title: An investigation of the formation of cytotoxic, protein-reactive and stable metabolites from carbamazepine in vitro.
abstract: The formation of chemically reactive metabolites from carbamazepine (CBZ) in the presence of mouse and human liver microsomes has been investigated using cytotoxicity and irreversible binding of radiolabelled compound as quantitative end-points. For comparison, the formation of the stable CBZ-10,11-epoxide (CBZ-10,11-E) has been measured. The formation of the cytotoxic, protein-reactive and stable metabolites of CBZ was increased by induction of the cytochrome P450 enzymes by phenobarbitone and reduced by co-incubation in vitro with ketoconazole (10-250 microM), suggesting that the formation of these metabolites is cytochrome P450 dependent. All human livers tested (N = 6) bioactivated CBZ to a protein-reactive metabolite, the mean covalent binding increasing from 0.08 +/- 0.01% (without NADPH) to 0.27 +/- 0.09% (with NADPH; P less than or equal to 0.05). The formation of the chemically reactive metabolites was reduced by a subphysiological concentration of reduced glutathione (GSH) (500 microM), while ascorbic acid (100 microM) had no effect. Neither compound affected the formation of CBZ-10,11-E. Microsomal epoxide hydrolase (mEH), but not cytosolic epoxide hydrolase, caused a concentration-dependent inhibition of cytotoxicity reaching a maximum of 60% at 100 U of mEH. Covalent binding was also reduced by 60% by 100 U mEH. The separated T- and B-lymphocytes showed no difference in sensitivity when incubated with CBZ and mouse microsomes. The study demonstrates that the balance between activation of CBZ by the cytochrome P450 enzymes to a chemically reactive arene oxide metabolite and its detoxification by mEH and GSH may contribute to individual susceptibility to CBZ idiosyncratic toxicity.
authors.affiliation: Department of Pharmacology and Therapeutics, University of Liverpool, U.K.
authors.forename: M
authors.initials: M
authors.lastname: Pirmohamed
authors.forename: N R
authors.initials: NR
authors.lastname: Kitteringham
authors.forename: T M
authors.initials: TM
authors.lastname: Guenthner
authors.forename: A M
authors.initials: AM
authors.lastname: Breckenridge
authors.forename: B K
authors.initials: BK
authors.lastname: Park
mesh_terms: D000818:Animals; D001205:Ascorbic Acid / Q000494:pharmacology; D001711:Biotransformation; D002220:Carbamazepine / Q000031:analogs & derivatives / Q000032:analysis / Q000378:metabolism* / Q000494:pharmacology; D016923:Cell Death / Q000187:drug effects; D003600:Cytosol / Q000201:enzymology; D004851:Epoxide Hydrolases / Q000096:biosynthesis; D005978:Glutathione / Q000494:pharmacology; D006801:Humans; D007654:Ketoconazole / Q000494:pharmacology; D008214:Lymphocytes / Q000187:drug effects; D051379:Mice; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D008956:Models, Chemical; D010634:Phenobarbital / Q000494:pharmacology; D011506:Proteins / Q000378:metabolism
chemical_list: D011506:Proteins; D002220:Carbamazepine; D004851:Epoxide Hydrolases; D005978:Glutathione; D001205:Ascorbic Acid; C012227:carbamazepine epoxide; D007654:Ketoconazole; D010634:Phenobarbital