Clozapine-induced hepatotoxicity in rat hepatocytes by gel entrapment and monolayer culture

Clozapine is limitedly used due to its adverse effect including agranulocytosis and hepatotoxicity. However, the mechanism of clozapine toxicity is still not clear. The previous in vitro studies on microsomes proposed a possible mediation of cytochrome P450 (CYP) in producing reactive metabolites. In this paper, clozapine toxicity was, respectively, examined in two cultures of rat hepatocytes. Gel entrapment culture of hepatocytes with higher expression on CYP activities showed higher sensitivity to clozapine treatment than hepatocyte monolayer, indicating the possible involvement of CYP in hepatotoxicity of clozapine. Moreover, in each culture, CYP inhibitors were used to confirm the possible mediation of CYP enzymes. Pretreatment of hepatocytes with CYP 3A inhibitor (ketoconazole), CYP 2E1 inhibitor (diethyldithiocarbamate, DDC) and non-specific inhibitor (cimetidine) significantly reduced the toxicity of clozapine. But the pretreatment with CYP 1A2 inhibitor (fluvoxamine) had no such protective effect indicative of non-function of CYP 1A2 in clozapine toxicity. In addition, glycyrrhizic acid (GA), a scavenger of reactive oxygen species (ROS), also inhibited the adverse response to clozapine, suggesting the positive involvement of oxidant pressure. Thus, it could be concluded that clozapine-induced toxicity was mediated by CYP, particularly CYP 3A and CYP 2E1, and oxidant pressure.