DIFFERENTIAL RESPONSE OF MICROWAVE IRRADIATION-INDUCED DIFFERENTIATION AND DAMAGE IN PC12 MUTANT AND PC12 PARENTAL CELLS

Abstract

The increasing use of mobile phone communication has raised concerns about possible health hazard effects of microwave irradiation. We investigated damage and differentiation of PC12 mutant cells (PC12m3 cells) caused by microwave irradiation. The frequency of neurite outgrowth induced by 2.45 GHz (200 W) of microwave irradiation was approximately 10-fold greater than that in non-irradiated control cells. Microwave treatment of PC12m3 cells resulted in an increased level of p38 MAPK activity. Similarly, CREB activity was seen in PC12m3 cells but not in PC12 parental cells. Heat shock treatment at 45°C had a strong toxic effect on PC12 parental cells, whereas PC12m3 cells showed stronger resistance than PC12 parental cells to temperatures of both 45°C and 42°C. Although microwave irradiation at 200 W had no toxic effect on PC12m3 cells, microwave irradiation led to toxicity in PC12 parental cells. Moreover, microwave irradiation at 100 W, rather than having a toxic effect, induced proliferation of PC12m3 cells despite having no effect in PC12 parental cells. These findings indicate that p38 MAPK is responsible for the survival and proliferation of PC12m3 cells and might induce neurite outgrowth via a CREB signaling pathway in PC12m3 cells subjected to microwave irradiation.

Introduction

High-frequency electromagnetic fields (EMFs) with frequencies ranging from 30 kHz to 300 GHz are widely generated by communication systems including broadcasting systems and mobile phones, by industrial processes, and by medical and domestic appliances. A high-frequency EMF produces millimetric or nanometric waves called short waves (frequency of less than 100 MHz) and microwaves (frequency of 900 MHz or higher). Industrial use of radiofrequency (RF) and microwave energy sources (nonionizing, high-frequency electromagnetic radiation) has been increasing on a global scale, and the number of workers being exposed to electromagnetic radiation has correspondingly been increasing¹⁾. Medical use of deep tissue heat produced by microwave radiation is called microwave diathermy²⁾.

Although epidemiological studies have shown no detrimental effects on human health, possible disturbance of cell physiology caused by high-frequency EMFs remains controversial. Accidental exposure to microwaves in industrial settings has led to limb necrosis and systemic effects¹⁾.

The extensive use of mobile phones has been accompanied by public debate about possible adverse effects on human health. The most widely used GSM (global system for mobile communication) cellular phones operate with carrier frequencies in the near-Gigahertz range, which are pulsed at 217 Hz. Exposure of the brain to continuous and pulsed microwaves has been reported to modulate electrophysiological activity in vivo^3,4,5) and in vitro^6,7,8) and to influence neurotransmitter systems^9,10,11) as well as signal transduction pathways^6,7,12).

To clarify the risks of microwave diathermy, we investigated cellular damage and differentiation of PC12 cells caused by microwave irradiation. PC12 cells are derived from a rat pheochromocytoma¹³⁾. These cells have high-affinity receptors for nerve growth factor (NGF), and they cease division in response to NGF and extend long neuronal processes that can support action potentials. PC12 cells have been used as models of neuronal cells maintained in tissue culture. To elucidate the mechanism of neural differentiation in PC12 cells induced by NGF, we developed a novel PC12 mutant cell line (PC12m3) that shows poor outgrowth of neuronal processes (dendrites and axons) despite normal sustained activation of MAP kinase by NGF treatment¹⁴⁾. In PC12m3 cells, outgrowth of neuronal processes is greatly stimulated by various inducers. PC12m3 cells treated with NGF showed enhancement of neurite outgrowth in response to various stimulants, including cAMP, calcimycin, heat shock and microwave irradiation^{14,15,16,17,18,19,20,21)}. The cells also exhibited sustained activation of p38 MAPK induced by various stimulants. In our previous study, we examined the effect of microwave irradiation for neuritogenesis on drug-hypersensitive PC12m3 cells¹⁵⁾. The frequency of neurite outgrowth induced by microwave irradiation was greatly enhanced compared to that in non-irradiated control cells. Incubation of PC12m3 cells with SB203580, a specific inhibitor of p38 MAPK, resulted in marked inhibition of the microwave radiation-induced neurite outgrowth. On the other hand, heat shock treatment at 45°C had a strong toxic effect on PC12m3 cells, whereas microwave treatment had no toxic effect on PC12m3 cells. However, the differences between the sensitivity to microwave irradiation of PC12 parental cells and that of PC12m3 cells were not examined in our previous study.

In this study, we found that microwave irradiation in a non-thermal condition at a dose of 2.45 GHz (200 W) for 30 or 60 min induced enhancement of neurite outgrowth of PC12m3 cells without any damage to the cells. However, the frequency of neurite outgrowth in PC12 parental cells induced by microwave irradiation at 2.45 GHz (200 W) in the presence of NGF was very low. Furthermore, although microwave irradiation had a toxic effect on PC12 parental cells, it was found for the first time that microwave irradiation promoted the growth of PC12m3 cells. The survival rate of PC12m3 cells increased due to the occurrence of mutations in the p38 MAPK pathway, but microwave irradiation to PC12 cells had a toxic effect because the p38 MAPK activity in PC12 cells is weak. We assume that the effect of microwave irradiation is also toxic in the human body because of the same actions as those on PC12 parental cells.

Materials and Methods

Cell culture

NGF (2.5 S) was purchased from Takara (Osaka, Japan). The p38 MAPK inhibitor SB203580 and ERK inhibitor U0126 were purchased from Sigma (St. Louis, MO). PC12 parental and PC12m3 mutant cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 0.35% glucose, 10% horse serum, 5% fetal bovine serum (FBS), and 100 units/ml kanamycin. All cells were grown at 37°C in 5% CO₂.

Determination of neurite outgrowth

Single-cell suspensions of PC12 parental cells and PC12m3 cells were obtained by trituration in DMEM. For experiments on neuritogenesis, the cells were plated in 25 cm² flasks at a density of 2–5 × 10⁵ cells per dish of serum-containing DMEM and then treated with NGF and/or exposed to microwave irradiation for various periods ranging from 10 to 60 min at 37°C using microthermy (OG GIKEN CO LTD., Okayama, Japan) and a cooling system (TOKYO RIKAKIKAI CO, LTD., Tokyo, Japan). After 7 days of incubation, the numbers of neurites were counted and their lengths were measured. Cells possessing at least one neurite with a length at least 1.5-fold greater than the diameter of the cell body were counted as previously described¹⁴⁾. Each value shown is the mean ± S.D. for 200 cells sampled from three independent experiments.

Detection of activated p38 MAPK and CREB

P38 MAPK and CREB activity was determined as described previously²²⁾. Briefly, PC12 parental and PC12 mutant cells were plated at a density of 1 × 10⁶ cells/25 cm² in a flask of serum-containing medium and cultured for 3 days. Then the culture medium was replaced with 0.5% FBS-containing medium and the cells were cultured for a further 48 h. PC12 parental and PC12m3 cells were then exposed to microwave irradiation for 10 to 60 min at 37°C. P38 MAPK and CREB activity in cell lysates was then assayed. The cells were lysed in a lysing buffer. Aliquots of the lysates (10–15 μg) from each sample were fractionated on SDS-10% polyacrylamide gel and transferred to polyvinylidene difluoride membranes. The blots were probed with antibodies specific to phospho-p38 MAPK, phospho-CREB, total p38 MAPK (New England BioLabs, Beverly, MA) at a dilution of 1:1000 in blocking buffer (5% nonfat dry milk) for 12 h at 4°C. The blots were then probed with a secondary antibody, horseradish peroxidase-linked antirabbit IgG, at a dilution of 1:2000 in blocking buffer for 60 min at room temperature. The blots were stained for 1 min using a nucleic acid chemiluminescence reagent (LumiGLO chemiluminescent reagent, Kirkegaard and Perry Laboratories) and exposed to X-ray film.

Survival assay

For cell survival studies, the cells were exposed to a temperature of 42°C or 45°C for various periods ranging from 10 to 60 min or the cells were exposed to microwave at closes of 2.45 GHz (100 W or 200 W) for various periods ranging from 10 to 60 min at 37°C. Survival fractions were determined by a standard colony formation assay. Twenty-five cm² flasks were seeded with an appropriate number of single cells (15,000 to 39,200) so that, accounting for the cloning efficiency (4,515) and toxicity of the particular treatment, 50 to 100 macroscopic colonies would develop when the cultures were fixed and stained, approximately 10 days later. The medium was changed five days after seeding. Three flasks were seeded with cells for each dose or time point, and colonies containing more than 50 cells were scored as survivors.

Statistical analysis

All data are shown as means ± standard deviation (SD). Statistical significance was determined by analysis of variance (ANOVA) using the SPSS 12.0 software package. The level for statistical differences was set at P < 0.05 or 0.01.

Results

In drug-hypersensitive PC12m3 cells treated with NGF, neurite outgrowth is stimulated by various drugs including calcimycin and FK506^14,16) as well as physical stimulations such as heat shock and osmotic shock^18,19,20,21). When cultures of NGF-treated PC12m3 cells were exposed for 1 h to microwave radiation at 2.45 GHz (200 W) under the temperature condition of 37°C, neurite outgrowth was significantly enhanced. However, the frequency of neurite outgrowth in PC12m3 cells induced by NGF alone was low (Fig. 1A). Furthermore, the frequency of neurite outgrowth gradually increased in a dose-dependent manner (Fig. 1B).

Fig. 1.

Results of microwave irradiation for stimulation of neurite outgrowth in PC12m3 cells in the presence of NGF (30 ng/mg). (A) Phase-contrast photomicrographs (× 200) of PC12 parental and PC12m3 cells were taken 7 days after treatment at 2.45 GHz (200 W) for 30 min at 37°C. (B) Frequencies of neurite outgrowth in PC12 parental and PC12m3 cells induced by microwave irradiation at a dose of 2.45 GHz (100 W or 200 W) for the indicated times in the presence of NGF. The frequency of neurite outgrowth induced by microwave irradiation is shown as the relative frequency against the frequency of neurite outgrowth of nontreated control cells with neurites set to 1. Each value is the mean ± S.E.M. for 200 cells sampled from three independent experiments. The data were presented as the mean and error bar (± SEM). *P < 0.05 and **P < 0.01 means statistically significant.

In PC12 parental cells treated with NGF, additional neurite outgrowth was not observed with microwave irradiation at 2.45 GHz (100 W). On the other hand, the frequency of neurite outgrowth in PC12m3 cells stimulated by microwave irradiation at 2.45 GHz (100 W) in the presence of NGF was significantly higher than that in cells treated with NGF alone. The frequency of neurite outgrowth in PC12 parental cells induced by microwave irradiation at 2.45 GHz (200 W) in the presence of NGF was low. However, when cultures of NGF-treated PC12m3 cells were exposed to microwave irradiation at 2.45 GHz (200 W), the frequency of neurite outgrowth was approximately 10-fold greater than that in non-irradiated control cells (Fig. 1B). The frequency of microwave irradiation-induced neurite outgrowth in both PC12 parental cells and PC12m3 cells without NGF was similar to that in NGF-treated cells (data not shown).

Since activation of p38 MAPK has been shown to play an important role in neuronal differentiation in PC12 cells^14,23), we examined whether the ability of microwave irradiation stimulus to induce neurite outgrowth of PC12m3 cells is a reflection of its effect on p38 MAPK activity. When cells were exposed to microwave radiation at 2.45 GHz (200 W) under the temperature condition of 37°C for 10 to 60 min, the extent of microwave radiation-induced phosphorylation of p38 MAPK was significantly greater in PC12m3 cells than in PC12 parental cells (Fig. 2). The level of p38 MAPK activity gradually increased with increase in irradiation time in both PC12m3 and PC12 parental cells. We have recently detected activity of CREB in PC12m3 cells stimulated by high osmolality¹⁹⁾. We therefore examined microwave irradiation-induced CREB activation in PC12 parental cells and PC12m3 cells. Cells were exposed to microwave irradiation at a dose of 2.45 GHz (200 W) for 10 to 60 min at 37°C, and CREB activity was detected by immunoblotting. Activation of CREB was enhanced by microwave irradiation at 2.45 GHz (200 W) for 10 to 60 min at 37°C in PC12m3 cells, but CREB activity was low in PC12 parental cells (Fig. 2).

Fig. 2.

Microwave irradiation-induced activation of p38 MAPK and the transcription factor CREB in PC12m3 cells. 2.45 GHz (200 W) microwave irradiation-induced activation of p38 MAPK and CREB. PC12 parental and PC12m3 cells were exposed to 2.45 GHz (200 W) microwave irradiation at 37°C for the indicated times. After treatment, the cells were lysed, and protein extracts were analyzed by Western blotting using antibodies specific for anti-phospho-p38 MAPK, anti-phospho CREB or anti-p38 MAPK. The data were presented as the mean and error bar (± SEM). **P < 0.01 means statistically significant.

Figure 3A-D shows survival curves of microwave-irradiated PC12 parental and PC12m3 cells and survival curves of heat shock-treated PC12 parental and PC12m3 cells. PC12m3 cells maintained at 37°C were exposed to microwave irradiation at 2.45 GHz or exposed to a temperature of 42°C or 45°C for various periods ranging from 10 to 60 min and then incubated for 10 days to determine colony-forming ability. The results showed that heat shock treatment at 45°C had a toxic effect on PC12m3 cells (Fig. 3D), whereas microwave treatment at 2.45 GHz (200 W) had no toxic effect on PC12m3 cells (Fig. 3B). Although microwave irradiation at 2.45 GHz (100 W) had a toxic effect on PC12 parental cells, it had a proliferative effect rather than a toxic effect on PC12m3 cells (Fig. 3A and 3B). In PC12m3 cells, the frequency of colony formation with microwave irradiation at 2.45 GHz (100 W) for 20 min was approximately 2-fold greater than that in non-irradiated control cells (Fig. 3B). Exposure to heat shock treatment at 45°C exerted a strong toxic effect on PC12 parental cells but only weak effect on PC12m3 cells (Fig. 3C and 3D).

Fig. 3.

Survival curve of microwave-irradiated PC12m3 cells. The cells were irradiated with the indicated dose of microwaves (A and B) or exposed to heat shock (C and D). PC12m3 cells or PC12 parental cells maintained at 37°C were exposed to 2.45 GHz (200 W) microwave irradiation at 37°C for the indicated times (A and B) or were exposed to a temperature of 42 or 45°C for various periods ranging from 10 to 60 min (C and D). The irradiated or heat shock-treated cells were incubated for 10 days until colony staining.

Discussion

Intensive use of mobile or cellular phones in the past 20 years has aroused concern about possible health problems that may be caused by the microwave irradiation that is associated with mobile phones²⁴⁾. The current safety standards for the use of mobile phones take into consideration mainly heating, which is induced by their electromagnetic field²⁵⁾. It has been shown that electromagnetic fields can affect living tissues by levels of energy that are much lower than those causing changes in the temperature of tissues. It has been shown that these temperature-insensitive responses can influence the physiology of cells in culture and in organisms, but whether this effect can cause cell damage in higher organisms is still unknown^26,27,28,29).

Mobile phones transmit electromagnetic waves in all directions, increasing the region of cells within the brain that are at risk for damage by microwave radiation penetrating the skull³⁰⁾. Although microwave radiation can result in thermal damage if energy absorption rates are high, it is more likely that deleterious effects of microwave radiation on cells of the brain are due to non-thermal effects induced by lower intensities of exposure³¹⁾.

We confirmed injury of PC12 parental cells caused by microwave irradiation in a non-thermal condition, but little injury was observed in PC12m3 mutant cells. The results for PC12 parental cells might raise concerns about the influence on the human body.

There have been some reports on the effects of microwave irradiation on animals including fruit flies (Drosophila) and rats. Lee et al.³²⁾ examined the effects of the mobile phone 835 MHz electromagnetic field (EMF) in the Drosophila model system. They found that when flies were exposed to a specific absorption rate (SAR) of 4.0 W/kg, about 50% of the flies survived after 18-h exposure and only 10% of the files survived after 30-h exposure. The EMF exposure activated ERK and JNK signaling but not p38 kinase signaling. It was reported that microwave radiation emitted from a 3G mobile phone induced DNA strand breaks and induced non-thermal activation of hsp27, hsp70 and p38 MAPK, leading to apoptotic cell death in the brains of rats³³⁾. These results indicate that 3G mobile phone radiation affects brain function and causes several neurological disorders. We have found in our research that cultured cells that are subjected to microwave irradiation can avoid effects of the stress by activating various intracellular signal transduction pathways. Since it has been reported that microwave irradiation in animal experiments results in activation of the same intracellular signal transduction pathways as those in experiments using cultured cells, it is thought that results of experiments using cultured cells can be applied to the effects of microwave irradiation in animals. Thus, results of experiments on the effects of stress using cultured cells and animals should be useful for investigating the effects of microwaves in humans. The fact that microwaves have effects in both cells and animals implies that they also have similar effects in humans.

Small animals can escape from heat and toxic substances. Since cultured cells cannot escape from such stresses, they have acquired genes that can resist stresses by mutations. In other words, it is thought that cells have developed systems for countering stresses by using intracellular signal transduction pathways. We have found cells (PC12m3 cells) that show strong resistance to stresses. It is thought that PC12m3 cells have stronger resistance than PC12 cells to stress due to their very high activity of p38 MAPK acquired by mutation, which functions to avoid stress (Fig. 2 and 3).

In studies in which the effects of microwaves on the human body were investigated, it was shown that microwaves induced necrosis in the feet¹⁾ and had adverse effects on cerebrospinal fluid³⁴⁾. In our studies, we found that microwaves acted on growth activity in PC12m3 cells, mutant cells that have mutations in the p38 MAPK pathway and high p38 MAPK activity, whereas microwaves had toxic effects in normal PC12 cells, which have weak p38 MAPK activity (Fig. 2). These findings indicate that microwaves have toxic effects in a human body with normal cells.

We found that p38 MAPK was activated by microwave irradiation and induced neurite outgrowth. We also found that neurite outgrowth induced by microwave irradiation was inhibited by a specific p38 MAPK inhibitor, SB203580, in the presence of NGF¹⁵⁾. These findings indicate that the p38 MAPK pathway plays a key role in neuronal differentiation of PC12 cells. Morook and Nishida showed that activation of p38 MAPK was involved in neuronal differentiation of PC12 cells²³⁾.

We have recently investigated the role of the p38 MAPK pathway in heat shock-induced or osmotic shock-induced neurite outgrowth of PC12m3 cells^18,19). When cultures of PC12m3 cells were exposed to heat or osmotic stress, activity of p38 MAPK increased and neurite outgrowth was greatly enhanced.

We found that both ERK and p38 MAPK in PC12 parental cells were activated by heat shock but that only p38 MAPK in PC12m3 cells was activated by heat shock. Furthermore, the extent of phosphorylation of p38 MAPK induced by heat shock in PC12m3 cells was much greater than that in PC12 parental cells¹⁹⁾. Heat shock-induced neurite extension was inhibited by treatment with the p38 MAPK inhibitor SB203580. These findings indicate that activation of the p38 MAPK pathway is necessary for heat or osmotic shock-induced and microwave irradiation-induced neuronal differentiation of PC12m3 cells. Takeda et al. examined the role of p38 MAPK in apoptosis signal-regulating kinase 1 (ASK1)-induced neurite outgrowth in PC12 cells³⁵⁾. They found that p38 MAPK, but not ERK, was activated by the expression of constitutively active ASK1 in PC12 cells. ASK1-induced neurite outgrowth was strongly inhibited by treatment with the p38 inhibitor SB203580 but not by treatment with an ERK inhibitor, suggesting that activation of p38 MAPK, rather than that of ERK, is required for the neurite outgrowth-inducing activity of ASK1 in PC12 cells. Given that NGF-induced differentiation of PC12 cells requires activation of the ERK pathway and is sensitive to ERK inhibitors, we speculate that a major set of signaling pathways mediating the neurite outgrowth-inducing activity of microwave irradiation, osmotic shock, heat shock and ASK1 differs from that activated in NGF-induced differentiation of PC12 cells.

The results of our present study suggested that the use of PC12m3 cells would enable more sensitive detection of the effects of microwaves since even low doses of microwave radiation such as that from cell phones can induce significant neurite formation and proliferative activity in PC12m3 cells. Since the signal transduction pathways that act on neurite formation and proliferative activity are the same in animals and humans, the use of PC12m3 cells should facilitate examination of the effects of electromagnetic interference including that from cell phones on the human body.

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