Copper neurotoxicity is dependent on dopamine-mediated copper uptake and one-electron reduction of aminochrome in a rat substantia nigra neuronal cell line

The mechanism of copper (Cu) neurotoxicity was studied in the RCSN-3 neuron al dopaminergic cell line, derived from substantia nigra of an adult rat. T he formation of a Cu-dopamine complex was accompanied by oxidation of dopam ine to aminochrome. We found that the Cu-dopamine complex mediates the upta ke of (CUSO4)-C-64 info the Raul Caviedes substantia nigra-clone 3 (RCSN3) cells, and it is inhibited by the addition of excess dopamine (2 mM) (63%, p < 0.001) and nomifensine (2 <mu>M) (77%, p < 0.001). Copper sulfate (1 mM ) alone was not toxic to RCSN-3 cells, but was when combined with dopamine or with dicoumarol (95% toxicity; p<0.001) which inhibits DPNH and TPNH (DT )-diaphorase. Electron spin resonance (ESR) spectrum of the 5,5-dimethylpyr roline-N-oxide (DMPO) spin trap adducts showed the presence of a C-centered radical when incubating cells with dopamine, CuSO4 and dicoumarol. A decre ase in the expression of CuZn-superoxide dismutase and glutathione peroxida se mRNA was observed when RCSN-3 cells were treated with CuSO4, dopamine, o r CuSO4 and dopamine. However, the mRNA expression of glutathione peroxidas e remained at control levels when the cells were treated with CuSO4, dopami ne and dicoumarol. The regulation of catalase was different since all the t reatments with CuSO4 increased the expression of catalase mRNA. Our results suggest that copper neurotoxicity is dependent on: (i) the formation of Cu -dopamine complexes with concomitant dopamine oxidation to aminochrome; (ii ) dopamine-dependent Cu uptake; and (iii) one-electron reduction of aminoch rome.