program and abstracts - swansea university · program and abstracts organised by section genetic...

European Environmental Mutagen Society

34th Annual Meeting

Pro

gra

m a

nd

ab

stra

cts

Organised by

section Genetic Toxicology of

the Netherlands Society of Toxicology

University of Maastricht

Sponsored by:

De

sig

n a

nd

pri

nt:

Un

igra

ph

ic,U

niv

ers

ite

it M

aa

stri

cht

Ph

oto

gra

ph

y:

Stu

dio

Pre

ss

Biopedricwww.biopedric.com

Eu

rop

ea

n E

nv

iron

me

nta

l Mu

tag

en

So

ciety

• 34th

An

nu

al M

ee

ting

Pro

gra

m a

nd

ab

stracts

European Environmental Mutagen Society

34th Annual Meeting

Program and Abstracts

3

abstractboek 20-10-2004 10:28 Pagina 3

Contents

Introduction 5

Map 6

Programme 8

Abstracts 14

Poster presentations 45

Author Index 127

4

Local Organising committee

Joost van Delft (President)

Jan van Benthem (vice-President)

Peter Weterings (Treasurer)

Bert van der Horst

Bert van Zeeland,

Cyrille Krul

Els van Vliet

Frederik-Jan van Schooten

Frederique van Acker

Gerrit Alink

Harry van Steeg

Harry Vrieling

Ineke Verspeek

Jos Kleinjans

Madeleine Nivard

Rob Baan

Editorial Board

Jan van Benthem (vice-President)

Peter Weterings (Treasurer)

Frederique van Acker

Carmen Hermans (Conference and Events Office)


Introduction

Dear participants,

Welcome to the 34th annual meeting of the European Environmental Mutagen Society with as central

theme “Genes and Environment, Bridging the Gap”. The Organising Committee is delighted to present to

you an exciting programme bringing many of the major scientific and regulatory topics and develop-

ments that currently are of interest for the Society’s members. DNA damage and repair, environmental

and dietary factors, genetic susceptibility, risk assessment and guidelines, are among them. To bridge the

gap between traditional and new technologies, special attention will be paid during the meeting to the

opportunities and achievements of genomics on R&D and on regulation.

Furthermore, to broaden the engagement of the Society as a whole and to specifically involve the new

members, the floor is given to many speakers that were selected on submitted abstracts. Hopefully, this

meeting will not only inspire your scientific life, but will also contribute to your social and Societal net-

work.

On behalf of the board of the Dutch EMS but also on behalf of all its members, I wish you a pleasant, fruit-

ful, informing and successful meeting. Furthermore, I hope you will enjoy your stay in Maastricht and

frequently bridge the gap between the two historic parts on both sides of the river Maas.

Finally, I would like to thank the Organising Committee and everyone else who was involved in settling

this meeting and making these days wonderful.

Joost van Delft

Chair of the Organising Committee

5


Map

Sponsors

1. Gentronix Ltd 7. TNO Voeding

2. Elsevier Science 8. Research Tox Center S.p.A.

3. Biopredic International 9. NOTOX B.V.

4. PerkinElmer 10. COVANCE LTD.

5. Snijders Scientific/ORDINA Technical 11. Centre International de Toxicologie (CIT)

6. Perceptive Instruments Ltd 12. Safepharm Laboratories Ltd

Legenda Poster Presentation

Bordeaux Foyer: PW 1001 - PW 2018

Foyer D’Alsace: PW 2019 - PW 3009

Bourgogne: PW 3010 - PW 7007

6

Céramique Ground Floor

Céramique 3 Céramique 2 Céramique 1

Céramique foyer

1 2

3

coffee/tea buffet


7

Bourgogne Souterrain

Beaune Pommard Macon

5

7

4

6

8 9 10

11

12

coffee/tea buffet

coffee/tea buffet

lunch buffet

lun

ch b

uff

et

Foyer D’Alsace

Bordeaux Ground Floor

Bordeaux Foyer


Programme

Saturday 4 September

15.00 Registration

17.00 Welcome and Opening

Ceramic Hall

Joost van Delft, president of the Organizing Committee, President of the Dutch EMS

Jo Ritzen, President of the Executive Board of Maastricht University

Young Scientist Award Lectures

Ceramic Hall

Chair: Krzysztof Szyfter, President of the EEMS

17.15 Oxidatively damaged DNA: from genes to populations

Marcus Cooke, University of Leicester, United Kingdom

17.45 Cell-type and DNA damage-specific response of human epidermal cells.

Mariarosaria D’Erico, Italian National Health Institute, Rome, Italy

18.15 Welcome Party, Crowne Plaza Hotel

Sunday 5 September

08.00 Registration

09.00 Symposium 1 “DNA Damage, repair and cell cycle control”

Ceramic Hall

Chair: Micheline Kirsch-Volders and Harry Vrieling

09.00 The effect of p53 mutations on cell cycle control and carcinogen-induced tumorigenesis.

Annemieke de Vries, National Institute of Public Health and the Environment, Bilthoven,

The Netherlands.

09.30 Dynamic organization of DNA damage repair proteins and chromosomes.

Roland Kanaar, Erasmus Medical Center, Rotterdam, The Netherlands.

10.00 Translesion synthesis and the Y-family of DNA polymerases.

Alan Lehmann, University of Sussex, Brighton, United Kingdom.

10.30 ATM: mobilizing the cellular response to genotoxic stress.

Yoshi Shiloh, Tel Aviv University, Israel.

11.00 Coffee/tea break

11.20 Poster session 1

Bourgogne/Bordeaux Foyer/Foyer D’Alsace

13.00 Lunch

14.00 Workshop 1 “Nutrigenomics for healthy and safe foods”

Bordeaux Hall

Chair: Ben van Ommen and Cyrille Krul

14.00 Nutrigenomics and nutritional systems biology.

Ben van Ommen, TNO Nutrition and Food Research, Zeist, The Netherlands.

14.30 The effect of plant phenols on AP-1 expression in mouse epidermis.

Wanda Baer-Dubowska, University of Medical Sciences, Poznan, Poland.

14.50 Natural Ah receptor agonists in the human diet: beneficial food components or unpercieved

risk factors?

Pim de Waard, Maastricht University, The Netherlands.

15.10 Folic acid metabolism and gene polymorphisms in the risk of non-hodgkin’s lymphomas.

Fabio Coppedè, University of Pisa, Italy.

15.30 Altered vegetable intake affects pivotal carcinogenesis pathways in colon mucosa from

adenoma patients and controls.

Simone van Breda, Maastricht University, The Netherlands.

8


14.00 Workshop 2A “DNA damage, repair and cell cycle control”

Ceramic Hall

Chair: Niels de Wind and Rob Baan

14.00 DNA translesion synthesis and mismatch repair in the response to endogenous and exoge-

nous DNA damage.

Niels de Wind, Leiden University Medical Center, The Netherlands.

14.30 Impact of mismatch repair on UVB-induced cell cycle arrest.

Gerdien Stout, Leiden University Medical Centre, The Netherlands.

14.50 Cadmium inhibits human DNA mismatch repair in vivo.

Lene Rasmussen, Roskilde University, Denmark.

15.10 Wild type p53 suppresses non-homologous end-joining of DSB but not overall repair profi-

ciency.

Jochen Dahm-Daphi, University of Hamburg, Germany.

15.30 Bystander apoptosis induced in non-irradiated cells needs of caspase 8 activity.

Mauro Grifalconi, University of Padova, Italy


16.10 Workshop 3 “Influence of genetic variation on cancer”

Ceramic Hall

Chair: Ari Hirvonen and Radim Sram

16.10 Molecular epidemiology of sporadic breast cancer: role of polymorphisms in the xenobiotic

metabolism and DNA repair genes.

Ari Hirvonen, Finnish Institute of Occupational Health, Helsinki, Finland.

16.40 Gene environment interactions and genetic damage introduction by ETS: the importance of

exposure levels.

Soterios Kyrtopoulos, National Hellenic Research Foundation, Athens, Greece.

17.00 Polymorphisms in DNA repair genes and risk of lung cancer in a Danish prospective cohort.

Ulla Vogel, National Institute of Occupational Health, Copenhagen, Denmark.

Cytogenetic biomarkers and human cancer risk (cancer risk biomarkers).

Hannu Norppa, Finnish Institute of Occupational Health, Helsinki, Finland.

17.40 Influence of GSTM1, GSTT1 and NAT2 genotypes on p53 mutational spectrum in bladder

tumors.

Charlotta Ryk, Karolinska Institute, Huddinge, Sweden (provisional).

16.10 Workshop 4 “Oxidative damage”

Bordeaux Hall

Chair: Anthony Lynch and Bert van Zeeland

16.10 Gene expression changes as biomarkers of oxidative damage.

Anthony Lynch, Glaxo Smith Kline, Herts, United Kingdom.

16.40 The multiple physiological and pathological consequences of oxidative DNA damage.

Alberto Izzotti, University of Genoa, Italy.

17.00 DNA damage by respirable quartz particles in rat lung target cells: the role of mitochondria.

Hui Li, IUF, Düsseldorf, Germany.

17.20 An hypothesis on the role of oxidative damage in neurodegenerative diseases.

Lucia Migliore, University of Pisa, Italy.

17.40 PARP1 together with Csb and Xpa accelerates the global repair of oxidative DNA base

damage.

Claudia Flohr, University of Mainz, Germany.

19.30 Concert in the St. Jans Kerk, Vrijthof, Maastricht

9


Monday 6 September

08:00 Registration

09.00 Symposium 2 “Toxicogenomics in genetic toxicology”

Ceramic Hall

Sponsored by LRI and organised by ECETOC.

chair: Awni Sarrif and Jos Kleinjans

09.00 Introduction.

Jos Kleinjans, Maastricht University, The Netherlands.

09.05 Experimental design, quality aspects of data collection and statistics.

Timothy Gant, University of Leicester, United Kingdom.

09.35 Discrimination of genotoxic from non-genotoxic carcinogens by expression profiling

in vitro.

Joost van Delft, Maastricht University, The Netherlands.

10.00 Toxicogenomic analysis of gene expression: an emerging approach for differentiating

genotoxic mechanisms.

Jiri Aubrecht, Pfizer Inc., Groton, USA.

10.25 Genotoxic and non-genotoxic carcinogens in rat liver.

Hans-Jurgen Ahr, Bayer Health Care AG, Wuppertal, Germany.

10.55 Final remarks.

Jos Kleinjans, Maastricht University, The Netherlands.


11.20 Poster session 2

Bourgogne

13.00 Lunch

14.00 Minisymposium “Biological clocks, chronotoxicology and therapy”

Bordeaux Hall

Chair: Bert van der Horst

14.00 The circadian clockwork: a central regulator of behaviour, physiology and disease.

Michael Hastings, University of Cambridge, United Kingdom.

14.30 Implications of biological clocks for cancer processes and their treatments.

Francis Levi, Université Paris XI Paul Brousse Hôpital, Villejuif, France.

15.00 Workshop 2B “DNA damage, repair and cell cycle control”

Bordeaux Hall

Chair: Niels de Wind and Rob Baan

15.00 Gene expression profiles revealed p53R2 protein plays a role in radiation-induced mutagenesis.

Eric Chuang, NCI/NIH, Bethesda, USA.

15.25 Extent of p53 binding to target genes in vivo and their expression.

Paola Menichini, National Institute for Cancer Research, IST, Genova, Italy.

14.00 Workshop 5 “Hazard identification by omics technologies”

Sponsored by LRI and organised by ECETOC

Ceramic Hall

Chair: Timothy Gant and Els van Vliet

14.00 Introduction.

Awni Sarrif, Du Pont de Nemours and Co., Newark, USA.

10


14.05 Use of genomics for regulatory science, industry perspective.

Silvio Albertini, Hoffmann-La Roche, Basel, Switzerland.

14.30 Identification of genotoxins by marker gene expression based on a supervised class predic-

tion approach.

Daniel Bauer, Novartis Pharma AG, Basel, Switzerland.

14.50 Effects of chlorpromazine with and without UV radiation on gene expression of HepG2

cells.

Roland Froetschl, Federal Institute for Drugs and Medical Devices, Bonn, Germany.

15.10 Rat liver transcriptomics after 28-day exposure to benzene and trichloroethylene (mix-

tures).

Wilbert Heijne, TNO Nutrition and Food Research, Zeist, The Netherlands.

15.30 Discussion and wrap up.

Silvio Albertini, Hoffmann-La Roche, Basel, Switzerland.


16.10 Workshop 6 “Health risk assessment; guidelines, harmonisation, validation, etc.”

Bordeaux Hall

Chair: Stephan Pfuhler and Frederique van Acker

16.10 New strategies in toxicological risk assessments: transparent and efficient approaches in

determining hazard and risk.

Bas Blaauboer, University of Utrecht, The Netherlands.

16.40 Overview of validation and international acceptance of new or updated test methods for

hazard assessment in the OECD.

Drew Wagner, OECD, Paris, France.

17.10 Evaluation of the performance of a small battery of in vitro tests in detecting rodent and

human carcinogens.

David Kirkland, Covance Laboratories Ltd., Harrogate, United Kingdom.

17.40 Frontloading genetic toxicology evaluation of DEREK, AmesII and Greenscreen as screening

tools.

Jacky van Gompel, Johnson & Johnson Pharmaceutical Research, Beerse, Belgium.

16.10 Workshop 7 “Genomic stability and aging”

Ceramic Hall

Chair: Ian Hickson and Madeleine Nivard

16.10 Role of the Bloom’s syndrome helicase in maintenance of genome stability.

Ian Hickson, University of Oxford, United Kingdom.

16.40 The studies on localization of human RECQ helicases fused with fluorescent proteins.

Marek Rusin, Center of Oncology, Gliwice, Poland.

17.00 Age related genome instability in DNA repair deficient mice.

Martijn Dollé, National Institute of Public Health and the Environment, Bilthoven, The

Netherlands.

17.20 DNA repair function, polymorphisms and genotoxicity in workers exposed to low dose

ionising radiation.

Peter van de Aka, Free University Brussels, Belgium.

17.20 DNA damage repair, apoptosis and necrosis in children, adults and old age humans.

Stelios Piperakis, NCSR Demokritos, Athens, Greece.

19.30 Reception at Town Hall, Maastricht

11


Tuesday 7 September

08.00 Registration

09.00 Symposium 3 “Genomic stability and aging”

Ceramic Hall

Chair: Peter Stambrook and Harry van Steeg

09.00 Aging, cancer and cellular responses to DNA damage.

Judy Campisi, Lawrence Berkeley National Laboratory, Berkeley, USA.

09.30 Interplay between telomeres and DNA repair: implications for cancer and aging.

Maria Blasco, Spanish National Cancer Center, Madrid, Spain.

10.00 Genome instability, aging and cancer.

Jan Vijg, University Texas Health Science Center, San Antonio, USA.

10.30 Stress, mutations and aging.

Tom Kirkwood, University of Newcastle, United Kingdom.


11.20 EEMS General Assembly

12.10 Fritz Sobels Award Lecture

How rare genetic diseases can help us understanding mutagenesis and carcinogenesis?

Alain Sarasin, Institut Gustave Roussy, Villejuif, France

Ceramic Hall

13.00 Lunch

14.00 Excursion to the caves of Maastricht and to the winery “Apostelhoeve”

19.30 Conference dinner at La Butte aux Bois, Belgium

Wednesday 8 September

08.00 Registration

09.00 Symposium 4 “Influence of genetic variation on cancer”

Ceramic Hall

Chair: Hannu Norppa and Frederik Jan van Schooten

09.00 Cancer: genes and the environment.

Kari Hemminki, German Cancer Research Center, Heidelberg, Germany.

09.30 Lung cancer and polymorphisms of genes involved in carcinogen metabolism and DNA

repair.

Federico Canzian, International Agency for Research on Cancer, Lyon, France.

10.00 Design and interpretation of studies on gene-environment interactions in carcinogenesis.

Paolo Vineis, University of Torino, Italy.

10.30 A significance of genetic factor in initiation and progression of laryngeal cancer.

Krzysztof Szyfter, Polish Academy of Sciences, Poznan, Poland.


12


11.20 Symposium 5 “Natural mutagens and carcinogens”

Ceramic Hall

Chair: Adela Lopez de Cerain and Gerrit Alink

11.20 Flavonoids and alkenylbenzenes; mechanisms of mutagenic action and carcinogenic risk.

Ivonne Rietjens, Wageningen University, The Netherlands.

11.50 Genotoxicity of phytoestrogens.

Helga Stopper, University of Würzburg, Germany.

12.20 Genotoxicology of heat processing of food.

Margaretha Jägerstad, Swedish University of Agricultural Sciences, Uppsala, Sweden.

12.50 Natural versus synthetic mutagens and carcinogens.

Lois Swirsky Gold, UC Berkeley and Lawrence Berkeley National Laboratory, Berkeley, USA.

13.20 Closure

Ceramic Hall

13.30 Lunch

13


Abstracts

S1 - Symposium 1

Oral Presentation

OS1001

ATM: MOBILIZING THE CELLULAR RESPONSE TO

DOUBLE-STRAND BREAKS

Y Shiloh

Tel Aviv University, TEL AVIV, Israel

The intricate signaling network mobilized by DNA

damage spans numerous pathways linking DNA

repair, cell cycle checkpoints and stress responses

that affect various aspects of cellular metabolism. A

prominent inducer of this response is the double

strand break (DSB). Formation of DSBs in the DNA

leads to rapid recruitment of sensor/activator pro-

teins to the damaged sites. Among them are the MRN

complex whose core contains the Mre11, Rad50 and

Nbs1 proteins and the BRCT proteins Brca1, 53BP1 and

Mdc1/Nfbd1. This process is required for the next step

in the DSB response, the activation of the nuclear pro-

tein kinase ATM - the primary transducer of the DSB

alarm. ATM, a member of the PI3-kinase-like protein

kinase (PIKK) family, is activated via autophosphory-

lation, and a portion of it binds to the DSB sites.

Activated ATM then phosphorylates key players in a

wide range of processes. Repeated cycles of phospho-

rylation at the DSB sites, which include as target the

histone H2AX, lead to further recruitment of the sen-

sor/activator proteins and the formation of promi-

nent protein foci at these sites. Deficiencies of ATM or

components of the MRN complex lead to genomic

instability syndromes such as ataxia-telangiectasia

(A-T), A-T-like disease (A-TLD) and the Nijmegen

breakage syndrome (NBS). We are investigating the

early events that precede ATM activation and are

seeking novel downstream processes represented by

new ATM substrates. Two such substrates will be dis-

cussed: the COP9 signalosome (CSN) protein complex

and the co-repressor protein KAP-1. These new func-

tional links in the web of the DNA damage response

add further dimension to the increasing complexity

and richness of this process.

S1 - Symposium 1

Oral Presentation

OS1002

DYNAMIC ORGANIZATION OF DNA DAMAGE

REPAIR PROTEINS AND CHROMOSOMES

R Kanaar

Erasmus Medical Center, ROTTERDAM,

The Netherlands

Homologous recombination, the exchange of DNA

sequence between homologous DNA molecules, is

essential for accurate genome duplication and preser-

vation of genome integrity. DNA double-strand

breaks (DSBs) and single-stranded gaps are efficient

initiators of homologous recombination, which

results in their accurate repair using an intact homol-

ogous template DNA in the same cell. Homologous

recombination requires the co-ordinated action of the

RAD52 group proteins, including Rad51, Rad52 and

Rad54. Upon treatment of mammalian cells with ion-

izing radiation, these proteins accumulate into foci at

sites of DSB induction. We probed the nature of the

DNA damage-induced foci in living cells with the use

of photobleaching techniques. These foci are not stat-

ic assemblies of DNA repair proteins. Instead, they

are dynamic structures of which Rad51 is a stable core

component, while Rad52 and Rad54 reversibly inter-

act with the structure. Furthermore, even though the

RAD52 group proteins colocalize in the DNA damage-

induced foci, the majority of the proteins are not part

of the same multi-protein complex in the absence of

DNA damage. Executing DNA transactions through

dynamic multi-protein complexes, rather than stable

holo-complexes, allows greater flexibility during the

transaction. In case of DNA repair, for example, it

allows cross talk between different DNA repair path-

ways and coupling to other DNA transactions, such as

replication.

Interactions between ends from different DSBs can

produce tumorigenic chromosome translocations.

Two theories for the juxtaposition of DSBs in translo-

cations, the static ‘contact-first’ and the dynamic

‘breakage-first’ theory, differ fundamentally in their

requirement for DSB mobility. To test whether or not

DSBs are mobile, we introduced linear tracks of DSB-

containing chromosome domains in nuclei. We

observed changes in track morphology within min-

utes after DSB induction indicating movement of the

domains. In a sub-population of cells domains

formed clusters. Juxtaposition of different DSB-con-

taining chromosome domains through clustering,

which was most extensive in G1 phase cells, suggests

an adhesion process in which we implicate the Mre11

protein complex. Our results support the ‘breakage-

first’ theory to explain the origin of chromosomal

translocations.

14


S1 - Symposium 1

Oral Presentation

OS1003

THE EFFECT OF P53 MUTATIONS ON CELL CYCLE

CONTROL AND CARCINOGEN-INDUCED TUMORI-

GENESIS

A de Vries

National Institute of Public Health and the

Environment, BILTHOVEN, The Netherlands

In vivo analysis of p53 function has been important to

confirm the importance of this gene in tumor sup-

pression. Homozygous p53 knockout mice develop

tumors with 100% incidence, primarily lymphomas.

As a more fine-tuned approach to study the role of

p53 in tumor suppression, we have generated

germline mutations in p53.

In the p53.R270H knock-in mouse model, an Arginine

to Histidine mutation was introduced in codon 270

(human 273) of the mouse p53 gene. This mutation is

a hot-spot mutation in several human and mouse

tumor types and is frequently found as a germ-line

mutation in the human Li-Fraumeni syndrome. In

addition to the mutation, a transcriptional stop-cas-

sette flanked by loxP sites was introduced in intron 1

of the p53 gene. As a result, expression of the R270H

protein can be induced through crosses with (tissue-

specific) Cre-transgenic mice. p53.R270H mice crossed

with mammary gland-specific WAP-Cre mice develop

mammary tumors with high incidence and short

latency time. In addition, chronic exposure of skin-

specific p53.R270H/ K14-Cre mice to UVB radiation

results in the same tumor phenotype. These experi-

ments show that the R270H mutation is an important

trigger for tumor development acting in a dominant-

negative manner.

Another mutation we generated is a Serine to

Alanine substitution at residue 389 (human 392).

Codon 389 is specifically phosphorylated after expo-

sure to UV radiation, whereas gamma radiation

involves phosphorylation of different residues. This

post-translational modification is envisioned to acti-

vate the p53 protein to exert its cellular functions.

Mice harboring the S389A mutation are sensitive to

UV- and 2-AAF induced tumor development, and

S389A cells display reduced p53-dependent cellular

responses. Additional in vivo and in vitro experi-

ments to further explore the phenotype of these p53

mutant mice are currently ongoing, and results

obtained will be presented.

Supported by Dutch Cancer Society and NIH/ NIEHS-

CMGCC.

S1 - Symposium 1

Oral Presentation

OS1004

TRANSLESION SYNTHESIS AND THE Y-FAMILY OF

DNA POLYMERASES

AR Lehmann, PL Kannouche

University of Sussex, BRIGHTON, United Kingdom

Most types of DNA damage block replication fork pro-

gression during DNA synthesis because replicative

DNA polymerases are unable to accommodate altered

DNA bases in their active sites. To overcome this

block, eukaryotic cells employ specialised translesion

synthesis (TLS) polymerases, which can insert

nucleotides opposite damaged bases. In particular,

TLS by DNA polymerase eta (pol eta) is the major

pathway for bypassing UV photoproducts. Pol eta is

deficient in individuals with the variant form of xero-

derma pigmentosum.

Pol eta is mostly localised uniformly in the nucleus,

but is associated with replication foci during S phase.

Following treatment of cells with UV-irradiation or

chemical carcinogens, the number of cells in which

pol eta accumulates into foci increases dramatically.

These foci are sites at which replication forks are

stalled at DNA damage. The C-terminal 120 aa are

needed for localisation in nuclei and into replication

foci. This region contains a nuclear localisation sig-

nal, a putative C2H2 zinc finger and a PCNA binding

domain, all of which are required for localisation of

pol eta into replication foci. Pol eta truncations lack-

ing the C-terminal 120 aa fail to correct the defects in

XP-variant cells.

How the cell switches from replicative to TLS poly-

merase at the site of blocked forks is unknown. In

human cells, PCNA becomes mono-ubiquitinated fol-

lowing UV-irradiation. Mono-ubiquitinated PCNA,

but not unmodified PCNA, specifically interacts with

pol eta and we have identified two motifs in pol eta

which are involved in this interaction. Our findings

provide an attractive mechanism by which mono-

ubiquitination of PCNA might mediate the switch

from replicative to TLS polymerase at the site of a

replication fork stalled at DNA damage.

15


S2 - Symposium 2

Oral Presentation

OS2001

DISCRIMINATION OF GENOTOXIC FROM NON-

GENOTOXIC CARCINOGENS BY EXPRESSION PRO-

FILING IN VITRO.

JHM van Delft, E van Agen, SGJ van Breda, MH van

Herwijnen, DM van Leeuwen, YCM Staal,

JCS Kleinjans

Maastricht University, MAASTRICHT, The Netherlands

Two general mechanisms are implicated in chemical

carcinogenesis. The first involves direct damage to

DNA, referred to as genotoxic (GTX), to which the cell

responds by repair of the damages, arrest of the cell

cycle or induction of apoptosis. The second is non-

DNA damaging, non-genotoxic (NGTX), in which a

wide variety of cellular processes may be involved.

Therefore, it is likely that modulation of the underly-

ing gene expression patterns is distinct between GTX

and NGTX carcinogens, and thus that expression pro-

filing can classify chemical carcinogens as GTX or

NGTX.

We investigated this hypothesis by analysing gene

regulation induced by 20 chemical carcinogens in

HepG2 cells with microarrays that contain 597 toxico-

logically relevant genes. A training data set was gen-

erated consisting of 16 treatments (9 GTX and 7

NGTX) and the validation set of 6 treatments (3 and

3). Several class discrimination models were applied,

based on nearest shrunken neighbours analyses.

Models were developed with data from the training

set, where after they were tested with all data. The

correct classification of the carcinogens from the

training and validation set were grossly similar,

namely 88 and 83% respectively. Exclusion of the

treatments with only marginal effects on the expres-

sion profiles, improved the correct classification for

the training and validation sets both to 100%. The

prediction strongly depends on treatment period and

type of cells used. Building a prediction model based

on 24 h treatment data, poorly predicts classes fol-

lowing a 6 h treatment. Similarly, a model based

HepG2 data, poorly predicts classes generated in

human lymphocytes. Interestingly, many of the dis-

criminating genes are involved in apoptosis and cell

cycle control, some in DNA-damage response, but

none in DNA-repair.

S2 - Symposium 2

Oral Presentation

OS2002

GENE EXPRESSION PROFILING OF GENOTOXIC

AND NON-GENOTOXIC CARCINOGENS IN RAT

LIVER

J Ahr, H Ellinger-Ziegelbauer

Bayer Health Care AG, WUPPERTAL, Germany

Application of the recently developed gene expres-

sion techniques using microarrays in toxicological

studies (toxicogenomics) facilitates the interpreta-

tion of a toxic compound´s mode of action and may,

vice versa, allow the prediction of selected toxic

effects based on gene expression changes. In order to

test this hypothesis we investigated whether carcino-

gens at doses known to induce liver tumors in the

two year rat bioassay deregulate characteristic sets of

genes in a short time in vivo study. Male Wistar rats

were treated for up to 14 days with genotoxic and

non-genotoxic carcinogens. After 1,3,7, and 14 days the

livers were taken for histopathology and for analysis

of the gene expression profiles. By using statistical

and clustering tools, characteristically deregulated

genes were extracted and then functionally classi-

fied. Both common as well as compound specific

effect with regard to the functional classes of deregu-

lated genes were observed. Common to genotoxic car-

cinogens were a DNA damage response and the

activation of proliferative and survival signaling

pathways. Non-genotoxic carcinogens generally

showed induction of oxidative stress genes as well as

signs of DNA replication and cell cycle progression. In

addition many of the gene alterations found with

non-genotoxic carcinogens imply compound specific

mechanisms. Based on this mechanistic analysis it

became evident that common gene expression

responses for both classes of carcinogens may exist.

Therefore, with gene ranking and classification algo-

rithms discriminating marker genes were identified

and evaluated for their value to classify genotoxic

versus non-genotoxic carcinogens. The results sug-

gest, that it may be possible to identify a carcinogenic

potential and to discriminate genotoxic from non

genotoxic mechanisms by applying such algorithms

on expression profiles from short time studies.

16


S2 - Symposium 2

Oral Presentation

OS2003

TOXICOGENOMIC ANALYSIS OF GENE EXPRES-

SION: AN EMERGING APPROACH FOR DIFFEREN-

TIATING GENOTOXIC MECHANISMS

J Aubrecht

Pfizer, GROTON, CT, United States of America

During the safety evaluation process of new drugs, a

battery of in vitro genotoxicity tests is conducted.

Positive results are not uncommon and their biologi-

cal relevance needs to be determined. Without this,

the risk assessment of genotoxic agents is generally

based on linear extrapolation methods, though there

is substantial evidence that some chemicals may

exhibit a clear thresholded dose-response. Hence,

gaining an insight into genotoxic mechanisms, i.e.,

differentiation of DNA-reactive vs. DNA non-reactive,

is essential for risk assessment to humans. This

results in laborious and time consuming follow-up

strategies. Genotoxic stress triggers a variety of bio-

logical responses including the transcriptional acti-

vation of genes regulating DNA repair, cell survival

and cell death. Thus, we evaluated the utility of gene

expression profile analysis in cultured cells in vitro

for gaining an insight into genotoxic mechanisms

using a set of model agents. The gene expression

changes were compared with micronucleus induc-

tion and direct DNA damage. We investigated

whether gene expression profiles can differentiate

between DNA reactive and DNA non-reactive mecha-

nisms of genotoxicity including those associated

with general toxic stress. Although, more experimen-

tal work including evaluation of a large number of

compounds and stresses is necessary to refine and

enhance our understanding of gene expression pro-

files and toxic pathways, our results suggest the

potential utility of gene expression profile analysis

for evaluating molecular mechanisms of action of

genotoxicants.

S2 - Symposium 2

Oral Presentation

OS2004

MICROARRAY EXPERIMENTAL DESIGN, QUALITY

ASPECTS OF DATA COLLECTION AND STATISTICS

TW Gant, SD Zhang

Medical Research Council, LEICESTER, United Kingdom

Biological systems, and their reactions to xenobiotic

stress, are complex. Until recently our view of this

complexity was obscured by the simplicity of the

analysis tools available. For example prior to microar-

rays the analysis methods of gene expression would

allow the determination of only a few genes in any

one study. Then the genome sequencing and high

throughput library screening projects commenced

and produced gene clones and sequence data which

previously had not been available. To exploit this new

resource the microarray was invented (1). Further

development has expanded the number of clones

contained on any one microarray a point where now

an individual researcher can determine the expres-

sion of many thousands of genes in a system in a few

days. What these data are revealing is the full com-

plexity of the gene expression response to stimuli

such as xenobiotic exposure. Toxicogenomics is seek-

ing to use the complexity of this response as a finger-

print or signature characteristic of xenobiotic

exposure (2,3). Molecular toxicology uses the detail of

individual differentially expressed genes to identify

mechanisms of toxicity. The challenge for both analy-

ses though is data quality (4). Gene expression has

always been difficult to accurately determine, a

reflection of technique deficiencies and natural

intrinsic variability. Thus statistically relevant data of

differential gene expression has always been difficult

to obtain. For microarrays this problem is multiplied

by the number of genes on the microarray itself. To

overcome this technical and experimental variability

correct experimental design is critical (5). We have

recently produced a framework for the design of

microarray experiments and statistical analysis of

microarray data (Zhang and Gant, In Press). This pres-

entation will cover aspects of this design framework

and data analysis.

1. Nature Genetics 21,15(1999)

2. Molecular Carcinogenesis 24,153(1999)

3. TIPS 23,388(2002)

4. PNAS 99,12975(2002)

5. Nature Genetics 32,490(2002)

17


S3 - Symposium 3

Oral Presentation

OS3001

STRESS, MUTATIONS AND AGING

TBL Kirkwood

University of Newcastle, NEWCASTLE UPON TYNE,

United Kingdom

As predicted by the disposable soma theory of aging,

evidence from many lines of research confirms that

senescence is a process of gradual accumulation of

damage in cells and tissues of the body, leading even-

tually to frailty and increased risk from a spectrum of

age-associated diseases. Multiple kinds of stress-

induced damage affect cells, ranging from mutations

in DNA to oxidative attack on proteins. Some of our

recent work has shown how this damage impairs the

function of tissue stem cells – specifically stem cells

of intestinal epithelium – as ageing proceeds. The key

to understanding the factors that regulate aging and

longevity is to be found in the network of cell main-

tenance systems that slow the accumulation of dam-

age. For example, long-lived species carried out repair

better than short-lived species and are endowed with

grater capacity to withstand stressors. A major chal-

lenge is therefore to understand how the diverse

mechanisms which make up this network interact

with each other. Using mathematical and computer

modeling of molecular mechanisms of aging, we are

developing a Biology of Aging e-Science Integration

and Simulation system (BASIS) to support the devel-

opment of an integrated understanding of the under-

lying cell and molecular mechanisms of age-related

frailty and disease.

S3 - Symposium 3

Oral Presentation

OS3002

GENOME INSTABILITY, AGING AND CANCER

J Vijg

University Texas Health Science Centre,

SAN ANTONIO, United States of America

Genomic instability as a consequence of the natural

limitations of DNA maintenance and repair has been

implicated as a major causal factor in both cancer and

aging. Defects in genome maintenance lead to

increased tumor formation and the premature appear-

ance of various symptoms of aging, possibly through

increased genome instability. Using a transgenic

mouse model with a chromosomally integrated lacZ

mutational target gene, we demonstrated that muta-

tions accumulate with age in an organ- and tissue-

specific manner. Depending on the organ, many of

the mutations accumulating during aging were

genome rearrangements rather than point muta-

tions, some of them involving millions of basepairs.

Furthermore, while accelerated mutation accumula-

tion was observed in mouse models of premature

aging, long-lived mutant mice, i.e., Ames dwarf mice,

were characterized by a delayed age-related increase

in mutation frequency. How can randomly accumu-

lating somatic mutations cause organ dysfunction at

old age? While only 2% of the genomic sequence

encodes proteins, almost half of it is transcribed and

possibly plays a role in the regulation of gene expres-

sion. Hence, random mutations, especially large

genome rearrangements, can have adverse effects on

normal patterns of gene regulation, which we specu-

late can result in a mosaic of cells at various stages on

a trajectory of functional decline eventually resulting

in cell death or neoplastic transformation. To further

investigate this possibility we are presently studying

single cells, directly obtained from organs of young

and old mice through enzymatic disassociation, for

changes in gene expression profiles using real-time

PCR and microarrays.

18


S3 - Symposium 3

Oral Presentation

OS3003

CROSSTALK BETWEEN TELOMERES AND DNA

DAMAGE REPAIR

MAB Blasco

Spanish National Cancer Centre (CNIO), MADRID,

Spain

Telomeres are specialized regions of the chromatin

that cap linear chromosomes. Telomere dysfunction,

either due to critical telomere shortening, or due to

loss of a proper telomere capping structure, leads to

end-to-end chromosome fusions and loss of cell via-

bility. Significantly, a number of mammalian pro-

teins have evolved dual roles in DNA repair and

telomere function. In particular, several DNA repair

proteins are components of the telomeric chromatin,

as well as have a role in processing and signaling of

dysfunctional telomeres. On the other hand, dysfunc-

tional telomeres have been shown to interfere with

proper DNA double strand break (DSB) repair, thus

modifying the sensitivity to genotoxic agents.

Significantly, telomere shortening is accelerated in

many DNA repair human syndromes, such as

Fanconi´s anemia (FA), suggesting that the proteins

involved could also be constitutive telomeric proteins

and have a role in telomere metabolism. To evaluate a

telomeric function for different DNA repair proteins,

we have studied telomere length and telomere

capping in various mouse models deficient for these

proteins.

S3 - Symposium 3

Oral Presentation

OS3004

AGING, CANCER AND CELLULAR RESPONSES TO

DNA DAMAGE

J Campisi

Lawrence Berkeley National Laboratory, BERKELEY,

United States of America

Environmentally-induced damage, particularly dam-

age to nuclear DNA, can elicit either of two cellular

tumor suppressor responses – apoptosis or cellular

senescence. Both of these responses evolved to pro-

tect complex multi-cellular organisms from develop-

ing cancer. Apoptosis culminates in cell death, and

thus protects the organism by eliminating potential

cancer cells. The senescence response, by contrast,

prevents cancer by permanently arresting the growth

of cells at risk for neoplastic transformation. In addi-

tion to suppressing cancer, several lines of evidence

suggest that both the apoptotic and senescence

responses also contribute to organismal aging and

age-related pathologies. Thus, these cellular tumor

suppressor mechanisms may be examples of evolu-

tionary antagonistic pleiotropy. That is, while these

responses protect organisms from malignant tumori-

genesis early in life, later in life – as tissues lose cells

due to apoptosis or accumulate dysfunctional senes-

cent cells – apoptosis and cellular senescence may

contribute to the age-related loss of tissue structure

and function and the development of certain age-

related pathologies. We have found that senescent

stromal fibroblasts have a striking influence on the

behavior of premalignant and normal epithelial cells.

Using the mammary gland and three dimensional

cultures as a model system, we find that senescent

fibroblasts stimulate the proliferation of mammary

epithelial cells. In addition, the senescent stroma dis-

rupts normal functional and morphological differen-

tiation of mammary epithelial cells. Together, these

findings support the idea that cellular senescence is

antagonistically pleiotropic, and that senescent cells

can contribute to age-related tissue dysfunction as

well as certain age-related pathologies, including

cancer.

19


FSAL - Fritz Sobels Award Lecture

Oral Presentation

OS3FS1

HOW RARE GENETIC DISEASES CAN HELP US

UNDERSTANDING MUTAGENESIS AND CARCINO-

GENESIS ?

AS Sarasin

Institut Gustave Roussy, VILLEJUIF, France

Cancer is a genetic disease due to the accumulation of

numerous mutations rendering the tumour cell

insensitive to control by the local environment and

by the whole organism. Analysis of the frequency of

human cancer as a function of age shows that

between 5 and 7 mutations in key genes are usually

necessary to produce most human cancers.

The major demonstration bridging the lack of DNA

repair, mutation induction and cancer development

is the existence of DNA repair-deficient diseases asso-

ciated with a very high frequency of cancer. For exam-

ple, xeroderma pigmentosum (XP) is a rare hereditary

disease recessively-transmitted. XP patients are char-

acterized by an extreme photosensitivity and a high

predisposition to skin cancer. Nucleotide excision

repair (NER) of UV-induced DNA lesions is deficient in

XP cells due mutations in one of the seven XP genes.

XP variant is another form of XP associated with a

late onset of clinical symptoms, due to increased

error-prone translesion synthesis, leading therefore,

to higher level of targeted point mutations.

Besides XP, genetic disorders on the RB or p53 genes

allowed the discovery of tumour suppressor genes.

The Fanconi's anaemia, the ataxia telangiectasia and

the SCID-like patients allowed to better understand-

ing homologous and illegitimate recombination as

well as the cascades of DNA lesion signalisation.

HNPCC patients allowed the discovery of the role of

mismatch repair in human tumours as well as on the

instability of several other repair genes. Finally,

BRCA1/2 genes and Cockayne's syndrome patients

allowed us connecting cancer-proneness or neurolo-

gical disorders to transcription-coupled repair of

oxidative lesions.

Rare genetic diseases do not concern only few thou-

sands affected patients, but represent beautiful

examples of human 'mutants' very useful for under-

standing the normal mutagenesis and carcinogene-

sis pathways leading to cancer including in the

general population.

S4 - Symposium 4

Oral Presentation

OS4001

CANCER: GENES AND THE ENVIRONMENT

KH Hemminki

DKFZ, HEIDELBERG, Germany

In the search for cancer etiology, the contribution

by heritable and environmental causes has been

debated. Cancer geneticists have emphasized the for-

mer while epidemiologists traditionally argued for

the latter. The literature on heritable cancers is full of

overstatements regarding their prevalence but recent

data from twin studies may offer scientifically based

estimates on the etiological apportioning of cancer

causation. With the emergence of single nucleotide

polymorphisms (SNPs) as versatile tools, studies on

‘gene-environment interactions’ have become popu-

lar, however with many embedded controversial

issues. Mass publication is going on and results are

reported without consideration of the functionality

of SNPs or tissue of expression of the relevant genes

in subgroups lacking any biological rationale. It is

ironical that the historical roles of epidemiologists

and geneticists appear to be completely changed: the

proponents of gene-environment interactions appear

to trust on the overwhelming importance of heritable

factors when they act in concert with environmental

factors while geneticists are raising concerns. There is

a common failure to recognize that SNPs are inherited

and that the related studies focus essentially on

heritable effects. Thus any cancer with a small

familial effect cannot show large effects in genetic

association studies on common polymorphisms.

Analogously, nesting of an association study in famil-

ial cancers is beneficial for statistical power. We hope

that those who plan association studies would take

notice of the familial risks and proportions to be

found in the literature.

20


S4 - Symposium 4

Oral Presentation

OS4002

LOW PENETRATION GENES AS RISK FACTORS IN

TOBACCO SMOKE-ASSOCIATED LARYNGEAL CAN-

CER

K Szyfter, M Gajecka, M Rydzanicz, M Wierzbicka,

K Szymoniak

Institute of Human Genetics, Pol Acad Sc, POZNAN,

Poland

Incidence of laryngeal cancer is strongly associated

with tobacco smoking and abusing of strong alco-

holic beverages. However, only a fraction of exposed

persons develops laryngeal cancer.

The attempts to identify a significance of genetic fac-

tor concern genetic polymorhism in genes coding car-

cinogen activation, detoxication and DNA repair. A

distribution of polymorhic genes and alleles was

studied in relation to CYP1A1, CYP 2E1 (activation),

GSTM1, GSTT1, GSTM3, NAT2 (detoxication) and XPD,

XRCC1, XGCC3 and OGG1 genes. Out of 11 examined

polymorhisms significant differences were estab-

lished in distribution of polymorphic variants of

CYP1A1, GSTM1 and NAT2 genes. A co-incidence of

some cancer risk variants was found as a factor mul-

tiplying a risk. A comparison of the subjects with a

single primary tumour with subjects with multiple

primary tumour has shown an involvement of the

same genes in risk estimate but an association was

stronger for MPT.

S4 - Symposium 4

Oral Presentation

OS4003

LUNG CANCER AND POLYMORPHISMS OF GENES

INVOLVED IN CARCINOGEN METABOLISM AND

DNA REPAIR

F Canzian, P Boffetta, F Gemignani, J Hall, RJ Hung,

S Landi, P Brennan

International Agency for Research on Cancer, LYON,

France

Even though lung cancer is predominantly caused by

tobacco, only a minority of smokers will develop lung

cancer. A possible explanation for this is that the

metabolization of carcinogenic products, the level of

internal dose and subsequent DNA repair and cell

cycle control mechanisms vary widely between indi-

viduals because of genetic factors.

We are investigating the role of over 100 genes which

are potentially involved in the susceptibility to lung

cancer, by means of a study conducted in six coun-

tries of Central and Eastern Europe, involving 2300

lung cancer cases and 2800 controls. This is a multi-

centric study, conducted with an identical protocol

involving the collection of high quality detailed infor-

mation on lifestyle and occupational history, as well

as blood collection for DNA extraction.

The genes comprise those involved in the metabolism

of tobacco products and other potential carcinogens

(e.g. CYPs, GSTs, MPO), as well as genes involved in

DNA repair (e.g. XRCC1, XRCC3, XPD, XPF), tumor sup-

pression (p53, p16, CCND1 ) and nicotine addiction

(dopamine D2 and D4 receptor genes).

We are genotyping 250 polymorphisms by DNA

microarrays in the subjects with young age at onset,

and a subset of 100 polymorphisms by TaqMan in the

whole sample set. Using this large sample size, we

aim at accurately measuring the overall effect of each

gene in lung cancer. Subsequently, the effect of com-

binations of genes will be measured (gene-gene

interaction), as well as the effect of individual genes

in specific subgroups identified by tobacco consump-

tion and occupational history (gene-environment

interaction). Statistical techniques include haplotype

reconstruction, empirical Bayes and semi-Bayes

analysis to control for false positive results, and mod-

eling of complex pathways. A first set of preliminary

results will be presented.

21


S4 - Symposium 4

Oral Presentation

OS4004

DESIGN AND INTERPRETATION OF STUDIES ON

GENE-ENVIRONMENT INTERACTIONS IN CAR-

CINOGENESIS

P Vineis

Imperial College London, LONDON, United Kingdom

The purpose of the presentation is to describe a spe-

cific study on gene-environment interactions, nested

within a large cohort. I will describe the practical

problems associated with the processing, storage,

and analysis of blood samples, laboratory validation

studies nested within the GenAir investigation.

Several large prospective investigations are under

way or are planned in different parts of the world,

aiming at the investigation of gene-environment

interactions for chronic diseases. Such studies are

extremely complex, involving the collection of hun-

dreds of thousands of biological samples within the

general population (e.g. 500,000 each in EPIC and UK-

Biobank). Technical, practical and ethical issues are

raised by such large investigations. I wish to describe

how such issues were dealt with within a case-con-

trol study nested in EPIC, a large European cohort, and

the kind of validation studies that have been set up.

The GenAir investigation aimed to measure the

effects of air pollution and ETS on human health in

EPIC with a nested design and with biological meas-

ures. Validation studies included (a) comparisons

between cotinine measurements, hemoglobin adducts

and questionnaire data; (b) an analysis of the deter-

minants of DNA adduct concentration; (c) comparison

among different genotyping methods; (d) an analysis

of the determinants of plasma DNA amounts.

S5 - Symposium 5

Oral Presentation

OS5001

FLAVONOIDS AND ALKENYLBENZENES: MECHA-

NISMS OF MUTAGENIC ACTION AND CARCINO-

GENIC RISK

IMCM Rietjens, GM Alink

Wageningen University, WAGENINGEN,

The Netherlands

In spite of a long history of use botanical or herb-

based preparations may contain individual ingredients

known to be toxic and even genotoxic and carcino-

genic. The present lecture focuses on two categories

of botanical ingredients, the flavonoids with quer-

cetin as an important bioactive representative, and

the alkenylbenzenes including safrole, methyl-

eugenol and estragole.

For quercetin a metabolic pathway for activation to

DNA-reactive species includes enzymatic and/or

chemical oxidation to quercetin ortho-quinone, fol-

lowed by isomerisation of the ortho-quinone to

quinone methides. These quinone methides are sug-

gested to be the active alkylating DNA-reactive inter-

mediates. Recent insights suggest that the transient

nature of quercetin quinone methide adducts

observed in vitro, may affect extrapolation of the

genotoxicity to the carcinogenicity in vivo.

For alkenylbenzenes the ultimate electrophilic and

carcinogenic metabolite is their 1'-sulfooxy-deriva-

tive. Identification of the cytochrome P450 isoenzymes

involved in bioactivation of the alkenylbenzenes iden-

tifies the groups within the population at higher risk,

due to life style factors or genetic polymorphisms.

Furthermore, a relative decrease in conversion to the

carcinogenic metabolite at lower doses, may provide

another important argument that has to be taken

into account in the risk assessment of alkenylben-

zenes.

Together the results presented stress that mechanis-

tic data have to be taken into account and that new

mechanism- and toxicokinetic-based models are

required for cancer risk extrapolation from high dose

experimental data and in vitro data to low dose car-

cinogenic risks. Also, in vitro mutagenicity studies

should put more emphasis on the reversible nature of

the DNA adducts responsible for the mutagenicity in

vitro, since the stability of the formed DNA adducts

may play an essential role in whether the genotoxicity

observed in vitro will have any impact in vivo.

22


S5 - Symposium 5

Oral Presentation

OS5002

GENOTOXICITY OF PHYTOESTROGENS

H Stopper

University of Wuerzburg, WUERZBURG, Germany

Plant extracts containing phytohormones are mar-

keted as a 'natural' medicine for many kinds of dis-

eases. On the other side, the in vivo carcinogenicity

of certain estrogens such as diethylstilbestrol is well

known. Here we review data on in vitro genotoxicity

of phytohormones. Genistein, coumestrol, quercetin,

zearalenon, and resveratrol exerted genotoxic effects

in in vitro test systems. Other phytoestrogens such as

lignans, the isoflavones daidzein and glycetein,

anthocyanidins, and the flavonol fisetin were found

to have only weak or no effects in vitro. However,

some metabolites of daidzein showed a genotoxic

activity. In hormone-responsive cells, an additional

pathway for induction of genomic damage may be

exerted through the stimulation of enhanced cell pro-

liferation, yielding cell cycle checkpoints less exact.

This has been demonstrated for estradiol, and could

now also be detected after treatment of cells with the

phytoestrogen daidzein. In conclusion, the safety of

concentrated formulations of phytoestrogens needs

to be investigated in more detail. Some concerns that

have been raised for endogenous or synthetic estro-

gens may also be true for phytoestrogens. However,

this should not lead to a reduction of a high intake of

plant food, which is still one of the best cancer pre-

vention strategies.

S5 - Symposium 5

Oral Presentation

OS5003

GENOTOXICITY OF HEAT PROCESSED FOODS

I Jagerstad

Swedish University of Agricultural Sc., UPPSALA,

Sweden

One of the first reports of food carcinogens generated

by cooking was reported in 1939 by a Swedish scien-

tist (Widmark), who prepared organic solvent extracts

of grilled horse meat and repeatedly painted them on

the skin of mice, resulting in tumours in the mam-

mary glands. During the 1960s and 1970s, much inter-

est was focussed on two new classes of chemicals

producing tumours in long-term animal studies -

polycyclic aromatic hydrocarbons (PAHs) and

N-nitrosocompounds. Since the early 1970s genotoxi-

city of cholesterol oxides also have been investigated.

In the late 1970s, a new, highly mutagenic class of

compounds, heterocyclic amines (HAs) was identified

in meat extract and grillled or broiled meat and fish.

More recently, reports on the presence of acrylamide

in a range of fried and oven-cooked foods have caused

worldwide concern because this compound has been

classified as probably carcinogenic in humans. The

occurrence in food of these mutagens/carcinogens is

very much dependent on the cooking condions and

the presence of precursors and modifiers. Following a

number of evaluations, the International Agency for

Research on Cancer (IARC) has come to the conclusion

that several of these food-borne genotoxic com-

pounds are probably or possibly carcinogenic to

human, based on both high-dose, long-term animal

studies and in vitro genotoxicity tests.Yet, there is

insufficient scientific evidence that these mutagens

really cause human cancer, and no limits have been

set for their presence in cooked foods. However, the

relevant authorities in most Western countries rec-

ommend minimising their occurrence. This presenta-

tion proviedes a brief overview of the state-of-the-art

research on some of these compounds, particularly

heterocyclic amines and acrylamide with respect to

their formation, occurrence, exposure, risk evaluation

and ways of minimising their levels in foods using

improved cooking methods and developments in

food technology.

23


S5 - Symposium 5

Oral Presentation

OS5004

NATURAL VS. SYNTHETIC CARCINOGENS: MAR-

GIN OF EXPOSURE BETWEEN RODENT CARCINO-

GENIC DOSE AND HUMAN EXPOSURE

LS Gold

UC Berkeley & LBNL, BERKELEY, United States of

America

The focus of cancer testing and regulatory policy is

synthetic chemicals; however, 99.9% of the chemicals

humans ingest are naturally-occurring. The human

diet contains an enormous background of natural

chemicals, e.g. plant pesticides and the products of

cooking, that are not a focus of carcinogenicity test-

ing. In the Carcinogenic Potency Database, half the

chemicals tested, whether natural or synthetic, are

carcinogenic; this high proportion may be due in part

to effects that are unique to high doses. Human expo-

sures to mutagens and rodent carcinogens are ubiq-

uitous.

A broad perspective on possible cancer hazards is pro-

vided by comparing the Margin of Exposure (MOE)

for a variety of humans exposures: MOE is the ratio of

the rodent carcinogenic dose of a chemical to the

human exposure (in mg/kg/day). The lower MOE, the

closer the human exposure is to the rodent carcino-

genic dose. Of the 100 human exposures investigated,

half are to chemicals that occur naturally in food.

MOE ranges a billion-fold across human exposures.

Several categories of exposure to both mutagenic and

nonmutagenic chemicals are investigated: occupa-

tional, pharmaceuticals and herbal supplements, nat-

ural pesticides, synthetic pesticide residues, cooking

and preparation of food, food additives, and environ-

mental contaminants. The lowest margins of expo-

sure are for some historically high exposures in the

workplace and some pharmaceuticals. Exposures to

natural chemicals occur throughout the range of

MOE, and pesticide residues have large MOEs. The

background of natural chemicals casts doubt on the

relative importance of low-dose exposures to syn-

thetic chemicals.

An increasing literature on mode of carcinogenic

action at the high doses used in animal cancer tests,

suggests that there are dose-dependent transitions in

the carcinogenic process and no-observed–adverse-

effect-levels for some chemicals, indicating a lack of

relevance of rodent results for assessing carcinogenic

hazards to humans at low dose.

MS - Minisymposium Biological clocks, chrono-

toxicology and therapy

Oral Presentation

OS6001

IMPLICATIONS OF BIOLOGICAL CLOCKS FOR CAN-

CER PROCESSES AND THEIR TREATMENTS

F Levi

INSERM E 0354 and Chronotherapy Unit, VILLEJUIF,

France

Genetic mutations and polymorphisms account for

differences in the susceptibility of malignant tumours

and normal tissues for anticancer medications. In

addition, most cellular and biochemical functions are

regulated at transcriptional and/or post transcrip-

tional levels by a molecular clock. As a result, 1) the

tolerability and the efficacy of anticancer agents can

vary as a function of circadian dosing time and 2) the

disruption of the circadian system can accelerate

malignant growth. Cancer chronotherapeutics involves

the adaptation of treatment delivery to circadian

rhythms and/or the administration of clock-targeted

therapies.

Circadian rhythms characterize many determinants

of anticancer drug pharmacology in bone marrow,

intestine or liver of rodents. Large 24-hour changes

were found for cell cycle, apoptosis (BCL-2 and BAX),

detoxication (dehydropyrimidine dehydrogenase,

glutathione) or DNA repair (O6 -alkylguanine-DNA

alkyltransferase) and partly related to the expression

patterns of clock genes.

The clinical relevance of cancer chronotherapeutics

has been investigated in over 2000 patients, regis-

tered in Phase I, II or III trials. Oxaliplatin safety and

antitumor activity against colorectal cancer were ini-

tially established based upon drug chronopharmacol-

ogy. In randomised clinical trials, we demonstrated

that the mucosal tolerability of 5-fluorouracil and the

neurological tolerability of oxaliplatin were improved

5-fold and 2 fold respectively, whereas the antitumor

activity was nearly doubled with chronotherapy as

compared with constant rate infusion. In two recent

EORTC trials, we confirmed the improved tolerability

of chronomodulated 5-fluorouracil and Platinum

complex as compared to constant rate infusion, and

found that 2-day infusions could ameliorate tolerabil-

ity as compared to more protracted infusional sched-

ules.

Conclusions: The circadian system plays a clinically

relevant role in the therapeutic index of cancer

treatments. Further development will stem out of a

circadian-based dynamic modelling approach of treat-

ment schedules.

Support: ARTBC, Hôpital Paul Brousse, Villejuif,

France.

24


MS - Minisymposium Biological clocks, chrono-

toxicology and therapy

Oral Presentation

OS6002

CIRCADIAN CLOCKS: NEURAL AND PERIPHERAL

PACEMAKERS

H Hastings, A Reddy, E Maywood

MRC Laboratory of Molecular Biology, CAMBRIDGE,

United Kingdom

Effective adaptation to the solar day is accomplished

by the action of internal timers; circadian clocks that

orchestrate an internal daily programme of physiolo-

gy and behaviour. The principal pacemaker is the

suprachiasmatic nuclei (SCN) of the hypothalamus.

This is entrained to the solar cycle by retinal innerva-

tion, but its ability to oscillate with a near 24-hour

period is an intrinsic property of its neurons. Signals

emanating from the SCN, both neural and neuroen-

docrine, control the activity of sub-ordinate oscilla-

tors in a wide range of peripheral tissues. The genes

encoding the SCN and peripheral oscillators have been

identified, and the core timing mechanism charac-

terised as an auto-regulatory, transcriptional/post-

translational negative feedback loop. This molecular

mechanism drives daily waves of gene expression

involving about 10% of the transcriptome of any one

tissue. The targets of the daily clock inclide cell cycle

factors, through which cell division is synchronised to

solar time.

W1 - Workshop 1

Oral Poster Presentation

OW1001

ALTERED VEGETABLE INTAKE AFFECTS PIVOTAL

CARCINOGENESIS PATHWAYS IN COLON MUCOSA

FROM ADENOMA PATIENTS AND CONTROLS

SGJ van Breda1, E van Agen1, LGJB Engels2,

JC Moonen1, JHM van Delft1

1 Maastricht University, MAASTRICHT,

The Netherlands2 Maasland Hospital, SITTARD, The Netherlands

Globally, cancer of the colon and rectum is the fourth

most common incident cancer and cause of death

from cancer. The evidence from epidemiological

and experimental studies that vegetables reduce the

risk of colorectal cancer is convincing. However, the

involved genes and genetic pathways are not clear.

Microarray technology makes it possible to assess the

effect of a specific vegetable diet on the expression of

multiple genes. Therefore, a human dietary interven-

tion study was carried out to identify the genes the

expression of which is modified in vivo in normal

human colorectal mucosa by vegetables. Twenty

female adenoma patients and eight healthy controls

were randomly split into two groups of ten and four

persons respectively, receiving either a decreased

(= 75 g/day) or increased (= 300 g/day) amount of veg-

etables for a period of two weeks. In order to assess

effects on gene expression at target level, before and

after the intervention colorectal biopsies from

healthy mucosa tissue were collected. Total RNA was

isolated from the biopsies to measure gene expres-

sion of 597 toxicologically relevant genes by means of

microarrays. Comparison of pre- and post-interven-

tion samples shows that 20 genes were modulated

which are related to (colon)carcinogenesis, including

c-fos proto-oncogene, ornithine decarboxylase and

cyclooxygenase-2. An increased intake of vegetables

resulted in down-regulation of genes promoting cell

proliferation and bioactivation of procarcinogens,

and in up-regulation of genes involved in cell growth

arrest; in contrast, a decreased intake of vegetables

resulted in down-regulation of genes inhibiting cell

growth and up-regulation of genes promoting cellu-

lar differentiation and bioactivation of procarcino-

gens. Some of the genes play a role in known colon

carcinogenic pathways and could thus be new targets

for chemoprevention.

25


W1 - Workshop 1

Oral Presentation

OW1002

THE EFFECT OF PLANT PHENOLS ON AP-1 EXPRES-

SION IN MOUSE EPIDERMIS

W Baer-Dubowska, V Krajka-Kuzniak, H Szaefer

Poznan University of Medical Sciences, POZNAN,

Poland

A wide array of phenolic substances present in edible

plants, have been reported to possess anticarcino-

genic and antimutagenic properties. These include

also polyphenol, tannic acid (TA), simple phenol, pro-

tocatechuic acid (PCA) and chlorogenic acid (CHA)

and phytoalexin 3,5,4’-trihydroxystilbene, resveratrol

(RES). In our previous studies we showed that these

potential chemopreventive compounds modulate the

12-O-tetradecanoylphorbol 13-acetate (TPA) - stimulat-

ed expression of inducible nitric oxide synthase and

cyclooxygenase-2. As the one of possible mechanism

of these activity, modulation of transcription factor,

the activator protein - 1 (AP-1) was postulated. AP-1

plays an important role as a mediator of tumour pro-

motion. AP-1 is a heterodimer formed by c-Jun and c-

Fos proteins. In the present study we evaluated the

expression of these proteins after topical application

of TPA (10 nmol per mice). Maximal expression of c-

Jun and c-Fos measured by immunoblot analysis was

observed 4 h after TPA application. Pretreatments

with TA, PCA, CHA and RES at a dose of 16 mmoles per

mice 15 minutes before the TPA application decreased

c-Jun and c-Fos expression. TA and PCA were the most

potent inhibitors of TPA – induced AP-1 activation.

These data suggest that modulation of AP-1 proteins

expression may be involved in antipromotional activ-

ity of the investigated plant phenols.

W1 - Workshop 1


OW1003

FOLIC ACID METABOLISM AND GENE POLYMOR-

PHISMS IN THE RISK OF NON-HODGKIN’S LYM-

PHOMAS

F Coppedè1, CF Skibola2, MT Smith2, MS Forrest2,

L Agana2, PM Bracci2, EA Holly2

1 University of Pisa, PISA, Italy2 University of California, BERKELEY, United States of

America

Folic acid is an important nutrient required for both

DNA synthesis and methylation. Impairments in the

synthesis of DNA could be responsible for mutations

critical for cancer outcome in rapidly dividing cells

such lymphocytes. We performed this study to evalu-

ate the role of several folate gene polymorphisms to

the risk of Non-Hodgkin’s Lymphomas (NHL). DNA

was extracted from 400 patients with NHL, and 800

healthy individuals, both groups collected in the San

Francisco Bay Area. The analysis of DNA polymor-

phisms was performed by real-time PCR for allelic

discrimination using an ABI PRISM 7700 sequencer

system. Among genes under study, thymidilate syn-

thase (TS) gene codes for an enzyme that catalyzes

the conversion of dUMP + 5,10- methylenetetrahydro-

folate (MTHF) to dTMP + dihydrofolate. A 28-bp tan-

dem repeat sequence upstream the TS gene initiation

start site is polymorphic, containing either two (2R) or

three repeats (3R); the 3R allele resulting in an in vitro

2.6-fold greater TS expression than the 2R. We also

studied a 6-bp deletion in the 3’UTR of TS gene, a

recently identified polymorphisms supposed to affect

the mRNA levels. We found that the TS 6bp++ geno-

type is associated with an increased risk of NHL.

Linkage disequilibrium was observed between the TS

28-bp 2R/3R tandem repeat and the 3’UTR 6bp dele-

tion polymorphisms (p<.001). Moreover interaction

was observed between the TS 28-bp 2R/3R tandem

repeat and the TS 6-bp deletion polymorphisms. The

TS 6bp++/3R3R genotype was associated with a

reduced risk of NHL while the 6bp--/3R3R genotype

with an increased risk. MTHF is reduced by methyl-

enetetraifolate-reductase (MTHFR) in the DNA

methylation pathway. The MTHFR C667T polymor-

phism was studied, and the 677C genotype was asso-

ciated with a reduced risk of NHL. Our data indicate

that the folic acid metabolic pathway plays an essen-

tial role in modulating the interindividual suscepti-

bility to NHL.

26


W1 - Workshop 1


OW1004

NATURAL AH RECEPTOR AGONISTS IN THE HUMAN

DIET: BENEFICIAL FOOD COMPONENTS OR UNPER-

CEIVED RISK FACTORS?

WJ de Waard1, LAP Hoogenboom2, A Peijnenburg2,

JMMJG Aarts3, TH de Kok1, FJ van Schooten1


The Netherlands2 RIKILT, WAGENINGEN, The Netherlands3 Wageningen University, WAGENINGEN,

The Netherlands

Activation of the omnipresent cytoplasmatic aromat-

ic hydrocarbon receptor (AhR) is thought to mediate

the toxic effects of dioxins like 2,3,7,8-tetrachloro-p-

dibenzodioxin (TCDD). Some fruits and vegetables

contain natural compounds which are also agonists

of the AhR, like indole-3-carbinol (I3C) in cruciferous

vegetables and furanocoumarins in citrus fruits.

However, some of these compounds are claimed to

be beneficial for human health, which promotes

increased consumption of food or pills containing

these compounds.

The objective of the project is to perform a food safe-

ty analysis of Natural AhR Agonists (NAhRA’s) in the

human diet through a genomics approach. Bioassays

measuring AhR activation (CALUX, EROD) were used

to determine the exposure level of some NAhRA’s pro-

ducing near maximum AhR activation. Microarray

gene expression profiles induced at these concentra-

tions in the human colon Caco-2 cell line by citrus

pulp and grapefruit juice extracts, and indolo[3,2-

b]carbazole (I3C metabolite) are compared to the

expression profile induced by TCDD. Benzo[a]pyrene

(B[a]P) is tested as positive control with a higher

metabolic rate compared to TCDD.

Gene expression profiles of the NAhRA’s showed

remarkable resemblance with that of TCDD and B[a]P.

Of the 29 genes up- or down-regulated more than 1.5

times by TCDD after 24 hours exposure, most genes

were regulated in the same direction by the NAhRA’s

and none in the opposite direction, and many are pos-

sibly involved in the toxic effects of TCDD.

Biomarkers will be chosen and a human intervention

study with diets of cruciferous vegetables and/or cit-

rus juices will be carried out.

W1 - Workshop 1

Oral Presentation

OW1005

NUTRIGENOMICS AND NUTRITIONAL SYSTEMS

BIOLOGY

B van Ommen

TNO Nutrition and Food Research, ZEIST,

The Netherlands

Nutritional sciences are discovering the application

of the so-called 'omics' sciences. Driven by the unrav-

elling of the human genome and its related techno-

logical developments, genotyping, transcriptomics,

proteomics and metabolomics are now available to

nutritional research. Screening tools can be devel-

oped for the selection of bioactive nutrients. New bio-

markers for the in vivo efficacy of nutrients in terms

of health promotion or disease prevention are aris-

ing. Better insights will be obtained on the influence

of genetic polymorphisms on nutrient metabolism.

However, are these promises just based on a biotech-

nological hype, or is a real fundamental change in

human nutritional sciences at hand? A new concept

of systems biology based biomarker is presented,

fully exploiting the multiple minor changes in

genomic responses (captured in patterns, profiles and

complex data sets) related to nutrition and health,

instead of single 'target' gene responses common in

drug therapy. It is concluded that systems biology

clearly provides new insights into the molecular

action of nutrients, without the need for a priori

knowledge on any mechanisms. Implications for the

development of biomarkers to predict human health

effects of nutrients are discussed.

27


W2A - Workshop 2A


OW2001

CADMIUM INHIBITS HUMAN DNA MISMATCH

REPAIR IN VIVO

LJR Rasmussen, A Lützen, SE Liberti

Roskilde University, ROSKILDE, Denmark

The heavy metal Cadmium (Cd) is a human carcino-

gen that inhibits DNA repair activities. We show that

DNA mismatch repair (MMR)-mediated cell cycle

arrest after alkylation damage is suppressed by expo-

sure to Cd and that this effect is reversed by preincu-

bation with excess of zinc (Zn). We show that

Cd-mediated inactivation of MMR activity is not

caused by disruption of complex formation between

the MMR proteins hEXO1-hMutSa and hEXO1-hMutLa

nor does Cd inhibit 5’-exonuclease activity of hEXO1 in

vitro. Thus, our studies show that exposure of human

cells to Cd suppresses MMR activity, a repair activity

known to play an important role in colon cancer and

that this effect can be reversed by Zn treatment.

W2A - Workshop 2A


OW2002

WILD-TYPE P53 SUPPRESSES NON-HOMOLOGOUS

END-JOINING OF DSB BUT NOT OVERALL REPAIR

PROFICIENCY

X Dahm-Daphi1, P Hubbe1, RA El-Awady1, SN Powell2,

H Willers2

1 University of Hamburg, HAMBURG, Germany2 Harvard Medical School, BOSTON, United States of

America

Non-homologous end-joining (NHEJ) of DNA double-

strand breaks (DSBs) can be an error-free or error-

prone repair process depending the structure of DNA

ends created. Complementary ends can be rejoined

by precise ligation (error-free or high-fidelity repair).

All non-compatible ends require DNA end modifica-

tion and hence sequence alteration prior to ligation

(error-prone). This error-prone repair may promote

chromosomal rearrangements and genomic instabil-

ity. The DNA-Pk dependent repair pathway is known

to largely control NHEJ. In this study, we asked

whether p53 can also affect NHEJ by dictating the

choice between error-free and error-prone mecha-

nisms. We applied a novel plasmid-based assay to

monitor the repair of chromosomal DSBs created by

the I-SceI endonuclease in isogenic mouse fibroblast

lines. Expression of wild-type p53 mediated a striking

inhibition of error-prone NHEJ events by > 3 orders of

magnitude compared to p53-null and mutated cells.

On the other hand, p53 appear to promote the precise

ligation of I-SceI breaks (error-free rejoining). p53 pre-

sumably exert a direct inhibitory effect through

restricting DNA end modification by blocking the

annealing of mismatched DNA strands along flank-

ing microhomologies. Importantly, p53 did not reduce

the overall levels of repair of I-SceI- or ionizing-radia-

tion-induced DSBs. We therefore conclude that p53

enhances repair fidelity without compromising

repair capacity. Our data support a model in which

p53 acts as a central regulator of recombinational

processes that maintains genomic stability by restrict-

ing both erroneous NHEJ and homologous recombina-

tion, as shown previously.

28


W2A - Workshop 2A


OW2003

IMPACT OF MISMATCH REPAIR ON UVB-INDUCED

CELL CYCLE ARREST

GJ Stout1, M van Oosten1, N de Wind1, FR de Gruijl1,

CMP Backendorf2, LHF Mullenders1

1 Leiden University Medical Center, LEIDEN,

The Netherlands2 Leiden Institute for Chemistry, LEIDEN,

The Netherlands

Nucleotide excision repair (NER), apoptosis and cell

cycle regulation are important defence mechanisms

against the carcinogenic effects of UVB radiation. The

impact of NER pathways on cell cycle progression and

apoptosis in the epidermis of UVB-exposed mice was

investigated previously in our laboratory. We then

found an UVB-induced G2 arrest in epidermal cells of

Xpc-/- mice (no GGR), but not in Xpa-/- (no TCR and no

GGR), nor wild type mice, which resolved without

apoptosis. We hypothesised that the observed G2

arrest in Xpc-/- mice is caused by replication of

genomic sequences containing large numbers of

lesions, giving rise to compound lesions ((mis-)incor-

porated bases opposite a lesion). We suggested that

compound lesions could be recognised by the mis-

match repair (MMR) giving rise to futile cycles of

breakage and resynthesis, which might be the signal

for a G2 arrest. Cell cycle checkpoint kinases may well

be involved in this UV-induced cell cycle arrest.

The present experiments show that the MMR system

indeed plays a role in the generation of the UVB-

induced G2-arrest in keratinocytes of Xpc-/- mice; in

cell cycle analysis with two independent we observed

17% G2-phase cells in Xpc-/-, compared to 11% in Xpc-

/-Msh2-/- mouse epidermis. We confirmed these

results in a recently developed in vitro system

analysing independent Xpc-/- and Xpc-/-Msh2-/-

deficient keratinocyte cell lines. Additional analysis

of artificially (calyculin A) induced premature chro-

mosome condensation of the cultured Xpc-/- ker-

atinocytes indicated that the delayed arrest of 4N

cells occurred in late-S instead of G2-phase.

Furthermore, in vitro pulse-chase experiments

revealed that, unlike Xpc-/-, Xpc-/-Msh2-/- cells finish

the cell division and re-enter G1. More interestingly,

Xpc-/-Msh2-/- cells display less UV sensitivity as com-

pared to Xpc-/- cells.

Based on these findings we suggest that mismatch

repair plays a role in the DNA damage S-phase check-

point.

W2A - Workshop 2A


OW2004

BYSTANDER APOPTOSIS INDUCED IN NON-IRRA-

DIATED CELLS NEEDS OF CASPASE 8 ACTIVITY

L Celotti, M Grifalconi, S Canova, M Mognato

University of Padova, PADOVA, Italy

Non-targeted responses to ionising radiation include

the bystander response, where cells neighbouring

those, which have been irradiated, respond to signals

released from the irradiated cells. Our study focused

on the apoptotic response induced in non-irradiated

TK6 cells by the medium from irradiated cells, with

the aim to analyse the mechanisms by which the

response has been activated. Culture medium was

renewed immediately after g-ray irradiation. From

irradiated and unirradiated cells the medium was

poured off donor flasks 6 h after irradiation, filtered

through a 0.22-mm filter and used to treat unirradiat-

ed TK6 cells. For detection of apoptotic morphology,

fixed cells were stained with DAPI 3.5 µg/µl in an

antifade solution which prevents fluorescence decay.

At least 2000 cells were scored for each time- and

dose-point by using fluorescence microscopy. Control

samples were treated with medium from unirradiat-

ed cells. Caspase-8 inhibitor (z-IETD-fmk) was added

to unirradiated cells to evaluate the involvement of

caspase-8 activity into inducing bystander apoptosis.

A significant increase of apoptotic index was observed

in TK6 cells after addition of medium from irradiated

cells, as well as in directly irradiated cells. No or little

increase of apoptotic index was measured when the

conditioned medium was added together with cas-

pase-8 inhibitor. z-IETD-fmk has no effect on apopto-

sis induction when added together with medium

g-irradiated without cells.

Our data suggest that the bystander signal, which

induces the apoptotic response in unirradiated cells,

needs of procaspase 8 activation. Moreover, the

bystander signal does not consist in reactive oxygen

species directly generated by irradiation, but derives

from the metabolism of irradiated cells.

As many indications exist on the bystander respons-

es in cellular models after exposure to low-LET radia-

tion, it is very important to take into consideration

this effect when radiation risk in environmental and

therapeutic exposures was evaluated.

29


W2B - Workshop2B


OW2005

GENE EXPRESSION PROFILES REVEALED P53R2

PROTEIN PLAYS A ROLE IN RADIATION-INDUCED

MUTAGENESIS

EY Chuang1, M-H Tsai1, X Chen1, CVR Gadisetti1,

CN Coleman1, JB Mitchell1, Y Chen2, HL Liber3, H Yan1,

S Zhao1

1 NCI/NIH, BETHESDA, United States of America2 NHGRI/NIH, BETHESDA, United States of America3 Colorado State University, FORT COLLINS, United

States of America

The p53 protein has been implicated in multiple cel-

lular responses related to DNA damage, including

apoptosis, cell cycle control, as well as DNA replica-

tion, transcription, and repair. Alterations in any of

these processes could be related to increased genomic

instability. Our previous study indicated that the lack

of wild-type p53 does not lead to increased mutabili-

ty. To investigate further how p53 is involved in regu-

lating mutational processes, we used 8K cDNA

microarrays to compare the patterns of gene expres-

sion among three closely related human cell lines

including TK6 (wild-type p53), NH32 (p53-null), and

WTK1 (mutant p53). Total RNA samples were collected

at different time points (1, 3, 6, 9, and 24h) after 10Gy

gamma-ray. After template-based clustering analysis

of the gene expression over the time course, our

results showed that the gene expression profiles

among these three cell lines revealed distinct pat-

terns after 10Gy irradiation. Furthermore, we found

several genes associated with DNA repair such as

DDB2, p53R2, XPC, PCNA, BTG2 and MSH2 were highly

induced in TK6 compared to WTK1 and NH32. Among

these DNA repair related genes, p53R2 showed the

highest expression level in TK6 cells. p53R2, which is

regulated by tumor suppressor p53, is a small subunit

of ribonucleotide reductase. To determine whether it

is involved in radiation-induced mutagenesis, p53R2

protein was knocked down by siRNA in TK6 cells fol-

lowing by 2Gy gamma-ray irradiation. The mutation

frequencies of siRNA transfected TK6 cells were

approximately equal as untreated TK6 cells at the TK

locus. Whereas, the siRNA transfected TK6 cells after

2Gy radiation were much more sensitive to gamma-

ray-induced mutation than the irradiated TK6 cells

without p53R2 knock down. Our result indicated that

p53R2 is induced by p53 protein and plays a pivotal

role in mutagenesis by repairing damaged DNA in

the nucleus.

W2B - Workshop2B


OW2006

EXTENT OF P53 BINDING TO TARGET GENES IN

VIVO AND THEIR EXPRESSION

T Menichini, R Magrini, D Russo, P Tamagno,

G Fronza

National Institute for Cancer Research, IST, GENOVA,

Italy

P53 is found mutated in about 50% of all tumor types.

Since p53 plays a crucial role in the activation of genes

important for cell cycle arrest, DNA repair and apop-

tosis, the question of which functions are affected

and which are retained by any p53 mutant protein is

an important issue in terms of the significance of p53

mutations in cancer.

Our aim was to determine the extent of binding of

wild type and p53 mutant proteins to p21, bax and

mdm2 promoters in vivo by Chromatin Immuno-

precipitation (ChIP) and, then, make a correlation with

the expression of the same genes by RT-PCR. Human

tumor cell lines carrying p53WT (A549), p53R280K

(SKMes1) and p53R273H (LX1) were used. Wild type p53

was induced by UV irradiation.

Our results show that in cells carrying p53WT the

binding to p21 and mdm2 promoters increased after

UV irradiation. For p21 there was correlation between

binding and expression. For mdm2 we did not observe

a relative increase in gene expression. The two mutat-

ed p53 proteins were not able to significantly bind p21

and mdm2 promoters in vivo and the low expression

of these genes correlated with binding data. Clearly,

for all cell lines the expression of bax was not p53-

binding dependent.

It has been shown that when p53 binds to the respon-

sive element of p21, the acetylation of histones bound

to its promoters, may influence p21 induction. Thus, to

measure histone acetylation at the p21 and mdm2

promoters before and after UV irradiation, we are

using ChIP with antibodies that recognize multiple

acetylated forms of H3 and H4 histones. Preliminary

results seem to indicate that the acetylation of H3 and

H4 histones is different at each promoter.

30


W2A - Workshop 2A

Oral Presentation

OW2007

DNA TRANSLESION SYNTHESIS AND MISMATCH

REPAIR IN THE RESPONSE TO ENDOGENOUS AND

EXOGENOUS DNA DAMAGE

N de Wind, V Borgdorff, JG Jansen, A Shtylik

Leiden University Medical Center, LEIDEN,

The Netherlands

The control of mutagenesis is seminal for the mainte-

nance of genomic integrity and therefore for averting

cancer and inherited disease. In mammals, two path-

ways determine DNA damage-induced and ‘sponta-

neous’ mutagenesis. DNA translesion synthesis (TLS)

polymerases introduce misincorporations while

replicating DNA damage. DNA mismatch repair

(MMR) recognizes and removes nucleotides, misin-

corporated by replicative DNA polymerases.

We have generated mouse cells and mice, mutated for

TLS polymerases and/or for MMR and have used these

to investigate interactions between MMR and TLS in

DNA damage responses including spontaneous and

induced mutagenesis.

Embryonic stem (ES) cells carrying a deletion in the

Rev1 TLS polymerase display reduced spontaneous

and short-wave Ultraviolet C (UVC)-induced point

mutagenesis, implying a role for the protein in repli-

cating both endogenous and exogenous (UVC-

induced) DNA damage. In contrast, exposure of ES

cells, deficient for the Msh2 MMR gene, to UVC results

in significantly higher mutation induction than in

wildtype ES cells. This suggests that MMR counter-

acts misincorporations introduced by TLS opposite

UVC-induced pyrimidine dimers. Truncation of the

Rev3 TLS polymerase results in embryonic growth

retardation, apoptosis and midterm lethality indicat-

ing an essential role of Rev3 in replicating endoge-

nous DNA damage. Remarkably, introduction of MMR

deficiency results in significant alleviation of the

Rev3 embryonic phenotype demonstrating that

MMR-dependent processing of DNA damage, rather

than the Rev3 deficiency itself, causes the apoptotic

response. We infer that the absence of Rev3 results in

replication of endogenous DNA damage by another,

more error-prone TLS polymerase. We hypothesize

that processing of these so-called compound lesions

by MMR results in either the repair of the TLS poly-

merase-induced misincorporations, or in provoking

an apoptotic response. We conclude that the action of

both MMR and TLS is essential in determining cellu-

lar responses to endogenous and exogenous DNA

damage.

W3 - Workshop 3


OW3001

GENE-ENVIRONMENT INTERACTIONS AND GENET-

IC DAMAGE INDUCTION BY ETS: THE IMPORTANCE

OF EXPOSURE LEVELS

SA Kyrtopoulos1, P Georgiadis1, J Topinka2,

D Vlachodimitropoulos3, G Stephanou1, M Stoikidou4,

H Autrup5, N Demopoulos3, K Katsouyianni4,

RJ Sram2

1 National Hellenic Research Foundation, ATHENS,

Greece2 Institute of Experimental Medicine AS CR, PRAGUE,

Czech Republic3 University of Patras, PATRAS, Greece4 Univ. of Athens Med. School, ATHENS, Greece5 University of Aarhus, AARHUS, Denmark

Current evidence regarding the influence of genetic

variation on genetic damage associated with environ-

mental tobacco smoke is contradictory. In the context of

the AULIS project, we examined the effects of different

CYP1A1, CYP1B1, GSTM1, GSTP1 and mEH genotypes on

lymphocyte bulky DNA adducts and % aberrant cells in

194 non-smokers, in whom recent personal exposure to

airborne PAH and ETS were also measured.

The levels of both biomarkers were found to respond, in

a parallel fashion to changes in exposure/ CYP1A1*2A or

CYP1A1*2B genotype combinations: Specifically, regard-

less of season, in subjects exposed to ETS for more than

3.25 hours during the last 4 days and to airborne B[a]P

within the range 0.28-0.93 ng/m3, biomarker levels

were increased (as compared to CYP1A1*1 carriers) in

CYP1A1*2A and combined CYP1A1*2A/ *2B carriers.

Outside these exposure limits, the differential effect in

CYP1A1*2 variants was lost. On the other hand, in the

same B[a]P exposure range and in subjects with ETS

exposure below the abovementioned level, biomarker

levels were consistently reduced in CYP1B1*2 carriers,

and enhanced in with CYP1B1*3/*4 carriers.

Genetic variations in mEH, GSTM1 and GSTP1 individual-

ly had no or limited effects on biomarker levels.

However, they enhanced significantly the effects of

CYP1A1 or CYP1B1 polymorphisms described above.

These results are interpreted as indicating

a) differential induction of CYP1A1 expression, and asso-

ciated DNA and chromosome damage, in CYP1A1*2A and

CYP1A1*2A/*2B carriers, by components of ETS-polluted

air at levels readily encountered by large sections of the

general population,

b) that, under conditions where CYP1A1 induction is

unlikely, CYP1B1-mediated metabolism dominates the

induction of genetic damage, and

c) that subjects with CYP1A1*2A and CYP1A1*2A/*2B car-

riers may have increased susceptibility to the genotoxic

effects of ETS.

The AULIS project was supported by the European

Union (ENV4V-CT96-0203 and IC20-CT96-0063). 31


W3 - Workshop 3


OW3002

POLYMORPHISMS IN DNA REPAIR GENES AND

RISK OF LUNG CANCER IN A DANISH PROSPEC-

TIVE COHORT

U Vogel1, BA Nexo2, H Autrup3, M Sorensen4, H Bak4,

H Wallin1, K Overvad5, A Tjønneland5, O Raaschou-

Nielsen4

1 National Institute of Occupational Health, COPEN-

HAGEN, Denmark2 Institute of Human Genetics, AARHUS, Denmark3 University of Aarhus, AARHUS, Denmark4 The Danish Cancer Society, COPENHAGEN,

Denmark5 Aalborg Hospital, AALBORG, Denmark

We have investigated the occurrence of lung cancer in

relation to polymorphisms in DNA repair genes in

case-cohort study nested in the prospective cohort

‘Diet, Cancer and Health’. Among 54,220 members of

the Danish prospective cohort study aged 50 to 65 at

entry, 265 lung cancer cases were identified and a

sub-cohort, matched by age, sex and duration of

smoking, comprising of 272 individuals was used for

comparison. 90 % of both cases and participants in

the comparison group were ever-smokers. The poly-

morphism XPA A-23G, XPC Lys939Gln and XPD

Lys751Gln in NER genes were associated with risk of

lung cancer. Carriers of risk alleles in two NER genes

were at higher risk of lung cancer than homozygous

carriers of the wild type alleles. The polymorphism

XRCC3 Thr241Met in the double strand DNA repair

gene XRCC3 was also associated with risk of lung can-

cer, whereas no associations were found with poly-

morphisms in the BER genes XRCC1 and OGG1. DNA

adduct levels were quantified by 32P-postlabelling

and associations between polymorphisms and DNA

adduct levels will be presented at the meeting.

W3 - Workshop 3


OW3003

CYTOGENETIC BIOMARKERS AND HUMAN CAN-

CER RISK (CANCERRISKBIOMARKERS)

H Norppa1, S Bonassi2, M Fenech3, I-L Hansteen4,

L Hagmar5, P Rössner6, C Lindholm7, M Kirsch-Volders8,

S Gundy9, J Lazutka10, A Cebulska-Wasilewska11,

E Fabiánová12, RJ Sram13, LE Knudsen14, R Barale15,

GJ Köteles16, E Mirkova17, A Fucic18, P Boffetta19

1 Finnish Institute of Occupational Health,

HELSINKI, Finland2 National Cancer Research Institute, GENOA, Italy3 CSIRO, ADELAIDE, Australia4 Telemark Hospital, SKIEN, Norway5 Lund University, LUND, Sweden6 Natl Inst of Public Health, PRAGUE, Czech Republic7 STUK, HELSINKI, Finland8 Vrije Universiteit Brussels, BRUSSELS, Belgium9 National Institute of Oncology, BUDAPEST, Hungary10 Vilnius University, VILNIUS, Lithuania11 Jagiellonian Univ Med College, KRAKOW, Poland12 Sate Health Institute, BANSKA BYSTRICA, Slovak

Republic13 Institute of Experimental Medicine AS CR,

PRAGUE, Czech Republic14 University of Copenhagen, COPENHAGEN, Denmark15 University of Pisa, PISA, Italy16 NRIRR, BUDAPEST, Hungary17 NCHMEN, SOFIA, Bulgaria18 Institute for Med Res Occup Health, ZAGREB,

Croatia19 International Agency for Research on Cancer,

LYON, France

Cytogenetic analyses of peripheral blood lympho-

cytes have been used for decades to show the geno-

toxic effects of chemical and radiation exposure in

humans. Nordic-Italian studies have revealed that a

high frequency of chromosome aberrations (CAs), but

not of sister chromatid exchanges (SCEs) or micronu-

clei (MN), in lymphocytes is predictive of an increased

cancer risk. Individual exposure assessment of the

cancer cases and their matched healthy controls indi-

cated that the association between CAs and cancer

risk was not explained by smoking or known occupa-

tional exposure to carcinogens. High CA level was

associated with increased cancer risk also in non-

smokers and in subjects with no history of occupa-

tional contact with carcinogens. Thus, high CA

frequency appeared to predict increased cancer risk

regardless of the reason for the elevated CAs.

Furthermore, the time between CA analysis and can-

cer outcome did not influence the association, sug-

gesting that the findings are not explained by

32


undetected cancer. An international collaborative

study (CancerRiskBiomarkers, QLK4-CT-2000-00628)

is presently going on to further characterise the rela-

tionship between cytogenetic biomarkers and cancer.

The project aims at clarifying, e.g., the cancer risk

predictivity of cytogenetic biomarkers in several new

European cohorts, the role of genetic polymorphisms

of carcinogen metabolism, DNA repair and folate

metabolism, the classes of chromosome alterations

having cancer predictive value, and the types of can-

cer predicted. The project also cooperates with the

International Collaborative Project on Micronucleus

Frequency in Human Populations (HUMN), to assess

the possible cancer risk predictivity of the cytokine-

sis-block MN assay.

W3 - Workshop 3


OW3004

INFLUENCE OF GSTM1, GSTT1, GSTP1 AND NAT2

GENOTYPES ON P53 MUTATIONAL SPECTRUM IN

BLADDER TUMOURS

CM Ryk1, P Berggren1, R Kumar2, P Larsson3,

G Steineck3, B Lambert1, S Hou1

1 Karolinska Institute, HUDDINGE, Sweden2 German Cancer Research Center, HEIDELBERG,

Germany3 Karolinska Hospital, STOCKHOLM, Sweden

Genetic polymorphisms affecting expression or activity

of the corresponding enzymes can influence the risk

of acquiring gene mutations and various cancers. We

have studied 327 bladder cancer patients with regard

to the functionally related polymorphisms of GSTM1,

GSTT1, GSTP1 and NAT2, and analysed the p53 muta-

tional status of their tumours. 50 p53 mutations, 26%

transversions and 74% transitions, were detected in

44 patients. P53 mutation frequency was significantly

higher in advanced than in less advanced tumours

(P<0.002). Also, patients with at least one variant

GSTP1 allele (Ile105Val) tended to have more advanced

tumours (P=0.057, Fisher’s exact test). Overall, there

was no significant difference in frequency of p53

mutation among patients with different genotypes.

With regard to the possible influence of genetic poly-

morphisms on the p53 mutational spectrum, several

trends were observed. Among patients with p53

mutation, transversions were significantly more fre-

quent in GSTM1 negative as compared to GSTM1 posi-

tive individuals (OR 5.18, CI 1.07-25.02). With one

exception, all tumours with the most common type

of transversion, G:C-C:G, occurred in GSTM1 negative

patients. Among smokers, carriers of the GSTP1 vari-

ant allele had a higher proportion of transversions

than non-carriers (P=0.022, Fisher’s exact test).

Samples carrying at least one variant GSTP1 allele had

more transitions at CpG sites than wild type samples

(OR 4.61, CI 0.82-26.04). No significant associations

were found for the NAT2 gene. Our results suggest

that impaired glutathione conjugation may affect the

mutation spectrum in critical target genes.

33


W3 - Workshop 3

Oral Presentation

OW3005

MOLECULAR EPIDEMIOLOGY OF SPORADIC

BREAST CANCER: POLYMORPHISMS IN THE

XENOBIOTIC METABOLISM AND DNA REPAIR

GENES

A Hirvonen

Finnish Institute of Occupational Health, HELSINKI,

Finland

The major known risk factors for female breast can-

cer are associated with prolonged exposure to

increased levels of oestrogen. The predominant theory

relates to effects of oestrogen on cell growth. Enhanced

cell proliferation, induced either by endogenous or

exogenous oestrogens, increases the number of cell

divisions and thereby the possibility for mutation.

However, current evidence also supports a role for

oxidative metabolites, in particular catechol oestro-

gens, in the initation of breast cancer. As observed in

drug and chemical metabolism, there is considerable

interindividual variability (polymorphism) in the

conjugation pathways of both oestrogen and catechol

oestrogens. These person-to-person differences,

which are attributed to polymorphisms in the genes

encoding for the respective xenobiotic metabolizing

enzymes, might define subpopulations of women

with higher lifetime exposure to hormone-depend-

ent growth promotion, or to cellular damage from

particular oestrogens and/or oestrogen metabolites.

Such variation could explain a portion of the cancer

susceptibility associated with reproductive effects

and hormone exposure. On the other hand, DNA

repair plays a central role in protecting the genome

against insults by genotoxic cancer-causing agents

such as tobacco smoke. Accordingly, it has been sug-

gested that individuals with reduced DNA repair

capacities might have altered risk of developing envi-

ronmentally induced malignancies, including breast

cancer. This presentation will give an overview of the

hitherto performed studies on the role of polymor-

phisms in genes coding for enzymes involved in

xenobiotic metabolism and DNA repair in individual

susceptibility to sporadic breast cancer.

W4 - Workshop 4


OW4001

DNA DAMAGE BY RESPIRABLE QUARTZ PARTICLES

IN RAT LUNG TARGET CELLS: THE ROLE OF MITO-

CHONDRIA

H Li, R Duffin, C Albrecht, T Shi, J Eicker, D Höhr,

PJA Borm, RPF Schins

IUF GmbH Düsseldorf, DUSSELDORF, Germany

Respirable quartz dust has been classified as human

carcinogen by the International Agency for Research

on Cancer (IARC), and chronic inhalation studies in

rats indicate that alveolar type II cells are key target

cells for quartz carcinogenesis. The aim of our studies

is to investigate the mechanism of DNA damage by

quartz. Quartz particles were found to elicit a dose-

dependent increase in DNA strand breakage (comet

assay) in primary rat lung epithelial cells as well as in

the rat lung cell line RLE. Using electron spin reso-

nance (ESR) with spin trapping we demonstrated that

quartz particles can generate hydroxyl-radicals and

immunohistochemical analysis showed that quartz

induces the formation of 8-hydroxydeoxuanosine.

However, transmission electron microscopy analysis

did not reveal quartz particles within the nucleus, e.g.

in close proximity with the nuclear DNA, indicating

that the observed DNA damage does not result form

particle surface-derived hydroxyl radicals directly.

Interestingly, DNA strand breakage by quartz could

be reduced in the presence of the mitochondrial

inhibitors rotenone, antimycin-A or tenoyltrifluo-

roacetone (TTFA). These inhibitors did not affect DNA

damage, but led to a reduced oxygen consumption by

the cells. Our observations suggest a role for the res-

piratory chain function in quartz-induced DNA dam-

age, but the exact mechanism remains to be

elucidated. This study is supported by the DFG

International Graduate College 738.

34


W4 - Workshop 4


OW4002

AN HYPOTHESIS ON THE ROLE OF OXIDATIVE

DAMAGE IN NEURODEGENERATIVE DISEASES

M Migliore, I Fontana, R Colognato, F Coppedè,

G Tognoni, G Siciliano

University of Pisa, PISA, Italy

Oxidative stress plays a key role in the degenerative

neuronal death and progression of many neurode-

generative diseases, such as Alzheimer’s disease (AD)

and Parkinson's disease (PD), although it is not clear if

it is the primary triggering event in the pathogenesis

of these disorders.

We have previously found chromosomal, primary

and oxidative DNA damage in peripheral lympho-

cytes of PD patients (Migliore et al., 2002). The present

study was performed on a group of AD patients and a

group of mild cognitive impairment (MCI) patients,

beeing the latter a clinical condition between normal

aging and AD, characterized by a memory deficit

without loss of general cognitive and functional abil-

ities. We performed this study by comet assay to eval-

uate the level of oxidative DNA damage in two groups

of MCI and AD patients, respectively, compared to

healthy controls. Data showed a significantly higher

level of primary DNA damage in leukocytes of AD and

also of MCI patients than in control individuals (aver-

age: 2.09±0.79 and 2.47±1.01, respectively for AD and

MCI, vs. 1.04±0.31). Moreover the amount of oxidised

DNA bases (either purines and pyrimidines) was

higher in the two groups of patients (AD and MCI)

compared with controls (p< 0.002 and p< 0.001 for

purines and pirimidines, respectively). Our results

give a further indication that oxidative stress, at least

at the DNA level, is an earlier event in the pathogene-

sis of AD.

W4 - Workshop 4


OW4003

THE MULTIPLE PHYSIOLOGICAL AND PATHOLOGI-

CAL CONSEQUENCES OF OXIDATIVE DNA DAMAGE

AI Izzotti, C Cartiglia, M. Longobardi, A Camoirano, M

Bagnasco, E Tampa, A Merello, S de Flora

University of Genoa, GENOA, Italy

We evaluated the consequences of oxidative DNA-

damage (ODD) in various experimental conditions by

monitoring 8-hydroxy-2’-deoxyguanosine by 32P-

postlabelling and gene-expression by cDNA array.

In mouse foetal liver, the low basal level of ODD was

increased following transplacental exposure to ciga-

rette smoke. The foetus counteracted ODD by increas-

ing the expression of genes inhibiting cell replication

and triggering apoptosis (FASEB J.,17:1127-9,2003).

Accordingly, smoke-induced ODD in foetus resulted

in growth retardation.

During the foetus-newborn transition, the acquisi-

tion of independent respiratory function triggered the

expression of genes involved in the removal of oxi-

dized proteins and detoxification of oxidative species

in mouse lung (Mutat.Res.Rev.,544:441-9,2004). These

alterations were attenuated by administering the

antioxidant N-acetyl-L-cysteine to the dams during

pregnancy.

A main problem of postgenomic analysis is whether

or not gene expression reflects protein amounts. We

demonstrated that the relationship between cDNA-

and antibody-microarray data in rat lung is not sig-

nificant under basal conditions, but becomes highly

significant in case of exposure to chromium(VI), an

ODD-inducing agent (Int.J.Oncol.,24,2004, in press).

In humans, we found that ODD is consistently

detectable in the aorta of atherosclerotic patients

(FASEB J.,11:1021-31,1997), being 4-fold higher in the

endothelium than in the medium layer.

To substantiate the hypothesis that ODD is related to

various chronic-degenerative diseases, we analyzed

the level of ODD in patients affected by primary open

angle glaucoma, the main cause of irreversible blind-

ness worldwide. We found that ODD is significantly

increased in the trabecular meshwork, the specialized

epithelium regulating the intraocular pressure, of

glaucoma patients as compared to unaffected con-

trols (Am.J.Med,114:638-46,2003). This situation leads

to an increase of the intraocular pressure resulting in

optic nerve alterations and visual field defects.

Altogether, these data support the hypothesis that

ODD is involved in a variety of physiological process

(e.g., birth) and pathological conditions (e.g., cancer,

atherosclerosis, glaucoma).

35


W4 - Workshop 4


OW4004

PARP1 TOGETHER WITH CSB AND XPA ACCELER-

ATES THE GLOBAL REPAIR OF OXIDATIVE DNA

BASE DAMAGE

CF Flohr1, I Schulz1, A Bürkle2, JP Radicella3, B Epe1

1 University of Mainz, MAINZ, Germany2 University of Konstanz, KONSTANZ, Germany3 CEA, FONTENAY-AUX-ROSES, France

In mammalian cells, oxidative DNA base modifica-

tions are repaired by the base excision repair (BER)

pathway. Whereas, in vitro only a few proteins are

necessary to accomplish BER, several other 'accessory'

proteins seem to be important for efficient DNA

repair in vivo. To elucidate the functions of those

accessory proteins on the repair of oxidative DNA

base modifications, we studied the repair rates of

oxidative purine modifications including 8-hydrox-

yguanine (8-oxoG) in cell lines defective in various

proteins, i.e. poly(ADP-ribose)polymerase 1 (PARP1),

Cockayne syndrome B (CSB), Xeroderma pigmentosum

A protein (XPA) and XRCC1. Low levels of 8-oxoG and

other oxidative purine modifications were induced

by the photosensitizer Ro 19-8022 plus visible light,

and their removal was followed by means of a modi-

fied alkaline elution assay in combination with the

repair glycosylase Fpg protein as a probe.

Repair rates of the induced oxidative DNA base modi-

fications were significantly slower in PARP-deficient,

CSB-deficient and XPA-deficient cells compared to

wild type cells. This retardation of BER could not be

observed in a cell-free assay measuring the incision

at an oligonucleotide containing a single 8-oxoG.

Interestingly, in cells that are deficient in CSB and

XPA, an additional inhibition of PARP activity did not

further reduce the repair rates. The loss of the plat-

form protein XRCC1 did not influence the repair rates

of oxidative base modifications, although the reseal-

ing of induced SSB was significantly slowed down.

Furthermore, in XRCC1 deficient cells the repair of

oxidative base modifications was reduced when PARP1

was inhibited.

The results suggest that PARP, CSB and XPA are

involved in a repair mechanism that stimulates BER

in vivo, possibly initiated by interaction with the

stalled transcription machinery. Details of the novel

pathway remain to be established.

W4 - Workshop 4

Oral Presentation

OW4005

GENE EXPRESSION CHANGES AS BIOMARKERS OF

OXIDATIVE DAMAGE

AM Lynch

GlaxoSmithKline, WARE, HERTS, United Kingdom

The differential gene expression (DGE) of selected

genes as part of the cellular response to oxidative

stress may be exploited to provide novel biomarkers

which may be predictive of developing toxicity with-

in a wide variety of tissues and organs. To test this

hypothesis, we have measured gene expression

changes in response to oxidative stress in vitro and in

vivo following treatment with various, well charac-

terised, reference compounds, using:

1. Real Time RT-PCR (TaqMan) in cultured Hep-G2

cells (Morgan et al, Toxicol Pathol. 30: 435-451,

2002),

2. Biophotonic imaging in HO-1.luc mice, transgenic

for the murine haem oxygenase-1 (HO-1) promoter

coupled to a luciferase reporter gene (Speight et al,

Mutagenesis 17: 569, 2002) and

3. RT-PCR to measure hepatic DGE in Sprague Dawley

rats.

In addition, traditional endpoints of toxicity were

assessed in parallel. These included the measurement

of the ratio of reduced (GSH) to oxidized (GSSG) glu-

tathione (GSH:GSSG ratio) in HepG2 cells and Sprague

Dawley rats. Clinical chemistry and pathology end-

points were also assessed in Sprague Dawley rats and

transgenic HO-1.luc mice along with RT-PCR analysis

of endogenous HO-1. The results of these studies con-

firm that real-time site-specific gene expression (or

expression of the transgene in the mouse model) may

be used as predictors of developing toxicity.

36


W5 - Workshop 5


OW5001

RAT LIVER TRANSCRIPTOMICS AFTER 28-DAY

EXPOSURE TO BENZENE AND TRICHLOROETHYL-

ENE (MIXTURES)

WHM Heijne, B van Ommen, JP Groten, M Bart,

D Jonker, AP Freidig, RH Stierum


The Netherlands

Trichloroethylene (TCE) is hepatotoxic, nephrotoxic,

and a suspected carcinogen. Benzene (B) is hemato-

toxic and myelotoxic, and can provoke leukemia. The

response to benzene, trichloroethylene (TCE) and

binary mixtures was analyzed in detail at the gene

expression level in liver. In the first study, rats were

dosed with B or TCE at three dose levels for 28-days.

Both compounds exerted (modest) toxic effects in

liver, which is not the primary target organ for toxic-

ity. Besides routine toxicological examinations,

oligonucleotide microarrays were used to find more

sensitive markers of effects and elucidate mecha-

nisms of response to B and TCE. Additionally, metabo-

lite profiling was used to analyse compound and dose

specific metabolite contents in urine. Transcriptome

data revealed compound-specific effects that enabled

to hypothesise on the mechanisms of (combined)

action of B and TCE. Drug-metabolizing enzymes and

genes involved in other biological processes like gly-

colysis and fatty acid metabolism were induced or

repressed by B, TCE or by both. Possible implications

of liver gene expression for toxic and carcinogenic

effects in other organs are discussed.

In a subsequent study, B and TCE and mixtures were

given to rats at two dose levels. Routine toxicological

examinations did not reveal marked hepatotoxicity.

Individual livers were analyzed using oliognucleotide

microarrays, testing the expression of around 5000

genes. Differences and similarities were observed in

expression changes by B and TCE, and for each gene,

the effect of the mixtures of B and TCE was deter-

mined. Many enyzmes responsible for the bioactivation

of B and TCE in liver were differentially expressed by the

treatments, with possible implications for the expo-

sures to mixtures. Genes important in diverse cellular

pathways were deregulated, and possible implica-

tions of combined action for toxicity are discussed.

W5 - Workshop 5


OW5002

EFFECTS OF CHLORPROMAZINE WITH AND WITH-

OUT UV IRRADIATION ON GENE EXPRESSION OF

HEPG2 CELLS

RF Froetschl1, S Weickhardt1, S Staszewski1,

G Kaufmann1, K Buss2, P Kasper1

1 Federal Institute for Drugs and Medical Devices,

BONN, Germany2 Memorec Biotec GmbH, COLOGNE, Germany

Data from standard genotoxicity tests provide only

little mechanistic insight into the mode of action of

genotoxic effects and labourious additional tests are

usually needed to gain such information. Microarray

technology has the potential to produce toxicity-

related pattern of gene expression changes and may

thus be used to classify genotoxic compounds with

different mechanisms of action.

Using HepG2 cells and PIQOR cDNA microarrays rep-

resenting 1089 genes related to e.g. DNA damage and

repair, cell cycle, transcription, metabolism and other

important cell functions we are currently investigat-

ing the gene expression profiles of several known

genotoxicans with different modes of action. This

presentation focus on data with the known photo-

mutagen chlorpromazine which itself is not genotox-

ic but upon UV irradiation (with a non-genotoxic

dose in the UVA range) produces reactive free radicals

with DNA damaging properties. Genotoxicity in

HepG2 cells has been assessed concomitantly to gene

expression profiling using the Comet assay.

The available results from repeat experiments show

significantly different gene expression pattern for

the relevant sample groups. In addition, time depend-

ent changes in gene expression were observed within

these groups. Genes showing differential expression

include stress induced SQSTM1, mitogen activated P-

kinase 6, mismatch repair protein1, apoptosis

inhibitor CARD4, thyroid receptor interacting protein

NFKBIB and others. Relationship of these genes to

treatment-related mode of action of genotoxicity will

be discussed.

We are continuing efforts to complete time and con-

centration kinetics with several other genotoxicants,

assess the reproducibility of the data generated and

apply further and different approaches in analysing

the profiling data.

37


W5 - Workshop 5


OW5003

IDENTIFICATION OF GENOTOXINS BY MARKER

GENE EXPRESSION BASED ON A SUPERVISED

CLASS PREDICTION APPROACH

D Bauer1, C McGinnis2, P Grass1, F Staedtler1, L Urban2,

W Suter1, L Mueller1

1 Novartis Pharma AG, BASEL, Switzerland2 Novartis Institutes (NIBRI), CAMBRIDGE,


Testing chemical compounds for genotoxicity is often

performed using a few highly standardized in vitro

assays. Nevertheless, relevance and predictivity

regarding in vivo results esp. in humans is limited. An

assay based on existing knowledge (e.g. well-known

reference substances) and being optimized on correct

classification would be of clear advantage. Therefore,

we performed a feasibility study in order to elucidate

whether an unbiased supervised class prediction

approach based on large scale gene expression pro-

files may lead to a stable prediction model for pre-

selected classes. In addition, the number of genes

taken into account for the discriminant function

should be reduced as far as possible in order to avoid

microarray experiments in a final testing situation.

Transcription profiles were obtained from TK6 human

lymphoblastoid cells treated with three known geno-

toxic compounds (cis-Platinum, Mitomycin C, Methyl

methansulfonate) and three compounds considered

non-genotoxic (trans-Platinum, Rifampicin, Sodium

chloride). Treatment was performed at nearly equicy-

totoxic conditions (cell count and Alamar Blue deter-

mination) using six replicates. Total RNA was

hybridized to Affymetrix HG-U133A microarrays

(~22000 probe sets) and raw expression data was fur-

ther analyzed using the software packages MAS5

(Affymetrix), Genespring 6 (Silicon Genetics) and

SIMCA-P 10 (Umetrics AB). In a supervised learning

approach discriminant functions involving the

expression pattern of only six genes were identified

that are capable of separating samples treated with

genotoxic and non genotoxic compounds without

any misclassification. According to validation by

response permutations it was found that the predic-

tive power of the model is strong and far from

random. Thus, this proof-of-concept study demon-

strates the general feasibility of a marker gene based

genotoxicity test system. Particularly, supervised

approaches using class prediction methods seem to

outperform conventional cluster analysis techniques.

Nevertheless, additional efforts are required in order

to accomplish a validated and predictive assay.

W5 - Workshop 5

Oral Presentation

OW5004

USE OF GENOMICS FOR REGULATORY SCIENCE –

INDUSTRY PERSPECTIVE

S Albertini, L Suter-Dick, S Ruepp, T Weiser

F. Hoffmann-La Roche AG, BASEL, Switzerland

Because of uncertainties how (toxico-)genomics data

will be used in regulatory decision industry was and

still is reluctant in submitting such data. To help clar-

ify the regulatory needs, FDA has initiated a guidance

document: ‘Guidance for Industry: Pharmacogenomic

Data Submission’. One clear statement is, that

‘because the field of pharmacogenomics is relatively

new, most experimental results may not be well

enough established to be suitable for regulatory deci-

sion making. Pharmaceutical companies, neverthe-

less use such data for internal decision making

during the drug development process. What is the

rational behind such an approach and where do we

stand? What are the issues the pharmaceutical indus-

try is facing to improve the attrition rate and thereby

increase the success rate?

It is believed, that toxicogenomics can significantly

contribute to this endeavour. Analysis of gene expres-

sion patterns seems suitable to detect organ-toxicity.

Fingerprints are used as working hypothesis to test

compounds in order to validate this technology as a

tool for prediction of possible toxicants, especially in

man. The major issue currently facing relates to the

interpretation of the obtained data in order to trans-

form the huge amount of information into knowl-

edge. Building a high quality reference database is

one of the major cornerstones for improving predic-

tivity by toxicogenomics. Approaches we followed

will be shown and insights in the complex data eval-

uation will be given focusing on hepatotoxicity pre-

diction.

Toxicogenomics represents an exciting, new

approach and has a great potential to influence posi-

tively the predictability of preclinical safety assess-

ments.

38


W6 - Workshop 6


OW6001

FRONTLOADING GENETIC TOXICOLOGY: EVALUA-

TION OF DEREK, AMESII AND GREENSCREEN AS

SCREENING TOOLS

JAJ van Gompel, D Beerens, F Woestenborghs, C Mackie

Johnson & Johnson Parmaceutical R&D, BEERSE,

Belgium

In the early Hit-to Lead phase, several assays are used

to obtain information on solubility, permeability,

metabolic stability, CYP450 interaction, cardiovascu-

lar safety pharmacology and genetic toxicology. With

5 mg of test compound, the AmesII assay gives a good

prediction towards the results in the downstream

GLP Ames assay. Due to the decision making position

of this assay, continuous monitoring of the predictive

value is a critical task. These downstream data are

normally only obtained for compounds entering the

next phase (Drug Evaluation) and whenever com-

pound quantities allow, agar based assays with TA98

and TA100 are performed. Since we lack eukaryotic

endpoints in this early phase, we are currently evalu-

ating GreenScreen, a yeast based assay (Cahill et al.

Mutagenesis 19,2, 2004). This assay detects a different

spectrum of compounds to the bacterial genotoxicity

assays and could therefore be complementary to the

AmesII bacterial screen. The bottleneck for the

assessment of this assay is however the more time-

consuming and cumbersome nature of downstream

assays (the mouse lymphoma assay and/or micronu-

cleus assay). Emerging structure-toxicity relation-

ships detected by these kind of medium throughput

assays can then be translated in new 'in-house' alerts

and fed back into in silico prediction tools like DEREK.

The combination of the ADME profiling data with

these in silico, prokaryotic and eukaryotic data pro-

vides a good hazard detection and allows the medici-

nal chemists to advance the most optimal hit series to

lead candidates.

W6 - Workshop 6

Oral Presentation

OW6002

NEW STRATEGIES IN TOXICOLOGICAL RISK ASSESS-

MENTS: TRANSPARENT AND EFFICIENT APPROACH-

ES IN DETERMINING HAZARD AND RISK

BJ Blaauboer

Utrecht University, UTRECHT, The Netherlands

The principles of risk assessment have been devel-

oped over the past 50 years and take into account the

intrinsic activity of a compound to cause a harmful

effect, the characterization of its hazard, i.e. the toxic

phenomena and the dose-effect relation and the

characteristics of exposure.

A prominent part of the data is derived from animal

models. These data ideally allow a quantification of

the dose-effect relationship, on the basis of which an

extrapolation can be made to humans, making use of

safety factor, which take into account the possible

higher sensitivity of human individuals.

These bioassays are highly standardised, laid down in

OECD guidelines. This has resulted in a system for

risk evaluation that allowed the relatively safe use of

chemicals.

However, this system meets criticism, mainly related

to its high degree of animal use and its relatively

fixed character. For instance, it is not easily allowing

adaptation of new scientific knowledge. The vast

increase in insight in the mechanisms of toxic action

should be taken into account.

In response to this criticism, proposals were made,

recently discussed in a report of the Health Council of

the Netherlands. Key is the use of schemes in which

knowledge is integrated from different domains, e.g.

structure, chemical functionalities, QSARs, mathe-

matical models for exposure and biokinetics and in

vitro data on toxicodynamics. Integrated approaches

need to be organized in the form of decision trees

with transparent criteria for expert judgments.

Examples of integrated approaches will be given and

the consequences for the hazard and risk assessment

in the fields of genotoxicity and carcinogenicity will

be discussed.

39


W6 - Workshop 6

Oral Presentation

OW6003

EVALUATION OF THE PERFORMANCE OF A SMALL

BATTERY OF IN VITRO TESTS IN DETECTING CAR-

CINOGENS

DJ Kirkland1, MJ Aardema2, LM Henderson3, L Müller4

1 Covance Laboratories Ltd., HARROGATE,

United Kingdom2 Procter & Gamble, CINCINNATI, United States of

America3 Henderson ScientificConsulting, NORTHWICH,

United Kingdom4 Novartis Pharma AG, BASEL, Switzerland

The performance of a battery of 3 in vitro genotoxici-

ty tests (Ames + MLA + in vitro MN) has been evalu-

ated for its ability to detect rodent carcinogens, from

the databases of Gold et al, NTP and IARC. Because

there are so few published data on the in vitro MN,

and the correlation between induction of MN and

chromosomal aberrations [CA] in vitro is so high, in

vitro chromosomal aberration data were also includ-

ed as relevant. The sensitivity of the 3-test battery

was high. Of the >400 chemicals for which there

were valid data, 97% gave positive results in at least 1

of the 3 tests. Only 14 carcinogens gave negative,

equivocal or inconclusive genotoxicity results in a full

3-test battery. All were either non-genotoxic carcino-

gens (liver enzyme inducers, peroxisome prolifera-

tors, hormonal carcinogens) many of which act via

mechanisms not relevant for humans, tumour pro-

moters (e.g. phenytoin), or were extremely weak

(presumed) genotoxic carcinogens (e.g. N-nitroso-

diphenylamine). The specificity of the 3-test battery

was poor. We identified about 200 chemicals that

were non-carcinogenic after testing in both male and

female rats and mice. For the approximately 50% of

non-carcinogens with relevant data, >80% gave posi-

tive results in 1 or more tests. Thus the specificity

(ability to accurately detect non-carcinogens as nega-

tive) was <20%. Some of the positive non-carcinogens

were aneugens or produced clastogenicity only at

high osmolality, but a large number of these positives

are unexplained. This performance highlights the

importance of understanding the mechanism by

which genotoxicity may be induced (whether it is rel-

evant for the whole animal or human) and using

weight of evidence approaches to assess the carcino-

genic risk from a positive genotoxicity signal.

W6 - Workshop 6

Oral Presentation

OW6004

OVERVIEW OF VALIDATION AND INTERNATIONAL

ACCEPTANCE OF TEST METHODS FOR HAZARD

ASSESSMENT IN THE OECD

AM Wagner

OECD, PARIS, France

Validation issues related to testing methods are

important to the chemical safety work of the

Organisation for Economic Co-Operation and Develop-

ment (OECD). In 1996 the OECD conducted the Solna

workshop, which resulted in a set of principles and

criteria for validation and regulatory acceptance of

new and alternative test methods that are important

for consideration during OECD Test Guideline devel-

opment. A Guidance Document has subsequently

been drafted (Guidance Document No 34 on the

'Validation and International Acceptance of New or

Updated Test Methods for Hazard Assessment'),

which provides specific guidance on the issues relat-

ed to validation of new or updated test methods, both

for in vivo or in vitro, and ecotoxicity tests. Validation

can be conducted prospectively (during the develop-

ment of a test method) or retrospectively (using exist-

ing data). The ultimate aim of GD34 is to facilitate the

development of OECD Test Guidelines and the inter-

national acceptance of data for hazard and risk

assessment. Test method validation is an evolving

area, and the finalisation of GD34 is now one of the

priority areas of the OECD. Test method validation is a

real issue for all areas in toxicology, including geno-

toxicity. Currently, a new draft Test Guideline for the

In Vitro Micronucleus Test is under consideration by

the OECD. A process of prospective validation was

undertaken for this test method, where several stud-

ies that investigated different protocol parameters

were conducted by various parties before the guide-

line was written, and the results of the validation

exercise were reviewed during an International

Meeting. In addition, a retrospective evaluation of

the validity of this in vitro genotoxicity assay, based

on available data, is currently underway and co-ordi-

nated by ECVAM (European Centre for the Validation

of Alternative Methods). Further information on the

OECD validation process will be described.

40


W7 - Workshop 7

Oral Presentation

OW7001

ROLE OF THE BLOOM’S SYNDROME HELICASE IN

MAINTENANCE OF GENOME STABILITY

ID Hickson

Institute of Molecular Medicine, OXFORD,

United Kingdom

The RecQ family of DNA helicases is highly conserved

in evolution from bacteria to man. There is a single

family member in unicellular organisms, but multi-

ple members in metazoans, with five being present in

humans. Of these, three are of particular interest

because germ-line mutations in these genes leads to

well-characterized disorders: BLM is mutated in

Bloom’s syndrome, WRN in Werner’s syndrome, and

RECQ4 in Rothmund-Thomson syndrome. These dis-

orders are associated with both cancer predisposition

(particularly evident in Bloom’s where cancers of all

types are seen) and premature aging (most notably in

Werner’s syndrome). Defects in RecQ family helicases

give rise to genomic instability in all organisms ana-

lyzed to date. This instability may take several forms,

but a consistent feature is aberrant genetic recombi-

nation – generally hyperrecombination between

homologous sequences and/or a failure to suppress

recombination between non-identical DNA sequences.

The focus of our work is the BLM gene product and its

homologue in budding yeast, Sgs1p. Mutant cells

lacking either BLM or Sgs1p show an increased fre-

quency of sister-chromatid exchanges and hypersen-

sitivity to agents that perturb DNA replication, such

as hydroxyurea. Moreover, BLM and Sgs1p act in con-

cert with a second protein, topoisomerase III, to effect

their functions. Our recent work has shown that BLM

and topoisomerase III cooperate to resolve late-stage

intermediates in the homologous recombination

repair pathway that contain Holliday junctions. Our

working model is that RecQ helicase and topoiso-

merase III act in a recombination–dependent path-

way that operates specifically at sites of damaged

replication forks.

W7 - Workshop 7


OW7002

THE STUDIES ON LOCALIZATION OF HUMAN

RECQ HELICASES FUSED WITH FLUORESCENT

PROTEINS

R Rusin1, M Krzesniak1, R Vaitiekunaite1,

D Butkiewicz1, M Mrzyglodzik1, CC Harris2

1 Center of Oncology, GLIWICE, Poland2 National Cancer Institute, NIH, BETHESDA,


The long-known, senescence-accelerating, cancer

prone diseases: Werner, Bloom and Rothmund-

Thomson syndromes are caused by mutations in

WRN, BLM and RTS genes, respectively, coding for

RecQ family helicases. In humans, there are other

members of RecQ helicases: RECQL and RECQ5. The

RecQ helicases are crucial for genomic stability and

DNA metabolism, however many of their functions

remain unknown. In order to better understand the

functioning of RecQ helicases, we generated the fluo-

rescently tagged proteins by attaching the monomer-

ic red fluorescent protein (mRFP1) cDNA to the 5’cDNA

end of WRN, BLM, and RECQL. Subsequently, we intro-

duced missense mutations in mRFP1-WRN by site-

directed mutagenesis and transfected the wild-type

and mutant genes into monkey cells (COS-7), human

lung carcinoma cells (NCI-H1299) and colon adeno-

carcinoma cells (HCT-116). Moreover, in order to per-

form the co-localization studies, we co-transfected

the helicase constructs with the plasmids producing

either fluorescently tagged PML protein, TRF1 protein

or selected proteins involved in DNA repair. We

noticed that, apart from the nucleolar and focal stain-

ing, the most remarkable feature of mRFP1-WRN

localization was its presence in donut-shaped struc-

tures of various sizes, some of them reaching 30%

diameter of cell nucleus. Neither helicase nor exonu-

clease activities are required for WRN entering nucle-

oli, 'donuts' and foci. The mutation of the key amino

acid residue in HRDC domain abolishes mRFP1-WRN

'donut' formation. Instead, mRFP1-WRN is present in

irregular, nuclear „speckles'. The mRFP1-WRN 'donuts'

resemble the PML bodies present in cells with alter-

native mechanism of telomere maintenance.

However, the cotransfection of mRFP1-WRN and

EGFP-PML constructs do not show any evidence of co-

localization in the 'donuts'. The nature and function

of these subnuclear structures remain unknown. The

mRFP1-WRN 'donuts' contain some other proteins

involved in DNA repair. We conclude that our fusion

constructs are useful tools for studying the structure-

function relationship in human RecQ helicases.

41


W7 - Workshop 7


OW7003

DNA DAMAGE-REPAIR, APOPTOSIS AND NECROSIS

IN CHILDREN, ADULTS AND OLD AGE HUMANS

G Karanastasi1, N Anagnostakis1, N Messini-

Nikolaki2, S Doudounakis3, S Tsilimigaki1,

SM Piperakis1

1 NCSR Demokritos, ATHENS, Greece2 University of Athens, ATHENS, Greece3 Aghia Sophia Hospital, ATHENS, Greece

Our aim using the comet assay, was:

1) To determine the amount of endogenous DNA

damage in human lymphocytes of children,

adult and old age human populations.

2) To find the sensitivity of their lymphocytes to the

exposure of increased concentrations of g-irradia-

tion and H2O2.

3) To investigate the DNA repair efficiency in these

populations, and finally,

4) To measure apoptosis and necrosis in the same

populations.

Our results indicate that:

1) The endogenous DNA damage is smaller in the

child than the other two population.

2) There is a difference in the sensitivity of the three

populations lymphocytes after been exposed to

g-irradiation or H2O2.

3) The DNA repair was more efficient in the child

than the other three populations, and finally,

4) The amount of apoptotic cells appears to be less in

the child than the other populations.

W7 - Workshop 7


OW7004

DNA REPAIR FUNCTION, POLYMORPHISMS AND

GENOTOXICITY IN WORKERS EXPOSED TO LOW

DOSE IONISING RADIATION

V Aka1, RAM Mateuca1, JP Buchet2, H Thierens3,

M Kirsch-Volders1

1 Vrije Universiteit Brussels, BRUSSELS, Belgium2 Catholic University of Louvain, BRUSSELS, Belgium3 University of Ghent, GHENT, Belgium

Identification of higher risk individuals carrying

genetic polymorphisms responsible for reduced DNA

repair capacity has substantial preventive implica-

tions as these individuals could be targeted for appro-

priate cancer prevention. We have conducted a study

in 32 male seasonal cleaners of a nuclear plant and 31

control workers to assess the predictivity of the OGG1,

XRCC1 and XRCC3 genotypes and the in vitro single

strand break repair phenotype for the induction of

genotoxic effects (DNA damage and micronuclei). At

the population level, a significant contribution of the

OGG1 genotypes to the in vitro DNA strand break

repair capacity was found. Genetic polymorphisms in

XRCC1 and XRCC3 did not significantly influence

repair capacity. At an individual level, the OGG1 vari-

ants Ser/Cys and Cys/Cys genotypes showed a slower

in vitro DNA repair than the Ser/Ser OGG1genotype. A

multivariate analysis performed with genotypes, age,

cumulative dose, exposure status and smoking as

independent variables indicated that in the control

population, repair capacity is influenced by age and

polymorphisms in OGG1. In the exposed population,

DNA damage is greater in older men and in smokers.

Repair capacity is slower in individuals with Ser/Cys

or Cys/Cys OGG1 genotypes compared to those with

the Ser/Ser OGG1 genotype. Micronuclei frequencies

increased with age and the cumulative dose of

gamma rays. Analysis of the total population

revealed that genetic polymorphisms in XRCC1 result-

ed in higher residual DNA values and the Met/Met

variant of XRCC3 gave an increased frequency of

micronuclei. A combined analysis of the three geno-

types, OGG1, XRCC1 and XRCC3 polymorphisms is

advised in order to assess individual susceptibility to

ionising radiation. As an alternative or complement,

the in vitro DNA strand break repair phenotype

which integrates several repair pathways is recom-

mended.

42


W7 - Workshop 7


OW7005

AGE-RELATED GENOME INSTABILITY IN DNA

REPAIR-DEFICIENT MICE

MET Dollé1, R Busuttil2, L Niedernhofer3, B van der

Horst3, J Hoeijmakers3, SWP Wijnhoven1, P Lohman1,

H van Steeg1, J Vijg4

1 Nat.Inst.of Publ. Health and Environment,

BILTHOVEN, The Netherlands2 Univ TX Health Science Center, SAN ANTONIO,

United States of America3 Erasmus University, ROTTERDAM, The Netherlands4 University Texas Health Science Centre,

SAN ANTONIO, United States of America

Genomic instability has been implicated as a major

cause of both cancer and aging. Previously we have

shown that mutations accumulate with age in an

organ-specific manner, using a transgenic mouse

model with a chromosomally integrated lacZ muta-

tional reporter gene. To examine the correlation

between age and mutation load, we are investigating

genomic instability in aging DNA repair-deficient

mice. Here we present our preliminary findings on

different mouse mutants with deficiencies in

nucleotide excision repair (NER) and DNA interstrand

crosslink (ICL) repair. NER removes a broad range of

lesions, such as UV-induced damage, other bulky

adducts and some forms of oxidative damage. Life

span studies and mutant frequency determinations

in liver and kidney were performed on male mice

with defects in global and/or transcription-coupled

repair (Xpa, Csb or Xpd(ttd)). We observed a reduced

life span for Xpa and Xpd(ttd), as compared to Csb and

wild type control animals. Conform to our initial

studies with just wild type mice, all NER-deficient

models showed an age-related increase of mutant

frequencies in liver and kidney. However, only the

Xpa-mice showed elevated mutant frequencies over

control animals in both organs. In addition, we exam-

ined mutant frequencies in liver of the very short-

lived Ercc1(*292) mouse. This mouse model lacks the

last 7 amino acids of Ercc1 and is deficient in both NER

and DNA ICL repair. We observed a two-fold increase

in mutant frequency over control mice at 23 weeks,

which is close to their maximum life span. A compar-

ison of mutation spectra in liver revealed that the

general NER-deficiency of XPA-mice resulted in point

mutations, predominantly consisting of one base pair

deletions. The increased mutant frequency in the

Ercc1(*292) mice was due to both point mutations and

translocations, the latter presumably reflecting the

additional cross-link repair deficiency compared to

Xpa-mice.

YSAL - Young Scientist Award Lectures

Oral Presentation

OYSAL1

OXIDATIVELY DAMAGED DNA: FROM GENES TO

POPULATIONS

MS Cooke

University of Leicester, LEICESTER, United Kingdom

There is an ever increasing number of physico-chem-

ical agents reported to induce oxidative damage to

DNA. Elevated levels of, and failure to repair, such

damage, has been associated with a wide variety of

pathological conditions. Nevertheless, proof of a

causative effect remains elusive. Attempts towards

obtaining a comprehensive understanding of the fac-

tors affecting the induction and repair of oxidatively

damaged DNA, have been facilitated by a diverse

range of approaches. For example, preferential

removal of lesions from transcriptionally active

genes, combined with transcribed strand-specific

repair can facilitate cell survival even when total

lesion levels would be expected to be toxic. This may,

in part, be responsible for the difficulties associating

elevated lesion levels with disease, and has prompted

the development of assays to examine damage

to individual genes. Furthermore, simultaneous

measurement of oxidative (e.g. 7,8-dihydro-8-oxo-

2'deoxyguanosine, 8-OHdG) and non-oxidative lesions

(cyclobutane thymine dimer, T<>T), has highlighted

similarities in the removal of both classes of lesion

from the genome, allowing conclusions to be drawn

regarding the processes involved. Indeed, it was the

measurement of multiple lesions, in various biologi-

cal matrices, including urine, that enabled the

hypothesis for the induction of DNA repair by vita-

min C, to be suggested. Although the proposal of a

number of confounding factors have meant urinary

8-OH-dG measurements are often considered to be no

more than reflective of whole body oxidative stress,

the potential exists for a non-invasive assay of DNA

repair.

The plethora of sources of oxidative damage to DNA

means that little precise exposure data can be gained

from studies which involve biomonitoring. However,

were these measurements able to provide informa-

tion concerning an individual's cellular defenses, and

DNA repair, for example, they would represent a far

more useful tool in human population studies.

43


YSAL - Young Scientist Award Lectures

Oral Presentation

OYSAL2

CELL-TYPE AND DNA DAMAGE-SPECIFIC RESPONSE

OF HUMAN EPIDERMAL CELLS

M D'Errico1, M Teson2, A Calcagnile1, T Nardo3,

P Jaruga4, G Zambruno2, M Stefanini3, M Dizdaroglu4,

E Dogliotti1

1 Italian National Health Institute, ROME, Italy2 I.D.I., IRCCS, ROME, Italy3 CNR, PAVIA, Italy4 NIST, GAITHERSBURG, United States of America

The epidermis is the primary target for environmen-

tal insults. In this study we present a systematic com-

parison of the response to UVB and X-rays of human

primary keratinocytes and fibroblasts. Nucleotide

excision repair (NER)-defective cells derived from XP-

C (no global genome repair, GGR) and CS-A (no tran-

scription-coupled repair, TCR) patients were used to

investigate the effects of alterations in the two NER

sub-pathways on DNA damage response.

A cell-type specific response was detected after UVB

treatment. Keratinocytes were more resistant to the

lethal effects of UVB than fibroblasts and removed

cyclobutane pyrimidine dimers (CPD) more efficient-

ly than fibroblasts. This improved repair was ascribed

to a more efficient GGR. A defect in TCR was associat-

ed with a strong apoptotic response in fibroblasts but

not in keratinocytes, whereas a defect in GGR had no

effect on the apoptotic response of either cell type.

We speculate that the persistence of CPD in the tran-

scribed sequences triggers apoptosis in fibroblasts

but not in keratinocytes where GGR operates as back-

up system to remove transcription-blocking lesions.

As observed for UVB, X-ray-induced cytotoxicity was

cell-type specific with keratinocytes more resistant to

the lethal effects of X-rays than fibroblasts. Similar

levels of DNA single-strand breaks and modified

nucleosides [8-hydroxy-2'-deoxyguanosine, 8-hydroxy-

2'-deoxyadenosine, (5'S)-8,5’-cyclo-2'-deoxyadenosine]

were detected in the two cell types after irradiation.

Normal keratinocytes repaired to completion modi-

fied DNA bases within one hour, whereas CSA ker-

atinocytes accumulated significant amounts of these

lesions. In contrast to UVB, the apoptotic response to

X-rays was not cell-type dependent. In both normal

and NER-defective skin cells apoptosis was triggered

at highly cytotoxic doses.

All together these results indicate that the response

to environmental agents is cell type- and DNA dam-

age-specific. Moreover, we provide evidence that CSA

has a cell-type specific impact in the response to UVB

and participates to repair oxidative DNA damage.

44


45

Poster presentations

PS1 - Poster Session 1

Poster Presentation

PW1001

EVALUATION OF THE MUTAGENIC PROPERTIES OF

PENTA® DRINKING WATER USING CYTOGENETIC

METHODS

AN Chebotarev, NV Kosyakova, VI Platonova

Research Center for Medical Genetics, MOSCOW,

Russia

The clastogenic properties of Penta® drinking water

has been performed using cytogenetic methods to

determine the chromosome aberration frequency

(CAF), sister chromatid exchange (SCE) number and

cell cycle duration. Penta® water samples have been

provided by Bio-Hydration Research Lab (USA).

Penta® water is manufactured using a process that

transforms cavitation acoustic energy into thermal

energy in a specially designed cavitation chamber

(US patent 6,521,248). Experiments have been con-

ducted on human lymphocytes that were cultured

using a standard protocol. The average number of cell

divisions in culture in the presence of 5BdU was

determined using the formula: (i x ni/2i-1)/( ni/2i-1),

where i = mitosis number and ni = cell number of i-

mitosis. Cell cycle duration was determined by 5BdU

presence time in cell media divided by the average

number of cell divisions. In addition to experiments

on spontaneous mutagenesis in various cell media,

induced clastogenic effects of mitomycin C (100, 200,

and 400 ng/ml) added 14 hours prior to the cell fixa-

tion have been studied. When Penta® water was used

in cell media preparation without a mutagen added,

the SCE number per cell = 3.38±0.12 and CAF = 0.92%.

These figures were lower than results seen with the

control: SCE number per cell = 4.01±0.15 and

CAF=2.50%. Cell cycle duration was the same for both

Penta' and deionized water based cell media (21.2

hours). The SCE number was the same for all concen-

trations of mutagen used in both types of waters in

cell media preparation. Regression analysis of the

mutagenic effect dependent on Mitomycin C concen-

tration has shown that the frequency of aberrant

metaphases is two times lower and the number of

chromosome breaks per cell is 1.5 times lower when

Penta® water is used compared to the control.


46


Poster Presentation

PW1002

ARISTOLOCHIC ACID AS A PROBABLE HUMAN

CANCER HAZARD IN HERBAL REMEDIES

VM Arlt1, GM Lord2, M Hollstein3, V Alunni-Perret4,

JP Cosyns5, HH Schmeiser3

1 Institute of Cancer Research, SUTTON, SURREY, United

Kingdom2 Hammersmith Hospital NHS Trust, LONDON, United

Kingdom3 German Cancer Research Center, HEIDELBERG,

Germany4 Hopital Pasteur, NICE, France5 Cliniques St. Luc, BRUSSELS, Belgium

The old herbal drug aristolochic acid (AA), derived

from Aristolochia spp., has been associated with the

development of a novel nephropathy, designated

aristolochic acid nephropathy (AAN), that predispos-

es patients to a high risk of urothelial carcinoma.

AAN was reported for the first time in a Belgian slim-

ming clinic. To date, more than 100 patients have

been identified in Belgium. All cases could be traced

to the herbal ingestion of a preparation containing

AA, namely Aristolochia fangchi. We found that the

adenosine adduct of aristolochic acid I [7-(deoxya-

denosin-N6-yl)aristolactam I, dA-AAI] is the predom-

inant DNA adduct detectable in urothelial tissue. In

the present study we describe the first fully docu-

mented cases of AAN in the UK and France.

Histopathologic and DNA adduct analyses performed

on autopsy samples revealed that these cases did

indeed represent authentic AAN outside Belgium. It is

of interest to note that some patients had developed

a transitional cell carcinoma in the urinary tract.

Given the availability of several tissue samples from

patient's post-mortem examination, we were able to

detect the dA-AAI adduct in various tissues outside

the urinary tract (kidney, bladder, ureter, liver, lung,

stomach, small intestine, spleen, adrenal, and brain)

indicating that other factors than DNA adduct forma-

tion by AA may also be critical for the high incidence

of urothelial tumours. Interestingly, a characteristic A

to T transversion mutation was found at the first ade-

nine of codon 139 (AAG) of the p53 gene in urothelial

tumour cells of one patient. A to T transversions are

the typical mutations observed in the H-ras gene of

AA-induced tumours in rodents and correspond with

DNA adducts at adenosine residues. These data indi-

cate the probable molecular mechanism whereby AA

causes urothelial tumours. These cases highlight the

need for continuing vigilance to ensure that AA is not

present in herbal medicinal remedies.

Good Food, Good

La passion de la


47


48


Poster Presentation

PW1003

CORRELATION BETWEEN STABILIZATION OF

CLEAVABLE COMPLEXES EVOKED BY ANTHRACY-

CLINES AND APOPTOSIS IN CELLS

BM Gruber1, EL Anuszewska1, W Priebe2, I Fokt2,

A Gozdzik1

1 National Institute of Public Health, WARSAW,

Poland2 M.D. Anderson Cancer Center, HOUSTON, United

States of America

Apoptosis is one of the possible mechanism responsi-

ble for cytotoxicity of anthracyclines. Last years

brought the new aspects of stimulating apoptotic

effects by these drugs including NFkappaB. As was

shown it can be activated by anthracyclines, leading

to apoptosis via various mechanisms. It is suggested

the role of DNA lesions generated through stabiliza-

tion of cleavable complexes DNA-topo II in activation

of NFkappaB and, indirectly, in apoptosis induction.

The aim of this study was to establish the possible

dependance between stabilization of cleavable com-

plexes by anthracyclines and intensity of apoptotic

effects. In the current work, the following cells differ-

ing in sensitivity to anthracyclines were used: human

melanoma cells(Me18); it's subline resistant to

DOX(Me18/R); human cervix carcinoma cells(HeLa);

it's subline resistant to vinblastine(KB-V1).Different

new anthracycline analogs synthetized in M.D.

Anderson Cancer Center, Houston, USA were tested.

As a result, quantitative differences in topo II protein

between the cells, in part correlated with sensitivity

to anthracyclines were observed. Also, some correla-

tion was shown between sensitivity to the tested

drugs, 'quenching' of topo II protein by these com-

pounds and the intensity of caspase dependent apop-

tosis in human cervix carcinoma cells.


Poster Presentation

PW1004

THE EFFECT OF RESVERATROL ON THE EXPRES-

SION OF ENZYMES METABOLIZING XENOBIOTICS

IN MOUSE EPIDERMIS

W Baer-Dubowska, V Krajka-Kuzniak, H Szaefer

Poznan University of Medical Sciences, POZNAN,

Poland

Resveratrol (3,4',5-trihydroxystilbene), a polyphenol

found at high levels in a wide variety of plant species

including peanuts, mulberries and grapes, has been

reported to exhibit a wide range of beneficial biolog-

ical properties including the modulation of multi-

stage carcinogenesis. This compound inhibited 7,12-

dimethylbenz[a]anthracene (DMBA)-induced preneo-

plastic lesion formation in mouse mammary organ

culture and reduced the incidence and multiplicity of

DMBA/TPA-induced papillomas in the two-stage

mouse skin model. Antimutagenic activity of resvera-

trol was also demonstrated against the foodborne

heterocyclic amine, 3-amino-1,4-dimethyl-5H-pyri-

do[4,3-b]indole (Trp-P-1) and 2-aminofluorene in

Salmonella bacterial tester strains. These chemical

carcinogens require metabolic activation by P450

dependent enzymes in order to exert their genotoxic

effect. However phase II enzymes, particularly GST

isoforms may modify their metabolic profile.

In the present study we examined the effect of

resveratrol on the expression of CYP1A1/1A2, CYP1B1

and GST theta in female Swiss mouse epidermis by

Western blot assays.

Resveratrol was applied topically to the shaved dorsal

skin at the doses of 8 or 16 mmoles in 0.2 ml of ace-

tone. A group treated with 5,6-benzoflavone served as

a positive control. A control group of mice received

acetone alone.

The constitutive expression of all tested enzymes was

observed, however their expression levels were differ-

ent. Topical application of resveratrol affected the

most GST theta. In both tested doses the significant

increase of this isoform protein was observed. This

compound was also a weak inducer of epidermis

CYP1B1. Treatment of mouse epidermis with resvera-

trol did not affect on the level of CYP1A1/1A2 in this

tissue.The results of our study indicate that although

moderate, the effect of resveratrol on expression of

CYP1A1/1A2, CYP1B1 and GST theta in mouse epider-

mis may play certain role in its anticarcinogenic

activity.


49


Poster Presentation

PW1005

CHARACTERIZATION OF HUMAN DIETARY COM-

PONENTS IN HUMAN DERIVED LIVER CELLS

F Darroudi1, S Knasmueller2, V Mersch-Sundermann3,

E Lhoste4, A Bader5


The Netherlands2 University of Vienna, VIENNA, Austria3 University of Giessen, GIESSEN, Germany4 INRA, JOUY EN JOSAS, France5 University of Leipzig, LEIPZIG, Germany

It is well documented that diet plays a crucial role in

the etiology of cancers in humans, and efforts were

also made to identify protective (anti-mutagenic and

anti-carcinogenic) substances in food. The traditional

risk assessment process applied successfully to food

additives relies on toxicology testing in vivo in ani-

mals at intake levels many times higher than is likely

in humans. The in vitro assays with metabolically

incompetent cells require addition of exogenous

enzyme homogenate (i.e. rat liver fractions) to cat-

alyze the activation of genotoxic food derived car-

cinogens. However, it became evident that certain

mode of action (i.e. detoxification process and to

detect protective / synergistic effects) are not repre-

sented adequately in the in vitro models.

Different biological assays were developed, validated

and applied using human derived liver cells (Hep G2

and hepatocytes) to detect the mutagenic potentials

of various classes of human food constituents (i.e.

polycyclic aromatic hydrocarbons, heterocyclic aro-

matic amines, nitrosamines and mycotoxins). HepG2

cells were used as the metabolic activation system as

well as target for evaluating DNA damage. Different

biological end-points, such as sister-chromatid

exchanges, micronuclei in binucleated cells, cytotoxi-

city, gene mutation (at HPRT locus) and Comet assay

were used. Furthermore, co- and anti-mutagenic

potential of human dietary food constituents (i.e.

vitamins, cruciferous vegetables, beverages and

heavy metals) was also investigated. The use of

human hepatoma (Hep G2) cell system in mutagenic-

ity testing further validated through gene expression

analysis and a comparison was made with human

hepatocytes.

The results indicate that the human hepatoma cell

systems reflect the activation / detoxification of

genotoxic carcinogens better than other indicator

cells that are currently being used and therefore have

an increased predictive value for the identification of

mutagenic, co- and anti-mutagenic constituents of

human foods.

GlaxoSmithKline is pleased to supportEEMS 2004


50


Poster Presentation

PW1006

THEOPHYLLINE INHIBITS POLY-(ADP-RIBOSE)POLY-

MERASE-1 (PARP-1) ACTIVITY IN HUMAN PUL-

MONARY EPITHELIAL CELLS

H.J.J. Moonen, L. Geraets, A. Vaarhorst, A. Bast,

G.J. Hageman

Maastricht University, MAASTRICHT,

The Netherlands

Background: Theophylline is still widely used in the

treatment of asthma and chronic obstructive pul-

monary disease (COPD), and is believed to exert anti-

inflammatory properties. Although several possible

mechanisms underlying the beneficial effects of

theophylline have been described, inhibition of the

nuclear enzyme poly(ADP-ribose)polymerase-1 (PARP-

1), which appears to be a major player in the patho-

physiology of chronic inflammatory diseases such as

COPD, has not been investigated in this perspective

before.

Objective: In this study, we investigated the effect of

theophylline on PARP-1 activity in human pulmonary

epithelial cells (A549).

Methods: PARP-1 activity in A549 cells was deter-

mined by measuring decreasing intracellular NAD+

levels upon exposure to 300 µM of hydrogen perox-

ide, with or without pre-incubation with either 0.1,

0.2, 1 or 10 mM theophylline. Electron spin resonance

(ESR) spectroscopy was used to investigate the antiox-

idant capacity of theophylline.

Results: Intracellular NAD+ levels dramatically

decreased upon exposure to hydrogen peroxide, indi-

cating strong induction of PARP-1 activity. Pre-incuba-

tion with theophylline was found to prevent the

decrease in NAD+ in a dose dependent manner, thus

showing that theophylline inhibits the activity of

PARP-1 in these cells. Furthermore, theophylline did

not show any antioxidant capacity, thus ruling out

scavenging of oxygen radicals.

Conclusions: Theophylline inhibits PARP-1 activity in

human pulmonary epithelial cells, and does not exert

any anti-oxidant effect in these cells. These findings

provide new insights into the therapeutic effect of

theophylline.


Poster Presentation

PW1007

GENOTOXIC EFFECTS OF OCHRATOXIN A ON THE

HUMAN KIDNEY HK-2 CELL LINE

A L C Lopez de Cerain1, S de Blaes2, O Ezpeleta1,

L Arbillaga1, A Lopez de Cerain1

1 University of Navarra, PAMPLONA, Spain2 Wageningen University, WAGENINGEN,

The Netherlands

Ochratoxin A (OTA) is a nephrotoxic mycotoxin classi-

fied by the International Agency for the Research on

Cancer (IARC) as a carcinogen category 2A. It is a

potent renal carcinogen in male rats although its

mechanism of action is not known. Few data on the

genotoxicity of OTA in human cells are available. The

aim of the present work was to investigate the muta-

genic effects of OTA using the micronucleus (MN)

assay in a human derived kidney cell line (HK-2).

A previous MTT cytototoxicity assay was carried out

with different concentrations of OTA and S9-mix in

order to select subtoxic OTA concentrations for the

mutagenicity assay. Cells were treated with OTA -

range 0.1- 100 µM- for 2, 4 and 6 h, in the presence of

rat kidney or liver S9-mix (4-10%). For the MN assay

the following conditions were decided: 10 and 40 µM,

5 hours, 4% S9-mix. The MN assay was performed

using the protocol of Fenech et al. (1999), modified for

HK-2 cells. Cytochalasin B (1 µM) was added for the

last 21-h of incubation. MN were identified in 500

binucleated cells, and scored according to the criteria

described by Bonassi et al. (2001). Data were statisti-

cally analysed with the Mann-Whitney U test (SPSS

program, version 11.0).

OTA caused an increase in the number of MN in the

absence of S9-mix (approximately 6-fold). In the pres-

ence of liver or kidney S9-mix, a 3-fold MN induction

was observed. Non significant differences were

obtained between the two concentrations tested.

These results show that OTA treatment induces the

production of MN in a human kidney derived cell

line. As we do not know the metabolic capability of

this cell line, the importance of the metabolic activa-

tion could not be completely assessed.


51


Poster Presentation

PW1008

DNA DAMAGE IN HUMAN CELLS TREATED WITH

DRINKING WATER DISTRIBUTED IN THREE NET-

WORKS

C Pellacani, A Buschini, M Furlini, P Poli, C Rossi

Universita di Parma, PARMA, Italy

Epidemiological studies and mutagenic short-term

bioassays have revealed the presence of genotoxic

disinfection by-products in drinking water. Few stud-

ies have been evaluated the influence of distribution

systems on mutagen formation. Mutagenic com-

pound could be leached from internal surface of

municipal water tanks and pipelines, which are fre-

quently coated with coal tar or from plastic pipelines.

The aim of this study is the evaluation of toxic and

genotoxic effects of drinking water in the networks of

three Italian towns with different water source sup-

plying systems. The drinking water have been sam-

pled in different pipelines points, after and before the

distribution systems. The water has been concentrat-

ed with semipermeable membrane devices (SPMDs),

membrane-based passive samplers, a promising tool

for the time-integrated monitoring of hydrophobic

pollutants in aquatic systems. SPMDs have been

exposed to water flow (1litre/min) during 30 days,

dialyzed in a ethyl acetate, acetone, and methanol

solution (1:1:1), and concentrated with a rotary evapo-

rator. The concentrated samples have been tested on

human leukocytes by the Comet assay for DNA dam-

age. Cell viability was checked by fluorescine diac-

etate/ethidium bromide-assay. All the water samples

were able to induce DNA damage even if with differ-

ent behaviour and efficacy. 'Town A' (groundwater):

DNA damage resulted hardly increased (≈15 times) in

cell treated with water concentrate equivalent to 12-

hour’s SPMD exposure before distribution system

with respect to the control; distribution system did

not increase water genotoxicity. 'Town B' (surface

water): before distribution-water induced a DNA

damage increase (≈3 times the control) whereas this

increase was ≈20 times for water sampled on the

pipeline farthest point. 'Town C' (surface water): DNA

damage was increased ≈15 times before the distribu-

tion and this effect become further greater (≈25

times) in tap water; in an other pipeline point it was

also observed a strong cytotoxic effect.


Poster Presentation

PW1009

ANTIGENOTOXIC EFFECTS OF ALOE AND ITS

MODE OF ACTION

BM Lee, E Jeong Yoo

Sungkyunkwan University, SUWON, South-Korea

Antigenotoxic effects of aloe were investigated in 3 dif-

ferent tests: sets of Ames, chromosome and micronuclei

tests. Aloe reduced in a dose-dependent manner the

mutational frequencies of Salmonella strains, chromo-

some aberration, and micronuclei formation in mouse

bone marrow induced by benzo(a)pyrene. In addition,

aloe significantly increased glutathione levels in cells

used for the tests, which might be involved in possi-

ble mechanisms of aloe antigenotoxicity.

These results suggest that aloe could be a chemopre-

ventive potential by modulating cellular glutathione

levels.


52


Poster Presentation

PW1010

CURCUMIN INTERACTS WITH INDUCTION OF

UROKINASE-TYPE PLASMINOGEN ACTIVATOR

(UPA) BY THE ALKILATING AGENT MNNG

JS Soric, J. Loniarek

Faculty of Pharmacy and Biochemistry, ZAGREB,

Croatia

We have demonstrated that the uPA gene is tran-

scriptionaly induced by MNNG, starting 24 hours post

treatment. ERK MAP kinase specific inhibitor did not

change neither constitutive neither basal uPA enzy-

matic activity, while p38 MAP kinase inhibitor could

partially abolish the uPA induction. Curcumin

appeared as most potent inhibitor of uPA induction,

decreasing basal level of uPA and abolishing the

inducible effect of MNNG. Curcumin addition and

removal from the cellular media at different time

points after MNNG treatment, showed that for sup-

pression of uPA by curcumin, the most relevant fact

was its constant presence in the growing medium.

Furthermore we observed its reversible effect, since

curcumin removal from the cell culture media abort-

ed its inhibiting ability. It indicated existence of an

persistent signal triggered by MNNG inducing uPA

expression and its inhibition by curcumin. Aditionally,

here we show that curcumin influences cellular prolif-

eration and disturbs the action of btubulin, as showed

by immunostaining. Although the precize mechanism

of curcumin action is currently unknown, the results

demonstrated, among possible other events, its addi-

tional action upon microtubular organization and

consequent induction of mitotic catastrophe (micro-

nucleation).


53


Poster Presentation

PW1011

32P-POSTLABELLING OF ACRYLAMIDE DNA AD-

DUCTS

K Segerbäck1, N Kotova1, T Juren1, P Rydberg2

1 Karolinska Institute, HUDDINGE, Sweden2 Stockholm University, STOCKHOLM, Sweden

An important issue in the discussions on the risk con-

nected to consumption of food containing the muta-

gen acrylamide is the dose-response relationship for

formation of biological effects in cancer tests in

rodents. DNA adducts is considered to be a marker of

biological significant dose and measurements of such

products could therefore be very useful for the risk

assessment of acrylamide. Acrylamide is metabolised

to the reactive epoxide glycidamide (GA) and it is

believed that most of the genotoxic effects of acry-

lamide is mediated via GA. As an alkylating agent GA

reacts primarily with ring nitrogens in DNA bases.

Besides the major product with N-7-guanine there are

also adducts at N-1- and N-3-adenine. We have previ-

ously been using the nuclease P1 enhancement of the

32P-postlabelling assay for analysis of the N-1-ade-

nine adduct of propylene oxide. In this work we man-

age to reach a sensitivity of 1 adduct per 10exp10

normal nucleotides. This was accomplished by con-

verting the N-1-adenine adduct to the N6-adenine

adduct and separation by HPLC with on-line radioiso-

tope detection. The same method was now developed

for the N-1-adenine adduct of GA. GA was reacted

with deoxyadenosine, deoxyadenosine-3'-monophos-

phate and deoxyadenosine-5'-monophosphate. The

N-1 products were isolated and characterized by UV

and mass spectrometry. The GA-N-1-deoxyadenosine-

3'-monophosphate standard was postlabelled and

analysed by HPLC. GA-reacted DNA was enzymatical-

ly digested and analysed by the developed postla-

belling assay. The results show high sensitivity and

specificity of the assay. This method will be used for

analysis of DNA from experimental animals exposed

to low doses of acrylamide.

The support of the EU CA Childrengenonetwork

QLK4-ct-2002-02198 is acknowledged.


Poster Presentation

PW1012

DNA-DAMAGE REDUCING EFFECTS OF FLAVONOID

SUPPLEMENTATION IN A HUMAN INTERVENTION

STUDY

LC Wilms, JCS Kleinjans


The Netherlands

Flavonoids are natural polyphenols, which are impor-

tant constituents of fruits, vegetables, nuts, tea and

red wine. Flavonoids are claimed to be able to protect

against cardiovascular disease, certain forms of can-

cer and ageing. In this project quercetin is used as a

model for the abundant group of flavonoids.

Aim of this study is to determine whether a four-

week period of increased quercetin intake through

foodstuffs enhances the anticarcinogenic defence

mechanism in healthy volunteers.

During a four-week period eight volunteers increased

their quercetin intake through consuming one litre of

a mixture of blueberry juice (50%) and apple juice

(50%). Blood was collected before, during and after

the intervention, and dietary flavonoid intake was

determined based on the subject’s dietary records.

Quercetin levels and antioxidative capacity were

determined in plasma. Peripheral blood lymphocytes

(PBL) were isolated and exposed ex vivo to an effec-

tive dose of benz(a)pyrene (B(a)P) and hydrogen per-

oxide. As indices for DNA damage BPDE-DNA adducts

and oxidative damage were measured by 32P-post-

labelling and COMET-assay, respectively.

Results: four weeks of supplementation of quercetin

through foods led to a significant increase in plasma

quercetin content (p=0.0277). Consequently, the total

antioxidative capacity of plasma, reflected by the

TEAC (trolox equivalent antioxidative capacity) value,

showed a significant increase (p=0.0425). The inter-

vention appeared to have protective effects on ex vivo

induced DNA damage in PBL. After the intervention

the level of oxidative damage upon exposure to H2O2

was decreased (p=0.0679, n=4). BPDE-DNA adduct

levels vary enormously between subjects, but in 5 out

of 7 subjects the DNA adduct level at t=4 is lower than

that at t=0. Further, large interindividual differences

in these effects and in absolute levels of damage were

observed, possibly indicating a role of polymor-

phisms in genes encoding anticarcinogenic/antiox-

idative defence.



Poster Presentation

PW1013

ANTIMUTAGENIC EFFECT OF PHENETHYL ISO-

THIOCYANATE AND ITS EFFECT ON THE IMMUNE

RESPONSE IN MICE

IVO Barta, P Smerak, Z Polfvkova, J Bártová, B Turek,

M Langová

Charles University, PRAGUE, Czech Republic

Phenethyl isothiocyanate (PEITC) was applied to mice

for three days in doses of 50 mg/kg, the third day a

carcinogen was also applied - either aflatoxin B1

(AFB1) 1 mg/kg, 2-amino-3-methylimidazo-4,5-f quino-

line (IQ) 20 mg/kg and N-nitroso-N-methylurea

(MNU) 20 mg/kg. PEITC demonstrably inhibits the

mutagenic activity of the three mutagenes: IQ, AFB1

and MNU in the mammalian model (micronucleus

test) in the bone marrow of laboratory mice. In the

procaryotic model (Ames test) a strong inhibition of

the mutagenic activity of IQ and AFB1 by PEITC has

been demonstrated. The protective effect of PEITC

was assessed by chemiluminiscence method and by

the blastic transformation method testing the func-

tional capacity of T-lymphocytes. The phagocytic

function of murine peritoneal granulocytes was sup-

pressed significantly by all 3 carcinogenes tested just

like blast formation from mitogen-stimulated

murine splenocytes. There was no statistically signif-

icant differences between groups of animals treated

with PEITC alone and those not treated, in chemilu-

miniscence follow-up and as regards blastic transfor-

mation. In all experiments PEITC to a significant

degree repaired the immunosuppressive effects of all

three carcinogenes tested. Results of this study indi-

cate that, PEITC may play an important role in pre-

vention of carcinogenesis by capability to modulate

immunosuppresion caused by carcinogenes.

Supported by grant IGA No. 6138 and Research Plan

No. MSM 111200001.


Poster Presentation

PW1014

IDENTIFICATION OF PROTEINS INVOLVED IN THE

PROTECTION AGAINST COLORECTAL CANCER BY

VEGETABLES

G Breikers, SGJ van Breda, FG Bouwman, MH van

Herwijnen, JW Renes, ECM Mariman, JCS Kleinjans,

JHM van Delft


The Netherlands

Colorectal cancer is one of the most common cancers

worldwide. There is abundant epidemiologic evi-

dence that colorectal cancer risk is decreased by con-

sumption of diets high in vegetables. Although the

evidence from animal studies is less clear, it was

found that vegetables or their components reduced

colorectal cancer risk. Vegetable components can,

directly or indirectly, influence gene expression lead-

ing to a change in transcription rate of a given gene.

Changes in gene expression will be translated into

the proteome. At present, however, the molecular tar-

gets at the genome and proteome level are largely

unknown. In our study, the effect of a vegetable rich

diet on protein expression in the colon mucosa of

healthy mice was studied by means of Proteomics.

We found that the expression of several proteins was

changed upon vegetable consumption, including

GAPDH, carbonic anhydrase I and high mobility

group protein 1 that are proteins involved in apopto-

sis or differentiation. In addition, we convincingly

showed that it is possible to link the up- or downreg-

ulation of certain proteins with their gene expres-

sion. In conclusion, these data indicate that vegetable

rich diets may exert their anti-carcinogenic effects by

changing gene- and protein expression profiles.

54


55


Poster Presentation

PW2001

THE ROLE OF PARP-1 AND XRCC1 IN CAMP-

TOTHECIN-INDUCED HOMOLOGOUS RECOMBI-

NATION

KM Parker, T Helleday

University of Sheffield, SHEFFIELD, United Kingdom

Poly (ADP-ribose) polymerase 1 (PARP-1) is a nuclear

protein responsible for detecting and signalling DNA

strand breaks and has a role in base excision repair

(BER) through interaction with XRCC1. We hypothe-

sise that the loss of PARP-1 or XRCC1 results in an

increase in homologous recombination, due to the

loss of a functional BER pathway.

In this study, we have used camptothecin (CPT), a

potent inhibitor of topoisomerase I (which creates

DNA double strand breaks (DSBs)), and derivatives of

which are used in the treatment of several cancers.

Recently it has been discovered that co-treatment

with CPT and PARP-1 inhibitors increases the cytotox-

icity of CPT. Here, we investigate the role of PARP-1

and XRCC1 in CPT toxicity, in the hope that this data

will prove useful in the future treatment of cancers.

We show that the treatment of PARP-1 -/- mouse

embryonic fibroblasts with CPT increases cell death,

and when treated with CPT for 24 hours, these cells

have an increased number of DSBs. The same effect

was seen when wildtype cells were co-treated with

CPT and PARP-1 inhibitors. We also show that the

number of RAD51 foci is spontaneously increased in

PARP-1 -/- cells and increases further after treatment

with CPT. We also observe that XRCC1 -/- cells have

increased sensitivity to CPT, have increased DSBs

after treatment with CPT, and have increased sponta-

neous and CPT-induced RAD51 foci.

We conclude that PARP-1’s role in BER protects CPT-

induced single strand breaks from causing DSBs,

through interaction with XRCC1 and allowing the BER

enzymes to repair the damage. In the absence of

either of these proteins, the single strand breaks

remain unrepaired, and DSBs are formed, leading to

homologous recombination. Therefore, co-treatment

with CPT and PARP-1 inhibitors increases cytotoxicity

through abolition of the BER pathway.


Poster Presentation

PW2002

IN-VIVO GENOTOXICITY OF THE SYNTHETIC

PYRETHROID PESTICIDE 'CYPERMETHRIN' IN

RATE LIVER CELLS BY COMET ASSAY

N El-Khatib1, M Abd El-Aziz2, Y Badr2, N Kamal1

1 Central Agricultral Pesticides Lab., GIZA, Egypt2 National Institute of Laser Sc, GIZA, Egypt

The comet assay (single-cell gel electrophoresis,

'SCGE') is a simple method for measuring deoxyri-

bonucleic acid (DNA) strand breaks in eukaryotic

cells. The assay has applications in testing different

chemical and physical agents for genotoxicity and

monitoring environmental contamination with

genotoxins. The objective of the present study was to

evaluate the genotoxic effects of the synthetic

pyrethroid pesticide 'cypermethrin', which is widely

used in Egypt in pest-control programs in agriculture

and in public health as well. Male rats were sacrificed

1, 7 or 14 days after administration of single oral dose

1/30, 1/10 or 1/5 LD50 of commercial formulation of

cypermethrin. Single liver cell suspensions were pre-

pared and a Comet assay was performed. With the

SCGE assay, a clear induction of DNA was observed. It

is generally noticed that all pesticide treatments

yielded statistically significant (p<0.0001) DNA dam-

age. In conclusion, cypermethrin induced a clear sig-

nificant positive dose-dependent increase in DNA

damage in the rat liver cells exposed to cypermethrin

as compared with controls. But the effects in the SCGE

were generally decreased with time after treatments.

The results of the present work suggested that comet

assay might be a suitable and sensitive endpoint in

genotoxicity evaluation of pesticides, but we confirm

that various tests should be used for detecting the

mutagenic activity of pesticides.



Poster Presentation

PW2003

DNA ADDUCTS AND MUTAGENIC SPECIFICITY OF

THE UBIQUITOUS ENVIRONMENTAL POLLUTANT

3-NITROBENZANTHRONE IN MUTA MOUSE

VM Arlt1, T Suzuki2, L Zhan2, HH Schmeiser3,

M Honma2, M Hayashi2, DH Phillips1

1 Institute of Cancer Research, SUTTON, SURREY,

United Kingdom2 Federal Institute of Health Sciences, TOKYO, Japan3 German Cancer Research Center, HEIDELBERG,

Germany

3-Nitrobenzanthrone (3-NBA) is an extremely potent

mutagen in the Ames assay and a suspected human

carcinogen identified in diesel exhaust and in ambi-

ent airborne particulate matter. To evaluate the in-

vivo mutagenicity of 3-NBA, we analysed the mutant

frequency (MF) in the cII gene of various organs in the

lambda/lacZ transgenic mouse (Muta Mouse) after

intraperitoneal treatment with 3-NBA (25 mg/kg

body weight per week for 4 weeks). Increases in MFs

were found in colon, liver and bladder, with 7.0-, 4.8-

and 4.1-fold increases above the control value, respec-

tively, whereas no increase in MF was found in lung,

kidney, spleen, and testis. The sequence alteration of

the cII gene recovered from 41 liver mutants from 3-

NBA-treated mice were compared with 32 sponta-

neous mutants from untreated mice. Base

substitution mutations predominated for both 3-

NBA-treated (80%) and untreated (81%) groups.

However, the proportion of G:C to T:A transversions in

the mutants from 3-NBA-treated mice was higher

(49% versus 6%) and the proportion of G:C to A:T tran-

sitions was lower (10% versus 66%) than in the

mutants from untreated mice. The increase in MF in

the liver was associated with strong DNA binding by

3-NBA, whereas in lung, in which there was no

increase in MF, a low level of DNA binding was

observed (268.0-282.7 versus 8.8-15.9 adducts per 10(8)

nucleotides). DNA adduct patterns with multiple

adducts spots (spots 1-6) were observed in all tissues

examined. Using HPLC co-chromatographic analysis

we confirmed that all major 3-NBA-DNA adducts pro-

duced in vivo in mice are derived from reductive

metabolites bound to purine bases (with 70-80%

deoxyguanosine and with 20-30% deoxyadenosine in

liver). These results suggest that G:C to T:A transver-

sions induced by 3-NBA are caused by misreplication

of adducted guanine residues through incorporation

of adenine opposite the adduct ('A'-rule).


Poster Presentation

PW2004

MOUSE MICRONUCLEUS TEST ON PERIPHERAL

BLOOD RETICULOCYTES: FLOW CYTOMETRIC

ANALYSIS AFTER MULTIPLE BLOOD SAMPLING

M de Boeck, BJ van der Leede, A de Smedt,

M Steemans, F van Goethem, A Lampo, PH Vanparys


Belgium

The current OECD and ICH guidelines for the mam-

malian erythrocyte micronucleus test consider flow

cytometry as an acceptable substitute for microscop-

ical evaluation, provided that the applied method is ade-

quately validated. The MicroFlow'PLUS Micronucleus

Analysis Kit (Litron Laboratories, Rochester, USA) con-

ceivably meets this requirement and determines

micronuclei in both reticulocytes and normochro-

matic erythrocytes of mouse peripheral blood using

flow cytometry. The method is based on the binding

of FITC-labelled antibodies to CD71 of immature reticu-

locytes and on parallel RNA degradation and propidi-

um iodide staining of DNA present as micronuclei.

A biological standard of mouse erythrocytes from ani-

mals infected with the malaria parasite Plasmodium

berghei is used to assure an accurate and reliable

flow cytometer set up.

The objective was to employ this method and to eval-

uate it for its utility and sensitivity following our in-

house experimental testing strategy. Time- and

dose-dependent induction of micronucleated reticu-

locytes was assessed in peripheral blood of mice

treated with nine reference compounds applying a

multiple blood sampling regimen of the same ani-

mals (i.e. before and at 48h and 72h after treatment).

Five known clastogens, two known aneugens and two

compounds previously reported to be inactive in the

mouse micronucleus test were evaluated. All known

mutagens produced a dose-dependent increase in

micronucleated reticulocytes with the highest

response at 48h after treatment, except in the case of 5-

fluorouracil which had its peak response at 72h. The

compounds previously shown to be inactive in the in

vivo micronucleus test were also negative using the

present methodology. Multiple blood sampling of the

same animal before and after treatment could be

applied without altering the sensitivity of the assay.

The flow cytometric assessment of MN-RET in mouse

peripheral blood using MicroFlow'PLUS is a sensitive

method with high analysis throughput and robust

quality control.

56



Poster Presentation

PW2005

INDUCTION OF EXCISION REPAIRABLE DNA

LESIONS IN LYMPHOCYTES EXPOSED TO AMINOLE-

VULINIC ACID IN-VITRO

Y Duydu, A Dur

Ankara University, ANKARA, Turkey

The inhibitory effect of lead compounds on ≠ -amino-

levulinic acid dehydrogenase (ALAD) accounts for an

accumulation of aminolevulinic acid (ALA) that can

generate reactive oxygen species (ROS) leading to an

oxidative DNA damage in lead exposure. Recently

this indirect mechanism has become more popular in

evaluating the genotoxic potential of lead com-

pounds because of the fast oxidation of ALA with con-

comitant generation of ROS. In the present study lead

and ALA were compared in terms of their ability to

induce excision repairable DNA lesions in lympho-

cytes. Because of its high sensitivity and specificity in

detecting agents inducing excsion repairable DNA

lesions, cytosine arabinoside/cytokinesis block

micronucleus (ARA-C/CBMN) assay was used in order

to convert excision repairable DNA lesions to MN.

Accordingly whole blood lymphocyte cultures were

exposed to lead and ALA at the moment of culture ini-

tiation (G0 phase) at three increasing doses (5, 10, 50

µM) with and without ARA-C treatment. The degree

of synergism (DS) between ALA and ARA-C was calcu-

lated as 0.87, 1.51 (p<0.01) and 1.78 (p<0.01) at doses of

5, 10, 50 µM ALA respectively. However the calculated

DS between lead and ARA-C was 0.83, 1.41 and 1.43 at

doses of 5, 10, 50 µM lead respectively. When this

results were taken into consideration 10 and 50 µM

ALA exposure clearly increased the number of lesions

converted to MN (DS=1.51, 1.78 respectively, 'p<0.01')

wheras the DS between lead and ARA-C was not sta-

tistically significant. Finally our results could be eval-

uated as a supportive evidance for the ALA mediated

indirect mechanism for genotoxic effects of lead

exposure.


Poster Presentation

PW2006

MUTATIONAL SPECIFICITY OF ESCHERICHIA COLI

DNAQ49 AND THE EFFECT OF UMUD(D’)C PRO-

TEINS

EG Grzesiuk, M Wrzesinski, JN Nieminuszczy,

A Nowosielska

Institute of Biochemistry and Biophysics PAS,

WARSZAWA, Poland

The high replication fidelity in Escherichia coli is

ensured by the proofreading activity of the ´ subunit

of the main replicative polymerase, pol III. Mutations

in encoding ´ - dnaQ lead to defective pol III, and are

strong mutators.

The contribution to translesion replication of pol III

and induced as a part of the SOS response umuDC-

encoded pol V has not been satisfactorily explained.

Some observations regarding E.coli dnaQ49 indicate

pol V as the only polymerase involved in replication

past cis-syn cyclobutane thymine dimers, whereas

others, indicate efficient TR past this lesion in strains

deficient in pol V and with an inactivated ´ subunit.

In the latter case it is evident that both polymerases,

pol V and pol III mutated in ´ , influence the level and

specificity of spontaneous mutations in E.coli.

The aim of this study was to analyse the mutational

specificity of dnaQ49 in umuDC+ and umuDC- back-

ground in the genetic system developed by J.H.Miller.

The system includes a set of 11 lacZ mutants and

allows to identify 6 substitutions and 5 specific

frameshifts. Using this system we established the

specificity of recessive dnaQ49 allele grown at per-

missive 28°C and non-permissive 37°C. We have found

that dnaQ49 grown at 37°C induces mainly GC:TA

transversions and +1A frameshifts; at 28°C AT:TA

transversions dramatically decreased and +1G pre-

vailed among the frameshifts. Introduction of

umuDC deletion into the dnaQ49 strain at 28°C result-

ed in noticeable increase in AT:TA transversions and

decrease in GC:AT transitions. At 37°C no such differ-

ences between dnaQ49 and dnaQ49dumuDC have

been observed. However, there were enormous differ-

ences in frameshifts between these strains. In

dnaQ49dumuDC there were only two groups of

frameshifts, -1G and +1A. At 28°C mutations resulted

mainly in -1G, whereas at 37°C both groups were

equally numerous.

57



Poster Presentation

PW2007

CONTRIBUTION OF E.COLI ALKB PROTEIN IN THE

ALKYLATING DNA REPAIR

JN Nieminuszczy, C Janion, M Wrzesinski, EG Grzesiuk

Institute of Biochemistry and Biophysics PAS,

WARSZAWA, Poland

Potentially mutagenic and genotoxic alkylating

agents are widely spread in nature. They are present

in the living cells and are major deleterious agents in

the environment. It is widely accepted that the muta-

genic effect of the alkylanes results from alkylation of

bases in DNA. All organisms possess mechanisms

that protect cells against the harmful effects of alky-

lanes. In E. coli these defence systems include: tag and

ogt genes, expressed constitutively; ada, alkB (form-

ing one operon) and alkA and aidB that are expressed

after induction of the adaptive response.

Even though AlkB protein is known since 1983 its role

in damaged DNA repair has been established recent-

ly. AlkB is a dioxygenase that oxidatively demethy-

lates N1meA and N3meC in DNA in a reaction

involving ketoglutarate, O2, and Fe II, resulting in

recovery of the natural A and C bases. In this work we

show that AlkB protects the cells against mutagenic

activity of MMS and non-repaired N1 meA and

N3meC being a potent inducers of mutations. In E. coli

AB1157 defective in alkB the frequency of MMS-

induced argE3:Arg+ reversion is much higher than in

AB1157 alkB+ strain, and depends on the type of alkB

mutation (point or disrupted). These Arg+ reversions

are mainly due to AT:TA transversions, and are fully

dependent on umuC encoded DNA polymerase V. In E.

coli AB1157alkB umuC36 double mutant the frequency

of argE3:Arg+ reversion is dramatically reduced.

Therefore, we infer that alkB protects the cells not

only against deleterious, but also mutagenic activity

of MMS. In Miller’s strains bearing point mutations,

we have found that specificity of MMS-induced

mutations depends on growth conditions. When

MMS-treated cells were incubated in minimal medi-

um (MM) deprived of proline, mutations arise mainly

by GC:AT transitions, whereas in MM enriched with

proline they arise mainly by GC:TA transversions.


Poster Presentation

PW2008

DAMAGE TO DNA AND ITS REPAIR IN RADIO-

IODINE TREATED PATIENTS WITH AUTONOMOUS

THYROID NODULE

EG Grzesiuk1, JN Nieminuszczy1, M Kruszewski2,

MT Plazinska3, W Grzesiuk3

1 Institute of Biochemistry and Biophysics PAS,

WARSZAWA, Poland2 Institute of Nuclear Chem. Technol, WARSZAWA,

Poland3 University Medical School, WARSZAWA, Poland

The purpose of this study is to evaluate the DNA

breakage and base damage with the use of comet assay

in radioiodine treated patients with autonomous

thyroid nodule.

In all the patients thyroid scintigram was performed

using 131-I. The thyroid scan showed a single 'hot nod-

ule' with suppressed radioactive iodine uptake in

remaining thyroid tissue. The dose of administered

131-I was from 14 to 16 mCi. Damage to DNA was esti-

mated in a tissue from 'hot nodule' by fine needle

aspiratory biopsy and in lymphocytes (PBL). Samples

were taken three times: before radioiodine treatment,

12 and 54 days after.

Preliminary results (on four patients) indicate a high

diversity in the level of DNA damage between indi-

vidual patients. Generally, in lymphocytes 12 days

after 131-I application significant level of DNA break-

age and base damage was still observed. However,

after 54 days the level of DNA damage in lymphocytes

was even lower than in the control. On the contrary,

in 'hot nodule' cells DNA damage persisted till 54th

day after 131-I treatment. Differences in the type of

damage between thyroid cells and lymphocytes were

also observed. In lymphocytes there were more base

damages while in nodule cells single strand DNA

breaks prevailed.

Our preliminary results indicate that the comet assay

can be a valuable tool for monitoring radioiodine

treated patients. It can also allowed to estimate prop-

er for each patient 131-I dose. Differences in the type

and persistence of DNA damage in lymphocytes and

thyroid nodule cells might indicate the different

mechanism of DNA damage induction and/or differ-

ences in DNA damage repair mechanisms.

58


59



Poster Presentation

PW2009

EVALUATION OF CONDENSATE EFFECTS FROM

STANDARD CIGARETTES USING IN VITRO ASSAYS

CA Andreoli, F Flamma, A Martino, A Nunziata

Eti SpA, ROME, Italy

Cigarette smoking is a known risk factor for the

development of several pathologies. In this context,

the study of cellular response induced by condensate

smoke cigarette (CSC) can be useful in view of better

understanding its biological activity.

In this study, CSC, collected on Cambridge filter and

dissolved in DMSO, was used. All experiments were

performed on Swiss/3T3 fibroblast. Cell survival, after

24h of treatment with 25, 50, 100, 150 mg/mL of CSC,

was evaluated employing NRU, MTT test and plating

efficiency cloning (PE). The same experimental condi-

tions were adopted to study the apoptosis induction

through an in situ marker to measure caspase activa-

tion followed by staining with Hoechst. Finally DNA

lesions were evaluated with comet assay treating

cells with 50, 100, 150, 180 mg/mL of CSC for 90min.

NRU and MTT assays demonstrated that CSC induced

dose-related cytotoxicity response, with IC50 values

of 80-85 mg/mL. The results from PE assay showed

that cells lost their ability of forming colonies after

treatment with 100mg/mL and the presence of giant

cells, morphologically like senescent cells with flat-

tened cytoplasm and multinucleated, was observed

at lower doses after 168h from the end of treatment.

Apoptosis studies highlighted cells caspase-positive

after CSC treatment. To examine whether activation

of caspases correlates with changes in nuclear mor-

phology, cells were labelled with Hoechst-33258

revealing the presence of apoptotic bodies and chro-

matin aggregation dose-depending. Preliminary

results from comet assay showed that CSC was able to

induce DNA strand breaks in a dose-dependent rela-

tionship.

These results show that cytotoxicity and genotoxicity

in vitro assays can be used for studying the biological

activity of complex mixtures, such as CSC. Moreover

these assays are a suitable strategy for measuring the

effects of the reduction or removal of hazardous com-

pounds and the progresses towards the production of

safer cigarettes.


Poster Presentation

PW2010

SYNTHESIS OF OLIGONUCLEOTIDE ANALOGUE

FOR DNA REPAIR

N Koissi, H Lönnberg

University of Turku, TURKU, Finland

The process by which normal cells become progres-

sively transformed to malignancy is now known to

require the sequential acquisition of mutations

which arise as a consequence of damage to the

genome. Mutagenic compounds act on DNA most

commonly by forming adducts; other types of dam-

age include oxidation, deamination and depurination

of the nucleosides.

DNA damages, mediated by bifunctional chemical

agents are known to form covalent cyclic adducts

with DNA bases. While cyclic adducts as such may be

classified as mutagenic DNA lesion, those bearing

additional functional groups that are able to interfere

with the base-pairing are of particular interest. Thus,

evolution has developed complex enzymatic path-

ways that recognize and repair DNA damage. The rate

of cleavage was found to differ with the lesion and

was also affected by neighbour sequences geometry

and the resulting local conformational changes,

which can be sequence-dependent.

For an understanding, the systematic study of the

biochemical and biological effects of these various

types of DNA damage, DNA in which the adduct or

other damage is placed in a structurally specific man-

ner in the DNA is needed. It has been our goal to char-

acterize adducts,1 and to develop convenient and

versatile synthetic approaches to oligodeoxynu-

cleotides containing structurally defined adducts.2

The adducted oligonucleotides have been useful not

only for establishing the biological effects of lesions,

and the fluorescent properties of the oligonucleotide,

but also for biophysical studies, in particular two-

dimensional NMR, with the goal of establishing the

structural basis for replication and other types of bio-

logical processing.

[1] Neuvonen K., Koissi N., Lönnberg H.: J. Chem. Soc.,

Perkin Trans. 2, 2002, 173

[2] Koissi N., Lönnberg H.: Nucleosides, Nucleotides &

Nucleic Acids, 2003, 22, 1135.

60



Poster Presentation

PW2011

SYNTHESIS AND CHARACTERIZATION OF DNA

ADDUCTS FROM MUTAGENIC 3-NITROBENZAN-

THRONE PRESENT IN ATMOSPHERIC ENVIRON-

MENT

TT Takamura1, M Kawanishi2, Y Nakagawa3,

T Watanabe3, T Hirayama3, K Wakabayashi1,

Y Hisamatsu4, T Yagi2

1 National Cancer Center Reserach Institute, TOYKO,

Japan2 Osaka Prefecture University, OSAKA, Japan3 Kyoto Pharmaceutical University, KYOTO, Japan4 National Institute Public Health, TOKYO, Japan

3-Nitrobenzanthrone (3-NBA) is a powerful mutagen

present in air-borne particulate matter and is sug-

gested to be a carcinogen in rodents. 3-NBA is also

reported to be metabolically activated to form several

DNA adducts when subjected to mammalian cell

lines or rats. However, the chemical structures of

these adducts are not fully understood. In this study,

we analyzed the chemical structures of several DNA

adducts derived from 3-NBA in vitro. We have already

reported an efficient preparation method of dG-C8

adducts with several amino/nitroarenes. Using this

method, it becomes possible to prepare general types

of DNA adducts with 3-NBA; ie. N-(2'-deoxyguanosin-

8-yl)-3-aminobenzanthrone (dG-C8-ABA), 2-(2'-deoxy-

guanosin-N2-yl)-3-aminobenzanthrone (dG-N2-ABA)

and their N8-acetyl counterparts (N-Ac-dG-C8-ABA

and N-Ac-dG-N2-ABA). With authentic samples, we

found that dG-C8-ABA and dG-N2-ABA were formed

in the ratio of 10/1 in the reaction mixture of dG with

N-acetoxy-3-aminobenzanthrone, which was pre-

pared by selective O-acetylation of 3-hydroxyamino-

benzanthrone. The other putative ultimate mutagen,

N-acetoxy-N-acetyl-3- aminobenzanthrone (N-Aco-N-

Ac-ABA), was also found to give N-Ac-dG-C8 ABA and N-

Ac-dG-N2-ABA, accompanied with an unusual C-C

bonded type of DNA adduct, 2-(2'-deoxyguanosin-8-yl)-3-

acetylaminobenzanthrone, whose formation has

already been reported by us. Moreover, N-Aco-N-Ac-

ABA was shown to easily react with 2'-deoxyadeno-

sine (dA) to give a novel type of DNA adduct in which

an aziridine ring formation took place between the

double bond of the 1,11b-position of 3-acety-

laminobenzanthrone and N6 of dA (1,11b-(2'-

deoxyadenosin-N6-yl)-3-acetylaminobenzanthrone).

The efficiency of the formation of this aziridine types

of adduct was similar to that of N-Ac-dG-C8-ABA. We

are now undataking detection of in vivo formation of

these DNA adducts.


Poster Presentation

PW2012

ROLE OF DAMAGE SPECIFIC DNA POLYMERASES

IN MUTAGENICITY OF LIPID PEROXIDATION-

INDUCED DNA ADDUCTS

B Tudek, B Rusin, B Tudek

Institute of Biochemistry and Biophysics, WARSAW,

Poland

Lipid peroxidation leads to the formation of a large

family of alkenals, among which trans-4-hydroxy-2-

nonenal (HNE) is one of the most ubiquitous. HNE

forms adducts to all four DNA bases, which are cyclic

propano- or ethenoadducts bearing a hexyl- or hep-

tyl- side chains. We were studying mutagenesis and

repair of HNE-DNA adducts in E. coli. dsM13 phage

DNA was modified in vitro with HNE, and transfected

into wild type E. coli as well as DNA repair and DNA

polymerases II, IV and V deficient mutants. The defi-

ciency of nucleotide excision repair, increased E. coli

susceptibility to HNE, as well as increased mutation

frequency in M13 lacZ gene. In uvrA strain about 44%

of mutations were recombinations between sequences

of M13 lacZ gene and lacZ gene of E. coli F’ factor, while

in the wild type the level of recombinations remained

low (8%). Additional dysfunction of DNA polymerase

IV in uvrA mutant dramatically decreased the survival,

but had no effect on mutation frequency in compari-

son to the wild type E. coli. In the strain lacking

UvrABC excinuclease and DNA polymerase V, the sur-

vival was higher than in uvrA din strain, but no HNE-

induced mutations were observed. In uvrA din polB

triple mutant, survival decreased dramatically, and

mutation frequency increased substantially in com-

parison to the wild type strain. These results suggest

that, in E. coli, HNE adducts to DNA bases are

processed by nucleotide excision repair system,

recombination and damage-specific DNA polymeras-

es. The data suggest that DNA polymerase II and

probably DNA polymerase IV are able to bypass HNE-

adducts in an error-proof mode, while DNA poly-

merase V is responsible for mutation induction on

HNE-damaged templates.

61


62


Poster Presentation

PW2013

INHIBITION OF MYELOPEROXIDASE ACTIVITY BY

NITRITE: IMPLICATIONS FOR NEUTROPHIL-MEDI-

ATED DNA STRAND BREAKAGE

AM Knaapen1, RPF Schins2, PJA Borm2, FJ van Schooten1


The Netherlands2 IUF GmbH Düsseldorf, DUSSELDORF, Germany

Hydrogen peroxide has been shown to be a crucial

factor in neutrophil-induced DNA strand breakage. In

general, H2O2 is consumed by myeloperoxidase (MPO)

to form HOCl. However, recently it was shown that

nitrite, also present at sites of inflammation, is a

potent MPO inhibitor. The consequences of this effect

for neutrophil-induced genotoxicity remain to be elu-

cidated. To this end, we co-incubated activated neu-

trophils with rat alveolar epithelial cells (RLE) in the

presence of nitrite (0-100 µM) or the specific MPO

inhibitor 4-amino benzoic acid hydrazide (4-ABAH, 0-

100 µM). DNA strand breakage in the RLE cells was

evaluated using single cell gel electrophoresis. H2O2

release and MPO activity were determined spec-

trophotometrically. Both nitrite and 4-ABAH were

found to inhibit MPO activity in a dose-dependent

manner. This was paralleled by a significant increase

in DNA damage in RLE cells, whereas no strand break-

age was induced upon incubation with nitrite or 4-

ABAH in the absence of neutrophils. To further

evaluate the underlying mechanisms, in vitro experi-

ments were performed in which H2O2 was allowed to

react with purified MPO before addition to the RLE

cells. In the presence of MPO no subsequent cellular

DNA damage could be detected. However, when

nitrite (100 µM) was added to these pre-incubations,

DNA damage in the RLE cells was comparable with

levels as induced by H2O2 alone. Inhibitory experi-

ments with catalase further confirmed that these

effects were H2O2 dependent. In conclusion, the pres-

ent data demonstrate that physiological levels of

nitrite increase the DNA damaging capacity of neu-

trophils. This effect is likely due to nitrite-induced

inhibition of MPO, which increases the availability of

H2O2. Further studies are needed to assess the impli-

cation of these observations for in vivo DNA damage

in relation to neutrophilic inflammation.



Poster Presentation

PW2014

MECHANISMS OF PRILOCAINE AND LIDOCAINE

CYTOTOXIC EFFECTS IN HEPG2 AND V79 CELL

LINES

GR Rodrigo, B Mayerhofer, E Richter

Ludwig Maximilian Universität, MUNICH, Germany

Prilocaine as lidocaine are amide-type local anes-

thetic agents that are used for regional, nerve block

and topical anesthesia. They are eliminated almost

exclusively by hepatic biotransformation and renal

excretion. In humans, the main metabolic pathway of

lidocaine is sequential N-deethylation to monoethyl-

glycinexylidide and glycinexylidide. Both metabolites

can be hydrolyzed to 2,6-dimethylaniline, which is

further oxidized to its main urinary metabolite 4-

hydroxyxylidine. Prilocaine is marketed as a racemic

mixture and both enantiomers have similar biologi-

cal activity. However, the S-(+) enantiomer is slowly

hydrolyzed, while the R-(-) enantiomer is quickly

hydrolyzed by hepatic amidase to 2-methylaniline (o-

toluidine). O-toluidine is metabolized in vivo into a

number of compounds, some of which are active

genotoxins. It produces DNA damage (single-strand

breaks and unscheduled DNA synthesis, UDS) and

causes cell transformation. Both 2,6-dimethylaniline

and o-toluidine have been shown to be carcinogenic

in animal studies. In vitro, metabolism of lidocaine as

well as o-toluidine has been shown to be mediated by

cytochrome P450 (CYP) 3A4 or CYP1A2. N-acetyltrans-

ferase (NAT) 1 in extra-hepatic tissues or NAT 2 in liver

could have a modulating effect in toxification of

o-toluidine.

Our study models are constituted of 2 cell lines, V79

and HepG2. V79 cell line derivated from Chinese ham-

ster lung fibroblasts has no CYP activity or hepatic

amidase expression. HepG2 cell line is derived from

human hepatocellular carcinoma and has the same

genetic potential as hepatocytes.

Accordingly, this study has the following aims:

1. Characterization of the metabolic pathways

involved in the activation of prilocaine and lido-

caine, leading to cytotoxic effects in both cell lines.

2. Evaluation of the effect of various modulators of

CYP activity on the cytotoxic potential of prilo-

caine and lidocaine in HepG2 cell line.

3. Determination of the metabolites involved in

adduct formation in both cell lines.


Poster Presentation

PW2015

GENOTOXICITY IN VITRO OF CHLORINATED

DRINKING WATERS OBTAINED FROM SURFACE

AND WATER TABLE

S Frigerio, S Radice, M Ferraris, E Chiesara, L Marabini

University of Milan, MILAN, Italy

We examined possible genotoxic effects of chlorinat-

ed drinking water concentrates in metabolically com-

petent human hepatoma cells (HepG2) using the

micronucleus assay (MN), single cell gel electrophore-

sis (comet assay) and cell cycle cytofluorometric

analysis.

The water samples were obtained from the aqueducts

of two towns with different types of water supplies:

town #1 uses water from a river, whereas town #2 is

supplied from a deep water table. In the case of town

#1 were also analysed the waters before conditioning.

Both towns use CLO2 for conditioning purpose. The

samples were concentrated by adsorption on silica

C18 cartridges and subsequently eluted.

In this study, the samples denominated RW (raw

waters) were obtained by concentrating the surface

water before conditioning, the A, R1 and R2 samples

were taken after conditioning at the point of admis-

sion into the aqueduct, and at two very distant from

each other points in distribution network respectively.

A preliminary toxicity evaluation was made using

NRU (neutral red uptake) and LDH (lactate dehydro-

genase) tests, wich revealed toxicity for concentrate

amounts starting from 0.5 Leq/ml of the A and R1

samples from town #2. No toxicity was found in all

other samples of both towns.

The short-term genotoxicity tests carried out with

waters samples at maximal non-cytotoxic doses

revealed a significant increase of tail moment (comet

assay) for point R1 of both town concentrates. For MN

test the results did not indicate statistically signifi-

cant effects for any examinated sample.

The results obtained indicate that non-cytotoxic

doses of water concentrates induce genotoxic dam-

age pointed out by comet assay and not by MN. These

evidences are not conflicting, because the two tests

have different genotoxic endpoints. Moreover these

data are in agreement with the unchanged cell cycle

distribution analysis.

63



Poster Presentation

PW2016

MITOCHONDRIAL DNA SEQUENCE ANALYSIS

AND MOLECULAR PHYLOGENY OF ORCHESTIA

CAVIMANA (CRUSTACEA) IN FRESHWATER GENO-

TOXICOLOGICAL STUDIES.

DD Davolos1, BM Pietrangeli1, N Maclean2

1 ISPESL, ROME, Italy2 University of Southampton, SOUTHAMPTON,

United Kingdom

The use of freshwater amphipods in toxicity assess-

ment is increasing since their usefulness has become

clear. However, various parameters must be consid-

ered when studying the toxicity of components to

wild life populations, taking into account the genetic

population structure and the evolutionary history of

the taxa. The talitrid amphipod Orchestia cavimana,

inhabiting beaches of freshwater lakes and rivers in

Europe and UK, is here chosen as a potential candi-

date for freshwater genotoxicology evaluations. We

initiated a molecular phylogenetic study of this semi-

terrestrial taxon based on the sequences of part of the

mitochondrial genes cytochrome oxidase subunit I

and subunit II (COI, COII). We analysed portions of the

COI and COII genes using neighbour-joining and

maximum parsimony inference using all nucleotides

plus their amino acid translations which have been

examined also by comparing the structural classes of

the COI and COII enzymes. In all the phylogenetic

trees, O. cavimana shows a basal placement with

respect to other talitrid ampihpods even of the same

genus (O. gammarellus, O. mediterranea). The low

genetic variation levels detected in samples of O. cav-

imana validate this crustacean for environmental

genotoxicity investigations at both micro- and

macrogeographic scales. Moreover this organism,

easily reared in laboratory terraria, could be used

under a variety of genotoxicological exposures. With

these considerations, molecular methods were con-

sidered for evaluating mitochondrial genotoxicity in

tissues of exposed bred organisms and populations

collected from contaminated sites.


Poster Presentation

PW2017

BREAST CANCER PATIENTS DNA DAMAGE AND

REPAIR EFFICIENCY

N Anagnostakis1, G Karanastasi1, N Messini-Nikolaki2,

K Gourgoulianis3, K Christou3, E Athanasiou3,

S Tsilimigaki1, SM Piperakis1

1 NCSR Demokritos, ATHENS, Greece2 University of Athens, ATHENS, Greece3 University of Thessaly, LARISA, Greece

In this study the effects of g-irradiation and ethanol

on lymphocytes from breast cancer patients was

examined.

Using the comet assay we estimated the DNA dam-

age and the repair efficiency on the above population

in comparison to controls.

The apoptotic and necrotic cell population was also

estimated.

Our results show a decreased DNA repair capacity in

lymphocytes from breast cancer patients. An increased

apoptosis was found if compared to healthy individuals.

64



Poster Presentation

PW2018

LUNG CANCER PATIENTS DNA DAMAGE AND

REPAIR EFFICIENCY

G Karanastasi1, N Anagnostakis1, N Messini-

Nikolaki2, K Gourgoulianis3, K Christou3,


1 NCSR Demokritos, ATHENS, Greece2 University of Athens, ATHENS, Greece3 University of Thessaly, LARISA, Greece

We examined the effects of ethanol and g-irradiation

in lymphocytes from lung cancer patients in compar-

ison to healthy individuals.

With the comet assay technique we estimated the

DNA damage and the repair capacity of the above

populations.

We also estimated apoptosis and necrosis in these

populations.

Our results show that lung cancer patients have a

reduced DNA repair capacity. Apoptosis was found

to increase in lung cancer patients if compared to

healthy individuals.


Poster Presentation

PW2019

VITAMINS' C AND E "PROTECTION" OF LYMPHO-

CYTES EXPOSED TO EXTERNAL AGENTS

K Maridaki1, G Christopoulos1, N Messini-Nikolaki2,


1 NCSR Demokritos, ATHENS, Greece2 University of Athens, ATHENS, Greece

In the present work we examined the 'protection'

offered in human lymphocytes by vitamins C and E

from the effects of UV and g-ray and H2O2.

We also measured the number of apoptotic and

necrotic cells after staining with acridine orange and

ethidium bromide.

Our results show that vitamin E and the mixture of

E+C show to 'protects' cells from the effects of UV,

g-ray and H2O2. Vitamin C alone however does not

show such clear 'protection'.

65



Poster Presentation

PW2020

GENOTOXICITY EVALUATION OF A DI-N-OXIDE

QUINOXALINE UNDER HYPOXIC CONDITIONS BY

THE COMET ASSAY

A Azqueta1, G Pachón2, A. Lopez de Cerain1

1 University of Navarra, PAMPLONA, Spain2 University of Barcelona, BARCELONA, Spain

Bio-reductive agents have being designed in order to

take advantage of the particular metabolic character-

istics of the resistant hypoxic regions of the solid

tumours. They are activated inside the hypoxic cells

to give active species that, in the presence of oxygen,

are oxidised back to the non-toxic parent compound.

Several quinoxalines 1,4-di-N- oxides have been

described as potential bio-reductive agent, among these,

the 7-cloro-3-[[(N,N-dimethylamino) propy]amino]-2-

quinoxalinecarbonitrile 1,4-di-N-oxide (Q-85), appears

the most promising.

In the present work the genotoxic effect of Q-85 in

hypoxia was studied in Caco-2 cells with the alkaline

comet assay. Cells were treated with 0.2 and 0.4 µM of

Q-85 during 2 hours under an hypoxic atmosphere.

The number of viable cells after the treatment was

determined by the Trypan blue exclusion method

and 18x104 cells/well were seeded in six-well plates.

After incubation at 37ºC and 5% CO2 for 24, 48, 72 and

168 hours, cells were trypsinized and counted with

the Tripan blue exclusion assay. The comet assay was

carried out just after the treatment and 24 and 48

hours later. Comets were classified by the visual scor-

ing method. All this procedure was carried out 3

times.

Just after treatment with 0.2 µM and 0.4 µM, viability

percentages were 95.6±4.7 and 88.2±8.9, but 24 h later,

these percentages decreased until 35.8±19.6 and

7.2±7.2, respectively. Only some cells treated with 0.2

µM were able to proliferate after 72 hours. The DNA

was very damaged just after the treatment (total

comet score, control = 42± 11; 0.2 µM = 343±30, 0.4 µM

= 399±1). After 24 and 48 hours, viable cells showed an

important repair of the DNA, which was reflected by

a decrease in the comet score, although control values

were not reached with either dose. These results indi-

cate that Q85 has a genotoxic effect.


Poster Presentation

PW2021

APNEA PATIENTS LYMPHOCYTES DNA DAMAGE

AND REPAIR CAPACITY

N Kontogianni1, N Messini-Nikolaki2, K Christou1,

C Pastaka1, K Gourgoulianis1, S Tsilimigaki3,

SM Piperakis3

1 University of Thessaly, LARISA, Greece2 University of Athens, ATHENS, Greece3 NCSR Demokritos, ATHENS, Greece

In this study we examined in a population of patients

with apnea, using the comet assay technique, the

DNA damage caused by g-irradiation and ethanol.

Furthermore, we studied apoptosis and necrosis.

Analysis of our results indicate that apnea patients

are more sensitive to the effects of external factors.

Apoptosis was found to be somehow higher in apnea

patients.

66


67


Poster Presentation

PW2022

DNA DAMAGE AND REPAIR EFFICIENCY IN COM-

MON VARIABLE IMMUNODEFICIENCY PATIENTS

LYMPHOCYTES

P Kanavetas1, N Messini-Nikolaki2, M Kanariou3,


1 NCSR Demokritos, ATHENS, Greece2 University of Athens, ATHENS, Greece3 Aghia Sophia Hospital, ATHENS, Greece

In the present study we examined with the comet

assay technique in a population of patients with

common variable immunodeficiency (CVID), the DNA

damage and repair efficiency after H2O2 and g-irradi-

ation treatment.

Apoptosis and necrosis were also investigated.

Analysis of our results indicates that CVID patients

are more sensitive to the effects of external factors.

Apoptosis was also found to be somehow higher in

CVID patients.


Poster Presentation

PW2023

PHARMACOLOGICAL BLOCKING OF ABC-TRANS-

PORTERS AND TOXICITY OF BENZO(A)PYRENE IN

MCF-7 CELLS

PK Myllynen1, T Kajosmaki1, L Vaskivuo1,

KV Vähäkangas2

1 University of Oulu, OULU, Finland2 University of Kuopio, KUOPIO, Finland

Benzo(a)pyrene, a polycyclic aromatic hydrocarbon

(PAH) present also in cigarette smoke, has a complex

metabolism in humans. The ultimate carcinogenic

metabolite, benzo(a)pyrene-7,8-diol-9,10-epoxide (BPDE),

can bind to DNA. ATP-binding cassette (ABC) trans-

porters protect cells from many foreign chemicals. In

addition to specific inhibitors many widely used

drugs inhibit function of these transporters. The aim

of this study is to clarify whether pharmacological

blocking of ABC-transporters affects benzo(a)pyrene

adduct formation. Also, it has been shown that the

level of BPDE-DNA- adducts correlates with the expres-

sion of p53 tumor suppressor protein. Therefore, p53

expression was also analysed.

MCF-7 breast adenocarcinoma cells were cultured

with benzo(a)pyrene (1microM) and the ABC-trans-

porter inhibitor verapamil (0.125 microM to 100

microM) for 24 and 48 hours. DNA was isolated from

samples using a phenol extraction/ethanol precipita-

tion method and BPDE-DNA adducts were analysed by

synchronous fluorescence spectrophotometry. The

level of p53 protein was studied with immunoblotting

using DO7 antibody.

Preliminary results show that BPDE-DNA adduct for-

mation after benzo(a)pyrene treatment was increased

by verapamil both at 24 and 48 hours (approximately

1.5 and 3.8 fold, respectively). Benzo(a)pyrene treat-

ment induced p53 protein as expected. We also have

some indications that low, but not high dose of vera-

pamil caused further increase in p53 expression at 48

hours.

According to these preliminary results it seems that

blocking of the ATP- binding cassette transporters

enhances benzo(a)pyrene toxicity in MCF-7 cells.



Poster Presentation

PW2024

SYNERGISTIC EFFECT OF CADMIUM AND ARO-

MATIC DNA ADDUCTS ON MUTAGENESIS DUR-

ING FETAL DEVELOPMENT

RWL Godschalk1, J Hogervorst1, H Albering1,

P Mercelina-Roumans2, FJ van Schooten1, J de Haan2,

JCS Kleinjans1


The Netherlands2 Academic Hospital Maastricht, MAASTRICHT,

The Netherlands

The foetus is exposed to xenobiotics via the mother’s

circulation, which is possibly involved in the develop-

ment of diseases in later life. Cadmium and lipophilic

genotoxins in umbilical cord blood of newborns may

have synergistic effects on mutagenesis in the hypox-

anthine-phosphoribosyl-transferase (HPRT) reporter

gene. Concentrations of zinc (Zn), lead (Pb) and cad-

mium (Cd) were determined in cord blood of 16 non-

smoking and 9 smoking healthy mothers by atomic

absorption spectrometry (AAS). Lipophilic-DNA

adducts in lymphocytes were determined in the same

subjects by 32P-postlabeling and the HPRT-mutant

frequency was assessed by selection of 6-thioguanine

resistant cells. Although the Cd/Zn ratio was 2.5 fold

higher in blood of smoking women than in non-

smoking women (0.10±0.02 and 0.04±0.01, respec-

tively, P=0.007), this difference could not be observed

in umbilical cord blood (0.03±0.01 and 0.03±0.01,

respectively, P=0.66). Similarly, mean DNA adduct

levels were increased in lymphocytes of smoking

women when compared to non-smoking controls

(0.99±0.31 adducts per 108 nucleotides and 0.43±0.12,

respectively, P=0.009), but were only marginally

higher in newborn children of smokers than in their

non-smoking counterparts (0.57±0.29 and 0.24±0.09,

respectively, P=0.38). Since Cd is known to effectively

inhibit DNA repair, we hypothesized that concomi-

tant exposure of neonates to Cd and genotoxic com-

pounds may result in an increased fixation of DNA

damage into somatic mutations. Indeed, taking both

biomarkers into account, the number of HPRT-muta-

tions per adduct correlated positively with the Cd

concentration in cord blood (r=0.61, P=0.001). These

data suggest a molecular link between DNA damage,

inhibition of DNA repair by Cd and in vivo mutagen-

esis during fetal development. Thus, simultaneous

exposures to heavy metals and DNA damaging com-

pounds act synergistically and may result in biologi-

cally relevant genotoxic effects in neonates.

68

BIOPREDIC INTERNATIONAL14-18, rue Jean Pecker - 35000 Rennes, France Tel: +33 (0)2 99 14 36 14Fax: +33 (0)2 99 54 44 72Web : www.biopredic.comE-mail : [email protected]

Biopredic international is a provider of assay systems for invitro toxicology:* S9mix* Peripheral Blood mononuclear cells (human origin)* Bcl2 recombinant CTLL2 cells, resistant against apoptosisand very valuable to discriminate between pro-apoptotic andmutagenic properties of xenobiotics,* and numerous other systems for drug discovery and drug developpment such as human and animal hepatocytes, kera-



Poster Presentation

PW2025

GENOTOXICITY OF FINE AND COARSE PARTICULATE

SAMPLES COLLECTED DURING CONTRASTING

POLLUTION SITUATIONS IN EUROPE (PAMCHAR)

R Duffin1, H Li1, ME Gerlofs-Nijland2, FR Cassee2,

FJ van Schooten3, RO Salonen4, PJA Borm5,

RPF Schins1

1 IUF, DUSSELDORF, Germany2 Nat.Inst.of Publ. Health and Environment,

BILTHOVEN, The Netherlands3 Maastricht University, MAASTRICHT,

The Netherlands4 KTL, KUOPIO, Finland5 IUF GmbH Düsseldorf, DUSSELDORF, Germany

Epidemiological studies have shown significant posi-

tive associations between ambient air particle (PM)

concentration and morbidity and mortality due to

cardiopulmonary diseases and lung cancer. These

adverse health effects have been consistently associ-

ated with inhalable (PM10) and fine (PM2.5) particles,

but the responsible biological mechanisms are large-

ly unknown. Coarse (PM10-2.5) and fine PM (PM2.5-

0.2) were collected using a high-volume cascade

impactor (HVCI) in six European cities: Duisburg

(autumn), Prague (winter), Amsterdam (winter),

Helsinki (spring), Barcelona (spring) and Athens

(summer). DNA strand breakage was assessed using

the comet assay and PAH-DNA adduct formation

was measured by 32-P postlabelling upon treatment

of A549 human lung epithelial cells. Considerable

sampling location/season and size fraction-specific

differences were observed in both endpoints.

Furthermore, a clear correlation with regard to the

fine PM was observed between particle PAH-content

and resulting DNA adduct formation. In ongoing

experiments, selected samples (fine and coarse PM

from Prague and fine PM from Duisburg), have been

used for intratracheal instillations in spontaneously

hypertensive rats (SHR). Subsequent genotoxicity

tests are carried out in isolated type II lung epithelial

cells from these animals. Preliminary results indicate

enhanced DNA strand breakage in animals instilled

with coarse PM. The contribution of pulmonary

inflammation to the observed DNA damage is cur-

rently under evaluation.


Poster Presentation

PW2026

UV-INDUCED SKIN TUMORIGENESIS IN MOUSE

STRAINS IMPAIRED IN THE INK4A/ARF LOCUS

AND DNA REPAIR

A van Schanke1, HJ van Kranen2, LHF Mullenders1,

FR de Gruijl1, LHF Mullenders1


The Netherlands2 National Institute of Public Health and the

Environment, BILTHOVEN, The Netherlands

We aimed to develop a mouse model for UV induced

malignant melanoma based on impairment of genes

involved in predisposition of humans to this disease

and environmental risk factors derived from epidemi-

ological data. Familial melanomas are often caused by

disruptions of CDKN2A (INK4a/ARF). Patients suffer-

ing the genetic disorder Xeroderma Pigmentosum are

also at high risk of developing melanoma, as a result

of dysfunctional DNA repair.

Mouse strains displaying such mutations were

exposed (neonatallly/adult) to ultraviolet (UV) light

and/or a chemical carcinogen and/or tumor promo-

tor (DMBA/TPA respectively). Ink4a/Arf ko (1), XPA ko

(2) and XPA* Ink4a/Arf double ko mice developed a

variety of skin lesions, most prominently papillomas

and nevi. Progression of benign nevi to malignant

melanomas was, however, not observed. The UV

exposure interval and dose with the highest yield of

nevi was also the most efficient inducer of

melanocyte proliferation in short-term experiments.

This regimen was used in further UV exposure exper-

iments with p16Ink4a specific mutant (3, with intact

p19Arf) and CDK4R24C mutant mice (4), both of

which developed spindle cell tumors but only few

nevi which is in contrast to previous experiments

with chemical carcinogens. A possible obstacle to the

formation of UV induced melanomas is the localiza-

tion of murine melanocytes, relatively deep in the

dermis where UVB light cannot penetrate efficiently.

Also the number of melanocytes may be low in nor-

mal murine skin. To circumvent the latter, pilot

experiments were started with K-14Slf mice (5), which

are strongly enriched in their epidermis for

melanocytes (due to K-14 driven expression of Steel

factor).

1. de Vries et al.(1995) Nature 377

2. Serrano et al.(1996) Cell 85

3. Krimpenfort et al (2001) Nature 413

4. Sotillo et al.(2001) EMBO 20

5. Kunisada et.al (1998) Development 125

(Dutch Cancer Society, UL2000-2303; European

Committee, QLK4-CT-199901084)

69



Poster Presentation

PW2027

IN VIVO GENOTOXIC EFFECTS OF A SYNTHETIC

PYRETHROID INSECTICIDE, CYHALOTHRIN IN

FISH

QM Khan, MA Haq, H Nisar

NIBGE, FAISALABAD, Pakistan

Lambda-cyhalothrin (Kerate) is one of the largest sell-

ing insecticides in Pakistan and many other coun-

tries. Pyrethroids can enter the aquatic environment

during agricultural use or by direct spraying of water

bodies. Thus study of their possible genotoxicity is of

immediate interest. The presence of genotoxins, even

in low doses, concerns aquatic as well as non aquatic

species through the food chain and drinking water.

The comet assay, can act as a biomarker of genetic

toxicity in fish.

The comet assay was used to test genotoxicity of

cyhalothrin in the common edible fresh water fish,

Cirrhinus mrigala (Mori), found through out the

Indian subcontinent. Six fish were exposed to each

concentration of cyhalothrin (0, 0.1, 0.2. 0.3 and 0.4

ug/L) for 36 h. The test concentrations of the insecti-

cide were based on LC50 doses of cyhalothrin for this

fish. Cyclophosphamide (5mg/L), was used as positive

control. Blood was collected from the caudal vein at 0,

4, 8, 24 and 36 h following cyhalothrin exposure and

comet assays were performed. A significant increase

in comet tail length, indicating DNA damage was

observed at all the doses with cyhalothrin when com-

pared to negative control. The mean tail length

showed a dose dependent increase. The maximum

increase in mean tail length for doses was observed

at 8 h. The reduction in tail length was evident after

24 h. After 36 h tail length values were near to nega-

tive control levels for all doses. This indicates repair of

damaged DNA. The results of the study suggest that

even low levels of cyhalothrin in the aquatic environ-

ment may have a significant genotoxic effect on fish

and accumulation of this pesticide in fish tissue could

also be of concern to human health.


Poster Presentation

PW2028

THE NOVEL UVS11 GENE OF CHLAMYDOMONAS

REINHARDTII AFFECTS CELL RESPONSE TO DNA

DAMAGE

EG Galova, A Sevcovicova, B Sviezena, M Slaninova,

D Vlcek

Comenius University FNS, BRATISLAVA, Slovak

Republic

The study of cell cycle regulation and checkpoints at

different levels has gained further insight into these

processes in different species. In comparison with

other lower eukaryotes there has been much less

progress in understanding cell cycle control in algae.

The first putative checkpoint mutant of green alga

Chlamydomonas reinhardtii have been isolated in

our lab and termed as uvs11. It has been predicted to

be UV-sensitive and repair-deficient. We assume now

that product of UVS11 gene is not required for DNA

repair itself but it is involved in cell response to DNA

damage.

In our work, we have assayed the activity of histone

H1 kinases presented in total protein extract and

those, mitosis specific ones bound to p13suc1-

Sepharose. The kinase activity was monitored in syn-

chronized cultures of untreated wild type and

mutant cells and compared with those irradiated by

UV-light (254nm) before mitosis. No substantial dif-

ferences between wild and mutant cells were found

in untreated cells. However, shortly after UV irradia-

tion the kinase activity in wild type has considerably

decreased, to recover about five hours later. The activ-

ity drop was accompanied by a prolongation of the

cell cycle due to a delay in mitoses, protoplast fis-

sions, and daughter cell release. The mutant strain

has shown no significant change in the course of

kinase activity and in other processes related to the

cell cycle after UV irradiation. Our present results

indicate that inhibition of histone H1 kinase could be

a way to arrest the initiation of mitosis in

Chlamydomonas and delay cell cycle progress in

response to DNA damage. We suggest that product of

the UVS11 gene affects cell response to DNA damage

and directly causes a decrease in kinase activity. This

implicates the role of UVS11 gene in regulation of the

cell cycle.

70


71


Poster Presentation

PW2029

ALKYLATING AGENTS INDUCE HOMOLOGOUS

RECOMBINATION IN ABSENCE OF DETECTABLE

DOUBLE STRAND BREAKS IN MAMMALIAN CELLS

CL Lundin1, F Johansson1, E Bolderson2, D Jenssen1,

K Erixon1, T Helleday2

1 Stockholm University, STOCKHOLM, Sweden2 University of Sheffield, SHEFFIELD, United Kingdom

Alkylating agents are potent inducers of sister-chro-

matid exchanges and chromosome aberrations, sug-

gesting that the subsequent lesions can be repaired

by homologous recombination (HR). Indeed, methyl

methanesulfonate (MMS) has been suggested to

induce DNA double-strand breaks (DSBs) in yeast and

is certainly inducing HR in that system. Here, we have

investigated the role of HR in repair of lesions induced

by MMS and N-methyl-N'-nitro-N-nitrosoguanidine

(MNNG) in mammalian cells. We show that the HR-

deficient hamster cell line irs1SF is equally sensitive

to MMS as the base excision repair (BER) defective

EM9 cell line and even more sensitive to MNNG,

showing that HR repairs alkylated DNA lesions. Both

agents induce HR at the hprt gene in the SPD8 cell

line, which we found is related to the amount of

O6-methylguanine produced by these agents.

Furthermore, inhibition of O6-methylguanine-DNA

methyltransferase increases recombination frequen-

cy following MMS or MNNG treatments, which alto-

gether suggest O6-methylguanine to be inducing HR.

In addition, and in contrast to what is believed to be

the case in yeast, we show that neither agent pro-

duces DSBs. We conclude that HR is a major pathway

in repair of O6-methylguanine, which is independent

on formation of a detectable DSB intermediate. We

speculate that HR is involved in repair of single-

strand break intermediates formed during BER.



Poster Presentation

PW2030

BCR/ABL ONCOGENIC TYROSINE KINASE STIMU-

LATES DNA REPAIR AND RESISTANCE TO DOX-

ORUBICINE IN K562 LEUKEMIC CELLS

IM Majsterek1, J Blasiak1, A Slupianek2, D Pytel1,

T Sliwinski1

1 University of Lodz, LODZ, Poland2 Temple University, PHILADELPHIA, United States

of America

Several fusion tyrosine kinases (FTKs) have been

detected in hematopoietic malignancies, one of the

most extensively studied being BCR/ABL, considered

as the pathogenic principle of Philadelphia (Ph) chro-

mosome-positive human leukemias. The BCR/ABL

fusion generated by a t(9;22) translocation mediates

its biological effects through deregulated, constitu-

tively active tyrosine kinase activity. BCR/ABL is

reported to participate in drug resistance in leukemo-

genesis. Our recent studies revealed a novel potential

mechanism of resistance in Ph-cells underlined by

the stimulation of DNA repair. In this work we exam-

ined a role of BCR/ABL in K562 cells derived from

human chronic myeloid leukemia in response to dox-

orubicin treatment. We used cells acquired resistance

to doxorubicin during long term exposure to the drug

in culture (K562R, resistant), and wild type K562 cells

(K562S, sensitive). The drug resistance was measured

by MTT assay. We found that K562R in contrast to

wild type K562S cells, exhibited accelerated kinetic of

DNA repair after doxorubicin treatment in the range

0.01–1 µM evaluated by the alkaline comet assay. Pre-

treatment of the K562R with a selective FTKs

inhibitor, imatinib (STI751) at 1 µM, reverted drug

resistance and slowed down DNA repair. Western blot

analysis with CRKL antibodies followed by cells cul-

ture in the presence of STI571 revealed inhibition of

the phosphorylation of the CRKL adaptor protein spe-

cific for Ph-leukemias confirming BCR/ABL inactiva-

tion by STI571. The results obtained suggest that

BCR/ABL in K562 cells may stimulate the repair of

DNA damages in response to the drug treatment and

be relevant to the resistance of the leukemic cells to

anti-cancer therapy.

Financial support by grant KBN 3P04 A03225.


Poster Presentation

PW2031

NEW APPROACHES TO IMPROVE HOMOLOGOUS

RECOMBINATION IN CHLAMYDOMONAS REIN-

HARDTII

M Slaninova1, L Fritsche2, M Pacanovska1, M Fusekova1,

G Treuner2, D Vlcek1, W Mages2

1 Comenius University FNS, BRATISLAVA, Slovak

Republic2 University Regensburg, REGENSBURG, Germany

The unicellular biflagellate green alga Chlamydomonas

reinhardtii is a popular model system used to study

photosynthesis, gametogenesis and mating, flagellar

assembly and cell division. The Chlamydomonas

genome which has recently been completed is cur-

rently being annotated and in the coming years func-

tional gene analysis in this organism will be in the

focus of interest. To this end, homologous recombina-

tion would be a highly desirable method, but unfor-

tunately Chlamydomonas (like higher organisms)

shows a very low rate of homologous recombination

by nature. Using two new dominant selectable

bacterial resistance markers directed against the

aminoglycoside antibiotics hygromycinB and paro-

momycin, respectively, we have constructed a trans-

genic Chlamydomonas strain as part of an in vivo

transformational readout system for homologous

recombination. In theory, this system allows positive

selection of homologous recombination events.

Currently we are trying to achieve this recombination

and in a second step we want to improve the rate of

recombination by pre- or post-treatment of trans-

formed cells with different physical or chemical stim-

uli.

In a parallel approach, we are trying to express a key

protein of bacterial recombination, the E. coli RecA

protein, in C. reinhardtii. For this purpose, we have

constructed a cassette in which the recA gene is

expressed from a (previously-published) strong C.

reinhardtii hybrid promoter consisting of constitu-

tive rbcs2 and inducible HSP70A promoter elements.

At the 3' end, the construct is terminated by the C. rein-

hardtii rbcs2 gene 3`region. This cassette is being used to

transform C. reinhardtii cells mutant in argininosucci-

nate lyase (arg2/arg7-8). Strains expressing the heterol-

ogous RecA protein will then be transformed again,

this time with truncated double stranded DNA

fragments of the argininosuccinate wild type gene

overlapping the arg2 mutation. Homologous recom-

bination should result in complementation of the

arg2 mutation in this latter strategy. Results will be

reported.

72



Poster Presentation

PW2032

REPLICATION BYPASS OF UV AND BPDE INDUCED

LESIONS IN REPAIR DEFICIENT CHINESE HAM-

STER CELL LINES

FJ Johansson, A Lagerqvist, K Erixon, D Jenssen

Stockholm University, STOCKHOLM, Sweden

The aim of the present study was to investigate the

kinetics of replication bypass of UV lesions and

adducts induced by benzo[a]pyrene-diol-epoxide

(BPDE) measured as replication fork progression in

Chinese hamster ovarian (CHO) cells. The fork pro-

gression was here measured by one of us developed

method based on the Alkaline DNA unwinding (ADU)

technique. The ADU technique is a method to meas-

ure single strand interruptions in cellular DNA, which

is based on the principle that strand separation in

alkali requires strand breaks. The fraction of single

stranded DNA will be a measurement of the number

of strand breaks. This method has been applied to

study replication fork progression, where each repli-

cation fork is expected to provide two single strand

breaks. Initially pulse labeled DNA is found in the sin-

gle stranded fraction, but with chase time it succes-

sively turns into double stranded DNA, due to fork

progression and replicon merging. Inhibition of fork

progression by DNA lesions will delay this process.

Using cells lines deficient in nucleotide excision

repair (NER) and homologous recombination repair

(HRR), the repair kinetics of UV- and BPDE-lesions

may reveal the type of bypass mechanisms involved

in restoration of forks. The results demonstrated that,

replication forks stalled by UV or BPDE induced

lesions were delayed in restoration and most pro-

nounced in NER-deficient cells. It was found that this

process was further blocked by post-treatment with

caffeine. Caffeine has been suggested to bind to sin-

gle stranded DNA at replication forks and thereby

stalling the post replication repair/translesion syn-

thesis. These results awoke the question whether also

deficiency in replication bypass in addition to repair

of damaged DNA could explain the sensitivity for UV

and BPDE, which will be further discussed in this

presentation.


Poster Presentation

PW2033

DNA DAMAGE, MUTAGEN SENSITIVITY AND DNA

REPAIR EFFICACY IN HELICOBACTER PYLORI-

INFECTED GASTRIC MUCOSA CELLS

M Arabski1, J Blasiak1, J Chojnacki2, J Drzewoski2,

P Kazmierczak2, G Klupinska2, M Kasprzak1,

M Wisniewska-Jarosinska2

1 University of Lodz, LODZ, Poland2 Medical University, LODZ, Poland

Helicobacter pylori is a common human pathogen

and its infection is believed to contribute to gastric

cancer. The malignant transformation of gastric

mucosa cells may be fueled up by impaired DNA

repair due to the accumulation of spontaneous muta-

tions in target genes and increasing susceptibility to

exogenous carcinogens. Moreover, the effectiveness

of DNA repair may contribute to failure of chemo-

therapy and resistance of gastric cancer cells to drugs.

To evaluate the role of the infection of H. pylori on

DNA repair we determined: 1) the kinetics of removal

of DNA damage induced by hydrogen peroxide and

the antibiotic amoxycillin, and 2) the level of basal,

oxidative and alkylative DNA damage in the H. pylori-

infected and non-infected gastric mucosa cells. The

level of DNA damage and the kinetics of DNA repair

were evaluated by alkaline single cell gel elec-

trophoresis (comet assay). Oxidative and alkylative

DNA damage were assayed with the use of DNA

repair enzymes formamidopyrimidine-DNA glycosy-

lase (Fpg) recognizing oxidized DNA bases and

3-methyladenine-DNA glycosylase II (AlkA) recogniz-

ing alkylated bases. We observed slower kinetics of

DNA repair after treatment with hydrogen peroxide

and amoxicillin in H. pylori-infected than non-infect-

ed gastric mucosa cells. The level of basal, oxidative

and alkylative DNA damage was higher in the infect-

ed compared to non-infected cells. Our results indi-

cate that H. pylori-infected gastric mucosa cells have

more damaged DNA and display decreased DNA

repair efficacy. Therefore, these features can be con-

sidered as risk markers for gastric cancer associated

with H. pylori infection and the comet assay may be

applied to evaluate these markers.

73



Poster Presentation

PW2034

CELL ADHESION MOLECULES ICAM-1 (CD54) IN

CANCER INVASION

MZ Milicevic1, B Bajic2, JC Ciric3

1 Vinca Institute of Nuclear Sciences, BELGRADE,

Yugoslavia2 Galenika Pharmaceuticals, BELGRADE, Yugoslavia3 Institute of Endocrinology, BELGRADE, Yugoslavia

Several families of adhesion molecules have been

identified over the past several years and their syn-

thesis and expression on the cell membrane studied

in relation to the invasive and metastatic behaviour

of cancers. These adhesion molecules may be deleted

selectively or exhibit specific patterns of expression.

They may also be differentially expressed as a metas-

tasis–associated phenomenon. Intercellular and cell-

matrix interactions are also a major element of

cancer cell invasion and dissemination. The expres-

sion of CD-54 in thyroid specimens from 10 cases of

papillary adenocarcinoma, 10 cases of follicular ade-

noma and 10 normal thyroid specimens was exam-

ined and quantified by an immunohystochemical

method, using frozen sections and a standard avidin-

biotin–peroxidase complex method. Thyroid epithe-

lial cells from all cases of papillary adenocarcinoma

expressed the CD54. The ICAM-positive staining was

detected predominantly on the apical site of malig-

nant thyroid epithelial cells, but not in the cytoplasm

of those cells. Intense expression of the ICAM-1 mole-

cule was detected on the lesion were malignant cells

were extended. Normal thyroid tissues showed weak

expression of ICAM-1 on capillary and post –capillary

endothelial cells but not on thyroid epithelial cells.

We found that carcinomas expressing CD54 had a sig-

nificantly higher incidence of lymph node metastasis

than tumours not expressing CD- 54. Those results

suggest that ICAM expression is up-regulated in thy-

roid adenocarcinoma cells and that ICAM-1 is

involved in malignant processes in thyroid gland.

Staining for this molecule might be useful adjunct for

distinguishing of benign and malignant thyroid dis-

eases. Increased expression of CD-54 correlated with

invasiveness of human thyroid papillary adenocarci-

noma. The study of CD-54 cell adhesion molecules has

added to our understanding of how alterations in

specific molecules may contribute to the neoplastic

phenotype. CD 54 may have much more specialized

roles in controlling cellular processes.


Poster Presentation

PW2035

LIFESPAN OF HUMAN LYMPHOCYTES EXAMINED

AFTER CHEMO- OR RADIOTHERAPY

S Gundy, M Baki, I Bodrogi, O Esik

National Institute of Oncology, BUDAPEST, Hungary

Estimates of the mean lifetimes of lymphocytes

based on the frequencies of unstable chromosomal

aberrations (CAs) seem to be ranged from 110 days to

10 years. Great differences in the estimations might

be accounted for since many circumstances influence

the aberration rate. The main objectives of our follow-

up study were to investigate the lifespan of human

lymphocytes in patients who had received either

radiotherapy in different volumes and locations of

the body, or chemotherapeutical treatment including

a combination of chemicals with a radiomimetic

drug causing the same variety of DNA damage as

radiation does. Cytogenetic effects of treatments

were examined prior to, during, immediately and up

to 7 years after the termination of different treat-

ments. The yields of unstable CAs showed a 3-fold

individual variability within the groups of patients

treated either with the same doses of chemicals or

radiotherapy. It was also found that not only the

extents of the irradiated volumes, but also the loca-

tions of lymph nodes play a role in both the yields

and the disappearance of unstable CAs. The average

half-life of the lymphocytes having suffered irradia-

tion is much shorter than reported so far. Our new

finding is that the disappearance of chemotherapy-

induced aberrations shows 2 peaks: the first one

occurs 3 years, and the second one occurs 6 years after

the end of treatment. It seems that the lifespan of

peripheral blood lymphocytes largely depends on the

nature of exposures. The amount of irreparable DNA

damage in precursor cells, which leads later to aber-

ration-formation is bigger when patients are chemo-,

rather than radiotherapy-treated. This phenomenon

must be taken into consideration when CAs are used

as biomarkers for the estimation of cancer risk.

74



Poster Presentation

PW2036

REACTIONS OF N,N-BIS(2-CHLOROETHYL)-P-AMINO-

PHENYLBUTYRIC ACID (CHLORAMBUCIL) WITH

2’-DEOXYCYTIDINE, 2’-DEOXY-5-METHYLCYTIDINE,

AND THYMIDINE

D Florea-Wang, J Hovinen

University of Turku, TURKU, Finland

N,N-Bis(2-chloroethyl)-p-aminophenylbutyric acid

(chlorambucil), alone or in combination with pred-

nisone, is routinely used in the chemotherapy of

chronic lymphocytic leukemia. Chlorambucil is also

used clinically for Hodgkin's lymphoma, non-Hodgkin's

lymphoma, Waldenström's macroglobulineamia, ovar-

ian and breast cancer, some other tumors, and certain

autoimmune diseases. Chlorambucil is an alkylating

agent that binds covalently to many types of cellular

molecules, such as DNA, RNA, and proteins. Because

the alkylating agents bind at sites of DNA that are

actively transcribed, they are more toxic to cancer

cells than to normal cells. However, like other alkylat-

ing agents, chlorambucil is potentially mutagenic,

teratogenic, and carcinogenic, and an increased inci-

dence of acute leukemias and other secondary malig-

nancies has been reported in patients who have

received this drug.

Chlorambucil was allowed to react with 2'-deoxycyti-

dine, 2’-deoxy-5-methylcytidine and thymidine at

physiological pH, and the reactions were followed by

HPLC-MS and HPLC-MS/MS techniques.1 Although

the predominant reaction observed was chlorambucil

hydrolysis, chlorambucil reacted with various het-

eroatoms of the nucleoside. The principal site of alky-

lation with all pyrimidines nucleosides was N3. Also,

several other adducts were detected. The N3, O2, 5’-O,

deglycosylated and deaminated derivatives were

characterized by means of MS/MS, UV, and 1H NMR in

the case of 2’-deoxycytidine and 2’-deoxy-5-methyl-

cytidine. C5-Adduct was also detected in the case of

2’-deoxycytidine. Thimidine was the least reactive

pyrimidine nucleoside studied, and in addition of the

N3 derivative, it reacted only at the carbohydrate

moiety.1 Diana Florea-Wang, Elina Haapala, Jorma Mattinen, Kristo

Hakala, Juhani Vilpo, and Jari Hovinen, Chemical Research

in Toxicology, Vol. 17, No. 3, pages 383-403, (2004)


Poster Presentation

PW2037

GENOTOXICITY AND NEPHROTOXICITY BY A

COMMON REACTIVE INTERMEDIATE OF ULTRA-

SHORT ACTING NON-DEPOLARIZING TROPINYL

DIESTER NEUROMUSCULAR BLOCKING AGENTS

FAA van Acker, DJ van den Dobbelsteen,

GJMJ Horbach

N.V. Organon, OSS, The Netherlands

Muscle relaxation is achieved during clinical anaes-

thesia by the intravenous administration of neuro-

muscular blocking agents (NMBAs). These agents

produce muscle paralysis, primarily by occupying

nicotinic acetylcholine receptors located in the motor

end-plate region of striated muscle. In general, recov-

ery of neuromuscular block is due to the rapid metab-

olism, enzymatic/chemical degradation or biliary or

renal clearance yielding pharmacologically inactive

metabolites.

From a series of di- and tri-substituted benzyl bisqua-

ternary ammonium derivatives of a bis (tropan-3a-0l)

diester, two compounds were selected for toxicological

evaluation prior to human testing. During these stud-

ies it was revealed that for Org A a reactive intermedi-

ate was formed, which is believed to be responsible

for the observed genotoxic and nephrotoxic proper-

ties. In non-rodent toxicity studies clinical chemistry

and histopathological investigations revealed that

kidney was the primary target organ even upon sin-

gle dosing. In addition, the in vitro chromosomal

aberration test showed clastogenic activity both in

the presence and absence of a metabolizing system.

Subsequently, the structural analogue Org B was eval-

uated for its genotoxic properties and consistent with

the genotoxicity of Org A, this derivative was also

found to be clastogenic.

The proposed mechanism of toxic action is the for-

mation of a quinone-methide product from the

chemical and enzymatic degradation of the phenolic

acetate function into a transient phenol which

undergoes Hofmann-like elimination.

Quinone-methides have been shown to engage in

electrophilic reactions resulting in toxicity by react-

ing with cellular macromolecules such as proteins

and DNA. Therefore, the quinone-methide metabo-

lites are believed to result in the clastogenic effect of

the NMBAs. Upon excretion in the urine and concen-

tration of the primary urine the proximal tubular

cells are targetted by this common reactive metabo-

lite.

75



Poster Presentation

PW2038

POLYNUCLEOTIDE KINASE PARTICIPATES IN NON-

HOMOLOGOUS END JOINING IN RESPONSE TO

IONIZING RADIATION

AR Rasouli-nia, F Karimi-Busheri, M Weinfeld

University of Alberta, EDMONTON, Canada

Human polynucleotide kinase (hPNK) catalyzes phos-

phorylation of 5'-DNA termini and dephosphoryla-

tion of 3'-DNA termini. The protein participates in

processing and rejoining of single- and double-

strand-break termini, and has been shown to interact

with XRCC1, DNA polymerase beta, DNA ligase III and

probably XRCC4. Down regulation of hPNK expres-

sion by a double stranded small-interfering RNA mol-

ecule (siRNA) in a human lung adenocarcinoma cell

line resulted in hypersensitivity to a wide variety of

genotoxic agents and an elevation in spontaneous

mutation frequency. Since double-strand breaks are

considered to contribute significantly towards

genomic instability and cell lethality induced by ion-

izing radiation, we sought further evidence for direct

involvement of hPNK in the non-homologous end

joining (NHEJ) pathway by comparing the influence

of hPNK down-regulation on the radiation response

of NHEJ-proficient and deficient cells. We observed

that while reduced expression of hPNK in the NHEJ-

proficient cell lines increased cellular sensitivity to

ionizing radiation, it had no effect on the response of

the NHEJ-deficient cell line. Together with an exami-

nation of the repair response of the cells, these data

provide evidence for hPNK participation in NHEJ.


Poster Presentation

PW2039

DAMAGE, REPAIR, DNA EVOLUTION

G de Buendia

Universidad Antonio Narinio, BOGOTA, Colombia

As many biological functions, DNA repair mecha-

nisms have also been evolving, so it is possible to

identify among the repair mechanisms those which

have appeared in the earlier stages of evolution.

Above all, evolution is a process which goes from sim-

plicity and unspecificity to complexity and specifici-

ty. From this point of view, translesion synthesis (TS),

non homologous recombination (NonHR) and the

addition and capture of telomeric sequences (ACTS)

can be considered as primeval DNA repair mecha-

nisms, and nucleotide excision repair (NER) and base

excision repair (BER) can be considered as evolved

DNA repair mechanisms.

TS, NonHR and ACTS are very simple mechanisms

because they consist of only one protein in the major-

ity of cases. They are general mechanisms because

they operate the same way in response to any lesion.

In contrast, NER and to a greater extent BER are very

specific for lesions and they recognize them before

the synthesis phase. In addition, they are complex

mechanisms because they need the operation of a

high number of proteins. In relation to complexity of

DNA sequences they can maintain, NER and BER

restitute the high variability of genetic information

with high fidelity, whereas TS, NonHR and ACST

reconstruct DNA chain continuity patching this with

monotonous sequences, as mutation spectra studies

as shown. The studies of DNA sequence analysis of

DNA repair genes in several organisms are also in

agreement with this model. It was found that the

genes of DNA polymerases which operate in TS in the

three superkingdoms (archaea, prokaryotes and

eukaryotes) are very similar, whereas genes of

methyl glycosylases which belong to BER vary from

one organism to another. This could be due to the fact

that they appeared recently in the evolution of the

analyzed organisms and so they do not have a com-

mon ancestor.

76



Poster Presentation

PW2040

ANALYSIS OF MICE WITH A SERINE TO ALANINE

SUBSTITUTION AT AA389 OF THE P53 GENE

W Bruins1, E Zwart1, E Hoogervorst1, LD Attardi2,

J van den Berg1, CTHM van Oostrom1, RB Beems1,

GJ van den Aardweg3, G. Lozano4, T Jacks5,

A de Vries1, H van Steeg1

1 National Institute of Public Health and the

Environment, BILTHOVEN, The Netherlands2 Stanford University Center, STANFORD,

United States of America3 Josephine Nefkens Institute, ROTTERDAM,

The Netherlands4 The University of Texas M.D., HOUSTON,

The Netherlands5 Center for Cancer Research,MIT, CAMBRIDGE,


It is well known that p53 plays an important role in

suppression of human cancer. Mice with a complete

loss of p53 or with an overexpression of mutant p53

have been extensively studied, but these models do

not fully recapitulate the human situation. To mimic

the human situation, we have generated a subtle

germline mutation in the p53 gene, changing Serine

389 (392 human) into Alanine. Presumably, phospho-

rylation of S389 activates p53 and enables the protein

to exert its functions in cell cycle arrest and/or apop-

tosis. S389 is specifically phosphorylated after UV-

light, whereas after gamma irradiation S389 shows

no phosphorylation.

Site-directed mutagenesis of relevant p53 exon

sequences, homologous recombination in ES cells and

cre-mediated excision of the selectable marker gene

have resulted in p53.S389A cells and mice. It is expect-

ed that this approach will only disturb specific

subsets of p53 protein functions rather than all. Aging

experiments showed no increased incidence of

tumors and no reduced lifespan up to the age of 2.5

years in p53.S389A mice. However, carcinogen-

induced tumorigenesis experiments showed an

increased incidence of 2-AAF induced bladder neo-

plastic lesions, and a decreased latency time of UV-

induced skin tumors in p53.S389A mice.

MEFs were treated with UV-light, and a reduction in

apoptosis, which was even more evident in MEFs iso-

lated from crosses between p53.S389A and DNA

repair deficient XPA-/- mice, was observed. Also,

p53 protein levels were reduced after UV. After

gamma-irradiation however, G1-arrest was compara-

ble to that in wild type cells. Thus far, our results indi-

cate that blocking phosphorylation at codon 389 does

not have an effect on p53 responses after DNA double

strand breaks, but does seem to affect responses after

UV-induced DNA damage. This work was supported

by the Dutch Cancer Society and the NIEHS

(Comparative Mouse Genomics Centers Consortium).

77



Poster Presentation

PW2041

CARCINOGEN-INDUCED TUMOR DEVELOPMENT

IN MICE WITH AN R270H MUTATION IN P53

SWP Wijnhoven1, D Tuveson2, E Zwart1, N Willis2,

K Olive2, E Speksnijder1, M Schaap1, CTHM van

Oostrom1, T Jacks2, A de Vries1, H van Steeg1


Environment, BILTHOVEN, The Netherlands2 MIT, BOSTON, United States of America

In the p53.R270H 'knock-in' mouse model, an

Arginine to Histidine mutation was introduced in

codon 270 of the mouse p53 gene. This mutation,

equivalent to human R273H, is a 'hot-spot' mutation

in several human and mouse tumor types (lung,

breast, colon) and is frequently found as a germ-line

mutation in the human Li-Fraumeni syndrome. In

addition, a transcriptional stop-cassette flanked by

loxP sites was introduced in intron 1 of the p53 gene.

As a consequence, the expression of the mutant p53

protein can be controlled by crossing the mice with

(tissue-specific) Cre-transgenic mice.

In order to study the effects of the R270H mutation on

DMBA-induced (mammary) tumorigenesis, p53.

R270H mice were bred to mammary gland-specific

WAP-Cre mice. DMBA-treated heterozygous p53.

R270H mice developed mammary tumors with a high

incidence and short latency time, whereas equally

treated wild type mice did not respond. This indicates

that the R270H point mutation is an important trig-

ger for mammary tumor development and can act in

a dominant-negative manner.

Another indication for a potential dominant-negative

phenotype of the R270H mutation was observed after

chronic exposure of skin-specific p53.R270H/ K14-Cre

mice to a subtoxic dose of UVB-irradiation. The laten-

cy time for UV-induced skin tumor development was

strongly reduced in p53.R270H mutant mice com-

pared to wild type mice, and moreover, the tumor

multiplicity seems to be increased in heterozygous

point mutant mice.

Finally, 2-AAF exposure in p53.R270H/ TgN-balancer

Cre mice with a ubiquitous but mosaic expression in

all tissues tested, only leads to minor differences in

tumor development between p53.R270H mutant and

wild type mice.

In conclusion, the p53.R270H point mutation seems to

explore a dominant-negative tumor phenotype after

carcinogen exposure in a highly tissue-specific man-

ner.

This work was supported in part by the Dutch Cancer

Society and the NIEHS (Comparative Mouse Genomics

Centers Consortium).


Poster Presentation

PW2042

USE OF METABOLIC COMPETENT CELLS TO EVALU-

ATE THE GENOTOXICITY OF TOBACCO CONDEN-

SATES

S Simi1, M Casella1, D Zampieri1, C Sorrentino2,

A di Muro2, L del Piano2, J Doehmer3, M Abet2, S Simi1

1 Institute of Clinical Physiology CNR, PISA, Italy2 Ist. Sperim. per il Tabacco, SCAFATI (SA), Italy3 GenPharmTox, MARTINSRIED/PLANEGG, Germany

Genetically engineered cells expressing cytochrome

P450 (CYP 450), a key enzyme for xenobiotic biotrans-

formation, have provided a new tool to investigate

both the genotoxicity and the P450 mediated meta-

bolic activation of a wide variety of chemicals includ-

ing drugs, environmental pollutants, mutagens and

carcinogens.

To study the genotoxicity of tobacco smoke, the major

cause of premature deaths in developed countries,

two Chinese hamster cell lines transfected with rat

cytochromes CYP 1A1 and CYP 1A2, known to be

induced by tobacco smoke, were exposed to two differ-

ent tobacco condensates.

Plating efficiency, sister chromatid exchanges and

chromosome aberrations were used as biomarkers of

genetic damage.

Preliminary results seem to show that all these end-

points are differently induced, suggesting also that

the two cytochromes operate in different way.

78



Poster Presentation

PW2043

DNA INSTABILITY IN DM1 MUSCLE CELLS

S Simi, M Casella, P Beffy, R del Carratore, S Simi,

M Simili

Institute of Clinical Physiology CNR, PISA, Italy

Myotonic Dystrophy type 1 (DM1) is an inherited

human disease whose molecular defect is the expan-

sion of a trinucleotide DNA sequence in the 3’UTR of

the DMPK gene. The triplet expansion is present in up

to 2,000 copies in DM patients while normal individ-

uals have 10–30 copies.

Since DM1 shares several features with fragile X syn-

drome, another 'unstable triplet syndrome' particularly

characterised by chromosome instability, cytogenetic

analyses (micronucleus assay, FISH assay) were per-

formed on human lymphocytes to verify if also DM1 is

prone to chromosome instability. A significant correla-

tion was found between the presence of the disease

and MN frequency increase, mainly due to MN bear-

ing centromere positive signals, thus indicating a

high incidence of chromosome loss.

Since it is known that the triplet expansion in muscle

cells is much higher than in lymphocytes, DM muscle

cells (DM-HFM, DM1 human foetal skeletal muscle

cells, 2800 CTG repeats) and control cells (C-HFM,

<200 CTG repeats) were kindly provided by J.

Puymirat (Quebec, Canada) and cultured to deter-

mine whether the chromosome instability occurred

also in muscle.

Cytogenetic analysis show that MN frequency in DM-

HFM is significantly higher than in C-HFM, strongly

suggesting a genomic instability phenomenon.

A possible mechanism of MN formation could involve

nuclear budding, a process which has been hypothe-

sised as the way by which amplified DNA selectively

localised to specific sites at the periphery of the

nucleus could be eliminated.

Our results of nuclear buds scoring show that the

buds frequency in DM-HFMC is significantly higher

than in C- HFM.

Experiments will be carried out to verify the presence

of amplified triplets in buds.


Poster Presentation

PW2044

DNA PROTECTIVE PROPERTIES OF A 1,4-DIHY-

DROPYRIDINE DERIVATIVE IN HUMAN CELLS IN

VITRO

NI Ryabokon1, NV Nikitchenko1, J Rzeszowska-

Wolny2, GJ Duburs3, RI Goncharova1

1 Institute of Genetics&Cytology, MINSK, Republic

of Belarus2 Center of Oncology, GLIWICE, Poland3 Latvian Institute of Organic, RIGA, Latvia

Beta-carbonyl-1,4-dihydropyridines (1,4-DHPs) are

synthetic analogs of dihydronicotinamide, i.e. the

hydrogen and electron transferring part of redox

coenzymes NADH and NAD(P)H, which are involved

in many reactions, including energy transduction,

signaling pathways, DNA repair and, thus, are crucial

for living cells. 1,4-DHPs are widely known and used

for their important chemical, biochemical and phar-

macological properties. They also have high efficiency

as antimutagenic compounds. So, among 12 1,4-DHPs

screened in the Antimutagenesis Laboratory, Institute

of Genetics and Cytology, Minsk, 6 showed antimuta-

genic activity, significantly reducing spontaneous

and alkylation-induced genetic damage in Drosophila

[Goncharova et al., 1980; Kuzhir, 1999], as well as radia-

tion-induced injuries in pond fish [Goncharova, 2000].

In this study, we revealed that AV-153, one of the most

effective 1,4-DHPs in experiments with animals, is

nontoxic in a wide range of concentrations for lym-

phocytes of healthy donors in vitro and culture lines

of human cells. It significantly reduces cell death fre-

quency and different types of DNA damage induced

spontaneously or by alkylating and oxidizing agents,

as well as by gamma- and X-rays. The reduction fac-

tors reach 70%. The analysis of DNA repair kinetics

showed that AV-153 increases the effectiveness of

repair process in human cells during the first 15–60

min after treatment. These data demonstrate that AV-

153 could be studied and used as a DNA protective

and, thus, a cancer preventive compound. The role of

AV-153 in DNA repair, as well as the mechanisms of its

action, are studied and will be discussed.

The work has been carried out in the framework of

collaborative research between the Institute of

Genetics and Cytology (Minsk) and the Center of

Oncology (Gliwice) (2003–2005) and partially sup-

ported by fellowship grants from the Association for

Supporting Cancer Research (Poland), UNESCO (Polish

brunch) and National Cancer Institute (Bethesda,

USA).

79



Poster Presentation

PW2045

ROLE FOR MISMATCH REPAIR IN THE CELLULAR

RESPONSE TO UV-C IRRADIATION

V Borgdorff, S van Hees-Stuivenberg, N de Wind

Leiden University Medical Center, LEIDEN,

The Netherlands

Mismatch repair is the cellular process responsible

for the correction of replication errors such as mis-

matches and insertion/deletion loops. This process is

essential for the maintenance of the cell’s genomic

integrity. To investigate whether mismatch repair, in

addition to removing misincorporations, plays a role

in the processing of lesions induced by genotoxic

agents, we studied the cellular response of Msh2-defi-

cient mouse embryonic stem (ES) cells to UV-C irradi-

ation. UV-C treatment induced fivefold more

mutations in Msh2-deficient ES cells than in wild-

type ES cells. This increased UV-induced mutagenesis

is dependent on the Msh2/Msh6 mismatch recogniz-

ing heterodimer, since it is also observed in Msh6-

deficient cells, whereas cells deficient for the

Msh2/Msh3 heterodimer show a similar mutation

induction as wild-type cells by UV exposure. No sig-

nificant differences in the UV-induced mutation spec-

trum were observed between Msh2-deficient- and

wildtype ES cells. However, we did observe UV-

induced mutation hotspots at unique sites in the

Msh2-deficient cells. In addition, Msh2-deficient cells

displayed a decreased S/G2 arrest accompanied by a

slightly reduced level of UV-C-induced apoptosis.

Taken together, our data demonstrate an important

role for mismatch repair in counteracting UV-induced

mutagenesis. We hypothesize that mismatch repair

mediates sequence context-dependent removal of

nucleotides misincorporated opposite to UV-induced

pyrimidine dimers (compound lesions). Alternatively,

the mismatch repair-dependent cell cycle arrest may

allow nucleotide excision repair to remove damage in

a DNA sequence context-dependent way, resulting in

reduced mutagenesis and an altered distribution of

mutations.

80



Poster Presentation

PW2046

BIOLUMINESCENT SALMONELLA REVERSE MUTA-

TION ASSAY: A HIGH THROUGHPUT SCREEN FOR

DETECTING MUTAGENICITY

J Aubrecht, JJ Osowski, WW Ku, JR Cheung,

JI Ackerman, SM Hoogendoorn, JF Blake, P Persaud

Pfizer, GROTON, CT, United States of America

Here, we describe the development and evaluation of

a novel bioluminescent high throughput Salmonella

reverse mutation assay applicable to the screening of

large numbers of small molecules including combi-

natorial libraries. The bioluminescent Salmonella assay

utilizes genetically engineered standard Salmonella

tester strains TA98 and TA100 expressing the luxCDABE

operon from Xenorhabdus luminescence. In princi-

ple, the assay employs bioluminescence as a sensor of

metabolic activities in living cells. The assay provides

highly concordant data with the outcome in the

standard Salmonella plate incorporation reverse

mutation assay. Since the results of the standard

Salmonella assay are required by various regulatory

agencies for approval of new drugs, the biolumines-

cent Salmonella assay can be effectively used for pri-

oritization of compounds in drug discovery. Because

of its high throughput attributes, the assay permits

effective, fast and economical screening of a large

series of structural analogs enabling the investiga-

tion of structure activity relationships. In general, the

application of bioluminescent sensors for detection

of metabolically active cells is expected to have

broader application in the design of other high

throughput clonogenic assays in various cell systems


Poster Presentation

PW2047

TP53 MUTATIONS IN EXPOSED WORKERS

KV Vähäkangas1, P Hainaut2

1 University of Kuopio, KUOPIO, Finland2 Intl. Agency for Research on Cancer, LYON, France

Kirsi Vähäkangas, Department of Pharmacology and

Toxicology, University of Kuopio, POB 1627, FIN-70211

Kuopio, Finland

Pierre Hainaut, International Agency for Research on

Cancer, Lyon, France

In some cases, evidence exists that carcinogenic

exposures contribute to the mutation spectrum of the

TP53 gene in human cancers. Most of this data comes

from dietary and environmental exposures, and very

few examples are available from occupational expo-

sures. In populations exposed to dietary Aflatoxin B1

with liver cancer (AFB1) and ultraviolet (UV)-radiation

with skin cancer, a single specific-looking TP53 muta-

tion has been described in some of the tumours.

Whether these fingerprints in the TP53 gene can be

used to reveal occupational etiology remains to be

shown. Uranium miners have probably been studied

enough to say that radon does not induce a specific

p53 mutation in lung cancer. In other cases, although

differences in the TP53 mutation spectrum exist, they

are more diffuse and difficult to interpret at this

point. Implications for a putatively specific p53 muta-

tion can be found in the literature for vinyl chloride,

mustard gas, metal industry and petrochemical

industry, and await further studies for confirmation.

Cigarette smoking seems to induce long-lasting

molecular footprints in TP53, but their use to rule out

other occupational exposures as etiological factors in

occupational cancers is still very questionable, espe-

cially due to the putatively synergistic effects of ciga-

rette smoke with other carcinogens. In IARC p53

mutation database (www.iarc.fr/p53) very few data

about occupations exist. Thus, although interesting

implications of possibly typical mutation spectra

among cancers with other occupational etiologies

exist, the data is scanty and awaits further develop-

ment of p53 mutation database.

81



Poster Presentation

PW2048

THE NOVEL UVS11 GENE OF CHLAMYDOMONAS

REINHARDTII AFFECTS CELL RESPONSE TO DNA

DAMAGE

M Slaninova, EG Galova, A Sevcovicova, B Sviezena,

D Vlcek

Comenius University FNS, BRATISLAVA,

Slovak Republic

The study of cell cycle regulation and checkpoints at

different levels has gained further insight into these

processes in different species. In comparison with

other lower eukaryotes there has been much less

progress in understanding cell cycle control in algae.

The first putative checkpoint mutant of green alga

Chlamydomonas reinhardtii have been isolated in

our lab and termed as uvs11. It has been predicted to

be UV-sensitive and repair-deficient. We assume now

that product of UVS11 gene is not required for DNA

repair itself but it is involved in cell response to DNA

damage.

In our work, we have assayed the activity of histone

H1 kinases presented in total protein extract and

those, mitosis specific ones bound to p13suc1-

Sepharose. The kinase activity was monitored in syn-

chronized cultures of untreated wild type and

mutant cells and compared with those irradiated by

UV-light (254nm) before mitosis. No substantial dif-

ferences between wild and mutant cells were found

in untreated cells. However, shortly after UV irradia-

tion the kinase activity in wild type has considerably

decreased, to recover about five hours later. The activ-

ity drop was accompanied by a prolongation of the

cell cycle due to a delay in mitoses, protoplast fis-

sions, and daughter cell release. The mutant strain

has shown no significant change in the course of

kinase activity and in other processes related to the

cell cycle after UV irradiation. Our present results

indicate that inhibition of histone H1 kinase could

be a way to arrest the initiation of mitosis in

Chlamydomonas and delay cell cycle progress in

response to DNA damage. We suggest that product of

the UVS11 gene affects cell response to DNA damage

and directly causes a decrease in kinase activity. This

implicates the role of UVS11 gene in regulation of the

cell cycle.


Poster Presentation

PW2049

DUAL EFFECT OF HTLV-1 TAX PROTEIN ON

NUCLEOTIDE EXCISION REPAIRIN HUMAN T-

CELLS

Y Schavinsky-Khrapunsky, E Priel, M Aboud

Ben Gurion University of the Negev, BEER SHEVA,

Israel

HTLV-I is the etiological agent of adult T-cell

leukemia. Its Tax oncoprotein is regarded as a key ele-

ment in initiating the viral-associated leukemogenic

process. Tax oncogenic potential is partially ascribed

to its interference with various modes of DNA repair,

which leads to genetic instability. In this study we

were particularly interested in elucidating Tax effect

on nucleotide excision repair (NER) in human T-cells,

since NER is one of the major repair pathways used by

eukaryotic cells to maintain their genomic integrity

and because T-cells are the main targets of HTLV-I in

human infection. We show here that at low doses Tax

rather stimulates NER, implying that the low Tax

level in the infected T-cells of latent HTLV-I carriers

might protect these cells from mutagenesis and con-

tribute, thereby, to the long lasting clinical latency of

HTLV-I infection. However at high doses Tax inhibits

this repair, suggesting that Tax must be elevated in

the carriers’ infected T-cells for initiating their

leukemogenic progression. This dual Tax effect proved,

in this study, to be mediated by NF-kappaB. Furthermore

we demonstrate that NER inhibition by Tax is exerted

through NF-kappaB-dependent interference with p53-

transcriptional activity and consequently with its role in

NER.

82



Poster Presentation

PW2050

NIEHS - COMPARATIVE MOUSE GENOMICS CENTERS

CONSORTIUM: UNDERSTANDING THE BIOLOGICAL

SIGNIFICANCE OF HUMAN POLYMORPHISMS

JP Packenham

NIEHS/NIH, RESEARCH TRIANGLE PARK,


The National Institute of Environmental Health

Sciences (NIEHS) Comparative Mouse Genomic

Centers Consortium (CMGCC) is a cross-disciplinary,

multi-Institutional, program that falls under the aus-

pices of the NIEHS Environmental Genome Project

(http://www.niehs.nih.gov/cmgcc). This program

was initiated with the goal of developing transgenic

and knockout mouse models based on human DNA

sequence variants in the environmentally responsive

genes discovered under phase I of the EGP. Ultimately,

these models will be used as tools to improve our

understanding of the biological significance of

human DNA polymorphisms and the role of such

variation in environmentally related diseases.

Initially, this program has focused on DNA polymor-

phisms that occur in DNA repair and cell cycle envi-

ronmentally responsive genes. The Consortium is

accomplishing its goals through, mouse model devel-

opment, technology development, and resources

developed by the Consortium. To date the Consortium

has over 32 mouse models under development and

has developed through its bioinformatics team a

Mouse Federated database, containing three major

components: mouse phenotypic assessment that

allows for functional prediction of gene variants to

model, collection and analysis of phenotypic data

including gene expression data, and mouse model

dissemination. Through this database, SNPs are ana-

lyzed for their functional impact using SNP location

and Haplotype frequency data, structural analysis

and prediction, literature mining and potential com-

binatorial effects through pathway data. Models are

generated and analyzed for pathology and other phe-

notypic endpoints under various environmental con-

ditions. This combined knowledge is then integrated

to generate knowledge of the human gene in the per-

turbed mouse background and map new information

onto existing knowledge and finally, the experimen-

tal data is used to refine and adapt models in order to

validate causality pathways. Validated mouse models

and resources developed within the CMGCC are avail-

able to the general scientific community without

charge.


Poster Presentation

PW2051

PRE-EXPOSURE: MODULATION OF FREQUENCIES

AND REPAIR OF DNA DAMAGE?

P Cramers1, AA van Zeeland2, LHF Mullenders2,

JCS Kleinjans1


The Netherlands2 Leiden University Medical Center, LEIDEN,

The Netherlands

Ionizing radiation (IR) interacts with DNA either by

direct hit, or by the production of reactive oxygen

inducing strand breaks and base modifications.

Alleviation of biological effects after pre-exposure to

low doses of IR has been described. Low dose expo-

sure might reduce damage induction from the high

dose, or might induce repair mechanisms.

The aim of this study was to test whether low radia-

tion doses can cause protection against high dose

effects by modulation of initial damage and repair.

By using confluent (G1 phase) human primary fibro-

blasts, we ruled out possible effects of the condition-

ing dose on the cell cycle.

Comet assay measurements showed that pre-expo-

sure of cells with 0.1 Gy prior to a challenge dose (0-8

Gy) resulted in a protective effect that was most pro-

nounced at higher challenge doses. No difference was

observed in repair kinetics between conditioned and

unconditioned cells.

DNA DSB were detected with gamma H2AX fluores-

cence. A linear dose response relationship was found

in the dose range 0-3 Gy. Damage induction experi-

ments have not yet been completed. No difference in

repair kinetics between conditioned and uncondi-

tioned cells was observed.

Taken together, our results indicate that in confluent

cells, pre-exposure gives a small modulation of DNA

damage induction and no enhancement of repair.

Another part of our project focuses on pre-exposure

to X-rays and the effects on UV-induced repair.

Recently several authors showed upregulation of

repair proteins after X-rays, including the NER pro-

tein XPC. It is therefore interesting to investigate

whether X-rays can trigger adaptation against UV

and vice versa. Repair replication experiments are

currently under way to study the inducibility of exci-

sion repair after low doses of ionizing radiation and

adaptation after both X-rays and UV conditioning

and challenging (cross-reactivity). Preliminary

results suggest an increased UV-repair in X-ray

pre-treated cells.

83



Poster Presentation

PW2052

THE ROLE OF LYS63-LINKED POLYUBIQUITIN

CHAINS IN REPAIR AND MUTAGENICITY OF

BENZO[A]PYRENE-DIOL-EPOXIDE (BPDE) DNA

ADDUCTS

SAS Langie, AM Knaapen, RK Chiu, RWL Godschalk,

CHMA Ramaekers, BG Wouters, FJ van Schooten


The Netherlands

Humans are continuously exposed to environmental

carcinogens including polycyclic aromatic hydrocar-

bons like benzo[a]pyrene (B[a]P) that can induce DNA

adducts. Since DNA adducts can not always be suc-

cessfully repaired prior to cell-division, cells have

evolved tolerance mechanisms to replicate lesion-

containing DNA. Template switching and lesion

bypass are two mechanisms that ensure this DNA

damage tolerance, and there is evidence for the

involvement of Lys63-linked multi-ubiquitination in

these pathways. We hypothesised that the absence

of Lys63-linked multi-ubiquitination will cause

increased mutagenicity upon exposure to benzo[a]

pyrene-diol-epoxide (BPDE). Therefore, A549 cells

(human epithelial lung carcinoma cells) overexpress-

ing ubiquitin in its Wt-form (Ub+) or as a K63R

mutant configuration, were applied to further study

the role of Lys63-linked multi-ubiquitination mediat-

ed DNA damage tolerance mechanisms in the muta-

genicity of B[a]P. 32Postlabeling results showed no

significant differences in decline of the BPDE-DNA

adduct levels between K63R and Ub+-cells over a peri-

od of 24 hours (P>0.5), indicating that Lys63-ubiquiti-

nation is not directly involved in DNA adduct

removal. Furthermore, we demonstrated that Ub+-

and K63R-cells, either exponentially growing or

arrested in G0/G1, show no differences in survival

upon exposure to low doses (( 1 µM) of BPDE, whereas

K63R-cells were more sensitive at concentrations

>1µM. G0/G1-arrested cells seemed to be more resist-

ant towards the toxic effects of (±)anti-BPDE. Finally,

we demonstrated that BPDE-exposure (0.5 or 1.0 µM)

causes higher mutation frequencies in the HPRT-gene

of K63R-cells, as compared to Ub+-cells. In conclusion,

our data show that absence of Lys63-multi-ubiquiti-

nation (K63R) causes enhanced BPDE-induced muta-

genicity. Thus, our data suggest that inhibition of

Lys63-linked multi-ubiquitination prevents error-free

template switching and shifts DNA damage tolerance

to error-prone lesion bypass.

84



Poster Presentation

PW3001

THE ROLE OF ALLELIC POLYMORPHISM IN COLO-

RECTAL CANCER RISK

CSA Csejtei1, A Tibold2, ZS Faluhelyi3, I Kiss2, I Ember2

1 Markusovszky Teaching Hospital, SZOMBATHELY,

Hungary2 Public Health Inst. Uni. PTcs, PTCS, Hungary3 Baranya County Hosp., PTCS, Hungary

Allelic polymorphisms of metabolyzing enzymes and

other genes (e.g.onco/supressor genes) might be able

to have an influence on colon cancer susceptibility. In

our present study we tested the effect of glutathione-

S-transferase M1 (GTSM1), and p53 polymorphisms on

the risk of colorectal cancer in the Hungarian popula-

tion. DNA was isolated from deparaffinized sections

of colorectal cancer samples, genotyped for 0 and +

allels (GSTM1 and T1) and Arg/Pro alleles (p53), and

compared to the data of healthy controls. The allelic

polymorphisms were determined by a simultaneous

polymerase chain reaction (GSTM1 and T1) in the pres-

ence of a positive control, and by allelic-specific poly-

merase chain reaction (p53). The allelic distributions

were compared, odds rations (OR) were calculated to

show the difference between the occurrence of „low-

risk” and „high-risk” alleles among cases and con-

trols. Our results indicated a statistically significant

connection between the occurrence of colorectal

tumours and GTSM1 0-allele, and p53 Pro allele.

Combination of the „high-risk” alleles further increased

the risk.

Certain alleles of metabolyzing enzymes and

onco/suppressor genes were proved to occure more

frequently in colon cancer patients than in healthy

controls. Therefore, genetic polymorphisms can be

used to gain useful data on the individual suscepti-

bility to colon cancer.


Poster Presentation

PW3002

CYP1B1 LEU432VAL POLYMORPHISM, SMOKING

HABITS AND THE RISK OF BREAST CANCER

PJ Sillanpää1, K Mitrunen1, V Kataja2, M Eskelinen2,

VM Kosma3, M Uusitupa3, H Vainio1, A Hirvonen1


HELSINKI, Finland2 Kuopio University Hospital, KUOPIO, Finland3 University of Kuopio, KUOPIO, Finland

Cytochrome P450 (CYP) 1B1 enzyme is involved in the

conversion of oestradiol and a number of PAHs to

mutagenic intermediates, capable of causing poten-

tial DNA damage. Several polymorphisms have been

described in the CYP1B1 gene.

Several previous studies have examined the associa-

tion between inherited differences in the CYP1B1 gene

and breast cancer risk with contrasting results. We

examined this issue further in a Finnish Caucasian

study population consisting of 483 breast cancer

patients and 482 healthy population controls. The

genotypes were determined by PCR-based allele-spe-

cific fluorogenic probes (Applied Biosystems). Odds

ratios (ORs) and 95% confidence limits (95% CIs) were

calculated by unconditional logistic regression analy-

ses adjusting for known or suspected risk factors for

breast cancer.

Overall, the frequencies of CYP1B1*1/*3 and *3/*3 geno-

types were not significantly different between cases

and controls (OR 1.19, 95% CI 0.89-1.59 and OR 1.17, 95 %

CI 0.77-1.78, respectively). However, when the data

was stratified according to smoking habits, an

increased risk for breast cancer was seen between

CYP1B1*3 alleles and number of cigarettes/day (p for

interaction 0.02). Women who carried at least one

CYP1B1*3 allele and smoked 1-9 cigarettes/day were at

3-fold increased risk of developing breast cancer (OR

3.06, 95% CI 1.32-7.12) compared to women who

smoked the same amount and had two CYP1B1*1 alle-

les. Moreover, a significant trend (p for trend 0.02) of

increasing risk with increasing number of CYP1B1*3

alleles was seen among women smoking 1-9 ciga-

rettes/day; ORs were 2.63 (95 % CI 1.07-6.46) and 5.09

(95% CI 1.30-19.9), for women with one or two copies

of the CYP1B1*3 variant alleles, respectively.

Our preliminary results suggest that the CYP1B1*3

allele may pose increased breast cancer risk among

light smoking Finnish women.

85



Poster Presentation

PW3003

GENETIC SUSCEPTIBILITY TO COPD AND LUNG

CANCER: COMBINED ANALYSIS OF GSTM1 AND MEH

POLYMORPHISMS

JS Salagovic, R Tkacova, V Habalova, J Zidzik,

B Fleischer, I Kalina

P. J. Safarik University, KOSICE, Slovak Republic

Objective: Cigarette smoking is the major risk factor

for the development of both chronic obstructive pul-

monary disease (COPD) and bronchogenic carcinoma,

but only 10-20% of heavy smokers develop the dis-

eases which suggests the presence of genetic suscep-

tibilty. The genetic susceptibility to lung cancer and

COPD might depend on variation in antioxidative

enzyme activities that detoxify cigarette smoke prod-

ucts such as microsomal epoxide hydrolase (mEH)

and glutathione S-transferase M1 (GSTM1).

Methods: The polymorphisms in GSTM1 gene and

mEH gene (EPHX1) were examined in 156 lung cancer

patients, 148 patients with COPD and 150 healthy con-

trol subjects using PCR and PCR-RFLP based methods.

The frequencies of polymorphic genotypes of mEH

and GSTM1 genes were compared both individually

and in combination in patients and healthy controls.

Results: Our findings suggest that the presence of at

least one mEH „slow allele” significantly increases

lung cancer risk (OR=1.71; 95% CI 1.02-2.89), especially

among smokers (OR=1.83; 95% CI 1.0-3.4). Moreover,

carriers of both slow alleles (homozygotes) with very

low mEH activity are at increased lung cancer risk

(OR=2.14; 95% CI 0.86-5.61), but significantly only

among nonsmokers. The odds ratio of individuals

with the both potentially risk genotypes: homozy-

gotes for slow allele in mEH gene and for null allele in

GSTM1 gene versus that of other genotypes combined

was 3.53 (95% CI 0.98-16.5; p=0.02, Fisher´s exact test).

The risk of development of COPD in lung cancer

patients was associated only with GSTM1 0/0 geno-

type (OR=2.98; 0.73-1.19).

Conclusions: Our study suggests that lung cancer risk

is associated with the presence of slow mEH allele

and GSTM1 0/0 genotype and that in patients with

lung cancer the presence of at least one active allele

in GSTM1 gene has a protective effect against the

development of COPD.


Poster Presentation

PW3004

THE INFLUENCE OF MPO, MTHFR, XRCC1 POLY-

MORPHISMS ON AROMATIC DNA ADDUCT LEVELS

IN BRONCHUS

E Gyorffy1, L Anna1, J Segesdi2, Z Gyori2, I Soltesz3,

S Kostic3, A Csekeo3, J Minarovits2, B Schoket1

1 National Center for Public Health, BUDAPEST,

Hungary2 National Center for Epidemiology, BUDAPEST,

Hungary3 National Institute of Pulmonology, BUDAPEST,

Hungary

There is a large database in the literature on the

impact of major metabolic polymorphisms on lung

cancer risk and on biomarkers of environmental

genotoxic exposures. However, there is little knowl-

edge and contradictory reports on the risk of more

recently introduced genetic polymorphisms, such as

MPO, MTHFR and XRCC1. The aim of our study was to

investigate the effect of these three polymorphisms

on smoking-related aromatic DNA adduct level in

bronchial tissue of lung patients. The study popula-

tion consisted of 215 Hungarian patients who under-

went lung resection. Smoking status was self-reported

in a questionnaire. Current smokers and those who

had given up smoking within one year before surgery

were considered as smokers (n=143). Life-time non-

smokers and those who had given up smoking in

more than one year were categorised as non-smokers

(n=72). Levels of aromatic DNA adducts in macroscop-

ically normal bronchial tissue were determined by

the 32P-postlabelling method with nuclease P1

enrichment. MPO G463A, MTHFR Ala222Val and

XRCC1 Arg399Gln genotypes were detected by PCR-

based methods. For MPO, there were no clear differ-

ences in DNA adduct levels in relation to its

polymorphism. For MTHFR, adduct levels were 12 to

33% lower in individuals with Ala/Val or Val/Val vari-

ant genotypes compared to the wild- type genotype

(P=0.078 in smokers and P=0.026 in non-smokers).

XRCC1 repair polymorphism did not affect the adduct

levels. However, in combination with MTHFR, adduct

levels were 30% lower in Gln/Gln subjects compared

to those with Arg/Arg (P=0.025) and Arg/Gln

(P=0.028), considering the sub-population of MTHFR

wild-type carrier and smoking individuals only. The

underlying molecular mechanisms need further

investigations.

The research project has been supported by the

Hungarian OTKA T 034616 research grant.

86



Poster Presentation

PW3005

DELTA-AMINOLEVULINIC ACID DEHYDRATASE

POLYMORPHISM AND SUSCEPTIBILITY TO LEAD

EXPOSURE IN OCCUPATIONALLY LEAD EXPOSED

WORKERS

Y Duydu, HS Suzen

Ankara University, ANKARA, Turkey

In this study, the cytogenetic response to lead expo-

sure in storage battery manufacturing workers

carrying different alleles of ≠ -aminolevulinic acid

dehydratase (ALAD 1 and ALAD 2) was evaluated. The

cytogenetic response was measured by analysis of

the frequency of sister chromatid exchange (SCE) and

the number of high-frequency cells (HFCs) in periph-

eral blood lymphocytes from workers occupationally

exposed to lead. A total of 71 voluntary male workers

were enrolled in the study. According to our genotype

analysis, 50 workers had the ALAD 1-1 genotype and 21

workers had the ALAD 1-2 genotype. When HFCs were

compared between ALAD 1-1 and ALAD 1-2 workers,

the percentage of HFC was statistically (T2-test,

P < 0.05) higher in ALAD 1-1 workers. However the dif-

ference in mean values of SCE/cell between ALAD 1-1

and ALAD 1-2 workers was statistically not signifi-

cant. On the basis of this result we suggest that ALAD

1-1 subjects might be more susceptible to cytogenetic

effects of lead exposure than ALAD 1-2 subjects.

This study was supported by Research Found of

Ankara University, Project no. 98.03.00.01.


Poster Presentation

PW3006

POLYMORPHISM IN DNA REPAIR GENE XPD AND

BREAST CANCER RISK IN SMOKING WOMEN

M Mitrunen1, V Kataja2, M Eskelinen2, VM Kosma3,

M Uusitupa3, A Hirvonen1

1 Finnish Institute of Occupational Health, HELSINKI,

Finland2 Kuopio University Hospital, KUOPIO, Finland3 University of Kuopio, KUOPIO, Finland

DNA repair is essential for protecting cells from the

genotoxic effects of carcinogenic exposures. The XPD

protein takes part in the nucleotide excision repair

pathway, which recognizes and repairs wide variety

of structurally unrelated lesions. Mutations in the

XPD gene can diminish the activity of the repair and

therefore lead to increased DNA damage and ulti-

mately to carcinogenesis. Four polymorphisms result-

ing in amino acid changes have been reported in the

XPD gene, of which the Lys751Gln change in exon 23

has been anticipated to have the most profound

effect on the activity of the enzyme. We examined if

this polymorphism was associated with breast cancer

risk in Finnish Caucasian population. A PCR-based

RFLP-analysis was used for the genotype Odds ratios

(OR) and 95% confidence intervals were calculated by

unconditional logistic regression. Our preliminary

statistical evaluations revealed no significant overall

association between the Lys751Gln polymorphism

and breast cancer risk; OR for subjects with two Gln

alleles was 1.13 (95% CI 0.76-1.66). Neither was any sig-

nificant difference seen by menopausal status,

whereas a statistically significant interaction was

seen with smoking (p for interaction 0.019); women

who reported ever smoking and had two Gln alleles

were at significantly increased risk of breast cancer

(OR 2.42, 95% CI 1.22 - 4.78) compared to women who

carried one or two Lys alleles. The risk was confined to

women who had smoked over 10 cigarettes/day.

These preliminary data suggests that the XPD

Lys751Gln polymorphism may be an important modi-

fier of breast cancer risk in smoking Finnish women.

This work was supported by the Academy of Finland

and by EVO funds from Kuopio University Hospital.

87



Poster Presentation

PW3007

THE FUNCTIONAL IMPACT OF POLYMORPHISMS

IN DNA REPAIR GENES: XPA, XPB AND RECQL1.

M Krzesniak1, D Butkiewicz1, M Mrzyglodzik1,

R Vaitiekunaite1, CC Harris2, M Rusin1

1 Center of Oncology, GLIWICE, Poland2 National Cancer Institute, NIH, BETHESDA,


Reduced capacity to repair DNA damage can lead to

cancer formation, which is examplified by many can-

cer-prone diseases caused by mutations in DNA

repair genes. The inter-individual variation in DNA

repair capacity and cancer risk are associated with

polymorphisms of DNA repair genes. Previously, we

and others have found sequence alterations within

the coding and non-coding regions of genes involved

in DNA metabolism. It is not known whether and

how these particular sequence alterations change the

functioning of the encoded proteins. We focused on

two genes involved in nucleotide excision repair sys-

tem (XPA, XPB) and on RECQL1, which encodes the

smallest known human helicase from the RecQ fami-

ly. This group of helicases contains at least four other

genes, three of which (WRN, BLM and RTS) are associ-

ated with cancer-prone genetic diseases. For our func-

tional analyses we have selected the common

polymorphism of XPA (-4G>A) located four residues

upstream the start codon, which in previous molecu-

lar epidemiological analyses showed significant

association with modulation of lung cancer risk. Two

relatively infrequent XPB polymorphisms (117:Lys>Arg;

402:Gly>Cys) were also selected due to their localiza-

tion within the evolutionary conserved regions of the

protein. Finally, we studied the functional impact of

three sequence alterations of RECQL1 helicase gene: 248:

Ala>Pro, 487:Lys>Thr (polymorphic allele), 566:Thr>Ala

(sequence alteration found in HeLa cells) arranged as 8

different sequence combinations. This helicase is not

well studied and its high expression in lung suggests

that its sequence alterations may potentially influ-

ence the lung cancer risk. Using the luciferase

reporter assay, host cell reactivation assay, in vivo

protein labeling by the fluorescent tags and Western

blotting we assessed the influence of the sequence

alterations on the expression level, activity and cellu-

lar localization of the studied DNA repair proteins.

The detailed results of the analyses will be presented.

88

Novartis Pharma AGwww.novartis.com



Poster Presentation

PW3008

EPHX1 GENE POLYMORPHISMS AND INDIVIDUAL

SUSCEPTIBILITY TO LUNG CANCER

AV Voho1, K Mitrunen1, S Anttila1, O Impivaara2,

J Jarvisalo3, H Vainio1, K Husgafvel-Pursiainen1,

A Hirvonen1

1 Finnish Institute of Occupational Health, HELSINKI,

Finland2 National Public Health Institute, TURKU, Finland3 Social Insurance Institution, TURKU, Finland

Microsomal epoxide hydrolase (EPHX1) is a widely

expressed enzyme involved in the first pass metabo-

lism of polyaromatic hydrocarbons (PAHs), especially

benzo(a)pyrene (BaP) from tobacco smoke. At least

two functional polymorphisms in the EPHX1 gene are

common in Caucasians. We conducted a case-control

study in a Finnish study population to investigate the

role of the EPHX1 Tyr113His and His139Arg polymor-

phism in lung cancer susceptibility, both alone and in

combination with glutathione S-transferase M1 (GSTM1)

genotypes. Subjects with homozygous Tyr113/Tyr113

genotype were at increased risk of lung cancer (OR,

1.47; 95% CI, 1.06-2.04) compared with those having

His113 allele-containing genotypes. Similarly, the

homozygous His139/His139 genotype posed an

increased risk for this malignancy (OR, 1.58; 95% CI,

1.08-2.30) compared with the Arg139 allele-contain-

ing genotypes. However, lung cancer risk was not

related to the EPHX1 phenotypes predicted from the

exon 3 and exon 4 genotype data. When combined

genotype effects were examined, the OR for EPHX1

Tyr113/Tyr113 genotype in combination with GSTM1

null genotype was 1.73 (95%CI, 1.09-2.74). Subjects who

had this genotype combination and a smoking histo-

ry of over 40 pack-years (PYs) showed a 4-fold risk of

lung cancer (OR, 4.05; 95%CI, 1.27-12.92). The combination

of GSTM1 null and EPHX1 His139/His139 genotypes was

also associated with significantly increased lung cancer

risk (OR, 1.79; 95%CI, 1.06-3.02). Moreover, subjects

with this genotype combination and over 40 PYs had

a 4.5-fold risk of lung cancer (95%CI, 1.28-15.8). Our

results support the hypothesis that combinations of

even modest risk genotypes may lead up to a remark-

ably increased risk of lung cancer among smokers.


Poster Presentation

PW3009

PREDICTING MULTIPLE GENE-ENVIRONMENT

INTERACTIONS USING HIGH THROUGHPUT SIN-

GLE NUCLEOTIDE POLYMORPHISM GENOTYPING

HB Ketelslegers, RWH Gottschalk, RWL Godschalk,

AM Knaapen, FJ van Schooten, JCS Kleinjans,

JHM van Delft


The majority of cancers in humans are caused by cig-

arette smoking. Cigarette smoke contains over 3500

chemicals, including carcinogenic polycyclic aromat-

ic hydrocarbons, n-nitrosamines and aromatic amines.

These compounds are metabolized in a complex path-

way of absorption, distribution, activation and detoxi-

fication leading to reactive compounds that can

interact with DNA, so called DNA adducts. Previous

studies have shown that there is a relationship

between cigarette smoke constituents, carcinogenic

DNA adducts formation and cancer. Moreover, there

is a positive relation between dose (number of ciga-

rettes per day) and effect (DNA adducts). However,

within this relationship there is a wide inter-individ-

ual variation in DNA adduct levels in individuals with

similar exposures. This variation can at least partly be

explained by polymorphisms in genes that code for

enzymes involved in metabolic pathways of the spe-

cific carcinogens.The majority of studies on these

biomarkers of susceptibility focused on single poly-

morphisms. But taking in account that cigarette smoke

is a complex mixture of a large number of carcinogens,

simultaneous assessment of multiple genotypes

seems to be necessary.

The current pilot study was performed to determine

whether multiple genotyping can indeed explain

part of the inter-individual variation. The susceptibil-

ity of smoking individuals towards the formation of

DNA adducts was investigated. 24 Single Nucleotide

Polymorphisms in 18 genes, involved in DNA repair,

oxidative stress and biotransformation were geno-

typed in 65 smoking individuals using the Snapshot

method. A significant relationship was found between

the sum of all 'bad' polymorphisms and the adduct

level adjusted for cigarettes per day. From this it was

concluded that the sum of putatively bad polymor-

phisms determines the efficiency of adduct forma-

tion at a certain dose.

89



Poster Presentation

PW3010

ASSOCIATION BETWEEN XRCC3 POLYMORPHISMS

AND BREAST CANCER RISK IN FINNISH POPULA-

TION

L Heikinheimo1, P Siivola1, V Kataja2, M Eskelinen2,

VM Kosma3, M Uusitupa3, K Mitrunen1, A Hirvonen1


HELSINKI, Finland2 Kuopio University Hospital, KUOPIO, Finland3 University of Kuopio, KUOPIO, Finland

Genetically determined variability in DNA repair

capacity has been suggested to contribute to inter-

individual differences in susceptibility to breast can-

cer. We explored this issue by evaluating the potential

association between the XRCC3 Thr241Met and intron

5 A>G polymorphisms and breast cancer risk in our

Finnish Caucasian study population consisting of 483

cancer patients and 482 healthy controls. PCR-RFLP and

Taqman analysis were used to determine the geno-

types. Odds ratios (ORs) and 95 % confidence intervals

(CIs) were estimated by unconditional logistic regres-

sion. No significant overall effect was seen between

these XRCC3 polymorphisms and breast cancer risk.

However, when stratified by the use of alcohol,

women with at least one Met allele were found to be

at 1.5-fold risk for this malignancy (OR 1.54, 95% CI

1.05–2.26) if they reported ever using alcohol. This

association was most evident among women who

reported using alcohol daily or weekly (OR 2.05, 95%

CI 1.03–4.09). A statistically significant interaction

was also found between the intron 5 polymorphism

and body mass index (BMI) (p for interaction = 0.046);

women who had BMI less than 25.4 and carried at

least one variant G allele were found to be at some-

what increased risk for breast cancer (OR 1.46, 95% CI

0.97–2.20). Similarly, women who had ever used HRT

and carried at least one variant G allele had a tenden-

cy of increased risk for breast cancer (OR 1.63, 95% CI

0.93–2.84). No association was found when stratified

by the use of alcohol or tobacco. Neither were com-

bined effects found for intron 5 and Thr241Met poly-

morphisms. The results suggest that XRCC3 Thr241Met

and intron 5 A>G polymorphisms may be modest mod-

ifiers of individual breast cancer risk in Finnish

women. This work was supported by the Academy of

Finland and EVO funds from Kuopio University

Hospital.


Poster Presentation

PW3011

EFFECT OF POLYMORPHISMS OF DNA REPAIR AND

FOLATE METABOLISM ON CHROMOSOMAL ABER-

RATIONS

I Heilimo, P Siivola, H Maunu, H Jarventaus, K Mitrunen,

A Hirvonen, H Norppa


Finland

The frequency of chromosomal aberrations (CAs) in

cultured peripheral blood lymphocytes has for many

years been applied as a biomarker of early effects of

genotoxic carcinogens. The relevance of CA level as an

indicator of cancer predisposition was further sup-

ported by epidemiological studies suggesting that a

high frequency of CAs is predictive of an increased

risk of cancer. The cancer risk predictivity of CAs was

not explained by tobacco smoking or occupational

carcinogen exposure and did not depend on the time

between CA analysis and cancer detection, indicating

that the association also concerns 'unexposed' sub-

jects but does not reflect undetected cancer. The find-

ings suggest a role for individual susceptibility to

chromosome damage. In particular, variation in DNA

repair capacity, possibly related to polymorphisms of

DNA repair proteins, might provide an explanation.

Several of such polymorphisms have been proposed

to be associated with an increased cancer risk.

However, very little is known about their effects on

cytogenetic biomarkers in humans. Our previous

findings have suggested that polymorphisms of

XRCC1 and XPD may affect the level of CAs in human

lymphocytes. In the present study, we have examined

a population of 170 occupationally unexposed

Finnish subjects. We have genotyped major genetic

polymorphisms in DNA repair genes XRCC1 (codons

194, 280 and 399), XRCC3 (codon 241), XPD (exon 23),

and hOGG1 (codon 326) using methods based on PCR-

RFLP. Additionally, two polymorphisms in folate

metabolism enzymes, MTHFR (C677T) and MS

(A2756G) were examined. A preliminary analysis sug-

gested that polymorphisms in XRCC1 codons 194 and

280 affect the frequency of chromosome-type aberra-

tions. Polymorphisms in XPD exon 23 and MTHFR

C677T appeared to influence the level of chromatid-

type aberrations. [Supported by QLK4-CT-2000-

00628].

90



Poster Presentation

PW3012

GENETIC POLYMORPHISMS IN XENOBIOTIC-

METABOLISING ENZYMES AND THEIR POSSIBLE

LINKS WITH CHROMOSOMAL ABERRATIONS

P Siivola, I Heilimo, K Mitrunen, H Jarventaus, H Maunu,

A Hirvonen, H Norppa


Finland

Genetic polymorphisms of various xenobiotic-

metabolising enzymes (XMEs) have been associated

with cancer risk. This association is assumed to

reflect the role of XMEs in the metabolic activation

and detoxification of environmental carcinogens. The

effect of genetic polymorphisms on susceptibility to

specific genotoxic exposures can be studied by exam-

ining biomarkers, such as chromosomal aberrations

(CAs), in exposed and unexposed subjects. For

instance, the GSTM1 null genotype has been associat-

ed with an increased CA level in smokers. Besides

affecting the frequency of CAs induced by genotoxic

exposures, genetic polymorphism may also modulate

the background level of CAs. This kind of an effect has

earlier been described for NAT2 slow and GSTT1 null

genotypes. In the present study, we examined a pop-

ulation of 170 occupationally unexposed subjects. We

analysed the major genetic polymorphisms in several

genes responsible for carcinogen metabolism (i.e.

GSTM1, GSTM3, GSTP1, GSTT1, NAT2, EPHX exon 3 and

4) using PCR-RFLP and fluorogenic allele-specific

probes –based genotyping methods. According to our

preliminary evaluation, EPHX genotype expected to

result in low enzyme activity was associated with an

elevated frequency of CAs, especially of the chro-

matid-type. [Supported by QLK4-CT-2000-00628].


Poster Presentation

PW3013

MICRONUCLEUS TEST AS AN INDEX OF INDIVID-

UAL CANCER SUSCEPTIBILITY: THE PLEURAL

MALIGNANT MESOTHELIOMA

C Bolognesi, E Perrone, E Giordano, P Roggieri,

R Filiberti, M Neri, R Andreatta, R Puntoni

National Cancer Research Institute, GENOA, Italy

Inherited or acquired genome instability character-

izes the cancer susceptibility in humans and increas-

es the sensitivity of DNA to various physical and

chemical carcinogenic agents. Malignant pleural

mesothelioma (MM) is a rare highly aggressive neo-

plasm. Although it is well established that asbestos is

the major risk for MM, accounting for about 80% of

cases, the molecular steps in the carcinogenic process

is unknown and other ethiological factors have been

suggested such as SV40, ionising radiation and genet-

ics. The evidence of a complex heterogeneity of struc-

tural chromosomal aberrations in MM seems reflect

an intrinsic predisposition of the cells to accumulate

genomic damage. A number of biomarkers have been

widely applied in molecular epidemiology for identi-

fying subjects at risk of developing cancer. They

should allow to evaluate environmental and occupa-

tional exposure and further give information on the

status of susceptibility. One of the most used method-

ologies for assessing genomic damage in human lym-

phocytes is the micronucleus assay (MN). This assay

seems to be an useful method for monitoring indi-

viduals with genetic instability and as a screening

test for carriers of specific mutations in evaluating

cancer susceptibility.

A biomonitoring study was carried out to evaluate

the MN frequency in PBLs of patients with MM and of

their first-degree relatives with respect to lung cancer

patients and two groups of controls. The study includ-

ed 47 patients with MM, 10 first-degree relatives, 48

patients with lung cancer, 53 risk controls and 88

healthy controls. Preliminary results revealed a signif-

icant increased MN frequency in patients with MM

and in their relatives: the mean MN is double in com-

parison with all the other groups. No association was

found between MN and asbestos exposure. The analy-

sis is ongoing: the final results will be presented.

91



Poster Presentation

PW3014

DNA REPAIR GENE POLYMORPHISMS AND GENO-

TOXICITY BIOMARKERS IN WORKERS EXPOSED

TO COBALT CONTAINING DUST

RAM Mateuca1, V Aka1, M de Boeck1,

M Kirsch-Volders1, D Lison2

1 Vrije Universiteit Brussels, BRUSSELS, Belgium2 Catholic University of Louvain, BRUSSELS, Belgium

The aim of this biomonitoring study was to identify

the link between relevant polymorphisms in DNA

repair genes (hOGG1, XRCC1 and XRCC3) and biomark-

ers of genotoxicity in workers exposed to cobalt metal

alone or in combination with tungsten carbide at a

mean level corresponding to the current TLV-TWA

(20µg Co/m3). The study comprised 21 workers exposed

to metallic Co alone, 26 workers exposed to a WC-Co

mixture and 26 matched controls. The genotoxicity

biomarkers represented both initial DNA damage

(8-hydroxydeoxyguanosine [8-OHdG] in urine and

comet assay on lymphocytes) and definitive chromo-

some breakage/loss (micronuclei in lymphocytes).

The predictivity of DNA repair genotypes was analysed

by direct comparisons between groups (Mann-Whitney

U test) and by multiple regression analysis taking into

account, age, exposure type, smoking, and interac-

tions between genotypes, smoking and exposure.

Multiple regression analysis indicated that in both

the exposed and total populations, MNMC frequen-

cies were higher in workers with variant hOGG1326

and XRCC3241 genotypes.

In the Co population, smokers with variant XRCC3241

genotypes had high frequencies of MNCB. In the WC-

Co exposed population, hOGG1 was predictive for

MNCB. Multiple regression analysis revealed that

workers with variant hOGG1326 and XRCC3241 geno-

types and smokers with variant hOGG1326 genotype

show a higher MNMC frequency. Direct comparison

between WC-Co exposed workers with Ser/Ser and

variant hOGG1326 genotypes showed a higher MNMC

frequency in the latter ones. Polymorphisms in

XRCC1280 resulted in an increased baseline DNA dam-

age in the total and exposed populations. There was

no influence of genetic polymorphisms on the uri-

nary levels of 8-OHdG.

It was concluded that for a better individual follow-

up of workers exposed to Co containing dust, and in

particular to hard metal dust, smoking should be

avoided and if unexpectedly high frequencies of MN

are observed at individual level, genotyping for

hOGG1326 and XRCC3241 should be advised.

92

www.numico-research.com



Poster Presentation

PW3015

POLYMORPHISMS OF TNF-A, IL-10 AND IL1RN

AND RISK OF NON-SMALL CELL LUNG CANCER

HL Lind, S Zienolddiny, AAGE Haugen

National Institute of Occupational Health, OSLO, Norway

Studies have indicated that inflammation and reac-

tive oxygen species induced by smoking, may play a

role in lung carcinogenesis. We have previously

shown that certain variants of interleukin-1 beta

(IL1B) and cyclooxygenase-2 (COX2) genes are associ-

ated with increased risk of non-small cell lung cancer.

We have now extended our genotyping studies to

include several other genes involved in the inflam-

matory pathway. Here, we report results from analy-

sis of SNPs in genes for tumor necrosis factor alpha

(TNF-a -308 G/A and +488 G/A), interleukin-10 (IL-10 -

1082 A/G and –592 C/A) and interleukin-1 receptor

antagonist (IL1RN 86 bp VNTR intron 2). The SNPs in

TNFa and IL-10 were determined using the TaqMan

methodolgy, while the 86 bp VNTR in intron 2 of the

IL1RN gene was determined using PCR and gel elec-

trophoresis. Both the cases and controls were of

Norwegian origin and were frequency matched on

the basis of age and smoking status. In summary, this

is the first study investigating polymorphisms in

TNF-a, IL-10 and IL1RN in relation to lung cancer.


Poster Presentation

PW3016

POLYMORPHISMS IN ARACHIDONIC ACID PATH-

WAY GENES AND FISH CONSUMPTION ARE MOD-

IFYING COLORECTAL ADENOMA RISK

HJ van Kranen1, CLE Siezen1, AIM van Leeuwen2,

NR Kram1, MEM Luken1, E Kampman2

1 National Institute of Publ. Health and Environment,

BILTHOVEN, The Netherlands2 WUR, WAGENINGEN, The Netherlands

The association between polymorphisms in genes

involved in lipid metabolism, particularly the arachi-

donic acid (AA) pathway, and colorectal adenomas

was investigated in a Dutch case control study includ-

ing 384 cases and 403 polyp-free controls. Twenty-one

polymorphisms in seven candidate genes were stud-

ied, and a potential modifying effect of fish con-

sumption was considered.

Inverse associations were found between colorectal

adenomas and the CT genotype of SNP H477H in

PPARg and the GC genotype of SNP V102V in COX-2

(OR, 0.63; 95%CI, 0.45-0.89 and OR, 0.65; 95%CI, 0.46-

0.92 respectively) compared to their homozygote

major genotypes. A positive association was observed

for the TC genotype of SNP c.2242T>C in COX-2 (OR,

1.47; 95%CI, 1.07-2.00) compared to the TT genotype.

Extended analysis with estimated haplotypes not

only confirmed these associations but also revealed

three additional associations between haplotypes in

COX-2, sPLA2 and 15LOX and colorectal adenomas.

Interactions between fish consumption and the

genotypes of COX-2 and PPAR d were observed. Firstly

for SNP c.-789C>T in PPAR d (P=0.01), an inverse asso-

ciation was observed for individuals with the CC

genotype and highest tertile (T3) of fish consumption

as compared to the lowest tertile (T1) of fish con-

sumption (OR, 0.65; 95%CI, 0.41-1.02). However, for

those with the CT or TT genotype, fish consumption

increased risk (OR T3 versus T1 and CC genotype, 2.22;

95%CI, 0.78-6.36). Secondly, inverse associations were

observed for SNPs V102V and c.2242T>C in COX-2. The

interaction between fish consumption and c.2242T>C

was statistically significant (p=0.01), with an OR for

the TT genotype and high fish consumption of 0.52

(95%CI, 0.27-1.01) as compared to low fish intake.

These results indicate that SNPs in various genes

involved in the AA-pathway are associated with

colorectal adenoma risk. Part of these associations is

modified by fish consumption.

93



Poster Presentation

PW3017

GSTM1, NUCLEOTIDE EXCISION REPAIR AND

APOPTOTIC CAPACITY POLYMORPHISMS ON LYM-

PHO-MONOCYTIC ANTI-BPDE-DNA ADDUCTS IN

COKE-OVEN WORKERS

SP Pavanello1, A Pulliero1, E Siwinska2, D Mielzynska2,

E Clonfero1

1 University of Padova, PADOVA, Italy2 Institute Occupational Health, SOSNOWIEC, Poland

Polymorphisms of nucleotide excision repair (NER)

genes (XPC-PAT +/-, XPA 5’ non-coding region -A23G,

XPD-exon 23 A35931C Lys751Gln, XPD-exon 10 G23591A

Asp312Asn), glutathione S-transferase µ 1 (GSTM1-active

or -null) and Fas-A670G (also know as Apo1/CD95 one of

the most important genes involved in the apoptotic

pathway) on anti-benzo[a]pyrenediolepoxide (BPDE)-

DNA adduct levels from the lympho-monocytes (LMF)

of highly PAH-exposed coke oven workers were eval-

uated. The sample cohort included 67 male Polish

coke-oven workers (67% current smokers), all belong-

ing to the high exposure group (individual urinary

post-shift 1-pyrenol >2.28 µmol/mol creatinine, the

proposed BEI). The bulky anti-BPDE-DNA adduct

levels were detected by HPLC/fluorescence analysis

and genotypes by PCR-RFLP.

The low DNA repair capacity of XPC-PAT +/+ and XPA-

A23A genotypes significantly (p<0.05) double up the

mean anti-BPDE-DNA adduct levels. As already

reported in our previous studies DNA adducts are also

strongly influenced by GSTM1 genotype (p<0.05). The

higher frequencies of workers with unfavourable XPC-

PAT +/+ and XPA- A23A NER genotypes, alone or com-

bined with GSTM1-null belonged to the tertile with the

highest adduct level (> 4.11 adducts/ 108 nucleotides,

p<0.01). The few subjects with the unfavourable geno-

typic combination, XPC- PAT +/+ or XPA- A23A jointly to

GSTM1-null and Fas A670A, all belonged to the tertile

with highest adduct level. No association of dietary

habits and tobacco smoking with anti-BPDE-DNA

adducts in the LMF of coke oven workers was found.

The modulation of anti-BPDE-DNA adducts in the

LMF, by GSTM1-null and some low-capacity geno-

types of NER, may be considered as genetic suscepti-

bility factors capable of modifying the individual

response to the high PAH(BaP) genotoxic exposure

and the consequent cancer risk in coke oven workers.


Poster Presentation

PW3018

GENETIC POLYMORPHISM OF XENOBIOTIC META-

BOLISING ENZYMES AND GENOTOXICITY OF

STYRENE

M Migliore1, A Naccarati1, F Mercati1, G de Palma2,

P Manini2, E Scotti3, R Andreoli3, A Mutti2

1 University of Pisa, PISA, Italy2 University of Parma, PARMA, Italy3 ISPESL, PARMA, Italy

We studied the modulating role of the genotypes

involving polymorphisms of xenobiotic metabolising

enzymes (XME) in the genotoxic responses associated

with occupational exposure to styrene.

A cross-sectional study was performed on 46 styrene-

exposed workers (26 males); 98 styrene unexposed

healthy subjects were also enrolled as control group.

Either the main [mandelic and phenylglyoxylic acids

(MA and PGA)] and the minor [4-vinylphenol (4-VP)]

styrene metabolites were determined by LC/MS/MS

on urine samples. The induction of MN on peripheral

blood lymphocytes was evaluated by the citokinesis-

block micronucleus assay. FISH analysis with a pan-

centromeric probe was used to assess the frequency

of centromere-positive MN (C+MN). Genetic polymor-

phism of GSTA1, GSTM1, GSTT1, GSTP1, EPHX1 and

NQO1 were characterised by PCR based methods on

workers’ blood samples only.

Workers expressed higher MN (but not C+MN) values

than controls. Among female workers, MN was

dependent from 4-VP values and NQO1 status, where-

as among males the only significant predictor was

age. The percentage of C+MN cells was independent

from exposure and modulated by age and GSTA sta-

tus in males and by alcohol, GSTM1, GSTT1, GSTP1 and

NQO1 in females.

In the present study, styrene exposure was associated

with clastogenic effects. Such effects were sex-

dependent, being related to urinary 4-VP and modu-

lated by the NQO1 polymorphism in female, but not

in male workers. Aneuploidogenic changes were

independent from styrene exposure and modulated

in the two sexes by different combinations of poly-

morphic XME. The limited sample size does not allow

firm conclusions on defined patterns of XME respon-

sible for such alterations.

94



Poster Presentation

PW3019

MUTAGEN SENSITIVITY OF PATIENTS WITH HEAD

AND NECK CANCER OF DIFFERENT ANATOMICAL

REGIONS

G Szekely, E Remenar, M Kasler, S Gundy


The bleomycin (BLM) sensitivity assay measures the

degree of cancer susceptibility on a genetic basis. The

elevated number of BLM-induced chromatid breaks

per cell (b/c) shows that patients with head and neck

squamous-cell carcinoma (HNSCC) are more sensitive

to the mutagen than the healthy controls. However,

there might be a heterogeneity of neoplastic process-

es in HNSCC of different anatomical regions, because

the areas have different duration of contacts with muta-

gens. The aim of this study was to examine whether the

mutagen sensitivity of patients is different with various

localisations of HNSCC. Conventional chromosome

analysis and BLM assay were carried out in HNSCC

patients, healthy non-smokers and non-drinkers, and

smokers but non-drinkers. The aberrant cell frequen-

cy in HNSCC patients (2.78%) and healthy smokers

(2.63%) was similar, and higher than that in non-

smoker controls (2.25%). These results indicate that

the spontaneous rate of aberrations and cancer risk

are clearly associated with mutagenic effect of tobac-

co use. Significant differences were also found in b/c

values between patients (1.11 ± 0.39) and controls (0.97

± 0.34) when overall BLM-sensitivity was studied, and

this phenomenon also demonstrated an increased

cancer risk (OR=1.83). Furthermore, patients with

HNSCC of different anatomical regions showed dif-

ferent mutagen sensitivities: b/c values of those with

oral cavity, oropharynx and hypopharynx cancers dif-

fered significantly from those of controls, but those of

patients with larynx tumour did not. Patients with

tumours of oral cavity (without lip) had the highest

cancer risk (OR=2.05) when compared to those with

other anatomical sites. These data suggest that the

mode of action of mutagens is more complex in upper

aerodigestive tract than it was previously thought.

Moreover, the concentration and the synergism of the

mutagens (alcohol, tobacco and dietary factors) is

probably more definitive in this location than in the

lower parts of the aerodigestive tract.


Poster Presentation

PW3020

ROLE OF GST AND NAT2 POLYMORPHISMS IN

THYROID CANCER PATIENTS

R Marcos1, A Hernandez1, N Xamena1, J Surralles1,

A Creus1, P Galofre2, R Marcos1

1 Universitat Autonoma Barcelona, CERDANYOLA

DEL VALLES, Spain2 Hosp. Unive. Vall d'Hebron, BARCELONA, Spain

The existence of different genetic polymorphisms in

human populations has proven to be a susceptibility

factor for different tumours. To link thyroid cancer

incidence to genetic susceptibility biomarkers, poly-

morphisms at the GSTM1, GSTT1, GSTP1 and NAT2

genes have been determined for 176 thyroid cancer

patients and 167 controls. The results indicate that,

according to the calculated odd-ratios, the frequencies

of the different GST genotypes found in the group of

cancer patients do not significantly differ from those

values obtained in the controls. Nevertheless, for the

NAT2 polymorphisms, the allele NAT2*5 is significant-

ly linked to cancer incidence with a high risk in

NAT2*5 +/+ individuals.

Since thyroid patients are therapeutically treated

with radioiodine, it was analysed whether the sensi-

tivity to radiation was modulated by genotype. In 30

patients, the MN frequency before and after exposure

was determined. The results indicate that none of the

polymorphisms studied show any kind of association

with the basal level of micronuclei. When the same

patients were followed after radioiodine exposure, a

significant increase in the frequency of MN was

observed in all of them, indicating genotoxic activity

of the ionising radiation exposure. The increase in the

MN frequency was not associated with any of the GST

polymorphisms evaluated. Nevertheless, the pres-

ence of slow acetylator genotypes and, in particular,

the presence of the NAT2*7 allele were significantly

associated with a lower increase of the MN frequency

after radioiodine treatment.

95



Poster Presentation

PW3021

DNA DAMAGE AND DNA REPAIR POLYMORPHISMS

IN HEALTHY INDIVIDUALS

A Zijno1, F Marcon1, P Leopardi1, S Rossi1, CA Andreoli2,

L Conti1, R Galati3, A Verdina3, R Crebelli1

1 Istituto Superiore di Sanita, ROME, Italy2 Eti SpA, ROME, Italy3 Istituto Regina Elena, ROME, Italy

The high variability observed among individuals in

DNA repair capacity (DRC) may result in different

response to DNA damage and thus different suscepti-

bility to environmental and occupational carcino-

gens. Apart from subjects affected by pathological

syndromes, healthy individuals also differ in intrinsic

DNA repair capacity that has been shown to be mod-

ulated by single nucleotide polymorphisms. These

polymorphisms are generally associated with only

moderate structural modifications of repair enzymes

but are prevalent in the population and may con-

tribute to the overall population risk of cancer. In the

present study, the relationships among polymor-

phisms in DNA repair genes and biomarkers of geno-

toxicity, such as SCE, micronuclei, single strand

breaks have been analysed in a healthy population.

The population (169 subjects) was genotyped for rele-

vant genes involved in different DNA repair path-

ways: APE1 (Asp148Glu) and XRCC1 (Arg399Gln and

Arg280His), key enzymes in base excision repair

(BER); XRCC3 (Thr241Met), involved in homologous

recombinational pathway for repairing double-

strand breaks; XPD (Lys751Gln) that participates in

nucleotide-excision repair (NER).

Results: Allelic frequencies were in the range

described for Caucasian population. All genotype

distribution was compatible with Hardy-Weinberg

equilibrium. Among the studied polymorphisms,

non-parametric test indicated that only the XPD poly-

morphism may modulate DNA damage. SCE frequen-

cies in double variant, heterozygous and wild type

were 5.63, 5.45 and 5.13, respectively (p=0.061). SCEs in

double variant were significantly higher than in wild

type (p=0.033) as well as in variant carriers (variant

homozygous plus heterozygous) respect to wild type

(p=0.32). A slight not significant increase of SCE in

relation to XPD polymorphism was observed in vari-

ant carriers respect to wild type in smoker group

(6.08 and 5.57, respectively, N=56, p=0.089). Overall

evaluation of these data indicated a slight role of the

studied DNA repair polymorphisms on basal and

smoking related DNA damage in the investigated

population.


Poster Presentation

PW3022

GERMLINE MUTATIONS OF HMSH2, HMLH1 AND

HMSH6 IN 48 CHINESE HEREDITARY NONPOLY-

POSIS COLORECTAL CANCER FAMILIES

H Yan, J HeiYing, S ShuHan, H Yan

Second Military Medical University, SHANGHAI,

China

Hereditary nonpolyposis colorectal cancer (HNPCC)

(MIM # 114500), also know as Lynch syndrome, is the

most frequent autosomal dominant predisposition to

the development of colorectal cancer. It is caused by

germline mutations in human homologues of the

bacterial mismatch repair (MMR) genes MutL and

MutS: hMSH2, hMLH1, hMSH6, hPMS1, hPMS2. Up to

date, more than 300 different predisposing muta-

tions have been identified; more than 90% of these

mutations were in hMLH1, hMSH2 and hMSH6. In

present study, we have screened 48 Chinese HNPCC

families fulfilling the Amsterdam criteria for muta-

tions in the DNA-mismatch repair genes hMLH1,

hMSH2 and hMSH6, using dHPLC and direct DNA

sequencing. The families were selected on the basis of a

family history of HNPCC-related tumors. Furthermore,

we required that tumor tissue from at least one indi-

vidual in the family had to display microsatellite

instability. We identified 10 novel germ-line muta-

tions in 10 families. Four families had mutations in

hMLH1, three mutations in hMLH2 and three muta-

tions in hMSH6. Among these mutations, 2 are splic-

ing site mutations, 4 are nonsence mutation and 4 are

frameshift mutations, the pathogenic effects of these

mutations in the process of carcinogenesis were also

analyzed.

96



Poster Presentation

PW3023

RELATIONSHIP BETWEEN SMOKING-RELATED

BULKY DNA ADDUCTS AND P53 ARG72PRO GENE-

TIC POLYMORPHISM IN LUNG CANCER PATIENTS

L Anna

National Center for Public Health, BUDAPEST,

Hungary

P53 tumour suppressor gene has a regulatory role in

various cellular mechanisms including DNA repair.

We hypothesize that its genetic polymorphism is a

potential modulator of the level of DNA damage

induced by smoking in pulmonary tissues. The aim of

the study was to investigate the association between

p53 Arg72Pro genetic polymorphism and smoking-

related bulky DNA adducts, and its modulation by

XRCC1 Arg399Gln DNA repair genotype in a study

population of lung cancer patients who underwent

lung resection (n=148). Smoking status was self-

reported by the patients. p53 Arg72Pro and XRCC1

Arg399Gln genotype was determined by PCR-RFLP

methods. Levels of bulky DNA adducts were deter-

mined by 32P-postlabelling in macroscopically nor-

mal bronchial tissues. Among current smokers and

short-term former smokers who stopped smoking

less than a year before surgery, those individuals who

were heterozygote or homozygote with the variant

Pro allele had elevated level of DNA adducts com-

pared to those who were of Arg/Arg genotype

(p=0.04). There was a statistically significant dose-

related increase of DNA adduct level up to 20 ciga-

rettes/day among current smokers with the variant

Pro allele (p=0.0004), whereas bronchial DNA adduct

level was not apparently influenced by cigarette

smoking in the subgroup of Arg/Arg homozygous

individuals. XRCC1 Arg399Gln polymorphism did not

apparently influence the impact of p53 genotypes on

bronchial DNA adduct levels. The results suggest that

p53 72Pro allele is a risk factor for increased primary

DNA damage in the bronchial tissue by smoking.

Further analyses are in progress to confirm the obser-

vations in a larger study population.

Supported by Hungarian OTKA T034616 and HU-FIN

S&T Research Fund SF-02/01.


Poster Presentation

PW4001

HYPERSENSITIVITY TO MITOMYCIN C-INDUCED

SISTER CHROMATID EXCHANGES (SCE) IN LYM-

PHOCYTES FROM HYPERBARIC OXYGEN EXPOSED

PATIENTS

Y Duydu1, A Aydin2, A Dur1, A Eken2, K Dundar2,

G Uzun2

1 Ankara University, ANKARA, Turkey2 Gata, Centre of HBOT, ANKARA, Turkey

Hyperbaric oxygen therapy (HBOT) has been success-

fully used for the treatment of a various clinical con-

ditions such as wound healing, carbon monoxide

poisoning, soft tissue infections, and radiation necro-

sis. However, as well known from the previously pub-

lished studies, HBO also leads to an increase in the

reactive oxygen species in the blood. This situation

resulted in an induction of DNA damage in leuco-

cytes as reported in many earlier studies. However,

studies on possible interactions between HBO and

other genotoxic chemicals are not available so far. In

this study, we investigated the SCE induction in

Mitomycin C (MMC) exposed and unexposed blood

samples from patients undergoing HBOT. Twelve vol-

untary patients (wound healing) were enrolled in

this study and exposed to 10 consecutive HBO treat-

ments (1 session/day). The patients inhaled 100% oxy-

gen under a pressure of 2.5 ATA. The mean SCE/Cell

level of patients were 5.92±1.37 before undergoing

HBOT. After the first session of HBOT the mean

SCE/Cell level reached the maximum (16.4±2.19). After

fifth (14.34±2.09) and last session (12.24±2.27) the

mean SCE/Cell levels were statistically lower (Mann-

Whitney U-test, p<0.05) then the first session of

HBOT. Nevertheless, the mean SCE/Cell level observed

after the last session of HBOT was still statistically

higher (Mann-Whitney U-test, p<0.05) than the

beginning of the HBOT. The same blood samples were

also exposed to MMC in-vitro at two doses (20, 40

ng/ml). MMC exposed samples (at both doses)

reached the maximum SCE/Cell levels after the first

session of HBOT as were observed in MMC unexposed

samples. However, there was no statistically signifi-

cant decrease in mean SCE/Cell levels of MMC

exposed samples after the fifth and even after the last

session of the HBOT. Such a result might be evaluated

as a hypersensitivity to MMC-induced SCE in HBOT.

97



Poster Presentation

PW4002

CYTO-AND GENOTOXIC POTENTIAL OF BETA-

CAROTENE CLEAVAGE PRODUCTS

PM Eckl1, A Alija1, N Bresgen1, O Sommerburg2,

W Siems3

1 University of Salzburg, SALZBURG, Austria2 University of Ulm, ULM, Germany3 Herzog-Julius Hospital, BAD HARZBURG, Germany

Free radical attack on b-carotene results in the forma-

tion of high amounts of cleavage products with

prooxidant activities. Since this finding may give an

explanation for the increased risk of lung cancer in

smokers observed in the Alpha-Tocopherol Beta-

carotene-Cancer prevention (ATBC) study and the

beta-Carotene and RETinol Efficacy (CARET) Trial a b-

carotene cleavage products mixture (CP), apo8'-

carotenal (apo8') and b-carotene were tested in the

highly sensitive primary rat hepatocyte genotoxicity

assay. The endpoints tested were: the mitotic index,

the percentage of necrotic and apoptotic cells,

micronucleated cells, chromosomal aberrations and

SCE (sister chromatid exchanges). Our results indicate

a genotoxic potential of both CP and apo8' already in

the concentration range of 100 nM and 1 µM, i.e. at

pathophysiologically relevant levels of b-carotene

and b-carotene breakdown products. A 3h treatment

with CP induced statistically significant levels of

micronuclei at concentrations of 0.1, 1 and 10µM, and

chromosomal aberrations at concentrations of 1, 5 and

10µM. Apo8' induced statistically significant levels of

micronuclei at concentrations of 0.1, 1 and 5µM, and

chromosomal aberrations at concentrations of 0.1, 1

and 10µM. In contrast, no significant cytotoxic effects

of these substances were observed. Since b-carotene

induced neither significant cytotoxic nor genotoxic

effects at concentrations ranging from 0.01 up to 10

µM, it can be concluded that most likely b-carotene

breakdown products are responsible for the occur-

rence of carcinogenic effects found for b-carotene in

clinical efficacy and cancer prevention trials.


Poster Presentation

PW4003

INFLUENCE OF TCDD ON 8-HYDROXY-2'-DEOXY-

GUANOSINE FORMATION IN HUMAN LIVER CELL

LINES

MO Eom, KP Byun, MS Park, OH Kim, HK Jung,

HI Kang

Korea Food and Drug Administration, SEOUL,

South-Korea

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is known

to induce a wide spectrum of biological responses,

including changes in gene expression, metabolism,

cell growth and differentiation. However, little is

known concerning the mechanisms that TCDD exerts

its effect at the cellular or molecular level. To enhance

our understanding of toxicity by TCDD at cellular

level, we investigated TCDD-induced responses in

two human liver cell lines, hepatoma HepG2 and non-

tumorigenic fetal hepatic WRL68 cells. We have

found that growth of HepG2 cell was decreased over

50% after treatment with over 1nM TCDD, but TCDD

does not have any effect on proliferation in WRL68

cell. Also, the levels of 8-hydroxy-2'-deoxyguanosine

(8-OHdG) was increased in a time dependent manner

after exposure to 0.1nM TCDD in HepG2 cell. The gene

expressions of 8-oxo-dGTPase (hMTH1) and adenine

DNA glycosylase (hMYH) were increased at 6h follow-

ing exposure to TCDD and then recovered at 24h. The

8-oxoguanine DNA glycosylase (hOGG1) gene expres-

sion was decreased at 72h after treatment with TCDD.

In WRL68 cells, the level of 8-OHdG increased 2 hours

after treatment with 0.1nM TCDD and then returned

to control level at 6 hours. Similarly, increase of the

repair enzymes hOGG1, hMTH and SOD expression

were observed at 6 hours and decreased at 12 hours in

WRL68 cells. These results suggest that the different

levels of 8-OHdG may be attributable to toxicity by

TCDD and enzymes related to oxidative stress may be

responsible to repair of 8-OHdG in our experimental

systems.

98



Poster Presentation

PW4004

ANTIMUTAGENIC PROPERTIES OF NATURAL

ANTIOXIDANTS IN THE ESCHERICHIA COLI K12

REVERSION ASSAY

B Nikolic, J Knezevic-Vukcevic, B Vukovic-Gacic,

D Mitic-Culafic, T Beric-Bjedov, J Stanojevic,

S Stankovic, D Simic

Faculty of Biology, BELGRADE, Yugoslavia

Natural antioxidants, widely distributed in plant

extracts, might protect genomic DNA from lesions

induced by reactive oxygen species, a significant

source of endogenous mutagenesis. The study of their

antimutagenic potential can promote the prevention

of carcinogenesis, immunodeficiency, neurodegener-

ation and aging.

The potential of vitamin E (alpha-tocopherol), essen-

tial oil (EO) of basil (Ocimum basilicum L.) and its

major constituent linalool to reduce the oxidative

mutagenesis, was studied in the E.coli K12 (arg- in

Arg+) reversion assay. Test A is created to evaluate the

antimutagenic potential against t-butyl hydroperox-

ide (t-BOOH)-induced oxidative mutagenesis and is

performed on repair proficient strain. Test B is per-

formed on mutator strains mutS (deficient in mis-

match repair) and mutT (deficient in removing

8-oxo-G); it is designed to determine the effect of

antioxidants on spontaneous mutagenesis and to

determine the molecular mechanism of its action.

The protective effect of model antioxidant vitamin E

against oxidative mutagenesis was detected in both

tests, but the strongest inhibition was detected in

mutS strain (76%). EO of basil, as well as linalool,

exhibited moderate inhibitory potential in Test A

(about 35%), while powerful inhibition was detected

with both test substances in mutS strain (over 60%).

EO of basil showed no effect on mutagenesis mediat-

ed by 8-oxo-G lesions, while investigation of linalool

in mutT strain is under way.


Poster Presentation

PW4005

ANTIMUTAGENIC PROPERTIES OF NATURAL ANTI-

OXIDANTS IN THE WP2 ANTIMUTAGENICITY TEST

B Vukovic-Gacic, J Stanojevic, J Knezevic-Vukcevic,

D Mitic-Culafic, T Beric-Bjedov, B Nikolic, S Stankovic,

D Simic

Faculty of Biology, BELGRADE, Yugoslavia

Oxidative damage to DNA by reactive oxygen species

is very important in mutagenic, carcinogenic, and

aging processes. The study of active substances from

medicinal and aromatic plants has revealed that

many of them possess antioxidative activity and may

be potent inhibitors of mutagenesis and carcinogen-

esis.

We tested essential oil (EO) of basil (Ocimum

basilicum L.) and its major monoterpene – linalool for

antimutagenesis due to their antioxidative proper-

ties. Vitamin E (alpha-tocopherol), potent scavenger

of reactive oxygen species, was used as the model

antioxidant.

The potential of antioxidant to reduce spontaneous

and t-butyl hydroperoxide (t-BOOH)-induced oxida-

tive mutagenesis were screened using strains E. coli

IC185 (WP2 lamB+) and IC202 (WP2 oxyRlamB+/pKM101).

Antimutagenic potential was observed by monitoring

trpE65 in Trp+ reversions. Strain IC202, highly sensi-

tive to oxidative damage due to its deficiency in the

OxyR function, shows increased mutability with

respect to the OxyR+ strain.

Vitamin E inhibited induced mutagenesis in both

strains (64% in IC202 and 43% in IC185). Slight inhibi-

tion of spontaneous mutagenesis was detected in

oxyR strain (17%). After validation with vitamin E, we

used highly sensitive oxyR strain for further investi-

gation.

A significant reduction of spontaneous mutagenesis

was obtained with both EO (63%) as well as linalool

(62%). The stronger antimutagenic potential against

induced mutagenesis was detected with EO (77%) in

respect to linalool (68%).

99



Poster Presentation

PW4006

EVALUATION OF OXIDATIVE DNA DAMAGE IN

ROME AIRPORT PERSONNEL

CLU Ursini1, D Cavallo1, C Chianese1, A di Francesco1,

M Gismondi2, S Iavicoli1

1 ISPESL, MONTEPORZIO CATONE, ROME, Italy2 Occup.Medic.Rome airport, ROME, Italy

Airport personnel is occupationally exposed on flight

lines and during aircraft routine maintenance proce-

dures to several polycyclic aromatic hydrocarbons

(PAH) produced by jet fuel, diesel fuel or kerosene

combustion. We evaluated, by enzyme Fpg-modified

comet test, direct and oxidative DNA damage on exfo-

liated buccal cells and lymphocytes of airport workers

(n=15) occupationally exposed to complex mixtures of

PAH and in a selected control group (n=7). For each

subject we calculated tail moments (product of relative

tail intensity and length) of 50 comets from fpg-

enzyme treated cells (TMenz) and from untreated

cells (TM), which indicate oxidative and direct DNA

damage respectively. For exfoliated cells, both TM and

TMenz mean values were higher in the exposed than

in the controls (TM p=0,12; TMenz p=0,05). For lym-

phocytes no significant differences were shown in

the exposed respect to control (TM p=0,4; TMenz

p=0,14). The presence of oxidative DNA damage was

evaluated in each subject considering TMenz/TM

ratio. When this ratio was higher than 1,5 the subject

was estimated to have oxidative damage. We found

the presence of oxidative DNA damage in 47% and in

13% respectively on lymphocytes and on exfoliated

cells of exposed in respect to the absence of oxidative

damage of controls. These results demonstrate a

great sensitivity of exfoliated cell to point out direct

DNA damage probably induced by substances con-

tained in the PAH mixture able to directly damage

DNA because they are the first target of interaction

with such substances. While the high presence of

subjects with oxidative DNA damage shown on lym-

phocytes could be related to effects of diol-epoxides

which are oxidant metabolites of PAH. The perform-

ing of enzyme-modified comet assay on both exfoli-

ated buccal cells and lymphocytes seems to be a

useful approach to study populations chronically

exposed to PAH.


Poster Presentation

PW4007

EXERCISE-INDUCED OXIDATIVE STRESS AND

PARP-1 ACTIVATION IN COPD PATIENTS BEFORE

AND AFTER REHABILITATION

GJ Hageman, EM Mercken, AMWJ Schols,

GJJM Haenen, EFM Wouters, A Bast


Previously, exercise-induced oxidative stress has

been shown after streneous exercise in patients with

COPD (chronic obstructive pulmonary disease). We

compared the exercise-induced systemic and local

oxidative stress response between a maximal and sub-

maximal cycle ergometry test and studied the influ-

ence of pulmonary rehabilitation. 11 (6M/5F) stable

COPD patients (FEV1=40 13%) were included. All sub-

jects performed an incremental cycle ergometry test

until exhaustion and a continuous work rate test at

60% of peak work load before and after 8 weeks pul-

monary rehabilitation. The standard Comet assay

and immunecytochemical staining for the presence

of poly(ADP-ribose) polymerase (PARP-1) polymers

were used to evaluate reactive oxygen species (ROS)-

induced DNA damage in peripheral blood lympho-

cytes. Breath condensate hydrogen peroxide was

measured as marker of pulmonary oxidative stress.

Measurements were done before, immediately after

and after 4h, and 24h.

Before rehabilitation: ROS-induced DNA strand

breaks were detected with the Comet assay immedi-

ately after both exercise tests (p<0.05), while breath

H(2)O(2) increased only immediately after and 4 h

after maximal exercise (p<0.05). The percentage of

PARP-polymer positive cells was significantly

increased only immediately after the submaximal

work load.

After rehabilitation: A significant improvement was

seen in peak work load (+24%, p<0.001) and in sub-

maximal exercise duration (+73%, p<0.002).

Remarkably, for the maximal exercise test a similar-

ly increased oxidative stress response was found with

the Comet assay, while no increase in ROS-induced

DNA damage or PARP-activation was detected after

the submaximal exercise test. These data indicate

that pulmonary rehabilitation positively influences

exercise-induced oxidative stress. Additional analy-

ses will possibly elucidate whether this positive effect

is caused by an improved antioxidant status.

Sponsored by a University Hospital Grant.

100



Poster Presentation

PW4008

SIGNIFICANT EFFECT OF ASCORBIC ACID ON DNA

CONFORMATION IN RELATION TO ITS ANTIOXI-

DANT ACTIVITY

YY Yoshikawa

Nagoya Bunri College, NAGOYA, Japan

Ascorbic acid is often regarded as an antioxidant in

vivo, where it protects against cancer by scavenging

DNA-damaging reactive oxygen species. In the pres-

ent paper, we will report another potent scenario on

the protective effect of ascorbic acid through signifi-

cant change in the higher order structure of DNA.

Recently, we examined the effect of ascorbic acid on

the higher-order structure of DNA through single

DNA observation with fluorescence microscopy, and

found that ascorbic acid generates a pearling struc-

ture in single giant DNA molecules, where elongated

and compact parts coexist along a molecular chain.[1]

The results of observations with electron microscopy

and atomic force microscopy indicate that the com-

pact parts assume a loosely packed conformation.

As the extension, here we study the protective effect

against double strand breakage by reactive oxygen at

different concentrations of ascorbic acid, in relation

to the change of the higher order structure of giant

DNA molecules. We have performed the real time

observation on the double strand breakage reaction

on individual giant DNA molecules by use of fluores-

cence microscopy. We have found that the double

strand break is markedly protected when coexisting

ascorbic acid is over millimolar concentrations. It is

found that such a protective effect of ascorbic acid

corresponds well to the above mentioned change on

the higher order structure of giant DNA molecules.

It has been reported that human circulating immune

cells, such as neutrophils, monocytes and lympho-

cytes, accumulate ascorbic acid in millimolar concen-

trations. Therefore, it is suggested that the ascorbic

acid concentration that induced the large conforma-

tional change in DNA in this study may be of physio-

logical significance.

Ref. [1] Y. Yoshikawa, et a., Eur.J.Biochem., 270,

3101(2003).


Poster Presentation

PW4009

THE MODULATING EFFECTS OF THYME AND ITS

MAJOR INGREDIENTS ON OXIDATIVE DNA DAM-

AGE

A Basaran, S Aydin

Hacettepe University Faculty of Pharmacy, ANKARA,

Turkey

Thyme has been commonly used in foods mainly for

its flavour, aroma and preservation and also in folk

medicine for years. Thyme essential oil and its ingre-

dients have been shown to exhibit a range of biologi-

cal activities. Antibacterial, antifungal, antitussive,

antispasmodic, antiplatelet aggregation and also

antioxidant activities were reported mostly with thy-

mol and carvacrol. On the other hand very few stud-

ies have been performed on the mutagenic and/or

antimutagenic effects of thyme and its ingredients.

In the present study the mutagenic potential of major

compound of thyme oil, i.e. thymol, carvacrol, and g-

terpinene and the methanolic extracts of thyme,

were investigated by single cell gel electrophoresis in

human lymphocytes and the effects of these sub-

stances on H2O2 (0.1 mM) induced oxidative DNA

damage were evaluated. No additional DNA strand

breakage was observed at thymol and g-terpinene

concentrations below 0.1 mM but at the higher con-

centrations of 0.2 mM significant increases were seen

in DNA damage. Carvacrol, which is an isomer of thy-

mol, seemed to induce DNA damage also in a dose-

manner and below concentrations of 0.1 mM no

damage in lymphocytes were seen whereas at the

higher concentrations carvacrol induced DNA dam-

age. The n-hexane and ethyl acetate fractions pre-

pared from the concentrated aqueous methanolic

extract of thyme showed no significant DNA damage

within the concentration range of 0.0075-7.5 mg/L,

and 10-50 mg/L respectively. Thymol and carvacrol at

the concentrations below 0.1 mM and 0.05 mM

respectively protected lymphocytes against H2O2

induced DNA damage but no protective effect was

observed with g-terpinene. N-Hexane and ethyl

acetate fractions of thyme containing phenolic, ter-

penic substances were also found to prevent the

oxidative DNA damage in human lymphocytes and it

can be concluded that phenolic compounds such as

thymol and carvacrol were responsible for the antiox-

idant effects of thyme.

101



Poster Presentation

PW4010

MODULATION OF OXIDATIVE CELL DAMAGE BY

POLYPHENOLIC APPLE JUICE COMPOUNDS/

EXTRACTS IN HUMAN COLON CELLS

CJ Janzowski1, S Schaefer1, M Baum1, F Will2,

H Dietrich2, G Eisenbrand1

1 University of Kaiserslautern, KAISERSLAUTERN,

Germany2 Institute of Enology and Beverages, GEISENHEIM,

Germany

Diets rich in fruits and vegetables are associated with

a lower risk of tumor induction in the intestine and

other sites. Many food ingredients have been claimed

to exhibit anticarcinogenic potential. We hypothe-

sized that apple juice with especially high amounts of

antioxidative polyphenols might be able to protect

the intestine and colon against oxidative cell damage.

We used the human colon cancer cell lines Caco-2 and

HT29. Cells were preincubated with selected polyphe-

nolic constituents/extracts for 24h. (Oxidative) DNA

damage was subsequently induced by treatment

with menadione (1h) and was detected by single cell

gel electrophoresis with/without additional treat-

ment with the repair enzyme formamidopyrimidine-

DNA-glycosylase (FPG). Lipid peroxidation was

induced by H2O2/ Fe2+ and monitored by HPLC/fluo-

rescence of the thiobarbituric acid derivative of mal-

ondialdehyde (MDA). Stability of parent compounds

during incubation was studied by HPLC/UV.

When incubating cells with menadione alone (1h,

37°C), higher concentrations were needed for HT29

(20µM) than for Caco-2 (3µM) cells to generate a simi-

lar extent of DNA damage. Preincubations with

quercetin and phloretin induced concentration

dependent modulation of DNA damage, HT29 being

more sensitive than Caco-2 cells. The respective glyco-

sides rutin and phloridzin (1-100µM) were not effec-

tive, epicatechin, chlorogenic acid and caffeic acid

were weakly effective. Preincubation with quercetin

increased the repair of oxidative DNA damage, the

extent of induced MDA formation was decreased.

During incubation of HT29, quercetin was rapidly

eliminated from the medium DMEM (t1/2: 117min);

rutin was eliminated much slower (t1/2>24h). Apple

juice extract efficiently diminished (oxidative) DNA

damage in HT29 and Caco-2 cells at 50µg/mL and

100µg/mL, respectively.

In conclusion, apple juice extract as well as quercetin

and phloretin are effective antioxidants, reducing

oxidative cell damage in human colon cells in a con-

centration dependent manner. (Support: BMBF grant

no. 01EA 0101).


Poster Presentation

PW4011

DNA DAMAGE ASSESSMENT IN TRANSGENIC

TOBACCO LINES AFTER EXPOSURE TO OXIDATIVE

STRESS

C Pellacani, A Mancini, FM Restivo, A Buschini, P Poli,

C Rossi

Universita di Parma, PARMA, Italy

Every year, environmental stress causes considerable

losses in crop quality and productivity. One of the

important mechanisms by which plants are damaged

during adverse environmental conditions is the

excessive production of ROS. Plants have evolved non-

enzymic and enzymic protection mechanisms that

efficiently scavenge ROS. Major ROS-scavenging

mechanisms of plants include superoxide dismutase

(SOD) and catalase (CAT). The aim of this work was to

verify the leaves cells sensitivity to oxidative stress in

terms of DNA damage in SR1 Nicotiana tabacum wt

line and in transgenic derivative lines (kindly provid-

ed by Dr Frank van Breusegem, Ghent University,

Belgium) CAT1, SOD+ and SOD-. CAT1 is an antisense

line for the N.plumbaginifolia catalase isoform 1 and

contains approximately 10% of normal catalase activ-

ity respect to SR1. SOD+ is an over-expressing line for

the N. plumbaginifolia mitochondrial SOD Mn-iso-

form, with enzyme activity ≈2 fold higher than in

untransformed plant. SOD- is a new transgenic line

obtained with antisense technology. After treatment

with oxygen peroxide for 2, 3 and 4hs at 25°C, the

leaves of 70 day-old plants were sliced to directly col-

lect nuclei for the Comet assay. DNA migration, as

DNA damage parameter, and cells with complete

DNA migration (GC), as necrosis/apoptosis parame-

ter, were recorded. Oxygen peroxide treatment

increased both the parameters in all the tobacco

lines. SOD+ showed behaviour similar to the wt line

SR1 both for DNA damage and GC induction; the oxi-

dant treatment produced GC more than DNA damage

in CAT1, whereas both the effects were evident in

SOD-. A time-dependent increase of DNA migration

values is observed still after 3h-exposure with subse-

quent decrease after 4h-exposure and simultaneous

increase of GC percentage. In conclusion, DNA dam-

age: CAT1 significantly less sensitive (P<0.05) than the

other lines; GC: SOD- more sensitive than SR1 and

SOD+ (P<0.01), and CAT1 than SR1 (P<0.05).

102



Poster Presentation

PW4012

GREEN TEA POLYPHENOLS, FLAVONOIDS, VITA-

MINS AND LACTOSERUM: PERSPECTIVES FOR COM-

BINATION CHEMOPREVENTION

TMCM de Kok, J de Jonge, MH van Herwijnen,

JJ Briedé, LC Wilms, LM Maas, P Kempers,

JCS Kleinjans


Many naturally occurring dietary compounds protect

against oxidative and alkylating DNA damage. These

chemopreventive effects can be induced either by the

inhibition of radical formation, direct radical scav-

enging, regeneration of antioxidants or modulation

of bioactivating or detoxifying enzymes. To achieve

maximal chemopreventive effects, a balanced mix-

ture of different compounds, combining several pro-

tective mechanisms, may give better results than the

individual factors. In this respect, Rivella green, a pop-

ular Swiss soft drink based on a combination of a

green tea, lactoserum, vitamin C and herbal and fruit

extracts, has in potential a promising chemopreven-

tive formula. Therefore, we investigated the effect of

Rivella green and its individual ingredients on in vitro

radical generation, induction of oxidative DNA dam-

age (8-oxodG) and DNA alkylation by benzo[a]pyrene

(BaP) and 2-amino-1-methyl-6-phenylimidazo[4,5-

b]pyridine (PhIP). The effects were compared to those

of two model green tea polyphenols, EGCG and ECG,

and quercetin as model flavonoid.

The green tea extract, lactoserum, sugar free base

(containing the herbal extract) and the combination

as present in the soft drink itself showed strong

inhibiting effects on superoxide radical formation

and induction of 8-oxodG by a superoxide generating

system. Even though lactoserum showed stimulation

of hydroxyl radical formation by hydrogen peroxide

(probably caused by stimulation of the Fenton reac-

tion by traces of transition metals in lactoserum), lev-

els of 8-oxodG were reduced; this indicates the

presence of other antioxidative agents. Rivella green,

the green tea extract, lactoserum and the sugar free

base showed furthermore inhibition of DNA alkyla-

tion by both BaP and PhIP. Based on these findings

and taking into account that green tea polyphenols

and flavonoids may also protect against DNA alkyla-

tion in vivo by modulation of phase I and II detoxify-

ing enzymes, a human dietary intervention study

appears relevant to evaluate the full potential of this

specific combination of chemopreventive com-

pounds.


Poster Presentation

PW4013

OXYGEN RADICAL SCAVENGING ACTIVITY AND

PREVENTION OF DNA STRAND BREAKAGE BY

QUERCETIN IN HUMAN LYMPHOCYTES

JJ Briedé, LC Wilms, S Peeters, EJC Moonen,

JCS Kleinjans


Quercetin is a dietary flavonoid present in plant-

derived nutrients, and reported to possess many bio-

logical activities including pro- and anti-oxidative

properties. Although the compound has been exten-

sively studied for its pro- and anti-oxidative proper-

ties in vitro, cellular (anti-)oxidative activity of

quercetin needs further investigation. Therefore, we

treated human peripheral blood lymphocytes with

quercetin and followed cellular uptake kinetics by

HPLC-UV detection. Subsequently, the oxygen free rad-

ical scavenging capacity and prevention of oxygen free

radical-induced DNA strand breakage of quercetin in

lymphocytes was determined. Incubations were per-

formed at concentrations ranging from physiological

relevant blood plasma levels up to its maximal solu-

bility in watery solvents (1 - 100 µM) and were fol-

lowed by several washing procedures. It is shown that

2% of extracellular quercetin is taken up by lympho-

cytes. Subsequently, quercetin pre-incubated lym-

phocytes were exposed to increased oxidative stress

by producing either hydroxyl or superoxide anion

radicals via respectively H2O2/Fe2+ or phenazine

methosulfate/NADH containing systems. Electron

spin resonance (ESR) spectrometry in combination

with spin trappings technique revealed that these

quercetin-treated cells have an increased capability

to scavenge extracellular produced hydroxyl- or

superoxide anion free radicals. Moreover, quercetin

pre-incubated lymphocytes challenged with these

oxygen free radical producing systems showed a

decrease in DNA strand breaks using the alkaline sin-

gle cell electrophoresis assay (COMET). Altogether,

these experiments show that the uptake of quercetin

by human blood lymphocytes results in improved cel-

lular anti-oxidant capacity and DNA protective prop-

erties in circumstances of increased oxidative stress.

103



Poster Presentation

PW4015

DNA DAMAGE IN COLON, LIVER AND LUNG AFTER

ORAL EXPOSURE TO DIESEL PARTICLES IN RATS

M Dybdahl1, AK Müller1, E Olatunde Farombi2,

P Møller3, H Wallin4, U Vogel4, L Risom3, H Autrup5,

LO Dragsted1, S Loft3, ML Binderup1

1 Danish Institute for Food and Vet. Research,

SØBORG, Denmark2 University of Nigeria, IBADAN, Nigeria3 University of Copenhagen, COPENHAGEN,

Denmark4 National Institute of Occupational Health,

COPENHAGEN, Denmark5 University of Aarhus, AARHUS, Denmark

Several chemical mutagens and carcinogens, includ-

ing polycyclic aromatic hydrocarbons (PAHs) and

nitrated PAHs, are adsorbed to the surface of diesel

exhaust particles (DEP). DEP can induce formation of

reactive oxygen species and cause oxidative DNA

damage as well as bulky DNA adducts. Lung tissue is

a target organ for DEP induced cancer following

inhalation, but recent studies have provided evidence

that the lung is also a target organ for DNA damage

and cancer after oral exposure to other complex mix-

tures of PAHs. The aim of the present study was to

investigate the genotoxic effect of DEP after oral

administration in terms of markers of DNA damage,

mutations and repair in Big Blue' rats fed a diet with

0.2, 0.8, 2, 8, 20 or 80 mg DEP/kg feed for 21 days.

DEP increased the formation of DNA adducts and

DNA strand breaks in colon, liver and lung. The num-

ber of oxidised DNA bases, measured as endoIII and

FPG sensitive sites, was increased in lung tissue, but

not in colon and liver. The lack of effect on DNA base

oxidation could be related to the increased expression

of the repair gene OGG1 observed in colon and liver,

but not in the lung. There was a significant increase

in ERCC1 mRNA in liver at the highest dose level, sug-

gesting a repair response to bulky DNA adducts.

There was no detectable increase in mutation fre-

quency in any of the investigated tissues.

It was shown by the present study that gastrointesti-

nal exposure to DEP at relatively low levels can induce

DNA adducts and oxidative stress resulting in DNA

strand breaks and increased expression of DNA repair

genes locally in colon and systemically in lung and

liver. The data indicate that consideration of oral

exposure should be included in risk assessment of

DEP.


Poster Presentation

PW5001

DIFFERENTIAL GENE EXPRESSION AFTER TREAT-

MENT WITH 2,3,7,8-TETRACHLORODIBENZO-P-

DIOXIN IN HAIRLESS MOUSE SKIN

TK Ryeom, HI Kang, MK Kang, MO Eom, MS Park,

SW Jee, KS Kwon, MH Kim, OH Kim

Korea Food and Drug Administration, SEOUL,

South-Korea

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) displays

high toxicity in animals and has been implicated in

human carcinogenesis. Despite extensive research,

the mechanisms of TCDD-induced carcinogenesis are

poorly understood. To enhance our understanding of

toxicity mediated through the pathway by which

TCDD stimulates gene expression, we have used two

different microarray hybridization to detect the tran-

scriptional signature in papillomas of hairless mouse

skin induced by two stage carcinogenesis protocol.

We found that 49 and 42 genes among 5,592 genes

(Digital genomics cDNA chip) associated with protein

synthesis, cell organization, lipid transport and oxida-

tive stress in tumor and surrounding regions were

up- or down- regulated two fold or more, respectively.

In addition, 53 genes among 96 genes (pathway-spe-

cific cDNA arrays, SupperArray) involved in cell cycle,

signal transduction, apoptosis, adhesion molecule,

angiogenesis, and invasion were up-regulated in the

tumor surrounding region. In the tumor region, only

a few genes related with cell cycle and oncogenes

were up-regulated but several genes encoding inte-

grin, Akt, Raf were down-regulated. Our data suggest

that TCDD promote to development of skin tumor in

hairless mouse by changes of multiple integrated

network of signaling pathway.

104



Poster Presentation

PW5002

HEPATOTOXICITY OF COUMARIN IN SANDWICH

CULTURED HEPATOCYTES INVESTIGATED BY

GENE EXPRESSION PROFILING

AS Kienhuis1, HM Wortelboer2, JCS Kleinjans1,

RH Stierum2, JHM van Delft1

1 Maastricht University, ZEIST, The Netherlands2 TNO Nutrition and Food Research, ZEIST,

The Netherlands

Primary hepatocyte cultures are often used to study

the hepatotoxicity of compounds. When primary

hepatocytes are cultured in sandwich configuration,

the viability and morphology of the cultures are

greatly enhanced compared to conventional mono-

layers, making the system more comparable to the in

vivo situation. Still, no ideal in vitro liver system exists.

Toxicogenomics can be used as a tool to analyse toxi-

cology at the molecular level without preconceived

notion on the molecular mechanisms involved in the

mode of action. This will make it possible to compare

the toxin-specific expression profiles in sandwich

cultures in vitro and rat livers in vivo and hence

determine the relevance of these in vitro models in

terms of their predictiveness of molecular alterations

associated with toxicity as they occur in vivo. To

investigate the in vitro - in vivo extrapolation para-

digm in toxicogenomics, coumarin is used as an

example.

In the present study, the in vitro hepatotoxicity of

coumarin was tested in rat hepatocytes cultured in

sandwich configuration. The hepatotoxic effects of

coumarin in rats are mediated by a cytochrome P450

(CYP) dependent conversion of coumarin to coumarin

epoxide and o-HPA. A major problem of in vitro hepa-

tocyte cultures is the rapid decline in CYP activity.

When hepatocytes were cultured in standard medi-

um, no cytotoxicity of coumarin up to 1.8 mM was

observed. Addition of an inducer mixture containing

low concentrations of model inducers phenobarbital,

b-naphthoflavone, and dexamthasone, significantly

increased CYP 2B and CYP 3A enzyme activities com-

pared to maintenance in standard medium. Besides,

the cytotoxicity of coumarin increased significantly

when cells were cultured in inducer-supplemented

medium as determined by MTT reduction and LDH

leakage assays. Future toxicogenomics studies will be

combined with this model to compare the gene

expression profiles of cells cultured in either standard

medium or inducer-supplemented medium.


Poster Presentation

PW5003

INFLUENCE OF SIX PAHS ON GENE-EXPRESSION

IN HEPG2 CELLS; RELATION TO DNA-ADDUCT

FORMATION AND CARCINOGENICITY

YCM Staal, MH van Herwijnen, FJ van Schooten,

JHM van Delft


Polycyclic aromatic hydrocarbons (PAHs) present in

the air originate from incomplete combustion and

some are known to cause cancer. To improve risk

assessment for PAHs, we investigated modulation of

gene expression in human cells after exposure to six

ambient PAHs. HepG2 cells were exposed to 3, 10 or

30µM of benzo[a]pyrene (B[a]P), benzo[b]fluoranthene

(B[b]F), fluoranthene (FA), dibenzo[a,h]anthracene

(DB[a,h]A), 1-methylphenanthrene (1-MPA), dibenzo[a,l]-

pyrene (DB[a,l]P) or solvent control during 6 hours in

duplicate experiments. RNA was isolated, indirectly

labelled with fluorophores and hybridised on a

Phase-I cDNA array containing 600 toxicologically

relevant genes. Samples were hybridised on the

microarray twice for each duplicate experiment, using

four fluorophores (Alexa 488, Alexa 594, Cyanine3 or

Cyanine5). Data were analysed with ImaGene and

GeneSight to identify changes in gene expression. Also,

DNA adducts were measured by 32P post-labelling.

All compounds, except 1-MPA, altered the gene

expression. In general, CYP1A1 (not 1-MPA) and CYP1A2

(not 1-MPA, FA) were up regulated. Insulin-like

growth factor binding protein 1, IgE binding protein

and Likely Ortholog of Rat Vacuole Membrane Protein

1 (VMP1) were up regulated by B[a]P, B[b]F and

DB[a,h]A (not VMP1). Histone 2A (B[a]P, B[b]F) and

Histone deacetylase (B[a]P, B[b]F, FA) were down regu-

lated. The high to low DNA-adduct forming capacity

for the PAHs was B[a]P≥DB[a,l]P≥B[b]F≥DB[a,h]A≥1-

MPA≥FA. This order is generally similar for the num-

ber of gene expression differences.

DNA adducts (2log) were correlated with (2log) gene-

expression differences for the significantly modulat-

ed genes. Significant correlations were found for 22 of

43 genes with a correlation up to 0.64 (CYP1A1). When

correlating the Potency Equivalency Factor (PEF) for

carcinogenicity (2log) values for B[a]P, B[b]F, DB[a,h]A

and DB[a,l]P with (2log) gene-expression differences,

significances were found for 20 of 43 genes with a

correlation up to 0.72 (Insulin-like growth factor I).

Different genes correlate with DNA adduct formation

or with PEF.

105



Poster Presentation

PW5004

DIFFERENTIAL GENE EXPRESSION IN HUMAN

PBMC INDUCED BY CIGARETTE SMOKE AND ITS

CONSTITUENTS

DM van Leeuwen, JCS Kleinjans, JHM van Delft


Background: Toxicogenomics is the application of

new technologies like microarrays in toxicology in

order to understand the molecular basis of the rela-

tionship between xenobiotic exposure (to e.g. car-

cinogenic agents) and early human response at

genome level, i.e. altered expression of large number of

genes that are analysed simultaneously. This project

focuses on this early response induced by tobacco

smoke related agents cigarette smoke condensate

(CSC), BaP, NNK, 4-ABP and H2O2. The aim of the study

was to research differential gene expression due to

exposure to carcinogenic compounds in human sur-

rogate cells from blood. In order to shed light on the

effects that may occur in vivo, we first conducted in

vitro studies in human PBMC.

Methods: Quiescent PBMC were exposed for 18 h to

several concentrations of the agents. Total RNA was

isolated and purified following Trizol and RNeasy

protocols. Derived cDNAs were indirectly labelled

with Cy3 or Cy5 fluorophores. Control and test sam-

ples were competitively hybridised to Phase-1

microarrays, containing 4 x 600 toxicologically rele-

vant genes. Data were analysed with the software

programs ImaGene and GeneSight to identify modu-

lated genes.

Results: Data on CSC, BaP, NNK, 4-ABP and H2O2 have

been generated. Significant modulations and >2 fold

up and down regulation of genes were clearly

detectable. Data are reported on total number of

genes modulated and genes that will be considered

as candidate biomarkers, i.e. senstively and conse-

quently responding genes. The inflammatory process

was the main pathway that was induced due to the

exposures.

Conclusions: This in vitro study showed clear gene

expression modulations after in vitro exposure of

PBMC to chemical carcinogens. Regarding

numbers of differentially expressed genes, the study

provides a basis for the selection of candidate genes

for the biomarker. However, in vivo data will be need-

ed to substantiate these results.


Poster Presentation

PW5005

ANALYSIS OF THE ILSI GENOTOXICITY WORKING

GROUP'S AFFYMETRIX MGU74V2 ARRAY DATA

USING ROSETTA RESOLVER

JS Harvey, J Kenny, R Rees, AM Lynch

GlaxoSmithKline, HERTFORDSHIRE, United Kingdom

The ILSI Genomics subcommittee was established to

evaluate the role of genomics in mechanism based

risk assessment. Here we present an analysis of the

Affymetrix MgU74Av2 gene expression data generat-

ed by members of the ILSI Genotoxicity working

group (Proctor and Gamble, Pfizer, Aventis, Glaxo

SmithKline). Mouse lymphoma cells were treated

with reference genotoxins (Etoposide, Hydroxyurea,

Taxol, Methyl Methanesulfonate, Cisplatin and

Mitomycin C), which induced robust genotoxicity and

cytotoxicity. Differential gene expression (DGE)

changes were measured 4 hrs and 24 hrs after treat-

ment using standard methodologies. Analysis of the

MgU74Av2 arrays (~12000 sequences) from replicate

studies for each compound was performed using

Rosetta Resolver software (Merck & Co, USA).

Supervised ratio analysis identified a number of DGE

changes related to compound treatment, concentra-

tion and time. There was, however, only a low num-

ber (generally < 50 ) of concentration dependent DGE

changes (>1.5 fold) for any particular treatment.

Furthermore there was limited reproducibility in the

DGE changes for any compound tested in any two

laboratories (generally <10 maximum observed ~100).

Unsupervised 2D-cluster analysis and Principal

Component Analysis failed to identify reproducible

or meaningful relationships between control and

treated DGE array data, either between laboratories

or more surprisingly, within a laboratory. Moreover,

numerous statistical methods were applied to the

data but failed to identify any DGE changes that

could be related to a compounds distinct mechanism

of action.

In conclusion, Rosetta Resolver analysis of the

Affymetrix MgU74Av2 data resulted in the identifica-

tion of a limited list of DGE changes in mouse lym-

phoma cells following treatment with various

genotoxins. Gene pathway analysis was non-inform-

ative for the majority of the genes identified.

Statistical analysis of the data (using numerous

methods), however, was unable to identify any DGE

changes that could be specifically related to a com-

pounds mechanism of action ie. direct or indirect

genotoxicity.

106



Poster Presentation

PW5006

IMPACT OF GENETIC POLYMORPHISMS ON BITH

WEIGHT

RJ Sram, J Dejmek, B Binkova, I Chvatalova,

A Milcova, M Dostal, P Rössner, I Solansky

Institute of Experimental Medicine AS CR, PRAGUE,

Czech Republic

All studies from Poland and the Czech Republic indi-

cate the relationship between ambient air pollution

and an increase of DNA adducts in maternal and cord

blood and/or in placentas, as well as the relationship

of these biomarkers to the development of newborns.

Therefore we studied the impact of genetic polymor-

phisms on pregnancy outcome as a part of CHIL-

DRENGENONETWORK project. DNA was isolated

from placenta samples collected in polluted regions

(Teplice and Prague) and control region (Prachatice).

Polymorphisms of metabolic genotypes (GSTM1, GSTP1,

GSTT1, EPHX3, EPHX4, CYP1A1 Ile/Val and CYP1A1-MspI)

were determined in 1014 placenta samples. The conse-

quence of genotypes to birth weight as well as catego-

ry LBW+prematurity (low birth weight < 2500 g,

prematurity < 37 weeks) was determined. Using mul-

tiple regression analysis, CYP1A1-MspI mutation

decreased the newborn birth weight of smoking

mothers in Teplice ( -315 g, P<0.05). GSTM1 nulll geno-

type significantly increased LBW+prematurity in all

three groups (AOR = 1.33, P<0.05, especially at Teplice

AOR = 1.54, P=0.01 and Teplice-nonsmokers AOR = 1.53,

P<0.05), CYP1A1 Ile/Val polymorphism in smoking

mothers in the first trimester (AOR = 2.89, P<0.05) and

smoking mothers in the first trimester at Teplice

(AOR = 2.91, P<0.05). Other significant predictors were

exposure to carc-PAHs, maternal smoking, passive

smoking. Combination of different polymorphisms is

further analyzed. Our results indicate that genetic

polymorphisms may affect birth weight in newborn

children, if combined with air pollution and/or

lifestyle (smoking).

Supported by the Czech Ministry of Environment

VaV/740/5/03 and by the EC QLK4-CT-2002-02198.


Poster Presentation

PW5007

BIOINFORMATICS FOR GENETIC TOXICOLOGY

PD Lewis1, S Abeysinghe2, I Khan3

1 Cardiff University, LLANELLI, United Kingdom2 UW College of Medicine, CARDIFF,

United Kingdom3 Biostat & Bioinformatics Unit, CARDIFF,

United Kingdom

Over the last decade Bioinformatics, as a discipline in

its own right, has developed in parallel with the

'omic' revolution, particularly genomics and pro-

teomics. In particular, huge efforts have been made to

develop novel algorithms and software tools to

analyse sequence and microarray data. Thus, many

bioinformaticists/bioinformaticians have focused

their research and skills within these areas. In con-

trast, little attention has been paid to developing and

implementing tools for analysis of mutation datasets

from dedicated databases such as the Human Gene

Mutation Database and the Mammalian Gene

Mutation Database. Such databases house mutation

data that is either inherited and disease-linked or

induced by known chemical/physical mutagens in

vitro or in transgenic animals. Such data is extremely

useful for trying to predict potential cause of genetic

disease although the tools to extract useful informa-

tion and make predictions from mutation data are

currently limited. We are currently developing algo-

rithms and software tools for mutation data retrieval,

statistical analysis, pattern discovery and prediction

of mutable DNA regions which will be made publicly

available. The software will be accessed using a web

site with a user friendly interface and state of the art

2D and 3D visualisation tools. Initial studies using

such tools have already allowed us to make predic-

tions as to a potential cause of lung cancer in smok-

ers.

107



Poster Presentation

PW5008

PROGRESS WITHIN THE CHILDRENGENONET-

WORK, A FP5 CONCERTED ACTION

LE Knudsen

Institute of Public Health, COPENHAGEN, Denmark

This Concerted Action (CA) form a European network

providing input and data for risk assessment at the

national and European level. The Children Genotoxicity

Network focus on gene-environment interactions dur-

ing the foetal, neonatal and infancy developmental

periods, concentrating on genotoxic exposures and

environmental factors with focus on air pollution (traf-

fic and tobacco). The aim of the Concerted Action is to

collect, validate and review the available, though few,

European and other studies (WP1) and study materi-

als on children’s exposure and susceptibility to geno-

toxicants (WP2), with focus on air pollution. A further

aim is to prepare protocols to conduct feasibility stud-

ies with perfused placenta (WP3) and with mothers

and their children (two siblings) (WP4), where

research will be financed by national or other

sources. Special attention is given to ethical, legal and

social issues related to environmental studies of chil-

dren (WP6). Progress witih the workpackages will be

described and the associations to the work on bio-

monitoring of children within Europe as initiated by

SCALE will be presented.


Poster Presentation

PW6001

CHEMICAL INDUCTION OF PREMATURE CEN-

TROMERE SEPARATION: A NEW PARAMETER OF

GENOTOXICITY?

B Bajic1, SP Biljana2, D Ninoslav3, L Zivkovic2

1 Galenika pharmaceuticals, BELGRADE, Yugoslavia2 School of Pharmacy, BELGRADE, Yugoslavia3 School of Veterinary Medicine, BELGRADE,

Yugoslavia

Premature Centromere Separation (PCS) is an uncom-

mon cytogenetic abnormality which is characterized

with chromosome chromatides distinctively separat-

ed before usual time. PCS can be viewed as a primary,

yet unspecific manifestation of chromosome insta-

bility that has been associated with various malig-

nancies and chromosome-breakage syndromes.

Recent investigations have found that PCS yields are

significantly higher in population exposed to mixed

chemicals, pesticides, crude oils and cytostatic drugs.

In order to evaluate PCS as a parameter of genotoxici-

ty we have used various cytostatics, such as an

inhibitor of protein synthesis, Cycloheximide (dose, 5

µg/ml; 10 µg/ml and 25 µg/ml), a cAMP analogue, 8-Cl

cAMP (1 µM; 5 µM and 15 µM), inductor of microtubu-

lar stabilization Taxol (0,01 µM; 0,05 µM and 0,2

µM)and alkylating agent Mitomycin C (dose 0,05 µM;

0,15 µM and 0,6 µM). All investigated cytostatics can

induce PCD when added into a culture of human

peripheral blood lymphocytes. In order to evaluate

these findings we have investigated the distribution

patterns of PCS involving less than two, more than

two and PCS involving all chromosomes compared to

increasing doses of our investigated substances. Also,

by comparing distribution patterns of PCS induced by

our investigating agents to there clastogenic and/or

aneugenic potential we tried to evaluate if PCS con-

tributes to there clastogenic potential. We have found

no correlation between means of total chromosome

aberration and micronucleus frequencies in human

peripheral blood lymphocytes. Our results suggest

that PCS induced by novel investigated substances

should be included as a parameter of genotoxicity in

pre clinical and clinical risk assessment analysis.

108



Poster Presentation

PW6002

GENOME DAMAGE CAUSED BY 5-NITROFURAN-

TOIN IN SUCKLING AND ADULT MICE DETECTED

BY MICRONUCLEUS ASSAY

A Fucic1, D Markovic2, Z Ferencic2, B Mildner2, AM

Jazbec3, J Bubic Spoljar2

1 Institute for Medical Research and Occupational

Health, ZAGREB, Croatia2 Pliva Research Institute Ltd, ZAGREB, Croatia3 Faculty for Forestry, ZAGREB, Croatia

Antibiotic 5-nitrofurantoin has widely been used in

paediatric patients to treat urosepsis for the last 20

years. Recent papers suggest the need for re-evalua-

tion of its safety due its potential genotoxic effects.

Taking into account possible differences in its metab-

olism and clearance between suckling and adult ani-

mals, we conducted a genotoxicological study to

reveal possible age-related genome damage caused

by this substance. In vivo micronucleus assay (MN)

was performed on 3 and 8- week- old mice. Each

group included 5 males and 5 females. Single doses of

5, 10 and 50 mg/kg of 5-nitrofurantoin were injected

intraperitonealy. Blood samples from tail vein were

taken before injection and 48h, 96h, 168h and 2 weeks

after the application. In each animal 1000 reticulo-

cytes were analysed.

5-nitrofurantoin caused a significantly increased MN

frequency in both suckling (mean group value 5,5 %)

and adult animals (mean group value 3,5%) than in

control animals. Mean MN frequency in suckling ani-

mals was higher than in adult animals for each dose

and sampling time. Micronucleus frequency remained

significantly higher in suckling animals even two

weeks after exposure to 5-nitrofurantoin. Our study

confirmed the genotoxic potency of 5-nitrofurantoin

in suckling and adult animals. We proved higher sen-

sitivity of suckling animals to this compound and a

significantly longer effect than in adults. This effect is

probably due to underdeveloped mechanisms of

detoxification and renal elimination of xenobiotics.

Our study confirmed the need for further analysis of

this compound and its cost-benefit ratio.


Poster Presentation

PW6003

ESTIMATED EXPOSURE TO PHTHALATES IN COS-

METICS AND RISK ASSESSMENT

BM Lee, HJ Koo


Some phthalates such as DEHP and DBP, and their

metabolites are suspected of having teratogenic and

endocrine disrupting effects. To predict possible

human exposure to phthalates in cosmetics, the lev-

els of di(2-ethylhexyl) phthalate (DEHP), di-ethyl

phthalate (DEP), di-butyl phthalate (DBP), and butyl-

benzyl phthalate (BBP) were determined by HPLC in

102 branded hair sprays, perfumes, deodorants and

nail polishes. DBP was detected in 19 of the 21 nail pol-

ishes, in 11 of the 42 perfumes, and DEP in 24 of the 42

perfumes and 2 of the 8 deodorants. Exposure levels

to phthalates in cosmetics by dermal absorption were

estimated to be 6.0x10-4 µg/kg bw/day for DEHP, 0.6

µg/kg bw/day for DEP, and 0.103 µg/kg bw/day for

DBP. However, if 100% absorption was assumed

through inhalation, the daily exposure levels to

phthalates in cosmetics were estimated to be 0.026

µg/kg bw/day for DEHP, 81.471 µg/kg bw/day for DEP,

and 22.917 µg/kg bw/day for DBP, which are far lower

than the regulation levels set by the Scientific

Committee on Toxicity, Ecotoxicity and the Environ-

ment (CSTEE) (37µg/kg bw/day, DEHP), Agency for

Toxic Substances and Disease Registry (ATSDR) (7000

µg/kg bw/day, DEP), and International Programme on

Chemical Safety (IPCS) (66 µg/kg bw/day, DBP),

respectively. Based on these data, hazard indices (HI,

daily exposure level / regulation level) were calculat-

ed to be 7.0x10-4 for DEHP, 0.012 for DEP, and 0.347 for

DBP, respectively. These data suggest that estimated

exposure to phthalates in the above mentioned cos-

metics are small. However, total exposure levels may

be greater and require further investigation.

109



Poster Presentation

PW6004

SETTING SAFE UPPER INTAKE LEVELS FOR

NUTRACEUTICALS

BM Lee, SC Kang


A nutraceutical is any food or food ingredient consid-

ered to provide medical or health benefits, including

the prevention and treatment of disease. Nutraceuticals

may be taken as the form of foods or of dietary supple-

ments. The scientific literature has shown that

nutraceuticals have not only beneficial properties, but

also adverse health effects. Nutraceuticals such as phy-

tosterols for cholesterol control, may be intended to be

consumed over a long period of time. However, for

many nutraceuticals including phytosterol, the safe

upper intake levels (SUIL) are not provided for con-

sumers and public concern on the safety issue has

been raised. The SUIL is the highest level of daily

nutrient intake that is likely to pose no risks of

adverse health effects to almost all individuals in the

general population. As intake of nutraceticals

increases above the SUIL, the risk of adverse effects

increases. The development of SUIL depends on the

establishment of an adequate uncertainty factor

between expected intake level and identified poten-

tial hazards. After careful review and analysis of pre-

vious reports, scientific judgement was employed to

determine how to establish SUIL. In this paper, a

methodology how to set safe upper intake levels for

nutraceuticals was proposed, based on a through

review of the scientific literature, the hazard identifi-

cation, dose-response assessment and uncertainty

factors.

(This work was supported by a grant from the

Ministry of Health & Welfare 03-PJ1-PG1-CH10-0002).


Poster Presentation

PW6005

GENOTOXIC STUDY OF SOME MEDICINAL PLANTS

USING THE WHITE/WHITE+ SMART ASSAY IN

DROSOPHILA

N Mezzoug1, M Idaomar1, J Abrini1, A Muñoz Serrano2,

A Alonso-Moraga2

1 Abdelmalek Essaadi University, TETOUAN, Maroc2 Universidad de Córdoba, CÓRDOBA, Spain

Since the plants often synthesize plus their major

components, the chemical substances which are

potentially genotoxic and/or carcinogenic; the evalu-

ation of the genotoxicity of the plants used in the tra-

ditional medicine can present a great importance.

The present study is carried out to assess the geno-

toxicity activity of Licorice (Glycyrrizha glabra),

Nutmeg (Myristica fragrans), Black cumin (Nigella

sativa), Oregano (Oreganum compactum), Rosemary

(Rosmarinus officinales) and Fenugreek (Trigonella

foenum graecum) using the w/w+ somatic assay in

Drosophila melanogaster. Oregon K-yellow females

were crossed to Oregon k-white males. The flies were

permitted to lay eggs in bottles for 3 days on food sup-

plemented with the aqueous extract of the plant at

different concentrations. Newly hatched flies were

counted and the females were transferred to fresh

medium; 2-6 days later their eyes were inspected for

the presence of white spots. The loss of heterozygosi-

ty of recessive marker used (white) can lead to the

formation of mutant clones of larval cells, which are

then expressed as spots on the eyes of the adult flies.

Negative solvent controls were included in all experi-

ments. For the evaluation of the genotoxic effect of

plants, the frequencies of spots per eyes of a treated

series are compared to its concurrent control using

the T2- test. Our results do not show any significant

genotoxic effect of the Black cumin, Oregano and

Fenugreek. But It was found that the Nutmeg,

Rosemary and Liquorice increase the rate of sponta-

neous mutations which remains no significant

(p>0.05) with Liquorice at 2%, significant (p<0.05)

with rosemary at 5% and highly significant (p<0.001)

when the Nutmeg is administered at 3%.

110



Poster Presentation

PW6006

GREENSCREEN EM - A YEAST BASED GENOTOXICITY

AND CYTOTOXICITY ASSAY FOR ENVIRONMEN-

TAL MONITORING

W Knight, PO Keenan, RM Walmsley

Umist, MANCHESTER, United Kingdom

GreenScreen, a new assay capable of simultaneously

measuring both genotoxicity and more general cyto-

toxicity in aqueous environmental samples, will be

described. There is currently a need for simple meth-

ods for the screening and monitoring of a wide range

of toxic contaminants in the aquatic environment.

For example, in assessing the effectiveness of waste

treatment, to control the toxicity of industrial efflu-

ent discharges, to enable compliance with pollution

prevention and control legislation, to protect water

treatment plants from toxic influents and for hazard

assessment where the deliberate release of chemical

armaments is suspected. GreenScreen allows rapid

determination of whole sample genotoxicity with

minimum sample pre-treatment.

The assay uses eukaryotic (yeast) cells, genetically

modified to express a green fluorescent protein when-

ever DNA damage, as a result of exposure to genotoxic

agents, is repaired. A measure of the reduction in cell

proliferation is used to characterise general toxicity

producing familiar EC50 and LOEC data. The assay pro-

tocol has been developed for use in the field by non-

skilled personnel, employing dedicated, portable

instrumentation. A range of environmentally relevant

substances has been evaluated using the assay, includ-

ing solutions of metal ions, solvents and pesticides.

Preliminary data from a year long direct toxicity

assessment validation programme will be presented,

comparing the assay’s response to that of standard

Daphnia and algal tests, in the analysis of 38 varied

industrial effluents. In addition, results from a project

funded by the European Commission 5th Framework

'Energy, Environment and Sustainable Development'

programme will also be discussed. In this work, a

novel at-line genotoxicity monitor has been devel-

oped and integrated with an effluent toxicity treat-

ment system. The sensitivity to a wide range of

substances and effluents suggests GreenScreen is a

useful tool for environmental toxicity monitoring

and hazard assessment.


Poster Presentation

PW6007

HUMAN STRESS EXPRESSION IN CONTEXT OF

RISK ASSESSMENT

FI Ingel

A.N.Sysin Research Institute of Human Ec, MOSCOW,

Russia

The fact, well known to oncologists, that strong emo-

tional trauma presents in each anamneses of cancer,

unfortunately, till now did not attract an attention of

specialists in risk assessment.

Here will be present the substantiation for new

approach for human risk assessment grounded on

evaluation of biomarkers of effect.

In 2 independent researches (60 adult men and 78

young children) we measured emotional stress

expression in parallel with evaluation of spectrum of

divided cells, level of micronuclei and apoptosis in

blood cells cultures (cytokinetic block) with and with-

out 1,0 Gy of gamma-irradiation in vitro as well as

adaptive response to irradiation.

Results demonstrated similar effects both for adults

and for children (P<0,05): positive correlation between

human stress expression and frequency of accelerately

divided cells; negative correlation between level of

apoptosis and stress expression; positive correlation

between stress expression and radiosensitivity as well

as type of adaptive response.

Because: stress is the first reaction of an organism to

any influence and therefore it cannot be avoided (Selye,

1936; Khaitsev et al, 1997); human stress expression

directly correlates with toxic and genotoxic exposure

(Ingel et al, 2001); abnormal stress expression connect-

ed with an increased level of chromosomal aberra-

tion and elevated individual genome susceptibility

(Ingel 1997-2002), we may conclude that human

stress expression is important indicator of risk of

oncological and other diseases connected with

appearance of genetic damage.

Study of mammalian and human genetical effects of

emotional stress demonstrated that measurement of

stress expression in complex with individual genome

susceptibility is one of key approach for creation of

the new system of risk assessment.

The algorithm of this approach will be present.

The study was partly supported with INTAS grant

1005.

111



Poster Presentation

PW6008

MEASUREMENT OF REACTIVE METABOLITES AS A

BASIS FOR CANCER RISK ASSESSMENT: APPLICA-

TION TO 1,3-BUTADIENE

CF Fred1, M Törnqvist1, A Kautiainen2, F Granath3

1 Stockholm University, STOCKHOLM, Sweden2 Preclinical R&D, Biovitrum AB, STOCKHOLM,

Sweden3 Karolinska Institute, STOCKHOLM, Sweden

1,3-Butadiene is a general air pollutant associated

with combustion of organic matter and is also an

extensively used monomer in polymer production.

The cancer risk estimation of 1,3-butadiene is encum-

bered with large uncertainties. Extrapolation from

tumour frequencies in long-term animal tests has led

to a relatively high figure for the risk associated with

1,3-butadiene exposure. This is mainly based on obser-

vations of very high tumour incidences in butadiene-

exposed mice, which in this respect are about 100

times more sensitive than rats. It has been hypothe-

sized that a high cancer risk from 1,3-butadiene could

be associated with its metabolism to the bifunctional

1,2:3,4-diepoxybutane (DEB) which, in comparison

with monofunctional epoxides, 1,2-epoxy-3-butene

(EB) and 1,2-epoxy-3,4-butanediol (EBdiol), is a highly

effective mutagen, i.e. cancer initiator. Measurement

of in vivo doses of DEB is therefore essential for the

risk assessment of 1,3-butadiene. Reaction products

with hemoglobin offer a possibility of measuring

reactive metabolites in vivo. Hemoglobin adducts

from EBdiol have in this study been measured with

available methods, which are, however, not applica-

ble to the bifunctional DEB.

This work presents a procedure for measurement of a

specific, ring-closed adduct, Pyr-Val, formed from the

reaction of DEB with N-terminal valines in hemoglo-

bin. It is based on LC-ESI-MS/MS analysis of the Pyr-

modified N-terminal peptides enriched after trypsin

digestion of globin. Mouse and rat could be compared

regarding the metabolism of EB, DEB and EBdiol.

From the data it was concluded that, in 1,3-butadiene

exposure, higher levels of DEB are formed in mice

compared to rats. Estimates of in vivo doses in pub-

lished cancer tests showed that carcinogenesis in

mice is mainly due to DEB, whereas in rat, and possi-

bly man, the monofunctional EBdiol is a larger risk

contributor. Preliminarily, the estimated cancer risk is

compatible with the epidemiology-based risk esti-

mate of US EPA.


Poster Presentation

PW6009

THE APPROACH OF REGIONAL MONITORING OF

CYTOGENETIC EFFECTS IN HUMAN DUE TO ENVI-

RONMENTAL POLLUTION CONSEQUENCES

VG Druzhinin, NV Mokrushina, VI Minina,

AN Volkov, TA Golovina

Kemerovo State University, KEMEROVO, Russia

The main task while planning and carrying out cyto-

genetic monitoring is to choose the strategy of study-

ing of clastogenic effects in human populations,

when genotoxic effects are either known or supposed

to be. First of all, the object of monitoring must be any

territory characterized by both specific climatic geo-

graphic conditions of the environment and a variety

of ecological situations. It can be accounted for by the

fact that the investigation of mutation processes in

local populations (a particular plant, city or village

dwellers) does not allow one to completely and cor-

rectly estimate the degree and orientation of geno-

toxic processes. On the other hand, monitoring of too

large territories is either difficult to be carried out or

it is practically impossible by one research team. It is

the approach of regional monitoring that allows one

to estimate the degree of genotoxic parameters of the

environment, which finally must promote the devel-

opment of ecogenetical approaches for a complex of

administrative and medical preventive measures to

improve the ecological situation and hence to protect

one's health.Within the framework of the regional

approach to monitoring we proposed the method of

cytogenetic cartography whose realization is based

on several basic principles such as the unification of

investigations; sanitary - hygienic examination pre-

ceding to monitoring; selectivity and priority; a com-

plete coverage of territories; the organization of

control points; the juxtaposition of maps (Druzhinin

et al., 2003). The experimental data of 15- year moni-

toring of chromosomal aberrations in cohorts of the

inhabitants of the large industrial region of Western

Siberia, i.e. the Kemerovskaya oblast' are submitted in

our paper. The potential of the method of cytogenetic

cartography in genetics monitoring is also discussed.

112



Poster Presentation

PW6010

BIOMARKERS OF AIR POLLUTION EXPOSURE -

FOLLOW -UP STUDY OF POLICEMEN IN PRAGUE

RJ Sram1, B Binkova1, O Beskid2, I Chvatalova1,

A Milcova1, P Rossner2, O Sevastyanova1,

Z Smerhovsky1, Z Stavkova1, J Topinka1

1 Institute of Experimental Medicine AS CR,

PRAGUE, Czech Republic2 Intitute of Experimental Medicine, PRAGUE, Czech

Republic

The effect of exposure to PAHs adsorbed onto res-

pirable air particles (<2.5 mm) on DNA adducts and

chromosomal aberrations was studied in a group of

policemen (males, aged 22-50 years) working in the

downtown area of Prague and spending >8 h out-

doors (EXP, N=53). The matched healthy volunteers

spending >90% daily time indoors were chosen as

Controls (CON, N=52). Ambient air particles (PM10,

PM2.5) and carcinogenic polycyclic aromatic hydro-

carbons (carc-PAHs) were monitored using VAPS sam-

pler, personal exposure was evaluated using personal

samplers during working shift. DNA adducts were

analyzed in lymphocytes by 32P-postlabeling assay,

chromosomal aberrations by conventional cytogenetic

analysis and fluorescent in situ hybridization (FISH).

Further biomarkers include cotinine in urine to con-

trol for exposure to tobacco smoke, plasma levels of

vitamins A, E and C, folic acid, cholesterol and trigly-

cerids. Polymorphisms of metabolic genotypes

(GSTM1, GSTP1, GSTT1, EPHX, CYP1A1-MspI) and DNA

repair genotypes (XRCC1 and XPD) were determined.

The level of 'like' B[a]P-derived DNA adduct was high-

er in exposed group (0.122±0.036 vs. 0.099±0.035

adducts/108 nucleotides, P=0.003). DNA adduct levels

were modified by XPD repair gene in exon 23 and

XRCC1 and GSTM1 genotypes. Using FISH technique

and probes for chromosomes 1 and 4 the genomic fre-

quency of translocations calculated as FG/100 was

1.72 and 1.24 for EXP and CON (P<0.05), respectively. To

see the dynamics of the observed changes reported

here, the prospective cohort study is going on. All the

biomarkers of exposure and effect are analyzed

repeatedly during the period of one year in three-

months intervals (4-times) to cover periods with high

(winter) and low (summer) levels of air pollution.

Supported by the Czech Ministry of Environment

VaV/340/2/00 and VaV/740/5/03 and by the EC IC

QLK4-2000-00091.


Poster Presentation

PW6011

CYTOGENETIC MONITORING OF OCCUPATIONAL

EXPOSURE TO ANTINEOPLASTIC AGENTS: INFLU-

ENCE OF GENETIC POLYMORPHISMS

R Cozzi1, A Testa2, M Giachelia3, F Festa1, G Tranfo4,

G Spagnoli4, D Tirindelli3, L Baccelliere5, C Sciutto5,

N la Rosa5

1 University, ROME, Italy2 ENEA CR Casaccia, ROME, Italy3 Casaccia, ENEA, ROME, Italy4 ISPESL, ROME, Italy5 S.Martino Hospital, GENOA, Italy

A multidisciplinary monitoring study was carried out

on hospital personnel exposed to antineoplastic drugs

to investigate the risk of occupational exposure to these

substances. In this project, a cytogenetic investigation

(comet assay, chromosomal aberrations and micronu-

clei analyses) was carried out on a group of 25 female

nurses employed in oncology units and on a control

group adequately selected. All subjects (mean age = 39

years) were asked to fill in the personal healthy ques-

tionnaire proposed by the International Commission

Protection against Environmental Mutagens and

Carcinogens. Workers were also selected using a ques-

tionnaire concerning the individual occupational expo-

sure.

Furthermore, as humans vary in their response to

chemicals, considering their specific genetic constitu-

tion, we characterised the subjects for the polymor-

phisms of four genes (metabolic genes: GSTM1, GSTT1;

DNA repair genes: XRCC1, XRCC3) in order to verify the

relationship between DNA damage and genetic vari-

ants.

Regarding to the cytogenetic assessment, the exposed

group showed a significantly higher frequency of

genetic damage where compared to the control group:

chromosome and chromatid aberrations frequencies

in workers (chromosome aberration 5.44 %, chromatid

aberration 5.48 %) appeared significantly higher

(p<0.001, p<0.01 respectively) than in controls (chromo-

some aberration 1.46 %, chromatid aberration 3.07 %).

Similarly, micronucleus frequencies appeared signifi-

cantly higher (p<0.001) in workers (16.1 ‰) than in con-

trols (7.95 ‰). Concerning the evaluation of DNA

primary damage in these workers, the results indicat-

ed significantly higher tail moment values in exposed

group than in controls (p=0.001).

Finally, regression multivariate analysis showed a rela-

tion between GSTT1 null genotype and chromatid aber-

rations. This research was supported by Ministry of

Public Health (Ricerca Finalizzata, bando 2001 : 'La valu-

tazione dei rischi nella manipolazione dei chemioter-

apici antiblastici in ambiente sanitario') and partially

by MIUR Project 'Biological basis on human suscepti-

bility'. 113



Poster Presentation

PW6012

THE AMES II™ MUTAGENICITY ASSAY: AN INTER-

NATIONAL VALIDATION STUDY PERFORMED

WITH NINETEEN CODED COMPOUNDS

SF Flueckiger-Isler1, K Braun2, G Engelhardt3,

M Baumeister4, H-G Wunderlich5, JAJ van Gompel6,

N Hasler-Nguyen7, R Reimann8, V Gervais9

1 Xenometrix by Endotell GmbH, ALLSCHWIL,

Switzerland2 Aventis Pharma Deutschland, HATTERSHEIM,

Germany3 BASF AG, LUDWIGSHAFEN, Germany4 Boehringer Ingelheim, BIBERACH, Germany5 Federal Environmental Agency, BAD ELSTER,

Germany6 Johnson & Johnson Parmaceutical R&D, BEERSE,

Belgium7 Novartis Consumer Health, NYON, Switzerland8 Schering AG, BERLIN, Germany9 Servier Group, ORLTANS-GIDY, France

An international collaborative validation study was

performed in nine laboratories to compare the Ames

II™ Mutagenicity Assay with the standard Salmonella

plate incorporation test. Nineteen coded chemicals

were tested for their mutagenic activity in the strains

TA98 and TAMix (TA7001-7006). The test compounds

were selected from a published study with a large

data set from the traditional Ames test. The results of

both assay systems were compared, and the inter-lab-

oratory consistency of the Ames II test was assessed.

Of the eight mutagens selected, six were correctly

identified with the Ames II assay by all laboratories,

one compound was judged positive by five of six

investigators and one by four of six laboratories. All

seven non-mutagenic samples were consistently neg-

ative in the Ames II assay. Of the four chemicals that

gave inconsistent results in the traditional Ames test,

three were uniformly classified as either positive or

negative in the present study, whereas one com-

pound gave equivocal results. A comparison of the

test outcome of the different investigators resulted in

an inter-laboratory consistency of 89.5%.

Owing to the high concordance between the two test

systems, and the low inter-laboratory variability in

the Ames II assay results, the Ames II is an effective

screening alternative to the standard Ames test for

screening new substances for their mutagenic poten-

tial.

Compared to the standard Ames test, the Ames II assay

offers higher speed format, the possibility of automa-

tion, the recording of all possible base-pair substitu-

tions in one culture, substantially lower amount of test

chemical, easy scoring being a colorimetric assay, easy

handling and ready to use reagents. These criteria are

essential to meet the constantly increasing number of

chemicals flowing through early phase development

and the increasing demand for early indications of

possible Mutagenicity.

114



Poster Presentation

PW6013

ABSENCE OF GENOTOXIC EFFECTS OF RADIO-

FREQUENCY ON HUMAN BLOOD BY USING

DIFFERENT CYTOGENETIC TESTS

A Testa, M Appolloni, E Cordelli, AM Fresegna,

C Marino, L Stronati, P Villani

ENEA CR Casaccia, ROME, Italy

The possible health hazard of environmental expo-

sure to radiofrequency (RF) signals became an issue

of considerable public concern. Despite the majority

of evidences suggest that radiofrequency has not

genotoxic potential, some studies report positive

results.

The aim of this study was to evaluate the potential

genotoxic effect of radiofrequency alone or in combi-

nation with X-rays (1 Gy) on human blood cells. Three

different conventional and molecular cytogenetic

tests: chromosome aberrations (CA), micronuclei

(MN) and alkaline comet assay were applied.

Whole blood samples from 6 healthy donors (35-45

years) were exposed to 935 MHz, 1 W/kg for 24 hours.

The exposure setup was based on two rectangular R9

waveguides (for sham and exposed cells radiated

simultaneously in blind way) in which only the fun-

damental TE01 mode can propagate for the frequency

bands of interest. In order to guarantee the same

environmental conditions (temperature, humidity,

CO2) for the exposed and sham cells, both waveguides

were placed inside the same incubator (exposure

system is provided by Foundation for Research

on Information Technologies in Society (IT’IS)

and described in J. Schuderer et al, In Vitro Exposure

Systems for RF Exposures at 900 MHz, submitted)

Results did not show any significant difference

between radiofrequency exposed and sham samples

for each cytogenetic endpoint analyzed. Similarly, the

combined exposure failed to indicate the presence of

any synergistic effect between radiofrequency and

X-rays.

This Project (PERFORM B) is partially supported by

Elettra 2000, MMF and GSM association.


Poster Presentation

PW6014

THE EFFECTS OF ACRYLONITRILE ON LEVEL OF

CHROMOSOMAL ABERRATIONS

O Beskid1, Z Dusek2, I Chvatalova2, M Dostal2,

Z Stavkova2, P Rössner2, RJ Sram2

1 Institute of Experimental Medicine, PRAGUE,

Czech Republic2 Institute of Experimental Medicine AS CR,

PRAGUE, Czech Republic

Acrylonitrile (ACN) is classified as possibly carcino-

genic to humans (group 2B) according to IARC. It is

used in production of plastics, synthetic fibers and

rubbers. We investigated the influence of ACN on the

level of unstable and stable aberrations measured by

conventional cytogenetic analysis and FISH (whole

chromosomal painting for chromosomes 1 and 4),

respectively. The investigation was carried out on a

group of 60 chemical plant workers and a control

group of 55 healthy volunteers from the same region.

The subjects were genotyped for glutatione-S-trans-

ferases (GST) M1, P1 and T1, EPHX, p53, XPD, XRCC1,

hOGG1, MTHFR, and MS to evaluate possible differ-

ences in individual susceptibility to xenobiotics due

to genetic polymorphism of metabolizing enzymes.

Further we analysed the levels of vitamin A, C, E in

plasma for determination of antioxidative status, and

the level of cotinine in urine to objectify smoking sta-

tus. The relationship between occupational exposure

and cytogenetic endpoints was tested by Kruskal-

Wallis test.

Occupational exposure to ACN significantly affected

the frequency of chromosomal aberrations detected

by conventional method. We observed an increase of

AB.C. (aberrant cells) in the exposed group (3.27±1.91%

AB.C.) in comparison with controls (2.05±1.53% AB.C.,

P<0.01). However, there was no significant difference

between these two groups in genomic frequency of

translocations (Fg/100) measured by FISH (1.88±1.52 in

the exposed subjects vs. 1.63±1.30 in controls). No rela-

tionship between smoking status and the frequency

of aberrant cells was found neither for stable nor

unstable aberrations. The frequency of AB.C evaluat-

ed by conventional method correlated with the level

of vitamins A and C, and p53 MspI polymorphism,

while Fg/100 revealed association to age, EPHX, XPD-

6 polymorphisms, and vitamin E level.

This study was supported by EC QLK4-CT-2000-02381.

115



Poster Presentation

PW6015

THE IMPACT OF OCCUPATIONAL EXPOSURE TO

IRRADIATION IN CZECH NUCLEAR POWER PLANT

WORKERS

Z Dusek1, O Beskid1, I Chvatalova1, J Schmuczerova1,

Z Stavkova1, A Milcova1, P Rossner1, J Rubes2,

Z Smerhovsky1, RJ Sram1

1 Institute of Experimental Medicine AS CR,

PRAGUE, Czech Republic2 Veterinary Research Institute, BRNO,

Czech Republic

The aim of our study was to identify occupational risk

of irradiation exposure or exposure to clastogenic

chemical compounds in the Czech nuclear plant

workers. The level of chromosomal aberrations is

known as a biomarker of early biological effects and a

predictor of cancer risk. Therefore, we applied classi-

cal cytogenetic analysis and FISH (whole chromo-

some painting for chromosomes 1 and 4, combined

with pancentromeric probe) in three groups: 123 sub-

jects in the Temelin plant, 114 subjects in the

Dukovany plant (15 years in use), and 53 matched con-

trols from Ceske Budejovice. Nuclear plant workers

were divided into two groups: subjects with admit-

tance into the controlled zone, and others. Further

factors were analysed: p53 expression, XPD, XRCC1,

hOGG1, p53, MTHFR, and MS gene polymorphisms,

levels of vitamin A, C, E in plasma, and level of coti-

nine in urine. Long-term exposure to ionizing radia-

tion in the controlled zone was 0.46±1.49 mSv in

Temelin and 5.68±9.51 mSv in Dukovany. Using con-

ventional cytogenetic analysis, we observed

1.90±0.95 and 1.82±1.19 %AB.C. in Temelin, and

2.39±1.01 and 2.33±1.04 %AB.C. in Dukovany, for con-

trolled zone workers and others, respectively. In the

control group, we found 2.25±0.82 %AB.C. Genomic

frequency of translocations Fg/100 measured by FISH

was 1.89±1.40 and 2.01±1.68 in Temelin, and 2.41±1.88

and 2.12±1.61 in Dukovany for controlled zone workers

and others, respectively. In the control group, Fg/100

was 1.83±1.19. As potential confounders were identi-

fied following factors: age, XPD-6, GSTP1, and GSTT1

genotypes, long-term medication, alcohol consump-

tion and smoking status. No association between

dose of irradiation and level of chromosomal aberra-

tions in any nuclear power plant was obtained nei-

ther by conventional cytogenetic analysis nor by

FISH.

This study was supported by the Czech Government.


Poster Presentation

PW6016

FLOW CYTOMETRIC ANALYSIS AND MANUAL

SCORING OF PERIPHERAL BLOOD MICRONUCLEUS

FREQUENCIES IN CYCLOPHOSPHAMIDE TREATED

MICE

BM van der Leede, M de Boeck, A de Smedt,

M Steemans, F van Goethem, A Lampo, PH Vanparys


Belgium

The in vivo rodent erythrocyte micronucleus test is

widely used to evaluate the clastogenic and/or aneu-

genic potential of chemicals and pharmaceuticals.

Micronucleus frequencies are traditionally determined

manually by microscopy, making the micronucleus test

relatively labour intensive and time consuming.

Investigators at Litron Laboratories (Rochester, NY)

developed a flow cytometric method to measure

micronuclei in both reticulocytes and normochro-

matic erythocytes of mouse peripheral blood. This

method has many advantages over manual scoring,

including high speed data acquisition and objective

analysis. In an international multi-laboratory study

co-ordinated by Litron Laboratories the flow cytomet-

ric method is currently being validated in nine partici-

pating laboratories. Phase I validation results have

been published (Torous et al., Environ. Mol. Mut. 38:59-

68, 2001) and demonstrated high transferability of the

flow cytometric method among the participating lab-

oratories. Phase II of the validation has focused on

comparing micronucleus frequencies obtained by

microscopy with those generated by flow cytometry.

Each of the participating laboratories was assigned

one compound for evaluation. Here we present the

data obtained on mice treated with cyclophos-

phamide (0, 2.5, 5, 10 or 20 mg/kg/day by oral gavage)

for 3 consecutive days as part of our contribution to

Phase II validation study. Wright stained blood smears

were used for the microscopic analysis. In addition,

results are presented of an in-house study with

cyclophosphamide (0 or 40 mg/kg by oral gavage)

using a single dose regime and acridine orange stain-

ing of blood smears. Cyclophosphamide induced a

dose-dependent increase in micronucleated reticulo-

cytes by both flow cytometric and microscopic analy-

sis. The results indicate a high correlation between

flow cytometry and microscopy, especially using acri-

dine orange staining and also between flow cytomet-

ric analysis of the same samples at two different

laboratories.

116



Poster Presentation

PW6017

THE GENOTOXICITY OF IPRONIDAZOLE (GASTRO-

GAL 10) ON CULTURED HUMAN LYMPHOCYTES

B Markovic, Z Stanimirovic, N Djelic, V Bajic

Faculty of Veterinary Medicine, BELGRADE,

Yugoslavia

The genotoxic effects of antimicrobial preparation

Gastrogal 10 (ICN Galenica, Belgrade, Pharmaceutical

Company, Serbia) has been investigated in vitro on

human peripheral blood lymphocytes by following

the capability of Gastrogal 10 to induce numerical

and structural chromosome changes. To test a possi-

ble effect of the investigated substance on DNA we

used the sister chromatid exchange (SCE) test in vitro.

The investigated substance Gastrogal 10 was tested

through three experimental concentrations: 25 µM;

50µM and 100µM. Negative control groups were

treated with physiological saline. Our results show

that all experimental doses of the investigated sub-

stance Gastrogal 10 shows the potential for transfor-

mation of the karyotype of human lymphocytes by

inducing numerical aberrations type aneuploidy and

polyploidy and structural aberrations type gaps and

lesions. The overall cytogenetic changes induced by

Gastrogal 10 in all three doses clearly shows a statisti-

cal high significant increase in respect to the control

group. We have also estimated a correlation between

an increase of dosage and cytogenetic changes.

Cytogentic changes and a dose-effect dependency

clearly show a genotoxic potential of Gastrogal 10 on

lymphocytes of human peripheral blood. All three

doses of Gastrogal 10 induced a highly statistically

increase in the frequency of SCE in respect to the

untreated control groups. High SCE frequencies

shows a possibility of the investigated substance to

generate changes in the DNA structure. Our results

classify Gastrogal 10 a clasogenic agent.


Poster Presentation

PW6018

AN ASSESSMENT OF CUMENE HYDROPEROXIDE

IN THE IN VIVO COMET ASSAY IN MOUSE SKIN

P Clay1, A Wolfreys2, B Elliott1, E Jones1

1 Syngenta CTL, MACCLESFIELD, United Kingdom2 SEAC, Unilever Colworth, SHARNBROOK,

United Kingdom

The Comet assay has been proposed as being an

appropriate assay for the assessment of the genotoxic

potential of chemicals that may be active at the site of

contact. One such class of chemicals is strong oxidis-

ing agents acting through reactive oxygen, many of

which are genotoxic in vitro. However, these chemi-

cals act via one of the mechanisms of mutagenicity

considered subject to a practical threshold and thus

there should be an effective 'non-mutagenic' dose of

this material.

The genotoxicity of cumene hydroperoxide, a strong

oxidising agent, has been assessed in an in vivo

mouse skin Comet assay following a single dermal

application. Small, but dose related, increases in the

group mean values for tail moment were observed in

all the treated groups. These increases reached statis-

tical significance (P<0.05) for the high dose group but

did not exceed twice the concurrent control value.

Although a clear positive response was not observed,

the reproducibility and dose relationship of the data

suggest that cumene hydroperoxide induces DNA

damage, as indicated by comet formation in the

mouse skin following treatment at both 2.5 and 5.0

mg/animal. This indicates that the threshold dose

lies below 2.5 mg/animal.

The maximum dose level tested was limited by

micropathological changes to the skin. The severity

of these effects was not reflected in clinical or macro-

scopic observations and underlines the importance of

micropathological assessment of the tissue of inter-

est when performing in vivo comet assays.

117



Poster Presentation

PW6019

THE GENOTOXICITY OF IPRONIDAZOLE (GASTRO-

GAL 10) ON CULTURED HUMAN LYMPHOCYTES

B Markovic1, Z Stanimirovic1, N Djelic1, B Bajic2

1 Faculty of Veterinary Medicine, BELGRADE,

Yugoslavia2 Galenika Pharmaceuticals, BELGRADE, Yugoslavia

The genotoxic effects of antimicrobial preparation

Gastrogal 10 (ICN Galenica, Belgrade, Pharmaceutical

Company, Serbia) has been investigated in vitro on

human peripheral blood lymphocytes by following

the capability of Gastrogal 10 to induce numerical

and structural chromosome changes. To test a possi-

ble effect of the investigated substance on DNA we

used the sister chromatid exchange (SCE) test in vitro.

The investigated substance Gastrogal 10 was tested

through three experimental concentrations: 25 µM,

50µM and 100µM. Negative control groups were

treated with physiological saline. Our results show

that all experimental doses of the investigated sub-

stance Gastrogal 10 shows the potential for transfor-

mation of the karyotype of human lymphocytes by

inducing numerical aberrations type aneuploidy and

polyploidy and structural aberrations type gaps and

lesions. The overall cytogenetic changes induced by

Gastrogal 10 in all three doses clearly shows a statisti-

cal high significant increase in respect to the control

group. We have also estimated a correlation between

an increase of dosage and cytogenetic changes.

Cytogentic changes and a dose-effect dependency

clearly show a genotoxic potential of Gastrogal 10 on

lymphocytes of human peripheral blood. All three

doses of Gastrogal 10 induced a highly statistically

increase in the frequency of SCE in respect to the

untreated control groups. High SCE frequencies

shows a possibility of the investigated substance to

generate changes in the DNA structure. Our results

classify Gastrogal 10 a clastogenic agent.


Poster Presentation

PW6020

PHOTOGENOTOXICITY TESTING; IN VIVO MICRONU-

CLEUS ASSAY IN RAT EPIDERMAL SKIN CELLS

CAM Krul, M-JST Steenwinkel, N de Vogel,

RNC van Meeuwen


The Netherlands

Photogenotoxicity testing is recommended for cos-

metic ingredients (e.g. sunscreens) and pharmaceuti-

cals (e.g. antimicrobal agents), that absorbs UV light

and reach the skin or eyes either by dermal or oral

administration. In recent years the demand for in

vitro photogenotoxicity assessment is remarkably

increased. Most photogenotoxicity assays are adopt-

ed from standard guideline assays, such as the Ames-

and chromosomal aberration test.

In the regular tiered approach for genotoxicity test-

ing, whenever an inconclusive response is observed

in vitro, an in vivo assay is performed to establish the

genotoxic potential in vivo. For the evaluation of the

photogenotoxic potential no follow-up assay in vivo

is available (except for a photocarcinogenicity study).

Particularly photo-clastogenic responses are observed

in photogenotoxicity assays in vitro. Therefore, a feasi-

bility study was performed to demonstrate the induc-

tion of micronuclei (MN) in rat epidermal skin cells.

Fisher 344 rats were shaved and dorsal skin (12 cm2)

was treated with 8-MOP (0.9 µg/cm2) 30 minutes

prior to UV-irradiation (1 MED) simulating sunlight.

Animals were sacrificed 48 and 72h after treatment,

epidermal skin cells were isolated and slides were

prepared. The incidence of micronucleated skin cells

(MNSC) was measured in 2000 skin cells (SC). The

induction of MNSC was highest at 72h and about

5-fold increased compared to the control (non-

exposed ventral side skin). Additionally, rats were

exposed to UV (2-3 MED) in the absence of a pho-

togenotox compound. The numbers of MNSC were

significantly increased compared to the controls. This

new developed photo-MN assay can be used to exam-

ine whether pharmaceutical compounds have the

ability to cause UV-induced MN in vivo.

The in vivo photo-MN assay in rats might be very

helpful bridging the gap between in vitro photogeno-

toxicity and in vivo photocarcinogenicity testing.

118



Poster Presentation

PW6021

CHROMOSOMAL ABERRATIONS AND RISK OF

CANCER IN A HUNGARIAN COHORT

A Kelecsenyi, S Gundy


The frequency of chromosomal aberrations (CAs) in

human peripheral blood lymphocytes (PBLs) has rou-

tinely been used to monitor occupational and envi-

ronmental exposures to genotoxic agents for over 3

decades. In 1990s a clear association was detected

between high CA frequencies and increased cancer

risk in a large collaborative project of Nordic and

Italian laboratories. In late 1990s another important

conclusion was drawn from the results that a high

level of CAs generally predicts an increased cancer

risk regardless of the reason for the elevated CAs. The

assay was introduced in Hungary in 70s. Under the

extension of Nordic-Italian 'Cancer Risk Biomarkers'

(CRB) program we also contribute in the project. The

results from other European cohorts will also be com-

bined with our ones and those already included into

the CRB project. The associations between total can-

cer incidence/mortality and the frequency of high,

medium, and low CAs, gender, age and time since

test, and exposures will be modelled by Cox's regres-

sion.

Between 1978 and 2003 we carried out more than

2000 CA analyses on cancer-free persons, but the data

of only 831 persons were fully available for the crite-

ria of the accepted protocol. CA data were tri-

chotomized into low (0-1 CA;) medium (2 CA), and

high (>2 CA). Out of 831 persons 52 cancer cases (6.3%)

occurred. The total of cancers observed in the low

(4.7%), medium (5.7%) and high (9.3%) CA groups dif-

fered significantly (log-rank test: p=0.0225). In conclu-

sion we may say that our findings also support the

hypothesis that an elevated CA frequency in PBLs pre-

dicts an increased future cancer risk, and the extent

of CAs in lymphocytes may reflect the same events,

which take place in other cells of target tissues of the

organism.

This study was supported by grant: QLK- CT-200-

00628.


Poster Presentation

PW6022

ANEUPLOIDY STUDIES IN MOUSE GERM CELLS

WITH BISPHENOL A

MS abdo Attia1, ID Adler1, U Eichenlaub-Ritter2,

R Ranaldi3, F Pacchierotti3

1 GSF, NEUHERBERG, Germany2 University of Bielefeld, BIELEFELD, Germany3 ENEA CR Casaccia, ROME, Italy

Bisphenol A (BP-A), widely used in polycarbonate-

plastics production, has estrogenic properties and is

aneugenic in vitro (Parry et al., 2002, Mutagenesis

17:509). Recently, Hunt et al. (2003, Cur. Biol. 13:546)

have associated spindle aberrations and chromosome

congression failure during meiosis of female mice

to BP-A leakage from damaged cages and/or water

bottles. Therefore, a series of coordinated experiments

were performed within the EU-Project 'Protection of the

European Population from Aneugenic Chemicals' (PEP-

FAC) to ascertain aneugenic effects of BP-A in mouse

germ cells. Meiotic delay and aneuploidy induction in

spermatocytes (Attia et al., 2002, Mutat. Res. 520:1) of

(102/ElxC3H/El)F1 males were studied after 6 daily

oral doses of 0.002-0.2 mg/kg BP-A in corn oil.

Aneuploidy induction was studied in a) in vitro

maturing oocytes of MF1-mice exposed to 50 to 400

ng/ml BP-A in medium, b) in vitro matured oocytes

(Eichenlaub-Ritter & Betzendahl, 1995, Mutagenesis

10:477) obtained from prepubertal (C57BlxCBA/Ca)F1

females exposed to 7 daily oral doses of 20 or 40 mg

/kg BP-A (Hunt et al., 2003), and c) ovulated oocytes

(Tiveron et al., 1992, Mutat. Res. 266:181) from C57Bl

females orally treated with single doses of 0.2 or 20

mg/kg or 7 daily doses of 0.04 mg/kg BP-A. No

increases in hyperhaploid or diploid sperm frequen-

cies were found. In vitro, BP-A did not increase the

hyperhaploidy rates but significantly increased

'diploid' metaphase II oocytes at 200 ng/ml BP-A. Ex

vivo, BP-A affected nuclear maturation to metaphase

II and spindle formation in oocytes but preliminary

data of the ex vivo and in vivo oocytes studies showed

no increase in hyperhaploidy rates. Thus, the aneu-

genic effects of BP-A forecasted by Hunt et al. could

not be demonstrated so far. Further studies are under

way.

Supported by EU-Contract No. QLK-CT 2000-00058

(PEPFAC).

119



Poster Presentation

PW6023

INTEGRATED CYTOTOXICITY ASSESSMENT IN A

DOWNSCALED IN VITRO MICRONUCLEUS TEST

FVG van Goethem, M de Boeck, B-J van der Leede,

A de Smedt, M Steemans, A Lampo, PH Vanparys


Belgium

Micronucleus formation is primarily considered as a

hallmark of genotoxicity. The in vitro micronucleus

test (MNT) is a well-established assay for the assess-

ment of the mutagenic properties of new chemical

entities during the early phases of drug development.

Its capacity to simultaneously detect clastogens and

aneugens and their potential effects on cell prolifera-

tion, the simplicity of scoring, the rapid performance

and the possibility for downscaling, have made it an

attractive method. It is well known that excessive

cytotoxicity of a test compound can indirectly cause

DNA strand breaks which renders the identification

of true genotoxins difficult. Recently, the expert group

from the International Workshop on Genotoxicity

Testing stated that assessment of toxicity should be

performed by determining cell proliferation (increased

cell counts, population doubling) and by determining

other markers of cytotoxicity (confluency, apoptosis,

necrosis) which can provide additional information

(Kirsch-Volders et al., Mutat. Res., 2003). Here we pres-

ent data of the concurrent assessment, in the same

cells, of genotoxicity and cytotoxicity using a down-

scaled version of the MNT in 96-well microplates and

Alamar Blue conversion. L5178Y cells were treated

with a number of genotoxic and non-genotoxic refer-

ence compounds. The proposed experimental set-up

allowed an increased screening throughput since the

cytotoxicity profile could be determined within min-

utes where after relevant concentrations were select-

ed for further processing in the MNT. A comparison

between the Alamar Blue fluorometric procedure and

relative cell counts to determine cytotoxicity for

selected compounds, learned that there was a good

correlation between the two approaches. In addition,

results showed that the present set-up was able to

identify false positive results due to cytotoxicity

interfering with the final outcome of the genotoxici-

ty testing. Moreover, the current protocol offers the

advantage that only 5 mg of test compound is

required.


Poster Presentation

PW6024

MICRNUCLEUS STUDIES IN MOUSE SOMATIC

CELLS WITH BISPHENOL-A (BP-A) AND NOCODA-

ZOLE (NOC)

MS abdo Attia1, A. Vanhauwaert2, M Kirsch-Volders2,

ID Adler1

1 GSF, NEUHERBERG, Germany2 Vrije Universiteit Brussels, BRUSSELS, Belgium

BP-A is widely used in polycarbonate-plastics produc-

tion, has estrogenic properties, and is aneugenic in

vitro (Parry et al., 2002, Mutagenesis 17:509). NOC is

used as an anti-tumor agent. It inhibited micro-

tubules function in vitro (De Brabander et al., 1976,

Cancer Res. 36:905) and induced chromosome loss,

non-disjunction and apoptosis human lymphocytes

(Elhajouji et al., 1995, Environ. Mol. Mutagenesis

26:292). Therefore, coordinated experiments were

performed within the EU-Project 'Protection of the

European Population from Aneugenic Chemicals’

(PEPFAC) to determine micronucleus (MN) induction

by these two aneugens in somatic cells in vivo.

Laboratory 1 used (102/ElxC3H/El)F1 males treated

with two daily oral doses of BP-A (0.2, 0.02, 0.002

mg/kg) or two daily ip doses of NOC (12.5, 25, 50

mg/kg), and performed the in vivo bone marrow MN-

test 24h after the last dosing. Laboratory 2 used (CD1)

SPF Swiss albino males treated with single oral doses

of BP-A (500, 1000, 2000 mg/kg) or single ip doses of

NOC (10, 40, 160 mg/kg), and performed the in vivo

bone marrow MN-test (only for BP-A) and the ex

vivo/in vitro MN-test with lymphocytes 24 and 48h

after exposure.

With BP-A, neither of the laboratories observed an

induction of MN in bone marrow. With NOC,

Laboratory 1 observed no induction of MN in bone

marrow but significant cytotoxicity with the highest

dose. Laboratory 2 found no MN induction with BP-A

or NOC in the ex vivo/in vitro test. Laboratory 2 will

have results from the in vivo gut MN-test performed

on the same animals soon.

The present data with NOC are in contrast to the

results reported by Tinwell and Ashby (1991;

Mutagenesis 6:193), which may be due to differences

in animals strains and/or dosing.

Supported by EU-Contract No. QLK-CT 2000-00058

(PEPFAC).

120



Poster Presentation

PW6025

EXPOSURE OF BIG BLUE MICE TO HC RED NO.3 DID

NOT INDUCE GENE MUTATIONS

S Pfuhler1, K Milne2, P Blackshear2, M Streicker2

1 Wella AG, DARMSTADT, Germany2 ILS, RESEARCH TRIANGLE PARK, United States of

America

HC Red No.3 is an aromatic amine which is used as

direct hair dye. It is known to trigger gene mutations

in the Ames test. In a 2-year bioassay in mice the dye

showed equivocal evidence with regard to hepatocel-

lular neoplasms in male animals. Therefore, the Big

Blue lacI/cII assay was used to clarify its potential to

induce gene mutations in cells of the liver and uri-

nary bladder.

HC Red No.3 and the vehicle control (1% CMC in deion-

ized water) were administered by gavage once daily

over a period of 8 weeks. The doses used for the

administration of the dye to six male mice per group

were 500 and 1000 mg/kg bw per day. The positive

control (50 mg/kg bw N'-N'-ethyl nitrosourea (ENU))

was applied as intraperitoneal injection for the first 5

days only. At the time of necropsy, samples of the

liver and urinary bladder were obtained for analysis

of mutations at the cII target genes of the shuttle vec-

tor. About one million plaque forming units were

evaluated per dose group. No significant increases in

cII transgene mutation frequency (MF) in the liver or

bladder compared with controls could be observed.

The MF for the bladder was 36.5 x 10-6 for the negative

control, 39.3 x 10-6 for the low dose group and 30.2

x -6 for the high dose group. For the liver the respec-

tive values were 46.8 x 10-6 , 37.0 x 10-6 and 34.5 x 10-6.

The positive control lead to a significant increase in

the MF (bladder:400 x 10-6; liver: 234 x 1010-6), con-

firming the validity of this study.

The result shows that HC Red No.3 does not cause

gene mutations in vivo and thus represents an impor-

tant additional element to assure the absence of a

carcinogenic hazard for humans.


Poster Presentation

PW6026

PREDICTIVITY OF THE MICRONUCLEUS (MN)

ASSAY FOR THE REGULATORY CHROMOSOME

ABERRATION TEST IN HUMAN LYMPHOCYTES

A Elhajouji, W Frieauff, W Suter

Novartis Pharma AG, BASEL, Switzerland

In the early phases of drug development a variety of

screening procedures are developed to better predict

the outcome of the regulatory assays required prior to

clinical phases and registration. As part of the geno-

toxicity package future drugs are assessed for their

potential to induce chromosome aberrations in mam-

malian cells. In our laboratories micronucleus test in

mammalian cell lines is used as a medium through-

put screening tool for the prediction of the time con-

suming chromosome aberration assay. Due to the low

throughput (manual analysis of the slides) of the pri-

mary human lymphocyte cytokinesis-blocked (CB)

MN assay, this test is used more for chemical class

effect clarification and further mechanistic evalua-

tion when combined with fluorescence in situ

hybridization. We have accumulated data from differ-

ent cell systems i.e. V79 Chinese hamster cells, L5178Y

mouse lymphoma cells, human lymphoblastoid TK6

cells and the primary human lymphocytes. For a com-

parative evaluation of the different test systems three

criteria were used: sensitivity, specificity and predic-

tion of the respective cell systems. TK6 cells showed

an improved prediction of the chromosome aberra-

tion assay when compared to the other two cell lines

V79 and L5178Y with 83% in sensitivity, 81% in speci-

ficity and 81% in predicitivity. However with the

human lymphocyte CB assay all tested compounds

that were also evaluated in the chromosome aberra-

tion assay showed the same genotoxicity profile.

Based on the data from the different screening assays

the primary human lymphocytes showed the highest

prediction for the chromosome aberration assay. An

improvement of the throughput of the human lym-

phocytes CB assay using image analysis would lead

to the most adequate in vitro cytogenetics screening

tool.

121



Poster Presentation

PW6027

EVALUATION OF THE MUTAGENIC EFFECTS OF

URBAN AND INDUSTRIAL WASTE WATER IN THE

CANARY ISLANDS

E de la Pena1, O Herrero1, S Aguayo2, A de la Torre2,

M Carballo2, MJ Mu-oz2

1 Centro de Ciencias Medioambientales CSIC,

MADRID, Spain2 CISA - INIA, MADRID, Spain

It is shown the importance of mutagenic studies of

wastewater due to the presence of polluting agents,

which are mainly organic micropollutants, that affect

the recipient water quality and which are not includ-

ed in the actual safety regulations. Eighteen waste

water effluents were studied: 5 urban, 5 industrial

and 8 mixed. Their physical and chemical characteri-

zation was realized and the acute toxicity of the

whole effluent was valued by means of different

assays recommended by Whole Effluent Toxicity. The

method based on Toxicological Identification

Assessment was used with certain effluents. As a part

of the toxicological assessment the organic extract

was evaluated with acute toxicity, mutagenic, terato-

genic and estrogenic assays. The mutagenic effects

are studied with the Salmonella typhimurium assay,

using the TA100 and TA98 strains analysis. The esti-

mation of Toxicity Units was done with the existing

information of the identified compounds. We show

no mutagenic effect in the preliminary data of the

analysed effluent samples. However, estrogenic and

teratogenic effects were detected in 4 mixed and 3

industrial effluents respectively. The organic part of

the samples could be the main responsible of this tox-

icity. The analysis of the organic compounds proves

the presence of different chemical remainders in

ng/L concentrations: surfactants (nonylphenol and

octylphenol), plastic softeners (phthalates, bisphenol

A), steroid hormones (estradiol) and synthetic ones

(ethinilestradiol), PHAs, fatty acids, insecticides

(diazinon), etc. We wish to emphasize the absence of

toxicological and environmental data for almost the

50% of the identified compounds. Project REN2002-

04162-C02-02


Poster Presentation

PW6028

BIOMONITORING OF CHILDREN - RESULTS FROM

THE WORK OF A SCALE SUBGROUP

LE Knudsen1, L Casteleyn2, C Sala3

1 Institute of Public Health, COPENHAGEN, Denmark2 Ministry of the Flemmish Community, BRUSSELS,

Belgium3 ARPA, LOMBARDIA, Italy

Monitoring of the quality of the environmental

media, air, water and soil, has a long tradition in

European countries and there is ample legislation

dealing with it. A step that is leading closer to evalu-

ating human health effects is human biological mon-

itoring, where the concentration of a pollutant - or its

metabolite(s) - is determined in a biological sample,

generally blood or urine. In a similar way, other types

of biomarkers can help assess the reaction of the

human body to environmental pollutants.

The various biomonitoring studies that are run in the

different European countries are generally not carried

out using the same methodological approach. More

harmonised biomonitoring survey programmes are

recommended. To study the cause-effect framework

and to document the level of scientific evidence of a

cause-effect link properly defined research studies

are needed. Therefore research projects should be

'grafted' on the surveillance framework where possi-

ble. Less invasive methods are to be considered

together with new techniques within genetic expres-

sion profiles.

An overview of the European research in children’s

health has identified some 100 studies including at

least 400,000 children participating in existing bio-

monitoring and or research activities conducted in

the member States and acceding Countries. Forty-

four studies dealt with exposure to heavy metals, 15

with dioxins/PCB, and 5 with exposure to endocrine

disruptors. Twenty-seven studies included the deter-

mination of biomarkers of asthma and allergy and

only a limited number investigated cytogenetic bio-

markers in relation to environmental pollution.

The ethical issues of doing good and no harm to the

participants and the information and dissemination

of project results are very important.

References to the 2 reports made by the group are

http://www.brussels-conference.org/Download/

baseline_report/BR_Biomonitoring_final.pdf

http://europa.eu.int/comm/environment/health/

finalreports_en.htm.

122



Poster Presentation

PW7001

AGEING AND THE SEQUENCE OF CENTROMERE

SEPARATION

B Bajic1, SP Biljana2, D Ninoslav3, L Zivkovic2,

MZ Milicevic4

1 Galenika Pharmaceuticals, BELGRADE, Yugoslavia2 School of Pharmacy, BELGRADE, Yugoslavia3 School of Veterinary Medicine, BELGRADE,

Yugoslavia4 Vinca Institute of Nuclear Sciences, BELGRADE,

Yugoslavia

Loss of control of the sequence of centromere separa-

tion or out-of-phase of centromere division is found

in ageing cells, Alzheimer’s disease patient’s (AD),

various chromosome instability syndromes and can-

cers. In ageing cells the main affected chromosome is

the X chromosome. X chromosome instability mani-

fests as premature separation of the centromere or

out-of-phase centromere division. Out-of-phase cen-

tromere division has been observed to correlate with

non-disjunction leading to aneuploidy of the X chro-

mosome in elderly human females and less in elderly

human males. Human sequence of centromere sepa-

ration is well known. In humans the earliest separat-

ing centromeres are from chromosome 18,17,2,10 and

12, respectively. Those separating last belong to chro-

mosomes 21,22,13,14 and 15. Other chromosomes and

the X chromosome separates between the two

extremes. Knowing that altered sequence of cen-

tromere separation in elderly people show that

X-chromosome separates before chromosome 18, we

used fluorescent in situ hybridization (FISH) for the a-

centromeric region of the X chromosome in order to

estimate more precisely the time when PCD arises in

the cell cycle. FISH analysis has revealed that X chro-

mosome expresses out-of-phase division in elderly

females 7.46% in metaphase of mitosis and 9.35% in

interphase nucleuses and in elderly males group

2.84% in metaphases and 5.54% in interphase nucle-

uses. Using FISH, our results show that out-of-phase

can occur much earlier than metaphase of mitosis, i.e.

in interphase of the cell cycle, immediately after

replication. Maintenance of sequential separation of

centromeres is genetically highly controlled thus sug-

gesting that out-of-phase of the X chromosome can

be viewed as a non-specific manifestation of chromo-

some instability affected by the ageing process in eld-

erly humans.


Poster Presentation

PW7002

TRIMETHYLTIN CHLORIDE INDUCES INTRACEL-

LULAR CALCIUM ELEVATION AND APOPTOSIS IN

HELA-S3 CELLS

AM Florea, D Büsselberg, AW Rettenmeier, ED Dopp

University Hospital Essen, ESSEN, Germany

Trimethyltin chloride (TMT) is found in the environ-

ment, and cases of human intoxication have been

reported. We have previously shown that TMT induces

elevated Ca2+ transients in HeLa cells (Florea et al.,

Europ. J. Physiol., 447, 2004, S127). Calcium-dependent

mechanisms are involved in the initiation of gene

expression, in cell proliferation, cell cycling, and

apoptosis. In this study, we further investigated if

TMT-induced [Ca2+]i rise is associated with the induc-

tion of apoptosis. Laser Scanning Microscopy (LSM)

and Fluorescence Activated Cell Sorting (FACS) were

used for the measurements. Intracellular calcium

changes were determined by using Fluo 4. Assessment

of apoptosis was proceeded using propidium iodide

and annexin V/FITC staining. The results show that

TMT perturbed calcium homeostasis in HeLa-S3 at

concentrations as low as 100 nM and that this pertur-

bation correlates with the induction of apoptosis. The

[Ca2+]i elevation is mainly due to a release from inter-

nal stores. Moreover, a [Ca2+]i rise in the nuclear

region of HeLa-S3 cells was also observed. Significant

apoptotic death was induced by TMT after 12 h at 2

µM and after 48 h at 0.5 and 2 µM. Parallel staining of

Fluo 4 and annexin V showed significant increases of

[Ca2+]i, however, the exteriorisation of phosphatidyl

serine was not significant. We propose that calcium

elevation is very important in TMT-induced cytotoxi-

city in HeLa-S3 cells. Moreover, the calcium rise in the

nuclei might be related to DNA damage and might

trigger apoptosis. The data suggest that TMT exerts

its effects at least at two different sites: at the cell

membrane and inside the cell resulting finally in cell

death.

123



Poster Presentation

PW7003

ELECTROPORATION INCREASES CELLULAR UPTAKE

AND GENOTOXIC EFFECTS OF ORGANOARSENIC

COMPOUNDS IN CHO CELLS

ED Dopp1, AM Florea1, LM Hartmann2, B Shokouhi1,

AV Hirner2, AW Rettenmeier1

1 University Hospital Essen, ESSEN, Germany2 University of Duisburg-Essen, ESSEN, Germany

We have shown in previous studies that the induction

of genotoxic effects caused by methylated arsenic

species primarily depend upon their ability to pene-

trate cell membranes (Dopp et al., Occup Medic, Social

Medic, Environm Medic 39 (2004) 4, 217). In the pres-

ent investigations cellular uptake of arsenic com-

pounds was enforced by electroporation to find out

whether an increased uptake leads to enhanced cyto-

toxic and genotoxic effects in CHO cells. We also

wanted to clarify if the arsenic compounds are intra-

cellularly bound to membranes, or if they are present

in the cytosol either bound to proteins or not.

Cytotoxicity was determined the by Trypan blue

extrusion test and genotoxicity by the micronucleus

assay. Measurements of the intracellular arsenic con-

centrations were carried out by ICP-MS in (1) whole

cell extracts, (2) membrane-free cell extracts and (3)

deproteinised cell extracts. The results show that, in

the concentration range of 0.1 µM-500 µM (exposure

period: 24 h), trivalent arsenic compounds (AsNaO2,

MeAs(OH)2, Me2AsOH) exert higher cyto- and geno-

toxicity compared to that of the pentavalent arsenic

species (AsHNa2O4·7 H2O, MeAsO(OH)2, Me2AsOOH,

Me3AsO). Three compounds with a low membrane

permeability (MeAsO(OH)2 = 0.05 %, Me2AsOOH =

0.02 %, Me3AsO = 0.13 %) were used in the electropo-

ration experiments. After an exposure period of 30

min and electroporation at 290 V for 40 µs, CHO cells

were loaded with 0.25 % MeAsO(OH)2, 0.33 %

Me2AsOOH, and 0.52 % Me3AsO, respectively, present

in the cell culture medium. Micronucleus formation

was significantly increased in arsenic-exposed CHO

cells after electroporation. The ICP-MS measurement

of the arsenic derivatives revealed that these com-

pounds were present in the cytosol and not bound to

proteins or intracellular membranes.


Poster Presentation

PW7004

GENOTOXIC POTENTIAL OF RESPIRATORY BEN-

TONITE PARTICLES WITH DIFFERENT QUARTZ

CONTENTS AND SURFACE MODIFICATIONS

S Geh1, D Höhr2, T Shi2, ED Dopp1, PJA Borm2,

AW Rettenmeier1

1 University Hospital Essen, ESSEN, Germany2 IUF GmbH Düsseldorf, DUSSELDORF, Germany

Crystalline silica has been classified as a human car-

cinogen, but there is still considerable controversy on

its fibrogenic and carcinogenic potential. In the pres-

ent study, we investigated the genotoxic potential of

bentonite particles (Ø 3 – 7 µm) with an a–quartz con-

tent of up to 6 % and different surface activation

(alkaline, acidic, organic). Human lung fibroblasts

(IMR-90) were incubated for 24 h, 48 h or 72 h with

bentonite in concentrations ranging from 1 to 100

µg/cm2. Cytotoxicity was studied by determination

of viable cells using flow cytometry and UV-

spectroscopy (Alamar blue test). Cellular uptake of

particles was estimated by transmission electron

microscopy (TEM). Genotoxicity was assessed using

the micronucleus (MN) assay and kinetochore analy-

sis. The generation of reactive oxygen species (ROS)

caused by bentonite particles was measured by the

electron spin resonance technique (ESR). Our results

show that surface- modified bentonite particles with

a quartz content of 5 % - 6 % are more cytotoxic than

untreated bentonites or bentonites with a quartz

content up to 5 %. The cellular uptake of surface-mod-

ified bentonites was significantly higher compared to

that of untreated (native) bentonite particles. The for-

mation of MN was only slightly increased (p<0.05)

after exposure of IMR-90 cells for 72 h to bentonite

samples with a higher quartz content (concentration:

15 µg/cm2). The surface modification of particles did

not influence MN formation. Clastogenic and/or

aneugenic effects were dependent upon the type of

bentonite samples applied. Generation of ROS corre-

lated with the observed genotoxic effects. We con-

clude, that the genotoxic potential of bentonite

particles depends upon the content of quartz and

available transition metals, in contrast to the cytotox-

ic potential which depends upon the chemical modi-

fication of particles and the surface structure.

124



Poster Presentation

PW7005

MUTS-DEPENDENT GASP PHENOMENON IN

MIXED CULTURES OF ESCHERICHIA COLI AND SAL-

MONELLA ENTERICA

V Bacun-Druzina1, K Gjuracic2, J Pesut1, J Franekioli1

1 Fac.of Food Technology and Biotechnology, ZAGREB,

Croatia2 Pliva, Research Division, ZAGREB, Croatia

The major mechanisms of microbial genome ecologi-

cal specialization are the loss of preexisting genes or

gene activities during evolution. The ability of

Escherichia coli to grow during carbon starvation in

stationary phase has been termed the Growth

Advantage in Stationary Phase (GASP) phenotype.

The GASP phenomenon has been observed in range

of microorganisms, including clinical isolates of E.

coli and S. enterica. The occurrence of mutators

among isolates of E. coli and S. enterica is up to 10%.

The resulting mutants with increased fitness express

GASP phenotype enabling them to grow and displace

the parent as the majority population. The amount of

MutS, homodimer which binds to mismatched bases,

is strongly down regulated as E. coli cells enter sta-

tionary phase. During the prolonged stationary phase

decreased amounts of MutS contributed to the

increased mutagenesis resulting in mutator pheno-

type.

The aim of our investigation was to analyze the

mixed populations of E. coli K12 and S. enterica

serovar Typhimurium during the prolonged period of

carbon starvation due to the evaluation of the GASP

phenomenon and its dependents of mutS gene. The

mixture of one-day-old culture of S. enterica and ten-

day-old cells of E. coli as minority showed the GASP

phenomenon when they are resistant to a growth

inhibitor (nalidixic acid or streptomycin). The same

results were obtained when the mixture consisted of

S. enterica as a minority cells. On the other hand, in

mixed populaton of mutS mutants of both bacteria, in

various ratios and age, only abortive GASP phenotype

was observed. In addition, in the cells with GASP phe-

notype the chromosomal rearrangements were

determined by PFGE.


Poster Presentation

PW7006

DIFFERENT NUCLEOTIDE EXCISION REPAIR-

DEFICIENT MICE SHOW DIVERGENT AGING

PHENOTYPES

SWP Wijnhoven1, RB Beems1, MET Dollé1, J Vijg2,

P Lohman1, J Hoeijmakers3, G van der Horst4,

H van Steeg1


Environment, BILTHOVEN, The Netherlands2 University of Texas, TEXAS, United States of America3 Erasmus University, ROTTERDAM, The Netherlands4 Erasmus Medical Center, ROTTERDAM,

The Netherlands

The accumulation of somatic DNA damage is consid-

ered to be a major cause of the aging process in vari-

ous species including mice and humans. Among the

sources of DNA damage, reactive oxygen species (ROS)

are often thought to be the ultimate cause of aging.

However, the mechanisms involved remain obscure.

To counteract the effects of DNA damage, an intricate

network of DNA repair pathways has evolved. One

major pathway is nucleotide excision repair (NER),

which removes a broad range of bulky lesions includ-

ing some forms of oxidative damage. Patients with a

defect in NER proteins like CSB and XPD, both

involved in repair as well as transcription of DNA,

appeared to have a decreased life span.

In order to investigate whether defects in genome

maintenance are correlated to accelerated aging, we

have successfully conducted several longevity and

cross sectional studies with mice having defect in

DNA repair and/or RNA transcription (i.e. Xpa-, Csb-,

Xpd(Ttd)- deficient mice as well as C57Bl/6 wild type

controls). The mean survival of female Xpd(Ttd) as

well as Xpa mice appeared to be much shorter (appr.

90 weeks) than those found for Csb and wild type lit-

termate controls (104-110 weeks). Full histopathology

has been performed in aged female mice, and gross

examination at autopsy revealed small posture,

kyphosis, large spleen, small thymus and abnormal

skin and hair especially in Xpd(Ttd) mice. The termi-

nal body weights in Xpd(Ttd) and Csb females were

decreased, with an increase in the relative weights of

several organs of Xpd(Ttd) mice, especially in the kid-

ney, spleen and the heart. Furthermore, lipofuscin

pigmentation (an aging feature, correlated to oxida-

tive damage) was found to be accumulated in the

liver of Xpd(Ttd) mice as compared to the other phe-

notypes.

This work was supported by NIH/NIA (1 PO1-AG17242).

125

^



Poster Presentation

PW7007

PREMATURE AGING IN XPC/CSB AND XPA/CSB

DOUBLE KNOCKOUT MICE

I van der Pluijm1, J de Wit1, RB Beems2, CTHM van

Oostrom2, A Maas1, H van Steeg2, J Hoeijmakers3,

G van der Horst1

1 Erasmus Medical Center, ROTTERDAM,

The Netherlands2 National Institute of Public Health and the

Environment, BILTHOVEN, The Netherlands3 Erasmus University, ROTTERDAM, The Netherlands

The Global Genome Repair subpathway of Nucleotide

Excision Repair (GG-NER) removes helix-distorting

DNA damage in the entire genome, whereas Trans-

cription Coupled Nucleotide Excision Repair (TC-NER)

removes transcription-blocking DNA lesions. Defects

in these pathways can have deleterious effects; XP-A

patients are totally devoid of NER activity, XP-C

patients only lack GG-NER. Both show cutaneous

abnormalities and increased skin cancer predisposi-

tion, often accompanied by neurological degenera-

tion. CS patients have a defect in TC-NER and show

postnatal growth failure and severe neurodysfunc-

tion. CS is a progeroid syndrome, revealing a strong

link between DNA repair deficiency and premature

aging.

Mouse models for XP-A, XP-C and CS-B mimic many

features of the human syndromes. XPA and XPC mice

are photosensitive and display skin-cancers after UV-

exposure. Aging features of CSB mice include retinal

degeneration and weight loss. These findings led us

to hypothesize that the aging features of CS are dis-

tinct from NER function. To test this we generated

double mutant mice and (XPC/CSB, XPA/CSB and

XPA/XPC). Surprisingly, the phenotype of CSB mice is

dramatically enhanced in an XPC or XPA deficient

background. Although embryos appear to develop

normally, XPC/CSB and XPA/CSB mice soon after birth

become severely growth retarded, ataxic and kyphot-

ic. The maximum age reached is 21 days. These mouse

models demonstrate that loss of CSB promotes an

aging phenotype, which is accelerated by a total NER-

deficiency, suggesting that the aging features are

caused by accumulation of endogenous DNA damage

that is not repaired in the absence of NER. Here we

present the generation and characterization of a con-

ditional XPA knockout mouse, allowing time- and tis-

sue dependent inactivation of the XPA gene in

XPA/CSB animals in order to further investigate the

onset of the XPA/CSB phenotype.

126


Author Index

Familyname Initials Prefix Abstractcodes

Aardema MJ OW6003

Aardweg GJ van den PW2040

Aarts JMMJG OW1004

Abd El-Aziz M PW2002

Abet M PW2042

Abeysinghe S PW5007

Aboud M PW2049

Abrini J PW6005

Acker FAA van PW2037

Ackerman JI PW2046

Adler ID PW6022, PW6024

Agana L OW1003

Agen E van OS2001, OW1001

Aguayo S PW6027

Ahr J OS2002

Aka V OW7004, PW3014

Albering H PW2024

Albertini S OW5004

Albrecht C OW4001

Alija A PW4002

Alink GM OS5001

Alonso-Moraga A PW6005

Alunni-Perret V PW1002

Anagnostakis N PW2017, PW2018,

OW7003

Andreatta R PW3013

Andreoli CA PW2009, PW3021

Andreoli R PW3018

Anna L PW3023, PW3004

Anttila S PW3008

Anuszewska EL PW1003

Appolloni M PW6013

Arabski M PW2033

Arbillaga L PW1007

Arlt VM PW2003, PW1002

Athanasiou E PW2017

Attardi LD PW2040

Attia MS abdo PW6022, PW6024

Aubrecht J OS2003, PW2046

Autrup H OW3001, OW3002,

PW4015

Aydin A PW4001

Aydin S PW4009

Azqueta A PW2020

Baccelliere L PW6011

Backendorf CMP OW2003

Bacun-Druzina V PW7005

Bader A PW1005

Badr Y PW2002

Baer-Dubowska W OW1002, PW1004

Bagnasco M OW4003

Bajic B PW6001, PW7001,

PW2034, PW6019

Bajic V PW6017

Bak H OW3002

Baki M PW2035

Barale R OW3003

Bart M OW5001

Barta IVO PW1013

Bártová J PW1013

Basaran A PW4009

Bast A PW4007

Bauer D OW5003

Baum M PW4010

Baumeister M PW6012

Beems RB PW7006, PW7007,

PW2040

Beerens D OW6001

Beffy P PW2043

Berg J van den PW2040

Berggren P OW3004

Beric-Bjedov T PW4004, PW4005

Beskid O PW6014, PW6010,

PW6015

Biljana SP PW6001, PW7001

Binderup ML PW4015

Binkova B PW6010, PW5006

Blaauboer BJ OW6002

Blackshear P PW6025

Blaes S de PW1007

Blake JF PW2046

Blasco MAB OS3003

Blasiak J PW2033, PW2030

Bodrogi I PW2035

Boeck M de PW2004, PW6016,

PW6023, PW3014

Boffetta P OS4003, OW3003

Bolderson E PW2029

Bolognesi C PW3013

Bonassi S OW3003

Borgdorff V PW2045, OW2007

Borm PJA OW4001, PW2013,

PW7004, PW2025

Bouwman FG PW1014

Bracci PM OW1003

Braun K PW6012

Breda SGJ van OW1001, OS2001,

PW1014

Breikers G PW1014

Brennan P OS4003

Bresgen N PW4002

Briedé JJ PW4013, PW4012

Bruins W PW2040

Bubic Spoljar J PW6002

Buchet JP OW7004

Buendia G de PW2039

Bürkle A OW4004

127


Buschini A PW1008, PW4011

Buss K OW5002

Busuttil R OW7005

Butkiewicz D PW3007, OW7002

Büsselberg D PW7002

Byun KP PW4003

Calcagnile A OYSAL2

Camoirano A OW4003

Campisi J OS3004

Canova S OW2004

Canzian F OS4003

Carballo M PW6027

Carratore R del PW2043

Cartiglia C OW4003

Casella M PW2042, PW2043

Cassee FR PW2025

Casteleyn L PW6028

Cavallo D PW4006

Cebulska-

Wasilewska A OW3003

Celotti L OW2004

Chebotarev AN PW1001

Chen X OW2005

Chen Y OW2005

Cheung JR PW2046

Chianese C PW4006

Chiesara E PW2015

Chiu RK PW2052

Chojnacki J PW2033

Christopoulos G PW2019

Christou K PW2017, PW2018,

PW2021

Chuang EY OW2005

Chvatalova I PW5006, PW6010,

PW6015, PW6014

Ciric JC PW2034

Clay P PW6018

Clonfero E PW3017

Coleman CN OW2005

Colognato R OW4002

Conti L PW3021

Cooke MS OYSAL1

Coppedè F OW1003, OW4002

Cordelli E PW6013

Cosyns JP PW1002

Cozzi R PW6011

Cramers P PW2051

Crebelli R PW3021

Creus A PW3020

Csejtei CSA PW3001

Csekeo A PW3004

D'Errico M OYSAL2

Dahm-Daphi X OW2002

Darroudi F PW1005

Davolos DD PW2016

Dejmek J PW5006

Delft JHM van OS2001, PW1014,

OW1001, PW5003,

PW5002, PW5004,

PW3009

Demopoulos N OW3001

Dietrich H PW4010

Dizdaroglu M OYSAL2

Djelic N PW6019, PW6017

Dobbelsteen DJ van den PW2037

Doehmer J PW2042

Dogliotti E OYSAL2

Dollé MET OW7005, PW7006

Dopp ED PW7003, PW7002,

PW7004

Dostal M PW6014, PW5006

Doudounakis S OW7003

Dragsted LO PW4015

Druzhinin VG PW6009

Drzewoski J PW2033

Duburs GJ PW2044

Duffin R PW2025, OW4001

Dundar K PW4001

Dur A PW4001, PW2005

Dusek Z PW6015, PW6014

Duydu Y PW4001, PW2005,

PW3005

Dybdahl M PW4015

Eckl PM PW4002

Eichenlaub-

Ritter U PW6022

Eicker J OW4001

Eisenbrand G PW4010

Eken A PW4001

El-Awady RA OW2002

El-Khatib N PW2002

Elhajouji A PW6026

Ellinger-

Ziegelbauer H OS2002

Elliott B PW6018

Ember I PW3001

Engelhardt G PW6012

Engels LGJB OW1001

Eom MO PW4003, PW5001

Epe B OW4004

Erixon K PW2029, PW2032

Esik O PW2035

Eskelinen M PW3006, PW3010,

PW3002

Ezpeleta O PW1007

Fabiánová E OW3003

Faluhelyi ZS PW3001

Fenech M OW3003

Ferencic Z PW6002

Ferraris M PW2015

Festa F PW6011

Filiberti R PW3013

128


Flamma F PW2009

Fleischer B PW3003

Flohr CF OW4004

Flora S de OW4003

Florea-Wang D PW2036

Florea AM PW7002, PW7003

Flueckiger-Isler SF PW6012

Fokt I PW1003

Fontana I OW4002

Forrest MS OW1003

Francesco A di PW4006

Franekioli J PW7005

Fred CF PW6008

Freidig AP OW5001

Fresegna AM PW6013

Frieauff W PW6026

Frigerio S PW2015

Fritsche L PW2031

Froetschl RF OW5002

Fronza G OW2006

Fucic A PW6002, OW3003

Furlini M PW1008

Fusekova M PW2031

Gadisetti CVR OW2005

Gajecka M OS4002

Galati R PW3021

Galofre P PW3020

Galova EG PW2028, PW2048

Gant TW OS2004

Geh S PW7004

Gemignani F OS4003

Georgiadis P OW3001

Gerlofs-Nijland ME PW2025

Gervais V PW6012

Giachelia M PW6011

Giordano E PW3013

Gismondi M PW4006

Gjuracic K PW7005

Godschalk RWL PW2024, PW3009,

PW2052

Goethem FVG van PW2004, PW6016,

PW6023,

Gold LS OS5004

Golovina TA PW6009

Gompel JAJ van OW6001, PW6012

Goncharova RI PW2044

Gottschalk RWH PW3009

Gourgoulianis K PW2017, PW2018,

PW2021

Gozdzik A PW1003

Granath F PW6008

Grass P OW5003

Grifalconi M OW2004

Groten JP OW5001

Gruber BM PW1003

Gruijl FR de PW2026,OW2003

Grzesiuk EG PW2006, PW2008,

PW2007

Grzesiuk W PW2008

Gundy S PW2035, PW3019,

PW6021, OW3003

Gyorffy E PW3004

Gyori Z PW3004

Haan J de PW2024

Habalova V PW3003

Haenen GJJM PW4007

Hageman GJ PW4007

Hagmar L OW3003

Hainaut P PW2047

Hall J OS4003

Hansteen I-L OW3003

Haq MA PW2027

Harris CC OW7002, PW3007

Hartmann LM PW7003

Harvey JS PW5005

Hasler-Nguyen N PW6012

Hastings H OS6002

Haugen AAGE PW3015

Hayashi M PW2003

Hees-

Stuivenberg S van PW2045

Heijne WHM OW5001

Heikinheimo L PW3010

Heilimo I PW3011, PW3012

HeiYing JIN PW3022

Helleday T PW2029, PW2001

Hemminki KH OS4001

Henderson LM OW6003

Hernandez A PW3020

Herrero O PW6027

Herwijnen MH van OS2001, PW1014,

PW4012, PW5003

Hickson ID OW7001

Hirayama T PW2011

Hirner AV PW7003

Hirvonen A PW3008, PW3010,

PW3011, PW3012,

OW3005, PW3006,

PW3002

Hisamatsu Y PW2011

Hoeijmakers J OW7005, PW7006,

PW7007

Hogervorst J PW2024

Höhr D PW7004, OW4001

Hollstein M PW1002

Holly EA OW1003

Honma M PW2003

Hoogenboom LAP OW1004

Hoogendoorn SM PW2046

Hoogervorst E PW2040

Horbach GJMJ PW2037

129


Horst B van der OW7005

Horst G van der PW7007, PW7006

Hou S OW3004

Hovinen J PW2036

Hubbe P OW2002

Hung RJ OS4003

Husgafvel-

Pursiainen K PW3008

Iavicoli S PW4006

Idaomar M PW6005

Impivaara O PW3008

Ingel FI PW6007

Izzotti AI OW4003

Jacks T PW2040, PW2041

Jagerstad I OS5003

Janion C PW2007

Jansen JG OW2007

Janzowski CJ PW4010

Jaruga P OYSAL2

Jarventaus H PW3011, PW3012

Jarvisalo J PW3008

Jazbec AM PW6002

Jee SW PW5001

Jenssen D PW2029,PW2032

Johansson F PW2029

Johansson FJ PW2032

Jones E PW6018

Jonge J de PW4012

Jonker D OW5001

Jung HK PW4003

Juren T PW1011

Kajosmaki T PW2023

Kalina I PW3003

Kamal N PW2002

Kampman E PW3016

Kanaar R OS1002

Kanariou M PW2022

Kanavetas P PW2022

Kang HI PW5001, PW4003

Kang MK PW5001

Kang SC PW6004

Kannouche PL OS1004

Karanastasi G PW2017, PW2018,

OW7003

Karimi-Busheri F PW2038

Kasler M PW3019

Kasper P OW5002

Kasprzak M PW2033

Kataja V PW3006, PW3010,

PW3002

Katsouyianni K OW3001

Kaufmann G OW5002

Kautiainen A PW6008

Kawanishi M PW2011

Kazmierczak P PW2033

Keenan PO PW6006

Kelecsenyi A PW6021

Kempers P PW4012

Kenny J PW5005

Ketelslegers HB PW3009

Khan I PW5007

Khan QM PW2027

Kienhuis AS PW5002

Kim MH PW5001

Kim OH PW4003, PW5001

Kirkland DJ OW6003

Kirkwood TBL OS3001

Kirsch-Volders M PW6024, PW3014,

OW3003, OW7004

Kiss I PW3001

Kleinjans JCS PW1014, PW5002,

PW5004, PW1012,

OS2001, PW3009,

PW2024, PW2051,

PW4012, PW4013

Klupinska G PW2033

Knaapen AM PW2013, PW3009,

PW2052

Knasmueller S PW1005

Knezevic-

Vukcevic J PW4004, PW4005

Knight W PW6006

Knudsen LE OW3003, PW5008,

PW6028

Koissi N PW2010

Kok TH de OW1004

Kok TMCM de PW4012

Kontogianni N PW2021

Koo HJ PW6003, PW6003

Kosma VM PW3006, PW3010,

PW3002

Kostic S PW3004

Kosyakova NV PW1001

Köteles GJ OW3003

Kotova N PW1011

Krajka-Kuzniak V OW1002, PW1004

Kram NR PW3016

Kranen HJ van PW2026, PW3016

Krul CAM PW6020

Kruszewski M PW2008

Krzesniak M PW3007, OW7002

Ku WW PW2046

Kumar R OW3004

Kwon KS PW5001

Kyrtopoulos SA dr. OW3001

Lagerqvist A PW2032

Lambert B OW3004

Lampo A PW2004, PW6016,

PW6023

Landi S OS4003

Langie SAS PW2052

Langová M PW1013

130


Larsson P OW3004

Lazutka J OW3003

Lee BM PW6003, PW6004,

PW1009

Leede BJ van der PW2004, PW6023

Leede BM van der PW6016

Leeuwen AIM van PW3016

Leeuwen DM van PW5004, OS2001

Lehmann AR OS1004

Leopardi P PW3021

Levi F OS6001

Lewis PD PW5007

Lhoste E PW1005

Li H PW2025, OW4001

Liber HL OW2005

Liberti SE OW2001

Lind HL PW3015

Lindholm C OW3003

Lison D PW3014

Loft S PW4015

Lohman P OW7005, PW7006

Longobardi M. OW4003

Loniarek J. PW1010

Lönnberg HARRI PW2010

Lopez de Cerain A PW1007, PW2020

Lord GM PW1002

Lozano G. PW2040

Luken MEM PW3016

Lundin CL PW2029

Lützen A OW2001

Lynch AM PW5005, OW4005

Maas A PW7007

Maas LM PW4012

Mackie C OW6001

Maclean N PW2016

Mages W PW2031

Magrini R OW2006

Majsterek IM PW2030

Mancini A PW4011

Manini P PW3018

Marabini L PW2015

Marcon F PW3021

Marcos R PW3020

Maridaki K PW2019

Mariman ECM PW1014

Marino C PW6013

Markovic B PW6019, PW6017

Markovic D PW6002

Martino A PW2009

Mateuca RAM PW3014, OW7004

Maunu H PW3011, PW3012

Mayerhofer B PW2014

Maywood E OS6002

McGinnis C OW5003

Meeuwen RNC van PW6020

Menichini T OW2006

Mercati F PW3018

Mercelina-

Roumans P PW2024

Mercken EM PW4007

Merello A OW4003

Mersch-

Sundermann V PW1005

Messini-

Nikolaki N PW2017, PW2018,

PW2019, PW2021,

PW2022, OW7003

Mezzoug N PW6005

Mielzynska D PW3017

Migliore M PW3018, OW4002

Milcova A PW6010, PW6015,

PW5006

Mildner B PW6002

Milicevic MZ PW2034, PW7001

Milne K PW6025

Minarovits J PW3004

Minina VI PW6009

Mirkova E OW3003

Mitchell JB OW2005

Mitic-Culafic D PW4004, PW4005

Mitrunen K PW3008, PW3010,

PW3011, PW3012,

PW3002

Mitrunen M PW3006

Mognato M OW2004

Mokrushina NV PW6009

Müller P PW4015

Moonen HJJ PW1006

Moonen EJC PW4013

Moonen JC OW1001

Mrzyglodzik M PW3007, OW7002

Mu-oz MJ PW6027

Mueller L OW5003

Mullenders LHF PW2026, PW2026,

PW2051, OW2003

Müller AK PW4015

Müller L OW6003

Muñoz serrano A PW6005

Muro A di PW2042

Mutti A PW3018

Müller L OW6003

Myllynen PK PW2023

Naccarati A PW3018

Nakagawa Y PW2011

Nardo T OYSAL2

Neri M PW3013

Nexo BA OW3002

Niedernhofer L OW7005

Nieminuszczy JN PW2007, PW2006,

PW2008

131


Nikitchenko NV PW2044

Nikolic B PW4004, PW4005

Ninoslav D PW6001, PW7001

Nisar H PW2027

Norppa H PW3011, PW3012,

OW3003

Nowosielska A PW2006

Nunziata A PW2009

Olatunde

Farombi E PW4015

Olive K PW2041

Ommen B van OW1005, OW5001

Oosten M van OW2003

Oostrom CTHM van PW2040, PW7007,

PW2041

Osowski JJ PW2046

Overvad K OW3002

Pacanovska M PW2031

Pacchierotti F PW6022

Pachón G PW2020

Packenham JP PW2050

Palma G de PW3018

Park MS PW5001

Park MS PW4003

Parker KM PW2001

Pastaka C PW2021

Pavanello SP PW3017

Peeters S PW4013

Peijnenburg A OW1004

Pellacani C PW1008, PW4011

Pena E de la PW6027

Perrone E PW3013

Persaud P PW2046

Peöut J PW7005

Pfuhler S PW6025

Phillips DH PW2003

Piano L del PW2042

Pietrangeli BM PW2016

Piperakis SM PW2017, PW2018,

PW2019, PW2021,

PW2022, OW7003

Platonova VI PW1001

Plazinska MT PW2008

Pluijm I van der PW7007

Polfvkova Z PW1013

Poli P PW1008, PW4011

Powell SN OW2002

Priebe W PW1003

Priel E PW2049

Pulliero A PW3017

Puntoni R PW3013

Pytel D PW2030

Raaschou-

Nielsen O OW3002

Radice S PW2015

Radicella JP OW4004

Ramaekers CHMA PW2052

Ranaldi R PW6022

Rasmussen LJR OW2001

Rasouli-nia AR PW2038

Reddy A OS6002

Rees R PW5005

Reimann R PW6012

Remenar E PW3019

Renes JW PW1014

Restivo FM PW4011

Rettenmeier AW PW7002, PW7003,

PW7004

Richter E PW2014

Rietjens IMCM OS5001

Risom L PW4015

Rodrigo GR PW2014

Roggieri P PW3013

Rosa N la PW6011

Rossi C PW1008, PW4011

Rossi S PW3021

Rössner P OW3003, PW5006,

PW6010, PW6014,

PW6015

Rubes J PW6015

Ruepp S OW5004

Rusin B PW2012

Rusin M PW3007

Rusin R OW7002

Russo D OW2006

Ryabokon NI PW2044

Rydberg P PW1011

Rydzanicz M OS4002

Ryeom TK PW5001

Ryk CM OW3004

Rzeszowska-

Wolny J PW2044

Sala C PW6028

Salagovic JS PW3003

Salonen RO PW2025

Sarasin AS OS3FS1

Schaap M PW2041

Schaefer S PW4010

Schanke A van PW2026

Schavinsky-

Khrapunsky Y PW2049

Schins RPF OW4001, PW2013,

PW2025

Schmeiser HH PW2003, PW1002

Schmuczerova J PW6015

Schoket B PW3004

Schols AMWJ PW4007

Schooten FJ van OW1004, PW5003,

PW3009, PW2052,

PW2013, PW2024,

PW2025

Schulz I OW4004

132


Sciutto C PW6011

Scotti E PW3018

Segerbäck K PW1011

Segesdi J PW3004

Sevastyanova O PW6010

Sevcovicova A PW2028, PW2048

Shi T OW4001, PW7004

Shiloh Y OS1001

Shokouhi B PW7003

Shtylik A OW2007

ShuHan SUN PW3022

Siciliano G OW4002

Siems W PW4002

Siezen CLE PW3016

Siivola P PW3010, PW3011,

PW3012

Sillanpää PJ PW3002

Simi S PW2042, PW2043,

PW2042, PW2043

Simic D PW4004, PW4005

Simili M PW2043

Siwinska E PW3017

Skibola CF OW1003

Slaninova M PW2031, PW2048,

PW2028

Sliwinski T PW2030

Slupianek A PW2030

Smedt A de PW2004, PW6016,

PW6023

Smerak P PW1013

Smerhovsky Z PW6010, PW6015

Smith MT OW1003

Solansky I PW5006

Soltesz I PW3004

Sommerburg O PW4002

Sorensen M OW3002

Soric JS PW1010

Sorrentino C PW2042

Spagnoli G PW6011

Speksnijder E PW2041

Sram RJ PW6010, PW5006,

OW3003, PW6015,

OW3001, PW6014

Staal YCM PW5003, OS2001

Staedtler F OW5003

Stanimirovic Z PW6019, PW6017

Stankovic S PW4004, PW4005

Stanojevic J PW4004, PW4005

Staszewski S OW5002

Stavkova Z PW6015, PW6010,

PW6014

Steeg H van PW7007, PW7006,

PW2040, OW7005,

PW2041

Steemans M PW2004, PW6016,

PW6023

Steenwinkel M-JST PW6020

Stefanini M OYSAL2

Steineck G OW3004

Stephanou G OW3001

Stierum RH PW5002, OW5001

Stoikidou M OW3001

Stopper H OS5002

Stout GJ OW2003

Streicker M PW6025

Stronati L PW6013

Surralles J PW3020

Suter-Dick L OW5004

Suter W PW6026, OW5003

Suzen HS PW3005

Suzuki T PW2003

Sviezena B PW2028, PW2048

Szaefer H OW1002, PW1004

Szekely G PW3019

Szyfter K OS4002

Szymoniak K OS4002

Takamura TT PW2011

Tamagno P OW2006

Tampa E OW4003

Teson M OYSAL2

Testa A PW6013, PW6011

Thierens H OW7004

Tibold A PW3001

Tirindelli D PW6011

Tjønneland A OW3002

Tkacova R PW3003

Tognoni G OW4002

Topinka J OW3001, PW6010

Törnqvist M PW6008

Torre A de la PW6027

Tranfo G PW6011

Treuner G PW2031

Tsai M-H OW2005

Tsilimigaki S PW2017, PW2018,

PW2019, PW2021,

PW2022, OW7003

Tudek B PW2012, PW2012,

PW1013

Tuveson D PW2041

Urban L OW5003

Ursini CLU PW4006

Uusitupa M PW3006, PW3010,

PW3002

Uzun G PW4001

Vähäkangas KV PW2047, PW2023

Vainio H PW3008, PW3002

Vaitiekunaite R OW7002, PW3007

Vanhauwaert A. PW6024

Vanparys PH PW2004, PW6016,

PW6023

Vaskivuo L PW2023

Verdina A PW3021

133


Vijg J OS3002, OW7005,

PW7006

Villani P PW6013

Vineis P OS4004

Vlachodimi-

tropoulos D OW3001

Vlcek D PW2031, PW2048,

PW2028

Vogel N de PW6020

Vogel U OW3002, PW4015

Voho AV PW3008

Volkov AN PW6009

Vries A de OS1003, PW2040,

PW2041

Vukovic-Gacic B PW4004, PW4005

Waard WJ de OW1004

Wagner AM OW6004

Wakabayashi K PW2011

Wallin H PW4015, OW3002

Walmsley RM PW6006

Watanabe T PW2011

Weickhardt S OW5002

Weinfeld M PW2038

Weiser T OW5004

Wierzbicka M OS4002

Wijnhoven SWP OW7005, PW2041,

PW7006

Will F PW4010

Willers H OW2002

Willis N PW2041

Wilms LC PW1012, PW4012,

PW4013

Wind N de OW2007, OW2003,

PW2045

Wisniewska-

Jarosinska M PW2033

Wit J de PW7007

Woestenborghs F OW6001

Wolfreys A PW6018

Wortelboer HM PW5002

Wouters BG PW2052

Wouters EFM PW4007

Wrzesinski M PW2006, PW2007

Wunderlich H-G PW6012

Xamena N PW3020

Yagi T PW2011

Yan H OW2005

Yan HE PW3022

Yan HONG PW3022

Yoo E jeong PW1009

Yoshikawa YY PW4008

Zambruno G OYSAL2

Zampieri D PW2042

Zeeland AA van PW2051

Zhan L PW2003

Zhang SD OS2004

Zhao S OW2005

Zidzik J PW3003

Zienolddiny S PW3015

Zijno A PW3021

Zivkovic L PW6001, PW7001

Zwart E PW2040, PW2041

134


program and abstracts - swansea university · program and abstracts organised by section genetic...

Documents