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Addition into the culture medium of the antioxidant N-acetylcysteine (NAC, 1 mM) in the presence of Cu2+ (0.0005-0.001 mM) induced intensive death of cultured rat cerebellar granule neurons, which was significantly decreased by the zinc ion chelator TPEN (N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine). However, the combined action of NAC and Zn2+ did not induce destruction of the neurons. Measurement of the relative intracellular concentration of Zn2+ with the fluorescent probe FluoZin-3 AM or of free radical production using a CellROX Green showed that incubation of the culture for 4 h with Cu2+ and NAC induced an intensive increase in the fluorescence of CellROX Green but not of FluoZin-3. Probably, the protective effect of TPEN in this case could be mediated by its ability to chelate Cu2+. Incubation of cultures in a balanced salt solution in the presence of 0.01 mM Cu2+ caused neuronal death already after 1 h if the NAC concentration in the solution was within 0.005-0.05 mM. NAC at higher concentrations (0.1-1 mM) together with 0.01 mM Cu2+ did not cause the death of neurons. These data imply that the antioxidant NAC can be potentially harmful to neurons even in the presence of nanomolar concentrations of variable valence metals.

Rat cultured cerebellar granule neurons (CGNs) were not sensitive to CuCl2 (1-10 µM, 24 h), whereas paraquat (150 µM) decreased neuronal survival to 79 ± 3% of control level. Simultaneous treatment of CGNs with paraquat and CuCl2 (2, 5, or 10 µM Cu2+/paraquat) caused significant copper dose-dependent death, lowering their survival to 56 ± 4, 37 ± 3, or 16 ± 2%, respectively, and stimulating elevated production of free radicals in CGNs. Introduction of vitamin E, a non-competitive antagonist of NMDA subtype of glutamate receptors (MK-801), and also removal of glutamine from the incubation medium decreased toxicity of Cu2+/paraquat mixture. However, addition of Cu2+ into the incubation medium did not affect CGNs death caused by glutamate. These data emphasize that excessive copper in the brain may trigger oxidative stress, which in turn results in release of glutamate, overstimulation of glutamate receptors, and neuronal death.

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