Multiassay analysis of the toxic potential of hydrogen peroxide on cultured neurons

To clarify discrepancies in the literature on the adverse effects of hydrogen peroxide on neurons, this study investigated the application of this peroxide to cultured cerebellar granule neurons with six assays frequently used to test for viability. Cultured neurons efficiently cleared exogenous H2O2. Although viability was not affected by exposure to 10 µM hydrogen peroxide, an exposure to the peroxide in higher concentrations rapidly lowered, within 15 min, the cellular 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltertrazolium bromide (MTT) reduction capacity to 53% ± 1% (100 µM) and 31% ± 1% (1,000 µM) and the 3-amino-7-dimethylamino-2-methyl-phenazine hydrochloride (neutral red; NR) uptake to 84% ± 6% (100 µM) and 33% ± 1% (1,000 µM) of control cells. The release of glycolytically generated lactate was stopped within 30 min in neurons treated with 1,000 µM peroxide. In contrast, even hours after peroxide application, the cell morphology, the number of propidium iodide-positive cells, and the extracellular activity of the cytosolic enzyme lactate dehydrogenase (LDH) were not significantly altered. The rapid loss in MTT reduction and NR uptake after exposure of neurons to H2O2 for 5 or 15 min correlated well with a strongly compromised MTT reduction and a very high extracellular LDH activity observed after further incubation in peroxide-free medium for a total incubation period of 24 hr. These data demonstrate that cultured neurons do not recover from damage that is inflicted by a short exposure to H2O2 and that the rapid losses in the capacities of neurons for MTT reduction and NR uptake are good predictors of delayed cell damage.