Lecture 5: Phase II Metabolism

Question 1

Glucuronidation*

Answer 1

Enzyme: Glucuronosyltransferases(UGT)
cofactor: urindine-5’-diphospho-D-glucuronic acid (UDP-GA)

Question 2

Sulfation*

Answer 2

Enzyme: Sulfotransferases (ST)
cofactor: 3’-phophoadenosine-5’-phosphosulfate (PAPS)

Question 3

Flutathione Conjugation*

Answer 3

Enzyme: Glutathione S-transferase
Cofactor: glutathione

Question 4

Acetylation*

Answer 4

Enzyme: acetyltransferase
Cofactor: acetyl coenzyme A

Question 5

Methylation*

Answer 5

Enzyme: methyl transferases
Cofactor: S-adenosylmethionine (SAM)

Question 6

Amino Acid Conjugation*

Answer 6

Cofactors: taurine, glycine and glutamine

Question 7

Glucuronidation

Answer 7

• Glucuronidation is a major pathway of xenobiotic biotransformation in mammalian species, except for the cat family.
• Glucuronidation requires UDP-GA and UGTs, located in the ER of liver, intestine, skin, brain, spleen and nasal mucosa.
• The site of glucuronidation is generally an electron-rich nucleophilic heteroatom (O, N, S).

Question 8

UGT is a Low Specificity, High Capacity Enzyme

Answer 8

• At low doses of xenobiotic, sulfate conjugates are predominant products.
• At high doses of xenobiotic, glucuronide conjugates predominate

Question 9

Synthesis of UDP-Glucuronic Acid

Answer 9

The cofactor UDP-GA is synthesized from glucose-1-phosphate and the linkage between GA and UDP has an α-configuration, which protects it from hydrolysis by β-glucuronidase.

Question 10

Enterohepatic Circulation of Glucuronides

Answer 10

–Xenobiotics conjugated by glucuronides have a β-configuration because of the nucleophilic attack by an electron rich atom on UDP-glucuronic acid, opposite to the linkage between glucuronic acid and UDP. -Enterohepatic circulation delays the elimination of xenobiotics and can increase toxicity.

Question 11

Sulfation

Answer 11

• Many xenobiotics and endogenous substrates that undergo O-glucuronidation also undergo sulfate conjugation.
• Sulfation occurs through sulfotransferases (SULT)—there are many isoforms of SULTs
• SULTS use phosphoadenosinephosphosulfate (PAPS) as a sulfate donor

Question 12

Sulfate conjugate excretion

Answer 12

• Most sulfate conjugates are excreted in the urine (actively excreted by organic anion transporters.(OAT)
• Some excreted in the bile may be hydrolyzed by arylsulfatases in gut microflora, which can contribute to enterohepatic circulation of certain xenobiotics. (occurs much less than gluconacidation)

Question 13

Sulfotransferase Genes

Answer 13

-There are nine genes encoding cytosolic sulfotransferases in humans, and they belong to the SULT1 or SULT2 gene families.

Question 14

ST Polymorphisms

Answer 14

SULT1A1, loss of function is associated with a 3.5-fold increase in esophageal cancer in high-risk males (alcohol, smoking).

Question 15

Glutathione Conjugation

Answer 15

• Substrates for glutathione conjugation include an enormous array of electrophilic xenobiotics, or xenobiotics biotransformed to electrophiles.
• Substrates for glutathione S-transferase (GST) share 3 common features: 1) hydrophobic; 2) electrophilic; 3) react nonenzymatically with glutathione (GSH) at a measurable rate.
• The concentration of GSH is very high in liver (10 mM) and GST makes up 10 % of total cellular protein.
• GSH is the co-factor for GST

Question 16

Aflatoxin

Answer 16

• Aflatoxins are naturally occurring mycotoxins that are produced by many species of Aspergillus, a fungus.
• They can be found on moldy peanuts, corn and other crops.
• Aflatoxin B1 is the most potent liver carcinogen.

Question 17

Glutathione S-transferase

Answer 17

GSTs are dimers composed of identical subunits of 23-29 kDa, although some form heterodimers. 95 % are soluble and 5 % are microsomal.
1.Microsomal
2.Soluble (4 Classes)
3.A: GSTA1 formerly 
		called ligandin; (basic pI)
-M: neutral pI
-P: acidic pI
-T: one enzyme

Question 18

Excretion of Glutathione Conjugates

Answer 18

Glutathione conjugates can be formed in the liver and can be excreted intact in bile or can be converted to mercapturic acids in the kidney and excreted in the urine.

Question 19

N-Acetyltransferases (NAT)

Answer 19

• N-acetylation of xenobiotics is performed by N-acetyltransferases (NAT)
• N-acetylation is a major route of biotransformation for xenobiotics containing an aromatic amine (R-NH2).
• Unlike other Phase II reactions, acetylation masks an amine with a nonionizable group and are less water soluble than the parent compound.
• NAT uses the co-factor acetyl-Coenzyme A (acetyl CoA)
• There are two N-acetyltransferases NAT1 and NAT2

Question 20

Polycyclic aromatic amines:

β-napthylamine

Answer 20

• 2-Naphthylamine (BNA) is an aromatic amine used to make azo dyes. It is a known human bladder carcinogen and has largely been replaced by less toxic compounds.
• BNA also is present in cigarette smoke

Question 21

Methylation

Answer 21

• Methylation is a common but generally minor pathway of xenobiotic transformation.
• Methylation differs from other conjugations because it generally decreases water solubility of the parents compound.
• An exception is the N-methylation of pyridine-containing xenobiotics such as nicotine, which produces quaternary ammonium ions are more water soluble and readily excreted.
• Another exception is the S-methylation of thioethers to form a positively charged sulfonium ion.
• There are many types of methyltransferases, e.g. catechol-O-methyltransferase (COMT), phenol-O-methyltransferase (POMT)
• Methyltransferases uses S-adenosylmethionine (SAM) as a co-factor