METABOLISM OF XENOBIOTICS

Raymond Oliver Cruz, M.D.

XENOBIOTICS
•A xenobiotic is a compound that is foreign to the body
•It may include drugs, food additives chemical used in
CYP450
the work place, industrial by-product that become
-named because it exhibit a distinct peak at 450nm after
environmental contaminants
in has been reduced and exposed to CO
 Metabolized in the body with the liver as the main
-metabolized appropriately 50% of drug ingested
organ involved
- are also involved in the metabolism of many
PHASES of XENOBIOTIC METABOLISM
endogenous compounds like steroids
PHASE I
-are hemoprotein
-major reaction is hydroxylation catalyzed by
- Present in highest amount in the membranes of the
monooxygenase or CYP 450
endoplasmic reticulum of the liver; also found in the
Other types of reaction are reduction, hydrolysis,
mitochondria and ER of adrenals where they play an
deamination, dehalogenation, etc.
important role in steroid synthesis
- adds or unmask a functional polar group; results in a
-uses NADPH
relatively small increase in hydrophilicity
- Enzyme involved in NADPH- CYP 450 reductase
PHASE II
- At least 6 closely related species of CYP450 present in
-Conjugation with glucoronic acid, sulfate, acetate,
liver ER with wide overlapping substrate specificities
glutathione or certain amino acid or by methylation
-lipids are component of CYP 450 system; preferred
- Often, but not always, to a functional group provided
lipid is phosphatidylcholine
by a phase I reaction
-are inducible; administration of phenobarbital causes
Increases water solubility and facilitates their excretion
hypertrophy of smooth ER and increase in CYP450
from the body
- Clinically important because it is one
biochemical mechanism of drug interaction
DETOXIFICATION
-sometimes refer to reactions involved in the
metabolism of xenobiotics
Not always appropriate since some reactions may
increase biologic activity and toxicity
- Metabolism may either decrease or increase toxicity
- Parent compound toxic  metabolites non-
toxic
- Parent compound non-toxic metabolites
NOMENCLATURE OF CYP 450
toxic
-those P450s that have 40% of their amino acids in the
PHASE I
same linear sequence are replaced into the same family
- Chief reaction is hydroxylation
designated by a no. following CYP
-responsible enzyme mono-oxygenase or cytochrome
- If the amino acid in the same family are identical 55%
P450
of the time, the 2 enzymes are in the same subfamily
which are designated by a letter following the family  The enzymes catalyzing these reactions are called
thus CYP 2E glutathione S-transferases
-then each individual enzyme in the subfamily is given a R+GSH→R—S—G
no. (i.e., CYP 2E1)  If potentially toxic xenobiotics were not conjugated to
GSH, they would be free to combine covalently with
CYP448 DNA, RNA or cell protein and lead to serious cell
-specific for the metabolism of polycyclic aromatic damage
hydrocarbon (PAH)  Glutathione conjugates are subjected to further
-also called aromatic hydrocarbon hydroxylase metabolism; Glu and Gly are removed, acetyl CoA
-involved in the conversion of inactive PAH inhaled in added and resulting compound is mercapturic acid
smoking to active carcinogen which is secreted in urine
 Other function of glutathione
o Participates in the decomposition of
PHASE II
potentially toxic hydrogen peroxide
-renders xenobiotics more water soluble and eventually catalyzed by glutathione peroxidase
excreted in the urine or bile

TYPES of PHASE II REACTION  Important intracellular reductant

o Maintain essential –SH groups in
GLUCORONIDATION
enzyme in reduced state
-most frequent conjugation reaction

-UDP-glucoronic acid is the glucoronyl donor with

glucoronyl transferase as catalyst
o G-S-S-G reduced to GSH by glutathione
-molecules excreted as glucoronides: 2- reductase
acetylaminoflourene, aniline, benzoic acid, o G-S-S-G + NADPH + H+ GSH + NADP
Important in the integrity of RBC membrane
meprobromate, phenols, steroids, glucuronide attached
to O, N, or S group

SULFATION

- Substrate: alcohol, arylamines, phenols

-sulfate donor: phosphoadenosine phosphosulfate

(PAPS) or active sulphate

CONJUGATION with GSH

GLUTATHIONE
•Glutathione is a tripeptide, y- glutamyl-cysteinylglycine
 Substrate are potentially toxic electrophilic Transports certain amino acids across membranes in
the kidney
xenobiotics
Amino acids + GSH
y- Glutamyl amino acid + cysteinyl glycine
ENZYME: y-glutamyl transferase
- present in membranes of renal tubular cells and
hepatocyte
-has diagnostic value in hepatobiliary disease
ACETYLATION
ACETYL CoA- is the acetyl donor
Catalysed by acetyl transferase
X+ acetyl CoA acetyl-x- CoA
E.g. ISONIAZID
- Slow and fast acetylators- influence the rate of
clearance of the drug
METHYLATION
-catalysed by ethyl transferase
- S-adenosyl methionine as donor
GENETIC POLYMORPHISM OF CYP450
- Responsible for the difference in the metabolism of
particular drugs among individuals
Greater expression of a specific allelic variants in an
ethnic population could lead to more frequent adverse
reaction with certain drugs
-can be caused by single nucleotide substitution,
frameshift or missense of generation of a termination
codon that produce a shorthand protein that is inactive
- variant alleles can code for proteins that contain an
altered substrate docking site CYP 450 reductase, which
reduces electron transfer  poor metabolizer
- can also occur when more than one active gene is
expressed or when 2 or more copies of a gene is
transcribed
- leads to greater amount of active protein and an
accelerated rate of metabolism-> rapid metabolizers
- Example: CYP 2D6
- metabolizes debrisoquine, asparteine,
amitriptyline, dextromethorphan and codeine
- Deficient in 5-10% of Caucasians and 8% of
Africans-African-American and 1% in Asians
- Catalyses methylation of codeine into
morphine; individuals who lack the normal genes do not
achieve the analgesic effects associated with codeine
- Example: CYP 2C9
- Deficient in 0.008% of Asians and 0.36% of
Caucasians
- metabolizes warfarin, an orally administered
drug used to inhibit blood coagulation
Individuals deficient in CYP2C9 may require only 0.5-1
mg of warfarin per week in contrast to the 4-5mg per
day for an individual who possess the normal active
form
TYPES OF TOXIC EFFECTS OF XENOBIOTICS
1. CELL INJURY
- can be severe enough to result in cell
- Mechanism:
- Covalent bonding to cell
macromolecules like, DNA, RNA and proteins
- affects critical cellular function such as
oxidative phosphorylation, regulation of
permeability of plasma membrane
2. ACTS AS HAPTEN
HAPTEN- molecule which by itself does not
stimulate antibody synthesis
- Reactive species of xenobiotic may bind to
protein modifying if and altering its antigenicity
and stimulating antibody formation
- Resulting antibody can damage cell by
immunologic mechanism
3. CHEMICAL CARCINOGENESIS
- may act either as indirect or direct carcinogen
- Some chemicals like benzo[a] pyrene
requires activation by monooxygenase to
become carcinogenic
- Other chemicals like alkylating agents
reacts directly with DNA
- Product of certain monooxygenase are
epoxides
- Epoxides are highly reactive and
mutagenic or carinogenic
- Epoxied hydrolase- converts epoxide
to less reactive dihydrodiols
FREE RADICALS AND ANTIOXIDANT
NUTRIENTS
FREE RADICALS
- are atoms and or group of atoms that have unpaired
electron
- associated with many conditions from cancer,
inflammatory conditions, artherosclerosis and aging
-are mostly products of oxygen metabolism
Includes superoxide, hydroxyl radicals and peroxy
radicals including hydrogen peroxide
-are collectively referred to as reactive oxygen species
-reactivity and destructiveness of individuals ROS varies
-H2O2 is less reactive than superoxide, which in turn is
less reactive than hydroxyl radicals
PROOXIDANTS AND ANTIOXIDANTS
PROOXIDANTS
-chemical compounds and reactions capable of
generating the above mentioned toxic oxygen species
ANTIOXIDANTS
- Compounds and reactions disposing of these toxic
species
-in a normal cell, there is a balance of the prooxidants
and antioxidants
OXIDATIVE STRESS
- Balance can be shifted towards the prooxidants when:
- The production of oxygen species is greatly
increased
- The levels of the antioxidant and diminished
- This state is called oxidative stress
-can result in cell damage or even cell death if massive
The ROS are extremely reactive and can react with or
chemically alter virtually compounds such as proteins,
DNA and lipids
DELETERIOUS EFFECTS OF FREE RADICALS
- Free radicals can react with the polyunsaturated fatty
acid found mostly in membrane in a process called lipid
peroxidation
- can lead to loss of membrane integrity not only to the
plasma membrane but also to the mitochondrial
membrane
-destruction of the mitochondria undermines the
function of the respiratory chain and the production of
ATP
-direct interaction of ROS with nucleotides in DNA can
cause misreading of the sequence and if left
uncorrected, can lead to mutation
- Damage to DNA in ovaries and testes can lead to
heritable mutations and in somatic cells can lead to
activation of proto-oncogenes to oncogenes resulting in
the initiation of cancer
-reactions with amino acids in proteins, either by direct
radical action or as a result of reaction with the
products of radical-induced lipid peroxidation leads to
modification of proteins that are recognized as non-self
by the immune system
- The resultant antibodies will also cross-react with
normal tissue proteins initiating autoimmune disease
- oxidation of the proteins or lipids in plasma LDL by
ROS leads to abnormal LDL that is not recognized by
liver LDL receptor and therefore not cleared by the liver
- The modified LDL is taken up by macrophages through
scavenger receptors. These macrophage cells that
infiltrates the endothelium of the blood vessels leading
to the development of atherosclerotic -many natural
processes in the cells that require enzyme-catalysed
oxidation of organic molecules by molecular oxygen
generate the reactive oxygen species
PRODUCTION OF FREE RADICALS
-Hydroxylation reaction that occurs in CYP 450 for the
detoxification of xenobiotics
-Synthesis of steroid hormone
- Degradation of purines to uric acid
-reoxidation of the prosthetic group of flavin-containing
enzyme
Transport of electron in the respiratory chain and the
reduction of oxygen which is the final acceptor of the
electron
 -generates malondialdehyde which by itself can also
covalently reacts with DNA, proteins and lipids forming
adducts that can produce more cellular damage

LIPID PEROXIDATION
-The O2 carried by RBC can accept an electron during
the conversion of the methemoglobin generating
superoxide free radicals

-When stimulated by bacterial, neutrophils exhibit a

respiratory burst and produce superoxide in a reaction
catalysed by NADPH OXIDASE MECHANISM FOR PROTECTION AGAINST FREE
RADICALS

- Metal Ions that can initiate free radical formations

-the generation of superoxide is one of the several ways are bound to proteins for which they provide the
neutrophils are able to kill bacteria prosthetic group or to their transport or storage
-pathways by which the less destructive ROS can proteins
transform to the more highly reactive ROS in the  Iron
presence of iron ions o Transferrin
1. FENTON REACTION, a non-enzymatic reaction, o Ferritin
ferrous ion can transform H2O2 to hydroxyl radical o Hemosiderin
2. Iron-catalyzed Haber-weiss reaction, hydroxyl radical - Most enzymes that produce and degrade free
can also be generated when superoxide and hydrogen radicals are confined in peroxisomes
peroxide reacts - Enzymes
 Superoxide Dismutase
o O2•- + O2•- + 2H+ → H2O2 + O2
 Catalase
o H2O2 → H2O + O2
 Glutathione Peroxidase
o GSH + ROOH → GSSH + H2O + ROH
- Vitamin and Minerals
 Vitamin A  Beta-Carotene
 Vitamin C  Selenium
 Vitamin E
- Phytochemicals
 Flavonoids and other polyphenols

-LIPID PEROXIDATION is a chain reaction involving

polyunsaturated fatty acids that produces a continuous
supply of more free radicals
POLYPHENOLS
- Compounds that contain several hydroxyl groups in
aromatic rings
- Occur in fruits, vegetables, tea, coffee, beer and
wine
- Generally present in plants to protect them against
UV radiations or aggression by pathogens
- Have Antioxidant properties
- Although a considerable body of epidemiological
evidence suggest
 Protective effects of anti-oxidant supplements
- Intervention trials proved otherwise
 Increased mortality rate with certain types of
cancer and Cardiovascular Diseases
- Anti-oxidants can act as Pro-oxidants
 Vitamin E reacts with lipid peroxidases in
lipoproteins
o Forms a stable Tocopheroxyl radical
 Causes radical damage if it persists long
enough to penetrate deeper into the
lipoproteins and tissues
 Vitamin C reacts with Superoxide and hydroxyl to
yield Monodegydroascorbate and Hydrogen
Peroxide or Water
 Source of superoxide radicals
o Reaction with Oxygen or Hydroxyl radicals by
reaction with Cupric ions
 β – Carotene
o A Radical-trapping Antioxidant under
condition of low partial pressure of
Oxygen
o At High Partial pressure oxygen
 Autocatalytic pro-oxidant
 Initiate radical damage to lipids and
proteins
Anti-oxidant Roles
Ascorbate + O2•- → H2O2 + monodehydroascorbate
Ascorbate + OH• → H2O + monodehydroascorbate
Pro-oxidant Roles
Ascorbate + O2 → O2•- + monodehydroascorbate
Ascorbate + Cu2+ → Cu2+ + monodehydroascorbate
Cu+ + H2O2 → Cu2+ + OH- + OH•