CHARACTERIZATION OF ZINC-INDUCED NEURONAL DEATH IN PRIMARY CULTURES OF RAT CEREBELLAR GRANULE CELLS

Although zinc is essential for the activity of numerous biological sys tems, and zinc deficiency has been associated with various pathologies , this metal can also exert direct neurotoxic action. In primary cultu res of rat cerebellar granule neurons, a brief, 15- to 30-min exposure to zinc (100-500 mu M) resulted in concentration-dependent delayed ne uronal death. The toxicity of zinc depended on the maturity of the neu ronal cultures-it was not apparent prior to Day 5 and it reached a pla teau at about 9-10 days in vitro. We assayed cell injury by measuring mitochondrial functioning (MTT assay) and cell death with the trypan b lue exclusion assay. Apoptosis was assayed by the morphological appear ance of cells following fluorescence staining with propidium iodide an d by the in situ TUNEL technique. Mitochondrial injury was an early re sult of zinc treatment. Actinomycin D, an inhibitor of macromolecular synthesis, attenuated delayed cell death. The calcium channel blockers nimodipine and amlodipine reduced both mitochondrial injury and cell death; the blockade of ionotropic glutamate receptors with MK-801 or C NQX was ineffective. These results suggest that calcium channel-blocke r-sensitive mitochondrial injury and DNA damage are operative in the p rotein-synthesis-dependent neurotoxicity of zinc. An imbalance of zinc homeostasis might play a role in the pathophysiology of apoptosis-ass ociated neurodegenerative disorders. (C) 1997 Academic Press.