In the neuronal cell line SH-SY5Y, oxidative stress-induced free radical overproduction causes cell death without any participation of intracellular Ca2+ increase

Adding the membrane-permeant oxidant tert-butylhydroperoxide (t-BOOH) to the incubation medium, in SH-SY5Y human neuroblastoma cells, induced a marked and progressive concentration-dependent (300, 500 and 1000 mu M) increase of free radical production, as evaluated by the fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) and of the intracellular Ca2+ ion concentrations [Ca2+](i). The removal of extracellular Ca2+ ions did not prevent t-BOOH-induced [Ca2+](i) elevation, whereas the intracellular Ca2+ ion chelator 1,2-bis(o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA) (10 mu M) was shown to be effective. Both t-BOOH-induced free radical formation and the [Ca2+](i) increase were completely prevented by the peroxyl scavenger alpha-tocopherol (50 mu M). t-BOOH induced a time-dependent SH-SY5Y cell injury, monitored by a 3-(4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) assay (approximately 25% at 1 h, 50% at 3 h, 80% at 5 h) and by fluorescein diacetate (FDA)-propidium iodide (PI) fluorescent staining. The entity of t-BOOH-induced cell damage was the same both in the absence and in the presence of the intracellular Ca2+ ion chelator BAPTA. By contrast, the peroxyl scavenger alpha-tocopherol (50 mu M) completely prevented cell injury due to oxidative stress. Finally, superoxide dismutase (SOD) (500 ng/ml) caused a 30% reduction of t-BOOH-induced 2',7'-dichlorofluorescein (DCF) fluorescence, whereas it did not modify the extent of cell injury produced by the oxidant. Collectively, the results of the present study demonstrated that in SH-SY5Y human neuroblastoma cells, the rise of [Ca2+](i) which occurs during oxidative stress is not involved in cell injury. Therefore, oxidative stress-induced cell death may be exclusively attributed to free radical overproduction. (C) 1999 Elsevier Science B.V. All rights reserved.