From the 1980s to the 2010s, all manned spaceflights have been taken place in low-Earth orbit with the protection of our planet’s geomagnetic field (GMF, about 50 μT). However, the environmental magnetic field in outer space decreases geometrically with the distance from the ground, which exposes the astronauts to space radiation during lengthy voyages outside the Earth’s magnetosphere. This GMF-eliminated environment is defined as a hypomagnetic field (HMF), to represent radiation situations in interplanetary space (6.6 nT) [1
], Moon surface (<300 nT) [2
], and above the Martian crust (<700 nT) [3
]. Although HMF and radiation are the two tightly intertwined ecological factors in outer space, their potential to compromise human health has not yet been fully elucidated.
Radiation-induced DNA lesions increase the cellular risk of malignant transformation that has been identified as one of the most harmful outcomes for human health. Among various cancer types, lung cancer is the largest potential health risk from space travel for both men and women astronauts [4
]. At the molecular level, radiation is known to negatively impact DNA integrity. To counteract DNA damage, cells have developed specific mechanisms that locate and repair DNA lesions. These mechanisms consist of a network of cellular proteins involved in DNA damage response pathways, such as cell cycle regulation, DNA repair, and apoptosis [5
]. Generally, cells can accommodate moderate DNA damage through different DNA repair processes, but in space flight with the lack of a magnetic field, these DNA repair processes may be interrupted by HMF leading to altered radiation sensitivity.
HMF has also been identified as a significant regulator of physical and behavioral functions on earth. Exposure to the HMF environment may induce a range of biological effects and adaptive alterations, such as interfered embryogenesis and brain function [6
], increased occurrences of developmental abnormalities in the newt and Xenopus laevis [10
], alteration of the circadian rhythm of birds [12
] and rats [13
], dysfunction in the learning and memory of Drosophila and chicks [14
], and a reduction in stress-induced analgesia in mice [16
]. It has been reported that even short-term HMF exposure can reduce the heart rate and increase capillary circulation in humans [18
]. At the cellular and molecular levels, in vitro and in vivo studies have shown that biological processes maintaining genomic stability, such as cell cycle progression, tubulin assembly, DNA super condensation, reactive oxygen species (ROS) production, and antioxidant capacity, can be modulated by HMF [19
]. It is therefore unsurprising that increasing research attention is being directed toward the biological effects of HMF, to provide knowledge that may be used to protect the astronauts.
Previous studies on radiation exposure and HMF have been usually conducted separately, so data regarding their combined effects remain quite limited. The present study was designed to explore the biological effect of HMF on DNA damage response in human bronchial epithelial cells upon exposure to ionizing radiation. To our knowledge, this is the first experiment that aimed to elucidate the joint biological effect of radiation and HMF on genotoxic endpoints in human lung cells.
The interplanetary magnetic field that the crew may be exposed to during manned flight is 103–104 times less than the regular GMF on the Earth surface, i.e., HMF. The importance of HMF in outer space lies in its impact on infiltration of intensive fluxes of radiation into the spacecraft, and most of which is blocked by GMF during the low-Earth orbit flights. Since radiation is known to induce genetic lesions to the cells and so increase human cancer risk, it is essential to understand the biological effect of HMF in combination with IR in terms of DNA damage and repair. In the present work, we revealed that, despite the fact that HMF alone showed no obvious effect on DNA integrity, it clearly enhanced the radiation resistant capacity of human bronchial epithelial cells by accelerating the decline of γH2AX foci after radiation. The result implied that the geomagnetic field shielding may stimulate cells to recover from IR induced DSB lesions. To confirm the inference, we further demonstrated that HMF could promote cellular DSB repair activity in response to IR by keeping the similar proportion of colocalization of γH2AX and 53BP1, compared with the GMF control.
The effects of HMF on biological systems vary according to magnetic shielding approach, remanence intensity being used, and duration of the exposure [8
]. The phosphorylation of histone H2AX on Ser139, which induces the designated γH2AX, has been used as an indicator of DSB. A recent study reported that γH2AX foci were markedly upregulated by magnetic deprivation in preputial skin fibroblasts [24
]. In the present study, HMF exposure brought about marginal alteration in spontaneous DNA damage (Figure 1
) and γH2AX foci formation (Figure 3
). The results are in accordance with a previous report on the repression of endogenous DNA oxidative damage by reactive oxygen species (ROS) in HMF cultured human neuroblastoma cells [25
], suggesting a minor genotoxic effect of HMF on DNA integrity.
Using the well-established colony formation assay, an increased cell survival rate in HMF group following IR treatment was observed (Figure 2
), indicating an enhanced radiation resistant capacity under HMF condition. Since DSB lesions are considered the most lethal type of DNA damage induced by radiation, this increased cell survival rate in HMF cultured cells might be resulted from either a decreased DSB induction or/and enhanced DSB repair capacity. To distinguish between these possibilities, a γH2AX foci kinetics assay was performed to monitor the functioning of the DSB response machinery [26
]. Our data showed that geomagnetic shielding resulted in a significant decline in γH2AX foci number per nucleus from 1h to 8h post IR, compared to the GMF controls. While at 30 min after IR, the maximum yield of γH2AX foci between HMF and GMF culture groups showed no statistical difference (Figure 3
B). These results suggested a strengthened DSB recovery efficiency due to the deprivation of geomagnetic field, as indicated by the increased cell survival rate.
Unlike γH2AX foci kinetics assay, the result from whole cell extracts detected by western blotting (Figure 5
) did not show significant changes between experimental and control groups. This may be accounted by the fact that multiple mechanisms have been proposed for eliminating γH2AX when DNA repair completes, including removal by histone exchange or dephosphorylation by a protein phosphatase [27
]. Thus, the histone H2AX may remain phosphorylated after foci disassembly and the kinetics of disappearance of γH2AX foci may be different from its cellular amount. Moreover, the level of γH2AX signal detected by Western blotting could be explained by the saturation of phosphorylated H2AX targeted for degradation [29
] and to the contribution of apoptotic DNA fragmentation.
DSBs can be repaired either through the error-prone Non-homologous end-joining (NHEJ) pathway, or through homologous recombination (HR) in the presence of a DNA donor template with high fidelity [31
]. The modification of H2A and H2AX triggers the binding of early phase DSB repair proteins, amongst which 53BP1 plays an important role in the DSB repair initiation and NHEJ/HR pathway selection [32
]. The presence of 53BP1 at the DSB normally results in the recruitment of proteins involved in NHEJ repair pathway, and inhibition of BRCA1 takes part in the HR repair pathway [33
]. The concurrence of γH2AX and 53BP1 at the DSBs initiates an on-site DSB repair process, which can be detected by colocalized antibody binding [23
]. The colocalization of 53BP1 and γH2AX foci in cells upon radiation has been found similar to the DSBs repair sites [24
]. In particular, the phosphorylation of H2AX resulting from DNA damage may enhance the interaction between γH2AX and 53BP1, leading to an increased accumulation of 53BP1 foci at the sites of DSB in IR exposed cells. Notably, our results presented a similar colocalization factor in IR exposed cells under both HMF and GMF culture conditions (Figure 6
B,C), indicating a higher ratio of 53BP1/γH2AX co-stained site among total γH2AX positive foci in HMF cultured cells, when taking account of the lower level of γH2AX foci in HMF cultured cells at 1–8 h after IR. Taken together, these data suggested a proficient repair of the IR-induced DSB damage in HMF cultured cells. However, the detailed mechanisms underlying its tumorigenesis are yet to be further elucidated.
In summary, our study has established, for the first time, that HMF can take part and interact with IR-induced DNA damage and DNA repair process, so as to provide an enhanced radiation resistant capability in human lung cells. As a previously unidentified regulator of radiation sensitivity, the new biological function of HMF may be potential in future astronaut’s radiation protection as well as in human cancer research.
4. Materials and Methods
4.1. Cell Culture
Human bronchial epithelial cell line BEAS-2B purchased from American Type Culture Collection were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Grand Island, NY, USA) supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco, Grand Island, NY, USA), 100 unit/mL penicillin and 100 mg/mL streptomycin and were grown in a monolayer in petri dishes (Corning Inc., Corning, NY, USA) and the medium was replaced every two days. The cells were used within 10–20 passages according to the requirements of ATCC, and were routinely tested for mycoplasma using mycoplasma detection kit (Beyotime Biotech, Haimen, China).
4.2. The HMF Conditions
HMF was achieved by a permalloy magnetic shield box (the 710th Research Institute of China Shipbuilding Industry Corporation, Yichang, China), and the residue magnetic field inside the magnetic shielding box was relatively uniform. The HMF-exposed cells were cultured within the shielding box where the residue magnetic field was lower than 50 nT. The shield box was put in a cell incubator (Thermo Fisher Scientific, Waltham, MA, USA) equipped with a circulating fan (Thermo Fisher Scientific, Waltham, MA, USA) to ensure the optimal conditions of cell culture (5% CO2, 37 °C). Cells of GMF control were cultured in a regular cell incubator (Thermo Fisher Scientific, Waltham, MA, USA), where the magnetic field was about 45 μT (i.e., GMF). The intensity of magnetic field was measured by a gaussmeter (Model BLD-630, Boland Magnetoelectric Technology, Beijing, China).
Cells were plated in Petri dishes and cultured for 24 h before irradiation and subsequently irradiated with X-rays, which were generated using an X-rays instrument (RS 2000 Xray Biological Irradiator, Rad Source Technologies, Suwanee, GA, USA) equipped with a tungsten target (160 kVp, 25 mA). The dose rate was 1 Gy/min. All irradiation treatments were carried out at room temperature.
4.4. Alkaline Comet Assay
The alkaline comet assay was performed to detect DNA damage at the single-cell level. Briefly, cells were collected by trypsinization and centrifugation at indicated time points after HMF condition culture and mixed with low melting point agarose to prepare a cell suspension in 0.1% agarose/phosphate-buffered saline (PBS). After gelation of the agarose, the cells were lysed. The resultant DNA samples were electrophoresed at 1 V/cm for 30 min in 0.3 M NaOH and 1 mM ethylenediamine-N,N,N’,N’- tetraacetic acid solution. After the DNA was stained with SYBR Green I, immunofluorescence images were captured on a fluorescence microscope. DNA damage was analyzed using the Comet software (Comet Assay IV, Perceptive Instruments, Suffolk, UK). At least 100 comets from each gel were analyzed. Tail length indicates the pixel length of the comet tail. The tail percentage indicates the percentage of tail content relative to comet content. The tail moment was calculated as follows:
Tail moment = (the distance between the center of the comet head and the center of the comet tail) × (Tail percentage)/100
4.5. Cell Survival Assay
The SF was measured using the colony formation assay to assess radiation sensitivity. Cells were inoculated into triplicate 60 mm plastic dishes to produce 20–100 colonies per dish post X-ray (0, 2, 4, 6 Gy) radiation. After 14 days of incubation, colonies were fixed with 20% methanol and stained with 0.2% crystal violet. Triplicate dishes of each dose point colony consisting of more than 50 cells were counted under a stereomicroscope. The SF at each dose point was determined as the ratio of live colonies in the treated dish relative to the number in the untreated/control.
4.6. Immunofluorescent Staining
Immunofluorescence staining was carried out as previously described [37
]. Primary mouse anti-γH2AX antibody (1:400, sc-517348, Santa Cruz Biotech, Dallas, TX, USA) and rabbit-anti-53BP1 antibody (1:300, ab21083, Abcam, Cambridge, MA, USA) were used. Alexa Fluor 555 –labelled donkey anti-mouse secondary antibody (1:1000, A0460, Beyotime Biotech, Haimen, China) and Alex Fluor 488 –labelled goat anti-rabbit secondary antibody (1:1000, A0423, Beyotime Biotech, Haimen, China) were used for visualization. Cell nuclei were stained with 4’, 6-diamidino-2-phenylindole (DAPI, Sigma, Milwaukee, WI, USA). Imaging of γH2AX foci was performed using a confocal microscope (Olympus FV1200, Tokyo, Japan) with a ×40 oil objective. At least 150 cells were scored randomly from 5 to 10 fields. The number of γH2AX foci per cell and the percentage of γH2AX foci–positive cells (at least 5 foci/nucleus) were used as the indicators of DNA damage. Each experiment was independently repeated three times.
The results from colocalization experiments for 53BP1 and γH2AX foci are quantitatively presented in terms of the colocalization factor (Pearson’s correlation coefficients and Manders’ overlap coefficients) [38
], which was analyzed using the Image Pro Plus 6.0 software (Media Cybernetics, Silver Spring, MD, USA).
4.7. Western Blotting
Cells were harvested and lysed using RIPA buffer. Samples were sonicated and centrifuged at 12,000× g for 15 min at 4 °C. The concentration of total protein was determined by using DC Protein Assay Kit I (Bio-Rad, Richmond, CA, USA). Then, the samples were denatured at 100 °C for 5 min. Total proteins were separated by 12% SDS-PAGE and transferred to a hybond nitrocellulose membrane (Amersham Biosciences, Pascataway, NJ, USA). The membrane was blocked with 5% nonfat milk powder (A600669, Sangon Biotech, Shanghai, China) in Tris-buffered saline which consisted of 20 mM Tris-HCl (1115GR500, Biofroxx, Einhausen, German) and 150 mM NaCl (10019318, Sinopharm, Shanghai, China) adjusted pH to 7.5 by using HCl. The membrane was then hybridized overnight with primary antibodies γH2AX (1:1000, 9718S, Cell Signaling Technology, Beverly, MA, USA), GAPDH (1:1000, RLM3029, Ruiyingbio, Suzhou, China), which were then detected with horseradish peroxidase-conjugated anti-IgG for 2 h at room temperature and visualized with an ECL kit (Millipore, Billerica, MA, USA). Protein expression levels were normalized to the loading controls basing on their intensity analyzed with Image J (Version 1.50i, National Institutes of Health, Bethesda, MD, USA). The target γH2AX/GAPDH value obtained from 0 Gy control cells was designated as “1”.
4.8. Statistical Analysis
All computations were performed with GraphPad Prism software (Version 8.0, GraphPad, La Jolla, CA, USA) for Windows. Data are presented as the means ± standard errors of the mean (SEM). After the normality test, statistical analysis was performed with analysis of variance (ANOVA) between HMF exposure and GMF exposure groups. p < 0.05 was considered statistically significant.