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Biotransformation Processes in the Detoxification of Xenobiotics

Xenobiotics are chemical compounds (drugs, pesticides or carcinogens) that are foreign to a
living organism. We are exposed to a great number of xenobiotics during the course of our
lifetime, including a variety of pharmaceuticals and food components. Following their
absorption, they are subjected to metabolic conversion in the body resulting in structural
changes. This metabolic process is called biotransformation. Biotransformation may occur in
any of the several body tissues and organs including skin, lung, intestine, liver and kidney. To
accomplish this task, our bodies have evolved complex systems of detoxification enzymes.
These enzyme systems generally function adequately to minimize the potential of damage from
xenobiotics. The detoxification systems are highly complex, show a great amount of individual
variability and are highly responsive to an individual’s environment, lifestyle and genetic
uniqueness. Biotransformation is thus a critical process in the body’s defense against the toxic
effects of a wide variety of xenobiotics.

Table 1: Tissues/Organs Involved in Biotransformation

S/N Organ Cells
1 Liver Parenchymal cells (hepatocytes)
2 Kidney Proximal tubular cells (S3 Segment)
3 Lung Clara cells, Type II alveolar cells
4 Intestine Mucosa lining cells
5 Skin Epithelial cells
6 Testes Seminiferous tubules, Sertoli cells

1.1. Hepatic Metabolism

The liver carries out majority of chemical reactions because it contains a large number of non-
specific enzymes capable of biotransforming xenobiotics. The enzymes involved in
biotransformation are mixed-function oxidases (MFO) commonly known as cytochrome P450
enzymes.
The cytochrome P450 system contains a heme-bound ion at the active site which is responsible
for binding with and metabolizing the xenobiotic and this is attached to a protein chain. It is so
named because of its location (cyto = cell) and the fact that the heme moiety absorbs colored
(chrome) light at a wavelength of 450 nm. The enzyme catalyzes the reaction whereby a
molecule of oxygen is split into its two atoms; one atom of oxygen is inserted into the substrate

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and one atom is reduced (i.e. combines with 2 hydrogen atoms to form water) and the reaction
is called a monooxygenase reaction. It uses a cofactor in NADPH-cytochrome P450 reductase
(CPR) also referred to as NADPH cytochrome c reductase to catalyze the oxidation of NADPH
to NADP+. Over 50 human cytochrome P450 enzymes have been identified and are classified
according to their number of shared amino acid sequences. Families are given the nomenclature
CYP1, CYP2 etc. Subfamilies can be further divided into isoforms CYP1A1, CYP1A2 etc.
Most xenobiotics are metabolized by more than one enzyme and genetic polymorphism do
exists.
Non-cytochrome P450 enzymes in the liver are also involved in the metabolism of xenobiotics
including esterases, flavin containing monooxygenases, amongst others.

1.2. Biotransformation Reactions

Biotransformation reactions are grouped as Phase 1 (functional group modification), and Phase
2 (conjugation).

Fig: Schematic Representation of Biotransformation Reactions

1.2.1. Phase 1 Reactions

Phase 1 reactions are classified into oxidation, reduction and hydrolysis. During these
reactions, certain groups are added so that the xenobiotic can undergo the second phase to
produce conjugation products. However, if the metabolite of phase 1 reactions is sufficiently
water soluble in nature, they can be readily excreted at this point.
1.2.2. Oxidation
Oxidation is the most common of the phase 1 reactions and it involves the initial insertion of a
single oxygen atom onto the foreign molecule. Phase 1 oxidative reactions catalyzed by
cytochrome P450 enzymes include hydroxylation, epoxidation, deakylation, deamination and
dehalogenation reactions. Oxygen stays on the foreign molecule with hydroxylation and
epoxidation reactions and leaves the alkyl group as aldehyde with deakylation reactions.

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Examples of drugs demonstrating oxidative metabolism include Acetaminophen
(paracetamol), codeine and omeprazole.
1.2.3. Reduction
Reduction reactions are the second type of phase 1 reactions. They are catalyzed by the
cytochrome P450 enzyme system and often take place under anaerobic conditions. An example
of a drug that undergoes reduction reactions is prednisone, a prodrug which is reduced to the
active glucocorticoid prednisolone.
1.2.4. Hydrolysis
Unlike the other two reactions, hydrolysis is catalyzed by esterases and amidases and not by
cytochrome P450 reactions.

1.3. Phase 2 Reactions

Phase 2 metabolism involves the complexing or conjugation of xenobiotics with relatively
large and highly water-soluble endogenous molecules. This significantly increases the water
solubility of the xenobiotic allowing excretion in the bile or urine. Compounds of molecular
weight of 250 Daltons are excreted in the urine while compounds of molecular weight >350
Daltons are excreted in the bile. Unlike phase 1 reactions, phase 2 reactions require cofactors
and endogenous substrates and are ATP dependent.

1.4. Enzyme System Involved in Detoxification

1.4.1. The Phase 1 System
The Phase 1 detoxification system composed mainly of cytochrome P450 supergene family of
enzymes is generally the first enzymic defense against foreign compounds. Most
pharmaceuticals are metabolized through phase 1 biotransformation. In a typical Phase 1
reaction, a cytochrome P450 enzyme uses oxygen and a cofactor NADPH to add a reactive
group such as hydroxyl radical. As a consequence of this step in the detoxification, reactive
molecules which may be more toxic than the parent molecule are produced. If these molecules
are not further metabolized by Phase 2 conjugation, they may cause damage to proteins, RNA
and DNA within the cell. An example of such can be seen in acetaminophen overdose.

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Biotransformation of Acetaminophen-The good, the bad and the ugly

Sulphation reactions catalyzed by phenol sulfotransferase using the cofactor 31 phospho

adenosine 51-phosphosulfate (PAPS) is responsible for the metabolism of about 40% of
acetaminophen. A further, 40% undergoes glucuronidation and this is catalyzed by the enzyme
uridyl-diphosphate (UDP)-glucoronosyl transferase. A small amount however undergoes N-
hydroxylation to N-acetyl-p-amino-benzoquinoneimine (NAPQI) which is toxic. Under
normal circumstances, NAPQI is rapidly conjugated with glutathione. In the case of
acetaminophen overdose, this later metabolic pathway becomes saturated and rapidly exhausts
the available supply of glutathione. NAPQI accumulates and reacts with thiol groups on liver
cell membranes. This results in the death of the liver cells. Normally, glutathione is rapidly
synthesized in the liver from various foodstuffs. However, in individuals deprived of food for
a few days, synthesis of glutathione will not occur and toxicity of acetaminophen increases by
more than 10-fold!
The treatment of acetaminophen overdose involves giving N-acetyl cysteine (NAC), a
precursor to glutathione.

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 1.4.2. The Phase 2 Enzyme system
Phase 2 conjugation reactions generally follow Phase 1 activation resulting in a xenobiotic that
has been transformed to a water-soluble compound that can be excreted through the urine or
bile. The four primary enzymes are glucoronosyl-transferase which transfers glucuronic acid,
which transfers glucuronic acid, sulfotransferase which transfers sulfate, glutathione-s-
transferase transfers glutathione (GSH) and acetyl transferase which transfers acetyl groups.
1.4.3. The Phase 3 Detoxification System
Recently, antiporter activity (P-glycoprotein or multidrug resistance) has been identified as the
Phase 3 detoxification system. The antiporter is an energy dependent efflux pump which pumps
xenobiotics out of the cell thereby decreasing the intracellular concentration of a xenobiotic.
P-glycoprotein is a membrane-bound protein encoded by the human multi-drug resistance-1
(MDR1) gene that acts as an efflux pump to transport a variety of compounds out of
mammalian cells. It is highly expressed on the apical (luminal) surface of the organs that have
excretory functions such as the bile canalicular membrane of hepatocytes and renal proximal
tubules. It is significantly expressed on the luminal surface of tissues that serve as barrier such
as the brush border of the small intestine and the capillary endothelial cells of the blood brain
barrier. Intestinal P-glycoprotein acts to limit the bioavailability of substrate drugs by pumping
them form the enterocyte back into the gut lumen. The anatomical location of P-glycoprotein
suggests a physiological role in detoxification and protection of the body against toxic
xenobiotics and metabolites. This it does by secreting these compounds into the bile, urine and
the intestinal lumen thereby preventing their circulation in the brain, testes and fetus.
Several mechanisms have been hypothesized to explain how P-glycoprotein performs their
function. In the conventional model of a transporter, P-glycoprotein binds substrates in the
cytoplasm and expels them directly into the extracellular medium using ATP hydrolysis as an
energy source.

1.5. Factors Affecting Xenobiotic Metabolism

Various physiological factors can increase or decrease the rate of metabolism of xenobiotics.
Physiological factors include; age, sex, individual variation/genetic polymorphism,
enterohepatic circulation, intestinal flora and nutrition. As a general rule, drugs are metabolized
more slowly in the neonate and elderly compared with other age group.
Nutritional status impacts on the biotransformation of acetaminophen as fasting for 1day has
been shown to decrease glutathione (GSH) levels by 50% in rats thus increasing toxicity. Liver

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pathologies will also influence its biotransformation capacity as will age, sex and species
differences.
Genetic polymorphism refers to the inherited differences in an enzyme structure among
individuals, groups or population. This difference can alter the way drugs are metabolized in
different individuals. One of the most common acetylation reactions is N-acetylation catalyzed
by the enzyme N-acetyl transferase. This is important as it was the first enzyme discovered to
exhibit genetic polymorphism. Genetic polymorphisms with N-acetyltransferases involved in
phase 2 metabolism produce fast and slow acetylators depending on the individual variation of
the enzyme. Slow acetylators are more at risk of dose dependent drug toxicity. In the case of
the cytochrome P450 system, variation can occur in up to 30% of people depending on their
ethnic background.