Humboldt State University ® Department of Chemistry

Richard A. Paselk

Chem 451

Biochemical Toxicology

Spring 2010

Lecture Notes:: 9 March

© R. Paselk 2008
 
     
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Reduction Reactions

Reductions take place in both the soluble and microsomal fractions under anaerobic conditions. Cytochrome P450 or a flavoprotein may be involved in the microsomal reactions, with NADPH as a required reductant.

Important reduction reactions may also take place in the gut by bacterial P450 reductases. An example is the reduction of the azo dye prontosil to give the antimicrobial drug sulfanilamide:

structural diagram for Prontosil reductive metabolism

Nitrobenzene toxicity to red blood cells occurs as a result of its reduction by the microbial P450/NADPH system. The nitrosobenzene and phenylhydroxylamine intermediates in this reduction are toxic to red cells:

Nitrobenzene reductive metabolism

Reductive Dehydrohalogenation

Under anerobic conditions can get dehalogenation using P450/NADPH. An example is the reaction of Halothane under anaerobic conditions:

structural diagram for reductive dehydrohalogenation

 

Hydrolysis Reactions

Esters and amides are commonly hydrolyzed:

structural diagram for the hydrolysis of esters

An example is the hydrolysis of the anesthetic, procaine, that is rapidly hydrolized by esterases in the plasma and by tissue microsomal esterases:

structural diagram for the hydrolysis of Procaine

The analogous amide, procainamide, is hydrolyzed more slowly by amidases in the microsomal fraction of tissue. It is not hydrolyzed at all in the plasma.

Phase Two Reactions

Phase two reactions involve the attachment of a generally polar, readily available in vivo, molecule to a susceptible functional group. Reactive functional groups are often, but not necessarily a result of Phase 1 reactions). The result is to make the whole molecule more polar, and thus more readily excreted.

Process in vivo reactant Product 
Glucuronide formation 
 Haworth structure for UDP-alpha-D-glucuronate
UDP--D-glucuronate
Haworth structure for Glucuronide
Glucuronide (X = O, N, or S)
 Glutathione conjugation
 glutathione
glutathione (Glu, Cys, Gly)
Mercapturic acid
 Mercapturic acid
 Sulfate conjugation
 Haworth structure for PAPS
PAPS = Adenosine-3'-P-5'-Phosphosulfate
alkyl sulfate
Aryl or alkyl sulfates and sulfamates
 Acetylation
 Acetyl Coenzyme A
Acetyl Coenzyme A
Acetylated derivative
Acetylated derivatives 
 Amino acid conjugation  Coenzyme A, ATP & e.g. glycine Acyl derivatives
 Methylation
 Haworth structure for S-Adenosylmethionine
S-Adenosylmethionine
CH3-R
Methyl derivatives

Let's look at examples for these different Phase 2 processes.

Glucuronide Formation: In order to form glucuronides we must first synthesize UDP-glucuronic acid by activating and oxidizing glucose.

Glucose + ATP right arrow Glucose-6-P + ADP

G-6-P right arrow G-1-P

G-1-P + UTP right arrow UDPG + PPi where PPi = inorganic pyrophosphate

PPi right arrow 2 Pi where Pi = inorganic phosphate

Note that the hydrolysis of PPi drives the reaction, since it is very favorable and the product of the previous reaction is removed. These rections take place in the cytosol.

Example: Conjugation of phenol gives an acetal glucuronide, phenyl-beta-D-glucuronide:

Haworth structure diagram for acetal glucuronide formation

Conjugation with benzoic acid gives an ester glucuronide, benzoyl-beta-D-glucuronide:

Haworth structure for benzoyl-beta-D-glucuronide

Examples with nitrogen and sulfur include the conjugation of aniline to give phenylamino-beta-D-glucuronide, and conjugation with 2-mercaptobenzothiazole to give the benzothiazole-2-thio-beta-D-glucuronide:

Haworth structure for phenylamino-beta-D-glucuronide, and benzothiazole-2-thio-beta-D-glucuronide

Glutathione conjugation: Glutathione is a tripeptide (gamma-glutamylcyteinylglycine, with the amino acid residues color coded in the structures below). It is used in cells as a recyclable anti-oxidant as well as a conjugating agent. When used in conjugation it is initially conjugated by a glutathione-S-transferase, or sometimes as a simple chemical reaction. Generally the conjugation occurs as a nucleophilic attack by the -SH group on a reactive electrophilic center. Conjugation is often followed by metabolic cleavage of the peptide to leave only the cysteinyl residue, which may then be acetylated to give a mercapturic acid.

Example:

1. Glutathione S-transferase:

Glutathione S-transferase reaction

2. -glutamyltranspeptidase:

gamma-glutamyltranspeptidase reaction

3. cysteinyl glycinase:

cysteinyl glycinase reaction

4. N-acetyl transferase:

N-acetyl transferase reaction

Of course not all glutathione derivatives are further metabolized, some may be excreted as is. However, if the derivative is excreted via the bile, it may be metabolized by the gut fauna and the toxin may be reabsorbed.

Sulfate Conjugation: This is the major Phase two process for a variety of hydroxyl groups and for some amines.

Haworth structure for  PAPS

PAPS synthesis via kinases

These enzymes are in the soluble fractions of a wide variety of tissues (e.g. liver, muscle, intestine, testes, etc.).

Example, sulfamate synthesis

sulfamate synthesis reaction

Acetylation: Acetyl transferases are found in the cytosol of hepatic endothelial cells, intestinal mucosal cells and white blood cells.

The "activated" acetyl group on acetyl CoA is used as the source of acetate, so it is readily available from normal metabolic sources. The enzyme mechanism is outlined below:

Acetyl transferase enzyme kinetic mechanism

Example: acetylation of a primary aryl amine:

acetylation of a primary aryl amine

There is evidence for more than one acetylase, which provides the genetic basis for "slow" and "fast" acetylators. Apparently there is polymorphism for various acetylases (we will see again later in comparative studies).

Slow acetylators are more susceptable to xenobiotics which are inactivated by acetylation. Turns out deacetylases are also present. The two processes show dominance in different organisms, varying the importance of acetylation.

Acylation: Acylation is similar to acetylation in the products excreted, but with an amino acid substituting for acetate (most commonly glycine; ornithine, taurine, and glutamine are also known). However, the synthetic strategy is "reversed" in that the xenobiotic is activated by adding coenzyme A, which then reacts with the amino acid.These reactions may be catalyzed by fatty acyl CoA synthetases:

Acylation reaction

This enzyme catalyses a two part reaction:

Fatty acyl CoA synthetase two stepp reaction sequence

The resulting acylCoA derivative can then react with an amino acid to give the acyl excretory product.

Example: Benzoic acid is first converted to benzoyl CoA, the benzoyl CoA then reacts with glycine to give the product, Hippuric acid:

benzoyl CoA synthesis

Hipp-uric acid synthesis from benzoyl CoA


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Last modified 9 March 2010

© RA Paselk 2001