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Biotransformation

of xenobiotics

Department of Biochemistry (J.D.) 2011

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Greek word ξένος [xenos] means strange

• xenobiotics do not occur in the body

• they enter body mainly with food or as medications

• Chemical industry – produces synthetic compounds which

do not occur in nature (plastics, pesticides, dyes, additives…)

• Pharmaceutical industry – produces substances of synthetic

and natural (plant) origin – do not occur in the body

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Entry of xenobiotics into body

• three principal entries: intestine, lungs, skin

• epithelium barrier between blood (ECF) and tissues (ICF) –

phospholipid bilayer

• penetration of xenobiotic depends on its physical and chemical

properties

• hydrophobicity facilitates the transport through cell membrane

4

Entry of xenobiotic into cells

• Simple diffusion – lipophilic substances, depends on concetration

gradient (liver – freely permeable, big pores in cell membrane, the

most affected in poisoning)

• Facilitated diffusion – transporters

• Active transport – primary, secondary

• Endocytosis

xenobiotics structurally similar with physiological

substrates can utilize all available transport systems

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Biotransformation of xenobiotics in cells

• mostly in liver

• I. Phase – predominantly hydroxylations,

product may be still biologically active

• II. Phase – conjugation, product usually inactive

• products of biotransformations are more polar - they can be

excreted from the body by urine and/or bile

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Excretion of xenobiotics from cell

• primary active transport – needs energy: ATP hydrolysis

• special ATP-ases called ABC (ATP binding cassettes)

• localized in cell membranes, export xenobiotics from cells into ECF

• MRP (multidrug resistence proteins) – in increased expresion, they

cause the resistance towards medicines

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• superfamily of transmembrane proteins, they have ATP-binding

domain(s), substrate binding domain, and transmembrane domain(s)

• after ATP binding, ABC can translocate a chemical species across

membrane

• ABC are located in cell membranes as well as in intracellular membranes

• lipids, cholesterol, peptides, drugs, toxins etc.

ABC = ATP binding cassettes

8

Excretion of xenobiotics from body

• chemically modified (more polar) xenobiotics are excreted

by urine, bile stool, or sweat

• volatile substance by lungs

• intestinal deconjugation and resorption sometimes occur -

enterohepatic circulation

• excretion into human milk

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I. phase of biotransformation: examples of reactions

Reaction Xenobiotic (example)

Hydroxylation (P-450)

Sulfooxidation

Dehydrogenation

Reduction

Hydrolysis

(hetero)aromatic compounds (Ar-H Ar-OH)

dialkylsulfide (R-S-R) sulfoxide (R-SO-R)

alcohol / aldehyde hydrate aldehyde / acid

nitrocompounds (R-NO2) amines (R-NH2)

ester acid + alcohol

Reactions occur mainly in ER, some in cytosol

Enzymes of I. phase are rather non-specific – advantage !!

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Cytochrome P450 (CYP)

• superfamily of heme enzymes (many isoforms)

• can catalyze different reaction types, mainly hydroxylation

• wide substrate specifity - advantage

• can be induced and inhibited

• occur in most tissues (except of muscles and erythrocytes)

• the highest amount in the liver (ER)

• exhibit genetic polymorphism ( atypical biotransformations)

Abbreviation: P = pigment, 450 = wave length (nm) of a absorption peak after

binding CO

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1 % other

4 % 1A2

11 % 2C

30 % 2D6

2 % 2E1

52 % 3A4

Contributions of CYP isoforms to drug metabolism

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Mechanism of CYP hydroxylation

• the formation of hydroxyl group

• monooxygenase: one O atom from O2 molecule is incorporated into

substrate between C and H (R-H R-OH )

• the second O atom + 2H from NADPH+H+ give water

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

2 e- + 2 H+

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NADP+

FAD

FADH2

Fe++

hem

Fe+++

hem

NADPH H++

2 H+

cyt. reduktasa cyt P-450

RH

R OH

O2

H2O

cytochrome P450 contains three cofactors and two enzymes:

• NADPH+H+, FAD, heme

• NADPH:CYP reductase (separates 2 H 2 e- + 2 H+)

• cytochrome P-450 (hydroxylase)

ER

Components of cytochrome P450

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Detailed scheme shows reductive activation of O2

cyt P-450

Fe+3

A H

cyt P-450

Fe+3

A H

cyt P-450

Fe+2

A HNADPH + H

NADP

O2e

e

cyt P-450

Fe+2

A H

O2

cyt P-450

Fe+2

A H

O2

2 H

H2O

A OH

hydroxylovanýsubstrát

substrátsubstrate A-H

hydroxylated product

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Hydroxylation by CYP450 occurs

in endogenous and exogenous substrates

• Endoplasmic reticulum:

squalene, cholesterol, bile acids, calciol,

FA desaturation, prostaglandins, xenobiotics

• Mitochondria:

steroidal hormones

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Compare various hydroxylations

Substrate Product Reagent Coreductant Other comp.

Phenylalanine

Xenobiotic

Proline

Dopamine

tyrosine

xen-OH

4-OH-Pro

noradrenaline

O2

O2

O2

O2

BH4 *

NADPH+H+

2-oxoglutarate

ascorbate

-

FAD, heme

Fe2+, ascorbate

Cu2+

* tetrahydrobiopterine

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Main isoforms of human cytochrome P450

CYP Substratea Inducera Inhibitora

CYP1A2

CYP2A6

CYP2C9

CYP2C19

CYP2D6

CYP2E1

CYP3A4

theophylline

methoxyflurane

ibuprofen

omeprazole

codeine

halothane

diazepam

tobacco smoke

phenobarbital

phenobarbital

phenobarbital

rifampicine

alcohol

phenobarbital

erythromycin

methoxsalem

sulfaphenazole

teniposide

quinidine

disulfiram

grapefruit

Various isoforms prefer different substrates, have different inducers and inhibitors

a Examples from many possible compounds.

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Inducers and inhibitors of CYP450

• some xenobiotics induce the synthesis of CYP – the metaboliccapacity of CYP is enhanced

• if administered inducer + drug, both metabolized by the same CYPisoform drug is metabolized faster drug is less effective

• some xenobiotics inhibit CYP

• the most common isoform CYP3A4 metabolizes more than 120different pharmaceutical drugs

• inhibitors of CYP3A4 are e.g. macrolide antibiotics, grapefruit(furanocoumarins), ketoconazole

• if administered inhibitor + drug increased drug level overdosing side effects

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Genetic polymorphism of CYP450

higher drug levelside effectsintoxication

poor metabolizer

no / insufficient effect

(ultra)rapidmetabolizer

extensive metabolizer

normal responseclinical effect of drug

usual drug dose

most of population

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I. Phase of biotransformation of benzene

H

hydroxylace(CYP 450)

O

H

Example

hydroxylation

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Biotransformation of polycyclic aromatichydrocarbons (PAH)

Oepoxid OH

HO

H2O

OH

HO

O

dihydrodiol

vazba na DNA, mutace

nádory (kůže, plíce)

benzo[a]pyrene

Example

reactive epoxide

interactions with DNA, mutations

tumours (skin, lungs)

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PAH in environment

• Industrial sources: combustion of fossil fuels (coal, petroleum oil,

etc.), production of coke, asphalt ...

• Non-industrial sources: forest fires, combustion of household

rubbish, cigarette smoke …

• Foods: fried, grilled, smoked, roasted foods, overheated fats and

oils, burnt (singed) bread, pastry …

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II. Phase of biotransformation

• conjugation – catalyzed by transferases

• synthesis = endergonic reaction, one of the reactants must be

activated

• xenobiotic after I. phase reacts with endogenous conjugation

reagent

• conjugate is more polar, less active, easily excreted by urine

and/or bile (stool)

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Conjugation reactions and reagents

Reaction Reagent Group in substrate

Glucuronidation

Sulfation

Methylation

Acetylation

Sulfide formation

Amide formation

UDP-glucuronate

PAPS

SAM

acetyl-CoA

glutathione

glycine, taurine

-OH, -COOH, -NH2

-OH, -NH2, -SH

-OH, -NH2

-OH, -NH2

Ar-halogen, Ar-epoxide

-COOH

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Biosynthesis of UDP-glucuronate

O

OHOH

OH

O

CH2OP

H

O

OOH

OH

O

CH2HO

PH

O

OOH

OH

O

CH2HO

UDPH

UTP

glukosa-6-P glukosa-1-P UDP-glukosa

H

O

OOH

OH

O

C

UDP

OO NAD+

H2ONAD

+

glukosiduronáty

UDP-glukuronát

glucose 6-P glucose 1-P UDP-glucose

UDP-glucuronate

(bis)glucosiduronates

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UDP-glucuronate

N

OHOH

O

NH

O

O

O

PO

O

O

PO

O

O

O

OH

O

COO

H

H

N-glycoside bondO-glycoside bond

of ester type

O

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Glucuronates are the most common conjugates

• O-glycosides

ether type (Ar-O-glucuronate, R-O-glucuronate)

ester type (Ar-COO-glucuronate)

• N-, S-glycosides

• Substrates: aromatic amines, amphetamines,

(acetyl)salicylic acid, drugs, flavonoids ...

• Endogenous substrates: bilirubin, steroids

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bilirubin bisglucosiduronate

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Biotransformation of amphetamine

Phase I reaction

amphetamine

Phase I reaction

4-hydroxyamphetamine

4-hydroxynorephedrine4-hydroxynorephedrine

4-O-glucosiduronate

4-hydroxyamphetamine4-O-glucosiduronate

Phase II reaction

Phase II reaction

Example

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PAPS phosphoadenosyl phosphosulfate

O

OHO

O

P

PO

O

OSO

O

O

O

O

O

N

N

N

N

NH2

Physiological sulfations:

Glycosaminoglycanesheparine, dermatane sulfate,keratane sulfate,chondroitine sulfate etc.

Sulfoglycosphingolipids(acidic glycolipids, sulfatides)

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Biotransformation of phenol

Example

konjugace

O glukuronát O sulfát

H

hydroxylace(CYP 450)

OH

conjugation

glucuronate SO3H

hydroxylation

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Glutathione – three functions

-glutamyl-cysteinyl-glycine

• Reductant = antioxidant (glutathione peroxidase)

• Conjugation agent (glutathione transferase)

endogenous substrates – leukotrienes

• AA Transport into cells (-glutamyltransferase, GMT)

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Glutathione (GSH)

HOOCN

N COOH

O

H

CH2

SH

O

H

NH2

R-X + GSH R-SG + XH (R-X epoxides, halogenalkanes)

nucleophilicgroup

electrophilicsite

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R-SG sulfide is converted to mercapturic acidsand excreted

N-acetyl-S-substituted cysteine

(mercapturic acid)

Example

GSH

acetyl-S-CoACoA-SH

Glu + Glyepoxide

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Methylations are involved in the inactivation of catecholamines

CH2HO

HO

CH2 NH2 CH2HO

HO

C

H

O

CH2HO

HO

C

OH

OCH2HO

O

C

OH

O

CH3

dopamine

MAO

- NH3

dihydroxyphenylacetic acid

COMT

homovanillic acid

MAO monoamine oxidase, COMT catechol-O-methyltransferase

SAM

Inactivation can proceed in the reverse order: first COMT, then MAO, product is the same.

dihydroxyphenylacetaldehyde

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Conjugation with amino acids(amide formation)

• glycine, taurine

• xenobiotics with -COOH groups

• amide bond formation

• endogenous example: conjugated bile acids

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Toluene biotransformation

CH3 CH2OH COOH

toluene benzylalcohol benzoic acid

C

OH

O glycin

C

NH

O

CH2 C

OH

O

benzoová kys. hippurová kyselina(N-benzoylglycin)

benzoic acid(activated by CoA)

glycine

hippuric acid(N-benzoylglycine)

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H3C C

H

H

O

H

+ NAD H3C C

H

O+

alkoholdehydrogenasa

aldehyddehydrogenasa

+ H2O H3C C

OH

H

O

H

H3C C

OH

ONAD- 2H

NADH+H

H3C C

H

O

acetaldehyd

aldehyd-hydrát octová kyselina

acetaldehyde dehydrogenase (AcD)

alcohol dehydrogenase (AD)

acetaldehyde

acetaldehyde hydrate acetic acid

NADH + H+

Biotransformation of ethanol in liver (cytosol)

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• Alcohol dehydrogenase (AD) – metalloenzyme (Zn), more

isoforms, in liver, lungs, kidneys, intestine, and other tissues

• some isoforms are less active in females

• Acetaldehyde dehydogenase (AcD) – more isoforms, liver,

cytosol and mitochondria

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Alternative pathways for alcohol metabolism

ER:

MEOS (microsomal ethanol oxidizing system, CYP2E1)

CH3-CH2-OH + O2 + NADPH+H+ CH3-CH=O + 2 H2O + NADP+

It is activated on higher alcohol levels (> 0.5 ‰) increased

production of acetaldehyde

Peroxisomes: oxidation of ethanol by hydrogen peroxide, catalase

CH3-CH2-OH + H2O2 CH3-CH=O + 2 H2O

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Metabolic consequences of EtOH biotransformation

Ethanol

AD

MEOS

acetaldehyde

(hangover)part. soluble

in membrane PL

toxic effectson CNS

adducts withproteinsnucleic acidsamines

acetate

acetyl-CoA

FA/TAG synth.liver steatosis

AD, AcD

excess of NADH in cytosol

reoxidation by pyruvate

excess of lactate acidosis

lack of pyruvate hypoglycaemia

various products

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Acetaldehyde reacts with biogenic amines

to tetrahydroisoquinoline derivatives (animal alkaloids)

NH2HO

HO

HC

O

CH3

HO

HO

N

CH3

H

salsolinol

6,7-dihydroxy-1-methyl-1,2,3,4-tetrahydroisoquinoline

dopamine- H2O

acetaldehyde

Neurotoxin ?

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Fatty acids ethyl esters (FAEE) appear in the blood in 12 – 18 h after drinking and can be

detected even 24 h after alcohol in blood is no more increased. However, traces of FAEEs are

deposited in hair for months and may serve as a measure of alcohol intake.

Ethyl glucosiduronate (EtG) increases in the blood synchronously with the decrease of

blood ethanol and can be detected (in the urine, too) after few days, even up to 5 days.

Phosphatidyl ethanol (PEth) is present in the blood of individuals, who have been drinking

moderate ethanol doses daily, in even 3 weeks after the last drink.

Carbohydrate-deficient transferrin (CDT). In the saccharidic component of each

transferrin molecules, there are 4 – 6 molecules of sialic acid. Drinking to excess disturbes the

process of transferrin glycosylation so that less sialylated forms of transferrin (with only two

or less sialyl residues per molecule, CDT) are detected in blood during approximately 4

weeks after substantial alcohol intake.

Tests for detection of ethanol intake

Liver enzymes: GTM, AST, ALT, GMD, CHS

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alcohol in blood (‰) =

Per milles of alcohol in blood

fm

m

body

alcohol

(kg)

(g)

0.67 (males)

0.55 (females)

‰ = per mille = 1/1000

Biological feature Males Females

Total body water 60 – 67 % 50 - 55 %

Total body fat 10 – 20 % 20 – 30 %

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Oxidation of ethylene glycol proceeds stepwise

with a number of intermediates

oxid.COOH

COOH

glyoxalovákyselina

CH2OH

CH2OH

CHO

CHO

COOH

CH2OH

oxid.CHO

CH2OH

COOH

CHO

ethylenglykol glykolaldehyd

glyoxal

glykolová kyselina

št'avelovákyselina

oxid.

oxid.

oxid.

oxid.

ethyleneglycol

glycolic acid

glycolaldehyde glyoxalicacid

oxalic acid

in kidneys calcium oxalate stones renal failure

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Tobacco

Substancesinvolved

nicotine, the products of incomplete combustion

Effects

euphoria, psychical relaxation, increase of pulse rate,vasoconstriction, stimulates adrenaline release (silent stress),increases salivary and gastric secretion, stimulates intestinalperistalsis (defecating effect of the first morning cigarette)

Symptomsof abuse

typical smell, yellow fingers and teeth

Riskslung diseases (COPD*, cancer), heart attack,erectile dysfunctions, premature wrinkles

* chronic obstructive pulmonary disease

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Nicotine is the principal alkaloid of tobacco

N

N

CH3

3-(1-methylpyrrolidine-2-yl)pyridine

more basic

pKB = 6,16

less basic

pKB = 10,96

1

2

3

1

2

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What happens during cigarette burning?

• temperature about 900 C

• dried tobacco undergoes incomplete combustion

• very complicated mixture of products

• nicotine partly passes to smoke, partly decomposes

Cigarette box

Nicotine: 0.9 mg/cig.

Tar: 11 mg/cig.

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Cigarette smoke contains

• free base of nicotine – binds to receptors in the brain

• CO – binds to hemoglobin to give carbonylhemoglobin (tissue ischemia)

• nitrogen oxides – may generate reactive radical species

• polycyclic aromatic hydrocarbons (PAH)

(pyrene, chrysene, benzo[a]pyrene …), main components of tar

they can attack and damage DNA, carcinogens

• other substances (N2, CO2, HCN, CH4, esters …)

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How to disclose a smoker?

1. saliva test

smoker’s saliva contains much higher level of thiocyanate

than saliva of non-smoker,

thiocyanate is generated from CN- → SCN-

reaction with Fe3+ ions give red complex

2. nicotine in urine

3. minor tobacco alkaloids in urine

(cotinine, nornicotine, anatabine, anabasine)

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Biotransformation of nicotine

N

NCH3

N

NH

N

NCH3

OH

N

NCH3

O

nicotine

nornicotine 5-hydroxynicotine

cotinine

nicotine-N-glucuronate

cotinine-N-glucuronate

Example

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Biotransformations of selected drugs

Drug Biotransformation Metabolite

Codeine

Bromhexin

Paracetamol

Aspirin

demethylation

hydroxylation + demethylation

conjugation, oxidation

hydrolysis, hydroxyl., conjug.

morphine (active, another way)

ambroxol (active, the same)

conjugates (mostly inactive)

conjugates (inactive)

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Antitussic codeine (3-O-methylmorphine) is transformed slowly into morphine

morphine(analgesic, an addictive drug)

O-demethylation

codeine(antitussic)

Bromohexin is the prodrug of an expectorant ambroxol

N-demethylationhydroxylation

bromohexin(prodrug)

ambroxol(expectorant)

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Acetaminophen (p-acetaminophenol, paracetamol)N-(4-hydroxyphenyl)acetamideprepared in 1893, common analgetic-antipyretic,overt the counter, without a prescription

~ 3 % excretedunchangedinto the urine

CONJUGATION

60 % as glucosiduronate30 % as sulfate ester

The amide bond is not hydrolyzed!

oxidation of only a small part toN-acetyl-p-benzoquinoneimide (NAPQI),unless the conjugating capacity is exhausted

cyt P450

mercapturic acid

if conjugation capacity

is limited,

unwanted side effects:– covalent bonding

to proteins,– oxidation of –SH groups

in enzymes,– depletion of GSH,– hepatotoxicity at

overdosing

GSH

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Acetylsalicylic acid (Aspirin)is an analgetic-antipyretic with antiinflammatory effect; over the counter,minute doses inhibit aggregation of blood platelets.

UDP-glucuronate

glycine

glycine

2,5-dihydroxyhippurate(gentisoylglycine,gentisuric acid)

gentisate

quinone(and products of its

polymerization)

esterase

salicylate

cyt P450

oxid.

acetylation of macromolecules(acetylation of COX inhibitsthe synthesis of prostaglandins)

o-hydroxyhippurate(salicyloylglycine,

salicyluric acid)

salicyl glucosiduronate salicyloyl glucosiduronate

and

UDP

O

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• it is proper to avoid application of too many different remedies together

• interactions between different drugs or their metabolites can cause

enhancement or inhibition of pharmacological effects

• the mixed type hydroxylases (cyt P450) are inducible, their activities

may increase many times in several days, so that the remedies are less efficient

• if the load of the detoxifying system is high, minor pathways of transformation can be

utilized and produce unwanted side-effects due to the formation of toxic metabolites

• intensive conjugation with glutathione can result in depletion of this important

reductant in the cells

Polypragmasy - application of multiple remedies simultaneously

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Selected biochemical markers of liver damage (in serum)

Analyte Reference values Change

ALT

GMD

GMT

Bilirubin

Ammonia

Urobilinogens (urine)

------------------------------

Pseudocholinesterase

Urea

Albumin

0,1 - 0,8 kat/l

0,1 - 0,7 kat/l

0,1 - 0,7 kat/l

5 - 20 mol/l

5 - 50 mol/l

up to 17 mol/l

---------------------

65 - 200 kat/l

3 - 8 mmol/l

35 - 53 g/l

-------------