Electromagnetic Biology and Medicine

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Role of NADPH oxidase in MAPK signaling

activation by a 50 Hz magnetic field in human
neuroblastoma cells

María Antonia Martínez, Alejandro Úbeda & María Ángeles Trillo

To cite this article: María Antonia Martínez, Alejandro Úbeda & María Ángeles Trillo
(2021) Role of NADPH oxidase in MAPK signaling activation by a 50 Hz magnetic field in
human neuroblastoma cells, Electromagnetic Biology and Medicine, 40:1, 103-116, DOI:
10.1080/15368378.2020.1851250

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ELECTROMAGNETIC BIOLOGY AND MEDICINE
2021, VOL. 40, NO. 1, 103–116
https://doi.org/10.1080/15368378.2020.1851250

Role of NADPH oxidase in MAPK signaling activation by a 50 Hz magnetic field in

human neuroblastoma cells
María Antonia Martínez, Alejandro Úbeda, and María Ángeles Trillo
Servicio BEM, Dept. Investigación, Hosp, Univ. Ramón Y Cajal– IRYCIS, Madrid, Spain

ABSTRACT ARTICLE HISTORY

Our previous studies have shown that intermittent exposure to a 50-Hz, 100-µT sine wave magnetic field Received 30 April 2020
(MF) promotes human NB69 cell proliferation, mediated by activation of the epidermal growth factor Accepted 1 November 2020
receptor (EGFR) and pathways MAPK-ERK1/2 and p38; being the effects on proliferation and p38 KEYWORDS
activation blocked by the chelator N-acetylcysteine. The present work investigates the MF effects on NB69; ELF; magnetic field;
free radical (FR) production, and the potential involvement of NADPH oxidase, the main source of MAPK; p67phox; ROS
reactive oxygen species (ROS), in the MF-induced activation of MAPK pathways. To this end, the field
effects on MAPK-ERK1/2, -p38 and -JNK activation in the presence or absence of the NADPH oxidase
inhibitor, diphenyleneiodonium chloride (DPI), as well as the expression of the p67phox subunit, were
analyzed. The results revealed that field exposure increases FR production and induces early, transient
expression of the cytosolic component of the NADPH oxidase, p67phox. Also, the MF-induced activation
of the MAPK-JNK pathway, but not that of -ERK1/2 or -p38 pathways, was prevented in the presence of
the DPI, which has been shown to significantly reduce p67phox expression. These data, together with
those from previous studies, identify various, FR-dependent or -independent mechanisms, involved in
the MF-induced proliferative response mediated by MAPK signaling activation.

Introduction
Although the epidemiological evidence of an association
between chronic exposure to extremely low frequency
(ELF) magnetic fields (MF) and the incidence of cancer is
limited (Carlberg et al. 2017; De Roos et al. 2001; Turner
et al. 2017), expert groups have considered such fields as
possible cancer promoters (IARC 2002; SCENIR 2009).
Also, there is a large body of experimental evidence that
various human cell types are responsive to short-term
exposure to weak ELF MF, having been the power fre­
quency (50–60 Hz) fields and their potential effects on
DNA damage (Ruiz-Gomez and Martinez-Morillo 2009),
cell proliferation (Sulpizio et al. 2011; Trillo et al. 2012), cell
differentiation (Ayşe et al. 2010) or apoptosis (Basile et al.
2011) extensively investigated. Nevertheless, other studies
have not obtained similar results (Santini et al. 2009) and,
in any case, the physical and biological mechanisms under­
lying the bioeffects induced by these MFs have not yet been
described and characterized sufficiently.
Oxidative stress due to disturbances in redox home­
ostasis that increases the levels of reactive oxygen species
(ROS) has been proposed as one of the potential mechan­
isms through which radiofrequency/microwave (RF/MW)
radiation and ELF-MF exposure affect cellular behaviour
(Consales et al. 2012; Falone et al. 2018; Friedman et al.
2007; Mattsson and Simkó 2014; Wang and Zhang 2017;
Yakymenko et al. 2016). For instance, in vitro exposure to
50 Hz MF at 0.1–0.4 mT induces rapid (15–30 min)
increases of total ROS or mitochondrial ROS in human
amniotic epithelial cells (Feng et al. 2016; Sun et al. 2018),
and activates cytoprotective mechanisms related to redox
systems, promoting cell proliferation and malignancy in
the SH-SY5Y human neuroblastoma line (Falone et al.
2017). Although most studies describe an increase in ROS
levels in response to MF, others report decreases or no
changes in ROS content (Hong et al. 2012; Song et al.
2018). It has been proposed that this variety of responses
could be due to differences in the experimental procedures
applied, including, among other factors, the MF frequency
or flux density, the exposure cycle or interval, the cell type,
the biological parameters examined or the presence of
exogenous stressors (Mattsson and Simkó 2014).
Previous studies by our group have shown that 50 Hz
MF at magnetic flux densities (B) of 10 or 100 µT, applied
intermittently (3 h On/3 h Off or 5 min On/10 min Off)
during 24–63 h hours, significantly increase DNA synthesis
and cell proliferation in two human cancer lines: hepato­
cellular carcinoma HepG2 and neuroblastoma NB69
(Martínez et al. 2012; Trillo et al. 2012). In NB69 cells the
reported effects were mediated by MF-induced alterations

CONTACT María Antonia Martínez. m.antonia.martinez@hrc.es Servicio BEM, Dept. Investigación, Hosp, Univ. Ramón Y Cajal– IRYCIS, Madrid, Spain.
Supplemental data for this article can be accessed on the publisher’s website.
© 2020 The Author(s). Published with license by Taylor & Francis Group, LLC.
This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License (http://creativecommons.org/licenses/by-nc-nd
/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited, and is not altered, transformed, or built upon
in any way.
104 M. A. MARTÍNEZ ET AL.
in the activation of pathways MAPK-ERK1/2 and p38
(Martínez et al. 2012, 2016). The results also showed that
the free radical (FR) chelator N-acetylcysteine (NAC)
blocks the MF effects on p38 activation and cell prolifera­
tion, but not those on ERK1/2 activation, which reveals
that FR intervene in some of these processes. More recent
results have shown that the MF-induced proliferative
response is mediated by the epidermal growth factor recep­
tor (EGFR) through the field effects on the aforementioned
signaling pathways (Martínez et al. 2019).
Reactive oxygen species are ubiquitous in mamma­
lian cells, and it is widely accepted that low levels of ROS
can act as subcellular messengers in regulatory routes of
gene expression and signal transduction, such as the
EGFR pathway (Tseng et al. 2012), which is involved
in relevant physiological processes, such as cell prolif­
eration and differentiation or apoptosis. On the other
hand, high levels of ROS can alter cellular physiological
processes by attacking membrane phospholipids, dis­
rupting mitochondrial functions or damaging proteins
and DNA (Schieber and Chandel 2014).
ROS are generated by both mitochondrial and non-
mitochondrial pathways. In the latter, NOX, the oxidase
of the nicotinamide adenine dinucleotide phosphate
(NADPH), is the main contributor to the production of
ROS. ROS derived from NOX activity have been reported
to be closely related to mechanisms involved in cancerous
processes (Paletta-Silva et al. 2013). The NOX complex is
formed by a membranal and a cytosolic component. The
membranal one, called cytochrome b558, is made up of
subunits p22phox and gp91phox (NOX2) and is the cata­
lytic part of the complex. The cytosolic component is
formed by three subunits: p40phox, p47phox and p67phox
that, together with the GTPase Rac, regulate the activity of
the catalytic complex. When activation takes place, the
cytosolic components migrate to the membranes, where
they associate with the membrane-bound components to
assemble the catalytically active oxidase responsible for
superoxide production (Babior et al. 1999). For the latter
to occur, the p67phox cytoplasmic subunit must interact
with NOX2 (Italiano et al. 2012). It has been proposed that
NOX could amplify the receptor-mediated tyrosine kinase
signal, contributing to carcinogenesis through regulation of
cell signaling pathways such as MAPK-p38, -ERK1/2 or -
JNK (Paletta-Silva et al. 2013; Rezatabar et al. 2019).
Diphenyleneiodonium (DPI), one of the most com­
monly used inhibitors of NOX activity, has been reported
to inhibit cytoplasmic and mitochondrial ROS production
(Altenhöfer et al. 2015; Li and Trush 1998) and to limit
tumor cell growth in vitro when administered at nanomo­
lar concentrations (Doroshow et al. 2013). Thus, on the
basis of our previous results on NB69 response to MF, and
since the NADPH oxidase complex has been shown to be
strongly involved in the regulation of cell proliferation, the
DPI inhibitor was used in the present study to investigate
a potential, NADPH-mediated mechanism, through which
a weak, power frequency MF could activate the MAPK
(ERK, p38 and JNK) pathway.
Methods
Cell culture
The neuroblastoma cell line NB69 (lot No. 03I019/2008,
item No. 99072802) was purchased from the European
Collection of Authenticated Cell Cultures (ECACC,
Salisbury, UK). The cells were periodically tested for myco­
plasma contamination (PCR) and response to chemical and
physical treatments. Cells were cultured for four days in
a cell incubator (Thermo Fisher Scientific, Waltham, MA,
USA) set at 37°C, 5% CO2 in moist air, using Dulbecco’s
Minimum Essential Medium (DMEM, Lonza, Verviers,
Belgium) supplemented with 10%, heat inactivated fetal
bovine serum (FBS, Gibco, Thermo Fisher Scientific),
2 mM L-glutamine and 100 U/ml Antibiotic-Antimycotic
(Gibco, Thermo Fisher Scientific). In each experimental
run the cell suspension was seeded (4.5 × 104 cells ml−1)
either directly on the bottom of the Petri dishes (Nunc,
LabClinics, Barcelona, Spain) or on 12-mm diameter glass
coverslips placed inside the dishes. When needed, black 96-
well microplates were used. A pre-incubation of 1 hour
before the MF exposure was carried out using the cell-
permeable fluorogenic ROS probe, 2´,7´-dichlorodihydr
ofluorescein diacetate (DCFH-DA, Sigma-Aldrich, St. Lou
is, MO, USA), the ROS scavenger, N-acetyl-L-cysteine
(NAC, Sigma-Aldrich), and the NADPH oxidase (NOX)
inhibitor, diphenyleneiodonium chloride (DPI, Santa Cruz
Biotechnology, Dallas, Texas, USA.). Stock compounds of
DCFH-DA, NAC and the NOX inhibitor DPI were dis­
solved in dimethyl sulfoxide (DMSO, Sigma-Aldrich),
milliQ water and ethanol, respectively. A volume of the
vehicle (DMSO, milliQ water-, or ethanol-only) equivalent
to that added to the experimental samples, was added to
control samples in each experimental run. The final con­
centration of vehicle in the culture was less than 0.1%.
Magnetic field exposure protocol
The cultures were exposed to a 50 Hz, sine wave, vertically
polarized MF, at a magnetic flux density BAC = 100 µT rms.
The exposure set-up has been described elsewhere (Trillo
et al. 2012). Briefly, the current flow was supplied by a wave
generator (Newtronic Model 200MSTPC, Madrid, Spain)
having a 3.53 mA DC offset (BDC = 15 µT rms). The
generator was connected to a pair of coils set in Helmholtz
configuration. One Helmholtz pair was placed inside each

of two magnetically shielded chambers (co-netic metal;
Amuneal Corp., Philadelphia, PA, USA) located within
two identical CO2 incubators (Thermo Fisher Scientific).
The electric current in the coils was monitored using
a multimeter (Hewlett Packard, model 974A, Loveland,
CO, USA) and the induced MF was routinely checked
using two magnetometers (EFA-3, Wandel and Golterma
nn, Eningen, Germany, and EMDEX II, Enertech
Consultants, Campbell, CA, USA). The background MF
inside the shielded chambers was BAC: 0.05 ± 0.02 µT (rms);
BDC: 0.04 ± 0.04 µT (rms). In each experimental run Petri
dishes (at least three per experimental group) or multiwells,
containing the cell samples, were stacked in the central
region of the Helmholtz coil gap to ensure uniformity of
MF exposure. In each experimental run only one set of coils
was energized, the samples in the unenergized set being
considered sham-exposed controls. Following a random
sequence, both coil sets and incubators were alternatively
used for MF- or sham-exposure. NB69 cultures were MF-
or sham-exposed for 5 to 30-minute intervals at day 4
postplating.
Free radical detection
The determination and quantification of FR production
were carried out using the fluorescent probe 2′,7-Dichloro
dihydrofluorescein diacetate (5 µM DCFH-DA, Sigma-
Aldrich), which is routinely used for the identification of
ROS (Piras et al. 2018). ROS production was assessed by
quantification of the intracellular oxidative transformation
of the oxidation-sensitive probe DCFH-DA into the fluor­
escent dye dichlorofluorescein (DCF). On day four post-
plating the induction assay was performed. Cells were
treated with or without 1 mM NAC and 5 µM DCFH-
DA and maintained in the dark at 37°C and 5% CO2
during 1-hour before MF- or sham-exposure. The used
DCFH-DA and NAC concentrations were selected after
comprehensive literature search (Martínez et al. 2016; Piras
et al. 2018). The samples were distributed in each of the two
sets of coils placed inside the MF-shielded chambers and
submitted to 10 minutes of MF- or sham-treatment fol­
lowed by 30 minutes of postincubation (10 min On/30 min
Off). Four different experimental conditions were assayed:
sham-exposure in the absence of NAC (controls); NAC
only; MF exposure only; NAC + MF. For DCFH-DA-
fluorescence spectroscopy analysis, cell cultures incubated
with DCFH-DA were established in black 96-well micro­
plates (3 × 103 cells/well). After treatment, the samples
were washed with PBS and read on a microplate reader
(TECAN SpectraFluor, Gödrig, Austria; λexc 490, λemi
535). The fluorescence values vs. background (fluorescence
in the absence of DCFH-DA) were processed for statistical
ELECTROMAGNETIC BIOLOGY AND MEDICINE 105
analysis. At least three independent replicates were per­
formed per experimental condition.
NADPH oxidase inhibition
On day four after seeding, one hour prior to MF- or sham-
exposure, the media were renewed and supplemented with
1 µM DPI, a concentration previously tested by other
authors (Alvarez-Maqueda et al. 2004). After 20 minutes
of MF exposure, the levels of phosphorylation of the
Mitogen-Activated Protein Kinases (MAPK-p38, -ERK1
/2 and -JNK) were assessed by Western blotting and
immunofluorescence analysis. In preliminary experiments
aiming to select optimal intervals for DPI treatment and
MF exposure, tests were performed to analyze, indepen­
dently and at different time intervals, the DPI effects on
p67phox expression and MAPK activation in the presence
of MF (supplementary files 1 and 2).
Western blotting
To assess the expression levels of subunit p67phox and the
phosphorylated proteins p-p38, p-ERK1/2 and p-JNK,
Western blotting was conducted as described elsewhere
(Martínez et al. 2012). Briefly, after MF- or sham-
exposure the cells were harvested in hypotonic lysis buffer
and proteins were separated and transferred to nitrocellu­
lose membranes (Hybond ECL, GE Healthcare, Little
Chalfont, Buckinghamshire, UK). The membranes were
blocked and incubated overnight at 4°C with mouse mono­
clonal antibody against p-p38 (1:1000 dilution, 44–684 G,
Thermo Fisher Scientific), rabbit polyclonal antibodies
against p-ERK1/2 (1:1000 dilution; 44–680 G, Thermo
Fisher Scientific), p-SAPK/JNK (1:1000 dilution; Cell
Signaling Technology, Inc, 4668, Danvers, MA, USA) or
goat polyclonal antibody against p67phox (1:500 dilution,
sc-7662, Santa Cruz Biotechnology). Monoclonal mouse
anti- β-Actin (1:5000; A5441, Sigma-Aldrich) was used as
loading control. After incubation with the indicated pri­
mary antibodies, the proteins of interest were detected
using peroxidase-conjugated secondary antibodies (GE
Healthcare) or fluorescently labeled secondary antibody
IRdye (LI-COR, Bioscience, Lincoln, NE, USA). ProXima
Imaging system (Isogen Life Science, Utrecht, Netherlands)
was used to detect ECL-chemiluminescence in the blots,
and fluorescence signal was revealed by infrared imaging
Odyssey (LI-COR). The bands obtained were evaluated by
densitometry (PDI Quantity One 4.5.2 software, BioRad,
Munich, Germany) and normalized against the corre­
sponding β-Actin band. At least four experimental repli­
cates were conducted for each protein.
106 M. A. MARTÍNEZ ET AL.

Immunofluorescence intervals (Figure 1(a, b)). In turn, the immunocyto­

chemical study revealed a significantly increased rate of
After MF- or sham-exposure of the samples cultured on
cells expressing p67phox (p67phox+) after 10 minutes of
coverslips, ERK1/2, p38 and JNK activation and p67phox
MF exposure (Figure 1(c, d)). The same increase in
expression were characterized by indirect immunofluores­
p67phox+ cells was observed at 15 minutes of exposure,
cence and computer-assisted image analysis. Cells were
although the effect was not accompanied by an increase
fixed with 4% formaldehyde, permeabilized with ethanol/
in the protein expression, which could be indicative of
acetic acid for 20 min at −4°C and blocked with PBS
a rapid drop in the amount of p67phox per cell. No
containing 10% goat serum for 1 hour at room tempera­
significant changes were found in the rate of p67phox+
ture. Coverslips were incubated overnight with goat pri­
cells in samples exposed for shorter or longer intervals.
mary antibody against p67phox (1:50; Santa Cruz
Biotechnology), with mouse monoclonal antibody against
p-p38 (1:100 dilution, Cell Signaling Technology), or with MF effects on free radical levels
rabbit antibody against p-ERK1/2 (1:100, Thermo Fisher
NB69 cells were MF- or sham-exposed and treated
Scientific) or p-JNK (1:100; Abcam, Cambridge, UK).
with DCFH-DA in the presence or absence of the
Secondary anti-goat conjugated with the fluorophore
free radical chelating agent NAC. This chelator has
Alexa Fluor 488 (Thermo Fisher Scientific), anti-mouse-
been shown adequate for assessment of the MF-
IgG conjugated to AlexaFluor® 546 or anti-rabbit IgG con­
induced response of the MAPK pathway in NB69
jugated to AlexaFluor® 488 (Molecular Probes, Eugene, OR,
cells (Martínez et al. 2016) and, apart from having
USA) were used to reveal the proteins of interest. The cell
a scavenging role, NAC can suppress superoxide gen­
nuclei were counterstained with Hoechst 33342 (Bisbenzi
eration in human neutrophils by restraining the trans­
mide, Sigma-Aldrich). Images were captured with a Nikon
location of p47phox or p67phox to the cell membrane
microscopy (Eclipse TE300, 1076 ER Amsterdam,
(Kitaoka et al. 2005). The fluorescent DCF due to
Netherlands) and analyzed by computer imaging Analy-
intracellular oxidation of DCFH-DA was analyzed by
SIS software (GMBH, Munich, Germany). In each of at
fluorescence spectroscopy and expressed in terms of
least three experimental runs, three coverslips were studied
ROS production. After 10 minutes of MF exposure
per experimental condition: sham-exposed controls, MF
followed by a 30-minute post-exposure incubation,
only and, when needed, the NOX inhibitor DPI + MF.
the intensity of fluorescent DCF and therefore, of FR
Fifteen microscope-fields per coverslip were randomly
production, were significantly increased (26.67% ±
selected for analysis. The total number of nuclei and
5.9% over controls, Figure 2). Such an increase was
the percent of p-p38, p-ERK1/2, or p67phox positive cells
not observed in samples exposed to the MF in the
per microscope-field were recorded.
presence of NAC.

Statistical analysis
All experimental procedures and analysis were con­
ducted blindly for treatment. Data were normalized
and expressed as means ± standard error (SEM) of at
least three independent experimental runs. Statistical
analyses were performed with Graph-Pad Prism 6.01
software (GraphPad Software, Inc., La Jolla, CA, USA).
The two-tailed Student’s t-test was used when compar­
ing two samples and the one-way ANOVA was applied
when comparing multiple samples. The limit of statisti­
cal significance was set at p < .05.
Results
MF effects on p67phox expression
The Western blotting analysis of the chronology of
p67phox expression showed significant protein overex­
pression at 10 minutes of MF exposure and no effects at
shorter (5 min) or longer (15, 20 or 30 min) exposure
MF effect on MAPK-p38 activation in the presence of
DPI
The potential role of NADPH activation in the MF-
induced expression of phosphorylated MAPK was inves­
tigated at 20 minutes of field exposure. This time-lapse
was selected on the basis of the results of preliminary
Western blotting tests, which revealed a simultaneous
and statistically significant activation peak for the three
MAPK proteins studied (p38, ERK1/2 and JNK) at
20 minutes of MF exposure, but not at shorter or longer
exposure intervals (supplementary file, Fig. S1). As
described in the methodological section, the DPI inhi­
bitor was added to the culture media 60 minutes before
the MF exposure onset. Therefore, although here we will
refer to the 20-minute treatments corresponding to the
interval of simultaneous exposure to DPI + MF, in fact,
the total period of treatment with the inhibitor, whether
applied independently or in combination with the MF,
was 80 minutes.
 ELECTROMAGNETIC BIOLOGY AND MEDICINE 107

MF C 5 min 10 min 15 min 20 min

Figure 1. Expression of protein p67phox and rate of p67phox+ cells after short-term MF exposure. (a) Western blotting quantification
of p67phox expression at different MF- or sham-exposure intervals (5–20 min). The data, normalized over the corresponding sham-
exposed controls, are means ± SEM of at least 4 experimental replicates per time interval, with 6 samples (3 MF- and 3 sham-exposed)
per replicate. The average raw values at t = 0 min (5521.0 ± 430.75) did not differ from those in controls sham-exposed during the
studied time interval (5876.0 ± 215.4 at 5–20 min). (b) Representative blots of p67phox at the different exposure (MF+) or sham-
exposure (MF-) intervals, using β-Actin as loading control. (c) Immunocytochemical and computer-assisted image quantification of the
rate of p67phox+ cells. The data, normalized over controls (line 100%), are means ± SEM of 4 experimental replicates. (d) Upper panel:
representative images of p67phox labeling (green) at the corresponding exposure intervals. Lower panel: Hoechst-stained nuclei (blue)
of the cells in the corresponding upper micrographs. Scale bar = 50 µm. **: 0.05 ≤p < .01 (ANOVA and Student’s t-test).

The results confirmed the preliminary observations of
significant p-p38 overexpression in cells exposed to MF
only (28.3% ± 5.9% above controls, p < .01, Figure 3(a, b)).
A similar overexpression of p-p38 (24.1% ± 10.3%, p < .05)
was observed in samples exposed to the MF in the presence
of DPI, while when DPI was applied in sham-exposure
conditions, a not statistically significant subexpression of
p38 was observed (16.11% ± 7.9% below controls). These
results are consistent with those from the immunocyto­
chemical analysis (Figure 3(c, d)), which revealed signifi­
cant increases in p-p38+ cell rate, induced by MF both in
the presence and absence of DPI (10.7% ± 3.4% and 10.9%
± 5.4% over controls, respectively; p < .05), while the
treatment with the inhibitor alone significantly decreased
the p-p38+ cell rate (11.44% ± 3.7% below controls;
p < .05). Taken together, these results indicate that
NADPH oxidase does not intervene in the MF-induced
activation of the p38 pathway.
MF effect on MAPK-ERK1/2 activation in the
presence of DPI
The Western blotting data summarized in Figure 4(a, b)
confirm preliminary data on significant p-ERK1/2 over­
expression in response to the exposure to MF only. Such
increase with respect to the expression levels in
untreated controls (MF-/DPI-) was not observed when
the MF was applied in the presence of DPI. This could be
indicative that the inhibitor, which in the absence of MF
induced significant p-ERK1/2 subexpression with respe
ct to untreated controls (−25.40% ± 4.8%; p < .001),
could partially block the field-induced overexpression.
108 M. A. MARTÍNEZ ET AL.
Figure 2. Impact of MF exposure (10 min On/30 min Off) on ROS production in the presence or absence of NAC (40 min), determined by
DCF-fluorescence microscopy. The data, normalized over sham-exposed controls (line 100%), are means ± SEM of 3 experimental
replicates, with 120 samples (30 sham-exposed, 30 MF, 30 NAC and 30 NAC+MF) per replicate. *: 0.01 ≤ p < .05; ***: p < .001 (ANOVA
and Student´s t-test).
However, the complementary immunocytochemical
study (Figure 4(c, d)) did not reveal significant changes
with respect to untreated controls in the rate of p-ERK1/
2+ cells when DPI was administered in the absence of
MF. On the other hand, MF exposure, both in the pre­
sence and in the absence of the inhibitor, significantly
increased the rate of cells expressing p-ERK1/2 com­
pared to untreated controls and to samples treated
with DPI only. Therefore, taken together, these data do
not support a potential implication of NADPH oxidase
in the MF-induced activation of ERK1/2.
MF effect on MAPK-JNK activation in the presence of
DPI
The Western blotting results illustrated in Figure 5(a, b)
confirmed previously reported data (Martínez et al.
2016) that, compared to untreated controls, MF expo­
sure induces significant overexpression of p-JNK.
However, the field effect was prevented by DPI, so that
under these conditions the p-JNK expression levels did
not differ from those of untreated controls nor from
those of samples treated with the inhibitor only. These
data were supported by the corresponding immunocy­
tochemical study (Figure 5(c, d)) and are indicative that
the NADPH oxidase complex is involved in the MF-
induced activation of the MAPK-JNK pathway.
Discussion
Previous studies by our group (Martínez et al. 2012,
2016, 2019) have shown that exposure to a 50 Hz, 100
µT MF activates the MAPK ERK1/2, p38 and JNK sig­
naling pathways in human neuroblastoma cells NB69.
Indeed, the activation of p38 and ERK1/2, as well as that
of the EGF receptor, are known to mediate the MF-
induced proliferation promotion in NB69, and the fact
that such proliferative response is blocked by NAC sug­
gests that free radicals can be involved in the field’s
effect. Evidence exists that NADPH oxidase, an enzy­
matic system involved in ROS generation, could be
a common mediator for various biological processes
including antioxidation/oxidative stress, various mito­
chondrial functions, calcium homeostasis, gene expres­
sion, immunological functions or cell aging and death
(Nauseef 2008). Protein p67phox, one of the cytosolic
subunits of NADPH oxidase, regulates the activity of
this enzyme complex through its interaction with the
membrane component of the complex (Italiano et al.
2012). The present study investigates the field effects
on free radical production and p67phox protein expres­
sion, as well as the potential involvement of NADPH
oxidase in the MF-induced activation of MAPK-p38, -
ERK1/2 and -JNK signal transduction pathways.
The obtained results show that the MF induces early
overexpression of the p67phox protein, which peaks at
 ELECTROMAGNETIC BIOLOGY AND MEDICINE 109

Figure 3. Expression of p-p38 and rate of p-p38+ cells after 20 min of MF exposure in the presence or absence of DPI. (a) Western blot
quantification of p-p38. The data, normalized over the corresponding controls (line 100%), are means ± SEM of 4 experimental
replicates with 12 samples (3 sham-exposed, 3 DPI, 3 MF and 3 DPI + MF) per replicate. (b) Representative blots of p-p38 at the
different exposure conditions (MF+) or sham-exposure (MF-), using β-Actin as loading control. (c). Immunofluorescence quantification
of p-p38+ cells by computer-assisted image analysis. The data, normalized over the corresponding controls (line 100%), are means ±
SEM of 3 experimental replicates. (d) Representative images of p-p38 labeling (upper panel) and Hoechst-stained nuclei of the cells in
the corresponding upper micrographs (lower panel). Scale bar = 50 µm. *: 0.01 ≤ p < .05; **: 0.05 ≤ p < .01 (ANOVA and Student’s
t-test).

10 minutes of exposure. The mechanism through which
the MF causes such a transient effect on this component
of the NADPH oxidase is unknown. Although some
studies, generally using chemical agents, have reported
translocation of the NADPH oxidase subunits at short
times similar to those reported here (Touyz et al. 2002;
Wang and Lou 2009), in most studies de novo protein
synthesis has been observed at longer times (Seo et al.
2012; Touyz et al. 2002). Therefore, it is conceivable the
effect described in the present study corresponds to the
initial phases of the MF response, involving
a translocation of preexisting p67phox from the cyto­
plasm to the plasma membrane, rather than a de-novo
expression of the protein. Although additional studies
would be necessary to test this hypothesis and determine
the location where the effect takes place, the possibility
of a membrane location would obtain partial support
from the immunocytochemical data illustrated in Figure
1(d), which showed a slight increase in p67phox labeling
at such level, both at 10 and 15 minutes of MF exposure.
In fact, as proposed in previous works (Martínez et al.
2019), the AC frequency (f = 50 Hz) and DC flux density
(BDC = 15 µT rms) parameters applied in this study
would meet the resonance conditions for the sulfur
ion, according to the ion parametric resonance model
IPR (Blackman et al. 1995; Blanchard and Blackman
1994). Therefore, it can be hypothesized that under
these conditions, a potential, MF-induced perturbation
on this ion could change the reactivity of the thiol or
thiolate groups present in the cysteine residues of the
p67phox subunit (Bizouarn et al. 2016; Fradin et al.
2018). Such an effect could promote the binding of
p67phox to NOX2 and/or a translocation of p67phox
to the plasma membrane, and subsequently activate the
NADPH oxidase.
On the other hand, increased expression of p67phox
does not necessarily imply activation of the NADPH
oxidase complex, which is made up of a set of subunits
110 M. A. MARTÍNEZ ET AL.

Figure 4. Expression of p-ERK1/2 and rate of p-ERK1/2+ cells after 20 min of MF exposure in the presence or absence of DPI. (a) Western
blot quantification of pERK1/2 expression. The data, normalized over the corresponding controls (line 100%) are means ± SEM of 4
experimental replicates with 12 samples (3 sham-exposed, 3 DPI, 3 MF and 3 DPI + MF) per replicate. (b) Representative blots of
p-ERK1/2, at the different exposure conditions (MF+) or sham-exposure (MF-), using β-Actin as loading control. (c)
Immunofluorescence quantification of p-ERK1/2+ cells by computer-assisted image analysis. Bars are means ± SEM of 4 experimental
replicates. Normalized values respect to sham-exposed controls (line 100%). (d) Representative images of p-ERK1/2 labeling (upper
panel) and Hoechst-stained nuclei of the cells in the corresponding upper micrographs (lower panel). Scale bar = 50 µm. *:
0.01 ≤ p < .05; ***: p < .001 (ANOVA and Student’s t-test).

whose specific functions and their interactions within
the complex have not been sufficiently elucidated yet. In
what concerns subunit p67phox, it is the key component
in the causation of a conformational remodeling of
NOX2 (Gorzalczany et al. 2000), and an activation
domain in p67phox has been found essential for the
oxidase activation, but not for the actual p67phox-
NOX2 interaction (Sumimoto 2008). Also, experimental
evidence exists supporting the hypothesis that the NOX2
dehydrogenase region (DHR) acts as a protein disulfide
isomerase (PDI) that leads to formation of disulfide
bonds with p67phox and ensures the stabilization of
the NADPH oxidase complex (Bechor et al. 2015;
Fradin et al. 2018).
The present results also reveal that ROS production is
significantly increased by a 10-minute exposure to the MF
followed by a 30-minute post-exposure interval, this effect
being prevented by NAC. This supports observations by
other authors reporting ROS level increases in various cell
types exposed to RF/MW (Durdik et al. 2019; Friedman
et al. 2007; Yakymenko et al. 2016) or DC – ELF MF
(Mattsson and Simkó 2014; Merla et al. 2019;
Poniedziałek et al. 2013; Rollwitz et al. 2004). In some of
these ELF studies, increases in ROS and cytoplasmic super­
oxides occurring after 5–45 minutes of exposure to flux
densities B ≥ 0.4 mT were inhibited by NADPH oxidase
inhibitors (Feng et al. 2016; Lupke et al. 2004; Merla et al.
2019). Although the present work does not address directly
the effects of DPI on ROS production, other studies have
shown that DPI concentrations similar to those used in the
present work do not cause a reduction in free radical levels.
Such studies show that DPI reverses the generation of
reactive oxygen species induced by ELF MF in SH-SY5Y
neuroblastoma cells (Merla et al. 2019) and does not inhibit
the increased production of free radicals or superoxide
radical anions induced by MF in non-stimulated Mono-
Mac-6 cells (Lupke el al. 2004). Overall, these MF-induced
increases in ROS levels were moderate and similar to those
 ELECTROMAGNETIC BIOLOGY AND MEDICINE 111

Figure 5. Expression of p-JNK and rate of p-JNK+ cells after 20-minute MF exposure in the presence or absence of DPI. (a) Western
blotting quantification of p-JNK expression. Each bar, normalized over the corresponding controls (line 100%), are means ± SEM of 4
experimental replicates with 12 samples (3 sham-exposed, 3 DPI, 3 MF and 3 DPI + MF) per replicate. (b) Representative blots of p-JNK
at the different exposure conditions (MF+) or sham-exposure (MF-), using β-Actin as loading control. (c) Immunofluorescence
quantification of p-JNK+ cells by computer-assisted image analysis. The data, normalized over the corresponding controls (line
100%), are means ± SEM of 3 experimental replicates. (d) Representative images of p-JNK labeling (upper panel) and Hoechst-stained
nuclei of the cells in the corresponding upper micrographs (lower panel). Scale bar = 50 µm. *: 0.01 ≤ p < .05; **: 0.05 ≤ p < .01; ***:
p < .001 (ANOVA and Student’s t-test).

obtained in the present study (about 26%). While high ROS
levels can cause severe cell dysfunction through DNA,
RNA and protein damage, moderate levels can act
as second messengers, activating signaling cascades and
triggering cellular responses affecting proliferation, apop­
tosis or gene expression (Schieber and Chandel 2014). In
any case, the mechanism or mechanisms through which
MF-induced ROS could affect specific biological systems
are not yet sufficiently characterized.
The present study applied inhibitor DPI to investigate
the potential involvement of NADPH oxidase in the
field-induced activation of MAPK-ERK1/2, -p38 and -
JNK. In the absence of MF, DPI caused significant sub­
expression of the cytosolic component of NADPH oxi­
dase, p67phox (supplementary file 2, Fig. S2), as well as
subexpression of p-ERK1/2 (Figure 4(a)) and decreased
rate of cells showing p38 activation (Figure 3(c)). These
data are consistent with those reporting that, through
their involvement in the regulation of p38 and ERK
activation, the ROS generated by NADPH oxidase
activation mediates the stimulation or blockade of cell
cycle progression in various cell types (Tormos et al.
2013; Venkatachalam et al. 2008). Concerning p67phox
expression, although no evidence of direct effects of DPI
has been reported so far, the possibility cannot be dis­
regarded that DPI may induce indirect effects through
an action on the p67phox-regulated flavin adenine dinu­
cleotide (FAD) redox center of NADPH oxidase (Han
and Lee 2000). From this, it can be stated that the field-
induced activation of ERK1/2 and p38 at 20 minutes of
exposure was not affected by the presence of DPI, which
indicates that such activation would not be mediated by
NADPH oxidase.
In the presence of the DPI inhibitor only, the phos­
phorylation levels of MAPK-p38 did not decrease sig­
nificantly with respect to those in controls. However,
after a 20-minute interval, during which the MF signifi­
cantly increased phosphorylation of this pathway (sup­
plementary Figure 1), the expression of the p67phox
subunit of NADPH oxidase was significantly reduced
112 M. A. MARTÍNEZ ET AL.
by DPI (supplementary Figure 2). These results are
indicative that a critical treatment time is necessary for
the response of each of the different MAPKs to DPI to be
detected. Indeed, while the field-induced phosphoryla­
tion in pathways p38 and JNK declines rapidly, that
induced in pathway ERK1/2 lasts longer (
Supplementary Figure 1), so a significant response of
this pathway to DPI was observed at 20 minutes of
treatment (Figure 2). It could be argued that since DPI
has been reported to have potential pro-oxidant effects
(Kučera et al. 2016), the possibility that such effects
could have increased the cell sensitivity to the MF in
our experiments cannot be ruled out. However, the
obtained results showing non-significant differences
with respect to controls indicate that the effect, if any,
would be rather weak.
In contrast to the above-described results, NADPH
oxidase has been reported to be involved in ERK1/2 activa­
tion in immortalized monkey fibroblasts (COS7) and in
human epithelial cells (HeLa) exposed in vitro to a 50 Hz, 1
mT MF (Kapri-Pardes et al. 2017). These cell types, on the
other hand, responded to the MF with early ERK or p38
activation peaks (at 5–17 min of exposure), which is con­
sistent with the herein reported early ERK and p38 activa­
tions in response to the 0.1 mT MF. Similar effects on ERK
have also been observed in response to short-term expo­
sure to RF electromagnetic fields. For instance, subthermal
exposure to an 875 MHz signal has been reported to induce
rapid ERK phosphorylation in isolated plasma membranes
from Rat1 and HeLa cells, this effect being mediated by
field-induced activation of NADH oxidase, which gener­
ates ROS at the plasma membrane (Friedman et al. 2007).
Regarding the activation of the MAPK-p38 pathway by
the MF, previous studies by our group had shown that this
pathway is inhibited by NAC (Martínez et al. 2016), which
together with the present results, suggests that the p38
activation by the MF would be mediated by a source of
FR, perhaps of mitochondrial origin, other than NADPH.
In fact, several studies have provided evidence of potential
involvement of mitochondrial electron transfer processes
in the RF/ELF-induced ROS generation (Consales et al.
2012; Destefanis et al. 2015; Feng et al. 2016; Kesari et al.
2016; Luukkonen et al. 2014; Wang and Zhang 2017). For
instance, Feng et al. (2016) reported that exposure to
a 50 Hz, 0.4 mT MF promotes ROS production in amniotic
cells through two pathways: the NADPH oxidase complex
and the mitochondria, the production of cytoplasmic per­
oxides (from 5 min of exposure) being earlier than that of
mitochondrial ROS (at 15 or 30 min). Besides, ROS inhibi­
tion by DPI prevented the MF-induced formation of EGFR
clusters. Recent studies by our group have revealed that
EGFR is involved in the MF-induced proliferative response
and activation of the MAPK pathways ERK, p38 and JNK
in the NB69 cell line (Martínez et al. 2019). However, the
possibility that a direct relationship exists between ROS
induction and EGFR activation in NB69 has not yet been
investigated. On the other hand, although the NADPH
oxidase complex (different NOX isoforms) has been
described to be involved in the regulation of EGFR activa­
tion in various cell types (Heppner and Van der Vliet
2016), such activation may also be mediated by mechan­
isms independent of ROS, including metalloproteinases or
G proteins capable of releasing ligands that are specific to
EGFR, tyrosine kinases or intracellular Ca2+ (Forrester
et al. 2016; George et al. 2013).
As for the activation of JNK by the MF, our results are
compatible with those showing that short-term exposure
(15–30 min) to 0.4 or 0.8 mT fields promotes activation of
the stress-activated protein kinase pathway (SAPK/JNK) in
the hamster lung fibroblast cell line CHL (Sun et al. 2001).
Nevertheless, the potential cellular responses deriving from
these changes occurring at the molecular level have not yet
been elucidated. Our previous studies on pharmacological
inhibition of the SAPK/JNK pathway have ruled out an
association between its activation and NB69 cell prolifera­
tion, both induced by the MF (Martínez et al. 2016).
However, the possibility cannot be ruled out that SAPK/
JNK activation may contribute to the proliferative response
through a potential increase in cell survival. In fact, JNK
pathway activation has been shown to be involved in
increased survival of human neuroblastoma cells and in
resistance to the treatment with chemical oncostatics, and
has been associated with poor prognosis in oncology
(Sheikh et al. 2016; Wu et al. 2019).
As regards the MAPK-JNK pathway activation, the
ROS generated by the activity of NADPH oxidase is
known to be involved in the activation of JNK in different
cell types, including liver or nervous system cells (Li et al.
2017; Yan et al. 2013). The present results showing that,
although the NADPH oxidase inhibitor alone does not
affect the expression of p-JNK at 20 min of treatment, it is
capable of preventing the MF-induced JNK activation,
due perhaps to underexpression of the p67phox subunit
at this time interval (Figure 2). These results suggest that
the field effect on the JNK pathway activation would be
mediated by NADPH oxidase.
Conclusion
In conclusion, the present results reveal that in vitro expo­
sure to a 50 Hz, 100 µT MF increases the total ROS levels in
NB69 cells and induces early, transient expression of the
cytosolic component of the NADPH oxidase, p67phox.
Also, the MF-induced activation of the MAPK-JNK path­
way, but not that of -ERK1/2 or -p38 pathways, was pre­
vented in the presence of the used NADPH inhibitor,
 ELECTROMAGNETIC BIOLOGY AND MEDICINE 113

Figure 6. Schematic representation, based on the present (boxed texts) and previously published results, of the molecular mechanism
that triggers the MF-induced proliferative response of the NB69 cell line, and of the involvement of free radicals (FR) in said mechanism.
Our previous studies reported that activation of the EGF receptor (EGFR) would trigger cell proliferation through phosphorylation of
pathways ERK1/2 (FR-independent) and p38 (FR-dependent). EGFR also activated the JNK pathway, which did not intervene in the
proliferative response to the MF (see the Discussion for detailed explanation). The present results show that the MF increases both the
expression levels of the p67phox protein, a component of the NADPH oxidase complex, and the total production of intracellular FR.
However, the analysis of the involvement of NOX in the activation of MAPK pathways revealed that only the JNK pathway was
prevented by DPI in the presence of MF. These data show that the NOX complex is not involved in the MF-induced proliferative effect
mediated by EGFR-p38-pERK.

which has been shown to significantly reduce p67phox
expression. Taken together with previously published
data (Martínez et al. 2016, 2019), the present results suggest
that the proliferative response of NB69 to the MF exposure
is mediated by free radicals originated from sources other
than NOX (Figure 6). On the other hand, since the JNK
pathway is known to be strongly involved in the regulation
of cell survival and apoptosis (Wu et al. 2019), NADPH is
likely to mediate the field effects on such processes, thus
contributing to the proliferative response of NB69 to
the MF.
Competing interests
The authors declare that they have no competing interests.
References
Altenhöfer, S., K. A. Radermacher, P. W. M. Kleikers,
K. Wingler, and H. H. H. W. Schmidt. 2015. Evolution of
NADPH oxidase inhibitors: Selectivity and mechanisms for
target engagement. Antioxid. Redox Signal. 23:406–27.
doi:10.1089/ars.2013.5814.
Alvarez-Maqueda, M., R. El Bekay, J. Monteseirín, G. Alba,
P. Chacón, A. Vega, C. Santa María, J. R. Tejedo,
J. Martín-Nieto, F. J. Bedoya, et al. 2004. Homocysteine
enhances superoxide anion release and NADPH oxidase
assembly by human neutrophils. Effects on MAPK activa­
tion and neutrophil migration. Atherosclerosis 172:229–38.
doi:10.1016/j.atherosclerosis.2003.11.005.
Ayşe, I. G., A. Zafer, O. Sule, I.-T. Işil, and T. Kalkan. 2010.
Differentiation of K562 cells under ELF-EMF applied at
different time courses. Electromagn. Biol. Med. 29:122–30.
doi:10.3109/15368378.2010.502451.
Babior, B. M. 1999. NADPH oxidase: An update. Blood
93:1464–76. https://www.ncbi.nlm.nih.gov/pubmed/
10029572.
Basile, A., R. Zeppa, N. Pasquino, C. Arra, M. Ammirante,
M. Festa, A. Barbieri, A. Giudice, M. Pascale, M. C. Turco,
et al. 2011. Exposure to 50 Hz electromagnetic field raises
the levels of the anti-apoptotic protein BAG3 in melanoma
cells. J Cell. Physiol. 226:2901–07. doi:10.1002/jcp.22641.
Bechor, E., I. Dahan, T. Fradin, Y. Berdichevsky, A. Zahavi,
A. F. Gross, M. Rafalowski, and E. Pick. 2015. The dehydro­
genase region of the NADPH oxidase component Nox2 acts
as a protein disulfide isomerase (PDI) resembling PDIA3
with a role in the binding of the activator protein p67
(phox.). Front. Chem. 3:3. doi:10.3389/fchem.2015.00003.
Bizouarn, T., G. Karimi, R. Masoud, H. Souabni, P. Machillot,
X. Serfaty, F. Wien, M. Réfrégiers, C. Houée-Levin, and
L. Baciou. 2016. Exploring the arachidonic acid-induced
structural changes in phagocyte NADPH oxidase p47
(phox) and p67(phox) via thiol accessibility and SRCD
spectroscopy. Febs J 283:2896–910. doi:10.1111/febs.13779.
Blackman, C. F., J. P. Blanchard, S. G. Benane, and
D. E. House. 1995. The ion parametric resonance model
predicts magnetic field parameters that affect nerve cells.
Faseb J 9:547–51. doi:10.1096/fasebj.9.7.7737464.
114 M. A. MARTÍNEZ ET AL.
Blanchard, J. P., and C. F. Blackman. 1994. Clarification and
application of an ion parametric resonance model for magnetic
field interactions with biological systems. Bioelectromagnetics
15:217–382. doi:10.1002/bem.2250150306.
Carlberg, M., T. Koppel, M. Ahonen, and L. Hardell. 2017.
Case-control study on occupational exposure to extremely
low frequency electromagnetic fields and glioma risk. Am.
J. Ind. Med. 60:494–503. doi:10.1002/ajim.22707.
Consales, C., C. Merla, C. Marino, and B. Benassi. 2012.
Electromagnetic fields, oxidative stress, and neurodegenerati
on. Int. J. Cell. Biol. (2012:683897. doi:10.1155/2012/683897.
De Roos, A. J., K. Teschke, D. A. Savitz, C. Poole, S. Grufferman,
B. H. Pollock, and A. F. Olshan. 2001. Parental occupational
exposures to electromagnetic fields and radiation and the inci­
dence of neuroblastoma in offspring. Epidemiol 12:508–17.
doi:10.1097/00001648-200109000-00008.
Destefanis, M., M. Viano, C. Leo, G. Gervino, A. Ponzetto, and
F. Silvagno. 2015. Extremely low frequency electromagnetic
fields affect proliferation and mitochondrial activity of
human cancer cell lines. Int. J. Radiat. Biol. 91:964–72.
doi:10.3109/09553002.2015.1101648.
Doroshow, J. H., S. Gaur, S. Markel, J. Lu, J. van Balgooy,
T. W. Synold, B. Xi, X. Wu, and A. Juhasz. 2013. Effects of
iodonium-class flavin dehydrogenase inhibitors on growth,
reactive oxygen production, cell cycle progression, NADPH
oxidase 1 levels, and gene expression in human colon can­
cer cells and xenografts. Free Radic. Biol. Med. 57:162–75.
doi:10.1016/j.freeradbiomed.2013.01.002.
Durdik, M., P. Kosik, E. Markova, A. Somsedikova,
B. Gajdosechova, E. Nikitina, E. Horvathova, K. Kozics,
D. Davis, and I. Belyaev. 2019. Microwaves from mobile
phone induce reactive oxygen species but not DNA damage,
preleukemic fusion genes and apoptosis in hematopoietic
stem/progenitor cells. Sci. Rep. 9:16182. doi:10.1038/
s41598-019-52389-x.
Falone, S., S. Jr, S. V. Cordone, P. Cesare, A. Bonfigli,
M. Grannonico, G. Di Emidio, C. Tatone, M. Cacchio,
and F. Amicarelli. 2017. Power frequency magnetic field
promotes a more malignant phenotype in neuroblastoma
cells via redox-related mechanisms. Sci. Rep. 7:11470.
doi:10.1038/s41598-017-11869-8.
Falone, S., S. Jr, S. V. Cordone, G. Di Emidio, C. Tatone,
M. Cacchio, and F. Amicarelli. 2018. Extremely
low-frequency magnetic fields and redox-responsive path­
ways linked to cancer drug resistance: Insights from
co-exposure-based in vitro studies. Front. Public Health.
6:33. doi:10.3389/fpubh.2018.00033.
Feng, B., A. Dai, L. Chen, L. Qiu, Y. Fu, and W. Sun. 2016.
NADPH oxidase-produced superoxide mediated a 50-Hz
magnetic field-induced epidermal growth factor receptor
clustering. Int. J. Radiat. Biol. 92:596–602. doi:10.1080/0955
3002.2016.1206227.
Forrester, S. J., T. Kawai, S. O’Brien, W. Thomas, R. C. Harris,
and S. Eguchi. 2016. Epidermal growth factor receptor
transactivation: Mechanisms, pathophysiology, and poten­
tial therapies in the cardiovascular system. Annu. Rev.
Pharmacol. Toxicol. 56:627–53. doi:10.1146/annurev-
pharmtox-070115-095427.
Fradin, T., E. Bechor, Y. Berdichevsky, W. Thomas,
R. C. Harris, and S. Eguchi. 2018. Binding of p67 phox to
Nox2 is stabilized by disulfide bonds between cysteines in
the 369 Cys-Gly-Cys 371 triad in Nox2 and in p67 phox.
J. Leukoc. Biol. 104:1023–39. doi:10.1002/jlb.4a0418-173r.
Friedman, J., S. Kraus, Y. Hauptman, Y. Schiff, and R. Seger.
2007. Mechanism of short-term ERK activation by electro­
magnetic fields at mobile phone frequencies. Biochem. J.
405:559–68. doi:10.1042/bj20061653.
George, A. J., R. D. Hannan, and W. G. Thomas. 2013.
Unravelling the molecular complexity of GPCR-mediated
EGFR transactivation using functional genomics
approaches. Febs J 280:5258–68. doi:10.1111/febs.12509.
Gorzalczany, Y., N. Sigal, M. Itan, O. Lotan, and E. Pick. 2000.
Targeting of Rac1 to the phagocyte membrane is sufficient
for the induction of NADPH oxidase assembly. J. Biol.
Chem. 275:40073–81. doi:10.1074/jbc.m006013200.
Han, C. H., and M. H. Lee. 2000. Activation domain in
p67phox regulates the steady state reduction of FAD in
gp91phox. J. Vet. Sci. 1:27–31.
Heppner, D. E., and A. Van der Vliet. 2016. Redox-dependent
regulation of epidermal growth factor receptor signaling.
Redox Biol 8:24–27. doi:10.1016/j.redox.2015.12.002.
Hong, M. N., N. K. Han, H. C. Lee, Y.-K. Ko, S.-G. Chi, Y.-
S. Lee, Y.-M. Gimm, S.-H. Myung, and J.-S. Lee. 2012.
Extremely low frequency magnetic fields do not elicit oxi­
dative stress in MCF10A cells. J. Radiat. Res. 53:79–86.
doi:10.1269/jrr.11049.
[IARC] International Agency for Research of Cancer. IARC
monograph on the evaluation of carcinogenic risks to
humans, Vol. 80. Non-ionizing radiation, Part 1: Static
and extremely low-frequency (ELF) electric and magnetic
fields. Lyon, France: IARC Press. (2002). Accessed on 15
July 2020: http://publications.iarc.fr/Book-And-Report-
Series/Iarc-Monographs-On-The-Identification-Of-
Carcinogenic-Hazards-To-Humans/Non-ionizing-
Radiation-Part-1-Static-And-Extremely-Low-frequency-
ELF-Electric-And-Magnetic-Fields-2002
Italiano, D., A. M. Lena, G. Melino, and E. Candi. 2012.
Identification of NCF2/p67phox as a novel p53 target
gene. Cell Cycle 11:4589–96. doi:10.4161/cc.22853.
Kapri-Pardes, E., T. Hanoch, G. Maik-Rachline, M. Murbach,
P. L. Bounds, N. Kuster, and R. Seger. 2017. Activation of
signaling cascades by weak extremely low frequency elec­
tromagnetic fields. Cell. Physiol. Biochem. 43:1533–46.
doi:10.1159/000481977.
Kesari, K. K., J. Juutilainen, J. Luukkonen, and J. Naarala.
2016. Induction of micronuclei and superoxide production
in neuroblastoma and glioma cell lines exposed to weak 50
Hz magnetic fields. J. R. Soc. Interface 13:20150995.
doi:10.1098/rsif.2015.0995.
Kitaoka, N., G. Liu, N. Masuoka, K. Yamashita, M. Manabe,
and H. Kodama. 2005. Effect of sulfur amino acids on
stimulus-induced superoxide generation and translocation
of p47phox and p67phox to cell membrane in human neu­
trophils and the scavenging of free radical. Clin. Chim. Acta.
353:109–16. doi:10.1016/j.cccn.2004.10.011.
Kučera, J., L. Binó, K. Štefková, J. Jaroš, O. Vašíček, J. Večeřa,
L. Kubala, and J. Pacherník. 2016. Apocynin and dipheny­
leneiodonium induce oxidative stress and modulate PI3K/
Akt and MAPK/Erk activity in mouse embryonic stem cells.
Oxid. Med. Cell. Longev. (2016:7409196. doi:10.1155/2016/
7409196.
Li, Y., and M. A. Trush. 1998. Diphenyleneiodonium, an
NAD(P)H oxidase inhibitor, also potently inhibits

mitochondrial reactive oxygen species production.
Biochem. Biophys. Res. Commun. 253:295–99. doi:10.1006/
bbrc.1998.9729.
Li, Y. Y., Z. M. Shi, X. T. Yu, P. Feng, and X. J. Wang. 2017.
The effects of urotensin II on migration and invasion are
mediated by NADPH oxidase-derived reactive oxygen spe­
cies through the c-Jun N-terminal kinase pathway in
human hepatoma cells. Peptides 88:106–14. doi:10.1016/j.
peptides.2016.12.005.
Lupke, M., J. Rollwitz, and M. Simkó. 2004. Cell activating
capacity of 50 Hz magnetic fields to release reactive oxygen
intermediates in human umbilical cord blood-derived
monocytes and in Mono Mac 6 cells. Free Radic. Res.
38:985–93. doi:10.1080/10715760400000968.
Luukkonen, J., A. Liimatainen, J. Juutilainen, and J. Naarala.
2014. Induction of genomic instability, oxidative processes,
and mitochondrial activity by 50Hz magnetic fields in
human SH-SY5Y neuroblastoma cells. Mutat. Res.
760:33–41. doi:10.1016/j.mrfmmm.2013.12.002.
Martínez, M. A., A. Úbeda, M. A. Cid, and M. A. Trillo. 2012.
The proliferative response of NB69 human neuroblastoma
cells to a 50 Hz magnetic field is mediated by ERK1/2
signaling. Cell. Physiol. Biochem. 29:675–86. doi:10.1159/
000178457.
Martínez, M. A., A. Úbeda, J. Moreno, and M. A. Trillo. 2016.
Power frequency magnetic fields affect the p38 MAPK
mediated regulation of NB69 cell proliferation implication of
free radicals. Int. J. Mol. Sci. 17:510. doi:10.3390/ijms17040510.
Martínez, M. A., A. Úbeda, and M. A. Trillo. 2019. Involvement of
the EGF receptor in MAPK signaling activation by a 50 Hz
magnetic field in human neuroblastoma cells. Cell. Physiol.
Biochem. 52:893–907. doi:10.33594/000000062.
Mattsson, M. O., and M. Simkó. 2014. Grouping of experi­
mental conditions as an approach to evaluate effects of
extremely low-frequency magnetic fields on oxidative
response in vitro studies. Front. Public. Health 2:132.
doi:10.3389/fpubh.2014.00132.
Merla, C., M. Liberti, C. Consales, A. Denzi, F. Apollonio,
C. Marino, and B. Benassi. 2019. Evidences of plasma
membrane-mediated ROS generation upon ELF exposure
in neuroblastoma cells supported by a computational multi­
scale approach. Biochim. Biophys. Acta Biomembr.
1861:1446–57. doi:10.1016/j.bbamem.2019.06.005.
Nauseef, W. M. 2008. Biological roles for the NOX family
NADPH oxidases. J. Biol. Chem 283:16961–65. https://
www.ncbi.nlm.nih.gov/pubmed/18420576.
Paletta-Silva, R., N. Rocco-Machado, and J. R. Meyer-
Fernandes. 2013. NADPH oxidase biology and the regula­
tion of tyrosine kinase receptor signaling and cancer drug
cytotoxicity. Int. J. Mol. Sci. 14:3683–704. doi:10.3390/
ijms14023683.
Piras, S., A. L. Furfaro, R. Caggiano, L. Brondolo, S. Garibaldi,
C. Ivaldo, U. M. Marinari, M. A. Pronzato, R. Faraonio, and
M. Nitti. 2018. MicroRNa-494 favors ho-1 expression in
neuroblastoma cells exposed to oxidative stress in a
Bach1-independent way. Front. Oncol. 8:199. doi:10.3389/
fonc.2018.00199.
Poniedziałek, B., P. Rzymski, J. Karczewski, F. Jaroszyk, and
K. Wiktorowicz. 2013. Reactive oxygen species (ROS) pro­
duction in human peripheral blood neutrophils exposed
in vitro to static magnetic field. Electromagn. Biol. Med.
32:560–68. doi:10.3109/15368378.2013.773910.
ELECTROMAGNETIC BIOLOGY AND MEDICINE 115
Rezatabar, S., A. Karimian, V. Rameshknia, H. Parsian,
M. Majidinia, T. A. Kopi, A. Bishayee, A. Sadeghinia,
M. Yousefi, M. Monirialamdari, et al. 2019. RAS/MAPK
signaling functions in oxidative stress, DNA damage
response and cancer progression. J. Cell. Physiol.
234:14951–65. doi:10.1002/jcp.28334.
Rollwitz, J., M. Lupke, and M. Simkó. 2004. Fifty-hertz magnetic
fields induce free radical formation in mouse bone
marrow-derived promonocytes and macrophages. Biochim.
Biophys. Acta. 1674:231–38. doi:10.1016/j.bbagen.2004.06.024.
Ruiz-Gómez, M. J., and M. Martínez-Morillo. 2009.
Electromagnetic fields and the induction of DNA strand
breaks. Electromagn. Biol. Med 28:201–14. doi:10.1080/
15368370802608696.
Santini, M. T., G. Rainaldi, and P. L. Indovina. 2009. Cellular
effects of extremely low frequency (ELF) electromagnetic
fields. Int. J. Radiat. Biol. 85:294–313. doi:10.1080/0955300
0902781097.
[SCENIHR]Scientific Committee on Emerging and Newly
Identified Health Risks: Health effects of exposure to
EMF. (2009). Accessed on 15 July 2020: https://ec.europa.
eu/health/ph_risk/committees/04_scenihr/docs/scenihr_o_
022.pdf
Schieber, M., and N. S. Chandel. 2014. ROS function in redox
signaling and oxidative stress. Curr. Biol. 24:R453–462.
doi:10.1016/j.cub.2014.03.034.
Seo, J. S., J. Y. Park, J. Choi, T. K. Kim, J. H. Shin, J. K. Lee, and
P. L. Han. 2012. NADPH oxidase mediates depressive beha­
vior induced by chronic stress in mice. NADPH oxidase
mediates depressive behavior induced by chronic stress in
mice. J. Neurosci 32:9690–99.
Sheikh, A., A. Takatori, M. S. Hossain, M. K. Hasan, M. Tagawa,
H. Nagase, and A. Nakagawara. 2016. Unfavorable neuroblas­
toma prognostic factor NLRR2 inhibits cell differentiation by
transcriptional induction through JNK pathway. Cancer Sci
107:1223–32. doi:10.1111/cas.13003.
Song, K., S. H. Im, Y. J. Yoon, H. M. Kim, H. J. Lee, and
G. S. Park. 2018. A 60 Hz uniform electromagnetic field
promotes human cell proliferation by decreasing intracel­
lular reactive oxygen species levels. PLoS One 13.
doi:10.1371/journal.pone.0199753.
Sulpizio, M., S. Falone, F. Amicarelli, M. Marchisio, F. Di
Giuseppe, E. Eleuterio, C. Di Ilio, and S. Angelucci. 2011.
Molecular basis underlying the biological effects elicited by
extremely low-frequency magnetic field (ELF-MF) on neuro­
blastoma cells. J. Cell. Biochem. 112:3797–806. doi:10.1002/
jcb.23310.
Sumimoto, H. 2008. Structure, regulation and evolution of
nox-family NADPH oxidases that produce reactive oxygen
species. Febs J 275:3249–77. doi:10.1111/j.1742-4658.2008.
06488.x.
Sun, L., L. Chen, L. Bai, Y. Xia, X. Yang, W. Jiang, and W. Sun.
2018. Reactive oxygen species mediates 50-Hz magnetic
field-induced EGF receptor clustering via acid sphingomye­
linase activation. Int. J. Radiat. Biol. 94:678–84. doi:10.1080/
09553002.2018.1466208.
Sun, W. J., H. Chiang, Y. T. Fu, Y. N. Yu, H. Y. Xie, and
D. Q. Lu. 2001. Exposure to 50 Hz electromagnetic fields
induces the phosphorylation and activity of stress-activated
protein kinase in cultured cells. Electro- and Magnetobiol
20:415–23. doi:10.1081/JBC-100108579.
116 M. A. MARTÍNEZ ET AL.
Tormos, A. M., R. Taléns-Visconti, A. R. Nebreda, and
J. Sastre. 2013. p38 MAPK: A dual role in hepatocyte pro­
liferation through reactive oxygen species. Free Radic. Res.
47:905–16. doi:10.3109/10715762.2013.821200.
Touyz, R. M., X. Chen, F. Tabet, G. Yao, G. He, M. T. Quinn,
P. J. Pagano, and E. L. Schiffrin. 2002. Expression of
a functionally active gp91phox-containing neutrophil-type
NAD(P)H oxidase in smooth muscle cells from human
resistance arteries: Regulation by angiotensin II. Circ. Res.
90:1205–13. doi:10.1161/01.res.0000020404.01971.2f.
Trillo, M. A., M. A. Martínez, M. A. Cid, J. Leal, and A. Úbeda.
2012. Influence of a 50 Hz magnetic field and of all-trans-
retinol on the proliferation of human cancer cell lines. Int.
J. Oncol. 40:1405–13. doi:10.3892/ijo.2012.1347.
Tseng, H. Y., Z. M. Liu, and H. S. Huang. 2012. NADPH
oxidase-produced superoxide mediates EGFR transactiva­
tion by c-Src in arsenic trioxide-stimulated human
keratinocytes. Arch. Toxicol. 86:935–45. doi:10.33594/
000000062.
Turner, M. C., G. Benke, J. D. Bowman, J. Figuerola,
S. Fleming, M. Hours, L. Kincl, D. Krewski, D. McLean,
M.-E. Parent, et al. 2017. Interactions between occupational
exposure to extremely low frequency magnetic fields and
chemicals for brain tumour risk in the INTEROCC study.
Occup. Environ. Med. 74:802–09. doi:10.1136/oemed-2016-
104080.
Venkatachalam, K., S. Mummidi, D. M. Cortez, S. D. Prabhu,
A. J. Valente, and B. Chandrasekar. 2008. Resveratrol inhi­
bits high glucose-induced PI3K/Akt/ERK-dependent
interleukin-17 expression in primary mouse cardiac
fibroblasts. Am. J. Physiol. Heart. Circ. Physiol. 294:
H2078–2087. doi:10.1152/ajpheart.01363.2007.
Wang, H., and X. Zhang. 2017. Magnetic fields and reactive
oxygen species. Int. J. Mol. Sci. 18:2175. doi:10.3390/
ijms18102175.
Wang, Y., and M. F. Lou. 2009. The regulation of NADPH
oxidase and its association with cell proliferation in human
lens epithelial cells. Invest. Ophthalmol. Vis. Sci.
50:2291–300. doi:10.1167/iovs.08-2568.
Wu, Q., W. Wu, B. Fu, L. Shi, X. Wang, and K. Kuca. 2019.
JNK signaling in cancer cell survival. Med. Res. Rev.
39:2082–104. doi:10.1002/med.21574.
Yakymenko, I., O. Tsybulin, E. Sidorik, D. Henshel,
O. Kyrylenko, and S. Kyrylenko. 2016. Oxidative mechan­
isms of biological activity of low-intensity radiofrequency
radiation. Electromagn. Biol. Med. 35:186–202. doi:10.3109/
15368378.2015.1043557.
Yan, L., S. Liu, C. Wang, F. Wang, Y. Song, N. Yan, S. Xi, Z. Liu,
and G. Sun. 2013. JNK and NADPH oxidase involved in
fluoride-induced oxidative stress in BV-2 microglia cells.
Mediators Inflamm 2013:895975. doi:10.1155/2013/895975.