Die Details zur Studie sind unter
http://www.bioelectromagnetics.org/doc/ ... tracts.pdf als
8 MB-Datei ab der "pdf-Seite" 132 zu finden.
Ich hab' mal die entpsrechenden 10 Seiten (nur den Text) herauskopiert.
P.S. Im Beitrag war ein Messtechniker zu sehen; im gleichen "Aufwasch" hat er offensichtlich auch Abschirmmaterialien verkauft, ich finde das unseriös - man sollte allenfalls Empfehlungen geben.
Generell ist es ratsam die Abschirmung erst mal zu testen, nicht sellten geht's den Leuten nicht besser (manchmal sogar schlechter) als vorher. Stichwörter dazu:
Reflektionen, CO2, niederfrequente E-Felder, Erd-Magnetfeldverzerrungen bei Abschirmsteinen mit hohem Magnetit-Anteil, Schimmel, ...und wer weiß welche unerforschten Wechselwirkungen mit natürlichen Feldern entstehen ?
Schöne Grüße
Mike
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SESSION 13: SYMPOSIUM I: PRESENTATION OF REFLEX RESULTS
Chair: Franz Adlkofer
13-1
INTRODUCTION IN THE REFLEX PROJECT. F. Adlkofer. Foundation for Behaviour and Environment,
Munich, Germany (Coordinator).
This symposium will present the results of experiments carried out in the EU-funded REFLEX project with data of
EMF exposure on genotoxicity, gene expression, and apoptosis. A project funded by the European Union under the
5th Framework Programme “Quality of Life and Management of Living Resources,” Key Action 4 “Environmental
Health”: QLK4-CT-1999-01574.
STUDENT
13-2
EXPOSURE SYSTEMS, DOSIMETRY AND QUALITY CONTROL. J. Schuderer, W. Oesch*, R. Mertens*, U.
Frauenknecht*, N. Kuster. Foundation for Research on Information Technologies in Society (IT'IS), Swiss Federal
Institute of Technology (ETH), Zurich, CH-8092 Zurich, Switzerland, *Schmid & Partner Engineering AG, CH-8004
Zurich, Switzerland.
OBJECTIVE: The objective was to meet the demands for exposure systems defined in [Kuster et al., 2000] for as
many setups utilized in the REFLEX project as possible. The requirements to be satisfied included field homogeneity,
high dynamic range, minimum temperature and vibration loads, double blind protocols, compact design with a large
loading volume and monitoring of all relevant exposure and environmental parameters during exposure. Furthermore,
the applied signals should represent worst-case environmental exposure conditions for GSM-DCS as well as ELF
magnetic field exposures in terms of field levels and amplitude modulation. Partner laboratories without any setup or
with existing ones that did not meet the standard had to be equipped with novel optimized exposure systems. An
additional requirement was that the quality of the exposure could be remotely monitored and maintained.
METHODS: Exposure setup evaluation, optimization and characterization was performed using numerical and
experimental techniques. The simulation platform SEMCAD including its thermal solver extension served for the
analysis of the RF setups. High resolution FDTD models including details such as precise meniscus models at the
solid/liquid interface as well as all plastic parts of the dishes and dish holder have been accounted for. Coupled
electro-thermal simulations have been performed in order to characterize field and temperature distributions as well as
heat flow processes during exposure. The ELF setups were analyzed and optimized with Mathematics by evaluating
the corresponding analytical equations based on the law of Biot-Savart. This procedure allows the calculation of the
B-field distribution resulting from a spatial current configuration of the coils. The numerical results were carefully
verified using the near-field scanner DASY3 equipped with free space E- and H-field as well as dosimetric field and
temperature probes for the RF system and with a 3-axis Hall meter and an ELF E-field probe for the ELF system
Additionally, vibrations were assessed with a 1-axis accelerometer.
RESULTS: Five RF and four ELF setups have been installed in the laboratories of the consortium. The novel RF
system is based on a dual resonant waveguide system installed in a standard incubator (carrier frequency 1800MHz;
dynamic range: a.1mW/kg - 100W/kg; deviation from uniformity of SAR < 30%; arbitrary modulation signals with a
16k point length and a frequency of less than 15 MHz; prediction of temperature load; blinded design). The ELF setup
consists of a dual 4-coil ELF system which also fits in a standard incubator (dynamic range: 0.02mTrms - 3.6mTrms
with frequency components from DC - 1.5kHz, deviation from B-field uniformity: 1.2%, stability and drift: <0.01%,
vibration; << 0.1g; E-fields < 2V/m; loading volume: 16cm x 16cm x 23cm; shielding: µ-metal box; monitoring of
current (field) and temperature; blinded design). The signal generation for the RF setup is based on an RF signal
generator (Rhode & Schwarz SML02) amplitude modulated by an arbitrary function generator (Agilent 33120A)
combined with a frame generator (SPEAG DCU). For the ELF setup the arbitrary function generator is utilized
together with a custom-made ELF current source. Several predefined GSM and powerline signal types can be applied.
The signals carp be additionally modulated by arbitrary field on/off cycles. The data acquisition system (Agilent
34970A) is used to multiplex the inputs from the field, temperature and airflow sensors. The software, written in
Visual C++, generates complicated environmental exposure schemes consisting of up to four independent random.
STUDENT
13-2 (Cont’d)
events, as well as controls and monitors all devices and sensors. Prior to the experiment, it performs redundant
verification checks to verify that all devices within the system operate within its specifications, During exposure, all
parameters are monitored every 10s. All communications between the computer and the devices are noted with time
stamps. This enables reconstruction of the entire experiment. Permanent quality control of the exposure is realized by
the analysis of the measurement data sent to Zurich in an encoded file.
In addition to these exposure systems, the wire patch cell setup [Laval et al., 1999] and the STUK resonator [Toivo et
al., 2001) are utilized for the experiments conducted at 900 MHz. Furthermore, two coil systems developed by
Insalud, Ramon y Cajal Hospital, Madrid as well as the University of Bologna are in use.
References:
Kuster et al., Recommended minimal requirements and development guidelines for exposure setups for bioexperiments
addressing the health risk concern of wireless communications, Bioelectromagnetics 21: 508 - 514
(2000).
Laval et al., A new in vitro exposure device for the mobile frequency of 900MHz, Bioelectromagnetics 20:1-9 (1999).
Toivo et al., Water-cooled waveguide chambers for exposure of cells in vitro at 900 MHz, proceedings of the 5th
International Congress of EBEA, 62-63 (2001).
13-3
GENOTOXIC EFFECTS OF ELF-EMF ON HUMAN CELLS IN VITRO. H.W. Ruediger*, S. Ivancsits*, E.
Diem*, O. Jahn*. Div. of Occupational Medicine, University of Vienna, Vienna, Austria.
Epidemiological data point to a weak association of extremely-low-frequency (ELF) electromagnetic fields with
increased risk of cancerous diseases albeit without clear dose-effect relations. Therefore, we studied genotoxic effects
of these electromagnetic fields on mainly human cells under controlled conditions in vitro.
METHODS: We used diploid fibroblasts of 6 different donors (age 6-81), blood lymphocytes, melanocytes, and
skeletal muscle cells of human origin and rat granulosa cells. The cells were exposed to an intermittent (5 min on/10
min off) vertical ELF electromagnetic field (50 Hz, sinusoidal, 1-24 h, 1000 µT). Occurrence of DNA single and
double strand breaks was determined using the alkaline and the neutral comet assay. In addition, induction of
micronuclei and chromosomal aberrations were evaluated.
RESULTS: Intermittent fields reproducibly induced a significant increase of DNA strand breaks with exposure time,
being largest at 15-19 hours. Comet assay levels declined thereafter, but did not return to basal levels. Fibroblasts
from older individuals exhibited more single and double strand breaks and, their DNA strand break levels started to
decline later than from younger donors. When exposure was terminated after 12-15 hours the comet factor returned to
basal levels after a repair time of 7 to 9 hours, comprising in a fast repair rate of DNA single strand breaks (< 1 hour)
and a slow repair rate of DNA double strand breaks (> 7 hours). Testing different tissues revealed that, rat granulosa
cells were most sensitive to ELF-EMF exposure (Figure 1) and that melanocytes also responded, but not as high as
fibroblasts or granulosa cells. In contrast, skeletal muscle cells and stimulated lymphocytes did not respond at all.
Exposure conditions producing maximum strand break levels also induced a significant increase of micronuclei and
chromosomal aberrations in human fibroblasts. In addition, a dose dependent response of comet tailfactors, beginning
already at 35 µT, could be demonstrated.
CONCLUSION: The time and dose dependent induction of DNA damage, which varied in cells of different tissues
and donors, may reflect specific differences in DNA repair efficiency of ELF-EMF induced damage. In summary, our
data strongly indicate a genotoxic and clastogenic potential of intermittent ELF-EMF.
13-3
GENOTOXIC EFFECTS OF ELF-EMF ON HUMAN CELLS IN VITRO. H.W. Ruediger*, S. Ivancsits*, E.
Diem*, O. Jahn*. Div. of Occupational Medicine, University of Vienna, Vienna, Austria.
Epidemiological data point to a weak association of extremely-low-frequency (ELF) electromagnetic fields with
increased risk of cancerous diseases albeit without clear dose-effect relations. Therefore, we studied genotoxic effects
of these electromagnetic fields on mainly human cells under controlled conditions in vitro.
METHODS: We used diploid fibroblasts of 6 different donors (age 6-81), blood lymphocytes, melanocytes, and
skeletal muscle cells of human origin and rat granulosa cells. The cells were exposed to an intermittent (5 min on/10
min off) vertical ELF electromagnetic field (50 Hz, sinusoidal, 1-24 h, 1000 µT). Occurrence of DNA single and
double strand breaks was determined using the alkaline and the neutral comet assay. In addition, induction of
micronuclei and chromosomal aberrations were evaluated.
RESULTS: Intermittent fields reproducibly induced a significant increase of DNA strand breaks with exposure time,
being largest at 15-19 hours. Comet assay levels declined thereafter, but did not return to basal levels. Fibroblasts
from older individuals exhibited more single and double strand breaks and, their DNA strand break levels started to
decline later than from younger donors. When exposure was terminated after 12-15 hours the comet factor returned to
basal levels after a repair time of 7 to 9 hours, comprising in a fast repair rate of DNA single strand breaks (< 1 hour)
and a slow repair rate of DNA double strand breaks (> 7 hours). Testing different tissues revealed that, rat granulosa
cells were most sensitive to ELF-EMF exposure (Figure 1) and that melanocytes also responded, but not as high as
fibroblasts or granulosa cells. In contrast, skeletal muscle cells and stimulated lymphocytes did not respond at all.
Exposure conditions producing maximum strand break levels also induced a significant increase of micronuclei and
chromosomal aberrations in human fibroblasts. In addition, a dose dependent response of comet tailfactors, beginning
already at 35 µT, could be demonstrated.
CONCLUSION: The time and dose dependent induction of DNA damage, which varied in cells of different tissues
and donors, may reflect specific differences in DNA repair efficiency of ELF-EMF induced damage. In summary, our
data strongly indicate a genotoxic and clastogenic potential of intermittent ELF-EMF.
© The Bioelectromagnetics Society
25th Annual Meeting, June 2003
130
13-4
GENOTOXIC EFFECTS OF RF-EMF ON CULTURED CELLS IN VITRO. K. Schlatterer, R. Gminski, R.
Tauber, R. Fitzner. Institut für Klinische Chemie und Pathobiochemie, Universitätsklinikum Benjamin Franklin, Freie
Universitaet Berlin, Hindenburgdamm 30, D-12200 Berlin, Germany
OBJECTIVES: The project is aimed at the investigation whether RF-EMF may have genotoxic effects in human cell
lines by causing damage to DNA either directly or indirectly.
METHODS: Damage to DNA by RF-EMF was studied in the human promyelocytic cell line HL-60 and was
assessed by use of the alkaline single cell gel electrophoresis (comet) assay and of the cytokinesis-block in vitro
micronucleus (MN) assay. The alkaline comet assay was carried out as described by Singh et al. 1988 according to the
guidelines developed by Tice at al. 1990, 2000; Fairbairn et al. 1995 and Klaude et al. 1996. The MN assay was
performed according to Darroudi and Natarajan (1991) and to the guidelines developed by Fenech (1986, 1993, 1994,
2000) and Garriott et al. (2002). Experiments were performed in a highly standardized RF-EMF exposure setup (N.
Kuster, Swiss Federal Institute of Technology, Zurich, Switzerland). HL-60 cells were cultured in RPMI 1640 with
10% FCS under temperature- and pH-controlled conditions, 37°C, in an atmosphere with 5%CO2 at an initial seeding
density of 7.5 x 105 cells per 35 mm petri dish. Cells were RF-EMF exposed (1800 MHz, continuous wave, 24 h) at
specific absorption rates (SAR) ranging from 0 to 2.0 W/kg or were sham-exposed under blinded conditions. Apart
from RF-EMF- and sham-exposure, positive (6 MeV γ-irradiation, hydrogen peroxide) and negative incubator
controls were examined. In addition, different exposure periods (2 h to 72 h) and RF-signals (1800 MHz, SAR 1,3
W/kg: continuous wave 5 min on/10 min off; continuous wave 217 Hz pulse; GSM talk) were examined. In order to
exclude possible in vitro cytotoxic effects of RF-EMF, cell viability was monitored with the trypan blue exclusion
method, flow cytometry tests that detect cells with reduced viability by binding of propidium iodide to DNA, and the
MTT assay. In the experiments on micronuclei induction, the ratio of binucleated (BNC) against total, i.e. mono-, bi-,
tri- and tetranucleated cells was determined as a measure of cell division and cell cycle progression. Induction of
apoptosis was examined by use of the TUNEL assay and the Annexin-V assay employing flow cytometry.
Differences were tested for significance employing the Student´s t-test.
RESULTS: RF-EMF (1800 MHz, continuous wave, 24h) exposure caused an increase both of MN frequencies and
DNA strand breaks in HL-60 cells in a SAR-dependent manner. Whereas at a SAR of 1.0 W/kg no significant
difference of MN frequencies or DNA strand breaks was found as compared to sham controls, MN frequencies were
almost tripled and and DNA strand breaks doubled at SAR of 1.3 W/kg and 1.6 W/kg. At higher SAR levels of 2.0
W/kg the induction of MN and of DNA strand breaks was less expressed. At the exposure conditions tested, no in
vitro cytotoxic effect of RF-EMF and no induction of apoptosis could be detected.
CONCLUSION: The findings clearly show that RF-EMF at the exposure conditions stated above cause the induction
of micronuclei as well as DNA strand breaks in HL-60 cells and, hence might have clastogenic effects in human cell
lines.
Supported by the European Union (QLK4-CT-1999-01574, REFLEX)
© The Bioelectromagnetics Society
25th Annual Meeting, June 2003
131
13-5
EFFECTS OF ELF-EMF ON GENE EXPRESSION OF VARIOUS CELL LINES. A.M. Wobus1, T. Nikolova1,
J. Czyz1, A. Rolletschek1, K. Meier1, T. Tölle1, S. Sommerfeld1, F. Clementi2, C. Gotti2, D. Fornasari2, R. Benfante2,
F, Bersani3, P. Mesirca3, C. Agostini3, C. Ventura4, M. Maioli4,Y. Azara4. 1Institute for Plant Genetics and Crop Plant
Research (IPK), D-06466 Gatersleben, Germany; 2Department of Pharmacology, University of Milan, I-20129 Milan,
Italy; 3Department of Physics, University of Bologna, I-40127 Bologna, Italy, and 4Department of Biochemistry,
University of Sassari, I-07100 Sassari, Italy.
OBJECTIVES: To determine whether in vitro exposure to 50 Hz ELF-EMF of various types of cell lines causes
changes in gene expression: (1) IPK/Germany: gene involved in cell proliferation and in early neuronal differentiation
processes, early genes, stress response genes, genes involved in apoptotic pathways. (2) Dept. Pharm./Italy: neuronal
nicotinic receptors subunits genes, gene and protein expression of the Dopamine beta-hydroxylase (DβH), the limiting
enzyme for the synthesis of norepinephrin, and of two homeodomain transcription factors (Phox 2a and Phox 2b)
responsible for the development of the autonomic nervous system and in the CNS of all noradrenergic centers (i.e.
locus coeruleus), where they are the main regulators of the expression of the DβH. (3 and 4) Dept. Phys./Italy: genes
involvement in the early commitment to the cardiac lineage of pluripotent embryonic stem (ES cells): GATA-4,
encoding for a zinc finger containing transcription factor; Nkx-2.5, encoding for a homeodomain essential for
cardiogenesis in different animal species.
MATERIAL AND METHODS: Three different experimental protocols were used: (1) IPK: embryonic stem (ES)
cells (p53+/+ and p53-/-) and neuronal progenitor cells derived from pluripotent ES cells, under different exposure
conditions: intermittent (5 min ON / 30 min OFF or 5 min ON / 10 min OFF) and continuous for 6 h and 48 h at
different field intensities (0.1 mT, 1 mT, 2 mT, 2.3 mT). Gene expression was analyze by RT-PCR. (2) Dep.t Pharm.:
neuroblastoma cell line SY5Y under different exposure conditions, in particular intermittent (5 min ON/5 min OFF,
16h) or continuous exposure for 16h and 48h at flux densities of 1 mT and 2 mT. (3 and 4) Dept. Phys./Italy: GTR1
ES cells, a derivative of R1 ES cells, bearing the puromycin-resistance gene driven by the cardiomyocyte-specific
MHC promoter (GTR1 cells were kindly provided by Dr. William L Stanford (University of Toronto and Centre for
Modeling Human Disease, Canada). To induce cardiac differentiation, cells were cultured in DMEM lacking
supplemental LIF. When spontaneous contractile activity was noticed, puromycin (2 µg/ml) was added to eliminate
non-cardiomyocytes. EBs, collected at several stages after plating, as well as puromycin-selected cells were processed
for gene expression and immunofluorescence analyses. Following LIF removal and throughout puromycin selection,
GTR1 cells were also exposed to magnetic fields (MF) (50 Hz, 0.8 mTrms).
RESULTS: (1) IPK: a) an intermittent exposure (5 min ON / 30 min OFF) short term (6h) high intensity (2.3 mT)
exposure to ELF-EMF resulted in a statistically significant up-regulation of 3 out of 5 tested regulatory genes (egr-1,
p21, c-jun) in p53-deficient ES cells, whereas in both cell lines a 48h exposure did not affect gene expression levels
during the differentiation time; b) for the intermittent and continuous exposure at lower flux densities (0.1 mT, 1 mT)
for 6h and 48h no significant effects with regard to the expression of regulatory genes in ES cells were observed; c) an
up-regulation of RNA transcript levels of the anti-apoptotic gene bcl-2 was observed in neuronal progenitor cells at 2
mT, intermittent (5 min ON / 30 min OFF) 48h exposure; d) the up-regulation of egr-1, p21 and c-jun mRNA in p53-
deficient ES cells are short term effects and do not persist during differentiation. (2) Dept. Pharm.: none of the
conditions tested showed an effect on nAchRs subunits, DßH, Phox2a and Phox2b, both at transcriptional and protein
level. (3 and 4) Dept. Phys.: RNase protection analysis of targeted mRNA revealed that exposure to EMF of GTR1 ES
cells following LIF removal and throughout puromycin selection led to a remarkable increase in GATA-4 gene
expression both at the stage of EBs formation and in puromycin-selected cardiomyocytes. Nuclear run-off
experiments performed in nuclei isolated from undifferentiated cells, as well as in nuclei from EBs and ES-derived
cardiomyocytes revealed that the EMF effects occurred at the transcriptional level.
CONCLUSIONS: The results suggest that 50 Hz ELF-EMF may affect gene expression in specific cell types.
13-6
CELLULAR RESPONSE TO MOBILE PHONE RADIATION APPEARS TO BE CELL GENOTYPEDEPENDENT.
D. Leszczynski1, F. Adlkofer2, J. Czyz3, K. Guan3, K. Jokela4, T. Kallonen1, R. Kuokka1, N. Kuster5,
A. Meister3, J. Reivinen1, J. Schuderer5, A.P. Sihvonen4, T. Toivo4, A.M. Wobus3, Q. Zeng3. 1Bio-NIR Research
Group, STUK-Radiation and Nuclear Safety Authority, Helsinki, Finland, 2VerUm Foundation, Munich, Germany,
3In vitro Differentiation Group, Institute for Plant Genetics (IPK), Gatersleben, Germany, 4NIR Laboratory, STUKRadiation
and Nuclear Safety Authority, Helsinki, Finland, 5IT’IS, Swiss Federal Institute of Technology (ETH),
Zurich, Switzerland.
BACKGROUND: Present status of the research suggests that the mobile phone radiation induces biological effects,
even though the biophysical mechanism still remains unknown. The use of high-throughput screening techniques
(HTST) of transcriptomics and proteomics will allow an educated prediction of all potentially hazardous effects of
mobile phone radiation. Also, HTST might be an efficient tool in determining similarities and differences in gene and
protein expression between exposure-responding and non-responding cells.
OBJECTIVE: To determine whether in vitro exposure of cells to mobile phone radiation causes changes in gene and
protein expression and whether these changes are cell genotype-dependent.
Material and Methods: STUK/Finland: human endothelial cell lines EA.hy926 and EA.hy926v1 (derived from
EA.hy926 by subcloning) were exposed for 1h to either 900 MHz or 1800 MHz GSM signal, at an average SAR 2.4
W/kg and SAR 2.0 W/kg, respectively. Temperature of cell cultures during the exposure period remained at 37 ±
0.3oC (900 MHz) and 37 ± 0.1oC (1800 MHz). Extracts of cellular mRNA and proteins were analyzed using cDNA
Expression Array (gene expression) and 2D-electrophoresis with PDQuest analysis (protein expression), respectively.
IPK/Germany: mouse embryonic pluripotent stem cells (p53+/+ and p53-/-) were exposed for 6h and 48h (5min.
on/30min. off) to 1800 MHz GSM signal at an average SAR 1.5 W/kg and 2.0 W/kg. Temperature of cell cultures
during the exposure period remained at 37 ± 0.1oC. Gene expression was analyzed by RT-PCR.
RESULTS: STUK/Finland: RF-EMF-induced changes in gene expression in human endothelial cell line EA.hy926
and its slow-growing variant EA.hy926v1 were examined. Cell cycle, apoptosis and proliferation ratio analyses have
demonstrated that both cell lines, in spite close relationship, differ in their growth pattern. Stress response to RF-EMF
radiation was different in both cell lines. Hsp27 expression and phosphorylation increased more in EA.hy926 cells as
compared with slow-growing EA.hy626v1 cells. Gene expression in both cell lines was altered differently in the
response to the RF-EMF exposure. In the EA.hy926 cells nearly 40 genes and in EA.hy926v1 less than 20 genes have
increased or decreased their expression by at least 3-folds. Among the affected genes were these involved in
regulation of cancer development, cell proliferation and apoptosis. In the fast growing variant was observed decline in
the expression of such genes involved in execution of apoptosis as p53 and genes involved in regulation of TNFα and
FAS pathways of programmed cell death, whereas anti-apoptotic bcl-2 expression remained unaltered. In the slow
growing variant the expression of p53 did not decline significantly whereas the anti-apoptotic bcl-2 expression
declined over 5-folds following RF-EMF exposure. Furthermore, expression of cancer-development-regulating rasrelated
genes was altered differently in both cell lines. In EA.hy926 cells ras-related genes were down-regulated
whereas in EA.hy926v1 cells they were up-regulated. Interestingly, in EA.hy926 cell line was observed an increase in
the expression of genes involved in DNA-repair process, what adds to the controversy of whether or whether not, RFEMF
is able to cause indirect DNA damage?
IPK/Germany: Exposure of p53+/+ stem cells to mobile phone radiation did not affect expression of genes involved in
regulation of stress response, cell cycle and apoptosis. However, the p53-/- stem cells responded to irradiation by a
small, but significant, up-regulation of c-jun, c-myc and p21 mRNA levels. Also, in p53-/- but not in p53+/+ cells, RFEMF
exposure has induced an increase in the expression of stress protein hsp70.
CONCLUSIONS: Our results suggest that mobile phone radiation causes changes in expression of various genes and
proteins. These changes were detected using varying irradiation times (1 - 48h), SAR levels (1.5 - 2.4 W/kg), signal
frequencies (900 and 1800 MHz) and cell systems (human and mice). This suggests that the observed effects are not a
peculiarity of a particular experimental set-up, but might have a broader biological significance. Finally, the observed
varying responses of cells with different genotype composition (EA.hy926 and EA.hy926v1; p53+/+ and p53-/- stem
cells) suggest that the response and possibly its severity might be influenced by the genotype. However, whether the
detected changes in gene and protein expression will be subsequently followed by physiological responses remains to
be determined.
13-7
EFFECTS OF ELF- AND RF-EMF ON CELL PROLIFERATION AND CELL DIFFERENTIATION. A.M.
Wobus1, M.A. Trillo2, A. Ubeda2, H.A. Kolb3; 1Institute for Plant Genetics (IPK), Gatersleben, Germany; 2Insalud,
Ramon y Cajal Hospital, Madrid, Spain; 3Institute of Biophysics, University of Hannover, Herrenhaeuserstrasse 2, D-
30419 Hannover, Germany
OBJECTIVE: Effects of EMF on growth and differentiation has been reported for several cell lines like murine
osteosarcoma cells [1] or neuronal stem cells [2]. In this study various cell lines and methods were used to evaluate
effects of EMF on cell differentiation and growth. An influence of ELF-EMF and RF-EMF on differentiation and
specialisation of neuronal precursor cells deriving from embryonic stem cells of mice was investigated by research
group 1. In addition, effects of ELF-EMF and RF-EMF on differentiation were studied on a human neuroblastoma
cell line and neural stem cells by research group 2. For analysis of interaction of cell growth with ELF-EMF exposure
cultured granulosa cells and fibroblasts were used by research group 3.
METHODS: Different experimental protocols were used: Embryonic stem cells from mice on the way to neuronal
differentiation were exposed to ELF-EMF (50 Hz, 2 mT, 48h, 5 min on/30 min off) and in preliminary studies also to
RF-EMF (1,5 W/kg, 48h, 5 min on/30 min off) at the stage when first progenitor cells proliferate and differentiate.
The expression of specific genes such as Nestin, GFAP, En-1, Nurr1 and TH that are upregulated by survival
promoting factors were analysed by RT-PCR (Reseach group 1). Human neuroblastoma NB69 were exposed to either
to ELF-EMF (50 Hz, 10 or 100 microT, 42 or 90h, 3h on/3 h off) or RF-EMF (1800MHz, GSM Basic, 2.0 W/kg, 5
min on/ 10 min off, 21h) Immunocytochemical and in situ hybridization studies were carried out 4 days after plating
(Research group 2). Cellular effects through ELF-EMF exposure (50 Hz powerline, 1.0 mT, 2.3 mT, 15 to 20h, 5 min
on, 10 min off) were studied in cultured GFSHR-17 granulosa cells of rat by recording [Ca2+]i , since [Ca2+]i is an
important indicator for effects on intracellular signal transduction which in turn characterise physiological/ pathophysiological
cell states. Cytoplasmic free calcium ([Ca2+]i) was determined by fluorescence spectroscopy of FURAAM
loaded cells. The amplitude and time course of [Ca2+]i was recorded in clusters of up to nine cells
simultaneously (Research group 3) .
RESULTS: By studying the expression of neuronal markers during in vitro differentiation and neuronal specialisation
no significant influence of ELF-EMF and with a high probability also of RF-EMF on neuronal precursor cells and on
differentiated neuronal cells that develop from embryonic stem cells could be observed [3]. This suggests that
neuronal differentiation and specialisation may not be affected by EMF (Research group 1). A growth promoting
effect on neuroblastoma cells was found after a 48h exposure to ELF-EMF at a flux density of 10 and 100 microT, but
not anymore after a 90h exposure at the same flux densities. The influence of RF-EMF on differentiation and growth
of human neuroblastoma cells was investigated by analysis of the expression of fibroblast growth factor (FGF)
receptors R1, R2 and R3. A significant reduction of FGF-R1 positive NB69 cells was observed after exposure to RFEMF
without significantly affecting the number of cell expressing FGF-R2 and FGF-R3. Similar results were
obtained on neural stem cells. For both cell lines, the total number of cells appears not to be affected by RF-EMF
exposure. Preliminary data indicate that the RF-EMF induced change of FGF-R1 expression depends on the age of the
cultured neural stem cells. Exposure to a GSM Talk signal did not have any effect on the expression of FGF-R1, -R2,
-R3 (Research group 2). ELF-EMF exposure did not induce any significant changes on [Ca2+]i , despite the
observation of a significant increase of single and double DNA strand breaks during the exposure period (Research
group 3). A similar observation was obtained for human diploid fibroblasts [4,5].
CONCLUSIONS: The variability of the observed effects on cell proliferation and cell differentiation caused by
exposure to ELF-EMF or RF-EMF which now not at all confirmed is until may indicate that the findings are strongly
dependent on cell phenotype, signal type and frequency, field intensity and application protocol. Comparison of
genotoxic effects through ELF-EMF exposure of cultured granulosa cells and fibroblasts with the corresponding
measurements of [Ca2+]i, as marker of intracellular signal transduction, may suggest that the genomic level rather
than the cellular level should be considered as the major target of ELF-EMF.
References.
Miyagi N, Sato K, Rong Y, Yamamura S, Katagiri H, Kobayashi K, Iwata H. 2000. Effects of PEMF on murine
osteosarcoma cell line: kein Ersatz-resitant (P-glycoprotein-positive) and non-resistant cells. Bioelectromag 21:112-121
Li Y, Zhao L, Xing X, Lou SJ, He C, Lu CL. 2002. Effects of different frequency electromagnetic fields on the
differentiation of midbrain neural stem cells. Space Med Med Eng 15:374-376
Rolletchek A, Chang H, Guan K, Czyz J, Meyer M, Wobus AM. 2001. Differences of embryonic stem cell-derived
dopaminergic neurons is enhanced by survival-promoting factors. Mech Develop 105:93-104
© The Bioelectromagnetics Society
25th Annual Meeting, June 2003
134
13-7 (Cont’d)
Ivancsits S, Diem E, Pilger A, Rüdiger HW, Jahn O. 2002. Induction of DANN strand breaks by intermittent exposure
to extremely-low-frequency electromagnetic fields in human diploid fibroblasts. Mutat Res 519:1-13
Pilger A, Ivancsits S, Diem E, Steffens M, Stranzl T, Kolb HA, Rüdiger HW.2002. No long lasting effects of
intermittent 50 Hz electromagnetic field on cytoplasmic free calcium and mitochondrial membrane potential in human
diploid fibroblasts (submitted)
13-8
EFFECTS OF ELF- AND RF-EMF ON THE APOPTOTIC PROCESS. I. Lagroye1, F. Bersani2, B. Billaudel1,
M. Capri2, J. Czyz3, P-E. Dulou1, K. Guan3, E. Haro1, S. Joenväärä4, R. Kuokka4, N. Kuster5, D. Leszczynski4, A.
Meister3, F. Poulletier de Gannes1, J. Reivinen‡, J. Schuderer5, B. Veyret1, A.M. Wobus3, Q. Zeng3.
1PIOM/Bioelectromagnetics Laboratory, ENSCPB/EPHE, Pessac, France. 2 University of Bologna, Italy. 3In vitro
Differentiation Group, IPK Gatersleben, Germany. 4STUK, Radiation and Nuclear Safety Authority, Helsinki,
Finland. 5ITIS, Zürich, Switzerland
OBJECTIVE: One of the objectives of the REFLEX consortium was to determine whether ELF or RF fields could
influence the apoptotic process in vitro, per se, or after previous treatment with known apoptosis inducers. In 4 out of
13 laboratories involved in the consortium, experiments were performed to assess apoptosis in different cell types
after exposure to ELF or RF fields.
METHODS: Depending on each laboratory’s experience and equipments, different cell types and electromagnetic
signals were used. Exposure lasted from 1 to 48 hours with or without a recovery period. RF exposure included GSM-
900 (frame) and GSM-1800 (basic, DTX, talk ) signals at SARs levels ranging from 0.1 to 2 W/kg. ELF exposure was
done at 50 Hz, at 0.1, 1 and 2.3 mT. Intermittence was introduced in some experiments.
Chemicals that were used to induce apoptosis were 2-deoxy-D-ribose, staurosporine, TNFα or camptothecin,
depending on the cell system used.
Markers that were used to detect apoptosis were (i) the loss of mitochondrial transmembrane potential, (ii) the
presence of phosphatidylserine on the outer leaflet of the plasma membrane, (iii) the appearance of a sub-G1
population, (iv) caspase-3 activity. Expression of apoptosis - related genes (bcl-2, hsp70, p21) was investigated using
Northern blotting.
RESULTS: RF fields (GSM-900 and GSM-1800) did not significantly affect apoptosis in cells of the immune system
(human peripheral blood mononuclear cells from young and elderly people, human U937 cells), in EA.hy926 human
endothelial cells and in cells from the nervous system (human U87 glioblastoma cells, human SHSY5Yneuroblastoma
cells, rat C6 glioma cells rat primary neurons (granule cells) and glial cells). The signaling
pathways involving blc-2 was not affected in either p53+/+ or p53-/- embryonic stem cells tested after exposure to RF
fields. When tested, the signal intermittence, the age of donors (human peripheral blood mononuclear cells), as well as
the cell genetics (embryonic stem cells) were shown to not substantially affect the occurrence of apoptosis. Moreover,
apoptosis induced by chemicals was not affected by further exposure to RF fields.
ELF magnetic fields were able to up-regulate bcl-2 mRNA expression when p53+/+ neuronal progenitors (embryonic
stem cells) were exposed for 48 hours to discontinuous ELF magnetic fields at the highest magnetic flux density
tested (2.3 mT, 5 min. ON, 30 min. OFF). This effect was transient since the level of bcl-2 went back to its basal level
after 18 hours of recovery. However, mRNA levels of other apoptosis-related genes (hsp70, p21), neuronal genes
(TH, Nurr1 and En-1) and tissue specific genes (GFAP, Nestin) were not affected by the applied magnetic field.
CONCLUSIONS: Overall, the results from the REFLEX programme do not bring evidence that RF fields could
interfere with the integrative apoptotic process in cultured cells. Although ELF magnetic fields were shown to upregulate
the anti-apoptotic bcl-2 gene at high magnetic flux density, no overall effect was detected on ESC neuronal
differentiation process, suggesting that the effect is compensated in some way.
The REFLEX project is supported by the 5th Framework Programme of the European Union.
CONCLUSION
SUMMARY OF THE FINDINGS OBTAINED IN THE REFLEX PROJECT AND FUTURE
PERSPECTIVES. F. Adlkofer, VERUM Foundation, Pettenkoferstr. 33, D-80336 Muenchen, Germany
INTRODUCTION: Exposure to electromagnetic fields (EMF) is a controversial topic throughout the industrial
world. Despite the fact that possible effects of EMF on processes controlling key cell functions have not been
investigated adequately to date, it has become a matter of concern that the rapidly increasing exposure to EMF may
cause, in addition to functional disorders, cancer and neurodegenerative diseases. This fear has triggered controversies
in communities especially in Europe with its high density of population and industry and the omnipresence of EMF in
infrastructures and consumer products. These controversies are affecting the siting of facilities, leading people to
relocate, schools to close or power lines to be resited, all at great expense. So far epidemiological and animal studies
have generated conflicting data and, thus, uncertainty regarding possible adverse health effects. Clearly, mere
continuation or replication of this kind of research without the introduction of innovative concepts will prolong the
uncertainty as to whether EMF does, or does not, represent a health risk. The causality between EMF exposure and
disease can never be regarded as proven without knowledge and understanding of the basic mechanisms possibly
triggered by EMF. To search for those basic mechanisms, state-of-the-art methods recently developed in toxicology
and molecular biology are being employed in the REFLEX project to investigate cellular and subcellular responses of
living cells exposed to EMF in vitro.
OBJECTIVES: The REFLEX project is a cooperation of 12 research institutions from 7 European countries. It is
funded by the EC within the 5th Framework Programme. By using the most powerful molecular biological tools
currently available, the REFLEX project will contribute to a better understanding of the biological effects of EMF, the
prerequisite for an objective assessment of potential health hazards and perhaps also of potential therapeutic effects.
The REFLEX project is aimed at the investigation of EMF-induced cellular, subcellular and/or molecular processes.
Although most, if not all chronic diseases, among them cancer, are of extremely diverse and heterogeneous origin, it
is suggested that underlying this variability lies a relatively small number of critical events, i.e. gene mutations,
deregulated cell proliferation and suppressed or exaggerated cell death or apoptosis. The convergence of these critical
events is required for the development of any and all chronic diseases. Gene mutations, cell proliferation and
apoptosis are caused by or result in an altered gene and protein expression. To reach the goal envisaged in the
REFLEX project, the following priority research areas have been selected:
1. Direct and indirect genotoxic effects of EMFs. 2. Effects of EMFs on differentiation and function of embryonic
stem cells. 3. Effects of EMFs on gene expression and protein targeting. 4. Effects of EMFs on the immune system. 5.
Effects of EMFs on cell transformation and apoptosis. To be able to compare the results of investigations carried out
in the different laboratories and to ensure the conclusiveness of the data obtained in the studies, the conditions of
exposure to EMFs are strictly controlled and the data are evaluated blindly.
RESULTS: Based on the data related to ELF-EMF, a genotoxic effect on primary cell cultures of human fibroblasts
was convincingly demonstrated. DNA strand breaks at a significant level followed by various types of chromosomal
aberrations are produced in a dose related manner. DNA damages can be demonstrated at a flux density as low as 35
µT, they are, however, rapidly and completely repaired, but - as it seems - not entirely errorfree. A strong correlation
between the increase in DNA strand breaks and the increase in micronucleus frequencies and chromosome aberrations
was observed. Since ELF-EMF did not affect cytoplasmic free calcium and mitochondrial membrane potential in
human diploid fibroblasts, it is suggested, that DNA strand breaks and the increase in micronuclei frequencies induced
by ELF-EMF are unlikely to be caused by intracellular changes which affect the free calcium level. Furthermore,
there is evidence that ELF-EMF influences the expression of genes in embryonic stem cells of mice, especially if
these cells are deficient of the p53 gene.
With respect to RF-EMF, data obtained clearly demonstrate that RF radiation produces genotoxic effects in living
cells, too. Single and double DNA strand breaks and the frequency in micronuclei are increased in primary cell
cultures of human fibroblasts, in granulosa cells of rats and in HL 60 cells, a promyelocytic hematopoetic human cell
line, after exposure to RF-EMF at a SAR at or even below 2 W/kg. In addition, RF-EMF at a SAR of 1.5 W/kg is able
to up-regulate the expression of early genes, such as hsp70, p21, c-jun and c-myc, in p53-deficient embryonic stem
cells of mice, but not in their healthy wildtype cells. After lowering the SAR value no influence on the mRNA levels
of these genes was observed anymore. Additional studies demonstrate that RF-EMF alters the expression of
numerous, yet largely unidentified proteins in a transformed human endothelial cell line.
SUMMARY AND CONCLUSIONS: While single and double DNA strand breaks and their repair to our knowledge
never have been shown in in vitro studies before as clearly as in the REFLEX project, the modulation of the gene
expression by EMFs has already been observed by several other authors. No evidence was obtained in the REFLEX
project to date, that these findings are in any way related to the vital processes of cell proliferation and apoptosis. The
further elucidation of these biological effects should help to determine whether or not any health hazards might be
associated with the use of EMF emitting devices at the presently allowed safety standards. There is no doubt, that the
scientific database of these standards is rather poor. Therefore, it cannot be excluded at present, that it might be
necessary one day to adjust them to a new reality. Our findings might show a way how science-based safety standards
could be created in the near future.
Fortunately, the present picture of EMF in vitro research, although far from being complete, is good enough to draw
conclusions where to future research efforts should be directed. The high-throughput screening techniques of
genomics, transcriptomics, and proteomics should enable the unravelling of biological effects of EMF that might
interfere with the functioning of cells and organs. Adequate research combined with adequate funding presupposed,
the probability is rather high that the basic scientific question can be resolved in a not too far future. This is: What do
the data obtained in the REFLEX project and in other comparable studies to date really mean? Are we dealing with
insignificant and more or less physiological biological effects of EMF or effects of EMF that are adverse to the health
of people?
Composition of the REFLEX Consortium:
Prof. Dr. F. Adlkofer, VERUM, Foundation for Behaviour and Environment, Munich, Germany (Coordinator); Prof.
Dr. R. Tauber, Free University of Berlin, Germany; Prof. Dr. H.W. Ruediger, University of Vienna, Austria; Dr. A.M.
Wobus, Institute for Plant Genetics, Gatersleben, Germany; Dr. A. Trillo, INSALUD, Madrid, Spain; Prof. Dr. D.
Leszczynski, Radiation and Nuclear Safety Authority, Helsinki, Finland; Prof. Dr. H.A. Kolb, University of
Hannover, Germany; Prof. Dr. F. Bersani, University of Bologna, Italy; Dr. I. Lagroye, PIOM, University of
Bordeaux, France; Prof. Dr. N. Kuster, Swiss Federal Institute of Technology, Zurich, Switzerland; Prof. Dr. F.
Clementi, University of Milan, Italy; Dr. C. Maercker, Resource Center/Primary Database, Heidelberg, Germany
Programme of the Symposium (The Symposium offers the opportunity to inform the scientific community on the state
of the REFLEX project after about 30 months of research):
INTRODUCTION IN THE REFLEX PROJECT - F. Adlkofer
EXPOSURE SYSTEMS, DOSIMETRY AND QUALITY CONTROL - J. Schuderer and N. Kuster
GENOTOXIC EFFECTS OF ELF-EMF ON HUMAN CELLS IN VITRO - H.W. Ruediger
GENOTOXIC EFFECTS OF RF-EMF ON CULTURED CELLS IN VITRO - R. Tauber
EFFECTS OF ELF-EMF ON GENE EXPRESSION IN MOUSE CELL LINES- A. Wobus
EFFECTS OF RF-EMF ON GENE AND PROTEIN EXPRESSION IN VITRO - D. Leszczynski
EFFECTS OF ELF- AND RF-EMF ON CELL PROLIFERATION AND CELL DIFFERENTIATION - H.-A. Kolb
EFFECTS OF ELF- AND RF-EMF ON THE APOPTOTIC PROCESS - I. Lagroye
SUMMARY OF THE FINDINGS OBTAINED IN THE REFLEX PROJECT AND FUTURE PERSPECTIVES - F.
Adlkofer and H. Dertinger
A project funded by the European Union under the programme "Quality of Life and Management of Living
Resources", Key Action 4 "Environment and Health": QLK4-CT-1999-01574
Die Details zur Studie sind unter http://www.bioelectromagnetics.org/doc/bems2003-abstracts.pdf als
8 MB-Datei ab der "pdf-Seite" 132 zu finden.
Ich hab' mal die entpsrechenden 10 Seiten (nur den Text) herauskopiert.
P.S. Im Beitrag war ein Messtechniker zu sehen; im gleichen "Aufwasch" hat er offensichtlich auch Abschirmmaterialien verkauft, ich finde das unseriös - man sollte allenfalls Empfehlungen geben.
Generell ist es ratsam die Abschirmung erst mal zu testen, nicht sellten geht's den Leuten nicht besser (manchmal sogar schlechter) als vorher. Stichwörter dazu:
Reflektionen, CO2, niederfrequente E-Felder, Erd-Magnetfeldverzerrungen bei Abschirmsteinen mit hohem Magnetit-Anteil, Schimmel, ...und wer weiß welche unerforschten Wechselwirkungen mit natürlichen Feldern entstehen ?
Schöne Grüße
Mike
------------------------------------------------------------------------------------------------------------------
SESSION 13: SYMPOSIUM I: PRESENTATION OF REFLEX RESULTS
Chair: Franz Adlkofer
13-1
INTRODUCTION IN THE REFLEX PROJECT. F. Adlkofer. Foundation for Behaviour and Environment,
Munich, Germany (Coordinator).
This symposium will present the results of experiments carried out in the EU-funded REFLEX project with data of
EMF exposure on genotoxicity, gene expression, and apoptosis. A project funded by the European Union under the
5th Framework Programme “Quality of Life and Management of Living Resources,” Key Action 4 “Environmental
Health”: QLK4-CT-1999-01574.
STUDENT
13-2
EXPOSURE SYSTEMS, DOSIMETRY AND QUALITY CONTROL. J. Schuderer, W. Oesch*, R. Mertens*, U.
Frauenknecht*, N. Kuster. Foundation for Research on Information Technologies in Society (IT'IS), Swiss Federal
Institute of Technology (ETH), Zurich, CH-8092 Zurich, Switzerland, *Schmid & Partner Engineering AG, CH-8004
Zurich, Switzerland.
OBJECTIVE: The objective was to meet the demands for exposure systems defined in [Kuster et al., 2000] for as
many setups utilized in the REFLEX project as possible. The requirements to be satisfied included field homogeneity,
high dynamic range, minimum temperature and vibration loads, double blind protocols, compact design with a large
loading volume and monitoring of all relevant exposure and environmental parameters during exposure. Furthermore,
the applied signals should represent worst-case environmental exposure conditions for GSM-DCS as well as ELF
magnetic field exposures in terms of field levels and amplitude modulation. Partner laboratories without any setup or
with existing ones that did not meet the standard had to be equipped with novel optimized exposure systems. An
additional requirement was that the quality of the exposure could be remotely monitored and maintained.
METHODS: Exposure setup evaluation, optimization and characterization was performed using numerical and
experimental techniques. The simulation platform SEMCAD including its thermal solver extension served for the
analysis of the RF setups. High resolution FDTD models including details such as precise meniscus models at the
solid/liquid interface as well as all plastic parts of the dishes and dish holder have been accounted for. Coupled
electro-thermal simulations have been performed in order to characterize field and temperature distributions as well as
heat flow processes during exposure. The ELF setups were analyzed and optimized with Mathematics by evaluating
the corresponding analytical equations based on the law of Biot-Savart. This procedure allows the calculation of the
B-field distribution resulting from a spatial current configuration of the coils. The numerical results were carefully
verified using the near-field scanner DASY3 equipped with free space E- and H-field as well as dosimetric field and
temperature probes for the RF system and with a 3-axis Hall meter and an ELF E-field probe for the ELF system
Additionally, vibrations were assessed with a 1-axis accelerometer.
RESULTS: Five RF and four ELF setups have been installed in the laboratories of the consortium. The novel RF
system is based on a dual resonant waveguide system installed in a standard incubator (carrier frequency 1800MHz;
dynamic range: a.1mW/kg - 100W/kg; deviation from uniformity of SAR < 30%; arbitrary modulation signals with a
16k point length and a frequency of less than 15 MHz; prediction of temperature load; blinded design). The ELF setup
consists of a dual 4-coil ELF system which also fits in a standard incubator (dynamic range: 0.02mTrms - 3.6mTrms
with frequency components from DC - 1.5kHz, deviation from B-field uniformity: 1.2%, stability and drift: <0.01%,
vibration; << 0.1g; E-fields < 2V/m; loading volume: 16cm x 16cm x 23cm; shielding: µ-metal box; monitoring of
current (field) and temperature; blinded design). The signal generation for the RF setup is based on an RF signal
generator (Rhode & Schwarz SML02) amplitude modulated by an arbitrary function generator (Agilent 33120A)
combined with a frame generator (SPEAG DCU). For the ELF setup the arbitrary function generator is utilized
together with a custom-made ELF current source. Several predefined GSM and powerline signal types can be applied.
The signals carp be additionally modulated by arbitrary field on/off cycles. The data acquisition system (Agilent
34970A) is used to multiplex the inputs from the field, temperature and airflow sensors. The software, written in
Visual C++, generates complicated environmental exposure schemes consisting of up to four independent random.
STUDENT
13-2 (Cont’d)
events, as well as controls and monitors all devices and sensors. Prior to the experiment, it performs redundant
verification checks to verify that all devices within the system operate within its specifications, During exposure, all
parameters are monitored every 10s. All communications between the computer and the devices are noted with time
stamps. This enables reconstruction of the entire experiment. Permanent quality control of the exposure is realized by
the analysis of the measurement data sent to Zurich in an encoded file.
In addition to these exposure systems, the wire patch cell setup [Laval et al., 1999] and the STUK resonator [Toivo et
al., 2001) are utilized for the experiments conducted at 900 MHz. Furthermore, two coil systems developed by
Insalud, Ramon y Cajal Hospital, Madrid as well as the University of Bologna are in use.
References:
Kuster et al., Recommended minimal requirements and development guidelines for exposure setups for bioexperiments
addressing the health risk concern of wireless communications, Bioelectromagnetics 21: 508 - 514
(2000).
Laval et al., A new in vitro exposure device for the mobile frequency of 900MHz, Bioelectromagnetics 20:1-9 (1999).
Toivo et al., Water-cooled waveguide chambers for exposure of cells in vitro at 900 MHz, proceedings of the 5th
International Congress of EBEA, 62-63 (2001).
13-3
GENOTOXIC EFFECTS OF ELF-EMF ON HUMAN CELLS IN VITRO. H.W. Ruediger*, S. Ivancsits*, E.
Diem*, O. Jahn*. Div. of Occupational Medicine, University of Vienna, Vienna, Austria.
Epidemiological data point to a weak association of extremely-low-frequency (ELF) electromagnetic fields with
increased risk of cancerous diseases albeit without clear dose-effect relations. Therefore, we studied genotoxic effects
of these electromagnetic fields on mainly human cells under controlled conditions in vitro.
METHODS: We used diploid fibroblasts of 6 different donors (age 6-81), blood lymphocytes, melanocytes, and
skeletal muscle cells of human origin and rat granulosa cells. The cells were exposed to an intermittent (5 min on/10
min off) vertical ELF electromagnetic field (50 Hz, sinusoidal, 1-24 h, 1000 µT). Occurrence of DNA single and
double strand breaks was determined using the alkaline and the neutral comet assay. In addition, induction of
micronuclei and chromosomal aberrations were evaluated.
RESULTS: Intermittent fields reproducibly induced a significant increase of DNA strand breaks with exposure time,
being largest at 15-19 hours. Comet assay levels declined thereafter, but did not return to basal levels. Fibroblasts
from older individuals exhibited more single and double strand breaks and, their DNA strand break levels started to
decline later than from younger donors. When exposure was terminated after 12-15 hours the comet factor returned to
basal levels after a repair time of 7 to 9 hours, comprising in a fast repair rate of DNA single strand breaks (< 1 hour)
and a slow repair rate of DNA double strand breaks (> 7 hours). Testing different tissues revealed that, rat granulosa
cells were most sensitive to ELF-EMF exposure (Figure 1) and that melanocytes also responded, but not as high as
fibroblasts or granulosa cells. In contrast, skeletal muscle cells and stimulated lymphocytes did not respond at all.
Exposure conditions producing maximum strand break levels also induced a significant increase of micronuclei and
chromosomal aberrations in human fibroblasts. In addition, a dose dependent response of comet tailfactors, beginning
already at 35 µT, could be demonstrated.
CONCLUSION: The time and dose dependent induction of DNA damage, which varied in cells of different tissues
and donors, may reflect specific differences in DNA repair efficiency of ELF-EMF induced damage. In summary, our
data strongly indicate a genotoxic and clastogenic potential of intermittent ELF-EMF.
13-3
GENOTOXIC EFFECTS OF ELF-EMF ON HUMAN CELLS IN VITRO. H.W. Ruediger*, S. Ivancsits*, E.
Diem*, O. Jahn*. Div. of Occupational Medicine, University of Vienna, Vienna, Austria.
Epidemiological data point to a weak association of extremely-low-frequency (ELF) electromagnetic fields with
increased risk of cancerous diseases albeit without clear dose-effect relations. Therefore, we studied genotoxic effects
of these electromagnetic fields on mainly human cells under controlled conditions in vitro.
METHODS: We used diploid fibroblasts of 6 different donors (age 6-81), blood lymphocytes, melanocytes, and
skeletal muscle cells of human origin and rat granulosa cells. The cells were exposed to an intermittent (5 min on/10
min off) vertical ELF electromagnetic field (50 Hz, sinusoidal, 1-24 h, 1000 µT). Occurrence of DNA single and
double strand breaks was determined using the alkaline and the neutral comet assay. In addition, induction of
micronuclei and chromosomal aberrations were evaluated.
RESULTS: Intermittent fields reproducibly induced a significant increase of DNA strand breaks with exposure time,
being largest at 15-19 hours. Comet assay levels declined thereafter, but did not return to basal levels. Fibroblasts
from older individuals exhibited more single and double strand breaks and, their DNA strand break levels started to
decline later than from younger donors. When exposure was terminated after 12-15 hours the comet factor returned to
basal levels after a repair time of 7 to 9 hours, comprising in a fast repair rate of DNA single strand breaks (< 1 hour)
and a slow repair rate of DNA double strand breaks (> 7 hours). Testing different tissues revealed that, rat granulosa
cells were most sensitive to ELF-EMF exposure (Figure 1) and that melanocytes also responded, but not as high as
fibroblasts or granulosa cells. In contrast, skeletal muscle cells and stimulated lymphocytes did not respond at all.
Exposure conditions producing maximum strand break levels also induced a significant increase of micronuclei and
chromosomal aberrations in human fibroblasts. In addition, a dose dependent response of comet tailfactors, beginning
already at 35 µT, could be demonstrated.
CONCLUSION: The time and dose dependent induction of DNA damage, which varied in cells of different tissues
and donors, may reflect specific differences in DNA repair efficiency of ELF-EMF induced damage. In summary, our
data strongly indicate a genotoxic and clastogenic potential of intermittent ELF-EMF.
© The Bioelectromagnetics Society
25th Annual Meeting, June 2003
130
13-4
GENOTOXIC EFFECTS OF RF-EMF ON CULTURED CELLS IN VITRO. K. Schlatterer, R. Gminski, R.
Tauber, R. Fitzner. Institut für Klinische Chemie und Pathobiochemie, Universitätsklinikum Benjamin Franklin, Freie
Universitaet Berlin, Hindenburgdamm 30, D-12200 Berlin, Germany
OBJECTIVES: The project is aimed at the investigation whether RF-EMF may have genotoxic effects in human cell
lines by causing damage to DNA either directly or indirectly.
METHODS: Damage to DNA by RF-EMF was studied in the human promyelocytic cell line HL-60 and was
assessed by use of the alkaline single cell gel electrophoresis (comet) assay and of the cytokinesis-block in vitro
micronucleus (MN) assay. The alkaline comet assay was carried out as described by Singh et al. 1988 according to the
guidelines developed by Tice at al. 1990, 2000; Fairbairn et al. 1995 and Klaude et al. 1996. The MN assay was
performed according to Darroudi and Natarajan (1991) and to the guidelines developed by Fenech (1986, 1993, 1994,
2000) and Garriott et al. (2002). Experiments were performed in a highly standardized RF-EMF exposure setup (N.
Kuster, Swiss Federal Institute of Technology, Zurich, Switzerland). HL-60 cells were cultured in RPMI 1640 with
10% FCS under temperature- and pH-controlled conditions, 37°C, in an atmosphere with 5%CO2 at an initial seeding
density of 7.5 x 105 cells per 35 mm petri dish. Cells were RF-EMF exposed (1800 MHz, continuous wave, 24 h) at
specific absorption rates (SAR) ranging from 0 to 2.0 W/kg or were sham-exposed under blinded conditions. Apart
from RF-EMF- and sham-exposure, positive (6 MeV γ-irradiation, hydrogen peroxide) and negative incubator
controls were examined. In addition, different exposure periods (2 h to 72 h) and RF-signals (1800 MHz, SAR 1,3
W/kg: continuous wave 5 min on/10 min off; continuous wave 217 Hz pulse; GSM talk) were examined. In order to
exclude possible in vitro cytotoxic effects of RF-EMF, cell viability was monitored with the trypan blue exclusion
method, flow cytometry tests that detect cells with reduced viability by binding of propidium iodide to DNA, and the
MTT assay. In the experiments on micronuclei induction, the ratio of binucleated (BNC) against total, i.e. mono-, bi-,
tri- and tetranucleated cells was determined as a measure of cell division and cell cycle progression. Induction of
apoptosis was examined by use of the TUNEL assay and the Annexin-V assay employing flow cytometry.
Differences were tested for significance employing the Student´s t-test.
RESULTS: RF-EMF (1800 MHz, continuous wave, 24h) exposure caused an increase both of MN frequencies and
DNA strand breaks in HL-60 cells in a SAR-dependent manner. Whereas at a SAR of 1.0 W/kg no significant
difference of MN frequencies or DNA strand breaks was found as compared to sham controls, MN frequencies were
almost tripled and and DNA strand breaks doubled at SAR of 1.3 W/kg and 1.6 W/kg. At higher SAR levels of 2.0
W/kg the induction of MN and of DNA strand breaks was less expressed. At the exposure conditions tested, no in
vitro cytotoxic effect of RF-EMF and no induction of apoptosis could be detected.
CONCLUSION: The findings clearly show that RF-EMF at the exposure conditions stated above cause the induction
of micronuclei as well as DNA strand breaks in HL-60 cells and, hence might have clastogenic effects in human cell
lines.
Supported by the European Union (QLK4-CT-1999-01574, REFLEX)
© The Bioelectromagnetics Society
25th Annual Meeting, June 2003
131
13-5
EFFECTS OF ELF-EMF ON GENE EXPRESSION OF VARIOUS CELL LINES. A.M. Wobus1, T. Nikolova1,
J. Czyz1, A. Rolletschek1, K. Meier1, T. Tölle1, S. Sommerfeld1, F. Clementi2, C. Gotti2, D. Fornasari2, R. Benfante2,
F, Bersani3, P. Mesirca3, C. Agostini3, C. Ventura4, M. Maioli4,Y. Azara4. 1Institute for Plant Genetics and Crop Plant
Research (IPK), D-06466 Gatersleben, Germany; 2Department of Pharmacology, University of Milan, I-20129 Milan,
Italy; 3Department of Physics, University of Bologna, I-40127 Bologna, Italy, and 4Department of Biochemistry,
University of Sassari, I-07100 Sassari, Italy.
OBJECTIVES: To determine whether in vitro exposure to 50 Hz ELF-EMF of various types of cell lines causes
changes in gene expression: (1) IPK/Germany: gene involved in cell proliferation and in early neuronal differentiation
processes, early genes, stress response genes, genes involved in apoptotic pathways. (2) Dept. Pharm./Italy: neuronal
nicotinic receptors subunits genes, gene and protein expression of the Dopamine beta-hydroxylase (DβH), the limiting
enzyme for the synthesis of norepinephrin, and of two homeodomain transcription factors (Phox 2a and Phox 2b)
responsible for the development of the autonomic nervous system and in the CNS of all noradrenergic centers (i.e.
locus coeruleus), where they are the main regulators of the expression of the DβH. (3 and 4) Dept. Phys./Italy: genes
involvement in the early commitment to the cardiac lineage of pluripotent embryonic stem (ES cells): GATA-4,
encoding for a zinc finger containing transcription factor; Nkx-2.5, encoding for a homeodomain essential for
cardiogenesis in different animal species.
MATERIAL AND METHODS: Three different experimental protocols were used: (1) IPK: embryonic stem (ES)
cells (p53+/+ and p53-/-) and neuronal progenitor cells derived from pluripotent ES cells, under different exposure
conditions: intermittent (5 min ON / 30 min OFF or 5 min ON / 10 min OFF) and continuous for 6 h and 48 h at
different field intensities (0.1 mT, 1 mT, 2 mT, 2.3 mT). Gene expression was analyze by RT-PCR. (2) Dep.t Pharm.:
neuroblastoma cell line SY5Y under different exposure conditions, in particular intermittent (5 min ON/5 min OFF,
16h) or continuous exposure for 16h and 48h at flux densities of 1 mT and 2 mT. (3 and 4) Dept. Phys./Italy: GTR1
ES cells, a derivative of R1 ES cells, bearing the puromycin-resistance gene driven by the cardiomyocyte-specific
MHC promoter (GTR1 cells were kindly provided by Dr. William L Stanford (University of Toronto and Centre for
Modeling Human Disease, Canada). To induce cardiac differentiation, cells were cultured in DMEM lacking
supplemental LIF. When spontaneous contractile activity was noticed, puromycin (2 µg/ml) was added to eliminate
non-cardiomyocytes. EBs, collected at several stages after plating, as well as puromycin-selected cells were processed
for gene expression and immunofluorescence analyses. Following LIF removal and throughout puromycin selection,
GTR1 cells were also exposed to magnetic fields (MF) (50 Hz, 0.8 mTrms).
RESULTS: (1) IPK: a) an intermittent exposure (5 min ON / 30 min OFF) short term (6h) high intensity (2.3 mT)
exposure to ELF-EMF resulted in a statistically significant up-regulation of 3 out of 5 tested regulatory genes (egr-1,
p21, c-jun) in p53-deficient ES cells, whereas in both cell lines a 48h exposure did not affect gene expression levels
during the differentiation time; b) for the intermittent and continuous exposure at lower flux densities (0.1 mT, 1 mT)
for 6h and 48h no significant effects with regard to the expression of regulatory genes in ES cells were observed; c) an
up-regulation of RNA transcript levels of the anti-apoptotic gene bcl-2 was observed in neuronal progenitor cells at 2
mT, intermittent (5 min ON / 30 min OFF) 48h exposure; d) the up-regulation of egr-1, p21 and c-jun mRNA in p53-
deficient ES cells are short term effects and do not persist during differentiation. (2) Dept. Pharm.: none of the
conditions tested showed an effect on nAchRs subunits, DßH, Phox2a and Phox2b, both at transcriptional and protein
level. (3 and 4) Dept. Phys.: RNase protection analysis of targeted mRNA revealed that exposure to EMF of GTR1 ES
cells following LIF removal and throughout puromycin selection led to a remarkable increase in GATA-4 gene
expression both at the stage of EBs formation and in puromycin-selected cardiomyocytes. Nuclear run-off
experiments performed in nuclei isolated from undifferentiated cells, as well as in nuclei from EBs and ES-derived
cardiomyocytes revealed that the EMF effects occurred at the transcriptional level.
CONCLUSIONS: The results suggest that 50 Hz ELF-EMF may affect gene expression in specific cell types.
13-6
CELLULAR RESPONSE TO MOBILE PHONE RADIATION APPEARS TO BE CELL GENOTYPEDEPENDENT.
D. Leszczynski1, F. Adlkofer2, J. Czyz3, K. Guan3, K. Jokela4, T. Kallonen1, R. Kuokka1, N. Kuster5,
A. Meister3, J. Reivinen1, J. Schuderer5, A.P. Sihvonen4, T. Toivo4, A.M. Wobus3, Q. Zeng3. 1Bio-NIR Research
Group, STUK-Radiation and Nuclear Safety Authority, Helsinki, Finland, 2VerUm Foundation, Munich, Germany,
3In vitro Differentiation Group, Institute for Plant Genetics (IPK), Gatersleben, Germany, 4NIR Laboratory, STUKRadiation
and Nuclear Safety Authority, Helsinki, Finland, 5IT’IS, Swiss Federal Institute of Technology (ETH),
Zurich, Switzerland.
BACKGROUND: Present status of the research suggests that the mobile phone radiation induces biological effects,
even though the biophysical mechanism still remains unknown. The use of high-throughput screening techniques
(HTST) of transcriptomics and proteomics will allow an educated prediction of all potentially hazardous effects of
mobile phone radiation. Also, HTST might be an efficient tool in determining similarities and differences in gene and
protein expression between exposure-responding and non-responding cells.
OBJECTIVE: To determine whether in vitro exposure of cells to mobile phone radiation causes changes in gene and
protein expression and whether these changes are cell genotype-dependent.
Material and Methods: STUK/Finland: human endothelial cell lines EA.hy926 and EA.hy926v1 (derived from
EA.hy926 by subcloning) were exposed for 1h to either 900 MHz or 1800 MHz GSM signal, at an average SAR 2.4
W/kg and SAR 2.0 W/kg, respectively. Temperature of cell cultures during the exposure period remained at 37 ±
0.3oC (900 MHz) and 37 ± 0.1oC (1800 MHz). Extracts of cellular mRNA and proteins were analyzed using cDNA
Expression Array (gene expression) and 2D-electrophoresis with PDQuest analysis (protein expression), respectively.
IPK/Germany: mouse embryonic pluripotent stem cells (p53+/+ and p53-/-) were exposed for 6h and 48h (5min.
on/30min. off) to 1800 MHz GSM signal at an average SAR 1.5 W/kg and 2.0 W/kg. Temperature of cell cultures
during the exposure period remained at 37 ± 0.1oC. Gene expression was analyzed by RT-PCR.
RESULTS: STUK/Finland: RF-EMF-induced changes in gene expression in human endothelial cell line EA.hy926
and its slow-growing variant EA.hy926v1 were examined. Cell cycle, apoptosis and proliferation ratio analyses have
demonstrated that both cell lines, in spite close relationship, differ in their growth pattern. Stress response to RF-EMF
radiation was different in both cell lines. Hsp27 expression and phosphorylation increased more in EA.hy926 cells as
compared with slow-growing EA.hy626v1 cells. Gene expression in both cell lines was altered differently in the
response to the RF-EMF exposure. In the EA.hy926 cells nearly 40 genes and in EA.hy926v1 less than 20 genes have
increased or decreased their expression by at least 3-folds. Among the affected genes were these involved in
regulation of cancer development, cell proliferation and apoptosis. In the fast growing variant was observed decline in
the expression of such genes involved in execution of apoptosis as p53 and genes involved in regulation of TNFα and
FAS pathways of programmed cell death, whereas anti-apoptotic bcl-2 expression remained unaltered. In the slow
growing variant the expression of p53 did not decline significantly whereas the anti-apoptotic bcl-2 expression
declined over 5-folds following RF-EMF exposure. Furthermore, expression of cancer-development-regulating rasrelated
genes was altered differently in both cell lines. In EA.hy926 cells ras-related genes were down-regulated
whereas in EA.hy926v1 cells they were up-regulated. Interestingly, in EA.hy926 cell line was observed an increase in
the expression of genes involved in DNA-repair process, what adds to the controversy of whether or whether not, RFEMF
is able to cause indirect DNA damage?
IPK/Germany: Exposure of p53+/+ stem cells to mobile phone radiation did not affect expression of genes involved in
regulation of stress response, cell cycle and apoptosis. However, the p53-/- stem cells responded to irradiation by a
small, but significant, up-regulation of c-jun, c-myc and p21 mRNA levels. Also, in p53-/- but not in p53+/+ cells, RFEMF
exposure has induced an increase in the expression of stress protein hsp70.
CONCLUSIONS: Our results suggest that mobile phone radiation causes changes in expression of various genes and
proteins. These changes were detected using varying irradiation times (1 - 48h), SAR levels (1.5 - 2.4 W/kg), signal
frequencies (900 and 1800 MHz) and cell systems (human and mice). This suggests that the observed effects are not a
peculiarity of a particular experimental set-up, but might have a broader biological significance. Finally, the observed
varying responses of cells with different genotype composition (EA.hy926 and EA.hy926v1; p53+/+ and p53-/- stem
cells) suggest that the response and possibly its severity might be influenced by the genotype. However, whether the
detected changes in gene and protein expression will be subsequently followed by physiological responses remains to
be determined.
13-7
EFFECTS OF ELF- AND RF-EMF ON CELL PROLIFERATION AND CELL DIFFERENTIATION. A.M.
Wobus1, M.A. Trillo2, A. Ubeda2, H.A. Kolb3; 1Institute for Plant Genetics (IPK), Gatersleben, Germany; 2Insalud,
Ramon y Cajal Hospital, Madrid, Spain; 3Institute of Biophysics, University of Hannover, Herrenhaeuserstrasse 2, D-
30419 Hannover, Germany
OBJECTIVE: Effects of EMF on growth and differentiation has been reported for several cell lines like murine
osteosarcoma cells [1] or neuronal stem cells [2]. In this study various cell lines and methods were used to evaluate
effects of EMF on cell differentiation and growth. An influence of ELF-EMF and RF-EMF on differentiation and
specialisation of neuronal precursor cells deriving from embryonic stem cells of mice was investigated by research
group 1. In addition, effects of ELF-EMF and RF-EMF on differentiation were studied on a human neuroblastoma
cell line and neural stem cells by research group 2. For analysis of interaction of cell growth with ELF-EMF exposure
cultured granulosa cells and fibroblasts were used by research group 3.
METHODS: Different experimental protocols were used: Embryonic stem cells from mice on the way to neuronal
differentiation were exposed to ELF-EMF (50 Hz, 2 mT, 48h, 5 min on/30 min off) and in preliminary studies also to
RF-EMF (1,5 W/kg, 48h, 5 min on/30 min off) at the stage when first progenitor cells proliferate and differentiate.
The expression of specific genes such as Nestin, GFAP, En-1, Nurr1 and TH that are upregulated by survival
promoting factors were analysed by RT-PCR (Reseach group 1). Human neuroblastoma NB69 were exposed to either
to ELF-EMF (50 Hz, 10 or 100 microT, 42 or 90h, 3h on/3 h off) or RF-EMF (1800MHz, GSM Basic, 2.0 W/kg, 5
min on/ 10 min off, 21h) Immunocytochemical and in situ hybridization studies were carried out 4 days after plating
(Research group 2). Cellular effects through ELF-EMF exposure (50 Hz powerline, 1.0 mT, 2.3 mT, 15 to 20h, 5 min
on, 10 min off) were studied in cultured GFSHR-17 granulosa cells of rat by recording [Ca2+]i , since [Ca2+]i is an
important indicator for effects on intracellular signal transduction which in turn characterise physiological/ pathophysiological
cell states. Cytoplasmic free calcium ([Ca2+]i) was determined by fluorescence spectroscopy of FURAAM
loaded cells. The amplitude and time course of [Ca2+]i was recorded in clusters of up to nine cells
simultaneously (Research group 3) .
RESULTS: By studying the expression of neuronal markers during in vitro differentiation and neuronal specialisation
no significant influence of ELF-EMF and with a high probability also of RF-EMF on neuronal precursor cells and on
differentiated neuronal cells that develop from embryonic stem cells could be observed [3]. This suggests that
neuronal differentiation and specialisation may not be affected by EMF (Research group 1). A growth promoting
effect on neuroblastoma cells was found after a 48h exposure to ELF-EMF at a flux density of 10 and 100 microT, but
not anymore after a 90h exposure at the same flux densities. The influence of RF-EMF on differentiation and growth
of human neuroblastoma cells was investigated by analysis of the expression of fibroblast growth factor (FGF)
receptors R1, R2 and R3. A significant reduction of FGF-R1 positive NB69 cells was observed after exposure to RFEMF
without significantly affecting the number of cell expressing FGF-R2 and FGF-R3. Similar results were
obtained on neural stem cells. For both cell lines, the total number of cells appears not to be affected by RF-EMF
exposure. Preliminary data indicate that the RF-EMF induced change of FGF-R1 expression depends on the age of the
cultured neural stem cells. Exposure to a GSM Talk signal did not have any effect on the expression of FGF-R1, -R2,
-R3 (Research group 2). ELF-EMF exposure did not induce any significant changes on [Ca2+]i , despite the
observation of a significant increase of single and double DNA strand breaks during the exposure period (Research
group 3). A similar observation was obtained for human diploid fibroblasts [4,5].
CONCLUSIONS: The variability of the observed effects on cell proliferation and cell differentiation caused by
exposure to ELF-EMF or RF-EMF which now not at all confirmed is until may indicate that the findings are strongly
dependent on cell phenotype, signal type and frequency, field intensity and application protocol. Comparison of
genotoxic effects through ELF-EMF exposure of cultured granulosa cells and fibroblasts with the corresponding
measurements of [Ca2+]i, as marker of intracellular signal transduction, may suggest that the genomic level rather
than the cellular level should be considered as the major target of ELF-EMF.
References.
Miyagi N, Sato K, Rong Y, Yamamura S, Katagiri H, Kobayashi K, Iwata H. 2000. Effects of PEMF on murine
osteosarcoma cell line: drug-resitant (P-glycoprotein-positive) and non-resistant cells. Bioelectromag 21:112-121
Li Y, Zhao L, Xing X, Lou SJ, He C, Lu CL. 2002. Effects of different frequency electromagnetic fields on the
differentiation of midbrain neural stem cells. Space Med Med Eng 15:374-376
Rolletchek A, Chang H, Guan K, Czyz J, Meyer M, Wobus AM. 2001. Differences of embryonic stem cell-derived
dopaminergic neurons is enhanced by survival-promoting factors. Mech Develop 105:93-104
© The Bioelectromagnetics Society
25th Annual Meeting, June 2003
134
13-7 (Cont’d)
Ivancsits S, Diem E, Pilger A, Rüdiger HW, Jahn O. 2002. Induction of DANN strand breaks by intermittent exposure
to extremely-low-frequency electromagnetic fields in human diploid fibroblasts. Mutat Res 519:1-13
Pilger A, Ivancsits S, Diem E, Steffens M, Stranzl T, Kolb HA, Rüdiger HW.2002. No long lasting effects of
intermittent 50 Hz electromagnetic field on cytoplasmic free calcium and mitochondrial membrane potential in human
diploid fibroblasts (submitted)
13-8
EFFECTS OF ELF- AND RF-EMF ON THE APOPTOTIC PROCESS. I. Lagroye1, F. Bersani2, B. Billaudel1,
M. Capri2, J. Czyz3, P-E. Dulou1, K. Guan3, E. Haro1, S. Joenväärä4, R. Kuokka4, N. Kuster5, D. Leszczynski4, A.
Meister3, F. Poulletier de Gannes1, J. Reivinen‡, J. Schuderer5, B. Veyret1, A.M. Wobus3, Q. Zeng3.
1PIOM/Bioelectromagnetics Laboratory, ENSCPB/EPHE, Pessac, France. 2 University of Bologna, Italy. 3In vitro
Differentiation Group, IPK Gatersleben, Germany. 4STUK, Radiation and Nuclear Safety Authority, Helsinki,
Finland. 5ITIS, Zürich, Switzerland
OBJECTIVE: One of the objectives of the REFLEX consortium was to determine whether ELF or RF fields could
influence the apoptotic process in vitro, per se, or after previous treatment with known apoptosis inducers. In 4 out of
13 laboratories involved in the consortium, experiments were performed to assess apoptosis in different cell types
after exposure to ELF or RF fields.
METHODS: Depending on each laboratory’s experience and equipments, different cell types and electromagnetic
signals were used. Exposure lasted from 1 to 48 hours with or without a recovery period. RF exposure included GSM-
900 (frame) and GSM-1800 (basic, DTX, talk ) signals at SARs levels ranging from 0.1 to 2 W/kg. ELF exposure was
done at 50 Hz, at 0.1, 1 and 2.3 mT. Intermittence was introduced in some experiments.
Chemicals that were used to induce apoptosis were 2-deoxy-D-ribose, staurosporine, TNFα or camptothecin,
depending on the cell system used.
Markers that were used to detect apoptosis were (i) the loss of mitochondrial transmembrane potential, (ii) the
presence of phosphatidylserine on the outer leaflet of the plasma membrane, (iii) the appearance of a sub-G1
population, (iv) caspase-3 activity. Expression of apoptosis - related genes (bcl-2, hsp70, p21) was investigated using
Northern blotting.
RESULTS: RF fields (GSM-900 and GSM-1800) did not significantly affect apoptosis in cells of the immune system
(human peripheral blood mononuclear cells from young and elderly people, human U937 cells), in EA.hy926 human
endothelial cells and in cells from the nervous system (human U87 glioblastoma cells, human SHSY5Yneuroblastoma
cells, rat C6 glioma cells rat primary neurons (granule cells) and glial cells). The signaling
pathways involving blc-2 was not affected in either p53+/+ or p53-/- embryonic stem cells tested after exposure to RF
fields. When tested, the signal intermittence, the age of donors (human peripheral blood mononuclear cells), as well as
the cell genetics (embryonic stem cells) were shown to not substantially affect the occurrence of apoptosis. Moreover,
apoptosis induced by chemicals was not affected by further exposure to RF fields.
ELF magnetic fields were able to up-regulate bcl-2 mRNA expression when p53+/+ neuronal progenitors (embryonic
stem cells) were exposed for 48 hours to discontinuous ELF magnetic fields at the highest magnetic flux density
tested (2.3 mT, 5 min. ON, 30 min. OFF). This effect was transient since the level of bcl-2 went back to its basal level
after 18 hours of recovery. However, mRNA levels of other apoptosis-related genes (hsp70, p21), neuronal genes
(TH, Nurr1 and En-1) and tissue specific genes (GFAP, Nestin) were not affected by the applied magnetic field.
CONCLUSIONS: Overall, the results from the REFLEX programme do not bring evidence that RF fields could
interfere with the integrative apoptotic process in cultured cells. Although ELF magnetic fields were shown to upregulate
the anti-apoptotic bcl-2 gene at high magnetic flux density, no overall effect was detected on ESC neuronal
differentiation process, suggesting that the effect is compensated in some way.
The REFLEX project is supported by the 5th Framework Programme of the European Union.
CONCLUSION
SUMMARY OF THE FINDINGS OBTAINED IN THE REFLEX PROJECT AND FUTURE
PERSPECTIVES. F. Adlkofer, VERUM Foundation, Pettenkoferstr. 33, D-80336 Muenchen, Germany
INTRODUCTION: Exposure to electromagnetic fields (EMF) is a controversial topic throughout the industrial
world. Despite the fact that possible effects of EMF on processes controlling key cell functions have not been
investigated adequately to date, it has become a matter of concern that the rapidly increasing exposure to EMF may
cause, in addition to functional disorders, cancer and neurodegenerative diseases. This fear has triggered controversies
in communities especially in Europe with its high density of population and industry and the omnipresence of EMF in
infrastructures and consumer products. These controversies are affecting the siting of facilities, leading people to
relocate, schools to close or power lines to be resited, all at great expense. So far epidemiological and animal studies
have generated conflicting data and, thus, uncertainty regarding possible adverse health effects. Clearly, mere
continuation or replication of this kind of research without the introduction of innovative concepts will prolong the
uncertainty as to whether EMF does, or does not, represent a health risk. The causality between EMF exposure and
disease can never be regarded as proven without knowledge and understanding of the basic mechanisms possibly
triggered by EMF. To search for those basic mechanisms, state-of-the-art methods recently developed in toxicology
and molecular biology are being employed in the REFLEX project to investigate cellular and subcellular responses of
living cells exposed to EMF in vitro.
OBJECTIVES: The REFLEX project is a cooperation of 12 research institutions from 7 European countries. It is
funded by the EC within the 5th Framework Programme. By using the most powerful molecular biological tools
currently available, the REFLEX project will contribute to a better understanding of the biological effects of EMF, the
prerequisite for an objective assessment of potential health hazards and perhaps also of potential therapeutic effects.
The REFLEX project is aimed at the investigation of EMF-induced cellular, subcellular and/or molecular processes.
Although most, if not all chronic diseases, among them cancer, are of extremely diverse and heterogeneous origin, it
is suggested that underlying this variability lies a relatively small number of critical events, i.e. gene mutations,
deregulated cell proliferation and suppressed or exaggerated cell death or apoptosis. The convergence of these critical
events is required for the development of any and all chronic diseases. Gene mutations, cell proliferation and
apoptosis are caused by or result in an altered gene and protein expression. To reach the goal envisaged in the
REFLEX project, the following priority research areas have been selected:
1. Direct and indirect genotoxic effects of EMFs. 2. Effects of EMFs on differentiation and function of embryonic
stem cells. 3. Effects of EMFs on gene expression and protein targeting. 4. Effects of EMFs on the immune system. 5.
Effects of EMFs on cell transformation and apoptosis. To be able to compare the results of investigations carried out
in the different laboratories and to ensure the conclusiveness of the data obtained in the studies, the conditions of
exposure to EMFs are strictly controlled and the data are evaluated blindly.
RESULTS: Based on the data related to ELF-EMF, a genotoxic effect on primary cell cultures of human fibroblasts
was convincingly demonstrated. DNA strand breaks at a significant level followed by various types of chromosomal
aberrations are produced in a dose related manner. DNA damages can be demonstrated at a flux density as low as 35
µT, they are, however, rapidly and completely repaired, but - as it seems - not entirely errorfree. A strong correlation
between the increase in DNA strand breaks and the increase in micronucleus frequencies and chromosome aberrations
was observed. Since ELF-EMF did not affect cytoplasmic free calcium and mitochondrial membrane potential in
human diploid fibroblasts, it is suggested, that DNA strand breaks and the increase in micronuclei frequencies induced
by ELF-EMF are unlikely to be caused by intracellular changes which affect the free calcium level. Furthermore,
there is evidence that ELF-EMF influences the expression of genes in embryonic stem cells of mice, especially if
these cells are deficient of the p53 gene.
With respect to RF-EMF, data obtained clearly demonstrate that RF radiation produces genotoxic effects in living
cells, too. Single and double DNA strand breaks and the frequency in micronuclei are increased in primary cell
cultures of human fibroblasts, in granulosa cells of rats and in HL 60 cells, a promyelocytic hematopoetic human cell
line, after exposure to RF-EMF at a SAR at or even below 2 W/kg. In addition, RF-EMF at a SAR of 1.5 W/kg is able
to up-regulate the expression of early genes, such as hsp70, p21, c-jun and c-myc, in p53-deficient embryonic stem
cells of mice, but not in their healthy wildtype cells. After lowering the SAR value no influence on the mRNA levels
of these genes was observed anymore. Additional studies demonstrate that RF-EMF alters the expression of
numerous, yet largely unidentified proteins in a transformed human endothelial cell line.
SUMMARY AND CONCLUSIONS: While single and double DNA strand breaks and their repair to our knowledge
never have been shown in in vitro studies before as clearly as in the REFLEX project, the modulation of the gene
expression by EMFs has already been observed by several other authors. No evidence was obtained in the REFLEX
project to date, that these findings are in any way related to the vital processes of cell proliferation and apoptosis. The
further elucidation of these biological effects should help to determine whether or not any health hazards might be
associated with the use of EMF emitting devices at the presently allowed safety standards. There is no doubt, that the
scientific database of these standards is rather poor. Therefore, it cannot be excluded at present, that it might be
necessary one day to adjust them to a new reality. Our findings might show a way how science-based safety standards
could be created in the near future.
Fortunately, the present picture of EMF in vitro research, although far from being complete, is good enough to draw
conclusions where to future research efforts should be directed. The high-throughput screening techniques of
genomics, transcriptomics, and proteomics should enable the unravelling of biological effects of EMF that might
interfere with the functioning of cells and organs. Adequate research combined with adequate funding presupposed,
the probability is rather high that the basic scientific question can be resolved in a not too far future. This is: What do
the data obtained in the REFLEX project and in other comparable studies to date really mean? Are we dealing with
insignificant and more or less physiological biological effects of EMF or effects of EMF that are adverse to the health
of people?
Composition of the REFLEX Consortium:
Prof. Dr. F. Adlkofer, VERUM, Foundation for Behaviour and Environment, Munich, Germany (Coordinator); Prof.
Dr. R. Tauber, Free University of Berlin, Germany; Prof. Dr. H.W. Ruediger, University of Vienna, Austria; Dr. A.M.
Wobus, Institute for Plant Genetics, Gatersleben, Germany; Dr. A. Trillo, INSALUD, Madrid, Spain; Prof. Dr. D.
Leszczynski, Radiation and Nuclear Safety Authority, Helsinki, Finland; Prof. Dr. H.A. Kolb, University of
Hannover, Germany; Prof. Dr. F. Bersani, University of Bologna, Italy; Dr. I. Lagroye, PIOM, University of
Bordeaux, France; Prof. Dr. N. Kuster, Swiss Federal Institute of Technology, Zurich, Switzerland; Prof. Dr. F.
Clementi, University of Milan, Italy; Dr. C. Maercker, Resource Center/Primary Database, Heidelberg, Germany
Programme of the Symposium (The Symposium offers the opportunity to inform the scientific community on the state
of the REFLEX project after about 30 months of research):
INTRODUCTION IN THE REFLEX PROJECT - F. Adlkofer
EXPOSURE SYSTEMS, DOSIMETRY AND QUALITY CONTROL - J. Schuderer and N. Kuster
GENOTOXIC EFFECTS OF ELF-EMF ON HUMAN CELLS IN VITRO - H.W. Ruediger
GENOTOXIC EFFECTS OF RF-EMF ON CULTURED CELLS IN VITRO - R. Tauber
EFFECTS OF ELF-EMF ON GENE EXPRESSION IN MOUSE CELL LINES- A. Wobus
EFFECTS OF RF-EMF ON GENE AND PROTEIN EXPRESSION IN VITRO - D. Leszczynski
EFFECTS OF ELF- AND RF-EMF ON CELL PROLIFERATION AND CELL DIFFERENTIATION - H.-A. Kolb
EFFECTS OF ELF- AND RF-EMF ON THE APOPTOTIC PROCESS - I. Lagroye
SUMMARY OF THE FINDINGS OBTAINED IN THE REFLEX PROJECT AND FUTURE PERSPECTIVES - F.
Adlkofer and H. Dertinger
A project funded by the European Union under the programme "Quality of Life and Management of Living
Resources", Key Action 4 "Environment and Health": QLK4-CT-1999-01574