The prevalence rate of senile cataracts among Americans 65-74 years old is 122.1/1000 and 229.0/1000 among those 75 and older. Studies from numerous laboratories particularly from the laboratory of Abraham Spector have established that oxidative stress is one of the likely initiating events in the development of the senile or maturity onset cataract. Lens proteins, cellular membranes and perhaps most importantly the DNA of the lens epithelium are targets for reactive oxygen species and hydrogen peroxide present in aqueous humor. There is evidence to suggest that compromise in the cellular function of lens epithelial cells may be directly related to cataract development particularly for cortical cataracts. Not only is cataract development a part of normal human aging it is also seen as a clinical manifestation of several heritable human progeroid syndromes including Werner, Cockayne, and Rothmund-Thomson syndromes. These entities are also characterized by DNA damage hypersensitivity and defects DNA repair. We are examining whether defects in the processing of oxidative DNA damage in lens epithelial cell lines plays a role in the multi-factorial etiology of cataracts.To assess the possible role of oxidative DNA damage and repair in premature cataractogenesis, we examined the cellular response of the premature cataract prone Nakano mouse lens epithelial cells (NKR11) to hydrogen peroxide-induced oxidative stress. NKR11 cells are more sensitive to H2O2 than normal mouse epithelial cells on the MTT-based cellular proliferation and viability assay. NKR11 cells also showed more apoptosis at 24 hrs after H2O2 treatment than the control cells. To examine whether this hypersensitivity is due to defective removal of DNA lesions induced by H2O2, we investigated the rate of removal of H2O2 induced DNA lesions in NKR11 and control cells, using the single cell gel electrophoresis (comet assay). NKR11 cells removed DNA lesions induced by 50-100uM H2O2, as efficiently as the control cell lines; however, they were deficient in the removal of lesions induced by 150uM H2O2. Comet Assay used with DNA repair enzymes as damage specific probes showed that NKR11 cells were defective in removal of both Endo III and fpg sensitive sites, suggesting defective base excision repair (BER). Western Analysis of BER related enzymes showed that NKR11 cells were deficient in flap endonuclease (FEN1) a protein that plays a role in BER and the maintenance of genomic stability. Other BER proteins including DNA Polymerase Beta, Proliferating cell nuclear antigen (PCNA) and AP endonuclease (APE1) were normal. Examination of chromosomal aberrations by premature chromatid condensation revealed that NKR11 cells had a higher level of breaks, gaps, fragments, and different modal number after treatment with 100uM H2O2 when compared with control cells, suggesting that these cells are indeed genetically unstable. Lens epithelial cells from the cataract prone Nakano mouse appear to have defect in processing DNA lesions induced by H2O2, suggesting that defective DNA repair could be one of the mechanisms of cataractogenesis. Further work on this project will involve assessing FEN-1 activity in Nakano cell extracts as well as expansion of the project to investigate the base excision repair capacity in the Emory Mouse known to develop maturity onset cortical cataracts at a chronological age similar to humans.Age-related cataract is not the only cause of visual decline in the elderly. Age-related macular degeneration is also an important entity about which even less in known in terms of the role of DNA damage and repair. Our future work will include a small project examining the role of oxidative damage and repair in age-related macular degeneration.
