Comparison of the Genotoxic Effects Induced by 50 Hz Extremely Low-Frequency Electromagnetic Fields and 1800 MHz Radiofrequency Electromagnetic Fields in GC-2 Cells
Duan W, Liu C, Zhang L, He M, Xu S, Chen C, Pi H, Gao P, Zhang Y, Zhong M, Yu Z, Zhou Z. Comparison of the Genotoxic Effects Induced by 50 Hz Extremely Low-Frequency Electromagnetic Fields and 1800 MHz Radiofrequency Electromagnetic Fields in GC-2 Cells. Radiat Res. 2015 Feb 17. [Epub ahead of print]
Extremely low-frequency electromagnetic fields (ELF-EMF) and radiofrequency electromagnetic fields (RF-EMF) have been considered to be possibly carcinogenic to humans. However, their genotoxic effects remain controversial. To make experiments controllable and results comparable, we standardized exposure conditions and explored the potential genotoxicity of 50 Hz ELF-EMF and 1800 MHz RF-EMF. A mouse spermatocyte-derived GC-2 cell line was intermittently (5 min on and 10 min off) exposed to 50 Hz ELF-EMF at an intensity of 1, 2 or 3 mT or to RF-EMF in GSM-Talk mode at the specific absorption rates (SAR) of 1, 2 or 4 W/kg. After exposure for 24 h, we found that neither ELF-EMF nor RF-EMF affected cell viability using Cell Counting Kit-8. Through the use of an alkaline comet assay and immunofluorescence against γ-H2AX foci, we found that ELF-EMF exposure resulted in a significant increase of DNA strand breaks at 3 mT, whereas RF-EMF exposure had insufficient energy to induce such effects. Using a formamidopyrimidine DNA glycosylase (FPG)-modified alkaline comet assay, we observed that RF-EMF exposure significantly induced oxidative DNA base damage at a SAR value of 4 W/kg, whereas ELF-EMF exposure did not. Our results suggest that both ELF-EMF and RF-EMF under the same experimental conditions may produce genotoxicity at relative high intensities, but they create different patterns of DNA damage. Therefore, the potential mechanisms underlying the genotoxicity of different frequency electromagnetic fields may be different.
With the pervasive presence of electromagnetic fields (EMFs) in daily life, concerns have been growing worldwide regarding the potential adverse effects of exposures to nonionizing radiation, particularly to extremely low-frequency electromagnetic fields (ELF-EMF) generated from use of electricity and radiofrequency
electromagnetic fields (RF-EMF) emitted from wireless devices such as cellular phones. A large number of epidemiological studies suggests an association between exposure to ELF-EMFs and an increased incidence of cancers particularly childhood leukemia and brain tumors (1–4). A meta-analysis has noted an association between mobile phone use and ipsilateral glioma and acoustic neuroma (5). However, other epidemiological results did not support an epidemiological association of adult cancers with ELF-EMF exposure (6, 7) and brain tumors with mobile phone use (8). In view of the controversial epidemiological studies and limited evidence in experimental animals, both ELF-EMF and RF-EMF were considered to be possibly carcinogenic to humans by the International Agency for Research on Cancer (IARC). However, the carcinogenic potential of EMF remains uncertain.
Genotoxic effects have been widely used to determine whether an environmental factor is a carcinogen. Of studies investigating potential genotoxic effects of ELF-EMF, 22% reported positive results, 46% reported negative results and 32% were inconclusive (9). Meanwhile, of studies investigating genotoxic effects of RF-EMF, 48.5% reported genotoxic effects, 42.5% reported no genotoxicity, and the remainder found that RF-EMF could act as a promoter or co-promoter of genotoxicity (10). In addition, approximately 50% of studies reported that exposure to EMF could cause DNA damage (10). Although a large number of studies have investigated the genotoxic effects of EMF, the aggregated results remain contradictory. There are various reasons that account for these noncomparable and controversial results, including nonstandardized exposure systems and conditions, varying assay sensitivities, different cell lines, and use of different EMF frequency, intensity, exposure duration and exposure mode (10–14). Therefore, a standardized exposure setup and a more sensitive cell line and methods are warranted to evaluate the genotoxicity of EMF.
A number of recent studies have revealed potential harmful effects of EMF exposure for male reproduction (15–19). Both ELF-EMF and RF-EMF exposure have been shown to induce DNA damage in sperm (20). Male germ cells, especially post-meiotic germ cells, including spermatocytes, spermatids and spermatozoa, are uniquely vulnerable to DNA damaging agents (21). Genetic effects in germ cells can result in genetic damage in next and subsequent generations (22). These cells have largely lost their cytoplasm, which possesses abundant antioxidant enzymes against free radical attack, and they have many targets for the induction of peroxidative damage, including polyunsaturated fatty acids and DNA (15, 22, 23). In light of these considerations, we selected a mouse spermatocytederived
cell line (GC-2) as a sensitive model to investigate genotoxicity of EMF ...
To examine and compare the genotoxic effects of ELF-EMF and RF-EMF, we used standardized exposure conditions. A mouse spermatocyte-derived GC-2 cell line was intermittently exposed (5 min on/10 min off) to 50 Hz ELF-EMF or 1800 MHz RF-EMF using an IT’IS Foundation-designed exposure system. After 24 h of
exposure, we assayed cell viability and the level of DNA damage ...
... At 24 h after cell seeding, the culture medium was renewed, and cells were exposed to 50 Hz sinusoidal ELF-EMF at magnetic flux densities of 1, 2 and 3 mT or to 1,800 MHz GSM-Talk signals at SAR values of 1, 2 and 4 W/kg at the same time with intermittency cycles of 5 min field on and 10 min field off for 24 h.
ELF: The temperature difference between the exposure and the sham-exposure chambers did not exceed 0.1 degrees C, so possible thermal effects could be ruled out. All exposure experiments were performed under blind conditions: the computer randomly decided which of the two chambers was exposed in each trial and all the data were encrypted in a file which were decoded by IT’IS foundation following the data analysis.
RF: The temperature difference between RF-exposed and sham-exposed chambers did not exceed 0.1 degrees C, so possible thermal effects could be ruled out. All exposure experiments were performed under blind conditions too.
In the current study, we investigated the potential genotoxicity of intermittent exposure to ELF-EMF or RF-EMF on mouse spermatocyte-derived cells. Using the same conditions, we found that ELF-EMF exposure can induce DNA strand breaks while RF-EMF exposure can cause oxidative DNA base damage. These results suggest that both ELF- and RF-EMF may produce genotoxicity, but they may differ in the type of DNA damage induced ...
To investigate the potential genotoxicity of ELF-ELF and RF-EMF, we first used an alkaline comet assay to detect single- and double-strand breaks ... We found
ELF exposure for 24 h can induce DNA strand breaks at an intensity of 3 mT, although exposure to ELF-EMF of this intensity does not affect cell viability. DNA double-strands breaks were observed ... Several other studies revealed that time-varying magnetic fields led to DNA double-strands breaks at several mT (40, 42). Therefore, our results support the idea that high-intensity ELF-EMF should be considered a genotoxic factor due to the resultant DNA breakage and damage (43, 44).
... we used an FPG-modified comet assay to detect whether oxidation is involved in ELF-induced DNA strand breaks. We failed to detect any oxidative DNA base damage induced by ELF exposure even at 3 mT, which suggests that oxidation is not involved in ELF-EMF induced DNA strand breaks.
Using the same experimental conditions, we found no detectable DNA strand breaks after RF exposure, which further confirms our previous study (34). The results suggest that RF-EMF exposure is insufficient to directly induce DNA strand breaks in male germ cells. In our opinion, RF-EMF may be genotoxic under certain conditions, including high frequencies or high-power intensities and in some cell types (human trophoblast HTR-8/SVneo cells, human leukocytes and spermatozoa) (15, 48–50) although many international expert groups consider that there are still no solid data in favor of this. For example, no DNA strand breaks were found in mouse fibroblast cells, HL-60 cells, human white blood cells, Molt-4 cells, human blood lymphocytes, human ES1 diploid fibroblasts or Chinese hamster V79 cells under the similar RF-EMF exposure conditions (51–55). Furthermore, the data from over 100 studies suggest that RF-EMF is not directly mutagenic (49).
Many previous studies have demonstrated that RF-EMF exposure increases ROS production in vitro and in vivo (45, 56–58). Therefore, we wondered whether DNA treated with low-energy RF-EMF might be more sensitive to base oxidation than to strand breakage. To test this hypothesis, we used an FPG-modified comet assay and found that RF-EMF exposure caused oxidative DNA base damage at a SAR value of 4 W/kg, as expressed as tail DNA (%). However, no significant difference was found in tail length between the RF-exposed and the sham-exposed groups... In our previous study, we demonstrated that RF-EMF exposure increased the level of ROS, which contributed to oxidative DNA base damage (34). This type of DNA damage may induce genome instability, chromosomal aberrations and even tumorigenesis (60). We have also demonstrated that melatonin could protect against mobile phone-induced DNA damage in GC-2 cells (61). These results suggest that RF-EMF might induce DNA damage and produce genotoxicity through the action of free radical species. Consistent with our results, De Iuliis et al. reported 1.8 GHz RF-EMF induced oxygen species production and DNA damage in human spermatozoa in vitro (15). Tomruk et al. reported that exposure to 1,800 MHz GSM-like RF radiation led to hepatic oxidative DNA damage in pregnant rabbits and their offspring (62).
In conclusion, our results clearly demonstrate that both ELF-EMF and RF-EMF have potential genotoxicity under the same conditions but that the underlying mechanism is different. The difference may be attributed to the different frequencies of ELF-EMF (1–300 Hz, mainly 50/60 Hz) and RF-EMF (10 kHz–300 GHz). The exposure levels of 3 mT for ELF-EMF and 4 W/kg for RF-EMF that produce DNA damage are higher than the current allowable levels, which may have no significance to actual human exposures but provide some mechanistic information about high-dose exposures. Consequently, there remains cause for concern
about the safety of exposure to high intensity of EMF. This issue deserves further study using both in vitro and in vivo models.
Joel M. Moskowitz, Ph.D., Director
Center for Family and Community Health
School of Public Health
University of California, Berkeley
Electromagnetic Radiation Safety
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