Furan-induced cytolethality in isolated rat hepatocytes: correspondence with in vivo dosimetry

Abstract

Furan, a rodent hepatotoxicant and hepatocarcinogen, produced incubation time- and concentration-dependent decreases in the glutathione (GSH) content and viability of freshly isolated F-344 rat hepatocytes in vitro. Since furan itself did not significantly react with GSH, these data indicate the formation of a reactive metabolite of furan in hepatocyte suspensions. Treatment of the hepatocyte suspensions with the cytochrome P450 inhibitor 1-phenylimidazole delayed GSH depletion but did not alter furan-induced (4 to 12 mM) cytolethality. The furan concentrations required to produce measurable hepatocyte cytolethality in vitro within 6 hr (4 to 12 mM) were several orders of magnitude greater than the predicted maximal liver concentrations of furan in vivo following hepatotoxic doses. In order to study the mechanisms involved in the cytolethality of furan toward hepatocytes in vitro at concentrations relevant to hepatotoxicity in vivo, a hepatocyte suspension/culture system was developed that utilized furan concentrations and incubation times similar to hepatic dosimetry in vivo. Freshly isolated rat hepatocytes in suspension (in Williams' Medium E) were incubated with furan (2 to 100 microM) for 1-4 hr and placed in culture, and viability was determined after 24 hr by lactate dehydrogenase release. Furan produced cytolethality (5 to 70%) and modest GSH depletion in an incubation time- and concentration-dependent manner. Both GSH depletion and cytolethality induced by furan were prevented by 1-phenylimidazole and enhanced by acetone pretreatment of the rats. These data show that oxidation of furan by cytochrome P450 is required for GSH depletion and cytolethality, indicating that a reactive metabolite is involved in cell death. The results of this study underscore the importance of using in vivo toxicant concentrations and exposure times for in vitro mechanistic studies of chemically induced cytolethality.