Anatomia, Histologia, Embryologia
ORIGINAL ARTICLE
Effect of Mobile Phone Radiation on Cardiovascular Development of Chick Embryo W. Ye1,2, F. Wang1, W. Zhang1, N. Fang1, W. Zhao1 and J. Wang2* Addresses of authors: 1 Medical College of Henan University, Kaifeng 475000, China; 2 Institute of Zoology, School of Life Science, Lanzhou University, Lanzhou 730000, China
*Correspondence: Tel.: +8609318912560; Fax: +8609318912561; e-mail: [email protected] With 9 figures and 1 table Received September 2014; accepted for publication July 2015 doi: 10.1111/ahe.12188
Summary The biological effects on cardiovascular development of chicken embryos were examined after radiation exposure using mobile phone (900 MHz; specific absorption rate~1.07 W/kg) intermittently 3 h per day during incubation. Samples were selected by morphological and histological methods. The results showed the rate of embryonic mortality and cardiac deformity increased significantly in exposed group (P < 0.05). No any histological pathological changes were observed on Day 5–7 (D5–D7) of incubation. A higher distribution of lipid droplets was unexpectedly present in myocardial tissue from the exposure groups on D10–D13. Soon afterwards, myofilament disruption, atrioventricular valve focal necrosis, mitochondria vacuolization and atrial natriuretic peptide (ANP) decrease appeared on D15-D21 of incubation. Comet assay data showed the haemocyte mean tail in the exposed group was significantly larger than that of the control (P < 0.01). The arterial vascular wall of exposed group was thicker (P < 0.05) than that of the control on D13, which was reversed to normal in later stages. Our findings suggest that long-term exposure of MPR may induce myocardium pathological changes, DNA damage and increased mortality; however, there was little effect on vascular development.
Introduction Altofrequent use of mobile phone (MP) causes public concern issues focused on whether its microwave radiation (MW: 300 MHz-3 GHz) is harmful to health or not. More and more epidemiological and experimental studies have examined this issue. The biological effects of MP microwave radiation are mainly based on non-thermal effects (Weisbrot et al., 2003; Diem et al., 2005; Girgert et al., 2005). Large numbers of experimental studies showed that microwave radiation causes damage not only to the nervous system, immune system and the lens of animals’ (Balci et al., 2007; De Gannes et al., 2009; Del Vecchio et al., 2009), but also to embryonic development (Salama et al., 2010; Roda et al., 2011). Permanent exposure to a GSM mobile phone during incubation, higher mortality rates occur than those of control (Bastide et al., 2002; Batellier et al., 2008). As a target organ, the © 2015 Blackwell Verlag GmbH Anat. Histol. Embryol. 45 (2016) 197–208
cardiovascular system is sensitive to microwave radiation and can cause different degrees of damage in cardiac structure and function, such as decreased heart rate, atrial and ventricular conduction delays and ECG waves changes (Jauchem, 1997). DNA damage plays an important role in cell mutation and death, tissue injury, cancer generation etc. The comet assay is now a well-established genotoxicity test for estimating DNA damage at a cell level both in vivo and in vitro (Garaj-Vrhovac and Orescanin, 2009). However, whether microwave exposure induces genetic alternation is inconclusive. Human fibroblasts and rat granulosa cells were exposed to 1800 MHz mobile phone signals at a specific absorption rate (SAR) of 1.2 and 2 W/kg, respectively, Diem found that after 16 h, DNA single- and double-strand breaks were induced in both cell types as measured by the comet assay (Diem et al., 2005). After acute exposure
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to a 1.7-GHz field, DNA double-strand breaks in mouse embryonic stem cells were noted in (Nikolova et al., 2005). In human lens epithelial cells DNA single-strand breaks appeared after 2 h of exposure to a 1.8-GHz field at SARs of 3 and 4 W/kg (Sun et al., 2006). However, partial studies hold a reverse idea that microwave radiation has little hereditary toxicity, for example long-term exposure (2 h/day, 5 days/week for 2 years) of rats to 900 MHz GSM signal at 0.3 and 0.9 W/kg did not significantly affect DNA strand breaks in cells (Verschaeve et al., 2006). Chemeris’ results were exactly the same, high-power microwave under the given exposure conditions did not induce DNA strand breaks, alkali-labile sites and incomplete excision repair sites of human wholeblood leucocytes and isolated lymphocytes exposed to 8.8-GHz field at SAR of 1.6 kW/kg for 40 min and 30 min, respectively (Chemeris et al., 2006). Further studies are needed to evaluate the genotoxic effects of an ordinary mobile phone radiation. In this study, a radial exposure model of the cardiovascular system in the chick embryo was built using actual GSM mobile phones. The secure SAR of MP is 2.0 W/kg recommended by the International Commission on Nonionizing Radiation Protection (ICNIRP 1998). The SAR in the present research is 1.07 W/kg. Our aim was to clarify whether DNA fragmentation, morphological and histological damage of chick embryo cardiovascular cells could be induced in non-thermal exposure from 900 MHz GSM MPR.
Materials and Methods Biological materials Experiments were performed with Hy-Line Brown Chicken eggs (brown eggshell), purchased from Lanzhou Institute of Biological Products. All chicken eggs had marked chamber, uniform stoma and complete shell. Temperature of the experimental incubation chamber was adjusted to 37.4 0.2°C, and the relative humidity was 70 5%. The eggs were turned automatically once every 2 h, and embryo development was observed regularly by egg candler. Experimental protocol Ten experimental replicates were carried out, and a total of 560 embryos were used for embryo mortality and cardiac deformity. For each experimental replicate, chicken eggs were randomly divided into a control group (no MPR) and exposed group with each group having 28 eggs. We randomly divided the exposed group eggs into four groups with each group of seven eggs. The seven eggs were placed in a circular permutation. Four mobile
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phones were used during the experiment. The phone was held horizontally, keyboard down, in the centre of each circular permutation, the keyboard of the phone being placed above the upper surface of the eggs. The distance between any two adjacent mobile phones was approximately 7 cm. Chicken eggs in the exposed group during period of incubation (from Day 2) were exposed discontinuously by the mobile phone in ‘connecting’ mode for a total 3 h per day. The eggs were given six calls every day by manual control, each connection lasted for 30 min. And then the phones hung up for 1–2 min, during the period, the phones were given a 90° clockwise turn. The aim of mobile phones rotary motion was to ensure that the eggs had approximately the same irradiation dose. That the exposure process could be designed as a total ‘dial + connection for 30 min + hung up for 2 min and gave a 90° turn’ sequence repeated six times every day. The control groups were incubated in the same conditions but less ‘connecting’. Type of mobile phone Our experiments were designed to assess the biological effects of microwave electromagnetic radiation at 900 MHz, emitted by GSM mobile phones. Four ordinary mobile phones of a commercial brand were used in the present study. The power density at 900 MHz of the radiation emitted by the MP was measured with Microwave Radiation Monitor, TriField Meter (America). An average intensity of MW radiation was 7.15 0.61 lW/cm2, estimated at 1 cm away from the surface of the phone and 0.052 0.003 lW/cm2 at the 10 cm away during ‘connecting’. The SAR of the mobile phones was approximately 1.07 W/kg according to the manufacturer. Microstructure and ultrastructure study As previously described, sampling was conducted on days 5, 7, 10, 13, 15 and 21 of incubation in the exposed and control group, respectively. Ten eggs were extracted at every group, and every group was repeated at least three times. Heart was extracted quickly after 30 min of irradiation, washed in PBS (0.1 mol/l), photographs taken timely by stereomicroscope to see whether the heart had exterior abnormalities. Then, the nondeformed hearts were fixed in Bouin’s fluid for 72 h, prepared for paraffin embedding, 5-lm-thick serial sections cut and H&E staining performed. All the sections were photographed and analysed by MOTIC IMAGES ADVANCED 3.0 software Motic Electric Group Co.,Ltd, Xiamen, China. © 2015 Blackwell Verlag GmbH Anat. Histol. Embryol. 45 (2016) 197–208
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Sampling on days 5, 15 and 21 of the exposed and control groups was prepared for electron microscopy sections, respectively. Then, the sections were observed under a transmission electron microscope (JEOL, JEM-1230).
Results
Blood samples were obtained from the heart on Day 15 of incubation in the exposed and control group, respectively, and the sampling were started at 30 min post-radiation. Comet assay was carried out as previously described (Tice et al., 2000). DNA ethidium-stained minigel electrophoresis was analysed by a fluorescent microscope with CASP software. At least three repeat experiments were set.
Clinical findings During the whole experimental period, embryo mortality of fertile eggs in the control group was 8.21%, and cardiac deformity rate was 2.41%. But in the exposed group embryo mortality of fertile eggs reached to 14.29%, and the cardiac deformity rate reached to 6.79%. Both embryo mortality and cardiac deformity rate increased significantly in the exposed group compared with the control group (P < 0.05) (Table 1). The abnormal development of the embryo hearts was demonstrated as follows: myocardial hypertrophy and cyst, atrium and ventricle inversion, interventricular septum hypoplasia and arch artery malformation (Fig. 1).
Morphological measurement of the arterial wall thickness
Effects of MPR on microstructure of heart in chick embryo
The heart samples were collected on days 10, 13 and 15 of the incubation, 10 samples at every stage from each group. The brachiocephalic artery (BA), left common carotid artery (LCCA), left subclavian artery (LSA), left pulmonary artery (LPA) and right pulmonary artery (RPA) were intercepted from the beginning of arterial arch branches. Then, the arteries were fixed in Bouin’s fluid for 72 h, prepared for paraffin sections, 7-lm-thick transverse serial sections cut and H&E staining performed. All the sections were measured by linear micrometer of MOTIC IMAGES ADVANCED 3.0 software. The thickest point of each arterial circular cross section was selected as the central point, and then, the arterial cross section was divided into equidistant five sections from this centre point. The wall thickness was measured at the five points (including the centre point), and the average value was obtained for statistical analyses.
Figures 2 and 3 shows the cardiac histological changes after exposure to MPR. On Day 5 to Day 7 of incubation, no differences were observed and radiation group, when compared with the control group, exhibited normal myocardial cell growth and fibre architecture (Fig. 2e and f). During this period, the heart started to become septated by forming the primary atrial septum and interventricular septum, while the myocardial fibre was irregular and loose arrangement (Fig. 2a and b). On D10 of incubation, lipid droplets were unexpectedly observed within cardiac tissue of exposed group, but were not in control group (Fig. 2c and g). Soon afterwards, cardiac development delay, inter-cellular space expansion, the disorderly arrangement of myocardial cells and a certain steatosis appeared in chick heart of exposed group on D13 of incubation (Fig. 2h). During the late embryonic stages (from Day 15 to Day 21), severe cardiac pathological changes appeared in chick embryo of the exposed group. It was characterized by myocardial fibres derangement with different degree of swelling and fracture, inter-cellular space significant expansion, a certain degree of atrioventricular valve fibrosis (Fig. 3d and e). Connective tissue proliferation, collagen and elastic fibres disorderly arrangement and focal lesion were seen in the leaflets of the atrioventricular valve (Fig. 3f). The characteristics of myocardium in control embryo exhibited normal development. The general cell shapes usually changed
Comet assay
Statistical analysis Data reported in this paper were analysed using the spss version 11.5 (IBM, New York, USA). Statistical significance was determined by the percentage of significant test and t-test. All data were presented as mean SD (Standard Deviation), and P < 0.05 was identified as significant difference.
Table 1. Statistical analysis of embryo mortality and cardiac deformity rate of all fertile eggs exposure to mobile phone in ‘connection’ state
Items
Number
Death
Mortality rate
P
Deformity
Deformity rate
P
Control Exposed
280 280
23 40
8.21% 14.29%
P < 0.05
6 19
2.14% 6.79%
P < 0.01
Statistically significant difference between the control group and exposed group at P < 0.05, and very significant difference at P < 0.01 by the percentage of significant test.
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Fig. 1. Effects of MPR on morphological teratogenicity of heart in chick embryo. a (control), c (exposed) for the ventral view of D15 embryo heart; b (control), d (exposed) for the dorsal view of D15 embryo heart, (4 9 1.5). Cardiac deformities show as follows: myocardial hypertrophy and cyst, interventricular septum hypoplasia and arch artery malformation in the exposed group.
from amoeboid in the early embryonic stages to somewhat elongated form in the mature heart. Myocardial fibres were changed into denser, and their arrangement had become more and more regular (Fig. 3a–c). Effects of MPR on ultrastructure of heart in chick embryo Figures 4, 5 and 6 shows the cardiac ultrastructure changes after exposure to MPR. Heart development on D10 of incubation, there was an increase of lipid droplets in cell cytoplasm and interstitial, and apoptotic myocardial cells were seen occasionally in exposed group as compared to the normal control group (Fig. 4). In contrast, marked differences were found later, on the Day 15 of incubation. Numerous vacuoles, abnormal lipid accumulations, partially degenerated myofibrils with disappeared sarcomere structure (Fig. 5d and e) and swelling mitochondria with
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invisible cristae were observed in myocardial cells of exposed group (Fig. 5f). And on D21 of incubation, more significant lesions of myocardial cells were observed in exposed group. It showed that nuclear alterations appeared and consisted of irregularity of the nuclear outline, the widened perinuclear space and irregular condensation of nuclear chromatin (Fig. 6e). Myocardial cells exhibited myofibrils disarray or loss of myofibrils and the Z-lines replaced by fatty tissue (Fig. 6f). And the mitochondria were severely swollen with reduced invisible cristae, vacuolization and even large myelin-like figures production (Fig. 6g). Worthy of mention was that atrial specific granules, atrial natriuretic peptide (ANP), decreased significantly in atrial muscle of embryo after exposure to MPR (Fig. 6h). However, electron micrographs of myocardial cells of control group exhibited typical patterns: normal nuclear and envelope, dense arrangement of myofibrils, zigzag-shaped Z-lines, mitochondria with well-arranged © 2015 Blackwell Verlag GmbH Anat. Histol. Embryol. 45 (2016) 197–208
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Fig. 2. Effects of MPR on microscopic structure of heart in early and middle embryonic stages. (a) (control), (e) (exposed) show the myocardium of D5 embryo; (b) (control), (f) (exposed) show the myocardium of D7 embryo; (c) (control), (g) (exposed) show the interventricular septum of D10 embryo, adipose tissue increased significantly in the exposed group (?); (d) (control), (h) (exposed) show the ventricular structure of D13 embryo, myocardial cells arranged disorderly, partially vacuolar degeneration appeared in the exposed group. HE.
Fig. 3. Effects of MPR on microscopic structure of heart in late embryonic stages. (a) (control), (d) (exposed) show the ventricular structure of D15 embryo, myocardial fibres with different degree of swelling and fracture in the exposed group;(b) (control), (e) (exposed) show the ventricular structure of D21 embryo, myocardial fibres with dissolution and rupture in the exposed group (?); (c) (control), (f) (exposed) for the right atrioventricular valve structure of D21 embryo, focal lesion in valve leaflet of the exposed group (↓). HE.
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cristae, rough endoplasmic reticulum and abundant atrial specific granules (Fig. 6a–d).
extremely significantly increased after exposure to MPR (P < 0.01). It indicated that DNA strand breaks occurred in the hemocytes after treatment with MPR.
Effects of MPR on DNA damage of the hemocytes in chick embryo
Effects of MPR on arterial wall thickness
The comet assay of genomic DNA was reported in Fig. 7 and the statistical analysis of data in Fig. 8 On Day 15 of incubation, the image analysis measurements of the overall fluorescent intensity in the control samples showed a circular head corresponding to the undamaged DNA. But in the exposed samples, the fluorescent images had long comet tail. The mean tail area of the hemocytes in the chick embryo from the exposed group was 38.91 4.06 lm2, and the mean tail area of the control group was 16.03 2.69 lm2. The tail area in cells was
The results about the effects of MPR on arterial wall thickness in different stages are showed in Fig. 9. On Day 10 of incubation, data analysis revealed no statistical difference between the control and exposed groups in wall thickness of all measured arteries, including brachiocephalic artery (BA), left common carotid artery (LCCA), left subclavian artery (LSA), left pulmonary artery (LPA) and right pulmonary artery (RPA). Surprisingly, when the embryo was on Day 13 of development, the wall thickness of BA, LCCA and LSA from the exposed group was
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Fig. 4. Effects of MPR on ultrastructure of Day 10 embryo myocardium. (a–c) For the left ventricle ultrastructure of embryo from the control group; (d–f) for the left ventricle ultrastructure from the exposed group, apoptotic cells ( ) and increased lipid droplets (↓) were observed in the exposed group. TEM.
significantly thicker (P < 0.05) than that from the control group. The three arteries showed obvious stress response after 12 days of exposure to MPR, but the wall thickness of the other arteries was increased slightly. However, as the embryo develops, on Day 15 of incubation, the wall thickness of the arteries in the exposed group returned to normal levels, there was no statistical difference between the control and exposed group. Discussion In this study, the SAR of the GSM mobile phones we used was approximately 1.07 W/kg and the discontinuous ‘connection’ state lasted for 3 h every day. Chick embryo could be used as a good model organism to investigate the biological effects of mobile phone radiation on the foetal heart development. The common mobile phones have average emitted power of 0.25 W with a maximum averaged SAR approximately 1.6 W/Kg, which results in a maximum temperature rise of 0.11°C in local tissue, and cannot produce obvious thermal effects on the body (Van Leeuwen et al., 1999). So, in this study, the mobile phones with SAR of 1.07 W/kg mainly induced non-thermal effects on chick embryo.
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Low-intensity microwave signals appear to effect many biological processes, including stimulation of cell proliferation (Kwee and Raskmark, 1998), alteration of protein conformation (De Pomerai et al., 2003) and increase in chromosome aberrations and micronuclei in human blood lymphocytes (Garaj-Vrhovac et al., 1991). Thus, microwave field can produce disruption of cellular function and affect the organism development (Weisbrot et al., 2003). These phenomena were also reflected in our study, we found that both embryo mortality and cardiac deformity rate increased significantly after radiation (P < 0.05), the embryo mortality increased from 8.21% to 14.29%, and the cardiac deformity rate increased from 2.41% to 6.79%. After hatching, some chickens from the exposed group showed worsening of behaviour status, such as abnormal hindlimbs, reduced activity, languishment, amblyopia or cataract, whereas these symptoms were seldom observed in the control group. Bastide exposed chick embryo over the entire period to 900 MHz GSM signal and found that the mortality in the exposed group increases significantly from 16% to 64% (Bastide et al., 2002). However, Tsybulin showed an increased embryo survival and hatchability in exposed groups of quail eggs after 14 days exposure to a GSM 900 MHz cell © 2015 Blackwell Verlag GmbH Anat. Histol. Embryol. 45 (2016) 197–208
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Fig. 5. Effects of MPR on ultrastructure of Day 15 embryo myocardium. (a–c) For the ventricle ultrastructure of embryo from the control group; (d–f) for the ventricle ultrastructure from the exposed group, myofibrils focal lesions, lipid droplets increase, mitochondria swelling (?), cristae derangement in the exposed group. TEM.
phone at 0.2 lW/cm2 as compared to sham-exposed groups (Tsybulin et al., 2012). These data of the effects on embryo mortality and deformity rate are so different, possibly because of the different radiation parameters and experimental conditions. At the histological level, no obvious pathological changes were observed in myocardium of embryo exposed to MP for 4 days. But after 9– 12 days of exposure (10–13 days of incubation), abnormal lipid droplets accumulations in myocardial cells were observed. After 14–20 days of exposure, the MPR caused partial myofibrils disruption and dissolution, myocardial steatosis, focal necrosis of the atrioventricular valve, mitochondria vacuolization and even myelin-like figures production. The histological studies suggested that there might be a positive correlation between the time of exposure and radiation damage. The myocardium continues to derive a large proportion of its energy from oxidative metabolism occurred in mitochondria, and the integrity of mitochondria cristae is important for energy metabolism. Under aerobic conditions, the predominant substrate used by the heart are free fatty acids, accounting for 60–90% of the energy generated, in pathological conditions, there is increased utilization by the myocardium of glucose as a substrate for energy production (Lee et al., 2004). In early study, it is © 2015 Blackwell Verlag GmbH Anat. Histol. Embryol. 45 (2016) 197–208
found that decimetre wave radiation can induce myocardial cell energy metabolism dysfunction (Bogoliubov et al., 1989). Another study showed that mitochondrial inhibitors significantly reduce the viability of myocardial cells, increase the glycogen consumption, suggesting that cardiac energy metabolism is switched from b-oxidation of fatty acid to glycolysis (Kakinuma et al., 2000). In this study, mitochondria swelling, cristae deletion and vacuolization were observed after more than 14 days of exposure to MPR at 3 h/days. Meanwhile, there was drastically increased distribution of lipid droplets in irradiated myocardial tissue. It could be speculated that MPR impaired mitochondrial function, induced a progressive reduction in mitochondrial b-oxidative capacity and ATP synthesis with free fatty acids substrate. Early study showed that electromagnetic radiation can modulate opioid peptides and b endorphin synthesis in myocardial cells, affect the endocrine function of the heart (Ventura et al., 2000). In the present study, we found that ANP decreased significantly in atrial muscle of embryo exposed to MPR. It implies that a significant endocrine functional alteration could exist in the heart after exposure to MP. It is important to note that most studies on the genotoxic effects of exposure use simulative mobile phone signal from a signal generator, while the present study
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Fig. 6. Effects of MPR on ultrastructure of Day 21 embryo myocardium. (a–d) For the right atrial ultrastructure from the control group; (e–h) for the right atrial ultrastructure from the exposed group, perinuclear space expansion, myocardial steatosis, mitochondria vacuolization and even myelin-like figures production (↓), ANP electron-dense granules located near Golgi apparatus decreased significantly in the exposed group ( ). TEM.
Fig. 7. Comet assay of genomic DNA of the hemocytes in the heart was performed on Single cell gel electrophoresis after 14 days of exposure to MPR. C (control), E (exposed), 9200.
added to demonstration of the effects using an actual mobile phone. The comet assay results showed that after 14 days of exposure (on D15 of incubation), the tail area
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in the hemocytes of the chick embryo was significantly larger than that from the control group (P < 0.01). It indicated that, although an ordinary mobile phone with © 2015 Blackwell Verlag GmbH Anat. Histol. Embryol. 45 (2016) 197–208
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accumulation of non-repaired DNA damage. Extensive DNA damage can result in cell death, tissue injuries, genetic alterations, subsequent cancer generation and other genetic diseases. It has been suggested that exposure to EMF may have genetic effects that cause the development of cancer, particularly lymphoma and leukaemia, and also birth defects such as Down’s syndrome (Savitz and Calle, 1987; Garson et al., 1991). Some studies suggested that the microwave radiation in reasonable power could be considered as the catalyst of cancer development (Loscher and Mevissen, 1994; Holmberg, 1995). However, some experiments exposed mouse models to simulative mobile phone microwave field for several weeks or 2 years, there were no positive effects of electromagnetic radiation for cancer development (Heikkinen et al., 2001; Bartsch et al., 2002; Shirai et al., 2005). And more, some studies suggested no evidence that exposure to radiofrequency fields affected gene expression or protein expression (Kim et al., 2010; Sekijima et al., 2010; Sakurai et al., 2011). The biological effects of microwave radiation may depend on the research model, radiation pattern, the radiation intensity, exposure time and so on. New works are required to elucidate the mechanisms of hereditary toxicity from MPR for improving prevention measures. Some studies suggested that oxidative stress and ROS production might be involved in the action of MW on biological system (Irmak et al., 2002; Ilhan et al., 2004). Microwave from mobile phones may affect biological systems by increasing free radicals, which in turn may enhance lipid peroxidation in the brain and lead to oxidative damage (Koylu et al., 2006). Oktem found that SOD activities of kidney tissue are reduced after 900-
Fig. 8. Statistical analysis of the DNA damages in chick embryo after 14 days of exposure to MPR. Data were presented as mean SD of at least three independent experiments.
SAR value was below ICNIRP’s exposure guidelines, longterm exposure could also induce DNA single-strand breaks. Paulraj and Behari reported that chronic exposure (25 days) of rats to 2.45 and 16.5 GHz low-intensity microwave with SAR of 1.0 and 2.01 W/kg, respectively, cause statistically significant (P < 0.001) increase in DNA single-strand breaks in brain cells (Paulraj and Behari, 2006). DNA double- or single-strand breaks and DNA chemical bonds breaks will not be repaired completely by cells themselves (Robison et al., 2002). DNA damage accumulates with time and the repair capacity decreases during long-term occupational exposure, although majority of DNA lesions are repaired in a few hours and days, a part of DNA damage induced persisted over time (Garaj-Vrhovac and Orescanin, 2009). It can be considered that microwave radiation induces an
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(c) Fig. 9. Arterial wall thickness analysis on days 10, 13 and 15 of incubation in the presence or in the absence of MPR are showed in a, b and c, respectively. All data were presented as mean SD of at least eight independent experiments. Brachiocephalic artery (BA), left common carotid artery (LCCA), left subclavian artery (LSA), left pulmonary artery (LPA) and right pulmonary artery (RPA).
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MHz electromagnetic radiation (EMR) exposure at 1.04 W/cm2 (30 min/day for 10 days) (Oktem et al., 2005). De Iuliis reported that exposing human spermatozoa to RF-EMR (at 1.8 GHz with SAR of 0.4 - 27.5 W/ kg) leads to significant declines in motility and vitality in concert with increases in ROS generation and DNA fragmentation (De Iuliis et al., 2009). So we speculated oxidative stress and ROS production could be one of key factors following MPR to induce mitochondrial alterations, DNA fragmentation, cell structural and functional lesions observed in this study, but we need to conduct further validation. What is puzzling is that some studies had shown no effects of radiofrequency radiation on ROS formation (Hook et al., 2004; Lantow et al., 2006; Simko et al., 2006; Zeni et al., 2007). The latest reports found a transient small increase in the ROS level after exposure of neuronal cells to multiple radiofrequency signals; however, the ROS level no prolonged or further increase (Kang et al., 2014). The present study confirmed MPR had little influence on vascular development. At early stage of development, there were no statistical differences of arterial wall thickness between the control group and exposed group. After 12 days of exposure to MP, the wall thickness of brachiocephalic artery, left common carotid artery and the left subclavian artery was significantly thicker (P < 0.05) than the control group. The stress response of the three arteries induced by MPR was observed on D13 of incubation, a period during which the body immunity is relatively low because of the chicken embryo normally developing feathers, beak and eyelids. However, on Day 15 of incubation, the wall thickness of the arteries in the exposed group returned to normal levels. It indicated that exposure to 900 MHz GSM mobile phone at SAR of 1.07 W/Kg (3 h/ days) did not obviously hinder the development of blood vessels. The MPR radiosensitivity of blood vessels was relatively lower than the heart. The microwave radiation tolerability and the repairing capability of blood vessels would be enhanced with the embryonic development. In summary, the present findings showed that longterm exposure to GSM mobile phone radiation could result in myocardial pathological changes, DNA damage and chick embryo mortality increase, but had little influence on vascular development. It would be necessary to re-evaluate the present mobile phone radiation safety standards. Acknowledgements The authors are grateful to Prof. Guangming Zhou, Ms. Guofen Wu, Ms. Yun Dong, Ms. Lifen Zhao, Mr. Yuanqing Xu and Mr. Guoqiang Song, for their kind help during the experimental period. This work was supported by
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grants-in-aid MMM200819 from the Chinese Educational Department Key Laboratory of Magnetism and Magnetic Materials of Lanzhou University. Conflict of Interest There are no any financial or personal interests that might be potentially viewed to influence the work presented. References Balci, M., E. Devrim, and I. Durak, 2007: Effects of mobile phones on oxidant/antioxidant balance in cornea and lens of rats. Curr. Eye Res. 32, 21–25. Bartsch, H., C. Bartsch, E. Seebald, F. Deerberg, K. Dietz, L. Vollrath, and D. Mecke, 2002: Chronic exposure to a GSMlike signal (mobile phone) does not stimulate the development of DMBA-induced mammary tumors in rats: results of three consecutive studies. Radiat. Res. 157, 183–190. Bastide, M., B. Youbicier-Simo, J. Lebecq, and J. Giaimis, 2002: Toxicologic study of electromagnetic radiation emitted by television and video display screens and cellular telephones on chickens and mice. Indoor Built. Environ. 10, 291–298. Batellier, F., I. Couty, D. Picard, and J. Brillard, 2008: Effects of exposing chicken eggs to a cell phone in “call” position over the entire incubation period. Theriogenology 69, 737– 745. Bogoliubov, V. M., I. D. Frenkel, S. M. Zubkova, I. Z. Liakhovetskii, and Z. A. Sokolova, 1989: Energy and plastic metabolism of the heart muscle in rabbits undergoing thyroid irradiation with decimeter waves. Biulleten’ eksperimental’noi biologii i meditsiny. 107, 570–572. Chemeris, N. K., A. B. Gapeyev, N. P. Sirota, O. Y. Gudkova, A. V. Tankanag, I. V. Konovalov, M. E. Buzoverya, V. G. Suvorov, and V. A. Logunov, 2006: Lack of direct DNA damage in human blood leukocytes and lymphocytes after in vitro exposure to high power microwave pulses. Bioelectromagnetics 27, 197–203. De Gannes, F. P., M. Taxile, S. Duleu, A. Hurtier, E. Haro, M. Geffard, G. Ruffie, B. Billaudel, P. Leveque, P. Dufour, I. Lagroye, and B. Veyret, 2009: A confirmation study of Russian and Ukrainian data on effects of 2450 MHz microwave exposure on immunological processes and teratology in rats. Radiat. Res. 172, 617–624. De Iuliis, G. N., R. J. Newey, B. V. King, and R. J. Aitken, 2009: Mobile phone radiation induces reactive oxygen species production and DNA damage in human spermatozoa in vitro. PLoS ONE 4, e6446. De Pomerai, D. I., B. Smith, A. Dawe, K. North, T. Smith, D. B. Archer, I. R. Duce, D. Jones, and P. M. Candido, 2003: Microwave radiation can alter protein conformation without bulk heating. FEBS Lett. 543, 93–97.
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Del Vecchio, G., A. Giuliani, M. Fernandez, P. Mesirca, F. Bersani, R. Pinto, L. Ardoino, G. A. Lovisolo, L. Giardino, and L. Calza, 2009: Continuous exposure to 900 MHz GSMmodulated EMF alters morphological maturation of neural cells. Neurosci. Lett. 455, 173–177. Diem, E., C. Schwarz, F. Adlkofer, O. Jahn, and H. Rudiger, 2005: Non-thermal DNA breakage by mobile-phone radiation (1800 MHz) in human fibroblasts and in transformed GFSH-R17 rat granulosa cells in vitro. Mutat. Res. 583, 178–183. Garaj-Vrhovac, V., and V. Orescanin, 2009: Assessment of DNA sensitivity in peripheral blood leukocytes after occupational exposure to microwave radiation: the alkaline comet assay and chromatid breakage assay. Cell Biol. Toxicol. 25, 33–43. Garaj-Vrhovac, V., D. Horvat, and Z. Koren, 1991: The relationship between colony-forming ability, chromosome aberrations and incidence of micronuclei in V79 Chinese hamster cells exposed to microwave radiation. Mutat. Res. Lett. 263, 143–149. Garson, O. M., T. L. McRobert, L. J. Campbell, B. A. Hocking, and I. Gordon, 1991: A chromosomal study of workers with long-term exposure to radio-frequency radiation. Med. J. Australia 155, 289–292. Girgert, R., H. Schimming, W. Korner, C. Grundker, and V. Hanf, 2005: Induction of tamoxifen resistance in breast cancer cells by ELF electromagnetic fields. Biochem. Biophys. Res. Commun. 336, 1144–1149. Heikkinen, P., V.-M. Kosma, T. Hongisto, H. Huuskonen, P. Hyysalo, H. Komulainen, T. Kumlin, T. Lahtinen, S. Lang, L. Puranen, and J. Juutilainen, 2001: Effects of mobile phone radiation on X-ray-induced tumorigenesis in mice. Radiat. Res. 156, 775–785. Holmberg, B., 1995: Magnetic-fields and cancer - animal and cellular evidence – an overview. Environ. Health Perspect. 103, 63–67. Hook, G. J., D. R. Spitz, J. E. Sim, R. Higashikubo, J. D. Baty, E. G. Moros, and J. L. Roti, 2004: Evaluation of parameters of oxidative stress after in vitro exposure to FMCW-and CDMA-modulated radiofrequency radiation fields. Radiat. Res. 162, 497–504. ICNIRP, 1998: Guidelines for limiting exposure to time-varying electric, magnetic, and electromagnetic fields (up to 300 GHz). International Commission on Non-Ionizing Radiation Protection. Health Phys. 74, 494– 522. Ilhan, A., A. Gurel, F. Armutcu, S. Kamisli, M. Iraz, O. Akyol, and S. Ozen, 2004: Ginkgo biloba prevents mobile phoneinduced oxidative stress in rat brain. Clin. Chim. Acta 340, 153–162. Irmak, M. K., E. Fadıllıoglu, M. G€ ulecß, H. Erdogan, M. Yagmurca, and O. Akyol, 2002: Effects of electromagnetic radiation from a cellular telephone on the oxidant and antioxidant levels in rabbits. Cell Biochem. Funct. 20, 279– 283.
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Jauchem, J. R., 1997: Exposure to extremely-low-frequency electromagnetic fields and radiofrequency radiation: cardiovascular effects in humans. Int. Arch. Occup. Environ. Health 70, 9–21. Kakinuma, Y., T. Miyauchi, K. Yuki, N. Murakoshi, K. Goto, and I. Yamaguchi, 2000: Mitochondrial dysfunction of cardiomyocytes causing impairment of cellular energy metabolism induces apoptosis, and concomitant increase in cardiac endothelin-1 expression. J. Cardiovasc. Pharmacol. 36, S201–S204. Kang, K. A., H. C. Lee, J. J. Lee, M. N. Hong, M. J. Park, Y. S. Lee, H. D. Choi, N. Kim, Y. G. Ko, and J. S. Lee, 2014: Effects of combined radiofrequency radiation exposure on levels of reactive oxygen species in neuronal cells. J. Radiat. Res. 55, 265–276. Kim, K. B., K. O. Byun, N. K. Han, Y. G. Ko, H. D. Choi, N. Kim, J. K. Pack, and J. S. Lee, 2010: Two-dimensional electrophoretic analysis of radio frequency radiation-exposed MCF7 breast cancer cells. J. Radiat. Res. 51, 205–213. Koylu, H., H. Mollaoglu, F. Ozguner, M. Nazyrodlu, and N. Delibalp, 2006: Melatonin modulates 900 MHz microwaveinduced lipid peroxidation changes in rat brain. Toxicol. Ind. Health 22, 211–216. Kwee, S., and P. Raskmark, 1998: Changes in cell proliferation due to environmental non-ionizing radiation: 2. Microwave radiation. Bioelectrochem. Bioenerg. 44, 251–255. Lantow, M., J. Schuderer, C. Hartwig, and M. Simko, 2006: Free radical release and HSP70 expression in two human immune-relevant cell lines after exposure to 1800 MHz radiofrequency radiation. Radiat. Res. 165, 88–94. Lee, L., J. Horowitz, and M. Frenneaux, 2004: Metabolic manipulation in ischaemic heart disease, a novel approach to treatment. Eur. Heart J. 25, 634–641. Loscher, W., and M. Mevissen, 1994: Animal studies on the role of 50/60-Hertz magnetic-fields in carcinogenesis. Life Sci. 54, 1531–1543. Nikolova, T., J. Czyz, A. Rolletschek, P. Blyszczuk, J. Fuchs, G. Jovtchev, J. Schuderer, N. Kuster, and A. M. Wobus, 2005: Electromagnetic fields affect transcript levels of apoptosisrelated genes in embryonic stem cell-derived neural progenitor cells. FASEB J. 19, 1686–1688. Oktem, F., F. Ozguner, H. Mollaoglu, A. Koyu, and E. Uz, 2005: Oxidative damage in the kidney induced by 900MHz-emitted mobile phone: protection by melatonin. Arch. Med. Res. 36, 350–355. Paulraj, R., and J. Behari, 2006: Single strand DNA breaks in rat brain cells exposed to microwave radiation. Mutat. Res. 596, 76–80. Robison, J. G., A. R. Pendleton, K. O. Monson, B. K. Murray, and K. L. O’Neill, 2002: Decreased DNA repair rates and protection from heat induced apoptosis mediated by electromagnetic field exposure. Bioelectromagnetics 23, 106–112. Roda, O., I. Garzon, V. Carriel, M. Alaminos, and I. SanchezMontesinos, 2011: Biological effects of low-frequency pulsed magnetic fields on the embryonic central nervous system
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development. A histological and histochemical study. Histol. Histopathol. 26, 873. Sakurai, T., T. Kiyokawa, E. Narita, Y. Suzuki, M. Taki, and J. Miyakoshi, 2011: Analysis of gene expression in a humanderived glial cell line exposed to 2.45 GHz continuous radiofrequency electromagnetic fields. J. Radiat. Res. 52, 185–192. Salama, N., T. Kishimoto, and H. Kanayama, 2010: Retracted: effects of exposure to a mobile phone on testicular function and structure in adult rabbit. Int. J. Androl. 33, 88–94. Savitz, D. A., and E. E. Calle, 1987: Leukemia and occupational exposure to electromagnetic fields: review of epidemiologic surveys. J. Occup. Med. 29, 47–51. Sekijima, M., H. Takeda, K. Yasunaga, N. Sakuma, H. Hirose, T. Nojima, and J. Miyakoshi, 2010: 2-GHz band CW and W-CDMA modulated radiofrequency fields have no significant effect on cell proliferation and gene expression profile in human cells. J. Radiat. Res. 51, 277–284. Shirai, T., M. Kawabe, T. Ichihara, O. Fujiwara, M. Taki, S. Watanabe, K. Wake, Y. Yamanaka, K. Imaida, M. Asamoto, and S. Tamano, 2005: Chronic exposure to a 1.439 GHz electromagnetic field used for cellular phones does not promote N-ethylnitrosourea induced central nervous system tumors in F344 rats. Bioelectromagnetics 26, 59–68. Simko, M., C. Hartwig, M. Lantow, M. Lupke, M. O. Mattsson, Q. Rahman, and J. Rollwitz, 2006: Hsp70 expression and free radical release after exposure to non-thermal radiofrequency electromagnetic fields and ultrafine particles in human Mono Mac 6 cells. Toxicol. Lett. 161, 73–82. Sun, L. X., Y. Ke, K. J. Wang, D. Q. Lu, H. J. Hu, X. W. Gao, B. H. Wang, W. Zheng, J. L. Lou, and W. Wu, 2006: Effects of 1.8 GHz radiofrequency field on DNA damage and expression of heat shock protein 70 in human lens epithelial cells. Mutat. Res. 602, 135–142.
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Tice, R., E. Agurell, D. Anderson, B. Burlinson, A. Hartmann, H. Kobayashi, Y. Miyamae, E. Rojas, J. Ryu, and Y. Sasaki, 2000: Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ. Mol. Mutagen. 35, 206–221. Tsybulin, O., E. Sidorik, S. Kyrylenko, D. Henshel, and I. Yakymenko, 2012: GSM 900 MHz microwave radiation affects embryo development of Japanese quails. Electromagn. Biol. Med. 31, 75–86. Van Leeuwen, G. M. J., J. J. W. Lagendijk, B. Van Leersum, A. P. M. Zwamborn, S. N. Hornsleth, and A. Kotte, 1999: Calculation of change in brain temperatures due to exposure to a mobile phone. Phys. Med. Biol. 44, 2367–2379. Ventura, C., M. Maioli, G. Pintus, G. Gottardi, and F. Bersani, 2000: Elf-pulsed magnetic fields modulate opioid peptide gene expression in myocardial cells. Cardiovasc. Res. 45, 1054–1064. Verschaeve, L., P. Heikkinen, G. Verheyen, U. Van Gorp, F. Boonen, F. Vander Plaetse, A. Maes, T. Kumlin, J. M€aki-Paakkanen, L. Puranen, and J. Juutilainen, 2006: Investigation of co-genotoxic effects of radiofrequency electromagnetic fields in vivo. Radiat. Res. 165, 598–607. Weisbrot, D., H. Lin, L. Ye, M. Blank, and R. Goodman, 2003: Effects of mobile phone radiation on reproduction and development in Drosophila melanogaster. J. Cell. Biochem. 89, 48–55. Zeni, O., R. Di Pietro, G. d’Ambrosio, R. Massa, M. Capri, J. Naarala, J. Juutilainen, and M. R. Scarfı, 2007: Formation of reactive oxygen species in L929 cells after exposure to 900 MHz RF radiation with and without co-exposure to 3chloro-4-(dichloromethyl)-5-hydroxy-2 (5H)-furanone. Radiat. Res. 167, 306–311.
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ORIGINAL ARTICLE
Effect of Mobile Phone Radiation on Cardiovascular Development of Chick Embryo W. Ye1,2, F. Wang1, W. Zhang1, N. Fang1, W. Zhao1 and J. Wang2* Addresses of authors: 1 Medical College of Henan University, Kaifeng 475000, China; 2 Institute of Zoology, School of Life Science, Lanzhou University, Lanzhou 730000, China
*Correspondence: Tel.: +8609318912560; Fax: +8609318912561; e-mail: [email protected] With 9 figures and 1 table Received September 2014; accepted for publication July 2015 doi: 10.1111/ahe.12188
Summary The biological effects on cardiovascular development of chicken embryos were examined after radiation exposure using mobile phone (900 MHz; specific absorption rate~1.07 W/kg) intermittently 3 h per day during incubation. Samples were selected by morphological and histological methods. The results showed the rate of embryonic mortality and cardiac deformity increased significantly in exposed group (P < 0.05). No any histological pathological changes were observed on Day 5–7 (D5–D7) of incubation. A higher distribution of lipid droplets was unexpectedly present in myocardial tissue from the exposure groups on D10–D13. Soon afterwards, myofilament disruption, atrioventricular valve focal necrosis, mitochondria vacuolization and atrial natriuretic peptide (ANP) decrease appeared on D15-D21 of incubation. Comet assay data showed the haemocyte mean tail in the exposed group was significantly larger than that of the control (P < 0.01). The arterial vascular wall of exposed group was thicker (P < 0.05) than that of the control on D13, which was reversed to normal in later stages. Our findings suggest that long-term exposure of MPR may induce myocardium pathological changes, DNA damage and increased mortality; however, there was little effect on vascular development.
Introduction Altofrequent use of mobile phone (MP) causes public concern issues focused on whether its microwave radiation (MW: 300 MHz-3 GHz) is harmful to health or not. More and more epidemiological and experimental studies have examined this issue. The biological effects of MP microwave radiation are mainly based on non-thermal effects (Weisbrot et al., 2003; Diem et al., 2005; Girgert et al., 2005). Large numbers of experimental studies showed that microwave radiation causes damage not only to the nervous system, immune system and the lens of animals’ (Balci et al., 2007; De Gannes et al., 2009; Del Vecchio et al., 2009), but also to embryonic development (Salama et al., 2010; Roda et al., 2011). Permanent exposure to a GSM mobile phone during incubation, higher mortality rates occur than those of control (Bastide et al., 2002; Batellier et al., 2008). As a target organ, the © 2015 Blackwell Verlag GmbH Anat. Histol. Embryol. 45 (2016) 197–208
cardiovascular system is sensitive to microwave radiation and can cause different degrees of damage in cardiac structure and function, such as decreased heart rate, atrial and ventricular conduction delays and ECG waves changes (Jauchem, 1997). DNA damage plays an important role in cell mutation and death, tissue injury, cancer generation etc. The comet assay is now a well-established genotoxicity test for estimating DNA damage at a cell level both in vivo and in vitro (Garaj-Vrhovac and Orescanin, 2009). However, whether microwave exposure induces genetic alternation is inconclusive. Human fibroblasts and rat granulosa cells were exposed to 1800 MHz mobile phone signals at a specific absorption rate (SAR) of 1.2 and 2 W/kg, respectively, Diem found that after 16 h, DNA single- and double-strand breaks were induced in both cell types as measured by the comet assay (Diem et al., 2005). After acute exposure
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to a 1.7-GHz field, DNA double-strand breaks in mouse embryonic stem cells were noted in (Nikolova et al., 2005). In human lens epithelial cells DNA single-strand breaks appeared after 2 h of exposure to a 1.8-GHz field at SARs of 3 and 4 W/kg (Sun et al., 2006). However, partial studies hold a reverse idea that microwave radiation has little hereditary toxicity, for example long-term exposure (2 h/day, 5 days/week for 2 years) of rats to 900 MHz GSM signal at 0.3 and 0.9 W/kg did not significantly affect DNA strand breaks in cells (Verschaeve et al., 2006). Chemeris’ results were exactly the same, high-power microwave under the given exposure conditions did not induce DNA strand breaks, alkali-labile sites and incomplete excision repair sites of human wholeblood leucocytes and isolated lymphocytes exposed to 8.8-GHz field at SAR of 1.6 kW/kg for 40 min and 30 min, respectively (Chemeris et al., 2006). Further studies are needed to evaluate the genotoxic effects of an ordinary mobile phone radiation. In this study, a radial exposure model of the cardiovascular system in the chick embryo was built using actual GSM mobile phones. The secure SAR of MP is 2.0 W/kg recommended by the International Commission on Nonionizing Radiation Protection (ICNIRP 1998). The SAR in the present research is 1.07 W/kg. Our aim was to clarify whether DNA fragmentation, morphological and histological damage of chick embryo cardiovascular cells could be induced in non-thermal exposure from 900 MHz GSM MPR.
Materials and Methods Biological materials Experiments were performed with Hy-Line Brown Chicken eggs (brown eggshell), purchased from Lanzhou Institute of Biological Products. All chicken eggs had marked chamber, uniform stoma and complete shell. Temperature of the experimental incubation chamber was adjusted to 37.4 0.2°C, and the relative humidity was 70 5%. The eggs were turned automatically once every 2 h, and embryo development was observed regularly by egg candler. Experimental protocol Ten experimental replicates were carried out, and a total of 560 embryos were used for embryo mortality and cardiac deformity. For each experimental replicate, chicken eggs were randomly divided into a control group (no MPR) and exposed group with each group having 28 eggs. We randomly divided the exposed group eggs into four groups with each group of seven eggs. The seven eggs were placed in a circular permutation. Four mobile
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phones were used during the experiment. The phone was held horizontally, keyboard down, in the centre of each circular permutation, the keyboard of the phone being placed above the upper surface of the eggs. The distance between any two adjacent mobile phones was approximately 7 cm. Chicken eggs in the exposed group during period of incubation (from Day 2) were exposed discontinuously by the mobile phone in ‘connecting’ mode for a total 3 h per day. The eggs were given six calls every day by manual control, each connection lasted for 30 min. And then the phones hung up for 1–2 min, during the period, the phones were given a 90° clockwise turn. The aim of mobile phones rotary motion was to ensure that the eggs had approximately the same irradiation dose. That the exposure process could be designed as a total ‘dial + connection for 30 min + hung up for 2 min and gave a 90° turn’ sequence repeated six times every day. The control groups were incubated in the same conditions but less ‘connecting’. Type of mobile phone Our experiments were designed to assess the biological effects of microwave electromagnetic radiation at 900 MHz, emitted by GSM mobile phones. Four ordinary mobile phones of a commercial brand were used in the present study. The power density at 900 MHz of the radiation emitted by the MP was measured with Microwave Radiation Monitor, TriField Meter (America). An average intensity of MW radiation was 7.15 0.61 lW/cm2, estimated at 1 cm away from the surface of the phone and 0.052 0.003 lW/cm2 at the 10 cm away during ‘connecting’. The SAR of the mobile phones was approximately 1.07 W/kg according to the manufacturer. Microstructure and ultrastructure study As previously described, sampling was conducted on days 5, 7, 10, 13, 15 and 21 of incubation in the exposed and control group, respectively. Ten eggs were extracted at every group, and every group was repeated at least three times. Heart was extracted quickly after 30 min of irradiation, washed in PBS (0.1 mol/l), photographs taken timely by stereomicroscope to see whether the heart had exterior abnormalities. Then, the nondeformed hearts were fixed in Bouin’s fluid for 72 h, prepared for paraffin embedding, 5-lm-thick serial sections cut and H&E staining performed. All the sections were photographed and analysed by MOTIC IMAGES ADVANCED 3.0 software Motic Electric Group Co.,Ltd, Xiamen, China. © 2015 Blackwell Verlag GmbH Anat. Histol. Embryol. 45 (2016) 197–208
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Sampling on days 5, 15 and 21 of the exposed and control groups was prepared for electron microscopy sections, respectively. Then, the sections were observed under a transmission electron microscope (JEOL, JEM-1230).
Results
Blood samples were obtained from the heart on Day 15 of incubation in the exposed and control group, respectively, and the sampling were started at 30 min post-radiation. Comet assay was carried out as previously described (Tice et al., 2000). DNA ethidium-stained minigel electrophoresis was analysed by a fluorescent microscope with CASP software. At least three repeat experiments were set.
Clinical findings During the whole experimental period, embryo mortality of fertile eggs in the control group was 8.21%, and cardiac deformity rate was 2.41%. But in the exposed group embryo mortality of fertile eggs reached to 14.29%, and the cardiac deformity rate reached to 6.79%. Both embryo mortality and cardiac deformity rate increased significantly in the exposed group compared with the control group (P < 0.05) (Table 1). The abnormal development of the embryo hearts was demonstrated as follows: myocardial hypertrophy and cyst, atrium and ventricle inversion, interventricular septum hypoplasia and arch artery malformation (Fig. 1).
Morphological measurement of the arterial wall thickness
Effects of MPR on microstructure of heart in chick embryo
The heart samples were collected on days 10, 13 and 15 of the incubation, 10 samples at every stage from each group. The brachiocephalic artery (BA), left common carotid artery (LCCA), left subclavian artery (LSA), left pulmonary artery (LPA) and right pulmonary artery (RPA) were intercepted from the beginning of arterial arch branches. Then, the arteries were fixed in Bouin’s fluid for 72 h, prepared for paraffin sections, 7-lm-thick transverse serial sections cut and H&E staining performed. All the sections were measured by linear micrometer of MOTIC IMAGES ADVANCED 3.0 software. The thickest point of each arterial circular cross section was selected as the central point, and then, the arterial cross section was divided into equidistant five sections from this centre point. The wall thickness was measured at the five points (including the centre point), and the average value was obtained for statistical analyses.
Figures 2 and 3 shows the cardiac histological changes after exposure to MPR. On Day 5 to Day 7 of incubation, no differences were observed and radiation group, when compared with the control group, exhibited normal myocardial cell growth and fibre architecture (Fig. 2e and f). During this period, the heart started to become septated by forming the primary atrial septum and interventricular septum, while the myocardial fibre was irregular and loose arrangement (Fig. 2a and b). On D10 of incubation, lipid droplets were unexpectedly observed within cardiac tissue of exposed group, but were not in control group (Fig. 2c and g). Soon afterwards, cardiac development delay, inter-cellular space expansion, the disorderly arrangement of myocardial cells and a certain steatosis appeared in chick heart of exposed group on D13 of incubation (Fig. 2h). During the late embryonic stages (from Day 15 to Day 21), severe cardiac pathological changes appeared in chick embryo of the exposed group. It was characterized by myocardial fibres derangement with different degree of swelling and fracture, inter-cellular space significant expansion, a certain degree of atrioventricular valve fibrosis (Fig. 3d and e). Connective tissue proliferation, collagen and elastic fibres disorderly arrangement and focal lesion were seen in the leaflets of the atrioventricular valve (Fig. 3f). The characteristics of myocardium in control embryo exhibited normal development. The general cell shapes usually changed
Comet assay
Statistical analysis Data reported in this paper were analysed using the spss version 11.5 (IBM, New York, USA). Statistical significance was determined by the percentage of significant test and t-test. All data were presented as mean SD (Standard Deviation), and P < 0.05 was identified as significant difference.
Table 1. Statistical analysis of embryo mortality and cardiac deformity rate of all fertile eggs exposure to mobile phone in ‘connection’ state
Items
Number
Death
Mortality rate
P
Deformity
Deformity rate
P
Control Exposed
280 280
23 40
8.21% 14.29%
P < 0.05
6 19
2.14% 6.79%
P < 0.01
Statistically significant difference between the control group and exposed group at P < 0.05, and very significant difference at P < 0.01 by the percentage of significant test.
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Fig. 1. Effects of MPR on morphological teratogenicity of heart in chick embryo. a (control), c (exposed) for the ventral view of D15 embryo heart; b (control), d (exposed) for the dorsal view of D15 embryo heart, (4 9 1.5). Cardiac deformities show as follows: myocardial hypertrophy and cyst, interventricular septum hypoplasia and arch artery malformation in the exposed group.
from amoeboid in the early embryonic stages to somewhat elongated form in the mature heart. Myocardial fibres were changed into denser, and their arrangement had become more and more regular (Fig. 3a–c). Effects of MPR on ultrastructure of heart in chick embryo Figures 4, 5 and 6 shows the cardiac ultrastructure changes after exposure to MPR. Heart development on D10 of incubation, there was an increase of lipid droplets in cell cytoplasm and interstitial, and apoptotic myocardial cells were seen occasionally in exposed group as compared to the normal control group (Fig. 4). In contrast, marked differences were found later, on the Day 15 of incubation. Numerous vacuoles, abnormal lipid accumulations, partially degenerated myofibrils with disappeared sarcomere structure (Fig. 5d and e) and swelling mitochondria with
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invisible cristae were observed in myocardial cells of exposed group (Fig. 5f). And on D21 of incubation, more significant lesions of myocardial cells were observed in exposed group. It showed that nuclear alterations appeared and consisted of irregularity of the nuclear outline, the widened perinuclear space and irregular condensation of nuclear chromatin (Fig. 6e). Myocardial cells exhibited myofibrils disarray or loss of myofibrils and the Z-lines replaced by fatty tissue (Fig. 6f). And the mitochondria were severely swollen with reduced invisible cristae, vacuolization and even large myelin-like figures production (Fig. 6g). Worthy of mention was that atrial specific granules, atrial natriuretic peptide (ANP), decreased significantly in atrial muscle of embryo after exposure to MPR (Fig. 6h). However, electron micrographs of myocardial cells of control group exhibited typical patterns: normal nuclear and envelope, dense arrangement of myofibrils, zigzag-shaped Z-lines, mitochondria with well-arranged © 2015 Blackwell Verlag GmbH Anat. Histol. Embryol. 45 (2016) 197–208
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Fig. 2. Effects of MPR on microscopic structure of heart in early and middle embryonic stages. (a) (control), (e) (exposed) show the myocardium of D5 embryo; (b) (control), (f) (exposed) show the myocardium of D7 embryo; (c) (control), (g) (exposed) show the interventricular septum of D10 embryo, adipose tissue increased significantly in the exposed group (?); (d) (control), (h) (exposed) show the ventricular structure of D13 embryo, myocardial cells arranged disorderly, partially vacuolar degeneration appeared in the exposed group. HE.
Fig. 3. Effects of MPR on microscopic structure of heart in late embryonic stages. (a) (control), (d) (exposed) show the ventricular structure of D15 embryo, myocardial fibres with different degree of swelling and fracture in the exposed group;(b) (control), (e) (exposed) show the ventricular structure of D21 embryo, myocardial fibres with dissolution and rupture in the exposed group (?); (c) (control), (f) (exposed) for the right atrioventricular valve structure of D21 embryo, focal lesion in valve leaflet of the exposed group (↓). HE.
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cristae, rough endoplasmic reticulum and abundant atrial specific granules (Fig. 6a–d).
extremely significantly increased after exposure to MPR (P < 0.01). It indicated that DNA strand breaks occurred in the hemocytes after treatment with MPR.
Effects of MPR on DNA damage of the hemocytes in chick embryo
Effects of MPR on arterial wall thickness
The comet assay of genomic DNA was reported in Fig. 7 and the statistical analysis of data in Fig. 8 On Day 15 of incubation, the image analysis measurements of the overall fluorescent intensity in the control samples showed a circular head corresponding to the undamaged DNA. But in the exposed samples, the fluorescent images had long comet tail. The mean tail area of the hemocytes in the chick embryo from the exposed group was 38.91 4.06 lm2, and the mean tail area of the control group was 16.03 2.69 lm2. The tail area in cells was
The results about the effects of MPR on arterial wall thickness in different stages are showed in Fig. 9. On Day 10 of incubation, data analysis revealed no statistical difference between the control and exposed groups in wall thickness of all measured arteries, including brachiocephalic artery (BA), left common carotid artery (LCCA), left subclavian artery (LSA), left pulmonary artery (LPA) and right pulmonary artery (RPA). Surprisingly, when the embryo was on Day 13 of development, the wall thickness of BA, LCCA and LSA from the exposed group was
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Fig. 4. Effects of MPR on ultrastructure of Day 10 embryo myocardium. (a–c) For the left ventricle ultrastructure of embryo from the control group; (d–f) for the left ventricle ultrastructure from the exposed group, apoptotic cells ( ) and increased lipid droplets (↓) were observed in the exposed group. TEM.
significantly thicker (P < 0.05) than that from the control group. The three arteries showed obvious stress response after 12 days of exposure to MPR, but the wall thickness of the other arteries was increased slightly. However, as the embryo develops, on Day 15 of incubation, the wall thickness of the arteries in the exposed group returned to normal levels, there was no statistical difference between the control and exposed group. Discussion In this study, the SAR of the GSM mobile phones we used was approximately 1.07 W/kg and the discontinuous ‘connection’ state lasted for 3 h every day. Chick embryo could be used as a good model organism to investigate the biological effects of mobile phone radiation on the foetal heart development. The common mobile phones have average emitted power of 0.25 W with a maximum averaged SAR approximately 1.6 W/Kg, which results in a maximum temperature rise of 0.11°C in local tissue, and cannot produce obvious thermal effects on the body (Van Leeuwen et al., 1999). So, in this study, the mobile phones with SAR of 1.07 W/kg mainly induced non-thermal effects on chick embryo.
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Low-intensity microwave signals appear to effect many biological processes, including stimulation of cell proliferation (Kwee and Raskmark, 1998), alteration of protein conformation (De Pomerai et al., 2003) and increase in chromosome aberrations and micronuclei in human blood lymphocytes (Garaj-Vrhovac et al., 1991). Thus, microwave field can produce disruption of cellular function and affect the organism development (Weisbrot et al., 2003). These phenomena were also reflected in our study, we found that both embryo mortality and cardiac deformity rate increased significantly after radiation (P < 0.05), the embryo mortality increased from 8.21% to 14.29%, and the cardiac deformity rate increased from 2.41% to 6.79%. After hatching, some chickens from the exposed group showed worsening of behaviour status, such as abnormal hindlimbs, reduced activity, languishment, amblyopia or cataract, whereas these symptoms were seldom observed in the control group. Bastide exposed chick embryo over the entire period to 900 MHz GSM signal and found that the mortality in the exposed group increases significantly from 16% to 64% (Bastide et al., 2002). However, Tsybulin showed an increased embryo survival and hatchability in exposed groups of quail eggs after 14 days exposure to a GSM 900 MHz cell © 2015 Blackwell Verlag GmbH Anat. Histol. Embryol. 45 (2016) 197–208
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Fig. 5. Effects of MPR on ultrastructure of Day 15 embryo myocardium. (a–c) For the ventricle ultrastructure of embryo from the control group; (d–f) for the ventricle ultrastructure from the exposed group, myofibrils focal lesions, lipid droplets increase, mitochondria swelling (?), cristae derangement in the exposed group. TEM.
phone at 0.2 lW/cm2 as compared to sham-exposed groups (Tsybulin et al., 2012). These data of the effects on embryo mortality and deformity rate are so different, possibly because of the different radiation parameters and experimental conditions. At the histological level, no obvious pathological changes were observed in myocardium of embryo exposed to MP for 4 days. But after 9– 12 days of exposure (10–13 days of incubation), abnormal lipid droplets accumulations in myocardial cells were observed. After 14–20 days of exposure, the MPR caused partial myofibrils disruption and dissolution, myocardial steatosis, focal necrosis of the atrioventricular valve, mitochondria vacuolization and even myelin-like figures production. The histological studies suggested that there might be a positive correlation between the time of exposure and radiation damage. The myocardium continues to derive a large proportion of its energy from oxidative metabolism occurred in mitochondria, and the integrity of mitochondria cristae is important for energy metabolism. Under aerobic conditions, the predominant substrate used by the heart are free fatty acids, accounting for 60–90% of the energy generated, in pathological conditions, there is increased utilization by the myocardium of glucose as a substrate for energy production (Lee et al., 2004). In early study, it is © 2015 Blackwell Verlag GmbH Anat. Histol. Embryol. 45 (2016) 197–208
found that decimetre wave radiation can induce myocardial cell energy metabolism dysfunction (Bogoliubov et al., 1989). Another study showed that mitochondrial inhibitors significantly reduce the viability of myocardial cells, increase the glycogen consumption, suggesting that cardiac energy metabolism is switched from b-oxidation of fatty acid to glycolysis (Kakinuma et al., 2000). In this study, mitochondria swelling, cristae deletion and vacuolization were observed after more than 14 days of exposure to MPR at 3 h/days. Meanwhile, there was drastically increased distribution of lipid droplets in irradiated myocardial tissue. It could be speculated that MPR impaired mitochondrial function, induced a progressive reduction in mitochondrial b-oxidative capacity and ATP synthesis with free fatty acids substrate. Early study showed that electromagnetic radiation can modulate opioid peptides and b endorphin synthesis in myocardial cells, affect the endocrine function of the heart (Ventura et al., 2000). In the present study, we found that ANP decreased significantly in atrial muscle of embryo exposed to MPR. It implies that a significant endocrine functional alteration could exist in the heart after exposure to MP. It is important to note that most studies on the genotoxic effects of exposure use simulative mobile phone signal from a signal generator, while the present study
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Fig. 6. Effects of MPR on ultrastructure of Day 21 embryo myocardium. (a–d) For the right atrial ultrastructure from the control group; (e–h) for the right atrial ultrastructure from the exposed group, perinuclear space expansion, myocardial steatosis, mitochondria vacuolization and even myelin-like figures production (↓), ANP electron-dense granules located near Golgi apparatus decreased significantly in the exposed group ( ). TEM.
Fig. 7. Comet assay of genomic DNA of the hemocytes in the heart was performed on Single cell gel electrophoresis after 14 days of exposure to MPR. C (control), E (exposed), 9200.
added to demonstration of the effects using an actual mobile phone. The comet assay results showed that after 14 days of exposure (on D15 of incubation), the tail area
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in the hemocytes of the chick embryo was significantly larger than that from the control group (P < 0.01). It indicated that, although an ordinary mobile phone with © 2015 Blackwell Verlag GmbH Anat. Histol. Embryol. 45 (2016) 197–208
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accumulation of non-repaired DNA damage. Extensive DNA damage can result in cell death, tissue injuries, genetic alterations, subsequent cancer generation and other genetic diseases. It has been suggested that exposure to EMF may have genetic effects that cause the development of cancer, particularly lymphoma and leukaemia, and also birth defects such as Down’s syndrome (Savitz and Calle, 1987; Garson et al., 1991). Some studies suggested that the microwave radiation in reasonable power could be considered as the catalyst of cancer development (Loscher and Mevissen, 1994; Holmberg, 1995). However, some experiments exposed mouse models to simulative mobile phone microwave field for several weeks or 2 years, there were no positive effects of electromagnetic radiation for cancer development (Heikkinen et al., 2001; Bartsch et al., 2002; Shirai et al., 2005). And more, some studies suggested no evidence that exposure to radiofrequency fields affected gene expression or protein expression (Kim et al., 2010; Sekijima et al., 2010; Sakurai et al., 2011). The biological effects of microwave radiation may depend on the research model, radiation pattern, the radiation intensity, exposure time and so on. New works are required to elucidate the mechanisms of hereditary toxicity from MPR for improving prevention measures. Some studies suggested that oxidative stress and ROS production might be involved in the action of MW on biological system (Irmak et al., 2002; Ilhan et al., 2004). Microwave from mobile phones may affect biological systems by increasing free radicals, which in turn may enhance lipid peroxidation in the brain and lead to oxidative damage (Koylu et al., 2006). Oktem found that SOD activities of kidney tissue are reduced after 900-
Fig. 8. Statistical analysis of the DNA damages in chick embryo after 14 days of exposure to MPR. Data were presented as mean SD of at least three independent experiments.
SAR value was below ICNIRP’s exposure guidelines, longterm exposure could also induce DNA single-strand breaks. Paulraj and Behari reported that chronic exposure (25 days) of rats to 2.45 and 16.5 GHz low-intensity microwave with SAR of 1.0 and 2.01 W/kg, respectively, cause statistically significant (P < 0.001) increase in DNA single-strand breaks in brain cells (Paulraj and Behari, 2006). DNA double- or single-strand breaks and DNA chemical bonds breaks will not be repaired completely by cells themselves (Robison et al., 2002). DNA damage accumulates with time and the repair capacity decreases during long-term occupational exposure, although majority of DNA lesions are repaired in a few hours and days, a part of DNA damage induced persisted over time (Garaj-Vrhovac and Orescanin, 2009). It can be considered that microwave radiation induces an
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(c) Fig. 9. Arterial wall thickness analysis on days 10, 13 and 15 of incubation in the presence or in the absence of MPR are showed in a, b and c, respectively. All data were presented as mean SD of at least eight independent experiments. Brachiocephalic artery (BA), left common carotid artery (LCCA), left subclavian artery (LSA), left pulmonary artery (LPA) and right pulmonary artery (RPA).
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MHz electromagnetic radiation (EMR) exposure at 1.04 W/cm2 (30 min/day for 10 days) (Oktem et al., 2005). De Iuliis reported that exposing human spermatozoa to RF-EMR (at 1.8 GHz with SAR of 0.4 - 27.5 W/ kg) leads to significant declines in motility and vitality in concert with increases in ROS generation and DNA fragmentation (De Iuliis et al., 2009). So we speculated oxidative stress and ROS production could be one of key factors following MPR to induce mitochondrial alterations, DNA fragmentation, cell structural and functional lesions observed in this study, but we need to conduct further validation. What is puzzling is that some studies had shown no effects of radiofrequency radiation on ROS formation (Hook et al., 2004; Lantow et al., 2006; Simko et al., 2006; Zeni et al., 2007). The latest reports found a transient small increase in the ROS level after exposure of neuronal cells to multiple radiofrequency signals; however, the ROS level no prolonged or further increase (Kang et al., 2014). The present study confirmed MPR had little influence on vascular development. At early stage of development, there were no statistical differences of arterial wall thickness between the control group and exposed group. After 12 days of exposure to MP, the wall thickness of brachiocephalic artery, left common carotid artery and the left subclavian artery was significantly thicker (P < 0.05) than the control group. The stress response of the three arteries induced by MPR was observed on D13 of incubation, a period during which the body immunity is relatively low because of the chicken embryo normally developing feathers, beak and eyelids. However, on Day 15 of incubation, the wall thickness of the arteries in the exposed group returned to normal levels. It indicated that exposure to 900 MHz GSM mobile phone at SAR of 1.07 W/Kg (3 h/ days) did not obviously hinder the development of blood vessels. The MPR radiosensitivity of blood vessels was relatively lower than the heart. The microwave radiation tolerability and the repairing capability of blood vessels would be enhanced with the embryonic development. In summary, the present findings showed that longterm exposure to GSM mobile phone radiation could result in myocardial pathological changes, DNA damage and chick embryo mortality increase, but had little influence on vascular development. It would be necessary to re-evaluate the present mobile phone radiation safety standards. Acknowledgements The authors are grateful to Prof. Guangming Zhou, Ms. Guofen Wu, Ms. Yun Dong, Ms. Lifen Zhao, Mr. Yuanqing Xu and Mr. Guoqiang Song, for their kind help during the experimental period. This work was supported by
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grants-in-aid MMM200819 from the Chinese Educational Department Key Laboratory of Magnetism and Magnetic Materials of Lanzhou University. Conflict of Interest There are no any financial or personal interests that might be potentially viewed to influence the work presented. References Balci, M., E. Devrim, and I. Durak, 2007: Effects of mobile phones on oxidant/antioxidant balance in cornea and lens of rats. Curr. Eye Res. 32, 21–25. Bartsch, H., C. Bartsch, E. Seebald, F. Deerberg, K. Dietz, L. Vollrath, and D. Mecke, 2002: Chronic exposure to a GSMlike signal (mobile phone) does not stimulate the development of DMBA-induced mammary tumors in rats: results of three consecutive studies. Radiat. Res. 157, 183–190. Bastide, M., B. Youbicier-Simo, J. Lebecq, and J. Giaimis, 2002: Toxicologic study of electromagnetic radiation emitted by television and video display screens and cellular telephones on chickens and mice. Indoor Built. Environ. 10, 291–298. Batellier, F., I. Couty, D. Picard, and J. Brillard, 2008: Effects of exposing chicken eggs to a cell phone in “call” position over the entire incubation period. Theriogenology 69, 737– 745. Bogoliubov, V. M., I. D. Frenkel, S. M. Zubkova, I. Z. Liakhovetskii, and Z. A. Sokolova, 1989: Energy and plastic metabolism of the heart muscle in rabbits undergoing thyroid irradiation with decimeter waves. Biulleten’ eksperimental’noi biologii i meditsiny. 107, 570–572. Chemeris, N. K., A. B. Gapeyev, N. P. Sirota, O. Y. Gudkova, A. V. Tankanag, I. V. Konovalov, M. E. Buzoverya, V. G. Suvorov, and V. A. Logunov, 2006: Lack of direct DNA damage in human blood leukocytes and lymphocytes after in vitro exposure to high power microwave pulses. Bioelectromagnetics 27, 197–203. De Gannes, F. P., M. Taxile, S. Duleu, A. Hurtier, E. Haro, M. Geffard, G. Ruffie, B. Billaudel, P. Leveque, P. Dufour, I. Lagroye, and B. Veyret, 2009: A confirmation study of Russian and Ukrainian data on effects of 2450 MHz microwave exposure on immunological processes and teratology in rats. Radiat. Res. 172, 617–624. De Iuliis, G. N., R. J. Newey, B. V. King, and R. J. Aitken, 2009: Mobile phone radiation induces reactive oxygen species production and DNA damage in human spermatozoa in vitro. PLoS ONE 4, e6446. De Pomerai, D. I., B. Smith, A. Dawe, K. North, T. Smith, D. B. Archer, I. R. Duce, D. Jones, and P. M. Candido, 2003: Microwave radiation can alter protein conformation without bulk heating. FEBS Lett. 543, 93–97.
© 2015 Blackwell Verlag GmbH Anat. Histol. Embryol. 45 (2016) 197–208
W. Ye et al.
Del Vecchio, G., A. Giuliani, M. Fernandez, P. Mesirca, F. Bersani, R. Pinto, L. Ardoino, G. A. Lovisolo, L. Giardino, and L. Calza, 2009: Continuous exposure to 900 MHz GSMmodulated EMF alters morphological maturation of neural cells. Neurosci. Lett. 455, 173–177. Diem, E., C. Schwarz, F. Adlkofer, O. Jahn, and H. Rudiger, 2005: Non-thermal DNA breakage by mobile-phone radiation (1800 MHz) in human fibroblasts and in transformed GFSH-R17 rat granulosa cells in vitro. Mutat. Res. 583, 178–183. Garaj-Vrhovac, V., and V. Orescanin, 2009: Assessment of DNA sensitivity in peripheral blood leukocytes after occupational exposure to microwave radiation: the alkaline comet assay and chromatid breakage assay. Cell Biol. Toxicol. 25, 33–43. Garaj-Vrhovac, V., D. Horvat, and Z. Koren, 1991: The relationship between colony-forming ability, chromosome aberrations and incidence of micronuclei in V79 Chinese hamster cells exposed to microwave radiation. Mutat. Res. Lett. 263, 143–149. Garson, O. M., T. L. McRobert, L. J. Campbell, B. A. Hocking, and I. Gordon, 1991: A chromosomal study of workers with long-term exposure to radio-frequency radiation. Med. J. Australia 155, 289–292. Girgert, R., H. Schimming, W. Korner, C. Grundker, and V. Hanf, 2005: Induction of tamoxifen resistance in breast cancer cells by ELF electromagnetic fields. Biochem. Biophys. Res. Commun. 336, 1144–1149. Heikkinen, P., V.-M. Kosma, T. Hongisto, H. Huuskonen, P. Hyysalo, H. Komulainen, T. Kumlin, T. Lahtinen, S. Lang, L. Puranen, and J. Juutilainen, 2001: Effects of mobile phone radiation on X-ray-induced tumorigenesis in mice. Radiat. Res. 156, 775–785. Holmberg, B., 1995: Magnetic-fields and cancer - animal and cellular evidence – an overview. Environ. Health Perspect. 103, 63–67. Hook, G. J., D. R. Spitz, J. E. Sim, R. Higashikubo, J. D. Baty, E. G. Moros, and J. L. Roti, 2004: Evaluation of parameters of oxidative stress after in vitro exposure to FMCW-and CDMA-modulated radiofrequency radiation fields. Radiat. Res. 162, 497–504. ICNIRP, 1998: Guidelines for limiting exposure to time-varying electric, magnetic, and electromagnetic fields (up to 300 GHz). International Commission on Non-Ionizing Radiation Protection. Health Phys. 74, 494– 522. Ilhan, A., A. Gurel, F. Armutcu, S. Kamisli, M. Iraz, O. Akyol, and S. Ozen, 2004: Ginkgo biloba prevents mobile phoneinduced oxidative stress in rat brain. Clin. Chim. Acta 340, 153–162. Irmak, M. K., E. Fadıllıoglu, M. G€ ulecß, H. Erdogan, M. Yagmurca, and O. Akyol, 2002: Effects of electromagnetic radiation from a cellular telephone on the oxidant and antioxidant levels in rabbits. Cell Biochem. Funct. 20, 279– 283.
© 2015 Blackwell Verlag GmbH Anat. Histol. Embryol. 45 (2016) 197–208
Radiation Research of the Mobile Phone
Jauchem, J. R., 1997: Exposure to extremely-low-frequency electromagnetic fields and radiofrequency radiation: cardiovascular effects in humans. Int. Arch. Occup. Environ. Health 70, 9–21. Kakinuma, Y., T. Miyauchi, K. Yuki, N. Murakoshi, K. Goto, and I. Yamaguchi, 2000: Mitochondrial dysfunction of cardiomyocytes causing impairment of cellular energy metabolism induces apoptosis, and concomitant increase in cardiac endothelin-1 expression. J. Cardiovasc. Pharmacol. 36, S201–S204. Kang, K. A., H. C. Lee, J. J. Lee, M. N. Hong, M. J. Park, Y. S. Lee, H. D. Choi, N. Kim, Y. G. Ko, and J. S. Lee, 2014: Effects of combined radiofrequency radiation exposure on levels of reactive oxygen species in neuronal cells. J. Radiat. Res. 55, 265–276. Kim, K. B., K. O. Byun, N. K. Han, Y. G. Ko, H. D. Choi, N. Kim, J. K. Pack, and J. S. Lee, 2010: Two-dimensional electrophoretic analysis of radio frequency radiation-exposed MCF7 breast cancer cells. J. Radiat. Res. 51, 205–213. Koylu, H., H. Mollaoglu, F. Ozguner, M. Nazyrodlu, and N. Delibalp, 2006: Melatonin modulates 900 MHz microwaveinduced lipid peroxidation changes in rat brain. Toxicol. Ind. Health 22, 211–216. Kwee, S., and P. Raskmark, 1998: Changes in cell proliferation due to environmental non-ionizing radiation: 2. Microwave radiation. Bioelectrochem. Bioenerg. 44, 251–255. Lantow, M., J. Schuderer, C. Hartwig, and M. Simko, 2006: Free radical release and HSP70 expression in two human immune-relevant cell lines after exposure to 1800 MHz radiofrequency radiation. Radiat. Res. 165, 88–94. Lee, L., J. Horowitz, and M. Frenneaux, 2004: Metabolic manipulation in ischaemic heart disease, a novel approach to treatment. Eur. Heart J. 25, 634–641. Loscher, W., and M. Mevissen, 1994: Animal studies on the role of 50/60-Hertz magnetic-fields in carcinogenesis. Life Sci. 54, 1531–1543. Nikolova, T., J. Czyz, A. Rolletschek, P. Blyszczuk, J. Fuchs, G. Jovtchev, J. Schuderer, N. Kuster, and A. M. Wobus, 2005: Electromagnetic fields affect transcript levels of apoptosisrelated genes in embryonic stem cell-derived neural progenitor cells. FASEB J. 19, 1686–1688. Oktem, F., F. Ozguner, H. Mollaoglu, A. Koyu, and E. Uz, 2005: Oxidative damage in the kidney induced by 900MHz-emitted mobile phone: protection by melatonin. Arch. Med. Res. 36, 350–355. Paulraj, R., and J. Behari, 2006: Single strand DNA breaks in rat brain cells exposed to microwave radiation. Mutat. Res. 596, 76–80. Robison, J. G., A. R. Pendleton, K. O. Monson, B. K. Murray, and K. L. O’Neill, 2002: Decreased DNA repair rates and protection from heat induced apoptosis mediated by electromagnetic field exposure. Bioelectromagnetics 23, 106–112. Roda, O., I. Garzon, V. Carriel, M. Alaminos, and I. SanchezMontesinos, 2011: Biological effects of low-frequency pulsed magnetic fields on the embryonic central nervous system
207
Radiation Research of the Mobile Phone
development. A histological and histochemical study. Histol. Histopathol. 26, 873. Sakurai, T., T. Kiyokawa, E. Narita, Y. Suzuki, M. Taki, and J. Miyakoshi, 2011: Analysis of gene expression in a humanderived glial cell line exposed to 2.45 GHz continuous radiofrequency electromagnetic fields. J. Radiat. Res. 52, 185–192. Salama, N., T. Kishimoto, and H. Kanayama, 2010: Retracted: effects of exposure to a mobile phone on testicular function and structure in adult rabbit. Int. J. Androl. 33, 88–94. Savitz, D. A., and E. E. Calle, 1987: Leukemia and occupational exposure to electromagnetic fields: review of epidemiologic surveys. J. Occup. Med. 29, 47–51. Sekijima, M., H. Takeda, K. Yasunaga, N. Sakuma, H. Hirose, T. Nojima, and J. Miyakoshi, 2010: 2-GHz band CW and W-CDMA modulated radiofrequency fields have no significant effect on cell proliferation and gene expression profile in human cells. J. Radiat. Res. 51, 277–284. Shirai, T., M. Kawabe, T. Ichihara, O. Fujiwara, M. Taki, S. Watanabe, K. Wake, Y. Yamanaka, K. Imaida, M. Asamoto, and S. Tamano, 2005: Chronic exposure to a 1.439 GHz electromagnetic field used for cellular phones does not promote N-ethylnitrosourea induced central nervous system tumors in F344 rats. Bioelectromagnetics 26, 59–68. Simko, M., C. Hartwig, M. Lantow, M. Lupke, M. O. Mattsson, Q. Rahman, and J. Rollwitz, 2006: Hsp70 expression and free radical release after exposure to non-thermal radiofrequency electromagnetic fields and ultrafine particles in human Mono Mac 6 cells. Toxicol. Lett. 161, 73–82. Sun, L. X., Y. Ke, K. J. Wang, D. Q. Lu, H. J. Hu, X. W. Gao, B. H. Wang, W. Zheng, J. L. Lou, and W. Wu, 2006: Effects of 1.8 GHz radiofrequency field on DNA damage and expression of heat shock protein 70 in human lens epithelial cells. Mutat. Res. 602, 135–142.
208
W. Ye et al.
Tice, R., E. Agurell, D. Anderson, B. Burlinson, A. Hartmann, H. Kobayashi, Y. Miyamae, E. Rojas, J. Ryu, and Y. Sasaki, 2000: Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ. Mol. Mutagen. 35, 206–221. Tsybulin, O., E. Sidorik, S. Kyrylenko, D. Henshel, and I. Yakymenko, 2012: GSM 900 MHz microwave radiation affects embryo development of Japanese quails. Electromagn. Biol. Med. 31, 75–86. Van Leeuwen, G. M. J., J. J. W. Lagendijk, B. Van Leersum, A. P. M. Zwamborn, S. N. Hornsleth, and A. Kotte, 1999: Calculation of change in brain temperatures due to exposure to a mobile phone. Phys. Med. Biol. 44, 2367–2379. Ventura, C., M. Maioli, G. Pintus, G. Gottardi, and F. Bersani, 2000: Elf-pulsed magnetic fields modulate opioid peptide gene expression in myocardial cells. Cardiovasc. Res. 45, 1054–1064. Verschaeve, L., P. Heikkinen, G. Verheyen, U. Van Gorp, F. Boonen, F. Vander Plaetse, A. Maes, T. Kumlin, J. M€aki-Paakkanen, L. Puranen, and J. Juutilainen, 2006: Investigation of co-genotoxic effects of radiofrequency electromagnetic fields in vivo. Radiat. Res. 165, 598–607. Weisbrot, D., H. Lin, L. Ye, M. Blank, and R. Goodman, 2003: Effects of mobile phone radiation on reproduction and development in Drosophila melanogaster. J. Cell. Biochem. 89, 48–55. Zeni, O., R. Di Pietro, G. d’Ambrosio, R. Massa, M. Capri, J. Naarala, J. Juutilainen, and M. R. Scarfı, 2007: Formation of reactive oxygen species in L929 cells after exposure to 900 MHz RF radiation with and without co-exposure to 3chloro-4-(dichloromethyl)-5-hydroxy-2 (5H)-furanone. Radiat. Res. 167, 306–311.
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