Cambios neurodegenerativos y apóptosis inducida

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Title: Neurodegenerative changes and apoptosis induced byintrauterine and extrauterine exposure of radiofrequency

radiation

Author: Goknur Guler Elcin Ozgur Hikmet Keles Arin

Tomruk Sevil Atalay Vural Nesrin Seyhan

PII: S0891-0618(15)00075-7

DOI: http://dx.doi.org/doi:10.1016/j.jchemneu.2015.10.006

Reference: CHENEU 1345

To appear in:

Received date: 14-7-2015

Revised date: 14-10-2015

Accepted date: 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

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http://dx.doi.org/doi:10.1016/j.jchemneu.2015.10.006http://dx.doi.org/10.1016/j.jchemneu.2015.10.006http://dx.doi.org/10.1016/j.jchemneu.2015.10.006http://dx.doi.org/doi:10.1016/j.jchemneu.2015.10.006


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

esearch Highlights


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Neurodegenerative changes and apoptosis induced by intrauterine and

extrauterine exposure of radiofrequency radiation

Gknur Glera

, Elcin Ozgura,*

, Hikmet Kelesb

, Arin Tomruka

, Sevil Atalay Vuralc

, NesrinSeyhana

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

Radiation Protection Center, 06500, Ankara, Turkey

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

03200, Afyon, Turkey

c

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

*Corresponding Author:

Elcin Ozgur, PhD

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

ANKARA / TURKEY

Tel: +90 312 202 46 02

Fax: +90 312 212 90 23

e-mail: [email protected]
http://ees.elsevier.com/cheneu/viewRCResults.aspx?pdf=1&docID=911&rev=2&fileID=42154&msid={510F15A4-6C1A-4CE2-BA31-0E7F1A8428E1}


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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

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

birth. Oxidative DNA damage and lipid peroxidation levels were investigated by measuring

the 8-hydroxy-2' -deoxyguanosine (8-OHdG) and malondialdehyde (MDA) levels

respectively. Histopathological changes were observed using by hematoxylin and eosin (HE)

staining. Apoptotic cells were detected in the examined organs by terminal deoxynucleotidyl

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 (p0.05), 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

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

all groups.

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


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1.

Introduction

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

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

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.)

and between countries (ICNIRP, 2015).

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

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

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

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

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-month-old 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.


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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

damage. DNA damage may be quantified by the level of 8-hydroxydeoxyguanosine (8-

OhdG), which is most widely used fingerprint of radical attack towards DNA (Marnett, 2000;

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

(Schraufstatter et al., 1988). One of the main biomarkers widely used in determination of

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

(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

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

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

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).


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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

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

2. Materials and Methods

2.1. Animals

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

study. The animals were obtained from the Laboratory Animals Breeding and Experimental

Research Center of Gazi University. The experimental protocol was reviewed and approvedby the Laboratory Animal Care Committee of Gazi University (G.U.ET-06.027). Thirty-six of

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

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

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

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 amplitude-

modulated 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.


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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

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

2.3. Experimental Design

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

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:

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.

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 ofthe gestational period).

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 (510 mg/kg, i.m.).

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


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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

(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

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

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 (++),

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.

2.6. Biochemical analysis

Brain tissues rinsed with ice-cold buffered saline and stored at30C (maximum 10 h) fordouble-blind biochemical analysis. After weighing, the brain was cut into small pieces and

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

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 tissueswas 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


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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

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

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

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.

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

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

data were expressed as mean+SEM.

3. Result

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.

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

3.1. 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


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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.

3.3. Histopathological and immunohistochemical results

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

results varied among experimental groups and among animals in each group.

Histopathologically; hyperaemia, neuronal necrobiosis, gliosis and mononuclear cells in

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

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

brain was not seen.

4.

Discussion

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

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


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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

effect chemical reaction. In other words, RFR may affect the metabolism by releasing

secondary messengers, such as reactive oxygen species (ROS), leading to oxidative

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

(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

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-

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, 8-

hydroxydeoxyguanosine (8-OHdG) (Marnett, 2000; Wiseman and Halliwell, 1996).

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 15thand

22nddays of the gestation. The coeval rabbits but non-pregnant ones were exposed to same

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 wereexposed 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 8-

OHdG 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. Similarwith the brain results of the newborns, hepatic levels of both 8-OHdG and MDA did not


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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

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;

Polk and Postow, 1986).

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

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

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

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.

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

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.


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Ac

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ripTablo 1Effects 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 (+)].

Group

GenderDescriptive

Statistics

Parameters

Group

GenderDescriptive

Statistics

Parameters

Group

GenderDescriptive

Statistics

Parameters

Group

GenderDescriptive

Statistics

Parameters

8OHdG

(nmol8OHdG/ 105

dG

MDA(nmol/g

tissue)

8OHdG

(nmol8OHdG

/ 105dG)

MDA(nmol/g

tissue)

8OHdG

(nmol8OHdG/

105dG)

MDA(nmol/g

tissue)

8OHdG

(nmol8OHdG

/105 dG

MDA(nmol/g

tissue)

I

Male

Mean 0.89 34.63

II

Male

Mean 0.89 34,793

III

Male

Mean 0.89 34.99

IV

Male

Mean 0.90 34.99

N 9.00 9.00 N 9.00 9.00 N 9.00 9.00 N 9.00 9.00

Minimum 0.87 33.79 Minimum 0.86 33.95 Minimum 0.87 34.90 Minimum 0.89 34.90

Maximum 0.91 35,014 Maximum 0.91 35.02 Maximum 0.92 35.03 Maximum 0.92 35.04

Std. Deviation 0.02 0.44 Std. Deviation 0.02 0.34 Std. Deviation 0.01 0.04 Std. Deviation 0.01 0.05

Median 0.89 34.70 Median 0.89 34.90 Median 0.89 35.01 Median 0.90 35.01

Female

Mean 0.88 34.92

Female

Mean 0.88 34.92

Female

Mean 0.89 34.98

Female

Mean 0.90 34.94

N 9.00 9.00 N 9.00 9.00 N 9.00 9.00 N 9.00 9.00

Minimum 0.87 34.74 Minimum 0.86 34.74 Minimum 0.86 34.85 Minimum 0.88 34.80

Maximum 0.90 35.02 Maximum 0.91 35.02 Maximum 0.91 35.03 Maximum 0.92 35.04

Std. Deviation 0.01 0.10 Std. Deviation 0.02 0.01 Std. Deviation 0.01 0.06 Std. Deviation 0.01 0.09

Median 0.90 34.96 Median 0.88 34.95 Median 0.89 35.00 Median 0.90 34.91

p 0.67 0.08 p 0.60 0.31 p 0.92 0.64 p 0.88 0.16

The significance value is less than 0.05 (p


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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

BRAIN

Hyperemia - - - - - + - +

Hemorrhage - - - - - - - -

Neuronal necrobiosis - - - ++ ++ - - -

Clarity of nissl substance in the

neurons

- - - - - - - -

Neuronophagie - - - - + - - +

Gliosis - - - - ++ ++ ++ ++

Mononuclear cells in the

perivascular areas

- - - - + + - -

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

severe (+++).

ble(s)


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AmplifierRF

Generator

Figure 1. Schematic view of exposure set-up

gure(s)


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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

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

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