Phases of Biotransformation: 2 Phases | Toxicants | Toxicology

The following points highlight the two main phases of biotransformation of toxicants. The phases are: 1. Phase I Reactions 2. Phase II Reactions.

1. Phase I Reactions:

There are three types of Phase I reactions:

i. Oxidation.

ii. Reduction.

iii. Hydrolysis.

1. Oxidation:

The biotransformation of great variety of xenobiotic compounds involves oxidation process. The most important enzyme system catalysing the involved processes are cytochrome P-450 and NADPH cytochrome P-450 reductase. In these reactions, one atom of molecular oxy gen is reduced to water and the other is incorporated into the substrate.

The cytochrome linked monooxygenases (oxidases) are located in the smooth endoplasmic reticulum. When a cell is homogenized, the endoplasmic reticulum splits into small vesicles known as microsomes. Because of the location of these enzymes and the great variety of chemicals that they may catalyze, these are also known as microsomal mixed-function oxidases (MMFO). In addition, oxidation of various toxicants is catalyzed by non-microsomal oxidoreductases that are located in the mitochondrial fraction.

Oxidation may take place in a variety of reaction forms and very often more than one metabolite is formed. Some examples are:

A. Microsomal Oxidations:

i. Aliphatic Oxidation:

Aliphatic oxidation involves oxidation of the aliphatic side-chains of aromatic chemicals:

Example:

n, propylbenzene → 3-phenylpropan-1, -ol, 3-Phenyl propan-2-ol, and 3-phenypropan- 3-ol

ii. Aromatic Hydroxylation:

Aromatic hydroxylation usually proceeds through an epoxide intermediate:

Example:

iii. Expoxidation:

Example:

Aldrin → Dieldrin

iv. Oxidative Deamination:

Example:

Amphetamine → Phenyl acetone

v. N-dealkylation:

vi. O-dealkylation:

Example:

p-nitroanisole →p-nitrophenol

vii. S-dealkylation:

Example:

6-Methyl thiopurine → 6-Mercaptothiopurine

viii. N-Oxidation:

Example:

Trimethylamine → Trimethylamine oxide

ix. N-Hydroxylation:

x. P-Oxidation:

Example:

xi. Sulphoxidation:

xii. Desulphuration:

Example:

Parathion → Paraoxon

B. Non-Microsomal Oxidations:

i. Amine Oxidation:

Monoamine oxidase located in mitochondria. Diamine oxidase is a soluble enzyme.

Both participate in the oxidation of the primary, secondary and tertiary amines, such as 5-hydroxytrypfamine and putrescine, into corresponding aldehydes:

ii Alcohol and Aldehyde Dehydrogenation:

It is catalysed by alcohol dehydrogenase and aldehyde dehydrogenase, respectively:

Example:

Ethanol → Acetaldehyde

Acetaldehyde → Acetic acid

Reaction showing Aldehyde dehydrogenation:

R – CHO^– + NAD⁺ → R – COOH^– + NAD + H⁺

ii. Reduction:

Xenobiotic chemicals may undergo reduction through the function of reductases. These reactions are actually less active in mammalian tissues but frequent in intestinal and intracellular bacteria. An important example is the reduction of prontosil to sulphanilamide.

Like oxidation, reduction may also be of two types, namely microsomal reduction and non-­microsomal reduction:

A. Microsomal Reductions:

I. Nitro Reduction:

II. Azo Reduction:

B. Non-Microsomal Reduction:

Non-microsomal reduction occurs via the reverse reaction of alcohol dehydrogenases:

iii. Hydrolysis:

Various toxicants are having ester-type bonds and are subjected to hydrolysis. These are essentially esters, amides, and compounds of phosphate. Mammalian tissues, including the plasma, contain a large number of non-specific esterases and amidases, which take part in hydrolysis.

Testa and Jenner (1976) reported that the esterases, usually located in the soluble fraction of the cell, may be broadly grouped into four classes:

a. Arylesterases:

These hydrolyse aromatic esters:

b. Cholinesterases:

These hydrolyse those esters in which the alcohol moiety is choline.

c. Carboxylesterases:

These hydrolyse aliphatic esters.

d. Acetylesterases:

These hydrolyse those esters in which the acid moiety is acetic acid.

In contrast to esterases, amidases cannot be grouped according to the substrate specificity. Furthermore, enzymatic hydrolysis of amides proceeds much slowly than that of esters, probably because of the lack of substrate specificity.

2. Phase II Reactions (Conjugation Reactions):

Phase II reactions involve several types of endogenous metabolites that may form conjugates with the xenobiotics or their metabolites. In general, these conjugates are more soluble in water and more readily excretable.

Examples of each type of conjugation have been illustrated below:

A. Glucuronide Formation:

This is very common and the most important type of conjugation. The enzyme catalyzing this reaction is UDPGT (Uridine Diphosphate Glucuronyl Transferase) and the coenzyme UDPGA (Uridine 5′- diphospho- α-D-glucuronic acid). This enzyme is also located in the endoplasmic reticulum.

There are four classes of chemical compounds capable of forming conjugates with glucuronic acid:

i. Aliphatic or aromatic alcohols,

ii. Carboxylic acid,

iii. Sulphydryl compounds, and

iv. Amines.

O-Glucuronide Formation:

B. Methylation:

This reaction is catalysed by methyl transferases. The coenzyme is the SAM (S-adenosylmethionine). However, methylation is not a major route of biotransformation of toxicants because of the broader availability of UDPGA, which leads to the formation of glucuronides. Futhermore, it does not always increase the water solubility of the methylated products.

Few reactions of methylations are:

C. Sulphate Conjugation:

This reaction is catalysed by sulphotransferases. These enzymes are located in the cytosolic fraction of liver, kidney and intestine. The coenzyme is PAPS (3′-phosphoadenosine-5′-phosphosulphate). The functional groups of the foreign compounds for sulphate transfer are phenols and aliphatic alcohols as well as aromatic amines.

Reactions for PAPS-mediated conjugation is shown below:

D. Acetylation:

Acetylation involves transfer of acetyl groups to primary aromatic amines, hydrazines, hydrazides, sulphonamides and certain primary aliphatic amines.

The enzyme and coenzyme involved, respectively, are N-acetyl transferases and acetyl coenzyme A:

E. Amino Acid Conjugation:

This conjugation is catalysed by amino conjugates and coenzyme A. Aromatic carboxylic acids, arylacetic acid, aryl-substituted acrylic acids can form conjugates with α-amino acids, mainly glycine, but also glutamine in humans and certain monkeys and ornithine in birds-

F. Glutathione Conjugation:

This important reaction is effected by glutathione S-transferases and the cofactor glutathione. Glutathione conjugates subsequently undergo enzymatic cleavage and acetylation forming N -acetylcystenine (mercapturic acid) derivatives of the toxicants, which are readily excreted. Glutathione can also conjugate with unsaturated aliphatic compounds and displace nitro groups in chemicals.

It is rather important to mention that in the process of biotransformation of xenobiotics, a number or highly reactive electrophilic compounds are formed. Some of these compounds can react with cellular constituents and cause cellular death or induce tumour formation.

The role of glutathione is to react with electrophilic compounds and thus prevent their harmful effects on the cells. However, exposure to very large amounts of such reactive substances can deplete the glutathione, thereby- resulting in marked toxic effects.

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