Superoxide radical-initiated apoptotic signalling pathway in selenite-treated HEPG(2) cells: Mitochondria serve as the main target

The exact role of superoxide radicals (O-2(.-)) in apoptosis is still a mat ter of debate. The main objective of the present study is to evaluate the a poptotic signalling pathway initiated by O-2(.-). The reductive reaction of sodium selenite with glutathione was used as the intracellular O-2(.-)-gen erating system. When cells were exposed to 5 to 25 muM selenite, a temporal pattern of apoptotic events was observed following the elevation of O-2(.- ), in which cytochrome c release and mitochondrial depolarization preceded caspase-3 activation and DNA fragmentation. The simultaneous treatment with N-acetylcysteine and 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl marked ly reduced O-2(.-) level and suppressed the mitochondrial changes and the d ownstream apoptotic events. Moreover, pretreatment with cyclosporin A plus trifluoperazine, two mitochondrial permeability transition (MPT) inhibitors , was capable of attenuating O-2(.-)-mediated cytochrome c release and mito chondrial depolarization, and subsequently inhibiting apoptosis. Thus, the present results provide convincing evidence that O-2(.-) generated from the reductive reaction of selenite with GSH is capable of triggering a mitocho ndria-dependent apoptotic pathway. Such knowledge may not only help to obta in a better understanding of the apoptotic effect of selenite per se, but o f the role of O-2(.-) in initiation and execution of apoptosis. (C) 2000 El sevier Science Inc.