Comet assay of UV-induced DNA damage in retinal pigment epithelial cells

The molecular mechanisms mediating photic injury to the retinal pigment epithelial (RPE) cell are not clearly understood. This study examined qualitative and quantitative aspects of DNA damage caused by broadband UVA and UVB radiation in RPE cells.

Cultured bovine RPE cells were exposed to doses of between 0 and 0.9 J/cm2 UVA or 0 and 0.09 J/cm2 UVB, as either a suspension or as an attached monolayer. The damage to DNA resulting in single-strand breaks was assessed by means of the comet assay in which the damaged DNA migrates out of the nucleus forming a tail, and this was quantified using image analysis. Two measurements were taken: the mean percentage of tail DNA, which reflects the overall level of DNA damage in the group of cells, and the Olive tail moment, which represents the extent of migration and thus the pattern of DNA damage in individual cells. Cells were processed by the comet assay immediately after UV exposure in acute experiments. To study the occurrence of DNA repair, RPE cells were first exposed to UVB and then incubated at 37 degrees C for either 1 or 24 hours before processing for the comet assay.

UVA- and UVB-exposed cells showed a mean percentage of tail DNA that was significantly greater than in unexposed cells. Olive tail moment was higher in cells exposed to larger doses of UVB. This parameter also showed a bimodal distribution when assessed 24 hours after exposure to UVB indicating the presence of two distinct subpopulations of cells with small and large tail moments. Cells with very large tail moments were not seen with doses below 0.045 J/cm2.

Relatively low doses of UVA and UVB induce the formation of DNA strand breaks in cultured RPE. The tail moment profiles for cells incubated for 24 hours after UVB irradiation are consistent with the occurrence of DNA repair in most cells exposed to low doses and apoptosis in a subpopulation of the cells exposed to high doses.