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| TITLE: | Effects and Mechanisms of EMF Signal Transduction | ||
| Principal Investigator |
Sek Wen Hui, Ph.D. | Roswell Park Cancer Institute | |
| Health Relevance |
Cancer | ||
| Research Categories |
Cellular Function | Gene Expression | Cell Proliferation |
| FY95 Funds | R01ES07091 $ 146,591 | Start Date 09/28/94 | End Date 08/31/98 |
| Rationale and Summary |
The purpose of this research grant is to investigate the effects of exposure to environmental level
electromagnetic fields (EMF) on cellular signal transduction, and possible effects on a known
model for carcinogenesis.
There are many conflicting reports that environmental EMF may cause cancer and other ailments, through mechanisms that are not understood at the present. There are also in vitro studies reporting conflicting results on environmental level EMF altering cellular functions, several of which may be related to carcinogenesis. The cellular functions may be better defined and controlled in a laboratory setting, and may shed light on the underlying mechanisms for reputed animal and human responses to environmental EMF. The goal of this project is to define a molecular mechanism for the reported effects of weak, low frequency electromagnetic field on cellular functions and if and how these effects are related to cancer etiology. The proposed work will begin with an attempt to reproduce the magnetic field exposure effect on the expression of proto-oncogenes in lymphoblastoid cells [Phillips et al., Biochem. Biophys. Acta. 1132:140-144, 1992]. After the effect is verified, so that a base line for parameter comparison is established, the hypothesis that electromagnetic field acts as a promoter rather than as an initiator will be tested using a two-stage carcinogenesis model system, i.e. the methylcholanthrene-initiated, retinoid suppressed C3H/10T1/2 fibroblasts cells. We will examine the reputed carcinogenic effect of electromagnetic field signal amplification through the secondary messenger system, such as induced calcium signal, protein kinase C activation and other downstream functions of the cell. The outcome of this study will help to explain, and to define the significance limits of elusive cellular effects of weak electromagnetic field corresponding to environmental exposures. | ||
| Experimental Design and Exposure Conditions |
The work will begin with an attempt to reproduce the 0.1 mT, 60 Hz magnetic field exposure effect on the expression of c-fos and c-myc proto-oncogenes in CEM-CM3 T-lymphoblastoid cells [Phillips et al., Biochem. Biophys. Acta. 1132:140-144, 1992], in order to bring the experimental conditions to the same baseline as certain published works. The magnetic field exposure is carried out in an exposure system duplicating the Phillips set up, with identical sham coils. After the effect is verified, further experiments employing 60 Hz electric fields of 2-200 mV/m, (corresponding to about 3-300 mA/m2 of current density in the culture medium), in addition to the magnetic field- induced current, will be performed. We then proceed to test the hypothesis that electromagnetic fields act as a promoter rather than as an initiator in carcinogenesis, using a two-stage carcinogenesis model system, i.e. the methylcholanthrene-initiated, retinoid suppressed C3H/10T1/2 fibroblasts. The timing and the extent of expression of the transformed phenotype, proto-oncogene expression, and proliferation of initiated cells will be determined. In order to identify the effective EMF signal and the amplification steps for the cellular responses, electromagnetic field-induced calcium signal, protein kinase C activation and other downstream functions will be measured in these cells. The origin and mechanism of inducing calcium signal by electromagnetic field will be investigated, by using channel blockers, calmodulin inhibitors, and calcium pump modulators, and by matching the response frequency windows with hypothetical resonance amplification by intra-, extra-, and trans-membrane circuits. | ||
| Quality Assurance Measures |
The magnetic field exposure system is constructed by Electric Research and Management, Inc. (Pittsburgh, PA) as a duplication of the Phillips set up at Loma Linda, CA. It consists of two identical, double wound Helmholtz sets encased in acrylic frames, such that each set can be energized in reinforcing or bucking current directions. Sham exposure is done in the bucking current mode, so the differences in thermal and vibrational environment between the exposed and sham-exposed samples can be minimized. The fields are ramped on/off with cells within the center portion of the Helmholtz set where deviation of field strength is within 1% accuracy, thus switching transients are eliminated. The AC and DC field strengths are monitored with a Bartington 3-axes magnetic field sensor. The earth magnetic field of 580 mG and 59 declination is identical in both incubators and is not shielded. Field exposed and sham exposed conditions were interchangeable in 2 identical incubators placed 3 m apart. The fan motors of the incubators have been replaced to minimize stray AC fields. The AC components of stray magnetic fields from the incubator heater and electronic control never exceed 3 mG within the sample volume. The control unit of the Helmholtz sets has a locked blind switch so that the experimenters do not know which is the sham set. The biochemical assays are also blind-coded. Exposures are always done in identical pairs with alternating incubators, so that slight differences between incubators are averaged. Results are normalized to internal housekeeping gene control for quantitating proto-oncogene expression. | ||
| Results and Discussion |
The EMF exposure system have been built and calibrated to duplicate the Phillips et al. experiment. CEM-CM3 T-lymphoid cells suspended at a density of 1.7x106 cells/ml in 4x75 cm2 culture flasks were either exposed to 0.1 mT, 50 Hz magnetic field or sham-exposed in bucking current coils for 30 min. The expressions of c-fos in field exposed samples were found to be about 2-3 times higher than those in sham-exposed samples in two repeating experiments. The results agree well with those reported by Phillips et al. The results on c-myc are not conclusive. We are in the process of repeating the experiments covering the entire exposure time range in the Phillips et al. paper. At least 10 repeating experiments will be performed. We also plan to examine if extremely low frequency electric field exposure has similar effects. We measured the electric field induced changes in intracellular calcium level in these cells by the fluorescence of fluo-3. No calcium influx is detected as a result of electric field stimulation up to 100 mV/cm, in agreement with the reported lack of voltage dependent calcium channels in this cell line. In the mean time, we have started the proposed experiment on the INIT-10T1/2 cells, to monitor their signaling process and carcinogenic progression as a result of EMF exposure. We will determine if any upstream signals such as calcium flux and PKC activation are connected to the enhanced c-fos and c-myc expressions in these cells. | ||
| Recent Publications |
Hui, S.W. "Electric Field Induced Calcium Flux and Changes in Cell Shape, Motility and Cytoskeleton". In: Biological Effects of Electric and Magnetic Fields (D.O. Carpenter, ed., Academic Press, 1994) vol. 2, pp. 83-101. | ||