Medical Radiology and Radiation Safety. 2025. Vol. 70. № 3

DOI:10.33266/1024-6177-2025-70-3-11-15

S.A. Korneva1, A.K. Chigasova1, 2, 3, A.A. Osipov2, M.A. Ignatov1, 2,
N.Yu. Vorobyova1, 2, V.O. Saburov4, E.I. Kazakov4, S.N. Koryakin4,
Yu.A. Fedotov1, 2, A.Yu. Bushmanov1, A.N. Osipov1, 2

Post-Irradiation Changes in the Number of γH2ax and patm Protein Foci in Human Mesenchymal Stem Cells Irradiated with 14.1 MeV Neutrons

1 A.I. Burnazyan Federal Medical Biophysical Center, Moscow, Russia

2 N.N. Semenov Federal Research Center for Chemical Physics, Moscow, Russia 

3 Institute of Biochemical Physics, Moscow, Russia

4 A.F. Tsyb Medical Radiological Research Center, Obninsk, Russia

Contact person: A.N. Osipov, e-mail: Этот адрес электронной почты защищен от спам-ботов. У вас должен быть включен JavaScript для просмотра.

 

Abstract

Purpose: Comparative analysis of dose-response dependences and kinetics of post-radiation changes in the number of γH2AX and pATM protein foci in human mesenchymal stem cells (MSCs) exposed to 14.1 MeV neutrons and cobalt-60 gamma-radiation.

Material and methods: The study used a primary culture of human MSCs obtained from the collection of BioloT LLC (Russia). The cells were irradiated using a neutron generator NG-14 (VNIIA, Russia), which provided neutron fluxes with an energy of 14.1 MeV, and a gamma-therapeutic device ROKUS-AM (JSC Ravenstvo, Russia; cobalt-60, dose rate 0.5 Gy/min) at doses of 0.1, 0.25 and 0.5 Gy. For quantitative assessment of γH2AX and pATM foci, immunocytochemical staining was done using antibodies to γH2AX and pATM, respectively. Statistical significance was assessed using analysis of variance (ANOVA).

Results: It was shown that the kinetics of post-radiation changes in the number of γH2AX foci in cells irradiated with neutrons is slower than after gamma irradiation. 24 h after irradiation with neutrons, ~ 62 % of γH2AX foci and ~ 52 % of pATM foci were recorded from their number 0.5 h after irradiation. These values were statistically significantly (p < 0.001) higher than the proportions of residual foci calculated after exposure to gamma-radiation: ~ 16 % and 6 %, respectively. The results obtained indicate that the proportion of complex, difficult-to-repair DNA damage in cells irradiated with neutrons is significantly higher than with gamma-radiation.

Keywords: mesenchymal stem cells, fast neutrons, gamma-radiation, γH2AX, рATM, DNA damage, DNA repair

For citation: Korneva SA, Chigasova AK, Osipov AA, Ignatov MA, Vorobyova NYu, Saburov VO, Kazakov EI, Koryakin SN, Fedotov YuA, Bushmanov AYu, Osipov AN. Post-Irradiation Changes in the Number of γH2AX and patm Protein Foci in Human Mesenchymal Stem Cells Irradiated with 14.1 MeV Neutrons. Medical Radiology and Radiation Safety. 2025;70(3):11–15. (In Russian). DOI:10.33266/1024-6177-2025-70-3-11-15

 

References

1. Nickoloff J.A., Sharma N., Allen C.P., Taylor L., Allen S.J., Jaiswal A.S., et al. Roles of Homologous Recombination in Response to Ionizing Radiation-Induced DNA Damage. Int J Radiat Biol. 2023;99;6:903-14. doi: 10.1080/09553002.2021.1956001.

2. Mladenov E., Mladenova V., Stuschke M., Iliakis G. New Facets of DNA Double Strand Break Repair: Radiation Dose as Key Determinant of HR Versus c-NHEJ Engagement. International Journal of Molecular Sciences. 2023;24;19:49-56. doi: 10.3390/ijms241914956.

3. Belov O., Chigasova A., Pustovalova M., Osipov A., Eremin P., Vorobyeva N., et al. Dose-Dependent Shift in Relative Contribution of Homologous Recombination to DNA Repair after Low-LET Ionizing Radiation Exposure: Empirical Evidence and Numerical Simulation. Current Issues in Molecular Biology. 2023;45;9:7352-73. doi: 10.3390/cimb45090465.

4. Krenning L., van den Berg J., Medema R.H. Life or Death after a Break: what Determines the Choice? Mol Cell. 2019;76;2:346-58. doi: 10.1016/j.molcel.2019.08.023.

5. Torgovnick A., Schumacher B. DNA Repair Mechanisms in Cancer Development and Therapy. Front Genet. 2015;6:157. doi: 10.3389/fgene.2015.00157.

6. White R.R., Vijg J. Do DNA Double-Strand Breaks Drive Aging? Mol Cell. 2016;63;5:729-38. doi: 10.1016/j.molcel.2016.08.004.

7. Jiang Y. Contribution of Microhomology to Genome Instability: Connection between DNA Repair and Replication Stress. International Journal of Molecular Sciences. 2022;23;21:129-37. doi: 10.3390/ijms232112937.

8. Sishc B.J., Davis A.J. The Role of the Core Non-Homologous End Joining Factors in Carcinogenesis and Cancer. Cancers (Basel). 2017;9;7:81. doi: 10.3390/cancers9070081.

9. Osipov A., Chigasova A., Yashkina E., Ignatov M., Vorobyeva N., Zyuzikov N., et al. Early and Late Effects of Low-Dose X-ray Exposure in Human Fibroblasts: DNA Repair Foci, Proliferation, Autophagy, and Senescence. International Journal of Molecular Sciences. 2024;25;15:8253. doi: 10.3390/ijms25158253.

10. Barbieri S., Babini G., Morini J., Friedland W., Buonanno M., Grilj V., et al. Predicting DNA Damage Foci and their Experimental Readout with 2D Microscopy: a Unified Approach Applied to Photon and Neutron Exposures. Sci Rep. 2019;9;1:14019. doi: 10.1038/s41598-019-50408-5.

11. Rothkamm K., Barnard S., Moquet J., Ellender M., Rana Z., Burdak-Rothkamm S. DNA Damage Foci: Meaning and Significance. Environ Mol Mutagen. 2015;56;6:491-504. doi: 10.1002/em.21944.

12. Penninckx S., Pariset E., Cekanaviciute E., Costes S.V. Quantification of Radiation-Induced DNA Double Strand Break Repair Foci to Evaluate and Predict Biological Responses to Ionizing Radiation. NAR Cancer. 2021;3;4:zcab046. doi: 10.1093/narcan/zcab046.

13. Belyaev I.Y. Radiation-Induced DNA Repair Foci: Spatio-Temporal Aspects of Formation, Application for Assessment of Radiosensitivity and Biological Dosimetry. Mutat Res. 2010;704;1-3:132-41. doi: 10.1016/j.mrrev.2010.01.011.

14. Wanotayan R., Chousangsuntorn K., Petisiwaveth P., Anuttra T., Lertchanyaphan W., Jaikuna T., et al. A Deep Learning Model (FociRad) for Automated Detection of Gamma-H2AX Foci and Radiation Dose Estimation. Sci Rep. 2022;12;1:5527. doi: 10.1038/s41598-022-09180-2.

15. Vorobyeva N.Y., Osipov A.A., Chigasova A.K., Yashkina E.I., Osipov A.N. Changes in the Number of Residual γH2AX Foci in Ki-67-Positive and Ki-67-Negative Human Fibroblasts Irradiated with X-Rays in Doses of 2-10 Gy. Bulletin of Experimental Biology and Medicine. 2023;175;4:450-3. doi: 10.1007/s10517-023-05883-2.

16. Vorobyeva N.Y., Astrelina T.A., Yashkina E.I., Chigasova A.K., Osipov A.A., Usupzhanova D.Y., et al. Effect of a Humic-Fulvic Acid Preparation on the Quantitative Yield of Residual γH2AX Foci and Proliferative Activity in Irradiated Human Mesenchymal Stromal Cells. Medical Radiology and Radiation Safety. 2023;68;2:11-5. doi: 10.33266/1024-6177-2023-68-2-11-15.

17. Falaschi A., Chiaramonte A., Testi S., Scarpato R. Dual Immunofluorescence of gammaH2AX and 53BP1 in Human Peripheral Lymphocytes. J Vis Exp. 2023;197:654-72. doi: 10.3791/65472.

18. Vorobyeva N.Y., Osipov A.A., Chigasova A.K., Pustovalova M.V., Kabanov D.I., Barchukov V.G., et al. 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. doi: 10.33266/1024-6177-2023-68-3-5-10.

19. Slonina D., Kowalczyk A., Janecka-Widla A., Kabat D., Szatkowski W., Biesaga B. Low-Dose Hypersensitive Response for Residual pATM and gammaH2AX Foci in Normal Fibroblasts of Cancer Patients. Int J Radiat Oncol Biol Phys. 2018;100;3:756-66. doi: 10.1016/j.ijrobp.2017.10.054.

20. Chigasova A.K., Pustovalova M.V., Osipov A.A., Korneva S.A., Eremin P.S., Yashkina E.I., et al. Post-Radiation Changes in The Number of Phosphorylated H2ax and Atm Protein Foci in Low Dose X-Ray Irradiated Human Mesenchymal Stem Cells. Medical Radiology and Radiation Safety. 2024;69;1:15-9. doi: 10.33266/1024-6177-2024-69-1-15-19.

21. Valente D., Gentileschi M.P., Guerrisi A., Bruzzaniti V., Morrone A., Soddu S., et al. Factors to Consider for the Correct Use of gammaH2AX in the Evaluation of DNA Double-Strand Breaks Damage Caused by Ionizing Radiation. Cancers (Basel). 2022;14;24:6204. doi: 10.3390/cancers14246204.

22. Shibata A., Jeggo P.A. ATM’s Role in the Repair of DNA Double-Strand Breaks. Genes (Basel). 2021;12;9:1370. doi: 10.3390/genes12091370.

23. Osipov A.N., Pustovalova M., Grekhova A., Eremin P., Vorobyova N., Pulin A., et al. Low Doses of X-Rays Induce Prolonged and ATM-Independent Persistence of GammaH2AX Foci in Human Gingival Mesenchymal Stem Cells. Oncotarget. 2015;6;29:27275-87. doi: 10.18632/oncotarget.4739.

24. Ozerov I.V., Osipov A.N. Kinetic Model of DNA Double-Strand Break Repair in Primary Human Fibroblasts Exposed to Low-LET Irradiation with Various Dose Rates. Computer Research and Modeling. 2015;7;1:159-76. doi: 10.20537/2076-7633-2015-7-1-159-176.

 

 

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Conflict of interest. The authors declare no conflict of interest.

Financing. The research was carried out within the framework of the state assignment of the Ministry of Science and Higher Education of the Russian Federation (No. 1023112000035-8, code “Cosmos-DNA”).

Contribution. Article was prepared with equal participation of the authors.

Article received: 20.02.2025. Accepted for publication: 25.03.2025.