Abstract
Purpose. To investigate the interrelationships between DNA damage, mitochondrial activity, and apoptosis in retinal pigment epithelial cells (RPE) after exposure to rod outer segments (ROS). Methods. After incubation of cultured human RPE with ROS, mitochondrial redox function was evaluated from MTT reduction. Mitochondrial (mt) and nuclear (n) DNA damage were determined by quantitative polymerase chain reactions (QPCR). Apoptotic RPE cells were detected by binding of annexin V to phosphatidyl serine (PS) using fluorescence microscopy. The expression of the pro-apoptotic proteins, p53 and p21 waf-1, and DNA repair enzymes, apurinic/apyrimidinic endonuclease (APE ref-1) and DNA polymerase ß (ß-pol) were quantitatively determined by Western blotting analysis. Results. Mitochondrial function decreased by 20 ± 5% and annexin V immunofluorscent binding was enhanced after exposure of cells to physiological levels of ROS (3.8 × 10 6 cm -2) for 4h. MtDNA was preferentially damaged after exposure to ROS with increased lesion frequencies of 1.49 ± 0.37 and 2.2 ± 0.14 per 10 kb base pairs (bp), respectively after 5 and 7h contact, compared to untreated controls (zero class damage). APE ref-1 expression increased more than 340% above controls after exposure to ROS for 7 and 24h. The expression of ß-pol in cultures increased 110% above controls after 24h contact with the ROS. The expression of p53 and p21 in cells increased 100 and 38% above controls after 24h exposure to the ROS. Conclusions. Exposure of ROS to ROS induced mtDNA damage and dysfunction and activated nDNA repair pathways, which did not prevent apoptosis.