Free radical production and changes in superoxide dismutases associated with hypoxia/reoxygenation-induced apoptosis of embryonic rat forebrain neurons in culture

Following hypoxia/reoxygenation (6h/96h), cultured neurons from the embryon ic rat forebrain undergo delayed apoptosis. To evaluate the participation o f oxidative stress and defense mechanisms, temporal evolution of intraneuro nal free radical generation was monitored by flow cytometry using dihydrorh odamine 123, in parallel with the study of transcriptional, translational, and activity changes of the detoxifying enzymes Cu/Zn-SOD and Mn-SOD. Two d istinct peaks of radical generation were depicted, at the time of reoxygena tion (+ 27%) and 48 h later (+ 25%), respectively. Radical production was u naffected by caspase inhibitors YVAD-CHO or DEVD-CHO, which prevented neuro nal damage, suggesting that caspase activation is not an upstream initiator of radicals in this model. Cell treatment by vitamin E (100 muM) displayed significant neuroprotection, whereas the superoxide generating system xant hine/xanthine oxidase induced apoptosis. Transcript and protein levels of b oth SODs were reduced 1 h after the onset of hypoxia, but activities were t ransiently stimulated. Reoxygenation was associated with an increased expre ssion (139%), but a decreased activity (21%) of the inducible Mn-SOD, where as Cu/Zn-SOD protein and activity were low and progressively increased unti l 48 h post-hypoxia, when the second rise in radicals occurred. In spite of a temporal regulation of SODs, which parallels radical formation, oxidativ e stress might account for neurotoxicity induced by hypoxia. (C) 2000 Elsev ier Science Inc.