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