Metabolic activation of troglitazone: identification of a reactive metabolite and mechanisms involved

Troglitazone (TGZ), the first glitazone used for the treatment of type II diabetes mellitus and removed from the market for liver toxicity, was shown to bind covalently to microsomal protein and glutathione (GSH) following activation by cytochrome P450 (P450). The covalent binding of (14)C-TGZ in dexamethasone-induced rat liver microsomes was NADPH-dependent and required the active form of P450; it was completely inhibited by ketoconazole (10 microM) and GSH (4 mM). The covalent binding in P450 3A4 Supersomes (9.2 nmol of TGZ Eq/nmol P450) was greater than that with P450 1A2 (0.7), 2C8 (3.7), 2C19 (1.4), 2E1 (0.6), and 2D6 (1.1) and 3A5 (3.0). The covalent binding in liver microsomes from rats pretreated with dexamethasone (5.3 nmol of TGZ Eq bound/nmol P450) was greater than that from rats pretreated with vehicle (3.5), beta-naphthoflavone (0.4), phenobarbital (1.1), or pyridine (2.5). A TGZ-GSH adduct was detected by liquid chromatography-tandem mass spectrometry and radioactivity detection with a deprotonated quasi-molecular ion [M-H](-) at m/z 745, with fragment ions at m/z 438 (deprotonated TGZ moiety), and at m/z 306 (deprotonated GSH moiety). The TGZ-GSH adduct was determined to be 5-glutathionyl-5-[4-(6-hydroxy-2,5,7,8-tetramethylchroman-2-ylmethoxy)benzyl]-thiazolidine-2,4-dione based on collision-induced dissociation fragmentation, and one- and two-dimensional NMR analysis of the isolated adduct. The synthetic 5-hydroxy TGZ and the benzylidene derivative of TGZ did not react with GSH or GSH ethyl ester. The mechanisms for metabolic activation of TGZ may involve an ultimate reactive sulfonium ion which could be formed from an initial sulfoxide followed by a formal Pummerer rearrangement, or a C5 thiazolidinedione radical or a sulfur cation radical.