Zinc status of human IMR-32 neuroblastoma cells influences their susceptibility to iron-induced oxidative stress

The current work tested the hypothesis that the zinc status of a cell influences its sensitivity to iron-induced oxidative stress. Human IMR-32 neuroblastoma cells were cultured for 24 h in nonchelated control media (5 μM zinc;4.5μMiron), or in media that was treated with DTPA to reduce its zinc content (chelated media). Chelated media was supplemented with zinc to achieve concentrations of 1.5-50 μM Zn. The media was then replaced with serum-free complex media (0.9 μM Zn) with either no added iron (0.6 μM Fe), or iron (FeCl₃) added at concentrations ranging from 15 to 100 μM. Cells were cultured for an additional 3- to 24-hour period. Over the 24-hour period, cells cultured in the control iron media had good viability, and they displayed the gross morphology typical of these cells in culture. With 100 μM iron, cell viability was low in all groups. After 24 h and at iron concentrations between 15-50 μM, cells that had been cultured in the low zinc-chelated media (1.5 μM Zn) showed a concentration-dependent increase in 5 (or 6)-carboxy-2′7′-dichlorodihydrofluorescein diacetate (DCDCDHF) fluorescence (oxidative stress) and decrease in cell viability. A positive correlation between both parameters was observed (r =0.92). These cells had altered morphology and high level of nucleosomes suggestive of cell death by apoptosis. These results support the concept that the zinc status of IMR-32 neuroblastoma cells modulates their sensitivity to iron overload.