title: Glutathione depletion and the production of reactive oxygen species in isolated hepatocyte suspensions.
abstract: Diethyl maleate (DEM) (5 mM) and ethyl methanesulfonate (EMS) (35 mM) treatments rapidly depleted cellular reduced glutathione (GSH) below detectable levels (1 nmol/10(6) 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 observed was not sufficient to induce lipid peroxidation or cell death. Instead, DEM- and EMS-induced lipid peroxidation and cell death were dependent on increased reactive oxygen species (ROS) production as measured by increases in dichlorofluorescein fluorescence. The addition of antioxidants (vitamin E succinate and deferoxamine) prevented lipid peroxidation and cell death, 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 against the loss of cell viability, suggesting a mitochondrial origin for the toxic lipid peroxidation event. In agreement with this conclusion, mitochondrial electron transport inhibitors (rotenone, thenoyltrifluoroacetone and antimycin A) increased EMS-induced lipid peroxidation and cell death, while the mitochondrial uncoupler, carbonyl cyanide m-chlorophenylhydrazone, blocked EMS- and DEM-mediated ROS production and lipid peroxidation. Furthermore, EMS treatment resulted in the significant loss of mitochondrial alpha-tocopherol shortly after its addition, and this loss preceded losses in cellular alpha-tocopherol levels. Treatment of hepatocytes with cyclosporin A, a mitochondrial 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 DEM and EMS induce cell death by a similar mechanism, which is dependent on the induction of ROS production and lipid peroxidation, and mitochondria are the major source for this toxic ROS generation. Cellular GSH depletion in itself does not appear to be responsible for the large increases in ROS production and lipid peroxidation observed.
authors.affiliation: Departments of Pharmaceutical Sciences and Pharmacy Practice, College of Pharmacy, Washington State University, Pullman, WA 99164-6510, USA.
authors.forename: M A
authors.initials: MA
authors.lastname: Tirmenstein
authors.forename: F A
authors.initials: FA
authors.lastname: Nicholls-Grzemski
authors.forename: J G
authors.initials: JG
authors.lastname: Zhang
authors.forename: M W
authors.initials: MW
authors.lastname: Fariss
mesh_terms: D000477:Alkylating Agents / Q000633:toxicity; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D016923:Cell Death / Q000187:drug effects; D002463:Cell Membrane Permeability / Q000187:drug effects; D065607:Cytochrome P-450 Enzyme Inhibitors; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005020:Ethyl Methanesulfonate / Q000633:toxicity; D005978:Glutathione / Q000378:metabolism*; D022781:Hepatocytes / Q000187:drug effects / Q000378:metabolism*; D007425:Intracellular Membranes / Q000187:drug effects / Q000378:metabolism; D015227:Lipid Peroxidation / Q000187:drug effects; D008297:Male; D008298:Maleates / Q000633:toxicity; D008930:Mitochondria, Liver / Q000187:drug effects / Q000378:metabolism; D011335:Proadifen / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000378:metabolism*; D017392:Thiobarbituric Acid Reactive Substances / Q000378:metabolism; D024505:Tocopherols; D014810:Vitamin E / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology
chemical_list: D000477:Alkylating Agents; D000975:Antioxidants; D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; D008298:Maleates; D017382:Reactive Oxygen Species; D017392:Thiobarbituric Acid Reactive Substances; D014810:Vitamin E; D005020:Ethyl Methanesulfonate; D011335:Proadifen; C014476:diethyl maleate; D005978:Glutathione; D024505:Tocopherols