Small Molecule Inhibitors”]

Similar results of DNA damage were observed as those directly exposed to HO.Rod outer segment treatment caused preferential mtDNA damage with increased lesion frequencies over the time of treatment. Since RPE cells in vivo are continuously exposed to light, primary RPE cells from human eye donors underwent blue light irradiation.Signicant mtDNA damage was observed, whereas nuclear DNA damage was not signicant. Several BER enzymes, including DNA glycosylase and AP endonuclease, have been isolated from mammalian mitochondria.The BER pathway is initiated by the action of DNA glycosylase, which removes the aberrant base by cleaving the glycosylic bond, thereby leaving a noncoding abasic site. The AP site is then recognized and cleaved by an AP endonuclease, which forms a onenucleotide gap.Finally, the gap is lled by DNA polymerase and the nick is sealed by DNA ligase.As mentioned earlier, we have shown that human RPE cells treated with HO resulted in preferential damage to mtDNA, but not nuclear DNA. In the same study, the rates of mtDNA repair were examined and it was found that the mtDNA sustained only limited repair after a min exposure of HO. A longer exposure resulted in persistent mtDNA damage, which was not repaired at all within hr.This was also observed in the transformed human broblast cells. The sustained mtDNA damage led to compromised mitochondrial redox function as indicated by MTT assay.These data suggest that mtDNA repair devices in the RPE cells are largely insufcient to overcome extensive DNA damage.This is supported by study showing that nitric oxide inhibited bacterial repair enzyme glycosylase. In addition, it is likely due to the vicious cycle of ROS production and damage as mentioned earlier, in which a damaged mitochondrion leads to increased ROS production and more mtDNA damage.Recent evidence suggests that bpolymerase may be induced in response to oxidative stress and thereby protect the DNA from ROS damage. We found that DNA polymeraseb, but not APE, was markedly increased after HO exposure. Interestingly, both enzymes were signicantly increased following rod outer segment treatment. It is unclear why APE expression is increased with outer segment treatment but not HO.Collectively, these data suggest that oxidative stress not only induces mtDNA damage but also activates DNA repair mechanisms in the RPE cells.We predict that exploration of mtDNA repair systems is going to be an important area of research in the coming years.This is particularly true for human RPE cells undergoing aging processes.It may be of interest to study mitochondrial repair capacity in aging human RPE cells and in RPE from eye donors with AMD.In surgically excised choroidal neovascular membrane from AMD patients and AMD donor eyes, apoptotic RPE cells were readily identied. It is hypothesized that oxidative stressinduced mitochondrial dysfunction plays a pivotal role in the series of molecular events culminating in apoptotic cell death.Our studies have shown that mitochondrial dysfunction is correlated with mtDNA damage. Because persistent mtDNA damage might be an early indicator of a decline of mitochondrial function, we sought to correlate mtDNA damage with mitochondrial function and apoptotic processes.We have found exposure of RPE cells to HO signicantly decreased mitochondrial respiratory function as measured by MTT assay and increased expression of proapoptotic proteins.

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