ENVR 132/TOXC 142ENVR 132/TOXC 142Biochemical & Molecular Biochemical & Molecular ToxicologyToxicologyInduction of Metabolism by ToxicantsInduction of Metabolism by ToxicantsInstructor:Edward LeCluyse, Ph.D.e-mail: [email protected]: Definitions and Induction: Definitions and PrinciplesPrinciples• 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 InductionP450 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 ToxicityAcetaminophen ToxicityProtein Adducts,GSH depletionPhase IIconjugatesInduced byEtOH andIsoniazidInternal Exposure to Natural andInternal Exposure to Natural andManMan--made Chemicalsmade 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 foodP450 Enzyme Induction:P450 Enzyme Induction:General PrinciplesGeneral 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 tissueCommon Outcomes of Common Outcomes of Enzyme InductionEnzyme 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 peroxisomesEffects of Inducers on Rodent Liver Effects of Inducers on Rodent Liver Physiology and FunctionPhysiology and FunctionSerrum triazolam (ng/ml)Rifampin Effects on Triazolam DispositionRifampin Effects on Triazolam DispositionVillikka et al., Clin Pharmacol Ther 1997;61:8-14.z Rifampin{ PlaceboInducible P450 Enzymes in Human LiverInducible P450 Enzymes in Human LiverP450 Enzyme InducersCYP1A2 Aromatic hydrocarbons, cigarette smoke,cruciferous vegetables, charbroiled meatCYP2A6 Anticonvulsants = rifampinCYP2B6 CLZ >> RIF ≥ phenytoin CYP2C8 Anticonvulsants > rifampinCYP2C9 Rifampin > AnticonvulsantsCYP2C19 Rifampin >> AnticonvulsantsCYP3A4 CLZ≈ Rifampin > AnticonvulsantsCYP2E1 Isoniazid, alcoholCYP2D6 Non-inducibleCYP4A11 Non-inducible (?)Types of Human P450 InducersTypes 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 InducersStructural Diversity of CYP3A InducersStructural Diversity of CYP3A InducersOHOHONNNMeNHOOOHOAcOMeOOHHOPO (O Et)2PO (O Et)2HOSR12813OHOCH2ButOOFHOODex-t-butylacetateRifampicinLovastatinOOHOHO OHClotrimazoleNNClOHOHONNNMeNHOOOHOAcOMeOOHHOPO (O Et)2PO (O Et)2HOSR12813OHOCH2ButOOFHOODex-t-butylacetateRifampicinLovastatinOOHOHO OHClotrimazoleNNClFactors Influencing P450 Enzyme Factors Influencing P450 Enzyme InductionInduction• Dose/Concentration of Inducer rifampin 600 mg/day 59% ↑ in antipyrine clearance1200 mg/day 125% ↑ in antipyrine clearance•Ageas age ↑, ability to induce metabolism ↔ or ↓• Liver Diseaseas hepatitis/cirrhosis ↑, ability to induce metabolism ↓• Time Course and P450 Enzymephenobarbital - 6-7 daysrifampin - 2 daysMolecular Mechanisms of P450 Molecular Mechanisms of P450 Enzyme InductionEnzyme InductionMechanisms of P450 InductionMechanisms of P450 Induction• Receptor-mediated transcriptional activation– Predominant mechanism– Specific nuclear receptors• mRNA stabilization• Protein stabilizationEnzyme InductionEnzyme InductionGeneral mechanism of enzyme inductionGeneral mechanism of enzyme inductionproteinproteinactivityactivitymRNAmRNAGene transcriptionGene transcriptionDrugDrugNuclear ReceptorNuclear ReceptorDDRRcytosolcytosolDDRRnucleusnucleusCYP1ACYP1ACYP2BCYP2BCYP3ACYP3ACYP2CCYP2CPP--gpgpMrp2Mrp2UGT1A1UGT1A1Nuclear Receptors Involved in Nuclear Receptors Involved in P450 Gene RegulationP450 Gene RegulationCYP4A (rodents)Fatty acid metabolismRXRPPARαCYP2BXenobiotic, Steroid metabolismRXRCARSteroids, DrugsGlitazones, FibratesCYP3AXenobiotic, Steroid metabolismRXRPXRSteroids, DrugsCYP1AXenobiotic metabolismAhRARNTPAHs, DrugsXREPBREM/XREMXREM/PXREPPREAminoCarboxyAF-1 DBD LBD AF-2Modulators interactwith some cofactorsBinding to responseelements of target genesLigand and coactivatorbinding pocketsRecruitment ofcoactivatorsA5’ 3’ 5’ 3’ 5’ 3’Monomers RXR Heterodimers HomodimersLBDDBDNR-LBDRXR-LBDDBD DBD DBD DBDNR-LBD NR-LBDRORTLXERRNGFI-BPXRCARPPARLXRFXRRARGRCOUP-TFHNF4Rev-ErbGCNFB5’ 3’nnnDRnIRnERnCYP2B Response elementsCYP2B6 TGTACT n=4 TGACCC CYP2b10 TGTACT n=4 TGACCTCYP2B1 TCTACT n=4 TGACCT CYP2B2 TGTACT n=5 TGACCTNR1sCYP2B6 TGGACT n=4 TGAACCCYP2b10 TCAACT n=4 TGACACCYP2B1 TCAACT n=4 TGACAC CYP2B2 TCAACT n=4 TGACACNR2sNR3CYP2B6 TGGACT n=4 TGACCC CYP3A Response elementsCYP3A4 TGAACT n=3 TGACCC CYP3A2 TGACCT n=3 TGAGCTCYP3A23 TGACCT n=4 TGAGTT CYP3A2 TGAACT n=3 TGAACTDRsCYP3A4 TGAAAT n=6 GGTTCA CYP3A4 TGAACT n=6 AGGTCACYP3A23 TTAACT n=6 AGGTCA CYP3A5 TGAACT n=6 AGGTAACYP3A7 TTAACT n=6 AGGTCA CYP3A7 TGAAAT n=6 AGTTCAERsOther GenesUGT1A1 TGAGTT n=4 TAACCT MDR1 TGAGAT n=6 AGTTCArMRP2 TGAACT n=8 AGTTCA CYP2C9 CAAACT n=4 TGACCTSimilar Binding of PXR and CAR Similar Binding of PXR and CAR to