Ionizing radiation-induced death in bovine lens epithelial cells: mechanisms and influence of irradiation dose rate.

We recently reported, in a series of patients receiving total body irradiation before transplant, an influence of dose rate (DR) on cataract formation. The aim of our present in vitro study was to investigate the influence of DR and the mechanisms of lens cell death in a bovine model. After a single fraction of 10 Gy, delivered using low (0.05 Gy/min) or high (2 Gy/min) DR (LDR and HDR, respectively), cells were incubated in media supplemented with two different fetal calf serum (FCS) concentrations (1% and 10%). Cell proliferation was evaluated using Hoechst 33342 (HO) probe and cell viability, with neutral red probe. These fluorimetric assays used a cold light cytofluorimeter. After HO assay, stained cells were examined with fluorescence microscopy to evaluate the nuclear changes related to apoptosis. Global comparison of the mean HO fluorescent values observed with LDR/controls (c) vs. HDR/c revealed a significant difference only after 96 hr (P = 0.036). In 1% FCS conditions, the difference between HDR/c and LDR/c was also statistically significant at 96 hr (P = 0.04). Pairwise multiple comparison using values observed in 1% FCS conditions after 96 hr incubation showed significant difference between HDR vs. c (P = 0.001) and HDR vs. LDR (P = 0.007). This difference, in terms of fluorescence, was correlated to the proportion of cells with nuclear apoptotic morphology. In contrast, cell viability was not influenced by DR whatever the FCS concentration used, from 24 to 96 hr after irradiation. We conclude that our fluorimetric methodology is adapted to evaluate intracellular DNA modifications and cell viability after x-ray irradiation. We observed that a single fraction of 10 Gy induces in vitro lens epithelial cell apoptosis, which is influenced by DR. In humans, HDR is considered more cataractogenic than LDR. Thus, we speculate that lens cell apoptosis could be one of the major mechanisms of radiation-induced cataract. Further investigations are necessary to study the other possible mechanisms of cataractogenesis. Int. J. Cancer (Radiat. Oncol. Invest.) 90, 138-144 (2000). Copyright 2000 Wiley-Liss, Inc.