A mechanism for zinc toxicity in neuroblastoma cells.

Zinc is an important component of proteins essential for normal functioning of the brain. However, it has been shown in vitro that this metal, at elevated levels, can be toxic to cells leading to their death. We investigated possible mechanisms of cell death caused by zinc: firstly, generation of reactive oxygen species, and secondly, the activation of the MAP-kinase pathway. Cell viability was assessed by means of the methyl-thiazolyl tetrazolium salt (MTT) assay and confirmed by tetramethylrhodamine methyl ester (TMRM) staining. We measured the phosphorylation status of Erk and p38 as indicators of MAP-kinase activity, using Western Blot techniques. A time curve was established when neuroblastoma (N2alpha) cells were exposed to 100 microM of zinc for 4, 12, and 24 h. Zinc caused a significant reduction in cell viability as early as 4 h, and indirectly stimulated the accumulation of reactive oxygen species as determined by 2.7 dichlorodihydrofluorescein diacetate (DCDHF) staining and confocal microscopy. Investigation of the MAP-kinase pathway indicated that Erk was downregulated, while p38 was stimulated. Our results therefore led us to conclude that in vitro, zinc toxicity involved the generation of reactive oxygen species and the activation of the MAP-kinase pathway.