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ISSN 0006-3509, Biophysics, 2020, Vol. 65, No. 4, pp. 625–630. © Pleiades Publishing, Inc., 2020.
Russian Text © The Author(s), 2020, published in Biofizika, 2020, V ol. 65, No. 4, pp. 735–740.
Decreased Production of the Superoxide Anion Radical in Neutrophils
Exposed to a Near-Null Magnetic Field
V. V. Novik ova, *, E. V. Yablokovaa, I. A. Shaeva, and E. E. Fesenkoa
aInstitute of Cell Biophysics, Subdivision of the Puschino Scientific Center for Biological Studies Federal Research Center,
Russian Academy of Sciences, Pushchino, Moscow Oblast, 142290 Russia
*e-mail: docmag@mail.ru
Received April 21, 2020; revised April 21, 2020; accepted May 7, 2020
Abstract—This paper reports that pre-incubation of a neutrophil suspension in the presence of a near-null
magnetic field produced using a system of magnetic shields (a residual constant magnetic f ield not greater
than 20 nT) results in a considerable decrease in the intensity of neutrophil lucigenin-dependent chemilumi-
nescence. The addition of the NADPH oxidase inhibitor diphenyliodonium to the incubation medium
reduced the chemiluminescence intensity in both the experimental and the control samples (geomagnetic
field). It should be noted that the differences observed between the groups, which were caused by the expo-
sure to a near-null magnetic field, are almost the same both at lower (2.5, 5, and 10 μM) and higher (50 and
100 μM) diphenyliodonium concentrations. In contrast, the addition of 2,4-dinitrophenol, an uncoupler of
oxidative phosphorylation in mitochondria, in concentrations starting from 5 μM and up to 200 μM almost
completely eliminated the difference between the control and experimental samples, which was observed at
low inhibitor concentrations, or in its absence.
Keywords: neutrophils, near-null magnetic f ield, superoxide anion radical, chemiluminescence, lucigenin,
NADPH oxidase, mitochondria
DOI: 10.1134/S0006350920040120
The literature data indicate that production of
reactive oxygen species (ROS) is reduced in various
types of cells exposed to hypomagnetic field [1–4]. We
have previously demonstrated that the exposure of
peritoneal mouse neutrophils to the hypomagnetic
field environment created by magnetic shielding
caused a decrease in the intracelullar production of
reactive oxygen species detected by the change in the
intensity of the fluorescence produced by the 2,7-
dichlorodihydrofluorescein and dihydrorhodamine
123 oxidation products [5–7]. Considering the fact
that this effect of a hypomagnetic field was observed in
experiments in which neutrophils were not subjected
to additional stimulation by any chemical respiratory
burst activator, and thus, was not a consequence of
impaired neutrophil response to these stimulators, we
performed a specially designed complex study using
nonactivated neutrophils in order to elucidate the pos-
sible molecular mechanisms that underlie the effects
of a near-zero magnetic field [6]. It has been demon-
strated that the reduced intensity of 2,7-dichlorodihy-
drofluorescein oxidation in nonactivated neutrophils
under hypomagnetic conditions is not associated with
calcium-dependent regulatory mechanisms, which
was supported by the fact that the intracellular calcium
ions chelator (1,2-bis(2-aminophenoxy)ethane-N,N,
N',N;-tetraacetic acid acetoxymethyl ester) had no
effect on the intensity of this process [6]. The observed
reduction is also not likely to be a result of a hypomag-
netic field affecting the phosphorylation of NADFH
oxidase components, since the addition of the protein
kinase C inhibitor (Ro 31-6233) had almost no impact
on the fluorescence intensity of the intracellular
dichlorodihydrofluorescein [6]. The addition of the
phospholipase C inhibitor (U73122) led to a slight,
and almost similar, decrease in the ROS production
both in the control and in the experiment [6]. The
possible involvement of the mitochondrial electron
transport chain in the mechanism underlying the
near-zero magnetic field effect is indicated by the
decrease in the ROS production in the presence of
rotenon, which was more profound in the experimen-
tal samples exposed to a hypomagnetic field [6].
All the above-mentioned results were obtained
using fluorescence spectroscopy with fluorescent
probes (2,7-dichlorodihydrofluorescein and dihy-
drorhodamine 123) that respond intensely to ROS, but
are not selective towards particular ROS types [8–10].
In the present work, we employed another technique
to assess the radical-producing ability of neutrophils
after exposure to a near-zero magnetic field, namely,
Abbreviations: ROS, reactive oxygen species; MF, magnetic
field; SAR, superoxide anion radical.
CELL BIOPHYSICS
626
BIOPHYSICS Vol. 65 No. 4 2020
NOVIKOV et al.
activated chemiluminescence using lucigenin, a selec-
tive probe for the superoxide anion [11, 12]. Using this
experimental model we also performed inhibition
analysis using diphenyliodonium, which is an
NADPH oxidase inhibitor [13, 14], and 2,4-dinitro-
phenol, an uncoupler of oxidative phosphorylation in
mitochondria [15, 16], in order to find potential
sources of superoxide production that would respond
to a hypomagnetic f ield.
MATERIALS AND METHODS
Preparation of neutrophil suspensions. The work
was performed using mouse peritoneal neutrophils.
Neutrophils were obtained from laboratory-kept
CD-1 line male mice with a weight of 24–26 g pro-
vided by the Laboratory Animal Incubator of the Pus-
chino Branch of the Institute of Bioorganic Chemistry
of the Russian Academy of Sciences (Puschino, Mos-
cow Oblast). Mice were injected with 150 μL of an
opsonized zymosan suspension at a concentration of
5 mg/mL (Zymozan A from Saccharomyces cerevisiae,
Sigma, United States) into the peritoneal cavity.
Twelve hours after the injection, the mice were eutha-
nized by cervical dislocation and their abdominal cav-
ity was washed with 4 mL of cooled Hank’s solution
without calcium. Peritoneal exudate was collected
using a pipette and centrifuged at 600 g for 5 min.
Supernatant was decanted and the pellet was resus-
pended in 4 mL of Hank’s solution without calcium
and left to stay for 1 h at 4°C. The number of isolated
cells was calculated using a Goryaev chamber. Cell
viability was assessed using the trypan blue vital stain.
The portion of living cells was not less than 98%.
Experimental samples were prepared by diluting the
neutrophil suspension with the standard Hank’s
medium (138 mM NaCl, 6 mM KCl, 1 mM MgSO4,
1mM Na
2HPO4, 5 mM NaHCO3, 5.5 mM glucose,
1 mM CaCl2, and 10 mM HEPES, pH 7.4; Sigma,
United States) to a concentration of 1 million
cells/mL.
Exposure of the neutrophil suspension to near-zero
and weak constant magnetic fields. Neutrophils at a
concentration of 1 million/mL were incubated in a
volume of 0.25 mL in round-bottom polystyrene
cuvettes (1.2 cm in diameter and 5.5 cm in length), in
which chemiluminescence was further measured, at
37.0 ± 0.2°C. The typical incubation time was 40 min.
The required temperature was maintained using a cir-
culating constant-temperature bath.
The control group samples were exposed to a local
geomagnetic field with a constant component of
~44 μT and a 50 Hz component of the magnetic back-
ground of 15–50 nT at the same temperature as the
experimental samples and simultaneously with them.
The experimental samples were placed into the hypo-
magnetic-environment simulator.
A specially designed experimental installation that
allows one to create a hypomargentic environment was
used, which made it possible to significantly weaken
the geomagnetic field, up to a reduction by
10000 times (the residual constant field did not exceed
20 nT), and to substantially reduce the variable tech-
nogenic noise (to several nT). This installation was
described in detail previously [7, 17]. It consists of
three coaxially nested cylindrical permalloy magnetic
shields (1-mm thick). The residual magnetic fields
inside the installation were determined by direct mea-
surement using a Mag-03 MS 100 flux gate magne-
tometer (Bartington, Great Britain). To produce an
experimental weak uniform constant magnetic field
(MF), a specially designed inducing coil (solenoid)
connected to a constant current supply was placed
inside the installation, in order to produce a weak con-
stant MF of different intensities (2.5, 7.0, and 44 μT)
used in a number of experiments. The size of the
experimental plot inside the shield system (a plot
diameter of 20 cm and a plot length of 40 cm) allowed
a sufficient number of experimental samples (not less
than six) to be simultaneously located in the uniform
weak magnetic field area. All experiments were per-
formed no less than three times.
Prior to exposure, a number of chemical com-
pounds, namely, diphenyliodonium chloride, which is
an inhibitor of NADPH oxidase (Sigma, United
States), in varying concentrations (2.5, 5.0, 10, 20, 50,
and 100 μM), which was dissolved in dimethylsulfox-
ide (Sigma, United States) prior to the experiment,
and 2,4-dinitrophenol, an uncoupler of oxidative
phosphorylation (Sigma, United States), in varying
concentrations (1.0, 4.0, 5.0, 10, 20, 200 μM, and
2 mM), also pre-dissolved in dimethylsulfoxide, were
added individually to certain samples. The individual
effects of dimethylsulfoxide in the final concentration
of 1 mM, which corresponded to its level in the sample
containing 10 μM diphenyliodonium, or 20 μM 2,4-
dinitrophenol, were estimated as well. In some exper-
iments, these inhibitors were added to the samples
immediately after the exposure was finished, rather
than prior to its start, but before the introduction of
lucigenin and chemiluminescence measurements.
Chemiluminescence measurement. When the expo-
sure ended, the intensity of the chemiluminescence
produced by neutrophil suspension samples in the
experimental and control cases was measured after the
addition of the lucigenin (Enzo Life Science, United
States) solution at a final concentration of 0.35 mM.
The Lum-1200 (OOO DISoft, Russia) chemilumi-
nometer was used to perform the measurements.
Power-Graph software was used to analyze the chemi-
luminescence data. Some results were provided in per-
cent relative to the chemiluminescence response
amplitude in the control, which was assumed to be
100%. The statistical processing of the measurement
results was performed using the Student’s t-test.
BIOPHYSICS Vol. 65 No. 4 2020
DECREASED PRODUCTION OF THE SUPEROXIDE ANION RADICAL 627
RESULTS AND DISCUSSION
Pre-incubation of the neutrophil suspension in the
near-zero magnetic field environment led to a sub-
stantial decrease in the intensity of the lucigenin-
dependent chemiluminescence (approximately by
30%) (Figs. 1 and 2). When the constant field magni-
tude was increased to 2.5 μT this effect disappeared; it
re-appeared again at 7 μT and disappeared again at
44 μT (this value corresponds to the constant MF
magnitude in the control) (Fig. 1). We also observed
the same multiple-peaked (polyextreme) pattern of
the response to a constant weak MF in previous exper-
iments using neutrophils where ROS production was
assessed by fluorescence measurement [7], as well as
when using other biological objects [18, 19].
The addition of diphenyliodonium to the incuba-
tion medium led to a decrease in the chemilumines-
cence intensity in both the experimental and control
samples (Figs. 2 and 3). The diphenyliodonium effect
showed a nearly linear dependence on its dose (it
increased with an increase in the concentration) in
both the control and experimental cases (Fig. 2). At
the same time, the difference between the two groups
caused by the effect of a near-zero magnetic field was
almost the same at both lower (2.5, 5.0, and 10 μM)
and higher (50 and 100 μM) diphenyliodonium con-
centrations. The absence of any significant impact of
dimethylsulfoxide (the difference between the samples
with the addition of 1 mM dimethylsulfoxide and the
corresponding control samples did not exceed 5%),
which is used as a solvent to prepare diphenyliodo-
nium solutions, allowed us to link the observed effects
with the activity of the studied inhibitor.
When diphenyliodonium was added after the incu-
bation immediately before the chemiluminescence
measurements, the direction of its effect exerted on
the experimental and control samples remained the
Fig. 1. The effects of a constant MF on the intensity of the
lucigenin-dependent chemiluminescence in the neutro-
phil suspension. X axis, constant MF magnitude in μT,
Yaxis, maximum chemiluminescence intensity in percent
relative to the control (mean values and standard devia-
tions, n = 8). Hypomagnetic field (HypoMF) corresponds
to the constant MF with the magnitude not greater than
0.02 μT; (1) control and (2) experiment. Statistically sig-
nificant differences from the control are indicated with an
asterisk (P < 0.05).
125
100
75
50
25
0
1
2
**
2.5 7.0 44.0
Chemiluminescence intensity, %
Magnetic field, µT
HypoMF
Fig. 2. The effects of diphenyliodonium on the intensity of the lucigenin-dependent chemiluminescence in a neutrophil suspen-
sion after exposure to a near-zero magnetic field. X axis, diphenyliodonium concentration in μM, Y axis, the maximum chemi-
luminescence intensity in percent relative to the control (mean values and standard deviations, n = 6). (1) Control, diphenyliodo-
nium addition prior to the incubation; (2) experiment, diphenyliodonium addition prior to the incubation; 3, control,
diphenyliodonium addition after the incubation, and 4, experiment, diphenyliodonium addition after the incubation. Statistically
significant differences from the control are indicated with an asterisk (P < 0.05).
125
100
75
50
25
0
0 2.5 5.0 10.0 20.0 50.0 100.0 1000.0
*
*
***
*
*
**
*
3
4
1
2
Chemiluminescence intensity, %
Diphenyliodonium concentration, µM
628
BIOPHYSICS Vol. 65 No. 4 2020
NOVIKOV et al.
same, although the size of the effect relative to the
inhibitor dose appeared to decrease (Fig. 2).
A substantially different result was obtained in the
experiments where 2,4-dinitrophenol, which uncou-
ples oxidation and phosphorylation, was used. In par-
ticular, the addition of this inhibitor in concentrations
starting from 5 μM and up to 200 μM almost com-
pletely eliminated the differences between the control
and experimental samples (Figs. 4 and 5). At the same
time, the intensity of the lucigenin-dependent chemi-
luminescence produced by the control neutrophil sus-
pension decreased proportionally to the 2,4-dinitro-
phenol concentration. On the basis of these data, the
conclusion may be drawn that dinitrophenol in certain
concentrations is able to completely eliminate the
effect of a near-zero MF. It may be suggested that the
most probable explanation for the observed dinitro-
phenol effects may be a certain similarity between its
mechanism of action and the action of a near-zero
magnetic field. In fact, in this case, as a result of sub-
strate competence, the chemical agent may be able to
suppress the effect of the factor of a physical nature.
An alternative hypothesis to explain this dinitrophenol
effect is that coupling of oxidation and phosphoryla-
tion in the neutrophil mitochondria is required to
enable the near-zero magnetic field effect, in other
words, certain ATP production levels are required.
However, the experiments where dinitrophenol was
added after the end of incubation in the near-zero
field, but not at the start of the experiment, which
demonstrated that the dinitrophenol effect can still be
observed in this case and showed no qualitative
changes (Fig. 4), provide evidence that supports the
first hypothesis.
It is well-known that the mechanism of action of
dinitrophenol, which is a well-characterized uncou-
pler of mitochondrial respiration, is based on its ability
to break the proton gradient formed as a result of elec-
tron transport [15, 16]. Disruption of the proton gradi-
Fig. 3. The kinetics of the chemiluminescence response of
the neutrophil suspension to lucigenin after exposure to a
near-zero MF in the absence, as well as in the presence, of
diphenyliodonium: (1) control; (2) experiment; (3) con-
trol, with the addition of 10 μM diphenyliodonium; and
(4) experiment, with the addition of 10 μM diphenyliodo-
nium.
8
7
6
5
4
3
2
1
0
800700500 6004003002001000
1
2
3
4
Chemiluminescence intensity, V
Time, s
Lucinogenin
Fig. 4. The effects of dinitrophenol on the intensity of the lucigenin-dependent chemiluminescence in a neutrophil suspension
after exposure to a near-zero MF. X axis, dinitrophenol concentration in μM, Y axis, maximum chemiluminescence intensity in
percent relative to the control (mean values and standard deviations, n = 6). (1) Control, dinitrophenol addition prior to the incu-
bation; (2) experiment, dinitrophenol addition prior to the incubation; (3) control, dinitrophenol addition after the incubation,
and (4) experiment, dinitrophenol addition after the incubation. Statistically significant differences from the control are indicated
with an asterisk (P < 0.05).
125
100
75
50
25
0
0 1 4 5 10 20 200 2000
****
3
4
1
2
Chemiluminescence intensity, %
Dinitrophenol concentration, µM
BIOPHYSICS Vol. 65 No. 4 2020
DECREASED PRODUCTION OF THE SUPEROXIDE ANION RADICAL 629
ent at the inner mitochondrial membrane does not
interfere with respiration, but it does inhibit ATP syn-
thesis, which is normally fueled by the proton gradi-
ent. This uncoupling abolishes the membrane poten-
tial across the inner mitochondrial membrane [15].
Previously, it was commonly thought that mature
neutrophils contain few functional mitochondria, if
they contain them at all [20]. This suggestion was
based on the fact that electron microscopy of fixed
cells is usually unable to detect intact mitochondria,
while those that can be observed are small with poorly
developed cristae and inner mitochondrial membrane
[20]. Moreover, it has been demonstrated that neutro-
phils mainly utilize glycolysis for energy production
[21], while the intensity of mitochondrial respiration
appears to be very low. This O2-independent energy
production mechanism is advantageous for neutro-
phils, since it enables their functioning in inflamma-
tion foci, or infection sites, where the O2 partial pres-
sure may be rather low [22]. During phagocytosis neu-
trophils utilize large amounts of molecular O2 for the
generation of superoxide anion and other ROS during
the respiratory burst catalyzed by NADPH oxidase
rather than for the mitochondrial respiration [23].
Thus, considering these morphological and biochem-
ical properties it seemed that neutrophils do not have
functionally active mitochondria and do not even
need them.
However, recently, new evidence for the important
role that functional mitochondria may play in the con-
trol of neutrophil apoptosis is emerging [24]. Using
the fluorescent indicators of mitochondrial function
in living cells it has been demonstrated that neutro-
phils possess a well-developed mitochondrial network
[25]. The membrane potential in these mitochondria
can be disrupted using the chemical uncouplers of
electron transport. It has been shown that mitochon-
dria in neutrophils do not participate in the rapid acti-
vation of the respiratory burst or phagocytosis; how-
ever, the intensity of these processes appeared to be
decreased in neutrophils pre-treated with the mito-
chondria inhibitors [25]. All these data, along with our
findings, indicate that mitochondria in neutrophils
may be investigated as potential targets towards which
the effects of a near-zero magnetic field may be
directed.
In neutrophils, there are several major systems in
which free radicals are formed as the main or a side
product. First, this regards NADPH oxidases, mem-
brane enzymes that produce the superoxide anion rad-
ical (SAR) by one-electron reduction [26]. Apart from
them, mitochondria may be an important SAR pro-
duction system. It is well known that SAR leakage
occurs at 11 sites of the mitochondrial inner mem-
brane, mostly in complexes I, II, and III, with SAR
being released both into the matrix and the intermem-
brane space [27].
Lucigenin is considered to be a selective probe for
SAR [11] and is thus intensively used to study ROS
production by both NADPH oxidase and mitochon-
dria [12]. The characteristics of the inhibitory effect of
diphenyliodonium (a non-specific NADPH oxidase
inhibitor) revealed in the present work using neutro-
phils exposed to a near-zero magnetic field put the
suggestion that NADPH oxidase is the main source of
SAR in response to a hypomagnetic field in doubt.
On the contrary, the experiments using dinitrophe-
nol, an uncoupler of oxidative phosphorylation, which
demonstrated almost complete elimination of the
effects of a near-zero magnetic field in the presence of
this compound, support the idea that it is mitochon-
dria as SAR producers that are the main target of this
physical factor. Certainly, this hyp othesis requires fur-
ther study.
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.
Conf lict of interest. Authors declare no conflict of inter-
est.
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Translated by E. Martynova
SPELL: 1. OK