γ-H2AX detection in macaque and human lymphocytes ex vivo
To evaluate the appropriateness of the macaque model for γ-H2AX biodosimeter studies in humans, we compared the kinetics of γ-H2AX foci incidence in macaque and human lymphocytes. Whole-blood samples were irradiated ex vivo with zero to 2 Gy and fixed 30 min to 48 hours later (). White cells were spotted on slides and stained for γ-H2AX. We restricted our γ-H2AX foci measurements to lymphocytes with spherical nuclei, because while lymphocytes generated a robust γ-H2AX response to these doses of irradiation, no substantial γ-H2AX signals were visible in the polymorphonuclear cells (, right panel).
γ-H2AX foci in macaque and human peripheral blood white cells exposed to IR ex vivo.
Like their human counterparts, macaque lymphocytes were found to respond robustly to irradiation over the range of 0.2 to 2 Gy, exhibiting a proportional relationship between the number of γ-H2AX foci and irradiation dose that was indistinguishable from that of the human lymphocytes (). In addition, the rates of γ-H2AX foci disappearance in macaque and human lymphocytes up to 48 hours after exposure to 2 Gy were indistinguishable (). However, γ-H2AX foci were still present 48-hours post exposure in both macaque and human lymphocytes at about 3% of the maximal level. While this value may seem low, it is still generally well above the control levels (0.75±0.04 at 48 hours vs. 0.03±0.01 in macaques). The virtually identical behavior of human and macaque lymphocytes indicates that the macaque makes an excellent model for developing γ-H2AX foci quantification as a biodosimeter for human radiation exposure.
γ-H2AX foci in lymphocytes after TBI of macaques
Individual macaques were subjected to TBI with a 60Co source at a rate of ~0.6 Gy per minute. Thirty individuals were placed in 5 groups, each receiving a different TBI dose of 0 (sham), 1, 3.5, 6.5 and 8.5 Gy. These doses were chosen to permit an evaluation of multiple biodosimetric assays over a broad dose range. At times ranging from before radiation exposure to 23 days after TBI, blood was drawn and separated into plasma and buffy coat fractions. Upon receipt, all buffy coat fractions were processed for γ-H2AX detection and 100–200 lymphocytes from each sample were used for γ-H2AX quantitation ().
Kinetics for γ-H2AX foci loss in macaque lymphocytes after total body irradiation.
shows the distribution graphs of γ-H2AX foci in the lymphocyte populations of different individual animals. These graphs of animals of the same cohort at the same time point overlay each other, indicating that the different animals responded similarly to TBI. However, their responses are not identical, giving rise to the SD values shown in . The distribution graphs of γ-H2AX foci in the two sham-treated animals in each cohort are also shown, revealing lymphocyte populations containing a few percent with one focus, and rarely a few cells with two foci (, red lines).
The mean values of foci per cell (fpc) for Sham-irradiated controls and TBI are listed in and respectively and are plotted vs
time after exposure () and vs
irradiation dose (). Values for 1-Gy exposure are significantly different than the corresponding values from the sham-irradiated controls at 0.3 and 1 day post exposure (, 1.69±0.25 and 0.43±0.14 respectively vs
0.10±0.02; p<0.05), but at 2 days post exposure, the fpc values had returned almost to control levels (, 0.17±0.07 vs
Values for γ-H2AX foci per cell in macaque lymphocytes for sham-irradiation at the indicated times in days (d).
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).
With both 3.5- and 6.5 Gy-TBI, γ-H2AX foci distribution differed significantly from those of the sham-treated animals up to 4 days after exposure (). The fpc values at 4 days post exposure were 0.82±0.15 after 3.5 Gy-TBI and 1.19±0.06 after 6.5 Gy-TBI, about five (±1)-fold and 15 (±2)-fold above the corresponding control values of 0.16±0.00 and 0.08±0.01 ( and and ). The subsequent sample was obtained at 16 days post-irradiation for 3.5 Gy-TBI and its value was statistically indistinguishable from the control value ( and and ). For 6.5 Gy, subsequent samples were obtained 9 and 16 days post-irradiation, and while the fpc values remained above the control values at these time points, the difference at 9 days post-irradiation was below the level of significance ( and and ).
For 8.5 Gy-TBI, residual foci were observed after each sample withdrawal for up to 14 days with all but 1 fpc value being statistically different from those of the corresponding sham-irradiated controls ( and and ; p<0.001 for days 1 and 2 post-IR; p<0.01 for days 3, 4, 7, 8 and 10 post-IR; p<0.05 for day 9 post-IR and 14 post-IR, ; p
0.053 for day 11 post-IR). The fpc values at 4 days were 1.34±0.46, well above the control values of 0.02±0.01 and at 9, 10, 11 and 14 days post-irradiation the values in the surviving animals were 0.39 ±0.13, 0.36 ±0.09, 0.32 ±0.15 and 0.35 ±0.0 respectively vs
0.02±0.00 for the sham-irradiated control animals. The two doses of 6.5 and 8.5 Gy were lethal to all the animals, and in contrast to the lower doses, the fpc values remained elevated for these two doses at two weeks post exposure (). Importantly, fpc values increase linearly with the irradiation dose (especially at doses greater than 1 Gy) and are substantially greater than the values for the sham-irradiation controls after 4 days ().
γ-H2AX in plucked hairs after TBI
Plucked hair bulbs retain many of the cells present in the intact follicle. These cells have been investigated as possible monitors of drug activity or diagnosis 
and to assess radiation exposure 
.When we examined macaque plucked hair bulbs for the presence of γ-H2AX foci after TBI, we found that hair bulbs plucked from the fur generally did not contain sufficient material for analysis (, top), however, those from the eyebrows and whiskers (, bottom) were amenable to γ-H2AX foci analysis. Hairs were plucked from animals, 1 day before, 1 and 2 days after 1, 3.5 and 6.5 Gy TBI and 1, 2, 3, 4 and 9 days after 8.5 Gy TBI. Plucked hairs taken from unirradiated (sham) animals contained few γ-H2AX foci (, left most images), with values of 0.04±0.03 indicating that the act of plucking in itself does not significantly induces γ-H2AX foci formation under these assay conditions.
Kinetics for γ-H2AX foci in macaque plucked hairs after total-body irradiation.
are representative images showing γ-H2AX foci in hairs plucked from unirradiated macaques and macaques after TBI. As previously observed with lymphocytes, the frequency of γ-H2AX residual foci in plucked hairs was dose dependent (). However, in contrast to lymphocytes, the incidence of γ-H2AX foci on days 1 and 2 were quite similar at 3.5 Gy and above (3D and 3E). With hairs plucked after 8.5 Gy exposure, the highest γ-H2AX signal were observed 1 and 2 days post-irradiation (2.47±0.80 and 1.70±0.57), followed by similar decreases on days 4 and 9 post-irradiation (0.58±0.19 and 0.56±0.19) ().
We also analyzed γ-H2AX levels relative to total H2AX levels by immunoblotting (). Analysis of plucked hairs by immunoblotting shows an increase of γ-H2AX levels in a dose dependent manner. Yet, the increase in γ-H2AX signals with doses, did not reach statistical difference. Surprisingly, the immunoblot results exhibited somewhat higher relative levels of γ-H2AX on day 2 compared to day 1. This may be explained at least in part by the accumulation of apoptotic cells in the hair bulbs following the larger TBI doses (). Apoptotic cells contain very much larger amounts of γ-H2AX compared to that contained in a focus, thus a relatively small number of apoptotic cells could account for the increased γ-H2AX signal 
These results suggest that plucked hair bulbs may be a useful tissue for assessing exposures to ionizing radiation, either in conjunction with lymphocytes or by themselves.