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Phase II: Conjugation

Phase II: Conjugation. Synthetic reaction of a xenobiotic (or of a Phase I metabolite of a xenobiotic) with an endogenous substance Results in introduction of polar, ionizable groups to enhance water solubility and hence excretion. Major Phase II reactions. Glucuronidation Sulfation

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Phase II: Conjugation

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  1. Phase II: Conjugation • Synthetic reaction of a xenobiotic (or of a Phase I metabolite of a xenobiotic) with an endogenous substance • Results in introduction of polar, ionizable groups to enhance water solubility and hence excretion

  2. Major Phase II reactions • Glucuronidation • Sulfation • Conjugation with amino acids • Conjugation with glutathione • Methylation • Acetylation

  3. Glucuronidation • Enzyme: glucuronyl transferase, or glucuronosyl transferase • Targets: • hydroxyl groups: Phenols, Alcohols, Dihydrodiols (ether glucuronides) • Carboxylic acids (ester glucuronides) • Amines (N-glucuronides) • Thiols (S-glucuronides) • Carbon (C-glucuronides, rare)

  4. Reaction Phenol Phenyl glucuronide

  5. Glucuronidation • Conjugating moiety: glucuronic acid, a sugar • Co-factor: UDP-glucuronic acid (UDPGA), derived from glycogen synthesis • Located in endoplasmic reticulum • Multiple families of isoforms:UGT1, UGT2 • UGT1.1 ..1.7, UGT2.1..2.4 • Inducible

  6. Uridine-5’-diphospho--D-glucuronic acid (UDPGA)

  7. GlucuronidationTypical substrates: • Phenol • 1-Naphthol • 4-Hydroxybiphenyl • 3-Hydroxybenzo[a]pyrene • Benzo[a]pyrene-7,8-dihydrodiol • 2-Naphthylamine • Bilirubin • Steroids

  8. Sulfation • Sulfotransferase ST,15 isoforms (xx-ST) • Targets • Hydroxyl groups (phenols, alcohols) • Amino groups • Thiols • Conjugating moiety: sulfuric acid, H2SO4 • Co-factor: 3’phosphoadenosine 5’phosphosulfate (PAPS), formed from ATP + sulfate • Located in cytosol, Probably not inducible

  9. SulfationTypical substrates • Ethanol • Phenol • 3-Hydroxybenzo[a]pyrene • Cholesterol • 2-Naphthylamine • N-hydroxy-2-naphthylamine

  10. Reaction PAPS PAP

  11. Conjugation with amino acids • Amino acid transferases • Targets: carboxylic acids • Conjugating moieties: Glycine, glutamine, alanine, taurine, histidine, ornithine • Co-factor: Acetyl CoA (CoASH) and ATP • In cytosol

  12. Reaction Benzoyl-CoA Hippuric acid Benzoic acid

  13. Conjugation with glutathione • Glutathione S-transferases (GST) • Targets: Epoxides, halogens • Conjugating moiety: Glutathione • Co-factor: None • Mainly in cytosol • Inducible • Multiple families of isoforms: GSTA, GSTM, GSTP, GSTT ()(αμπθ)

  14. Glutathione A tripeptide Glutamic acid (Glu) Glycine (Gly) Cysteine (Cys)

  15. Reaction

  16. Typical substrates • Organic halides, e.g methyl iodide, benzyl chloride • Alkenes e.g. diethyl maleate • Epoxides

  17. Mercapturic acid pathway

  18. Methylation • Methyltransferases • Target: Hydroxyl groups, amines, thiols • Substrates mainly endogenous: Catechols, noradrenalin, histamine • Conjugating moiety: Methyl group • Co-factor: S-adenosylmethionine

  19. S-adenosylmethionine

  20. MethylationReaction Substrate: Catechol Enzyme: Catechol-O-methyltransferase (COMT)

  21. Acetylation • N-acetyltransferases (NAT) • Target: Aromatic amines, sulfonamides • Conjugating moiety: Acetyl group • Co-factor: Acetyl-CoA • Few forms: NAT1, NAT2. NAT3: mice • Genetic polymorphisms: “slow and fast acetylators”

  22. AcetylationReaction 2-Naphthylamine 2-Aminonaphthalene 2-Acetylaminonaphthalene 2-Acetamidonaphthalene

  23. “Other” detoxication mechanisms • P-glycoprotein: ATP-dependent carrier that removes molecules from cells • Multidrug resistance associated protein MDR • Multispecific organic anion transporter MOAT

  24. Reactive Oxygen Species (ROS) • Peroxides • Hydrogen peroxide HOOH • Peroxynitrite OONO- • Lipid hydroperoxide LOOH • Free radicals • Superoxide anion O2•- • Hydroxyl radical HO• • Nitric oxide NO•

  25. Non-enzymic reaction with anti-oxidants • Ascorbic acid (Vitamin C) • alpha-Tocopherol (Vitamin E) • Glutathione

  26. Superoxide dismutase Converts superoxide anions to hydrogen peroxide O2•- +O2•- + 2H+ O2 + H2O2

  27. Peroxidases Couple reduction of hydrogen peroxide (or other peroxide) to oxidation of another substrate (co-oxidation) ROOH + R’H ROH + R’OH

  28. Peroxidases • Catalase • Prostaglandin synthetase • Myeloperoxidase • Lactoperoxidase • Glutathione peroxidase

  29. Glutathione peroxidase GSH + GSH GSSG HOOH HOH + HOH

  30. Metabolic Activation/Metabolic Detoxication • “Metabolism is a double-edged sword” • Generation of (re)active intermediates • Detoxication of (re)active intermediates Pharmacologically active Chemically reactive

  31. Major reactive species Electrophiles Epoxides (Epoxide hydrolase Glutathione S-transferase) Carbonium ions Arylnitrenium ions Reactive Oxygen Species

  32. Genetic Toxicology Reading material: Casarett and Doull Chapter 9, Timbrell, Chapter 6, pp. 259-279

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