Effect of iron and manganese on hydroxyl radical production by 6-hydroxydopamine: mediation of antioxidants

6-Hydroxydopamine (6-OHDA) neurotoxicity has often been related to the generation of free radicals. Here we examined the effect of the presence of iron (Fe2+ and Fe3+) and manganese and the mediation of ascorbate, L-cysteine (CySH), glutathione (GSH), and N-acetyl-CySH on hydroxyl radical (√OH) production during 6-OHDA autoxidation. In vitro, the presence of 800 nM iron increased (> 100%) the production of √OH by 5 μM 6-OHDA while Mn2+ caused a significant reduction (72%). The presence of ascorbate (100 μM) induced a continuous generation of √OH while the presence of sulfhydryl reductants (100 μM) limited this production to the first minutes of the reaction. In general, the combined action of metal + antioxidant increased the √OH production, this effect being particularly significant (> 400%) with iron + ascorbate. In vivo, tyrosine hydroxylase immunohistochemistry revealed that intrastriatal injections of rats with 6-OHDA (30 nmol) + ascorbate (600 nmol), 6-OHDA + ascorbate + Fe2+ (5 nmol), and 6-OHDA + ascorbate + Mn2+ (5 nmol) caused large striatal lesions, which were markedly reduced (60%) by the substitution of ascorbate by CySH. Injections of Fe2+ or Mn2+ alone showed no significant difference to those of saline. These results clearly demonstrate the role of ascorbate as an essential element for the neurotoxicity of 6-OHDA, as well as the diminishing action of sulfhydryl reductants, and the negliglible effect of iron and manganese on 6-OHDA neurotoxicity.