| Troglitazone, an oral anti-diabetic drug belonging to the 2,4-thiazolidnedione (TZD) structural class, has proven to lower plasma glucose levels in patients with insulin-dependant glucose metabolism in muscle and adipose tissue and inhibits hepatic glucose production. The mode of the action is believed to be through binding to and activation of the peroxisome proliferator-activated y receptor (PPARr) , which results in induction of DNA transcription and stimulation of glucose kinase promoter. However, use of troglitazone is associated with hepatotoxicity in diabetic patients. During clinical trails of the drug, 1.9% of the troglitazone treated patients had liver enzymes level increase compared to 0.6% of the patients on placebo. Evaluation of liver biopsies from patients that developed liver dysfunction was consistent with an idiosyncratic drug reaction. Since it introduction to the US market in 1997 until its recent withdrawal, troglitazone has been linked to 26 deaths and several patients required liver transplants following troglitazone induced hepatic injure. While the mechanism of its serious hepatotoxicity remains unknown. The goal of the present study is to investigate the effects of troglitazone on the viability and functionality in established cell lines and rat hepatocytes in primary cultures to discriminate the possible mechanisms of the cytotoxicity. Part one Cytotoxic effects and oxidative damage of troglitazone in primary cultured rat hepatocytes Aim: Primary cultured rat hepatocytes and established cell lines (hepatoma HepG2 cell line, non-hepatic Hela cell line and Balb/c 3T3 cell line) were selected for the investigation and comparison of troglitazone cytotoxicity. The effects of D,L-buthionine-S,R-sulfoximine (BSO) and N-acetylcysteine (NAC) treatment on theheptotoxicity of troglitazone were also under observation. Whether troglitazone induces oxidative damage in primary cultured rat hepatocytes was evaluated either. Methods: 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-l,3-benzene disulfonate (WST-1) assay and lactate dehydrogenase (LDH) release assay were used as endpoints to evaluate cytotoxicity after troglitazone exposure. Formation of reactive oxygen species (ROS), Rh123 up-take, adenosine triphosphate (ATP) bioluminescence, reduced glutathione (GSH) levels, Annexin-V-FLUOS/ Propidium iodide (PI) staining and Hoechst 33342 staining were conducted to investigate the effects of troglitazone on the induction of ROS, the cellular GSH depletion and the functionality of mitochondria, i.e., mitochomdrial membrane potential (MMP), ATP synthesis. Results: Troglitazone produced concentration- and time-dependant decrease in cell viability and displayed the lowest IC50 value in primary cultured rat hepatocytes among the four selected cell models. The cytotoxic effect of troglitazone on HepG2 cells was relatively weaker in comparison with primary cultures. Whereas troglitazone displayed 4 times and 8 times higher IC50 values in Hela cells and 3T3 cells, respectively. BSO treatment significantly potentitated troglitazone cytotoxic effects on primary rat hepatocytes while NAC showed protective effect on the hepatotoxicity. Troglitazone induced ROS formation, decreased both of the ATP and MMP levels at the concentration of 50 uM; cellular GSH levels also decreased in dose- and time-dependent manners after troglitazone treatment. Annexin V-positive hepatocytes, Pi-positive hepatocytes and Hoechst 33342-positive nucleus increased dose-dependently after 5 hr exposure to troglitazone Conclusion: Troglitazone is more toxic to rat primary cultured hepatocytes. The hepatotoxicity of troglitazone may have direct relationship with cytochrome P450 (CYP450, CYP) oxidative enzymes and cellular GSH/GSSG redox state. Troglitazone induces ROS and depletes...
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