Stress-related endocrinological and psychopathological effects of short- and long-term 50 Hz electromagnetic field exposure in rats

Brain Research Bulletin 81 (2010) 92–99

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Stress-related endocrinological and psychopathological effects of short- and long-term 50 Hz electromagnetic field exposure in rats Renáta Szemerszky a , Dóra Zelena b , István Barna b , György Bárdos c,∗ a

Department of Physiology and Neurobiology, Institute of Biology, Eötvös Loránd University, Budapest, Hungary Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest, Hungary c Department of Behavioral Sciences, Institute of Health Promotion and Sport Sciences, Eötvös Loránd University, Budapest, Hungary b

a r t i c l e

i n f o

Article history: Received 10 June 2009 Received in revised form 15 October 2009 Accepted 21 October 2009 Available online 31 October 2009 Keywords: Electromagnetic field HPA axis Corticosterone Anxiety Depression

a b s t r a c t It is believed that different electromagnetic fields do have beneficial and harmful biological effects. The aim of the present work was to study the long-term consequences of 50 Hz electromagnetic field (ELF-EMF) exposure with special focus on the development of chronic stress and stress-induced psychopathology. Adult male Sprague–Dawley rats were exposed to ELF-EMF (50 Hz, 0.5 mT) for 5 days, 8 h daily (short) or for 4–6 weeks, 24 h daily (long). Anxiety was studied in elevated plus maze test, whereas depression-like behavior of the long-treated group was examined in the forced swim test. Some days after behavioral examination, the animals were decapitated among resting conditions and organ weights, blood hormone levels as well as proopiomelanocortin mRNA level from the anterior lobe of the pituitary gland were measured. Both treatments were ineffective on somatic parameters, namely none of the changes characteristic to chronic stress (body weight reduction, thymus involution and adrenal gland hypertrophy) were present. An enhanced blood glucose level was found after prolonged ELF-EMF exposure (p = 0.013). The hormonal stress reaction was similar in control and short-term exposed rats, but significant proopiomelanocortin elevation (p < 0.000) and depressive-like behavior (enhanced floating time; p = 0.006) were found following long-term ELF-EMF exposure. Taken together, long and continuous exposure to relatively high intensity electromagnetic field may count as a mild stress situation and could be a factor in the development of depressive state or metabolic disturbances. Although we should stress that the average intensity of the human exposure is normally much smaller than in the present experiment. © 2009 Elsevier Inc. All rights reserved.

1. Introduction Along technological advance using more and more electrical devices, exposure to extremely low frequency (50 Hz) electromagnetic field (ELF-EMF) has significantly been enhanced in both intensity and duration and seems to affect public and occupational health more and more [36,38]. Contrary to high frequency and high energy ionizing electromagnetic radiation, the harmful features of ELF-EMF have not been proven unambiguously although biological effects were demonstrated in many studies of different sort [24]. 50/60 Hz electromagnetic field is generated by the power lines, transformers and electrical devices induced with current flow, thus it can be found in our living environment, which has raised some concerns about the effects of ELF-EMF on human health [38].

∗ Corresponding author at: Department of Physiology and Neurobiology, Eötvös Loránd University, H-1117 Budapest, Pázmány Péter Sétány 1/C, Hungary. Tel.: +36 1 381 2181; fax: +36 1 381 2182. E-mail address: [email protected] (G. Bárdos). 0361-9230/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.brainresbull.2009.10.015

Over the past decades, experimental data have shown that ELF-EMF can act on the emotional state of people and on the anxiety-related behavior of animals. For example, epidemiological studies suggested an association between chronic ELF-EMF exposure and depression [5,51]. It was also reported that residential exposure to ELF-EMF could increase trait anxiety in women [10]. Moreover, repetitive transcranial magnetic stimulation (rTMS), a magnetic field exposure used for therapeutic purposes, was reported to cause anxiety in normal volunteers [22]. In accordance with this, Isogawa et al. [28,29] observed an anxiogenic effect of rTMS in rats on the elevated plus maze (EPM). Although results from behavioral studies are rather ambiguous, according to some studies, ELF-EMF enhanced the anxiety-like behavior in rats by increasing the time spent with thigmotaxis (tendency to stay close to the sidewall of the open field box), by increasing the frequency of grooming behavior in the open field test [15,38], and by decreasing the open arm entries and the time spent in the open arm or on the central platform of the EPM [15,62]. The precise mechanisms of alterations in the anxiety level caused by ELF-EMF exposure are not fully understood.

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It is well known that activation of the central corticotropinreleasing factor (CRF) system and the glucocorticoid secretion can evoke negative emotional state and can potentiate fear- and anxiety-related behaviors [39]. Therefore, it is reasonable to speculate that elevation in the anxiety level may be attributed to the stimulating effect of ELF-EMF on the hypothalamic–pituitary– adrenal (HPA) system function. To our best knowledge, the effect of ELF-EMF exposure on the HPA axis function has not yet been studied extensively. Some reports suggest that long-term ELF-EMF exposure may elevate the plasma corticosterone level and the mean lipid volume in the zona glomerulosa of adrenal cortex in rodents [18,46]. These data suggest that the ELF-EMF exposure may act as a chronic stressor [18]. According to another study [70], however, chronic ELF-EMF exposure was accompanied by markedly depressed level of circulating corticosterone in young chickens. Based on the above-mentioned studies, we hypothesized that long term, permanent ELF-EMF exposure would result in a chronic stress state accompanied by the permanent activation of HPA axis with consequent anxious and depressive-like behavior. Activation of the HPA axis is a fundamental component of stressadaptation and survival. It leads to an enhanced secretion of CRF from the hypothalamus provoking the splicing of adrenocorticotropin (ACTH) from proopiomelanocortin (POMC) precursor in the anterior lobe of the pituitary gland. ACTH triggers the secretion of glucocorticoids from the adrenal cortex. Overall functioning is controlled by several negative feedback loops (for an overview, see [16,64]). The consequences of the physiologic response are generally adaptive in the short run but can be damaging when stress is chronic and long lasting [21,42,56]. The chronic stressinduced inhibition of weight gain [32,44] can be explained by CRF rises, as CRF is an anorexigenic molecule, therefore it reduces food intake and body weight [14,37,41,71]. The plasma ACTH level was found to be unchanged in different chronic stress situations, but the more proximal level of the axis (POMC mRNA level) showed enhanced activity [72]. In animal models, exposure to chronic stress increases adrenal weight [44,55] due to resting corticosterone/cortisol hypersecretion [2,3,59,71]. Studies have found complex and bi-directional regulatory interactions between the immune and HPA systems [6]. Due to persistent excess of glucocorticoids in chronic stress state, cellular immunity is suppressed [7,19] and thymus is involuted in rats [20,63]. Corticosterone increases plasma glucose level, too; e.g. chronic restraint stress caused permanent increase in fasting basal plasma glucose levels [71]. What is more interesting, stress may induce diabetes mellitus in humans [54,60] or different kinds of stressors could evoke or inhibit type 1 diabetes in different experimental models of the disease [13,26]. Exposure to chronic stress in rodent-models induces several emotional or behavioral changes including anxiety, anhedonia, enhanced fear, and depression-like states [8,9,17,68,69], probably due to neuronal atrophy in the hippocampus [43,67] and to impaired glucocorticoid receptor function in the forebrain [11] caused by the enhanced glucocorticoid secretion. It is not yet clear, however, how mood changes develop in time and how the length of ELF-EMF exposure affects short- and longterm stress adaptation. The aim of our present work was, therefore, to study the consequences of short- and long-term exposure to ELF-EMF (durations of 5 × 8 h and 4–6 weeks, respectively, 50 Hz frequency, 0.5 mT intensity) on anxiety- and depression-related behavior of rats in connection with the development of chronic stress state. Anxiety was studied in EPM test both after shortand long-term EMF exposure, respectively, whereas depression like behavior of the long-exposed group was examined in the forced swim test (FST). Finally, to assess chronic stress-related endocrine changes, changes of the adrenal gland and thymus weights as well as of the body weight of the animals, blood hormone levels of ACTH and corticosterone, POMC mRNA level from the pituitary gland, and

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Fig. 1. Scheme of Exp. 1 (A) and Exp. 2 (B). Abbreviations: EPM = elevated plus maze; Blood A = blood samples from the tail for measuring ACTH and corticosterone level after EPM test; Blood B = blood samples after decapitation for measuring basal ACTH and corticosterone level, blood glucose level and haematocrit; Organ weights = measuring the weight of the thymus and the adrenal gland; Hypophysis = taking out the pituitary gland for the ACTH precursor POMC mRNA level determination. FST = Forced swim test. Numbers indicate the length of the procedure. (C) Helmholtz-coil apparatus for the ELF-EMF exposure with the plastic box containing rats between the coils.

blood glucose and haematocrit levels were measured both after short- and long-term EMF exposure. 2. Materials and methods 2.1. Animals Subjects were male Sprague–Dawley rats weighing 300–370 g in Group 5 × 8 h and 120–140 g in Group Chronic at the start of the experiment (EGIS Pharmaceuticals Budapest, Hungary). Each group consisted of 8 rats, alltogether 32 animals were used. Rats were kept in a 12:12 h day–night schedule (lights on at 7:00 a.m.) under normal laboratory conditions (temperature: 22 ± 2 ◦ C). Standard laboratory chow and tap water were available ad libitum. The experimental design had been approved by the Eötvös Loránd University Animal Use and Care Committee and by the Hungarian National Animal Health Care Authority and was in accordance with regulations set by the European Communities Council Directive of 24 November 1986 (86/609/EEC). 2.2. Apparatus 2.2.1. Device for electromagnetic field exposure ELF-EMF was generated by a standard Helmholtz-coil apparatus. Two round coils were used, spaced apart at a distance equal to their radii (21 cm) on a common axis, with equal currents flowing in the same direction (Fig. 1C). Helmholtz coils provide a fairly homogenous field in the space between them. The coils were constructed of glaze-insulated copper wire (d = 1.4 mm) and had 240 turns (DC resistance was 2.9 ). 50 Hz EMF frequency was generated by sinusoidal current (1.6 A in each coil) at the output of the circuit driven by a 230 V, 180 VA adjustable thoroid-transformer. EMF was measured by a hand-held Electric and Magnetic Field Meter (Maschek-ESM-100) and the value of the magnetic field was fixed at 0.5 ± 0.025 mT. The electric gradient was between 525 and 575 V/m. The ambient background level of the magnetic field was <0.01 mT. Subjects were placed in the center of the Helmholtz-coil apparatus. Experiments were carried out at ambient room temperature and no significant temperature change was detected between the two activated Helmholtz coils (24 ± 0.5 ◦ C).

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Table 1 Changes of somatic and endocrine parameters after ELF-EMF exposure. Experiment 1 Group Control Initial body weight (g) Final body weight (g) Thymus weight (mg) Weight of adrenal gland (mg) Haematocrit Blood glucose (mmol/l)

353.3 383.6 569.1 53.5 48.75 6.06

± ± ± ± ± ±

6.4 7.2 38.3 1.5 1.67 0.14

Experiment 2 Group 5 × 8 h 338.1 373.8 607.3 52.5 48.37 6.13

± ± ± ± ± ±

10.1 10.8 43.1 1.5 2.05 0.17

Significance

Group Control

t(14) = 1.27; p = 0.23 t(14) = 0.76; p = 0.46 t(14) = −0.66; p = 0.52 t(14) = 0.48; p = 0.64 t(14) = 0.15; p = 0.89 t(14) = −0.30; p = 0.78

128.3 341.0 475.8 59.7 52.0 4.97

± ± ± ± ± ±

2.6 7.2 17.9 3.74 3.2 0.4

Group Chronic 128.9 336.4 517.9 54.4 53.2 6.14

± ± ± ± ± ±

2.4 9.4 41.7 3.0 2.8 0.2*

Significance t(14) = −0.17; p = 0.86 t(14) = 0.39; p = 0.70 t(14) = −0.93; p = 0.38 t(14) = 1.12; p = 0.28 t(14) = −0.28; p = 0.78 t(14) = 2.860; p = 0.013; d = 1.43

Values are means ± SEM of 8 rats in each group. * p < 0.05 significant difference versus control group.

2.2.2. Elevated plus maze test Experiments were run based on the method of Pellow et al. [48]. The apparatus consisted of two open arms (10 cm × 50 cm (length)) and two closed arms (10 cm × 50 cm (length) × 40 cm (height)) extending from a common central platform (10 cm × 10 cm). The brightly illuminated maze was made of wood with black floor and walls, elevated to 50 cm above the floor. The intensity of the white light on the surface of the open arms was 640 lx. At the beginning of an experiment the rat was placed on the central platform with the head directed toward the open arm. It was allowed to freely explore the apparatus for 5 min. Behavior was recorded with an overhead video camera. An arm entry was defined as all four legs entered into one of the arms. The number of transitions between the arms, the time spent in the open arms, the ratio of the open/total arm entries and the number of rearings, of head scans (i.e. an animal explores an open arm from the central part with its head) and of the stretched attend postures (i.e. the animal stretches forward and retracts to its original position without stepping forward or moving its hind paws [66]) were measured or calculated. 2.2.3. Forced swim test The forced swim test was performed according to a modified version of the method of Porsolt et al. [52]. A vertical glass cylinder (40 cm high, 20 cm in diameter) was filled with 24 ± 1 ◦ C water to a depth of 30 cm. The water depth was chosen to force the animals to swim or to float without their hind legs touching the bottom. The rats were first subjected to a pre-test by placing them in the cylinder for 15 min. A second 5 min testing took place 24 h after the pre-test. The duration of floating (i.e. only small movements necessary to keep the rat’s head above the water), of swimming (i.e. making active swimming motions, more than necessary to merely keep the head above water) and of struggling (i.e. forceful efforts to get out from the water) were recorded. Water was changed between each rat. The animals were gently dried after removal from the bath and were returned to their home-cage. 2.3. Experimental design (Fig. 1) After 3 days of habituation to the laboratory environment, the animals were put either into a Helmholtz-coil apparatus or into a similar device without EMF (control). A 0.5 mT magnetic induction was applied modeling the human tolerable limit determined by the European Union which is 0.5 mT at work for 8 h (and 0.1 mT at home for 24 h) [27]. Animals were kept in pairs in opaque plastic boxes (35 cm × 35 cm × 17 cm (height)) with a perforated Plexiglas cover and with wood chip bedding. Rats were removed daily from their boxes for cleaning and for replacing food and water. In Experiment 1: rats had been exposed to ELF-EMF for 8 h per day for five consecutive days, i.e. altogether 40 h (Group 5 × 8 h). Tests were performed with a considerable delay (48 h after the last exposure) because possible lasting consequences were studied. First a 5-min long EPM test was carried out. Immediately and 15 min following the EPM test blood samples were collected from the tail of the rats during a short (less than 1 min) restraint for measuring ACTH and corticosterone level (Fig. 1A: ‘Blood A’). Two days later, in a resting state, the animals were decapitated to gauge the basal corticosterone and ACTH levels (Fig. 1A: ‘Blood B’) and the weight of the thymus and adrenal gland. The blood glucose level and haematocrit were also measured. In addition, pituitary gland was taken out and frozen on dry ice in embedding medium and was stored at −70 ◦ C until cutting for the ACTH precursor POMC mRNA level determination. Changes of the body weight of the animals were monitored throughout the electromagnetic treatment, too. In Experiment 2: ELF-EMF exposure was continuous for 6 weeks (Group Chronic). One hour after disrupting EMF exposure at the 4th week, FSTs were carried out, than EMF exposure had been continued. On the 6th week, an EPM test was run to determine anxiety state of the rats, with collecting blood samples for measuring ACTH and corticosterone level (Fig. 1B: ‘Blood A’), then EMF exposure had been continued again. The further experimental procedure – measuring organ weights, basal blood hormone levels (Fig. 1B: ‘Blood B’) and POMC mRNA level – was the same as in Experiment 1.

In all experiments, controls had been treated exactly like the experimental animals except EMF exposure. An extra care was taken to keep them out of any EMF range thus the level of EMF in their boxes was equal to the background field intensity. Therefore, the only difference between our control and exposed groups was the EMF exposure. Previous experiments [72–74] with cage controls have shown that the procedure used in this work could reliably detect differences in both stress reactivity and resting hormonal levels and the latter are barely influenced by the EPM test after a two days recovery period. 2.4. Hormone analysis Plasma was collected in K2 -EDTA containing tubes on ice. After centrifugation, the plasma was stored on −20 ◦ C until hormone measurement. Plasma ACTH was measured with radioimmunoassay (RIA) in 50 ␮l unextracted plasma as described earlier [74]. The intraassay coefficients of variation was 6.4%. Plasma corticosterone was measured from 10 ␮l unextracted plasma with RIA using a specific antiserum developed in the Institute of Experimental Medicine [74]. The intraassay coefficients of variation was 9.6%. All samples from one experiment were run in the same assay. 2.5. In situ hybridization Pituitaries were cut in 12 ␮m sections on a cryostat-microtome. POMC mRNA levels were quantified by riboprobes complementary to the exonic sequences of the POMC gene labeled by 35 S-UTP (the plasmid containing the 1.2 kb template was a generous gift of Dr. J. Eberwine, University of Pennsylvania). The hybridization technique was derived from that described by Simmons [58] and was described in detailes elsewhere [73]. After the hybridization process, slides were exposed to imaging plates for 3 h. The plates were scanned by a fluorescent image analyzer (FLA 3000, Fujifilm, resolution 50 ␮m). Radiograms were evaluated by the ImageJ software (NIH, USA). The boundary of the anterior lobe was outlined and the average grayness value was corrected for the background taken from the neighboring hypothalamic tissue. 2.6. Statistical analysis Data are presented as mean ± SEM. After detecting and excluding outliers with Grubbs’ test, a Kolmogorov–Smirnov test was performed to determine whether data were sampled from a Gaussian distribution. Since all the data series passed the normality test (with the exception of variable ‘Open/total entries’ in EPM test in the Group Chronic), parametric tests were performed afterwards. (‘Open/total entries’ were compared by Mann–Whitney non-parametric analysis.) Corticosterone and ACTH changes were analyzed by a two-way mixed ANOVA (time × EMF-treatment) followed by Bonferroni post hoc tests to compare the means of the control and exposed groups. For all other variables, differences between the two groups were analyzed by unpaired t tests using the GraphPad PRISM 5.00 software package (GraphPad Software, San Diego, CA, USA). If equal variances could not have been assumed, Welch’s corrections were performed. The level of statistical significance was set at p < 0.05 in all statistical analyses. Each group consisted of 8 rats.

3. Results 3.1. Somatic parameters We examined the typical somatic parameters known to be changed in chronic stress situations (Table 1). There was no difference between the initial weight of the control and EMF-exposed groups. During the examination period, all rats gained weight, the magnitude of which was independent from EMF exposure both in

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Fig. 2. HPA-axis changes after short-term ELF-EMF exposure (Exp. 1). The ACTH precursor proopiomelanocortin (POMC) mRNA levels in the anterior lobe of the pituitary gland is presented on part (A). Plasma concentrations of ACTH (B) and corticosterone (C) were measured among resting conditions (basal) and right after performing a 5-min long EPM test (EPM5) as well as 15 min later (EPM20); n = 8, ## p < 0.01 vs. basal levels, ++ p < 0.01 vs. levels at the end of EPM.

the first (5 × 8 h) and the second (chronic exposure for 6 weeks) experiments. There were no significant differences either in thymus involution or in adrenal gland hypertrophy between control and short or long EMF-exposed groups (Table 1). There were no significant differences between control and EMFtreated groups in haematocrit levels, and short-term ELF-EMF exposure has not induced any change in blood glucose levels either (Table 1.). However, basal, unfasting level of blood glucose was significantly elevated after 6 weeks of ELF-EMF exposure (t(14) = 2.860; p = 0.013; d = 1.43). 3.2. Endocrine changes POMC mRNA level in the anterior lobe of the pituitary gland (Figs. 2A and 3A) as well as baseline plasma ACTH (Figs. 2B and 3B) and corticosterone levels (Figs. 2C and 3C) were measured in a resting state of the rats (see Fig. 1A and B: ‘Blood B’). The only difference was found in POMC level, which was elevated in the chronically (6 weeks) exposed group (t(14) = 5.808; p < 0.0001; d = 2.90), but not after short ELF-EMF exposure. EPM test in itself, as a stress situation, induced a significant rise both in ACTH and corticosterone levels (effect of time factor; short

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Fig. 3. HPA-axis changes after long-term ELF-EMF exposure (Exp. 2). The ACTH precursor proopiomelanocortin (POMC) mRNA levels in the anterior lobe of the pituitary gland is presented on part (A). Plasma concentrations of ACTH (B) and corticosterone (C) were measured among resting conditions (basal) and right after performing a 5-min long EPM test (EPM5) as well as 15 min later (EPM20); n = 8, *** p < 0.000 vs. control, ## p < 0.01 vs. basal levels, ++ p < 0.01 vs. levels at the end of EPM.

ELF-EMF: ACTH F(2,28) = 31.4; p < 0.01; corticosterone F(2,28) = 35.5; p < 0.01; long ELF-EMF: ACTH F(2,26) = 30.69; p < 0.000; corticosterone F(2,28) = 17.8; p < 0.01; Figs. 2 and 3) whereas neither the effect of treatment nor its interaction with time were significant in either experiment for any hormone. 3.3. Behavioral tests 3.3.1. Elevated plus maze test The time an animal spend in the open arm of the EPM apparatus reflects its anxiety level. There was no difference in this variable among the control and EMF-exposed groups either after short or long treatment (Figs. 4A and 5A). The locomotion-independent anxiety, calculated from the open/total arm entry ratio (Figs. 4B and 5B) had not shown any effect of short- or long-term ELF-EMF exposure, either. We had also not found differences between control and EMF-exposed groups in the risk assessment behavior reflected by the head scans and stretched attend postures (Figs. 4D, E and 5D, E). ELF-EMF exposure could not induce any changes in locomotion of the animals (estimated by the number of closed arm entries and of rears, see Figs. 4C, F and 5C, F).

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Fig. 4. Performance in the elevated plus maze (EPM) test after short-term (5 × 8 h) ELF-EMF exposure. The time spent in the open arm (A), the locomotion-independent open arm entries (B), the risk assessment behaviors (D and E) and the estimation of the locomotion (C and F) are presented; n = 8: no significant difference was found.

3.3.2. Forced swim test In the classical Porsolt-method, the first day of the test (15 min swimming) is supposed to induce a depression-like state and only the second test (5 min) is used for measurement [52]. However, the chronic (4 wk) ELF-EMF exposure might have induced a depressionlike state per se, therefore, we have analyzed the behavior of the animals already in the first session, too. The most characteristic behavior of an animal in the FST is floating. We could not detect any significant differences between the control and the exposed rats in either variable studied during the first 15 min section (floating time, control: 62.9 ± 4%; EMF-exposed: 57.5 ± 4%). However, the analysis of the second test revealed a significantly higher floating time (Fig. 5G; t(13) = 3.245; p = 0.006; d = 1.72) and a tendency of shorter struggling time (Fig. 5H) in the long-exposed group. 4. Discussion The present study showed that short-term, repeated (5 × 8 h) ELF-EMF exposure did not induce any sign of chronic stress or any symptom of anxiety-like behavior with the examined parameters in rats. Thus, only the effects of the long-term (4–6 weeks) ELF-EMF exposure will be discussed in details. After continuous long-term ELF-EMF treatment, elevated blood glucose levels, elevated POMC mRNA level in the anterior lobe of the pituitary gland, and enhanced susceptibility for depression-like behavior in the FST were detected. Stress-induced psychopathology is a major health concern nowadays. Therefore it was a key question if ELF-EMF exposure could induce anxiety- and depression-like behavioral abnormalities. Contrary to the results of several studies [15,28,29,62], we could not detect any effect of ELF-EMF exposure on anxiety-related behavior in the EPM test. This was in accordance with the possibly underlying stress-related hormonal reactions, too. Namely, neither the elevation of ACTH level nor that of the corticosterone level was larger in the exposed group compared to the control group following the EPM test (i.e. after a stress situation).

A large number of recent publications have confirmed that chronic stress induces a depressive behavioral state in rodents (e.g. [44,68]). In accordance with this, enhanced depression-like behavior (significantly longer floating time and a tendency of shorter struggling time in FST) was detected in the present study. From among the endocrine parameters measured, this behavioral change was only supported by the elevation of the level of ACTH-precursor POMC in the pituitary gland, whereas basal serum ACTH and corticosterone levels remained normal and had not shown significant variability among groups. This discrepancy between POMC and ACTH changes may arise from the fact that actual concentration of ACTH in the plasma is not as optimal marker of chronic stress state as is the gene expression of POMC that increases slowly after permanent stimulation and thus may be the most reliable indicator of the actual state of the HPA axis [1,72]. On the other hand, there are several studies that, similarly to us, have not found elevated levels of basal corticosterone or ACTH after prolonged stress situations, whereas effects of stress were reflected in other parameters, such as depressive state, anhedonia, changes in immune processes, changes in plasma renin activity etc. [4,44,49]. In the study of Silberman et al. [57], chronic mild stress exposure induced an early increase in corticosterone levels that returned to normal values after 3 weeks, and similarly, the study of Zardooz et al. [71] showed that fasting plasma corticosterone concentrations returned to baseline values on the 30th day of the experiment. These results suggest that rats exposed repeatedly to the same stress acquire partial habituation and adaptation [25,34]. It is widely accepted that, through the regulation of glucocorticoid secretion, the HPA axis is a major modulator of immune functions [33] and there is a direct relation between prolonged high glucocorticoid levels and thymus involution as well as adrenal gland hypertrophy. Although these parameters are used as the best somatic markers of chronic stress, we had not found any changes in the weight of thymus or adrenal glands following long-term ELF-EMF exposure, probably because of the lack of long-lasting

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Fig. 5. Effects of long-term (4–6 wks) ELF-EMF exposure on performance in the elevated plus maze (EPM) test and in the forced swim test (FST). Time spent in the open arm (A), locomotion-independent open arm entries (B), risk assessment behaviors (D and E) and estimated amount of locomotion (C and F) on EPM are presented, as well as the comparison of the time spent with floating (G) and struggling (H) are presented during the 5 min FST on the second test. The animals spent the remaining time with swimming; n = 8, ** p < 0.01 vs. control.

elevation of corticosterone secretion. On the other hand, although some data suggests an immunosuppressive effect of different acute or repeated strong stressors [7,19,40], many animal studies have found a higher immune activity after prolonged mild stressful situations [30,31,35,45] which, obviously, did not involve thymus involution. The long-term ELF-EMF exposure might be such a mild stressful situation, too. A large body of animal studies support the notion that stressful situations reliably produce hyperglycemia [61] via the metabolic effects of increased corticosterone secretion. In the present study, elevation of basal blood glucose levels was detected following the presumably stressful long-term ELF-EMF exposure, while the glucocorticoid overexposure was not present. The role of glucocorticoid regulation in gluconeogenesis and in blood glucose level in healthy subjects or in animals was inconclusive in other studies as well [65]. Increased glucose levels observed during stress may be the result of the interaction of two mechanisms, i.e. the hyperglycemic effect of glucocorticoids and catecholamines released by the activation of the HPA-axis and the sympatho-adreno-

medullary system, respectively [12,23,61], or the ␣-adrenergic inhibition of insulin secretion through activation of the sympathoadreno-medullary system [50,53]. Our results suggest the latter mechanism, although only unfasting blood glucose levels were measured and sympathetic activity had not been examined. Results of this study show that continuous long-term (4–6 week) ELF-EMF treatment induced some signs of chronic-stress or HPA-axis activation (elevated blood glucose level, elevated POMC mRNA level and enhanced depression-like behavior in FST), though other markers of chronic stress (elevated basal ACTH and corticosterone secretion, adrenal gland hypertrophy, thymus involution, loss of weight gain and anxiety-like behavior in EPM test) were not observed. These inconsistencies might be attributable to the fact that stress exposure evokes variable changes in several physiological functions dependent on the exact nature of the actual stress situation [47]. Moreover, ELF-EMF exposure may count as a mild stress situation, thus its effects may be weak and uncertain, as well. Our results are in accordance with the recommendations of the European Union, since the tolerable limit of the exposure at

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the workplace (0.5 mT for 8 h long per day) proved to be completely harmless, while the same intensity (which is 5 times higher than the limit for home) continuously and in the long run caused some detectable disturbances. Although our short- and long-term ELF-EMF-treatments are not fully comparable with each other (intermittent vs. continuous), they reflect to the real life situations (workplace vs. all day exposure) which improves the overall external validity of the study. We propose that a certain exposure duration may be important to the accumulation of the effect of EMF exposure, similarly to that demonstrated in the study of Liu et al. [38]. Our results may allow the conclusion that long and continuous exposure to relatively high intensity ELF-EMFs could be a factor in the development of depressive state or metabolic disturbances, although we must stress that the average intensity of the exposure in a home or general workplace setting is only about one hundredth of that applied in the present experiment.

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Conflict of interest

[23]

The authors hereby declare that they have no conflict of interest of any sort.

[24] [25]

Acknowledgements [26]

This work has been supported by grants from the Hungarian National Scientific Research Fund (OTKA) Nos. T 047170, K76880 and NN71629 and an ETT grant 59/2006.

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