Role of bile acids and compensatory hepatic transport proteins in troglitazone-mediated hepatotoxicity

Drug-induced hepatotoxicity is the most common reason pharmaceuticals are removed from clinical use. Understanding mechanisms of hepatotoxicity, and in vitro models to predict hepatotoxicity in humans, are essential for drug development and patient safety. The drug troglitazone (TRO) was removed from the market because of hepatotoxicity, but preclinical testing failed to predict toxicity in humans. One hypothesized mechanism for TRO's hepatotoxicity is impairment of bile acid (BA) transport, causing cholestasis and subsequent hepatocellular apoptosis or necrosis. The goal of this research was to demonstrate that inhibition of BA transport and compensatory transport proteins contribute to the hepatotoxicity of TRO by causing intracellular accumulation of BAs and altering the BA pool composition. In human sandwich-cultured hepatocytes (SCH) and suspended hepatocytes, TRO inhibited uptake and biliary efflux of [3H] taurocholic acid ([3H]TCA), consistent with published reports in rat SCH; however, intracellular accumulation of [3H]TCA was not observed in either species. Because BAs differ in affinity for transport proteins, it was hypothesized that TRO causes intracellular accumulation of more cytotoxic BAs, such as chenodeoxycholic acid (CDCA). Indeed, TRO caused significant intracellular accumulation of [14C]CDCA species in rat SCH. In suspended rat hepatocytes, TRO inhibited [3H]TCA uptake more potently than [14C]CDCA uptake. This differential effect on individual BA disposition was hypothesized to shift the intracellular BA pool toward more toxic species. However, 24-h exposure of rat and human SCH to TRO had no significant effect on the concentration or composition of BAs in medium, cell, or bile, although the SCH model reflected reasonably well the in vivo BA pool composition in rats and humans. TRO-sulfate (TS), the major TRO metabolite, inhibited TCA transport in plasma membrane vesicles overexpressing the efflux protein multidrug resistance-associated protein 4 (MRP4). Accumulation of TS may inhibit BA efflux and increase intrahepatic accumulation of cytotoxic BAs in susceptible patients. This may explain, in part, the idiosyncratic nature of TRO hepatotoxicity. This research indicates that preclinical drug testing should include in vitro screens for drug-induced transport inhibition of multiple BAs, not just TCA, to fully characterize the effects of a compound on BA homeostasis