combined effects of 872 mhz radiofrequency radiation...
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Combined Effects of 872 MHz Radiofrequency Radiation and Known Genotoxic Agents
on DNA Damage in Rat Primary Astrocytes
MUSTAFA, EHAB
Combined Effects of 872 MHz Radiofrequency Radiation and Known Genotoxic Agents on
DNA Damage in Rat Primary Astrocytes
General Toxicology and Environmental Health Risk Assessment
Department of Environmental and biological Sciences, Radiation and Chemicals Research
group, University of Eastern Finland, Kuopio.
June 2017
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UNIVERSITY OF EASTERN FINLAND, Faculty of Science and Forestry
Master degree programme, General Toxicology and Environmental Health Risk Assessment
Ehab Mustafa: Combined Effects of 872 MHz Radiofrequency Radiation and Known
Genotoxic Agents on DNA Damage in Rat Primary Astrocytes
MSc thesis 56 pages, 3 appendixes (59 pages)
Supervisors: Mikko Herrala, MSc and Jonne Naarala, PhD, Docent.
June 2017
Key words: electromagnetic fields, radiofrequency fields, DNA damage, genotoxicity, comet
assay, micronucleus assay, primary astrocytes
ABSTRACT
The aim of this thesis is to investigate a possible co-genotoxic effect of radiofrequency
radiation. The DNA damaging effect of known genotoxic agents (menadione and methyl
methanesulfonate) was combined with the effect of radiofrequency radiation; modulated
(GSM) or unmodulated (CW). Rat primary astrocytes were exposed to radiofrequency
radiation at 872 MHz frequency and specific absorption rate level of 0.6 W/kg for 24 hours
followed by 3 hours chemical exposure or incubation without chemical. Genotoxicity was
assessed by alkaline comet assay immediately after chemical exposure or by micronucleus
assay six days after end of exposure. Comet assay results showed that modulated
radiofrequency radiation significantly decreased DNA damaging effect of menadione. While
micronucleus assay results showed that radiofrequency radiation (modulated and unmodulated)
increased the tendency of both menadione and methyl methanesulfonate to form micronuclei.
When compared to cells exposed to chemicals alone, such effect was statistically different in
case of methyl methanesulfonate combined with unmodulated radiofrequency radiation. This
thesis concluded that radiofrequency radiation was able to modify the genotoxic effects of
menadione and methyl methanesulfonate. Such finding greatly depended on the radiofrequency
signal modulation, method utilized to assess genotoxicity and the time point at which the DNA
damage was assessed.
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Acknowledgements
I want to thank all members of the Radiation and Chemicals Research group for their academic
professionalism. I am deeply grateful to Jukka Luukkonen, Kajal Kumari and Hanne Säppi for
their technical assistance and warm cooperation. My deepest gratitude is to my supervisors:
Mikko Herrala and Jonne Naarala for their constructive comments, excellent advice, kind
regards, and giving me the opportunities to work under their supervision.
The research leading to these results has received funding from the European Community’s
Seventh Framework Programme (FP7/2007-2013) under grant agreement no 603794 – the
GERONIMO project. Without such support, this work would not have been possible.
Finally, to my family, I am grateful for your support, understanding, and prayers. I owe you
everything.
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LIST OF ABBREVIATIONS
°C Celsius Centigrade (Unit of Temperature)
4-NQO 4-Nitroquinolone 1-oxide
8-oxoG 8-Oxoguanine
A Adenine
AC Alternative Current
C Cytosine
CA Chromosomal Aberration
Co-A Alkaline Comet Assay
CW Continuous Waves
DC Direct Current
DLS Dynamic Light Scattering (Visualization Technique)
DMSO Dimethyle sulphoxide
DNA Deoxyribonucleic Acid
E Electric Field
EDTA Ethylenediaminetetraacetic Acid
ELF Extremely Low Frequency (Electric and Magnetic fields)
EMA Ethidium Monoazide
EMF Electromagnetic Field
EU European Union
f Frequency
FBS Fetal Bovine Serum
FISH Fluorescence in Situ Hybridization (DNA Damage Assessment
tool)
FM Frequency Modulation (Radio Technology)
FPG-Co Formamidopyrimidine DNA Glycosylase Modified Comet
Assay
G Guanine
g a measurement of the gravitational force
GHz Giga-Hertz (Unit of Frequency)
GSM Global System for Mobile Communications
H Magnetic Field
HF High Frequency (Electric and Magnetic Fields)
hPBLs Human Peripheral Blood Lymphocytes
hr Hour (Unit of Time)
HSFs Human Skin Fibroblasts
Hz Hertz (Unit of Frequency)
IARC International Agency for Research on Cancer
ICNIRP International Commission on Non-Ionizing Radiation
Protection
IF Intermediate Frequency (Electric and Magnetic fields)
IGEPAL Octylphenoxypolyethoxyethanol
IR Infrared Radiation
5
kg Kilogram (Unit of Weight)
kHz Kilo-Hertz (Unit of Frequency)
LMPA Low Melting Point Agarose
M Molar (Unit of Concentration)
MF Medium Frequency (Electric and Magnetic Fields)
MHz Mega-Hertz (Unit of Frequency)
min Minute (Unit of Time)
MMC Mitomycin C
MMS Methyl methanesulphonate
MN Micronucleus
MQ Menadione
NMPA Normal Melting Point Agarose
OGG1 8-Oxoguanine DNA glycosylase-1
OTM Olive Tail Moment
PBS Phosphate Buffer Saline
pCA Plant Chromosomal Aberration Assay
PM Pulse Modulated
RF Radiofrequency (Electric and Magnetic Fields)
RFR Radiofrequency Radiation
RNA Ribonucleic Acid
SAR Specific Absorption Rate
SCE Sister Chromatid Exchange
SEM Standard Error of the Mean
SF Static Fields
SiRNA Small Interfering RNA
STUK Finnish Radiation and Nuclear Safety Authority
T Thymine
TRIS Tris(hydroxymethyl)aminomethane
UVA Ultraviolet A (315–400nm)
UVB Ultraviolet B (280–315nm)
UVC Ultraviolet C (200–280nm)
W Watt (unit of power)
VIS Visible Light
ζ Zeta potential
λ Wavelength
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Table of Contents 1. INTRODUCTION ................................................................................................................. 8
2. LITERATURE REVIEW ...................................................................................................... 9
2.1. Electromagnetic fields .................................................................................................... 9
2.2. RF fields ........................................................................................................................ 11
2.2.1. Exposure to RF fields............................................................................................. 11
2.3. Health effects of related to RFR ................................................................................... 12
2.4. DNA and DNA damage ................................................................................................ 13
2.4.1. RF radiation induced DNA damage ....................................................................... 15
3. OBJECTIVES ...................................................................................................................... 27
4. MATERIALS AND METHODS ......................................................................................... 28
4.1. Cell line and cell culture ............................................................................................... 28
4.2. Exposure of the cells ..................................................................................................... 28
4.2.1. RF exposure ........................................................................................................... 28
4.2.2. Chemical treatment ................................................................................................ 30
4.3. DNA damage assessment .............................................................................................. 32
4.3.1. Comet assay ........................................................................................................... 32
4.3.2. Micronucleus assay ................................................................................................ 34
4.4. Data analysis and statistical methods ............................................................................ 38
5. RESULTS ............................................................................................................................ 39
5.1. Comet assay .................................................................................................................. 39
5.1.1. GSM + MQ ............................................................................................................ 39
5.1.2. GSM + MMS ......................................................................................................... 39
5.1.3. CW + MQ .............................................................................................................. 40
5.1.4. CW + MMS ........................................................................................................... 41
5.2. Micronucleus assay ....................................................................................................... 41
5.2.1. GSM + MQ ............................................................................................................ 41
5.2.2. GSM + MMS ......................................................................................................... 42
5.2.3. CW + MQ .............................................................................................................. 43
5.2.4. CW + MMS ........................................................................................................... 43
6. DISCUSSION ...................................................................................................................... 45
7. CONCLUSION .................................................................................................................... 49
REFERENCES ........................................................................................................................ 50
APPENDICES ......................................................................................................................... 57
Appendix 1: Reagents .......................................................................................................... 57
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Appendix 2: Preparation of chemicals and buffers .............................................................. 57
2.1. Preparation of menadione ......................................................................................... 57
2.2. Preparation of methyl methanesulphonate. ............................................................... 58
2.3. Preparation of alkaline lysis buffer ........................................................................... 58
2.4. Preparation of electrophoresis buffer ........................................................................ 58
2.5. Preparation of TRIS neutralization buffer ................................................................ 58
2.6. Preparation of ethidium monoazide (EMA) solution. ............................................... 58
2.7. Preparation of micronucleus assay lysis buffer 1 ..................................................... 58
2.8. Preparation of micronucleus assay lysis buffer 2 ..................................................... 59
Appendix 3: Preparation of normal melting point agarose slides ........................................ 59
Appendix 4: BD FACSCanto II flow cytometer check performance .................................. 59
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1. INTRODUCTION
In 2016, there were 7,377 million mobile-cellular telephone subscriptions and 3,654 million
active mobile-broadband subscriptions worldwide (International Telecommunication Union,
2017). These numbers continue to increase year-by-year introducing radiofrequency radiation
(RFR) a one of the most predominant environmental agents on Earth. Besides, humans –on
daily basis- are exposed intentionally and unintentionally to a wide range of environmental
agents (e.g. chemicals and solar radiation) that might lead to undesired effects on health
including genotoxicity, which could be enhanced by RFR.
Elevation of tissue temperature is the most well established biological effect resulted from RFR
coupling with body of living organism. This was the reason that the great majority of studies
investigating the biological potential of the electromagnetic nature of RFR utilize experimental
conditions where temperature is controlled. Non-cancer related effects e.g. neurocognitive
effects, cardiovascular function and reproductive toxicities due to exposure to RFR have been
studied extensively providing neither evident nor consistent results. Based on epidemiological
findings suggesting a positive association between RFR exposure and increased risk of glioma
and acoustic neuroma, International Agency for Research on Cancer (IARC) in 2013 classified
RFR as possibly carcinogenic to humans (Group 2B). In spite of criticism on susceptibility to
bias- due to recall error and selection for participation, such findings could not be dismissed
especially when experimental animal studies and other relevant data were taken into
consideration.
Because photons of RFR do not have enough quantum energy to ionize biological molecule,
RFR is not considered to cause DNA damage at least under non-thermal exposure conditions.
An interesting question would be whether RF radiation is able to enhance the genotoxicity of
known genotoxic agents (e.g. menadione and methyl methanesulfonate used in this thesis).
In different parts of the world mobile phone networks utilize different frequency bands and
different form of RF signal modulation that enable phones to carry information. In Europe,
mobile phones work on 900 and 1800 MHz bands. RF signal is pulse modulated at 217 Hz
according to the standardization of global system of mobile communication (GSM). It was
suggested that RFR biological effects might depend on the modulation characteristics.
Therefore, possible modulation dependent effects were taken into account in this thesis.
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2. LITERATURE REVIEW
2.1. Electromagnetic fields
The electromagnetic nature of both natural environment and living creatures has been
questionable in literature over decades. Living creatures exist in harmony with natural fields
and other different sources of radiation as long as their normal levels are not exceeded and they
are not subject to dramatic changes. The inevitable interaction between human beings and such
fields that is capable of shaping health and well-being has been an initiative for conducting
many studies.
When the wire of an appliance is plugged into electricity outlet, an instantaneous electric field
(E) in the space surrounding the appliance and its wire is produced, though there is no need for
the appliance to be switched on. Electric field exists whenever there is a difference in voltage
even without current flow. The greater the voltage, the sharper is the created E. Contrarily; a
flowing current is necessary for the existence of a magnetic field (H). The higher the current,
the more intensely magnetic field is produced. At this moment, electric field and magnetic field
are contemporary and orthogonally travel in unison forming so-called electromagnetic field
(EMF).
Electric current is the flow of charged particles, explicitly electrons in the case of endeavoring
to reveal the difference between direct current (DC) and alternative current (AC). The
substantial difference is the direction of the flow. DC constantly moves in only one direction
resulting in the creation of static magnetic field, which does not vary over time. On the other
hand, AC generates time variant electromagnetic fields; this is because AC reverses its
direction in oscillating repetitions over regular intervals of times. In most of European Union
(EU) countries, electricity changes direction 50 times per second (50 Hertz). Equitably, the
resultant time variant electromagnetic field changes its orientation in the frequency of 50 Hertz
(Hz).
Radiation as a term simply refers to energy being emitted or transmitted in the form of waves
or particles through space or through a material medium. In the context of electromagnetic
fields, radiation convoy electromagnetic waves that are harmonized waving of electric and
magnetic fields propagating concomitantly at the speed of light. The most known form of
electromagnetic radiation is sunlight. Based on frequency, radiation ensuing from the sun
divides the electromagnetic spectrum into two segments; ionizing and non-ionizing radiation.
The ionizing segment consists of X-rays, gamma rays and ultraviolet C radiation (UVC).
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Ionizing radiation bears photon energy enough to ionize and break biological bonds. Non-
ionizing radiation (Table: 1) does not have enough energy to invoke such ionization and it
includes ultraviolet B radiation (UVB) and ultraviolet A radiation (UVA), visible light (VIS),
infrared radiation (IR), radiofrequency fields (RF), intermediate frequency fields (IF),
extremely low frequency fields (ELF) and static fields (SF).
Table 1: electromagnetic spectrum of non-ionizing radiation
λ/ f ULRAVIOLET VIS (IR) Microwaves (RF) (IF) (LF)
and
(ELF)
(SF)
UVC UVB UVA
Wave
length (λ)
200
–
280
nm
280
–
315
nm
315
–
400
nm
400
–
780
nm
780
nm
– 1
mm
1 mm – 33
cm
33
cm –
3
km
30 m
–
1000
km
1000
km –
∞
∞
Frequency
(f)
1500
–
1071
THz
1071
–
952
THz
952
–
750
THz
750
–
385
THz
385
THz
–
300
GHz
300 GHz – 1
GHz
1
GHz
–
100
kHz
10
MHz
–
300
Hz
300
Hz –
> 0
Hz
0
Hz
All living organisms are exposed to a wide range of different forms of natural radiation
including terrestrial electric and magnetic fields, cosmic microwaves and gamma radiation,
infrared, visible and ultraviolet radiation from the sun and radioactive substances (radon,
uranium, etc.). Biological investigations have shown that living creatures from unicellular
organisms to humans are sensitive to natural EMFs and any variation of their intensity resulting
in having an impact on several vital biological processes, such as neurohumoral regulation,
circadian rhythm, reproduction, development and even the ability of living organisms to orient
themselves in the space (Presman, 1977).
Humans are constantly exposed to a wide spectrum of different patterns of artificial (manmade)
EMFs. Ranging in frequency from ELF to microwaves, exposure has tended to increase over
time since the inauguration of the twentieth century and even earlier.
Low frequency electric and magnetic fields are known for their ability to induce electric current
in tissues. In contrast to magnetic fields which penetrate the body easily and in turn more
potential to interact with human body, electric fields inside the body are very weak. In addition,
the geometry of the induced electric current in both cases is quite different. In both cases
(electric and magnetic fields), the strength of the internal induced electric field depends on the
strength of the external field and its frequency.
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Regarding optical radiation (IR, VIS, UV), at those high frequencies (or very short wave
lengths) there is no deep penetration into the body and everything is absorbed at the skin surface
or near the skin surface. Then two things can happen; generation of heat and photochemical
reactions because of the high energy of the photon.
Figure 1 is an overview of the biological interactions and health effects of radiation based on
frequency.
Figure 1: Overview of the biological interactions and health effects of radiation.
2.2. RF fields
Typically, the term radiofrequency is giving to the electric and magnetic fields locating in the
frequency band between 100 kilohertz and 300 gigahertz including so-called microwaves that
range between 300 MHz and 300 GHz. RF fields are known for being extensively used in
wireless communication systems.
2.2.1. Exposure to RF fields
Until the last two decades concerns about exposure to RFR were limited to occupational setting
where only finite subgroups of population were affected. However, the ubiquity and
tremendous rapid growth of using mobile phones render RFR as one of the most predominant
environmental agents to which humans are exposed. Other sources also exist, for instance;
medical applications (magnetic resonance imaging and other imaging techniques), navigation
and forecasting applications (traffic radar, weather radar), industrial applications (microwaves
ovens, induction and dielectric heating), wireless communications (mobile phone base stations,
cordless phones, Bluetooth technology) and broadcasting (MF, HF, FM radio, television
signal).
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In different parts of the world, mobile networks operate in different frequency ranges. The three
predominant frequency ranges are 1) 824 MHz - 896 MHz many times termed as 850 MHz
band, 2) 1850 MHz – 1990 MHz frequently termed as 1.9 GHz band, and 3) 2.45 GHz. Some
mobile networks employ only one frequency range, others employ combination of these. To
utilize a mix of two or three frequency ranges, mobile devices should be designed to utilize
dual or triple band range. Mobile networks’ digital technology is not only meant for carrying
voice but also digital transferring of different forms of data. Therefore, RF signal is pulse
modulated to tote information. Biological effects of the pulse modulation of RF signal have
been sporadically studied and conclusions are contradictory. In 2011, Juutilainen et al.
suggested some evidence on modulation-dependent effects.
The continuously increasing public concerns about potential health outcomes arising from
exposure to RFR heartened the International Commission on Non-Ionizing Radiation
Protection (ICNIRP) to inaugurate international regulatory ground rules on exposure to RF
fields aiming to protect people (Table: 2). Exposure limits are commonly established in the
terms of Specific Absorption Rate (SAR), which refers to rate by which energy is taken into
tissues of human body when exposed to RF fields. The SAR values for mobile phones in market
are up to 1.6 W/kg (average over 10 g of tissues).
Table 2: Basic restrictions for exposure to RFR (ICNIRP, 1998)
Exposure Whole body
average SAR
(W/kg)
Localized SAR
(head and trunk)
(W/kg)
Localized SAR
(limbs) (W/kg)
Occupational
exposure
0.4 10 20
General public
exposure
0.08 2 4
2.3. Health effects related to RFR
Biological effects of RFR have been investigated in vitro, in vivo and on humans. Most of the
studies used high levels of RFR focusing on its thermal effect. RFR is able to penetrate human
body. The extent to which penetration occurs depends on frequency, the higher the frequency,
the less penetration capability is. RFR body penetration leads to vibration of biological
molecules, subsequently friction and heat generation. Human body has a capacity to regulate
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internal heat. However, if the level of heat exceeds a certain threshold, tissue damage will be
inventible. Recently, it has been suggested that research has to focus on the non-thermal effects
of RFR and their relevance to human health including neurocognitive effects, cardiovascular
function, reproductive toxicities, and cancer. Tissue heating effect should be considered as a
potential confounding factor because of the fact that utilizing high SAR level of the RFR
increases the likelihood of positive findings. Non-thermal effects that were studied extensively
showed no conclusive evidence of adverse health effects. Probably, the only consistent finding
with in the regard that RFR can induce minor changes in brain activity that were detected by
electroencephalography (Hamblin and Wood, 2002).
In May 2011, 30 scientists from 14 countries gathered at IARC headquarters to evaluate the
current situation regarding the raised concerns about the carcinogenicity of RFR. After
reviewing several epidemiological studies, animal studies, and other relevant data, they ended
up that there is a limited evidence on the carcinogenicity of RFR in human. Accordingly, RFR
was classified as possibly carcinogenic to humans (Group 2B) (Baan et al. 2011). The
classification was based on positive association between use of mobile phone and increased
risk of glioma and acoustic neuroma. One cohort study (Schuz et al., 2006), three early case-
control studies (Muscat, 2000, Inskip et al., 2001, Auvinen et al., 2002), one multicenter case-
control study (Brain tumour risk in relation to mobile telephone use: results of the
INTERPHONE international case–control study, 2011), and one pooled analysis of case-
control studies (Hardell et al., 2011) were all considered regarding judging potential association
between use of mobile phones and glioma. Same multicenter case-control study and the pooled
analysis, in addition to a Japanese case-control study (Sato et al., 2011), were all considered
regarding judging potential association between use of mobile phones and acoustic neuroma.
2.4. DNA and DNA damage
Deoxyribonucleic acid (DNA) is a biological molecule present in the cells of all living
organisms, it carries the hereditary instructions that make every species unique controlling how
it functions, develops, and adapts to the surrounding environment and it transfers to the
offspring through reproduction.
Nucleotides are the basic building blocks of DNA structure. Every nucleotide is built of 3
components: a sugar, a phosphate group, a nitrogenous base. DNA sugar is five carbons ribose
sugar, in which one of the hydroxyl, or OH, groups on the second carbon is missing; this is
why it is named 2-deoxyribose. Nitrogenous base of every nucleotide is one of four bases:
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adenine (A), guanine (G), thymine (T) and cytosine(C). A single DNA strand is formed when
the sugar of one nucleotide is linked with the phosphate group of an adjacent nucleotide through
phosphodiester bond. The double stranded structure of DNA occurs through complementary
base pairing. Every adenine and thymine bases are held together by double hydrogen bond.
Every guanine and cytosine bases are held together by triple hydrogen bond as illustrated in
Figure 2.
Figure 2: Schematic diagram of complementary double stranded structure and forming bases
of DNA.
The way how DNA bases are sequenced forms genes. Genes are the functional units of
hereditary. Genes are decoded into proteins; complex biological molecules that take care of
most of work in the cell.
Human DNA is under stress all the time with a daily rate of 105 lesions per cell (Hoeijmakers,
2009). Chemical events behind DNA damage involve hydrolysis, oxidative stress and other
forms of interaction with different types of active metabolites. This can be caused not only by
a wide range of chemical and physical exogenous agents but also as a consequence of many
endogenous processes including metabolism. It is also important to notice that DNA damage
and DNA mutations are fundamentally different. DNA damage is a change in the chemical
nature of DNA or its physical configuration. DNA damage is most often recognized by DNA
repairing enzymes, and thus repaired. Contrarily, DNA mutation is a permanent change in the
base sequence that, in case it is present in on both DNA strands, it is not liable anymore to be
recognized or repaired. However, DNA damage and DNA mutation are still related as it is
certain that the retention of DNA damage without remedy or improper restoration of DNA
damage will eventually lead to mutations.
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There are many types of DNA damage but generally they are classified into three major groups;
DNA base damages, DNA backbone damages and DNA cross-links. DNA base damage
includes: (1) O6 methylguanine; a derivatization process in which guanine nucleobase is
attached to a methyl group through the oxygen atom at position 6. The derivative base tends to
base pair with thymine rather than cytosine, The resulted O6 methylguanine-thymine complex
may raise to G:C to A:T transition mutation (De Bont and Larebeke 2004), (2) Thymine glycol;
an oxidative stress induced DNA damage in which thymine nucleobase is modified to end up
as 5,6-dihydroxy 5,6-dihydrothymine or “thymine glycol”. DNA sequences embeds thymine
glycol show resistance to be replicated as long as DNA polymerase is not able to bypass the
sequence of lesion (Basu et. al. 1989), (3) Base adducts; They vary from alkylating agent simple
adducts to polyaromatic hydrocarbons bulky adducts, (4) Oxidation, reduction and
fragmentation of bases triggered by reactive oxygen species, heavy metals mediated reactions,
ionizing radiation and UV radiation.
DNA backbone damage includes: (1) Abasic sites; locations on the strands of DNA where
either purinic or pyrimidinic bases are missed. They may result spontaneously from the
intermediates of base excision repair or from monofunctional alkylating agents generated DNA
adducts, (2) Single-strand breaks; 1-30 nucleotides gap single-strand breaks may emerge as
intermediates during base excision repair and nucleotide excision repair. Moreover, single-
strand breaks exist as a consequence of directly damaging agents, (3) Double-strand breaks;
they are distinctively associated with ionizing radiation induced damage but also they are
intermediates of genetic recombination during meiosis and mitosis. Electron deficient reactive
intermediate of bi-functional alkylating agents such as cisplatin and nitrogen mustard
covalently link with the nucleophilic DNA producing DNA-DNA intra-strand and inter-strand
cross-links rising to form roadblocks that might interfere with transcription and replication.
Different classes of DNA lesions bring out different pathways that the cell can get along in
order to respond. These pathways, however, operate collectively and share many components.
Any defect in the harmonized way these pathways serve may give rise to genomic instability
(Jackson and Bartek, 2009).
2.4.1. RF radiation induced DNA damage
A recent review in 2016 by Manna and Ghosh approached effects of radiofrequency radiation
in cultured mammalian cells in the past 20- 25 years with an interest on cellular morphology,
proliferation, growth profile, cellular signaling cell cycle arrest, cell death mechanism, cellular
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metabolism, gene expression and genetic toxicology. They scrutinized variant responses at the
same irradiation frequency, but at different power densities, signal modulation and exposure
times. The possibility of occurrence of DNA damage was increased at higher SAR values and
longer exposure times. Interestingly, authors figured out that the sensitivity of the cells differed
on exposure to differently polarized radiofrequency radiation. This may be related to the
asymmetry of biological molecules, most importantly in the DNA. Co-treatment impact
differed by using different agents. While radiofrequency radiation enhanced the DNA
damaging effect of bleomycin, doxorubicin, UV radiation and heat, such effect was lacking
with mitomycin C and X-rays.
From genotoxicological point of view, Verschaeve and Maes in 1998 reviewed the literature
on RFR paying a special attention to frequencies utilized by mobile phones. The vast majority
of studies asserted that RFR is not genotoxic in vitro or in vivo at non-thermal conditions and
it is not likely to induce cancer. The most consistent biological findings was the potentiality of
RFR to elevate tissue temperature by 1°C or more at SAR level 2 or more.
Again, in 2010 Verschaeve et al. reviewed the literature on RFR induced genotoxicity. This
time attention was paid to studies combined exposure to RFR with chemicals or other physical
agents. The reviewers criticized articles for matters that could hinder a clear evaluation of data
including; unrealistic hyperthermal exposure condition, indigent dosimetry, improper control
over temperature, poor description of experimental details, and inappropriate statistical
procedures. Repeatedly, No consistent overall picture was drawn. Authors suggested that
findings from studies used co-exposure approach should be emphasized.
Luukkonen in 2011 reviewed in-vitro and in-vivo studies published in 5 past years with a
primary interest on selected endpoints relevant to the mechanisms of cancer; oxidative stress,
cell death, cellular proliferation and genotoxicity. A total of 34-genotoxicity studies were
reported. Effects of radiofrequency radiation were found in 14 studies. Among all co-exposure
experiments, notably one of the positive studies reported that instead of increasing the
genotoxic effect, exposure to radiofrequency radiation decreased the genotoxic effect of
mitomycin c. Consistently with other reviewing work, most of positive studies used a relatively
high level of SAR (2 W/kg or above).
Vijayalaxmi's meta-analysis in 2012 paid an attention to effect on DNA damage in human cells
through reviewing data from 88 articles published from 1990 to 2011. Vijayalaxmi indicated
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that the emergence of statistically significant difference between exposed and non-exposed
cells in the level of DNA damage was exceptional and of small magnitude. Such difference
was majorly observed in studies used small sample size and probably was driven by publication
bias. The difference was lacking in most of studies utilized the recommended guidelines of
exposure to RFR.
This chapter of the thesis addresses experimental in-vitro studies published in year 2011 or
later with genotoxicity as only concern. Total of 16-genotoxicity studies provided inconsistent
outcomes. In comparison to previous reviewing efforts, It was interesting in this review that
the number of studies where radiofrequency radiation had an effect (Esmekaya et al. 2011,
Karaca et al. 2011, Xu et al. 2013, Hekmat et al. 2013, Liu et al. 2013 a & b, Duan et al. 2015,
Zalata et al. 2015, Wang et al. 2015, Qureshi et al. 2016, Ji et al. 2016) is higher than number
of studies where it did not have any effects (Bourthoumieu et al. 2011, Hintzsche et al. 2012,
Waldmann et al. 2013, Speit et al. 2013, Kumar et al. 2015).
Investigators used wide range experimental set ups to evaluate the genotoxic potential of
radiofrequency radiation including different signal modulation, exposure durations, exposure
temperatures and different attitudes towards combined exposures with chemical or physical
agents. While gingko biloba (Esmekaya et al. 2011), α-tocopherol (Liu et al. 2013 a), and
melatonin (Liu et al. 2013 b) were studied for their potential protective role, 8-oxoguanine
DNA glycosylase-1 SiRNA was investigative for its radiofrequency radiation induced DNA
damage enhancing effect (Wang et al. 2015).
Notably, radiofrequency radiation DNA damaging effect was observed at SAR level as low as
0.04 W/kg (Hekmat et al. 2013), 0.21 W/kg (Esmekaya et al. 2011), 0.5 W/kg (Wang et al.
2015), 0.58 W/kg (Liu et al. 2013 b), and 0.725 W/kg (Karaca et al. 2011).
Different tools of assessing genotoxicity were utilized including alkaline comet assay
(Waldmann et al. 2013, Speit et al. 2013, Liu et al. 2013 b, Duan et al. 2015, Kumar et al. 2015,
Wang et al. 2015, Ji et al. 2016), modified alkaline comet assay (Duan et al. 2015 and Wang et
al. 2015), micronucleus assay (Karaca et al. 2011, Hintzsche et al. 2012, Waldmann et al. 2013,
Speit et al. 2013), chromosomal aberration assay (Waldmann et al. 2013 and Qureshi et al.
2016), assay of change in the rate of aneuploidy of chromosomes (Bourthoumieu et al. 2011),
sister chromatid exchange assay (Esmekaya et al. 2011 and Waldmann et al. 2013), testing
induction of γH2AX foci formation ( Xu et al. 2013, Duan et al. 2015, Ji et al. 2016), along
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with microscopic and spectroscopic observation of DNA structure (Hekmat et al. 2013). In
addition, determination of the level of 8-oxoguanine DNA adduct as a way of assessing
oxidative DNA damage (Liu et al. 2013 a).
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Table 3: RF radiation induced DNA damage (In vitro studies: 2011 -2016)
RF radiation exposure In vitro model Assay endpoint Co-exposure Response Reference
Signal Exposure power/ SAR
(W/kg)
Exposure duration
Exposure temperature
GSM-900 MHz
0.25, 1, 2 and 4
24hrs temperature range: 36.3- 39.78 8 °C
Human amniotic cells
Change in the rate of aneuploidy of
chromosomes 11 and 17 determined by
interphase Fluorescence In Situ Hybridization (FISH)
No No effect Bourthoumieu et al. 2011
The signal was used at 10.715 GHz
oscillator frequency
with 8.0mW power output
0.725 6hrs per day for 3 days
25 °C Wistar albino mouse brain
cells
DNA damage: micronucleus assay
(MN)
No Rate of MN frequency
increased by 11 folds
Karaca et al. 2011
GSM-1800 MHz
(pulsed at 217 Hz)
0.21 6,8,24 and 48hrs
37 °C Human peripheral
blood lymphocytes
(hPBLs)
mutagenic potential: sister chromatid exchange (SCE)
and change in the chromosome
structure visualized by electron microscopy
Ginkgo biloba
(EGb761) (72hrs
before RF radiation,
RFR, exposure)
Significant increase in
SCE frequency compared to
sham controls
EGb761 pre-treatment
significantly decreased SCE
Esmekaya et al. 2011
20
900 MHz (CW and
GSM)
field strength
values of 0 (sham), 5, 10, 30 and
90 V/m
Not reported 30mins and 22hrs
37 °C HaCaT cells (human cells) and AL cells
(human-hamster hybrid
cells)
DNA damage (MN) No No effect Hintzsche et al. 2012
GSM-1800 MHz
(pulsed at 217 Hz)
3 1 and 24hrs (intermittent exposure of
5mins on and 10mins off)
37 °C Chinese hamster lung cells, Human
skin fibroblasts (HSFs), Primary
newborn Sprague-
Dawley rat astrocytes,
Human amniotic
epithelial cells and Human
umbilical vein endothelial
cells
DNA damage: induction of γH2AX
foci formation
No Exposure to RFR for 24hrs significantly
induced γH2AX foci
formation in Chinese
hamster lung cells and
HSFs, but not the other cells
Xu et al. 2013
940 MHz 0.04 45mins 37 °C extracted calf thymus DNA
Changes in the structure of DNA
using diverse range of
spectroscopic techniques: UV
radiation-visible light
No UV–VIS studies:
significant alteration in
the DNA structure.
Hekmat et al. 2013
21
investigation studies (UV–VIS),
fluorescence studies, dynamic light
scattering (DLS) and zeta potential (ζ)
Fluorescence studies: more fluorescence intensity due
to RFR exposure
DLS and ζ-potential:
increment in the size and
surface charge of DNA
due to RFR exposure
GSM-1800 MHz
(pulsed at 217 Hz)
0.2, 2 and 10 28hrs (intermittent exposure of
5mins on and 10mins off)
37 °C hPBLs Genotoxicity: chromosomal
aberration (CA), MN, SCE and alkaline
comet assay (Co-A)
No No effect Waldmann et al. 2013
GSM-1800 MHz
1,2 and 4 24hrs (intermittent exposure of
5mins on and 10mins off)
37 °C Mouse spermatocyte-
derived cell line (GC-2)
oxidative DNA base damage: Flow
cytometry analysis for DNA adduct 8-
oxoguanine (8-oxoG)
α-tocopherol (24hrs
before RFR exposure)
Level of 8-oxoG was significantly
increased at a SAR level of 4
W/kg
This phenomenon
was significantly mitigated by co-treatment
Liu et al. 2013 (a)
22
with the antioxidant α-
tocopherol
CW-1800 MHz
1.3 24hrs (intermittent exposure of
5mins on and 10mins off)
37 °C Human lymphoblastoid cell line (HL-60)
DNA damage: Co-A and MN
No No effect Speit et al. 2013
GSM-1800 MHz
(pulsed at 217 Hz)
The exposure device used in this study
was a commercial
mobile phone:
handset-Motorola
XT300
The highest SAR value for
the human head was
0.585 and it was 0.813 for
the whole body
according to the
manufacturer
Four modes were
examined: Standby,
listen, dialed and dialing
Each
mode was performed
once for 1min once
every 20mins for a total duration of 24hrs
37 °C Mouse spermatocyte-derived GC-2
cell line
DNA damage: Co-A Melatonin (2hrs before
RFR exposure)
Significant increase in
DNA damage in listening, dialed and
dialing modes
Significantly higher
increases in the dialed and
dialing modes than in
the listen mode
DNA damage effects in the dialing mode
were significantly
attenuated by melatonin
pretreatment.
Liu et al. 2013 (b)
23
GSM-1800 MHz
1, 2 and 4 24hrs (intermittent exposure of
5mins on and 10mins off)
37 °C Mouse spermatocyte-
derived GC-2 cell line
DNA damage: Co-A and induction of
γH2AX foci formation
Oxidative DNA base damage:
formamidopyrimidine DNA
glycosylase modified alkaline comet assay
(FPG-Co)
No No effect (Co-A and γH2AX
foci formation)
FPG-Co: RFR
exposure significantly
induced oxidative DNA base damage at a SAR value
of 4 W/kg
Duan et al. 2015
850 MHz The exposure device used in this study
was a commercial
mobile phone
with SAR value of 1.46 according to
the manufacturer
1hr Room temperature
Human sperms Sperm DNA fragmentation:
fluorescent flow cytometry
No Significant increase in sperm DNA
fragmentation in the semen
samples exposed to
RFR compared with non-exposed samples
Zalata et al. 2015
Experiment 1 (E1): CW-
900 MHz
Experiment 2 (E2): 1800
MHz (CW and PM)
E1: 2 and 10 (at bone
marrow level)
E2: 2.5 and 12.4 (at bone marrow level)
E1: 1.5hr
E2: 2hrs
37±1 °C Excised long bones of
young male Sprague
Dawley rats
DNA damage in bone marrow lymphoblast
(Co-A)
No No effect in all experimental
setups
Kumar et al. 2015
24
GSM-900 MHz
0.5, 1, 2 24hrs 37 °C Mouse neuroblastoma cell line (neuro-
2a)
DNA damage: Co-A
Oxidative DNA base damage: FPG-Co
8-oxoG DNA glycosylase-1
siRNA (OGG1-
siRNA); a small
interfering RNA
molecule that inhibits
the expression of OGG1 gene
which plays a pivotal role
in base excision repair
(24hrs
before RFR exposure)
No effect (Co-A)
FPG-Co: RFR exposure
significantly induced
oxidative DNA base damage at a SAR value
of 2 W/kg
Inhibition of OGG1 gene significantly induce DNA
base damage at SAR value as low as 1
W/kg
Wang et al. 2015
GSM-900 MHz (Nokia
GSM set, model: X2-
00) and 3.31 GHz
(HP laptop, model: 430
core i5)
Not reported 24 and 48hrs Room temperature
Dry seeds of chickpea
DNA damage: Plant chromosomal
aberration assay (pCA)
No Significant increase in the level of
DNA damage in all
treatment setups
compared to untreated
control
Qureshi et al. 2016
25
DNA damages increase with
increasing duration of
RFR exposure
48hrs laptop treatment has
the most negative
effect
CW-900 MHz
Not reported (power
intensity was adjusted to
120μW/cm2)
4hrs per day for 5 days
37±0.5 °C Mouse bone-marrow
stromal cells
DNA damage: Co-A (immediately after end of exposure as well as after 30, 60, 90 and 120mins to
determine the kinetics of repair of
strand breaks)
Induction of γH2AX foci formation (only
after 120mins)
Acute dose of 1.5Gy of
gamma-radiation given at 4 hour after
RFR last exposure
No significant differences in level of DNA
damage between RFR exposed and control cells
Significant increase in
level of DNA damage in
cells exposed to gamma radiation
RFR followed
by gamma radiation exposure
significantly decreased
Ji et al. 2016
26
level of DNA damage and resulted in
faster kinetics of repair of
DNA damage compared to
gamma radiation
alone
27
3. OBJECTIVES
Aims of the thesis are to investigate a possible genotoxic effect of RFR with frequency of 872
MHz and SAR level of 0.6 W/kg in rat primary astrocytes and to study whether the effect is
modulation dependent. The thesis focuses on co-exposure settings combining RFR with
chemicals known to be genotoxic (menadione and methyl methanesulfonate).
The thesis is aiming at accepting or rejecting the following hypotheses:
Null hypothesis 1: exposure to 872 MHz RFR at SAR level of 0.6 W/kg does not increase
genotoxic agent-induced DNA damage in rat primary astrocytes.
Null hypothesis 2: the possible genotoxic induced effect is not modulation dependent.
Alternative hypothesis 1: Exposure to 872 MHz RFR increases genotoxic agent-induced DNA
damage in rat primary astrocytes.
Alternative hypothesis 2: the possible genotoxic induced effect is modulation dependent.
28
4. MATERIALS AND METHODS
4.1. Cell culture
Primary astrocytes isolated from 1-3 days old RccHan:WIST rats are used in the study. In
partnership with neurons, astrocytes are known for their major supportive functions in the
central nervous system of mammalians and their implications of many pathological processes
as well. Cells were cultured in Dulbecco’s modified eagle medium (DMEM) containing 1 g/l
glucose, L-glutamine and pyruvate (Gibco, Paisley, UK). Cell culture medium was treated by
10% heat inactivated fetal bovine serum (FBS), penicillin (50 U/ml) and streptomycin
(50μg/ml). Cells maintenance was in cell culture flasks with culture area of 75 cm2 (Thermo
Fisher Scientific, Roskilde, Denmark). Humidified incubation of the cell culture was carried
out in 5% CO2 and 37 °C adjusted incubator (Sanyo, Japan). During passaging cells phosphate
buffer saline (PBS) was used for rinsing, while trypsinization (2.5% trypsin in 0.02% EDTA
in Ca+2 and Mg+2 free phosphate buffer saline) was used to detach cells. Detached cells were
extracted by centrifuging (Biofuge Primo, Heraeus instruments, France) at 363 g for 8 mins
after being transferred to suitable size cylinder tube. At end of centrifuging, cells were in form
of a pellet in the bottom of the tube, old medium was withdrawn and pellet to be dissolved in
a 1 ml of new medium and mixed well for subsequent use. For cell counting, 10 µl was
withdrawn from the cells suspension and mixed with 990 µl of PBS, subsequently after dilution
75 µl of the mix was introduced to an automated cell counter (MOXI Z, ORFLO technologies,
USA). Cells at passage numbers 4-14 were used for experiments. The cells were plated 24
hours prior to exposures in petri dishes of 60 mm diameter with a culture area of 21.5 cm2
(Thermo Fisher Scientific, Roskilde, Denmark). Dishes for micronucleus assay experiments
were seeded with 0.2×106 cells per dish, while dishes for comet assay experiments were seeded
with 0.25×106 cells per dish.
4.2. Exposure of the cells
4.2.1. RF exposure
The RF exposure system (Figure: 3 and 4) was designed and built by Radiation and Nuclear
Safety Authority, STUK, Helsinki, Finland. It consists of two separate and identical aluminum
chambers; a waveguide exposure chamber in which the RF power is fed by a monopole antenna
and a sham exposure chamber. Each is of 420 mm (length), 248 mm (width), 175 mm (height).
In addition to signal generator (9 kHz – 2080 GHz, SMYO2, ROHDE & SCHWARZ,
29
Germany), directional power meter (Neuwirht/Bick, ROHDE & SCHWARZ, Germany), and
RF amplifier (400 – 1000 MHz, WIDBAND, R720FC, Fairview Microwave, USA) were used.
Figure 3: RF exposure system with two separate identical exposure chambers.
Figure 4: RF exposure system; amplifier (bottom), signal generator (middle), directional
power meter (top)
30
Isothermal conditions in the exposure system (+ 37 ± 0.3 °C) were achieved through equipping
both of the chambers by water circulation based heat exchanger. Dishes were placed on the
glassy surface of the heat exchanger where elimination of temperature elevation was allowed.
The chambers were ventilated by temperature-controlled mixture of 5% carbon dioxide in air
coming from a modified cell culture incubator (HERA cell, Heraeus instruments, France).
STUK calibrated the exposure system that it guarantees a uniform distribution of SAR (± 35%
in the cell cultures).
Cells were exposed to 872 MHz RF radiation with modulation (GSM modulation, pulsed at
217 Hz) or modulation free (continuous wave; CW) at a SAR of 0.6 W/kg. The capacity of the
exposure system did not allow using more than four Petri dishes in single experiment; two
dishes were exposed to RF radiation being placed in the waveguide exposure. Whilst two other
dishes were simultaneously in the sham exposure chamber. Cells were divided in four different
groups: (1) RF radiation, (2) RF radiation + chemical treatment, (3) sham exposed, (4) sham +
chemical treatment (Figure: 5).
Figure 5: Experimental design in the two chambers of the exposure system.
4.2.2. Chemical treatment
Menadione (MQ, free radical-producing agent) having the concentration of 15 μM (Appendix:
2.1) and methyl methanesulphonate (MMS, alkylating agent) having the concentration of 40
µg/ml (Appendix: 2.2) were used for chemical treatment.
For comet assay experiments (Table: 4), after 24hrs of sham or RF exposure cells were treated
with chemicals or incubated without chemical treatment for 3hrs and then assayed (Figure: 6).
31
Figure 6: Temporal sequence for performing comet assay.
Table 4: types of experimental replicates performed within the context of comet assay.
Exposure/ treatment No. of replicates
GSM, GSM + MQ, sham, sham + MQ 5
GSM, GSM + MMS, sham, sham + MMS 3
CW, CW + MQ, sham, sham + MQ 3
CW, CW + MMS, sham, sham + MMS 3
For MN assay experiments (Table: 5), the 24hrs sham and RF exposed cells were subjected to
a 3hrs incubation or 3hrs chemical exposure respectively. Following this, cells were washed to
remove the chemical and further cultivated in fresh medium for 6 days before the micronucleus
assay (Figure: 7).
Figure 7: Temporal sequence for performing MN assay.
32
Table 5: types of experimental replicates performed within the context of MN assay.
Exposure/ treatment No. of replicates
GSM, GSM + MQ, sham, sham + MQ 3
GSM, GSM + MMS, sham, sham + MMS 3
CW, CW + MQ, sham, sham + MQ 3
CW, CW + MMS, sham, sham + MMS 3
4.3. DNA damage assessment
4.3.1. Comet assay
Comet assay is an investigatory laboratory tool meant for the examination of DNA damage and
DNA repair and studying the underlying mechanisms of genetic toxicity triggered by a variety
of experimental conditions. The principle of comet assay is based on the negatively charged
DNA fragments liability to be pulled through an agarose gel upon application of electric
current. When electric field is encountered, damaged DNA is drifted away from the nucleus
towards the positively charged anode deputed like “comets”. The magnitude of DNA damage
is one of the major considering factors determine how far fragments migrate. Meanwhile
migration farther away from the nucleus, DNA fragments form “tail”. The size, shape and
content of the tail is directly proportional to the extent of DNA damage, this is subsequently
visualized and quantified utilizing fluorescent microscopy and software developed digitized
images (Fairbairn et al. 1995). Microgel electrophoresis, single cell gel assay or comet assay
was firstly introduced in 1984 by Östling and Johansen while they were trying to develop a
microelectrographic technique to measure radiation induced DNA damage in individual
mammalian cells. Östling and Johansen’s assay was neutral where both electrophoresis and
lysis were performed in neutral conditions. The conditions used by these authors appeared to
be effective in detecting double strand DNA breaks causing relaxation of DNA supercoils.
Alkaline modification by Singh et al. in 1988 made it possible to detect low level of DNA
damage of different types of strands breaks with higher sensitivity.
Immediately after the end of chemical exposure, petri dishes were gotten away from the
incubator and medium was removed by suction. Trypsinization was utilized for cell detachment
via using 1.5 ml of 2.5% trypsin in 0.02% EDTA in Ca+2 and Mg+2 free phosphate buffer saline
33
(PBS). Trypsinization was ceased by adding 3 ml of fresh medium. The suspensions of the
detached cells were then transferred to 15 ml tubes to be centrifuged at 438 g and +4°C for 8
mins (Biofuge Strator, Heraeus Instruments, France). After centrifugation, supernatant was
aspirated and each cell pellet was resuspended in 250 µl of Ca+2 and Mg+2 free phosphate buffer
saline, and the samples were placed on ice. Beforehand, low melting point agarose (LMPA)
was melted by placing the LMPA containing Eppendorf tube in boiling water and 75 µl of
melted agarose were allocated into each appropriately labeled Eppendorf tube to be kept in 37
°C block heater. 15 µl of each PBS-cells mixture to be analyzed suspensions were dispensed
to every LMPA Eppendorf tube. Following mixing, 80 µl of cells-LMPA suspension were
moved from every tube onto previously prepared normal melting point agarose (NMPA) slides
(Appendix: 3). The transferred amount of the suspension was spread on the slides using a cover
slip, and then the slides were placed immediately on ice for at least for 5 mins. Further, coves
slips were removed and slides were moved to a dark box to be poured gently by a previously
prepared alkaline lysis buffer (Appendix: 2.3) and kept covered in refrigerator for an hour. 10
to 15 mins prior to electrophoresis, the already prepared electrophoresis buffer (Appendix: 2.4)
was poured to the electrophoresis tank to stabilize. The electrophoresis tank was covered by
aluminum foil throughout the whole experiment to avoid light. After one hour, the slides were
gotten out from the refrigerator, lysis buffer was aspirated and slides were moved to the
electrophoresis tank ensuring that slides were well covered by solution and were not on top of
one another. Slides were kept in the electrophoresis buffer for 25 mins without connecting the
electricity for unwinding the DNA. Afterwards, the electrophoresis unit was switched on for
30 mins at 380 milli-Amperes and 24 Volts. After electrophoresis, the slides were transferred
into an opaque covered box where they are immersed in TRIS neutralizing buffer (Appendix:
2.5) for 5 mins, this washing step was repeated for three times. After washing, the neutralizing
buffer was aspirated and 50 ml of 96% ethanol was added for one minute to fix the cells. After
fixing bottom of slides were wiped and slides were moved to a tray to be kept in dark at least
24hrs prior to analysis.
Staining of the slides was carried out in dark conditions with well vortexed 75 µl of diluted
ethidium bromide solution prepared by adding 100 µl of ethidium bromide stock solution (0.2
mg of ethidium bromide in 1 ml of water) to 900 µl of milliQ water. For fixation, stain was in
contact with the slides at least 30mins prior to microscopic analysis.
34
The samples were blinded by coding and analyzed using Carl Zeiss AxioImager A1
epifluorescence microscope (Axio Imager A1, Carl Zeiss, Göttingen, Germany) and Perceptive
Instruments Comet Assay IV programme package (Perceptive Instruments, Haverhill, UK).
Olive tail moments (OTM) of 100 nuclei were scored per slide.
Tail Length is the distance of DNA migration from the body of the nuclear core (Figure: 8).
OTM is defined as the product of the tail length and the fraction of total DNA in the tail. Tail
moment incorporates a measure of both the smallest detectable size of migrating DNA
(reflected in the comet tail length) and the number of relaxed / broken pieces (represented by
the intensity of DNA in the tail).
Figure 8: Comet IV live video image introducing mouse single click automatic scoring (a),
and Comet IV scoring including background correction, determination of head and tail
regions and computation of all parameters (b).
4.3.2. Micronucleus assay
When a eukaryotic cell undergoes division, it replicates its genetic material which is then
divided equally between two newly produced daughter cells. If this process is affected or the
chromosomes are broken or damaged via the action of chemical or physical agents, it is possible
that the distribution of the genetic material to the new daughter cells gets disturbed that
fragments or entire chromosomes fail to be incorporated in the nucleus of any of these newly
formed cells. At telophase, the remnant genetic material not included into the nuclei of the new
cells tends to be wrapped with a nuclear envelope and taking into consideration that it is smaller
in size than the main nucleus, it is given the name “micronucleus” (Figure: 9).
35
Figure 9: Disruption of chromosomes leads to lagging fragments or entire chromosomes from
being incorporated in the nuclei of the daughter cells during cell division and hence
micronuclei formation.
Based on that, micronucleus can act as an indicator for both chromosome breaks where the
centromere is lacking and chromosome loss where a whole chromosome is incapable of being
drawn to the spindle poles during mitosis without discrimination. Easiness of scoring and
higher statistical power obtained by scoring larger number of cells provide micronucleus assay
a major advantage over the classical metaphase chromosome analysis which is complex and
more laborious. However, it is obvious that the assay cannot be used as an indicator for
genotoxicity in non-dividing cells or cells with lack of understanding of their division (Fenech
2000).
Flow cytometrical automated scoring of micronuclei based on 2-color labeling technique was
used in this thesis. Through this staining technique, the differentiation micronuclei and
chromatin of apoptotic and necrotic cells could be assured. Therefore, the reliability of
micronuclei measurement was guaranteed even with high number of dying/dead cells. Nucleic
acid dye A (ethidium monoazide, EMA) crossed the compromised plasma membrane of both
apoptotic and necrotic cells (EMA positive cells) and covalently bound to their DNA via photo
activation. Later on, a detergent-based buffer was used to lyse cell membrane so that nuclei
and any possible micronuclei were liberated. Nucleic acid dye B (SYTOX Green) was then
introduced to label all chromatin allowing the differential staining of healthy chromatin versus
that of apoptotic and necrotic cells (Figure: 10).
36
Figure 10: Sequential staining of cells during the micronucleus assay.
Along with micronuclei determination, other endpoints could be measured conveying more
information about cytotoxicity, one of those was “relative survival” that represents a multi-
parametric tool of evaluating health of cells. Familiarity with extent of cytotoxicity is elemental
in the evaluation of genotoxicity assays. Determination of such extent can be achieved by the
addition of fluorescent latex microspheres or what so called “counting beads”. The presence of
such beads at known and consistent density provides information about the total volume of
suspension that passes through the flow cytometer. This allows the determination of nuclei
(EMA negative cells) –to– beads ratios and these values can be used to calculate relative
survival (Avlasevich et al., 2010).
At the end of the six-day incubation period after exposures, petri dishes were taken away from
the incubator and placed on ice making sure that ice made direct contact with their bottom.
Medium was removed and ice incubation lasted 20 mins. Meanwhile, EMA solution was
prepared (Appendix: 2.6). Afterwards, 1.5 ml of the freshly prepared EMA solution was added
to each petri dish. Visualization of apoptotic and necrotic cells was performed through photo-
activation under light at 15 cm distance from a white lamp where petri dishes were maintained
on ice without lids for 30 mins. Meanwhile lysis buffer 1 (Appendix: 2.7) and lysis buffer 2
(Appendix: 2.8) were prepared. Afterwards, the lamp was turned off, EMA solution was
aspirated away and every petri dish was washed by 1.5 ml of PBS-FBS solution and 1 ml of
37
the freshly prepared lysis buffer 1 was added to each petri dish (+4 °C). Petri dishes were then
incubated for 1 hour at +37 °C. Subsequently, every petri dish was seeded with 1 ml of the
freshly prepared lysis buffer 2 prior to a 30 mins dark incubation period at room temperature.
Finally, the petri dish contents were moved to labeled falcon tubes to immediately start the
flow cytometric analysis.
Flow cytometry was performed by BD FACSCanto II flow cytometer (Becton Dickinson, BD
Biosciences, San Jose, California, USA) provided with BD FACSDiva Software. Flow
cytometer was allowed to warm up for 20-30 mins prior to running samples. Meanwhile,
software was initiated, fluidics system was started up, fluids leaks and liquid containers were
checked, cleanness of the system was confirmed, and quality control check performance was
carried out (Appendix: 4). Afterwards, samples were run, the software acquisition dashboard
was used to adjust number of events to be recorded and flow rate of sample to be analyzed.
Number of beads, nuclei and micronuclei were written down to be imported later on to another
statistical software for analysis. Figure 11 shows the areas on the scatter plot of FACSDiva
Software generated during the flow cytometry experiment; gates. Through gates, types of cells
with an intention to be analyzed are decided; micronuclei, hypodiploids, and nucleated cells.
After running the samples, the fluidics system was rinsed and cleaned preceding switching off
the software and the machine.
Figure 11: Gates of micronuclei, hypodiploids, and nucleated cells of two different
experiments. It is noticeable that micronuclei gate in experiment (a) is less rich in content
than micronuclei gate of experiment (b) where the genotoxic MMS was applied.
38
4.4. Data analysis and statistical methods
The statistical analysis was performed with the aid of GraphPad Prism, 5th edition (GraphPad
Software, Inc., La Jolla, California, USA) and Microsoft Excel, 2016 edition (Microsoft,
Redmond, Washington, USA). One-way ANOVA statistical model was use to estimate
variance in the level of DNA damage, micronucleus frequency and cell relative survival in
different settings of exposure. A p-value less than 0.05 was considered statistically significant.
39
5. RESULTS
5.1. Comet assay
5.1.1. GSM + MQ
Figure 12 is for combination of five replicates. It shows that cells exposed to sham + MQ
produced the highest OTM value (5.90 ± 0.27) which is statistically significant higher when
compared to OTM value produced by cells exposed to GSM + MQ (4.76 ± 0.27). OTM value
for cells exposed to sham (1.45 ± 0.13) was higher than one produced by GSM (1.81 ± 0.11),
difference was not statistically significant though.
ShamGSM
Sham +
MQ
GSM +
MQ
0
2
4
6
8***
OTM
Val
ue
Figure 12: Mean comet olive tail moments of primary astrocytes exposed to different setting
of GSM RFR exposure and chemical treatment with MQ. Error bars denote SEM, and ***
p<0.001, n=5
5.1.2. GSM + MMS
Figure 13 is for combination of three replicates. It shows that cells exposed to GSM + MMS
produced the highest OTM value (9.79 ± 0.38) compared to ones produced by exposure to
sham + MMS (9.21 ± 0.30), sham (1 ± 0.11), and GSM (0.80 ± 0.10). Differences between
sham and GSM, or sham + MQ and GSM + MQ show no statistical significance.
40
Sham
GSM
Sham
+ M
MS
GSM
+ M
MS
0
5
10
15
OT
M V
alu
e
Figure 13: Mean comet olive tail moments of primary astrocytes exposed to different setting
of GSM RFR exposure and chemical treatment with MMS. Error bars denote SEM, n=3
5.1.3. CW + MQ
Figure 14 is for combination of three replicates. It shows that cells exposed to sham + MQ
produced the highest OTM value (6.64 ± 0.43) compared to ones produced by exposure to CW
+ MQ (6.51 ± 0.43), sham (1.50 ± 0.22), and CW (1.41 ± 0.15). Differences between sham and
GSM, or sham + MQ and GSM + MQ show no statistical significance.
Sham CW
Sham +
MQ
CW +
MQ
0
2
4
6
8
OTM
Val
ue
Figure 14: Mean comet olive tail moments of primary astrocytes exposed to different setting
of CW RFR exposure and chemical treatment with MQ. Error bars denote SEM, n=3
41
5.1.4. CW + MMS
Figure 15 is for combination of three replicates. It show that cells exposed to sham + MMS
produced the highest OTM value (9.38 ± 0.22) compared to one produced by exposure to CW
+ MMS (8.94 ± 0.25), sham (1.63 ± 0.14), and CW (1.48 ± 0.15). Differences between sham
and GSM, or sham + MQ and GSM + MQ show no statistical significance.
Sham CW
Sham +
MM
S
CW
+ M
MS
0
5
10
15
OT
M V
alu
e
Figure 15: Mean comet olive tail moments of primary astrocytes exposed to different setting
of CW RF exposure and chemical treatment with MMS. Error bars denote SEM, n=3
5.2. Micronucleus assay
In the regard of results introduced in this thesis, “MN%” refers micronucleus frequency as a
percentage of micronuclei detected to the total observed number of nuclei. While, percentage
of nuclei detected to the total number of observed number of beads in sham exposure is always
set to be 100%, and “relative survival%” of other exposure groups is the divide product of
nuclei/beads in certain exposure group as a percentage of sham exposure.
5.2.1. GSM + MQ
Figure 16 shows that cells exposed to GSM + MQ produced the highest micronucleus
frequency (2.52 ± 0.92) compared to ones produced by exposure to sham + MQ (1.37 ± 0.61),
GSM (0.55 ± 0.08), and sham (0.50 ± 0.08). In addition, it shows that cells exposed to GSM +
MQ experienced the highest relative survival (108.9 ± 71.15) compared to ones experienced
due to exposure to sham (100 ± 0.00), GSM (98 ± 21.24), and sham + MQ (72.55 ± 23.91). In
42
the regards of either MN% or relative survival%, differences between sham and GSM, or sham
+ MQ and GSM + MQ show no statistical significance.
Figure 16: micronucleus frequency (a) and relative survival (b) of primary astrocytes exposed
to different setting of GSM RFR exposure and chemical treatment with MQ. Error bars
denote SEM, n=3
5.2.2. GSM + MMS
Figure 17 shows that cells exposed to GSM + MMS produced the highest micronucleus
frequency (1.73 ± 0.29) compared to ones produced by exposure to sham + MMS (1.36 ± 0.51),
GSM (0.69 ± 0.26), and sham (0.66 ± 0.21). In addition, it shows that cells exposed to GSM
experienced the highest relative survival (107 ± 7.82) compared to ones experienced due to
exposure to sham (100 ± 0.00), GSM + MMS (92.97 ± 25.91), and sham + MMS (64.41 ±
15.36). In the regards of either MN% or relative survival%, differences between sham and
GSM, or sham + MMS and GSM + MMS show no statistical significance.
Figure 17: micronucleus frequency (a) and relative survival (b) of primary astrocytes exposed
to different setting of GSM RF exposure and chemical treatment with MMS. Error bars
denote SEM, n=3
43
5.2.3. CW + MQ
Figure 18 shows that cells exposed to CW + MQ produced the highest micronucleus frequency
(2.26 ± 0.96) compared to ones produced by exposure to sham + MQ (1.86 ± 0.62), sham (0.54
± 0.16), and CW (0.53 ± 0.16). In addition, it shows that cells exposed to sham experienced the
highest relative survival (100 ± 0.00) compared to ones experienced due to exposure to CW +
MQ (59.31 ± 13.68), sham + MQ (50.58 ± 8.89), and CW (50.51 ± 13.39). In the regards of
either MN% or relative survival%, differences between sham and CW, or sham + MQ and CW
+ MQ show no statistical significance.
Figure 18: micronucleus frequency (a) and relative survival (b) of primary astrocytes exposed
to different setting of GSM RF exposure and chemical treatment with MQ. Error bars denote
SEM, n=3
5.2.4. CW + MMS
Figure 19 shows that cells exposed to CW + MMs produced the highest micronucleus
frequency (1.5 ± 0.04) which was significantly higher that one produced by sham + MMS (0.95
± 0.08). Cells exposed to sham (0.57 ± 0.08) produced higher micronucleus frequency than one
produced by cells exposed to CW (0.56 ± 0.13), difference was not statistically significant
though. In addition, the figure shows that cells exposed to sham + MMS experienced the
highest relative survival (114.2 ± 75.66) compared to ones experienced due to exposure to CW
(108.7 ± 32.71), sham (100 ± 0.00), and CW + MMS (64.03 ± 9.72). In the regard of relative
survival, differences between sham and GSM, or sham + MMS and GSM + MMS show no
statistical significance.
44
Figure 19: micronucleus frequency (a) and relative survival (b) of primary astrocytes exposed
to different setting of CW RF exposure and chemical treatment with MMS. Error bars denote
SEM, and ** p<0.01, n=3
45
6. DISCUSSION
This thesis focuses on co-exposure setting i.e. combining RFR with agents known to cause
damage to DNA and investigating whether RFR could modifies the genotoxic effect of them.
Co-exposure approach is interesting and of high relevance as it resembles daily life situations
where humans are exposed to a variety of environmental agents. Reviewing the scientific
literature revealed that such approach was exploited overwhelmingly. Different studies used
different co-exposure agents including chemicals and forms of radiation other than RFR. Maes
et al. in 1996, 1997, 2001 and 2006, and Baohong et al. in 2005 used mitomycin C (MMC) as
a co-exposure agent. Difference in experimental setups between Maes’ work in 1996 and 1997
is clear example on how poor dosimetry could affect the outcomes. In 1996, Maes et al used
GSM base station antenna to expose whole blood samples, a significant synergistic effect of
MMC was observed. Again, in 1997 with almost same experimental conditions, but more
precise calculation of SAR through exposing the samples in transverse electromagnetic
transmission cell “TEM” cell, where generation of waves is more homogenous, such
synergistic effect was lacking. Bleomycin was used by Koyama et al. in 2003, 2004 and 2007,
and Baohong et al. in 2005. 4-Nitroquinoline 1-oxide (4-NQO) was used by Baohong et al. in
2005 and Kim et al. in 2008. In addition, MMS, cyclophosphamide, MQ, doxorubicin, ferrous
chloride and SiRNA were used by Baohong et al. in 2005, Kim et al. in 2008, Luukkonen et al.
in 2009, Zhijian et al. in 2010, Luukkonen et al. in 2011 and Wang et al. in 2015, respectively.
Forms of radiation other than RFR were also used as co-exposure agent e.g. X-rays (Maes et
al. 2001, Stronati et al. 2006 and Zhijian et al. 2009), UVC (Baohong et al. 2007), and gamma
radiation (Figueiredo et al. 2004 and Ji et al. 2016). Interestingly, Figueiredo et al. and Ji et al.
findings did not support the hypothesis that RFR might enhance direct genotoxic effect of
gamma radiation. Contrarily, Figueiredo et al. could not find significant co-genotoxic effect in
human blood lymphocytes. Moreover, Ji et al. implied that RFR significantly decreased gamma
radiation induced genotoxicity and resulted in faster DNA kinetics in mouse bone marrow
stromal cells.
In addition, studies used different methods for genetic toxicology assessment. Comet assay was
the method of choice by Maes et al. 1997, 2001 and 2006, Baohong et al. 2005 and 2007,
Stronati et al. 2006, Kim et al. 2008, Luukkonen 2009 and 2011, Zhijian et al. 2009 and 2010,
Wang et al. 2015, and Ji et al. 2016. It is noticeable that the study by Maes et al. in 2006 is
among few where cells exposure to RFR was not under laboratorial conditions but occupational
settings. Occupational settings allow studying effects at real and relatively high level of SAR,
46
mutagenic and MMC co-mutagenic effects in peripheral blood lymphocytes were lacking
though. Similarly, to our findings, Baohong et al. work in 2007 is an example on how co-
genotoxic effects could differ when assessed in different time point. The study group studied
the co-exposure effect of 1.8 GHz RFR at SAR level of 3 W/kg in human blood lymphocytes.
In combination with UVC exposure, they found that RFR significantly decreased DNA damage
when assessed 1.5hrs after end of exposure, but it the damaging effect significantly increased
after 4hrs. CA was an indicator for genotoxicity by Maes et al. 1997, 2001 and 2006, Figueiredo
et al. 2004, Stronati et al. 2006, and Kim et al. 2008. Consistently with Figueiredo et al. finding
on gamma radiation, combining x-rays and 935 MHz GSM RFR at different level of SAR had
no significant difference when compared to genetic damage caused by exposure to X-rays
alone. SCE was a tool for assessing DNA damage by Maes et al. 1996, 1997, 2001 and 2006,
and Stronati et al. 2006. The MN assay was utilized Koyama et al. 2003 and 2004, and Stronati
et al. 2006. In addition, HPRT mutation, FPG-Co, and induction of γH2AX foci formation were
the means to assess the genetic damage by Koyama et al. 2007, Wang et al. 2015, and Ji et al.
2016 respectively.
Among all, the most comparable studies to this thesis work are the two carried by Baohong et
al. in 2005 and Luukkonen et al. in 2009. They both maintained the temperature of the cultures
during the experiments at 37 °C and utilized comet assay for assessing DNA damage. While
Baohong et al. in 2005 used MMS, Luukkonen et al. in 2009 used MQ for co-exposure.
Results of this thesis work show that RFR could modify the DNA damaging effect of MQ and
MMS in rat primary astrocytes. Only GSM RFR had an observable impact on the DNA
damaging effect of MQ. GSM RFR was able to significantly decrease the DNA damaging of
MQ measured immediately after exposure by comet assay. Compared to Luukkonen et al.
findings in 2009, CW RFR but not GSM RFR significantly increased the damaging effect of
MQ. Such conflicts of outcomes can be explained on the basis of using different cell line and
experimental setups. While we used primary cell line of rat astrocytes (more relevant to
physiology than secondary cell lines), Luukkonen et al. used secondary human SH-SY5Y
neuroblastoma cells. Both studies utilized RF signal with a frequency adjusted to 872 MHz.
However, SAR values and RFR exposure duration differ from 0.6 W/kg (typically correlated
to daily life use of mobile phones) and 24hrs in our work to 5 W/kg and 1hr in Luukkonen et
al. study.
47
In the context of immediate assessment of DNA damage by comet assay, neither CW RFR nor
GSM RFR had a noticeable impact on the DNA damaging effect of MMS. This complies with
Baohong et al. findings in 2005. Baohong et al. invested whether 2hrs of exposure to GSM-
1800 MHz RFR could modify the genotoxic effect of MMS in human blood lymphocytes at
SAR level of 3 W/kg. They figured out that the effect of RFR was not obvious at concentrations
of 12.5, 25 and 50 µM of MMS.
Micronuclei detection needs cells to divide; this is the reason why MN is an indicator for
genotoxicity at a later point of time. There was a clear trend that RF radiation (with and without
modulation) increased the tendency to form micronuclei for both MQ and MMS when assessed
6 days after end of exposure. Difference was only significant in case of CW + MMS. This was
compliant with Koyama et al. findings in 2003 when they observed 245 MHz RFR increased
the micronuclei formation induced by bleomycin at SAR levels of 78 and 100 W/kg in CHO-
K1 cells. Contrarily, such potentiation effect was lacking when 935 GSM RFR at SAR levels
of 1 and 2 W/kg combined the effect of X-rays in human blood lymphocytes (Koyama et al.
2003).
The capacity of the exposure system was the major limitation of our study. Each chamber of
waveguide exposure and sham exposure fitted only two petri dishes. Taking into account the
time given for the thesis work, this hindered our abilities to perform more replicates for
different exposure setups especially in the regards of MN. Maintaining cell cultures at
isothermal conditions during RFR exposure was one the hardest challenges and probably it was
the main origin of uncertainty in this thesis, taking into account the variability in temperature
in that time of year when the work was carried out and checking the validity of the chambers
ventilation every time before exposure started. Besides, using different batches of MQ and
MMS with different expiry dates could be one of the reasons explaining discrepancies in the
biological responses of the cells while performing MN. It happened because of the necessity to
prepare new ampoules of the chemicals when the insufficient initial amount of ampules run
out.
Taking into account the relatively long duration of the life span of primary astrocytes, it is
important to notice despite the fact that comet assay and micronucleus assay are carried out in
different points of time; they are both tools for assessing the immediate DNA damaging effect
after end of exposure. Comet assay is a tool to assess both single-strand breaks and double
strand breaks. On the other hand, micronuclei are formed when correct repair to double strand
48
breaks fails; this is why micronucleus assay is considered as indicator for the integrity of the
genome after DNA repair. The ability of the RFR to decrease the chemically induced
immediate DNA damage measured at time 0 (assessed by comet assay), and then increase it
after 6 days (assessed by micronucleus assay) is interesting. It arises questions whether RFR
could increase the vulnerability to DNA damage and whether it could impair the repair
responses. As future aspects of researches, this thesis suggests studies on assessing the effect
of RFR on MQ and MMS induced DNA damage at different time points other than 0 and 6
days after end of exposure with a focus on the dynamics of DNA repair. It is also suggested to
study delayed genotoxic effects and possible induced genomic instability several cell
generations after exposure.
49
7. CONCLUSION
This thesis showed that RFR was able to modify the genotoxic effects of MQ and MMs. Such
finding greatly depended on the RF signal modulation, method utilized to assess genotoxicity
and the time point at which the DNA damage was assessed. At zero time after end of exposure,
comet assay revealed that GSM significantly decreased the DNA damaging effect of MQ. Six
days after end of exposure, MN revealed that RFR increased the DNA damaging effect of MQ
and MMS, difference was statistically significant only in the case of CW + MMS. Further
studies on the toxicological mechanisms including assessing genotoxicity at different time
points and on patterns of DNA repair are needed to explain such variability. In a wider frame,
the suggestion that RFR is not likely to cause genotoxic effects prevails, at least at relatively
low SAR levels relevant to human environmental exposure. Findings on how RFR could
synergize the genotoxic of mutagens and carcinogens are inconclusive. Further exploration
using this co-exposure approach with environmentally relevant agents is still needed.
50
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APPENDICES
Appendix 1: Reagents
Citric acid (Riedel-de Haën, Germany).
Ethanol (Altia, Rajamäki, Finland).
Ethidium bromide (Sigma, USA).
Ethidium monoazide (EMA) (Molecular Probes, USA).
Ethylenediaminetetraacetic acid (EDTA) (Merck, Netherlands)
Fetal bovine serum (Gibco, South America).
Low melting point agarose (Sigma, USA).
Menadione (MQ) (Sigma Aldrich, China)
Methyl methanesulfonate (MMS) (Sigma Aldrich, USA)
N-Lauroylsarcosine sodium salt (Sigma, UK).
Normal melting point agarose (Biowhittaker Molecular Application, Rockland, Maine, USA).
Nucleic acid dye (SYTOX green) (Molecular Probes, USA).
Octylphenoxypolyethoxyethanol (IGEPAL CA-630) (Sigma, USA).
Penicillin (Gibco, USA).
Phosphate buffer saline (PBS) (Oy Reagena LTD, Kuopio, Finland).
RNAase A (Sigma, UK).
Sodium chloride (Fisher Scientific, UK)
Sodium citrate (Riedel-de Haën, Germany).
Sodium hydroxide (VWR Chemicals Prolabo, Czech Republic).
Streptomycin (Gibco, USA).
Sucrose (MP Biomedicals, Germany).
Tris(hydroxymethyl)aminomethane (TRIS) (Sigma, USA).
Triton X100 (DOW chemical, Midland, Michigan, USA).
Trypsin (Gibco, UK).
Appendix 2: Preparation of chemicals and buffers
2.1. Preparation of menadione
Menadione stock solution was provided with the concentration of 100 mM in 10µl batches in
Eppendorf’s tubes. Dilution of 1:100 was done when 990 µls of fresh medium were added to
the tube. 165 µl of the resultant dilution were withdrawn to be added to 10.835 ml (11 ml – 165
µl) of fresh medium, where the final concentration was 15 μM in every 5 ml of the solution.
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2.2. Preparation of methyl methanesulphonate.
Methyl methanesulphonate stock solution was provided with the concentration of 10 mg/ml in
100µl batches in Eppendorf’s tubes. Dilution of 1:10 was done when 900 µl of fresh medium
were added to the tube. 440 µl of the resultant dilution were withdrawn to be added to 10.56
ml (11 ml – 440 µl) of fresh medium, where the final concentration of the solution was 40
µg/ml if the total volume was 5ml.
2.3. Preparation of alkaline lysis buffer
10 ml of milliQ water and 1 ml of Triton X-100 were added to 89 ml of lysis buffer stock
solution (NaCl, EDTA, TRIS and N-Lauroylsarcosine sodium salt) in a suitable size beaker
which was properly shacked and then covered with a piece of parafilm to be stored in the
refrigerator for at least one prior to its use in the assay. Lysis buffer stock solution is made of
2.5M NaCl, 100 mM EDTA, 10 mM TRIS and 1% N-Lauroylsarcosine sodium salt, and water
was the solvent.
2.4. Preparation of electrophoresis buffer
60 ml of 10N NaOH and 10 ml of 200 mM EDTA were added to 1930 ml of milliQ water in a
suitable size beaker which was properly shacked and then covered with a piece of parafilm to
be stored in the refrigerator for at least one hour prior to its use in the assay.
2.5. Preparation of TRIS neutralization buffer
It was prepared with the concentration of 0.4 M and pH of 7.5, water was the solvent.
2.6. Preparation of ethidium monoazide (EMA) solution.
Firstly, PBS-FBS buffer solution was prepared by adding 1 ml fetal bovine serum (FBS) to 49
ml of phosphate buffer saline (PBS) w/o Ca+2, Mg+2. EMA solution was prepared by adding 7
ml of PBS-FBS buffer solution to 70 µl of EMA stock solution. The mixer was kept on ice or
in refrigerator covered from light. EMA stock solution is with the concentration of 0.85 mg/ml,
ethyl alcohol was the solvent.
2.7. Preparation of micronucleus assay lysis buffer 1
20 µl of SYTOX green stock solution and 250 µl of RNAase A solution were added to 5 ml of
lysis buffer 1 stock solution. The mixture was kept on ice or in refrigerator covered from light.
Lysis 1 buffer stock solution is made of 0.584 mg/ml NaCl, 1 mg/ml Na-citrate and 0.3 µl/ml
59
octylphenoxypolyethoxyethanol (IGEPAL CA-630), water was the solvent. SYTOX green
stock solution is made of 0.1 Mm SYTOX green in, dimethyl sulphoxide (DMSO) was the
solvent
2.8. Preparation of micronucleus assay lysis buffer 2
20 µl of SYTOX green stock solution was added to 5 ml of lysis buffer 2 stock solution. The
mixture was kept in room temperature covered from light. One drop of counting beads
(PeakFlow™ Green flow cytometry 6 µm sodium azide reference beads, Life technologies,
Eugen, Oregon, USA) had been added just before the mixture was used. Lysis buffer 2 was
made of 85.6 mg/ml sucrose and 15 mg/ml citric acid, water was the solvent.
Appendix 3: Preparation of normal melting point agarose slides
0.5 grams of normal melting point agarose was weight in 50 ml beaker where 50 ml of PBS
w/o Ca+2 Mg+2 were added. The mixture was heated up in a microwave till it was homogenous.
For coating, every slide used in the assay was dipped in the agarose solution three times,
afterwards, the bottom was wiped by a soft tissue, and then slides were transferred to a tray to
be stored in a freezer for a week. One day prior to their use in the assay, they were gotten away
from the freezer to dry up in a dark dry place.
Appendix 4: BD FACSCanto II flow cytometer check performance
One drop of CST beads (BD FACSDiva™ CS&T research beads, 0.5%
Bis(trimethylsilyl)acetamide and 0.1% sodium azide, , Becton, Dickinson and company, BD
Biosciences, San Jose, California, USA) was mixed with 350 µl of PBS w/o Ca+2, Mg+2. The
mixture was vortexed and its running was used in the quality control check performance of the
flow cytometer utilized the CST function in the BD FACSDiva Software.