Purpose: To evaluate the influence of a humic-fulvic acid substance on the quantitative yield of residual foci of the DNA double-strand break (DSB) repair protein-marker - phosphorylated histone H2AX (γH2AX) and proliferation activity in a culture of human mesenchymal stromal cells (MSCs) 24, 48, and 72 h after exposure to X-ray radiation at doses of 2, 4 and 10 Gy. Material and methods: Through 24 hours after incubation of MSCs with a substance of humic-fulvic acids (Humic Complex, OOO Sistema-BioTechnologies, Russia) at a dilution of 1/1000. Cells were irradiated on an X-ray biological device RUB RUST-M1 at a voltage of 200 kV, beam current 2×5 mA, aluminum filter 1.5 mm, absorbed dose rate 0.85 Gy/min. Immunocytochemical staining was used to quantify the residual γH2AX foci and the percentage of proliferating cells using antibodies to γH2AX and Ki-67 (a marker protein for cell proliferation), respectively. Statistical analysis of the obtained data was carried out using the statistical software package Statistica 8.0 (StatSoft). To assess the significance of differences between samples, Student’s t-test was used. Results and conclusion: The conducted studies showed that on the cell model used and under the above experimental conditions, the humic-fulvic acid substance does not affect the efficiency of repair of radiation-induced DNA DSBs, however, it significantly reduces the proliferation activity of both irradiated and non-irradiated MSCs. It is advisable to conduct detailed studies of the molecular and cellular mechanisms of the antiproliferative effect of humic and fulvic acids.

Purpose: The evaluation of the repair efficiency of DNA double-strand breaks (DSB), proliferative activity and the yield of cytogenetic disorders in human tumor HeLa cells which survived and gave stable growth after acute irradiation at a dose of 15 Gy. Material and methods: HeLa human tumor cell line (cervical carcinoma) was used. Cells were irradiated on an X-ray biological installation RUST-M1 (Russia), equipped with two X-ray emitters, at a dose rate of 0.85 Gy / min, a voltage of 200 kV, a total current of 10 mA, and a 1.5 mm Al filter. To obtain clones of surviving cells (HeLaRR), after acute irradiation at a dose of 15 Gy, cell cultures were incubated under standard CO2 incubator conditions (37 °C, 5 % CO2) for several weeks until well proliferating cells were obtained. Immunocytochemical staining of the foci of the phosphorylated H2AX protein (γH2AX) was used to quantitatively evaluate the residual foci of DNA DSB repair. The micronuclei number was assessed in cytochalasin-B cytokinesis-blocked binucleated cells stained with acridine orange with luminescence microscopy. The doubling time of the cell population was analyzed by the cell growth curves obtained by daily cell counting for five days. The cell cycle stages distribution was assessed by flow cytometry using the propidium iodide dye. All quantitative indicators of the studies were processed using the Student’s t-test for independent samples and the Kolmogorov – Smirnov test. Results: It was revealed that acute irradiation at a high dose leads to the selection of cells with a higher reparative capacity which is confirmed by the low yield of residual foci of DNA DSB repair and MN after testing irradiation at doses of 5 and 10 Gy. A significant decrease in the proliferative activity of cells that survived after acute X-ray irradiation at a dose of 15 Gy was revealed. The doubling time of the population of unirradiated cells at the stage of exponential growth was ~18 hours while for cells that survived after irradiation at a dose of 15 Gy ~42 hours. A change in the cell cycle phases distribution was observed. Conclusion: Thus, acute irradiation at a high dose leads to the selection of cells with a higher reparative capacity which is confirmed by the low yield of residual γH2AX foci and MN after testing irradiation at doses of 5 and 10 Gy. The decrease in proliferative activity was accompanied by a change in the cell cycle phases distribution.