Mitochondrial permeability transition in hepatocytes induced by t -butylhydroperoxide: nad(p)h and reactive oxygen species. Nieminen, Anna-Liisa, Aaron M. Byrne, Brian Herman, and John J. Lemasters. Department of Anatomy, School of Medicine, Case Western Reserve University, Cleveland, Ohio and Department of Cell Biology & Anatomy, School of Medicine, University of North Carolina, Chapel Hill, North Carolina
APStracts 3:0366C, 1996.
Tert-butylhydroperoxide (t-BuOOH), induces the mitochondrial permeability transition (MPT) in hepatocytes, leading to cell death. Here using confocal microscopy, we visualized pyridine nucleotide oxidation and reactive oxygen species (ROS) formation induced by t -BuOOH. Reduced mitochondrial pyridine nucleotides (NADH and NADPH) were imaged by autofluorescence. Mitochondrial membrane potential, ROS, onset of the MPT and cell death were monitored using tetramethylrhodamine methylester (TMRM), dichlorofluorescin, calcein, and propidium iodide, respectively. t-BuOOH rapidly oxidized mitochondrial NAD(P)H. Oxidation was biphasic, and the second slower phase occurred during mitochondrial ROS generation. Subsequently, the MPT took place, mitochondria depolarized, and cells died. [beta] -Hydroxybutyrate, which reduces mitochondrial NAD&, delayed cell killing, but lactate, which reduces cytosolic NAD&, did not. Trifluoperazine, which inhibits the MPT, did not block the initial oxidation of NAD(P)H, but prevented the second phase of oxidation, partially blocked ROS formation and preserved cell viability. The antioxidants, desferal and DPPD, also prevented the second phase of NAD(P)H oxidation. They also blocked ROS formation nearly completely and stopped cell killing. Both antioxidants also prevented the mitochondrial permeability transition and subsequent mitochondrial depolarization. In conclusion, NAD(P)H oxidation and ROS formation are critical events promoting the MPT in oxidative injury and death of hepatocytes. 

Received 7 June 1996; accepted in final form 1 November 1996.
APS Manuscript Number C321-6.
Article publication pending Am. J. Physiol. (Cell Physiology).
ISSN 1080-4757 Copyright 1996 The American Physiological Society.
Published in APStracts on 31 December 1996