1. Ecotoxicology Biotransformation

2. RÉSUMÉ UPTAKE IN ORGANISM DEPENDS ON: Concentration Route of uptake Molecular size Lipophilicity (polarization, ionization) UPTAKE IN ORGANS DEPENDS ON: Vascularization Binding mechanisms in blood Lipophilicity Binding sites in the cells of the organ

3. Bound Free Bound Free Bound Free Target organ Depot Absorption Excretion Adipose tissues Inert membranes Lipoprotein micells Lysosomes Skeleton (endo or exo) Urine Faeces (gall) Lungs or gills Secretion from surface

4. Organism’s defence against xenobiotics Fast excretion Deposition in less susceptible organs (fat depots, skeleton) Deposition in intracellular organelles Formation of complexes (i.e. metallothionin and Se/Hg) Biotransformation

5. Uptake and excretion of hydrophilic and lipophilic compounds UPTAKE ORGAN EXCRETION ORGAN UPTAKE EXCRETION Primarily biotransformation makes lipophilic compounds more hydrophilic

6. BLOOD XENOBIOTIC BIOTRANS- FORMATION EXCRETION DNA damage Somatic effect Detoxification Activation Non-toxic metabolite Toxic metabolite Definition Biotransformation is the sum of all processes, whereby a compound is transformed chemically within a living organism

7. XENOBIOTIC PRIMARY PRODUCT SECUNDARY PRODUCT Expose or add functional group Oxidation Reduction Hydrolysis PHASE IPHASE II EXCRETION Conjugation LIPOPHILICHYDROPHILIC Phase I and phase II reactions

8. Phase I reactions

9. Mixed function oxidase enzymes (P450) are located in the endoplasmic reticulum (SER)

10. Important phase I enzymes EnzymeCo-factorSubstrate Mixed-function oxidasesNADPHMost lipophilic substances (cytochrome P-450)(NADH)with M.wt < 800 Carboxyl esterasesUnknownLipophilic carboxyl esters ’A’ esterasesCa ++ Organophosphate esteres Epoxide hydrolasesUnknownOrganic epoxids ReduktasesNADHOrganic nitrous compounds NADPHOrganic halogens

11. P-450 system in the endoplasmic reticulum

12. 2,000 1,500 1,000 250 80 17 III I IV BA C D E AB XIX XXI CI XI LI II I XVII Classification and evolution of the P-450 gene-family Millioner år før nutid I-IV involved in phase I reactions XI, XVII, XIX, XXI participate in the biosynthesis of steroid hormones

13. Xenobiotic NADPH NADP+ Fp oxidized Fp reduced (RH)-P450-(Fe2+) (RH)-P450-(Fe3+) (RH)-P450-(Fe2+) ·O2 P450 (Fe3+) O2 RH ROH + H2O e - e - Cytochrome P-450’s catalytic cycle CYT P- 450 Fe3+ NADPH NADP CYT P-450 reductase + e - Fe2+ O2 Fe2+ e - NADPH NADP Fe3+ H2OH2O

14. Aliphatic hydroxylation Aromatic hydroxylation RR OHOH R - CH CH - R’ R - CH - CH - R’ Epoxidation O N-, O-, or S-dealkylation R - (N, O, S) - CH 3 H N - hydroxylation Deamination R - C - H + NH 3 O O O R - NH - C – CH 3 R - NOH - C – CH 3 R - C - H R - C - OH R - C - H + HX O XX HH R - S - R’ Sulphur oxidation S R 1 R 2 P - X R 1 R 2 P - X + S O De-sulphurnation Oxidative dehalogenation Examples of oxidations catalysed by P-450 R - CH 2 – CH 2 – CH 3 R – CH 2 – CHOH – CH 3 R – (NH 2, OH, SH) + CH 2 O R – CH 2 – NH 2

15. Other phase I enzymes N N O P CH 2 O C2H5C2H5 C2H5C2H5 N CH 3 S MO N N O P CH 2 O C2H5C2H5 C2H5C2H5 N CH 3 O Diazinone Diazoxon N N O P CH 2 O C2H5C2H5 C2H5C2H5 N CH 3 O Diazoxon N N OH P C2H5C2H5 C2H5C2H5 N CH 3 CH 2 O OH O + ’A’ esterase

16. Other phase I enzymes O COOCH 2 Cl COOH Cl O HOH 2 C ’B’ esterase Permethrine O OH Epoxide hydrolase Benzo(a)pyrene 7,8 oxide NO 2 NH 2 Nitroreductase Nitropyrene

17. Phase II reactions

18. XENOBIOTIC PRIMARY PRODUCT SECUNDARY PRODUCT Expose or add functional group Oxidation Reduction Hydrolysis PHASE IPHASE II EXCRETION Conjugation LIPOPHILICHYDROPHILIC

19. O OH CH 2 O P O O O O S O N NN NH 2 O OH CH 2 O P O O O HO O OH COOH O O P N HN O O Two important co-factors in phase II conjugations UDP and PAPS Uridine-5’-diphospho- α-D-glucuronic acid (UDP-GA) 3’-Phosphoadenosine- 5’-phosphosulfate (PAPS)

20. R – OH + UDP O HO O OH COOH Glucuronyl transferase O HO O OH COOH R + UDP Glucuronyl transferase conjugations UDP (uridin diphosphate) delivers the energy to the conjugation process Important phase II reactions for both exo- and endogenous compounds Many forms with a wide range of substrates Localised in SER in close connection with the MFO-system The resulting glucuronides are excreted in urine and faeces

21. Examples of Glucuronide conjugations O-Glucuronid Alcohol AliphaticTrichloroethanol AlicyclicHexobarbital PhenolicEstrone Carboxyl acid Aliphaticα-Ethylhexanoic acid Aromatico-Aminobenzoic acid α,β-Unsaturated ketoneProgesterone N-Glucuronide CarbamateMeprobamate SulfonamideSulfadimethoxine S-Glucuronide Ar – S - GAryl thiolThiophenol C-Glucuronide 1,3-Dicarbonyl systemPhenylbutazone - C – O - G - C - O - G O - CH = C – O - G - O – C – N - G OH R – SO 2 – N - G H - C - G

22. Sulfotransferase conjugation R – OH + PAPS R – O – S – O + ADP O O Sulfo- transferase PAPS (Phosphoadenine phosphosulphate) delivers the energy Localised in the cytosol Adds sulphate to OH-groups (phenols and aliphatic alcohols) Also important for the transformation of endogenous low-molecular compounds (catacholamins, hydroxy-steroids, bile salts) The conjugates are primarily excreted in the urine

23. N O H O H N S H O N O O H H O H H H + + N O O O H H O H H N O S H H O H N O H H O H Glutamic acid Cysteine Glycine Glutathione

24. Glutathione S-transferase CH - CH O + GSH CH – CH - SG OH Glutathion S-transferase 1,2-Epoxyetylbenzene GSH = reduced glutathione (tripeptide) glutathione’s – SH group attacks electrophilic (reactive) C-atoms predominantly localised in the cytosol several enzymatic cleavages of glutathione after conjugation ends with a derivate of mercapturic acid, which is excreted in the urine R – SCH 2 CHCOOH HNCCH 3 O

25. Glutathione S-transferase reactions NO 2 Cl Glutathione S-alkyltransferase CH 3 I + GSH CH 3 -SG + HI Methyl iodide Glutathione S-aryltransferase NO 2 Cl SG + GSH + HCl 3,4-Dichloronitrobenzen CH 2 Cl Glutathione S-aralkyltransferase CH 2 SG + GSH + HCl Benzyl chloride Glutathione S-alkenetransferase CHCOOC 2 H 5 + GSH CH 2 COOC 2 H 5 GS-CHCOOC 2 H 5 Diethyl maleate O GSH SH OH P-450 Glutathione S-aryl epoxidetransferase Naphthalene oxide

26. Induction of biotransformation enzymes

27. Characteristics of the hepatic effects of Phenobarbital and Benzo[a]pyren (PAH) CHARACTERISTICSPHENOBARBITALPAH Onset of effect8-12 hours3-6 hours Time of maximum effects3-5 days24-48 hours Persistence of induction5-7 days5-12 days Liver enlargementmarkedslight Protein synthesislarge increasesmall increase Phosphorlipid synthesismarked increaseno effect Liver blood flowincreasedno effects Biliary flowincreasedno effect Enzyme components Cytochrome P-450increasedno effect Cytochrome P-448no effectincreased NADRH-cytochrome reductaseincreasedno effect Substrate specificity N-Demethylationincreasedno effect Aliphatic hydroxylationincreasedno effect PAH hydroxylationsmall increaseincreased Glucuronidationincreasedsmall increase Glutathione conjugationsmall increasesmall increase Epoxide hydrolaseincreasedsmall increase

28. Examples of other inducers Halogenated pesticides (DDT, aldrin, lindan, chlordan) PCB Steroids Chlorinated dioxins (TCDD) Alcohol and acetone

29. HC (inducer) Ah receptor-hsp90 HC P450 mRNAP450 protein Bioactivation Detoxification Toxicity Elimination Cell HC-AhR hsp90 HC-AhR XRE P450 gen Nucleus Induction of cytochrome P-450 HC: Hydrocarbon (inducer) XRC: Regulator gene (stimulates transcription of P-450 gene)

30. Bioactivation

31. Bioactivation is define as: Enzymatically formed metabolites, which are more reactive than the mother substance and excreted metabolites The most significant toxicological effects of xenobiotics are reactive metabolites - can react with nucleophilic sites - SH groups (glutathione, cystein) - NH 2 and – COOH groups (DNA, RNA, proteins) Imbalance between formation and detoxification of reactive metabolites can arise from: - enzyme induction (increased biotransformation and formation of reactive metabolites) - high dose of xenobiotic depletion of cellular defence mechanisms saturation of non-toxic pathways

32. Examples of bioactivating compounds Reactive pathwayFactors increasing Stofor intermediate producttoxicity AcetaminophenN-hydroxylationSulphate and GSH depletion AcetylhydrazineN-hydroxylation Aflatoxin BEpoxidation BenzenEpoxidation Benzo[a]pyrenEpoxidationFurther metabolism PCBEpoxidationGSH depletion TetrachlorcarbonFree radicalsReductive metabolism HalotaneFree radicals Reductive metabolism ParathionOxidation with sulphur formation

33. HNCOCH 3 OH NADPH O2O2 Activation of cyt. P-450 Sulfotransferase Glucuronosyl- transferase Acetaminophen (Paracetamol) HNCOCH 3 O CONJUGATE NCOCH 3 O N-Acetyl-p-Benzoquinoneimin *+ HONCOCH 3 OH Activation of Paracetamol HNCOCH 3 OH Cellular macro molecule cellular macro molecule Liver damage HNCOCH 3 O Mercapturic acid GSH At overdose Glutathione (GSH) is depleted 95% 5%