Hydrogen peroxide-induced DNA damage in bovine lens epithelial cells.
The present investigation was undertaken to determine the types and extent of DNA damage resulting from incubation of primary cultures of bovine lens epithelial cells with hydrogen peroxide. Significant numbers of DNA single-strand breaks were detected by alkaline elution after exposure to as little as 25 microM H2O2 for 5 min at 37 degrees C. The extent of single-strand breakage was concentration dependent and linear from 25 to 200 microM H2O2. The observed single-strand breaks appear primarily due to the action of the hydroxyl radical via a Fenton reaction as both an iron chelator, 1,10-phenanthroline and OH. scavengers, including DMSO, KI and glycerol, significantly inhibited the DNA-damaging effect of H2O2. Diethyldithiocarbamate, an inhibitor of superoxide dismutase, further potentiated the DNA-damaging effects of H2O2, presumably by increasing the steady-state concentration of Fe2+. DNA-protein cross-linking was not observed. In addition, significant levels of 5,6-saturated thymine residues or pyrimidine dimers were not detected after modification of the alkaline elution methodology to allow the use of either E. coli endonuclease III or bacteriophage T4 endonuclease V, respectively. No double-strand breaks were detected after incubation of epithelial cell cultures with H2O2 concentrations of up to 400 microM for 10 min and subsequent neutral filter elution. Since, in vivo, the lens epithelium contains populations of both quiescent and dividing cells, the degree of susceptibility to oxidative damage was also studied in actively growing and plateau-phase cultures. Reduced levels of single-strand breakage were observed when plateau-phase cultures were compared to actively growing cells. In contrast, essentially no differences in repair rates were noted at equitoxic doses of H2O2. The above results suggest that lens epithelial cells may be particularly sensitive to oxidative damage and thus are a good model system in which to study the effects of oxidative stress.
Kleiman
N J
NJ
Department of Ophthalmology, College of Physicians and Surgeons, Columbia University, New York, NY 10032.
Wang
R R
RR
Spector
A
A
D000818:Animals; D002417:Cattle; D002453:Cell Cycle; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient; D003432:Cross-Linking Reagents; D004249:DNA Damage; D004277:DNA, Single-Stranded / Q000187:drug effects*; D004720:Endonucleases; D004848:Epithelium / Q000187:drug effects; D006861:Hydrogen Peroxide / Q000633:toxicity*; D006863:Hydrogen-Ion Concentration; D007502:Iron Chelating Agents; D007908:Lens, Crystalline / Q000187:drug effects*; D010618:Phenanthrolines
D003432:Cross-Linking Reagents; D004277:DNA, Single-Stranded; D007502:Iron Chelating Agents; D010618:Phenanthrolines; D006861:Hydrogen Peroxide; D004720:Endonucleases; C025205:1,10-phenanthroline