Lower intracellular hydrogen peroxide levels in cells overexpressing CuZn-superoxide dismutase

Abstract

Transfection of V79 Chinese hamster cells produced clones in which CuZn-superoxide dismutase (CuZn-SOD) activities were 2.2- to 3. 5-fold higher than in the parental cells. An overall reduction of antioxidant enzyme activities and both total and oxidized glutathione levels had been found in these clones. Aconitase activities in these cells were determined to indirectly measure the O2- steady-state levels. As expected, in cells overexpressing CuZn-SOD, both total and cytosolic aconitase activities have increased. Because these clones showed reduced oxidized glutathione contents, it is unlikely that they present higher H2O2 steady-state levels as a consequence of the higher SOD levels. This was confirmed by measuring H2O2 steady-state levels in cells by flow cytometric analysis of 2',7'-dichlorofluorescein diacetate-treated cells. Despite the decreased antioxidant defenses, three of the clones overexpressing CuZn-SOD showed reduced H2O2 steady-state levels. These reduced H2O2 steady-state levels were found even when the cells were treated with the O2- generator 2,3-dimethoxy-1, 4-naphthoquinone. These data provide in vivo support for the hypothesis proposed by Liochev and Fridovich [Liochev, S. I. & Fridovich, I. (1994) Free Radical Biol. Med. 16, 29-33] that O2- dismutation prevents the formation of higher H2O2 levels by other reactions.

Figure 1

Kinetics of H₂O₂ formation after treatment with 100 μM DMNQ. Cell suspensions were treated with 100 μM DMNQ. Aliquots were taken at different times and further incubated with the probe DCFH-DA for 5 min. Cellular fluorescence was then immediately determined by flow cytometric analysis. (A and B) Results of representative kinetics assays.