Metabolism of XenobioticsMetabolism of Xenobiotics

Xiao Li

※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.

IntroductionIntroduction

※Endogenous: Pigments , hormone

※nonendogenous : Such as drugs , food ad

ditives, pollutants, toxin, etc Most of these compounds are subject to

metabolism (biotransformation) in human body.

Definition of theDefinition of the biotransformationbiotransformation

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.

1. facilitates excretion: Converts lipophilic to h

ydrophilic compounds

2. Detoxification/inactivation: converts chemic

als to less toxic forms

3. Metabolic activation: converts chemicals to

more toxic active forms

Purpose of biotransformation

Spider lentigoHepatic palm

Chronic hepatitis and cirrhosis of liver

Increase of estrogen, aldosterone and antidiuretin

benzpyrene

Detoxification ≠ biotransformation

3,4-Benzypyrene

Sites of Sites of bbiotransformationiotransformation

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 glands

Knowledge of the metabolism of xenobiotics:

rational understanding of pharmacology and therapeutics , pharmacy, toxicology, cancer research , and drug addiction .

It is convenient to consider the metabolism of xenobiotics in two phases– phase Ⅰand phase Ⅱ

General Metabolic PathwaysGeneral Metabolic Pathways

Approximately 30 different enzymes catalyze reactions involved in xenobiotic metabolism; however, this note will only cover a selected group of them.

Phase I reactionsPhase 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 reactionsPhase II reactions

★ 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

♣♣ Conjugation

Comparing Phase I & Phase IIComparing Phase I & Phase II

Enzyme Phase I Phase II

Types of reactions HydrolysisOxidationReduction

Conjugations

Increase inhydrophilicity

Small Large

General mechanism Exposes functionalgroup

Polar compound addedto functional group

Consquences May result inmetabolic activation

Facilitates excretion

Phase : OxidationⅠ1. Hydroxylation

RH: drugs, cacinogens, pesticides, petroleum products,

pollutants, steroids, eicosanoids, fatty acids, retinoids, etc. Enzyme:

Cytochrome P450s-dependent monooxygenase

Addition of an oxygen atom or bond

Require NADH or NADPH and O2 as cofactors

RH + O2 + NADPH + H+ R-OH + H2O + NADP+

HydroxylationHydroxylation ：： OO22

   * 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+

★Microsomal drug oxidations require

cytochrome P450,

cytochrome P450 reductase,

NADPH , & O2

The actual reaction mechanism is as follows:

Cytochrome P450s-dependent monooxygenase

-----the most versatile biocatalyst

----Works on a large number of diverse compounds

CYP or Cytochrome P-450CYP or Cytochrome P-450

Cytochrome P450s-dependent monooxygenase

★ Heme proteins

★ Iron containing porphyrin - binds O2

★ 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

What is a Heme Protein?

Cytochrome P-450, Hemoglobin, & Myoglobin ALL Heme Proteins!

Cytochrome P-450Cytochrome 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

CCytochrome P450: ytochrome P450: IsozymesIsozymes

★ 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

CCytochrome P450ytochrome 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.

SubstratesSubstrates

H2C

CH

Aromatic Benzyl

Vinyl

Allyl

Aliphatic Alicyclic

2. Monoamine oxidase, MAO

RCH2NH2+O2+H2O2RCHO+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 3. ADH and ALDH

Alcohol Dehydrogenase belongs to the oxidored

uctase family of enzymes.

high concentrations within the liver and kidney.

ALDH aldehyde dehydrogenase

ADHADH alcohol dehydrogenasealcohol dehydrogenase

FunctionFunction The primary and most common role of ADH in hum

ans 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.

R CH2OH R CHO R CO2H

NAD+ NADH NAD+ NADH

ADHADH ALDHALDH

R CH2OH R CHO R CO2H

NAD+ NADH NAD+ NADH

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).

ADHADH ALDHALDH

NADH participates in numerous other metabolic reactions, passing on the hydrogen to other compounds or electron transfer chain.

AbsorptionAbsorption Soluble in waterSoluble in water

Small size - Small size - penetrates everywhere, penetrates everywhere, easily crosses all bio membraneseasily crosses all bio membranes

Rapidly absorbed from GIRapidly absorbed from GI

20%

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.

CH3CH2OH + NADPH + O2 + H+ CH3CHO

+ NADP+ + 2H2O

MEOS

CH3CHOALDHALDH

CH3COOH

MEOS: Microsomal Ethanol-Oxidizing System , is also called Cytochrome P450-dependent Microsomal Ethanol Oxidizing System. converts alcohol to acetaldehyde

◆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.

CH3CH2OH + NADPH + O2 + H+ CH3CHO

+ NADP+ + 2H2O

MEOS

This reaction also relies on oxygen and NADPH, and results in the formation of NADP and water.

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.

4. Nitro and Azo Reduction4. 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.

Ar NO

O

+Ar N O Ar NHOH Ar NH2

N Ar'NAr NH

Ar NH

Ar' NH2 Ar NH2 Ar'+

Phase : ReductionⅠ

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chili patse

chloromycetin

nitroreductase

5. Hydrolysis5. Hydrolysis

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 participateO CH3

CO2H

O

OH

CO2H CH3

O

OH

ASA

Acetylsalicylic Acetylsalicylic AcidAcid, ASA, ASA

esterase

salicylic acidsalicylic acid

Substrates: esters , amide , glycoside , amide , glycoside, etc.

Phase : ReductionⅠ

PPhasehase II: II: ConjugationConjugation

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 II

nontoxic metabolite

ProtectionElimination

Phase I

excrectionexcrectionReactive metabolite

Cell injury Antibody product mutation

cancerCell injury

FFive types of phase ive types of phase IIII reactions reactions

A.      Glucuronidation

B.      Sulfation

C. Conjugation with glutathione

D. Acetylation

E. Methylation

1. G1. Glucuronidationlucuronidation 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…Oxygen glucuronides cont…

Alcohol hydroxyl example

Phenol hydroxyl example

NH

CH3

OH

O

NH

CH3

O

O

O

HOHOH

HO2C

OH

AcetaminophenAPAPN-acetyl-p-aminophenol

NH

ClO2N

OH

OHO

Cl NH

ClO2N

OH

OO

Cl

O

HOHOH

HO2C

OHChloramphenicol - Chloromycetin® -

2. Sulfate Conjugation2. 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

OHH2O3PO

OAdenine

OP

O

OH

OS

O

O

-O

HX Drug

X DrugS

O

O

-O

PAPS

sulfotransferase

Sulfate ConjugationSulfate Conjugation

estrone

O

OH

＋ PAPS+PAP

estrone sulfate

O

HO3SO

OH

OH

CH3

NH2

CO2H

O

OH

CH3

NH2

CO2H

S

O

O

-O

alpha-MethyldopaAldomet® - MerckAntihypertensive

3. Conjugation with glutathione3. Conjugation with glutathione

glutamic acid, cysteine, glycine

R + GSH R-S-GGST

R: epoxides and halogenides

GST: Glutathione S-Transferases (Liver and kidney)

where R= an electrophilic xenobiotics

Glutathione (GSH) ConjugationGlutathione (GSH) Conjugation

DETOXIFICATION of electrophiles!Electrophilic chemicals cause:

– Tissue necrosis– Carcinogenicity– Mutagenicity– Teratogenicity

The thiol (SH group) ties up potent electrophiles

GST + glutathione

Inactive DNA DNA reactive;reactive;

lung and skin lung and skin

tumorstumors

Glutathione S-transferase Glutathione S-transferase

HO

HOHO

OH

(+)benzo[a]pyrene-7,8-dihydrodiol-9-10-epoxide

glu

tathio

ne

DETOXIFICATION

O

oxidation

Epoxide or anArene Oxide

OH

SGHSG

H+

Benzene

aflatoxin b-1 epoxide aflatoxin b-1 epoxide

Glutathione (GSH) ConjugationGlutathione (GSH) Conjugation

   X + Acetyl-CoA - - - - >Acetyl-X + CoS     where X represents a xenobiotics. (for: aromatic amines)

4. Acetylation4. Acetylation

• Enzyme: acetyltransferases

• present in the cytosol of various tissues, particularly in liver.

isoniazid

sulfanilamidesulfanilamide

Important for drugs with primary amino groupsGenerally, metabolites are nontoxic and inacti

veAcetylation does NOT increase water solubilityDetoxification or termination of drug activity

MethylationMethylationA few xenobiotics are subject to

methylation by methyltransferase,

emplyoing S-adenosylmethione(SAM) as the methyl donor.

SAM

catecholcatechol

Metabolism via MethylationMetabolism 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 drugsMethylation does NOT increase water solubilityMost methylated products are inactive

Factors that influence Factors that influence metabolismmetabolism

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 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

ALCOHOLALCOHOLMechanism of Fatty LiverMechanism 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.