Stimulation and inhibition of respiratory burst in neutrophils as a result of action of weak combined magnetic fields adjusted to ICR of protonated water forms

Abstract

Weak combined collinear magnetic fields (CMFs) composed of constant component, 60 μT and low-frequency alternating field, 100 nT, used in 40 min pretreatment, were shown to have diverse effects (alleviation/stimulation) depending on the frequency of the alternating component on respiratory burst intensity in neutrophil suspension after activation by N-formyl-Met-Leu-Phe recorded by luminol-dependent chemiluminescence. About 12.6 Hz frequency formally corresponding to ion cyclotron resonance (ICR) frequency of hydrated hydronium ion (Н9О4 ⁺) had notable stimulating effect. On the contrary, treatment with 48.5 Hz frequency corresponding to hydronium ion (Н3О⁺) was accompanied by significant alleviation of respiratory burst intensity. CMFs-pretreated water conducted only stimulating effect of CMFs when the field was adjusted to ICR of hydrated hydronium ion form, which is direct proof of participation of water in the mechanism of this effect of CMFs.

Full text

Stimulation and inhibition of respiratory burst in neutrophils as a result of action
of weak combined magnetic elds adjusted to ICR of protonated water forms
Vadim V. Novikov, Elena V. Yablokova, and Eugenii E. Fesenko
Institute of Cell Biophysics of Russian Academy of Sciences, Pushchino, Russia�
ABSTRACT
Weak combined collinear magnetic elds (CMFs) composed of constant component, 60 μT and low-
frequency alternating eld, 100 nT, used in 40 min pretreatment, were shown to have diverse
eects (alleviation/stimulation) depending on the frequency of the alternating component on
respiratory burst intensity in neutrophil suspension after activation by N-formyl-Met-Leu-Phe
recorded by luminol-dependent chemiluminescence. About 12.6 Hz frequency formally corre-
sponding to ion cyclotron resonance (ICR) frequency of hydrated hydronium ion (Н
9
О
4+
) had
notable stimulating eect. On the contrary, treatment with 48.5 Hz frequency corresponding to
hydronium ion (Н
3
О
+
) was accompanied by signicant alleviation of respiratory burst intensity.
CMFs-pretreated water conducted only stimulating eect of CMFs when the eld was adjusted to
ICR of hydrated hydronium ion form, which is direct proof of participation of water in the mechan-
ism of this eect of CMFs.
ARTICLE HISTORY
Received 3 March 2020
Accepted 16 August 2020
KEYWORDS
Weak combined magnetic
fields; respiratory burst;
neutrophils; water
Introduction
Some authors regard the possible effect of magnetic
fields on production of free radicals and other reactive
oxygen species as a promising approach to analyze the
mechanisms of their biological action (Barnes and
Greenebaum 2015; Lai 2019; Mattsson and Simkó 2014).
It is well-known that the main producers of reactive
oxygen species (ROS) in mammalian blood are the cells
performing phagocytosis, particularly, neutrophils
(Lindena et al. 1987). ROS generation in activated neu-
trophils is mainly initiated by a multienzyme complex,
NADPH-oxidase, localized in plasma membrane, that
produces superoxide anion radical (Lambeth 2004).
Polymorphonuclear neutrophilic granulocytes (neu-
trophils) are the most abundant leukocytes of human
blood. Phagocytosis and respiratory burst are known as
the main functions of these cells (Dahlgren and Karlsson
1999). During phagocytosis and response to soluble
mediators, such as bacterial peptide N-formyl-Met-Leu-
Phe (fMLF) or phorbol ester phorbol-12-myristate-13-
acetate (PMA), the neutrophils generate reactive oxygen
species, such as superoxide anion radical, hydrogen per-
oxide and hydroxyl radical. These molecules participate
in disintegration of the phagocytized objects. One of the
most sensitive methods for ROS detection is based on
measurement of chemiluminescence generated during
luminol oxidation, so-called luminol-enhanced chemi-
luminescence (Dahlgren and Karlsson 1999).
“Respiratory burst” is a massive ROS efflux during acti-
vation of neutrophils. It is a sum of processes depending
on a complex of membrane and cytosolic proteins which
are gathering to react on many factors (El-Benna et al.
2008,2016; Vignais 2002). The neutrophil is capable of
regulating its reactivity, for example, of deactivating or
priming its propensity to generate respiratory bursts
(Mayansky 2007). Pretreatment of the cell by
a priming agent leads to subsequent enhancement of
response to activation.
A number of data in the literature shows effect of
relatively weak low-frequency MFs on kinetics of reac-
tive oxygen species formation in suspension of neutro-
phils (Table 1). As our previous results showed, the
respiratory burst in neutrophils is sensitive to variations
of magnetic parameters preceding this event (Novikov
et al. 2016b). Priming effect (pre-activation of respira-
tory burst in neutrophils) of combined weak constant
(42 μT) and collinear low-frequency alternating (sum of
frequencies 4.4 and 16.5 Hz, total amplitude 0.86 μT)
was shown in one work as more significant enhance-
ment of chemiluminescence of neutrophil suspension in
response to application of a bacterial peptide fMLF, or
a phorbol ester PMA, in presence of luminol (Novikov
et al. 2016b). In the mentioned work, the parameters of
the alternating component of the field were adjusted
based on the data obtained before on mouse model
inoculated with Ehrlich Ascites Carcinoma (Novikov
CONTACT Elena V. Yablokova, e.v.yablokova@mail.ru Institute of Cell Biophysics of Russian Academy of Sciences, Pushchino 142290, Russia
ELECTROMAGNETIC BIOLOGY AND MEDICINE
https://doi.org/10.1080/15368378.2020.1813158
© 2020 Taylor & Francis Group, LLC

et al. 2009a,2009b,2010). To generate the alternating
magnetic signal, the principle of tuning of the field to
cyclotron resonance frequency values for a number of
simple and complex ions (NAD
+
, ionic form of glutamic
acid, K
+
) was realized in the algorithm: 1.0, 4.4 and
16.5 Hz corresponded to these ions, the induction of
the static field was 42 μT. As the experiments showed,
such a three-frequency combined signal possessed the
most significant anti-tumor activity compared to several
isolated monofrequencies involved in its generation
(Novikov et al. 2009a,2009b,2010).
On the contrary, when using other parameters of
CMFs, for example, the frequency tuned to the cyclo-
tron resonance of Fe
3+
ion, deactivation of respiratory
burst was demonstrated (Novikov et al. 2020). In this
case, 60 μT static magnetic field and the collinear alter-
nating low-frequency magnetic field, 49.5 Hz and
60–180 nT, caused, after 40 min pretreatment, signifi-
cant alleviation of respiratory burst intensity in suspen-
sion of neutrophils in response to the activator fMLF,
recorded by luminol-dependent chemiluminescence
assay.
Use of magnetic field parameters causing pre-
activation of respiratory burst (three-frequency
magnetic signal) showed only slight increase of
lipid peroxidation (LPO) in neutrophils after treat-
ment by CMFs of this mode (Novikov et al. 2017a).
The relation of such elevation of LPO to functional
pre-activation of neutrophils resulting from the
action of this CMF was not revealed, because LPO
inhibitor, ionol, did not decrease neutrophil prim-
ing index (Novikov et al. 2017a). Low concentra-
tions of intracellular calcium chelator, BAPTA AM
(~1 µM), blocked this stimulating effect of the CMF
(Novikov et al. 2017b). As was shown by further
experiments, one of key elements of the mechanism
of pre-activation of the neutrophils in weak CMFs is
strong dependence of the amplitude of this effect on
concentration of atmospheric gases (Novikov and
Fesenko 2001�). Preceding mild degassing of the
suspension at 640 Torr pressure led to essential
(fourfold) decrease of response to the CMFs, but it
did not alter the ability of the cells to generate
respiratory burst in response to the activator
(N-formyl-Met-Leu-Phe) in control (partially
degassed suspension of neutrophils) (Novikov and
Fesenko 2001). This allowed to suppose that the
properties of water solution itself can be changed
under CMFs. To analyze such a possibility experi-
mentally, special experiments listed in this work
were designed.
Selection of parameters (frequencies and amplitude)
of alternating component of the MFs in the current study
was made taking into account the most recent experi-
mental data on the effect of combined magnetic fields
corresponding to ion cyclotron resonance (ICR) condi-
tion (Liboff 1985) in pure water for hydronium ions (H
3
O
+
) (protonated water) and its hydrated form (H
3
O
+
*
3H
2
O) (D’Emilia et al. 2017,2015). These works showed
that the treatment of water by very weak magnetic field
stimulates short-term changes in refraction coefficient,
pH and provides emission of stable transition magnetic
signal on 48.5 Hz frequency for at least several tens of
minutes after the CMF treatment is stopped, in absence
of any other measurable field. When adjusting the para-
meters of the alternating frequency of CMFs in these
experiments (D’Emilia et al. 2017,2015), the authors of
this study took into account our earlier results (Novikov
1994,1996; Novikov and Zhadin 1994; Zhadin et al.
1998), showing short-term increase of conductivity of
protonated form of glutamic acid (GluH
+
) in water solu-
tion under combined magnetic fields satisfying ICR
Table 1. The effect of weak combined magnetic fields on the production of ROS by neutrophils.
Object Reference
Frequency
Hz
B
AC
μT
B
DC
μT Response
Rat neutrophil
suspension
Roy et al. (1995) 60 141 ~50 Increased fluorescence of DCF. Activator, FMA
Mouse neutrophil
suspension
Belova et al.
(2010)
31
ICR for Ca
2+
74.7 40.6 Decreased luminol-dependent
chemiluminescence. Activator, FMA.
Increased luminol-dependent
chemiluminescence.
Activator, fMLF
Human blood
neutrophils
Poniedzialek et al.
(2013)
Pulse signal modulated by the frequency of ICR
for Ca
2+
10
60
50 Increased fluorescence of DCF and DHR-123.
Activator, FMA
Mouse neutrophil
suspension
Novikov et al.
(2016a)
1, 4.4, 16.5
Complex signal simultaneously tuned to ICR
for NAD
+
, Glu
+
, K
+
0.86 42 Increased luminol-dependent
chemiluminescence.
Activators, fMLF or FMA
Mouse neutrophil
suspension
Novikov et al.
(2020)
49.5
ICR for Fe
3+
0.12 60 Decreased luminol-dependent
chemiluminescence. Activator, fMLF
2V. V. NOVIKOV ET AL.

condition, that are of special interest because of the low
value of the alternating magnetic field induction, being
effective at ~50 nT amplitude.
Materials and methods
Generation of CMFs
The experiments were carried out using relatively weak
magnetic fields (comparable to geomagnetic field and
weaker) in a frequency band close to the utility fre-
quency present in laboratory facilities (50 Hz). Due to
that, the experiments involved special equipment. The
setup for CMFs treatment of neutrophils (Figure 1)
included a system of protective magnetic screens allow-
ing high degree of alleviation of geomagnetic field, up to
10,000-folds (the residual field did not exceed 10 nT),
and essential alleviation of alternating technogenic
interference (to several nT). The setup was composed
of three coaxial cylindric magnetic screens made of
permalloy (d = 1 mm) closed by lids with openings for
measuring and thermostabilizing devices (inner dia-
meter 22 cm, length 42 cm). The residual fields in the
setup were measured directly by ferroprobe magnet-
ometer Mag-03 MS 100 (Bartington Instruments Ltd,
United Kingdom). To generate weak uniform collinear
static and alternating components of the CMFs, a special
inductor (solenoid) was installed inside the system. It
was attached to direct current power supply to get static
field and to alternating low-frequency generator to
generate the alternating component of the field.
Solenoid dimensions were: diameter, 18 cm; length,
36 cm (720 coils of copper wire, diameter 1 mm; the
resistance of solenoid was 7.5 Ohm.). They allowed to
form the experimental area inside the screens sufficient
for simultaneous placement of experimental samples
(six and more) into the zone of the uniform weak com-
bined magnetic fields.
The frequencies of the alternating component of
CMFs corresponded to cyclotron resonance values for
hydronium ions, Н
3
О
+
and for complex ions of its
hydrated form, Н
3
О
+
(3Н
2
О), calculated by standard
equation:
fc¼qBDC=2
where qand mare the charge and mass of the ion,
respectively, B
DC
is the induction of the static compo-
nent of CMF (60 μT). The calculated frequencies were
48.5 Hz for Н
3
О
+
and 12.6 Hz for Н
9
О
4+
. The para-
meters of alternating MF (frequencies and amplitude)
were set digitally using digital-analog converter (DAC)
of L 791 card (L-Card Company, Moscow, Russia). This
provided high precision of their adjustment. Generation
of alternating magnetic field was provided by sinusoidal
alternating current passing through the solenoid:
I tð Þ¼ Acosωt
,
ω¼2πf; fis frequency, Hz,
Figure 1. Magnetic treatment setup. 1- magneitc screens; 2-solenoid; 3-thermostabilized cuvette; 4-experimental samples.
ELECTROMAGNETIC BIOLOGY AND MEDICINE 3

which produced alternating magnetic field (MF) of
100 nT.
The waveforms of currents passing through the sole-
noid to form the set values of static and alternating
components of CMFs are presented in Figure 2.
Obtaining neutrophil suspension
The work was carried out on murine peritoneal
neutrophils. Male laboratory mice CD-1 (body-
weight 22–25 g, obtained from the nursery of the
Shemyakin – Ovchinnikov Institute of Bioorganic
Chemistry, Russian Academy of Sciences) were
used for isolation of the cells. The animals received
intraperitoneal injections (150 μl, 5 mg/ml) of opso-
nized Zymozan A from Saccharomyces cerevisiae
(Sigma, USA). The mice were killed by cervical dis-
location 12 hours after the injection and the abdom-
inal cavity was washed with four milliliters of
calcium-free Hank’s solution. The exudate was col-
lected by a pipette and centrifuged for 5 min at
600 g. The supernatant was decanted, and the pellet
was dissolved in 4 ml of calcium-free solution. The
number of isolated cells was calculated in Goryaev’s
chamber. Cell viability was tested using a vital dye,
Trypan Blue. It was at least 98%. The samples for
the experiments were obtained by dilution of the
neutrophil suspension by Hank’s solution (138 mM
NaCl, 6 mM KCl, 1 mM MgSO
4
, 1 mM Na
2
HPO
4
, 5
mM NaHCO
3
, 5.5 mM glucose, 1 mM CaCl
2
,
10 mM HEPES, pH 7.4, Sigma, USA) to final con-
centration of 10
6
cells/ml.
The procedures followed were approved by the ethics
committee for guidance for care and use of laboratory
animals № 57.30.12.2011 of the institution and in accor-
dance with the Guidelines for Ethical Conduct in the
Care and Use of Animals.
Exposure of the neutrophil suspension
The neutrophils were incubated at 37.0±0.2°C at 10
6
cells/ml concentration. Samples (0.25 ml each) were
kept in round-bottle polystyrene cuvettes (d = 1.2 cm,
l = 5.5 cm), which were further used for chemilumines-
cence measurements. Typical incubation time was
40 minutes. The set temperature was maintained by
circulation thermostat UH 4 (MLW, Germany).
The samples of the main control groups were
placed in local geomagnetic field (GMF) with con-
stant component of 44 μT and background alternat-
ing field of 15–50 nT on 50 Hz frequency at the same
temperature and the same time as experimental
group (GMF control). A group of samples incubated
inside the experimental CMFs-generating setup
described above at 60 μT constant field with the
alternating field switched off was used as sham con-
trol. The experimental samples were exposed inside
the experimental setup at set CMFs parameters. The
experiments were replicated at least three times. The
overall scheme of the experiments is shown in
Figure 3.
After incubation of the specimens, as well as before
it, the vitality of the cells in experimental (CMFs-
exposed) and control groups was tested with trypan
blue. The content of living cells in all the groups after
incubation remained as high as before, comprising at
least 98%.
pH of neutrophil suspension in saline was tested on
a pH-meter SevenEasy pH (Mettler Toledo, Switzerland);
conductivity was measured by a conductivity meter OK-
102/1 (Radelkis, Hungary) in experimental and control
groups. The data on pH and conductivity of neutrophil
suspension before and after exposure to CMFs for each of
the tested frequencies and for corresponding control
groups are shown in Table 2.
13.9
14
14.1
14.2
14.3
0 0.5 1
I, mA
Time, s
13.9
14
14.1
14.2
14.3
0 0.5 1
I, mA
Time, s
ab
Figure 2. Waveforms of the currents passing through the solenoid to form the set values of constant and alternating components of
CMFs. A-12.6 Hz frequency; B-48.5 Hz frequency.
4V. V. NOVIKOV ET AL.

Chemiluminescence recording
After the incubation of the neutrophil suspension, che-
miluminescence of the samples was measured in control
and experimental groups after addition of 0.35 mM
luminol (Enzo Life Sciences, USA) and 1 μM ROS gen-
eration activator, chemotactic formylated peptide
N-formyl-Met-Leu-Phe (Sigma, USA). 12-channel che-
miluminometer Lum-1200 (DISoft LLC, Russia) was
used in the work. The values of luminescence intensity
corresponded to light flow, i.e. number of photons per
time unit: 1 Volt corresponded to ≈ 1000 photons/s.
Chemiluminescence data were processed in
PowerGraph program. Part of the results was presented
in relative values (percentage of the amplitude of che-
miluminescent response in the main control taken
for 100%).
Figure 3. The overall scheme of the experiment.
Table 2. Conductivity and pH of saline solution in neutrophil
suspension before and after exposure to CMFs.
Control
before
exposition
Control after
exposition
(t = 37ºC,
40 min)
Exposed
12.6 Hz
Exposed
48.5 Hz
pH 7.40 ± 0.05 7.42 ± 0.05 7.42 ± 0.05 7.42 ± 0.05
Conductivity
(mS.
cm
−1
)
16.9 ± 0.1 16.9 ± 0.1 16.9 ± 0.1 17.0 ± 0.1
ELECTROMAGNETIC BIOLOGY AND MEDICINE 5

Pretreatment of water by CMFs and testing its
biological activity
Beside the aforementioned experiments on direct treat-
ment of neutrophil suspension by CMFs, the same experi-
ments were carried out for high-purity water (Milli-Q
standard, 18 MOhm * cm, Arium pro (Sartorius,
Germany), prepared 20 hours before the experiment) in
the same setup at room temperature (23–24°C). For this
purpose, water samples were placed into the CMFs gener-
ating setup (Figure 1) in optic glass cubic cuvettes
(V = 18 ml). First the water was added to the neutrophils
(as a saline solution) right after CMFs treatment. Series of
additional experiments were carried out, where CMFs-
treated water was added to the neutrophils 5 min after
termination of water exposure to CMFs and 40 min after it.
Water samples before addition of luminol and
formylated peptide pre-incubated in CMFs were
tested for their ability to perform pre-activation
(priming) or deactivation of neutrophils. At this
stage, Hank’s medium was prepared by mixing 8
volume parts of water pre-incubated in CMFs with
1 volume part of concentrated Hank’s solution (mix-
ing was performed right before addition of saline to
neutrophils). The neutrophils were incubated in this
medium under GMF at 37±0.1°C for 40 min at 10
6
cells/ml (0.25 ml samples in cuvettes for further
chemiluminescence measurements). The cells were
treated with CMFs-exposed water (as in case of direct
CMFs exposure) 1 hour after the isolation; during
this time, they were kept on ice to minimize the
spontaneous chemiluminescence and to achieve the
identical functional state of the cells (Belova et al.
2010). Right after finishing incubation at 37ºC (in
CMFs, in presence of CMFs-treated water, and in
control) the polystyrene vials with the cells were
transferred to the chemiluminometer cuvette holders
pre-heated at 37°C, and chemiluminescence registra-
tion started. After 1 minute of registration, luminol
solution was added, and right after that, in several
seconds, fMLF activator solution was added. After
these manipulations, chemiluminescence was
recorded for at least 5 minutes.
After the incubation of the neutrophils in the media
containing experimental water samples, chemilumines-
cence was measured after addition of luminol solution
and ROS generation activator, i.e. N-formylated peptide,
in exactly the same way as in aforementioned experiments
on direct CMFs treatment of neutrophil suspensions.
Samples prepared with non-CMFs-treated Milli-Q water
were used as controls corresponding to CMFs-treated
experimental samples in this series of experiments.
Control and experimental samples were incubated in
GMF simultaneously. The experiments were replicated at
least three times.
Statistical analysis
The data were evaluated using the Student’s t-test.
Pvalues < 0.05 were considered significant. All values
were expressed as means ± SD.
Results
First of all, the direct action of CMFs with static compo-
nent of 60 µT and collinear alternating component with
48.5 Hz or 12.6 Hz frequency and 100 nT amplitude on
neutrophil suspension was studied.
Comparison of data from two control groups, group 1
in geomagnetic field with constant component of 44 µT
in presence of background technogenic interference
(15–50 nT, 50 Hz) and group 2 placed into CMFs gen-
erating setup with alternating field switched off did not
reveal any difference in respiratory burst intensities
between these sample groups (Table 3). Thus, taking
into account the possibility of simultaneous incubation
with experimental samples, the samples exposed in GMF
(GMF control) were used as the main control.
Pre-incubation of neutrophil suspension in CMFs
with 12.6 Hz frequency of alternating component caused
significant activation of respiratory burst in the neutro-
phils (Figures 4 and 5,Table 4). Due to this, the intensity
of chemiluminescence of the neutrophil suspension
increased by around 40%. On the contrary, 48.5 Hz
frequency had a significant (~20%) inhibitory effect
(Figures 4 and 5,Table 4).
Rapid addition (right after termination of CMFs
exposure) of water pre-treated by CMFs to saline and
then to neutrophil suspension led to different results for
two studied frequencies. Water pre-treated with 12.6 Hz
CMFs had a remarkable stimulating effect (Figures 6 and
7,Table 5), which was even more significant than the
direct treatment of neutrophil suspension with CMFs at
this frequency. Chemiluminescence intensity of neutro-
phil suspension was increased by ~ 70% when using this
supplement. Contrary to this, water pre-treated with
Table 3. Comparison of chemiluminescence intensity of neutro-
phil suspension after incubation in geomagnetic field (GMF
control) and inside the setup (Sham control) in the absence of
alternating component of the CMFs.
Control Intensity of chemiluminescence, V pValue
Geomagnetic field 3.38 ± 0.33
n = 6
Sham control 3.50 ± 0.42
n = 6
p= .631974
6V. V. NOVIKOV ET AL.

48.5 Hz field has no apparent effect (Figures 6 and 7,
Table 5). Chemiluminescence intensity did not actually
change after addition of these water samples to neutro-
phil suspension.
The delayed (by 5 and 40 minutes) addition of CMFs-
exposed water to neutrophil suspension (only if the
frequency of exposure was 12.6 Hz) caused almost the
same prominent stimulating effect as rapid addition of
such water (Table 6).
Discussion
As it was earlier mentioned, selection of parameters
(frequencies and amplitude) of alternating component
of MF for the samples was made taking into account the
most recent experimental data on the effects observed in
pure water after the exposure to combined magnetic
fields satisfying ICR condition (Liboff 1985) for hydro-
nium ion H
3
O
+
(protonated water) and its hydrated
forms (D’Emilia et al. 2017,2015).
Our experiments on direct action of CMFs on the
neutrophil suspension showed that pre-treatment with
CMFs adjusted to ICR frequency for hydronium ion
(48.5 Hz) inhibits respiratory burst, whereas CMFs
tuned to the hydrated form of this complex ion
(12.6 Hz), on the contrary, activates this process. The
inhibiting effect of 48.5 Hz frequency of CMFs could be
explained by proximity of this frequency corresponding
to ICR of hydronium ion (48.5 Hz) to three-valent iron
ion (Fe
3+
), 49.5 Hz (when the constant MF induction
was 60 µT), which had earlier shown a significant (~
60%) decrease of respiratory burst intensity in response
to activator fMLF (Novikov et al. 2020). One cannot
definitely suggest that the action of these CFMs is related
to this exact ion yet. More reliable experimental proofs
are required. However, this seems to be quite possible. In
this regard we should note that the principle of the
technique used in the work for CMFs effect detection,
luminol-dependent chemiluminescence, is based on
production of a large amount of different pro-oxidants
by activated neutrophils, which can interact with
0
25
50
75
100
125
150
175
∗
Intensity of chemiluminescence, %
Sham control 12.6 Hz 48.5 Hz
∗
Figure 4. Effect of combined magnetic fields with 60 µT static MF
on intensity of neutrophils chemiluminescence. Ordinate, che-
miluminescence intensity (maximal values) as percentage of the
main control (mean values ± standard deviations). The asterisks
show statistically significant differences from control group
values (P< .05). Abscissa, group numbers: 1 – setup control;
2 – experiment (12.6 Hz, 100 nT amplitude); 3 – experiment
(48.5 Hz, 100 nT amplitude).
Table 4. Intensity of chemiluminescence suspension of the neu-
trophils after direct action of the CMFs (60 µT static MF, 100 nT
alternating MF).
Alternating MF
Intensity of chemiluminescence, V
Control Exposed pValue
12.6 Hz 4.23 ± 0.13
n = 6
5.78 ± 0.50
n = 6
p= .000079
48.5 Hz 4.33 ± 0.29
n = 6
3.53 ± 0.30
n = 6
p= .001846
0 200 400 600 800
0
1
2
3
4
5
6
7
luminol, fMLF
Intensity of chemiluminescence, V
Time, s
a
control
exposed
0 200 400 600 800
0
1
2
3
4
5
control
exposed
Time, s
Intensity of chemiluminescence, V
b
luminol, fMLF
Figure 5. Effect of combined magnetic fields with 60 µT static MF on kinetics and intensity of chemiluminescence of neutrophils under
stimulation by fMLF peptide in presence of luminol (averaged curves, n = 6). A) 12.6 Hz frequency. B) 48.5 Hz frequency.
ELECTROMAGNETIC BIOLOGY AND MEDICINE 7

luminol and generate powerful chemiluminescence, its
intensity being dependent on functional state of the cells
(Vladimirov and Proskurina 2009). Special experiments
showed that one of the most powerful oxidants of lumi-
nol in this system is hypochlorite (Roshchupkin et al.
2006), which is produced by iron-containing myeloper-
oxidase enzyme that plays a direct role in generating
luminol-dependent chemiluminescence during respira-
tory burst (Vladimirov and Proskurina 2009). Partial
inhibition of this enzyme could explain the decrease of
respiratory burst intensity in the current work, i.e. the
effect of CMF close to resonance frequency of Fe
3+
ion.
It is important to note that this inhibiting regime of
CMFs is non-toxic for the neutrophils. At least the
viability was not decreased after such treatment and
remained as high as in control cases (more than 98%).
Neither did pH and conductivity of the solution used for
cell incubation (Table 2) change after such action. As the
measurement showed (Table 2), the modified Hank’s
solution supplied with 10 mM HEPES used in the
work has sufficient buffering capacity to compensate
possible changes of pH and conductivity arising in pro-
cess of short-term cultivation of neutrophils and possi-
ble harmful action of CMFs on these parameters.
To analyze the mechanism of action of CMFs at
48.5 Hz frequency, we must note that the inhibiting
effect of direct exposure to the CMFs on the cells is not
transduced through pre-treated water. Absence of the
effect of CMF at this frequency (48.5 Hz) on pure water
in the current experiments could be the consequence of
the action of this external CMFs on inner processes of
intrinsic electromagnetic oscillations in water having the
same frequency (48.5 Hz), as was shown by other
authors (D’Emilia et al. 2015). At least, one can suggest
(independent on the interpretation) that such CMFs
mode (static MF = 60 µT, alternating MF = 100 nT,
frequency = 48.5 Hz) has no apparent water-dependent
component in the mechanism of action on the neutro-
phil suspension.
On the contrary, the experiments of the current study
showed that the mode having stimulating effect on
respiratory burst intensity in neutrophil suspension
when used in preliminary treatment is CMFs tuned to
hydrated form of hydronium ions, Н
9
О
4+
(12.6 Hz).
This stimulating effect of CMFs is transduced through
the water (during pre-treatment of the water with CMFs
and its subsequent addition as a water-salt solution to
saline suspension of the neutrophils) similar to
a number of results of our earlier experiments
0
25
50
75
100
125
150
175 ∗
Intensity of chemiluminescence, %
Control 12.6 Hz 48.5 Hz
Figure 6. Effect of water pre-treated with combined magnetic
field with 60 µT constant component and 100 nT alternating MF
on neutrophils chemiluminescence intensity. Ordinate, chemilu-
minescence intensity (maximal values) as percentage of the
main control (mean values ± standard deviations). The asterisks
show statistically significant differences from control group
values (P< .05). Abscissa, group numbers: 1 – setup control;
2 – experiment (12.6 Hz); 3 – experiment (48.5 Hz).
0 200 400 600 800
0
1
2
3
4
5
6
7
Time, s
luminol, fMLF
a
control
exposed
Intensity of chemiluminescence, V
0 200 400 600 800
0
1
2
3
4
5
Time, s
luminol, fMLF
control
exposed
b
Intensity of chemiluminescence, V
Figure 7. Effect of water pre-treated with combined magnetic fields with 60 µT constant component and 100 nT alternating MF on
neutrophils chemiluminescence (averaged curves, n = 6). Rapid addition right after exposure to CMFs. A) 12.6 Hz frequency. B) 48.5 Hz
frequency.
8V. V. NOVIKOV ET AL.

(Fesenko et al. 2000,2002; Novikov and Fesenko 2001;
Novikov et al. 1999,2002) and numerous reports of
other authors (Ayrapetyan et al. 1994,2004; Foletti
et al. 2017; Fukushima et al. 2002). Such effects were
also recorded under action of more high-frequency elec-
tromagnetic fields (GHz band) (Fesenko et al. 1995�;
Fesenko and Gluvstein 1995�).
Preservation of stimulating properties of water
after termination of CMF treatment for at least sev-
eral tens of minutes (at least 40 minutes, as revealed
in the current study) presupposes the presence of
structural rearrangements or stable products of che-
mical reactions (maybe hydrogen peroxide) resulting
from such an action. Currently, this ability of water
to form structure at ambient temperature cannot be
considered as well-studied. The experimental data on
presence of long-term electromagnetic oscillations in
water which take place after termination of action of
external physical factors (weak magnetic field, GHz
radiation) can be promising (D’Emilia et al. 2015;
Fesenko and Gluvstein 1995; Liboff et al. 2017).
Considering the duration of the oscillations (tens of
minutes) and our data on the preservation of the
ability to stimulate respiratory burst in neutrophils
for at least 40 minutes after termination of exposure
to CMFs with 12.6 Hz frequency, it is tempting to
suppose that in this case changes in water structure
and generation of certain electromagnetic fields and
their action on a biological object take place.
In the context of the current study, it can be admitted
that the stimulating CMFs mode (static MF = 60 µT,
alternating MF = 100 nT, frequency = 12.6 Hz) has
explicit water component in the mechanism of its action
on neutrophil suspension. Transduction of stimulating
effect through the pre-treated water is the direct proof of
water participation in such effect of CMFs.
Considering the mechanism of action of CMFs
and CMFs-treated water on respiratory burst in neu-
trophils, it should be noted that the effect of different
priming (pre-activating) agents on the neutrophils
usually leads to intensification of phosphorylation of
molecular components of NADPH-oxidase, their
incorporation into the membrane and partial assem-
bly of the functional oxidase (El-Benna et al. 2008,
2016). The influence of factors studied in the work
on these processes could explain the observed effects.
The results of our experiments showed that water
pre-treated with physical factor as a certain CMFs
mode changed its properties (increased chemilumi-
nescence intensity of neutrophils suspension by 70%
compared to control samples). This gives an overall
evidence on the applicability of the approach under
current development to the analysis of the role of
water medium in perception of external physical fac-
tors. Their results could be conserved for a long time
(at least tens of minutes).
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