1Metabolism of XenobioticsSuggested Reading:• Hodgson E, Goldstein J.A. Metabolism of Toxicants Phase I Reactions and Pharmacogenetics. In: Hodgson E , Smart R.C. INTRODUCTION TO BIOCHEMICAL TOXICOLOGY 3rded. Chapter 5, pp.67-113 ( 2001)• Rose, RL, Hodgson E. Adaptation to Toxicants. Chemical and Environmental Factors Affecting Metabolism of Xenobiotics In: Hodgson E, Smart RC. INTRODUCTION TO BIOCHEMICAL TOXICOLOGY 3rd ed. Chapter 8 , pp. 163-198 (2001) • Guengerich FP. Cytochromes P450, Drugs, and Diseases. Molecular Interventions 3: 194-204 (2003)Optional Reading:• Shimada T. Xenobiotic-Metabolizing Enzymes involved in Activation and Detoxification of Carcinogenic Polycyclic Aromatic Hydrocarbons. Drug Metab. Pharmacokinet 21: 257- 276 (2006)21. Xenobiotic Metabolism: DefinitionXenobiotic = "foreign"Synonyms: Biotransformation; Drug MetabolismNot: Detoxication Reactions2. Phase I and Phase II Reactions: ClassificationPhase I Reactions: Oxidation, Reduction, HydrolysisIntroduce one of following groups into the initial compound: -OH, -COOH, -NH2, or –SHPhase II Reactions: ConjugationIntroduces a highly hydrophilic group to promote excretionExamples:Phase I: Benzene -(oxidation)Æ epoxide intermediate -(rearrangement)Æ phenol with pKa=10 (<1% ionized at pH 7.4)Phase II: Phenol -(glucuronidation)Æ phenyl glucuroide with pHa=3.4 (very water soluble, greater than 99% ionized at pH 7.43. Phase I Oxidations (at least in part) by microsomal system3.1. Aromatic Hydroxylation (via epoxide)Benzene -(aromatic hydroxylation)Æ epoxide. Then either of 2 possible reactions:(a) epoxide -(nonenzymatic rearrangement)Æ phenol(b) epoxide -(epoxide hydrolyase)Æ 1,2-dihydro-1,2-diolalso 1,2-dihydro-1,2-diol -(cytosolic dehydrogenase)Æ catechol33. Phase I Oxidations (at least in part) by microsomal system3.1. Aromatic Hydroxylation (via epoxide)NIH Shift = intramolecular migration of substituent group at the site of oxidation that moves to an adjacent ring position.If R group is notreadily ionizable (-CH3, -OCH3, -phenyl, -halo, -nitro), then 40-65% migration of DIf R group is readily ionizable (-OH, -NH2), then 0-6% migration of DGroups capable of migration: D or deuterium, 3H or tritium, chloro, methyl3. Phase I Oxidations (at least in part) by microsomal system3.1. Aromatic Hydroxylation (via epoxide)Other substrates subject to Aromatic Hydroxylation (via epoxidation):Bromobenzene Æ bromobenzene epoxideChlorobenzene Æ chlorobenzene epoxide Æ ortho-chlorophenol, andpara-chlorophenol43.2. Aromatic Hydroxylation via O-insertionChlorobenzene Æ meta-chlorophenolAniline Æ para-hydroxyaniline3.3. Aliphatic Hydroxylationn-propyl-benzene Æ phenyl-CH2-CH2-CH2OH omega hydroxylationand phenyl-CH2-CHOH-CH3 omega minus 1 hydroxylationand phenyl-CHOH-CH2-CH3 alpha hydroxylationThe products from Omega and Omega-minus 1 hydroxylations are always the major metabolites. If a substrate can be metabolized either by aliphatic hydroxylation or aromatic hydroxylation, aliphatic hydroxylation is always predominant.53.4. N-Dealkylation (Oxidation of the Alkyl Group)R-NH-CH3 (oxidation of the C) Æ R-NH2 + HCHO (methyl group is oxidized to formaldehyde)R-NH-C(R2) (oxidation of the carbon) Æ RNH2 + ketone (e.g., acetone)Also O-Dealkylation and S-Dealkylation (always oxidation of the carbon)3.5 N-Oxidation (Oxidation of N)Aromatic primary and secondary amines yield aromatic hydroxylamineProducts very reactive and toxicAliphatic tertiary amines yield N-Oxide(CH3)3N Æ (CH3)3N=OAromatic tertiary amines Æ N-oxideTwo pathways leading to N-oxidation:• Microsomal FAD-containing monooxygenase (FMO)Not inhibited by CO• Cytochrome P450Inhibited by CO63.6. S-OxidationBoth FMO and cytochrome P450 are active sulfides, sulfoxide sulfonethioethers (major product)4. Non-Microsomal Oxidations4.1. Alcohol OxidationAliphatic alcohol Æ Aldehyde Æ Carboxylic AcidADH = alcohol dehydrogenaseALDH = aldehyde dehydrogenaseRate of metabolism: primary alcohols > secondary alcohols >>> tertiary alcohols (latter not metabolized by alcohol dehydrogenase)ETHANOL METABOLISM75. Phase I Reactions: ReductionsFavored by anaerobic conditions. Do occur in mammalian tissues where oxygen concentration is low.In vitro conditions: replace air or oxygen with nitrogenNitro Reduction (Nitro Reductase)Azo Reduction (Azo Reductase)Nitro Reduction: 3 important enzyme systems(a) Cytochrome P450 (e.g., in liver). Inhibited by CO(b) DT-diaphorase: cytosolic flavoprotein (in liver) = NAD(P)H quinone oxidoreductase(c) Bacterial intestinal enzymes86. Phase I Reaction: HydrolysisSubstrates: (a) carboxylic acid ester, amides - same enzyme(b) phosphate ester(c) epoxideEnzyme:Epoxide hydrolase (former names: Epoxide hydratase, epoxide hydrase)Most of enzyme in microsomes, but also in cytosol. Different gene products.Product is always trans hydroxyls7. Monooxygenase or Mixed Function Oxidase (MFO)(a) Monooxygenase = 1 atom of molecular oxygen is added to substrate(b) Mixed function oxidase = 1 atom of molecular oxygen is added to substrate, and 1 atom of oxygen is converted to water(c) Terms in (a) and (b) have been used interchangeably, but term monooxygenase is probably the primary term now. Overall reaction: -SH + O2 + 2 e- Æ SOH + H2Oreduced substrate + molecular oxygen + two one-electron transfer Æ oxidized substrate + waterSystem also requires lipid. Lipid-soluble compounds are better substrates for cytochrome P450 than water-soluble substrates98. Cytochrome P450: Terminal Oxidase in Xenobiotic MetabolismCytochrome P450s family ("superfamily') of similar hemoproteins, and is critically important in xenobiotic metabolism.The human genome encodes 57 P450 proteins: (Guengerich, 2003)• 15 involved in metabolism of xenobiotic chemicals (i.e., chemicals, such as drugs, not normally found in the body)• 14 primarily involved in the metabolism of sterols (including bile acids);• 4 that oxidize fat-soluble vitamins; and• 9 involved in the metabolism of fatty acids and eicosanoids. Substrates are essentially unknown for the remaining 15 of the 57.8. Cytochrome P450: Terminal Oxidase in Xenobiotic MetabolismSubstrates:"The cytochrome P450 gene superfamily encodes many [isozymes] that are unusual in the variety of chemical reactions catalyzed and the number of substrates [metabolized]. The [substrates] include physiologically important substances such as steroids, eicosanoids,
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