Office of Biological and Environmental Research

DOE Lowdose Radiation Program Workshop V

Abstract

Title: Low dose and low dose-rate irradiation for identification of individuals of high, low and intermediate radiosensitivities using the gamma-H2AX assay.

Authors: Takamitsu Kato¹, Hatsumi Nagasawa¹, Michael Weil¹, Paula Genik¹, Yuanlin Peng¹, J. B. Little², and J. S. Bedford1 ¹

Institutions: ¹Department of Environmental and Radiological Health Sciences, Colorado State University. Fort Collins, CO 80523, ²Center for Radiation Sciences and Environmental Health, Harvard School of Public Health, Boston MA 02115

We have investigated the use of the sensitive gamma-H2AX assay, reflecting the presence of DNA double strand breaks, as a potential means for identifying not only individuals who are at the extremes of hypersensitivity to effects of ionizing radiation, relative to normal wild-types, but individuals who are of intermediate with respect to this phenotype. By using this assay under conditions of low dose-rate gamma irradiation at 10 cGy/hour, significant differences in the levels of gammaH2AX foci per cell were repeatedly observed in synchronized G1 cells derived from four AT heterozygous mice relative to cells from four ATM +/+ mice, and the levels were intermediate between that for four AT -/- mice. The differences were seen in every case. The mean frequencies (+/- SEM) after 24 hours exposure at this low dose-rate were 1.77 (+/- 0.30) foci /cell for normal ATM+/+ cells, 4.75 (+/- 0.20) foci /cell for the ATM+/- cells, and 11.10 (+/- 0.58) foci per cell for the ATM-/- cells. The distributions of foci per G1 cell were, in all cases, not significantly different from Poisson, and for each of the 4-group sets, the means were not significantly different from each other. Similar comparisons are now being made for two human AT families including parents, probands, and unaffected siblings, and the results from these experiments will also be reported. We are also testing the limits of the gamma-H2AX assay for detection of differences following low acute gamma-ray doses in normal cells and cells known to be mildly or severely hypersensitive to radiation. Our initial studies have involved irradiation of low passage normal human fibroblasts in G2 or mitosis over a range of doses followed by assays for gamma-H2AX foci 0.5 hours later when the foci per cell reach a maximum, and 1.0 hours later to allow an additional repair time. Acute doses of 3.1 cGy, 6.2 cGy, 9.3 cGy, 12.4 cGy and 20.7 cGy of gamma rays were delivered, and the dose response curves were not significantly different from linear throughout this range. In all cases the numbers of foci per cell were significantly higher that that measured for 0 cGy control cells. The slope of the curve was 60.1 foci per cell/Gy (0.60 foci per cell/cGy) for the G2 and the mitotic cells, and agrees well with the expected values based on our repeated observations of about 30 foci per cell/Gy for synchronized G1 or G0 cells. With the additional 0.5 hours of repair time, the G2 chromatin also showed a linear dose response, but with a shallower slope of 28 foci per cell/Gy (0.28 foci per cell/cGy). Results for other cells will also be reported.

This work was supported by grant DE-FG03-01ER63235 from the U.S. Department of Energy