Hepatotoxicity of flunitrazepam and alcohol in vitro

Flunitrazepam (FNZ) is a benzodiazepine derivative more potent than diazepa m. FNZ abuse in the US has emerged in the last few years and has a growing popularity among young people and drug abusing populations. Ethanol (EtOH) consumption with FNZ enhances euphoria and onset of action. It is postulate d that FNZ and EtOH cause liver cell injury. In this study, hepatocytes are employed to study the hepatotoxicity of FNZ, EtOH and their combination (F NZ-EtOH). Hepatocytes (2 x 10(6) cells/ml) isolated from male Sprague-Dawle y rats were exposed to saline, FNZ, EtOH or FNZ-EtOH in combination. The up take of 0.4% trypan blue and the leakage of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) enzymes into the incubation media were used to assess cell membrane damage of hepatocytes. Where metabolism of FN Z is nearly complete through several hepatic pathways, animal pretreatment with Phenobarbital was used to study the effect of microsomal enzyme induct ion on cellular injury. FNZ (0.16 mM), EtOH (32.56 mM) or their combination caused a significant (P < 0.05) decrease in fell viability. Compared with control. FNZ and FNZ-EtON in combination caused significant AST leakage ove r the 2-hour incubation period. EtOH alone caused significant AST leakage a fter 2 hours of incubation. The leakage of ALT enzyme was significant for F NZ, EtOH and FNZ-EtOH over the 2-hour incubation period. While FNZ alone di d not produce any significant enzymatic leakage in the Phenobarbital pretre ated groups, the leakage of ALT and AST were significant for FNZ-EtOH in co mbination as early as 30 minutes of incubation. A sig significant depletion (P < 0.05) of glutathione (GSH) was observed for EtOH and FNZ-EtOH in comb ination treated samples. This investigation suggests that FNZ and EtOH caus e hepatotoxicity, and their combinations have an additive effect in increas ing liver toxicity. Induction of microsomal enzymes revealed that FNZ is mo re hepatotoxic than the metabolites. And FNZ alone has no effect on GSH con tent. (C) 1999 Elsevier Science Ltd. All rights reserved.