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ENVR 432/TOXC 442/BIOC442 Biochemical & Molecular Toxicology Induction of Metabolism by Toxicants Instructor: Stephen S. Ferguson, Ph.D. e-mail: [email protected] Induction: Definitions and Principles • The process of increasing the amount or the activity of a protein. • A homeostatic mechanism for regulating enzyme production in a barrier organ, such as the liver, intestine, kidney. • In enzymology, an inducer usually combines with and deactivates/activates a regulatory protein which leads to increased gene expression. P450 Enzyme Induction • Induction can cause marked increases in P450 composition (>20-fold) and chemical clearance or bioactivation. • As a result, induction can increase tolerance to some toxicants while enhancing the toxicity of others. • Induction can decrease the therapeutic effect of drugs by increasing the rate and pattern of metabolism. • Xenobiotics are known to induce enzymes that play a major or no role in their biotransformation (e.g., omeprazole vs. ethanol). Why Is It Important to Assess Enzyme Induction? • Failure of therapy (e.g. OC’s, epilepsy, HIV) Inhibition-Induction • Drug tolerance with auto-induction Toxic / side-effect level • Xenobiotic toxicity potentiated Therapeutic Window (drug efficacy) Concentration • Complicated dosing regimen • Chemical carcinogenesis potentiated • Perturbation of endogenous substrate metabolism/homeostasis • Hepatomegaly & proliferation of cellular ER & peroxisomes Ineffective level Time 6 Invitrogen Proprietary & Confide Internal Exposure to Natural and Man-made Chemicals • • • • • • drugs industrial chemicals pesticides pollutants alkaloids cigarette smoke • cruciferous vegetables (indole-3-carbinol) • secondary plant metabolites • toxins produced by molds, plants, and animals • pyrolysis products in cooked food Types of P450 Inducers • Many “prototypical” inducers of specific families or subfamilies of P450 enzymes – CYP1A inducers: 3-MC, BNF, omeprazole, TCDD – CYP3A inducers: rifampin, dexamethasone, troglitazone – CYP2B inducers: phenobarbital, PCBs, phenytoin – CYP4A inducers: fibrates – CYP2E1 inducers: ethanol, isoniazid • Some overlap in “specificities” of inducers • An inducer for one family of enzymes may also suppress another family (e.g., BNF) Induction of Rat Liver P450 Enzymes by Prototypical Inducers In Vivo In Vivo Induction in Male Rats P450 Enzyme Inducer Control Activity Induced Activity 152 27 3,320 183 24 4 1,460 180 PCN 2,460 780 12,693 2,255 CLO 489 52 10,693 620 CYP1A BNF CYP2B PB CYP3A CYP4A CYP1A, EROD; CYP2B, PROD; CYP3A, testosterone 6b-hydroxylation; CYP4A, lauric acid 12-hydroxylation. Induction and Inhibition of P450 in Mice Treated with PB or SKF525A: [14C-methyl]aminopyrine Serrum triazolam (ng/ml) Rifampin Effects on Triazolam Disposition Villikka et al., Clin Pharmacol Ther 1997;61:8-14. Rifampin Placebo Consequences of Cytochrome P450 Enzyme Induction • Increased toxic effect – Acetaminophen – Bromobenzene, CCl4 Alcohol, 3-MC Phenobarbital • Increased bioactivation – Cyclophosphamide Macrolides, pesticides • Increased tumor formation – Altered disposition of endogenous substrates • Altered cellular and physiological function – proliferation of peroxisomes and SER – increased liver weight – endocrine disruption • Porphyria, chloracne – PCDDs, azobenzenes, biphenyls (PCBs), naphthalene Effects of Inducers on Rodent Liver Physiology and Function Acetaminophen Metabolism and Toxicity HN COCH 3 ~60% HN O COCH 3 O OH CO 2 H OH HO ~35% CYP2E* CYP1A CYP3A N COCH 3 OH HN O COCH 3 SO 3 H *induced by ethanol, isoniazid, phenobarbital Protein adducts, Oxidative stress, Toxicity O NAPQI N-acetyl-p-benzoquinone imine Endocrine Disruption Molecular Mechanisms of P450 Enzyme Induction General 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 Induction General mechanism of hepatic enzyme induction X XR cytosol XR nucleus nucleus Nuclear Receptor cytoplasm Gene transcription mRNA Phase1 Phase 2 transporters protein activity Hepatocyte NR’s and P450 Induction PXR CAR Transcription I SRC-1 RNA poly II RXR NR TFs XREM PBREM Promoter P450 mRNA Translation CYP450 gene P450 Drug DrugOH Increased Drug Metabolism Complex Transcriptional Machinery precursor mRNA mature mRNA mRNA degradation micro RNA protein translation protein folding protein degradation 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 in drug metabolism and excretion (e.g., CYP’s, UGT’s, GST’s, transporter proteins). • These target genes represent redundant but distinct layers of defense. • There are overlapping similarities and distinct differences in species’ response to activators of NR’s. Receptors Involved in the Regulation of CYP Gene Expression Transcription factor Dimerization partner AHR ARNT CAR Examples of ligands Genes Regulated Dioxins, non-ortho PCBs, some PAHs, bilirubin, etc. CYP1A, CYP1B GST, UGT, NQO RXR Phenobarbital (PB), TCPOBOP, chlorinated pesticides, ortho-PCBs, androstanol/ androstenol (inhibits) CYP2B, CYP3A GST, ABC transporters PXR (SXR) RXR CYP3A, CYP2B, CYP7A PB, ortho-PCBs, organochlorine (repression) pesticides, dexamethasone, pregnenalone, corticosterone, bile acids GST, ABC transporters (lithocholic acid) PPAR RXR Fibrate drugs, phthalate esters, linoleic acid, arachidonic acid, CYP4A, CYP7A (repression), CYP8B, LXR, HMGCS2 LXR RXR Cholesterol; (24 S)- hydroxycholesterol CYP7A, ABC transporters, LXR FXR RXR Bile acids, chenodeoxycholic acid Represses CYP7A, BSEP (ABCB11), CYP8B, CYP27A ER ER Structurally diverse xenoestrogens CYP19 Coordinate Regulation of P450’s, UGT’s and Transporters by NR’s UGT’s MRP3 Modified from Kast, H. R. et al. J. Biol. Chem. 277:2908-2915, 2002 What is Relevant Induction? Potency and Efficacy Dose-Response ‘Window’ (Position → potency) Emax Magnitude of Response (Efficacy) 1. Efficacy (e.g. % of PC) EC50 2. Potency (e.g. EC50) PAH Inducers in Rat vs. Human EC50 = 0.00767 +/- 0.00409 EC50 = 0.00767 +/- 0.00409 CYP1A1 mRNA Hu497 Rat TCDD 1A1 mRNA EC50 = 0.0107 +/- 0.043 EC50 10X Difference Relationship between In Vitro Potency and Induction In Vivo EC50 Cmax [Cmax]/EC50 Clinical Relevance Nifedione 8 0.008 0.001 No known Lovastatin 1-6 0.008 0.008-0.002 No known Rosiglitazone 5-10 0.3-1.2 0.05-0.12 No known Simvastatin 0.14 0.024 0.17 No known Troglitazone 3-6 7 2.3 Yes Phenytoin 25 80 3.2 Yes Avasimibe 0.2 1-6 5-30 Yes Rifampicin 0.8 14 17.5 Yes Carbamazepine 0.9 25 28 Yes Clotrimazole 1-5 Topical (Inhibition) [Cmax]/EC50 < 0.1, induction not likely 1< [Cmax]/ EC50 < 0.1, induction possible [Cmax]/ EC50 > 1, induction likely Aryl Hydrocarbon Receptor (AhR) • Aryl hydrocarbon receptor (AHR) is a basic helixloop-helix (bHLH) protein belonging to the PerArnt-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 Pathway Nucleus Cytoplasm X 90 90 X X X 90 90 90 90 90 90 L L L AhR L or L Arnt L From: Anne Mullen, Advanced Pharmacology, McMaster University, Ontario, CA AhR Signaling Pathway AhR/Arnt heterodimer Increased expression CYP1A1 protein + mRNA Translation IC XRE promoter gene (CYP1A1) TNGCGTG Increased expression of other gene products Nuclear Hormone Receptors AF-1 DBD LBD AF-2 Carboxy Amino Modulators interact with some cofactors Binding to response elements of target genes LBD 5’ DBD 3’ Monomers ROR TLX ERR NGFI-B 5’ NR-LBD RXR-LBD DBD DBD Ligand and coactivator binding pockets 3’ RXR Heterodimers PXR CAR PPAR LXR FXR RAR 5’ Translocase activity NR-LBD NR-LBD DBD DBD Homodimers GR ER RXR COUP-TF HNF4 Rev-Erb GCNF 3’ 5’ 3’ n n DRn IRn n ERn CYP2B Response elements CYP3A Response elements NR1s DRs CYP2B6 CYP2b10 CYP2B1 CYP2B2 TGTACT n=4 TGACCC TGTACT n=4 TGACCT TCTACT n=4 TGACCT TGTACT n=5 TGACCT CYP3A4 CYP3A2 CYP3A23 CYP3A2 NR2s CYP2B6 CYP2b10 CYP2B1 CYP2B2 ERs TGGACT n=4 TGAACC TCAACT n=4 TGACAC TCAACT n=4 TGACAC TCAACT n=4 TGACAC NR3 CYP2B6 TGAACT n=3 TGACCC TGACCT n=3 TGAGCT TGACCT n=4 TGAGTT TGAACT n=3 TGAACT CYP3A4 CYP3A4 CYP3A23 CYP3A5 CYP3A7 CYP3A7 TGAAAT n=6 GGTTCA TGAACT n=6 AGGTCA TTAACT n=6 AGGTCA TGAACT n=6 AGGTAA TTAACT n=6 AGGTCA TGAAAT n=6 AGTTCA MDR1 CYP2C9 TGAGAT n=6 AGTTCA CAAACT n=4 TGACCT TGGACT n=4 TGACCC Other Genes UGT1A1 rMRP2 TGAGTT n=4 TAACCT TGAACT n=8 AGTTCA Nuclear Receptor PXR PB RIF ? PXR translocation? -mouse-yes -human-no cytoplasm RXR nucleus XREM Human Rat Mouse Activator/Agonist RIF PCN PCN CYP3A CYP Target CYP3A4 CYP3A1/2 Cyp3a11 Nuclear Receptor CAR: PB Induction-Constitutively Active PB ? CAR CCRP CCRP PP2A cytoplasm nucleus PBREM Human Rat Mouse Activator/Agonist CITCO, PB, DPH PB, TCPOBOP PB, TCPOBOP CYP2B Inhibitor/Antagonist Clotrimazole?, Miclizine? Androstenol Androstenol In cell lines spontaneously translocates to the nucleus CYP Target CYP2B6 CYP2B1 Cyp2b10 Similar Binding of PXR and CAR to Promoter Response Elements Goodwin et al., Mol. Pharmacol., 2001 Differential Binding of PXR and CAR to Other Promoter Regions NR3-2B6 PXR CAR RXR + + + + ER6-3A4 + + + + + + + + + + + + + + + + + + + + PXR/RXR CAR/RXR GR/Dex Role in Basal & Induced P450 Expression via CAR/PXR (master regulator) Role of CAR/PXR in lipid metabolism, synthesis, and uptake Moreau et al. 2007 Mol. Pharmaceutics PXR & CAR role in Glucose Homeostasis Molecular Basis for the Species Differences in Enzyme Induction 10mM SR12813 10mM DTBA 5mM PCN 10mM Rifampicin 0.1% DMSO Species Differences in the Regulation of CYP3A Enzymes CYP3A4 Human CYP3A6 Rabbit CYP3A23 Rat Species Differences in CYP2B Induction by Phenobarbital Species Differences in CYP1A Induction by Xenobiotics Phenacetin O-Dealkylation (pmol/min/mg) Treatment with Drug 'X' 1.2 300 200 100 1% 0. ( l tr o DM ) SO M 3- C 2µ M ug Dr 2µ 0. ' 'X M ug Dr µ '2 'X ug Dr µ '6 'X ug Dr '2 'X M 0µ 'X '2 0µ M 'X '6 µM ru g D ru g 'X '2 µM g D M D g ru D M ru C 'X '0 .6µ M 2µ M SO ) DM Co nt ro D D ru g ru g C 3M DM 0. 1% Co nt ro l( 0.2 0.0 n Co 400 0 'X '0 .6 µM 1µ M 0 0.4 500 l( 0. 1% 100 600 'X '2 0µ M 200 0.6 g 300 ru 400 0.8 D 500 'X '6 µM 600 ru g 700 1.0 D 800 'X '2 µM CYP1A2 Activity (nmol/min/mg) 900 3M 1000 SO ) Phenacetin O-Dealkylation (pmol/min/mg) CYP1A1/2 Activity in Rat Hepatocytes a Function CYP1A Activity Dog Hepatocytes as a Function of CYP1A2asActivity in Human Hepatocytes as a inFunction of of Treatment with Drug 'X' Treatment with Drug 'X' Species Differences in CYP4A Induction by Clofibric Acid Rat Hepatocytes Human Hepatocytes Rat 40 30 20 50 Human 4 40 CYP4A11 Fold Induction 50 60 CYP4A11 Fold Induction acid 12-hydroxylation LauricCYP4A1 Fold Induction 60 30 20 3 2 1 0 10 10 CTL 1 10 100 500 1000 Clofibric Acid (µM) 0 0 CTL 1 10 100 500 Clofibric Acid (µM) 1000 CTL 1 10 100 500 Clofibric Acid (µM) 1000 Observations 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 speciesspecific responses? • What is the significance of these differences to predicting human toxicity? Transfection Assay for P450 Enzyme Induction CV-1 HuH7 cell PXR RXR PXR Expression Plasmid RXR PXR PXRE Reporter Gene Reporter Plasmid Drug Differential Activation of Human, Rabbit, and Rat PXR by CYP3A Inducers O H CN HO AcO PCN HO HO OH O OH MeO rifampicin NH N O N HO O NMe OH O O O H O O H lovastatin Cl N N clotrimazole 0 20 40 60 80 100 300 350 Normalized Reporter Activity 400 PXR Sequence Homology 1 41 141 1 41 Rabbit PXR1 107 141 38 104 138 96 Rat PXR1 1 38 1 104 37 Xenopus ONR1 138 102 24 431 76 136 69 1 431 76 96 Mouse PXR1 434 82 94 1 434 Ligand DNA Human PXR1 Human VDR 107 386 42 89 122 63 427 37 Variation in ligand binding domain consistent with in vivo species differences in response to inducers Amino Acid Differences in the Ligand Binding Domain of PXR Val184 Val210 Glu263 Glu333 His260 Arg333 Thr414 rPXR Asp178 Ser203 Phe184 Leu210 Asp263 Lys334 Tyr260 Arg333 Ser414 mPXR Gly178 Arg203 Ser187 Leu213 Asp266 Glu337 Tyr263 His333 hPXR Gly181 Leu206 Zhang et al., Arch. Biochem. Biophys., 1999 Ile417 Rat CYP4A1 Response Elements -10kb -2kb +1 (gene) Rat CYP4A1 -4850 -4466 DR1 (9/12) 384 bp DR1 (9/12) ATTTAAGGAAAgGGGTCAGACC------AACTAGGGTAaAGTTCAGTG Element 1 not functional Element 2 is a Functional PPRE Proximal PPRE Identified by Aldridge et. al. Biochem. J. 306, 473-479, 1995 Analysis of the Human CYP4A11 Gene Kawashima et. al., Archives of Biochemistry and Biophysics (2000) 378(2), 333-339 Sequenced -2251 bp upstream of gene, no PPRE identified. DR1 (8/12) AAAAGTGGGCAAAGGATATGCA -7238 -10kb -7217 -7kb -5kb -2kb +1 Human CYP411 -4493 -4472 DR1 (8/12) AAACAAGGGAATAGCCCAAAAG Upstream analysis of the CYP4A11 gene located on chromosome 1 revealed two possible PPRE’s Gel Shift Assay Rat PPARa + - .5 RXRa - + .5 PPRE/PPARa/RXR Human -4.5 kb 1 2 1 2 + - + .5 1 2 .5 1 2 Human -7.5 kb + - + .5 1 2 .5 1 2 Summary • 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 ligandbinding domain of the receptors are mainly responsible for the species differences in the induction 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 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. Additional 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 Pharmacol 56:1329-1339. • 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.