New strategy for oxidative stress studies with a newly developed device for O_2^- generation

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Application of the device to cell experimentsWe examined the effect of reactive oxygen species on several cultured cells using the original device. When it was applied to HEK293 cells, apparent cell death took place after 30h. When it was used for CHO cells, cell growth was suppressed but cell death did not occur at the same concentration. On the other hand, the device did not affect growth or viability of Hela cells. These results suggested that the effect of reactive oxygen species varies depending on cell types and carcinoma cells were fairly resistant to reactive oxygen species. Using SOD or catalase in the experiments with HEK293 cells we found that reactive oxygen species that is actually effective is hydrogen peroxide.Development of device II, the second version of the original deviceUnfortunately, the original device was found to be unstable in cell culture media. Therefore, we tried to improve the device in stability by chemical cross-linking. The device, somewhat diluted, was treated with a cross-linker EDC and sulfo-NHS. The resulting device became very stable in a culture medium (e.g.MEM). The half-life of the activity was over 3h and the device showed 40% activity even after 5h. The new device was referred to as "device II".Application of device II to oxidative stress experimentsThe new device was applied to human HL-60 cells and examined the effect of reactive oxygen on the cells. After incubation with device II and NADPH at 22h, cell death was observed and the cell number decreased to 30% of the initial. When the device was added without NADPH, such phenomena was not seen. SOD did not influence the effect much, but catalase blocked the effect mostly, indicating that the actual reacting species is also hydrogen peroxide.