Accepted Manuscript

Title: Neurodegenerative changes and apoptosis induced by

intrauterine and extrauterine exposure of radiofrequency
radiation
Author: Goknur Guler Elcin Ozgur Hikmet Keles Arin
Tomruk Sevil Atalay Vural Nesrin Seyhan
PII:
DOI:
Reference:

S0891-0618(15)00075-7
http://dx.doi.org/doi:10.1016/j.jchemneu.2015.10.006
CHENEU 1345

To appear in:
Received date:
Revised date:
Accepted date:

14-7-2015
14-10-2015
15-10-2015

Please cite this article as: Guler, G., Ozgur, E., Keles, H., Tomruk, A., Vural, S.A.,
Seyhan, N.,Neurodegenerative changes and apoptosis induced by intrauterine and
extrauterine exposure of radiofrequency radiation, Journal of Chemical Neuroanatomy
(2015), http://dx.doi.org/10.1016/j.jchemneu.2015.10.006
This is a PDF file of an unedited manuscript that has been accepted for publication.
As a service to our customers we are providing this early version of the manuscript.
The manuscript will undergo copyediting, typesetting, and review of the resulting proof
before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that
apply to the journal pertain.

*Research Highlights

Ac

ce
pt

ed

an

us

cr

ip
t

Apoptosis and oxidative damage in brain due to mobile phone radiation investigated.

Page 1 of 19

Neurodegenerative changes and apoptosis induced by intrauterine and

extrauterine exposure of radiofrequency radiation
Gknur Glera, Elcin Ozgura,*, Hikmet Kelesb, Arin Tomruka, Sevil Atalay Vuralc, Nesrin

ip
t

Seyhana

Department of Biophysics, Gazi University School of Medicine and Gazi Non-Ionizing

Radiation Protection Center, 06500, Ankara, Turkey

Department of Pathology, Faculty of Veterinary Medicine, Afyon Kocatepe University,

cr

03200, Afyon, Turkey

Department of Pathology, Faculty of Veterinary Medicine, Ankara University, 06110,

us

ce
pt

ed

an

Ankara, Turkey

Corresponding Author:

Elcin Ozgur, PhD

Gazi niversitesi Tp Fakltesi Biyofizik Abd, Dekanlk Binas 5. Kat 06500 Beevler

Ac

ANKARA / TURKEY

Tel: +90 312 202 46 02

Fax: +90 312 212 90 23

e-mail: elcin.ozgur@gmail.com

Page 2 of 19

ABSTRACT
Adverse health effects of radiofrequency radiation (RFR) on the ongoing developmental
stages of children from conception to childhood are scientifically anticipated subject. This
study was performed to identify the effects of global system for mobile communications
(GSM) modulated mobile phone like RFR in 1800 MHz frequency on oxidative DNA damage
and lipid peroxidation beside the apoptotic cell formation, using histopathological and

ip
t

immunohistochemical methods in the brain tissue of 1-month-old male and female New
Zealand White rabbits that were exposed to these fields at their mothers womb and after the

cr

birth. Oxidative DNA damage and lipid peroxidation levels were investigated by measuring
the 8-hydroxy-2' -deoxyguanosine (8-OHdG) and malondialdehyde (MDA) levels

us

respectively. Histopathological changes were observed using by hematoxylin and eosin (HE)
staining. Apoptotic cells were detected in the examined organs by terminal deoxynucleotidyl

an

transferase-mediated dUTP nick end-labelling (TUNEL) staining.

For both male and female infants; 8-OHdG levels increased in the group exposed to RFR in
both intrauterine and extrauterine periods compared to the infants that were never exposed to

RFR and the ones were exposed when they reached one month of age (p<0.05). MDA results
were different for male and female rabbits. There was no difference between all female infant

ed

groups (p>0.05), while only intrauterine exposure significantly causes MDA level increase for
the male infants. HE staining revealed mild lessions in neuronal necrobiosis in brain tissues of
female rabbits that had only intaruterine exposure and male rabbits had only extrauterine

ce
pt

exposure. Gliosis were mildly positive in brain tissues of rabbits that are exposed only
intrauterine period, also the group exposed both intrauterine and extrauterine periods.
However, there was no apoptotic change detected by TUNEL staining in the brain tissues of

Ac

all groups.

Keywords: Mobile phone, apoptosis, DNA damage, 8-OhdG, lipid peroxidation, TUNEL

Page 3 of 19

1. Introduction
Part of the electromagnetic spectrum comprising the frequency range from 100 kHz to 300

ip
t

GHz may be named as high frequency (HF) or radiofrequency (RF) radiation. Mobile phones
operate in this range of the electromagnetic spectrum, from several hundred MHz to several

cr

GHz, to enable wireless phone calls and data transfer, including communication through the
internet. The exact frequency band used differs between technologies (GSM, UMTS, 4G, etc.)

us

and between countries (ICNIRP, 2015).

Increasing use of mobile phones cause to ascend the public concern about the possible ill-

an

effects of mobile phone radiation especially on children and teenagers, beside the sensitive
people such as pregnant women and the babies. Although it may be stated as if there is
scientific uncertainty potential health hazard of low-energy radiofrequency radiation (RFR)

emitted by mobile phones, International Agency for Research on Cancer (IARC) published a
release in France at May 31, 2011 has classified radiofrequency electromagnetic fields as

ed

possibly carcinogenic to humans (Group 2B), based on an increased risk for glioma, a
malignant type of brain cancer, associated with wireless phone use (Hietanen, 2006). At the
time of the IARC review it was known that when mobile phone use began as a teenager, the

ce
pt

risks were higher than when use began as an adult (Hardell and Carlberg, 2009; Hardell et al.,
2006). Since then, additional evidence has accrued of an increased risk to children (Morgan et
al., 2015).

Our previous studies revealed the evidence on the possible biological effects in several

Ac

tissues of both non-pregnant and pregnant New Zealand White rabbits and in their newborns
(Guler et al., 2010; Guler et al., 2011; Tomruk et al., 2010; Kismali et al., 2012) and 1-monthold infants (Guler et al. 2012; Ozgur et al., 2013) that are exposed to whole body 1800 MHz
GSM-like RFR.
This study is also designed to study the same level of RFR on the oxidative DNA damage,
lipid peroxidation levels and the apoptotic cell formation by using histopathological and
immunohistochemical methods in the brain tissues of 1-month-old infant rabbits. Here, we
focused on two exposure scenarios: intrauterine (IU) (pre-natal) and extrauterine (EU)
(postnatal) exposure to mobile phone-like RFR.

Page 4 of 19

Oxidative stress is defined as an imbalance between production of free radicals and

reactive metabolites, so-called oxidants or reactive oxygen species (ROS), and their
elimination by protective mechanisms, referred to as antioxidants. This imbalance leads to
damage of important biomolecules and cells, with potential impact on the whole organism
(Durackova, 2009; Reuter et al., 2010). Since repair of almost all of the biomolecules depends
on the information coded in the DNA, there is a postulated importance of oxidative DNA

ip
t

damage. DNA damage may be quantified by the level of 8-hydroxydeoxyguanosine (8OhdG), which is most widely used fingerprint of radical attack towards DNA (Marnett, 2000;

cr

Wiseman and Halliwell, 1996). Proteins and lipids are also significant targets for oxidative
attack, and modification of these molecules can increase the risk of mutagenesis
One of the main biomarkers widely used in determination of

us

(Schraufstatter et al., 1988).

oxidative destruction on lipids mediated by second messengers is malondialdehyde (MDA)

an

(Nair et al., 1986; Draper and Hadley, 1990). Levels of 8-OhdG and MDA were analyzed in
the present study in order to identify the oxidative DNA damage and lipid peroxidation.
Cancer initiation and progression has been linked to oxidative stress by increasing DNA

mutations or inducing DNA damage, genome instability, and cell proliferation (Visconti and
Grienco, 2009). Dysregulated cell proliferation rate, in other words dysfunction in apoptosis is

ed

directly related to tumor development. Apoptosis, also termed programmed cell death is
the necessary mechanism complementary to proliferation that ensures homeostasis of all
tissues (Larsson et al., 2010). Our previous reports showing histopathological changes due to

ce
pt

1800 MHz RFR exposure were observed in the brain, eyes, liver, kidneys, lung, heart, and
spleen of non-pregnant and pregnant rabbits and their newly born babies (Guler et al., 2011).
In this study, brain tissue was histopathologically examined by haematoxylin-eosin (HE)
staining in the brain tissues of the one-month-old infants of the pregnant rabbits. Apoptotic

Ac

cell formations were detected by terminal deoxynucleotidyl transferase-mediated dUTP nick

end-labeling (TUNEL) staining.
To clarify the possible link between RFR and health effects, scientists have been
investigating this problem more than 20 years. Most of the recent epidemiological and
experimental (in vivo/in vitro) studies have indicated that acute or chronic exposure in
different frequency ranges may alter biological responses including cell cycle (Clearly et al.,
1996), cell proliferation (Clearly et al., 1990; Kwee and Raskmark, 1998; Velizarov et al.,
1999), apoptosis (Marinelli et al., 2004; Zhao et al., 2007), and DNA damage (Diem et al.,
2005; Lai and Singh, 1995; Lai and Singh, 1996; Lai and Singh, 1997; Tice et al., 2002).

Page 5 of 19

In the present study, the principal aim was to design the continual RF exposure and
investigate the possible bio-effects of RF radiation on the ongoing developmental stages of
children from conception to childhood. The levels of lipid peroxidation and DNA damage
based on free radical attacks were analyzed, beside the histopathological examination and

2.

Materials and Methods

2.1.

Animals

ip
t

apoptosis detection carried out in the brain tissues of baby rabbits aged one month.

cr

A total of 72 one-month-old female and male New Zealand white rabbits were used in this

us

study. The animals were obtained from the Laboratory Animals Breeding and Experimental
Research Center of Gazi University. The experimental protocol was reviewed and approved
by the Laboratory Animal Care Committee of Gazi University (G.U.ET-06.027). Thirty-six of

an

the infant rabbits were exposed to 1800 MHz GSM-like RF radiation for 15 min/day during a
week in the intrauterine period (between 15th and 22nd days of the gestational period when

the transition from embryogenesis to organogenesis takes place) whereas others were not
exposed.

After birth, all 72 infant rabbits were kept with their mothers until they reached one month

ed

of age. They were breastfed and their optimum growth was obtained during this one-month
period. Baby rabbits aged one month were housed under the same conditions in a temperature

ce
pt

and humidity-controlled room (20 1 C, 50 10% relative humidity) and 14/16 h light/dark
cycle conditions. The animals were provided with tap water and standard pelletized food ad
libitum except during exposure periods. Only one animal was placed in each cage during each
radiofrequency radiation (RFR) exposure period because placing more than one animal in a

Ac

cage could have created stress.

2.2. Exposure level and quality control

GSM-like signals in 1800 MHz frequency were formed by using a signal generator
(Agilent Technologies 8648C, 9 kHz3.2 GHz) with the integrated pulse modulation unit and
horn antenna (Schwarzbeck, Doppelsteg Breitband Horn antenna BBHA 9120 L3F, 0.52.8
GHz). The generated power was controlled by a spectrum analyzer (Agilent Technologies
N9320A, 9 kHz3 GHz) integrated to the signal generator. The signals were amplitudemodulated by rectangular pulses with a repetition frequency of 217 Hz and a duty cycle of 1:8
(pulse width 0.576 ms), corresponding to the dominant modulation component of the GSM.

Page 6 of 19

RFR generator provided 0.1 W (20 dBm) during the exposure period. The signal was
controlled by means of the spectrum analyzer connected to the signal generator, and NARDA
EMR 300 and type 26.1 probe were used for measurement of the output radiation.
Measurements were taken during the entire experiment and the data was saved in the
computer which was connected to the device via fiber optic cable. The evaluated data was 14

ip
t

0.5 V/m. Estimated SAR value is calculated as18 mW/kg.

2.3. Experimental Design

cr

A total of 72 one-month-old female and male New Zealand white rabbits were used.
Thirty-six females were exposed to RF radiation for 15 min/day during 7 days, whereas 36

us

males were exposed to the same level of radiation for 15 min/day during 14 days. Female and
male infant rabbits were randomly divided into four groups:

an

Group I [Intrauterine exposure (-) ; Extrauterine exposure (-)]: Sham exposure which
means rabbits were exposed to 1800 MHz GSM-like RF signals neither in the intrauterine
(IU) nor in the extrauterine (EU) periods.

Group II [Intrauterine exposure (-) ; Extrauterine exposure (+)]: Infant rabbits were
exposed to 1800 MHz GSM-like RF signals when they reached one month of age.

ed

Group III [Intrauterine exposure (+); Extrauterine exposure (-) ]: Infant rabbits were
exposed to 1800 MHz GSM-like RF signals in the IU period (between 15th and 22nd days of
the gestational period).

ce
pt

Infant rabbits were exposed to 1800 MHz GSM-like RF signals both in the IU period
(between 15th and 22nd days of the gestational period) and in the EU period when they
reached one month of age. The day after the last exposure, baby rabbits were anesthetized and
sacrificed with ketamine (35 mg/kg, i.m.) and xylazine (5 10 mg/kg, i.m.).

Ac

2.4. Euthanasia and histopathological examination

The day after the last exposure, all rabbits were anesthetised by the injections of ketamine
(35 mg/kg, i.m.) and xylazine (5-10 mg/kg, i.m.) and killed by cervical dislocation. The brain
tissues were removed, fixed in 10% buffered formalin, processed, and embedded in paraffin.
The sections were cut at 5 m and stained with haematoxylin-eosin (HE)
2.5. Assessment of apoptotic cells
Apoptotic cells were detected by terminal deoxynucleotidyl transferasemediated dUTP
nick end-labeling (TUNEL) staining using a commercial ready-to-use kit (in situ cell death

Page 7 of 19

detection kit, POD, Roche, Germany). The procedure and control stainings were carried out
according to the manufacturers instruction. After deparaffinisation and rehydration, tissue
sections were digested with proteinase K (20 g/mL, 30 min) and methanol with 3%
hydrogen peroxide in PBS (5 min). Then the sections were incubated in a humidified chamber
in 200 l of TUNEL mixture (TdT and label solution) at 370C for 60 min and with POD
converter at 370C for 30 min. The sections were then treated with 3-amino-9-ethylcarbasole

ip
t

(AEC) as a chromogen (Dako, USA) for 5 min, washed with PBS (pH 7.4), and
counterstained with Mayers haematoxylin. TUNEL sections were blindly examined by two

cr

pathologists under light microscope (Leica DM 4000B) interfaced with a camera (Leica, DFC
80). TUNEL positivity was evaluated by a semi-quantitative scoring system according to Xu

us

et al. (40) with minor modifications. Ten different fields on each slide were examined at high
magnification. The intensity of staining was scored as negative (-), mild (+), moderate (++),

an

and severe (+++). The extent of staining was scored as - (0%-5%), + (6%-25%), ++ (26%50%), and +++ (51% and higher) according to the percentage of positively stained cells. Each
field was graded according to the score and then the total score was divided by ten. This way

the average score was calculated for each slide.

ed

2.6. Biochemical analysis

Brain tissues rinsed with ice-cold buffered saline and stored at 30C (maximum 10 h) for
double-blind biochemical analysis. After weighing, the brain was cut into small pieces and

ce
pt

then homogenized in four volumes of ice-cold Tris-HCl buffer (50 mmol/l, pH 7.4) by using
homogenizer (Disperser T10 basic D-79219, IKA-WERKE, GmbH, Staufer). MDA levels
were analyzed in the brain homogenate. The principle of MDA determination method is based
on the spectrophotometric measurement of the color generated by the reaction of

Ac

thiobarbituric acid (TBA) with MDA (Draper and Hadley 1990). For the measurement of
oxidative DNA damage (lesions/10 6 DNA nucleosides), the genomic DNA of brain tissues
was extracted by Roche DNA extraction kit, and it was denatured by heating for 3 min at
95C and then cooled on ice. 100 l 2 mmol/l desferrioxamine-B mesylate (DFAM) and 20
mmol/l acetate buffer (pH = 5) were added to the denaturated DNA. DNA content was
analyzed spectrophotometrically at 260 nm and then hydrolyzed to nucleotides by incubation
with 4 l of 3.3 mg/ml suspension of nuclease P1. The Tris-HCl buffer (pH = 8.5) was added
to the mixture and hydrolyzed to the corresponding nucleosides by incubation with calf
intestine alkaline phosphatase for 1 h at 37C. After adding up acetate buffer and 50 mmol/l
ethylenediaminetetraacetic acid (EDTA) /10 mmol/l DFAM solution, the mixture was filtered

Page 8 of 19

through a 0.22 m Millipore filter unit (UltraFree, Bedford, MA, USA) and then centrifuged
at

10.000g

for

20

min

at

4C.

Reverse-phase

high

pressure

liquid

chromatography/electrochemical detection (HPLC-EC) was performed as described by Floyd

et al. (1986). The DNA hydrolysate was injected onto a Waters C18 reverse-phase column (5
m, 0.46 cm 25 cm; Waters Assoc., Milford, MA, USA) at a flow rate of 1 ml/min. The
mobile phase was 50 mmol/l phosphate buffer (pH = 5.5) with 5% methanol (Halliwell and

ip
t

Dizdaroglu, 1992; Hamilton et al., 1999). The eluant was monitored at 290 nm for the
ultraviolet detection of deoxyguanosine (dG) and at 0.6 V for the electrochemical detection of

cr

8-OHdG. The system was calibrated with authentic dG and 8-OHdG standards (Sigma
Chemical,St. Louis, MO, USA). dG had a retention time of 1012 min and 8-OHdG had a

us

retention time of 8.713.8 min. Standards were run after every fifth sample for verification,
and the data were expressed as the ratio of 8-OHdG to 10 6 dG.

an

2.7. Statistical analysis

Statistical analyses were carried out using SPSS software (SPSS 11.5 for windows, SPSS

Inc., Chicago, USA). The one-way analysis of variance (ANOVA) and post hoc multiple
comparison tests (LSD) were performed on the data of biochemical variables to examine the

ed

difference among groups. A p-value of < 0.05 was considered as statistically signicant. All

3.

Result

ce
pt

data were expressed as mean+SEM.

MDA and 8-OHdG results for both female and male infant rabbits are shown in Tables I.
No difference related to gender difference on the biochemical parameters was found.

3.1.

Ac

Histopathologic findings (HxE) are shown in Table II respectively.

Results for DNA damage for both male and female rabbits

8-OHdG levels of Group IV were significantly increased with respect to Group I and
Group II for both male and female infant rabbits. In other words, both intrauterine and
extrauterine exposure to 1800 MHz RFR cause to increase in 8-OHdG levels compared to the
infants which were exposed to RFR neither in the intrauterine nor in the extrauterine periods
and the other group which were exposed to RFR when they reached one month of age
(p<0.05).
3.2. MDA levels for both male and female rabbits

Page 9 of 19

Results for MDA showed that there was no difference between all female infant groups
(p>0.05). However, for the male infants; significant increase was detected in Group IV
compared to Group I and Group II. Also, difference between Group I and Group III was
statistically significant. In this case, it may be stated that intrauterine exposure significantly
causes MDA level increase.

ip
t

3.3. Histopathological and immunohistochemical results

Analyzed and scored results of histopathological methods are presented in Tables II. These

cr

results varied among experimental groups and among animals in each group.
Histopathologically; hyperaemia, neuronal necrobiosis, gliosis and mononuclear cells in

us

perivascular areas were detected in the brain (Table II). Neuronal necrobiosis was seen in
Group II, male rabbits exposed only after birth, female infants exposed only intaruterine

an

period respectively. Mild neuronophagie was seen in female animals of Group III. Gliosis was
obtained in female and male infants of Group III and Group IV that contains the rabbits
exposed to RFR in both periods. Mononuclear cells in the perivascular areas were mildly seen

in all infants of Group III. In case of the TUNEL staining; evident TUNEL positivity in the

4.

Discussion

ed

brain was not seen.

ce
pt

Experimental data obtained in this study revealed that prenatal and post natal exposure of
female and male rabbits aged one-month-old to 1800 MHz GSM-like RFR (18 mW/kg SAR)
resulted to cause oxidative destruction in lipids and DNA molecules in brain tissues. In
addition, histological findings showed mild lesions by HE staining, even though there was no

Ac

TUNEL positivity in the brain tissues of all groups.

Possible health effects related to RFR emitted by mobile phones are still unclear and
debated since the results of relevant clinical and epidemiological studies have been
inconsistent. Health effects of RFR are mainly classified as thermal and non-thermal
effects. Although the current international safety standards are based on the thermal effects of
RFR in acute exposure, people are chronically exposed to these fields at the non-thermal
levels. Some scientists mention that no biophysical mechanism has been identified so far
which would speak in favor of such effects since the quantum energy in the frequency range
used for mobile communication is far too low to break chemical bonds. They report that only
accepted mechanism by which RF-EMF could be harmful is heating which is prevented at the

Page 10 of 19

current exposure limits for the general population (specific absorption rate (SAR) 0.08 W/kg
whole body; 2 W/kg local exposure (Lerchl et al., 2015). However, other researchers note that
chemical bonds in biologically systems are broken and reformed constantly because metabolic
energy and enzymes can manage this process. He considers that it is very nave belief that
RFR in non-thermal range cant break chemical bond because its energy is too low. RFR
exposure may affect metabolism and other chemical reactions such as the Fenton reaction to

ip
t

effect chemical reaction. In other words, RFR may affect the metabolism by releasing
secondary messengers, such as reactive oxygen species (ROS), leading to oxidative

cr

destruction in lipids and DNA molecules. H2O2, an example of ROS, may be formed either by
dismutation from superoxide anion or spontaneously in peroxisomes from molecular oxygen

us

(Lerchl et al., 2015; Mates and Sanchez-Jimenez, 2000). H2O2 plays an important role in
carcinogenesis due to its diffusion capability throughout the mitochondria and across cell

an

membranes. It may produce many types of cellular injury (Mates and Sanchez-Jimenez, 2000;
Ray and Husain, 2002). On the other hand, ROS injuries in mammalian cells are mediated by
the hydroxyl radical (OH) which has a very unstable electron structure (Marnett, 2000; Valko

et al., 2004). The majority of OH in vivo is produced in the presence of reduced transition
metals such as iron, mainly via the Fenton reaction when Fe2+ contacts H2O2. The OH-

ed

derived DNA damage includes the generation of 8-hydroxyguanosine (8-OHG), the

hydrolysis product of which is the mostly used signifier of radical attack towards DNA, 8hydroxydeoxyguanosine (8-OHdG) (Marnett, 2000; Wiseman and Halliwell, 1996).

ce
pt

Oxidative DNA damage and lipid peroxidation levels were also investigated in the brain
tissues of 1-month-old infants. In our previous study, the 13-month-old mother rabbits were
exposed to 1800 MHz GSM modulated RFR in their pregnancy period, between the 15th and
22nd days of the gestation. The coeval rabbits but non-pregnant ones were exposed to same

Ac

level of RFR in the same period. The brain tissue levels of 8-OHdG and MDA for adult
rabbits significantly increased whereas no difference was found in the newborns which were
exposed as fetus decapitated when they reach 2-day-old (Guler et al., 2010). In the lights of
these results, it may be discussed that extrauterine exposure is effective for DNA and
oxidative damage for brain tissue. Alongside of these data, we analyzed the hepatic level of 8OHdG and MDA of pregnant, non-pregnant, newborns (Tomruk et al., 2010) and 1-month-old
male and female infants (Guler et al., 2012). Whole body exposure of 1800 MHz GSM like
RFR was not affected the hepatic level of 8-OHdG with respect to the non-exposed adult
rabbits, while MDA and FOX levels were statistically different compared to controls. Similar
with the brain results of the newborns, hepatic levels of both 8-OHdG and MDA did not

Page 11 of 19

change due to the RFR exposure during intrauterine periods (Tomruk et al., 2010).
Extrauterine exposure of female infants caused to increase the levels of 8-OHdG while lipid
peroxidation levels in the liver tissues of female and male infant rabbits increased under RFR
exposure. Overall results showed that brain tissue is more affected than liver due to the fact
that head of the animals is closer to the RFR source during the whole body exposure in our
exposure set-up. These findings may also be interpreted as a result of the depth of penetration

ip
t

phenomenon. As RFR propagates in the tissue medium, energy is absorbed by the tissue,
resulting in a progressive reduction of RFR as it advances in the tissue (Guler et al., 2010;

cr

Polk and Postow, 1986).

The data above reported that oxidative damage occurs even though DNA damage does not

us

occur. Similarly, studies have shown that RFR emitted from cellular phones could increase
the release of free radicals. Meral et al. (2007) revealed that RFR radiation generated from

an

cellular phones (12 h/day, 30 days) may produce oxidative stress by increasing lipid
peroxidation of brain tissues in guinea pigs. Ozgur et al. (2010) showed that GSM-like
radiation can cause significant modification in the activities of liver antioxidant enzymes.

However, the administration of an external antioxidant has a protective effect against the RF
radiation by boosting the antioxidant activity. In addition to these, RFR may cause oxidative

ed

damage in the fetus; this could be caused by melatonin pathway disruption in the mother
(Wakatsuki et al., 1999; Wakatsuki et al., 2001).
Histopathological data revealed mild lesions in the brain tissue of the exposed infants.

ce
pt

Moreover, our previous data showed the formation of apoptotic cells in neurons, meningeal
cells, and glial cells was observed after TUNEL staining. Histopathologically, some
alterations such as hyperaemia, haemorrhage, neuronal necrobiosis, clarity of

Nissl

substance, gliosis, and mononuclear cells infiltration in perivascular areas were detected in the

Ac

brain tissue of exposed adult rabbits (Guler et al., 2011). These results are parallel with the
biochemical data which show DNA damage.
To sum up, daily exposed level of RFR in nonthermal region may cause oxidative and
DNA damage in the brain tissue of baby rabbits which are exposed while they are fetus and
after they are 1-month-old. In conclusion, these data may be effective for protecting children
and babies from environmental RFR exposure by drawing the attention of decision-makers
and finally succeed in the establishment of international standards for the protection of
children.

Page 12 of 19

References
Cleary, R.E., Cau, G., Liu, L.M., 1996. Effects of isothermal 45 GHz microwave radiation on the mammalian
cell cycle: comparison with the effects of isothermal 27 MHz radiofrequency radiation exposure.
Bioeletrochem. Bioenerg. 39, 167-173.
Cleary, S.F., Liu, L.M., Merchant, R.E., 1990. Glioma proliferation modulated in vitro by isothermal
radiofrequency radiation exposure. Radiat. Res.121, 38-45.
Diem, E., Shwarz, C., Adlkofer, F., Jahn, O., Rudiger, H., 2005. Non-thermal DNA breakage by mobile-phone

ip
t

radiation (1800 MHz) in human fibroblasts and in transformed GFSH-R17 rat granulosa cells in vitro.
Mutat. Res.583, 178-183.

Draper, H.H., Hadley, M., 1990. Malondialdehyde determination as index of lipid peroxidation. Methods

cr

Enzymol. 186,421431.

Durackova, Z., 2009. Some current insights into oxidative stress. Physiol. Res. 59(4), 459-469.

us

Guler, G., Ozgur, E., Keles, H., Tomruk, A., Atalay Vural, S., Seyhan, N.,2011. Apoptosis resulted by
Radiofrequency Radiaton exposure on pregnant rabbits and their infants. Bulletin Veterinary in Pulawy. 55,
127-134.

an

Guler, G., Tomruk, A., Ozgur, E., Sahin, D., Sepici, A., Altan, N., Seyhan, N., 2012. The effect of
radiofrequency radiation on DNA and lipid damage in female and male infant rabbits. International Journal
of Radiation Biology. 88(4), 367-73.

Guler, G., Tomruk, A., Ozgur, E., Seyhan, N., 2010. The Effect of radiofrequency radiation on DNA and lipid
damage in non-pregnant and pregnant rabbits and their newborns. General Physiology and Biophysics.
29(1), 59-66.

ed

Halliwell, B., Dizdaroglu, M., 1992. Commentary. The measurement of oxidative damage to DNA by HPLC and
GC/MS techniques. Free Radical Res Commun. 16, 7587.
Hamilton, M.L., Guo, Z.M., Fuller, C.D., Van Remmen, H.,Ward, W.F., Austad, S.N., Troyer, D.A., Thompson,

ce
pt

I., Richardson A., 1999. A reliable assesment of 8-oxo-2-deoxyguanosine levels in nuclear and
mitochondrial DNA using the sodium iodide method to isolate DNA. Nucleic Acids Res. 29, 21172126.
Hardell, L., Carlberg, M., 2009. Mobile phones, cordless phones and the risk for brain tumours. Int J Oncol. 35,
517.

Ac

Hardell, L., Carlberg, M., Hansson Mild, K., 2006. Pooled analysis of two casecontrol studies on the use of
cellular and cordless telephones and the risk of benign brain tumours diagnosed during 19972003. Int J
Oncol. 28, 509518.

Hietanen, M., 2006. Health risks of exposure to non-ionizing radiation myths or science-based evidence. La
medicina del lavoro. 97(2), 184-188.
International

Commussion

for

Non-Ionizing

Radiation

Protection

Center,

ICNIRP

website,

http://www.icnirp.org/en/home/index.html, 2015
Kismali, G., Ozgur, E., Gler, G., Akcay, A., Sel, T., Seyhan, N., 2012. The influence of 1800 MHz GSM-like
signals on blood chemistry and oxidative stress in non-pregnant and pregnant rabbits. International Journal
of Radiation Biology. 88(5), 414-9.
Kwee, S., Raskmark, P., 1998. Changes in cell proliferation due to environmental non-ionizing radiation: 2.
Microwave radiation. Bioelectrochem Bioenerg.44, 251-255.

Page 13 of 19

Lai, H., Singh, N.P., 1995. Acute low-intensity microwave exposure increases DNA single-strand breaks in rat
brain cells. Bioelectromagnetics. 16, 207-210.
Lai, H., Singh, N.P., 1996. DNA single- and double strand breaks in rat brain cells after acute exposure to lowlevel radiofrequency electromagnetic radiation. Int J Radiat Biol. 69, 513-521.
Lai, H., Singh, N.P., 1997. Melatonin and a spin-trap compound block radiofrequency electromagnetic radiationinduced DNA strand breaks in rat brain cells. Bioelectromagnetics. 18, 446-454.
Larsson, D.E., Wickstrm, M., Hassan, S., Oberg, K., Granberg, D., 2010.The cytotoxic agents NSC-95397,

ip
t

brefeldin A, bortezomib and sanguinarine induce apoptosis in neuroendocrine tumors in vitro. Anticancer
Res. 30(1),149-56.

Lerchl, A., Klose, M., Grote, K., Wilhelm, A.F., Spathmann, O., Fiedler, T., Streckert, J., Hansen, V., Clemens,

cr

M., 2015. Tumor promotion by exposure to radiofrequency electromagnetic fields below exposure limits for
humans. Biochem Biophys Res Commun. 459(4):585-90. 35

us

Marinelli, F., La Sala, D., Cicciotti, G., Cattini, L., Trimarchi, C., Putti, S., Zamparelli, A., Giuliani, L.,
Tomasetti, G., Cinti, C., 2004. Exposure to 900 MHz electromagnetic field induces an unbalance between
proapoptotic and pro-survival signals in T-lymphoblastoid leukemia CCRF-CEM cells. J Cell Physiol.198,

an

324-332.

Marnett, L.J., 2000. Oxyradicals and DNA damage. Carcinogenesis. 21361370.

therapy. Int J Biochem Cell Biol.32:157170.

Mates, J.M., Sanchez-Jimenez, F.M., 2000. Role of reactive oxygen species in apoptosis: implications for cancer

Meral, I., Mert, H., Mert, N., Deger, Y., Yoruk, I., Yetkin, A., Keskin, S., 2007. Effects of 900 MHz

Brain Res. 1169, 120124.

ed

electromagnetic field emitted from cellular phone on brain oxidative stress and some levels of guinea pigs.

Morgan, L.L., Miller, A.B., Sasco, A., Davis, D.L., 2015. Mobile phone radiation causes brain tumors and
should be classified as a probable human carcinogen (2A) (review). Int J Oncol. 46(5), 1865-71.

ce
pt

Nair, V., Cooper, C. S., Vietti, D. E., Turner, G. A., 1986. The chemistry of lipid peroxidation metabolites:
crosslinking reactions of malondialdehyde. Lipids. 21, 69.
Ozgur E, Kismali G, Guler G, Akcay A, Sel T, Seyhan N., 2013. Effects of prenatal and postnatal exposure to
GSM-like radiofrequency on blood chemistry and oxidative stress in infant rabbits, an experimental study.
Cell Biochemistry and Biophysics. 67, 743751

Ac

Ozgur, E., Gler, G., Seyhan, N., 2010. Mobile phone-radiation-induced free radical damage in the liver is
inhibited by the antioxidants n-acetyl cysteine and epigallocatechin-gallate. International Journal of
Radiation Biology. 86, 935 945.
Polk, C., Postow, E., 1986. CRC Handbook of Biological Effects of Electromagnetic Fields. CRC Press, Boston
Ray, G., Husain, S.A., 2002. Oxidants, antioxidants and carcinogenesis. Indian J Exp Biol. 40:1213 1232.
Reuter,S., Gupta, S.C., Chaturvedi, M.M., Aggarwal B.B., 2010. Oxidative stress, inflammation, and cancer:
How are they linked? Free Radic Biol Med. 49(11), 16031616.
Schraufstatter, I., Hyslop, P.A., Jackson, J.H., Cochrane, C.G., 1988. Oxidant-induced DNA damage of target
cells. J Clin Invest.82, 10401050.

Page 14 of 19

Tice, R.R., Hook, G.G., Donner, M., McRee, D.I., Guy, A.W., 2002. Genotoxicity of radiofrequency signals. I.
Investigation

of

DNA

damage

and

micronuclei

induction

in

cultered

human

blood

cells.

Bioelectromagnetics. 23,113-126.
Tomruk, A., Gler, G., Dincel Sepici, A., 2010. The infl uence of 1800 MHz GSM-like signals on hepatic
oxidative DNA and lipid damage in nonpregnant, pregnant, and newly born rabbits. Cell Biochemistry and
Biophysics. 56, 39 47.
Valko, M., Izakovic, M., Mazur, M., Rhodes, C.J., Telser, J., 2004. Role of oxygen radicals in DNA damage and

ip
t

cancer incidence. Mol Cell Biochem. 2004. 266, 3756.

Velizarov, S., Raskmark, P., Kwee, S., 1999. The effects of radiofrequency fields on cell proliferation are nonthermal. Bioelectrochem Bioenerg. 48, 177-180.

cr

Visconti, R., Grieco, D., 2009. New insights on oxidative stress in cancer. Curr Opin Drug Discov Devel. 12,
240245.

us

Wakatsuki, A., Okatani, Y., Izumiya, C., Ikenoue, N., 1999. Melatonin protects against ischemia and
reperfusion-induced oxidative lipid and DNA damage in fetal rat brain. Journal of Pineal Research. 26, 147
152.

an

Wakatsuki, A., Okatani, Y., Shinohara, K., Ikenoue, N., Kaneda, C., Fukaya, T., 2001. Melatonin protects fetal
rat brain against oxidative mitochondrial damage. Journal of Pineal Research. 30, 22 28.
Wiseman, H., Halliwell, B., 1996. Damage to DNA by reactive oxygen and nitrogen species: role in

inflammatory disease and progression to cancer. Biochem J. 313, (Pt 1):1729.

Zhao, T.Y., Zou, S.P., Knapp, P.E., 2007. Exposure to cell phone radiation up-regulates apoptosis genes in

Ac

ce
pt

ed

primary cultures of neurons and astrocytes. Neurosci Lett. 412, 34-38.

Page 15 of 19

cr

ip
t

Table(s)

Female

MDA
(nmol/g
tissue)

Mean

0.89

34.63

Mean

9.00

9.00

Minimum

0.87

33.79

Minimum

0.91

35,014

Std. Deviation

0.02

0.44

Median

0.89

34.70

Mean

0.88

34.92

9.00

9.00

Minimum

0.87

34.74

Maximum

0.90

35.02

Std. Deviation

0.01

Median

8OHdG
(nmol
8OHdG
/ 105dG)

MDA
(nmol/g
tissue)

0.89

34,793

9.00

9.00

0.86

33.95

Maximum

Gender

Male

Maximum

0.91

35.02

Std. Deviation

0.02

0.34

Median

0.89

34.90

II

Female

Mean

0.88

34.92

9.00

9.00

Minimum

0.86

34.74

Maximum

0.91

35.02

0.10

Std. Deviation

0.02

0.90

34.96

Median

0.67

0.08

Group

8OHdG
(nmol
8OHdG/ 105
dG

Descriptive
Statistics

ep
te

Male

Group

Gender

Parameters

Ac
c

Group

Parameters
Descriptive
Statistics

Parameters
Descriptive
Statistics

Gender

Male

III

Female

8OHdG
(nmol
8OHdG/
105dG)

MDA
(nmol/g
tissue)

Mean

0.89

34.99

9.00

9.00

Minimum

0.87

34.90

Maximum

0.92

35.03

Std. Deviation

0.01

0.04

Median

0.89

35.01

Mean

0.89

34.98

9.00

9.00

Minimum

0.86

34.85

Parameters
Group

an

us

Tablo 1
Effects of 1800 MHz GSM-like radiation on 8-hydroxy-2 ' -deoxyguanosine (8-OHdG, (nmol 8OHdG / 105dG) ) and Malondialdehyde (MDA, nmol/g tissue) levels in (a)
female infant rabbits ( n=36) and (b) male infant rabbits ( n = 36). I. Group I [Intrauterine exposure (-) ; Extrauterine exposure (-) ]; II. Group II [Intrauterine
exposure (-); Extrauterine exposure (+) ]; III. Group III [Intrauterine exposure (+ ) ; Extrauterine exposure ( - ) ]; IV. Group IV [Intrauterine exposure (+) ;
Extrauterine exposure (+)].

Descriptive
Statistics

Gender

Male

IV

Female

8OHdG
(nmol
8OHdG
/105 dG

MDA
(nmol/g
tissue)

Mean

0.90

34.99

9.00

9.00

Minimum

0.89

34.90

Maximum

0.92

35.04

Std. Deviation

0.01

0.05

Median

0.90

35.01

Mean

0.90

34.94

9.00

9.00

Minimum

0.88

34.80

Maximum

0.92

35.04

Maximum

0.91

35.03

0.01

Std. Deviation

0.01

0.06

Std. Deviation

0.01

0.09

0.88

34.95

Median

0.89

35.00

Median

0.90

34.91

0.60

0.31

0.92

0.64

0.88

0.16

The significance value is less than 0.05 (p<0.05)

No differences were found between the experimental groups both in terms of parameters (8OHdG and MDA) and gender (female/male)

Page 16 of 19

Table(s)

Table 2
Histopathologic findings (HxE) of brain tissues of RFR exposed and control groups*
Group I

Group II

Group III

Group IV

Female

Male

Female Male

Female Male

Female

Male

Hyperemia

Hemorrhage

Neuronal necrobiosis

++

++

Clarity of nissl substance in the neurons

Neuronophagie

Gliosis

the -

cr
-

us

an

++

++

++

++

in

ed

Mononuclear cells
perivascular areas

ip
t

BRAIN

Ac

severe (+++).

ce
pt

* HE stained sections semiquantitatively scored as no lesion (-), mild (+), moderate (++) and

Page 17 of 19

Figure(s)

Amplifier

an

us

cr

ip
t

RF
Generator

Ac

ce
pt

ed

Figure 1. Schematic view of exposure set-up

Page 18 of 19

Ethical statement

The authors declare that they have no conflict of interest. All experimental protocols reported
here were in accordance with the EU Directive (2010/63/EU) for animal experiments. The
animals were obtained from the Laboratory Animals Breeding and Experimental Research
Center of Gazi University. The experimental protocol was reviewed and approved by the

Ac

ce
pt

ed

an

us

cr

ip
t

Laboratory Animal Care Committee of Gazi University (G.U.ET-06.027).

Page 19 of 19