Top

EJNMMI Research

Published in:

Open Access 01-12-2020 | Original research

Quantitative γ-H2AX immunofluorescence method for DNA double-strand break analysis in testis and liver after intravenous administration of ¹¹¹InCl₃

Authors: Anna Stenvall, Erik Larsson, Bo Holmqvist, Sven-Erik Strand, Bo-Anders Jönsson

Published in: EJNMMI Research | Issue 1/2020

Abstract

Background

It is well known that a severe cell injury after exposure to ionizing radiation is the induction of DNA double-strand breaks (DSBs). After exposure, an early response to DSBs is the phosphorylation of the histone H2AX molecule regions adjacent to the DSBs, referred to as γ-H2AX foci. The γ-H2AX assay after external exposure is a good tool for investigating the link between the absorbed dose and biological effect. However, less is known about DNA DSBs and γ-H2AX foci within the tissue microarchitecture after internal irradiation from radiopharmaceuticals. Therefore, in this study, we aimed to develop and validate a quantitative ex vivo model using γ-H2AX immunofluorescence staining and confocal laser scanning microscopy (CLSM) to investigate its applicability in nuclear medicine dosimetry research. Liver and testis were selected as the organs to study after intravenous administration of ¹¹¹InCl₃.

Results

In this study, we developed and validated a method that combines ex vivo γ-H2AX foci labeling of tissue sections with in vivo systemically irradiated mouse testis and liver tissues. The method includes CLSM imaging for intracellular cell-specific γ-H2AX foci detection and quantification and absorbed dose calculations. After exposure to ionizing radiation from ¹¹¹InCl₃, both hepatocytes and non-hepatocytes within the liver showed an absorbed dose-dependent elevation of γ-H2AX foci, whereas no such correlation was seen for the testis tissue.

Conclusion

It is possible to detect and quantify the radiation-induced γ-H2AX foci within the tissues of organs at risk after internal irradiation. We conclude that our method developed is an appropriate tool to study dose–response relationships in animal organs and human tissue biopsies after internal exposure to radiation.

Bonner WM, Redon CE, Dickey JS, Nakamura AJ, Sedelnikova OA, Solier S, et al. GammaH2AX and cancer. Nat Rev Cancer. 2008;8:957–67. https://doi.org/10.1038/nrc2523.CrossRefPubMedPubMedCentral

Rogakou EP, Pilch DR, Orr AH, Ivanova VS, Bonner WM. DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139. J Biol Chem. 1998;273:5858–68.CrossRef

Hamer G, Roepers-Gajadien HL, van Duyn-Goedhart A, Gademan IS, Kal HB, van Buul PP, et al. DNA double-strand breaks and gamma-H2AX signaling in the testis. Biol Reprod. 2003;68:628–34.CrossRef

Vasireddy RS, Tang MM, Mah LJ, Georgiadis GT, El-Osta A, Karagiannis TC. Evaluation of the spatial distribution of gammaH2AX following ionizing radiation. J Vis Exp. 2010. https://doi.org/10.3791/2203.

Löbrich M, Shibata A, Beucher A, Fisher A, Ensminger M, Goodarzi AA, et al. gammaH2AX foci analysis for monitoring DNA double-strand break repair: strengths, limitations and optimization. Cell Cycle. 2010;9:662–9. https://doi.org/10.4161/cc.9.4.10764.CrossRefPubMed

Kühne M, Riballo E, Rief N, Rothkamm K, Jeggo PA, Löbrich M. A double-strand break repair defect in ATM-deficient cells contributes to radiosensitivity. Cancer Res. 2004;64:500–8.CrossRef

Mariotti LG, Pirovano G, Savage KI, Ghita M, Ottolenghi A, Prise KM, et al. Use of the gamma-H2AX assay to investigate DNA repair dynamics following multiple radiation exposures. PLoS One. 2013;8:e79541. https://doi.org/10.1371/journal.pone.0079541.CrossRefPubMedPubMedCentral

Qvarnström OF, Simonsson M, Johansson KA, Nyman J, Turesson I. DNA double strand break quantification in skin biopsies. Radiother Oncol. 2004;72:311–7. https://doi.org/10.1016/j.radonc.2004.07.009.CrossRefPubMed

Redon CE, Nakamura AJ, Sordet O, Dickey JS, Gouliaeva K, Tabb B, et al. Gamma-H2AX detection in peripheral blood lymphocytes, splenocytes, bone marrow, xenografts, and skin. DNA damage detection in situ, ex vivo, and in vivo: methods and protocols. 2011;682:249–70. https://doi.org/10.1007/978-1-60327-409-8_18.

10.

Ivashkevich AN, Martin OA, Smith AJ, Redon CE, Bonner WM, Martin RF, et al. Gamma H2AX foci as a measure of DNA damage: a computational approach to automatic analysis. Mutat Res Fundam Mol Mech Mutag. 2011;711:49–60. https://doi.org/10.1016/j.mrfmmm.2010.12.015.CrossRef

11.

Firsanov D, Vasilishina A, Kropotov A, Mikhailov V. Dynamics of gammaH2AX formation and elimination in mammalian cells after X-irradiation. Biochimie. 2012;94:2416–22. https://doi.org/10.1016/j.biochi.2012.06.019.CrossRefPubMed

12.

Lassmann M, Hanscheid H, Gassen D, Biko J, Meineke V, Reiners C, et al. In vivo formation of gamma-H2AX and 53BP1 DNA repair foci in blood cells after radioiodine therapy of differentiated thyroid cancer. J Nucl Med. 2010;51:1318–25. https://doi.org/10.2967/jnumed.109.071357.CrossRefPubMed

13.

Eberlein U, Nowak C, Bluemel C, Buck AK, Werner RA, Scherthan H, et al. DNA damage in blood lymphocytes in patients after (177)Lu peptide receptor radionuclide therapy. Eur J Nucl Med Mol Imaging. 2015;42:1739–49. https://doi.org/10.1007/s00259-015-3083-9.CrossRefPubMedPubMedCentral

14.

Kristiansson A, Ahlstedt J, Holmqvist B, Brinte A, Tran TA, Forssell-Aronsson E, et al. Protection of kidney function with human antioxidation protein alpha(1)-microglobulin in a mouse Lu-177-DOTATATE radiation therapy model. Antioxis Redox Sign. 2019;30:1746–59. https://doi.org/10.1089/ars.2018.7517.CrossRef

15.

ICRP. 2012 ICRP Statement on Tissue Reactions / Early and Late Effects of Radiation in Normal Tissues and Organs – Threshold Doses for Tissue Reactions in a Radiation Protection Context. ICRP Publication 118. Ann. ICRP 41(1/2).

16.

Jönsson B-A, Strand S-E, Emanuelsson H, Larsson BS. Tissue, cellular, and subcellular distribution of indium radionuclides in the rat. Biophysical aspects of Auger processes, AAPM Symposium Series No 8. Woodbury: American Institute of Physics; 1992. p. 249–72.

17.

Jönsson BA, Strand SE, Larsson BS. A quantitative autoradiographic study of the heterogeneous activity distribution of different indium-111-labeled radiopharmaceuticals in rat tissues. J Nucl Med. 1992;33:1825–33.PubMed

18.

Grafström G, Jönsson BA, El Hassan AM, Tennvall J, Strand SE. Rat testis as a radiobiological in vivo model for radionuclides. Radiat Prot Dosimetry. 2006;118:32–42. https://doi.org/10.1093/rpd/nci328.CrossRefPubMed

19.

Nettleton JS, Lawson RS, Prescott MC, Morris ID. Uptake, localization, and dosimetry of In-111 and Tl-201 in human testes. J Nucl Med. 2004;45:138–46.PubMed

20.

Hoyes KP, Nettleton JS, Lawson RS, Morris ID. Transferrin-dependent uptake and dosimetry of Auger-emitting diagnostic radionuclides in human spermatozoa. J Nucl Med. 1998;39:895–9.PubMed

21.

Reilly RM, Sandhu J, Alvarez-Diez TM, Gallinger S, Kirsh J, Stern H. Problems of delivery of monoclonal antibodies. Pharmaceutical and pharmacokinetic solutions. Clin Pharmacokinet. 1995;28:126–42. https://doi.org/10.2165/00003088-199528020-00004.CrossRefPubMed

22.

Sun HZ, Li HY, Sadler PJ. Transferrin as a metal ion mediator. Chem Rev. 1999;99:2817–42. https://doi.org/10.1021/Cr980430w.CrossRefPubMed

23.

Oakberg EF. A description of spermiogenesis in the mouse and its use in analysis of the cycle of the seminiferous epithelium and germ cell renewal. Am J Anat. 1956;99:391–413. https://doi.org/10.1002/aja.1000990303.CrossRefPubMed

24.

Meistrich ML, Samuels RC. Reduction in sperm levels after testicular irradiation of the mouse: a comparison with man. Radiat Res. 1985;102:138–47.CrossRef

25.

Rowley MJ, Leach DR, Warner GA, Heller CG. Effect of graded doses of ionizing radiation on the human testis. Radiat Res. 1974;59:665–78.CrossRef

26.

ICRP. Nonstochastic effects of ionizing radiation. ICRP Publication 41. Ann ICRP. 1984;14.

27.

Jackson C, Jackson H, Bock M, Morris ID, Sharma HL. Metal radionuclides and the testis. Int J Rad Biol. 1991;60:851–8.CrossRef

28.

Oakberg EF. Duration of spermatogenesis in the mouse and timing of stages of the cycle of the seminiferous epithelium. Am J Anat. 1956;99:507–16. https://doi.org/10.1002/aja.1000990307.CrossRefPubMed

29.

Meistrich ML. Critical components of testicular function and sensitivity to disruption. Biol Reprod. 1986;34:17–28. https://doi.org/10.1095/biolreprod34.1.17.CrossRefPubMed

30.

Rao DV, Sastry KSR, Govelitz G, Grimmond H, Hill H. Radiobiological effects of Auger electron emitters in vivo: spermatogenesis in mice as an experimental model. Radiation Protection Dosimetry. 1988;13:245–8.CrossRef

31.

Rao DV, Narra V, Howell RW, Lanka V, Sastry KSR. Induction of sperm head abnormalities by incorporated radionuclides: dependance on subcellular distribution, type of radiation, dose rate, and presence of radioprotectors. Radiat Res. 1991;125:89–97.CrossRef

32.

Rao DV, Govelitz G, Sastry KSR. Radiotoxicity of thallium-201 in mouse testes: inadequacy of conventional dosimetry. J Nuclear Med. 1983;24:145–53.

33.

Rao DV, Sastry KS, Grimmond HE, Howell RW, Govelitz GF, Lanka VK, et al. Cytotoxicity of some indium radiopharmaceuticals in mouse testes. J Nucl Med. 1988;29:375–84.PubMed

34.

Hacker U, Schumann J, Göhde W. Mammalian spermatogenesis as a new system for biologic dosimetry of ionizing irradiation. Acta Radiologica Oncologica. 1982;21:349–51.CrossRef

35.

Inagaki A, Schoenmakers S, Baarends WM. DNA double strand break repair, chromosome synapsis and transcriptional silencing in meiosis. Epigenetics. 2010;5:255–66.CrossRef

36.

Blanco-Rodriguez J. gammaH2AX marks the main events of the spermatogenic process. Microsc Res Tech. 2009;72:823–32. https://doi.org/10.1002/jemt.20730.CrossRefPubMed

37.

Emami B, Lyman J, Brown A, Coia L, Goitein M, Munzenrider JE, et al. Tolerance of normal tissue to therapeutic irradiation. Int J Radiat Oncol Biol Phys. 1991;21:109–22.CrossRef

38.

Pan CC, Kavanagh BD, Dawson LA, Li XA, Das SK, Miften M, et al. Radiation-associated liver injury. Int J Radiat Oncol Biol Phys. 2010;76:S94–100. https://doi.org/10.1016/j.ijrobp.2009.06.092.CrossRefPubMedPubMedCentral

39.

Authors on behalf of I, Stewart FA, Akleyev AV, Hauer-Jensen M, Hendry JH, Kleiman NJ, et al. ICRP publication 118: ICRP statement on tissue reactions and early and late effects of radiation in normal tissues and organs--threshold doses for tissue reactions in a radiation protection context. Ann ICRP. 2012;41:1–322. https://doi.org/10.1016/j.icrp.2012.02.001.CrossRef

40.

ICRU. International Commission on Radiation and Units. Absorbed-dose specification in nuclear medicine (Report 67). J ICRU. 2002;2:1–110.

41.

Larsson E, Meerkhan SA, Strand S-E, Jönsson B-A. A small-scale anatomic model for testicular radiation dosimetry for radionuclides localized in the human testes. J Nuclear Med. 2012;53:72–81. https://doi.org/10.2967/jnumed.111.095133.CrossRef

42.

Stenvall A, Larsson E, Strand SE, Jönsson BA. A small-scale anatomical dosimetry model of the liver. Phys Med Biol. 2014;59:3353–71. https://doi.org/10.1088/0031-9155/59/13/3353.CrossRefPubMed

43.

Meerkhan SA, Sjögreen-Gleisner K, Larsson E, Strand SE, Jönsson BA. Testis dosimetry in individual patients by combining a small-scale dosimetry model and pharmacokinetic modeling-application of In-111-Ibritumomab Tiuxetan (Zevalin (R)). Phys Med Biol. 2014;59:7889–904. https://doi.org/10.1088/0031-9155/59/24/7889.CrossRefPubMed

44.

Larsson E, Strand SE, Ljungberg M, Jönsson BA. Mouse S-factors based on Monte Carlo simulations in the anatomical realistic Moby phantom for internal dosimetry. Cancer Biother Radiopharm. 2007;22:438–42. https://doi.org/10.1089/cbr.2006.320.CrossRefPubMed

45.

Segars WP, Tsui BMW, Frey EC, Johnson GA, Berr SS. Development of a 4-D digital mouse phantom for molecular imaging research. Mol Imag Biol. 2004;6:149–59. https://doi.org/10.1016/j.mibio.2004.03.002.CrossRef

46.

Rasband WS. ImageJ. U.S. National Institutes of Health, Bethesda, Maryland, USA, http://imagej.nih.gov/ij/, 1997-2014.

47.

Du G, Drexler GA, Friedland W, Greubel C, Hable V, Krucken R, et al. Spatial dynamics of DNA damage response protein foci along the ion trajectory of high-LET particles. Radiat Res. 2011;176:706–15.CrossRef

48.

Soille P, Vincent L. Determining watersheds in digital pictures via flooding simulations. Visual Communications and Image Processing 90, Pts 1-3. 1990;1360:240-250. doi: https://doi.org/10.1117/12.24211.

49.

{R Core Team}. R: a language and environment for statistical computing. Vienna: R Foundation for Statistical Computing; 2014.

50.

van Oorschot B, Hovingh S, Dekker A, Stalpers LJ, Franken NA. Predicting radiosensitivity with gamma-H2AX foci assay after single high-dose-rate and pulsed dose-rate ionizing irradiation. Radiat Res. 2016;185:190–8. https://doi.org/10.1667/RR14098.1.CrossRefPubMed

51.

Hall EJ, Giaccia AJ. Radiobiology for the radiologist. Eighth edition. ed. Philadelphia: Wolters Kluwer; 2019.

52.

Gardin I, Colas-Linhart N, Petiet A, Bok B. Dosimetry at the cellular level of Kupffer cells after technetium-99m-sulphur colloid injection. J Nucl Med. 1992;33:380–4.PubMed

53.

Hindie E, Colas-Linhart N, Petiet A, Bok B. Microautoradiographic study of technetium-99m colloid uptake by the rat liver. J Nucl Med. 1988;29:1118–21.PubMed

54.

Makrigiorgos GM, Ito S, Baranowska-Kortylewicz J, Vinter DW, Iqbal A, Van den Abbeele AD, et al. Inhomogeneous deposition of radiopharmaceuticals at the cellular level: experimental evidence and dosimetric implications. J Nucl Med. 1990;31:1358–63.PubMed

55.

Knight JC, Koustoulidou S, Cornelissen B. Imaging the DNA damage response with PET and SPECT. Eur J Nucl Med Mol Imag. 2017;44:1065–78. https://doi.org/10.1007/s00259-016-3604-1.CrossRef

Title: Quantitative γ-H2AX immunofluorescence method for DNA double-strand break analysis in testis and liver after intravenous administration of 111InCl3
Authors: Anna Stenvall
Erik Larsson
Bo Holmqvist
Sven-Erik Strand
Bo-Anders Jönsson
Publication date: 01-12-2020
Publisher: Springer Berlin Heidelberg
Published in: EJNMMI Research / Issue 1/2020
Electronic ISSN: 2191-219X
DOI: https://doi.org/10.1186/s13550-020-0604-8

At a glance: The ONWARDS insulin icodec trials

Springer Medicine

Quantitative γ-H2AX immunofluorescence method for DNA double-strand break analysis in testis and liver after intravenous administration of ¹¹¹InCl₃

Abstract

Background

Results

Conclusion

At a glance: The ONWARDS insulin icodec trials

Springer Medicine

Abstract

Background

Results

Conclusion

Please log in to get access to this content

Other articles of this Issue 1/2020

[18F]MPPF and [18F]FDG μPET imaging in rats: impact of transport and restraint stress

Targeted alpha therapy with astatine-211-labeled anti-PSCA A11 minibody shows antitumor efficacy in prostate cancer xenografts and bone microtumors

Quantitative 18F-FDG PET-CT scan characteristics correlate with tuberculosis treatment response

Diagnostic value of [18F]FDG PET/MRI for staging in patients with ovarian cancer

Correlation of positron emission tomography ventilation-perfusion matching with CT densitometry in severe emphysema

A DROP-IN beta probe for robot-assisted 68Ga-PSMA radioguided surgery: first ex vivo technology evaluation using prostate cancer specimens