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February 16, 2010

Intermittent extremely low frequency electromagnetic fields cause DNA damage in a dose-dependent way

Filed under: Xanya Sofra Weiss — Tags: — Dr. Xanya @ 7:39 am

Sabine Ivancsits Æ Elisabeth Diem Æ Oswald Jahn
Hugo W. Ru¨ diger

ORIGINAL ARTICLE
Intermittent extremely low frequency electromagnetic fields cause
DNA damage in a dose-dependent way

Received: 23 October 2002 / Accepted: 7 March 2003 / Published online: 12 June 2003
 Springer-Verlag 2003

Abstract Objectives: Epidemiological studies have
reported an association between exposure to extremely
low frequency electromagnetic fields (ELF-EMFs) and
increased risk of cancerous diseases, albeit without
dose–effect relationships. The validity of such findings
can be corroborated only by demonstration of dosedependent
DNA-damaging effects of ELF-EMFs in cells
of human origin in vitro. Methods: Cultured human
diploid fibroblasts were exposed to intermittent ELF
electromagnetic fields. DNA damage was determined by
alkaline and neutral comet assay. Results: ELF-EMF
exposure (50 Hz, sinusoidal, 1–24 h, 20–1,000 lT, 5 min
on/10 min off) induced dose-dependent and timedependent
DNA single-strand and double-strand
breaks. Effects occurred at a magnetic flux density as
low as 35 lT, being well below proposed International
Commission of Non-Ionising Radiation Protection
(ICNIRP) guidelines. After termination of exposure the
induced comet tail factors returned to normal within
9 h. Conclusion: The induced DNA damage is not based
on thermal effects and arouses concern about environmental
threshold limit values for ELF exposure.
Keywords ELF-EMF Æ 50-Hz sinusoidal Æ Intermittent
exposure Æ Comet assay
Introduction
The issue of adverse health effects of extremely low
frequency electromagnetic fields (ELF-EMFs) is highly
controversial. Numerous contradictory results regarding
the carcinogenic potential of ELF-EMFs have been
reported in the literature. Some epidemiological studies
indicate that exposure to ELF-EMFs may lead to an
increased risk of certain types of adult and childhood
cancer, including leukaemia, cancer of the central nervous
system, and lymphoma (Wertheimer and Leeper
1979; Savitz et al. 1988; Feychting et al. 1997; Li et al.
1997), while others (Verkasalo et al. 1993; Tomenius
1986; Schreibner et al. 1993) did not find such an association.
Interpretation of these studies is complicated,
due to different and unreliable methods of exposure
assessment. Therefore, in vitro studies with defined
exposure conditions and with genotoxic effect markers
as endpoints could provide evidence for a carcinogenic
potential of ELF-EMFs.
Up to date, several comprehensive reviews regarding
in vivo and in vitro laboratory studies on ELF-EMFs
have been published (McCann et al. 1993, 1998; Murphy
et al. 1993; Moulder 1998). Conflicting results have been
reported, with genotoxic endpoints such as sister chromatid
exchange (SCE), micronuclei (MN), chromosome
aberrations (CA) and assessment of DNA strand breaks
at exposure levels ranging from 1 lT to 10 mT. The
majority of these studies, however, did not show any
EMF-related genotoxic effects. Several studies with
whole-body exposure of rodents to ELF-EMFs revealed
DNA single-strand and double-strand breaks in the
brain (Lai and Singh 1997; Singh and Lai 1998;
Svedenstal et al. 1999a, 1999b). These results, in the first
place, gave rise to the classification of ELF magnetic
fields as being possibly carcinogenic to humans
(group 2B) by the International Agency for Research on
Cancer (IARC 2002).
As previously reported (Ivancsits et al. 2002a) we
were able to corroborate these findings by demonstration
of an increase in DNA single-strand breaks (SSBs)
and double-strand breaks (DSBs) in cultured human
diploid fibroblasts upon intermittent exposure to a
50-Hz magnetic field, using comet assay under alkaline
(detection of SSBs + DSBs) and neutral conditions
Int Arch Occup Environ Health (2003) 76: 431–436
DOI 10.1007/s00420-003-0446-5
Sabine Ivancsits Æ Elisabeth Diem Æ Oswald Jahn
Hugo W. Ru¨ diger
S. Ivancsits (&)
Division of Occupational Medicine, University Hospital/AKH,
Waehringer Guertel 18–20, 1090 Vienna, Austria
E-mail: Sabine.Ivancsits@akh-wien.ac.at
Tel.: +43-1-404004022
Fax: +43-1-4088011
S. Ivancsits Æ E. Diem Æ O. Jahn Æ H. W. Ru¨ diger
Division of Occupational Medicine
University of Vienna, Vienna, Austria
(detection of DSBs). The extent of EMF-induced DNA
damage was variable in relation to the setting of on and
off times, and was highest at an intermittence of 5 min
on/10 min off. No effects were detected during continuous
exposure.
Here we report studies on the influence of exposure
time and of magnetic-flux densities on the induction of
DNA strand breaks with human fibroblast cultures of
three healthy donors.
Materials and methods
ELF-EMF exposure conditions and cell culture
Human diploid fibroblast strains of donors with different ages
(ES1, male, 6 years old; IH9, female, 28 years old; KE1, male, 43
years old) were initiated from skin biopsies from healthy donors
and maintained in culture as previously described (Ivancsits et al.
2002a). The cells were seeded into 35-mm Petri dishes at a density
of 5·104 cells/3 ml, 24 h prior to ELF-EMF exposure.
The exposure system was built and provided by the Foundation
for Information Technologies in Society (IT’IS foundation), Zurich,
Switzerland, http://ww.itis.ethz.ch). The set-up, which generated
a vertical EMF, consisted of two four-coil systems (two coils
with 56 windings, two coils with 50 windings), each of which was
placed inside a l-metal box. The currents in the bi-filar coils could
be switched parallel for field exposure or non-parallel for control
(sham-exposure). The residual magnetic field in the sham chamber
was at least 150 times (43 dB) lower than the applied field in the
exposure chamber. In addition, both chambers were not completely
insulated from the earth’s magnetic field, which remained at 20–
50 lT. Both systems were placed inside a commercial incubator
(BBD 6220, Kendro, Vienna, Austria) to ensure constant environmental
conditions (37C, 5% CO2, 95% humidity). Two fans
per l-metal box ensured atmospheric exchange of the chambers. A
PC controlled and continuously monitored the exposure set-up.
Data (temperature, current) were collected and stored in an encoded
file. The temperature was monitored at the location of the
dishes during exposure and was maintained at 36.5–37.5C. The
temperature difference between the chambers did not exceed 0.3C.
A current source based on four audio-amplifiers (Agilent Technologies,
Zurich, Switzerland) allowed magnetic fields up to
2.3 mT. The field could be varied in the frequency range from DC
to 1.5 kHz by a computer-controlled function generator. To enable
blind exposures, the computer randomly determined which of the
two chambers was exposed. This information was provided to the
investigator by the IT’IS foundation in Zurich via e-mail in exchange
with the transmission of comet assay results. All experiments
were performed at a frequency of 50 Hz sinusoidal at
intermittent exposure (5 min field on/10 min field off). Timedependent
effects were studied at a magnetic flux density of 1 mT;
for dose–response effects, the magnetic flux density was varied
between 20 and 1,000 lT (5 min field on/10 min field off) at a
constant exposure time of 15 h. After exposure the fibroblasts were
detached with trypsin and suspended in fresh culture medium. To
study repair kinetics, we post-incubated fibroblasts at 37C for 0.5–
9 h. Each exposure level was tested in duplicate.
Comet assay analysis
We followed the technique described by O¨ stling and Johanson
(1984) with minor modifications by Singh et al. (1988, 1991). ELFexposed
and sham-exposed cells (10,000–30,000) were mixed with
100 ll low-melting agarose (0.5%, 37C), and this cell suspension
was pipetted onto 1.5% normal-melting agarose pre-coated slides,
spread with a cover slip, and kept on a cold flat tray for approximately
10 min to solidify. After the cover slip had been removed, a
third layer of 0.5% low-melting agarose was added and allowed to
solidify. The slides were then immersed in freshly prepared cold
lysis solution (2.5 mol/l NaCl, 100 mmol/l Na2EDTA, 10 mmol/l
Tris, pH 10, 1% sodium sarcosinate, 1% Triton X-100, 10%
DMSO, pH 10) and lysed for 90 min at 4C. Subsequently, the
slides were drained and placed in a horizontal gel electrophoresis
tank, side by side and nearest the anode. The tank was filled with
fresh electrophoresis buffer (1 mmol/l Na2EDTA, 300 mmol/l
NaOH, pH>13 or pH 12.1 in the case of alkaline comet assay, and
100 mmol/l Tris, 300 mmol/l sodium acetate, 500 mmol/l sodium
chloride, pH 8.5 in the case of neutral comet assay) to a level
approximately 0.4 cm above the slides. For both alkaline and
neutral comet assay, the slides were left in the solution for 40 min
to allow equilibration and unwinding of the DNA before electrophoresis.
Electrophoresis conditions (25 V, 300 mA, 4C, 20 min,
field strength 0.8 V/cm) were the same for neutral and alkaline
comet assay. All steps were performed under dimmed light to
prevent the occurrence of additional DNA damage. After electrophoresis
the slides were washed three times with Tris buffer
(0.4 mol/l Tris, pH 7.5), to be neutralized, then air-dried and stored
until required for analysis. Comets were visualized by ethidium
bromide staining (20 lg/ml, 30 s) and examined at 400· magnification
with a fluorescence microscope (Axiophot, Zeiss, Germany).
One thousand DNA spots from each sample were classified into
five categories corresponding to the amount of DNA in the tail, in
accordance with Anderson et al. (1994). The proposed classification
system provides a fast and inexpensive method for genotoxic
monitoring. Due to the classification to different groups by eye, no
special imaging software is required. The technique becomes more
sensitive, because many cells can be scored in a short time (1,000
cells instead of 50–100 cells with image analysing). The subsequent
calculation of a ‘‘comet tail factor’’ allows DNA damage to be
quantified as a single figure, which makes it easier for results to be
compared. Due to the scoring of 1,000 cells in one experiment,
which are ten times the cells processed with image analysing,
standard deviations are very low. Reproducibility has been thoroughly
checked.
Results were expressed as ‘‘comet tail factors’’, calculated in
accordance with Diem, with modifications as previously described
(Diem et al. 2002; Ivancsits et al. 2002a, 2002b). The same investigator
performed all analyses. Figure 1 shows the five classification
groups, with the group averages, and the microphotograph.
Fig. 1 Comet assay classification groups and respective microscopic
appearance (cell line ES-1)
432
Tail factors were calculated according to the following formula:
tailfactor% ¼
A  FA þ B  FB þ C  FC þ D  FD þ E  FE
1000
where
A = the number of cells classified to group A and FA
= the average of group A (=2.5)
B = the number of cells classified to group B and FB
= the average of group B (=12.5)
C = the number of cells classified to group C and FC
= the average of group C (=30)
D = the number of cells classified to group D and FD
= the average of group D (=67.5)
E = the number of cells classified to group E and FE
= the average of group E (=97.5)
Statistical analysis
Statistical analysis was performed with STATISTICA V. 5.0
package (Statsoft, Tulsa, USA). All data are presented as mean ±
SD. The differences between exposed and sham-exposed, as well as
between different exposure conditions, were tested for significance
with an independent Student’s t-test. A difference at P< 0.01 was considered statistically significant. Results Fibroblast cultures of three healthy donors were exposed to ELF-EMFs (50 Hz sinusoidal, 1,000 lT, intermittent 5 min on/10 min off) for 1 to 24 h. Alkaline and neutral comet tail factors increased with exposure time, being largest at 15–19 h (Fig. 2). Comet assay levels declined thereafter, but did not return to basal levels. The different cell donors exhibited different basal levels, different maxima, and different end levels. When exposure was terminated after 15 h the comet factor returned to basal levels after a repair time of 7 to 9 h (Fig. 3a), which comprised a fast repair rate of DNA SSBs (<1 h) and a slow repair rate of DNA DSBs (>7 h). The marked comet peak value between 12 and
17 h and the following repair kinetics could also be detected
when ELF exposure was terminated after 12 h
(Fig. 3b). However, it disappeared when comet assay
was performed at pH 12.1 instead of pH>13, thereby
eliminating the cleavage of alkali labile sites in the DNA
(Fig. 4).
Fig. 2a, b Influence of exposure
time on formation of DNA
SSBs and DSBs in three human
fibroblast strains (ES-1, IH-9,
KE-1) determined with comet
assay (1 mT, 5 min on/10 min
off cycles). a Alkaline
conditions; b neutral conditions
433
When magnetic flux densities were varied between 20
and 1,000 lT (cell strain ES-1) we observed a dosedependent
increase of comet factor at alkaline and
neutral conditions, which had already become significant
at 35 lT (Fig. 5, Table 1).
Discussion
Exposure to thermal stress may result in alterations in
the integrity of DNA, comprising DNA strand breaks or
Fig. 3a, b Repair kinetics of
DNA SSBs and DSBs in human
fibroblasts after termination of
ELF-EMF exposure (cell strain
ES-1, 1 mT, 5 min on/10 min
off cycles) determined with
alkaline and neutral comet
assay. a Repair after 15-h ELFEMF
exposure; b repair after
12-h ELF-EMF exposure
Fig. 4 Comet assay of exposed
human fibroblasts was
performed at different pH
(1 mT, intermittent 5 min on/
10 min off)
434
apoptosis (Fairbairn et al. 1995). The induced DNA
damage depends on the extent and duration of the applied
heat stress. Taking these findings into account, we
consider it highly unlikely that, in our experiments, the
observed genotoxic damage is caused non-specifically by
spots of increased temperature within the cell layer as a
secondary effect of the electromagnetic field. If so, the
damage would increase with prolongation of the on time
during the intermittent exposure and would be largest at
continuous exposure. It has previously been shown,
however, that the largest effects are obtained at 5¢ on/10¢
off cycles, and that continuous exposure has no effect at
all (Ivancsits et al. 2002a). Therefore, we conclude that
the observed induction of DNA SSBs and DSBs is a
direct consequence of an intermittent exposure to ELFEMFs.
We observed an increase in DNA breaks up to 15 h
of exposure and then a decline to a ‘‘steady-state level’’
of approximately 1.5-times the base line. This unexpected
finding can be explained if the exposure activates
DNA repair processes and this activation takes a time of
10 to 12 h. After this time the DNA damage is repaired
at an enhanced rate, which leads to a reduction in DNA
breaks, albeit not to a normalisation. This explanation is
supported experimentally by the observation that the
single-strand DNA breaks (alkaline conditions) are repaired
after approximately 30 min, and double-strand
breaks 7 to 9 h after shut down of the exposure (Fig. 3).
The repair process itself also leads to a temporary increase
of alkali-sensitive sites in the DNA, which are
detected as a peak at hours 12 to 17 at comet assay
conditions of pH>13, but not of pH 12.1, the latter not
being able cleave the alkali-sensitive sites (Fig. 4).
It is well known that the repair of SSBs is a fast and
almost error-free process, while the repair of more
complex DNA damage (i.e. DNA DSBs) by homologous
recombination, single-strand annealing or nonhomologous
end joining requires more time and is error
prone in part (Van den Bosch et al. 2002). Therefore,
DNA DSBs may affect the integrity of the genome and
can lead to cell death, uncontrolled cell growth, or
cancer (Van Gent at al. 2001).
In addition, we demonstrate here an increase in DNA
SSBs and DSBs in relation to an increasing magnetic
flux density, which becomes significant at 35 lT at 15 h
of intermittent ELF-EMF exposure. This threshold is
well below the guidelines of the International Commission
of Non-Ionising Radiation Protection (ICNIRP
1998), which propose 500 lT per working day for
occupational exposures and 100 lT per 24 h for the
general population. However, no proposal with regard
to intermittent exposures has been made by the ICNIRP
as yet.
In conclusion, our findings strongly indicate a genotoxic
potential of intermittent ELF-EMFs. The induced
DNA damage was time-dependent and dose-dependent
and points to the need for consideration of environmental
and occupational threshold limit values for ELFEMFs,
in particular with regard to intermittent exposures.
Acknowledgements This study was funded by the European Union
under the programme ‘‘Quality of Life and Management of Living
Resources’’, Key Action 4 ‘‘Environment and Health’’: QLK4-CT-
01574.
References
Anderson D, Yu TW, Phillips BJ, Schmerzer P (1994) The effect of
various antioxidants and other modifying agents on oxygenradical-
generated DNA damage in human lymphocytes in the
comet assay. Mutat Res 307:261–271
Diem E, Ivancsits S, Ru¨ diger HW (2002) Basal levels of DNA
strand breaks in human leukocytes determined by comet assay.
J Toxicol Environ Health A 65:641–648
Fairbairn JJ, Khan MW, Ward KJ, Loveridge BW, Fairbairn DW,
O’Neill KL (1995) Induction of apoptotic cell DNA fragmentation
in human cells after treatment with hyperthermia. Cancer
Lett 89:183–188
Fig. 5 Dose-dependent formation of DNA SSBs and DSBs
determined with comet assay under alkaline and neutral conditions
with cell strain ES-1 (exposure time 15 h, 5 min on/10 min off
cycles)
Table 1 Alkaline and neutral comet assay levels of ELF-exposed
(20–1,000 lT, 15 h, 5 min on/10 min off) and non-exposed human
diploid fibroblasts (ES-1)
Magnetic
flux density
Alkaline comet assay Neutral comet assay
(lT) Comet tail
factor (%)
(± SD) Comet tail
factor (%)
(± SD)
0 4.105 0.028 3.797 0.011
20 4.100 0.092 3.906 0.027
35 5.807a 0.025 4.455a 0.127
50 8.985a 0.170 5.812a 0.018
70 12.450a 0.134 7.605a 0.103
100 14.494a 0.012 8.799a 0.004
1,000 16.213a 0.124 9.311a 0.063
aSignificant differences (P<0.01) between exposed and non-exposed 435 Feychting M, Forssen U, Floderus B (1997) Occupational and residential magnetic field exposure and leukemia and central nervous system tumors. Epidemiology 8:384–389 International Agency for Research on Cancer (2002) Non-ionizing radiation, part 1: static and extremely low-frequency (ELF) electric and magnetic fields. IARC Monogr Eval Carcinog Risks Hum 80:1–395 International Commission on Non-Ionizing Radiation Protection (1998) Guidelines for limiting exposure to time-varying, magnetic, and electromagnetic fields up to 300 GHz. Health Phys 74:494–522 Ivancsits S, Diem E, Pilger A, Ru¨ diger HW, Jahn O (2002a) Induction of DNA strand breaks by exposure to extremely-lowfrequency electromagnetic fields in human diploid fibroblasts. Mutat Res 519:1–13 Ivancsits S, Pilger A, Diem E, Schaffer A, Ru¨ diger HW (2002b) Vanadate induces DNA strand breaks in cultured human fibroblasts at doses relevant to occupational exposure. Mutat Res 519:25–35 Lai H, Singh N (1997) Acute exposure to a 60 Hz magnetic field increases DNA strand breaks in rat brain cells. Bioelectromagnetics 18:156–165 Li CY, Theriault G, Lin RS (1997) Residential exposure to 60 Hz magnetic fields and adult cancers in Taiwan. Epidemiology 8:25–30 McCann J, Dietrich F, Rafferty C, Martin AO (1993) A critical review of the genotoxic potential of electric and magnetic fields. Mutat Res 297:61–95 McCann J, Dietrich F, Rafferty C (1998) The genotoxic potential of electric and magnetic fields: an update. Mutat Res 411:45–86 Moulder JE (1998) Power-frequency fields and cancer. Crit Rev Biomed Eng 26:1–116 Murphy JC, Kaden DA, Warren J, Sivak A (1993) Power frequency electric and magnetic fields: a review of genetic toxicology. Mutat Res 296: 221–240 O¨ stling O, Johanson KJ (1984) Microelectrophoretic study of radiation-induced DNA damages in individual mammalian cells. Biochem Biophys Res Commun 123:291–298 Savitz DA, Wachtel H, Barnes FA, John EM, Tvrdik JG (1988) Case control study of childhood cancer and exposure to 60 Hz magnetic fields. Am J Epidemiol 128:21–38 Schreibner GH, Swaen GMH, Meijers JMM, Slangen JJM, Sturmans F (1993) Cancer mortality and residence near electricity transmission equipment: a retrospective cohort study. Int J Epidemiol 22:9-15 Singh N, Lai H (1998) 60 Hz magnetic field exposure induces DNA crosslinks in rat brain cells. Mutat Res 400:313–320 Singh NP, McCoy MT, Tice RR, Schneider EL (1988) A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res 175:184–191 Singh NP, Tice RR, Stephens RE, Schneider EL (1991) A microgelelectrophoresis technique for direct quantitation of DNA damage and repair in individual fibroblasts cultured on microscope slides. Mutat Res 252:289–296 Svedenstal BM, Johanson KJ, Mild K H (1999a) DNA damage induced in brain cells of CBA mice exposed to magnetic fields. In Vivo 13:551–552 Svedenstal BM, Johanson KJ, Mattsson MO, Paulsson LE (1999b) DNA damage, cell kinetics and ODC activities studied in CBA mice exposed to electromagnetic fields generated by transmission lines. In Vivo 13:507–513 Tomenius L (1986) 50 Hz electromagnetic environment and the incidence of childhood tumors in Stockholm County. Bioelectromagnetics 7:191–207 Van den Bosch M, Lohman PHM, Pastink (2002) A DNA double strand break repair by homologous recombination. Rev Biol Chem 383:873–892 Van Gent DC, Hoeijmakers JH, Kanaar R (2001) Chromosomal stability and the DNA double-strand break connection. Nat Rev Genet 2:196–206 Verkasalo PK, Pukkala E, Hongisto MY, Valjus JE, Ja¨ rvinen PJ, Heikkila¨ KV, Koskenvuo M (1993) Risk of cancer in Finnish children living close to power lines. Br Med J 307:895–899 Wertheimer N, Leeper E (1979) Electrical wiring configurations and childhood cancer. Am J Epidemiol 109: 273–284 436

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