ENVR 132 TOXC 142 BIOC142 Biochemical Molecular Toxicology Induction of Metabolism by Toxicants Instructor Edward LeCluyse Ph D e mail ed lecluyse 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 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 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 CYP1A BNF CYP2B PB CYP3A CYP4A 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 EROD CYP2B PROD CYP3A testosterone 6 hydroxylation CYP4A lauric acid 12 hydroxylation Induction and Inhibition of P450 in Mice Treated with PB or SKF525A 14C methyl aminopyrine Unwanted Side effects of Enzyme Induction Failure of drug therapy Drug tolerance with auto induction Xenobiotic toxicity potentiated Chemical carcinogenesis potentiated Endogenous substrate metabolism perturbed Proliferation of cellular ER and peroxisomes 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 60 35 CYP2E CYP1A CYP3A induced by ethanol isoniazid phenobarbital Protein adducts Oxidative stress Toxicity NAPQI N acetyl p benzoquinone imine Endocrine Disruption UG T1 A Molecular Mechanisms of P450 Enzyme 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 to a receptor mRNA stabilization Protein stabilization Enzyme Induction General mechanism of enzyme induction Drug DR cytosol DR nucleus Nuclear Receptor Gene transcription mRNA Phase1 Phase 2 transporters protein activity Enzyme Induction General mechanism of enzyme induction X XR cytosol XR nucleus Gene transcription nucleus Nuclear Receptor cytoplasm mRNA Phase1 Phase 2 transporters protein activity Hepatocyte 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 transporters These target genes represent redundant but distinct layers of defense There are distinct and overlapping species differences in 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 UGT s MRP3 Modified from Kast H R et al J Biol Chem 277 2908 2915 2002 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 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 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 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 TGGACT n 4 TGACCC 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 UGT1A1 rMRP2 TGAGTT n 4 TAACCT TGAACT n 8 AGTTCA 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 Molecular Basis