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BIOTRANSFORMATION OF TOXINS/DRUGS
Biotransformation is the metabolic conversion of compounds (toxins, drugs etc.) to more water
soluble compounds and less lipid soluble compounds as a result of enzymatic activities in the
bodies of living organisms. Biotransformation may result in bio-activation leading to more toxic
compounds (metabolites) than the parent compounds or in bio-inactivation (detoxification)
leading to less toxic and water-soluble compounds for easy excretion in urine.
Foreign compounds absorbed into a biological system by passive diffusion are generally lipid
soluble and consequently not ideally suited for excretion. For example, very lipophilic substances
such as DDT and the polychlorinated biphenyls (PCBs) are very poorly excreted and hence
remain in the animal’s body for many years.
The consequences of metabolism are increased excretion, shortened half-life and reduced
accumulation and exposure of the biological system to potentially toxic compounds. Metabolism
of a chemical is determined by its structure, properties and available enzymes. Molecules are
transformed to more polar metabolites readily soluble in water for easy elimination out of the
body.
The majority of these reactions are catalyzed by a superfamily of enzymes, the cytochrome P450
mono-oxygenase system which is located in the smooth endoplasmic reticulum of the cells and
containing heme as a cofactor. The liver has the highest concentration of this enzyme although it
can be found in most, if not all tissues. This is a collection of isoenzymes (at least forty in
humans). The enzyme protein is designated CYP. Cytochrome P450 enzymes in the liver
converts steroid hormones into water-soluble metabolites to be excreted. In mammals, these
proteins oxidize steroids, fatty acids, toxins, drugs and are important for the clearance of various
compounds, as well as for hormone synthesis and breakdown. CYP enzymes have been identified
in all kingdoms of life: animals, plants, fungi, protists, bacteria, and archaea, as well as
in viruses. However, they are not omnipresent; for example, they have not been found
in Escherichia coli.
The term "P450" is derived from the spectrophotometric peak at the wavelength of the absorption
maximum of the enzyme (450 nm) when it is in the reduced state and complexed with carbon
monoxide.
The overall metabolic pathway of biotransformation is divided into two phases. A third phase may
sometimes be involved in the biotransformation of xenobiotics and drugs.
PHASE I
Phase I reactions involve hydrolysis, reduction, and oxidation of toxins/drugs.
As a result of phase I reactions, reactive intermediates including epoxides and free radical
species (unpaired electrons) are formed that are short-lived, highly reactive and may cause
damage to body if not conjugated immediately in the phase II of biotransformation.
Toxicology Instructor: Dr. M. Attaullah
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Main steps involved in phase I of biotransformation include:

➢ introduction of functional groups (—OH, —NH2, —SH, or —COOH etc.)
➢ hydrophilicity increases slightly
➢ may inactivate or activate original compound
➢ major player is CYP (Cytochrome P450) or mixed function oxygenase (MFO)
system in conjunction with NAD(P)H. These enzymes introduce reactive or polar
groups into xenobiotics or drugs.
➢ location of reactions is smooth endoplasmic reticulum
A common Phase I oxidation involves conversion of a C-H bond to a C-OH i.e. an inactive
compound to active one. By the same token, Phase I can turn a nontoxic molecule into a toxic
one (toxification).
➢ Oxidation
➢ Hydroxylation (addition of -OH group)
➢ N- and O- Dealkylation (removal of -CH side chains)
➢ Deamination (removal of -NH side chains)
➢ Epoxidation (formation of epoxides)
➢ Oxygen addition (sulfoxidation, N-oxidation)
➢ Hydrogen removal
➢ Reduction
➢ Hydrogen addition (unsaturated bonds to saturated)
➢ Donor molecules include GSH, FAD, NAD(P)H
➢ Oxygen removal
➢ Hydrolysis
➢ Splitting of C-NC-C (amide) and C-O2-C (ester) bonds

PHASE II
Phase II reactions involve conjugation of the metabolites formed in phase I.
The activated xenobiotic metabolites from phase I (free radicals or reactive oxygen species ROS)
are conjugated with charged species (antioxidants) such as sulfate, glycine, glutathione,
or glucuronic acid to polar compounds. Sites on drugs or toxins where conjugation reactions occur
include carboxyl (-COOH), hydroxyl (-OH), amino (-NH2), and sulfhydryl (-SH) groups.
Products of conjugation reactions have
➢ increased molecular weight
➢ the compounds tend to be less active than their substrates, unlike Phase I reactions which
often produce active metabolites.
These reactions are catalysed by transferase enzymes.
Toxicology Instructor: Dr. M. Attaullah
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Main events of phase II include:

• conjugation with endogenous molecules or antioxidants (Glutathione-GSH; glycine;
cysteine; glucuronic acid–glucuronidation; sulfate–sulfation; amino acid conjugation;
phosphate conjugation; methylation etc.)
• hydrophilicity increases substantially
• neutralization of active metabolic intermediates
• facilitation of elimination
• location of reactions is cytoplasm

Diagram of the overall biotransformation reactions

PHASE III: – further modification and excretion of toxins/drugs

After phase II reactions, the xenobiotic conjugates may be further metabolised. A common
example is the processing of glutathione conjugates to acetylcysteine (mercapturic acid)
conjugates.

Toxicology Instructor: Dr. M. Attaullah

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Quantitatively, the smooth endoplasmic reticulum of the liver cells is the principal organ of
drug metabolism, although every biological tissue has some ability to metabolize drugs.
Factors responsible for the liver's contribution to biotransformation of toxins/drugs include:
• it is a large organ,
• it is the first organ perfused by chemicals absorbed in the gut,
• and there are very high concentrations of most drug-metabolizing enzyme systems
relative to other organs e.g. cytochrome P450 monoxygenases, mixed function oxidases
(MFOs) etc.
Other sites of drug metabolism include epithelial cells of the gastrointestinal tract, lungs, kidneys,
and the skin. These sites are usually responsible for localized toxicity reactions.
Biotransformation (metabolism) often converts lipophilic compounds into hydrophilic products
that are more readily excreted.

Phases of biotransformation

Adverse effects of reactive metabolites on living organisms

Toxicology Instructor: Dr. M. Attaullah
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METABOLIC ENZYMES
1. Microsomal: Enzymes occurring in endoplasmic reticulum (microsomes)
specially smooth ER of the cells. They are mainly found in liver cells
i. CYP450 monooxygenases
ii. Flavin monooxygenase
2. Non-microsomal: Enzymes occurring in organelles/sites other than endoplasmic
reticulum are called non-microsomal enzymes. These are usually present in the
cytoplasm, mitochondria, etc. and occur mainly in the liver, GI tract, plasma and
other tissues.
i. Alcohol dehydrogenases
ii. Aldehyde dehydrogenases
iii. Monoamine and diamine oxidases
3. Mixed Function Oxidases (MFOs) containing both microsomal and non-
microsomal enzymes.
i. Esterases and Amidases
ii. Prostaglandin synthases
iii. Peroxidases

Role of cytochrome P450 and peroxidases in the activation of benzene to myelotoxic

metabolite – leukemia causing metabolite (PHS = prostaglandin H synthase)

Toxicology Instructor: Dr. M. Attaullah