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. Glucuronidation Typical 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, H 2 SO 4 Co-factor: 3’phosphoadenosine 5’phosphosulfate (PAPS), formed from ATP + sulfate Located in cytosol, Probably not inducible

9. Sulfation Typical 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 Benzoic acid Benzoyl-CoAHippuric 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 Glutamic acid (Glu) Glycine (Gly) Cysteine (Cys) A tripeptide

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

21. Methylation Reaction Substrate: Catechol Enzyme: Catechol-O- methyltransferase (COMT)

22. 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”

23. Acetylation Reaction 2-Naphthylamine 2-Aminonaphthalene 2-Acetylaminonaphthalene 2-Acetamidonaphthalene

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

25. Reactive Oxygen Species (ROS) Peroxides –Hydrogen peroxide HOOH –Peroxynitrite OONO - –Lipid hydroperoxide LOOH Free radicals –Superoxide anion O 2 - –Hydroxyl radical HO –Nitric oxide NO

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

27. Superoxide dismutase Converts superoxide anions to hydrogen peroxide O 2- + O 2- + 2H + O 2 + H 2 O 2

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

29. Peroxidases Catalase Prostaglandin synthetase Myeloperoxidase Lactoperoxidase Glutathione peroxidase