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.
Mayer
D
D
IIIrd Institute of Biological Chemistry and Nutrition, University of Hohenheim, Stuttgart, Germany.
Mühlhöfer
A
A
Biesalski
H K
HK
D000975:Antioxidants / Q000494:pharmacology*; D001681:Biological Assay / Q000379:methods*; D002460:Cell Line; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005452:Fluoresceins / Q000737:chemistry / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism*; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D010084:Oxidation-Reduction; D018384:Oxidative Stress / Q000187:drug effects*; D017382:Reactive Oxygen Species / Q000378:metabolism; D014810:Vitamin E / Q000494:pharmacology
D000975:Antioxidants; D004791:Enzyme Inhibitors; D005452:Fluoresceins; D005456:Fluorescent Dyes; D017382:Reactive Oxygen Species; D014810:Vitamin E; D006861:Hydrogen Peroxide