Comment;
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.
DOI: 10.1667/RR13851.1
Comparison of the Genotoxic Effects Induced by 50 Hz Extremely Low-
Frequency Electromagnetic Fields and 1800 MHz Radiofrequency
Electromagnetic Fields in GC-2 Cells
Weixia Duan,1 Chuan Liu,1 Lei Zhang, Mindi He, Shangcheng Xu, Chunhai Chen, Huifeng Pi, Peng Gao, Yanwen
Zhang, Min Zhong, Zhengping Yu and Zhou Zhou2
Department of Occupational Health, Third Military Medical University, Chongqing 400038, People’s Republic of China
Duan, W. X., Liu, C., Zhang, L., He, M. D., Xu, S. C., Chen,
C. H., Pi, H. F., Gao, P., Zhang, Y. W., Zhong, M., Yu, Z. P. and
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. 183, 305–314 (2015).
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 contro-
versial. 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 c-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 electro-
magnetic fields may be different. 2015 by Radiation Research
Society
INTRODUCTION
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 extreme-
ly low-frequency electromagnetic fields (ELF-EMF) gener-
ated 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 experimen-
tal 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 investi-
gating 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, approximate-
ly 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 contro-
versial results, including nonstandardized exposure systems
and conditions, varying assay sensitivities, different cell
lines, and use of different EMF frequency, intensity,
1 These authors contributed equally to the study.
2 Address for correspondence: Department of Occupational Health,
Third Military Medical University, Chongqing 400038, People’s
Republic of China; e-mail: lunazhou00@163.com.
305
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 sper-
matocytes, 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 spermatocyte-
derived cell line (GC-2) as a sensitive model to investigate
genotoxicity of EMF.
The comet assay is a well-established genotoxicity test for
detection of DNA damage both in vivo and in vitro (24, 25).
In mammals, one of the earliest biomarkers of DNA double-
strand breaks (DSBs) is phosphorylation of serine 139 of
H2AX (26). The c-H2AX assay for DNA double-strands
break is 100-fold more sensitive than the alkaline comet
assay (27). While the alkaline comet assay detects both
single- and double-strands breaks, the c-H2AX assay
primarily detects double-strands breaks. The formamido-
pyrimidine DNA glycosylase (FPG)-modified comet assay
for oxidative DNA base damage is 1,000-fold more
sensitive than the immunoassay for 8-oxoG; the sensitivities
of these two assays are ;1 adduct/108 nucleobases and ;1
adduct/105 nucleobases, respectively (28). The FPG enzyme
mainly converts the most prevalent oxidized purines, which
primarily derive from guanine, such as the premutagenic 8-
oxoguanine lesion and the open ring formamidopyrimidine
(FAPY), into DNA strand breaks (29). In addition, the FPG-
modified comet assay can provide important insights into
the mechanisms of DNA damage (30, 31). Therefore, the
combined use of these methods would be better able to
detect DNA damage caused by EMFs.
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.
MATERIALS AND METHODS
Cell Culture
A mouse spermatocyte-derived cell line (GC-2) was purchased from
the American Tissue Culture Collection (ATCC; Rockville, MD) and
incubated at 378C in a 5% CO2 humidified atmosphere. The GC-2
cells were maintained in Dulbecco’s modified Eagle medium (DMEM,
HyClone, Logan, UT) containing 4.5 g/L glucose, 0.5 mM L-
glutamine, 110 mg/L sodium pyruvate and supplemented with 10%
fetal bovine serum (FBS, HyClone) and 100 U/ml penicillin and
streptomycin (Beyotime Institute of Biotechnology, Haimen, China).
The cell doubling time of GC-2 cells were 24 h at 378C (32). Two ml
of cell suspension were seeded into a 35 mm Petri dish (Corning, NY,
US) at a density of 1 3 105 cells/ml. 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-EMF Exposure
The ELF-EMF exposure system (shown in Fig. 1A) was purchased
from the Foundation for Information Technologies in Society (IT’IS-
foundation, Zurich, Switzerland), detailed descriptions were given by
Focke et al. and Schuderer et al. (12, 13). Briefly, the system primarily
consisted of a computer, an ELF generator and two mu-metal box
chambers. Each chamber contained 2 coils with 56 windings, 2 coils
with 50 windings, two fans, a petri dish holder and a temperature
sensor (PT100 probe). Constant environmental conditions (378C, 5%
CO2, 95% humidity) were ensured by two fans in both chambers,
which were placed inside a commercial incubator (Heracell 240i,
Thermo scientific). The setup has been optimized for homogeneous
field distribution, maximum field strength, minimum temperature
FIG. 1. ELF-EMF and RF-EMF exposure system. Panel A: (a) The
ELF-EMF exposure system consisted of two mu-metal box chambers
installed in an incubator (one lid removed). Panel A: (b) Each chamber
contained 2 coils with 56 windings, 2 coils with 50 windings, two
fans, a petri dish holder and a temperature sensor (PT100 probe).
Panel B: The RF-EMF exposure system consisted of two rectangular
waveguide chambers installed in an incubator (one lid removed).
306 DUAN ET AL.
increase and minimum vibrations. A current source was developed
based on four audio amplifiers (Agilent Technologies, Zurich,
Switzerland) and allowed magnetic fields up to 3.5 mT. The field
could be arbitrarily varied in the frequency range from 3–1,250 Hz by
a computer controlled arbitrary function generator. Nonuniformity of
the magnetic field was ,1% (SD) for all possible petri dish locations.
Fields in sham exposure were less than 43 dB (,0.05%) compared
to field exposure. Pt100 probes continuously monitored the temper-
ature at the location of the dishes and maintained at 37.0–37.58C
during exposure. The temperature difference between the exposure
and the sham-exposure chambers did not exceed 0.18C, 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-EMF Exposure
The RF-EMF exposure system (shown in Fig. 1B) was purchased
from the IT’IS foundation (Zurich, Switzerland), as described in a
previous study (34). Briefly, the system primarily consisted of a
computer, a RF generator and two rectangular waveguide chambers.
Both chambers were placed inside a commercial CO2 incubator. One
of the chambers was excited by an EMF signal mimicking the basic
pulse structure of the GSM signal at 1,800 MHz. The other chamber
was used for sham exposure. The sensors and fans of the exposure
system were connected to a computer that monitored the SAR value
during exposure and maintained a constant environment within the
chambers (378C, 5% CO2, 95% humidity). Six petri dishes (d ¼35
mm) were simultaneously placed in the H-field maxima and exposed
to a polarized E-field (an electric field that is perpendicular to the H-
field). This system operated at a steady frequency of 1,800 MHz and
controlled the SAR variability below 6%. The temperature of this
system was uniformly distributed without ‘‘hot spots’’ and a
maximum rise of ,0.038C/(W/kg) for monolayer cells was docu-
mented. The temperature difference between RF-exposed and sham-
exposed chambers did not exceed 0.18C, so possible thermal effects
could be ruled out. All exposure experiments were performed under
blind conditions too.
Cell Viability Assay
Cell viability was measured using the Cell Counting Kit-8 (CCK-8,
Dojindo Molecular Technologies, Kumamoto, Japan) according to the
manufacturer’s instructions. First, to ensure the assay performed
appropriately, cells treated with 240 lM H2O2 for 15 min were served
as positive controls and unexposed cells were served as negative
controls. GC-2 cells were seeded into 35 mm Petri dishes at a
concentration of 1 3 105/ml in complete medium and grown for 24 h
prior to ELF- and RF-EMF exposure. Immediately after 24 h of
exposure, the medium was discarded and cells were washed 3 times
with phosphate buffered saline (PBS). Then 1 ml of CCK-8 working
solution (10% CCK-8 reagent in cell culture medium) was added to
each dish and further cultured at 378C for 2 h. In the CCK-8 kit, a
novel tetrazolium salt WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-
nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt]
produced a water-soluble formazan dye upon bioreduction in the
presence of an electron carrier 1-methoxy PMS. Finally, the
supernatant was added to a new 96-well plate and the optical density
(OD) value of each well was measured using a microplate reader
(Infinite M200, Tecan, Austria) with a test wavelength of 450 nm. To
determine prolonged effects of EMF exposure, cells were further
incubated in absence of any EMF signals for 24 h after 24 h of ELF-
and RF-EMF exposure, and then cell viability was measured. Results
were expressed as optical density 450 relative to that sham-exposed
cells or negative control cells. All experiments were performed in
triplicate on three separate occasions.
Alkaline Comet Assay and FPG-modified Comet Assay
An alkaline comet assay (the single cell gel electrophoresis assay)
was performed and optimized under alkaline conditions according to
instructions as previously described (35). First, to ensure the comet
assay performed appropriately, cells treated with 30 lM H2O2 for 15
min to induce massive DNA strand breaks were used as positive
controls, and unexposed cells were negative controls. Immediately
after cells were exposed to ELF- or RF-EMF, they were detached by
trypsin digestion and diluted to a concentration of 3–5 3 105/ml. Ten
ll of cell suspensions were mixed with 100 ll 0.65% prewarmed low-
melting point agarose at 378C. The cell/agarose mixture (60 ll) was
then laid onto Trevigen Comet Slides (Trevigen, Gaithersburg, MD)
precoated with 1.0% normal-melting point agarose. After the slides
had been chilled at 48C, they were immersed into freshly prepared
lysis solution (2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris, 1%
Triton X-100, 10% DMSO, pH 10) for 2 h at 48C to lyse cells and
allow DNA unfolding. Then the slides were rinsed with double-
distilled water (ddH2O) 2 times and placed in fresh alkaline buffer (1
mM Na2EDTA, 300 mM NaOH, pH 13) for 30 min at room
temperature to allow DNA unwinding and expose alkali-labile sites.
Subsequently, electrophoresis was conducted in the alkaline buffer at
48C for 40 min at 1 V/cm. Then the slides were immersed twice in
ddH2O for 5 min each and in 75% ethanol for 5 min. The resulting
comets were stained by diluted SYBR Green I (Invitrogen, Carlsbad,
CA) and visualized at 2003 magnification with a Leica microscope
(DM 6000 B, Wetzlar, Germany). All steps after exposure were
performed in the dark.
To determine the level of FPG-sensitive sites, the modified comet
assay was applied according to a previous study (36). After the lysing
step and before unwinding, the slides were washed three times for 5
min each in enzyme buffer solution (40 mM HEPES, 0.1 M KCl, 0.5
mM Na2EDTA, 0.2 mg/ml bovine serum albumin, pH 8.0) at 48C to
remove the remaining lysis buffer. One slide was then treated with 50
ll enzyme buffers only, and the other was treated with 50 ll FPG
enzyme solutions (FPG enzyme diluted in enzyme buffer at 1:1,000)
(New England Biolabs, Ipswich, MA) and cultured in a humidity
chamber at 378C for 30 min. After 10 min of solidification at 48C, the
standard alkaline comet assay procedure was performed as described
above.
Approximately 100 cells from each sample were analyzed in at least
three independent experiments. The edges and damaged parts of the
gel were excluded from analysis. The following two comet parameters
were used to express the level of DNA damage (30, 37): 1. the tail
DNA%: the ratio of the DNA present in the tail to the total DNA
content; 2. the tail length (lm): the distance from the nuclear core to
the end of DNA migration. The DNA damage parameters were
calculated using a computer-based image analysis system (Comet
Assay Software Project, CASP Lab, Poland) (38).
Immunofluorescence Staining
Immunofluorescence detection of c-H2AX was performed accord-
ing to a previous study (39) with minor modifications. To validate the
protocol, cells not treated with any genotoxic agent were used as
negative controls, while cells treated with 2 lg/ml etoposide for 2 h
were used as positive controls. After exposure, cells were immediately
fixed in 4% paraformaldehyde for 10 min and then permeabilized with
0.5% Triton-100 for 15 min at room temperature. Nonspecific binding
sites were blocked with goat serum (Zhongshan Golden Bridge
Biotechnology Co., Ltd.) for 30 min at room temperature. The cells
were then incubated with a mouse monoclonal anti-phosphoserine-139
H2AX antibody (Abcam, Cambridge, MA), at 1:500 dilutions at 48C
overnight in a humidified chamber. The next day, the cells were
incubated with Alexa Fluor 488-conjugated goat anti-mouse second-
ary antibodies (diluted 1:100) (Invitrogen) at room temperature for 1
h. Thereafter, the nuclei were stained with 40, 6-diamidino-2-
phenylindole (DAPI) (Beyotime) for 15 min. Each step was followed
COMPARISON OF GENOTOXICITY OF ELF-EMF AND RF-EMF 307
by 3 washes for 5 min with 0.5% Tween-20 in PBS. Finally, cells were
mounted in a mounting medium containing glycerol (Beyotime) and
visualized under a Leica confocal laser-scanning microscope (TCS
SP2; Germany). c-H2AX positive cells were defined as those
containing more than ten c-H2AX foci per cell and at least 200 cells
per sample were quantified. Each experiment was independently
performed at least four times.
Statistical Analysis
All the data were analyzed with the SPSS 16.0 software package.
First of all, we tested all the data for normal distribution using a
Shapiro-Wilk test. Then, we used independent samples t test to
compare the differences between the sham control and exposure
groups, or between negative control and positive control groups. For
data of FPG-modified comet assay, the independent samples t test was
used to compare differences between FPG (–) and FPG (þ) of the
same group. All exposure experiments were repeated at least three
times on independent samples. The results were presented as mean 6
SEM (standard error of three independent experimental means). A
statistical difference was accepted when P , 0.05.
RESULTS
Neither ELF nor RF-EMF Exposure Affected Cell Viability
As shown in Fig. 2, H2O2 treatment (positive controls)
significantly inhibited the cell viability compared with
negative control groups, suggesting the assay is capable of
detecting the effects on cell viability. After ELF and RF-
EMF exposure, the cell viability was above 90% in all of the
exposed groups and no significant differences were
observed either in ELF- or RF-EMF exposed groups
compared with their respective sham-exposed group (Fig.
2A and B). Meanwhile, no significant changes were found
between exposed groups and sham-exposed groups either in
ELF- or RF-EMF exposure after 24 h prolonged incubation
in absence of EMF exposure (Fig. 2C and D).
ELF-EMF Exposure Induced Detectable DNA Strand
Breakage, but RF-EMF Did Not
To evaluate the possible impact of ELF- and RF-EMF
exposure on DNA, the alkaline comet assay was performed.
The H2O2 treatment significantly increased the percentage
of tail DNA and tail length compared with negative control
groups, which suggested that the dose of H2O2-induced
dramatic DNA damage and validated the comet assay. After
24 h exposure to ELF-EMF, the tail DNA (%) and tail
length increased in a dose-dependent manner and were
significantly elevated at the intensity of 3 mT compared to
the sham-exposed group (P , 0.05, Fig. 3A and B).
However, no statistically significant differences in these
comet parameters were observed between the RF-EMF
exposed and the sham-exposed groups at any SAR values
FIG. 2. Effects of ELF-EMF and RF-EMF exposure on cell
viability in GC-2 cells. ELF-EMF (panel A) and RF-EMF (panel B)
exposure for 24 h did not affect GC-2 cell viability. Prolonged 24 h
incubation in the absence of EMF exposure after 24 h of ELF- and RF-
EMF exposure, neither ELF-EMF (panel C) nor RF-EMF (panel D)
affected the cell viability in GC-2 cells. The error bars represent the
means 6 SEM from three independent experiments. *P , 0.05. NC:
negative control (Cells were cultured at an incubator without ELF-
EMF or RF-EMF signal); PC: positive control (240 lM H2O2 for 15
min).
FIG. 3. Effects of ELF-EMF and RF-EMF exposure on DNA
strand breaks in GC-2 cells. The DNA migration pattern of GC-2 cells
after exposure to ELF-EMF (1, 2, 3 mT) or RF-EMF (1, 2, 4 W/kg) for
24 h or H2O2 (30 lM for 15 min), as evaluated by an alkaline comet
assay. DNA strand breaks were expressed as the tail DNA (%) and tail
length. ELF-EMF: The tail DNA (%) (panel A) and tail length (lm)
(panel B); RF-EMF: The tail DNA (%) (panel C) and tail length (lm)
(panel D). At least 100 nuclei were analyzed for each group of each
experiment. Bars represent the means 6 SEM from three independent
experiments. *P , 0.05. NC: negative control (Cells were cultured at
an incubator without ELF-EMF or RF-EMF signal); PC: positive
control (30 lM H2O2 for 15 min).
308 DUAN ET AL.
(Fig. 3C and D). As shown in the representative comet
images in Fig. 4, DNA migration clearly increased
following either H2O2 treatment or ELF-EMF exposure at
3 mT.
ELF-EMF Exposure Increased Nuclear c-H2AX Foci
Formation, but RF-EMF Exposure Did Not
To further confirm the impact of EMF on DNA, we used
immunofluorescence detection of c-H2AX foci formation,
which was evidenced by green staining. The representative
pictures show that there was strong c-H2AX staining in the
positive control (Etoposide) (Fig. 5A). After exposure to
ELF-EMF for 24 h, the formation of c-H2AX foci
increased, particularly at the 3 mT exposure level, as
evidenced by moderate c-H2AX foci staining intensity (Fig.
5B) and significantly increasing c-H2AX positive cells
compared with those of the sham-exposed group (Fig. 5D).
The images of positive c-H2AX cells at 3 mT were
magnified (Fig. 5B). However, RF-EMF exposure did not
significantly increase c-H2AX foci formation at any SAR
value compared with the sham-exposed group (Fig. 5C and
E).
RF-EMF Exposure Induced Oxidative DNA Base Damage,
but ELF-EMF Exposure Did Not
To further explore whether EMF exposure-induced
oxidative DNA base damage, the FPG-modified comet
assay was used. As shown in Fig. 6, the FPG-treated cells
exhibited significant increases in tail DNA (%) and tail
length after H2O2 treatment compared with buffer-treated
cells. The cells exposed to ELF-EMF did not exhibit
significant increases in these comet parameters at any
intensity when comparing the FPG-treated group with the
buffer-treated group (Fig. 6A and B). However, after cells
were exposed to RF-EMF at a SAR value of 4 W/kg, the tail
DNA (%) was significantly increased in the FPG-treated
group compared with the buffer-treated group (P , 0.05)
(Fig. 6C). But the tail length was not significantly increased
in the FPG-treated cells after exposed to RF-EMF at 4 W/kg
(Fig. 6D).
DISCUSSION
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.
Both ELF- and RF-EMF are forms of nonionizing
radiation, so their induced cellular damage may be subtle.
To enable comparison between experiments under con-
trolled and standardized conditions, we exposed the same
passage of GC-2 cells to the same exposure setup (IT’IS-
foundation) with the same exposure mode (5 min on and 10
min off) for 24 h for both ELF-EMF and RF-EMF. In
addition, to make the genotoxic effects induced by ELF-
EMF and RF-EMF easily comparable, the same end points
were detected using the same methods at the same time for
both ELF-EMF and RF-EMF. Additionally, the temperature
difference between sham-exposed and exposed chambers
remained less than 0.18C, which ensured the experiment
was not confounded by thermal effects.
The intermittent exposure mode (5 min on and 10 min
off) may more closely mimic real life exposure conditions
not only to mobile phones, but also to the time-varying
magnetic fields that are the primary sources of ELF-EMFs.
Although living organisms seem to have defense mecha-
nisms against natural electromagnetic fields and adapt to
them more easily, they may be less tolerant of large
variations in natural EMF and unnatural (manmade) EMF,
which are mostly time varying (19). Although most people
FIG. 4. Representative comet images from ELF-EMF exposure at
an intensity of 3 mT and RF exposure at 4 W/kg, as measured by an
alkaline comet assay. The negative control (NC, cells were cultured in
an incubator without ELF-EMF or RF-EMF signal) and positive
control (PC, 30 lM H2O2 for 15 min) were performed to ensure the
comet assay performed appropriately.
COMPARISON OF GENOTOXICITY OF ELF-EMF AND RF-EMF 309
are normally exposed to very low intensity ELF-EMF in
daily life, sometimes occupational workers may occasion-
ally be exposed to a few militesla (mT) of ELF-EMF
generated from induction furnaces, welding machines, high-
voltage transmission lines and electrical public transport
systems (40). Therefore, the potentially adverse effects of
high intensity of ELF-EMF on human health should not be
ignored. Thus, for our studies the intensity levels of ELF
FIG. 5. Effects of ELF-EMF and RF-EMF on nucleus c-H2AX foci formation in GC-2 cells. c-H2AX foci (green) and nuclei (blue) were
visualized by confocal microscopy. Panel A: Representative images of negative control (NC, cells were cultured in an incubator without ELF-
EMF or RF-EMF signal) and positive control (PC, 2 lg/ml etoposide for 2 h). Panel B: Representative images of ELF-EMF exposure at intensity
levels of 1, 2, 3 mT and magnified positive c-H2AX cells at 3 mT. The bottom figures were the magnified picture of 3 mT, which also provided
clear descriptions of our criteria for c-H2AX positive cells as representative pictures. Panel C: Representative images of RF-EMF exposure at SAR
values of 1, 2, 4 W/kg. Panels D and E: The frequency of c-H2AX positive cells was quantified following ELF-EMF and RF-EMF exposure,
respectively. c-H2AX positive cells were defined as those containing more than ten c-H2AX foci per cell and at least 200 cells per sample were
quantified. Bars represent the means 6 SEM from four independent experiments. **P , 0.01.
310 DUAN ET AL.
exposure were chosen as 1, 2 and 3 mT. The SAR values
selected in the current study were based on the guidelines of
exposure to RF, which are 1.6 W/kg in U.S. and 2.0 W/kg
in Europe, and the accepted SAR threshold for thermal
effects is 4 W/kg (11). Therefore, in our studies the
exposure doses of 1, 2 and 4 W/kg were selected to avoid
thermal effects.
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. The percentage of DNA in
the tail has been considered to be the most generally useful
parameter since it uses a quantitative measure of damage.
When low levels of damage are present, the tail length is
useful (41). Therefore, we evaluated the level of DNA
damage using the tail DNA (%) and tail length. 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 by measuring formation of c-H2AX
foci. Therefore, the combined results of the comet assay and
immunofluorescence for c-H2AX strongly demonstrate that
exposure of male germ cells to ELF-EMF induces DNA
strand breaks. But it was not certain whether ELF-EMF at 3
mT induced apoptosis and this will be assessed in our future
investigations. 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).
However, the mechanism by which ELF-EMF induces
DNA strand breaks has not been clearly elucidated. Several
studies proposed that increased free radical activity accounts
for the genotoxic effects of electromagnetic fields (45).
Therefore, 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. Consistent with our results, Kim et al. (40)
demonstrated that DNA DSBs induced by 7 mT ELF were
not the direct effect of ROS. Focke et al. (12) reported that
DNA fragmentation caused by exposure to ELF-EMF at 1
mT did not cause oxidative damage. On the other hand,
some results reported that long-term exposure to ELF-EMF
was able to induce oxidative DNA damage in HL-60
leukemia cells and WI-38 diploid fibroblasts (46, 47).
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 condi-
tions, 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.
These inconsistent results may be due to the tail length only
FIG. 6. Effects of ELF-EMF and RF-EMF exposure on oxidative
DNA base damage in GC-2 cells. Oxidative DNA base damage was
expressed as the tail DNA (%) and tail length after exposure to ELF-
EMF (1, 2, 3 mT) and RF-EMF (1, 2, 4 W/kg) for 24 h or to H2O2 (30
lM) for 15 min, as measured by FPG-modified alkaline comet assay.
ELF-EMF: The tail DNA (%) (panel A), tail length (lm) (panel B);
RF-EMF: The tail DNA (%) (panel C), tail length (lm) (panel D). At
least 100 nuclei were analyzed for each group of each experiment.
Bars represent the means 6 SEM from three independent experiments.
*P , 0.05, compared FPG (þ) with FPG (–) of the same group. NC:
negative control (Cells were cultured at an incubator without ELF-
EMF or RF-EMF signal); PC: positive control (30 lM H2O2 for 15
min). FPG (–) (white columns): buffer-treated group; FPG (þ) (black
columns): FPG enzyme-treated group.
COMPARISON OF GENOTOXICITY OF ELF-EMF AND RF-EMF 311
represents the length of the damaged DNA, whereas the tail
DNA (%) showed more significant discrepancies and better
correlations than that of the tail length (59). 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.
ACKNOWLEDGMENTS
We thank Professor Wei Sun, Dr. Liting Wang and Hongjuan Wu for
their technical help. This work was supported by the National Basic
Research Program of China (National 973 Program) (grant no.
2011CB503700), the National Natural Science Foundation of China
(grant no. 31170800) and Natural Science Foundation from Chongqing
science and technology commission (grant no. cstc2011jjA10009).
Received: July 1, 2014; accepted: December 12, 2014; published online:
February 17, 2015
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