Effect of 50Hz EMF on Neuroblastoma Morphology

International Journal of Integrative Biology

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ISSN 0973-8363

Effect of 50Hz Magnetic field exposure on Neuroblastoma

Morphology
D.Pozzi 1, S.Grimaldi 2,*, M. Ledda 2, F. De Carlo 2, A.Modesti 3,
S.Scarpa 1 , A. Foletti 4 and A. Lisi 2
1
Dipartimento di Medicina Sperimentale e Patologia, Università La Sapienza Rome Italy,
2
Istituto di Neurobiogia e Medicina Molecolare CNR Rome Italy,
3
Dipartimento di Medicina Sperimentale Università di Tor Vergata Rome Italy
4
BIT Milano Italy

Received 20 Jul., 2007; Accepted 10 August 2007

Abstract:
Neuroblastoma is one of the most common paediatric solid tumors originating from the sympathoadrenal lineage
of neural crest. This tumor shows extremely different clinical phenotypes such as spontaneous regression on one
hand and aggressive growth on the other hand. Undifferentiated neuroblastoma cell line (Lan-5) represent a good
model to study neuronal differentiation induced by a variety of stimuli such as retinoic acid treatment. At
scanning electron microscopy analysis this cells showed rare neurite-like processes containing a small amount of
neurosecretory granules and a low expression of the neurofilaments polypeptides. In our experiments the
exposure for 5 days to 50 Hz, 2 mT magnetic field inhibits cell growth proliferation with a decrease of 30 % in
exposed cells respect to the control. Moreover at scanning electron microscopy exposed cells showed a neuronal
morphology aspect and development of a larger amount of neuritic like processes than in the control cells. Also
the immunofluorescent analysis of 50 Hz, 2mT exposed cells shows an enhancement on the expression of
NF200. These results suggest that exposition of Lan-5 to 50 Hz, 2mT magnetic field can interfere with cell
growth process and induce a neuronal like cell morphology.

Keywords: Neuroblastoma, Lan-5, magnetic field, low-frequency EMF

INTRODUCTION: laboratories is difficult. The possible

Reports of biological effects of low-frequency
EMF have increased in recent years (Chiabrera
et al., 1985; Blank et al., 1987; Tenforde et al.,
1995; Lacy-Hulbert et al., 1998; Zhadin et al.,
2001). Particularly debated is the question of the
epidemiological evidence of adverse effects of
an extremely low frequency magnetic field
(ELF-MF) (Hatch, 1998; Day, 1999). The
experimental approach to the effects of
electromagnetic fields on living systems is
complicated by reported nonlinearities (intensity,
frequency and time windows of the fields) and
other variables (cell type, age, and treatment) so
that extrapolation or replication among
*Corresponding author: Settimio Grimaldi, Ph.D.
e-mail.: settimio.grimaldi@artov.inmm.cnr.it
mechanisms of the induced effects are not known,
although a number of theoretical models have
been proposed (Glaser, 1992; Liburdy, 1992;
Barnes, 1996).
We studied the effect of ELF on the
neuroblastic tumor (neuroblastoma) (NB) , with
the aim to evaluate how ELF can interfere with
the ability of this tumor to differentiate and
proliferate. Neuroblastoma is one of the most
common pediatric tumors and originates from
the sympathoadrenal lineage derived from the
neural crest (Bolande, 1974), furthermore it is
characterized by its potential for spontaneous
resolution by neuronal differentiation in vivo
(Brodeur, 2003). Neuroblastoma cells are usually
highly undifferentiated, and they are often
induced to maturation in vitro and in vivo into
terminally mature non proliferating cells by the

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Effect of 50Hz EMF on Neuroblastoma Morphology
utilization of differentiation protocols with
morphogens, growth factors and cytokines
(Lovat et al., 1997; Nakagawara, 1998; Maris et
al., 1999; Ponzoni et al., 1992). The
neuroblastoma cell line LAN-5 is a widely
utilized model for the study of neuritic
outgrowth phase of neuronal differentiation
induced by a variety of stimuli among which
retinoic acid treatment (Neuroni et al., 1991).
LAN-5 is an undifferentiated NB cell line with
rare neurite-like processes containing a small
amount of neurosecretory granules and a low
positivity for the three major neurofilament
polypeptides (NF 160, NF200, NF 60).
To evaluate the effect of 50 Hz, 2mT magnetic
field exposure on LAN-5 proliferation and neural
differentiation, we have performed cell growth
curves, scanning electron microscopy and
immunofluorescent staining for neurofilaments.
The results obtained showed that 5 days
exposure induced a decrease in the rate of cell
replication and cell differentiation toward a more
neuronal phenotype, such as the one induced by
retinoic acid stimuli.
MATERIALS AND METHODS:
Exposure solenoid
Cells were exposed for 5 days to a sinusoidal 50
Hz magnetic field at a flux density of 2 mT (rms)
in a temperature regulate solenoid. Five percent
of CO2 was provided. Temperature within the
center of the solenoid was continuously recorded
by Hanna HI 9274 OC printing thermometer and
was maintained in 37 C° +- 0.3 range.
The solenoid was placed in a cell incubator
while the control cells were run in a second
incubator of the same make and in the same
conditions. As a double check, in a subsequent
experiment the control sample was placed in the
cell incubator containing the solenoid with no
field, in the same condition of the exposed one.
No differences were detected between control
and sham cells. All experiments are made in
blind condition. The solenoid manufacture has
been published elsewhere (Santoro et al., 1997).
The main body of the solenoid is a concrete –
asbestos cylinder 2 cm thick , with a diametre of
20 cm and height of 40 cm. It is made of 1200
turns of 2mm diameter copper wire wound in
three layers in continuous forward-backward
fashion. The solenoid was energised from a 50
International Journal of Integrative Biology
Hz power mains through variable
autotransformer and provided aflux density of 2
mT (rms) for an applied voltage of 12 V (rms).
The solenoid is then placed into a cell incubator
with the centre ventilated by the fan for
appropriate air circulation. The modest heat due
to Joule effect is efficiently dispersed by the
continuous forced ventilation in the total mass of
the CO2 incubator. As stated above, in all
experiment the temperature in the solenoid,
measured at the position of the sample, was
never out of the range 37± 0,3 °C. Since, in
exposed samples, long exposure at temperatures
higher than physiological, will induce synthesis
of heat shock ( HSP) Western blot analysis of
HSP was performed. Western blotting
experiments on control and exposed Lan-5 cells
showed no difference in HSP expression,
suggesting that exposed cells are not undergoing
stress induced by heat effect (data not shown).
Field intensity (B), measured with a calibrated
Hall probe, is homogeneous within -5% of the
value at the center of the cylindrical exposure
volume (11cm by 17cmalong the solenoid axis).
The measured geomagnetic ambient field is 32
μT (vertical component ) and 16μT ( horizontal
component ). Stray ambient ac field are below
0.1μT. An often overlooked fact related to
solenoid is the presence of an almost
homogeneous electric field oriented parallel to
the magnetic field (Chute et al., 1981) unless
adequate electric shielding is used (Stauffer et al.,
1994). An approximate estimate of this field may
be obtained from the voltage across the
extremes of the inner of the six layers and its
length. The corresponding field induced by
capacitative coupling in the culture medium will
be in the order of 0.01 V/cm (Foster et al., 1995).
1800
1600
1400
Cell Concentration (cell*10 /ml)
3
1200
1000
800
600
400
200
0
0 20 40 60 80 100 120
Culture Time (Hrs)
Figure 1: Growth curve of non exposed (▼) and 50 Hz five
days exposed Lan-5 cells (●)
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Effect of 50Hz EMF on Neuroblastoma Morphology

Cell cultures Immunofluorescence

LAN-5 cells were grown in RPMI (Gibco
Laboratories, Scotland) supplemented with 10%
Fetal Calf Serum (Gibco Laboratories, Scotland)
and antibiotics (110 IU/ml of penicillin and 0.1
mg/ml of streptomycin) at 37 ±- 0.3°C, and 5%
CO2 as carbon source and sub-cultured twice a
week at a 1:5 ratio. For every experiment,
control and exposed cells were taken from the
same flask.
For immunofluorescence staining the cells were
grown in Labtek chamber slides. The cells were
then washed with PBS with Ca/Mg and fixed
with absolute ethanol for 5 minutes, then
incubated with the specific monoclonal
antibodies, anti-160 KDa, anti-200 KDa
neurofilaments (Sigma) appropiately diluted for
1 hour at room temperature. Cells were then
washed three times with PBS and incubated with
fluoresceinated anti-mouse IgGF(ab')2 fragment
(Sigma), appropriately diluted for 1 hour at room
temperature.

RESULTS

EMF effect on LAN-5 cell growth

Fig.2 Light microcopy of control ( A) and 50 Hz exposed
Lan-5 cells (B,C,D ). Objective magnification:40x.
The cell growth rate was analyzed by the Trypan
bleu staining both in LAN-5 cells as control (not
exposed) or exposed to the field. An inhibition in
the cell growth in the exposed cells started to be
statistically (p < 0.01) significant after 5 days
exposure and reached the maximum extent (25%)
at 5 days exposure. (Fig.1)

SEM conditions
Control and LFMF-exposed cells were washed
in PBS and fixed with 2.5% glutaraldheyde in
0.1M Millonig's phosphate buffer for 1 hr at 4°C .
Samples were post-fixed in 1% OsO4,
dehydrated through a graded acetone series, and
critical point dried with CO2 in a Balzers CPD
030 critical point drier. Specimens were coated
with gold in a Balzers SCD 050 sputter
instrument and observed on a Cambridge S240
scanning electron microscope.

Cells growth curves

LAN-5 cells were exposed in the solenoid region
where the field gradient was within 5% of 50
Hz ,2 mT (rms) For each experiment LAN-5
cells were plated into 25 ml 4.2 x 5.2 cm base
Corning flasks (2.0 x 105 /ml cells in a total
Fig.3 Scanning electron microscopy of control ( A,B ) , 50
volume of 5 ml). The flasks were kept in the Hz 2mT exposed ( C,D) and AR treated ( E,F) Lan-5 cells.
exposure system continuously for up to 5 days
with or without MF. Cells were then counted
from the third to the fifth day and viability EMF effect on LAN-5 cell morphology
determined by Tripan Blue dye exclusion. The By phase contrast and scanning electron
experiment was repeated three times. microscopy LAN-5 control cells (Fig.2a and Fig.

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Effect of 50Hz EMF on Neuroblastoma Morphology
CTR
Fig.4 Indirect immunofluorescent analysis
3 a-b) appeared small, polygonal, without
neurite-like structures. The exposure to magnetic
field (50 Hz, 2mT) induced morphological
changes toward a more neuronal phenotype: the
cells were stretched out and rich of neurite-like
structures (Fig. 2c-d and Fig.3c-d) with blebs,
mimicking the same effect induced by retinoic
acid treatment (Fig.2b, Fig.3e-f)
EMF effect on neurofilament expression
Fig. 4 shows the indirect immunofluorescent
analysis of control and exposed Lan-5 cells with
anti 200 and 160 neurofilaments. While control
cell were little or not positive for NF 160 (CTR),
the neurofilament protein become more
fluorescent after exposure to the field (EXP).
The same effect was much more evident for NF-
200.
SUMMARY
Low frequency electromagnetic fields at 50 or 60
Hz indeed are reported to stimulate nerve
regeneration (Rusovan et al., 1992), alter gene
transcription (Phillips et al., 1992) and they may
also play a synergistic role in cellular processes
that are already activated, such as cell
proliferation (Walleczeck, 1992 ; Manni et al.,
International Journal of Integrative Biology
EXP
NF-160
NF 200
2002). Despite an increasing number of
publications demonstrate an effect of very low
frequencies EM field on biological systems,
other in vivo and in vitro studies suggest
opposite results; in addition the possible
interaction mechanism is not yet completely
understood.
A possible mechanism evoked to explain the
mechanism of EM field action to biological
system is involving Ca2+ transport across cell
membrane, to trigger the signal transduction
cascade (Walleczeck, 1992 ; Karabakhtsian et al.,
1994).
Electromagnetic therapeutic potential can be
seen in the proven efficacy of low-energy pulsed
magnetic fields in non-union bone fracture
healing, confirming that under certain conditions
non-ionizing electro-magnetic energy can
influence physiological processes in organisms.
Physiological paradigms for non-ionizing
radiation effects are required. Clues may be
found in the mechanisms by which EM field
interacts with cultured cells under controlled
laboratory conditions and by correlating in vivo
evidence with in vitro data (Glaser, 1992).
Brain maturation depends on a sequence of
postnatal events (Vignes et al., 1997; Mulle et
al., 1998). Brushart et al., 2002 found that
IJIB, 2007, Vol. 1, No. 1, 15
Effect of 50Hz EMF on Neuroblastoma Morphology
electrical stimulation at 20 Hz, promote
motoneuron regeneration, confirming previous
finding of the use of electric field for the
orientation and growth of neurite (Patel et al.,
1982).
Five days continuous exposure to 50 Hz low
frequency 2 mT magnetic field has significant
effects on cells proliferation leading to a 30%
inhibition of cell growth (Fig. 1). In addition
with the impairment in cell growth
electromagnetic field exposure, generated a
morphological change as reported by the
scanning electron microscopy (SEM) study
showed in Fig. 2.
In particular SEM analysis showed a more
neuronal morphology characterized by the
development of neuritic like processes in the
exposed cells compared to control.
Immunofluorescence using monoclonal
antibodies for the major neurofilament proteins
NF 160 and NF 200 unequivocally demonstrated
an increase in synthesis and accumulation of
these neuronal proteins in the exposed cells.
Taken together all these data support an evident
effect of the electromagnetic field of driving
neuroblastoma cells toward a neuronal
differentiation, which resembles the effect
determined by morphogens, such as retinoic acid.
This induction of cell maturation correlates with
the described inhibition of cell proliferation,
suggesting a profound effect of electromagnetic
field on neuroblastoma cell metabolism.
One mechanism that can be evoked to explain
the action of electromagnetic field on LAN-5
morphological and biochemical effect is an
effect of the field on membrane potential. In our
experiments cells are exposed both to a weak
electric field generated together with a magnetic
component at 50 Hz, and to an intracytoplasmic
very weak electric current induced by the
magnetic component. The action of both electric
component, across the cell membrane, can affect
the membrane potential and consequently could
bias ionic conductance, enzyme activity or
activating genome sequences. Rapid signaling in
neurons requires fast voltage sensitive
mechanisms for closing and opening ions
channel. Anything that interferes with the
membrane voltage can alter channel gating and
comparatively small changes in the gating
properties of a channel can have profound effects.
From a theoretical analysis (King et al., 1998)
demonstrated that for a perfectly spherical cells,
International Journal of Integrative Biology
the electric component of an electro-magnetic
field is effectively shielded by the cell membrane,
in contrast for a non spherical cells ( which is
supposed to be longer compared to its radius)
the cell membrane has only a partial shielding
effect. Since LAN-5 are far to be considered as
perfect sphere, the applied electric component of
the applied electromagnetic field can enter the
cells producing microvolt changes in membrane
potential, consequently being responsible for a
morphological and biochemical effect.
Acknowledgements:
This work has been partially supported by grant
ISPESL B1 n°4/ DIPIA/ 05 and by grant from
NAMED.
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