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ORIGINAL ARTICLE
Experimental model for ELF-EMF
exposure: Concern for human health
C. D’Angelo
a,*
, E. Costantini
a
, M.A. Kamal
b
, M. Reale
a
a
Dept. Experimental and Clinical Sciences, Immunodiagnostic and Molecular Pathology Section, University ‘‘G. d’Annunzio’’
Chieti-Pescara, Italy
b
King Fahd Medical Research Center, King Abdulaziz University, P.O. Box 80216, Jeddah 21589, Saudi Arabia
Received 2 June 2014; revised 16 July 2014; accepted 17 July 2014
Dedicated to the memory of our dear friend and colleague, Giovina Vianale.
KEYWORDS
ELF-EMF;
HaCaT;
K562;
MCP-1;
SH-SY5Y;
THP-1
Abstract Low frequency (LF) electromagnetic fields (EMFs) are abundantly present in modern
society and in the last 20 years the interest about the possible effect of extremely low frequency
(ELF) EMFs on human health has increased progressively. Epidemiological studies, designed to
verify whether EMF exposure may be a potential risk factor for health, have led to controversial
results. The possible association between EMFs and an increased incidence of childhood leukemia,
brain tumors or neurodegenerative diseases was not fully elucidated. On the other hand, EMFs are
widely used, in neurology, psychiatry, rheumatology, orthopedics and dermatology, both in diag-
nosis and in therapy.
In vitro studies may help to evaluate the mechanism by which LF-EMFs affect biological systems.
In vitro model of wound healing used keratinocytes (HaCaT), neuroblastoma cell line (SH-SY5Y)
as a model for analysis of differentiation, metabolism and functions related to neurodegenerative
processes, and monocytic cell line (THP-1) was used as a model for inflammation and cytokines
production, while leukemic cell line (K562) was used as a model for hematopoietic differentiation.
MCP-1, a chemokine that regulates the migration and infiltration of memory T cells, natural
killer (NK), monocytes and epithelial cells, has been demonstrated to be induced and involved in
various diseases.
Abbreviations: AD, Alzheimer’s disease; ELF, extremely low fre-
quency; EMFs, electromagnetic fields; HD, Huntington disease; LF,
low frequency; MCP-1, monocyte chemoattractant protein-1; PMA,
phorbol-12-myristate-13-acetate; PEMF, pulsed EMF
*Corresponding author.
E-mail address: chiara_dangelo@hotmail.it (C. D’Angelo).
Peer review under responsibility of King Saud University.
Production and hosting by Elsevier
Saudi Journal of Biological Sciences (2014) xxx, xxx–xxx
King Saud University
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Please cite this article in press as: D’Angelo, C. et al., Experimental model for ELF-EMF exposure: Concern for human health. Saudi Journal of Biological Sciences
(2014), http://dx.doi.org/10.1016/j.sjbs.2014.07.006
Since, varying the parameters of EMFs different effects may be observed, we have studied MCP-1
expression in HaCaT, SH-SY5Y, THP-1 and K562 exposed to a sinusoidal EMF at 50 Hz fre-
quency with a flux density of 1 mT (rms).
Our preliminary results showed that EMF-exposure differently modifies the expression of MCP-1
in different cell types. Thus, the MCP-1 expression needs to be better determined, with additional
studies, with different parameters and times of exposure to ELF-EMF.
ª2014 Production and hosting by Elsevier B.V. on behalf of King Saud University.
1. Introduction
Exposure to electromagnetic fields (EMF) is a phenomenon
that has always existed, nevertheless, during the 20th century,
this is steadily increasing due to environmental exposure to
man-made electromagnetic fields. Growing electricity demand,
ever-advancing technologies and changes in social behaviors
have created more and more artificial sources. Thus, both at
home and at work everyone are exposed to a complex mix of
weak electric and magnetic fields, arising from the generation
and transmission of electricity by domestic appliances and
industrial equipment, by telecommunications and
broadcasting.
Generally the extremely low frequency (ELF) region of the
electromagnetic spectrum is defined by frequencies from 3 to
3000 Hz (Poole and Ozonoff, 1996). These fields are produced
by electrical devices, high tension electrical distribution net-
works, from residential and occupational sources and by
power lines. 60 Hz (in the USA) and 50 Hz sine wave signals
resemble the household alternating current electrical power
supply in Europe and a large part of the world. Low-frequency
electric fields influence all systems characterized by charged
particles as the human body. In fact tiny electrical currents
exist in the human body due to the chemical reactions that
occur as part of the normal bodily functions, even in the
absence of external electric fields. For example, nerves transmit
signals through electrical impulses. Most biochemical reac-
tions, from digestion to brain activities, are complying with
the rearrangement of charged particles.
For several years, it has been considered that both residen-
tial and occupational exposures to ELF magnetic fields (MF)
could be a possible carcinogen, based on several epidemiolog-
ical studies reporting childhood leukemia and brain tumors in
adult and leukemia following chronic exposure to MF (IARC
2002). Epidemiologic studies on EMF effect, reported evidence
of association among childhood leukemia and postnatal expo-
sures above 0.4lT. Previous studies concluded that residential
exposures to EMFs carry an increased risk of leukemia,
although other studies showed that there is no significant risk
(Leitgeb, 2011). In contrast with earlier studies (Wertheimer
and Leeper, 1979; Savitz et al., 1988; London et al., 1991),
but in accord with others (Jirik et al., 2012; Auvinen et al.,
2000) that have shown no significant increase in risk of the
Acute Lymphoblastic Leukemia (ALL) for children exposed
to residential levels of magnetic fields, Linet et al. show a lack
of association between electromagnetic field exposure and
ALL (Linet et al., 1997.
Harmful effect of EMF exposure on living tissue depends
primarily on the frequency (wavelength) and density of the
field and on the exposure time. Further important risk factors
are the functional state and the sensibility of the exposed
organism. The vascularization of the irradiated parts and the
distance from the radiation source must be considered, too.
EMFs of the magnitude to which we are now regularly
exposed, have been implicated as a contributory factor to the
childhood cancer incidence, particularly leukemia and brain
cancer.
There are numerous publications describing various in vitro
effects of EMF exposure, although the significance of these
observations for clinical interpretation is unsubstantiated. A
fundamental interaction mechanism between weak ELF mag-
netic fields and cells is also lacking, although several candidate
mechanisms have been proposed. Numerous hypotheses have
been suggested (IARC 2002; Davanipour et al., 2007; Draper
et al., 2005; Gottwald et al., 2007), although none is convincingly
supported by experimental data. A large number of cellular
components, systems and processes such as proliferation, (Tsai
et al., 2007) morphology, (Noriega-Luna et al., 2011) apoptosis,
(Grassi et al., 2004) gene expression (Mayer-Wagner et al., 2011)
and differentiation (Piacentini et al., 2008), can conceivably be
affected by EMF exposure (Simko
`and Mattsson, 2004).
Although the role of increased intracellular Ca
2+
was already
well documented more than 20 years ago (Walleczek, 1992),
recent studies have confirmed the role of increased intracellular
Ca
2+
following EMF exposure. Recently, it was suggested that a
possible early biological response to EMF exposure, is the for-
mation and prolonged survival of reactive oxygen species and
other free radicals (Mannerling et al., 2010).
Different types of magnetic and electromagnetic fields are
now used effectively in medicine (Markov 2007), such as in
diagnostic (e.g. magnetic resonance imaging-MRI, scanner
and microwave imaging) or therapy (Consales et al., 2012).
Electromagnetic therapy carries the promise to be used in dif-
ferent diseases, in fact magnetotherapy provides an easy and
non invasive method to treat the site of injury (Markov
2007). Pulsed electromagnetic fields in low frequency and
intensity range (Gauss or micro-Tesla) increase oxygenation
to the blood, improve circulation and cell metabolism, improve
function, pain and fatigue from fibromyalgia (Sutbeyaz et al.,
2009), help patients with treatment-resistant depression
(Martiny et al., 2010), and may reduce symptoms from multi-
ple sclerosis (Lappin et al., 2003). EMFs have been commonly
used for the treatment of some pathological conditions to stim-
ulate tissue regeneration and repair (Bertolino et al., 2006).
Application in the area of orthopedics for the treatment of
non-union fractures and failed fusions, takes advantage of
the evidence that pulsed EMF (PEMF) accelerates the re-
establishment of normal potentials in damaged cells (Fiorani
et al., 1997), promotes the proliferation and differentiation
of osteoblasts (Wei et al., 2008) and improves the osteogenic
2 C. D’Angelo et al.
Please cite this article in press as: D’Angelo, C. et al., Experimental model for ELF-EMF exposure: Concern for human health. Saudi Journal of Biological Sciences
(2014), http://dx.doi.org/10.1016/j.sjbs.2014.07.006
phase of the healing process (Cane
`et al., 1993). Long-lasting
relief of pelvic pain of gynecological origin has been obtained
consistently by short exposures of affected areas with the
application of a magnetic induction device, producing short,
sharp, magnetic-field pulses of minimal amplitude (Jorgensen
et al., 1994). EMFs improve cell survival and reduce ischemic
damage (Grant et al., 1994).
2. EMFs and skin injury
Being the skin the largest organ that covers the surface of our
body, it is frequently subject to the action of non ionizing MF.
Keratinocytes, those are able to release immunomodulators
and to play a key role in immune system function may be used
as an in vitro model to evaluate the biological effects of non
ionizing electromagnetic field on the skin.
Wound healing is a highly coordinated and complex pro-
cess involving the proliferation and migration of various cell
types (epidermal, dermal as well as inflammatory cell), chemi-
cal mediators and the surrounding extracellular matrix, result-
ing in a tightly orchestrated re-establishment of tissue integrity
by specific cytokines. Wounds can be categorized as acute or
chronic according to their healing time-frame. Acute wounds
repair themselves and heal normally following the correct
pathway. The chronic condition derives from non-healing
wounds in a timely and orderly manner, that determines ulcers
(Lazarus et al., 1994). Ischemia, diabetes mellitus, venous sta-
sis and pressure can be at the root of the majority of non-heal-
ing wounds that are prone to complications including
functional limitations, infections and malignant transforma-
tion (Eltorai et al., 2002; Chraibi et al., 2004).
Although there are many experimental and clinical evidences
supporting the use of magnetic fields to help bone healing, its
application for soft tissue healing, including skin and tendons
is still ambiguous. Several authors, however, showed the ability
of PEMFs in reducing the wound healing durations (Cheing
et al., 2014; Athanasiou et al., 2007; Strauch et al., 2007) and
improving tensile strength of scars (Goudarzi et al., 2010).
Roland et al. used pulsed magnetic energy to stimulate neovas-
cularization in a rat model (Roland et al., 2000). Weber et al.
showed that rat groin composite flap survival increases when
supported by an arterial loop, thus confirming that PEMFs pro-
mote neovascularization (Weber et al., 2004). The most rapid
wound healing exposed to EMF may be dependent on the
anti-inflammatory effects caused by the change in the coagula-
tion system, in the improvement of microcirculation and in
immunological reactiveness (Matic et al., 2009). Conversely,
Milgram found that PEMF did not produce any beneficial
effects on wound healing. So the effects of PEMF on wound clo-
sure varied among the studies, possibly due to different treat-
ment protocols that were applied (Milgram et al., 2004).
Callaghan et al. (2008) confirmed the results of Tepper et al.
(2004), that demonstrated the increase in proliferation and tubu-
lization of endothelial cell cultures and the increase in the expres-
sion of fibroblast growth factor 2 (FGF-2), a potent stimulator
of angiogenesis, after exposure to electromagnetic field.
Vianale et al. showed that ELF-EMF modulates produc-
tion of RANTES, MCP-1, MIP-1aand IL-8, and keratinocyte
growth through the inhibition of the NF-kB signaling pathway
and they hypothesized that ELF-EMF may inhibit inflamma-
tory processes (Vianale et al., 2008).
Recently, several reports have supported the anti-inflamma-
tory effects of EMFs on tissue repair. Pesce et al. reviewed the
effect of EMFs on cytokines that drive the transition from a
chronic pro-inflammatory to an anti-inflammatory state of the
healing process (Pesce et al., 2013). Patruno’s results showed
the ability of ELF-EMF to induce keratinocyte proliferation
and to up modulate NOS activities and to down-regulate
COX-2 expression and PGE-2 production, involved in the mod-
ulation of inflammatory reaction (Patruno et al., 2010). In vitro
study of Huo et al. showed that the noninvasive EMFs have a
strong effect on normal human keratinocytes and fibroblast
migration while only weakly promote keratinocyte proliferation
(Huo et al., 2009). The observations of Manni et al. confirm the
hypothesis that ELF-EMF (50 Hz) may modify cell membrane
morphology and interfere with initiation of the signal cascade
pathway and cellular adhesion (Manni et al., 2002). ELF-
EMF application modifies the biochemical properties of human
keratinocytes (HaCaT) associated with different actin distribu-
tions as demonstrated by Lisi et al. (2006).
3. EMFs and neurodegenerative diseases
The term neurodegeneration indicates the progressive loss of
neuronal function and structure until the neuron death. Many
neurodegenerative diseases such as Parkinson disease (PD),
Alzheimer’s disease (AD), Huntington disease (HD), and
Amyotrophic Lateral Sclerosis (ALS) result from neurodegen-
erative processes and many of these are classified as patholo-
gies due to the aggregation of misfolded proteins. PD is a
disorder of the central nervous system resulting from the death
of dopaminergic cells in the substantia nigra. The basis of this
mechanism may consist of an abnormal accumulation of the
protein alpha-synuclein that forms insoluble fibrils, in the
damaged cells. The beta-amyloid peptide (Ab)is a small pep-
tide that comes from the cleavage of a larger transmembrane
protein called amyloid precursor protein (APP). Abis the
major component of plaques in the cerebral cortex of AD
and is critical to neuron growth, survival and is involved in
the loss of synapses and the neuron death, as well as hyper-
phosphorylated tau protein, the main component of neurofi-
brillary tangles in AD brain. Sobel and Davanipour
hypothesized the ability of the ELF-EMFs to increase the
intracellular calcium concentration levels that are positively
correlated with the cleavage of the APP to give the soluble
Ab(Sobel and Davanipour, 1996). Several studies seem to sug-
gest a potential association between occupational exposure to
ELF-EMFs (typical of electric power installers and repairers,
power plant operators, electricians, telephone line technicians,
welders, carpenters, and machinists) and AD onset (Garcı
`a
et al., 2008; Ro
¨o
¨sli, 2008), although their biological nexuses
remain unknown.
HD is a progressive neurodegenerative disorder whose
underlying genetic defect lies in expanded trinucleotide
(CAG)n of the Huntington ubiquitous protein. In HD, an
autosomal dominant disease, the mutated gene leads neuronal
dysfunction and degeneration, even though the mechanisms by
which it acts are not fully understood. The potential correla-
tion between EMF exposure and HD pathogenesis is not sus-
tained by epidemiological evidence, while there is evidence that
the improvement in behavior and the neuroprotective effect of
ELF-EMF exposure may be due to enhanced neurotrophic
Experimental model for ELF-EMF exposure 3
Please cite this article in press as: D’Angelo, C. et al., Experimental model for ELF-EMF exposure: Concern for human health. Saudi Journal of Biological Sciences
(2014), http://dx.doi.org/10.1016/j.sjbs.2014.07.006
factor levels, and reduced both oxidative damage. The Amyo-
trophic Lateral Sclerosis is a fatal neurodegenerative disorder
characterized by progressive degeneration of motor neurons
in the spinal cord, motor cortex, and brainstem. Approxi-
mately 20% of patients were found to show mutation of the
gene encoding the antioxidant Cu
2+
/Zn
2+
SOD (SOD1)
(Julien and Kriz 2006), confirming the central role of oxidative
stress in neurodegenerative diseases (Chang et al., 2008). On
the basis of epidemiologic findings, evidence shows an associ-
ation between amyotrophic lateral sclerosis and occupational
EMF exposure although there is confounding (Davanipour
et al., 1997; Savitz et al., 1998). The investigations of EMF
effects on neurodegenerative diseases are now very interesting,
although not well developed, in fact the experimental findings
supporting this link are still controversial due to the field fre-
quency applied and the disease investigated (Consales et al.,
2012). Crasson et al. indicated that 50 Hz EMF may have
slight influence on event-related potential and reaction time
under specific circumstances of sustained attention in healthy
male volunteers. (Crasson et al., 1999). Trimmel’s study shows
a reduction of cognitive performances in attention, perception
and memory performances by a 50 Hz EMF of 1 mT (Trimmel
and Schweiger, 1998).
Sulpizio et al. have demonstrated that ELF-MF exposure
triggers significant changes in the protein global profile of
SH-SY5Y cell line, experimental model for neurodegenerative
disorders. In particular, the expression levels of common pro-
tein spots involved in cellular defense mechanisms, organiza-
tion, and biogenesis increased as a consequence of ELF-
EMF treatment. In ELF-EMF treated samples was observed
the over-expression of proteins related to a high malignant
potential, drug resistance, cytoskeleton re-arrangement, and
enhanced defense against oxidative stress, in association with
higher proliferative activity (Sulpizio et al., 2011; Xie et al.,
2010). In vivo study showed that exposure to environmental
ELF-EMF did not change the expression of a3, a5 and a7 nic-
otinic cholinergic receptors impaired in AD (Antonini et al.,
2006). Falone et al. have demonstrated that a 50 Hz magnetic
field induced a significant enhancement of the antioxidant
defenses together with a major shift of redox homeostasis
and they previously established that ELF-MF exposure
improves cellular viability and induces significant adaptations
in the redox-related biochemical machinery of the human neu-
roderived SH-SY5Y cell line (Falone et al., 2007).
4. EMFs and immune cells
Cells of the immune system regulate health on a systemic level,
thus are plausible study targets. .In response to a pathogen chal-
lenge they must respond in a very sensitive, swift and effective
way. Immune cells produce cytokines, important signaling mol-
ecules, which are key regulators of cell activation and inhibition.
Monocytes and macrophages have an important function
as the first line of defense against pathogens and can act as
antigen-presenting cells to trigger a specific response from lym-
phocytes, and are capable of producing several cytokines
including interleukin-1 beta (IL-1b), tumor necrosis factor-
alpha (TNF-a), and interleukin-10 (IL-10). Their recruitment
to inflammatory sites and neoplastic tissues and their activa-
tion induce a wide range of intracellular signaling pathways
and are crucial to the success of an immune reaction. Cytokine
production and secretion patterns are modified upon differen-
tiation of monocytes into macrophages (Bouwens et al., 2012).
Several papers have demonstrated that the in vitro exposure of
immune cells to nonthermal ELF-EMF can elicit molecular
and cellular changes that might be relevant to the activity of
the immune system in vivo. Years ago, it was demonstrated
that only mutagen-activated lymphocytes are responsive to
EMF exposure, in fact EMF do not interfere with activation
and committeemen of cells (Cadossi et al., 1992). Nindl et al.
have showed that 60 Hz sinusoidal EMFs induce an increase
in anti-CD3 binding to T cell receptors (TcRs) of Jurkat cells,
a T lymphocyte cell line, and that can regulate lymphocyte
proliferation in vitro and in vivo (Nindl et al., 2000). Reale
et al. have demonstrated that upon ELF-EMF activation,
monocytes/macrophages increase the production of chemo-
kines, peroxidases, cytolytic proteases, and nitric oxide (NO)
enhancing their microbicidal/tumoricidal capacity (Reale
et al., 2006). Akan et al. have demonstrated that field applica-
tion increased NO, cGMP, and HSP levels, and caused a slight
decrease in apoptosis (Akan et al., 2010; Frahm et al., 2010).
Many in vitro and in vivo studies have evaluated the expression
of free radicals in human monocytes and mouse macrophages
after exposure to 50 Hz, 1 mT ELF-EMF (Simko
`et al., 2001;
Lupke et al., 2004; Rollwitz et al., 2004). NO, a free radical, is
an important intracellular and intercellular signaling molecule,
and an important host defense effector for the phagocytic cells
of the immune system (Fo
¨rstermann and Kleinert, 1995).
Several studies have been conducted to evaluate the effect of
ELF-EMF exposure on cytokine profiles, consistent and inde-
pendently replicated laboratory evidence to support modula-
tion of cytokines expression and production has not been
obtained (Ikeda et al., 2003; Luceri et al., 2005; Miller et al.,
1999; Natarajan et al., 2006; Reale et al., 2006; Lupke et al.,
2006; Murabayashi et al., 2004). Ays
ße et al. demonstrated that
in vitro effect of ELF-EMF on the differentiation of K562 cells
is time dependent. In fact single exposure to ELF-EMF resulted
in a decrease in differentiation; ELF-EMF applied everyday for
1 h caused an increase in differentiation. These results imply
that the time-course of application is an important parameter
determining the physiological response of cells to ELF-EMF
(Ays
ße et al., 2010) and other authors have supported the
hypothesis that the effect of ELF-EMF on biological
systems depends on the conditions of the cell (Garip and
Akan, 2010).
Numerous studies are still underway to try to understand
the mechanism behind these alterations investigated as the
gaming is very complex. Conflicting conclusion were showed
in many EMF in vivo studies, due to small numbers of subject,
distance from EMF source, time exposure or concomitant
environmental risks. The in vivo study of Boscolo did find dif-
ferences in cytokine levels in serum of subject exposed to ELF-
EMF (Boscolo et al., 2001). In a set of experiments evaluating
time courses for immediate early genes, stress response, cell
proliferation and apoptotic genes, Kirschenlohr et al. showed
no consistent response profiles after repeated ELF-EMF expo-
sures (Kirschenlohr et al. 2012).
5. Cell line in vitro models
Cell lines have some advantages over human primary cells
such as (a) homogeneous genetic background that minimizes
4 C. D’Angelo et al.
Please cite this article in press as: D’Angelo, C. et al., Experimental model for ELF-EMF exposure: Concern for human health. Saudi Journal of Biological Sciences
(2014), http://dx.doi.org/10.1016/j.sjbs.2014.07.006
the degree of variability in the cell phenotype, a trait particu-
larly important when studying the biological function with
high variability; (b) ability to be stored indefinitely in liquid
nitrogen to guarantee sufficient cells for DNA, RNA and pro-
tein; (c) reduced variability compared to primary cells; and (d)
reproducibility of the results obtained.
SH-SY5Y cells were derived from immature neoplastic neu-
ral crest cells that exhibit properties of stem cells. The SH-
SY5Y cell line is a thrice-cloned subline of SK-N-SH cells that
were originally established from a bone marrow biopsy of a
neuroblastoma patient and were widely used as model of neu-
rons since the early 1980’s (Biedler et al., 1973). These cells
possess the capability of proliferating in culture for long peri-
ods without contamination, a prerequisite for the development
of an in vitro cell model, posses many biochemical and func-
tional properties of neurons, exhibits neuronal marker enzyme
activity, express neurofilament proteins and also express opi-
oid, muscarinic, and nerve growth factor receptors
(Ciccarone et al., 1989). Consequently, the SH-SY5Y cell line
has been widely used in experimental neurological studies,
including analysis of processes related to neurodegeneration,
neuroprotection and neurotoxicity. The processes of keratino-
cyte proliferation and differentiation represent the central and
final event in tissue regeneration leading to the formation of a
massive bulk of cells, necessary to cover the wounded area. It
is widely accepted that in vitro keratinocyte model systems,
such as HaCaT cell line, at low and high density can be com-
pared with early and late phases of the re-epithelialization pro-
cess. HaCaT cells are in vitro spontaneously transformed
keratinocytes from histologically normal skin. Thus keratino-
cytes are the most likely cells to be impacted by electromag-
netic radiation.
THP-1 is single, round suspension cells that after exposure
to phorbol-12-myristate-13-acetate (PMA) or 1a,25-
dihydroxyvitamin D3 (1a,25(OH)2D3) may start to adhere
to culture plates accompanied by phenotype change into a
macrophage. Based on phenotypic and functional features
with human microglial cells, human monocyte-derived macro-
phages were called brain macrophages (Ulvestad et al., 1994).
THP-1 cells, due to their functional and morphological simi-
larities, have been widely used as a model of human mono-
cytes/macrophages or microglia (Tsuchiya et al., 1982;
Tsuchiya et al., 1980; McDonald et al., 1998) or as a valid
model to mimic proliferation, adhesion and migration of
monocytes and macrophages in the vasculature.
The human K562 cell line has been isolated and character-
ized by Lozzio (Lozzio and Lozzio, 1975) from a patient with
chronic myelogenous leukemia (CML) in blast crisis. K562
has been used as a model of common progenitor of erythro-
blasts and megakaryocytes and can be differentiated into ery-
throid and megakaryocytic lineages thus has been used
extensively as a model for the study of leukemia differentiation,
molecular mechanism(s) regulating the expression of genes
(Iyamu et al., 2000), as well as to determine the therapeutic
potential of new differentiation-inducing compounds (Bianchi
et al., 2001).
6. Effects of ELF-EMF exposure on MCP-1
Chemokines are low molecular weight chemotactic cytokines
that have been shown to play a relevant role in inflammatory
events, such as transendothelial migration and accumulation
of leucocytes at the site of damage. In addition, they modulate
a number of biological responses, including enzyme secretion,
cellular adhesion, cytotoxicity and T-cell activation and tissue
regeneration (Vianale et al. 2008).
The monocyte chemoattractant protein-1 (MCP-1/CCL2)
is a member of the C–C chemokine family and is a potent che-
motactic factor for monocytes. Located on chromosome 17
(chr.17, q11.2), human MCP-1 is composed of 76 amino acids
and is 13 kDa in size (Van Coillie et al., 1999). A variety of cell
types including endothelial, fibroblasts, epithelial, smooth
muscle, mesangial, astrocytic, monocytic, and microglial cells
(Cushing et al., 1990; Standiford et al., 1991; Brown et al.,
1992; Barna et al., 1994), are able to produce MCP-1, either
constitutively or after induction by oxidative stress, cytokines,
or growth factors. Rolling of monocytes on endothelial cells is
dependent on the binding of E-selectin and sialyl Lewis X, and
adhesion to the endothelium is dependent on the interaction of
integrin on monocytes and adhesion molecules on the endothe-
lial cells. Although leukocytes have been considered the main
targets for chemokines, recent evidence indicates that the
actions of these proteins are not restricted to these cell types.
The main function of MCP-1 consists of the establishment of
chemotaxis driving the recruitment of cells at sites of inflam-
mation, by integrin activation. Specifically MCP-1 attracts
monocytes, natural killer cell and memory T cells, and influ-
ences expression of cytokines related to T helper responses.
Its expression occurs in a variety of diseases characterized by
mononuclear cell infiltration, and there is substantial biologi-
cal and genetic evidence suggesting that it may contribute to
the inflammatory component of diseases such as atherosclero-
sis, multiple sclerosis, Alzheimer’s disease, or rheumatoid
arthritis. In the central nervous system (CNS), MCP-1 is
involved in the recruitment of the main resident immune cell
types of the brain (astrocytes and microglia) and of infiltrating
monocytes from the systemic bloodstream. There is strong evi-
dence that MCP-1 plays a major role in myocarditis, ischemia/
reperfusion injury in the heart, in transplant rejection, and in
cardiac repair. After 24 h of chronic exposure to 50 Hz,
1 mT EMF, MCP-1 levels were reduced significantly in
PHA-stimulated cells, while in non-stimulated cells no signifi-
cant differences in MCP-1 levels were observed. The authors
speculate the anti-inflammatory potency of electromagnetic
fields and suggest that the inhibitory effect on MCP-1 release,
evaluated by the ELISA assay, could be one of the mechanisms
by which ELF-EMF is therapeutic in inflammatory diseases
(Di Luzio et al., 2001).
Previous studies have suggested that magnetic field is
involved in NO production. Thus, Reale et al. exposed LPS-
stimulated peripheral blood adherent mononuclear cells to
50 Hz EMF. Results of RT-PCR showed that, while both
mRNA and protein levels of MCP-1 were up-regulated, iNOS
was down-regulated. The increase in MCP-1 is related to NF-
kB protein expression and in agreement with previous results
showing that the inhibition of nitric oxide production in endo-
thelial cells increased the expression of MCP-1. The changes in
MCP-1 and iNOS expression, evaluated through RT-PCR,
after ELF-EMF are very interesting for their roles in the devel-
opment of inflammatory responses. The authors suggest a non
pharmacological role of EMF in maintaining the balance
between MCP-1 and NO in inflammatory reaction (Reale
et al., 2006).
Experimental model for ELF-EMF exposure 5
Please cite this article in press as: D’Angelo, C. et al., Experimental model for ELF-EMF exposure: Concern for human health. Saudi Journal of Biological Sciences
(2014), http://dx.doi.org/10.1016/j.sjbs.2014.07.006
Since the EMF effect is cell type-dependent and MCP-1 is
produced which acts on different cell types, and very little is
known about the influence of ELF-EMF on MCP-1 expression
in different cell types, we studied the effect of ELF-EMF on
MCP-1 expression and production in HaCaT, SH-5YSY,
THP-1 and K562 cells.
In our ELF-EMF the flux density of 1 mT (rms) was pro-
duced by an electromagnetic generator (Agilent Technologies,
Santa Clara, CA, mod. 33220A) with stability higher than 1%
both in frequency and in amplitude. The generator was con-
nected to a power amplifier (Nad Electronics Ltd, London,
U.K., mod. 216). An oscilloscope (ISO-TECH mod. ISR658,
Vicenza, Italy) was dedicated to the monitoring of output sig-
nals from the Gaussmeter (MG-3D, Walker Scientific Inc.,
Worcester, MA). A current flux passed through a 160 turns
solenoid (22 cm length, 6 cm radius, copper wire diameter of
1.25 ·10
5
cm) generating a horizontal magnetic field. The
achieved MF intensity (1 mT/rms) was measured continuously
during exposure using a Hall-effect probe connected to the
Gaussmeter. The solenoid was then placed inside the incuba-
tor. The environmental magnetic noise inside the incubator
was related to the geomagnetic field (40 mT), and to the
50 Hz disturbance associated with the working incubator
[7 mT (rms)]. The built-in digital thermometer of the incuba-
tor monitored the internal temperature, which resulted con-
stant at 37 ± 0.38 C. In addition, another digital
thermometer (HD 2107.2; Delta OHM, Padova, Italy) was
placed inside the solenoid and near the cell cultures to record
local temperature variations. No significant temperature
change related to applied ELF fields was observed
(DT0.18C). However, no thermal effect on cells can be hypoth-
esized for temperatures around 37.8 C, because EMF interac-
tions with biological molecules are known to be non thermal in
nature. Low-level Joule heating was dissipated inside the incu-
bator by a fan system. In all experiments cells were placed in
the central part of the solenoid, which presented the highest
degree of the field homogeneity (98%).
All experiments are performed at the same conditions of
EMF intensity, frequency, chronical exposure, and tempera-
ture. In Table 1, we report the effects of the ELF-EMF expo-
sure on MCP-1 expression in different cell lines.
In HaCaT cells, using RT-PCR we have evidenced a
decrease in MCP-1 expression from 4 to 72 h in EMF-exposed
cells with respect to non-exposed cells. This decrease was con-
firmed by additional Real Time PCR (basal exposed
0.9 ± 0.02 vs. basal non-exposed 1.6 ± 0.05). Also the ELISA
immunoassay, performed to evaluate the release of MCP-1,
confirmed the expression results. Since it is well accepted that
an excessive or prolonged inflammatory response may interfere
with wound healing and cause reduction of the inflammatory
chemokines by ELF-EMF exposure, represents an interesting
and new therapeutic approach in delayed healing.
In SH-SY5Y cell cultures exposed to ELF-EMF, genes
involved in the stress response, cell growth and differentiation
or protein metabolism have been reported to be generally
down-regulated. Genes involved in Ca
2+
metabolism, the
PI3-kinase pathway are up-regulated. Likewise, key mediators
of the inflammatory response appear susceptible to swift mod-
ulation, in SH-SY5Y.
MCP-1 is involved in the neuroinflammatory processes
associated with diseases characterized by neuronal degenera-
tion. To characterize the impact of ELF-EMF on early ongo-
ing cellular processes, MCP-1 gene expression in SH-SY5Y,
was evaluated in the presence and absence of ELF-EMF expo-
sure by RT-PCR. After 24 h of ELF-EMF exposure MCP-1
expression was not significantly affected. Albeit our results
on MCP-1 expression, despite differences in experimental con-
ditions, are in line with several other ELF-EMF exposure
results, while they are not in accord with a study reporting that
ELF-EMF promotes cellular neurodifferentiation, as exempli-
fied by neurite extension and number (Falone et al., 2007). In
conclusion, our results showed that ELF-EMF exposure is well
tolerated and has no relevant impact on MCP-1 gene
expression.
The role of MCP-1 in human disease has been demon-
strated by immunohistochemical studies in fact the adhesion
of cells to the endothelium was induced by expression of adhe-
sion molecules and chemotactic proteins, such as MCP-1. We
have analyzed the effects of EMF on the expression of MCP-1
also in THP-1 cells. Since in THP-1 exposed to ELF-EMF no
increase in basal levels of MCP-1 was observed, cells were trea-
ted or not with LPS and exposed to 50 Hz, 1 mT EMF for
24hr. Our data indicate that the presence of 10 lg/ml of LPS
leads to an increase in expression of MCP-1 in both THP-1
cells non exposed or exposed to EMF. Thus, we hypothesized
that MCP-1 mediated THP-1 migration is not affected by
EMF exposure, and consequently the exposure to the fields
is not a risk factor in diseases in which microglial migration
plays a crucial role, such as atherosclerosis, multiple sclerosis
and other neuroinflammatory diseases.
Although it is known that intracellular redox status modu-
lates MCP-1 expression and that ELF-EMF exposure can act
on redox state of K562 cells. No studies have evaluated the
influence of EMF exposure on MCP-1 expression in K562 cell
line. In K562 exposed to the ELF-EMF spontaneous expres-
sion of MCP-1, detected by RT-PCR, was not modulated in
comparison to cells not exposed. PMA induces monocytic or
megakaryocytic differentiation of K562 cells through the acti-
vation of MAP kinases. When PMA-stimulated cells were
exposed to the field, we noticed a slight increase in the expres-
sion of the chemokines and particularly the increase in MCP-1.
Next step of this study will be to evaluate if ELF-EMF expo-
sure is able to modulate the activation of MAP kinases in com-
parison with PMA.
Table 1 Effects of the ELF-EMF exposure on MCP-1 in
different cell types.
Cell lines Effect on MCP-1 expression
HaCaT
Basal Decreased
SH-SY5Y
Basal Decreased
THP-1
Basal No effect
LPS-stimulated No effect
K562
Basal No effect
PMA-stimulated Increased
6 C. D’Angelo et al.
Please cite this article in press as: D’Angelo, C. et al., Experimental model for ELF-EMF exposure: Concern for human health. Saudi Journal of Biological Sciences
(2014), http://dx.doi.org/10.1016/j.sjbs.2014.07.006
7. Conclusions
The results of in vivo and in vitro studies suggest that EMF
may modulate the expression of some inflammatory molecules.
The understanding of the influences of EMF on transcriptional
events will lead to a better understanding of their mechanisms
and to therapeutic interventions for diseases in which these
inflammatory molecules play a key role. In spite of the fact
that the mechanisms of action of EMF are still under investi-
gation, some authors have supposed that exposure to ELF-
EMF affects cell function through mechanical action on both
intracellular and membrane proteins, which includes ion chan-
nels, membrane receptors and enzymes. All studies agree that
the effect of the sinusoidal ELF-EMF varies in relation to cell
type and other parameters, such as frequency, flux density and
time exposure.
Our data confirm the cell-type dependent effects; in fact we
observed increase, decrease or no effect on the MCP-1 expres-
sion in different cell lines grown under the same conditions
(sinusoidal 50 Hz, 1 mT, 37 C, 5% CO
2
).
In order to assess if ELF-EMFs, associated with both indus-
trial and domestic use, may play a role as adjuvant or causative
factor in disease development or may play a role as therapeutic
and diagnostic tool, further studies to evaluate a more complete
list of genes that may be up- or down-regulated by ELF-EMF
exposure must be preformed. New studies designed to evaluate
the actions of ELF-EMF under multiple conditions, including
chronic or sporadic exposure, in combination with common
stressors pertinent to real life, appear warranted and may aid
our understanding of their true biological impact.
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