Investigation of the role of the thiazolidinedione ring of troglitazone in inducing hepatotoxicity.

Troglitazone (TGZ) is an orally active hypoglycemic agent which is used for the treatment of non-insulin-dependent diabetes mellitus. It had been associated with severe drug-induced liver failure which resulted in its withdrawal from the market in 2000. While the exact mechanism of its toxicity remains unknown, it has been postulated that the formation of toxic reactive metabolites (RMs) may play an important role in the hepatotoxicity of TGZ. The purpose of this study is to investigate the role of sulfur moiety of thiazolidinedione (TZD) nucleus in inducing liver toxicity via the formation of RMs. An analogue of TGZ, trosuccinimide (TSN), was synthesized chemically where the sulfur moiety of thiazolidinedione ring was replaced by a methylene group. Both compounds were incubated independently with human liver microsomes enriched with glutathione (GSH) and normal human hepatocytes (THLE-2 cell lines) to profile GSH-adducts using ultra performance liquid chromatography tandem mass spectrometry (UPLC/MS/MS). Four RM-GSH conjugates of TGZ were identified during the profiling experiments of which three were related to the sulfur moiety of the TZD ring, whereas no RM of TSN was detected in both microsomes and hepatocytes. MTT, GSH and protein carbonyl (PC) assays were performed using THLE-2 hepatocytes to measure the levels of toxicity of TGZ and TSN in vitro. Finally, peroxisome proliferator activated receptor gamma (PPAR(gamma)) binding activity was measured to check the binding affinities of both TGZ and TSN. The calculated binding affinities of TGZ and TSN were 332.2 and 1106.0 microM, respectively. Our results indicated collectively that TSN (EC(50)=138.5+/-7.32 microM) was less toxic than TGZ (EC(50)=27.2+/-4.8 microM) in THLE-2 hepatocytes. As both compounds were shown to bind to PPAR(gamma), the substitution of the TZD moiety may be beneficial from a drug design perspective. In conclusion, our study confirmed that the TZD ring of TGZ may be partially responsible for its liver toxicity in humans via the formation of RMs.