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ISSN 0006-3509, Biophysics, 2021, Vol. 66, No. 3, pp. 434–437. © Pleiades Publishing, Inc., 2021.
Russian Text © The Author(s), 2021, published in Biofizika, 2021, Vol. 66, No. 3, pp. 511–515.
The Kinetics of the Production of Reactive Oxygen Species
by Neutrophils after Incubation in a Hypomagnetic Field
V. V. Novikova, *, E. V. Yablokovaa, I. A. Shaeva, and E. E. Fesenkoa
aInstitute of Cell Biophysics, Russian Academy of Sciences, Moscow Region, 142290 Russia
*e-mail: docmag@mail.ru
Received February 15, 2021; revised February 26, 2021; accepted February 27 02, 2021
Abstract—It has been shown that 30-min incubation of neutrophils in the presence of a near null magnetic
field produced with the use of permalloy for magnetic shielding (a residual static magnetic field not greater
than 20 nT) leads to a significant decrease (by 48%) in the intensity of lucigenin-dependent chemilumines-
cence measured directly after removal of the hypomagnetic field. At 20 min after being in hypomagnetic con-
ditions (followed by a 20 min of incubation of neutrophils in the geomagnetic field), the degree of the differ-
ences between the control and experimental samples is completely preserved. When the time periods of incu-
bation of experimental samples in the geomagnetic field (static magnetic field 44 μT) were extended (40 min
and 60 min) after exposure to a near-null magnetic field, the differences between experimental and appropri-
ate control groups of samples were smaller, by up to 32 and 22%.
Keywords: hypomagnetic field, geomagnetic field, neutrophils, reactive oxygen species, lucigenin, chemilu-
minescence
DOI: 10.1134/S000635092103012X
INTRODUCTION
Free radicals and other reactive oxygen species
(ROS) can be potential molecules for modulating bio-
logical functions in response to a “zero” magnetic
field [1]. The literature reports a decrease in ROS pro-
duction under hypomagnetic conditions in various
types of cells [2–5]. We have previously shown that the
exposure of peritoneal neutrophils in mice under mag-
netic screening under hypomagnetic conditions causes
a decrease in intracellular ROS production, which is
recorded by a change in the fluorescence intensity of
the oxidation products of 2,7-dichlorodihydrofluores-
cein and dihydrorhodamine 123 [6–8]. This effect of
the hypomagnetic field was manifested in experiments
on neutrophils without additional stimulation by
chemical activators of the respiratory burst and, there-
fore, is not due to an impaired response of neutrophils
to these stimuli [6, 7]. To assess the radical-producing
ability of neutrophils after the action of a zero field, we
also used another method, that is, the method of acti-
vated chemiluminescence using lucigenin, a selective
superoxide anion probe [9, 10], which showed a signif-
icant decrease in the intensity of their lucigenin-
dependent chemiluminescence under these condi-
tions [11].
When analyzing the effects of hypomagnetic con-
ditions on the production of ROS by biological
objects, one of the important and practically unex-
plored issues is the experimental assessment of the
duration of the aftereffect of these conditions, i.e., the
determination of the time period in which the residual
effects of the zero magnetic field (MF) appear after the
termination of its direct actions. To study the temporal
dynamics of ROS production in this work, we used the
method of lucigenin-dependent chemiluminescence,
which has already proved itself well in previous studies
on this topic [11]; it allows a relatively short period
(several minutes) to assess the change in the rate of
ROS production [9–11].
MATERIALS AND METHODS
Obtaining a suspension of neutrophils. The work
was performed on peritoneal neutrophils of mice. To
obtain neutrophils, laboratory mice, males of the CD-
1 line weighing 24–26 g from the nursery of laboratory
animals of the Institute of Cell Biophysics of the Rus-
sian Academy of Sciences (Pushchino, Moscow
region), were used; 150 μL of a suspension of
opsonized zymosan with a concentration of 5 mg/mL
(Zymozan A from Saccharomyces carevisiae, Sigma,
United States) was injected into the peritoneal cavity
of the mouse. Twelve hours later, the animals were
euthanized by the method of cervical dislocation and
their abdominal cavity was washed with 4 mL of
chilled Hanks solution without calcium. The exudate
was collected with a pipette and centrifuged for 5 min
at 600 g. The supernatant was decanted and the pre-
cipitate was diluted in 4 mL of calcium-free Hanks
CELL BIOPHYSICS
BIOPHYSICS Vol. 66 No. 3 2021
THE KINETICS OF THE PRODUCTION OF REACTIVE OXYGEN SPECIES 435
solution and left for at least 1 hour at 4°C (but not
more than 3 hours, since longer storage in this case
reduced the chemiluminescent response of neutro-
phils to the subsequent addition of lucigenin). This
procedure allowed reduction of the spontaneous che-
miluminescence of neutrophils and transfer of these
cells to a “uniform” state characterized by their uni-
form chemiluminescent response, which made it pos-
sible to work with them during the experimental day.
The number of isolated cells was counted in a Goryaev
chamber. Cell viability was determined using trypan
blue vital dye. The content of living cells was at least
98%. For the experiments, samples were obtained by
diluting a suspension of neutrophils with Hanks
medium (final composition of the 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 1 million cells/mL.
Exposure of a neutrophil suspension in a zero and a
geomagnetic f ield. Neutrophils were incubated at
37.0 ± 0.1°С at a concentration of 1 mln cells/mL,
0.25 mL each in round-bottom polystyrene cuvettes
(diameter, 1.2 cm; length, 5.5 cm) in which chemilu-
minescence was measured later.
The set temperature was maintained using a circu-
lating water thermostat. Samples of the control groups
were in a local geomagnetic field (GMF) with a con-
stant component of ~44 μT and a magnetic back-
ground level at 50 Hz of 15–50 nT at the same tem-
perature regime as the experimental samples, and
simultaneously with them (the subsequent registration
of chemiluminescence of control and experimental
samples in experiments with the same duration of their
incubation was also carried out simultaneously).
Experimental samples were placed in a device for the
formation of hypomagnetic conditions for 30 min, the
lucigenin-dependent chemiluminescence was
recorded in the samples (immediately after the end of
incubation in a zero MF, experiments with an afteref-
fect time of 0 min).
Other experimental groups of samples were trans-
ferred to incubation conditions in a geomagnetic field
for 20, 40, and 60 min immediately after the end of the
zero field, followed by registration of chemilumines-
cence after the end of incubation.
Each experimental group of samples corresponded
to its own control group, incubated simultaneously
with the experimental one, but only under GMF con-
ditions.
Special research equipment was used in the exper-
iments, that is, a device for the formation of hypomag-
netic conditions, which allowed us to obtain a high
degree of attenuation of the GMF of up to 10 000 times
(the residual constant field did not exceed 20 nT) and
significantly weakened the variable technogenic inter-
ference (up to a few nT). This setup was described in
detail in [8, 12]. The setup consisted of three cylindri-
cal magnetic screens made of permalloy (1-mm thick)
inserted coaxially into one another.
The determination of the residual fields inside the
setup was carried out by direct measurement using a
Mag-03 MS 100 fluxgate magnetometer (Bartington,
Great Britain). The dimensions of the experimental
section inside the system of screens (diameter, 20 cm;
length, 40 cm) made it possible to simultaneously
place a sufficient number of experimental samples (at
least six) in the zone of a uniform weak magnetic field.
The experiments were repeated at least three times.
The registration of chemiluminescence. After incu-
bation of the neutrophil suspension, the intensity of
chemiluminescence of the samples in control and
experimental cases after the addition of a lucigenin
solution (Enzo Life Sciences, United States) at a final
concentration of 0.35 mM was measured. A 12-chan-
nel Lum-1200 chemiluminometer (DISoft, Russia)
was used in the work. The PowerGraph program was
used to analyze the chemiluminescence data. Some of
the results are presented as a percentage in relation to
the amplitudes of the chemiluminescent response in
the control, taken as 100%.
The results were statistically processed using the
Student’s t-test.
RESULTS AND DISCUSSION
A 30-minute incubation of a suspension of neutro-
phils in a zero magnetic field led to a significant
decrease in the intensity of its lucigenin-dependent
Fig. 1. The influence of a zero magnetic field on the inten-
sity of lucigenin-dependent chemiluminescence of a sus-
pension of neutrophils, depending on the duration of sub-
sequent incubation in the geomagnetic field. The ordinate
is the maximum intensity of chemiluminescence in condi-
tional units (mean values and standard deviations, n = 6),
the x-axis is the incubation time in the geomagnetic field
after being in a zero MF. Light bars, control; dark bars,
experiment. The asterisk marks significant differences
from the control (P < 0.05).
Intensity of chemiluminescence, CU
Time, min
6
5
4
3
2
1
0
0204060
436
BIOPHYSICS Vol. 66 No. 3 2021
NOVIKOV et al.
chemiluminescence, as determined immediately after
the end of exposure to hypomagnetic conditions (by
approximately 48%) (Fig. 1–3). At 20 min after expo-
sure to hypomagnetic conditions (with subsequent
20 min incubation in GMF), the degree of manifesta-
tion of differences between the control and experi-
mental samples is completely preserved (the differ-
ences are 49%) (Fig. 1–3). With an increase in the
duration of the subsequent incubation of experimental
samples in the GMF after being in the zero MF up to
40 and 60 min, the differences between them and the
corresponding control groups of samples decreased to
32 and 22%, respectively (Fig. 1–3). It should be
noted that an increase in the incubation time both in
the control and in the experiment itself is accompa-
nied by a decrease in the intensity of lucigenin-depen-
dent chemiluminescence of the neutrophil suspen-
sion, which may be associated with the activation of
antioxidant systems in these cells [13, 14] or the con-
sumption of endogenous reduction equivalents
(NADH , NADPH) [10]. Subsequent experiments
Fig. 2. The time dynamics of the aftereffect of the zero
magnetic field in CU normalized relative to control values
(mean values and standard deviations, n = 6). The abscissa
is the time of incubation in the geomagnetic f ield after
being in the zero MF. Light bars, control; dark bars, exper-
iment. The asterisk marks significant differences from the
control (P < 0.05).
Intensity of chemiluminescence
100
80
60
40
20
0
Time, min
0 204060
Fig. 3. The kinetic curves of the chemiluminescent response of a suspension of neutrophils to lucigenin after exposure to a zero
MF at different times of subsequent incubation in a geomagnetic field: (a) 0 min (without incubation in GMF); (b) 20 min;
(c) 40 min; (d), 60 min; 1, control; 2, experiment.
Intensity of chemiluminescence, CU
Lucigenin
Lucigenin
Lucigenin
Lucigenin
1
1
1
1
2
2
2
2
6(a) (b)
(c) (d)
5
4
3
2
1
0
Intensity of chemiluminescence, CU
2.0
1.5
1.0
0.5
Intensity of chemiluminescence, CU
1.6
1.2
0.8
0.4
00
Intensity of chemiluminescence, CU
3.0
2.5
2.0
1.5
1.0
0.5
0
0 200 400 600 800 0 200 400 600 800
0 200 400 600 800
Time, s
Time, s
Time, s
0 200 400 600 800
Time, s
BIOPHYSICS Vol. 66 No. 3 2021
THE KINETICS OF THE PRODUCTION OF REACTIVE OXYGEN SPECIES 437
will help to establish whether these natural processes
are related to the mechanisms of zero field action.
However, this is contradicted by the dynamics of the
zero field effect with the response maxima precisely at
the early periods of observations.
Our data on the presence of at least a 20-minute
time period of the aftereffect of hypomagnetic condi-
tions on the rate of ROS production by neutrophils, in
which the degree of severity of the effect of the action
of the zero field (decrease in ROS production) is fully
preserved, has a pronounced applied (methodologi-
cal) aspect, in addition to fundamental importance.
Indeed, in a number of cases it is extremely difficult to
carry out a direct study of the action of a zero field
using standard research equipment, since this equip-
ment itself is a source of magnetic interference. More-
over, the extraction of samples from hypomagnetic
conditions into the conditions of the surrounding
GMF leads to a significant change in their magnetic
environment. The relatively long period of the afteref-
fect of the discovered physical factor, in principle,
allows planning and conducting experiments with the
separation (for a certain time) of the exposure of the
research objects to hypomagnetic conditions and
recording the effect of their action using measuring
equipment, which can contribute to progress in this
field of research.
In conclusion, it should be noted that it is espe-
cially important and promising to study the effects of a
zero magnetic field in general for bioelectromagnetic
research. Along with the applied aspects of the already
discovered unfavorable effects of hypomagnetic con-
ditions on the processes of embryonic development
[15–7], morphogenesis [18–0], and behavioral reac-
tions [21, 22], the study of these effects can help to
determine the primary working targets of the action of
a weak magnetic field in biological objects [1, 5, 23,
24].
CONFLICT OF INTEREST
The authors declare that they have no conflict of inter-
est.
COMPLIANCE WITH ETHICAL STANDARDS
Statement on the welfare of animals. All applicable inter-
national, national, and/or institutional guidelines for the
care and use of animals were followed.
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Translated by P. Kuchina
SPELL: 1. OK
