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Decrease of reactive oxygen species (ROS) production by neutrophils after incubation in hypomagnetic conditions

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Incubation of the suspension of neutrophils in hypomagnetic field generated by a system of magnetic shields (residual constant magnetic field not exceeding 20 nT) leads to significant decrease in reactive oxygen species (ROS) production compared to control (geomagnetic field 44 μT), which was recorded by lucigenin-dependent chemiluminescence and fluorescent spectroscopy with 2,7-dichlorodihydrofluorescein diacetate (H2DCF-DA). During increase of constant magnetic field (CMF) in 0.02-44 μT range, polyextreme character of the response of the neutrophils to this action was observed: the minima of ROS production were at 0.02 μT and 7.0 μT, alternating with 2.5 μT and 30 μT values, at which the used test system does not react to the exposure to CMF.
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Decrease of reactive oxygen species (ROS) production by neutrophils
after incubation in hypomagnetic conditions
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XIV International Conference "Space and Biosphere" (Space and Biosphere 2021)
IOP Conf. Series: Earth and Environmental Science 853 (2021) 012008
IOP Publishing
doi:10.1088/1755-1315/853/1/012008
1
Decrease of reactive oxygen species (ROS) production by
neutrophils after incubation in hypomagnetic conditions
V V Novikov1*, E V Yablokova1 and I A Shaev1
1Institute of Cell Biophysics, Subdivision of the Pushchino Scientific Center for
Biological Studies Federal Research Center, Russian Academy of Sciences,
Pushchino, Moscow oblast, 142290 Russia
* docmag@mail.ru
Abstract. Incubation of the suspension of neutrophils in hypomagnetic field generated by a
system of magnetic shields (residual constant magnetic field not exceeding 20 nT) leads to
significant decrease in reactive oxygen species (ROS) production compared to control
(geomagnetic field 44 T), which was recorded by lucigenin-dependent chemiluminescence
and fluorescent spectroscopy with 2,7-dichlorodihydrofluorescein diacetate (H2DCF-DA).
During increase of constant magnetic field (CMF) in 0.02-44 T range, polyextreme character
of the response of the neutrophils to this action was observed: the minima of ROS production
were at 0.02 μT and 7.0 μT, alternating with 2.5 μT and 30 μT values, at which the used test
system does not react to the exposure to CMF.
1. Introduction
Many authors regard the possibility of effect of weak magnetic fields on reactive oxygen species
(ROS) production and generation of other free radicals as a promising approach to the analysis of
mechanisms of their biological action [13].
There is a number of reports in the scientific literature about the effect of weak low-frequency MFs
on the kinetics of ROS formation in the suspension of neutrophils activated by chemical stimulators of
respiratory burst [48]. It was also reported on the effect of hypomagnetic conditions (HMC) on ROS
production in different cells [913].
We have earlier shown that exposure of murine peritoneal neutrophils to magnetic shielding in
HMC causes the decrease of intracellular ROS production recorded by change of fluorescence
intensity of the products of 2,7-dichlorodihydrofluorescein and dihydrorhodamine 123 [14]. In these
experiments, we found that the effect of the hypomagnetic field is apparent on neutrophils without
their additional stimulation by the chemical activators of respiratory burst, and, consequently, the
mechanism of such action can be not related to the impairment of the response of neutrophils to these
stimuli. Thus, in this work we carried out a complex study on non-activated neutrophils to determine
possible molecular mechanisms and targets of the “zero” magnetic field action.
We must note that the aforementioned results were obtained using the method of fluorescence
spectroscopy with intensely reacting fluorescent probes that are not selective to certain ROS types
(2,7- dichlorodihydrofluorescein diacetate and dihydrorhodamine 123) [1518]. In this work, we also
used another method, activated chemiluminescence with selective probe for superoxide anion,
lucigenin [19,20], to estimate the radical producing ability of the neutrophils after exposure to HMC.
XIV International Conference "Space and Biosphere" (Space and Biosphere 2021)
IOP Conf. Series: Earth and Environmental Science 853 (2021) 012008
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doi:10.1088/1755-1315/853/1/012008
2
Attention was paid to determination of residual constant magnetic field (CMF) values at which the
effects of HMC can be reproduced.
2. Materials and methods
The work was carried out on murine peritoneal neutrophils. Male laboratory mice CD-1 (bodyweight
22-25 g) obtained from the nursery of the ShemyakinOvchinnikov Institute of Bioorganic
Chemistry, Russian Academy of Sciences were used for isolation of the cells. The mice were
administered opsonized Zymozan A suspension (150 μl i.p., 5 mg/ml) (Zymozan A from
Saccharomyces cerevisiae, Sigma, USA). After that, the animals were brought to an end by cervical
dislocation 12 hours after administration, four milliliters of calcium-free Hank’s solution were injected
into their abdominal cavity for washing [5]. The exudate was collected by pipetting, and cells
wereharvested by centrifugation for 5 min at 600 g. The supernatant was decanted, and the cell pellet
was diluted in 4 ml of calcium-free Hank’s solution and incubated for 1 hour at 4°C [5]. The isolated
cells were counted in Goryaev chamber. Cell viability was determined using the vital dye, trypan blue
(Sigma, USA). Content of living cells comprised at least 98%. The experimental samples were
obtained by dilution of the suspension with Hank’s 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, USA) to 1∙106 cells/ml concentration.
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 accordance with the Guidelines for
Ethical Conduct in the Care and Use of Animals.
The neutrophils were incubated at 37.00.2C at 106 cells/ml concentration in 0.25 ml aliquots in
round-bottom polystyrene cuvettes (d = 1.2 cm, l = 5.5 cm) (Sarstend, Germany), in which further
fluorescence measurements were taken. Typical incubation time was 40 min. The set temperature was
maintained by a circulation thermostat UH 4 (MLW, Germany).
The control samples were placed in local geomagnetic field (GMF) with a constant component
around 44 μT and 50 Hz technogenic magnetic background around 15-50 nT at the same time and
temperature as the experimental ones. The experimental specimens were placed into an appliance for
maintaining hypomagnetic conditions.
An appliance for creation of HMC used in the work was capable of decreasing the magnetic field
by four orders of magnitude (the residual field did not exceed 20 nT) and it significantly mitigated
alternating technogenic noise (to several nT) [8]. The appliance was composed of three coaxial
cylindric magnetic shields made of 1 mm thick permalloy. The residual fields inside the appliance
were measured directly with ferroprobe magnetometer, Mag 03 MS 100 (Bartington, UK). The
experimental weak uniform constant magnetic field was generated by a special inductor (solenoid)
installed inside the appliance. The solenoid could be attached to a direct current source to form weak
magnetic field of various intensities (2.5; 7; 30; 44 μT) used in a series of experiments. Solenoid
dimensions were: diameter, 18 cm; length, 38 cm (720 coils of copper wire, diameter 1 mm; the
resistance of solenoid was 7.5 Ohm). The size of experimental area inside the shields allowed
simultaneous placement of a number of samples sufficient for the experiment (at least 6) into the
uniform CMF zone. The experiments were performed in triplicates.
After the incubation of the neutrophil suspension in control and experimental groups, a solution of
lucigenin (Enzo Life Sciences, USA) was added at a final concentration of 0.35 mM. The
measurements of luminescence were performed on a 12-channel chemiluminometer Lum-1200
(DISoft LLC, Russia). There was a linear correlation between values of luminescence intensity and
number of photons per time unit: 1 Volt of recorded voltage corresponded to 1000 photons/s.
Chemiluminescence records were analyzed in PowerGraph program. Some results were expressed in
relative units (percent of control chemiluminescence amplitude, which was taken for 100%). After 40
min incubation, a part of the samples of neutrophil suspension was supplied with fluorescent probe for
intracellular ROS, 2,7-dichlorodihydrofluorescein diacetate (Sigma, USA) to a final concentration of
0.01 mg/ml. The samples were incubated for extra 25 min at 37C in the dark to minimize the
XIV International Conference "Space and Biosphere" (Space and Biosphere 2021)
IOP Conf. Series: Earth and Environmental Science 853 (2021) 012008
IOP Publishing
doi:10.1088/1755-1315/853/1/012008
3
photooxidation of the dye. Then, the cells were transferred to Eppendorf tubes, washed by
centrifugation at 600 g for 5 min in Hank’s solution. The pellet was then resuspended in 1 ml of the
medium and fluorescence spectra of the specimens were recorded on Thermo Scientific Lumina
Fluorescence Spectrometer (Thermo Fisher Scientific, USA) at 488 nm excitation wavelength. A part
of the results was represented as percentage of fluorescence intensity at 528 nm in the control group,
which was taken for 100 %.
The data were evaluated using Student’s test. P-values < 0.05 were considered significant. All
values were expressed as means ± SD.
3. Results and discussion
Pre-incubation of the neutrophil suspension in “zero” magnetic field (<0.02 μT) led to significant
decrease of the intensity of lucigenin-dependent chemiluminescence (by around 30%) (figure 1, figure
2). When the constant field was increased to 2.5 μT, this effect vanished, but at CMF value = 7 μT it
appeared again; at 30 and 44 μT, the effect was absent (the value corresponded to CMF value in
control) (figure 2). Such polyextreme character of the dependence of response to weak CMF was also
noticed in the experiment on neutrophils during ROS production registration by fluorescence
spectroscopy (figure 3, figure 4). It is important that this result was obtained by two different methods,
lucigenin-dependent chemiluminescence and fluorescence spectroscopy, which does apparently
increase its reliability level.
0200 400 600 800
0
1
2
3
4
5
6
7
8
- - - -
Chemiluminescence intensity, V
Time, s
Control
Exposed
Lucigenin
0
20
40
60
80
100
120
Control <0.02 2.5 7.0 30 44
Magnetic field, T
Chemiluminescence intensity, %
Figure 1. The kinetics of the
chemiluminescence response of the
neutrophil suspension to lucigenin after
exposure to “zero” magnetic field (CMF
< 0.02 μT).
Figure 2. The influence of a constant MF
on the intensity of the lucigenin-
dependent chemiluminescence in the
neutrophil suspension. Statistically
significant differences from the control
values are pointed by an asterisk (P <
0.05).
The discovered dependence of ROS production in neutrophils on the value of constant magnetic
field in the studied induction range (0.02 - 44 μT) evidencing on high sensitivity of such processes to
changes of magnetic conditions appears to be of high information value. The revealed anisotropy of
the response of biological system on such an action could give certain data for analyzing the physical
mechanisms of non-specific magnetoreception. In the current moment, a model of non-specific (not
related to certain receptors) magnetoreception becomes more popular [21]. It is based on the analysis
of precession of magnetic moments and molecular rotations in weak MF, and our data could be useful
XIV International Conference "Space and Biosphere" (Space and Biosphere 2021)
IOP Conf. Series: Earth and Environmental Science 853 (2021) 012008
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doi:10.1088/1755-1315/853/1/012008
4
for its realistic validation. Moreover, these data could be useful for estimation of quantum mechanical
model based on application of spin chemistry for the cases of weak MFs [22], which gives a prediction
on reaction product (peroxyradical) yield depending on MF parameters, allows to estimate the
contribution of weak MF to this process, and shows the polyextreme character of the response of
biological system depending on the magnitude of MF.
500 550 600 650
0
5000
10000
15000
20000
25000
30000
35000
Exposed
- - - - Control
Fluorescence intensity, arb. un
Wave length, nm
0
25
50
75
100
125
Control <0.02 2.5 7.0 30 44
Magnetic field, T
Fluorescence intensity, %
Figure 4. The influence of a constant MF
on the intensity of dichlorofluorescein
fluorescence in the neutrophil suspension.
Statistically significant differences from
the control values are pointed by an
asterisk (P < 0.05).
From the practical point of view, the result showing the absence of biological action of weak CMF
at 2.5 μT, while the neutrophil suspension reacts both at lower (0.02 μT) and higher (7 μT)
magnitudes, is important. This fact is also interesting because earlier the similar result (absence of
action) was shown at close CMF value, 3 μT, on another experimental model, division of planaria
Dugesia tigrina [23].
There are several cellular systems generating free radicals as main products or byproducts. First of
all, such systems include NADPH oxidases, membrane-bound enzymes producing superoxide anion
radical (SAR) via one-electron reduction reaction [24]. Beside that, an important system of SAR
production are mitochondria. The leakage of SAR was shown to occur in 11 sites of their inner
membrane, mainly in complexes I, II and III, SAR being produced both into matrix and into
intermembrane space [25]. Lucigenin is considered as a selective probe for SAR [19], this is why it is
actively used for studying ROS production by both NADPH oxidase and mitochondria [20]. We have
previously shown [26] that the addition of a NADPH-oxidase inhibitor diphenyliodonium to the
incubation medium leads to the decreased intensity of chemiluminescence in both experimental
(hypomagnetic conditions) and control (geomagnetic field) samples. The differences between the
groups caused by action of “zero” magnetic field were observed in a wide range of concentrations of
this inhibitor (2.5-100 μM) to the similar extent. Contrary to that, addition of an agent uncoupling
oxidation and phosphorylation in mitochondria, 2,4-dinitrophenol, from 5 μM and up to 200 μM,
almost completely leveled the differences between control and experimental group, that were
observed in absence or at lower concentrations of this inhibitor. These data show the prospect of
studying neutrophil mitochondria as potential targets reacting on changes of CMF values.
On the whole, the obtained data show high sensitivity of biological processes to HMC and
variations of weak static magnetic fields.
XIV International Conference "Space and Biosphere" (Space and Biosphere 2021)
IOP Conf. Series: Earth and Environmental Science 853 (2021) 012008
IOP Publishing
doi:10.1088/1755-1315/853/1/012008
5
4. Conclusion
Two different methods, fluorescence spectroscopy determining intracellular ROS production, and
chemiluminescence analysis with lucigenin, a SAR-specific chemiluminescence activator, have shown
a decrease in ROS production by neutrophils under hypomagnetic conditions. This effect of
hypomagnetic field depends on the magnitude of the residual constant magnetic field in the same way
for both research methods used. It must be noted that both methods for ROS detection used in the
work revealed one value of weak constant magnetic field, 2.5 µT, under which the studied system
(neutrophil suspension) makes no significant response to the field compared to ROS production under
natural geomagnetic field (44 µT). Meanwhile, the changes of weak constant field value in the area
close to 2.5 µT (switching to 0.02 µT or 7 µT) led to decrease in ROS production also detectable by
both methods. Since the value of the acting magnetic field can be related to the value of magnetic
moment of the field sensor and its lifetime, these results could be useful for discovering the sensors of
weak magnetic field in the studied biological system.
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IOP Conf. Series: Earth and Environmental Science 853 (2021) 012008
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doi:10.1088/1755-1315/853/1/012008
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... В этой связи перспективны исследования действия МП на нативных тканях (приспособленных к жизнедеятельности в условиях слабого ГМП), в частности, на крови [20,21] и на ее клеточных компонентах, в том числе фагоцитирующих клетках -нейтрофилах (одних из основных продуцентов АФК в крови), при их кратковременной инкубации вне организма в определенных (создаваемых и четко контролируемых) магнитных условиях [22][23][24]. На этих объектах удалось получить устойчивые и выраженные результаты влияния изменений параметров магнитного поля (комбинированных МП; «нулевого», постоянного и импульсного МП) на продукцию АФК [25][26][27][28], что позволяет исследовать биофизические молекулярные механизмы действия этого физического фактора [29][30][31]. ...
... Поэтому при наличии детально снятой экспериментальной зависимости величины эффекта от величины ослабленного остаточного постоянного МП в диапазоне близком к «нулю», есть вероятность удачного расчета и определения мишени, ответственной за конкретный магнитобиологический эффект. В этой связи можно упомянуть результаты наших экспериментов по действию ослабленного постоянного МП на продукцию АФК нейтрофилами, в которых действительно выявлена анизотропия ответа в зависимости от величины остаточного постоянного МП [27,41,42]. Можно надеяться, что при наличии более детальной экспериментально определенной зависимости такие расчеты удастся произвести, и определить соответствующие первичные мишени этого магнитобиологического эффекта. ...
... В наших недавних работах были представлены результаты влияния гипомагнитных условий [24,27,41,42] и комбинированных магнитных полей [22,23] на продукцию АФК перитонеальными нейтрофилами мыши. Как более простая модель, отвечающая на воздействие гипомагнитных условий, использовались неактивированные клетки, что позволило исключить из рассмотрения дополнительные факторы, связанные с перестройкой метаболического режима работы нейтрофила при его химической стимуляции активаторами респираторного взрыва [24,41,42]. ...
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