Mitochondrial and glycolytic dysfunction in lethal injury to hepatocytes by t-butylhydroperoxide: protection by fructose, cyclosporin A and trifluoperazine

• Published in: The Journal of pharmacology and experimental therapeutics Vol. 265; no. 1; p. 392
• Main Authors: Imberti, R, Nieminen, A L, Herman, B, Lemasters, J J
• Format: Journal Article
• Language: English
• Published: United States 01-04-1993
• Subjects: Adenosine Triphosphate - metabolism; Animals; Cell Survival - drug effects; Cells, Cultured; Cyclosporine - pharmacology; Fructose - pharmacology; Glycolysis; Intracellular Membranes - drug effects; Intracellular Membranes - physiology; Lactates - biosynthesis; Lactic Acid; Liver - cytology; Liver - drug effects; Liver - metabolism; Male; Membrane Potentials - drug effects; Mitochondria, Liver - drug effects; Mitochondria, Liver - metabolism; Oligomycins - pharmacology; Oxidative Phosphorylation; Peroxides - antagonists & inhibitors; Peroxides - toxicity; Pyruvates - metabolism; Pyruvic Acid; Rats; Rats, Sprague-Dawley; tert-Butylhydroperoxide; Trifluoperazine - pharmacology
• ISSN: 0022-3565

In isolated mitochondria, t-butylhydroperoxide (t-BuOOH) and other pro-oxidants cause a permeability transition characterized by increased permeability to small ions, swelling and loss of membrane potential. Cyclosporin A and trifluoperazine inhibit this permeability transition. Here, we investigated the role of the mitochondrial permeability transition in lethal cellular injury from t-BuOOH. Hepatocytes from fasted rats were isolated by collagenase perfusion, and cell viability was assessed by propidium iodide fluorescence. t-BuOOH caused dose- and time-dependent cell killing. Fructose, a substrate for glycolytic ATP formation, protected at lower (< or = 100 microM), but not at higher concentrations of t-BuOOH. In fructose-treated cells, oligomycin (10 micrograms/ml) delayed cell killing after 100 to 300 microM t-BuOOH, whereas cyclosporin A (0.5 microM) plus trifluoperazine (5 microM) even more potently reduced lethal injury. In hepatocyte suspensions, 100 microM t-BuOOH caused mitochondrial depolarization as determined by release of rhodamine 123. Cyclosporin A plus trifluoperazine in the presence of fructose substantially reduced release of rhodamine 123. Similarly, in single cultured hepatocytes viewed by laser scanning confocal microscopy, t-BuOOH caused leakage of rhodamine 123 from mitochondria, an event which preceded cell death and which was delayed by fructose in combination with cyclosporin A plus trifluoperazine. At 1 mM, t-BuOOH inhibited glycolysis, and fructose in combination with either oligomycin or cyclosporin A plus trifluoperazine had only a short-lived protective effect. In conclusion, t-BuOOH toxicity was progressive with increasing dosages. At low t-BuOOH (< or = 50 microM), mitochondrial ATP synthetic capacity was inhibited, but not uncoupled.