MITOCHONDRIA AS A SOURCE OF REACTIVE OXYGEN SPECIES DURING REDUCTIVE STRESS IN RAT HEPATOCYTES

Cell killing, oxygen consumption, and hydroperoxide formation were det ermined in rat hepatocytes after glycolytic and respiratory inhibition . These conditions model the ATP depletion and reductive stress of ano xia (''chemical hypoxia''). Glycolysis was inhibited with iodoacetate, and mitochondrial electron transfer was blocked with sodium azide, cy anide, or myxothiazol. Cell killing, hydroperoxide formation, and inhi bitor-insensitive oxygen consumption were greater after azide than aft er myxothiazol or cyanide. Desferrioxamine, an inhibitor of iron-catal yzed hydroxyl radical formation, delayed cell killing after each of th e respiratory inhibitors. Anoxia also delayed cell killing during chem ical hypoxia. However, during anoxic incubations, desferrioxamine did not delay the onset of cell death. These findings indicate that reacti ve oxygen species participate in lethal cell injury during chemical hy poxia. In isolated mitochondria, previous studies have shown that myxo thiazol inhibits Q cycle-mediated ubisemiquinone formation in complex III (ubiquinol-cytochrome c oxidoreductase) and that ubisemiquinone ca n react with molecular oxygen to form superoxide. Decreased killing of hepatocytes with myxothiazol compared with azide suggests, therefore, that mitochondrial oxygen radical formation by complex III is involve d in cell killing during reductive stress. In support of this hypothes is, myxothiazol reduced rates of cell killing and hydroperoxide format ion in hepatocytes incubated with azide or cyanide. This mitochondrial mechanism for oxygen radical formation may be important in relative b ut not absolute hypoxia.