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ISSN 0006-3509, Biophysics, 2017, Vol. 62, No. 5, pp. 759–763. © Pleiades Publishing, Inc., 2017.
Original Russian Text © V.V. Novikov, E.V. Yablokova, G.V. Novikov, E.E. Fesenko, 2017, published in Biofizika, 2017, Vol. 62, No. 5, pp. 926–931.
The Role of Lipid Peroxidation and Myeloperoxidase in Priming
a Respiratory Burst in Neutrophils under the Action of Combined
Constant and Alternating Magnetic Fields
V. V. Novikov*, E. V. Yablokova, G. V. Novikov, an d E . E . Fesenko
Institute of Cell Biophysics, Russian Academy of Sciences, Pushchino, Moscow oblast, 142290 Russia
*e-mail: docmag@mail.ru
Received June 5, 2017
Abstract⎯The enhancement of lipid peroxidation in neutrophils (the content of malonic dialdehyde
increased by 10.2%) has been shown after a 1-h exposure to a combined constant (42 μT) magnetic field and
a weak low-frequency magnetic field (1.0, 4.4, and 16.5 Hz; 860 nT) collinear to it. No correlation was found
between this effect and the process of functional pre-activation (priming) of neutrophils as a result of the
combined action of magnetic fields detected by chemiluminescence enhancement in response to the intro-
duction of the bacterial peptide N-formyl–Met–Leu–Phe in the presence of luminol, since ionol (10 μM),
an inhibitor of lipid peroxidation, did not reduce the neutrophil priming index in this case. Preliminary addi-
tion of histidine (0.1 and 1.0 mM), a singlet oxygen scavenger, also did not decrease the priming index. A
myeloperoxidase inhibitor, sodium azide (0.1 mM), exerted a signif icant inhibitory effect on the chemilumi-
nescence intensity of the neutrophil suspension; priming did not develop in the presence of this inhibitor after
the action of combined magnetic fields.
Keywords: weak magnetic field, neutrophils, free radicals, lipid peroxidation, chemiluminescence, myeloper-
oxidase, singlet oxygen
DOI: 10.1134/S0006350917050165
INTRODUCTION
Analysis of experimental data on the effects of low-
frequency magnetic fields of different intensities on
oxidative responses of mammalian cells and cell lines
associated with the generation of free radicals and
other reactive oxygen species shows the prevalence of
studies with strong magnetic fields with an induction
of more than 1 mT [1]. There are some reports in the
literature on the influence of relatively weak (tens of
μT) low-frequency magnetic fields on the kinetics of
the formation of active oxygen species in a suspension
of neutrophils [2–4]. We have shown the increased
generation of free radicals and other reactive oxygen
species in experiments on the whole blood of mam-
mals [5–7] and separate cell subpopulations (neutro-
phils) [8, 9] as a result of the action of combined con-
stant and low-frequency alternating magnetic fields
(CCAMFs) with a very weak variable component (less
than 1 μT) using methods of activated chemilumines-
cence and fluorescence spectroscopy. In these experi-
ments the priming effect of a weak combined constant
(42 μT) magnetic field and a low-frequency alternat-
ing (1.0, 4.4, and 16.5 Hz; 0.86 μT) magnetic field col-
linear to it was recorded, which was manifested as a
more pronounced increase in chemiluminescence of a
neutrophil suspension after CCAMF pre-treatment in
the response to the introduction of the bacterial pep-
tide N-formyl–Met–Leu–Phe or a phorbolic ether
phorbol-12-maristat-13-acetate in the presence of
luminol. It was shown that a low concentration of the
intracellular calcium chelator BAPTA-AM blocked
the effect of the weak CCAMFs [10]. At the same
time, the level of extracellular calcium virtually did not
affect the degree of the respiratory explosion priming
[10]. It follows from this that a redistribution of intra-
cellular calcium in neutrophils is possible under the
action of weak CCAMFs. Apparently, an increased
outflow of calcium ions into the cytosol from intracel-
lular depots may be a key point of the mechanism of
weak CCAMF action. This mechanism differs from
that observed in the priming effect on neutrophils of
another well-studied relatively weak physical factor,
low-intensity laser radiation. According to the
hypothesis of Yu. A. Vladimirov [11], which has been
confirmed experimentally [12, 13], low-intensity laser
radiation increases lipid peroxidation in the mem-
brane, thereby increasing its permeability for calcium
ions, and as a result activates intracellular processes,
Abbreviations: CCAMF, combined constant and low-frequency
alternating magnetic fields; LPO, lipid peroxidation; fMLP,
peptide N-formyl–Met–Leu–Phe.
CELL BIOPHYSICS
760
BIOPHYSICS Vol. 62 No. 5 2017
NOVIKOV et al.
causing priming of phagocytes. In our case of the
action of weak CCAMFs with a very weak variable
component, the processes of lipid peroxidation (LPO)
in the membranes of neutrophils and their role in the
functional activation of the cells has not yet been stud-
ied. In this regard, the aim of this work was to study the
relationship of LPO processes in neutrophil mem-
branes and subsequent changes in the functional activ-
ity of the cells under weak CCAMF action.
MATERIALS AND METHODS
Obtaining neutrophil suspensions. This work was
performed on the peritoneal neutrophils of mice. Lab-
oratory male mice of the Balb line weighing 22–25 g
were used to obtain peritoneal neutrophils; 150 μL of
an opsonizing zymosan suspension at a concentration
of 5 mg/mL (Zymozan A from Saccharomyces carevi-
siae, Sigma, United States) was injected into the peri-
toneal cavity of the mouse. After 12 h, the animals
were killed by ulnar dislocation and their peritoneal
cavity was washed with 3 mL of chilled Hanks solution
without calcium. The exudate was collected with a
pipette and centrifuged for 5 min at 600 g. The super-
natant was decanted and the residue was dissolved in
1 mL of calcium-free Hanks solution and left for
40 min at 4°C. The number of isolated cells was
counted in a Goryaev hemocytometer. Cell viability
was determined using the vital dye trypan blue. The
content of living cells was no less than 98%. The sam-
ples for experiments were obtained by diluting the sus-
pension of neutrophils to a concentration of
106cells/mL in a standard Hanks 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).
The exposure of the neutrophil suspension to a mag-
netic field. The 0.25-mL samples of neutrophils at a
concentration of 2 · 106 cells/mL were incubated in
polypropylene tubes at 37 ± 0.1°C. The typical incu-
bation time was 1 h. The temperature was maintained
by a circulating thermostat. Before the start of incuba-
tion, an inhibitor of lipid peroxidation ionol (2,6-Di-
tert-butyl-4-methylphenol) (Sigma, United States) at
a concentration of 10 μM or the singlet oxygen scaven-
ger histidine (Sigma, United States) at concentrations
of 0.1 and 1.0 mM were added to a part of the samples.
Sodium azide at concentrations of 0.1 and 1.0 mM was
used as a non-specific inhibitor of intracellular and
extracellular activity of myeloperoxidase [14], which
was also added to some samples before the incubation.
The samples of the control groups were in the local
geomagnetic field with a constant component of
approximately 42 μT and the level of the magnetic
background at 50 Hz of 15–50 nT that corresponded
to these indices in the experimental groups, except
that it was artificially given the variable component of
the field.
The apparatus for the exposure to weak magnetic
fields consisted of two pairs of coaxially arranged
Helmholtz rings with a diameter of 140 cm oriented
along the geomagnetic field vector. A direct current
was applied to one pair of the rings to form a preset
value of the constant magnetic field component of
42 ± 0.1 μT. An electric current from a generator of
sinusoidal signals was applied to the second pair of the
rings for the formation of the variable field compo-
nent. The base amplitude of the variable component
was of 860 ± 10 nT. A three-frequency signal of 1.0,
4.4, and 16.5 Hz, with the amplitudes of the individual
frequencies of 600, 100, and 160 nT, respectively,
which showed activity in previous studies [5–10, 15],
were used in the experiments. The magnetic field val-
ues were determined by direct measurement using a
Mag-03 MS 100 f luxgate sensor (Bartington, UK).
Determination of lipid peroxidation by the thiobarbi-
turic test for malonic dialdehyde. A modified spectro-
fluorometric method [13, 16] was used to determine
the accumulation of thiobarbiturate-active lipid oxi-
dation products in the neutrophils suspension. Four
samples from each group (control and experimental)
were placed into centrifuge tubes after cultivation;
1.5 mL of 1% phosphoric acid (pH 2.0) and 0.5 mL of
0.67% thiobarbituric acid were added to each tube.
The tubes were shaken for 1 min. The samples were
incubated for 45 min in a boiling water bath. The tubes
were cooled in a current of cold water to room tem-
perature; 2 mL of butanol was added to each tube and
the tube content was intensively stirred at an angle of
45° for 3 min. Phase separation was performed by cen-
trifugation at 1500 g for 10 min. The upper butanol
phase was sampled for the investigation. To determine
the concentration of thiobarbiturate-active products,
the fluorescence spectrum of butanol extract was
recorded on a Lumina Fluorescence Spectrometer
(Thermo Fisher Scientific, United States), at exci-
tation and emission wavelengths of 515 and 553 nm,
respectively.
For the assessments, some of the results were pre-
sented as the ratio of the maximum fluorescence
intensity in the experiments to the baseline control,
taken as 100%. The experiments were repeated three
times.
Recording chemiluminescence. After 1 hour of
incubation, the chemiluminescence intensity was
measured in the control and experimental samples
after the addition of a 0.35 mM luminol solution
(Enzo Life Sciences, United States) and an activator
of the generation of reactive oxygen species, the che-
motactic peptide N-formyl–Met–Leu–Phe (fMLP)
(Sigma, United States) at a concentration of 1 μM. A
Lum-5773 chemiluminometer (Disoft, Russia) was
used. PowerGraph software was used for chemilumi-
nescence data analysis. Part of the results are pre-
sented as the ratio to the amplitudes of the chemilumi-
nescent response in the control, which was taken as
BIOPHYSICS Vol. 62 No. 5 2017
THE ROLE OF LIPID PEROXIDATION AND MYELOPEROXIDASE 761
100%. For clarity, an index of priming (prestimula-
tion) of the respiratory burst is used, which is equal to
the ratio of the maximum chemiluminescence inten-
sity in the experiment to the corresponding value in
the control.
The results were statistically processed using the
student’s t-test.
RESULTS AND DISCUSSION
As can be seen from Fig. 1, the impact of weak
CCAMFs led to a small increase of 10.2% (110.2 ±
5.1% relative to control values constituting 100 ±
2.1%, P < 0.05) in the content of malondialdehyde in
neutrophils. It follows from this that the process of
lipid peroxidation in the neutrophil membranes was
slightly accelerated under the action of CCAMF.
However, preliminary addition of the lipid peroxida-
tion inhibitor ionol at 10 μM to the suspension of neu-
trophils does not affect the result of functional activa-
tion of neutrophils by weak CCAMFs (Fig. 2), as eval-
uated by the chemiluminescence enhancement in the
presence of fMLP and luminol. The priming indices
of the respiratory explosion in neutrophils under the
CCAMF action in this series of experiments, with and
without ionol addition, were 2.9 and 3.6, respectively.
In the control cases, 10 μM ionol addition caused
some (22.5%) reduction of the chemiluminescence
intensity, which was not observed in the experimental
samples (Fig. 2). These results do not confirm the
hypothesis that a decisive influence of LPO occurs on
the priming of neutrophils under the action of weak
CCAMFs. In this experiment, under weak CCAMF
action only small (approximately 10%) changes in
LPO and the lack of the inhibitory effect of the inhib-
itor LPO on the reactive oxygen species production
were detected. This result is in contrast to the situation
for low-intensity laser irradiation, when it was shown
that this treatment led to a significant (several times)
increase in the content of malondialdehyde in the
neutrophil membranes [13] and this process was
linked with the functional activation of the cells, since
LPO inhibitors reduced the degree of the priming
effect [13].
The influence of the singlet oxygen scavenger histi-
dine on the functional activation of neutrophils under
weak CCAMF action was studied in a separate series
of experiments. As seen in Fig. 3, histidine in the
tested concentrations (0.1 and 1.0 mM) had little
effect on the chemiluminescence intensity of neutro-
phils, which increased after exposure to the CCAMFs.
The priming index of the respiratory explosion in
these experiments without prior addition of histidine
was 2.3, while after histidine addition at 0.1 and
1.0 mM it was 2.5 and 2.8, respectively. Some increase
of the priming index in the presence of histidine is due
to its stronger inhibitory effect on the control samples.
With the addition of 0.1 mM histidine, the intensity of
chemiluminescence was reduced by 18.5% in the con-
trol and by 12% in the experiment. With the addition
of 1 mM histidine, the intensity of chemilumines-
cence in the control continued to decline to 70.4% of
the initial value; while a further decline practically was
not observed in the experiement. These data do not
support a special role of singlet oxygen in the mecha-
nism of the weak CCAMF action on neutrophil prim-
ing. Singlet oxygen along with hydroxyl radical is the
most prominent initiator of LPO in the membranes
[17, 18]. Previously, we obtained similar results in
Fig. 1. The fluorescence spectra of the thiobarbiturate-
active lipid oxidation products in a butanol extract from a
suspension of neutrophils at the excitation wavelength of
515 n m in the co ntr ol (1) and experiment (2) after the
action of weak combined magnetic fields.
0
35 000
30 000
25 000
20 000
15 000
10 000
5000
40 000
1
2
625550525 575 600
Wavelength, nm
F
luorescence intensity, arb. un.
Fig. 2. The ionol effect on the chemiluminescence inten-
sity of neutrophils upon fMLP stimulation of cells in the
presence of luminol; the control (1) and the experimental
(2) (CCAMF treatment) samples were incubated without
and with ionol for 60 min at 37°C before the chemilumi-
nescence measurements. The data for chemiluminescence
intensity (maximum values) are expressed in percent rela-
tive to the baseline control (mean values and standard
deviations). The asterisk indicates significant differences
from the control groups (P < 0.05).
0
1
1
22
300
250
200
150
100
50
350
Chemiluminescence intensity, %
010
Ionol concentration, µM
**
762
BIOPHYSICS Vol. 62 No. 5 2017
NOVIKOV et al.
experiments with the hydroxyl radical scavenger
dimethyl sulfoxide [10]. Given the small LPO changes
detected in the present work, along with lack of a rela-
tionship between the LPO intensity and the neutrophil
preactivation processes in the weak CCAMF (no
inhibitory effect of the LPO inhibitor on the func-
tional activation of neutrophils), this result is not
unexpected.
The effect of sodium azide, which is an inhibitor of
myeloperoxidase, on the chemiluminescence intensity
of neutrophils in the control and the experiment (after
the CCAMFs) is shown in Fig. 4. Both studied con-
centrations of 0.1 and 1.0 mM cause a sharp decrease
of this indicator. The chemiluminescence intensity
was reduced by approximately five times in the con-
trol. Chemiluminescence was reduced to the same val-
ues in the suspension of neutrophils in the experiment.
The respiratory explosion priming index changed from
3.75 (without the addition of sodium azide) to 1.0.
Previously we showed a similar effect of apocynin,
which is an inhibitor of NADPH oxidase, on these
processes [6].
Apparently, intracellular regulatory processes (in
particular, activation of protein kinase C and phos-
pholipase C), which provide calcium-dependent pre-
activation of the key enzymes of the respiratory burst
(NADPH-oxidase, myeloperoxidase, etc.), can be
sensitive to weak CCAMFs directly (or via the hydra-
tion envelopes of proteins), in contrast, for example, to
the mechanism of the laser-radiation action that is
mediated mainly through the cell membrane. The
small increase of the LPO intensity observed in our
experiments may be a consequence of the transmem-
brane leakage of electrons in the NADPH oxidase
activation [19], or can be explained in terms of spin
chemistry for the case of weak alternating magnetic
fields [20].
It is tempting to suggest that the CCAMFs used in
this work affect a biological object via inf luence on the
structure formation processes in the aqueous phase of
a living organism. The similarity of the CCAMF
parameters that were used in this study to those that
cause effects in relatively simple aquatic systems sup-
ports this possibility. These effects include an inf lu-
ence on structure formation in water under the action
of weak magnetic fields and xenon (the formation of
micron size gas-hydrate crystals) [21] and the occur-
rence of the fluorescent fractions of associates after
the CCAMF action and gel filtration [22, 23];
CCAMFs affect ionic currents in aqueous solutions of
amino acids [24–31] and, as recently shown,
CCAMFs change the refractive index in pure water
under the action of the hydronic ion and its hydrated
form at cyclotron frequencies [32]. A number of exper-
imental data provide a strong argument in favor of the
role of the aqueous phase in the biological effects of
weak magnetic f ields and high-frequency electromag-
netic fields ([33–40] and others). In this regard, the
development of a methodical basis for the study of the
role of the aquatic environment in the reception and
response of an organism to the action of a weak mag-
netic field is topical.
Fig. 3. The histidine effect on the chemiluminescence
intensity of neutrophils upon fMLP stimulation of cells in
the presence of luminol; the control (1) and experiment
(2) (CCAMF treatment) samples incubated without and
with histidine for 60 min at 37°C before the chemilumines-
cence measurements. The data for chemiluminescence
intensity (maximum values) are expressed in percent rela-
tive to the baseline control (mean values and standard
deviations). The asterisk indicates significant differences
from the control groups (P < 0.05).
0
1
1
1
2
2
2
300
250
200
150
100
50
350
Chemiluminescence intensity, %
01.00.1
Histazine concentration, µM
*
*
*
Fig. 4. The effect of sodium azide on the chemilumines-
cence intensity of neutrophils upon fMLP stimulation of
cells in the presence of luminol; the control (1) and exper-
iment (2) (CCAMF treatment) samples incubated without
and with sodium azide for 60 min at 37°C before the che-
miluminescence measurements. The data for chemilumi-
nescence intensity (maximum values) are expressed in per-
cent relative to the baseline control (mean values and stan-
dard deviations). The asterisk indicates significant
differences from the control groups (P < 0.05).
0
1
122
1
2
300
250
200
150
100
50
350
Chemiluminescence intensity, %
0 0.50.1
Sodium azide concentration, µM
*
BIOPHYSICS Vol. 62 No. 5 2017
THE ROLE OF LIPID PEROXIDATION AND MYELOPEROXIDASE 763
ACKNOWLEDGMENTS
This work was supported by the Russian Founda-
tion for Basic Research and the Ministry of Invest-
ments and Innovations of Moscow Region, project
no. 14-44-03676P_center_a.
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Translated by E. Puchkov
