Fig. 5. Production and regulation of ROS during apoptosis. Superoxide (O^-₂) production was measured by the fluorescent dye DHE. The association of increased fluorescence intensity to ROS production was confirmed with the positive control xanthine/xanthine oxidase (see D). (A) Large increases in fluorescence intensity were detected after 9–10-hour treatment in the serum-free medium. This chronic increase in ROS was effectively prevented in the presence of 5 mM pyruvate or 20 mM succinate. Bar, 20 µm. (B) Superoxide production was increased after 9–10-hour serum deprivation. Pyruvate (5 mM) prevented the O₂^- production; succinate (20 mM) showed a less but significant inhibitory effect on superoxide stress. (C) Staurosporine (0.1 µM, 9-10 hours) induced a robust increase in O₂^- production; the increase was prevented or attenuated by co-applied 5 mM pyruvate and 20 mM succinate, respectively. (D) Measured in cell-free culture solution, the antioxidant scavenger property of pyruvate was confirmed by a marked reduction in DHE fluorescence intensity induced by xanthine/xanthine oxidase. Succinate was not effective. n=211-330 cells for serum deprivation experiments; n=92-148 cells for staurosporine experiments; n=128 wells in D. *P<0.05 compared with controls; #P<0.05 compared with serum deprivation or staurosporine alone.

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