RESUMO
Leukoaminochrome o-semiquinone radical is generated during one-electron reduction of dopamine oxidation product aminochrome when DT-diaphorase is inhibited. Incubation of 100 microM aminochrome with 100 microM dicoumarol, an inhibitor of DT-diaphorase during 2 h, induces 56% cell death (P < 0.001) with concomitant formation of (i) intracellular hydroperoxides (4.2-fold increase compared to control; P < 0.001); (ii) hydroxyl radicals, detected with ESR and spin trapping agents (2.4-fold increase when cells were incubated with aminochrome in the presence of dicoumarol compared to aminochrome alone); (iii) intracellular edema, and cell membrane deterioration determined by transmission electron microscopy; (iv) absence of apoptosis, supported by using anexin-V with flow cytometry; (v) a strong decrease of mitochondrial membrane potential determined by the fluorescent dye 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanineiodide (P < 0.01); (vi) swelling and disruption of outer and inner mitochondrial membranes determined by transmission electron microscopy. These results support the proposed role of leukoaminochrome o-semiquinone radical as neurotoxin in Parkinson's disease neurodegeneration and DT-diaphorase as neuroprotective enzyme.
