MITOCHONDRIAL PERMEABILITY TRANSITION IN HEPATOCYTES INDUCED BY T-BUOOH - NAD(P)H AND REACTIVE OXYGEN SPECIES

Tert-butyl hydroperoxide (t-BuOOH) induces the mitochondrial permeabil ity transition (MPT) in hepatocytes, leading to cell death. Using conf ocal microscopy, we visualized pyridine nucleotide oxidation and react ive oxygen species (ROS) formation induced by t-BuOOH. Reduced mitocho ndrial pyridine nucleotides (NADH and NADPH) were imaged by autofluore scence. Mitochondrial membrane potential, ROS, onset of MPT, and cell death were monitored with tetramethylrhodamine methyl ester (TMRM), di chlorofluorescin, calcein, and propidium iodide, respectively. t-BuOOH rapidly oxidized mitochondrial NAD(P)H. Oxidation was biphasic, and t he second slower phase occurred during mitochondrial ROS generation. S ubsequently, MPT took place, mitochondria depolarized, and cells died. beta-Hydroxybutyrate, which reduces mitochondrial NAD(+), delayed cel l killing, but lactate, which reduces cytosolic NAD(+), did not. Trifl uoperazine, which inhibits MPT, did not block the initial oxidation of NAD(P)H but prevented the second phase of oxidation, partially blocke d ROS formation, and preserved cell viability. The antioxidants, defer oxamine and diphenylphenylenediamine, also prevented the second phase of NAD(P)H oxidation. They also blocked ROS formation nearly completel y and stopped cell killing. Both antioxidants also prevented the mitoc hondrial permeability transition and subsequent mitochondrial depolari zation. In conclusion, NAD(P)H oxidation and ROS formation are critica l events promoting MPT in oxidative injury and death of hepatocytes.