Figure 1.
Images showing foci of γH2AX, 53BP1 and MRE11 DSB-marking proteins in nuclei of peripheral blood lymphocytes of minipigs.
(A–C) Images of minipig PBL immunostained for γH2AX (green), 53BP1 (red) and nuclei (DNA, blue; DAPI). (A) Control cells without IR exposure and DNA damage foci. (B) Minipig PBL 4 h post 49 Gy irradiation revealing fluorescent foci of γH2AX and 53BP1 marking DSBs. Foci usually contain variable amounts of both DSB markers leading to reddish – greenish colored foci due to color overlap (see insets in C,D). A cell with pan-γH2AX staining shows heavy overall green labeling (arrow). (C) PBL nuclei obtained 4 h post IR. A cell with 8 foci is seen in the image center (arrow). Inset: fluorescence intensity profiles across 5 foci in 3 PBL nuclei revealing colocalization of green γH2AX and red 53BP1 fluorescence intensity peaks. (D) PBLs of a 4 h sample showing colocalization of the DNA repair protein MRE11 (red) and γH2AX (green) at DNA damage foci, those appear whitish due to color overlap. The inset shows fluorescence intensity profiles across 4 foci in 2 PBL nuclei revealing colocalization of green γH2AX and red MRE11 fluorescence intensity peaks. Magnification 630× in the original micrographs.
Figure 2.
Mean γH2AX, 53BP1 and MRE11 foci numbers in peripheral blood lymphocytes from 13 minipigs detected in control and blood samples obtained 4, 24 and 168 h post IR.
(A) Foci per cell values are greatly increased 4 h after 49 Gy partial body exposure and decline with passing time. Error bars represent SE. (B) Box plot showing variation of the different γH2AX fpc values for the 13 minipigs. Whiskers represent minimum and maximum values; a box comprises 50% of the data points.
Table 1.
Mean γH2AX fpc values in control and post IR minipig PBL.
Table 2.
Mean 53BP1 fpc values in control and post IR minipig PBL.
Table 3.
Mean MRE11 fpc values in control and post IR minipig PBL.
Figure 3.
Distribution of γH2AX foci in minipig PBL samples.
(A) Average frequency (%) of PBL cells without (w/o) and with (w) γH2AX foci. While in controls nearly all cells were without foci, cells displaying foci were significantly increased 4, 24 and 168 h post IR; differences amongst all time points were highly significant (p<0.0001). (B) Average foci numbers in DSB-positive PBL nuclei. Average frequency (%) of foci-expressing PBL with ≥1 γH2AX focus in 13 investigated minipigs before (control) and 4, 24, and 168 h after PBI. In controls most γH2AX-positive nuclei showed 1 focus, while at 4 h post IR numerous cells contained 2 or 3 foci, with a few cells showing up to 8 foci. Cells with higher foci numbers are absent at later time points. Error bars represent SD.
Figure 4.
Decline of mean fpc values in PBL of ∼49 Gy PBI γ-exposed minipigs.
Mean fpc values ± SD are shown. While the study comprises only 3 time points the shape of the obtained function is consistent with that known for DNA repair after in vitro irradiation of minipig cells [9].
Figure 5.
Dispersion analysis (u) of foci distribution in 13 minipig samples at the time post IR indicated.
(A) U values outside the red dotted lines indicate over- and underdispersion, which is seen for all values 4 h post IR. In vitro 240 kV X ray irradiation of peripheral pig blood (4 h X in vitro) with 0.1, 0.5, 1, 2 and 4 Gy revealed a Poisson distribution for foci in 11 out of 15 samples of three pigs. (B) A linear dose response was obtained for X-irradiated PBL from three pigs obtained 4 h post IR. Mean fpc values (± SE) from three pigs are sown (900 cells per time point).
Figure 6.
Mean γH2AX fpc values in keratinocytes of the minipig skin facing the beam 4 h after 50 Gy Co-60 γ irradiation.
Average of three minipigs (± SD) is shown. Keratinocytes 4 h post IR display >20 γH2AX foci/cell (data taken from [18]).
Figure 7.
Frequency of pan-γH2AX labeled PBL nuclei in minipig blood.
(A) Image of γH2AX (green) and 53BP1 (red) stained PBL showing a pan-γH2AX-positive nucleus (arrow) with numerous 53BP1 (red) foci. DNA is stained in blue (DAPI). (B) Percentage of minipig PBL carrying a pan-γH2AX signal (compare also Fig. 1B). Mean and SD of samples from 13 minipigs is shown.
Table 4.
Antibodies, dilutions and sources.