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Induction and Inhibition of P450 in Mice Treated Induction and Inhibition of P450 in Mice Treated with PB or SKF525A: [with PB or SKF525A: [1414CC--methyl]aminopyrinemethyl]aminopyrine
Serr
umtri
azol
am (n
g/m
l)
Rifampin Effects on Triazolam DispositionRifampin Effects on Triazolam Disposition
Villikka et al., Clin Pharmacol Ther 1997;61:8-14.
RifampinPlacebo
Consequences of Cytochrome P450 Enzyme Induction
Consequences of Cytochrome Consequences of Cytochrome P450 Enzyme InductionP450 Enzyme Induction
Effects of Inducers on Rodent Liver Effects of Inducers on Rodent Liver Physiology and FunctionPhysiology and Function
Acetaminophen Metabolism and ToxicityAcetaminophen Metabolism and Toxicity
~60% ~35%
CYP2E*CYP1A CYP3A
NAPQIN-acetyl-p-benzoquinone imine
*induced by ethanol, isoniazid, phenobarbital
Protein adducts,Oxidative stress,Toxicity
HNCOCH 3
OH
HNCOCH 3
OSO 3H
HNCOCH 3
OO CO 2H
OHOH
HON
O
COCH 3
Endocrine Disruption Endocrine Disruption • Many xenobiotics can mimic certain hormones and
bind to target cellular sites receptive to natural hormones
• Modes of endocrine disruption can result from agonistic or antagonistic receptor binding affecting biosynthesis, transport, storage, release, and clearance of hormones
• Some pesticides have been identified as endocrine disruptors, in particular the thyroid hormone can be affected by: acetochlor, alachlor, fipronil (Frontline), heptachlor, maneb, methomyl, and zineb
• PCBs, mercury, pentachlorophenol are some of the thyroid hormone disruptors that are no longer used as pesticides
• DDT, dieldrin, lindane, methoxychlor, triadimefon are thought to be estrogenic-type environmental endocrine disruptors (EEDs) while atrazine, vinclozolin, and procymidone are thought to be andogenic EEDs
UGT1A
Molecular Mechanisms of P450 Molecular Mechanisms of P450 Enzyme InductionEnzyme Induction
General Mechanisms of P450 InductionGeneral 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 to a receptor.
• mRNA stabilization• Protein stabilization
Coordinates: Kumar R, Thompson EB (1999). "The structure of the nuclear hormone receptors". Steroids 64 (5): 310–9
Enzyme InductionEnzyme InductionGeneral mechanism of hepatic enzyme inductionGeneral mechanism of hepatic enzyme induction
Receptors Involved in the Regulation of Receptors Involved in the Regulation of CYP Gene ExpressionCYP Gene Expression
Modified from Kast, H. R. et al. J. Biol. Chem. 277:2908-2915, 2002
Coordinate Regulation of P450Coordinate Regulation of P450’’s, s, UGTUGT’’ss and and Transporters by NRTransporters by NR’’ss
UGT’s
MRP3
Role of CAR/PXR in lipid metabolism, Role of CAR/PXR in lipid metabolism, synthesis, and uptake & glucose homeostasissynthesis, and uptake & glucose homeostasis
Moreau et al. 2007 Mol. Pharmaceutics
Induction in cultures of primary Induction in cultures of primary human hepatocyteshuman hepatocytes
CYP2B6 Activity and mRNA with PB & RIF
• Saturable, sigmoidal responses
What is Relevant Induction?What is Relevant Induction?Potency and EfficacyPotency and Efficacy
Dose-Response ‘Window’
(Position → potency)
Magnitude of Response (Efficacy)
EC50
1. Efficacy (e.g. % of PC)
2. Potency (e.g. EC50)
Emax
Relationship between In Vitro Potency and Induction In VivoEC50 Cmax [Cmax]/EC50 Clinical
• Aryl hydrocarbon receptor (AHR) is a basic helix-loop-helix (bHLH) protein belonging to the Per-Arnt-Sim (PAS) family of transcription factors
• It transcriptionally induces expression of hepatic CYP1A1, CYP1A2, and CYP1B1 , as well as several other genes, including some phase II metabolizing enzymes
• AHR ligands include PAHs and TCDD
AhR Signaling PathwayAhR 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
GR/Dexamethasone Role in Basal & Induced Expression GR/Dexamethasone Role in Basal & Induced Expression via CAR/PXR (Master Regulator)via CAR/PXR (Master Regulator)
Pascussi et al. 2001, Eur J. Biochem, v. 268, p.6346
Wang & LeCluyse 2003, Clin Pharmacokin, v. 32, p.1331
Molecular Basis for the Species Molecular Basis for the Species Differences in Enzyme InductionDifferences in Enzyme Induction
Rabbit
Human
Rat
0.1%
DM
SO
5μM
PC
N
10μM
Rifa
mpi
cin
10μM
SR
1281
3
10μM
DTB
A
CYP3A6
CYP3A4
CYP3A23
Species Differences in the Regulation of CYP3A Enzymes
Species Differences in the Regulation Species Differences in the Regulation of CYP3A Enzymesof CYP3A Enzymes
Species Differences in CYP2B Species Differences in CYP2B Induction by PhenobarbitalInduction by Phenobarbital
Species Differences in CYP1A Species Differences in CYP1A Induction by XenobioticsInduction 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%
DMSO)
3-MC 1µ
M
Drug 'X
' 0.6µ
M
Drug 'X
' 2µM
Drug 'X
' 6µM
Drug 'X
' 20µ
M
Phen
acet
in O
-Dea
lkyl
atio
n (p
mol
/min
/mg
CYP1A Activity in Dog Hepatocytes as a Function of Treatment with Drug 'X'
0
100
200
300
400
500
600
Contro
l (0.1%
DMSO)
3-MC 2µ
M
Drug 'X
' 0.6µ
M
Drug 'X
' 2µM
Drug 'X
' 6µM
Drug 'X
' 20µ
M
Phen
acet
in O
-Dea
lkyl
atio
n (p
mol
/min
/mg)
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
' 0.2µM
Drug 'X
' 2µM
Drug 'X
' 6µM
Drug 'X
' 20µ
M
CY
P1A
2 A
ctiv
ity (n
mol
/min
/mg
Species Differences in CYP4A Species Differences in CYP4A Induction by Clofibric AcidInduction by Clofibric Acid
Rat Human
CTL 1 10 100 500 1000
CYP
4A1
Fold
Indu
ctio
n
0
10
20
30
40
50
60
Clofibric Acid (µM)
CTL 1 10 100 500 1000
CYP
4A11
Fol
d In
duct
ion
0
10
20
30
40
50
60
Clofibric Acid (µM)
CTL 1 10 100 500 1000
CYP
4A11
Fol
d In
duct
ion
0
1
2
3
4
Clofibric Acid (µM)
Rat Hepatocytes Human Hepatocytes
Laur
ic a
cid
12-h
ydro
xyla
tion
PAH Inducers in Rat vs. Human
Rat TCDD 1A1 mRNA
EC50 = 0.00767 +/- 0.00409
EC50 = 0.0107 +/- 0.043
CYP1A1 mRNA Hu497
Species difference in potency and efficacy
EC50 = 0.00767 +/- 0.00409
Observations and QuestionsObservations and Questions• Significant species differences are observed
in response to inducers.• All major subfamilies of inducible CYP’s
(CYP1A, CYP2B, CYP3A, CYP4A) exhibit this behavior.
• What is the molecular basis of the species-specific responses?
• What is the significance of these differences to predicting human toxicity?
PXR ExpressionPlasmid
RXR PXR
PXRE Reporter Gene
Drug
Reporter Plasmid
Transfection Assay for P450 Transfection Assay for P450 Enzyme InductionEnzyme Induction
CV-1HuH7 cell
PXRRXR
Differential Activation of Differential Activation of Human,Human, Rabbit,Rabbit,andand RatRat PXR by CYP3A InducersPXR by CYP3A Inducers
SummarySummary• Induction of metabolism is caused by many
structurally unrelated xenobiotics.• Induction occurs mainly by transcriptional
regulation of metabolizing enzymes and transporter proteins.
• Nuclear receptors mediate the induction response by most xenobiotics.
• Amino acid differences in the ligand-binding domain of the receptors are mainly responsible for the species differences in the induction of CYP450 enzymes.
Additional ReadingAdditional 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 of cytochrome 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. and Berkenstam, A. (1998) Identification of a human nuclear receptor defines a new signaling pathway for CYP3A induction. Proc. Natl. Acad. USA.95:12208-12213.
• Blumberg, B., and Evans, R.M. (1998) Orphan nuclear receptors – new ligands and new possibilities. Genes Dev. 12:3149-3155.
• Geick A., Eichelbaum M., and Burk O. (2001) Nuclear receptor response elements mediate induction of intestinal MDR1 by rifampin. J Biol Chem.276(18):14581-14587.
• Moreau, A, Vilarem, MJ, Maurel, P; and Pascussi, JM. (2007) Xenoreceptors CAR and PXR Activation and Consequences on Lipid Metabolism, Clucose Homeostasis, and Inflammatory Response. Mol. Pharmaceutics 5(1):35-41
Additional ReadingAdditional Reading• Goodwin B., Hodgson E., and Liddle C. (1999) The orphan human
pregnane X receptor mediates the transcriptional activation of CYP3A4 by rifampicin through a distal enhancer module. Mol Pharmacol56:1329-1339.
• Honkakoski P. and Negishi M. (1998) Regulatory DNA elements of phenobarbital-responsive cytochrome P450 CYP2B genes. J Biochem Mol 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, a promiscuous xenobiotic receptor that has diverged during evolution. Mol. Endocrinol. 14: 27-39.
• Wang, H., and LeCluyse E. L. 2003. Role of orphan nuclear receptors in the regulation of drug metabolising enzymes. Clin. Pharmacokinet. 42: 1331-1357.