Mechanisms of acetaminophen-induced hepatotoxicity: role of oxidative stress and mitochondrial permeability transition in freshly isolated mouse hepatocytes.
Freshly isolated mouse hepatocytes were used to determine the role of mitochondrial permeability transition (MPT) in acetaminophen (APAP) toxicity. Incubation of APAP (1 mM) with hepatocytes resulted in cell death as indicated by increased alanine aminotransferase in the media and propidium iodide fluorescence. To separate metabolic events from later events in toxicity, hepatocytes were preincubated with APAP for 2 h followed by centrifugation of the cells and resuspension of the pellet to remove the drug and reincubating the cells in media alone. At 2 h, toxicity was not significantly different between control and APAP-incubated cells; however, preincubation with APAP followed by reincubation with media alone resulted in a marked increase in toxicity at 3 to 5 h that was not different from incubation with APAP for the entire time. Inclusion of cyclosporine A, trifluoperazine, dithiothreitol (DTT), or N-acetylcysteine (NAC) in the reincubation phase prevented hepatocyte toxicity. Dichlorofluorescein fluorescence increased during the reincubation phase, indicating increased oxidative stress. Tetramethylrhodamine methyl ester perchlorate fluorescence decreased during the reincubation phase indicating a loss of mitochondrial membrane potential. Inclusion of cyclosporine A, DTT, or NAC decreased oxidative stress and loss of mitochondrial membrane potential. Confocal microscopy studies with the dye calcein acetoxymethyl ester indicated that MPT had also occurred. These data are consistent with a hypothesis where APAP-induced cell death occurs by two phases, a metabolic phase and an oxidative phase. The metabolic phase occurs with GSH depletion and APAP-protein binding. The oxidative phase occurs with increased oxidative stress, loss of mitochondrial membrane potential, MPT, and toxicity.
Department of Pharmacology and Toxicology, Slot 638, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA.
Angela B
AB
Reid
Richard C
RC
Kurten
Sandra S
SS
McCullough
Robert W
RW
Brock
Jack A
JA
Hinson
D000082:Acetaminophen / Q000037:antagonists & inhibitors / Q000633:toxicity*; D000410:Alanine Transaminase / Q000378:metabolism; D018712:Analgesics, Non-Narcotic / Q000037:antagonists & inhibitors / Q000633:toxicity*; D000818:Animals; D002469:Cell Separation; D056486:Chemical and Drug Induced Liver Injury / Q000473:pathology*; D005456:Fluorescent Dyes; D022781:Hepatocytes / Q000187:drug effects*; D066298:In Vitro Techniques; D008297:Male; D051379:Mice; D018613:Microscopy, Confocal; D008930:Mitochondria, Liver / Q000187:drug effects*; D018384:Oxidative Stress / Q000187:drug effects / Q000502:physiology*; D010539:Permeability / Q000187:drug effects; D013050:Spectrometry, Fluorescence
D018712:Analgesics, Non-Narcotic; D005456:Fluorescent Dyes; D000082:Acetaminophen; D000410:Alanine Transaminase