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 irradiat ion 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 wi th two different fetal calf serum (FCS) concentrations (1% and 10%). Cell p roliferation was evaluated using Hoechst 33342 CHO) probe and cell viabilit y, with neutral red probe. These fluorimetric assays used a cold light cyto fluorimeter. After HO assay, stained cells were examined with fluorescence microscopy to evaluate the nuclear changes related to apoptosis. Global com parison 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 statisti cally significant at 96 hr (P = 0.04). Pairwise multiple comparison using v alues observed in 1% FCS conditions after 96 hr incubation showed significa nt difference between HDR vs. c (P = 0.001) and HDR vs. LDR (P = 0.007), Th is difference, in terms of fluorescence, was correlated to the proportion o f 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. me observed that a single fraction of 10 Gy induces in vitro lens epithelial cell apoptosis, which is influenced by DR. In humans, HDR i s considered more cataractogenic than LDR. Thus, we speculate that lens cel l apoptosis could be one of the major mechanisms of radiation-induced catar act. Further investigations are necessary to study the other possible mecha nisms of cataractogenesis. (C) 2000 Wiley-Liss, Inc.