MITOCHONDRIAL AND GLYCOLYTIC DYSFUNCTION IN LETHAL INJURY TO HEPATOCYTES BY T-BUTYLHYDROPEROXIDE - PROTECTION BY FRUCTOSE, CYCLOSPORINE-A AND TRIFLUOPERAZINE

In isolated mitochondria, t-butylhydroperoxide (t-BuOOH) and other pro -oxidants cause a permeability transition characterized by increased p ermeability to small ions, swelling and loss of membrane potential. Cy closporin A and trifluoperazine inhibit this permeability transition. Here, we investigated the role of the mitochondrial permeability trans ition in lethal cellular injury from t-BuOOH. Hepatocytes from fasted rats were isolated by collagenase perfusion, and cell viability was as sessed by propidium iodide fluorescence. t-BuOOH caused dose- and time -dependent cell killing. Fructose, a substrate for glycolytic ATP form ation, protected at lower (less-than-or-equal-to 1 00 muM), but not at higher concentrations of t-BuOOH. In fructose-treated cells, oligomyc in (10 mug/ml) delayed cell killing after 100 to 300 muM t-BuOOH, wher eas cyclosporin A (0.5 muM) plus trifluoperazine (5 muM) even more pot ently reduced lethal injury. In hepatocyte suspensions, 100 muM t-BuOO H caused mitochondrial depolarization as determined by release of rhod amine 123. Cyclosporin A plus trifluoperazine in the presence of fruct ose substantially reduced release of rhodamine 123. Similarly, in sing le cult, d hepatocytes viewed by laser scanning confocal microscopy, t -BuOOH caused leakage of rhodamine 123 from mitochondria, an event whi ch preceded cell death and which was delayed by fructose in combinatio n with cyclosporin A plus trifluoperazine. At 1 mM, t-BuOOH inhibited glycolysis, and fructose in combination with either oligomycin or cycl osporin A plus trifluoperazine had only a short-lived protective effec t. In conclusion, t-BuOOH toxicity was progressive with increasing dos ages. At low t-BuOOH (less-than-or-equal-to 50 muM), mitochondrial ATP synthetic capacity was inhibited, but not uncoupled. At higher concen trations, mitochondria became uncoupled, an event which seemed to be a ssociated 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 ce llular ATP generation is a common final pathway leading to cell death after exposure to t-BuOOH.