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

2. S-adenosylmethionine

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

5. 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”

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

7. Non-polar (lipophilic) Hydrophobic Lipophobic Hydrophilic (Polar) XENOBIOTIC INTERMEDIATE METABOLITE ELIMINATION WATER-SOLUBLE METABOLITE May be reactive/toxic Can accumulate in tissues Phase I Metabolism Oxidation Phase II Metabolism Conjugation Solubility in lipids Solubility in water Phase III (?) Transport into bile, urine

8. Transporters (Phase III) Organic Anion Transporters OAT1 OAT2 OAT3 OAT4 Antiport (dicarboxylates eg αKG) Pump into cell Organic Anion Transporting Polypeptides OATP1A1 OATP-A (1A2) Antiport (eg GSH) Pump into cell Organic Cation Transporters OCT1 OCT2 OCT3 Uniport Proton Multidrug Resistance Transporters MRP1, MRP2 MRP4, MRP6 MDR1, MDR3 ATP pump Glucurinide, glutathione, sulfate conjugates Pump out of cell

9. Lethal Synthesis Methylation of Mercury: Methylmercury CH 3 -Hg + Dimethylmercury CH 3 -Hg-CH 3 http://www.chm.bris.ac.uk/motm/dimethylmercury/dmmh.htm Glutathione conjugation of 1,2-dihaloalkanes GSH + Br-CH 2 -CH 2 -BrGS-CH 2 -CH 2 -Br Ethylene dibromide Episulfonium ion CH 2 GS+ CH 2

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

11. 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

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

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

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

15. Peroxidases Catalase Prostaglandin synthetase Myeloperoxidase Lactoperoxidase Glutathione peroxidase

16. GSH + GSHGSSG HOOH HOH + HOH

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

19. The main purpose of this pilot study is to identify an optimal concentration of diesel exhaust which can be used to study the risks of cardiac changes in healthy older subjects. Results from this study may increase the understanding of how gaseous and particulate air pollutants (which causes the haze seen in some polluted cities) may adversely affect the functioning of the human cardiovascular and respiratory systems. This pilot research will study the responses to diesel exhaust in healthy older subjects with the specific GSTM1 and GSTP1 genotype. For more information, please visit our web site at www.epastudies.org or call Westat EPA Recruiting at 966-0604. Approved June 08, 2007 by the Committee on the Protection of the Rights of Human Subjects Biomedical Institutional Review Board. IRB # 07-0190 (GCRC #2579). This email is sponsored by: U.S. Environmental Protection Agency Human Studies Division located on the UNC-Chapel Hill campus. This email is sponsored by: U.S. Environmental Protection Agency Human Studies Division This email is sponsored by: CEMALBwww.epastudies.org

20. Another example Organophosphate Insecticides: Parathion Malathion

21. Parathion

22. Malathion

23. Hydrolysis enzymes Serum cholinesterase BChE Serum paraoxonase PON1 Polymorphisms in PON1 – differential sensitivity Heart disease Atherosclerosis Gulf War Syndrome

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

25. Target organs What makes a particular organ a target for toxicity / infection ? What makes a particular organ or species susceptible ?

26. Portal of entry to Blood to Target Organ e.g. Intestine to hepatic portal vein to liver to vena cava to heart to lungs back to heart to aorta to rest of body Location, location, location

27. Intestines Hepatic portal vein Liver Vena cavaAorta Lungs

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

29. Reaction Glucuronidation

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

31. Enterohepatic recirculation (EHC) Liver Intestine

32. Presence/absence of receptors –Estrogen receptors, Ah receptor Presence/absence of transporters/carriers –Resistance to chemotherapy Presence of repair mechanisms –DNA repair Balance of metabolic activation/detoxication

33. 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)

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

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