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Electromagnetic radiation 2450 MHz exposure causes cognition
deficit with mitochondrial dysfunction and activation of intrinsic
pathway of apoptosis in rats
SUKESH KUMAR GUPTA
1
,MANOJ KUMAR MESHARAM
2
and SAIRAM KRISHNAMURTHY
1
*
1
Neurotherapeutics Laboratory, Department of Pharmaceutical Engineering and Technology,
Indian Institute of Technology, Banaras Hindu University,
Varanasi 221 005, India
2
Department of Electronics Engineering, Indian Institute of Technology, Banaras Hindu University,
Varanasi 221 005, India
*Corresponding author (Email, ksairam.phe@iitbhu.ac.in)
MS received 19 August 2017; accepted 9 February 2018; published online 24 March 2018
Electromagnetic radiation (EMR) can induce or modulate several neurobehavioral disorders. Duration and frequency of
exposure of EMR is critical to develop cognitive disorders. Even though EMR-2450 is widely used, its effects on cognition
in relation to mitochondrial function and apoptosis would provide better understanding of its pathophysiological effects.
Therefore, a comparative study of different frequencies of EMR exposure would give valuable information on effects of
discrete frequencies of EMR on cognition. Male rats were exposed to EMR (900, 1800 and 2450 MHz) every day for 1 h
for 28 consecutive days. The cognitive behavior in terms of novel arm entries in Y-maze paradigm was evaluated every
week after 1 h to last EMR exposure. Animals exposed to EMR-2450 MHz exhibited significant cognitive deficits. EMR-
2450 MHz caused loss of mitochondrial function and integrity, an increase in amyloid beta expression. There was release of
cytochrome-c and activation of apoptotic factors such as caspase-9 and -3 in the hippocampus. Further, there was decrease
in levels of acetylcholine, and increase in activity of acetyl cholinesterase, indicating impairment of cholinergic system.
Therefore, exposure of EMR-2450 in rats caused cognitive deficit with related pathophysiological changes in mitochondrial
and cholinergic function, and amyloidogenesis.
Keywords. Apoptosis; cognition; electromagnetic radiation – 900, 1800, 2450 MHz; hippocampus; mitochondria
1. Introduction
Mobile phones are now an integral part of modern
telecommunications. There is an estimated 6.9 billion sub-
scriptions globally (Brahima 2015). It has been reported that
mobile phones like 2G and 3G emit radiofrequency of non-
ionizing electromagnetic radiation (EMR) in the range of
869–900 MHz (Code division multiple access; CDMA),
935–960 MHz (Global system for mobile communication;
GSM-900), 1805–1880 MHz (GSM-1800) and
2110–2450 MHz (3G and Wi-Fi) (Dogan et al. 2012;
Naziroglu et al. 2012). Since it is widely used by the pop-
ulation in various applications, it may lead to potential
irradiation into the environment and associated health haz-
ards to humans. There are reports to suggest that occupa-
tional exposure to EMR may increase risk of
neurodegenerative diseases (Jiang et al. 2013). There is no
consensus regarding whether EMR exposure could cause
potential detrimental effects in whole animals or isolated
brain regions. The use of the new generation mobile phones
has been reported to initiate symptoms like headache, sleep
anomalies (Hossmann and Hermann 2003) and cognitive
impairment (Levin 2015).
Earlier studies have reported that long term low fre-
quency of EMR exposure altered the cognitive behavior in
rats. This leads to a marked hindrance in learning and
recall of memory tasks and may cause a further risk of
developing Alzheimer’s disease (AD) (Dogan et al. 2012;
Naziroglu et al. 2012; Jiang et al. 2013). Similarly, recent
reports observed that EMR-900 MHz and -1800 MHz may
lead to cognitive impairments due to decrease in the level
of neurotransmitters (Narayanan et al. 2015). However, till
date there is no report on the comparative effects of EMR
(900, 1800 and 2450 MHz) exposure on the hippocampus.
Therefore, the present study was designed to compare the
effect of different EMR radiation (900, 1800 and
2450 MHz) on mitochondrial-linked hippocampus-directed
cognitive behavior. It has already been shown that EMR-
http://www.ias.ac.in/jbiosci 263
J Biosci Vol. 43, No. 2, June 2018, pp. 263–276 ÓIndian Academy of Sciences
DOI: 10.1007/s12038-018-9744-7
2.45 GHz at low intensity (50–230HZ) for short time
period (15–120 min daily for 7 days) decreases the level of
acetylcholine (Ach) with increase in enzymatic activity of
acetylcholineterase (AchE) in the hippocampus. This could
lead to cognitive disorders or memory dysfunction in rats
(Lai et al. 1987; Wang and Lai 2000; Afrasiabi et al.
2014).
Mitochondria are essential for typical cognitive functions
in the experimental animals (Joshi et al. 2014; Tanaka et al.
2008). Mitochondrial function can regulate synaptic release
of acetylcholine (Pochynyuk et al. 2002). It has been shown
that mitochondrial dysfunction leads to decrease in acetyl-
choline release (Lykhmus et al. 2014). Mitochondria
impairment leads to production of reactive oxygen species
(ROS) (Guo et al. 2013). Several studies report that amyloid
beta (Ab) is localized to mitochondrial membranes (Glenner
and Wong 1984; Villemagne et al. 2013). Oxidative stress
and accumulation of Ab1–40 are implicated in the patho-
genesis of AD (Chen and Yan 2010;Fuet al. 2016; Zhang
et al. 2016). In particular, the Ab1–40 has been circum-
stantially linked to the neurotoxic principle causing cell
death in the disease (Neve et al. 1990). Alternatively,
deposition of amyloid beta in cases such as AD may alter
mitochondrial morphology leading to impaired neurotrans-
mission of Ach, which may ultimately result in synaptic
damage and neurodegeneration causes cognitive impairment
(Chen and Yan 2010; Pinho et al. 2014). However, there are
very few reports on the effects of EMR on mitochondrial
function and related physiological effects. Microwave fre-
quency exposure has been reported to alter the hippocampal
mitochondrial cristae morphology observed by histopatho-
logical analysis (Zhao et al. 2012). Mitochondrial stress and
ensuing dysfunction could lead to activation of apoptotic
factors leading to cell death (Ott et al. 2007). These apop-
totic events are initiated by over activation of caspases-9/3,
which has been suggested to be a hallmark in the induction
of apoptotic cell death, and may further lead to cognitive
impairments (Ja¨nicke et al. 1998). However, the effect of
EMR on mitochondrial-linked cell death is yet to be
explored (Yang et al. 2015). Therefore, study of effects of
EMR on mitochondrial function and apoptosis would pro-
vide valuable information to understand the pathophysio-
logical mechanisms of cognitive dysfunction.
Therefore, the present study evaluated the effects of dis-
crete frequencies of EMR on cognitive behavior and asso-
ciated pathophysiological mechanisms. Mitochondrial
integrity, complex enzyme and oxidative stress were evalu-
ated to understand the effects of EMR on mitochondrial
function. The expression of Ab1–40 was studied to explore
the effect of EMR on amyloidogenesis. Further, the effect of
EMR on the level of Ach and the activity of AChE were
investigated. Furthermore, the mitochondrial mediated
apoptosis was assessed by the expression of cytochrome-c,
caspase-9 and caspase-3.
2. Materials and methods
2.1 Animals
Inbred Charles–Foster albino male rats (150–180 g) were
collected from Institute of Medical Sciences, Banaras Hindu
University and were housed at 26±2°C, relative humidity
44–56% and light:dark cycle of 12:12 h. Animals were
provided with standard rodent pellet diet (Hind liver) and
water ad libitum. The experiment was conducted in accor-
dance with the Committee for the Purpose of Control and
Supervision of Experiments on Animals (CPCSEA-2010;
Institute of Medical Sciences, Banaras Hindu University)
guidelines (Approval No.: Dean/2015/CAEC/1414).
2.2 Chemicals
AchE, Choline oxidase, horse radish peroxidase, nitroblue
tetrazolium (NBT) and Griess reagent were procured from
sigma aldrich (St. Louis, MO, USA). Tetramethyl rhodamine
methyl ester (TMRM) was purchased from Thermofischer.
All antibodies used in this experiment were purchased from
Abcam (UK). All other chemicals and reagents of high
performance liquid chromatography (HPLC) and analytical
grade were procured from Hi-media, Mumbai, India.
2.3 Electromagnetic radiation exposure system
and design
The electromagnetic radiation exposure system and design
are shown schematically in figure 1. The animals were kept
in a wooden cage having dimension of 46930940 cm
having sufficient free space for propagation of EMR for
maintaining low dielectric constant (1.25). Hence, the
wooden cage was divided into 6 compartments with 2 rows
of 3 compartments with dimension 15914.5940 cm, having
ventilation and restrainer (to keep animal immobile) facility
during the exposure of EMR. The compartment was
designed in such a way which facilitated the propagation of
radiation parallel to the electric field. The upper surface and
the base of the cage were wrapped with lining of carbon
sheets impregnated with Styrofoam absorber in order to
minimize the reflections of electromagnetic radiation.
Thereafter, the wooden cage was placed and fixed to an
anechoic chamber of dimension 65965960 cm, impreg-
nated with Styrofoam radiation absorber (to reduce the
reflection of radiation towards horn antenna). Then walls of
the anechoic chamber were surrounded by cones and foam to
shield the back reflections same as reported by (Holloway
et al. 2002). The horn antenna (Scientific Instrument Co.
Ltd. SB-771, India) was kept in such a manner that it
received uninterrupted the radiations from the source. The
264 Sukesh Kumar Gupta et al.
temperature of anechoic chamber was kept constant 25±2°C
throughout the exposure period.
The horn antenna was connected to the generator, which
emitted continuous radiofrequency (RF) signals of 900, 1800
and 2450 MHz. The repetition time, frequency and ampli-
tude was continuously monitored by the Analog Signal
Generator (Model No. MXG N5183A manufactured by
Agilent Technologies, USA) covering the frequency range
from 100 KHz to 20 GHz. The exposure system also has a
microwave amplifier (Model no. 8349B Hewlett Packard
Co) connected with waveguide transition, 20 dB cross
coupler, E-plane bend and pyramidal horn antenna (made of
brass and coated with silver). The set-up also has a power
meter with power sensor (Model No. 836A manufactured by
Agilent technologies) to measure the maximum output
power. This was measured to be 19.9 dBm which was
delivered to the horn antenna and amplifier. The power meter
has a range from 30 lW to 3 W (Model no. 8481H, Hewlett
Packard Co.). The antenna used has neck and opening
aperture dimensions 7.2 cm 93.2 cm and 9.0 95.0 cm
respectively while its axial length from the neck is 10 cm.
Comparison method was used to measure the gain of the
horn antenna (Gt), which was found to be 1.965, 2.862 and
4.8657 (for 900, 1800 and 2450 MHz). The distance
between the pyramidal horn and the mid plane of body of the
animal was estimated by far field formulae (RC2D
2
/k)tobe
25 cm. The rats were kept inside the cage and positioned at
distance of 8, 11 and 15 cm from the antenna for exposure to
900, 1800 and 2450 MHz respectively to maintain the
required power density, which is described in the following
section. The cross-sectional dimensions of the animal cage
in E- and H-planes (electric and magnetic field planes,
respectively) were designed on the basis of E=6.798, 6.742
and 6.754 V/m and H=0.01803, 0.01806 and 0.018.8 A/m
for 900, 1800 and 2450 MHz fields in corresponding planes
of horn (Balanis 1997; Fontolliet 1996; Langrange et al.
Pyramidal
Horn
Antenna
Holes for Ventilation
Anechoic chamber with
Animal cage
EMR Exposure
Source
Isolater Power Amplifier Wave Guide
Transition
Power Sensor with
Power Meter
Matched load
Figure 1. Schematic representation of the EMR exposure set-up and the position of animal cages during exposure. Experimental rats were
exposed to 900, 1800 and 2450 MHz EMR exposure radiation for 1 h/day for each group continuously for 28 days by using EMR source
and pyramidal horn antenna.
Effect of EMR on cognition 265
1999). The power input to the horn antenna and the reflec-
tions were measured by using a 20 dB cross-coupler, power
sensor and a meter of range 30 lm to 3 W (Model no.
8481H, Hewlett Packard Co., USA). The power that was
transmitted (Pt) to the antenna was estimated by subtracting
the reflected power with input power, which was found to be
50.19, 65.15 and 71.43 mW for 900, 1800 and 2450 MHz,
respectively.
2.4 Calculation of power density and specific
absorption rate
The power density obtained by using the following formula:
Power Density=P
t
G
t
/4pR
2
, where P
t
is the power transmitted
into the cage, G
t
is the gain of the horn and R is the distance
between the horn aperture and mid plane of the rat in the
cage. The animals after being exposed to 900, 1800 and
2450 MHz EMR, the average power density was found to be
0.1227 W/m
2
. While the overall value of whole body aver-
age SAR was found to be approximately, 0.0227, 0.030 and
0.0616 W/kg for 900, 1800 and 2450 MHz respectively. The
measurement of SAR value was dependent upon arrange-
ment of rat in animal cage (Paxinos and Watson 1986;
Gandhi 1990; Repacholi et al. 1997). Experimental rats
(n=6) were subjected to continuous modulated electromag-
netic radiation exposure of 900, 1800 and 2450 MHz (for
modulation: modulating signal 217 Hz, modulation index
0.1%), for daily 1 h for each group for 28 days between
10.00 AM to 1.00 PM. Control group was placed in ane-
choic chamber without any exposure. The average rectal
temperatures of rat were observed to be 33.71±0.56 (con-
trol), 35.3±0.63 (EMR-900 MHz), 35.48±0.67 (EMR-
1800 MHz) and 36.96±0.55 (EMR-2450 MHz) before and
33.91±0.82 (control), 35.5±0.51 (EMR-900 MHz),
35.66±0.60 (EMR-1800 MHz) and 37±0.78 (EMR-
2450 MHz) just after completion of exposure. These chan-
ges were not statistically significant as analysed by Two-
way ANOVA.
2.5 Experimental design
The rats were randomly divided into four groups with six
animals each, namely: (i) control (animals not exposed to
EMR radiation but kept under same conditions as that of
other groups), (ii) animals irradiated at EMR-900, (iii) ani-
mals irradiated at EMR-1800 and (iv) animals irradiated at
EMR-2450 MHz. The animals were exposed to EMR of 900,
1800 and 2450 MHz frequencies between 10 am to 2 pm for
1 h from D-1 (day-1) to D-28 (day-28) of experimental
schedule. One hour after EMR exposure on D-1, 7, 14, 21
and 28 of experimental protocol all the animals were sub-
jected to Y-maze paradigm. All the behavioral observations
were recorded and evaluated using ANY-maze
TM
(version-
3.72, USA) video tracking system. On D-28 day of EMR
exposure immediately after behavioral experiment, the rat
brains were dissected out immediately after decapitation. The
hippocampal regions were quickly isolated using the coor-
dinates from rat brain atlas (Paxinos and Watson 1986) over
ice (4°C) for further molecular studies.
2.6 Assessment of behavioral performance
2.6.1 Evaluation of spatial recognition memory in Y-maze
test: In the Y-maze paradigm, general exploratory attitude
(curiosity), spatial recognition memory and anxiety-like
behavior were assessed as the total number of entries in all
arms (15 min for trial 1 and 5 min for trial 2). The per-
centage of entries in known and novel arms for the 5 min
period of trial 2 (for the general exploratory attitude), the
percentage ratio of time spent in novel arm to time spent in
all the arms (spatial recognition memory) and at the center of
the apparatus during trial 2 were taken into count to assess
the anxiety-like behavior (Krishnamurthy et al.2013).The
three identical arms (50 cm long, 16 cm wide and 32 cm
high) of Y-maze at 120 angles to each other, radiating out
from a central point were used to assess the above behavioral
tasks. Visual cues were made from colored construction
paper and laboratory glassware was placed around the
perimeter of the maze and above the top of the black Plex-
iglas sides. These cues were not repeated for each test to
maintain novelty to the animals. The floor of the maze was
covered with animal bedding. The Y-maze novel arm was
blocked and rats were allowed to visit the other two arms of
the maze for 15 min. Four hours after the first phase, the
novel arm was unblocked and animals had free access to all
three arms for 5 min. The number of entries in each arm was
recorded for a 5-min period. The dependent variables such as
the total number of entries in all arms (for the trial 1 and 2),
the percentage of entries in known and novel arms for the
5 min period of trial 2 and the percentage ratio of time spent
in novel arm to time spent in all the arms and at the center of
the apparatus during trial 2 were measured. The total number
of entries in the trial 1 and 2 is a sign of general exploration
attitude (curiosity) and the percentage of entries in known
versus novel arms in trial 2 was appraised as a measure of
arm discrimination (spatial recognition memory). Coping
strategy or behavior to novel environment was assessed by
the percentage of time spent in the novel arm to time spent in
all arms and at the center of the apparatus during trial 2. An
increase in anxiety-like behavior was confirmed by decrease
in the coping behavior to novel environment (Poimenova
et al. 2010). An arm entry was counted when the head and
two front paws were inside the arm, and duration of an arm
visit was ended when the head and two front paws were
outside the arm again.
266 Sukesh Kumar Gupta et al.
2.7 Assessment of mitochondrial function, integrity
and oxidative stress
2.7.1 Isolation of mitochondria from rat brain: The mito-
chondria were isolated from hippocampus by following
standard protocol (Pedersen et al. 1978). The mitochondrial
protein content was estimated using the method of Lowry
et al. (1951).
2.7.2 Estimation of mitochondrial function: The (3-(4,
5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide;
MTT) reduction was used to assess the mitochondrial
function by estimating formazan formed at 595 nm (Kamboj
et al. 2008). Results were expressed as mg formazan formed/
min/mg protein.
2.7.3 Evaluation of mitochondrial membrane potential
(MMP): The MMP was assessed with the help of fluorescent
cationic dye tetramethylrhodamine methyl ester (TMRM;
Catalogue number Thermo fischer T668) to assess the
mitochondrial integrity. The peak fluorescence intensity was
recorded around 570±5 nm. The results were expressed as
fluorescence intensity value/mg protein (Hitachi, F-2500,
Japan) (Huang 2002).
2.7.4 Estimation of NADH dehydrogenase (complex-I)
activity: Activity of NADH dehydrogenase was evaluated at
excitation and emission wavelength of 350 nm and 470 nm
respectively (Shapiro et al. 1979). Results were expressed as
nano-mole NADH oxidized/min/mg protein.
2.7.5 Assessment of succinate dehydrogenase (complex-II)
activity: The activity of mitochondrial succinate dehydro-
genase (SDH) was determined at 570 nm (Sally and Mar-
garet 1989). Results were reported as micro-mole formazan
produced/min/mg protein.
2.7.6 Estimation of cytochrome c oxidase (complex-IV)
activity: The activity of complex-IV was assessed as per
method (Storrie and Madden 1990). The decrease in absor-
bance was measured at 550 nm for 3 min. Results were
expressed as nmole cytochrome c oxidized/min/mg protein
(e550=19.6 mmol
-1
cm
-1
).
2.7.7 Estimation of F1F0 ATP synthase (complex-V) activ-
ity: Mitochondrial F1F0 synthase was measured according to
the method of Griffiths and Houghton (1974) the inorganic
phosphate concentration was measured by the method of
Fiske and Subbarao (1925). Results were expressed as n
mole ATP hydrolysed/min/mg protein.
2.7.8 Estimation of lipid peroxidation: Assessment of
mitochondrial malonaldehyde (MDA) was measured (Sun-
dermann et al. 1985). The absorbance of sample was
measured spectrophotometrically at 532 nm and results were
expressed as lmol MDA/mg of protein (Ohkawa et al.
1979).
2.7.9 Assessment of nitric oxide level: The NO level was
estimated by following standard protocol (Green et al. 1982)
using Griess reagent (0.1% at 540 nm) and results were
expressed as nano-moles of NO/mg protein.
2.7.10 Assay of superoxide dismutase: Mitochondrial
superoxide dismutase (SOD) was estimated by the reduction
of NBT in the presence of NADH, Phenazine Methosulphate
at 560 nm (Kakkar et al. 1984). One unit of the enzyme was
represented as 50% inhibition of NBT reduction/minute/mg
of protein of assay conditions. The absorbance of sample
was measured at 560 nm using n-butanol as blank. The
results were expressed as units [U] of SOD activity/mg of
protein.
2.7.11 Assay of catalase activity: Decomposition of H
2
O
2
in
the presence of catalase was determined at 240 nm for 3 min
at 30 s interval by the method of Beers and Sizer (1952).
Results were expressed as units [U] of Catalase activity/mg
of protein.
2.8 Evaluation of cholinergic system
2.8.1 Preparation of the samples and Estimation of acetyl-
choline using spectrofluorometer: The hippocampus tissue
was homogenized and kept in the polypropylene tubes for
15 min after which 50 lL of 4 M potassium acetate was
added to adjust the pH to 4.0 followed by centrifugation for
15 min at 4000 g(Muthuraju et al. 2009). The Ach level
was measured by using amplex red kit (Molecular Probes
Inc. USA) in a hippocampus as per the protocol (Zoukhri
and Kublin 2001). The AChE activity was estimated using
the Amplex red AchE assay kit (Molecular Probes Inc. USA)
in terms of fluorescence with the help of spectrofluorometer
at 530 nm excitation wavelength and 590 nm emission
wavelength and the protein content was measured by Lowry
method (Lowry et al. 1951).
2.9 Western blotting analysis
For Western blot analysis, the hippocampus was lysed in
buffer containing complete protease inhibitor cocktail. Pro-
tein concentrations were determined according to Bradford’s
method (Bradford 1976). A standard plot was generated
using bovine serum albumin. An aliquot of each sample
were electrophoresed in 10% SDS-PAGE gels for Ab,
cytochrome-c, caspase-9 and active caspase-3 proteins,
transferred to polyvinylidene fluoride membranes and
Effect of EMR on cognition 267
probed with specific antibodies. The membrane was incu-
bated overnight with, rabbit anti-beta Amyloid (1:500,
87 kDa; ab62658; Abcam plc., India), rabbit anti-cy-
tochrome-c (1:500, 15 kDa; ab13575; Abcam plc., India),
rabbit anti- Caspase-9 (1:200, 45 kDa; ab47537; Abcam
plc., India) and rabbit active anti-caspase-3 (1:1000, 32 kDa;
ab90437; Abcam plc., India) polyclonal primary antibodies.
After detection with the desired antibodies against the pro-
teins of interest the membrane was stripped with stripping
buffer (mercaptoethanol, Tris-Hcl and SDS) for 30 min at
room temperature and re-probed overnight with rabbit anti
b-actin (1:500, 42 kDa; ab93027; Abcam plc., India) poly-
clonal primary antibody to confirm equal loading of protein.
Further, membrane was probed with corresponding sec-
ondary antibodies. Immunoreactive band of proteins were
detected by chemiluminescence using enhanced chemilu-
minescence (ECL) reagents (Amersham Bioscience, USA).
Quantification of the results was performed by densitometric
scan of films. The immunoreactive area was determined by
densitometric analysis using Biovis gel documentation
software.
2.10 Statistical analysis
All values were expressed as mean±standard error of mean
(S.E.M.). Two-way ANOVA followed by Bonferroni post
hoc test was performed to estimate total arm entries in trials
1 and 2 and percentage entries into known and novel arm in
trial-2 in Y-maze test. Arm discrimination behavior between
known and novel arm was assessed by using Two-way
ANOVA with Bonferroni test in Y-maze paradigm. All other
statistical analysis of data was performed by using One-way
ANOVA with Newman–Keuls post hoc test. P\0.05 was
considered as significant.
3. Results
3.1 EMR attenuated the spatial recognition memory
in Y-maze paradigm
Figure 2depicts the effect of EMR (900, 1800 and
2450 MHz) induced alterations in the exploratory behavior
(curiosity) in trial 1 and 2 depicted in figure 2A and B
respectively. Figure 2C shows the coping behavior to novel
arm (anxiety-like behavior) in Y-maze test. Repeated mea-
sure two-way ANOVA revealed significant differences for
curiosity in trial-1 [F (3,100)=45.60; P\0.0001] among
groups, time [F (4,100)=11.11; P\0.0001] and a significant
interaction between groups and time [F (12,100)=9.982;
P\0.0001]. Similarly, there was significant differences for
curiosity in trial-2 [F (3,100)=36.65; P\0.0001] among
groups, time [F (4,100)=13.87; P\0.0001] and a significant
interaction between groups and time [F (12,100)=13.73;
P\0.0001] respectively. Furthermore, there were significant
differences in coping behavior among groups [F
(3,100)=14.94; P\0.0001], time [F (4,100)=3.728;
P\0.0072] and an interaction between group and time [F
(12,100)=3.917; P\0.0001]. Post hoc analysis using Bon-
ferroni test revealed that the effect of EMR 900 and
1800 MHz did not change the curiosity both in trial-1 and 2
and, in the coping behavior (P[0.05). Whereas EMR
(2450 MHz) significantly decreased the curiosity and
increased coping behavior on D-21 and this effect was
observed up to D-28 compared to vehicle administered rats.
Figure 2. The effect of EMR (900, 1800, and 2450 MHz)
exposure ensuing alterations in the total arm entries in trials 1
and 2 (curiosity; A), total novel arm entries (spatial recognition
memory; B), and coping behavior to novel arm (anxiety-like
behavior; C) in Y-maze paradigm. All results are expressed as
mean±SEM, (n=6).
a
P\0.05 compared to control,
b
P\0.05 com-
pared to EMR-900 and
c
P\0.05 compared to EMR-1800. Repeated
measure two-way ANOVA followed by Bonferroni test for
curiosity analysis and percentage entries into known and novel arm.
268 Sukesh Kumar Gupta et al.
EMR (900, 1800 and 2450 MHz)-induced alterations in
the spatial recognition memory on D-1 (A), D-7 (B), D-14
(C), D-21 (D) and D-28 (E) are depicted in figure 3.Two
way ANOVA showed significant differences in arm dis-
crimination behavior during trial-2 among groups on D-1
[F(3,40)=1.497; P\0.2299 ], D-7 [ F(3,40)=6.025;
P\0.0017], D-14 [F(3,40)=4.486; P\0.0083], D-21 [
F(3,40)=19.39; P\0.0001] and D-28 [F(3,40)=59; P\0.0001
respectively]. Significant effect for known and novel arms
entries on D-1 [F (1, 40)=31.68; P\0.0001], D-7 [F (1,
40)=34.92; P\0.0001], D-14 [F (1, 40)=25.53; P\0.0001],
D-21 [F (1, 40)=13.53; P\0.0007] and D-28
[F(1,40)=3.944; P\0.0439 respectively]. There was signifi-
cant interaction between group and total arm entries on D-1
[F(3,40)=0.1068; P\0.9546], D-7 [F(3,40)=2.589;
P\0.0663], D-14 [F(3,40)=1.830; P\0.1571], D-21
[F(3,40)=8.396; P\0.0002] and D-28 [F(3,40)=0.7988;
P\0.05019 respectively] in Y-maze paradigms. Post hoc
analysis revealed that EMR-2450 MHz exposed experi-
mental animals showed cognitive impairments in terms of
Figure 3. The effect of EMR (900, 1800, and 2450 MHz) exposure ensuing alterations in arm discrimination behavior during Y-maze test
paradigm on D-1 (A), D-7 (B), D-14 (C), D-21 (D) and D-28 (E) of the experimental schedule. All values are expressed as mean±SEM,
(n=6).
*
P\0.05 compared to corresponding known arm entries (two-way ANOVA followed by Bonferroni test).
a
P\0.05 compared to
control,
b
P\0.05 compared to EMR-900 and
c
P\0.05 compared to EMR-1800 (repeated measure two-way ANOVA followed by
Bonferroni test).
Effect of EMR on cognition 269
decrease in novel arm entries on D-21 and this effect was
persisted up to D-28. However, EMR (900, 1800 MHz) did
not show significant changes in novel arm entries on all days
of experimental protocol compared to control rodents.
3.2 Estimation of Ach and AchE levels in EMR-exposed
rats
Figure 4illustrates the effect of EMR (900, 1800 and
2450 MHz)-induced changes in concentration of Ach and
AchE activity in hippocampus. One-way ANOVA revealed that
there were significant differences in (A) Ach [F (3, 20)=14.8;
P\0.0001] and (B) AchE [F (3, 20)=34.6; P\0.0001] among
groups. Post hoc analysis revealed that EMR (2450 MHz)
significantly decreased levels of Ach and increased the AchE
levels compared to control rats respectively.
3.3 Quantization of expression of b-Amyloid
in hippocampus of EMR-subjected rats
The effect of EMR (900, 1800 and 2450 MHz)-induced
changes in the level of bAmyloid in brain tissues of rodents
is depicted in figure 5. There were significant differences in
bAmyloid [F (3, 8)=9.3; P\0.0050] among groups. Post
hoc analysis demonstrated that EMR at the frequency of
2450 MHZ increased the expression of bAmyloid compared
to control rats.
3.4 EMR mitigated the mitochondrial integrity
in hippocampus of animals
The effect of discrete range of EMR induced alterations in
MMP in EMR subjected rats is depicted in figure 6. One
way ANOVA revealed that there were significant differences
in MMP [F (3, 20)=36.91; P\0.0001] among groups. Post
hoc analysis revealed that EMR (2450 MHz) significantly
decreased the intensity of TMRM indicating loss of mito-
chondrial integrity.
3.5 EMR showed the increase in expression
of cytoplasmic cytochrome-c, caspase-9 and caspase-3
in hippocampus
Figure 7depicts the effect of EMR (900, 1800 and
2450 MHz)-induced alterations in the levels of (A) cy-
tochrome-C, (B) caspase-9 and (C) caspase-3 in the hip-
pocampus. There were significant differences in expression
of cytochrome-C [F (3, 8)=22.4; P\0.0003], caspase-9 [F (3,
8)=15.8; P\0.0010] and caspase-3 [F (3, 8)=7.9; P\0.0090]
among groups. Post hoc test revealed that EMR (2450 MHz)
Figure 4. Effect of EMR (900, 1800 and 2450 MHz) induced
changes in the level of Ach (A) and activity of AChE (B) in HIP.
All values are Mean±SEM.
a
P\0.05 compared to control,
b
P\0.05
compared to EMR-900 and
c
P\0.05 compared to EMR-1800 (one-
way ANOVA followed by Student–Newman–Keuls test).
Figure 5. The effect of EMR (900, 1800 and 2450 MHz)
exposure induced changes in the level of expression of Ab1–40
in hippocampus. The blots are representative of Ab1–40 (A) in HIP.
The results in the histogram are expressed as ratio of relative
intensity of levels of protein expression of Ab1–40 to b-Actin (B).
All values are Mean±SEM.
@
p\0.05compared to Hippocampus of
respective group,
a
P\0.05 compared to control,
b
P\0.05 compared
to EMR-900 and
c
P\0.05 compared to EMR-1800 (one-way
ANOVA followed by Student–Newman–Keuls test).
270 Sukesh Kumar Gupta et al.
showed significant increase in the expression of cytochrome-
C, caspase-9 and caspase-3 compared to control rodents.
3.6 Evaluation of EMR-exposed modulations
in mitochondrial enzyme activities
The effect of EMR (900, 1800 and 2450 MHz)-induced
changes in the activities of mitochondrial complex-I, II, IV
and V in hippocampus is depicted in table 1. One way
ANOVA statistical analysis revealed that there were signif-
icant differences in mitochondrial complex-I, II, IV, V
enzyme activities among groups [F (3, 20)=12.25,
P\0.0023], [F (3, 20)=1.521; P\0.02821], [F (3, 20)=4.528;
P\0.0389] and [F (3, 20)=4.911; P\0.0223] respectively.
Post hoc test illustrated that EMR (2450 MHz) showed the
significantly decreased complex-I, II, IV and V activities in
hippocampus compared to control animals.
3.7 Assessment of the mitochondrial oxidative
and nitrosative stress markers in EMR-induced animals
Table 2shows the effect of EMR (900, 1800 and
2450 MHz)-induced alterations in the levels of (A) LPO,
(B) NO, (C) SOD and (D) Catalase in the brain tissues.
There were significant differences in LPO [F (3, 20)=17.74;
P\0.0007], NO [F (3, 20)=3.341; P\0.013], SOD [F (3,
Figure 6. Effect of EMR (900, 1800 and 2450 MHz) exposure on
alterations in the mitochondrial function (A) and integrity (B)in
hippocampus. All values are Mean±SEM.
a
P\0.05 compared to
control,
b
P\0.05 compared to EMR-900 and
c
P\0.05 compared to
EMR-1800 (one-way ANOVA followed by Student–Newman–
Keuls test).
Figure 7. The effect of EMR (900, 1800 and 2450 MHz) exposure on modifications in the level of expression of cytochrome-c, casapse-3
and -9 in the hippocampus. The blots are representative of cytochrome-c, casapse-3 and -9 expressions (A). The results in the histogram are
expressed as ratio of relative intensity of levels of protein expression of cytochrome-c, casapse-3 and -9 in the hippocampus to that of b-
actin (B). All values are Mean±SEM.
a
P\0.05 compared to control,
b
P\0.05 compared to EMR-900 and
c
P\0.05 compared to EMR-1800
(one-way ANOVA followed by Student–Newman–Keuls test).
Effect of EMR on cognition 271
20)=3.588; P\0.044] and catalase [F (3, 20)=4.085;
P\0.0252] among groups. Post hoc test revealed that the
concentration of MDA and NO significantly increased by
highest frequency of EMR compared to control animals.
Furthermore, EMR (2450 MHz) modulated the activities of
SOD and catalase in hippocampus of rodents.
4. Discussion
In the present study, experimental animals exposed to EMR
at frequency of 2450 MHz exhibited cognitive dysfunction.
For the first time we report that the cognitive deficit was
associated with hippocampal mitochondrial dysfunction and
amyloidogenesis. In contrast, EMR-900 and EMR-1800 did
not show any effect on cognitive function.
EMR-2450 exposure significantly induced cognitive def-
icit in rats. Y-maze paradigm is commonly used for the
assessment of cognitive impairments (McEwen et al. 1997).
Y-Maze accentuates spatial recognition memory, visuospa-
tial tasks in addition to hippocampus dependent tasks (Cai
et al. 2013). In the present study, we found that EMR-2450-
subjected rats showed abnormalities in number of behavioral
indices in the Y-maze paradigm like spatial recognition
memory (increase in percentage of entries into known arm)
and coping behavior (anxiety-like behavior) to novel envi-
ronment (decrease in time spent in novel arm to time spent in
all arms and in the center) on D-21 and D-28. However,
EMR-900 and EMR-1800 exposed rats did not show sig-
nificant differences in novel arm entries on D-21 and D-28
indicating the fact that these frequencies did not induce any
significant cognitive deficits. Earlier study (Choi and Choi
2016) reported that long term (10 weeks) exposure to elec-
tromagnetic radiation (smartphones) may induce delayed
hyperactivity like behavior without affecting spatial working
memory through Y-maze in brain. This task allows the
simultaneous assessment of hyperactivity independent of
spatial memory and it exploits the natural inclination of rats
to investigate their environment. The differences in obser-
vations may be due to the use of continuous frequency
exposure whereas the other study had pulsed exposure.
Previous studies have reported biological differences with
pulsed and continuous EMR exposure. Pulsed microwaves
alter not only the EEG but also regional cerebral blood flow,
and continuous exposure of EMR causes alterations in brain
physiology (Huber et al. 2005).
In consonant with our results, a report (Dubreuil et al.
2003) suggested that continuous GSM (900/1800 MHz)
electromagnetic radiations do not alter memory of rat in
spatial and non-spatial tasks. In contrast, other studies have
Table 1. The effect of EMR (900, 1800 and 2450 MHz) exposure on changes in the level of mitochondrial enzyme activities in the
hippocampus
S. No. Groups
Complex-I activity Complex-II activity Complex-IV activity Complex-V activity
(nmolNADH
oxidized/min/mg/protein)
(lmolformazan/
min/mg/protein)
(nmol cytochrome
c oxidized/min/mg/protein)
ATP hydrolysed
protein
1 Control 5.36±.27 0.49±.11 0.81±.08 12.41±.52
2 EMR900 5.21±.42 0.46±.07 0.76±.05 12.19±.57
3 EMR1800 5.14±.19 0.42±.09 0.72±.08 12.05±.47
4 EMR2450 3.10±.30
a,b,c
0.26±.05
a,b,c
0.47±.07
a,b,c
8.86±1.09
a,b,c
Values are Mean±SEM.
a
p\0.05 compared to control,
b
p\0.05 compared to EMR-900 group and
c
p\0.05 compared to EMR-1800 group (one-way ANOVA followed by
Student–Newman–Keuls test).
Table 2. The effect of EMR (900, 1800 and 2450 MHz) exposure on changes in the level of mitochondrial oxidative and nitrosative stress
markers in hippocampus
S. No. Groups
LPO level Brain nitrite level Catalase level SOD activity
(lM MDA/mg protein) (nM NO/mg protein) (nm at H-
2
O
2
/min/mg of protein) (units/min/mg of protein)
1 Control 0.78±.15 1.24±.85 0.42±.10 0.61±.0.09
2 EMR-900 0.75±.17 1.18±.71 0.38±.13 0.54±.11
3 EMR1800 0.71±.12 1.14±.77 0.36±.11 0.51±.07
4 EMR2450 2.09±.19
a,b,c
1.85±.66
a,b,c
0.15±.21
a,b,c
0.25±0.05
a,b,c
Values are Mean±SEM.
a
p\0.05 compared to control,
b
p\0.05 compared to EMR-900 group and
c
p\0.05 compared to EMR-1800 group (one-way ANOVA followed by
Student–Newman–Keuls test).
272 Sukesh Kumar Gupta et al.
reported learning and memory deficit in rats with the Morris
water maze (MWM) using pulsed 2450 MHz (Wang and Lai
2000). The MWM is a memory test, frequently used for
demonstrating visuospatial navigation, topographic disori-
entations and motivational deficits and to examine the
facilitations of content dependent behavior and reference
memory in rodents (Vorhees and Williams 2006). However,
in our study we have shown cognitive deficits using the
Y-Maze test. The Y-maze task is dependent on the clinically
relevant factors such as the speed of information processing
and psychomotor ability, executive functions, learning,
memory and general cognitive treatments (Conrad et al.
1997).
Earlier, a study has reported that exposure of EMR
900 MHz for 2 h daily over 28 days in rats with SAR
average in between 0.52–1.08 W/kg led to reduction of
synapses and decreased the postsynaptic density of neuron in
the hippocampus CA1 region, resulting spatial learning and
reference memory deficit in rats (Li et al. 2012). Similarly, it
has been shown that exposure of microwave radiation for
6 min (3 times in a week up to 6 weeks) causes shrinkage
and loss of dendritic spines in hippocampus lead to cognitive
impairments (Zhi et al. 2017). However, in our study we did
not observe cognitive deficit with EMR 900 MHz. The
reason may that the experimental design and duration of
exposure was different. Moreover, it has been shown that
loss of mitochondrial function could cause hippocampal
synaptic dysfunction that leads to memory deficits (Santini
and Turner 2015). Mitochondria are considered as the
principal site of the oxidative and nitrosative stress (Boru-
taite et al. 2013). Oxidative stress in addition to alteration in
functional changes like reducing complex activities and also
facilitates deposition of Abin mitochondria (Diana et al.
2008; Zuo et al. 2015). In present study EMR-2450 signif-
icantly increased the level of LPO and NO resulting in the
decrease in the level of antioxidant property causing damage
to hippocampus. In contrast, EMR-900 and 1800 MHz did
not show any changes in the level of LPO and NO. A pre-
vious report suggests that long term exposure of EMR-
2100 MHz increases LPO and NO production and decreases
the level of catalase and SOD (Hidisoglu et al. 2016). In the
present study, decrease in level of catalase and SOD activ-
ities in the 2450-MHz group indicated excessive level of
hydrogen peroxide and less decomposition of superoxide
radicals in the hippocampus. However, EMR-900 and
1800 MHz did not show any significant changes in oxidative
stress markers. Therefore, EMR-2450 MHz induces oxida-
tive stress in contrast to EMR-900 and 1800 MHz.
Mitochondrial membrane potential (MMP) is a key factor
for bioenergetics as it regulates the energy needs of cell (Ott
et al. 2007). MMP was significantly decreased with long
term exposure of EMR-2450 MHz while no significant
changes were observed with exposure of EMR-900
and1800 MHz. It is interesting to note that marked decrease
of fluorescence intensity was observed reflecting compro-
mised mitochondrial membrane integrity. However, in vitro
study has revealed that EMR-1800 MHz causes neurotoxi-
city due to mitochondrial DNA toxicity in neuron cultures
(Xu et al. 2010). This may be due to the compensatory
mechanisms stabilizing the mitochondria in vivo and also
may be due to difference in intensity and duration of EMR
exposure. Earlier study reported that exposure of microwave
radiation ranging from 300 MHz to 300 GHz causes alter-
ation of brain energy metabolism due to oxidative phos-
phorylation (Hao et al. 2015). Hence, exposure of EMR-
2450 MHz causes alteration of MMP and interferes with the
complexes-I, II, IV and V activities that predisposes to loss
of mitochondrial integrity leading to mitochondrial
dysfunction.
Aggregation of Abis observed in AD along with cogni-
tive deficits (Pozueta et al. 2013). The cellular concentration
of Abis kept within a precise range by balancing its syn-
thesis and degradation (Pinho et al. 2014). It has also been
suggested that Abis found in the several compartments of
the cell including mitochondria (Villemagne et al. 2013). In
the current study, EMR-2450 MHz shows increase in
expression of Abin contrast to the effect of EMR-900 and
1800 MHz in the hippocampus. It has been reported that
EMR 900 MHz exposure for ten months induced over
expression of Abin rats (Suleyman et al. 2012). In sum-
mary, 28 day’s exposure of EMR-2450 MHz induced
expression of hippocampus Ab, which may be one of the
reasons for EMR-induced cognitive deficits in rats.
Abaccumulation in the mitochondria can lead to irregu-
larities in the secretion of neurotransmitters such as acetyl-
choline. This may ultimately cause synaptic damage
followed by neurodegeneration and cognitive deficits (Grill
and Cummings 2010). The cholinergic system plays a crit-
ical role in performing cognitive tasks (Himmelheber et al.
2000). The activity of AChE regulates sustained extracel-
lular ACh release which plays a crucial role in restoring
cognitive function (Chen et al. 2016). Our results showed
that ACh and the activity of AChE were not altered by
EMR-900 and EMR-1800 animals. However, EMR-2450
significantly decreased the level of ACh with increase in the
enzymatic activity of AChE in the hippocampus. It has been
reported that EMR 2450 MHz exposure leads to neurode-
generation of cholinergic neurons followed by decrease in
level of Ach and increase in the activity of AChE with (Lai
et al. 1987). On the basis of neurochemical changes, it can
be presumed that EMR-2450 causes alteration in the
cholinergic neurotransmission which can lead to cognitive
deficits.
The mitochondrial pathway of apoptosis regulated by
mitochondrial integrity may lead to mitochondrial swelling
and opening of mitochondrial transition pore (Webster
2012). Apoptosis is an organized, energy-dependent process
in which mitochondria plays a pivotal role as regulators of
Effect of EMR on cognition 273
cell death (Samaiya et al. 2016). In the current study, EMR-
2450 exposure led to increase in expression of cytochrome-
c, indicating opening of mitochondrial transition pore due to
loss of mitochondrial integrity. Cytochrome-c activated the
proapoptotic factors caspase-9 and caspase-3. However,
EMR-900 and EMR-1800 exposed rats did not show sig-
nificant differences in mitochondrial dysfunction and
expression of apoptotic markers. Exposure to mobile phone
radiation has been reported to up-regulate apoptotic genes in
primary cultures of neurons and astrocytes (Zhao et al.
2007). Therefore, exposure to EMR-2450 caused mito-
chondrial dysfunctions leading to leakage of cytochrome-c
from the mitochondria and activation of intrinsic pathway of
apoptosis. Apoptosis has been reported in cognitive
impairment (Man et al. 2015) and therefore mitochondrial-
linked apoptosis may be one of the major factors involved in
EMR induced cognitive changes.
In summary, EMR at 2450 MHz induced cognitive
behavioral deficit with concomitant loss in mitochondrial
function. Alteration in the activity of mitochondrial complex
enzyme systems caused oxidative stress and decrease in
MMP, which ultimately lead to loss of mitochondrial integ-
rity. Further, mitochondrial stress as observed from increases
in cytochrome-c activated the expression of caspase-9 and
caspase-3, indicating mitochondrial-linked apoptosis. Fur-
thermore, exposure with EMR-2450 increased expression of
hippocampal Aband decreased cholinergic neurotransmis-
sion in the hippocampus, which are considered to be
important factors for development of cognitive dysfunction.
Acknowledgements
SKG is thankful to Indian Institute of Technology–Banaras
Hindu University (IIT-BHU), Varanasi, India, for the fel-
lowship as teaching assistant. All animal experiments were
carried out according to the principles stated in guidelines of
laboratory animal care (National Research Council US
Committee for the Update of the Guide for the Care and Use
of Laboratory Animals 2011 guidelines). All the experi-
mental methods were approved by the Institutional animal
ethical committee, Banaras Hindu University (Approval No.:
Dean/2015/CAEC/1414).
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Corresponding editor: NEERAJ JAIN
276 Sukesh Kumar Gupta et al.
