Introduction
Nonionizing electromagnetic radiation is energy that is given off from energy sources, including power lines, mobile phones, common electrical devices, and some types of machinery. This type of radiation differs from ionizing radiation, such as gamma rays, X-rays, and ultraviolet light, which exhibit high-frequency waves and have enough energy to liberate an electron from molecules
[1]. Although nonionizing radiation has a lower frequency and is generally considered safe, accumulating evidence suggests that some types of nonionizing electromagnetic radiation have enough energy to harm living tissues. Especially, in recent years, the number of smartphone users has tremendously increased; thus, concerns and debates regarding the effects on human health of nonionizing radiation from smartphones have emerged. Importantly, unless some protective measures, such as hands-free or Bluetooth, are employed, the majority of people speak over smartphones with them near the users' heads. Therefore, it is an urgent issue to elucidate the effect of nonionizing radiation from smartphones or mobile phones on development and brain function.
In this study, to elucidate the effect of chronic exposure to electromagnetic radiation from smartphones, we examined adult progenitor cell proliferation in the hippocampus, as well as special learning ability using a Y maze in mice.
Materials and methods
2.1 Animals
Male and female C57BL/6 mice (8 wk old, Koatec, Kyoungki-do, Korea) were housed at a standard temperature (22 ± 1°C), humidity (50 ± 5%), and in a light-controlled environment (lights on from 8:00 am to 8:00 pm) with ad libitum access to food and water. The experimental protocol was approved by the Institutional Animal Care and Use Committee of the Catholic University of Daegu (IACUC-2012-34).
2.2 Exposure to electromagnetic radiation
Mice were divided into two groups: animals from the exposed group were housed in a cage that was placed on the smartphone (Galaxy K, Samsung Electronics, South Korea) and it was maintained on operating mode throughout the study. To imitate the condition of daily smartphone use in life, we called and let mice hear the sound from the smartphone for 10 minutes a day. In the control group, animals were housed under normal conditions without electromagnetic radiation exposure. Mice were exposed to electromagnetic radiation for 9 weeks, and spontaneous alternation was tested. To verify the effect of recovery following electromagnetic radiation exposure, female mice were further exposed to electromagnetic radiation for 2 more weeks. After the smartphone was removed, female mice were housed under normal conditions for 4 weeks to provide a recovery period, and spontaneous alternation was tested (
Figure 1).
2.3 Spontaneous alternation in a Y maze
A Y maze was made from black Plexiglas and consisted of three arms with an angle of 120° between each arm. Spontaneous alternation consists of sequential entry into all three arms. Percent alternation was calculated by dividing the number of alternations by the number of possible alternations [number of alternation/(number of total arm entries − 2)].
2.4 Injection of 5-bromo-2′-deoxyuridine and immunohistochemistry
To label proliferating cells, mice received an intraperitoneal (i.p.) injection of 5-bromo-2′-deoxyuridine (BrdU; 100 mg/kg, dissolved in saline; Sigma Aldrich, St. Louis, MO, USA) and were sacrificed 1 day later. A BrdU immunohistochemistry method was described by Choi et al
[2]. To label glial fibrillary acidic protein (GFAP) and CD68, sections were blocked with 10% normal goat serum, followed by overnight incubation with a mouse monoclonal anti-GFAP antibody (1:1,000; Millipore, Temecula, CA, USA) or a monoclonal anti-CD68 antibody (1:500; Biolegend, San Diego, CA, USA). After several washes with phosphate buffered saline, sections were incubated (2 h at room temperature) with secondary antibodies conjugated with horseradish peroxidase (1:1,000; Jackson Immunoresearch, West Grove, PA, USA) and developed using diaminobenzidine (0.1%, Sigma Aldrich) and hydrogen proxidase (0.005%, Junsei Chemical, Tokyo, Japan).
2.5 Cell quantitation
The density of BrdU-positive cells was quantitated as described by Choi et al
[2]. Briefly, cells were counted (bilaterally) in three dorsal hippocampal sections (AP coordinate of the first dorsal-most section: –1.40 μm) separated by 160-μm intervals and averaged for each animal. To measure the granule cell layer/subgranular zone area, photomicrographs of sections were captured (10×) and quantitation was performed using Photoshop (Adobe Systems Incorporated, San Jose, CA, USA). The granule cell layers/subgranular zones of both the upper and lower blades were outlined and the area was measured. Volume was counted in three sections using the coordinates described above. The number of cells was expressed as the mean ± standard error of the mean from five mice for each group. Cell counts were analyzed statistically using the Student
t test, and significance was accepted at
p < 0.05.
Results
3.1 The effect of electromagnetic radiation on the Y maze task
First, to elucidate the effect of nonionizing radiation from smartphones on spatial working memory, we measured spontaneous alternations using a Y maze. For this purpose, 4-week-old mice were randomly separated into two groups, and animals from one group were exposed to electromagnetic radiation emitted by a smartphone for 9 weeks (exposed group: 5 males and 8 females). The control group mice (5 males and 10 females) were housed under standard conditions without any electromagnetic radiation from the smartphone. In this experiment, the percent of alternation was not significantly different between the two groups (
Figure 2A). In addition, there was no significant difference in arm entry (
Figure 2B). These data indicate that chronic electromagnetic radiation exposure from smartphones for 9 weeks does not affect spatial working memory in mice.
3.2 The effect of electromagnetic radiation on progenitor cell proliferation in the dentate gyrus
Next, to elucidate whether electromagnetic radiation emitted by a smartphone affects progenitor cell proliferation in the dentate gyrus, male mice from both groups were injected with BrdU (100 mg/kg) 1 day after the Y maze test and transcardially perfused 24 hours later. As shown in
Figure 3, although the density of BrdU in the subgranular zone of the exposed group was slightly higher than that of the control animals, there was no significant difference between the two groups. These data also indicate that exposure to electromagnetic radiation for 9 weeks does not have any significant influence on progenitor cell proliferation in the subgranular zone.
3.3 The effect of electromagnetic radiation on glial reaction in the hippocampus
Next, to identify whether chronic exposure to electromagnetic radiation has an effect on glial reaction immunoreactivity to GFAP, a marker of astrocytes, and CD68, a marker of microglia/macrophages, was examined. For this study, sections from the controls or from the mice exposed to electromagnetic radiation for 9 weeks were stained, and, as shown in
Figure 4, increased immunoreactivity to GFAP was observed in the hippocampus, especially in the CA1 subfield. However, there was no difference in immunoreactivity to CD68 (data not shown).
3.4 The effect of recovery after electromagnetic radiation on the Y maze task
Finally, we tested whether recovery following chronic exposure to electromagnetic radiation affects spatial working memory. For this purpose, female mice tested using the Y maze above were further exposed to electromagnetic radiation emitted from a smartphone for 2 more weeks (total of 11 weeks); then, smartphones were removed and mice were housed under normal conditions for 4 weeks to provide a recovery period. As shown in
Figure 5, the percentage of spontaneous alternation was not significantly different compared with that measured after exposure to electromagnetic radiation for 9 weeks. However, the number of arm entries was significantly increased. These data indicate that chronic exposure to electromagnetic radiation may induce hyperactivity-like behavior later on.
Discussion
At present, people are constrainedly exposed to the electromagnetic radiation given off from the electronic devices, including mobile phones and many types of machinery. As electromagnetic waves have been known to be capable of breaking chemical bonds and harming living tissues, extensive research has been focused on elucidating the impact of electromagnetic radiation on human health. However, in view of the results thus far achieved, it is quite controversial. For example, radiation from mobile phones or electromagnetic fields of generators increased the intracellular concentration of reactive oxygen species, activated apoptotic signaling, and induced marked cell death
3,
4,
5,
6,
7. On the other hand, exposure to electromagnetic fields does not alter apoptosis and even enhances progenitor cell proliferation and its survival in the hippocampus
8,
9,
10. In human studies, as the majority so far has been focused on human health following acute to subchronic exposure to electromagnetic radiation, it is not easy to translate results to the circumstance of smartphone use in real life. Of note is that many people carry smartphones in their pockets all day long and sleep with them next to them or even under the pillow. That being said, it is urgent to evaluate carefully the physiological effect of long-term exposure to the electromagnetic radiation from smartphones. To this end, male and female mice were exposed to the electromagnetic field for 9 weeks and 11 weeks, respectively, and spatial working memory and progenitor cell proliferation in the dentate gyrus were examined.
To assess spatial working memory, we employed a spontaneous alternation test with a Y maze apparatus. In this experiment, exposure to the radiation field for 9 weeks did not significantly change the percentage of spontaneous alternation or the number of arm entries in both male and female animals. In addition, there was no significant difference in progenitor cell proliferation in the dentate gyrus. As adult neurogenesis in the dentate gyrus has been well known to play an important role in spatial working memory, these behavioral and physiological data indicate that chronic exposure to electromagnetic radiation does not affect spatial working memory in mice.
Recently, it was reported that prenatal exposure to electromagnetic radiation resulted in hyperactivity in the open field test or the light-dark box test in rodents
11,
12. In addition, prenatal and, to a lesser degree, postnatal exposure to cell phones is associated with hyperactivity problems around the age of school entry
[13]. These reports led us to test whether recovery after chronic electromagnetic radiation exposure affects spatial working memory and/or animal behavior. To answer these questions, we exposed electromagnetic radiation to female mice for 11 weeks and provided a recovery period under normal housing condition for 4 weeks. To our surprise, although the percentage of spontaneous alternation was not affected, the number of arm entries was significantly increased by providing a recovery for 4 weeks following an 11-week-long period of exposure to a radiation field. These data further support the previous evidence showing that exposure to electromagnetic radiation may be able to induce delayed hyperactivity-like behavior.
In our study, there was no significant difference in progenitor cell proliferation in the hippocampus and in spatial working memory. However, some reports indicate that electromagnetic radiation influences progenitor cell proliferation and/or spatial working memory
14,
15. The reason for the discord is unknown, although it is worth noting that we provided exposure to electromagnetic radiation for a relatively long term. Interestingly, recent data indicate that although electromagnetic fields influence learning and memory in rodents, the animals can adapt to long-term exposure
[16]. In addition, long-term whole-body exposure to an electromagnetic field from a mobile phone does not cause any adverse effects on memory function and development
[17]. Therefore, it is tempting to hypothesize that our nervous system can adapt to long-term exposure to electromagnetic radiation from smartphones. However, we cannot exclude the possibility that acute whole-body exposure to electromagnetic radiation changes working memory and/or hippocampal progenitor cell proliferation. Further study will be needed to elucidate this hypothesis.
In summary, we provided herein further solid evidence supporting the hypothesis that chronic exposure to electromagnetic radiation may induce delayed hyperactivity-like behavior without affecting spatial working memory and hippocampal progenitor cell proliferation.
