ENVR 132/TOXC 142/BIOC142 Biochemical & Molecular Toxicology Induction of Metabolism by Toxicants Instructor: Stephen S. Ferguson, Ph.D. e-mail: stephen.ferguson@lifetech.com

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) Drug tolerance with auto-induction Xenobiotic toxicity potentiated Complicated dosing regimen Chemical carcinogenesis potentiated Perturbation of endogenous substrate metabolism/homeostasis Hepatomegaly & proliferation of cellular ER & peroxisomes

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 Control Activity Induced Activity CYP1A BNF 152  27 3,320  183 CYP2B PB 24  4 1,460  180 CYP3A PCN 2,460  780 12,693  2,255 CYP4A CLO 489  52 10,693  620 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

Rifampin Effects on Triazolam Disposition  Placebo Serrum triazolam (ng/ml) Villikka et al., Clin Pharmacol Ther 1997;61:8-14.

Consequences of Cytochrome P450 Enzyme Induction Increased toxic effect Acetaminophen Alcohol, 3-MC Bromobenzene, CCl4 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 ~60% ~35% CYP2E* CYP1A CYP3A *induced by ethanol, isoniazid, phenobarbital Protein adducts, Oxidative stress, Toxicity NAPQI N-acetyl-p-benzoquinone imine

Endocrine Disruption UGT1A

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 protein activity mRNA Gene transcription X Nuclear Receptor XR cytosol XR nucleus Phase1 Phase 2 transporters cytoplasm nucleus Hepatocyte

NR’s and P450 Induction P450 mRNA Transcription I Translation XREM CAR PXR RNA poly II SRC-1 Transcription P450 mRNA I TFs RXR NR Translation P450 XREM PBREM Promoter CYP450 gene Drug-OH Drug 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

Coordinate Regulation of P450’s, UGT’s and Transporters by NR’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) Efficacy (e.g. % of PC) Potency (e.g. EC50) 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 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 Pathway Cytoplasm Nucleus 90 X L 90 X AhR 90 X L or 90 X Arnt L From: Anne Mullen, Advanced Pharmacology, McMaster University, Ontario, CA

AhR Signaling Pathway + + AhR/Arnt heterodimer Increased expression CYP1A1 protein Increased expression of other gene products Translation mRNA IC XRE promoter gene (CYP1A1) TNGCGTG

Nuclear Hormone Receptors AF-1 DBD LBD AF-2 Amino Carboxy Modulators interact with some cofactors Binding to response elements of target genes Ligand and coactivator binding pockets Translocase activity LBD NR-LBD RXR-LBD NR-LBD NR-LBD DBD DBD DBD DBD DBD 5’ 3’ 5’ 3’ 5’ 3’ Monomers RXR Heterodimers Homodimers ROR TLX ERR NGFI-B PXR CAR PPAR LXR FXR RAR GR ER RXR COUP-TF HNF4 Rev-Erb GCNF

n DRn IRn ERn CYP2B Response elements CYP3A Response elements 5’ 3’ n DRn IRn ERn CYP2B Response elements CYP3A Response elements NR1s DRs CYP2B6 TGTACT n=4 TGACCC CYP2b10 TGTACT n=4 TGACCT CYP2B1 TCTACT n=4 TGACCT CYP2B2 TGTACT n=5 TGACCT CYP3A4 TGAACT n=3 TGACCC CYP3A2 TGACCT n=3 TGAGCT CYP3A23 TGACCT n=4 TGAGTT CYP3A2 TGAACT n=3 TGAACT NR2s ERs CYP2B6 TGGACT n=4 TGAACC CYP2b10 TCAACT n=4 TGACAC CYP2B1 TCAACT n=4 TGACAC CYP2B2 TCAACT n=4 TGACAC CYP3A4 TGAAAT n=6 GGTTCA CYP3A4 TGAACT n=6 AGGTCA CYP3A23 TTAACT n=6 AGGTCA CYP3A5 TGAACT n=6 AGGTAA CYP3A7 TTAACT n=6 AGGTCA CYP3A7 TGAAAT n=6 AGTTCA NR3 CYP2B6 TGGACT n=4 TGACCC Other Genes UGT1A1 TGAGTT n=4 TAACCT MDR1 TGAGAT n=6 AGTTCA rMRP2 TGAACT n=8 AGTTCA CYP2C9 CAAACT n=4 TGACCT

Nuclear Receptor PXR ? cytoplasm nucleus Activator/Agonist CYP Target RIF PB ? PXR HSP90 translocation? -mouse-yes -human-no PXR cytoplasm RXR nucleus CAR RXR Receptor Conserved across species, but there are differences PXR XREM CYP3A Activator/Agonist CYP Target Human RIF CYP3A4 Rat PCN CYP3A1/2 Mouse PCN Cyp3a11

Nuclear Receptor CAR: PB Induction-Constitutively Active ? HSP90 CAR CCRP CCRP PP2A HSP90 OA CAR cytoplasm nucleus RXR In cell lines spontaneously translocates to the nucleus RXR CAR PBREM CYP2B Emphasize Constitutive Activity and Translocation…need inhibitors and sequestration to study properly Activator/Agonist Inhibitor/Antagonist CYP Target Human CITCO, PB, DPH Clotrimazole?, Miclizine? CYP2B6 Rat PB, TCPOBOP Androstenol CYP2B1 Mouse PB, TCPOBOP Androstenol 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 ER6-3A4 PXR + + + + + + CAR + + + + + + RXR + + + + + + + + + + + + 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

Species Differences in the Regulation of CYP3A Enzymes 10mM Rifampicin 10mM SR12813 0.1% DMSO 10mM DTBA 5mM PCN CYP3A4 Human CYP3A6 Rabbit CYP3A23 Rat

Species Differences in CYP2B Induction by Phenobarbital

Species Differences in CYP1A Induction by Xenobiotics

Species Differences in CYP4A Induction by Clofibric Acid Rat Hepatocytes Human Hepatocytes Rat Human Lauric acid 12-hydroxylation Data are mean plus or minus standard deviation of three replicates in the TaqMan assay. Rat data are consistent with literature reports of EC50 for rat CYP4A induction by clofibric acid. Human data are consistent with literature reports of EC50 for human CYP4A induction by clofibric acid. Need to perform this experiment in dog hepatocytes when CYP4A primers and probes are validated.

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 species-specific 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

Normalized Reporter Activity Differential Activation of Human, Rabbit, and Rat PXR by CYP3A Inducers PCN rifampicin lovastatin clotrimazole 20 40 60 80 100 300 350 400 Normalized Reporter Activity

PXR Sequence Homology 1 41 107 141 434 Human PXR1 DNA Ligand 1 41 107 141 434 Variation in ligand binding domain consistent with in vivo species differ-ences in response to inducers Rabbit PXR1 94 82 1 38 104 138 431 Rat PXR1 96 76 1 38 104 138 431 Mouse PXR1 96 76 1 37 102 136 386 Xenopus ONR1 69 42 1 24 89 122 427 Human VDR 63 37

Amino Acid Differences in the Ligand Binding Domain of PXR Val184 Val210 Glu263 Glu333 Thr414 rPXR Asp178 Ser203 His260 Arg333 Phe184 Leu210 Asp263 Lys334 Ser414 mPXR Gly178 Arg203 Tyr260 Arg333 Ser187 Leu213 Asp266 Glu337 Ile417 hPXR Gly181 Leu206 Tyr263 His333 Zhang et al., Arch. Biochem. Biophys., 1999

Rat CYP4A1 Response Elements -10kb -2kb +1 (gene) Rat CYP4A1 -4850 -4466 DR1 (9/12) 384 bp DR1 (9/12) ATTTAAGGAAAgGGGTCAGACC------AACTAGGGTAaAGTTCAGTG In 1995 Aldridge identified two response elements approximately 4 Kb upstream of the CYP4A1 gene. The authors were able to show through gel shift and transfection studies (review this paper) that the more distal response element was non functional whereas the proximal element was functional and was necessary for induction of rat CYP4A1. 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. +1 Human CYP411 -2kb -5kb AAACAAGGGAATAGCCCAAAAG -4493 DR1 (8/12) -4472 -7kb -10kb AAAAGTGGGCAAAGGATATGCA -7238 -7217 While the CYP4A11 gene sequence was reported in 2000, Kawashima was not able to identify a PPRE upstream of the CYP4A11 gene. However, at the time of the report, data from the human genome project was not as accessible as it is today. Analysis of the upstream region of human CYP4A11 on chromosome 1 reveals several potential PPREs with the two best (according to consensus) shown here. Interestingly, there is a potential response element in the same region as the two rat response elements, however a second element is not apparent in the human sequence. Upstream analysis of the CYP4A11 gene located on chromosome 1 revealed two possible PPRE’s

Gel Shift Assay PPARa + - .5 1 2 + - .5 1 2 + - .5 1 2 RXRa - + .5 1 2 Rat Human -4.5 kb Human -7.5 kb PPARa + - .5 1 2 + - .5 1 2 + - .5 1 2 RXRa - + .5 1 2 - + .5 1 2 - + .5 1 2 PPRE/PPARa/RXR Data are mean plus or minus standard deviation of three replicates in the TaqMan assay. Rat data are consistent with literature reports of EC50 for rat CYP4A induction by clofibric acid. Human data are consistent with literature reports of EC50 for human CYP4A induction by clofibric acid. Need to perform this experiment in dog hepatocytes when CYP4A primers and probes are validated.

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 ligand-binding 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.