Phase II Metabolism Conjugation Reactions

Phase II Metabolism

Conjugation reactions add an organic molecule to a xenobiotic to make it more soluble, recognizable and overall more easily excreted. Two General Types of Conjugation Reactions

Mike Hooper
Based on an earlier version by Richard Dickerson

Type I Xenobiotic + Reactive Conjugating Ligand Type II Reactive Xenobiotic + Conjugating Ligand

Conjugated Product Conjugated Product

Examples of Type I Conjugation Methylation

Examples of Type I Conjugation Glucuronidation and Sulfation

*
+ SAM

Acetaminophen Sulfate

Acetaminophen

Acetaminophen Glucuronide

* SAM = S-adenosyl methionine

PAPS= 3-Phosphoadenosine5-phosphosulfate

UDP-Glucuronic Acid = Uridine-5-diphospho--Dglucuronic acid

Examples of Type I Conjugation Acetylation

Example of Type II Conjugation Peptide Conjugation *

Example of Type II Conjugation Glutathione Conjugation

Phase II Metabolism Requires The Presence of a Reactive Group

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

Direct

Indirect

Cofactors are Required In Enzyme-Mediated Conjugation Reactions

Type I Reactive/Activated Cofactor
UDP-Glucuronic Acid and UDP-glucose PAPS Acetyl CoA SAM

Phase II Cofactors
Activated Cofactors

Type II Reactive Xenobiotic

Glutathione Amino Acids (Glycine, glutamine, taurine)
Target of Activated Xenobiotics

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.

Glycogen O CH2OH O OH OH OH O O P OH OH Uridine triphosphate (UTP)

HN N

O O O O O P O P O P O CH2 O O O O HO

Glucose-1-phosphate OH

UDP-Glucuronic Acid Synthesis

UDP-glucose pyrophosphorylase

O O P O

O P OH

O O Pyrophosphate (PPi)

O CH2OH O OH OH OH O O O O O O
UDP-glucose dehydrogenase

HN N
2NAD+ H2O 2NADH 2H+

COOH O OH HO O-UDP OH

O P O P O CH2

Uridine diphosphate glucose (UDP-glucose) HO

OH

UDP-Glucuronic Acid

O COOH O HN

Alcohol Glucuronidation
COOH O OH HO
+
O

OH OH

O O

O O

O O

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.

O P O P O CH2 OH

O-UDP OH

+
Phenol

UDP-Glucuronic Acid

HO

OH

UDP-Glucuronic Acid

UDP activates glucuronic acid by inducing partial positive charge on carbon.

HO

COOH O OH

Nucleophilic Attack

O-UDP OH
UDP

Note alpha configuration and inversion to beta configuration Gut bacteria contain betagalactosidase that hydrolyzes glucuronide conjugates can lead to entero-hepatic recirculation

UDP-Glucuronosyl Transferase

COOH OH HO OH O O

Glucuronidated Phenol

Types of Glucuronidation Reactions

The site of glucuronidation is generally an electron-rich nucleophilic heteroatom (O,N,S) O-glucuronides (alcohols):
Naphthol, chloramphenicol, acetaminophen, codeine, DES, estrone, hexobarbital etc

Glucuronide Excretion
MW < 250 = urine 350 > MW > 250 = either MW > 350 = bile MW cutoff is somewhat species dependent

O-glucuronides (carboxylic acids & esters):

Bilirubin, diclofenac, naproxen, valproic acid, etc

N-glucuronides:
Aniline, amitryptiline, Nhydroxyarylamines, 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

Characteristics of Glucuronide Metabolism

Products are susceptible to enterohepatic circulation
a. Intestinal flora have -glucuronidase activity b. Cleaved aglycones can be re-adsorbed

Genetic and developmental deficiencies of glucuronidation

Human-specific deficiency in glucuronidation Perinatal predisposition for jaundice Increased risk of toxicity from chloramphenicol and other drugs Criglar-Najjar Syndrome and Gilberts Disease lmpaired glucuronidation of bilirubin and some xenobiotics Hyperbilirubinemia

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

Sulfate Conjugation
Biotransforms xenobiotics as well as endogenous compounds Biosynthesis and excretion of thyroid and steroid hormones Also some proteins and peptides Occurs in vertebrates, invertebrates, fungi and bacteria Generally a detoxification mechanism but has been implicated in the formation of reactive intermediates that lead to cancer and tissue damage Sulfotransferase-mediated sulfation reactions generally are high affinity and low capacity. Low levels of PAPS limits this pathway. Despite sulfotransferase as the name, this enzyme catalyzes the transfer of sulfonate, not sulfate (i.e., SO3 , not SO4-)

Cofactor for Sulfation Reaction

Formation of PAPS
Formed in a two step reaction using inorganic sulfate and ATP
Sulfation

Common Acceptors for Sulfotransferases

Hydroxyl groups of phenols, alcohols and Nsubstituted hydroxylamines Thiols and amines

Phosphorylation

General Sulfate Conjugation Reaction Example of Sulfate Conjugation

Example of Reactive Tumorigenic Metabolites Formed via Sulfation Pathway

Sulfotransferases
1. 2. 3. 4. Cytosolic Found in liver, GI and kidney with high activity Some phenol forms found in blood platelets Forms often named by their activity:
a. aryl sulfotransferase- phenols, catechols (or monoamines), hydroxylamines b. hydroxysteroid sulfotransferase- hydroxysteroids and some primary or secondary alcohols c. estrone sulfotransferase-phenolic steroids d. bile salt sulfotransferase-bile acids

5.
DNA binding and tumor formation

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 2,6dichloro-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

Methylation
Common but minor pathway of xenobiotic biotransformation Makes substrates slightly less water soluble and masks available functional groups for conjugation Wide variety of acceptor substrates
Proteins, lipids, phospholipids and nucleic acids SAM most important for xenobiotics containing N, S or O nucleophiles

Cofactor for Methylation Reaction

SAM synthesis
L-methionine + ATP (ATP:L-methionine-S-methyl transferase) = SAM

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 be mono- and di-methylated Inorganic selenium can be trimethylated

Example Compounds that are Methylated

Two Enzymes Catalyze O-Methylation

Phenol O-methyltransferase (POMT), a microsomal enzyme, methylates phenols but not catechols Catechol-O-methyltransferase (COMT), both a cytosolic and microsomal enzyme, methylates catechols but not phenols
Important substrates: epinephrine, norepinephrine, dopamine, L-DOPA, catechol estrogens COMT is polymorphic in humans. High activity is associated with poor therapeutic management of Parkinsons 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 in RBCs

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 sulfhydrylcontaining 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-induced myelotoxicity in cancer patients Patients with high TPMT activity must be given higher doses

Species Differences in Methylation

Guinea pigs have unusually high capacity to methylate histamine and xenobiotics

Thiol methyltransferase (TMT)

Polymorphic in humans

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-NHCOCH3) 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 have low 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 Caucasian populations, and low in Asian populations Now known to be due to genetic polymorphism in NAT2 gene

10

Isoniazid is a Substrate for NAT2

Plasma Isoniazid is increased in slow acetylators

Number of Subjects
Blood levels of Isoniazid, a few hours after standard dose, are 4-6 times higher in slow acetylators

O O C NH NH2

O CoA SH

O C

NH NH C CH3

CoA S C CH3

N-acetyl transferase (NAT 2)

[Blood isoniazid] (ug/ml)

Isoniazid Resistance
Some organisms, including Mycobacterium tuberculosis, express an NAT2-like enzyme With increased expression, the bacterium becomes 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

11

N-OH-2AF and 2AAF are acetylated, and can break down to form reactive ions that bind 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 acylCoA:amino acid N-acyltransferase Amino acids = glycine, glutamine, arginine, taurine in mammals and primates; ornithine in reptiles and birds Activity herbivores>omnivores>carnivores

Amino Acid Conjugation

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 acyl-CoA thioether that reacts 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

12

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

Significance of Glutathione Conjugation for Toxicology Cofactor for glutathione conjugation is a tripeptide
Electrophilic substrates are potentially toxic species that can bind to critical nucleophiles, such as proteins and nucleic acids, and cause cell damage and genetic mutations Glutathione is a cofactor for glutathione peroxidase, which protects cells against lipid peroxidation High glutathione peroxidase levels have been linked to: DDT resistance in insects Corn resistance to atrazine Cancer cells to chemotherapeutic agents

13

Significance of Glutathione Conjugation for Toxicology (contd)

GSTs are major determinants of differential chemicalinduced 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

14

Enhancement of Toxicity by Glutathione Conjugation

See Figure 6-59 in Casarett and Doull, and explanatory text

Integration of Principles:
Acetaminophen

15