The use of gamma-H2AX as a biodosimeter for total-body radiation exposure in non-human primates.

There is a crucial shortage of methods capable of determining the extent of accidental exposures of human beings to ionizing radiation. However, knowledge of individual exposures is essential for early triage during radiological incidents to provide optimum possible life-sparing medical procedures to each person.We evaluated immunocytofluorescence-based quantitation of γ-H2AX foci as a biodosimeter of total-body radiation exposure ((60)Co γ-rays) in a rhesus macaque (Macaca mulatta) model. Peripheral blood lymphocytes and plucked hairs were collected from 4 cohorts of macaques receiving total body irradiation doses ranging from 1 Gy to 8.5 Gy. Each cohort consisted of 6 experimental and 2 control animals. Numbers of residual γ-H2AX foci were proportional to initial irradiation doses and statistically significant responses were obtained until 1 day after 1 Gy, 4 days after 3.5 and 6.5 Gy, and 14 days after 8.5 Gy in lymphocytes and until 1 day after 1 Gy, at least 2 days after 3.5 and 6.5 Gy, and 9 days after 8.5 Gy in plucked hairs.These findings indicate that quantitation of γ-H2AX foci may make a robust biodosimeter for analyzing total-body exposure to ionizing radiation in humans. This tool would help clinicians prescribe appropriate types of medical intervention for optimal individual outcome. These results also demonstrate that the use of a high throughput γ-H2AX biodosimeter would be useful for days post-exposure in applications like large-scale radiological events or radiation therapy. In addition, this study validates a possibility to use plucked hair in future clinical trials investigating genotoxic effects of drugs and radiation treatments

Kinetics for γ-H2AX foci loss in macaque lymphocytes after total body irradiation.

<p>(<b>A</b>) Representative images of macaque lymphocytes in Sham-irradiated (Sham) and 0.3, 1, 2, 3, 7 and 10 days (d) after 8.5 Gy-TBI. As previously observed with blood samples irradiated <i>ex-vivo</i>, the γ-H2AX foci were fewer and fainter in neutrophils than in lymphocytes. Green, γ-H2AX; red, DNA stained with PI. Arrowheads indicate neutrophils. (<b>B</b>) γ-H2AX foci distribution in lymphocytes taken from individual animals subjected to TBI. The rows from top to bottom show the focal distributions from individual animals after 8.5 Gy-TBI (6 macaques E25 to E30) and Sham-TBI (2 macaques E23 and E24, noted S), 6.5 Gy-TBI (6 macaques E15 toE20) and Sham-TBI (2 macaques E21 and E22, noted S), 3.5 Gy-TBI (6 macaques E01to E06) and Sham-TBI (2 macaques E07and E08, noted S), and 1 Gy-TBI (6 macaques E09 to E14) and sham-TBI (2 macaques E21 and E22, noted S). Results from the sham-IR are shown with red lines (noted S). The columns from left to right correspond to the different sampling times post-TBI, 0.3 d (1 and 3.5 Gy), 1 d (1 , 3.5 , 6.5 and 8.5 Gy), 2 d (1 , 3.5 , 6.5 and 8.5 Gy), and 4 d (3.5 , 6.5 and 8.5 Gy). (<b>C</b>) Values for γ-H2AX foci per cell <i>vs</i> days after the indicated TBI doses. The data for the TBI treated cohorts (blue bars) and sham treated controls for each cohort (red bars) are plotted on a log scale to bring out the details concerning differences between the TBI and sham treated animals. The data are shown as mean numbers of γ-H2AX foci per lymphocyte from each cohort ± standard deviations (n = 2 for Sham irradiated samples; n = 6 for TBI-irradiation, except 16 days after 3.5 Gy, n = 3 and 11 days after 8.5 Gy; no SD was determined 14 days after 8.5 Gy as data were prepared from 1 animal). (<b>D</b>) Foci per cell values in lymphocytes taken at the noted time (Pre = the day before TBI) and plotted <i>vs</i> TBI dose. Data are means ± standard deviations (n = 6). Equations for trendlines and R-squared values are y = −0.005x+0.096 / R² = 0.225 for Pre-IR; y = 3.167x−0.578 / R² = 0.980 for 0.3 d; y = 0.672x−0.034 / R² = 0.996 for 1 d; y = 0.286x+0.014 / R² = 0.993 for 2 d and y = 0.158x+0.097 / R² = 0.956 for 4 d. Asterisks in (<b>C</b>) denote statistically difference between TBI-and sham-irradiated macaques (P<0.05, t-test). Detailed statistical analysis is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015544#pone-0015544-t002" target="_blank"><b>Table 2</b></a>. Numbers in (<b>C</b>) for the 6.5 and 8.5 Gy panels correspond to the numbers of animals alive after TBI at the indicated time (no number indicates that all 6 TBI-irradiated animals are alive).</p

Kinetics for γ-H2AX foci in macaque plucked hairs after total-body irradiation.

<p>(<b>A</b>) Representatives images of plucked hairs obtained from the fur and the head (upper panel) and from eyebrows and whiskers obtained from the face (lower panels). Plucked eyebrow hairs and whiskers with morphologies similar to the ones shown in the lower panels were found suitable for γ-H2AX detection. The black squares in the lower panel indicate the region of interest for γ-H2AX microscopy. (<b>B and C</b>) Representative images of sampled hair bulb regions of eyebrow hairs and whiskers taken from NHPs at the indicated times after 8.5 Gy-TBI (<b>B</b>) and at 1 day after the indicated TBI doses (<b>C</b>) are shown. Images in the bottom row are enlargements of the regions inside the white squares in the upper images. The rightmost image in (<b>C</b>) is an enlargement of the apoptotic region in the adjacent image. Arrows denote apoptotic cells in the other images. Green, γ-H2AX; red, DNA stained with PI. (<b>D</b>) Kinetics for γ-H2AX foci loss in plucked hairs for pre-irradiation (Pre IR), sham-irradiation (Sham IR) and at time points after the indicated TBI dose. Images of the hair apex were taken using laser-scanning confocal microscopy and γ-H2AX foci were counted in 100–500 cells. fpc: foci per cell. (<b>E</b>) The incidence of γ-H2AX foci at 1 and 2 days post TBI obtained by counting foci. Error bars signify standard deviations (n≥3). fpc: foci per cell. Least squares regression analysis show a direct relationship between radiation doses and γ-H2AX signals (slopes = 0.260103 and 0.194473 and correlation coefficients r>0, r = 0.844 and 0.880 for 1 d and 2 d post-IR respectively (p<0.001)). (<b>F</b>) Incidence of γ-H2AX relative amounts at 1 and 2 days post TBI obtained by immunoblotting. Error bars signify standard deviations (n≥2). Relative amounts of γ-H2AX in each sample were determined by comparison with total H2AX.</p

γ-H2AX foci in macaque and human peripheral blood white cells exposed to IR <i>ex vivo</i>.

<p>(<b>A</b>) <i>Left panel</i>, Incidence of γ-H2AX foci in human and macaque blood samples irradiated <i>ex vivo</i>. Blood samples were irradiated with zero, 0.2, 0.6, 1 and 2 Gy, incubated at 37°C for 30 min, then processed for γ-H2AX foci counting. Values are average numbers of γ-H2AX foci per cell (fpc). Error bars indicate standard deviations (n = 3). <i>Right panel</i>, Representative images of white cell preparations used for fpc determinations shown in the left panel. Green, γ-H2AX; red, DNA stained with PI. Arrowheads denote some of the polymorphonuclear cells. (<b>B</b>) <i>Left panel</i>, Incidence of γ-H2AX foci in macaque and human lymphocytes at various times after exposure to 2 Gy. Experimental procedures were as described above. Data presented as averages ± standard deviations (n = 3). <i>Right panel</i>, Representative images of white cell preparations used for the fpc determinations shown in the left panel. Green, γ-H2AX; red, DNA stained with propidium iodide (PI).</p

Values for γ-H2AX foci per cell in macaque lymphocytes for sham-irradiation at the indicated times in days (d).

<p>Blanks indicate no data for the corresponding times. Pre-IR: values for γ-H2AX foci per cell prior to irradiation.</p

Values for γ-H2AX foci per cell in macaque lymphocytes after total body irradiation with doses of 1, 3.5, 6.5 and 8,5 Gy at the indicated times in days (d).

<p>Blanks indicate no data for the corresponding times and doses. Pre-IR: values for γ-H2AX foci per cell prior to irradiation. Asterisks denote statistically difference between TBI-and sham-irradiated macaques ((*): P<0.05, (**): P<0.01, (***): P<0.001; t-test).</p