Medical Radiology and Radiation Safety. 2023. Vol. 68. № 3
DOI: 10.33266/1024-6177-2023-68-3-5-10
N.Yu. Vorobyeva1, 2, A.A. Osipov2, A.K. Chigasova3, M.V. Pustovalova1, 4,
D.I. Kabanov1, V.G. Barchukov1, O.A. Kochetkov1, A.N. Osipov1, 2
Comparative Study of Changes in the γh2ax and 53bp1 Foci Number in Human Mesenchymal Stromale Cells Incubated with 3H-thymidine or Tritiated Water
1 A.I. Burnazyan Federal Medical Biophysical Center, Moscow, Russia
2 N.N. Semenov Federal Research Center for Chemical Physics, Russian Academy of Sciences Moscow, Russia
3 Institute of Biochemical Physics of the Russian Academy of Sciences, Moscow, Russia
4 Moscow Institute of Physics and Technology, Dolgoprudny, Moscow region, Russia
Contact person: N.Yu. Vorobyeva, e-mail: This email address is being protected from spambots. You need JavaScript enabled to view it.
ABSTRACT
Purpose: Comparative study of changes in the number of foci of DNA (DSB) marker proteins (γH2AX and 53BP1) in human mesenchymal stromal cells (MSCs) incubated with 3H-thymidine or HTO for 24, 48, and 72 h.
Material and methods: We used the primary culture of human MSCs of passage 5–6, obtained from the collection of LLC “BioloT” (Russia). A sterile solution of 3H-thymidine or HTO with a specific activity of 100 to 400 MBq/l was added to the nutrient medium and incubated under standard conditions of a CO2 incubator for 24, 48, and 72 hours. To quantify γH2AX foci and the proportion of proliferating cells using antibodies to γH2AX, 53BP1 and Ki67 (a marker protein for cell proliferation), were used, 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: Incubation of MSCs with 3H-thymidine with a specific radioactivity of 100-400 MBq/l in the first 24 hours leads to a dose-dependent increase in the number of γH2AX and 53BP1 foci. With a further increase in the incubation time to 48 h and 72 h, a saturation effect is observed ‒ the number of foci reaches a plateau. A statistically significant increase in the number of γH2AX and 53BP1 foci in MSCs incubated with HTO was observed only in actively proliferating cells during the first 24 h of incubation in a medium with specific radioactivity of 300 and 400 MBq/l, after which, with a decrease in proliferative activity, it decreased to control values. Calculations made on the basis of the results of a quantitative analysis of γH2AX and 53BP1 foci after 24 h of incubation of MSCs with tritium compounds obtained in the course of the work show, that under the influence of 3H-thymidine ~ 6 times more DNA double-strand breaks are induced than under the influence of HTO.
Keywords: mesenchymal stromal cells, γH2AX, 53BP1, DNA double-strand breaks, cell proliferation, tritium, incubation
For citation: Vorobyeva NYu, Osipov AA, Chigasova AK, Pustovalova MV, Kabanov DI, Barchukov V., Kochetkov OA, Osipov AN. Comparative Study of Changes in the γh2ax and 53bp1 Foci Number in Human Mesenchymal Stromale Cells Incubated with 3h-thymidine or Tritiated Water. Medical Radiology and Radiation Safety. 2023;68(3):5–10. (In Russian). DOI: 10.33266/1024-6177-2023-68-3-5-10
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Conflict of interest. The authors declare no conflict of interest.
Financing. The work was carried out with the support of the RGNF (project No. 22-2400490).
Contribution. Article was prepared with equal participation of the authors.
Article received: 20.01.2022. Accepted for publication: 25.02.2023.