Effects of mobile phone radiation on parotid gland: immunohistochemical study

INTRODUCTION

Nowadays, non-ionizing radiation is widely spread; its most important source is the microwave-radiation produced by mobile phone devices, which in recent years have been used by most of the world’s population [1]. For many years, an intense dispute has been carried out about the seriousness of mobile phone radiation, and researchers have not come yet to a final conclusion about the adverse role of this radiation entering the human body, but what is agreed, is the presence of this radiation in various health issues [2]. The International Agency for Research on Cancer (IARC) has classified electromagnetic fields caused by mobile phones as group 2b, i.e., they may be carcinogenic to humans [3, 4]. Studies are under way to assess long-term health effects of mobile phone use. Regarding the large number of mobile phone users, it is important to perform all necessary studies on these phones to understand and monitor their potential public health impacts [5]. Since 2000, several studies have been conducted on mobile phones influence on public health, especially carcinogenic effects on the brain, salivary glands, and other neoplasms in the head and neck region [6]. Microwave radiation has a frequency ranging 0.3-300 GHz and is located between radiology and infrared fields [7]. Micro-wave radiation is not ionized and have high tissue temperature effects, and in view of the frequencies used by mobile phones, most of the energy is absorbed by the skin and other surface tissues, which results in very little rise in temperature of the brain and other parts of the body [8]. Many studies on the non-thermal effects of microwave-radiation on the biological system have been conducted during the past thirty years, and some of these studies indicated: • an increase in oxidation intensity [9], • an increase in the permeability of the erythrocyte membrane and an increase in its dissolution [10], • an effect on the cerebral vasoconstriction with an increase in the flow of calcium carbonate [11], • an influence of the monocyte-dependent immunoregulatory mechanisms responsible for the initiation of immune response [12]. Since the parotid gland is placed directly under the skin of the face, it is one of the most exposed organs to the effect of radiation emitted from a mobile phone [4, 13]. Therefore, there are studies assessing a relationship between an exposure to microwave-radiation and neoplasms of the parotid gland, and several studies indicated no association of parotid gland neoplasms with an exposure to mobile phone radiation [14]. However, these studies are still inadequate because of limited exposure period [15]. Ki-67 is a human nuclear protein expression, which is associated with cell proliferation used as a proliferation marker to measure the division of cells in human neoplasms [16]. It is a nuclear protein expressed during the interstitial phase (G1-S-G2) within the nucleus, while in the division phase (M) is expressed on the surfaces of chromosomes. There is no expression during the phase g0 because this protein is destroyed rapidly when the cell enters the non-proliferation state [17, 18].

OBJECTIVES

This current study aims to investigate the potential effects of microwave-radiation on the proliferation activity of glandular cells in the parotid gland by studying Ki67 protein levels.

MATERIAL AND METHODS

ANIMALS

An approval was gained from the ethics committee of the Damascus University to conduct this study (approval No: 2033/2016). The sample consisted of sixteen parotid glands removed from sixteen adult male New Zealand white rabbits that were divided evenly into two groups: experimental group (g1) rabbits exposed to microwave-radiation and control group (g2) rabbits not exposed to microwave-radiation.

EXPERIMENTAL PROCEDURES

A third generation (3G) device Samsung Galaxy J1 Mini Prime (model SM-J106H/DS) was used. This 3G mobile phone supports four Universal Mobile Telecommunications System (UMTS) bands (B1, B2, B5, and B8 with 2100, 1900, 850, and 900 MHz, respectively). In the present study, it has been confirmed which band is active in the country of this study depending on the “country-based mobile phone network frequency bands coverage guide”, which indicates that these devices run on B1 mode (2100 MHz). Also, the same was confirmed after contacting the telecommunications company (Syria¬tel Mobile Telecom S.A.). The 3G mobile phone devices emit much more electromagnetic radiation, since they run on a higher frequency compared to second generation phones [19]. During experimental time, the rabbits exposed to microwaves were kept in glass cages [20] equipped with ventilation openings specially designed for this research, according to a uniform size (30 cm in length, 12 cm in width, and 18 cm in height). The rabbits were an average size, which did not allow a rotational movement, but a minimal lateral movement of the head and forward or backward movement of the rabbit’s body [21]. The distance between the mobile device and the side face of the rabbit was 5 cm [22], and the distance between the two devices was 35 cm (Figure 1). Mobile phones were without vibration with a silent mode to reduce the stress of the animals. The head specific absorption rate (SAR) was 0.873 W/kg as mentioned by the manufacturer and in the “specific absorption rate certification information” (quick start guide), which is included with the device when purchased. The power of radiation was 4 W (Watt), and the daily total dose of radiation was 14,400 J (Joule). A virtual phone call was made between the two devices, i.e. sender and receiver. At the beginning of the call, animals were placed in the glass cages and daily calls were simulated [19]. The rabbits in the experimental group were exposed to mobile phone devices (call mode) for one continuous hour daily, 6 days per week, for 6 months. The rabbits in the control group were exposed to mobile phone devices (switched off mode, no microwave radiation) for the same period. After the end of the exposure period, rabbits were sacrificed according to ethical methods, and the parotid glands were removed unilaterally from each rabbit’s side, which was exposed to the aforementioned mobile phone devices. As for animals in the control group, they were subjected to the same conditions, considering that mobile devices were switched off. Therefore, 16 parotid glands isolated from 16 rabbits on the same side were obtained.

IMMUNOHISTOCHEMICAL STAINING

Immunohistochemical (IHC) detection of Ki-67 was carried out by using DAKO-LSAB-2HRP detection system. For IHC staining, the sections were cut at 4 µm thickness using manual microtome. For IHC, sections were placed on pre-coated slides and incubated for 1 hour at 60°C in an incubator. For antigen retrieval, the sections were placed in a 1 mM citrate buffer (pH 6), and microwave was used with cycles of high, medium high, low, and very low, each lasting for 5 minutes and then cooled down to room temperature. The endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 10 minutes, followed by washing in 0.05 mM Tris-buffered saline (TBS) at pH 7.4. The sections were incubated with precisely diluted mouse monoclonal antibodies against Ki67 (MIB-1 Dako, Japan) as primary antibodies for 1 hour at 37°C. After washing in TBS, the sections were incubated with a secondary antibody conjugated with peroxidase- labelled dextran polymers (Envision + Dual Link/HRP System, Dako) for 30 minutes at room temperature. After rinsing with TBS, they were treated with 0.5 mg/ml3, 3’-diaminobenzidine solution containing 0.001% hydrogen peroxide to visualize reaction products, and counterstained with hematoxylin for 3 minutes. For positive control, sections of the colon for Ki-67 were taken.

PROTOCOL OF HISTOPATHOLOGICAL STUDY

The staining score of Ki-67 was evaluated under light microscope (OLYMPUS CX21) in glandular regions of the parotid gland as the percentage of glandular cells that exhibited a positive nuclear immunoreactivity to Ki-67 out of the total number of glandular cells in the field. The percentage of positive cells was determined by counting four non-overlapping microscopic fields (four random fields) at ×400 magnification according to the following equation: Percentage of positive stained cells = number of posi¬tive cells ÷ number of total cells × 100. Every brown-staining glandular cell was considered positive, and no counts were performed in areas of necrosis or inflammation.

STATISTICAL ANALYSIS

SPSS software (v.20; IBM, Armonk, New York) was used to analyze the data obtained statistically, MS Excel 2019 program to perform the graphs, and G*power to calculate the sample size. The normality distribution was checked using the Kolmogorov-Smirnov test. T-test was used for independent samples to study significance of the difference in mean cell positive ratio between the exposed and unexposed group of rabbits to microwave-radiation.

RESULTS

Descriptive statistics are shown in the Figure 2. The expression of Ki-67 in the parotid glands of both the control and the experimental sample was investigated. Different expressions were observed in glandular cells. However, all samples were positive in both groups as in the forms (Figure 3). To study the differences in Ki-67 expression between the control and the experimental group, the t-test of independent samples was applied. Table 1 shows that the value of the significance level was considerably less than the value of 0.05, regardless of the studied stain; at 95% confidence level, there were statistically significant differences in the mean positive ratio of the Ki-67 protein between the exposed and unexposed group of rabbits to microwave-radiation.

DISCUSSION

Most experimental studies on salivary glands focused on traditional hematoxylin and eosin. In the study by Aydogan et al. [23], the authors concluded that the exposure to the microwave-radiation from a mobile phone of third generation, with the frequency of 2,100 MHz, results in changes in the histopathological structure of parotid gland in rats, and these changes were represented in the epithelial cells, connective tissue, interstitial spaces, vascular system, size of nuclei, and gaps in cytoplasm [23]. Previous studies investigated the non-thermal effects of microwave-radiation supported the hypothesis of increased oxidative stress in the tissue exposed to this radiation. Akdag et al. [24] supposed that the exposure to such a radiation leads to an escalated level of free radicals, and since they are chemically unstable compounds, they seek stabilization by pilfering electrons from a stable molecule. The molecule itself becomes a free radical, and therefore strikes another stable molecule, which causes an imbalance between free radicals and antioxidants and eventually harming cells [25-27]. Consequently, these studies have used different types of antioxidants. Anan et al. [28], who used vitamin E in his research concluded that an exposure to microwave-radiation leads to regressive changes similar to changes reported in Aydogan et al. study [23]; however, these changes decreased when rats were injected with vitamin E. In another context, chemical studies have examined the flow rate of saliva. Goldwein and Aframian [29] concluded that the exposure to microwave-radiation increased salivary flow rate, but this increase was low in protein secretion due to continuous and cumulative damage to the glands. During a literature review, we found only one immunohistochemical study by Helal and Abdelrahman [30], who used Ki-67 in exposed 25 male guinea pigs to 900 MHz microwave-radiation. This exposure was of two types, acute and chronic. The acute exposure was for two hours a day for 3 months, while the chronic exposure was two hours a week for the same period. At the end of exposure period, they collected the parotid and submandibular glands from the same exposed side, studied the expression of Ki-67, and concluded that there was a gradual increase in Ki-67 expression associated with increased exposure time. Moreover, the exposure to microwave-radiation produced proliferative effects in the glandular tissue. The results of our study were convergent with the Helal and Abdelrahman [30] in terms of statistically significant differences, although the size of sample and the period of exposure varied. The results of current study indicate an important increase in immunoexpression of Ki67 protein in the glandular tissue cells. This suggests an important increase in the ability of these cells for mitosis and division in response to the damage that is interpreted based on the theory of oxidative stress.

CONCLUSIONS

Within the limits of this study, we conclude that exposure to microwave-radiation from a third-generation mobile phones lead to an increased proliferation activity in the parotid gland in response to damage to the tissue structure.

ACKNOWLEDGEMENT

We want to point out that the laboratory tests were done at the research laboratory in the Department of Oral Pathology, Faculty of Dentistry at the Damascus University.

CONFLICT OF INTEREST

The authors declare no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

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