Application and modulation of a permanent hydrogen peroxide-induced oxidative stress to cultured astroglial cells

Hydrogen peroxide is often applied to cultured brain cells to investigate functions of these cells under oxidative stress. However, this peroxide might be quickly detoxified by cultured neural cells. At least cultured astroglial cells dispose of exogenous H(2)O(2) with a half-time in the minute range [R. Dringen, B. Hamprecht, Involvement of glutathione peroxidase and catalase in the disposal of exogenous hydrogen peroxide by cultured astroglial cells, Brain. Res. 759 (1997) pp. 67-75]. Therefore, application of hydrogen peroxide to astroglial cells leads only to a period of transient oxidative stress which depends on the ability of the cells to detoxify the peroxide. In order to apply a permanent H(2)O(2)-induced oxidative stress to astroglial cells the generation of H(2)O(2) by the coupled reactions of xanthine oxidase (XO) and superoxide dismutase (SOD) was studied and this system was applied to cultured astroglial cells. In the presence of astroglial cells, an almost constant steady state concentration of H(2)O(2) in the range up to 100 microM was generated, which depended on the activity of the XO applied. These steady state levels of H(2)O(2) were: (i) elevated by application of additional XO, (ii) slowly reduced by application of the XO inhibitor allopurinol, and (iii) immediately reduced to zero by application of catalase or a combination of allopurinol plus catalase. In conclusion, the method presented allows the application of an almost constant exogenous H(2)O(2) stress to cultured cells.