Medical Radiology and Radiation Safety. 2018. Vol. 63. No. 1. P. 28-34

RADIATION BIOLOGY

DOI: 10.12737/article_5a855c9d5b1211.49546901

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

N.Yu. Vorobyeva, V.V. Uyba, O.A. Kochetkov, T.A. Astrelina, M.V. Pustovalova, A.K. Grekhova, T.M. Blokhina, E.I. Yashkina, D.I. Kabanov, V.A. Nikitina, Yu.B. Suchkova, I.V. Kobzeva, A.N. Osipov

A.I. Burnasyan Federal Medical Biophysical Center of Federal Medical Biological Agency, Moscow, Russia, e-mail: This email address is being protected from spambots. You need JavaScript enabled to view it.

N.Yu. Vorobyeva – Senior Researcher, PhD Biol.; V.V. Uyba – Head of the Federal Medical Biological Agency of Russia, Dr. Sc. Med., Prof.; O.A. Kochetkov – Head of Lab., PhD Tech.; T.A. Astrelina – Head of Center for Biomedical Technologies, Dr. Sc. Med.; M.V. Pustovalova – Research Fellow; A.K. Grekhova – Junior Researcher; T.M. Blokhina – Research Fellow; E.I. Yashkina – Junior Researcher; D.I. Kabanov – Engineer; V.A. Nikitina – Leading Researcher, PhD Med.; Yu.B. Suchkova – Senior Researcher, PhD Med.; I.V. Kobzeva – Senior Researcher, PhD Med.; A.N. Osipov – Head of Dep., Dr. Biol. Sc., Prof.

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 – phosphorylated histone (γH2AX) foci 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.

Key words: ³H-thymidine, DNA double strand breaks, γH2AX foci, mesenchymal stem cells

REFERENCES

1. Kotzer T, Trivedi A. Dosimetric implications of atmospheric dispersal of tritium near a heavy-water research reactor facility. Radiat. Prot. Dosim. 2001;93:61-6.
2. Okada S, Momoshima N. Overview of tritium. characteristics, sources, and problems. Health Phys. 1993;65:595-609.
3. Alloni D, Cutaia C, Mariotti L, et al. Modeling dose deposition and DNA damage due to low-energy beta(-) emitters. Radiat. Res. 2014;182:322-30.
4. Melintescu A, Galeriu D. Uncertainty of current understanding regarding OBT formation in plants. J. Environ. Radioact. 2017;167:134-49.
5. Dingwall S, Mills CE, Phan N, et al. Human health and the biological effects of tritium in drinking water. Prudent policy through science  addressing the ODWAC new recommendation. Dose Response. 2011;9:6-31.
6. Umata T. Estimation of biological effects of tritium. J. UOEH. 2017;39:25-33.
7. Little MP, Lambert BE. Systematic review of experimental studies on the relative biological effectiveness of tritium. Radiat. Environ. Biophys. 2008;47:71-93.
8. Task Group on Radiation Quality Effects in Radiological Protection CoREICoRP. Relative biological effectiveness (RBE), quality factor (Q), and radiation weighting factor (w(R)). A report of the International Commission on Radiological Protection. Ann ICRP. 2003;33:1-117.
9. Harrison J. Doses and risks from tritiated water and environmental organically bound tritium. J. Radiol. Prot. 2009;29:335-49.
10. Balonov MI, Muksinova KN, Mushkacheva GS. Tritium radiobiological effects in mammals. review of experiments of the last decade in Russia. Health Phys. 1993;65:713-26.
11. Balonov MI, Chipiga LA. [Estimation of the dose from the intake of tritium oxide in the human body. The role of inclusion of tritium in the organic matter of tissues]. Radiats. Gigiena [Radiation Hygiene]. 2016;9(4):16-25. DOI: 10.21514/1998-426X-2016-9-4-16-25. Russian.
12. Kochetkov OA, Monastyrskaya SG, Kabanov DI. [Problems of rationing technogenic tritium]. Saratov Journal of Medical Scientific Research. 2013;9(4):815-19. (English abstracts. Available from: https://cyberleninka.ru/article/n/problemy-normirovaniya-tehnogennogo-tritiya). Russian.
13. Korzeneva IB, Kostuyk SV, Ershova LS, et al. Human circulating plasma DNA significantly decreases while lymphocyte DNA damage increases under chronic occupational exposure to low-dose gamma-neutron and tritium β-radiation. Mutation Res./Fundamental and Molecular Mechanisms of Mutagenesis. 2015;779:1-15.
14. Osipov AN, Grekhova A, Pustovalova M, et al. Activation of homologous recombination DNA repair in human skin fibroblasts continuously exposed to X-ray radiation. Oncotarget. 2015;6:26876-85.
15. Osipov AN, Pustovalova M, Grekhova 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;6:27275-87.
16. Kotenko KV, Bushmanov AY, Ozerov IV, et al. Changes in the number of double-strand DNA breaks in Chinese hamster V79 cells exposed to gamma-radiation with different dose rates. Int. J. Mol. Sci. 2013;14:13719-26.
17. Halazonetis TD, Gorgoulis VG, Bartek J. An oncogene-induced DNA damage model for cancer development. Science. 2008;319:1352-55.
18. Chen J. Estimated yield of double-strand breaks from internal exposure to tritium. Radiat. Environ. Biophys. 2012;51:295-302.
19. Moiseenko VV, Waker AJ, Hamm RN, Prestwich WV. Calculation of radiation-induced DNA damage from photons and tritium beta-particles. Part II. Tritium RBE and damage complexity. Radiat. Environ. Biophys. 2001;40:33-8.
20. Vignard J, Mirey G, Salles B. Ionizing-radiation induced DNA double-strand breaks. A direct and indirect lighting up. Radiother. Oncol. 2013;108(3):362-9.
21. Sharma A, Singh K, Almasan A. Histone H2AX phosphorylation. a marker for DNA damage. Methods Mol. Biol. 2012;920:613-26.
22. Tsvetkova A, Ozerov IV, Pustovalova M, et al. γH2AX, 53BP1 and Rad51 protein foci changes in mesenchymal stem cells during prolonged X-ray irradiation. Oncotarget. 2017;8(38):64317-29.
23. Grekhova AK, Eremin PS, Osipov AN, et al. [Delayed processes of γH2AX foci formation and degradation in human skin fibroblasts irradiated with x-rays in low doses]. Radiats. Biol. Radioekologia (‘Radiation biology. Radioecology’). 2015;55(4):395-401. (English abstracts. PubMed PMID: 26601539). Russian.
24. Pustovalova M, Grekhova A, Astrelina Ь et al. Accumulation of spontaneous γH2AX foci in long-term cultured mesenchymal stromal cells. Aging (Albany NY). 2016;8(12):3498-506.
25. Pustovalova M, Astrelina с, Grekhova A, et al. Residual γH2AX foci induced by low dose x-ray radiation in bone marrow mesenchymal stem cells do not cause accelerated senescence in the progeny of irradiated cells. Aging (Albany NY). 2017;9(11):2397-410.
26. Paull TT, Rogakou EP, Yamazaki V, et al. A critical role for histone H2AX in recruitment of repair factors to nuclear foci after DNA damage. Curr. Biol. 2000;10:886-95.
27. Lobrich M, Shibata A, Beucher A, et al. GammaH2AX foci analysis for monitoring DNA double-strand break repair. strengths, limitations and optimization. Cell Cycle. 2010;9:662-9.
28. Gonen RGU, Alfassi ZB, Priel E. Production of DNA double strand breaks in human cells due to acute exposure to tritiated water (HTO). Conference of the Nuclear Societies in Israel; 2014 Feb 11-13. Dead Sea (Israel). p. 69-73.
29. Saintigny Y, Roche S, Meynard D, Lopez BS. Homologous recombination is involved in the repair response of mammalian cells to low doses of tritium. Radiat. Res. 2008;170:172-83.
30. Ozerov IV, Osipov AN. [Kinetic model of repair of DNA double-strand breaks in primary human fibroblasts under the action of rare-ionizing radiation with different dose rates]. Kompiuternye issledovania i modelirovanie [Computer Studies and Modeling]. 2015;7(1):159-76. (English abstracts. Available from: http://crm-en.ics.org.ru/journal/article/2237/). Russian.
31. Ozerov IV, Eremin PS, Osipov AN, et al. [Features of changes in the number of foci of proteins γH2AX and Rad51 in human fibroblasts subjected to prolonged exposure to low-intensity X-rays]. Saratov Scientific-Medical Journal. 2014;10(4):739-43. (English abstracts. Available from: https://cyberleninka.ru/article/n/osobennosti-izmeneniya-chisla-fokusov-belkov-un2ah-i-rad51-v-fibroblastah-kozhi-cheloveka-podvergavshihsya-prolongirovannomu). Russian.
32. Ozerov IV, Bushmanov AYu, Anchishkina NA, et al. [Induction and repair of DNA double-strand breaks in V79 cells under long-term exposure to low-intensity γ-radiation]. Saratov Scientific-Medical Journal. 2013;9(4):787-91.(English abstracts. Available from: https://cyberleninka.ru/article/n/induktsiya-i-reparatsiya-dvunitevyh-razryvov-dnk-v-kletkah-linii-v79-pri-dlitelnom-vozdeystvii-nizkointensivnogo-u-izlucheniya). Russian.
33. Duque A, Rakic P. Different effects of BrdU and ³H-Thymidine incorporation into DNA on cell proliferation, position and fate. J. Neurosci. 2011;31(42):15205-17.
34. Hoy CA, Lewis ED, Schimke RT. Perturbation of DNA replication and cell cycle progression by commonly used [³H]thymidine labeling protocols. Mol. Cell Biol. 1990 Apr;10(4):1584-92.
35. Hu VW, Black GE, Torres-Duarte A, Abramson FP. ³H-thymidine is a defective tool with which to measure rates of DNA synthesis. FASEB J. 2002;16(11):1456-7.
36. Jurikova M, Danihel L, Polak S. Varga I. Ki67, pcna, and mcm proteins. Markers of proliferation in the diagnosis of breast cancer. Acta Histochemica 2016;118:544-52.

For citation: Vorobyeva NYu, Uyba VV, Kochetkov OA, Astrelina TA, Pustovalova MV, Grekhova AK, Blokhina TM, Yashkina EI, Kabanov DI, Nikitina VA, Suchkova YuB, Kobzeva IV, Osipov AN. ³H-Thymidine Influence on DNA Double Strand Breaks Induction in Cultured Human Mesenchymal Stem Cells. Medical Radiology and Radiation Safety. 2018;63(1):28-34. DOI: 10.12737/article_5a855c9d5b1211.49546901. Russian.

PDF (RUS) Full-text article (in Russian)