Biotransformation

Xenobiotic metabolism
“Essentials of Toxicology”
by Klaassen Curtis D. and Watkins John B
Chapter 6
 Biotransformation
• Water soluble xenobiotics are easier to eliminate
( t1/2)
– Urine, feces but not exhalation
– If within barrier, no out
• Multiple enzymes (families)
– Constitutively expressed
– Inducible
– Broad specificity
– Polymorphic (allelic variants)
– Stereo-isomer specificity: 6-OH in hormones:
CYP2A1 6-OH
CYP3A 6-OH
 Biotransformation

Potentially toxic xenobiotic Relatively harmless

Metabolic
Detoxification activation

Inactive metabolite Reactive intermediate

Converting lipophilic to water
soluble compounds
Lipophilic
Xenobiotic (non-polar)
Phase I - Activation

Reactive intermediate

Phase II - Conjugation

Conjugate
Water soluble
Excretion (polar)
 Phase I

• introduction of functional group

• hydrophilicity increases slightly

• may inactivate or activate original compound
• major player is CYP or mixed function oxygenase
(MFO) system in conjunction with NAD(P)H
• location of reactions is smooth endoplasmic reticulum
 Phase II

• conjugation with endogenous molecules

(GSH, glycine, cystein, glucuronic acid)

• hydrophilicity increases substantially

• neutralization of active metabolic intermediates
• facilitation of elimination
• location of reactions is cytoplasm
 Phase I reactions Table 6.1

 Oxidation
 Hydroxylation (addition of -OH group)
 N- and O- Dealkylation (removal of -CH side chains)
 Deamination (removal of -NH side chains) O epoxide
 Epoxidation (formation of epoxides) C C
 Oxygen addition (sulfoxidation, N-oxidation)
 Hydrogen removal

 Reduction
 Hydrogen addition (unsaturated bonds to saturated)
 Donor molecules include GSH, FAD, NAD(P)H
 Oxygen removal

C O
 Hydrolysis
 Splitting of C-N-C (amide) and C-O-C (ester) bonds

See also Chapter 6 of Casarett and Doull’s “Toxicology”

 Biotransformation
• Activation of xenobiotics is a key element
(e.g. benzene, vinyl chloride)
– Reactive intermediates include epoxides and free
radical species (unpaired electrons) that are short-lived
and hence highly reactive
– Protection is provided by
• endogenous antioxidant substances, e.g. GSH
• vitamins C and E
• antioxidant enzymes, SOD, GPX, CAT in coupled reactions
– Antioxidant molecules are oxidized in the process but
have the capacity to regenerate the reduced form from
the oxidized - NAD(P)H is a key player

See also p. 40-44 of Casarett and Doull’s “Toxicology”

 Cytochrome P450 (CYP)
Mixed Function Oxidases (MFO)
• Located in many tissues but highly in liver ER
• Human: 16 gene families
• CYP 1,2,3 perform drug metabolism
• >48 genes sequenced
• Key forms: CYP1A2, CYP2C9, CYP2C19, CYP2D6,
CYP2E1, and CYP3A4
• Highly inducible
– Alcohol CYP2E1
– Dioxin/PCBs CYP1A
– Barbiturates CYP2B
• CYP genes have multiple alleles (2D6 has 53, and 2E1 has
13)
The CYP-450 reaction cycle
A

G
(B)

D
Oxidation of vinyl chloride to an
epoxide
 Metabolic enzymes
1. Microsomal:
1. CYP450 monooxygenases
2. Flavin monooxygenase
2. Non-microsomal
1. Alcohol dehydrogenase
2. Aldehyde dehydrogenase
3. Monoamine and diamine oxidases
3. Both
1. Esterases and Amidases
2. Prostaglandin synthase
3. Peroxidases
Cooxidation of
acetaminophen
by prostaglandin
endoperoxide
synthetase

Compare to fig. 6-2

Hydrolysis of esters and amides
Hydrolysis of organophosphates
Hydrolysis of epoxides
Stereoselective
hydroxylation
Metabolism of
benzo(a)pyrene to
9,10 epoxide:
Potent mutagen
that binds DNA
Azo- and nitro- reduction
 Intestinal flora as part of biotransformation

Ready for elimination

n Flora action
tio
r p
o
a bs
re
Oxidation reactions
 Benzene trasformation to
leukemia-causing metabolite
Flavin mono-oxygenases
(FMO) catalyzed reactions

Nitrogen compounds
 Phase II reactions
• Glycoside conjugation - glucuronidation
• Sulfate - sulfation
• Glutathione (GSH)
• Methylation
• Acylation
– Acetylation
– Amino acid conjugation
– Deacetylation
• Phosphate conjugation
Glucuronidation of phenol
Sulfation of phenol and toluene
GSH conjugation of
acetaminophen
 Glutathione

-glutamyl-cysteinyl-glycine

Active site of a GST: