Biotransformation

A xenobiotic (toxicant) may be absorbed into an animal via different routes. After absorption it is
distributed to different parts of the body and, finally, is available for excretion. Many xenobiotics
are known to undergo biotransformation while in the organs and tissues. Biotransformation is
also termed metabolic transformation.
Definition of Biotransformation:
Biotransformation may be defined as “the biologically catalysed conversions of chemicals, other
than the normal body constituents into other chemicals”.
Biotransformation is a process that, in general, converts the parent xenobiotic compounds or
toxicants into their metabolites and then form conjugates and facilitate their release from the
body.
Or, 
The biochemical modification of the xenobiotic (toxicant) molecules through the living cell is
termed biotransformation or metabolic transformation.
Or,
The act of reduction in the potentiality of the toxicant by the internal system of animal may be
termed biotransformation.
During biotransformation, generally the parent toxic compounds undergo detoxification i.e., the
formed metabolites and, finally, conjugates prove to be lesser toxic than the original or parent
toxicants. But in certain cases the metabolites have been proved more toxic than the parent
xenobiotics (toxicants) and such reaction is termed bioactivation.

Biotransformation Sites:
The organ/tissue wherein the biotransformation takes place is called biotransformation site. The
biotransformation of xenobiotics are often catalyzed by the enzymes which chiefly occur in the
liver of vertebrates. In the vertebrates these enzymes also occur in the skin, kidney, lungs,
intestine, placenta, gonads, embryonic liver, aorta, lymphocytes, blood platelets, adrenal cortex
and medulla but not in nervous system, though their activity have been anticipated. In insects,
such enzymes have been reported in mid-gut, fat body and Malpighian tubules.
The most important biotransformation sites in the vertebrates including human are- liver,
kidney, intestine, skin and lungs where the biotransformation takes place in the following
order:
Liver > Kidney > Intestine > Lungs > Skin
Principal Objectives of Biotransformation:
The principal objectives of the biotransformation processes are to detoxify the toxicants and
their elimination from the body.
It includes:
1. Conversion of water-insoluble substances to water-soluble form,
2. Emulsification of substances with bile, so as to facilitate elimination in colloidal form with
feces,
3. Incorporation of toxic molecules within inactive proteins, for example, metal molecules in
metallothionien protein and formation of metalloprotein for elimination.
Nature of Biotransformation Enzymes:
Actually the enzymes involved in biotransformation of xenobiotic compounds (toxicants) have
relatively low degree of substrate specificity in comparison to those involved in the metabolism
of constitutive chemicals.
The responsible enzymes in biotransformation of xenobiotics are monooxygenases or
cytochrome P-450 species.
The reaction catalyzed by a mono-oxygenase is:
RH + O2 + NADP + H+ → R-OH + HOH + NADP+
RH represents a very wide variety of xenobiotics including endogenous compounds such as
steroids.
The reaction catalyzed by cytochrome P-450 may also be represented as:
Reduced cyt. P-450 → Oxidized cyt. P-450.
RH + O2 → R-OH + HOH
The major mono-oxygenases in the ER are cytochrome P-450 species. Cytochrome P-450 is
notable because of the fact that approximately 50% of the drugs ingested are metabolized by
species of cytochrome P-450. The same enzyme also acts on various carcinogens and
pollutants.
Salient Features of Cytochrome P-450 Species:
1. These are hemoproteins.
2. These are present in high concentration in the membranes of the endoplasmic reticulum (ER)
or liver.
3. These are also present in the mitochondria as well as in the ER of the adrenal gland.
4. Six closely related species of cytochrome P-450 present in the liver ER that act on a wide
variety of drugs, carcinogens, and other xenobiotics. In recent years, the genes for these
species of cytochrome P-450 have been isolated and studied.
5. The enzyme that uses NADPH in the reaction mechanism of cytochrome P-450 is called the
NADPH-Cytochrome P-450 reductase.
6. The phosphatidyl choline, the major lipid found in the membranes of ER, is the principal
component of the cytochrome P-450 system.

7. Most species of cytochrome P-450 are inducible e.g., the administration of phenobarbital (PB)
causes a hypertrophy of the smooth ER and a 3 to 4 fold increase of the amount of cytochrome
P-450 within 4-5 days. The mechanism of induction involves increased transcription of mRNA
for cytochrome P-450.
8. Another species called cytochrome P-448 is specific for the metabolism polycyclic aromatic
hydrocarbons (PAHs) and related molecules; hence it is said to be aromatic hydrocarbon
hydroxylase (AHH). This enzyme is very important in the metabolism of PAHs and in
carcinogenesis produced by these agents. Researches reveal that smokers have higher levels
of this enzyme in some of their cells and tissues than do non-smokers. It is noticed that the
activity of this enzyme may be increased in the placentae of women who smoke, thus altering
the quantities of metabolites of PAHs to which the fetus is exposed.
The biological significance for the presence of these enzymes may be to evolve a protective
device against xenobiotic compounds.
Mechanism of Biotransformation:
R. T. Williams, in 1959, firstly studied the mechanism of biotransformation of xenobiotics
and divided the entire steps taking part in enzymatic biotransformation into two phases:
1. Phase I reactions or non-synthetic reactions involving oxidation, reduction and hydrolysis.
2. Phase II reactions or synthetic reactions involving the formation of a conjugate, i.e.,
metabolites of parent toxicant + endogenous polar or ionic moiety.
To sum up, biotransformation is such a process in which the parent xenobiotic compound is
converted into metabolites and then conjugates are formed. For example, benzene undergoes
oxidation, a phase I reaction, to form phenol, which conjugates with sulphate, a phase II
reaction. However, some chemicals form conjugates without proceeding in phase I reaction.
For example phenol may conjugate with sulphate without proceeding in phase 1 reactions. The
metabolites and conjugates are usually more water-soluble and polar, hence more readily
excretable. Biotransformation can, therefore, be considered in general as a mechanism of
detoxification by the host animal.
The type and rate of biotransformation of any xenobiotic compound differs from one species of
animal to another and even from one strain to another, which often accounts for the difference
of toxicity in animals. Apart from age and sex of the animal, exposures to other chemicals may
also alter the biotransformation. Knowledge of such factors is important in designing the
toxicological studies and in also the interpretation of possible health hazards to humans.
Complex Nature of Biotransformation:
Certain toxicants in their mode of action during biotransformation show a great degree of
complexity because they generally undergo various types of biotransformation, forming a variety
of metabolites and conjugates. Some of the metabolites and conjugates of bromobenzene and
carbaryl are shown in Figs. 21.1 and 21.2, respectively.
WHO (1971) reported that organophosphorus insecticides, viz. fenitrothion, omethoate, etc., can
be metabolised through dealkylation, oxidation, hydrolysis or desulphuration, yielding ten or
more different metabolites. Parathion, which is an organophosphorus pesticide, is bio-activated
in the liver to paraoxon, which is much more potent cholinesterase inhibitor.
Factors Affecting Biotransformation:
The liver is the main organ wherein biotransformation of xenobiotics takes, place. However,
diseases such as acute and chronic hepatitis, cirrhosis of the liver, and hepatic necrosis often
decrease the biotransformation.
The biotransformation capability of infants of premature births is extremely low in comparison to
the adults.
Certain other factors that affect the efficiency of liver to bio-transform the toxicant/drug are-
nutritional status, sex, age, procedure of administration of the drug and duration of drug
administered.
Starvation of organisms results in decrease in the levels of biotransformation enzymes.
Therefore, starved animals are often more sensitive to toxicants than the normally fed
individuals.
The biotransformation of toxicants is catalyzed by the MMFO. A deficiency of essential fatty
acids generally depresses MMFO activities. This is also true with protein deficiency. Deficiency
of certain vitamins like vits. A, C and E depresses the MMFO. However, thiamine deficiency has
the opposite effect.
Some foods contain appreciable amount of chemicals that are actually strong inducers of the
MMFO, e.g., safrole, xanthines, and indoles. In addition, potent inducers such as DDT and PCB
are present as contaminants in many foods.
The rate of biotransformation of xenobiotics varies according to the sex of the organism. For
example, adult male rats bio-transform xenobiotics at high rates than those of adult females.
Environmental factors like temperature and ionizing radiations also affect the biotransformation
process. For instance, exposure of rodents to ionizing radiations reduce the rate of biotrans-
formation of xenobiotics. It is because ionizing radiations have been reported to reduce the
hydroxylation of steroids, desulphuration activity and glucuronide formation.
Bioactivation:
The conversion of certain chemically stable compounds to highly chemically reactive
metabolites is termed bioactivation. In other words, bioactivation is the biotransformation in
which the formed metabolite proves to be highly toxic than the parent compound, i.e., toxicant or
drug. These reactive compounds become covalently bound to tissue macromolecules and
cause injury. In addition, other types of metabolites produce deleterious effects via other
mechanism also.
The biotransformation of bromobenzene to its epoxide and subsequent reactions serve as an
interesting example of bioactivation and its consequences. Although bromobenzene epoxide
may become covalently bound to tissue macromolecules and cause injury, the alternative routes
of metabolism may prevent or reduce the injury.
Likewise parathion, an organophosphorous pesticide, is bioactivated in the liver to paraoxon,
which is much more potent cholinesterase inhibitor.
Important site of bioactivation of many toxicants is liver; hence it is a common target organ.
Liver is regarded as the largest metabolic gland. However, if the metabolites are sufficiently
stable, they may affect other organs after being transported there (e.g., bromobenzene on the
kidney). In rare cases, other sites may be lungs, stomach, adrenal, retina and bone marrow.
Phases of Biotransformation: 2 Phases

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:
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.