Ty of the lens. From a radiation protection perspective, radiation cataracts are currently viewed as a threshold effect within the context of a linear-no-threshold interpretation [18,25,26]. It was, however, unknown whether epithelial cells in the lens itself show a linear dose-response by measuring, for instance, markers of DSBs such as gH2AX, 53BP1, RAD51 and cyclin D1. To address such questions, a low-dose IR exposure model was developed in response to recent ICRP recommendations [22] using mice exposed to 20 mGy? Gy X-rays and sacrificed after 1, 3 or 24 h or 10 months post-irradiation. This was a `pilot’ study with the key aim of identifying appropriate study methods for low-dose dose-responses in early lens changes, although the 10 month time point also allowed effects on lens morphology to be studied. The results of this study strongly suggest that the eye lens is correctly identified as a radiosensitive tissue, but the data also suggest differential responses dependent upon both IR dose and the location of the epithelial cells within the lens epithelium. Specifically, we demonstrate that the increased radiosensitivity is associated with unusually slow repair of DNA damage in the peripheral region of the lens. When analysed for expression of gH2AX, RAD51 and 53BP1, the peripheral zone demonstrated linear dose-response, but was significantly more sensitive within the low-dose range than cells in the central region and circulating blood lymphocytes. These differences were furthermore correlated with specific low-dose effects upon cyclin D1 levels, EdU labelling and cell density changes in the lens periphery and finally, after 10 months, alteration to lens shape. These data provide evidence of nonlinear effects in the low-dose range of IR that are lens region specific.rsob.PD168393 web royalsocietypublishing.org Open Biol. 5:3. Material and methods3.1. Animal irradiation studiesSix-week-old C57BL/6J mice (Harlan, UK), in groups of two males and two females, were exposed to single doses of IR in an X-ray chamber irradiator (250 kVp, with Gulway generator (AGO Ltd, model no.: CD160/1 Serial no.: 1032?109; copper- and aluminium-filtered 250kVp X-rays; dose rates of 5 mGy min21 for doses up to 250 mGy and 500 mGy min21 for the 100 and 250, 1000 and 2000 mGy dose points; both dose rates for 100 and 250 mGy). Each animal received a single intraperitoneal injection of EdU (Jena Bioscience GmbH, Germany) at a dose of 90 mg kg21 body weight, 1 h before irradiation. All Tulathromycin A cancer procedures strictly followed the UK Animals (Scientific Procedures) Act 1986 and had ethical approval of the UK Home Office and local AWERB (Animal Welfare and Ethical Review Body) Committee. Animals were returned to their home cages following X-irradiation for the duration of the experiment and were provided with standard maintenance diet and water ad libitum. For short-term effects, the doses were 0, 20, 100 and 1000 mGy and the animals were sacrificed at 1, 3 or 24 h post-irradiation. For long-term effects, the doses were 0, 50, 100, 250, 1000 and 2000 mGy and the animals were sacrificed after 1, 3 or 24 h or 10 months post-irradiation.central + regionposterior lens capsule flapsM199 media (Gibco Life Technologies, UK) and images recorded for each lens (Nikon SMZ1500). Two measurements of the lens diameter at right angles were made, the ratio providing the aspect ratio for each lens. Cataract incidence in this strain of mice at 47 weeks is reported to be as high as 60 [44], making the.Ty of the lens. From a radiation protection perspective, radiation cataracts are currently viewed as a threshold effect within the context of a linear-no-threshold interpretation [18,25,26]. It was, however, unknown whether epithelial cells in the lens itself show a linear dose-response by measuring, for instance, markers of DSBs such as gH2AX, 53BP1, RAD51 and cyclin D1. To address such questions, a low-dose IR exposure model was developed in response to recent ICRP recommendations [22] using mice exposed to 20 mGy? Gy X-rays and sacrificed after 1, 3 or 24 h or 10 months post-irradiation. This was a `pilot’ study with the key aim of identifying appropriate study methods for low-dose dose-responses in early lens changes, although the 10 month time point also allowed effects on lens morphology to be studied. The results of this study strongly suggest that the eye lens is correctly identified as a radiosensitive tissue, but the data also suggest differential responses dependent upon both IR dose and the location of the epithelial cells within the lens epithelium. Specifically, we demonstrate that the increased radiosensitivity is associated with unusually slow repair of DNA damage in the peripheral region of the lens. When analysed for expression of gH2AX, RAD51 and 53BP1, the peripheral zone demonstrated linear dose-response, but was significantly more sensitive within the low-dose range than cells in the central region and circulating blood lymphocytes. These differences were furthermore correlated with specific low-dose effects upon cyclin D1 levels, EdU labelling and cell density changes in the lens periphery and finally, after 10 months, alteration to lens shape. These data provide evidence of nonlinear effects in the low-dose range of IR that are lens region specific.rsob.royalsocietypublishing.org Open Biol. 5:3. Material and methods3.1. Animal irradiation studiesSix-week-old C57BL/6J mice (Harlan, UK), in groups of two males and two females, were exposed to single doses of IR in an X-ray chamber irradiator (250 kVp, with Gulway generator (AGO Ltd, model no.: CD160/1 Serial no.: 1032?109; copper- and aluminium-filtered 250kVp X-rays; dose rates of 5 mGy min21 for doses up to 250 mGy and 500 mGy min21 for the 100 and 250, 1000 and 2000 mGy dose points; both dose rates for 100 and 250 mGy). Each animal received a single intraperitoneal injection of EdU (Jena Bioscience GmbH, Germany) at a dose of 90 mg kg21 body weight, 1 h before irradiation. All procedures strictly followed the UK Animals (Scientific Procedures) Act 1986 and had ethical approval of the UK Home Office and local AWERB (Animal Welfare and Ethical Review Body) Committee. Animals were returned to their home cages following X-irradiation for the duration of the experiment and were provided with standard maintenance diet and water ad libitum. For short-term effects, the doses were 0, 20, 100 and 1000 mGy and the animals were sacrificed at 1, 3 or 24 h post-irradiation. For long-term effects, the doses were 0, 50, 100, 250, 1000 and 2000 mGy and the animals were sacrificed after 1, 3 or 24 h or 10 months post-irradiation.central + regionposterior lens capsule flapsM199 media (Gibco Life Technologies, UK) and images recorded for each lens (Nikon SMZ1500). Two measurements of the lens diameter at right angles were made, the ratio providing the aspect ratio for each lens. Cataract incidence in this strain of mice at 47 weeks is reported to be as high as 60 [44], making the.
