KWARA STATE UNIVERSITY, MALETE

DEPARTMENT OF BIOCHEMISTRY
BCH421: XENOBIOTICS (2 UNITS)

BIOTRANSFORMATION OF XENOBIOTICS
I n nature, the organisms are often exposed to a wide variety of chemicals, which are
foreign to their body. Of these, the lipophilic substances easily penetrate the lipoprotein bilayers
of their cell membranes and finally reach the target cells. If such chemicals are continuously or
intermittently exposed to organisms and are gradually absorbed by the organisms but not
eliminated from their body, they tend to accumulate. Thus, they may produce lethal effects.
However, quite often during the course of their transport, they pass through certain
tissues/organs (such as, liver) having some activity against those xenobiotics. Consequently, the
lipophilic chemicals are biocatalytically converted into their hydrophilic forms that are easily
excreted from the body of organisms. The biologically catalyzed conversion of one form of
xenobiotic compound into another form may be simply termed as biotransformation.
Almost all biochemical reactions in the biological system take place under the influence of
certain enzymes and the biological conversion of one compound into another is termed as
metabolism. But, the term biotransformation is used preferably to denote the biological
conversion of xenobiotics and metabolism is referred to the biological conversion of useful and
endogenous substances, such as nutrients or body constituents, i.e. carbohydrates, fats (lipids),
proteins, nucleic-acids, etc.
The biotransformation of one xenobiotic compound into another often involves several
changes in the parent compound. These changes may occur through a series of sequential
reactions leading to one or more products. The new compound has distinct physical and chemical
properties, hence different pharmacological and toxicological properties. Thus,
biotransformation may lead to following alterations in the toxicological properties of the
xenobiotics:
(i)Conversion of an active chemical into another inactive chemical;
(ii) Conversion of an active chemical into another active chemical;
(iii)Conversion of an inactive chemical into another active chemical; and
(iv)Conversion of an inactive chemical into another inactive chemical.
From the examples of above four types of reactions, it is obvious that although the basic
purpose of biotransformation reactions is inactivation or detoxification of potent xenobiotic
compounds to facilitate their elimination from the body of organisms. Sometimes, the
biotransformation may also lead to the conversion of inactive or less active chemicals into more
active products, which are not easily excreted and thus, highly toxic. Usually the hydrophilic
substances are easily and quickly eliminated from the body of organisms. On the other hand,
lipophilic substances are not easily eliminated from the body of organisms. Thus, quite often the
biotransformation may cause biological conversion of lipophilic substances into their hydrophilic
forms and facilitates their excretion. In other words, it may be stated that the biotransformations
are often prerequisites to the excretion of xenobiotics.
(A) Biotransformation sites All organs and tissues are the sites of xenobiotics metabolism, including
biotransformation. As xenobiotics are harmful to the organism, biotransformation of these xenobiotics
can be considered as a defense mechanism, which may expedite the process of termination of the
xenobiotics from the body. Thus, the process of biotransformation if viewed as a defense mechanism
can be best administered as soon as the xenobiotic exposure occurred through the portal, i.e. skin, lung
and gastro-intestinal tract. This biotransformation process is catalyzed in the presence of enzymes
present in the tissues of the above organs but this is best administered in the liver of vertebrates. The
hepatocytes in the liver are the major site of xenobiotic biotransformation. These contain relatively high
volume of

endoplasmic reticulum (ca 15%) and approximately 20 % of the hepatocyte protein, which are essential
for most enzymes needed for biotransformation.

Kidney also receives a bulk of xenobiotics despite the fact it is not amongst the portals. It
receives the toxicants during excretion process and is also rich in xenobiotic metabolizing
enzymes. These enzymes also occur in adrenal cortex and medulla, placenta, testis, ovary, foetal
and embryonic liver, corpus luteum, aorta and blood platelets. In man, these enzymes have been
demonstrated in foetal and adult liver, placenta, kidney, testis, foetal and adult adrenal gland,
skin, blood platelets and lymphocytes. In insects, such enzymes have been reported from the
mid-gut, fat body and Malpighian tubules. Generally, the midgut is the site of greatest
concentration of these enzymes in insects. It is also important to mention that these enzymes are
not uniformly distributed within an organ. For instance, enzymes are most abundant in the
centrilobular region of the liver and nonciliated branchiolar or Clara cells of lungs than the other
cell types.
Thus, it is clear that almost every tissue has some activities against xenobiotics, but major
enzyme systems are localized mainly in the liver of vertebrates. The primary function of liver is
to process the chemicals received through portal circulation before they are distributed to other
tissues. Because of this property, the liver has capacity to extract xenobiotics readily from portal
circulation and modifies many of them before they are stored, secreted into the bile or released
into the general circulation. Other tissues often also biotransform the xenobiotics, but they have
limited capacity with respect to diversity of chemicals they can handle. Hence, their contribution
to the overall biotransformation of xenobiotics is limited. The biotransformation of xenobiotics
in extrahepatic tissue may have an important toxicological implication for that particular tissue.
(B) Nature of biotransformation enzymes One of the basic properties of enzymes is substrate
specificity. But, the enzymes involved in biotransformation of xenobiotics have relatively low
degree of substrate specificity in comparison to those involved in the metabolism of constitutive
chemicals. Because these enzymes catalyze reactions of a wide variety of chemicals after the
recognition of functional groups of the molecules rather than the recognition of entire molecules.
The biological significance for the presence of these enzymes may be either to evolve as a
protective device against chemical insults encountered in the environment or they are relatively
nonspecific biological catalysts, which normally biotransform certain body constituents, but have
relatively low substrate specificity.
(I) Microsomal mixed function oxidases
The biotransformation of xenobiotics is accomplished by several remarkable enzyme
systems. The biotransformation reactions are broadly of two types: (i) Phase I reactions, and (ii)
Phase II reactions.
 In phase I reactions, the functional groups (such as -OH, -SH, NH2, -COOH) of
xenobiotics are either added or exposed. These include simple reactions such as oxidation,
reduction and hydrolysis. The phase II reactions consist of relatively complex conjugation and
synthetic reactions. Phase I reactions are very important and predominant reactions in the
biotransformation of toxicants and are catalyzed by enzyme systems, such as cytochrome P-450
and cytochrome P-450 reductase and these enzyme systems are termed as mixed function
oxidase (MFO) system. It is also termed as monooxygenases because they catalyze the
incorporation of one atom of molecular oxygen into the substrate. But, the term mixed function
oxidase is preferred. Some other hydrolytic enzymes, such as esterases and amidases, also
expose the functional groups of xenobiotics.
The enzymes catalyzing phase I reactions are principally located in the endoplasmic
reticulum, which is a continuous anastomosing network of lipoprotein membranes between
plasma membrane, nucleus and mitochondria. The endoplasmic reticulum is of two types: (i)
rough, and (ii) smooth. Rough endoplasmic reticulum (ER) is studded ribosomes on its outer
surface whereas smooth endoplasmic reticulum is devoid of ribosomes. Although both these
types contain all the components of mixed function oxidase system; the smooth form is rich in
oxidative enzymes, hence the oxidative activity.
As a result of translocation, the lipophilic substances ultimately reach the lipoprotein
bilayers of membranes. Hence, the presence of phase I enzymes within lipoprotein matrix of
membranes, the actual site of biotransformation, is of great significance. The wide distribution of
cytochrome P-450 system among various animal species and in various organs of a species along
with its capability to catalyze incorporation of oxygen atoms into a wide variety of xenobiotics,
make it the most important group of enzymes associated with the biotransformation of
xenobiotics.
To isolate mixed function oxidase system, liver is taken out from the organisms and
homogenized. On account of homogenization the endoplasmic reticulum breaks up and the
fragments unite to form microvesicles often referred to as microsomes. The microsomes are
isolated by the differential centrifugation of liver homogenate. The liver homogenate is first
centrifuged at 9000 X g to remove nuclei, mitochondria, ribosomes, unbroken cells, etc. and the
resultant supernatant is again centrifuged at 1,05,000 X g, which yields pellets highly enriched in
microsomes. The microsome contains mixed function oxidase system, i.e. catalysts of phase I
reaction. The mixed function oxidases are also referred to as, microsomal mixed function
oxidases. The supernatant obtained after second step of centrifugation is referred to as cytosol,
which contains soluble enzymes of Phase II reactions of biotransformation. Therefore, these
enzymes may also be termed as cytosolic enzymes.
(a) Constituent enzymes of microsomal mixed function oxidase
A number of research workers have suggested the mixed function oxidation of xenobiotics
by the system consisted of cytochrome P-450, NADPH-cytochrome P-450 reductase and
phosphatidylcholine. This system oxidizes xenobiotics in the presence of NADPH and oxygen,
but this does not mean that:
(a) all xenobiotics are oxidized by this minimal enzyme system, and
(b) other microsomal constituents, such as cytochrome b5 system, mixed function amine
oxidase, etc. may not catalyze xenobiotic oxidations, although the role of cytochrome b5 system
in such oxidations is not well established.
(i) Cytochrome P-450 system The most important enzyme system of microsomal mixed function
oxidase is cytochrome P-450. It is a coupled enzyme system consisted of:
(a) a haeme containing enzyme, cytochrome P-450, and
(b) NADPH-cytochrome P-450 reductase, which prefers NADPH as its cofactor.
Cytochrome P-450 is a haemoprotein of cytochrome b5 type with unique redox potential
and is named from the unique wave-length of absorption maximum (at 450 nm) of carbon
monoxide derivative of the reduced form (reduced cytochrome P-450 (Fe2+) forms a ligand with
carbon monoxide). Only intact and biocatalytically functional cytochrome P-450 possesses this
spectral property while denatured form loses its spectral peak and achieves maximum absorbance
at 420 nm. At least 10 different forms of cytochrome P-450 have been isolated from rat liver
microsomes. The different forms of enzyme differ in the structure of polypeptide chain as also
the specificity of reactions catalyzed by them. Treatment of rats with certain xenobiotics shifts
the absorption maximum of the enzyme. The types and amounts of cytochrome P-450 also vary
with the species, age, sex and health of organisms. On the other hand, cytochrome P-450
reductase has been isolated from single source and its concentration varies between one tenth to
one thirteenth of cytochrome P-450. Therefore, it is believed that this enzyme mediates reduction
of different forms of cytochrome P-450.
(ii) Cytochrome b5 system Associated with cytochrome P-450 system, there is another
cytochrome system consisted of cytochrome b5 and cytochrome b5 reductase, but the function of
this system in cytochrome P-450 mediated reactions is not properly understood.
(iii) Mixed function amine oxidase Another important oxidative enzyme is referred to as mixed
function amine oxidase. It is also present in endoplasmic reticulum and oxidizes nucleophilic
nitrogen and sulphur atoms. This enzyme is not solely an amine oxidase, but it should be
appropriately termed as microsomal flavin containing monooxygenase. Human beings and pigs
possess remarkably high amounts of this enzyme while rats possess low level of the same.
(II) Microsomal hydrolytic enzymes
(i) Epoxide hydrolase Epoxide hydrolase is an important hydrolytic enzyme and is believed to be
located in the microsomal fractions, i.e. in close proximity of microsomal cytochrome P-450
systems. It has been recorded in a wide variety of tissues, such as liver, testis, ovary, lung,
kidney, skin, intestine, colon, spleen, thymus, brain and heart. The activity of this enzyme differs
in various tissues. For example, clara cells of lung possess three to four times more activity of
epoxide hydrolase than alveolar type I cells.
A distinct cytosolic epoxide hydrolase has also been reported in a number of animal
tissues. The cytosolic enzyme has high levels in mice and rabbits in comparison to those of rats.
Epoxide hydrolase catalyses the hydration reactions of aliphatic and aromatic epoxides of
xenobiotics; and thus inactivates a number of highly reactive epoxides. Hence, epoxide hydrolase
is considered as a detoxication enzyme. The occurrence of enzyme in close proximity to the site
of formation of its substrate (i.e. oxides of xenobiotics) may have great toxicological
significance. In fact, it has been suggested that epoxide hydrolase has been evolved to detoxify
various aliphatic and aromatic epoxides of xenobiotics.
(ii) Esterases and amidases A large number of nonspecific esterase and amidase enzymes occur
in various mammalian tissues. Both of these hydrolyze ester and amide linkages of xenobiotics.
These enzymes occur both in the microsomal and cytosolic fractions. The cytosolic esterases are
usually specific in reaction, such as acetylcholinesterase and pseudocholinesterases. On the other
hand, the microsomal esterases are relatively nonspecific and can catalyze the hydrolytic
reactions of ester of a diverse group of xenobiotics.
The amidases are comparatively more specific enzymes. Generally, the enzymatic
hydrolysis of amides takes place more slowly than those of esters. The slow hydrolysis of amides
by amidases has been ascribed to the comparatively high specificity of amidases.
(iii) Glutathione-S-transferases It is one of the most important enzymes of phase II
biotransformation reaction. The initial step of glutathione conjugation to xenobiotics is catalyzed
by a group of enzymes called as glutathione-S-transferases. These enzymes are present both in
the soluble and microsomal fractions of tissues. But, their high concentration occurs principally
in the soluble fractions. These enzymes are widely distributed in the animal kingdom, for
instance, in protozoans (e.g. Acanthamoeba), insects, aquatic invertebrates, fishes and mammals.
Besides, they are also known to be present in certain bacteria.
Various forms of glutathione-S-transferases have been reported from the liver of rats, mice
and human-beings as also in insects. At least five different forms of enzyme have been reported
from the soluble fraction of rat- and human liver.
Glutathione-S-transferases have been classified in accordance with their substrate
specificity and pH optima. They may be grouped and named as follows:
(i) Glutathione-S-alkyltransferase,
(ii) Glutathione-S-aryltransferase,
(iii) Glutathione-S-aralkyltransferase,
(iv) Glutathione-S-epoxidetransferase,
As glutathione-S-transferases catalyze the conjugation of toxic electrophilic compounds
with an endogenous molecule, glutathione, they protect endogenous critical nucleophiles, such as
proteins and nucleic acids.
(IV) Factors Affecting Biotransformation of Xenobiotics
It is obvious that biotransformation of xenobiotics may take place by different pathways
involving phase I and II reactions and they may give rise to different biotransformation products.
Various factors related to the chemical, the environment and the physiological state of organisms
affect the rate and relative importance of biotransformation reactions of xenobiotics. These
factors can thus be divided into three categories: (1) Chemical factors, (2) Environmental factors,
and (3) Factors related to organisms. The first two factors may also be referred to as abiotic
factors and the third one as biotic factors.
(1) Chemical Factors
(i) Concentration The most important factor related to the chemical is that which affect
rate and route of enzymatic modification of active concentration of the chemical at specific target
site. Some of xenobiotics having immediate effects may cause pronounced damage to the tissue
and thus the biotransformation enzymes may be inhibited on account of their binding with active
sites. Therefore, such substances are believed to inhibit the xenobiotic biotransformation. On the
other hand, there are certain substances, which may enhance the activity of these enzymes, hence
may enhance their biotransformation. Such activator substances may increase the rate of
biotransformation of xenobiotics in the body of organisms.
 (ii) Chemical nature Another important factor related to the chemical is the nature of
xenobiotics, i.e. whether lipophilic or hydrophilic or polar. The xenobiotics ionized at
physiological pH have poor lipid solubility, hence they cannot easily cross lipoprotein layers of
biological membranes. On the other hand, lipophilic chemicals are more easily absorbed through
membranes. If such chemicals are inhibitors of biotransformation enzymes, they may either
affect the process or at least slow down.
(iii) Interaction with endogenous molecules The xenobiotics interact with intracellular
and extracellular proteins. This interaction reduces the concentration of xenobiotics at the active
sites of enzymes involved in their biotransformation. This binding of xenobiotics with certain
proteins has a definite effect on the intrinsic clearance of xenobiotics.
(2) Environmental factors
In vitro effects of light, temperature, etc. on xenobiotic biotransformation enzymes are
similar to those of other enzymes. In vivo effects of environmental factors, such as temperature,
light, moisture, ionizing radiation, etc. on the biotransformation of xenobiotics are being given
under this section.
(i) Temperature It is often expected that variations in ambient temperature would not
affect the biotransformation of xenobiotics in homeotherms, but actually it is not so.
Temperature variations may work as a kind of stress and thereby produce changes mediated by
hormonal variations. Seasonal changes in temperature also have a definite effect on hormonal
levels. It is now known that changes in temperature clearly affect the biotransformation of
xenobiotics. But, it is not clear whether these changes directly affect the biotransformation or
through some other physiological mechanism.
(ii) Light Many xenobiotic biotransformation enzymes show a diurnal pattern in relation
to light cycles rather than light intensity. Continuous darkness causes maintenance of high
concentrations of hydroxy indol-O-methyl transferase. This enzyme has diurnal rhythm with
greatest activity at night. Microsomal mixed function oxidase system also shows diurnal rhythm
with greatest activity at the beginning of the dark phase. The activity of these enzymes may be
correlated with the biotransformation of xenobiotics.
(iii) Moisture Effect of moisture on the biotransformation of xenobiotics in vertebrates
does not seem to be investigated, but similar studies in insects have been made. It has been
reported that housefly larvae reared at 40% moisture content have four times more activity of
epoxidation of heptachlor than those of larvae reared on water saturated medium.
(iv) Ionizing radiation Exposure of rodents (i.e. rats and mice) to ionizing radiation
reduces the rate of biotransformation of xenobiotics. This view has been supported by the fact
that exposure to ionizing radiations reduce the hydroxylation of steroids, desulphuration activity
and glucuronide formation. Inhibition in pseudocholinesterase activity following exposure to
ionizing radiation has also been reported to be reduced.
(3) Factors related to organisms
(i) Species Different species differ in the toxicity of xenobiotics. These differences may
be related to differences in the translocation and biotransformation of xenobiotics. The species
differences in biotransformation of xenobiotics are quantitative rather than qualitative. These
quantitative differences in biotransformation of xenobiotics may be ascribed to species
differences in the enzyme concentration or its kinetics, availability of cofactors and the
concentration of substrate in tissues. For instance, rats have higher cytochrome P-450
concentrations than other mammalian species. It is found that rodents are well bestowed with the
activity of enzyme sulphotransferase and relatively deficient in activity of enzyme glutathione
transferase. The quantitative differences suggest that different biotransformation routes are
favoured in different species, which may lead to different pharmacological and toxicological
activity. In addition, the metabolic rates of a xenobiotic may be similar in some closely related
species but the end product is different. For example, oxidative metabolism of amphetamines by
cytochrome P-450 produces largely the deamination products in rabbit but it forms phenyl
hydroxylation products in rats. Human beings, however, are capable to carry out almost all the
metabolic transformation and do not show any particular difference in enzymatic pathways.
(ii) Strains Different strains of an organism differ in biotransformation of xenobiotics and
these differences are under genetic control. The differences in biotransformation often may lead
to variations in biological responses to xenobiotics. The strain-wise variation in phase I
biotransformation reactions may be attributed to marked differences in cytochrome P-450 related
enzymes activities. Variation in phase II biotransformation reactions in various strains are also
known. For instance, Sprague- Dawley strain of rat and Fischer strain of rat are very similar but
Gunn strain of rat shows a remarkable deficiency in one of the two major classes of UDP-
glucuronosyl transferase activity. The cytochrome P-450 concentration in al three strains of rats
is however very similar.
In humans, there is a tendency to respond differently towards the metabolism of various
drugs. For instance, different populations of humans have different response to the
antituberculosis drug, isoniazid. Variation in the acetylator phenotypes in humans is a reason of
this differential response. High percent of Asian and Eskimo populations are fast acetylators and
here the biotransformation of isoniazid drug leads to reactive metabolites which cause liver
toxicity (hepatic necrosis). On the contrary, there is almost no effect on the metabolism of the
isoniazid in the human population of slow acetylators. The unchanged drug accumulates causing
the toxicity of peripheral neuropathy, which results from pyridoxal phosphate depletion by the
unmetabolized drug. This accumulation of drug may cause bladder cancer when it gets exposed
with arylamine compounds.
(iii) Sex The rate of biotransformation of xenobiotics varies according to sex of organisms.
Gender-wise differences in biotransformation of xenobiotics by mammalian liver appear with the
onset of puberty and are usually maintained throughout the adult life. Meager studies of sex
dependent biotransformation in humans have shown that females have a better tendency for
oxidative metabolism rates than the males. Adult male rats biotransform xenobiotics at higher
rates than those of adult females. The differences in biotransformation of xenobiotics between
males and females may be ascribed to differential activity of microsomal enzymes, which are
normally under control of sex hormones.
(iv) Age Foetal and new born individuals have limited ability to biotransform xenobiotics,
which provides the basis for increased sensitivity of young animals to the xenobiotics. But, all
the pathways of biotransformation are not absent in new born individuals. The enzyme systems
develop rapidly after the birth of individuals. For instance, new born rats possess almost
negligible activity of cytochrome P-450 system that develops rapidly and reaches at peaks by 30
days of age. Thereafter, activity starts gradually declining with increase in the age of individuals.
The age related changes in the activities of biotransformation enzymes may be ascribed to
biochemical differentiation in hepatocytes.
 In humans, the premature and neonatal infants have low activities of microsomal and
nonmicrosomal metabolizing enzymes. The biotransformation o my exotic chemicals are fastest
in adolescents.
(v) Diet The nutritional status of organisms also plays an important role in the
biotransformation of xenobiotics. Human diets usually contain nutrients which are needed for the
body growth and development. Apart from these they may contain pesticides residues and food
additives, which can enter our physiological and biochemical systems and alter the activities of
drug metabolizing enzymes. The flavonoids, naringin and quercetin present in the grape fruit
juices have a tendency of inhibiting the Cytochrome P450 3A. There are other flavonoids, like
catechin, myricetin and rutin, which can induce Phase II enzymes and provide protection against
biotransformed products. Starvation of organims results in decrease in the levels of
biotransformation enzymes. Therefore, starved animals are often more sensitive to toxicants than
the normally fed individuals. Similarly the mineral and vitamin deficiencies in the diet decrease
the levels of cytochrome P-450 system that in turn may decrease the rate of biotransformation of
xenobiotics. Low protein and protein free diet also decrease the levels of microsomal enzymes.
After the supply of nutrient rich diet to these animals, the enzyme activities return to normal
levels. Burned carbon containing materials, like cigar, cigarette and charcoal broiled foods may
largely modify and alter the biotransformation tendencies of enzymes.
(vi) Health The health status of organisms affects the biotransformation of xenobiotics. It
is obvious from the previous accounts that the liver is most important organ and the site of
biotransformation of xenobiotics. Any type of damage (either due to certain infections or due to
the action of chemicals) may have pronounced effects on the levels of xenobiotic
biotransforming enzyme systems, hence the biotransformation of xenobiotics. Hepatitis patients
have been reported to show impaired ability to biotransform xenobiotics via microsomal mixed
function oxidase system.
(vii) Circadian rhythms Daily and seasonal changes in the photocycle and other
parameters are known to affect the endocrine functions of organisms. The biotransformation
enzymes are under control of neuroendocrines. Therefore, changes in the season and the time of
the day may have great influence on the levels of biotransformation enzymes, hence on the
biotransformation of xenobiotics.
(V) Bioactivation
The biotransformation of xenobiotics may involve either the conversion of an active
chemical to more active chemical or an inactive chemical to an active chemical. As a
consequence to such biotransformation, i.e. conversion of an inactive chemical to an active
chemical, the toxicity of parent compound is increased. The process of bioconversion of inactive
xenobiotics into more reactive products is termed as bioactivation. Such biological conversions
may have great pharmacological and toxicological consequences. One promising example of
bioactivation is the conversion of organophosphorus pesticides belonging to the groups;
phosphorothionates and phosphorodithioates having P=S group. These compounds are poor
inhibitors of target enzyme, AChE. But, in the body of organisms both in the mammals and
insects, they are biotransformed into more active form of organophosphates (having P=O group)
by the microsomal mixed function oxidases, which are potent inhibitors of AChE, hence more
toxic. For instance, less active malathion (having P=S group) is bioconverted into its oxygen
analogue malaoxon (having P=O group) and parathion into paraoxon. Similarly, various other
xenobiotics are also bioactivated by the organisms.