Phase II: Conjugation

• Synthetic reaction of a xenobiotic (or of a Phase I metabolite of a xenobiotic) with an endogenous substance

• Results in introduction of polar, ionizable groups to enhance water solubility and hence excretion

Major Phase II reactions

• Glucuronidation

• Sulfation

• Conjugation with amino acids

• Conjugation with glutathione

• Methylation

• Acetylation

Glucuronidation• Enzyme: glucuronyl transferase, or

glucuronosyl transferase• Targets:

– hydroxyl groups: Phenols, Alcohols, Dihydrodiols (ether glucuronides)

– Carboxylic acids (ester glucuronides)– Amines (N-glucuronides)– Thiols (S-glucuronides)– Carbon (C-glucuronides, rare)

Reaction

OHo o

OH

HO

OH

COOH

PhenolPhenyl glucuronide

Glucuronidation

• Conjugating moiety: glucuronic acid, a sugar

• Co-factor: UDP-glucuronic acid (UDPGA), derived from glycogen synthesis

• Located in endoplasmic reticulum• Multiple families of isoforms:UGT1, UGT2

– UGT1.1 ..1.7, UGT2.1..2.4

• Inducible

Uridine-5’-diphospho--D-glucuronic acid (UDPGA)

O

O

OH

OH

COO-

P

OH

OHO

OP

OH

O

OH

CH2O

OH

HN

N

O

O

O

GlucuronidationTypical substrates:

• Phenol• 1-Naphthol• 4-Hydroxybiphenyl• 3-Hydroxybenzo[a]pyrene• Benzo[a]pyrene-7,8-dihydrodiol• 2-Naphthylamine• Bilirubin• Steroids

Sulfation• Sulfotransferase ST,15 isoforms (xx-ST)

• Targets– Hydroxyl groups (phenols, alcohols)– Amino groups– Thiols

• Conjugating moiety: sulfuric acid, H2SO4

• Co-factor: 3’phosphoadenosine 5’phosphosulfate (PAPS), formed from ATP + sulfate

• Located in cytosol, Probably not inducible

SulfationTypical substrates

• Ethanol

• Phenol

• 3-Hydroxybenzo[a]pyrene

• Cholesterol

• 2-Naphthylamine

• N-hydroxy-2-naphthylamine

Reaction

OH OSO3-

PAPS

PAP

Conjugation with amino acids

• Amino acid transferases

• Targets: carboxylic acids

• Conjugating moieties: Glycine, glutamine, alanine, taurine, histidine, ornithine

• Co-factor: Acetyl CoA (CoASH) and ATP

• In cytosol

Reaction

C OH

O

CS CoA

OCoASCOCH3+ ATP

- AMP

CH3COO-

CNH2

CH2 COO-

O+ NH2CH2COO

-

-CoASH

Benzoic acid Benzoyl-CoA Hippuric acid

Conjugation with glutathione

• Glutathione S-transferases (GST)• Targets: Epoxides, halogens• Conjugating moiety: Glutathione• Co-factor: None• Mainly in cytosol• Inducible• Multiple families of isoforms: GSTA, GSTM,

GSTP, GSTT ()(αμπθ)

Glutathione

NH2CH

-OOC

CH2CH2

CNH CH

CH2

CNH

CH2COO

-

SH

OO

Glutamic acid (Glu)

Glycine (Gly)

Cysteine (Cys)

A tripeptide

Reaction

OSG

HOH

+ GSH

H

Typical substrates

• Organic halides, e.g methyl iodide, benzyl chloride

• Alkenes e.g. diethyl maleate

• Epoxides

Mercapturic acid pathway

C ClH

H H

C SH

+ GSH Glu

Cys

Gly

Gly

Cys

Glu

C S

H

H C S

H

H C

H

H-Cys

Glu

SCys

NCOCH3H

Methylation

• Methyltransferases

• Target: Hydroxyl groups, amines, thiols

• Substrates mainly endogenous: Catechols, noradrenalin, histamine

• Conjugating moiety: Methyl group

• Co-factor: S-adenosylmethionine

S-adenosylmethionine

N

N

NH2

O

OH

CH2S+

CH3

(CH2)2CH

-OOC

H2N

HO

MethylationReaction

OH

OH

OH

O CH3

SAM

Substrate:

Catechol

Enzyme:

Catechol-O-methyltransferase (COMT)

Acetylation

• N-acetyltransferases (NAT)

• Target: Aromatic amines, sulfonamides

• Conjugating moiety: Acetyl group

• Co-factor: Acetyl-CoA

• Few forms: NAT1, NAT2. NAT3: mice

• Genetic polymorphisms: “slow and fast acetylators”

AcetylationReaction

NH2 N CH3

OH

Acetyl CoA

CoASH

C

2-Naphthylamine

2-Aminonaphthalene

2-Acetylaminonaphthalene

2-Acetamidonaphthalene

“Other” detoxication mechanisms

• P-glycoprotein: ATP-dependent carrier that removes molecules from cells

• Multidrug resistance associated protein MDR

• Multispecific organic anion transporter MOAT

Reactive Oxygen Species (ROS)

• Peroxides– Hydrogen peroxide HOOH– Peroxynitrite OONO-

– Lipid hydroperoxide LOOH

• Free radicals– Superoxide anion O2

•- – Hydroxyl radical HO•

– Nitric oxide NO•

Non-enzymic reaction with anti-oxidants

• Ascorbic acid (Vitamin C)

• alpha-Tocopherol (Vitamin E)

• Glutathione

O

OHOH

O

C HHO

C H

OH

H

O CH3

CH3

H3C

HO

CH3

CH3CH3 CH3

CH3

Superoxide dismutase

Converts superoxide anions to hydrogen peroxide

O2•- + O2

•- + 2H+ O2 + H2O2

Peroxidases

Couple reduction of hydrogen peroxide (or other peroxide) to oxidation of another substrate

(co-oxidation)

ROOH + R’H ROH + R’OH

Peroxidases

• Catalase

• Prostaglandin synthetase

• Myeloperoxidase

• Lactoperoxidase

• Glutathione peroxidase

Glutathione peroxidase

GSH + GSH GSSG

HOOH HOH + HOH

Metabolic Activation/Metabolic Detoxication

• “Metabolism is a double-edged sword”

• Generation of (re)active intermediates

• Detoxication of (re)active intermediates

Pharmacologically active

Chemically reactive

Major reactive species

Electrophiles

Epoxides

(Epoxide hydrolaseGlutathione S-transferase)

Carbonium ions

Arylnitrenium ions

Reactive Oxygen Species

Genetic Toxicology

Reading material:

Casarett and Doull Chapter 9,

Timbrell, Chapter 6, pp. 259-279