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1. Introduction





Types of biotransformation
microbial biotransformation
Chemical Reactions
Categorization of the Biotransformation reactions
Phase I Reactions
a) Oxidation
b) Reduction
c) Hydrolysis
d) Cytochrome P450 system
Phase II Reactions
a) Glucuronide conjugation
b) Sulfate conjugation
Biotransformation Sites
Modifiers of Biotransformation
a) Age
b) Genetic variability in biotransforming capability
c) Poor nutrition
d) Enzyme inhibition and enzyme induction
e) Dose level

Biotransformation is the process whereby a substance is changed from one chemical to another
(transformed) by a chemical reaction within the body. Metabolism or metabolic transformations
are terms frequently used for the biotransformation process. However, metabolism is sometimes
not specific for the transformation process but may include other phases of toxico-kinetics.
Biotransformation is vital to survival in that it transforms absorbed nutrients (food, oxygen, etc.)
into substances required for normal body functions. For some pharmaceuticals, it is a metabolite
that is therapeutic and not the absorbed drug. For example, phenoxy-benzamine, a drug given to
relieve hypertension, is biotransformed into a metabolite, which is the active agent.
Biotransformation also serves as an important defense mechanism in those toxic xenobiotics and
body wastes are converted into less harmful substances and substances that can be excreted from
the body.
Toxicants that are lipophilic, non-polar, and of low molecular weight are readily absorbed
through the cell membranes of the skin, gastrointestinal (GI) tract, and lungs. These same
chemical and physical properties control the distribution of a chemical throughout the body and
its penetration into tissue cells. Lipophilic toxicants are hard for the body to eliminate and can
accumulate to hazardous levels. However, most lipophilic toxicants can be transformed into
hydrophilic metabolites that are less likely to pass through membranes of critical cells.
Hydrophilic chemicals are easier for the body to eliminate than lipophilic substances.
Biotransformation is thus a key body defense mechanism.
Fortunately, the human body has a well-developed capacity to biotransform most xenobiotics as

well as body wastes. An example of a body waste that must be eliminated is hemoglobin, the
oxygen-carrying iron-protein complex in red blood cells. Hemoglobin is released during the
normal destruction of red blood cells. Under normal conditions hemoglobin is initially
biotransformed to bilirubin, one of a number of hemoglobin metabolites. Bilirubin is toxic to the
brain of newborns and, if present in high concentrations, may cause irreversible brain injury.
Biotransformation of the lipophilic bilirubin molecule in the liver results in the production of
water-soluble (hydrophilic) metabolites excreted into bile and eliminated via the feces. The
biotransformation process is not perfect. When biotransformation results in metabolites of lower
toxicity, the process is known as detoxification. In many cases, however, the metabolites are
more toxic than the parent substance. This is known as bio-activation.
Occasionally, biotransformation can produce an unusually reactive metabolite that may interact
with cellular macromolecules (e.g., DNA). This can lead to very serious health effects, for
example, cancer or birth defects. An example is the biotransformation of vinyl chloride to vinyl
chloride epoxide, which covalently binds to DNA and RNA, a step leading to cancer of the liver.
Types of transformations/Bioconversion

1) Microbial transformation

2) Plant cell culture transformation

3) Animal cell culture transformation

Microbial transformation

When the transformation of the organic compounds is carried out by microorganism then the
process is called as microbial transformation.

The microorganisms have got the ability to chemically modify a wide variety of organic
compounds. These microbes during the bioconversion provide enzymes which act upon and
convert the organic compound into other compounds or modify it.

E.g. production of Vinegar: the oldest and most established transformation process.

Fig2: biotransformation of xenobiotics

A xenobiotic is a chemical which is found in an organism but which is not normally produced or
expected to be present in it. It can also cover substances which are present in much higher
concentrations than are usual. Specifically, drugs such as antibiotics are xenobiotics in humans
because the human body does not produce them itself, nor are they part of a normal diet.
The body removes xenobiotics by xenobiotic metabolism. This consists of the deactivation and
the secretion of xenobiotics, and happens mostly in the liver. Secretion routes are urine, faeces,
breath, and sweat. Hepatic enzymes are responsible for the metabolism of xenobiotics by first
activating them (oxidation, reduction, hydrolysis and/or hydration of the xenobiotic), and then
conjugating the active secondary metabolite with glucuronic or sulphuric acid, or glutathione,
followed by excretion in bile or urine. An example of a group of enzymes involved in xenobiotic
metabolism is hepatic microsomal cytochrome P450. These enzymes that metabolize xenobiotics
are very important for the pharmaceutical industry, because they are responsible for the
breakdown of medications.
Chemical Reactions
Chemical reactions are continually taking place in the body. They are a normal aspect of life,
participating in the building up of new tissue, tearing down of old tissue, conversion of food to
energy, disposal of waste materials, and elimination of toxic xenobiotics. Within the body is a
magnificent assembly of chemical reactions, which is well-orchestrated and called upon as
needed. Most of these chemical reactions occur at significant rates only because specific
proteins, known as enzymes, are present to catalyze them, that is, accelerate the reaction. A
catalyst is a substance that can accelerate a chemical reaction of another substance without itself
undergoing a permanent chemical change. Enzymes are the catalysts for nearly all biochemical
reactions in the body. Without these enzymes, essential biotransformation reactions would take
place slowly or not at all, causing major health problems.
An example is the inability of persons that have phenylketonuria (PKU) to use the artificial
sweetener, aspartame (in Equal). Aspartame is basically phenylalanine, a natural constituent of
most protein-containing foods. Some persons are born with a genetic condition in which the
enzyme that can biotransform phenylalanine to tyrosine (another amino acid), is defective. As
the result, phenylalanine can build up in the body and cause severe mental retardation. Babies are
routinely checked at birth for PKU. If they have PKU, they must be given a special diet to
restrict the intake of phenylalanine in infancy and childhood.
These enzymatic reactions are not always simple biochemical reactions. Some enzymes require
the presence of cofactors or co-enzymes in addition to the substrate before their catalytic activity
can be exerted.

Phase I Reactions
Phase I biotransformation reactions are simple reactions as compared to Phase II reactions. In
Phase I reactions, a small polar group (containing both positive and negative charges) is either
exposed on the toxicant or added to the toxicant. The three main Phase I reactions are oxidation,
reduction, and hydrolysis.
1. Oxidation
Oxidation is a chemical reaction in which a substrate loses electrons. There are a number of
reactions that can achieve the removal of electrons from the substrate. Addition of oxygen was
the first of these reactions discovered and thus the reaction was named oxidation. However,
many of the oxidizing reactions do not involve oxygen. The simplest type of oxidation reaction is
dehydrogenation that is the removal of hydrogen from the molecule. Another example of
oxidation is electron transfer that consists simply of the transfer of an electron from the substrate.
Examples of these types of oxidizing reactions are illustrated below:

The specific oxidizing reactions and oxidizing enzymes are numerous. Most of the reactions are
self-evident from the name of the reaction or enzyme involved. Few of them are listed below: Oxidizing reactions.
Alcohol dehydrogenation
Aldehyde dehydrogenation
Alkyl/acyclic hydroxylation
Aromatic hydroxylation
Deamination / Desulfuration

Theoretical aspects of microbial transformations

1. Conversion of uncommon substrates.
2. Interference with metabolic pathways
3. Interference with biotransformation

4. Interference with biosynthesis.

5. Rate of conversion depends upon the structure of substrate
6. Specificity of enzyme reaction.

Biotransformation: Enzymes

Few Examples of other Phase II reactions are listed below:1. Methylation

2. Amino acid conjugation

Biotransformation Sites
Biotransforming enzymes are widely distributed throughout the body. However, the liver is the
primary biotransforming organ due to its large size and high concentration of biotransforming
enzymes. The kidneys and lungs are next with 10-30% of the liver's capacity. A low capacity
exists in the skin, intestines, testes, and placenta. Since the liver is the primary site for
biotransformation, it is also potentially quite vulnerable to the toxic action of a xenobiotic that is
activated to a more toxic compound.
Within the liver cell, the primary subcellular components that contain the transforming enzymes
are the microsomes (small vesicles) of the endoplasmic reticulum and the soluble fraction of the
cytoplasm (cytosol). The mitochondria, nuclei, and lysosomes contain a small level of
transforming activity.
Microsomal enzymes are associated with most Phase I reactions. Glucuronidation enzymes,
however, are contained in microsomes. Cytosolic enzymes are non-membrane-bound and occur
free within the cytoplasm. They are generally associated with Phase II reactions, although some
oxidation and reduction enzymes are contained in the cytosol. The most important enzyme
system involved in Phase I reactions it the cytochrome P-450 enzyme system. This system is
frequently referred to as the "mixed function oxidase (MFO) system. It is found in microsomes
and is responsible for oxidation reactions of a wide array of chemicals.
The fact that the liver biotransforms most xenobiotics and that it receive blood directly from the
gastrointestinal tract renders it particularly susceptible to damage by ingested toxicants. Blood
leaving the gastrointestinal tract does not directly flow into the general circulatory system.
Instead, it flows into the liver first via the portal vein. This is known as the "first pass"

phenomena. Blood leaving the liver is eventually distributed to all other areas of the body;
however, much of the absorbed xenobiotic has undergone detoxication or bioactivation. Thus,
the liver may have removed most of the potentially toxic chemical. On the other hand, some
toxic metabolites are in high concentration in the liver.

Modifiers of Biotransformation
The relative effectiveness of biotransformation depends on several factors, including species,
age, gender, genetic variability, nutrition, disease, exposure to other chemicals that can inhibit or
induce enzymes, and dose levels. Differences in species capability to biotransform specific
chemicals are well known. Such differences are normally the basis for selective toxicity, used to
develop chemicals effective as pesticides but relatively safe in humans. For example, malathion
in mammals is biotransformed by hydrolysis to relatively safe metabolites, but in insects, it is
oxidized to malaoxon, which is lethal to insects.
Safety testing of pharmaceuticals, environmental and occupational substances is conducted with
laboratory animals. Often, differences between animal and human biotransformation are not
known at the time of initial laboratory testing since information is lacking in humans. Humans
have a higher capacity for glutamine conjugation than laboratory rodents. Otherwise, the types of
enzymes and biotransforming reactions are basically comparable. For this reason, determination
of biotransformation of drugs and other chemicals using laboratory animals is an accepted
procedure in safety testing.
1. Age:
Age may affect the efficiency of biotransformation. In general, human fetuses and neonates
(newborns) have limited abilities for xenobiotic biotransformations. This is due to inherent
deficiencies in many, but not all, of the enzymes responsible for catalyzing Phase I and Phase II
biotransformations. While the capacity for biotransformation fluctuates with age in adolescents,
by early adulthood the enzyme activities have essentially stabilized. Biotransformation capability
is also decreased in the aged. Gender may influence the efficiency of biotransformation for
specific xenobiotics. This is usually limited to hormone-related differences in the oxidizing
cytochrome P-450 enzymes.
2. Genetic variability in biotransforming capability :
Genetic variability in biotransforming capability accounts for most of the large variation among
humans. The Phase II acetylation reaction in particular is influenced by genetic differences in
humans. Some persons are rapid and some are slow acetylators. The most serious drug-related
toxicity occurs in the slow acetylators, often referred to as "slow metabolizers". With slow
acetylators, acetylation is so slow that blood or tissue levels of certain drugs (or Phase I
metabolites) exceeds their toxic threshold.
3. Poor nutrition:
Poor nutrition can have a detrimental effect on biotransforming ability. This is related to
inadequate levels of protein, vitamins, and essential metals. These deficiencies can decrease the
ability to synthesize biotransforming enzymes. Many diseases can impair an individual's capacity
to biotransform xenobiotics. A good example, is hepatitis (a liver disease), which is well known
to reduce hepatic biotransformation to less than half normal capacity.
4. Enzyme inhibition and enzyme induction:
Enzyme inhibition and enzyme induction can be caused by prior or simultaneous exposure to
xenobiotics. In some situations exposure to a substance will inhibit the biotransformation

capacity for another chemical due to inhibition of specific enzymes. A major mechanism for the
inhibition is competition between the two substances for the available oxidizing or conjugating
enzymes is the presence of one substance uses up the enzyme that is needed to metabolize the
second substance.
Enzyme induction is a situation where prior exposure to certain environmental chemicals and
drugs results in an enhanced capability for biotransforming a xenobiotic. The prior exposures
stimulate the body to increase the production of some enzymes. This increased level of enzyme
activity results in increased biotransformation of a chemical subsequently absorbed. Examples of
enzyme inducers are alcohol, isoniazid, polycyclic halogenated aromatic hydrocarbons (e.g.,
dioxin), phenobarbital, and cigarette smoke. The most commonly induced enzyme reactions
involve the cytochrome P-450 enzymes.
5. Dose level:
Dose level can affect the nature of the biotransformation. In certain situations, the
biotransformation may be quite different at high doses versus that seen at low dose levels. This
contributes to the existence of a dose threshold for toxicity. The mechanism that causes this
dose-related difference in biotransformation usually can be explained by the existence of
different biotransformation pathways. At low doses, a xenobiotic may follow a biotransformation
pathway that detoxifies the substance. However, if the amount of xenobiotic exceeds the specific
enzyme capacity, the biotransformation pathway is "saturated". In that case, it is possible that the
level of parent toxin builds up. In other cases, the xenobiotic may enter a different
biotransformation pathway that may result in the production of a toxic metabolite.
An example of a dose-related difference in biotransformation occurs with acetaminophen. At
normal doses, approximately 96% of acetaminophen is biotransformed to non-toxic metabolites
by sulfate and glucuronide conjugation. At the normal dose, about 4% of the acetaminophen is
oxidized to a toxic metabolite; however, that toxic metabolite is conjugated with glutathione and
excreted. With 7-10 times the recommended therapeutic level, the sulphate and glucuronide
conjugation pathways become saturated and more of the toxic metabolite is formed. In addition,
the glutathione in the liver may also be depleted so that the toxic metabolite is not detoxified and
eliminated. It can react with liver proteins and cause fatal liver damage.

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