ENVR 132 TOXC 142 Biochemical Molecular Toxicology Induction of Metabolism by Toxicants Instructor Edward LeCluyse Ph D e mail edl cellzdirect 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 Acetaminophen Toxicity Induced by EtOH and Isoniazid Protein Adducts GSH depletion Phase II conjugates Internal Exposure to Natural and ManMan 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 P450 Enzyme Induction General Principles Xenobiotics can Induce more than one P450 enzyme Induce non P450 proteins Phase II transporters Undergo auto induction Stimulate metabolism of other chemicals Act in more than one particular tissue Common Outcomes of Enzyme 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 Liver Physiology and Function Serrum triazolam ng ml Rifampin Effects on Triazolam Disposition Villikka et al Clin Pharmacol Ther 1997 61 8 14 z Rifampin Placebo Inducible P450 Enzymes in Human Liver P450 Enzyme Inducers CYP1A2 Aromatic hydrocarbons cigarette smoke cruciferous vegetables charbroiled meat CYP2A6 Anticonvulsants rifampin CYP2B6 CYP2C8 CLZ RIF phenytoin Anticonvulsants rifampin CYP2C9 CYP2C19 Rifampin Anticonvulsants Rifampin Anticonvulsants CYP3A4 CYP2E1 CLZ Rifampin Anticonvulsants Isoniazid alcohol CYP2D6 CYP4A11 Non inducible Non inducible Types of Human 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 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 Structural Diversity of CYP3A Inducers HO HO AcO HO OH O OH NH MeO N O O O O O H O O H N OH Lovastatin NMe Rifampicin O Cl N O PO O Et 2 N HO PO O Et HO O OH CH2But 2 F O Clotrimazole SR12813 Dex t butylacetate Factors Influencing P450 Enzyme Induction Dose Concentration of Inducer rifampin 600 mg day 1200 mg day 59 in antipyrine clearance 125 in antipyrine clearance Age as age ability to induce metabolism or Liver Disease as hepatitis cirrhosis ability to induce metabolism Time Course and P450 Enzyme phenobarbital 6 7 days rifampin 2 days Molecular Mechanisms of P450 Enzyme Induction Mechanisms of P450 Induction Receptor mediated transcriptional activation Predominant mechanism Specific nuclear receptors mRNA stabilization Protein stabilization Enzyme Induction General mechanism of enzyme induction Drug DR cytosol Nuclear Receptor DR nucleus Gene transcription CYP1A CYP2B CYP3A CYP2C P gp Mrp2 UGT1A1 mRNA protein activity Nuclear Receptors Involved in P450 Gene Regulation CYP1A Xenobiotic metabolism AhR ARNT PAHs Drugs XRE CYP2B Xenobiotic Steroid metabolism CAR RXR Steroids Drugs PBREM XREM PXR Steroids Drugs CYP3A Xenobiotic Steroid metabolism RXR XREM PXRE Glitazones Fibrates CYP4A rodents Fatty acid metabolism PPAR RXR PPRE A AF 1 DBD LBD AF 2 Carboxy Amino Modulators interact with some cofactors Binding to response elements of target genes Ligand and coactivator binding pockets Recruitment of coactivators B 5 LBD NR LBD RXR LBD DBD DBD DBD 3 Monomers ROR TLX ERR NGFI B 5 NR LBD 3 RXR Heterodimers PXR CAR PPAR LXR FXR RAR 5 DBD NR LBD DBD Homodimers GR COUP TF HNF4 Rev Erb GCNF 3 5 3 n n n DRn IRn 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 TGAACT n 3 TGACCC TGACCT n 3 TGAGCT TGACCT n 4 TGAGTT TGAACT n 3 TGAACT ERs TGGACT n 4 TGAACC TCAACT n 4 TGACAC TCAACT n 4 TGACAC TCAACT n 4 TGACAC NR3 CYP2B6 TGGACT n 4 TGACCC UGT1A1 rMRP2 TGAGTT n 4 TAACCT TGAACT n 8 AGTTCA 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 Other Genes Similar Binding of PXR and CAR to Some Promoter Regions Goodwin et al Mol Pharmacol 2001 Differential Binding of PXR and CAR to Other Promoter Regions XREM 2B6 PXR CAR RXR ER6 3A4 PXR RXR CAR RXR AhR ARNT Aryl hydrocarbon Receptor CYP1A UGT1A1 SULT1A1 XRE DRE CAR RXR CYP2B CYP3A CYP1A CYP2A CYP2Cs OATP2 UGT1A1 MRP2 SULT1A1 Constitutive Androstane Receptor XREM PBREM PXR RXR CYP3A CYP2B CYP2Cs CYP7A MDR1 MRP2 OATP2 GSTA 2 UGT1A1 AldHs Carboxyesterase 2 3 Pregnane X Receptor XREM PXRE colon PBL thymus prostate testis ovary small intestine spleen skeletal muscle pancreas kidney lung liver placenta brain heart Human PXR Tissue Expression 9 5 7 5 4 4 2 4 1 4 PXR Activation by Drugs Nucleus RXR PXR Target gene Cytoplasm Ligand CAR Activation by Drugs Nucleus RXR CAR Target gene CAR Cytoplasm Ligand Nuclear Receptor Binding Sites in the CYP3A4 MDR1 Promoters 7836 CYP3A4 XREM 362 7208 1 CYP3A4 TGAACTtgcTGACCC 7975 TGAACTcaaaggAGGTCA TGAAATcatgtcGGTTCA MDR1 XREM 7013 MDR1 1974 281 bp TGAACTaactTGACCT Nuclear Receptor Binding Sites in the CYP3A4 2B6 Promoters 7836 CYP3A4 XREM 362 7208 1 CYP3A4 TGAACTtgcTGACCC TGAACTcaaaggAGGTCA TGAAATcatgtcGGTTCA 8500 8600 CYP2B6 XREM PBREM 1733 1683 bp NR1 TGGACTttccTGACCC TGTACTttccTGACCC NR2 CYP2B6 Synergy between the Distal and Proximal Enhancer Modules 60 Luciferase activity fold over control 50 40 control PB 30 20 10 0 B 1 6k B 1 6k PBREM B