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 (≤100 μM), but not at higher concentrations of t-BuOOH. In fructose- treated cells, oligomycin (10 μg/ml) delayed cell killing after 100 to 300 μM t-BuOOH, whereas cyclosporin A (0.5 μM) plus trifluoperazine (5 μM) even more potently reduced lethal injury. In hepatocyte suspensions, 100 μM 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 (≤50 μM) mitochondrial ATP synthetic capacity was inhibited, but not uncoupled. At higher concentrations, mitochondria became uncoupled, an event which seemed to be associated with a mitochondrial permeability transition. At the highest concentrations examined (1 mM), glycolytic ATP formation also became inhibited. These findings support the hypothesis that inhibition of cellular ATP generation is a common final pathway leading to cell death after exposure to t-BuOOH.
|Number of pages||9|
|Journal||Journal of Pharmacology and Experimental Therapeutics|
|Publication status||Published - 1993|
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