Mitochondrial and glycolytic dysfunction in lethal injury to hepatocytes by t-butylhydroperoxide

Protection by fructose, cyclosporin A and trifluoperazine

R. Imberti, A. L. Nieminen, B. Herman, J. J. Lemasters

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196 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)392-400
Number of pages9
JournalJournal of Pharmacology and Experimental Therapeutics
Volume265
Issue number1
Publication statusPublished - 1993

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tert-Butylhydroperoxide
Trifluoperazine
Fructose
Cyclosporine
Hepatocytes
Permeability
Rhodamine 123
Adenosine Triphosphate
Wounds and Injuries
Mitochondria
Cell Death
Oligomycins
Propidium
Collagenases
Glycolysis
Confocal Microscopy
Membrane Potentials
Reactive Oxygen Species
Cell Survival
Suspensions

ASJC Scopus subject areas

  • Pharmacology

Cite this

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title = "Mitochondrial and glycolytic dysfunction in lethal injury to hepatocytes by t-butylhydroperoxide: Protection by fructose, cyclosporin A and trifluoperazine",
abstract = "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.",
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T1 - Mitochondrial and glycolytic dysfunction in lethal injury to hepatocytes by t-butylhydroperoxide

T2 - Protection by fructose, cyclosporin A and trifluoperazine

AU - Imberti, R.

AU - Nieminen, A. L.

AU - Herman, B.

AU - Lemasters, J. J.

PY - 1993

Y1 - 1993

N2 - 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.

AB - 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.

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