Metabolism of XenobioticsPhase I reactionsOverview of oxidations, reductions, hydrolysesOxidation reactionsSlide 5Slide 6Slide 7ReductionHydrolysisPrincipal Phase I enzymesCytochrome P450Slide 12Overall reactionSlide 14Ferric protoporphyrin IXProtoporphyrin IXCatalytic cycle of cytochrome P450Slide 18P450 and reductase in endoplasmic reticulumThe P450 gene superfamilySub- Family family GeneSlide 22Slide 23Slide 24Sub- Family family GeneSlide 26Slide 27Slide 28Changes in P450 levels with age RatsFlavin monooxygenaseSlide 31Monoamine oxidaseEsterasesAmidasesHydrolasesReductases, dehydrogenases, oxidasesMetabolism of XenobioticsII. Phase 1 MetabolismAug 31, 2010L.M. BallRosenau [email protected]/TOXC 442 Fall 2010Phase I reactions•Chemical modification of xenobiotics•Introduces or uncovers polar functional groups that provide sites for Phase II metabolism•Major classes of reaction:–Oxidation–Reduction–HydrolysisOverview of oxidations, reductions, hydrolyses•Oxidation–Loss of electrons M M+ + e-–Gain of oxygen R + O ROOxidation reactionsHydroxylationStyrene Styrene oxideHCCH2HC CH2OEpoxidationOHHBenzo[a]pyreneBenzo[a]pyrene 7,8-oxideDemethylationDeethylationONH2CCH3H C CH3OOHNH C CH3O+PhenacetinAcetaminophenC CH3oHAcetaldehydeOCH3OHCOHH+AnisolePhenolFormaldehydeOverview of oxidations, reductions, hydrolyses•Reduction–Gain of electrons M+ + e- M–Loss of oxygen RO R + O–Gain of hydrogen R + H RHReduction•Nitro to amino group•Chromium VI to Chromium IIINONOHNO2NHHHCr6+ + 3 e- Cr3+Hydrolysis•Addition of water–Cleavage of R-O or R-N bond accompanied by addition of H2OR’-O-R + H2O R’-O-H + R-OHR’-N-R + H2O R’-N-H + R-OH H HPrincipal Phase I enzymes•Cytochrome P450•Flavin monooxygenase•Monoamine oxidase•Esterases•Amidases•Hydrolases•Reductases, dehydrogenases, oxidasesCytochrome P450•Heme protein•Terminal oxidase of the mixed-function oxidase (MFO) electron-transfer system•Located in the smooth endoplasmic reticulum of all major organs and tissues•Uses NADPH as a source of reducing equivalents•InducibleCytochrome P450•Heme protein•Terminal oxidase of the mixed-function oxidase (MFO) electron-transfer system•Located in the smooth endoplasmic reticulum of all major organs and tissues•Uses NADPH as a source of reducing equivalents•InducibleOverall reactionR-H + O2 + NADPH + H+R-OH + H2O + NADP+Ferric protoporphyrin IXNN-N-NFe+3CH3HCCH2H3CCH3CH3CHCH2CH2H2CCOOHH2CH2CCOOHProtoporphyrin IXCatalytic cycle of cytochrome P450from NADPH-cytC reductase Fe3+Fe3+-RHFe2+-RH O2[Fe2+-RH] HO2-[Fe2+-RH] HO22-Fe3+-RH+ RH+ e-+O2ROHH+ + e-NADPHNADHH2OH2O2H+H+O2-.P450 and reductase in endoplasmic reticulumThe P450 gene superfamily•Format of nomenclature: CYPFamily/Subfamily/Gene•Family = 1, 2, …150 and counting–~40% aa similarity•Subfamily = A, B,…H…–55-65% aa similarity•Gene = 1, 2..10 or above–>97% aa similarity (allelic variants)•Families grouped in ClansSub-Family family GeneCYP1 A 1 BaP hydroxylation, O-deethyl’n(PAC-inducible 2 N-hydroxylation, O-deethylationCYP2 A 1 Testosterone 7-hydroxylation2 Testosterone 15-hydroxylation B 1 Aliphatic hydroxylation2 O-deethylation C 1 - 20+2C19, mephenytoin hydroxylaseDemethylationDeethylationONH2CCH3H C CH3OOHNH C CH3O+PhenacetinAcetaminophenC CH3oHAcetaldehydeOCH3OHCOHH+AnisolePhenolFormaldehydeSub-Family family GeneCYP1 A 1 BaP hydroxylation, O-deethyl’n(PAC-inducible 2 N-hydroxylation, O-deethylationCYP2 A 1 Testosterone 7-hydroxylation2 Testosterone 15-hydroxylation B 1 Aliphatic hydroxylation2 O-deethylation C 1 - 20+2C19, mephenytoin hydroxylaseSub- Family family GeneCYP2 D 1 - 6+2D6, debrisoquine hydroxylase E 1 C- and N-hydroxylationsmall molecules2 F 1CYP3 A 1-43A4CYP4 A 1 Lauric acid - and -1 hydroxylationSub-Family family GeneCYP11 (mito) A 1 Steroid 11-hydroxylationCYP17 A 1 Steroid 17-hydroxylationCYP21 A 1 Steroid 21-hydroxylationCYP51 A 1 (Plants, yeast)CYP52-66 A Yeasts, fungiCYP71-99, 701 PlantsCYP101 A 1 Pseudomonas putida P450camCYP102-132 A BacteriaChanges in P450 levels with ageRats2A12C63A2M: 2C6, 2C11, 3A2F: 2A1, 2C6, 2C12Flavin monooxygenase•Flavoprotein•Mixed-function amine oxidase•Located in smooth endoplasmic reticulum, in human, pig, rabbit liver, guinea-pig lung, human kidney•Uses NADPH as a source of reducing equivalents•Not inducibleOverall reactionR-H + O2 + NADPH + H+R-OH + H2O + NADP+Monoamine oxidase•Metabolizes endogenous monoamine neurotransmitters•Uses NADPH as a source of reducing equivalents•Found in the endoplasmic reticulum and in mitochondria, of nerve endings and liverEsterases•Hydrolyse esters to carboxylic acid and alcohol functional groups•Non-specific esterases in plasma, more substrate-specific forms in liver cytosolH3CCOH2CCH3OH3CCOOHHOH2CCH3+Ethyl acetateAcetic acidEthanol+ H2OAmidases•Hydrolyse amides to carboxylic acids and amines (or ammonia)•Found in plasma and in liver cytosolRCONHHR COOH+H NHH+ H2OHydrolases•Hydrolyse ethersH3CH2COH2CCH3H3CH2COH HOH2CCH3++ H2ODiethyl etherEthanol EthanolReductases, dehydrogenases, oxidases•In cytosol, endoplasmic reticulum, mitochondriaH3C C OHHHH3C COHH3C COOHEthanolAcetaldehydeAcetic acidNAD+NADH + H+NAD+NADH + H+Alcohol dehydrogenase Aldehyde
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