Figure 1.
γ-H2AX foci in macaque and human peripheral blood white cells exposed to IR ex vivo.
(A) Left panel, Incidence of γ-H2AX foci in human and macaque blood samples irradiated ex vivo. 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). Right panel, 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) Left panel, 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). Right panel, 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).
Figure 2.
Kinetics for γ-H2AX foci loss in macaque lymphocytes after total body irradiation.
(A) 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 ex-vivo, 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) 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). (C) Values for γ-H2AX foci per cell vs 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). (D) Foci per cell values in lymphocytes taken at the noted time (Pre = the day before TBI) and plotted vs 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 (C) denote statistically difference between TBI-and sham-irradiated macaques (P<0.05, t-test). Detailed statistical analysis is shown in Table 2. Numbers in (C) 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).
Table 1.
Values for γ-H2AX foci per cell in macaque lymphocytes for sham-irradiation at the indicated times in days (d).
Table 2.
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).
Figure 3.
Kinetics for γ-H2AX foci in macaque plucked hairs after total-body irradiation.
(A) 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 and C) 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) and at 1 day after the indicated TBI doses (C) are shown. Images in the bottom row are enlargements of the regions inside the white squares in the upper images. The rightmost image in (C) 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. (D) 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. (E) 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)). (F) 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.