1

International Journal of Radiation Biology, 2013; Early Online: 1–8© 2013 Informa UK, Ltd.ISSN 0955-3002 print / ISSN 1362-3095 onlineDOI: 10.3109/09553002.2013.809170

Correspondence: Zülal Atlı Sekeroglu, Department of Biology, Faculty of Art and Sciences, Ordu University, 52200, Ordu, Turkey. Tel: 90 45223 45010/1667. Fax: 90 452 233 9149. E-mail: zulalas@odu.edu.tr or zulalatli@hotmail.com

(Received 13 October 2012; revised 14 May 2013; accepted 16 May 2013)

Evaluation of the cytogenotoxic damage in immature and mature rats exposed to 900 MHz radiofrequency electromagnetic fields

Zülal Atlı Şekeroğlu1, Ayşegül Akar2 & Vedat Şekeroğlu1

1Department of Biology, Faculty of Science and Letters, Ordu University, Ordu, Turkey, and 2Department of Biophysics, Faculty of Medicine, Ondokuz Mayıs University, Samsun, Turkey

Introduction

Non-ionizing radiation is used in industry, commerce, research, medicine and private homes. Everyone is exposed to radiofrequency electromagnetic fields (RF-EMF), a type of non-ionizing radiation at high frequency, from communica-tion devices such as cell phones, cordless phones, cellular antennas, base stations and broadcast transmission towers (Hardell and Sage 2008). Cellular mobile communication

networks such as base stations, handsets and cell phones are in the range of 890–960 MHz for the global system for mobile communication (GSM) 900 systems (Valberg et al. 2007). According to the reports of International Commis-sion on Non-Ionizing Radiation Protection (ICNIRP), while cellular mobile communication networks cause on average low level electromagnetic fields (EMF) to the general pub-lic, handsets and cell phones may cause significantly higher peak levels of exposure during use (ICNIRP 2009). Although the use of mobile phones and other devices emitting RF-EMF has increased significantly (Verschaeve et al. 2010) very little is known about personal EMF exposure in everyday life. Personal EMF exposure of the general public is assessed nowadays with personal exposure meters (exposimeters) whose use has been recommended. Several countries have performed measurement studies in this area of research (Frei et al. 2010, Joseph et al. 2010).

In recent years there has been increasing scientific evidence for, and public concern on, potential health risks from RF-EMF (Hardell and Sage 2008). Amongst many bio-logical targets, the DNA molecule has received the greatest attention in respect to potential EMF-induced damage, because of its relevance for cell function, proliferation, viability, mutation and cancer (Franzellitti et al. 2010). In May 2011, the International Agency for Research on Cancer (IARC) classified EMF electromagnetic fields as possibly carcinogenic to humans (Group 2.B) (Baan et al. 2011). A large number of RF-EMF genotoxicity studies, using cul-tured human and other mammalian cells, have yielded inconsistent and often conflicting information (Speit et al. 2007). Several reviews have concluded that EMF is not directly mutagenic and probably does not increase DNA damage or induce carcinogenesis of known physical or chemical genotoxic agents (Vijayalaxmi 2004, Verschaeve 2005, Otto and von Muhlendahl 2007, Speit et al. 2007, Franzellitti et al. 2010). However, some laboratory investi-gations and biomonitoring studies have provided positive findings (Maes et al. 1997, Koyama et al. 2003, Simi et al. 2008, Phillips et al. 2009, Franzellitti et al. 2010). Recent

AbstractPurpose: One of the most important issues regarding radio­frequency electromagnetic fields (RF­EMF) is their effect on genetic material. Therefore, we investigated the cytogeno­toxic effects of 900 MHz radiofrequency electromagnetic fields (RF­EMF) and the effect of a recovery period after exposure to RF­EMF on bone marrow cells of immature and mature rats.Materials and methods: The immature and mature rats in treat­ment groups were exposed to RF­EMF for 2 h/day for 45 days. Average electrical field values for immature and mature rats were 28.1 4.8 V/m and 20.0 3.2 V/m, respectively. Whole­body specific absorption rate (SAR) values for immature and mature rats were in the range of 0.38–0.78 W/kg, and 0.31–0.52 W/kg during the 45 days, respectively. Two recovery groups were kept for 15 days after RF­EMF exposure.Results: Significant differences were observed in chromosome aberrations (CA), micronucleus (MN) frequency, mitotic index (MI) and ratio of polychromatic erythrocytes (PCE) in all treat­ment and recovery groups. The cytogenotoxic damage in immature rats was statistically higher than the mature rats. The recovery period did not reduce the damage to the same extent as the corresponding control groups.Conclusions: The exposure of RF­EMF leads to cytotoxic and genotoxic damage in immature and mature rats. More sensitive studies are required to elucidate the possible carcinogenic risk of EMF exposure in humans, especially children.

Keywords: Radiofrequency electromagnetic field, chromosomal aberrations, micronucleus, cytotoxicity, rat bone marrow

Int J

Rad

iat B

iol D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y M

onas

h U

nive

rsity

on

09/0

9/13

For

pers

onal

use

onl

y.

2 Z. Atlı Şekeroglu et al.

data have suggested that the results of non-thermal EMF studies vary depending on a variety of biological and physical parameters (Belyaev 2010) which may account for the inconsistency between different studies. Since posi-tive and negative results, with respect to genotoxic effects of EMF, have been found in published studies, new studies are necessary in order to provide assurance whether EMF are really safe and will not cause any unwanted health side effects.

It is very important to understand the possible effects of EMF on DNA damage and repair, and to determine safe limits of RF-EMF for the environment and human exposure, especially for children. Dosimetry plays an important role in determining safe limits of human exposure to RF-EMF (ICNIRP 2009). Specific absorption rate (SAR) is used in risk evaluation. The SAR is expressed as watts per kilogram in different biological tissues (ICNIRP 1998, 2009). Accord-ing to ICNIRP data, the basic SAR limits for time-varying electric and magnetic fields for frequencies up to 10 GHz in the general public exposure are 0.08 W/kg for whole-body, 4 W/kg for limbs and 2 W/kg for head and trunk (ICNIRP 1998). Literature review indicated that comparison of cytotoxic and genotoxic effects on immature and mature animals exposed to long-term RF-EMF and investigation of the effects of a recovery period have not been sufficiently studied.

The aims of this study were to investigate the potential cytotoxic and genotoxic effects of long-term exposure (45 days) to 900 MHz RF-EMF on bone marrow cells in imma-ture and mature rats; to determine the effect of a recovery period of 15 days after exposure to RF-EMF and to compare whether there is any difference between immature and mature rats in terms of genotoxic damage. Two cytogenetic assays – chromosome aberrations (CA) and micronucleus test (MN) – were used in order to determine the genotoxic effects of EMF in rat bone marrow cells. Mitotic index (MI) and the ratio of polychromatic erythrocytes (PCE) to nor-mochromatic erythrocytes (NCE) were also evaluated in order to determine possible cytotoxic effects.

Materials and methods

AnimalsThis study was conducted at Ondokuz Mayıs University, Experimental Research Center with the permission granted by the Local Animal Studies Ethical Board (HADYEK/53, 2009/45). Twenty-four 2-week-old (immature) and twenty-four 10-week-old (mature) Wistar albino rats were obtained from the Experimental Research Center of University of Ondokuz Mayıs. The animals were procured, maintained and used in accordance with the Animal Welfare Act and the Guide for the Care and Use of Laboratory Animals prepared by the Institute of Laboratory Animal Resources, National Research Council, USA. They were housed in cages under laboratory conditions (12 h day/12 h night cycle, air temperature of 21 1°C). The animals were fed with com-mercial rat pellet (Bil-Yem Co, Ankara, Turkey) and were provided with a distilled water system to set an ad libitum care environment.

Experimental designThe animals were randomly divided into six groups each containing eight rats:

IMC (Immature control): Two-week-old immature rats were kept in the normal conditions of the laboratory for 45 days as an immature sham-exposed group. They were kept in the pie cage restrainer but did not receive any treatment.IMT (Immature treatment): Two-week-old immature rats were exposed to 900 MHz RF-EMF for 2 h/day for 45 days.IMR (Immature recovery): Immature rats were kept for a recovery period of 15 days without being exposed to RF-EMF after exposure to RF-EMF for 45 days.MC (Mature control): Ten-week-old mature rats were kept in the normal conditions of the laboratory for 45 days as the mature sham-exposed group. They were kept in the pie cage restrainer but did not receive any treatment.MT (Mature treatment): Ten-week-old mature rats were exposed to 900 MHz RF-EMF for 2 h/day for 45 days.MR (Mature recovery): Mature rats were kept for a recovery period of 15 days without being exposed to RF-EMF after exposure to RF-EMF for 45 days.

All treatments were performed each day between 10:00 and 12:00 h during the 45-day period. All the rats were euthanized by cervical dislocation on the 45th day (control and RF-EMF treatment groups) and 60th day (recovery groups).

Exposure system and SAR calculationThe design of RF-EMF was performed by using the procedure described by Koyu et al. (2005) and Dasdag et al. (2009) in the Department of Biophysics at the Ondokuz Mayıs University. During the study period, Department of Electrical and Electronic Engineering of that University performed measurements to detect quality controls and electric field measurements. For exposure of RF-EMF, Ever-est GSM Simulator type with a continuous wave RF source (900CW4 type; Everest Co. Adapazari, Turkey) was used. The background levels of other RF sources in the laboratory were measured with a spectrum analyzer/satellite receiver (Promax MC-877C, Barcelona, Spain) and an electrical field meter (PMM 8053 Portable Field Meter, Costruzioni Elettron-iche Centro Misure Radioelettriche Srl., Cisano Sul Neva, Italy). The stability of the frequency system was checked every week by using a spectrum analyzer. 900CW4 type GSM Simulator which had an 850–950 MHz band interval. A galva-nized plate with 1 mm thick was placed at the bottom of the pie cage restrainer for grounding of the static field.

The rats in the treatment groups were exposed to 900 MHz RF-EMF with continuous wave signal in a spe-cially designed pie cage PlexiglasÒ restrainer. The pie cage PlexiglasÒ restrainer consisted of 12 slices and every one of the rats was located in one slice during exposure. The antenna heights were approximately 15 cm. To supply an equal dis-tribution of the electric field to rats, the antenna was located in the middle of the pie cage restrainer. It was fixed as closely as possible to the whole body of the rat (about 6 cm from the head). Due to the movements of immature and mature rats, the distance between the animals and the antenna ranged from 3–6 cm. To reduce the stress of rats in the cage and supply

Int J

Rad

iat B

iol D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y M

onas

h U

nive

rsity

on

09/0

9/13

For

pers

onal

use

onl

y.

Radiofrequency electromagnetic field and cytogenotoxic damage 3

air, a series of air holes, 1 cm in diameter, were created at the top of restrainer. The generator was set to 2 W and the rats in the treatment groups were exposed to RF-EMF for 2 h/day, at the same time every day, for 45 days. A PMM 8053 Portable Field Meter and an EP-330 electric field probe (Costruzioni Elettroniche Centro Misure Radioelettriche Srl., Cisano Sul Neva, Italy) were used to measure electrical field exposure.

In this study, every week, the background effects of other RF sources in the environment were measured with the spectrum analyzer/satellite receiver while the RF source was turned off. To calculate the SAR, during exposure of rats for 45 days, the electric field values were measured every week daily at the same time while the rats were growing up. Every week, whole body electric field measurements were obtained from head, dorsal, tail of one rat at one minute for each region. Electric field values applied during the period of 45 days to rats were calculated using a statistical package for the Social Sciences (SPSS for Windows, version 15.0, SPSS Inc., Chicago, IL, USA). Average electric field values, which were calculated from a total of 102 electric field values for each of immature or mature rats, were expressed as means standard deviations (SD). The average electrical field val-ues for immature and mature rats were 28.1 4.8 V/m and 20.0 3.2 V/m, respectively. Due to the movement of imma-ture and mature rats, the 45 day electric field values varied from 19.8–28.6 V/m and from 18.2–23.6 V/m, respectively. The SAR value can be approximately calculated according to the formula from previous studies (Ferreira et al. 2006a, Dasdag et al. 2009, Esmekaya et al. 2010, Avci et al. 2012):

SARE

(W/kg)2

σ⋅ρRMS (1)

where E2RMS is the root mean square value of the electrical

field, s is the mean electrical conductivity of the tissues, and r is the mass density. For immature and mature rats, whole body SAR were calculated using the SAR equation, assuming r 1040 kg/m3. Dielectric values for immature rats are dif-ferent than the mature rats. The mean electrical conductivity value at 900 MHz was obtained from Peyman et al. (2001) for immature rats. The whole body SAR value for immature rats was found to be 0.76 W/kg from average electric field values, adopting s 1 S/m (siemens per metre) for 45 days. Dielectric data at 900 MHz for mature rats was obtained from Federal Communications Commission (http://transition.fcc.gov/oet/rfsafety/dielectric.html). The whole body SAR value for mature rats was found to be 0.37 W/kg using average electric field values of mature rats and, adopting s 0.969 S/m. Because of the movement of rats, whole-body SAR val-ues for immature and mature rats were in the range of 0.38– 0.78 W/kg, and 0.31–0.52 W/kg during the 45 days of exposure, respectively.

CA analysisFour animals were used in each group for CA analysis. Bone marrow preparation for the analysis of CA in metaphase cells was conducted according to the method described by Preston et al. (1981), with slight modifications. An aqueous solution of colchicine (2 mg/kg b.w.) obtained from Sigma (St Louis, MO, USA) was injected intraperitoneally 2 h prior

to scheduled euthanasia by cervical dislocation. Both femurs were dissected out and cleaned of any adhering muscle. Bone marrow cells were collected from the femurs by flushing in 0.9% isotonic sodium chloride (Sigma Chemicals Co., St Louis, MO, USA) solution in the centrifuge tube. The material was centrifuged at 204 g for 10 min and the pellet was re-suspended in 0.56% potassium chloride (Sigma Chemicals Co., St Louis, MO, USA) solution and incubated at 37°C for 25 min. Cells were re-centrifuged for 10 min and then fixed in chilled Carnoy’s fixative (acetic acid: methanol, 1:3, v/v). Centrifugation and fixation (in the cold) were repeated five times at interval of 20 min. Fixed cells were resuspended in a small volume of the fixative and dropped onto chilled slides which were coded for blind analysis, air dried, and stained on the following day in 5% buffered Giemsa (pH 6.8). All chemicals used for fixation and staining were obtained from Merck (Darmstadt, Germany).

MN testThe remaining four rats from each group were employed for the bone marrow MN test. The test was performed as described by Schmid (1975), with minor modifications. Bone marrows were flushed out from the both femurs using 2 ml of fetal calf serum (Biological Industries, Beit Haemek, Israel) and collected in centrifuge tubes containing 2 ml of fetal calf serum. The tubes were centrifuged at 204 g for 10 min. The supernatant was removed with Pasteur pipette. The pellet was resuspended in a small amount of fetal calf serum and spread on clean slides. The preparations were then air dried and fixed in absolute methanol for 10 min. After fixation, the coded slides were stained in May-Grünwald for 3 min and Giemsa (5%) for 12 min at pH 6.8. All chemicals used for fixation and staining were obtained from Merck (Darmstadt, Germany).

ScoringAll slides were examined using a Leica DM2500 light microscope (Leica Microsystems, Wetzlar, Germany) at 1000 magnification. MI was calculated as the number of metaphases in 2000 cells per animal for a total of 8000 cells for each group. Structural and numerical CA were analyzed from 100 well spread, intact metaphases per animal for a total of 400 metaphases for each group. They were identified according to criteria described by Savage (1976). As recom-mended by Mace et al. (1978), gaps were not considered CA. The number of each type of aberration, the total and percentage of CA were recorded and summarized.

For the analysis of MN frequencies, 2000 PCE were scored per animal for a total of 8000 PCE for each group. Two hun-dred erythrocytes (immature and mature cells) per animal for a total of 800 cells for each group were scored to determine the ratio of PCE to NCE. PCE can be distinguished from NCE by different staining. PCE, the young erythrocytes, are blue stained cells due to the presence of ribonucleic acid. While the PCE are maturing the ribonucleic acid gradually disap-pears. For this reason mature erythrocytes (NCE) stain pink.

Statistical analysisThe data are expressed as the mean standard deviation (SD). Differences between groups were assessed by one way

Int J

Rad

iat B

iol D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y M

onas

h U

nive

rsity

on

09/0

9/13

For

pers

onal

use

onl

y.

4 Z. Atlı Şekeroglu et al.

Figure 1. Different types of chromosome aberrations in rat bone marrow cells after long-term EMF exposure. (A) Fragment, (B) Chromatid break, (C) Chromosome break, (D) Sister chromatid union, (E) Centric fusion, and (F) Polyploidy.

Table I. Effects of 900 MHz EMF for 45 days on mitotic index (MI), chromosome aberrations (CA), micronucleus (MN) frequency and polychromatic erythrocytes (PCE) ratio in rat bone marrow cells.

Treatment groups MI % SD P SCU B′ B′′ AF CF Total CA CA % SD

MN frequency

(MN/2000 PCE)

PCE ratio

(PCE/200 erythrocytes)

IMC 4.72 0.13 1 – 1 – 1 – 3 0.75 0.50 0.5 0.57 101.8 6.0IMT 1.81 0.22a 12 5 7 2 33 1 60 15 2.94a,b,d 11.75 2.2a,d 63.75 3.77 a, b, d

IMR 2.41 0.6a,e 15 1 1 – 25 1 43 10.75 2.2a,e 9.5 1.3a,e 77.25 3.86 a, e

MC 4.53 0.12 1 – 1 – 2 – 4 1.00 0.0 0.75 0.5 100.5 7.14MT 1.63 0.27a,c 10 1 6 1 19 1 38 9.50 1.73a,c 8.5 1.3a,c 81.25 3.4 a

MR 3.60 0.36a 1 – 9 1 10 1 22 5.50 1.3a 5.25 1.7a 88.25 1.7 a

P, Polyploidy; SCU, Sister chromatid unions; B′, Chromatid breaks; B′′, Chromosome breaks; AF, Acentric fragments; CF, Centric fusions. aIMT, IMR, MT and MR groups compared to the corresponding control group. bIMT group compared to IMR group.cMT group compared to MR group. dIMT group compared to MT group. eIMR group compared to MR group. (p 0.05).

analysis of variance (ANOVA) using the SPSS software pack-age for Windows. The Tukey post hoc testing was performed for inter-group comparisons. Statistical significance was set at p 0.05.

Results

Table I represents the effects of long-term RF-EMF exposure and a recovery period after RF-EMF exposure on the MI, CA, MN and PCE ratio in rat bone marrow cells. The frequencies of CA and MN significantly increased in the all treatment groups of immature and mature rats compared with the corresponding control group (p 0.05). Figure 1 presents different types of CA and Figure 2 shows a PCE with a MN in rat bone marrow cells after long-term RF-EMF exposure. The major type of CA observed in all the treatment and recovery groups was acentric fragment. Significant differ-ences were also observed in CA and MN frequencies between

the recovery groups and the corresponding control group in rats (p 0.05). In addition, the frequencies of CA and MN obtained for the treatment and recovery groups of immature rats were statistically higher than the mature rats (p 0.05) (Table I). Although the formation of CA and MN decreased in the recovery groups of rats with respect to EMF-exposed animals, the 15-day recovery period did not reduce the CA and MN frequencies to the same extent as the correspond-ing control groups. The CA and MN frequencies of the treat-ment groups were found significantly different from those in the recovery groups (p 0.05) except the MN frequency of immature treatment group (Table I).

With regard to the cytotoxic effects of the RF-EMF exposure on bone marrow cells, as measured by MI and PCE ratio, in all the treatment and recovery groups of immature and mature rats the MI and the PCE values were signifi-cantly reduced both compared to the corresponding control group (p 0.05) (Table I). Significant difference between the

Int J

Rad

iat B

iol D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y M

onas

h U

nive

rsity

on

09/0

9/13

For

pers

onal

use

onl

y.

Radiofrequency electromagnetic field and cytogenotoxic damage 5

treatment groups and recovery groups was found in MI value of mature rats and in PCE value of immature rats (p 0.05). The values of PCE obtained for the treatment and recovery groups of immature rats were found statistically differ-ent from those in mature rats, whereas the MI values were only found different in recovery groups of immature and mature rats (p 0.05). Although the 15-day recovery period increased the MI and PCE values in recovery groups of rats, these values did not increase to the same extent as the cor-responding control groups (Table I).

The results of this study indicate that in vivo exposure of bone marrow cells of immature and mature rats to RF-EMF had cytotoxic and genotoxic effects. In addition, the 15-day recovery period was insufficient for the improvement of the chromosomal damage, MI and PCE ratios in the bone mar-row of mature and immature rats under the present experi-mental conditions.

Discussion

Over the past decade, the use of mobile phones and other devices emitting RF-EMF has increased significantly. Along with this increase, concerns about possible hazards related to cell phone exposure have also been growing. Amongst many biological targets, the DNA molecule has received the greatest attention in respect to potential RF-EMF dam-age, because of its relevance for cell function, proliferation, viability, mutation and cancer. Besides cancer, genetic effects are a major concern (Vijayalaxmi 2004, Franzellitti et al. 2010, Verschaeve et al. 2010). No convincing evidence that RF-EMF causes significant damage to the DNA molecule or induces carcinogenesis was found in some studies per-formed to determine a potential genotoxic effect of exposure to RF-EMF (Vijayalaxmi et al. 2001a, 2001b, Bisht et al. 2002, Gorlitz et al. 2005, Verschaeve et al. 2006, Juutilainen et al. 2007, Ziemann et al. 2009). However, other studies have shown that exposure of laboratory animals in vivo and of cultured cells in vitro to various RF-EMF signals induced DNA damage (Phillips et al. 2009, Ruediger 2009, Franzellitti et al. 2010, Verschaeve et al. 2010). Although most of the posi-tive findings could be attributed to thermal effects, following low level (non-thermal) exposure conditions, there may be some indirect effects on, for example, the replication and/or

transcription of genes and, some new studies have reopened this discussion (ICNRP 2009). It has been stated that the results of non-thermal RF-EMF studies vary depending on physical and biological parameters such as laboratory condi-tions, power density and specific absorption rate, duration of exposure and post-exposure time, endpoint measured, cell type and density, individual variations, genotype, gender, age and individual variations (Belyaev 2010). Because these parameters may be different in various laboratories, both positive and negative findings may be obtained by different authors.

Bone marrow cell toxicity is normally indicated by sub-stantial and statistically significant decreases in the MI and the ratio of PCE. A very large decrease in the ratios of these parameters would be indicative of cytostatic or cytotoxic effects in parallel with other biological endpoints (Suzuki et al. 1989, Hamada et al. 2001). In addition, the relationship between cytotoxicity and tumor promotion was also indi-cated in several studies (Albert and Magee 2000). In the pres-ent study we found that the long-term 900 MHz RF-EMF in vivo exposure of immature and mature rats was an inhibitor of cell proliferation and showed cytotoxic effects by decreas-ing of the MI and the PCE ratios in bone marrow cells. Such decreases could be due to inhibition of DNA synthesis, microtubule alternation or blocks induced in the G2 phase of the cell cycle, preventing the cells from entering mitosis. It has been stated that when V79 cells were exposed to 935 MHz RF-EMF (SAR 0.12 W/kg), the microtubule structure was altered after 3 h of irradiation and significantly decreased growth was noted in cells exposed for 3 h three days after irradiation (Pavicic and Trosic 2008).

Genotoxicity endpoints have been considered to be poten-tial biomarkers in relation to cancer risk. The rodent bone marrow MN and CA tests are the most widely used in vivo assays for identification of genotoxic effects such as chromo-some damage and aneuploidy, associated with mutagenesis and carcinogenesis (Heddle 1973, Schmid 1973). During the last few years, a number of studies, performed to determine a potential genotoxic effect of exposure to RF-EMF, showed increased genetic damage although others were negative. As noted before, these different results are probably due to differences in physical, biological and methodological parameters. Vijayalaxmi et al. (2001a, 2001b) did not find increased CA and MN frequencies in human blood lympho-cytes exposed to 835.6 and 847.7 MHz RF-EMF for 24 h. Bisht et al. (2002) reported that exposure to 835.6 and 847.7 MHz RF-EMF for 3, 8, 16 or 24 h did not increase MN frequency in C3H 10T1/2 mouse fibroblast cells. Gorlitz et al. (2005) did not observe an increase in the number of micronuclei in erythrocytes of the peripheral blood and bone marrow cells, keratinocytes and spleen lymphocytes of mice exposed to 900 and 1800 MHz RF-EMF for 2 h per day over periods of one and six weeks. Verschaeve et al. (2006) investigated the possible combined genotoxic effects of 900 MHz RF-EMF with the drinking water mutagen and carcinogen 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) in rats exposed to RF-EMF for a period of 2 years for 2 h per day, 5 days per week. Co-exposures to MX and RF-EMF did not significantly increase the DNA damage and MN in rat blood,

Figure 2. A polychromatic erythrocyte with a micronucleus in rat bone marrow cells after long-term EMF exposure.

Int J

Rad

iat B

iol D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y M

onas

h U

nive

rsity

on

09/0

9/13

For

pers

onal

use

onl

y.

6 Z. Atlı Şekeroglu et al.

RF-EMF at SAR of 0.37 W/kg and 0.49 W/kg for 2 h/day for 45 days. The cytogenotoxic damage was more remarkable in immature rats and, the recovery period did not improve this damage in immature rats (Sekeroglu et al. 2012). The results of the present study were similar, especially for immature rats.

Several studies have suggested that the RF-EMF exposure can produce an increase in DNA damage directly or affect the rate of DNA repair processes (Phillips et al. 1998, Ruediger 2009). It seems that indirect mechanisms such as increased free radical activity may be responsible for this damage rather than direct mechanisms. It has been stated that the exposure of RF-EMF causes an increase free radical produc-tion, and contributes to human carcinogenesis through the production of genetic mutations that are associated with the initiation and progression of cancer and with changes in cell proliferation (Phillips et al. 1998, Ruediger 2009, Aydin and Akar 2011, Avci et al. 2012, (Sekeroglu et al. 2012). Aydin and Akar (2011) focused on oxidative stress in the major lymphoid organs of immature and mature rats and found that levels of irreversible oxidative damage were much higher in immature rats than mature rats. In addition, a 15-day recovery period was insufficient for the normalization of some enzyme activi-ties in the lymphoid organs of rats. These results support our findings in which RF-EMF affected immature rats more than mature rats and the 15-day recovery period after exposure to RF-EMF for 45 days was insufficient suggesting that RF-EMF may affect DNA repair processes.

Some peripheral blood lymphocyte cultures were exposed to magnetic resonance imaging for different periods and dif-ferent variable magnetic fields in the radiofrequency domain and, they were left at room temperature for 24 h, to test for possible damage recovery. Twenty-four h of recovery seemed to allow a certain degree of repair by modulating the MN fre-quency (Simi et al. 2008). Although a 15 min to 24 h recovery period was sufficient to allow repair after in vitro exposure of RF-EMF, a 15-day recovery period after in vivo exposure to RF-EMF for 45 days was insufficient for DNA repair in the present study. Belyaev et al. (2009) reported that universal global telecommunications system mobile phone micro-waves affect chromatin and inhibit formation of DNA dou-ble-strand breaks (DSB) as measured by the co-localization of 53BP1/gamma-H2AX DNA repair foci in human lympho-cytes from hypersensitive and healthy persons. The effects of microwaves on 53BP1/gamma-H2AX foci persisted up to 72 h following exposure of cells, even longer than the stress response following heat shock. According to the results, they stated that a decrease in 53BP1/gamma-H2AX foci could be a manifestation of the inhibitory effects of RF-EMF on repair of spontaneous DSB. Markova et al. (2010) detected 915 and 1947.4 MHz EMF from mobile phones inhibited formation of 53BP1 foci caused by DSB in human primary fibroblasts, mesenchymal stem cells and lymphocytes. Contrary to fibroblasts, stem cells did not adapt to chronic exposure during the 2 weeks. Inhibitory effects of RF-EMF exposure on DSB repair in stem cells may result in formation of CA and cancer induction. Stem cells in children are more active than in adults and are more sensitive to RF-EMF expo-sure. Stem cells react to more RF-EMF frequencies than do

liver and brain cells. Juutilainen et al. (2007) reported that RF-EMF had no significant effect on MN frequency in poly-chromatic or normochromatic erythrocytes in mice exposed to 902.5 MHz EMF for 78 weeks (1.5 h per day, 5 days per week) and in K2 transgenic and non-transgenic mice exposed to digital mobile phone signals for 52 weeks. Ziemann et al. (2009) did not find a significant increase in the frequency of MN in erythrocytes of mice exposed to 902 MHz or 1747 MHz RF-EMF for 2 h per day, 5 days per week for 2 years.

Although there is no consensus on whether RF-EMF causes genetic damage, the results of the present study and some previous reports have indicated that RF-EMF may be capable of damaging to DNA. A slight increase in the CA frequency was observed in human white blood cells exposed to an RF-EMF at 954 MHz for 2 h (Maes et al. 1995). Significantly increased DNA damage was determined using the Comet assay in human Molt-4 T-lymphoblas-toid cells exposed to 813.5 MHz RF-EMF for 2 h and 21 h (Phillips et al. 1998). A significant increase in the frequency of micronucleated lymphocytes was detected in human blood cells exposed to 837 MHz and 1909.8 MHz RF-EMF for 24 h (Tice et al. 2002). Although the frequencies of CA and sister chromatid exchange (SCE) showed a significant increase, no change in cell cycle progression was noted among mobile phone users exposed to 900 MHz band for maximum 4–5 h per day during 2 years (Gadhia et al. 2003). An increase in aneuploidy of chromosome 17 was observed in human lymphocytes exposed to 830 MHz EMF for 72 h (Mashevich et al. 2003). An increased MN frequency was observed in rat bone marrow cells exposed to 910 MHz RF-EMF for 2 h/day per 30 consecutive days (Demsia et al. 2004). Thirty min and 1 h exposure to 895 and 915 MHz RF-EMF sig-nificantly affected chromatin conformation in transformed human blood lymphocytes (Sarimov et al. 2004). The exposure to microwaves from a GSM mobile phone (915 MHz) caused significant condensation of chromatin in human blood lymphocytes (Belyaev et al. 2005). The exposure to RF-EMF of a frequency range between 890 MHz and 915 MHz RF-EMF for 72 h affected chromatin conformation, p53 binding protein 1 (53BP1) and phosphorylated histone H2AX (gamma-H2AX) DNA repair foci in human blood lymphocytes (Markova et al. 2005). Increased DNA damage and MN frequency were detected in peripheral blood lymphocytes of mobile phone users exposed to RF-EMF frequency ranging from 800–2000 MHz (Gandhi 2005). The exposure of pregnant rats to a cellu-lar phone antenna (834 MHz) from the first day of pregnancy for 8.5 h per day significantly increased the MN frequency in erythrocytes in newborn pups from pregnant rats (Ferreira et al. 2006b). Increased levels of aneuploidy were detected in human blood lymphocytes exposed to 800 MHz RF-EMF for 72 h (Mazor et al. 2008). Increased micronuclei frequency in exfoliated buccal mucosa cells was found in mobile phone users exposed to RF-EMF frequency ranging from 900–1800 MHz (Yadav and Sharma 2008). Increased DNA damage in rat leukocytes exposed to 915 MHz microwaves for 30 min was detected by using the Comet assay (Gajski and Garaj-Vrhovac 2009). In our previous research, we found that increased MN and CA frequencies and, decreased MI and ratio of PCE were observed in immature and mature rats exposed to 1800 MHz

Int J

Rad

iat B

iol D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y M

onas

h U

nive

rsity

on

09/0

9/13

For

pers

onal

use

onl

y.

Radiofrequency electromagnetic field and cytogenotoxic damage 7

Belyaev IY, Hillert L, Protopopova M, Tamm C, Malmgren LO, Persson BR, Selinova G, Harms-Ringdahl M. 2005. 915 MHz microwaves and 50 Hz magnetic field affect chromatin conformation and 53BP1 foci in human lymphocytes from hypersensitive and healthy persons. Bioelectromagnetics 26:173–184.

Bisht KS, Moros EG, Straube WL, Baty JD, Roti Roti JL. 2002. The effect of 835.62 MHz FDMA or 847.74 MHz CDMA modulated radiofrequency radiation on the induction of micronuclei in C3H 10T(1/2) cells. Radiation Research 157:506–515.

Dasdag S, Akdag Z, Ulukaya E, Uzunlar AK, Yegin D, Dasdag S, Akdag MZ, Ulukaya E, Uzunlar AK, Ocak AR. 2009. Effect of mobile phone exposure on apoptotic glial cells and status of oxidative stress in rat brain. Electromagnetic Biology and Medicine 28:342–354.

Demsia G, Vlastos D, Matthopoulos DP. 2004. Effect of 910-MHz electromagnetic field on rat bone marrow. Scientific World Journal 4:48–54.

Esmekaya MA, Seyhan N, Omeroglu S. 2010. Pulse modulated 900 MHz radiation induces hypothyroidism and apoptosis in thyroid cells: A light, electron microscopy and immunohistochemical study. International Journal of Radiation Biology 86:1106–1116.

Federal Communications Commission. 2012. Radio frequency safety, Body tissue dielectric parameters tool. Available from: http://transi-tion.fcc.gov/oet/rfsafety/dielectric.html.

Frei P, Mohler E, Burgi A, Frohlich J, Neubauer G, Braun-Fahrlander C, Roosli M. 2010. Classification of personal exposure to radio frequency electromagnetic fields (RF-EMF) for epidemiological research: Evaluation of different exposure assessment methods. Environment International 36:714–720.

Ferreira AR, Bonatto F, de Bittencourt Pasquali MA, Polydoro M, Dal-Pizzol F, Fernandez C, de Salles AA, Moreira JC. 2006a. Oxidative stress effects on the central nervous system of rats after acute exposure to ultra high frequency electromagnetic fields. Bioelectromagnetics 27:487–493.

Ferreira AR, Knakievicz T, Pasquali MA, Gelain DP, Dal-Pizzol F, Fernandez CE, de Salles AA, Ferreira HB, Moreira JC. 2006b. Ultra high frequency-electromagnetic field irradiation during pregnancy leads to an increase in erythrocytes micronuclei incidence in rat offspring. Life Sciences 80:43–50.

Franzellitti S, Valbonesia P, Ciancaglinia N, Biondib C, Contina A, Bersanic F, Fabbria E. 2010. Transient DNA damage induced by high-frequency electromagnetic fields (GSM 1.8 GHz) in the human trophoblast HTR-8/SVneo cell line evaluated with the alkaline comet assay. Mutation Research 683:35–42.

Gadhia P, Shah T, Mistry A, Pithawala M, Tamakuwala D. 2003. A pre-liminary study to assess possible chromosomal damage among users of digital mobile phones. Electromagnetic Biology and Medicine 22:149–159.

Gajski G, Garaj-Vrhovac V. 2009. Radioprotective effects of honeybee venom (Apis mellifera) against 915-MHz microwave radiation- induced DNA damage in Wistar rat lymphocytes: In vitro study. International Journal of Toxicology 28:88–98.

Gandhi AG. 2005. Genetic damage in mobile phone users: Some preliminary findings. Indian Journal of Human Genetics 11:99–104.

Gorlitz BD, Muller M, Ebert S, Hecker H, Kuster N, Dasenbrock C. 2005. Effects of 1-week and 6-week exposure to GSM/DCS radiofrequency radiation on micronucleus formation in B6C3F1 mice. Radiation Research 164:431–439.

Hamada S, Yamasaki K, Nakanishi S, Omori T, Serikawa T, Hayashi M. 2001. Evaluation of the general suitability of the rat for the micro-nucleus assay: The effect of cyclophosphamide in 14 strains. Mutation Research 495:127–134.

Hardell L, Sage C. 2008. Biological effects from electromagnetic field exposure and public exposure standards. Biomedicine & Pharmaco-therapy 62:104–109.

Heddle JA. 1973. A rapid in vivo test for chromosomal damage. Mutation Research 18:187–190.

International Commission on Non-Ionizing Radiation Protection (ICNIRP), 1998. ICNIRP Guidelines; For Limiting Exposure to Time-Varying Electric, Magnetic and Electromagnetic Fields (Up to 300 GHz). Published in: Health Physics 74:494–522. Available from: http://www.icnirp.de/documents/emfgdl.pdf.

International Commission on Non-Ionizing Radiation Protection (ICNRP), 2009. Exposure to high frequency electromagnetic fields, biological effects and health consequences (100 kHz–300 GHz), ICNIRP 16/2009. Munich, Germany. Available from: http://www.icnirp.de/documents/RFReview.pdf.

Joseph W, Frei P, Roosli M, Thuroczy G, Gajsek P, Trcek T, Bolte J, Vermeeren G, Mohler E, Juhasz P, Finta V, Martens L. 2010.

differentiated cells and this may be important for cancer risk assessment. These sensitivities of stem cells may help clarify the differences between results obtained in leukemia studies with children and adults exposed to RF-EMF (Markova et al. 2010). In agreement with these results, we have found that much higher and irreversible cytogenotoxic damage was observed in immature rats than in mature rats in the pres-ent study as well as in our previous study ((Sekeroglu et al. 2012).

Our in vivo study suggests that the exposure of 900 MHz RF-EMF for 45 days has a cytotoxic and genotoxic potential in immature and mature rats, and the damage in the treat-ment and recovery groups of immature rats is statistically higher than the mature rats. In addition, the recovery period is insufficient for the reversal of this cytogenotoxic damage in bone marrow cells after the exposure to RF-EMF. Because the results of RF-EMF investigations are often conflicting and contradictory, more detailed studies are necessary to eluci-date the molecular mechanisms of RF-EMF on cell-cycle kinetics, DNA damage and repair processes, to determine the possible carcinogenic risk and to make a risk assessment of RF-EMF exposure in human, especially for children.

Acknowledgements

We thank Seval Kontas, Muhammet Aksoy (Ordu University) and Mustafa Ince (Ondokuz Mayıs University) for contribu-tions to the laboratory studies. We would also like to thank Prof. Dr Güven Önbilgin and his assistants, Ertugrul Sunan (Ondokuz Mayıs University), who helped us in the electrical field measurements.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

ReferencesAlbert RE, Magee PS. 2000. The tumorigenicity of mutagenic contact

sensitizing chemicals. Risk Analysis 20:317–325.Avci B, Akar A, Bilgici B, Tunçel OK. 2012. Oxidative stress induced by

1.8 Ghz radio frequency electromagnetic radiation and effects of the garlic extract in rats. International Journal of Radiation Biology 88:799–805.

Aydin B, Akar A. 2011. Effects of a 900-MHz electromagnetic field on oxidative stress parameters in rat lymphoid organs, polymorpho-nuclear leukocytes and plasma. Archives of Medical Research 42: 261–267.

Baan R, Grosse Y, Lauby-Secretan B, El Ghissassi F, Bouvard V, Benbrahim-Tallaa L, Guha N, Islami F, Galichet L, Straif K; WHO International Agency for Research on Cancer Monograph Working Group. 2011. Carcinogenicity of radiofrequency electromagnetic fields. The Lancet Oncology 12:624–626.

Belyaev IY. 2010. Dependence of non-thermal biological effects of microwaves on physical and biological variables: Implications for reproducibility and safety standards. In: Giuliani L, Soffritti M, editors. Non-thermal effects and mechanisms of interaction between electromagnetic fields and living matter. European Journal of Oncology Library, An ICEMS Monograph, Vol. 5. Italy: Bologna. pp. 187–218.

Belyaev IY, Markova E, Hillert L, Malmgren LOG, Persson BRR. 2009. Microwaves from UMTS/GSM mobile phones induce long-lasting inhibition of 53BP1/g-H2AX DNA repair foci in human lymphocytes. Bioelectromagnetics 30:129–141.

Int J

Rad

iat B

iol D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y M

onas

h U

nive

rsity

on

09/0

9/13

For

pers

onal

use

onl

y.

8 Z. Atlı Şekeroglu et al.

Savage JRK. 1976. Classification and relationships of induced chromosomal structural changes. Journal of Medical Genetics 13:103–122.

Schmid W. 1973. Chemical mutagen testing on in vivo somatic mammalian cells. Agents Actions 3:77–85.

Schmid W. 1975. The micronucleus test. Mutation Research 31:9–15.Sekeroglu V, Akar A, Atlı Sekeroglu Z. 2012. Cytotoxic and genotoxic

effects of high-frequency electromagnetic fields (GSM 1800 MHz) on immature and mature rats. Ecotoxicology and Environmental Safety 80:140–144.

Simi S, Ballardin M, Casella M, De Marchi D, Hartwig V, Giovannetti G, Vanello N, Gabbriellini S, Landini L, Lombardi M. 2008. Is the geno-toxic effect of magnetic resonance negligible? Low persistence of micronucleus frequency in lymphocytes of individuals after cardiac scan. Mutation Research 645:39–43.

Speit G, Schütz P, Hoffmann H. 2007. Genotoxic effects of exposure to radiofrequency electromagnetic fields (RF-EMF) in cultured mam-malian cells are not independently reproducible. Mutation Research 626:42–47.

Suzuki Y, Nagae Y, Li J, Sabaka H, Mozawa K, Takahashi A, Shimizu H. 1989. The micronucleus test and erythropoiesis: Effects of erythropoietin and a mutagen on the ratio of polychro-matic to normochromatic erythrocytes (P:N ratio). Mutagenesis 4:420–424.

Tice RR, Hook GG, Donner M, McRee DI, Guy AW. 2002. Genotoxic-ity of radiofrequency signals. I. Investigation of DNA damage and micronuclei induction in cultured human blood cells. Bioelectro-magnetics 23:113–126.

Valberg PA, van Deventer TE, Repacholi MH. 2007. Workgroup report: Base stations and wireless networks – radiofrequency (RF) expo-sures and health consequences. Environmental Health Perspectives 115:416–24.

Verschaeve L. 2005. Genetic effects of radiofrequency radiation (RFR). Toxicology and Applied Pharmacology 207:336–341.

Verschaeve L, Heikkinen P, Verheyen G, Van GU, Boonen F, Vander, PF, Maes A, Kumlin T, Maki-Paakkanen J, Puranen L, Juutilainen J. 2006. Investigation of co-genotoxic effects of radiofrequency electromag-netic fields in vivo. Radiation Research 165:598–607.

Verschaeve L, Juutilainen J, Lagroye I, Miyakoshi J, Saunders R, de Seze R, Tenforde T, van Rongen E, Veyret B, Xu Z. 2010. In vitro and in vivo genotoxicity of radiofrequency fields. Mutation Research 705:252–268.

Vijayalaxmi, Bisht KS, Pickard WF, Meltz MJ, Roti Roti JL, Moros EG. 2001a. Chromosome damage and micronucleus formation in human blood lymphocytes exposed in vitro to radiofrequency radiation at a cellular telephone frequency (847.74 MHz, CDMA). Radiation Research 156:430–432.

Vijayalaxmi, Leal BZ, Meltz ML, Pickard WF, Bisht KS, Roti Roti JL, Straube WL, Moros EG. 2001b. Cytogenetic studies in human blood lymphocytes exposed in vitro to radiofrequency radiation at a cel-lular telephone frequency (835.62 MHz, FDMA). Radiation Research 155:113–121.

Vijayalaxmi OG. 2004. Controversial cytogenetic observations in mammalian somatic cells exposed to radiofrequency radiation. Radiation Research 162:481–496.

Yadav O, Sharma MK. 2008. Increased frequency of micronucleated exfoliated cells among humans exposed in vivo to mobile telephone radiations. Mutation Research 650:175–180.

Ziemann C, Brockmeyer H, Reddy SB, Vijayalaxmi, Prihoda TJ, Kuster N, Tillmann T, Dasenbrock C. 2009. Absence of geno-toxic potential of 902 MHz (GSM) and 1747 MHz (DCS) wireless communication signals: In vivo two-year bioassay in B6C3F1 mice. International Journal of Radiation Biology 85:454–464.

Comparison of personal radio frequency electromagnetic field exposure in different urban areas across Europe. Environmental Research 110:658–663.

Juutilainen J, Heikkinen P, Soikkeli H, Maki-Paakkanen J. 2007. Micronucleus frequency in erythrocytes of mice after long-term exposure to radiofrequency radiation. International Journal of Radi-ation Biology 83:213–220.

Koyama S, Nakahara T, Wake K, Taki M, Isozumi Y, Miyakoshi J. 2003. Effects of high frequency electromagnetic fields on micronucleus formation in CHO-K1 cells. Mutation Research 541:81–89.

Koyu A, Cesur G, Ozguner F, Akdogan M, Mollaoglu H, Ozen S. 2005. Effects of 900 MHz electromagnetic field on TSH and thyroid hor-mones in rats. Toxicology Letters 157:257–262.

Mace ML Jr, Daskal Y, Wray W. 1978. Scanning-electron microscopy of chromosome aberrations. Mutation Research 52:199–206.

Maes A, Collier M, Slaets D, Verschaeve L. 1995. Cytogenetic effects of microwaves from mobile communication frequencies (954 MHz). Electro and Magnetobiology 14:91–98.

Maes A, Collier M, Van Gorp U, Vandoninck S, Verschaeve L. 1997. Cytogenetic effects of 935.2-MHz (GSM) microwaves alone and in combination with mitomycin C. Mutation Research 393:151–156.

Markova E, Hillert L, Malmgren LO, Persson BR, Belyaev IY. 2005. Microwaves from GSM mobile telephones affect 53BP1 and gamma-H2AX foci in human lymphocytes from hypersensitive and healthy persons. Environmental Health Perspectives 113:1172–1177.

Markova E, Malmgren LOG, Belyaev IY. 2010. Microwaves from mobile phones inhibit 53BP1 focus formation in human stem cells more strongly than in differentiated cells: Possible mechanistic link to cancer risk. Environmental Health Perspectives 118:394–399.

Mashevich M, Folkman D, Kesar A, Barbul A, Korenstein R, Jerby E, Avivi L. 2003. Exposure of human peripheral blood lymphocytes to electromagnetic fields associated with cellular phones leads to chro-mosomal instability. Bioelectromagnetics 24:82–90.

Mazor R, Korenstein-Ilan A, Barbul A, Eshet Y, Shahadi A, Jerby E, Korenstein R. 2008. Increased levels of numerical chromosome aberrations after in vitro exposure of human peripheral blood lym-phocytes to radiofrequency electromagnetic fields for 72 hours. Radiation Research 169:28–37.

Otto M, von Muhlendahl KE. 2007. Electromagnetic fields (EMF): Do they play a role in children’s environmental health (CEH)? Interna-tional Journal of Hygiene and Environmental Health 210:635–644.

Pavicic I, Trosic I. 2008. In vitro testing of cellular response to ultra high frequency electromagnetic field radiation. Toxicology in Vitro 22:1344–1348.

Peyman A, Rezazadeh A, Gabriel C. 2001. Changes in the dielectric properties of rat tissue as a function of age at microwave frequencies. Physics in Medicine and Biology 46:1617–1629.

Phillips J, Ivaschuk O, Ishida-Jones T, Jones R, Campbell-Beachler M, Haggren W. 1998. DNA damage in Molt-4 T lymphoblastoid cells exposed to cellular telephone radiofrequency fields in vitro. Bioelec-trochemistry and Bioenergetics 45:103–110.

Phillips JL, Singh NP, Lai H. 2009. Electromagnetic fields and DNA damage. Pathophysiology 16:79–88.

Preston RJ, Au W, Bender MA, Brewen JG, Carrano AV, Heddle JA, McFee AF, Wolff S, Wassom JS. 1981. Mammalian in vivo and in vitro cytogenetic assays: A report of the USEPA’s gene-tox programme. Mutation Research 87:143–188.

Ruediger HW. 2009. Genotoxic effects of radiofrequency electromag-netic fields. Pathophysiology 16:89–102.

Sarimov R, Malmgren LO, Markova E, Persson BR, Belyaev IY. 2004. Nonthermal GSM microwaves affect chromatin conformation in human lymphocytes similar to heat shock. IEEE Transactions on Plasma Science 32:1600–1608.

Int J

Rad

iat B

iol D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y M

onas

h U

nive

rsity

on

09/0

9/13

For

pers

onal

use

onl

y.