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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
Binding sites in the cells of the organ
Absorption
Target organ
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
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
Uptake and excretion of hydrophilic and lipophilic compounds
UPTAKE
UPTAKE
ORGAN
ORGAN
EXCRETION
EXCRETION
UPTAKE
BIOTRANSFORMATION
EXCRETION
Primarily biotransformation makes
lipophilic compounds more hydrophilic
XENOBIOTIC
BLOOD
EXCRETION
Somatic
effect
BIOTRANSFORMATION
DNA
damage
Non-toxic
metabolite
Activation
Detoxification
Toxic
metabolite
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
XENOBIOTIC
Oxidation
Reduction
Hydrolysis
PRIMARY
PRODUCT Conjugation
SECUNDARY
PRODUCT
EXCRETION
LIPOPHILIC
HYDROPHILIC
Phase I reactions
Mixed function oxidase enzymes (P450) are
located in the endoplasmic reticulum (SER)
Important phase I enzymes
Enzyme
Co-factor
Substrate
Mixed-function oxidases
(cytochrome P-450)
NADPH
(NADH)
Most lipophilic substances
with M.wt < 800
Carboxyl esterases
Unknown
Lipophilic carboxyl esters
’A’ esterases
Epoxide hydrolases
Ca++
Unknown
Organophosphate esteres
Organic epoxids
Reduktases
NADH
NADPH
Organic nitrous compounds
Organic halogens
P-450 system in the endoplasmic reticulum
Millioner år før nutid
Classification and evolution of the P-450 gene-family
2,000
1,500
1,000
CI
II
II
A B
C E
D
250
III IV XIX
XXI
XVII
LI XI
A B
80
17
I-IV involved in phase I reactions
XI, XVII, XIX, XXI participate in the biosynthesis of steroid hormones
Cytochrome P-450’s catalytic cycle
Xenobiotic
Fe3+
Fe3+
CYT P- 450
NADPH
NADP
e-
CYT P-450 reductase
+
Fe2+
Fe3+
O2
H2O
Fe3+
NADPH
e-
Fe2+
NADP
Fp oxidized
NADPH
e
NADP+
(RH)-P450-(Fe2+)
Fp reduced
(RH)-P450-(Fe3+)
O2
e
(RH)-P450-(Fe2+) ·O2
RH
P450 (Fe3+)
ROH
+
H2O
Examples of oxidations catalysed by P-450
Aliphatic hydroxylation
R - CH2 – CH2 – CH3
Sulphur oxidation
R – CH2 – CHOH – CH3
Aromatic hydroxylation
R
OH
Deamination
R – CH2 – NH2
R - CH - CH - R’
R - NH - C – CH3
R 1R2P - X + S
X
X
R-C-H
R - C - OH
H
R – (NH2, OH, SH) + CH2O
O
R - C - H + NH3
N - hydroxylation
O
R1R2P - X
Oxidative dehalogenation
O
N-, O-, or S-dealkylation
H
R - (N, O, S) - CH3
O
S
R
CH - R’
R - S - R’
De-sulphurnation
Epoxidation
R - CH
R - S - R’
O
R - NOH - C – CH3
H
O
R - C - H + HX
Other phase I enzymes
S
CH2O
O
P
O
CH2O
N
N
CH2O
C2H5
C2H5
MO
P
O
CH2O
N
C2H5
N
C2H5
N
N
CH3
CH3
Diazinone
Diazoxon
O
CH2O
O
CH2O
CH2O
P
O
N
N
C2H5
C2H5
’A’ esterase
P OH
CH2O
+
OH
N
N
N
N
CH3
CH3
Diazoxon
C2H5
C2H5
Other phase I enzymes
Cl
Cl
’B’ esterase
COOCH2
Cl
Cl
COOH
HOH2C
O
Permethrine
Epoxide
hydrolase
OH
O
OH
Benzo(a)pyrene 7,8 oxide
NO2
NH2
Nitroreductase
Nitropyrene
O
Phase II reactions
PHASE I
PHASE II
Expose or add
functional group
XENOBIOTIC
Oxidation
Reduction
Hydrolysis
PRIMARY
PRODUCT Conjugation
SECUNDARY
PRODUCT
EXCRETION
LIPOPHILIC
HYDROPHILIC
Two important co-factors in phase II conjugations
UDP and PAPS
O
NH2
HN
COOH
O
HO
OH
O
O O
O P P O CH2
O O
N
N
O
O O
N
O S P O CH2
O O
N
O
OH
OH OH
Uridine-5’-diphosphoα-D-glucuronic acid (UDP-GA)
OH OH
3’-Phosphoadenosine5’-phosphosulfate (PAPS)
Glucuronyl transferase conjugations
R – OH +
COOH
COOH
O
O
O UDP
OH
Glucuronyl
transferase
HO
O
OH
R + UDP
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
Examples of Glucuronide conjugations
O-Glucuronid
-C–O-G
-C-O-G
O
- CH = C – O - G
Alcohol
Aliphatic
Alicyclic
Phenolic
Carboxyl acid
Aliphatic
Aromatic
α,β-Unsaturated
ketone
Trichloroethanol
Hexobarbital
Estrone
α-Ethylhexanoic acid
o-Aminobenzoic acid
Progesterone
N-Glucuronide
-O–C–N-G
O
Carbamate
Meprobamate
Sulfonamide
Sulfadimethoxine
Aryl thiol
Thiophenol
1,3-Dicarbonyl
system
Phenylbutazone
H
R – SO2 – N - G
H
S-Glucuronide
Ar – S - G
C-Glucuronide
-C-G
Sulfotransferase conjugation
O
R – OH + PAPS
Sulfotransferase
R – O – S – O + ADP
O
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
Glutathione
H
H
N
Glutamic
acid
O
H
H
N
O
H
O
H
N
O
H
O
S
H
H
O
S
O
H
O
+
Cysteine
H
H
N
O
H
N
O
H
H
Glycine
+
N
O
H
H
Glutathione
O
H
O
O
Glutathione S-transferase
O
OH
CH – CH - SG
CH - CH
+ GSH
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 – SCH2CHCOOH
HNCCH3
O
Glutathione S-transferase reactions
Glutathione S-alkyltransferase
CH3I + GSH
Glutathione S-alkenetransferase
CH3-SG + HI
CHCOOC2H5
CHCOOC2H5
Methyl iodide
CH2COOC2H5
+ GSH
GS-CHCOOC2H5
Diethyl maleate
Glutathione S-aryltransferase
Cl
Cl
Cl
Glutathione S-aryl epoxidetransferase
SG
+ GSH
NO2
+ HCl
GSH
P-450
NO2
Naphthalene
3,4-Dichloronitrobenzen
Glutathione S-aralkyltransferase
CH2SG
CH2Cl
+ GSH
Benzyl chloride
+ HCl
OH
O
Naphthalene
oxide
SH
Induction of biotransformation enzymes
Characteristics of the hepatic effects of
Phenobarbital and Benzo[a]pyren (PAH)
CHARACTERISTICS
PHENOBARBITAL
PAH
Onset of effect
Time of maximum effects
Persistence of induction
Liver enlargement
Protein synthesis
Phosphorlipid synthesis
Liver blood flow
Biliary flow
Enzyme components
Cytochrome P-450
Cytochrome P-448
NADRH-cytochrome reductase
Substrate specificity
N-Demethylation
Aliphatic hydroxylation
PAH hydroxylation
Glucuronidation
Glutathione conjugation
Epoxide hydrolase
8-12 hours
3-5 days
5-7 days
marked
large increase
marked increase
increased
increased
3-6 hours
24-48 hours
5-12 days
slight
small increase
no effect
no effects
no effect
increased
no effect
increased
no effect
increased
no effect
increased
increased
small increase
increased
small increase
increased
no effect
no effect
increased
small increase
small increase
small increase
Examples of other inducers
Halogenated pesticides (DDT, aldrin, lindan, chlordan)
PCB
Steroids
Chlorinated dioxins (TCDD)
Alcohol and acetone
Induction of cytochrome P-450
Cell
Ah receptor-hsp90
HC
(inducer)
HC
Nucleus
HC-AhR
HC-AhR
P450 gen
hsp90
XRE
P450 protein
• Bioactivation
• Detoxification
Elimination
P450 mRNA
Toxicity
HC: Hydrocarbon (inducer)
XRC: Regulator gene (stimulates
transcription of P-450 gene)
Bioactivation
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
Examples of bioactivating compounds
Stof
Reactive pathway
or intermediate product
Factors increasing
toxicity
Acetaminophen
N-hydroxylation
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
Sulphate and GSH depletion
Activation of Paracetamol
HNCOCH 3
Acetaminophen
(Paracetamol)
HNCOCH 3
Sulfotransferase
95%
GlucuronosylOH
NADPH
O2
transferase
O
Activation of
cyt. P-450
CONJUGATE
5%
HONCOCH3
HNCOCH3
Mercapturic acid
O
OH
NCOCH 3
At overdose
Glutathione (GSH)
is depleted
HNCOCH3
*
+
O
N-Acetyl-p-Benzoquinoneimin
OH
Cellular
macro molecule Liver
damage