Biotransformation Xenobiotic metabolism “Essentials of Toxicology” by Klaassen Curtis D. and Watkins John B Chapter 6

Biotransformation Water soluble xenobiotics are easier to eliminate ( t 1/2 ) –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 Inactive metabolite Relatively harmless Reactive intermediate Detoxification Metabolic activation

Converting lipophilic to water soluble compounds Xenobiotic Reactive intermediate Conjugate Phase I - Activation Phase II - Conjugation Excretion Lipophilic (non-polar) Water soluble (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  Oxidation Hydroxylation (addition of -OH group) N- and O- Dealkylation (removal of -CH side chains) Deamination (removal of -NH side chains) Epoxidation (formation of epoxides) Oxygen addition (sulfoxidation, N-oxidation) Hydrogen removal  Reduction Hydrogen addition (unsaturated bonds to saturated) Donor molecules include GSH, FAD, NAD(P)H Oxygen removal  Hydrolysis Splitting of C-N-C (amide) and C-O-C (ester) bonds OCCOCCC O C See also Chapter 6 of Casarett and Doull’s “Toxicology” Table 6.1 epoxide

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 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 –AlcoholCYP2E1 –Dioxin/PCBs CYP1A –BarbituratesCYP2B CYP genes have multiple alleles (2D6 has 53, and 2E1 has 13)

The CYP-450 reaction cycle A E D C F G (B)

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

Flora action Intestinal flora as part of biotransformation reabsorption Ready for elimination

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: