Increased γ-H2A.X Intensity in Response to Chronic Medium-Dose-Rate γ-Ray Irradiation

<div><h3>Background</h3><p>The molecular mechanisms of DNA repair following chronic medium-dose-rate (MDR) γ-ray-induced damage remain largely unknown.</p> <h3>Methodology/Principal Findings</h3><p>We used a cell function imager to quantitatively measure the fluorescence intensity of γ<em>-</em>H2A.X foci in MDR (0.015 Gy/h and 0.06 Gy/h) or high-dose-rate (HDR) (54 Gy/h) γ<em>-</em>ray irradiated embryonic fibroblasts derived from DNA-dependent protein kinase mutated mice (scid/scid mouse embryonic fibroblasts (scid/scid MEFs)). The obtained results are as follows: (1) Automatic measurement of the intensity of radiation-induced γ<em>-</em>H2A.X foci by the cell function imager provides more accurate results compared to manual counting of γ<em>-</em>H2A.X foci. (2) In high-dose-rate (HDR) irradiation, γ<em>-</em>H2A.X foci with high fluorescence intensity were observed at 1 h after irradiation in both scid/scid and wild-type MEFs. These foci were gradually reduced through de-phosphorylation at 24 h or 72 h after irradiation. Furthermore, the fluorescence intensity at 24 h increased to a significantly greater extent in scid/scid MEFs than in wild-type MEFs in the G₁ phase, although no significant difference was observed in G₂/M-phase MEFs, suggesting that DNA-PKcs might be associated with non-homologous-end-joining-dependent DNA repair in the G₁ phase following HDR γ<em>-</em>ray irradiation. (3) The intensity of γ<em>-</em>H2A.X foci for continuous MDR (0.06 Gy/h and 0.015 Gy/h) irradiation increased significantly and in a dose-dependent fashion. Furthermore, unlike HDR-irradiated scid/scid MEFs, the intensity of γ<em>-</em>H2A.X foci in MDR-irradiated scid/scid MEFs showed no significant increase in the G₁ phase at 24 h, indicating that DNA repair systems using proteins other than DNA-PKcs might induce cell functioning that are subjected to MDR γ<em>-</em>ray irradiation.</p> <h3>Conclusions</h3><p>Our results indicate that the mechanism of phosphorylation or de-phosphorylation of γ<em>-</em>H2A.X foci induced by chronic MDR γ<em>-</em>ray irradiation might be different from those induced by HDR γ<em>-</em>ray irradiation.</p> </div

Time-course-dependent changes in the intensity of γ-H2A.X foci in response to MDR (0.015 Gy/h) γ-ray irradiation.

<p>A. The left side shows relative ratios (IR/non-IR) at 1, 24, and 72 h for MDR (0.015 Gy/h, and total doses are 0.015, 0.36 and 1.08 Gy, respectively) γ<i>-</i>ray irradiations in both wild-type and scid/scid MEFs from the G₁-phase fraction. A. The right side is a box plot representation of the same values. B. The left side shows relative ratios (IR/non-IR) at 1, 24, and 72 h for MDR (0.015 Gy/h, and total doses are 0.015, 0.36, and 1.08 Gy, respectively) γ<i>-</i>ray irradiations in both wild-type and scid/scid MEFs from the G₂/M-phase fraction. B. The right side is a box plot representation of the same data. Closed circles (•) indicate wild-type MEFs, and open circles (○) indicate scid/scid MEFs. The numbers next to the circles indicate statistical significance compared to non-irradiated cells by <i>t-test</i>. The numbers in the box plot indicate statistical significance between wild-type MEFs and scid/scid MEFs by <i>t-test</i>. Each averaged I/A was calculated from 20 obtained values (4 independent experiments with 5 points taken in each experiment) for detecting relative ratio.</p

Time-course-dependent changes in the intensity of γ-H2A.X foci in response to HDR (54 Gy/h) γ-ray irradiation.

<p>A. The left side shows the relative ratios (IR/non-IR), at 1, 24, and 72 h after HDR (0.9 Gy/min, and total dose was 4.32 Gy) γ<i>-</i>ray irradiations in both wild-type and scid/scid MEFs from the G₁-phase fraction. The right side shows the same data using a box plot, which depicts groups of numerical data through their five-number summaries, i.e., smallest observation, lower quartile, median, upper quartile and largest observation. B. The left side shows relative ratios (IR/non-IR) at 1, 24, and 72 h after HDR (0.9 Gy/min and total dose was 4.32 Gy) γ<i>-</i>ray irradiation in both wild-type and scid/scid MEFs from the G₂/M-phase fraction. The right side shows a box plot representation of the same data. Closed circles (•) indicate wild-type MEFs, and opened circles (○) indicate scid/scid MEFs. The numbers next to the circles indicate statistical significance compared to non-irradiated cells by <i>t-test</i>. The numbers in the box plot indicate statistical significance between wild-type MEFs and scid/scid MEFs by <i>t-test</i>. Each averaged I/A was calculated from 20 replicate experiments (4 independent experiments with 5 points taken in each experiment) for detecting relative ratios.</p

Increased intensity of γ-H2A.X foci induced by HDR γ-ray irradiation in scid/scid MEFs.

<p>A. Histogram of cell cycle fractionation of scid/scid MEFs, where the G₁ and G₂/M phases are indicated by black bars. B. Relation between averaged I/A (γ<i>-</i>H2A.X foci per nuclear area) and total doses (0, 0.54, 1.08, 1.67, 2.16, and 3.24 Gy) by HDR irradiation, in the G₁-phase fraction from scid/scid MEFs. C. Relation between averaged I/A (γ<i>-</i>H2A.X foci per nuclear area) and total doses (0, 0.54, 1.08, 1.67, 2.16, and 3.24 Gy) by HDR irradiation in the G2/M phase fraction from scid/scid MEFs. Each averaged I/A was calculated from 10 replicate experiments (2 independent experiments with 5 points taken in each experiment) for detecting relative ratios.</p

Screening of biomarkers for liver adenoma in low-dose-rate γ-ray-irradiated mice

<p><b>Purpose:</b> Chronic low-dose-rate (20 mGy/day) γ-irradiation increases the incidence of hepatocellular adenomas (HCA) in female B6C3F1 mice. The purpose of this study is to identify potential serum biomarkers for these HCAs by a new approach.</p> <p><b>Material and methods:</b> Microarray analysis were performed to compare the gene expression profiles of HCAs from mice exposed to low-dose-rate γ-rays with those of normal livers from non-irradiated mice. From the differentially expressed genes, those for possibly secretory proteins were selected. Then, the levels of the proteins in sera were analysed by ELISA.</p> <p><b>Results:</b> Microarray analysis identified 4181 genes differentially expressed in HCAs (>2.0-fold). From these genes, those for α-fetoprotein (Afp), α-1B-glycoprotein (A1bg) and serine peptidase inhibitor Kazal type-3 (Spink3) were selected as the genes for candidate proteins. ELISA revealed that the levels of Afp and A1bg proteins in sera significantly increased and decreased, respectively, in low-dose-rate irradiated mice with HCAs and also same tendency was observed in human patients with hepatocellular carcinomas.</p> <p><b>Conclusion:</b> These results indicate that A1bg could be a new serum biomarker for liver tumor. This new approach of using microarray to select genes for secretory proteins is useful for prediction of novel tumor markers in sera.</p