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ENVR 132/TOXC 142/BIOC142Biochemical & Molecular Toxicology
Induction of Metabolism by Toxicants
Instructor:Edward LeCluyse, Ph.D.
e-mail: [email protected]
Induction: Definitions and Principles
• The process of increasing the amount or theactivity of a protein.
• A homeostatic mechanism for regulating enzymeproduction in a barrier organ, such as the liver,intestine, kidney.
• In enzymology, an inducer usually combines withand deactivates/activates a regulatory proteinwhich leads to increased gene expression.
P450 Enzyme Induction
• Induction can cause marked increases in P450composition (>20-fold) and chemical clearance orbioactivation.
• As a result, induction can increase tolerance to sometoxicants while enhancing the toxicity of others.
• Induction can decrease the therapeutic effect of drugsby increasing the rate and pattern of metabolism.
• Xenobiotics are known to induce enzymes that play amajor or no role in their biotransformation (e.g.,omeprazole vs. ethanol).
Internal Exposure to Natural andMan-made Chemicals
• drugs
• industrial chemicals
• pesticides
• pollutants
• alkaloids
• cigarette smoke
• cruciferous vegetables(indole-3-carbinol)
• secondary plantmetabolites
• toxins produced bymolds, plants, andanimals
• pyrolysis products incooked food
Induction of Rat Liver P450 Enzymes byPrototypical Inducers In Vivo
CLO
PCN
PB
BNF
Inducer
10,693 ± 620489 ± 52CYP4A
12,693 ± 2,2552,460 ± 780CYP3A
1,460 ± 18023.8 ± 4.2CYP2B
3,320 ± 183152 ± 27CYP1A
Induced ActivityControl Activity
In vivo Induction in Male Rats
P450 Enzyme
CYP1A, EROD; CYP2B, PROD; CYP3A, testosterone 6β-hydroxylation;CYP4A, lauric acid 12-hydroxylation.
Induction and Inhibition of P450 in Mice Treatedwith PB or SKF525A: [14C-methyl]aminopyrine
Unwanted Side-effects ofEnzyme Induction
• Failure of drug therapy
• Chemical tolerance with auto-induction
• Xenobiotic toxicity potentiated
• Chemical carcinogenesis potentiated
• Endogenous substrate metabolism perturbed
• Proliferation of cellular ER and peroxisomes
Effects of Inducers on Rodent LiverPhysiology and Function
Serr
um tr
iazo
lam
(ng
/ml)
Rifampin Effects on Triazolam Disposition
Villikka et al., Clin Pharmacol Ther 1997;61:8-14.
Rifampin Placebo
Acetaminophen Toxicity
ProteinAdducts,
GSHdepletion
Phase IIconjugates
Induced byEtOH andIsoniazid
Disruption of Thyroid Function
UGT1A
Types of P450 Inducers
• Many “prototypical” inducers of specificfamilies or subfamilies of P450 enzymes– CYP1A inducers: 3-MC, BNF, omeprazole, TCDD
– CYP3A inducers: rifampin, dexamethasone,troglitazone
– CYP2B inducers: phenobarbital, PCBs, phenytoin
– CYP2E1 inducers: ethanol, isoniazid
• Some overlap in “specificities” of inducers
• An inducer for one family of enzymes mayalso suppress another family (e.g., BNF)
Molecular Mechanisms of P450Enzyme Induction
Mechanisms of P450 Induction• Receptor-mediated transcriptional activation
– Receptor• A macromolecule with which a hormone, drug, or other
chemical interacts to produce a characteristic effect.
– Two key features:• chemical recognition• signal transduction
– Ligand: A chemical that exhibits specific binding toa receptor.
• mRNA stabilization
• Protein stabilization
Enzyme InductionGeneral mechanism of enzyme induction
protein
activity
mRNA
Gene transcription
Drug
Nuclear Receptor
DR cytosol DR nucleus
CYP1ACYP2BCYP3ACYP2C
P-gpMrp2
UGT1A1
Transcription factor
Dimerization partner
Examples of ligands
Genes Regulated
AHR ARNT Dioxins, non -ortho PCBs, some PAHs, bilirubin, etc.
CYP1A, CYP1B GST, UGT, NQO
CAR
RXR
Phenobarbital (PB), TCPOBOP, chlorinated pesticides, ortho -PCBs, androstanol / androstenol (inhibits)
CYP2B , CYP3A GST, ABC transporters
PXR (SXR)
RXR
PB, ortho-PCBs, organochlorine pesticides, dexamethasone, pregnenalone, corticosterone, bile acids (lithocholic acid)
CYP3A , CYP2B, CYP7A (repression) GST, ABC transporters
PPAR
RXR
Fibrate drugs, phthalate esters, linoleic acid, arachidonic acid
CYP4A , CYP7A (repression), CYP8B, LXR
LXR RXR Cholesterol; (24 S) - hydroxycholesterol CYP7A, ABC transporters, LXR
FXR RXR Bile acids, chenodeoxycholic acid Represses CYP7A, CYP8B , CYP27A
ER ER Structurally diverse xenoestrogens CYP19
Receptors Involved in the Regulation ofCYP Gene Expression
AhR Signaling Pathway
Cytoplasm Nucleus
9090
X
AhR
L
L
9090
X
L
9090
X
L
L
9090
X
L
or Arnt
From: Anne Mullen, Advanced Pharmacology, McMaster University, Ontario, CA
AhR Signaling Pathway
XRE promoter gene(CYP1A1)
Translation
Increased expressionCYP1A1 protein
Increased expression ofother gene products
+
AhR/Arntheterodimer
mRNA
IC
+
Co-regulation of Target Genes by NR’s• Complementary roles of NR’s in protection against
xenobiotic exposure.
• Increased expression of the hepatic genes involved indrug metabolism and excretion (e.g., CYP’s, UGT’s,GST’s, transporters).
• These target genes represent redundant but distinctlayers of defense.
• There are distinct and overlapping species differencesin response to activators of NR’s.
Modified from Kast, H. R. et al. J. Biol. Chem. 277:2908-2915, 2002
Coordinate Regulation of P450’s, UGT’s andTransporters by PXR and CAR
UGT’s
MRP3
Amino Carboxy
AF-1 DBD LBD AF-2
Modulators interactwith some cofactors
Binding to responseelements of target genes
Ligand and coactivatorbinding pockets
Translocaseactivity
5’ 3’ 5’ 3’ 5’ 3’
Monomers RXR Heterodimers Homodimers
LBD
DBD
NR-LBD RXR-LBD
DBD DBD DBD DBD
NR-LBD NR-LBD
RORTLXERRNGFI-B
PXRCARPPARLXRFXRRAR
GRCOUP-TFHNF4Rev-ErbGCNF
5’ 3’
n
n
n
DRn
IRn
ERn
CYP2B Response elements
CYP2B6 TGTACT n=4 TGACCCCYP2b10 TGTACT n=4 TGACCTCYP2B1 TCTACT n=4 TGACCTCYP2B2 TGTACT n=5 TGACCT
NR1s
CYP2B6 TGGACT n=4 TGAACCCYP2b10 TCAACT n=4 TGACACCYP2B1 TCAACT n=4 TGACAC CYP2B2 TCAACT n=4 TGACAC
NR2s
NR3
CYP2B6 TGGACT n=4 TGACCC
CYP3A Response elements
CYP3A4 TGAACT n=3 TGACCC CYP3A2 TGACCT n=3 TGAGCT CYP3A23 TGACCT n=4 TGAGTT CYP3A2 TGAACT n=3 TGAACT
DRs
CYP3A4 TGAAAT n=6 GGTTCACYP3A4 TGAACT n=6 AGGTCACYP3A23 TTAACT n=6 AGGTCACYP3A5 TGAACT n=6 AGGTAACYP3A7 TTAACT n=6 AGGTCACYP3A7 TGAAAT n=6 AGTTCA
ERs
Other Genes
UGT1A1 TGAGTT n=4 TAACCT MDR1 TGAGAT n=6 AGTTCA rMRP2 TGAACT n=8 AGTTCA CYP2C9 CAAACT n=4 TGACCT
Similar Binding of PXR and CARto Promoter Response Elements
Goodwin et al., Mol. Pharmacol., 2001
Differential Binding of PXR andCAR to Other Promoter Regions
XREM-2B6 ER6-3A4
PXR + + + + + +CAR + + + + + +RXR + + + + + + + + + + + +
PXR/RXRCAR/RXR
CYP2B, CYP3A, CYP1A,CYP2A, CYP2Cs, OATP2UGT1A1, MRP2, SULT1A1
CYP1A, UGT1A1, SULT1A1
CAR RXR
XREM / PBREM
AhR ARNT
XRE / DRE
PXR RXR
XREM / PXRE
CYP3A, CYP2B, CYP2Cs,CYP7A, MDR1, MRP2,OATP2, GSTA-2, UGT1A1,AldHs, Carboxyesterase 2, 3
Aryl hydrocarbonReceptor
ConstitutiveAndrostane Receptor
Pregnane XReceptor
Molecular Basis for the SpeciesDifferences in Enzyme Induction
Rabbit
Human
Rat
0.1%
DM
SO
5µ
M P
CN
10µ
M R
ifam
pici
n
10µ
M S
R12
813
10µ
M D
TB
A
CYP3A6
CYP3A4
CYP3A23
Species Differences in the Regulationof CYP3A Enzymes
Species Differences in CYP2BInduction by PB
Species Differences in CYP1AInduction by Xenobiotics
CYP1A1/2 Activity in Rat Hepatocytes as a Function of Treatment with Drug 'X'
0
100
200
300
400
500
600
700
800
900
1000
Contro
l (0.1
% D
MSO)
3-M
C 1µM
Drug
'X' 0
.6µM
Drug
'X' 2µM
Drug
'X' 6µM
Drug
'X' 2
0µM
Phe
nace
tin
O-D
ealk
ylat
ion
(pm
ol/m
in/m
g)
CYP1A Activity in Dog Hepatocytes as a Function of Treatment with Drug 'X'
0
100
200
300
400
500
600
Contro
l (0.1
% D
MSO)
3-M
C 2µM
Drug
'X' 0
.6µM
Drug
'X' 2µM
Drug
'X' 6µM
Drug
'X' 2
0µM
Phe
nace
tin
O-D
ealk
ylat
ion
(pm
ol/m
in/m
g)
CYP1A2 Activity in Human Hepatocytes as a Function of Treatment with Drug 'X'
0.0
0.2
0.4
0.6
0.8
1.0
1.2
Contro
l (0.1
% D
MSO)
3-MC 2µM
Drug '
X' 0.2µ
M
Drug '
X' 2µM
Drug '
X' 6µM
Drug '
X' 20µ
M
CY
P1A
2 A
ctiv
ity
(nm
ol/m
in/m
g)
Observations and Questions• Significant species differences are observed
in response to inducers.
• All major subfamilies of inducible CYP’s(CYP1A, CYP2B, CYP3A, CYP4A) exhibitthis behavior.
• What is the molecular basis of the species-specific responses?
• What is the significance of these differencesto predicting human toxicity?
PXR ExpressionPlasmid
PXR
RXR PXR
PXRE Reporter Gene
RXR
Drug
Reporter Plasmid
Transfection Assay for P450Enzyme Induction
CV-1HuH7 cell
Differential Activation of Human, Rabbit,and Rat PXR by CYP3A Inducers
PCN
rifampicin
lovastatin
clotrimazole
Normalized Reporter Activity
0 20 40 60 80 100 300 350 400
OHOHONNNMeNHOOOHOAcOMeOOHHO
NNCl
OOHOHOOH
HOOHCN
PXR Sequence Homology1 41 107 141 434
Human PXR1
Rat PXR11 38 104 138 431
Xenopus ONR11 37 102 136 386
Human VDR 1 24 89 122 427
LigandDNA
96
69
63 37
76
42
Mouse PXR11 38 104 138 431
96 76
Rabbit PXR11 41 107 141 434
8294 Variation in ligandbinding domainconsistent with invivo species differ-ences in responseto inducers
Amino Acid Differences in theLigand Binding Domain of PXR
(Zhang et al., Arch. Biochem. Biophys., 1999)
Ser187 Leu213 Asp266 Glu337 Ile417
Gly181 Leu206 Tyr263 His333hPXR
Phe184 Leu210 Asp263 Lys334 Ser414
Gly178 Arg203 Tyr260 Arg333mPXR
Val184 Val210 Glu263 Glu333 Thr414
Asp178 Ser203 His260 Arg333rPXR
Summary• Induction of metabolism is caused by many
structurally unrelated xenobiotics.• Induction occurs mainly by transcriptional
regulation of metabolizing enzymes andtransporters.
• Nuclear receptors mediate the inductionresponse by most xenobiotics.
• Amino acid differences in the ligand-binding domain of the receptors are mainlyresponsible for the species differences in theinduction of CYP450 enzymes.
Additional Reading• Parkinson, A.: Biotransformation of xenobiotics. In: Casarett and
Doull’s Toxicology. The Basic Science of Poisons. Sixth edition(edited by C.D. Klaassen). McGraw Hill, New York, 2001.
• Wang, H. and Negishi, M. (2003) Transcriptional regulation ofcytochrome p450 2B genes by nuclear receptors. Curr Drug Metab.4(6):515-25.
• Bertilsson, G., Heidrich, J., Svensson, K., Asman, M., Jendeberg, L.,Sydowbackman, M., Ohlsson, R., Postlind, H., Blomquist, P. andBerkenstam, A. (1998) Identification of a human nuclear receptordefines a new signaling pathway for CYP3A induction. Proc. Natl.Acad. USA. 95:12208-12213.
• Blumberg, B., and Evans, R.M. (1998) Orphan nuclear receptors – newligands and new possibilities. Genes Dev. 12:3149-3155.
• Geick A., Eichelbaum M., and Burk O. (2001) Nuclear receptorresponse elements mediate induction of intestinal MDR1 by rifampin. JBiol Chem. 276(18):14581-14587.
Additional Reading• Goodwin B., Hodgson E., and Liddle C. (1999) The orphan human
pregnane X receptor mediates the transcriptional activation of CYP3A4by rifampicin through a distal enhancer module. Mol Pharmacol56:1329-1339.
• Honkakoski P. and Negishi M. (1998) Regulatory DNA elements ofphenobarbital-responsive cytochrome P450 CYP2B genes. J BiochemMol Toxicol 12:3-9.
• Jones, S. A., Moore, L. B., Shenk, J. L., Wisely, G.B., Hamilton, G. A.,McKee, D. D., Tomkinson, N. C. O., LeCluyse, E. L., Wilson, T. M.,Kliewer, S. A. and Moore, J. T. 2000. The pregnane X receptor, apromiscuous xenobiotic receptor that has diverged during evolution.Mol. Endocrinol. 14: 27-39.
• Wang, H., and LeCluyse E. L. 2003. Role of orphan nuclear receptorsin the regulation of drug metabolising enzymes. Clin. Pharmacokinet.42: 1331-1357.