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Research Article |
Department of Reproductive Endocrinology,3 Women's Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang 310006, China
Department of Public Health,4 Institute for Toxicology,
Bioelectromagnetics Laboratory,5 Research Center for Environmental Genomics, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310031, China
ABSTRACT
Effects of electromagnetic fields (EMFs) on DNA damage in mammals are still controversial. In the present study, the effects of EMFs on DNA damage in preimplantation mouse embryos in vitro were investigated by using gammaH2AX foci formation, a new sensitive indicator for detecting DNA double-strand breaks (DSBs). The data obtained demonstrated that EMFs decreased the cleavage rate of preimplantation mouse embryos. This decreasing effect of EMFs was related to the DNA-damaging effect indicated by the induction of gammaH2AX foci formation in preimplantation mouse embryos. The inducing effects of EMFs on gammaH2AX foci formation could be inhibited by the treatment of noise MFs or wortmannin, a phosphatidylinositol 3-kinase (PI3K) family inhibitor. Furthermore, the data obtained also showed that EMFs could activate the DNA damage-repair mechanism by recruiting repair factor Rad50 to the damaged DNA sites to repair the corresponding DNA damage. These findings suggest that EMFs could cause DNA damage in preimplantation embryos in vitro and that the adverse effects of EMFs on development might at least partly act through DNA damage. The DNA damage induced by EMFs could be at least partly repaired by the natural activation of DNA damage-repair mechanism or prevented by the simultaneous treatment of noise magentic fields.
electromagnetic fields, embryo, environment, 
H2AX, preimplantation embryos, toxicology
INTRODUCTION
Both occupational and general exposure to extremely low frequency electromagnetic fields (ELF-EMFs), generated by sources such as electric appliances or electric power lines, have dramatically increased over the past 20-year period [1]. This has created social concerns about the possible adverse effects of magnetic fields (MFs) on human health. To date, concerns about the potential health effects of exposure to ELF-EMFs have focused on damage to DNA (the genetic material) and mutations leading to diseases such as cancer [2]. Indeed, DNA damage has been correlated with carcinogenicity [3], cell death [4], aging [5], and neurodegenerative diseases [6]. It has been reported that 50/60-Hz EMF exposure may indirectly affect the structure of DNA, causing strand breaks and other chromosomal aberrations [7, 8]. Conversely, several in vitro studies found that exposure to 50/60-Hz MFs was not able to induce mutations [9]. Up to now, the DNA-damaging effects of EMFs remain controversial, and it is necessary to revaluate these adverse effects of EMFs with highly specific and sensitive measurement methods and biological systems.
Recently, H2AX foci formation has been suggested as a sensitive way to detect DNA damage, especially for DNA double-strand breaks (DSBs). H2AX (also known as H2AFX) is one of the variants of H2A, representing about 2% to 25% of total H2A [10]. H2AX has an SQ motif with the serine (Ser 139) four residues from C-terminus, which is highly conserved during evolution. The serine residue in the SQ motif is phosphorylated (termed H2AX), and H2AX forms "foci" in response to DSBs induced by many stimuli, including ionizing radiation (IR), replication stresses, and cellular programmed DSBs (such as apoptosis, V[D]J recombination, and retroviral DNA integration) [10–12]. In an elegantly designed experiment, Rothkamm and Lobrich [13] have shown that the number of H2AX foci detected by immunofluorescence is quantitatively the same as DSBs, suggesting that H2AX may be used as an indicator for DSBs. In addition, H2AX seems to play an important role in the processing and repair of DSBs, which is crucial for the maintenance of genome integrity and stability, because H2AX–/– mice exhibited radiation sensitivity, growth retardation, immune deficiency, and infertility in mutant males [14, 15]. H2AX also determines the recruitment of many other repair factors to damaged DNA sites, including Rad50, Rad51, and BRCA1 [16]. The mechanisms underlying the phosphorylation of H2AX have also been investigated, and it has been shown that members of the phosphatidylinositol 3-kinase family (PI3K), such as ATM (ataxia telangiectasia mutated), ATR (ATM and Rad3-related), and DNA-PK (DNA-dependent protein kinase), are involved in the phosphorylation of H2AX [17–19]. Although H2AX is regarded as a specific indicator for DSBs, many genotoxic agents, which do not cause DSBs directly, have also been shown to induce H2AX foci formation [20], suggesting that H2AX might be a more general indicator for DNA damage than previously thought.
The preimplantation mammalian embryo is sensitive to the environment in which it develops, either in vitro or in vivo [21]. This sensitivity may lead to long-term alterations in the characteristics of fetal and/or postnatal growth and phenotype, which may be important for clinical and biotechnological applications [21]. Also, with the development of in vitro fertilization (IVF) techniques in human-assisted reproduction and in livestock improvement, the opportunity for exposure to potential environmental adverse factors including EMFs increases dramatically.
Previous studies have shown that ELF-EMFs can disturb the development of mouse embryos in vitro; however, the mechanism remains unknown [22]. In this study, we investigated the effects of 50-Hz EMFs on DNA in mouse preimplantation embryos by using H2AX as an indicator. As reported here, EMF exposure did induce H2AX foci formation, indicating the presence of DNA damage, which may partially contribute to the development-related effects of EMFs.
MATERIALS AND METHODS
Animals, Embryo Culture, and Analysis
Animal care and use procedures were in accordance with the institutional guide of the laboratory animal established by the Animal Care and Use Committee (ACUC), and were approved by the ACUC of Zhejiang University, School of Medicine. Female ICR mice (Zhejiang University Animal Center, Hangzhou, China) were housed in 12L:12D (light on from 0:00 to 12:00) at 25 ± 0.5°C and 50% to 60% of humidity. The mice were synchronized and superovulated by intraperitoneal injection with 7.5 IU eCG (Intervet), and 48 h later, they were injected with human chorionic gonadotropin (hCG; Pregnyl, Organon, the Netherlands). They were bred with proven breeder males and were checked the next day for a copulation plug (designated as Day 0 of gestation). Females were killed on Day 1 (d1) or Day 2 (d2), and two-cell- or eight-cell-stage embryos were collected by flushing modified human tubal fluid (MHTF; Irvine Scientific, Irvine, CA) through the oviducts and/or uteri [23]. The embryos were rinsed 3 times in MHTF and then transferred into the culture medium. Embryos were cultured in 400-µl droplets of human tubal fluid (HTF; Irvine Scientific) medium, covered with mineral oil, and maintained in a humidified atmosphere of 5% CO2 at 37°C. Daily microscopic evaluation of embryos was performed with the aim of recording any delay and/or abnormality that might arise during their development. Blastomere cleavage, occurring every 12 to 24 h in preimplantation mouse embryos, was monitored daily.
Exposure System
The 50-Hz sinusoidal EMF cell-exposure system used in the present study was the same as the one described in the previous studies [24, 25]. In brief, the exposure system is composed of three groups of copper coils, each with 36 cm width and 8 cm height, in a CO2 incubator (model 3164, Forma) at 37°C. The upper, middle, and lower coils have 168, 60, and 168 turns, respectively, and are spaced 8 cm apart from each other. Two power regulators control these coils, and a voltage stabilizer is used to remove electrical spikes and transients. The system generates highly uniform magnetic fields in the center of a 10 x 10 x 10-cm cube when the coils are energized. It can generate a magnetic flux density as high as 0.8 mT, which is measured by a power frequency field meter (EFA-2, W&G, Eningen, Germany).
To generate a noise MF, this system was double-wrapped with two lines of copper wires. One of the double wires was provided with a 30- to 90-Hz white-noise signal (generated through software designed by Dr. Penafiel, Catholic University of America) after magnifying through power amplifier (CROWN 1400CSL). The other wire was fed with sinusoidal MF during combined MF exposure (MF + noise MF). The amplitude of the noise MF was adjusted to produce an MF strength (rms) equal to the sinusoidal fields used in the experiments.
The electromagnetic waveforms of different MFs were monitored by an oscillograph. The AC background field in the incubator was 12 µT, and total static magnetic field was 18.5 µT with 14.1-µT horizontal and 12.0-µT vertical components.
The sham exposure system was the same as exposure system but without current or signal input. Temperature in all the exposure systems was monitored with a thermocouple probe (sensitivity, ±0.1°C) near the plates containing samples and kept at 37 ± 0.2°C throughout the entire experiment. The exposure system was turned on at least 2 h before the experiment to achieve system stability and thermal equilibrium. After exposure, plates were removed from the container while the system was still on.
Embryo Exposure
Four-well plates were used in the present study. Each four-well plate contained 30 embryos (eight-cell embryo groups) or 40 embryos (two-cell embryo groups), respectively. The plates were placed coaxially with the center line in the central area of the coils, and the EMF was perpendicular to the plates. To maintain a uniform exposure, two to four plates of embryos were exposed in the system in one experiment.
The experiments were conducted in nine treatment groups with four dishes in each group: a) sham exposure, b) 0.3-mT EMF for 24 h, c) 0.3-mT EMF for 48 h, d) 0.5-mT EMF for 24 h, e) 0.5-mT EMF for 48 h, f) pretreated with 200-µM wortmannin for 30 min and 0.5-mT EMF for 24 h; g) 0.5-mT EMF for 24 h, and no exposure for another 24 h; h) 0.5-mT noise MFs alone for 24 h; and i) 0.5-mT EMF and 0.5-mT noise MF for 24 h. The exposed embryos included both two-cell embryos and eight-cell embryos. N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), an alkylating agent, could strongly induce H2AX foci formation in several cell lines [26, 27]. The treatment of embryos with MNNG was used as a positive control in the present study. To examine the effects of wortmannin, a fungal PI3K inhibitor, on the phosphorylation of H2AX, embryos were preincubated with 200-µM wortmannin for 30 min before EMF exposure.
Immunofluorescent Analysis
Embryos were washed extensively in protein-free HEPES-HTF before fixation in 4% paraformaldehyde (Sigma, St. Louis, MO) at 25°C for 1 h. They were permeabilized with 1% Tween 20 in PBS containing 0.1% BSA at 4°C for 60 min. Heat-inactivated sheep serum in PBS (30% v/v) with 2% BSA was used to block nonspecific binding at 25°C for 30 min. Embryos were incubated in primary antibody at 4°C for 24 h, followed by FITC-labeled secondary antibody at 37°C for 30 min. For nuclear staining, 250 ng/µl DAPI or 200 ng/µl PI was added to 10 µl of antifade medium (50% v/v glycerol in PBS) before a whole embryo was placed on the glass slide. A coverslip was gently pressed onto petroleum jelly strips placed on two sides of the slide until the embryos bulged slightly. Excessive fluid was removed with tissue, and the coverslip was sealed around the edges with varnish.
An epifluorescent microscope (Olympus, Japan) fitted with 10x and 20x objectives was used to visualize immunostaining of embryos. Every experiment had several negative control samples: no primary antibody but fluorescent secondary antibody was added.
The primary antibodies included a mouse monoclonal anti-H2AX and a rabbit monoclonal anti-Rad50 used at dilutions of 1:2000 and 1:4000, respectively. The secondary antibodies included FITC-conjugated rabbit-anti-mouse secondary antibody and Cy3-conjugated goat-anti-rabbit secondary antibody, used at dilutions of 1:400 and 1:500, respectively.
Data Statistics
In the experiments of the effects of 50-Hz EMF exposure on the development of embryos, data were collected by counting the numbers of cleaved embryos in the total embryos in each group (80 embryos in the two-cell group or 60 embryos in the eight-cell group in each experiment). In the experiments on the effect of 50-Hz EMF exposure on the formation of H2AX foci in embryos, data were collected by counting H2AX-positive embryos in the total embryos in each group (80 embryos in the two-cell group or 60 embryos in the eight-cell group in each experiment). All the experiments were repeated at least 3 times. Data are presented as the mean ± SEM. Statistical analysis was performed with the 
2 test. A probability level of P < 0.05 was considered significant.
RESULTS
Effects of 50-Hz EMF Exposure on the Development of Mouse Embryos
To evaluate the effects of EMF exposure on development, the cleavage rate of the two- and eight-cell embryos during EMF exposure for 24 h was examined. As shown in Table 1, 90.0% of the two-cell embryos in the control group reached four-cell stage after 24 h, whereas 81.3% of the two-cell embryos reached four-cell stage in the 0.3-mT EMF–exposed group. However, in the 0.5-mT EMF–exposed group, only 37.5% of two-cell embryos reached the four-cell stage. Similarly, in the eight-cell embryo groups, only 16.7% of embryos reached the morula stage after they were exposed to 0.5-mT EMF for 24 h, significantly lower than that in the control group (93.3%) and the 0.3-mT EMF–exposed group (90%).
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Effects of 50-Hz EMF Exposure on H2AX Foci Formation in Mouse Embryos
In our preliminary study, it was found that 50-Hz sinusoidal EMF exposure at 0.5 mT induced H2AX foci formation in mouse preimplantation two-cell embryos after 24-h exposure. Based on this observation, the effects of 50-Hz EMF exposure at 0.3 mT or 0.5 mT for 24 h and 48 h on H2AX foci formation in preimplantation two- and eight-cell embryos were examined with immunofluorescent microscopy. MNNG, a positive control, induced H2AX foci in almost all embryos (Fig. 1, A and B). No H2AX foci formation was detected in embryos treated with vehicle. Compared with the control group, exposure of embryos to 50-Hz EMF at 0.3 mT for 24 h did not induce a significant increase in H2AX foci formation in two- or eight-cell embryos (Fig. 1B). H2AX foci formation was significantly increased in embryos after embryos were exposed to 50-Hz EMF at 0.5 mT for 24 h (Fig. 1, A and B). Similar results were also observed in 48-h exposure (Fig. 1B).
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Delaying Effects on Development Consistent with DNA-damaging Effects of EMF on Two- and Eight-cell Embryos
Further analysis demonstrated that the percentage of embryos with H2AX-positive labeling was mostly equal to the percentage of the embryos remaining in the two- or eight-cell stage in each group (Table 1). This result suggests that the delayed development of embryos exposed to EMF may result from DNA damage in two- and eight-cell embryos in vitro.
Effects of a PI3K Inhibitor, Wortmannin, on H2AX Foci Formation in EMF-treated Mouse Embryos
Earlier studies showed that wortmannin, a fungal PI3K inhibitor, can inhibit phosphorylation of H2AX in response to IR and replication stress [11, 28]. In the present study, the effects of wortmannin on EMF-induced H2AX phosphorylation in two-cell preimplantation embryos were examined. Preincubation of two-cell preimplantation embryos with 200- µM wortmannin for 30 min significantly inhibited the H2AX foci formation in the embryos exposed to 0.5-mT, 50-Hz EMF for 24 h (Fig. 2, A and B).
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Formation of Rad50 Foci and Their Co-localization with H2AX Foci after EMF Exposure
Rad50 is a component of the MRN (Mre11-Rad50-Nbs1) complex. MRN has been shown to play a vital role in both the processing and assembly of foci that facilitate efficient DSB responses [29]. Furthermore, Rad50 has been observed to colocalize with H2AX after DNA damage [16]. Thus the formation of Rad50 foci and the colocalization with H2AX was examined in the preimplantation two-cell embryos exposed to 0.5-mT 50-Hz EMF for 24 h. As shown in Figure 3, A and B, in sham-treated embryos, no Rad50 foci formation occurred; however, after 24 h of EMF exposure, Rad50 foci were readily detected. Furthermore, the Rad50 foci and H2AX foci were found colocalized. Because Rad50 is implicated in the repair of DBSs, we further conducted an experiment in which a group of two-cell embryos was cultured for another 24 h after 24-h EMF exposure. A significant decrease was found in the percentage of embryos remained in the two-cell stage in the group cultured for another 24 h without EMF exposure compared with the group cultured for 48 h with continuous EMF exposure (Fig. 4A). The result was consistent with the decrease in the percentage of H2AX-positive embryos (Fig. 4B).
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Effects of Noise MFs on EMF-induced H2AX Foci Formation in Mouse Embryos
Previous studies have shown that superimposing a spatially coherent but random-noise MF on a coherent EMF can abolish the bioeffects induced by the latter at intensities of 0.0001 to 0.05 mT [30–33]. To determine whether noise MFs can also interfere with the EMF-induced H2AX foci, preimplantation two-cell embryos were superimposed with a noise field when exposed to 0.5-mT 50-Hz EMF. Twenty-four hours later, the formation of H2AX foci was examined. It was found that noise MF alone had no significant effects on H2AX foci formation, and it did inhibit H2AX foci formation induced by EMF exposure in preimplantation embryos (Fig. 5).
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DISCUSSION
Several studies have demonstrated that EMF has genotoxic effects on human and animals. For example, significant increases in the numbers of micronuclei have been found in human peripheral lymphocytes exposed to 75-µT and 150-µT EMF at 32 Hz in vitro [34]. A dose-dependent increase in DNA strand breaks also was noted in SD rat brain cells after whole-body exposure to EMF (60 Hz, 0.1 mT, 0.25 mT, and 0.5 mT) [35]. Recently, the Report of the European Union's REFLEX (Risk Evaluation of Potential Environmental Hazards from Low Frequency Electromagnetic Field Exposure Using Sensitive In Vitro Methods) Project was released, which showed that ELF-EMFs had genotoxic effects on primary human fibroblasts as well as in other cell lines (e.g., ELF-EMFs generated significant DNA strand breaks at a flux density as low as 35 µT [36]). However, many other studies have also reported a negative outcome for the genotoxicity of 50/60-Hz EMFs [37–39]. In this regard, the different exposure conditions, cell types, and end points used may have contributed to the inconsistent observations. However, the method used to detect DNA damage may be rather important, as different methods have quite variable sensitivity for detecting DNA damage and may reflect different mechanisms underlying DNA damage.
It should be noted that, in a number of these studies, the genotoxic effects of EMFs were assessed by the micronucleus assay, which represents a rather profound change in DNA. The comet assay is a relatively sensitive method for detecting DNA strand breaks; nonetheless, the effects of EMFs on DNA may still be below the detection limit of the comet assay. H2AX foci formation is emerging as a new sensitive method for detecting DNA damage. The formation of H2AX has been identified as an early event after the production of DSBs. Within minutes of damage, H2AX in a 2-Mb region around the break site becomes phosphorylated on serine 139, producing foci that are microscopically visible when labeled with a specific antibody [16, 40]. The potential to detect a single focus within the nucleus makes this the most sensitive method currently available for detecting DSBs. Ever since H2AX was first reported, it has been applied in many studies to demonstrate DSB formation induced by various chemicals, viruses, or even heat shock [20, 41–43]. In our study, using H2AX foci formation as an indicator, we found that 50-Hz EMF at 0.5 mT did induce DNA damage in the preimplantation embryos. The results suggest that the detection of H2AX foci formation, which is sensitive and can be conducted on individual embryos, may be a useful method for DSB evaluation in embryos and may provide a molecular marker that is qualitative.
The genotoxic effect of EMF appears to be dose related in the present study. The onset of genotoxic effects in our study was at a magnetic flux density of 0.5 mT, but not at 0.3 mT, for continuous exposure, which is consistent with the exposure limit of 0.5 mT for the general population from the guidelines of the International Commission on Non-Ionizing Radiation Protection (ICNIRP 1998). However, previous study has shown that continuous exposure of human fibroblasts to 50-Hz EMF at 0.5 mT does not induce genotoxicity [36]. This obvious difference may be due to the different experimental systems used (e.g., fibroblasts vs. embryos). More importantly, the different observations may have resulted from the different methods used for detecting DNA damage. With a well-known genotoxic agent, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), we had systematically compared the sensitivity of both the comet assay and H2AX foci in detecting DNA damages, and found that H2AX was more sensitive in detecting DNA damages induced by low concentrations of MNNG than the comet assay [26]. Because EMFs may be a very weak genotoxic agent, it is very possible that the comet assay cannot detect the DNA damage caused by EMF exposure at such level.
The kinases responsible for the phosphorylation of H2AX have been identified as members of the PI3K family, including DNA-PK, ATM, and ATR. It has been shown that these kinases are activated under DNA-damaging conditions, leading to the phosphorylation of H2AX. Inhibition of these kinases by using specific inhibitors, such as wortmannin, may inhibit the phosphorylation of H2AX [11, 28]. Therefore we also tested the involvement of PI3K in EMF-induced H2AX foci formation. As reported here, preincubation of cells with wortmannin significantly inhibited the formation of H2AX foci after EMF exposure, suggesting that members of the PI3K family may be involved in the phosphorylation of H2AX under such conditions. However, further study should be conducted to clarify which member is the most important one involved in this process.
The disappearance of H2AX foci (dephosphorylation of H2AX) has been suggested as an indication of the repair of DSBs taking place [44]. The two most important pathways in repairing DSBs are the homologous recombination (HR) or nonhomologous end joining (NHEJ). MRN complex has been shown to be involved in both pathways [45, 46]. In mammalian cells, the MRN complex appears to be one of the earliest sensors of DSBs. MRN is one of the first factors to be localized to the DNA lesion, where it may have a structural role by tethering together, and therefore stabilizing, broken chromosomes [29]. MRN binding likely facilitates the tethering of DSB ends via a zinc-hook structure in Rad50 [47, 48]. Thus the possible involvement of MRN complex in the repair of EMF-induced DNA damage was also examined. By using immunofluorescent microscopy, we found that Rad50 protein also formed foci in EMF-exposed embryos; furthermore, the Rad50 foci colocalized with H2AX foci (Fig. 3A), indicating that the MRN complex probably participated in the repair process by binding to the damaged DNA sites. In the present study, we also found that if the exposed embryos culture for another cleavage cycle of time without exposure, a significant decrease occurred in both the percentage of embryos remaining in the two-cell stage and the percentage of H2AX foci-positive embryos. The results suggest that the DNA-damaging effects and the embryo development-delaying effects of EMF may be revisable.
It is proposed that living cells are affected only by EMF that is spatially coherent over their surface. However, as with all physical detection systems, cells are subject to the laws of conventional physics and can be confused by noise. Litovitz et al. [49] suggested that a spatially coherent but temporally random noise field superimposed on a coherent EMF signal might defeat the mechanism of discrimination against noise and that any observed field-induced bioeffects would be suppressed. This hypothesis was supported by a line of experimental evidence. By measuring different biological end points, accumulating data showed that superimposing a spatially coherent but random noise field on a coherent ELF-MF could abolish the bioeffects induced by the ELF-MF at intensities of 0.0001 to 0.05 mT. Those bioeffects include cell growth [30], chicken embryo development [49], c-myc transcription [50], 5'-nucleotidase and ornithine decarboxylase (ODC) activity [32, 33], and neurotransmitter synthesis (dopamine) [51]. With a similar exposure system, we also explored the possible disruptive effects of the superimposed noise MFs on the formation of H2AX foci induced by EMFs. Although noise MF itself did not significantly induce H2AX foci formation, it strongly inhibited the formation of H2AX foci induced by EMF exposure. The present study provides another piece of evidence supporting Litovitz's temporal-and-spatial coherence hypothesis [49] and justifying noise MFs as a potential approach to prevent EMF-induced hazardous effects as well.
If the DNA damage is not so severe, the damage is repaired by cell-cycle arrest, that is, the "time-out" response activated by the so-called cell-cycle checkpoints [52, 53]. In our study, we found that the cleavage rate of the exposed two- and eight-cell embryos was significantly delayed when compared with that of the control embryos. Additionally, it should be noted that, in the present work, we found that the delaying effects of EMFs on development of embryos are consistent with the DNA-damaging effects of EMFs on embryos. The results strongly suggest that the adverse effects of EMFs on development of embryos may, at least partly, act through DNA damage and subsequent DNA repair. The delay in cleavage rate may be the result of cell-cycle arrest; however, determining at which phase the cells are arrested requires further investigation.
For most species, the embryonic stage of development, compared with the somatic cell stage, is the life stage most sensitive to the toxic effects of environment. Direct analysis of preimplantation embryos presents many advantages for mechanistic studies. Any sublethal changes of the embryos may impair the quality of the embryos, causing preimplantation loss, implantation failure, spontaneous abortion, or developmental abnormality. An increased risk of congenital malformations is currently being recorded both in humans and in livestock [21]. In the present study, we found that a significant delay of development in EMF-exposed embryos. With the development of in vitro fertilization (IVF) technology, the chances for EMF exposure of the embryos increase significantly. In addition, exposure strength of EMFs in the environment also increased by severalfold during the last two or three decades, which may also increase the exposure risk for pregnant women. Therefore our results should be considered when evaluating the hazardous effects of environmental EMFs on reproduction and when searching for an effective approach for shielding from them.
However, it is noted that several in vivo studies have indicated a lack of sensitivity to EMF with respect to fertility and litter size [54–56], which contradicts our findings. Still, considering the complexity of a biological system, different or even conflicting results could be obtained from in vitro and in vivo studies. For in vitro studies, the system has been simplified, and very few other interfering factors are involved—except the EMF exposure. Conversely, during in vivo studies, many unwanted factors may be involved, which would complicate the results. This is quite obvious during the study of many chemotherapeutic drugs, which have shown great promise in in vitro studies, but never succeeded in in vivo studies. In addition, even though the animal was exposed to a certain amount of EMFs, it is unclear exactly how much the uterus was exposed. Finally, the exposure strength, duration, stages of embryos, and testing end point were somewhat different between these studies and our experimental settings. Therefore it is believed that the inconsistent results may reflect more a difference of experimental system rather than a fundamental difference of response to the same stimulus.
In summary, by using H2AX foci formation as an indicator, we report for the first time that 50-Hz, 0.5-mT EMF exposure can induce DNA damage in preimplantation mouse embryos in vitro, accompanied with a decrease in the cleavage rate of the embryos. Preincubation of cells with wortmannin significantly inhibited the formation of H2AX foci after EMF exposure. Noise MFs strongly inhibited the formation of H2AX foci induced by EMF exposure. These data suggest that the genotoxic effects of EMFs on preimplantation embryonic development do exist and appear to be dose dependent. The PI3K family is involved in H2AX foci formation, and H2AX may be a better indicator for detecting DNA damage induced by EMFs, which can reflect the damage at the molecular level. It is postulated that noise MFs might be used to prevent the genotoxic effect of EMFs on preimplantation embryonic development.
ACKNOWLEDGMENTS
We thank Deqiang Lu for his helpful work for the EMF exposure experiment and Prof. Huai Jiang for her helpful suggestions about this manuscript.
FOOTNOTES
2 Correspondence: FAX: 86 571 8721 7044; huanghefg{at}hotmail.com 
1 Supported in part by National Natural Science Foundation of China (NO.30471642) and Basic Research Program of China (NO.973:2006CB504004).
Received: 9 March 2006.
First decision: 24 April 2006.
Accepted: 17 July 2006.
REFERENCES
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