
modules in molecular networks Proceedings of the National Academy
of Sciences of the United States of America 100 12123ndash12128
Staudinger J Liu Y Madan A Habeebu S amp Klaassen C D
(2001) Coordinate regulation of xenobiotic and bile acid homeo-
stasis by pregnane X receptor Drug Metabolism and Disposition
29 1467ndash1472
Sun L Z Ji Z L Chen X Wang J F amp Chen Y Z (2002) ADME-
AP A database of ADME associated proteins Bioinformatics 18
1699ndash1700
Suter L Babiss L E amp Wheeldon E B (2004) Toxicogenomics in
predictive toxicology in drug development Chemistry and Biology 11
161ndash171
Synold T W Dussault I amp Forman B M (2001) The orphan nuclear
receptor SXR coordinately regulates drug metabolism and efflux
Nature Medicine 7 584ndash590
Tabuchi Y amp Kondo T (2003) cDNA microarray analysis reveals chop-
10 plays a key role in Sertoli cell injury induced by bisphenol A
Biochemical and Biophysical Research Communications 305 54ndash61
Tamai I Nezu J Uchino H Sai Y Oku A Shimane M et al (2000)
Molecular identification and characterization of novel members of the
human organic anion transporter (OATP) family Biochemical and
Biophysical Research Communications 273 251ndash260
Taylor S W Fahy E Zhang B Glenn G M Warnock D E Wiley S
et al (2003) Characterization of the human heart mitochondrial
proteome Nature Biotechnology 21 281ndash286
Terasaka S Aita Y Inoue A Hayashi S Nishigaki M Aoyagi K
et al (2004) Using a customized DNA microarray for expression
profiling of the estrogen-responsive genes to evaluate estrogen activity
among natural estrogens and industrial chemicals Environmental
Health Perspectives 112 773ndash781
Thomas D M Francescutti-Verbeem D M Liu X amp Kuhn D M
(2004) Identification of differentially regulated transcripts in mouse
striatum following methamphetamine treatmentmdashan oligonucleotide
microarray approach Journal of Neurochemistry 88 380ndash393
Thomas R S Rank D R Penn S G Zastrow G M Hayes K R
Pande K et al (2001) Identification of toxicologically predictive
gene sets using cDNA microarrays Molecular Pharmacology 60
1189ndash1194
Thompson K L Afshari C A Amin R P Bertram T A Car B
Cunningham M et al (2004) Identification of platform-independent
gene expression markers of cisplatin nephrotoxicity Environmental
Health Perspectives 112 488ndash494
Tirona R G Leake B F Merino G amp Kim R B (2001)
Polymorphisms in OATP-C Identification of multiple allelic
variants associated with altered transport activity among European-
and African-Americans Journal of Biological Chemistry 276
35669ndash35675
Tirona R G Leake B F Wolkoff A W amp Kim R B (2003)
Human organic anion transporting polypeptide-C (SLC21A6) is a
major determinant of the rifampin-mediated pregnane X receptor
activation Journal of Pharmacology and Experimental Therapeutics
304 223ndash228
Tong W Cao X Harris S Sun H Fang H Fuscoe J et al (2003)
ArrayTrackmdashsupporting toxicogenomic research at the US Food and
Drug Administration National Center for Toxicological Research
Environmental Health Perspectives 111 1819ndash1826
Tornow S amp Mewes H W (2003) Functional modules by relating
protein interaction networks and gene expression Nucleic Acids
Research 31 6283ndash6289
Trocho C Escola-Gil J C Ribas V Benitez S Martin-Campos J M
Rotllan N et al (2004) Phenytoin treatment reduces atherosclerosis in
mice through mechanisms independent of plasma HDL-cholesterol
concentration Atherosclerosis 174 275ndash285
Ueda A Hamadeh H K Webb H K Yamamoto Y Sueyoshi T
Afshari C A et al (2002) Diverse roles of the nuclear orphan
receptor CAR in regulating hepatic genes in response to phenobarbital
Molecular Pharmacology 61 1ndash6
Ulrich R G (2003) The toxicogenomics of nuclear receptor agonists
Current Opinion in Chemical Biology 7 505ndash510
Ulrich R G Rockett J C Gibson G G amp Pettit S D (2004)
Overview of an interlaboratory collaboration on evaluating the effects
of model hepatotoxicants on hepatic gene expression Environmental
Health Perspectives 112 423ndash427
Unami A Shinohara Y Ichikawa T amp Baba Y (2003) Biochemical
and microarray analyses of bupivacaine-induced apoptosis Journal of
Toxicological Sciences 28 77ndash94
Vasquez A Flammini A Maritan A amp Vespignani A (2003) Global
protein function prediction from proteinndashprotein interaction networks
Nature Biotechnology 21 697ndash700
Vezina C M Walker N J amp Olson J R (2004) Subchronic Exposure to
TCDD PeCDF PCB126 and PCB153 Effect on hepatic gene
expression Environmental Health Perspectives 112 1636ndash1644
Vo T D Greenberg H J amp Palsson B O (2004) Reconstruction and
functional characterization of the human mitochondrial metabolic
network based on proteomic and biochemical data Journal of Bio-
logical Chemistry 279 39532ndash39540
Wang H amp LeCluyse E L (2003) Role of orphan nuclear receptors in
the regulation of drug-metabolising enzymes Clinical Pharmacoki-
netics 42 1331ndash1357
Wang N Silver D L Thiele C amp Tall A R (2001) ATP-
binding cassette transporter A1 (ABCA1) functions as a choles-
terol efflux regulatory protein Journal of Biological Chemistry 276
23742ndash23747
Waring J F Cavet G Jolly R A McDowell J Dai H Ciurlionis R
et al (2003) Development of a DNA Microarray for toxicology based
on hepatoxin-regulated sequences Environmental Health Perspectives
111 863ndash870
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6666
Waring J F Gum R Morfitt D Jolly R A Ciurlionis R Heindel M
et al (2002) Identifying toxic mechanisms using DNA microarrays
Evidence that an experimental inhibitor of cell adhesion molecule
expression signals through the aryl hydrocarbon nuclear receptor
Toxicology 181ndash182 537ndash550
Waring J F Jolly R A Ciurlionis R Lum P Y Praestgaard J T
Morfitt D C et al (2001) Clustering of hepatotoxins based on
mechanism of toxicity using gene expression profiles Toxicology and
Applied Pharmacology 175 28ndash42
Waters M Boorman G Bushel P Cunningham M Irwin R Merrick
A et al (2003) Systems toxicology and the Chemical Effects in
Biological Systems (CEBS) knowledge base EHP Toxicogenomics
111 15ndash28
Wei M Arnold L Cano M amp Cohen S M (2005) Effects of co-
administration of antioxidants and arsenicals on the rat urinary bladder
epithelium Toxicology Sciences 83 237ndash245
Weinshilboum R (2003) Inheritance and drug response New England
Journal of Medicine 348 529ndash537
Weinshilboum R amp Wang L (2004) Pharmacogenetics Inherited
variation in amino acid sequence and altered protein quantity Clinical
Pharmacology and Therapeutics 75 253ndash258
Werner E (2003) In silico multicellular systems biology and minimal
genomes Drug Discovery Today 8 1121ndash1127
Wong J S amp Gill S S (2002) Gene expression changes induced in
mouse liver by di(2-ethylhexyl) phthalate Toxicology and Applied
Pharmacology 185 180ndash196
Xie T Tong L McCann U D Yuan J Becker K G Mechan A O
et al (2004) Identification and characterization of metallothionein-1
and -2 gene expression in the context of ()34-methylenedioxymetham-
phetamine-induced toxicity to brain dopaminergic neurons Journal of
Neuroscience 24 7043ndash7050
Yadetie F Laegreid A Bakke I Kusnierczyk W Komorowski J
Waldum H L et al (2003) Liver gene expression in rats in response
to the peroxisome proliferator-activated receptor-alpha agonist ciprofi-
brate Physiological Genomics 15 9ndash19
Yamashita S Nomoto T Abe M Tatematsu M Sugimura T amp
Ushijima T (2004) Persistence of gene expression changes in stomach
mucosae induced by short-term N-methyl-NV-nitro-N-nitrosoguanidinetreatment and their presence in stomach cancers Mutation Research
549 185ndash193
Yan Q amp Sadee W (2000) Human membrane transporter database A
web-accessible relational database for drug transport studies and
pharmacogenomics AAPS Pharmsci 2 E20
Yeger-Lotem E amp Margalit H (2003) Detection of regulatory circuits by
integrating the cellular networks of proteinndashprotein interactions and
transcription regulation Nucleic Acids Research 31 6053ndash6061
Yoon B I Li G -X Kitada K Kawasaki Y Igarashi K amp Kodama
Y et al (2003) Mechanisms of benzene-induced hematotoxicity and
leukemogenicity cDNA microarray analyses using mouse bone marrow
tissue Environmental Health Perspectives 111 1411ndash1420
Young M B DiSilvestro M R Sendera T J Freund J Kriete A amp
Magnuson S R (2003) Analysis of gene expression in carbon
tetrachloride-treated rat livers using a novel bioarray technology
Pharmacogenomics Journal 3 41ndash52
Yu H Zhu X Greenbaum D Karro J amp Gerstein M (2004) TopNet
A tool for comparing biological sub-networks correlating protein
properties with topological statistics Nucleic Acids Research 32
328ndash337
Zhang E Y Fu D J Pak Y A Stewart T Mukhopadhyay N
Wrighton S A et al (2004) Genetic polymorphisms in human proton-
dependent dipeptide transporter PEPT1 Implications for the functional
role of Pro586 Journal of Pharmacology and Experimental Therapeu-
tics 310 437ndash445
Zhang E Y Knipp G T Ekins S amp Swaan P W (2002)
Structural biology and function of solute transporters Implications
for identifying and designing substrates Drug Metabolism Reviews
34 709ndash750
Zhang E Y Phelps M A Cheng C Ekins S amp Swaan P W (2002)
Modeling of active transport systems Advanced Drug Delivery
Reviews 54 329ndash354
- Systems-ADMETox Resources and network approaches
-
- Introduction
- Data available
- Network analysis and databases
-
- Network applications
-
- The role of transporters
-
- Clinical relevance of transporters
- Transporter network examples ABCA1
- Transporter network examples OATP
- Transporter microarray data
-
- Applications to enzymes
- Future network applications
-
- Discussion
- Acknowledgements
- References
-
Table 5
Datasets acquired from publications and other sources and used to evaluate two databasepathway tools KEGG and metaCore
Dataset Number of objects
genes proteins in list
Number of objects
mapped with KEGG
Number of objects mapped
with MetaCore on networks
Reference for microarray
data gene list
Tox Chip 10 (NIEHS) 1853 405 1498 httpsdir-appsniehsnihgov
mapsguestclonesrchcfm
4-Hydroxytamoxifen and
estrogen
1617 434 1343 (Hodges et al 2003)
Mitochondrial proteins 722 156 388 (Gaucher et al 2004
Taylor et al 2003)
Bromobenzene
(24 and 48 h)
130 41 89 (Heijne et al 2004)
Acetaminophen 30 19 23 (Heinloth et al 2004)
Acetaminophen 84 29 64 (Huang et al 2004)
Furan 185 64 139 (Huang et al 2004)
Tetrodotoxin 116 31 86 (Raghavendra Prasad et al
2004)
Benzene 73 16 62 (Yoon et al 2003)
Benzene 76 5 53 (Faiola et al 2004)
Carbon tetrachloride 37 8 26 (Young et al 2003)
Estrogen 94 33 90 (Lobenhofer et al 2002)
Trovafloxacin 142 20 82 (Liguori et al 2005)
Phenobarbital 37 13 28 (Ueda et al 2002)
L-742694 (liver)+ 45 17 19 (Hartley et al 2004)
L-742694 (intestine)+ 23 11 10 (Hartley et al 2004)
A-277249 21 7 9 (Waring et al 2002)
All files were converted to Locuslink identifiers except as noted () unigene () Affymetrix (+) Genbank identifiers were used
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 51
proteins across cellular membranes These may function as
passive processes or active processes energized by the
hydrolysis of ATP or coupling to the co-transport of counter
ions down an electrochemical gradient such as Na+ H+ and
Cl There are many thousands of transporters which can be
classified into distinct superfamilies One of these the solute
carrier class (SLC) is rapidly expanding and contains over
30 families and 200 members The ATP-binding cassette
(ABC) contains 7 families and over 48 members including
P-glycoprotein (P-gp) and MRP subfamilies (Zhang Knipp
Ekins amp Swaan 2002a Zhang Phelps Cheng Ekins amp
Swaan 2002b) Transporters have a key role in clinical
pharmacology with many drugs specifically targeting them
Numerous drugs share transport pathways with nutrients
and transporters have a role in oral absorption drug
bioavailability drug resistance excretion and ultimately
pharmacokinetics and pharmacodynamics
Polymorphism of drug transporters may be a key factor
in drug interactions and lack of effectiveness This field has
become known as pharmacogenomics and is focused on
understanding of inherited DNA sequence variations (poly-
morphisms and mutations) revealed by xenobiotics (Evans
amp McLeod 2003 Weinshilboum 2003) Over the last few
decades many genes have been directly linked to the
mechanisms of response (Evans amp McLeod 2003 Wein-
shilboum 2003) such that 20ndash95 of variability to drug
response is inheritable (Evans amp McLeod 2003) This
phenotypical variability is mainly caused by single nucleo-
tide polymorphisms (SNPs) present in anywhere from 1 to
50 of the population resulting in either lower protein
activity incorrect folding or rapid degradation via proteo-
somes (Weinshilboum amp Wang 2004) A number of
structurally diverse molecules bind to P-gp which is
expressed in many tissues and has numerous SNPs one of
which (C3435T) affects the expression level in the
duodenum and therefore can impact the absorption of
molecules which would be substrates for this transporter
(Sakaeda Nakamura amp Okumura 2002) The human
proton-dependent dipeptide transporter (hPEPT1) can also
affect the absorption of molecules in the intestine and
recently 9 SNPs were found with only one displaying a
reduced transport capacity (Zhang et al 2004) The sodium-
dependent carnitine cotransporter OCTN2 can possess
mutations and these result in primary carnitine deficiency
which impacts fatty acid oxidation and is characterized by
many clinical manifestations (Lahjouji Mitchel amp Qureshi
2001) The organic cation transporter 1 (OCT1) is also
important in the transport of numerous xenobiotics and
endobiotics Recently 4 SNPs were identified in the
Japanese population and when functionally characterized
in vitro the uptake of cations was reduced significantly for
some of these mutations indicating that this would likely
contribute to inter-individual variations in metabolism of
drugs which were transported via OCT1 (Sakata et al
2004)
3111 Clinical relevance of transporters The pregnane
X-receptor (PXR) is a transcriptional regulator of the
enzyme human MDR1 (P-gp) MRPs and OATP (Wang amp
LeCluyse 2003) CYP3A (Bertilsson et al 1998 Blumberg
et al 1998 Kliewer et al 1998) and CYP2C89 (Synold
Dussault amp Forman 2001) as well as many other genes
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6652
involved in the transport metabolism and biosynthesis of
bile acids (Staudinger Liu Madan Habeebu amp Klaassen
2001) However the additional receptors CAR FXR LXR
and other nuclear receptors take part in a complex network
of interactions to control these and other proteins Thus
elucidation of the regulatory networks which control the
expression of these transporters is also important To date
most of the research has centered on efflux transporters but
there has been considerable interest in uptake transporters
such as the organic anion transporter polypeptide (OATP
((Kim 2003) see also later Section on OATP)
There are several specific examples of the importance of
drug transporters to the clinical development of drugs One
example is the insulin sensitizer troglitazone which was
withdrawn due to hepatotoxicity although the precise
mechanism appears to have been unclear until recently
The major metabolite is a sulfated species and is suspected
of being responsible for the observed toxicity The recent
assessment of the organic anion transporting polypeptides
OATP-C and OATP8 expressed on the hepatocyte baso-
lateral membrane indicated that sulphated troglitazone has a
high affinity for the former and possibly lower affinity for
the latter (Nozawa et al 2004) This metabolite would
therefore be expected to accumulate in hepatocytes and
inhibit the bile salt export pump and Mrp2 Because
polymorphisms have been shown for OATP-C (Tirona
Leake Merino amp Kim 2001) it is also possible that these
may result in the accumulation of the metabolite and in turn
elicit idiosyncratic toxicity A second relevant example of
the impact of transporters is the clinically significant drugndash
drug interaction between cerivastatin and cyclosporine A
which occurs via the OATP-C transporter (Shitara Itoh
Sato Li amp Sugiyama 2003) A third example are the HIV
protease inhibitors saquinavir ritonavir and indinavir which
are transported by MRP2 in vitro and other drugs such as
probenecid and sulfinpyrazone are able to enhance this
transport Transport by MRP2 suggests that these com-
pounds will have decreased bioavailability due to increased
clearance and other drugs could aggravate this situation by
further enhancing transport (Huisman et al 2002) Sim-
ilarly the rifampicin mediated induction via PXR of MRP2
and P-gp in healthy subjects was found to significantly
decrease the AUC and also correlated with intestinal
expression of these transporters This transporter is also
inducible by cisplatin 2-AAF and phenobarbital (Schrenk
et al 2001) indicating multiple mechanisms may be
involved In other species such as rat commonly used as a
toxicity model orthologs of the transporters such as OATP2
are expressed and can be induced with ligands for PXR like
PCN (Guo Choudhuri amp Klaassen 2002) This is useful
knowledge because the advent of microarray technology
allows one to dose a rat with a xenobiotic and assess
thousands of genes simultaneously in a particular tissue For
instance animals dosed with known nephrotoxins have
shown some upregulation of the NandashKndashCl transporter
however the authors suggested genomic responses are
stronger soon after exposure before declining (Fleck et
al 2003) Some transporters may therefore be specifically
targeted by drugs in one tissue such as the CNS but these
same transporters may also be expressed elsewhere in the
body hence off-target effects may result in toxicity The
serotonin transporter is one such example which is
expressed in the lungs and brain Some substrates for this
channel like fenfluramine can result in primary pulmonary
hypertension as they accumulate in pulmonary cells (Roth-
man Ayestas Dersch amp Baumann 1999) Similarly P-gp is
expressed at the blood brain barrier and intestine impacting
the efficacy and bioavailability of drugs
3112 Transporter network examples ABCA1 The
ABCA1 transporter mediates the first step of cholesterol
transport Mutations in this gene cause Tangier disease
which results in severe HDL deficiency cholesterol
accumulation in macrophages and attendant atherosclerosis
This transporter represents a drug target for upregulation
modulating cholesterol metabolism and prevention of
cardiovascular disease (Oram amp Lawn 2001) In vitro
ABCA1 can be inhibited by the sulfonylurea glybenclamide
(Field Burn amp Mathur 2004 Wang Silver Thiele amp Tall
2001) In order to illustrate the advantages of mapping drug
transporters as networks onto functional models alongside
other proteins one can consider the example of ABCA1
The query of the MetaCorei database shows that this
transporter appears on three manually curated pathway maps
representing the Fstate of the art_ knowledge derived from
reliable high quality literature sources One can also use the
individual maps as an interface to access the underlying
layers of information about the transporter including the list
of encoding genessplice variants with known SNPs In
addition to browsing MetaCorei a user can also build
custom networks around ABCA1 using the network-
construction tool (Fig 5) Such a visualization utility may
be very helpful for identification of all putative pathways
around a particular transporter or compound of interest The
ABCA1 network created by this tool shows that this
transporter at the time of writing is linked directly to
twenty-five other objects such as APOE1 and LXR Many
of its neighbors have their own SNPs that could be
important in determining interactions between transport of
a drug and normal human transport of endogenous ligands
in health or disease
3113 Transporter network examples OATP The
OATPs are key membrane bound transporters expressed in
many organs including intestine liver lung choroid plexus
blood brain barrier and other organs (Tamai et al 2000)
This family of transporters is capable of mediating the
sodium-independent transport of a diverse array of mole-
cules such as steroid conjugates organic anions and
xenobiotics by coupling uptake with efflux of bicarbonate
(Satlin Amin amp Wolkoff 1997) glutathione or its
conjugates (Hagenbuch amp Meier 2004) The inhibition of
Fig 5 The network of direct gene interactions around the ABCA1 transporter gene (centre) generated with MetaCorei The interation types between nodes are shown as small colored hexagons eg unspecified
allosteric regulation binding cleavage competition covalent modification dephosphorylation phosphorylation transcription regulation transformation When applicable interactions also have a positive or
negative effect and direction Ligands (purple) linked to other proteins (blue blobs) transfactors (red) enzymes (orange) (For interpretation of the references to colour in this figure legend the reader is referred to
the web version of this article)
SEkin
sJournalofPharm
acologica
landToxico
logica
lMeth
ods53(2006)38ndash66
53
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6654
this transporterrsquos hepatic uptake of other compounds may be
important for reported drugndashdrug interactions (Kim 2003)
described earlier (Shitara et al 2003) as well as cerivastatin
with gemfibrozil (Shitara Hirano Sato amp Sugiyama 2004)
OATP1B1 (previous names OATP-C LST-1 OATP2
SLC21A6) represents the most studied human OATP to
date (Meier amp Stieger 2000) and is expressed on the
basolateral plasma membrane of hepatocytes Several single
nucleotide polymorphisms have been identified in the
OATP1B1 gene in European-Americans African-Americans
(Tirona et al 2001) and Japanese (Nozawa et al 2002)
dramatically impacting the transport of ligands such as
pravastatin (Mwinyi Johne Bauer Roots amp Gerloff 2004
Nishizato et al 2003) estrone-3-sulfate (Nozawa et al
2002 Tirona et al 2001) Rifampin (Tirona Leake
Wolkoff amp Kim 2003) and estradiol 17h-d-glucuronide(Tirona et al 2001)
The regulation of SLCO may be affected during
extrahepatic cholestasis bile duct ligation bile salt induced
Fig 6 Networks generated with the autoexpand algorithm in MetaCorei to illustr
be interconnected with other protein regulatory signaling information Informa
unspecified allosteric regulation binding cleavage competition covalent m
transformation When applicable interactions also have a positive or negative e
transfactors (red) enzymes (orange) (For interpretation of the references to colour
cholestatic hepatitis (Dumont et al 1997 Meier amp Stieger
2000 Rost et al 2003) and primary sclerosing cholangitis
(Oswald Kullack-Ublick Paumgarter amp Beuer 2001) In
particular OATP1B1 appears to be regulated by the liver-
enriched transcription factor hepatocyte nuclear factor 1a
(HNF1a) which binds to the promoter region of this
transporter (Jung et al 2001) Site directed mutagenesis
of this binding site resulted in inactivation suggesting the
critical nature of the interaction with HNF1a Bile acids
such as CDCA have been shown to transcriptionally repress
HNF1a in vitro via inhibition of the transactivating effect of
HNF4a on HNF1a (Jung amp Kullak-Ublick 2003) After
screening many rat and human uptake transporters in vitro
OATP1B1 was also shown to modulate the PXR response
by controlling rifampin retention in the cell and therefore
affecting the induction of CYP3A4 and other gene products
such as P-gp (Tirona et al 2003)
Some of the literature for OATP1B1 human substrate
data has been annotated into MetaCorei to illustrate the
ate how ligands for the human OATP-C (OATP1B1) transporter (centre) can
tion on the type of interaction between objects is hidden for clarity eg
odification dephosphorylation phosphorylation transcription regulation
ffect and direction Ligands (purple) linked to other proteins (blue blobs)
in this figure legend the reader is referred to the web version of this article)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 55
visualization of the complex interconnections between this
transporter its ligands regulatory factors and signaling
molecules already in the database (Fig 6) The network was
generated with the autoexpand algorithm in the software
representing one of multiple available algorithms for
connecting genes ligands and other objects in the database
Clearly if more ligands and their connections are added to
the database the complexity of the network will increase
considerably The OATP-C (OATP1B1) gene details can be
viewed upon querying the database and links are provided
to other public databases This page can be used to highlight
the multiple synonyms for this gene as well as links to the
multiple SNPs identified to date
3114 Transporter microarray data Microarrays have
generally been limited in the number of transporters present
on them (Annereau et al 2004) however they have been used
in an attempt to correlate pharmacokinetic properties with
gene expression for valacyclovir (Landowski et al 2003) as
well as understand the expression profile in different tissues
or cell lines upon food component or xenobiotic treatment
(Anderle Huang amp Sadee 2004) This lack of transporters
on microarrays has prompted some groups to produce their
own arrays with a heavier emphasis on transporters These
arrays have for example then been used to demonstrate the
upregulation of ABC transporters and down-regulation of
GST-Pi in cell lines resistant to colchicines or 9-nitro-
camptothecin (Annereau et al 2004) The genes that were
significantly up or down-regulated in this particular study
were used to build networks with MetaCore (Fig 7A B) and
the similarities between them were assessed (Fig 7C)
Although there were only a small number of significantly
changed genes in common (IL-8 Fos GST-Pi Calpactin and
Ubiquitin hydrolase) it is perhaps likely that there is a much
larger common gene network that is important for drug
resistance although a much larger number of cell lines and
drug treatments need to be evaluated to produce a definitive
drug resistance signature involving transporters enzymes
and transcriptional regulators
312 Applications to enzymes
As we have already described it may be particularly
valuable to visualize enzymes as networks to show
interactions with transcriptional regulators and ligands
For example a key enzyme is CYP3A4 which metabolizes
40ndash45 of all drugs and has relatively few SNPs
(Ingelman-Sundberg 2004) Using a second software suite
MetaDrugi (wwwgenegocom) it is possible to construct
a custom network around this or other drug metabolizing
enzymes (Fig 8) In this case the gene network for
CYP3A4 highlights all of the major transcriptional
regulators and several more distant linked proteins and
ligands connected on the network that may be useful for
further study (Fig 8) Substrates inhibitors as well as
regulatory factors and other enzymes can be observed
connected on this network Due to the many hundreds of
known inhibitors and substrates only a small number are
shown here for clarity We assume that if a perturbation in
a pathway (eg due to a nonfunctional enzyme) is linked
to a certain pathologic condition a similar perturbation
caused by the interference from xenobiotic metabolism
(eg competitive inhibition of the same enzyme) may
result in identical effects As microarray gene expression
data is increasingly generated the role of enzyme
regulation in toxicity of certain xenobiotics will become
more apparent from either in vivo or in vitro studies The
visualization of such signature gene networks involving
transporters and enzymes their ligands and regulatory
factors will also be important for future toxicity prediction
methods We have recently generated visualizations of
microarray data from MCF-7 cells treated with 4-hydrox-
ytamoxifen to show that some of the key genes involved in
metabolism and transport are upregulated (Ekins et al
2005d) In addition we have made predictions with various
QSAR models in MetaDrug to indicate the involvement of
PXR CYP3A4 and P-gp (Ekins et al 2005e) Therefore it
appears likely that 4-hydroxytamoxifen could increase its
own metabolism as well as efflux from cells via P-gp
which can be visualized on networks Any decrease of
function of these enzymes or transporters in a population
would likely result in changes in the metabolism and
transport of this active metabolite potentially impacting
the clinical effect This represents one example of how
both pathway tools QSAR models and network building
algorithms can be used with different types of predicted
and experimental data to allow visualization of potential
compound interactions or toxicity
313 Future network applications
As the population ages an increasing incidence and
prevalence of systemic diseases especially chronic diseases
have occurred among older adults This has resulted in an
increase in medications used concomitantly by this
population which presents challenges for drugndashdrug
interactions Physiologically elderly patients may behave
differently to the younger patients for which the drugs are
initially developed for and tested on Many pharmacoki-
netic investigations in the elderly population reveal
decreased clearance of lipophilic drugs metabolized by
the cytochrome P450 enzymes however few studies have
evaluated aging-dependent or gender-related changes in
specific P450 enzymes (Hunt Westerkam amp Slave 1992)
Age-related physiological changes such as a reduction in
liver mass hepatic metabolizing enzyme activity liver
blood flow and alterations in plasma drug binding may
account for the decreased elimination of some metabolized
drugs in the elderly It is particularly difficult to separate an
effect of aging from the variation in the rate of metabolism
due to factors such as individual metabolic phenotype
(slow or fast metabolizer due to SNPs) environmental
influences concomitant disease states and drug intake
(drugndashdrug interactions) The available data suggest that
Fig 7 Networks for drug resistant cell lines generated with the Auto expand algorithm in MetaCore A KB-3-1 vs KB-8-5 B ATC-DU-145 vs RCO1 C node overlap of A and B with initial nodes highlighted
with blue or red solid circles (Initial gene lists derived from (Annereau et al 2004)) Information on the type of interaction between objects is hidden for clarity eg unspecified allosteric regulation binding
cleavage competition covalent modification dephosphorylation phosphorylation transcription regulation transformation When applicable interactions also have a positive or negative effect and direction
Ligands (purple) linked to other proteins (blue blobs) transfactors (red) enzymes (orange) (For interpretation of the references to colour in this figure legend the reader is referred to the web version of this article)
SEkin
sJournalofPharm
acologica
landToxico
logica
lMeth
ods53(2006)38ndash66
56
Fig7(continued)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 57
Fig7(continued)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6658
Fig 8 The network of gene interactions around the CYP3A4 enzyme (centre) from MetaCorei Information on the type of interaction between objects is
hidden for clarity eg unspecified allosteric regulation binding cleavage competition covalent modification dephosphorylation phosphorylation
transcription regulation transformation When applicable interactions also have a positive or negative effect and direction Ligands (purple) linked to other
proteins (blue blobs) transfactors (red) enzymes (orange) (For interpretation of the references to colour in this figure legend the reader is referred to the web
version of this article)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 59
the initial doses of drugs metabolized by these enzymes
should be reduced in older patients according to the clinical
response In most published studies the elderly appear at
least as responsive as the young to inducers or inhibitors of
P450s (Durnas Loi amp Cusack 1990) More recently there
has been some suggestion that there are age related
reductions in function of some specific P450s such as
CYP3A4 (Patki et al 2004) and this could occur at the
level of regulation However earlier studies with the same
enzyme showed no change in clearance with age (Hunt et
al 1992) To date there has been even less examination of
the transporter functions and any changes with age
(Kinirons amp OrsquoMahony 2004) so the current understand-
ing of the effects of aging on metabolism and transport is
anything but transparent This represents an extreme
challenge for the pharmaceutical industry how to predict
whether a drug has an affinity for an enzyme or transporter
and whether this may be clinically important if it shows a
decline in expression or function with aging Any decrease
of function of these enzymes or transporters in an elderly
population would result in changes in the metabolism and
transport of metabolites potentially impacting the clinical
effect This represents another example of how computa-
tional approaches may perhaps be used with different types
of predicted and experimental data to allow visualization of
potential compound interactions or toxicity in elderly
populations We may see different gene networks high-
lighted as humans age and these may be modified by drug
treatment and coadministration This represents an area
were network analysis could be applied in the future and is
in need of considerable further research
The collection of microarray data in databases such as
CEBS ArrayTrack and EDGE represents a future resource
for computational gene network analysis One could
envisage that ultimately in each case such data is converted
to one or more networks that are also displayed for the user
and can be used to compare treatments from in vivo and in
vitro experiments This would represent a different approach
to clustering the data as currently implemented in one of
these efforts (Hayes et al 2005) and may condense large
amounts of experimentally derived data into a readily
interpreted network
4 Discussion
Previously in this journal the progress of many
research groups in predicting human ADME parameters
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6660
in silico (Ekins et al 2000b) and approaches for drug
metabolism (Ekins Ring Grace McRobie-Belle amp
Wrighton 2000a) have been described Both of these
reviews commented on moving HT assays for ADME
Tox much earlier in drug discovery which had also been
indicated by other groups The initial reviews also
highlighted the likely wealth of data that would become
available and how this could be used for structure
activity relationships alongside the bioactivity data in
computational models It was noted that there was a
paucity of predictive metabolism tools at that time In
addition a growing number of efforts to model whole
cells and organs now a field called systems biology
were recognized as models that could be integrated with
the in silico ADME approaches In summary since these
past reviews virtually all pharmaceutical companies have
attempted earlier high throughput screening for ADME
Tox properties and to some extent the wider application
of computational approaches for physicochemical proper-
ties Systems biology is being quite widely acknowledged
as the new paradigm for understanding complex bio-
logical datasets derived from high throughput technolo-
gies and the accumulated knowledge on human protein
interactions (Hartwell Hopfield Leibler amp Murray 1999
Hood 2003b) Therefore systems biology can be defined
as the integration of genetic proteomic transcriptomic
and metabonomic data using computational methods
(Nicholson amp Wilson 2003) When taken together
information on molecular processes derived from different
sources represents a lsquolsquouniversersquorsquo of putative biological
functionality of which only a small fraction of it will be
realized in a cell at any given time To date systems
biology has been driven by academia and funding bodies
such as the NIH rather than the pharmaceutical compa-
nies Presently there is a great deal of interest from
scientists of all backgrounds in identifying the networks
of cellular pathways and the corresponding physically
interacting proteins
The network building software for systems biology
described in this current review will be valuable to query
high throughput data and known literature interactions in
order to predict potential toxicity in different species In the
future the compilation of published toxicogenomics data-
sets characteristic of different types of toxicity will likely
be available in these software systems to act as a reference
database It is also feasible that we will be able to generate
the annotated datasets which specifically address the
differences between human and rat networks implicated
in toxicity The identification of sub-network modules
conserved between human and rat distinct for toxicity
types or predictive for toxic end-points in human will be
possible Such signature gene networks (Nikolsky et al
2005) can then be verified with other experimentally
derived data prospectively or from preexisting databases
The combined Fhigh throughput datandashin silico network_approaches therefore represents a novel method for access-
ing complex tissue-level processes as networks integrating
all data types based on functional interactions The data-
bases developed and used in this approach will certainly
benefit from further annotation around the drug metaboliz-
ing enzymes and transporters as described herein in terms
of transcriptional regulation and the ligands associated with
them which frequently appear in the literature It will be
important to capture disparities in the assignment of ligands
to enzymes transporters and other proteins as well as
negative data
In summary although there have been numerous
toxicogenomics studies published there is presently a
relatively small number of datasets that are freely available
to perform network analysis of microarray data The
number of studies identifying large numbers of proteins
which are affected by molecule treatment are even scarcer
still while there are several examples of some published
studies that combine such data It is hoped that the
numerous database initiatives for high content and toxicol-
ogy data that are being undertaken will improve the
situation for other researchers that are not currently
equipped to do such microarray studies themselves The
addition of requirements by journals to deposit such raw
data in a freely accessible resource will aid these initiatives
ADMETox groups have seen new technologies and
approaches developed over the last decade that have all
been applied to identify poor compounds earlier (Fig 2)
The latest technologies integrate network building tools
with high content data and databases The current review
described the limited number of networks generated for
ADMETox at present and one hopes that the impact of
such analyses will be commonplace in the future Systems
biology is however more than just applying a network
approach and hence systems-ADMETox will have to
evolve due to the continual pressure to develop newer
technologies This current paradigm combining empirical
data and computational methods should integrate the
complex data already generated making it readily inter-
pretable and valuable for identifying the most promising
compounds in the future
Acknowledgements
Dr Maggie AZ Hupcey is gratefully acknowledged for
editorial assistance Dr Peter W Swaan (University of
Maryland) and Dr Cheng Chang (Ohio State University)
Dr Steve Wright (University of Arizona) Dr K Sandy
Pang (University of Toronto) and Dr Craig Giroux (Wayne
State University) are kindly acknowledged for their support
and discussions My colleagues at GeneGo Sergey
Andreyev Andy Ryabov Eugene Kirillov Eugene A
Rakhmatulin Svetlana Sorokina Andrej Bugrim Tatiana
Nikolskaya Yuri Nikolsky John Metz and Julie Bryant are
all thanked for their considerable contributions to software
development and data annotation
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 61
References
Abe M Yamashita S Kuramoto T Hirayama Y Tsukamoto T Ohta
T et al (2003) Global expression analysis of N-methyl-NV-nitro-N-nitrosoguanidine-induced rat stomach carcinomas using oligonucleotide
microarrays Carcinogenesis 24 861ndash867
Adachi T Ono Y Koh K B Takashima K Tainaka H Matsuno Y
et al (2004) Long-term alteration of gene expression without
morphological change in testis after neonatal exposure to genistein in
mice Toxicogenomic analysis using cDNA microarray Food and
Chemical Toxicology 42 445ndash452
Akerman G S Rosenzweig B A Domon O E McGarrity L J
Blankenship L R Tsai C A et al (2004) Gene expression profiles
and genetic damage in benzo(a)pyrene diol epoxide-exposed TK6 cells
Mutation Research 549 43ndash64
Aksenov S V Church B Dhiman A Georgieva A Sarangapani R
Helmlinger G et al (2005) An integrated approach for inference and
mechanistic modeling for advancing drug development FEBS Letters
579 1878ndash1883
Anderle P Huang Y amp Sadee W (2004) Intestinal membrane transport
of drugs and nutrients Genomics of membrane transporters using
expression microarrays European Journal of Pharmaceutical Sciences
21 17ndash24
Annereau J P Szakacs G Tucker C J Arciello A Cardarelli C
Collins J et al (2004) Analysis of ATP-binding cassette transporter
expression in drug-selected cell lines by a microarray dedicated to
multidrug resistance Molecular Pharmacology 66 1397ndash1405
Apic G Ignjatovic T Boyer S amp Russell R B (2005) Illuminating drug
discovery with biological pathways FEBS Letters 579 1872ndash1877
Baker V A Harries H M Waring J F Duggan C M Ni H A Jolly
R A et al (2004) Clofibrate-induced gene expression changes in rat
liver A cross-laboratory analysis using membrane cDNA arrays
Environmental Health Perspectives 112 428ndash438
Balakin K V Ekins S Bugrim A Ivanenkov Y A Korolev D amp
Nikolsky Y et al (2004a) Quantitative structurendashmetabolism relation-
ship modeling of the metabolic N-dealkylation rates Drug Metabolism
and Disposition 32 1111ndash1120
Balakin K V Ekins S Bugrim A Ivanenkov Y A Korolev D amp
Nikolsky Y et al (2004b) Kohonen maps for prediction of binding to
human cytochrome P450 3A4 Drug Metabolism and Disposition 32
1183ndash1189
Barabasi A -L amp Oltvai Z N (2004) Network biology Under-
standing the cellrsquos functional organization Nature Reviews Genetics
5 101ndash113
Bartosiewicz M Penn S G amp Buckpitt A (2001) Applications of gene
arrays in environmental toxicology Fingerprints of gene regulation
associated with cadmium chloride benzo(a)pyrene and trichloro-
ethylene Environmental Health Perspectives 109 71ndash74
Bertilsson G Heidrich J Svensson K Asman M Jendeberg L
Sydow-Backman M et al (1998) Identification of a human nuclear
receptor defines a new signaling pathway for CYP3A induction
Proceedings of the National Academy of Sciences of the United States
of America 95 12208ndash12213
Blumberg B Sabbagh Jr W Juguilon H Bolado Jr J van Meter
C M Ong E S et al (1998) SXR a novel steroid and xenobiotic-
sensing nuclear receptor Genes and Development 12 3195ndash3205
Borodina Y Rudik A Filimonov D Kharchevnikova N Dmitriev A
Blinova V et al (2004) A new statistical approach to predicting
aromatic hydroxylation sites Comparison with model-based
approaches Journal of Chemical Information and Computer Sciences
44 1998ndash2009
Borodina Y Sadym A Filimonov D Blinova V Dmitriev A amp
Poroikov V (2003) Predicting biotransformation potential from
molecular structure Journal of Chemical Information and Computer
Sciences 43 1636ndash1646
Bottone Jr F G Martinez J M Collins J B Afshari C A amp Eling
T E (2003) Gene modulation by the cyclooxygenase inhibitor
sulindac sulfide in human colorectal carcinoma cells Possible link to
apoptosis Journal of Biological Chemistry 278 25790ndash25801
Boyer S amp Zamora I (2002) New methods in predictive metabolism
Journal of Computer-Aided Molecular Design 16 403ndash413
Bredel M amp Jacoby E (2004) Chemogenomics An emerging strategy
for rapid target and drug discovery Nature Reviews Genetics 5
262ndash275
Bugrim A Nikolskaya T amp Nikolsky Y (2004) Early prediction of drug
metabolism and toxicity Systems biology approach and modeling
Drug Discovery Today 9 127ndash135
Bulera S J Eddy S M Ferguson E Jatkoe T A Reindel J F
Bleavins M R et al (2001) RNA expression in the early character-
ization of hepatotoxicants in Wistar rats by high-density DNA micro-
arrays Hepatology 33 1239ndash1258
Butcher E C Berg E L amp Kunkel E J (2004) Systems biology in drug
discovery Nature Biotechnology 22 1253ndash1259
Butte A (2002) The use and analysis of microarray data Naturalist
Review of Drug Discovery 1 951ndash960
Cary M P Bader G D amp Sander C (2005) Pathway information for
systems biology FEBS Letters 579 1815ndash1820
Cheok M H Yang W Pui C H Downing J R Cheng C Naeve
C W et al (2003) Treatment-specific changes in gene expression
discriminate in vivo drug response in human leukemia cells Nature
Genetics 34 85ndash90
Cleary J D Rogers P D amp Chapman S W (2001) Differential
transcription factor expression in human mononuclear cells in response
to amphotericin B Identification with complementary DNA microarray
technology Pharmacotherapy 21 1046ndash1054
Coen M Ruepp S U Lindon J C Nicholson J K Pognan F Lenz
E M et al (2004) Integrated application of transcriptomics and
metabonomics yields new insight into the toxicity due to paracetamol in
the mouse Journal of Pharmaceutical and Biomedical Analysis 35
93ndash105
Cornwell P D De Souza A T amp Ulrich R G (2004) Profiling of
hepatic gene expression in rats treated with fibric acid analogs
Mutation Research 549 131ndash145
Csermely P Agoston V amp Pongor S (2005) The efficiency of multi-
target drugs The network approach might help drug design Trends in
Pharmacological Sciences 26 178ndash182
Cunningham M J Liang S Fuhrman S Seilhamer J J amp
Somogyi R (2000) Gene expression microarray data analysis for
toxicology profiling Annals of the New York Academy of Sciences 919
52ndash67
de Longueville F Atienzar F A Marcq L Dufrane S Evrard S
Wouters L et al (2003) Use of a low density microarray for studying
gene expression patterns induced by hepatotoxicants on primary
cultures of rat hepatocytes Toxicological Sciences 75 378ndash392
Dobrin R Beg Q K Barabasi A L amp Oltvai Z N (2004)
Aggregation of topological motifs in the Escherichia coli transcriptional
regulatory network BMC Bioinformatics 5 10
Donald S Verschoyle R D Edwards R Judah D J Davies R
Riley J et al (2002) Hepatobiliary damage and changes in hepatic
gene expression caused by the antitumor drug ecteinascidin-743 (ET-
743) in the female rat Cancer Research 62 4256ndash4262
Dumont M Jacquemin E DrsquoHont C Descout C Cresteil D Haouzi
D et al (1997) Expression of the liver Na+-independent organic anion
transporting polypeptide (oatp-1) in rats with bile duct ligation Journal
of Hepatology 27 1051ndash1056
Durnas C Loi C M amp Cusack B J (1990) Hepatic drug metabolism
and aging Clinical Pharmacokinetics 19 359ndash389
Eisen M B Spellman P T Brown P O amp Botstein D (1998) Cluster
analysis and display of genome-wide expression patterns Proceedings
of the National Academy of Sciences of the United States of America
95 14863ndash14868
Ekins S Andreyev S Ryabov A Kirilov E Rakhmatulin E A
Bugrim A et al (in press) Computational Prediction of Human Drug
Metabolism Exp Opin Drug Metab Toxicol
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6662
Ekins S Boulanger B Swaan P W amp Hupcey M A Z (2002)
Towards a new age of virtual ADMETOX and multidimensional drug
discovery Journal of Computer-Aided Molecular Design 16 381ndash401
Ekins S Bugrim A Nikolsky Y amp Nikolskaya T (2005) Systems
biology Applications in drug discovery In S Gad (Ed) Drug
Discovery Handbook (pp 123ndash183) Hoboken NJrsquo Wiley and Sons
Ekins S Giroux C N Nikolsky Y Bugrim A amp Nikolskaya T
(2005) A signature gene network approach to toxicity The Toxoco-
logists 84
Ekins S Kirillov E Rakhmatulin E amp Nikolskaya T (2005) A novel
method for visualizing nuclear hormone receptor networks relevant to
drug metabolism Drug Metabolism and Disposition 33 474ndash481
Ekins S amp McGowan R J (2001) The limits of reductionism The
shifting genomic paradigmrsquos impact on industry and academia
Philsophy in Science 9 1ndash23
Ekins S Mirny L amp Schuetz E G (2002) A ligand-based approach to
understanding selectivity of nuclear hormone receptors PXR CAR
FXR LXRa and LXRb Pharmaceutical Research 19 1788ndash1800
Ekins S Nikolsky Y amp Nikolskaya T (2005) Techniques Application
of systems biology to absorption distribution metabolism excretion
and toxicity Trends in Pharmacological Sciences 26 202ndash209
Ekins S Ring B J Grace J McRobie-Belle D J amp Wrighton S
A (2000) Present and future in vitro approaches for drug
metabolism Journal of Pharmacological and Toxicological Methods
44 313ndash324
Ekins S Waller C L Swaan P W Cruciani G Wrighton S A amp
Wikel J H (2000) Progress in predicting human ADME parameters in
silico Journal of Pharmacological and Toxicological Methods 44
251ndash272
Erhardt P W (2003) A human drug metabolism database Potential roles
in the quantitative predictions of drug metabolism and metabolism-
related drugndashdrug interactions Current Drug Metabolism 4 411ndash422
Evans W E amp McLeod H L (2003) Pharmacogenomicsmdashdrug
disposition drug targets and side effects New England Journal of
Medicine 348 538ndash549
Ezendam J Staedtler F Pennings J Vandebriel R J Pieters R
Boffetta P et al (2004) Toxicogenomics of subchronic hexachlor-
obenzene exposure in Brown Norway rats Environmental Health
Perspectives 112 782ndash791
Faiola B Fuller E S Wong V A amp Recio L (2004) Gene expression
profile in bone marrow and hematopoietic stem cells in mice exposed to
inhaled benzene Mutation Research 549 195ndash212
FDA (2004) Innovation stagnation Challenge and opportunity on the
critical path to new medicinal products
Fiehn O (2001) Combining genomics metabolome analysis and bio-
chemical modelling to understand metabolic networks Comparative
Functional Genomics 2 155ndash168
Field F J Born E amp Mathur S N (2004) LXRRXR ligand activation
enhances basolateral efflux of beta-sitosterol in CaCo-2 cells Journal of
Lipid Research 45 905ndash913
Fleck C Sutter L Appenroth D Koch B Meinhold T Pitack M
et al (2003) Use of gene chip technology for the characterisation of
the regulation of renal transport processes and of nephrotoxicity in
rats Experimental and Toxicologic Pathology 54 401ndash410
Fliri A F Loging W T Thadeio P F amp Volkmann R A (2005)
Biological spectra analysis Linking biological activity profiles to
molecular structure Proceedings of the National Academy of Sciences
of the United States of America 102 261ndash266
Fountoulakis M (2004) Application of proteomics technologies in the
investigation of the brain Mass Spectrometry Reviews 23 231ndash258
Gaucher S P Taylor S W Fahy E Zhang B Warnock D E Ghosh
S S et al (2004) Expanded coverage of the human heart
mitochondrial proteome using multidimensional liquid chromatography
coupled with tandem mass spectrometry Journal of Proteome
Research 3 495ndash505
Gerhold D Lu M Xu J Austin C Caskey C T amp Rushmore T
(2001) Monitoring expression of genes involved in drug metabolism
and toxicology using DNA microarrays Physiological Genomics 5
161ndash170
Guo G L Choudhuri S amp Klaassen C D (2002) Induction profile of
rat organic anion transporting polypeptide 2 (oatp2) by prototypical
drug-metabolizing enzyme inducers that activate gene expression
through ligand-activated transcription factor pathways Journal of
Pharmacology and Experimental Therapeutics 300 206ndash212
Hagenbuch B amp Meier P J (2004) Organic anion transporting
polypeptides of the OATP SLC21 family Phylogenetic classification
as OATP SLCO superfamily new nomenclature and molecularfunc-
tional properties Pflugers Archiv 447 653ndash665
Hamadeh H K amp Afshari C A (2004) Toxicogenomics principles and
applications Hobokenrsquo John Wiley and Sons
Hamadeh H K Bushel P R Jayadev S Martin K DiSorbo O Sieber
S et al (2002) Gene expression analysis reveals chemical-specific
profiles Toxicological Sciences 67 219ndash231
Hamadeh H K Jayadev S Gaillard E T Huang Q Stoll R
Blanchard K et al (2004) Integration of clinical and gene expression
endpoints to explore furan-mediated hepatotoxicity Mutation Research
549 169ndash183
Hamadeh H K Knight B L Haugen A C Sieber S Amin R P
Bushel P R et al (2002) Methapyrilene toxicity Anchorage of
pathologic observations to gene expression alterations Toxicologic
Pathology 30 470ndash482
Han J D Bertin N Hao T Goldberg D S Berriz G F Zhang L V
et al (2004) Evidence for dynamically organized modularity in the
yeast proteinndashprotein interaction network Nature 430 88ndash93
Hanisch D Zien A Zimmer R amp Lengauer T (2002) Co-clustering of
biological networks and gene expression data Bioinformatics 18
S145ndashS154
Harris A J Dial S L amp Casciano D A (2004) Comparison of basal
gene expression profiles and effects of hepatocarcinogens on gene
expression in cultured primary human hepatocytes and HepG2 cells
Mutation Research 549 79ndash99
Harrison S C (2004) Whither structural biology Nature Structural
Molecular Biology 11 12ndash15
Hartley D P Dai X He Y D Carlini E J Wang B Huskey S E
et al (2004) Activators of the rat pregnane X receptor differentially
modulate hepatic and intestinal gene expression Molecular Pharma-
cology 65 1159ndash1171
Hartwell L H Hopfield J J Leibler S amp Murray A W (1999) From
molecular to modular cell biology Nature 402 C47ndashC52
Hasmall S Orphanides G James N Pennie W Hedley K Soames
A et al (2002) Downregulation of lactoferrin by PPARalpha ligands
Role in perturbation of hepatocyte proliferation and apoptosis
Toxicology Sciences 68 304ndash313
Hayase T Yamamoto Y Yamamoto K Muso E amp Shiota K (2004)
Microarray profile analysis of toxic cocaine-induced alterations in the
expression of mouse brain gene sequences A possible rsquoprotectiversquo
effect of buprenorphine Journal of Applied Toxicology 24 15ndash20
Hayes K R Vollrath A L Zastrow G M McMillan B J Craven M
Jovanovich S et al (2005) EDGE A centralized resource for the
comparison analysis and distribution of toxicogenomic information
Molecular Pharmacology 67 1360ndash1368
Heijne W H Slitt A L van Bladeren P J Groten J P Klaassen C D
Stierum R H et al (2004) Bromobenzene-induced hepatotoxicity at
the transcriptome level Toxicology Sciences 79 411ndash422
Heijne W H Stierum R H Slijper M van Bladeren P J amp van
Ommen B (2003) Toxicogenomics of bromobenzene hepatotoxicity
A combined transcriptomics and proteomics approach Biochemical
Pharmacology 65 857ndash875
Heinloth A N Irwin R D Boorman G A Nettesheim P Fannin
R D Sieber S O et al (2004) Gene expression profiling of rat
livers reveals indicators of potential adverse effects Toxicology
Sciences 80 193ndash202
Hodges L C Cook J D Lobenhofer E K Li L Bennett L Bushel
P R et al (2003) Tamoxifen functions as a molecular agonist inducing
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 63
cell cycle-associated genes in breast cancer cells Molecular Cancer
Research 1 300ndash311
Holleman A Cheok M H den Boer M L Yang W Veerman KM
A J Kazemier et al (2004) Gene-expression patterns in drug-resistant
acute lymphoblastic leukemia cells and response to treatment New
England Journal of Medicine 351 533ndash542
Hong Y Muller U R amp Lai F (2003) Discriminating two classes of
toxicants through expression analysis of HepG2 cells with DNA arrays
Toxicology in Vitro 17 85ndash92
Hood E (2003) Proteomics Characterizing the cogs in the machinery of
life Environmental Health Perspectives 111 A816ndashA825
Hood L (2003) Systems biology Integrating technology biology and
computation Mechanism of Ageing and Development 124 9ndash16
Hood L amp Galas D (2003) The digital code of DNA Nature 421
444ndash448
Hood L amp Perlmutter R M (2004) The impact of systems approaches on
biological problems in drug discovery Nature Biotechnology 22
1215ndash1217
Hu T Gibson D P Carr G J Torontali S M Tiesman J P Chaney
J G et al (2004) Identification of a gene expression profile that
discriminates indirect-acting genotoxins from direct-acting genotoxins
Mutation Research 549 5ndash27
Huang Q Dunn II R T Jayadev S DiSorbo O Pack F D Farr S B
et al (2001) Assessment of cisplatin-induced nephrotoxicity by
microarray technology Toxicology Sciences 63 196ndash207
Huang Q Jin X Gaillard E T Knight B L Pack F D Stoltz J H et
al (2004) Gene expression profiling reveals multiple toxicity endpoints
induced by hepatotoxicants Mutation Research 549 147ndash167
Huisman M T Smit J W Crommentuyn K M Zelcer N Wiltshire
H R Beijnen J H et al (2002) Multidrug resistance protein 2
(MRP2) transports HIV protease inhibitors and transport can be
enhanced by other drugs Aids 16 2295ndash2301
Hunt C M Westerkam W R amp Stave G M (1992) Effect of age and
gender on the activity of human hepatic CYP3A Biochemical
Pharmacology 44 275ndash283
Ideker T Ozier O Schwikowski B amp Siegal A F (2002) Discovering
regulatory and signalling circuits in molecular interaction networks
Bioinformatics 18 S233ndashS240
Ideker T Thorsson V Ranish J A Christmas R Buhler J Eng J K
et al (2001) Integrated genomic and proteomic analyses of a system-
atically perturbed network Science 292 929ndash934
Iida M Anna C H Hartis J Bruno M Wetmore B Dubin J R et al
(2003) Changes in global gene and protein expression during early
mouse liver carcinogenesis induced by non-genotoxic model carcino-
gens oxazepam and Wyeth-14643 Carcinogenesis 24 757ndash770
Ingelman-Sundberg M (2004) Pharmacogenetics of cytochrome P450 and
its applications in drug therapy The past present and future Trends in
Pharmacological Sciences 25 193ndash200
Jeong H Mason S P Barabasi A L amp Oltvai Z N (2001) Lethality
and centrality in protein networks Nature 411 41ndash42
Jeong H Tombor B Albert R Oltvai Z N amp Barabasi A L
(2000) The large-scale organization of metabolic networks Nature
407 651ndash654
Jung D Hagenbuch B Gresh L Pontoglio M Meier P J amp Kullak-
Ublick G A (2001) Characterization of the human OATP-C
(SLC21A6) gene promoter and regulation of liver-specific OATP genes
by hepatocyte nuclear factor 1 alpha Journal of Biological Chemistry
276 37206ndash37214
Jung D amp Kullak-Ublick G A (2003) Hepatocyte nuclear factor 1
alpha A key mediator of the effect of bile acids on gene expression
Hepatology 37 622ndash631
Jung J -W Park J -S Hwang J -W Kang K -S YS L Song B
-S et al (2004) Gene expression analysis of peroxisome prolifer-
ators- and phenytoin-induced hepatotoxicity using cDNA microarray
Journal of Veterinary Medical Science 66 1329ndash1333
Kato N Shibutani M Takagi H Uneyama C Lee K -Y Takiami S
et al (2004) Gene expression profile in the livers of rats orally
administered ethinylestradiol for 28 days using a microarray technique
Toxicology 200 179ndash192
Kier L D Neft R Tang L Suizu R Cook T Onsurez K et al
(2004) Applications of microarrays with toxicologically relevant genes
(tox genes) for the evaluation of chemical toxicants in Sprague Dawley
rats in vivo and human hepatocytes in vitro Mutation Research 549
101ndash113
Kijima K Toyosawa K Yasuba M Matsuoka N Adachi T
Komiyama M et al (2004) Gene expression analysis of the rat testis
after treatment with di(2-ethylhexyl) phthalate using cDNA microarray
and real-time RT-PCR Toxicology and Applied Pharmacology 200
103ndash110
Kim R B (2003) Organic anion-transporting polypeptide (OATP) trans-
porter family and drug disposition European Journal of Clinical
Investigation 33 1ndash5
Kinirons M T amp OrsquoMahony M S (2004) Drug metabolism and ageing
British Journal of Clinical Pharmacology 57 540ndash544
Kitano H (2002a) Computational systems biology Nature 420 206ndash210
Kitano H (2002b) Systems biology A brief overview Science 295
1662ndash1664
Kliewer S A Moore J T Wade L Staudinger J L Watson M
A Jones S A et al (1998) An orphan nuclear receptor activated
by pregnanes defines a novel steroid signalling pathway Cell 92
73ndash82
Korolev D Balakin K V Nikolsky Y Kirillov E Ivanenkov Y A
Savchuk N P et al (2003) Modeling of human cytochrome p450-
mediated drug metabolism using unsupervised machine learning
approach Journal of Medicinal Chemistry 46 3631ndash3643
Kramer J A Pettit S D Amin R P Bertram T A Car B
Cunningham M et al (2004) Overview on the application of
transcription profiling using selected nephrotoxicants for toxicology
assessment Environmental Health Perspectives 112 460ndash464
Kultima K Nystrom A -M Scholz B Gustafson A -L Dencker L
amp Stigson M (2004) Valproic acid teratogenicity A toxicogenomics
approach Environmental Health Perspectives 112 1225ndash1235
Kutuzova G D amp Deluca H F (2004) Gene expression profiles in rat
intestine identify pathways for 125-dihydroxyvitamin D(3) stimulated
calcium absorption and clarify its immunomodulatory properties
Archives of Biochemistry and Biophysics 432 152ndash166
Kwak M K Wakabayashi N Itoh K Motohashi H Yamamoto M amp
Kensler T W (2003) Modulation of gene expression by cancer
chemopreventive dithiolethiones through the Keap1-Nrf2 pathway
Identification of novel gene clusters for cell survival Journal of
Biological Chemistry 278 8135ndash8145
Lahjouji K Mitchell G A amp Qureshi I A (2001) Carnitine transport
by organic cation transporters and systemic carnitine deficiency
Molecular Genetics and Metabolism 73 287ndash297
Landowski C P Sun D Foster D R Menon S S Barnett J L
Welage L S et al (2003) Gene expression in the human intestine and
correlation with oral valacyclovir pharmacokinetic parameters Journal
of Pharmacology and Experimental Therapeutics 306 778ndash786
Lee G -J Lee W -S Jeon K -S Um C -H Kim S -J Lee C -H
et al (2004) cDNA microarray gene expression analysis and
toxicological phenotype for anticancer drug Journal of Veterinary
Medical Science 66 1339ndash1345
Lee M Kwon J Kim S N Kim J E Koh W S Kim E J et al
(2003) cDNA microarray gene expression profiling of hydroxyurea
paclitaxel and p-anisidine genotoxic compounds with differing
tumorigenicity results Environmental and Molecular Mutagenesis
42 91ndash97
Li S Armstrong C M Bertin N Ge H Milstein S Boxem M et al
(2004) A map of the interactome network of the metazoan C elegans
Science 303 540ndash543
Liguori M J Anderson L M Bukofzer S McKim J Pregenzer J F
Retief J et al (2005) Microarray analysis in human hepatocytes
suggests a mechanism for hepatotoxicity induced by trovafloxacin
Hepatology 41 177ndash186
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6664
Lobenhofer E K Bennett L Cable P L Li L Bushel P R amp Afshari
C A (2002) Regulation of DNA replication fork genes by 17beta-
estradiol Molecular Endocrinology 16 1215ndash1229
Locker J Tian J Carver R Concas D Cossu C Ledda-Columbano
G M et al (2003) A common set of immediate-early response genes
in liver regeneration and hyperplasia Hepatology 38 314ndash325
Mattes W B Pettit S D Sansone S A Bushel P R amp Waters M D
(2004) Database development in toxicogenomics Issues and efforts
Environmental Health Perspectives 112 495ndash505
McMillian M Nie A Y Parker J B Leone A Bryant S Kemmerer
M et al (2004) A gene expression signature for oxidant stressreactive
metabolites in rat liver Biochemical Pharmacology 68 2249ndash2261
Meier P J amp Stieger B (2000) Molecular mechanisms in bile formation
News in Physiological Sciences 15 89ndash93
Meneses-Lorente G de Longueville F Dos Santos-Mendes S Bonnert
T P Jack A Evrard S et al (2003) An evaluation of a low-density
DNA microarray using cytochrome P450 inducers Chemical Research
in Toxicology 16 1070ndash1077
Meneses-Lorente G Guest P C Lawrence J Muniappa N Knowles
M R Skynner H A et al (2004) A proteomic investigation of drug-
induced steatosis in rat liver Chemical Research in Toxicology 17
605ndash612
Merrill C L Ni H Yoon L W Tirmenstein M A Narayanan P
Benavides G R et al (2002) Etomoxir-induced oxidative stress in
HepG2 cells detected by differential gene expression is confirmed
biochemically Toxicology Sciences 68 93ndash101
Milo R Shen-Orr S Itzkovitz S Kashtan N Chklovskii D amp Alon
U (2002) Network motifs Simple building blocks of complex
networks Science 298 824ndash827
Morgan K T Ni H Brown H R Yoon L Qualls C W Crosby L
M et al (2002) Application of cDNA microarray technology to in
vitro toxicology and the selection of genes for a real-time RT-PCR-
based screen for oxidative stress in Hep-G2 cells Toxicologic
Pathology 30 435ndash451
Mwinyi J Johne A Bauer S Roots I amp Gerloff T (2004) Evidence
for inverse effects of OATP-C (SLC21A6) 5 and 1b haplotypes on
pravastatin kinetics Clinical Pharmacology and Therapeutics 75
415ndash421
Nakata K Yukawa M Komiyama N Nakano T amp Kaminuma T
(2002) A nuclear receptor database that maps pathways to diseases
Genome Infomatics 13 515ndash516
Nicholson J K Holmes E Lindon J C amp Wilson I D (2004) The
challenges of modeling mammalian biocomplexity Nature Biotechnol-
ogy 22 1268ndash1274
Nicholson J K amp Wilson I D (2003) Understanding rsquoglobal systems
biology Metabonomics and the continuum of metabolism Nature
Reviews on Drug Discovery 2 668ndash676
Nikitin A Egorov S Daraselia N amp Mazo I (2003) Pathway studiomdash
the analysis and navigation of molecular networks Bioinformatics 19
2155ndash2157
Nikolsky Y Ekins S Nikolskaya T amp Bugrim A (2005) A novel
method for generation of signature networks as biomarkers from
complex high throughput data Toxicology Letters 158 20ndash29
Nikolsky Y Nikolskaya T amp Bugrim A (2005) Biological networks
and analysis of experimental data in drug discovery Drug Discovery
Today 10 653ndash662
Nishizato Y Ieiri I Suzuki H Kimura M Kawabata K Hirota T
et al (2003) Polymorphisms of OATP-C (SLC21A6) and OAT3
(SLC22A8) genes Consequences for pravastatin pharmacokinetics
Clinical Pharmacology and Therapeutics 73 554ndash565
Nozawa T Nakajima M Tamai I Noda K Nezu J Sai Y et al
(2002) Genetic polymorphisms of human organic anion transporters
OATP-C (SLC21A6) and OATP-B (SLC21A9) Allele frequencies in
the Japanese population and functional analysis Journal of Pharmacol-
ogy and Experimental Therapeutics 302 804ndash813
Nozawa T Sugiura S Nakajima M Goto A Yokoi T Nezu J et al
(2004) Involvement of organic anion transporting polypeptides in the
transport of troglitazone sulfate Implications for understanding trogli-
tazone hepatotoxicity Drug Metabolism and Disposition 32 291ndash294
Oliver D E Rubin D L Stuart J M Hewett M Klein T E amp
Altman R B (2002) Ontology development for a pharmacogenetics
knowledge base Package Symposion 88ndash99
Oram J F amp Lawn R M (2001) ABCA1 The gatekeeper for eliminating
excess tissue cholesterol Journal of Lipid Research 42 1173ndash1179
Oswald M Kullak-Ublick G A Paumgartner G amp Beuers U (2001)
Expression of hepatic transporters OATP-C and MRP2 in primary
sclerosing cholangitis Liver 21 247ndash253
Parsons A B Brost R I Ding H Li Z Zhang C Sheikh B et al
(2004) Integration of chemical-genetic and genetic interaction data
links bioactive compounds to cellular target pathways Nature Bio-
technology 22 62ndash69
Patki K C von Moltke L L Harmatz J S Hesse L M Court M H
amp Greenblatt D J (2004) Effect of age on in vitro triazolam
biotransformation in male human liver microsomes Journal of
Pharmacology and Experimental Therapeutics 308 874ndash879
Pereira-Leal J B Enright A J amp Ouzounis C A (2004) Detection of
functional modules from protein interaction networks Proteins 54
49ndash57
Plant N (2004) Interaction networks Coordinating responses to xeno-
biotic exposure Toxicology 202 21ndash32
Raghavendra Prasad H S Qi Z Srinivasan K N amp Gopalakrish-
nakone P (2004) Potential effects of tetrodotoxin exposure to
human glial cells postulated using microarray approach Toxicon 44
597ndash608
Reilly T P Bourdi M Brady J N Pise-Masison C A Radonovich M
F George J W et al (2001) Expression profiling of acetaminophen
liver toxicity in mice using microarray technology Biochemical and
Biophysical Research Communications 282 321ndash328
Rives A W amp Galitski T (2003) Modular organization of cellular
networks Proceedings of the National Academy of Sciences of the
United States of America 100 1128ndash1133
Rogers P D Pearson M M Cleary J D Sullivan D C amp Chapman
S W (2002) Differential expression of genes encoding immunomodu-
latory proteins in response to amphotericin B in human mononuclear
cells identified by cDNA microarray analysis Journal of Antimicrobial
Chemotherapy 50 811ndash817
Rosen M B Wilson V S Schmid J E amp Gray L E (2005) Gene
expression analysis in the ventral prostate of rats exposed to vinclozolin
or procymidone Reproductive Toxicology 19 367ndash379
Rost D Herrmann T Sauer P Schmidts H L Stieger B Meier P J
et al (2003) Regulation of rat organic anion transporters in bile salt-
induced cholestatic hepatitis Effect of ursodeoxycholate Hepatology
38 187ndash195
Rothman R B Ayestas M A Dersch C M amp Baumann M H (1999)
Aminorex fenfluramine and chlorphentermine are serotonin transporter
substrates Implications for primary pulmonary hypertension Circula-
tion 100 869ndash875
Ruepp S U Tonge R P Shaw J Wallis N amp Pognan F (2002)
Genomics and proteomics analysis of acetaminophen toxicity in mouse
liver Toxicology Sciences 65 135ndash150
Sakaeda T Nakamura T amp Okumura K (2002) MDR1 genotype-
related pharmacokinetics and pharmacodynamics Biological amp Phar-
maceutical Bulletin 25 1391ndash1400
Sakata T Anzai N Shin H J Noshiro R Hirata T Yokoyama H
et al (2004) Novel single nucleotide polymorphisms of organic cation
transporter 1 (SLC22A1) affecting transport functions Biochemical
and Biophysical Research Communications 313 789ndash793
Satlin L M Amin V amp Wolkoff A W (1997) Organic anion
transporting polypeptide mediates organic anionHCO3-exchange
Journal of Biological Chemistry 272 26340ndash26345
Satomi Y Tsuchiya W Mihara K Ota M Kasahara Y amp Akahori F
(2004) Gene expression analysis of the lung following paraquat
administration in rats using DNA microarray Journal of Toxicological
Sciences 29 91ndash100
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 65
Schrenk D Baus P R Ermel N Klein C Vorderstemann B amp
Kauffmann H M (2001) Up-regulation of transporters of the MRP
family by drugs and toxins Toxicology Letters 120 51ndash57
Segal E Shapira M Regev A Persquoer D Botstein D Koller D et al
(2003) Module networks Identifying regulatory modules and their
condition-specific regulators from gene expression data Nature
Genetics 34 166ndash176
Segal E Wang H amp Koller D (2003) Discovering molecular pathways
from protein interaction and gene expression Bioinformatics 19
i264ndash i272
Seidel S D Kan H L Stott W T Schisler M R amp Gollapudi B B
(2003) Identification of transcriptome profiles for the DNA-damaging
agents bleomycin and hydrogen peroxide in L5178Y mouse lymphoma
cells Environmental and Molecular Mutagenesis 42 19ndash25
Shan L Yu Y Schut H A J amp Snyderwine E G (2004) Susceptibility
of rats to mammary gland carcinogenesis by the food-derived
carcinogen 2-Amino-1-Methyl-6-Phenylimidazo[45-b]Pyridine (PhIP)
varies with age and is associated with the induction of differential gene
expression American Journal of Pathology 165 191ndash202
Sharom J R Bellows D S amp Tyers M (2004) From large networks to
small molecules Current Opinion in Chemical Biology 8 81ndash90
Shitara Y Hirano M Sato H amp Sugiyama Y (2004) Gemfibrozil and
its glucuronide inhibit the OATP2(OATP1B1 SLC21A6)-mediated
hepatic uptake and CYP2C8-mediated metabolism of cerivastatinmdash
Analysis of the mechanism of the clinically relevant drugndashdrug
interaction between cerivastatin and gemfibrozil Journal of Pharma-
cology and Experimental Therapeutics
Shitara Y Itoh T Sato H Li A P amp Sugiyama Y (2003) Inhibition of
transporter-mediated hepatic uptake as a mechanism for drugndashdrug
interaction between cerivastatin and cyclosporin A Journal of
Pharmacology and Experimental Therapeutics 304 610ndash616
Somogyi R Fuhrman S amp Wen X (2001) Genetic network inference in
computational models and applications to large-scale gene expression
data In M Bower amp H Bolouri (Eds) Computational modeling of
genetic and biochemical networks (pp 119ndash157) Cambridge MArsquoThe
MIT Press
Spirin V amp Mirny L A (2003) Protein complexes and functional
modules in molecular networks Proceedings of the National Academy
of Sciences of the United States of America 100 12123ndash12128
Staudinger J Liu Y Madan A Habeebu S amp Klaassen C D
(2001) Coordinate regulation of xenobiotic and bile acid homeo-
stasis by pregnane X receptor Drug Metabolism and Disposition
29 1467ndash1472
Sun L Z Ji Z L Chen X Wang J F amp Chen Y Z (2002) ADME-
AP A database of ADME associated proteins Bioinformatics 18
1699ndash1700
Suter L Babiss L E amp Wheeldon E B (2004) Toxicogenomics in
predictive toxicology in drug development Chemistry and Biology 11
161ndash171
Synold T W Dussault I amp Forman B M (2001) The orphan nuclear
receptor SXR coordinately regulates drug metabolism and efflux
Nature Medicine 7 584ndash590
Tabuchi Y amp Kondo T (2003) cDNA microarray analysis reveals chop-
10 plays a key role in Sertoli cell injury induced by bisphenol A
Biochemical and Biophysical Research Communications 305 54ndash61
Tamai I Nezu J Uchino H Sai Y Oku A Shimane M et al (2000)
Molecular identification and characterization of novel members of the
human organic anion transporter (OATP) family Biochemical and
Biophysical Research Communications 273 251ndash260
Taylor S W Fahy E Zhang B Glenn G M Warnock D E Wiley S
et al (2003) Characterization of the human heart mitochondrial
proteome Nature Biotechnology 21 281ndash286
Terasaka S Aita Y Inoue A Hayashi S Nishigaki M Aoyagi K
et al (2004) Using a customized DNA microarray for expression
profiling of the estrogen-responsive genes to evaluate estrogen activity
among natural estrogens and industrial chemicals Environmental
Health Perspectives 112 773ndash781
Thomas D M Francescutti-Verbeem D M Liu X amp Kuhn D M
(2004) Identification of differentially regulated transcripts in mouse
striatum following methamphetamine treatmentmdashan oligonucleotide
microarray approach Journal of Neurochemistry 88 380ndash393
Thomas R S Rank D R Penn S G Zastrow G M Hayes K R
Pande K et al (2001) Identification of toxicologically predictive
gene sets using cDNA microarrays Molecular Pharmacology 60
1189ndash1194
Thompson K L Afshari C A Amin R P Bertram T A Car B
Cunningham M et al (2004) Identification of platform-independent
gene expression markers of cisplatin nephrotoxicity Environmental
Health Perspectives 112 488ndash494
Tirona R G Leake B F Merino G amp Kim R B (2001)
Polymorphisms in OATP-C Identification of multiple allelic
variants associated with altered transport activity among European-
and African-Americans Journal of Biological Chemistry 276
35669ndash35675
Tirona R G Leake B F Wolkoff A W amp Kim R B (2003)
Human organic anion transporting polypeptide-C (SLC21A6) is a
major determinant of the rifampin-mediated pregnane X receptor
activation Journal of Pharmacology and Experimental Therapeutics
304 223ndash228
Tong W Cao X Harris S Sun H Fang H Fuscoe J et al (2003)
ArrayTrackmdashsupporting toxicogenomic research at the US Food and
Drug Administration National Center for Toxicological Research
Environmental Health Perspectives 111 1819ndash1826
Tornow S amp Mewes H W (2003) Functional modules by relating
protein interaction networks and gene expression Nucleic Acids
Research 31 6283ndash6289
Trocho C Escola-Gil J C Ribas V Benitez S Martin-Campos J M
Rotllan N et al (2004) Phenytoin treatment reduces atherosclerosis in
mice through mechanisms independent of plasma HDL-cholesterol
concentration Atherosclerosis 174 275ndash285
Ueda A Hamadeh H K Webb H K Yamamoto Y Sueyoshi T
Afshari C A et al (2002) Diverse roles of the nuclear orphan
receptor CAR in regulating hepatic genes in response to phenobarbital
Molecular Pharmacology 61 1ndash6
Ulrich R G (2003) The toxicogenomics of nuclear receptor agonists
Current Opinion in Chemical Biology 7 505ndash510
Ulrich R G Rockett J C Gibson G G amp Pettit S D (2004)
Overview of an interlaboratory collaboration on evaluating the effects
of model hepatotoxicants on hepatic gene expression Environmental
Health Perspectives 112 423ndash427
Unami A Shinohara Y Ichikawa T amp Baba Y (2003) Biochemical
and microarray analyses of bupivacaine-induced apoptosis Journal of
Toxicological Sciences 28 77ndash94
Vasquez A Flammini A Maritan A amp Vespignani A (2003) Global
protein function prediction from proteinndashprotein interaction networks
Nature Biotechnology 21 697ndash700
Vezina C M Walker N J amp Olson J R (2004) Subchronic Exposure to
TCDD PeCDF PCB126 and PCB153 Effect on hepatic gene
expression Environmental Health Perspectives 112 1636ndash1644
Vo T D Greenberg H J amp Palsson B O (2004) Reconstruction and
functional characterization of the human mitochondrial metabolic
network based on proteomic and biochemical data Journal of Bio-
logical Chemistry 279 39532ndash39540
Wang H amp LeCluyse E L (2003) Role of orphan nuclear receptors in
the regulation of drug-metabolising enzymes Clinical Pharmacoki-
netics 42 1331ndash1357
Wang N Silver D L Thiele C amp Tall A R (2001) ATP-
binding cassette transporter A1 (ABCA1) functions as a choles-
terol efflux regulatory protein Journal of Biological Chemistry 276
23742ndash23747
Waring J F Cavet G Jolly R A McDowell J Dai H Ciurlionis R
et al (2003) Development of a DNA Microarray for toxicology based
on hepatoxin-regulated sequences Environmental Health Perspectives
111 863ndash870
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6666
Waring J F Gum R Morfitt D Jolly R A Ciurlionis R Heindel M
et al (2002) Identifying toxic mechanisms using DNA microarrays
Evidence that an experimental inhibitor of cell adhesion molecule
expression signals through the aryl hydrocarbon nuclear receptor
Toxicology 181ndash182 537ndash550
Waring J F Jolly R A Ciurlionis R Lum P Y Praestgaard J T
Morfitt D C et al (2001) Clustering of hepatotoxins based on
mechanism of toxicity using gene expression profiles Toxicology and
Applied Pharmacology 175 28ndash42
Waters M Boorman G Bushel P Cunningham M Irwin R Merrick
A et al (2003) Systems toxicology and the Chemical Effects in
Biological Systems (CEBS) knowledge base EHP Toxicogenomics
111 15ndash28
Wei M Arnold L Cano M amp Cohen S M (2005) Effects of co-
administration of antioxidants and arsenicals on the rat urinary bladder
epithelium Toxicology Sciences 83 237ndash245
Weinshilboum R (2003) Inheritance and drug response New England
Journal of Medicine 348 529ndash537
Weinshilboum R amp Wang L (2004) Pharmacogenetics Inherited
variation in amino acid sequence and altered protein quantity Clinical
Pharmacology and Therapeutics 75 253ndash258
Werner E (2003) In silico multicellular systems biology and minimal
genomes Drug Discovery Today 8 1121ndash1127
Wong J S amp Gill S S (2002) Gene expression changes induced in
mouse liver by di(2-ethylhexyl) phthalate Toxicology and Applied
Pharmacology 185 180ndash196
Xie T Tong L McCann U D Yuan J Becker K G Mechan A O
et al (2004) Identification and characterization of metallothionein-1
and -2 gene expression in the context of ()34-methylenedioxymetham-
phetamine-induced toxicity to brain dopaminergic neurons Journal of
Neuroscience 24 7043ndash7050
Yadetie F Laegreid A Bakke I Kusnierczyk W Komorowski J
Waldum H L et al (2003) Liver gene expression in rats in response
to the peroxisome proliferator-activated receptor-alpha agonist ciprofi-
brate Physiological Genomics 15 9ndash19
Yamashita S Nomoto T Abe M Tatematsu M Sugimura T amp
Ushijima T (2004) Persistence of gene expression changes in stomach
mucosae induced by short-term N-methyl-NV-nitro-N-nitrosoguanidinetreatment and their presence in stomach cancers Mutation Research
549 185ndash193
Yan Q amp Sadee W (2000) Human membrane transporter database A
web-accessible relational database for drug transport studies and
pharmacogenomics AAPS Pharmsci 2 E20
Yeger-Lotem E amp Margalit H (2003) Detection of regulatory circuits by
integrating the cellular networks of proteinndashprotein interactions and
transcription regulation Nucleic Acids Research 31 6053ndash6061
Yoon B I Li G -X Kitada K Kawasaki Y Igarashi K amp Kodama
Y et al (2003) Mechanisms of benzene-induced hematotoxicity and
leukemogenicity cDNA microarray analyses using mouse bone marrow
tissue Environmental Health Perspectives 111 1411ndash1420
Young M B DiSilvestro M R Sendera T J Freund J Kriete A amp
Magnuson S R (2003) Analysis of gene expression in carbon
tetrachloride-treated rat livers using a novel bioarray technology
Pharmacogenomics Journal 3 41ndash52
Yu H Zhu X Greenbaum D Karro J amp Gerstein M (2004) TopNet
A tool for comparing biological sub-networks correlating protein
properties with topological statistics Nucleic Acids Research 32
328ndash337
Zhang E Y Fu D J Pak Y A Stewart T Mukhopadhyay N
Wrighton S A et al (2004) Genetic polymorphisms in human proton-
dependent dipeptide transporter PEPT1 Implications for the functional
role of Pro586 Journal of Pharmacology and Experimental Therapeu-
tics 310 437ndash445
Zhang E Y Knipp G T Ekins S amp Swaan P W (2002)
Structural biology and function of solute transporters Implications
for identifying and designing substrates Drug Metabolism Reviews
34 709ndash750
Zhang E Y Phelps M A Cheng C Ekins S amp Swaan P W (2002)
Modeling of active transport systems Advanced Drug Delivery
Reviews 54 329ndash354
- Systems-ADMETox Resources and network approaches
-
- Introduction
- Data available
- Network analysis and databases
-
- Network applications
-
- The role of transporters
-
- Clinical relevance of transporters
- Transporter network examples ABCA1
- Transporter network examples OATP
- Transporter microarray data
-
- Applications to enzymes
- Future network applications
-
- Discussion
- Acknowledgements
- References
-
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6652
involved in the transport metabolism and biosynthesis of
bile acids (Staudinger Liu Madan Habeebu amp Klaassen
2001) However the additional receptors CAR FXR LXR
and other nuclear receptors take part in a complex network
of interactions to control these and other proteins Thus
elucidation of the regulatory networks which control the
expression of these transporters is also important To date
most of the research has centered on efflux transporters but
there has been considerable interest in uptake transporters
such as the organic anion transporter polypeptide (OATP
((Kim 2003) see also later Section on OATP)
There are several specific examples of the importance of
drug transporters to the clinical development of drugs One
example is the insulin sensitizer troglitazone which was
withdrawn due to hepatotoxicity although the precise
mechanism appears to have been unclear until recently
The major metabolite is a sulfated species and is suspected
of being responsible for the observed toxicity The recent
assessment of the organic anion transporting polypeptides
OATP-C and OATP8 expressed on the hepatocyte baso-
lateral membrane indicated that sulphated troglitazone has a
high affinity for the former and possibly lower affinity for
the latter (Nozawa et al 2004) This metabolite would
therefore be expected to accumulate in hepatocytes and
inhibit the bile salt export pump and Mrp2 Because
polymorphisms have been shown for OATP-C (Tirona
Leake Merino amp Kim 2001) it is also possible that these
may result in the accumulation of the metabolite and in turn
elicit idiosyncratic toxicity A second relevant example of
the impact of transporters is the clinically significant drugndash
drug interaction between cerivastatin and cyclosporine A
which occurs via the OATP-C transporter (Shitara Itoh
Sato Li amp Sugiyama 2003) A third example are the HIV
protease inhibitors saquinavir ritonavir and indinavir which
are transported by MRP2 in vitro and other drugs such as
probenecid and sulfinpyrazone are able to enhance this
transport Transport by MRP2 suggests that these com-
pounds will have decreased bioavailability due to increased
clearance and other drugs could aggravate this situation by
further enhancing transport (Huisman et al 2002) Sim-
ilarly the rifampicin mediated induction via PXR of MRP2
and P-gp in healthy subjects was found to significantly
decrease the AUC and also correlated with intestinal
expression of these transporters This transporter is also
inducible by cisplatin 2-AAF and phenobarbital (Schrenk
et al 2001) indicating multiple mechanisms may be
involved In other species such as rat commonly used as a
toxicity model orthologs of the transporters such as OATP2
are expressed and can be induced with ligands for PXR like
PCN (Guo Choudhuri amp Klaassen 2002) This is useful
knowledge because the advent of microarray technology
allows one to dose a rat with a xenobiotic and assess
thousands of genes simultaneously in a particular tissue For
instance animals dosed with known nephrotoxins have
shown some upregulation of the NandashKndashCl transporter
however the authors suggested genomic responses are
stronger soon after exposure before declining (Fleck et
al 2003) Some transporters may therefore be specifically
targeted by drugs in one tissue such as the CNS but these
same transporters may also be expressed elsewhere in the
body hence off-target effects may result in toxicity The
serotonin transporter is one such example which is
expressed in the lungs and brain Some substrates for this
channel like fenfluramine can result in primary pulmonary
hypertension as they accumulate in pulmonary cells (Roth-
man Ayestas Dersch amp Baumann 1999) Similarly P-gp is
expressed at the blood brain barrier and intestine impacting
the efficacy and bioavailability of drugs
3112 Transporter network examples ABCA1 The
ABCA1 transporter mediates the first step of cholesterol
transport Mutations in this gene cause Tangier disease
which results in severe HDL deficiency cholesterol
accumulation in macrophages and attendant atherosclerosis
This transporter represents a drug target for upregulation
modulating cholesterol metabolism and prevention of
cardiovascular disease (Oram amp Lawn 2001) In vitro
ABCA1 can be inhibited by the sulfonylurea glybenclamide
(Field Burn amp Mathur 2004 Wang Silver Thiele amp Tall
2001) In order to illustrate the advantages of mapping drug
transporters as networks onto functional models alongside
other proteins one can consider the example of ABCA1
The query of the MetaCorei database shows that this
transporter appears on three manually curated pathway maps
representing the Fstate of the art_ knowledge derived from
reliable high quality literature sources One can also use the
individual maps as an interface to access the underlying
layers of information about the transporter including the list
of encoding genessplice variants with known SNPs In
addition to browsing MetaCorei a user can also build
custom networks around ABCA1 using the network-
construction tool (Fig 5) Such a visualization utility may
be very helpful for identification of all putative pathways
around a particular transporter or compound of interest The
ABCA1 network created by this tool shows that this
transporter at the time of writing is linked directly to
twenty-five other objects such as APOE1 and LXR Many
of its neighbors have their own SNPs that could be
important in determining interactions between transport of
a drug and normal human transport of endogenous ligands
in health or disease
3113 Transporter network examples OATP The
OATPs are key membrane bound transporters expressed in
many organs including intestine liver lung choroid plexus
blood brain barrier and other organs (Tamai et al 2000)
This family of transporters is capable of mediating the
sodium-independent transport of a diverse array of mole-
cules such as steroid conjugates organic anions and
xenobiotics by coupling uptake with efflux of bicarbonate
(Satlin Amin amp Wolkoff 1997) glutathione or its
conjugates (Hagenbuch amp Meier 2004) The inhibition of
Fig 5 The network of direct gene interactions around the ABCA1 transporter gene (centre) generated with MetaCorei The interation types between nodes are shown as small colored hexagons eg unspecified
allosteric regulation binding cleavage competition covalent modification dephosphorylation phosphorylation transcription regulation transformation When applicable interactions also have a positive or
negative effect and direction Ligands (purple) linked to other proteins (blue blobs) transfactors (red) enzymes (orange) (For interpretation of the references to colour in this figure legend the reader is referred to
the web version of this article)
SEkin
sJournalofPharm
acologica
landToxico
logica
lMeth
ods53(2006)38ndash66
53
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6654
this transporterrsquos hepatic uptake of other compounds may be
important for reported drugndashdrug interactions (Kim 2003)
described earlier (Shitara et al 2003) as well as cerivastatin
with gemfibrozil (Shitara Hirano Sato amp Sugiyama 2004)
OATP1B1 (previous names OATP-C LST-1 OATP2
SLC21A6) represents the most studied human OATP to
date (Meier amp Stieger 2000) and is expressed on the
basolateral plasma membrane of hepatocytes Several single
nucleotide polymorphisms have been identified in the
OATP1B1 gene in European-Americans African-Americans
(Tirona et al 2001) and Japanese (Nozawa et al 2002)
dramatically impacting the transport of ligands such as
pravastatin (Mwinyi Johne Bauer Roots amp Gerloff 2004
Nishizato et al 2003) estrone-3-sulfate (Nozawa et al
2002 Tirona et al 2001) Rifampin (Tirona Leake
Wolkoff amp Kim 2003) and estradiol 17h-d-glucuronide(Tirona et al 2001)
The regulation of SLCO may be affected during
extrahepatic cholestasis bile duct ligation bile salt induced
Fig 6 Networks generated with the autoexpand algorithm in MetaCorei to illustr
be interconnected with other protein regulatory signaling information Informa
unspecified allosteric regulation binding cleavage competition covalent m
transformation When applicable interactions also have a positive or negative e
transfactors (red) enzymes (orange) (For interpretation of the references to colour
cholestatic hepatitis (Dumont et al 1997 Meier amp Stieger
2000 Rost et al 2003) and primary sclerosing cholangitis
(Oswald Kullack-Ublick Paumgarter amp Beuer 2001) In
particular OATP1B1 appears to be regulated by the liver-
enriched transcription factor hepatocyte nuclear factor 1a
(HNF1a) which binds to the promoter region of this
transporter (Jung et al 2001) Site directed mutagenesis
of this binding site resulted in inactivation suggesting the
critical nature of the interaction with HNF1a Bile acids
such as CDCA have been shown to transcriptionally repress
HNF1a in vitro via inhibition of the transactivating effect of
HNF4a on HNF1a (Jung amp Kullak-Ublick 2003) After
screening many rat and human uptake transporters in vitro
OATP1B1 was also shown to modulate the PXR response
by controlling rifampin retention in the cell and therefore
affecting the induction of CYP3A4 and other gene products
such as P-gp (Tirona et al 2003)
Some of the literature for OATP1B1 human substrate
data has been annotated into MetaCorei to illustrate the
ate how ligands for the human OATP-C (OATP1B1) transporter (centre) can
tion on the type of interaction between objects is hidden for clarity eg
odification dephosphorylation phosphorylation transcription regulation
ffect and direction Ligands (purple) linked to other proteins (blue blobs)
in this figure legend the reader is referred to the web version of this article)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 55
visualization of the complex interconnections between this
transporter its ligands regulatory factors and signaling
molecules already in the database (Fig 6) The network was
generated with the autoexpand algorithm in the software
representing one of multiple available algorithms for
connecting genes ligands and other objects in the database
Clearly if more ligands and their connections are added to
the database the complexity of the network will increase
considerably The OATP-C (OATP1B1) gene details can be
viewed upon querying the database and links are provided
to other public databases This page can be used to highlight
the multiple synonyms for this gene as well as links to the
multiple SNPs identified to date
3114 Transporter microarray data Microarrays have
generally been limited in the number of transporters present
on them (Annereau et al 2004) however they have been used
in an attempt to correlate pharmacokinetic properties with
gene expression for valacyclovir (Landowski et al 2003) as
well as understand the expression profile in different tissues
or cell lines upon food component or xenobiotic treatment
(Anderle Huang amp Sadee 2004) This lack of transporters
on microarrays has prompted some groups to produce their
own arrays with a heavier emphasis on transporters These
arrays have for example then been used to demonstrate the
upregulation of ABC transporters and down-regulation of
GST-Pi in cell lines resistant to colchicines or 9-nitro-
camptothecin (Annereau et al 2004) The genes that were
significantly up or down-regulated in this particular study
were used to build networks with MetaCore (Fig 7A B) and
the similarities between them were assessed (Fig 7C)
Although there were only a small number of significantly
changed genes in common (IL-8 Fos GST-Pi Calpactin and
Ubiquitin hydrolase) it is perhaps likely that there is a much
larger common gene network that is important for drug
resistance although a much larger number of cell lines and
drug treatments need to be evaluated to produce a definitive
drug resistance signature involving transporters enzymes
and transcriptional regulators
312 Applications to enzymes
As we have already described it may be particularly
valuable to visualize enzymes as networks to show
interactions with transcriptional regulators and ligands
For example a key enzyme is CYP3A4 which metabolizes
40ndash45 of all drugs and has relatively few SNPs
(Ingelman-Sundberg 2004) Using a second software suite
MetaDrugi (wwwgenegocom) it is possible to construct
a custom network around this or other drug metabolizing
enzymes (Fig 8) In this case the gene network for
CYP3A4 highlights all of the major transcriptional
regulators and several more distant linked proteins and
ligands connected on the network that may be useful for
further study (Fig 8) Substrates inhibitors as well as
regulatory factors and other enzymes can be observed
connected on this network Due to the many hundreds of
known inhibitors and substrates only a small number are
shown here for clarity We assume that if a perturbation in
a pathway (eg due to a nonfunctional enzyme) is linked
to a certain pathologic condition a similar perturbation
caused by the interference from xenobiotic metabolism
(eg competitive inhibition of the same enzyme) may
result in identical effects As microarray gene expression
data is increasingly generated the role of enzyme
regulation in toxicity of certain xenobiotics will become
more apparent from either in vivo or in vitro studies The
visualization of such signature gene networks involving
transporters and enzymes their ligands and regulatory
factors will also be important for future toxicity prediction
methods We have recently generated visualizations of
microarray data from MCF-7 cells treated with 4-hydrox-
ytamoxifen to show that some of the key genes involved in
metabolism and transport are upregulated (Ekins et al
2005d) In addition we have made predictions with various
QSAR models in MetaDrug to indicate the involvement of
PXR CYP3A4 and P-gp (Ekins et al 2005e) Therefore it
appears likely that 4-hydroxytamoxifen could increase its
own metabolism as well as efflux from cells via P-gp
which can be visualized on networks Any decrease of
function of these enzymes or transporters in a population
would likely result in changes in the metabolism and
transport of this active metabolite potentially impacting
the clinical effect This represents one example of how
both pathway tools QSAR models and network building
algorithms can be used with different types of predicted
and experimental data to allow visualization of potential
compound interactions or toxicity
313 Future network applications
As the population ages an increasing incidence and
prevalence of systemic diseases especially chronic diseases
have occurred among older adults This has resulted in an
increase in medications used concomitantly by this
population which presents challenges for drugndashdrug
interactions Physiologically elderly patients may behave
differently to the younger patients for which the drugs are
initially developed for and tested on Many pharmacoki-
netic investigations in the elderly population reveal
decreased clearance of lipophilic drugs metabolized by
the cytochrome P450 enzymes however few studies have
evaluated aging-dependent or gender-related changes in
specific P450 enzymes (Hunt Westerkam amp Slave 1992)
Age-related physiological changes such as a reduction in
liver mass hepatic metabolizing enzyme activity liver
blood flow and alterations in plasma drug binding may
account for the decreased elimination of some metabolized
drugs in the elderly It is particularly difficult to separate an
effect of aging from the variation in the rate of metabolism
due to factors such as individual metabolic phenotype
(slow or fast metabolizer due to SNPs) environmental
influences concomitant disease states and drug intake
(drugndashdrug interactions) The available data suggest that
Fig 7 Networks for drug resistant cell lines generated with the Auto expand algorithm in MetaCore A KB-3-1 vs KB-8-5 B ATC-DU-145 vs RCO1 C node overlap of A and B with initial nodes highlighted
with blue or red solid circles (Initial gene lists derived from (Annereau et al 2004)) Information on the type of interaction between objects is hidden for clarity eg unspecified allosteric regulation binding
cleavage competition covalent modification dephosphorylation phosphorylation transcription regulation transformation When applicable interactions also have a positive or negative effect and direction
Ligands (purple) linked to other proteins (blue blobs) transfactors (red) enzymes (orange) (For interpretation of the references to colour in this figure legend the reader is referred to the web version of this article)
SEkin
sJournalofPharm
acologica
landToxico
logica
lMeth
ods53(2006)38ndash66
56
Fig7(continued)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 57
Fig7(continued)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6658
Fig 8 The network of gene interactions around the CYP3A4 enzyme (centre) from MetaCorei Information on the type of interaction between objects is
hidden for clarity eg unspecified allosteric regulation binding cleavage competition covalent modification dephosphorylation phosphorylation
transcription regulation transformation When applicable interactions also have a positive or negative effect and direction Ligands (purple) linked to other
proteins (blue blobs) transfactors (red) enzymes (orange) (For interpretation of the references to colour in this figure legend the reader is referred to the web
version of this article)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 59
the initial doses of drugs metabolized by these enzymes
should be reduced in older patients according to the clinical
response In most published studies the elderly appear at
least as responsive as the young to inducers or inhibitors of
P450s (Durnas Loi amp Cusack 1990) More recently there
has been some suggestion that there are age related
reductions in function of some specific P450s such as
CYP3A4 (Patki et al 2004) and this could occur at the
level of regulation However earlier studies with the same
enzyme showed no change in clearance with age (Hunt et
al 1992) To date there has been even less examination of
the transporter functions and any changes with age
(Kinirons amp OrsquoMahony 2004) so the current understand-
ing of the effects of aging on metabolism and transport is
anything but transparent This represents an extreme
challenge for the pharmaceutical industry how to predict
whether a drug has an affinity for an enzyme or transporter
and whether this may be clinically important if it shows a
decline in expression or function with aging Any decrease
of function of these enzymes or transporters in an elderly
population would result in changes in the metabolism and
transport of metabolites potentially impacting the clinical
effect This represents another example of how computa-
tional approaches may perhaps be used with different types
of predicted and experimental data to allow visualization of
potential compound interactions or toxicity in elderly
populations We may see different gene networks high-
lighted as humans age and these may be modified by drug
treatment and coadministration This represents an area
were network analysis could be applied in the future and is
in need of considerable further research
The collection of microarray data in databases such as
CEBS ArrayTrack and EDGE represents a future resource
for computational gene network analysis One could
envisage that ultimately in each case such data is converted
to one or more networks that are also displayed for the user
and can be used to compare treatments from in vivo and in
vitro experiments This would represent a different approach
to clustering the data as currently implemented in one of
these efforts (Hayes et al 2005) and may condense large
amounts of experimentally derived data into a readily
interpreted network
4 Discussion
Previously in this journal the progress of many
research groups in predicting human ADME parameters
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6660
in silico (Ekins et al 2000b) and approaches for drug
metabolism (Ekins Ring Grace McRobie-Belle amp
Wrighton 2000a) have been described Both of these
reviews commented on moving HT assays for ADME
Tox much earlier in drug discovery which had also been
indicated by other groups The initial reviews also
highlighted the likely wealth of data that would become
available and how this could be used for structure
activity relationships alongside the bioactivity data in
computational models It was noted that there was a
paucity of predictive metabolism tools at that time In
addition a growing number of efforts to model whole
cells and organs now a field called systems biology
were recognized as models that could be integrated with
the in silico ADME approaches In summary since these
past reviews virtually all pharmaceutical companies have
attempted earlier high throughput screening for ADME
Tox properties and to some extent the wider application
of computational approaches for physicochemical proper-
ties Systems biology is being quite widely acknowledged
as the new paradigm for understanding complex bio-
logical datasets derived from high throughput technolo-
gies and the accumulated knowledge on human protein
interactions (Hartwell Hopfield Leibler amp Murray 1999
Hood 2003b) Therefore systems biology can be defined
as the integration of genetic proteomic transcriptomic
and metabonomic data using computational methods
(Nicholson amp Wilson 2003) When taken together
information on molecular processes derived from different
sources represents a lsquolsquouniversersquorsquo of putative biological
functionality of which only a small fraction of it will be
realized in a cell at any given time To date systems
biology has been driven by academia and funding bodies
such as the NIH rather than the pharmaceutical compa-
nies Presently there is a great deal of interest from
scientists of all backgrounds in identifying the networks
of cellular pathways and the corresponding physically
interacting proteins
The network building software for systems biology
described in this current review will be valuable to query
high throughput data and known literature interactions in
order to predict potential toxicity in different species In the
future the compilation of published toxicogenomics data-
sets characteristic of different types of toxicity will likely
be available in these software systems to act as a reference
database It is also feasible that we will be able to generate
the annotated datasets which specifically address the
differences between human and rat networks implicated
in toxicity The identification of sub-network modules
conserved between human and rat distinct for toxicity
types or predictive for toxic end-points in human will be
possible Such signature gene networks (Nikolsky et al
2005) can then be verified with other experimentally
derived data prospectively or from preexisting databases
The combined Fhigh throughput datandashin silico network_approaches therefore represents a novel method for access-
ing complex tissue-level processes as networks integrating
all data types based on functional interactions The data-
bases developed and used in this approach will certainly
benefit from further annotation around the drug metaboliz-
ing enzymes and transporters as described herein in terms
of transcriptional regulation and the ligands associated with
them which frequently appear in the literature It will be
important to capture disparities in the assignment of ligands
to enzymes transporters and other proteins as well as
negative data
In summary although there have been numerous
toxicogenomics studies published there is presently a
relatively small number of datasets that are freely available
to perform network analysis of microarray data The
number of studies identifying large numbers of proteins
which are affected by molecule treatment are even scarcer
still while there are several examples of some published
studies that combine such data It is hoped that the
numerous database initiatives for high content and toxicol-
ogy data that are being undertaken will improve the
situation for other researchers that are not currently
equipped to do such microarray studies themselves The
addition of requirements by journals to deposit such raw
data in a freely accessible resource will aid these initiatives
ADMETox groups have seen new technologies and
approaches developed over the last decade that have all
been applied to identify poor compounds earlier (Fig 2)
The latest technologies integrate network building tools
with high content data and databases The current review
described the limited number of networks generated for
ADMETox at present and one hopes that the impact of
such analyses will be commonplace in the future Systems
biology is however more than just applying a network
approach and hence systems-ADMETox will have to
evolve due to the continual pressure to develop newer
technologies This current paradigm combining empirical
data and computational methods should integrate the
complex data already generated making it readily inter-
pretable and valuable for identifying the most promising
compounds in the future
Acknowledgements
Dr Maggie AZ Hupcey is gratefully acknowledged for
editorial assistance Dr Peter W Swaan (University of
Maryland) and Dr Cheng Chang (Ohio State University)
Dr Steve Wright (University of Arizona) Dr K Sandy
Pang (University of Toronto) and Dr Craig Giroux (Wayne
State University) are kindly acknowledged for their support
and discussions My colleagues at GeneGo Sergey
Andreyev Andy Ryabov Eugene Kirillov Eugene A
Rakhmatulin Svetlana Sorokina Andrej Bugrim Tatiana
Nikolskaya Yuri Nikolsky John Metz and Julie Bryant are
all thanked for their considerable contributions to software
development and data annotation
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 61
References
Abe M Yamashita S Kuramoto T Hirayama Y Tsukamoto T Ohta
T et al (2003) Global expression analysis of N-methyl-NV-nitro-N-nitrosoguanidine-induced rat stomach carcinomas using oligonucleotide
microarrays Carcinogenesis 24 861ndash867
Adachi T Ono Y Koh K B Takashima K Tainaka H Matsuno Y
et al (2004) Long-term alteration of gene expression without
morphological change in testis after neonatal exposure to genistein in
mice Toxicogenomic analysis using cDNA microarray Food and
Chemical Toxicology 42 445ndash452
Akerman G S Rosenzweig B A Domon O E McGarrity L J
Blankenship L R Tsai C A et al (2004) Gene expression profiles
and genetic damage in benzo(a)pyrene diol epoxide-exposed TK6 cells
Mutation Research 549 43ndash64
Aksenov S V Church B Dhiman A Georgieva A Sarangapani R
Helmlinger G et al (2005) An integrated approach for inference and
mechanistic modeling for advancing drug development FEBS Letters
579 1878ndash1883
Anderle P Huang Y amp Sadee W (2004) Intestinal membrane transport
of drugs and nutrients Genomics of membrane transporters using
expression microarrays European Journal of Pharmaceutical Sciences
21 17ndash24
Annereau J P Szakacs G Tucker C J Arciello A Cardarelli C
Collins J et al (2004) Analysis of ATP-binding cassette transporter
expression in drug-selected cell lines by a microarray dedicated to
multidrug resistance Molecular Pharmacology 66 1397ndash1405
Apic G Ignjatovic T Boyer S amp Russell R B (2005) Illuminating drug
discovery with biological pathways FEBS Letters 579 1872ndash1877
Baker V A Harries H M Waring J F Duggan C M Ni H A Jolly
R A et al (2004) Clofibrate-induced gene expression changes in rat
liver A cross-laboratory analysis using membrane cDNA arrays
Environmental Health Perspectives 112 428ndash438
Balakin K V Ekins S Bugrim A Ivanenkov Y A Korolev D amp
Nikolsky Y et al (2004a) Quantitative structurendashmetabolism relation-
ship modeling of the metabolic N-dealkylation rates Drug Metabolism
and Disposition 32 1111ndash1120
Balakin K V Ekins S Bugrim A Ivanenkov Y A Korolev D amp
Nikolsky Y et al (2004b) Kohonen maps for prediction of binding to
human cytochrome P450 3A4 Drug Metabolism and Disposition 32
1183ndash1189
Barabasi A -L amp Oltvai Z N (2004) Network biology Under-
standing the cellrsquos functional organization Nature Reviews Genetics
5 101ndash113
Bartosiewicz M Penn S G amp Buckpitt A (2001) Applications of gene
arrays in environmental toxicology Fingerprints of gene regulation
associated with cadmium chloride benzo(a)pyrene and trichloro-
ethylene Environmental Health Perspectives 109 71ndash74
Bertilsson G Heidrich J Svensson K Asman M Jendeberg L
Sydow-Backman M et al (1998) Identification of a human nuclear
receptor defines a new signaling pathway for CYP3A induction
Proceedings of the National Academy of Sciences of the United States
of America 95 12208ndash12213
Blumberg B Sabbagh Jr W Juguilon H Bolado Jr J van Meter
C M Ong E S et al (1998) SXR a novel steroid and xenobiotic-
sensing nuclear receptor Genes and Development 12 3195ndash3205
Borodina Y Rudik A Filimonov D Kharchevnikova N Dmitriev A
Blinova V et al (2004) A new statistical approach to predicting
aromatic hydroxylation sites Comparison with model-based
approaches Journal of Chemical Information and Computer Sciences
44 1998ndash2009
Borodina Y Sadym A Filimonov D Blinova V Dmitriev A amp
Poroikov V (2003) Predicting biotransformation potential from
molecular structure Journal of Chemical Information and Computer
Sciences 43 1636ndash1646
Bottone Jr F G Martinez J M Collins J B Afshari C A amp Eling
T E (2003) Gene modulation by the cyclooxygenase inhibitor
sulindac sulfide in human colorectal carcinoma cells Possible link to
apoptosis Journal of Biological Chemistry 278 25790ndash25801
Boyer S amp Zamora I (2002) New methods in predictive metabolism
Journal of Computer-Aided Molecular Design 16 403ndash413
Bredel M amp Jacoby E (2004) Chemogenomics An emerging strategy
for rapid target and drug discovery Nature Reviews Genetics 5
262ndash275
Bugrim A Nikolskaya T amp Nikolsky Y (2004) Early prediction of drug
metabolism and toxicity Systems biology approach and modeling
Drug Discovery Today 9 127ndash135
Bulera S J Eddy S M Ferguson E Jatkoe T A Reindel J F
Bleavins M R et al (2001) RNA expression in the early character-
ization of hepatotoxicants in Wistar rats by high-density DNA micro-
arrays Hepatology 33 1239ndash1258
Butcher E C Berg E L amp Kunkel E J (2004) Systems biology in drug
discovery Nature Biotechnology 22 1253ndash1259
Butte A (2002) The use and analysis of microarray data Naturalist
Review of Drug Discovery 1 951ndash960
Cary M P Bader G D amp Sander C (2005) Pathway information for
systems biology FEBS Letters 579 1815ndash1820
Cheok M H Yang W Pui C H Downing J R Cheng C Naeve
C W et al (2003) Treatment-specific changes in gene expression
discriminate in vivo drug response in human leukemia cells Nature
Genetics 34 85ndash90
Cleary J D Rogers P D amp Chapman S W (2001) Differential
transcription factor expression in human mononuclear cells in response
to amphotericin B Identification with complementary DNA microarray
technology Pharmacotherapy 21 1046ndash1054
Coen M Ruepp S U Lindon J C Nicholson J K Pognan F Lenz
E M et al (2004) Integrated application of transcriptomics and
metabonomics yields new insight into the toxicity due to paracetamol in
the mouse Journal of Pharmaceutical and Biomedical Analysis 35
93ndash105
Cornwell P D De Souza A T amp Ulrich R G (2004) Profiling of
hepatic gene expression in rats treated with fibric acid analogs
Mutation Research 549 131ndash145
Csermely P Agoston V amp Pongor S (2005) The efficiency of multi-
target drugs The network approach might help drug design Trends in
Pharmacological Sciences 26 178ndash182
Cunningham M J Liang S Fuhrman S Seilhamer J J amp
Somogyi R (2000) Gene expression microarray data analysis for
toxicology profiling Annals of the New York Academy of Sciences 919
52ndash67
de Longueville F Atienzar F A Marcq L Dufrane S Evrard S
Wouters L et al (2003) Use of a low density microarray for studying
gene expression patterns induced by hepatotoxicants on primary
cultures of rat hepatocytes Toxicological Sciences 75 378ndash392
Dobrin R Beg Q K Barabasi A L amp Oltvai Z N (2004)
Aggregation of topological motifs in the Escherichia coli transcriptional
regulatory network BMC Bioinformatics 5 10
Donald S Verschoyle R D Edwards R Judah D J Davies R
Riley J et al (2002) Hepatobiliary damage and changes in hepatic
gene expression caused by the antitumor drug ecteinascidin-743 (ET-
743) in the female rat Cancer Research 62 4256ndash4262
Dumont M Jacquemin E DrsquoHont C Descout C Cresteil D Haouzi
D et al (1997) Expression of the liver Na+-independent organic anion
transporting polypeptide (oatp-1) in rats with bile duct ligation Journal
of Hepatology 27 1051ndash1056
Durnas C Loi C M amp Cusack B J (1990) Hepatic drug metabolism
and aging Clinical Pharmacokinetics 19 359ndash389
Eisen M B Spellman P T Brown P O amp Botstein D (1998) Cluster
analysis and display of genome-wide expression patterns Proceedings
of the National Academy of Sciences of the United States of America
95 14863ndash14868
Ekins S Andreyev S Ryabov A Kirilov E Rakhmatulin E A
Bugrim A et al (in press) Computational Prediction of Human Drug
Metabolism Exp Opin Drug Metab Toxicol
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6662
Ekins S Boulanger B Swaan P W amp Hupcey M A Z (2002)
Towards a new age of virtual ADMETOX and multidimensional drug
discovery Journal of Computer-Aided Molecular Design 16 381ndash401
Ekins S Bugrim A Nikolsky Y amp Nikolskaya T (2005) Systems
biology Applications in drug discovery In S Gad (Ed) Drug
Discovery Handbook (pp 123ndash183) Hoboken NJrsquo Wiley and Sons
Ekins S Giroux C N Nikolsky Y Bugrim A amp Nikolskaya T
(2005) A signature gene network approach to toxicity The Toxoco-
logists 84
Ekins S Kirillov E Rakhmatulin E amp Nikolskaya T (2005) A novel
method for visualizing nuclear hormone receptor networks relevant to
drug metabolism Drug Metabolism and Disposition 33 474ndash481
Ekins S amp McGowan R J (2001) The limits of reductionism The
shifting genomic paradigmrsquos impact on industry and academia
Philsophy in Science 9 1ndash23
Ekins S Mirny L amp Schuetz E G (2002) A ligand-based approach to
understanding selectivity of nuclear hormone receptors PXR CAR
FXR LXRa and LXRb Pharmaceutical Research 19 1788ndash1800
Ekins S Nikolsky Y amp Nikolskaya T (2005) Techniques Application
of systems biology to absorption distribution metabolism excretion
and toxicity Trends in Pharmacological Sciences 26 202ndash209
Ekins S Ring B J Grace J McRobie-Belle D J amp Wrighton S
A (2000) Present and future in vitro approaches for drug
metabolism Journal of Pharmacological and Toxicological Methods
44 313ndash324
Ekins S Waller C L Swaan P W Cruciani G Wrighton S A amp
Wikel J H (2000) Progress in predicting human ADME parameters in
silico Journal of Pharmacological and Toxicological Methods 44
251ndash272
Erhardt P W (2003) A human drug metabolism database Potential roles
in the quantitative predictions of drug metabolism and metabolism-
related drugndashdrug interactions Current Drug Metabolism 4 411ndash422
Evans W E amp McLeod H L (2003) Pharmacogenomicsmdashdrug
disposition drug targets and side effects New England Journal of
Medicine 348 538ndash549
Ezendam J Staedtler F Pennings J Vandebriel R J Pieters R
Boffetta P et al (2004) Toxicogenomics of subchronic hexachlor-
obenzene exposure in Brown Norway rats Environmental Health
Perspectives 112 782ndash791
Faiola B Fuller E S Wong V A amp Recio L (2004) Gene expression
profile in bone marrow and hematopoietic stem cells in mice exposed to
inhaled benzene Mutation Research 549 195ndash212
FDA (2004) Innovation stagnation Challenge and opportunity on the
critical path to new medicinal products
Fiehn O (2001) Combining genomics metabolome analysis and bio-
chemical modelling to understand metabolic networks Comparative
Functional Genomics 2 155ndash168
Field F J Born E amp Mathur S N (2004) LXRRXR ligand activation
enhances basolateral efflux of beta-sitosterol in CaCo-2 cells Journal of
Lipid Research 45 905ndash913
Fleck C Sutter L Appenroth D Koch B Meinhold T Pitack M
et al (2003) Use of gene chip technology for the characterisation of
the regulation of renal transport processes and of nephrotoxicity in
rats Experimental and Toxicologic Pathology 54 401ndash410
Fliri A F Loging W T Thadeio P F amp Volkmann R A (2005)
Biological spectra analysis Linking biological activity profiles to
molecular structure Proceedings of the National Academy of Sciences
of the United States of America 102 261ndash266
Fountoulakis M (2004) Application of proteomics technologies in the
investigation of the brain Mass Spectrometry Reviews 23 231ndash258
Gaucher S P Taylor S W Fahy E Zhang B Warnock D E Ghosh
S S et al (2004) Expanded coverage of the human heart
mitochondrial proteome using multidimensional liquid chromatography
coupled with tandem mass spectrometry Journal of Proteome
Research 3 495ndash505
Gerhold D Lu M Xu J Austin C Caskey C T amp Rushmore T
(2001) Monitoring expression of genes involved in drug metabolism
and toxicology using DNA microarrays Physiological Genomics 5
161ndash170
Guo G L Choudhuri S amp Klaassen C D (2002) Induction profile of
rat organic anion transporting polypeptide 2 (oatp2) by prototypical
drug-metabolizing enzyme inducers that activate gene expression
through ligand-activated transcription factor pathways Journal of
Pharmacology and Experimental Therapeutics 300 206ndash212
Hagenbuch B amp Meier P J (2004) Organic anion transporting
polypeptides of the OATP SLC21 family Phylogenetic classification
as OATP SLCO superfamily new nomenclature and molecularfunc-
tional properties Pflugers Archiv 447 653ndash665
Hamadeh H K amp Afshari C A (2004) Toxicogenomics principles and
applications Hobokenrsquo John Wiley and Sons
Hamadeh H K Bushel P R Jayadev S Martin K DiSorbo O Sieber
S et al (2002) Gene expression analysis reveals chemical-specific
profiles Toxicological Sciences 67 219ndash231
Hamadeh H K Jayadev S Gaillard E T Huang Q Stoll R
Blanchard K et al (2004) Integration of clinical and gene expression
endpoints to explore furan-mediated hepatotoxicity Mutation Research
549 169ndash183
Hamadeh H K Knight B L Haugen A C Sieber S Amin R P
Bushel P R et al (2002) Methapyrilene toxicity Anchorage of
pathologic observations to gene expression alterations Toxicologic
Pathology 30 470ndash482
Han J D Bertin N Hao T Goldberg D S Berriz G F Zhang L V
et al (2004) Evidence for dynamically organized modularity in the
yeast proteinndashprotein interaction network Nature 430 88ndash93
Hanisch D Zien A Zimmer R amp Lengauer T (2002) Co-clustering of
biological networks and gene expression data Bioinformatics 18
S145ndashS154
Harris A J Dial S L amp Casciano D A (2004) Comparison of basal
gene expression profiles and effects of hepatocarcinogens on gene
expression in cultured primary human hepatocytes and HepG2 cells
Mutation Research 549 79ndash99
Harrison S C (2004) Whither structural biology Nature Structural
Molecular Biology 11 12ndash15
Hartley D P Dai X He Y D Carlini E J Wang B Huskey S E
et al (2004) Activators of the rat pregnane X receptor differentially
modulate hepatic and intestinal gene expression Molecular Pharma-
cology 65 1159ndash1171
Hartwell L H Hopfield J J Leibler S amp Murray A W (1999) From
molecular to modular cell biology Nature 402 C47ndashC52
Hasmall S Orphanides G James N Pennie W Hedley K Soames
A et al (2002) Downregulation of lactoferrin by PPARalpha ligands
Role in perturbation of hepatocyte proliferation and apoptosis
Toxicology Sciences 68 304ndash313
Hayase T Yamamoto Y Yamamoto K Muso E amp Shiota K (2004)
Microarray profile analysis of toxic cocaine-induced alterations in the
expression of mouse brain gene sequences A possible rsquoprotectiversquo
effect of buprenorphine Journal of Applied Toxicology 24 15ndash20
Hayes K R Vollrath A L Zastrow G M McMillan B J Craven M
Jovanovich S et al (2005) EDGE A centralized resource for the
comparison analysis and distribution of toxicogenomic information
Molecular Pharmacology 67 1360ndash1368
Heijne W H Slitt A L van Bladeren P J Groten J P Klaassen C D
Stierum R H et al (2004) Bromobenzene-induced hepatotoxicity at
the transcriptome level Toxicology Sciences 79 411ndash422
Heijne W H Stierum R H Slijper M van Bladeren P J amp van
Ommen B (2003) Toxicogenomics of bromobenzene hepatotoxicity
A combined transcriptomics and proteomics approach Biochemical
Pharmacology 65 857ndash875
Heinloth A N Irwin R D Boorman G A Nettesheim P Fannin
R D Sieber S O et al (2004) Gene expression profiling of rat
livers reveals indicators of potential adverse effects Toxicology
Sciences 80 193ndash202
Hodges L C Cook J D Lobenhofer E K Li L Bennett L Bushel
P R et al (2003) Tamoxifen functions as a molecular agonist inducing
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 63
cell cycle-associated genes in breast cancer cells Molecular Cancer
Research 1 300ndash311
Holleman A Cheok M H den Boer M L Yang W Veerman KM
A J Kazemier et al (2004) Gene-expression patterns in drug-resistant
acute lymphoblastic leukemia cells and response to treatment New
England Journal of Medicine 351 533ndash542
Hong Y Muller U R amp Lai F (2003) Discriminating two classes of
toxicants through expression analysis of HepG2 cells with DNA arrays
Toxicology in Vitro 17 85ndash92
Hood E (2003) Proteomics Characterizing the cogs in the machinery of
life Environmental Health Perspectives 111 A816ndashA825
Hood L (2003) Systems biology Integrating technology biology and
computation Mechanism of Ageing and Development 124 9ndash16
Hood L amp Galas D (2003) The digital code of DNA Nature 421
444ndash448
Hood L amp Perlmutter R M (2004) The impact of systems approaches on
biological problems in drug discovery Nature Biotechnology 22
1215ndash1217
Hu T Gibson D P Carr G J Torontali S M Tiesman J P Chaney
J G et al (2004) Identification of a gene expression profile that
discriminates indirect-acting genotoxins from direct-acting genotoxins
Mutation Research 549 5ndash27
Huang Q Dunn II R T Jayadev S DiSorbo O Pack F D Farr S B
et al (2001) Assessment of cisplatin-induced nephrotoxicity by
microarray technology Toxicology Sciences 63 196ndash207
Huang Q Jin X Gaillard E T Knight B L Pack F D Stoltz J H et
al (2004) Gene expression profiling reveals multiple toxicity endpoints
induced by hepatotoxicants Mutation Research 549 147ndash167
Huisman M T Smit J W Crommentuyn K M Zelcer N Wiltshire
H R Beijnen J H et al (2002) Multidrug resistance protein 2
(MRP2) transports HIV protease inhibitors and transport can be
enhanced by other drugs Aids 16 2295ndash2301
Hunt C M Westerkam W R amp Stave G M (1992) Effect of age and
gender on the activity of human hepatic CYP3A Biochemical
Pharmacology 44 275ndash283
Ideker T Ozier O Schwikowski B amp Siegal A F (2002) Discovering
regulatory and signalling circuits in molecular interaction networks
Bioinformatics 18 S233ndashS240
Ideker T Thorsson V Ranish J A Christmas R Buhler J Eng J K
et al (2001) Integrated genomic and proteomic analyses of a system-
atically perturbed network Science 292 929ndash934
Iida M Anna C H Hartis J Bruno M Wetmore B Dubin J R et al
(2003) Changes in global gene and protein expression during early
mouse liver carcinogenesis induced by non-genotoxic model carcino-
gens oxazepam and Wyeth-14643 Carcinogenesis 24 757ndash770
Ingelman-Sundberg M (2004) Pharmacogenetics of cytochrome P450 and
its applications in drug therapy The past present and future Trends in
Pharmacological Sciences 25 193ndash200
Jeong H Mason S P Barabasi A L amp Oltvai Z N (2001) Lethality
and centrality in protein networks Nature 411 41ndash42
Jeong H Tombor B Albert R Oltvai Z N amp Barabasi A L
(2000) The large-scale organization of metabolic networks Nature
407 651ndash654
Jung D Hagenbuch B Gresh L Pontoglio M Meier P J amp Kullak-
Ublick G A (2001) Characterization of the human OATP-C
(SLC21A6) gene promoter and regulation of liver-specific OATP genes
by hepatocyte nuclear factor 1 alpha Journal of Biological Chemistry
276 37206ndash37214
Jung D amp Kullak-Ublick G A (2003) Hepatocyte nuclear factor 1
alpha A key mediator of the effect of bile acids on gene expression
Hepatology 37 622ndash631
Jung J -W Park J -S Hwang J -W Kang K -S YS L Song B
-S et al (2004) Gene expression analysis of peroxisome prolifer-
ators- and phenytoin-induced hepatotoxicity using cDNA microarray
Journal of Veterinary Medical Science 66 1329ndash1333
Kato N Shibutani M Takagi H Uneyama C Lee K -Y Takiami S
et al (2004) Gene expression profile in the livers of rats orally
administered ethinylestradiol for 28 days using a microarray technique
Toxicology 200 179ndash192
Kier L D Neft R Tang L Suizu R Cook T Onsurez K et al
(2004) Applications of microarrays with toxicologically relevant genes
(tox genes) for the evaluation of chemical toxicants in Sprague Dawley
rats in vivo and human hepatocytes in vitro Mutation Research 549
101ndash113
Kijima K Toyosawa K Yasuba M Matsuoka N Adachi T
Komiyama M et al (2004) Gene expression analysis of the rat testis
after treatment with di(2-ethylhexyl) phthalate using cDNA microarray
and real-time RT-PCR Toxicology and Applied Pharmacology 200
103ndash110
Kim R B (2003) Organic anion-transporting polypeptide (OATP) trans-
porter family and drug disposition European Journal of Clinical
Investigation 33 1ndash5
Kinirons M T amp OrsquoMahony M S (2004) Drug metabolism and ageing
British Journal of Clinical Pharmacology 57 540ndash544
Kitano H (2002a) Computational systems biology Nature 420 206ndash210
Kitano H (2002b) Systems biology A brief overview Science 295
1662ndash1664
Kliewer S A Moore J T Wade L Staudinger J L Watson M
A Jones S A et al (1998) An orphan nuclear receptor activated
by pregnanes defines a novel steroid signalling pathway Cell 92
73ndash82
Korolev D Balakin K V Nikolsky Y Kirillov E Ivanenkov Y A
Savchuk N P et al (2003) Modeling of human cytochrome p450-
mediated drug metabolism using unsupervised machine learning
approach Journal of Medicinal Chemistry 46 3631ndash3643
Kramer J A Pettit S D Amin R P Bertram T A Car B
Cunningham M et al (2004) Overview on the application of
transcription profiling using selected nephrotoxicants for toxicology
assessment Environmental Health Perspectives 112 460ndash464
Kultima K Nystrom A -M Scholz B Gustafson A -L Dencker L
amp Stigson M (2004) Valproic acid teratogenicity A toxicogenomics
approach Environmental Health Perspectives 112 1225ndash1235
Kutuzova G D amp Deluca H F (2004) Gene expression profiles in rat
intestine identify pathways for 125-dihydroxyvitamin D(3) stimulated
calcium absorption and clarify its immunomodulatory properties
Archives of Biochemistry and Biophysics 432 152ndash166
Kwak M K Wakabayashi N Itoh K Motohashi H Yamamoto M amp
Kensler T W (2003) Modulation of gene expression by cancer
chemopreventive dithiolethiones through the Keap1-Nrf2 pathway
Identification of novel gene clusters for cell survival Journal of
Biological Chemistry 278 8135ndash8145
Lahjouji K Mitchell G A amp Qureshi I A (2001) Carnitine transport
by organic cation transporters and systemic carnitine deficiency
Molecular Genetics and Metabolism 73 287ndash297
Landowski C P Sun D Foster D R Menon S S Barnett J L
Welage L S et al (2003) Gene expression in the human intestine and
correlation with oral valacyclovir pharmacokinetic parameters Journal
of Pharmacology and Experimental Therapeutics 306 778ndash786
Lee G -J Lee W -S Jeon K -S Um C -H Kim S -J Lee C -H
et al (2004) cDNA microarray gene expression analysis and
toxicological phenotype for anticancer drug Journal of Veterinary
Medical Science 66 1339ndash1345
Lee M Kwon J Kim S N Kim J E Koh W S Kim E J et al
(2003) cDNA microarray gene expression profiling of hydroxyurea
paclitaxel and p-anisidine genotoxic compounds with differing
tumorigenicity results Environmental and Molecular Mutagenesis
42 91ndash97
Li S Armstrong C M Bertin N Ge H Milstein S Boxem M et al
(2004) A map of the interactome network of the metazoan C elegans
Science 303 540ndash543
Liguori M J Anderson L M Bukofzer S McKim J Pregenzer J F
Retief J et al (2005) Microarray analysis in human hepatocytes
suggests a mechanism for hepatotoxicity induced by trovafloxacin
Hepatology 41 177ndash186
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6664
Lobenhofer E K Bennett L Cable P L Li L Bushel P R amp Afshari
C A (2002) Regulation of DNA replication fork genes by 17beta-
estradiol Molecular Endocrinology 16 1215ndash1229
Locker J Tian J Carver R Concas D Cossu C Ledda-Columbano
G M et al (2003) A common set of immediate-early response genes
in liver regeneration and hyperplasia Hepatology 38 314ndash325
Mattes W B Pettit S D Sansone S A Bushel P R amp Waters M D
(2004) Database development in toxicogenomics Issues and efforts
Environmental Health Perspectives 112 495ndash505
McMillian M Nie A Y Parker J B Leone A Bryant S Kemmerer
M et al (2004) A gene expression signature for oxidant stressreactive
metabolites in rat liver Biochemical Pharmacology 68 2249ndash2261
Meier P J amp Stieger B (2000) Molecular mechanisms in bile formation
News in Physiological Sciences 15 89ndash93
Meneses-Lorente G de Longueville F Dos Santos-Mendes S Bonnert
T P Jack A Evrard S et al (2003) An evaluation of a low-density
DNA microarray using cytochrome P450 inducers Chemical Research
in Toxicology 16 1070ndash1077
Meneses-Lorente G Guest P C Lawrence J Muniappa N Knowles
M R Skynner H A et al (2004) A proteomic investigation of drug-
induced steatosis in rat liver Chemical Research in Toxicology 17
605ndash612
Merrill C L Ni H Yoon L W Tirmenstein M A Narayanan P
Benavides G R et al (2002) Etomoxir-induced oxidative stress in
HepG2 cells detected by differential gene expression is confirmed
biochemically Toxicology Sciences 68 93ndash101
Milo R Shen-Orr S Itzkovitz S Kashtan N Chklovskii D amp Alon
U (2002) Network motifs Simple building blocks of complex
networks Science 298 824ndash827
Morgan K T Ni H Brown H R Yoon L Qualls C W Crosby L
M et al (2002) Application of cDNA microarray technology to in
vitro toxicology and the selection of genes for a real-time RT-PCR-
based screen for oxidative stress in Hep-G2 cells Toxicologic
Pathology 30 435ndash451
Mwinyi J Johne A Bauer S Roots I amp Gerloff T (2004) Evidence
for inverse effects of OATP-C (SLC21A6) 5 and 1b haplotypes on
pravastatin kinetics Clinical Pharmacology and Therapeutics 75
415ndash421
Nakata K Yukawa M Komiyama N Nakano T amp Kaminuma T
(2002) A nuclear receptor database that maps pathways to diseases
Genome Infomatics 13 515ndash516
Nicholson J K Holmes E Lindon J C amp Wilson I D (2004) The
challenges of modeling mammalian biocomplexity Nature Biotechnol-
ogy 22 1268ndash1274
Nicholson J K amp Wilson I D (2003) Understanding rsquoglobal systems
biology Metabonomics and the continuum of metabolism Nature
Reviews on Drug Discovery 2 668ndash676
Nikitin A Egorov S Daraselia N amp Mazo I (2003) Pathway studiomdash
the analysis and navigation of molecular networks Bioinformatics 19
2155ndash2157
Nikolsky Y Ekins S Nikolskaya T amp Bugrim A (2005) A novel
method for generation of signature networks as biomarkers from
complex high throughput data Toxicology Letters 158 20ndash29
Nikolsky Y Nikolskaya T amp Bugrim A (2005) Biological networks
and analysis of experimental data in drug discovery Drug Discovery
Today 10 653ndash662
Nishizato Y Ieiri I Suzuki H Kimura M Kawabata K Hirota T
et al (2003) Polymorphisms of OATP-C (SLC21A6) and OAT3
(SLC22A8) genes Consequences for pravastatin pharmacokinetics
Clinical Pharmacology and Therapeutics 73 554ndash565
Nozawa T Nakajima M Tamai I Noda K Nezu J Sai Y et al
(2002) Genetic polymorphisms of human organic anion transporters
OATP-C (SLC21A6) and OATP-B (SLC21A9) Allele frequencies in
the Japanese population and functional analysis Journal of Pharmacol-
ogy and Experimental Therapeutics 302 804ndash813
Nozawa T Sugiura S Nakajima M Goto A Yokoi T Nezu J et al
(2004) Involvement of organic anion transporting polypeptides in the
transport of troglitazone sulfate Implications for understanding trogli-
tazone hepatotoxicity Drug Metabolism and Disposition 32 291ndash294
Oliver D E Rubin D L Stuart J M Hewett M Klein T E amp
Altman R B (2002) Ontology development for a pharmacogenetics
knowledge base Package Symposion 88ndash99
Oram J F amp Lawn R M (2001) ABCA1 The gatekeeper for eliminating
excess tissue cholesterol Journal of Lipid Research 42 1173ndash1179
Oswald M Kullak-Ublick G A Paumgartner G amp Beuers U (2001)
Expression of hepatic transporters OATP-C and MRP2 in primary
sclerosing cholangitis Liver 21 247ndash253
Parsons A B Brost R I Ding H Li Z Zhang C Sheikh B et al
(2004) Integration of chemical-genetic and genetic interaction data
links bioactive compounds to cellular target pathways Nature Bio-
technology 22 62ndash69
Patki K C von Moltke L L Harmatz J S Hesse L M Court M H
amp Greenblatt D J (2004) Effect of age on in vitro triazolam
biotransformation in male human liver microsomes Journal of
Pharmacology and Experimental Therapeutics 308 874ndash879
Pereira-Leal J B Enright A J amp Ouzounis C A (2004) Detection of
functional modules from protein interaction networks Proteins 54
49ndash57
Plant N (2004) Interaction networks Coordinating responses to xeno-
biotic exposure Toxicology 202 21ndash32
Raghavendra Prasad H S Qi Z Srinivasan K N amp Gopalakrish-
nakone P (2004) Potential effects of tetrodotoxin exposure to
human glial cells postulated using microarray approach Toxicon 44
597ndash608
Reilly T P Bourdi M Brady J N Pise-Masison C A Radonovich M
F George J W et al (2001) Expression profiling of acetaminophen
liver toxicity in mice using microarray technology Biochemical and
Biophysical Research Communications 282 321ndash328
Rives A W amp Galitski T (2003) Modular organization of cellular
networks Proceedings of the National Academy of Sciences of the
United States of America 100 1128ndash1133
Rogers P D Pearson M M Cleary J D Sullivan D C amp Chapman
S W (2002) Differential expression of genes encoding immunomodu-
latory proteins in response to amphotericin B in human mononuclear
cells identified by cDNA microarray analysis Journal of Antimicrobial
Chemotherapy 50 811ndash817
Rosen M B Wilson V S Schmid J E amp Gray L E (2005) Gene
expression analysis in the ventral prostate of rats exposed to vinclozolin
or procymidone Reproductive Toxicology 19 367ndash379
Rost D Herrmann T Sauer P Schmidts H L Stieger B Meier P J
et al (2003) Regulation of rat organic anion transporters in bile salt-
induced cholestatic hepatitis Effect of ursodeoxycholate Hepatology
38 187ndash195
Rothman R B Ayestas M A Dersch C M amp Baumann M H (1999)
Aminorex fenfluramine and chlorphentermine are serotonin transporter
substrates Implications for primary pulmonary hypertension Circula-
tion 100 869ndash875
Ruepp S U Tonge R P Shaw J Wallis N amp Pognan F (2002)
Genomics and proteomics analysis of acetaminophen toxicity in mouse
liver Toxicology Sciences 65 135ndash150
Sakaeda T Nakamura T amp Okumura K (2002) MDR1 genotype-
related pharmacokinetics and pharmacodynamics Biological amp Phar-
maceutical Bulletin 25 1391ndash1400
Sakata T Anzai N Shin H J Noshiro R Hirata T Yokoyama H
et al (2004) Novel single nucleotide polymorphisms of organic cation
transporter 1 (SLC22A1) affecting transport functions Biochemical
and Biophysical Research Communications 313 789ndash793
Satlin L M Amin V amp Wolkoff A W (1997) Organic anion
transporting polypeptide mediates organic anionHCO3-exchange
Journal of Biological Chemistry 272 26340ndash26345
Satomi Y Tsuchiya W Mihara K Ota M Kasahara Y amp Akahori F
(2004) Gene expression analysis of the lung following paraquat
administration in rats using DNA microarray Journal of Toxicological
Sciences 29 91ndash100
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 65
Schrenk D Baus P R Ermel N Klein C Vorderstemann B amp
Kauffmann H M (2001) Up-regulation of transporters of the MRP
family by drugs and toxins Toxicology Letters 120 51ndash57
Segal E Shapira M Regev A Persquoer D Botstein D Koller D et al
(2003) Module networks Identifying regulatory modules and their
condition-specific regulators from gene expression data Nature
Genetics 34 166ndash176
Segal E Wang H amp Koller D (2003) Discovering molecular pathways
from protein interaction and gene expression Bioinformatics 19
i264ndash i272
Seidel S D Kan H L Stott W T Schisler M R amp Gollapudi B B
(2003) Identification of transcriptome profiles for the DNA-damaging
agents bleomycin and hydrogen peroxide in L5178Y mouse lymphoma
cells Environmental and Molecular Mutagenesis 42 19ndash25
Shan L Yu Y Schut H A J amp Snyderwine E G (2004) Susceptibility
of rats to mammary gland carcinogenesis by the food-derived
carcinogen 2-Amino-1-Methyl-6-Phenylimidazo[45-b]Pyridine (PhIP)
varies with age and is associated with the induction of differential gene
expression American Journal of Pathology 165 191ndash202
Sharom J R Bellows D S amp Tyers M (2004) From large networks to
small molecules Current Opinion in Chemical Biology 8 81ndash90
Shitara Y Hirano M Sato H amp Sugiyama Y (2004) Gemfibrozil and
its glucuronide inhibit the OATP2(OATP1B1 SLC21A6)-mediated
hepatic uptake and CYP2C8-mediated metabolism of cerivastatinmdash
Analysis of the mechanism of the clinically relevant drugndashdrug
interaction between cerivastatin and gemfibrozil Journal of Pharma-
cology and Experimental Therapeutics
Shitara Y Itoh T Sato H Li A P amp Sugiyama Y (2003) Inhibition of
transporter-mediated hepatic uptake as a mechanism for drugndashdrug
interaction between cerivastatin and cyclosporin A Journal of
Pharmacology and Experimental Therapeutics 304 610ndash616
Somogyi R Fuhrman S amp Wen X (2001) Genetic network inference in
computational models and applications to large-scale gene expression
data In M Bower amp H Bolouri (Eds) Computational modeling of
genetic and biochemical networks (pp 119ndash157) Cambridge MArsquoThe
MIT Press
Spirin V amp Mirny L A (2003) Protein complexes and functional
modules in molecular networks Proceedings of the National Academy
of Sciences of the United States of America 100 12123ndash12128
Staudinger J Liu Y Madan A Habeebu S amp Klaassen C D
(2001) Coordinate regulation of xenobiotic and bile acid homeo-
stasis by pregnane X receptor Drug Metabolism and Disposition
29 1467ndash1472
Sun L Z Ji Z L Chen X Wang J F amp Chen Y Z (2002) ADME-
AP A database of ADME associated proteins Bioinformatics 18
1699ndash1700
Suter L Babiss L E amp Wheeldon E B (2004) Toxicogenomics in
predictive toxicology in drug development Chemistry and Biology 11
161ndash171
Synold T W Dussault I amp Forman B M (2001) The orphan nuclear
receptor SXR coordinately regulates drug metabolism and efflux
Nature Medicine 7 584ndash590
Tabuchi Y amp Kondo T (2003) cDNA microarray analysis reveals chop-
10 plays a key role in Sertoli cell injury induced by bisphenol A
Biochemical and Biophysical Research Communications 305 54ndash61
Tamai I Nezu J Uchino H Sai Y Oku A Shimane M et al (2000)
Molecular identification and characterization of novel members of the
human organic anion transporter (OATP) family Biochemical and
Biophysical Research Communications 273 251ndash260
Taylor S W Fahy E Zhang B Glenn G M Warnock D E Wiley S
et al (2003) Characterization of the human heart mitochondrial
proteome Nature Biotechnology 21 281ndash286
Terasaka S Aita Y Inoue A Hayashi S Nishigaki M Aoyagi K
et al (2004) Using a customized DNA microarray for expression
profiling of the estrogen-responsive genes to evaluate estrogen activity
among natural estrogens and industrial chemicals Environmental
Health Perspectives 112 773ndash781
Thomas D M Francescutti-Verbeem D M Liu X amp Kuhn D M
(2004) Identification of differentially regulated transcripts in mouse
striatum following methamphetamine treatmentmdashan oligonucleotide
microarray approach Journal of Neurochemistry 88 380ndash393
Thomas R S Rank D R Penn S G Zastrow G M Hayes K R
Pande K et al (2001) Identification of toxicologically predictive
gene sets using cDNA microarrays Molecular Pharmacology 60
1189ndash1194
Thompson K L Afshari C A Amin R P Bertram T A Car B
Cunningham M et al (2004) Identification of platform-independent
gene expression markers of cisplatin nephrotoxicity Environmental
Health Perspectives 112 488ndash494
Tirona R G Leake B F Merino G amp Kim R B (2001)
Polymorphisms in OATP-C Identification of multiple allelic
variants associated with altered transport activity among European-
and African-Americans Journal of Biological Chemistry 276
35669ndash35675
Tirona R G Leake B F Wolkoff A W amp Kim R B (2003)
Human organic anion transporting polypeptide-C (SLC21A6) is a
major determinant of the rifampin-mediated pregnane X receptor
activation Journal of Pharmacology and Experimental Therapeutics
304 223ndash228
Tong W Cao X Harris S Sun H Fang H Fuscoe J et al (2003)
ArrayTrackmdashsupporting toxicogenomic research at the US Food and
Drug Administration National Center for Toxicological Research
Environmental Health Perspectives 111 1819ndash1826
Tornow S amp Mewes H W (2003) Functional modules by relating
protein interaction networks and gene expression Nucleic Acids
Research 31 6283ndash6289
Trocho C Escola-Gil J C Ribas V Benitez S Martin-Campos J M
Rotllan N et al (2004) Phenytoin treatment reduces atherosclerosis in
mice through mechanisms independent of plasma HDL-cholesterol
concentration Atherosclerosis 174 275ndash285
Ueda A Hamadeh H K Webb H K Yamamoto Y Sueyoshi T
Afshari C A et al (2002) Diverse roles of the nuclear orphan
receptor CAR in regulating hepatic genes in response to phenobarbital
Molecular Pharmacology 61 1ndash6
Ulrich R G (2003) The toxicogenomics of nuclear receptor agonists
Current Opinion in Chemical Biology 7 505ndash510
Ulrich R G Rockett J C Gibson G G amp Pettit S D (2004)
Overview of an interlaboratory collaboration on evaluating the effects
of model hepatotoxicants on hepatic gene expression Environmental
Health Perspectives 112 423ndash427
Unami A Shinohara Y Ichikawa T amp Baba Y (2003) Biochemical
and microarray analyses of bupivacaine-induced apoptosis Journal of
Toxicological Sciences 28 77ndash94
Vasquez A Flammini A Maritan A amp Vespignani A (2003) Global
protein function prediction from proteinndashprotein interaction networks
Nature Biotechnology 21 697ndash700
Vezina C M Walker N J amp Olson J R (2004) Subchronic Exposure to
TCDD PeCDF PCB126 and PCB153 Effect on hepatic gene
expression Environmental Health Perspectives 112 1636ndash1644
Vo T D Greenberg H J amp Palsson B O (2004) Reconstruction and
functional characterization of the human mitochondrial metabolic
network based on proteomic and biochemical data Journal of Bio-
logical Chemistry 279 39532ndash39540
Wang H amp LeCluyse E L (2003) Role of orphan nuclear receptors in
the regulation of drug-metabolising enzymes Clinical Pharmacoki-
netics 42 1331ndash1357
Wang N Silver D L Thiele C amp Tall A R (2001) ATP-
binding cassette transporter A1 (ABCA1) functions as a choles-
terol efflux regulatory protein Journal of Biological Chemistry 276
23742ndash23747
Waring J F Cavet G Jolly R A McDowell J Dai H Ciurlionis R
et al (2003) Development of a DNA Microarray for toxicology based
on hepatoxin-regulated sequences Environmental Health Perspectives
111 863ndash870
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6666
Waring J F Gum R Morfitt D Jolly R A Ciurlionis R Heindel M
et al (2002) Identifying toxic mechanisms using DNA microarrays
Evidence that an experimental inhibitor of cell adhesion molecule
expression signals through the aryl hydrocarbon nuclear receptor
Toxicology 181ndash182 537ndash550
Waring J F Jolly R A Ciurlionis R Lum P Y Praestgaard J T
Morfitt D C et al (2001) Clustering of hepatotoxins based on
mechanism of toxicity using gene expression profiles Toxicology and
Applied Pharmacology 175 28ndash42
Waters M Boorman G Bushel P Cunningham M Irwin R Merrick
A et al (2003) Systems toxicology and the Chemical Effects in
Biological Systems (CEBS) knowledge base EHP Toxicogenomics
111 15ndash28
Wei M Arnold L Cano M amp Cohen S M (2005) Effects of co-
administration of antioxidants and arsenicals on the rat urinary bladder
epithelium Toxicology Sciences 83 237ndash245
Weinshilboum R (2003) Inheritance and drug response New England
Journal of Medicine 348 529ndash537
Weinshilboum R amp Wang L (2004) Pharmacogenetics Inherited
variation in amino acid sequence and altered protein quantity Clinical
Pharmacology and Therapeutics 75 253ndash258
Werner E (2003) In silico multicellular systems biology and minimal
genomes Drug Discovery Today 8 1121ndash1127
Wong J S amp Gill S S (2002) Gene expression changes induced in
mouse liver by di(2-ethylhexyl) phthalate Toxicology and Applied
Pharmacology 185 180ndash196
Xie T Tong L McCann U D Yuan J Becker K G Mechan A O
et al (2004) Identification and characterization of metallothionein-1
and -2 gene expression in the context of ()34-methylenedioxymetham-
phetamine-induced toxicity to brain dopaminergic neurons Journal of
Neuroscience 24 7043ndash7050
Yadetie F Laegreid A Bakke I Kusnierczyk W Komorowski J
Waldum H L et al (2003) Liver gene expression in rats in response
to the peroxisome proliferator-activated receptor-alpha agonist ciprofi-
brate Physiological Genomics 15 9ndash19
Yamashita S Nomoto T Abe M Tatematsu M Sugimura T amp
Ushijima T (2004) Persistence of gene expression changes in stomach
mucosae induced by short-term N-methyl-NV-nitro-N-nitrosoguanidinetreatment and their presence in stomach cancers Mutation Research
549 185ndash193
Yan Q amp Sadee W (2000) Human membrane transporter database A
web-accessible relational database for drug transport studies and
pharmacogenomics AAPS Pharmsci 2 E20
Yeger-Lotem E amp Margalit H (2003) Detection of regulatory circuits by
integrating the cellular networks of proteinndashprotein interactions and
transcription regulation Nucleic Acids Research 31 6053ndash6061
Yoon B I Li G -X Kitada K Kawasaki Y Igarashi K amp Kodama
Y et al (2003) Mechanisms of benzene-induced hematotoxicity and
leukemogenicity cDNA microarray analyses using mouse bone marrow
tissue Environmental Health Perspectives 111 1411ndash1420
Young M B DiSilvestro M R Sendera T J Freund J Kriete A amp
Magnuson S R (2003) Analysis of gene expression in carbon
tetrachloride-treated rat livers using a novel bioarray technology
Pharmacogenomics Journal 3 41ndash52
Yu H Zhu X Greenbaum D Karro J amp Gerstein M (2004) TopNet
A tool for comparing biological sub-networks correlating protein
properties with topological statistics Nucleic Acids Research 32
328ndash337
Zhang E Y Fu D J Pak Y A Stewart T Mukhopadhyay N
Wrighton S A et al (2004) Genetic polymorphisms in human proton-
dependent dipeptide transporter PEPT1 Implications for the functional
role of Pro586 Journal of Pharmacology and Experimental Therapeu-
tics 310 437ndash445
Zhang E Y Knipp G T Ekins S amp Swaan P W (2002)
Structural biology and function of solute transporters Implications
for identifying and designing substrates Drug Metabolism Reviews
34 709ndash750
Zhang E Y Phelps M A Cheng C Ekins S amp Swaan P W (2002)
Modeling of active transport systems Advanced Drug Delivery
Reviews 54 329ndash354
- Systems-ADMETox Resources and network approaches
-
- Introduction
- Data available
- Network analysis and databases
-
- Network applications
-
- The role of transporters
-
- Clinical relevance of transporters
- Transporter network examples ABCA1
- Transporter network examples OATP
- Transporter microarray data
-
- Applications to enzymes
- Future network applications
-
- Discussion
- Acknowledgements
- References
-
Fig 5 The network of direct gene interactions around the ABCA1 transporter gene (centre) generated with MetaCorei The interation types between nodes are shown as small colored hexagons eg unspecified
allosteric regulation binding cleavage competition covalent modification dephosphorylation phosphorylation transcription regulation transformation When applicable interactions also have a positive or
negative effect and direction Ligands (purple) linked to other proteins (blue blobs) transfactors (red) enzymes (orange) (For interpretation of the references to colour in this figure legend the reader is referred to
the web version of this article)
SEkin
sJournalofPharm
acologica
landToxico
logica
lMeth
ods53(2006)38ndash66
53
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6654
this transporterrsquos hepatic uptake of other compounds may be
important for reported drugndashdrug interactions (Kim 2003)
described earlier (Shitara et al 2003) as well as cerivastatin
with gemfibrozil (Shitara Hirano Sato amp Sugiyama 2004)
OATP1B1 (previous names OATP-C LST-1 OATP2
SLC21A6) represents the most studied human OATP to
date (Meier amp Stieger 2000) and is expressed on the
basolateral plasma membrane of hepatocytes Several single
nucleotide polymorphisms have been identified in the
OATP1B1 gene in European-Americans African-Americans
(Tirona et al 2001) and Japanese (Nozawa et al 2002)
dramatically impacting the transport of ligands such as
pravastatin (Mwinyi Johne Bauer Roots amp Gerloff 2004
Nishizato et al 2003) estrone-3-sulfate (Nozawa et al
2002 Tirona et al 2001) Rifampin (Tirona Leake
Wolkoff amp Kim 2003) and estradiol 17h-d-glucuronide(Tirona et al 2001)
The regulation of SLCO may be affected during
extrahepatic cholestasis bile duct ligation bile salt induced
Fig 6 Networks generated with the autoexpand algorithm in MetaCorei to illustr
be interconnected with other protein regulatory signaling information Informa
unspecified allosteric regulation binding cleavage competition covalent m
transformation When applicable interactions also have a positive or negative e
transfactors (red) enzymes (orange) (For interpretation of the references to colour
cholestatic hepatitis (Dumont et al 1997 Meier amp Stieger
2000 Rost et al 2003) and primary sclerosing cholangitis
(Oswald Kullack-Ublick Paumgarter amp Beuer 2001) In
particular OATP1B1 appears to be regulated by the liver-
enriched transcription factor hepatocyte nuclear factor 1a
(HNF1a) which binds to the promoter region of this
transporter (Jung et al 2001) Site directed mutagenesis
of this binding site resulted in inactivation suggesting the
critical nature of the interaction with HNF1a Bile acids
such as CDCA have been shown to transcriptionally repress
HNF1a in vitro via inhibition of the transactivating effect of
HNF4a on HNF1a (Jung amp Kullak-Ublick 2003) After
screening many rat and human uptake transporters in vitro
OATP1B1 was also shown to modulate the PXR response
by controlling rifampin retention in the cell and therefore
affecting the induction of CYP3A4 and other gene products
such as P-gp (Tirona et al 2003)
Some of the literature for OATP1B1 human substrate
data has been annotated into MetaCorei to illustrate the
ate how ligands for the human OATP-C (OATP1B1) transporter (centre) can
tion on the type of interaction between objects is hidden for clarity eg
odification dephosphorylation phosphorylation transcription regulation
ffect and direction Ligands (purple) linked to other proteins (blue blobs)
in this figure legend the reader is referred to the web version of this article)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 55
visualization of the complex interconnections between this
transporter its ligands regulatory factors and signaling
molecules already in the database (Fig 6) The network was
generated with the autoexpand algorithm in the software
representing one of multiple available algorithms for
connecting genes ligands and other objects in the database
Clearly if more ligands and their connections are added to
the database the complexity of the network will increase
considerably The OATP-C (OATP1B1) gene details can be
viewed upon querying the database and links are provided
to other public databases This page can be used to highlight
the multiple synonyms for this gene as well as links to the
multiple SNPs identified to date
3114 Transporter microarray data Microarrays have
generally been limited in the number of transporters present
on them (Annereau et al 2004) however they have been used
in an attempt to correlate pharmacokinetic properties with
gene expression for valacyclovir (Landowski et al 2003) as
well as understand the expression profile in different tissues
or cell lines upon food component or xenobiotic treatment
(Anderle Huang amp Sadee 2004) This lack of transporters
on microarrays has prompted some groups to produce their
own arrays with a heavier emphasis on transporters These
arrays have for example then been used to demonstrate the
upregulation of ABC transporters and down-regulation of
GST-Pi in cell lines resistant to colchicines or 9-nitro-
camptothecin (Annereau et al 2004) The genes that were
significantly up or down-regulated in this particular study
were used to build networks with MetaCore (Fig 7A B) and
the similarities between them were assessed (Fig 7C)
Although there were only a small number of significantly
changed genes in common (IL-8 Fos GST-Pi Calpactin and
Ubiquitin hydrolase) it is perhaps likely that there is a much
larger common gene network that is important for drug
resistance although a much larger number of cell lines and
drug treatments need to be evaluated to produce a definitive
drug resistance signature involving transporters enzymes
and transcriptional regulators
312 Applications to enzymes
As we have already described it may be particularly
valuable to visualize enzymes as networks to show
interactions with transcriptional regulators and ligands
For example a key enzyme is CYP3A4 which metabolizes
40ndash45 of all drugs and has relatively few SNPs
(Ingelman-Sundberg 2004) Using a second software suite
MetaDrugi (wwwgenegocom) it is possible to construct
a custom network around this or other drug metabolizing
enzymes (Fig 8) In this case the gene network for
CYP3A4 highlights all of the major transcriptional
regulators and several more distant linked proteins and
ligands connected on the network that may be useful for
further study (Fig 8) Substrates inhibitors as well as
regulatory factors and other enzymes can be observed
connected on this network Due to the many hundreds of
known inhibitors and substrates only a small number are
shown here for clarity We assume that if a perturbation in
a pathway (eg due to a nonfunctional enzyme) is linked
to a certain pathologic condition a similar perturbation
caused by the interference from xenobiotic metabolism
(eg competitive inhibition of the same enzyme) may
result in identical effects As microarray gene expression
data is increasingly generated the role of enzyme
regulation in toxicity of certain xenobiotics will become
more apparent from either in vivo or in vitro studies The
visualization of such signature gene networks involving
transporters and enzymes their ligands and regulatory
factors will also be important for future toxicity prediction
methods We have recently generated visualizations of
microarray data from MCF-7 cells treated with 4-hydrox-
ytamoxifen to show that some of the key genes involved in
metabolism and transport are upregulated (Ekins et al
2005d) In addition we have made predictions with various
QSAR models in MetaDrug to indicate the involvement of
PXR CYP3A4 and P-gp (Ekins et al 2005e) Therefore it
appears likely that 4-hydroxytamoxifen could increase its
own metabolism as well as efflux from cells via P-gp
which can be visualized on networks Any decrease of
function of these enzymes or transporters in a population
would likely result in changes in the metabolism and
transport of this active metabolite potentially impacting
the clinical effect This represents one example of how
both pathway tools QSAR models and network building
algorithms can be used with different types of predicted
and experimental data to allow visualization of potential
compound interactions or toxicity
313 Future network applications
As the population ages an increasing incidence and
prevalence of systemic diseases especially chronic diseases
have occurred among older adults This has resulted in an
increase in medications used concomitantly by this
population which presents challenges for drugndashdrug
interactions Physiologically elderly patients may behave
differently to the younger patients for which the drugs are
initially developed for and tested on Many pharmacoki-
netic investigations in the elderly population reveal
decreased clearance of lipophilic drugs metabolized by
the cytochrome P450 enzymes however few studies have
evaluated aging-dependent or gender-related changes in
specific P450 enzymes (Hunt Westerkam amp Slave 1992)
Age-related physiological changes such as a reduction in
liver mass hepatic metabolizing enzyme activity liver
blood flow and alterations in plasma drug binding may
account for the decreased elimination of some metabolized
drugs in the elderly It is particularly difficult to separate an
effect of aging from the variation in the rate of metabolism
due to factors such as individual metabolic phenotype
(slow or fast metabolizer due to SNPs) environmental
influences concomitant disease states and drug intake
(drugndashdrug interactions) The available data suggest that
Fig 7 Networks for drug resistant cell lines generated with the Auto expand algorithm in MetaCore A KB-3-1 vs KB-8-5 B ATC-DU-145 vs RCO1 C node overlap of A and B with initial nodes highlighted
with blue or red solid circles (Initial gene lists derived from (Annereau et al 2004)) Information on the type of interaction between objects is hidden for clarity eg unspecified allosteric regulation binding
cleavage competition covalent modification dephosphorylation phosphorylation transcription regulation transformation When applicable interactions also have a positive or negative effect and direction
Ligands (purple) linked to other proteins (blue blobs) transfactors (red) enzymes (orange) (For interpretation of the references to colour in this figure legend the reader is referred to the web version of this article)
SEkin
sJournalofPharm
acologica
landToxico
logica
lMeth
ods53(2006)38ndash66
56
Fig7(continued)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 57
Fig7(continued)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6658
Fig 8 The network of gene interactions around the CYP3A4 enzyme (centre) from MetaCorei Information on the type of interaction between objects is
hidden for clarity eg unspecified allosteric regulation binding cleavage competition covalent modification dephosphorylation phosphorylation
transcription regulation transformation When applicable interactions also have a positive or negative effect and direction Ligands (purple) linked to other
proteins (blue blobs) transfactors (red) enzymes (orange) (For interpretation of the references to colour in this figure legend the reader is referred to the web
version of this article)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 59
the initial doses of drugs metabolized by these enzymes
should be reduced in older patients according to the clinical
response In most published studies the elderly appear at
least as responsive as the young to inducers or inhibitors of
P450s (Durnas Loi amp Cusack 1990) More recently there
has been some suggestion that there are age related
reductions in function of some specific P450s such as
CYP3A4 (Patki et al 2004) and this could occur at the
level of regulation However earlier studies with the same
enzyme showed no change in clearance with age (Hunt et
al 1992) To date there has been even less examination of
the transporter functions and any changes with age
(Kinirons amp OrsquoMahony 2004) so the current understand-
ing of the effects of aging on metabolism and transport is
anything but transparent This represents an extreme
challenge for the pharmaceutical industry how to predict
whether a drug has an affinity for an enzyme or transporter
and whether this may be clinically important if it shows a
decline in expression or function with aging Any decrease
of function of these enzymes or transporters in an elderly
population would result in changes in the metabolism and
transport of metabolites potentially impacting the clinical
effect This represents another example of how computa-
tional approaches may perhaps be used with different types
of predicted and experimental data to allow visualization of
potential compound interactions or toxicity in elderly
populations We may see different gene networks high-
lighted as humans age and these may be modified by drug
treatment and coadministration This represents an area
were network analysis could be applied in the future and is
in need of considerable further research
The collection of microarray data in databases such as
CEBS ArrayTrack and EDGE represents a future resource
for computational gene network analysis One could
envisage that ultimately in each case such data is converted
to one or more networks that are also displayed for the user
and can be used to compare treatments from in vivo and in
vitro experiments This would represent a different approach
to clustering the data as currently implemented in one of
these efforts (Hayes et al 2005) and may condense large
amounts of experimentally derived data into a readily
interpreted network
4 Discussion
Previously in this journal the progress of many
research groups in predicting human ADME parameters
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6660
in silico (Ekins et al 2000b) and approaches for drug
metabolism (Ekins Ring Grace McRobie-Belle amp
Wrighton 2000a) have been described Both of these
reviews commented on moving HT assays for ADME
Tox much earlier in drug discovery which had also been
indicated by other groups The initial reviews also
highlighted the likely wealth of data that would become
available and how this could be used for structure
activity relationships alongside the bioactivity data in
computational models It was noted that there was a
paucity of predictive metabolism tools at that time In
addition a growing number of efforts to model whole
cells and organs now a field called systems biology
were recognized as models that could be integrated with
the in silico ADME approaches In summary since these
past reviews virtually all pharmaceutical companies have
attempted earlier high throughput screening for ADME
Tox properties and to some extent the wider application
of computational approaches for physicochemical proper-
ties Systems biology is being quite widely acknowledged
as the new paradigm for understanding complex bio-
logical datasets derived from high throughput technolo-
gies and the accumulated knowledge on human protein
interactions (Hartwell Hopfield Leibler amp Murray 1999
Hood 2003b) Therefore systems biology can be defined
as the integration of genetic proteomic transcriptomic
and metabonomic data using computational methods
(Nicholson amp Wilson 2003) When taken together
information on molecular processes derived from different
sources represents a lsquolsquouniversersquorsquo of putative biological
functionality of which only a small fraction of it will be
realized in a cell at any given time To date systems
biology has been driven by academia and funding bodies
such as the NIH rather than the pharmaceutical compa-
nies Presently there is a great deal of interest from
scientists of all backgrounds in identifying the networks
of cellular pathways and the corresponding physically
interacting proteins
The network building software for systems biology
described in this current review will be valuable to query
high throughput data and known literature interactions in
order to predict potential toxicity in different species In the
future the compilation of published toxicogenomics data-
sets characteristic of different types of toxicity will likely
be available in these software systems to act as a reference
database It is also feasible that we will be able to generate
the annotated datasets which specifically address the
differences between human and rat networks implicated
in toxicity The identification of sub-network modules
conserved between human and rat distinct for toxicity
types or predictive for toxic end-points in human will be
possible Such signature gene networks (Nikolsky et al
2005) can then be verified with other experimentally
derived data prospectively or from preexisting databases
The combined Fhigh throughput datandashin silico network_approaches therefore represents a novel method for access-
ing complex tissue-level processes as networks integrating
all data types based on functional interactions The data-
bases developed and used in this approach will certainly
benefit from further annotation around the drug metaboliz-
ing enzymes and transporters as described herein in terms
of transcriptional regulation and the ligands associated with
them which frequently appear in the literature It will be
important to capture disparities in the assignment of ligands
to enzymes transporters and other proteins as well as
negative data
In summary although there have been numerous
toxicogenomics studies published there is presently a
relatively small number of datasets that are freely available
to perform network analysis of microarray data The
number of studies identifying large numbers of proteins
which are affected by molecule treatment are even scarcer
still while there are several examples of some published
studies that combine such data It is hoped that the
numerous database initiatives for high content and toxicol-
ogy data that are being undertaken will improve the
situation for other researchers that are not currently
equipped to do such microarray studies themselves The
addition of requirements by journals to deposit such raw
data in a freely accessible resource will aid these initiatives
ADMETox groups have seen new technologies and
approaches developed over the last decade that have all
been applied to identify poor compounds earlier (Fig 2)
The latest technologies integrate network building tools
with high content data and databases The current review
described the limited number of networks generated for
ADMETox at present and one hopes that the impact of
such analyses will be commonplace in the future Systems
biology is however more than just applying a network
approach and hence systems-ADMETox will have to
evolve due to the continual pressure to develop newer
technologies This current paradigm combining empirical
data and computational methods should integrate the
complex data already generated making it readily inter-
pretable and valuable for identifying the most promising
compounds in the future
Acknowledgements
Dr Maggie AZ Hupcey is gratefully acknowledged for
editorial assistance Dr Peter W Swaan (University of
Maryland) and Dr Cheng Chang (Ohio State University)
Dr Steve Wright (University of Arizona) Dr K Sandy
Pang (University of Toronto) and Dr Craig Giroux (Wayne
State University) are kindly acknowledged for their support
and discussions My colleagues at GeneGo Sergey
Andreyev Andy Ryabov Eugene Kirillov Eugene A
Rakhmatulin Svetlana Sorokina Andrej Bugrim Tatiana
Nikolskaya Yuri Nikolsky John Metz and Julie Bryant are
all thanked for their considerable contributions to software
development and data annotation
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 61
References
Abe M Yamashita S Kuramoto T Hirayama Y Tsukamoto T Ohta
T et al (2003) Global expression analysis of N-methyl-NV-nitro-N-nitrosoguanidine-induced rat stomach carcinomas using oligonucleotide
microarrays Carcinogenesis 24 861ndash867
Adachi T Ono Y Koh K B Takashima K Tainaka H Matsuno Y
et al (2004) Long-term alteration of gene expression without
morphological change in testis after neonatal exposure to genistein in
mice Toxicogenomic analysis using cDNA microarray Food and
Chemical Toxicology 42 445ndash452
Akerman G S Rosenzweig B A Domon O E McGarrity L J
Blankenship L R Tsai C A et al (2004) Gene expression profiles
and genetic damage in benzo(a)pyrene diol epoxide-exposed TK6 cells
Mutation Research 549 43ndash64
Aksenov S V Church B Dhiman A Georgieva A Sarangapani R
Helmlinger G et al (2005) An integrated approach for inference and
mechanistic modeling for advancing drug development FEBS Letters
579 1878ndash1883
Anderle P Huang Y amp Sadee W (2004) Intestinal membrane transport
of drugs and nutrients Genomics of membrane transporters using
expression microarrays European Journal of Pharmaceutical Sciences
21 17ndash24
Annereau J P Szakacs G Tucker C J Arciello A Cardarelli C
Collins J et al (2004) Analysis of ATP-binding cassette transporter
expression in drug-selected cell lines by a microarray dedicated to
multidrug resistance Molecular Pharmacology 66 1397ndash1405
Apic G Ignjatovic T Boyer S amp Russell R B (2005) Illuminating drug
discovery with biological pathways FEBS Letters 579 1872ndash1877
Baker V A Harries H M Waring J F Duggan C M Ni H A Jolly
R A et al (2004) Clofibrate-induced gene expression changes in rat
liver A cross-laboratory analysis using membrane cDNA arrays
Environmental Health Perspectives 112 428ndash438
Balakin K V Ekins S Bugrim A Ivanenkov Y A Korolev D amp
Nikolsky Y et al (2004a) Quantitative structurendashmetabolism relation-
ship modeling of the metabolic N-dealkylation rates Drug Metabolism
and Disposition 32 1111ndash1120
Balakin K V Ekins S Bugrim A Ivanenkov Y A Korolev D amp
Nikolsky Y et al (2004b) Kohonen maps for prediction of binding to
human cytochrome P450 3A4 Drug Metabolism and Disposition 32
1183ndash1189
Barabasi A -L amp Oltvai Z N (2004) Network biology Under-
standing the cellrsquos functional organization Nature Reviews Genetics
5 101ndash113
Bartosiewicz M Penn S G amp Buckpitt A (2001) Applications of gene
arrays in environmental toxicology Fingerprints of gene regulation
associated with cadmium chloride benzo(a)pyrene and trichloro-
ethylene Environmental Health Perspectives 109 71ndash74
Bertilsson G Heidrich J Svensson K Asman M Jendeberg L
Sydow-Backman M et al (1998) Identification of a human nuclear
receptor defines a new signaling pathway for CYP3A induction
Proceedings of the National Academy of Sciences of the United States
of America 95 12208ndash12213
Blumberg B Sabbagh Jr W Juguilon H Bolado Jr J van Meter
C M Ong E S et al (1998) SXR a novel steroid and xenobiotic-
sensing nuclear receptor Genes and Development 12 3195ndash3205
Borodina Y Rudik A Filimonov D Kharchevnikova N Dmitriev A
Blinova V et al (2004) A new statistical approach to predicting
aromatic hydroxylation sites Comparison with model-based
approaches Journal of Chemical Information and Computer Sciences
44 1998ndash2009
Borodina Y Sadym A Filimonov D Blinova V Dmitriev A amp
Poroikov V (2003) Predicting biotransformation potential from
molecular structure Journal of Chemical Information and Computer
Sciences 43 1636ndash1646
Bottone Jr F G Martinez J M Collins J B Afshari C A amp Eling
T E (2003) Gene modulation by the cyclooxygenase inhibitor
sulindac sulfide in human colorectal carcinoma cells Possible link to
apoptosis Journal of Biological Chemistry 278 25790ndash25801
Boyer S amp Zamora I (2002) New methods in predictive metabolism
Journal of Computer-Aided Molecular Design 16 403ndash413
Bredel M amp Jacoby E (2004) Chemogenomics An emerging strategy
for rapid target and drug discovery Nature Reviews Genetics 5
262ndash275
Bugrim A Nikolskaya T amp Nikolsky Y (2004) Early prediction of drug
metabolism and toxicity Systems biology approach and modeling
Drug Discovery Today 9 127ndash135
Bulera S J Eddy S M Ferguson E Jatkoe T A Reindel J F
Bleavins M R et al (2001) RNA expression in the early character-
ization of hepatotoxicants in Wistar rats by high-density DNA micro-
arrays Hepatology 33 1239ndash1258
Butcher E C Berg E L amp Kunkel E J (2004) Systems biology in drug
discovery Nature Biotechnology 22 1253ndash1259
Butte A (2002) The use and analysis of microarray data Naturalist
Review of Drug Discovery 1 951ndash960
Cary M P Bader G D amp Sander C (2005) Pathway information for
systems biology FEBS Letters 579 1815ndash1820
Cheok M H Yang W Pui C H Downing J R Cheng C Naeve
C W et al (2003) Treatment-specific changes in gene expression
discriminate in vivo drug response in human leukemia cells Nature
Genetics 34 85ndash90
Cleary J D Rogers P D amp Chapman S W (2001) Differential
transcription factor expression in human mononuclear cells in response
to amphotericin B Identification with complementary DNA microarray
technology Pharmacotherapy 21 1046ndash1054
Coen M Ruepp S U Lindon J C Nicholson J K Pognan F Lenz
E M et al (2004) Integrated application of transcriptomics and
metabonomics yields new insight into the toxicity due to paracetamol in
the mouse Journal of Pharmaceutical and Biomedical Analysis 35
93ndash105
Cornwell P D De Souza A T amp Ulrich R G (2004) Profiling of
hepatic gene expression in rats treated with fibric acid analogs
Mutation Research 549 131ndash145
Csermely P Agoston V amp Pongor S (2005) The efficiency of multi-
target drugs The network approach might help drug design Trends in
Pharmacological Sciences 26 178ndash182
Cunningham M J Liang S Fuhrman S Seilhamer J J amp
Somogyi R (2000) Gene expression microarray data analysis for
toxicology profiling Annals of the New York Academy of Sciences 919
52ndash67
de Longueville F Atienzar F A Marcq L Dufrane S Evrard S
Wouters L et al (2003) Use of a low density microarray for studying
gene expression patterns induced by hepatotoxicants on primary
cultures of rat hepatocytes Toxicological Sciences 75 378ndash392
Dobrin R Beg Q K Barabasi A L amp Oltvai Z N (2004)
Aggregation of topological motifs in the Escherichia coli transcriptional
regulatory network BMC Bioinformatics 5 10
Donald S Verschoyle R D Edwards R Judah D J Davies R
Riley J et al (2002) Hepatobiliary damage and changes in hepatic
gene expression caused by the antitumor drug ecteinascidin-743 (ET-
743) in the female rat Cancer Research 62 4256ndash4262
Dumont M Jacquemin E DrsquoHont C Descout C Cresteil D Haouzi
D et al (1997) Expression of the liver Na+-independent organic anion
transporting polypeptide (oatp-1) in rats with bile duct ligation Journal
of Hepatology 27 1051ndash1056
Durnas C Loi C M amp Cusack B J (1990) Hepatic drug metabolism
and aging Clinical Pharmacokinetics 19 359ndash389
Eisen M B Spellman P T Brown P O amp Botstein D (1998) Cluster
analysis and display of genome-wide expression patterns Proceedings
of the National Academy of Sciences of the United States of America
95 14863ndash14868
Ekins S Andreyev S Ryabov A Kirilov E Rakhmatulin E A
Bugrim A et al (in press) Computational Prediction of Human Drug
Metabolism Exp Opin Drug Metab Toxicol
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6662
Ekins S Boulanger B Swaan P W amp Hupcey M A Z (2002)
Towards a new age of virtual ADMETOX and multidimensional drug
discovery Journal of Computer-Aided Molecular Design 16 381ndash401
Ekins S Bugrim A Nikolsky Y amp Nikolskaya T (2005) Systems
biology Applications in drug discovery In S Gad (Ed) Drug
Discovery Handbook (pp 123ndash183) Hoboken NJrsquo Wiley and Sons
Ekins S Giroux C N Nikolsky Y Bugrim A amp Nikolskaya T
(2005) A signature gene network approach to toxicity The Toxoco-
logists 84
Ekins S Kirillov E Rakhmatulin E amp Nikolskaya T (2005) A novel
method for visualizing nuclear hormone receptor networks relevant to
drug metabolism Drug Metabolism and Disposition 33 474ndash481
Ekins S amp McGowan R J (2001) The limits of reductionism The
shifting genomic paradigmrsquos impact on industry and academia
Philsophy in Science 9 1ndash23
Ekins S Mirny L amp Schuetz E G (2002) A ligand-based approach to
understanding selectivity of nuclear hormone receptors PXR CAR
FXR LXRa and LXRb Pharmaceutical Research 19 1788ndash1800
Ekins S Nikolsky Y amp Nikolskaya T (2005) Techniques Application
of systems biology to absorption distribution metabolism excretion
and toxicity Trends in Pharmacological Sciences 26 202ndash209
Ekins S Ring B J Grace J McRobie-Belle D J amp Wrighton S
A (2000) Present and future in vitro approaches for drug
metabolism Journal of Pharmacological and Toxicological Methods
44 313ndash324
Ekins S Waller C L Swaan P W Cruciani G Wrighton S A amp
Wikel J H (2000) Progress in predicting human ADME parameters in
silico Journal of Pharmacological and Toxicological Methods 44
251ndash272
Erhardt P W (2003) A human drug metabolism database Potential roles
in the quantitative predictions of drug metabolism and metabolism-
related drugndashdrug interactions Current Drug Metabolism 4 411ndash422
Evans W E amp McLeod H L (2003) Pharmacogenomicsmdashdrug
disposition drug targets and side effects New England Journal of
Medicine 348 538ndash549
Ezendam J Staedtler F Pennings J Vandebriel R J Pieters R
Boffetta P et al (2004) Toxicogenomics of subchronic hexachlor-
obenzene exposure in Brown Norway rats Environmental Health
Perspectives 112 782ndash791
Faiola B Fuller E S Wong V A amp Recio L (2004) Gene expression
profile in bone marrow and hematopoietic stem cells in mice exposed to
inhaled benzene Mutation Research 549 195ndash212
FDA (2004) Innovation stagnation Challenge and opportunity on the
critical path to new medicinal products
Fiehn O (2001) Combining genomics metabolome analysis and bio-
chemical modelling to understand metabolic networks Comparative
Functional Genomics 2 155ndash168
Field F J Born E amp Mathur S N (2004) LXRRXR ligand activation
enhances basolateral efflux of beta-sitosterol in CaCo-2 cells Journal of
Lipid Research 45 905ndash913
Fleck C Sutter L Appenroth D Koch B Meinhold T Pitack M
et al (2003) Use of gene chip technology for the characterisation of
the regulation of renal transport processes and of nephrotoxicity in
rats Experimental and Toxicologic Pathology 54 401ndash410
Fliri A F Loging W T Thadeio P F amp Volkmann R A (2005)
Biological spectra analysis Linking biological activity profiles to
molecular structure Proceedings of the National Academy of Sciences
of the United States of America 102 261ndash266
Fountoulakis M (2004) Application of proteomics technologies in the
investigation of the brain Mass Spectrometry Reviews 23 231ndash258
Gaucher S P Taylor S W Fahy E Zhang B Warnock D E Ghosh
S S et al (2004) Expanded coverage of the human heart
mitochondrial proteome using multidimensional liquid chromatography
coupled with tandem mass spectrometry Journal of Proteome
Research 3 495ndash505
Gerhold D Lu M Xu J Austin C Caskey C T amp Rushmore T
(2001) Monitoring expression of genes involved in drug metabolism
and toxicology using DNA microarrays Physiological Genomics 5
161ndash170
Guo G L Choudhuri S amp Klaassen C D (2002) Induction profile of
rat organic anion transporting polypeptide 2 (oatp2) by prototypical
drug-metabolizing enzyme inducers that activate gene expression
through ligand-activated transcription factor pathways Journal of
Pharmacology and Experimental Therapeutics 300 206ndash212
Hagenbuch B amp Meier P J (2004) Organic anion transporting
polypeptides of the OATP SLC21 family Phylogenetic classification
as OATP SLCO superfamily new nomenclature and molecularfunc-
tional properties Pflugers Archiv 447 653ndash665
Hamadeh H K amp Afshari C A (2004) Toxicogenomics principles and
applications Hobokenrsquo John Wiley and Sons
Hamadeh H K Bushel P R Jayadev S Martin K DiSorbo O Sieber
S et al (2002) Gene expression analysis reveals chemical-specific
profiles Toxicological Sciences 67 219ndash231
Hamadeh H K Jayadev S Gaillard E T Huang Q Stoll R
Blanchard K et al (2004) Integration of clinical and gene expression
endpoints to explore furan-mediated hepatotoxicity Mutation Research
549 169ndash183
Hamadeh H K Knight B L Haugen A C Sieber S Amin R P
Bushel P R et al (2002) Methapyrilene toxicity Anchorage of
pathologic observations to gene expression alterations Toxicologic
Pathology 30 470ndash482
Han J D Bertin N Hao T Goldberg D S Berriz G F Zhang L V
et al (2004) Evidence for dynamically organized modularity in the
yeast proteinndashprotein interaction network Nature 430 88ndash93
Hanisch D Zien A Zimmer R amp Lengauer T (2002) Co-clustering of
biological networks and gene expression data Bioinformatics 18
S145ndashS154
Harris A J Dial S L amp Casciano D A (2004) Comparison of basal
gene expression profiles and effects of hepatocarcinogens on gene
expression in cultured primary human hepatocytes and HepG2 cells
Mutation Research 549 79ndash99
Harrison S C (2004) Whither structural biology Nature Structural
Molecular Biology 11 12ndash15
Hartley D P Dai X He Y D Carlini E J Wang B Huskey S E
et al (2004) Activators of the rat pregnane X receptor differentially
modulate hepatic and intestinal gene expression Molecular Pharma-
cology 65 1159ndash1171
Hartwell L H Hopfield J J Leibler S amp Murray A W (1999) From
molecular to modular cell biology Nature 402 C47ndashC52
Hasmall S Orphanides G James N Pennie W Hedley K Soames
A et al (2002) Downregulation of lactoferrin by PPARalpha ligands
Role in perturbation of hepatocyte proliferation and apoptosis
Toxicology Sciences 68 304ndash313
Hayase T Yamamoto Y Yamamoto K Muso E amp Shiota K (2004)
Microarray profile analysis of toxic cocaine-induced alterations in the
expression of mouse brain gene sequences A possible rsquoprotectiversquo
effect of buprenorphine Journal of Applied Toxicology 24 15ndash20
Hayes K R Vollrath A L Zastrow G M McMillan B J Craven M
Jovanovich S et al (2005) EDGE A centralized resource for the
comparison analysis and distribution of toxicogenomic information
Molecular Pharmacology 67 1360ndash1368
Heijne W H Slitt A L van Bladeren P J Groten J P Klaassen C D
Stierum R H et al (2004) Bromobenzene-induced hepatotoxicity at
the transcriptome level Toxicology Sciences 79 411ndash422
Heijne W H Stierum R H Slijper M van Bladeren P J amp van
Ommen B (2003) Toxicogenomics of bromobenzene hepatotoxicity
A combined transcriptomics and proteomics approach Biochemical
Pharmacology 65 857ndash875
Heinloth A N Irwin R D Boorman G A Nettesheim P Fannin
R D Sieber S O et al (2004) Gene expression profiling of rat
livers reveals indicators of potential adverse effects Toxicology
Sciences 80 193ndash202
Hodges L C Cook J D Lobenhofer E K Li L Bennett L Bushel
P R et al (2003) Tamoxifen functions as a molecular agonist inducing
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 63
cell cycle-associated genes in breast cancer cells Molecular Cancer
Research 1 300ndash311
Holleman A Cheok M H den Boer M L Yang W Veerman KM
A J Kazemier et al (2004) Gene-expression patterns in drug-resistant
acute lymphoblastic leukemia cells and response to treatment New
England Journal of Medicine 351 533ndash542
Hong Y Muller U R amp Lai F (2003) Discriminating two classes of
toxicants through expression analysis of HepG2 cells with DNA arrays
Toxicology in Vitro 17 85ndash92
Hood E (2003) Proteomics Characterizing the cogs in the machinery of
life Environmental Health Perspectives 111 A816ndashA825
Hood L (2003) Systems biology Integrating technology biology and
computation Mechanism of Ageing and Development 124 9ndash16
Hood L amp Galas D (2003) The digital code of DNA Nature 421
444ndash448
Hood L amp Perlmutter R M (2004) The impact of systems approaches on
biological problems in drug discovery Nature Biotechnology 22
1215ndash1217
Hu T Gibson D P Carr G J Torontali S M Tiesman J P Chaney
J G et al (2004) Identification of a gene expression profile that
discriminates indirect-acting genotoxins from direct-acting genotoxins
Mutation Research 549 5ndash27
Huang Q Dunn II R T Jayadev S DiSorbo O Pack F D Farr S B
et al (2001) Assessment of cisplatin-induced nephrotoxicity by
microarray technology Toxicology Sciences 63 196ndash207
Huang Q Jin X Gaillard E T Knight B L Pack F D Stoltz J H et
al (2004) Gene expression profiling reveals multiple toxicity endpoints
induced by hepatotoxicants Mutation Research 549 147ndash167
Huisman M T Smit J W Crommentuyn K M Zelcer N Wiltshire
H R Beijnen J H et al (2002) Multidrug resistance protein 2
(MRP2) transports HIV protease inhibitors and transport can be
enhanced by other drugs Aids 16 2295ndash2301
Hunt C M Westerkam W R amp Stave G M (1992) Effect of age and
gender on the activity of human hepatic CYP3A Biochemical
Pharmacology 44 275ndash283
Ideker T Ozier O Schwikowski B amp Siegal A F (2002) Discovering
regulatory and signalling circuits in molecular interaction networks
Bioinformatics 18 S233ndashS240
Ideker T Thorsson V Ranish J A Christmas R Buhler J Eng J K
et al (2001) Integrated genomic and proteomic analyses of a system-
atically perturbed network Science 292 929ndash934
Iida M Anna C H Hartis J Bruno M Wetmore B Dubin J R et al
(2003) Changes in global gene and protein expression during early
mouse liver carcinogenesis induced by non-genotoxic model carcino-
gens oxazepam and Wyeth-14643 Carcinogenesis 24 757ndash770
Ingelman-Sundberg M (2004) Pharmacogenetics of cytochrome P450 and
its applications in drug therapy The past present and future Trends in
Pharmacological Sciences 25 193ndash200
Jeong H Mason S P Barabasi A L amp Oltvai Z N (2001) Lethality
and centrality in protein networks Nature 411 41ndash42
Jeong H Tombor B Albert R Oltvai Z N amp Barabasi A L
(2000) The large-scale organization of metabolic networks Nature
407 651ndash654
Jung D Hagenbuch B Gresh L Pontoglio M Meier P J amp Kullak-
Ublick G A (2001) Characterization of the human OATP-C
(SLC21A6) gene promoter and regulation of liver-specific OATP genes
by hepatocyte nuclear factor 1 alpha Journal of Biological Chemistry
276 37206ndash37214
Jung D amp Kullak-Ublick G A (2003) Hepatocyte nuclear factor 1
alpha A key mediator of the effect of bile acids on gene expression
Hepatology 37 622ndash631
Jung J -W Park J -S Hwang J -W Kang K -S YS L Song B
-S et al (2004) Gene expression analysis of peroxisome prolifer-
ators- and phenytoin-induced hepatotoxicity using cDNA microarray
Journal of Veterinary Medical Science 66 1329ndash1333
Kato N Shibutani M Takagi H Uneyama C Lee K -Y Takiami S
et al (2004) Gene expression profile in the livers of rats orally
administered ethinylestradiol for 28 days using a microarray technique
Toxicology 200 179ndash192
Kier L D Neft R Tang L Suizu R Cook T Onsurez K et al
(2004) Applications of microarrays with toxicologically relevant genes
(tox genes) for the evaluation of chemical toxicants in Sprague Dawley
rats in vivo and human hepatocytes in vitro Mutation Research 549
101ndash113
Kijima K Toyosawa K Yasuba M Matsuoka N Adachi T
Komiyama M et al (2004) Gene expression analysis of the rat testis
after treatment with di(2-ethylhexyl) phthalate using cDNA microarray
and real-time RT-PCR Toxicology and Applied Pharmacology 200
103ndash110
Kim R B (2003) Organic anion-transporting polypeptide (OATP) trans-
porter family and drug disposition European Journal of Clinical
Investigation 33 1ndash5
Kinirons M T amp OrsquoMahony M S (2004) Drug metabolism and ageing
British Journal of Clinical Pharmacology 57 540ndash544
Kitano H (2002a) Computational systems biology Nature 420 206ndash210
Kitano H (2002b) Systems biology A brief overview Science 295
1662ndash1664
Kliewer S A Moore J T Wade L Staudinger J L Watson M
A Jones S A et al (1998) An orphan nuclear receptor activated
by pregnanes defines a novel steroid signalling pathway Cell 92
73ndash82
Korolev D Balakin K V Nikolsky Y Kirillov E Ivanenkov Y A
Savchuk N P et al (2003) Modeling of human cytochrome p450-
mediated drug metabolism using unsupervised machine learning
approach Journal of Medicinal Chemistry 46 3631ndash3643
Kramer J A Pettit S D Amin R P Bertram T A Car B
Cunningham M et al (2004) Overview on the application of
transcription profiling using selected nephrotoxicants for toxicology
assessment Environmental Health Perspectives 112 460ndash464
Kultima K Nystrom A -M Scholz B Gustafson A -L Dencker L
amp Stigson M (2004) Valproic acid teratogenicity A toxicogenomics
approach Environmental Health Perspectives 112 1225ndash1235
Kutuzova G D amp Deluca H F (2004) Gene expression profiles in rat
intestine identify pathways for 125-dihydroxyvitamin D(3) stimulated
calcium absorption and clarify its immunomodulatory properties
Archives of Biochemistry and Biophysics 432 152ndash166
Kwak M K Wakabayashi N Itoh K Motohashi H Yamamoto M amp
Kensler T W (2003) Modulation of gene expression by cancer
chemopreventive dithiolethiones through the Keap1-Nrf2 pathway
Identification of novel gene clusters for cell survival Journal of
Biological Chemistry 278 8135ndash8145
Lahjouji K Mitchell G A amp Qureshi I A (2001) Carnitine transport
by organic cation transporters and systemic carnitine deficiency
Molecular Genetics and Metabolism 73 287ndash297
Landowski C P Sun D Foster D R Menon S S Barnett J L
Welage L S et al (2003) Gene expression in the human intestine and
correlation with oral valacyclovir pharmacokinetic parameters Journal
of Pharmacology and Experimental Therapeutics 306 778ndash786
Lee G -J Lee W -S Jeon K -S Um C -H Kim S -J Lee C -H
et al (2004) cDNA microarray gene expression analysis and
toxicological phenotype for anticancer drug Journal of Veterinary
Medical Science 66 1339ndash1345
Lee M Kwon J Kim S N Kim J E Koh W S Kim E J et al
(2003) cDNA microarray gene expression profiling of hydroxyurea
paclitaxel and p-anisidine genotoxic compounds with differing
tumorigenicity results Environmental and Molecular Mutagenesis
42 91ndash97
Li S Armstrong C M Bertin N Ge H Milstein S Boxem M et al
(2004) A map of the interactome network of the metazoan C elegans
Science 303 540ndash543
Liguori M J Anderson L M Bukofzer S McKim J Pregenzer J F
Retief J et al (2005) Microarray analysis in human hepatocytes
suggests a mechanism for hepatotoxicity induced by trovafloxacin
Hepatology 41 177ndash186
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6664
Lobenhofer E K Bennett L Cable P L Li L Bushel P R amp Afshari
C A (2002) Regulation of DNA replication fork genes by 17beta-
estradiol Molecular Endocrinology 16 1215ndash1229
Locker J Tian J Carver R Concas D Cossu C Ledda-Columbano
G M et al (2003) A common set of immediate-early response genes
in liver regeneration and hyperplasia Hepatology 38 314ndash325
Mattes W B Pettit S D Sansone S A Bushel P R amp Waters M D
(2004) Database development in toxicogenomics Issues and efforts
Environmental Health Perspectives 112 495ndash505
McMillian M Nie A Y Parker J B Leone A Bryant S Kemmerer
M et al (2004) A gene expression signature for oxidant stressreactive
metabolites in rat liver Biochemical Pharmacology 68 2249ndash2261
Meier P J amp Stieger B (2000) Molecular mechanisms in bile formation
News in Physiological Sciences 15 89ndash93
Meneses-Lorente G de Longueville F Dos Santos-Mendes S Bonnert
T P Jack A Evrard S et al (2003) An evaluation of a low-density
DNA microarray using cytochrome P450 inducers Chemical Research
in Toxicology 16 1070ndash1077
Meneses-Lorente G Guest P C Lawrence J Muniappa N Knowles
M R Skynner H A et al (2004) A proteomic investigation of drug-
induced steatosis in rat liver Chemical Research in Toxicology 17
605ndash612
Merrill C L Ni H Yoon L W Tirmenstein M A Narayanan P
Benavides G R et al (2002) Etomoxir-induced oxidative stress in
HepG2 cells detected by differential gene expression is confirmed
biochemically Toxicology Sciences 68 93ndash101
Milo R Shen-Orr S Itzkovitz S Kashtan N Chklovskii D amp Alon
U (2002) Network motifs Simple building blocks of complex
networks Science 298 824ndash827
Morgan K T Ni H Brown H R Yoon L Qualls C W Crosby L
M et al (2002) Application of cDNA microarray technology to in
vitro toxicology and the selection of genes for a real-time RT-PCR-
based screen for oxidative stress in Hep-G2 cells Toxicologic
Pathology 30 435ndash451
Mwinyi J Johne A Bauer S Roots I amp Gerloff T (2004) Evidence
for inverse effects of OATP-C (SLC21A6) 5 and 1b haplotypes on
pravastatin kinetics Clinical Pharmacology and Therapeutics 75
415ndash421
Nakata K Yukawa M Komiyama N Nakano T amp Kaminuma T
(2002) A nuclear receptor database that maps pathways to diseases
Genome Infomatics 13 515ndash516
Nicholson J K Holmes E Lindon J C amp Wilson I D (2004) The
challenges of modeling mammalian biocomplexity Nature Biotechnol-
ogy 22 1268ndash1274
Nicholson J K amp Wilson I D (2003) Understanding rsquoglobal systems
biology Metabonomics and the continuum of metabolism Nature
Reviews on Drug Discovery 2 668ndash676
Nikitin A Egorov S Daraselia N amp Mazo I (2003) Pathway studiomdash
the analysis and navigation of molecular networks Bioinformatics 19
2155ndash2157
Nikolsky Y Ekins S Nikolskaya T amp Bugrim A (2005) A novel
method for generation of signature networks as biomarkers from
complex high throughput data Toxicology Letters 158 20ndash29
Nikolsky Y Nikolskaya T amp Bugrim A (2005) Biological networks
and analysis of experimental data in drug discovery Drug Discovery
Today 10 653ndash662
Nishizato Y Ieiri I Suzuki H Kimura M Kawabata K Hirota T
et al (2003) Polymorphisms of OATP-C (SLC21A6) and OAT3
(SLC22A8) genes Consequences for pravastatin pharmacokinetics
Clinical Pharmacology and Therapeutics 73 554ndash565
Nozawa T Nakajima M Tamai I Noda K Nezu J Sai Y et al
(2002) Genetic polymorphisms of human organic anion transporters
OATP-C (SLC21A6) and OATP-B (SLC21A9) Allele frequencies in
the Japanese population and functional analysis Journal of Pharmacol-
ogy and Experimental Therapeutics 302 804ndash813
Nozawa T Sugiura S Nakajima M Goto A Yokoi T Nezu J et al
(2004) Involvement of organic anion transporting polypeptides in the
transport of troglitazone sulfate Implications for understanding trogli-
tazone hepatotoxicity Drug Metabolism and Disposition 32 291ndash294
Oliver D E Rubin D L Stuart J M Hewett M Klein T E amp
Altman R B (2002) Ontology development for a pharmacogenetics
knowledge base Package Symposion 88ndash99
Oram J F amp Lawn R M (2001) ABCA1 The gatekeeper for eliminating
excess tissue cholesterol Journal of Lipid Research 42 1173ndash1179
Oswald M Kullak-Ublick G A Paumgartner G amp Beuers U (2001)
Expression of hepatic transporters OATP-C and MRP2 in primary
sclerosing cholangitis Liver 21 247ndash253
Parsons A B Brost R I Ding H Li Z Zhang C Sheikh B et al
(2004) Integration of chemical-genetic and genetic interaction data
links bioactive compounds to cellular target pathways Nature Bio-
technology 22 62ndash69
Patki K C von Moltke L L Harmatz J S Hesse L M Court M H
amp Greenblatt D J (2004) Effect of age on in vitro triazolam
biotransformation in male human liver microsomes Journal of
Pharmacology and Experimental Therapeutics 308 874ndash879
Pereira-Leal J B Enright A J amp Ouzounis C A (2004) Detection of
functional modules from protein interaction networks Proteins 54
49ndash57
Plant N (2004) Interaction networks Coordinating responses to xeno-
biotic exposure Toxicology 202 21ndash32
Raghavendra Prasad H S Qi Z Srinivasan K N amp Gopalakrish-
nakone P (2004) Potential effects of tetrodotoxin exposure to
human glial cells postulated using microarray approach Toxicon 44
597ndash608
Reilly T P Bourdi M Brady J N Pise-Masison C A Radonovich M
F George J W et al (2001) Expression profiling of acetaminophen
liver toxicity in mice using microarray technology Biochemical and
Biophysical Research Communications 282 321ndash328
Rives A W amp Galitski T (2003) Modular organization of cellular
networks Proceedings of the National Academy of Sciences of the
United States of America 100 1128ndash1133
Rogers P D Pearson M M Cleary J D Sullivan D C amp Chapman
S W (2002) Differential expression of genes encoding immunomodu-
latory proteins in response to amphotericin B in human mononuclear
cells identified by cDNA microarray analysis Journal of Antimicrobial
Chemotherapy 50 811ndash817
Rosen M B Wilson V S Schmid J E amp Gray L E (2005) Gene
expression analysis in the ventral prostate of rats exposed to vinclozolin
or procymidone Reproductive Toxicology 19 367ndash379
Rost D Herrmann T Sauer P Schmidts H L Stieger B Meier P J
et al (2003) Regulation of rat organic anion transporters in bile salt-
induced cholestatic hepatitis Effect of ursodeoxycholate Hepatology
38 187ndash195
Rothman R B Ayestas M A Dersch C M amp Baumann M H (1999)
Aminorex fenfluramine and chlorphentermine are serotonin transporter
substrates Implications for primary pulmonary hypertension Circula-
tion 100 869ndash875
Ruepp S U Tonge R P Shaw J Wallis N amp Pognan F (2002)
Genomics and proteomics analysis of acetaminophen toxicity in mouse
liver Toxicology Sciences 65 135ndash150
Sakaeda T Nakamura T amp Okumura K (2002) MDR1 genotype-
related pharmacokinetics and pharmacodynamics Biological amp Phar-
maceutical Bulletin 25 1391ndash1400
Sakata T Anzai N Shin H J Noshiro R Hirata T Yokoyama H
et al (2004) Novel single nucleotide polymorphisms of organic cation
transporter 1 (SLC22A1) affecting transport functions Biochemical
and Biophysical Research Communications 313 789ndash793
Satlin L M Amin V amp Wolkoff A W (1997) Organic anion
transporting polypeptide mediates organic anionHCO3-exchange
Journal of Biological Chemistry 272 26340ndash26345
Satomi Y Tsuchiya W Mihara K Ota M Kasahara Y amp Akahori F
(2004) Gene expression analysis of the lung following paraquat
administration in rats using DNA microarray Journal of Toxicological
Sciences 29 91ndash100
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 65
Schrenk D Baus P R Ermel N Klein C Vorderstemann B amp
Kauffmann H M (2001) Up-regulation of transporters of the MRP
family by drugs and toxins Toxicology Letters 120 51ndash57
Segal E Shapira M Regev A Persquoer D Botstein D Koller D et al
(2003) Module networks Identifying regulatory modules and their
condition-specific regulators from gene expression data Nature
Genetics 34 166ndash176
Segal E Wang H amp Koller D (2003) Discovering molecular pathways
from protein interaction and gene expression Bioinformatics 19
i264ndash i272
Seidel S D Kan H L Stott W T Schisler M R amp Gollapudi B B
(2003) Identification of transcriptome profiles for the DNA-damaging
agents bleomycin and hydrogen peroxide in L5178Y mouse lymphoma
cells Environmental and Molecular Mutagenesis 42 19ndash25
Shan L Yu Y Schut H A J amp Snyderwine E G (2004) Susceptibility
of rats to mammary gland carcinogenesis by the food-derived
carcinogen 2-Amino-1-Methyl-6-Phenylimidazo[45-b]Pyridine (PhIP)
varies with age and is associated with the induction of differential gene
expression American Journal of Pathology 165 191ndash202
Sharom J R Bellows D S amp Tyers M (2004) From large networks to
small molecules Current Opinion in Chemical Biology 8 81ndash90
Shitara Y Hirano M Sato H amp Sugiyama Y (2004) Gemfibrozil and
its glucuronide inhibit the OATP2(OATP1B1 SLC21A6)-mediated
hepatic uptake and CYP2C8-mediated metabolism of cerivastatinmdash
Analysis of the mechanism of the clinically relevant drugndashdrug
interaction between cerivastatin and gemfibrozil Journal of Pharma-
cology and Experimental Therapeutics
Shitara Y Itoh T Sato H Li A P amp Sugiyama Y (2003) Inhibition of
transporter-mediated hepatic uptake as a mechanism for drugndashdrug
interaction between cerivastatin and cyclosporin A Journal of
Pharmacology and Experimental Therapeutics 304 610ndash616
Somogyi R Fuhrman S amp Wen X (2001) Genetic network inference in
computational models and applications to large-scale gene expression
data In M Bower amp H Bolouri (Eds) Computational modeling of
genetic and biochemical networks (pp 119ndash157) Cambridge MArsquoThe
MIT Press
Spirin V amp Mirny L A (2003) Protein complexes and functional
modules in molecular networks Proceedings of the National Academy
of Sciences of the United States of America 100 12123ndash12128
Staudinger J Liu Y Madan A Habeebu S amp Klaassen C D
(2001) Coordinate regulation of xenobiotic and bile acid homeo-
stasis by pregnane X receptor Drug Metabolism and Disposition
29 1467ndash1472
Sun L Z Ji Z L Chen X Wang J F amp Chen Y Z (2002) ADME-
AP A database of ADME associated proteins Bioinformatics 18
1699ndash1700
Suter L Babiss L E amp Wheeldon E B (2004) Toxicogenomics in
predictive toxicology in drug development Chemistry and Biology 11
161ndash171
Synold T W Dussault I amp Forman B M (2001) The orphan nuclear
receptor SXR coordinately regulates drug metabolism and efflux
Nature Medicine 7 584ndash590
Tabuchi Y amp Kondo T (2003) cDNA microarray analysis reveals chop-
10 plays a key role in Sertoli cell injury induced by bisphenol A
Biochemical and Biophysical Research Communications 305 54ndash61
Tamai I Nezu J Uchino H Sai Y Oku A Shimane M et al (2000)
Molecular identification and characterization of novel members of the
human organic anion transporter (OATP) family Biochemical and
Biophysical Research Communications 273 251ndash260
Taylor S W Fahy E Zhang B Glenn G M Warnock D E Wiley S
et al (2003) Characterization of the human heart mitochondrial
proteome Nature Biotechnology 21 281ndash286
Terasaka S Aita Y Inoue A Hayashi S Nishigaki M Aoyagi K
et al (2004) Using a customized DNA microarray for expression
profiling of the estrogen-responsive genes to evaluate estrogen activity
among natural estrogens and industrial chemicals Environmental
Health Perspectives 112 773ndash781
Thomas D M Francescutti-Verbeem D M Liu X amp Kuhn D M
(2004) Identification of differentially regulated transcripts in mouse
striatum following methamphetamine treatmentmdashan oligonucleotide
microarray approach Journal of Neurochemistry 88 380ndash393
Thomas R S Rank D R Penn S G Zastrow G M Hayes K R
Pande K et al (2001) Identification of toxicologically predictive
gene sets using cDNA microarrays Molecular Pharmacology 60
1189ndash1194
Thompson K L Afshari C A Amin R P Bertram T A Car B
Cunningham M et al (2004) Identification of platform-independent
gene expression markers of cisplatin nephrotoxicity Environmental
Health Perspectives 112 488ndash494
Tirona R G Leake B F Merino G amp Kim R B (2001)
Polymorphisms in OATP-C Identification of multiple allelic
variants associated with altered transport activity among European-
and African-Americans Journal of Biological Chemistry 276
35669ndash35675
Tirona R G Leake B F Wolkoff A W amp Kim R B (2003)
Human organic anion transporting polypeptide-C (SLC21A6) is a
major determinant of the rifampin-mediated pregnane X receptor
activation Journal of Pharmacology and Experimental Therapeutics
304 223ndash228
Tong W Cao X Harris S Sun H Fang H Fuscoe J et al (2003)
ArrayTrackmdashsupporting toxicogenomic research at the US Food and
Drug Administration National Center for Toxicological Research
Environmental Health Perspectives 111 1819ndash1826
Tornow S amp Mewes H W (2003) Functional modules by relating
protein interaction networks and gene expression Nucleic Acids
Research 31 6283ndash6289
Trocho C Escola-Gil J C Ribas V Benitez S Martin-Campos J M
Rotllan N et al (2004) Phenytoin treatment reduces atherosclerosis in
mice through mechanisms independent of plasma HDL-cholesterol
concentration Atherosclerosis 174 275ndash285
Ueda A Hamadeh H K Webb H K Yamamoto Y Sueyoshi T
Afshari C A et al (2002) Diverse roles of the nuclear orphan
receptor CAR in regulating hepatic genes in response to phenobarbital
Molecular Pharmacology 61 1ndash6
Ulrich R G (2003) The toxicogenomics of nuclear receptor agonists
Current Opinion in Chemical Biology 7 505ndash510
Ulrich R G Rockett J C Gibson G G amp Pettit S D (2004)
Overview of an interlaboratory collaboration on evaluating the effects
of model hepatotoxicants on hepatic gene expression Environmental
Health Perspectives 112 423ndash427
Unami A Shinohara Y Ichikawa T amp Baba Y (2003) Biochemical
and microarray analyses of bupivacaine-induced apoptosis Journal of
Toxicological Sciences 28 77ndash94
Vasquez A Flammini A Maritan A amp Vespignani A (2003) Global
protein function prediction from proteinndashprotein interaction networks
Nature Biotechnology 21 697ndash700
Vezina C M Walker N J amp Olson J R (2004) Subchronic Exposure to
TCDD PeCDF PCB126 and PCB153 Effect on hepatic gene
expression Environmental Health Perspectives 112 1636ndash1644
Vo T D Greenberg H J amp Palsson B O (2004) Reconstruction and
functional characterization of the human mitochondrial metabolic
network based on proteomic and biochemical data Journal of Bio-
logical Chemistry 279 39532ndash39540
Wang H amp LeCluyse E L (2003) Role of orphan nuclear receptors in
the regulation of drug-metabolising enzymes Clinical Pharmacoki-
netics 42 1331ndash1357
Wang N Silver D L Thiele C amp Tall A R (2001) ATP-
binding cassette transporter A1 (ABCA1) functions as a choles-
terol efflux regulatory protein Journal of Biological Chemistry 276
23742ndash23747
Waring J F Cavet G Jolly R A McDowell J Dai H Ciurlionis R
et al (2003) Development of a DNA Microarray for toxicology based
on hepatoxin-regulated sequences Environmental Health Perspectives
111 863ndash870
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6666
Waring J F Gum R Morfitt D Jolly R A Ciurlionis R Heindel M
et al (2002) Identifying toxic mechanisms using DNA microarrays
Evidence that an experimental inhibitor of cell adhesion molecule
expression signals through the aryl hydrocarbon nuclear receptor
Toxicology 181ndash182 537ndash550
Waring J F Jolly R A Ciurlionis R Lum P Y Praestgaard J T
Morfitt D C et al (2001) Clustering of hepatotoxins based on
mechanism of toxicity using gene expression profiles Toxicology and
Applied Pharmacology 175 28ndash42
Waters M Boorman G Bushel P Cunningham M Irwin R Merrick
A et al (2003) Systems toxicology and the Chemical Effects in
Biological Systems (CEBS) knowledge base EHP Toxicogenomics
111 15ndash28
Wei M Arnold L Cano M amp Cohen S M (2005) Effects of co-
administration of antioxidants and arsenicals on the rat urinary bladder
epithelium Toxicology Sciences 83 237ndash245
Weinshilboum R (2003) Inheritance and drug response New England
Journal of Medicine 348 529ndash537
Weinshilboum R amp Wang L (2004) Pharmacogenetics Inherited
variation in amino acid sequence and altered protein quantity Clinical
Pharmacology and Therapeutics 75 253ndash258
Werner E (2003) In silico multicellular systems biology and minimal
genomes Drug Discovery Today 8 1121ndash1127
Wong J S amp Gill S S (2002) Gene expression changes induced in
mouse liver by di(2-ethylhexyl) phthalate Toxicology and Applied
Pharmacology 185 180ndash196
Xie T Tong L McCann U D Yuan J Becker K G Mechan A O
et al (2004) Identification and characterization of metallothionein-1
and -2 gene expression in the context of ()34-methylenedioxymetham-
phetamine-induced toxicity to brain dopaminergic neurons Journal of
Neuroscience 24 7043ndash7050
Yadetie F Laegreid A Bakke I Kusnierczyk W Komorowski J
Waldum H L et al (2003) Liver gene expression in rats in response
to the peroxisome proliferator-activated receptor-alpha agonist ciprofi-
brate Physiological Genomics 15 9ndash19
Yamashita S Nomoto T Abe M Tatematsu M Sugimura T amp
Ushijima T (2004) Persistence of gene expression changes in stomach
mucosae induced by short-term N-methyl-NV-nitro-N-nitrosoguanidinetreatment and their presence in stomach cancers Mutation Research
549 185ndash193
Yan Q amp Sadee W (2000) Human membrane transporter database A
web-accessible relational database for drug transport studies and
pharmacogenomics AAPS Pharmsci 2 E20
Yeger-Lotem E amp Margalit H (2003) Detection of regulatory circuits by
integrating the cellular networks of proteinndashprotein interactions and
transcription regulation Nucleic Acids Research 31 6053ndash6061
Yoon B I Li G -X Kitada K Kawasaki Y Igarashi K amp Kodama
Y et al (2003) Mechanisms of benzene-induced hematotoxicity and
leukemogenicity cDNA microarray analyses using mouse bone marrow
tissue Environmental Health Perspectives 111 1411ndash1420
Young M B DiSilvestro M R Sendera T J Freund J Kriete A amp
Magnuson S R (2003) Analysis of gene expression in carbon
tetrachloride-treated rat livers using a novel bioarray technology
Pharmacogenomics Journal 3 41ndash52
Yu H Zhu X Greenbaum D Karro J amp Gerstein M (2004) TopNet
A tool for comparing biological sub-networks correlating protein
properties with topological statistics Nucleic Acids Research 32
328ndash337
Zhang E Y Fu D J Pak Y A Stewart T Mukhopadhyay N
Wrighton S A et al (2004) Genetic polymorphisms in human proton-
dependent dipeptide transporter PEPT1 Implications for the functional
role of Pro586 Journal of Pharmacology and Experimental Therapeu-
tics 310 437ndash445
Zhang E Y Knipp G T Ekins S amp Swaan P W (2002)
Structural biology and function of solute transporters Implications
for identifying and designing substrates Drug Metabolism Reviews
34 709ndash750
Zhang E Y Phelps M A Cheng C Ekins S amp Swaan P W (2002)
Modeling of active transport systems Advanced Drug Delivery
Reviews 54 329ndash354
- Systems-ADMETox Resources and network approaches
-
- Introduction
- Data available
- Network analysis and databases
-
- Network applications
-
- The role of transporters
-
- Clinical relevance of transporters
- Transporter network examples ABCA1
- Transporter network examples OATP
- Transporter microarray data
-
- Applications to enzymes
- Future network applications
-
- Discussion
- Acknowledgements
- References
-
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6654
this transporterrsquos hepatic uptake of other compounds may be
important for reported drugndashdrug interactions (Kim 2003)
described earlier (Shitara et al 2003) as well as cerivastatin
with gemfibrozil (Shitara Hirano Sato amp Sugiyama 2004)
OATP1B1 (previous names OATP-C LST-1 OATP2
SLC21A6) represents the most studied human OATP to
date (Meier amp Stieger 2000) and is expressed on the
basolateral plasma membrane of hepatocytes Several single
nucleotide polymorphisms have been identified in the
OATP1B1 gene in European-Americans African-Americans
(Tirona et al 2001) and Japanese (Nozawa et al 2002)
dramatically impacting the transport of ligands such as
pravastatin (Mwinyi Johne Bauer Roots amp Gerloff 2004
Nishizato et al 2003) estrone-3-sulfate (Nozawa et al
2002 Tirona et al 2001) Rifampin (Tirona Leake
Wolkoff amp Kim 2003) and estradiol 17h-d-glucuronide(Tirona et al 2001)
The regulation of SLCO may be affected during
extrahepatic cholestasis bile duct ligation bile salt induced
Fig 6 Networks generated with the autoexpand algorithm in MetaCorei to illustr
be interconnected with other protein regulatory signaling information Informa
unspecified allosteric regulation binding cleavage competition covalent m
transformation When applicable interactions also have a positive or negative e
transfactors (red) enzymes (orange) (For interpretation of the references to colour
cholestatic hepatitis (Dumont et al 1997 Meier amp Stieger
2000 Rost et al 2003) and primary sclerosing cholangitis
(Oswald Kullack-Ublick Paumgarter amp Beuer 2001) In
particular OATP1B1 appears to be regulated by the liver-
enriched transcription factor hepatocyte nuclear factor 1a
(HNF1a) which binds to the promoter region of this
transporter (Jung et al 2001) Site directed mutagenesis
of this binding site resulted in inactivation suggesting the
critical nature of the interaction with HNF1a Bile acids
such as CDCA have been shown to transcriptionally repress
HNF1a in vitro via inhibition of the transactivating effect of
HNF4a on HNF1a (Jung amp Kullak-Ublick 2003) After
screening many rat and human uptake transporters in vitro
OATP1B1 was also shown to modulate the PXR response
by controlling rifampin retention in the cell and therefore
affecting the induction of CYP3A4 and other gene products
such as P-gp (Tirona et al 2003)
Some of the literature for OATP1B1 human substrate
data has been annotated into MetaCorei to illustrate the
ate how ligands for the human OATP-C (OATP1B1) transporter (centre) can
tion on the type of interaction between objects is hidden for clarity eg
odification dephosphorylation phosphorylation transcription regulation
ffect and direction Ligands (purple) linked to other proteins (blue blobs)
in this figure legend the reader is referred to the web version of this article)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 55
visualization of the complex interconnections between this
transporter its ligands regulatory factors and signaling
molecules already in the database (Fig 6) The network was
generated with the autoexpand algorithm in the software
representing one of multiple available algorithms for
connecting genes ligands and other objects in the database
Clearly if more ligands and their connections are added to
the database the complexity of the network will increase
considerably The OATP-C (OATP1B1) gene details can be
viewed upon querying the database and links are provided
to other public databases This page can be used to highlight
the multiple synonyms for this gene as well as links to the
multiple SNPs identified to date
3114 Transporter microarray data Microarrays have
generally been limited in the number of transporters present
on them (Annereau et al 2004) however they have been used
in an attempt to correlate pharmacokinetic properties with
gene expression for valacyclovir (Landowski et al 2003) as
well as understand the expression profile in different tissues
or cell lines upon food component or xenobiotic treatment
(Anderle Huang amp Sadee 2004) This lack of transporters
on microarrays has prompted some groups to produce their
own arrays with a heavier emphasis on transporters These
arrays have for example then been used to demonstrate the
upregulation of ABC transporters and down-regulation of
GST-Pi in cell lines resistant to colchicines or 9-nitro-
camptothecin (Annereau et al 2004) The genes that were
significantly up or down-regulated in this particular study
were used to build networks with MetaCore (Fig 7A B) and
the similarities between them were assessed (Fig 7C)
Although there were only a small number of significantly
changed genes in common (IL-8 Fos GST-Pi Calpactin and
Ubiquitin hydrolase) it is perhaps likely that there is a much
larger common gene network that is important for drug
resistance although a much larger number of cell lines and
drug treatments need to be evaluated to produce a definitive
drug resistance signature involving transporters enzymes
and transcriptional regulators
312 Applications to enzymes
As we have already described it may be particularly
valuable to visualize enzymes as networks to show
interactions with transcriptional regulators and ligands
For example a key enzyme is CYP3A4 which metabolizes
40ndash45 of all drugs and has relatively few SNPs
(Ingelman-Sundberg 2004) Using a second software suite
MetaDrugi (wwwgenegocom) it is possible to construct
a custom network around this or other drug metabolizing
enzymes (Fig 8) In this case the gene network for
CYP3A4 highlights all of the major transcriptional
regulators and several more distant linked proteins and
ligands connected on the network that may be useful for
further study (Fig 8) Substrates inhibitors as well as
regulatory factors and other enzymes can be observed
connected on this network Due to the many hundreds of
known inhibitors and substrates only a small number are
shown here for clarity We assume that if a perturbation in
a pathway (eg due to a nonfunctional enzyme) is linked
to a certain pathologic condition a similar perturbation
caused by the interference from xenobiotic metabolism
(eg competitive inhibition of the same enzyme) may
result in identical effects As microarray gene expression
data is increasingly generated the role of enzyme
regulation in toxicity of certain xenobiotics will become
more apparent from either in vivo or in vitro studies The
visualization of such signature gene networks involving
transporters and enzymes their ligands and regulatory
factors will also be important for future toxicity prediction
methods We have recently generated visualizations of
microarray data from MCF-7 cells treated with 4-hydrox-
ytamoxifen to show that some of the key genes involved in
metabolism and transport are upregulated (Ekins et al
2005d) In addition we have made predictions with various
QSAR models in MetaDrug to indicate the involvement of
PXR CYP3A4 and P-gp (Ekins et al 2005e) Therefore it
appears likely that 4-hydroxytamoxifen could increase its
own metabolism as well as efflux from cells via P-gp
which can be visualized on networks Any decrease of
function of these enzymes or transporters in a population
would likely result in changes in the metabolism and
transport of this active metabolite potentially impacting
the clinical effect This represents one example of how
both pathway tools QSAR models and network building
algorithms can be used with different types of predicted
and experimental data to allow visualization of potential
compound interactions or toxicity
313 Future network applications
As the population ages an increasing incidence and
prevalence of systemic diseases especially chronic diseases
have occurred among older adults This has resulted in an
increase in medications used concomitantly by this
population which presents challenges for drugndashdrug
interactions Physiologically elderly patients may behave
differently to the younger patients for which the drugs are
initially developed for and tested on Many pharmacoki-
netic investigations in the elderly population reveal
decreased clearance of lipophilic drugs metabolized by
the cytochrome P450 enzymes however few studies have
evaluated aging-dependent or gender-related changes in
specific P450 enzymes (Hunt Westerkam amp Slave 1992)
Age-related physiological changes such as a reduction in
liver mass hepatic metabolizing enzyme activity liver
blood flow and alterations in plasma drug binding may
account for the decreased elimination of some metabolized
drugs in the elderly It is particularly difficult to separate an
effect of aging from the variation in the rate of metabolism
due to factors such as individual metabolic phenotype
(slow or fast metabolizer due to SNPs) environmental
influences concomitant disease states and drug intake
(drugndashdrug interactions) The available data suggest that
Fig 7 Networks for drug resistant cell lines generated with the Auto expand algorithm in MetaCore A KB-3-1 vs KB-8-5 B ATC-DU-145 vs RCO1 C node overlap of A and B with initial nodes highlighted
with blue or red solid circles (Initial gene lists derived from (Annereau et al 2004)) Information on the type of interaction between objects is hidden for clarity eg unspecified allosteric regulation binding
cleavage competition covalent modification dephosphorylation phosphorylation transcription regulation transformation When applicable interactions also have a positive or negative effect and direction
Ligands (purple) linked to other proteins (blue blobs) transfactors (red) enzymes (orange) (For interpretation of the references to colour in this figure legend the reader is referred to the web version of this article)
SEkin
sJournalofPharm
acologica
landToxico
logica
lMeth
ods53(2006)38ndash66
56
Fig7(continued)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 57
Fig7(continued)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6658
Fig 8 The network of gene interactions around the CYP3A4 enzyme (centre) from MetaCorei Information on the type of interaction between objects is
hidden for clarity eg unspecified allosteric regulation binding cleavage competition covalent modification dephosphorylation phosphorylation
transcription regulation transformation When applicable interactions also have a positive or negative effect and direction Ligands (purple) linked to other
proteins (blue blobs) transfactors (red) enzymes (orange) (For interpretation of the references to colour in this figure legend the reader is referred to the web
version of this article)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 59
the initial doses of drugs metabolized by these enzymes
should be reduced in older patients according to the clinical
response In most published studies the elderly appear at
least as responsive as the young to inducers or inhibitors of
P450s (Durnas Loi amp Cusack 1990) More recently there
has been some suggestion that there are age related
reductions in function of some specific P450s such as
CYP3A4 (Patki et al 2004) and this could occur at the
level of regulation However earlier studies with the same
enzyme showed no change in clearance with age (Hunt et
al 1992) To date there has been even less examination of
the transporter functions and any changes with age
(Kinirons amp OrsquoMahony 2004) so the current understand-
ing of the effects of aging on metabolism and transport is
anything but transparent This represents an extreme
challenge for the pharmaceutical industry how to predict
whether a drug has an affinity for an enzyme or transporter
and whether this may be clinically important if it shows a
decline in expression or function with aging Any decrease
of function of these enzymes or transporters in an elderly
population would result in changes in the metabolism and
transport of metabolites potentially impacting the clinical
effect This represents another example of how computa-
tional approaches may perhaps be used with different types
of predicted and experimental data to allow visualization of
potential compound interactions or toxicity in elderly
populations We may see different gene networks high-
lighted as humans age and these may be modified by drug
treatment and coadministration This represents an area
were network analysis could be applied in the future and is
in need of considerable further research
The collection of microarray data in databases such as
CEBS ArrayTrack and EDGE represents a future resource
for computational gene network analysis One could
envisage that ultimately in each case such data is converted
to one or more networks that are also displayed for the user
and can be used to compare treatments from in vivo and in
vitro experiments This would represent a different approach
to clustering the data as currently implemented in one of
these efforts (Hayes et al 2005) and may condense large
amounts of experimentally derived data into a readily
interpreted network
4 Discussion
Previously in this journal the progress of many
research groups in predicting human ADME parameters
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6660
in silico (Ekins et al 2000b) and approaches for drug
metabolism (Ekins Ring Grace McRobie-Belle amp
Wrighton 2000a) have been described Both of these
reviews commented on moving HT assays for ADME
Tox much earlier in drug discovery which had also been
indicated by other groups The initial reviews also
highlighted the likely wealth of data that would become
available and how this could be used for structure
activity relationships alongside the bioactivity data in
computational models It was noted that there was a
paucity of predictive metabolism tools at that time In
addition a growing number of efforts to model whole
cells and organs now a field called systems biology
were recognized as models that could be integrated with
the in silico ADME approaches In summary since these
past reviews virtually all pharmaceutical companies have
attempted earlier high throughput screening for ADME
Tox properties and to some extent the wider application
of computational approaches for physicochemical proper-
ties Systems biology is being quite widely acknowledged
as the new paradigm for understanding complex bio-
logical datasets derived from high throughput technolo-
gies and the accumulated knowledge on human protein
interactions (Hartwell Hopfield Leibler amp Murray 1999
Hood 2003b) Therefore systems biology can be defined
as the integration of genetic proteomic transcriptomic
and metabonomic data using computational methods
(Nicholson amp Wilson 2003) When taken together
information on molecular processes derived from different
sources represents a lsquolsquouniversersquorsquo of putative biological
functionality of which only a small fraction of it will be
realized in a cell at any given time To date systems
biology has been driven by academia and funding bodies
such as the NIH rather than the pharmaceutical compa-
nies Presently there is a great deal of interest from
scientists of all backgrounds in identifying the networks
of cellular pathways and the corresponding physically
interacting proteins
The network building software for systems biology
described in this current review will be valuable to query
high throughput data and known literature interactions in
order to predict potential toxicity in different species In the
future the compilation of published toxicogenomics data-
sets characteristic of different types of toxicity will likely
be available in these software systems to act as a reference
database It is also feasible that we will be able to generate
the annotated datasets which specifically address the
differences between human and rat networks implicated
in toxicity The identification of sub-network modules
conserved between human and rat distinct for toxicity
types or predictive for toxic end-points in human will be
possible Such signature gene networks (Nikolsky et al
2005) can then be verified with other experimentally
derived data prospectively or from preexisting databases
The combined Fhigh throughput datandashin silico network_approaches therefore represents a novel method for access-
ing complex tissue-level processes as networks integrating
all data types based on functional interactions The data-
bases developed and used in this approach will certainly
benefit from further annotation around the drug metaboliz-
ing enzymes and transporters as described herein in terms
of transcriptional regulation and the ligands associated with
them which frequently appear in the literature It will be
important to capture disparities in the assignment of ligands
to enzymes transporters and other proteins as well as
negative data
In summary although there have been numerous
toxicogenomics studies published there is presently a
relatively small number of datasets that are freely available
to perform network analysis of microarray data The
number of studies identifying large numbers of proteins
which are affected by molecule treatment are even scarcer
still while there are several examples of some published
studies that combine such data It is hoped that the
numerous database initiatives for high content and toxicol-
ogy data that are being undertaken will improve the
situation for other researchers that are not currently
equipped to do such microarray studies themselves The
addition of requirements by journals to deposit such raw
data in a freely accessible resource will aid these initiatives
ADMETox groups have seen new technologies and
approaches developed over the last decade that have all
been applied to identify poor compounds earlier (Fig 2)
The latest technologies integrate network building tools
with high content data and databases The current review
described the limited number of networks generated for
ADMETox at present and one hopes that the impact of
such analyses will be commonplace in the future Systems
biology is however more than just applying a network
approach and hence systems-ADMETox will have to
evolve due to the continual pressure to develop newer
technologies This current paradigm combining empirical
data and computational methods should integrate the
complex data already generated making it readily inter-
pretable and valuable for identifying the most promising
compounds in the future
Acknowledgements
Dr Maggie AZ Hupcey is gratefully acknowledged for
editorial assistance Dr Peter W Swaan (University of
Maryland) and Dr Cheng Chang (Ohio State University)
Dr Steve Wright (University of Arizona) Dr K Sandy
Pang (University of Toronto) and Dr Craig Giroux (Wayne
State University) are kindly acknowledged for their support
and discussions My colleagues at GeneGo Sergey
Andreyev Andy Ryabov Eugene Kirillov Eugene A
Rakhmatulin Svetlana Sorokina Andrej Bugrim Tatiana
Nikolskaya Yuri Nikolsky John Metz and Julie Bryant are
all thanked for their considerable contributions to software
development and data annotation
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 61
References
Abe M Yamashita S Kuramoto T Hirayama Y Tsukamoto T Ohta
T et al (2003) Global expression analysis of N-methyl-NV-nitro-N-nitrosoguanidine-induced rat stomach carcinomas using oligonucleotide
microarrays Carcinogenesis 24 861ndash867
Adachi T Ono Y Koh K B Takashima K Tainaka H Matsuno Y
et al (2004) Long-term alteration of gene expression without
morphological change in testis after neonatal exposure to genistein in
mice Toxicogenomic analysis using cDNA microarray Food and
Chemical Toxicology 42 445ndash452
Akerman G S Rosenzweig B A Domon O E McGarrity L J
Blankenship L R Tsai C A et al (2004) Gene expression profiles
and genetic damage in benzo(a)pyrene diol epoxide-exposed TK6 cells
Mutation Research 549 43ndash64
Aksenov S V Church B Dhiman A Georgieva A Sarangapani R
Helmlinger G et al (2005) An integrated approach for inference and
mechanistic modeling for advancing drug development FEBS Letters
579 1878ndash1883
Anderle P Huang Y amp Sadee W (2004) Intestinal membrane transport
of drugs and nutrients Genomics of membrane transporters using
expression microarrays European Journal of Pharmaceutical Sciences
21 17ndash24
Annereau J P Szakacs G Tucker C J Arciello A Cardarelli C
Collins J et al (2004) Analysis of ATP-binding cassette transporter
expression in drug-selected cell lines by a microarray dedicated to
multidrug resistance Molecular Pharmacology 66 1397ndash1405
Apic G Ignjatovic T Boyer S amp Russell R B (2005) Illuminating drug
discovery with biological pathways FEBS Letters 579 1872ndash1877
Baker V A Harries H M Waring J F Duggan C M Ni H A Jolly
R A et al (2004) Clofibrate-induced gene expression changes in rat
liver A cross-laboratory analysis using membrane cDNA arrays
Environmental Health Perspectives 112 428ndash438
Balakin K V Ekins S Bugrim A Ivanenkov Y A Korolev D amp
Nikolsky Y et al (2004a) Quantitative structurendashmetabolism relation-
ship modeling of the metabolic N-dealkylation rates Drug Metabolism
and Disposition 32 1111ndash1120
Balakin K V Ekins S Bugrim A Ivanenkov Y A Korolev D amp
Nikolsky Y et al (2004b) Kohonen maps for prediction of binding to
human cytochrome P450 3A4 Drug Metabolism and Disposition 32
1183ndash1189
Barabasi A -L amp Oltvai Z N (2004) Network biology Under-
standing the cellrsquos functional organization Nature Reviews Genetics
5 101ndash113
Bartosiewicz M Penn S G amp Buckpitt A (2001) Applications of gene
arrays in environmental toxicology Fingerprints of gene regulation
associated with cadmium chloride benzo(a)pyrene and trichloro-
ethylene Environmental Health Perspectives 109 71ndash74
Bertilsson G Heidrich J Svensson K Asman M Jendeberg L
Sydow-Backman M et al (1998) Identification of a human nuclear
receptor defines a new signaling pathway for CYP3A induction
Proceedings of the National Academy of Sciences of the United States
of America 95 12208ndash12213
Blumberg B Sabbagh Jr W Juguilon H Bolado Jr J van Meter
C M Ong E S et al (1998) SXR a novel steroid and xenobiotic-
sensing nuclear receptor Genes and Development 12 3195ndash3205
Borodina Y Rudik A Filimonov D Kharchevnikova N Dmitriev A
Blinova V et al (2004) A new statistical approach to predicting
aromatic hydroxylation sites Comparison with model-based
approaches Journal of Chemical Information and Computer Sciences
44 1998ndash2009
Borodina Y Sadym A Filimonov D Blinova V Dmitriev A amp
Poroikov V (2003) Predicting biotransformation potential from
molecular structure Journal of Chemical Information and Computer
Sciences 43 1636ndash1646
Bottone Jr F G Martinez J M Collins J B Afshari C A amp Eling
T E (2003) Gene modulation by the cyclooxygenase inhibitor
sulindac sulfide in human colorectal carcinoma cells Possible link to
apoptosis Journal of Biological Chemistry 278 25790ndash25801
Boyer S amp Zamora I (2002) New methods in predictive metabolism
Journal of Computer-Aided Molecular Design 16 403ndash413
Bredel M amp Jacoby E (2004) Chemogenomics An emerging strategy
for rapid target and drug discovery Nature Reviews Genetics 5
262ndash275
Bugrim A Nikolskaya T amp Nikolsky Y (2004) Early prediction of drug
metabolism and toxicity Systems biology approach and modeling
Drug Discovery Today 9 127ndash135
Bulera S J Eddy S M Ferguson E Jatkoe T A Reindel J F
Bleavins M R et al (2001) RNA expression in the early character-
ization of hepatotoxicants in Wistar rats by high-density DNA micro-
arrays Hepatology 33 1239ndash1258
Butcher E C Berg E L amp Kunkel E J (2004) Systems biology in drug
discovery Nature Biotechnology 22 1253ndash1259
Butte A (2002) The use and analysis of microarray data Naturalist
Review of Drug Discovery 1 951ndash960
Cary M P Bader G D amp Sander C (2005) Pathway information for
systems biology FEBS Letters 579 1815ndash1820
Cheok M H Yang W Pui C H Downing J R Cheng C Naeve
C W et al (2003) Treatment-specific changes in gene expression
discriminate in vivo drug response in human leukemia cells Nature
Genetics 34 85ndash90
Cleary J D Rogers P D amp Chapman S W (2001) Differential
transcription factor expression in human mononuclear cells in response
to amphotericin B Identification with complementary DNA microarray
technology Pharmacotherapy 21 1046ndash1054
Coen M Ruepp S U Lindon J C Nicholson J K Pognan F Lenz
E M et al (2004) Integrated application of transcriptomics and
metabonomics yields new insight into the toxicity due to paracetamol in
the mouse Journal of Pharmaceutical and Biomedical Analysis 35
93ndash105
Cornwell P D De Souza A T amp Ulrich R G (2004) Profiling of
hepatic gene expression in rats treated with fibric acid analogs
Mutation Research 549 131ndash145
Csermely P Agoston V amp Pongor S (2005) The efficiency of multi-
target drugs The network approach might help drug design Trends in
Pharmacological Sciences 26 178ndash182
Cunningham M J Liang S Fuhrman S Seilhamer J J amp
Somogyi R (2000) Gene expression microarray data analysis for
toxicology profiling Annals of the New York Academy of Sciences 919
52ndash67
de Longueville F Atienzar F A Marcq L Dufrane S Evrard S
Wouters L et al (2003) Use of a low density microarray for studying
gene expression patterns induced by hepatotoxicants on primary
cultures of rat hepatocytes Toxicological Sciences 75 378ndash392
Dobrin R Beg Q K Barabasi A L amp Oltvai Z N (2004)
Aggregation of topological motifs in the Escherichia coli transcriptional
regulatory network BMC Bioinformatics 5 10
Donald S Verschoyle R D Edwards R Judah D J Davies R
Riley J et al (2002) Hepatobiliary damage and changes in hepatic
gene expression caused by the antitumor drug ecteinascidin-743 (ET-
743) in the female rat Cancer Research 62 4256ndash4262
Dumont M Jacquemin E DrsquoHont C Descout C Cresteil D Haouzi
D et al (1997) Expression of the liver Na+-independent organic anion
transporting polypeptide (oatp-1) in rats with bile duct ligation Journal
of Hepatology 27 1051ndash1056
Durnas C Loi C M amp Cusack B J (1990) Hepatic drug metabolism
and aging Clinical Pharmacokinetics 19 359ndash389
Eisen M B Spellman P T Brown P O amp Botstein D (1998) Cluster
analysis and display of genome-wide expression patterns Proceedings
of the National Academy of Sciences of the United States of America
95 14863ndash14868
Ekins S Andreyev S Ryabov A Kirilov E Rakhmatulin E A
Bugrim A et al (in press) Computational Prediction of Human Drug
Metabolism Exp Opin Drug Metab Toxicol
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6662
Ekins S Boulanger B Swaan P W amp Hupcey M A Z (2002)
Towards a new age of virtual ADMETOX and multidimensional drug
discovery Journal of Computer-Aided Molecular Design 16 381ndash401
Ekins S Bugrim A Nikolsky Y amp Nikolskaya T (2005) Systems
biology Applications in drug discovery In S Gad (Ed) Drug
Discovery Handbook (pp 123ndash183) Hoboken NJrsquo Wiley and Sons
Ekins S Giroux C N Nikolsky Y Bugrim A amp Nikolskaya T
(2005) A signature gene network approach to toxicity The Toxoco-
logists 84
Ekins S Kirillov E Rakhmatulin E amp Nikolskaya T (2005) A novel
method for visualizing nuclear hormone receptor networks relevant to
drug metabolism Drug Metabolism and Disposition 33 474ndash481
Ekins S amp McGowan R J (2001) The limits of reductionism The
shifting genomic paradigmrsquos impact on industry and academia
Philsophy in Science 9 1ndash23
Ekins S Mirny L amp Schuetz E G (2002) A ligand-based approach to
understanding selectivity of nuclear hormone receptors PXR CAR
FXR LXRa and LXRb Pharmaceutical Research 19 1788ndash1800
Ekins S Nikolsky Y amp Nikolskaya T (2005) Techniques Application
of systems biology to absorption distribution metabolism excretion
and toxicity Trends in Pharmacological Sciences 26 202ndash209
Ekins S Ring B J Grace J McRobie-Belle D J amp Wrighton S
A (2000) Present and future in vitro approaches for drug
metabolism Journal of Pharmacological and Toxicological Methods
44 313ndash324
Ekins S Waller C L Swaan P W Cruciani G Wrighton S A amp
Wikel J H (2000) Progress in predicting human ADME parameters in
silico Journal of Pharmacological and Toxicological Methods 44
251ndash272
Erhardt P W (2003) A human drug metabolism database Potential roles
in the quantitative predictions of drug metabolism and metabolism-
related drugndashdrug interactions Current Drug Metabolism 4 411ndash422
Evans W E amp McLeod H L (2003) Pharmacogenomicsmdashdrug
disposition drug targets and side effects New England Journal of
Medicine 348 538ndash549
Ezendam J Staedtler F Pennings J Vandebriel R J Pieters R
Boffetta P et al (2004) Toxicogenomics of subchronic hexachlor-
obenzene exposure in Brown Norway rats Environmental Health
Perspectives 112 782ndash791
Faiola B Fuller E S Wong V A amp Recio L (2004) Gene expression
profile in bone marrow and hematopoietic stem cells in mice exposed to
inhaled benzene Mutation Research 549 195ndash212
FDA (2004) Innovation stagnation Challenge and opportunity on the
critical path to new medicinal products
Fiehn O (2001) Combining genomics metabolome analysis and bio-
chemical modelling to understand metabolic networks Comparative
Functional Genomics 2 155ndash168
Field F J Born E amp Mathur S N (2004) LXRRXR ligand activation
enhances basolateral efflux of beta-sitosterol in CaCo-2 cells Journal of
Lipid Research 45 905ndash913
Fleck C Sutter L Appenroth D Koch B Meinhold T Pitack M
et al (2003) Use of gene chip technology for the characterisation of
the regulation of renal transport processes and of nephrotoxicity in
rats Experimental and Toxicologic Pathology 54 401ndash410
Fliri A F Loging W T Thadeio P F amp Volkmann R A (2005)
Biological spectra analysis Linking biological activity profiles to
molecular structure Proceedings of the National Academy of Sciences
of the United States of America 102 261ndash266
Fountoulakis M (2004) Application of proteomics technologies in the
investigation of the brain Mass Spectrometry Reviews 23 231ndash258
Gaucher S P Taylor S W Fahy E Zhang B Warnock D E Ghosh
S S et al (2004) Expanded coverage of the human heart
mitochondrial proteome using multidimensional liquid chromatography
coupled with tandem mass spectrometry Journal of Proteome
Research 3 495ndash505
Gerhold D Lu M Xu J Austin C Caskey C T amp Rushmore T
(2001) Monitoring expression of genes involved in drug metabolism
and toxicology using DNA microarrays Physiological Genomics 5
161ndash170
Guo G L Choudhuri S amp Klaassen C D (2002) Induction profile of
rat organic anion transporting polypeptide 2 (oatp2) by prototypical
drug-metabolizing enzyme inducers that activate gene expression
through ligand-activated transcription factor pathways Journal of
Pharmacology and Experimental Therapeutics 300 206ndash212
Hagenbuch B amp Meier P J (2004) Organic anion transporting
polypeptides of the OATP SLC21 family Phylogenetic classification
as OATP SLCO superfamily new nomenclature and molecularfunc-
tional properties Pflugers Archiv 447 653ndash665
Hamadeh H K amp Afshari C A (2004) Toxicogenomics principles and
applications Hobokenrsquo John Wiley and Sons
Hamadeh H K Bushel P R Jayadev S Martin K DiSorbo O Sieber
S et al (2002) Gene expression analysis reveals chemical-specific
profiles Toxicological Sciences 67 219ndash231
Hamadeh H K Jayadev S Gaillard E T Huang Q Stoll R
Blanchard K et al (2004) Integration of clinical and gene expression
endpoints to explore furan-mediated hepatotoxicity Mutation Research
549 169ndash183
Hamadeh H K Knight B L Haugen A C Sieber S Amin R P
Bushel P R et al (2002) Methapyrilene toxicity Anchorage of
pathologic observations to gene expression alterations Toxicologic
Pathology 30 470ndash482
Han J D Bertin N Hao T Goldberg D S Berriz G F Zhang L V
et al (2004) Evidence for dynamically organized modularity in the
yeast proteinndashprotein interaction network Nature 430 88ndash93
Hanisch D Zien A Zimmer R amp Lengauer T (2002) Co-clustering of
biological networks and gene expression data Bioinformatics 18
S145ndashS154
Harris A J Dial S L amp Casciano D A (2004) Comparison of basal
gene expression profiles and effects of hepatocarcinogens on gene
expression in cultured primary human hepatocytes and HepG2 cells
Mutation Research 549 79ndash99
Harrison S C (2004) Whither structural biology Nature Structural
Molecular Biology 11 12ndash15
Hartley D P Dai X He Y D Carlini E J Wang B Huskey S E
et al (2004) Activators of the rat pregnane X receptor differentially
modulate hepatic and intestinal gene expression Molecular Pharma-
cology 65 1159ndash1171
Hartwell L H Hopfield J J Leibler S amp Murray A W (1999) From
molecular to modular cell biology Nature 402 C47ndashC52
Hasmall S Orphanides G James N Pennie W Hedley K Soames
A et al (2002) Downregulation of lactoferrin by PPARalpha ligands
Role in perturbation of hepatocyte proliferation and apoptosis
Toxicology Sciences 68 304ndash313
Hayase T Yamamoto Y Yamamoto K Muso E amp Shiota K (2004)
Microarray profile analysis of toxic cocaine-induced alterations in the
expression of mouse brain gene sequences A possible rsquoprotectiversquo
effect of buprenorphine Journal of Applied Toxicology 24 15ndash20
Hayes K R Vollrath A L Zastrow G M McMillan B J Craven M
Jovanovich S et al (2005) EDGE A centralized resource for the
comparison analysis and distribution of toxicogenomic information
Molecular Pharmacology 67 1360ndash1368
Heijne W H Slitt A L van Bladeren P J Groten J P Klaassen C D
Stierum R H et al (2004) Bromobenzene-induced hepatotoxicity at
the transcriptome level Toxicology Sciences 79 411ndash422
Heijne W H Stierum R H Slijper M van Bladeren P J amp van
Ommen B (2003) Toxicogenomics of bromobenzene hepatotoxicity
A combined transcriptomics and proteomics approach Biochemical
Pharmacology 65 857ndash875
Heinloth A N Irwin R D Boorman G A Nettesheim P Fannin
R D Sieber S O et al (2004) Gene expression profiling of rat
livers reveals indicators of potential adverse effects Toxicology
Sciences 80 193ndash202
Hodges L C Cook J D Lobenhofer E K Li L Bennett L Bushel
P R et al (2003) Tamoxifen functions as a molecular agonist inducing
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 63
cell cycle-associated genes in breast cancer cells Molecular Cancer
Research 1 300ndash311
Holleman A Cheok M H den Boer M L Yang W Veerman KM
A J Kazemier et al (2004) Gene-expression patterns in drug-resistant
acute lymphoblastic leukemia cells and response to treatment New
England Journal of Medicine 351 533ndash542
Hong Y Muller U R amp Lai F (2003) Discriminating two classes of
toxicants through expression analysis of HepG2 cells with DNA arrays
Toxicology in Vitro 17 85ndash92
Hood E (2003) Proteomics Characterizing the cogs in the machinery of
life Environmental Health Perspectives 111 A816ndashA825
Hood L (2003) Systems biology Integrating technology biology and
computation Mechanism of Ageing and Development 124 9ndash16
Hood L amp Galas D (2003) The digital code of DNA Nature 421
444ndash448
Hood L amp Perlmutter R M (2004) The impact of systems approaches on
biological problems in drug discovery Nature Biotechnology 22
1215ndash1217
Hu T Gibson D P Carr G J Torontali S M Tiesman J P Chaney
J G et al (2004) Identification of a gene expression profile that
discriminates indirect-acting genotoxins from direct-acting genotoxins
Mutation Research 549 5ndash27
Huang Q Dunn II R T Jayadev S DiSorbo O Pack F D Farr S B
et al (2001) Assessment of cisplatin-induced nephrotoxicity by
microarray technology Toxicology Sciences 63 196ndash207
Huang Q Jin X Gaillard E T Knight B L Pack F D Stoltz J H et
al (2004) Gene expression profiling reveals multiple toxicity endpoints
induced by hepatotoxicants Mutation Research 549 147ndash167
Huisman M T Smit J W Crommentuyn K M Zelcer N Wiltshire
H R Beijnen J H et al (2002) Multidrug resistance protein 2
(MRP2) transports HIV protease inhibitors and transport can be
enhanced by other drugs Aids 16 2295ndash2301
Hunt C M Westerkam W R amp Stave G M (1992) Effect of age and
gender on the activity of human hepatic CYP3A Biochemical
Pharmacology 44 275ndash283
Ideker T Ozier O Schwikowski B amp Siegal A F (2002) Discovering
regulatory and signalling circuits in molecular interaction networks
Bioinformatics 18 S233ndashS240
Ideker T Thorsson V Ranish J A Christmas R Buhler J Eng J K
et al (2001) Integrated genomic and proteomic analyses of a system-
atically perturbed network Science 292 929ndash934
Iida M Anna C H Hartis J Bruno M Wetmore B Dubin J R et al
(2003) Changes in global gene and protein expression during early
mouse liver carcinogenesis induced by non-genotoxic model carcino-
gens oxazepam and Wyeth-14643 Carcinogenesis 24 757ndash770
Ingelman-Sundberg M (2004) Pharmacogenetics of cytochrome P450 and
its applications in drug therapy The past present and future Trends in
Pharmacological Sciences 25 193ndash200
Jeong H Mason S P Barabasi A L amp Oltvai Z N (2001) Lethality
and centrality in protein networks Nature 411 41ndash42
Jeong H Tombor B Albert R Oltvai Z N amp Barabasi A L
(2000) The large-scale organization of metabolic networks Nature
407 651ndash654
Jung D Hagenbuch B Gresh L Pontoglio M Meier P J amp Kullak-
Ublick G A (2001) Characterization of the human OATP-C
(SLC21A6) gene promoter and regulation of liver-specific OATP genes
by hepatocyte nuclear factor 1 alpha Journal of Biological Chemistry
276 37206ndash37214
Jung D amp Kullak-Ublick G A (2003) Hepatocyte nuclear factor 1
alpha A key mediator of the effect of bile acids on gene expression
Hepatology 37 622ndash631
Jung J -W Park J -S Hwang J -W Kang K -S YS L Song B
-S et al (2004) Gene expression analysis of peroxisome prolifer-
ators- and phenytoin-induced hepatotoxicity using cDNA microarray
Journal of Veterinary Medical Science 66 1329ndash1333
Kato N Shibutani M Takagi H Uneyama C Lee K -Y Takiami S
et al (2004) Gene expression profile in the livers of rats orally
administered ethinylestradiol for 28 days using a microarray technique
Toxicology 200 179ndash192
Kier L D Neft R Tang L Suizu R Cook T Onsurez K et al
(2004) Applications of microarrays with toxicologically relevant genes
(tox genes) for the evaluation of chemical toxicants in Sprague Dawley
rats in vivo and human hepatocytes in vitro Mutation Research 549
101ndash113
Kijima K Toyosawa K Yasuba M Matsuoka N Adachi T
Komiyama M et al (2004) Gene expression analysis of the rat testis
after treatment with di(2-ethylhexyl) phthalate using cDNA microarray
and real-time RT-PCR Toxicology and Applied Pharmacology 200
103ndash110
Kim R B (2003) Organic anion-transporting polypeptide (OATP) trans-
porter family and drug disposition European Journal of Clinical
Investigation 33 1ndash5
Kinirons M T amp OrsquoMahony M S (2004) Drug metabolism and ageing
British Journal of Clinical Pharmacology 57 540ndash544
Kitano H (2002a) Computational systems biology Nature 420 206ndash210
Kitano H (2002b) Systems biology A brief overview Science 295
1662ndash1664
Kliewer S A Moore J T Wade L Staudinger J L Watson M
A Jones S A et al (1998) An orphan nuclear receptor activated
by pregnanes defines a novel steroid signalling pathway Cell 92
73ndash82
Korolev D Balakin K V Nikolsky Y Kirillov E Ivanenkov Y A
Savchuk N P et al (2003) Modeling of human cytochrome p450-
mediated drug metabolism using unsupervised machine learning
approach Journal of Medicinal Chemistry 46 3631ndash3643
Kramer J A Pettit S D Amin R P Bertram T A Car B
Cunningham M et al (2004) Overview on the application of
transcription profiling using selected nephrotoxicants for toxicology
assessment Environmental Health Perspectives 112 460ndash464
Kultima K Nystrom A -M Scholz B Gustafson A -L Dencker L
amp Stigson M (2004) Valproic acid teratogenicity A toxicogenomics
approach Environmental Health Perspectives 112 1225ndash1235
Kutuzova G D amp Deluca H F (2004) Gene expression profiles in rat
intestine identify pathways for 125-dihydroxyvitamin D(3) stimulated
calcium absorption and clarify its immunomodulatory properties
Archives of Biochemistry and Biophysics 432 152ndash166
Kwak M K Wakabayashi N Itoh K Motohashi H Yamamoto M amp
Kensler T W (2003) Modulation of gene expression by cancer
chemopreventive dithiolethiones through the Keap1-Nrf2 pathway
Identification of novel gene clusters for cell survival Journal of
Biological Chemistry 278 8135ndash8145
Lahjouji K Mitchell G A amp Qureshi I A (2001) Carnitine transport
by organic cation transporters and systemic carnitine deficiency
Molecular Genetics and Metabolism 73 287ndash297
Landowski C P Sun D Foster D R Menon S S Barnett J L
Welage L S et al (2003) Gene expression in the human intestine and
correlation with oral valacyclovir pharmacokinetic parameters Journal
of Pharmacology and Experimental Therapeutics 306 778ndash786
Lee G -J Lee W -S Jeon K -S Um C -H Kim S -J Lee C -H
et al (2004) cDNA microarray gene expression analysis and
toxicological phenotype for anticancer drug Journal of Veterinary
Medical Science 66 1339ndash1345
Lee M Kwon J Kim S N Kim J E Koh W S Kim E J et al
(2003) cDNA microarray gene expression profiling of hydroxyurea
paclitaxel and p-anisidine genotoxic compounds with differing
tumorigenicity results Environmental and Molecular Mutagenesis
42 91ndash97
Li S Armstrong C M Bertin N Ge H Milstein S Boxem M et al
(2004) A map of the interactome network of the metazoan C elegans
Science 303 540ndash543
Liguori M J Anderson L M Bukofzer S McKim J Pregenzer J F
Retief J et al (2005) Microarray analysis in human hepatocytes
suggests a mechanism for hepatotoxicity induced by trovafloxacin
Hepatology 41 177ndash186
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6664
Lobenhofer E K Bennett L Cable P L Li L Bushel P R amp Afshari
C A (2002) Regulation of DNA replication fork genes by 17beta-
estradiol Molecular Endocrinology 16 1215ndash1229
Locker J Tian J Carver R Concas D Cossu C Ledda-Columbano
G M et al (2003) A common set of immediate-early response genes
in liver regeneration and hyperplasia Hepatology 38 314ndash325
Mattes W B Pettit S D Sansone S A Bushel P R amp Waters M D
(2004) Database development in toxicogenomics Issues and efforts
Environmental Health Perspectives 112 495ndash505
McMillian M Nie A Y Parker J B Leone A Bryant S Kemmerer
M et al (2004) A gene expression signature for oxidant stressreactive
metabolites in rat liver Biochemical Pharmacology 68 2249ndash2261
Meier P J amp Stieger B (2000) Molecular mechanisms in bile formation
News in Physiological Sciences 15 89ndash93
Meneses-Lorente G de Longueville F Dos Santos-Mendes S Bonnert
T P Jack A Evrard S et al (2003) An evaluation of a low-density
DNA microarray using cytochrome P450 inducers Chemical Research
in Toxicology 16 1070ndash1077
Meneses-Lorente G Guest P C Lawrence J Muniappa N Knowles
M R Skynner H A et al (2004) A proteomic investigation of drug-
induced steatosis in rat liver Chemical Research in Toxicology 17
605ndash612
Merrill C L Ni H Yoon L W Tirmenstein M A Narayanan P
Benavides G R et al (2002) Etomoxir-induced oxidative stress in
HepG2 cells detected by differential gene expression is confirmed
biochemically Toxicology Sciences 68 93ndash101
Milo R Shen-Orr S Itzkovitz S Kashtan N Chklovskii D amp Alon
U (2002) Network motifs Simple building blocks of complex
networks Science 298 824ndash827
Morgan K T Ni H Brown H R Yoon L Qualls C W Crosby L
M et al (2002) Application of cDNA microarray technology to in
vitro toxicology and the selection of genes for a real-time RT-PCR-
based screen for oxidative stress in Hep-G2 cells Toxicologic
Pathology 30 435ndash451
Mwinyi J Johne A Bauer S Roots I amp Gerloff T (2004) Evidence
for inverse effects of OATP-C (SLC21A6) 5 and 1b haplotypes on
pravastatin kinetics Clinical Pharmacology and Therapeutics 75
415ndash421
Nakata K Yukawa M Komiyama N Nakano T amp Kaminuma T
(2002) A nuclear receptor database that maps pathways to diseases
Genome Infomatics 13 515ndash516
Nicholson J K Holmes E Lindon J C amp Wilson I D (2004) The
challenges of modeling mammalian biocomplexity Nature Biotechnol-
ogy 22 1268ndash1274
Nicholson J K amp Wilson I D (2003) Understanding rsquoglobal systems
biology Metabonomics and the continuum of metabolism Nature
Reviews on Drug Discovery 2 668ndash676
Nikitin A Egorov S Daraselia N amp Mazo I (2003) Pathway studiomdash
the analysis and navigation of molecular networks Bioinformatics 19
2155ndash2157
Nikolsky Y Ekins S Nikolskaya T amp Bugrim A (2005) A novel
method for generation of signature networks as biomarkers from
complex high throughput data Toxicology Letters 158 20ndash29
Nikolsky Y Nikolskaya T amp Bugrim A (2005) Biological networks
and analysis of experimental data in drug discovery Drug Discovery
Today 10 653ndash662
Nishizato Y Ieiri I Suzuki H Kimura M Kawabata K Hirota T
et al (2003) Polymorphisms of OATP-C (SLC21A6) and OAT3
(SLC22A8) genes Consequences for pravastatin pharmacokinetics
Clinical Pharmacology and Therapeutics 73 554ndash565
Nozawa T Nakajima M Tamai I Noda K Nezu J Sai Y et al
(2002) Genetic polymorphisms of human organic anion transporters
OATP-C (SLC21A6) and OATP-B (SLC21A9) Allele frequencies in
the Japanese population and functional analysis Journal of Pharmacol-
ogy and Experimental Therapeutics 302 804ndash813
Nozawa T Sugiura S Nakajima M Goto A Yokoi T Nezu J et al
(2004) Involvement of organic anion transporting polypeptides in the
transport of troglitazone sulfate Implications for understanding trogli-
tazone hepatotoxicity Drug Metabolism and Disposition 32 291ndash294
Oliver D E Rubin D L Stuart J M Hewett M Klein T E amp
Altman R B (2002) Ontology development for a pharmacogenetics
knowledge base Package Symposion 88ndash99
Oram J F amp Lawn R M (2001) ABCA1 The gatekeeper for eliminating
excess tissue cholesterol Journal of Lipid Research 42 1173ndash1179
Oswald M Kullak-Ublick G A Paumgartner G amp Beuers U (2001)
Expression of hepatic transporters OATP-C and MRP2 in primary
sclerosing cholangitis Liver 21 247ndash253
Parsons A B Brost R I Ding H Li Z Zhang C Sheikh B et al
(2004) Integration of chemical-genetic and genetic interaction data
links bioactive compounds to cellular target pathways Nature Bio-
technology 22 62ndash69
Patki K C von Moltke L L Harmatz J S Hesse L M Court M H
amp Greenblatt D J (2004) Effect of age on in vitro triazolam
biotransformation in male human liver microsomes Journal of
Pharmacology and Experimental Therapeutics 308 874ndash879
Pereira-Leal J B Enright A J amp Ouzounis C A (2004) Detection of
functional modules from protein interaction networks Proteins 54
49ndash57
Plant N (2004) Interaction networks Coordinating responses to xeno-
biotic exposure Toxicology 202 21ndash32
Raghavendra Prasad H S Qi Z Srinivasan K N amp Gopalakrish-
nakone P (2004) Potential effects of tetrodotoxin exposure to
human glial cells postulated using microarray approach Toxicon 44
597ndash608
Reilly T P Bourdi M Brady J N Pise-Masison C A Radonovich M
F George J W et al (2001) Expression profiling of acetaminophen
liver toxicity in mice using microarray technology Biochemical and
Biophysical Research Communications 282 321ndash328
Rives A W amp Galitski T (2003) Modular organization of cellular
networks Proceedings of the National Academy of Sciences of the
United States of America 100 1128ndash1133
Rogers P D Pearson M M Cleary J D Sullivan D C amp Chapman
S W (2002) Differential expression of genes encoding immunomodu-
latory proteins in response to amphotericin B in human mononuclear
cells identified by cDNA microarray analysis Journal of Antimicrobial
Chemotherapy 50 811ndash817
Rosen M B Wilson V S Schmid J E amp Gray L E (2005) Gene
expression analysis in the ventral prostate of rats exposed to vinclozolin
or procymidone Reproductive Toxicology 19 367ndash379
Rost D Herrmann T Sauer P Schmidts H L Stieger B Meier P J
et al (2003) Regulation of rat organic anion transporters in bile salt-
induced cholestatic hepatitis Effect of ursodeoxycholate Hepatology
38 187ndash195
Rothman R B Ayestas M A Dersch C M amp Baumann M H (1999)
Aminorex fenfluramine and chlorphentermine are serotonin transporter
substrates Implications for primary pulmonary hypertension Circula-
tion 100 869ndash875
Ruepp S U Tonge R P Shaw J Wallis N amp Pognan F (2002)
Genomics and proteomics analysis of acetaminophen toxicity in mouse
liver Toxicology Sciences 65 135ndash150
Sakaeda T Nakamura T amp Okumura K (2002) MDR1 genotype-
related pharmacokinetics and pharmacodynamics Biological amp Phar-
maceutical Bulletin 25 1391ndash1400
Sakata T Anzai N Shin H J Noshiro R Hirata T Yokoyama H
et al (2004) Novel single nucleotide polymorphisms of organic cation
transporter 1 (SLC22A1) affecting transport functions Biochemical
and Biophysical Research Communications 313 789ndash793
Satlin L M Amin V amp Wolkoff A W (1997) Organic anion
transporting polypeptide mediates organic anionHCO3-exchange
Journal of Biological Chemistry 272 26340ndash26345
Satomi Y Tsuchiya W Mihara K Ota M Kasahara Y amp Akahori F
(2004) Gene expression analysis of the lung following paraquat
administration in rats using DNA microarray Journal of Toxicological
Sciences 29 91ndash100
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 65
Schrenk D Baus P R Ermel N Klein C Vorderstemann B amp
Kauffmann H M (2001) Up-regulation of transporters of the MRP
family by drugs and toxins Toxicology Letters 120 51ndash57
Segal E Shapira M Regev A Persquoer D Botstein D Koller D et al
(2003) Module networks Identifying regulatory modules and their
condition-specific regulators from gene expression data Nature
Genetics 34 166ndash176
Segal E Wang H amp Koller D (2003) Discovering molecular pathways
from protein interaction and gene expression Bioinformatics 19
i264ndash i272
Seidel S D Kan H L Stott W T Schisler M R amp Gollapudi B B
(2003) Identification of transcriptome profiles for the DNA-damaging
agents bleomycin and hydrogen peroxide in L5178Y mouse lymphoma
cells Environmental and Molecular Mutagenesis 42 19ndash25
Shan L Yu Y Schut H A J amp Snyderwine E G (2004) Susceptibility
of rats to mammary gland carcinogenesis by the food-derived
carcinogen 2-Amino-1-Methyl-6-Phenylimidazo[45-b]Pyridine (PhIP)
varies with age and is associated with the induction of differential gene
expression American Journal of Pathology 165 191ndash202
Sharom J R Bellows D S amp Tyers M (2004) From large networks to
small molecules Current Opinion in Chemical Biology 8 81ndash90
Shitara Y Hirano M Sato H amp Sugiyama Y (2004) Gemfibrozil and
its glucuronide inhibit the OATP2(OATP1B1 SLC21A6)-mediated
hepatic uptake and CYP2C8-mediated metabolism of cerivastatinmdash
Analysis of the mechanism of the clinically relevant drugndashdrug
interaction between cerivastatin and gemfibrozil Journal of Pharma-
cology and Experimental Therapeutics
Shitara Y Itoh T Sato H Li A P amp Sugiyama Y (2003) Inhibition of
transporter-mediated hepatic uptake as a mechanism for drugndashdrug
interaction between cerivastatin and cyclosporin A Journal of
Pharmacology and Experimental Therapeutics 304 610ndash616
Somogyi R Fuhrman S amp Wen X (2001) Genetic network inference in
computational models and applications to large-scale gene expression
data In M Bower amp H Bolouri (Eds) Computational modeling of
genetic and biochemical networks (pp 119ndash157) Cambridge MArsquoThe
MIT Press
Spirin V amp Mirny L A (2003) Protein complexes and functional
modules in molecular networks Proceedings of the National Academy
of Sciences of the United States of America 100 12123ndash12128
Staudinger J Liu Y Madan A Habeebu S amp Klaassen C D
(2001) Coordinate regulation of xenobiotic and bile acid homeo-
stasis by pregnane X receptor Drug Metabolism and Disposition
29 1467ndash1472
Sun L Z Ji Z L Chen X Wang J F amp Chen Y Z (2002) ADME-
AP A database of ADME associated proteins Bioinformatics 18
1699ndash1700
Suter L Babiss L E amp Wheeldon E B (2004) Toxicogenomics in
predictive toxicology in drug development Chemistry and Biology 11
161ndash171
Synold T W Dussault I amp Forman B M (2001) The orphan nuclear
receptor SXR coordinately regulates drug metabolism and efflux
Nature Medicine 7 584ndash590
Tabuchi Y amp Kondo T (2003) cDNA microarray analysis reveals chop-
10 plays a key role in Sertoli cell injury induced by bisphenol A
Biochemical and Biophysical Research Communications 305 54ndash61
Tamai I Nezu J Uchino H Sai Y Oku A Shimane M et al (2000)
Molecular identification and characterization of novel members of the
human organic anion transporter (OATP) family Biochemical and
Biophysical Research Communications 273 251ndash260
Taylor S W Fahy E Zhang B Glenn G M Warnock D E Wiley S
et al (2003) Characterization of the human heart mitochondrial
proteome Nature Biotechnology 21 281ndash286
Terasaka S Aita Y Inoue A Hayashi S Nishigaki M Aoyagi K
et al (2004) Using a customized DNA microarray for expression
profiling of the estrogen-responsive genes to evaluate estrogen activity
among natural estrogens and industrial chemicals Environmental
Health Perspectives 112 773ndash781
Thomas D M Francescutti-Verbeem D M Liu X amp Kuhn D M
(2004) Identification of differentially regulated transcripts in mouse
striatum following methamphetamine treatmentmdashan oligonucleotide
microarray approach Journal of Neurochemistry 88 380ndash393
Thomas R S Rank D R Penn S G Zastrow G M Hayes K R
Pande K et al (2001) Identification of toxicologically predictive
gene sets using cDNA microarrays Molecular Pharmacology 60
1189ndash1194
Thompson K L Afshari C A Amin R P Bertram T A Car B
Cunningham M et al (2004) Identification of platform-independent
gene expression markers of cisplatin nephrotoxicity Environmental
Health Perspectives 112 488ndash494
Tirona R G Leake B F Merino G amp Kim R B (2001)
Polymorphisms in OATP-C Identification of multiple allelic
variants associated with altered transport activity among European-
and African-Americans Journal of Biological Chemistry 276
35669ndash35675
Tirona R G Leake B F Wolkoff A W amp Kim R B (2003)
Human organic anion transporting polypeptide-C (SLC21A6) is a
major determinant of the rifampin-mediated pregnane X receptor
activation Journal of Pharmacology and Experimental Therapeutics
304 223ndash228
Tong W Cao X Harris S Sun H Fang H Fuscoe J et al (2003)
ArrayTrackmdashsupporting toxicogenomic research at the US Food and
Drug Administration National Center for Toxicological Research
Environmental Health Perspectives 111 1819ndash1826
Tornow S amp Mewes H W (2003) Functional modules by relating
protein interaction networks and gene expression Nucleic Acids
Research 31 6283ndash6289
Trocho C Escola-Gil J C Ribas V Benitez S Martin-Campos J M
Rotllan N et al (2004) Phenytoin treatment reduces atherosclerosis in
mice through mechanisms independent of plasma HDL-cholesterol
concentration Atherosclerosis 174 275ndash285
Ueda A Hamadeh H K Webb H K Yamamoto Y Sueyoshi T
Afshari C A et al (2002) Diverse roles of the nuclear orphan
receptor CAR in regulating hepatic genes in response to phenobarbital
Molecular Pharmacology 61 1ndash6
Ulrich R G (2003) The toxicogenomics of nuclear receptor agonists
Current Opinion in Chemical Biology 7 505ndash510
Ulrich R G Rockett J C Gibson G G amp Pettit S D (2004)
Overview of an interlaboratory collaboration on evaluating the effects
of model hepatotoxicants on hepatic gene expression Environmental
Health Perspectives 112 423ndash427
Unami A Shinohara Y Ichikawa T amp Baba Y (2003) Biochemical
and microarray analyses of bupivacaine-induced apoptosis Journal of
Toxicological Sciences 28 77ndash94
Vasquez A Flammini A Maritan A amp Vespignani A (2003) Global
protein function prediction from proteinndashprotein interaction networks
Nature Biotechnology 21 697ndash700
Vezina C M Walker N J amp Olson J R (2004) Subchronic Exposure to
TCDD PeCDF PCB126 and PCB153 Effect on hepatic gene
expression Environmental Health Perspectives 112 1636ndash1644
Vo T D Greenberg H J amp Palsson B O (2004) Reconstruction and
functional characterization of the human mitochondrial metabolic
network based on proteomic and biochemical data Journal of Bio-
logical Chemistry 279 39532ndash39540
Wang H amp LeCluyse E L (2003) Role of orphan nuclear receptors in
the regulation of drug-metabolising enzymes Clinical Pharmacoki-
netics 42 1331ndash1357
Wang N Silver D L Thiele C amp Tall A R (2001) ATP-
binding cassette transporter A1 (ABCA1) functions as a choles-
terol efflux regulatory protein Journal of Biological Chemistry 276
23742ndash23747
Waring J F Cavet G Jolly R A McDowell J Dai H Ciurlionis R
et al (2003) Development of a DNA Microarray for toxicology based
on hepatoxin-regulated sequences Environmental Health Perspectives
111 863ndash870
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6666
Waring J F Gum R Morfitt D Jolly R A Ciurlionis R Heindel M
et al (2002) Identifying toxic mechanisms using DNA microarrays
Evidence that an experimental inhibitor of cell adhesion molecule
expression signals through the aryl hydrocarbon nuclear receptor
Toxicology 181ndash182 537ndash550
Waring J F Jolly R A Ciurlionis R Lum P Y Praestgaard J T
Morfitt D C et al (2001) Clustering of hepatotoxins based on
mechanism of toxicity using gene expression profiles Toxicology and
Applied Pharmacology 175 28ndash42
Waters M Boorman G Bushel P Cunningham M Irwin R Merrick
A et al (2003) Systems toxicology and the Chemical Effects in
Biological Systems (CEBS) knowledge base EHP Toxicogenomics
111 15ndash28
Wei M Arnold L Cano M amp Cohen S M (2005) Effects of co-
administration of antioxidants and arsenicals on the rat urinary bladder
epithelium Toxicology Sciences 83 237ndash245
Weinshilboum R (2003) Inheritance and drug response New England
Journal of Medicine 348 529ndash537
Weinshilboum R amp Wang L (2004) Pharmacogenetics Inherited
variation in amino acid sequence and altered protein quantity Clinical
Pharmacology and Therapeutics 75 253ndash258
Werner E (2003) In silico multicellular systems biology and minimal
genomes Drug Discovery Today 8 1121ndash1127
Wong J S amp Gill S S (2002) Gene expression changes induced in
mouse liver by di(2-ethylhexyl) phthalate Toxicology and Applied
Pharmacology 185 180ndash196
Xie T Tong L McCann U D Yuan J Becker K G Mechan A O
et al (2004) Identification and characterization of metallothionein-1
and -2 gene expression in the context of ()34-methylenedioxymetham-
phetamine-induced toxicity to brain dopaminergic neurons Journal of
Neuroscience 24 7043ndash7050
Yadetie F Laegreid A Bakke I Kusnierczyk W Komorowski J
Waldum H L et al (2003) Liver gene expression in rats in response
to the peroxisome proliferator-activated receptor-alpha agonist ciprofi-
brate Physiological Genomics 15 9ndash19
Yamashita S Nomoto T Abe M Tatematsu M Sugimura T amp
Ushijima T (2004) Persistence of gene expression changes in stomach
mucosae induced by short-term N-methyl-NV-nitro-N-nitrosoguanidinetreatment and their presence in stomach cancers Mutation Research
549 185ndash193
Yan Q amp Sadee W (2000) Human membrane transporter database A
web-accessible relational database for drug transport studies and
pharmacogenomics AAPS Pharmsci 2 E20
Yeger-Lotem E amp Margalit H (2003) Detection of regulatory circuits by
integrating the cellular networks of proteinndashprotein interactions and
transcription regulation Nucleic Acids Research 31 6053ndash6061
Yoon B I Li G -X Kitada K Kawasaki Y Igarashi K amp Kodama
Y et al (2003) Mechanisms of benzene-induced hematotoxicity and
leukemogenicity cDNA microarray analyses using mouse bone marrow
tissue Environmental Health Perspectives 111 1411ndash1420
Young M B DiSilvestro M R Sendera T J Freund J Kriete A amp
Magnuson S R (2003) Analysis of gene expression in carbon
tetrachloride-treated rat livers using a novel bioarray technology
Pharmacogenomics Journal 3 41ndash52
Yu H Zhu X Greenbaum D Karro J amp Gerstein M (2004) TopNet
A tool for comparing biological sub-networks correlating protein
properties with topological statistics Nucleic Acids Research 32
328ndash337
Zhang E Y Fu D J Pak Y A Stewart T Mukhopadhyay N
Wrighton S A et al (2004) Genetic polymorphisms in human proton-
dependent dipeptide transporter PEPT1 Implications for the functional
role of Pro586 Journal of Pharmacology and Experimental Therapeu-
tics 310 437ndash445
Zhang E Y Knipp G T Ekins S amp Swaan P W (2002)
Structural biology and function of solute transporters Implications
for identifying and designing substrates Drug Metabolism Reviews
34 709ndash750
Zhang E Y Phelps M A Cheng C Ekins S amp Swaan P W (2002)
Modeling of active transport systems Advanced Drug Delivery
Reviews 54 329ndash354
- Systems-ADMETox Resources and network approaches
-
- Introduction
- Data available
- Network analysis and databases
-
- Network applications
-
- The role of transporters
-
- Clinical relevance of transporters
- Transporter network examples ABCA1
- Transporter network examples OATP
- Transporter microarray data
-
- Applications to enzymes
- Future network applications
-
- Discussion
- Acknowledgements
- References
-
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 55
visualization of the complex interconnections between this
transporter its ligands regulatory factors and signaling
molecules already in the database (Fig 6) The network was
generated with the autoexpand algorithm in the software
representing one of multiple available algorithms for
connecting genes ligands and other objects in the database
Clearly if more ligands and their connections are added to
the database the complexity of the network will increase
considerably The OATP-C (OATP1B1) gene details can be
viewed upon querying the database and links are provided
to other public databases This page can be used to highlight
the multiple synonyms for this gene as well as links to the
multiple SNPs identified to date
3114 Transporter microarray data Microarrays have
generally been limited in the number of transporters present
on them (Annereau et al 2004) however they have been used
in an attempt to correlate pharmacokinetic properties with
gene expression for valacyclovir (Landowski et al 2003) as
well as understand the expression profile in different tissues
or cell lines upon food component or xenobiotic treatment
(Anderle Huang amp Sadee 2004) This lack of transporters
on microarrays has prompted some groups to produce their
own arrays with a heavier emphasis on transporters These
arrays have for example then been used to demonstrate the
upregulation of ABC transporters and down-regulation of
GST-Pi in cell lines resistant to colchicines or 9-nitro-
camptothecin (Annereau et al 2004) The genes that were
significantly up or down-regulated in this particular study
were used to build networks with MetaCore (Fig 7A B) and
the similarities between them were assessed (Fig 7C)
Although there were only a small number of significantly
changed genes in common (IL-8 Fos GST-Pi Calpactin and
Ubiquitin hydrolase) it is perhaps likely that there is a much
larger common gene network that is important for drug
resistance although a much larger number of cell lines and
drug treatments need to be evaluated to produce a definitive
drug resistance signature involving transporters enzymes
and transcriptional regulators
312 Applications to enzymes
As we have already described it may be particularly
valuable to visualize enzymes as networks to show
interactions with transcriptional regulators and ligands
For example a key enzyme is CYP3A4 which metabolizes
40ndash45 of all drugs and has relatively few SNPs
(Ingelman-Sundberg 2004) Using a second software suite
MetaDrugi (wwwgenegocom) it is possible to construct
a custom network around this or other drug metabolizing
enzymes (Fig 8) In this case the gene network for
CYP3A4 highlights all of the major transcriptional
regulators and several more distant linked proteins and
ligands connected on the network that may be useful for
further study (Fig 8) Substrates inhibitors as well as
regulatory factors and other enzymes can be observed
connected on this network Due to the many hundreds of
known inhibitors and substrates only a small number are
shown here for clarity We assume that if a perturbation in
a pathway (eg due to a nonfunctional enzyme) is linked
to a certain pathologic condition a similar perturbation
caused by the interference from xenobiotic metabolism
(eg competitive inhibition of the same enzyme) may
result in identical effects As microarray gene expression
data is increasingly generated the role of enzyme
regulation in toxicity of certain xenobiotics will become
more apparent from either in vivo or in vitro studies The
visualization of such signature gene networks involving
transporters and enzymes their ligands and regulatory
factors will also be important for future toxicity prediction
methods We have recently generated visualizations of
microarray data from MCF-7 cells treated with 4-hydrox-
ytamoxifen to show that some of the key genes involved in
metabolism and transport are upregulated (Ekins et al
2005d) In addition we have made predictions with various
QSAR models in MetaDrug to indicate the involvement of
PXR CYP3A4 and P-gp (Ekins et al 2005e) Therefore it
appears likely that 4-hydroxytamoxifen could increase its
own metabolism as well as efflux from cells via P-gp
which can be visualized on networks Any decrease of
function of these enzymes or transporters in a population
would likely result in changes in the metabolism and
transport of this active metabolite potentially impacting
the clinical effect This represents one example of how
both pathway tools QSAR models and network building
algorithms can be used with different types of predicted
and experimental data to allow visualization of potential
compound interactions or toxicity
313 Future network applications
As the population ages an increasing incidence and
prevalence of systemic diseases especially chronic diseases
have occurred among older adults This has resulted in an
increase in medications used concomitantly by this
population which presents challenges for drugndashdrug
interactions Physiologically elderly patients may behave
differently to the younger patients for which the drugs are
initially developed for and tested on Many pharmacoki-
netic investigations in the elderly population reveal
decreased clearance of lipophilic drugs metabolized by
the cytochrome P450 enzymes however few studies have
evaluated aging-dependent or gender-related changes in
specific P450 enzymes (Hunt Westerkam amp Slave 1992)
Age-related physiological changes such as a reduction in
liver mass hepatic metabolizing enzyme activity liver
blood flow and alterations in plasma drug binding may
account for the decreased elimination of some metabolized
drugs in the elderly It is particularly difficult to separate an
effect of aging from the variation in the rate of metabolism
due to factors such as individual metabolic phenotype
(slow or fast metabolizer due to SNPs) environmental
influences concomitant disease states and drug intake
(drugndashdrug interactions) The available data suggest that
Fig 7 Networks for drug resistant cell lines generated with the Auto expand algorithm in MetaCore A KB-3-1 vs KB-8-5 B ATC-DU-145 vs RCO1 C node overlap of A and B with initial nodes highlighted
with blue or red solid circles (Initial gene lists derived from (Annereau et al 2004)) Information on the type of interaction between objects is hidden for clarity eg unspecified allosteric regulation binding
cleavage competition covalent modification dephosphorylation phosphorylation transcription regulation transformation When applicable interactions also have a positive or negative effect and direction
Ligands (purple) linked to other proteins (blue blobs) transfactors (red) enzymes (orange) (For interpretation of the references to colour in this figure legend the reader is referred to the web version of this article)
SEkin
sJournalofPharm
acologica
landToxico
logica
lMeth
ods53(2006)38ndash66
56
Fig7(continued)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 57
Fig7(continued)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6658
Fig 8 The network of gene interactions around the CYP3A4 enzyme (centre) from MetaCorei Information on the type of interaction between objects is
hidden for clarity eg unspecified allosteric regulation binding cleavage competition covalent modification dephosphorylation phosphorylation
transcription regulation transformation When applicable interactions also have a positive or negative effect and direction Ligands (purple) linked to other
proteins (blue blobs) transfactors (red) enzymes (orange) (For interpretation of the references to colour in this figure legend the reader is referred to the web
version of this article)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 59
the initial doses of drugs metabolized by these enzymes
should be reduced in older patients according to the clinical
response In most published studies the elderly appear at
least as responsive as the young to inducers or inhibitors of
P450s (Durnas Loi amp Cusack 1990) More recently there
has been some suggestion that there are age related
reductions in function of some specific P450s such as
CYP3A4 (Patki et al 2004) and this could occur at the
level of regulation However earlier studies with the same
enzyme showed no change in clearance with age (Hunt et
al 1992) To date there has been even less examination of
the transporter functions and any changes with age
(Kinirons amp OrsquoMahony 2004) so the current understand-
ing of the effects of aging on metabolism and transport is
anything but transparent This represents an extreme
challenge for the pharmaceutical industry how to predict
whether a drug has an affinity for an enzyme or transporter
and whether this may be clinically important if it shows a
decline in expression or function with aging Any decrease
of function of these enzymes or transporters in an elderly
population would result in changes in the metabolism and
transport of metabolites potentially impacting the clinical
effect This represents another example of how computa-
tional approaches may perhaps be used with different types
of predicted and experimental data to allow visualization of
potential compound interactions or toxicity in elderly
populations We may see different gene networks high-
lighted as humans age and these may be modified by drug
treatment and coadministration This represents an area
were network analysis could be applied in the future and is
in need of considerable further research
The collection of microarray data in databases such as
CEBS ArrayTrack and EDGE represents a future resource
for computational gene network analysis One could
envisage that ultimately in each case such data is converted
to one or more networks that are also displayed for the user
and can be used to compare treatments from in vivo and in
vitro experiments This would represent a different approach
to clustering the data as currently implemented in one of
these efforts (Hayes et al 2005) and may condense large
amounts of experimentally derived data into a readily
interpreted network
4 Discussion
Previously in this journal the progress of many
research groups in predicting human ADME parameters
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6660
in silico (Ekins et al 2000b) and approaches for drug
metabolism (Ekins Ring Grace McRobie-Belle amp
Wrighton 2000a) have been described Both of these
reviews commented on moving HT assays for ADME
Tox much earlier in drug discovery which had also been
indicated by other groups The initial reviews also
highlighted the likely wealth of data that would become
available and how this could be used for structure
activity relationships alongside the bioactivity data in
computational models It was noted that there was a
paucity of predictive metabolism tools at that time In
addition a growing number of efforts to model whole
cells and organs now a field called systems biology
were recognized as models that could be integrated with
the in silico ADME approaches In summary since these
past reviews virtually all pharmaceutical companies have
attempted earlier high throughput screening for ADME
Tox properties and to some extent the wider application
of computational approaches for physicochemical proper-
ties Systems biology is being quite widely acknowledged
as the new paradigm for understanding complex bio-
logical datasets derived from high throughput technolo-
gies and the accumulated knowledge on human protein
interactions (Hartwell Hopfield Leibler amp Murray 1999
Hood 2003b) Therefore systems biology can be defined
as the integration of genetic proteomic transcriptomic
and metabonomic data using computational methods
(Nicholson amp Wilson 2003) When taken together
information on molecular processes derived from different
sources represents a lsquolsquouniversersquorsquo of putative biological
functionality of which only a small fraction of it will be
realized in a cell at any given time To date systems
biology has been driven by academia and funding bodies
such as the NIH rather than the pharmaceutical compa-
nies Presently there is a great deal of interest from
scientists of all backgrounds in identifying the networks
of cellular pathways and the corresponding physically
interacting proteins
The network building software for systems biology
described in this current review will be valuable to query
high throughput data and known literature interactions in
order to predict potential toxicity in different species In the
future the compilation of published toxicogenomics data-
sets characteristic of different types of toxicity will likely
be available in these software systems to act as a reference
database It is also feasible that we will be able to generate
the annotated datasets which specifically address the
differences between human and rat networks implicated
in toxicity The identification of sub-network modules
conserved between human and rat distinct for toxicity
types or predictive for toxic end-points in human will be
possible Such signature gene networks (Nikolsky et al
2005) can then be verified with other experimentally
derived data prospectively or from preexisting databases
The combined Fhigh throughput datandashin silico network_approaches therefore represents a novel method for access-
ing complex tissue-level processes as networks integrating
all data types based on functional interactions The data-
bases developed and used in this approach will certainly
benefit from further annotation around the drug metaboliz-
ing enzymes and transporters as described herein in terms
of transcriptional regulation and the ligands associated with
them which frequently appear in the literature It will be
important to capture disparities in the assignment of ligands
to enzymes transporters and other proteins as well as
negative data
In summary although there have been numerous
toxicogenomics studies published there is presently a
relatively small number of datasets that are freely available
to perform network analysis of microarray data The
number of studies identifying large numbers of proteins
which are affected by molecule treatment are even scarcer
still while there are several examples of some published
studies that combine such data It is hoped that the
numerous database initiatives for high content and toxicol-
ogy data that are being undertaken will improve the
situation for other researchers that are not currently
equipped to do such microarray studies themselves The
addition of requirements by journals to deposit such raw
data in a freely accessible resource will aid these initiatives
ADMETox groups have seen new technologies and
approaches developed over the last decade that have all
been applied to identify poor compounds earlier (Fig 2)
The latest technologies integrate network building tools
with high content data and databases The current review
described the limited number of networks generated for
ADMETox at present and one hopes that the impact of
such analyses will be commonplace in the future Systems
biology is however more than just applying a network
approach and hence systems-ADMETox will have to
evolve due to the continual pressure to develop newer
technologies This current paradigm combining empirical
data and computational methods should integrate the
complex data already generated making it readily inter-
pretable and valuable for identifying the most promising
compounds in the future
Acknowledgements
Dr Maggie AZ Hupcey is gratefully acknowledged for
editorial assistance Dr Peter W Swaan (University of
Maryland) and Dr Cheng Chang (Ohio State University)
Dr Steve Wright (University of Arizona) Dr K Sandy
Pang (University of Toronto) and Dr Craig Giroux (Wayne
State University) are kindly acknowledged for their support
and discussions My colleagues at GeneGo Sergey
Andreyev Andy Ryabov Eugene Kirillov Eugene A
Rakhmatulin Svetlana Sorokina Andrej Bugrim Tatiana
Nikolskaya Yuri Nikolsky John Metz and Julie Bryant are
all thanked for their considerable contributions to software
development and data annotation
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 61
References
Abe M Yamashita S Kuramoto T Hirayama Y Tsukamoto T Ohta
T et al (2003) Global expression analysis of N-methyl-NV-nitro-N-nitrosoguanidine-induced rat stomach carcinomas using oligonucleotide
microarrays Carcinogenesis 24 861ndash867
Adachi T Ono Y Koh K B Takashima K Tainaka H Matsuno Y
et al (2004) Long-term alteration of gene expression without
morphological change in testis after neonatal exposure to genistein in
mice Toxicogenomic analysis using cDNA microarray Food and
Chemical Toxicology 42 445ndash452
Akerman G S Rosenzweig B A Domon O E McGarrity L J
Blankenship L R Tsai C A et al (2004) Gene expression profiles
and genetic damage in benzo(a)pyrene diol epoxide-exposed TK6 cells
Mutation Research 549 43ndash64
Aksenov S V Church B Dhiman A Georgieva A Sarangapani R
Helmlinger G et al (2005) An integrated approach for inference and
mechanistic modeling for advancing drug development FEBS Letters
579 1878ndash1883
Anderle P Huang Y amp Sadee W (2004) Intestinal membrane transport
of drugs and nutrients Genomics of membrane transporters using
expression microarrays European Journal of Pharmaceutical Sciences
21 17ndash24
Annereau J P Szakacs G Tucker C J Arciello A Cardarelli C
Collins J et al (2004) Analysis of ATP-binding cassette transporter
expression in drug-selected cell lines by a microarray dedicated to
multidrug resistance Molecular Pharmacology 66 1397ndash1405
Apic G Ignjatovic T Boyer S amp Russell R B (2005) Illuminating drug
discovery with biological pathways FEBS Letters 579 1872ndash1877
Baker V A Harries H M Waring J F Duggan C M Ni H A Jolly
R A et al (2004) Clofibrate-induced gene expression changes in rat
liver A cross-laboratory analysis using membrane cDNA arrays
Environmental Health Perspectives 112 428ndash438
Balakin K V Ekins S Bugrim A Ivanenkov Y A Korolev D amp
Nikolsky Y et al (2004a) Quantitative structurendashmetabolism relation-
ship modeling of the metabolic N-dealkylation rates Drug Metabolism
and Disposition 32 1111ndash1120
Balakin K V Ekins S Bugrim A Ivanenkov Y A Korolev D amp
Nikolsky Y et al (2004b) Kohonen maps for prediction of binding to
human cytochrome P450 3A4 Drug Metabolism and Disposition 32
1183ndash1189
Barabasi A -L amp Oltvai Z N (2004) Network biology Under-
standing the cellrsquos functional organization Nature Reviews Genetics
5 101ndash113
Bartosiewicz M Penn S G amp Buckpitt A (2001) Applications of gene
arrays in environmental toxicology Fingerprints of gene regulation
associated with cadmium chloride benzo(a)pyrene and trichloro-
ethylene Environmental Health Perspectives 109 71ndash74
Bertilsson G Heidrich J Svensson K Asman M Jendeberg L
Sydow-Backman M et al (1998) Identification of a human nuclear
receptor defines a new signaling pathway for CYP3A induction
Proceedings of the National Academy of Sciences of the United States
of America 95 12208ndash12213
Blumberg B Sabbagh Jr W Juguilon H Bolado Jr J van Meter
C M Ong E S et al (1998) SXR a novel steroid and xenobiotic-
sensing nuclear receptor Genes and Development 12 3195ndash3205
Borodina Y Rudik A Filimonov D Kharchevnikova N Dmitriev A
Blinova V et al (2004) A new statistical approach to predicting
aromatic hydroxylation sites Comparison with model-based
approaches Journal of Chemical Information and Computer Sciences
44 1998ndash2009
Borodina Y Sadym A Filimonov D Blinova V Dmitriev A amp
Poroikov V (2003) Predicting biotransformation potential from
molecular structure Journal of Chemical Information and Computer
Sciences 43 1636ndash1646
Bottone Jr F G Martinez J M Collins J B Afshari C A amp Eling
T E (2003) Gene modulation by the cyclooxygenase inhibitor
sulindac sulfide in human colorectal carcinoma cells Possible link to
apoptosis Journal of Biological Chemistry 278 25790ndash25801
Boyer S amp Zamora I (2002) New methods in predictive metabolism
Journal of Computer-Aided Molecular Design 16 403ndash413
Bredel M amp Jacoby E (2004) Chemogenomics An emerging strategy
for rapid target and drug discovery Nature Reviews Genetics 5
262ndash275
Bugrim A Nikolskaya T amp Nikolsky Y (2004) Early prediction of drug
metabolism and toxicity Systems biology approach and modeling
Drug Discovery Today 9 127ndash135
Bulera S J Eddy S M Ferguson E Jatkoe T A Reindel J F
Bleavins M R et al (2001) RNA expression in the early character-
ization of hepatotoxicants in Wistar rats by high-density DNA micro-
arrays Hepatology 33 1239ndash1258
Butcher E C Berg E L amp Kunkel E J (2004) Systems biology in drug
discovery Nature Biotechnology 22 1253ndash1259
Butte A (2002) The use and analysis of microarray data Naturalist
Review of Drug Discovery 1 951ndash960
Cary M P Bader G D amp Sander C (2005) Pathway information for
systems biology FEBS Letters 579 1815ndash1820
Cheok M H Yang W Pui C H Downing J R Cheng C Naeve
C W et al (2003) Treatment-specific changes in gene expression
discriminate in vivo drug response in human leukemia cells Nature
Genetics 34 85ndash90
Cleary J D Rogers P D amp Chapman S W (2001) Differential
transcription factor expression in human mononuclear cells in response
to amphotericin B Identification with complementary DNA microarray
technology Pharmacotherapy 21 1046ndash1054
Coen M Ruepp S U Lindon J C Nicholson J K Pognan F Lenz
E M et al (2004) Integrated application of transcriptomics and
metabonomics yields new insight into the toxicity due to paracetamol in
the mouse Journal of Pharmaceutical and Biomedical Analysis 35
93ndash105
Cornwell P D De Souza A T amp Ulrich R G (2004) Profiling of
hepatic gene expression in rats treated with fibric acid analogs
Mutation Research 549 131ndash145
Csermely P Agoston V amp Pongor S (2005) The efficiency of multi-
target drugs The network approach might help drug design Trends in
Pharmacological Sciences 26 178ndash182
Cunningham M J Liang S Fuhrman S Seilhamer J J amp
Somogyi R (2000) Gene expression microarray data analysis for
toxicology profiling Annals of the New York Academy of Sciences 919
52ndash67
de Longueville F Atienzar F A Marcq L Dufrane S Evrard S
Wouters L et al (2003) Use of a low density microarray for studying
gene expression patterns induced by hepatotoxicants on primary
cultures of rat hepatocytes Toxicological Sciences 75 378ndash392
Dobrin R Beg Q K Barabasi A L amp Oltvai Z N (2004)
Aggregation of topological motifs in the Escherichia coli transcriptional
regulatory network BMC Bioinformatics 5 10
Donald S Verschoyle R D Edwards R Judah D J Davies R
Riley J et al (2002) Hepatobiliary damage and changes in hepatic
gene expression caused by the antitumor drug ecteinascidin-743 (ET-
743) in the female rat Cancer Research 62 4256ndash4262
Dumont M Jacquemin E DrsquoHont C Descout C Cresteil D Haouzi
D et al (1997) Expression of the liver Na+-independent organic anion
transporting polypeptide (oatp-1) in rats with bile duct ligation Journal
of Hepatology 27 1051ndash1056
Durnas C Loi C M amp Cusack B J (1990) Hepatic drug metabolism
and aging Clinical Pharmacokinetics 19 359ndash389
Eisen M B Spellman P T Brown P O amp Botstein D (1998) Cluster
analysis and display of genome-wide expression patterns Proceedings
of the National Academy of Sciences of the United States of America
95 14863ndash14868
Ekins S Andreyev S Ryabov A Kirilov E Rakhmatulin E A
Bugrim A et al (in press) Computational Prediction of Human Drug
Metabolism Exp Opin Drug Metab Toxicol
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6662
Ekins S Boulanger B Swaan P W amp Hupcey M A Z (2002)
Towards a new age of virtual ADMETOX and multidimensional drug
discovery Journal of Computer-Aided Molecular Design 16 381ndash401
Ekins S Bugrim A Nikolsky Y amp Nikolskaya T (2005) Systems
biology Applications in drug discovery In S Gad (Ed) Drug
Discovery Handbook (pp 123ndash183) Hoboken NJrsquo Wiley and Sons
Ekins S Giroux C N Nikolsky Y Bugrim A amp Nikolskaya T
(2005) A signature gene network approach to toxicity The Toxoco-
logists 84
Ekins S Kirillov E Rakhmatulin E amp Nikolskaya T (2005) A novel
method for visualizing nuclear hormone receptor networks relevant to
drug metabolism Drug Metabolism and Disposition 33 474ndash481
Ekins S amp McGowan R J (2001) The limits of reductionism The
shifting genomic paradigmrsquos impact on industry and academia
Philsophy in Science 9 1ndash23
Ekins S Mirny L amp Schuetz E G (2002) A ligand-based approach to
understanding selectivity of nuclear hormone receptors PXR CAR
FXR LXRa and LXRb Pharmaceutical Research 19 1788ndash1800
Ekins S Nikolsky Y amp Nikolskaya T (2005) Techniques Application
of systems biology to absorption distribution metabolism excretion
and toxicity Trends in Pharmacological Sciences 26 202ndash209
Ekins S Ring B J Grace J McRobie-Belle D J amp Wrighton S
A (2000) Present and future in vitro approaches for drug
metabolism Journal of Pharmacological and Toxicological Methods
44 313ndash324
Ekins S Waller C L Swaan P W Cruciani G Wrighton S A amp
Wikel J H (2000) Progress in predicting human ADME parameters in
silico Journal of Pharmacological and Toxicological Methods 44
251ndash272
Erhardt P W (2003) A human drug metabolism database Potential roles
in the quantitative predictions of drug metabolism and metabolism-
related drugndashdrug interactions Current Drug Metabolism 4 411ndash422
Evans W E amp McLeod H L (2003) Pharmacogenomicsmdashdrug
disposition drug targets and side effects New England Journal of
Medicine 348 538ndash549
Ezendam J Staedtler F Pennings J Vandebriel R J Pieters R
Boffetta P et al (2004) Toxicogenomics of subchronic hexachlor-
obenzene exposure in Brown Norway rats Environmental Health
Perspectives 112 782ndash791
Faiola B Fuller E S Wong V A amp Recio L (2004) Gene expression
profile in bone marrow and hematopoietic stem cells in mice exposed to
inhaled benzene Mutation Research 549 195ndash212
FDA (2004) Innovation stagnation Challenge and opportunity on the
critical path to new medicinal products
Fiehn O (2001) Combining genomics metabolome analysis and bio-
chemical modelling to understand metabolic networks Comparative
Functional Genomics 2 155ndash168
Field F J Born E amp Mathur S N (2004) LXRRXR ligand activation
enhances basolateral efflux of beta-sitosterol in CaCo-2 cells Journal of
Lipid Research 45 905ndash913
Fleck C Sutter L Appenroth D Koch B Meinhold T Pitack M
et al (2003) Use of gene chip technology for the characterisation of
the regulation of renal transport processes and of nephrotoxicity in
rats Experimental and Toxicologic Pathology 54 401ndash410
Fliri A F Loging W T Thadeio P F amp Volkmann R A (2005)
Biological spectra analysis Linking biological activity profiles to
molecular structure Proceedings of the National Academy of Sciences
of the United States of America 102 261ndash266
Fountoulakis M (2004) Application of proteomics technologies in the
investigation of the brain Mass Spectrometry Reviews 23 231ndash258
Gaucher S P Taylor S W Fahy E Zhang B Warnock D E Ghosh
S S et al (2004) Expanded coverage of the human heart
mitochondrial proteome using multidimensional liquid chromatography
coupled with tandem mass spectrometry Journal of Proteome
Research 3 495ndash505
Gerhold D Lu M Xu J Austin C Caskey C T amp Rushmore T
(2001) Monitoring expression of genes involved in drug metabolism
and toxicology using DNA microarrays Physiological Genomics 5
161ndash170
Guo G L Choudhuri S amp Klaassen C D (2002) Induction profile of
rat organic anion transporting polypeptide 2 (oatp2) by prototypical
drug-metabolizing enzyme inducers that activate gene expression
through ligand-activated transcription factor pathways Journal of
Pharmacology and Experimental Therapeutics 300 206ndash212
Hagenbuch B amp Meier P J (2004) Organic anion transporting
polypeptides of the OATP SLC21 family Phylogenetic classification
as OATP SLCO superfamily new nomenclature and molecularfunc-
tional properties Pflugers Archiv 447 653ndash665
Hamadeh H K amp Afshari C A (2004) Toxicogenomics principles and
applications Hobokenrsquo John Wiley and Sons
Hamadeh H K Bushel P R Jayadev S Martin K DiSorbo O Sieber
S et al (2002) Gene expression analysis reveals chemical-specific
profiles Toxicological Sciences 67 219ndash231
Hamadeh H K Jayadev S Gaillard E T Huang Q Stoll R
Blanchard K et al (2004) Integration of clinical and gene expression
endpoints to explore furan-mediated hepatotoxicity Mutation Research
549 169ndash183
Hamadeh H K Knight B L Haugen A C Sieber S Amin R P
Bushel P R et al (2002) Methapyrilene toxicity Anchorage of
pathologic observations to gene expression alterations Toxicologic
Pathology 30 470ndash482
Han J D Bertin N Hao T Goldberg D S Berriz G F Zhang L V
et al (2004) Evidence for dynamically organized modularity in the
yeast proteinndashprotein interaction network Nature 430 88ndash93
Hanisch D Zien A Zimmer R amp Lengauer T (2002) Co-clustering of
biological networks and gene expression data Bioinformatics 18
S145ndashS154
Harris A J Dial S L amp Casciano D A (2004) Comparison of basal
gene expression profiles and effects of hepatocarcinogens on gene
expression in cultured primary human hepatocytes and HepG2 cells
Mutation Research 549 79ndash99
Harrison S C (2004) Whither structural biology Nature Structural
Molecular Biology 11 12ndash15
Hartley D P Dai X He Y D Carlini E J Wang B Huskey S E
et al (2004) Activators of the rat pregnane X receptor differentially
modulate hepatic and intestinal gene expression Molecular Pharma-
cology 65 1159ndash1171
Hartwell L H Hopfield J J Leibler S amp Murray A W (1999) From
molecular to modular cell biology Nature 402 C47ndashC52
Hasmall S Orphanides G James N Pennie W Hedley K Soames
A et al (2002) Downregulation of lactoferrin by PPARalpha ligands
Role in perturbation of hepatocyte proliferation and apoptosis
Toxicology Sciences 68 304ndash313
Hayase T Yamamoto Y Yamamoto K Muso E amp Shiota K (2004)
Microarray profile analysis of toxic cocaine-induced alterations in the
expression of mouse brain gene sequences A possible rsquoprotectiversquo
effect of buprenorphine Journal of Applied Toxicology 24 15ndash20
Hayes K R Vollrath A L Zastrow G M McMillan B J Craven M
Jovanovich S et al (2005) EDGE A centralized resource for the
comparison analysis and distribution of toxicogenomic information
Molecular Pharmacology 67 1360ndash1368
Heijne W H Slitt A L van Bladeren P J Groten J P Klaassen C D
Stierum R H et al (2004) Bromobenzene-induced hepatotoxicity at
the transcriptome level Toxicology Sciences 79 411ndash422
Heijne W H Stierum R H Slijper M van Bladeren P J amp van
Ommen B (2003) Toxicogenomics of bromobenzene hepatotoxicity
A combined transcriptomics and proteomics approach Biochemical
Pharmacology 65 857ndash875
Heinloth A N Irwin R D Boorman G A Nettesheim P Fannin
R D Sieber S O et al (2004) Gene expression profiling of rat
livers reveals indicators of potential adverse effects Toxicology
Sciences 80 193ndash202
Hodges L C Cook J D Lobenhofer E K Li L Bennett L Bushel
P R et al (2003) Tamoxifen functions as a molecular agonist inducing
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 63
cell cycle-associated genes in breast cancer cells Molecular Cancer
Research 1 300ndash311
Holleman A Cheok M H den Boer M L Yang W Veerman KM
A J Kazemier et al (2004) Gene-expression patterns in drug-resistant
acute lymphoblastic leukemia cells and response to treatment New
England Journal of Medicine 351 533ndash542
Hong Y Muller U R amp Lai F (2003) Discriminating two classes of
toxicants through expression analysis of HepG2 cells with DNA arrays
Toxicology in Vitro 17 85ndash92
Hood E (2003) Proteomics Characterizing the cogs in the machinery of
life Environmental Health Perspectives 111 A816ndashA825
Hood L (2003) Systems biology Integrating technology biology and
computation Mechanism of Ageing and Development 124 9ndash16
Hood L amp Galas D (2003) The digital code of DNA Nature 421
444ndash448
Hood L amp Perlmutter R M (2004) The impact of systems approaches on
biological problems in drug discovery Nature Biotechnology 22
1215ndash1217
Hu T Gibson D P Carr G J Torontali S M Tiesman J P Chaney
J G et al (2004) Identification of a gene expression profile that
discriminates indirect-acting genotoxins from direct-acting genotoxins
Mutation Research 549 5ndash27
Huang Q Dunn II R T Jayadev S DiSorbo O Pack F D Farr S B
et al (2001) Assessment of cisplatin-induced nephrotoxicity by
microarray technology Toxicology Sciences 63 196ndash207
Huang Q Jin X Gaillard E T Knight B L Pack F D Stoltz J H et
al (2004) Gene expression profiling reveals multiple toxicity endpoints
induced by hepatotoxicants Mutation Research 549 147ndash167
Huisman M T Smit J W Crommentuyn K M Zelcer N Wiltshire
H R Beijnen J H et al (2002) Multidrug resistance protein 2
(MRP2) transports HIV protease inhibitors and transport can be
enhanced by other drugs Aids 16 2295ndash2301
Hunt C M Westerkam W R amp Stave G M (1992) Effect of age and
gender on the activity of human hepatic CYP3A Biochemical
Pharmacology 44 275ndash283
Ideker T Ozier O Schwikowski B amp Siegal A F (2002) Discovering
regulatory and signalling circuits in molecular interaction networks
Bioinformatics 18 S233ndashS240
Ideker T Thorsson V Ranish J A Christmas R Buhler J Eng J K
et al (2001) Integrated genomic and proteomic analyses of a system-
atically perturbed network Science 292 929ndash934
Iida M Anna C H Hartis J Bruno M Wetmore B Dubin J R et al
(2003) Changes in global gene and protein expression during early
mouse liver carcinogenesis induced by non-genotoxic model carcino-
gens oxazepam and Wyeth-14643 Carcinogenesis 24 757ndash770
Ingelman-Sundberg M (2004) Pharmacogenetics of cytochrome P450 and
its applications in drug therapy The past present and future Trends in
Pharmacological Sciences 25 193ndash200
Jeong H Mason S P Barabasi A L amp Oltvai Z N (2001) Lethality
and centrality in protein networks Nature 411 41ndash42
Jeong H Tombor B Albert R Oltvai Z N amp Barabasi A L
(2000) The large-scale organization of metabolic networks Nature
407 651ndash654
Jung D Hagenbuch B Gresh L Pontoglio M Meier P J amp Kullak-
Ublick G A (2001) Characterization of the human OATP-C
(SLC21A6) gene promoter and regulation of liver-specific OATP genes
by hepatocyte nuclear factor 1 alpha Journal of Biological Chemistry
276 37206ndash37214
Jung D amp Kullak-Ublick G A (2003) Hepatocyte nuclear factor 1
alpha A key mediator of the effect of bile acids on gene expression
Hepatology 37 622ndash631
Jung J -W Park J -S Hwang J -W Kang K -S YS L Song B
-S et al (2004) Gene expression analysis of peroxisome prolifer-
ators- and phenytoin-induced hepatotoxicity using cDNA microarray
Journal of Veterinary Medical Science 66 1329ndash1333
Kato N Shibutani M Takagi H Uneyama C Lee K -Y Takiami S
et al (2004) Gene expression profile in the livers of rats orally
administered ethinylestradiol for 28 days using a microarray technique
Toxicology 200 179ndash192
Kier L D Neft R Tang L Suizu R Cook T Onsurez K et al
(2004) Applications of microarrays with toxicologically relevant genes
(tox genes) for the evaluation of chemical toxicants in Sprague Dawley
rats in vivo and human hepatocytes in vitro Mutation Research 549
101ndash113
Kijima K Toyosawa K Yasuba M Matsuoka N Adachi T
Komiyama M et al (2004) Gene expression analysis of the rat testis
after treatment with di(2-ethylhexyl) phthalate using cDNA microarray
and real-time RT-PCR Toxicology and Applied Pharmacology 200
103ndash110
Kim R B (2003) Organic anion-transporting polypeptide (OATP) trans-
porter family and drug disposition European Journal of Clinical
Investigation 33 1ndash5
Kinirons M T amp OrsquoMahony M S (2004) Drug metabolism and ageing
British Journal of Clinical Pharmacology 57 540ndash544
Kitano H (2002a) Computational systems biology Nature 420 206ndash210
Kitano H (2002b) Systems biology A brief overview Science 295
1662ndash1664
Kliewer S A Moore J T Wade L Staudinger J L Watson M
A Jones S A et al (1998) An orphan nuclear receptor activated
by pregnanes defines a novel steroid signalling pathway Cell 92
73ndash82
Korolev D Balakin K V Nikolsky Y Kirillov E Ivanenkov Y A
Savchuk N P et al (2003) Modeling of human cytochrome p450-
mediated drug metabolism using unsupervised machine learning
approach Journal of Medicinal Chemistry 46 3631ndash3643
Kramer J A Pettit S D Amin R P Bertram T A Car B
Cunningham M et al (2004) Overview on the application of
transcription profiling using selected nephrotoxicants for toxicology
assessment Environmental Health Perspectives 112 460ndash464
Kultima K Nystrom A -M Scholz B Gustafson A -L Dencker L
amp Stigson M (2004) Valproic acid teratogenicity A toxicogenomics
approach Environmental Health Perspectives 112 1225ndash1235
Kutuzova G D amp Deluca H F (2004) Gene expression profiles in rat
intestine identify pathways for 125-dihydroxyvitamin D(3) stimulated
calcium absorption and clarify its immunomodulatory properties
Archives of Biochemistry and Biophysics 432 152ndash166
Kwak M K Wakabayashi N Itoh K Motohashi H Yamamoto M amp
Kensler T W (2003) Modulation of gene expression by cancer
chemopreventive dithiolethiones through the Keap1-Nrf2 pathway
Identification of novel gene clusters for cell survival Journal of
Biological Chemistry 278 8135ndash8145
Lahjouji K Mitchell G A amp Qureshi I A (2001) Carnitine transport
by organic cation transporters and systemic carnitine deficiency
Molecular Genetics and Metabolism 73 287ndash297
Landowski C P Sun D Foster D R Menon S S Barnett J L
Welage L S et al (2003) Gene expression in the human intestine and
correlation with oral valacyclovir pharmacokinetic parameters Journal
of Pharmacology and Experimental Therapeutics 306 778ndash786
Lee G -J Lee W -S Jeon K -S Um C -H Kim S -J Lee C -H
et al (2004) cDNA microarray gene expression analysis and
toxicological phenotype for anticancer drug Journal of Veterinary
Medical Science 66 1339ndash1345
Lee M Kwon J Kim S N Kim J E Koh W S Kim E J et al
(2003) cDNA microarray gene expression profiling of hydroxyurea
paclitaxel and p-anisidine genotoxic compounds with differing
tumorigenicity results Environmental and Molecular Mutagenesis
42 91ndash97
Li S Armstrong C M Bertin N Ge H Milstein S Boxem M et al
(2004) A map of the interactome network of the metazoan C elegans
Science 303 540ndash543
Liguori M J Anderson L M Bukofzer S McKim J Pregenzer J F
Retief J et al (2005) Microarray analysis in human hepatocytes
suggests a mechanism for hepatotoxicity induced by trovafloxacin
Hepatology 41 177ndash186
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6664
Lobenhofer E K Bennett L Cable P L Li L Bushel P R amp Afshari
C A (2002) Regulation of DNA replication fork genes by 17beta-
estradiol Molecular Endocrinology 16 1215ndash1229
Locker J Tian J Carver R Concas D Cossu C Ledda-Columbano
G M et al (2003) A common set of immediate-early response genes
in liver regeneration and hyperplasia Hepatology 38 314ndash325
Mattes W B Pettit S D Sansone S A Bushel P R amp Waters M D
(2004) Database development in toxicogenomics Issues and efforts
Environmental Health Perspectives 112 495ndash505
McMillian M Nie A Y Parker J B Leone A Bryant S Kemmerer
M et al (2004) A gene expression signature for oxidant stressreactive
metabolites in rat liver Biochemical Pharmacology 68 2249ndash2261
Meier P J amp Stieger B (2000) Molecular mechanisms in bile formation
News in Physiological Sciences 15 89ndash93
Meneses-Lorente G de Longueville F Dos Santos-Mendes S Bonnert
T P Jack A Evrard S et al (2003) An evaluation of a low-density
DNA microarray using cytochrome P450 inducers Chemical Research
in Toxicology 16 1070ndash1077
Meneses-Lorente G Guest P C Lawrence J Muniappa N Knowles
M R Skynner H A et al (2004) A proteomic investigation of drug-
induced steatosis in rat liver Chemical Research in Toxicology 17
605ndash612
Merrill C L Ni H Yoon L W Tirmenstein M A Narayanan P
Benavides G R et al (2002) Etomoxir-induced oxidative stress in
HepG2 cells detected by differential gene expression is confirmed
biochemically Toxicology Sciences 68 93ndash101
Milo R Shen-Orr S Itzkovitz S Kashtan N Chklovskii D amp Alon
U (2002) Network motifs Simple building blocks of complex
networks Science 298 824ndash827
Morgan K T Ni H Brown H R Yoon L Qualls C W Crosby L
M et al (2002) Application of cDNA microarray technology to in
vitro toxicology and the selection of genes for a real-time RT-PCR-
based screen for oxidative stress in Hep-G2 cells Toxicologic
Pathology 30 435ndash451
Mwinyi J Johne A Bauer S Roots I amp Gerloff T (2004) Evidence
for inverse effects of OATP-C (SLC21A6) 5 and 1b haplotypes on
pravastatin kinetics Clinical Pharmacology and Therapeutics 75
415ndash421
Nakata K Yukawa M Komiyama N Nakano T amp Kaminuma T
(2002) A nuclear receptor database that maps pathways to diseases
Genome Infomatics 13 515ndash516
Nicholson J K Holmes E Lindon J C amp Wilson I D (2004) The
challenges of modeling mammalian biocomplexity Nature Biotechnol-
ogy 22 1268ndash1274
Nicholson J K amp Wilson I D (2003) Understanding rsquoglobal systems
biology Metabonomics and the continuum of metabolism Nature
Reviews on Drug Discovery 2 668ndash676
Nikitin A Egorov S Daraselia N amp Mazo I (2003) Pathway studiomdash
the analysis and navigation of molecular networks Bioinformatics 19
2155ndash2157
Nikolsky Y Ekins S Nikolskaya T amp Bugrim A (2005) A novel
method for generation of signature networks as biomarkers from
complex high throughput data Toxicology Letters 158 20ndash29
Nikolsky Y Nikolskaya T amp Bugrim A (2005) Biological networks
and analysis of experimental data in drug discovery Drug Discovery
Today 10 653ndash662
Nishizato Y Ieiri I Suzuki H Kimura M Kawabata K Hirota T
et al (2003) Polymorphisms of OATP-C (SLC21A6) and OAT3
(SLC22A8) genes Consequences for pravastatin pharmacokinetics
Clinical Pharmacology and Therapeutics 73 554ndash565
Nozawa T Nakajima M Tamai I Noda K Nezu J Sai Y et al
(2002) Genetic polymorphisms of human organic anion transporters
OATP-C (SLC21A6) and OATP-B (SLC21A9) Allele frequencies in
the Japanese population and functional analysis Journal of Pharmacol-
ogy and Experimental Therapeutics 302 804ndash813
Nozawa T Sugiura S Nakajima M Goto A Yokoi T Nezu J et al
(2004) Involvement of organic anion transporting polypeptides in the
transport of troglitazone sulfate Implications for understanding trogli-
tazone hepatotoxicity Drug Metabolism and Disposition 32 291ndash294
Oliver D E Rubin D L Stuart J M Hewett M Klein T E amp
Altman R B (2002) Ontology development for a pharmacogenetics
knowledge base Package Symposion 88ndash99
Oram J F amp Lawn R M (2001) ABCA1 The gatekeeper for eliminating
excess tissue cholesterol Journal of Lipid Research 42 1173ndash1179
Oswald M Kullak-Ublick G A Paumgartner G amp Beuers U (2001)
Expression of hepatic transporters OATP-C and MRP2 in primary
sclerosing cholangitis Liver 21 247ndash253
Parsons A B Brost R I Ding H Li Z Zhang C Sheikh B et al
(2004) Integration of chemical-genetic and genetic interaction data
links bioactive compounds to cellular target pathways Nature Bio-
technology 22 62ndash69
Patki K C von Moltke L L Harmatz J S Hesse L M Court M H
amp Greenblatt D J (2004) Effect of age on in vitro triazolam
biotransformation in male human liver microsomes Journal of
Pharmacology and Experimental Therapeutics 308 874ndash879
Pereira-Leal J B Enright A J amp Ouzounis C A (2004) Detection of
functional modules from protein interaction networks Proteins 54
49ndash57
Plant N (2004) Interaction networks Coordinating responses to xeno-
biotic exposure Toxicology 202 21ndash32
Raghavendra Prasad H S Qi Z Srinivasan K N amp Gopalakrish-
nakone P (2004) Potential effects of tetrodotoxin exposure to
human glial cells postulated using microarray approach Toxicon 44
597ndash608
Reilly T P Bourdi M Brady J N Pise-Masison C A Radonovich M
F George J W et al (2001) Expression profiling of acetaminophen
liver toxicity in mice using microarray technology Biochemical and
Biophysical Research Communications 282 321ndash328
Rives A W amp Galitski T (2003) Modular organization of cellular
networks Proceedings of the National Academy of Sciences of the
United States of America 100 1128ndash1133
Rogers P D Pearson M M Cleary J D Sullivan D C amp Chapman
S W (2002) Differential expression of genes encoding immunomodu-
latory proteins in response to amphotericin B in human mononuclear
cells identified by cDNA microarray analysis Journal of Antimicrobial
Chemotherapy 50 811ndash817
Rosen M B Wilson V S Schmid J E amp Gray L E (2005) Gene
expression analysis in the ventral prostate of rats exposed to vinclozolin
or procymidone Reproductive Toxicology 19 367ndash379
Rost D Herrmann T Sauer P Schmidts H L Stieger B Meier P J
et al (2003) Regulation of rat organic anion transporters in bile salt-
induced cholestatic hepatitis Effect of ursodeoxycholate Hepatology
38 187ndash195
Rothman R B Ayestas M A Dersch C M amp Baumann M H (1999)
Aminorex fenfluramine and chlorphentermine are serotonin transporter
substrates Implications for primary pulmonary hypertension Circula-
tion 100 869ndash875
Ruepp S U Tonge R P Shaw J Wallis N amp Pognan F (2002)
Genomics and proteomics analysis of acetaminophen toxicity in mouse
liver Toxicology Sciences 65 135ndash150
Sakaeda T Nakamura T amp Okumura K (2002) MDR1 genotype-
related pharmacokinetics and pharmacodynamics Biological amp Phar-
maceutical Bulletin 25 1391ndash1400
Sakata T Anzai N Shin H J Noshiro R Hirata T Yokoyama H
et al (2004) Novel single nucleotide polymorphisms of organic cation
transporter 1 (SLC22A1) affecting transport functions Biochemical
and Biophysical Research Communications 313 789ndash793
Satlin L M Amin V amp Wolkoff A W (1997) Organic anion
transporting polypeptide mediates organic anionHCO3-exchange
Journal of Biological Chemistry 272 26340ndash26345
Satomi Y Tsuchiya W Mihara K Ota M Kasahara Y amp Akahori F
(2004) Gene expression analysis of the lung following paraquat
administration in rats using DNA microarray Journal of Toxicological
Sciences 29 91ndash100
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 65
Schrenk D Baus P R Ermel N Klein C Vorderstemann B amp
Kauffmann H M (2001) Up-regulation of transporters of the MRP
family by drugs and toxins Toxicology Letters 120 51ndash57
Segal E Shapira M Regev A Persquoer D Botstein D Koller D et al
(2003) Module networks Identifying regulatory modules and their
condition-specific regulators from gene expression data Nature
Genetics 34 166ndash176
Segal E Wang H amp Koller D (2003) Discovering molecular pathways
from protein interaction and gene expression Bioinformatics 19
i264ndash i272
Seidel S D Kan H L Stott W T Schisler M R amp Gollapudi B B
(2003) Identification of transcriptome profiles for the DNA-damaging
agents bleomycin and hydrogen peroxide in L5178Y mouse lymphoma
cells Environmental and Molecular Mutagenesis 42 19ndash25
Shan L Yu Y Schut H A J amp Snyderwine E G (2004) Susceptibility
of rats to mammary gland carcinogenesis by the food-derived
carcinogen 2-Amino-1-Methyl-6-Phenylimidazo[45-b]Pyridine (PhIP)
varies with age and is associated with the induction of differential gene
expression American Journal of Pathology 165 191ndash202
Sharom J R Bellows D S amp Tyers M (2004) From large networks to
small molecules Current Opinion in Chemical Biology 8 81ndash90
Shitara Y Hirano M Sato H amp Sugiyama Y (2004) Gemfibrozil and
its glucuronide inhibit the OATP2(OATP1B1 SLC21A6)-mediated
hepatic uptake and CYP2C8-mediated metabolism of cerivastatinmdash
Analysis of the mechanism of the clinically relevant drugndashdrug
interaction between cerivastatin and gemfibrozil Journal of Pharma-
cology and Experimental Therapeutics
Shitara Y Itoh T Sato H Li A P amp Sugiyama Y (2003) Inhibition of
transporter-mediated hepatic uptake as a mechanism for drugndashdrug
interaction between cerivastatin and cyclosporin A Journal of
Pharmacology and Experimental Therapeutics 304 610ndash616
Somogyi R Fuhrman S amp Wen X (2001) Genetic network inference in
computational models and applications to large-scale gene expression
data In M Bower amp H Bolouri (Eds) Computational modeling of
genetic and biochemical networks (pp 119ndash157) Cambridge MArsquoThe
MIT Press
Spirin V amp Mirny L A (2003) Protein complexes and functional
modules in molecular networks Proceedings of the National Academy
of Sciences of the United States of America 100 12123ndash12128
Staudinger J Liu Y Madan A Habeebu S amp Klaassen C D
(2001) Coordinate regulation of xenobiotic and bile acid homeo-
stasis by pregnane X receptor Drug Metabolism and Disposition
29 1467ndash1472
Sun L Z Ji Z L Chen X Wang J F amp Chen Y Z (2002) ADME-
AP A database of ADME associated proteins Bioinformatics 18
1699ndash1700
Suter L Babiss L E amp Wheeldon E B (2004) Toxicogenomics in
predictive toxicology in drug development Chemistry and Biology 11
161ndash171
Synold T W Dussault I amp Forman B M (2001) The orphan nuclear
receptor SXR coordinately regulates drug metabolism and efflux
Nature Medicine 7 584ndash590
Tabuchi Y amp Kondo T (2003) cDNA microarray analysis reveals chop-
10 plays a key role in Sertoli cell injury induced by bisphenol A
Biochemical and Biophysical Research Communications 305 54ndash61
Tamai I Nezu J Uchino H Sai Y Oku A Shimane M et al (2000)
Molecular identification and characterization of novel members of the
human organic anion transporter (OATP) family Biochemical and
Biophysical Research Communications 273 251ndash260
Taylor S W Fahy E Zhang B Glenn G M Warnock D E Wiley S
et al (2003) Characterization of the human heart mitochondrial
proteome Nature Biotechnology 21 281ndash286
Terasaka S Aita Y Inoue A Hayashi S Nishigaki M Aoyagi K
et al (2004) Using a customized DNA microarray for expression
profiling of the estrogen-responsive genes to evaluate estrogen activity
among natural estrogens and industrial chemicals Environmental
Health Perspectives 112 773ndash781
Thomas D M Francescutti-Verbeem D M Liu X amp Kuhn D M
(2004) Identification of differentially regulated transcripts in mouse
striatum following methamphetamine treatmentmdashan oligonucleotide
microarray approach Journal of Neurochemistry 88 380ndash393
Thomas R S Rank D R Penn S G Zastrow G M Hayes K R
Pande K et al (2001) Identification of toxicologically predictive
gene sets using cDNA microarrays Molecular Pharmacology 60
1189ndash1194
Thompson K L Afshari C A Amin R P Bertram T A Car B
Cunningham M et al (2004) Identification of platform-independent
gene expression markers of cisplatin nephrotoxicity Environmental
Health Perspectives 112 488ndash494
Tirona R G Leake B F Merino G amp Kim R B (2001)
Polymorphisms in OATP-C Identification of multiple allelic
variants associated with altered transport activity among European-
and African-Americans Journal of Biological Chemistry 276
35669ndash35675
Tirona R G Leake B F Wolkoff A W amp Kim R B (2003)
Human organic anion transporting polypeptide-C (SLC21A6) is a
major determinant of the rifampin-mediated pregnane X receptor
activation Journal of Pharmacology and Experimental Therapeutics
304 223ndash228
Tong W Cao X Harris S Sun H Fang H Fuscoe J et al (2003)
ArrayTrackmdashsupporting toxicogenomic research at the US Food and
Drug Administration National Center for Toxicological Research
Environmental Health Perspectives 111 1819ndash1826
Tornow S amp Mewes H W (2003) Functional modules by relating
protein interaction networks and gene expression Nucleic Acids
Research 31 6283ndash6289
Trocho C Escola-Gil J C Ribas V Benitez S Martin-Campos J M
Rotllan N et al (2004) Phenytoin treatment reduces atherosclerosis in
mice through mechanisms independent of plasma HDL-cholesterol
concentration Atherosclerosis 174 275ndash285
Ueda A Hamadeh H K Webb H K Yamamoto Y Sueyoshi T
Afshari C A et al (2002) Diverse roles of the nuclear orphan
receptor CAR in regulating hepatic genes in response to phenobarbital
Molecular Pharmacology 61 1ndash6
Ulrich R G (2003) The toxicogenomics of nuclear receptor agonists
Current Opinion in Chemical Biology 7 505ndash510
Ulrich R G Rockett J C Gibson G G amp Pettit S D (2004)
Overview of an interlaboratory collaboration on evaluating the effects
of model hepatotoxicants on hepatic gene expression Environmental
Health Perspectives 112 423ndash427
Unami A Shinohara Y Ichikawa T amp Baba Y (2003) Biochemical
and microarray analyses of bupivacaine-induced apoptosis Journal of
Toxicological Sciences 28 77ndash94
Vasquez A Flammini A Maritan A amp Vespignani A (2003) Global
protein function prediction from proteinndashprotein interaction networks
Nature Biotechnology 21 697ndash700
Vezina C M Walker N J amp Olson J R (2004) Subchronic Exposure to
TCDD PeCDF PCB126 and PCB153 Effect on hepatic gene
expression Environmental Health Perspectives 112 1636ndash1644
Vo T D Greenberg H J amp Palsson B O (2004) Reconstruction and
functional characterization of the human mitochondrial metabolic
network based on proteomic and biochemical data Journal of Bio-
logical Chemistry 279 39532ndash39540
Wang H amp LeCluyse E L (2003) Role of orphan nuclear receptors in
the regulation of drug-metabolising enzymes Clinical Pharmacoki-
netics 42 1331ndash1357
Wang N Silver D L Thiele C amp Tall A R (2001) ATP-
binding cassette transporter A1 (ABCA1) functions as a choles-
terol efflux regulatory protein Journal of Biological Chemistry 276
23742ndash23747
Waring J F Cavet G Jolly R A McDowell J Dai H Ciurlionis R
et al (2003) Development of a DNA Microarray for toxicology based
on hepatoxin-regulated sequences Environmental Health Perspectives
111 863ndash870
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6666
Waring J F Gum R Morfitt D Jolly R A Ciurlionis R Heindel M
et al (2002) Identifying toxic mechanisms using DNA microarrays
Evidence that an experimental inhibitor of cell adhesion molecule
expression signals through the aryl hydrocarbon nuclear receptor
Toxicology 181ndash182 537ndash550
Waring J F Jolly R A Ciurlionis R Lum P Y Praestgaard J T
Morfitt D C et al (2001) Clustering of hepatotoxins based on
mechanism of toxicity using gene expression profiles Toxicology and
Applied Pharmacology 175 28ndash42
Waters M Boorman G Bushel P Cunningham M Irwin R Merrick
A et al (2003) Systems toxicology and the Chemical Effects in
Biological Systems (CEBS) knowledge base EHP Toxicogenomics
111 15ndash28
Wei M Arnold L Cano M amp Cohen S M (2005) Effects of co-
administration of antioxidants and arsenicals on the rat urinary bladder
epithelium Toxicology Sciences 83 237ndash245
Weinshilboum R (2003) Inheritance and drug response New England
Journal of Medicine 348 529ndash537
Weinshilboum R amp Wang L (2004) Pharmacogenetics Inherited
variation in amino acid sequence and altered protein quantity Clinical
Pharmacology and Therapeutics 75 253ndash258
Werner E (2003) In silico multicellular systems biology and minimal
genomes Drug Discovery Today 8 1121ndash1127
Wong J S amp Gill S S (2002) Gene expression changes induced in
mouse liver by di(2-ethylhexyl) phthalate Toxicology and Applied
Pharmacology 185 180ndash196
Xie T Tong L McCann U D Yuan J Becker K G Mechan A O
et al (2004) Identification and characterization of metallothionein-1
and -2 gene expression in the context of ()34-methylenedioxymetham-
phetamine-induced toxicity to brain dopaminergic neurons Journal of
Neuroscience 24 7043ndash7050
Yadetie F Laegreid A Bakke I Kusnierczyk W Komorowski J
Waldum H L et al (2003) Liver gene expression in rats in response
to the peroxisome proliferator-activated receptor-alpha agonist ciprofi-
brate Physiological Genomics 15 9ndash19
Yamashita S Nomoto T Abe M Tatematsu M Sugimura T amp
Ushijima T (2004) Persistence of gene expression changes in stomach
mucosae induced by short-term N-methyl-NV-nitro-N-nitrosoguanidinetreatment and their presence in stomach cancers Mutation Research
549 185ndash193
Yan Q amp Sadee W (2000) Human membrane transporter database A
web-accessible relational database for drug transport studies and
pharmacogenomics AAPS Pharmsci 2 E20
Yeger-Lotem E amp Margalit H (2003) Detection of regulatory circuits by
integrating the cellular networks of proteinndashprotein interactions and
transcription regulation Nucleic Acids Research 31 6053ndash6061
Yoon B I Li G -X Kitada K Kawasaki Y Igarashi K amp Kodama
Y et al (2003) Mechanisms of benzene-induced hematotoxicity and
leukemogenicity cDNA microarray analyses using mouse bone marrow
tissue Environmental Health Perspectives 111 1411ndash1420
Young M B DiSilvestro M R Sendera T J Freund J Kriete A amp
Magnuson S R (2003) Analysis of gene expression in carbon
tetrachloride-treated rat livers using a novel bioarray technology
Pharmacogenomics Journal 3 41ndash52
Yu H Zhu X Greenbaum D Karro J amp Gerstein M (2004) TopNet
A tool for comparing biological sub-networks correlating protein
properties with topological statistics Nucleic Acids Research 32
328ndash337
Zhang E Y Fu D J Pak Y A Stewart T Mukhopadhyay N
Wrighton S A et al (2004) Genetic polymorphisms in human proton-
dependent dipeptide transporter PEPT1 Implications for the functional
role of Pro586 Journal of Pharmacology and Experimental Therapeu-
tics 310 437ndash445
Zhang E Y Knipp G T Ekins S amp Swaan P W (2002)
Structural biology and function of solute transporters Implications
for identifying and designing substrates Drug Metabolism Reviews
34 709ndash750
Zhang E Y Phelps M A Cheng C Ekins S amp Swaan P W (2002)
Modeling of active transport systems Advanced Drug Delivery
Reviews 54 329ndash354
- Systems-ADMETox Resources and network approaches
-
- Introduction
- Data available
- Network analysis and databases
-
- Network applications
-
- The role of transporters
-
- Clinical relevance of transporters
- Transporter network examples ABCA1
- Transporter network examples OATP
- Transporter microarray data
-
- Applications to enzymes
- Future network applications
-
- Discussion
- Acknowledgements
- References
-
Fig 7 Networks for drug resistant cell lines generated with the Auto expand algorithm in MetaCore A KB-3-1 vs KB-8-5 B ATC-DU-145 vs RCO1 C node overlap of A and B with initial nodes highlighted
with blue or red solid circles (Initial gene lists derived from (Annereau et al 2004)) Information on the type of interaction between objects is hidden for clarity eg unspecified allosteric regulation binding
cleavage competition covalent modification dephosphorylation phosphorylation transcription regulation transformation When applicable interactions also have a positive or negative effect and direction
Ligands (purple) linked to other proteins (blue blobs) transfactors (red) enzymes (orange) (For interpretation of the references to colour in this figure legend the reader is referred to the web version of this article)
SEkin
sJournalofPharm
acologica
landToxico
logica
lMeth
ods53(2006)38ndash66
56
Fig7(continued)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 57
Fig7(continued)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6658
Fig 8 The network of gene interactions around the CYP3A4 enzyme (centre) from MetaCorei Information on the type of interaction between objects is
hidden for clarity eg unspecified allosteric regulation binding cleavage competition covalent modification dephosphorylation phosphorylation
transcription regulation transformation When applicable interactions also have a positive or negative effect and direction Ligands (purple) linked to other
proteins (blue blobs) transfactors (red) enzymes (orange) (For interpretation of the references to colour in this figure legend the reader is referred to the web
version of this article)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 59
the initial doses of drugs metabolized by these enzymes
should be reduced in older patients according to the clinical
response In most published studies the elderly appear at
least as responsive as the young to inducers or inhibitors of
P450s (Durnas Loi amp Cusack 1990) More recently there
has been some suggestion that there are age related
reductions in function of some specific P450s such as
CYP3A4 (Patki et al 2004) and this could occur at the
level of regulation However earlier studies with the same
enzyme showed no change in clearance with age (Hunt et
al 1992) To date there has been even less examination of
the transporter functions and any changes with age
(Kinirons amp OrsquoMahony 2004) so the current understand-
ing of the effects of aging on metabolism and transport is
anything but transparent This represents an extreme
challenge for the pharmaceutical industry how to predict
whether a drug has an affinity for an enzyme or transporter
and whether this may be clinically important if it shows a
decline in expression or function with aging Any decrease
of function of these enzymes or transporters in an elderly
population would result in changes in the metabolism and
transport of metabolites potentially impacting the clinical
effect This represents another example of how computa-
tional approaches may perhaps be used with different types
of predicted and experimental data to allow visualization of
potential compound interactions or toxicity in elderly
populations We may see different gene networks high-
lighted as humans age and these may be modified by drug
treatment and coadministration This represents an area
were network analysis could be applied in the future and is
in need of considerable further research
The collection of microarray data in databases such as
CEBS ArrayTrack and EDGE represents a future resource
for computational gene network analysis One could
envisage that ultimately in each case such data is converted
to one or more networks that are also displayed for the user
and can be used to compare treatments from in vivo and in
vitro experiments This would represent a different approach
to clustering the data as currently implemented in one of
these efforts (Hayes et al 2005) and may condense large
amounts of experimentally derived data into a readily
interpreted network
4 Discussion
Previously in this journal the progress of many
research groups in predicting human ADME parameters
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6660
in silico (Ekins et al 2000b) and approaches for drug
metabolism (Ekins Ring Grace McRobie-Belle amp
Wrighton 2000a) have been described Both of these
reviews commented on moving HT assays for ADME
Tox much earlier in drug discovery which had also been
indicated by other groups The initial reviews also
highlighted the likely wealth of data that would become
available and how this could be used for structure
activity relationships alongside the bioactivity data in
computational models It was noted that there was a
paucity of predictive metabolism tools at that time In
addition a growing number of efforts to model whole
cells and organs now a field called systems biology
were recognized as models that could be integrated with
the in silico ADME approaches In summary since these
past reviews virtually all pharmaceutical companies have
attempted earlier high throughput screening for ADME
Tox properties and to some extent the wider application
of computational approaches for physicochemical proper-
ties Systems biology is being quite widely acknowledged
as the new paradigm for understanding complex bio-
logical datasets derived from high throughput technolo-
gies and the accumulated knowledge on human protein
interactions (Hartwell Hopfield Leibler amp Murray 1999
Hood 2003b) Therefore systems biology can be defined
as the integration of genetic proteomic transcriptomic
and metabonomic data using computational methods
(Nicholson amp Wilson 2003) When taken together
information on molecular processes derived from different
sources represents a lsquolsquouniversersquorsquo of putative biological
functionality of which only a small fraction of it will be
realized in a cell at any given time To date systems
biology has been driven by academia and funding bodies
such as the NIH rather than the pharmaceutical compa-
nies Presently there is a great deal of interest from
scientists of all backgrounds in identifying the networks
of cellular pathways and the corresponding physically
interacting proteins
The network building software for systems biology
described in this current review will be valuable to query
high throughput data and known literature interactions in
order to predict potential toxicity in different species In the
future the compilation of published toxicogenomics data-
sets characteristic of different types of toxicity will likely
be available in these software systems to act as a reference
database It is also feasible that we will be able to generate
the annotated datasets which specifically address the
differences between human and rat networks implicated
in toxicity The identification of sub-network modules
conserved between human and rat distinct for toxicity
types or predictive for toxic end-points in human will be
possible Such signature gene networks (Nikolsky et al
2005) can then be verified with other experimentally
derived data prospectively or from preexisting databases
The combined Fhigh throughput datandashin silico network_approaches therefore represents a novel method for access-
ing complex tissue-level processes as networks integrating
all data types based on functional interactions The data-
bases developed and used in this approach will certainly
benefit from further annotation around the drug metaboliz-
ing enzymes and transporters as described herein in terms
of transcriptional regulation and the ligands associated with
them which frequently appear in the literature It will be
important to capture disparities in the assignment of ligands
to enzymes transporters and other proteins as well as
negative data
In summary although there have been numerous
toxicogenomics studies published there is presently a
relatively small number of datasets that are freely available
to perform network analysis of microarray data The
number of studies identifying large numbers of proteins
which are affected by molecule treatment are even scarcer
still while there are several examples of some published
studies that combine such data It is hoped that the
numerous database initiatives for high content and toxicol-
ogy data that are being undertaken will improve the
situation for other researchers that are not currently
equipped to do such microarray studies themselves The
addition of requirements by journals to deposit such raw
data in a freely accessible resource will aid these initiatives
ADMETox groups have seen new technologies and
approaches developed over the last decade that have all
been applied to identify poor compounds earlier (Fig 2)
The latest technologies integrate network building tools
with high content data and databases The current review
described the limited number of networks generated for
ADMETox at present and one hopes that the impact of
such analyses will be commonplace in the future Systems
biology is however more than just applying a network
approach and hence systems-ADMETox will have to
evolve due to the continual pressure to develop newer
technologies This current paradigm combining empirical
data and computational methods should integrate the
complex data already generated making it readily inter-
pretable and valuable for identifying the most promising
compounds in the future
Acknowledgements
Dr Maggie AZ Hupcey is gratefully acknowledged for
editorial assistance Dr Peter W Swaan (University of
Maryland) and Dr Cheng Chang (Ohio State University)
Dr Steve Wright (University of Arizona) Dr K Sandy
Pang (University of Toronto) and Dr Craig Giroux (Wayne
State University) are kindly acknowledged for their support
and discussions My colleagues at GeneGo Sergey
Andreyev Andy Ryabov Eugene Kirillov Eugene A
Rakhmatulin Svetlana Sorokina Andrej Bugrim Tatiana
Nikolskaya Yuri Nikolsky John Metz and Julie Bryant are
all thanked for their considerable contributions to software
development and data annotation
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 61
References
Abe M Yamashita S Kuramoto T Hirayama Y Tsukamoto T Ohta
T et al (2003) Global expression analysis of N-methyl-NV-nitro-N-nitrosoguanidine-induced rat stomach carcinomas using oligonucleotide
microarrays Carcinogenesis 24 861ndash867
Adachi T Ono Y Koh K B Takashima K Tainaka H Matsuno Y
et al (2004) Long-term alteration of gene expression without
morphological change in testis after neonatal exposure to genistein in
mice Toxicogenomic analysis using cDNA microarray Food and
Chemical Toxicology 42 445ndash452
Akerman G S Rosenzweig B A Domon O E McGarrity L J
Blankenship L R Tsai C A et al (2004) Gene expression profiles
and genetic damage in benzo(a)pyrene diol epoxide-exposed TK6 cells
Mutation Research 549 43ndash64
Aksenov S V Church B Dhiman A Georgieva A Sarangapani R
Helmlinger G et al (2005) An integrated approach for inference and
mechanistic modeling for advancing drug development FEBS Letters
579 1878ndash1883
Anderle P Huang Y amp Sadee W (2004) Intestinal membrane transport
of drugs and nutrients Genomics of membrane transporters using
expression microarrays European Journal of Pharmaceutical Sciences
21 17ndash24
Annereau J P Szakacs G Tucker C J Arciello A Cardarelli C
Collins J et al (2004) Analysis of ATP-binding cassette transporter
expression in drug-selected cell lines by a microarray dedicated to
multidrug resistance Molecular Pharmacology 66 1397ndash1405
Apic G Ignjatovic T Boyer S amp Russell R B (2005) Illuminating drug
discovery with biological pathways FEBS Letters 579 1872ndash1877
Baker V A Harries H M Waring J F Duggan C M Ni H A Jolly
R A et al (2004) Clofibrate-induced gene expression changes in rat
liver A cross-laboratory analysis using membrane cDNA arrays
Environmental Health Perspectives 112 428ndash438
Balakin K V Ekins S Bugrim A Ivanenkov Y A Korolev D amp
Nikolsky Y et al (2004a) Quantitative structurendashmetabolism relation-
ship modeling of the metabolic N-dealkylation rates Drug Metabolism
and Disposition 32 1111ndash1120
Balakin K V Ekins S Bugrim A Ivanenkov Y A Korolev D amp
Nikolsky Y et al (2004b) Kohonen maps for prediction of binding to
human cytochrome P450 3A4 Drug Metabolism and Disposition 32
1183ndash1189
Barabasi A -L amp Oltvai Z N (2004) Network biology Under-
standing the cellrsquos functional organization Nature Reviews Genetics
5 101ndash113
Bartosiewicz M Penn S G amp Buckpitt A (2001) Applications of gene
arrays in environmental toxicology Fingerprints of gene regulation
associated with cadmium chloride benzo(a)pyrene and trichloro-
ethylene Environmental Health Perspectives 109 71ndash74
Bertilsson G Heidrich J Svensson K Asman M Jendeberg L
Sydow-Backman M et al (1998) Identification of a human nuclear
receptor defines a new signaling pathway for CYP3A induction
Proceedings of the National Academy of Sciences of the United States
of America 95 12208ndash12213
Blumberg B Sabbagh Jr W Juguilon H Bolado Jr J van Meter
C M Ong E S et al (1998) SXR a novel steroid and xenobiotic-
sensing nuclear receptor Genes and Development 12 3195ndash3205
Borodina Y Rudik A Filimonov D Kharchevnikova N Dmitriev A
Blinova V et al (2004) A new statistical approach to predicting
aromatic hydroxylation sites Comparison with model-based
approaches Journal of Chemical Information and Computer Sciences
44 1998ndash2009
Borodina Y Sadym A Filimonov D Blinova V Dmitriev A amp
Poroikov V (2003) Predicting biotransformation potential from
molecular structure Journal of Chemical Information and Computer
Sciences 43 1636ndash1646
Bottone Jr F G Martinez J M Collins J B Afshari C A amp Eling
T E (2003) Gene modulation by the cyclooxygenase inhibitor
sulindac sulfide in human colorectal carcinoma cells Possible link to
apoptosis Journal of Biological Chemistry 278 25790ndash25801
Boyer S amp Zamora I (2002) New methods in predictive metabolism
Journal of Computer-Aided Molecular Design 16 403ndash413
Bredel M amp Jacoby E (2004) Chemogenomics An emerging strategy
for rapid target and drug discovery Nature Reviews Genetics 5
262ndash275
Bugrim A Nikolskaya T amp Nikolsky Y (2004) Early prediction of drug
metabolism and toxicity Systems biology approach and modeling
Drug Discovery Today 9 127ndash135
Bulera S J Eddy S M Ferguson E Jatkoe T A Reindel J F
Bleavins M R et al (2001) RNA expression in the early character-
ization of hepatotoxicants in Wistar rats by high-density DNA micro-
arrays Hepatology 33 1239ndash1258
Butcher E C Berg E L amp Kunkel E J (2004) Systems biology in drug
discovery Nature Biotechnology 22 1253ndash1259
Butte A (2002) The use and analysis of microarray data Naturalist
Review of Drug Discovery 1 951ndash960
Cary M P Bader G D amp Sander C (2005) Pathway information for
systems biology FEBS Letters 579 1815ndash1820
Cheok M H Yang W Pui C H Downing J R Cheng C Naeve
C W et al (2003) Treatment-specific changes in gene expression
discriminate in vivo drug response in human leukemia cells Nature
Genetics 34 85ndash90
Cleary J D Rogers P D amp Chapman S W (2001) Differential
transcription factor expression in human mononuclear cells in response
to amphotericin B Identification with complementary DNA microarray
technology Pharmacotherapy 21 1046ndash1054
Coen M Ruepp S U Lindon J C Nicholson J K Pognan F Lenz
E M et al (2004) Integrated application of transcriptomics and
metabonomics yields new insight into the toxicity due to paracetamol in
the mouse Journal of Pharmaceutical and Biomedical Analysis 35
93ndash105
Cornwell P D De Souza A T amp Ulrich R G (2004) Profiling of
hepatic gene expression in rats treated with fibric acid analogs
Mutation Research 549 131ndash145
Csermely P Agoston V amp Pongor S (2005) The efficiency of multi-
target drugs The network approach might help drug design Trends in
Pharmacological Sciences 26 178ndash182
Cunningham M J Liang S Fuhrman S Seilhamer J J amp
Somogyi R (2000) Gene expression microarray data analysis for
toxicology profiling Annals of the New York Academy of Sciences 919
52ndash67
de Longueville F Atienzar F A Marcq L Dufrane S Evrard S
Wouters L et al (2003) Use of a low density microarray for studying
gene expression patterns induced by hepatotoxicants on primary
cultures of rat hepatocytes Toxicological Sciences 75 378ndash392
Dobrin R Beg Q K Barabasi A L amp Oltvai Z N (2004)
Aggregation of topological motifs in the Escherichia coli transcriptional
regulatory network BMC Bioinformatics 5 10
Donald S Verschoyle R D Edwards R Judah D J Davies R
Riley J et al (2002) Hepatobiliary damage and changes in hepatic
gene expression caused by the antitumor drug ecteinascidin-743 (ET-
743) in the female rat Cancer Research 62 4256ndash4262
Dumont M Jacquemin E DrsquoHont C Descout C Cresteil D Haouzi
D et al (1997) Expression of the liver Na+-independent organic anion
transporting polypeptide (oatp-1) in rats with bile duct ligation Journal
of Hepatology 27 1051ndash1056
Durnas C Loi C M amp Cusack B J (1990) Hepatic drug metabolism
and aging Clinical Pharmacokinetics 19 359ndash389
Eisen M B Spellman P T Brown P O amp Botstein D (1998) Cluster
analysis and display of genome-wide expression patterns Proceedings
of the National Academy of Sciences of the United States of America
95 14863ndash14868
Ekins S Andreyev S Ryabov A Kirilov E Rakhmatulin E A
Bugrim A et al (in press) Computational Prediction of Human Drug
Metabolism Exp Opin Drug Metab Toxicol
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6662
Ekins S Boulanger B Swaan P W amp Hupcey M A Z (2002)
Towards a new age of virtual ADMETOX and multidimensional drug
discovery Journal of Computer-Aided Molecular Design 16 381ndash401
Ekins S Bugrim A Nikolsky Y amp Nikolskaya T (2005) Systems
biology Applications in drug discovery In S Gad (Ed) Drug
Discovery Handbook (pp 123ndash183) Hoboken NJrsquo Wiley and Sons
Ekins S Giroux C N Nikolsky Y Bugrim A amp Nikolskaya T
(2005) A signature gene network approach to toxicity The Toxoco-
logists 84
Ekins S Kirillov E Rakhmatulin E amp Nikolskaya T (2005) A novel
method for visualizing nuclear hormone receptor networks relevant to
drug metabolism Drug Metabolism and Disposition 33 474ndash481
Ekins S amp McGowan R J (2001) The limits of reductionism The
shifting genomic paradigmrsquos impact on industry and academia
Philsophy in Science 9 1ndash23
Ekins S Mirny L amp Schuetz E G (2002) A ligand-based approach to
understanding selectivity of nuclear hormone receptors PXR CAR
FXR LXRa and LXRb Pharmaceutical Research 19 1788ndash1800
Ekins S Nikolsky Y amp Nikolskaya T (2005) Techniques Application
of systems biology to absorption distribution metabolism excretion
and toxicity Trends in Pharmacological Sciences 26 202ndash209
Ekins S Ring B J Grace J McRobie-Belle D J amp Wrighton S
A (2000) Present and future in vitro approaches for drug
metabolism Journal of Pharmacological and Toxicological Methods
44 313ndash324
Ekins S Waller C L Swaan P W Cruciani G Wrighton S A amp
Wikel J H (2000) Progress in predicting human ADME parameters in
silico Journal of Pharmacological and Toxicological Methods 44
251ndash272
Erhardt P W (2003) A human drug metabolism database Potential roles
in the quantitative predictions of drug metabolism and metabolism-
related drugndashdrug interactions Current Drug Metabolism 4 411ndash422
Evans W E amp McLeod H L (2003) Pharmacogenomicsmdashdrug
disposition drug targets and side effects New England Journal of
Medicine 348 538ndash549
Ezendam J Staedtler F Pennings J Vandebriel R J Pieters R
Boffetta P et al (2004) Toxicogenomics of subchronic hexachlor-
obenzene exposure in Brown Norway rats Environmental Health
Perspectives 112 782ndash791
Faiola B Fuller E S Wong V A amp Recio L (2004) Gene expression
profile in bone marrow and hematopoietic stem cells in mice exposed to
inhaled benzene Mutation Research 549 195ndash212
FDA (2004) Innovation stagnation Challenge and opportunity on the
critical path to new medicinal products
Fiehn O (2001) Combining genomics metabolome analysis and bio-
chemical modelling to understand metabolic networks Comparative
Functional Genomics 2 155ndash168
Field F J Born E amp Mathur S N (2004) LXRRXR ligand activation
enhances basolateral efflux of beta-sitosterol in CaCo-2 cells Journal of
Lipid Research 45 905ndash913
Fleck C Sutter L Appenroth D Koch B Meinhold T Pitack M
et al (2003) Use of gene chip technology for the characterisation of
the regulation of renal transport processes and of nephrotoxicity in
rats Experimental and Toxicologic Pathology 54 401ndash410
Fliri A F Loging W T Thadeio P F amp Volkmann R A (2005)
Biological spectra analysis Linking biological activity profiles to
molecular structure Proceedings of the National Academy of Sciences
of the United States of America 102 261ndash266
Fountoulakis M (2004) Application of proteomics technologies in the
investigation of the brain Mass Spectrometry Reviews 23 231ndash258
Gaucher S P Taylor S W Fahy E Zhang B Warnock D E Ghosh
S S et al (2004) Expanded coverage of the human heart
mitochondrial proteome using multidimensional liquid chromatography
coupled with tandem mass spectrometry Journal of Proteome
Research 3 495ndash505
Gerhold D Lu M Xu J Austin C Caskey C T amp Rushmore T
(2001) Monitoring expression of genes involved in drug metabolism
and toxicology using DNA microarrays Physiological Genomics 5
161ndash170
Guo G L Choudhuri S amp Klaassen C D (2002) Induction profile of
rat organic anion transporting polypeptide 2 (oatp2) by prototypical
drug-metabolizing enzyme inducers that activate gene expression
through ligand-activated transcription factor pathways Journal of
Pharmacology and Experimental Therapeutics 300 206ndash212
Hagenbuch B amp Meier P J (2004) Organic anion transporting
polypeptides of the OATP SLC21 family Phylogenetic classification
as OATP SLCO superfamily new nomenclature and molecularfunc-
tional properties Pflugers Archiv 447 653ndash665
Hamadeh H K amp Afshari C A (2004) Toxicogenomics principles and
applications Hobokenrsquo John Wiley and Sons
Hamadeh H K Bushel P R Jayadev S Martin K DiSorbo O Sieber
S et al (2002) Gene expression analysis reveals chemical-specific
profiles Toxicological Sciences 67 219ndash231
Hamadeh H K Jayadev S Gaillard E T Huang Q Stoll R
Blanchard K et al (2004) Integration of clinical and gene expression
endpoints to explore furan-mediated hepatotoxicity Mutation Research
549 169ndash183
Hamadeh H K Knight B L Haugen A C Sieber S Amin R P
Bushel P R et al (2002) Methapyrilene toxicity Anchorage of
pathologic observations to gene expression alterations Toxicologic
Pathology 30 470ndash482
Han J D Bertin N Hao T Goldberg D S Berriz G F Zhang L V
et al (2004) Evidence for dynamically organized modularity in the
yeast proteinndashprotein interaction network Nature 430 88ndash93
Hanisch D Zien A Zimmer R amp Lengauer T (2002) Co-clustering of
biological networks and gene expression data Bioinformatics 18
S145ndashS154
Harris A J Dial S L amp Casciano D A (2004) Comparison of basal
gene expression profiles and effects of hepatocarcinogens on gene
expression in cultured primary human hepatocytes and HepG2 cells
Mutation Research 549 79ndash99
Harrison S C (2004) Whither structural biology Nature Structural
Molecular Biology 11 12ndash15
Hartley D P Dai X He Y D Carlini E J Wang B Huskey S E
et al (2004) Activators of the rat pregnane X receptor differentially
modulate hepatic and intestinal gene expression Molecular Pharma-
cology 65 1159ndash1171
Hartwell L H Hopfield J J Leibler S amp Murray A W (1999) From
molecular to modular cell biology Nature 402 C47ndashC52
Hasmall S Orphanides G James N Pennie W Hedley K Soames
A et al (2002) Downregulation of lactoferrin by PPARalpha ligands
Role in perturbation of hepatocyte proliferation and apoptosis
Toxicology Sciences 68 304ndash313
Hayase T Yamamoto Y Yamamoto K Muso E amp Shiota K (2004)
Microarray profile analysis of toxic cocaine-induced alterations in the
expression of mouse brain gene sequences A possible rsquoprotectiversquo
effect of buprenorphine Journal of Applied Toxicology 24 15ndash20
Hayes K R Vollrath A L Zastrow G M McMillan B J Craven M
Jovanovich S et al (2005) EDGE A centralized resource for the
comparison analysis and distribution of toxicogenomic information
Molecular Pharmacology 67 1360ndash1368
Heijne W H Slitt A L van Bladeren P J Groten J P Klaassen C D
Stierum R H et al (2004) Bromobenzene-induced hepatotoxicity at
the transcriptome level Toxicology Sciences 79 411ndash422
Heijne W H Stierum R H Slijper M van Bladeren P J amp van
Ommen B (2003) Toxicogenomics of bromobenzene hepatotoxicity
A combined transcriptomics and proteomics approach Biochemical
Pharmacology 65 857ndash875
Heinloth A N Irwin R D Boorman G A Nettesheim P Fannin
R D Sieber S O et al (2004) Gene expression profiling of rat
livers reveals indicators of potential adverse effects Toxicology
Sciences 80 193ndash202
Hodges L C Cook J D Lobenhofer E K Li L Bennett L Bushel
P R et al (2003) Tamoxifen functions as a molecular agonist inducing
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 63
cell cycle-associated genes in breast cancer cells Molecular Cancer
Research 1 300ndash311
Holleman A Cheok M H den Boer M L Yang W Veerman KM
A J Kazemier et al (2004) Gene-expression patterns in drug-resistant
acute lymphoblastic leukemia cells and response to treatment New
England Journal of Medicine 351 533ndash542
Hong Y Muller U R amp Lai F (2003) Discriminating two classes of
toxicants through expression analysis of HepG2 cells with DNA arrays
Toxicology in Vitro 17 85ndash92
Hood E (2003) Proteomics Characterizing the cogs in the machinery of
life Environmental Health Perspectives 111 A816ndashA825
Hood L (2003) Systems biology Integrating technology biology and
computation Mechanism of Ageing and Development 124 9ndash16
Hood L amp Galas D (2003) The digital code of DNA Nature 421
444ndash448
Hood L amp Perlmutter R M (2004) The impact of systems approaches on
biological problems in drug discovery Nature Biotechnology 22
1215ndash1217
Hu T Gibson D P Carr G J Torontali S M Tiesman J P Chaney
J G et al (2004) Identification of a gene expression profile that
discriminates indirect-acting genotoxins from direct-acting genotoxins
Mutation Research 549 5ndash27
Huang Q Dunn II R T Jayadev S DiSorbo O Pack F D Farr S B
et al (2001) Assessment of cisplatin-induced nephrotoxicity by
microarray technology Toxicology Sciences 63 196ndash207
Huang Q Jin X Gaillard E T Knight B L Pack F D Stoltz J H et
al (2004) Gene expression profiling reveals multiple toxicity endpoints
induced by hepatotoxicants Mutation Research 549 147ndash167
Huisman M T Smit J W Crommentuyn K M Zelcer N Wiltshire
H R Beijnen J H et al (2002) Multidrug resistance protein 2
(MRP2) transports HIV protease inhibitors and transport can be
enhanced by other drugs Aids 16 2295ndash2301
Hunt C M Westerkam W R amp Stave G M (1992) Effect of age and
gender on the activity of human hepatic CYP3A Biochemical
Pharmacology 44 275ndash283
Ideker T Ozier O Schwikowski B amp Siegal A F (2002) Discovering
regulatory and signalling circuits in molecular interaction networks
Bioinformatics 18 S233ndashS240
Ideker T Thorsson V Ranish J A Christmas R Buhler J Eng J K
et al (2001) Integrated genomic and proteomic analyses of a system-
atically perturbed network Science 292 929ndash934
Iida M Anna C H Hartis J Bruno M Wetmore B Dubin J R et al
(2003) Changes in global gene and protein expression during early
mouse liver carcinogenesis induced by non-genotoxic model carcino-
gens oxazepam and Wyeth-14643 Carcinogenesis 24 757ndash770
Ingelman-Sundberg M (2004) Pharmacogenetics of cytochrome P450 and
its applications in drug therapy The past present and future Trends in
Pharmacological Sciences 25 193ndash200
Jeong H Mason S P Barabasi A L amp Oltvai Z N (2001) Lethality
and centrality in protein networks Nature 411 41ndash42
Jeong H Tombor B Albert R Oltvai Z N amp Barabasi A L
(2000) The large-scale organization of metabolic networks Nature
407 651ndash654
Jung D Hagenbuch B Gresh L Pontoglio M Meier P J amp Kullak-
Ublick G A (2001) Characterization of the human OATP-C
(SLC21A6) gene promoter and regulation of liver-specific OATP genes
by hepatocyte nuclear factor 1 alpha Journal of Biological Chemistry
276 37206ndash37214
Jung D amp Kullak-Ublick G A (2003) Hepatocyte nuclear factor 1
alpha A key mediator of the effect of bile acids on gene expression
Hepatology 37 622ndash631
Jung J -W Park J -S Hwang J -W Kang K -S YS L Song B
-S et al (2004) Gene expression analysis of peroxisome prolifer-
ators- and phenytoin-induced hepatotoxicity using cDNA microarray
Journal of Veterinary Medical Science 66 1329ndash1333
Kato N Shibutani M Takagi H Uneyama C Lee K -Y Takiami S
et al (2004) Gene expression profile in the livers of rats orally
administered ethinylestradiol for 28 days using a microarray technique
Toxicology 200 179ndash192
Kier L D Neft R Tang L Suizu R Cook T Onsurez K et al
(2004) Applications of microarrays with toxicologically relevant genes
(tox genes) for the evaluation of chemical toxicants in Sprague Dawley
rats in vivo and human hepatocytes in vitro Mutation Research 549
101ndash113
Kijima K Toyosawa K Yasuba M Matsuoka N Adachi T
Komiyama M et al (2004) Gene expression analysis of the rat testis
after treatment with di(2-ethylhexyl) phthalate using cDNA microarray
and real-time RT-PCR Toxicology and Applied Pharmacology 200
103ndash110
Kim R B (2003) Organic anion-transporting polypeptide (OATP) trans-
porter family and drug disposition European Journal of Clinical
Investigation 33 1ndash5
Kinirons M T amp OrsquoMahony M S (2004) Drug metabolism and ageing
British Journal of Clinical Pharmacology 57 540ndash544
Kitano H (2002a) Computational systems biology Nature 420 206ndash210
Kitano H (2002b) Systems biology A brief overview Science 295
1662ndash1664
Kliewer S A Moore J T Wade L Staudinger J L Watson M
A Jones S A et al (1998) An orphan nuclear receptor activated
by pregnanes defines a novel steroid signalling pathway Cell 92
73ndash82
Korolev D Balakin K V Nikolsky Y Kirillov E Ivanenkov Y A
Savchuk N P et al (2003) Modeling of human cytochrome p450-
mediated drug metabolism using unsupervised machine learning
approach Journal of Medicinal Chemistry 46 3631ndash3643
Kramer J A Pettit S D Amin R P Bertram T A Car B
Cunningham M et al (2004) Overview on the application of
transcription profiling using selected nephrotoxicants for toxicology
assessment Environmental Health Perspectives 112 460ndash464
Kultima K Nystrom A -M Scholz B Gustafson A -L Dencker L
amp Stigson M (2004) Valproic acid teratogenicity A toxicogenomics
approach Environmental Health Perspectives 112 1225ndash1235
Kutuzova G D amp Deluca H F (2004) Gene expression profiles in rat
intestine identify pathways for 125-dihydroxyvitamin D(3) stimulated
calcium absorption and clarify its immunomodulatory properties
Archives of Biochemistry and Biophysics 432 152ndash166
Kwak M K Wakabayashi N Itoh K Motohashi H Yamamoto M amp
Kensler T W (2003) Modulation of gene expression by cancer
chemopreventive dithiolethiones through the Keap1-Nrf2 pathway
Identification of novel gene clusters for cell survival Journal of
Biological Chemistry 278 8135ndash8145
Lahjouji K Mitchell G A amp Qureshi I A (2001) Carnitine transport
by organic cation transporters and systemic carnitine deficiency
Molecular Genetics and Metabolism 73 287ndash297
Landowski C P Sun D Foster D R Menon S S Barnett J L
Welage L S et al (2003) Gene expression in the human intestine and
correlation with oral valacyclovir pharmacokinetic parameters Journal
of Pharmacology and Experimental Therapeutics 306 778ndash786
Lee G -J Lee W -S Jeon K -S Um C -H Kim S -J Lee C -H
et al (2004) cDNA microarray gene expression analysis and
toxicological phenotype for anticancer drug Journal of Veterinary
Medical Science 66 1339ndash1345
Lee M Kwon J Kim S N Kim J E Koh W S Kim E J et al
(2003) cDNA microarray gene expression profiling of hydroxyurea
paclitaxel and p-anisidine genotoxic compounds with differing
tumorigenicity results Environmental and Molecular Mutagenesis
42 91ndash97
Li S Armstrong C M Bertin N Ge H Milstein S Boxem M et al
(2004) A map of the interactome network of the metazoan C elegans
Science 303 540ndash543
Liguori M J Anderson L M Bukofzer S McKim J Pregenzer J F
Retief J et al (2005) Microarray analysis in human hepatocytes
suggests a mechanism for hepatotoxicity induced by trovafloxacin
Hepatology 41 177ndash186
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6664
Lobenhofer E K Bennett L Cable P L Li L Bushel P R amp Afshari
C A (2002) Regulation of DNA replication fork genes by 17beta-
estradiol Molecular Endocrinology 16 1215ndash1229
Locker J Tian J Carver R Concas D Cossu C Ledda-Columbano
G M et al (2003) A common set of immediate-early response genes
in liver regeneration and hyperplasia Hepatology 38 314ndash325
Mattes W B Pettit S D Sansone S A Bushel P R amp Waters M D
(2004) Database development in toxicogenomics Issues and efforts
Environmental Health Perspectives 112 495ndash505
McMillian M Nie A Y Parker J B Leone A Bryant S Kemmerer
M et al (2004) A gene expression signature for oxidant stressreactive
metabolites in rat liver Biochemical Pharmacology 68 2249ndash2261
Meier P J amp Stieger B (2000) Molecular mechanisms in bile formation
News in Physiological Sciences 15 89ndash93
Meneses-Lorente G de Longueville F Dos Santos-Mendes S Bonnert
T P Jack A Evrard S et al (2003) An evaluation of a low-density
DNA microarray using cytochrome P450 inducers Chemical Research
in Toxicology 16 1070ndash1077
Meneses-Lorente G Guest P C Lawrence J Muniappa N Knowles
M R Skynner H A et al (2004) A proteomic investigation of drug-
induced steatosis in rat liver Chemical Research in Toxicology 17
605ndash612
Merrill C L Ni H Yoon L W Tirmenstein M A Narayanan P
Benavides G R et al (2002) Etomoxir-induced oxidative stress in
HepG2 cells detected by differential gene expression is confirmed
biochemically Toxicology Sciences 68 93ndash101
Milo R Shen-Orr S Itzkovitz S Kashtan N Chklovskii D amp Alon
U (2002) Network motifs Simple building blocks of complex
networks Science 298 824ndash827
Morgan K T Ni H Brown H R Yoon L Qualls C W Crosby L
M et al (2002) Application of cDNA microarray technology to in
vitro toxicology and the selection of genes for a real-time RT-PCR-
based screen for oxidative stress in Hep-G2 cells Toxicologic
Pathology 30 435ndash451
Mwinyi J Johne A Bauer S Roots I amp Gerloff T (2004) Evidence
for inverse effects of OATP-C (SLC21A6) 5 and 1b haplotypes on
pravastatin kinetics Clinical Pharmacology and Therapeutics 75
415ndash421
Nakata K Yukawa M Komiyama N Nakano T amp Kaminuma T
(2002) A nuclear receptor database that maps pathways to diseases
Genome Infomatics 13 515ndash516
Nicholson J K Holmes E Lindon J C amp Wilson I D (2004) The
challenges of modeling mammalian biocomplexity Nature Biotechnol-
ogy 22 1268ndash1274
Nicholson J K amp Wilson I D (2003) Understanding rsquoglobal systems
biology Metabonomics and the continuum of metabolism Nature
Reviews on Drug Discovery 2 668ndash676
Nikitin A Egorov S Daraselia N amp Mazo I (2003) Pathway studiomdash
the analysis and navigation of molecular networks Bioinformatics 19
2155ndash2157
Nikolsky Y Ekins S Nikolskaya T amp Bugrim A (2005) A novel
method for generation of signature networks as biomarkers from
complex high throughput data Toxicology Letters 158 20ndash29
Nikolsky Y Nikolskaya T amp Bugrim A (2005) Biological networks
and analysis of experimental data in drug discovery Drug Discovery
Today 10 653ndash662
Nishizato Y Ieiri I Suzuki H Kimura M Kawabata K Hirota T
et al (2003) Polymorphisms of OATP-C (SLC21A6) and OAT3
(SLC22A8) genes Consequences for pravastatin pharmacokinetics
Clinical Pharmacology and Therapeutics 73 554ndash565
Nozawa T Nakajima M Tamai I Noda K Nezu J Sai Y et al
(2002) Genetic polymorphisms of human organic anion transporters
OATP-C (SLC21A6) and OATP-B (SLC21A9) Allele frequencies in
the Japanese population and functional analysis Journal of Pharmacol-
ogy and Experimental Therapeutics 302 804ndash813
Nozawa T Sugiura S Nakajima M Goto A Yokoi T Nezu J et al
(2004) Involvement of organic anion transporting polypeptides in the
transport of troglitazone sulfate Implications for understanding trogli-
tazone hepatotoxicity Drug Metabolism and Disposition 32 291ndash294
Oliver D E Rubin D L Stuart J M Hewett M Klein T E amp
Altman R B (2002) Ontology development for a pharmacogenetics
knowledge base Package Symposion 88ndash99
Oram J F amp Lawn R M (2001) ABCA1 The gatekeeper for eliminating
excess tissue cholesterol Journal of Lipid Research 42 1173ndash1179
Oswald M Kullak-Ublick G A Paumgartner G amp Beuers U (2001)
Expression of hepatic transporters OATP-C and MRP2 in primary
sclerosing cholangitis Liver 21 247ndash253
Parsons A B Brost R I Ding H Li Z Zhang C Sheikh B et al
(2004) Integration of chemical-genetic and genetic interaction data
links bioactive compounds to cellular target pathways Nature Bio-
technology 22 62ndash69
Patki K C von Moltke L L Harmatz J S Hesse L M Court M H
amp Greenblatt D J (2004) Effect of age on in vitro triazolam
biotransformation in male human liver microsomes Journal of
Pharmacology and Experimental Therapeutics 308 874ndash879
Pereira-Leal J B Enright A J amp Ouzounis C A (2004) Detection of
functional modules from protein interaction networks Proteins 54
49ndash57
Plant N (2004) Interaction networks Coordinating responses to xeno-
biotic exposure Toxicology 202 21ndash32
Raghavendra Prasad H S Qi Z Srinivasan K N amp Gopalakrish-
nakone P (2004) Potential effects of tetrodotoxin exposure to
human glial cells postulated using microarray approach Toxicon 44
597ndash608
Reilly T P Bourdi M Brady J N Pise-Masison C A Radonovich M
F George J W et al (2001) Expression profiling of acetaminophen
liver toxicity in mice using microarray technology Biochemical and
Biophysical Research Communications 282 321ndash328
Rives A W amp Galitski T (2003) Modular organization of cellular
networks Proceedings of the National Academy of Sciences of the
United States of America 100 1128ndash1133
Rogers P D Pearson M M Cleary J D Sullivan D C amp Chapman
S W (2002) Differential expression of genes encoding immunomodu-
latory proteins in response to amphotericin B in human mononuclear
cells identified by cDNA microarray analysis Journal of Antimicrobial
Chemotherapy 50 811ndash817
Rosen M B Wilson V S Schmid J E amp Gray L E (2005) Gene
expression analysis in the ventral prostate of rats exposed to vinclozolin
or procymidone Reproductive Toxicology 19 367ndash379
Rost D Herrmann T Sauer P Schmidts H L Stieger B Meier P J
et al (2003) Regulation of rat organic anion transporters in bile salt-
induced cholestatic hepatitis Effect of ursodeoxycholate Hepatology
38 187ndash195
Rothman R B Ayestas M A Dersch C M amp Baumann M H (1999)
Aminorex fenfluramine and chlorphentermine are serotonin transporter
substrates Implications for primary pulmonary hypertension Circula-
tion 100 869ndash875
Ruepp S U Tonge R P Shaw J Wallis N amp Pognan F (2002)
Genomics and proteomics analysis of acetaminophen toxicity in mouse
liver Toxicology Sciences 65 135ndash150
Sakaeda T Nakamura T amp Okumura K (2002) MDR1 genotype-
related pharmacokinetics and pharmacodynamics Biological amp Phar-
maceutical Bulletin 25 1391ndash1400
Sakata T Anzai N Shin H J Noshiro R Hirata T Yokoyama H
et al (2004) Novel single nucleotide polymorphisms of organic cation
transporter 1 (SLC22A1) affecting transport functions Biochemical
and Biophysical Research Communications 313 789ndash793
Satlin L M Amin V amp Wolkoff A W (1997) Organic anion
transporting polypeptide mediates organic anionHCO3-exchange
Journal of Biological Chemistry 272 26340ndash26345
Satomi Y Tsuchiya W Mihara K Ota M Kasahara Y amp Akahori F
(2004) Gene expression analysis of the lung following paraquat
administration in rats using DNA microarray Journal of Toxicological
Sciences 29 91ndash100
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 65
Schrenk D Baus P R Ermel N Klein C Vorderstemann B amp
Kauffmann H M (2001) Up-regulation of transporters of the MRP
family by drugs and toxins Toxicology Letters 120 51ndash57
Segal E Shapira M Regev A Persquoer D Botstein D Koller D et al
(2003) Module networks Identifying regulatory modules and their
condition-specific regulators from gene expression data Nature
Genetics 34 166ndash176
Segal E Wang H amp Koller D (2003) Discovering molecular pathways
from protein interaction and gene expression Bioinformatics 19
i264ndash i272
Seidel S D Kan H L Stott W T Schisler M R amp Gollapudi B B
(2003) Identification of transcriptome profiles for the DNA-damaging
agents bleomycin and hydrogen peroxide in L5178Y mouse lymphoma
cells Environmental and Molecular Mutagenesis 42 19ndash25
Shan L Yu Y Schut H A J amp Snyderwine E G (2004) Susceptibility
of rats to mammary gland carcinogenesis by the food-derived
carcinogen 2-Amino-1-Methyl-6-Phenylimidazo[45-b]Pyridine (PhIP)
varies with age and is associated with the induction of differential gene
expression American Journal of Pathology 165 191ndash202
Sharom J R Bellows D S amp Tyers M (2004) From large networks to
small molecules Current Opinion in Chemical Biology 8 81ndash90
Shitara Y Hirano M Sato H amp Sugiyama Y (2004) Gemfibrozil and
its glucuronide inhibit the OATP2(OATP1B1 SLC21A6)-mediated
hepatic uptake and CYP2C8-mediated metabolism of cerivastatinmdash
Analysis of the mechanism of the clinically relevant drugndashdrug
interaction between cerivastatin and gemfibrozil Journal of Pharma-
cology and Experimental Therapeutics
Shitara Y Itoh T Sato H Li A P amp Sugiyama Y (2003) Inhibition of
transporter-mediated hepatic uptake as a mechanism for drugndashdrug
interaction between cerivastatin and cyclosporin A Journal of
Pharmacology and Experimental Therapeutics 304 610ndash616
Somogyi R Fuhrman S amp Wen X (2001) Genetic network inference in
computational models and applications to large-scale gene expression
data In M Bower amp H Bolouri (Eds) Computational modeling of
genetic and biochemical networks (pp 119ndash157) Cambridge MArsquoThe
MIT Press
Spirin V amp Mirny L A (2003) Protein complexes and functional
modules in molecular networks Proceedings of the National Academy
of Sciences of the United States of America 100 12123ndash12128
Staudinger J Liu Y Madan A Habeebu S amp Klaassen C D
(2001) Coordinate regulation of xenobiotic and bile acid homeo-
stasis by pregnane X receptor Drug Metabolism and Disposition
29 1467ndash1472
Sun L Z Ji Z L Chen X Wang J F amp Chen Y Z (2002) ADME-
AP A database of ADME associated proteins Bioinformatics 18
1699ndash1700
Suter L Babiss L E amp Wheeldon E B (2004) Toxicogenomics in
predictive toxicology in drug development Chemistry and Biology 11
161ndash171
Synold T W Dussault I amp Forman B M (2001) The orphan nuclear
receptor SXR coordinately regulates drug metabolism and efflux
Nature Medicine 7 584ndash590
Tabuchi Y amp Kondo T (2003) cDNA microarray analysis reveals chop-
10 plays a key role in Sertoli cell injury induced by bisphenol A
Biochemical and Biophysical Research Communications 305 54ndash61
Tamai I Nezu J Uchino H Sai Y Oku A Shimane M et al (2000)
Molecular identification and characterization of novel members of the
human organic anion transporter (OATP) family Biochemical and
Biophysical Research Communications 273 251ndash260
Taylor S W Fahy E Zhang B Glenn G M Warnock D E Wiley S
et al (2003) Characterization of the human heart mitochondrial
proteome Nature Biotechnology 21 281ndash286
Terasaka S Aita Y Inoue A Hayashi S Nishigaki M Aoyagi K
et al (2004) Using a customized DNA microarray for expression
profiling of the estrogen-responsive genes to evaluate estrogen activity
among natural estrogens and industrial chemicals Environmental
Health Perspectives 112 773ndash781
Thomas D M Francescutti-Verbeem D M Liu X amp Kuhn D M
(2004) Identification of differentially regulated transcripts in mouse
striatum following methamphetamine treatmentmdashan oligonucleotide
microarray approach Journal of Neurochemistry 88 380ndash393
Thomas R S Rank D R Penn S G Zastrow G M Hayes K R
Pande K et al (2001) Identification of toxicologically predictive
gene sets using cDNA microarrays Molecular Pharmacology 60
1189ndash1194
Thompson K L Afshari C A Amin R P Bertram T A Car B
Cunningham M et al (2004) Identification of platform-independent
gene expression markers of cisplatin nephrotoxicity Environmental
Health Perspectives 112 488ndash494
Tirona R G Leake B F Merino G amp Kim R B (2001)
Polymorphisms in OATP-C Identification of multiple allelic
variants associated with altered transport activity among European-
and African-Americans Journal of Biological Chemistry 276
35669ndash35675
Tirona R G Leake B F Wolkoff A W amp Kim R B (2003)
Human organic anion transporting polypeptide-C (SLC21A6) is a
major determinant of the rifampin-mediated pregnane X receptor
activation Journal of Pharmacology and Experimental Therapeutics
304 223ndash228
Tong W Cao X Harris S Sun H Fang H Fuscoe J et al (2003)
ArrayTrackmdashsupporting toxicogenomic research at the US Food and
Drug Administration National Center for Toxicological Research
Environmental Health Perspectives 111 1819ndash1826
Tornow S amp Mewes H W (2003) Functional modules by relating
protein interaction networks and gene expression Nucleic Acids
Research 31 6283ndash6289
Trocho C Escola-Gil J C Ribas V Benitez S Martin-Campos J M
Rotllan N et al (2004) Phenytoin treatment reduces atherosclerosis in
mice through mechanisms independent of plasma HDL-cholesterol
concentration Atherosclerosis 174 275ndash285
Ueda A Hamadeh H K Webb H K Yamamoto Y Sueyoshi T
Afshari C A et al (2002) Diverse roles of the nuclear orphan
receptor CAR in regulating hepatic genes in response to phenobarbital
Molecular Pharmacology 61 1ndash6
Ulrich R G (2003) The toxicogenomics of nuclear receptor agonists
Current Opinion in Chemical Biology 7 505ndash510
Ulrich R G Rockett J C Gibson G G amp Pettit S D (2004)
Overview of an interlaboratory collaboration on evaluating the effects
of model hepatotoxicants on hepatic gene expression Environmental
Health Perspectives 112 423ndash427
Unami A Shinohara Y Ichikawa T amp Baba Y (2003) Biochemical
and microarray analyses of bupivacaine-induced apoptosis Journal of
Toxicological Sciences 28 77ndash94
Vasquez A Flammini A Maritan A amp Vespignani A (2003) Global
protein function prediction from proteinndashprotein interaction networks
Nature Biotechnology 21 697ndash700
Vezina C M Walker N J amp Olson J R (2004) Subchronic Exposure to
TCDD PeCDF PCB126 and PCB153 Effect on hepatic gene
expression Environmental Health Perspectives 112 1636ndash1644
Vo T D Greenberg H J amp Palsson B O (2004) Reconstruction and
functional characterization of the human mitochondrial metabolic
network based on proteomic and biochemical data Journal of Bio-
logical Chemistry 279 39532ndash39540
Wang H amp LeCluyse E L (2003) Role of orphan nuclear receptors in
the regulation of drug-metabolising enzymes Clinical Pharmacoki-
netics 42 1331ndash1357
Wang N Silver D L Thiele C amp Tall A R (2001) ATP-
binding cassette transporter A1 (ABCA1) functions as a choles-
terol efflux regulatory protein Journal of Biological Chemistry 276
23742ndash23747
Waring J F Cavet G Jolly R A McDowell J Dai H Ciurlionis R
et al (2003) Development of a DNA Microarray for toxicology based
on hepatoxin-regulated sequences Environmental Health Perspectives
111 863ndash870
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6666
Waring J F Gum R Morfitt D Jolly R A Ciurlionis R Heindel M
et al (2002) Identifying toxic mechanisms using DNA microarrays
Evidence that an experimental inhibitor of cell adhesion molecule
expression signals through the aryl hydrocarbon nuclear receptor
Toxicology 181ndash182 537ndash550
Waring J F Jolly R A Ciurlionis R Lum P Y Praestgaard J T
Morfitt D C et al (2001) Clustering of hepatotoxins based on
mechanism of toxicity using gene expression profiles Toxicology and
Applied Pharmacology 175 28ndash42
Waters M Boorman G Bushel P Cunningham M Irwin R Merrick
A et al (2003) Systems toxicology and the Chemical Effects in
Biological Systems (CEBS) knowledge base EHP Toxicogenomics
111 15ndash28
Wei M Arnold L Cano M amp Cohen S M (2005) Effects of co-
administration of antioxidants and arsenicals on the rat urinary bladder
epithelium Toxicology Sciences 83 237ndash245
Weinshilboum R (2003) Inheritance and drug response New England
Journal of Medicine 348 529ndash537
Weinshilboum R amp Wang L (2004) Pharmacogenetics Inherited
variation in amino acid sequence and altered protein quantity Clinical
Pharmacology and Therapeutics 75 253ndash258
Werner E (2003) In silico multicellular systems biology and minimal
genomes Drug Discovery Today 8 1121ndash1127
Wong J S amp Gill S S (2002) Gene expression changes induced in
mouse liver by di(2-ethylhexyl) phthalate Toxicology and Applied
Pharmacology 185 180ndash196
Xie T Tong L McCann U D Yuan J Becker K G Mechan A O
et al (2004) Identification and characterization of metallothionein-1
and -2 gene expression in the context of ()34-methylenedioxymetham-
phetamine-induced toxicity to brain dopaminergic neurons Journal of
Neuroscience 24 7043ndash7050
Yadetie F Laegreid A Bakke I Kusnierczyk W Komorowski J
Waldum H L et al (2003) Liver gene expression in rats in response
to the peroxisome proliferator-activated receptor-alpha agonist ciprofi-
brate Physiological Genomics 15 9ndash19
Yamashita S Nomoto T Abe M Tatematsu M Sugimura T amp
Ushijima T (2004) Persistence of gene expression changes in stomach
mucosae induced by short-term N-methyl-NV-nitro-N-nitrosoguanidinetreatment and their presence in stomach cancers Mutation Research
549 185ndash193
Yan Q amp Sadee W (2000) Human membrane transporter database A
web-accessible relational database for drug transport studies and
pharmacogenomics AAPS Pharmsci 2 E20
Yeger-Lotem E amp Margalit H (2003) Detection of regulatory circuits by
integrating the cellular networks of proteinndashprotein interactions and
transcription regulation Nucleic Acids Research 31 6053ndash6061
Yoon B I Li G -X Kitada K Kawasaki Y Igarashi K amp Kodama
Y et al (2003) Mechanisms of benzene-induced hematotoxicity and
leukemogenicity cDNA microarray analyses using mouse bone marrow
tissue Environmental Health Perspectives 111 1411ndash1420
Young M B DiSilvestro M R Sendera T J Freund J Kriete A amp
Magnuson S R (2003) Analysis of gene expression in carbon
tetrachloride-treated rat livers using a novel bioarray technology
Pharmacogenomics Journal 3 41ndash52
Yu H Zhu X Greenbaum D Karro J amp Gerstein M (2004) TopNet
A tool for comparing biological sub-networks correlating protein
properties with topological statistics Nucleic Acids Research 32
328ndash337
Zhang E Y Fu D J Pak Y A Stewart T Mukhopadhyay N
Wrighton S A et al (2004) Genetic polymorphisms in human proton-
dependent dipeptide transporter PEPT1 Implications for the functional
role of Pro586 Journal of Pharmacology and Experimental Therapeu-
tics 310 437ndash445
Zhang E Y Knipp G T Ekins S amp Swaan P W (2002)
Structural biology and function of solute transporters Implications
for identifying and designing substrates Drug Metabolism Reviews
34 709ndash750
Zhang E Y Phelps M A Cheng C Ekins S amp Swaan P W (2002)
Modeling of active transport systems Advanced Drug Delivery
Reviews 54 329ndash354
- Systems-ADMETox Resources and network approaches
-
- Introduction
- Data available
- Network analysis and databases
-
- Network applications
-
- The role of transporters
-
- Clinical relevance of transporters
- Transporter network examples ABCA1
- Transporter network examples OATP
- Transporter microarray data
-
- Applications to enzymes
- Future network applications
-
- Discussion
- Acknowledgements
- References
-
Fig7(continued)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 57
Fig7(continued)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6658
Fig 8 The network of gene interactions around the CYP3A4 enzyme (centre) from MetaCorei Information on the type of interaction between objects is
hidden for clarity eg unspecified allosteric regulation binding cleavage competition covalent modification dephosphorylation phosphorylation
transcription regulation transformation When applicable interactions also have a positive or negative effect and direction Ligands (purple) linked to other
proteins (blue blobs) transfactors (red) enzymes (orange) (For interpretation of the references to colour in this figure legend the reader is referred to the web
version of this article)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 59
the initial doses of drugs metabolized by these enzymes
should be reduced in older patients according to the clinical
response In most published studies the elderly appear at
least as responsive as the young to inducers or inhibitors of
P450s (Durnas Loi amp Cusack 1990) More recently there
has been some suggestion that there are age related
reductions in function of some specific P450s such as
CYP3A4 (Patki et al 2004) and this could occur at the
level of regulation However earlier studies with the same
enzyme showed no change in clearance with age (Hunt et
al 1992) To date there has been even less examination of
the transporter functions and any changes with age
(Kinirons amp OrsquoMahony 2004) so the current understand-
ing of the effects of aging on metabolism and transport is
anything but transparent This represents an extreme
challenge for the pharmaceutical industry how to predict
whether a drug has an affinity for an enzyme or transporter
and whether this may be clinically important if it shows a
decline in expression or function with aging Any decrease
of function of these enzymes or transporters in an elderly
population would result in changes in the metabolism and
transport of metabolites potentially impacting the clinical
effect This represents another example of how computa-
tional approaches may perhaps be used with different types
of predicted and experimental data to allow visualization of
potential compound interactions or toxicity in elderly
populations We may see different gene networks high-
lighted as humans age and these may be modified by drug
treatment and coadministration This represents an area
were network analysis could be applied in the future and is
in need of considerable further research
The collection of microarray data in databases such as
CEBS ArrayTrack and EDGE represents a future resource
for computational gene network analysis One could
envisage that ultimately in each case such data is converted
to one or more networks that are also displayed for the user
and can be used to compare treatments from in vivo and in
vitro experiments This would represent a different approach
to clustering the data as currently implemented in one of
these efforts (Hayes et al 2005) and may condense large
amounts of experimentally derived data into a readily
interpreted network
4 Discussion
Previously in this journal the progress of many
research groups in predicting human ADME parameters
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6660
in silico (Ekins et al 2000b) and approaches for drug
metabolism (Ekins Ring Grace McRobie-Belle amp
Wrighton 2000a) have been described Both of these
reviews commented on moving HT assays for ADME
Tox much earlier in drug discovery which had also been
indicated by other groups The initial reviews also
highlighted the likely wealth of data that would become
available and how this could be used for structure
activity relationships alongside the bioactivity data in
computational models It was noted that there was a
paucity of predictive metabolism tools at that time In
addition a growing number of efforts to model whole
cells and organs now a field called systems biology
were recognized as models that could be integrated with
the in silico ADME approaches In summary since these
past reviews virtually all pharmaceutical companies have
attempted earlier high throughput screening for ADME
Tox properties and to some extent the wider application
of computational approaches for physicochemical proper-
ties Systems biology is being quite widely acknowledged
as the new paradigm for understanding complex bio-
logical datasets derived from high throughput technolo-
gies and the accumulated knowledge on human protein
interactions (Hartwell Hopfield Leibler amp Murray 1999
Hood 2003b) Therefore systems biology can be defined
as the integration of genetic proteomic transcriptomic
and metabonomic data using computational methods
(Nicholson amp Wilson 2003) When taken together
information on molecular processes derived from different
sources represents a lsquolsquouniversersquorsquo of putative biological
functionality of which only a small fraction of it will be
realized in a cell at any given time To date systems
biology has been driven by academia and funding bodies
such as the NIH rather than the pharmaceutical compa-
nies Presently there is a great deal of interest from
scientists of all backgrounds in identifying the networks
of cellular pathways and the corresponding physically
interacting proteins
The network building software for systems biology
described in this current review will be valuable to query
high throughput data and known literature interactions in
order to predict potential toxicity in different species In the
future the compilation of published toxicogenomics data-
sets characteristic of different types of toxicity will likely
be available in these software systems to act as a reference
database It is also feasible that we will be able to generate
the annotated datasets which specifically address the
differences between human and rat networks implicated
in toxicity The identification of sub-network modules
conserved between human and rat distinct for toxicity
types or predictive for toxic end-points in human will be
possible Such signature gene networks (Nikolsky et al
2005) can then be verified with other experimentally
derived data prospectively or from preexisting databases
The combined Fhigh throughput datandashin silico network_approaches therefore represents a novel method for access-
ing complex tissue-level processes as networks integrating
all data types based on functional interactions The data-
bases developed and used in this approach will certainly
benefit from further annotation around the drug metaboliz-
ing enzymes and transporters as described herein in terms
of transcriptional regulation and the ligands associated with
them which frequently appear in the literature It will be
important to capture disparities in the assignment of ligands
to enzymes transporters and other proteins as well as
negative data
In summary although there have been numerous
toxicogenomics studies published there is presently a
relatively small number of datasets that are freely available
to perform network analysis of microarray data The
number of studies identifying large numbers of proteins
which are affected by molecule treatment are even scarcer
still while there are several examples of some published
studies that combine such data It is hoped that the
numerous database initiatives for high content and toxicol-
ogy data that are being undertaken will improve the
situation for other researchers that are not currently
equipped to do such microarray studies themselves The
addition of requirements by journals to deposit such raw
data in a freely accessible resource will aid these initiatives
ADMETox groups have seen new technologies and
approaches developed over the last decade that have all
been applied to identify poor compounds earlier (Fig 2)
The latest technologies integrate network building tools
with high content data and databases The current review
described the limited number of networks generated for
ADMETox at present and one hopes that the impact of
such analyses will be commonplace in the future Systems
biology is however more than just applying a network
approach and hence systems-ADMETox will have to
evolve due to the continual pressure to develop newer
technologies This current paradigm combining empirical
data and computational methods should integrate the
complex data already generated making it readily inter-
pretable and valuable for identifying the most promising
compounds in the future
Acknowledgements
Dr Maggie AZ Hupcey is gratefully acknowledged for
editorial assistance Dr Peter W Swaan (University of
Maryland) and Dr Cheng Chang (Ohio State University)
Dr Steve Wright (University of Arizona) Dr K Sandy
Pang (University of Toronto) and Dr Craig Giroux (Wayne
State University) are kindly acknowledged for their support
and discussions My colleagues at GeneGo Sergey
Andreyev Andy Ryabov Eugene Kirillov Eugene A
Rakhmatulin Svetlana Sorokina Andrej Bugrim Tatiana
Nikolskaya Yuri Nikolsky John Metz and Julie Bryant are
all thanked for their considerable contributions to software
development and data annotation
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 61
References
Abe M Yamashita S Kuramoto T Hirayama Y Tsukamoto T Ohta
T et al (2003) Global expression analysis of N-methyl-NV-nitro-N-nitrosoguanidine-induced rat stomach carcinomas using oligonucleotide
microarrays Carcinogenesis 24 861ndash867
Adachi T Ono Y Koh K B Takashima K Tainaka H Matsuno Y
et al (2004) Long-term alteration of gene expression without
morphological change in testis after neonatal exposure to genistein in
mice Toxicogenomic analysis using cDNA microarray Food and
Chemical Toxicology 42 445ndash452
Akerman G S Rosenzweig B A Domon O E McGarrity L J
Blankenship L R Tsai C A et al (2004) Gene expression profiles
and genetic damage in benzo(a)pyrene diol epoxide-exposed TK6 cells
Mutation Research 549 43ndash64
Aksenov S V Church B Dhiman A Georgieva A Sarangapani R
Helmlinger G et al (2005) An integrated approach for inference and
mechanistic modeling for advancing drug development FEBS Letters
579 1878ndash1883
Anderle P Huang Y amp Sadee W (2004) Intestinal membrane transport
of drugs and nutrients Genomics of membrane transporters using
expression microarrays European Journal of Pharmaceutical Sciences
21 17ndash24
Annereau J P Szakacs G Tucker C J Arciello A Cardarelli C
Collins J et al (2004) Analysis of ATP-binding cassette transporter
expression in drug-selected cell lines by a microarray dedicated to
multidrug resistance Molecular Pharmacology 66 1397ndash1405
Apic G Ignjatovic T Boyer S amp Russell R B (2005) Illuminating drug
discovery with biological pathways FEBS Letters 579 1872ndash1877
Baker V A Harries H M Waring J F Duggan C M Ni H A Jolly
R A et al (2004) Clofibrate-induced gene expression changes in rat
liver A cross-laboratory analysis using membrane cDNA arrays
Environmental Health Perspectives 112 428ndash438
Balakin K V Ekins S Bugrim A Ivanenkov Y A Korolev D amp
Nikolsky Y et al (2004a) Quantitative structurendashmetabolism relation-
ship modeling of the metabolic N-dealkylation rates Drug Metabolism
and Disposition 32 1111ndash1120
Balakin K V Ekins S Bugrim A Ivanenkov Y A Korolev D amp
Nikolsky Y et al (2004b) Kohonen maps for prediction of binding to
human cytochrome P450 3A4 Drug Metabolism and Disposition 32
1183ndash1189
Barabasi A -L amp Oltvai Z N (2004) Network biology Under-
standing the cellrsquos functional organization Nature Reviews Genetics
5 101ndash113
Bartosiewicz M Penn S G amp Buckpitt A (2001) Applications of gene
arrays in environmental toxicology Fingerprints of gene regulation
associated with cadmium chloride benzo(a)pyrene and trichloro-
ethylene Environmental Health Perspectives 109 71ndash74
Bertilsson G Heidrich J Svensson K Asman M Jendeberg L
Sydow-Backman M et al (1998) Identification of a human nuclear
receptor defines a new signaling pathway for CYP3A induction
Proceedings of the National Academy of Sciences of the United States
of America 95 12208ndash12213
Blumberg B Sabbagh Jr W Juguilon H Bolado Jr J van Meter
C M Ong E S et al (1998) SXR a novel steroid and xenobiotic-
sensing nuclear receptor Genes and Development 12 3195ndash3205
Borodina Y Rudik A Filimonov D Kharchevnikova N Dmitriev A
Blinova V et al (2004) A new statistical approach to predicting
aromatic hydroxylation sites Comparison with model-based
approaches Journal of Chemical Information and Computer Sciences
44 1998ndash2009
Borodina Y Sadym A Filimonov D Blinova V Dmitriev A amp
Poroikov V (2003) Predicting biotransformation potential from
molecular structure Journal of Chemical Information and Computer
Sciences 43 1636ndash1646
Bottone Jr F G Martinez J M Collins J B Afshari C A amp Eling
T E (2003) Gene modulation by the cyclooxygenase inhibitor
sulindac sulfide in human colorectal carcinoma cells Possible link to
apoptosis Journal of Biological Chemistry 278 25790ndash25801
Boyer S amp Zamora I (2002) New methods in predictive metabolism
Journal of Computer-Aided Molecular Design 16 403ndash413
Bredel M amp Jacoby E (2004) Chemogenomics An emerging strategy
for rapid target and drug discovery Nature Reviews Genetics 5
262ndash275
Bugrim A Nikolskaya T amp Nikolsky Y (2004) Early prediction of drug
metabolism and toxicity Systems biology approach and modeling
Drug Discovery Today 9 127ndash135
Bulera S J Eddy S M Ferguson E Jatkoe T A Reindel J F
Bleavins M R et al (2001) RNA expression in the early character-
ization of hepatotoxicants in Wistar rats by high-density DNA micro-
arrays Hepatology 33 1239ndash1258
Butcher E C Berg E L amp Kunkel E J (2004) Systems biology in drug
discovery Nature Biotechnology 22 1253ndash1259
Butte A (2002) The use and analysis of microarray data Naturalist
Review of Drug Discovery 1 951ndash960
Cary M P Bader G D amp Sander C (2005) Pathway information for
systems biology FEBS Letters 579 1815ndash1820
Cheok M H Yang W Pui C H Downing J R Cheng C Naeve
C W et al (2003) Treatment-specific changes in gene expression
discriminate in vivo drug response in human leukemia cells Nature
Genetics 34 85ndash90
Cleary J D Rogers P D amp Chapman S W (2001) Differential
transcription factor expression in human mononuclear cells in response
to amphotericin B Identification with complementary DNA microarray
technology Pharmacotherapy 21 1046ndash1054
Coen M Ruepp S U Lindon J C Nicholson J K Pognan F Lenz
E M et al (2004) Integrated application of transcriptomics and
metabonomics yields new insight into the toxicity due to paracetamol in
the mouse Journal of Pharmaceutical and Biomedical Analysis 35
93ndash105
Cornwell P D De Souza A T amp Ulrich R G (2004) Profiling of
hepatic gene expression in rats treated with fibric acid analogs
Mutation Research 549 131ndash145
Csermely P Agoston V amp Pongor S (2005) The efficiency of multi-
target drugs The network approach might help drug design Trends in
Pharmacological Sciences 26 178ndash182
Cunningham M J Liang S Fuhrman S Seilhamer J J amp
Somogyi R (2000) Gene expression microarray data analysis for
toxicology profiling Annals of the New York Academy of Sciences 919
52ndash67
de Longueville F Atienzar F A Marcq L Dufrane S Evrard S
Wouters L et al (2003) Use of a low density microarray for studying
gene expression patterns induced by hepatotoxicants on primary
cultures of rat hepatocytes Toxicological Sciences 75 378ndash392
Dobrin R Beg Q K Barabasi A L amp Oltvai Z N (2004)
Aggregation of topological motifs in the Escherichia coli transcriptional
regulatory network BMC Bioinformatics 5 10
Donald S Verschoyle R D Edwards R Judah D J Davies R
Riley J et al (2002) Hepatobiliary damage and changes in hepatic
gene expression caused by the antitumor drug ecteinascidin-743 (ET-
743) in the female rat Cancer Research 62 4256ndash4262
Dumont M Jacquemin E DrsquoHont C Descout C Cresteil D Haouzi
D et al (1997) Expression of the liver Na+-independent organic anion
transporting polypeptide (oatp-1) in rats with bile duct ligation Journal
of Hepatology 27 1051ndash1056
Durnas C Loi C M amp Cusack B J (1990) Hepatic drug metabolism
and aging Clinical Pharmacokinetics 19 359ndash389
Eisen M B Spellman P T Brown P O amp Botstein D (1998) Cluster
analysis and display of genome-wide expression patterns Proceedings
of the National Academy of Sciences of the United States of America
95 14863ndash14868
Ekins S Andreyev S Ryabov A Kirilov E Rakhmatulin E A
Bugrim A et al (in press) Computational Prediction of Human Drug
Metabolism Exp Opin Drug Metab Toxicol
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6662
Ekins S Boulanger B Swaan P W amp Hupcey M A Z (2002)
Towards a new age of virtual ADMETOX and multidimensional drug
discovery Journal of Computer-Aided Molecular Design 16 381ndash401
Ekins S Bugrim A Nikolsky Y amp Nikolskaya T (2005) Systems
biology Applications in drug discovery In S Gad (Ed) Drug
Discovery Handbook (pp 123ndash183) Hoboken NJrsquo Wiley and Sons
Ekins S Giroux C N Nikolsky Y Bugrim A amp Nikolskaya T
(2005) A signature gene network approach to toxicity The Toxoco-
logists 84
Ekins S Kirillov E Rakhmatulin E amp Nikolskaya T (2005) A novel
method for visualizing nuclear hormone receptor networks relevant to
drug metabolism Drug Metabolism and Disposition 33 474ndash481
Ekins S amp McGowan R J (2001) The limits of reductionism The
shifting genomic paradigmrsquos impact on industry and academia
Philsophy in Science 9 1ndash23
Ekins S Mirny L amp Schuetz E G (2002) A ligand-based approach to
understanding selectivity of nuclear hormone receptors PXR CAR
FXR LXRa and LXRb Pharmaceutical Research 19 1788ndash1800
Ekins S Nikolsky Y amp Nikolskaya T (2005) Techniques Application
of systems biology to absorption distribution metabolism excretion
and toxicity Trends in Pharmacological Sciences 26 202ndash209
Ekins S Ring B J Grace J McRobie-Belle D J amp Wrighton S
A (2000) Present and future in vitro approaches for drug
metabolism Journal of Pharmacological and Toxicological Methods
44 313ndash324
Ekins S Waller C L Swaan P W Cruciani G Wrighton S A amp
Wikel J H (2000) Progress in predicting human ADME parameters in
silico Journal of Pharmacological and Toxicological Methods 44
251ndash272
Erhardt P W (2003) A human drug metabolism database Potential roles
in the quantitative predictions of drug metabolism and metabolism-
related drugndashdrug interactions Current Drug Metabolism 4 411ndash422
Evans W E amp McLeod H L (2003) Pharmacogenomicsmdashdrug
disposition drug targets and side effects New England Journal of
Medicine 348 538ndash549
Ezendam J Staedtler F Pennings J Vandebriel R J Pieters R
Boffetta P et al (2004) Toxicogenomics of subchronic hexachlor-
obenzene exposure in Brown Norway rats Environmental Health
Perspectives 112 782ndash791
Faiola B Fuller E S Wong V A amp Recio L (2004) Gene expression
profile in bone marrow and hematopoietic stem cells in mice exposed to
inhaled benzene Mutation Research 549 195ndash212
FDA (2004) Innovation stagnation Challenge and opportunity on the
critical path to new medicinal products
Fiehn O (2001) Combining genomics metabolome analysis and bio-
chemical modelling to understand metabolic networks Comparative
Functional Genomics 2 155ndash168
Field F J Born E amp Mathur S N (2004) LXRRXR ligand activation
enhances basolateral efflux of beta-sitosterol in CaCo-2 cells Journal of
Lipid Research 45 905ndash913
Fleck C Sutter L Appenroth D Koch B Meinhold T Pitack M
et al (2003) Use of gene chip technology for the characterisation of
the regulation of renal transport processes and of nephrotoxicity in
rats Experimental and Toxicologic Pathology 54 401ndash410
Fliri A F Loging W T Thadeio P F amp Volkmann R A (2005)
Biological spectra analysis Linking biological activity profiles to
molecular structure Proceedings of the National Academy of Sciences
of the United States of America 102 261ndash266
Fountoulakis M (2004) Application of proteomics technologies in the
investigation of the brain Mass Spectrometry Reviews 23 231ndash258
Gaucher S P Taylor S W Fahy E Zhang B Warnock D E Ghosh
S S et al (2004) Expanded coverage of the human heart
mitochondrial proteome using multidimensional liquid chromatography
coupled with tandem mass spectrometry Journal of Proteome
Research 3 495ndash505
Gerhold D Lu M Xu J Austin C Caskey C T amp Rushmore T
(2001) Monitoring expression of genes involved in drug metabolism
and toxicology using DNA microarrays Physiological Genomics 5
161ndash170
Guo G L Choudhuri S amp Klaassen C D (2002) Induction profile of
rat organic anion transporting polypeptide 2 (oatp2) by prototypical
drug-metabolizing enzyme inducers that activate gene expression
through ligand-activated transcription factor pathways Journal of
Pharmacology and Experimental Therapeutics 300 206ndash212
Hagenbuch B amp Meier P J (2004) Organic anion transporting
polypeptides of the OATP SLC21 family Phylogenetic classification
as OATP SLCO superfamily new nomenclature and molecularfunc-
tional properties Pflugers Archiv 447 653ndash665
Hamadeh H K amp Afshari C A (2004) Toxicogenomics principles and
applications Hobokenrsquo John Wiley and Sons
Hamadeh H K Bushel P R Jayadev S Martin K DiSorbo O Sieber
S et al (2002) Gene expression analysis reveals chemical-specific
profiles Toxicological Sciences 67 219ndash231
Hamadeh H K Jayadev S Gaillard E T Huang Q Stoll R
Blanchard K et al (2004) Integration of clinical and gene expression
endpoints to explore furan-mediated hepatotoxicity Mutation Research
549 169ndash183
Hamadeh H K Knight B L Haugen A C Sieber S Amin R P
Bushel P R et al (2002) Methapyrilene toxicity Anchorage of
pathologic observations to gene expression alterations Toxicologic
Pathology 30 470ndash482
Han J D Bertin N Hao T Goldberg D S Berriz G F Zhang L V
et al (2004) Evidence for dynamically organized modularity in the
yeast proteinndashprotein interaction network Nature 430 88ndash93
Hanisch D Zien A Zimmer R amp Lengauer T (2002) Co-clustering of
biological networks and gene expression data Bioinformatics 18
S145ndashS154
Harris A J Dial S L amp Casciano D A (2004) Comparison of basal
gene expression profiles and effects of hepatocarcinogens on gene
expression in cultured primary human hepatocytes and HepG2 cells
Mutation Research 549 79ndash99
Harrison S C (2004) Whither structural biology Nature Structural
Molecular Biology 11 12ndash15
Hartley D P Dai X He Y D Carlini E J Wang B Huskey S E
et al (2004) Activators of the rat pregnane X receptor differentially
modulate hepatic and intestinal gene expression Molecular Pharma-
cology 65 1159ndash1171
Hartwell L H Hopfield J J Leibler S amp Murray A W (1999) From
molecular to modular cell biology Nature 402 C47ndashC52
Hasmall S Orphanides G James N Pennie W Hedley K Soames
A et al (2002) Downregulation of lactoferrin by PPARalpha ligands
Role in perturbation of hepatocyte proliferation and apoptosis
Toxicology Sciences 68 304ndash313
Hayase T Yamamoto Y Yamamoto K Muso E amp Shiota K (2004)
Microarray profile analysis of toxic cocaine-induced alterations in the
expression of mouse brain gene sequences A possible rsquoprotectiversquo
effect of buprenorphine Journal of Applied Toxicology 24 15ndash20
Hayes K R Vollrath A L Zastrow G M McMillan B J Craven M
Jovanovich S et al (2005) EDGE A centralized resource for the
comparison analysis and distribution of toxicogenomic information
Molecular Pharmacology 67 1360ndash1368
Heijne W H Slitt A L van Bladeren P J Groten J P Klaassen C D
Stierum R H et al (2004) Bromobenzene-induced hepatotoxicity at
the transcriptome level Toxicology Sciences 79 411ndash422
Heijne W H Stierum R H Slijper M van Bladeren P J amp van
Ommen B (2003) Toxicogenomics of bromobenzene hepatotoxicity
A combined transcriptomics and proteomics approach Biochemical
Pharmacology 65 857ndash875
Heinloth A N Irwin R D Boorman G A Nettesheim P Fannin
R D Sieber S O et al (2004) Gene expression profiling of rat
livers reveals indicators of potential adverse effects Toxicology
Sciences 80 193ndash202
Hodges L C Cook J D Lobenhofer E K Li L Bennett L Bushel
P R et al (2003) Tamoxifen functions as a molecular agonist inducing
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 63
cell cycle-associated genes in breast cancer cells Molecular Cancer
Research 1 300ndash311
Holleman A Cheok M H den Boer M L Yang W Veerman KM
A J Kazemier et al (2004) Gene-expression patterns in drug-resistant
acute lymphoblastic leukemia cells and response to treatment New
England Journal of Medicine 351 533ndash542
Hong Y Muller U R amp Lai F (2003) Discriminating two classes of
toxicants through expression analysis of HepG2 cells with DNA arrays
Toxicology in Vitro 17 85ndash92
Hood E (2003) Proteomics Characterizing the cogs in the machinery of
life Environmental Health Perspectives 111 A816ndashA825
Hood L (2003) Systems biology Integrating technology biology and
computation Mechanism of Ageing and Development 124 9ndash16
Hood L amp Galas D (2003) The digital code of DNA Nature 421
444ndash448
Hood L amp Perlmutter R M (2004) The impact of systems approaches on
biological problems in drug discovery Nature Biotechnology 22
1215ndash1217
Hu T Gibson D P Carr G J Torontali S M Tiesman J P Chaney
J G et al (2004) Identification of a gene expression profile that
discriminates indirect-acting genotoxins from direct-acting genotoxins
Mutation Research 549 5ndash27
Huang Q Dunn II R T Jayadev S DiSorbo O Pack F D Farr S B
et al (2001) Assessment of cisplatin-induced nephrotoxicity by
microarray technology Toxicology Sciences 63 196ndash207
Huang Q Jin X Gaillard E T Knight B L Pack F D Stoltz J H et
al (2004) Gene expression profiling reveals multiple toxicity endpoints
induced by hepatotoxicants Mutation Research 549 147ndash167
Huisman M T Smit J W Crommentuyn K M Zelcer N Wiltshire
H R Beijnen J H et al (2002) Multidrug resistance protein 2
(MRP2) transports HIV protease inhibitors and transport can be
enhanced by other drugs Aids 16 2295ndash2301
Hunt C M Westerkam W R amp Stave G M (1992) Effect of age and
gender on the activity of human hepatic CYP3A Biochemical
Pharmacology 44 275ndash283
Ideker T Ozier O Schwikowski B amp Siegal A F (2002) Discovering
regulatory and signalling circuits in molecular interaction networks
Bioinformatics 18 S233ndashS240
Ideker T Thorsson V Ranish J A Christmas R Buhler J Eng J K
et al (2001) Integrated genomic and proteomic analyses of a system-
atically perturbed network Science 292 929ndash934
Iida M Anna C H Hartis J Bruno M Wetmore B Dubin J R et al
(2003) Changes in global gene and protein expression during early
mouse liver carcinogenesis induced by non-genotoxic model carcino-
gens oxazepam and Wyeth-14643 Carcinogenesis 24 757ndash770
Ingelman-Sundberg M (2004) Pharmacogenetics of cytochrome P450 and
its applications in drug therapy The past present and future Trends in
Pharmacological Sciences 25 193ndash200
Jeong H Mason S P Barabasi A L amp Oltvai Z N (2001) Lethality
and centrality in protein networks Nature 411 41ndash42
Jeong H Tombor B Albert R Oltvai Z N amp Barabasi A L
(2000) The large-scale organization of metabolic networks Nature
407 651ndash654
Jung D Hagenbuch B Gresh L Pontoglio M Meier P J amp Kullak-
Ublick G A (2001) Characterization of the human OATP-C
(SLC21A6) gene promoter and regulation of liver-specific OATP genes
by hepatocyte nuclear factor 1 alpha Journal of Biological Chemistry
276 37206ndash37214
Jung D amp Kullak-Ublick G A (2003) Hepatocyte nuclear factor 1
alpha A key mediator of the effect of bile acids on gene expression
Hepatology 37 622ndash631
Jung J -W Park J -S Hwang J -W Kang K -S YS L Song B
-S et al (2004) Gene expression analysis of peroxisome prolifer-
ators- and phenytoin-induced hepatotoxicity using cDNA microarray
Journal of Veterinary Medical Science 66 1329ndash1333
Kato N Shibutani M Takagi H Uneyama C Lee K -Y Takiami S
et al (2004) Gene expression profile in the livers of rats orally
administered ethinylestradiol for 28 days using a microarray technique
Toxicology 200 179ndash192
Kier L D Neft R Tang L Suizu R Cook T Onsurez K et al
(2004) Applications of microarrays with toxicologically relevant genes
(tox genes) for the evaluation of chemical toxicants in Sprague Dawley
rats in vivo and human hepatocytes in vitro Mutation Research 549
101ndash113
Kijima K Toyosawa K Yasuba M Matsuoka N Adachi T
Komiyama M et al (2004) Gene expression analysis of the rat testis
after treatment with di(2-ethylhexyl) phthalate using cDNA microarray
and real-time RT-PCR Toxicology and Applied Pharmacology 200
103ndash110
Kim R B (2003) Organic anion-transporting polypeptide (OATP) trans-
porter family and drug disposition European Journal of Clinical
Investigation 33 1ndash5
Kinirons M T amp OrsquoMahony M S (2004) Drug metabolism and ageing
British Journal of Clinical Pharmacology 57 540ndash544
Kitano H (2002a) Computational systems biology Nature 420 206ndash210
Kitano H (2002b) Systems biology A brief overview Science 295
1662ndash1664
Kliewer S A Moore J T Wade L Staudinger J L Watson M
A Jones S A et al (1998) An orphan nuclear receptor activated
by pregnanes defines a novel steroid signalling pathway Cell 92
73ndash82
Korolev D Balakin K V Nikolsky Y Kirillov E Ivanenkov Y A
Savchuk N P et al (2003) Modeling of human cytochrome p450-
mediated drug metabolism using unsupervised machine learning
approach Journal of Medicinal Chemistry 46 3631ndash3643
Kramer J A Pettit S D Amin R P Bertram T A Car B
Cunningham M et al (2004) Overview on the application of
transcription profiling using selected nephrotoxicants for toxicology
assessment Environmental Health Perspectives 112 460ndash464
Kultima K Nystrom A -M Scholz B Gustafson A -L Dencker L
amp Stigson M (2004) Valproic acid teratogenicity A toxicogenomics
approach Environmental Health Perspectives 112 1225ndash1235
Kutuzova G D amp Deluca H F (2004) Gene expression profiles in rat
intestine identify pathways for 125-dihydroxyvitamin D(3) stimulated
calcium absorption and clarify its immunomodulatory properties
Archives of Biochemistry and Biophysics 432 152ndash166
Kwak M K Wakabayashi N Itoh K Motohashi H Yamamoto M amp
Kensler T W (2003) Modulation of gene expression by cancer
chemopreventive dithiolethiones through the Keap1-Nrf2 pathway
Identification of novel gene clusters for cell survival Journal of
Biological Chemistry 278 8135ndash8145
Lahjouji K Mitchell G A amp Qureshi I A (2001) Carnitine transport
by organic cation transporters and systemic carnitine deficiency
Molecular Genetics and Metabolism 73 287ndash297
Landowski C P Sun D Foster D R Menon S S Barnett J L
Welage L S et al (2003) Gene expression in the human intestine and
correlation with oral valacyclovir pharmacokinetic parameters Journal
of Pharmacology and Experimental Therapeutics 306 778ndash786
Lee G -J Lee W -S Jeon K -S Um C -H Kim S -J Lee C -H
et al (2004) cDNA microarray gene expression analysis and
toxicological phenotype for anticancer drug Journal of Veterinary
Medical Science 66 1339ndash1345
Lee M Kwon J Kim S N Kim J E Koh W S Kim E J et al
(2003) cDNA microarray gene expression profiling of hydroxyurea
paclitaxel and p-anisidine genotoxic compounds with differing
tumorigenicity results Environmental and Molecular Mutagenesis
42 91ndash97
Li S Armstrong C M Bertin N Ge H Milstein S Boxem M et al
(2004) A map of the interactome network of the metazoan C elegans
Science 303 540ndash543
Liguori M J Anderson L M Bukofzer S McKim J Pregenzer J F
Retief J et al (2005) Microarray analysis in human hepatocytes
suggests a mechanism for hepatotoxicity induced by trovafloxacin
Hepatology 41 177ndash186
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6664
Lobenhofer E K Bennett L Cable P L Li L Bushel P R amp Afshari
C A (2002) Regulation of DNA replication fork genes by 17beta-
estradiol Molecular Endocrinology 16 1215ndash1229
Locker J Tian J Carver R Concas D Cossu C Ledda-Columbano
G M et al (2003) A common set of immediate-early response genes
in liver regeneration and hyperplasia Hepatology 38 314ndash325
Mattes W B Pettit S D Sansone S A Bushel P R amp Waters M D
(2004) Database development in toxicogenomics Issues and efforts
Environmental Health Perspectives 112 495ndash505
McMillian M Nie A Y Parker J B Leone A Bryant S Kemmerer
M et al (2004) A gene expression signature for oxidant stressreactive
metabolites in rat liver Biochemical Pharmacology 68 2249ndash2261
Meier P J amp Stieger B (2000) Molecular mechanisms in bile formation
News in Physiological Sciences 15 89ndash93
Meneses-Lorente G de Longueville F Dos Santos-Mendes S Bonnert
T P Jack A Evrard S et al (2003) An evaluation of a low-density
DNA microarray using cytochrome P450 inducers Chemical Research
in Toxicology 16 1070ndash1077
Meneses-Lorente G Guest P C Lawrence J Muniappa N Knowles
M R Skynner H A et al (2004) A proteomic investigation of drug-
induced steatosis in rat liver Chemical Research in Toxicology 17
605ndash612
Merrill C L Ni H Yoon L W Tirmenstein M A Narayanan P
Benavides G R et al (2002) Etomoxir-induced oxidative stress in
HepG2 cells detected by differential gene expression is confirmed
biochemically Toxicology Sciences 68 93ndash101
Milo R Shen-Orr S Itzkovitz S Kashtan N Chklovskii D amp Alon
U (2002) Network motifs Simple building blocks of complex
networks Science 298 824ndash827
Morgan K T Ni H Brown H R Yoon L Qualls C W Crosby L
M et al (2002) Application of cDNA microarray technology to in
vitro toxicology and the selection of genes for a real-time RT-PCR-
based screen for oxidative stress in Hep-G2 cells Toxicologic
Pathology 30 435ndash451
Mwinyi J Johne A Bauer S Roots I amp Gerloff T (2004) Evidence
for inverse effects of OATP-C (SLC21A6) 5 and 1b haplotypes on
pravastatin kinetics Clinical Pharmacology and Therapeutics 75
415ndash421
Nakata K Yukawa M Komiyama N Nakano T amp Kaminuma T
(2002) A nuclear receptor database that maps pathways to diseases
Genome Infomatics 13 515ndash516
Nicholson J K Holmes E Lindon J C amp Wilson I D (2004) The
challenges of modeling mammalian biocomplexity Nature Biotechnol-
ogy 22 1268ndash1274
Nicholson J K amp Wilson I D (2003) Understanding rsquoglobal systems
biology Metabonomics and the continuum of metabolism Nature
Reviews on Drug Discovery 2 668ndash676
Nikitin A Egorov S Daraselia N amp Mazo I (2003) Pathway studiomdash
the analysis and navigation of molecular networks Bioinformatics 19
2155ndash2157
Nikolsky Y Ekins S Nikolskaya T amp Bugrim A (2005) A novel
method for generation of signature networks as biomarkers from
complex high throughput data Toxicology Letters 158 20ndash29
Nikolsky Y Nikolskaya T amp Bugrim A (2005) Biological networks
and analysis of experimental data in drug discovery Drug Discovery
Today 10 653ndash662
Nishizato Y Ieiri I Suzuki H Kimura M Kawabata K Hirota T
et al (2003) Polymorphisms of OATP-C (SLC21A6) and OAT3
(SLC22A8) genes Consequences for pravastatin pharmacokinetics
Clinical Pharmacology and Therapeutics 73 554ndash565
Nozawa T Nakajima M Tamai I Noda K Nezu J Sai Y et al
(2002) Genetic polymorphisms of human organic anion transporters
OATP-C (SLC21A6) and OATP-B (SLC21A9) Allele frequencies in
the Japanese population and functional analysis Journal of Pharmacol-
ogy and Experimental Therapeutics 302 804ndash813
Nozawa T Sugiura S Nakajima M Goto A Yokoi T Nezu J et al
(2004) Involvement of organic anion transporting polypeptides in the
transport of troglitazone sulfate Implications for understanding trogli-
tazone hepatotoxicity Drug Metabolism and Disposition 32 291ndash294
Oliver D E Rubin D L Stuart J M Hewett M Klein T E amp
Altman R B (2002) Ontology development for a pharmacogenetics
knowledge base Package Symposion 88ndash99
Oram J F amp Lawn R M (2001) ABCA1 The gatekeeper for eliminating
excess tissue cholesterol Journal of Lipid Research 42 1173ndash1179
Oswald M Kullak-Ublick G A Paumgartner G amp Beuers U (2001)
Expression of hepatic transporters OATP-C and MRP2 in primary
sclerosing cholangitis Liver 21 247ndash253
Parsons A B Brost R I Ding H Li Z Zhang C Sheikh B et al
(2004) Integration of chemical-genetic and genetic interaction data
links bioactive compounds to cellular target pathways Nature Bio-
technology 22 62ndash69
Patki K C von Moltke L L Harmatz J S Hesse L M Court M H
amp Greenblatt D J (2004) Effect of age on in vitro triazolam
biotransformation in male human liver microsomes Journal of
Pharmacology and Experimental Therapeutics 308 874ndash879
Pereira-Leal J B Enright A J amp Ouzounis C A (2004) Detection of
functional modules from protein interaction networks Proteins 54
49ndash57
Plant N (2004) Interaction networks Coordinating responses to xeno-
biotic exposure Toxicology 202 21ndash32
Raghavendra Prasad H S Qi Z Srinivasan K N amp Gopalakrish-
nakone P (2004) Potential effects of tetrodotoxin exposure to
human glial cells postulated using microarray approach Toxicon 44
597ndash608
Reilly T P Bourdi M Brady J N Pise-Masison C A Radonovich M
F George J W et al (2001) Expression profiling of acetaminophen
liver toxicity in mice using microarray technology Biochemical and
Biophysical Research Communications 282 321ndash328
Rives A W amp Galitski T (2003) Modular organization of cellular
networks Proceedings of the National Academy of Sciences of the
United States of America 100 1128ndash1133
Rogers P D Pearson M M Cleary J D Sullivan D C amp Chapman
S W (2002) Differential expression of genes encoding immunomodu-
latory proteins in response to amphotericin B in human mononuclear
cells identified by cDNA microarray analysis Journal of Antimicrobial
Chemotherapy 50 811ndash817
Rosen M B Wilson V S Schmid J E amp Gray L E (2005) Gene
expression analysis in the ventral prostate of rats exposed to vinclozolin
or procymidone Reproductive Toxicology 19 367ndash379
Rost D Herrmann T Sauer P Schmidts H L Stieger B Meier P J
et al (2003) Regulation of rat organic anion transporters in bile salt-
induced cholestatic hepatitis Effect of ursodeoxycholate Hepatology
38 187ndash195
Rothman R B Ayestas M A Dersch C M amp Baumann M H (1999)
Aminorex fenfluramine and chlorphentermine are serotonin transporter
substrates Implications for primary pulmonary hypertension Circula-
tion 100 869ndash875
Ruepp S U Tonge R P Shaw J Wallis N amp Pognan F (2002)
Genomics and proteomics analysis of acetaminophen toxicity in mouse
liver Toxicology Sciences 65 135ndash150
Sakaeda T Nakamura T amp Okumura K (2002) MDR1 genotype-
related pharmacokinetics and pharmacodynamics Biological amp Phar-
maceutical Bulletin 25 1391ndash1400
Sakata T Anzai N Shin H J Noshiro R Hirata T Yokoyama H
et al (2004) Novel single nucleotide polymorphisms of organic cation
transporter 1 (SLC22A1) affecting transport functions Biochemical
and Biophysical Research Communications 313 789ndash793
Satlin L M Amin V amp Wolkoff A W (1997) Organic anion
transporting polypeptide mediates organic anionHCO3-exchange
Journal of Biological Chemistry 272 26340ndash26345
Satomi Y Tsuchiya W Mihara K Ota M Kasahara Y amp Akahori F
(2004) Gene expression analysis of the lung following paraquat
administration in rats using DNA microarray Journal of Toxicological
Sciences 29 91ndash100
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 65
Schrenk D Baus P R Ermel N Klein C Vorderstemann B amp
Kauffmann H M (2001) Up-regulation of transporters of the MRP
family by drugs and toxins Toxicology Letters 120 51ndash57
Segal E Shapira M Regev A Persquoer D Botstein D Koller D et al
(2003) Module networks Identifying regulatory modules and their
condition-specific regulators from gene expression data Nature
Genetics 34 166ndash176
Segal E Wang H amp Koller D (2003) Discovering molecular pathways
from protein interaction and gene expression Bioinformatics 19
i264ndash i272
Seidel S D Kan H L Stott W T Schisler M R amp Gollapudi B B
(2003) Identification of transcriptome profiles for the DNA-damaging
agents bleomycin and hydrogen peroxide in L5178Y mouse lymphoma
cells Environmental and Molecular Mutagenesis 42 19ndash25
Shan L Yu Y Schut H A J amp Snyderwine E G (2004) Susceptibility
of rats to mammary gland carcinogenesis by the food-derived
carcinogen 2-Amino-1-Methyl-6-Phenylimidazo[45-b]Pyridine (PhIP)
varies with age and is associated with the induction of differential gene
expression American Journal of Pathology 165 191ndash202
Sharom J R Bellows D S amp Tyers M (2004) From large networks to
small molecules Current Opinion in Chemical Biology 8 81ndash90
Shitara Y Hirano M Sato H amp Sugiyama Y (2004) Gemfibrozil and
its glucuronide inhibit the OATP2(OATP1B1 SLC21A6)-mediated
hepatic uptake and CYP2C8-mediated metabolism of cerivastatinmdash
Analysis of the mechanism of the clinically relevant drugndashdrug
interaction between cerivastatin and gemfibrozil Journal of Pharma-
cology and Experimental Therapeutics
Shitara Y Itoh T Sato H Li A P amp Sugiyama Y (2003) Inhibition of
transporter-mediated hepatic uptake as a mechanism for drugndashdrug
interaction between cerivastatin and cyclosporin A Journal of
Pharmacology and Experimental Therapeutics 304 610ndash616
Somogyi R Fuhrman S amp Wen X (2001) Genetic network inference in
computational models and applications to large-scale gene expression
data In M Bower amp H Bolouri (Eds) Computational modeling of
genetic and biochemical networks (pp 119ndash157) Cambridge MArsquoThe
MIT Press
Spirin V amp Mirny L A (2003) Protein complexes and functional
modules in molecular networks Proceedings of the National Academy
of Sciences of the United States of America 100 12123ndash12128
Staudinger J Liu Y Madan A Habeebu S amp Klaassen C D
(2001) Coordinate regulation of xenobiotic and bile acid homeo-
stasis by pregnane X receptor Drug Metabolism and Disposition
29 1467ndash1472
Sun L Z Ji Z L Chen X Wang J F amp Chen Y Z (2002) ADME-
AP A database of ADME associated proteins Bioinformatics 18
1699ndash1700
Suter L Babiss L E amp Wheeldon E B (2004) Toxicogenomics in
predictive toxicology in drug development Chemistry and Biology 11
161ndash171
Synold T W Dussault I amp Forman B M (2001) The orphan nuclear
receptor SXR coordinately regulates drug metabolism and efflux
Nature Medicine 7 584ndash590
Tabuchi Y amp Kondo T (2003) cDNA microarray analysis reveals chop-
10 plays a key role in Sertoli cell injury induced by bisphenol A
Biochemical and Biophysical Research Communications 305 54ndash61
Tamai I Nezu J Uchino H Sai Y Oku A Shimane M et al (2000)
Molecular identification and characterization of novel members of the
human organic anion transporter (OATP) family Biochemical and
Biophysical Research Communications 273 251ndash260
Taylor S W Fahy E Zhang B Glenn G M Warnock D E Wiley S
et al (2003) Characterization of the human heart mitochondrial
proteome Nature Biotechnology 21 281ndash286
Terasaka S Aita Y Inoue A Hayashi S Nishigaki M Aoyagi K
et al (2004) Using a customized DNA microarray for expression
profiling of the estrogen-responsive genes to evaluate estrogen activity
among natural estrogens and industrial chemicals Environmental
Health Perspectives 112 773ndash781
Thomas D M Francescutti-Verbeem D M Liu X amp Kuhn D M
(2004) Identification of differentially regulated transcripts in mouse
striatum following methamphetamine treatmentmdashan oligonucleotide
microarray approach Journal of Neurochemistry 88 380ndash393
Thomas R S Rank D R Penn S G Zastrow G M Hayes K R
Pande K et al (2001) Identification of toxicologically predictive
gene sets using cDNA microarrays Molecular Pharmacology 60
1189ndash1194
Thompson K L Afshari C A Amin R P Bertram T A Car B
Cunningham M et al (2004) Identification of platform-independent
gene expression markers of cisplatin nephrotoxicity Environmental
Health Perspectives 112 488ndash494
Tirona R G Leake B F Merino G amp Kim R B (2001)
Polymorphisms in OATP-C Identification of multiple allelic
variants associated with altered transport activity among European-
and African-Americans Journal of Biological Chemistry 276
35669ndash35675
Tirona R G Leake B F Wolkoff A W amp Kim R B (2003)
Human organic anion transporting polypeptide-C (SLC21A6) is a
major determinant of the rifampin-mediated pregnane X receptor
activation Journal of Pharmacology and Experimental Therapeutics
304 223ndash228
Tong W Cao X Harris S Sun H Fang H Fuscoe J et al (2003)
ArrayTrackmdashsupporting toxicogenomic research at the US Food and
Drug Administration National Center for Toxicological Research
Environmental Health Perspectives 111 1819ndash1826
Tornow S amp Mewes H W (2003) Functional modules by relating
protein interaction networks and gene expression Nucleic Acids
Research 31 6283ndash6289
Trocho C Escola-Gil J C Ribas V Benitez S Martin-Campos J M
Rotllan N et al (2004) Phenytoin treatment reduces atherosclerosis in
mice through mechanisms independent of plasma HDL-cholesterol
concentration Atherosclerosis 174 275ndash285
Ueda A Hamadeh H K Webb H K Yamamoto Y Sueyoshi T
Afshari C A et al (2002) Diverse roles of the nuclear orphan
receptor CAR in regulating hepatic genes in response to phenobarbital
Molecular Pharmacology 61 1ndash6
Ulrich R G (2003) The toxicogenomics of nuclear receptor agonists
Current Opinion in Chemical Biology 7 505ndash510
Ulrich R G Rockett J C Gibson G G amp Pettit S D (2004)
Overview of an interlaboratory collaboration on evaluating the effects
of model hepatotoxicants on hepatic gene expression Environmental
Health Perspectives 112 423ndash427
Unami A Shinohara Y Ichikawa T amp Baba Y (2003) Biochemical
and microarray analyses of bupivacaine-induced apoptosis Journal of
Toxicological Sciences 28 77ndash94
Vasquez A Flammini A Maritan A amp Vespignani A (2003) Global
protein function prediction from proteinndashprotein interaction networks
Nature Biotechnology 21 697ndash700
Vezina C M Walker N J amp Olson J R (2004) Subchronic Exposure to
TCDD PeCDF PCB126 and PCB153 Effect on hepatic gene
expression Environmental Health Perspectives 112 1636ndash1644
Vo T D Greenberg H J amp Palsson B O (2004) Reconstruction and
functional characterization of the human mitochondrial metabolic
network based on proteomic and biochemical data Journal of Bio-
logical Chemistry 279 39532ndash39540
Wang H amp LeCluyse E L (2003) Role of orphan nuclear receptors in
the regulation of drug-metabolising enzymes Clinical Pharmacoki-
netics 42 1331ndash1357
Wang N Silver D L Thiele C amp Tall A R (2001) ATP-
binding cassette transporter A1 (ABCA1) functions as a choles-
terol efflux regulatory protein Journal of Biological Chemistry 276
23742ndash23747
Waring J F Cavet G Jolly R A McDowell J Dai H Ciurlionis R
et al (2003) Development of a DNA Microarray for toxicology based
on hepatoxin-regulated sequences Environmental Health Perspectives
111 863ndash870
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6666
Waring J F Gum R Morfitt D Jolly R A Ciurlionis R Heindel M
et al (2002) Identifying toxic mechanisms using DNA microarrays
Evidence that an experimental inhibitor of cell adhesion molecule
expression signals through the aryl hydrocarbon nuclear receptor
Toxicology 181ndash182 537ndash550
Waring J F Jolly R A Ciurlionis R Lum P Y Praestgaard J T
Morfitt D C et al (2001) Clustering of hepatotoxins based on
mechanism of toxicity using gene expression profiles Toxicology and
Applied Pharmacology 175 28ndash42
Waters M Boorman G Bushel P Cunningham M Irwin R Merrick
A et al (2003) Systems toxicology and the Chemical Effects in
Biological Systems (CEBS) knowledge base EHP Toxicogenomics
111 15ndash28
Wei M Arnold L Cano M amp Cohen S M (2005) Effects of co-
administration of antioxidants and arsenicals on the rat urinary bladder
epithelium Toxicology Sciences 83 237ndash245
Weinshilboum R (2003) Inheritance and drug response New England
Journal of Medicine 348 529ndash537
Weinshilboum R amp Wang L (2004) Pharmacogenetics Inherited
variation in amino acid sequence and altered protein quantity Clinical
Pharmacology and Therapeutics 75 253ndash258
Werner E (2003) In silico multicellular systems biology and minimal
genomes Drug Discovery Today 8 1121ndash1127
Wong J S amp Gill S S (2002) Gene expression changes induced in
mouse liver by di(2-ethylhexyl) phthalate Toxicology and Applied
Pharmacology 185 180ndash196
Xie T Tong L McCann U D Yuan J Becker K G Mechan A O
et al (2004) Identification and characterization of metallothionein-1
and -2 gene expression in the context of ()34-methylenedioxymetham-
phetamine-induced toxicity to brain dopaminergic neurons Journal of
Neuroscience 24 7043ndash7050
Yadetie F Laegreid A Bakke I Kusnierczyk W Komorowski J
Waldum H L et al (2003) Liver gene expression in rats in response
to the peroxisome proliferator-activated receptor-alpha agonist ciprofi-
brate Physiological Genomics 15 9ndash19
Yamashita S Nomoto T Abe M Tatematsu M Sugimura T amp
Ushijima T (2004) Persistence of gene expression changes in stomach
mucosae induced by short-term N-methyl-NV-nitro-N-nitrosoguanidinetreatment and their presence in stomach cancers Mutation Research
549 185ndash193
Yan Q amp Sadee W (2000) Human membrane transporter database A
web-accessible relational database for drug transport studies and
pharmacogenomics AAPS Pharmsci 2 E20
Yeger-Lotem E amp Margalit H (2003) Detection of regulatory circuits by
integrating the cellular networks of proteinndashprotein interactions and
transcription regulation Nucleic Acids Research 31 6053ndash6061
Yoon B I Li G -X Kitada K Kawasaki Y Igarashi K amp Kodama
Y et al (2003) Mechanisms of benzene-induced hematotoxicity and
leukemogenicity cDNA microarray analyses using mouse bone marrow
tissue Environmental Health Perspectives 111 1411ndash1420
Young M B DiSilvestro M R Sendera T J Freund J Kriete A amp
Magnuson S R (2003) Analysis of gene expression in carbon
tetrachloride-treated rat livers using a novel bioarray technology
Pharmacogenomics Journal 3 41ndash52
Yu H Zhu X Greenbaum D Karro J amp Gerstein M (2004) TopNet
A tool for comparing biological sub-networks correlating protein
properties with topological statistics Nucleic Acids Research 32
328ndash337
Zhang E Y Fu D J Pak Y A Stewart T Mukhopadhyay N
Wrighton S A et al (2004) Genetic polymorphisms in human proton-
dependent dipeptide transporter PEPT1 Implications for the functional
role of Pro586 Journal of Pharmacology and Experimental Therapeu-
tics 310 437ndash445
Zhang E Y Knipp G T Ekins S amp Swaan P W (2002)
Structural biology and function of solute transporters Implications
for identifying and designing substrates Drug Metabolism Reviews
34 709ndash750
Zhang E Y Phelps M A Cheng C Ekins S amp Swaan P W (2002)
Modeling of active transport systems Advanced Drug Delivery
Reviews 54 329ndash354
- Systems-ADMETox Resources and network approaches
-
- Introduction
- Data available
- Network analysis and databases
-
- Network applications
-
- The role of transporters
-
- Clinical relevance of transporters
- Transporter network examples ABCA1
- Transporter network examples OATP
- Transporter microarray data
-
- Applications to enzymes
- Future network applications
-
- Discussion
- Acknowledgements
- References
-
Fig7(continued)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6658
Fig 8 The network of gene interactions around the CYP3A4 enzyme (centre) from MetaCorei Information on the type of interaction between objects is
hidden for clarity eg unspecified allosteric regulation binding cleavage competition covalent modification dephosphorylation phosphorylation
transcription regulation transformation When applicable interactions also have a positive or negative effect and direction Ligands (purple) linked to other
proteins (blue blobs) transfactors (red) enzymes (orange) (For interpretation of the references to colour in this figure legend the reader is referred to the web
version of this article)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 59
the initial doses of drugs metabolized by these enzymes
should be reduced in older patients according to the clinical
response In most published studies the elderly appear at
least as responsive as the young to inducers or inhibitors of
P450s (Durnas Loi amp Cusack 1990) More recently there
has been some suggestion that there are age related
reductions in function of some specific P450s such as
CYP3A4 (Patki et al 2004) and this could occur at the
level of regulation However earlier studies with the same
enzyme showed no change in clearance with age (Hunt et
al 1992) To date there has been even less examination of
the transporter functions and any changes with age
(Kinirons amp OrsquoMahony 2004) so the current understand-
ing of the effects of aging on metabolism and transport is
anything but transparent This represents an extreme
challenge for the pharmaceutical industry how to predict
whether a drug has an affinity for an enzyme or transporter
and whether this may be clinically important if it shows a
decline in expression or function with aging Any decrease
of function of these enzymes or transporters in an elderly
population would result in changes in the metabolism and
transport of metabolites potentially impacting the clinical
effect This represents another example of how computa-
tional approaches may perhaps be used with different types
of predicted and experimental data to allow visualization of
potential compound interactions or toxicity in elderly
populations We may see different gene networks high-
lighted as humans age and these may be modified by drug
treatment and coadministration This represents an area
were network analysis could be applied in the future and is
in need of considerable further research
The collection of microarray data in databases such as
CEBS ArrayTrack and EDGE represents a future resource
for computational gene network analysis One could
envisage that ultimately in each case such data is converted
to one or more networks that are also displayed for the user
and can be used to compare treatments from in vivo and in
vitro experiments This would represent a different approach
to clustering the data as currently implemented in one of
these efforts (Hayes et al 2005) and may condense large
amounts of experimentally derived data into a readily
interpreted network
4 Discussion
Previously in this journal the progress of many
research groups in predicting human ADME parameters
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6660
in silico (Ekins et al 2000b) and approaches for drug
metabolism (Ekins Ring Grace McRobie-Belle amp
Wrighton 2000a) have been described Both of these
reviews commented on moving HT assays for ADME
Tox much earlier in drug discovery which had also been
indicated by other groups The initial reviews also
highlighted the likely wealth of data that would become
available and how this could be used for structure
activity relationships alongside the bioactivity data in
computational models It was noted that there was a
paucity of predictive metabolism tools at that time In
addition a growing number of efforts to model whole
cells and organs now a field called systems biology
were recognized as models that could be integrated with
the in silico ADME approaches In summary since these
past reviews virtually all pharmaceutical companies have
attempted earlier high throughput screening for ADME
Tox properties and to some extent the wider application
of computational approaches for physicochemical proper-
ties Systems biology is being quite widely acknowledged
as the new paradigm for understanding complex bio-
logical datasets derived from high throughput technolo-
gies and the accumulated knowledge on human protein
interactions (Hartwell Hopfield Leibler amp Murray 1999
Hood 2003b) Therefore systems biology can be defined
as the integration of genetic proteomic transcriptomic
and metabonomic data using computational methods
(Nicholson amp Wilson 2003) When taken together
information on molecular processes derived from different
sources represents a lsquolsquouniversersquorsquo of putative biological
functionality of which only a small fraction of it will be
realized in a cell at any given time To date systems
biology has been driven by academia and funding bodies
such as the NIH rather than the pharmaceutical compa-
nies Presently there is a great deal of interest from
scientists of all backgrounds in identifying the networks
of cellular pathways and the corresponding physically
interacting proteins
The network building software for systems biology
described in this current review will be valuable to query
high throughput data and known literature interactions in
order to predict potential toxicity in different species In the
future the compilation of published toxicogenomics data-
sets characteristic of different types of toxicity will likely
be available in these software systems to act as a reference
database It is also feasible that we will be able to generate
the annotated datasets which specifically address the
differences between human and rat networks implicated
in toxicity The identification of sub-network modules
conserved between human and rat distinct for toxicity
types or predictive for toxic end-points in human will be
possible Such signature gene networks (Nikolsky et al
2005) can then be verified with other experimentally
derived data prospectively or from preexisting databases
The combined Fhigh throughput datandashin silico network_approaches therefore represents a novel method for access-
ing complex tissue-level processes as networks integrating
all data types based on functional interactions The data-
bases developed and used in this approach will certainly
benefit from further annotation around the drug metaboliz-
ing enzymes and transporters as described herein in terms
of transcriptional regulation and the ligands associated with
them which frequently appear in the literature It will be
important to capture disparities in the assignment of ligands
to enzymes transporters and other proteins as well as
negative data
In summary although there have been numerous
toxicogenomics studies published there is presently a
relatively small number of datasets that are freely available
to perform network analysis of microarray data The
number of studies identifying large numbers of proteins
which are affected by molecule treatment are even scarcer
still while there are several examples of some published
studies that combine such data It is hoped that the
numerous database initiatives for high content and toxicol-
ogy data that are being undertaken will improve the
situation for other researchers that are not currently
equipped to do such microarray studies themselves The
addition of requirements by journals to deposit such raw
data in a freely accessible resource will aid these initiatives
ADMETox groups have seen new technologies and
approaches developed over the last decade that have all
been applied to identify poor compounds earlier (Fig 2)
The latest technologies integrate network building tools
with high content data and databases The current review
described the limited number of networks generated for
ADMETox at present and one hopes that the impact of
such analyses will be commonplace in the future Systems
biology is however more than just applying a network
approach and hence systems-ADMETox will have to
evolve due to the continual pressure to develop newer
technologies This current paradigm combining empirical
data and computational methods should integrate the
complex data already generated making it readily inter-
pretable and valuable for identifying the most promising
compounds in the future
Acknowledgements
Dr Maggie AZ Hupcey is gratefully acknowledged for
editorial assistance Dr Peter W Swaan (University of
Maryland) and Dr Cheng Chang (Ohio State University)
Dr Steve Wright (University of Arizona) Dr K Sandy
Pang (University of Toronto) and Dr Craig Giroux (Wayne
State University) are kindly acknowledged for their support
and discussions My colleagues at GeneGo Sergey
Andreyev Andy Ryabov Eugene Kirillov Eugene A
Rakhmatulin Svetlana Sorokina Andrej Bugrim Tatiana
Nikolskaya Yuri Nikolsky John Metz and Julie Bryant are
all thanked for their considerable contributions to software
development and data annotation
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 61
References
Abe M Yamashita S Kuramoto T Hirayama Y Tsukamoto T Ohta
T et al (2003) Global expression analysis of N-methyl-NV-nitro-N-nitrosoguanidine-induced rat stomach carcinomas using oligonucleotide
microarrays Carcinogenesis 24 861ndash867
Adachi T Ono Y Koh K B Takashima K Tainaka H Matsuno Y
et al (2004) Long-term alteration of gene expression without
morphological change in testis after neonatal exposure to genistein in
mice Toxicogenomic analysis using cDNA microarray Food and
Chemical Toxicology 42 445ndash452
Akerman G S Rosenzweig B A Domon O E McGarrity L J
Blankenship L R Tsai C A et al (2004) Gene expression profiles
and genetic damage in benzo(a)pyrene diol epoxide-exposed TK6 cells
Mutation Research 549 43ndash64
Aksenov S V Church B Dhiman A Georgieva A Sarangapani R
Helmlinger G et al (2005) An integrated approach for inference and
mechanistic modeling for advancing drug development FEBS Letters
579 1878ndash1883
Anderle P Huang Y amp Sadee W (2004) Intestinal membrane transport
of drugs and nutrients Genomics of membrane transporters using
expression microarrays European Journal of Pharmaceutical Sciences
21 17ndash24
Annereau J P Szakacs G Tucker C J Arciello A Cardarelli C
Collins J et al (2004) Analysis of ATP-binding cassette transporter
expression in drug-selected cell lines by a microarray dedicated to
multidrug resistance Molecular Pharmacology 66 1397ndash1405
Apic G Ignjatovic T Boyer S amp Russell R B (2005) Illuminating drug
discovery with biological pathways FEBS Letters 579 1872ndash1877
Baker V A Harries H M Waring J F Duggan C M Ni H A Jolly
R A et al (2004) Clofibrate-induced gene expression changes in rat
liver A cross-laboratory analysis using membrane cDNA arrays
Environmental Health Perspectives 112 428ndash438
Balakin K V Ekins S Bugrim A Ivanenkov Y A Korolev D amp
Nikolsky Y et al (2004a) Quantitative structurendashmetabolism relation-
ship modeling of the metabolic N-dealkylation rates Drug Metabolism
and Disposition 32 1111ndash1120
Balakin K V Ekins S Bugrim A Ivanenkov Y A Korolev D amp
Nikolsky Y et al (2004b) Kohonen maps for prediction of binding to
human cytochrome P450 3A4 Drug Metabolism and Disposition 32
1183ndash1189
Barabasi A -L amp Oltvai Z N (2004) Network biology Under-
standing the cellrsquos functional organization Nature Reviews Genetics
5 101ndash113
Bartosiewicz M Penn S G amp Buckpitt A (2001) Applications of gene
arrays in environmental toxicology Fingerprints of gene regulation
associated with cadmium chloride benzo(a)pyrene and trichloro-
ethylene Environmental Health Perspectives 109 71ndash74
Bertilsson G Heidrich J Svensson K Asman M Jendeberg L
Sydow-Backman M et al (1998) Identification of a human nuclear
receptor defines a new signaling pathway for CYP3A induction
Proceedings of the National Academy of Sciences of the United States
of America 95 12208ndash12213
Blumberg B Sabbagh Jr W Juguilon H Bolado Jr J van Meter
C M Ong E S et al (1998) SXR a novel steroid and xenobiotic-
sensing nuclear receptor Genes and Development 12 3195ndash3205
Borodina Y Rudik A Filimonov D Kharchevnikova N Dmitriev A
Blinova V et al (2004) A new statistical approach to predicting
aromatic hydroxylation sites Comparison with model-based
approaches Journal of Chemical Information and Computer Sciences
44 1998ndash2009
Borodina Y Sadym A Filimonov D Blinova V Dmitriev A amp
Poroikov V (2003) Predicting biotransformation potential from
molecular structure Journal of Chemical Information and Computer
Sciences 43 1636ndash1646
Bottone Jr F G Martinez J M Collins J B Afshari C A amp Eling
T E (2003) Gene modulation by the cyclooxygenase inhibitor
sulindac sulfide in human colorectal carcinoma cells Possible link to
apoptosis Journal of Biological Chemistry 278 25790ndash25801
Boyer S amp Zamora I (2002) New methods in predictive metabolism
Journal of Computer-Aided Molecular Design 16 403ndash413
Bredel M amp Jacoby E (2004) Chemogenomics An emerging strategy
for rapid target and drug discovery Nature Reviews Genetics 5
262ndash275
Bugrim A Nikolskaya T amp Nikolsky Y (2004) Early prediction of drug
metabolism and toxicity Systems biology approach and modeling
Drug Discovery Today 9 127ndash135
Bulera S J Eddy S M Ferguson E Jatkoe T A Reindel J F
Bleavins M R et al (2001) RNA expression in the early character-
ization of hepatotoxicants in Wistar rats by high-density DNA micro-
arrays Hepatology 33 1239ndash1258
Butcher E C Berg E L amp Kunkel E J (2004) Systems biology in drug
discovery Nature Biotechnology 22 1253ndash1259
Butte A (2002) The use and analysis of microarray data Naturalist
Review of Drug Discovery 1 951ndash960
Cary M P Bader G D amp Sander C (2005) Pathway information for
systems biology FEBS Letters 579 1815ndash1820
Cheok M H Yang W Pui C H Downing J R Cheng C Naeve
C W et al (2003) Treatment-specific changes in gene expression
discriminate in vivo drug response in human leukemia cells Nature
Genetics 34 85ndash90
Cleary J D Rogers P D amp Chapman S W (2001) Differential
transcription factor expression in human mononuclear cells in response
to amphotericin B Identification with complementary DNA microarray
technology Pharmacotherapy 21 1046ndash1054
Coen M Ruepp S U Lindon J C Nicholson J K Pognan F Lenz
E M et al (2004) Integrated application of transcriptomics and
metabonomics yields new insight into the toxicity due to paracetamol in
the mouse Journal of Pharmaceutical and Biomedical Analysis 35
93ndash105
Cornwell P D De Souza A T amp Ulrich R G (2004) Profiling of
hepatic gene expression in rats treated with fibric acid analogs
Mutation Research 549 131ndash145
Csermely P Agoston V amp Pongor S (2005) The efficiency of multi-
target drugs The network approach might help drug design Trends in
Pharmacological Sciences 26 178ndash182
Cunningham M J Liang S Fuhrman S Seilhamer J J amp
Somogyi R (2000) Gene expression microarray data analysis for
toxicology profiling Annals of the New York Academy of Sciences 919
52ndash67
de Longueville F Atienzar F A Marcq L Dufrane S Evrard S
Wouters L et al (2003) Use of a low density microarray for studying
gene expression patterns induced by hepatotoxicants on primary
cultures of rat hepatocytes Toxicological Sciences 75 378ndash392
Dobrin R Beg Q K Barabasi A L amp Oltvai Z N (2004)
Aggregation of topological motifs in the Escherichia coli transcriptional
regulatory network BMC Bioinformatics 5 10
Donald S Verschoyle R D Edwards R Judah D J Davies R
Riley J et al (2002) Hepatobiliary damage and changes in hepatic
gene expression caused by the antitumor drug ecteinascidin-743 (ET-
743) in the female rat Cancer Research 62 4256ndash4262
Dumont M Jacquemin E DrsquoHont C Descout C Cresteil D Haouzi
D et al (1997) Expression of the liver Na+-independent organic anion
transporting polypeptide (oatp-1) in rats with bile duct ligation Journal
of Hepatology 27 1051ndash1056
Durnas C Loi C M amp Cusack B J (1990) Hepatic drug metabolism
and aging Clinical Pharmacokinetics 19 359ndash389
Eisen M B Spellman P T Brown P O amp Botstein D (1998) Cluster
analysis and display of genome-wide expression patterns Proceedings
of the National Academy of Sciences of the United States of America
95 14863ndash14868
Ekins S Andreyev S Ryabov A Kirilov E Rakhmatulin E A
Bugrim A et al (in press) Computational Prediction of Human Drug
Metabolism Exp Opin Drug Metab Toxicol
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6662
Ekins S Boulanger B Swaan P W amp Hupcey M A Z (2002)
Towards a new age of virtual ADMETOX and multidimensional drug
discovery Journal of Computer-Aided Molecular Design 16 381ndash401
Ekins S Bugrim A Nikolsky Y amp Nikolskaya T (2005) Systems
biology Applications in drug discovery In S Gad (Ed) Drug
Discovery Handbook (pp 123ndash183) Hoboken NJrsquo Wiley and Sons
Ekins S Giroux C N Nikolsky Y Bugrim A amp Nikolskaya T
(2005) A signature gene network approach to toxicity The Toxoco-
logists 84
Ekins S Kirillov E Rakhmatulin E amp Nikolskaya T (2005) A novel
method for visualizing nuclear hormone receptor networks relevant to
drug metabolism Drug Metabolism and Disposition 33 474ndash481
Ekins S amp McGowan R J (2001) The limits of reductionism The
shifting genomic paradigmrsquos impact on industry and academia
Philsophy in Science 9 1ndash23
Ekins S Mirny L amp Schuetz E G (2002) A ligand-based approach to
understanding selectivity of nuclear hormone receptors PXR CAR
FXR LXRa and LXRb Pharmaceutical Research 19 1788ndash1800
Ekins S Nikolsky Y amp Nikolskaya T (2005) Techniques Application
of systems biology to absorption distribution metabolism excretion
and toxicity Trends in Pharmacological Sciences 26 202ndash209
Ekins S Ring B J Grace J McRobie-Belle D J amp Wrighton S
A (2000) Present and future in vitro approaches for drug
metabolism Journal of Pharmacological and Toxicological Methods
44 313ndash324
Ekins S Waller C L Swaan P W Cruciani G Wrighton S A amp
Wikel J H (2000) Progress in predicting human ADME parameters in
silico Journal of Pharmacological and Toxicological Methods 44
251ndash272
Erhardt P W (2003) A human drug metabolism database Potential roles
in the quantitative predictions of drug metabolism and metabolism-
related drugndashdrug interactions Current Drug Metabolism 4 411ndash422
Evans W E amp McLeod H L (2003) Pharmacogenomicsmdashdrug
disposition drug targets and side effects New England Journal of
Medicine 348 538ndash549
Ezendam J Staedtler F Pennings J Vandebriel R J Pieters R
Boffetta P et al (2004) Toxicogenomics of subchronic hexachlor-
obenzene exposure in Brown Norway rats Environmental Health
Perspectives 112 782ndash791
Faiola B Fuller E S Wong V A amp Recio L (2004) Gene expression
profile in bone marrow and hematopoietic stem cells in mice exposed to
inhaled benzene Mutation Research 549 195ndash212
FDA (2004) Innovation stagnation Challenge and opportunity on the
critical path to new medicinal products
Fiehn O (2001) Combining genomics metabolome analysis and bio-
chemical modelling to understand metabolic networks Comparative
Functional Genomics 2 155ndash168
Field F J Born E amp Mathur S N (2004) LXRRXR ligand activation
enhances basolateral efflux of beta-sitosterol in CaCo-2 cells Journal of
Lipid Research 45 905ndash913
Fleck C Sutter L Appenroth D Koch B Meinhold T Pitack M
et al (2003) Use of gene chip technology for the characterisation of
the regulation of renal transport processes and of nephrotoxicity in
rats Experimental and Toxicologic Pathology 54 401ndash410
Fliri A F Loging W T Thadeio P F amp Volkmann R A (2005)
Biological spectra analysis Linking biological activity profiles to
molecular structure Proceedings of the National Academy of Sciences
of the United States of America 102 261ndash266
Fountoulakis M (2004) Application of proteomics technologies in the
investigation of the brain Mass Spectrometry Reviews 23 231ndash258
Gaucher S P Taylor S W Fahy E Zhang B Warnock D E Ghosh
S S et al (2004) Expanded coverage of the human heart
mitochondrial proteome using multidimensional liquid chromatography
coupled with tandem mass spectrometry Journal of Proteome
Research 3 495ndash505
Gerhold D Lu M Xu J Austin C Caskey C T amp Rushmore T
(2001) Monitoring expression of genes involved in drug metabolism
and toxicology using DNA microarrays Physiological Genomics 5
161ndash170
Guo G L Choudhuri S amp Klaassen C D (2002) Induction profile of
rat organic anion transporting polypeptide 2 (oatp2) by prototypical
drug-metabolizing enzyme inducers that activate gene expression
through ligand-activated transcription factor pathways Journal of
Pharmacology and Experimental Therapeutics 300 206ndash212
Hagenbuch B amp Meier P J (2004) Organic anion transporting
polypeptides of the OATP SLC21 family Phylogenetic classification
as OATP SLCO superfamily new nomenclature and molecularfunc-
tional properties Pflugers Archiv 447 653ndash665
Hamadeh H K amp Afshari C A (2004) Toxicogenomics principles and
applications Hobokenrsquo John Wiley and Sons
Hamadeh H K Bushel P R Jayadev S Martin K DiSorbo O Sieber
S et al (2002) Gene expression analysis reveals chemical-specific
profiles Toxicological Sciences 67 219ndash231
Hamadeh H K Jayadev S Gaillard E T Huang Q Stoll R
Blanchard K et al (2004) Integration of clinical and gene expression
endpoints to explore furan-mediated hepatotoxicity Mutation Research
549 169ndash183
Hamadeh H K Knight B L Haugen A C Sieber S Amin R P
Bushel P R et al (2002) Methapyrilene toxicity Anchorage of
pathologic observations to gene expression alterations Toxicologic
Pathology 30 470ndash482
Han J D Bertin N Hao T Goldberg D S Berriz G F Zhang L V
et al (2004) Evidence for dynamically organized modularity in the
yeast proteinndashprotein interaction network Nature 430 88ndash93
Hanisch D Zien A Zimmer R amp Lengauer T (2002) Co-clustering of
biological networks and gene expression data Bioinformatics 18
S145ndashS154
Harris A J Dial S L amp Casciano D A (2004) Comparison of basal
gene expression profiles and effects of hepatocarcinogens on gene
expression in cultured primary human hepatocytes and HepG2 cells
Mutation Research 549 79ndash99
Harrison S C (2004) Whither structural biology Nature Structural
Molecular Biology 11 12ndash15
Hartley D P Dai X He Y D Carlini E J Wang B Huskey S E
et al (2004) Activators of the rat pregnane X receptor differentially
modulate hepatic and intestinal gene expression Molecular Pharma-
cology 65 1159ndash1171
Hartwell L H Hopfield J J Leibler S amp Murray A W (1999) From
molecular to modular cell biology Nature 402 C47ndashC52
Hasmall S Orphanides G James N Pennie W Hedley K Soames
A et al (2002) Downregulation of lactoferrin by PPARalpha ligands
Role in perturbation of hepatocyte proliferation and apoptosis
Toxicology Sciences 68 304ndash313
Hayase T Yamamoto Y Yamamoto K Muso E amp Shiota K (2004)
Microarray profile analysis of toxic cocaine-induced alterations in the
expression of mouse brain gene sequences A possible rsquoprotectiversquo
effect of buprenorphine Journal of Applied Toxicology 24 15ndash20
Hayes K R Vollrath A L Zastrow G M McMillan B J Craven M
Jovanovich S et al (2005) EDGE A centralized resource for the
comparison analysis and distribution of toxicogenomic information
Molecular Pharmacology 67 1360ndash1368
Heijne W H Slitt A L van Bladeren P J Groten J P Klaassen C D
Stierum R H et al (2004) Bromobenzene-induced hepatotoxicity at
the transcriptome level Toxicology Sciences 79 411ndash422
Heijne W H Stierum R H Slijper M van Bladeren P J amp van
Ommen B (2003) Toxicogenomics of bromobenzene hepatotoxicity
A combined transcriptomics and proteomics approach Biochemical
Pharmacology 65 857ndash875
Heinloth A N Irwin R D Boorman G A Nettesheim P Fannin
R D Sieber S O et al (2004) Gene expression profiling of rat
livers reveals indicators of potential adverse effects Toxicology
Sciences 80 193ndash202
Hodges L C Cook J D Lobenhofer E K Li L Bennett L Bushel
P R et al (2003) Tamoxifen functions as a molecular agonist inducing
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 63
cell cycle-associated genes in breast cancer cells Molecular Cancer
Research 1 300ndash311
Holleman A Cheok M H den Boer M L Yang W Veerman KM
A J Kazemier et al (2004) Gene-expression patterns in drug-resistant
acute lymphoblastic leukemia cells and response to treatment New
England Journal of Medicine 351 533ndash542
Hong Y Muller U R amp Lai F (2003) Discriminating two classes of
toxicants through expression analysis of HepG2 cells with DNA arrays
Toxicology in Vitro 17 85ndash92
Hood E (2003) Proteomics Characterizing the cogs in the machinery of
life Environmental Health Perspectives 111 A816ndashA825
Hood L (2003) Systems biology Integrating technology biology and
computation Mechanism of Ageing and Development 124 9ndash16
Hood L amp Galas D (2003) The digital code of DNA Nature 421
444ndash448
Hood L amp Perlmutter R M (2004) The impact of systems approaches on
biological problems in drug discovery Nature Biotechnology 22
1215ndash1217
Hu T Gibson D P Carr G J Torontali S M Tiesman J P Chaney
J G et al (2004) Identification of a gene expression profile that
discriminates indirect-acting genotoxins from direct-acting genotoxins
Mutation Research 549 5ndash27
Huang Q Dunn II R T Jayadev S DiSorbo O Pack F D Farr S B
et al (2001) Assessment of cisplatin-induced nephrotoxicity by
microarray technology Toxicology Sciences 63 196ndash207
Huang Q Jin X Gaillard E T Knight B L Pack F D Stoltz J H et
al (2004) Gene expression profiling reveals multiple toxicity endpoints
induced by hepatotoxicants Mutation Research 549 147ndash167
Huisman M T Smit J W Crommentuyn K M Zelcer N Wiltshire
H R Beijnen J H et al (2002) Multidrug resistance protein 2
(MRP2) transports HIV protease inhibitors and transport can be
enhanced by other drugs Aids 16 2295ndash2301
Hunt C M Westerkam W R amp Stave G M (1992) Effect of age and
gender on the activity of human hepatic CYP3A Biochemical
Pharmacology 44 275ndash283
Ideker T Ozier O Schwikowski B amp Siegal A F (2002) Discovering
regulatory and signalling circuits in molecular interaction networks
Bioinformatics 18 S233ndashS240
Ideker T Thorsson V Ranish J A Christmas R Buhler J Eng J K
et al (2001) Integrated genomic and proteomic analyses of a system-
atically perturbed network Science 292 929ndash934
Iida M Anna C H Hartis J Bruno M Wetmore B Dubin J R et al
(2003) Changes in global gene and protein expression during early
mouse liver carcinogenesis induced by non-genotoxic model carcino-
gens oxazepam and Wyeth-14643 Carcinogenesis 24 757ndash770
Ingelman-Sundberg M (2004) Pharmacogenetics of cytochrome P450 and
its applications in drug therapy The past present and future Trends in
Pharmacological Sciences 25 193ndash200
Jeong H Mason S P Barabasi A L amp Oltvai Z N (2001) Lethality
and centrality in protein networks Nature 411 41ndash42
Jeong H Tombor B Albert R Oltvai Z N amp Barabasi A L
(2000) The large-scale organization of metabolic networks Nature
407 651ndash654
Jung D Hagenbuch B Gresh L Pontoglio M Meier P J amp Kullak-
Ublick G A (2001) Characterization of the human OATP-C
(SLC21A6) gene promoter and regulation of liver-specific OATP genes
by hepatocyte nuclear factor 1 alpha Journal of Biological Chemistry
276 37206ndash37214
Jung D amp Kullak-Ublick G A (2003) Hepatocyte nuclear factor 1
alpha A key mediator of the effect of bile acids on gene expression
Hepatology 37 622ndash631
Jung J -W Park J -S Hwang J -W Kang K -S YS L Song B
-S et al (2004) Gene expression analysis of peroxisome prolifer-
ators- and phenytoin-induced hepatotoxicity using cDNA microarray
Journal of Veterinary Medical Science 66 1329ndash1333
Kato N Shibutani M Takagi H Uneyama C Lee K -Y Takiami S
et al (2004) Gene expression profile in the livers of rats orally
administered ethinylestradiol for 28 days using a microarray technique
Toxicology 200 179ndash192
Kier L D Neft R Tang L Suizu R Cook T Onsurez K et al
(2004) Applications of microarrays with toxicologically relevant genes
(tox genes) for the evaluation of chemical toxicants in Sprague Dawley
rats in vivo and human hepatocytes in vitro Mutation Research 549
101ndash113
Kijima K Toyosawa K Yasuba M Matsuoka N Adachi T
Komiyama M et al (2004) Gene expression analysis of the rat testis
after treatment with di(2-ethylhexyl) phthalate using cDNA microarray
and real-time RT-PCR Toxicology and Applied Pharmacology 200
103ndash110
Kim R B (2003) Organic anion-transporting polypeptide (OATP) trans-
porter family and drug disposition European Journal of Clinical
Investigation 33 1ndash5
Kinirons M T amp OrsquoMahony M S (2004) Drug metabolism and ageing
British Journal of Clinical Pharmacology 57 540ndash544
Kitano H (2002a) Computational systems biology Nature 420 206ndash210
Kitano H (2002b) Systems biology A brief overview Science 295
1662ndash1664
Kliewer S A Moore J T Wade L Staudinger J L Watson M
A Jones S A et al (1998) An orphan nuclear receptor activated
by pregnanes defines a novel steroid signalling pathway Cell 92
73ndash82
Korolev D Balakin K V Nikolsky Y Kirillov E Ivanenkov Y A
Savchuk N P et al (2003) Modeling of human cytochrome p450-
mediated drug metabolism using unsupervised machine learning
approach Journal of Medicinal Chemistry 46 3631ndash3643
Kramer J A Pettit S D Amin R P Bertram T A Car B
Cunningham M et al (2004) Overview on the application of
transcription profiling using selected nephrotoxicants for toxicology
assessment Environmental Health Perspectives 112 460ndash464
Kultima K Nystrom A -M Scholz B Gustafson A -L Dencker L
amp Stigson M (2004) Valproic acid teratogenicity A toxicogenomics
approach Environmental Health Perspectives 112 1225ndash1235
Kutuzova G D amp Deluca H F (2004) Gene expression profiles in rat
intestine identify pathways for 125-dihydroxyvitamin D(3) stimulated
calcium absorption and clarify its immunomodulatory properties
Archives of Biochemistry and Biophysics 432 152ndash166
Kwak M K Wakabayashi N Itoh K Motohashi H Yamamoto M amp
Kensler T W (2003) Modulation of gene expression by cancer
chemopreventive dithiolethiones through the Keap1-Nrf2 pathway
Identification of novel gene clusters for cell survival Journal of
Biological Chemistry 278 8135ndash8145
Lahjouji K Mitchell G A amp Qureshi I A (2001) Carnitine transport
by organic cation transporters and systemic carnitine deficiency
Molecular Genetics and Metabolism 73 287ndash297
Landowski C P Sun D Foster D R Menon S S Barnett J L
Welage L S et al (2003) Gene expression in the human intestine and
correlation with oral valacyclovir pharmacokinetic parameters Journal
of Pharmacology and Experimental Therapeutics 306 778ndash786
Lee G -J Lee W -S Jeon K -S Um C -H Kim S -J Lee C -H
et al (2004) cDNA microarray gene expression analysis and
toxicological phenotype for anticancer drug Journal of Veterinary
Medical Science 66 1339ndash1345
Lee M Kwon J Kim S N Kim J E Koh W S Kim E J et al
(2003) cDNA microarray gene expression profiling of hydroxyurea
paclitaxel and p-anisidine genotoxic compounds with differing
tumorigenicity results Environmental and Molecular Mutagenesis
42 91ndash97
Li S Armstrong C M Bertin N Ge H Milstein S Boxem M et al
(2004) A map of the interactome network of the metazoan C elegans
Science 303 540ndash543
Liguori M J Anderson L M Bukofzer S McKim J Pregenzer J F
Retief J et al (2005) Microarray analysis in human hepatocytes
suggests a mechanism for hepatotoxicity induced by trovafloxacin
Hepatology 41 177ndash186
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6664
Lobenhofer E K Bennett L Cable P L Li L Bushel P R amp Afshari
C A (2002) Regulation of DNA replication fork genes by 17beta-
estradiol Molecular Endocrinology 16 1215ndash1229
Locker J Tian J Carver R Concas D Cossu C Ledda-Columbano
G M et al (2003) A common set of immediate-early response genes
in liver regeneration and hyperplasia Hepatology 38 314ndash325
Mattes W B Pettit S D Sansone S A Bushel P R amp Waters M D
(2004) Database development in toxicogenomics Issues and efforts
Environmental Health Perspectives 112 495ndash505
McMillian M Nie A Y Parker J B Leone A Bryant S Kemmerer
M et al (2004) A gene expression signature for oxidant stressreactive
metabolites in rat liver Biochemical Pharmacology 68 2249ndash2261
Meier P J amp Stieger B (2000) Molecular mechanisms in bile formation
News in Physiological Sciences 15 89ndash93
Meneses-Lorente G de Longueville F Dos Santos-Mendes S Bonnert
T P Jack A Evrard S et al (2003) An evaluation of a low-density
DNA microarray using cytochrome P450 inducers Chemical Research
in Toxicology 16 1070ndash1077
Meneses-Lorente G Guest P C Lawrence J Muniappa N Knowles
M R Skynner H A et al (2004) A proteomic investigation of drug-
induced steatosis in rat liver Chemical Research in Toxicology 17
605ndash612
Merrill C L Ni H Yoon L W Tirmenstein M A Narayanan P
Benavides G R et al (2002) Etomoxir-induced oxidative stress in
HepG2 cells detected by differential gene expression is confirmed
biochemically Toxicology Sciences 68 93ndash101
Milo R Shen-Orr S Itzkovitz S Kashtan N Chklovskii D amp Alon
U (2002) Network motifs Simple building blocks of complex
networks Science 298 824ndash827
Morgan K T Ni H Brown H R Yoon L Qualls C W Crosby L
M et al (2002) Application of cDNA microarray technology to in
vitro toxicology and the selection of genes for a real-time RT-PCR-
based screen for oxidative stress in Hep-G2 cells Toxicologic
Pathology 30 435ndash451
Mwinyi J Johne A Bauer S Roots I amp Gerloff T (2004) Evidence
for inverse effects of OATP-C (SLC21A6) 5 and 1b haplotypes on
pravastatin kinetics Clinical Pharmacology and Therapeutics 75
415ndash421
Nakata K Yukawa M Komiyama N Nakano T amp Kaminuma T
(2002) A nuclear receptor database that maps pathways to diseases
Genome Infomatics 13 515ndash516
Nicholson J K Holmes E Lindon J C amp Wilson I D (2004) The
challenges of modeling mammalian biocomplexity Nature Biotechnol-
ogy 22 1268ndash1274
Nicholson J K amp Wilson I D (2003) Understanding rsquoglobal systems
biology Metabonomics and the continuum of metabolism Nature
Reviews on Drug Discovery 2 668ndash676
Nikitin A Egorov S Daraselia N amp Mazo I (2003) Pathway studiomdash
the analysis and navigation of molecular networks Bioinformatics 19
2155ndash2157
Nikolsky Y Ekins S Nikolskaya T amp Bugrim A (2005) A novel
method for generation of signature networks as biomarkers from
complex high throughput data Toxicology Letters 158 20ndash29
Nikolsky Y Nikolskaya T amp Bugrim A (2005) Biological networks
and analysis of experimental data in drug discovery Drug Discovery
Today 10 653ndash662
Nishizato Y Ieiri I Suzuki H Kimura M Kawabata K Hirota T
et al (2003) Polymorphisms of OATP-C (SLC21A6) and OAT3
(SLC22A8) genes Consequences for pravastatin pharmacokinetics
Clinical Pharmacology and Therapeutics 73 554ndash565
Nozawa T Nakajima M Tamai I Noda K Nezu J Sai Y et al
(2002) Genetic polymorphisms of human organic anion transporters
OATP-C (SLC21A6) and OATP-B (SLC21A9) Allele frequencies in
the Japanese population and functional analysis Journal of Pharmacol-
ogy and Experimental Therapeutics 302 804ndash813
Nozawa T Sugiura S Nakajima M Goto A Yokoi T Nezu J et al
(2004) Involvement of organic anion transporting polypeptides in the
transport of troglitazone sulfate Implications for understanding trogli-
tazone hepatotoxicity Drug Metabolism and Disposition 32 291ndash294
Oliver D E Rubin D L Stuart J M Hewett M Klein T E amp
Altman R B (2002) Ontology development for a pharmacogenetics
knowledge base Package Symposion 88ndash99
Oram J F amp Lawn R M (2001) ABCA1 The gatekeeper for eliminating
excess tissue cholesterol Journal of Lipid Research 42 1173ndash1179
Oswald M Kullak-Ublick G A Paumgartner G amp Beuers U (2001)
Expression of hepatic transporters OATP-C and MRP2 in primary
sclerosing cholangitis Liver 21 247ndash253
Parsons A B Brost R I Ding H Li Z Zhang C Sheikh B et al
(2004) Integration of chemical-genetic and genetic interaction data
links bioactive compounds to cellular target pathways Nature Bio-
technology 22 62ndash69
Patki K C von Moltke L L Harmatz J S Hesse L M Court M H
amp Greenblatt D J (2004) Effect of age on in vitro triazolam
biotransformation in male human liver microsomes Journal of
Pharmacology and Experimental Therapeutics 308 874ndash879
Pereira-Leal J B Enright A J amp Ouzounis C A (2004) Detection of
functional modules from protein interaction networks Proteins 54
49ndash57
Plant N (2004) Interaction networks Coordinating responses to xeno-
biotic exposure Toxicology 202 21ndash32
Raghavendra Prasad H S Qi Z Srinivasan K N amp Gopalakrish-
nakone P (2004) Potential effects of tetrodotoxin exposure to
human glial cells postulated using microarray approach Toxicon 44
597ndash608
Reilly T P Bourdi M Brady J N Pise-Masison C A Radonovich M
F George J W et al (2001) Expression profiling of acetaminophen
liver toxicity in mice using microarray technology Biochemical and
Biophysical Research Communications 282 321ndash328
Rives A W amp Galitski T (2003) Modular organization of cellular
networks Proceedings of the National Academy of Sciences of the
United States of America 100 1128ndash1133
Rogers P D Pearson M M Cleary J D Sullivan D C amp Chapman
S W (2002) Differential expression of genes encoding immunomodu-
latory proteins in response to amphotericin B in human mononuclear
cells identified by cDNA microarray analysis Journal of Antimicrobial
Chemotherapy 50 811ndash817
Rosen M B Wilson V S Schmid J E amp Gray L E (2005) Gene
expression analysis in the ventral prostate of rats exposed to vinclozolin
or procymidone Reproductive Toxicology 19 367ndash379
Rost D Herrmann T Sauer P Schmidts H L Stieger B Meier P J
et al (2003) Regulation of rat organic anion transporters in bile salt-
induced cholestatic hepatitis Effect of ursodeoxycholate Hepatology
38 187ndash195
Rothman R B Ayestas M A Dersch C M amp Baumann M H (1999)
Aminorex fenfluramine and chlorphentermine are serotonin transporter
substrates Implications for primary pulmonary hypertension Circula-
tion 100 869ndash875
Ruepp S U Tonge R P Shaw J Wallis N amp Pognan F (2002)
Genomics and proteomics analysis of acetaminophen toxicity in mouse
liver Toxicology Sciences 65 135ndash150
Sakaeda T Nakamura T amp Okumura K (2002) MDR1 genotype-
related pharmacokinetics and pharmacodynamics Biological amp Phar-
maceutical Bulletin 25 1391ndash1400
Sakata T Anzai N Shin H J Noshiro R Hirata T Yokoyama H
et al (2004) Novel single nucleotide polymorphisms of organic cation
transporter 1 (SLC22A1) affecting transport functions Biochemical
and Biophysical Research Communications 313 789ndash793
Satlin L M Amin V amp Wolkoff A W (1997) Organic anion
transporting polypeptide mediates organic anionHCO3-exchange
Journal of Biological Chemistry 272 26340ndash26345
Satomi Y Tsuchiya W Mihara K Ota M Kasahara Y amp Akahori F
(2004) Gene expression analysis of the lung following paraquat
administration in rats using DNA microarray Journal of Toxicological
Sciences 29 91ndash100
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 65
Schrenk D Baus P R Ermel N Klein C Vorderstemann B amp
Kauffmann H M (2001) Up-regulation of transporters of the MRP
family by drugs and toxins Toxicology Letters 120 51ndash57
Segal E Shapira M Regev A Persquoer D Botstein D Koller D et al
(2003) Module networks Identifying regulatory modules and their
condition-specific regulators from gene expression data Nature
Genetics 34 166ndash176
Segal E Wang H amp Koller D (2003) Discovering molecular pathways
from protein interaction and gene expression Bioinformatics 19
i264ndash i272
Seidel S D Kan H L Stott W T Schisler M R amp Gollapudi B B
(2003) Identification of transcriptome profiles for the DNA-damaging
agents bleomycin and hydrogen peroxide in L5178Y mouse lymphoma
cells Environmental and Molecular Mutagenesis 42 19ndash25
Shan L Yu Y Schut H A J amp Snyderwine E G (2004) Susceptibility
of rats to mammary gland carcinogenesis by the food-derived
carcinogen 2-Amino-1-Methyl-6-Phenylimidazo[45-b]Pyridine (PhIP)
varies with age and is associated with the induction of differential gene
expression American Journal of Pathology 165 191ndash202
Sharom J R Bellows D S amp Tyers M (2004) From large networks to
small molecules Current Opinion in Chemical Biology 8 81ndash90
Shitara Y Hirano M Sato H amp Sugiyama Y (2004) Gemfibrozil and
its glucuronide inhibit the OATP2(OATP1B1 SLC21A6)-mediated
hepatic uptake and CYP2C8-mediated metabolism of cerivastatinmdash
Analysis of the mechanism of the clinically relevant drugndashdrug
interaction between cerivastatin and gemfibrozil Journal of Pharma-
cology and Experimental Therapeutics
Shitara Y Itoh T Sato H Li A P amp Sugiyama Y (2003) Inhibition of
transporter-mediated hepatic uptake as a mechanism for drugndashdrug
interaction between cerivastatin and cyclosporin A Journal of
Pharmacology and Experimental Therapeutics 304 610ndash616
Somogyi R Fuhrman S amp Wen X (2001) Genetic network inference in
computational models and applications to large-scale gene expression
data In M Bower amp H Bolouri (Eds) Computational modeling of
genetic and biochemical networks (pp 119ndash157) Cambridge MArsquoThe
MIT Press
Spirin V amp Mirny L A (2003) Protein complexes and functional
modules in molecular networks Proceedings of the National Academy
of Sciences of the United States of America 100 12123ndash12128
Staudinger J Liu Y Madan A Habeebu S amp Klaassen C D
(2001) Coordinate regulation of xenobiotic and bile acid homeo-
stasis by pregnane X receptor Drug Metabolism and Disposition
29 1467ndash1472
Sun L Z Ji Z L Chen X Wang J F amp Chen Y Z (2002) ADME-
AP A database of ADME associated proteins Bioinformatics 18
1699ndash1700
Suter L Babiss L E amp Wheeldon E B (2004) Toxicogenomics in
predictive toxicology in drug development Chemistry and Biology 11
161ndash171
Synold T W Dussault I amp Forman B M (2001) The orphan nuclear
receptor SXR coordinately regulates drug metabolism and efflux
Nature Medicine 7 584ndash590
Tabuchi Y amp Kondo T (2003) cDNA microarray analysis reveals chop-
10 plays a key role in Sertoli cell injury induced by bisphenol A
Biochemical and Biophysical Research Communications 305 54ndash61
Tamai I Nezu J Uchino H Sai Y Oku A Shimane M et al (2000)
Molecular identification and characterization of novel members of the
human organic anion transporter (OATP) family Biochemical and
Biophysical Research Communications 273 251ndash260
Taylor S W Fahy E Zhang B Glenn G M Warnock D E Wiley S
et al (2003) Characterization of the human heart mitochondrial
proteome Nature Biotechnology 21 281ndash286
Terasaka S Aita Y Inoue A Hayashi S Nishigaki M Aoyagi K
et al (2004) Using a customized DNA microarray for expression
profiling of the estrogen-responsive genes to evaluate estrogen activity
among natural estrogens and industrial chemicals Environmental
Health Perspectives 112 773ndash781
Thomas D M Francescutti-Verbeem D M Liu X amp Kuhn D M
(2004) Identification of differentially regulated transcripts in mouse
striatum following methamphetamine treatmentmdashan oligonucleotide
microarray approach Journal of Neurochemistry 88 380ndash393
Thomas R S Rank D R Penn S G Zastrow G M Hayes K R
Pande K et al (2001) Identification of toxicologically predictive
gene sets using cDNA microarrays Molecular Pharmacology 60
1189ndash1194
Thompson K L Afshari C A Amin R P Bertram T A Car B
Cunningham M et al (2004) Identification of platform-independent
gene expression markers of cisplatin nephrotoxicity Environmental
Health Perspectives 112 488ndash494
Tirona R G Leake B F Merino G amp Kim R B (2001)
Polymorphisms in OATP-C Identification of multiple allelic
variants associated with altered transport activity among European-
and African-Americans Journal of Biological Chemistry 276
35669ndash35675
Tirona R G Leake B F Wolkoff A W amp Kim R B (2003)
Human organic anion transporting polypeptide-C (SLC21A6) is a
major determinant of the rifampin-mediated pregnane X receptor
activation Journal of Pharmacology and Experimental Therapeutics
304 223ndash228
Tong W Cao X Harris S Sun H Fang H Fuscoe J et al (2003)
ArrayTrackmdashsupporting toxicogenomic research at the US Food and
Drug Administration National Center for Toxicological Research
Environmental Health Perspectives 111 1819ndash1826
Tornow S amp Mewes H W (2003) Functional modules by relating
protein interaction networks and gene expression Nucleic Acids
Research 31 6283ndash6289
Trocho C Escola-Gil J C Ribas V Benitez S Martin-Campos J M
Rotllan N et al (2004) Phenytoin treatment reduces atherosclerosis in
mice through mechanisms independent of plasma HDL-cholesterol
concentration Atherosclerosis 174 275ndash285
Ueda A Hamadeh H K Webb H K Yamamoto Y Sueyoshi T
Afshari C A et al (2002) Diverse roles of the nuclear orphan
receptor CAR in regulating hepatic genes in response to phenobarbital
Molecular Pharmacology 61 1ndash6
Ulrich R G (2003) The toxicogenomics of nuclear receptor agonists
Current Opinion in Chemical Biology 7 505ndash510
Ulrich R G Rockett J C Gibson G G amp Pettit S D (2004)
Overview of an interlaboratory collaboration on evaluating the effects
of model hepatotoxicants on hepatic gene expression Environmental
Health Perspectives 112 423ndash427
Unami A Shinohara Y Ichikawa T amp Baba Y (2003) Biochemical
and microarray analyses of bupivacaine-induced apoptosis Journal of
Toxicological Sciences 28 77ndash94
Vasquez A Flammini A Maritan A amp Vespignani A (2003) Global
protein function prediction from proteinndashprotein interaction networks
Nature Biotechnology 21 697ndash700
Vezina C M Walker N J amp Olson J R (2004) Subchronic Exposure to
TCDD PeCDF PCB126 and PCB153 Effect on hepatic gene
expression Environmental Health Perspectives 112 1636ndash1644
Vo T D Greenberg H J amp Palsson B O (2004) Reconstruction and
functional characterization of the human mitochondrial metabolic
network based on proteomic and biochemical data Journal of Bio-
logical Chemistry 279 39532ndash39540
Wang H amp LeCluyse E L (2003) Role of orphan nuclear receptors in
the regulation of drug-metabolising enzymes Clinical Pharmacoki-
netics 42 1331ndash1357
Wang N Silver D L Thiele C amp Tall A R (2001) ATP-
binding cassette transporter A1 (ABCA1) functions as a choles-
terol efflux regulatory protein Journal of Biological Chemistry 276
23742ndash23747
Waring J F Cavet G Jolly R A McDowell J Dai H Ciurlionis R
et al (2003) Development of a DNA Microarray for toxicology based
on hepatoxin-regulated sequences Environmental Health Perspectives
111 863ndash870
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6666
Waring J F Gum R Morfitt D Jolly R A Ciurlionis R Heindel M
et al (2002) Identifying toxic mechanisms using DNA microarrays
Evidence that an experimental inhibitor of cell adhesion molecule
expression signals through the aryl hydrocarbon nuclear receptor
Toxicology 181ndash182 537ndash550
Waring J F Jolly R A Ciurlionis R Lum P Y Praestgaard J T
Morfitt D C et al (2001) Clustering of hepatotoxins based on
mechanism of toxicity using gene expression profiles Toxicology and
Applied Pharmacology 175 28ndash42
Waters M Boorman G Bushel P Cunningham M Irwin R Merrick
A et al (2003) Systems toxicology and the Chemical Effects in
Biological Systems (CEBS) knowledge base EHP Toxicogenomics
111 15ndash28
Wei M Arnold L Cano M amp Cohen S M (2005) Effects of co-
administration of antioxidants and arsenicals on the rat urinary bladder
epithelium Toxicology Sciences 83 237ndash245
Weinshilboum R (2003) Inheritance and drug response New England
Journal of Medicine 348 529ndash537
Weinshilboum R amp Wang L (2004) Pharmacogenetics Inherited
variation in amino acid sequence and altered protein quantity Clinical
Pharmacology and Therapeutics 75 253ndash258
Werner E (2003) In silico multicellular systems biology and minimal
genomes Drug Discovery Today 8 1121ndash1127
Wong J S amp Gill S S (2002) Gene expression changes induced in
mouse liver by di(2-ethylhexyl) phthalate Toxicology and Applied
Pharmacology 185 180ndash196
Xie T Tong L McCann U D Yuan J Becker K G Mechan A O
et al (2004) Identification and characterization of metallothionein-1
and -2 gene expression in the context of ()34-methylenedioxymetham-
phetamine-induced toxicity to brain dopaminergic neurons Journal of
Neuroscience 24 7043ndash7050
Yadetie F Laegreid A Bakke I Kusnierczyk W Komorowski J
Waldum H L et al (2003) Liver gene expression in rats in response
to the peroxisome proliferator-activated receptor-alpha agonist ciprofi-
brate Physiological Genomics 15 9ndash19
Yamashita S Nomoto T Abe M Tatematsu M Sugimura T amp
Ushijima T (2004) Persistence of gene expression changes in stomach
mucosae induced by short-term N-methyl-NV-nitro-N-nitrosoguanidinetreatment and their presence in stomach cancers Mutation Research
549 185ndash193
Yan Q amp Sadee W (2000) Human membrane transporter database A
web-accessible relational database for drug transport studies and
pharmacogenomics AAPS Pharmsci 2 E20
Yeger-Lotem E amp Margalit H (2003) Detection of regulatory circuits by
integrating the cellular networks of proteinndashprotein interactions and
transcription regulation Nucleic Acids Research 31 6053ndash6061
Yoon B I Li G -X Kitada K Kawasaki Y Igarashi K amp Kodama
Y et al (2003) Mechanisms of benzene-induced hematotoxicity and
leukemogenicity cDNA microarray analyses using mouse bone marrow
tissue Environmental Health Perspectives 111 1411ndash1420
Young M B DiSilvestro M R Sendera T J Freund J Kriete A amp
Magnuson S R (2003) Analysis of gene expression in carbon
tetrachloride-treated rat livers using a novel bioarray technology
Pharmacogenomics Journal 3 41ndash52
Yu H Zhu X Greenbaum D Karro J amp Gerstein M (2004) TopNet
A tool for comparing biological sub-networks correlating protein
properties with topological statistics Nucleic Acids Research 32
328ndash337
Zhang E Y Fu D J Pak Y A Stewart T Mukhopadhyay N
Wrighton S A et al (2004) Genetic polymorphisms in human proton-
dependent dipeptide transporter PEPT1 Implications for the functional
role of Pro586 Journal of Pharmacology and Experimental Therapeu-
tics 310 437ndash445
Zhang E Y Knipp G T Ekins S amp Swaan P W (2002)
Structural biology and function of solute transporters Implications
for identifying and designing substrates Drug Metabolism Reviews
34 709ndash750
Zhang E Y Phelps M A Cheng C Ekins S amp Swaan P W (2002)
Modeling of active transport systems Advanced Drug Delivery
Reviews 54 329ndash354
- Systems-ADMETox Resources and network approaches
-
- Introduction
- Data available
- Network analysis and databases
-
- Network applications
-
- The role of transporters
-
- Clinical relevance of transporters
- Transporter network examples ABCA1
- Transporter network examples OATP
- Transporter microarray data
-
- Applications to enzymes
- Future network applications
-
- Discussion
- Acknowledgements
- References
-
Fig 8 The network of gene interactions around the CYP3A4 enzyme (centre) from MetaCorei Information on the type of interaction between objects is
hidden for clarity eg unspecified allosteric regulation binding cleavage competition covalent modification dephosphorylation phosphorylation
transcription regulation transformation When applicable interactions also have a positive or negative effect and direction Ligands (purple) linked to other
proteins (blue blobs) transfactors (red) enzymes (orange) (For interpretation of the references to colour in this figure legend the reader is referred to the web
version of this article)
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 59
the initial doses of drugs metabolized by these enzymes
should be reduced in older patients according to the clinical
response In most published studies the elderly appear at
least as responsive as the young to inducers or inhibitors of
P450s (Durnas Loi amp Cusack 1990) More recently there
has been some suggestion that there are age related
reductions in function of some specific P450s such as
CYP3A4 (Patki et al 2004) and this could occur at the
level of regulation However earlier studies with the same
enzyme showed no change in clearance with age (Hunt et
al 1992) To date there has been even less examination of
the transporter functions and any changes with age
(Kinirons amp OrsquoMahony 2004) so the current understand-
ing of the effects of aging on metabolism and transport is
anything but transparent This represents an extreme
challenge for the pharmaceutical industry how to predict
whether a drug has an affinity for an enzyme or transporter
and whether this may be clinically important if it shows a
decline in expression or function with aging Any decrease
of function of these enzymes or transporters in an elderly
population would result in changes in the metabolism and
transport of metabolites potentially impacting the clinical
effect This represents another example of how computa-
tional approaches may perhaps be used with different types
of predicted and experimental data to allow visualization of
potential compound interactions or toxicity in elderly
populations We may see different gene networks high-
lighted as humans age and these may be modified by drug
treatment and coadministration This represents an area
were network analysis could be applied in the future and is
in need of considerable further research
The collection of microarray data in databases such as
CEBS ArrayTrack and EDGE represents a future resource
for computational gene network analysis One could
envisage that ultimately in each case such data is converted
to one or more networks that are also displayed for the user
and can be used to compare treatments from in vivo and in
vitro experiments This would represent a different approach
to clustering the data as currently implemented in one of
these efforts (Hayes et al 2005) and may condense large
amounts of experimentally derived data into a readily
interpreted network
4 Discussion
Previously in this journal the progress of many
research groups in predicting human ADME parameters
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6660
in silico (Ekins et al 2000b) and approaches for drug
metabolism (Ekins Ring Grace McRobie-Belle amp
Wrighton 2000a) have been described Both of these
reviews commented on moving HT assays for ADME
Tox much earlier in drug discovery which had also been
indicated by other groups The initial reviews also
highlighted the likely wealth of data that would become
available and how this could be used for structure
activity relationships alongside the bioactivity data in
computational models It was noted that there was a
paucity of predictive metabolism tools at that time In
addition a growing number of efforts to model whole
cells and organs now a field called systems biology
were recognized as models that could be integrated with
the in silico ADME approaches In summary since these
past reviews virtually all pharmaceutical companies have
attempted earlier high throughput screening for ADME
Tox properties and to some extent the wider application
of computational approaches for physicochemical proper-
ties Systems biology is being quite widely acknowledged
as the new paradigm for understanding complex bio-
logical datasets derived from high throughput technolo-
gies and the accumulated knowledge on human protein
interactions (Hartwell Hopfield Leibler amp Murray 1999
Hood 2003b) Therefore systems biology can be defined
as the integration of genetic proteomic transcriptomic
and metabonomic data using computational methods
(Nicholson amp Wilson 2003) When taken together
information on molecular processes derived from different
sources represents a lsquolsquouniversersquorsquo of putative biological
functionality of which only a small fraction of it will be
realized in a cell at any given time To date systems
biology has been driven by academia and funding bodies
such as the NIH rather than the pharmaceutical compa-
nies Presently there is a great deal of interest from
scientists of all backgrounds in identifying the networks
of cellular pathways and the corresponding physically
interacting proteins
The network building software for systems biology
described in this current review will be valuable to query
high throughput data and known literature interactions in
order to predict potential toxicity in different species In the
future the compilation of published toxicogenomics data-
sets characteristic of different types of toxicity will likely
be available in these software systems to act as a reference
database It is also feasible that we will be able to generate
the annotated datasets which specifically address the
differences between human and rat networks implicated
in toxicity The identification of sub-network modules
conserved between human and rat distinct for toxicity
types or predictive for toxic end-points in human will be
possible Such signature gene networks (Nikolsky et al
2005) can then be verified with other experimentally
derived data prospectively or from preexisting databases
The combined Fhigh throughput datandashin silico network_approaches therefore represents a novel method for access-
ing complex tissue-level processes as networks integrating
all data types based on functional interactions The data-
bases developed and used in this approach will certainly
benefit from further annotation around the drug metaboliz-
ing enzymes and transporters as described herein in terms
of transcriptional regulation and the ligands associated with
them which frequently appear in the literature It will be
important to capture disparities in the assignment of ligands
to enzymes transporters and other proteins as well as
negative data
In summary although there have been numerous
toxicogenomics studies published there is presently a
relatively small number of datasets that are freely available
to perform network analysis of microarray data The
number of studies identifying large numbers of proteins
which are affected by molecule treatment are even scarcer
still while there are several examples of some published
studies that combine such data It is hoped that the
numerous database initiatives for high content and toxicol-
ogy data that are being undertaken will improve the
situation for other researchers that are not currently
equipped to do such microarray studies themselves The
addition of requirements by journals to deposit such raw
data in a freely accessible resource will aid these initiatives
ADMETox groups have seen new technologies and
approaches developed over the last decade that have all
been applied to identify poor compounds earlier (Fig 2)
The latest technologies integrate network building tools
with high content data and databases The current review
described the limited number of networks generated for
ADMETox at present and one hopes that the impact of
such analyses will be commonplace in the future Systems
biology is however more than just applying a network
approach and hence systems-ADMETox will have to
evolve due to the continual pressure to develop newer
technologies This current paradigm combining empirical
data and computational methods should integrate the
complex data already generated making it readily inter-
pretable and valuable for identifying the most promising
compounds in the future
Acknowledgements
Dr Maggie AZ Hupcey is gratefully acknowledged for
editorial assistance Dr Peter W Swaan (University of
Maryland) and Dr Cheng Chang (Ohio State University)
Dr Steve Wright (University of Arizona) Dr K Sandy
Pang (University of Toronto) and Dr Craig Giroux (Wayne
State University) are kindly acknowledged for their support
and discussions My colleagues at GeneGo Sergey
Andreyev Andy Ryabov Eugene Kirillov Eugene A
Rakhmatulin Svetlana Sorokina Andrej Bugrim Tatiana
Nikolskaya Yuri Nikolsky John Metz and Julie Bryant are
all thanked for their considerable contributions to software
development and data annotation
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 61
References
Abe M Yamashita S Kuramoto T Hirayama Y Tsukamoto T Ohta
T et al (2003) Global expression analysis of N-methyl-NV-nitro-N-nitrosoguanidine-induced rat stomach carcinomas using oligonucleotide
microarrays Carcinogenesis 24 861ndash867
Adachi T Ono Y Koh K B Takashima K Tainaka H Matsuno Y
et al (2004) Long-term alteration of gene expression without
morphological change in testis after neonatal exposure to genistein in
mice Toxicogenomic analysis using cDNA microarray Food and
Chemical Toxicology 42 445ndash452
Akerman G S Rosenzweig B A Domon O E McGarrity L J
Blankenship L R Tsai C A et al (2004) Gene expression profiles
and genetic damage in benzo(a)pyrene diol epoxide-exposed TK6 cells
Mutation Research 549 43ndash64
Aksenov S V Church B Dhiman A Georgieva A Sarangapani R
Helmlinger G et al (2005) An integrated approach for inference and
mechanistic modeling for advancing drug development FEBS Letters
579 1878ndash1883
Anderle P Huang Y amp Sadee W (2004) Intestinal membrane transport
of drugs and nutrients Genomics of membrane transporters using
expression microarrays European Journal of Pharmaceutical Sciences
21 17ndash24
Annereau J P Szakacs G Tucker C J Arciello A Cardarelli C
Collins J et al (2004) Analysis of ATP-binding cassette transporter
expression in drug-selected cell lines by a microarray dedicated to
multidrug resistance Molecular Pharmacology 66 1397ndash1405
Apic G Ignjatovic T Boyer S amp Russell R B (2005) Illuminating drug
discovery with biological pathways FEBS Letters 579 1872ndash1877
Baker V A Harries H M Waring J F Duggan C M Ni H A Jolly
R A et al (2004) Clofibrate-induced gene expression changes in rat
liver A cross-laboratory analysis using membrane cDNA arrays
Environmental Health Perspectives 112 428ndash438
Balakin K V Ekins S Bugrim A Ivanenkov Y A Korolev D amp
Nikolsky Y et al (2004a) Quantitative structurendashmetabolism relation-
ship modeling of the metabolic N-dealkylation rates Drug Metabolism
and Disposition 32 1111ndash1120
Balakin K V Ekins S Bugrim A Ivanenkov Y A Korolev D amp
Nikolsky Y et al (2004b) Kohonen maps for prediction of binding to
human cytochrome P450 3A4 Drug Metabolism and Disposition 32
1183ndash1189
Barabasi A -L amp Oltvai Z N (2004) Network biology Under-
standing the cellrsquos functional organization Nature Reviews Genetics
5 101ndash113
Bartosiewicz M Penn S G amp Buckpitt A (2001) Applications of gene
arrays in environmental toxicology Fingerprints of gene regulation
associated with cadmium chloride benzo(a)pyrene and trichloro-
ethylene Environmental Health Perspectives 109 71ndash74
Bertilsson G Heidrich J Svensson K Asman M Jendeberg L
Sydow-Backman M et al (1998) Identification of a human nuclear
receptor defines a new signaling pathway for CYP3A induction
Proceedings of the National Academy of Sciences of the United States
of America 95 12208ndash12213
Blumberg B Sabbagh Jr W Juguilon H Bolado Jr J van Meter
C M Ong E S et al (1998) SXR a novel steroid and xenobiotic-
sensing nuclear receptor Genes and Development 12 3195ndash3205
Borodina Y Rudik A Filimonov D Kharchevnikova N Dmitriev A
Blinova V et al (2004) A new statistical approach to predicting
aromatic hydroxylation sites Comparison with model-based
approaches Journal of Chemical Information and Computer Sciences
44 1998ndash2009
Borodina Y Sadym A Filimonov D Blinova V Dmitriev A amp
Poroikov V (2003) Predicting biotransformation potential from
molecular structure Journal of Chemical Information and Computer
Sciences 43 1636ndash1646
Bottone Jr F G Martinez J M Collins J B Afshari C A amp Eling
T E (2003) Gene modulation by the cyclooxygenase inhibitor
sulindac sulfide in human colorectal carcinoma cells Possible link to
apoptosis Journal of Biological Chemistry 278 25790ndash25801
Boyer S amp Zamora I (2002) New methods in predictive metabolism
Journal of Computer-Aided Molecular Design 16 403ndash413
Bredel M amp Jacoby E (2004) Chemogenomics An emerging strategy
for rapid target and drug discovery Nature Reviews Genetics 5
262ndash275
Bugrim A Nikolskaya T amp Nikolsky Y (2004) Early prediction of drug
metabolism and toxicity Systems biology approach and modeling
Drug Discovery Today 9 127ndash135
Bulera S J Eddy S M Ferguson E Jatkoe T A Reindel J F
Bleavins M R et al (2001) RNA expression in the early character-
ization of hepatotoxicants in Wistar rats by high-density DNA micro-
arrays Hepatology 33 1239ndash1258
Butcher E C Berg E L amp Kunkel E J (2004) Systems biology in drug
discovery Nature Biotechnology 22 1253ndash1259
Butte A (2002) The use and analysis of microarray data Naturalist
Review of Drug Discovery 1 951ndash960
Cary M P Bader G D amp Sander C (2005) Pathway information for
systems biology FEBS Letters 579 1815ndash1820
Cheok M H Yang W Pui C H Downing J R Cheng C Naeve
C W et al (2003) Treatment-specific changes in gene expression
discriminate in vivo drug response in human leukemia cells Nature
Genetics 34 85ndash90
Cleary J D Rogers P D amp Chapman S W (2001) Differential
transcription factor expression in human mononuclear cells in response
to amphotericin B Identification with complementary DNA microarray
technology Pharmacotherapy 21 1046ndash1054
Coen M Ruepp S U Lindon J C Nicholson J K Pognan F Lenz
E M et al (2004) Integrated application of transcriptomics and
metabonomics yields new insight into the toxicity due to paracetamol in
the mouse Journal of Pharmaceutical and Biomedical Analysis 35
93ndash105
Cornwell P D De Souza A T amp Ulrich R G (2004) Profiling of
hepatic gene expression in rats treated with fibric acid analogs
Mutation Research 549 131ndash145
Csermely P Agoston V amp Pongor S (2005) The efficiency of multi-
target drugs The network approach might help drug design Trends in
Pharmacological Sciences 26 178ndash182
Cunningham M J Liang S Fuhrman S Seilhamer J J amp
Somogyi R (2000) Gene expression microarray data analysis for
toxicology profiling Annals of the New York Academy of Sciences 919
52ndash67
de Longueville F Atienzar F A Marcq L Dufrane S Evrard S
Wouters L et al (2003) Use of a low density microarray for studying
gene expression patterns induced by hepatotoxicants on primary
cultures of rat hepatocytes Toxicological Sciences 75 378ndash392
Dobrin R Beg Q K Barabasi A L amp Oltvai Z N (2004)
Aggregation of topological motifs in the Escherichia coli transcriptional
regulatory network BMC Bioinformatics 5 10
Donald S Verschoyle R D Edwards R Judah D J Davies R
Riley J et al (2002) Hepatobiliary damage and changes in hepatic
gene expression caused by the antitumor drug ecteinascidin-743 (ET-
743) in the female rat Cancer Research 62 4256ndash4262
Dumont M Jacquemin E DrsquoHont C Descout C Cresteil D Haouzi
D et al (1997) Expression of the liver Na+-independent organic anion
transporting polypeptide (oatp-1) in rats with bile duct ligation Journal
of Hepatology 27 1051ndash1056
Durnas C Loi C M amp Cusack B J (1990) Hepatic drug metabolism
and aging Clinical Pharmacokinetics 19 359ndash389
Eisen M B Spellman P T Brown P O amp Botstein D (1998) Cluster
analysis and display of genome-wide expression patterns Proceedings
of the National Academy of Sciences of the United States of America
95 14863ndash14868
Ekins S Andreyev S Ryabov A Kirilov E Rakhmatulin E A
Bugrim A et al (in press) Computational Prediction of Human Drug
Metabolism Exp Opin Drug Metab Toxicol
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6662
Ekins S Boulanger B Swaan P W amp Hupcey M A Z (2002)
Towards a new age of virtual ADMETOX and multidimensional drug
discovery Journal of Computer-Aided Molecular Design 16 381ndash401
Ekins S Bugrim A Nikolsky Y amp Nikolskaya T (2005) Systems
biology Applications in drug discovery In S Gad (Ed) Drug
Discovery Handbook (pp 123ndash183) Hoboken NJrsquo Wiley and Sons
Ekins S Giroux C N Nikolsky Y Bugrim A amp Nikolskaya T
(2005) A signature gene network approach to toxicity The Toxoco-
logists 84
Ekins S Kirillov E Rakhmatulin E amp Nikolskaya T (2005) A novel
method for visualizing nuclear hormone receptor networks relevant to
drug metabolism Drug Metabolism and Disposition 33 474ndash481
Ekins S amp McGowan R J (2001) The limits of reductionism The
shifting genomic paradigmrsquos impact on industry and academia
Philsophy in Science 9 1ndash23
Ekins S Mirny L amp Schuetz E G (2002) A ligand-based approach to
understanding selectivity of nuclear hormone receptors PXR CAR
FXR LXRa and LXRb Pharmaceutical Research 19 1788ndash1800
Ekins S Nikolsky Y amp Nikolskaya T (2005) Techniques Application
of systems biology to absorption distribution metabolism excretion
and toxicity Trends in Pharmacological Sciences 26 202ndash209
Ekins S Ring B J Grace J McRobie-Belle D J amp Wrighton S
A (2000) Present and future in vitro approaches for drug
metabolism Journal of Pharmacological and Toxicological Methods
44 313ndash324
Ekins S Waller C L Swaan P W Cruciani G Wrighton S A amp
Wikel J H (2000) Progress in predicting human ADME parameters in
silico Journal of Pharmacological and Toxicological Methods 44
251ndash272
Erhardt P W (2003) A human drug metabolism database Potential roles
in the quantitative predictions of drug metabolism and metabolism-
related drugndashdrug interactions Current Drug Metabolism 4 411ndash422
Evans W E amp McLeod H L (2003) Pharmacogenomicsmdashdrug
disposition drug targets and side effects New England Journal of
Medicine 348 538ndash549
Ezendam J Staedtler F Pennings J Vandebriel R J Pieters R
Boffetta P et al (2004) Toxicogenomics of subchronic hexachlor-
obenzene exposure in Brown Norway rats Environmental Health
Perspectives 112 782ndash791
Faiola B Fuller E S Wong V A amp Recio L (2004) Gene expression
profile in bone marrow and hematopoietic stem cells in mice exposed to
inhaled benzene Mutation Research 549 195ndash212
FDA (2004) Innovation stagnation Challenge and opportunity on the
critical path to new medicinal products
Fiehn O (2001) Combining genomics metabolome analysis and bio-
chemical modelling to understand metabolic networks Comparative
Functional Genomics 2 155ndash168
Field F J Born E amp Mathur S N (2004) LXRRXR ligand activation
enhances basolateral efflux of beta-sitosterol in CaCo-2 cells Journal of
Lipid Research 45 905ndash913
Fleck C Sutter L Appenroth D Koch B Meinhold T Pitack M
et al (2003) Use of gene chip technology for the characterisation of
the regulation of renal transport processes and of nephrotoxicity in
rats Experimental and Toxicologic Pathology 54 401ndash410
Fliri A F Loging W T Thadeio P F amp Volkmann R A (2005)
Biological spectra analysis Linking biological activity profiles to
molecular structure Proceedings of the National Academy of Sciences
of the United States of America 102 261ndash266
Fountoulakis M (2004) Application of proteomics technologies in the
investigation of the brain Mass Spectrometry Reviews 23 231ndash258
Gaucher S P Taylor S W Fahy E Zhang B Warnock D E Ghosh
S S et al (2004) Expanded coverage of the human heart
mitochondrial proteome using multidimensional liquid chromatography
coupled with tandem mass spectrometry Journal of Proteome
Research 3 495ndash505
Gerhold D Lu M Xu J Austin C Caskey C T amp Rushmore T
(2001) Monitoring expression of genes involved in drug metabolism
and toxicology using DNA microarrays Physiological Genomics 5
161ndash170
Guo G L Choudhuri S amp Klaassen C D (2002) Induction profile of
rat organic anion transporting polypeptide 2 (oatp2) by prototypical
drug-metabolizing enzyme inducers that activate gene expression
through ligand-activated transcription factor pathways Journal of
Pharmacology and Experimental Therapeutics 300 206ndash212
Hagenbuch B amp Meier P J (2004) Organic anion transporting
polypeptides of the OATP SLC21 family Phylogenetic classification
as OATP SLCO superfamily new nomenclature and molecularfunc-
tional properties Pflugers Archiv 447 653ndash665
Hamadeh H K amp Afshari C A (2004) Toxicogenomics principles and
applications Hobokenrsquo John Wiley and Sons
Hamadeh H K Bushel P R Jayadev S Martin K DiSorbo O Sieber
S et al (2002) Gene expression analysis reveals chemical-specific
profiles Toxicological Sciences 67 219ndash231
Hamadeh H K Jayadev S Gaillard E T Huang Q Stoll R
Blanchard K et al (2004) Integration of clinical and gene expression
endpoints to explore furan-mediated hepatotoxicity Mutation Research
549 169ndash183
Hamadeh H K Knight B L Haugen A C Sieber S Amin R P
Bushel P R et al (2002) Methapyrilene toxicity Anchorage of
pathologic observations to gene expression alterations Toxicologic
Pathology 30 470ndash482
Han J D Bertin N Hao T Goldberg D S Berriz G F Zhang L V
et al (2004) Evidence for dynamically organized modularity in the
yeast proteinndashprotein interaction network Nature 430 88ndash93
Hanisch D Zien A Zimmer R amp Lengauer T (2002) Co-clustering of
biological networks and gene expression data Bioinformatics 18
S145ndashS154
Harris A J Dial S L amp Casciano D A (2004) Comparison of basal
gene expression profiles and effects of hepatocarcinogens on gene
expression in cultured primary human hepatocytes and HepG2 cells
Mutation Research 549 79ndash99
Harrison S C (2004) Whither structural biology Nature Structural
Molecular Biology 11 12ndash15
Hartley D P Dai X He Y D Carlini E J Wang B Huskey S E
et al (2004) Activators of the rat pregnane X receptor differentially
modulate hepatic and intestinal gene expression Molecular Pharma-
cology 65 1159ndash1171
Hartwell L H Hopfield J J Leibler S amp Murray A W (1999) From
molecular to modular cell biology Nature 402 C47ndashC52
Hasmall S Orphanides G James N Pennie W Hedley K Soames
A et al (2002) Downregulation of lactoferrin by PPARalpha ligands
Role in perturbation of hepatocyte proliferation and apoptosis
Toxicology Sciences 68 304ndash313
Hayase T Yamamoto Y Yamamoto K Muso E amp Shiota K (2004)
Microarray profile analysis of toxic cocaine-induced alterations in the
expression of mouse brain gene sequences A possible rsquoprotectiversquo
effect of buprenorphine Journal of Applied Toxicology 24 15ndash20
Hayes K R Vollrath A L Zastrow G M McMillan B J Craven M
Jovanovich S et al (2005) EDGE A centralized resource for the
comparison analysis and distribution of toxicogenomic information
Molecular Pharmacology 67 1360ndash1368
Heijne W H Slitt A L van Bladeren P J Groten J P Klaassen C D
Stierum R H et al (2004) Bromobenzene-induced hepatotoxicity at
the transcriptome level Toxicology Sciences 79 411ndash422
Heijne W H Stierum R H Slijper M van Bladeren P J amp van
Ommen B (2003) Toxicogenomics of bromobenzene hepatotoxicity
A combined transcriptomics and proteomics approach Biochemical
Pharmacology 65 857ndash875
Heinloth A N Irwin R D Boorman G A Nettesheim P Fannin
R D Sieber S O et al (2004) Gene expression profiling of rat
livers reveals indicators of potential adverse effects Toxicology
Sciences 80 193ndash202
Hodges L C Cook J D Lobenhofer E K Li L Bennett L Bushel
P R et al (2003) Tamoxifen functions as a molecular agonist inducing
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 63
cell cycle-associated genes in breast cancer cells Molecular Cancer
Research 1 300ndash311
Holleman A Cheok M H den Boer M L Yang W Veerman KM
A J Kazemier et al (2004) Gene-expression patterns in drug-resistant
acute lymphoblastic leukemia cells and response to treatment New
England Journal of Medicine 351 533ndash542
Hong Y Muller U R amp Lai F (2003) Discriminating two classes of
toxicants through expression analysis of HepG2 cells with DNA arrays
Toxicology in Vitro 17 85ndash92
Hood E (2003) Proteomics Characterizing the cogs in the machinery of
life Environmental Health Perspectives 111 A816ndashA825
Hood L (2003) Systems biology Integrating technology biology and
computation Mechanism of Ageing and Development 124 9ndash16
Hood L amp Galas D (2003) The digital code of DNA Nature 421
444ndash448
Hood L amp Perlmutter R M (2004) The impact of systems approaches on
biological problems in drug discovery Nature Biotechnology 22
1215ndash1217
Hu T Gibson D P Carr G J Torontali S M Tiesman J P Chaney
J G et al (2004) Identification of a gene expression profile that
discriminates indirect-acting genotoxins from direct-acting genotoxins
Mutation Research 549 5ndash27
Huang Q Dunn II R T Jayadev S DiSorbo O Pack F D Farr S B
et al (2001) Assessment of cisplatin-induced nephrotoxicity by
microarray technology Toxicology Sciences 63 196ndash207
Huang Q Jin X Gaillard E T Knight B L Pack F D Stoltz J H et
al (2004) Gene expression profiling reveals multiple toxicity endpoints
induced by hepatotoxicants Mutation Research 549 147ndash167
Huisman M T Smit J W Crommentuyn K M Zelcer N Wiltshire
H R Beijnen J H et al (2002) Multidrug resistance protein 2
(MRP2) transports HIV protease inhibitors and transport can be
enhanced by other drugs Aids 16 2295ndash2301
Hunt C M Westerkam W R amp Stave G M (1992) Effect of age and
gender on the activity of human hepatic CYP3A Biochemical
Pharmacology 44 275ndash283
Ideker T Ozier O Schwikowski B amp Siegal A F (2002) Discovering
regulatory and signalling circuits in molecular interaction networks
Bioinformatics 18 S233ndashS240
Ideker T Thorsson V Ranish J A Christmas R Buhler J Eng J K
et al (2001) Integrated genomic and proteomic analyses of a system-
atically perturbed network Science 292 929ndash934
Iida M Anna C H Hartis J Bruno M Wetmore B Dubin J R et al
(2003) Changes in global gene and protein expression during early
mouse liver carcinogenesis induced by non-genotoxic model carcino-
gens oxazepam and Wyeth-14643 Carcinogenesis 24 757ndash770
Ingelman-Sundberg M (2004) Pharmacogenetics of cytochrome P450 and
its applications in drug therapy The past present and future Trends in
Pharmacological Sciences 25 193ndash200
Jeong H Mason S P Barabasi A L amp Oltvai Z N (2001) Lethality
and centrality in protein networks Nature 411 41ndash42
Jeong H Tombor B Albert R Oltvai Z N amp Barabasi A L
(2000) The large-scale organization of metabolic networks Nature
407 651ndash654
Jung D Hagenbuch B Gresh L Pontoglio M Meier P J amp Kullak-
Ublick G A (2001) Characterization of the human OATP-C
(SLC21A6) gene promoter and regulation of liver-specific OATP genes
by hepatocyte nuclear factor 1 alpha Journal of Biological Chemistry
276 37206ndash37214
Jung D amp Kullak-Ublick G A (2003) Hepatocyte nuclear factor 1
alpha A key mediator of the effect of bile acids on gene expression
Hepatology 37 622ndash631
Jung J -W Park J -S Hwang J -W Kang K -S YS L Song B
-S et al (2004) Gene expression analysis of peroxisome prolifer-
ators- and phenytoin-induced hepatotoxicity using cDNA microarray
Journal of Veterinary Medical Science 66 1329ndash1333
Kato N Shibutani M Takagi H Uneyama C Lee K -Y Takiami S
et al (2004) Gene expression profile in the livers of rats orally
administered ethinylestradiol for 28 days using a microarray technique
Toxicology 200 179ndash192
Kier L D Neft R Tang L Suizu R Cook T Onsurez K et al
(2004) Applications of microarrays with toxicologically relevant genes
(tox genes) for the evaluation of chemical toxicants in Sprague Dawley
rats in vivo and human hepatocytes in vitro Mutation Research 549
101ndash113
Kijima K Toyosawa K Yasuba M Matsuoka N Adachi T
Komiyama M et al (2004) Gene expression analysis of the rat testis
after treatment with di(2-ethylhexyl) phthalate using cDNA microarray
and real-time RT-PCR Toxicology and Applied Pharmacology 200
103ndash110
Kim R B (2003) Organic anion-transporting polypeptide (OATP) trans-
porter family and drug disposition European Journal of Clinical
Investigation 33 1ndash5
Kinirons M T amp OrsquoMahony M S (2004) Drug metabolism and ageing
British Journal of Clinical Pharmacology 57 540ndash544
Kitano H (2002a) Computational systems biology Nature 420 206ndash210
Kitano H (2002b) Systems biology A brief overview Science 295
1662ndash1664
Kliewer S A Moore J T Wade L Staudinger J L Watson M
A Jones S A et al (1998) An orphan nuclear receptor activated
by pregnanes defines a novel steroid signalling pathway Cell 92
73ndash82
Korolev D Balakin K V Nikolsky Y Kirillov E Ivanenkov Y A
Savchuk N P et al (2003) Modeling of human cytochrome p450-
mediated drug metabolism using unsupervised machine learning
approach Journal of Medicinal Chemistry 46 3631ndash3643
Kramer J A Pettit S D Amin R P Bertram T A Car B
Cunningham M et al (2004) Overview on the application of
transcription profiling using selected nephrotoxicants for toxicology
assessment Environmental Health Perspectives 112 460ndash464
Kultima K Nystrom A -M Scholz B Gustafson A -L Dencker L
amp Stigson M (2004) Valproic acid teratogenicity A toxicogenomics
approach Environmental Health Perspectives 112 1225ndash1235
Kutuzova G D amp Deluca H F (2004) Gene expression profiles in rat
intestine identify pathways for 125-dihydroxyvitamin D(3) stimulated
calcium absorption and clarify its immunomodulatory properties
Archives of Biochemistry and Biophysics 432 152ndash166
Kwak M K Wakabayashi N Itoh K Motohashi H Yamamoto M amp
Kensler T W (2003) Modulation of gene expression by cancer
chemopreventive dithiolethiones through the Keap1-Nrf2 pathway
Identification of novel gene clusters for cell survival Journal of
Biological Chemistry 278 8135ndash8145
Lahjouji K Mitchell G A amp Qureshi I A (2001) Carnitine transport
by organic cation transporters and systemic carnitine deficiency
Molecular Genetics and Metabolism 73 287ndash297
Landowski C P Sun D Foster D R Menon S S Barnett J L
Welage L S et al (2003) Gene expression in the human intestine and
correlation with oral valacyclovir pharmacokinetic parameters Journal
of Pharmacology and Experimental Therapeutics 306 778ndash786
Lee G -J Lee W -S Jeon K -S Um C -H Kim S -J Lee C -H
et al (2004) cDNA microarray gene expression analysis and
toxicological phenotype for anticancer drug Journal of Veterinary
Medical Science 66 1339ndash1345
Lee M Kwon J Kim S N Kim J E Koh W S Kim E J et al
(2003) cDNA microarray gene expression profiling of hydroxyurea
paclitaxel and p-anisidine genotoxic compounds with differing
tumorigenicity results Environmental and Molecular Mutagenesis
42 91ndash97
Li S Armstrong C M Bertin N Ge H Milstein S Boxem M et al
(2004) A map of the interactome network of the metazoan C elegans
Science 303 540ndash543
Liguori M J Anderson L M Bukofzer S McKim J Pregenzer J F
Retief J et al (2005) Microarray analysis in human hepatocytes
suggests a mechanism for hepatotoxicity induced by trovafloxacin
Hepatology 41 177ndash186
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6664
Lobenhofer E K Bennett L Cable P L Li L Bushel P R amp Afshari
C A (2002) Regulation of DNA replication fork genes by 17beta-
estradiol Molecular Endocrinology 16 1215ndash1229
Locker J Tian J Carver R Concas D Cossu C Ledda-Columbano
G M et al (2003) A common set of immediate-early response genes
in liver regeneration and hyperplasia Hepatology 38 314ndash325
Mattes W B Pettit S D Sansone S A Bushel P R amp Waters M D
(2004) Database development in toxicogenomics Issues and efforts
Environmental Health Perspectives 112 495ndash505
McMillian M Nie A Y Parker J B Leone A Bryant S Kemmerer
M et al (2004) A gene expression signature for oxidant stressreactive
metabolites in rat liver Biochemical Pharmacology 68 2249ndash2261
Meier P J amp Stieger B (2000) Molecular mechanisms in bile formation
News in Physiological Sciences 15 89ndash93
Meneses-Lorente G de Longueville F Dos Santos-Mendes S Bonnert
T P Jack A Evrard S et al (2003) An evaluation of a low-density
DNA microarray using cytochrome P450 inducers Chemical Research
in Toxicology 16 1070ndash1077
Meneses-Lorente G Guest P C Lawrence J Muniappa N Knowles
M R Skynner H A et al (2004) A proteomic investigation of drug-
induced steatosis in rat liver Chemical Research in Toxicology 17
605ndash612
Merrill C L Ni H Yoon L W Tirmenstein M A Narayanan P
Benavides G R et al (2002) Etomoxir-induced oxidative stress in
HepG2 cells detected by differential gene expression is confirmed
biochemically Toxicology Sciences 68 93ndash101
Milo R Shen-Orr S Itzkovitz S Kashtan N Chklovskii D amp Alon
U (2002) Network motifs Simple building blocks of complex
networks Science 298 824ndash827
Morgan K T Ni H Brown H R Yoon L Qualls C W Crosby L
M et al (2002) Application of cDNA microarray technology to in
vitro toxicology and the selection of genes for a real-time RT-PCR-
based screen for oxidative stress in Hep-G2 cells Toxicologic
Pathology 30 435ndash451
Mwinyi J Johne A Bauer S Roots I amp Gerloff T (2004) Evidence
for inverse effects of OATP-C (SLC21A6) 5 and 1b haplotypes on
pravastatin kinetics Clinical Pharmacology and Therapeutics 75
415ndash421
Nakata K Yukawa M Komiyama N Nakano T amp Kaminuma T
(2002) A nuclear receptor database that maps pathways to diseases
Genome Infomatics 13 515ndash516
Nicholson J K Holmes E Lindon J C amp Wilson I D (2004) The
challenges of modeling mammalian biocomplexity Nature Biotechnol-
ogy 22 1268ndash1274
Nicholson J K amp Wilson I D (2003) Understanding rsquoglobal systems
biology Metabonomics and the continuum of metabolism Nature
Reviews on Drug Discovery 2 668ndash676
Nikitin A Egorov S Daraselia N amp Mazo I (2003) Pathway studiomdash
the analysis and navigation of molecular networks Bioinformatics 19
2155ndash2157
Nikolsky Y Ekins S Nikolskaya T amp Bugrim A (2005) A novel
method for generation of signature networks as biomarkers from
complex high throughput data Toxicology Letters 158 20ndash29
Nikolsky Y Nikolskaya T amp Bugrim A (2005) Biological networks
and analysis of experimental data in drug discovery Drug Discovery
Today 10 653ndash662
Nishizato Y Ieiri I Suzuki H Kimura M Kawabata K Hirota T
et al (2003) Polymorphisms of OATP-C (SLC21A6) and OAT3
(SLC22A8) genes Consequences for pravastatin pharmacokinetics
Clinical Pharmacology and Therapeutics 73 554ndash565
Nozawa T Nakajima M Tamai I Noda K Nezu J Sai Y et al
(2002) Genetic polymorphisms of human organic anion transporters
OATP-C (SLC21A6) and OATP-B (SLC21A9) Allele frequencies in
the Japanese population and functional analysis Journal of Pharmacol-
ogy and Experimental Therapeutics 302 804ndash813
Nozawa T Sugiura S Nakajima M Goto A Yokoi T Nezu J et al
(2004) Involvement of organic anion transporting polypeptides in the
transport of troglitazone sulfate Implications for understanding trogli-
tazone hepatotoxicity Drug Metabolism and Disposition 32 291ndash294
Oliver D E Rubin D L Stuart J M Hewett M Klein T E amp
Altman R B (2002) Ontology development for a pharmacogenetics
knowledge base Package Symposion 88ndash99
Oram J F amp Lawn R M (2001) ABCA1 The gatekeeper for eliminating
excess tissue cholesterol Journal of Lipid Research 42 1173ndash1179
Oswald M Kullak-Ublick G A Paumgartner G amp Beuers U (2001)
Expression of hepatic transporters OATP-C and MRP2 in primary
sclerosing cholangitis Liver 21 247ndash253
Parsons A B Brost R I Ding H Li Z Zhang C Sheikh B et al
(2004) Integration of chemical-genetic and genetic interaction data
links bioactive compounds to cellular target pathways Nature Bio-
technology 22 62ndash69
Patki K C von Moltke L L Harmatz J S Hesse L M Court M H
amp Greenblatt D J (2004) Effect of age on in vitro triazolam
biotransformation in male human liver microsomes Journal of
Pharmacology and Experimental Therapeutics 308 874ndash879
Pereira-Leal J B Enright A J amp Ouzounis C A (2004) Detection of
functional modules from protein interaction networks Proteins 54
49ndash57
Plant N (2004) Interaction networks Coordinating responses to xeno-
biotic exposure Toxicology 202 21ndash32
Raghavendra Prasad H S Qi Z Srinivasan K N amp Gopalakrish-
nakone P (2004) Potential effects of tetrodotoxin exposure to
human glial cells postulated using microarray approach Toxicon 44
597ndash608
Reilly T P Bourdi M Brady J N Pise-Masison C A Radonovich M
F George J W et al (2001) Expression profiling of acetaminophen
liver toxicity in mice using microarray technology Biochemical and
Biophysical Research Communications 282 321ndash328
Rives A W amp Galitski T (2003) Modular organization of cellular
networks Proceedings of the National Academy of Sciences of the
United States of America 100 1128ndash1133
Rogers P D Pearson M M Cleary J D Sullivan D C amp Chapman
S W (2002) Differential expression of genes encoding immunomodu-
latory proteins in response to amphotericin B in human mononuclear
cells identified by cDNA microarray analysis Journal of Antimicrobial
Chemotherapy 50 811ndash817
Rosen M B Wilson V S Schmid J E amp Gray L E (2005) Gene
expression analysis in the ventral prostate of rats exposed to vinclozolin
or procymidone Reproductive Toxicology 19 367ndash379
Rost D Herrmann T Sauer P Schmidts H L Stieger B Meier P J
et al (2003) Regulation of rat organic anion transporters in bile salt-
induced cholestatic hepatitis Effect of ursodeoxycholate Hepatology
38 187ndash195
Rothman R B Ayestas M A Dersch C M amp Baumann M H (1999)
Aminorex fenfluramine and chlorphentermine are serotonin transporter
substrates Implications for primary pulmonary hypertension Circula-
tion 100 869ndash875
Ruepp S U Tonge R P Shaw J Wallis N amp Pognan F (2002)
Genomics and proteomics analysis of acetaminophen toxicity in mouse
liver Toxicology Sciences 65 135ndash150
Sakaeda T Nakamura T amp Okumura K (2002) MDR1 genotype-
related pharmacokinetics and pharmacodynamics Biological amp Phar-
maceutical Bulletin 25 1391ndash1400
Sakata T Anzai N Shin H J Noshiro R Hirata T Yokoyama H
et al (2004) Novel single nucleotide polymorphisms of organic cation
transporter 1 (SLC22A1) affecting transport functions Biochemical
and Biophysical Research Communications 313 789ndash793
Satlin L M Amin V amp Wolkoff A W (1997) Organic anion
transporting polypeptide mediates organic anionHCO3-exchange
Journal of Biological Chemistry 272 26340ndash26345
Satomi Y Tsuchiya W Mihara K Ota M Kasahara Y amp Akahori F
(2004) Gene expression analysis of the lung following paraquat
administration in rats using DNA microarray Journal of Toxicological
Sciences 29 91ndash100
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 65
Schrenk D Baus P R Ermel N Klein C Vorderstemann B amp
Kauffmann H M (2001) Up-regulation of transporters of the MRP
family by drugs and toxins Toxicology Letters 120 51ndash57
Segal E Shapira M Regev A Persquoer D Botstein D Koller D et al
(2003) Module networks Identifying regulatory modules and their
condition-specific regulators from gene expression data Nature
Genetics 34 166ndash176
Segal E Wang H amp Koller D (2003) Discovering molecular pathways
from protein interaction and gene expression Bioinformatics 19
i264ndash i272
Seidel S D Kan H L Stott W T Schisler M R amp Gollapudi B B
(2003) Identification of transcriptome profiles for the DNA-damaging
agents bleomycin and hydrogen peroxide in L5178Y mouse lymphoma
cells Environmental and Molecular Mutagenesis 42 19ndash25
Shan L Yu Y Schut H A J amp Snyderwine E G (2004) Susceptibility
of rats to mammary gland carcinogenesis by the food-derived
carcinogen 2-Amino-1-Methyl-6-Phenylimidazo[45-b]Pyridine (PhIP)
varies with age and is associated with the induction of differential gene
expression American Journal of Pathology 165 191ndash202
Sharom J R Bellows D S amp Tyers M (2004) From large networks to
small molecules Current Opinion in Chemical Biology 8 81ndash90
Shitara Y Hirano M Sato H amp Sugiyama Y (2004) Gemfibrozil and
its glucuronide inhibit the OATP2(OATP1B1 SLC21A6)-mediated
hepatic uptake and CYP2C8-mediated metabolism of cerivastatinmdash
Analysis of the mechanism of the clinically relevant drugndashdrug
interaction between cerivastatin and gemfibrozil Journal of Pharma-
cology and Experimental Therapeutics
Shitara Y Itoh T Sato H Li A P amp Sugiyama Y (2003) Inhibition of
transporter-mediated hepatic uptake as a mechanism for drugndashdrug
interaction between cerivastatin and cyclosporin A Journal of
Pharmacology and Experimental Therapeutics 304 610ndash616
Somogyi R Fuhrman S amp Wen X (2001) Genetic network inference in
computational models and applications to large-scale gene expression
data In M Bower amp H Bolouri (Eds) Computational modeling of
genetic and biochemical networks (pp 119ndash157) Cambridge MArsquoThe
MIT Press
Spirin V amp Mirny L A (2003) Protein complexes and functional
modules in molecular networks Proceedings of the National Academy
of Sciences of the United States of America 100 12123ndash12128
Staudinger J Liu Y Madan A Habeebu S amp Klaassen C D
(2001) Coordinate regulation of xenobiotic and bile acid homeo-
stasis by pregnane X receptor Drug Metabolism and Disposition
29 1467ndash1472
Sun L Z Ji Z L Chen X Wang J F amp Chen Y Z (2002) ADME-
AP A database of ADME associated proteins Bioinformatics 18
1699ndash1700
Suter L Babiss L E amp Wheeldon E B (2004) Toxicogenomics in
predictive toxicology in drug development Chemistry and Biology 11
161ndash171
Synold T W Dussault I amp Forman B M (2001) The orphan nuclear
receptor SXR coordinately regulates drug metabolism and efflux
Nature Medicine 7 584ndash590
Tabuchi Y amp Kondo T (2003) cDNA microarray analysis reveals chop-
10 plays a key role in Sertoli cell injury induced by bisphenol A
Biochemical and Biophysical Research Communications 305 54ndash61
Tamai I Nezu J Uchino H Sai Y Oku A Shimane M et al (2000)
Molecular identification and characterization of novel members of the
human organic anion transporter (OATP) family Biochemical and
Biophysical Research Communications 273 251ndash260
Taylor S W Fahy E Zhang B Glenn G M Warnock D E Wiley S
et al (2003) Characterization of the human heart mitochondrial
proteome Nature Biotechnology 21 281ndash286
Terasaka S Aita Y Inoue A Hayashi S Nishigaki M Aoyagi K
et al (2004) Using a customized DNA microarray for expression
profiling of the estrogen-responsive genes to evaluate estrogen activity
among natural estrogens and industrial chemicals Environmental
Health Perspectives 112 773ndash781
Thomas D M Francescutti-Verbeem D M Liu X amp Kuhn D M
(2004) Identification of differentially regulated transcripts in mouse
striatum following methamphetamine treatmentmdashan oligonucleotide
microarray approach Journal of Neurochemistry 88 380ndash393
Thomas R S Rank D R Penn S G Zastrow G M Hayes K R
Pande K et al (2001) Identification of toxicologically predictive
gene sets using cDNA microarrays Molecular Pharmacology 60
1189ndash1194
Thompson K L Afshari C A Amin R P Bertram T A Car B
Cunningham M et al (2004) Identification of platform-independent
gene expression markers of cisplatin nephrotoxicity Environmental
Health Perspectives 112 488ndash494
Tirona R G Leake B F Merino G amp Kim R B (2001)
Polymorphisms in OATP-C Identification of multiple allelic
variants associated with altered transport activity among European-
and African-Americans Journal of Biological Chemistry 276
35669ndash35675
Tirona R G Leake B F Wolkoff A W amp Kim R B (2003)
Human organic anion transporting polypeptide-C (SLC21A6) is a
major determinant of the rifampin-mediated pregnane X receptor
activation Journal of Pharmacology and Experimental Therapeutics
304 223ndash228
Tong W Cao X Harris S Sun H Fang H Fuscoe J et al (2003)
ArrayTrackmdashsupporting toxicogenomic research at the US Food and
Drug Administration National Center for Toxicological Research
Environmental Health Perspectives 111 1819ndash1826
Tornow S amp Mewes H W (2003) Functional modules by relating
protein interaction networks and gene expression Nucleic Acids
Research 31 6283ndash6289
Trocho C Escola-Gil J C Ribas V Benitez S Martin-Campos J M
Rotllan N et al (2004) Phenytoin treatment reduces atherosclerosis in
mice through mechanisms independent of plasma HDL-cholesterol
concentration Atherosclerosis 174 275ndash285
Ueda A Hamadeh H K Webb H K Yamamoto Y Sueyoshi T
Afshari C A et al (2002) Diverse roles of the nuclear orphan
receptor CAR in regulating hepatic genes in response to phenobarbital
Molecular Pharmacology 61 1ndash6
Ulrich R G (2003) The toxicogenomics of nuclear receptor agonists
Current Opinion in Chemical Biology 7 505ndash510
Ulrich R G Rockett J C Gibson G G amp Pettit S D (2004)
Overview of an interlaboratory collaboration on evaluating the effects
of model hepatotoxicants on hepatic gene expression Environmental
Health Perspectives 112 423ndash427
Unami A Shinohara Y Ichikawa T amp Baba Y (2003) Biochemical
and microarray analyses of bupivacaine-induced apoptosis Journal of
Toxicological Sciences 28 77ndash94
Vasquez A Flammini A Maritan A amp Vespignani A (2003) Global
protein function prediction from proteinndashprotein interaction networks
Nature Biotechnology 21 697ndash700
Vezina C M Walker N J amp Olson J R (2004) Subchronic Exposure to
TCDD PeCDF PCB126 and PCB153 Effect on hepatic gene
expression Environmental Health Perspectives 112 1636ndash1644
Vo T D Greenberg H J amp Palsson B O (2004) Reconstruction and
functional characterization of the human mitochondrial metabolic
network based on proteomic and biochemical data Journal of Bio-
logical Chemistry 279 39532ndash39540
Wang H amp LeCluyse E L (2003) Role of orphan nuclear receptors in
the regulation of drug-metabolising enzymes Clinical Pharmacoki-
netics 42 1331ndash1357
Wang N Silver D L Thiele C amp Tall A R (2001) ATP-
binding cassette transporter A1 (ABCA1) functions as a choles-
terol efflux regulatory protein Journal of Biological Chemistry 276
23742ndash23747
Waring J F Cavet G Jolly R A McDowell J Dai H Ciurlionis R
et al (2003) Development of a DNA Microarray for toxicology based
on hepatoxin-regulated sequences Environmental Health Perspectives
111 863ndash870
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6666
Waring J F Gum R Morfitt D Jolly R A Ciurlionis R Heindel M
et al (2002) Identifying toxic mechanisms using DNA microarrays
Evidence that an experimental inhibitor of cell adhesion molecule
expression signals through the aryl hydrocarbon nuclear receptor
Toxicology 181ndash182 537ndash550
Waring J F Jolly R A Ciurlionis R Lum P Y Praestgaard J T
Morfitt D C et al (2001) Clustering of hepatotoxins based on
mechanism of toxicity using gene expression profiles Toxicology and
Applied Pharmacology 175 28ndash42
Waters M Boorman G Bushel P Cunningham M Irwin R Merrick
A et al (2003) Systems toxicology and the Chemical Effects in
Biological Systems (CEBS) knowledge base EHP Toxicogenomics
111 15ndash28
Wei M Arnold L Cano M amp Cohen S M (2005) Effects of co-
administration of antioxidants and arsenicals on the rat urinary bladder
epithelium Toxicology Sciences 83 237ndash245
Weinshilboum R (2003) Inheritance and drug response New England
Journal of Medicine 348 529ndash537
Weinshilboum R amp Wang L (2004) Pharmacogenetics Inherited
variation in amino acid sequence and altered protein quantity Clinical
Pharmacology and Therapeutics 75 253ndash258
Werner E (2003) In silico multicellular systems biology and minimal
genomes Drug Discovery Today 8 1121ndash1127
Wong J S amp Gill S S (2002) Gene expression changes induced in
mouse liver by di(2-ethylhexyl) phthalate Toxicology and Applied
Pharmacology 185 180ndash196
Xie T Tong L McCann U D Yuan J Becker K G Mechan A O
et al (2004) Identification and characterization of metallothionein-1
and -2 gene expression in the context of ()34-methylenedioxymetham-
phetamine-induced toxicity to brain dopaminergic neurons Journal of
Neuroscience 24 7043ndash7050
Yadetie F Laegreid A Bakke I Kusnierczyk W Komorowski J
Waldum H L et al (2003) Liver gene expression in rats in response
to the peroxisome proliferator-activated receptor-alpha agonist ciprofi-
brate Physiological Genomics 15 9ndash19
Yamashita S Nomoto T Abe M Tatematsu M Sugimura T amp
Ushijima T (2004) Persistence of gene expression changes in stomach
mucosae induced by short-term N-methyl-NV-nitro-N-nitrosoguanidinetreatment and their presence in stomach cancers Mutation Research
549 185ndash193
Yan Q amp Sadee W (2000) Human membrane transporter database A
web-accessible relational database for drug transport studies and
pharmacogenomics AAPS Pharmsci 2 E20
Yeger-Lotem E amp Margalit H (2003) Detection of regulatory circuits by
integrating the cellular networks of proteinndashprotein interactions and
transcription regulation Nucleic Acids Research 31 6053ndash6061
Yoon B I Li G -X Kitada K Kawasaki Y Igarashi K amp Kodama
Y et al (2003) Mechanisms of benzene-induced hematotoxicity and
leukemogenicity cDNA microarray analyses using mouse bone marrow
tissue Environmental Health Perspectives 111 1411ndash1420
Young M B DiSilvestro M R Sendera T J Freund J Kriete A amp
Magnuson S R (2003) Analysis of gene expression in carbon
tetrachloride-treated rat livers using a novel bioarray technology
Pharmacogenomics Journal 3 41ndash52
Yu H Zhu X Greenbaum D Karro J amp Gerstein M (2004) TopNet
A tool for comparing biological sub-networks correlating protein
properties with topological statistics Nucleic Acids Research 32
328ndash337
Zhang E Y Fu D J Pak Y A Stewart T Mukhopadhyay N
Wrighton S A et al (2004) Genetic polymorphisms in human proton-
dependent dipeptide transporter PEPT1 Implications for the functional
role of Pro586 Journal of Pharmacology and Experimental Therapeu-
tics 310 437ndash445
Zhang E Y Knipp G T Ekins S amp Swaan P W (2002)
Structural biology and function of solute transporters Implications
for identifying and designing substrates Drug Metabolism Reviews
34 709ndash750
Zhang E Y Phelps M A Cheng C Ekins S amp Swaan P W (2002)
Modeling of active transport systems Advanced Drug Delivery
Reviews 54 329ndash354
- Systems-ADMETox Resources and network approaches
-
- Introduction
- Data available
- Network analysis and databases
-
- Network applications
-
- The role of transporters
-
- Clinical relevance of transporters
- Transporter network examples ABCA1
- Transporter network examples OATP
- Transporter microarray data
-
- Applications to enzymes
- Future network applications
-
- Discussion
- Acknowledgements
- References
-
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6660
in silico (Ekins et al 2000b) and approaches for drug
metabolism (Ekins Ring Grace McRobie-Belle amp
Wrighton 2000a) have been described Both of these
reviews commented on moving HT assays for ADME
Tox much earlier in drug discovery which had also been
indicated by other groups The initial reviews also
highlighted the likely wealth of data that would become
available and how this could be used for structure
activity relationships alongside the bioactivity data in
computational models It was noted that there was a
paucity of predictive metabolism tools at that time In
addition a growing number of efforts to model whole
cells and organs now a field called systems biology
were recognized as models that could be integrated with
the in silico ADME approaches In summary since these
past reviews virtually all pharmaceutical companies have
attempted earlier high throughput screening for ADME
Tox properties and to some extent the wider application
of computational approaches for physicochemical proper-
ties Systems biology is being quite widely acknowledged
as the new paradigm for understanding complex bio-
logical datasets derived from high throughput technolo-
gies and the accumulated knowledge on human protein
interactions (Hartwell Hopfield Leibler amp Murray 1999
Hood 2003b) Therefore systems biology can be defined
as the integration of genetic proteomic transcriptomic
and metabonomic data using computational methods
(Nicholson amp Wilson 2003) When taken together
information on molecular processes derived from different
sources represents a lsquolsquouniversersquorsquo of putative biological
functionality of which only a small fraction of it will be
realized in a cell at any given time To date systems
biology has been driven by academia and funding bodies
such as the NIH rather than the pharmaceutical compa-
nies Presently there is a great deal of interest from
scientists of all backgrounds in identifying the networks
of cellular pathways and the corresponding physically
interacting proteins
The network building software for systems biology
described in this current review will be valuable to query
high throughput data and known literature interactions in
order to predict potential toxicity in different species In the
future the compilation of published toxicogenomics data-
sets characteristic of different types of toxicity will likely
be available in these software systems to act as a reference
database It is also feasible that we will be able to generate
the annotated datasets which specifically address the
differences between human and rat networks implicated
in toxicity The identification of sub-network modules
conserved between human and rat distinct for toxicity
types or predictive for toxic end-points in human will be
possible Such signature gene networks (Nikolsky et al
2005) can then be verified with other experimentally
derived data prospectively or from preexisting databases
The combined Fhigh throughput datandashin silico network_approaches therefore represents a novel method for access-
ing complex tissue-level processes as networks integrating
all data types based on functional interactions The data-
bases developed and used in this approach will certainly
benefit from further annotation around the drug metaboliz-
ing enzymes and transporters as described herein in terms
of transcriptional regulation and the ligands associated with
them which frequently appear in the literature It will be
important to capture disparities in the assignment of ligands
to enzymes transporters and other proteins as well as
negative data
In summary although there have been numerous
toxicogenomics studies published there is presently a
relatively small number of datasets that are freely available
to perform network analysis of microarray data The
number of studies identifying large numbers of proteins
which are affected by molecule treatment are even scarcer
still while there are several examples of some published
studies that combine such data It is hoped that the
numerous database initiatives for high content and toxicol-
ogy data that are being undertaken will improve the
situation for other researchers that are not currently
equipped to do such microarray studies themselves The
addition of requirements by journals to deposit such raw
data in a freely accessible resource will aid these initiatives
ADMETox groups have seen new technologies and
approaches developed over the last decade that have all
been applied to identify poor compounds earlier (Fig 2)
The latest technologies integrate network building tools
with high content data and databases The current review
described the limited number of networks generated for
ADMETox at present and one hopes that the impact of
such analyses will be commonplace in the future Systems
biology is however more than just applying a network
approach and hence systems-ADMETox will have to
evolve due to the continual pressure to develop newer
technologies This current paradigm combining empirical
data and computational methods should integrate the
complex data already generated making it readily inter-
pretable and valuable for identifying the most promising
compounds in the future
Acknowledgements
Dr Maggie AZ Hupcey is gratefully acknowledged for
editorial assistance Dr Peter W Swaan (University of
Maryland) and Dr Cheng Chang (Ohio State University)
Dr Steve Wright (University of Arizona) Dr K Sandy
Pang (University of Toronto) and Dr Craig Giroux (Wayne
State University) are kindly acknowledged for their support
and discussions My colleagues at GeneGo Sergey
Andreyev Andy Ryabov Eugene Kirillov Eugene A
Rakhmatulin Svetlana Sorokina Andrej Bugrim Tatiana
Nikolskaya Yuri Nikolsky John Metz and Julie Bryant are
all thanked for their considerable contributions to software
development and data annotation
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 61
References
Abe M Yamashita S Kuramoto T Hirayama Y Tsukamoto T Ohta
T et al (2003) Global expression analysis of N-methyl-NV-nitro-N-nitrosoguanidine-induced rat stomach carcinomas using oligonucleotide
microarrays Carcinogenesis 24 861ndash867
Adachi T Ono Y Koh K B Takashima K Tainaka H Matsuno Y
et al (2004) Long-term alteration of gene expression without
morphological change in testis after neonatal exposure to genistein in
mice Toxicogenomic analysis using cDNA microarray Food and
Chemical Toxicology 42 445ndash452
Akerman G S Rosenzweig B A Domon O E McGarrity L J
Blankenship L R Tsai C A et al (2004) Gene expression profiles
and genetic damage in benzo(a)pyrene diol epoxide-exposed TK6 cells
Mutation Research 549 43ndash64
Aksenov S V Church B Dhiman A Georgieva A Sarangapani R
Helmlinger G et al (2005) An integrated approach for inference and
mechanistic modeling for advancing drug development FEBS Letters
579 1878ndash1883
Anderle P Huang Y amp Sadee W (2004) Intestinal membrane transport
of drugs and nutrients Genomics of membrane transporters using
expression microarrays European Journal of Pharmaceutical Sciences
21 17ndash24
Annereau J P Szakacs G Tucker C J Arciello A Cardarelli C
Collins J et al (2004) Analysis of ATP-binding cassette transporter
expression in drug-selected cell lines by a microarray dedicated to
multidrug resistance Molecular Pharmacology 66 1397ndash1405
Apic G Ignjatovic T Boyer S amp Russell R B (2005) Illuminating drug
discovery with biological pathways FEBS Letters 579 1872ndash1877
Baker V A Harries H M Waring J F Duggan C M Ni H A Jolly
R A et al (2004) Clofibrate-induced gene expression changes in rat
liver A cross-laboratory analysis using membrane cDNA arrays
Environmental Health Perspectives 112 428ndash438
Balakin K V Ekins S Bugrim A Ivanenkov Y A Korolev D amp
Nikolsky Y et al (2004a) Quantitative structurendashmetabolism relation-
ship modeling of the metabolic N-dealkylation rates Drug Metabolism
and Disposition 32 1111ndash1120
Balakin K V Ekins S Bugrim A Ivanenkov Y A Korolev D amp
Nikolsky Y et al (2004b) Kohonen maps for prediction of binding to
human cytochrome P450 3A4 Drug Metabolism and Disposition 32
1183ndash1189
Barabasi A -L amp Oltvai Z N (2004) Network biology Under-
standing the cellrsquos functional organization Nature Reviews Genetics
5 101ndash113
Bartosiewicz M Penn S G amp Buckpitt A (2001) Applications of gene
arrays in environmental toxicology Fingerprints of gene regulation
associated with cadmium chloride benzo(a)pyrene and trichloro-
ethylene Environmental Health Perspectives 109 71ndash74
Bertilsson G Heidrich J Svensson K Asman M Jendeberg L
Sydow-Backman M et al (1998) Identification of a human nuclear
receptor defines a new signaling pathway for CYP3A induction
Proceedings of the National Academy of Sciences of the United States
of America 95 12208ndash12213
Blumberg B Sabbagh Jr W Juguilon H Bolado Jr J van Meter
C M Ong E S et al (1998) SXR a novel steroid and xenobiotic-
sensing nuclear receptor Genes and Development 12 3195ndash3205
Borodina Y Rudik A Filimonov D Kharchevnikova N Dmitriev A
Blinova V et al (2004) A new statistical approach to predicting
aromatic hydroxylation sites Comparison with model-based
approaches Journal of Chemical Information and Computer Sciences
44 1998ndash2009
Borodina Y Sadym A Filimonov D Blinova V Dmitriev A amp
Poroikov V (2003) Predicting biotransformation potential from
molecular structure Journal of Chemical Information and Computer
Sciences 43 1636ndash1646
Bottone Jr F G Martinez J M Collins J B Afshari C A amp Eling
T E (2003) Gene modulation by the cyclooxygenase inhibitor
sulindac sulfide in human colorectal carcinoma cells Possible link to
apoptosis Journal of Biological Chemistry 278 25790ndash25801
Boyer S amp Zamora I (2002) New methods in predictive metabolism
Journal of Computer-Aided Molecular Design 16 403ndash413
Bredel M amp Jacoby E (2004) Chemogenomics An emerging strategy
for rapid target and drug discovery Nature Reviews Genetics 5
262ndash275
Bugrim A Nikolskaya T amp Nikolsky Y (2004) Early prediction of drug
metabolism and toxicity Systems biology approach and modeling
Drug Discovery Today 9 127ndash135
Bulera S J Eddy S M Ferguson E Jatkoe T A Reindel J F
Bleavins M R et al (2001) RNA expression in the early character-
ization of hepatotoxicants in Wistar rats by high-density DNA micro-
arrays Hepatology 33 1239ndash1258
Butcher E C Berg E L amp Kunkel E J (2004) Systems biology in drug
discovery Nature Biotechnology 22 1253ndash1259
Butte A (2002) The use and analysis of microarray data Naturalist
Review of Drug Discovery 1 951ndash960
Cary M P Bader G D amp Sander C (2005) Pathway information for
systems biology FEBS Letters 579 1815ndash1820
Cheok M H Yang W Pui C H Downing J R Cheng C Naeve
C W et al (2003) Treatment-specific changes in gene expression
discriminate in vivo drug response in human leukemia cells Nature
Genetics 34 85ndash90
Cleary J D Rogers P D amp Chapman S W (2001) Differential
transcription factor expression in human mononuclear cells in response
to amphotericin B Identification with complementary DNA microarray
technology Pharmacotherapy 21 1046ndash1054
Coen M Ruepp S U Lindon J C Nicholson J K Pognan F Lenz
E M et al (2004) Integrated application of transcriptomics and
metabonomics yields new insight into the toxicity due to paracetamol in
the mouse Journal of Pharmaceutical and Biomedical Analysis 35
93ndash105
Cornwell P D De Souza A T amp Ulrich R G (2004) Profiling of
hepatic gene expression in rats treated with fibric acid analogs
Mutation Research 549 131ndash145
Csermely P Agoston V amp Pongor S (2005) The efficiency of multi-
target drugs The network approach might help drug design Trends in
Pharmacological Sciences 26 178ndash182
Cunningham M J Liang S Fuhrman S Seilhamer J J amp
Somogyi R (2000) Gene expression microarray data analysis for
toxicology profiling Annals of the New York Academy of Sciences 919
52ndash67
de Longueville F Atienzar F A Marcq L Dufrane S Evrard S
Wouters L et al (2003) Use of a low density microarray for studying
gene expression patterns induced by hepatotoxicants on primary
cultures of rat hepatocytes Toxicological Sciences 75 378ndash392
Dobrin R Beg Q K Barabasi A L amp Oltvai Z N (2004)
Aggregation of topological motifs in the Escherichia coli transcriptional
regulatory network BMC Bioinformatics 5 10
Donald S Verschoyle R D Edwards R Judah D J Davies R
Riley J et al (2002) Hepatobiliary damage and changes in hepatic
gene expression caused by the antitumor drug ecteinascidin-743 (ET-
743) in the female rat Cancer Research 62 4256ndash4262
Dumont M Jacquemin E DrsquoHont C Descout C Cresteil D Haouzi
D et al (1997) Expression of the liver Na+-independent organic anion
transporting polypeptide (oatp-1) in rats with bile duct ligation Journal
of Hepatology 27 1051ndash1056
Durnas C Loi C M amp Cusack B J (1990) Hepatic drug metabolism
and aging Clinical Pharmacokinetics 19 359ndash389
Eisen M B Spellman P T Brown P O amp Botstein D (1998) Cluster
analysis and display of genome-wide expression patterns Proceedings
of the National Academy of Sciences of the United States of America
95 14863ndash14868
Ekins S Andreyev S Ryabov A Kirilov E Rakhmatulin E A
Bugrim A et al (in press) Computational Prediction of Human Drug
Metabolism Exp Opin Drug Metab Toxicol
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6662
Ekins S Boulanger B Swaan P W amp Hupcey M A Z (2002)
Towards a new age of virtual ADMETOX and multidimensional drug
discovery Journal of Computer-Aided Molecular Design 16 381ndash401
Ekins S Bugrim A Nikolsky Y amp Nikolskaya T (2005) Systems
biology Applications in drug discovery In S Gad (Ed) Drug
Discovery Handbook (pp 123ndash183) Hoboken NJrsquo Wiley and Sons
Ekins S Giroux C N Nikolsky Y Bugrim A amp Nikolskaya T
(2005) A signature gene network approach to toxicity The Toxoco-
logists 84
Ekins S Kirillov E Rakhmatulin E amp Nikolskaya T (2005) A novel
method for visualizing nuclear hormone receptor networks relevant to
drug metabolism Drug Metabolism and Disposition 33 474ndash481
Ekins S amp McGowan R J (2001) The limits of reductionism The
shifting genomic paradigmrsquos impact on industry and academia
Philsophy in Science 9 1ndash23
Ekins S Mirny L amp Schuetz E G (2002) A ligand-based approach to
understanding selectivity of nuclear hormone receptors PXR CAR
FXR LXRa and LXRb Pharmaceutical Research 19 1788ndash1800
Ekins S Nikolsky Y amp Nikolskaya T (2005) Techniques Application
of systems biology to absorption distribution metabolism excretion
and toxicity Trends in Pharmacological Sciences 26 202ndash209
Ekins S Ring B J Grace J McRobie-Belle D J amp Wrighton S
A (2000) Present and future in vitro approaches for drug
metabolism Journal of Pharmacological and Toxicological Methods
44 313ndash324
Ekins S Waller C L Swaan P W Cruciani G Wrighton S A amp
Wikel J H (2000) Progress in predicting human ADME parameters in
silico Journal of Pharmacological and Toxicological Methods 44
251ndash272
Erhardt P W (2003) A human drug metabolism database Potential roles
in the quantitative predictions of drug metabolism and metabolism-
related drugndashdrug interactions Current Drug Metabolism 4 411ndash422
Evans W E amp McLeod H L (2003) Pharmacogenomicsmdashdrug
disposition drug targets and side effects New England Journal of
Medicine 348 538ndash549
Ezendam J Staedtler F Pennings J Vandebriel R J Pieters R
Boffetta P et al (2004) Toxicogenomics of subchronic hexachlor-
obenzene exposure in Brown Norway rats Environmental Health
Perspectives 112 782ndash791
Faiola B Fuller E S Wong V A amp Recio L (2004) Gene expression
profile in bone marrow and hematopoietic stem cells in mice exposed to
inhaled benzene Mutation Research 549 195ndash212
FDA (2004) Innovation stagnation Challenge and opportunity on the
critical path to new medicinal products
Fiehn O (2001) Combining genomics metabolome analysis and bio-
chemical modelling to understand metabolic networks Comparative
Functional Genomics 2 155ndash168
Field F J Born E amp Mathur S N (2004) LXRRXR ligand activation
enhances basolateral efflux of beta-sitosterol in CaCo-2 cells Journal of
Lipid Research 45 905ndash913
Fleck C Sutter L Appenroth D Koch B Meinhold T Pitack M
et al (2003) Use of gene chip technology for the characterisation of
the regulation of renal transport processes and of nephrotoxicity in
rats Experimental and Toxicologic Pathology 54 401ndash410
Fliri A F Loging W T Thadeio P F amp Volkmann R A (2005)
Biological spectra analysis Linking biological activity profiles to
molecular structure Proceedings of the National Academy of Sciences
of the United States of America 102 261ndash266
Fountoulakis M (2004) Application of proteomics technologies in the
investigation of the brain Mass Spectrometry Reviews 23 231ndash258
Gaucher S P Taylor S W Fahy E Zhang B Warnock D E Ghosh
S S et al (2004) Expanded coverage of the human heart
mitochondrial proteome using multidimensional liquid chromatography
coupled with tandem mass spectrometry Journal of Proteome
Research 3 495ndash505
Gerhold D Lu M Xu J Austin C Caskey C T amp Rushmore T
(2001) Monitoring expression of genes involved in drug metabolism
and toxicology using DNA microarrays Physiological Genomics 5
161ndash170
Guo G L Choudhuri S amp Klaassen C D (2002) Induction profile of
rat organic anion transporting polypeptide 2 (oatp2) by prototypical
drug-metabolizing enzyme inducers that activate gene expression
through ligand-activated transcription factor pathways Journal of
Pharmacology and Experimental Therapeutics 300 206ndash212
Hagenbuch B amp Meier P J (2004) Organic anion transporting
polypeptides of the OATP SLC21 family Phylogenetic classification
as OATP SLCO superfamily new nomenclature and molecularfunc-
tional properties Pflugers Archiv 447 653ndash665
Hamadeh H K amp Afshari C A (2004) Toxicogenomics principles and
applications Hobokenrsquo John Wiley and Sons
Hamadeh H K Bushel P R Jayadev S Martin K DiSorbo O Sieber
S et al (2002) Gene expression analysis reveals chemical-specific
profiles Toxicological Sciences 67 219ndash231
Hamadeh H K Jayadev S Gaillard E T Huang Q Stoll R
Blanchard K et al (2004) Integration of clinical and gene expression
endpoints to explore furan-mediated hepatotoxicity Mutation Research
549 169ndash183
Hamadeh H K Knight B L Haugen A C Sieber S Amin R P
Bushel P R et al (2002) Methapyrilene toxicity Anchorage of
pathologic observations to gene expression alterations Toxicologic
Pathology 30 470ndash482
Han J D Bertin N Hao T Goldberg D S Berriz G F Zhang L V
et al (2004) Evidence for dynamically organized modularity in the
yeast proteinndashprotein interaction network Nature 430 88ndash93
Hanisch D Zien A Zimmer R amp Lengauer T (2002) Co-clustering of
biological networks and gene expression data Bioinformatics 18
S145ndashS154
Harris A J Dial S L amp Casciano D A (2004) Comparison of basal
gene expression profiles and effects of hepatocarcinogens on gene
expression in cultured primary human hepatocytes and HepG2 cells
Mutation Research 549 79ndash99
Harrison S C (2004) Whither structural biology Nature Structural
Molecular Biology 11 12ndash15
Hartley D P Dai X He Y D Carlini E J Wang B Huskey S E
et al (2004) Activators of the rat pregnane X receptor differentially
modulate hepatic and intestinal gene expression Molecular Pharma-
cology 65 1159ndash1171
Hartwell L H Hopfield J J Leibler S amp Murray A W (1999) From
molecular to modular cell biology Nature 402 C47ndashC52
Hasmall S Orphanides G James N Pennie W Hedley K Soames
A et al (2002) Downregulation of lactoferrin by PPARalpha ligands
Role in perturbation of hepatocyte proliferation and apoptosis
Toxicology Sciences 68 304ndash313
Hayase T Yamamoto Y Yamamoto K Muso E amp Shiota K (2004)
Microarray profile analysis of toxic cocaine-induced alterations in the
expression of mouse brain gene sequences A possible rsquoprotectiversquo
effect of buprenorphine Journal of Applied Toxicology 24 15ndash20
Hayes K R Vollrath A L Zastrow G M McMillan B J Craven M
Jovanovich S et al (2005) EDGE A centralized resource for the
comparison analysis and distribution of toxicogenomic information
Molecular Pharmacology 67 1360ndash1368
Heijne W H Slitt A L van Bladeren P J Groten J P Klaassen C D
Stierum R H et al (2004) Bromobenzene-induced hepatotoxicity at
the transcriptome level Toxicology Sciences 79 411ndash422
Heijne W H Stierum R H Slijper M van Bladeren P J amp van
Ommen B (2003) Toxicogenomics of bromobenzene hepatotoxicity
A combined transcriptomics and proteomics approach Biochemical
Pharmacology 65 857ndash875
Heinloth A N Irwin R D Boorman G A Nettesheim P Fannin
R D Sieber S O et al (2004) Gene expression profiling of rat
livers reveals indicators of potential adverse effects Toxicology
Sciences 80 193ndash202
Hodges L C Cook J D Lobenhofer E K Li L Bennett L Bushel
P R et al (2003) Tamoxifen functions as a molecular agonist inducing
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 63
cell cycle-associated genes in breast cancer cells Molecular Cancer
Research 1 300ndash311
Holleman A Cheok M H den Boer M L Yang W Veerman KM
A J Kazemier et al (2004) Gene-expression patterns in drug-resistant
acute lymphoblastic leukemia cells and response to treatment New
England Journal of Medicine 351 533ndash542
Hong Y Muller U R amp Lai F (2003) Discriminating two classes of
toxicants through expression analysis of HepG2 cells with DNA arrays
Toxicology in Vitro 17 85ndash92
Hood E (2003) Proteomics Characterizing the cogs in the machinery of
life Environmental Health Perspectives 111 A816ndashA825
Hood L (2003) Systems biology Integrating technology biology and
computation Mechanism of Ageing and Development 124 9ndash16
Hood L amp Galas D (2003) The digital code of DNA Nature 421
444ndash448
Hood L amp Perlmutter R M (2004) The impact of systems approaches on
biological problems in drug discovery Nature Biotechnology 22
1215ndash1217
Hu T Gibson D P Carr G J Torontali S M Tiesman J P Chaney
J G et al (2004) Identification of a gene expression profile that
discriminates indirect-acting genotoxins from direct-acting genotoxins
Mutation Research 549 5ndash27
Huang Q Dunn II R T Jayadev S DiSorbo O Pack F D Farr S B
et al (2001) Assessment of cisplatin-induced nephrotoxicity by
microarray technology Toxicology Sciences 63 196ndash207
Huang Q Jin X Gaillard E T Knight B L Pack F D Stoltz J H et
al (2004) Gene expression profiling reveals multiple toxicity endpoints
induced by hepatotoxicants Mutation Research 549 147ndash167
Huisman M T Smit J W Crommentuyn K M Zelcer N Wiltshire
H R Beijnen J H et al (2002) Multidrug resistance protein 2
(MRP2) transports HIV protease inhibitors and transport can be
enhanced by other drugs Aids 16 2295ndash2301
Hunt C M Westerkam W R amp Stave G M (1992) Effect of age and
gender on the activity of human hepatic CYP3A Biochemical
Pharmacology 44 275ndash283
Ideker T Ozier O Schwikowski B amp Siegal A F (2002) Discovering
regulatory and signalling circuits in molecular interaction networks
Bioinformatics 18 S233ndashS240
Ideker T Thorsson V Ranish J A Christmas R Buhler J Eng J K
et al (2001) Integrated genomic and proteomic analyses of a system-
atically perturbed network Science 292 929ndash934
Iida M Anna C H Hartis J Bruno M Wetmore B Dubin J R et al
(2003) Changes in global gene and protein expression during early
mouse liver carcinogenesis induced by non-genotoxic model carcino-
gens oxazepam and Wyeth-14643 Carcinogenesis 24 757ndash770
Ingelman-Sundberg M (2004) Pharmacogenetics of cytochrome P450 and
its applications in drug therapy The past present and future Trends in
Pharmacological Sciences 25 193ndash200
Jeong H Mason S P Barabasi A L amp Oltvai Z N (2001) Lethality
and centrality in protein networks Nature 411 41ndash42
Jeong H Tombor B Albert R Oltvai Z N amp Barabasi A L
(2000) The large-scale organization of metabolic networks Nature
407 651ndash654
Jung D Hagenbuch B Gresh L Pontoglio M Meier P J amp Kullak-
Ublick G A (2001) Characterization of the human OATP-C
(SLC21A6) gene promoter and regulation of liver-specific OATP genes
by hepatocyte nuclear factor 1 alpha Journal of Biological Chemistry
276 37206ndash37214
Jung D amp Kullak-Ublick G A (2003) Hepatocyte nuclear factor 1
alpha A key mediator of the effect of bile acids on gene expression
Hepatology 37 622ndash631
Jung J -W Park J -S Hwang J -W Kang K -S YS L Song B
-S et al (2004) Gene expression analysis of peroxisome prolifer-
ators- and phenytoin-induced hepatotoxicity using cDNA microarray
Journal of Veterinary Medical Science 66 1329ndash1333
Kato N Shibutani M Takagi H Uneyama C Lee K -Y Takiami S
et al (2004) Gene expression profile in the livers of rats orally
administered ethinylestradiol for 28 days using a microarray technique
Toxicology 200 179ndash192
Kier L D Neft R Tang L Suizu R Cook T Onsurez K et al
(2004) Applications of microarrays with toxicologically relevant genes
(tox genes) for the evaluation of chemical toxicants in Sprague Dawley
rats in vivo and human hepatocytes in vitro Mutation Research 549
101ndash113
Kijima K Toyosawa K Yasuba M Matsuoka N Adachi T
Komiyama M et al (2004) Gene expression analysis of the rat testis
after treatment with di(2-ethylhexyl) phthalate using cDNA microarray
and real-time RT-PCR Toxicology and Applied Pharmacology 200
103ndash110
Kim R B (2003) Organic anion-transporting polypeptide (OATP) trans-
porter family and drug disposition European Journal of Clinical
Investigation 33 1ndash5
Kinirons M T amp OrsquoMahony M S (2004) Drug metabolism and ageing
British Journal of Clinical Pharmacology 57 540ndash544
Kitano H (2002a) Computational systems biology Nature 420 206ndash210
Kitano H (2002b) Systems biology A brief overview Science 295
1662ndash1664
Kliewer S A Moore J T Wade L Staudinger J L Watson M
A Jones S A et al (1998) An orphan nuclear receptor activated
by pregnanes defines a novel steroid signalling pathway Cell 92
73ndash82
Korolev D Balakin K V Nikolsky Y Kirillov E Ivanenkov Y A
Savchuk N P et al (2003) Modeling of human cytochrome p450-
mediated drug metabolism using unsupervised machine learning
approach Journal of Medicinal Chemistry 46 3631ndash3643
Kramer J A Pettit S D Amin R P Bertram T A Car B
Cunningham M et al (2004) Overview on the application of
transcription profiling using selected nephrotoxicants for toxicology
assessment Environmental Health Perspectives 112 460ndash464
Kultima K Nystrom A -M Scholz B Gustafson A -L Dencker L
amp Stigson M (2004) Valproic acid teratogenicity A toxicogenomics
approach Environmental Health Perspectives 112 1225ndash1235
Kutuzova G D amp Deluca H F (2004) Gene expression profiles in rat
intestine identify pathways for 125-dihydroxyvitamin D(3) stimulated
calcium absorption and clarify its immunomodulatory properties
Archives of Biochemistry and Biophysics 432 152ndash166
Kwak M K Wakabayashi N Itoh K Motohashi H Yamamoto M amp
Kensler T W (2003) Modulation of gene expression by cancer
chemopreventive dithiolethiones through the Keap1-Nrf2 pathway
Identification of novel gene clusters for cell survival Journal of
Biological Chemistry 278 8135ndash8145
Lahjouji K Mitchell G A amp Qureshi I A (2001) Carnitine transport
by organic cation transporters and systemic carnitine deficiency
Molecular Genetics and Metabolism 73 287ndash297
Landowski C P Sun D Foster D R Menon S S Barnett J L
Welage L S et al (2003) Gene expression in the human intestine and
correlation with oral valacyclovir pharmacokinetic parameters Journal
of Pharmacology and Experimental Therapeutics 306 778ndash786
Lee G -J Lee W -S Jeon K -S Um C -H Kim S -J Lee C -H
et al (2004) cDNA microarray gene expression analysis and
toxicological phenotype for anticancer drug Journal of Veterinary
Medical Science 66 1339ndash1345
Lee M Kwon J Kim S N Kim J E Koh W S Kim E J et al
(2003) cDNA microarray gene expression profiling of hydroxyurea
paclitaxel and p-anisidine genotoxic compounds with differing
tumorigenicity results Environmental and Molecular Mutagenesis
42 91ndash97
Li S Armstrong C M Bertin N Ge H Milstein S Boxem M et al
(2004) A map of the interactome network of the metazoan C elegans
Science 303 540ndash543
Liguori M J Anderson L M Bukofzer S McKim J Pregenzer J F
Retief J et al (2005) Microarray analysis in human hepatocytes
suggests a mechanism for hepatotoxicity induced by trovafloxacin
Hepatology 41 177ndash186
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6664
Lobenhofer E K Bennett L Cable P L Li L Bushel P R amp Afshari
C A (2002) Regulation of DNA replication fork genes by 17beta-
estradiol Molecular Endocrinology 16 1215ndash1229
Locker J Tian J Carver R Concas D Cossu C Ledda-Columbano
G M et al (2003) A common set of immediate-early response genes
in liver regeneration and hyperplasia Hepatology 38 314ndash325
Mattes W B Pettit S D Sansone S A Bushel P R amp Waters M D
(2004) Database development in toxicogenomics Issues and efforts
Environmental Health Perspectives 112 495ndash505
McMillian M Nie A Y Parker J B Leone A Bryant S Kemmerer
M et al (2004) A gene expression signature for oxidant stressreactive
metabolites in rat liver Biochemical Pharmacology 68 2249ndash2261
Meier P J amp Stieger B (2000) Molecular mechanisms in bile formation
News in Physiological Sciences 15 89ndash93
Meneses-Lorente G de Longueville F Dos Santos-Mendes S Bonnert
T P Jack A Evrard S et al (2003) An evaluation of a low-density
DNA microarray using cytochrome P450 inducers Chemical Research
in Toxicology 16 1070ndash1077
Meneses-Lorente G Guest P C Lawrence J Muniappa N Knowles
M R Skynner H A et al (2004) A proteomic investigation of drug-
induced steatosis in rat liver Chemical Research in Toxicology 17
605ndash612
Merrill C L Ni H Yoon L W Tirmenstein M A Narayanan P
Benavides G R et al (2002) Etomoxir-induced oxidative stress in
HepG2 cells detected by differential gene expression is confirmed
biochemically Toxicology Sciences 68 93ndash101
Milo R Shen-Orr S Itzkovitz S Kashtan N Chklovskii D amp Alon
U (2002) Network motifs Simple building blocks of complex
networks Science 298 824ndash827
Morgan K T Ni H Brown H R Yoon L Qualls C W Crosby L
M et al (2002) Application of cDNA microarray technology to in
vitro toxicology and the selection of genes for a real-time RT-PCR-
based screen for oxidative stress in Hep-G2 cells Toxicologic
Pathology 30 435ndash451
Mwinyi J Johne A Bauer S Roots I amp Gerloff T (2004) Evidence
for inverse effects of OATP-C (SLC21A6) 5 and 1b haplotypes on
pravastatin kinetics Clinical Pharmacology and Therapeutics 75
415ndash421
Nakata K Yukawa M Komiyama N Nakano T amp Kaminuma T
(2002) A nuclear receptor database that maps pathways to diseases
Genome Infomatics 13 515ndash516
Nicholson J K Holmes E Lindon J C amp Wilson I D (2004) The
challenges of modeling mammalian biocomplexity Nature Biotechnol-
ogy 22 1268ndash1274
Nicholson J K amp Wilson I D (2003) Understanding rsquoglobal systems
biology Metabonomics and the continuum of metabolism Nature
Reviews on Drug Discovery 2 668ndash676
Nikitin A Egorov S Daraselia N amp Mazo I (2003) Pathway studiomdash
the analysis and navigation of molecular networks Bioinformatics 19
2155ndash2157
Nikolsky Y Ekins S Nikolskaya T amp Bugrim A (2005) A novel
method for generation of signature networks as biomarkers from
complex high throughput data Toxicology Letters 158 20ndash29
Nikolsky Y Nikolskaya T amp Bugrim A (2005) Biological networks
and analysis of experimental data in drug discovery Drug Discovery
Today 10 653ndash662
Nishizato Y Ieiri I Suzuki H Kimura M Kawabata K Hirota T
et al (2003) Polymorphisms of OATP-C (SLC21A6) and OAT3
(SLC22A8) genes Consequences for pravastatin pharmacokinetics
Clinical Pharmacology and Therapeutics 73 554ndash565
Nozawa T Nakajima M Tamai I Noda K Nezu J Sai Y et al
(2002) Genetic polymorphisms of human organic anion transporters
OATP-C (SLC21A6) and OATP-B (SLC21A9) Allele frequencies in
the Japanese population and functional analysis Journal of Pharmacol-
ogy and Experimental Therapeutics 302 804ndash813
Nozawa T Sugiura S Nakajima M Goto A Yokoi T Nezu J et al
(2004) Involvement of organic anion transporting polypeptides in the
transport of troglitazone sulfate Implications for understanding trogli-
tazone hepatotoxicity Drug Metabolism and Disposition 32 291ndash294
Oliver D E Rubin D L Stuart J M Hewett M Klein T E amp
Altman R B (2002) Ontology development for a pharmacogenetics
knowledge base Package Symposion 88ndash99
Oram J F amp Lawn R M (2001) ABCA1 The gatekeeper for eliminating
excess tissue cholesterol Journal of Lipid Research 42 1173ndash1179
Oswald M Kullak-Ublick G A Paumgartner G amp Beuers U (2001)
Expression of hepatic transporters OATP-C and MRP2 in primary
sclerosing cholangitis Liver 21 247ndash253
Parsons A B Brost R I Ding H Li Z Zhang C Sheikh B et al
(2004) Integration of chemical-genetic and genetic interaction data
links bioactive compounds to cellular target pathways Nature Bio-
technology 22 62ndash69
Patki K C von Moltke L L Harmatz J S Hesse L M Court M H
amp Greenblatt D J (2004) Effect of age on in vitro triazolam
biotransformation in male human liver microsomes Journal of
Pharmacology and Experimental Therapeutics 308 874ndash879
Pereira-Leal J B Enright A J amp Ouzounis C A (2004) Detection of
functional modules from protein interaction networks Proteins 54
49ndash57
Plant N (2004) Interaction networks Coordinating responses to xeno-
biotic exposure Toxicology 202 21ndash32
Raghavendra Prasad H S Qi Z Srinivasan K N amp Gopalakrish-
nakone P (2004) Potential effects of tetrodotoxin exposure to
human glial cells postulated using microarray approach Toxicon 44
597ndash608
Reilly T P Bourdi M Brady J N Pise-Masison C A Radonovich M
F George J W et al (2001) Expression profiling of acetaminophen
liver toxicity in mice using microarray technology Biochemical and
Biophysical Research Communications 282 321ndash328
Rives A W amp Galitski T (2003) Modular organization of cellular
networks Proceedings of the National Academy of Sciences of the
United States of America 100 1128ndash1133
Rogers P D Pearson M M Cleary J D Sullivan D C amp Chapman
S W (2002) Differential expression of genes encoding immunomodu-
latory proteins in response to amphotericin B in human mononuclear
cells identified by cDNA microarray analysis Journal of Antimicrobial
Chemotherapy 50 811ndash817
Rosen M B Wilson V S Schmid J E amp Gray L E (2005) Gene
expression analysis in the ventral prostate of rats exposed to vinclozolin
or procymidone Reproductive Toxicology 19 367ndash379
Rost D Herrmann T Sauer P Schmidts H L Stieger B Meier P J
et al (2003) Regulation of rat organic anion transporters in bile salt-
induced cholestatic hepatitis Effect of ursodeoxycholate Hepatology
38 187ndash195
Rothman R B Ayestas M A Dersch C M amp Baumann M H (1999)
Aminorex fenfluramine and chlorphentermine are serotonin transporter
substrates Implications for primary pulmonary hypertension Circula-
tion 100 869ndash875
Ruepp S U Tonge R P Shaw J Wallis N amp Pognan F (2002)
Genomics and proteomics analysis of acetaminophen toxicity in mouse
liver Toxicology Sciences 65 135ndash150
Sakaeda T Nakamura T amp Okumura K (2002) MDR1 genotype-
related pharmacokinetics and pharmacodynamics Biological amp Phar-
maceutical Bulletin 25 1391ndash1400
Sakata T Anzai N Shin H J Noshiro R Hirata T Yokoyama H
et al (2004) Novel single nucleotide polymorphisms of organic cation
transporter 1 (SLC22A1) affecting transport functions Biochemical
and Biophysical Research Communications 313 789ndash793
Satlin L M Amin V amp Wolkoff A W (1997) Organic anion
transporting polypeptide mediates organic anionHCO3-exchange
Journal of Biological Chemistry 272 26340ndash26345
Satomi Y Tsuchiya W Mihara K Ota M Kasahara Y amp Akahori F
(2004) Gene expression analysis of the lung following paraquat
administration in rats using DNA microarray Journal of Toxicological
Sciences 29 91ndash100
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 65
Schrenk D Baus P R Ermel N Klein C Vorderstemann B amp
Kauffmann H M (2001) Up-regulation of transporters of the MRP
family by drugs and toxins Toxicology Letters 120 51ndash57
Segal E Shapira M Regev A Persquoer D Botstein D Koller D et al
(2003) Module networks Identifying regulatory modules and their
condition-specific regulators from gene expression data Nature
Genetics 34 166ndash176
Segal E Wang H amp Koller D (2003) Discovering molecular pathways
from protein interaction and gene expression Bioinformatics 19
i264ndash i272
Seidel S D Kan H L Stott W T Schisler M R amp Gollapudi B B
(2003) Identification of transcriptome profiles for the DNA-damaging
agents bleomycin and hydrogen peroxide in L5178Y mouse lymphoma
cells Environmental and Molecular Mutagenesis 42 19ndash25
Shan L Yu Y Schut H A J amp Snyderwine E G (2004) Susceptibility
of rats to mammary gland carcinogenesis by the food-derived
carcinogen 2-Amino-1-Methyl-6-Phenylimidazo[45-b]Pyridine (PhIP)
varies with age and is associated with the induction of differential gene
expression American Journal of Pathology 165 191ndash202
Sharom J R Bellows D S amp Tyers M (2004) From large networks to
small molecules Current Opinion in Chemical Biology 8 81ndash90
Shitara Y Hirano M Sato H amp Sugiyama Y (2004) Gemfibrozil and
its glucuronide inhibit the OATP2(OATP1B1 SLC21A6)-mediated
hepatic uptake and CYP2C8-mediated metabolism of cerivastatinmdash
Analysis of the mechanism of the clinically relevant drugndashdrug
interaction between cerivastatin and gemfibrozil Journal of Pharma-
cology and Experimental Therapeutics
Shitara Y Itoh T Sato H Li A P amp Sugiyama Y (2003) Inhibition of
transporter-mediated hepatic uptake as a mechanism for drugndashdrug
interaction between cerivastatin and cyclosporin A Journal of
Pharmacology and Experimental Therapeutics 304 610ndash616
Somogyi R Fuhrman S amp Wen X (2001) Genetic network inference in
computational models and applications to large-scale gene expression
data In M Bower amp H Bolouri (Eds) Computational modeling of
genetic and biochemical networks (pp 119ndash157) Cambridge MArsquoThe
MIT Press
Spirin V amp Mirny L A (2003) Protein complexes and functional
modules in molecular networks Proceedings of the National Academy
of Sciences of the United States of America 100 12123ndash12128
Staudinger J Liu Y Madan A Habeebu S amp Klaassen C D
(2001) Coordinate regulation of xenobiotic and bile acid homeo-
stasis by pregnane X receptor Drug Metabolism and Disposition
29 1467ndash1472
Sun L Z Ji Z L Chen X Wang J F amp Chen Y Z (2002) ADME-
AP A database of ADME associated proteins Bioinformatics 18
1699ndash1700
Suter L Babiss L E amp Wheeldon E B (2004) Toxicogenomics in
predictive toxicology in drug development Chemistry and Biology 11
161ndash171
Synold T W Dussault I amp Forman B M (2001) The orphan nuclear
receptor SXR coordinately regulates drug metabolism and efflux
Nature Medicine 7 584ndash590
Tabuchi Y amp Kondo T (2003) cDNA microarray analysis reveals chop-
10 plays a key role in Sertoli cell injury induced by bisphenol A
Biochemical and Biophysical Research Communications 305 54ndash61
Tamai I Nezu J Uchino H Sai Y Oku A Shimane M et al (2000)
Molecular identification and characterization of novel members of the
human organic anion transporter (OATP) family Biochemical and
Biophysical Research Communications 273 251ndash260
Taylor S W Fahy E Zhang B Glenn G M Warnock D E Wiley S
et al (2003) Characterization of the human heart mitochondrial
proteome Nature Biotechnology 21 281ndash286
Terasaka S Aita Y Inoue A Hayashi S Nishigaki M Aoyagi K
et al (2004) Using a customized DNA microarray for expression
profiling of the estrogen-responsive genes to evaluate estrogen activity
among natural estrogens and industrial chemicals Environmental
Health Perspectives 112 773ndash781
Thomas D M Francescutti-Verbeem D M Liu X amp Kuhn D M
(2004) Identification of differentially regulated transcripts in mouse
striatum following methamphetamine treatmentmdashan oligonucleotide
microarray approach Journal of Neurochemistry 88 380ndash393
Thomas R S Rank D R Penn S G Zastrow G M Hayes K R
Pande K et al (2001) Identification of toxicologically predictive
gene sets using cDNA microarrays Molecular Pharmacology 60
1189ndash1194
Thompson K L Afshari C A Amin R P Bertram T A Car B
Cunningham M et al (2004) Identification of platform-independent
gene expression markers of cisplatin nephrotoxicity Environmental
Health Perspectives 112 488ndash494
Tirona R G Leake B F Merino G amp Kim R B (2001)
Polymorphisms in OATP-C Identification of multiple allelic
variants associated with altered transport activity among European-
and African-Americans Journal of Biological Chemistry 276
35669ndash35675
Tirona R G Leake B F Wolkoff A W amp Kim R B (2003)
Human organic anion transporting polypeptide-C (SLC21A6) is a
major determinant of the rifampin-mediated pregnane X receptor
activation Journal of Pharmacology and Experimental Therapeutics
304 223ndash228
Tong W Cao X Harris S Sun H Fang H Fuscoe J et al (2003)
ArrayTrackmdashsupporting toxicogenomic research at the US Food and
Drug Administration National Center for Toxicological Research
Environmental Health Perspectives 111 1819ndash1826
Tornow S amp Mewes H W (2003) Functional modules by relating
protein interaction networks and gene expression Nucleic Acids
Research 31 6283ndash6289
Trocho C Escola-Gil J C Ribas V Benitez S Martin-Campos J M
Rotllan N et al (2004) Phenytoin treatment reduces atherosclerosis in
mice through mechanisms independent of plasma HDL-cholesterol
concentration Atherosclerosis 174 275ndash285
Ueda A Hamadeh H K Webb H K Yamamoto Y Sueyoshi T
Afshari C A et al (2002) Diverse roles of the nuclear orphan
receptor CAR in regulating hepatic genes in response to phenobarbital
Molecular Pharmacology 61 1ndash6
Ulrich R G (2003) The toxicogenomics of nuclear receptor agonists
Current Opinion in Chemical Biology 7 505ndash510
Ulrich R G Rockett J C Gibson G G amp Pettit S D (2004)
Overview of an interlaboratory collaboration on evaluating the effects
of model hepatotoxicants on hepatic gene expression Environmental
Health Perspectives 112 423ndash427
Unami A Shinohara Y Ichikawa T amp Baba Y (2003) Biochemical
and microarray analyses of bupivacaine-induced apoptosis Journal of
Toxicological Sciences 28 77ndash94
Vasquez A Flammini A Maritan A amp Vespignani A (2003) Global
protein function prediction from proteinndashprotein interaction networks
Nature Biotechnology 21 697ndash700
Vezina C M Walker N J amp Olson J R (2004) Subchronic Exposure to
TCDD PeCDF PCB126 and PCB153 Effect on hepatic gene
expression Environmental Health Perspectives 112 1636ndash1644
Vo T D Greenberg H J amp Palsson B O (2004) Reconstruction and
functional characterization of the human mitochondrial metabolic
network based on proteomic and biochemical data Journal of Bio-
logical Chemistry 279 39532ndash39540
Wang H amp LeCluyse E L (2003) Role of orphan nuclear receptors in
the regulation of drug-metabolising enzymes Clinical Pharmacoki-
netics 42 1331ndash1357
Wang N Silver D L Thiele C amp Tall A R (2001) ATP-
binding cassette transporter A1 (ABCA1) functions as a choles-
terol efflux regulatory protein Journal of Biological Chemistry 276
23742ndash23747
Waring J F Cavet G Jolly R A McDowell J Dai H Ciurlionis R
et al (2003) Development of a DNA Microarray for toxicology based
on hepatoxin-regulated sequences Environmental Health Perspectives
111 863ndash870
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6666
Waring J F Gum R Morfitt D Jolly R A Ciurlionis R Heindel M
et al (2002) Identifying toxic mechanisms using DNA microarrays
Evidence that an experimental inhibitor of cell adhesion molecule
expression signals through the aryl hydrocarbon nuclear receptor
Toxicology 181ndash182 537ndash550
Waring J F Jolly R A Ciurlionis R Lum P Y Praestgaard J T
Morfitt D C et al (2001) Clustering of hepatotoxins based on
mechanism of toxicity using gene expression profiles Toxicology and
Applied Pharmacology 175 28ndash42
Waters M Boorman G Bushel P Cunningham M Irwin R Merrick
A et al (2003) Systems toxicology and the Chemical Effects in
Biological Systems (CEBS) knowledge base EHP Toxicogenomics
111 15ndash28
Wei M Arnold L Cano M amp Cohen S M (2005) Effects of co-
administration of antioxidants and arsenicals on the rat urinary bladder
epithelium Toxicology Sciences 83 237ndash245
Weinshilboum R (2003) Inheritance and drug response New England
Journal of Medicine 348 529ndash537
Weinshilboum R amp Wang L (2004) Pharmacogenetics Inherited
variation in amino acid sequence and altered protein quantity Clinical
Pharmacology and Therapeutics 75 253ndash258
Werner E (2003) In silico multicellular systems biology and minimal
genomes Drug Discovery Today 8 1121ndash1127
Wong J S amp Gill S S (2002) Gene expression changes induced in
mouse liver by di(2-ethylhexyl) phthalate Toxicology and Applied
Pharmacology 185 180ndash196
Xie T Tong L McCann U D Yuan J Becker K G Mechan A O
et al (2004) Identification and characterization of metallothionein-1
and -2 gene expression in the context of ()34-methylenedioxymetham-
phetamine-induced toxicity to brain dopaminergic neurons Journal of
Neuroscience 24 7043ndash7050
Yadetie F Laegreid A Bakke I Kusnierczyk W Komorowski J
Waldum H L et al (2003) Liver gene expression in rats in response
to the peroxisome proliferator-activated receptor-alpha agonist ciprofi-
brate Physiological Genomics 15 9ndash19
Yamashita S Nomoto T Abe M Tatematsu M Sugimura T amp
Ushijima T (2004) Persistence of gene expression changes in stomach
mucosae induced by short-term N-methyl-NV-nitro-N-nitrosoguanidinetreatment and their presence in stomach cancers Mutation Research
549 185ndash193
Yan Q amp Sadee W (2000) Human membrane transporter database A
web-accessible relational database for drug transport studies and
pharmacogenomics AAPS Pharmsci 2 E20
Yeger-Lotem E amp Margalit H (2003) Detection of regulatory circuits by
integrating the cellular networks of proteinndashprotein interactions and
transcription regulation Nucleic Acids Research 31 6053ndash6061
Yoon B I Li G -X Kitada K Kawasaki Y Igarashi K amp Kodama
Y et al (2003) Mechanisms of benzene-induced hematotoxicity and
leukemogenicity cDNA microarray analyses using mouse bone marrow
tissue Environmental Health Perspectives 111 1411ndash1420
Young M B DiSilvestro M R Sendera T J Freund J Kriete A amp
Magnuson S R (2003) Analysis of gene expression in carbon
tetrachloride-treated rat livers using a novel bioarray technology
Pharmacogenomics Journal 3 41ndash52
Yu H Zhu X Greenbaum D Karro J amp Gerstein M (2004) TopNet
A tool for comparing biological sub-networks correlating protein
properties with topological statistics Nucleic Acids Research 32
328ndash337
Zhang E Y Fu D J Pak Y A Stewart T Mukhopadhyay N
Wrighton S A et al (2004) Genetic polymorphisms in human proton-
dependent dipeptide transporter PEPT1 Implications for the functional
role of Pro586 Journal of Pharmacology and Experimental Therapeu-
tics 310 437ndash445
Zhang E Y Knipp G T Ekins S amp Swaan P W (2002)
Structural biology and function of solute transporters Implications
for identifying and designing substrates Drug Metabolism Reviews
34 709ndash750
Zhang E Y Phelps M A Cheng C Ekins S amp Swaan P W (2002)
Modeling of active transport systems Advanced Drug Delivery
Reviews 54 329ndash354
- Systems-ADMETox Resources and network approaches
-
- Introduction
- Data available
- Network analysis and databases
-
- Network applications
-
- The role of transporters
-
- Clinical relevance of transporters
- Transporter network examples ABCA1
- Transporter network examples OATP
- Transporter microarray data
-
- Applications to enzymes
- Future network applications
-
- Discussion
- Acknowledgements
- References
-
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 61
References
Abe M Yamashita S Kuramoto T Hirayama Y Tsukamoto T Ohta
T et al (2003) Global expression analysis of N-methyl-NV-nitro-N-nitrosoguanidine-induced rat stomach carcinomas using oligonucleotide
microarrays Carcinogenesis 24 861ndash867
Adachi T Ono Y Koh K B Takashima K Tainaka H Matsuno Y
et al (2004) Long-term alteration of gene expression without
morphological change in testis after neonatal exposure to genistein in
mice Toxicogenomic analysis using cDNA microarray Food and
Chemical Toxicology 42 445ndash452
Akerman G S Rosenzweig B A Domon O E McGarrity L J
Blankenship L R Tsai C A et al (2004) Gene expression profiles
and genetic damage in benzo(a)pyrene diol epoxide-exposed TK6 cells
Mutation Research 549 43ndash64
Aksenov S V Church B Dhiman A Georgieva A Sarangapani R
Helmlinger G et al (2005) An integrated approach for inference and
mechanistic modeling for advancing drug development FEBS Letters
579 1878ndash1883
Anderle P Huang Y amp Sadee W (2004) Intestinal membrane transport
of drugs and nutrients Genomics of membrane transporters using
expression microarrays European Journal of Pharmaceutical Sciences
21 17ndash24
Annereau J P Szakacs G Tucker C J Arciello A Cardarelli C
Collins J et al (2004) Analysis of ATP-binding cassette transporter
expression in drug-selected cell lines by a microarray dedicated to
multidrug resistance Molecular Pharmacology 66 1397ndash1405
Apic G Ignjatovic T Boyer S amp Russell R B (2005) Illuminating drug
discovery with biological pathways FEBS Letters 579 1872ndash1877
Baker V A Harries H M Waring J F Duggan C M Ni H A Jolly
R A et al (2004) Clofibrate-induced gene expression changes in rat
liver A cross-laboratory analysis using membrane cDNA arrays
Environmental Health Perspectives 112 428ndash438
Balakin K V Ekins S Bugrim A Ivanenkov Y A Korolev D amp
Nikolsky Y et al (2004a) Quantitative structurendashmetabolism relation-
ship modeling of the metabolic N-dealkylation rates Drug Metabolism
and Disposition 32 1111ndash1120
Balakin K V Ekins S Bugrim A Ivanenkov Y A Korolev D amp
Nikolsky Y et al (2004b) Kohonen maps for prediction of binding to
human cytochrome P450 3A4 Drug Metabolism and Disposition 32
1183ndash1189
Barabasi A -L amp Oltvai Z N (2004) Network biology Under-
standing the cellrsquos functional organization Nature Reviews Genetics
5 101ndash113
Bartosiewicz M Penn S G amp Buckpitt A (2001) Applications of gene
arrays in environmental toxicology Fingerprints of gene regulation
associated with cadmium chloride benzo(a)pyrene and trichloro-
ethylene Environmental Health Perspectives 109 71ndash74
Bertilsson G Heidrich J Svensson K Asman M Jendeberg L
Sydow-Backman M et al (1998) Identification of a human nuclear
receptor defines a new signaling pathway for CYP3A induction
Proceedings of the National Academy of Sciences of the United States
of America 95 12208ndash12213
Blumberg B Sabbagh Jr W Juguilon H Bolado Jr J van Meter
C M Ong E S et al (1998) SXR a novel steroid and xenobiotic-
sensing nuclear receptor Genes and Development 12 3195ndash3205
Borodina Y Rudik A Filimonov D Kharchevnikova N Dmitriev A
Blinova V et al (2004) A new statistical approach to predicting
aromatic hydroxylation sites Comparison with model-based
approaches Journal of Chemical Information and Computer Sciences
44 1998ndash2009
Borodina Y Sadym A Filimonov D Blinova V Dmitriev A amp
Poroikov V (2003) Predicting biotransformation potential from
molecular structure Journal of Chemical Information and Computer
Sciences 43 1636ndash1646
Bottone Jr F G Martinez J M Collins J B Afshari C A amp Eling
T E (2003) Gene modulation by the cyclooxygenase inhibitor
sulindac sulfide in human colorectal carcinoma cells Possible link to
apoptosis Journal of Biological Chemistry 278 25790ndash25801
Boyer S amp Zamora I (2002) New methods in predictive metabolism
Journal of Computer-Aided Molecular Design 16 403ndash413
Bredel M amp Jacoby E (2004) Chemogenomics An emerging strategy
for rapid target and drug discovery Nature Reviews Genetics 5
262ndash275
Bugrim A Nikolskaya T amp Nikolsky Y (2004) Early prediction of drug
metabolism and toxicity Systems biology approach and modeling
Drug Discovery Today 9 127ndash135
Bulera S J Eddy S M Ferguson E Jatkoe T A Reindel J F
Bleavins M R et al (2001) RNA expression in the early character-
ization of hepatotoxicants in Wistar rats by high-density DNA micro-
arrays Hepatology 33 1239ndash1258
Butcher E C Berg E L amp Kunkel E J (2004) Systems biology in drug
discovery Nature Biotechnology 22 1253ndash1259
Butte A (2002) The use and analysis of microarray data Naturalist
Review of Drug Discovery 1 951ndash960
Cary M P Bader G D amp Sander C (2005) Pathway information for
systems biology FEBS Letters 579 1815ndash1820
Cheok M H Yang W Pui C H Downing J R Cheng C Naeve
C W et al (2003) Treatment-specific changes in gene expression
discriminate in vivo drug response in human leukemia cells Nature
Genetics 34 85ndash90
Cleary J D Rogers P D amp Chapman S W (2001) Differential
transcription factor expression in human mononuclear cells in response
to amphotericin B Identification with complementary DNA microarray
technology Pharmacotherapy 21 1046ndash1054
Coen M Ruepp S U Lindon J C Nicholson J K Pognan F Lenz
E M et al (2004) Integrated application of transcriptomics and
metabonomics yields new insight into the toxicity due to paracetamol in
the mouse Journal of Pharmaceutical and Biomedical Analysis 35
93ndash105
Cornwell P D De Souza A T amp Ulrich R G (2004) Profiling of
hepatic gene expression in rats treated with fibric acid analogs
Mutation Research 549 131ndash145
Csermely P Agoston V amp Pongor S (2005) The efficiency of multi-
target drugs The network approach might help drug design Trends in
Pharmacological Sciences 26 178ndash182
Cunningham M J Liang S Fuhrman S Seilhamer J J amp
Somogyi R (2000) Gene expression microarray data analysis for
toxicology profiling Annals of the New York Academy of Sciences 919
52ndash67
de Longueville F Atienzar F A Marcq L Dufrane S Evrard S
Wouters L et al (2003) Use of a low density microarray for studying
gene expression patterns induced by hepatotoxicants on primary
cultures of rat hepatocytes Toxicological Sciences 75 378ndash392
Dobrin R Beg Q K Barabasi A L amp Oltvai Z N (2004)
Aggregation of topological motifs in the Escherichia coli transcriptional
regulatory network BMC Bioinformatics 5 10
Donald S Verschoyle R D Edwards R Judah D J Davies R
Riley J et al (2002) Hepatobiliary damage and changes in hepatic
gene expression caused by the antitumor drug ecteinascidin-743 (ET-
743) in the female rat Cancer Research 62 4256ndash4262
Dumont M Jacquemin E DrsquoHont C Descout C Cresteil D Haouzi
D et al (1997) Expression of the liver Na+-independent organic anion
transporting polypeptide (oatp-1) in rats with bile duct ligation Journal
of Hepatology 27 1051ndash1056
Durnas C Loi C M amp Cusack B J (1990) Hepatic drug metabolism
and aging Clinical Pharmacokinetics 19 359ndash389
Eisen M B Spellman P T Brown P O amp Botstein D (1998) Cluster
analysis and display of genome-wide expression patterns Proceedings
of the National Academy of Sciences of the United States of America
95 14863ndash14868
Ekins S Andreyev S Ryabov A Kirilov E Rakhmatulin E A
Bugrim A et al (in press) Computational Prediction of Human Drug
Metabolism Exp Opin Drug Metab Toxicol
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6662
Ekins S Boulanger B Swaan P W amp Hupcey M A Z (2002)
Towards a new age of virtual ADMETOX and multidimensional drug
discovery Journal of Computer-Aided Molecular Design 16 381ndash401
Ekins S Bugrim A Nikolsky Y amp Nikolskaya T (2005) Systems
biology Applications in drug discovery In S Gad (Ed) Drug
Discovery Handbook (pp 123ndash183) Hoboken NJrsquo Wiley and Sons
Ekins S Giroux C N Nikolsky Y Bugrim A amp Nikolskaya T
(2005) A signature gene network approach to toxicity The Toxoco-
logists 84
Ekins S Kirillov E Rakhmatulin E amp Nikolskaya T (2005) A novel
method for visualizing nuclear hormone receptor networks relevant to
drug metabolism Drug Metabolism and Disposition 33 474ndash481
Ekins S amp McGowan R J (2001) The limits of reductionism The
shifting genomic paradigmrsquos impact on industry and academia
Philsophy in Science 9 1ndash23
Ekins S Mirny L amp Schuetz E G (2002) A ligand-based approach to
understanding selectivity of nuclear hormone receptors PXR CAR
FXR LXRa and LXRb Pharmaceutical Research 19 1788ndash1800
Ekins S Nikolsky Y amp Nikolskaya T (2005) Techniques Application
of systems biology to absorption distribution metabolism excretion
and toxicity Trends in Pharmacological Sciences 26 202ndash209
Ekins S Ring B J Grace J McRobie-Belle D J amp Wrighton S
A (2000) Present and future in vitro approaches for drug
metabolism Journal of Pharmacological and Toxicological Methods
44 313ndash324
Ekins S Waller C L Swaan P W Cruciani G Wrighton S A amp
Wikel J H (2000) Progress in predicting human ADME parameters in
silico Journal of Pharmacological and Toxicological Methods 44
251ndash272
Erhardt P W (2003) A human drug metabolism database Potential roles
in the quantitative predictions of drug metabolism and metabolism-
related drugndashdrug interactions Current Drug Metabolism 4 411ndash422
Evans W E amp McLeod H L (2003) Pharmacogenomicsmdashdrug
disposition drug targets and side effects New England Journal of
Medicine 348 538ndash549
Ezendam J Staedtler F Pennings J Vandebriel R J Pieters R
Boffetta P et al (2004) Toxicogenomics of subchronic hexachlor-
obenzene exposure in Brown Norway rats Environmental Health
Perspectives 112 782ndash791
Faiola B Fuller E S Wong V A amp Recio L (2004) Gene expression
profile in bone marrow and hematopoietic stem cells in mice exposed to
inhaled benzene Mutation Research 549 195ndash212
FDA (2004) Innovation stagnation Challenge and opportunity on the
critical path to new medicinal products
Fiehn O (2001) Combining genomics metabolome analysis and bio-
chemical modelling to understand metabolic networks Comparative
Functional Genomics 2 155ndash168
Field F J Born E amp Mathur S N (2004) LXRRXR ligand activation
enhances basolateral efflux of beta-sitosterol in CaCo-2 cells Journal of
Lipid Research 45 905ndash913
Fleck C Sutter L Appenroth D Koch B Meinhold T Pitack M
et al (2003) Use of gene chip technology for the characterisation of
the regulation of renal transport processes and of nephrotoxicity in
rats Experimental and Toxicologic Pathology 54 401ndash410
Fliri A F Loging W T Thadeio P F amp Volkmann R A (2005)
Biological spectra analysis Linking biological activity profiles to
molecular structure Proceedings of the National Academy of Sciences
of the United States of America 102 261ndash266
Fountoulakis M (2004) Application of proteomics technologies in the
investigation of the brain Mass Spectrometry Reviews 23 231ndash258
Gaucher S P Taylor S W Fahy E Zhang B Warnock D E Ghosh
S S et al (2004) Expanded coverage of the human heart
mitochondrial proteome using multidimensional liquid chromatography
coupled with tandem mass spectrometry Journal of Proteome
Research 3 495ndash505
Gerhold D Lu M Xu J Austin C Caskey C T amp Rushmore T
(2001) Monitoring expression of genes involved in drug metabolism
and toxicology using DNA microarrays Physiological Genomics 5
161ndash170
Guo G L Choudhuri S amp Klaassen C D (2002) Induction profile of
rat organic anion transporting polypeptide 2 (oatp2) by prototypical
drug-metabolizing enzyme inducers that activate gene expression
through ligand-activated transcription factor pathways Journal of
Pharmacology and Experimental Therapeutics 300 206ndash212
Hagenbuch B amp Meier P J (2004) Organic anion transporting
polypeptides of the OATP SLC21 family Phylogenetic classification
as OATP SLCO superfamily new nomenclature and molecularfunc-
tional properties Pflugers Archiv 447 653ndash665
Hamadeh H K amp Afshari C A (2004) Toxicogenomics principles and
applications Hobokenrsquo John Wiley and Sons
Hamadeh H K Bushel P R Jayadev S Martin K DiSorbo O Sieber
S et al (2002) Gene expression analysis reveals chemical-specific
profiles Toxicological Sciences 67 219ndash231
Hamadeh H K Jayadev S Gaillard E T Huang Q Stoll R
Blanchard K et al (2004) Integration of clinical and gene expression
endpoints to explore furan-mediated hepatotoxicity Mutation Research
549 169ndash183
Hamadeh H K Knight B L Haugen A C Sieber S Amin R P
Bushel P R et al (2002) Methapyrilene toxicity Anchorage of
pathologic observations to gene expression alterations Toxicologic
Pathology 30 470ndash482
Han J D Bertin N Hao T Goldberg D S Berriz G F Zhang L V
et al (2004) Evidence for dynamically organized modularity in the
yeast proteinndashprotein interaction network Nature 430 88ndash93
Hanisch D Zien A Zimmer R amp Lengauer T (2002) Co-clustering of
biological networks and gene expression data Bioinformatics 18
S145ndashS154
Harris A J Dial S L amp Casciano D A (2004) Comparison of basal
gene expression profiles and effects of hepatocarcinogens on gene
expression in cultured primary human hepatocytes and HepG2 cells
Mutation Research 549 79ndash99
Harrison S C (2004) Whither structural biology Nature Structural
Molecular Biology 11 12ndash15
Hartley D P Dai X He Y D Carlini E J Wang B Huskey S E
et al (2004) Activators of the rat pregnane X receptor differentially
modulate hepatic and intestinal gene expression Molecular Pharma-
cology 65 1159ndash1171
Hartwell L H Hopfield J J Leibler S amp Murray A W (1999) From
molecular to modular cell biology Nature 402 C47ndashC52
Hasmall S Orphanides G James N Pennie W Hedley K Soames
A et al (2002) Downregulation of lactoferrin by PPARalpha ligands
Role in perturbation of hepatocyte proliferation and apoptosis
Toxicology Sciences 68 304ndash313
Hayase T Yamamoto Y Yamamoto K Muso E amp Shiota K (2004)
Microarray profile analysis of toxic cocaine-induced alterations in the
expression of mouse brain gene sequences A possible rsquoprotectiversquo
effect of buprenorphine Journal of Applied Toxicology 24 15ndash20
Hayes K R Vollrath A L Zastrow G M McMillan B J Craven M
Jovanovich S et al (2005) EDGE A centralized resource for the
comparison analysis and distribution of toxicogenomic information
Molecular Pharmacology 67 1360ndash1368
Heijne W H Slitt A L van Bladeren P J Groten J P Klaassen C D
Stierum R H et al (2004) Bromobenzene-induced hepatotoxicity at
the transcriptome level Toxicology Sciences 79 411ndash422
Heijne W H Stierum R H Slijper M van Bladeren P J amp van
Ommen B (2003) Toxicogenomics of bromobenzene hepatotoxicity
A combined transcriptomics and proteomics approach Biochemical
Pharmacology 65 857ndash875
Heinloth A N Irwin R D Boorman G A Nettesheim P Fannin
R D Sieber S O et al (2004) Gene expression profiling of rat
livers reveals indicators of potential adverse effects Toxicology
Sciences 80 193ndash202
Hodges L C Cook J D Lobenhofer E K Li L Bennett L Bushel
P R et al (2003) Tamoxifen functions as a molecular agonist inducing
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 63
cell cycle-associated genes in breast cancer cells Molecular Cancer
Research 1 300ndash311
Holleman A Cheok M H den Boer M L Yang W Veerman KM
A J Kazemier et al (2004) Gene-expression patterns in drug-resistant
acute lymphoblastic leukemia cells and response to treatment New
England Journal of Medicine 351 533ndash542
Hong Y Muller U R amp Lai F (2003) Discriminating two classes of
toxicants through expression analysis of HepG2 cells with DNA arrays
Toxicology in Vitro 17 85ndash92
Hood E (2003) Proteomics Characterizing the cogs in the machinery of
life Environmental Health Perspectives 111 A816ndashA825
Hood L (2003) Systems biology Integrating technology biology and
computation Mechanism of Ageing and Development 124 9ndash16
Hood L amp Galas D (2003) The digital code of DNA Nature 421
444ndash448
Hood L amp Perlmutter R M (2004) The impact of systems approaches on
biological problems in drug discovery Nature Biotechnology 22
1215ndash1217
Hu T Gibson D P Carr G J Torontali S M Tiesman J P Chaney
J G et al (2004) Identification of a gene expression profile that
discriminates indirect-acting genotoxins from direct-acting genotoxins
Mutation Research 549 5ndash27
Huang Q Dunn II R T Jayadev S DiSorbo O Pack F D Farr S B
et al (2001) Assessment of cisplatin-induced nephrotoxicity by
microarray technology Toxicology Sciences 63 196ndash207
Huang Q Jin X Gaillard E T Knight B L Pack F D Stoltz J H et
al (2004) Gene expression profiling reveals multiple toxicity endpoints
induced by hepatotoxicants Mutation Research 549 147ndash167
Huisman M T Smit J W Crommentuyn K M Zelcer N Wiltshire
H R Beijnen J H et al (2002) Multidrug resistance protein 2
(MRP2) transports HIV protease inhibitors and transport can be
enhanced by other drugs Aids 16 2295ndash2301
Hunt C M Westerkam W R amp Stave G M (1992) Effect of age and
gender on the activity of human hepatic CYP3A Biochemical
Pharmacology 44 275ndash283
Ideker T Ozier O Schwikowski B amp Siegal A F (2002) Discovering
regulatory and signalling circuits in molecular interaction networks
Bioinformatics 18 S233ndashS240
Ideker T Thorsson V Ranish J A Christmas R Buhler J Eng J K
et al (2001) Integrated genomic and proteomic analyses of a system-
atically perturbed network Science 292 929ndash934
Iida M Anna C H Hartis J Bruno M Wetmore B Dubin J R et al
(2003) Changes in global gene and protein expression during early
mouse liver carcinogenesis induced by non-genotoxic model carcino-
gens oxazepam and Wyeth-14643 Carcinogenesis 24 757ndash770
Ingelman-Sundberg M (2004) Pharmacogenetics of cytochrome P450 and
its applications in drug therapy The past present and future Trends in
Pharmacological Sciences 25 193ndash200
Jeong H Mason S P Barabasi A L amp Oltvai Z N (2001) Lethality
and centrality in protein networks Nature 411 41ndash42
Jeong H Tombor B Albert R Oltvai Z N amp Barabasi A L
(2000) The large-scale organization of metabolic networks Nature
407 651ndash654
Jung D Hagenbuch B Gresh L Pontoglio M Meier P J amp Kullak-
Ublick G A (2001) Characterization of the human OATP-C
(SLC21A6) gene promoter and regulation of liver-specific OATP genes
by hepatocyte nuclear factor 1 alpha Journal of Biological Chemistry
276 37206ndash37214
Jung D amp Kullak-Ublick G A (2003) Hepatocyte nuclear factor 1
alpha A key mediator of the effect of bile acids on gene expression
Hepatology 37 622ndash631
Jung J -W Park J -S Hwang J -W Kang K -S YS L Song B
-S et al (2004) Gene expression analysis of peroxisome prolifer-
ators- and phenytoin-induced hepatotoxicity using cDNA microarray
Journal of Veterinary Medical Science 66 1329ndash1333
Kato N Shibutani M Takagi H Uneyama C Lee K -Y Takiami S
et al (2004) Gene expression profile in the livers of rats orally
administered ethinylestradiol for 28 days using a microarray technique
Toxicology 200 179ndash192
Kier L D Neft R Tang L Suizu R Cook T Onsurez K et al
(2004) Applications of microarrays with toxicologically relevant genes
(tox genes) for the evaluation of chemical toxicants in Sprague Dawley
rats in vivo and human hepatocytes in vitro Mutation Research 549
101ndash113
Kijima K Toyosawa K Yasuba M Matsuoka N Adachi T
Komiyama M et al (2004) Gene expression analysis of the rat testis
after treatment with di(2-ethylhexyl) phthalate using cDNA microarray
and real-time RT-PCR Toxicology and Applied Pharmacology 200
103ndash110
Kim R B (2003) Organic anion-transporting polypeptide (OATP) trans-
porter family and drug disposition European Journal of Clinical
Investigation 33 1ndash5
Kinirons M T amp OrsquoMahony M S (2004) Drug metabolism and ageing
British Journal of Clinical Pharmacology 57 540ndash544
Kitano H (2002a) Computational systems biology Nature 420 206ndash210
Kitano H (2002b) Systems biology A brief overview Science 295
1662ndash1664
Kliewer S A Moore J T Wade L Staudinger J L Watson M
A Jones S A et al (1998) An orphan nuclear receptor activated
by pregnanes defines a novel steroid signalling pathway Cell 92
73ndash82
Korolev D Balakin K V Nikolsky Y Kirillov E Ivanenkov Y A
Savchuk N P et al (2003) Modeling of human cytochrome p450-
mediated drug metabolism using unsupervised machine learning
approach Journal of Medicinal Chemistry 46 3631ndash3643
Kramer J A Pettit S D Amin R P Bertram T A Car B
Cunningham M et al (2004) Overview on the application of
transcription profiling using selected nephrotoxicants for toxicology
assessment Environmental Health Perspectives 112 460ndash464
Kultima K Nystrom A -M Scholz B Gustafson A -L Dencker L
amp Stigson M (2004) Valproic acid teratogenicity A toxicogenomics
approach Environmental Health Perspectives 112 1225ndash1235
Kutuzova G D amp Deluca H F (2004) Gene expression profiles in rat
intestine identify pathways for 125-dihydroxyvitamin D(3) stimulated
calcium absorption and clarify its immunomodulatory properties
Archives of Biochemistry and Biophysics 432 152ndash166
Kwak M K Wakabayashi N Itoh K Motohashi H Yamamoto M amp
Kensler T W (2003) Modulation of gene expression by cancer
chemopreventive dithiolethiones through the Keap1-Nrf2 pathway
Identification of novel gene clusters for cell survival Journal of
Biological Chemistry 278 8135ndash8145
Lahjouji K Mitchell G A amp Qureshi I A (2001) Carnitine transport
by organic cation transporters and systemic carnitine deficiency
Molecular Genetics and Metabolism 73 287ndash297
Landowski C P Sun D Foster D R Menon S S Barnett J L
Welage L S et al (2003) Gene expression in the human intestine and
correlation with oral valacyclovir pharmacokinetic parameters Journal
of Pharmacology and Experimental Therapeutics 306 778ndash786
Lee G -J Lee W -S Jeon K -S Um C -H Kim S -J Lee C -H
et al (2004) cDNA microarray gene expression analysis and
toxicological phenotype for anticancer drug Journal of Veterinary
Medical Science 66 1339ndash1345
Lee M Kwon J Kim S N Kim J E Koh W S Kim E J et al
(2003) cDNA microarray gene expression profiling of hydroxyurea
paclitaxel and p-anisidine genotoxic compounds with differing
tumorigenicity results Environmental and Molecular Mutagenesis
42 91ndash97
Li S Armstrong C M Bertin N Ge H Milstein S Boxem M et al
(2004) A map of the interactome network of the metazoan C elegans
Science 303 540ndash543
Liguori M J Anderson L M Bukofzer S McKim J Pregenzer J F
Retief J et al (2005) Microarray analysis in human hepatocytes
suggests a mechanism for hepatotoxicity induced by trovafloxacin
Hepatology 41 177ndash186
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6664
Lobenhofer E K Bennett L Cable P L Li L Bushel P R amp Afshari
C A (2002) Regulation of DNA replication fork genes by 17beta-
estradiol Molecular Endocrinology 16 1215ndash1229
Locker J Tian J Carver R Concas D Cossu C Ledda-Columbano
G M et al (2003) A common set of immediate-early response genes
in liver regeneration and hyperplasia Hepatology 38 314ndash325
Mattes W B Pettit S D Sansone S A Bushel P R amp Waters M D
(2004) Database development in toxicogenomics Issues and efforts
Environmental Health Perspectives 112 495ndash505
McMillian M Nie A Y Parker J B Leone A Bryant S Kemmerer
M et al (2004) A gene expression signature for oxidant stressreactive
metabolites in rat liver Biochemical Pharmacology 68 2249ndash2261
Meier P J amp Stieger B (2000) Molecular mechanisms in bile formation
News in Physiological Sciences 15 89ndash93
Meneses-Lorente G de Longueville F Dos Santos-Mendes S Bonnert
T P Jack A Evrard S et al (2003) An evaluation of a low-density
DNA microarray using cytochrome P450 inducers Chemical Research
in Toxicology 16 1070ndash1077
Meneses-Lorente G Guest P C Lawrence J Muniappa N Knowles
M R Skynner H A et al (2004) A proteomic investigation of drug-
induced steatosis in rat liver Chemical Research in Toxicology 17
605ndash612
Merrill C L Ni H Yoon L W Tirmenstein M A Narayanan P
Benavides G R et al (2002) Etomoxir-induced oxidative stress in
HepG2 cells detected by differential gene expression is confirmed
biochemically Toxicology Sciences 68 93ndash101
Milo R Shen-Orr S Itzkovitz S Kashtan N Chklovskii D amp Alon
U (2002) Network motifs Simple building blocks of complex
networks Science 298 824ndash827
Morgan K T Ni H Brown H R Yoon L Qualls C W Crosby L
M et al (2002) Application of cDNA microarray technology to in
vitro toxicology and the selection of genes for a real-time RT-PCR-
based screen for oxidative stress in Hep-G2 cells Toxicologic
Pathology 30 435ndash451
Mwinyi J Johne A Bauer S Roots I amp Gerloff T (2004) Evidence
for inverse effects of OATP-C (SLC21A6) 5 and 1b haplotypes on
pravastatin kinetics Clinical Pharmacology and Therapeutics 75
415ndash421
Nakata K Yukawa M Komiyama N Nakano T amp Kaminuma T
(2002) A nuclear receptor database that maps pathways to diseases
Genome Infomatics 13 515ndash516
Nicholson J K Holmes E Lindon J C amp Wilson I D (2004) The
challenges of modeling mammalian biocomplexity Nature Biotechnol-
ogy 22 1268ndash1274
Nicholson J K amp Wilson I D (2003) Understanding rsquoglobal systems
biology Metabonomics and the continuum of metabolism Nature
Reviews on Drug Discovery 2 668ndash676
Nikitin A Egorov S Daraselia N amp Mazo I (2003) Pathway studiomdash
the analysis and navigation of molecular networks Bioinformatics 19
2155ndash2157
Nikolsky Y Ekins S Nikolskaya T amp Bugrim A (2005) A novel
method for generation of signature networks as biomarkers from
complex high throughput data Toxicology Letters 158 20ndash29
Nikolsky Y Nikolskaya T amp Bugrim A (2005) Biological networks
and analysis of experimental data in drug discovery Drug Discovery
Today 10 653ndash662
Nishizato Y Ieiri I Suzuki H Kimura M Kawabata K Hirota T
et al (2003) Polymorphisms of OATP-C (SLC21A6) and OAT3
(SLC22A8) genes Consequences for pravastatin pharmacokinetics
Clinical Pharmacology and Therapeutics 73 554ndash565
Nozawa T Nakajima M Tamai I Noda K Nezu J Sai Y et al
(2002) Genetic polymorphisms of human organic anion transporters
OATP-C (SLC21A6) and OATP-B (SLC21A9) Allele frequencies in
the Japanese population and functional analysis Journal of Pharmacol-
ogy and Experimental Therapeutics 302 804ndash813
Nozawa T Sugiura S Nakajima M Goto A Yokoi T Nezu J et al
(2004) Involvement of organic anion transporting polypeptides in the
transport of troglitazone sulfate Implications for understanding trogli-
tazone hepatotoxicity Drug Metabolism and Disposition 32 291ndash294
Oliver D E Rubin D L Stuart J M Hewett M Klein T E amp
Altman R B (2002) Ontology development for a pharmacogenetics
knowledge base Package Symposion 88ndash99
Oram J F amp Lawn R M (2001) ABCA1 The gatekeeper for eliminating
excess tissue cholesterol Journal of Lipid Research 42 1173ndash1179
Oswald M Kullak-Ublick G A Paumgartner G amp Beuers U (2001)
Expression of hepatic transporters OATP-C and MRP2 in primary
sclerosing cholangitis Liver 21 247ndash253
Parsons A B Brost R I Ding H Li Z Zhang C Sheikh B et al
(2004) Integration of chemical-genetic and genetic interaction data
links bioactive compounds to cellular target pathways Nature Bio-
technology 22 62ndash69
Patki K C von Moltke L L Harmatz J S Hesse L M Court M H
amp Greenblatt D J (2004) Effect of age on in vitro triazolam
biotransformation in male human liver microsomes Journal of
Pharmacology and Experimental Therapeutics 308 874ndash879
Pereira-Leal J B Enright A J amp Ouzounis C A (2004) Detection of
functional modules from protein interaction networks Proteins 54
49ndash57
Plant N (2004) Interaction networks Coordinating responses to xeno-
biotic exposure Toxicology 202 21ndash32
Raghavendra Prasad H S Qi Z Srinivasan K N amp Gopalakrish-
nakone P (2004) Potential effects of tetrodotoxin exposure to
human glial cells postulated using microarray approach Toxicon 44
597ndash608
Reilly T P Bourdi M Brady J N Pise-Masison C A Radonovich M
F George J W et al (2001) Expression profiling of acetaminophen
liver toxicity in mice using microarray technology Biochemical and
Biophysical Research Communications 282 321ndash328
Rives A W amp Galitski T (2003) Modular organization of cellular
networks Proceedings of the National Academy of Sciences of the
United States of America 100 1128ndash1133
Rogers P D Pearson M M Cleary J D Sullivan D C amp Chapman
S W (2002) Differential expression of genes encoding immunomodu-
latory proteins in response to amphotericin B in human mononuclear
cells identified by cDNA microarray analysis Journal of Antimicrobial
Chemotherapy 50 811ndash817
Rosen M B Wilson V S Schmid J E amp Gray L E (2005) Gene
expression analysis in the ventral prostate of rats exposed to vinclozolin
or procymidone Reproductive Toxicology 19 367ndash379
Rost D Herrmann T Sauer P Schmidts H L Stieger B Meier P J
et al (2003) Regulation of rat organic anion transporters in bile salt-
induced cholestatic hepatitis Effect of ursodeoxycholate Hepatology
38 187ndash195
Rothman R B Ayestas M A Dersch C M amp Baumann M H (1999)
Aminorex fenfluramine and chlorphentermine are serotonin transporter
substrates Implications for primary pulmonary hypertension Circula-
tion 100 869ndash875
Ruepp S U Tonge R P Shaw J Wallis N amp Pognan F (2002)
Genomics and proteomics analysis of acetaminophen toxicity in mouse
liver Toxicology Sciences 65 135ndash150
Sakaeda T Nakamura T amp Okumura K (2002) MDR1 genotype-
related pharmacokinetics and pharmacodynamics Biological amp Phar-
maceutical Bulletin 25 1391ndash1400
Sakata T Anzai N Shin H J Noshiro R Hirata T Yokoyama H
et al (2004) Novel single nucleotide polymorphisms of organic cation
transporter 1 (SLC22A1) affecting transport functions Biochemical
and Biophysical Research Communications 313 789ndash793
Satlin L M Amin V amp Wolkoff A W (1997) Organic anion
transporting polypeptide mediates organic anionHCO3-exchange
Journal of Biological Chemistry 272 26340ndash26345
Satomi Y Tsuchiya W Mihara K Ota M Kasahara Y amp Akahori F
(2004) Gene expression analysis of the lung following paraquat
administration in rats using DNA microarray Journal of Toxicological
Sciences 29 91ndash100
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 65
Schrenk D Baus P R Ermel N Klein C Vorderstemann B amp
Kauffmann H M (2001) Up-regulation of transporters of the MRP
family by drugs and toxins Toxicology Letters 120 51ndash57
Segal E Shapira M Regev A Persquoer D Botstein D Koller D et al
(2003) Module networks Identifying regulatory modules and their
condition-specific regulators from gene expression data Nature
Genetics 34 166ndash176
Segal E Wang H amp Koller D (2003) Discovering molecular pathways
from protein interaction and gene expression Bioinformatics 19
i264ndash i272
Seidel S D Kan H L Stott W T Schisler M R amp Gollapudi B B
(2003) Identification of transcriptome profiles for the DNA-damaging
agents bleomycin and hydrogen peroxide in L5178Y mouse lymphoma
cells Environmental and Molecular Mutagenesis 42 19ndash25
Shan L Yu Y Schut H A J amp Snyderwine E G (2004) Susceptibility
of rats to mammary gland carcinogenesis by the food-derived
carcinogen 2-Amino-1-Methyl-6-Phenylimidazo[45-b]Pyridine (PhIP)
varies with age and is associated with the induction of differential gene
expression American Journal of Pathology 165 191ndash202
Sharom J R Bellows D S amp Tyers M (2004) From large networks to
small molecules Current Opinion in Chemical Biology 8 81ndash90
Shitara Y Hirano M Sato H amp Sugiyama Y (2004) Gemfibrozil and
its glucuronide inhibit the OATP2(OATP1B1 SLC21A6)-mediated
hepatic uptake and CYP2C8-mediated metabolism of cerivastatinmdash
Analysis of the mechanism of the clinically relevant drugndashdrug
interaction between cerivastatin and gemfibrozil Journal of Pharma-
cology and Experimental Therapeutics
Shitara Y Itoh T Sato H Li A P amp Sugiyama Y (2003) Inhibition of
transporter-mediated hepatic uptake as a mechanism for drugndashdrug
interaction between cerivastatin and cyclosporin A Journal of
Pharmacology and Experimental Therapeutics 304 610ndash616
Somogyi R Fuhrman S amp Wen X (2001) Genetic network inference in
computational models and applications to large-scale gene expression
data In M Bower amp H Bolouri (Eds) Computational modeling of
genetic and biochemical networks (pp 119ndash157) Cambridge MArsquoThe
MIT Press
Spirin V amp Mirny L A (2003) Protein complexes and functional
modules in molecular networks Proceedings of the National Academy
of Sciences of the United States of America 100 12123ndash12128
Staudinger J Liu Y Madan A Habeebu S amp Klaassen C D
(2001) Coordinate regulation of xenobiotic and bile acid homeo-
stasis by pregnane X receptor Drug Metabolism and Disposition
29 1467ndash1472
Sun L Z Ji Z L Chen X Wang J F amp Chen Y Z (2002) ADME-
AP A database of ADME associated proteins Bioinformatics 18
1699ndash1700
Suter L Babiss L E amp Wheeldon E B (2004) Toxicogenomics in
predictive toxicology in drug development Chemistry and Biology 11
161ndash171
Synold T W Dussault I amp Forman B M (2001) The orphan nuclear
receptor SXR coordinately regulates drug metabolism and efflux
Nature Medicine 7 584ndash590
Tabuchi Y amp Kondo T (2003) cDNA microarray analysis reveals chop-
10 plays a key role in Sertoli cell injury induced by bisphenol A
Biochemical and Biophysical Research Communications 305 54ndash61
Tamai I Nezu J Uchino H Sai Y Oku A Shimane M et al (2000)
Molecular identification and characterization of novel members of the
human organic anion transporter (OATP) family Biochemical and
Biophysical Research Communications 273 251ndash260
Taylor S W Fahy E Zhang B Glenn G M Warnock D E Wiley S
et al (2003) Characterization of the human heart mitochondrial
proteome Nature Biotechnology 21 281ndash286
Terasaka S Aita Y Inoue A Hayashi S Nishigaki M Aoyagi K
et al (2004) Using a customized DNA microarray for expression
profiling of the estrogen-responsive genes to evaluate estrogen activity
among natural estrogens and industrial chemicals Environmental
Health Perspectives 112 773ndash781
Thomas D M Francescutti-Verbeem D M Liu X amp Kuhn D M
(2004) Identification of differentially regulated transcripts in mouse
striatum following methamphetamine treatmentmdashan oligonucleotide
microarray approach Journal of Neurochemistry 88 380ndash393
Thomas R S Rank D R Penn S G Zastrow G M Hayes K R
Pande K et al (2001) Identification of toxicologically predictive
gene sets using cDNA microarrays Molecular Pharmacology 60
1189ndash1194
Thompson K L Afshari C A Amin R P Bertram T A Car B
Cunningham M et al (2004) Identification of platform-independent
gene expression markers of cisplatin nephrotoxicity Environmental
Health Perspectives 112 488ndash494
Tirona R G Leake B F Merino G amp Kim R B (2001)
Polymorphisms in OATP-C Identification of multiple allelic
variants associated with altered transport activity among European-
and African-Americans Journal of Biological Chemistry 276
35669ndash35675
Tirona R G Leake B F Wolkoff A W amp Kim R B (2003)
Human organic anion transporting polypeptide-C (SLC21A6) is a
major determinant of the rifampin-mediated pregnane X receptor
activation Journal of Pharmacology and Experimental Therapeutics
304 223ndash228
Tong W Cao X Harris S Sun H Fang H Fuscoe J et al (2003)
ArrayTrackmdashsupporting toxicogenomic research at the US Food and
Drug Administration National Center for Toxicological Research
Environmental Health Perspectives 111 1819ndash1826
Tornow S amp Mewes H W (2003) Functional modules by relating
protein interaction networks and gene expression Nucleic Acids
Research 31 6283ndash6289
Trocho C Escola-Gil J C Ribas V Benitez S Martin-Campos J M
Rotllan N et al (2004) Phenytoin treatment reduces atherosclerosis in
mice through mechanisms independent of plasma HDL-cholesterol
concentration Atherosclerosis 174 275ndash285
Ueda A Hamadeh H K Webb H K Yamamoto Y Sueyoshi T
Afshari C A et al (2002) Diverse roles of the nuclear orphan
receptor CAR in regulating hepatic genes in response to phenobarbital
Molecular Pharmacology 61 1ndash6
Ulrich R G (2003) The toxicogenomics of nuclear receptor agonists
Current Opinion in Chemical Biology 7 505ndash510
Ulrich R G Rockett J C Gibson G G amp Pettit S D (2004)
Overview of an interlaboratory collaboration on evaluating the effects
of model hepatotoxicants on hepatic gene expression Environmental
Health Perspectives 112 423ndash427
Unami A Shinohara Y Ichikawa T amp Baba Y (2003) Biochemical
and microarray analyses of bupivacaine-induced apoptosis Journal of
Toxicological Sciences 28 77ndash94
Vasquez A Flammini A Maritan A amp Vespignani A (2003) Global
protein function prediction from proteinndashprotein interaction networks
Nature Biotechnology 21 697ndash700
Vezina C M Walker N J amp Olson J R (2004) Subchronic Exposure to
TCDD PeCDF PCB126 and PCB153 Effect on hepatic gene
expression Environmental Health Perspectives 112 1636ndash1644
Vo T D Greenberg H J amp Palsson B O (2004) Reconstruction and
functional characterization of the human mitochondrial metabolic
network based on proteomic and biochemical data Journal of Bio-
logical Chemistry 279 39532ndash39540
Wang H amp LeCluyse E L (2003) Role of orphan nuclear receptors in
the regulation of drug-metabolising enzymes Clinical Pharmacoki-
netics 42 1331ndash1357
Wang N Silver D L Thiele C amp Tall A R (2001) ATP-
binding cassette transporter A1 (ABCA1) functions as a choles-
terol efflux regulatory protein Journal of Biological Chemistry 276
23742ndash23747
Waring J F Cavet G Jolly R A McDowell J Dai H Ciurlionis R
et al (2003) Development of a DNA Microarray for toxicology based
on hepatoxin-regulated sequences Environmental Health Perspectives
111 863ndash870
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6666
Waring J F Gum R Morfitt D Jolly R A Ciurlionis R Heindel M
et al (2002) Identifying toxic mechanisms using DNA microarrays
Evidence that an experimental inhibitor of cell adhesion molecule
expression signals through the aryl hydrocarbon nuclear receptor
Toxicology 181ndash182 537ndash550
Waring J F Jolly R A Ciurlionis R Lum P Y Praestgaard J T
Morfitt D C et al (2001) Clustering of hepatotoxins based on
mechanism of toxicity using gene expression profiles Toxicology and
Applied Pharmacology 175 28ndash42
Waters M Boorman G Bushel P Cunningham M Irwin R Merrick
A et al (2003) Systems toxicology and the Chemical Effects in
Biological Systems (CEBS) knowledge base EHP Toxicogenomics
111 15ndash28
Wei M Arnold L Cano M amp Cohen S M (2005) Effects of co-
administration of antioxidants and arsenicals on the rat urinary bladder
epithelium Toxicology Sciences 83 237ndash245
Weinshilboum R (2003) Inheritance and drug response New England
Journal of Medicine 348 529ndash537
Weinshilboum R amp Wang L (2004) Pharmacogenetics Inherited
variation in amino acid sequence and altered protein quantity Clinical
Pharmacology and Therapeutics 75 253ndash258
Werner E (2003) In silico multicellular systems biology and minimal
genomes Drug Discovery Today 8 1121ndash1127
Wong J S amp Gill S S (2002) Gene expression changes induced in
mouse liver by di(2-ethylhexyl) phthalate Toxicology and Applied
Pharmacology 185 180ndash196
Xie T Tong L McCann U D Yuan J Becker K G Mechan A O
et al (2004) Identification and characterization of metallothionein-1
and -2 gene expression in the context of ()34-methylenedioxymetham-
phetamine-induced toxicity to brain dopaminergic neurons Journal of
Neuroscience 24 7043ndash7050
Yadetie F Laegreid A Bakke I Kusnierczyk W Komorowski J
Waldum H L et al (2003) Liver gene expression in rats in response
to the peroxisome proliferator-activated receptor-alpha agonist ciprofi-
brate Physiological Genomics 15 9ndash19
Yamashita S Nomoto T Abe M Tatematsu M Sugimura T amp
Ushijima T (2004) Persistence of gene expression changes in stomach
mucosae induced by short-term N-methyl-NV-nitro-N-nitrosoguanidinetreatment and their presence in stomach cancers Mutation Research
549 185ndash193
Yan Q amp Sadee W (2000) Human membrane transporter database A
web-accessible relational database for drug transport studies and
pharmacogenomics AAPS Pharmsci 2 E20
Yeger-Lotem E amp Margalit H (2003) Detection of regulatory circuits by
integrating the cellular networks of proteinndashprotein interactions and
transcription regulation Nucleic Acids Research 31 6053ndash6061
Yoon B I Li G -X Kitada K Kawasaki Y Igarashi K amp Kodama
Y et al (2003) Mechanisms of benzene-induced hematotoxicity and
leukemogenicity cDNA microarray analyses using mouse bone marrow
tissue Environmental Health Perspectives 111 1411ndash1420
Young M B DiSilvestro M R Sendera T J Freund J Kriete A amp
Magnuson S R (2003) Analysis of gene expression in carbon
tetrachloride-treated rat livers using a novel bioarray technology
Pharmacogenomics Journal 3 41ndash52
Yu H Zhu X Greenbaum D Karro J amp Gerstein M (2004) TopNet
A tool for comparing biological sub-networks correlating protein
properties with topological statistics Nucleic Acids Research 32
328ndash337
Zhang E Y Fu D J Pak Y A Stewart T Mukhopadhyay N
Wrighton S A et al (2004) Genetic polymorphisms in human proton-
dependent dipeptide transporter PEPT1 Implications for the functional
role of Pro586 Journal of Pharmacology and Experimental Therapeu-
tics 310 437ndash445
Zhang E Y Knipp G T Ekins S amp Swaan P W (2002)
Structural biology and function of solute transporters Implications
for identifying and designing substrates Drug Metabolism Reviews
34 709ndash750
Zhang E Y Phelps M A Cheng C Ekins S amp Swaan P W (2002)
Modeling of active transport systems Advanced Drug Delivery
Reviews 54 329ndash354
- Systems-ADMETox Resources and network approaches
-
- Introduction
- Data available
- Network analysis and databases
-
- Network applications
-
- The role of transporters
-
- Clinical relevance of transporters
- Transporter network examples ABCA1
- Transporter network examples OATP
- Transporter microarray data
-
- Applications to enzymes
- Future network applications
-
- Discussion
- Acknowledgements
- References
-
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6662
Ekins S Boulanger B Swaan P W amp Hupcey M A Z (2002)
Towards a new age of virtual ADMETOX and multidimensional drug
discovery Journal of Computer-Aided Molecular Design 16 381ndash401
Ekins S Bugrim A Nikolsky Y amp Nikolskaya T (2005) Systems
biology Applications in drug discovery In S Gad (Ed) Drug
Discovery Handbook (pp 123ndash183) Hoboken NJrsquo Wiley and Sons
Ekins S Giroux C N Nikolsky Y Bugrim A amp Nikolskaya T
(2005) A signature gene network approach to toxicity The Toxoco-
logists 84
Ekins S Kirillov E Rakhmatulin E amp Nikolskaya T (2005) A novel
method for visualizing nuclear hormone receptor networks relevant to
drug metabolism Drug Metabolism and Disposition 33 474ndash481
Ekins S amp McGowan R J (2001) The limits of reductionism The
shifting genomic paradigmrsquos impact on industry and academia
Philsophy in Science 9 1ndash23
Ekins S Mirny L amp Schuetz E G (2002) A ligand-based approach to
understanding selectivity of nuclear hormone receptors PXR CAR
FXR LXRa and LXRb Pharmaceutical Research 19 1788ndash1800
Ekins S Nikolsky Y amp Nikolskaya T (2005) Techniques Application
of systems biology to absorption distribution metabolism excretion
and toxicity Trends in Pharmacological Sciences 26 202ndash209
Ekins S Ring B J Grace J McRobie-Belle D J amp Wrighton S
A (2000) Present and future in vitro approaches for drug
metabolism Journal of Pharmacological and Toxicological Methods
44 313ndash324
Ekins S Waller C L Swaan P W Cruciani G Wrighton S A amp
Wikel J H (2000) Progress in predicting human ADME parameters in
silico Journal of Pharmacological and Toxicological Methods 44
251ndash272
Erhardt P W (2003) A human drug metabolism database Potential roles
in the quantitative predictions of drug metabolism and metabolism-
related drugndashdrug interactions Current Drug Metabolism 4 411ndash422
Evans W E amp McLeod H L (2003) Pharmacogenomicsmdashdrug
disposition drug targets and side effects New England Journal of
Medicine 348 538ndash549
Ezendam J Staedtler F Pennings J Vandebriel R J Pieters R
Boffetta P et al (2004) Toxicogenomics of subchronic hexachlor-
obenzene exposure in Brown Norway rats Environmental Health
Perspectives 112 782ndash791
Faiola B Fuller E S Wong V A amp Recio L (2004) Gene expression
profile in bone marrow and hematopoietic stem cells in mice exposed to
inhaled benzene Mutation Research 549 195ndash212
FDA (2004) Innovation stagnation Challenge and opportunity on the
critical path to new medicinal products
Fiehn O (2001) Combining genomics metabolome analysis and bio-
chemical modelling to understand metabolic networks Comparative
Functional Genomics 2 155ndash168
Field F J Born E amp Mathur S N (2004) LXRRXR ligand activation
enhances basolateral efflux of beta-sitosterol in CaCo-2 cells Journal of
Lipid Research 45 905ndash913
Fleck C Sutter L Appenroth D Koch B Meinhold T Pitack M
et al (2003) Use of gene chip technology for the characterisation of
the regulation of renal transport processes and of nephrotoxicity in
rats Experimental and Toxicologic Pathology 54 401ndash410
Fliri A F Loging W T Thadeio P F amp Volkmann R A (2005)
Biological spectra analysis Linking biological activity profiles to
molecular structure Proceedings of the National Academy of Sciences
of the United States of America 102 261ndash266
Fountoulakis M (2004) Application of proteomics technologies in the
investigation of the brain Mass Spectrometry Reviews 23 231ndash258
Gaucher S P Taylor S W Fahy E Zhang B Warnock D E Ghosh
S S et al (2004) Expanded coverage of the human heart
mitochondrial proteome using multidimensional liquid chromatography
coupled with tandem mass spectrometry Journal of Proteome
Research 3 495ndash505
Gerhold D Lu M Xu J Austin C Caskey C T amp Rushmore T
(2001) Monitoring expression of genes involved in drug metabolism
and toxicology using DNA microarrays Physiological Genomics 5
161ndash170
Guo G L Choudhuri S amp Klaassen C D (2002) Induction profile of
rat organic anion transporting polypeptide 2 (oatp2) by prototypical
drug-metabolizing enzyme inducers that activate gene expression
through ligand-activated transcription factor pathways Journal of
Pharmacology and Experimental Therapeutics 300 206ndash212
Hagenbuch B amp Meier P J (2004) Organic anion transporting
polypeptides of the OATP SLC21 family Phylogenetic classification
as OATP SLCO superfamily new nomenclature and molecularfunc-
tional properties Pflugers Archiv 447 653ndash665
Hamadeh H K amp Afshari C A (2004) Toxicogenomics principles and
applications Hobokenrsquo John Wiley and Sons
Hamadeh H K Bushel P R Jayadev S Martin K DiSorbo O Sieber
S et al (2002) Gene expression analysis reveals chemical-specific
profiles Toxicological Sciences 67 219ndash231
Hamadeh H K Jayadev S Gaillard E T Huang Q Stoll R
Blanchard K et al (2004) Integration of clinical and gene expression
endpoints to explore furan-mediated hepatotoxicity Mutation Research
549 169ndash183
Hamadeh H K Knight B L Haugen A C Sieber S Amin R P
Bushel P R et al (2002) Methapyrilene toxicity Anchorage of
pathologic observations to gene expression alterations Toxicologic
Pathology 30 470ndash482
Han J D Bertin N Hao T Goldberg D S Berriz G F Zhang L V
et al (2004) Evidence for dynamically organized modularity in the
yeast proteinndashprotein interaction network Nature 430 88ndash93
Hanisch D Zien A Zimmer R amp Lengauer T (2002) Co-clustering of
biological networks and gene expression data Bioinformatics 18
S145ndashS154
Harris A J Dial S L amp Casciano D A (2004) Comparison of basal
gene expression profiles and effects of hepatocarcinogens on gene
expression in cultured primary human hepatocytes and HepG2 cells
Mutation Research 549 79ndash99
Harrison S C (2004) Whither structural biology Nature Structural
Molecular Biology 11 12ndash15
Hartley D P Dai X He Y D Carlini E J Wang B Huskey S E
et al (2004) Activators of the rat pregnane X receptor differentially
modulate hepatic and intestinal gene expression Molecular Pharma-
cology 65 1159ndash1171
Hartwell L H Hopfield J J Leibler S amp Murray A W (1999) From
molecular to modular cell biology Nature 402 C47ndashC52
Hasmall S Orphanides G James N Pennie W Hedley K Soames
A et al (2002) Downregulation of lactoferrin by PPARalpha ligands
Role in perturbation of hepatocyte proliferation and apoptosis
Toxicology Sciences 68 304ndash313
Hayase T Yamamoto Y Yamamoto K Muso E amp Shiota K (2004)
Microarray profile analysis of toxic cocaine-induced alterations in the
expression of mouse brain gene sequences A possible rsquoprotectiversquo
effect of buprenorphine Journal of Applied Toxicology 24 15ndash20
Hayes K R Vollrath A L Zastrow G M McMillan B J Craven M
Jovanovich S et al (2005) EDGE A centralized resource for the
comparison analysis and distribution of toxicogenomic information
Molecular Pharmacology 67 1360ndash1368
Heijne W H Slitt A L van Bladeren P J Groten J P Klaassen C D
Stierum R H et al (2004) Bromobenzene-induced hepatotoxicity at
the transcriptome level Toxicology Sciences 79 411ndash422
Heijne W H Stierum R H Slijper M van Bladeren P J amp van
Ommen B (2003) Toxicogenomics of bromobenzene hepatotoxicity
A combined transcriptomics and proteomics approach Biochemical
Pharmacology 65 857ndash875
Heinloth A N Irwin R D Boorman G A Nettesheim P Fannin
R D Sieber S O et al (2004) Gene expression profiling of rat
livers reveals indicators of potential adverse effects Toxicology
Sciences 80 193ndash202
Hodges L C Cook J D Lobenhofer E K Li L Bennett L Bushel
P R et al (2003) Tamoxifen functions as a molecular agonist inducing
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 63
cell cycle-associated genes in breast cancer cells Molecular Cancer
Research 1 300ndash311
Holleman A Cheok M H den Boer M L Yang W Veerman KM
A J Kazemier et al (2004) Gene-expression patterns in drug-resistant
acute lymphoblastic leukemia cells and response to treatment New
England Journal of Medicine 351 533ndash542
Hong Y Muller U R amp Lai F (2003) Discriminating two classes of
toxicants through expression analysis of HepG2 cells with DNA arrays
Toxicology in Vitro 17 85ndash92
Hood E (2003) Proteomics Characterizing the cogs in the machinery of
life Environmental Health Perspectives 111 A816ndashA825
Hood L (2003) Systems biology Integrating technology biology and
computation Mechanism of Ageing and Development 124 9ndash16
Hood L amp Galas D (2003) The digital code of DNA Nature 421
444ndash448
Hood L amp Perlmutter R M (2004) The impact of systems approaches on
biological problems in drug discovery Nature Biotechnology 22
1215ndash1217
Hu T Gibson D P Carr G J Torontali S M Tiesman J P Chaney
J G et al (2004) Identification of a gene expression profile that
discriminates indirect-acting genotoxins from direct-acting genotoxins
Mutation Research 549 5ndash27
Huang Q Dunn II R T Jayadev S DiSorbo O Pack F D Farr S B
et al (2001) Assessment of cisplatin-induced nephrotoxicity by
microarray technology Toxicology Sciences 63 196ndash207
Huang Q Jin X Gaillard E T Knight B L Pack F D Stoltz J H et
al (2004) Gene expression profiling reveals multiple toxicity endpoints
induced by hepatotoxicants Mutation Research 549 147ndash167
Huisman M T Smit J W Crommentuyn K M Zelcer N Wiltshire
H R Beijnen J H et al (2002) Multidrug resistance protein 2
(MRP2) transports HIV protease inhibitors and transport can be
enhanced by other drugs Aids 16 2295ndash2301
Hunt C M Westerkam W R amp Stave G M (1992) Effect of age and
gender on the activity of human hepatic CYP3A Biochemical
Pharmacology 44 275ndash283
Ideker T Ozier O Schwikowski B amp Siegal A F (2002) Discovering
regulatory and signalling circuits in molecular interaction networks
Bioinformatics 18 S233ndashS240
Ideker T Thorsson V Ranish J A Christmas R Buhler J Eng J K
et al (2001) Integrated genomic and proteomic analyses of a system-
atically perturbed network Science 292 929ndash934
Iida M Anna C H Hartis J Bruno M Wetmore B Dubin J R et al
(2003) Changes in global gene and protein expression during early
mouse liver carcinogenesis induced by non-genotoxic model carcino-
gens oxazepam and Wyeth-14643 Carcinogenesis 24 757ndash770
Ingelman-Sundberg M (2004) Pharmacogenetics of cytochrome P450 and
its applications in drug therapy The past present and future Trends in
Pharmacological Sciences 25 193ndash200
Jeong H Mason S P Barabasi A L amp Oltvai Z N (2001) Lethality
and centrality in protein networks Nature 411 41ndash42
Jeong H Tombor B Albert R Oltvai Z N amp Barabasi A L
(2000) The large-scale organization of metabolic networks Nature
407 651ndash654
Jung D Hagenbuch B Gresh L Pontoglio M Meier P J amp Kullak-
Ublick G A (2001) Characterization of the human OATP-C
(SLC21A6) gene promoter and regulation of liver-specific OATP genes
by hepatocyte nuclear factor 1 alpha Journal of Biological Chemistry
276 37206ndash37214
Jung D amp Kullak-Ublick G A (2003) Hepatocyte nuclear factor 1
alpha A key mediator of the effect of bile acids on gene expression
Hepatology 37 622ndash631
Jung J -W Park J -S Hwang J -W Kang K -S YS L Song B
-S et al (2004) Gene expression analysis of peroxisome prolifer-
ators- and phenytoin-induced hepatotoxicity using cDNA microarray
Journal of Veterinary Medical Science 66 1329ndash1333
Kato N Shibutani M Takagi H Uneyama C Lee K -Y Takiami S
et al (2004) Gene expression profile in the livers of rats orally
administered ethinylestradiol for 28 days using a microarray technique
Toxicology 200 179ndash192
Kier L D Neft R Tang L Suizu R Cook T Onsurez K et al
(2004) Applications of microarrays with toxicologically relevant genes
(tox genes) for the evaluation of chemical toxicants in Sprague Dawley
rats in vivo and human hepatocytes in vitro Mutation Research 549
101ndash113
Kijima K Toyosawa K Yasuba M Matsuoka N Adachi T
Komiyama M et al (2004) Gene expression analysis of the rat testis
after treatment with di(2-ethylhexyl) phthalate using cDNA microarray
and real-time RT-PCR Toxicology and Applied Pharmacology 200
103ndash110
Kim R B (2003) Organic anion-transporting polypeptide (OATP) trans-
porter family and drug disposition European Journal of Clinical
Investigation 33 1ndash5
Kinirons M T amp OrsquoMahony M S (2004) Drug metabolism and ageing
British Journal of Clinical Pharmacology 57 540ndash544
Kitano H (2002a) Computational systems biology Nature 420 206ndash210
Kitano H (2002b) Systems biology A brief overview Science 295
1662ndash1664
Kliewer S A Moore J T Wade L Staudinger J L Watson M
A Jones S A et al (1998) An orphan nuclear receptor activated
by pregnanes defines a novel steroid signalling pathway Cell 92
73ndash82
Korolev D Balakin K V Nikolsky Y Kirillov E Ivanenkov Y A
Savchuk N P et al (2003) Modeling of human cytochrome p450-
mediated drug metabolism using unsupervised machine learning
approach Journal of Medicinal Chemistry 46 3631ndash3643
Kramer J A Pettit S D Amin R P Bertram T A Car B
Cunningham M et al (2004) Overview on the application of
transcription profiling using selected nephrotoxicants for toxicology
assessment Environmental Health Perspectives 112 460ndash464
Kultima K Nystrom A -M Scholz B Gustafson A -L Dencker L
amp Stigson M (2004) Valproic acid teratogenicity A toxicogenomics
approach Environmental Health Perspectives 112 1225ndash1235
Kutuzova G D amp Deluca H F (2004) Gene expression profiles in rat
intestine identify pathways for 125-dihydroxyvitamin D(3) stimulated
calcium absorption and clarify its immunomodulatory properties
Archives of Biochemistry and Biophysics 432 152ndash166
Kwak M K Wakabayashi N Itoh K Motohashi H Yamamoto M amp
Kensler T W (2003) Modulation of gene expression by cancer
chemopreventive dithiolethiones through the Keap1-Nrf2 pathway
Identification of novel gene clusters for cell survival Journal of
Biological Chemistry 278 8135ndash8145
Lahjouji K Mitchell G A amp Qureshi I A (2001) Carnitine transport
by organic cation transporters and systemic carnitine deficiency
Molecular Genetics and Metabolism 73 287ndash297
Landowski C P Sun D Foster D R Menon S S Barnett J L
Welage L S et al (2003) Gene expression in the human intestine and
correlation with oral valacyclovir pharmacokinetic parameters Journal
of Pharmacology and Experimental Therapeutics 306 778ndash786
Lee G -J Lee W -S Jeon K -S Um C -H Kim S -J Lee C -H
et al (2004) cDNA microarray gene expression analysis and
toxicological phenotype for anticancer drug Journal of Veterinary
Medical Science 66 1339ndash1345
Lee M Kwon J Kim S N Kim J E Koh W S Kim E J et al
(2003) cDNA microarray gene expression profiling of hydroxyurea
paclitaxel and p-anisidine genotoxic compounds with differing
tumorigenicity results Environmental and Molecular Mutagenesis
42 91ndash97
Li S Armstrong C M Bertin N Ge H Milstein S Boxem M et al
(2004) A map of the interactome network of the metazoan C elegans
Science 303 540ndash543
Liguori M J Anderson L M Bukofzer S McKim J Pregenzer J F
Retief J et al (2005) Microarray analysis in human hepatocytes
suggests a mechanism for hepatotoxicity induced by trovafloxacin
Hepatology 41 177ndash186
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6664
Lobenhofer E K Bennett L Cable P L Li L Bushel P R amp Afshari
C A (2002) Regulation of DNA replication fork genes by 17beta-
estradiol Molecular Endocrinology 16 1215ndash1229
Locker J Tian J Carver R Concas D Cossu C Ledda-Columbano
G M et al (2003) A common set of immediate-early response genes
in liver regeneration and hyperplasia Hepatology 38 314ndash325
Mattes W B Pettit S D Sansone S A Bushel P R amp Waters M D
(2004) Database development in toxicogenomics Issues and efforts
Environmental Health Perspectives 112 495ndash505
McMillian M Nie A Y Parker J B Leone A Bryant S Kemmerer
M et al (2004) A gene expression signature for oxidant stressreactive
metabolites in rat liver Biochemical Pharmacology 68 2249ndash2261
Meier P J amp Stieger B (2000) Molecular mechanisms in bile formation
News in Physiological Sciences 15 89ndash93
Meneses-Lorente G de Longueville F Dos Santos-Mendes S Bonnert
T P Jack A Evrard S et al (2003) An evaluation of a low-density
DNA microarray using cytochrome P450 inducers Chemical Research
in Toxicology 16 1070ndash1077
Meneses-Lorente G Guest P C Lawrence J Muniappa N Knowles
M R Skynner H A et al (2004) A proteomic investigation of drug-
induced steatosis in rat liver Chemical Research in Toxicology 17
605ndash612
Merrill C L Ni H Yoon L W Tirmenstein M A Narayanan P
Benavides G R et al (2002) Etomoxir-induced oxidative stress in
HepG2 cells detected by differential gene expression is confirmed
biochemically Toxicology Sciences 68 93ndash101
Milo R Shen-Orr S Itzkovitz S Kashtan N Chklovskii D amp Alon
U (2002) Network motifs Simple building blocks of complex
networks Science 298 824ndash827
Morgan K T Ni H Brown H R Yoon L Qualls C W Crosby L
M et al (2002) Application of cDNA microarray technology to in
vitro toxicology and the selection of genes for a real-time RT-PCR-
based screen for oxidative stress in Hep-G2 cells Toxicologic
Pathology 30 435ndash451
Mwinyi J Johne A Bauer S Roots I amp Gerloff T (2004) Evidence
for inverse effects of OATP-C (SLC21A6) 5 and 1b haplotypes on
pravastatin kinetics Clinical Pharmacology and Therapeutics 75
415ndash421
Nakata K Yukawa M Komiyama N Nakano T amp Kaminuma T
(2002) A nuclear receptor database that maps pathways to diseases
Genome Infomatics 13 515ndash516
Nicholson J K Holmes E Lindon J C amp Wilson I D (2004) The
challenges of modeling mammalian biocomplexity Nature Biotechnol-
ogy 22 1268ndash1274
Nicholson J K amp Wilson I D (2003) Understanding rsquoglobal systems
biology Metabonomics and the continuum of metabolism Nature
Reviews on Drug Discovery 2 668ndash676
Nikitin A Egorov S Daraselia N amp Mazo I (2003) Pathway studiomdash
the analysis and navigation of molecular networks Bioinformatics 19
2155ndash2157
Nikolsky Y Ekins S Nikolskaya T amp Bugrim A (2005) A novel
method for generation of signature networks as biomarkers from
complex high throughput data Toxicology Letters 158 20ndash29
Nikolsky Y Nikolskaya T amp Bugrim A (2005) Biological networks
and analysis of experimental data in drug discovery Drug Discovery
Today 10 653ndash662
Nishizato Y Ieiri I Suzuki H Kimura M Kawabata K Hirota T
et al (2003) Polymorphisms of OATP-C (SLC21A6) and OAT3
(SLC22A8) genes Consequences for pravastatin pharmacokinetics
Clinical Pharmacology and Therapeutics 73 554ndash565
Nozawa T Nakajima M Tamai I Noda K Nezu J Sai Y et al
(2002) Genetic polymorphisms of human organic anion transporters
OATP-C (SLC21A6) and OATP-B (SLC21A9) Allele frequencies in
the Japanese population and functional analysis Journal of Pharmacol-
ogy and Experimental Therapeutics 302 804ndash813
Nozawa T Sugiura S Nakajima M Goto A Yokoi T Nezu J et al
(2004) Involvement of organic anion transporting polypeptides in the
transport of troglitazone sulfate Implications for understanding trogli-
tazone hepatotoxicity Drug Metabolism and Disposition 32 291ndash294
Oliver D E Rubin D L Stuart J M Hewett M Klein T E amp
Altman R B (2002) Ontology development for a pharmacogenetics
knowledge base Package Symposion 88ndash99
Oram J F amp Lawn R M (2001) ABCA1 The gatekeeper for eliminating
excess tissue cholesterol Journal of Lipid Research 42 1173ndash1179
Oswald M Kullak-Ublick G A Paumgartner G amp Beuers U (2001)
Expression of hepatic transporters OATP-C and MRP2 in primary
sclerosing cholangitis Liver 21 247ndash253
Parsons A B Brost R I Ding H Li Z Zhang C Sheikh B et al
(2004) Integration of chemical-genetic and genetic interaction data
links bioactive compounds to cellular target pathways Nature Bio-
technology 22 62ndash69
Patki K C von Moltke L L Harmatz J S Hesse L M Court M H
amp Greenblatt D J (2004) Effect of age on in vitro triazolam
biotransformation in male human liver microsomes Journal of
Pharmacology and Experimental Therapeutics 308 874ndash879
Pereira-Leal J B Enright A J amp Ouzounis C A (2004) Detection of
functional modules from protein interaction networks Proteins 54
49ndash57
Plant N (2004) Interaction networks Coordinating responses to xeno-
biotic exposure Toxicology 202 21ndash32
Raghavendra Prasad H S Qi Z Srinivasan K N amp Gopalakrish-
nakone P (2004) Potential effects of tetrodotoxin exposure to
human glial cells postulated using microarray approach Toxicon 44
597ndash608
Reilly T P Bourdi M Brady J N Pise-Masison C A Radonovich M
F George J W et al (2001) Expression profiling of acetaminophen
liver toxicity in mice using microarray technology Biochemical and
Biophysical Research Communications 282 321ndash328
Rives A W amp Galitski T (2003) Modular organization of cellular
networks Proceedings of the National Academy of Sciences of the
United States of America 100 1128ndash1133
Rogers P D Pearson M M Cleary J D Sullivan D C amp Chapman
S W (2002) Differential expression of genes encoding immunomodu-
latory proteins in response to amphotericin B in human mononuclear
cells identified by cDNA microarray analysis Journal of Antimicrobial
Chemotherapy 50 811ndash817
Rosen M B Wilson V S Schmid J E amp Gray L E (2005) Gene
expression analysis in the ventral prostate of rats exposed to vinclozolin
or procymidone Reproductive Toxicology 19 367ndash379
Rost D Herrmann T Sauer P Schmidts H L Stieger B Meier P J
et al (2003) Regulation of rat organic anion transporters in bile salt-
induced cholestatic hepatitis Effect of ursodeoxycholate Hepatology
38 187ndash195
Rothman R B Ayestas M A Dersch C M amp Baumann M H (1999)
Aminorex fenfluramine and chlorphentermine are serotonin transporter
substrates Implications for primary pulmonary hypertension Circula-
tion 100 869ndash875
Ruepp S U Tonge R P Shaw J Wallis N amp Pognan F (2002)
Genomics and proteomics analysis of acetaminophen toxicity in mouse
liver Toxicology Sciences 65 135ndash150
Sakaeda T Nakamura T amp Okumura K (2002) MDR1 genotype-
related pharmacokinetics and pharmacodynamics Biological amp Phar-
maceutical Bulletin 25 1391ndash1400
Sakata T Anzai N Shin H J Noshiro R Hirata T Yokoyama H
et al (2004) Novel single nucleotide polymorphisms of organic cation
transporter 1 (SLC22A1) affecting transport functions Biochemical
and Biophysical Research Communications 313 789ndash793
Satlin L M Amin V amp Wolkoff A W (1997) Organic anion
transporting polypeptide mediates organic anionHCO3-exchange
Journal of Biological Chemistry 272 26340ndash26345
Satomi Y Tsuchiya W Mihara K Ota M Kasahara Y amp Akahori F
(2004) Gene expression analysis of the lung following paraquat
administration in rats using DNA microarray Journal of Toxicological
Sciences 29 91ndash100
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 65
Schrenk D Baus P R Ermel N Klein C Vorderstemann B amp
Kauffmann H M (2001) Up-regulation of transporters of the MRP
family by drugs and toxins Toxicology Letters 120 51ndash57
Segal E Shapira M Regev A Persquoer D Botstein D Koller D et al
(2003) Module networks Identifying regulatory modules and their
condition-specific regulators from gene expression data Nature
Genetics 34 166ndash176
Segal E Wang H amp Koller D (2003) Discovering molecular pathways
from protein interaction and gene expression Bioinformatics 19
i264ndash i272
Seidel S D Kan H L Stott W T Schisler M R amp Gollapudi B B
(2003) Identification of transcriptome profiles for the DNA-damaging
agents bleomycin and hydrogen peroxide in L5178Y mouse lymphoma
cells Environmental and Molecular Mutagenesis 42 19ndash25
Shan L Yu Y Schut H A J amp Snyderwine E G (2004) Susceptibility
of rats to mammary gland carcinogenesis by the food-derived
carcinogen 2-Amino-1-Methyl-6-Phenylimidazo[45-b]Pyridine (PhIP)
varies with age and is associated with the induction of differential gene
expression American Journal of Pathology 165 191ndash202
Sharom J R Bellows D S amp Tyers M (2004) From large networks to
small molecules Current Opinion in Chemical Biology 8 81ndash90
Shitara Y Hirano M Sato H amp Sugiyama Y (2004) Gemfibrozil and
its glucuronide inhibit the OATP2(OATP1B1 SLC21A6)-mediated
hepatic uptake and CYP2C8-mediated metabolism of cerivastatinmdash
Analysis of the mechanism of the clinically relevant drugndashdrug
interaction between cerivastatin and gemfibrozil Journal of Pharma-
cology and Experimental Therapeutics
Shitara Y Itoh T Sato H Li A P amp Sugiyama Y (2003) Inhibition of
transporter-mediated hepatic uptake as a mechanism for drugndashdrug
interaction between cerivastatin and cyclosporin A Journal of
Pharmacology and Experimental Therapeutics 304 610ndash616
Somogyi R Fuhrman S amp Wen X (2001) Genetic network inference in
computational models and applications to large-scale gene expression
data In M Bower amp H Bolouri (Eds) Computational modeling of
genetic and biochemical networks (pp 119ndash157) Cambridge MArsquoThe
MIT Press
Spirin V amp Mirny L A (2003) Protein complexes and functional
modules in molecular networks Proceedings of the National Academy
of Sciences of the United States of America 100 12123ndash12128
Staudinger J Liu Y Madan A Habeebu S amp Klaassen C D
(2001) Coordinate regulation of xenobiotic and bile acid homeo-
stasis by pregnane X receptor Drug Metabolism and Disposition
29 1467ndash1472
Sun L Z Ji Z L Chen X Wang J F amp Chen Y Z (2002) ADME-
AP A database of ADME associated proteins Bioinformatics 18
1699ndash1700
Suter L Babiss L E amp Wheeldon E B (2004) Toxicogenomics in
predictive toxicology in drug development Chemistry and Biology 11
161ndash171
Synold T W Dussault I amp Forman B M (2001) The orphan nuclear
receptor SXR coordinately regulates drug metabolism and efflux
Nature Medicine 7 584ndash590
Tabuchi Y amp Kondo T (2003) cDNA microarray analysis reveals chop-
10 plays a key role in Sertoli cell injury induced by bisphenol A
Biochemical and Biophysical Research Communications 305 54ndash61
Tamai I Nezu J Uchino H Sai Y Oku A Shimane M et al (2000)
Molecular identification and characterization of novel members of the
human organic anion transporter (OATP) family Biochemical and
Biophysical Research Communications 273 251ndash260
Taylor S W Fahy E Zhang B Glenn G M Warnock D E Wiley S
et al (2003) Characterization of the human heart mitochondrial
proteome Nature Biotechnology 21 281ndash286
Terasaka S Aita Y Inoue A Hayashi S Nishigaki M Aoyagi K
et al (2004) Using a customized DNA microarray for expression
profiling of the estrogen-responsive genes to evaluate estrogen activity
among natural estrogens and industrial chemicals Environmental
Health Perspectives 112 773ndash781
Thomas D M Francescutti-Verbeem D M Liu X amp Kuhn D M
(2004) Identification of differentially regulated transcripts in mouse
striatum following methamphetamine treatmentmdashan oligonucleotide
microarray approach Journal of Neurochemistry 88 380ndash393
Thomas R S Rank D R Penn S G Zastrow G M Hayes K R
Pande K et al (2001) Identification of toxicologically predictive
gene sets using cDNA microarrays Molecular Pharmacology 60
1189ndash1194
Thompson K L Afshari C A Amin R P Bertram T A Car B
Cunningham M et al (2004) Identification of platform-independent
gene expression markers of cisplatin nephrotoxicity Environmental
Health Perspectives 112 488ndash494
Tirona R G Leake B F Merino G amp Kim R B (2001)
Polymorphisms in OATP-C Identification of multiple allelic
variants associated with altered transport activity among European-
and African-Americans Journal of Biological Chemistry 276
35669ndash35675
Tirona R G Leake B F Wolkoff A W amp Kim R B (2003)
Human organic anion transporting polypeptide-C (SLC21A6) is a
major determinant of the rifampin-mediated pregnane X receptor
activation Journal of Pharmacology and Experimental Therapeutics
304 223ndash228
Tong W Cao X Harris S Sun H Fang H Fuscoe J et al (2003)
ArrayTrackmdashsupporting toxicogenomic research at the US Food and
Drug Administration National Center for Toxicological Research
Environmental Health Perspectives 111 1819ndash1826
Tornow S amp Mewes H W (2003) Functional modules by relating
protein interaction networks and gene expression Nucleic Acids
Research 31 6283ndash6289
Trocho C Escola-Gil J C Ribas V Benitez S Martin-Campos J M
Rotllan N et al (2004) Phenytoin treatment reduces atherosclerosis in
mice through mechanisms independent of plasma HDL-cholesterol
concentration Atherosclerosis 174 275ndash285
Ueda A Hamadeh H K Webb H K Yamamoto Y Sueyoshi T
Afshari C A et al (2002) Diverse roles of the nuclear orphan
receptor CAR in regulating hepatic genes in response to phenobarbital
Molecular Pharmacology 61 1ndash6
Ulrich R G (2003) The toxicogenomics of nuclear receptor agonists
Current Opinion in Chemical Biology 7 505ndash510
Ulrich R G Rockett J C Gibson G G amp Pettit S D (2004)
Overview of an interlaboratory collaboration on evaluating the effects
of model hepatotoxicants on hepatic gene expression Environmental
Health Perspectives 112 423ndash427
Unami A Shinohara Y Ichikawa T amp Baba Y (2003) Biochemical
and microarray analyses of bupivacaine-induced apoptosis Journal of
Toxicological Sciences 28 77ndash94
Vasquez A Flammini A Maritan A amp Vespignani A (2003) Global
protein function prediction from proteinndashprotein interaction networks
Nature Biotechnology 21 697ndash700
Vezina C M Walker N J amp Olson J R (2004) Subchronic Exposure to
TCDD PeCDF PCB126 and PCB153 Effect on hepatic gene
expression Environmental Health Perspectives 112 1636ndash1644
Vo T D Greenberg H J amp Palsson B O (2004) Reconstruction and
functional characterization of the human mitochondrial metabolic
network based on proteomic and biochemical data Journal of Bio-
logical Chemistry 279 39532ndash39540
Wang H amp LeCluyse E L (2003) Role of orphan nuclear receptors in
the regulation of drug-metabolising enzymes Clinical Pharmacoki-
netics 42 1331ndash1357
Wang N Silver D L Thiele C amp Tall A R (2001) ATP-
binding cassette transporter A1 (ABCA1) functions as a choles-
terol efflux regulatory protein Journal of Biological Chemistry 276
23742ndash23747
Waring J F Cavet G Jolly R A McDowell J Dai H Ciurlionis R
et al (2003) Development of a DNA Microarray for toxicology based
on hepatoxin-regulated sequences Environmental Health Perspectives
111 863ndash870
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6666
Waring J F Gum R Morfitt D Jolly R A Ciurlionis R Heindel M
et al (2002) Identifying toxic mechanisms using DNA microarrays
Evidence that an experimental inhibitor of cell adhesion molecule
expression signals through the aryl hydrocarbon nuclear receptor
Toxicology 181ndash182 537ndash550
Waring J F Jolly R A Ciurlionis R Lum P Y Praestgaard J T
Morfitt D C et al (2001) Clustering of hepatotoxins based on
mechanism of toxicity using gene expression profiles Toxicology and
Applied Pharmacology 175 28ndash42
Waters M Boorman G Bushel P Cunningham M Irwin R Merrick
A et al (2003) Systems toxicology and the Chemical Effects in
Biological Systems (CEBS) knowledge base EHP Toxicogenomics
111 15ndash28
Wei M Arnold L Cano M amp Cohen S M (2005) Effects of co-
administration of antioxidants and arsenicals on the rat urinary bladder
epithelium Toxicology Sciences 83 237ndash245
Weinshilboum R (2003) Inheritance and drug response New England
Journal of Medicine 348 529ndash537
Weinshilboum R amp Wang L (2004) Pharmacogenetics Inherited
variation in amino acid sequence and altered protein quantity Clinical
Pharmacology and Therapeutics 75 253ndash258
Werner E (2003) In silico multicellular systems biology and minimal
genomes Drug Discovery Today 8 1121ndash1127
Wong J S amp Gill S S (2002) Gene expression changes induced in
mouse liver by di(2-ethylhexyl) phthalate Toxicology and Applied
Pharmacology 185 180ndash196
Xie T Tong L McCann U D Yuan J Becker K G Mechan A O
et al (2004) Identification and characterization of metallothionein-1
and -2 gene expression in the context of ()34-methylenedioxymetham-
phetamine-induced toxicity to brain dopaminergic neurons Journal of
Neuroscience 24 7043ndash7050
Yadetie F Laegreid A Bakke I Kusnierczyk W Komorowski J
Waldum H L et al (2003) Liver gene expression in rats in response
to the peroxisome proliferator-activated receptor-alpha agonist ciprofi-
brate Physiological Genomics 15 9ndash19
Yamashita S Nomoto T Abe M Tatematsu M Sugimura T amp
Ushijima T (2004) Persistence of gene expression changes in stomach
mucosae induced by short-term N-methyl-NV-nitro-N-nitrosoguanidinetreatment and their presence in stomach cancers Mutation Research
549 185ndash193
Yan Q amp Sadee W (2000) Human membrane transporter database A
web-accessible relational database for drug transport studies and
pharmacogenomics AAPS Pharmsci 2 E20
Yeger-Lotem E amp Margalit H (2003) Detection of regulatory circuits by
integrating the cellular networks of proteinndashprotein interactions and
transcription regulation Nucleic Acids Research 31 6053ndash6061
Yoon B I Li G -X Kitada K Kawasaki Y Igarashi K amp Kodama
Y et al (2003) Mechanisms of benzene-induced hematotoxicity and
leukemogenicity cDNA microarray analyses using mouse bone marrow
tissue Environmental Health Perspectives 111 1411ndash1420
Young M B DiSilvestro M R Sendera T J Freund J Kriete A amp
Magnuson S R (2003) Analysis of gene expression in carbon
tetrachloride-treated rat livers using a novel bioarray technology
Pharmacogenomics Journal 3 41ndash52
Yu H Zhu X Greenbaum D Karro J amp Gerstein M (2004) TopNet
A tool for comparing biological sub-networks correlating protein
properties with topological statistics Nucleic Acids Research 32
328ndash337
Zhang E Y Fu D J Pak Y A Stewart T Mukhopadhyay N
Wrighton S A et al (2004) Genetic polymorphisms in human proton-
dependent dipeptide transporter PEPT1 Implications for the functional
role of Pro586 Journal of Pharmacology and Experimental Therapeu-
tics 310 437ndash445
Zhang E Y Knipp G T Ekins S amp Swaan P W (2002)
Structural biology and function of solute transporters Implications
for identifying and designing substrates Drug Metabolism Reviews
34 709ndash750
Zhang E Y Phelps M A Cheng C Ekins S amp Swaan P W (2002)
Modeling of active transport systems Advanced Drug Delivery
Reviews 54 329ndash354
- Systems-ADMETox Resources and network approaches
-
- Introduction
- Data available
- Network analysis and databases
-
- Network applications
-
- The role of transporters
-
- Clinical relevance of transporters
- Transporter network examples ABCA1
- Transporter network examples OATP
- Transporter microarray data
-
- Applications to enzymes
- Future network applications
-
- Discussion
- Acknowledgements
- References
-
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 63
cell cycle-associated genes in breast cancer cells Molecular Cancer
Research 1 300ndash311
Holleman A Cheok M H den Boer M L Yang W Veerman KM
A J Kazemier et al (2004) Gene-expression patterns in drug-resistant
acute lymphoblastic leukemia cells and response to treatment New
England Journal of Medicine 351 533ndash542
Hong Y Muller U R amp Lai F (2003) Discriminating two classes of
toxicants through expression analysis of HepG2 cells with DNA arrays
Toxicology in Vitro 17 85ndash92
Hood E (2003) Proteomics Characterizing the cogs in the machinery of
life Environmental Health Perspectives 111 A816ndashA825
Hood L (2003) Systems biology Integrating technology biology and
computation Mechanism of Ageing and Development 124 9ndash16
Hood L amp Galas D (2003) The digital code of DNA Nature 421
444ndash448
Hood L amp Perlmutter R M (2004) The impact of systems approaches on
biological problems in drug discovery Nature Biotechnology 22
1215ndash1217
Hu T Gibson D P Carr G J Torontali S M Tiesman J P Chaney
J G et al (2004) Identification of a gene expression profile that
discriminates indirect-acting genotoxins from direct-acting genotoxins
Mutation Research 549 5ndash27
Huang Q Dunn II R T Jayadev S DiSorbo O Pack F D Farr S B
et al (2001) Assessment of cisplatin-induced nephrotoxicity by
microarray technology Toxicology Sciences 63 196ndash207
Huang Q Jin X Gaillard E T Knight B L Pack F D Stoltz J H et
al (2004) Gene expression profiling reveals multiple toxicity endpoints
induced by hepatotoxicants Mutation Research 549 147ndash167
Huisman M T Smit J W Crommentuyn K M Zelcer N Wiltshire
H R Beijnen J H et al (2002) Multidrug resistance protein 2
(MRP2) transports HIV protease inhibitors and transport can be
enhanced by other drugs Aids 16 2295ndash2301
Hunt C M Westerkam W R amp Stave G M (1992) Effect of age and
gender on the activity of human hepatic CYP3A Biochemical
Pharmacology 44 275ndash283
Ideker T Ozier O Schwikowski B amp Siegal A F (2002) Discovering
regulatory and signalling circuits in molecular interaction networks
Bioinformatics 18 S233ndashS240
Ideker T Thorsson V Ranish J A Christmas R Buhler J Eng J K
et al (2001) Integrated genomic and proteomic analyses of a system-
atically perturbed network Science 292 929ndash934
Iida M Anna C H Hartis J Bruno M Wetmore B Dubin J R et al
(2003) Changes in global gene and protein expression during early
mouse liver carcinogenesis induced by non-genotoxic model carcino-
gens oxazepam and Wyeth-14643 Carcinogenesis 24 757ndash770
Ingelman-Sundberg M (2004) Pharmacogenetics of cytochrome P450 and
its applications in drug therapy The past present and future Trends in
Pharmacological Sciences 25 193ndash200
Jeong H Mason S P Barabasi A L amp Oltvai Z N (2001) Lethality
and centrality in protein networks Nature 411 41ndash42
Jeong H Tombor B Albert R Oltvai Z N amp Barabasi A L
(2000) The large-scale organization of metabolic networks Nature
407 651ndash654
Jung D Hagenbuch B Gresh L Pontoglio M Meier P J amp Kullak-
Ublick G A (2001) Characterization of the human OATP-C
(SLC21A6) gene promoter and regulation of liver-specific OATP genes
by hepatocyte nuclear factor 1 alpha Journal of Biological Chemistry
276 37206ndash37214
Jung D amp Kullak-Ublick G A (2003) Hepatocyte nuclear factor 1
alpha A key mediator of the effect of bile acids on gene expression
Hepatology 37 622ndash631
Jung J -W Park J -S Hwang J -W Kang K -S YS L Song B
-S et al (2004) Gene expression analysis of peroxisome prolifer-
ators- and phenytoin-induced hepatotoxicity using cDNA microarray
Journal of Veterinary Medical Science 66 1329ndash1333
Kato N Shibutani M Takagi H Uneyama C Lee K -Y Takiami S
et al (2004) Gene expression profile in the livers of rats orally
administered ethinylestradiol for 28 days using a microarray technique
Toxicology 200 179ndash192
Kier L D Neft R Tang L Suizu R Cook T Onsurez K et al
(2004) Applications of microarrays with toxicologically relevant genes
(tox genes) for the evaluation of chemical toxicants in Sprague Dawley
rats in vivo and human hepatocytes in vitro Mutation Research 549
101ndash113
Kijima K Toyosawa K Yasuba M Matsuoka N Adachi T
Komiyama M et al (2004) Gene expression analysis of the rat testis
after treatment with di(2-ethylhexyl) phthalate using cDNA microarray
and real-time RT-PCR Toxicology and Applied Pharmacology 200
103ndash110
Kim R B (2003) Organic anion-transporting polypeptide (OATP) trans-
porter family and drug disposition European Journal of Clinical
Investigation 33 1ndash5
Kinirons M T amp OrsquoMahony M S (2004) Drug metabolism and ageing
British Journal of Clinical Pharmacology 57 540ndash544
Kitano H (2002a) Computational systems biology Nature 420 206ndash210
Kitano H (2002b) Systems biology A brief overview Science 295
1662ndash1664
Kliewer S A Moore J T Wade L Staudinger J L Watson M
A Jones S A et al (1998) An orphan nuclear receptor activated
by pregnanes defines a novel steroid signalling pathway Cell 92
73ndash82
Korolev D Balakin K V Nikolsky Y Kirillov E Ivanenkov Y A
Savchuk N P et al (2003) Modeling of human cytochrome p450-
mediated drug metabolism using unsupervised machine learning
approach Journal of Medicinal Chemistry 46 3631ndash3643
Kramer J A Pettit S D Amin R P Bertram T A Car B
Cunningham M et al (2004) Overview on the application of
transcription profiling using selected nephrotoxicants for toxicology
assessment Environmental Health Perspectives 112 460ndash464
Kultima K Nystrom A -M Scholz B Gustafson A -L Dencker L
amp Stigson M (2004) Valproic acid teratogenicity A toxicogenomics
approach Environmental Health Perspectives 112 1225ndash1235
Kutuzova G D amp Deluca H F (2004) Gene expression profiles in rat
intestine identify pathways for 125-dihydroxyvitamin D(3) stimulated
calcium absorption and clarify its immunomodulatory properties
Archives of Biochemistry and Biophysics 432 152ndash166
Kwak M K Wakabayashi N Itoh K Motohashi H Yamamoto M amp
Kensler T W (2003) Modulation of gene expression by cancer
chemopreventive dithiolethiones through the Keap1-Nrf2 pathway
Identification of novel gene clusters for cell survival Journal of
Biological Chemistry 278 8135ndash8145
Lahjouji K Mitchell G A amp Qureshi I A (2001) Carnitine transport
by organic cation transporters and systemic carnitine deficiency
Molecular Genetics and Metabolism 73 287ndash297
Landowski C P Sun D Foster D R Menon S S Barnett J L
Welage L S et al (2003) Gene expression in the human intestine and
correlation with oral valacyclovir pharmacokinetic parameters Journal
of Pharmacology and Experimental Therapeutics 306 778ndash786
Lee G -J Lee W -S Jeon K -S Um C -H Kim S -J Lee C -H
et al (2004) cDNA microarray gene expression analysis and
toxicological phenotype for anticancer drug Journal of Veterinary
Medical Science 66 1339ndash1345
Lee M Kwon J Kim S N Kim J E Koh W S Kim E J et al
(2003) cDNA microarray gene expression profiling of hydroxyurea
paclitaxel and p-anisidine genotoxic compounds with differing
tumorigenicity results Environmental and Molecular Mutagenesis
42 91ndash97
Li S Armstrong C M Bertin N Ge H Milstein S Boxem M et al
(2004) A map of the interactome network of the metazoan C elegans
Science 303 540ndash543
Liguori M J Anderson L M Bukofzer S McKim J Pregenzer J F
Retief J et al (2005) Microarray analysis in human hepatocytes
suggests a mechanism for hepatotoxicity induced by trovafloxacin
Hepatology 41 177ndash186
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6664
Lobenhofer E K Bennett L Cable P L Li L Bushel P R amp Afshari
C A (2002) Regulation of DNA replication fork genes by 17beta-
estradiol Molecular Endocrinology 16 1215ndash1229
Locker J Tian J Carver R Concas D Cossu C Ledda-Columbano
G M et al (2003) A common set of immediate-early response genes
in liver regeneration and hyperplasia Hepatology 38 314ndash325
Mattes W B Pettit S D Sansone S A Bushel P R amp Waters M D
(2004) Database development in toxicogenomics Issues and efforts
Environmental Health Perspectives 112 495ndash505
McMillian M Nie A Y Parker J B Leone A Bryant S Kemmerer
M et al (2004) A gene expression signature for oxidant stressreactive
metabolites in rat liver Biochemical Pharmacology 68 2249ndash2261
Meier P J amp Stieger B (2000) Molecular mechanisms in bile formation
News in Physiological Sciences 15 89ndash93
Meneses-Lorente G de Longueville F Dos Santos-Mendes S Bonnert
T P Jack A Evrard S et al (2003) An evaluation of a low-density
DNA microarray using cytochrome P450 inducers Chemical Research
in Toxicology 16 1070ndash1077
Meneses-Lorente G Guest P C Lawrence J Muniappa N Knowles
M R Skynner H A et al (2004) A proteomic investigation of drug-
induced steatosis in rat liver Chemical Research in Toxicology 17
605ndash612
Merrill C L Ni H Yoon L W Tirmenstein M A Narayanan P
Benavides G R et al (2002) Etomoxir-induced oxidative stress in
HepG2 cells detected by differential gene expression is confirmed
biochemically Toxicology Sciences 68 93ndash101
Milo R Shen-Orr S Itzkovitz S Kashtan N Chklovskii D amp Alon
U (2002) Network motifs Simple building blocks of complex
networks Science 298 824ndash827
Morgan K T Ni H Brown H R Yoon L Qualls C W Crosby L
M et al (2002) Application of cDNA microarray technology to in
vitro toxicology and the selection of genes for a real-time RT-PCR-
based screen for oxidative stress in Hep-G2 cells Toxicologic
Pathology 30 435ndash451
Mwinyi J Johne A Bauer S Roots I amp Gerloff T (2004) Evidence
for inverse effects of OATP-C (SLC21A6) 5 and 1b haplotypes on
pravastatin kinetics Clinical Pharmacology and Therapeutics 75
415ndash421
Nakata K Yukawa M Komiyama N Nakano T amp Kaminuma T
(2002) A nuclear receptor database that maps pathways to diseases
Genome Infomatics 13 515ndash516
Nicholson J K Holmes E Lindon J C amp Wilson I D (2004) The
challenges of modeling mammalian biocomplexity Nature Biotechnol-
ogy 22 1268ndash1274
Nicholson J K amp Wilson I D (2003) Understanding rsquoglobal systems
biology Metabonomics and the continuum of metabolism Nature
Reviews on Drug Discovery 2 668ndash676
Nikitin A Egorov S Daraselia N amp Mazo I (2003) Pathway studiomdash
the analysis and navigation of molecular networks Bioinformatics 19
2155ndash2157
Nikolsky Y Ekins S Nikolskaya T amp Bugrim A (2005) A novel
method for generation of signature networks as biomarkers from
complex high throughput data Toxicology Letters 158 20ndash29
Nikolsky Y Nikolskaya T amp Bugrim A (2005) Biological networks
and analysis of experimental data in drug discovery Drug Discovery
Today 10 653ndash662
Nishizato Y Ieiri I Suzuki H Kimura M Kawabata K Hirota T
et al (2003) Polymorphisms of OATP-C (SLC21A6) and OAT3
(SLC22A8) genes Consequences for pravastatin pharmacokinetics
Clinical Pharmacology and Therapeutics 73 554ndash565
Nozawa T Nakajima M Tamai I Noda K Nezu J Sai Y et al
(2002) Genetic polymorphisms of human organic anion transporters
OATP-C (SLC21A6) and OATP-B (SLC21A9) Allele frequencies in
the Japanese population and functional analysis Journal of Pharmacol-
ogy and Experimental Therapeutics 302 804ndash813
Nozawa T Sugiura S Nakajima M Goto A Yokoi T Nezu J et al
(2004) Involvement of organic anion transporting polypeptides in the
transport of troglitazone sulfate Implications for understanding trogli-
tazone hepatotoxicity Drug Metabolism and Disposition 32 291ndash294
Oliver D E Rubin D L Stuart J M Hewett M Klein T E amp
Altman R B (2002) Ontology development for a pharmacogenetics
knowledge base Package Symposion 88ndash99
Oram J F amp Lawn R M (2001) ABCA1 The gatekeeper for eliminating
excess tissue cholesterol Journal of Lipid Research 42 1173ndash1179
Oswald M Kullak-Ublick G A Paumgartner G amp Beuers U (2001)
Expression of hepatic transporters OATP-C and MRP2 in primary
sclerosing cholangitis Liver 21 247ndash253
Parsons A B Brost R I Ding H Li Z Zhang C Sheikh B et al
(2004) Integration of chemical-genetic and genetic interaction data
links bioactive compounds to cellular target pathways Nature Bio-
technology 22 62ndash69
Patki K C von Moltke L L Harmatz J S Hesse L M Court M H
amp Greenblatt D J (2004) Effect of age on in vitro triazolam
biotransformation in male human liver microsomes Journal of
Pharmacology and Experimental Therapeutics 308 874ndash879
Pereira-Leal J B Enright A J amp Ouzounis C A (2004) Detection of
functional modules from protein interaction networks Proteins 54
49ndash57
Plant N (2004) Interaction networks Coordinating responses to xeno-
biotic exposure Toxicology 202 21ndash32
Raghavendra Prasad H S Qi Z Srinivasan K N amp Gopalakrish-
nakone P (2004) Potential effects of tetrodotoxin exposure to
human glial cells postulated using microarray approach Toxicon 44
597ndash608
Reilly T P Bourdi M Brady J N Pise-Masison C A Radonovich M
F George J W et al (2001) Expression profiling of acetaminophen
liver toxicity in mice using microarray technology Biochemical and
Biophysical Research Communications 282 321ndash328
Rives A W amp Galitski T (2003) Modular organization of cellular
networks Proceedings of the National Academy of Sciences of the
United States of America 100 1128ndash1133
Rogers P D Pearson M M Cleary J D Sullivan D C amp Chapman
S W (2002) Differential expression of genes encoding immunomodu-
latory proteins in response to amphotericin B in human mononuclear
cells identified by cDNA microarray analysis Journal of Antimicrobial
Chemotherapy 50 811ndash817
Rosen M B Wilson V S Schmid J E amp Gray L E (2005) Gene
expression analysis in the ventral prostate of rats exposed to vinclozolin
or procymidone Reproductive Toxicology 19 367ndash379
Rost D Herrmann T Sauer P Schmidts H L Stieger B Meier P J
et al (2003) Regulation of rat organic anion transporters in bile salt-
induced cholestatic hepatitis Effect of ursodeoxycholate Hepatology
38 187ndash195
Rothman R B Ayestas M A Dersch C M amp Baumann M H (1999)
Aminorex fenfluramine and chlorphentermine are serotonin transporter
substrates Implications for primary pulmonary hypertension Circula-
tion 100 869ndash875
Ruepp S U Tonge R P Shaw J Wallis N amp Pognan F (2002)
Genomics and proteomics analysis of acetaminophen toxicity in mouse
liver Toxicology Sciences 65 135ndash150
Sakaeda T Nakamura T amp Okumura K (2002) MDR1 genotype-
related pharmacokinetics and pharmacodynamics Biological amp Phar-
maceutical Bulletin 25 1391ndash1400
Sakata T Anzai N Shin H J Noshiro R Hirata T Yokoyama H
et al (2004) Novel single nucleotide polymorphisms of organic cation
transporter 1 (SLC22A1) affecting transport functions Biochemical
and Biophysical Research Communications 313 789ndash793
Satlin L M Amin V amp Wolkoff A W (1997) Organic anion
transporting polypeptide mediates organic anionHCO3-exchange
Journal of Biological Chemistry 272 26340ndash26345
Satomi Y Tsuchiya W Mihara K Ota M Kasahara Y amp Akahori F
(2004) Gene expression analysis of the lung following paraquat
administration in rats using DNA microarray Journal of Toxicological
Sciences 29 91ndash100
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 65
Schrenk D Baus P R Ermel N Klein C Vorderstemann B amp
Kauffmann H M (2001) Up-regulation of transporters of the MRP
family by drugs and toxins Toxicology Letters 120 51ndash57
Segal E Shapira M Regev A Persquoer D Botstein D Koller D et al
(2003) Module networks Identifying regulatory modules and their
condition-specific regulators from gene expression data Nature
Genetics 34 166ndash176
Segal E Wang H amp Koller D (2003) Discovering molecular pathways
from protein interaction and gene expression Bioinformatics 19
i264ndash i272
Seidel S D Kan H L Stott W T Schisler M R amp Gollapudi B B
(2003) Identification of transcriptome profiles for the DNA-damaging
agents bleomycin and hydrogen peroxide in L5178Y mouse lymphoma
cells Environmental and Molecular Mutagenesis 42 19ndash25
Shan L Yu Y Schut H A J amp Snyderwine E G (2004) Susceptibility
of rats to mammary gland carcinogenesis by the food-derived
carcinogen 2-Amino-1-Methyl-6-Phenylimidazo[45-b]Pyridine (PhIP)
varies with age and is associated with the induction of differential gene
expression American Journal of Pathology 165 191ndash202
Sharom J R Bellows D S amp Tyers M (2004) From large networks to
small molecules Current Opinion in Chemical Biology 8 81ndash90
Shitara Y Hirano M Sato H amp Sugiyama Y (2004) Gemfibrozil and
its glucuronide inhibit the OATP2(OATP1B1 SLC21A6)-mediated
hepatic uptake and CYP2C8-mediated metabolism of cerivastatinmdash
Analysis of the mechanism of the clinically relevant drugndashdrug
interaction between cerivastatin and gemfibrozil Journal of Pharma-
cology and Experimental Therapeutics
Shitara Y Itoh T Sato H Li A P amp Sugiyama Y (2003) Inhibition of
transporter-mediated hepatic uptake as a mechanism for drugndashdrug
interaction between cerivastatin and cyclosporin A Journal of
Pharmacology and Experimental Therapeutics 304 610ndash616
Somogyi R Fuhrman S amp Wen X (2001) Genetic network inference in
computational models and applications to large-scale gene expression
data In M Bower amp H Bolouri (Eds) Computational modeling of
genetic and biochemical networks (pp 119ndash157) Cambridge MArsquoThe
MIT Press
Spirin V amp Mirny L A (2003) Protein complexes and functional
modules in molecular networks Proceedings of the National Academy
of Sciences of the United States of America 100 12123ndash12128
Staudinger J Liu Y Madan A Habeebu S amp Klaassen C D
(2001) Coordinate regulation of xenobiotic and bile acid homeo-
stasis by pregnane X receptor Drug Metabolism and Disposition
29 1467ndash1472
Sun L Z Ji Z L Chen X Wang J F amp Chen Y Z (2002) ADME-
AP A database of ADME associated proteins Bioinformatics 18
1699ndash1700
Suter L Babiss L E amp Wheeldon E B (2004) Toxicogenomics in
predictive toxicology in drug development Chemistry and Biology 11
161ndash171
Synold T W Dussault I amp Forman B M (2001) The orphan nuclear
receptor SXR coordinately regulates drug metabolism and efflux
Nature Medicine 7 584ndash590
Tabuchi Y amp Kondo T (2003) cDNA microarray analysis reveals chop-
10 plays a key role in Sertoli cell injury induced by bisphenol A
Biochemical and Biophysical Research Communications 305 54ndash61
Tamai I Nezu J Uchino H Sai Y Oku A Shimane M et al (2000)
Molecular identification and characterization of novel members of the
human organic anion transporter (OATP) family Biochemical and
Biophysical Research Communications 273 251ndash260
Taylor S W Fahy E Zhang B Glenn G M Warnock D E Wiley S
et al (2003) Characterization of the human heart mitochondrial
proteome Nature Biotechnology 21 281ndash286
Terasaka S Aita Y Inoue A Hayashi S Nishigaki M Aoyagi K
et al (2004) Using a customized DNA microarray for expression
profiling of the estrogen-responsive genes to evaluate estrogen activity
among natural estrogens and industrial chemicals Environmental
Health Perspectives 112 773ndash781
Thomas D M Francescutti-Verbeem D M Liu X amp Kuhn D M
(2004) Identification of differentially regulated transcripts in mouse
striatum following methamphetamine treatmentmdashan oligonucleotide
microarray approach Journal of Neurochemistry 88 380ndash393
Thomas R S Rank D R Penn S G Zastrow G M Hayes K R
Pande K et al (2001) Identification of toxicologically predictive
gene sets using cDNA microarrays Molecular Pharmacology 60
1189ndash1194
Thompson K L Afshari C A Amin R P Bertram T A Car B
Cunningham M et al (2004) Identification of platform-independent
gene expression markers of cisplatin nephrotoxicity Environmental
Health Perspectives 112 488ndash494
Tirona R G Leake B F Merino G amp Kim R B (2001)
Polymorphisms in OATP-C Identification of multiple allelic
variants associated with altered transport activity among European-
and African-Americans Journal of Biological Chemistry 276
35669ndash35675
Tirona R G Leake B F Wolkoff A W amp Kim R B (2003)
Human organic anion transporting polypeptide-C (SLC21A6) is a
major determinant of the rifampin-mediated pregnane X receptor
activation Journal of Pharmacology and Experimental Therapeutics
304 223ndash228
Tong W Cao X Harris S Sun H Fang H Fuscoe J et al (2003)
ArrayTrackmdashsupporting toxicogenomic research at the US Food and
Drug Administration National Center for Toxicological Research
Environmental Health Perspectives 111 1819ndash1826
Tornow S amp Mewes H W (2003) Functional modules by relating
protein interaction networks and gene expression Nucleic Acids
Research 31 6283ndash6289
Trocho C Escola-Gil J C Ribas V Benitez S Martin-Campos J M
Rotllan N et al (2004) Phenytoin treatment reduces atherosclerosis in
mice through mechanisms independent of plasma HDL-cholesterol
concentration Atherosclerosis 174 275ndash285
Ueda A Hamadeh H K Webb H K Yamamoto Y Sueyoshi T
Afshari C A et al (2002) Diverse roles of the nuclear orphan
receptor CAR in regulating hepatic genes in response to phenobarbital
Molecular Pharmacology 61 1ndash6
Ulrich R G (2003) The toxicogenomics of nuclear receptor agonists
Current Opinion in Chemical Biology 7 505ndash510
Ulrich R G Rockett J C Gibson G G amp Pettit S D (2004)
Overview of an interlaboratory collaboration on evaluating the effects
of model hepatotoxicants on hepatic gene expression Environmental
Health Perspectives 112 423ndash427
Unami A Shinohara Y Ichikawa T amp Baba Y (2003) Biochemical
and microarray analyses of bupivacaine-induced apoptosis Journal of
Toxicological Sciences 28 77ndash94
Vasquez A Flammini A Maritan A amp Vespignani A (2003) Global
protein function prediction from proteinndashprotein interaction networks
Nature Biotechnology 21 697ndash700
Vezina C M Walker N J amp Olson J R (2004) Subchronic Exposure to
TCDD PeCDF PCB126 and PCB153 Effect on hepatic gene
expression Environmental Health Perspectives 112 1636ndash1644
Vo T D Greenberg H J amp Palsson B O (2004) Reconstruction and
functional characterization of the human mitochondrial metabolic
network based on proteomic and biochemical data Journal of Bio-
logical Chemistry 279 39532ndash39540
Wang H amp LeCluyse E L (2003) Role of orphan nuclear receptors in
the regulation of drug-metabolising enzymes Clinical Pharmacoki-
netics 42 1331ndash1357
Wang N Silver D L Thiele C amp Tall A R (2001) ATP-
binding cassette transporter A1 (ABCA1) functions as a choles-
terol efflux regulatory protein Journal of Biological Chemistry 276
23742ndash23747
Waring J F Cavet G Jolly R A McDowell J Dai H Ciurlionis R
et al (2003) Development of a DNA Microarray for toxicology based
on hepatoxin-regulated sequences Environmental Health Perspectives
111 863ndash870
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6666
Waring J F Gum R Morfitt D Jolly R A Ciurlionis R Heindel M
et al (2002) Identifying toxic mechanisms using DNA microarrays
Evidence that an experimental inhibitor of cell adhesion molecule
expression signals through the aryl hydrocarbon nuclear receptor
Toxicology 181ndash182 537ndash550
Waring J F Jolly R A Ciurlionis R Lum P Y Praestgaard J T
Morfitt D C et al (2001) Clustering of hepatotoxins based on
mechanism of toxicity using gene expression profiles Toxicology and
Applied Pharmacology 175 28ndash42
Waters M Boorman G Bushel P Cunningham M Irwin R Merrick
A et al (2003) Systems toxicology and the Chemical Effects in
Biological Systems (CEBS) knowledge base EHP Toxicogenomics
111 15ndash28
Wei M Arnold L Cano M amp Cohen S M (2005) Effects of co-
administration of antioxidants and arsenicals on the rat urinary bladder
epithelium Toxicology Sciences 83 237ndash245
Weinshilboum R (2003) Inheritance and drug response New England
Journal of Medicine 348 529ndash537
Weinshilboum R amp Wang L (2004) Pharmacogenetics Inherited
variation in amino acid sequence and altered protein quantity Clinical
Pharmacology and Therapeutics 75 253ndash258
Werner E (2003) In silico multicellular systems biology and minimal
genomes Drug Discovery Today 8 1121ndash1127
Wong J S amp Gill S S (2002) Gene expression changes induced in
mouse liver by di(2-ethylhexyl) phthalate Toxicology and Applied
Pharmacology 185 180ndash196
Xie T Tong L McCann U D Yuan J Becker K G Mechan A O
et al (2004) Identification and characterization of metallothionein-1
and -2 gene expression in the context of ()34-methylenedioxymetham-
phetamine-induced toxicity to brain dopaminergic neurons Journal of
Neuroscience 24 7043ndash7050
Yadetie F Laegreid A Bakke I Kusnierczyk W Komorowski J
Waldum H L et al (2003) Liver gene expression in rats in response
to the peroxisome proliferator-activated receptor-alpha agonist ciprofi-
brate Physiological Genomics 15 9ndash19
Yamashita S Nomoto T Abe M Tatematsu M Sugimura T amp
Ushijima T (2004) Persistence of gene expression changes in stomach
mucosae induced by short-term N-methyl-NV-nitro-N-nitrosoguanidinetreatment and their presence in stomach cancers Mutation Research
549 185ndash193
Yan Q amp Sadee W (2000) Human membrane transporter database A
web-accessible relational database for drug transport studies and
pharmacogenomics AAPS Pharmsci 2 E20
Yeger-Lotem E amp Margalit H (2003) Detection of regulatory circuits by
integrating the cellular networks of proteinndashprotein interactions and
transcription regulation Nucleic Acids Research 31 6053ndash6061
Yoon B I Li G -X Kitada K Kawasaki Y Igarashi K amp Kodama
Y et al (2003) Mechanisms of benzene-induced hematotoxicity and
leukemogenicity cDNA microarray analyses using mouse bone marrow
tissue Environmental Health Perspectives 111 1411ndash1420
Young M B DiSilvestro M R Sendera T J Freund J Kriete A amp
Magnuson S R (2003) Analysis of gene expression in carbon
tetrachloride-treated rat livers using a novel bioarray technology
Pharmacogenomics Journal 3 41ndash52
Yu H Zhu X Greenbaum D Karro J amp Gerstein M (2004) TopNet
A tool for comparing biological sub-networks correlating protein
properties with topological statistics Nucleic Acids Research 32
328ndash337
Zhang E Y Fu D J Pak Y A Stewart T Mukhopadhyay N
Wrighton S A et al (2004) Genetic polymorphisms in human proton-
dependent dipeptide transporter PEPT1 Implications for the functional
role of Pro586 Journal of Pharmacology and Experimental Therapeu-
tics 310 437ndash445
Zhang E Y Knipp G T Ekins S amp Swaan P W (2002)
Structural biology and function of solute transporters Implications
for identifying and designing substrates Drug Metabolism Reviews
34 709ndash750
Zhang E Y Phelps M A Cheng C Ekins S amp Swaan P W (2002)
Modeling of active transport systems Advanced Drug Delivery
Reviews 54 329ndash354
- Systems-ADMETox Resources and network approaches
-
- Introduction
- Data available
- Network analysis and databases
-
- Network applications
-
- The role of transporters
-
- Clinical relevance of transporters
- Transporter network examples ABCA1
- Transporter network examples OATP
- Transporter microarray data
-
- Applications to enzymes
- Future network applications
-
- Discussion
- Acknowledgements
- References
-
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6664
Lobenhofer E K Bennett L Cable P L Li L Bushel P R amp Afshari
C A (2002) Regulation of DNA replication fork genes by 17beta-
estradiol Molecular Endocrinology 16 1215ndash1229
Locker J Tian J Carver R Concas D Cossu C Ledda-Columbano
G M et al (2003) A common set of immediate-early response genes
in liver regeneration and hyperplasia Hepatology 38 314ndash325
Mattes W B Pettit S D Sansone S A Bushel P R amp Waters M D
(2004) Database development in toxicogenomics Issues and efforts
Environmental Health Perspectives 112 495ndash505
McMillian M Nie A Y Parker J B Leone A Bryant S Kemmerer
M et al (2004) A gene expression signature for oxidant stressreactive
metabolites in rat liver Biochemical Pharmacology 68 2249ndash2261
Meier P J amp Stieger B (2000) Molecular mechanisms in bile formation
News in Physiological Sciences 15 89ndash93
Meneses-Lorente G de Longueville F Dos Santos-Mendes S Bonnert
T P Jack A Evrard S et al (2003) An evaluation of a low-density
DNA microarray using cytochrome P450 inducers Chemical Research
in Toxicology 16 1070ndash1077
Meneses-Lorente G Guest P C Lawrence J Muniappa N Knowles
M R Skynner H A et al (2004) A proteomic investigation of drug-
induced steatosis in rat liver Chemical Research in Toxicology 17
605ndash612
Merrill C L Ni H Yoon L W Tirmenstein M A Narayanan P
Benavides G R et al (2002) Etomoxir-induced oxidative stress in
HepG2 cells detected by differential gene expression is confirmed
biochemically Toxicology Sciences 68 93ndash101
Milo R Shen-Orr S Itzkovitz S Kashtan N Chklovskii D amp Alon
U (2002) Network motifs Simple building blocks of complex
networks Science 298 824ndash827
Morgan K T Ni H Brown H R Yoon L Qualls C W Crosby L
M et al (2002) Application of cDNA microarray technology to in
vitro toxicology and the selection of genes for a real-time RT-PCR-
based screen for oxidative stress in Hep-G2 cells Toxicologic
Pathology 30 435ndash451
Mwinyi J Johne A Bauer S Roots I amp Gerloff T (2004) Evidence
for inverse effects of OATP-C (SLC21A6) 5 and 1b haplotypes on
pravastatin kinetics Clinical Pharmacology and Therapeutics 75
415ndash421
Nakata K Yukawa M Komiyama N Nakano T amp Kaminuma T
(2002) A nuclear receptor database that maps pathways to diseases
Genome Infomatics 13 515ndash516
Nicholson J K Holmes E Lindon J C amp Wilson I D (2004) The
challenges of modeling mammalian biocomplexity Nature Biotechnol-
ogy 22 1268ndash1274
Nicholson J K amp Wilson I D (2003) Understanding rsquoglobal systems
biology Metabonomics and the continuum of metabolism Nature
Reviews on Drug Discovery 2 668ndash676
Nikitin A Egorov S Daraselia N amp Mazo I (2003) Pathway studiomdash
the analysis and navigation of molecular networks Bioinformatics 19
2155ndash2157
Nikolsky Y Ekins S Nikolskaya T amp Bugrim A (2005) A novel
method for generation of signature networks as biomarkers from
complex high throughput data Toxicology Letters 158 20ndash29
Nikolsky Y Nikolskaya T amp Bugrim A (2005) Biological networks
and analysis of experimental data in drug discovery Drug Discovery
Today 10 653ndash662
Nishizato Y Ieiri I Suzuki H Kimura M Kawabata K Hirota T
et al (2003) Polymorphisms of OATP-C (SLC21A6) and OAT3
(SLC22A8) genes Consequences for pravastatin pharmacokinetics
Clinical Pharmacology and Therapeutics 73 554ndash565
Nozawa T Nakajima M Tamai I Noda K Nezu J Sai Y et al
(2002) Genetic polymorphisms of human organic anion transporters
OATP-C (SLC21A6) and OATP-B (SLC21A9) Allele frequencies in
the Japanese population and functional analysis Journal of Pharmacol-
ogy and Experimental Therapeutics 302 804ndash813
Nozawa T Sugiura S Nakajima M Goto A Yokoi T Nezu J et al
(2004) Involvement of organic anion transporting polypeptides in the
transport of troglitazone sulfate Implications for understanding trogli-
tazone hepatotoxicity Drug Metabolism and Disposition 32 291ndash294
Oliver D E Rubin D L Stuart J M Hewett M Klein T E amp
Altman R B (2002) Ontology development for a pharmacogenetics
knowledge base Package Symposion 88ndash99
Oram J F amp Lawn R M (2001) ABCA1 The gatekeeper for eliminating
excess tissue cholesterol Journal of Lipid Research 42 1173ndash1179
Oswald M Kullak-Ublick G A Paumgartner G amp Beuers U (2001)
Expression of hepatic transporters OATP-C and MRP2 in primary
sclerosing cholangitis Liver 21 247ndash253
Parsons A B Brost R I Ding H Li Z Zhang C Sheikh B et al
(2004) Integration of chemical-genetic and genetic interaction data
links bioactive compounds to cellular target pathways Nature Bio-
technology 22 62ndash69
Patki K C von Moltke L L Harmatz J S Hesse L M Court M H
amp Greenblatt D J (2004) Effect of age on in vitro triazolam
biotransformation in male human liver microsomes Journal of
Pharmacology and Experimental Therapeutics 308 874ndash879
Pereira-Leal J B Enright A J amp Ouzounis C A (2004) Detection of
functional modules from protein interaction networks Proteins 54
49ndash57
Plant N (2004) Interaction networks Coordinating responses to xeno-
biotic exposure Toxicology 202 21ndash32
Raghavendra Prasad H S Qi Z Srinivasan K N amp Gopalakrish-
nakone P (2004) Potential effects of tetrodotoxin exposure to
human glial cells postulated using microarray approach Toxicon 44
597ndash608
Reilly T P Bourdi M Brady J N Pise-Masison C A Radonovich M
F George J W et al (2001) Expression profiling of acetaminophen
liver toxicity in mice using microarray technology Biochemical and
Biophysical Research Communications 282 321ndash328
Rives A W amp Galitski T (2003) Modular organization of cellular
networks Proceedings of the National Academy of Sciences of the
United States of America 100 1128ndash1133
Rogers P D Pearson M M Cleary J D Sullivan D C amp Chapman
S W (2002) Differential expression of genes encoding immunomodu-
latory proteins in response to amphotericin B in human mononuclear
cells identified by cDNA microarray analysis Journal of Antimicrobial
Chemotherapy 50 811ndash817
Rosen M B Wilson V S Schmid J E amp Gray L E (2005) Gene
expression analysis in the ventral prostate of rats exposed to vinclozolin
or procymidone Reproductive Toxicology 19 367ndash379
Rost D Herrmann T Sauer P Schmidts H L Stieger B Meier P J
et al (2003) Regulation of rat organic anion transporters in bile salt-
induced cholestatic hepatitis Effect of ursodeoxycholate Hepatology
38 187ndash195
Rothman R B Ayestas M A Dersch C M amp Baumann M H (1999)
Aminorex fenfluramine and chlorphentermine are serotonin transporter
substrates Implications for primary pulmonary hypertension Circula-
tion 100 869ndash875
Ruepp S U Tonge R P Shaw J Wallis N amp Pognan F (2002)
Genomics and proteomics analysis of acetaminophen toxicity in mouse
liver Toxicology Sciences 65 135ndash150
Sakaeda T Nakamura T amp Okumura K (2002) MDR1 genotype-
related pharmacokinetics and pharmacodynamics Biological amp Phar-
maceutical Bulletin 25 1391ndash1400
Sakata T Anzai N Shin H J Noshiro R Hirata T Yokoyama H
et al (2004) Novel single nucleotide polymorphisms of organic cation
transporter 1 (SLC22A1) affecting transport functions Biochemical
and Biophysical Research Communications 313 789ndash793
Satlin L M Amin V amp Wolkoff A W (1997) Organic anion
transporting polypeptide mediates organic anionHCO3-exchange
Journal of Biological Chemistry 272 26340ndash26345
Satomi Y Tsuchiya W Mihara K Ota M Kasahara Y amp Akahori F
(2004) Gene expression analysis of the lung following paraquat
administration in rats using DNA microarray Journal of Toxicological
Sciences 29 91ndash100
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 65
Schrenk D Baus P R Ermel N Klein C Vorderstemann B amp
Kauffmann H M (2001) Up-regulation of transporters of the MRP
family by drugs and toxins Toxicology Letters 120 51ndash57
Segal E Shapira M Regev A Persquoer D Botstein D Koller D et al
(2003) Module networks Identifying regulatory modules and their
condition-specific regulators from gene expression data Nature
Genetics 34 166ndash176
Segal E Wang H amp Koller D (2003) Discovering molecular pathways
from protein interaction and gene expression Bioinformatics 19
i264ndash i272
Seidel S D Kan H L Stott W T Schisler M R amp Gollapudi B B
(2003) Identification of transcriptome profiles for the DNA-damaging
agents bleomycin and hydrogen peroxide in L5178Y mouse lymphoma
cells Environmental and Molecular Mutagenesis 42 19ndash25
Shan L Yu Y Schut H A J amp Snyderwine E G (2004) Susceptibility
of rats to mammary gland carcinogenesis by the food-derived
carcinogen 2-Amino-1-Methyl-6-Phenylimidazo[45-b]Pyridine (PhIP)
varies with age and is associated with the induction of differential gene
expression American Journal of Pathology 165 191ndash202
Sharom J R Bellows D S amp Tyers M (2004) From large networks to
small molecules Current Opinion in Chemical Biology 8 81ndash90
Shitara Y Hirano M Sato H amp Sugiyama Y (2004) Gemfibrozil and
its glucuronide inhibit the OATP2(OATP1B1 SLC21A6)-mediated
hepatic uptake and CYP2C8-mediated metabolism of cerivastatinmdash
Analysis of the mechanism of the clinically relevant drugndashdrug
interaction between cerivastatin and gemfibrozil Journal of Pharma-
cology and Experimental Therapeutics
Shitara Y Itoh T Sato H Li A P amp Sugiyama Y (2003) Inhibition of
transporter-mediated hepatic uptake as a mechanism for drugndashdrug
interaction between cerivastatin and cyclosporin A Journal of
Pharmacology and Experimental Therapeutics 304 610ndash616
Somogyi R Fuhrman S amp Wen X (2001) Genetic network inference in
computational models and applications to large-scale gene expression
data In M Bower amp H Bolouri (Eds) Computational modeling of
genetic and biochemical networks (pp 119ndash157) Cambridge MArsquoThe
MIT Press
Spirin V amp Mirny L A (2003) Protein complexes and functional
modules in molecular networks Proceedings of the National Academy
of Sciences of the United States of America 100 12123ndash12128
Staudinger J Liu Y Madan A Habeebu S amp Klaassen C D
(2001) Coordinate regulation of xenobiotic and bile acid homeo-
stasis by pregnane X receptor Drug Metabolism and Disposition
29 1467ndash1472
Sun L Z Ji Z L Chen X Wang J F amp Chen Y Z (2002) ADME-
AP A database of ADME associated proteins Bioinformatics 18
1699ndash1700
Suter L Babiss L E amp Wheeldon E B (2004) Toxicogenomics in
predictive toxicology in drug development Chemistry and Biology 11
161ndash171
Synold T W Dussault I amp Forman B M (2001) The orphan nuclear
receptor SXR coordinately regulates drug metabolism and efflux
Nature Medicine 7 584ndash590
Tabuchi Y amp Kondo T (2003) cDNA microarray analysis reveals chop-
10 plays a key role in Sertoli cell injury induced by bisphenol A
Biochemical and Biophysical Research Communications 305 54ndash61
Tamai I Nezu J Uchino H Sai Y Oku A Shimane M et al (2000)
Molecular identification and characterization of novel members of the
human organic anion transporter (OATP) family Biochemical and
Biophysical Research Communications 273 251ndash260
Taylor S W Fahy E Zhang B Glenn G M Warnock D E Wiley S
et al (2003) Characterization of the human heart mitochondrial
proteome Nature Biotechnology 21 281ndash286
Terasaka S Aita Y Inoue A Hayashi S Nishigaki M Aoyagi K
et al (2004) Using a customized DNA microarray for expression
profiling of the estrogen-responsive genes to evaluate estrogen activity
among natural estrogens and industrial chemicals Environmental
Health Perspectives 112 773ndash781
Thomas D M Francescutti-Verbeem D M Liu X amp Kuhn D M
(2004) Identification of differentially regulated transcripts in mouse
striatum following methamphetamine treatmentmdashan oligonucleotide
microarray approach Journal of Neurochemistry 88 380ndash393
Thomas R S Rank D R Penn S G Zastrow G M Hayes K R
Pande K et al (2001) Identification of toxicologically predictive
gene sets using cDNA microarrays Molecular Pharmacology 60
1189ndash1194
Thompson K L Afshari C A Amin R P Bertram T A Car B
Cunningham M et al (2004) Identification of platform-independent
gene expression markers of cisplatin nephrotoxicity Environmental
Health Perspectives 112 488ndash494
Tirona R G Leake B F Merino G amp Kim R B (2001)
Polymorphisms in OATP-C Identification of multiple allelic
variants associated with altered transport activity among European-
and African-Americans Journal of Biological Chemistry 276
35669ndash35675
Tirona R G Leake B F Wolkoff A W amp Kim R B (2003)
Human organic anion transporting polypeptide-C (SLC21A6) is a
major determinant of the rifampin-mediated pregnane X receptor
activation Journal of Pharmacology and Experimental Therapeutics
304 223ndash228
Tong W Cao X Harris S Sun H Fang H Fuscoe J et al (2003)
ArrayTrackmdashsupporting toxicogenomic research at the US Food and
Drug Administration National Center for Toxicological Research
Environmental Health Perspectives 111 1819ndash1826
Tornow S amp Mewes H W (2003) Functional modules by relating
protein interaction networks and gene expression Nucleic Acids
Research 31 6283ndash6289
Trocho C Escola-Gil J C Ribas V Benitez S Martin-Campos J M
Rotllan N et al (2004) Phenytoin treatment reduces atherosclerosis in
mice through mechanisms independent of plasma HDL-cholesterol
concentration Atherosclerosis 174 275ndash285
Ueda A Hamadeh H K Webb H K Yamamoto Y Sueyoshi T
Afshari C A et al (2002) Diverse roles of the nuclear orphan
receptor CAR in regulating hepatic genes in response to phenobarbital
Molecular Pharmacology 61 1ndash6
Ulrich R G (2003) The toxicogenomics of nuclear receptor agonists
Current Opinion in Chemical Biology 7 505ndash510
Ulrich R G Rockett J C Gibson G G amp Pettit S D (2004)
Overview of an interlaboratory collaboration on evaluating the effects
of model hepatotoxicants on hepatic gene expression Environmental
Health Perspectives 112 423ndash427
Unami A Shinohara Y Ichikawa T amp Baba Y (2003) Biochemical
and microarray analyses of bupivacaine-induced apoptosis Journal of
Toxicological Sciences 28 77ndash94
Vasquez A Flammini A Maritan A amp Vespignani A (2003) Global
protein function prediction from proteinndashprotein interaction networks
Nature Biotechnology 21 697ndash700
Vezina C M Walker N J amp Olson J R (2004) Subchronic Exposure to
TCDD PeCDF PCB126 and PCB153 Effect on hepatic gene
expression Environmental Health Perspectives 112 1636ndash1644
Vo T D Greenberg H J amp Palsson B O (2004) Reconstruction and
functional characterization of the human mitochondrial metabolic
network based on proteomic and biochemical data Journal of Bio-
logical Chemistry 279 39532ndash39540
Wang H amp LeCluyse E L (2003) Role of orphan nuclear receptors in
the regulation of drug-metabolising enzymes Clinical Pharmacoki-
netics 42 1331ndash1357
Wang N Silver D L Thiele C amp Tall A R (2001) ATP-
binding cassette transporter A1 (ABCA1) functions as a choles-
terol efflux regulatory protein Journal of Biological Chemistry 276
23742ndash23747
Waring J F Cavet G Jolly R A McDowell J Dai H Ciurlionis R
et al (2003) Development of a DNA Microarray for toxicology based
on hepatoxin-regulated sequences Environmental Health Perspectives
111 863ndash870
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6666
Waring J F Gum R Morfitt D Jolly R A Ciurlionis R Heindel M
et al (2002) Identifying toxic mechanisms using DNA microarrays
Evidence that an experimental inhibitor of cell adhesion molecule
expression signals through the aryl hydrocarbon nuclear receptor
Toxicology 181ndash182 537ndash550
Waring J F Jolly R A Ciurlionis R Lum P Y Praestgaard J T
Morfitt D C et al (2001) Clustering of hepatotoxins based on
mechanism of toxicity using gene expression profiles Toxicology and
Applied Pharmacology 175 28ndash42
Waters M Boorman G Bushel P Cunningham M Irwin R Merrick
A et al (2003) Systems toxicology and the Chemical Effects in
Biological Systems (CEBS) knowledge base EHP Toxicogenomics
111 15ndash28
Wei M Arnold L Cano M amp Cohen S M (2005) Effects of co-
administration of antioxidants and arsenicals on the rat urinary bladder
epithelium Toxicology Sciences 83 237ndash245
Weinshilboum R (2003) Inheritance and drug response New England
Journal of Medicine 348 529ndash537
Weinshilboum R amp Wang L (2004) Pharmacogenetics Inherited
variation in amino acid sequence and altered protein quantity Clinical
Pharmacology and Therapeutics 75 253ndash258
Werner E (2003) In silico multicellular systems biology and minimal
genomes Drug Discovery Today 8 1121ndash1127
Wong J S amp Gill S S (2002) Gene expression changes induced in
mouse liver by di(2-ethylhexyl) phthalate Toxicology and Applied
Pharmacology 185 180ndash196
Xie T Tong L McCann U D Yuan J Becker K G Mechan A O
et al (2004) Identification and characterization of metallothionein-1
and -2 gene expression in the context of ()34-methylenedioxymetham-
phetamine-induced toxicity to brain dopaminergic neurons Journal of
Neuroscience 24 7043ndash7050
Yadetie F Laegreid A Bakke I Kusnierczyk W Komorowski J
Waldum H L et al (2003) Liver gene expression in rats in response
to the peroxisome proliferator-activated receptor-alpha agonist ciprofi-
brate Physiological Genomics 15 9ndash19
Yamashita S Nomoto T Abe M Tatematsu M Sugimura T amp
Ushijima T (2004) Persistence of gene expression changes in stomach
mucosae induced by short-term N-methyl-NV-nitro-N-nitrosoguanidinetreatment and their presence in stomach cancers Mutation Research
549 185ndash193
Yan Q amp Sadee W (2000) Human membrane transporter database A
web-accessible relational database for drug transport studies and
pharmacogenomics AAPS Pharmsci 2 E20
Yeger-Lotem E amp Margalit H (2003) Detection of regulatory circuits by
integrating the cellular networks of proteinndashprotein interactions and
transcription regulation Nucleic Acids Research 31 6053ndash6061
Yoon B I Li G -X Kitada K Kawasaki Y Igarashi K amp Kodama
Y et al (2003) Mechanisms of benzene-induced hematotoxicity and
leukemogenicity cDNA microarray analyses using mouse bone marrow
tissue Environmental Health Perspectives 111 1411ndash1420
Young M B DiSilvestro M R Sendera T J Freund J Kriete A amp
Magnuson S R (2003) Analysis of gene expression in carbon
tetrachloride-treated rat livers using a novel bioarray technology
Pharmacogenomics Journal 3 41ndash52
Yu H Zhu X Greenbaum D Karro J amp Gerstein M (2004) TopNet
A tool for comparing biological sub-networks correlating protein
properties with topological statistics Nucleic Acids Research 32
328ndash337
Zhang E Y Fu D J Pak Y A Stewart T Mukhopadhyay N
Wrighton S A et al (2004) Genetic polymorphisms in human proton-
dependent dipeptide transporter PEPT1 Implications for the functional
role of Pro586 Journal of Pharmacology and Experimental Therapeu-
tics 310 437ndash445
Zhang E Y Knipp G T Ekins S amp Swaan P W (2002)
Structural biology and function of solute transporters Implications
for identifying and designing substrates Drug Metabolism Reviews
34 709ndash750
Zhang E Y Phelps M A Cheng C Ekins S amp Swaan P W (2002)
Modeling of active transport systems Advanced Drug Delivery
Reviews 54 329ndash354
- Systems-ADMETox Resources and network approaches
-
- Introduction
- Data available
- Network analysis and databases
-
- Network applications
-
- The role of transporters
-
- Clinical relevance of transporters
- Transporter network examples ABCA1
- Transporter network examples OATP
- Transporter microarray data
-
- Applications to enzymes
- Future network applications
-
- Discussion
- Acknowledgements
- References
-
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash66 65
Schrenk D Baus P R Ermel N Klein C Vorderstemann B amp
Kauffmann H M (2001) Up-regulation of transporters of the MRP
family by drugs and toxins Toxicology Letters 120 51ndash57
Segal E Shapira M Regev A Persquoer D Botstein D Koller D et al
(2003) Module networks Identifying regulatory modules and their
condition-specific regulators from gene expression data Nature
Genetics 34 166ndash176
Segal E Wang H amp Koller D (2003) Discovering molecular pathways
from protein interaction and gene expression Bioinformatics 19
i264ndash i272
Seidel S D Kan H L Stott W T Schisler M R amp Gollapudi B B
(2003) Identification of transcriptome profiles for the DNA-damaging
agents bleomycin and hydrogen peroxide in L5178Y mouse lymphoma
cells Environmental and Molecular Mutagenesis 42 19ndash25
Shan L Yu Y Schut H A J amp Snyderwine E G (2004) Susceptibility
of rats to mammary gland carcinogenesis by the food-derived
carcinogen 2-Amino-1-Methyl-6-Phenylimidazo[45-b]Pyridine (PhIP)
varies with age and is associated with the induction of differential gene
expression American Journal of Pathology 165 191ndash202
Sharom J R Bellows D S amp Tyers M (2004) From large networks to
small molecules Current Opinion in Chemical Biology 8 81ndash90
Shitara Y Hirano M Sato H amp Sugiyama Y (2004) Gemfibrozil and
its glucuronide inhibit the OATP2(OATP1B1 SLC21A6)-mediated
hepatic uptake and CYP2C8-mediated metabolism of cerivastatinmdash
Analysis of the mechanism of the clinically relevant drugndashdrug
interaction between cerivastatin and gemfibrozil Journal of Pharma-
cology and Experimental Therapeutics
Shitara Y Itoh T Sato H Li A P amp Sugiyama Y (2003) Inhibition of
transporter-mediated hepatic uptake as a mechanism for drugndashdrug
interaction between cerivastatin and cyclosporin A Journal of
Pharmacology and Experimental Therapeutics 304 610ndash616
Somogyi R Fuhrman S amp Wen X (2001) Genetic network inference in
computational models and applications to large-scale gene expression
data In M Bower amp H Bolouri (Eds) Computational modeling of
genetic and biochemical networks (pp 119ndash157) Cambridge MArsquoThe
MIT Press
Spirin V amp Mirny L A (2003) Protein complexes and functional
modules in molecular networks Proceedings of the National Academy
of Sciences of the United States of America 100 12123ndash12128
Staudinger J Liu Y Madan A Habeebu S amp Klaassen C D
(2001) Coordinate regulation of xenobiotic and bile acid homeo-
stasis by pregnane X receptor Drug Metabolism and Disposition
29 1467ndash1472
Sun L Z Ji Z L Chen X Wang J F amp Chen Y Z (2002) ADME-
AP A database of ADME associated proteins Bioinformatics 18
1699ndash1700
Suter L Babiss L E amp Wheeldon E B (2004) Toxicogenomics in
predictive toxicology in drug development Chemistry and Biology 11
161ndash171
Synold T W Dussault I amp Forman B M (2001) The orphan nuclear
receptor SXR coordinately regulates drug metabolism and efflux
Nature Medicine 7 584ndash590
Tabuchi Y amp Kondo T (2003) cDNA microarray analysis reveals chop-
10 plays a key role in Sertoli cell injury induced by bisphenol A
Biochemical and Biophysical Research Communications 305 54ndash61
Tamai I Nezu J Uchino H Sai Y Oku A Shimane M et al (2000)
Molecular identification and characterization of novel members of the
human organic anion transporter (OATP) family Biochemical and
Biophysical Research Communications 273 251ndash260
Taylor S W Fahy E Zhang B Glenn G M Warnock D E Wiley S
et al (2003) Characterization of the human heart mitochondrial
proteome Nature Biotechnology 21 281ndash286
Terasaka S Aita Y Inoue A Hayashi S Nishigaki M Aoyagi K
et al (2004) Using a customized DNA microarray for expression
profiling of the estrogen-responsive genes to evaluate estrogen activity
among natural estrogens and industrial chemicals Environmental
Health Perspectives 112 773ndash781
Thomas D M Francescutti-Verbeem D M Liu X amp Kuhn D M
(2004) Identification of differentially regulated transcripts in mouse
striatum following methamphetamine treatmentmdashan oligonucleotide
microarray approach Journal of Neurochemistry 88 380ndash393
Thomas R S Rank D R Penn S G Zastrow G M Hayes K R
Pande K et al (2001) Identification of toxicologically predictive
gene sets using cDNA microarrays Molecular Pharmacology 60
1189ndash1194
Thompson K L Afshari C A Amin R P Bertram T A Car B
Cunningham M et al (2004) Identification of platform-independent
gene expression markers of cisplatin nephrotoxicity Environmental
Health Perspectives 112 488ndash494
Tirona R G Leake B F Merino G amp Kim R B (2001)
Polymorphisms in OATP-C Identification of multiple allelic
variants associated with altered transport activity among European-
and African-Americans Journal of Biological Chemistry 276
35669ndash35675
Tirona R G Leake B F Wolkoff A W amp Kim R B (2003)
Human organic anion transporting polypeptide-C (SLC21A6) is a
major determinant of the rifampin-mediated pregnane X receptor
activation Journal of Pharmacology and Experimental Therapeutics
304 223ndash228
Tong W Cao X Harris S Sun H Fang H Fuscoe J et al (2003)
ArrayTrackmdashsupporting toxicogenomic research at the US Food and
Drug Administration National Center for Toxicological Research
Environmental Health Perspectives 111 1819ndash1826
Tornow S amp Mewes H W (2003) Functional modules by relating
protein interaction networks and gene expression Nucleic Acids
Research 31 6283ndash6289
Trocho C Escola-Gil J C Ribas V Benitez S Martin-Campos J M
Rotllan N et al (2004) Phenytoin treatment reduces atherosclerosis in
mice through mechanisms independent of plasma HDL-cholesterol
concentration Atherosclerosis 174 275ndash285
Ueda A Hamadeh H K Webb H K Yamamoto Y Sueyoshi T
Afshari C A et al (2002) Diverse roles of the nuclear orphan
receptor CAR in regulating hepatic genes in response to phenobarbital
Molecular Pharmacology 61 1ndash6
Ulrich R G (2003) The toxicogenomics of nuclear receptor agonists
Current Opinion in Chemical Biology 7 505ndash510
Ulrich R G Rockett J C Gibson G G amp Pettit S D (2004)
Overview of an interlaboratory collaboration on evaluating the effects
of model hepatotoxicants on hepatic gene expression Environmental
Health Perspectives 112 423ndash427
Unami A Shinohara Y Ichikawa T amp Baba Y (2003) Biochemical
and microarray analyses of bupivacaine-induced apoptosis Journal of
Toxicological Sciences 28 77ndash94
Vasquez A Flammini A Maritan A amp Vespignani A (2003) Global
protein function prediction from proteinndashprotein interaction networks
Nature Biotechnology 21 697ndash700
Vezina C M Walker N J amp Olson J R (2004) Subchronic Exposure to
TCDD PeCDF PCB126 and PCB153 Effect on hepatic gene
expression Environmental Health Perspectives 112 1636ndash1644
Vo T D Greenberg H J amp Palsson B O (2004) Reconstruction and
functional characterization of the human mitochondrial metabolic
network based on proteomic and biochemical data Journal of Bio-
logical Chemistry 279 39532ndash39540
Wang H amp LeCluyse E L (2003) Role of orphan nuclear receptors in
the regulation of drug-metabolising enzymes Clinical Pharmacoki-
netics 42 1331ndash1357
Wang N Silver D L Thiele C amp Tall A R (2001) ATP-
binding cassette transporter A1 (ABCA1) functions as a choles-
terol efflux regulatory protein Journal of Biological Chemistry 276
23742ndash23747
Waring J F Cavet G Jolly R A McDowell J Dai H Ciurlionis R
et al (2003) Development of a DNA Microarray for toxicology based
on hepatoxin-regulated sequences Environmental Health Perspectives
111 863ndash870
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6666
Waring J F Gum R Morfitt D Jolly R A Ciurlionis R Heindel M
et al (2002) Identifying toxic mechanisms using DNA microarrays
Evidence that an experimental inhibitor of cell adhesion molecule
expression signals through the aryl hydrocarbon nuclear receptor
Toxicology 181ndash182 537ndash550
Waring J F Jolly R A Ciurlionis R Lum P Y Praestgaard J T
Morfitt D C et al (2001) Clustering of hepatotoxins based on
mechanism of toxicity using gene expression profiles Toxicology and
Applied Pharmacology 175 28ndash42
Waters M Boorman G Bushel P Cunningham M Irwin R Merrick
A et al (2003) Systems toxicology and the Chemical Effects in
Biological Systems (CEBS) knowledge base EHP Toxicogenomics
111 15ndash28
Wei M Arnold L Cano M amp Cohen S M (2005) Effects of co-
administration of antioxidants and arsenicals on the rat urinary bladder
epithelium Toxicology Sciences 83 237ndash245
Weinshilboum R (2003) Inheritance and drug response New England
Journal of Medicine 348 529ndash537
Weinshilboum R amp Wang L (2004) Pharmacogenetics Inherited
variation in amino acid sequence and altered protein quantity Clinical
Pharmacology and Therapeutics 75 253ndash258
Werner E (2003) In silico multicellular systems biology and minimal
genomes Drug Discovery Today 8 1121ndash1127
Wong J S amp Gill S S (2002) Gene expression changes induced in
mouse liver by di(2-ethylhexyl) phthalate Toxicology and Applied
Pharmacology 185 180ndash196
Xie T Tong L McCann U D Yuan J Becker K G Mechan A O
et al (2004) Identification and characterization of metallothionein-1
and -2 gene expression in the context of ()34-methylenedioxymetham-
phetamine-induced toxicity to brain dopaminergic neurons Journal of
Neuroscience 24 7043ndash7050
Yadetie F Laegreid A Bakke I Kusnierczyk W Komorowski J
Waldum H L et al (2003) Liver gene expression in rats in response
to the peroxisome proliferator-activated receptor-alpha agonist ciprofi-
brate Physiological Genomics 15 9ndash19
Yamashita S Nomoto T Abe M Tatematsu M Sugimura T amp
Ushijima T (2004) Persistence of gene expression changes in stomach
mucosae induced by short-term N-methyl-NV-nitro-N-nitrosoguanidinetreatment and their presence in stomach cancers Mutation Research
549 185ndash193
Yan Q amp Sadee W (2000) Human membrane transporter database A
web-accessible relational database for drug transport studies and
pharmacogenomics AAPS Pharmsci 2 E20
Yeger-Lotem E amp Margalit H (2003) Detection of regulatory circuits by
integrating the cellular networks of proteinndashprotein interactions and
transcription regulation Nucleic Acids Research 31 6053ndash6061
Yoon B I Li G -X Kitada K Kawasaki Y Igarashi K amp Kodama
Y et al (2003) Mechanisms of benzene-induced hematotoxicity and
leukemogenicity cDNA microarray analyses using mouse bone marrow
tissue Environmental Health Perspectives 111 1411ndash1420
Young M B DiSilvestro M R Sendera T J Freund J Kriete A amp
Magnuson S R (2003) Analysis of gene expression in carbon
tetrachloride-treated rat livers using a novel bioarray technology
Pharmacogenomics Journal 3 41ndash52
Yu H Zhu X Greenbaum D Karro J amp Gerstein M (2004) TopNet
A tool for comparing biological sub-networks correlating protein
properties with topological statistics Nucleic Acids Research 32
328ndash337
Zhang E Y Fu D J Pak Y A Stewart T Mukhopadhyay N
Wrighton S A et al (2004) Genetic polymorphisms in human proton-
dependent dipeptide transporter PEPT1 Implications for the functional
role of Pro586 Journal of Pharmacology and Experimental Therapeu-
tics 310 437ndash445
Zhang E Y Knipp G T Ekins S amp Swaan P W (2002)
Structural biology and function of solute transporters Implications
for identifying and designing substrates Drug Metabolism Reviews
34 709ndash750
Zhang E Y Phelps M A Cheng C Ekins S amp Swaan P W (2002)
Modeling of active transport systems Advanced Drug Delivery
Reviews 54 329ndash354
- Systems-ADMETox Resources and network approaches
-
- Introduction
- Data available
- Network analysis and databases
-
- Network applications
-
- The role of transporters
-
- Clinical relevance of transporters
- Transporter network examples ABCA1
- Transporter network examples OATP
- Transporter microarray data
-
- Applications to enzymes
- Future network applications
-
- Discussion
- Acknowledgements
- References
-
S Ekins Journal of Pharmacological and Toxicological Methods 53 (2006) 38ndash6666
Waring J F Gum R Morfitt D Jolly R A Ciurlionis R Heindel M
et al (2002) Identifying toxic mechanisms using DNA microarrays
Evidence that an experimental inhibitor of cell adhesion molecule
expression signals through the aryl hydrocarbon nuclear receptor
Toxicology 181ndash182 537ndash550
Waring J F Jolly R A Ciurlionis R Lum P Y Praestgaard J T
Morfitt D C et al (2001) Clustering of hepatotoxins based on
mechanism of toxicity using gene expression profiles Toxicology and
Applied Pharmacology 175 28ndash42
Waters M Boorman G Bushel P Cunningham M Irwin R Merrick
A et al (2003) Systems toxicology and the Chemical Effects in
Biological Systems (CEBS) knowledge base EHP Toxicogenomics
111 15ndash28
Wei M Arnold L Cano M amp Cohen S M (2005) Effects of co-
administration of antioxidants and arsenicals on the rat urinary bladder
epithelium Toxicology Sciences 83 237ndash245
Weinshilboum R (2003) Inheritance and drug response New England
Journal of Medicine 348 529ndash537
Weinshilboum R amp Wang L (2004) Pharmacogenetics Inherited
variation in amino acid sequence and altered protein quantity Clinical
Pharmacology and Therapeutics 75 253ndash258
Werner E (2003) In silico multicellular systems biology and minimal
genomes Drug Discovery Today 8 1121ndash1127
Wong J S amp Gill S S (2002) Gene expression changes induced in
mouse liver by di(2-ethylhexyl) phthalate Toxicology and Applied
Pharmacology 185 180ndash196
Xie T Tong L McCann U D Yuan J Becker K G Mechan A O
et al (2004) Identification and characterization of metallothionein-1
and -2 gene expression in the context of ()34-methylenedioxymetham-
phetamine-induced toxicity to brain dopaminergic neurons Journal of
Neuroscience 24 7043ndash7050
Yadetie F Laegreid A Bakke I Kusnierczyk W Komorowski J
Waldum H L et al (2003) Liver gene expression in rats in response
to the peroxisome proliferator-activated receptor-alpha agonist ciprofi-
brate Physiological Genomics 15 9ndash19
Yamashita S Nomoto T Abe M Tatematsu M Sugimura T amp
Ushijima T (2004) Persistence of gene expression changes in stomach
mucosae induced by short-term N-methyl-NV-nitro-N-nitrosoguanidinetreatment and their presence in stomach cancers Mutation Research
549 185ndash193
Yan Q amp Sadee W (2000) Human membrane transporter database A
web-accessible relational database for drug transport studies and
pharmacogenomics AAPS Pharmsci 2 E20
Yeger-Lotem E amp Margalit H (2003) Detection of regulatory circuits by
integrating the cellular networks of proteinndashprotein interactions and
transcription regulation Nucleic Acids Research 31 6053ndash6061
Yoon B I Li G -X Kitada K Kawasaki Y Igarashi K amp Kodama
Y et al (2003) Mechanisms of benzene-induced hematotoxicity and
leukemogenicity cDNA microarray analyses using mouse bone marrow
tissue Environmental Health Perspectives 111 1411ndash1420
Young M B DiSilvestro M R Sendera T J Freund J Kriete A amp
Magnuson S R (2003) Analysis of gene expression in carbon
tetrachloride-treated rat livers using a novel bioarray technology
Pharmacogenomics Journal 3 41ndash52
Yu H Zhu X Greenbaum D Karro J amp Gerstein M (2004) TopNet
A tool for comparing biological sub-networks correlating protein
properties with topological statistics Nucleic Acids Research 32
328ndash337
Zhang E Y Fu D J Pak Y A Stewart T Mukhopadhyay N
Wrighton S A et al (2004) Genetic polymorphisms in human proton-
dependent dipeptide transporter PEPT1 Implications for the functional
role of Pro586 Journal of Pharmacology and Experimental Therapeu-
tics 310 437ndash445
Zhang E Y Knipp G T Ekins S amp Swaan P W (2002)
Structural biology and function of solute transporters Implications
for identifying and designing substrates Drug Metabolism Reviews
34 709ndash750
Zhang E Y Phelps M A Cheng C Ekins S amp Swaan P W (2002)
Modeling of active transport systems Advanced Drug Delivery
Reviews 54 329ndash354
- Systems-ADMETox Resources and network approaches
-
- Introduction
- Data available
- Network analysis and databases
-
- Network applications
-
- The role of transporters
-
- Clinical relevance of transporters
- Transporter network examples ABCA1
- Transporter network examples OATP
- Transporter microarray data
-
- Applications to enzymes
- Future network applications
-
- Discussion
- Acknowledgements
- References
-