Phase II Metabolism

8/3/2019 Phase II Metabolism

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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


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


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

Phase II Metabolism

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