phase ii metabolism
TRANSCRIPT
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Phase II Metabolism
Mike Hooper
Based on an earlier version by Richard Dickerson
Phase II Metabolism Conjugation Reactions
Two General Types of Conjugation Reactions
Type I
Xenobiotic + Reactive Conjugating Ligand ConjugatedProduct
Type II
Reactive Xenobiotic + Conjugating Ligand ConjugatedProduct
Conjugation reactions add an organic molecule to axenobiotic to make it more soluble, recognizable and overallmore easily excreted.
Examples of Type I Conjugation
Methylation
+ SAM *
* SAM = S-adenosyl methionine
Examples of Type I Conjugation
Glucuronidation and Sulfation
AcetaminophenAcetaminophen
Sulfate
Acetaminophen
Glucuronide
UDP-Glucuronic Acid =
Uridine-5-diphospho--D-
glucuronic acid
PAPS=
3-Phosphoadenosine-
5-phosphosulfate
* *
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Examples of
Type I Conjugation
Acetylation
*
Example ofType II Conjugation
Peptide Conjugation
*
Example of Type II Conjugation
Glutathione Conjugation
Indirect
Direct
Hydroxyl group (R-OH)
Amino group (R-NH2)
Carboxyl group (R-COOH)
Epoxide group (R1-COC-R2)
Thiol group (R-SH)
Halogen group (R-X)
Electrophiles
Some others
Phase II Metabolism Requires
The Presence of a Reactive Group
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Type I Reactive/Activated Cofactor
UDP-Glucuronic Acid and UDP-glucose
PAPS
Acetyl CoA
SAM
Type II Reactive Xenobiotic
Glutathione
Amino Acids (Glycine, glutamine, taurine)
Cofactors are Required
In Enzyme-Mediated Conjugation Reactions
Activated
Cofactors
Target of
Activated
Xenobiotics
Phase II
Cofactors
Glycoside Formation
Glucuronidation & Glucosidation
Most important and widespread form of
conjugation
1. Found in plants, animals and
microorganisms 2. Reactive intermediates formed from
glucose
3. Supply unlikely to be depleted
(typically high capacity, low affinity
reactions)
4. Ability to react with wide range of
functional groups.
N
NH
O
O
P
O
O
O O P
O
OO
OHOH
CH2
OP
O
O
O
O
OH
OH
OH
CH2OH
OH
P
O
OHO
P
O
O
O O P
O
O
OH
O
OH
OH
OH
CH2OH
O
P
O
OO
N
NH
O
O
O
OHOH
CH2
OP
O
O
O
O-UDP
OH
OH
OH
COOH
2NAD+
H2O
2NADH2H+
Glycogen
Glucose-1-phosphate
Uridine triphosphate(UTP)
UDP-glucosepyrophosphorylase
Uridine diphosphate glucose(UDP-glucose)
Pyrophosphate (PPi)
UDP-glucosedehydrogenase
UDP-Glucuronic Acid
+
UDP-Glucuronic Acid
Synthesis
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O
O-UDP
OH
OH
OH
COOH
UDP-Glucuronic Acid
+
+
O
Phenol
OO
O-UDP
OH
OH
OH
COOH
+
O
OH
OH
OH
COOH
O Glucuronidated
Phenol
UDP-Glucuronosyl
Transferase
NucleophilicAttack
UDP
O
OH
OH
OHO
P
O
OO
N
NH
O
O
O
OHOH
CH2
OP
O
O
COOH
UDP-Glucuronic Acid
+
Alcohol Glucuronidation
Note alpha configuration andinversion to beta configuration
Gut bacteria contain beta-
galactosidase that hydrolyzes
glucuronide conjugates can lead
to entero-hepatic recirculation
UDP activates glucuronic acid
by inducing partial positive
charge on carbon.
Species Differences
in Glycosylation Reactions
Glucuronidation is a major pathway in
vertebrates (except for cat family!).
Glucosidation is a major pathway in plants
and invertebrates.
However, some glucosidation occurs in
mammals.
Types of Glucuronidation Reactions
The site of glucuronidation is generally anelectron-rich nucleophilic heteroatom(O,N,S)
O-glucuronides (alcohols): Naphthol, chloramphenicol,
acetaminophen, codeine, DES, estrone,hexobarbital etc
O-glucuronides (carboxylic acids & esters): Bilirubin, diclofenac, naproxen, valproic
acid, etc
N-glucuronides: Aniline, amitryptiline, N-
hydroxyarylamines, benzidine,imipramine, etc
S-glucuronides: Diethyldithiocarbamate, thiophenol etc
C-glucuronides: Phenylbutazone, sulfinpyrazone
(Blue arrow indicates site of conjugation)
See Figure 6-47
In C&D
Glucuronide Excretion
MW < 250 = urine
350 > MW > 250 = either
MW > 350 = bile
MW cutoff is somewhat species dependent
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Characteristics of
Glucuronide Metabolism
Products are susceptible to enterohepatic
circulation
a. Intestinal flora have -glucuronidase activity
b. Cleaved aglycones can be re-adsorbed
Glucuronides can be cleaved by acid or base
UDP-GTs are inducible by 3-MC, PB, TCDD,
Aroclors, dietary cabbage and brussel sprouts,
smoking Subject to inter-individual variation, including
genetic and developmental deficiencies
Genetic and developmental
deficiencies of glucuronidation
Human-specific deficiency in glucuronidation
Perinatal predisposition for jaundice
Increased risk of toxicity fromchloramphenicol and other drugs
Criglar-Najjar Syndrome and Gilberts Disease
lmpaired glucuronidation of bilirubin and somexenobiotics
Hyperbilirubinemia
Sulfate Conjugation
Biotransforms xenobiotics as well as endogenouscompounds
Biosynthesis and excretion of thyroid and steroidhormones
Also some proteins and peptides Occurs in vertebrates, invertebrates, fungi and bacteria
Generally a detoxification mechanism but has beenimplicated in the formation of reactive intermediates thatlead to cancer and tissue damage
Sulfotransferase-mediated sulfation reactions generallyare high affinity and low capacity. Low levels of PAPSlimits this pathway.
Despite sulfotransferase as the name, this enzymecatalyzes the transfer of sulfonate, not sulfate (i.e., SO3 ,not SO4-)
Cofactor for Sulfation Reaction
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Formation of PAPS
Formed in a two step reaction using
inorganic sulfate and ATP
Sulfation
Phosphorylation
Common Acceptors for Sulfotransferases
Hydroxyl groups of phenols, alcohols and N-
substituted hydroxylamines
Thiols and amines
General Sulfate Conjugation Reaction
Example of Sulfate Conjugation
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Example of
ReactiveTumorigenic
Metabolites
Formed via
Sulfation
Pathway
DNA binding and tumor
formation
Sulfotransferases
1. Cytosolic
2. Found in liver, GI and kidney with high activity
3. Some phenol forms found in blood platelets
4. Forms often named by their activity:
a. aryl sulfotransferase- phenols, catechols (ormonoamines), hydroxylamines
b. hydroxysteroid sulfotransferase- hydroxysteroidsand some primary or secondary alcohols
c. estrone sulfotransferase-phenolic steroids
d. bile salt sulfotransferase-bile acids
5. Five gene families (alternative nomenclature)
Xenobiotics and Endogenous Compounds
that Undergo Sulfate Conjugation
Primary alcohols: Chloramphenicol, ethanol, hydroxymethyl
PAHs, polyethylene glycols
Secondary alcohols: Bile acids, 2-butanol, cholesterol,
dehydroepiandrosterone, doxaminol
Phenols: Acetaminophen, estrone, ethinylestradiol, naphthol,
pentachlorophenol, phenol, picenadol, salicylamide,
trimetrexate
Catechols: Dopamine, ellagic acid, methyl-DOPA
N-oxides: Minoxidil
Aliphatic amines: 2-amino-3,8-dimethylimidazo [4,5,-f]-
quinoxaline (MeIQx), 2-amino-3-methylinidaz0-[4,5-f]-
quinoline (IQ), 2-cyanoethyl-N-hydroxythioacetaminde,
despramine
Aromatic amines: 2-aminonaphthalene, aniline
Aromatic hydroxylamines: N-hydroxy-2-aminonaphthalene
Aromatic hydroxyamides: N-hydroxy-2-acethylaminoflurorene
Other characteristics of ST reactions
Sulfotransferase activity is low in pigs, but high in cats
High sulfotransferase activity in cats offsets their low capacity to
conjugate xenbiotics with glucuronic acid
Activity is higher in male rats, compared to females
Sex differences due to complex interplay between gonadal, thyroidal,
and pituitary hormones
Sulfation can determine the rate of elimination of thyroid hormones in
some species
Unlike UGTs, STs are not readily inducible
Low levels of one of the phenolsulfotransferases predisposes
individuals to diet-induced migraine headaches
STs can be experimentally inhibited with pentachlorophenol and 2,6-
dichloro-4-nitrophenol
Products generally secreted in the urine
Sulfate conjugates excreted in bile may be hydrolyzed by aryl sulfatases
present in gut microflora, therefore subject to enterohepatic
recirculation
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Methylation
Common but minor pathway of xenobioticbiotransformation
Makes substrates slightly less water soluble andmasks available functional groups forconjugation
Wide variety of acceptor substrates
Proteins, lipids, phospholipids and nucleic acids
SAM most important for xenobiotics containing N,
S or O nucleophiles SAM synthesis
L-methionine + ATP (ATP:L-methionine-S-methyltransferase) = SAM
Cofactor for Methylation Reaction
Functional groups for methylation
Phenols,
Catechols
Aliphatic and aromatic amines
N-heterocyclics Sulfhydryl-containing compounds
Metals can also be methylated
Inorganic mercury and arsenic can both bemono- and di-methylated
Inorganic selenium can be trimethylated
Example Compounds that are Methylated
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Two Enzymes Catalyze O-Methylation
Phenol O-methyltransferase (POMT), amicrosomal enzyme, methylates phenols but notcatechols
Catechol-O-methyltransferase (COMT), both acytosolic and microsomal enzyme, methylatescatechols but not phenols
Important substrates: epinephrine, norepinephrine,dopamine, L-DOPA, catechol estrogens
COMT is polymorphic in humans. High activity isassociated with poor therapeutic management ofParkinsons disease
N-Methylation
Phenylethanolamine N-methyltransferase (PNMT) Catalyzes the N-methylation of norepinephrine to form
epinephrine
Histamine N-methyltransferase (HNMT) Methylates the imidazole ring of histamine and closely
related compounds
Genetic polymorphism in humans, can be measured inRBCs
Nicotinamide N-methyltransferase (NNMT)
Methylates compounds containing a pyridine ring Examples: nicotinamide and nicotine
Methylates compounds containing an indole ring Examples: tryptophan and serotonin
S-Methylation
Important biotransformation pathway for sulfhydryl-containing xenobiotics Examples:
D-penicillamine (antirheumatic agent)
6-mercaptopurin (antineoplastic and imunosuppressive) Disulfuram (antibuse)
S-methylation catalyzed by 2 enzymes Thiopurine methyltransferase (TPMT)
Polymorphic in humans
Low TPMT activity increases risk of thiopurine-inducedmyelotoxicity in cancer patients
Patients with high TPMT activity must be given higher doses
Thiol methyltransferase (TMT) Polymorphic in humans
Species Differences in Methylation
Guinea pigs have unusually high capacity to
methylate histamine and xenobiotics
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Acetylation
Major route of biotransformation for xenobiotics
containing an aromatic amine (R-NH2) or a
hydrazine group (R-NH-NH2)
These are converted to aromatic amides (R-NH-
COCH3) and hydrazides (R-NH-NH-COCH3),
respectively
Cofactor for Acetylation Reaction
Characteristics of Acetylases
Cytosolic
Liver and many other mammalian tissues
Wide species variability: dog and fox are
unable to acetylate xenobiotics, cats havelow activity
Humans, rats, and hamsters express two
N-acetyltransferases (NAT-1 and NAT-2)
Mice express three forms
Fast and Slow Acetylators
Documented in 1950s by the differential
metabolism of isoniazid (anti-tuberculosis drug)
Incidence of slow acetylator is high in Middle
Eastern populations, intermediate in Caucasianpopulations, and low in Asian populations
Now known to be due to genetic polymorphism in
NAT2 gene
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Isoniazid is a Substrate for NAT2
N
C
O NH NH C
O
CH3
N
C
O NH NH2
CH3
C
O
SCoA SHCoA
N-acetyl transferase(NAT 2)
Plasma Isoniazid is increased
in slow acetylators
[Blood isoniazid] (ug/ml)
NumberofSubjects Blood levels of Isoniazid, a
few hours after standard
dose, are 4-6 times higher
in slow acetylators
Isoniazid Resistance
Some organisms, including Mycobacterium
tuberculosis, express an NAT2-like enzyme
With increased expression, the bacteriumbecomes resistant to standard treatment
Risk factors associated with rates of
acetylation in humans
Slow NAT2 acetylators are at increased risk:
nerve damage (peripheral neuropathy) from isoniazid
and dapsone
Systemic lupus erythematosis from hydralazine and
procainamide Various drug interactions
Bladder cancer from cigarette smoking and
occupational exposure to bicyclic aromatic amines
Fast NAT2 acetylators are at increased risk:
Myelotoxic effects of the antineoplastic drug, amonafide
(N-acetylation slows clearance)
Colon cancer from heterocyclic aromatic amines
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N-OH-2AF
and 2AAFare
acetylated,
and can
break down
to form
reactive
ions thatbind to DNA
N+
H
Amino Acid Conjugation
Activation of carboxylic acid xenobiotic to CoA
derivative using acid:CoA ligase
Acyl CoA derivative reacts with an amino acid
giving an acylated amino acid conjugate plus
CoA. This reaction is catalyzed by acyl-
CoA:amino acid N-acyltransferase
Amino acids = glycine, glutamine, arginine,
taurine in mammals and primates; ornithine inreptiles and birds
Activity herbivores>omnivores>carnivores
Amino Acid ConjugationAmino Acid Conjugation
Activation of carboxylic acid xenobiotic to
CoA derivative using acid:CoA ligase
Acyl CoA derivative reacts with an amino
acid giving an acyl-CoA thioether thatreacts with the amino group of an amino
acid to form an amide linkage. This
reaction is catalyzed by acyl-CoA:amino
acid N-acyltransferase
Example: Benzoic acid and glycine
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Amino Acid Conjugation
Predominant amino acids:
Glycine, glutamine, arginine, taurine in
mammals and primates; ornithine in reptiles
and birds
Activity herbivores>omnivores>carnivores
Glutathione Conjugation
Substrates share features
Hydrophobic
Contain an electrophilic atom (+ or partial+)
React nonenzymatically with glutathione to
some degree. Glutathione transferases
(GSTs) increase the rate of this reaction
Concentration of glutathione is high in
cells (10 mM)
Some GST substrates are also inducers
Cofactor for
glutathione
conjugation
is atripeptide
Significance of Glutathione Conjugation
for Toxicology
Electrophilic substrates are potentially toxicspecies that can bind to critical nucleophiles,such as proteins and nucleic acids, and causecell damage and genetic mutations
Glutathione is a cofactor for glutathioneperoxidase, which protects cells against lipidperoxidation
High glutathione peroxidase levels have beenlinked to:
DDT resistance in insects
Corn resistance to atrazine
Cancer cells to chemotherapeutic agents
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Significance of Glutathione
Conjugation for Toxicology (contd)
GSTs are major determinants of differential chemical-
induced toxicity
Example: Rats are more sensitive to aflatoxin B1
toxicity than mice due to high levels of a GST form
GST is polymorphic in humans
Individuals with null alleles for GST-M1 are at
increased risk for cigarette smoking-induced lung and
bladder cancer Individuals with deletion of GST-T1 are at increased
risk for development of astrocytoma, meningioma, and
myelodysplasia
Risks due to polymorphisms are small, but additive
Direct Conjugation with Glutathione
Initial Step in GST-Catalyzed
Glutathione Conjugation
Glutathion Degradation
and
Mercapturic Acid Synthesis
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Enhancement of Toxicity
by Glutathione Conjugation
See Figure 6-59 in Casarett and Doull, and
explanatory textIntegration
of Principles:Acetaminophen