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
OOH
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
- AMPCH3COO-
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
-OOCCH2
CH2
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
SCysNCOCH3H
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 CH3SAM
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
CH3 CH3 CH3CH3
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
