Radiation exposure lymphocyte damage assessed by γ-H2AX level using flow cytometry

DNA double-strand breaks (DSBs) are considered the most relevant lesions to the DNA damage of ionizing radiation (IR), and γ-H2AX foci in peripheral blood lymphocytes are regarded as an adequate marker for DSB quantitative studies. This study aimed to investigate IR-induced DNA damage in mice through γ-H2AX fluorescence analyses by flow cytometry (FCM). The levels of γ-H2AX in CD4/CD8/B220-positive lymphocytes were quantified by FCM through mean fluorescence intensity (MFI) values. Peripheral venous blood samples were collected for evaluation, and all the control groups were restrained from irradiation. For external irradiation experiments, the dose-dependency of MFI values and temporal alternations were assessed both in vitro and in vivo. External radiation exposure damage was positively correlated with the absorbed radiation dose, and the lymphocyte recovered from damage within 3 days. I-131 sodium iodide solution (74 MBq) was injected into the mice intraperitoneally for internal irradiation experiments. Gamma counting and γH2AX foci analyses were performed at 1 h and 24 h by the group. The blood-to-blood S values (Sblood←blood) were applied for the blood-absorbed dose estimation. Internal low-dose-irradiation-induced damage was proved to recover within 24 h. The FCM method was found to be an effective way of quantitatively assessing IR-induced DNA damage.


Linear positive correlation between γ-H2AX levels and doses
The in vivo and in vitro experiments of external irradiation showed that the ratio of MFI'/MFI was positively correlated with the radiation doses.The results of the linear regression analysis are shown in Table 2. Notably, B220 + cells (B-cells) represented the greatest linear correlation with DNA damage in the in vitro experiments, whereas in vivo experiment results showed that CD4 + and CD8 + lymphocytes (T-cells) had the best linear correlation.

Temporal alternation
In the external irradiation experiments, no significant difference between the ratio of MFI' to MFI on days 3 and 7 was observed regardless of the radiation doses; however, differences between days 0 and 3 were proved significant in the experimental groups that accepted radiation dose of more than 0.10 Gy.The t-test results are shown in Table 3.
The internal irradiation (D blood = 0.16 Gy) experiments demonstrated that the formation of γ-H2AX was reversed within 24 h.The MFI'/MFI ratios of the experimental and control groups did not differ significantly after 24 h.The t-test results are shown in Table 4.
Meanwhile, the absorbed doses represented in the percentages of accumulated radioactivity to the injected dose per gram gradually decreased to a level close to the controls with time.The specific details are shown in Table 5.

Discussion and conclusion
The present study proposed an innovative evaluation method of IR-induced DNA damage that quantitatively presents damage levels through γ-H2AX foci flow cytometry analyses.In contrast to previous studies 4, [16][17][18][19] which have primarily focused on the examination of either the dose dependence or the temporal alternation of radiation damage or the examination of one aspect of external or internal irradiation, this study confirmed the dose and time dependence of radiation damage caused by both internal and external irradiation under consistent laboratory conditions.
Various methods are available for detecting DSB, each with distinct characteristics that suit different purposes.Pulsed-field gel electrophoresis is the most commonly used method in analyzing DSBs; however, it has limitations in monitoring persisting DSBs 20 .Microscopic observation of the γ-H2AX level is another commonly used method; however, it can be cumbersome and time-consuming.In comparison, FCM allows for a rapid, sensitive, and quantitative evaluation of DSBs 5,21 .Notably, γ-H2AX flow cytometry analyses were considered inadequate for detecting DNA damage induced by low-dose irradiation 14 .This study found that exposure to a radiation dose of ≤ 0.10 Gy resulted in no significant alteration in γ-H2AX levels, suggesting that FCM may be suitable for assessing the presence of detectable damage in radiation-exposed individuals and estimating the absorbed blood dose in a single-point test.
In the study, we found that DNA damage in lymphocytes is linearly positively correlated with the irradiation doses of external radiation received by mice.The results further revealed that the CD45R/B220 lymphocyte subset is relatively more sensitive to radiation 7 , as indicated by the equation in Table 2.However, linear regression analyses of the in vivo internal irradiation experiment were not conducted due to the high-dose exposure to the blood of external irradiation in mice compared to the low doses of radiopharmaceuticals injected in the mice.This is because internal irradiation does not result in a higher radiation dose as the radioisotope transfer from the blood is completed quickly after administration.Considering that the degree of DSB repair varies depending on the radiation type and the damage increases with the radiation dose, it is conceivable that similar linearity could Table 1.t-Test results of γ-H2AX cell count analyses in external exposure experiments.*0 Gy versus 1 Gy, P < 0.05.

P-value of ANOVA
Count ratio 0.18 ± 0.03* 0.32 ± 0.03 * < 0.05 be observed in internal irradiation.Due to ethical reasons, high-dose administration to the mice was precluded.
When evaluating DNA damage induced by internal radiation exposure to blood with FCM, it is challenging to detect low-dose-induced DSBs, and the accuracy of linearity is expected to vary with the evaluation time.
The study first investigated β-ray-induced DSB in mice.IR-induced DNA damage assessment through the FCM method has been reported 10,22 ; however, radiation emitted by the Lu-177 or Ra-223 nuclide-conjugated  www.nature.com/scientificreports/radioactive pharmaceuticals is mainly α-rays 18,22 , not the β-rays emitted by I-131 that we used in this study.Besides, Lenka Zárybnická et al. reported that H2AX phosphorylation of lymphocyte subsets in response to in vivo irradiation in rats had the potential to be applied to bio-dosimetry 5 ; however, related studies in mice had never been reported before, which highlights the need for exploring the utility of H2AX phosphorylation as a biomarker and further research to investigate biodosimetry in mice.www.nature.com/scientificreports/Due to the technical reason that if the same mouse is blood-collected twice or more within 3 days, severe anemia or the death of the experimental individual will occur, leading to deviations in results, blood samples between 0 and 3 days were not included in our study.The results of the present animal experiment also showed that there was no significant difference in the level of γ-H2AX 3 days and 7 days after irradiation.A relevant clinical study reported that DNA repair detected in mononuclear cells has been almost completed 24 h after receiving γ or X-rays 19 .A Bayesian model to predict individual radiosensitivity in patients also reported that the number of DSB foci is positively correlated with the doses 23 , and the focus count was expected to enter a stable state in 24 h.As Schumann et al. reported no data after 24 h and significantly higher average numbers of foci per cell after 24 h than the baseline, it was reasonable to conclude that the DNA damage repair may be complete within three days, and more data were required between the time points of 24 h and 72 h.
Although the colocalization of γ-H2AX/53BP1 is currently considered the most reliable marker of DSB, considering the dephosphorylation of γ-H2AX was reported to represent recovery from DNA damage, γ-H2AX foci analyses proved to be efficient enough in providing quantitative DSB evaluations 16,24 .Additionally, the 53BP1 protein is not induced by radiation but instead undergoes relocalization to the sites of double-strand breaks (DSBs).Consequently, this molecular marker cannot be utilized for the detection of DSBs through conventional flow cytometry (FCM) methods 4,25 .Imaging Flow Cytometry (IFC) was reported to be able to perform the colocalization of γ-H2AX/53BP1 4 , but the instrument and analysis system required were still not used extensively throughout the world.The present study also showed that the FCM method is a valid method for the quantitative evaluation of IR-induced DNA damage in radiosensitive lymphocytes.
In conclusion, DNA damage in lymphocytes caused by external γ-ray exposure damage was proved to be positively dependent on the absorbed radiation dose.The recovery from IR-induced damage was confirmed to be less than 3 days in the present study, while low-dose internal irradiation caused damage recuperated relatively rapidly in 24 h.Our study demonstrated that the FCM method could quantitatively assess IR-induced lymphocyte damage rapidly and effectively.

Animals
Wild-type mice (BALB/cCrSlc) were obtained from the Jackson Laboratory and bred in-house.Male mice aged 8-12 weeks old and weighing 23.0-25.0g were included in the study.Each experimental group consisted of six mice.Peripheral venous blood was collected into the heparin-containing solution using the experimental siphon tubes (final concentration 100 International Unit (UI)/ml, Mochida Pharmaceutical Co., LTD., Tokyo, Japan).The Ethics Committee of Kanazawa University approved the study (AP-184027), and the study is conducted and reported in compliance with the national legislation and the ARRIVE guidelines.Euthanasia via carbon dioxide inhalation was performed after the experiment as a humane method of animal sacrifice.

Irradiation and peripheral blood collection
Ionizing irradiation was provided using an X-ray Generator System (MBR-1520R-3; Hitachi Power Solutions Co. Ltd., Ibaraki, Japan).Before X-ray exposure, air kerma rate measurements were performed.The dose rate at the location where the subject is placed and the dose rate at the monitoring location for accumulated dose during irradiation were premeasured.The dose administered to the subject was then automatically calculated based on the ratio between these two rates, and irradiation is automatically stopped.Mice were anesthetized with isoflurane prior to blood collection.The volume of each blood sample was 500 μL.

External irradiation experiment: in vitro
For FCM, the peripheral venous blood samples of eight experimental groups (6 mice/group) collected were exposed to the following irradiation doses: 0.10, 0.25, 0.75, 1.00, 1.25, 1.50, 1.75, and 2.00 Gy (tube voltage, 150 kV; tube current, 20 mA; filter, 0.5 mm Al and 0.2 mm Cu; distance between focus and target, 320 mm).Blood samples excluded from irradiation were set as controls.For immunohistochemistry (IHC) staining, one experimental group (3 mice/group) were irradiated with doses of 1 Gy and one control group of 3 mice were set for observation and analyses.

External irradiation experiment: in vivo
The mice of eight experimental groups (6 mice/group) received whole-body irradiation at doses of 0.10, 0.25, 0.5, 0.75, 1.00, 1.25, 1.5, 2.00 Gy, respectively for each observation time point (tube voltage, 150 kV; tube current, 15 mA; filter, 0.5 mm Al and 0.5 mm Cu; distance between focus and target, 400 mm).During irradiation, mice were confined to a small cage (fan-shaped; radius 10 cm, height 4.5 cm, angle 30°) specific for X-irradiation.Nonirradiated animals served as controls.Blood samples were collected on day 0, day 3, day 7, and day 14 after radiation exposure.

Internal irradiation
Fourteen mice were injected with I-131 sodium iodide solution (74 MBq) intraperitoneally (five mice/group), and four mice injected with stroke-physiological saline solution were set as the control group.Blood samples were collected at the time points of 1 h and 24 h by group (5 mice/group).

Sample preparation
Collected peripheral blood samples were lysed using red blood cell lysis reagents (RBC Lysis Buffer).Each sample was divided into two aliquots of 50 μl each at a density of 1 × 10 6 cells/50 μl for the following T-and B-cell

Figure 1 .
Figure 1.Illustration of positive dose-dependency changes in MFI values in CD4 + Lymphocytes.The y-axis represents the cells count.*Autofluorescence of unstained cells.

Table 2 .
Radioactive correlation linear fit in external exposure experiments.

Table 3 .
t-Test results of external exposure in in vivo experiments.
*The significant difference between day 0 and day 7 at a dose of 0.10 Gy was observed exclusively in the B220(+) lymphocytes.No significant differences were noted between day 0 and day 7 at a dose of 0.10 Gy in other lymphocyte subtypes.

Table 4 .
t-Test results of internal exposure experiments.

Table 5 .
Percentages of accumulated radioactivity to the injected dose per gram (%ID/g) were used to measure absorbed doses.