DNA damage and repair in rabbit lens epithelial cells following UVA radiation

Abstract

Since ultraviolet light may be a contributing factor to cataractogenesis, we investigated the response of the lens epithelium, a potential target for W insult, to WA radiation, Cell survival and the induction and repair of DNA single-strand breaks (SSBs) were measured in cultured rabbit lens epithelial cells following WA exposure. The light was passed through a filter which eliminated wavelengths below 335 nm in order to ensure that the cells were exposed only to WA. In order to study the effect of various fluences of WA on cell survival, 2 × 10⁶ cells suspended in mode's buffer were exposed to WA. During all irradiations the cells were maintained at 0.5 °C in order to minimize DNA repair. Following WA treatment, 200 cells were cultured in minimal essential medium containing 10% rabbit serum, and a colony forming assay was used to quantify cell survival. WA induced cell death in a dose-dependent manner. In additional experiments, confluent epithelial cells on glass slides immersed in Tyrode's buffer were irradiated and SSBs were quantified using the alkaline elution technique.

A 30 min exposure to WA (180 KJ/m²) induced measurable SSBs. An increase in WA fluence brought about an increase in the number of DNA SSBs. Rejoining of SSBs was measured after the cells were irradiated in Tyrode's for 2 hrs and allowed to repair in the dark for 4 hrs at 36 °C in MEM containing 10% serum. Eighty percent of the DNA SSBs were repaired within 4 hrs as determined by analysis of the alkaline elution profile. The repair kinetics were biphasic with an initial fast and subsequently slower component. The results indicate that UVA can induce SSBs in lens epithelial cells, that the cells can repair most UVA-induced SSBs, and that UVA treatment can be toxic to the epithelium.