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Association between Mobile Phone Using and DNA Damage of Epithelial Cells of the Oral Mucosa

Exposure to high levels of radiofrequency radiation can potentially cause tissue damage characteristic of many diseases including cancer. This study explored relationship between use of mobile phone and DNA damage in oral mucosal cells. One hundred individuals completed a questionnaire were grouped according to frequency and duration of mobile usage. Comet and TUNEL assays were used to determine DNA damage and rate of apoptosis, respectively. Number of damaged cells in right cheek was significantly higher than in left cheek in right ear phone dominant users, but not in left ear dominant people. Years of phone use was not positively correlated to degree of DNA damage, however, damage increased with increased frequency of phone use. Apoptotic cells were highest in medium (3060 min/day) phone users. Although no significant correlation was observed between degree of apoptosis and cumulative year of mobile phone use, phone use must be minimized to reduce health effects.

Furthermore, the increasing use of mobile phones in children has been associated with emotional and behavioral disorders [5] and high frequency of hearing loss [9]. The 5G mobile networking technology (which is projected to use mainly the higher microwave frequencies part of the spectrum in the highest performance mode) will affect not only the skin and eyes, but will have adverse systemic effects as well [10].
The frequency of electromagnetic waves emitted from mobile phones significantly increased the human response to stimulus time i.e. slower reaction of the subjects [7]. The loss of mental attention [2] and accidents caused by distracted driving [11] have been highlighted as a public health concern. The inconsistent results between similar studies and the same research groups have made it very difficult to make any comprehensive interpretation [12].
The limit of mobile phone use is the SAR of 2 W/kg for the human head [13]. Depending on the different type of mobile phones, the maximum local SAR values ranged between 0.2 and 1.5 W/kg on an average for 10 grams of tissue [14]. 40% of which is absorbed in the head and neck region [15]. The oral cavity and other extraoral structures including the salivary glands, dental appliances and dental restorations are the closest organs and tissues of the body for mobile phone use.
Consequently, they are expected to be the most exposed to mobile phone emitted radiations during the conversation period [14,16].
The knowledge of the effects of RFR on oral mucosal cells may give a perspective about what kind of effects of the radiation could have on other organs. Several studies have confirmed the genotoxic effect of mobile phone radiation on oral mucosa [15,16], but others have apparently denied such effects [17,18]. In the light of the contradictory results obtained from previous studies, the reevaluation of the effect of mobile radiation on the oral epithelium has been kindled. The aim of the present cross-sectional study was comparing high-, medium-, and low-RFR exposure persons to generate information about association of this exposure with DNA damage and cell death in oral mucosal cells of people exposed to RFR from mobile phones.

Subjects
This cross-sectional study was approved by a specialized Human Research Ethics Committee of Yarmouk University. A written informed consent was obtained from each participant. A sample of 100 university student volunteers in the age range of [18][19][20][21][22][23][24][25][26][27][28][29][30] years was randomly recruited. Before providing the oral mucosal cells, each participant was interviewed by the same research staff to standardize data collection data regarding age, gender, locality of residence, type of mobile used, duration of daily mobile phone usage (min a day), the overall period of exposure (number of years) and the of use of headsets by completing a specially constructed detailed questionnaire. The study excluded subjects with smoking habit, or receiving drug therapy in the last three months, suffering any illness including cancer, subjected to radiotherapy including dental procedures, dietary supplements, and regular mouthwash users. Nobody dropped out of the study and was ready to answer any query that may arise at any time during the study. Participants were randomly stratified into three groups based on the frequency and intensity of phone use: Group 1: Low mobile phone users (less than 30 min/day), Group 2: Medium mobile phone users (30-60min/day) and Group 3: Heavy mobile phone users (more than 60 min/day). The participants were further divided into three categories on the basis of the history of phone use; short-, mediate-, and long-duration users for those who used their phones for periods less than 5 years, 5-10 years and longer than 10 years, respectively.
Data collection and results analysis of all experimental work were performed under blind code.

Collecting cells
Before collecting the oral mucosal cells from the subject, he/she was asked not to eat and drink an hour.  (Figure 1).

Apoptosis assay (TUNEL assay)
The DeadEnd TM colorimetric TUNEL kit (Promega, Madison, WI, USA) was used to measure apoptosis-

Results
The study group included 100 subjects. All of mobile phones used by participants were within the most common SAR levels (in the range between 0.244 to Analysis of the pooled data from both sexes ( Table 2) showed that in general, among the three groups of total daily calling times there were no statistically significant differences in the percentage of damaged cells, estimated by comet assay, neither on the left-nor on the right-ear preferring mobile phone users (P-values equal 0.46 and 0.96, respectively). However, the average percent was slightly higher on the right side relative to the left side; 7.57 ± 0.635% and 6.17 ± 0.503%, respectively. A similar trend was noted regarding the duration of use ( Table 3).
The percentage of damaged cells on the right side was significantly higher than on the left side for the participants who preferred to use phone on the right ear (P-value 0.02). Whereas, no significant differences were noticed between the two cheeks for the left ear dominant users (P-value 0.76). DNA damage in 100 cells exposed to RF fields detected by comet assay and estimated by measuring the DNA extent of the migration toward anode pole is summarized in Figure 3. The results of comet analysis are summarized in Tables 4 and 5. In

Discussion
The principal finding of the present study was the absence of significant difference in the effect of RFR fields between the two cheeks. Another major finding was the increase in tail moment (tail length × tail density) and in tail density (DNA% in tail) as well as Consistent with our results are those reported earlier on the effect of mobile phone on oral mucosa using MN assay [15,16]. Our data also are a long with previous reports that an 1800-MHz RFR caused DNA damage in human lens epithelial cells [22,23], lymphocytes [24] and hair root stem cells located around the ear [25,26] as evidenced by comet technique. It was observed that leucocytes in those living in close proximity to base stations had a significant increase in their DNA migration length, damage frequency, and damage indexes. These findings are parallel with previous studies [26,27] which reported that the DNA damage measured as tail DNA intensity increased with higher dose of radiation. DNA damage, in terms of tail moment of comet in peripheral blood lymphocytes and MN formation in buccal cells, was significantly increased in human populations exposed to radiation from mobile towers [3]. Residing within a perimeter of 80 meters of mobile base stations showed significantly higher frequency of MN when compared to the control group, residing 300 meters away from the mobile base stations [28]. It has been reported that phone radiation is capable of disturbing the DNA repair mechanism, and this can continue for several hours after the phone use [5]. It is known that DNA damage that can occur is related to the cell type and experimental setup (Exposure time, RF frequency, SAR, continuous or pulsed wave, exposure as mobile phone user, etc.) [29,30]. Mobile phone radiation may increase oxidative stress indices [3,28,31,32,33] and increase oxidative DNA damage (8-Oxo-7, 8-dihydro-2ʹ-deoxyguanosine) formation [34,35]. These free radicals could cause membrane and macromolecule damage by three basic mechanisms: lipid peroxidation, DNA fragmentation, and protein oxidation. When DNA damage reaches an unrepairable level or it is not repaired in the right way, it might lead to apoptosis mutation, aging, and cancer [31,36].
Contradictory results were obtained, where mobile phone-associated electromagnetic fields did not induce significant increase in MN formation in the human oral cavity's mucosa cells [18,37,38] and in cells of the  [14,[46][47][48][49]. Two groups of researchers [37,46] considered the increased nuclear fragmentation as a parameter which leads to a lower transcription activity or eventual disintegration of the nucleus.
To our knowledge, the present investigation is the first study in which putative effects (e.g., influence on apoptosis rate) of RFR were investigated in oral mucosal cells. One limitation of the study is that it depended on the student's response to health problems rather than making sure if they are already affecting the results or not. The limitation is due to the nature of the study and its basic drawings of the problem. Cluster

Conclusion
The present findings indicate that current RFR wavelengths we are exposed to appear to may act as a toxin to biological systems. Our data did not predict any statistically significant differences in cellular damage