These results are in agreement with those previously described showing that DHM is a respiratory chain complex I inhibitor in isolated mitochondria (Mingatto et al., 2007). The incubation of MCT at concentrations such as 10 mM with hepatocytes isolated from normal rats did not produce toxic effects (results not shown). Thus, in order to stimulate the production of MCT metabolites by isolated hepatocytes, rats were previously treated with dexamethasone, an inducer of cytochrome P-450 3A (Gonzales, 1990). Metabolism of MCT

has been attributed to this cytochrome (Reid et al., 1998). The addition of increasing concentrations of MCT to hepatocytes OSI-906 cell line of rats pre-treated with dexamethasone resulted in decreased cell viability, as assessed by ALT leakage into the incubation medium (Fig. 2A). ALT leakage was concentration- and time-dependent, with a significant increase being observed at MCT concentrations of 5 and 7.5 mM at 60 min incubation. In Epigenetics inhibitor a previous report, we showed that the exposure of isolated perfused liver to MCT results in bioenergetic metabolism failure, which may reflect cell death due to decreased cellular ATP (Mingatto et al., 2008). In the current study, the incubation of isolated hepatocytes with MCT promoted a gradual decrease in ATP levels, which appeared to correlate closely with cell death (Fig. 2B). After 90 min incubation with 5 mM or more of drug, when almost all cells lost

viability, ATP was almost completely depleted. In order to investigate the mechanisms involved in the cytotoxicity of MCT we evaluated the protective effects of fructose (20 mM) and DTT (10 mM) using 5 mM MCT. Fructose is an efficient substrate for 3-oxoacyl-(acyl-carrier-protein) reductase glycolytic ATP formation in hepatocytes and protects against the loss of cell viability due to mitochondrial impairment. Such protection implies that cytotoxicity involves the inhibition of nonglycolytic mitochondrial ATP formation (Mingatto et al., 2002).

Pre-treatment of hepatocytes with fructose prevented the decrease of cell viability caused by MCT (Fig. 3A). After 15 min pre-incubation with fructose, the intracellular levels of ATP were decreased to 30% of the control levels. This effect would be a consequence of the action of fructokinase producing fructose-1-phosphate with ATP consumption (Nakagawa et al., 1996). The addition of MCT did not further decrease the ATP levels, which remained constant for the 90-min incubation period (Fig. 3B). Because protein thiols and cellular thiol groups have long been described as important targets for reactive intermediates derived from some chemicals including MCT (Moore et al., 1985, Reed, 1990, Yan and Huxtable, 1996 and Lamé et al., 2005), we also investigated the effects of DTT, a thiol reductant, on the MCT-induced cytotoxicity. Both the cytotoxicity and loss of intracellular ATP caused by 5 mM MCT were prevented by the addition of DTT (Fig.