THE CONSEQUENCES OF LOW FREQUENCY AND INTENSITY

ELECTROMAGNETIC FIELDS ON THE FREQUENCY OF

MICRONUCLEI IN HeLa CELLS

Cosmin Teodor Mihai

, Gabriela Căpraru

, Elena Truţă

, Pincu Rotinberg

and Daniela Gherghel

Faculty of Biology,“Al. I. Cuza” University, Bdul Carol I nr. 20A, Iasi, Romania

Biological Research Institute, Str. Lascar Catargi nr. 47, Iasi, Romania

Keywords: Low frequency electromagnetic fields, HeLa neoplastic cells, Micronuclei assay, Genetic effects.

Abstract: The treatment of HeLa neoplastic cells with low frequency and intensity electromagnetic field has

determined modifications of the micronuclei number, this impact being correlated with the application

manner of the electromagnetic field (continuous or discontinuous). Thus, the continuous electromagnetic

field has reduced the frequency of the micronuclei formation (2.91 ± 0.015 ‰), as compared to the value of

control group (3.93 ± 0.023 ‰), while the discontinuously applied electromagnetic field has increased the

number of micronuclei (4.92± 0.012 ‰). These variations in micronuclei number suggested that low

frequency electromagnetic field interfere in different ways with the genetic material of cancerous cells,

indicating that the cEMF had a protective effect upon DNA molecule, while dcEMF had a genotoxic impact.

Also, the estimation of the micronuclei area has revealed that the area of micronuclei generated by dcEMF

was smaller than that of cEMF.

1 INTRODUCTION

Low frequency and intensity electromagnetic fields

are a part of our life due to the large scale utilization

of computers, home appliances, radio

communications or of the other electrical devices.

Now, everyone is living in a mix of weak electric

and magnetic fields, the impact upon our organism

being still under investigation. The majority of

studies have investigated the possible negative

effects of low frequency and intensity

electromagnetic fields upon humans (Hardell &

Sage, 2008; Heynick, Johnston & Mason, 2003;

Johansson, 2009; Kavet, 1996; Verschaeve et al.,

2006).

A few studies have been oriented towards the

investigation of the impact of the electromagnetic

fields upon the cancerous cells and the evaluation of

consequences of the exposure on this type of cells to

the low frequency and intensity EMF (Falone et al.,

2007; Girgert, Gründker, Emons & Hanf, 2008;

Ronchetto et al., 2004; Tenuzzo et al., 2006). The

findings obtained on this experimental model are

contradictory and full of gaps due to the lack of an

unitary exposure setup, protocol or used biological

material.

The necessity to understand and investigate the

possible functional interactions between

electromagnetic fields and cancerous cells is

determined by the fact that electromagnetic fields

are incriminated to facilitate the carcinogenicity

(Juutilainen & Lang, 1997; Juutilainen, Kumlin &

Naarala, 2006; Mairs et al., 2007; Meltz, 2003;

Simkó, Kriehuber, Weiss & Luben, 1998; Thun-

Battersby, Mevissen & Löscher, 1999). From this

point of view is important to identify the most

dangerous characteristics (frequency, intensity,

amplitude modulations, time of exposure) of

electromagnetic fields to limit the influence upon

healthy or tumor bearing persons. One of the ways

to estimate the impact of electromagnetic fields upon

cells is represented by quantification of micronuclei

occurrence in the exposed cells.

The micronuclei (MN) test is the most frequent

technique used to detect chromosome breakage or

mitotic interference of different xenobiotics, events

thought to be associated with increased risk for

cancer or with a supplementary genetic

destabilization of already mutated cells (like

cancerous ones) (Wolff & Muller, 2005).

440

Mihai C., C

apraru G., Tru¸t

a E., Rotinberg P. and Gherghel D..

THE CONSEQUENCES OF LOW FREQUENCY AND INTENSITY ELECTROMAGNETIC FIELDS ON THE FREQUENCY OF MICRONUCLEI IN HeLa

CELLS.

DOI: 10.5220/0003292804400443

In Proceedings of the International Conference on Biomedical Electronics and Devices (BIODEVICES-2011), pages 440-443

ISBN: 978-989-8425-37-9

 2011 SCITEPRESS (Science and Technology Publications, Lda.)

This work presents an initial investigation about

the impact of the low frequency and intensity

electromagnetic field upon the integrity of HeLa's

genetic material by registration of the variations in

the frequency of the micronuclei occurrence.

2 MATERIAL AND METODS

The biological material used in the in vitro

experiments was represented by mycoplasm-

negative HeLa cellular cultures of human neoplastic

origin.

HeLa cells were cultured in DMEM medium

(Dulbeco's Modified Eagle’s Medium, Biochrom

AG, Germany, FG 0415), supplemented with 10.0%

fetal bovine serum (Sigma, Germany, F9665), 100

µg/mL streptomycin (Biochrom AG, Germany,A

331-26), 100 IU/mL penicillin (Biochrom AG,

Germany, A 321-44) and 50 µg/mL antimycotic

amphotericin B (Biochrom AG, Germany, A 2612),

at a density of 5 x 10

cells / 25 cm

flask, at 37

When the cells reached confluence in the monolayer

stage, the cultures were divided into control and

electromagnetic treated cell cultures.

The electromagnetic field (EMF) of continuous

or discontinuous type (cEMF, dcEMF) was

generated by an IBF magnetodiaflux device. This

presents two circular coils (29 cm in diameter,

placed at a distance of 14.5 cm) disposed on a

cardboard cylinder, which delimits the place where

the culture flasks are maintained during the

electromagnetic treatment. The intensity and

frequency of the generated electromagnetic field

were of 5.5 mT and 100 Hz.

Single EMF was applied continuously or

discontinuously (with breaks of 1 second and action

3 seconds) to the cell cultures, for a period up to 60

minutes. Simultaneous experiments skipping the

electromagnetic field were also performed on the

control cultures. During the real or blind treatment

the cell cultures were removed from the incubator

and placed into magnetodiaflux, where the

temperature has reached up to 30

After the electromagnetic treatment, the cell

cultures were left for another 24 hours in incubator

and then were subjected to quantification of the

micronuclei number. First, the medium was

discarded, the cell layer was rinsed with PBS

(phosphate buffer saline) and than was detached

from the surface of the flasks with a solution of

trypsin (Biochrom AG, Germany).

The cell suspension was resuspended in normal

growth medium and centrifuged at 1800 rpm for 2

minutes. Over the pellet was added fresh and cooled

Carnoy's fixative. The slides preparation was made

by air-dry method and then observed at a Nikon

Eclipse 600 microscope, at the magnification of

4000x.

The frequency of micronuclei was calculated as

the number of micronuclei at 1000 read interphases.

The determination of the micronuclei area was

realized with ImageJ software and the calculation of

the approximate surface of micronuclei was based

on the circle area formula.

The results are expressed as mean ± SE and were

statistically analyzed by Students‘t’ test. The p value

<0.05 was considered significant.

3 RESULTS AND DISCUSSIONS

The evaluation of the consequences determined by

the exposure of HeLa cells to the continuously or

discontinuously applied electromagnetic fields upon

the genetic material integrity was based on the

estimation of the micronuclei occurrence frequency

to 1000 interphases, as compared to the specific MN

frequency of the control group (Figure 1). The

micronuclei occurrence frequency in the control

group was of 3.93 ± 0.023 ‰, value which was

considered by us as reference and was equated to

100%.

Figure 1: Micronuclei occurrence frequency (expressed as

procentual values) in the case of the HeLa neoplastic cell

cultures untreated or treated with continuous or

discontinuous electromagnetic field (100 Hz, 5.5 mT) for

60 minutes. Significantly different from control: *p<0.05,

**p<0.01, ***p<0.001.

As compared to the control group, the

electromagnetic treatment applied continuously has

determined a reduction of the micronuclei frequency

to the value of 2.91 ± 0.015 ‰, corresponding to a

procentual depletion of 25.84%.

THE CONSEQUENCES OF LOW FREQUENCY AND INTENSITY ELECTROMAGNETIC FIELDS ON THE

FREQUENCY OF MICRONUCLEI IN HeLa CELLS

441

Contrary, the discontinuous electromagnetic field

has induced an augmentation of the micronuclei

frequency, the registered value (4.92± 0.012 ‰)

being with 25.20% over the reference.

Another approach of the research was the

estimation of the micronuclei total area, determined

using photos, as an attempt to quantify the amount

of genetic material expelled with micronuclei (Table

1).

Table 1: Mean micronuclei area (μm

) and procentual

variations specific to the control group and to the treated

groups either with cEMF or with dcEMF.

Experimental

group

Micronuclei total area

( μm

)

% variation

Control 13.01 100

cEMF 50.10 385.09

dcEMF 20.59 158.26

The mean area of the micronuclei of the control

group was of 13.01 μm

, while the groups treated

with cEMF and dcEMF, respectively, have presented

a mean area of 50.10 μm

and 20.59 μm

respectively.

Ionizing radiations along with other numerous

chemical mutagens causes structural chromosomal

aberrations, some of them being visible at the light-

microscope level. The aberrations from the level of

chromosome can generate chromosome fragments

without spindle attachment organelles, being called

acentric fragments. When the cell divides, some of

these fragments are excluded from the main

daughter nuclei and form small extra nuclei within

the cytoplasm, either on their own, or in conjunction

with other fragments. Such "micronuclei" (MN) can

appear in the cytoplasm of either, or both, daughter

cells (Savage, 2000)

The electromagnetic field treatment,

continuously or discontinuously applied, has

determined, as compared to the reference value,

fluctuations in the micronuclei occurrence

frequency. These effects, specific for every type of

electromagnetic field, are due to probably different

patterns of interaction between EMFs and the

genetic material of the HeLa cells.

Our presumption regarding the reduced

frequency of micronuclei in the experimental group

treated with cEMF is that cEMF either modifies the

electric charge of DNA molecule, followed by a

stabilization of the genetic material, or intensifies

the activity of molecular mechanisms responsible for

maintaining the DNA integrity. Also, changes in the

electrical charge of DNA macromolecule could

explain the higher area of micronuclei, as compared

to control group and dcEMF. In this case, the

breakage of the DNA structure could occur in the

zones where the fragility of DNA is higher,

generating fragments of genetic material.

The elevated number of micronuclei in cells

treated with dcEMF suggests the weakening of the

DNA integrity either by induction by dcEMF of

microoscilations in the structure of DNA, with the

generation of suplimentary breakages, or by

modification of the intracellular microenvironoment

constants, with brutal and destructive consequences

upon genetic material integrity (Davies et al., 1999).

As in the case of cEMF, the area of micronuclei after

dcEMF action was higher than that of the reference

group, but smaller than of cEMF, suggesting the

release of shorter DNA fragments.

From the above presented data, we can conclude

the existence of an inverse relationship between

frequency of micronuclei and their area, suggesting

different sites of interaction of EMF with DNA, with

immediate consequences upon its integrity.

Our experimental data, obtained in this

experimental frame, are overlapping with those from

the specialty literature (that contains limited

references to cancerous cells) - which has signaled

the sporadic existence (Hee Cho & Chung Won,

2003; Juutilainen, Heikkinen, Soikkeli & Mäki-

Paakkanen, 2007) or even absence (Pasquini et al.,

2003; Speit, Schütz & Hoffmann, 2007; Verschaeve

et al., 2006) of some genotoxic effects induced by

electromagnetic fields, generator of micronuclei.

4 CONCLUSIONS

The EMF interaction with neoplastic HeLa cells has

generated an increase (dcEMF) or a decrease

(cEMF) of the micronuclei occurrence frequency,

suggesting different sites and ways of action upon

the genetic material of cancerous cells.

cEMF expressed a protector effect upon genetic

integrity of HeLa cells, while the dcEMF had a

genotoxic impact upon DNA molecule.

Software analysis of the expelled micronuclei

area has showed that EMF has generated

micronuclei with higher areas than those in the case

of the control group and allowed the establishment

of an inverse relationship between micronuclei

occurrence frequency and their areas.

BIODEVICES 2011 - International Conference on Biomedical Electronics and Devices

442

ACKNOWLEDGEMENTS

This study was possible with financial support from

the Sectoral Operational Programme for Human

Resources Development, project “Developing the

innovation capacity and improving the impact of

research through post-doctoral programmes”,

POSDRU/89/1.5/S/49944

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