FEDERAL STATE BUDGETARY INSTITUTION OF SCIENCE
2 UNDER CONTRACT ON SCIENTIFIC COOPERATION BETWEEN FSBSI PAVLOV INSTITUTE OF PHYSIOLOGY OF THE RUSSIAN ACADEMY OF SCIENCE AND AIRES HUMAN GENOME RESEARCH FOUNDATION “Study of exposure to high frequency electromagnetic radiation and Aires resonators influence on behavior, genetic and epigenetic processes in cells of midline and peripheral organs (Rattus norvegicus and Apis mellifera L. models)”
THE SECOND STAGE (October 2016 – April 2017): study of exposure to electromagnetic
radiation of a router and Aires resonators influence on stability of genetic apparatus in bone
marrow cells of rats, originating from lines with different nervous system excitability levels.
Technical progress, increased power generation, communication systems development involve
wide practical application of electromagnetic radiations of different frequencies, which affect
living organisms. It is known that EMR enhancement, caused by common use of household
appliances, mobile phones and wireless routers, affects genetic apparatus of cells in different
organs of humans and animals. At the moment, accumulated evidence indicates that
electromagnetic fields of different ranges, including mobile phones and tiFi ranges, can induce
a wide spectrum of genetic damages, modify genes expression, affect structural and functional
properties of cell nuclei (Kryukov, 2000; Deynekina, 2002; Boyko et al., 2010, Gapeev, 2006,
Garaj-srhovac et al., 1992, Tice et al., 1999,2002, et al.) tays of EMR influence on genetic
apparatus of cells and organism on the whole are still not quite clear (Gapeev et al., 2000, 2001,
Gapeev, 2006). Individual peculiarities of response to electromagnetic fields exposure, role of
the nervous system’s functional state in mechanisms of responsiveness/ resistance to effect of
such factors are hardly known.
The relevant task is to create systems, protecting from destructive EMR influence, and to study
mechanisms of EMR influence on genetic processes in the cells of midline and peripheral organs
of model objects subject to inherent properties of nervous processes, in particular, nervous
system excitability. At present, Aires Company has created devices, which, based on fractal-
matrix nanotechnologies of EMR transformation, efficiently redistribute EMR and have
therapeutical value (http://www.aires.spb.ru/nano.html, http://airestech.ru). However, no studies
on mechanisms of protective influence of the indicated devices on cells’ chromosome apparatus,
providing for peculiarities of genetically determined nervous system excitation level, are
available.
Purpose and objectives of the second stage:
1) to carry out the second replication of the experiment on assessment of EMR-caused
impairment upon 6 h/day 4 day operation of a standard tiFi router, and protective influence of
3 Aires Defender fractal-matrix resonators on genetic apparatus of cycling bone marrow cells in
male sistar line rats.
2) comparative study of EMR influence on state of genetic apparatus of cycling bone marrow
cells in male rates of sP and NP lines, differing in the level of the nervous system excitation,
upon 6 h/day 4 day operation of a standard tiFi router.
3) comparative assessment of Aires Defender fractal-matrix resonators on state of genetic
apparatus of cycling bone marrow cells upon EMR router damaging action in male rates of sP
and NP lines, differing in the level of the nervous system excitation.
MATERIAL AND METHODS.
Subject if the study was rats of two lines, selected subject to nervous system excitability
threshold – sP and NP (high and low excitability threshold correspondingly) (saido, 2000), and
of initial sistar line (body weight 300 – 350 g). sP and NP line rats were bred in the standard
conditions of the animal facility in the higher nervous activity genetics laboratory of Pavlov
Institute of Physiology of the Russian Academy of Science. The rats had a free access to water
and food. sistar line rats came from the central animal facility of FSBSI Pavlov Institute of
Physiology of the Russian Academy of Science. After arrival the animals spent at least two
weeks in conditions of the laboratory animal facility to ensure adjustment thereof. The males
were kept in groups of six in standard cages with standard diet.
International principles of the Declaration of Helsinki on humane treatment of animals were
observed upon work with animals.
Description of sP and NP line rats.
To study genetically determined connections between functional state of nervous system
(excitability), brain functioning and a wide range of behavioral characteristics, breeding program
was started in the seventies of the last century. The purpose of the program was to breed rat lines
with different levels of nervous system excitability (saido, Sitdikov, 1979). sistar line rats
(Rappolovo) served as a starting material. sistar rats are outbred line of albino rats (Rattus
norvegicus species), they were bred in 1906. Currently, this is a widely spread line, used in
medical and biological studies.
Direct selection was based on threshold of neuromuscular excitability upon electric
current stimulation (rectangular electrical 2 ms pulses) of tibial nerve (n. Tibialis). 4 lines of rats
with different excitability thresholds were bred: sP1, NP1, sP2, NP2 (high and low thresholds,
1 – 2: selection program number) (saido, Sitdikov, 1979). At present, two lines, which show the
most different excitability thresholds, are maintained: sP1(sP) and NP2(NP). The indicated
4 lines have a history of over 70 selection generations. Unidirectional changes of peripheral and
central nervous system divisions excitation (mostly, in subcortical system) are detected in these
rats. Influence of nervous system excitability, and corresponding differences in manifestation of
instinctive reactions and behavior characteristics, emotionality, aggressiveness, altruism,
peculiarities of behavior strategy, ability to develop conditioned reflexes are found. Different
elements of hormonal control systems, transmitters metabolism, ion channels functioning,
structural and functional characteristics of nerve cell membranes change in rats of the indicated
lines. Molecular and genetic studies show divergence of lines by the level of some genes
expression as well. The lines have different stress responsiveness in deprivation of paradoxical
phase, immobilization, short-term and long-term emotional and noxious stress tests. Long-term
post-stress behavioral changes, persisting for 6 months, are demonstrated for the first time upon
exposure of sP and NP rats to long-term emotional and noxious stress sensu K. Geht. Disorders
of higher nervous activity are present in lines with different nervous system excitation level,
however, each line presents specific disorders: sP line with low excitability – development of
depression-like condition, increase of excitability, aggression, disruption of plastic processes; NP
line with high excitability – appearance and preservation of compulsive movements, thus
enabling to use these lines as a model to study post-stress pathology (depressed mood, post-
trauma stress disorder (PTSD), compulsive disorder (CD)). Long-term stress effects are based
on neuron structural changes in different brain structures, differential post-stress modifications of
chromosomes in neurons and cells of peripheral organs, which is shown in characteristics studies
of heterochromatin (condensed chromatin) and different epigenetic modifications of DNA and
histone proteins in neurons (saido et al., 2009). At present, these lines are used to study genetic
and epigenetic mechanisms of adaptive and pathological brain state in extreme conditions, and to
search for nervous system excitability correlates (Dyuzhikova et al., 2015).
In the work presented here a standard tiFi router (LinkSys E1200-EE/RU wireless
router) was used. The specifications thereof are as follows: wireless coupling frequency:
2.4GHz, number and type of antennae: 2 internal antennae, standard antenna(e) gain factor, dBi:
4 dBi.
To study EMR router influence, “home” cage with the animals was placed into the
Faraday cage (Fig. 2), the router was placed under the upper lid of the cage on the removable
shelf, in the center of the cage. Experimental groups were under the router influence during 4
days, 6 hours each day (8 am – 2 pm). Controls included groups of rats, who were placed into the
Faraday cage without the router for the same period of time, and intact animals.
Aires Defender fractal-matrix resonators – transformers (special annular diffraction grids)
were also used in the experiment (whabrev et al., 2005). Interaction of electromagnetic field with
5 Aires Defender results in structural transformation, partial suppression of the former, which does
not hinder distance data exchange between message devices (the stage report of Gedeminas
silnius Technical University).
To assess resonators influence on damaging action of router EMR, 6 resonators-
transformers were used. They were placed in the center of each face of the Faraday cage (Fig. 2).
One of the experimental groups of each tested line was exposed to the router effect in the
Faraday cage with resonators during 4 days, 6 h per day. Moreover, one of the experimental
groups of sistar line was used to assess the effect of resonators as such (without the router
action) on the studied parameters of the bone marrow cells.
Special preliminary measurements of tiFi router radiation energy were made in four
points on the floor of the cage without and with the resonators. Kharchenko antenna was used for
measurements (Fig 1).
Fig. 1. Kharchenko antenna
Fig 2 shows the faraday cage and tiFi router and antenna layout therein.
Faraday cage for studies
2-level shelf for the tiFi router (400x600)
Defender Location areas – centers of all faces
A – front panel
Removable lid
6
Fig. 2. Faraday cage and layout of the tiFi router and Kharchenko antenna therein. Points of the antenna location (1-4) and signal value (ms) at the antenna output without and with (in the brackets) resonators are shown.
The signal, received by the antenna, in layout points (1, 2, 3, 4, Fig. 2) is as follows, Table 1.
Table 1.
Layout point 1 2 3 4
tithout resonators
2ms 20ms 20ms 2.5 ms
tith resonators 1.5ms 15ms 15ms 1.7 ms
Bone marrow cells sample preparation. Upon extraction from femoral bones, 24 hours after the
end of exposure, bone marrow was fixed in the fresh Clarke’s solution (1 part of glacial acetic
acid: 3 parts of ethanol) and stored at +4ᴼWITH. Bone marrow samples were tinted with 2% solution
of aceto-orcein and squash preparations were made according to the standard method (Makarov,
Safronov, 1978; Dev et al., 2009).
Bone marrow samples analysis. Squash preparations of bone marrow cells were analyzed, using
Mikromed-3 microscope at 640-1600x magnification. Chromosome aberrations at the anaphase –
telophase stages (standard ana-telophase method) were recorded subject to additional
recommendations (Daev et al., 2009). Min 200 cells of each animal were analyzed. Record was
made of the number of normal and aberrant anaphases – telophases with the following
deviations: single rearrangements (fragment, bridge, laggard), multiple rearrangements (two and
more abnormalities of any type per cell).
Statistical analysis. Frequency of detected mitosis distortions was calculated in percents. Data
was checked for homogeneity and normality of tested values distribution (Glotov et al., 1982).
The shelf for the tiFi router
Floor of the cage
A – front panel
2 ms (1,5ms) 2,5 ms (1,7ms)
7 Based on the check individual data were combined in groups. To determine significance of
differences between variants the following was used: ANOsA test, Student t-test (upon normal
distribution of data within groups), or Man-thitney U test (upon abnormal distribution of data
within groups); software applied - Statgraphics Centurion us11 and Statistica 6.0.
FINDINGS
Repeated experiments on assessment of the router’s electromagnetic radiation effect and
influence of Aires resonators on stability of genetic apparatus in marrow bone cells of sistar line
rats enabled to confirm that router’s action during 4 days, 6 h a day, causes disruption of genetic
apparatus of cycling bone marrow cells: total frequency of mitotic disorders increases fourfold in
comparison with control 2 (Faraday cage, 4 days x 6 hours) and with intact control 1 (Fig. 3).
Resonators application resulted in decrease of chromosome disorders by 2.8 times against the
proper router’s action (Fig. 3). However, it is to be noted that in the repeated experiments
combined effect of the router and resonators did not result in complete restoration of
chromosome aberration level to the control level. Frequency of mitosis disorders at the
background of significant decrease in comparison with the router’s effect statistically exceeded
values of control 1 and 2. Animals exposure in the Faraday cage with resonators only did not
cause alteration of chromosome aberrations frequency in comparison with both control groups,
however it was lower than in the “router + resonators” group.
Control 1 Control 2 Router Router + resonators
Chromosome aberrations frequency, %
8 Fig.3. Frequency of chromosome aberrations in bone marrow cells of sistar line male rats after exposure to electromagnetic radiation of the router and resonators. Median values, medium, boundaries of the first and third quartiles, min and max values of the sampling are presented.
Legend: Control 1 – intact rats, control 2 – rats, placed in the Faraday cage, * - differences against each group of “control 1”, “control 2”, “router+resonators” and “resonators” are statistically significant (p<0.0005), brackets denote significantly different values, r- significance point.
During the second stage of studies under the contract it was shown that router’s high frequency
electromagnetic radiation upon exposure within 4 days, 6 h a day, caused disruption of genetic
apparatus of cycling bone marrow cells in male rats of both lines under study - sP and NP: total
frequency of mitotic disorders in low excitation line animals increases by 1.6 times in
comparison with control 2 (Faraday cage, 4 days x 6 hours) and by 1.8 times in comparison with
intact control 1 (Fig. 4). In rats of highly excitable NP line in the same conditions aberration
frequency increases by 2.6 times in comparison with control 2 (Faraday cage, 4 days x 6 hours)
and by 3.4 times in comparison with intact control 1 (Fig. 4), the indicated results are
characterized by high confidence according to all applied statistical criteria. Even though no
statistically significant interstrain differences in chromosome disorders after exposure to the
router’s EMR were detected in the used samples, however degree of bone marrow cells genome
instability is higher in rats of NP line against sP line (Fig. 4), which may be indicative of their
higher sensitivity to effect of EMR of the range under study.
It is important to note that after the animals exposure in the Faraday cage (control 2), a higher
level of chromosome disorders was detected in sP line rats in comparison with the alternative
NP line rats, however, no differences with relevant intact control groups were revealed.
Aires Defender fractal-matrix resonators’ protective effect was assessed in conditions of the
router action during 4 days, 6 h a day, which, as previously shown (First stage report), produce
the most damaging effect on the chromosome apparatus of bone marrow cells of sistar line rats.
Resonators application during the router’s action results in lower frequency of mitosis damage in
cycling bone marrow cells in comparison with the router’s action without protection: sP line rats
– by 1.9 times, NP line rats – by 3.8 times. The indicated level is comparable to control 2
(faraday cage, 4 days, 6 h a day) and intact control 1 (Fig. 4). It is to be noted that in spite of
absence of statistically significant interstrain differences with regard to frequency of
chromosomes damage upon joint router and resonators action, based on rate of decrease of
mitosis disorders, caused by the router’s EMR, efficiency of the resonators’ protective effect is
higher in NP line rats with high excitability against NP line rats with low excitability.
Conspicuous is the tendency of the test items values decrease upon exposition with resonators in
both lines rats against control groups.
9
Fig.4. Frequency of chromosome aberrations in bone marrow cells of sP and NP lines male rats after exposure to electromagnetic radiation of the router and resonators. Median values, medium, boundaries of the first and third quartiles, min and max values of the sampling are presented
Legend: no hatching – sP line, hatching – NP line. Control 1 – intact rats, control 2 – rats, placed into the Faraday cage. * - differences with the group of control 1 and control 2 of the relevant line are statistically significant (p<0,05), brackets denote significantly different values, r- significance point.
DISCUSSION
Findings of the repeated assessment of chromosome aberrations level in bone marrow cells of
sistar line male rats conformed that the router’s electromagnetic radiation, upon exposure
thereto during 4 days, 6 h a day, has a cytogenetic activity and induces chromosome aberrations.
te have shown for the first time (First stage report) and it was confirmed by findings of the
second research stage, that Aires Defender fractal matrix resonators – transformers have a
protective effect on genetic apparatus of cycling bone marrow cells in sistar line male rats
against the router’s EMR action. However, efficiency thereof was different in the first and the
Control 1 Control 2 Router Router + resonators
Chromosome aberrations frequency, %
10 second stages of the experiment, which could be caused by initially higher frequency of
chromosome disorders in control groups of rats, used in the first experiment, and/or other
seasonal and weather factors. It is to be highlighted that the resonators as such do not influence
the level of chromosome aberrations in bone marrow cells of sistar line rats in comparison with
control groups.
Findings of evaluation of chromosome aberration level in bone marrow cells of sP and NP lines
male rats with contrasting excitability of nervous system have shown that the router’s
electromagnetic radiation during 4 days, 6 h a day, induces mitotic disorders in animals of both
lines. No statistically significant interstrain differences in chromosome disorders frequency were
revealed, however, rate of bone marrow cells genome instability increase is larger in NP line rats
against sP rats, which demonstrates dependency of sensitivity thereof to EMR exposure under
study on functional state of nervous system. Sensitivity of bone marrow cells in was higher in
sistar rats than in sP line rats by all applied statistical criteria (t=4,36, r=0,0018; F=19,04,
p=0,0018,t=-15, p=0,008), however it was not different from and was comparable with NO
line. Therefore, character of EMR damaging effect on chromosome apparatus of bone marrow
cells depends on genotype and is connected with excitability level of animals’ nervous system.
Chromosome aberrations (bridges and fragments), detected in anaphase and telophase of mitosis
are caused by incorrect repair of DNA damages at the interphase stage and/or replication errors
(Aguilera, Garsia-Muse, 2013). Average cell cycle duration makes 23 – 24 h for bone marrow
cells of a rat and a mouse (Mejer et al., 2013). Apparently, damaging effect of EMR factors and
DNA disorders formation occur long before its implementation at the chromosome level, at
initial stages of EMR action.
Change of stability of cycling bone marrow cells genetic apparatus when exposed to the router’s
EMR, may be caused by cell oxidative stress. Mutagenic activity thereof is based on genotoxic
action of endopathic causes of humoral nature and free-radical products of peroxidation
(Achudume et al., 2010). It is known that key neuroendocrine factors of DNA stress damage
induction are corticosterone and catecholamines: adrenaline and noradrenaline (Flint et al., 2007,
Hara et al., 2011).
Revealed chromosome disorders may affect functioning of immune system components, related
to bone marrow processes, cause immune system suppression, immunopoiesis and hematopoiesis
arrest (Flach et al., 2014), which will have an effect on the organism state and vital capacity
thereof.
At present, ways of EMR influence on somatic cells genome remain under-explored. The
following hypothesis have been advanced and partially sustained: effects of EMR influence on
the cell within EHF are related to change of intracellular signal systems activity, effect on
11 secondary messengers, DNA, ferments (Gapeev, Chemeris, 2000; Gapeev et al., 2001). Problems
of physical and chemical mechanisms of EMR reception are still mostly unsettled. It is suggested
that EHF EMR influences weak electrostatic bonds, participating in maintenance of
biomolecules and submolecular structures conformation (Gapeev, 2006). It is also suggested that
neuroendocrine system is involved in EMR reception, mediating effect thereof on functioning of
individual organs and the organism on the whole (Gapeev, 2006).
Studies of individual peculiarities of sensibility/ resistance to EMR effect and means of
protection against damaging effect of EMR on genetic apparatus are also of considerable
interest. This aspect is directly related to development of EMR-caused disorders prevention and
correction methods based on individual approach. It is well known that genotype influences
degree of chromosomes damage, caused by mutagenes, genotoxicants, stress factors of different
nature (e.g. Rosenfeld et al., 2001, Semenov et al., 2010, Kovaleva, 2008). However, little is
known of genotype-specificity of EMR influence (Irons et al., 2012, sereschako, 2015). This
paper shows dependence of chromosome aberrations frequency increase under the influence of
wireless router’s EMR on the line (sistar, NP, sP). Given that, chromosome apparatus of NP
line highly-excitable animals is more prone to EMR damaging effect against sP line. Previously,
we have shown that NP line highly-excitable rats are more sensitive to cyclophosphan mutagen
and to psychoemotional stress than sP line rats, it is also evidenced by increase of cytogenetic
disorders in bone marrow cells (Bykovskaya et al., 1994, Dyuzhikova et al., 1996).
During the second stage of the work we have shown that efficiency of resonators’ protective
effect may depend on functional state of animals’ nervous system, on genotype of the rats under
study. Thus, high excitability of nervous system determines a more prominent decrease of
mitotic disorders under resonators’ influence, when the router is used. It is worth noting that
there is a tendency of chromosome anomalies decrease upon both lines rats exposure with
resonators in comparison with the control.
Mechanism of resonators protective effect is being actively studied currently. It is known that
interaction of electromagnetic field with Aires Defender results in structural transformation of
the former (whabrev et al., 2005). Electromagnetic field voltage is concentrated along the count
directions, while considerably decreasing along other directions, which, on the whole, results in
attenuation of total electromagnetic exposure, exceeding organism’s threshold sensitivity to
electromagnetic radiation. Thus, resonators’ protective features are, apparently, based on
restructurization (transformation) of incident EMR, which decreases damaging effect hereof on
cycling cells.
12 On the whole, findings of the performed study enable to conclude that it is more promising to
use lines with already known characteristics of nervous system to reveal EMR mechanisms and
study ways of attenuation of damaging action thereof.
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