Master's thesis of Engineering: Examining changes in sensitivity and functionality of mechanosensitive ion channel protein Piezo 1 exposed to low-level radiofrequency radiation

Examining changes in sensitivity and functionality of mechanosensitive ion channel protein Piezo 1 exposed to Low-Level Radiofrequency Radiation

A thesis submitted in fulfilment of the requirements for the degree of Master of Engineering

Azadeh Torkan

Bachelor of Microbiology

Bachelor of

University of Isfahan

School of Engineering

College of Science, Technology, Engineering and Maths

RMIT University

March 2021

Declaration

I certify that except where due acknowledgement has been made, the work is that of the author alone;

the work has not been submitted previously, in whole or in part, to qualify for any other academic award;

the content of the thesis is the result of work which has been carried out since the official commencement

date of the approved research program; any editorial work, paid or unpaid, carried out by a third party is

acknowledged; and, ethics procedures and guidelines have been followed. I acknowledge the support I

have received for my research through the provision of an Australian Government Research Training

Program Scholarship.

Name : Azadeh Torkan Date: 06 March 2021

Acknowledgment

I would like to acknowledge my supervisors, Professor Elena Pirogova, School of

Engineering and Dr Sara Baratchi, School of Health and Biomedical Sciences, for

giving me the opportunity to complete this project under their supervision, for their

ongoing support, guidance, academic advice and encouragement throughout my

candidature.

Also, I would like to dedicate this thesis to my family and thank them for their

patience, help and encouragement. They have supported me throughout this research

journey and thesis preparation.

Furthermore, I am thankful for the research services and facilities presented by the

School of Engineering, and especially by the Mechanobiology & Microfluidics

laboratory, School of Health and Biomedical Science, STEM College, RMIT

University.

Declaration ………………………………………………………………………… i ii Acknowledgements ……………………………………………………………… v List of Tables ………………………………………………… vi List of Figures …………………………………………………….....

Abstract…………………………………………………………………………….. 1

1.0 Chapter 1: Introduction 1.1 Motivation…………………………………………………………………………... 2 Electromagnetic fields (EMFs)…………………………………………………….. 5 1.2 8 Research Objective……………………………………………………………..….. 1.3 1.4 Experimental Study.………………………………………………………………... 9 1.4.1 Selected cells and target mechanosensitive ion channels, TRPV4 and

Piezo1…………………………………………………………………………….… 9 10 1.4.2 Source of RF radiation………………………………...…………………...….…... Computational Study..……………………………………………………………... 1.5 11 Research Hypothesis and Research Questions ………………………...………..… 11 1.6 Thesis Composition………………………………………………………………… 13 1.7 References …………………………………………………………………............ 14 1.8

2.0 Chapter 2: Literature Review Background …………………………………………………………………......... 2.1 2.2 Electromagnetic Spectrum ……………………………………………………….. 2.2.1 Ionizing Radiation ………………………………………………………..….…… 2.2.2 Non-Ionizing Radiation …………………………………………………......…… RF-EMR used in communication technologies ………………………………….. 2.3 In silico molecular modelling studies …………………………………………….. 2.4 In vitro studies ………………………………………………………………….… 2.5 In vivo animal and clinical human studies ……………...………………………... 2.6 Effects of radiofrequency exposure on mechanoreceptors …………………….... 2.7 Summary ……………………………………………………………………….... 2.8 References ………………………………………………………………………... 2.9 16 17 19 20 20 22 23 29 33 35 37

3.0 Chapter 3: Experimental Study

46 48 48 48

III

Investigating the effect of low-level radio-frequency radiation on activation of mechanosensitive ion channel Piezo 1 and TRPV4 Introduction ……………………………………………………………………..... 3.1 3.2 Materials and Methods ……………………………………………………………. 3.2.1 Reagents and buffers ……………………………………………………………... 3.2.2 Cell Culture ……………………………………………………………………..... 3.2.3 Experimental set up using a mobile phone to investigate the short-term effects of electromagnetic radiation ……………………………………………………….... 3.2.4 Calcium imaging and confocal microscopy ……………………………….....…... 3.2.5 Image analysis ………………….………………………………………………… 3.2.6 Transverse electromagnetic (TEM) cell exposure system ………………...…….. 3.2.7 RNA extraction and RT qPCR …………………………………………………… 48 49 50 50 53

53 53 53 3.2.8 Intracellular Ca2+ measurement …………………………………………………... 3.2.9 Statistical Analysis ………………………………………………………..……… 3.3 Results ………………………………….………………………………………… 3.3.1 A low-level electromagnetic field activates the mechanosensitive ion channel

54 57

Piezo1 …………………………………………………………………………….. 3.3.2 Low-level electromagnetic field activate THP-1 cells …………………………… 3.3.3 The response of HEK293-piezo 1 to low-level electromagnetic radiation is Piezo1 specific ……………………………………………………………………………. 60

3.3.4 Long-term radiation does not affect the expression of inflammatory cytokines in THP1 cells ………………………………………………………………………... 61 3.3.5 4 hours of radiation did not affect the expression of the mechanosensitive ion 63 channel TRPV4 and Piezo1 in THP1 cells ………………………………………………... 3.3.6 Low-level electromagnetic field desensitizing the response of Piezo1 to 10uM Yoda-1 ……………………………………………………………………………. 64 3.4.1 Low-level electromagnetic field desensitizing the response of Piezo1 67

70 endogenously expressed in THP1 to 10uM Yoda-1 ……………………………… 3.4.2 Desensitization effect of low-level electromagnetic radiation on HEK293-piezo1 to Yoda1 is absent in parental HEK293 cells …………………………………….. 3.4.3 2 hours of radiation did not affect the response of HEK293-piezo1 cell to different 72

the concentration of Yoda1 …………………………………………………………... Summary ……………………………………………………………. References ……………………………………………………………………...... 3.5 3.6 74 75

4.0 Chapter 4: Computational Study

Simulating RF field exposures emitted by mobile phone headset using CST microwave studio ………………………………………………………………... Background ……………………………………………………………………… Computer Simulation Technology Microwave Studio…………………………. Factors affecting the field strength (power) ……………………………………

76 78 81 82 82 83 84 4.1 4.2 4.3 4.3.1 Antenna parameters ………………………………………………………………. 4.3.2 Position of the RF field source ………………………………………………….... 4.3.3 Permittivity, permeability and conductivity of material ………………………….. 4.3.4 Correlation and comparison of RF field simulation with its physical measurement ………………………………………………………………………

4.3.5 Identifying Experimental field parameters ……………………………………….. Simulation of RF fields using identified parameters ………………………….. 4.4 4.5 Different positions of a mobile phone device (irradiation source) …………… 4.5.1 Simulation of the RF radiation at position 1 ………………………….………….. 4.5.2 Simulation of the RF radiation at position 2 ………………………….………….. 4.5.3 Simulation of the RF radiation at position 3 ………………………….………….. 4.6 4.7 Summary ……………………………………………………………. References ……………………………………………………………………….. 85 90 98 99 99 103 105 113 116

5 Chapter 5: Conclusions and Future Work 127

List of Tables

3.1 Positions of mobile phone against the 24-well plate …………………………..…. 49

4.1 Parameters of Corning 24-Well Plate (Balanis)…………………………………... 97

List of Figures

1.1

2.1 2.2 18 3.1 51 3.2

51 3.3 55 3.4 56 3.5

58 59 3.6 3.7 60 3.8 62 3.9 The electromagnetic spectrum: the range of frequencies, their relevant wavelengths and photon energies ……………………………………………... 5 Electromagnetic wave propagation……………………………………………. 18 Electromagnetic spectrum showing the entire range of wavelength and frequency …………………………………………………………………….... Experimental set up showing exposure camera, signal generator and temperature controller …………………………………………………...……. The position of the sample and the direction of the electric field inside the TEM cell. a) The vertical distance from the top of the cell to the sample is 22 cm, b) Field pattern at the position of the sample (top view)……..……….…. Low-level RF radiation activates the mechanosensitive ion channel Piezo- 1……………………………………………………………………….. Low-level RF radiation increases the [Ca2+]i of HEK293-Piezo-1 cells……………………………………………………………………………. Low-level RF radiation emitted by two mobile phones activates THP-1 cells…………………………………………………………………… Low-level RF radiation increases the [Ca2+]i of THP1 cells…………. The increase in [Ca2+]i post-exposure to the low-level RF radiation is Piezo-1 dependent…………………………………………………………… Effects of Low-level RF radiation on the expression of inflammatory cytokines in THP-1 cells ……………………………………... Effects of Low-level RF radiation on the expression of mechanosensitive ion channels in THP-1 cells ……………………………… 63 3.10 Low-level RF radiation desensitizes the response of the mechanosensitive ion channel Piezo-1 to Yoda-1…………………………... 65 3.11 Low-level RF radiation desensitizes the response of the mechanosensitive ion channel Piezo-1 to Yoda-1………………………….. 66

3.12 Low-level RF radiation desensitizes the response of endogenous Piezo-1 to Yoda-1…………………………………………………………….. 68 3.13 Low-level RF radiation desensitizes the response the endogenously expressed Piezo-1 to Yoda-1…………………………………. 69 3.14 The desensitization of HEK293-Piezo-1 cells to Yoda-1 is dependent on the expression of Piezo-1……………………………………………………... 71

3.15 Effects of Long-term exposure to low-level RF radiation on the Piezo-1 response to Yoda-1……………………………………………… 50-ohm microstrip Linefeed calculation…………………………………….

Return Loss Result Comparison…………………………………………….. Radiation Pattern Result Comparison @ 870 MHz………………………… Radiation Pattern Result Comparison @ 1850 MHz……………………….. Radiation Pattern Result Comparison @ 3400 MHz……………………….. Radiation Pattern Result Comparison @ 5400 MHz……………………….. SAR (1g) Results comparison………………………………………………...

4.1 4.2 Waveguide Port Extension Coefficient Calculation in CST………………… 4.3 4.4 4.5 4.6 4.7 4.8 4.9 Vodafone coverage at RMIT Bundoora West campus…………………….. 4.10 Nearest Cell Tower from RMIT Bundoora West campus………………… 73 86 86 87 87 88 89 89 90 91 91

92 4.11 Setup with wideband antenna (left) and Huawei Mate 9 phone (right)…... 93 Idle stage frequency measurement………………………………………….. 4.12 93 Initialising stage frequency measurement………………………………….. 4.13 95 4.14 In-call stage frequency measurement………………………………………. 95 4.15 Huawei Mate 9 radiated power level………………………………………... 96 4.16 Calculated antenna dimension (left) and S11 (right)………………………. 97 4.17 Conductivity measurement of buffer solution……………………………… 98 4.18 Dimension of a 24-well plate and Huawei mate 9 used in experiments…… 99 4.19 Simulated point of interest…………………………………………………... 100 4.20 Experimental set up- position 1 of exposure……………………………….. 101 4.21 Simulated Electric field strength/power in position 1……………………… 101 4.22 Simulated power density loss in position 1…………………………………. 102 4.23 Simulated energy density in position 1……………………………………… 102 4.24 Simulated RF radiation pattern at position 1………………………………. 103 4.25 Experimental set up- position 2 of exposure………………………………... 103 4.26 Simulated Electric field intensity in position 2……………………………... 104 4.27 Simulated power density loss in position 2………………………………….. 4.28 Simulated energy density in position 2……………………………………… 104 4.29 Simulated RF radiation pattern at position 2…………………………………... 105 106 4.30 Experimental set up- position 3 of exposure………………………………... 106 4.31 Simulated Electric field intensity in position 3……………………………... 4.32 Simulated power density loss in position 3…………………………………. 107 107 4.33 Simulated energy density in position 3……………………………………… 4.34 Simulated RF radiation pattern at position 3…………………………………... 108 4.35 The 3D radiation pattern of the position 1, 2 and 3……………………………. 108 4.36 Comparison of the electric field strength between three positions of the 110

111

VII

112 mobile exposure device ……………………………………………………… 4.37 Comparison of the power density loss between three positions of the mobile exposure device………………………………………………………. 4.38 Comparison of the energy density plots between three positions of the mobile exposure devise……………………………………………………….

ABSTRACT

The worldwide increase in the use of wireless telecommunications devices, mainly mobile

phones, has resulted in increased human exposure to radiofrequency (RF) radiation. RF

exposure imparts heat on the body through energy deposition that may interact with other

mechanosensitive pathways, such as through the mechanosensitive ion channels, which are

essential for maintaining normal physiological processes such as balance and touch.

This project is focused on studying the biological effects of low-power RF radiation on the

expression and function of mechanosensitive ion channel protein (Piezo-1). To evaluate the

effects of long-term exposures, cells were exposed at the frequency of 1800 MHz and power

of 17 dBm for a period of 2 and 4 hrs using the transverse electromagnetic (TEM) cell exposure

system. To study the effects of short-term RF radiation (10 min), exposures emitted from the

mobile phone headset at the frequency of 845 MHz and powers of -15 dBm and -5 dBm were

applied to cell cultures.

The obtained results demonstrate that non-thermal exposures at specific frequencies and

powers modulate the biological activity of studied ion channels. The findings of this study will

assist in identifying the thresholds of microwave (MW) exposures affecting these selected

proteins and be useful in providing information on appropriate exposure guidelines.

CHAPTER 1

INTRODUCTION

1.1 Motivation

Modern technology and advanced knowledge are intertwined with all aspects of human life and

can have two opposing perspectives, positive and negative. The most essential tools and

achievements of wireless technology can be referred to as mobile and/or smartphones.

Smartphones have two distinct roles. One is to make life easier – it also enables a better and

faster exchange of information on a broader scale – this is a useful and effective achievement.

But unfortunately, with the increase in the number of these smart devices, there have been a

large number of concerns raised associated with their widespread use. Among the concerns

raised by the use of mobile phones and other smart meters, are biological and possible health

effects of radiofrequency (RF) and MW radiation emitted from these devices on humans and

also its overall environmental effect (known as electrosmog). The dramatic increase in the use

of smart meters has been related to the prevalence of brain tumours in particular and other types

of cancer (Naeem 2014, Bor 2016). Reduced level of children’s creativity, along with less

physical activity, has also been reported (Hardell 2018).

Research studies were focused predominantly on the effects of long-term and short-term

exposures of high power RF and MW radiation, i.e. heating effects on the body and tissues.

These effects, along with the mechanism of action, underlying these effects, are well

understood and extensively reported. This knowledge formed the basis for developing currently

used standards that govern the use of mobile and smart devices and deployment of mobile

phones and National Broadband Network (NBN) base stations.

However, it is known that wireless communication devices emit only low-power RF/MW

radiation, a type of non-ionizing radiation. Energy levels associated with it are not strong

enough to enable the ionization of atoms and molecules. But it was also reported that RF/MW

can produce resonant interactions with ions and charged macromolecules, and such interactions

can signiﬁcantly alter biochemical functions. A large body of research suggests that MW/RF

promotes the production of free radicals and reactive oxidant species in living tissues and that

this increased oxidant stress can damage DNA. As reported, this damage can and does occur at

the power levels well below those levels that could produce damage by established thermal

mechanisms. Also, up to date, studies of biological and health effects of low-power RF and

MW radiation are often conflicting, ranging from reports of “no effects” or “negligible effects”

to “detrimental effects”. In addition, studies focused on the effects of long-term exposure to

RF and MW radiation on human physiology and mental health is limited or inadequate

(Gaestel, 2010).

Overall, effects caused by applied radiation depend upon:

 Value of radiation absorbed by the body (the dose)

 The radiation source (ionizing or non-ionizing radiation)

 The type of irradiance (internal or external)

 The characteristics of an exposure (wavelength/frequency, power, power density)

 The nature of exposure (continuous or intermittently)

 Proximity to the source

 The exposure time (duration)

A growing number of mobile phone users stimulates a need to further study and evaluate the

effects of long-term exposure to low-power RF and MW radiation on various biological

systems. The statistics show that between 2005 and 2015 the number of mobile phone users

increased by 200%. It is anticipated that growth will continue substantially so that in 2020, the

worldwide penetration of mobile phones will reach 100%, meaning that the number of mobile

phone subscriptions will be equal to the population (Statista, 2015).

In essence, this Masters by Research project is focused on investigating the biological effects

of low-level radiation produced by mobile phones at the cellular level by examining changes

in sensitivity and functionality of mechanosensitive ion channel proteins expressed in

endothelial and THP-1 monocytic cells. The study has two arms, experimental and

computational investigation.

1.2 Electromagnetic fields (EMFs)

Electromagnetic waves are produced by synchronized and repetitive vibration of electric and

magnetic fields. Electromagnetic radiation is transmitted through empty space at 3×108 meters

per second (300 thousand kilometers per second). The different forms of electromagnetic

radiation are distinguished from each other by (i) their wavelength (frequency) and (ii) the

amount of energy they transfer. These properties also determine their ability to travel through

objects, their heating effects and their effect on living tissue. The electromagnetic spectrum,

ranging from low to high frequencies (longest to shortest wavelength), consists of radio waves

(e.g., commercial radio and television, microwaves, radar), infrared radiation, visible light,

ultraviolet radiation, X-rays, and gamma rays as shown in Figure 1.1).

Figure 1.1. The electromagnetic spectrum: the range of frequencies, their relevant wavelengths

and photon energies (Wood, 2012).

RF radiation is electromagnetic radiation in the frequency range of 3 kHz to 300 GHz. RF

exposure is usually specified in terms of modulation (continuous wave or pulsed), incident

electric-field and magnetic-field strengths (power), incident power density (when appropriate),

source frequency, type and zone of exposure, and duration of exposure. The coupling of RF

energy into biological systems may be quantified by the induced electric and magnetic fields,

power deposition, energy absorption, and the distribution and penetration into biological

tissues. These quantities are all functions of their relationship to the physical configuration and

dimension of the biological body. Important to note that exposure of a whole body to a given

field power could have outcomes far different for partial body or localized exposure at the same

power. The spatially averaged field power, depending on the region of space over which the

fields are averaged, may vary widely for a given body. The current understanding is that

induced fields are the primary cause for the biological effects of RF exposures, regardless of

the mechanism.

It is important to improve our knowledge of the biological effects of low-power RF/MW

radiations used produced by mobile phone devices, operating at the frequencies used in 3G and

4G mobile networks on molecules and cells. Moreover, it is essential to confirm that currently

used safety standards for the operating frequency range are indeed appropriate to protect living

organisms from ever-increasing electromagnetic pollution. In Australia, the current mobile

frequency bands can be broken into 800, 900, 1800, and 2100 and 2300 MHz. From the early

years, of deployment of wireless mobile communication in Australia, GSM was on the

900/1800 MHz frequencies. Increased demand for the mobile internet and coverage inspired

the carriers to introduce a 3G mobile network with frequencies of 850 and 900 MHz with 4G

network later being introduced using the 2100 MHz frequency. With the introduction of the 4G

mobile network, the carriers have turned off their GSM 1800 service. From 2019, the telecom

industry in Australia has introduced a new 5G network, which still will use 1.8, 2.1, 2.3, 2.6

GHz (frequencies used in 4G network), and 6 GHz and above (Guideline, 1998, Banik,

Bandyopadhyay et al., 2003, Communications and Authority 2013).

The Australian Radiation Protection and Nuclear Safety Agency (ARPANSA) have imposed

restrictions on the frequencies that can be used in mobile phones (Levels, 2002). The Institute

of Electrical and Electronics Engineers (IEEE) (Lin, 2006), the International Commission on

Non-Ionizing Radiation Protection (ICNIRP) and the American National Standards Institute

(ANSI) have developed the Public safety limits and standards that the communication industry

have to comply with (ICNIRP 1998, ICNIRP 2009, Protection 2009). However, these

restrictions alone might not be sufficient as several published studies have provided evidence

on the effects of electromagnetic radiation emitted by mobile phones on brain tumours,

inflammatory responses and cardiovascular systems (Guideline 1998).

The ARPANSA standard specifies exposure limits to RF exposure for mobile phone handsets

in terms of the specific absorption rate (SAR). In the ARPANSA standard, the SAR limit for

mobile phone handsets is 2 watts per kilogram (W/kg) of tissue (averaged over 10 grams). A

SAR of 4 W/kg is associated with a 1oC temperature rise in humans. In practice, a mobile phone

will only induce a very small temperature rise, which is unlikely to be noticed compared with

the normal daily variations in body temperature (ARPANSA 2016).

1.3 Research Objective

The major objective of conducting this research project is to evaluate whether low-power RF

exposures can affect the biological activity of selected proteins. In particular, it was of interest

to study the changes in sensitivity of mechanosensitive (MS) ion channel proteins to low-power

RF radiation emitted by a mobile phone headset. The computational study has been also

conducted to visualise the generated field distribution at the exposures used in the experimental

study. The following sub-studies have been conducted and reported in this thesis:

1. To investigate the effects of low-power RF radiation emitted from mobile phones on

Piezo-1 functions using calcium imaging.

2. To understand the long-term effect of electromagnetic radiation on activation, expression

of Piezo-1 and TRPV4 (another critical mechanosensitive ion channels) and expression

of inflammatory genes in monocytes.

3. To investigate the effect of low-level RF radiation on Piezo-1 response to its selective

agonist Yoda-1.

4. To visualise the distribution of generated fields along with the well plate with cell culture, and

5. To evaluate field properties, including field strength, power and energy densities, at each

well which will aid in understanding the effects of applied exposures on the bioactivity

of irradiated endothelial cells and THP-1 cells.

1.4 Experimental Study

1.4.1 Selected cells and target mechanosensitive ion channels, TRPV4 and Piezo1

Within the experimental in vitro studies, the effects of low-power RF exposures have been

evaluated on the Piezo-1 and TRPV4 ion channel proteins expressed in HEK293 epithelial cell

lines. Human embryonic kidney 293 (HEK293) cells were isolated from human embryonic

kidney cells grown in tissue culture. These cells are widely used in research studies due to their

propensity for easy transfection and reliable and valid growth (Van der Eb, 2012). Another cell

line used in this research project is THP1, a human monocytic cell line that exhibits a single-

cell morphology with a large size, and round shape. THP1 has originally been isolated from

the peripheral blood of a 1-year-old human male with critical monocytic leukemia disease and

is commercially available (Tsuchiya, Yamabe et al., 1980). HEK293 and THP1 cell lines are

of particular importance to this project because they express the mechanosensitive ion channel

Piezo-1.

The Piezo channel-group family of mechanoreceptors is a relatively new class of non-selective

cation channels comprised of two members, Piezo-1 and Piezo-2. These proteins exhibit

characteristic sensitivity to generic MS channel blockers (streptomycin, Gd3+, Ru3+, and the

spider toxin GsMTx4). Piezo-1 is expressed in different cell types, such as in red blood cells,

leukocytes, epithelial cells, and endothelial cells. Piezo channels have a crucial role – they

conduct and transfer mechanical stimuli into electrical and chemical signals to strongly

influence development, regeneration, and homeostasis (Coste, Mathur et al., 2010, Coste, Xiao

et al, 2012, Zarychanski, Schulz et al., 2012).

Another group of ion channel proteins, TRP ion channels, are of particular interest to this study

because they play a key role in cellular calcium signalling and homeostasis and are at the

forefront of sensory physiology. Four of the TRP ion channels are heat-activated (TRPV1-4),

and one of them, TRPV4, is of importance for this project because of its sensitivity to

temperatures in the range of 27oC to 42oC. TRPV4 is a non-selective cation channel that is

permeable to Ca2+. TRPV4 is activated (sensitized) by agonists (GSK1016790A,

Epoxyeicosanoids), mechanical stimuli (hypotonicity, shear stress) and thermal stimuli.

TRPV4 can be expressed in different tissues such as epithelial cells, endothelial cells, vascular

endothelium, kidney, sensory and motor neurons, and chondrocytes. TRPV4 play multiple

roles, including regulation of systemic osmotic pressure in the brain, vascular homeostasis,

function of the liver, intestine, renal system, and sensing of pain (Liedtke, Choe et al.,

1.4.2 Source of RF radiation

Effects of long-term exposures

2000).

The commercial Transverse Electro-Magnetic (TEM) cell (No. TC-5062A UHF-TEM Cell)

was used in this study to irradiate the selected cells for a long period of exposure. A TEM cell

is an enclosed box made of a conductor material, with its dimensions varied depending on the

operating frequency used. The exposure system consists of Transverse Electro-Magnetic

(TEM) TC-5062AUHF TEM cell (100 kHz to 3 GHz) from TESCOM Ltd, and the signal

generator (Wiltron 68247B) with an operating range of 10 MHz to 20 GHz. The exposure

system was connected to a signal generator from which the external signal was applied to the

cells inside the TEM Cell. The generated field inside the TEM cell was calibrated using a

broadband electric field probe to determine the electric field the sample received inside the

system for given input power (Figure 4.2). The calibration test result showed the estimated

uncertainty of ±1–3%, depending on the input signal frequency. To evaluate the effects of long-

term exposures, cells were exposed to the frequency of 1800 MHz and powers of 17 dBm for

a period of 2 and 4 hrs.

Effects of short-term exposures

Mobile phone Huawei 9 Mate was used to study the effects of short-term RF radiation (10 min).

The frequency 845 MHz and powers -15 dBm and -5 dBm were used in this experiment. Effects

of RF exposures emitted from the mobile phone headset at different positions to a well plate

with cell culture were evaluated using qualitative and quantitative assessments.

1.5 Computational Study

In the computational or in silico study, factors that can influence the generated RF field’s

strength are studied aiming to accurately simulate RF exposures emitted by a mobile phone

handset that was utilized in the experimental study presented in Chapter 3. To validate the RF

exposure patterns and ensure that the electromagnetic simulation stays true to the actual in vitro

experiment conducted on endothelial cells, both the phone chassis and antenna are considered

in combination due to the effect of the ground plane on the antenna performance.

The permittivity of the instrument used in the experimental setup is also accounted for in the

simulation. Since the electric field strength/power has an inversely proportional relation to the

conductivity of a material, the conductivity of the buffer used to maintain the pH level of the

cells is included in the simulation too, since the intensity level of the electric field will influence

the SAR value, crucial parameter affecting field exposures on cell cultures. Because the

characteristics of the RF field vary in different field regions, the distance between the antenna

and the measurement point (cell cultures in well plate) is also taken into account with respect

to these different regions. Hence, the computational study was conducted to: (i) visualise the

distribution of generated fields along the well plate with cell culture, and (ii) evaluate field

properties, including field strength (power), power and energy densities, at each well, which

will aid in understanding the effects of applied exposures on the bioactivity of irradiated cells.

1.6 Research Hypothesis and Research Questions

It has been demonstrated that shear stress, cyclic stretch, pressure and magnetic fields can

actuate and interfere with the function of mechanosensitive ion channels (Soloperto, Boccaccio

et al., 2018). Therefore, this project hypothesizes that both electric and magnetic components

of RF radiation can affect the biological activity of mechanosensitive ion channels expressed

in cell membranes because the RF radiation emitted by mobile phones and other wireless

devices may result in more superficial deposition of energy in tissues.

The main research objective of this project is to understand the short-term and long-term effects

of RF radiation on the expression and function of mechanosensitive ion channels Piezo-1 in

stable cell lines and primary cells.

To address this objective, the following research questions were formulated:

1) Whether low-power RF radiation can activate and affect the function of the

mechanosensitive ion channels?

2) Does low-power RF radiation affect the mechanosensitive ion channel responses to their

selective agonist?

3) Does low-power RF radiation affect TRPV4 and Piezo-1 that endogenously expressed in

THP-1 cells?

4) Are monocytes that express mechanosensitive ion channels at the high level, sensitive

(within a limited range) to such RF exposures?

5) Can we visualise the distribution of generated fields along the well plate with cell culture?

6) Can we evaluate field properties, including field strength, power and energy densities, at

each well which will aid in understanding the effects of applied exposures on the bioactivity

of irradiated THP-1 cells?

1.7 Thesis Composition

This master thesis is focused on improving our understanding of the effects of low-power RF

radiation at the frequencies used in the 3G and 4G mobile networks on selected biomolecules

and cells. An outline of the thesis’s chapters is presented below:

Chapter 1 - Introduction

Chapter 2 - Literature review

Chapter 3 - Experimental Study: Investigating the effect of low-level radiofrequency radiation

on activation of mechanosensitive ion channels Piezo-1 and TRPV4

Chapter 4 - Computational Study: Simulating RF field exposures emitted by mobile phone

headset using Computer Simulated Technology (CST) microwave studio

Chapter 5 - Conclusions and Future Work

1.8 References

ARPANSA (2016). Maximum Exposure Levels to Radiofrequency Fields — 3 kHz to 300 GHz A. R. P. A. N. S. AGENCY.

Banik, S., S. Bandyopadhyay and S. Ganguly (2003). "Bioeffects of microwave––a brief review." Bioresource technology 87(2): 155-159.

Bor, D. (2016). "Cep telefonlari sagligimiza zararli mi?/Are mobile phones dangerous to our health." Turkish Journal of Radiology 35(3): 85-88.

Communications, A. and M. Authority (2013). Like, post, share young Australians’ experience of social media (Quantitative Research Report).

Coste, B., J. Mathur, M. Schmidt, T. J. Earley, S. Ranade, M. J. Petrus, A. E. Dubin and A. Patapoutian (2010). "Piezo1 and Piezo2 are essential components of distinct mechanically activated cation channels." Science 330(6000): 55-60.

Coste, B., B. Xiao, J. S. Santos, R. Syeda, J. Grandl, K. S. Spencer, S. E. Kim, M. Schmidt, J. Mathur and A. E. Dubin (2012). "Piezo proteins are pore-forming subunits of mechanically activated channels." Nature 483(7388): 176.

Gaestel, M. (2010). "Biological monitoring of non‐thermal effects of mobile phone radiation: recent approaches and challenges." Biological Reviews 85(3): 489-500.

The guideline, I. (1998). "Guidelines for limiting exposure to time-varying electric, magnetic, and electromagnetic fields (up to 300 GHz)." Health Phys 74(4): 494-522.

Hardell, L. (2018). "Effects of mobile phones on children's and adolescents’ health: A commentary." Child development 89(1): 137-140.

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CHAPTER 2

LITERATURE REVIEW

2.1 Background

The use of mobile phones is becoming more and more popular every day, and consequently,

there is an increasing public interest in the biological and possible health effects of RF

radiation. The biological effects of RF exposures have been categorized into thermal and non-

thermal effects (Israel, Zaryabova et al., 2013). The International Commission of Non-Ionising

Radiation Protection (ICNIRP) has classified radiofrequency electromagnetic radiation (RF-

EMR) into category 2B as possibly carcinogenic to humans (IARC 2013, IARC 2019).

Many countries, including Australia, have set exposure limits for low-power radiation emitted

by mobile phone devices. ARPANSA is the Australian Government’s primary authority on

radiation protection and nuclear safety This agency aims to protect the Australian people and

the environment from the harmful effects of radiation through a range of initiatives such as

understanding risks, practice regulation, research, policy, services, partnerships and engaging

with the community. It is well known that exposure to a sufficiently high power of RF EMR

can heat biological tissue and potentially cause tissue damage (example of a microwave oven).

ARPANSA states that the amount of environmental RF EMR generated by a mobile phone and

encountered by the general public is sufficiently low to induce significant heating or increased

body temperature. At low levels of exposure to RF EMR (i.e. power is at the lower level, not

sufficient to produce a measurable heating effect) the evidence for the production of harmful

biological effects is unproven. However, there have been an increasing amount of studies

reporting a wide range of biological effects at low levels (the so-called non-thermal effects).

Despite these indications, there is not enough evidence to prove that such effects might

constitute a human health hazard. Epidemiological studies have suggested a correlation

between heavy mobile and cordless phone use and brain cancer (most pronounced for glioma)

(Hardell, Carlberg et al., 2013, Coureau, Bouvier et al., 2014).

2.2 Electromagnetic spectrum

According to a definition, an electromagnetic field (EMF) is a physical field produced by

charged objects. The electromagnetic field is generated from the interaction of the electric field

produced by stationary charged particles and the magnetic field produced by moving charged

particles. The interaction between the electromagnetic field, charges and currents are defined

by the Lorentz force law shown in Equation 2.1.

F = q(E + v × B) (2.1)

where F is the force applied on a particle, of, q is the electric charge of the particle, v is the

particle’s velocity, E is the electric field, and B is the magnetic field.

Electromagnetic radiation (EMR) is a specific outline of the extra typical electromagnetic field,

where photons are released and absorbed by stimulating elements, which moves and spread out

through space as a wave (Bakshi, 2009) (Figure 2.1). A time-varying electric and magnetic

field is generated by accelerated atomic elements (Saliev, Begimbetova et al., 2019). EMR

includes both magnetic and electric field mechanisms, oscillating with a 90o degree level

alteration from other vertical directions of wave transmission or energy (Chongchitpaisan,

Wiwatanadate et al., 2019). The most important physical properties of RF-EMF are frequency

(f), wavelength (λ), and photon energy (e) (Mehta, 2011). Frequency is described as the number

of pulsation or cycles per second, and wavelength is described as the distance between the

following waves (Terzi, Ozberk et al., 2016).

Figure 2.1 Electromagnetic wave propagation (The image derived from University Physics

Volume 2 by OpenStax University Physics)

According to the corresponding wavelength (meters) and frequency (Hertz), EM waves are

mapped onto the electromagnetic spectrum (Keqian and Dejie, 2001, Tasić, Jeremijić et al.,

2019). The electromagnetic spectrum is separated into seven broad groups of Radio waves,

Microwave, Infrared, Visible light, Ultraviolet, X-rays, and Gamma rays and some further are

divided into different subgroups (Figure 2.2).

Figure 2.2 Electromagnetic spectrum showing the entire range of wavelength and frequency

(Lai 1996). The image source is https://www.trzcacak.rs/myfile/full/40-

408426_the-electromagnetic-spectrum-is-the-entire-range-of.png.

The combination of an electric field and a magnetic field is considered as the electromagnetic

field (EMF) (Purcell and Morin, 2013). The voltage gradient generates the electrical field that

is measured in volts per meter (V/m). The magnetic field is produced by any flow of current

and is calculated in Tesla (T) (ICNIRP 2009). The rate at which energy is consumed or

produced is electromagnetic power, and it is the result of voltage and current (Paulraj and

Behari, 2006, Panagopoulos and Margaritis, 2008). Furthermore, power density is the power

flux density is a distribution of power over a particular area (mW/cm2). As mentioned above,

the key characteristic of the field exposure used to calculate how much the body absorbs EMR is

the SAR. It is measured in units of watts per kilogram (W/kg) or mill watts per gram (mW/g)

(Sun and Hynynen, 1998). The electromagnetic spectrum (shown in Figure 2.2) is divided into

ionizing and non-ionizing radiation, ranging from the lowest to the highest frequency (longest

to shortest wavelength) and include the region of radio waves produced by commercial radio

2.2.1 Ionizing radiation

and television, microwaves, radars, smart meters and mobile phones. (Elert, 1998).

Ionizing radiation is radiation that transfers enough energy to separate electrons (ions) from

atoms or molecules at high speeds and thereby ionizing them (Hansen, Swartz et al., 2019).

Ionizing radiation with high frequency and short wavelength, including Gamma rays and X-

rays, is used in fields such as nuclear power, construction, research, medicine, manufacturing,

and many other areas, however, there are health hazards associated with ionizing radiation

(Parasuraman, Xin et al., 2018). For example, a study by (Werner, Alter et al., 2019) has

demonstrated that exposure of lung epithelial cells to ionizing radiation activates cholesterol

biosynthesis by up-regulating the expression of four enzymes in the cholesterol biosynthesis

pathway. Another study showed that exposure to ionizing radiofrequency causes cell and tissue

2.2.2 Non-ionizing radiation

damage, cancer, radiation sickness, radiation burns, and even death (Lee, Jeong et al., 2017).

This Masters by Research project is focused on the non-ionising part of the electromagnetic

spectrum, RF radiation, that includes radio frequencies and microwaves. Non-ionizing

radiation does not carry sufficient energy to ionize molecules (Paulraj and Behari, 2006, Nandi,

Futera et al., 2016). The non-ionizing radiation does not have enough energy to break chemical

bonds, and the mechanism by which non-ionizing radiation, particularly at low powers,

interacts with biological material is still not clear. It is considered that tissues nearest to the

mobile antenna can absorb this energy, which may contribute to changes in the physiological

functions of the living organism (Lai, 1996, Oncul, Cuce et al., 2016, Terzi, Ozberk et al.,

2016). Furthermore, there are many various applications established based on the heating

effects created by high-power RF/MW radiation which are successfully used in the food

industry, protection and safety, and various medical applications (i.e. RF ablation). However,

high-power RF radiation can induce detrimental biological and health effects.

2.3 RF-EMR used in communication technologies

Of particular relevance to this study is RF radiation which is non-ionising radiation. Exposure

to artificial radiofrequency electromagnetic fields (EMFs) is increasing in recent years

(Kaszuba-Zwoińska, Gremba et al., 2015). There is a big concern about potential health risks

for low-power RF and microwave radiation emissions produced by wireless communication

devices, such as mobile phones, cordless telephones, smart meters, computers and radars

(Hardell, 2018). Radiowaves have a frequency ranging from 3 kHz to 300 GHz, with

wavelengths ranging from 100km to 1mm. RF radiation can be natural or be artificially

generated, controlled, transmitted and received. Moreover, RF radiation can penetrate through

haze, rain, cloud, snow, and smoke without any distortion, so they are used for transferring data

and audio/video signals. The radio waves produce very low power EMR and consequently a

very low almost negligible heating effect. Mobile phones operating at different radio frequency

bands and low powers generate RF energy, which is absorbed by surface tissues and skin,

causing an insignificant temperature increase in any body part and brain in close proximity to

the mobile phone device (Behari, 2019).

When a mobile phone is in use, RF exposure emitted by a mobile phone can penetrate only a

few centimetres through a human head, and will be located on the side of the head, the anatomic

area closest to the antenna inbuilt in the mobile phone. It has been shown that at the side of the

head where the phone is used, 50-60% of the total RF energy is absorbed in the temporal lobe,

and the average SAR is maximum in the temporal lobe and the cerebellum (Cardis, Deltour et

al., 2008). Therefore, analysis of the location of the tumour concerning the location of radiation

is of importance. There is extensive research that investigates the relationship between brain

tumour risk and mobile phone use. World Health Organisation (WHO) is leading research

efforts in this area and have organized a comprehensive and extensive study which includes

research labs and institutes from 13 participating countries, coordinated by International

Agency for Research on Cancer (IARC) (Krewski, Glickman et al., 2007). There is

reasonable evidence that even at very low powers RF and MW radiation can modify the regular

biological processes in the human body.

2.4 In silico molecular modelling studies

Computational or in silico studies showed to be potent tools that allow a mechanistic

understanding of interfaces between external fields and molecules or cells (Nandi, Futera et al.,

2016). In silico techniques are effective in studying the impacts of electromagnetic fields on

proteins’ denaturation and stability. It is worth mentioning here that the experimental approach

to monitoring these effects is very challenging because of the short time-frame of a nanosecond

(ns) (MacKerell Jr, Banavali et al., 2000, Du, Han et al., 2007, Hess, Kutzner et al., 2008,

Singh, Munshi et al., 2013, Astrakas, Gousias et al., 2015). Furthermore, it has been shown that

external static electric fields of 3 V/nm affect protein folding (Singh, Orsat et al., 2013).

Currently, molecular modelling is planned for identifying the dynamics, structure, surface

properties, and thermodynamics of inorganic, polymeric and biological systems. Recently, for

better comprehension of the effects of the power of static electric fields at the molecular and

cellular levels, in silico studies were performed to examine the effects of variable, static on

particular proteins and peptides (Singh, Orsat et al., 2013, Astrakas, Gousias et al.,2015, Nandi,

Futera et al., 2016) and oscillating electric fields (Ojeda-May and Garcia, 2010, Fallah, Jamali

et al., 2016, Karim, Indei et al., 2016). For example, it was shown that exposure to the

continuous external electric field could directly cause a dramatic conformational modification

in the secondary structure of proteins. Misfolding of a protein (Native β-Sheet) happens while

is maintained in a localised minimum of the potential energy surface (PES), where the

confirmation differs from the native-state structure. Through the degree of the field is improved,

the local dipole moments of the amino acids begin to modify their direction (Ojeda- May and

Garcia, 2010). Fernandes, de Carvalho et al., 2015 has verified that model Photon Laser III

(DMC, São Carlos, SP—Brazil) with a wavelength of 660 nm, and variable power

from 30 to 100 mW can increase in the percentage of live sperm cells in Nellore bulls (Bos

taurus indicus), with ages ranging from 24 to 50 months, in comparison with the control group.

Molecular Dynamics (MD) modelling, is an influential computational tool used to verify the

interaction of atoms and molecules under external stimuli for a stable time. There are just

insufficient reports in biological sciences executed using the MD method that has prepared a

complete explanation of the effects for practical exposures at the atomic level within the

nanoseconds (Marracino, Apollonio et al., 2013, Singh, Munshi et al., 2013, Nandi, Futera et

al., 2016). For example, a study has found that static external electric fields of strength 0.001

V/nm and 0.002 V/nm induce effects on the structural stability of gliadin protein. This study

demonstrated that external electrical fields can induce conformational modifications in the

protein via the formation of hydrogen bonds between amino acid residues (Singh, Munshi et

al., 2013).

2.5 In vitro studies

Experimental research of the effects of RF radiation includes both studies of cell cultures and

tissues (in vitro) and laboratory animals (in vivo), as well as human subjects (in vivo clinical

studies). A number of these studies were focused on functional changes in the brain and the

effects of RF fields on cognition in humans. Section 2.5 will summarise the findings of

experimental in vitro studies.

Research into the biological effects of RF radiation is not a new research area; it spans a few

decades but the research interest has been significantly intensified in the early 1990s. For

example, the study conducted by Dutta, Subramoniam et al., 1992, investigated the effects

of RF radiation at the frequency of 915 MHz and SAR of 1mW/g on human neuroblastoma.

The researchers reported a significant increase in the efflux of calcium ions in studied cells.

Their results showed the increased end-tidal CO2 excretion. Shckorbatov et al., 2002

investigated RF in a range of 837 to 1909.8 MHz, with SAR of 5 mW/g on chromatin expressed

in human cells. They reported that the applied RF radiation of human cells induces a significant

increase of heterochromatin granules’ quantity parameters.

Frequency-dependent effects of non-thermal MW from GSM mobile phones on 53BP1/γ-

H2AX foci and chromatin conformation in human lymphocytes were observed and reported by

(Belyaev, Hillert et al., 2005). The findings showed that MW radiation induces significant

adverse effects in human lymphocytes, similar to effects of heat shock and GSM MWs at

particular frequencies. The obtained results were in line with the hypothesis that MW radiation

may affect cells more efficiently than GSM MWs, because of the nature of the signal (Belyaev,

Hillert et al., 2005). The effects of microwaves from mobile phones on 53BP1/γ-H2AX foci

persisted up to 72 hrs following exposure of lymphocytes. This long-lasting adverse effect on

these critical cells of the immune system can impose a health risk to humans from mobile

telephony technology (Belyaev, Markovà et al., 2009).

A significant reduction of colony growth compared to non-irradiated yeast strains after all

exposure times was reported in the study by Vrhovac, Hrascan et al., 2010, where

Saccharomyces cerevisiae yeast samples were exposed at the frequency of 905 MHz and SAR

0.5 mW/g. A number of studies reported that RF radiation can alter the proliferation rate of

cells, as well as the rate of DNA, RNA, and protein synthesis (French, Penny et al., 2001,

Leszczynski, Joenväärä et al., 2002, Breckenkamp, Berg et al., 2003, Elwood, 2003, Hardell,

Carlberg et al., 2006, Vander Vorst, Rosen et al., 2006, Jeffrey, 2011). The biochemical

processes are strongly affected by changes in cytosolic ion concentrations (especially calcium).

It was reported that such changes could be induced by RF radiation (Alekseev and Ziskin, 1995,

Zhao, Ma et al., 2003). In vitro studies showed that membrane structure and its functionality

could be altered upon exposure to RF fields (Volkow, Tomasi et al., 2011), and hence, it can

be suggested that low-power RF radiation may affect a biological system without necessarily

causing an adverse change in health (Barnes and Greenebaum, 2006).

It is important to note that there are a number of studies showing the biological effects of RF

exposure. In contrast, studies focused on investigating the direct health effects of RF radiation

are inconclusive. The possibility of a direct relationship between mobile phone use and

carcinogenic processes, reproduction and development, the cardiovascular system and

longevity, are ruled out by a good number of researchers. These studies have found minimal

and reversible biological and physiological effects which do not necessarily lead to diseases or

injuries. In addition, the research findings on changes at the molecular level associated with the

development of cancer are inconsistent and contradictory (2006). However, it should be noted

that in vitro studies of non-thermal effects of RF also often report conflicting results (Malyapa,

Ahern et al., 1997, Phillips, Ivaschuk et al., 1998, Panagopoulos, Karabarbounis et al., 2004).

Evidently, the biological consequences of most of the changed genes/proteins are still unclear

and need to be further explored to make an evidence-based conclusion on their health effects.

There is a lack of understanding of the long-term accumulating effects of RF radiation at the

genetic and protein levels, which might lead to health effects (Zeni, Schiavoni et al., 2003,

Valbonesi, Franzellitti et al., 2015, Areti K. Manta, Deppie Papadopoulou et al., Tomomi

Kurashige, Mika Shimamura et al., 2016).

Interestingly, in the last five years, an increased number of in vitro studies have been conducted

to evaluate the biological and health effects of low-power RF radiation (Megha, Deshmukh et

al., 2015, Black, Granja-Vazquez et al., 2016, Jain, Vojisaveljevic et al., 2016, Sahin, Ozgur et

al., 2016, Al-Serori, Kundi et al., 2017) on different living systems. Of note, a more significant

number of published studies have reported health effects associated with mobile phone radiation

as opposed to studies reporting NO effects. Several in vitro studies have demonstrated that RF-

EMR induces oxidative stress, activation of heat shock proteins as well as the change in the cell

membrane and transmission potential (Oncul, Cuce et al., 2016, Havas, 2017, Zeni, Simkó et

al., 2017, Santini, Cordone et al., 2018).

It is understood in the bioelectromagnetics research community, that effects induced by applied

RF radiation on living organisms are complex and dependent on the frequency, power,

exposure duration, and SAR. Long-term exposures can induce quite different effects than short-

term exposures, and it is important to study both to understand whether accumulative effects

can induce permanent change on biological activity in studied living organisms. The

temperature should be always monitored to confirm whether long-term exposures can

contribute to temperature elevation and thus result in heating effects. In the study by

Fragopoulou, Grigoriev et al., 2010, the RF exposures at different SAR values 1, 2 and 4 W/kg

and exposure duration of 1, 2, and 3 days, respectively, at the temperature of 37.06 ± 0.5°C on

mRNA and protein expression of proneural genes NGN1 and NEUROD were studied. It was

reported that radiation decreased protein expression of these genes, and affected upregulation

of their inhibitor HES1. Neurite outgrowth of eNSC differentiated neurons was inhibited after

RF exposures for 3 days at SAR 4 W/kg.

In another study (Veeldersa, Brücknerb et al., 2010) the effects of the RF radiation at the power

density 50 mW/cm2, temperature 37.0°C, were studied on mRNA of blood-brain barrier (BBB)

proteins. The findings showed the structure of BBB has been damaged, and the permeability

of ions and low molecular weight molecules were increased. The authors also reported the

decrease in occluding mRNA and protein along with increased Tyrosine (Tyr) phosphorylation.

It was also reported by Aydogan, Unlu et al., 2015 that microwave radiation at the power

densities of 10, 30, 50 and 100 mW/cm (temperature 37±0.5 oC) induces apoptosis in the neural

cell through the mitochondria-mediated caspase-3 pathway. Chen, Ma et al., 2014 studied the

effects of RF radiation on embryonic stem cells at the exposures SAR of 0.607 W/kg for 4 and

24 hrs at 37 oC. Their results showed that cell viability was decreased; cell proliferation was

inhibited and apoptosis induced, as well as the mitochondrial membrane potential was

decreased.

The potential sources of inconsistency in reporting of research findings include differences in

experimental protocols, temperature control, exposure parameters, cytogenetic

techniques, and sensitivity of different cell types to applied radiation. Different cell types

respond differently to applied EMRs produced by mobile phones, which may lead to changes

in various biological processes through both thermal and non-thermal biological mechanisms

(Banik, Bandyopadhyay et al., 2003). Of note, in vitro studies of non-thermal effects of RF

regularly convey incompatible results because there is a deficiency of perception of the long-

term accumulating impacts of RF radiation at the genetic and protein levels, which can lead to

health effects (Malyapa, Ahern et al., 1997, Phillips, Ivaschuk et al., 1998, Panagopoulos,

Karabarbounis et al., 2004). Furthermore, it was reported that mobile-phone radiation at 1800

MHz has a non-thermal effect on DNA breakage of the human fibroblasts (Diema, Schwarza

et al. 2005). Therefore, further investigation is needed to elucidate the mechanism behind the

observed various biological effects of low-level RF exposures on molecular and cellular

biological systems.

Any measurable change in a biological system initiated by a specific stimulus is referred to as

the biological effect of the stimuli. However, it is not necessary for every biological effect to

lead to a biological or health hazard. Several reports suggest that EMR from mobile phones at

non-thermal levels might induce a biological effect in target cells or tissues. Whether or not

these biological effects lead to adverse health effects (including cancer) is unclear. To date,

there is limited scientific evidence of health issues and no mechanism by which mobile phone

radiation could influence cancer development (Dutta, Das et al., 1992, Peinnequina, Piriou et

al., 2000, Diema, Schwarza et al., 2005, Zeni, Romanò et al., 2005, Vander Vorst, Rosen et al.,

2006). The research findings on changes at the molecular level associated with the development

of cancer are inconsistent and contradictory. Nevertheless, other biological effects of low-

power RF radiation are neither rejected nor denied.

Kwon et al. (Kwon, Vorobyev et al., 2011) developed a theoretical mechanism by which RF

radiation from mobile phones could induce cancer, via the chronic activation of the heat shock

response. Upregulation of heat shock proteins (HSPs) is a standard defence response to cellular

stress. However, chronic expression of HSPs is known to induce or promote oncogenesis,

metastasis and resistance to anti-cancer drugs. The authors suggest that repeated exposure to

mobile phone radiation might serve as repetitive stress causing a continuous expression of

HSPs in exposed cells, which in turn affects their normal regulation, and thus cancer can result.

This hypothesis provides the possibility of a direct relationship between mobile phone use and

cancer, and thus provides a principal focus for future investigation (French, Penny et al. 2001,

Cancer 2011).

2.6 In vivo animal and human studies

Although it is verified that the exposure level to RF radiation produced by mobile base stations,

mobile phones, smart meters and other wireless communication devices on the population is

lower than the set standard safety exposure level (SAR 2 w/kg, 1 mW/cm2) (Pareja-Pena,

Burgos-Molina et al., 2020), it cannot be confidently concluded that continuous exposure to

low-level of RF radiation does not induce any health effect. For example, human studies have

found that RF-EMR alters the cerebral blood flow, brain physiology and stem cell function

(Volkow, Tomasi et al., 2011, Bhargav, Srinivasan et al., 2015). As mentioned in the

Introduction section, SAR is the rate of energy absorption per unit of mass and is expressed, as

watts per kilogram (W/kg) or mill watts per gram (mW/g). For example, the relationship

between SAR and brain tumour malignancy has been reported before and has shown to be

dependent on location, frequency of exposure and antenna configuration (Kaburcuk, 2019).

It was also reported that MW exposures create heating in tissues that can be sensed by thermal

receptors expressed by different tissues, including skin fibroblasts and the central nervous

system (CNS) (Behari, 2019). As reported, increasing use of smartphone devices can cause a

health effect by targeting biochemical processes and biological processes in the human body

(Megha, Deshmukh et al., 2015).

In vivo studies have revealed that the long-term and short-term radiation by mobile phones has

no significant effect on the average survival of radiated groups of animals in 96% of studies

(Megha, Deshmukh et al., 2015, Black, Granja-Vazquez et al., 2016, Yüksel, Nazıroğlu et al.,

2016). However, it was also reported that mobile phone radiation at 900 and 1800 MHz and

Wi-Fi radiation at 2450 MHz affects the uterine oxidative stress and serum progesterone and

estrogen levels in maternal rats and their offspring (Megha, Deshmukh et al., 2015, Black,

Granja-Vazquez et al., 2016, Yüksel, Nazıroğlu et al., 2016).

In the study by Adey, Bawin et al., 1982, researchers tested the effects of applied RF exposures

at 450 MHz and SAR of 0.29 mW/g on calcium efflux from awake cat cerebral cortex. In

another report, double- and single-strand DNA damages were detected in brain cells of rats

exposed to pulsed and continuous MWs at 2.45 GHz at the power density of 2 mW/cm2 (Paulraj

and Behari, 2006). The study by Deshmukh, Megha et al., 2013, which investigated the effects

of exposures at 1800MHz and SAR 0.06 mW/g on deoxyribonucleic acid damage vis-à-vis

genotoxicity in the brain of fischer rats, demonstrated that chronic exposure to low-level RF

radiation induces DNA damage in brain cells. Paulraj and Behari, 2012 studied the effects of

radiation at 9.9 GHz and SAR of 1 mW/g on biochemical changes in the rat brain. The authors

concluded that applied radiation resulted in the decreased activity of protein kinase.

The RF radiation emitted by a mobile phone for 30 mins at SAR 0.15 W/kg on ovaries of 4

days-old female Drosophila melanogaster resulted in the increased cellular oxidative stress

level, with 168 differentially expressed genes (Manta, Papadopoulou et al., 2017). In another

study, the effects of 935 MHz RF radiation were investigated on fertilization and embryonic

development in mice. The ovulation of mice was decreased in response to 4 hours of radiation

per day during three successive days (Chen, Ma et al., 2014). Lower-power MW radiation and

the resulting DNA damage might be the source of cancer and also the loss of fertility, as

reported previously (Hussein, El-Saba et al., 2016).

The study by Tang, Zhang et al., 2015, investigated the effects of RF radiation at 900 MHz and

SAR of 0.016 W/Kg on spatial memory and BBB permeability in rats. The authors reported

that exposures impaired spatial memory and damaged BBB permeability in experimental

animals. Another research group studied the effects of RF radiation at 900 MHz and power

density of 608 mW/m2 special mamoty in rats. The authors showed the applied radiation

affected significantly learning capacity and special memory in rats (Eris, Kiziltan et al. 2015).

Interestingly, similar results are reported in the study by Li, Peng et al. 2015, where the

researchers show that long-term, chronic MW exposure at the frequency of 2.856 GHz and

power densities 5, 10, 20 mW/cm2 induce a dose-dependent deficit in learning and spatial

memory in rats. Aydogan, Unlu et al. 2015 studied the effects of radiation at 2.1 GHz and SAR

0.4 W/kg on the salivary gland. They concluded that applied exposures cause salivary gland

damage to some extent and especially with a more prolonged exposure duration. Senavirathna

and Asaeda, 2018 evaluated exposure at 2.45 GHz and power density in the range of 1.9 to 2.1

W/m2 and showed that RF radiation alters burn injury-evoked electric potential in Nicotiana

benthamiana. It was also reported that exposures at power density 492.3 ± 21.43 mW/m2 and

frequency of 2350 MHz incite cyto- and genotoxic effects in root meristems of Allium cepa

(Chandel, Kaur et al. 2019). Another recent study (Zong, Gao et al. 2019) demonstrated that

RF radiation at 900 MHz power density of 120 μW/cm2 activates NF-κB in mouse bone marrow

stromal cells.

In human studies, the effective parameters are generally considered for various demographic

data such as gender, age, dietary pattern, smoking habit, alcohol consumption, duration of

mobile phone use and average daily mobile phone usage (Elwood, 2003, Belyaev, Hillert et al.,

2005, Cancer 2011, Bhargav, Srinivasan et al., 2015, Al-Serori, Kundi et al., 2017). Some

investigations reported the effects of RF radiation on embryo development, behaviour, and

biochemical processes and immune systems in animals and humans. Though, the health

concerns of these biological alterations are still unclear and need to be further investigated

(Sage and Burgio, 2018). This review study examined different research studies and scientific

reports and summarized findings on main cellular responses to EMFs and the published efforts

at replication. The original review paper that discussed quantitative features of exposures to

RF-EMFs, connected the occurrence of cancers with such low-level RF fields (Lacy-Hulbert,

Metcalfe et al., 1998).

Several members of the TRP family of ion channels are important in cellular responses to

thermal and mechanical stimulations. For example, TRPV4 mRNA is extremely enhanced in

colonic sensory neurons compared to other visceral and somatic sensory neurons. TRPV4

protein was discovered in colonic nerve fibres from patients with inflammatory bowel disease

and is specified in a subset of fibres with the sensory neuropeptide CGRP in mice (Brierley,

Page et al., 2008). TRPV1 ion channel protein has been examined for long-time radiation in 24

adult rats which were divided into control and test groups and radiated at 900 and 1800 MHz

exposures. Animals were radiated for 6 min per day, 5 days per week, for one year. The study

determined that the TRPV1 activation post-exposure to EMR leads to mitochondrial oxidative

stress and apoptosis (Yüksel, Nazıroğlu et al., 2016).

2.7 Effects of radiofrequency exposure on mechanoreceptors

Mechanotransduction is a conversion of mechanical forces into biochemical responses; it is the

process by which cells can sense and respond to their physical surrounding. In many tissues,

including muscle, blood vessel, bone, ligament and cartilage, mechanotransduction plays

critical roles in tissue health and function. Mechanoreceptors are a class of proteins that detect

mechanical stimuli resulted from vibration, pressure, touch, sound. They play a crucial role in

transmitting mechanical inputs into electrical signals (Adrian and Umrath, 1929).

One important class of mechanoreceptors are mechanosensitive ion channels. An important

characteristic of mechanosensitive ion channels is that they are expressed in mechanosensitive

cells gated by mechanical forces. Mechanosensitive ion channels are essential for our senses

of touch, hearing, and balance. Furthermore, mechanosensitive ion channels have a significant

role in regulating osmotic pressure in cells, as well as blood pressure in arteries and veins, heart

electrophysiology and also micturition (Kloda and Martinac, 2002). Mechanosensitive ion

channels are a diverse class of membrane proteins that depending on the structure of their pore

can be permeable to a specific class of ions (Martinac, Saimi et al., 2008).

Calcium (Ca2+) plays a crucial role in the translation of such forces to biochemical signals that

control various biological processes fundamental in muscle development. It was reported that

RF radiation could affect cell membrane proteins and trigger an increase in intracellular Ca2+

ions (FJ 1990, J 1992, Pilla, 2012). It was shown that changes in Ca2+ signalling occur almost

immediately after EMR exposure. The thermal mechanisms that may convey the detection of

microwaves by mammals are the heating of tissues, which can be detected by thermal receptors

in the skin and elsewhere in the body and central nervous system (CNS) (Barnes and

Greenebaum, 2006). It has been shown that RF fields modulated by extremely low frequencies

(ELF) decrease cytosolic Ca2+ concentration (Blackman, Elder et al., 1979). In some

experiments, this effect was at the maximum power densities between 0.6 and 1 mW/cm2. In

one study (Adey, Bawin et al. 1982), the GSM signals tested were the RF carrier signals pulsed

at ELF and the power densities ranging from 0.436 to 0.060 mW/cm2. It is known that cell

proliferation, DNA, RNA, and protein synthesis are connected with increased cytosolic ion

concentrations (especially calcium) and with depolarization of the plasma membrane. The

effects of external RF fields on the cytosolic ion concentrations appear to be connected with

the interaction between the external field and the cation channels of the plasma membrane,

which results in irregular gating of these channels. A biophysical mechanism for this

interaction has been proposed (Adey, Bawin et al., 1982). According to this mechanism, RF

fields of the order of a few V/m can gate electro-sensitive channels of a cell’s plasma membrane

irregularly and disrupt cell function. In addition, it was also reported that pulsed fields are more

bioactive than continuous ones. Therefore, according to one study (Chiang, Hu et al., 2002),

the ELF modulation component of a GSM RF signal, at the pulse repetition frequency at 217

Hz, with a mean electric field of 6 V/m, can disrupt cell function and consequently impact the

reproductive ability of a living organism. Two significant findings of these studies are that the

effects of RF fields are waveform-specific and cell type-specific (Campisi, Gulino et al., 2010).

The recognition of a family of transient receptor potential (TRP) ion channels which are gated

by particular temperatures, has been important progress in the clarification of the molecular

procedures of thermo-sensitivity (Filingeri, 2011). Research has discovered a family of TRP

proteins that sense heat and cold at the cellular level (Patapoutian, Peier et al., 2003).

Recent studies (Jain, Vojisaveljevic et al., 2016), on the effects of low-power MW radiation at

1800 and 2100 MHz on yeast cells’ growth demonstrated that RF-EMR at low-level powers in

the frequency range of 900 - 2600 MHz (which are used in 3G and 4G mobile networks) can

induce changes in the gating function of TRPV channels, affect the growth rate of yeast cells

and modulate the biological activities of various enzymes. As reported by Jain, et al., 2017,

TRPV4 were exposed at 1.8 GHz and power 17 dBm and the response of TRPV4 channel

gating with its selective agonist GSK1016790A was investigated in comparison to the control

group by using Ca 2+ using imaging and confocal microscopy. It was shown that applied RF

exposures can modify intracellular calcium homeostasis. These findings suggest that specific

non-thermal exposures can induce changes in the biological activity of TRPV4.

2.8 Summary

This chapter summarised the fundamental concepts of electromagnetic radiation with an

emphasis on non-ionizing RF radiation. Recent works on the effect of RF radiation (the

frequency range used in mobile telecommunication) at cellular and tissue levels have been

presented. Based on the current body of knowledge, the author conversed a study on the

biological and possible health effects and protocols for service providers to use such radiation

possessively. Mobile phones operating at high-frequency bands, which are used in modern 5G

networks, generate RF energy which is absorbed by surface tissues and skin, causing an

insignificant temperature increase in any body part and brain (Bernardi, Cavagnaro et al., 2000,

Hirata, 2005). The power of radiation produced by mobile phones and other telecom

technologies is at a low level and conforms with safety standards (no heating effect necessity,

<1ºC in surface heating).

Since the radio waves emit very low-power radiation and consequently produce a very low or

negligible heating effect; therefore, the focus of this Masters by Research project is towards

the possible impact of non-thermal effects of RF radiation on ion channel proteins (TRPV4

and Piezo-1) expressed in different cells. This project is aimed to study the effects (in vitro

and in silico) of RF-EMR on a mechanoreceptor Piezo-1 ion channel protein that is a non-

selective cation channel. Piezo-1 is expressed in various tissues, including epithelial and

endothelial cells, which are mechanically gated (Nilius 2010, Douguet, Patel et al. 2019,

Ridone, Vassalli et al. 2019).

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CHAPTER 3 EXPERIMENTAL STUDY ____________________________________________

Investigating the effects of low-level radiofrequency radiation on

activation of mechanosensitive ion channel Piezo-1 and TRPV4

3.1. Introduction

Cells inside our body experience a variety of mechanical forces from their physical

environment; this includes thermal agitation of molecules to shear stress and osmotic cell

swelling. A low-level electromagnetic field emitted by a mobile phone is one example of an

external mechanical force that cells would experience (Miller, Sears et al, 2019).

Mechanosensitive ion channels are pore-forming membrane proteins, that can directly or

indirectly gate in response to mechanical forces and contribute to different physiological

responses (Martinac, 2004).

Piezo-1 is a novel example of the mechanically activated ion channel protein that is present in

various tissues and responds to static pressure, shear stress and membrane stretch. Apart from

activation by mechanical signalling, several synthetic molecules have been identified to activate

Piezo-1 channels. Among them, Yoda-1 is a highly potent channel agonist that has

revolutionized research in this field (Botello-Smith, Jiang et al., 2019). Piezo-1 plays a

physiological role in several tissues, including vascular endothelium and immune cells such as

monocytes (Bird, 2019, Lhomme, Gilbert et al., 2019).

The first aim of this sub-study is to understand the short-term effects of low-level RF radiation

on the activation of mechanosensitive ion channel Piezo-1. The second aim is to understand

the long-term effect of RF radiation on activation, expression of Piezo-1 and TRPV4 (another

critical mechanosensitive ion channel) and expression of inflammatory genes in monocytes.

The final aim is to investigate the effect of low-level RF radiation on Piezo-1 response to its

selective agonist Yoda-1.

To address these aims, a series of experimental studies were conducted; two different types of

equipment as a source of RF radiation were used. First, I have used the mobile phone set up to

generate/emit EMR in the radiofrequency range for investigating the short-term effects (10 min

exposure) of RF exposures on the function of Piezo-1. Second, I have used the Transverse

Electro-Magnetic (TEM) Cell Exposure System to investigate the long-term effect (2hr and 4hr

exposures) of mobile phone radiation on Piezo-1 and TRPV4.

3.2 Material and Methods

3.2.1 Reagents and buffers

Calcium imaging buffer was Hanks' Balanced Salt Solution (HBSS) (Life Technologies, VIC,

Australia) buffer with 10mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 1mM

magnesium chloride (MgCl2), and 2 mM calcium chloride (CaCl2), adjusted to pH 7.4. Calcium

free buffer was prepared with HBSS without MgCl2 and CaCl2 supplemented with 2 mM ethylene

glycol tetraacetic acid (EGTA). Stock solutions of Piezo-1 selective agonist Yoda1 (Tocris

Bioscience, Bristol, UK), thapsigargin (Sigma-Aldrich, Missouri, USA), Ruthenium red (Sigma-

Aldrich, Missouri, USA), were diluted in HBSS buffer to the desired concentrations using

3.2.2 Cell culture

previously reported concentrations (Baratchi, Almazi et al., 2016).

HEK293 cells stably expressing piezo-1 (Piezo-1-HEK293) (a kind gift from Charles Cox, Victor

Chang) cells were cultured in DMEM supplemented with 10% foetal bovine serum, 300 µl (1

µg/ml) puromycin and cultures under 5% CO2 at 37°C inside a humidified incubator. THP1 cells

(ATCC® TIB-202™) cultured in RPMI supplemented with 10% foetal bovine serum, 1%

3.2.3 Experimental set up using a mobile phone to investigate the short-

term effects of electromagnetic radiation

penicillin-streptomycin (PS) and cultured under 5% CO2 at 37°C inside a humidified incubator.

Mobile phone Huawei 9 Mate (frequency 845 MHz, powers -15dBm and -5dBm) was used to

investigate the short-term effects of RF radiation, 10 min exposures, on mechanosensitive ion

channel Piezo-1 at settings illustrated in Table 3.1. This set up enabled us to investigate the effect

of RF electromagnetic radiation emitted from mobile phone headset(s) placed at different distance

from the well-plate with cells.

Table 3.1 Positions of mobile phone headset against the 24-well plate

(1) One Phone at 12 cm (2) One Phone at 1 cm

(3) One Phone at 4 cm on top of the (4) Two phones placed at 4cm and 12

well-plate cm

3.2.4 Calcium imaging and confocal microscopy

HEK293-Piezo-1 and THP-1 cells were cultured on 24-well plates coated with Poly-L-lysine

(0.1% (w/v) in H2O) (Sigma-Aldrich, Missouri, USA) at a density of 2.5×105 cells per well. Before

each experiment, cells were loaded for 30 minutes at 37°C with 0.33 µM Fluo-4 AM ester (Life

Technologies, VIC, Australia) in an imaging buffer. For experiments involving inhibitors, cells

were preincubated with a specific inhibitor concentration for at least 30 min before the calcium

imaging experiment. The optimum concentration of the inhibitor was selected based on available

literature. Calcium imaging was performed on a stage Nikon A1 confocal laser scanning inverted

microscope (Nikon Instruments, Inc., New York, USA) equipped with the temperature controller

and motorized stage.

For each experiment, cells were stimulated with mobile phone radiation for 10 min at different

positions as described in Table 3.1, and fluorescence emissions were detected using a

photomultiplier tube following a 525/50 nm band-pass ﬁlter and a PlanFluor 20× objective.

Change in [Ca2+]i was measured as an increase in the ﬂuorescent intensity of Fluo-4 AM and

normalized to the ﬂuorescent intensity of resting cells. All imaging experiments were performed

3.2.5 Image analysis

at 37°C unless otherwise stated.

The cell area was measured by automatically acquiring the region of interest (ROIs) around each

cell using NIS element analysis software (Nikon Instruments Inc). To quantify changes in [Ca2+]i,

the average intensity of at least 50 ROIs was measured, and results were reported as the ratio of

F1/F0. Data are shown as mean ± standard error of the mean (SEM) of at least four independent

3.2.6 Transverse Electro-Magnetic (TEM) Cell Exposure System

experiments.

The commercial Transverse Electro-Magnetic (TEM) cell (No. TC-5062A UHF-TEM Cell) was

used in this study to irradiate the selected cells for an extended period of time (2 and 4 hrs). A

TEM cell is an enclosed box made of a conductor material, with its dimensions varied depending

on the operating frequency used. The exposure system consists of Transverse Electro-Magnetic

(TEM) TC-5062AUHF TEM cell (100 kHz to 3 GHz) from TESCOM Ltd, and the signal

generator (Wiltron 68247B) operating range 10 MHz to 20 GHz. The TEM cell was connected to

a signal generator from which the external signal was applied to the cells inside the TEM Cell.

Figure 3.1 shows the experimental setup of the TEM cell exposure system.

Figure 3.1 Experimental set-up showing exposure camera, signal generator and temperature

controller

Before each experiment, HEK-293-Piezo-1 cells or THP-1 cells were seeded on a 24-well plate at

a density of 2.5×105 cells per well overnight. Figure 3.2 shows the sample's position and the

direction of the electric field inside the TEM cell. The generated field inside the TEM cell was

calibrated using a broadband electric field probe to determine the electric field the sample received

inside the system for given input power (Figure 3.2).

The calibration test result showed the estimated uncertainty of ±1-3%, depending on the input

signal frequency. For each experiment, cells were exposed to the frequency of 1.8 GHz, and power

of 17 dBm for the period of 2 to 4 hrs.

Figure 3.2 The position of the sample and the direction of the electric field inside the TEM cell.

a) The vertical distance from the top of the cell to the sample is 22 cm, b) Field pattern at the

position of the sample (top view).

3.2.7 RNA extraction and RT-qPCR

RNA was isolated from THP-1 cells using the RNAeasy Micro Kit (Qiagen) according to the

manufacturer's instructions and quantified using a NanoDrop spectrometer (Life Technologies,

California, USA). To investigate the effect of radiation on mRNA expression, RNA was

converted into cDNA using the High-Capacity cDNA Reverse Transcription Kit (Applied

Biosciences) and amplified by using validated TaqMan primers and TaqMan Fast Advanced

Master mix on a QuantStudio 7 Flex system (Thermo Fisher Scientific). The mRNA levels

were normalized to GAPDH levels, as the expression of GAPDH is not modulated by

mechanical stimulations.

3.2.8 Intracellular Ca2+ measurement

Intracellular [Ca2+]i was measured by fluorescence with a Clariostar plate reader (Molecular

Devices). 50,000 cells per well into poly-l-lysine–coated 96-well plates, grew them overnight,

and then loaded cells with 2 µM Fluo4AM (Molecular Probes) for 30 min. Loading and

experiments were performed in HEBSS buffer with NaCl and MgCl2 containing 10 mM

HEPES, pH 7.4. We measured emission intensity at 520 nm in response to the synthetic Piezo-

1 agonists Yoda-1 after 2 hrs of exposure to 1.8 GHz at16 dBm. Increases in [Ca2+]i..The peak

3.2.9 Statistical analysis

values were plotted to construct concentration-response curves.

The data from three or four independent experiments with duplicate or triplicate determinations

at each point were combined and expressed as mean ± SEM. Intracellular calcium peaks in

cells exposed to radiation were compared to those from control groups using a student's t-test,

One-Way or Two-way ANOVA as indicated in the result section. Calcium levels in

unstimulated cells were analyzed using a one-way analysis of variance with a Tukey's multiple

comparison post-test. GraphPad Prism 8 has been used for statistical analyses.

3.3. Results

3.3.1 A low-level electromagnetic field activates the mechanosensitive

ion channel Piezo-1

In this sub-study, the effects of low-level RF radiation on the activation of mechanosensitive

ion channel Piezo-1 were investigated. For this, I used HEK293 cells stably expressing Piezo-

1 (HEK293-Piezo-1) and exposed them at 845 MHz and 5 and -15 dBm using one phone

positioned at 1 cm, 4 cm and 12 cm distance from the cell culture plate and two phones-settings

(at 4 cm and 12 cm distance) for a maximum of 10 min.

I found that exposures of cells to RF radiation emitted from the mobile phone in both one

phone, positioned at 1 cm, and two-phone setting, position at 4 cm, lead to the transient increase

in the [Ca2+]i that was stable for the duration of radiation (Figure 3.3 a-e). Contrary, cells

exposed to radiation by one phone at 4 and 12 cm distances did not induce any response in cells

(Figure 3.3 f-g). Overall, stimulation of HEK293-Piezo-1 cells to 10 min RF radiation in one-

phone positioned at 1 cm and two-phone setting (4cm) led to a statistically significant increase

in the intracellular calcium level of HEK293-Piezo-1 cells (P<0.0001, N=4) and (P<0.001,

N=4), respectively that was not observed in the non-stimulated control group (Figure 3.4).

Figure 3.3 Low-level RF radiation activates the mechanosensitive ion channel Piezo-1. (a-

b) Representative microscopic images of HEK293-Piezo-1 cells in control and cells exposed

to the low-level RF electromagnetic field emitted from two mobile phones. (c-g) Single cells

profile of change in [Ca2+]i of HEK293-Piezo-1 cells in the control group and after exposure to

the low-level RF field emitted by two phones-setting (positioned at 4 cm and 12 cm) (d), one

phone (1 cm) (e), one phone (4 cm) (f) and one phone (12 cm) (g) positions. Each black line

represents one cell's intensity profile, and the red line represents the average intensity profile.

Figure 3.4 Low-level RF radiation increases the [Ca2+]i of HEK293-Piezo-1 cells. A

summary graph showing the max increase in [Ca2+]i of HEK293-Piezo-1 cells after exposure

to low-level electromagnetic filed emitted by two phones or one phone at 1, 4 and 12 cm

positions. Data presented here represent the mean ± SEM of four different experiments

analyzed with one-way ANOVA. ***P<0.001 and ****P<0.0001.

3.3.2 A low-level electromagnetic field activates THP-1 cells

Next, I investigated the effect of low-level RF field exposures on the function of endogenously

expressed Piezo-1 channel proteins. For this, I used a human monocytic cell line known as

THP-1 cells. Monocytes are the largest leukocytic cell type of circulating blood that controls

the innate immune responses and therefore, an important class of cells to study (Chiu and

Bharat, 2016). Here, I found that exposure of THP-1 cells to the low-level electromagnetic field

emitted for 10 min by two phones activated the cells and led to the sustained increase in the

[Ca2+]i (Figure 3.5 a-d). On the contrary, exposure of THP-1 cells to the electromagnetic field

emitted by one-phone-near field, one-phone-far field, and one-phone on top setting did not

affect [Ca2+]i level of THP-1 cells (Figure 3.5 e-g).

Overall, I found that exposure of THP-1 cells to the low-level RF field emitted by two phones

(4 cm and 12 cm distances to cell well) and one-phone (1cm distance to the cell well) leads to

a significant increase in [Ca2+]i, P<0.001 and p<0.01 respectively, N=4 (Figure 3.6).

Figure 3.5 Low-level RF radiation emitted by two mobile phones activates THP-1 cells.

(a-b) Representative microscopic images of THP-1 cells in the control group and cells

exposed to the low-level electromagnetic field emitted from two mobile phones. (c-g) Single

cells profile of change in [Ca2+]i of THP-1 cells in the control group and after exposure to

the low-level electromagnetic field emitted by two phones (d) and one phone at 1 cm (e), 4

cm (f) and 12 cm (g) positions. Each black line represents one cell's intensity profile, and

the red line represents the average intensity profile.

Figure 3.6 Low-level RF radiation increases the [Ca2+]i of THP1 cells. A summary graph

showing the Max increase in [Ca2+]i of THP-1 cells after exposure to low-level

electromagnetic filed emitted by two phones or one phone at 1, 4 and 12 cm positions. Data

presented here represent the mean ± SEM of four different experiments analyzed with one-

way ANOVA. **P<0.01 and ****P<0.0001

3.3.3 The response of HEK293-Piezo-1 to Low-level electromagnetic radiation

is Piezo-1 specific

Next, to confirm the response observed in HEK293-Piezo-1 cells are specific to Piezo-1, I

repeated the experiment using the one-phone near-field setting. Using this approach, I found

that low-level electromagnetic radiation at one-phone near-field setting does not have any

effect on [Ca2+]i of HEK293 parental cells, while at the same experimental setting low-level

electromagnetic radiation leads to the significant increase in [Ca2+]i of HEK293-Piezo-1,

P<0.05, N=4 (Figure 3.7).

Figure 3.7 The increase in [Ca2+]i post-exposure to the low-level RF radiation is Piezo-1

dependent. Summary graphs showing changes in [Ca2+]i of parental HEK293 cells (a) and

HEK-Piezo-1cells (b) after stimulation with a low-level electromagnetic field emitted from one

mobile phone (1 cm). Data presented here represent mean ± SEM of four different experiments

and have been analyzed using a student's t-test *P<0.05.

3.3.4 Long-term radiation has no effects on the expression of inflammatory

cytokines in THP1 cells

In this sub-study, I investigated the effect of long term exposures to low-level electromagnetic

radiation on monocytes immune response. As mentioned previously, monocytes are

inflammatory cells controlling innate immune responses. One of the hallmarks of monocyte

activation is an increase in inflammatory cytokines and chemokines' expression levels.

Cytokines are signalling molecules that expressed by immune cells and control inflammatory

reactions. In this regard, TNF-α, IL1β, IL6, IL10 and Interferon β1 (INFβ1) are an important class

of inflammatory cytokines expressed by monocytes (Kurokawa, Araujo et al., 2007). Following

expression, these cytokines and chemokines further contribute to the development of

downstream inflammatory responses.

Therefore, I initially investigated the induction of inflammatory cytokine after exposure in

THP-1 cells to the RF electromagnetic field. For this, THP-1 cells were exposed to 1.8 GHz

and 17 dBm RF radiation for 4 hrs. Followed by that, THP-1 cells were incubated for 24 hrs in

the humidified incubator and on the following day, the expression level of inflammatory

cytokines, TNF-α, IL1β, IL6, IL10 and Interferon β1 (INFβ1) were assessed using qPCR.

Surprisingly, exposure of THP-1 cells at 1.8 GHz and 17 dBm of RF radiation did not have any

effect on the expression of inflammatory markers (Figure 3.8).

Figure 3.8 Effects of Low-Level RF radiation on the expression of inflammatory cytokines

in THP-1 cells. A summary graph of qPCR experiments showing the expression level of IL6,

TNFα, IL1β and IL10 in THP-1 cells after exposure at 1.8 GHz and 17 dBm for 4 hrs. Data

presented here represents the mean ± SEM of seven different experiments and has been

analyzed using a student's t-test.

3.3.5 4 hr radiation did not affect the expression of mechanosensitive ion

channels TRPV4 and Piezo-1 in THP-1 cells

Next, I investigated the long term effect of low-level RF radiation on the expression of

mechanosensitive ion channels Piezo-1 and TRPV4. For this, THP-1 cells were exposed at 1.8

GHz and 17 dBm radiation for 4 hrs.

Followed by that, THP-1 cells were incubated for 24 hrs in the humidified incubator and on the

following day, the expression level of TRPV4 and Piezo-1 were assessed using qPCR. Using

this approach, I found that exposure of THP-1 cells for 4 hrs at 1.8 GHz and 17 dBm does not

have any consequent effects on the expression of Piezo-1 and TRPV4 (Figure 3.9)

Figure 3.9 Effects of Low-level RF radiation on the expression of mechanosensitive ion

channels in THP-1 cells. Summary graphs of qPCR experiments showing the expression level

of TRPV4 (a) and Piezo-1 (b) in THP-1 cells after exposure at 1.8 GHz and 17 dBm for 4 hrs.

Data presented here represents the mean ± SEM of seven different experiments and has been

analyzed using a student's t-test.

3.3.6 Low-level electromagnetic field desensitizing the response of Piezo-1 to

10 µM Yoda-1

For the next step, I investigated the short-term effect (10 min exposure duration) of low-level

RF field (mobile phone settings) on Piezo-1 response to its selective agonist Yoda-1. Yoda-1

is a chemical compound that has been developed to study the molecular pharmacology of

Piezo-1(Davies, Lopresto et al. 2019) and the discovery of this drug has revolutionized research

in the field of Piezo-1 mediated mechanosensitivity.

Here, I found that 10 min radiation of HEK293-Piezo-1 cells in all conditions desensitized the

response of Piezo-1 to Yoda-1 (Figure 3.10). Specifically, electromagnetic radiation in two-

phone setting (4cm and 12 cm distance to cell well) leads to a significant decrease by 1.1±0.1,

P<0.001, in one-phone setting 1, 4 and 12 cm leads to 2.8±0.1, p<0.0001, 1.4±0.1 fold, P<0.01

and 1.2±0.1 fold, P<0.01 decreases respectively compared to the control group (Figure 3.11).

Figure 3.10 Low-level RF radiation desensitizes the response of the mechanosensitive ion

channel Piezo-1 to Yoda-1. (a-b) Representative microscopic images of HEK293-Piezo-1

cells in control and cells exposed to the low-level electromagnetic field emitted from two

mobile phones in the presence of Piezo-1 selective agonist, Yoda-1. (c-g) Single cells profile

of change in [Ca2+]i of HEK293-Piezo-1 cells response to Yoda-1 in the control group and after

exposure low-level electromagnetic field emitted by two phones (d) or one phone at 1 cm (e),

4 cm (f) and 12 cm (g) positions. Each black line represents the intensity profile of one cell and

the red line represent the average intensity profile.

Figure 3.11 Low-level RF radiation desensitizes the response of the mechanosensitive ion

channel Piezo-1 to Yoda-1. A summary graph showing the max increase in [Ca2+]i of

HEK293-Piezo-1 cells to 10 µM Yoda-1 after 10 min exposure to low-level RF field emitted

by two phones, one phone at 1, 4 and 12 cm positions. Data presented here represent the mean

± SEM of four different experiments analyzed with one-way ANOVA. ***P<0.001 and

****P<0.0001.

3.4.1 Low-level electromagnetic field desensitizing the response of Piezo-1

endogenously expressed in THP-1 to 10 µM Yoda-1

Next, I investigated the effect of low-level RF radiation of endogenous Piezo-1 response to its

selective agonist Yoda-1. Similar to the HEK293-Piezo-1 data, here I found that exposure of

THP-1 to RF radiation in two-phone, one-phone at 1, 4 and 12cm distance settings from the

cell wells induce effects in Piezo-1 ion channel (Figure 3.12). Specifically, radiation of THP-

1 cells by the low-level RF field at all conditions significantly desensitized the cellular

responses to Yoda-1 (P<0.001) (Figure 3.13).

Figure 3.12 Low-level RF radiation desensitizes the response of endogenous Piezo-1 to

Yoda-1. (a-b) Representative microscopic images of THP-1 cells in the control group and cells

exposed to the low-level electromagnetic field emitted from two mobile phones in the presence

of Piezo-1 selective agonist, Yoda-1. (c-g) Single cells profile of change in [Ca2+]i of HEK293-

Piezo-1 cells response to Yoda-1 in the control group and after exposure to the low-level

electromagnetic field emitted by two phones (d) or one phone at 1 cm (e), 4 cm (f) and 12 cm

(g) positions. Each black line represents the intensity profile of one cell, and the red line

represents the average intensity profile.

Figure 3.13 Low-level RF radiation desensitizes the response of the endogenously

expressed Piezo-1 to Yoda-1. A summary graph showing the max increase in [Ca2+]i of THP-

1 cells to 10 µM Yoda-1 after 10 min exposure to low-level electromagnetic filed emitted by two

phones, one phone at 1, 4 and 12 cm positions. Data presented here are representative of the

mean ± SEM of four different experiments that have been analyzed with one-way ANOVA.

*P<0.05 and ****P<0.0001

3.4.2 Desensitization effect of low-level electromagnetic radiation on HEK293-

Piezo-1 to Yoda1 is absent in parental HEK293 cells

Next, I investigated the specificity of Piezo-1 response to Yoda-1. For this, the parental

HEK293 cells were exposed to low-level RF radiation using the one-phone near-field setting

and compared the result to the one obtained from HEK293-Piezo-1 cells. Using this approach,

I found that parental HEK293 cells are not responsive to Yoda-1 and stimulation of HEK293

cells does not affect their response to Yoda-1.

As expected, HEK293-Piezo-1 cells were responsive to Yoda-1 and stimulation with low-level

RF radiation desensitized the response of Piezo-1 to Yoda-1 (P<0.0001) (Figure 3.14). Further,

the experiment was performed for HEK293-Piezo-1 and HEK293-NT cell lines at the same

condition and realized that 10 min radiation using one mobile phone within the talking mode

in the near field setting at 1 cm distance from Petri dish desensitized the response of Piezo-1

mechanosensitive ion channel to its selective agonist, 10 µM Yoda-1in HEK293-Piezo-1

(Figure 3.14b). Although 10 min of radiation using one mobile phone within the talking mode

in the near field setting at 1 cm distance from the Petri dish has no significant effect on the

intracellular level and also the response of Piezo-1 mechanosensitive ion channels in HEK293-

NT (Figure 3.14a).

Figure 3.14 The desensitization of HEK293-Piezo-1 cells to Yoda-1 is dependent on the

expression of Piezo-1.

Summary graphs showing changes in [Ca2+]i of parental HEK293 cells (a) and HEK-Piezo-1 cells

(b) after stimulation with 10 µM Yoda1 and in the presence of a low-level electromagnetic field

emitted from one mobile phone (1 cm). Data presented here are representative of the mean ±

SEM of four different experiments and have been analyzed using

a student's t-test ****p<0.0001.

3.4.3 2 hrs radiation did not affect the response of HEK293-Piezo-1 cells to

different concentrations of Yoda-1

Next, I investigated the effect of 2 hrs exposure to low-level RF radiation on HEK293-Piezo-

1 response sensitivity and response to its selective agonist Yoda-1. For this, HEK293-Piezo1

cells were exposed at 1.8 GHz and 17 dBm of radiation for 2 hrs. Followed by that, cells were

treated to different concentrations of Yoda-1 ranging from 0.1 to 10 µM. Using this approach,

I did not find any significant effect on the Piezo-1 response to its selective agonist on any of the

concentrations (Figure 3.15).

The seeded cells at a density of 0.5×106 cells/ml and 100 µl volume were loaded in three rows

of 96-well plates, then radiated in the TEM enclosed box by the signal generator. All

experiments were conducted at 37C with a frequency of 1.8 GHz and a maximum power of

16 dBm for 2 hrs.

Figure 3.15 Effects of Long term exposure to low-level RF radiation on the Piezo-1

response to Yoda-1. Calcium imaging experiment showing dynamics of changes in [Ca2+] of

HEK293-Piezo-1 cells in response to vehicle control or 0.1 to 10 µM of Yoda-1 in the control

group and after exposure 1.8 GHz, 16 dBm RF for 2 hrs (a-e). Summary graph showing the

pick response of HEK293-Piezo-1 to vehicle control or 0.1 to 10 µM of Yoda-1 in the control

group and after exposure 1.8 GHz, 16 dBm RF for 2 hrs (f). Data presented here represents the

mean ± SEM of four different experiments and have been analyzed using a two-way ANOVA.

3.5 Summary

This study aimed to examine the effects of short-term exposures of low-level RF radiation on

the mechanosensitive ion channel Piezo-1, focusing on the frequencies emitted by mobile

phones. Overall, the findings of this chapter allowed me to address Aims 1, 2 and 3 of this

project as follows:

First, I showed that low-level RF radiation of 10 min leads to short term activation of the

mechanosensitive ion channel Piezo-1, leading to an increase in intracellular Ca2+ in both

HEK293-Piezo-1 and THP-1 cells.

Second, I showed that the cellular responses to low-level electromagnetic radiation emitted by

mobile phones depend on the distance between the mobile phone headset and the cell culture

plate. I found that the response of HEK293-Piezo-1 to mobile phone radiation is dependent on

the expression of Piezo-1 as the response was absent in parental HEK293 cells.

Third, I investigated the effect of long-term exposures to low-level electromagnetic radiation.

I showed that exposure of THP-1 monocytic cells to the RF field emitted by TEM cell (4 hrs)

do not affect the expression of Piezo-1 and TRPV4 channels as not inducing the expression of

inflammatory cytokines and chemokines in THP-1 cells.

Forth, I showed that low-level electromagnetic radiation desensitizes HEK293-Piezo-1 and

THP-1 cells' response to Yoda-1 and this effect was dependent on the distance of the phone

from the cell culture plate.

3. 6 References

Bird, L. (2019). "Monocytes feel the pressure." Nature Reviews Immunology 19(10): 595- 595.

Chiu, S. and A. Bharat (2016). "Role of monocytes and macrophages in regulating immune response following lung transplantation." Current opinion in organ transplantation 21(3): 239-245.

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Martinac, B. (2004). "Mechanosensitive ion channels: molecules of mechanotransduction." Journal of Cell Science 117(12): 2449-2460.

CHAPTER 4

COMPUTATIONAL STUDY

Simulating RF field exposures emitted by mobile phone headset

using Computer Simulation Technology (CST) Microwave Studio

In this Chapter, factors that can influence the electromagnetic field’s (EMF) strength are

studied aiming to accurately simulate RF exposures emitted by a mobile phone handset that

was used in the experimental study described in Chapter 3, where changes in sensitivity and

functionality of mechanosensitive ion channel proteins, expressed in endothelial cells and THP-

1 cells (a model for human monocytes), were induced by applied exposures and discussed in

detail. To validate the RF exposure patterns and ensure that the electromagnetic simulation

stays true to the actual in vitro experiment conducted on endothelial cells, both the phone chassis

and antenna have to be considered in combination due to the effect of the ground plane on the

antenna performance. The permittivity of the instrument used in the experimental setup should

also be accounted for in the simulation. Since the electric field strength/power has an inversely

proportional relation to the conductivity of a material, the conductivity of the buffer used to

maintain the pH level of the cells should be accounted for in the simulation too, since the

intensity level of the electric field will influence the SAR value, crucial parameter affecting

field exposures on cell cultures. Because the characteristics of the RF field differ in different

field regions, the distance between the antenna and the measurement point (cell

cultures in well plate) should be taken into account with respect to these different regions. This

distance between the antenna and measurement points will affect the power density. As the

distance increases, the power density will be reduced due to the path loss, meaning that a lesser

dose of irradiation will be given to cells.

The aims of this computational study are to:

(i) visualise the distribution of generated RF fields along the well plate with cell

culture, and

(ii) evaluate field properties, including field strength, power and energy densities, at

each well which will aid in understanding the effects of applied exposures on the

bioactivity of irradiated endothelial cells and THP-1 cells

Since antenna performance is dependent on its ground plane, both the phone chassis and

antenna should be evaluated in combination to obtain a reliable result. In addition, as the

emitted RF field is different in different positions (regions) depending on the position of the

irradiation source (mobile phone headset) from cell culture wells, the distance between the

antenna and the measurement point should be taken into account with respect to these different

positions. Furthermore, the permittivity, permeability and conductivity of the instrument and

material used in the experimental setup should be accounted for to ensure an accurate

simulation of the RF field’s strength/power.

The results of this computational study show that positioning of the mobile phone(s) is a very

important factor for producing a RF field for irradiating cells which can induce different

modulating effects or no effects at all. Findings reveal that different placements of the handset

with respect to the wells plate generate the FR filed of different strength/ intensity, power

density and energy density. With a single phone placed at the side of the well plate, a linear

decrease in field properties is observed. When the phone is placed directly on top of the wells,

peaks for all the results are centralised in the middle due to the Omni-direction radiation pattern

of the antenna. With two phones placed at the opposite ends of a measurement point, destructive

interference attributed to the phones' electromagnetic waves can be observed. These findings are

presented in detail in the section below.

4.1 Background

In the ever advancing fast-paced world of wireless communication, one facet has always been

a constant – concerns for adverse health effects of radiation emitted by mobile phone devices.

Since the late 1950s, when radar and radio devices became prevalent, many concerns have been

raised regarding possible biological and consequent health effects of non-ionizing radiation

produced by such devices (Cook 1951, Barron and Baraff 1958, Baldwin, Bach et al. 1960).

Non-ionizing radiation which ranges from Radiofrequency to Ultraviolet frequency in the

electromagnetic spectrum is a low energy level radiation that does not produce sufficient energy

to cause ionization or break covalent bonds in molecules (WHO). In contrast, ionizing radiation,

such as X-rays and Gamma-rays, has enough energy to remove electrons from an atom, thus

causing it to be ionized or charged (WHO). The majority of wireless devices such as mobile

phones, laptops and tablets developed in recent years emit electromagnetic fields in the radio

frequency (RF) range of 20 kHz to 300 GHz (Commission).

While direct biological effects caused by ionizing radiation at higher frequencies have been

well established over the years, there is a lack of conclusive evidence regarding the non-

ionizing radiation in the RF range and its possible biological effect and health effects, in

particular the so called “non-thermal” effects (Protection. 2018). Due to the difference in

experimental setups used by different research groups, many results reported in research studies

are inconsistent and even contradictory (Miyakoshi 2013). In addition, a vast majority of

studies relating to health effects of non-ionizing radiation have been conducted with

considerably lower exposures than experiments which show impaired health effect (Protection.

2018). The different power levels (field strengths) and frequencies of non-ionizing radiation

exposures would result in different measured outcomes and thus, contribute to a pool of

erroneous results as a consequence. Due to the lack of a guideline for non-ionizing radiation

exposure in the field of bioelectromagnetic, studies on possible biological effects caused by

mobile phones have, therefore, remains inconclusive today. As such, this computational study

aims to resolve the perplexity caused by the lack of guidelines for non-ionizing radiation

measurement specifically for the FR field produced by mobile phones. This investigation was

focused on simulating exposures produced by the mobile phone handset used in the previous

experimental study (Chapter 3) where endothelial cells and THP-1 cells, a model for human

monocytes, were irradiated. Cell cultures were exposed to the mobile phone(s) located at the

specific distance and positions to the cell culture wells, with the results showing that emitted

RF radiation at different measuring points can induce different amounts of calcium level in the

exposed ion channel proteins expressed in endothelial cells. However, due to the micro-scale

of cell culture, even the smallest existing field probe with a 25mm diameter (Holloway, Gordon

et al. 2014) is unable to accurately determine the field strength acting on the cells

underexposure. Therefore, the EMF simulation study was proposed here to overcome this

challenge faced by the physical measurements of the field strength generated by the mobile

phone handset. However, the accuracy of the EMF simulation is highly dependent on many

factors which are discussed in detail in the sections below.

Most existing studies that investigate biological or health effects caused by exposures from a

mobile or any other wireless communication devices employ a Specific Absorption Rate (SAR)

value to define and quantify the applied exposures. SAR is a unit of measurement which

indicates the energy absorption rate on a body (living organism) exposed by the RF field

emitted from mobile phones (Commission 2017) and is represented by the following

relationship:

Where 𝜎 is the conductivity of the tissue (𝑠/𝑐𝑚);

𝐸2 is the induced electric field strength (𝑉2/𝑐𝑚2);

𝜌 is the tissue density (𝑔/𝑐𝑚3);

𝑐 represents the specific heat capacity of the tissue (𝐽/𝑔/℃);

∆𝑇 is the change in temperature with respect to time (𝑊/𝑘𝑔), and

∆𝑡 represents the change in time (𝑠).

To achieve an accurate simulation of the RF field true to its physical form generated

experimentally and evaluate how the actual electromagnetic field (EMF) emitted from a mobile

device influences the bioactivity of ion channel protein in exposed cell culture, understanding

of the RF field’s properties or characteristics are essential. SAR is prominently used in most

studies investigating the biological effects of mobile phones and other RF devices, therefore

the factors that impact SAR in evaluating generated RF fields will be discussed in this chapter.

4.2 Computer Simulation Technology Microwave Studio

Computer Simulation Technology (CST) Microwave Studio (MWS) is a 3D electromagnetic

simulation software. CST MWS is one component of the CST STUDIO SUITE™ package,

which includes CST DESIGN ENVIRONMENT™, CST DESIGN STUDIO™, CST EM

STUDIO™, and CST PARTICLE STUDIO™. CST MWS presents the culmination of many

years of research and development into the most efficient and accurate computational solutions

to 3D electromagnetic designs. CST MWS specializes in providing rapid and accurate 3D

electromagnetic modelling of high-frequency problems (RF and MW fields). The product offers

users shorter development cycles by virtual prototyping before physical trials and optimization

instead of experimentation. CST MWS provides a link between MATLAB® and CST MWS's

VBA macro language. This interface allows CST MWS users to take advantage of the data

manipulation, signal processing, and graphics capabilities provided in MATLAB. COM and

ActiveX interfaces allow behind the scenes data transfer and tight integration between the two

programs.

The highlights of the software module include:

(i) Complete technology for 3D EM simulation

(ii) Time-domain solver for performance and efficiency in complex structures

(iii) Frequency domain solvers for highly resonant or periodic structures

(iv) Various CAD file imports

(v) Easy-to-use interface, and

(vi) Distributed networking support

4.3 Factors Affecting the Field Strength (Power)

4.3.1 Antenna parameters

For different brands and models of mobile phones, antennas within the phone chassis differ in

terms of types, size, location, quantity and frequency range due to design constraints. To

compare SAR between different types of antennas, the study's authors (Zhao, Zhang et al.,

2013) have analysed the SAR difference between a half-wavelength dipole, quarter-wavelength

monopole, whip and planar inverted D antenna (PIFA). Both whip and PIFA antennas are

extensively used in actual mobile phones, while the half-wavelength dipole and quarter-

wavelength monopole are widely referred to in literature. In both vertical and tiled positions,

the phone model with PIFA mounted at the sides of the phone chassis produces the highest

SAR as shown in the conducted measurements (Bernardi, Cavagnaro et al. 2000). Similarly,

the position, where the antenna is placed in the phone chassis, would affect the resultant EMF

as well. Another study (Zhao, Zhang et al. 2013) has shown that SAR values vary according to

the phone chassis length, feeding port position and antenna height with respect to a simulated

head model. When dual elements within the phone work simultaneously, the peak SAR position

would differ with respect to the chassis length under the following relationship (Zhao, Zhang

et al. 2013):

𝑆𝑃𝐿𝑆𝑅 = (𝑆𝐴𝑅1 + 𝑆𝐴𝑅2)/𝐷

Since the electrical characteristic of an antenna depends greatly on the ground plane, which it

is mounted on, the phone chassis that usually consists of the RF shield and printed circuitry will

influence the performance of the antenna and its SAR substantially. A study (Kivekas,

Ollikainen et al. 2004) concluded that the parameters of the phone chassis, i.e. length, height,

thickness and distance between head to the phone would influence the bandwidth, efficiency

and SAR characteristics of the device. Therefore, for accurate simulation of the RF field, both

the phone antenna and chassis should be considered in combination.

4.3.2 Position of the RF field source

Between the source of electromagnetic energy and a point of interest (in our case it is a well

plate with cells), there exist three possible regions: reactive near-field, radiating near-field

(Fresnel) and far-field (Fraunhofer). The reactive near-field region is the region closest to the

antenna form within a radius of:

where D is defined as the largest dimension of the antenna. In this region, the electric and

magnetic field is 90 degrees out of phase to one another. Next, the Fresnel region is sandwiched

between the reactive near-field and far-field region and forms within

In this region, the radiating field starts to emerge. And lastly, in the far-field region at the

outermost region, is defined by:

This region is dominated by the radiating field and the EMF (RF field) strength reduces as a

function of 1/R and its power density reduces as 1/R2 (Balanis). A study (Hirata 2005) has

concluded that SAR measurements are highly dependent on the region the EMF is measured

at. The authors used different radiofrequency from both near and far-field exposures and a

significant difference between the measured SAR was observed. The decrease in SAR with

regards to the increasing distance from the source is much more rapid in the near-field region

compared to the far-field region (Hirata 2005). Thereby, supporting the importance of

identifying these regions as the generated EMF is off different characteristics and, thus can

produce different effects on an object underexposure. Thus, the distance between the antenna

and the cell culture wells in the experimental set-up will play a big part in determining the

4.3.3 Permittivity, Permeability and Conductivity of material

actual strength of the RF field acting on the cells.

In the vast majority of studies investigating possible biological effects caused by low-level

radiation from mobile phones, the permittivity, permeability and conductivity of the instrument

and material used were neglected or regarded as negligible. While cell culture well plates and

dishes used in the lab are typically made from non-conducting plastics or glass, they still hold

the ability to store energy in the electric and magnetic field much like a polymer in a capacitor

(C. Furse, 2009). Most Petri dishes and well plates are made of polystyrene and plexiglass with

relative permittivity around 2.5 which can be compounded depending on the structure of the

dish or plate. Theoretically, a 96-well plate would be able to store much more energy compared

to a 24-well plate due to its dense structure. In addition, the buffer solution used to culture cells

for in vitro experiments would also influence the measured EMF. Buffers are used in

experiments for maintaining the desired pH level required for living cells to thrive (survive and

grow) (Eagle, 1971). Cell solutions with higher metal and water content tissues are found to

contain more free charges than insulating material due to higher electrons and ions present in

the solution. The movement of these free charges results in different levels of conductivity

which in turn affects the measured EMF within the solution (C. Furse, 2009).

As the permittivity, permeability and conductivity of instruments and materials used in in vitro

setup could influence EMF results at the specific point of interest in the simulation, extra care

4.3.4 Correlation and comparison of RF field simulation with its physical

measurement

is required to ensure these factors are taken into consideration.

For accuracy of the simulations of the RF fields and their comparison with the measurement

results from the actual experiment (radiofrequencies used are 870, 1850, 3400, 5400 MHz), our

simulation protocol is designed based on the study reported in (Ma, Yin et al. 2010). This study

is selected due to the similarity of the mobile phone antenna used in (Ma, Yin et al., 2010) and

our experimental study.

While most parameters and dimensions are stated in the paper, some key parameters were not

and an empirical method was used to determine these missing parameters which result in slight

differences between our findings and results reported in (Ma, Yin et al. 2010). The following

figures show the initial calculation for the linefeed and waveguide port extension coefficient

used in the report.

Figure 4.1 50-ohm Microstrip Linefeed Calculation (Pasternack)

Figure 4.2. Waveguide Port Extension Coefficient Calculation in CST

In the reference paper (Ma, Yin et al. 2010), a microstrip mobile antenna is simulated in CST

STUDIO SUITE and its antenna’s return loss, SAR, electric field, E, and magnetic field, H,

plane radiation patterns were shown. From Figures 4.3 to 4.8, we can compare the results

presented in the reference paper with the simulated results obtained for our case study.

As mentioned earlier, the reference study (Ma, Yin et al., 2010) is selected due to the similarity

of the mobile phone antenna used in Ma, Yin et al., 2010 and our experimental study. Minor

differences can be observed between the results from the reference paper and our simulation

results. Fig 4.3 presents the results from our simulation for Return loss, S11, the parameter for

the microstrip antenna used in the experimental study with mobile phone headset Huawei Mate

9 (experiments reported in Chapter 3) and in the paper by Ma, Yin et al., 2010. As can be seen

from Fig. 4.3, the Return loss parameter shows the same pattern and almost identical values

(within a small calculation error). We also simulated the electric field, E, and magnetic field,

H, pattern at the selected frequencies of 870 MHz (Fig. 4.4), 1850 MHz (Fig. 4.5), 3400 MHz

(Fig. 4.6), 5400 MHz (Fig. 4.7) and compared our simulation results with the reference paper

by Ma, Yin et al., 2010. As evident, our obtained simulation results closely match with the

results reported in the reference paper. Further, we also simulated the SAR (as shown in Fig.

4.8), at the selected frequencies and achieved the SAR values very similar to the values reported

by Ma, Yin et al., 2010. In summary, there are only minor differences for all microstrip antenna

parameters between our simulated results and the values reported in the reference paper. These

differences could be caused by the factors such as the mesh size used in the simulation, antenna

parameters used and the difference in a head model used for determining SAR values, etc.

Figure 4.3 Return Loss Result Comparison

Figure 4.4 RF Radiation Pattern Result Comparison @ 870 MHz

Figure 4.5 RF Radiation Pattern Result Comparison @ 1850 MHz

Figure 4.6 RF Radiation Pattern Result Comparison @ 3400 MHz

Figure 4.7 RF Radiation Pattern Result Comparison @ 5400 MHz

Figure 4.8 SAR (1g) Results Comparison

Overall, a good agreement between the results of our simulation and the reference paper is

observed. Taking into account the simulation parameters used, a realistic reflection of the actual

experiment can be perceived from the subsequent simulations. However, minor tolerance for

4.3.5 Identifying Experimental Field Parameters

error should be realistically expected due to the nature of the simulation software.

Following the successful correlation of the results above, parameters pertaining to the actual in

vitro experiment are sourced and measured and presented in this section. Huawei Mate 9 phone

connected to Vodafone as the service provider was utilized in the experiment aiming to

determine the effects of RF radiation emitted by mobile phones on endothelial cells. In

particular, we aimed to evaluate changes if any in calcium production level in ion channel

proteins expressed in epithelial cells. According to the Vodafone Coverage Checker, 4G

network availability was present at RMIT University Bundoora West Campus, the location of

the experiment (Vodafone). Vodafone 4G network operates at 2100, 1800 and 850 MHz

frequencies, while its 3G network operates at 2100 MHz for the metropolitan areas and 900

MHz for the regional areas (Vodafone).

Figure 4.9 Vodafone Coverage at RMIT Bundoora West Campus [(Vodafone)]

Figure 4.10 Nearest Cell Tower from RMIT Bundoora West Campus (RFNSA)

As the 4G operation frequency on Vodafone is not fixed and varies from location to location,

depending on the nearest cell tower, the phone operating frequency has to be measured

physically to determine the exact frequency used in our experimental work. Since a field

strength (power) is highly dependent on the field region, the frequency that determines the

radius of these regions have to be identified.

Hence, to identify the operating frequency of the Huawei Mate 9 mobile device, a wideband

antenna connected to a Frequency Analyzer was set up in the RMIT Bundoora West Campus

(as shown in Fig. 4.11).

Figure 4.11 Setup with the Wideband Antenna (left) and Huawei Mate 9 phone (right)

Measurements for 3 stages of the mobile operators were recorded during the monitoring. The

3 stages of mobile operation are idle, initializing and in-call. These three stages present

different frequency and power outputs which will be discussed further below.

Figure 4.12 Idle Stage Frequency Measurement

In the idle stage, shown in Figure 4.12, the noise floor is observed below -65 dBm on average

with a couple of noises located at 63, 780 and 958 MHz. These frequency signals are generally

observed below -60 dBm, which could be generated by the electronic equipment found in the

lab.

Figure 4.13 Initialising Stage Frequency Measurement

In the initializing stage, the handshaking process takes place between the mobile device and

the cell tower. This process establishes the transmission frequency for the call by determining

the highest signal strength of the nearby cell tower (G. Miao, 2016). The whole initializing

process only constitutes less than a second. In our measurement, RF signals at 845 MHz, 1.757

GHz and 2.421 GHz are observed as shown in Figure 4.13. The range of these three signals

correlates well with the Vodafone 4G network frequency, which the mobile phone used in the

measurement was operating on. The variation observed between the network frequency

declared by Vodafone and the measurements could be due to the antennas dimension and

modulation used in the cell tower.

When the calls are connected, only one frequency signal would be maintained as the

transmission frequency (G. Miao, 2016). In our measurement, the signal at 840-845 MHz was

established as the transmission frequency after the handshaking process. The signal strength

was observed at -15 dBm approximately as shown in Figure 4.14.

The power of the irradiation emitted from the mobile phone was also measured by placing an

antenna in close proximity to the phone chassis. A reading of -5.46 dBm was observed as shown

in Figure 4.15 below. With the measured radiated power level, the input current to the antenna

can be identified using the CST software as 0.003A approximately.

Figure 4.14 In-Call Stage Frequency Measurement

Figure 4.15 Huawei Mate 9 Radiated Power Level

Unfortunately, due to a patent’s rights, the exact antenna dimension used in Huawei Mate 9

was not revealed to the public. Nevertheless, the dimension can be easily calculated from its

operating frequency due to its simple monopole structure. Figure 4.16 shown below, illustrates

the calculated dimension and its simulated operating frequency.

Figure 4.16 Calculated Antenna Dimension (Left) and S11 (Right)

The conductivity of the buffer solution used to maintain the pH level of the cell culture was

also measured using a conductivity meter as shown in Figure 4.17. Hank’s Balanced Salt

Solution was used as the buffer solution in the experiment. An average of 12.52 mS/cm was

observed at 21.3°C. This value coincides with the conductivity of blood which ranges between

10 to 20 mS/cm (Hirsch, Texter et al. 1950). In the experiment, the buffer solution occupies

1.5 mm of the height of the well while the cell culture has a thickness of 50 µm approximately.

Figure 4.17 Conductivity measurement of buffer solution

Corning Costar TC-Treated 24-well plate was used in the experiments to hold the cell cultures

and its buffer solution. This variety of 24-well plates is made of polystyrene polymer with a

relative permittivity ranging between 2.5 to 2.59. Its electric conductivity is extremely small at

the range of 10e-15 to 10e-19. The table below shows other parameters of the 24-well plate.

Table 4.1 Parameters of Corning 24-well Plate (Balanis)

Parameters of Corning 24-well plate Values

Relative Permittivity 2.5 to 2.59

Electric Conductivity 10e-15 to 10e-19

Specific heat capacity 1.17

Thermal Conductivity 0.13

Mass Density 1029 to 1071

Loss tangent @ 100MHz 0.0001

Loss tangent @ 3GHz 0.000333

4.4 Simulation of generated RF fields using identified parameters

Once the research paper is co-related with the simulation, the final rounds of simulation

including the identified parameters used in the experimental setup can be inserted to determine

the field strength acting on the cell culture. The following section shows the set-up positions

and results of the final simulations.

Figure 4.18 Dimensions of a 24-well Plate and Huawei Mate 9 used in experiments

The following values indicated in the power loss density and energy density model are obtained

at specific points on the well plate. The points are specified in the middle of the buffer height

at 0.75 mm from the base of the well. Each of the points in each individual well is indicated by

their row number and length on the horizontal line as shown in Figure 4.19.

Figure 4.19 Simulated Point of interest

4.5 Different positions of a mobile phone device (irradiation source)

4.5.1 Simulation of the RF radiation at Position 1

In the experiment, the mobile phone is placed at different positions around a 24-well-plate. At

its first position (Position 1), the Huawei Mate 9 phone is placed 4 cm away from the side of

the 24-well-plate as shown in Figure 4.20. Taking into account the operating frequency of the

phone (845 MHz), it can be calculated that the first column of the 24-well plate, nearest to the

mobile device, works within the nearfield region (< λ/2π), while the rest of the well plate is in

the far-field region.

Since part of the first column of the well plate, nearest to the mobile phone is located in the

near field region, the electric and magnetic field measured in this column would experience a

100

spike

in the field strength (power) before the far-field region. This is observed in the electric field

intensity plot shown in Figure 4.21. Values shown for each well represent the calculated electric

field intensity. These values vary because of the location of each well relative to the position

of the antenna of the mobile phone. Of particular interest, the raw two of the well-plated where

the cell culture was placed.

Figure 4.20 Experimental set up - Position 1 of exposure

Both power density loss (calculated for each well) and energy density plot (calculated for each

well) show a linear decline as the distance increases, shown in Figures 4.22 and 4.23,

respectively. As can be observed from these figures, the power density level values drop to near

“zero” at the gaps between the wells as polystyrene polymer (plastic) is almost non-conductive

and is not able to release power easily. Energy density values, which is reliant on permittivity,

101

do not drop to “zero” at the gaps as polystyrene have a relative permittivity

between 2.5 and 2.59 that allows the well plate to store some amount of energy (C. Furse,

2009).

Figure 4.21 Simulated Electric Field Strength/Power (V/m) for the phone

in Position 1

102

Figure 4.22 Simulated Power Density Loss in Position 1

Figure 4.23 Simulated Energy Density in Position 1

The pattern of the generated electric field, E, and magnetic field, H, at the frequency 845 MHz are shown in Fig. 4.24. As can be seen, the mobile phone headset generates a homogenous uniform field.

103

Figure 4.24 Simulated RF Radiation Pattern at Position 1

4.5.2 Simulation of the RF radiation at Position 2

In position 2, the mobile phone is placed directly above the 24-well plate with a gap of 20.27

mm in between the chassis of the phone and the top of the well plate as shown in Figure 4.25.

Since the RF field is being irradiated from above, the electric field strength/power is observed

at its peak in the centre of the well plate as the antenna is omnidirectional (Balanis). Power

density loss and energy density are observed to peak at the centre as well, as shown in Fig. 4.27

and 4.28.

Figure 4.25 Experimental set up - Position 2 of exposure

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Figure 4.26 Simulated Electric Field Intensity in Position 2

Values shown for each well (Fig. 4.26) represent the calculated electric field intensity. These

values vary because of the location of each well relative to the position of the antenna of the

mobile phone. Of particular interest, the raw two of the well-plated where the cell culture was

placed.

Figure 4.27 Simulated Power Density Loss in Position 2

Figure 4.28 Simulated Energy Density in Position 2

Both power density loss (calculated for each well) and energy density plot (calculated for each

105

well) values are shown in Fig. 4.27 and Fig. 4.28, respectively.

Figure 4.29 Simulated Radiation Pattern at Position 2

The pattern of the generated electric field, E, and magnetic field, H, at the frequency 845 MHz

are shown in Fig. 4.29. As can be seen, the mobile phone headset generates a homogenous

4.5.3 Simulation of the RF radiation at Position 3

uniform field.

In position 3, the two phones are placed exactly 40mm away from both sides of the 24-well plate

as shown in Figure 4.30. It is observed that the simulated results are similar in nature to the

results obtained for the mobile phone in Position 1 of exposure since in both positions, the

106

mobile phones are placed at a fixed distance on the side of the well plate.

Figure 4.30 Experimental set up - Position 3 of exposure

Figure 4.31 Simulated Electric Field Intensity in Position 3

Values shown for each well (Fig. 4.31) represent the calculated electric field intensity. These

values vary because of the location of each well relative to the position of the antenna of the

mobile phone (1 and 2, respectively). Of particular interest, the raw two of the well-plated

where the cell culture was placed. As can be seen, the electric field intensity values are different

(0.325 and 0.172 for each well with the cell culture, as shown above) because of their location

107

to the phones generating the RF field.

Both power density loss (calculated for each well) and energy density plot (calculated for each well)

values are shown in Fig. 4.32 and Fig. 4.33, respectively.

Figure 4.32 Simulated Power Density Loss in Position 3

108

Figure 4.33 Simulated Energy Density in Position 3

Figure 4.34 Simulated Radiation Pattern at Position 3

The pattern of the generated electric field, E, and magnetic field, H, at the frequency 845 MHz are shown in Fig. 4.34 above. The 3D radiation pattern is shown in Fig. 4.35

109

Figure 4.35 3D Radiation Pattern of the Positions 1, 2, and 3

From the electric field intensity plots shown in Figure 4.36, it can be observed that the field

strength/power is the strongest when the phone is placed directly above the 24-well plate

as shown in Position 2. This is because the distance between the phone’s antenna and the well

plate in Position 2 is much closer compared to the other two positions. As the distance between

antennas increases, a field strength/power will be reduced due to a path loss (Erceg, Greenstein

et al. 1999). The peak in the centre in Position 2 is due to the omnidirectional nature of the

antenna which 3D radiation pattern can be observed in Figure 4.35. Electric field

strength/power in Positions 1 and 3 are similar in nature, except at a higher field strength on the

leftmost side of Position 3. This is due to an additional mobile phone being placed on the left

for Position 3, which increases the magnitude of the field strength to the leftmost side of the

well plate.

According to the Guass Law, the electric field inside a perfect conductor is “zero” (C. Furse

2009). Since the buffer solution is much more conductive compared to the polystyrene well

plate, a sharp fall of the field strength/power can be observed at the location of the wells filled

110

with the buffer solution as can be seen from Figure 4.36.

Figure 4.36 Comparison of the electric field strength between three positions of the mobile

exposure device

While the power density loss plot, shown in Figure 4.37, has a similar relation to the electric

field intensity plot, some slight differences are observed. The power density shown at the

leftmost side of the well plate has a higher density when the phone is placed in Position 1

compared to Position 3. A higher power density loss can also be observed at the leftmost of the

well plate. This could be due to the destructive interference (Balanis) attributed to the two

111

phones electromagnetic waves in Position 3.

Figure 4.37 Comparison of the power density loss between three positions of the mobile

exposure device

Similarly to the previous plots, the energy density (Fig. 4.38), shown when the phone is in

position 2, exhibits the highest magnitude compared to the other positions 1 and 3. This is

because of the higher amount of energy radiated into the well plate due to its closer proximity

to the antenna in position 2. Energy density observed when the phones are placed in positions

1 and 3 are close to zero as more energy disperses as distance increases. Therefore, only a small

amount of energy is stored in wells furthers from the phone. In addition, a large spike in energy

density can be observed at the boundary between the buffer solution and well plate in position

2. This observation is mainly due to the electromagnetic surface traction between the materials

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as the energy transfer from one material to another (Costen and Adamson 1965).

Figure 4.38 Comparison of energy density plots between three positions of the mobile

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exposure device

4.6 Summary

To validate the RF exposure patterns and ensure that the electromagnetic simulation stays true

to the actual in vitro experiment conducted on endothelial cells, both the phone chassis and

antenna have to be considered in combination due to the effect of the ground plane on the

antenna performance. The permittivity of the instrument used in the experimental setup should

also be accounted for in the simulation. The reason for this is because the permittivity of a

material defines its ability to store electric field within itself, which influences the energy

density that could affect cell activity in the experiment. In addition, since the electric field

strength/power has an inversely proportional relation to the conductivity of a material, the

conductivity of the buffer used to maintain the pH level of the cells should be accounted in the

simulation too, since the intensity level of the electric field will influence the SAR value, crucial

parameter affecting field exposures on cell cultures.

Lastly, as the characteristics of the RF field differs in different field regions, the distance

between the antenna and the measurement point (cell cultures in well plate) should be taken

into account with respect to these different regions. This distance between the antenna and

measurement points would also affect the power density. As the distance increases, the power

density will be reduced due to the path loss, meaning that a lesser dose of irradiation will be

given to cells.

With regards to the simulated results pertaining to the in vitro experiments conducted with

endothelial cells, it has been observed that the mobile phone positioned in three different

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positions generates different results for the electric field intensity, power density and energy

density. In position 1, where the phone is placed 40 mm to the right of the 24-well plate, the

electric field observed at the rightmost wells, nearest to the phone, experiences a peak in the

field magnitude. This peak is observed as a portion of the rightmost wells coincides within the

near-field region. The power and energy density plot generated when the phone is placed in

Position 1, shows a linear decline as the distance increases due to the path loss and the field

absorption by the preceding wells and buffer solution.

In position 2, where the phone is placed directly above the 24-well plate, peak values for the

electric field intensity, power and energy density are observed to be centralized at the middle

of the well plate and diminishes as it approaches the side of the well plate. This observation is

likely due to the monopole structure of the phone antenna used. Monopole antenna is

omnidirectional with a doughnut-shaped radiation pattern being produced that has a higher

power radiated at its center compared to its sides. In addition, due to the closer proximity to the

well plate, a higher level of magnitude in regards to the electric field strength, power and energy

densities can also be observed in Position 2 of exposure. Energy density spikes at the boundary

between the buffer solution and the wells are also observed in Position 2 due to the increase in

the electromagnetic surface traction between the materials caused by the increase in power

radiated from the mobile phone’s antenna.

In position 3, where two phones are placed 40mm away from the opposite side of the 24-well

plate, the results of our simulations are similar in nature to results obtained for Position 1 of

exposure. Comparing the results obtained for radiation at Position 1 to 3, the destructive

interference attributed by the two phones electromagnetic waves can be observed. At the

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leftmost of Position 3’s power density plot, the peak power density can be observed which

reduces in the middle of the well and increases again as it reaches the rightmost side of the well

plate. This effect is caused by the field cancellation as the field, generated by the two phones

placed at the opposite ends of the well plate, cancel each other and produce the displacement

as shown in the power density plot.

For the future outlook of this project, methods to measure field strength in the micro-scale can

be investigated. Moreover, a thin sensitive optical field probe can be procured to verify the

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results of the experiment further and ensure the accuracy and precision of the simulations.

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CHAPTER 5 CONCLUSIONS AND FUTURE WORK

In recent years, exposure to RF radiation has dramatically increased due to advancements and

penetration of communication technology, medical and food-processing technology, and other

industrial applications. Biological and possible health effects of RF radiation as well as its non-

thermal effects have received considerable attention from media and the public, and remain the

subject of intense debate in the scientific community. Non-thermal effects of RF radiation have

been postulated to result from a direct interaction of the electric field with specific (polar)

molecules in the reaction medium that is not related to a macroscopic temperature effect. These

effects depend on several physical parameters and biological variables. Therefore, only results

obtained under the same experimental conditions of RF exposures should be compared in

“replication” studies.

Essential features of non-thermal RF radiation effects include:

(i) effects of resonance type within specific frequency windows;

(ii) dependence on the type of signal, modulation, and polarization;

(iii) decreasing power density (PD) by orders of magnitude can be compensated by an

increase in exposure time.

Therefore, duration of exposure may have a more significant role as compared to Power

Density (PD); (iv) cell density - radical scavengers/antioxidants have a potential to abolish RF

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radiation effects, and (v) genomic differences influence responses to RF exposures.

This Masters by Research project was aimed at investigating the effects of low-power RF

radiation on the selected mechanosensitive ion channel proteins expressed in two different cell

types, with a specific focus on the frequencies emitted by mobile phones. The frequencies

selected for this investigation are used in 3G (945 MHz) and 4G (1800 MHz) mobile networks.

The experimental in vitro and simulation investigations were conducted.

The following studies were completed within the Masters by Research project:

1. Investigating the effects of low-level radiofrequency radiation on activation of

mechanosensitive ion channel Piezo-1 and TRPV4 (Chapter 3)

This sub-study examined the effects of short-term and long-term low-level RF radiation on the

mechanosensitive ion channel Piezo-1, focusing on the frequencies emitted via mobile phones

(3G and 4G mobile networks). The obtained results allowed us to draw the following

conclusions.

 Findings show that short-term (10 min) RF exposures emitted by mobile phones (945

MHz) lead to short-term activation of the mechanosensitive ion channel Piezo-1, thus

leading to an increase in intracellular Ca2+ in both HEK293-Piezo-1 and THP-1 cells.

 The results show that cellular responses to RF radiation from mobile phones depend on

the distance between the mobile phone handset and the cell culture plate. I found that

the response of HEK293-Piezo-1 to mobile phone radiation is dependent on the

expression of Piezo-1 as the response was absent in parental HEK293 cells.

 I investigated the effects of long-term exposures (2 and 4 hrs) to low-level RF radiation.

I showed that exposure of THP-1 monocytic cells to the RF field generated by the TEM

cell (1800 MHz) does not change the expression of Piezo-1 and TRPV4 channels and

not inducing changes in the expression of inflammatory cytokines and chemokines in

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THP-1 cells.

 I showed that low-level RF radiation desensitizes HEK293-Piezo-1 and THP-1 cells'

response to Yoda-1 and this effect is dependent on the distance between the mobile

phone device and the cell culture plate.

2. Simulating RF field exposures emitted by mobile phone headset using CST Microwave

Studio (Chapter 4)