Purpose: To estimate the impact of 3H-thymidine on DNA double strand breaks (DSBs) induction in cultured human mesenchymal stem cells (MSC). Material and methods: Isolation and cultivation of human bone marrow MSC was carried out according to a standard procedure. A sterile solution of 3H-thymidine with different specific radioactivity was added to the cell culture and incubated under the conditions of the CO2 incubator for 24 hours. The specific radioactivity of 3H-thymidine in the incubation medium was 50–1600 kBq/ml. To evaluate quantitatively the DSBs, an immunocytochemical analysis of the DSB marker – γH2AX foci histone was used. Additionally, the proportion of dividing cells was estimated using an immunocytochemical analysis of the cell proliferation marker, the Ki67 protein. Results: It was shown that 24 h incubation of human MSC in a culture medium results in a dose-dependent increase in γH2AX foci. There is a linear increase in the foci γH2AX in the range of 50–400 kBq/ml, after which the relative quantitative yield of foci per unit of specific radioactivity begins to decrease. In general, the dose-effect relationship is approximated by the quadratic function y = 3.13 + 50.80x – 12.38x2 (R2 = 0.99), where y is the number of foci γH2AX in the cell nucleus, and x is the specific radioactivity in 1000 kBq/ml. It was found that incubation of human MSC in a culture medium containing 800 and 1600 kBq/ml of 3H-thymidine resulted in a statistically significant decrease in the cells proliferative activity compared to the control of ~1.25 and 1.41 respectively. The peculiar biological limitation of tritium accumulation in the cell nucleus explains well the nonlinear character of the dependence of the formation of DSBs on the specific radioactivity of 3H-thymidine in the culture medium observed in our study. Conclusion: Quantitative analysis of γH2AX foci has proved to be a highly reproducible and highly sensitive method for evaluating the induction of DSBs in living cells under the action of 3H-thymidine. An analysis of the foci of γH2AX will be useful for accurate estimating the quantitative yield of DBS in living cells per dose of 3H-thymidine β-radiation. To do this, it is necessary to make a correct calculation of the doses received by the cells taking into account the microdistribution of 3H-thymidine in the cell volume and its accumulation in the DNA of living cells.

The review considers investigations presenting experimental data on the embryotoxic and teratogenic effects of exposure to ionizing radiation in zebrafish (Danio rerio) which is a convenient model for experimental embryology and radiation biology. The molecular mechanisms involved in response to the ionizing radiation influence as well as determining the embryonic death level, development disorders of embryos are examined. The data on acute and chronic effects of ionizing radiation on embryos of various stages of development with wide range of dose rates are presented. It was shown that the influence of γ-radiation on the death and development of zebrafish embryos is nonmonotonic and depends both on the irradiation conditions and on the stage of embryogenesis. The results of such studies are extremely important for understanding the mechanisms of radiation-induced biological effects formation in the embryogenesis of vertebrates, including humans, as well as for developing methods and approaches to assessing radiation risk for a developing organism.

Purpose: To conduct a comparative assessment of the effect of irradiation of human mesenchymal stem cells (MSC)at ultrahigh doses at liquid nitrogen temperature (–196 °C) and room temperature (+22 °C) on the yield of “residual” DNA double-strand breaks (DSB) and proliferative activity of thawed MSC. Material and methods: Isolation and cultivation of MSC was carried out according to standard methods. Dimethyl sulfoxide (DMSO) at a final concentration of 10 % was used for cells cryopreservation. The cells were irradiated with bremsstrahlung photon radiation with photon nominal energy 5 MeV, the accelerator UELR-10-100-T-100 (Russia). Cells were irradiated at the doses of 50 and 500 Gy at a temperature of 22 °C and –196 °C. The yield of “residual” DNA DSB was assessed using an immunocytochemical analysis of the foci of the protein-marker DSB – γH2AX. To assess the proliferative activity, the number of Ki67 (protein marker of cell proliferation) of positive cells was analyzed. Results: The results of γH2AX foci assessment in MSCs after 48 hours irradiation at a dose of 50 Gy showed that the number of “residual” γH2AX foci in the MSC nuclei irradiated at 22 °C is about 3.2 times (p = 0.0002) higher than in the MSC nuclei irradiated at –196 °C. Analysis of the proliferative activity of cells using the molecular marker of cell proliferation of the Ki67 protein showed that cells irradiated at a dose of 50 Gy at a temperature of 22 °C, completely lost their ability to proliferate. The proliferative activity of cells irradiated at the same dose, but at a temperature of –196 °C, is significantly reduced, but some of the cells (3.5 ± 1.1 %) still retain the ability to proliferate. After irradiation with a dose of 500 Gy at –196 °C, the cells completely lose their ability to proliferate, but partially retain the ability to adhere. The integral fluorescence conjugated with the flurochrome of antibodies to γH2AX of MSC nuclei irradiated at a dose of 500 Gy at a temperature of –196 °C is 1.8 times lower than that of nuclei irradiated at a temperature of 22 °C. Conclusion: The results of the studies indicate that cryopreserved MSCs irradiated at liquid nitrogen temperature (–196 °C) in a preservation medium containing 10 % DMSO can tolerate the effects of ionizing radiation in large doses (up to 50 Gy). However, there is a rather high yield of “residual” DSB DNA and a very low proliferative activity, which makes them unsuitable in clinical practice. It seems promising to use a quantitative analysis of γH2AX foci to assess genome damage and the functional state of cells irradiated in a cryopreserved state.