Metabolic Brain Disease, Vol. 19, Nos. 1/2, June 2004 ( C  2004) A Mechanism for Zinc Toxicity in Neuroblastoma Cells Willie M. U. Daniels, 1,4 Jacobus Hendricks, 1 Ruduwaan Salie, 2 and Susan J. van Rensburg 3 Received September 16, 2003; accepted December 8, 2003 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 (N 2α ) cells were exposed to 100 μM 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. Key words: Zinc toxicity; MAP-kinase pathway; Erk; p38; MTT; reactive oxygen species. INTRODUCTION Zinc is required as a catalyst for over 300 enzymes. It is important as a structural and regulatory ion for enzymes, proteins, and transcription factors (Vallee and Falchuk, 1993). Within the brain zinc is an essential nutrient for normal neurological functions. For example, high levels of zinc in the limbic system regulate both inhibitory and excitatory synaptic transmission in the hippocampus and therefore may play an important role in the processing of memory formation (Takeda, 2000). Zinc uptake into neuronal cells occurs at the level of the cell body as well as neuron terminal. It is incorporated into zinc-binding proteins and transported into vesicles (Cole et al., 1999). The concentration of zinc in these vesicles is approximately 300–350 μM (Takeda, 2000). Zinc is therefore sequestered at high concentrations in these presynaptic boutons (Perez-Clausell and Dansher, 1985), and when released with neuronal activity, is estimated to achieve peak synaptic concentrations of several hundred micromolar (Howell et al., 1984). It has not yet been established why 1 Department of Medical Physiology, University of Stellenbosch, Tygerberg, South Africa. 2 The MRC Diabetic Research Group, Parow, South Africa. 3 Department of Chemical Pathology, University of Stellenbosch, Tygerberg, South Africa. 4 To whom correspondence should be addressed at Department of Medical Physiology, University of Stellenbosch, P.O. Box 19063, Tygerberg 7505, Western Cape, South Africa. E-mail: wd@sun.ac.za 79 0885-7490/04/0600-0079/0 C  2004 Plenum Publishing Corporation