A modified system to evaluate the potency of anti-oxidative compounds in different cell types in vitro

Common assays for evaluation of antioxidative capacity of different compounds are usually performed in cell-free systems. By this approach, cell-specific regulatory mechanisms upon distinct stimuli are not taken into account. Therefore, there is a need to measure anti-oxidative capacity in a cellular setting. - We now developed a valid method that provides monitoring of anti-oxidative capacities of compounds in different cell types. Oxidative stress, induced by 100 microM H subset2O subset2 in human microvascular endothelial cells (HMEC-1), was quantified by the generation of oxidized, fluorescent C-DCF from C-H subset2DCF-DA/AM. As DCF-production could be almost completely blocked by diethyldithiocarbamate (DEDTC), which inhibits intracellular Cu/Zn superoxide dismutase (SOD), mainly intracellular production of C-DCF was assumed. Preincubation with alpha-tocopherol resulted in a dose-dependent reduction of both spontaneous and H subset2O subset2-induced C-DCF-production (maximal inhibition by 41.6% at 75 microM). A synergistic effect was observed with co-incubation with vitamin C (maximal inhibition 46.8% at 10 microM vitamin C and 50 microM alpha-tocopherol). In this way compounds with different modes of action and subcellular localization can be evaluated concomitantly in respect of their anti-oxidative capacities. As this method was established on 24- and 48-well plates in other cell lines (Caco-2, HFP-1), too, screening of a large array of antioxidative compounds in different cell lines can be performed.