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Determination of dose-response calibration curves for gamma radiation using gamma-H2AX immunofluorescence based biodosimetry

Solomon Raj Jose1, Peace Balasingh Timothy2, Suganthy J3, Selvamani Backianathan2, Soosai Manickam Amirtham4, Sandya Rani5, Rabi Singh2

Abstract

Background: Gamma-H2AX immunofluorescence assay has gained popularity as a DNA double strand break marker. In this work, we have investigated the potential use of gamma H2AX immunofluorescence assay as a biological dosimeter for estimation of dose in our institution.

Materials and methods: Seven healthy individuals were selected for the study and the blood samples collected from the first five individuals were irradiated to low doses (0–10 cGy) and high doses (50–500 cGy) in a telecobalt unit. All the samples were processed for gamma-H2AX immunofluorescence assay and the dose-response calibration curves for low and high doses were determined. In order to validate the determined dose-response calibration curves, the blood samples obtained from the sixth and seventh subjects were delivered a test dose of 7.5 cGy and 250 cGy. In addition, time and cost required to complete the assay were also reported.

Results: The goodness of fit (R2) values was found to be 0.9829 and 0.9766 for low and high dose-response calibration curves. The time required to perform the gamma-H2AX immunofluorescence assay was found to be 7 hours and 30 minutes and the estimated cost per sample was 5000 rupees (~ 60 USD).

Conclusion: Based on this study we conclude that the individual dose-response calibration curves determined with gamma-H2AX immunofluorescence assay for both low and high dose ranges of gamma radiation can be used for biological dosimetry. Further, the gamma-H2AX immunofluorescence assay can be used as a rapid cost-effective biodosimetric tool for institutions with an existing confocal microscope facility.

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References

  1. Sorokine-Durm I, Durand V, Le Roy A, et al. Is FISH painting an appropriate biological marker for dose estimates of suspected accidental radiation overexposure? A review of cases investigated in France from 1995 to 1996. Environ Health Perspect. 1997; 105 Suppl 6(Suppl 6): 1427–1432.
  2. International Atomic Energy Agency. Cytogenetic Dosimetry: Applications in Preparedness for and Response to Radiation Emergencies, Emergency Preparedness and Response . IAEA, Vienna 2011: 247.
  3. Macià I Garau M, Lucas Calduch A, López EC. Radiobiology of the acute radiation syndrome. Rep Pract Oncol Radiother. 2011; 16(4): 123–130.
  4. Boice JD. The linear nonthreshold (LNT) model as used in radiation protection: an NCRP update. Int J Radiat Biol. 2017; 93(10): 1079–1092.
  5. Gnanasekaran TS. Cytogenetic biological dosimetry assays: recent developments and updates. Radiat Oncol J. 2021; 39(3): 159–166.
  6. Ryan TL, Pantelias AG, Terzoudi GI, et al. Use of human lymphocyte G0 PCCs to detect intra- and inter-chromosomal aberrations for early radiation biodosimetry and retrospective assessment of radiation-induced effects. PLoS One. 2019; 14(5): e0216081.
  7. Eberlein U, Peper M, Fernández M, et al. Calibration of the γ-H2AX DNA double strand break focus assay for internal radiation exposure of blood lymphocytes. PLoS One. 2015; 10(4): e0123174.
  8. Turner HC, Brenner DJ, Chen Y, et al. Adapting the γ-H2AX assay for automated processing in human lymphocytes. 1. Technological aspects. Radiat Res. 2011; 175(3): 282–290.
  9. Moquet J, Barnard S, Rothkamm K. Gamma-H2AX biodosimetry for use in large scale radiation incidents: comparison of a rapid '96 well lyse/fix' protocol with a routine method. PeerJ. 2014; 2: e282.
  10. Wanotayan R, Wongsanit S, Boonsirichai K, et al. Quantification of histone H2AX phosphorylation in white blood cells induced by ex vivo gamma irradiation of whole blood by both flow cytometry and foci counting as a dose estimation in rapid triage. PLoS One. 2022; 17(3): e0265643.
  11. Chang DS, Lasley FD, Das IJ, Mendonca MS, Dynlacht JR. Basic Radiotherapy Physics and Biology [Internet]. Cham: Springer International Publishing; 2021. http://link.springer.com/10.1007/978-3-030-61899-5 (18.01.2023).
  12. Vignard J, Mirey G, Salles B. Ionizing-radiation induced DNA double-strand breaks: a direct and indirect lighting up. Radiother Oncol. 2013; 108(3): 362–369.
  13. Arya G, Schlick T. A tale of tails: how histone tails mediate chromatin compaction in different salt and linker histone environments. J Phys Chem A. 2009; 113(16): 4045–4059.
  14. Foster ER, Downs JA. Histone H2A phosphorylation in DNA double-strand break repair. FEBS J. 2005; 272(13): 3231–3240.
  15. Johansson P, Muslimovic A, Hultborn R, et al. In-solution staining and arraying method for the immunofluorescence detection of γH2AX foci optimized for clinical applications. Biotechniques. 2011; 51(3): 185–189.
  16. Kuo LJ, Yang LX. Gamma-H2AX - a novel biomarker for DNA double-strand breaks. In Vivo. 2008; 22(3): 305–309.
  17. Redon CE, Nakamura AJ, Sordet O, et al. γ-H2AX detection in peripheral blood lymphocytes, splenocytes, bone marrow, xenografts, and skin. Methods Mol Biol. 2011; 682: 249–270.
  18. Chaurasia RK, Bhat NN, Gaur N, et al. Establishment and multiparametric-cytogenetic validation of Co-gamma-ray induced, phospho-gamma-H2AX calibration curve for rapid biodosimetry and triage management during radiological emergencies. Mutat Res Genet Toxicol Environ Mutagen. 2021; 866: 503354.
  19. Ricoul M, Gnana Sekaran TS, Brochard P, et al. γ-H2AX Foci Persistence at Chromosome Break Suggests Slow and Faithful Repair Phases Restoring Chromosome Integrity. Cancers (Basel). 2019; 11(9).
  20. Schindelin J, Arganda-Carreras I, Frise E, et al. Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012; 9(7): 676–682.
  21. Visweswaran S, Joseph S, Dhanasekaran J, et al. Exposure of patients to low doses of X-radiation during neuro-interventional imaging and procedures: Dose estimation and analysis of γ-H2AX foci and gene expression in blood lymphocytes. Mutat Res Genet Toxicol Environ Mutagen. 2020; 856-857: 503237.
  22. Parris CN, Adam Zahir S, Al-Ali H, et al. Enhanced γ-H2AX DNA damage foci detection using multimagnification and extended depth of field in imaging flow cytometry. Cytometry A. 2015; 87(8): 717–723.
  23. Moquet J, Barnard S, Staynova A, et al. The second gamma-H2AX assay inter-comparison exercise carried out in the framework of the European biodosimetry network (RENEB). Int J Radiat Biol. 2017; 93(1): 58–64.
  24. Grudzenski S, Raths A, Conrad S, et al. Inducible response required for repair of low-dose radiation damage in human fibroblasts. Proc Natl Acad Sci U S A. 2010; 107(32): 14205–14210.
  25. Abu Shqair A, Lee US, Kim EH. Computational modelling of γ-H2AX foci formation in human cells induced by alpha particle exposure. Sci Rep. 2022; 12(1): 14360.
  26. Redon CE, Nakamura AJ, Gouliaeva K, et al. The use of gamma-H2AX as a biodosimeter for total-body radiation exposure in non-human primates. PLoS One. 2010; 5(11): e15544.



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