Biotransformation
of
toxicants
DR. NADIA NOUR
Introduction
Xenobiotics
Endogenous:
Pigments ,
hormone

nonendogenous :
Such as drugs ,
food additives,
pollutants, toxin,
etc
Most of these compounds are subject to metabolism
(biotransformation) in human body.
Definition of the biotransformation
 Conversion of lipophilic xenobiotics to
water-soluble chemicals by a process
catalyzed by enzymes in the liver and other
tissues.
 In most cases, biotransformation lessens the
toxicity of xenobiotics, but many must
undergo the process to exert their toxic effects.
Purpose of biotransformation
1. facilitates excretion: Converts
lipophilic to hydrophilic compounds
2. Detoxification/inactivation:
converts chemicals to less toxic
forms
3. Metabolic activation: converts
chemicals to more toxic active
forms
Chronic hepatitis and cirrhosis of liver
Hepatic palm
Spider lentigo
Increase of estrogen, aldosterone and antidiuretin
Biotransformation
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.
Biotransformation
Biotransformation
Physical properties
More POLAR (hydrophilic)
Chemical Modification
Detoxification
Bio-activation
Inactive
metabolites
Active
metabolites
Non-Toxic
Toxic
Biotransformation
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.
Sites of biotransformation
• Liver
– Primary site! Rich in enzymes
– Acts on endogenous and exogenous compounds
 Hepatocytes contain wide variety of enzymes to process xenobiotics
 Enzymes are present in cytosol, endoplasmic reticulum and to
lesser extent in other organelles
 Each enzyme represents a large family of gene product
 Each gene product may be induced by different xenobiotics
• Extrahepatic metabolism sites
– Intestinal wall
• Sulfate conjugation
• Esterase and lipases - important in prodrug metabolism
– Lungs, kidney, placenta, brain, skin, adrenal glands
Overview of
biotransformation reactions
 Phase 1 reactions can limit the toxicity of a xenobiotics.
 Phase 1 reactions can also convert xenobiotics from inactive
to biologically active compounds (Metabolic activation). In
these instances, the original xenobiotics are referred to as
"procarcinogens.“
 Phase 2/conjugation reactions can convert the active products
of phase 1 reactions to less active or inactive species, which
are subsequently excreted in the urine or bile.
 In a very few cases, conjugation may actually increase the
biologic activity of a xenobiotic (Metabolic activation).
Comparing Phase I & Phase II
Enzyme
Phase I
Phase II
Types of reactions
Hydrolysis
Oxidation
Reduction
Small
Conjugations
Exposes functional
group
Polar compound added
to functional group
May result in
metabolic activation
Facilitates excretion
Increase in
hydrophilicity
General mechanism
Consquences
Large
Biotransformation
enzymes
Intercellular localization of
phase I CYTOCHROME
p450 enzymes with SER
(Smooth endoplasmic
reticulum ) in the cell
Phase I and Phase II enzymes
are integral proteins within
the phospholipd bilayer of
SER
Biotransformation Pathways
 Phase I reaction:
Polar functional groups are either
introduced into the molecule or modified by oxidation,
reduction or hydrolysis.

Or convert lipophilic molecules into more polar molecules
by introducing or exposing polar functional groups.

E.g. aromatic and aliphatic hydroxylation or reduction of
ketones and aldehydes to alcohols.
• Phase Ⅰ: Oxidation
Addition of oxygen or removal of hydrogen.
Normally the first and most common step involved in the xenobiotic
metabolism
Majority of oxidation occurs in the liver and it is possible to occur in
intestinal mucosa, lungs and kidney.
Most important enzyme involved in this type of oxidation is
cytochrome P450
Increased polarity of the oxidized products (metabolites) increases
their water solubility and reduces their tubular reabsorption, leading
to their excretion in urine.
These metabolites are more polar than their parent compounds and
might undergo further metabolism by phase II pathways
Phase Ⅰ: Oxidation
Hydroxylation
RH + O2 + NADPH + H+  R-OH + H2O + NADP+
Addition
of an oxygen atom or bond
Require NADH or NADPH and O2 as cofactors
RH: drugs, carcinogens, pesticides, petroleum products,
pollutants, steroids, fatty acids, etc.
 Enzyme:
Cytochrome P450s-dependent monooxygenase
Oxidation by CYP450
Many oxidation reactions are carried out by a group of
enzymes known as Cytochrome P 450 (CYP450)
Cytochrome P450: a superfamily of enzymes bound to the
phospholipid bilayer of the mitochondria and endoplasmic
reticulum. They are hemoproteins (contain heme cofactors) which
catalyze mainly the oxidation of organic substrates.
CYP enzymes have many isoforms (for example CYP1A1,
CYP2E1…) which are categorized into families and subfamilies
according to their amino acid sequence similarities: families have
40% structural similarities while isoforms in the same subfamily
have >60% structural similarities
Properties of Human
Cytochrome P450s(Contd.)
 Liver contains highest amounts, but found in most if not all tissues,
including small intestine, brain, and lung
 Located in the smooth endoplasmic reticulum or in mitochondria
(steroidogenic hormones)
 In some cases, their products are mutagenic or carcinogenic
 Many have a molecular mass of about 55 kDa
 Many are inducible, resulting in one cause of drug interactions
 Many are inhibited by various drugs or their metabolic products,
providing another cause of drug interactions
 Some exhibit genetic polymorphisms, which can result in atypical
drug metabolism
Oxidation by CYP450
Requirements for CYP450 activity:
1)The enzymes must be membrane-bound (the soluble form
loses its activity)
2)A single molecule of O2
3)NADPH-CYP450 Reductase …? …therefore CYP-450 is
known as a Mixed Function Oxygenase (MFO)…
Mixed
Function
Oxygenase
(MFO) O2
molecule
½ molecule to oxidize
the xenobiotic
½ molecule is reduced
to water
Oxidation by CYP450
Main Reactions types catalyzed by MFO
1) Hydroxylation of aromatic carbon
2) Epoxidation of double bond
3)Oxidative Deamination
4) Dehydrogenation
5) Heteroatom (O, S, N dealkylation)
Other Reactions Catalyzed by CYP450
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.
Phase Ⅰ: Reduction
is the converse of oxidation (i.e. removal of oxygen or addition of
hydrogen).
E.g. reduction of aldehydes and ketones, reduction of nitro and azo
compounds.
It is less common than oxidation, but the aim is same to create polar
functional groups that can be eliminated in the urine.
Cytochrome P450 system is involved in some reaction. Other reactions
are catalyzed by reductases enzymes present in different sites within
the body.
Phase Ⅰ: Reduction
Nitro and Azo Reduction
• NADPH
dependent microsomal and nitro-reductase
enzymes.
• Bacterial reductases play a role in enterohepatic
recirculation of nitro or azo containing drugs.
+ O
Ar N
O
Ar N N Ar'
Ar N O
Ar N N Ar'
H H
Ar NHOH
H2N Ar
Ar NH2
+
H2N Ar'
Phase Ⅰ: Reduction
Hydrolysis
Substrates: esters , amide , glycoside, etc.
 Catalyzed by widely distributed hydrolytic enzymes
 Hydrolysis of esters  major metabolic pathway for
ester drugs
☻Non-specific esterases (liver, kidney, and intestine)
☻Plasma pseudocholinesterases also participate
Acetylsalicylic
Acid, ASA
esterase
CO2H
O
CH3
CO2H
OH
CH3
O
O
salicylic acid
ASA
HO
Phase 1 reactions- Enzymes
 Mainly Catalyzed by members of a class of enzymes referred to
as Monooxygenases, Mixed Function oxidases or
Cytochrome P450s.
 Other enzymes of significance areo Aldehyde and alcohol dehydrogenase
o Deaminases
o Esterases
o Amidases
o Epoxide hydrolases
Phase II: Conjugation
• In phase Ⅰ reactions, xenobiotics are generally
converted to more polar, hydroxylated derivatives.
• In phase Ⅱ reactions, these derivatives are conjugated
with molecules such as glucuronic acid, sulfate, or
glutathione.
• This renders them even more water-soluble, and they
are eventually excreted in the urine or bile.
Five types of phase II reactions
A.
B.
C.
D.
E.
Glucuronidation
Sulfation
Conjugation with glutathione
Acetylation
Methylation
1. Glucuronidation
• the most frequent conjugation reaction.
• UDP-glucuronic acid (UDPGA) is the glucuronyl
donor
• UDP-glucuronyl transferases (UGT), present in
both the endoplasmic reticulum(ER) and cytosol,
are the catalysts.
– Liver, lung, kidney, skin, brain and intestine


Attachment sites are hydroxyls
– Alcohols, phenols, enols, N-hydroxyls,
acids
Oxygen site often from Phase I
2. Sulfate Conjugation
• Some alcohols, arylamines, and phenols are sulfated.
• Catalyzed by sulfotransferases
– liver, kidney and intestine
• Sulfate donor: adenosine 3’-phosphate-5’-phosphosulfate
(PAPS); this compound is called “active sulfate.”
• Leads to inactive water-soluble metabolites
• Glucuronate conjugation often more competitive process
HX Drug
sulfotransferase
PAPS
O
O
-O S O P O
O
OH
H2O3PO
O
Adenine
OH
O
-O S
O
X Drug
3. Conjugation with glutathione
Glutathione (Υ-glutamyl-cysteinylglycine) is a tripeptide consisting of
glutamic acid, cysteine, and glycine
 Glutathione is commonly abbreviated GSH (because of the
sulfhydryl group of its cysteine, which is the business part of the
molecule).
 A number of potentially toxic electrophilic xenobiotics (such as certain
carcinogens) are conjugated to the nucleophilic GSH in reactions that can
be represented as follows:
GST
R + GSH
R-S-G
where R= an electrophilic
xenobiotics
R: epoxides and halogenides
GST: Glutathione S-Transferases (Liver and kidney)
Glutathione (GSH) Conjugation
 A variety of glutathione S-transferases are present in human
tissue. They exhibit different substrate specificities and can
be separated by electrophoretic and other techniques.
 If the potentially toxic xenobiotics were not conjugated to
GSH, they would be free to combine covalently with DNA,
RNA, or cell protein and could thus lead to serious cell
damage.
 GSH is therefore an important defense mechanism against
certain toxic compounds, such as some drugs and
carcinogens.
Glutathione (GSH)
Conjugation
• DETOXIFICATION of electrophiles!
• Electrophilic chemicals cause:
– Tissue necrosis
– Carcinogenicity
– Mutagenicity
– Teratogenicity
• The thiol (SH group) ties up potent
electrophiles
4. Acetylation
Acetylation: N-acetyl transferases
N-acetylating of xenbioticsis catalyzed by
cytosolicN-acetyl transferasesand require a co substareas acetyl co A. It involves metabolism of
amines , sulphonamides, hydrazines
X + Acetyl-CoA - - - - >Acetyl-X +
where X represents a xenobiotics.
(for: aromatic amines)
CoS
• Enzyme: acetyltransferases
• present in the cytosol of various tissues,
particularly in liver.
5-Methylation
• A few xenobiotics are subject to methylation by
methyltransferase, which transfer methyl group to
functional group on the xenobiotic using
S-adenosylmethione(SAM) as the methyl donor.
Example :
-Amino, hydroxy,thiolgroups can be methylated.
-Certain metals as Mercury in microorganism in aquatic system .
Metabolism via Methylation
• Key for biosynthesis of many compounds
• Important in the inactivation of physiologically
active biogenic amines  neurotransmitters
– norepinephrine, dopamine, serotonin,
histamine
• Minor pathway in the metabolism of drugs
• Methylation does NOT increase water
solubility
• Most methylated products are inactive
Effects of Xenobiotics
Effects of Xenobiotics
Metabolism of a xenobiotic can result in cell
injury, immunologic damage, or cancer.
 Cell injury (cytotoxicity), can be severe
enough to result in cell death.
These macromolecular targets include DNA,
RNA, and protein.
The reactive species of a xenobiotic may bind
to a protein, altering its antigenicity
The resulting antibodies can then damage the
cell by several immunologic mechanisms that
grossly perturb normal cellular biochemical
processes.
Effects of Xenobiotics
Reactions of activated species of chemical carcinogens
with DNA are of great importance in chemical
carcinogenesis
 Some chemicals (eg, benzo[α]pyrene) require activation
by monooxygenases in the endoplasmic reticulum to
become carcinogenic (they are thus called indirect
carcinogens).
 The products of the action of certain monooxygenases
on some procarcinogen substrates are epoxides.
 Epoxides are highly reactive and mutagenic or
carcinogenic or both.
 Epoxide hydrolase—like cytochrome P450acts on these
compounds, converting them into much less reactive
dihydrodiols.
Modifiers of biotransformation
Factors affecting metabolism and
disposition
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.
Modifiers of Biotransformation
• A-Species differences
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.
B-Sex differences
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.
In general ; Male metabolize foreign
compounds more rapidly than female
C- 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 drugrelated 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.
GENETICS
• Activity of xenobiotic metabolizing enzymes can be
vary between individual
• Population can be divided to RAPID METABOLIZER
and SLOW METABOLIZERS
Variation in toxicant metabolism
Variation of toxicant level in the body
Variation of toxicant response/toxicity
D-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.
E- 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.
F- 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.
G- 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.
Summary
•
•
•
Xenobiotic, Biotransformation
Phase I reactions:
– Purpose:
• Enhances elimination
• Converts chemical to less toxic forms (detoxification)
• Converts chemicals to more toxic active forms
(activation)
– Functionalization:
• Oxidation: monooxygenase, CYP450
• Reduction: ADH, ALDH
• Hydrolytic reactions: esterase
Phase II: Conjugation Rx
– Purpose: more water-soluble, excreted in the urine or bile
– Functionalization:
• Glucuronidation, Sulfation, Conjugation with