Cellular lysis in freshly isolated hepatocytes incubated with varying concentrations of selenite could be related to the reductive metabolism of selenite. A decrease in intracellular GSH levels was observed concomitant with an increased rate of accumulation of oxidized glutathione in the incubation medium. Pretreatment of hepatocytes with an inhibitor of GSSG-reductase (1,3-bis(2-chloroethyl)-1-nitrosourea), prior to the addition of 50 microM selenite, resulted in substantially lower GSH-levels. The rate of GSSG reductase-catalyzed metabolism of selenite (50 microM) could be estimated to approximately 7 nmoles reduced/h per 10(6) cells. The results indicate that this was the major metabolic pathway for toxic concentrations of selenite in isolated hepatocytes. Furthermore, selenite considerably decreased cellular NADPH levels. In hepatocytes isolated from starved rats, the presence of alanine and glucose in the incubation medium protected against selenite-mediated cellular lysis. These observations suggest that an insufficient NADPH generation could be critical for selenite reduction and toxicity in isolated hepatocytes.