Abstract
Purpose
We aimed to evaluate the effect of 2100 MHz radiofrequency radiation on the parotid gland of rats in short and relatively long terms.
Material and methods
Thirty Wistar albino rats were divided into four groups. Groups A and B served as the control groups (for 10 days and 40 days, respectively), and each group included six rats. Groups C and D were composed of nine rats each, and they were the exposure groups. The rats were exposed to 2100 MHz radiofrequency radiation emitted by a generator, simulating a third generation mobile phone for 6 hours/day, 5 days/week, for 10 or 40 days. Following exposure, the rats were sacrificed and parotid glands were removed. Histopathological and biochemical examinations were performed.
Results
Although there were no histopathological changes in the control groups except for two animals in group A and three animals in group B, the exposure groups C (10 days) and D (40 days) showed numerous histopathological changes regarding salivary gland damage including acinar epithelial cells, interstitial space, ductal system, vascular system, nucleus, amount of cytoplasm and variations in cell size. The histopathological changes were more prominent in group D compared to group C. There was statistically significant different parameter regarding variation in cell size between the groups B and D (p = 0.036).
Conclusion
The parotid gland of rats showed numerous histopathological changes after exposure to 2100 MHz radiofrequency radiation, both in the short and relatively long terms. Increased exposure duration led to an increase in the histopathological changes.
1
Introduction
Recently, mobile phones have been incrementally used both at work and the social life for communication. The third-generation (3G) is the newest version of the mobile phone technologies. In particular 3G-mobile phones are important means of wireless communication that satisfy the needs of modern societies, and their use has been increased rapidly worldwide. They are associated with networks providing internet access, data transmission and multimedia applications, requiring high speed and bandwidth .
Mobile phones generate heat and emit radiofrequency radiation as non-ionizing electromagnetic radiations in the range of 800–2200 MHz . 3G mobile phones run on 2100 MHz radiofrequency. They emit much more electromagnetic radiation since they run on a higher frequency compared to second-generation phones. In addition, 3G-mobile phones can emit continuous electromagnetic radiation to the environment .
Electromagnetic radiation is a non-ionizing radiation which has thermal and non-thermal effects on biological systems . There is great concern regarding the potential adverse health effects of heat and radiofrequency radiation produced from mobile phones .
Electromagnetic field of mobile phones may affect biological systems by increasing free radicals (enhancing lipid peroxidation), and by changing the antioxidant defense systems of tissues, thus leading to oxidative stress . Reactive oxygen species produced by natural consequences of oxidative cell metabolism include partially reduced forms of oxygen. Their generation is controlled through biological antioxidant defense systems .
Parotid gland is the largest salivary gland in the human body and located 4–10 mm deep to the skin surface in front of the ear and behind the ramus of the mandible . Due to its location, parotid gland appears to be in considerable contact with mobile phones during their use . It may be vulnerable to possible adverse effects of mobile phone use .
This study aimed to evaluate histopathological and biochemical influence of 2100 MHz radiofrequency radiation emitted by a generator, simulating a 3G-mobile phone, on the parotid gland of rats in short term (10 days) and relatively long term (40 days). Experimental animal studies can provide more reliable data since the duration and frequency of electromagnetic field exposure can be set. To our knowledge, no studies investigated both histopathological and biochemical effects of 2100-MHz frequency mobile phone radiation on the parotid gland up to date.
2
Materials and methods
The rats were obtained from the Laboratory Animals Breeding and Experimental Research Center of Gazi University. The experimental design of this study was approved by the Gazi University Ethics Research Committee of Animal Experiments (G.Ü. ET-13.046). This study was in compliance with experimental ethical principles and animal protection laws according to the rules and regulations in Turkey, and was performed in the Laboratory Animals Breeding and Experimental Research Center of Gazi University. The animals were kept in cages with free access to food and water at a temperature of 20–22°C with artificial lighting in a period of 12 hours. They were given pellets (2700 ME kcal/kg, 23% HP) and water ad libitum, and used after 1 week of quarantine and acclimatization.
In this study, parotid tissues of the rats were obtained from other animal study in which we investigated the effects of 2100 MHz radiofrequency radiation on the nasal mucosa and mucociliary clearance. Thirty healthy female Wistar albino rats, weighing 200–256 g each were used. The animals were randomly divided into four groups (groups A, B, C, D). Groups A and B served as the control groups (for 10 days and 40 days, respectively) and each group included six rats. Groups C and D were the exposure groups and each group composed of nine rats.
2.1
Exposure conditions
Digitally modulated 2100 MHz 3G signals were produced by a vector signal generator (Rohde &Schwarz SMBV 100A, 9 kHz–3.2 GHz, München, Germany), and a horn antenna (Schwarzbeck, Doppelsteg Breitband Horn antenna BBHA 9120 L3F, 0.5–2.8 GHz, Schönau, Germany) in a temperature-controlled shielded room. The output power and the frequency were controlled by a spectrum analyzer (Rohde &Schwarz, FSH 18, 10 MHz–18 GHz, München, Germany) integrated to the signal generator. Measurements were taken during the entire experiment, and the data were saved in a computer which was connected to the device via a fiberoptic cable. The distance between the horn antenna and the heads of the rats were 12 cm to provide far-field condition. The direction of the radiofrequency propagation was perpendicular to the plexiglass cage (40 × 25 × 20 cm). Output level of radiofrequency radiation was measured as 16 V/m during the exposure period. Whole body radiofrequency radiation exposure levels of the rats were measured as 0.4 W/kg using the Finite Domain Time Difference (FDTD) method .
The exposure groups were exposed to 2100 MHz radiofrequency radiation emitted by a generator, simulating a 3G mobile phone for 6 h/day, 5 consecutive days/wk, at the same time of the day (between 9 am and 3 pm), for 10 days (group C) and 40 days (group D).
Two days after the last exposure, rats were anesthetized by ketamine hydrochloride (50 mg/kg, intramuscular). Groups A and C were sacrificed following an exposure duration of 10 days, and groups B and D were sacrificed following an exposure duration of 40 days. Right parotid glands of the rats were removed and divided into two parts. One part was used for histopathological evaluation, and the other part for biochemical evaluation. Malondialdehyde (MDA) levels, and activities of xanthine oxidase (XO), glutathione peroxidase (GSH-Px), catalase (CAT), superoxide dismutase (SOD) were measured in the parotid tissue.
2.2
Histopathological analysis
For histopathological evaluation, specimens were fixed in 10% buffered formaldehyde solution, and embedded in paraffin. Next, 5-μm-thick sections were cut from the paraffin blocks. All cross-sections were stained with hematoxylin and eosin, approximately 10 slides per specimen were studied. They were examined under the light microscope by a single pathologist blinded to the study. The tissue reactions were evaluated in all samples.
The histopathological changes regarding salivary gland damage including acinar epithelial cells (vacuolization, perivascular inflammation, necrosis, atrophy), interstitial space (periductal fibrosis, periductal infiltration), ductal system (duct ectasia, squamous metaplasia), vascular system (sclerosis, stenosis), nuclear variability (small shapes, large single, large double), amount of cytoplasm, and variation in cell size were semiquantitatively scored using a three-point scale, as follows: 0 = absent, 1 = mild, 2 = moderate.
2.3
Biochemical analysis
The tissues were first homogenized in physiological saline, and then centrifuged at 4000 x g. Upper clear supernatants were removed, and used in the analyses. Protein levels of the supernatants were measured as mg/mL, by using the Lowry’s method . They were adjusted to equal concentrations before analyses. MDA levels were measured by the thiobarbituric acid reactive substances method . XO activity was determined by measuring uric acid formation from xanthine substrate at 293 nm . GSH-Px activity was measured by following changes in nicotinamide adenine dinucleotide phosphate (NADPH) absorbance at 340 nm . CAT activity was determined by measuring decrease of hydrogen peroxide (H 2 O 2 ) absorbance at 240 nm . In the activity calculations (IU-international unit), extinction coefficients of uric acid, H 2 O 2 and NADPH were used for XO, CAT and GSH-Px, respectively. SOD activity was measured by the method based on nitro blue tetrazolium (NBT) reduction rate . One unit for SOD activity was expressed as the enzyme protein amount causing 50% inhibition in the NBT reduction rate.
2.4
Statistical methods
Statistical analysis were performed using SPSS for Windows Version 21.0 package program. Numerical variables were summarized as mean ± standard deviation and median [minimum–maximum]. Differences between the exposure and control groups were evaluated using Mann Whitney U test. Significance level was considered as p < 0.05.
2
Materials and methods
The rats were obtained from the Laboratory Animals Breeding and Experimental Research Center of Gazi University. The experimental design of this study was approved by the Gazi University Ethics Research Committee of Animal Experiments (G.Ü. ET-13.046). This study was in compliance with experimental ethical principles and animal protection laws according to the rules and regulations in Turkey, and was performed in the Laboratory Animals Breeding and Experimental Research Center of Gazi University. The animals were kept in cages with free access to food and water at a temperature of 20–22°C with artificial lighting in a period of 12 hours. They were given pellets (2700 ME kcal/kg, 23% HP) and water ad libitum, and used after 1 week of quarantine and acclimatization.
In this study, parotid tissues of the rats were obtained from other animal study in which we investigated the effects of 2100 MHz radiofrequency radiation on the nasal mucosa and mucociliary clearance. Thirty healthy female Wistar albino rats, weighing 200–256 g each were used. The animals were randomly divided into four groups (groups A, B, C, D). Groups A and B served as the control groups (for 10 days and 40 days, respectively) and each group included six rats. Groups C and D were the exposure groups and each group composed of nine rats.
2.1
Exposure conditions
Digitally modulated 2100 MHz 3G signals were produced by a vector signal generator (Rohde &Schwarz SMBV 100A, 9 kHz–3.2 GHz, München, Germany), and a horn antenna (Schwarzbeck, Doppelsteg Breitband Horn antenna BBHA 9120 L3F, 0.5–2.8 GHz, Schönau, Germany) in a temperature-controlled shielded room. The output power and the frequency were controlled by a spectrum analyzer (Rohde &Schwarz, FSH 18, 10 MHz–18 GHz, München, Germany) integrated to the signal generator. Measurements were taken during the entire experiment, and the data were saved in a computer which was connected to the device via a fiberoptic cable. The distance between the horn antenna and the heads of the rats were 12 cm to provide far-field condition. The direction of the radiofrequency propagation was perpendicular to the plexiglass cage (40 × 25 × 20 cm). Output level of radiofrequency radiation was measured as 16 V/m during the exposure period. Whole body radiofrequency radiation exposure levels of the rats were measured as 0.4 W/kg using the Finite Domain Time Difference (FDTD) method .
The exposure groups were exposed to 2100 MHz radiofrequency radiation emitted by a generator, simulating a 3G mobile phone for 6 h/day, 5 consecutive days/wk, at the same time of the day (between 9 am and 3 pm), for 10 days (group C) and 40 days (group D).
Two days after the last exposure, rats were anesthetized by ketamine hydrochloride (50 mg/kg, intramuscular). Groups A and C were sacrificed following an exposure duration of 10 days, and groups B and D were sacrificed following an exposure duration of 40 days. Right parotid glands of the rats were removed and divided into two parts. One part was used for histopathological evaluation, and the other part for biochemical evaluation. Malondialdehyde (MDA) levels, and activities of xanthine oxidase (XO), glutathione peroxidase (GSH-Px), catalase (CAT), superoxide dismutase (SOD) were measured in the parotid tissue.
2.2
Histopathological analysis
For histopathological evaluation, specimens were fixed in 10% buffered formaldehyde solution, and embedded in paraffin. Next, 5-μm-thick sections were cut from the paraffin blocks. All cross-sections were stained with hematoxylin and eosin, approximately 10 slides per specimen were studied. They were examined under the light microscope by a single pathologist blinded to the study. The tissue reactions were evaluated in all samples.
The histopathological changes regarding salivary gland damage including acinar epithelial cells (vacuolization, perivascular inflammation, necrosis, atrophy), interstitial space (periductal fibrosis, periductal infiltration), ductal system (duct ectasia, squamous metaplasia), vascular system (sclerosis, stenosis), nuclear variability (small shapes, large single, large double), amount of cytoplasm, and variation in cell size were semiquantitatively scored using a three-point scale, as follows: 0 = absent, 1 = mild, 2 = moderate.
2.3
Biochemical analysis
The tissues were first homogenized in physiological saline, and then centrifuged at 4000 x g. Upper clear supernatants were removed, and used in the analyses. Protein levels of the supernatants were measured as mg/mL, by using the Lowry’s method . They were adjusted to equal concentrations before analyses. MDA levels were measured by the thiobarbituric acid reactive substances method . XO activity was determined by measuring uric acid formation from xanthine substrate at 293 nm . GSH-Px activity was measured by following changes in nicotinamide adenine dinucleotide phosphate (NADPH) absorbance at 340 nm . CAT activity was determined by measuring decrease of hydrogen peroxide (H 2 O 2 ) absorbance at 240 nm . In the activity calculations (IU-international unit), extinction coefficients of uric acid, H 2 O 2 and NADPH were used for XO, CAT and GSH-Px, respectively. SOD activity was measured by the method based on nitro blue tetrazolium (NBT) reduction rate . One unit for SOD activity was expressed as the enzyme protein amount causing 50% inhibition in the NBT reduction rate.
2.4
Statistical methods
Statistical analysis were performed using SPSS for Windows Version 21.0 package program. Numerical variables were summarized as mean ± standard deviation and median [minimum–maximum]. Differences between the exposure and control groups were evaluated using Mann Whitney U test. Significance level was considered as p < 0.05.
3
Results
The animals tolerated the electromagnetic field well throughout the study. No differences were observed between the control and radiofrequency radiation exposure groups regarding the body weights (weight loss or gain), or water and food consumption of the rats.
3.1
Histopathological analysis
Comparison of groups did not reveal statistically significant difference concerning histopathological parameters except for variation in cell size. There was statistically significant difference for this parameter between groups B and D (p = 0.036). Statistical results are summarized in Table 1 .
| Control groups (n = 6) | Exposure groups (n = 9) | Among groups p | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Group A (10 days) | Group B (40 days) | Control Groups p | Group C (10 days) | Group D (40 days) | Exposure Groups p | Groups A and C 10 days | Groups B and D 40 days | |||||
| Mean ± SD | Median [Min–Max] | Mean ± SD | Median [Min–Max] | Mean ± SD | Median [Min–Max] | Mean ± SD | Median [Min–Max] | |||||
| Acinar epithelial cells | ||||||||||||
| Vacuolization | 0 ± 0 | 0 [0–0] | 0.17 ± 0.41 | 0 [0–1] | 0.699 | 0.22 ± 0.44 | 0 [0–1] | 0.56 ± 0.88 | 0 [0–2] | 0.605 | 0.529 | 0.529 |
| Perivascular inflammation | 0 ± 0 | 0 [0–0] | 0.17 ± 0.41 | 0 [0–1] | 0.699 | 0.11 ± 0.33 | 0 [0–1] | 0.33 ± 0.71 | 0 [0–2] | 0.666 | 0.776 | 0864 |
| Necrosis | 0 ± 0 | 0 [0–0] | 0 ± 0 | 0 [0–0] | 1.000 | 0 ± 0 | 0 [0–0] | 0.11 ± 0.33 | 0 [0–1] | 0.730 | 1.000 | 0.776 |
| Atrophy | 0.11 ± 0.33 | 0 [0–1] | 0 ± 0 | 0 [0–0] | 0.699 | 0.11 ± 0.33 | 0 [0–1] | 0.22 ± 0.44 | 0 [0–1] | 0.730 | 0.864 | 0.529 |
| Interstitial space | ||||||||||||
| Periductal fibrosis | 0 ± 0 | 0 [0–0] | 0 ± 0 | 0 [0–0] | 1.000 | 0.22 ± 0.67 | 0 [0–2] | 0.44 ± 0.73 | 0 [0–2] | 0.489 | 0.776 | 0.328 |
| Periductal infiltration | 0 ± 0 | 0 [0–0] | 0 ± 0 | 0 [0–0] | 1.000 | 0.11 ± 0.33 | 0 [0–1] | 0.22 ± 0.44 | 0 [0–1] | 0.730 | 0.776 | 0.529 |
| Ductal system | ||||||||||||
| Duct ectasia | 0 ± 0 | 0 [0–0] | 0 ± 0 | 0 [0–0] | 1.000 | 0.11 ± 0.33 | 0 [0–1] | 0.11 ± 0.33 | 0 [0–1] | 1.000 | 0.776 | 0.776 |
| Squamous metaplasia | 0 ± 0 | 0 [0–0] | 0 ± 0 | 0 [0–0] | 1.000 | 0 ± 0 | 0 [0–0] | 0 ± 0 | 0 [0–0] | 1.000 | 1.000 | 1.000 |
| Vascular system | ||||||||||||
| Sclerosis | 0 ± 0 | 0 [0–0] | 0 ± 0 | 0 [0–0] | 1.000 | 0 ± 0 | 0 [0–0] | 0.11 ± 0.33 | 0 [0–1] | 0.730 | 1.000 | 0.776 |
| Stenosis | 0 ± 0 | 0 [0–0] | 0 ± 0 | 0 [0–0] | 1.000 | 0 ± 0 | 0 [0–0] | 0 ± 0 | 0 [0–0] | 1.000 | 1.000 | 1.000 |
| Cell outlines | ||||||||||||
| Variation in cell size | 0 ± 0 | 0 [0–0] | 0 ± 0 | 0 [0–0] | 1.000 | 0.11 ± 0.33 | 0 [0–1] | 0.67 ± 0.50 | 1 [0–1] | 0.050 | 0.776 | 0.036 |
| Amount of cytoplasm | ||||||||||||
| Scant | 0.17 ± 0.41 | 0 [0–1] | 0 ± 0 | 0 [0–0] | 0.699 | 0.11 ± 0.33 | 0 [0–1] | 0.33 ± 0.71 | 0 [0–2] | 0.666 | 0.864 | 0.529 |
| Nuclear variability | ||||||||||||
| Small shapes | 0 ± 0 | 0 [0–0] | 0.17 ± 0.41 | 0 [0–1] | 0.699 | 0.56 ± 0.88 | 0 [0–2] | 0.67 ± 1.00 | 0 [0–2] | 0.931 | 0.328 | 0.529 |
| Large single | 0 ± 0 | 0 [0–0] | 0 ± 0 | 0 [0–0] | 1.000 | 0.22 ± 0.67 | 0 [0–2] | 0.67 ± 0.87 | 0 [0–2] | 0.297 | 0,776 | 0.181 |
| Large double | 0 ± 0 | 0 [0–0] | 0 ± 0 | 0 [0–0] | 1.000 | 0.11 ± 0.33 | 0 [0–1] | 0.33 ± 0.50 | 0 [0–1] | 0.436 | 0.776 | 0.328 |
Stay updated, free articles. Join our Telegram channel
Full access? Get Clinical Tree
