The effect of 50 Hz magnetic fields on cellular sensitivity of mouse spermatogenic cell lines to hydrogen peroxide

Abstract

With the widespread application of electromagnetic technology, electromagnetic fields (EMFs) emitted from various electric and electronic devices have significantly altered the electromagnetic environment. This has raised concerns about the potential health impacts of EMFs. Previous studies have indicated that EMFs may influence male infertility, with oxidative stress proposed as a key factor; however, the underlying mechanisms remain unclear. In this study, we aimed to determine whether EMFs enhance the impact of oxidative stress on male infertility. We investigated the effects of 50 Hz magnetic fields (MFs) on the sensitivity of mouse spermatogenic cell lines (GC-1 spg and GC-2 spd) to low-dose hydrogen peroxide (H₂O₂, 5 and 10 μM). Our findings revealed that pre-exposure to 2.0 mT 50 Hz MFs for 24 h increased the sensitivity of GC-2 spd cells to low-dose H₂O₂ in terms of γH2AX foci formation, a marker for DNA damage repair. However, no significant changes were observed in DNA fragmentation, cell viability, or cell cycle progression in either GC-1 spg or GC-2 spd cells. In conclusion, our results suggest that 50 Hz MFs do not significantly enhance the sensitivity of mouse spermatogenic cell lines to low-dose H₂O₂.

Introduction

Electric energy has become an indispensable component of modern life, bringing significant convenience. However, the increasing exposure to electromagnetic fields (EMFs) from power transmission lines and electric devices has raised public concerns about potential health hazards.¹ In 1983, a retrospective study reported that male high-voltage switchyard workers faced a higher risk of abnormal pregnancy outcomes,² which heightened concerns about the effects of extremely low frequency magnetic fields (ELF-MFs) on male fertility over the past few decades.³

Some studies have reported a significant correlation between ELF-MFs and male infertility; however, the results have been inconsistent across different studies. For instance, a population-based case–control study indicated that ELF-MF exposure has an adverse effect on sperm quality,⁴ while no such effects were observed in other studies.^5,6 Some experimental research demonstrated that ELF-MF exposure increases testicular germ cell apoptosis, particularly in spermatogonia, and reduces sperm count in rats,^7,8 whereas other studies reported no significant impacts.⁹ The debate continues.

Oxidative stress is one of the major threats to male fertility,^10,11 and can be induced by exposure to ELF-MFs in male germ cells or tissues.^12,13 However, this effect has not been consistently reliable.¹⁴ Combined effects of ELF-MFs with other factors have also been observed, often correlated with oxidative stress, such as enhanced drug activity¹⁵ and toxicity¹⁶ by increasing oxidative stress, reduced damage from airborne particulate matter¹⁷ by decreasing oxidative stress. Nonetheless, some studies have found no significant combined effects.^18,19 Therefore, the role and mechanisms of oxidative stress in the relationship between ELF-MFs and male infertility remain unclear.

Oxidative stress is not only a harmful condition induced by external stimuli but also an inherent attribute of aerobic metabolism, involving the formation of free radicals and redox reactions that play crucial roles in various biological processes.^20–22 Under oxidative stress, cellular responses play a critical role in determining biological outcomes.^23–25 Thus, it is imperative to understand the potential impact of EMFs on the susceptibility of germ cells to oxidative stress which remains unknown.

In this study, we investigated the effect of hydrogen peroxide (H₂O₂) on cell viability, cell cycle progression, and DNA damage in two mouse spermatogenic cell lines (GC-1 spg and GC-2 spd), both with and without exposure to 50 Hz MFs. This was done to determine whether ELF-MFs influence the sensitivity of germ cells to oxidative stress.

Methods and materials

Cell lines and cultures

GC-1 and GC-2 cell lines were purchased from Guangzhou Jennio Biotech Co.,Ltd (Guangzhou, China). The cells were maintained in 10 cm-diameter Petri dishes (Corning, Shanghai, China) with 10 mL Roswell Park Memorial Institute 1640 (RPMI1640, Corning) supplemented with 15% (v/v) fetal bovine serum (HyClone; Thermo Scientific, Shanghai, China) at 37 °C in a humidified atmosphere containing 5% CO₂.

Exposure system

The exposure system was designed and provided by the IT’IS Foundation (Zurich, Switzerland) and is detailed in reference.²⁶ Briefly, the setup was housed within a commercial incubator (Hera Cell 240, Thermo Scientific, Waltham, MA) and comprised two-shielded, four-coil systems enclosed in two μ-metal box chambers. The bi-filar coils could be parallel-switched for field exposure non-parallel for control (sham). This system enabled MF exposures to B fields up to 3.6 mT (root mean square amplitude) within the frequency range of 3 Hz to 1.25 kHz. Artifacts were characterized by IT’IS Foundation as follows: sham exposure levels were < −43 dB, parasitic incident E fields <1 V/m, no measurable temperature differences in the media between exposure and sham conditions, and vibrations of the mechanically decoupled dish holder <0.1 m/s² (=0.01 g). The applied signal in this experiment was a 50 Hz sinusoidal waveform. During exposure, the coil current and chamber temperature were continuously monitored using HS50 low-temperature-coefficient power resistors (Arcol, Cornwall, United Kingdom) and Pt100 temperature sensors (Labfacility, West Sussex, United Kingdom), which were fixed on the inner side of the shield. Temperature differences between exposed and sham-exposed cells were maintained below 0.1 °C.

Immunofluorescence staining with γH2AX

The detailed protocol of immunofluorescent staining of γH2AX could be obtained in our pervious publication.²⁷ Briefly, immediately after treatment, cells were fixed in 4% paraformaldehyde solution at 4 °C for 15 min and permeabilized with 0.2% Triton X-100 at 4 °C for 15 min. Non-specific binding was blocked using blocking serum (Zhongshan Golden Bridge Biotechnology, Beijing, China) at room temperature (25 °C) for 2 h. Cells were then incubated with a primary anti-γH2AX antibody (Upstate, Millipore, Temecula, CA) at room temperature for 2 h, followed by incubation with a tetramethyl rhodamine isothiocyanate-conjugated goat-anti-mouse secondary antibody (Zhongshan Golden bridge Biotechnology Co., Ltd, Beijing, China) for 1 h at room temperature. Nuclei were stained with 1 μg/mL 4′, 6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich, St. Louis, MO) for 15 min. Finally, a coverslip was mounted on a glass slide for visualization.

Immunofluorescence images of γH2AX foci in the nuclei were captured using a fluorescent microscope (AX70, Olympus, Tokyo, Japan) equipped with a 40 × oil immersion objective. Approximately 20 cells per sample were analyzed. The average number of γH2AX foci per cell were calculated.

Exposure protocol

Before exposure, cells were sub-cultured into 35-mm-diameter Petri dishes (1 × 10⁵ cells per dish) for 12 h. Then, cells were exposed to 50 Hz MFs at 2.0 or 3.0 mT for 24 h (5 min on/10 min off). For MFs + H₂O₂ exposure, cells were exposed to H₂O₂ for 15 min immediately after the 50 Hz MFs exposure. For cell cycle analysis, cells were exposed to H₂O₂ for 6 h after the 50 Hz MFs exposure.

Alkaline single-cell gel electrophoresis (comet assay)

Cells were immediately harvested and re-suspended following treatment. Cells were mixed with 0.65% low-melting agarose and then added onto slides pre-coated with 0.65% normal-melting agarose.²⁷ The slides were then immersed in ice-cold lysis buffer (2.5 M NaCl, 1% sodium N-lauroyl sarcosinate, 0.1 M Na₂EDTA, 10 mM Tris–HCl, pH 10.0) containing 1% Triton X-100 for 1 h. Subsequently, the slides were incubated in DNase-free proteinase K (0.5 mg/mL, Amresco, OH) in lysis buffer without Triton X-100 for 2 h at 37 °C. Prior to electrophoresis, the slides were placed in alkaline electrophoresis solution (0.3 M NaOH, 0.1% 8-hydroxyquinolines, 2% DMSO, 10 mM tetrasodium EDTA, pH 13.0) for 20 min to unwind DNA. Electrophoresis was performed at constant electric field of 100 V/m for 20 min, followed by neutralization in Tris buffer (0.4 M, pH 7.5) for 15 min. DNA “comets” were stained with Gel-Red (Biotium, Fremont, CA) and visualized under a fluorescence microscope (Nikon, Tokyo, Japan) equipped with a 10 × objective lens. Approximately 100 comets for each slide were analyzed using CASP 1.2.2 software (Krzysztof Konca, Wroclaw, Poland).

Cell cycle analysis

After exposure, cells were harvested and re-suspended in pre-cooled 70% ethanol at −20 °C for 24 h. The cells were then washed with PBS and re-suspended in PBS containing 50 μg/mL propidium iodide (PI, Sigma-Aldrich, St. Louis, MO) and 50 μg/mL RNase A (Thermo Scientific, Shanghai, China) at room temperature for 30 min. The PI signal was detected by flow cytometry (FC500MCL, Beckman Coulter, CA), and the percentage of cells in the G0/G1, S, and G2/M phases were determined using Wincycle32 software (Beckman Coulter, CA). A total of 10,000 events per sample were acquired.

Cell viability analysis

After treatment, 10 μL of Cell Counting Kit-8 (CCK-8, Dojindo Molecular Technologies, Kumamoto, Japan) reagent was added to each well and incubated for 3 h at 37 °C, as previously reported.²⁸ The optical density (OD) value of each well was measured using a microplate reader (Varioskan Flash, Thermo Scientific, MA) at a test wavelength of 450 nm.

Statistical analysis

All experiments and data analyses were conducted in double blind manner. Data are presented as mean ± SEM (standard error of the mean) or as boxplots (boxes indicate the 25th–75th percentiles, whiskers show the minimum and maximum values). Statistical analysis was performed using One-way ANOVA, Two-way ANOVA, and Student’s t test for comparisons between different groups. P < 0.05 was considered statistically significant. The sample size (N) is indicated in the figure legends and supplementary data.

Results

Effects of 50 Hz MFs alone-exposure on cell proliferation and DNA damage in GC-1 spg cells

First, we exposed GC-1 spg cells to 50 Hz MFs at 2.0 mT and 3.0 mT for 24 h to determine the effects of MF exposure on cell viability. The results showed that 2.0 mT MFs did not significantly alter cell viability, whereas 3.0 mT MFs significantly increased cell viability compared to the sham group (Fig. 1A). This suggests that high-intensity 50 Hz MFs can enhance cell viability in GC-1 spg cells. However, exposure to 50 Hz MFs at either 2.0 or 3.0 mT did not significantly affect cell cycle progression compared to the sham group (Fig. 1B), indicating that 50 Hz MFs alone may not significantly influence cell proliferation.

To evaluate the genotoxic effects of 50 Hz MFs, we examined DNA fragmentation and γH2AX foci formation. The results indicated that 2.0 mT MFs significantly reduced DNA fragmentation levels in GC-1 cells compared to the sham group (Fig. 1C), suggesting a potential protective effect against endogenous DNA damage at low intensity. In contrast, 3.0 mT MFs had no significant effect on DNA fragmentation. Additionally, exposure to 50 Hz MFs at either 2.0 or 3.0 mT did not significantly affect γH2AX foci formation in GC-1 spg cells compared to the sham group (Fig. 1D), consistent with the comet assay results (Fig. 1C), further supporting the lack of significant genotoxic effects.

Effects of 50 Hz MFs alone-exposure on cell proliferation and DNA damage and GC-2 spd cells

In GC-2 spd cells, exposure to 50 Hz MFs at either 2.0 or 3.0 mT for 24 h did not induce significant changes in cell viability (Fig. 2A) or cell cycle progression (Fig. 2B) compared to the sham group, indicating that 50 Hz MFs alone do not significantly affect cell proliferation in GC-2 spd cells.

Regarding genotoxicity, 2.0 mT MFs did not induce significant changes in DNA fragmentation, but 3.0 mT MFs significantly increased DNA fragmentation as indicated by comet tail length compared to the sham group (Fig. 2C). This suggests that high-intensity 50 Hz MFs can induce DNA damage in GC-2 spd cells. However, exposure to 50 Hz MFs at either 2.0 or 3.0 mT did not significantly affect γH2AX foci formation in GC-2 spd cells compared to the sham group (Fig. 2D).

Based on these findings, we speculate that under our experimental conditions, 2.0 mT 50 Hz MFs may represent the threshold for significant effects on cell proliferation and DNA damage in both GC-1 spg and GC-2 spd cells. Therefore, 2.0 mT 50 Hz MFs were used for subsequent experiments.

Effect of H₂O₂ on cell viability, cell cycle and DNA damage in GC-1 spg and GC-2 spd cells

To determine the toxicity of H₂O₂, we exposed both GC-1 spg and GC-2 spd cells to 0–100 μM H₂O₂. We found that <50 μM H₂O₂ did not significantly affect cell viability compared to 0 μM H₂O₂ control group (Fig. 3A). Cell cycle analysis also showed no significant differences at these concentrations (< 50 μM) (Fig. 3B). To evaluate the DNA damage induced by H₂O₂, we observed that 5 and 10 μM H₂O₂ were near the threshold for inducing significant DNA damage in both GC-1 spg and GC-2 spd cells (Fig. 3C). Based on these findings, we selected 5 and 10 μM H₂O₂ for subsequent low-dose treatments.

Pre-exposure to 50 Hz MFs did not sensitize GC-1 spg cells to low-dose H₂O₂

To determine the effect of 50 Hz MFs on cellular sensitivity to oxidative stress, we treated GC-1 spg cells with 5 or 10 μM H₂O₂ after exposure to 50 Hz MFs at 2.0 mT for 24 h. The results showed that pre-exposure to 50 Hz MFs did not significantly alter cell viability (Fig. 4A) or cell cycle progression (Fig. 4B) compared to the sham exposure group. This suggests that 50 Hz MFs do not sensitize GC-1 spg cells to low-dose H₂O₂ in terms of cell proliferation. Additionally, pre-exposure to 50 Hz MFs did not significantly affect DNA fragmentation (Fig. 4C) or γH2AX foci formation (Fig. 4D) compared to the sham group. These findings indicate that pre-exposure to 50 Hz MFs does not sensitize GC-1 spg cells to low-dose H₂O₂-induced DNA damage.

Pre-exposure to 50 Hz MFs sensitized GC-2 spd cells to low-dose H₂O₂ on γH2AX foci formation but not DNA fragmentation

In GC-2 spd cells treated with low-dose H₂O₂, pre-exposure to 50 Hz MFs did not significantly affect cell viability (Fig. 5A) or cell cycle progression (Fig. 5B) compared to the sham exposure group. This suggests that 50 Hz MFs do not sensitize GC-2 spd cells to low-dose H₂O₂ in terms of cell proliferation. For DNA damage, pre-exposure to 50 Hz MFs did not significantly alter DNA fragmentation (Fig. 5C) but led to a significant increase in γH2AX foci formation (Fig. 5D) compared to the sham exposure group. These findings indicate that 50 Hz MFs may have a weak sensitizing effect on GC-2 spd cells to low-dose H₂O₂-induced DNA damage.

Discussion

Male fertility has become a significant concern due to the observed systematic decline in male semen parameters over recent decades, with reductions linked to environmental factors.^3,29–32 Oxidative stress is a well-established threat to male germ cells and fertility,^33,34 and exposure to EMFs may potentially induce oxidative stress in germ cells,^12,13 thereby affecting reproductive health. However, this hypothesis has not been fully substantiated, and the underlying mechanisms remain unclear.^3,14 Cellular responses play a crucial role in determining cell fate under oxidative stress,²³ but it remains unknown whether EMFs influence germ cell responses to oxidative stress. In this study, we investigated whether exposure to 50 Hz MFs affects the response of two mouse spermatogenic cell lines (GC-1 spg and GC-2 spd) to low-dose H₂O₂. Our findings indicate that pre-exposure to 50 Hz MFs did not affect GC-1 spg cells but enhanced γH2AX foci formation in GC-2 spd cells in response to low-dose H₂O₂.

GC-1 spg and GC-2 spd were derived from mouse spermatogonia and spermatocytes, respectively, representing two consecutive stages during spermatogenesis. These cell lines serve as typical models for investigating the potential effects of ELF-MFs on male reproductive health in several previous in vitro studies. Liu et al. reported that ELF-MFs exposure can affect genome methylation and miRNA expression in GC-2 spd.^35–37 Duan et al. found that exposure to 50 Hz MFs at 3.0 mT can induce significant DNA damage in GC-2 spd.³⁸ Solek et al. demonstrated that exposure to 2–120 Hz MFs at 2.5 mT induce oxidative and nitrosative stress-mediated DNA damage, leading to p53/p21-dependent cell cycle arrest and apoptosis in GC-1 spg and GC-2 spd cells.³⁹ These studies suggested that ELF-MFs exposure alone can influence DNA damage and other biological process in these cell lines. In our study, we also found that exposure to 50 Hz MFs at 2.0 mT for 24 h significantly decreased the DNA fragmentation in GC-1 spg cells (Fig. 1C), while exposure to 50 Hz MFs at 3.0 mT for 24 h significantly increased cell viability in GC-1 spg (Fig. 1A) and increased DNA fragmentation in GC-2 spd cells (Fig. 2C). However, there are some discrepancies between our results and those previously published. These differences may be attributed to variations in exposure systems and protocols. For example, the exposure system used in our study differs from that employed by Solek et al.³⁹ Additionally, experimental protocols varied; in our study, cells were sub-cultured for 12 h without renewing the culture median before exposure, where as in Duan et al’.s study,³⁸ cells were sub-cultured for 24 h with a renewal of the culture medium prior to exposure. Further investigation is required to determine whether these differences in exposure systems or protocols lead to inconsistent results. Despite the inconsistent results among different studies, our findings indicate that exposure to 50 Hz MFs 3.0 mT significantly increased cell viability in GC-1 spd cells (Fig. 1A) but not in GC-2 spd cells (Fig. 2A). Moreover, pre-exposure to 50 Hz MFs enhanced the effect of low-dose H₂O₂ on γH2AX foci formation in GC-2 spd cells (Fig. 5D) but not in GC-1 spg cells (Fig. 4D). These observations suggest that different spermatogenic cell lines respond differently to 50 Hz MFs under the same experimental conditions.

The combined effect of ELF-MFs and H₂O₂ co-exposure on DNA damage has been investigated in several previous in vitro studies. Koyama et al. reported that exposure to 60 Hz MFs at 5.0 mT simultaneously with H₂O₂ (1 μM) treatment for 4 h potentiated H₂O₂-induced mutation in pTN89 plasmids.⁴⁰ Yoon et al. reported that exposure to 60 Hz MFs at 2.0 mT for 6 h did not enhance γH2AX expression or foci formation when combined with H₂O₂ (0.05–0.1 mM) in human lung fibroblast WI-38 cells and human lung epithelial L132 cells.⁴¹ Cantoni et al. also demonstrated that exposure to 50 Hz MFs at up to 0.2 mT did not affect the rate of repair of H₂O₂-induced DNA damage.⁴² In these studies, cells were simultaneously exposed to ELF-MFs and H₂O₂, where as in our study, cells were pre-exposed to 50 Hz MFs before subsequent H₂O₂ exposure. Markkanen et al. observed that pre-exposure to ELF-MFs can alter cellular response to other agents, but no effects were noted from ELF-MFs exposures after or simultaneous with other agents.⁴³ This suggests that the combined effect of ELF-MFs exposure with other agents depends on the order of exposure. Therefore, under co-exposure conditions, different orders of exposure may lead to inconsistent outcomes.

The mechanism by which pre-exposure to 50 Hz MFs enhances γH2AX foci formation induced by low-dose H₂O₂ in GC-2 spd cells (Fig. 5D) remains unclear. γH2AX foci formation is an early event in the DNA double-strand break (DSB) damage response, indicating that DSBs have occurred and that the DNA repair pathway has been activated.⁴⁴ Therefore, it is plausible that DNA repair mechanisms are activated in GC-2 spd cells pre-exposed to 50 Hz MFs. Chow et al. reported that pre-exposure to 50 Hz MFs can enhance DNA repair through inducing DnaK/J synthesis,⁴⁵ whereas Robison et al. found that exposure to 60 Hz MFs decrease DNA repair efficiency.⁴⁶ The effect of ELF-MFs on DNA repair remains inconclusive, and further investigations are necessary to elucidate this relationship.

γH2AX foci formation and the alkaline comet assay were both employed to assess DNA damage, and some differences were observed between the results of these two methods. Pre-exposure to 50 Hz MFs enhanced the effect of low-dose H₂O₂ exposure on γH2AX foci formation (Fig. 5D) but not DNA fragmentation (Fig. 5C) in GC-2 spd cells. The reason for this discrepancy is currently unknown, but it may be explained by the differing sensitivities of the two assays. γH2AX foci formation assay is generally considered more sensitive than the alkaline comet assay in detecting DNA damage.⁴⁷ Therefore, pre-exposure to 50 Hz MFs may enhance the subtle effects of low-dose H₂O₂ exposure on DNA damage in GC-2 spd cells, which are detectable by the γH2AX foci formation assay but too subtle to be captured by the comet assay.

The MF flux densities of 2 mT and 3 mT used in our experiments exceeded the exposure limits set by the International Commission on Non-Ionizing Radiation Protection (ICNIRP) for both the general public (0.2 mT) and occupational settings (1.0 mT).⁴⁸ Based on our experimental results, ELF-MFs do not appear to pose a significant threat to human health as long as exposure remains within these limits, even under conditions of oxidative stress.

In conclusion, under the current experimental conditions, pre-exposure to 50 Hz MFs does not significantly affect the sensitivity of GC-1 spg and GC-2 spd cells to low-dose H₂O₂ in terms of cell viability, cell cycle progression, and DNA fragmentation.

Author contributions

Conceptualization, G. C. and C. S.; methodology, all authors; validation, C. S. and X. W.; investigation, X. W. and L. Z.; resources, X. Z.; writing-original draft preparation, X. W. and C. S.; writing-review and editing, all authors; Supervision, G. C.; all authors have read and approved the manuscript.

Funding

This research was supported by the Zhejiang Provincial Program for the Cultivation of High-Level Innovative Health Talents (No. 2020–18).

Conflicts of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

Raw data that support this article have been deposited in Zenodo (10.5281/zenodo.14905603) that will be available on request.

References

Author notes