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ISSN 0006-3509, Biophysics, 2019, Vol. 64, No. 2, pp. 209–213. © Pleiades Publishing, Inc., 2019.
Russian Text © The Author(s), 2019, published in Biofizika, 2019, Vol. 64, No. 2, pp. 290–295.
The Effects of Various Chemical Agents on Priming of Neutrophils
Exposed to Weak Combined Magnetic Fields
V. V. Novikova, *, E. V. Yablokovaa, N. I. Novikovab, and E. E. Fesenkoa
aInstitute of Cell Biophysics, Division of Federal Research Center Pushchino Scientific Center of Biological Investigations
of the Russian Academy of Sciences, Institutskaya ul. 3, Pushchino, Moscow oblast, 142290 Russia
bBranch of Shemiakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences,
prosp. Nauki 6, Pushchino, Moscow oblast, 142290 Russia
*e-mail: docmag@mail.ru
Received January 18, 2019; revised January 18, 2019; accepted January 29, 2019
Abstract⎯It has been shown that various chemical agents (ethylenediaminetetraacetic acid, zinc sulphate,
ethyl alcohol, and rotenone) have different effects on preactivation (priming) of neutrophils that develop
under weak combined collinear static and alternating magnetic fields (combined magnetic fields: a static field
of 42 μT and an alternating field of 0.86 μT with the sum of frequencies 1.0, 4.4 and 16.5 Hz). Low concen-
trations (0.05%) of ethylenediaminetetraacetic acid decrease the intensity of luminol dependent chemilumi-
nescence of neutrophils in response to an activator of the respiratory burst the peptide N-formyl-Met–Leu–
Phe under the action of combined magnetic fields to a lesser extent than in the control. In contrast, ethyl alco-
hol (0.45%) and zinc sulphate (0.1 mM) have a greater effect on this process under the action of combined
magnetic fields. Rotenone (1 μM) has a weak effect on neutrophil chemiluminescence both under the action
of combined magnetic fields and in the control.
Keywords: weak magnetic field, neutrophils, respiratory burst, free radicals, reactive oxygen species, ethylene-
diaminetetraacetic acid, rotenone, zinc sulphate, ethyl alcohol, chemiluminescence
DOI: 10.1134/S000635091902012X
INTRODUCTION
A number of authors have considered the possibil-
ity of the influence of magnetic fields on the produc-
tion of reactive oxygen species as a promising
approach to the analysis of the mechanisms of their
biological effects [1, 2]. We previously found an
increase in the generation of free radicals and reactive
oxygen species as a result of action of the combined
constant and low-frequency alternating magnetic
fields (CMF) with a very weak variable component
(less than 1 μT) [3–7] in experiments on whole blood
and neutrophils by methods of activated chemilumi-
nescence and fluorescence spectroscopy. In particu-
lar, in these studies, the priming effect (preactivation
of respiratory burst in neutrophils) was found of a
combined weak constant field (42 μT) and a low-fre-
quency alternating (1.0, 4.4 and 16.5 Hz; 0.86 μT)
magnetic field collinear to it, which was manifested as
a more pronounced increase in chemiluminescence of
the suspension of neutrophils after their pretreatment
by CMF in response to the introduction of the bacte-
rial peptide N-formyl-Met–Leu–Phe or a phorbol
ester phorbol 12-meristat-13-acetate in the presence
of luminol [5]. Only a small increase in lipid peroxida-
tion in neutrophils after 1 hour action of CMF was
detected [7]. There was no correlation between this
increase in the intensity of lipid peroxidation and the
process of functional preactivation of neutrophils as a
result of the action of CMF, since an inhibitor of lipid
peroxidation ionol (10 μM) did not reduce the prim-
ing index in this case. The priming index was also not
reduced by the preliminary addition of a singlet oxy-
gen scavenger histidine (0.1 and 1 mM) [7] or a
hydroxyl radical scavenger dimethyl sulfoxide at con-
centrations up to 1 mM [8]. A significant inhibitory
effect on the intensity of chemiluminescence of the
suspension of neutrophils was exerted by the myelop-
eroxidase inhibitor sodium azide (0.1 mM), while
priming in its presence did not develop [7]. Chemilu-
minescence was also effectively reduced by the inhibi-
tor of NADPH oxidase apocynin and the hypochlorite
scavenger edaravone [4]. It has been shown that low
concentrations of intracellular calcium chelator
BAPTA-AM blocked this effect of CMF [8]. At the
same time, the level of extracellular calcium practi-
cally did not affect the severity of respiratory burst
priming. It follows that one of the key points of the
mechanism of action of weak CMF on neutrophils
may be an increased outflow of calcium ions into the
Abbreviations: CMF, combined magnetic fields (static and alter-
nating of low frequency).
CELL BIOPHYSICS
210
BIOPHYSICS Vol. 64 No. 2 2019
NOVIKOV et al.
cytosol from intracellular depots. The second of the
experimentally established key moments of the mech-
anism of neutrophil preactivation in a weak CMF is
the pronounced dependence of the expression of this
effect on the concentration of atmospheric gases [9]. It
was shown that the preliminary mild partial degassing
of the suspension of neutrophils at atmospheric gas
pressure of 640 mm Hg leads to a significant (four-
fold) decrease in the degree of influence of
CMF, but practically did not affect the ability of cells
to generate a respiratory burst in response to the acti-
vator (n-formyl-Met–Leu–Phe peptide) in the con-
trol [9].
In this regard, for a comprehensive analysis of the
mechanisms of action of weak CMF on neutrophils, it
is important to further study this effect of CMF in the
presence of chemical additives that block various links
in the regulation of respiratory burst and neutrophil
metabolism.
MATERIALS AND METHODS
Preparation of the Suspension of Neutrophils
The work was performed on murine peritoneal
neutrophils. To obtain peritoneal neutrophils, we used
laboratory male mice of the CD-1 line weighing 22–
25 g obtained from the laboratory of animal nursery
Pushchino (BIBCh RAS, Pushchino, Moscow
oblast). A suspension of opsonized zimozan (150 μL)
at a concentration of 5 mg/mL (Zymozan A from Sac-
charomyces carevisiae, Sigma, United States) was
injected into the peritoneal cavity of the mice. The
animals were killed 12 hours later by ulnar dislocation
and their abdominal cavity was washed with 3 mL of
chilled Hanks’ solution without calcium. The exudate
was collected by the pipette and centrifuged for 10 min
at 600 g. The supernatant was decanted and the pre-
cipitate was diluted in 2 mL of Hanks’ calcium-free
solution and left for 60 min at 4°C. The number of iso-
lated cells was counted with a Goryaev chamber. Cell
viability was determined using the vital dye trypan
blue. The content of living cells was not less than 98%.
The samples for the experiments were obtained by
diluting the suspension of neutrophils in a standard
Hanks’ medium (138 mM NaCl, 6 mM KCl, 1 mM
MgSO4, 1 mM Na2HPO4, 5 mM NaHCO3, 5.5 mM
glucose, 1 mM CaCl2, 10 mM HEPES, pH 7.4;
Sigma, United States) to a concentration of
106cells/mL.
Exposure of the Suspension of Neutrophils
to a Magnetic Field
The samples of neutrophils (0.25 mL) at a concen-
tration of 106 cells/mL were incubated at 37.0 ± 0.2°C
in cuvettes for subsequent chemiluminescence mea-
surement. The typical incubation time was 1 hour. The
required temperature was maintained by a circulation
thermostat.
The samples of the control groups were kept in a
local geomagnetic field with a constant component of
~42 μT and a magnetic background level at 50 Hz of
15–50 nT corresponding to these parameters in the
experimental groups, except for the component of the
alternating field, which was set artificially in the exper-
iments.
The installation for the action of weak magnetic
fields consisted of two pairs of coaxially arranged
Helmholtz rings with a diameter of 140 cm (the dis-
tance between the rings of one pair was 70 cm) ori-
ented so that the directions of the magnetic fields
formed by them were located along the vector of the
geomagnetic field. A constant current was applied to
one pair of the rings to form a predetermined magnifi-
cation of the constant component of the magnetic
field of 42 ± 0.1 μT. An electric current from the pro-
grammable generator of sinusoidal signals (digital-to-
analog converter circuit board L-791 of the company
L-Card, Russia) was applied to the second pair of
rings for the formation of the alternating component
of the field. The base amplitude of the alternating
component was 860 ± 10 nT. A three-frequency signal
of 1.0, 4.4, and 16.5 Hz was used in the experiments,
which showed activity in previous experiments [10,
11], with amplitudes of individual frequencies of 600,
100 and 160 nT, respectively. The acting magnetic
fields were directly measured with a Mag-03 MS100
flux-gate sensor (Bartington, UK).
Prior to incubation, various chemical additives
were added to a part of the samples separately: the
nonselective metal chelator ethylenediaminetetra-
acetic acid disodium salt dihydrate (Na2-EDTA)
(Sigma, United States) at concentrations of 0.05 and
0.5%; the mitochondrial respiratory chain inhibitor
rotenone (Sigma, United States) at a concentration of
1 μM; a blocker of proton channels (the channel
VSOP/HV1) ZnSO4 at a concentration of 100 μM [12,
13] or a scavenger of hydroxyl radicals ethyl alcohol at
a concentration of 0.45%.
The Recording of Chemiluminescence
After a 1-hour incubation of the suspension of neu-
trophils, the intensity of chemiluminescence of the
samples was measured in control and experimental
cases after the addition of a solution of luminol (Enzo
Life Sciences, United States) at a concentration of
0.35 mM and an activator for the generation of reactive
oxygen species the chemotactic peptide N-formyl-
Met–Leu–Phe (Sigma, United States) at a concen-
tration of 1 μM. A Lum-5773 chemiluminometer
(LLC Disoft, Russia) was used in the work. The Pow-
erGraph program was used to analyze the chemilumi-
nescence data. Some of the results are presented as a
percentage relative to the amplitudes of the chemilu-
BIOPHYSICS Vol. 64 No. 2 2019
THE EFFECTS OF VARIOUS CHEMICAL AGENTS 211
minescent response in the control taken for 100%. For
clarity, the priming index equal to the ratio of the max-
imum intensity of chemiluminescence in the experi-
ment to the corresponding value in the control was
used.
The results were statistically processed using the
student’s t-test.
RESULTS AND DISCUSSION
Pre-incubation of the suspension of neutrophils in
the combined magnetic field caused a significant pre-
activation of the respiratory burst (the index of the
priming of neutrophils was ~1.5) (Fig. 1 and 2). The
addition of the metal chelator EDTA to the incubation
medium resulted in a significant decrease in the che-
miluminescence intensity in both the experimental
and control samples (Fig. 2). A high concentration of
EDTA (0.5%) reduced the intensity of chemilumines-
cence in the control by approximately five times and
eliminated the differences between the control and
experimental samples (Fig. 2). At low concentrations
of ETDA (0.05%), the intensity of chemilumines-
cence of the samples was also reduced, but to a greater
extent in the control cases (by 66%). Chemilumines-
cence intensity decreased in the experiment (the pre-
liminary action of CMF) by only 50%. This distinc-
tion led to the growth of the index of priming of the
neutrophils under the action of CMF in the presence
of 0.05% ETDA from 1.47 to 2.18 (48%). This result
corresponds to our earlier observations on the weak
dependence of the priming effect of CMF on the con-
centration of extracellular calcium after the addition of
various amounts of calcium to the cultivation medium
of neutrophils and the leading role of intracellular cal-
cium in these processes [8].
The addition of rotenone to the incubation
medium of neutrophils had little effect on the intensity
of the respiratory burst in both the control and exper-
iment (Fig. 3). The differences between the groups due
to the action of CMF fully remained in this case
(Fig. 3). This does not allow one to draw a conclusion
about the participation of the mitochondrial respira-
tory chain in this effect under the used CMF regime.
The addition of zinc sulfate, a blocker of
VSOP/HV1 proton channels, reduced the intensity of
chemiluminescence of neutrophils in the control by
22% and in the experiment by 34% (Fig. 4). The
higher degree of attenuation of the respiratory burst in
the experiment after the action of CMF under the
inhibition of proton channels seems to be due to their
role in maintaining the production of a superoxide and
the supply of calcium into the cells by preventing
depolarization of the membrane and cellular acidifi-
cation caused by the electrogenicity of NADPH oxi-
dase carrying out electron transfer through the mem-
brane [12, 13]. Obviously, with the increased load as a
result of the pre-activation process (experimental
group), the blocking of these channels can occur to a
greater extent than in the less active state (control
group), which was observed in the experiment. Appar-
ently, in this regard, the index of priming of neutro-
phils under the action of the CMF in the presence of
100 mM zinc sulfate decreased by 16%.
The addition of ethyl alcohol (0.45%) to the
medium for neutrophil incubation reduced the inten-
Fig. 1. The impact of the CMF on the kinetics and inten-
sity of chemiluminescence of neutrophils from the perito-
neal cavity of the mouse after stimulation of the cells with
1 μM N-formyl-Met–Leu–Phe in the presence of lumi-
nol: 1, control; 2, experiment.
0
1
2
3
4
5
6
7
8000 200 400 600
Time, s
Luminol, N-formyl-Met–Leu–Phe
2
1
Chemiluminescence intensity, arb.un
Fig. 2. The effect of EDTA on the intensity of chemilumi-
nescence of neutrophils: control, gray column; experiment
(action of CMF), dark column. The abscissa is the con-
centration of EDTA; the ordinate is the intensity of chemi-
luminescence (maximum value) in percent in relation to
the control (mean values and standard deviations). The
asterisk marks significant differences from the control
groups (P < 0.05).
0
150
100
50
200
*
*
0 0.05 0.50
Concentration of EDTA, %
Chemiluminescence intensity, %
212
BIOPHYSICS Vol. 64 No. 2 2019
NOVIKOV et al.
sity of their chemiluminescence by 45% in the control
and by 60% in the experiment (Fig. 5). Ethyl alcohol
in the used concentration is a well-studied scavenger
of hydroxyl radicals [14]. Previously, we used dimethyl
sulfoxide for this purpose [8] and did not detect its
effect up to a concentration of 1 mM, although it is
known that dimethyl sulfoxide is able to effectively
scavenge hydroxyl radicals at relatively low concentra-
tions (0.02–2.5 mM) [15, 16]. We obtained new data
in the experiments with ethyl alcohol. It was shown
that the priming index of neutrophils under the action
of CMF in the presence of 0.45% alcohol was reduced
by 27%. It is not yet clear whether this effect of ethyl
alcohol is due to the scavenging of hydroxyl radicals
and their role in the mechanism of action of weak
CMF or, no less likely, due to its other physical and
chemical properties. However, it is obvious that ethyl
alcohol in the studied concentration significantly
reduced the effectiveness of CMF.
Because the processes and mechanisms of short-
term priming of neutrophils are well studied [17–20],
there is a high probability of identifying the main, and
possibly initiating, links of the priming effect of weak
CMF. It is obvious that the increase in the production
of free radicals by neutrophils as a result of the action
of a CMF is primarily due to the resonance-like reac-
tions that have been long studied by biophysici sts [21–
37] and is hardly due to the mechanism of radical pairs
in its classical version [2, 38] or the magnetochemical
mechanism [39], as supported by both the small value
of the alternating component of CMF and the depen-
dence of the effect on its frequencies.
Fig. 3. The effect of rotenone on the intensity of chemilu-
minescence of neutrophils: control, gray column; experi-
ment (action of CMF), dark column. The abscissa is the
concentration of rotenon; the ordinate is the intensity of
chemiluminescence (maximum value) in percent in rela-
tion to the control (mean values and standard deviations).
The asterisk marks significant differences from the control
groups (P < 0.05).
0
150
100
50
200
*
*
01
Concentration of rotenone, µM
Chemiluminescence intensity, %
Fig. 4. The effect of zinc sulfate on the intensity of chemi-
luminescence of neutrophils: control, gray column; exper-
iment (action of CMF), dark column. The abscissa is the
concentration of ZnSO4; the ordinate is the intensity of
chemiluminescence (maximum value) in percent in rela-
tion to the control (mean values and standard deviations).
0
150
100
50
200
*
*
01
Concentration of ZnSO4, mM
Chemiluminescence intensity, %
Fig. 5. The effect of ethyl alcohol on the intensity of che-
miluminescence of neutrophils: control, gray column;
experiment (action of CMF), dark column. The abscissa is
the concentration of C2H5OH rotenon; the ordinate is the
intensity of chemiluminescence (maximum value) in per-
cent in relation to the control (mean values and standard
deviations).
0
150
100
50
200
*
*
0 0.45
Concentration of ethanol, %
Chemiluminescence intensity, %
BIOPHYSICS Vol. 64 No. 2 2019
THE EFFECTS OF VARIOUS CHEMICAL AGENTS 213
COMPLIANCE WITH ETHICAL STANDARDS
@Conflict of interests.@ The authors declare that
they have no conflict of interest.
@Statement on the welfare of animals.@ All appli-
cable international, national, and/or institutional
guidelines for the care and use of animals were fol-
lowed.
REFERENCES
1. M. O. Mattsson and M. Simkó, Frontiers Publ. Health
2, 132 (2014).
2. F. S. Barnes and B. Greenebaum, Bioelectromagnetics
36, 45 (2015).
3. V. V. Novikov, E. V. Yablokova, and E. E. Fesenko, Bio-
physics (Moscow) 60 (3), 429 (2015).
4. V. V. Novikov, E. V. Yablokova, and E. E. Fesenko, Bio-
physics (Moscow) 61 (1), 105 (2016).
5. V. V. Novikov, E. V. Yablokova, and E. E. Fesenko, Bio-
physics (Moscow) 61 (3), 429 (2016).
6. V. V. Novikov, E. V. Yablokova, and E. E. Fesenko, Bio-
physics (Moscow) 61 (6), 959 (2016).
7. V. V. Novikov, E. V. Yablokova, G. V. Novikov, and E.
E. Fesenko, Biophysics (Moscow) 62 (5), 759 (2017).
8. V. V. Novikov, E. V. Yablokova, and E. E. Fesenko, Bio-
physics (Moscow) 62 (3), 440 (2017).
9. V. V. Novikov, E. V. Yablokova, and E. E. Fesenko, Bio-
physics (Moscow) 63 (2), 193 (2018).
10. V. V. Novikov, G. V. Novikov, and E. E. Fesenko, Bio-
electromagnetics 30, 343 (2009).
11. V. V. Novikov, V. O. Po no marev, G . V. No vi ko v, et al. ,
Biophysics (Moscow) 55 (4), 565 (2010).
12. A. El. Chemaly, Y. Okochi, M. Sasaki, et al., J. Exp.
Med. 207 (1), 129 (2010).
13. N. Demaurex, WIREs Membr. Transp. Signal. 1, 3
(2012).
14. V. N. Mal’tseva, N. V. Avkhacheva, B. F. Santalov, and
V. G. Safonova, Tsitologiya 48 (12), 1000 (2006).
15. Yu. A. Vladimirov and E. V. Proskurina, Usp. Biol.
Khim. 49, 341 (2009).
16. D. I. Roshchupkin, N. S. Belakina, and M. A. Murina,
Biophysics (Moscow) 51 (1), 79 (2006).
17. G. I. Klebanov and Yu. A. Vladimirov, Usp. Sovrem.
Biol. 119 (5), 462 (1999).
18. A. N. Mayanskii, Tsitokines Inflamm. 6 (3), 3 (2007).
19. J. El-Benna, P. M. Dang, and M. A. Gougerot-Pocid-
alo, Semin. Immunopathol. 30, 279 (2008).
20. J. El-Benna, M. Hurtado-Nedelec, V. Marzaioli, et al.,
Immunol. Rev. 273 (1), 180 (2016).
21. C. F. Blackman, S. G. Benane, D. E. House, and
W. T. Joines, Bioelectromagnetics 6 (1), 1 (1985).
22. A. R. Liboff, J. Biol. Phys. 13, 99 (1985).
23. V. V. Lednev, Bioelectromagnetics 12 (2), 71 (1991).
24. V. V. Novikov and M. N. Zhadin, Biofizika 39 (1), 45
(1994).
25. V. V. Novikov, Biofizika 39 (5), 825 (1994).
26. V. V. Novikov, Biofizika 41 (5), 973 (1996).
27. M. N. Zhadin, V. V. Novikov, F. S. Barnes, and
N.\ F. Pergola, Bioelectromagnetics 19, 41 (1998).
28. V. V. Novikov and E. E. Fesenko, Biophysics (Moscow)
46 (2), 233 (2001).
29. N. A. Belova and V. A. Panchelyuga, Biophysics (Mos-
cow) 55 (4), 661 (2010).
30. E. D’Emilia, L. Giuliani, M. Ledda, et al., Electro-
magn. Biol. Med. 36 (1), 55 (2017).
31. A. Pazur, Electromagn. Biol. Med. 37 (2), 100 (2018).
32. N. V. Bobkova, V. V. Novikov, N. I. Medvinskaya, et al.,
Electromagn. Biol. Med. 37 (3), 127 (2018).
33. E. G. Novoselova, V. V. Novikov, S. M. Lunin, et al.,
Electromagn. Biol. Med. 38 (1), 74 (2019).
https://doi.org/10.1080/15368378.2018.1545667
34. G. Khokhlova, T. Abashina, N. Belova, et al., Bioelec-
tromagnetics 39 (6), 485 (2018).
35. V. N. Binhi, Bioelectromagnetics 21, 34 (2000).
36. V. N. Binhi and F. S. Prato, Bioelectromagnetics 38, 41
(2017).
37. V. N. Binhi and F.S. Prato, Sci. Reports 8, 13495
(2018).
38. P. J. Hore and H. Mouritsen, Annu. Rev. Biophys. 45,
299 (2016).
39. V. N. Binhi, Principles of Electromagnetic Biophysics
(Fizmatlit, Moscow, 2011) [in Russian].
Translated by E. Puchkov
SPELL: 1. ЩЛ
