The effect of cell-phone radiation on rabbits: Lymphocyte enzyme-activity data

Abstract

The effect of a GSM 900/1800 mobile phone, which is a widespread source of electromagnetic radiation of the microwave frequency in the environment, on rabbits was studied at power densities of 5–7 μW/cm2. The biological effect was recorded by a sensitive method for the detection of the physiological regulation of enzyme activity inside lymphocytes in blood smears. Succinate dehydrogenase, which is the most powerful energy-supply enzyme in mitochondria, and lactate dehydrogenase, which is an enzyme of glycolysis, were measured. The lactate dehydrogenase to succinate dehydrogenase activity ratio was also calculated as an analog of the Warburg effect, which demonstrates the relationship between glycolysis and respiration. After 60 min of mobile-phone exposure each day for 11 days at a moderate dose, the emitted radiation induced a threefold increase in succinate dehydrogenase activity and a twofold decrease in lactate dehydrogenase activity. As a result, the lactate dehydrogenase/succinate dehydrogenase activity ratio falls from 15 to 5, thus indicating that respiration is predominant over glycolysis. The changes develop already after the first exposure and reach a maximum in 4 days. The predominance of respiration is usually considered as a beneficial state of an organism. However, continuous activation of respiration by mobile phone exposure may cause damage to the normal restorative processes that are supported by glycolysis during periods of rest.

Full text

ISSN 00063509, Biophysics, 2016, Vol. 61, No. 1, pp. 100–104. © Pleiades Publishing, Inc., 2016.
Original Russian Text © M.V. Zakharchenko, A.V. Kovzan, N.V. Khunderyakova, T.V. Yachkula, O.V. Krukova, R.G. Khlebopros, P.M. Shvartsburd, N.I. Fedotcheva,
E.G. Litvinova, M.N. Kondrashova, 2016, published in Biofizika, 2016, Vol. 61, No. 1, pp. 120–125.
100
Natural and technogenic electromagnetic fields are
important environmental factors that have large bio
logical effects. The most widespread sources of micro
wave electromagnetic fields are portable communica
tion devices that create a uniform radio coverage area
via base transceiver stations and user terminals. In
recent years, the cell phone has become an indispens
able communications tool [1, 2]. Studies have shown
that the electromagnetic waves that are generated by
cell phones have a number of effects at different levels
of organization of biological systems, from organismal
to molecular, which arouses concern.
The protection of cells from the stress that is
induced by extreme exposure to microwave electro
magnetic radiation (EMR) includes various nonspe
Abbreviations
: EMR, electromagnetic radiation; SDH, succi
nate dehydrogenase; LDH, lactate dehydrogenase; ICA, isoc
itric acid.
cific protective mechanisms, such as heatshockpro
tein expression and excessive production of reactive
oxygen species [3–10].
It seems important to find the physiological and
biochemical limits of adaptation under changing envi
ronmental conditions and indicators that are sensitive
enough to assess the biological effects that are associ
ated with the consequences of exposure of an organ
ism to EMR. It has been shown that cell membranes
are sensitive to microwave EMR due to deformation
instability, which occurs at a high strength of an elec
tric field in a membrane. This instability leads to a
drastic change in the diffusion “transparency” of the
cell’s plasma membrane [11, 12].
The hematopoietic system is one of the systems
that respond strongly to variations of environmental
conditions. Some works have demonstrated changes in
blood and bone marrow cells in response to EMR that
The Effect of CellPhone Radiation on Rabbits:
Lymphocyte EnzymeActivity Data
M. V. Zakharchenko
a
, A. V. Kovzan
b
, N. V. Khunderyakova
a
, T. V. Yachkula
a
,
O. V. Krukova
c
, R. G. Khlebopros
b
, P. M. Shvartsburd
a
, N. I. Fedotcheva
a
,
E. G. Litvinova
a
, and M. N. Kondrashova
a
a
Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences,
ul. Institutskaya 3, Pushchino, Moscow oblast, 142290 Russia
b
Institute of Economics, Management and Environmental Management, Siberian Federal Univesity,
Svobodnyi pr. 79, Krasnoyarsk, 660041 Russia
c
Krasnoyarsk Scientific Center, Siberian Branch, Russian Academy of Sciences,
Akademgorodok 50, Krasnoyarsk, 660036 Russia
email: mkondrashova23@inbox.ru
Received November 4, 2015
Abstract
—The effect of a GSM 900/1800 mobile phone, which is a widespread source of electromagnetic
radiation of the microwave frequency in the environment, on rabbits was studied at power densities of 5–
7
µ
W/cm
2
. The biological effect was recorded by a sensitive method for the detection of the physiological reg
ulation of enzyme activity inside lymphocytes in blood smears. Succinate dehydrogenase, which is the most
powerful energysupply enzyme in mitochondria, and lactate dehydrogenase, which is an enzyme of glycol
ysis, were measured. The lactate dehydrogenase to succinate dehydrogenase activity ratio was also calculated
as an analog of the Warburg effect, which demonstrates the relationship between glycolysis and respiration.
After 60 min of mobilephone exposure each day for 11 days at a moderate dose, the emitted radiation
induced a threefold increase in succinate dehydrogenase activity and a twofold decrease in lactate dehydro
genase activity. As a result, the lactate dehydrogenase/succinate dehydrogenase activity ratio falls from 15 to
5, thus indicating that respiration is predominant over glycolysis. The changes develop already after the first
exposure and reach a maximum in 4 days. The predominance of respiration is usually considered as a bene
ficial state of an organism. However, continuous activation of respiration by mobile phone exposure may
cause damage to the normal restorative processes that are supported by glycolysis during periods of rest.
Keywords
: electromagnetic radiation, mobile phone communication, mitochondria, lymphocytes, succinate
dehydrogenase, lactate dehydrogenase
DOI:
10.1134/S0006350916010279
CELL BIOPHYSICS

BIOPHYSICS Vol. 61 No. 1 2016
THE EFFECT OF CELLPHONE RADIATION ON RABBITS 101
is generated at the operating frequency of cell phones
[13–15].
In this work we attempted to reveal the effects of
cell phone EMR using the rabbit as the test object. The
activities of succinate dehydrogenase (SDH), the most
powerful enzyme in the mitochondrial energysupply
system, which oxidizes succinic acid, and the indica
tor of glycolytic activity lactate dehydrogenase (LDH)
were selected as response indicators. The enzyme
activities were measured in lymphocytes in blood
smears (the method was improved by the authors).
This method combines the advantages and avoids the
disadvantages of cytochemical and biochemical
detection and has been named the cytobiochemical
method.
This technique allows the enzyme to be better
maintained in an isolated preparation in the same state
as in the body compared to other methods [16–20].
Therefore, its results better demonstrate the changes
in the body compared to other enzymatic methods.
There are several other methodical advantages in
this work. The experiments with rabbits (which have a
ratio of adrenergic/cholinergic regulation that is simi
lar to that of human beings) are of great importance. In
small animals, adrenergic regulation (i.e., excitation)
is predominant, in contrast to humans. In humans, it
is more equilibrated by cholinergic regulation. This
important difference provides a better course of resto
ration processes and higher resistance to strong and
pathogenic impacts [21–26].
This role of cholinergic regulation was discovered
in the works by I.A. Arshavsky in the 1980s [21]. It is
determined by acetylcholine, which occurs in the
blood not in its role as a wellknown synaptic media
tor. The formation of nonneuronal acetylcholine was
discovered independently in the early 2000s [22, 23].
In addition, the protective and restorative role of ace
tylcholine centers of the vagus nerve has been demon
strated [24]. A global network of cholinergic regula
tion was revealed in the brain [25]. The relationship
between melatonin (a known regulator of the restora
tion processes) and cholinergic regulation was shown
[26]. Thus, the latest studies have disclosed the novel
role of cholinergic regulation: the involvement in all
adaptation processes and the intensification of resto
ration processes in the body. Therefore, it is very
important to conduct research on animals with well
marked cholinergic regulation.
Among the available laboratory animals, guinea
pigs and rabbits are close to humans in their level of
cholinergic regulation. Studies on rabbits, in addition
to the wellmarked cholinergic regulation, have the
advantage of allowing repeated experiments using the
same animal, since their auricular veins are suitable for
injection of different substances. This provides highly
reproducible results and the ability to compare cyto
biochemical studies and individual physiological
characteristics of animals.
In addition to the above two important advantages
of working with rabbits, we have found another advan
tage of substantial value. Rabbits, even genetically
similar individuals of the same brood, clearly demon
strate two types of behavior: quiet (cholinergic) and
spontaneously excited (adrenergic). Hence, it is possi
ble to easily compare cytobiochemical values and
behavioral traits. The abovementioned advantages of
the studies on rabbits make them individualized, in
contrast to the average statistical featureless data for
small animals.
MATERIALS AND METHODS
Animals and procedures.
The experiments were car
ried out with Chinchilla rabbits (males, 7 months old,
3.8–4.1 kg) that were grown in the vivarium of the
Institute of Theoretical and Experimental Biophysics,
Russian Academy of Sciences. The animals demon
strated substantial differences in behavior before the
beginning of their interaction with the experimental
ist. One of the rabbits was quite excitable and shy. The
second and third rabbits were initially quieter. How
ever, after the animals had been habituated to being
handled, their behavior stabilized and their excitation
diminished. They were not excited during the proce
dures.
The rabbits were exposed to radiation for 11 days,
1 h per day, at the same time (10:00). The source of
microwave electromagnetic radiation was a portable
communication device with the GSM 900/1800 stan
dard (an energyflux density of 5–7
μ
W/cm
2
) that was
fixed on the rabbit’s back with a breastband. The cell
phone worked in the voice communication regime
(but without sound), i.e., transmitted and received a
signal continuously for 1 h. After the exposure, the cell
phone and the breastband were removed and blood
samples were taken during 5–20 min at the following
periods: day 1, immediately before the exposure;
day 1, after the exposure; day 4, 7, and 11 (the last day
of the exposure); and day 14 (3 days after the last expo
sure). Thus, there were six blood samples from each
animal altogether.
Measurement of dehydrogenase activities in lym
phocytes.
The DH activities were measured in a blood
smear that was taken after auricular vein puncture,
without adding anticoagulants. The newly prepared
blood smears were fixed for 30 s with 60% acetone
buffered with 10 mM HEPES at room temperature,
pH 5.2–5.4. They were then rinsed with distilled water
and dried in the air.
The enzyme activities were measured by the reduc
tion of nitro blue tetrazolium in a newly prepared
blood smear by the method that was described previ
ously [17] in a medium containing: 125 mM KCl
(Sigma, United States), 10 mM HEPES (Sigma,
United States), 1.22 mM nitro blue tetrazolium chlo

102
BIOPHYSICS Vol. 61 No. 1 2016
ZAKHARCHENKO et al.
ride (Dudley Chemical Corporation), with the addi
tives listed below, for 1 h at 37
°
C, pH 7.20 ± 0.05.
The dehydrogenase activities were measured with a
probe set that formed a complex response that was
regarded as a certain type or “pattern” of the state of
mitochondrial dehydrogenases. The resulting patterns
make it possible to analyze the biochemical mecha
nisms of the regulation of dehydrogenase activities.
The basic probe that characterizes the activity of
SDH and the degree of adrenergic regulation in an
organism is succinic acid (5 mM). The increase in
SDH activity reflects first the physiological activation
of adrenergic regulation and then the pathogenic
hyperactivation reaching pathological inhibition.
These stages are revealed in the next test with isocitric
acid (ICA).
Isocitric acid was added to succinic acid (5 mM) at
a concentration of 5 mM. The absence of an effect of
ICA on SDH activity is characteristic of the quiescent
state. The decrease or increase in SDH activity after
the addition of ICA demonstrates the activation of
adrenergic regulation or the inhibition that develops in
an organism under adrenalininduced stress, respec
tively.
Lactate (5 mM) + malonate (5 mM) + NAD
(0.5 mM) is a probe that characterizes the activity of
glycolysis, namely, lactate dehydrogenase. The simul
taneous measurement of succinate dehydrogenase and
lactate dehydrogenase activities provides a much more
complete picture of oxidative metabolism in a cell,
which is not possible when studying isolated mito
chondria.
After the incubation, the smears were stained with
a nuclear neutral red dye (0.5%) for 8 min.
After the dye color had developed, the smears were
observed using a LeicaDM 2000 microscope with a
Leica DFC 425 color camera at a magnification of
100
×
with oil immersion. The cells were sought and
captured using the specialized Bloodrunner software
package; 50–100 lymphocytes were taken from each
smear. The Cell Composer software package, which is
specially designed for the quantitative morphological
analysis of color images, made it possible to determine
the amount and distribution of dye in a cell by calcu
lating the dye decomposition via the color space data,
as well as the basic morphological parameters of a cell.
Thus, the concentration of the reaction product
(Diformazan) in each cell was calculated.
RESULTS AND DISCUSSION
The response was assessed by measuring the SDH
and LDH activities and the effect of isocitric acid on
SDH activity, which makes it possible to define the
functional state of the enzyme more precisely, as well
as the LDH/SDH ratio.
Previously it has been shown that the addition of
isocitric acid (the precursor of
α
ketoglutarate) makes
it possible to distinguish the active and hyperactive
states of SDH from the inhibited one. The activated
states are reduced after the addition of ICA, while the
inhibited states are increased. ICA has no effect on the
quiescent state. The mechanism of action of isocitric
acid on the activity of SDH is based both on its well
known reduction by respiratory chain oxidation and
by the disregarded conjugating effect of guanosine
triphosphate that is formed via substrate phosphoryla
tion during the oxidation of
α
ketoglutarate [17–19].
According to our concept, the LDH/SDH ratio is
used as the ratio of the activity of respiration and glyc
olysis. This parameter is an analog of the Warburg
effect, which was described approximately a century
ago and recently has again attracted the attention of
researchers as an index of the intensification of glyso
clysis in tumor cells [27–29]. It is not an unambiguous
indicator of just tumor growth and allows for quantifi
cation of the respiration/glycolysis ratio in different
states of an organism. The proposed LDH/SDH
activity ratio is applicable for such studies.
The dehydrogenase activity measurement data
averaged for three animals are presented in the figure.
It is notable that the results of the measurement for the
three rabbits are in good agreement. The SDH activity
is observed to have increased in all rabbits after the first
exposure to radiation. It increased twofold until day 4
and threefold after the 7th day and until the end of the
course. Internal inhibition occurred on day 4 as isoc
itric acid highly increased its activity. Further exposure
to radiation led to the development of hyperactivation,
because the addition of ICA reduced the activity.
After the exposure, the activity of SDH was nearly two
times higher compared to the initial state. At the
same time, its activity is constrained by internal inhi
bition, because the addition of isocitric acid slightly
increases it.
After the exposure, the LDH activity was reduced
by nearly two times relative both to the initial value and
to its slight increase at the end of the course. As a
result, the change in the LDH/SDH activity ratio is
particularly strong. It was approximately 15 initially
and approximately 5 after the exposure.
The data are similar at all points after the exposure.
A large spread of the data is observed only in the initial
state. This is in agreement with the greater differences
in the behavior of animals in the initial state compared
to that after the exposure. During the exposure, the
spread of the LDH/SDH activity ratio was insignifi
cant; it already dropped sharply after the first session
and on day 4 it reached a value that corresponds to that
after the course. The decrease in this value demon
strates that glycolysis is better inhibited by active respi
ration than in the quiescent state.
The intensification of glycolysis inhibition by respi
ration is usually regarded as a favorable indicator for
aerobic cells, the socalled Pasteur Effect. However, it
is unclear whether it can be used to explain the

BIOPHYSICS Vol. 61 No. 1 2016
THE EFFECT OF CELLPHONE RADIATION ON RABBITS 103
observed effect of radiation. All the same, it activates
mitochondria in a quiescent animal. We have previ
ously considered the loss of the quiescent state by the
SDH enzyme as the very first sign of prepathology,
including that without any clinical manifestations. We
arrived at this conclusion based on the results of exam
ining patients with hypertension and allergic eating
disorders and animals under stress [18, 19]. One of the
potential impairments of normal cell functions during
such a shift in the quiescent state may be the weaken
ing of biosynthetic restoration processes that occur
during the activity cutoff in quiescent periods. If elec
tromagnetic radiation maintains the active state of the
cells, restoration seems to be disturbed. This fact is
also in agreement with the decline in this index analo
gous to the Warburg effect, viz., from 15 in the quies
cent state to 5 during and after the exposure. Such a
point of view is supported also by the data in [30],
where it was concluded that aerobic glycolysis is not a
weak point of tumor cells but is rather a property that
is necessary for their intensive growth.
As a result of this study we can conclude that expo
sure to cellphone radiation at a very moderate dose
(for 1 hour once a day) causes a distinct activation of
the basic enzyme of energy supply, viz., succinate
dehydrogenase. This activation is also coupled with
suppression of the activity of the glycolytic enzyme
lactate dehydrogenase. However, thus far it is too early
to make any conclusions on the physiological signifi
cance of the observed shift in enzyme activities. One of
the possible consequences of such activation is the
impairment of biosynthetic restoration processes,
including cell division.
ACKNOWLEDGMENTS
This work was supported by the Russian Founda
tion for Basic Research (project no. 130401049a).
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Translated by E. Makeeva