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ISSN 0006-3509, Biophysics, 2018, Vol. 63, No. 3, pp. 365–368. © Pleiades Publishing, Inc., 2018.
Original Russian Text © V.V. Novikov, E.V. Yablokova, E.E. Fesenko, 2018, published in Biofizika, 2018, Vol. 63, No. 3, pp. 484–488.
The Effect of a “Zero” Magnetic Field on the Production of Reactive
Oxygen Species in Neutrophils
V. V. Novikova, *, E. V. Yablokovaa, and E. E. Fesenkoa
aInstitute of Cell Biophysics, Russian Academy of Sciences, ul. Institutskaya 3, Pushchino, Moscow oblast, 142290 Russia
*e-mail: docmag@mail.ru
Re ceived Mar ch 15 , 2018
Abstract⎯Exposure of mouse peritoneal neutrophils to hypomagnetic conditions (magnetic shielding, a
residual static magnetic field of 20 nT) for 1.5 h decreased the level of intracellular reactive oxygen species as
recorded by changes in the fluorescence intensity of 2,7-dichlorodihydrofluorescein and dihydrorhodamine
123 oxidation products. The effect of a hypomagnetic field was similarly observed after adding a respiratory
burst activator (the formylated peptide N-formyl–Met–Leu–Phe or phorbol 12-meristate-13-acetate) to a
low concentration.
Keywords: hypomagnetic field, neutrophils, reactive oxygen species, fluorescence, respiratory burst
DOI: 10.1134/S000635091803017X
INTRODUCTION
The possibility of affecting the production of reac-
tive oxygen species (ROS) is thought to provide a
promising approach to understanding the mecha-
nisms of the biological effects of weak magnetic fields
[1–4]. Our previous experiments have been performed
with mammalian whole blood and individual cell pop-
ulations (neutrophils) by activated chemilumines-
cence and f luorescence spectroscopy and have shown
that free radicals and other reactive oxygen and chlo-
rine species are generated to higher levels on exposure
to static and low-frequency alternating combined
magnetic fields with an extremely weak alternating
component (less than 1 μT) [5–11]. It was of interest
to study how hypomagnetic conditions affect these
processes.
A recent review [12] have summarized the results of
more than 100 original experimental works focusing
on the effects of hypomagnetic fields and have com-
pared several hypotheses to explain the effects. As
noted, hypomagnetic f ields are likely to exert detect-
able effects because the dynamics of all magnetic
moments involved in precession are inf luenced in
hypomagnetic conditions (precession arrest) in view
of the fact that quantum-energy levels of magnetic
moments are not split into Zeeman sublevels in a
“zero” magnetic field. Studies of the problem, as well
as studies of the effects exerted by combined fields,
may help to identify the primary targets and biochem-
ical mechanisms involved in transduction of a mag-
netic signal in a neutrophil suspension.
At this step, the effects of hypomagnetic conditions
were investigated by fluorescence spectroscopy with
two known cell-penetrating fluorescent probes used to
detect ROS, namely, 2,7-dichlorodihydrofluorescein
diacetate (H2DCF-DA) and dihydrorhodamine 123
[13–16]. H2DCF-DA penetrates the cell and is con-
verted to H2DCF by intracellular esterases. H2DCF
fluoresces weakly; however, its reaction with oxidizers
yields dichlorofluorescein, which has high fluores-
cence intensity. Like H2CDF-DA, dihydrorhodamine
is a lipophilic compound that easily penetrates into the
cell. Oxidation of dihydrorhodamne 123 yields fluo-
rescent rhodamine 123. One of its amino groups
acquires a charge upon oxidation, thus preventing the
oxidized dye from leaving the cell.
MATERIALS AND METHODS
Neutrophil suspensions. Mouse peritoneal neutro-
phils were used in experiments. Peritoneal neutrophils
were obtained from male CD-1 laboratory mice with a
body weight of 24–26 g, which were obtained from the
laboratory animal breeding facility of the Institute of
Bioorganic Chemistry Branch (Pushchino, Moscow
oblast). Mice were injected intraperitoneally with
150 μL of 5 mg/mL opsonized Saccharomyces ceveri-
siae zymosan A (Sigma, United States) and sacrificed
by ulnar dislocation 12 h later. The peritoneal cavity
was washed with 3 mL of chilled calcium-free Hanks’
solution. Exudation fluid was collected with a pipette
and centrifuged at 600 g for 10 min. The supernatant
Abbreviations: ROS, reactive oxygen species; H2DCF-DA, 2,7-
dichlorodihydrofluorescein diacetate; fMLF, N-formyl–Met–
Leu–Phe; PMA, phorbol 12-meristate-13-acetate.
CELL BIOPHYSICS
366
BIOPHYSICS Vol. 63 No. 3 2018
NOVIKOV et al.
was discarded by decantation and the pellet was resus-
pended in 2 mL of calcium-free Hanks’ solution and
incubated at 4°C for 1 h. Isolated cells were counted in
a Goryaev chamber. Cell viability was assayed with the
vital dye Trypan Blue and was observed to be no less
than 98%. To obtain test samples, the neutrophil sus-
pension was diluted to 1 · 106 cells/mL 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, and 10 mM HEPES, pH 7.4;
Sigma, United States).
Exposure of neutrophil suspensions in a hypomag-
netic field. Neutrophils (250 μL, 1 · 106 cells/mL) were
incubated in Eppendorf-type polypropylene tubes at
37.0 ± 0.2°C with light deprivation. The temperature
was maintained in a circulation thermostat. The typi-
cal incubation time was 1.5 h. Control samples were
placed in the local geomagnetic field with a static
component of ~42 μT and a background magnetic
field strength of 15–50 nT at 50 Hz.
The device used to expose samples to hypomag-
netic conditions consisted of three coaxial cylindrical
magnetic shields of 1-mm permalloy, each covered
with a lid. The residual static magnetic field did not
exceed 20 nT. The actual magnetic field strengths were
measured with a Mag-03MS100 ferroprobe instru-
ment (Bartington, United Kingdom). Ten control
(geomagnetic f ield) and ten test (hypomagnetic con-
ditions) samples were incubated simultaneously. The
experiments were carried out at least in triplicate.
Fluorescence detection of intracellular ROS. After a
1.5-h incubation in hypomagnetic conditions, a neu-
trophil suspension was supplemented with
0.01 mg/mL ROS-detecting f luorescent probe
(H2DCF-DA or dihyrorhodamine 123). In some
experiments, control and test samples were addition-
ally supplemented with 0.5 μM chemotactic formy-
lated peptide N-formyl–Met–Leu–Phe (fMLF;
Sigma, United States) or 0.05 μM phorbol 12-meri-
state-13-acetate (PMA; Sigma, United States) to trig-
ger ROS production. The samples were further incu-
bated at 37°C in the dark to minimize dye photooxida-
tion. Incubation was carried out for 30 min in the case
of H2DCF-DA and 10 min in the case of dihydror-
hodamine 123. Cells were washed with Hanks’ solu-
tion at room temperature and centrifuged at 600 g for
10 min. The pellet was resuspended in 1 mL of the
medium, and a fluorescence spectrum was recorded
using a Lumina fluorescence spectrometer (Thermo
Scientific, United States) with excitation at 488 nm.
The results were analyzed using Student’s t-test. In
some cases, test results were expressed as the percent
ratio of the maximum fluorescence intensity in a test
sample to the baseline intensity measured in a control
sample in the absence of the activators; the latter was
taken as 100%.
RESULTS AND DISCUSSION
Exposure of peritoneal neutrophils in hypomag-
netic conditions (the geomagnetic field weakened by a
factor of ~2000) substantially (by 25%) reduced the
fluorescence intensity of intracellular dichlorofluores-
cein (Figs. 1, 2). Approximately the same difference
(~23%) between control (geomagnetic field) and test
(hypomagnetic field) samples was observed when cells
were additionally stimulated with fMLF. Mere stimu-
lation of neutrophils with a relatively low fMLF dose
(0.5 μM) increased the fluorescence intensity of the
ROS-detecting probe by 18% in control samples and
16% in test samples (Fig. 2). Neutrophil stimulation
with PMA (0.05 μM) increased the fluorescence
intensity of intracellular dichlorofluorescein in con-
trol samples by 30%, on average. When cells were
stimulated after exposure to hypomagnetic conditions,
the increase was 22% relative to the respective control
(Fig. 2). It is important to note that the dichlorofluo-
rescein fluorescence intensities observed in test sam-
ples after stiulation with fMLF or PMA approached
the intensities observed in control samples without
stimulation.
The rhodamine 123 fluorescence intensity in test
samples (hypomagnetic conditions without additional
stimulation) was substantially (by 35%) lower than in
control samples (Figs. 3, 4). The effects of additional
stimulation with fMLF were estimated at 23% in the
control and 35% in the test samples. The difference
between the control and test samples was 20% in this
case (stimulation with fMLF). Similar results were
obtained for additional neutrophil stimulation with
PMA (Figs. 3, 4).
Thus, exposure to hypomagnetic conditions
decreased the baseline production of intracellular
ROS in neutrophils, as was observed in all experiments
with both of the fluorescent probes. The effect of a
hypomagnetic field was similarly detected in experi-
Fig. 1. Dichlorofluorescein fluorescence spectra of
(1) control neutrophil suspensions and (2) suspensions
exposed to a hypomagnetic field. Standard deviations are
shown with dashed lines.
0
35 000
30 000
25 000
20 000
15 000
10 000
5000
650550 600500
Wavelength, nm
1
2
Fluorescence intensity,
conv. units
BIOPHYSICS Vol. 63 No. 3 2018
THE EFFECT OF A “ZERO” MAGNETIC FIELD 367
ments where neutrophils were additionally stimulated
with the respiratory burst activators fMLF and PMA.
The effect was determined to a greater extent by the
initial difference in ROS production than by a dis-
torted neutrophil response to the activators in this
case.
Phagosomes are well-known sites of intracellular
ROS production in neutrophils, However, ROS can
be generated as well in cytochrome b-containing gran-
ules outside the phagosomes [17]. Phagocytes possess
not only NADPH oxidase, but also other ROS-gener-
ating systems, such as NADPH oxidases with other
members of the NOX family [18], NO synthases [19],
and single mitochondria [20]. However, the NOX2-
containing oxidase complex yields ROS in far greater
amounts compared with other cell oxidases in phago-
cytes [17]. When oxidase is assembled on the plasma
membrane, ROS are released into the surrounding
extracellular space (extracellular ROS); when oxidase
is assembled on intracellular membranes, ROS remain
within membrane organelles (intracellular ROS).
Both intracellular and extracellular ROS are generated
in cells activated by some stimulus [21, 22].
The extent to which NADPH oxidase subunits are
phosphorylated plays a key role in regulating NADHP
oxidase activity of in neutrophils by determining
whether the enzyme complex is assembled on a mem-
brane from several specific cytoplasmic and mem-
brane proteins [23–25]. Phosphorylation of NADPH
oxidase subunits is controlled primarily by protein
kinase C; protein kinase C activity strongly depends on
the intracellular Ca2+ level [23–25] and is associated
with phospholipase C activity, which provides the
proper levels of inositol triphosphate (which stimu-
lates a release of calcium ions from the endoplasmic
reticulum) and diacylglycerol (which directly activates
protein kinase C). Our findings make it possible to
assume that phosphorylation of NADPH oxidase sub-
units is inhibited in hypomagnetic conditions, leading
eventually to lower ROS production. In contrast,
combined magnetic fields with certain parameters
[26–28] cause pre-activation (priming) of neutro-
phils; this priming is abolished by low concentrations
Fig. 2. The effect of a hypomagnetic field on dichloroflu-
orescein fluorescence intensity in a neutrophil suspension
in the presence or absence of respiratory-burst activators.
Ordinate, the percentage of the maximum fluorescence
intensity relative to the baseline fluorescence observed in
control samples without the activators (means and stan-
dard deviations are shown, n = 10). Abscissa, group:
1, control and test samples without activators; 2, control
and test samples with 0.5 μM fMLP; and 3, control and
test samples with 0.05 μM PMA . The dif ferences from the
respective controls were significant at (*) P < 0.05.
0
100
75
50
25
125
150
*
*
*
123
Group
Fluorescence intensity, %
Fig. 3. The rhodamine 123 fluorescence spectra of (1) con-
trol neutrophil suspensions and (2) suspensions exposed to
a hypomagnetic field. Standard deviations are shown with
dashed lines.
0
35 000
40 000
30 000
25 000
20 000
15 000
10 000
5000
650550 600500
Wavelength, nm
1
2
Fluorescence intensity,
conv. units
Fig. 4. The effect of a hypomagnetic field on rhodamine
123 fluorescence intensity in a neutrophil suspension in
the presence or absence of respiratory burst activators.
Ordinate, the percentage of the maximum fluorescence
intensity relative to the baseline fluorescence observed in
control samples without the activators (means and stan-
dard deviations are shown, n = 10). Abscissa, group:
1, control and test samples without activators; 2, control
and test samples with 0.5 μM fMLP; and 3, control and
test samples with 0.05 μM PMA . The differences from the
respective controls were significant at (*) P < 0.05.
0
100
75
50
25
125
150
*
*
*
123
Group
Fluorescence intensity, %
368
BIOPHYSICS Vol. 63 No. 3 2018
NOVIKOV et al.
of an intracellular Ca2+ chelator [7, 10]. These data
also implicate NADPH oxidase phosphorylation in
the mechanism that sustains the biological effect of
weak magnetic fields. These issues are certainly wor-
thy of further investigation.
A decrease in ROS production in hypomagnetic
conditions has been described in the literature for
other cells and longer exposures [29–31]. Our experi-
mental model has several advantages, e.g., the mecha-
nisms of ROS generation in neutrophils are well
understood and relatively simple and the effect
becomes detectable after a short exposure. It is of prin-
cipal importance to determine how the effect depends
on the hypomagnetic field strength in such studies,
because such data may provide further information
about the molecular targets of the effect [12]. A dis-
crete character of the response to exposure to an
extremely weak static magnetic field has been
observed in our previous experiments, wherein planar-
ian division and regeneration was used as a model, i.e.,
two peak responses were observed in a range of 0–
1.5 μT, with their separation being especially distinct
at ~500 nT [32, 33]. We believe it is important to per-
form similar measurements with neutrophil suspen-
sions.
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Translated by T. Tkacheva