Metabolism of Xenobiotics

Xiao Li
 Introduction
※Xenobiotics: is a compound that is foreign
to the body ; is a chemical which is found in a
n organism but which is not normally produce
d or expected to be present in body.
※Endogenous: Pigments , hormone
※nonendogenous : Such as drugs , food addi
tives, pollutants, toxin, etc
 Most of these compounds are subject to
metabolism (biotransformation) in human body.
 Definition of the biotransformation

 Conversion of lipophilic xenobiotics to wat

er-soluble chemicals by a process catalyzed
by enzymes in the liver and other tissues.
 In most cases, biotransformation lessens th
e toxicity of xenobiotics, but many must un
dergo the process to exert their toxic effects.
Purpose of biotransformation

1. facilitates excretion: Converts lipophilic to h

ydrophilic compounds

2. Detoxification/inactivation: converts chemic

Chronic hepatitis and cirrhosis of liver

Spider lentigo

als to less toxic forms

Hepatic palm

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 metaboli
sm
– Lungs, kidney, placenta, brain, skin, adrenal gl
ands
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 Conjugations
Oxidation
Reduction
Increase in Small Large
hydrophilicity
General mechanism Exposes functional Polar compound added
group to functional group

Consquences May result in Facilitates excretion

metabolic activation
 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 “mixed-
function oxidases”.
RH + O2 + NADPH + H+  R-OH + H2O + NADP+
Cytochrome P450s-dependent monooxygenase
-----the most versatile biocatalyst
----Works on a large number of diverse compou
nds
★ 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 disr
upted
– 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 monoa

mines.
★ Oxygen is used to remove an amine group from a mo
lecule, resulting in the corresponding aldehyde and am
monia.
★ MAO are found bound to the outer membrane of mi
tochondria in most cell types in the body. They belong t
o protein family of flavin containing amine oxidoreduct
ases.
3. ADH and ALDH
ADH alcohol dehydrogenase

ALDH aldehyde dehydrogenase

 Alcohol Dehydrogenase belongs to the oxidored

uctase family of enzymes.
 high concentrations within the liver and kidney.
 Function
 The primary and most common role of ADH in hum
ans is to detoxify incoming ethanol by converting it i
nto aldehyde.

 The resulting aldehyde, a more toxic molecule than e

thanol, is quickly converted into acetate by aldehyde
dehydrogenase (ALDH) and other molecules easily u
tilized by the cell.

ADH ALDH
NAD+ NADH NAD+ NADH

R CH2OH R CHO R CO2H

 ADH ALDH
NAD+ NADH NAD+ NADH

R CH2OH R CHO R CO2H

During this reaction, hydrogen is removed from the alc

ohol and transferred to a molecule called nicotinamide adeni
ne dinucleotide (NAD), converting it to reduced NAD (NA
DH).

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

 Rapidly absorbed from GI

80%
  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.
MEOS
CH3CH2OH + NADPH + O2 + H+ CH3CHO
+ NADP+ + 2H2O
ALDH
CH3CHO CH3COOH

MEOS: Microsomal Ethanol-Oxidizing System , is also

called Cytochrome P450-dependent Microsomal Ethanol
Oxidizing System. converts alcohol to acetaldehyde
 MEOS
CH3CH2OH + NADPH + O2 + H +
CH3CHO
+ NADP+ + 2H2O
This reaction also relies on oxygen and NADPH, and resu
lts in the formation of NADP and water.

◆consume oxygens of liver and NADPH

◆ As byproducts of these reactions, oxygen radicals or reac
tive oxygen species (ROS) are generated. These ROS can cont
ribute 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 activ
ity resulting from high alcohol consumption also can alter the
metabolism of those medications.
This may contribute to harmful interactions between alcoho
l and those medications or otherwise influence the activity of t
hose 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 rec
irculation of nitro or azo containing drugs.
+ O
Ar N Ar N O Ar NHOH Ar NH2
O

Ar N N Ar' Ar N N Ar' H2N Ar + H2N Ar'

H H
2005 , sudan red incident

chili patse

sudan red
 nitroreductase

chloromycetin
 Phase Ⅰ: Reduction
5. Hydrolysis
Substrates: esters , amide , glycoside,
glycoside etc.
 Catalyzed by widely distributed hydrolytic enzymes
 Hydrolysis of esters  major metabolic pathway for este
r drugs

☻Non-specific esterases (liver, kidney, and intestin

e)
☻Plasma
CO2Hpseudocholinesterases
esterase CO H
also participate
2
O CH3 HO CH3
Acetylsalicylic OH
Acid, ASA O O
salicylic acid
ASA
 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
excrection Elimination

Reactive
metabolite nontoxic
metabolite
Cell injury Antibody product mutation

cancer
Cell injury
Five types of phase II reactions

A.      Glucuronidation
B.      Sulfation
C. Conjugation with glutathione
D. Acetylation
E. 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 Cl
OH Cl H
H N
N O 2N Cl
O2N Cl O
O HO2C O O
HO
HO
Chloramphenicol - Chloromycetin® - HO HO H

Phenol hydroxyl example

CH3
CH3 O NH
O NH

HO2C O O
OH HO
HO HO H
Acetaminophen
APAP
N-acetyl-p-aminophenol
 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 O
O O -O S X Drug
-O S O P O O
PAPS Adenine
O OH O

H2O3PO OH
 Sulfate Conjugation
O O

＋ PAPS
+PAP

HO HO3SO

estrone estrone sulfate

O
CH3 CH3
HO -O S O
CO2H O CO2H
H2N H2N
HO HO
alpha-Methyldopa
Aldomet® - Merck
Antihypertensive
3. Conjugation with glutathione

glutamic acid, cysteine, glycine

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

 DETOXIFICATION of electrophiles!
 Electrophilic chemicals cause:
– Tissue necrosis
– Carcinogenicity
– Mutagenicity
– Teratogenicity
 The thiol (SH group) ties up potent electrop
hiles
 Glutathione S-transferase

glu
(+)benzo[a]pyrene-
7,8-dihydrodiol-

tath
9-10-epoxide

ion
e
HO

HO GST
HO
OH + glutathione

DNA reactive; Inactive

lung and skin
tumors
DETOXIFICATION
Glutathione (GSH) Conjugation
Epoxide or an
Arene Oxide
oxidation

HSG SG
O
Benzene OH
H+

aflatoxin b-1 epoxide

 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 inacti
ve
 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  ne
urotransmitters
– 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:
 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 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.