ENVR 132/TOXC 142/BIOC142ENVR 132/TOXC 142/BIOC142Biochemical & Molecular ToxicologyBiochemical & Molecular ToxicologyInduction of Metabolism by ToxicantsInduction of Metabolism by ToxicantsInstructor:Stephen S. Ferguson, Ph.D.e-mail: [email protected]: Definitions and PrinciplesInduction: 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 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).Invitrogen Proprietary & Confid6Inhibition-InductionTimeConcentrationIneffective levelTherapeutic Window(drug efficacy)Toxic / side-effect levelWhy Is It Important to Assess Enzyme Why Is It Important to Assess Enzyme Induction?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 & peroxisomesInternal 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 foodTypes of P450 InducersTypes 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 Induction of Rat Liver P450 Enzymes by Prototypical Inducers Prototypical Inducers In VivoIn VivoCLOPCNPBBNFInducer10,693 ± 620489 ± 52CYP4A12,693 ± 2,2552,460 ± 780CYP3A1,460 ± 18024 ± 4CYP2B3,320 ± 183152 ± 27CYP1AInduced ActivityControl ActivityIn Vivo Induction in Male RatsP450 EnzymeCYP1A, EROD; CYP2B, PROD; CYP3A, testosterone 6β-hydroxylation;CYP4A, lauric acid 12-hydroxylation.Induction and Inhibition of P450 in Mice Treated Induction and Inhibition of P450 in Mice Treated with PB or SKF525A: [with PB or SKF525A: [1414CC--methyl]aminopyrinemethyl]aminopyrineSerrum triazolam (ng/ml)Rifampin Effects on Triazolam DispositionRifampin Effects on Triazolam DispositionVillikka et al., Clin Pharmacol Ther 1997;61:8-14.z Rifampin{ PlaceboConsequences of Cytochrome P450 Enzyme InductionConsequences of Cytochrome Consequences of Cytochrome P450 Enzyme InductionP450 Enzyme Induction• Increased toxic effect– Acetaminophen Alcohol, 3-MC– Bromobenzene, CCl4Phenobarbital• 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), naphthaleneEffects of Inducers on Rodent Liver Effects of Inducers on Rodent Liver Physiology and FunctionPhysiology and FunctionAcetaminophen Metabolism and ToxicityAcetaminophen Metabolism and Toxicity~60%~35%CYP2E*CYP1A CYP3A NAPQIN-acetyl-p-benzoquinone imine*induced by ethanol, isoniazid, phenobarbital Protein adducts,Oxidative stress,ToxicityHNCOCH3OHHNCOCH3OSO3H HNCOCH3OOCO2HOHOHHONOCOCH3Endocrine Disruption Endocrine Disruption • Many xenobiotics can mimic certain hormones and bind to target cellular sites receptive to natural hormones• Modes of endocrine disruption can result from agonistic or antagonistic receptor binding affecting biosynthesis, transport, storage, release, and clearance of hormones• Some pesticides have been identified as endocrine disruptors, in particular the thyroid hormone can be affected by: acetochlor, alachlor, fipronil (Frontline), heptachlor, maneb, methomyl, and zineb• PCBs, mercury, pentachlorophenol are some of the thyroid hormone disruptors that are no longer used as pesticides• DDT, dieldrin, lindane, methoxychlor, triadimefon are thought to be estrogenic-type environmental endocrine disruptors (EEDs) while atrazine, vinclozolin, and procymidone are thought to be andogenic EEDsUGT1AMolecular Mechanisms of P450 Molecular Mechanisms of P450 Enzyme InductionEnzyme InductionGeneral Mechanisms of P450 InductionGeneral 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 stabilizationCoordinates: Kumar R, Thompson EB (1999). "The structure of the nuclear hormone receptors". Steroids 64 (5): 310–9Enzyme InductionEnzyme InductionGeneral mechanism of hepatic enzyme inductionGeneral mechanism of hepatic enzyme inductionproteinproteinactivityactivitymRNAmRNAGene transcriptionGene transcriptionXXNuclear ReceptorNuclear ReceptorXXRRcytosolcytosolXXRRnucleusnucleusPhase1Phase1Phase 2 Phase 2 transporterstransporterscytoplasmnucleusHepatocyteNRNR’’s and P450 Inductions and P450 InductionCYP450 genePromoter XREM PBREM RNA poly IITranscriptionP450mRNATranslationP450Increased Drug MetabolismDrug-OHDrugTFsPXRCARRXR NRSRC-1IComplex Transcriptional MachineryComplex Transcriptional Machineryprecursor mRNAmature mRNAmRNA
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