Download BIOTRANSFORMATION PHASE I Phase II

BIOTRANSFORMATION
•Enzymatic processes in liver and other tissues that modify the chemical
structure of xenobiotics, render them more water-soluble, increase their
elimination, decrease their half-life
•Biotransformed
Biotransformed metabolites are chemically different from the parent molecule
Xenobiotic
PHASE I
Expose or Add
functional groups
Oxidation
Reduction
H d l i
Hydrolysis
Lipophilic
Primary
products
PHASE II
Biosynthetic
conjugation
Secondary
products
Excretion
Hydrophilic
(Ionizable)
e.g. barbital (water soluble) vs hexobarbital (highly liposoluble)
t1/2= 55h (theoretical, real) vs t1/2 of months, real t1/2= 5hr
PHASE I
Add or expose
functional groups:
-OH, -SH, -NH2,
-COOH,
COOH …
(small increase in
hydrophilicity)
Phase II
Conjugation with
endogenous molecules:
glucuronic acid, sulfate,
glutathione, ...
covalent bonds formed
(large increase in
hydrophilicity)
Table 6.1 C&D
1
Major organ of biotransformation
LIVER (hepatocytes)
Secondary organs of biotransformation
kidney
lungs
testes
skin
intestines
Preparation of Phase I enzymes (e.g. liver)
endoplasmic reticulum (microsomes) and cytosol
Liver homogenate
Centrifuge at 9 000g
(separation of mitochondria
mitochondria, lysosomes,
lysosomes nuclei,
nuclei broken cells)
Supernatant at 105 000g
Precipitate
(Phase I)
Supernatant
(Phase II)
Biotransformation
PHASE I
Hydrolysis
(chemical reaction of a compound with water, usually resulting in the formation
of one or more new compounds)
•
Carboxylesterases, cholinesterases, organophosphotases
•
Peptidases
•
Epoxide Hydrolase (EH)
-detoxifying
detoxifying enzyme for epoxides
-formation of diols
-EH present in many tissues
-epoxides: electrophilic,
tend to bind to proteins,
to nucleic acids
Fig. 6.4 C&D, Hydrolyation of epoxides by EH
2
Role of EH in the biotransformation of benzo[a]pyrene B[a]P
•Inactivation of benzo[a]pyrene 4,5-oxide
•Conversion of B[a]P to tumorigenic diolepoxide
Fig. 6.6
C&D
REDUCTION
e.g Azo- and Nitro-Reductions
Azo-reduction
N=N
Prontosil
[4H]
-NH2
1,2,4-triaminobenzene
-NH2
Sulfanilamide
Fig. 6.8 C&D
Azo- and Nitro-reductions can be catalyzed
•by enzymes of intestinal flora
• by cytochrome P450 (usually oxidizing enzyme),
has the capacity to reduce xenobiotics under low oxygen or anaerobic
conditions (substrate rather than oxygen, accept electrons and is reduced)
•interactions with reducing agents (reduced forms of glutathione, NADP,…)
3
REDUCTION – Role of intestinal microbial flora in biotransformation
•
•Oxidation by cytochrome P450
•Conjugation with glucuronic
acid by UDP-GT
UDP GT
•Excretion of glucuronide in
bile
•Reduction by microbial flora
under anaerobic conditions
•Reabsorption from intestine
•Hydroxylation of amine group
•Conjugation with sulfate or
acetate
•Reactive metabolites
•Tumorigenic
Fig. 6.9 C&D
OXIDATION
•Monoamine oxidase (MAO), Multiple function oxidase (MFO)
•Flavin Monooxygenases (nicotine, cocaine,…)
•Alcohol dehydrogenase (liver ADH, gastric ADH)
oxidation of alcohols to aldehydes
low ADH links to high blood alcohol levels
lower gastric ADH in women/men
ADH activity lower after fasting
clinical alcoholics have lower gastric ADH
racial differences in gastric ADH
•Aldehyde dehydrogenase (ALDH)
oxidation of aldehydes to carboxylic acid
ADH
R-CH2OH
NAD+
ALDH
R-C=O
H
NADH + H+
NAD+ + H2O
R-C=O
OH
NADH + H+
Fig. 6.19
C&D
4
Biotransformation of alcohol
Fig. 6.20C&D
CYTOCHROME P450
•
•
Table 6.6 C&D
•
most versatile oxidative enzyme
heme-containing protein
ferric Fe3+ reduced to ferrous Fe2+
reduced cytP450 can bind oxygen
6.35 C&D
5
Nomenclature of cytochrome P450 – use of recombinant DNA
CYP
Amino acid sequence known for many cytP450s
<40% a.a. homology, assigned to different genes
(CYP1 CYP2
(CYP1,
CYP2, CYP3
CYP3,..))
40-55% a.a. homology, different subfamily
(CYP2A, CYP2B, CYP2C,…)
>55% a.a. homology, different members of same subfamily
(CYP2A1, CYP2A2,…)
Use of Knockout Mice
Inhibition of cytochrome P450
(aminobenzotriazole – inhibits synthesis of cytP450)
detergents, CO, …
Fig. 6.35 C&D
6
Cytochrome P450
-can oxidize a substrate (e.g. a xenobiotic)
-can transport electrons
-many isoforms of cytP450 isolated (in human, rat, others)
-importance of NADPH-cytochrome P450 reductase
(embedded in ER membrane with cytP450)
-catalytic
catalytic cycle
-inducible by specific substrates (e.g. phenobarbital,
benzo[a]pyrene, methylcholanthrene)
Simplified reaction:
Substrate (RH) + O2 + NADPH + H+
Product (ROH) + H2O + NADP+
How can we demonstrate a role for cytP450 in biotransformation of a XB?
-cytP450 activity increases
-enzymatic inhibitors of cytP450 (e.g. aminobenzotriazole)
-absorption spectrum of cytP450
-reaction can be reconstituted
Hydroxylation of substrates by cytochrome P450
Fig. 6.36 C&D
Deactivation (detoxication)
7
Role of cytochrome P450 and peroxidase in activation of benzene in
bone marrow
Fig. 6.29 C&D
PHS – prostaglandin H synthase
Halothane hepatitis and activation by oxidation and reduction
Fig. 6.16 C&D
8
Reactions catalyzed by cytochrome P450
Fig. 6.43 C&D
Experimental studies with biotransformation enzymes
1. Induction of enzymes
exposure to inducing agents
phenobarbital, PAH
stimulation of protein synthesis “de novo”
Characteristics
Time of max effect
Persistence of induction
Protein synthesis
Biliairy flow
Cytochrome P450
Cytochrome P448
NADPH-cytochrome-c
Reductase
PAH hydroxylation
Reductive dehalogenation
Epoxide hydrolase
….
phenobarbital
3-5 days
5-7 days
large increase
increase
increase
no effect
PAH
1-2 days
5-12 days
small increase
no effect
no effect
increase
increase
small increase
increase
increase
no effect
increase
no effect
small increase
2. Inhibition of biotransformation enzymes
e.g. inhibition cytP450 (see graph)
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BIOTRANSFORMATION
•Enzymatic processes in liver and other tissues that modify the chemical
structure of xenobiotics, render them more water-soluble, increase their
elimination, decrease their half-life
•Biotransformed
Biotransformed metabolites are chemically different from the parent molecule
Xenobiotic
PHASE I
Expose or Add
functional groups
Oxidation
Reduction
H d l i
Hydrolysis
Lipophilic
Primary
products
PHASE II
Biosynthetic
conjugation
Secondary
products
Excretion
Hydrophilic
(Ionizable)
Structure of cofactors for Phase II biotransformation
PHASE II REACTIONS
•biosynthesis of polar,
hydrophilic metabolites
eliminated in urine
(small M.W.,250)
or bile (M.W.>350)
•requires cofactors and
endogenous substrates
•ATP dependent
Fig. 6.45 C&D
10
Substrates that are glucuronidated
1. Glucuronidation
major Phase II pathway in all mammals,
except cats
•
•
conjugation
j g
with g
glucuronic acid
mediated by UDP-GT
Liver>>>>kidney, lung, brain
localized in ER
(other Phase II enzymes in cytosol)
•
•
detoxication or bioactivation
large increase in hydrophilicity
Fig. 6.47 C&D
Bioactivation of xenobiotics by Phase II reactions (glucuronidation)
Fig. 6.49 C&D
11
bile
Fig. 6.9 C&D
2. SULFATION
Bioactivation by sulfation
•cofactor:
PAPS
(3’-phosphoadenosine-5Phosphosulfate)
•enzyme
enzyme
Sulfotransferase
•detoxication or
bioactivation
3. Methylation
•cofactor: SAM
(s-adenosyl methionine)
•enzyme
methyl transferase
(minor, ~hydrophilicity)
4. Acetylation
Fig. 6.50 C&D
12
Toxicity/Damage
– alteration of the regulatory or maintenance function of the cell in target tissue
Fig. 3.10
5. GLUTATHIONE CONJUGATION
•cofactor: GSH (tripeptide glycine, glutamic acid, cysteine)
•enzyme: glutathione-S-transferase (GST)
localized in cytoplasm>>>ER
liver>kidney, testes, intestine, adrenal
GST
GSH + Substrate (XB)
polar metabolites
•substrates for GST must
have affinity for GSH
be hydrophobic
contain electrophilic carbon
•glutathione conjugates are
excreted in the bile (intact)
excreted as mercapturic acid in urine
13
Biotransformation of acetaminophen – the good, the bad and the ugly
Fig.6.28 C&D
Biotransformation of acetaminophen
Phase I
Phase II:
At low dose of Acetaminophen:
90% of dose excreted as sulfates
(sulfotransferase has strong affinity, low capacity)
no toxic intermediates, no toxicity
At high dose, Phase II enzymes capacity is surpassed
43% of dose excreted as sulfates
glucuronides, glutahione cojugates (low affinity, strong capacity)
accumulation
l i off toxic
i iintermediates,
di
toxicity
i i
•Protective effect of GSH supplementation, e.g. NAC
•Fasting for 1 day decreases liver GSH levels by 50% in rats
(increase in toxicity of acetaminophen) – effect of nutritional status
•Liver pathologies will also influence its biotransformation capacity, as will
age, sex, species differences
14
Biotransformation and difficulties of predicting effects of mixtures
Fig. 6.14
15
Toxicokinetics : study of modeling and mathematical description of the time
course of xenobiotics in the whole organism
•Dilution and distribution of agent A
•Routes of excretion
•Theoretical graphs of blood
concentrations of A against
time (at time A, B and C)
Compare a mammal
to fish
to amphibian
Stocking DDT, PCB in adipose tissue
Stocking Pb, St in bone
Ecobichon, p.13
C = f(t) C = dependent variable
t = independent variable
dC
dt
if > 0 → accumulation
if < 0 → elimination
Ecobichon
16
Classical approach to toxicokinetics:
administer XB in a bolus mode (e.g. iv)
take blood samples
measure XB in blood
•equilibrium reached rapidly between blood
and tissues
•body behaves as a homogenous unit
C = Co x e –kel x t
•more complex pattern of distribution
•two (or more) compartments involved
C = A x e –α x t + B x e –ß x t
where A and B are
proportionality constants of the
change in C
α= constant of distribution
ß= constant of elimination
Fig.7.1
One compartment model
log of plasma concentrations vs time
Fig.7.2
C = Co x e –kel x t
straight line
(monoexponential model)
Log C = - (kel/2.303)
/2 303) x t + log Co (logarithmic equation)
Kel = elimination constant (elimination includes biotransformation, excretion and
exhalation)
C=plasma concentration
Co=concentration at t= 0 (y intercept )
How does the dose or the remaining concentration in the plasma influence elimination?
17