Evidence for a lack of DNA double-strand break repair in human cells exposed to very low x-ray doses

Abstract

DNA double-strand breaks (DSBs) are generally accepted to be the most biologically significant lesion by which ionizing radiation causes cancer and hereditary disease. However, no information on the induction and processing of DSBs after physiologically relevant radiation doses is available. Many of the methods used to measure DSB repair inadvertently introduce this form of damage as part of the methodology, and hence are limited in their sensitivity. Here we present evidence that foci of gamma-H2AX (a phosphorylated histone), detected by immunofluorescence, are quantitatively the same as DSBs and are capable of quantifying the repair of individual DSBs. This finding allows the investigation of DSB repair after radiation doses as low as 1 mGy, an improvement by several orders of magnitude over current methods. Surprisingly, DSBs induced in cultures of nondividing primary human fibroblasts by very low radiation doses (approximately 1 mGy) remain unrepaired for many days, in strong contrast to efficient DSB repair that is observed at higher doses. However, the level of DSBs in irradiated cultures decreases to that of unirradiated cell cultures if the cells are allowed to proliferate after irradiation, and we present evidence that this effect may be caused by an elimination of the cells carrying unrepaired DSBs. The results presented are in contrast to current models of risk assessment that assume that cellular responses are equally efficient at low and high doses, and provide the opportunity to employ gamma-H2AX foci formation as a direct biomarker for human exposure to low quantities of ionizing radiation.

Figure 1

DSB induction and repair in repair-proficient (MRC-5) and repair-deficient (180BR) primary human fibroblasts. (A) γ-H2AX foci (green) in MRC-5 cells; nuclei were stained with 4,6 diamidino-2-phenylindole (blue); scale bar = 10 μm. (B) γ-H2AX foci in 180BR cells. (C) Distribution of MRC-5 cells with n foci either 3 min (filled columns) or 24 h (shaded columns) after irradiation. (D) Distribution of 180BR cells with n foci either 3 min (filled columns) or 24 h (shaded columns) after irradiation. (E) Mean number of foci per cell for various repair times in irradiated MRC-5 (shaded columns) or 180BR (filled columns) cells. (F) Time course for the repair of DSBs obtained with PFGE measurements in irradiated MRC-5 (shaded columns) or 180BR (filled columns) cells. The dotted and solid lines in C and D represent Poisson distributions with a mean number of foci that was calculated from the experimental distribution of cells with n foci.

Figure 2

DSB induction in MRC-5 cells. γ-H2AX foci were counted 3 min after irradiation, and the mean values of foci per cell are shown (circles). Triangles represent DSB induction data obtained from PFGE analysis. The line is a linear fit to the data points with a slope of 35 DSBs per cell per Gy.

Figure 3

DSB repair after low doses of IR. (A) Mean number of foci per cell for various repair times in irradiated MRC-5 cells. The IR-induced foci are drawn on top of the background value obtained in a parallel sample (filled columns). (B) Distribution of MRC-5 cells with n foci for an unirradiated sample (filled columns) and for cells irradiated with 1.2 mGy and incubated for various repair times (hatched and open columns). (C) Mean number of foci per cell. The legend indicates the cell line, passage number, and time in confluency before irradiation. The IR-induced foci are drawn on top of the value of the corresponding control sample (filled columns).

Figure 4

DSB repair after long incubation times. (A) Mean number of foci per cell for various repair times in irradiated MRC-5 cells. (B) Mean number of foci per cell after repeated daily irradiations of MRC-5 or HSF1 cells. (C) Mean number of foci per cell for various repair times in MRC-5 cells irradiated and allowed to grow. The IR-induced foci are drawn on top of the background values obtained in parallel samples (filled columns).