Modulating Role of Endogenous Reduced Glutathione in Tert-Butyl Hydroperoxide-Induced Cell Injury in Isolated Rat Hepatocytes

Abstract

The role of endogenous reduced glutathione (GSH) in tert-butyl hydroperoxide (TBHP)-induced cell injury was examined in isolated rat hepatocytes. When liver cell injury was estimated from release of transaminases from hepatocytes into the incubation medium, cell injury in hepatocytes (2 × 10 6 cells/ml) incubated in Hanks' balanced salt solution (pH 7.2) containing 1.0 mM TBHP at 37°C was potentiated with enhanced lipid peroxidation by prior depletion of intracellular GSH which was induced by diethylmaleate, a GSH depletor. GSH-depleted hepatocytes were incubated with γ-glutamylcysteinylethyl ester ( γ-ECOEt), which is known to be converted to GSH via glutathione synthetase after its hydrolysis by esterase, at concentrations of 1.0 to 10 mM in order to replenish intracellular GSH. Although TBHP-induced cell injury and lipid peroxidation were enhanced in GSH-depleted hepatocytes, these enhancements were prevented with the consumption of intracellular GSH in GSH-depleted hepatocytes pretreated with 5.0 mM γ-ECOEt. These preventive effects were observed at any time point during the TBHP treatment over a 60 min period and depended on the concentration of γ-ECOEt used. But, no preventive effect was found in GSH-depleted hepatocytes pretreated with 5.0 mM GSH. No prevention of the potentiation of TBHP-induced cell injury found in GSH-depleted hepatocytes occurred in GSH-depleted hepatocytes pretreated with both 5.0 mM γ-ECOEt and 250 μM bis-( p-nitrophenyl)phosphate, a nonspecific esterase inhibitor. γ-ECOEt treatment caused an increase in intracellular GSH content in GSH-depleted hepatocytes, while treatments of both γ-ECOEt and the esterase inhibitor caused no increase in intracellular GSH content in the cells. These results indicate that endogenous GSH modulates TBHP-induced cell injury and lipid peroxidation in isolated rat hepatocytes. The present results suggest that endogenous GSH should play a critical role in TBHP-induced cell injury in isolated rat hepatocytes and that in rat hepatocytes treated with TBHP, enhanced lipid peroxidation with the consumption of intracellular GSH could be associated with the initiation of cell injury.