Rod outer segments mediate mitochondrial DNA damage and apoptosis in humanretinal pigment epithelium

Purpose. To investigate the interrelationships between DNA damage, mitochon drial activity, and apoptosis in retinal pigment epithelial cells (R-PE) af ter exposure to rod outer segments (ROS). Methods. After incubation of cultured human RPE with ROS, mitochondrial red ox function was evaluated from MTT reduction, Mitochondrial (mt) and nuclea r (n) DNA damage were determined by quantitative polymerase chain reactions (QPCR). Apoptotic RPE cells were detected by binding of annexin V to phosp hatidyl serine (PS) using fluorescence microscopy. The expression of the pr o-apoptotic proteins, p53 and p21(waf-1) and DNA repair enzymes, apurinic/a pyrimidinic endonuclease (APE(ref-1)) and DNA polymerase beta (beta -pol) w ere quantitatively determined by Western blotting analysis. Results. Mitochondrial function decreased by 20 +/-5% and annexin V immunof luorscent binding was enhanced after exposure of cells to physiological lev els of ROS (3.8 x 10(6) cm(-2)) for 4h. MtDNA was preferentially damaged af ter 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 7 h 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 beta -pol in cultures increased 110% above controls aft er 24h contact with the ROS. The expression of p53 and p21 in cells increas ed 100 and 38% above controls after 24h exposure to the ROS. Conclusions. Exposure of ROS to ROS induced mtDNA damage and dysfunction an d activated nDNA repair pathways, which did not prevent apoptosis.