ULTRAVIOLET-LIGHT INDUCED DNA DAMAGE AND REPAIR IN BOVINE LENS EPITHELIAL-CELLS

DNA damage caused by UV-B and UV-A irradiation and the rate of repair of such damage was quantitated in bovine lens epithelial cell cultures using a modified alkaline elution methodology. Two enzymes, bacteriophage T4 endonuclease V, which cleaves at the the site of pyrimidine dimers, and E. coli endonuclease III, which cleaves at the site of thymine glycols, were utilized. Pyrimidine dimers were not detected after UV-A irradiation of lens cultures with up to 400 J/m2. In contrast, after exposure to as little as 2 J/m2 of UV-B irradiation, large numbers of pyrimidine dimers were observed. At higher fluences, thymine glycols were also found. Significant levels of DNA-DNA crosslinking were suggested by reduced rates of elution of DNA from cells treated with both UV-B irradiation and H2O2 in comparison to treatment with H2O2 alone. Protein-DNA crosslinks, in contrast, were not observed. The rate of repair of UV-B induced DNA damage was quantitated by harvesting cells at various times after the UV-B exposure. Single-strand breaks were never observed immediately after UV-B exposure but appeared later during the repair phase. In contrast to the repair of H2O2 induced DNA damage, which is largely completed within 30 min of exposure, more than 50% of the UV-B light induced DNA damage remained unrepaired five hours after exposure. This difference between the rate of repair of H2O2 and UV-B induced DNA damage could provide valuable insights into the nature of DNA damaging agents in the lens environment and may reflect underlying differences in the potential for epithelial cell DNA mutation in response to various DNA damaging insults.