3H-Thymidine Influence on DNA Double Strand Breaks Induction in Cultured Human Mesenchymal Stem Cells

Abstract

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.