Biochemistry of Xenobiotics

lecture outlines

xenobiotics overview
 Biotransformation of xenobiotics
Overview of cytochrome P450
Conjugation of xenobiotics
 Mercapturic acid pathway of
xenobiotics
 Xenobiotics
Xenobiotics (Greek xenos “foreign”; bios “life”

• Xenobiotics are agents that are foreign to the body or

a biological system

• Xenobiotics are defined as chemicals to which an

organism is exposed that are extrinsic to the normal
metabolism of that organism

• The substances if present more concentration then

usual are also grouped under xenobiotics
 Xenobiotics…cont’d
Xenobiotic can originate from:
air, water,
food additive and dietary supplements
drugs, antioxidants
emulsifiers, cosmetics, soaps, perfumes
Metabolic metabolites
therapeutic drugs
Industrial chemicals (soaps, detergents, dyes,
bleaching agents)
Pesticides
Food additives
 bacterial and fungi toxins
 Xenobiotics…cont’d
Xenobiotics enter to the body as result of intentional
or accidental exposure via:

• Ingestion(gastrointestinal tract): intestinal- blood-

liver

• Inhalation (respiratory tract): Lungs –blood

• Dermal contact -skin – blood

• Parenteral (circulation/muscles) -liver

 Xenobiotics…cont’d
Chemical nature of Lipophilic
xenobiotics • Poorly soluble in water

Hydrophilic • Need a blood transporter

(albumin)
• Water soluble
• Freely diffuse through
• Difficult to transport
membranes
through membrane
• Can be stored in membranes
• Rapidly eliminated with • Slowly eliminated from body
urine and bile
Xenobiotics…Cont’d
 Xenobiotics…cont’d
Biological Damages Caused by Xenobiotics
1. Alteration of protein conformation
• Xenobiotics may bind to the body protein to alter its
conformation, which may cause functional inactivation of the
protein

2. RBCs Agglutination

Lectins from plants agglutinate red blood cells

3. Damage to DNA and RNA

• Binding of xenobiotics to DNA may result in mutation often
with serious consequences
 Xenobiotics…Cont’d
4. Antagonists

• Some xenobiotics exhibit structural similarity to a

natural metabolite, therefore, act as antagonists.
For example, dicumarol, a structural analogue of
vitamin K, antagonizes action of this vitamin.

5. Neurotoxin

• Many bacterial products act as neurotoxins

Xenobiotics
metabolism
 Xenobiotics metabolism

Xenobiotics could be undergo through different pathway

either:
1. unchanged-hydrophilic compounds eliminated in urine
2. Lipophilic compounds eliminated through faeces
3. retained in the body unchanged(e.g. lead and
cadmium)
4. undergo spontaneous transformation or
5. may be metabolized enzymatically(most common
pathway)
Xenobiotics metabolism …Cont’d

xenobiotic can be:

a parent compound
intermediate or product of a metabolic pathway
Xenobiotics are metabolized by:
Xenobiotic- Metabolizing Enzymes(XME)
XMEs can be either:
Phase I enzymes(activation)
Phase II enzymes (conjugation)
 Phase III enzymes (Transporter enzymes).
 Xenobiotics metabolism …Cont’d

There are three major steps in the xenobiotics

metabolism mechanism:
1. Biotransformation – Phase I Reaction
2. Conjugation – Phase II Reactions
3. Mercapturic acid pathway – Phase III Reactions
 Biotransformation-Phase I Reactions
Introduces reactive functional groups

Non polar metabolites to polar metabolites

It can be beneficial or harmful

Biotransformation consequence:

• Change in solubility of substances

• Detoxification

• Metabolic activation
 Biotransformation-Phase I Reactions…cont’d

Detoxification: Toxic xenobiotics converted into less harmful

and excretable, water soluble substances

Bioactivaton: Metabolic activation of xenobiotics /drugs into

reactive or toxic compounds

• Xenobiotics converted to more toxic metabolites

 Role of liver in xenobiotics metabolism
Phase III
Toxins
Metabolic end Waste products
products Phase I Phase II (water soluble)
• Microorganism
• Food additives
• Insecticides
• Pesticides
• Drugs
• Alcohols
• Contaminants /
pollutant
Phase I Reactions
 Biotransformation-Phase I Reactions …Cont’d
There are three major types of reactions occurring in Phase I:

• Oxidation reactions catalyzed By enzymes like:

cytochrome P450 monooxygenase, Flavin-containing
monooxygenase, Alcohol dehydrogenase, aldehyde
dehydrogenase, monoamine oxidase and peroxidases
• Reduction reactions catalyzed by NADPH cytochrome
P450 reductase and reduced cytochrome P450
• Hydrolysis reactions catalyzed by esterases, amidases
and hydrolases
Biotransformation-Phase I Reactions …Cont’d
 Biotransformation-Phase I Reactions …Cont’d
The end-products of biotransformation reactions are
charged molecules
They are either electrophilic or nucleophilic as compared
with before the biotransformation
most common biotransformation reaction is the addition
of polar groups to the foreign molecule( i.e. addition of a
hydroxyl group (-OH)) catalyzed by the cytochrome P450
dependent mixed function oxidase system
It introduces or exposes a functional group that serves as
the active center for sequential conjugation in phase II
reactions
 Biotransformation-Phase I Reactions …Cont’d

Cytochrome P450 dependent mixed function

oxidase acts to add an atom of oxygen into a non-
activated xenobiotic, notably a hydrocarbon molecule,
to produce a more polar molecule

The product of this reaction is the hydroxylated

xenobiotic, which is highly reactive and more polar
than the starting xenobiotic
 Biotransformation-Phase I Reactions …Cont’d

The cell is capable of adding a Sulphur (-S) and nitrogen (-N)

atom in biotransformation reaction.
This will result in the dealkylation (removal of alkyl groups such
as methyl or ethyl group from the xenobiotic) by addition of a
sulfhydryl group (-SH) or an amino group (-NH).
The products of biotransformation reactions are normally
electrophilic or nucleophilic in nature, such as sulphides and
amines.
 The products of biotransformation reactions such as the oxides,
hydroxylated metabolites, sulphides and amines are now primed
for conjugation reactions
 Biotransformation-Phase I Reactions …Cont’d

1. Hydroxylation reaction
• Most common reaction of phase I metabolism ,which
introduces hydroxyl group in substrates
NADPH + H+
Xenobiotic +O2 xenobiotic-OH + H2O
NADP+
• Detoxification of a number of drugs and steroids occur by
hydroxylation
• These reactions are catalyzed by cytochrome P450
dependent monooxygenase
 Biotransformation-Phase I Reactions …Cont’d
Human cytochromes P450.
They are hemoprotein
It contains heme prosthetic group in the active site
The heme iron is liganded to both a conserved cysteine residue and a
water molecule
Tissue location: Mainly found in the liver but also in kidney, lungs,
intestine .etc.
Cellular location: located either in the mitochondria or endoplasmic
reticulum.
Cyt P450 in endoplasmic reticulum membrane associated with:
1. NADPH
2. Cytochrome P-450 reductase
 Biotransformation-Phase I Reactions …Cont’d

Properties of human cytochromes P450 Enzymes

1. Cytochrome P-450 are highly inducible
• Tts substrates (and other xenobiotic agents) often induce
the enzyme synthesis, usually by increasing the rate of
transcription.
• Such self-induction provides an efficient mechanism of
detoxification
• However, it also results in rapid development of tolerance
against drugs/xenobiotics
 Biotransformation-Phase I Reactions …Cont’d

2. The Most Versatile Biocatalysts

• The most versatile biocatalysts that can oxidize any
conceivable foreign molecule
• They can be broadly divided into two varieties:

1. Lipid metabolizing variety: P-450 flavoproteins has a

tight substrate specificity
2. Drug metabolizing variety: These cytochromes are
present in microsomal where they co-localize with
NADPH and an enzyme called cytochrome P-450
reductase
 Biotransformation-Phase I Reactions …Cont’d

3. Isoforms of Cytochrome P-450

• Large number of isoforms of cytochrome P-450 (about
150) have been identified

• They are encoded by at least 12 gene families

• Three of these gene families designated CYP1, CYP2,

and CYP3—share responsibility for xenobiotic
metabolism
• All P-450 Families Subfamilies Isoform
cytochromes
 Properties of human cytochrome P 450.

• CYP-mediated monooxygenation reactions involve

incorporation of one atom of oxygen into the substrate, while
the other is reduced to water
 Biotransformation-Phase I Reactions …Cont’d

2. Oxidation reaction

• Most of the foreign substances are detoxified by

oxidation

• These includes alcohols, aldehydes, amines,

aromatic hydrocarbons and Sulphur compounds

• Aliphatic compounds are more easily oxidized

than aromatic ones
 Biotransformation-Phase I Reactions …Cont’d

2. Oxidation reaction

• Indole and Skatole are produced from tryptophan,

by the action of microbes

They undergo oxidation

• Indole →Inodoxyl

• Skatole → Skatoxyl
 Biotransformation-Phase I Reactions …Cont’d

2. Oxidation reaction
ALCOHOLS
• Both aromatic and aliphatic alcohols undergo oxidation to form
their corresponding acids
Example:
• Methanol → formaldehyde → formic acid
• Ethanol→Acetaldehyde → acetic acid
• Benzyl alcohol →Benz aldehyde → benzoic acid
• Halogenated Alcohol ------------> Halogenated acid
• Ethylene glycol -----------------------> oxalic acid
 Biotransformation-Phase I Reactions …Cont’d

Methanol toxicity

• It is metabolized in the liver

• In the first step of degradation, methanol is

transformed to formaldehyde via enzyme ADH

• Transformation of formaldehyde to formic acid via

aldehyde dehydrogenase is faster
 Biotransformation-Phase I Reactions …Cont’d

• The metabolism of formic acid is very slow.

• Thus it often accumulates in the body ,which

results in metabolic acidosis.

• The major damage occurs to the optic nerve.

• Ethanol is given as an antidote, since it is the

substrate of ADH, methanol is spared
Biotransformation-Phase I Reactions …Cont’d

Methanol Toxicity

• Metabolic
acidosis
• Tissue injury
 Biotransformation-Phase I Reactions …Cont’d
2. Oxidation reaction
Aldehydes
• Aldehydes are oxidized to corresponding acids

• Acid thus formed is further conjugated in phase II

Example: Benz aldehyde → benzoic acid.

• Benzoic acid is conjugated with glycine to form

Hippuric acid
 Biotransformation-Phase I Reactions …Cont’d

2. Oxidation reaction
Aromatic hydrocarbons
• Aromatic hydrocarbons are oxidized to phenolic
compounds.
• Which can further conjugated with glucuronic acid
or sulphuric acid in phase II reactions, so as to be
excreted through urine.

• Benzene Mono, Di, Tri hydroxy phenol

 Biotransformation-Phase I Reactions …Cont’d
2. Oxidation reaction
Amines
• Many primary aliphatic amines undergo oxidation to
form the corresponding acids and nitrogen is
converted to urea
Example: Benzyl amine →Benzoic acid + urea
• While aromatic amines are oxidized to phenols
Aniline → P-Amino phenol
 Biotransformation-Phase I Reactions …Cont’d
2. Oxidation reaction
Anilids and sulphur containing compounds
• Anilids are oxidized to corresponding phenols
• Acetanilide is oxidized in the body to form P-acetyl
amino phenol.
• Acetanilide is a constituent of analgesic drug

Sulfur compounds
• Organic sulfur is oxidized to sulfuric acid
 Conjugation – Phase II Reactions…cont’d
Phase II reaction involves the conjugation of the bio-transformed
xenobiotics to cellular endogenous compounds

Conjugation may occur with the following compounds:

Glucuronic acid
Sulphate,
Amino acids (glycine, glutamine, histidine)
 Glutathione
Acetate

Conjugation reaction make bio-transformed xenobiotics more

strongly hydrophilic to form easily excretable products
Conjugation – Phase II Reactions…cont’d
 Conjugation – Phase II Reactions…cont’d
1. Glutathione(GSH) conjugate reaction

GSH is tripeptide (glutamic acid, cysteine and glycine,

linked covalent bond

Bio-transformed xenobiotics conjugated with

glutathione (glutamyl-glycinyl-cysteine)

Enzyme: Glutathione S- Transferases (GSTs)

 Conjugation – Phase II Reactions…cont’d
 Conjugation occurs with the sulfhydryl group of the cysteine
residue; and the glutamate and glycine residues are split off
 Conjugation – Phase II Reactions…cont’d
2. Glucuronic acid conjugation reaction
Glucuronyl moiety of UDP-glucuronic acid is added
to bio-transformed xenobiotics

Enzyme: UDP-glucuronyl transferases

 Conjugation – Phase II Reactions…cont’d

Glutamine-glycine conjugate reaction

• Some xenobiotics are converted to the
corresponding thioester(R−C−S−R) form in phase I
and then conjugated with glycine/glutamine
 Mercapturic acid pathway – Phase III
Reactions
This mechanism involves reactions leading to the removal
or excretion of the conjugated xenobiotics from the cell
Three enzymes are involved in the process:
1.Gamma-glutamyltranspeptidase(GGT): cleavage of
glutamic acid residue of glutathione
2. Dipeptidase: cleaves the glycine residue
3. N-acetyltransferase: attaches an acetic acid group to the
remaining xenobiotic conjugate
 Mercapturic acid pathway – Phase III
Reactions
The acidified conjugate is finally excreted from the cell as
a mercapturic acid