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A Mechanism for Zinc Toxicity in Neuroblastoma Cells

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Abstract

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 (N2α) 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.

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references

  • Abe, K., and Saito, H. (2000). Amyloid β neurotoxicity not mediated by the mitogen-activated protein kinase cascade in cultured rat hippocampal and cortical neurons. Neurosci. Lett. 292:1-4.

    Google Scholar 

  • Armstrong, C., Leong, W., and Lees, G.J. (2001). Comparative effects of metal chelating agents on the neuronal cytotoxicity induced by copper (Cu2+), iron (Fe3+) and zinc in the hippocampus. Brain Res. 892:51-62.

    Google Scholar 

  • Bradford, M.M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254.

    Google Scholar 

  • Brown, A.M., Kristal, B.S., Effron, M.S., Shestopalov, A.I., Ullucci, P.A., Sheu, K.F., Blass, J.P., and Cooper, A.L. (2000). Zn2+ inhibits α-ketoglutarate-stimulated mitochondrial respiration and the isolated α-ketoglutarate dehydrogenase complex. J. Biol. Chem. 275:13441-13447.

    Google Scholar 

  • Bruins, M.R., Kapil, S., and Oehme, F.W. (2000). Microbial resistance to metals in the environment. Ecotoxicol. Environ. Saf. 45:198-207.

    Google Scholar 

  • Cassarino, D.S., and Bennett, J.P., Jr. (1999). An evaluation of the role of mitochondria in neurodegenerative diseases: Mitochondrial mutations and oxidative pathology, protective nuclear responses, and cell death in neurodegeneration. Brain Res. Brain Res. Rev. 29:1-25.

    Google Scholar 

  • Clerk, A., Fuller, S.J., Michael, A., and Sugden, P.H. (1997). Stimulation of “stress-regulated” mitogen-activated protein kinases (stress-activated protein kinases/c-Jun N-terminal kinases and p38-mitogen-activated protein kinases) in perfused rat hearts by oxidative and other stresses. J. Biol. Chem. 273:7228-7234.

    Google Scholar 

  • Cole, T.B., Wenzel, H.J., Kafer, K.E., Schwartzkroin, P.A., and Palmiter, R.D. (1999). Elimination of zinc from synaptic vesicles in the intact mouse brain by disruption of the ZnT3 gene. Proc. Natl. Acad. Sci. U.S.A. 96:1716-1721.

    Google Scholar 

  • Crawford, I.L., and Connor, J.D. (1973). Localization and release of glutamic acid in relation to the hippocampal mossy fiber pathway. Nature 244:422-423.

    Google Scholar 

  • Denizot, F., and Lang, R. (1986). Rapid colorimetric assay for cell growth and survival. Modifications to the tetrazolium dye procedure giving improved sensitivity and reliability. J. Immunol. Methods 89:271-277.

    Google Scholar 

  • Dugan, L.L., Creedon, D.J., Johnson, E.M., Jr, and Holtzman, D.M. (1997). Rapid suppression of free radical formation by nerve growth factor involves the mitogen-activated protein kinase pathway. Proc. Natl. Acad. Sci. U.S.A. 94:4086-4091.

    Google Scholar 

  • Halliwell, B., and Gutteridge, J.M.C. (1989). Free Radicals in Biology and Medicine, Clarendon Press, Oxford.

    Google Scholar 

  • Howell, G.A., Welch, M.G., and Fredrickson, C.J. (1984). Stimulation-induced uptake and release of zinc in hippocampal slices. Nature 308:736-738.

    Google Scholar 

  • Kisilevsky, R. (2000). Review: Amyloidogenesis—Unquestioned answers and unanswered questions. J. Struct. Biol. 130:99-108.

    Google Scholar 

  • Li, P.-F., Maasch, C., Haller, H., Dietz, R., and von Harsdorf, R. (1999). Requirement for protein kinase C in reactive oxygen species-induced apoptosis of vascular smooth muscle cells. Circulation 100:967-973.

    Google Scholar 

  • Lovell, M.A., Xie, C., and Markesbery, W.R. (1999). Protection against amyloid beta peptide toxicity by zinc. Brain Res. 823:88-95.

    Google Scholar 

  • Mattson, M.P., Barger, S.W., Begley, J.G., and Mark, R.J. (1995). Calcium, free radicals, and excitotoxic neuronal death in primary cell culture. Methods Cell Biol. 46:187-216.

    Google Scholar 

  • May, M.J., and Ghosh, S. (1998). Signal transduction through NF-ϰB. Immunol Today 19:80-88.

    Google Scholar 

  • Mielke, K., and Herdegen, T. (2000). JNK and p38 stresskinases—degenerative effectors of signal-transduction-cascades in the nervous system. Prog Neurobiol. 61:45-60.

    Google Scholar 

  • Mosmann, T. (1983). Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J. Immunol. Methods 65:55-63.

    Google Scholar 

  • New, L., and Han, J. (1998). The p38 MAP Kinase pathway and its biological function. Trends Cardiovasc. Med. 8:220-229.

    Google Scholar 

  • Perez-Clausell, J., and Danscher, G. (1985). Intravesicular localization of zinc in rat telencephalic boutons. A histochemical study. Brain Res. 337:91-98.

    Google Scholar 

  • Peters, S., Koh, J., and Choi, D.W. (1987). Zinc selectively blocks the action of N-Methyl-D-Aspartate on cortical neurons. Science 236:589-593.

    Google Scholar 

  • Potocnik, F.C.V., Van Rensburg, S.J., Taljaard, J.J.F., and Emsley, R.A. (1997). Zinc and platelet membrane microviscosity in Alzheimer's disease. The in vivo effect of zinc on platelet membranes and cognition. S. Afr. Med. J. 87:1116-1119.

    Google Scholar 

  • Samanta, S., Perkinton, M.S., Morgan, M., and Williams, R.J. (1998). Hydrogen peroxide enhances signal-responsive arachidonic acid release from neurons: Role of mitogen-activated protein kinase. J. Neurochem. 70:2082-2090.

    Google Scholar 

  • Sensi, S.L., Yin, H.Z., Carriedo, S.G., Rao, S.S., and Weiss, J.H. (1999). Preferential Zn2+ influx through Ca2+-permeable AMPA/kainate channels triggers prolonged mitochondrial superoxide production. Proc. Natl. Acad. Sci. U.S.A. 96:2414-2419.

    Google Scholar 

  • Spiridon, M., Kamm, D., Billups, B., Mobbs, P., and Attwell, D. (1998). Modulation by zinc of the glutamate transporters in glial cells and cones isolated from the tiger salamander retina. J. Physiol. 506:363-376.

    Google Scholar 

  • Takeda, A. (2000). Movement of zinc and its functional significance in the brain. Brain Res. Brain Res. Rev. 34:137-148.

    Google Scholar 

  • Vallee, B.L., and Falchuk, K.H. (1993). The biochemical basis of zinc physiology. Physiol. Rev. 73:79-118.

    Google Scholar 

  • Weiss, J.H., Hartley, D.M., Koh, J.Y., and Choi, D.W. (1993). AMPA receptor activation potentiates zinc neurotoxocity. Neuron 10:43-49.

    Google Scholar 

  • Weiss, J.H., Sensi, S.L., and Koh, J.Y. (2000). Zn2+: A novel ionic mediator of neural injury in brain disease. Trends Pharmacol. Sci. 21:395-401.

    Google Scholar 

  • Xue, L., Murray, J.H., and Tolkovsky, A.M. (2000). The Ras/phosphatidylinositol 3-kinase and Ras/ERK pathways function as independent survival modules each of which inhibits a distinct apoptotic signaling pathway in sympathetic neurons. J. Biol. Chem. 275:8817-8824.

    Google Scholar 

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Authors and Affiliations

  1. Department of Medical Physiology, University of Stellenbosch, Tygerberg, South Africa

    Willie M. U. Daniels & Jacobus Hendricks

  2. The MRC Diabetic Research Group, Parow, South Africa

    Ruduwaan Salie

  3. Department of Chemical Pathology, University of Stellenbosch, Tygerberg, South Africa

    Susan J. van Rensburg

Authors
  1. Willie M. U. Daniels
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  2. Jacobus Hendricks
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  3. Ruduwaan Salie
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  4. Susan J. van Rensburg
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Correspondence to Willie M. U. Daniels.

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Daniels, W.M.U., Hendricks, J., Salie, R. et al. A Mechanism for Zinc Toxicity in Neuroblastoma Cells. Metab Brain Dis 19, 79–88 (2004). https://doi.org/10.1023/B:MEBR.0000027419.79032.bd

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