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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online August 22, 2005 as doi:10.1096/fj.04-3549fje. |
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* Institute of Plant Genetics and Crop Plant Research (IPK), Gatersleben, Germany;
Foundation for Research on Information Technologies in Society (IT’IS), Zurich, Switzerland
2 Correspondence: In Vitro Differentiation Group, Institute of Plant Genetics and Crop Plant Research (IPK), Correnstr.3, D-06466 Gatersleben, Germany. E-mail: wobusam{at}ipk-gatersleben.de
SPECIFIC AIMS
In the present study we aimed to investigate the effects of radiofrequency (RF) and extremely low frequency (ELF) electromagnetic fields (EMF) on the transcript level of cell cycle regulatory and apoptosis-related genes, on proliferation, apoptosis and chromosomal damage in neural progenitors generated from pluripotent mouse embryonic stem cells in vitro.
PRINCIPAL FINDINGS
1. ELF-EMF exposure affected bcl-2, bax, and GADD45 transcript levels in embryonic stem (ES) cell-derived neural progenitors
Pluripotent mouse ES cells were cultured as aggregates ("embryoid bodies," EBs) for 4 days followed by differentiation induction into neural progenitor and neuronal cells (Fig. 1
). Differentiation of 4d EBs resulted in up to 85% of nestin-positive neural progenitor cells 4–6 days after plating. The cells were exposed to 50 Hz powerline ELF-EMF for 48 h at day 4+4, differentiated into the neuronal lineage, and analyzed at various time points. Magnetic flux density of 2 mT was applied with 5 min ON/ 30 min OFF intermittency cycles. The ELF exposure setup allowed studies under "blind" conditions and the control of temperature differences (±0.2°C) for sham- and ELF-EMF-exposed cultures. Quantitative (Q) RT-PCR analysis (Fig. 2
) showed a relative increase of bcl-2 and bax mRNA levels at stage 4+11d relative to transcript levels of GAPDH, used as an internal standard. Transcript levels of the "growth arrest and DNA damage-inducible gene" GADD45 were down-regulated at terminal stage (4+23d, Fig. 2A
). Immunofluorescence analysis of EMF- and sham-exposed cells, however, showed no differences in the intracellular distribution and number of cells expressing neuronal (ßIII-tubulin, tyrosin hydroxylase, TH) or astrocytic (glial fibrillary acidic protein, GFAP) proteins.
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2. RF-EMF exposure affected Nurr1, bax, and GADD45 transcript levels in neural progenitor cells
According to the experimental protocol described above, ES-derived neural cells were exposed to RF-EMF at stage 4+4d for 48 h. The RF-EMF setup was defined to operate at 1.71 GHz, which is within the up-link band of the GSM1800 system, also named GSM DCS [Digital Communication System]. The GSM signals were amplitude modulated by rectangular pulses with a repetition frequency of 217 Hz corresponding to the dominant modulation component of GSM. Signals were applied to the cells at time-averaged specific absorption rate (SAR) values of 1.5 W/kg with intermittency cycles of 5 min ON/30 min OFF.
Exposure of ES-derived cells to RF-EMF revealed an up-regulation of bax at stage 4+17d and of GADD45 mRNA level at stage 4+23d (Fig. 2B
). Transcript levels of Nurr1 (a transcription factor implicated in the development of dopaminergic neurons) were significantly down-regulated at stage 4+7d, whereas IF analysis showed that the distribution and abundance of neuronal and glial proteins were not affected.
3. RF-EMF induced transient double-strand DNA breaks in neural progenitor cells at low level
To study primary DNA damage in neural progenitor cells, we applied 6 h and 48 h EMF exposure followed by the alkaline (detecting single-strand breaks) and neutral (detecting double-strand breaks) COMET assay. We found a low but statistically significant increase of DNA double-strand breaks in ES-derived progenitor cells immediately after 6 h RF-EMF exposure (Fig. 3
). The exposure of cells to 48 h RF-EMF did not result in DNA break induction suggesting the activation of short-term and transient responses by RF-EMF (similarly, 6 and 48 h ELF-EMF exposure did not induce single- or double-strand DNA breaks).
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4. RF- and ELF-EMF exposure did not induce cytogenetic effects or detectable changes of cell proliferation and apoptosis
Metaphase analysis of EMF-exposed ES-derived neural cells did not present evidence for the induction of chromosomal aberrations (CA) or sister chromatid exchanges (SCE). Determination of BrdU-positive/nestin-positive (BrdU+/nestin+) cells (as % of Hoechst 33342-labeled cells) and the estimation of the number of cells in the 1st, 2nd, and 3rd mitosis (M1: M2: M3 ratio) did not indicate changes in the proliferation rate of EMF-exposed cells. The mitochondrion-selective dye (Mitotracker CM-H2X ROS) applied to detect loss of mitochondrial membrane potential, as an early marker of apoptosis, did not show EMF-induced effects. FACS analysis revealed no significant differences between EMF- and sham-exposed cells with respect to the percentage values of the "subG1 fraction" (=hypodiploid DNA content).
CONCLUSIONS AND SIGNIFICANCE
Epidemiological studies and experimental models using mammalian cells in vivo and in vitro performed to elucidate potential hazardous effects of ELF and RF EMF raised controversies about the involvement of electromagnetic fields in the origin of cancer and/or neurological disorders.
In the present experiments using the ES cell differentiation model, we detected specific changes of transcript levels of various regulatory genes in ES-derived neural progenitor cells exposed to either extremely low electromagnetic fields or high-frequency GSM signals.
ELF-EMF induced a significant up-regulation of transcript levels of two opposing members of the bcl-2 family, bcl-2 and bax. Because the increase of the (anti-apoptotic) bcl-2 level was only slightly higher in comparison to (proapoptotic) bax level, we would not expect significant effects on apoptosis in the neural progenitor cells. Microscopic analysis of selectively stained active mitochondria and FACS analysis of nuclear apoptosis did not show effects on the apoptotic process in EMF- and sham-exposed cells. The down-regulation of transcript levels of GADD45 might be considered as an indication of subtle stimulatory effects on cell proliferation. However, neither the BrdU incorporation into nestin-positive cells, nor the M1: M2: M3 ratio indicated changes in the proliferation rate of EMF-exposed cells. Our finding that neuron (Nurr1, TH)- and glial (GFAP) -specific transcript and protein abundance was not altered, lead us to conclude that ELF-EMF exposure does not affect the process of neural differentiation.
RF-EMF exposure resulted in down-regulation of transcript levels of the neural-specific gene Nurr1 at early stage. Because this effect was observed only after RF-EMF exposure, we may conclude that specific cellular responses are dependent on the electromagnetic field frequency. RF-EMF induced also up-regulation of bax at intermediate and GADD45 mRNA levels at the terminal stage of differentiation. However, similar to ELF-EMF data, we did not detect effects on proliferation and apoptosis. Despite the low induction of DNA double strand breaks in neural progenitor cells after short-term exposure to GSM signals, RF-EMF did not increase the spontaneous frequency (=sham exposure) of chromosomal aberrations and sister chromatid exchanges in ES-derived neural cells.
In summary, we conclude that EMF signals are able to trigger responses at the transcript level of cell cycle regulatory and apoptosis-related genes in neural progenitor cells derived from pluripotent ES cells in vitro. However, due to the lack of effects on other cellular processes including proliferation, chromosomal stability and apoptosis, we postulate that EMF responses at the mRNA level may be gradually compensated at the translational and post-translational level and do not lead to detectable changes of cell physiology.
FOOTNOTES
1 Present address: Department of Cell Biology, Faculty of Biotechnology, Jagiellonian University, Cracow, Poland. 
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-3549fje;
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