Ecotoxicology. Biotransformation

Transcription

1 Ecotoxicology Biotransformation

2 RÉSUMÉ UPTAKE I RGAISM DEPEDS : Lipophilicity (polarization, ionization) Route of uptake Concentration Molecular size UPTAKE I RGAS DEPEDS : Vascularization Binding mechanisms in blood Lipophilicity Binding sites in the cells of the organ

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

4 rganism 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/g) Biotransformation

5 Uptake and excretion of hydrophilic and lipophilic compounds UPTAKE UPTAKE UPTAKE RGA RGA BITRAS- FRMATI EXCRETI EXCRETI EXCRETI Primarily biotransformation makes lipophilic compounds more hydrophilic

6 XEBITIC EXCRETI on-toxic metabolite BLD BITRAS- FRMATI Activation Detoxification Toxic metabolite Somatic effect DA damage 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 PASE I PASE II Expose or add functional group XEBITIC xidation Reduction ydrolysis PRIMARY PRDUCT Conjugation SECUDARY PRDUCT EXCRETI LIPPILIC YDRPILIC

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 ADP Most lipophilic substances (cytochrome P-450) (AD) with M.wt < 800 Carboxyl esterases Unknown Lipophilic carboxyl esters A esterases Ca ++ rganophosphate esteres Epoxide hydrolases Unknown rganic epoxids Reduktases AD rganic nitrous compounds ADP rganic halogens

11 P-450 system in the endoplasmic reticulum REDUCTASE P-450 P-450

12 Classification and evolution of the P-450 gene-family Millioner år før nutid 2,000 1,500 1,000 LI I II III IV XIX D A B C E XXI XVII CI XI A B 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 Xenobiotic Fe3+ Fe3+ CYT P- 450 ADP ADP + e - CYT P-450 reductase Fe3+ Fe Fe3+ eṉadp Fe2+ ADP ADP ADP+ Fp oxidized e - Fp reduced (R)-P450-(Fe2+) (R)-P450-(Fe3+) 2 e - R (R)-P450-(Fe2+) 2 P450 (Fe3+) R + 2

14 Examples of oxidations catalysed by P-450 Aliphatic hydroxylation Sulphur oxidation R - C 2 C 2 C 3 R C 2 C C 3 R -S-R R -S-R Aromatic hydroxylation R R Epoxidation R - C C - R R - C - C - R -, -, or S-dealkylation R - (,, S) - C 3 R ( 2,, S) + C 2 De-sulphurnation S R 1 R 2 P - X R 1 R 2 P - X + S xidative dehalogenation X X R - C - R - C - R - C - + X Deamination R C 2 2 R - C hydroxylation R - - C C 3 R - - C C 3

15 ther phase I enzymes C 2 C 2 S P C 2 5 C 2 5 M C 2 C 2 P C 2 5 C 2 5 C 3 Diazinone C 3 Diazoxon C 2 C 2 P C 2 5 C 2 5 A esterase C 2 C 2 P + C 2 5 C 2 5 C 3 C 3 Diazoxon

16 ther phase I enzymes Cl Cl CC 2 B esterase Cl Cl C 2 C Permethrine Epoxide hydrolase Benzo(a)pyrene 7,8 oxide 2 2 itroreductase itropyrene

17 Phase II reactions

18 PASE I PASE II Expose or add functional group XEBITIC xidation Reduction ydrolysis PRIMARY PRDUCT Conjugation SECUDARY PRDUCT EXCRETI LIPPILIC YDRPILIC

19 Two important co-factors in phase II conjugations UDP and PAPS C P P C 2 S P C 2 2 Uridine-5 -diphosphoα-d-glucuronic acid (UDP-GA) 3 -Phosphoadenosine- 5 -phosphosulfate (PAPS)

20 Glucuronyl transferase conjugations C C R + UDP Glucuronyl transferase R + UDP 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 MF-system The resulting glucuronides are excreted in urine and faeces

21 Examples of Glucuronide conjugations -Glucuronid -C -G -C - -G - C = C - G -Glucuronide - C -G Alcohol Aliphatic Alicyclic Phenolic Carboxyl acid Aliphatic Aromatic α,β-unsaturated ketone Carbamate Trichloroethanol exobarbital Estrone α-ethylhexanoic acid o-aminobenzoic acid Progesterone Meprobamate R S 2 -G Sulfonamide Sulfadimethoxine S-Glucuronide Ar S - G Aryl thiol Thiophenol C-Glucuronide -C -G 1,3-Dicarbonyl system Phenylbutazone

22 Sulfotransferase conjugation R + PAPS Sulfotransferase R S + ADP PAPS (Phosphoadenine phosphosulphate) delivers the energy Localised in the cytosol Adds sulphate to -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 Glutathione Glutamic acid Cysteine Glycine S + + S Glutathione

24 Glutathione S-transferase C - C C C - SG + GS Glutathion S-transferase 1,2-Epoxyetylbenzene GS = reduced glutathione (tripeptide) glutathione s S 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 SC 2 CC CC 3

25 Glutathione S-transferase reactions Glutathione S-alkyltransferase Glutathione S-alkenetransferase C 3 I + GS C 3 -SG + I Methyl iodide Glutathione S-aryltransferase CCC 2 5 CCC GS C 2 CC 2 5 GS-CCC 2 5 Diethyl maleate Cl Cl Glutathione S-aryl epoxidetransferase Cl SG + GS + Cl P-450 GS S 2 3,4-Dichloronitrobenzen 2 aphthalene aphthalene oxide Glutathione S-aralkyltransferase C 2 Cl C 2 SG + GS + Cl Benzyl chloride

26 Induction of biotransformation enzymes

27 Characteristics of the hepatic effects of Phenobarbital and Benzo[a]pyren (PA) CARACTERISTICS PEBARBITAL PA nset of effect 8-12 hours 3-6 hours Time of maximum effects 3-5 days 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 ADR-cytochrome reductase increased no effect Substrate specificity -Demethylation increased no effect Aliphatic hydroxylation increased no effect PA hydroxylation small increase increased Glucuronidation increased small increase Glutathione conjugation small increase small increase Epoxide hydrolase increased small increase

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

29 Induction of cytochrome P-450 Ah receptor-hsp90 Cell C (inducer) C C-AhR hsp90 C-AhR ucleus P450 gen XRE P450 protein P450 mra Bioactivation Detoxification Toxicity Elimination C: ydrocarbon (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 - S groups (glutathione, cystein) - 2 and C groups (DA, RA, 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 -hydroxylation Sulphate and GS depletion Acetylhydrazine Aflatoxin B Benzen -hydroxylation Epoxidation Epoxidation Benzo[a]pyren Epoxidation Further metabolism PCB Epoxidation GS depletion Tetrachlorcarbon Free radicals Reductive metabolism alotane Free radicals Reductive metabolism Parathion xidation with sulphur formation

33 Activation of Paracetamol Acetaminophen (Paracetamol) ADP 2 CC 3 Activation of cyt. P-450 5% CC 3 Sulfotransferase 95% Glucuronosyltransferase CC 3 CJUGATE CC 3 Mercapturic acid CC 3 GS cellular macro molecule At overdose Glutathione (GS) is depleted CC 3 *+ -Acetyl-p-Benzoquinoneimin Cellular macro molecule Liver damage