Mitochondrial and glycolytic dysfunction in lethal injury to hepatocytes by t-butylhydroperoxide: protection by fructose, cyclosporin A and trifluoperazine.
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.(ABSTRACT TRUNCATED AT 250 WORDS)
Imberti
R
R
Department of Cell Biology & Anatomy, School of Medicine, University of North Carolina, Chapel Hill.
Nieminen
A L
AL
Herman
B
B
Lemasters
J J
JJ
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D016572:Cyclosporine / Q000494:pharmacology*; D005632:Fructose / Q000494:pharmacology*; D006019:Glycolysis; D007425:Intracellular Membranes / Q000187:drug effects / Q000502:physiology; D007773:Lactates / Q000096:biosynthesis; D019344:Lactic Acid; D008099:Liver / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects; D008930:Mitochondria, Liver / Q000187:drug effects* / Q000378:metabolism; D009840:Oligomycins / Q000494:pharmacology; D010085:Oxidative Phosphorylation; D010545:Peroxides / Q000037:antagonists & inhibitors / Q000633:toxicity*; D011773:Pyruvates / Q000378:metabolism; D019289:Pyruvic Acid; D051381:Rats; D017207:Rats, Sprague-Dawley; D014268:Trifluoperazine / Q000494:pharmacology*; D020122:tert-Butylhydroperoxide
D007773:Lactates; D009840:Oligomycins; D010545:Peroxides; D011773:Pyruvates; D014268:Trifluoperazine; D005632:Fructose; D019344:Lactic Acid; D016572:Cyclosporine; D019289:Pyruvic Acid; D000255:Adenosine Triphosphate; D020122:tert-Butylhydroperoxide