Regular Paper
J. Radiat. Res., 48, 69–75 (2007)
Effects of 2.45 GHz Electromagnetic Fields with a Wide Range
of SARs on Bacterial and
HPRT
Gene Mutations
Shin KOYAMA
1,2, Yoshio TAKASHIMA
1, Tomonori SAKURAI
1,
Yukihisa SUZUKI
3, Masao TAKI
3and Junji MIYAKOSHI
1*
High-frequency electromagnetic fields (HFEMF)/Bacterial mutation/Hypoxanthine-guanine phos-phoribosyl transferase (HPRT) gene mutation/Chinese hamster ovary (CHO)-K1 cells.
Present day use of mobile phones is ubiquitous. This causes some concern for human health due to exposure to high-frequency electromagnetic fields (HFEMF) from mobile phones. Consequently, we have examined the effects of 2.45 GHz electromagnetic fields on bacterial mutations and the hypoxanthine-gua-nine phosphoribosyl transferase (HPRT) gene mutations. Using the Ames test, bacteria were exposed to HFEMF for 30 min at specific absorption rates (SARs) from 5 to 200 W/kg. In all strains, there was no significant difference in the frequency of revertant colonies between sham exposure and HFEMF-exposed groups. In examination of mutations of the HPRT gene, Chinese hamster ovary (CHO)-K1 cells were exposed to HFEMF for 2 h at SARs from 5 to 200 W/kg. We detected a combination effect of simulta-neous exposure to HFEMF and bleomycin at the respective SARs. A statistically significant difference was observed between the cells exposed to HFEMF at the SAR of 200 W/kg. Cells treated with the com-bination of HFEMF at SARs from 50 to 200 W/kg and bleomycin exhibited increased HPRT mutations. As the exposure to HFEMF induced an increase in temperature, these increases of mutation frequency may be a result of activation of bleomycin by heat. We consider that the increase of mutation frequency may be due to a thermal effect.
INTRODUCTION
In industrialized countries, people are exposed to many kinds of electromagnetic fields. Exposure to high-frequency electromagnetic fields (HFEMF) or radiofrequency (RF) electromagnetic fields has spread through radio communica-tions in various areas. In particular, the use of mobile phones that emit HFEMF is increasing rapidly. However, it remains controversial as to whether or not exposure to HFEMF elic-its adverse results towards human health. Richter et al.1) found that HFEMF exposure exhibits an epidemiologic effect on cancer risk. No effects of HFEMF have been dem-onstrated on human health2–5); thus, if there are any effects,
they are very weak. Moulder et al.6) reviewed the current state of evidence concerning the carcinogenic-potential of HFEMF energy used for wireless communication. In this review, most studies indicated no relationship between the HFEMF energy and cancer, although further investigations of this field are required. In this decade, the world-wide spread of mobile phones is accelerating. Consequently, con-cern about potential health risk by HFEMF exposure from mobile phone is increasing. Epidemiological studies have produced contradictory results7–11) that were likely due to the short-term mobile phone use and little evidence.
Laboratory studies have also indicated contradictory results. Lai and Singh12,13) demonstrated that acute, low-intensity microwave (whole body average SAR (specific absorption rate) 1.2W/kg) exposure and radiofrequency electromagnetic radiation increased DNA strand breaks. Marinelli et al.14) reported that unmodulated 900 MHz elec-tromagnetic field at the SAR of 3.5 mW/kg induced DNA breaks and early activation of both p53-dependent and – independent apoptotic pathways. Similarly, a positive find-ing was reported by Erdinc et al.15) that exposure to a 900 MHz electromagnetic field might affect the developing brain in prepubertal mice injected with pentylenetetrazole. An increase in frequency of micronuclei has also been demon-strated following exposure to radiofrequency electromagnet-*Corresponding author: Phone: +81-172-39-5964,
Fax: +81-172-33-2830,
E-mail: [email protected]
1Department of Radiological Technology, School of Health Sciences, Fac-ulty of Medicine, Hirosaki University, 66-1 Hon-cho, Hirosaki, 036-8564, Japan; 2Department of Interdisciplinary Environment, Graduate School of Human and Environmental Studies, Kyoto University, Yoshida-Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan; and 3Department of Electrical Engineer-ing, Graduate School of EngineerEngineer-ing, Tokyo Metropolitan University, 1-1 Minami Ohsawa, Hachioji, Tokyo 192-0397, Japan.
S. Koyama et al. 70
ic field16) and the potential hazards of HFEMF at a SAR of 2.1 µW/kg exposure have been discussed under non-thermal condition.17) However, Vijayalaxmi et al.18) suggested that 2450 MHz radiofrequency exposure at a whole body average SAR of 12 W/kg exhibited no effect on the induction of micronuclei in peripheral blood and bone marrow cells of rats. Malyapa et al.19,20) also investigated the effect of expo-sure to 2450 MHz radiofrequency (SARs; 0.7 and 1.9 W/kg) by comet assay and demonstrated no significant differences between the tested groups and the controls after irradiation. Moulder et al.21) suggested in their review, that in the labo-ratory studies cell phone HFEMF radiation generally exhib-its no genotoxic or epigenetic activity, and that the cell phone HFEMF radiation-cancer connection is physically implausible. While, Leszczynski22) reported that mobile phone radiation (average SAR 2 W/kg) caused activation of the HSP27/p38MAPK stress pathway. They hypothesized that this activation may facilitate the development of brain cancer or cause an increase in blood-brain barrier permeabil-ity through stabilization of endothelial cell fibers. Nylund and Leszczynski23) reported that mobile phone radiation at an average SAR of 2.4 W/kg affected protein expression. However, it has also been reported that 1800 MHz radiofre-quency radiation at the SARs of 1.4 and 2.0 W/kg does not affect apoptosis or HSP70 levels in peripheral blood.24) Vijayalaxmi and Obe25) reviewed that a large number of investigations were conducted using rodents, cultured rodent and human cells, and freshly collected human blood lym-phocytes to determine the genotoxic potential of exposure to HFEMF. The results of most of these studies did not indicate increased damage to the genetic material (assessed from DNA strand breaks, incidence of chromosomal aberrations, micronuclei and sister chromatid exchanges) in cells exposed to HFEMF compared to sham-exposed and/or unex-posed cells. Some investigations reported an increase in such damage in cells exposed to HFEMF.
Thus, although many studies have indicated that there is no relationship between radiofrequency radiation and adverse outcomes, such as genotoxicity, mutagenicity, etc., an epidemiological study has indicated that HFEMF from mobile phones may exhibit adverse effects on humans7) and the mechanism of the effects of HFEMF on living organisms remains uncertain. National Radiological Protectiopn Board (NRPB)26) published the advice on limiting exposure to elec-tromagnetic fields from 0 to 300 GHz and summarized the exposure guidelines of International Commission on Non-Ionizing Radiation Protection (ICNIRP). In the guidelines, occupational and general public people are recommended to be restricted to 0.4 or 0.08 W/kg whole-body average SAR (specific absorption rate), respectively. Alhough we are exposed to HFEMF much less than ICNIRP guidelines, pub-lic anxiety about exposure to HFEMF still persists. To inves-tigate these controversial issues, we examined bacterial mutagenicity and 6-thioguanine (6-TG)-resistant mutations
in the hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene following exposure to HFEMF. We have cho-sen 2.45 GHz electromagnetic fields because of the near fre-quency to the mobile phone type which is going to be used in next generation. In our previous study, we detected that an increase in micronucleus formation following exposure to continuous wave of 2.45 GHz at the SAR of from 50 to 200 W/kg might be connected to a rise in temperature by the exposure of HFEMF.27) Mutagenicity was determined by standard bacterial mutation assays that are commonly used for screening of chemicals and environmental contaminants. Mutation frequency was detected by counting 6-TG resistant colonies in Chinese hamster ovary (CHO)-K1 cells. This method also commonly used for genotoxicity assay. Simul-taneously, the effect of heat on mutation frequency was examined with the rise in temperature during HFEMF expo-sure.
MATERIALS AND METHODS
Bacterial strains
Five routinely used Ames testing strains of Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and
Escherichia coli (WP2 uvrA) were used (Table 1). S. typh-imurium were originally provided by Dr. B. N. Ames of the University of California, Berkeley while E. coli was origi-nally provided by Japan Bioassay center, Kanagawa, Japan and these strains were provided by Dr. M. Ikehata of the Railway Technical Research Institute of Tokyo, Japan. Table 1 shows the strain genotypes.
Cells were grown on nutrient broth medium (Oxioid, Nutrient broth No. 2; Unipath, Hampshire, UK) at 37°C to the late-logarithmic growth phase. Cells were harvested and resuspended in phosphate buffer at a density of 1 × 108 cells/ ml. Aliquots (20.1ml) of the cell suspension were placed into a special culture dish designed to allow exposure to an elec-tromagnetic field, and were exposed to HFEMF for 30 min according to the protocol of pre-incubation method of Ames test.28) Three chemical mutagens were used as positive con-trol. Sodium azide and 2-(2-Furyl)-3-(5-nitro-2-furyl) acry-lamide (AF-2) were purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan) and 4-nitroquinoline-N-oxide (4-NQO) was purchased from Sigma-Aldrich Co. (St. Louis, MO). For E. coli WP2 uvrA, 0.01 µg/plate of AF-2 was used. For S. typhimurium TA 1535, 0.5 µg/plate of sodium azide was used. For S. typhimurium TA98 and TA1537, 0.2 µg/ plate of 4-NQO were used while 0.025 µg/plate of 4-NQO were used for S. typhimurium TA100.
Mammalian cell line
CHO-K1 cells were obtained from the Japanese Cancer Research Bank, Tokyo. Cells were maintained in Ham’s F-12 medium (Nikken, Kyoto, Japan) supplemented with 10% fetal bovine serum (Invitrogen Corp., Carlsbad, CA) at 37°C
HFEMF on Bacterial and HPRT Gene Mutations 71
in 95% air and 5% CO2. Aliquots (20.1 ml) of the cellular suspensions (concentration of 5 × 105 cells/ml) were seeded on a section of a special culture dish designed to allow expo-sure to an electromagnetic field, and were exposed to HFEMF for 2 h.27)
Exposure system
Details of the exposure system have been described pre-viously.27) In brief, the exposure setup is based on the rect-angular wave-guide. The electromagnetic wave is propagat-ed along the waveguide with TE10 mode. To generate standing waves in the wave-guide, one end of it is terminated with a short-circuiting plate. A frequency of 2.45GHz is used for exposure to HFEMF in an acrylic CO2 incubator, in which the atmosphere is humidified 95% air and 5% CO2, that is installed into inner space of the core. A culture dish is placed on two slits that are bored on the wave-guide. Cells are exposed to the HFEMF through these slits. SAR distri-bution of the exposure setup was measured.
Mutagenicity assay in bacteria
For the detection of mutagenicity following exposure to HFEMF, the plate incubation method was used.28) Bacteria were inoculated and incubated for 1 h, in which most bac-teria were sinking on the bottom of culture dishes. Bacbac-teria were exposed to HFEMF for 30 min at SARs from 5 to 200 W/kg. Exposed cells were mixed with phosphate buffer and top agar, plated onto minimal glucose agar plates and incu-bated at 37°C in a conventional incubator for 48 h. Revertant colonies were then counted.
HPRT assay in mammalian cells
CHO-K1 cells were seeded at a concentration of 5 × 105 cells/ml onto special culture plates before exposure and cul-tured for 12 h. Cells were exposed to HFEMF for 2 h at SARs from 5 to 200 W/kg. Exposed cells were washed with Phosphate Buffered Saline (PBS) three times and cultured for 24 h with new medium. The cells were re-plated in 75
cm2 flasks and subcultured to subconfluence for 3 days. However, as a positive control or co-mutagenic treatment, bleomycin-treated cells were exposed to bleomycin (final concentration 10 µg/ml) for 1 h before HFEMF-exposure. After the exposure, cells were washed three times with PBS and were re-plated in 75 cm2 flasks for subculture to sub-confluence for 3 days. Cells (2 × 105 cells) were then plated in 10 cm dishes containing medium with 15 µM of 6-TG. Ten days later, the cells were stained with Giemsa’s solution. For the determination of cloning efficiency, about 100 cells were seeded on 6 cm dishes after 3 days-subculturing and colonies were counted after 6 days. Mutation frequencies were calculated according to the formula of mean scored colonies/2 × 105/mean scored colonies in cloning efficiency
× 100.
Heat treatment
CHO-K1 cells for heat treatment were prepared as the same as for HFEMF exposure method. Cells were incubated for 2 h at 39, 41 and 44°C corresponding to the heat induc-tion of about 50, 100 and 200 W/kg, respectively. Co-mutagenic treatment, such as heat and bleomycin, was also performed. HPRT gene assay was done the same way as the above-described method.
Statistical analysis
Statistical analysis of the data was conducted using ANO-VA (analysis of variance) followed by Fisher’s PLSD test from the three independent experiments.
RESULTS
Mutagenicity of HFEMF
Mutagenicity of HFEMFs were tested using five different strains; E.coli WP uvrA, S.typhimurium TA98, TA100, TA1535 and TA1537. In all strains, there was no significant difference in the frequency of revertant colonies between sham control and HFEMF-exposed groups; although
posi-Table 1. Genotypes of the tester strains used in this study
Strain Amino acid marker Other relevant mutations
Plasmid Mutation Type of mutation Main DNA target DNA repair Cell wall
S. typhimurium
TA98 hisD 3052 Frameshift GC uvrB rfa pKM101
TA100 his G 46 Base pair substitution GC uvrB rfa pKM101
TA1535 his G 46 Base pair substitution GC uvrB rfa –
TA1537 his C 3076 Frameshift GC uvrB rfa –
E. coli
S. Koyama et al. 72
tive controls showed statistically significant differences compared with sham exposure (Table 2). These results sug-gested that exposure to HFEMF ranging from 5 to 200 W/ kg did not effect mutagenicity in these five experimental strains.
Mutation frequency of the HPRT gene following expo-sure to HFEMF and heat treatment
Mutation frequency of the HPRT gene by HFEMF was determined and the results are presented in Fig. 1. An effect of temperature on HPRT was also detected, due to the
Table 2. Mutagenicity of HFEMFs by Ames test
Strain Revertant colonies/plate
Sham 5W/kg 50W/kg 100W/kg 200W/kg Positive controlb
S. typhimurium TA98 30±5a 23±9 28±6 29±3 29±8 69±6* TA100 111±28 149±7 98±12 107±2 111±12 430±46* TA1535 18±7 16±6 19±4 32±4 12±4 540±24* TA1537 32±5 37±1 34±2 32±3 28±2 56±7* E.coli WP2 uvrA 40±11 32±3 25±3 47±9 36±9 963±96* a
Data are given as mean ± S.D. from the three independent experiments. Statistical analysis of the data in the sham control and experimental groups was conducted using ANOVA (analysis of variance) followed by Fisher’s PLSD test.
bChemical mutagen was used as positive control. For S. typhimurium TA 98, TA 100 and TA 1537, 4NQO
was used. For S. typhimurium TA 1535, sodium azide was used. For E. coli WP2 uvrA, AF 2 was used. *Statistically significant differences compared with sham control (p < 0.01).
Fig. 1. Mutation frequency of the HPRT gene following exposure to HFEMF. Columns represent the mean of mutation frequency calculated from 6-TG resistant colonies. Bars represent the standard deviation from at least three independent experiments. Statistical analysis of the data in the sham exposure and experimental groups was conducted using ANOVA (analysis of variance) followed by Fisher’s PLSD test. * Statistically significant differences compared with sham exposure (p < 0.05).
HFEMF on Bacterial and HPRT Gene Mutations 73
increase in temperature induced by HFEMF-exposure. The effects of 39, 41 and 44°C exposure were investigated and the combination of the effects of heat and bleomycin were determined simultaneously (Fig. 2). Statistically significant differences were conducted with comparing of HFEMF-exposed and sham HFEMF-exposed samples, and also comparing of HFEMF + bleomycin treatment and bleomycin treatment alone samples. Statistically significant differences were also conducted with comparing of heat treated samples and sham exposed samples, and also comparing of heat + bleomycin treatment and bleomycin treatment alone samples. There was no difference between HFEMF-exposure ranging from 5 to 100 W/kg and sham exposure using the HPRT mutation assay. Nevertheless HFEMF-exposure of 200W/kg elicited a statistically significant induction of HPRT mutants. In the heat experiment, an increase in temperature to 44°C, corre-sponding to the 200W/kg HFEMF-exposure, induced statis-tically significant mutation compared with sham exposure. However, exposure to 39 and 41°C did not exhibit any effect on the HPRT gene compared with sham exposure. In the experiment of combined exposures to HFEMF and bleomy-cin, the mutation frequencies at 5, 10 and 20 W/kg were the same as bleomycin treated alone control. However, com-bined exposures of 50, 100 and 200 W/kg HFEMF and ble-omycin resulted in a dose-dependent increase of mutation frequency. A hundred and 200 W/kg HFEMF + bleomycin induced statistically significant mutation compared with
ble-omycin treatment alone. In the experiment of combined treatment of heat and bleomycin, the mutation frequencies at 39°C were the same as sham controls. However, combined treatment of 41 or 44°C + bleomycin induced statistically significant mutation compared with bleomycin treatment alone.
DISCUSSION
In the current study, to determine the effect of HFEMF, we performed Ames test to detect the mutagenic activity of a mutagen and measured the mutation frequency of the
HPRT gene. Using the Ames test, there was no effect of HFEMF. Mineta et al.29) concluded that bacterial mutation is not increased by 270 MHz radiofrequency radiation. We also observed no mutagenic effect of HFEMF using the bacterial mutation assay.
In this study, we determined the mutation frequency induced by HFEMF and heat. We treated cells with a radio-mimetic agent, bleomycin, that elicits DNA damage by free radical mechanism(s), as a positive control.30,31) Bleomycin also functions as a positive control for the HPRT test.32,33) Cells treated with bleomycin in this assay exhibited muta-tion of the HPRT gene, compared with the sham exposure control (Fig. 1). HFEMF exposure up to SAR 100 W/kg did not affect the mutation frequency. However, at 200 W/kg SAR the increase of mutation frequency was statistically
Fig. 2. Mutation frequency of the HPRT gene following heat treatment. Columns represent the mean mutation frequency calculated from 6-TG resistant colonies. Bars represent standard deviation from at least three independent experiments. Statistical analysis of the data in the sham exposure and experimental groups was conducted using ANOVA (analysis of variance) followed by Fisher’s PLSD test. * Statistically significant differences compared with sham exposure (p < 0.01). #
Statisti-cally significant differences compared with bleomycin treatment alone (p < 0.05) ** StatistiStatisti-cally sig-nificant differences compared with bleomycin treatment alone (p < 0.01)
S. Koyama et al. 74
significant. In heat treatment, we demonstrated an increase of mutation frequency at 44°C, but not at 39 and 41°C (Fig. 2). We have previously demonstrated the relationship bet-ween increases in temperature and 2.45 GHz HFEMF expo-sure.27) Exposure to HFEMF at the 200 W/kg SAR corre-sponds to an increase in temperature of about 44°C. This suggests that increasing the temperature > 44°C may induce
HPRT gene mutation. Although there have been few inves-tigations concerning HPRT mutation and heat, high temper-ature may induce increased HPRT gene mutation, indicating that the mutational process in the presence of azaserine was influenced by temperature.34) Recently, Dewhirst et al.35)
summarized the carcinogenic effects of hyperthermia con-sidering the induction of heat by HFEMF exposure. Baner-jee et al.36) suggested that the frequency of HPRT mutants was increased ~ 3fold when Chinese hamster V79 cells were heated at 45°C for 10 min or at 43.5°C for 30 min.
The combined effects of HFEMF and bleomycin were also detected. The mutation frequency increased in a dose-dependent manner from SARs of 50 W/kg and higher (Fig. 1). We suggested that this was caused by the activation of bleomycin itself by the increase in temperature. This tenden-cy was also detected with the combination of bleomycin and heat (Fig. 2). With heat treatment, it would appear that the mutation frequency is higher than treatment with HFEMF exposure. This may be a result of conditions in the dish. The increase of temperature in the special HFEMF exposure dish was heterogeneous, primarily in the middle of exposed area,27) whereas the heat treatment increased the temperature constantly at every part of the culture dish.
In the present investigation, we could not detect an increase of bacterial mutation after exposure to HFEMF even the temperature elevated by the exposure. Barnett et al.37) that no changes were observed in survival, in the inci-dence of mutants following irradiation with ultrasound intensities of 4.5 W/cm2 at temperatures ranging from 37 degree to 43 degree C. They demonstrated that rise in tem-perature also might not affect bacterial mutations. Several studies38–41) also indicated that no increase in mutation was observed by heating or microwave radiation in bacteria. We also concluded that the increase of mutation frequency may be induced by the increase in temperature. The reason for the increase of mutation frequency may be the thermal effect.
During the past decade, mobile phone use has spread world-wide and a huge number of people are now exposed to radiofrequency radiation from mobile phones or base sta-tions. Almost all investigations have indicated the absence of any relation between exposure to RF radiation and adverse health effects. However, there may still be some positive data. In this study, we could not demonstrate any effect of HFEMF exposure using the Ames test. We also could not demonstrate any mutation of the HPRT gene as a result of exposure to HFEMF alone, except for 200 W/kg. However, we were able to demonstrate a combined effect of HFEMF
exposure and bleomycin. We concluded that the effects of HFEMF on HPRT gene mutation exposed to high SARs were increased by heat induced by the HFEMF exposure. However, experiments should still be performed to confirm the safety of high-frequency radiation.
ACKNOWLEDGEMENTS
This work was supported in part by the Committee to Pro-mote Research on the Possible Biological Effects of Electro-magnetic Fields, Ministry of Internal Affairs and Telecom-munications, Japan.
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Received on October 3, 2006 Revision received on November 14, 2006 Accepted on November 15, 2006 J-STAGE Advance Publication Date: December 19, 2006
