Glutathione depletion and the production of reactive oxygen species in isolated hepatocyte suspensions

Diethyl maleate (DEM) (5 mM) and ethyl methanesulfonate (EMS) (35 mM) treat ments rapidly depleted cellular reduced glutathione (GSH) below detectable levels (1 nmol/106 cells), and induced lipid peroxidation and necrotic cell death in freshly isolated rat hepatocytes. In hepatocytes incubated with 2 .5 mM DEM and 10 mM EMS, however, the complete depletion of cellular GSH ob served was not sufficient to induce lipid peroxidation or cell death. Inste ad, DEM- and EMS-induced lipid peroxidation and cell death were dependent o n increased reactive oxygen species (ROS) production as measured by increas es in dichlorofluorescein fluorescence. The addition of antioxidants (vitam in E succinate and deferoxamine) prevented lipid peroxidation and cell deat h, suggesting that lipid peroxidation is involved in the sequence of events leading to necrotic cell death induced by DEM and EMS. To investigate the subcellular site of ROS generation, the cytochrome P450 inhibitor, SKF525A, was found to reduce EMS-induced lipid peroxidation but did not protect aga inst the loss of cell viability, suggesting a mitochondrial origin for the toxic lipid peroxidation event. In agreement with this conclusion, mitochon drial electron transport inhibitors (rotenone, thenoyltrifluoroacetone and antimycin A) increased EMS-induced lipid peroxidation and cell death, while the mitochondrial uncoupler, carbonyl cyanide m-chlorophenylhydrazone, blo cked EMS- and DEM-mediated ROS production and lipid peroxidation. Furthermo re, EMS treatment resulted in the significant loss of mitochondrial cc-toco pherol shortly after its addition, and this loss preceded losses in cellula r cc-tocopherol levels. Treatment of hepatocytes with cyclosporin A, a mito chondrial permeability transition inhibitor. oxypurinol, a xanthine oxidase inhibitor, or BAPTA-AM, a calcium chelator, provided no protection against EMS-induced cell death or lipid peroxidation. Our results indicate that DE M and EMS induce cell death by a similar mechanism, which is dependent on t he induction of ROS production and lipid peroxidation, and mitochondria are the major source for this toxic ROS generation. Cellular GSH depiction in itself does not appear to be responsible for the large increases in ROS pro duction and lipid peroxidation observed. (C) 2000 Published by Elsevier Sci ence ireland Ltd. All rights reserved.