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

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 111 InCl 3 .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 111 InCl 3 , both hepatocytes and non-hepatocytes within the liver showed an absorbed dosedependent 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 doseresponse relationships in animal organs and human tissue biopsies after internal exposure to radiation.

H2A.X Phosphorylation in Oxidative Stress and Risk Assessment in Plasma Medicine

“…Hence, radiosensitive tumor cell lines retain phosphorylated H2A.X longer than radioresistant counterparts, making the former more sensitive to apoptosis than the latter [ 102 ]. In addition to various cells in vitro, γ H2A.X detection was also used to quantify the effects of IR in tissues exposed ex vivo or in vivo [ 109 , 110 ]. With a growing range of applications, γ H2A.X analysis expands beyond its traditional field of radiobiology, mainly because of its increasingly recognized role in ROS-related conditions.…”

7-Amino-6H-anthra[9,1-cd] Isothiazol-6-one-Casein Nanosystem for Live Cell Staining and Augmenting Therapeutic Effectiveness in Triple Negative Breast Cancer

“…It allows us to visualize and quantify the presence and localization of significant proteins responsible for DNA damage, providing insights into its repair mechanisms and cellular processes. We have used a red fluorochrome-conjugated Alexa secondary antibody to see the expression of γH2AX with and without treatment related to DNA damage and cell death . We observed that 4T1 cells treated with CAAT NPs showed more significant red fluorescence than the untreated and AAT control groups due to the expression of γH2AX (Figure A,B).…”

“…We have used a red fluorochromeconjugated Alexa secondary antibody to see the expression of γH2AX with and without treatment related to DNA damage and cell death. 45 We observed that 4T1 cells treated with CAAT NPs showed more significant red fluorescence than the untreated and AAT control groups due to the expression of γH2AX (Figure 9A,B). From this observation, it can be suggested that CAAT NPs cause cell death by triggering DNA damage and apoptosis.…”

Mechanisms of the DNA Damage Repair Gene Work on Elevated α/β ratio after Hypofractionated Radiotherapy for Prostate Cancer

“…foci can roughly re ect the number of DSB and was consistent with DSB at the beginning after irradiation. After a period of time, due to DNA repair, the numbers of γ-H 2 AX foci reduced and the expression level decreased(8, 9,22). Therefore, observing and counting γ-H 2 AX foci in irradiated cells was a widely used method to verify the presence of DSB in DNA and to analyze its repair (23).…”