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

2. Major reactive species Electrophiles Epoxides Carbonium ions Arylnitrenium ions Reactive Oxygen Species

3. Epoxide hydrolase Several isoforms Inducible Mainly in endoplasmic reticulum, also in cytosol and in nuclear envelope Catalyse trans addition of water to epoxides

4. Epoxide hydrolase Reaction

5. trans-dihydrodiols Detoxication products from epoxides (PAH) Targets for Phase II metabolism (glucuronidation, sulfation) Oxidized by dihydrodiol dehydrogenases to quinones On terminal rings, oxidized to diol- epoxides

6. Dioxygenases Microbial enzymes Form cis-dihydrodiols

7. Complete mineralization cis-dihydrodiol to catechol ring-cleavage,  -oxidation, formation of CO 2

8. 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

9. Catalytic cycle of cytochrome P450 from NADPH-cytC reductase Fe 3+ Fe 3+ -RH Fe 2+ -RH O 2 [Fe 2+ -RH] HO 2 - [Fe 2+ -RH] HO 2 2- Fe 3+ -RH + RH + e - +O 2 ROH H + + e - NADPH NADH H2OH2O H2O2H2O2 H+H+ H+H+ O 2 -.

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

11. Superoxide dismutase Converts superoxide radicals to hydrogen peroxide O 2- + O 2- + 2H + O 2 + H 2 O 2

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

13. Peroxidases Catalase Prostaglandin synthetase Myeloperoxidase Lactoperoxidase Glutathione peroxidase

14. GSH + GSHGSSG HOOH HOH + HOH

15. Gut flora Reductions –nitro to amine Hydrolyses –Cleavage of glucuronides

16. Reaction Glucuronidation

17. Reaction OH o o OH HO OH COOH De-glucuronidation  -glucuronidase AglyconeConjugate

18. Enterohepatic recirculation (EHC) Liver Intestine

19. Gastrointestinal tract

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

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

22. Factors affecting xenobiotic metabolism Intrinsic –Species, strain, gender, age, genotype Physiological status –Temperature, time of day, season, –Health status, disease, stress –Diet, nutritional status Related to exposure –Route of administration, frequency and size of dose, co-exposures (induction, inhibition

23. Changes in P450 levels with age Rats 2A1 2C6 3A2 M: 2C6, 2C11, 3A2 F: 2A1, 2C6, 2C12

24. Data determined in experimental animals (often rodents) Information needed about target species (usually humans)

25. Cross-species extrapolation What factors are similar ? What factors are different ? The basic problem: data determined in experimental animals Information needed about target species (usually humans) Differences between individuals (interindividual variation)

26. Genetic polymorphisms CYP2D6 Debrisoquine hydroxylation (poor and extensive metabolizers) Acetylation (fast and slow acetylators) GSTM null genotype

27. Effect is the outcome of interaction between susceptibility and exposure

28. What makes a particular organ or species susceptible ?

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