1. INTRODUCTION
1.1 The electromagnetic spectrum
The electromagnetic (EM) spectrum is the range of all possible electromagnetic
radiations. Conventionally the EM is divided into several ranges according to the
peculiar wavelengths, as shown in Figure 1.1.1.
Figure 1.1.1 The electromagnetic spectrum. RF and MW were highlighted, in particular mobile phone
frequency
The electromagnetic fields are generally classified in ionizing and non-ionizing
radiations (cfr. Figure 1.1.1). An ionizing radiation has energy enough to remove bound
electrons from the external shell of an atom, effectively causing the atom ionization.
Conversely a non-ionizing radiation has not this ability. Until recently it has been
thought that non-ionizing radiations had only a thermal effect on living being; however,
in the last few years several researches have disproven this belief, as it will be explained
in the next sections. In this work we show how non-ionizing radiation, in the
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microwaves range (0.9-2 GHz), determine kinetic and structural modifications in
biological macromolecules.
1.2 The effects induced by microwaves on biological systems
The enormous increase in the use of mobile phones has prompted a corresponding
increase in the study of its effect on man’s health. The mechanisms of interaction have
been described as thermal effects, with rise in body/tissue temperature of more than 1°C,
non thermal effects, with no evident increase in body temperature and microthermal
with thermoelastic expansion in the brain giving rise to microwave effect on hearing,
have been also investigated. The microwaves produce thermal effects on biological
systems at high power levels. Some of the thermal effects reported include cataract
formation, foetal abnormalities, decreased thyroid function (through
hypothalamichypophyseal- thyroid axis inhibition), suppression of behavioural
responses, gonadal disfunction and natural killer cell activity, increase in the number of
complement receptor positive cells and increase phagocytic activity of peritoneal
macrophages [1-2]. At non thermal levels (<0.5 C° rise in rectal temperature)
stimulation of thyroid, increased susceptibility of the organism to bacterial infections,
decline in neutrophil and complement activity, increased lymphoblastoid transformation
of lymphocytes, abnormalities in the erythrocyte/lymphocyte precursors in bone marrow
are some of the reported effects [3-4].
However, it is important to distinguish between acute and chronic effects. The first are
due to continual but short-time exposure, the second are due to non-continual but time-
repeated exposure. It has been demonstrated that exposure of rats for 45 min to 2450-
MHz RFR [5-6] causes an increase in the endogenous opioid activity and in the
concentration of benzodiazepine receptors in cerebral cortex, without significant effect
in the hippocampus and cerebellum. A decrease in high affinity cholinergic uptake in
frontal cortex and hippocampus has been also observed [7]. Further study [8] showed
the response depends on the duration of exposure. A shorter exposure time (20 min)
actually increased, rather than decreased the activity. They stated that different brain
areas have different sensitivities to RFR with respect to cholinergic responses. In
addition, repeated exposure could lead to some rather long lasting changes in the
system, since the number of acetylcholine receptors increases or decreases after
repeated exposure to RFR for 45 min or 20 min sessions respectively. Increase in single
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and double strand breaks in DNA has also been reported [9-10]. Changes in blood brain
barrier, neurotransmitter functions, cellular metabolism, calcium efflux, altered
sensitivity to microwaves upon treatment with certain drugs [11] and a more common
arise of Alzheimer’s disease in electrical workers [12], are some of the effects.
Furthermore experiments were conducted [13] to elucidate the effects of chronic low
power-level microwave irradiation on the immunological systems of rabbits. Fourteen
male Belgian white rabbits were exposed to microwave radiation at 5 mW/cm
2
, 2.1
GHz, 3 h daily, 6 days/wk for 3 months in two batches of 7 each (exposed and control
group) in specially designed miniature anechoic chambers. Significant suppression of T
lymphocyte numbers was noted in the exposed group at 2 months. From these results it
was speculated that the T lymphocytes are sequestered to various lymphoid organs
under the influence of microwaves. However, as we will explain in section 1.4, the
evaluation of risk due to chronic exposure is very difficult given the enormous
complexity of biological systems.
Besides, some acute effects observed within “frequency windows” are known, as
those shown in repairing of radiation-induced DNA breaks in E. coli K12 AB1157 [14].
These effects display a pronounced resonance character. In particular the power density
(PD, mWatt/cm
2
) absorbed by the system rapidly decreases with little changes of
irradiating frequency.
1.3 The risk evaluation and the law limits
The method generally used to quantify the effect of electromagnetic field on biological
system is the Specific Absorption Rate (S.A.R.). SAR is defined as amount of
electromagnetic energy absorbed by the mass unity in function of the induced electric
field. The rigorous definition of the S.A.R is obtained as follows:
dW
ρdV W
SAR ;
tK
⎛⎞
∂
⎜⎟
g
⎡ ⎤
⎝⎠
=
⎢ ⎥
∂
⎣ ⎦
(1)
where dW is the energy increment and ρdV is the mass element contained in a volume
V with energy density ρ. However it is difficult to actually use this expression to
calculate the S.A.R., but it is quite simple to obtain another equation easier to apply:
2
σ E
W
SAR= ;
ρ Kg
⎡ ⎤
⎢ ⎥
⎣ ⎦
(2)
3
where σ (Ohm
-1
cm
-1
) is the medium conductivity, ρ (mass/volume) is the medium
density and E (Volt/m) is the emitted electric field. This parameter is experimentally
obtained by the technique called microwaves dosimetry on model systems.
Most often, the current safety standards are based on the thermal effects of MWs
obtained in short-term (acute) exposure. In some countries, such as Russia, the NT MW
effects, especially those induced during prolonged (chronic) exposures, are accepted and
taken into account for establishment of the national safety standards [15-17]. It should
be stressed that, in contrast to the ICNIRP (International Commission for Non-Ionizing
Radiation Protection) safety standards [18], which are based on the acute thermal effects
of MWs, the standards adopted by the Russian National Committee on Non-Ionizing
Radiation Protection (RNCNIRP) are based on the experimental data from chronic (up
to 4 month) exposures of animals to MWs at various physical parameters including
intensity, frequency and modulation. Nowadays, most part of population is chronically
exposed to MW signals from various sources including mobile phones and base
stations. These exposures are characterized by low intensities, varieties of signals, and
long-term durations of exposure comparable with a life span. So far, the “dose”
(accumulated absorbed energy which is measured in radiobiology, as the dose rate
multiplied by the exposure time) is not adopted for the MW exposures and SAR is
usually used for the guidelines. Actually, it is not known up to what degree SAR/PD can
be applied to the nowadays NT MW chronic exposures and the current state of research
demands re-evaluation of the safety standards [19]. Thus, the safety standards
significantly vary, up to 1000 times, between countries as a consequence of such
various treatments of the experimental data.
1.4 The complexity of biological systems: the need to work at a very basic molecular
level
A biological system can be defined as a precise assembly of different chemical
components; each of these molecules has got a specific and peculiar structure and
function to ensure the growth, regulation and reproduction of the whole system.
Therefore, it is clear it is very difficult to estimate SAR in a real biological system. The
main reason is that, in a biological system (i.e. cells, tissues, organs and animals), σ and
ρ are not constant thus making it impossible to quantify exactly SAR applying equation
(2). Furthermore this problem becomes more complicated if we consider that, when a
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biological system is under stress, it carries out hardly measurable reparation
mechanisms. Recently, it has been proven that MWs irradiation can affect the kinetic of
the folding process of some globular proteins, such as myoglobin [20] and that it
induces changes in fluorescence of Green Fluorescent Protein [21]. Furthermore, in our
laboratory it has been shown as MWs significantly alter kinetic and structure of
acetylcholinesterase, an important enzyme of the brain [22]. Supported by these
experimental evidences, we supposed that biological damages, caused by exposure to
MWs, could start at “ molecular level” involving progressively higher levels of the
biological assembling. We hypothesized that conformational and/or kinetic alteration in
proteins and enzymes, can be propagated, for instance modifying biochemical reactive
pathways or membranes activity. We also supposed that the irreproducibility of the
same “in vivo” experiments can be attributed to the enormous complexity of biological
system. On the contrary, limiting experiments to homogeneous water solution of highly
purified proteins we obtained reproducible results. It is worth to stress out that in a
homogeneous solution σ and ρ are known and constant, so there is a direct relationship
between the SAR and the electric field emitted.
1.5 The extremely variability of a mobile phone as a radiation source: the need to use a
controlled exposure system
A mobile telephone is a long-range, portable electronic device
used for mobile communication. The radiation emitted by both
mobile phones and the network antennas vary significantly from
provider to provider, and from country to country. However,
communication are established through electromagnetic
microwaves with a cell site base station and the antennas (Figure
1.5.1), which are usually mounted on a tower, pole or building.
Figure 1.5.1 A repeater antenna
There are three major technical standards for the current generation of mobile phones
and networks, and two major standards for the next generation 3G phones and networks.
European, Asian and many African countries have adopted a single system, GSM
(Global System of Mobile communication), which is the only technology available for
over 74% of all subscribers to mobile networks.
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In the United States, Australia, Brazil, India, Japan, and South Korea GSM co-exists
with other internationally adopted standards such as CDMA and TDMA, as well as
national standards such as iDEN in the USA and PDC in Japan. Over the past five years
several dozen mobile operators (carriers)
have abandoned networks on TDMA and
CDMA technologies switching over to
GSM. None has switched away from
GSM. There are two frequency bands
dedicated to GSM mobile phones, one at
900 MHz, and one at 1800 MHz. GSM
uses a combination of frequency division
multiple access (FDMA) and time division
multiple access (TDMA).
Delay (t/T)
BT = 0.20
BT = 0.25
BT = 0.30
Amplitude
Figure 1.5.2 GMSK frequent shaping pulse for
various normalized filter bandwidths BT [24]
That means that within each band there are a hundred or so available carrier frequencies
on 200 kHz spacing (the FDMA bit), and each carrier is broken up into time-slots so as
to support 8 separate conversations (the TDMA bit) [23]. The GSM modulation is
Gaussian minimum shift keying (GMSK) with time bandwidth product of 0.3. The
modulator band pass has a cut-off frequency of 0.3 times [24] (cfr. Figure 1.5.2). In
Table 1.5.1 are reported the frequencies and power used by the common GSM mobile
phone.
FREQUENCY BASE STATION HAND SET PEAK POWER
GSM 900 (935-960) MHz (890-915) MHz 2 Watt
GSM 1800 1805-1880 1710-1785 1 Watt
Table 1.5.1 Frequency and power of GSM [24]
It is evident from these considerations that the mobile phone emission is an extremely
variable radiation source. Therefore it is necessary to use a controlled exposure system
(with frequency and power exactly determined), although it could be probably more
realistic to use a commercial mobile phone. In this work we carried out several
experiments using a controlled exposure system (cfr. section 3.1) but also some
experiments using a commercial mobile phone as a radiation source in order to compare
the effects due to both the radiation sources, as it will be shown in section 4.4.
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2. THESIS AIM
The aim of this study is to establish a direct relationship between mobile phone
emission and biological effects. As it has already been explained, we carried out our
experiments using highly purified macromolecules in order to simplify the problem,
supposing the possible damages can be propagated to more complex systems. In this
manner we realize an “in vitro” model system able to provide data useful for planning
further experiments using a more complex “ex vivo” and” in vivo” systems. Therefore
we chose several macromolecules with peculiar structural and functional characteristics
in order to detect those which are more sensitive to microwaves emission. Among all
the macromolecules of significant biological interest, we analyzed metallo-proteins and
metallo-enzymes which catalyze electron-transfer reactions. First of all, we supposed
that these systems could be sensitive to microwaves irradiation given their magnetic
properties (paramagnetic metal centres and reaction intermediates). Afterwards we
carried out similar experiments on non-electron transfer reactions in order to verify
whether or not a real interference of microwaves radiations with analogous enzymatic
reactions mechanism is possible.
As explained in section 1.5, it has been necessary to use a certified exposure system
operating at frequency and power exactly determined. Nevertheless, we carried out
some experiments comparing the effects due to commercial mobile phone and those
observed by using controlled exposure system.
Finally we wondered whether microwaves radiation emitted by this kind of sources
can produce a instantaneous thermal effects which can originate biological damages.
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8
3. MATERIALS AND METHODS
3.1 Irradiation systems
At the beginning of this work we carried out our experiments irradiating the samples
with commercial mobile phone. Afterwards we carried out similar experiments using a
controlled exposure system as a radiation source (cfr. section 1.5).
3.1.1 Mobile phone
The mobile phone is a low power radio transmitter which works on microwaves range
as it is described in section 1.5. The telephone used in our experiments is a GSM dual
band mobile phone (frequencies: 915 – 1822 MHz), whose emitted electromagnetic
field measurements are shown in Figure 3.1.1.1 as obtained by means of PMM 8053
test instrument [22]. In the calling mode the maximum intensity is about 25 V/m.
Conversely in the receiving mode the maximum intensity is about 9 V/m. In the first 10
s (red line), both the intensities of the emitted electric field increase, since the mobile
phone needs sufficient energy to link to the nearest relay station. Both in the cases, as
soon as, the connection is established the intensity remains constant at about 3 V/m. We
carried out all experiments in the receiving mode by calling the phone used in our
experiments from another remote mobile phone. Figure 3.2.1.2 shows a schematic
representation of the irradiation procedure we used throughout all the experiments.
Figure 3.1.1.1 Electric field emitted from mobile phone used in our experiments [22]
20
15
10
5
0
0 5 10 15 20 25
RECEIVING MODE
CALLING MODE
I
E
(V/m)
t (s)
25
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