The potential applicability of γ-H2AX foci labeling, detection, and quantification in cells and tissues after acute external irradiation has previously been demonstrated both in vitro and ex vivo, as presented in the introduction. However, the distribution of DNA double-strand breaks within the tissue microarchitecture of liver and testis after internal irradiation from a radionuclide or radiopharmaceutical has not been previously shown. Our study proposes a suitable ex vivo method that enables γ-H2AX foci immunolabeling of internally irradiated tissue sections combined with confocal laser scanning microscopy imaging that provides quantitative digital analysis of intranuclear γ-H2AX foci. In the current analysis of tissue sections, we demonstrate an absorbed dose-dependent elevation of γ-H2AX foci after internal exposure from 111InCl3. The method was validated by using two tissues with major differences in proliferation rate and radiosensitivity, the testis and the liver. The spermatogenesis has for long and repeatedly been used as a suitable in vivo experimental model in basic radiobiological research [18, 31,32,33,34]. However, one disadvantage is that most models are based on the long-time cycle of spermatogonial cells becoming whole spermheads, to find the minimum spermhead count in survival studies as a measure of the depletion of the proliferating tissue of the testis. The novel ex vivo γ-H2AX method developed and evaluated in the present study could further expand the testis as a suitable experimental model. Moreover, our results in the liver do indicate that the ex vivo method may be suitable for analyses of induced γ-H2AX foci in other tissues.
As expected, the endogenous level (background) and variation of intranuclear foci for non-exposed animals was significantly lower in the liver cells than in the spermatogonia and primary spermatocytes. For hepatocytes and non-hepatocytes, the average number of endogenous foci per nuclei was only 0.5 ± 0.2 (mean ± SD) and 0.3 ± 0.3, respectively, whereas for the spermatogonia and primary spermatocytes, the average endogenous value of foci per nuclei was high, 19 ± 3, in accordance with published studies [3, 35, 36].
In hepatocytes, we illustrated a significant increase in the number of γ-H2AX foci per nuclei occurred after exposure. In contrast, there were no differences in the γ-H2AX foci count between exposed and non-exposed animals in the nuclei of spermatogonia or primary spermatocytes in the testis. This is consistent with the fact that a lower endogenous level of γ-H2AX foci and a higher absorbed dose, as seen in the liver, leads to increased separation between exposed and non-exposed animals . The results also concur, with the conclusion by Firsanov et al. , that the efficiency of γ-H2AX foci formation correlates with the proliferation capabilities of the tissues.
It is known that the dose rate of the irradiation and the time point after an exposure will affect the measured number of γ-H2AX foci. Continuous radiation emitted from radionuclides, where the dose rate usually is low compared with acute exposures, will provide time for repair of sub-lethal DNA-damage to occur during the time of irradiation. This will cause a lower foci-count directly after a protracted exposure than after a single high-dose rate exposure with the same total absorbed dose. However, the foci count after a 24 h protracted irradiation is expected to be higher than 24 h after an acute exposure, as shown by van Oorschot et al. . In addition, the magnitude of the dose-rate effect does vary between cell types, due to the inherent radiosensitivity, in our study represented by two contrasting tissues, related to the degree of differentiation and proliferation but also the repair time of sub-lethal DNA damage. Cells with a high inherent radiosensitivity and a slow repair rate of sublethal damage will be less affected by the dose rate effect . Kühne et al.  measured the maximum number of γ-H2AX foci in human fibroblasts 3 min after 2 Gy X-ray irradiation. However, the foci at that time were small, and the authors suggested performing quantification 15–30 min post-exposure, despite the risk that some foci may be repaired during that time. Within the liver, Firsanov et al.  showed a decrease in the fraction of γ-H2AX-positive nuclei from 1 to 24 h after acute X-ray irradiation, indicating inception of DNA DSB repair after X-ray irradiation. When DNA repair mechanisms are activated, a smaller induction of γ-H2AX foci per unit absorbed dose was shown in a split dose experiment by Mariotti et al. , where the time between the two fractions was less than 12 h. Hence, it follows that the correlation between absorbed dose and the number of γ-H2AX foci after and between exposures is complex and will depend on the tissue of interest, the time point studied, the effective half-life of the radiopharmaceutical, the dose rate of the exposure, and the repair rate of the studied cells. After continuous internal irradiation from 131I, Lassmann et al.  exhibited the highest number of irradiation-induced foci in leukocytes 2 h after administration (0.2 excess foci per nucleus). Afterwards, despite subsequently increasing absorbed dose, the foci count decreased. However, Eberlein et al.  showed a linear relation between the absorbed dose to the blood and the average number of radiation-induced γ-H2AX foci per cell for peripheral blood lymphocytes up to 5 h after administration of 177Lu labeled DOTATATE/DOTATOC, with 0.55 excess foci per cell at 4 h for the average absorbed dose of 34 ± 13 mGy. In all, more systems of cells and variations of irradiations must be studied before a clear dose–effect relationship can be formulated.
When studying a variety of cells within a tissue, cells can be active in the cell cycle, wherein duplication of DNA occurs and radiosensitivity varies. It is well known that ionizing radiation may activate cell cycle checkpoints, which may delay the movement of cells through the cell cycle phases. Cells in the G2/M phase will, per unit absorbed dose, express double the number of γ-H2AX foci than cells in the G1/G0 phase, consistent with the doubling of DNA content . Our results, obtained at two time points after 111InCl3 administration, are limited by not knowing the underlying cell cycle progression and possible alteration of progression, which may affect the results seen 25 h P.I.
The calculated average absorbed doses to the testis, assuming a mono-exponential elimination, was 20 mGy for animals 4 h P.I. and 0.1 Gy for animals 25 h P.I. If no sacrifice had been carried out, the total absorbed dose to the testis would have been 0.4 Gy, which is the median lethal dose (LD50) for mice spermatogonia . The human testis is much more sensitive, where 0.15 Gy causes a pronounced depression of sperm counts (oligospermia), and temporary sterility (azoospermia) has been reported at 0.3 Gy [25, 26]. However, no significant increase in the number of γ-H2AX foci were seen in the mice exposed to these absorbed doses. However, within the liver of the same mice, the absorbed doses gave rise to a significant increase in the number of γ-H2AX foci per nuclei, even though the absorbed doses to the liver, 0.5 Gy and 3.2 Gy for animals exposed for 4 h and 25 h, respectively, were small in comparison to the absorbed doses held accountable for irradiation effects within the human liver. If no sacrifice had been carried out, the total absorbed dose to the liver would have been 12 Gy, which still is far below the 30–35 Gy from which a 5% incidence of RILD has been observed within the human liver [15, 37, 38].
In this study, because no complete biokinetic data was collected, the absorbed dose calculation accounted only for self-absorbed doses in the testis and the liver itself; it did not include any cross-dose component originating from activity in the surrounding tissue. For both the testis and the liver, the self-absorbed dose is considered a good estimate of the total absorbed dose. It is known that 111In is taken up within the tubuli of the testis, more specifically in the basal layer containing the spermatogonia, after systemic administration of 111InCl3 [16, 17, 33]. Absorbed doses from such a heterogeneous activity distribution can be accounted for using small-scale dosimetry models. For the human testis , the self-absorbed dose from 111In to the layer of spermatogonia could be a factor of two higher than the corresponding average absorbed dose to the whole testis. In addition, since 111In is a radionuclide emitting low energy conversion electrons and Auger electrons with high linear energy transfer (LET), the cellular and subcellular distribution will affect the absorbed dose to the nuclei. Rao et al. have shown a likely intranuclear distribution of 111In radiopharmaceuticals within the testicular cells and shown that the subcellular decay sites of high LET Auger-electron emitters primarily determine their radiotoxicity [31, 33]. An intranuclear localization of 111In, with decay sites close to the DNA, would further increase the absorbed dose to the DNA. Furthermore, within the liver, published studies [52,53,54] have shown a heterogeneous radiopharmaceutical distribution after intravenous injections, where radiolabeled colloids such as metal–plasma protein complexes [17, 20,21,22] tend to accumulate within the liver macrophages, i.e., Kupffer cells. According to a small-scale anatomical model of the human liver tissue for radiation dosimetry , the locally absorbed dose close to the source of the activity (e.g., the Kupffer cells) would increase slightly (5–10%) for 10% of the hepatocytes, whereas the self-absorbed dose to the Kupffer cells themselves would be 25 times higher than the average absorbed dose. Hence, a non-uniform radionuclide distribution within the studied tissues is likely present, both on a cellular and on a subcellular level. This, in combination with the high LET from the Auger emissions from 111In, may result in a non-uniform absorbed dose distribution to the different cells within the tissues, which may affect the accuracy of our results.
γ-H2AX immunofluorescence labeling methods and quantitative analyses
In this study, the two different primary antibodies used to target the γ-H2AX epitope showed identical labeling results that are in agreement with the results described in different previous reports [3, 35, 36]. There may be room for certain experimental improvements; for example, the amount of detergent and dilutions used for the antibody incubations can be optimized, thereby influencing antibody targeting. Additionally, to account for the large variation in cellular characteristics in different parts of the testis tubuli and to better understand the differences seen between the testis and the liver, γ-H2AX immunofluorescence labeling could be used for double immunofluorescence labeling, for instance, for correlations with specific cellular phenotypes, apoptosis/cell death, and cell proliferation. This may add both spatial and temporal information about the relation of γ-H2AX to specific cell types and of DSBs in other cellular mechanisms.
Automated image digital analyses for identification of structures in biological tissue samples still require the operator to supply criteria and define regions of interest for the analysis to be carried out, which will, as discussed by Ivashkevich et al. , inevitably affect the outcome of the segmentation and hence the counting of nuclei and foci. Therefore, all cell-specific analyses in this work were performed with strong criteria in terms of histology and morphology, and no true automatic segmentation method is presented. The random selection of testis tubuli for the CLSM intranuclear analysis of γ-H2AX foci was performed after localization of high-intensity γ-H2AX-positive tubuli in both exposed and non-exposed animals. This was further motivated by the purpose of the analysis, to investigate whether any divergence between exposed and non-exposed animals was detectable. Hence, only tubular regions with high-intensity γ-H2AX foci regions from the two groups of animals were included in the selection. For further optimization of the quantification method, use of a grid system for random selection of tubuli may be helpful.
For future research, it would be of interest to use high spatial resolution autoradiography techniques to study the corresponding micro-distribution of the radionuclides, which could provide valuable information for more detailed absorbed dose calculations using small-scale dosimetry models. Furthermore, it would be of interest to study the spatial and temporal distribution of γ-H2AX foci throughout larger tissue areas with our suggested method. These could then be correlated to data on a macroscopic level with recently developed PET- or SPECT-imaging of DNA damage repair proteins . Together, these techniques could further refine our understanding of dose–effect relationship.