Effect of mobile phones on micronucleus frequency in human exfoliated oral mucosal cells

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1 (2012) 18, doi: /j x Ó 2012 John Wiley & Sons A/S All rights reserved ORIGINAL ARTICLE Effect of mobile phones on micronucleus frequency in human exfoliated oral mucosal cells I Ros-Llor, M Sanchez-Siles, F Camacho-Alonso, P Lopez-Jornet Department of Oral Medicine, Morales Meseguer Hospital, University of Murcia, Murcia, Spain OBJECTIVE: In the last two decades, the use of mobile phones has increased enormously all over the world. The controversy regarding whether radiofrequency (RF) fields exert effects upon biological systems is a concern for the general population. An evaluation is made of DNA damage and cytokinetic defects, proliferative potential, and cell death because of RF radiation emitted by mobile phones in healthy young users. STUDY DESIGN: This cohort study was carried out in 50 Caucasian mobile phone users. We collected two cell samples from each subject (a total of 100 cell samples), corresponding to the right and left cheek mucosa, respectively. Case histories and personal information were assessed, including age, gender, body height and weight, history of cancer, smoking and alcohol consumption, exposure to chemical carcinogens or radiation, and dietary habits. Sampling comprised cell collection from both cheeks with a cytobrush, centrifugation, slide preparation, fixation, and staining, followed by fluorescent microscopic analysis. A total of 2000 exfoliated cells were screened for nuclear abnormalities, especially micronucleus. RESULTS: No statistically significant changes were recorded in relation to age, gender, body mass index, or smoking status. A comparison of the results vs the control area according to the side of the face on which the mobile phone was placed, and in relation to the duration of exposure (years) to mobile phone radiation in the total 100 samples, yielded no significant differences. CONCLUSIONS: No genotoxic effects because of RF exposure were observed in relation to any of the study parameters. (2012) 18, Keywords: exfoliated oral mucosal cells; cell death; DNA damage; micronucleus test; mobile cellular phone; proliferative potential Correspondence: Pía Lopez-Jornet, MD, DDS, PhD, Department of Oral Medicine, Morales Meseguer Hospital, Marqués de los Vélez s n, Murcia, Spain. Tel: , Fax: , majornet@um.es Received 17 February 2012; revised 7 April 2012; accepted 16 April 2012 Introduction In the last two decades, the use of mobile phones has increased enormously all over the world. We mainly use mobile phones for calls and messages, but data transfer, music, games, and other applications are becoming increasingly popular, especially among young people. This technology is based upon electromagnetic radiation in the microwave frequency range [radiofrequency (RF) waves and microwaves]. The radiation frequency and modulation standards vary in the range of MHz, depending on the region in the world. The most important second generation standard is the Global System for Mobile Communication (GSM), which uses frequencies of around 900 and 1800 MHz. Higher frequencies of around 2100 MHz are used by the most common third generation standard, the universal mobile telecommunication system (UMTS). For all these frequencies (up to 300 GHz), the International Commission on Non-Ionizing Radiation Protection (ICNIRP) has established guidelines for exposure limitations (ICNIRP, 2009) that have been adopted by national regulations in many countries. In the case of biological systems, the extent of RF field exposure depends on the amount of energy deposited in tissue and is measured by the specific absorption rate (SAR), the amount of energy absorbed per unit time per unit mass of tissue, and is expressed in Wkg )1. The SAR limit recommended by the ICNIRP is 2.0 W kg )1. This value has been averaged over 10 g of body tissue. The guidelines incorporate a substantial margin of safety to ensure the protection of all persons, regardless of age and health. Epidemiological studies have suggested that exposure to the low energy, ultra-high-frequency electromagnetic field (UHF-EMF) emitted by a mobile phone may have biological effects in living organisms, though at present no definitive association can be obtained to the incidences of cancer or to other genetic and non-genetic pathological conditions. While some authors do see a link (Schwarz et al, 2008; Khurana et al, 2009), most investigations have been unable to confirm increased risk (Moulder et al, 2005; Schuz et al, 2009). Some studies have reported alterations in enzyme activity (Friedman et al, 2007; Ammari et al, 2008), while others

2 have recorded no changes (Huang et al, 2008). The same applies to the contradictory increase in free radical formation, based on the assumption that reactive oxygen species (ROS) are implicated in several types of tissue injury (Guney et al, 2007; Hoyto et al, 2008), cell proliferation (Capri et al, 2004; Lee et al, 2008), and many other biological end points. DNA damage (micronuclei and or nuclear buds), cytokinetic defects (binucleated cells), proliferative potential (frequency of basal cells), and or cell death (condensed chromatin, karyorrhexis, pyknotic and karyolytic cells) have been related to a high risk of cancer, aging and neurovegetative disorders, and to the carcinogenic process, with increased genetic instability (Fenech, 2007; Thomas et al, 2009). These biomarkers can be testing in exfoliated cells (Majer et al, 2001), especially in oral mucosal cells, using a rapid, easy, inexpensive, and non-invasive test (Fenech, 2007; Thomas et al, 2009), or in cultured blood cells such as lymphocytes and erythrocytes (Holland et al, 2007; Wang et al, 2010). Micronuclear (MN) studies are frequently used in mammalian species, including humans, to monitor and investigate genotoxic effects of malignancies, pesticides, smoking, pollution, work exposures, and other influences (Pitarque et al, 2002; Bonassi et al, 2009; Bortoli et al, 2009). Micronuclear can originate spontaneously or in response to clastogenic and aneugenic agents. MN arise from either acentric chromosomes (fragments formed by unpaired double-stranded breaks or by misrepair of various DNA lesions) in the case of a clastogenic event, or missing whole chromosomes that fail to be incorporated into daughter nuclei during cytokinesis, in the case of an aneugenic event (Tucker and Preston, 1996; Holland et al, 2008). Cells affected in this way are characterized by the presence of both a main nucleus and one or more smaller nuclear structures called micronuclei. The MN must be located within the cytoplasm of the cells, with a round or oval shape, and their diameter should range between 1 3 and 1 16 of the diameter of the main nucleus. MN have the same staining intensity and texture as the main nucleus. Most cells with MN contain only one MN, but it is possible to find cells with two or more MN. According to recent suggestions, other anomalies such as nucleoplasmic bridges or nuclear buds may be scored in parallel to micronuclei, thus converting the micronucleus test into a Ôcytomic assay (Fenech, 2009). About 90% of all human cancers are carcinomas, perhaps because most cell proliferation in the body occurs in epithelia, or because epithelial tissues are most frequently exposed to various forms of physical, chemical, and radiation damage that favors the development of cancer. The epithelial cells of the oral mucosa were used in the present study, as these cells are more closely exposed to the radiation of mobile phone devices. The aim of this study was to evaluate DNA damage and cytokinetic defects, proliferative potential, and cell death because of RF radiation emitted by mobile phones in healthy young users. Materials and methods This cohort study was carried out in 50 Caucasian patients. We collected two cell samples from each subject (for a total of 100 cell samples), corresponding to the right and left cheek, respectively. We planned to compare cell samples with the preferential side used during phone calls (comparing buccal mucosa exposed with buccal mucosa not exposed). Case histories and personal information were assessed, including age, gender, body height and weight, history of cancer, smoking and alcohol consumption, exposure to chemical carcinogens or radiation, and dietary habits. A questionnaire addressing the duration of daily mobile phone use, overall period of exposure, preferential side, and the use of headsets was completed. All subjects were fully informed of the purpose of the study and gave their consent to take part. All subjects were using mobile phone models from different manufacturers, that is, Nokia, L.G., Samsung, Tata, Reliance, Motorola, etc. The SAR value for these handsets in our investigation ranged from 0.34 in Nokia 9210 (Type RAE-3N) to 0.95 in Nokia N70 (Type RM-84) as provided by the manufacturer s manual (ICNIRP). Inclusion criteria Healthy people between the ages of 20 and 40 years. Exclusion criteria The study excluded people with dietary supplements, regular mouthwash users, patients receiving drug therapy or suffering any illness, subjects over the age of 40 years, healthy non-mobile phone users between the ages of 20 and 40 years, or any subjects who had been exposed to radiation or other carcinogens. Cell sampling and preparation Exfoliated oral mucosal cells (MCs) were collected from each subject by a single practitioner. According to Thomas et al (2009), prior to cell collection, the mouth was rinsed with water to remove saliva, food particles, and any other debris. The inside of each cheek was brushed using conventional toothbrushes and applying a circular motion 20 times, covering a wide area without damaging the cheek mucosa. Sample sites for cell collection were uniform for all the subjects. Separate brushes were used for each cheek. Two 30-ml yellowcapped containers (one for each cheek) were prepared containing 20 ml of oral mucosal cell buffer (EDTA 0.1 M, Tris HCl 0.01 M, NaCl 0.02 M, ph = 7) (E6758; Sigma-Aldrich, Madrid, Spain). The brushes were placed in their respective buffer containers and rotated repeatedly to dislodge the cells and release them into the buffer medium. The cells were then transferred to centrifuge tubes and centrifuged for 10 min at 581 g. After centrifuging, the supernatant was aspirated, and the cells were resuspended in another 5 ml of oral 787

3 788 mucosal cell buffer, followed by repeat centrifugation. The process was repeated once again to eliminate bacteria and inactivate enzymes. The cells were transferred using a pipette, placing ll of cell suspension onto two clean and labeled microscope slides. After drying, the slides were placed in an oven at 55 C for 15 min and were then fixed with 50% methanol (E-08211; Panreac SAU, Barcelona, Spain) at 0 C for 15 min. DAPI staining Cell samples were stained with DAPI (40,6-diamidino -2-phenylindole dihydrochloride) (D9542; Sigma-Aldrich) at a concentration of 200 lg ml )1, during 15 min. DAPI is a fluorescent dye that binds strongly to DNA. It is a DNA-specific stain that reduces the risk of false positive readings (keratin bodies) that could be misinterpreted as corresponding to micronuclei when a non-specific DNA stain such as the Giemsa stain is used. The slides were then washed in Milli-Q water. Slides were scored using a Leica DRMB fluorescence microscope equipped with a DAPI band filter [excitation wavelength filter set (BP ), dichroic filters RKP400, and emission filters LP425] under 100 magnification. Scoring method We scored 1000 cells per subject for the various cells types (basal cells, binucleated cells) and cell death parameters (pyknotic and karyolytic cells). Micronuclei were scored over 2000 cells. Only basal and normal differentiated cells were scored for micronuclei (MNs), and their scores were combined to yield the overall incidence. The purpose of scoring criteria is to classify oral mucosal cells into categories that distinguish between normal cells and cells that are considered abnormal on the basis of cytological and nuclear features indicative of DNA damage, cytokinetic failure, or cell death. A more detailed description referred to the cell types is provided below. Basal cells have a larger nucleus-to-cytoplasm ratio than differentiated oral mucosal cells. Basal cells have a uniformly stained nucleus and are smaller in size and more oval in shape when compared with the more angular and flat differentiated oral mucosal cells (Figure 1a). Normal differentiated cells have a uniformly stained nucleus, which is oval or round in shape. They are distinguished from basal cells by their larger size and by a smaller nucleus-to-cytoplasm ratio (Figure 1b). Cells with micronuclei are characterized by the presence of both a main nucleus and one or more smaller nuclear structures (micronuclei), as commented above (Figure 1c). Binucleated cells are cells containing two main nuclei instead of only one. The nuclei are usually very close together and may come into contact. They usually have the same morphological features as the nucleus seen in normal cells (Figure 1d). Pyknotic cells are characterized by a small shrunken nucleus, with a high density of nuclear material that is uniformly but intensely stained. The nuclear diameter is usually one- to two-thirds that of the nucleus in normal differentiated cells (Figure 1e). Karyolytic cells are cells in which the nucleus is completely depleted of DNA and produces a ghost-like image (Figure 1f). Statistical analysis Data were analyzed using the SPSS version 12.0 statistical package (SPSS Ò Inc., Chicago, IL, USA). A descriptive study was made of each variable. We used the Student t-test for two independent samples in application to quantitative variables, in each case determining whether the variances were homogeneous. The Kolmogorov Smirnov normality test was applied, and when the data showed a skewed distribution, they were analyzed using a nonparametric ranking test; in this sense, we used the Kruskal Wallis test (for more than two samples) and the Mann Whitney U-test (for two independent samples), in application to quantitative variables. Statistical significance was accepted for P Results (a) (b) Figure 1 (a) binucleated cell; (b) micronucleated cell We evaluated 50 healthy individuals, collecting cells from the right and left cheek, for a total of 100 analyzed samples. Table 1 shows the most important

4 Table 1 Characteristics of the study population Characteristics of the sample Patients: n 50 Age: mean ± s.d ± 4.25 Sex: n (%) Male 16 (32) Female 34 (68) Body mass index (Kg m )2 ): n (%) <20 13 (26) (46) >25 14 (28) Smoking status: n (%) Non-smoker 38 (76) <10 9 (18) 10 3 (6) Drinking habits: n (%) No 16 (32) Daily 0 (0) Sometimes 34 (68) Overall period of exposure: n (%) <5 years 2 (4) 5 10 years 24 (48) >10 years 24 (48) Duration of phone use (h week )1 ): n (%) 0 0 (0) (60) (22) (10) (6) (2) Side of the face in which the mobile phone is placed: n (%) Right 37 (74) Left 13 (26) s.d., standard deviation. characteristic of this cohort. The study population consisted of men (32%) and women (68%) aged between 20 and 39 years. The body mass index (BMI) ranged from to kg m )2 (mean ± 3.59 kg m )2 ). The primary study parameter was the duration (hours) of mobile phone use per week, with a mean of 2.27 ± 2.32 (range ); the mean total period of exposure in this study was 9.17 ± 3.04 years (range 3 18). We recorded no statistically significant changes in micronucleus frequency according to age, gender, body mass index, drinking habits, or smoking status (see Table 2). Table 2 Cytological observations for buccal mucosa exposed and controls (Student t-test) Cytological observations Buccal mucosa exposed (n =50) mean ± s.d. Buccal mucosa no exposed (n =50) mean ± s.d. P-value DNA damage and cytokinetic defects Micronuclei 1.38 ± ± Binucleated cells 4.76 ± ± Proliferative potential Basal cells ± ± Cell death Pyknotic cells 2.08 ± ± Karyolytic cells ± ± s.d., standard deviation. Table 3 Cytological observations in relation to duration of exposure (years) to mobile phone radiation in the buccal mucosa exposed (n = 50) (Mann Whitney U-test) Cytological observations 10 years (n =26) median (range) >10 years (n =24) median (range) P-value DNA damage and cytokinetic defects Micronuclei 1 (0 6) 1 (0 3) Binucleated cells 5 (1 14) 3 (1 10) Proliferative potential Basal cells 20 (8 92) 30 (10 70) Cell death Pyknotic cells 2 (0 8) 2 (0 6) Karyolytic cells (10 125) 78 (40 105) Table 4 Frequencies of micronuclei in the buccal mucosa exposed (n = 50) depending on demographic characteristics and various lifestyle factors (Kruskal Wallis and Mann Whitney U tests) Demographic characteristics and various lifestyle factors Micronuclei median (range) P-value Age 25 (n = 17) 1 (0 6) >25 (n = 33) 1 (0 3) Sex Male (n = 16) 1 (0 6) Female (n = 34) 1 (0 6) Body mass index (Kg m )2 ) <20 (n = 13) 1 (0 4) (n = 23) 1 (0 6) >25 (n = 14) 1 (0 3) Smoking habits Yes (n = 38) 1 (0 2) No (n = 12) 1 (0 6) Drinking habits Yes (n = 34) 1 (0 6) No (n = 16) 1 (0 6) Our study allowed the identification of biomarkers for DNA damage, cytokinetic defects, proliferative potential, and cell death in oral mucosal cells of mobile phone users. A comparison of the results vs the control area according to the side of the face on which the mobile phone was placed yielded no differences in any of the variables studied. The results obtained are shown in Table 3. We also compared the duration of exposure (years) to mobile phone radiation in the exposed oral mucosa. No significant differences were found in DNA damage or cytokinetic defects between the subjects exposed for 10 years or less and those exposed for 10 years or more. As can be seen in Table 4, there was an increase in karyolytic cells in the group with the longest duration of exposure, though statistical significance was not reached. We only found significant differences in proliferative potential, with an increase in basal cells with a greater number of years of exposure. Discussion The connection between mobile phone use and cancer is a concern for the world population. Apart from the well-documented and widely accepted heating effect of 789

5 790 RF at higher radiation power settings, the controversy over whether such radiation exerts effects upon biological systems has been going on for a long time and is almost as old as the widespread use of mobile phone technology. Although a large number of studies ranging from tests in cell systems to studies in animals and large epidemiological surveys have been carried out, this issue has not been conclusively settled to date. Most studies do not report effects, but the scientific community is far from reaching a consensus on this topic (Khurana et al, 2009; Hintzsche and Stopper, 2010). Only a few studies have been based on exfoliated cells. We chose exfoliated oral mucosal cells because the oral cavity is located within the area of mobile phone radiation exposure, and epithelial tissue is the target tissue for carcinogenetic damage. The test used is rapid and easy to use, as oral mucosal cells do not need to be cultivated like different types of blood cells especially lymphocytes, which must be stimulated to undergo mitosis. In this study, we used a sensitive, non-invasive, and low cost technique to identify the potential biomarkers associated to DNA damage in the oral mucosal cells of mobile phone users. Our study recorded no statistically significant changes in micronucleus frequency according to subject age, gender, body mass index, drinking habits, or smoking status. This is in agreement with most of the studies that have used the oral mucosa micronucleus test (Holland et al, 2008; Hintzsche and Stopper, 2010). We decide not to include volunteers older than 40 years old as the trend of age is highly significant for the micronucleus frequency (Bonassi et al, 2011). In 2010, Hintzsche and Stopper investigated the effect of mobile phone use on genomic instability in human oral mucosa cells. A total of 131 individuals (13 nonusers, 85 users for 3 h or less a week, and 33 users for more than 3 h a week) were evaluated for the frequency of MN and other nuclear aberrations, applying the MN test. No significant differences were observed, however. In 2008, Yadav and Sharma studied exfoliated oral mucosal cells in 109 subjects (85 users and 24 controls), applying the BMNcyt assay. A slight increase in frequencies was observed, but the difference failed to reach statistical significance. In both studies, the staining techniques differed. Whereas Hintzsche and Stopper used chromomycin A3, which is a DNA-specific stain; Yadav and Sharma used orceine, which is not a DNA-specific stain and thus might not only stain DNA-containing micronuclei but also other artifacts not associated to genomic instability. In contrast, we used DAPI, another DNA-specific stain involving a very simple sample elaboration process. We compared the results of exposed mucosa vs the control mucosa, according to the side of the face on which the mobile phone was placed, in a total of 100 samples, but found no significant differences. It is theoretically possible to differentiate between exposure in the left and right cheek to determine side-related differences that might be considered informative, because some epidemiological studies have suggested an ipsilateral increase in brain cancers, but not on the contralateral side (Khurana et al, 2009). However, such discrimination has not been previously carried out. Hintzsche and Stopper detected no influence on the part of mobile phone use referred to either the duration of weekly use or to the overall period of exposure. Yadav and Sharma did not distinguish between different durations of weekly mobile phone use; rather, the study individuals were grouped according to the overall period of exposure. The authors reported a positive correlation between the overall period of exposure and micronuclei. We also compared the duration of exposure (total years) to mobile phone radiation and only found a significant increase in basal cells with a longer duration of exposure. Weekly frequency of use is only important if the overall duration of exposure is long. The difference in SAR index comparing a phone s model with other model does not imply that lower level is safer because all phones emitted under 2.0 w kg )1 recommended by European Community. SAR is different depend on the model of the terminal and regulational agency in each country. The effects in organism occur in a long time. Several studies involving human fibroblasts, lymphocytes, and erythrocytes (d Ambrosio et al, 2002; Vijayalaxmi et al, 2003; Zeni et al, 2003, 2008; Ferreira et al, 2006; Scarfi et al, 2006; Verschaeve et al, 2006; Juutilainen et al, 2007; Ziemann et al, 2009; Yildirim et al, 2010; Kesari et al, 2011) have described the effects of exposure to RF-electromagnetic fields at frequencies used for communication with mobile phones. These studies, mentioned below, evaluated the presence of MN in cultivated blood cells by comet assay, MN test, and or BMNcyt assay. In 2010, Yildirim et al evaluated the effects of mobile phone based stations on MN frequency and chromosomal aberrations in human blood cells. There were no significant differences in results between the two study groups. (Zeni et al, 2003, 2008) in turn evaluated MN formation in incubated leukocytes applying the cytokinesis-block MN assay and also the comet assay. Their results indicate that intermittent exposure of human lymphocytes does not increase MN frequency. No evidence of the existence of genotoxicity in lymphocyte cultures was found by Scarfi et al in a study published in 2006, applying the cytokinesis-block MN assay. Similar results were recorded by d Ambrosio et al, On the other hand, different studies in laboratory animals (Vijayalaxmi et al, 2003; Ferreira et al, 2006; Verschaeve et al, 2006; Juutilainen et al, 2007; Ziemann et al, 2009) have yielded no evidence of increased genotoxicity in rats or mice exposed to RF radiation. However, in a 2011 study carried out in Wistar rats, Kesari et al concluded that RF might affect the fertilizing potential of spermatozoa. In conclusion, no genotoxic effects because of RF exposure were observed in relation to any of the study parameters. Funding There were no sources of funding for the present study.

6 Competing interests The authors report no competing interests. Author contributions Study concepts and design P. Lo pez Jornet, F. Camacho Alonso, M. Sanchez Siles, I. Ros Llor. Data acquisition: I. Ros-Llor, data analysis Camacho Alonso. All authors read and approved the final manuscript. References Ammari M, Lecomte A, Sakly M, Abdelmelek H, de-seze R (2008). Exposure to GSM 900MHz electromagnetic fields affects cerebral cytochrome oxidase activity. Toxicology 250: Bonassi S, Biasotti B, Kirsch-Volders M et al (2009). HUM- NXL Project Consortium. State of the art survey of the buccal micronucleus assay a first stage in the HUMN(XL) project initiative. Mutagenesis 24: Bonassi S, Coskun E, Ceppi M et al (2011). The HUman MicroNucleus project on exfoliated buccal cells (HUMN(XL)): the role of life-style, host factors, occupational exposures, health status, and assay protocol. Mutat Res 28: Bortoli GM, Azevedo MB, Silva LB (2009). 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