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Ecotoxicology

Ecotoxicology. Biotransformation. RÉSUMÉ. UPTAKE IN ORGANISM DEPENDS ON:. Lipophilicity (polarization, ionization). Route of uptake. Concentration. Molecular size. UPTAKE IN ORGANS DEPENDS ON:. Vascularization. Binding mechanisms in blood. Lipophilicity.

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Ecotoxicology

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  1. Ecotoxicology Biotransformation

  2. RÉSUMÉ UPTAKE IN ORGANISM DEPENDS ON: Lipophilicity (polarization, ionization) Route of uptake Concentration Molecular size 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 Absorption Target organ Depot 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 UPTAKE UPTAKE BIOTRANS- ORGAN ORGAN FORMATION EXCRETION EXCRETION EXCRETION Uptake and excretion of hydrophilic and lipophilic compounds Primarily biotransformation makes lipophilic compounds more hydrophilic

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

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

  8. Phase I reactions

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

  10. Important phase I enzymes Enzyme Co-factor Substrate Mixed-function oxidases NADPH Most lipophilic substances (cytochrome P-450) (NADH) with M.wt < 800 Carboxyl esterases Unknown Lipophilic carboxyl esters ’A’ esterases Ca++ Organophosphate esteres Epoxide hydrolases Unknown Organic epoxids Reduktases NADH Organic nitrous compounds NADPH Organic halogens

  11. P-450 system in the endoplasmic reticulum

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

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

  14. Examples of oxidations catalysed by P-450 Aliphatic hydroxylation Sulphur oxidation R - CH2 – CH2 – CH3 R – CH2 – CHOH – CH3 R - S - R’ R - S - R’ Aromatic hydroxylation De-sulphurnation O S R R OH R R P - X R R P - X + S 1 2 1 2 Oxidative dehalogenation Epoxidation O O X X R - CH CH - R’ R - CH - CH - R’ R - C - H R - C - OH R - C - H + HX N-, O-, or S-dealkylation H H H R – (NH2, OH, SH) + CH2O R - (N, O, S) - CH 3 Deamination O R – CH2 – NH2 R - C - H + NH3 N - hydroxylation O O R - NH - C – CH3 R - NOH - C – CH3

  15. Other phase I enzymes N N N N N N N N S O CH2O CH2O C2H5 C2H5 P P O N O N CH2O CH2O C2H5 C2H5 CH3 CH3 O CH2O O OH P CH2O CH2O C2H5 C2H5 P + OH O N N CH2O C2H5 C2H5 CH3 CH3 MO Diazoxon Diazinone ’A’ esterase Diazoxon

  16. Other phase I enzymes Cl Cl Cl COOH Cl HOH2C O COOCH2 O OH O OH NO2 NH2 ’B’ esterase Permethrine Epoxide hydrolase Benzo(a)pyrene 7,8 oxide Nitroreductase Nitropyrene

  17. Phase II reactions

  18. PHASE I PHASE II Expose or add functional group PRIMARY PRODUCT SECUNDARY PRODUCT XENOBIOTIC Conjugation Oxidation Reduction Hydrolysis EXCRETION LIPOPHILIC HYDROPHILIC

  19. HN O N N N N COOH O O O O P CH2 O P OH HO O O OH O O O OH OH Two important co-factors in phase II conjugations UDP and PAPS NH2 O O O P CH2 S O O O OH OH Uridine-5’-diphospho- α-D-glucuronic acid (UDP-GA) 3’-Phosphoadenosine- 5’-phosphosulfate (PAPS)

  20. O O Glucuronyl transferase conjugations COOH COOH Glucuronyl transferase O O UDP R +UDP R – OH + OH OH HO HO OH OH 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 -C – O - G - C - O - G O - CH = C – O - G - O – C – N - G O H R – SO2 – N - G H - C - G O-Glucuronid Alcohol Aliphatic Trichloroethanol Alicyclic Hexobarbital Phenolic Estrone Carboxyl acid Aliphatic α-Ethylhexanoic acid Aromatic o-Aminobenzoic acid α,β-Unsaturated ketone Progesterone N-Glucuronide Carbamate Meprobamate Sulfonamide Sulfadimethoxine S-Glucuronide Ar – S - GAryl thiol Thiophenol C-Glucuronide 1,3-Dicarbonyl system Phenylbutazone

  22. O Sulfo- transferase R – OH + PAPSR – O – S – O + ADP O Sulfotransferase conjugation 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. H H N O H O O H O H H N O O H Glutathione Glutamic acid H H N O H H O N + H O O H H S H N H N O Cysteine S O O H O + H Glutathione Glycine

  24. Glutathione S-transferase O OH CH - CH CH – CH - SG Glutathion S-transferase + GSH 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 – SCH2CHCOOH HNCCH3 O

  25. Glutathione S-transferase reactions Cl Cl O Cl SG NO2 NO2 CH2Cl CH2SG Glutathione S-alkyltransferase Glutathione S-alkenetransferase CHCOOC2H5 CH2COOC2H5 CH3I + GSH CH3-SG + HI + GSH GS-CHCOOC2H5 CHCOOC2H5 Methyl iodide Diethyl maleate Glutathione S-aryltransferase Glutathione S-aryl epoxidetransferase OH SH + GSH + HCl GSH P-450 Naphthalene Naphthalene oxide 3,4-Dichloronitrobenzen Glutathione S-aralkyltransferase + HCl + GSH Benzyl chloride

  26. Induction of biotransformation enzymes

  27. Characteristics of the hepatic effects of Phenobarbital and Benzo[a]pyren (PAH) CHARACTERISTICS PHENOBARBITAL PAH Onset of effect 8-12 hours 3-6 hours Time of maximum effects 3-5 days 24-48 hours Persistence of induction 5-7 days 5-12 days Liver enlargement marked slight Protein synthesis large increase small increase Phosphorlipid synthesis marked increase no effect Liver blood flow increased no effects Biliary flow increased no effect Enzyme components Cytochrome P-450 increased no effect Cytochrome P-448 no effect increased NADRH-cytochrome reductase increased no effect Substrate specificity N-Demethylation increased no effect Aliphatic hydroxylation increased no effect PAH hydroxylation small increase increased Glucuronidation increased small increase Glutathione conjugation small increase small increase Epoxide hydrolase increased small increase

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

  29. P450 protein P450 mRNA • Bioactivation • Detoxification Toxicity Elimination Induction of cytochrome P-450 Cell Ah receptor-hsp90 HC (inducer) HC Nucleus HC-AhR HC-AhR P450 gen hsp90 XRE 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) • NH2 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 pathway Factors increasing Stof or intermediate product toxicity Acetaminophen N-hydroxylation Sulphate and GSH depletion Acetylhydrazine N-hydroxylation Aflatoxin B Epoxidation Benzen Epoxidation Benzo[a]pyren Epoxidation Further metabolism PCB Epoxidation GSH depletion Tetrachlorcarbon Free radicals Reductive metabolism Halotane Free radicals Reductive metabolism Parathion Oxidation with sulphur formation

  33. Activation of Paracetamol HNCOCH3 Mercapturic acid GSH At overdose Glutathione (GSH) is depleted O cellular macro molecule HNCOCH 3 Cellular Liver damage macro molecule OH HNCOCH HNCOCH Sulfotransferase 3 3 Acetaminophen (Paracetamol) 95% Glucuronosyl- transferase OH O Activation of NADPH CONJUGATE cyt. P-450 O2 5% HON COCH 3 OH NCOCH 3 * + O N-Acetyl-p-Benzoquinoneimin

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