BIOCHEMICAL TOXICOLOGY

BY

DR J.O.AREOLA
 What is Biochemical toxicology ?
• Biochemical toxicology is concerned with the
mechanisms underlying toxicity of xenobiotic,
particularly the events at the molecular level
and the factors which determine and affect
toxicity.
• Xenobiotic are generally described as
substances that are foreign to living cells.
These are the substances that were absent in
organisms from their origin.
 The nature of xenobiotics
• The interaction of a foreign compound with a biological
system is two-fold: there is the effect of the organism
on the compound and the effect of the compound on
the organism. These effects are determined by
absorption, distribution, metabolism and excretion of
xenobiotics.
• Foreign and potentially toxic compounds absorbed into
biological system are generally lipophilic substances.
They are therefore not ideally suited for excretion as
they will be reabsorbed in the kidney or from the
gastrointestinal tract after biliary excretion.
 Biotransformation
• The biotransformation of foreign compounds,
attempts to convert such lipophilic substances
into more polar and consequently more readily
excreted metabolites.
• The exposure of the body to the compound is
reduced and potential toxicity decreased. This
process of biotransformation is therefore a
crucial aspect of the disposition of toxic
compounds by animals.
• Biotransformation may also increase the toxicity
of some compounds.
 The primary results of
biotransformation (PRB)
• The parent compound is transformed into a
more polar metabolite, usually by the addition
of ionizable groups.
• The molecular weight and size are commonly
increased.
• The excretion is facilitated and hence
elimination of the compound from the tissues
and the body is increased.
 Examples of PRB
• For example, the analgesic drug, paracetamol has
a renal clearance value of 12 ml/min whereas one
of its major metabolites, the sulphate conjugate
is cleared at the rate of 170 ml/min.
• However, biotransformation may not always
increase water solubility. For example many
sulphonamide drugs are acetylated in vivo, but
the acetylated metabolites are less water soluble.
The metabolite precipitate in the kidney tubules
and cause toxicity.
 Biotransformation of Drugs
• As the chemical structure is changed from that of the
parent compound, there may be consequential changes to
the pharmacological and toxicological activity of the
compound.
• For some drugs, the pharmacological activity resides in the
metabolites rather than the parent compound. A classic
example of this is the antibacterial drug sulphanilamide
which is released from the parent compound prontosil after
bacterial metabolism in the gut.
• For other drugs, it is the parent compound that is active as
is the case with the muscle relaxant drug succinyl choline.
The action of this drug normally last for a few minutes
because metabolism rapidly cleaves the molecule to yield
inactive metabolite.
 Effects of Biotransformation
• Although biotransformation is usually regarded
as a detoxification process but this is not always
so, thus the toxicological activity may be greater
than that of the parent compound after
metabolism. When this happens, it is called
bioactivation.
• Metabolism is therefore an important deter -
minant of the activity of xenobiotic, the duration
of that activity and the half- life of the
metabolites in the body.
 Enzymes of xenobiotics
• The enzymes involved in the metabolism of foreign
compounds are flexible and non-specific, i.e. the
foreign compounds are not exclusively metabolized
by specific enzymes. They are metabolized by
endogenous enzymes that also metabolize other
compounds. The organ most commonly used for the
biotransformation of xenobiotic is the liver, because
of its position, blood supply and function.
• The metabolism processes are divided into two;
phase 1 and phase 2 metabolism.
 Phase 1 metabolism
• Phase 1 is the alteration of the original foreign
compound by the addition of a functional group
which can then be conjugated in phase 2.
• Example 1, Benzene is a xenobiotic that is highly
lipophilic molecule which is not readily excreted from
the body except in the expired air as it is volatile.
Phase 1 metabolism converts benzene into a variety
of metabolites but the major product is phenol (Fig
1). The addition of hydroxyl group allows a phase 2
conjugation reaction to take place
 Phase 1 Methabolism
• with the polar sulphate group being added.
Phenyl sulphate the final metabolite is water
soluble and it is readily excreted in urine (fig 1)
Metabolism of benzene to
phenylsulphate(fig 1)
 Phase 1 reactions
• Phase 1 Reactions - The major types of phase
1 reactions are oxidation, reduction and
hydrolysis.
Oxidation
Oxidation reactions are involved in the
catabolism of many xenobiotic, the reactions
are catalyzed by mono-oxygenase enzymes
found in the smooth endoplasmic reticulum, it
is also known as microsomal enzymes.
 Oxidation reactions
The major oxidation reaction is hydroxylation;
there are various types of hydroxylation
reactions such as: Aromatic, Aliphatic, Alicyclic
Oxidative dealkylation, oxidative
desulphuration and deamination.
 Hydroxylation
• Heterocyclic hydroxylation
• Aromatic hydroxylation
In aromatic hydroxylation, the major product
of this rxn is phenol but catechol and quinol
may also be formed from further metabolism.
One of the toxic effects of benzenes is; it
causes plastic anemia which is believed to be
due to an intermediate metabolite possibly
hydroquinone (fig 2).
Aromatic hydroxylation of benzene
(fig 2)
 Aliphatic Hydroxylation
• In aliphatic hydroxylation, the unsaturated aliphatic
compounds such as vinyl chloride are metabolized by
epoxidation.
• Saturated aliphatic compounds can also undergo
oxidation. The initial products are primary &
secondary alcohols, for example the solvent n-
hexane is known to be metabolized to the secondary
alcohol hexan-2-ol alcohol, further to hexan – 2, 5-
dione in occupationally exposed humans. The latter
metabolite is believed to be
 Hydroxylation
• responsible for neuropathy caused by the
solvent Other toxicologically important examples are
the nephrotoxic fuel consti- tuents 2, 2, 4, and 2, 3, 4-
trimetyl pentane which are hydroxylated to yield
primary and tertiary alcohols (fig 3.)
Aliphatic hydroxylation of
nHexane(fig 3)
 O-Dealkylation
Deakylation- This is the removal of alkyl groups from
nitrogen, sulphur and oxygen atoms, the reaction is
catalyzed by the microsomal enzymes. Aromatic
methyl and ethyl ethers may be metabolized to give
phenol and corresponding aldelyde.
• O-De-ethylation
• Aromatic methyl and ethyl ethers may be
metabolized to give the phenol and corresponding
aldelyde as illustrated by the de-ethylation of
phenacetin to produce paracetamol (fig 4)
Metabolism of phenacetin (fig 4)
 Desulphuration
• The replacement of sulphur with oxygen is known to
occur in a number of cases and the oxygenation of
the insecticide parathion to give the more toxic
paraxon is a good example of desulphuration. This is
also important for other phosphothionate insect-
• icides. The toxicity depends upon the inhibition of
choline sterases and the oxidized product is much
potent in this respect. The reaction is catalyzed by
either CP450 or the FAD containing monooxygenases
and therefore require NADPH and Oxygen (fig 5).
Desulphuration of parathion (fig 5)
 Oxidative Dehalogenation
• Halogen atoms may be removed from xenobiotics in
an oxidative reaction catalyzed by Cp450. For
example the anaesthetic halothane is metabolized to
trifluoro acetic acid via several steps, which involves
the insertion of an oxygen atom and the loss of
chlorine and bromine.
This is the major metabolic pathway in man and it is
believed to be involved in the hepatotoxicity of the
drug. Trifuoroacetyl chloride is thought to be the
reactive intermediate.
 Reduction
• The enzymes responsible for reduction may be
located in both the microsomal fraction and
the soluble cell fraction (Ultracentrifugation of
cell homogenate). There are number of
different reductases which can catalyze the
reduction of Azo and nitro compounds. The
cytochrome P450 present in the microsomal
fraction is the major enzyme, FAD alone may
also catalyze reduction by acting as an
electron donor.
 Reduction of azo
• Reduction of the azo dye prontosil to produce
the antibacterial drug sulphani- lamide is a
well known example of azo reduction. This
reaction is catalysed by cytochrome P450 and
the reduction in the gut by bacteria. The
reduction of azo groups in food colouring dyes
such as amaranth is catalysed by several
enzymes, including CP450, NADPH, cyto P450
reductase.
Reduction of azo group (fig 6)
 Reduction of nitro groups
• The reduction of nitro groups may also be
catalysed by microsomal reductases and gut
bacterial enzymes. The reduction pusses
through several stages to yield the fully
reduced primary amine e.g nitrobenzene, the
intermediates are nitrosobenzene and
phennylhydroxy amine which are also reduced
in the microsomal system.
 Hydrolysis of hydrazides

• The drug isoniazid is hydrolyzed in vivo to the

corresponding hydrolysed acid and hydrazine as
shown in the reaction below. However, in man, in
vivo hydrolysis of the acetylated metabolite,
acetylisoniazid is quantitatively more important and
toxicologically more significant. This hydrolysis
reactions account for about 45% of the
acetylisoniazid produced. These hydrolysis reactions
are probably catalysed by amidases and are inhibited
by organophosphorus compounds.
Hydrolysis of isoniazid (fig7)
 Phase 2 conjugation rxns
• Conjugation reactions involve the addition to
xenobiotic of endogenous groups which are
generally polar and readily available in vivo.
These groups are added to a suitable functional
group present on the xenobiotics or added by
phase 1 now. This renders the whole molecule
more polar and less lipid soluble. The
endogenous compounds donated in conjugation
include glucuronide, amino acids, glutathione and
sulphate.
 Phase 2 reactions
• This renders the whole molecule more polar
and less lipid soluble. The endogenous
compounds donated in conjugation include
glucuronide, amino acids, glutathiones and
sulphate. The mechanism commonly involves
formation of a high energy intermediates
where either the endogenous metabolite or
the xenobiotic is activated type 1 and type 2
respectively.
 Glucuronide formation (gf)
• This is the major type of conjugation in phase II,
the reaction also occur in most species with a wide
variety of substrates. It involves the transfer of
glucuronic acid in an activated form as uridine
diphosphate glucuronic acid (UDPGA) to hydroxyl,
carboxyl, sulphur, Nitrogen and occasionally carbon
atom. This UDPGA is formed in the cytosol from
Glucosephosphate in a two step reaction. The first
reaction is the addition of UDP catalysed by
 Gf continued
• UDP glucose pyrophosphorylase and the 2nd
step is catalyzed by UDP glucose dehydro-
genase. The enzyme catalyzing the
conjugation reaction is UDP gllucuronosyl
transferase. It exists in possibly four or more
forms, each with different substrate
specificities. The enzymes are located in the
endoplasmic reticulum and are found in many
tissues including the liver.
Formation of uridine diphosphate
glucuronic acid (fig 8)
Examples of glucuronide
conjugation (fig 9)
More examples of glucuronide
conjugation reactions (fig 10)
 Sulphate conjugation (activation)
• The formation of suplhate esters is a major route of
conjugation for various types of hydroxyl group amino
groups, aliphatic alcoholics, phenols aromatic amines,
steroids and carbohydrates. The sulphate donor for this
reaction is an activated form – 3- phosphoadenosyl.5.
phosphosulphate (PAPS) it is formed from inorganic
sulphate and ATP.
• S042 + ATP Adenoosyl – 5 – Phosphosulphate (PAPS) +
PPO
• PAPS + ATP 3 Phospoadenosyl -5 –
phosposulphate (PAB) + ADP
Fig 11
 Sulphate conjugation
• Sulphate conjugation is catalysed by a sulphotransferase
enzyme which is located in the cytosol and is found
particularly in the liver, gastrointestinal mucosa and kidney.
The inorganic suphate precursor of PAPS may become
deplete if large amount of xenobiotic is conjugated with
sulphate as in the case of overdose, such as paracetamol .
• The list of other endogenous compounds involved in phase
2 conjugation reaction are as follows: Glutathione,
Cysteine, acetylation, methylation and amino acids –
(glycine is the most commonly used amino acids, others are
arginine, glutamine, ornithine and taurine.