In the neuronal cell line SH-SY5Y, oxidative stress-induced free radical overproduction causes cell death without any participation of intracellular Ca(2+) 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 microM) increase of free radical production, as evaluated by the fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) and of the intracellular Ca(2+) ion concentrations [Ca(2+)](i). The removal of extracellular Ca(2+) ions did not prevent t-BOOH-induced [Ca(2+)](i) elevation, whereas the intracellular Ca(2+) ion chelator 1,2-bis(o-aminophenoxy) ethane-N,N, N',N'-tetraacetic acid (BAPTA) (10 microM) was shown to be effective. Both t-BOOH-induced free radical formation and the [Ca(2+)](i) increase were completely prevented by the peroxyl scavenger alpha-tocopherol (50 microM). 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 Ca(2+) ion chelator BAPTA. By contrast, the peroxyl scavenger alpha-tocopherol (50 microM) 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 [Ca(2+)](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.
Amoroso
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S
Section of Pharmacology, Department of Neuroscience, School of Medicine, 'Federico II' University of Naples, Via S. Pansini 5, 80131, Naples, Italy. amoroso@unina.it
Gioielli
A
A
Cataldi
M
M
Di Renzo
G
G
Annunziato
L
L
D002118:Calcium / Q000032:analysis / Q000378:metabolism*; D016923:Cell Death; D002470:Cell Survival; D002614:Chelating Agents / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D005452:Fluoresceins; D005609:Free Radicals / Q000032:analysis / Q000378:metabolism*; D016257:Fura-2; D006801:Humans; D015227:Lipid Peroxidation; D009447:Neuroblastoma; D018384:Oxidative Stress / Q000187:drug effects; D013482:Superoxide Dismutase / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D014810:Vitamin E / Q000494:pharmacology; D020122:tert-Butylhydroperoxide
D002614:Chelating Agents; D005452:Fluoresceins; D005609:Free Radicals; D014810:Vitamin E; D004533:Egtazic Acid; D020122:tert-Butylhydroperoxide; D013482:Superoxide Dismutase; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium; D016257:Fura-2