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Metabolism of Xenobiotics
Xiao Li
Introduction
※Xenobiotics: is a compound that is foreign
to the body ; is a chemical which is found in an
organism but which is not normally produced
or expected to be present in body.
※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
Chronic hepatitis and cirrhosis of liver
Hepatic palm
Spider lentigo
Increase of estrogen, aldosterone and antidiuretin
3. Metabolic activation: converts chemicals to
more toxic active forms
Detoxification ≠
biotransformation
benzpyrene
3,4-Benzypyrene
Sites of biotransformation

Liver
– Primary site! Rich in enzymes
– Acts on endogenous and exogenous compounds

Extrahepatic metabolism sites
– Intestinal wall


Sulfate conjugation
Esterase and lipases - important in prodrug
metabolism
– Lungs, kidney, placenta, brain, skin, adrenal
glands
Knowledge of the metabolism of xenobiotics:
rational understanding of
pharmacology and therapeutics ,
pharmacy, toxicology, cancer research ,
and drug addiction .
General Metabolic Pathways

Approximately 30 different enzymes catalyze
reactions involved in xenobiotic metabolism;
however, this note will only cover a selected
group of them.
 It
is convenient to consider the
metabolism of xenobiotics in two phases
– phase Ⅰand phase Ⅱ
Phase I reactions
♣♣ Functionalization
– Oxidation
– Reduction
– Hydrolytic reactions
Purpose
Introduction of polar functional groups in a
molecules
♣ Increase a molecule’s polarity
♣ Does provide a site for phase II metabolism
Phase II reactions
♣♣ Conjugation
★
Purpose
– Introduce highly polar conjugates:
☻☻Glucuronic acid ☻☻ Sulfate
– Detoxification
 Glycine or other Amino Acids (some solubility),
Acetyl , Methylations , Glutathione
★ Site of attachment often introduced in Phase I
Hydroxyl , Carboxylate , Amino
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
 Phase Ⅰ: Oxidation
1. 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, cacinogens, pesticides, petroleum products,
pollutants, steroids, eicosanoids, fatty acids, retinoids, etc.
 Enzyme:
Cytochrome P450s-dependent monooxygenase
Hydroxylation：O2
* It has been shown by the use of O2 that one
atom of oxygen enters R-OH and one atom
enters H2O.
* This dual fate of the oxygen accounts for the
former naming of monooxygenases as “mixedfunction oxidases”.
RH + O2 + NADPH + H+  R-OH + H2O + NADP+
Cytochrome P450s-dependent monooxygenase
-----the most versatile biocatalyst
----Works on a large number of diverse
compounds
★ Microsomal drug oxidations require
cytochrome P450,
cytochrome P450 reductase,
NADPH , & O2
The actual reaction mechanism is as follows:
Cytochrome P450s-dependent monooxygenase
CYP or Cytochrome P-450
★ Heme proteins
★ Iron containing porphyrin - binds O2
What is a Heme Protein?
Cytochrome P-450, Hemoglobin, & Myoglobin ALL Heme Proteins!
★ The name cytochrome P450 is derived from the
spectral properties of this hemoprotein  in its reduced
(ferrous, Fe2+) form, it binds CO to give a complex that
absorbs light maximally at 450 nm
Cytochrome P-450

Enzymes isolated by disruption of the liver cells
– Endoplasmic reticulum - microsomes when
disrupted
– Enzymes are membrane bound
– Explains why lipophilic drugs are processed
– Catalytic process  heme binds O2
Cytochrome P450: Isozymes
★ Isozymes - multiple forms of an enzyme
★ Supergene family
- More than 8,000 P450 genes as of November/2007
- More than 368 gene families, 814 subfamilies
- Human: 18 families, 43 subfamilies, 57 sequenced
genes
★ Nomenclature
CYP1A2
family
subfamily
individual member
of that subfamily
Cytochrome P450

This enzyme is very important. approximately
50% of the drugs that humans ingest are
metabolized by isoforms of cytochrome P450.

These enzymes also act on various carcinogens
and pollutants.
Substrates
CH
Allyl
H2
C
Vinyl
Aromatic
Benzyl


Aliphatic
Alicyclic
2. Monoamine oxidase, MAO
RCH2NH2+O2+H2O2
RCHO+NH3+H2O
★ MAO catalyze the oxidative deamination of
monoamines.
★ Oxygen is used to remove an amine group from a
molecule, resulting in the corresponding aldehyde and
ammonia.
★ MAO are found bound to the outer membrane of
mitochondria in most cell types in the body. They
belong to protein family of flavin containing amine
oxidoreductases.
3. ADH and ALDH
ADH alcohol dehydrogenase
ALDH aldehyde dehydrogenase

Alcohol Dehydrogenase belongs to the
oxidoreductase family of enzymes.

high concentrations within the liver and kidney.
Function

The primary and most common role of ADH in
humans is to detoxify incoming ethanol by
converting it into aldehyde.

The resulting aldehyde, a more toxic molecule than
ethanol, is quickly converted into acetate by
aldehyde dehydrogenase (ALDH) and other
molecules easily utilized by the cell.
ALDH
ADH
NAD+
R CH2OH
NADH
NAD+
R CHO
NADH
R CO2H
ADH
NAD+
R CH2OH
ALDH
NADH
NAD+
R CHO
NADH
R CO2H
During this reaction, hydrogen is removed from the
alcohol and transferred to a molecule called nicotinamide
adenine dinucleotide (NAD), converting it to reduced NAD
(NADH).
NADH participates in numerous other metabolic
reactions, passing on the hydrogen to other compounds
or electron transfer chain.
Absorption
20%

Soluble in water

Small size - penetrates everywhere,
easily crosses all bio membranes
80%

Rapidly absorbed from GI
 In people who consume alcohol at moderate levels and/or
only occasionally, most of the alcohol is broken down by
ADH and ALDH.
after higher alcohol consumption, The MEOS plays
a role in alcohol metabolism.
CH3CH2OH + NADPH + O2 +
+ NADP+ + 2H2O
CH3CHO
ALDH
H+
MEOS
CH3CHO
CH3COOH
MEOS: Microsomal Ethanol-Oxidizing System , is also
called Cytochrome P450-dependent Microsomal Ethanol
Oxidizing System. converts alcohol to acetaldehyde
CH3CH2OH + NADPH + O2 +
+ NADP+ + 2H2O
H+
MEOS
CH3CHO
This reaction also relies on oxygen and NADPH, and
results in the formation of NADP and water.
◆consume oxygens of liver and NADPH
◆ As byproducts of these reactions, oxygen radicals or
reactive oxygen species (ROS) are generated. These ROS can
contribute to liver damage through a variety of mechanisms.
Although the rate at which ADH breaks down alcohol
generally stays the same, the activity of the MEOS can be
increased (induced) by alcohol consumption.
Because the MEOS metabolizes not only alcohol but also
other compounds (certain medications), enhanced MEOS
activity resulting from high alcohol consumption also can alter
the metabolism of those medications.
This may contribute to harmful interactions between
alcohol and those medications or otherwise influence the
activity of those medications.
Alcoholism leads to fat accumulation in the
liver, hyperlipidemia, and ultimately cirrhosis.
 Phase Ⅰ: Reduction
4. 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'
2005 , sudan red incident
chili patse
sudan red
nitroreductase
chloromycetin
 Phase Ⅰ: Reduction
5. 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
CO2H
O
esterase
CH3
CO2H
OH
CH3
O
O
salicylic acid
ASA
HO
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.
xenobiotic
Phase I
Phase II
Protection
Elimination
excrection
Reactive
metabolite
Cell injury
Antibody product
Cell injury
nontoxic
metabolite
mutation
cancer
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
Oxygen glucuronides cont…
Alcohol hydroxyl example
OH
OH
Cl
H
N
O 2N
O 2N
Cl
HO
HO
Chloramphenicol - Chloromycetin® -
O
HO
HO H
Phenol hydroxyl example
CH3
CH3
O
O
NH
NH
HO2C
OH
Acetaminophen
APAP
N-acetyl-p-aminophenol
HO
HO
Cl
O
HO 2C
O
Cl
H
N
O
HO H
O
O
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
Sulfate Conjugation
O
O
＋PAPS
+PAP
HO 3SO
HO
estrone sulfate
estrone
CH3
HO
HO
H2 N
CO2H
alpha-Methyldopa
Aldomet® - Merck
Antihypertensive
O
-O S O
O
HO
CH3
H2N
CO2H
3. Conjugation with glutathione
glutamic acid, cysteine,
R + GSH
GST
glycine
R-S-G
where R= an electrophilic xenobiotics
R: epoxides and halogenides
GST: Glutathione S-Transferases (Liver and kidney)
Glutathione (GSH) Conjugation

DETOXIFICATION of electrophiles!
 Electrophilic chemicals cause:
– Tissue necrosis
– Carcinogenicity
– Mutagenicity
– Teratogenicity
 The thiol (SH group) ties up potent
electrophiles
Glutathione S-transferase
(+)benzo[a]pyrene7,8-dihydrodiol9-10-epoxide
HO
GST
HO
OH
DNA reactive;
+ glutathione
HO
Inactive
lung and skin
tumors
DETOXIFICATION
Glutathione (GSH) Conjugation
Epoxide or an
Arene Oxide
oxidation
Benzene
O
H+
aflatoxin b-1 epoxide
SG
HSG
OH
4. Acetylation
X + Acetyl-CoA - - - - >Acetyl-X +
CoS
where X represents a xenobiotics.
(for: aromatic amines)
• Enzyme: acetyltransferases
• present in the cytosol of various
tissues, particularly in liver.
isoniazid
sulfanilamide

Important for drugs with primary amino groups
 Generally, metabolites are nontoxic and
inactive
 Acetylation does NOT increase water solubility
 Detoxification or termination of drug activity
Methylation

A few xenobiotics are subject to
methylation by methyltransferase,
emplyoing S-adenosylmethione(SAM) as the
methyl donor.
SAM
catechol
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
Factors that influence
metabolism

Age
– older people less efficient at metabolism

Sex
– Linked to hormonal differences

Heredity
– Genetic differences can influence amounts and
efficiency of metabolic enzymes

Disease states
– Liver, cardiac, kidney disease
Summary

Xenobiotic, Biotransformation

Phase I reactions:
–
Purpose:



–
Functionalization:




Enhances elimination
Converts chemical to less toxic forms (detoxification)
Converts chemicals to more toxic active forms (activation)
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 glutathione,
Acetylation, Methylation
ALCOHOL
Mechanism of Fatty Liver

The likelihood of hypoglycemia is also
increased in alcoholics when they fast, as they
often have low hepatic stores of glycogen
because of poor nutrition.
 The shift in the NADH/NAD+ ratio also inhibits
β-oxidation of fatty acids and promotes
triglyceride synthesis; this increases hepatic
synthesis of VLDL, and the remaining excess
triglyceride is deposited in the liver.