title: Mechanisms of chloroform-induced hepatotoxicity: oxidative stress and mitochondrial permeability transition in freshly isolated mouse hepatocytes.
abstract: The role of mitochondrial permeability transition (MPT) and oxidative stress in chloroform toxicity was determined in freshly isolated female B6C3F1 mouse hepatocytes. Incubation of chloroform (12 mM) with hepatocytes resulted in cell death (alanine aminotransferase release and propidium iodide fluorescence). Chloroform had volatilized from the incubation and glutathione was depleted by 1 h; however, toxicity was not significantly different between control and chloroform-incubated cells. Hepatocytes were washed and reincubated in fresh media at 1 h. Subsequent reincubation of chloroform-treated hepatocytes resulted in significant toxicity at 3-5 h. Inclusion of the MPT inhibitor cyclosporine A or the antioxidant N-acetylcysteine (NAC) in the reincubation media at 1 h prevented toxicity. Confocal microscopy studies with the dye calcein AM indicated MPT that was blocked by cyclosporine A or NAC. Fluorescence microscopy studies utilizing JC-1 indicated loss of mitochondrial membrane potential, which was also blocked by cyclosporine A or NAC. Dichlorofluorescein fluorescence increased during the reincubation phase, indicating increased oxidative stress, and the increase was blocked by cyclosporine A. Since oxidative stress may occur by peroxynitrite, its role in toxicity was examined. Either of the nitric oxide synthase inhibitors N(G)-methyl-L-arginine (L-NMMA) and 7-nitroindazole (7-NI) at 1 h blocked toxicity. Western blot analysis of hepatocytes for 3-nitrotyrosine in proteins, a biomarker of peroxynitrite, indicated one major nitrated protein at 81 kD. Nitration of this protein was inhibited by cyclosporine A, L-NMMA, 7-NI, or NAC. The data indicate that chloroform-induced cell death occurs in two phases: a metabolic phase characterized by glutathione depletion, and an oxidative phase characterized by MPT and protein nitration.
authors.affiliation: Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205, USA.
authors.forename: Angela S
authors.initials: AS
authors.lastname: Burke
authors.forename: Kelly
authors.initials: K
authors.lastname: Redeker
authors.forename: Richard C
authors.initials: RC
authors.lastname: Kurten
authors.forename: Laura P
authors.initials: LP
authors.lastname: James
authors.forename: Jack A
authors.initials: JA
authors.lastname: Hinson
mesh_terms: D000111:Acetylcysteine / Q000494:pharmacology; D000410:Alanine Transaminase / Q000378:metabolism; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D002478:Cells, Cultured; D002725:Chloroform / Q000633:toxicity*; D016572:Cyclosporine / Q000494:pharmacology; D005260:Female; D005978:Glutathione / Q000378:metabolism; D022781:Hepatocytes / Q000187:drug effects* / Q000378:metabolism; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D051379:Mice; D008815:Mice, Inbred Strains; D008930:Mitochondria, Liver / Q000187:drug effects* / Q000502:physiology; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors; D018384:Oxidative Stress / Q000187:drug effects; D030421:Peroxynitrous Acid / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism
chemical_list: D000975:Antioxidants; D017382:Reactive Oxygen Species; D030421:Peroxynitrous Acid; C002744:3-nitrotyrosine; D014443:Tyrosine; D002725:Chloroform; D016572:Cyclosporine; D019001:Nitric Oxide Synthase; D000410:Alanine Transaminase; D005978:Glutathione; D000111:Acetylcysteine