http://informahealthcare.com/ebm ISSN: 1536-8378 (print), 1536-8386 (electronic) Electromagn Biol Med, Early Online: 1–10 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/15368378.2014.987923

ORIGINAL ARTICLE

The developmental effects of extremely low frequency electric fields on visual and somatosensory evoked potentials in adult rats Deniz Kantar Gok1, Deniz Akpinar1, Enis Hidisoglu1, Sukru Ozen2, Aysel Agar3, and Piraye Yargicoglu1 Department of Biophysics, Faculty of Medicine, 2Department of Electrical and Electronics Engineering, Engineering Faculty, and 3Department of Physiology, Faculty of Medicine, Akdeniz University, Antalya, Turkey

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Abstract

Keywords

The purpose of our study was to investigate the developmental effects of extremely low frequency electric fields (ELF-EFs) on visual evoked potentials (VEPs) and somatosensoryevoked potentials (SEPs) and to examine the relationship between lipid peroxidation and changes of these potentials. In this context, thiobarbituric acid reactive substances (TBARS) levels were determined as an indicator of lipid peroxidation. Wistar albino female rats were divided into four groups; Control (C), gestational (prenatal) exposure (Pr), gestational+ postnatal exposure (PP) and postnatal exposure (Po) groups. Pregnant rats of Pr and PP groups were exposed to 50 Hz electric field (EF) (12 kV/m; 1 h/day), while those of C and Po groups were placed in an inactive system during pregnancy. Following parturition, rats of PP and Po groups were exposed to ELF-EFs whereas rats of C and Pr groups were kept under the same experimental conditions without being exposed to any EF during 68 days. On postnatal day 90, rats were prepared for VEP and SEP recordings. The latencies of VEP components in all experimental groups were significantly prolonged versus C group. For SEPs, all components of PP group, P2, N2 components of Pr group and P1, P2, N2 components of Po group were delayed versus C group. As brain TBARS levels were significantly increased in Pr and Po groups, retina TBARS levels were significantly elevated in all experimental groups versus C group. In conclusion, alterations seen in evoked potentials, at least partly, could be explained by lipid peroxidation in the retina and brain.

Developmental period, electric field, lipid peroxidation, somatosensory evoked potentials, visual evoked potentials

Introduction Most public exposure to extremely low frequency electric fields (ELF-EFs) comes from electrical appliances, household wiring and alternating current (AC) transmission and distribution lines. So, people have been constantly exposed to ELF electric and magnetic fields in different intensities, exposure periods and directions. One of the greatest sources of ELFEFs exposure is transformers and power lines, which produce higher levels of field strength in comparison with environmental field strength. Electric field (EF) strength may be as high as 12 kV/m around AC transmission lines and 16 kV/m around electricity generating stations (Kheifets et al., 2010; Valberg et al., 1997). It is still uncertain whether ELF-EF may constitute a risk to human health at current pollution levels. However, previous studies suggest that there is a possible association between ELF exposure and increased risk of cardiovascular disease, cancers and neurodegenerative disorders (Guler et al., 2006, 2007).

Address correspondence to Piraye Yargicoglu, Department of Biophysics, Faculty of Medicine, Akdeniz University, Arapsuyu, 07070 Antalya, Turkey. Tel: 0 090-242-2496906 (Work). Fax: 0 090242-2274495. E-mail: [email protected]

History Received 15 July 2014 Revised 4 November 2014 Accepted 11 November 2014 Published online 11 December 2014

The most important effect of ELF-EFs has been claimed to be production of free radicals that may lead to several diseases. In this manner, several biochemical studies indicated that ELF- EFs caused changes in lipid peroxidation (Benov et al., 1994; Guler et al., 2006; Seyhan and Guler, 2006; Watanabe et al., 1997; Yokus et al., 2005) and antioxidant enzymes (Bediz et al., 2006; Guler et al., 2006; Harakawa et al., 2005; Seyhan and Guler, 2006) of different tissues and plasma. The deleterious effects of an imbalance between free radical production and the available antioxidant defense capacity, termed oxidative stress has been implicated in the harmful effects of ELF-EFs. Since the retina and brain tissues include high content of polyunsaturated fatty acids, free radical reactions produce marked damage to the structure and function of cell membranes in these tissues (Jain et al., 1991; Matsumoto et al., 1999). Thus, it could be expected that ELFEFs induced lipid peroxidation may cause alterations in brain and retina functions. Therefore, in the present study, developmental effects of ELF-EFs exposure on visual and somatosensory systems were assessed by means of visual evoked potentials (VEPs) and somatosensory evoked potentials (SEPs), respectively. Visual evoked potentials (VEPs) is widely used to investigate the physiology and pathophysiology of the visual

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system, including the visual pathway and the visual cortex. Electrophysiologic recording of these potentials provide a powerful objective tool for identifying the earliest alterations in the visual system (Celesia, 1984; Chiappa and Ropper, 1982; Hudnell and Boyes, 1991; Lehman and Harrison, 2002) are often employed for clinical assessment (Chiappa, 1983; Halliday et al., 1972). VEPs are shown to be a sensitive method detecting the involvement of optic pathways in optic neuritis, ischemic optic neuropathy and demyelinating diseases (Chiappa, 1983; Halliday et al., 1972) and sensitive indicators of the neurotoxic effects caused by a broad range of compounds (Otto and Hudnell, 1993; Otto et al., 1988). Early components of the VEPs reflect the integrity of the optic nerve (Holder, 2004), whereas latter components reflect processing by higher cortical centers (Aminoff and Goodin, 1994; Tobimatsu and Celesia, 2006). Furthermore, they have been extensively used in pharmacological experiments and in assessing the neuroprotective effects of compounds, as it provides a means of monitoring neural activity and sensory processing in vivo (Iwamura et al., 2003). In the present paper, we recorded the flash-evoked potentials (FEPs), which is a complex electrical response of neural pathways that are activated during the photic stimulation. It can be easily recorded from both cortical and subcortical sites in rodents (Hudetz et al., 2009; Mazzucchelli et al., 1995; Onofrj et al., 1985; Ridder and Nusinowitz, 2006; Strain and Tedford, 1993) even in animal model of diseases, such as central nervous system myelin pathologies (Gambi et al., 1996; Lehman and Harrison, 2002). In the current study, we also recorded SEPs which are used extensively to evaluate afferent pathways following electrical stimulation of peripheral nerves (Cracco, 1973; Desmedt and Cheron, 1980). The components of SEP waves are characterized by the latency, reflecting the conduction velocity of different stages of afferent pathway and, the amplitude, corresponding to the postsynaptic response to the quantity of sensory input (Canu et al., 2003; Herr et al., 2007; Shapiro, 2002). The recording of these potentials is now a well established technique and has assumed a valuable role in the diagnosis, prognosis and monitoring of a number of disorders of the nervous system, such as neuropathy, cerebral dysfunction and complications caused by neurotoxic agents (Araki and Murata, 1993; Chiappa, 1983). In a number of studies, SEP values have been correlated with neurological deficit scores (Cracco, 1973; Dorfman et al., 1980). Power lines cause a greater risk for people living around transmission lines and electricity generating stations due to an increased use of electromagnetic energy together with the distorted urbanization in developing countries. So this has raised public health concerns and accelerated research to identify possible biological effects associated with exposure to ELF fields which is produced by power lines (Repacholi and Greenebaum, 1999; Valberg et al., 1997). So in our earlier studies (Akpinar et al., 2012; Gok et al., 2014) EF at 12 kV/m (around transmission lines) and 18 kV/m strengths (around electricity generating stations) were used to investigate biochemical and electrophysiological alterations in the adult central nervous system. Hence, based on earlier investigations (Akpinar et al., 2012; Benov et al., 1994; Cossarizza et al., 1993; Gok et al., 2014; Margonato et al., 1993;

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Marino et al., 1986) EF at 12 kV/m was used in the present study to investigate the effects of ELF-EFs on VEPs and SEPs recorded from developing rats. On the other hand, the effects of magnetic component of ELF electromagnetic field on biological systems have been reported in many researches (Graham et al., 1999; Kheifets et al., 2010; Lupke et al., 2004; Lyskov et al., 1993; Rollwitz et al., 2004; Simko´ et al., 2001), but there are limited data on brain tissues related to the effect of pure ELF-EF without magnetic field. Hence, 12 kV/m field exposure similar to those to which we are exposed around power lines was used in pre and post-natal periods of developing rats. There are a limited number of studies about the effects of ELF-EFs on brain. Few electrophysiological studies of animal nervous system activity have been reported; however, and surprisingly little is known about exposure effects on electroencephalographic EEG measures of sensory functions. Previous investigations (Benov et al., 1994; Cossarizza et al., 1993; Margonato et al., 1993; Marino et al., 1986) indicated that an increment of strength and duration of ELF-EFs had greater effects on living organisms. Consistent with this point, in our previous study (Akpinar et al., 2012; Gok et al., 2014), we also showed that when the intensity of the ELF-EFs was increased, lipid peroxidation increased proportionally. Additionally, our results demonstrated that ELF-EFs caused prolongation of VEPs resulted from an increased peroxidation. Hence, based on earlier investigations (Akpinar et al., 2012; Benov et al., 1994; Cossarizza et al., 1993; Margonato et al., 1993; Marino et al., 1986), electric field (EF) at 12 kV/m was used in the present study to investigate the effects of ELF-EFs on animal development. To date there have been no studies investigating the developmental effects of ELF-EFs exposure on VEPs and SEPs. Therefore, the aims of the present study are to further investigate the developmental effect of ELF-EFs exposure on VEPs and SEPs and to elucidate the role of lipid peroxidation in the alterations of these potentials. In this context, thiobarbituric acid reactive substances (TBARS) were used as an indicator of lipid peroxidation.

Materials and methods Animals All experimental protocols conducted on rats were performed in accordance with the standards established by the Institutional Animal Care and Use Committee at Akdeniz University Medical School. Animals were maintained at 12 h light-dark cycles and a constant temperature of 23 ± 1  C at all times. The experiments were performed between 09:00 am and 05:00 pm. In our study, female Wistar albino rats aged four months were mated (two females with each male). Pregnancy was determined by vaginal smear test. Pregnant rats were removed and kept in separate cages. They were divided into four groups as follows: control (C; sham-exposed), prenatal (gestational) exposure (Pr), postnatal exposure (Po), and prenatal + postnatal exposure (PP) groups. Pregnant rats of Pr and PP groups were exposed to EF, while pregnant rats of C and Po groups were placed in an inactive system for the same period of time. Following parturition, rats of Po and PP

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groups were exposed to EF whereas rats of C and Pr groups were placed in an inactive system during 3 months. At the end of 22 days (postnatal day (PND) 22), female rats were separated from their mothers. The age of rats was recorded as zero day on the day of birth. Each group included 10 female rats which were taken from 10 mothers. Experimental groups were exposed to 50 Hz EF at 12 kV/m intensity (1 h/day).

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Electric field exposure system Parallel plate capacitor was used to generate EF. Custommade parallel copper plates (50  80 cm) were plated with zinc (2 mm thickness). In order to produce uniform EF, the corners of parallel plates were rounded, plates were placed upright on wooden stands and positioned parallel to each other. Cables were connected to the center of the plates on their outer surfaces to preserve EF homogeneity. Plastic cage (425  265 mm  185 mm) placed between plates. Four animals in one cage were exposed at one time. Plastic cage was suitable for free movement of rats and therefore homogenous shielding of each other (Aydin et al., 2006). The cage was placed in the center between the two plates and equal distance from the plates. The EF strength was calculated according to the equation E ¼ V/d, where V is electric potential between the plates, d is the distance, and E is the EF strength in volt/m. A custom-made step-up transformer rated 220 Vrms/6000 Vrms was used for the 50 Hz AC EF. The plates were spaced at 50 cm in distance for 12 kV/m EF calculated via the equation above. Max 3000 TRMS Multimeter (Chauvin Arnoux, Paris, France) was used for voltage measurements. Primary and secondary voltage of the source was 218.5– 226.5 V, and 5850–5945 V, respectively. Electric field strength for 50 cm distances between the plates were in the voltage range was 11,700–11,890 V/m. Digital Gauss/Tesla Meter (Unilab, Blackburn, UK) was used to test the purity of EF from background magnetic fields. Maximum magnetic field density was 0.1 mT. Uniformity and homogeneity of the EF was tested by using HI-3804 Electromagnetic Field Survey Meter-Industrial Compliance Meter and its probe (Holaday Industries, Maintan, UK). Maximum variation was less than 1%. VEP recordings On postnatal day 90, rats were deprived of food for 24 h and then prepared for VEP recording. Visual evoked potentials were recorded between 09:00 am and 02:00 pm. As reported in our earlier studies (Agar et al., 2000; Akpinar et al., 2007; Aydin et al., 2005; Derin et al., 2009; Yargicoglu et al., 1999, 2003), VEPs were recorded with stainless steel subdermal electrodes (NE 223 S, Nihon Kohden, Tokyo, Japan) under ether anesthesia between 09:00 am and 02:00 pm. The reference and active electrodes were placed 0.5 cm in front of and behind bregma, respectively. The active electrode was also placed 0.4 cm lateral to the midline over area 17 of visual cortex. A ground electrode was placed on the animal’s tail. After 5 min of dark adaptation, a photic stimulator (NovaStrobe AB, Biopac System, Santa Barbara, CA) at the lowest strength setting was used to provide the flash stimulus at a distance of 15 cm, which allowed lighting of the entire optic papilla from the temporal visual field. Repetition rate of flash

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stimulus was 1 Hz and flash energy was 0.1 J. The estimate flash duration was 10 ms. Visual evoked potential recordings from both right and left eye were obtained, and throughout the experiments the eye not under investigation was occluded by appropriate black carbon paper and cotton. Body temperature was maintained between 37.5 and 38  C (Hetzler et al., 1988) and monitored via a heating pad and a rectal thermometer probe (Homoeothermic Blanket Control Unit, Harvard Apparatus, Edenbridge, UK). The averaging of 100 responses was accomplished with the averager of Biopac MP100 data acquisition equipment (Biopac System, Santa Barbara, CA). Analysis time was 300 ms. The frequency bandwidth of the amplifier was 1–100 Hz. The gain was selected to be 20 mV/div. The sampling rate of the VEP recording was 2000 samples/s. The microprocessor was programmed to reject any sweeps contaminated with larger artifacts, and at least two averages were obtained to ensure response reproducibility. Peak latencies of the components were measured from the stimulus artifact to the peak in milliseconds. Amplitudes were measured as the voltage between successive peaks. Measurements were made on three positive (P1, P2, P3) and two negative (N1, N2) potentials which are seen in all groups. SEP recordings On postnatal day 90, rats were deprived of food for 24 h and then SEPs were recorded. SEPs were recorded with stainless steel subdermal electrodes (Medelec 017K024, Medelec Manor Way, Old Woking Surrey, UK) under ether anesthesia. The active electrode was placed over the left somatosensory area of the cerebral cortex (0.4 cm to the left of bregma); the reference was 1.0 cm anterior to bregma on the midline. A ground electrode was placed on the animal’s tail (Hall & Lindholm, 1974; Kanda et al., 1986; Kucukatay et al., 2003). SEPs were recorded using Biopac MP100 data acquisition equipment (BiopacSystem, Inc.) The electrical stimulus was a square-wave, constant-voltage impulse delivered at a rate of 1/s transcutaneously to the right posterior tibial nerve at the ankle. The stimulus duration was 0.5 ms, at an intensity sufficient to the produce a definite twitch of the big toe. Analysis time was set to 150 ms, the sampling rate was 1000 Hz, and the frequency bandwidth of the amplifier was 1–3000 Hz. The gain was selected as 20 mV/div. The body temperature of rats was maintained between 37 and 37.5  C using a heating pad during the SEP recording (Panjwani et al., 1991). Two hundred responses were averaged. Sweeps contaminated with large artifacts were rejected by the computer. To ensure the response reproducibility, at least two averages were obtained. Peak latencies of the components were measured from the stimulus artifact to the peak in ms. Amplitudes were measured as the voltage (mV) between two successive peaks. Biochemical investigations After recordings, animals were sacrificed the next day at the same time interval (09:00 am and 02:00 pm). Animals were anesthetized with the diethyl ether and sacrificed by exsanguination via cardiac puncture. Brain and retina tissues were removed immediately. The isolated retina and brain tissues

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were placed in 2 ml and 4 ml of 50 mM potassium phosphate buffer, pH 7.4 at 0–4  C. All tissues were rapidly sonicated in a thermally regulated sonicator (Branson Sonifier 250, G. Heinemann Ultraschall- und Labortechnik, Germany) for 1 min. The sonicated samples were stored frozen at 80  C until assay determinations. Sonicated samples were centrifuged at 14,000 g for 10 min at 4  C in an eppendorf microcentrifuge (Biofuge 15R, Heraeus Sepatech, Osterode, Germany). The supernatant of centrifuged samples was used for the assay of TBARS measurements.

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TBARS assay Levels of TBARS were measured by a fluorimetric method described by Wasowicz et al. (1993), using 1,1,3,3-tetraethoxypropane as a standard. Tissue samples (50 ml) were introduced into a tube containing 29 mmol/l thiobarbituric acid (TBA) in acetic acid (8.75 mol/l), samples were placed in a water bath and heated for 1 h at 95–100  C. After the samples were cooled, 25 ml of 5 M HCl was added and the reaction mixture was extracted by agitation for 5 min with 3.5 ml n-butanol. After centrifugation, the butanol phase was separated and the fluorescence of the butanol extract was measured in a spectrofluorometer (Shimadzu RF-5500, Kyoto, Japan) using wavelengths of 525 nm for excitation, and 547 nm for emission. The results are given as mmol/g protein. Determination of protein Protein concentrations in brain tissues were spectrophotometrically measured (Shimadzu RF-5500, Kyoto, Japan) by a protein assay reagent kit (Pierce, Rockford, IL) via a modified Bradford method (Bradford, 1976). Bovine serum albumin was used as a standard. Statistical analysis The statistical analysis of the obtained data was performed by SPSS (SPSS 18.0, SPSS Inc., Chicago, IL) software for

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Windows. Statistical comparisons between groups for latencies and amplitudes of VEPs, SEPs and TBARS levels were performed by using the analysis of variance test (ANOVA) and post-hoc Tukey’s test. Results are expressed as mean ± standard deviation (SD). Significance levels were set at p50.05.

Results The general health of the animals, such as grooming, appearance and behavioral status did not appear significantly different. No significant difference could be observed in the body weight change among different experimental groups. Representative waveforms of VEPs for the all groups are presented in Figure 1. Measurement were made on three positive and two negative potentials, which were seen in all of the groups. We did not find significant differences in latencies and amplitudes between the right and left eyes but between groups. Therefore, based on the analysis, data obtained from stimulating each eye were averaged. The means and standard deviations of peak latencies and peak-to-peak amplitudes of VEP components (mean of both eyes) of the four groups are shown in Tables 1 and 2. Analysis of data revealed that there was a statistically significant difference between groups for the mean latencies of P1 (F(3,36) ¼ 21.81, p50.001), N1 (F(3,36) ¼ 10.80, p50.001), P2 (F(3,36) ¼ 5.53, p50.01), N2 (F(3,36) ¼ 5.99, p50.01) and P3 (F(3,36) ¼ 12.75, p50.001) components. Tukey’s post-hoc analysis showed that all components of Pr, Po and PP groups were significantly increased with respect to the C group (Table 1). As seen in Table 2, no significant difference was observed in the recorded amplitudes among the different experimental groups. Characteristic waveforms of SEPs for all groups are presented in Figure 2. In all groups, two positive (P1, P2) and two negative (N1, N2) components were used for the analysis. The means and standard deviations of peak latencies of each SEP component recorded from all experimental groups are shown in Table 3. Analysis of data showed that there was a statistically significant difference between groups

Figure 1. Representative waveforms of VEPs in control and all experimental groups. The two tracings in each panel are the overlay of representative VEP recordings from both the left and right eye. Three positive (P1, P2, P3) and two negative (N1, N2).

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Table 1. The means and standard deviations of peak latencies of VEP components in all experimental groups. Groups C Pr Po PP

P1 (ms)

N1 (ms)

P2 (ms)

N2 (ms)

P3 (ms)

20.05 ± 1.53 24.49 ± 2.08*** 25.17 ± 2.29*** 26.14 ± 1.47***

31.96 ± 2.60 38.03 ± 2.84*** 39.68 ± 2.29*** 38.08 ± 1.52***

48.68 ± 2.63 54.35 ± 3.10* 56.03 ± 2.67** 53.31 ± 2.80*

70.16 ± 2.63 74.75 ± 2.90* 74.30 ± 2.94** 73.91 ± 3.35**

89.98 ± 1.99 96.61 ± 2.95** 96.48 ± 2.25** 101.9 ± 2.99***

The results are presented as mean ± SD, n ¼ 10 for each group. *p50.05, **p50.01, ***p50.001 versus control groups.

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Table 2. The means and standard deviations of peak-to-peak amplitudes of VEPs in all experimental groups. Groups

P1N1 (mV)

N1P2 (mV)

P2N2 (mV)

N2P3 (mV)

C Pr Po PP

7.02 ± 1.40 6.72 ± 2.90 8.81 ± 3.84 7.10 ± 2.25

8.60 ± 1.42 8.82 ± 3.75 8.98 ± 2.55 8.58 ± 2.84

8.55 ± 3.40 8.06 ± 2.71 8.84 ± 2.64 8.46 ± 2.60

8.12 ± 3.53 8.63 ± 4.84 8.76 ± 4.47 7.47 ± 3.08

for the mean latencies of P1 (F(3,33) ¼ 11.58, p50.001), N1 (F(3,33) ¼ 7.00, p50.01), P2 (F(3,33) ¼ 8.22, p50.001) and N2 (F(3,33) ¼ 18.07, p50.001) components. It was seen that P2, N2 latencies of the Pr group and P1, P2 and N2 latencies of the Po group were significantly longer than the C group. While, all SEP components of the PP group were delayed in comparison with the C group, P2 and N2 latencies were prolonged versus the Pr group. Table 4 shows the mean SEP amplitude changes of each group over the course of the study. Analysis of the amplitudes demonstrated that there was a significant difference between groups for amplitudes of P1N1 (F(3,30) ¼ 4.88, p50.01), P2N2 (F(3,29) ¼ 3.69, p50.05) components, but no significant difference for amplitude of N1P2 components (F(3,30) ¼ 1.86, not significant). No significant differences were observed when the peak-to-peak amplitude values of Po and Pr groups were compared with the C group. P1N1 and P2N2 amplitudes of PP group were significantly increased compared with the C group. P1N1 amplitude was also higher in this group than those of other groups. Lipid peroxidation was measured as the amount of TBARS. TBARS values of the brain tissues of control and experimental groups are given in Figure 3. There was a statistically significant difference between groups (F(3,36) ¼ 17.41, p50.001). Brain TBARS levels were statistically significantly increased in the Pr and Po groups with respect to the C group. However, no significant difference was observed in brain TBARS levels in the PP group versus the control group. TBARS levels of the retina tissues from control and experimental groups are shown in Figure 4. There was a statistically significant difference between groups (F(3,33) ¼ 27.09, p50.001). Retina TBARS levels were statistically significantly elevated in the Pr, Po and PP groups with respect to the C group.

Discussion The purpose of the study was to examine the developmental effects of ELF-EFs exposure on the components of VEPs and SEPs and elucidate the role of oxidant tissue injury on these

potentials. To our knowledge, this is the first report aimed at assessing the developmental effect of 50 Hz EFs on the generation of these potentials. Several studies have addressed possible effects of ELF-EFs on reproduction and development in rats, using field strengths from 10 to 150 kV/m (Rommereim et al., 1987, 1989, 1990; Seto et al., 1984; Sikov et al., 1984). In general, the studies involved large group sizes and exposure over multiple generations did not report any consistent adverse effects. Malformations were increased and fertility was decreased in one experiment (Rommereim et al., 1987). These effects were not confirmed in the second experiment of the same study or in further studies of the same group. Exposure to 50 Hz EFs at 50 kV/m did not induce significant effects on growth and development in 8-week-old male rats exposed 8 h/day for 4 weeks or rabbits exposed 16 h/day from the last 2 weeks of gestation to 6 weeks after birth (Portet and Cabanes, 1988). Consistent with these data, we also not observed any development changes in rats of experimental groups exposed to 50 Hz EF at 12 kV/m. On the other hand, Marino et al. (1976) reported that mice exposed to 60 Hz, 15 kV/m vertical EF showed decreased body weight and increased mortality over all three generations of their study. Taken as a whole, experimental studies have not found strong adverse effects on development. However, additional studies might increase our understanding of sensitivity of biological organism to ELF-EFs. The amplitudes and latencies of VEPs (Dyer et al., 1987; Sisson and Siegel, 1989) and SEP components (Dowman and Rosenfeld, 1985; Kanda et al., 1986; Todorova et al., 1992) found in our laboratory are generally in agreement with those found in other laboratories. In the present study, our data indicate that the mean latencies of all VEP components of experimental groups were significantly prolonged with respect to the C group. In view of the fact that VEP is a sensitive and reliable method to evaluate the earliest changes in the visual system (Chiappa and Ropper, 1982; Halliday, 1976; Hudnell and Boyes, 1991; Lehman and Harrison, 2002), our results probably indicate that ELF-EFs affects the visual system in all developmental periods. On the other hand, prolongation of all SEP components in the PP group, while some components in Pr (P2 and N2) and Po (P1, P2 and N2) groups also showed that ELF-EFs exposure caused changes in somatosensory pathways. Our data clearly indicated that ELFEF caused similar effect as seen in the prolongation of some components of these potentials. These results could be partially attributed to delayed transmission of sensory information to appropriate cortical centers by ELF exposure. In a previous study, Jaffe et al. (1980) reported a detailed analysis of VEP component latencies, peak-to-peak

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Figure 2. Representative waveforms of SEPs in control and all experimental groups. Two positive (P1, P2) and two negative (N1, N2).

Table 3. The means and standard deviations of peak latencies of SEP components in all experimental groups. Groups C Pr Po PP

P1 (ms)

N1 (ms)

22.5 ± 2.70 25.5 ± 1.57 27.0 ± 1.95*** 26.9 ± 2.21***

40.2 ± 2.56 44. 3 ± 2.20 44.6 ± 1.73 50.5 ± 2.64***

P2 (ms) 63.3 ± 1.13 67.4 ± 1.70* 69.6 ± 1.79* 78.5 ± 2.80***,

N2 (ms)

x

82.6 ± 1.94 90.2 ± 2.75* 100.3 ± 1.57*** 106.59 ± 2.65***,

xx

The results are presented as mean ± SD, n ¼ 10 for each group. *p50.05, **p50.01, ***p50.001 versus control group; xp50.05. xxp50.01 versus prenatal group. Table 4. The means and standard deviations of peak-to-peak amplitudes of SEPs in all experimental groups. Groups C Pr Po PP

P1N1 (mV)

N1P2 (mV)

P2N2 (mV)

2.8 ± 0.88 2.8 ± 0.58 2.7 ± 0.23 4.7 ± 0.73*,x,

3.8 ± 1.12 3.0 ± 0.57 3.3 ± 0.62 5.5 ± 0.45

2.1 ± 0.55 3.1 ± 0.53 4. 4 ± 0.56 5.3 ± 0.17*

z

The results are presented as mean ± SD, n ¼ 10 for each group. *p50.05 versus control group; xp50.05 versus prenatal group; zp50.05 versus postnatal group.

amplitude, and power spectra which failed to reveal any consistent, statistically significant effect of postnatal exposure to 60-Hz EFs. On the other hand, Wolpaw and associates (1987) also examined evoked potentials (EPs) in pig-tailed macaques exposed to combined electric and magnetic fields. In their studies, an attenuation of the late components of the SEPs was demonstrated in exposed animals to 60 Hz electric and magnetic field. As in Jaffe’s studies, the VEPs and auditory evoked potentials (AEP) showed no changes caused by exposure. The results of our study were not in agreement with these data. Because, we observed significant increments of P1N1 and P2N2 amplitudes of SEPs, whereas there were no significant changes in amplitudes of VEPs components. Inconsistent findings in previous studies investigating the effect of ELF-EFs on ERP are quite reasonable if we consider the complex mechanisms underlying the ERP generation and modulation. So ELF-EFs dependence of all observed alterations in this work is not easy to explain. But, it is worth to

emphasize that contradictory results, which might be related to variations in experimental designs and methodology among studies, makes it diffucult to understand the exact alterations in different sensory systems. Although firm conclusions cannot yet be made regarding potential impacts from ELFEFs effects on EPs, putting together all these results in the present study, it seems reasonable to suggest that 50 Hz ELFEFs can alter the perception mechanisms. Much work remains to be accomplished before the observed effects and their biological consequences are clearly understood. Somatosensory-evoked potentials to hand and foot stimulation have been used extensively for evaluation of corresponding afferent pathways (Cracco, 1973; Desmedt and Cheron, 1980). The latency of the first component is considered to reflect conduction velocity from the peripheral to central nervous system (Shaw and Synek, 1985; Tsuji et al., 1984). Significant prolongation of this component in Po and PP groups clearly showed that ELF-EFs exposure slowed peripheral nerve conduction velocity in the Po and PP groups. The most important effect of ELF-EFs has been on the production of free radicals that can cause considerable damage to biomolecules, such as DNA, lipids and proteins. It is well known that the brain and retina tissues are vulnerable to oxidative stress because they contain high concentration of easily peroxidizable fatty acids (Lee et al., 2004). Because of this elevated susceptibility, lipid peroxidation causes many damages in a cell, such as decreases in membrane fluidity, elevated sensitivity to oxidant stress and changes in enzyme activities (Fukui et al., 2001, 2002; Matsumoto et al., 1999). In the current study, we found that

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Figure 3. The means of TBARS levels of the brain tissues of control and experimental groups. The results are presented as mean ± SD, n ¼ 10 for each group (*p50.05 versus control group; zp50.05 versus postnatal group).

Figure 4. The means of TBARS levels of the retina tissues of control and experimental groups. The results are presented as mean ± SD, n ¼ 10 for each group (*p50.05, ***p50.001 versus control group; xxxp 50.001 versus prenatal group; #p50.05 versus prenatal + postnatal group).

retina TBARS levels were elevated in all experimental groups, whereas brain TBARS levels were increased only in the pre- and postnatal exposure to 12 kV/m ELF (1 h/day). These results of our study are also consistent with the previous studies showing that ELF-EFs caused the tissue damage (Dundar et al., 2009) and lipid peroxidation in rat brain (Akpinar et al., 2012; Romodanova et al., 1990) and different tissues and plasma (Benov et al., 1994; Guler et al., 2006; Seyhan and Guler, 2006; Watanabe et al., 1997; Yokus et al., 2005). Interestingly, while the magnitude of brain lipid peroxidation returned back to the control level in the PP group, retina TBARS levels remained elevated in the same group. So it could be suggested that ELF-EF in longer exposure period may probably alter antioxidant ability of central nervous system. This thought was further supported by a previous report showing that high EFs activate antioxidant activity of living organism (Cieslar et al., 2003). On the other hand, the other possible reason for this result is that retina tissue can be more sensitive than brain to external stressors like EF exposure. From VEP data it could be hypothesized that lipid peroxidation might be one of the possible factors that affects latencies of these potentials in all developmental periods. High TBARS levels in retina tissue in all ELF-EF applied groups probably caused significant increase in VEP latencies of all experimental groups versus the control group. This finding may reflect defective retinal transmission or even synaptic abnormalities that are caused by lipid peroxidation. On the other hand, we found significant electrophysiological changes for SEPs in the PP group even if brain TBARS levels

in this group was not different from those in the control group. Unchanged brain TBARS levels in the PP group makes it difficult to consider that the SEP alterations seen in this exposure group resulted from high lipid peroxidation in the somatosensory cortex. These discordant findings could be explained in two ways. First, from our data, it is conceivable to suggest that there may be a threshold level of EF-induced lipid peroxidation that leads SEP alterations. It is possible that enhanced production of superoxide radicals might cause adaptive reactions to counteract free radical effect of ELF-EF that might be a defense response to protect brain tissues from oxidative stress. So, it is possible that lipid peroxidative effect of ELF-EF might be masked by some adaptive changes. Second, in addition to lipid peroxidation, other modulatory factors may also mediate the effects of ELF fields on evoked potential alterations. It is likely that ELF fields may cause alterations of several functional properties of neurons, neuronal networks and/or may affect oscillatory activity by influencing neurotransmitter systems. This opinion is also in accordance with some reports indicating that several structural and functional properties of membranes may be altered in response to induced ELF fields at or below 100 mV/m (Sienkiewicz et al., 1993; Tenforde, 1993). Functional changes in tissues have also been reported to occur in response to induced EF of 10 mV/m (Tenforde, 1996). These effects become progressively more severe as the induced current density is increased (Bernhardt, 1979; Tenforde, 1991). Our data clearly revealed that 50 Hz EF exposure led to changes of VEPs and SEPs in all exposure groups. However,

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it is noteworthy that the largest effect of EF on these potentials was observed in the PP group, even if the level of brain TBARS was smaller than other groups. Therefore, it is difficult to conclude that electrophysiological alterations resulted only from increased lipid peroxidation. The possibility of other unexplored factors that may affect VEP and SEP must be considered. As no comparable report is available in the literature concerning the effect of ELF-EFs on VEP, further work is required to understand the functional significance and mechanisms underlying the observed changes in the pre- and postnatal group. The most striking finding of the current study was that the effect of ELF-EF on TBARS levels was not the same for prenatal, postnatal and pre + post natal exposure periods. Yet, the relationship between ELF-EF exposure and electrophysiological alterations is not clearly defined. So it could be suggested that alterations seen in EPs, at least partly, be explained by increased lipid peroxidation. However, other unexplored factors cannot be ruled out since there have been many factors that affect visual and somatosensory systems. Consequently, the effects of ELF-EF exposure may be complex and subtle and precise relationship between ELF-EF effects on EPs needs further study.

Declaration of interest The authors report no conflict of interest. The authors alone are responsible for the content and writing of the paper. This study was supported by a grant from Akdeniz University Research Foundation, Turkey (Grant No: 2008.01.0103.008).

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