The normal phagocytosis of outer segments of photoreceptor by RPE is known to accumulate H
2O
2, which is detoxified by antioxidant enzymes. We therefore assessed the protective effect of SOD2 against oxidation-induced apoptosis after exposure to H
2O
2. To detect apoptosis, we analyzed by fluorescence microscopy the occurrence of DNA fragmentation in the cells
(Fig. 3) . The number of TUNEL-positive cells increased with H
2O
2 treatment in a dose- and time-dependent manner (data not shown).
Figure 3shows that when RPE cells were exposed to a 300-μM concentration of H
2O
2 for 1 hour and then recultured for 16 hours, apoptotic cell death increased compared with that in untreated control cells (without H
2O
2). The number of TUNEL-positive RPE cells from HET mice was much greater than that in cells derived from WT or HEMI mice. Furthermore, in cells derived from HEMI mice, in which the SOD2 level was nearly 3.4 times that of WT, apoptosis was suppressed. It is notable that the least number of TUNEL-positive cells was present in the cells derived from the HEMI mice. To obtain further confirmation of peroxide-induced apoptosis, additional experiments were performed for quantitative assessment. RPE cells from the three groups of mice were exposed to different doses of H
2O
2 for 1 hour and recultured for various times, and apoptotic cell death was measured by cell-death–detection ELISA kit
(Figs. 4 5) . H
2O
2-induced apoptosis was both dose and time dependent.
Figure 4shows cell death as a function of the concentration of H
2O
2 ranging from 0 to 500 μM. The cell death in SOD2-deficient cells (HET) was significantly greater than in those from WT and HEMI mice at all concentrations of H
2O
2 higher than 200 μM. The time dependency of H
2O
2-induced apoptosis was also related to the cellular level of SOD2
(Fig. 5) . The cells overexpressing SOD2 (HEMI) had a greater protective effect against apoptosis induced by H
2O
2 than did those from WT or HET mice.