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ISSN 0006-3509, Biophysics, 2019, Vol. 64, No. 4, pp. 571–575. © Pleiades Publishing, Inc., 2019.
Russian Text © The Author(s), 2019, published in Biofizika, 2019, Vol. 64, No. 4, pp. 720–725.
On the Molecular Mechanisms of the Effect of a Zero Magnetic Field
on the Production of Reactive Oxygen Species
in Inactivated Neutrophils1
V. V. Novikov a, *, E. V. Yablokovaa, E. R. Valeevaa, and E. E. Fesenkoa
aInstitute of Cell Biophysics, Russian Academy of Sciences, Pushchino, Moscow oblast, 142290 Russia
*e-mail: docmag@mail.ru
Received March 17, 2019 ; revised April 15, 2 019; accepted Ap ril 24, 2019
Abstract—It is shown that the lower intensity of 2,7-dichlorodihydrofluorescein oxidation processes in inac-
tivated neutrophils exposed to hypomagnetic field (the residual static magnetic field 20 nT) is not related to
calcium-mediated mechanisms as shown by the absence of the effect of cell-permeant Ca2+ chelators, such
as 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester), on the inten-
sity of the process. This decrease is hardly likely to be caused by effects of a hypomagnetic field on phosphor-
ylation of NADPH-oxidase components, because addition of the protein kinase C inhibitor Ro 316233
decreases the fluorescence intensity of intracellular dichlorodihydrofluorescein little, if at all. Addition of
phospholipase C inhibitor U73122 causes a negligible decrease in ROS production in the control and exper-
iment, almost equally. Different concentrations of apocynin increase ROS production in nonactivated neu-
trophils and this effect is approximately two times lower under hypomagnetic conditions. The decrease in
ROS production is more pronounced in cells treated with a hypomagnetic field with the presence of rote-
none, indicating that the mitochondrial electron-transport chain is involved in the mechanism of the effect
of hypomagnetism.
Keywords: hypomagnetic field, neutrophils, reactive oxygen species, fluorescence, calcium ions, protein
kinase C, phospholipase C, mitochondria
DOI: 10.1134/S0006350919040122
There were several reports on lower production of
reactive oxygen species (ROS) under hypomagnetic
conditions in various cell types and at various exposure
regimes [1–4]. Previously, we showed that the mag-
netic screening of murine peritoneal neutrophils
(residual constant magnetic field below 20 nT) for
1.5 h caused a decrease in basal intracellular ROS pro-
duction, assessed by changes in the fluorescence of
oxidation products of 2,7-dichlorodihydrofluorescein
and dihydrorhodamine 123 [5]. This effect of a hypo-
magnetic (“zero”) field remained with the presence of
small concentrations of the respiratory burst activator,
formylated peptide N-formyl-Met-Leu-Phe or phor-
bol 12-myristate 13-acetate [5]. With regard to the fact
that the hypomagnetism effect manifested itself in
neutrophils without additional stimulation, thus being
caused by other factors than the impaired response of
neutrophils to respiratory burst activators, we under-
took a series of experiments with nonactivated neutro-
phils to identify putative molecular mechanisms that
underlie the effect of zero field. In contrast, experi-
ments with combined magnetic fields with certain
parameters [6, 7] reveal a stimulatory effect of these
magnetic fields on ROS production in neutrophil sus-
pension [8]. In this regard, comparison of the key
aspects of the molecular mechanisms mediating the
action of combined magnetic fields [9–12] and zero
magnetic field is of special interest.
At this phase of the study we employed fluores-
cence spectrometry with the well-studied cell-per-
meant ROS probe, 2,7-dichlorodihydrofluorescein
diacetate (H2DCF-DA) [13–16] for studying the
effects of hypomagnetism. The probe penetrates into
the cell, where it is deacetylated to H2DCF by intra-
cellular esterases. H2DCF fluoresces weakly, but its
reactions with oxidizers yield intensely fluorescing
dichlorofluorescein.
The chief ROS producers in nonactivated neutro-
phils are the NOX2-containing phagocyte oxidase
complex and occasional mitochondria [17–19]. The
NADPH oxidase assembly and activity directly
depend on the activities of phospholipase C and pro-
Abbreviations: ROS, reactive oxygen species, H2DCFDA, 2,7-
dichlorodihydrofluorescein diacetate; BAPTA AM, 1,2-bis(2-
aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(ace-
toxymethyl ester).
CELL BIOPHYSICS
572
BIOPHYSICS Vol. 64 No. 4 2019
NOVIKOV et al.
tein kinase C. They are often conjugated with calcium-
mediated regulatory mechanisms [20]. Therefore, we
used appropriate chemical inhibitors for analysis of
the molecular mechanism of the effect of hypomagne-
tism on ROS production in nonactivated neutrophils.
MATERIALS AND METHODS
Preparation of the neutrophil suspension. Experi-
ments were carried out with peritoneal neutrophils
isolated from 24–26 g male CD-1 laboratory mice.
The mice were received from the vivarium of the
Branch of the Shemyakin–Ovchinnikov Institute of
Bioorganic Chemistry, Pushchino, Moscow oblast,
Russia. Opsonized zymosan (Zymosan A from Sac-
charomyces cerevisiae, Sigma, United States;
5 mg/mL, 150 μL) was injected intraperitoneally.
Twelve hours later, the animals were euthanized by
ulnar dislocation and the abdominal cavity was
flushed with 3 mL of cooled calcium-free Hanks’
solution. The exudate was collected with a pipette and
centrifuged at 600 g for 10 min. The supernatant was
poured off and the sediment was dissolved in 2 mL of
calcium-free Hanks’ solution and incubated at 4°C for
1 h. Cells were counted in a hemocytometer. Cell via-
bility was tested with the vital stain trypan blue. The
percentage of living cells was no less than 98%. Sam-
ples for experiments were prepared by diluting neutro-
phil suspensions with standard Hanks’ solution
(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 the concentration 106 cells/mL.
Exposure of neutrophil suspension to hypomagnetic
field. Neutrophils were incubated in Eppendorf-com-
patible polypropylene tubes at the concentration of
106/mL in the volume 250 μL in darkness. The tem-
perature was controlled with a circulation bath. Typi-
cally, the incubation lasted for 1.5 h. Control samples
were kept in the local geomagnetic field with the
steady component of approximately 42 μT and mag-
netic noise 15–50 nT at 50 Hz.
The device that provided the hypomagnetic condi-
tions consisted of three 1-mm thick coaxial cylindrical
permalloy magnetic field screens. The residual con-
stant magnetic field measured with a Mag-03 MS 100
ferroprobe (Bartington, United Kingdom) did not
exceed 20 nT. Control (geomagnetic f ield) and exper-
imental (hypomagnetic field) samples, ten in each
group, were incubated simultaneously. The experi-
ments were conducted in no less than three replica-
tions.
Prior to incubation, some samples were individu-
ally supplemented with various chemicals: cell-per-
meant calcium-chelating agent 1,2-bis(2-aminophe-
noxy)ethane-N,N,N',N'-tetraacetic acid tetra-
kis(acetoxymethyl ester) (BAPTA AM; Sigma, United
States), final concentrations 2 or 4 μM; inhibitor of
the mitochondrial electron transport chain rotenone
(Sigma, United States), 1 μM; inhibitor of NADPH
oxidase apocynin, 20 or 500 μM; phospholipase C
inhibitor U73122 (Sigma, United States), 2 μM; and
protein kinase C inhibitor bisindolylmaleimide IV (Ro
31-6233, Sigma, United States), 1 μM.
Fluorescence assay of intracellular ROS. After 1.5-
h hypomagnetic treatment of neutrophil suspension,
the ROS probe H2DCF-DA (Sigma, United States)
was added to the final concentration of 0.01 mg/mL.
The samples were further incubated in darkness, to
minimize dye photooxidation, at 37°C for 30 min. The
cells were then washed with Hanks’ solution by centri-
fuging them at 600 g and room temperature for
10 min. One milliliter of the medium was added to the
sediment and resuspended. Fluorescence spectra were
recorded with a Thermo Scientific Lumina Fluores-
cence Spectrometer (Thermo Fisher Scientific,
United States) with excitation at 488 nm.
Statistical evaluation of the results was performed
using the Student’s t test. Some results are presented at
the maximum fluorescence intensity at 528 nm with
reference to the basal control (TN: neutrophils with-
out additives not exposed to zero field), taken to be
100%.
RESULTS AND DISCUSSION
Hypomagnetic treatment of peritoneal neutrophils
(approximately 2000-fold geomagnetic field weaken-
ing) significantly (by 25%) decreased the f luorescence
of intracellular dichlorofluorescein (Figs. 1 and 2). We
note that the spectrum shape and the wavelength of
the maximum probe fluorescence in the zero magnetic
field were the same as in the control. They also
remained the same with the presence of chemicals
used in subsequent experiments.
Addition of 2 or 4 μM BAPTA AM as the cell-per-
meant calcium chelating agent to the neutrophil incu-
bation medium exerted practically no effect on the
production of intracellular ROS by neutrophils in
experimental (hypomagnetic field) or control (local
magnetic field) samples (Fig. 2). This observation
indicates the independence of the effect of hypomag-
netism on the intracellular calcium level or on cal-
cium-dependent mechanisms in general. It differenti-
ates this effect from that of combined magnetic fields
on the same object, neutrophils, where it is entirely
inhibited even by low BAPTA AM concentrations [9].
Addition of 1 μM rotenone, an inhibitor of mito-
chondrial electron transport chain, reduced intracel-
lular ROS production by 20% in the control and much
more, by 35%, in the experiment (Fig. 3). This obser-
vation indicates that mitochondria are involved in the
effect of the hypomagnetic field and confirms their
role as one of the main ROS producers in nonactivated
BIOPHYSICS Vol. 64 No. 4 2019
ON THE MOLECULAR MECHANISMS OF THE EFFECT OF A ZERO MAGNETIC FIELD 573
neutrophils [17, 18]. It should be mentioned in this
regard that the effect of combined magnetic fields does
not depend on the addition of this inhibitor to the neu-
trophil culture medium [12].
Addition of the phospholipase C inhibitor U73122
reduced ROS production in the experiment and con-
trol approximately equally, by 20% (Fig. 4). Addition
of the protein kinase C inhibitor Ro 31-6233 exerted
practically no effect on intracellular dichlorofluores-
cein fluorescence (Fig. 5). These data are consistent
with the aforementioned independence of the zero-
field effect on calcium-mediated regulatory mecha-
nisms. They do not confirm the involvement of phos-
phorylation of NADPH oxidase components in the
mechanisms of the effect.
Addition of various concentrations of the specific
NADPH oxidase inhibitor apocynin caused a para-
doxical increase in ROS production in nonactivated
neutrophils. The increase was in direct proportion to
inhibitor concentration and was approximately twice
as large as that in the control samples (Fig. 6). This
phenomenon can be explained on the basis of the fact
that the NADPH oxidase-inhibiting activity of
apocynin requires its dimerization, which occurs in
the presence of peroxidases and hydrogen peroxide
[21, 22], e.g., in activated neutrophils. In nonactivated
cells, apocynin can enhance ROS production owing to
its prior oxidation, generating transient free radicals
[23]. Concerning our experiments, it is pertinent to
mention that the rate of this process under experimen-
tal conditions (after hypomagnetic treatment)
decreases significantly.
To sum up, our results point to a decrease in the
rates of oxidation of the H2DCF fluorescence probe
in neutrophils under hypomagnetic treatment,
involvement of mitochondria and their electron-
transport chain in the zero-field effect, putative slow-
down of the oxidation of some other compounds (e.g.,
apocynin), and independence of the zero-field effect
on calcium-dependent regulatory mechanisms. Thus,
Fig. 1. Dichlorofluorescein fluorescence spectra in neu-
trophil suspensions: control (curve 1) and exposed to
hypomagnetic field (curve 2). Dashed curves indicate
standard deviations.
40 000
30 000
20 000
10 000
0500 600 650550
Wavelength, nm
Fluorescence, a. u.
1
2
Fig. 2. The effect of a hypomagnetic field on dichlorof lu-
orescein fluorescence in neutrophil suspensions with and
without the presence of the cell-permeant calcium chelat-
ing agent BAPTA AM. Y-axis: maximum fluorescence
intensity as percentage of the basal control (mean values
and standard deviations, n = 10). X-axis: 1, control group;
2, experiment. Open bars: no additives; gray bars: 2 μM
BAPTA AM; dark bars: 4 μM BAPTA AM. * The differ-
ences between groups are significant at p < 0.05.
125
100
75
50
25
012
Fluorescence, %
Group No.
Fig. 3. The effect of hypomagnetic field on dichlorofluo-
rescein f luorescence in neutrophil suspensions with and
without the presence of rotenone. Y-axis: maximum fluo-
rescence intensity as the percentage of the basal control
(mean values and standard deviations, n = 10). X-axis:
1,control group; 2, experiment. Op en bar s: no additives;
gray bars: 1 μM rotenone. *Differences between groups
significant at p < 0.05. **Differences within a group signif-
icant at p < 0.05.
125
100
75
50
25
012
Fluorescence, %
Group No.
574
BIOPHYSICS Vol. 64 No. 4 2019
NOVIKOV et al.
a metabolic basis may be conjectured for this effect. In
all these features, the zero-field effect differs dramati-
cally from the effects of combined magnetic fields with
certain parameters [6, 7], which show an association
with regulatory calcium-dependent mechanisms [9]
that control the respiratory burst in neutrophils.
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ON THE MOLECULAR MECHANISMS OF THE EFFECT OF A ZERO MAGNETIC FIELD 575
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Translated by Victor Gulevich
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