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| TITLE: | Mechanism of EMF Action in Human Breast Epithelial Cells | ||
| Principal Investigator |
David L. McCormick, Ph.D. | IIT Research Institute | |
| Health Relevance |
Cancer | ||
| Research Categories |
Cellular Function | Gene Expression | Cell Proliferation |
| FY95 Funds | R01ES07093 $ 213,542 | Start Date 09/30/94 | End Date 08/31/98 Rationale and |
| Rationale and Summary |
Increased exposure to power frequency magnetic fields (EMF) has been proposed as a causal factor in the rising incidence of breast cancer in the United States. However, plausible biological mechanisms for the putative tumor enhancing effects of EMF at the level of the breast epithelial cell are essentially lacking. The overall hypothesis being investigated in this program is that the effects of EMF on the breast are epigenetic in nature, and are effected through influences on the action of hormones and other regulators of breast epithelial cell differentiation and proliferation. Using primary cultures of normal human breast epithelial cells and the non-transformed human breast epithelial cell strain HBL-100 as in vitro model systems, the program will: a) determine the influence of EMF + hormones on proliferation of human breast epithelial cells; b) investigate alterations in expression of breast cancer-associated genes as a mechanism for EMF + hormone effects; and c) evaluate the role of local secondary electric fields and currents as mediators of EMF effects. Using an extensively validated in vitro EMF exposure system, initial studies focus on the effects of EMF alone on cell proliferation and the expression of the genes HER-2/neu, c-myc, p53, and cyclin E. Dose-response and time-response parameters will be determined to investigate the reversibility of induced effects, and the hypothesized existence of field strength "windows." After studies with EMF alone are completed, a series of addition and deletion experiments will be performed to study the interactions between EMF and estradiol, prolactin, and melatonin; these studies will be directed towards the identification of one or more endocrine mediators of EMF action in the breast. Additional experiments using annular ring plates will determine the importance of the secondary induction of electric fields and currents as mediators of the biological effects of EMF in the breast epithelium. Finally, should EMF be found to have no influence on the expression of the battery of breast cancer-associated genes heretofore evaluated, subtraction hybridization studies will be performed with sham- and EMF-exposed cells in order to identify genes other than HER-2/neu, c-myc, p53, and cyclin E whose expression is altered by EMF exposure. The results of these studies should identify one or more specific genes whose altered expression is associated with the proliferation of human breast epithelial cells, and will evaluate the role of endocrine status and the induction of local electric currents and fields as effectors of the biological activity of EMF. | ||
| Experimental Design and Exposure Conditions |
1. Experimental Model System -- Normal (non-transformed) human breast epithelial cells
(primary cultures), and non-transformed HBL-100 human breast epithelial cells.
2. Experimental Endpoints -- The influence of exposure to 60 Hz magnetic fields + mammotropic hormones on mammary epithelial cell proliferation and the expression of cancer- associated genes (at the mRNA and protein level) will be assessed. Cell proliferation/cytotoxicity of EMF will be quantitated by the MTT and/or XTT assays. The influence of EMF on gene expression will be evaluated by Northern and Western blot assays, and by RNAse protection assays. Appropriate positive and negative control materials will be included in all assays. 3. Magnetic Field Polarization, Frequency, and Harmonic Content -- Linearly polarized, pure 60 Hz fields; < 3% total harmonic distortion. 4. Magnetic Field Intensity Range -- 10 mG to 10 G. 5. Relative Orientation of AC and DC Magnetic Fields -- AC fields vertical (parallel to primary component of DC field). 6. Associated Electric Fields -- < 10 Volts/meter. 7. Field Spatial Homogeneity -- Uniform to + 5% throughout exposure volume. 8. Field Temporal Homogeneity -- No variations expected (experimental fields are derived from function generator, independent of line voltage). Field temporal homogeneity is determined periodically throughout research program. 9. Field Monitoring -- Continuous throughout program (during both exposures and field-off conditions; monitoring includes both field strength and waveform). 10. Geometry of Cell Culture System -- Magnetic field exposure will be vertical (perpendicular to monolayer cultures of human mammary epithelial cells). 11. Environmental Conditions -- Temperature in all exposure chambers will be maintained at 37 + 0.2 oC, in an environment held at 5.0 + 0.5% CO2. Environmental conditions in all EMF exposure chambers are controlled by a single incubator located remotely from the exposure chambers. 12. Stray Fields -- Laboratory site fully mapped for ambient 60 Hz and geomagnetic (dc) fields. Temporal characteristics of ambient magnetic fields have been determined; ambient magnetic fields are < 1 mG. No DC field cancellation. All incubators are Faraday-shielded. 13. Sham Exposure Conditions (including cross-talk) -- 60 Hz fields in sham control incubator are < 1 mG (total of fields generated by cross-talk from energized experimental module + fields generated by energized sham). 14. Blinding -- All studies conducted using fully blinded design. |
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| Quality Assurance Measures |
1. Ambient 60 Hz and Static Magnetic Fields -- Fully mapped by IITRI engineers prior to
installation of exposure system and incubators. No DC field cancellation.
2. Magnetic Field Monitoring -- Field strength and waveform monitored continuously throughout program (both during exposure periods and periods of module shutdown). 3. Study Blinding -- All studies conducted using fully blinded design. Selection of sham:exposure, exposure:sham, or sham:sham design is made by computer; cell and molecular biology staff are unaware of exposure regimen until exposures are uncoded after completion of endpoint analyses. 4. Positive Controls -- Agents with known activity in modulating expression of cancer- associated genes (tumor promoters, hormones, and chemopreventive agents) are included in the design of all studies. Housekeeping genes and loading controls are included in all studies to ensure no systematic bias in study design or performance. |
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| Results and Discussion |
The magnetic field exposure and monitoring system has been installed in a laboratory that is dedicated to the program, and validation processes have been completed. Where necessary, hardware modifications have been performed to optimize system performance, and software for exposure and monitoring systems has been written. Preliminary studies to validate system operation have been completed, and background studies to optimize the model systems and to generate appropriate positive and negative control data are in progress. | ||
| Recent Publications |
None to date. | ||