Ecotoxicology
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
Bound
Free
Bound
Free
Bound
Free
Absorption
Excretion
Target organ
Depot
Urine
Faeces (gall)
Lungs or gills
Secretion from surface
Adipose tissues
Inert membranes
Lipoprotein micells
Lysosomes
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)
Uptake and excretion of
hydrophilic
and lipophilic
compounds
ORGAN UPTAKE EXCRETION UPTAKE EXCRETION BIOTRANS-FORMATION UPTAKE ORGAN EXCRETIONPrimarily biotransformation makes
lipophilic compounds more hydrophilic
XENOBIOTIC
BLOOD
BIOTRANS-FORMATION
DNA
damage
Somatic
effect
Detoxification Activation Non-toxicmetabolite metaboliteToxic
EXCRET
IO
N
Definition
Biotransformation
is the sum of all processes,
whereby a compound is transformed chemically
within a living organism
Phase I and phase II reactions
PHASE I
PHASE II
Expose or add functional group PRIMARY PRODUCT SECUNDARY PRODUCT EXCRETION Conjugation XENOBIOTIC Oxidation Reduction HydrolysisLIPOPHILIC
HYDROPHILIC
Mixed function oxidase enzymes (P450) are
located in the endoplasmic reticulum (SER)
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
P-450 system in the endoplasmic reticulum
REDUCTASE
P-450
P-450
Classification and evolution of the P-450 gene-family
2,000 1,500 1,000 250 80 17 III I IV B A C E D A B XIX XXI CI XI LI II I XVIIMillioner å
r før nutid
I-IV involved in phase I reactions
Cytochrome P-450’s catalytic cycle
Xenobiotic CYT P- 450 Fe3+ Fe3+ NADPH NADP CYT P-450 reductase + e -Fe2+ O2 Fe2+ e-NADPH NADP Fe3+ H2O Fe3+ NADPH NADP+ Fp oxidized Fp reduced (RH)-P450-(Fe2+) (RH)-P450-(Fe3+) (RH)-P450-(Fe2+) ·O2 P450 (Fe3+) O2 RH ROH + H2O e -e-Examples of oxidations catalysed by P-450
Aliphatic hydroxylation Aromatic hydroxylation R R OH R - CH CH - R’ R - CH - CH - R’ Epoxidation O N-, O-, or S-dealkylation R - (N, O, S) - CH3 H N - hydroxylation Deamination R - C - H + NH3 O O O R - NH - C – CH3 R - NOH - C – CH3 Sulphur oxidation R – CH2– CHOH – CH3 R - CH2– CH2– CH3 R - S - R’ R - S - R’ De-sulphurnation S R1R2P - X R1R2P - X + S O Oxidative dehalogenation R - C - H R - C - OH R - C - H + HX O X X H H R – (NH2, OH, SH) + CH2O R – CH2– NH2Other phase I enzymes
N N O P CH2O CH2O C2H5 C2H5 N CH3 S N N O P CH2O CH2O C2H5 C2H5 N CH3 O MO Diazoxon Diazinone N N OH P C2H5 C2H5 N CH3 CH2O CH2O OH O + N N O P CH2O CH2O C2H5 C2H5 N CH3 O ’A’ esterase DiazoxonOther phase I enzymes
COOH Cl Cl O HOH2C O COOCH2 Cl Cl ’B’ esterase Permethrine OH OH O Epoxide hydrolase Benzo(a)pyrene 7,8 oxide NO2 NH2 Nitroreductase NitropyrenePHASE I
PHASE II
Expose or add functional group PRIMARY PRODUCT SECUNDARY PRODUCT EXCRETION Conjugation XENOBIOTIC Oxidation Reduction HydrolysisLIPOPHILIC
HYDROPHILIC
Two important co-factors in phase II conjugations
UDP and PAPS
O OH OH CH2 O P O O O O S O N N N NH2 O OH OH CH2 O P O O O HO O OH OH COOH O O P N HN O O
Uridine-5’-diphospho-α-D-glucuronic acid (UDP-GA)
3’-Phosphoadenosine-5’-phosphosulfate (PAPS)
Glucuronyl transferase conjugations
R – OH + O UDP HO O OH OH COOH Glucuronyl transferase O HO O OH OH COOH R + UDPUDP (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
Examples of Glucuronide conjugations
O-Glucuronid Alcohol Aliphatic Trichloroethanol Alicyclic Hexobarbital Phenolic Estrone Carboxyl acidAliphatic α-Ethylhexanoic acid
Aromatic o-Aminobenzoic acid
α,β-Unsaturated ketone Progesterone N-Glucuronide Carbamate Meprobamate Sulfonamide Sulfadimethoxine S-Glucuronide
Ar – S - G Aryl thiol Thiophenol
C-Glucuronide 1,3-Dicarbonyl system Phenylbutazone - C – O - G - C - O -G O - CH = C – O - G - O – C – N - G O H R – SO2 – N - G H - C - G
Sulfotransferase conjugation
R
– OH +
PAPS
R
– O –
S
–
O
+
ADP
O
O
Sulfo-transferasePAPS
(
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)
Glutathione
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
Glutathione
Glycine
Glutathione S-transferase
CH - CH O CH – CH - SG OH 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
– SCH
2CHCOOH
HNCCH
3Glutathione S-transferase reactions
Glutathione S-alkyltransferase Glutathione S-alkenetransferase
CHCOOC2H5 CHCOOC2H5 CH2COOC2H5 GS-CHCOOC2H5 CH3I + GSH CH3-SG + HI + GSH Methyl iodide Diethyl maleate Glutathione S-aryltransferase NO2 Cl Cl NO2 Cl SG
Glutathione S-aryl epoxidetransferase
O GSH OH SH + GSH + HCl P-450 Naphthalene Naphthalene oxide 3,4-Dichloronitrobenzen Glutathione S-aralkyltransferase CH2SG CH2Cl + HCl + GSH Benzyl chloride
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
