Effects of extremely low-frequency electric fields at different intensities and exposure durations on mismatch negativity.

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EFFECTS OF EXTREMELY LOW-FREQUENCY ELECTRIC FIELDS AT DIFFERENT INTENSITY AND EXPOSURE DURATION ON MISMATCH NEGATIVITY Q1 D. KANTAR GOK, a D. AKPINAR, a P. YARGICOGLU, a*

in the amplitudes of ERP between the responses to the standard and the deviant tones in all groups. When peak-to-peak amplitude of the difference curves was evaluated, MMN amplitude was significantly decreased in the E18-4 group compared with the C4 group. Additionally, the amount of 4-HNE was increased in all experimental groups compared with the control group. Consequently, it could be concluded that electric field decreased MMN amplitudes possibly induced by lipid peroxidation. Ó 2014 Published by Elsevier Ltd. on behalf of IBRO.

S. OZEN, b M. ASLAN, c N. DEMIR, d N. DERIN a AND A. AGAR e

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a Department of Biophysics, Faculty of Medicine, Akdeniz University, Arapsuyu, 07070 Antalya, Turkey

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b Department of Electrical and Electronics Engineering, Engineering Faculty, Akdeniz University, Arapsuyu, 07070 Antalya, Turkey

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Department of Biochemistry, Faculty of Medicine, Akdeniz University, Arapsuyu, 07070 Antalya, Turkey

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d Department of Histology, Faculty of Medicine, Akdeniz University, Arapsuyu, 07070 Antalya, Turkey

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Department of Physiology, Faculty of Medicine, Akdeniz University, Arapsuyu, 07070 Antalya, Turkey

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Abstract—The effects of extremely low-frequency electric fields (ELF-EFs, 3–300 Hz) on lipid peroxidation levels and antioxidant enzyme activities have been shown in many tissues and plasma after exposure to 50-Hz alternating current (AC) electric fields. However, similar studies investigating brain lipid peroxidation status are limited. Moreover and as far as we know, no study has been conducted to examine mismatch negativity (MMN) response in rats following exposure to a 50-Hz AC electric field. Therefore, the purQ2 pose of the study was to investigate different intensity and exposure duration of ELF-EFs on MMN component of event-related potentials (ERPs) as well as apoptosis and oxidative brain damage in rats. Ninety male rats, aged 3 months were used in our study. A total of six groups, composed of 15 animals each, was formed as follows: sham-exposed rats for 2 weeks (C2), sham-exposed rats for 4 weeks (C4), rats exposed to 12-kV/m and 18-kV/m electric fields for 2 weeks (E12-2 and E18-2), rats exposed to 12- and 18-kV/m electric fields for 4 weeks (E12-4 and E18-4). At the end of the experimental period, MMN responses were recorded in urethane-anesthetized rats by electrodes positioned stereotaxically to the surface of the dura. After MMN recordings, animals were killed by exsanguination and their brain tissues were removed for 4-hydroxy-2-nonenal (4-HNE), protein carbonyl and TUNEL analysis. In the current study, different change patterns in ERP parameters were observed dependent on the intensity and exposure duration of ELF-EFs. There were differences

Key words: electric field, mismatch negativity, 4-hydroxy-2nonenal, protein carbonyl, apoptosis. 19

INTRODUCTION During the last half century, people have been constantly exposed to extremely low-frequency (3–300 Hz) electric (EF) and magnetic fields (MF), which have dramatically increased in our environment. Extremely low-frequency electric fields (ELF-EFs) come from electrical appliances, alternating current (AC) transmission and distribution lines. One of the greatest sources of ELFEFs exposure is transformers and power lines, which produce higher levels of field strength as high as 12-kV/ m around AC transmission lines and 16-kV/m around electricity-generating stations in comparison with environmental field strength (Valberg et al., 1997; Repacholi and Greenebaum, 1999; Kheifets et al., 2010). Therefore, due to increased electricity together with distorted urbanization in developing countries, power lines cause greater risk for people living around passing transmission lines and electricity-generating stations. So, this has raised public health concerns and accelerated research to identify possible biological effects associated with exposure to high-level ELF-EFs, which are produced by power lines (Repacholi and Greenebaum, 1999). On the other hand, it is still uncertain not only whether power lines but also current pollution levels of ELF-EFs may constitute a risk to human health. 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). Hence, biological effects of ELF-EFs are receiving increasing scientific interest since humans are chronically exposed to ELF-EFs in varying degrees (Repacholi and Greenebaum, 1999).

*Corresponding author. Tel: +90-242-2496906; fax: +90-2422274482. E-mail address: [email protected] (P. Yargicoglu). Abbreviations: 4-HNE, 4-hydroxy-2-nonenal; AC, alternating current; ANOVA, analysis of variance; ELF-EFs, extremely low- frequency electric fields; ERPs, event- related potentials; MMN, mismatch negativity; PBS, phosphate- buffered saline; SOD, synthesis of Q3 superoxide dismutase; StbD, standard before deviant; TUNEL, terminal deoxynucleotidyl transferase. http://dx.doi.org/10.1016/j.neuroscience.2014.04.056 0306-4522/Ó 2014 Published by Elsevier Ltd. on behalf of IBRO. 1

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The brain and central nervous system are considered as the most likely sites of interaction between biological systems and ELF-EFs. Some controlled laboratory studies have demonstrated subtle effects of ELF-EFs on human cerebral functioning, such as modifications of performances, alterations in the latency and amplitude of event-related potentials (ERPs) (Graham et al., 1987, 1994, 1999; Cook et al., 1992). It is well known that ERPs reflect the reception and processing of sensory information as well as higher level processing that involves selective attention, memory updating, semantic comprehension, and other types of cognitive activity (Picton, 1992; Demiralp et al., 1999; Polish, 1999). The mismatch negativity (MMN) component of ERPs is regarded as a bioelectric correlate of a result of the mismatch between a sensory memory trace and an incoming stimulus (Naatanen et al., 1978; Naatanen, 1990). MMN is elicited using the so-called oddball stimulus paradigm, in which a series of repeated stimuli (standards) are interrupted by an occasional, slightly different stimulus (the deviant). It can be recorded in the condition only if the distinguishing feature(s) of the standard relative to the deviant are successfully represented neurally, stored in transient auditory memory, and compared to the auditory input by the deviant (Naatanen et al., 1978; Naatanen, 1990; Jacobsen and Schroger, 2001). This component is thought to reflect an automatic process, since it occurs in the absence of attention and even during sleep or under anesthesia, and which makes it useful in the assessment of very young or impaired participants (Duncan et al., 2009). MMN, higher amplitude responses to deviants than standards have been reported several times in animals (Cse´pe et al., 1987, 1989, 1994; Javitt et al., 1992; Kraus et al., 1994; Ruusuvirta et al., 1996, 1998, 2007; Astikainen et al., 2005, 2006; Eriksson and Villa, 2005; Umbricht et al., 2005; Tikhonravov et al., 2008). Animal studies have been conducted in order to understand the neural mechanisms involved in generating MMN. It has been recorded cortically and/or subcortically in several animal species including rats (Ruusuvirta et al., 1998; Tikhonravov et al., 2008), primates (Javitt et al., 1992, 1994), rabbits (Ruusuvirta et al., 1996), cats (Cse´pe et al., 1989, 1994), mice (Umbricht et al., 2005) and guinea pigs (Kraus et al., 1994). In awake and anesthetized rats, the MMN has been found to occur in the latency range of 30–250 ms (Ruusuvirta et al., 1998; Astikainen et al., 2006; Nakamura et al., 2011; Tikhonravov et al., 2008; Ahmed et al., 2011). Many researches have been focused on adverse health effects in biological systems resulting from exposure to 50-Hz ELF-EFs. Consistent with this point, we also showed that when the intensity of the ELF-EFs was increased, lipid peroxidation increased proportionally (Akpinar et al., 2012). Hence, based on these data (Akpinar et al., 2012) and previous investigations (Marino et al., 1986; Cossarizza et al., 1993; Margonato et al., 1993; Benov et al., 1994) indicating that an increment of strength and duration of ELF-EFs had greater effects on living organisms, EFs at 12- and 18-kV/m strengths were used in the present study to investigate biochemical and electrophysiological

alterations in the central nervous system. To date, there have been no studies investigating the MMN changes in rats, which were exposed to 50-Hz AC electric fields. Therefore, the purpose of the study was to examine MMN alterations as well as oxidative brain damage and apoptosis in rats exposed to 50-Hz ELF-EFs in different strengths and periods. In order to evaluate the relationship between oxidative cell injury induced by ELF-EFs and differences in ERP parameters, 4-hydroxy-2-nonenal (4-HNE) levels and protein carbonyl values of the brain tissue were determined in the present research. Additionally, apoptotic cells in the brain tissue of control and experimental groups were analyzed by the terminal deoxynucleotidyl transferase (TUNEL) assay. On the other hand, the effects of magnetic component of ELF electromagnetic fields on biological systems have been reported in many researches, but there are limited data on brain tissues related to the effect of pure ELF-EFs without magnetic field. Hence, particular field strengths (Harakawa et al., 2005; Guler et al., 2008) and exposure durations (Aydin et al., 2006) were selected in this study.

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EXPERIMENTAL PROCEDURES

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Animals

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All experiments were approved by the Akdeniz University Animal Care and Use Committee and were performed in accordance with the European Community directive. All effort was taken to minimize the number of animals used and their suffering. Animals were maintained at 12-h light–dark cycles and a constant temperature of 23 ± 1 °C at all times. In our study, Male albino Wistar rats aged 3 months, weighing 300–350 g were housed in stainless steel cages in groups of four rats per cage and given food and water ad libitum. Rats were divided into six groups of 15 animals each: Group 1: shamexposed rats for 2 weeks (C2); Group 2: sham-exposed rats for 4 weeks (C4); Group 3: rats exposed to 12-kV/m EF for 2 weeks (E12-2); Group 4: rats exposed to 18-kV/m EF for 2 weeks (E18-2); Group 5: rats exposed to 12-kV/m EF for 4 weeks (E12-4); Group 6: rats exposed to 18-kV/m EF for 4 weeks (E18-4). Experimental groups were exposed to 50-Hz EF at a given intensity for 1 h per day. Animals of C2 and C4 groups were kept under the same experimental conditions without being exposed to any EF for 2 and 4 weeks, respectively. Experiments were performed between 09:00 am and 05:00 pm.

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Electric field exposure system

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The exposure system is presented in Fig. 1. Parallel plate capacitor was used to generate EF. Custom-made 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. A plastic cage was placed between plates. Plastic cage was suitable for free movement of

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rats and therefore homogeneous shielding of each other (Aydin et al., 2006). The EF strength was calculated according to the equation E = V/d where V is electric potential between the plates, d is the distance, and E is the EF strength in volt/meter. A custom-made step-up transformer (rated 220 Vrms/6000 Vrms) was used to produce 50-Hz AC EF. 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 voltages of the source were 218.5–226.5 and 5850–5945 V respectively. Electric field strength for 50-cm distances between the plates were in the voltage range of 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 lT. Uniformity and homogeneity of the EF were 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%.

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MMN recordings

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At the end of the experimental period, adult male rats aged 3-months were deprived of food for 24 h and then prepared for ERP recordings. ERPs were recorded between 09:00 am and 05:00 pm. Rats were anesthetized (24 g/100 ml) with intraperitoneal injections of urethane (1.2 g/kg, Sigma–Aldrich, St Louis, MO, USA). The head of the anesthetized animal was attached to the standard stereotaxic frame and two screws were placed in the skull. Two small holes (1.5 mm diameter) were drilled for the placement of the stainless steel electrodes. A unilateral craniotomy exposed a 5 5 mm region with the rostral edge, 3 mm posterior to the bregma and the medial edge 5 mm lateral to the midline, positioned over the auditory cortex in the left hemisphere. The tip of a stainless steel wire was positioned to the surface of the dura on the basis of online-recorded potentials to click stimuli. A skull screw on the right cerebellar cortex served as an indifferent reference electrode. After the surgery, the ear bars were

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removed. The anesthetized animal was moved into a sound-attenuated recording room. Mean background noise level of the recording room measured 46 dB with a sound level meter (Testo 816 Sound Level Meter, Germany). ERPs were recorded using the oddball condition and an experimental set-up adapted and modified from Ruusuvirta et al. (1998) and Tikhonravov et al. (2008, 2010). In the oddball condition for auditory stimuli, frequencies of standard and deviant tones were 2000 and 2500 Hz, respectively. Deviant tones were pseudorandomized to occur at a 10% probability (900 standard tones, 100 deviant tones) in a sequence of standard tones presented at the inter-stimulus interval of 500 ms. The tones were ordered pseudorandomly in their series with the restriction that there were no less than two standards between consecutive deviants. The duration of the 85-dB tones was 50 ms and the tones were presented through a loudspeaker at a distance of 15 cm from the right ear of the animal. Electroencephalogram signal was amplified (Brainamp EEG/EP Amplifier, Brain Products, Munich, Germany), band-pass filtered (0.1–1000 Hz), and digitized at a 1000-Hz sampling rate (Brainvision Recorder, Brain Products, Munich, Germany). Data recorded during oddball condition were filtered (0.1–36 Hz) and baseline corrected (the average amplitude of a 100-ms period preceding stimulus onset) (EEGLab7). The following averaged curves were computed for each animal and then for all groups of animals: Standard before deviant (StbD) (ERPs to standard tones preceding deviant tones), Deviant (Dev) (ERPs to all deviant tones during the oddball paradigm) and difference wave (Dev minus StbD). The peak-topeak amplitudes and latencies of the P1, N1, P2 and N2 components of the ERPs were determined for each rat from the averaged StbD, Dev and Difference curves were within the time period of 0–500 ms from stimulus onset.

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Data analysis. Time window around each ERP component was chosen (P1: 20–70 ms, N1: 40–110 ms, P2: 70–145 ms, N2: 155–250 ms) and the maximum (or

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minimum) peak in the corresponding window was identified. For MMN response, three different analysis windows were used based on visual inspection of the peaks of StbD and Dev stimuli: 35–70 ms, 80–140 ms, 150–250 ms from stimulus onset. The latencies and peak to peak amplitudes of P1, N1, P2 and N2 components of the ERPs were determined for each rat and condition from the averaged StbD and Dev waveforms. We prefered to measure peak to peak amplitudes (Grau et al., 2001; Linka et al., 2005; ZionGolumbic et al., 2007) to obtain amplitude values that were free from residual noise, DC shifts, and other confounding artifacts that may exist in a pre-stimulus baseline (Picton et al., 2000). Mixed-design MANOVA was used to examine the latencies and peak-to-peak amplitudes of ERPs with between subjects factors (duration, intensity) and within subjects factor (stimulus). Differences in latencies and amplitudes of ERP components for each stimulus type (Dev, StbD) were determined separately by using 2 3 factorial analysis of variance (ANOVA) with three levels of intensity (sham, 12 kV/m, 18 kV/m) and two levels of duration (2 and 4 weeks). Greenhouse–Geisser degrees of freedom were used whenever the sphericity assumption was violated. Bonferroni post hoc test was used to further analyze the main effect of intensity. Then for more detailed examination of simple main effects, separate ANOVAs with three levels of intensity for the two duration levels and stimulus were applied (Bonferroni post hoc test). The comparisons between the two duration levels for each intensity level were made using the independent sample t-test. For MMN response, three different analysis windows were used based on visual inspection of the peaks of the deviant and standard amplitude differences in ERP curves for StbD and Dev stimuli: 35–70, 80–140, 150–250 ms from Q5 stimulus onset. The MMN response was defined as the part of the ERP wave that response to the deviant stimulus significantly different from the response to the standard stimulus. ERP curves in these time windows were submitted to repeated measures ANOVA to determine the MMN response. Since the significant difference was in only the 150–250-ms time window, this time window was selected for further analysis and reporting. MMN response was measured from difference wave as the peak to peak amplitude of the maximum deflection (the small positive peak followed by a larger negative response) within 150–250-ms time window. This time window was selected based on the determined significant effect of stimulus on the response amplitudes. MMN responses were submitted to 2 3 Factorial ANOVA with the factors duration and intensity (Bonferroni post hoc test). Then two one-way ANOVAs were conducted to examine each duration level. Comparisons of MMN response between the two duration levels for each intensity level were made using the independent sample t-test.

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Chemical analysis

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After ERP recordings, animals were sacrificed at the same time interval (09:00 am and 05:00 pm) by exsanguination via cardiac puncture. Brain tissues were

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removed immediately. The isolated brain tissues were stored frozen at 80 °C until assay determinations.

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4-HNE. Levels of 4-HNE were determined as an indicator of lipid peroxidation. Western Blot analysis was performed as previously described (Aslan et al., 2007). Briefly, supernatants were boiled for 5 min in Laemmli sample buffer (Bio-Rad Laboratories, Munchen, Germany) and proteins (40 mg/well) were separated on a 7.5% polyacrylamide gel. Proteins separated by sodium dodecyl sulfate polyacrylamide were used as loading standard. Gel electrophoresis (SDS–PAGE) was either transferred to nitrocellulose membranes (Amersham Biosciences, Buckinghamshire, UK) or was visualized by Electro-Blue staining solution (Qbiogene, Heidelberg, Germany) to verify equal protein loading for Western blot analysis. A rabbit polyclonal antibody against 4-HNE (1:1000 dilution) was used for Western blot analysis. Horseradish peroxidase-conjugated goat anti-rabbit IgG (1:10,000 dilution; Zymed Laboratories, San Francisco, CA, USA) was used as a secondary antibody and immunoreactive proteins were visualized on high-performance chemiluminescence film via ECL reagent (Amersham Pharmacia Biotech, Buckinghamshire, UK). Densitometric analysis of immunoblots was performed using NIH image 1.61 software for windows (Scion Corporation, Frederick, MD, USA).

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Measurement of tissue protein carbonyl content. Protein-bound carbonyls were measured via a protein carbonyl assay kit (Cat. #1005020 Cayman Chemical, Ann Arbor, MI, USA). The utilized method was based on the covalent reaction of the carbonylated protein side chain with 2,4-dinitrophenylhydrazine (DNPH) and detection of the produced protein-hydrazone at an absorbance of 370 nm. Results were calculated using the extinction coefficient of 22 mM 1 cm 1 for aliphatic hydrazones and were expressed as nmol/mg protein.

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TUNEL assay. Apoptotic cells in the brain tissues of control and experimental groups were analyzed by the TUNEL assay. TUNEL was conducted using a Cell Death Detection kit (Roche, Cat No. 11684795910, Mannheim, Germany) and performed according to the manufacturer’s instructions. Briefly, paraffin sections of 5-lm thickness from brain tissues were cut and taken onto slides covered with poly-L-lysine. Slides were washed twice in phosphate-buffered saline (PBS) for 5 min. Following the incubation of slides with the permeabilisation solution (0.1% Triton X-100 in 0.1% sodium citrate) for 8 min at 4 °C and washing twice with PBS for 5 min, the labelling reaction was performed using 50 ll TUNEL reagent for each sample, except negative controls, in which reagent without enzyme was added and incubated for 1 h at 37 °C. Following PBS washings, slides were mounted with Vectashild Mounting Medium for fluorescence with DAPI (H-1200; Vector Lab. Inc. Burlingame, CA, USA). Slides were examined using a Zeiss Axioplan microscope and photographs were taken with Spot Advanced Software.

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Statistical analysis

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The statistical analysis of the obtained data was performed by SPSS 18.0 (SPSS, Chicago, IL, USA) software for Windows. Statistical comparisons between groups for 4-HNE and protein carbonyl levels were performed by using Kruskal–Wallis one-way analysis of variance and the Mann–Whitney U test. Results are expressed as mean ± standard error of mean (SEM). Significance levels were set at p < 0.05.

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RESULTS

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P1, N1, P2 and N2 components of ERPs to StbD and Dev tones in the oddball condition for all experimental groups are presented in Fig. 2. Difference waveforms (DW) obtained by subtracting standard from deviant responses are also shown in the same figure. Measurements were made on two positive and two negative potentials, which were seen in all groups. Mean ± SEM of peak latencies of ERPs components (P1, N1, P2, N2) in response to StbD and Dev tones are given in Table 1. The analysis of the latencies revealed no significant main effect of stimulus (F(4,50) = 1.02; not significant), stimulus duration interaction (F(4,50) = 0.76; not significant) and stimulus intensity interaction (F(8,102) = 1.06; not significant). The between subjects test indicates a significant main effect for intensity (F(8,102) = 2.11, p < .05) and intensity duration interaction (F(8,102) = 2.49, p < .05), but no significant main effect for duration (F(4,50) = 0.80; not significant) for all ERP components. Because the interaction was disordinal, main effects could not be sensibly interpreted. Separate analysis of responses to Dev and StbD stimulus showed that there was a significant main effect of intensity (F(8,102)StbD = 2.50, F(8,102)Dev = 2.39, p < .05) and intensity duration interaction (F(8,102)StbD = 2.50, F(8,102)Dev = 2.91, p < .05) but no significant main effect for duration (F(4,50)StbD = 0.75, F(4,50)Dev = 0.76, not significant) for all components. However, because the interaction was again disordinal, we did not interpret the main effects which would be misleading. For a 2-week duration, subsequent analysis of data obtained for StbD stimulus revealed that there was a statistically significant difference between groups for latencies of P1 (F(2,24) = 5.39, p < .05) and N1 (F(2,24) = 5.26, p < .05), P2 (F(2,24) = 7.81, p < .05) and N2 (F(2,24) = 6.20, p < .05) components. Latencies of all components were significantly increased in the E18-2 group versus the C2 group (p < .05). Also, latencies of P2 and N2 components were significantly increased in the E18-2 group compared to the E12-2 groups (p < .05). For Dev stimulus, there was a statistically significant difference between groups for latencies of P2 (F(2,24) = 6.96, p < .05) and N2 (F(2,24) = 6.31, p < .05) components for Dev stimulus. While, P2 and N2 component latencies were significantly increased in the E18-2 group versus C2

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and E12-2 groups (p < .05), the same increment trend in P1, N1 components was also viewed in the E18-2 group for the Dev stimulus, but it failed to reach statistical significance (p > .05). For a 4-week duration, subsequent analysis of data obtained for both stimuli showed that there was a statistically significant difference between groups for latencies of P1 (F(2,29)StbD = 5.68, F(2,29)Dev = 4.96, p < .05) and N1 (F(2,29)StbD = 4.77, F(2,29)Dev = 5.03, p < .05) and P2 (F(2,29)StbD = 4,17, F(2,29)Dev = 5.84, p < .05) components. For both stimulus, the latencies of P1, N1 and P2 components were significantly increased in the E12-4 group compared to the C4 group (p < .05) whereas latencies of these components were significantly decreased in E18-4 group relative to the E12-4 group (p < .05). In spite of the observed similarities in latency changes of the N2 component, these changes did not reach a statistical significance (p > .05). For the same intensity level in different duration periods, further t-test revealed that there was no significant difference in latencies of all components between C2/C4 and E12-2/E12-4 groups for both stimuli. Latencies of all components significantly decreased in the E18-4 group versus the E18-2 group (StbD: t(17)P1 = 3.72, t(17)N1 = 3.05, t(17)P2 = 3.44, t(17)N2 = 2.93; Dev: t(17)P1 = 2.62, t(17)N1 = 2,13, t(17)P2 = 3.26, t(17)N2 = 1.92, p < .05). Mean ± SEM of peak-to-peak amplitudes of ERP components (P1N1, N1P2, P2N2) in response to StbD and Dev tones are shown in Table 2. The analysis of the amplitudes demonstrated a significant main effect of the stimulus (F(1,53) = 1167.2, p < .001), stimulus duration interaction (F(1,53) = 9.53, p< .01), stimulus intensity interaction (F(1,53) = 15.00, p < .001) for only P2N2 amplitude. The between subjects test indicates a significant main effect for duration (F(3,51) = 16.50, p < .05), intensity (F(3,51) = 15.94, p < .001) and intensity duration interaction (F(3,51) = 13.56, p < .001) for all amplitudes. Factorial ANOVA for each stimulus revealed that there was a significant main effect of duration (F(3,51)StbD = 8.34, F(3,51)Dev = 18.01; p < .05), intensity (F(6,104)StbD = 12.85, F(6,104)Dev = 15.19; p < .05) and intensity duration interaction (F(6,104)StbD = 10.91, F(6, 104)Dev = 10.62; p < .05) for all ERPs amplitudes. Again, because the interaction was significant and disordinal, main effects could not be sensibly interpreted. For a 2-week duration, analysis showed that there was a statistically significant difference between groups for amplitudes of P1N1 (F(2,24)StbD = 103.71, F(2, 24)Dev = 41.24, p < .001) and N1P2 (F(2,24) = 23.16, F(2,24)Dev = 64.72, p < .001) and P2N2 (F(2,24)StbD = 4,06, F(2,24)Dev = 18.56, p < .05) components. P1N1 amplitude was significantly increased with intensity for both stimuli (p < .05). Beside, N1P2 amplitude was significantly increased in the E18-2 group versus the E12-2 and the C2 groups (p < .05), there was a slight increase in N1P2 amplitude of the E12-2 group compared to the C2 group but it did not reach a significance level for both stimuli (p > .05). While there


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Fig. 2. P1, N1, P2 and N2 components of grand-averaged ERPs to standard (StbD) and deviants (Dev) tones in the oddball condition for all groups. 488 489 490 491 492 493 494 495 496 497 498 499 500

was no significant difference in P2N2 amplitude for the StbD stimulus (p > .05), P2N2 amplitude was significantly decreased in the E12-2 group compared with the C2 and E18-2 groups for Dev stimulus (p < .05). For a 4-weeks duration, analysis revealed that there was a statistically significant difference between groups for amplitudes of P1N1 (F(2,29)StbD = 21.68, F(2, 29)Dev = 13.14, p < .001) and N1P2 (F(2,29)StbD = 16.71, F(2,29)Dev = 15.24, p < .001) and P2N2 (F(2, 29)StbD = 11.17, F(2,29)Dev = 55.92, p < .001) components. For both stimuli, P1N1 amplitude of E12-4 and E18-4 groups were significantly increased compared with the C4 group (p < .05). For StbD stimulus, N1P2

amplitude of the E12-4 group was significantly higher than those of C4 and E18-4 groups and N1P2 amplitude of the E18-4 group was significantly decreased to the control levels versus the E12-4 group (p < .05). For the Dev stimulus, N1P2 amplitudes of E12-4 and E18-4 groups were significantly increased versus the C4 group and similarly N1P2 amplitude of the E18-4 group was slightly attenuated compared to the E12-4 group but failed to reach significance. Also, P2N2 amplitude in response to the Dev stimulus was significantly decreased by increasing intensity levels. The same trend was observed for the StbD stimulus but difference between groups did not reach a significance level.


501 502 503 504 505 506 507 508 509 510 511 512 513

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184.28 ± 8.41n 170.90 ± 7.10n

189.70 ± 11.79

176.50 ± 5.80

#,n

97.30 ± 3.85

#,n

54.90 ± 4.39

#,n

51.90 ± 3.39

#,n

7

Further paired sample t-tests revealed that P2N2 amplitude in response to Dev tones was increased compared with P2N2 responses to StbD tones in withingroup comparison in all groups (t(19) = 2.7, p < .01). For the same EF intensity level in different duration periods, further t-test revealed that there was no significant difference in amplitudes of all components between C2/C4 and E12-2/E12-4 groups for both stimuli (p > .05). Amplitudes of all components were significantly decreased in the E18-4 group versus the E18-2 group (StbD: t(17)P1N1 = 6.97, t(17)N1P2 = 7.42, t(17)P2N2 = 7.18; Dev: t(17)P1N1 = 3.01, t(17)N1P2 = 14.34, t(17)P2N2 = 16.09, p < .05). When peak to peak amplitudes of the difference wave were evaluated, factorial ANOVA showed that there was a significant main effect of the duration (F(1,53) = 27.94, p < .001), intensity (F(2,53) = 58.17, p < .001), and duration intensity interaction (F(2,53) = 34.25, p < .001). Further one-way ANOVA analyses for a 4week duration showed a significant difference between groups (F(2,29) = 12.75, p < .001). Post hoc tests revealed that MMN peak amplitude was significantly decreased in the E18-4 group compared with the E12-4 and C4 groups (Fig. 3B) (p < .001). MMN amplitude of E12-4 group was slightly attenuated versus the C4 group but it did not reach the significance level (p > .05). However, no statistical difference was found in the MMN peak amplitudes between all groups for a 2week duration (F(2,24) = 3.23, not significant). Further t-test revealed that there was no significant difference in amplitudes of MMN between C2/C4 and E12-2/E12-4 groups (p > .05). Amplitudes of MMN significantly decreased in the E18-4 group versus the E18-2 group (t(17) = 14.04, p < .001). Analysis of MMN latency values yielded a significant main effect of intensity (F(2,53) = 6.30, p < .001) and duration intensity of interaction (F(2,53) = 4.06, p < .05). One-way ANOVA analyses for a 2-week duration showed that there was a significant difference between groups (F(2,24) = 11.14, p < .001). Further post hoc tests revealed that MMN peak latencies of E12-2 and E18-2 groups were significantly prolonged compared with the C2 groups (Fig. 3A) (p < .001). MMN latency of E18-2 group was slightly increased versus the E12-2 group but it did not reach the significance level (p > .05). However, no statistical difference was found in the MMN peak latencies between all groups for a 4-week duration (F(2,29) = 0.12, p > .05). Further t-test revealed that there was no significant difference in latencies of MMN between C2/C4 and E12-2/E12-4 groups (p > .05). The latency of MMN significantly increased in the E18-2 group versus the E18-4 group (t(17) = 2.33, p < .05). 4-HNE (Fig. 4A) western blot and densitometric analysis (Fig. 4B) of brain tissues from control and experimental groups are shown in Fig. 4. There was a statistically significant difference between groups (H(4) = 19.944, p < .01). 4-HNE protein levels were elevated in all experimental groups compared with the control group (Fig. 4B) (U = .000, p < .05). An increase in 4-HNE level was detected in the E18-4 group versus

n

#

p < 0.05 p < 0.05 p < 0.05 p < 0.05

*

E18-4

E12-4

C4

E18-2

E12-2

C2

versus versus versus versus

respective control groups. respective E12-2 groups. respective E12-4 groups. E18-2 group).

25.71 ± 2.45 #,n

23.70 ± 2.92

126.70 ± 11.05* 88.10 ± 10.86* 82.00 ± 11.17* 52.10 ± 8.67* 49.40 ± 8.48*

29.00 ± 4.14

32.58 ± 5.31

51.91 ± 6.72

57.16 ± 6.27

96.66 ± 7.37

133.23 ± 8.33*,# 97.66 ± 5.08 130.90 ± 9.89* 99.10 ± #,n 4.52 135.84 ± 10.62*, 83.44 ± 11.31 97.44 ± 12.35* 53.22 ± 8.90 61.77 ± 8.83*

110.00 ± 8.66 70.90 ± 9.27 66.00 ± 7.48 41.00 ± 5.97 36.80 ± 5.86

174.08 ± 4.60 192.80 ± 11.76

218.07 ± 9.32*,

184.30 ± 4.93

171.62 ± 7.67

166.87 ± 9.74 182.10 ± 5.38 221.11 ± 10.58*, 83.12 ± 2.72 109.90 ± 11.94 88.20 ± 7.28 51.75 ± 5.65 50.12 ± 5.23 32.50 ± 5.15 28.75 ± 3.66

N2

Dev P2

StbD Dev

N1

StbD Dev StbD

P1 Groups

Table 1. The mean and standard errors of peak latencies of ERP components in response to Standard and the deviant tones in all experimental groups

StbD

Dev



514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574

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Q11 Table 2. The means and standard errors of peak-to-peak amplitudes of ERPs for each component P1N1

C2 E12-2 E18-2 C4 E12-4 E18-4 a *

n

N1P2

StbD

Dev

StbD

Dev

StbD

Dev

2.24 ± 0.81 5.23 ± 2.17* 9.53 ± 1.56*, 2.70 ± 0.56 5.92 ± 1.88* 4.17 ± 0.66*,n

2.52 ± 0.62 5.96 ± 2.39* 7.51 ± 1.37*, 4.34 ± 1.11 6.61 ± 1.65* 6.25 ± 1.07*,n

6.42 ± 1.58 8.33 ± 1.87 12.29 ± 2.55*, 6.11 ± 0.97 9.23 ± 2.00* 6.74 ± 1.48#,n

4.46 ± 0.99 5.18 ± 1.24 13.18 ± 2.87*, 4.41 ± 1.35 7.92 ± 1.56* 6.58 ± 1.08n

9.56 ± 0.86 8.57 ± 1.01 9.87 ± 1.66 9.95 ± 1.75 8.45 ± 1.55 7.42 ± 1.73n

18.35 ± 1.12a 15.86 ± 1.00a* 17.42 ± 1.86a, 18.49 ± 1.97a 15.80 ± 1.03a* a,⁄,#,n 11.28 ± 0.85

p < 0.05 versus the StbD response. p < 0.05 versus respective control groups. p < 0.05 versus the E12-2 group. # p < 0.05 versus the E12-4 group. p < 0.05 versus the E18-2 group.

Fig. 3. The mean and standard errors of MMN latencies (A) and amplitudes (B) of ERPs in difference curves. Results are presented as mean ± SEM, n = 15 for each group (⁄p < 0.05 versus control groups).

575 576 577 578 579 580 581 582 583 584

P2N2

all other groups (U = .000, p < .05). The amount of 4HNE was increased in the E12-4 group with respect to E12-2 group (U = .000, p < .05). Loading controls applied for western blot analysis show equal protein loading in each lane (Fig. 4A). Protein carbonyl values of the brain tissues of control and test groups are given in Fig. 5. There was a statistically significant difference between groups (H(5) = 23.660, p < .01). Protein carbonyl contents of E12-4 (U = 4.000, p < .01), E18-2 (U = 4.000,

p < .05) and E18-4 (U = 4.000, p < .01) groups were significantly increased with respect to their corresponding control groups. In addition, it was found that a statistically significant increase in protein carbonyl values of E12-4 (U = 4.000, p < .01), E18-2 (U = 6.000, p < .01) and E18-4 (U = 8.000, p < .05) groups in comparison with the E12-2 group. Apoptotic cells that undergo extensive DNA degradation during the late stages of apoptosis in the brain tissues of control and experimental groups were analyzed by the TUNEL assay. Apoptotic cells were not seen in the hippocampus, auditory and frontal cortex layers of all control and experimental groups (data not shown).

585

DISCUSSION

599

There are a limited number of studies about ELF-EFs effects on the living system (Harakawa et al., 2005; Seyhan and Guler, 2006; Guler et al., 2007) particularly on oxidant status and lipid peroxidation. Therefore, in the current study we investigated the effect of ELF-EF exposure on the MMN component of ERPs and elucidate the role of oxidant tissue injury on the generation of this potential. To our knowledge, this is the first report aimed at assessing the effect of 50-Hz electric fields at different exposure periods and intensities on the generation of the MMN response in anaesthetized rats. In the current study, we recorded MMN response defined as a significant difference between deviant and standard evoked potentials observed in the 150–250 ms following stimulus onset. Mismatch responses in rats have been found to be higher in amplitude to deviant tones than to standard tones in all experimental groups. MMN activity recorded in the present study is also in agreement with previous studies (Ruusuvirta et al., 1998, 2013; Eriksson and Villa, 2005; Tikhonravov et al., 2010; Astikainen et al., 2011). The most important effect of ELF-EFs has been thought to be the production of free radicals that can cause considerable damage to biomolecules such as DNA, lipids and proteins. 4-HNE levels as an index of lipid peroxidation were significantly increased in the brain of rats exposed to 12- and 18-kV/m strength of ELF for 2- and 4-week application periods (1-h/day).

600


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This study is consistent with the previous studies showing that ELF electric fields caused lipid peroxidation in rat brain (Romodanova et al., 1990; Akpinar et al., 2012) and in different tissues and plasma. (Benov et al., 1994; Watanabe et al., 1997; Yokus et al., 2005; Guler et al., 2006; Seyhan and Guler, 2006). Additionally, previous studies (Benov et al., 1994; Akpinar et al., 2012) also showed that when the intensity and duration of the electric field was increased, lipid peroxidation increased proportionally. Protein and amino acids are defined as major targets for free-radical attacks and oxidative damage, since they constitue a large proportion of cell and tissue composition. The usage of protein carbonyl groups as a marker may have some advantages in comparison with other oxidation products because of the early formation and the relative stability of carbonylated proteins (DalleDonne et al., 2003; Stadtman and Oliver, 1991). Carbonyls, formed following reactive oxygen species mediated oxidation of sugar and membrane lipids, are able to form adducts commonly known as CO-proteins (proteins

9

Fig. 5. Protein carbonyl values of the brain tissues of control and experimental groups. Results are presented as mean ± SD, n = 15 for each group. (⁄p < 0.05 versus related controls, p < 0.05 versus E12-2 group).

bearing carbonyl groups) with structural proteins, causing alterations in their biological activities (Shacter, 2000). Reactive carbonyl groups on proteins can also be formed by direct oxidation of protein side-chains (Reznick and

Fig. 4. (A) Western blot and loading control of 4-HNE analysis. 1, control2; 2, control4; 3, E12-21; 4,E12-22; 5, E12-41; 6, E12-42; 7, E18-21; 8, E1822; 9, E18-23; 10, E18-41; 11, E18-42; 12, E18-43. (B) Densitometric analysis of 4-HNE proteins. Results are mean ± SD, n = 2–3.Data are expressed as fold increase in which the control is defined as 1,0. (⁄p < 0.05 versus control group, p < 0.05 versus E12-2 group, #p < 0.05 versus E12-4 group, np < 0.05 versus E18-2) group. Please cite this article in press as: Kantar Gok D et al. Effects of extremely low-frequency electric fields at different intensity and exposure duration on mismatch negativity. Neuroscience (2014), http://dx.doi.org/10.1016/j.neuroscience.2014.04.056

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Packer, 1994). Reactive oxygen species may oxidize amino acid side-chains into ketone or aldehyde derivatives. In the statistical evaluation of all experimental groups, protein carbonyl contents of the E12-4, E18-2 and E18-4 groups were significantly increased compared with the control group. No information is available about possible effect of ELF-EFs on brain carbonyl content. Therefore, we compared our data with previous studies of Guler et al. (2009a,b) done in liver and lung tissues Q6 of guinea pigs exposed to EF. They showed that while protein carbonyl levels in the lung, insignificant changes were observed in liver for EF (12 kV/m, 7 days/for 8 h/ day) exposure groups compared to the control group. The increase in carbonyl content in liver tissues of guinea pigs exposed to EF is in agreement with our findings found in brain tissues of the exposure groups. Together with previously reported results (Akpinar et al., 2012), the present observations lead to the conclusion that 50-Hz EF can affect brain functions by increasing free radicals. In the present study, our data indicated that P1N1, N1P2 amplitudes were significantly raised with increasing ELF-EFs intensity for a 2-week duration. The same trend in P1N1, N1P2 amplitudes were also observed in E18-4 and E12-4 groups versus the C4 group. Our results were in agreement with some reports showing alterations in amplitudes of ERPs in both animal (Blanchi et al., 1973) and human subjects (Cook et al., 1992; Graham et al., 1994) exposed to 9-kV/m EFs and 20 microTesla⁄⁄. One of the possible causes of the increase in P1N1, N1P2 amplitudes could be linked to changes in neuromodulatory systems that resulted from ELF field exposure. Previous studies revealed that ELF fields increased norepinephrine levels (Mose, 1978; Zecca et al., 1998), decreased dopamine, serotonin levels (Wolpaw et al., 1987) and acetylcholineesterase activity (Fathi and Farahzadi, 2012). Studies on the relationship between ERPs and neurotransmitter systems showed that enhancement in epinephrine levels and decrease in serotonergic, dopaminergic and cholinergic neuronal transmission may cause an increase in some ERP amplitudes (Jaaskelainen et al., 1999; Berntson et al., 2003; Moxon et al., 2003; Manjarrez et al., 2005; Klinkenberg et al., 2013). Compatible with these reports, amplitude alterations due to ELF-EFs exposure, at least partly, are explained by changes in one or multiple neurotransmitter systems. However, other unexplored factors cannot be ruled out which may have possible effects on ERPs. On the other hand, alteration pattern of P2N2 amplitude was remarkably different than other amplitudes. A decrement trend with increasing intensity was detected in P2N2 amplitude for 4 weeks. However, the same reduction in P2N2 amplitude was not observed in the E18-2 group. Based on this change pattern in P2N2 amplitude proQ7 duced by ELF-EFs, It could be suggested that modulation of the neural network subserves the generation of the P2N2 appears to be relatively different than the other amplitudes. The cause of these data is unclear at present and further research is needed to provide the answer. MMN systematically decreases with increasing ELFEFs exposure duration as well as with exposure

intensity. While the intensity-related decrease is more pronounced after longer exposure period, the exposurerelated decrease is more apparent for higher intensity level. Briefly, a general trend to lower than normal MMN amplitude in all experimental groups exposed to ELFEFs was observed. However, it did not reach statistical significance. MMN, which is elicited by the deviant tone in the oddball paradigm, was significantly decreased only in the E18-4 group in comparison with the corresponding control group. This finding suggests that ELF-EFs caused a shift in physiological state in the E18-4 group. In view of the fact that MMN is shown to be a sensitive and reliable method to evaluate sensory short-term memory (Naatanen et al., 1978; Naatanen, 1990), these results probably indicate that 50-Hz EF significantly affects the auditory processing in this group. Thus, it can be concluded that the marked and consistent changes in the function of the brain resulting from the oxidative damage caused by 50-Hz ELF electric fields (4-week exposure to ELF-EFs with intensity of 18 kV/m) might be responsible in the decline of auditory sensory (‘echoic’) memory abilities. Several studies suggested that brain tissues are considered likely sites of interaction with ELF-EFs. This is because the brain is an aerobic organ with one of the Q8 highest oxygen consumption rates in the body and contains a large amount of phospholipids rich in polyunsaturated fatty acids that are susceptible to peroxidation by free radicals (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 (Matsumoto et al., 1999; Fukui et al., 2001, 2002). Therefore, it could be concluded that lipid peroxidation might have a role in altered MMN responses in rats. This view is partially supported by our data indicating that the MMN amplitude was decreased in the E18-4 group with respect to the control group. However, we found no significant changes in other experimental groups even if lipid peroxidation was increased. In earlier studies, functional changes have also been reported to occur in response to induced electric fields of 100 mV/m or less (Sienkiewicz et al., 1993; Tenforde, 1993). Functional effects become progressively more, as the strength and duration of induced EF is increased. From our data, which is also in line with these findings, it is conceivable to suggest that there may be a threshold level of EF-induced lipid peroxidation that leads a reduction in MMN response. The possibility of other unexplored factors that may affect MMN must be considered. As no comparable report is available in the literature concerning the effect of ELFEFs on MMN, further work is required to understand the functional significance and mechanisms underlying the observed MMN changes in the E18-4 group. It is found that the latencies of ERP components were increased in E12-2 and E18-2 groups with respect to their control groups. Our results indicated that latencies of all ERP components showed a tendency for prolongation in response to the increasing level of intensity for a 2-week duration. A trend of prolongation of ERP latencies was seen. A similar increase in latencies of all components


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of ERPs was also observed in E12-4, but not in E18-4 groups compared with the C4 group. These results could be partially attributed to delayed or distorted transmission of sensory information to appropriate cortical centers by a 2-week ELF exposure. The other interpretation is that prolonged latencies of MMN response of ERPs observed in these groups may be an indication of an impaired change detection mechanism. On the other hand, latencies of all components returned to the control values in the E18-4 group. So the most striking finding of the current study was that the effect of ELF-EF on ERP components was not the same for Q9 different application periods. From these results , it could be suggested that the longer duration period of E18-kV/m EF probably modifies the antioxidant ability of the nervous system. This view was further supported by a previous report showing that high EFs activate the antioxidant activity of a living organism (Cieslar et al., 2003). It is possible that enhanced production of superoxide radicals might increase the synthesis of superoxide dismutase (SOD) presenting a compensatory reaction against oxidative stress. There are also some studies supporting this view (Trastman et al., 1991; Yeh et al., 1997; Guler et al., 2006). Therefore, it could be suggested that SOD has a pivotal role especially in protecting the brain against the damaging effects of super oxide radicals, which at least transiently increases in response to oxidative stress in biological systems as a defence. It is well known that the other important effect of free radicals is to induce apoptosis (Stoian et al., 1996). Excessive oxidative stress may cause apoptotic or necrotic cell death, depending on its strength. Therefore, apoptotic cells in the brain tissues of control and experimental groups were examined. However, TUNEL-positive cells in experimental groups did not differ from the C group. So our data might indicate that apoptosis was not induced by increased free radicals in the brain of rats exposed to 12- and 18-kV/m strength of ELF-EF for 2and 4-week application periods (1 h/day). However, having no TUNEL-positive cells detected in all groups does not necessarily mean that the experimental group did not differ from the control group. No doubt, further studies that include more comprehensive methods are needed to verify these results. In summary, our finding may have an important implication only for the 4-week exposure to ELF EF with an intensity of 18-kV/m, which decreases the MMN amplitude indicating reducing effect on the auditory processing. Thus, it could be suggested that lipid peroxidation might have a role in the reduction of auditory information processing if it increases to the threshold level, which must be effective. On the other hand, in our current study the exposure periods of 12-kV/m and 18-kV/m were at 4 weeks, adaptive changes might occur to counteract free-radical effects of ELF EF that might induce a defense response to protect brain tissues from oxidative damage. Therefore, from our findings, it could be concluded that the cognitive effects of ELF electric fields depend on the dosage and duration of ELF exposure.

11

1. UNCITED REFERENCE (Pereira et al., 2013).

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Acknowledgment—This study was supported by a grant from the Akdeniz University Research Foundation, Turkey (Grant No. 2008.01.0103.008).

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(Accepted 26 April 2014) (Available online xxxx)


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Mismatch Negativity Affects Muscle Fatigue during Repeated Contraction Trials of Different Durations.

Effects of day-time exposure to different light intensities on light-induced melatonin suppression at night.

Effects of extremely low-frequency electromagnetic fields (ELF-EMF) exposure on B6C3F1 mice.

Different effects of alcohol on automatic detection of colour, location and time change: a mismatch negativity study.

The developmental effects of extremely low frequency electric fields on visual and somatosensory evoked potentials in adult rats.

Perspectives on health effects of electric and magnetic fields.

Effects of electric fields on fibroblast contractility and cytoskeleton.

M-wave potentiation after voluntary contractions of different durations and intensities in the tibialis anterior.

Data on the auditory duration mismatch negativity for different sound pressure levels and visual perceptual loads.

Considering effects of nanosecond pulsed electric fields on proteins.

Effects of nanosecond pulse electric fields on cellular elasticity.

Effects of higher-order aberration correction on stereopsis at different viewing durations.

Mismatch negativity: translating the potential.

Biological effects of exposure to static electric fields in humans and vertebrates: a systematic review.

Memantine Effects On Sensorimotor Gating and Mismatch Negativity in Patients with Chronic Psychosis.

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A neurocomputational model of the mismatch negativity.

Dosimetric extrapolations of extremely-low-frequency electric and magnetic fields across biological systems.

Demonstration of mismatch negativity in the monkey.

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