[ EMF-Link Home
| Online Store
| Comments
| Up One Level ]
| TITLE: | Perturbation of Cell Processes by ELF Electric Fields | ||
| Principal Investigator |
Kenneth J. McLeod, Ph.D. | State University of New York, Stony Brook, NY 11794-8181 | |
| Health Relevance |
Other: Biophysics of Cellular Function | ||
| Research Categories |
Cellular Function | Cell Proliferation | Cell Cycle Kinetics |
| FY95 Funds | R01ES06287 $ 150,929 | Start Date 09/01/93 | End Date 08/31/96 Rationale and |
| Rationale and Summary |
Exposure to electric and magnetic fields (EMF) has been linked to various forms of cancer in several epidemiologic studies. However, laboratory-based studies have yet to determine a definitive biological mechanism which would explain the epidemiologic findings. The principal objective of this work is to address the hypothesis that the response of cells to extremely low frequency EMF exposure is dependent on the physical relationship between the cells and the induced field. More specifically, we have proposed that the interaction of EMF with living cells is through the electric polarization forces developed at the surface of the cells, a hypothesis which predicts that cell size, shape, and surface charge density will critically affect field-cell interactions. Moreover, this hypothesis predicts that the characteristics of the induced electric field, including frequency, intensity, and duration will have specific effects on the responsiveness of exposed cells. To test this hypothesis, four specific aims have been proposed: 1) to determine whether cells respond in a dose dependent manner to the magnitude of an induced electric field; 2) whether the morphologic characteristics of a cell can dictate the responsiveness of the cell to an electric field; 3) whether the biological response of a cell to induced electric field exposure is directly proportional to the mechanical perturbation imposed on the cell by electric polarization forces; and, 4) whether the responsiveness of a cell line to electric field exposure can be predicted from its morphology. | ||
| Experimental Design and Exposure Conditions |
In these experiments, cells are exposed to electric fields through both magnetic induction and by
direct application via electrodes. Magnetic induction is accomplished through a solenoidal
exposure apparatus. This exposure system ensures a uniform magnetic field exposure for all cells
within an exposed sample and provides a rigorous sham exposure. The individual solenoid coils
used for both control, sham and treatment exposures consist of 4 cm diameter, 25 cm long hollow
cores with two windings. Because the coils are of identical construction, any coil can be used in a
treatment, sham or control configuration. In the treatment configuration, the two windings on the
solenoid coil are attached in series so as to produce aiding magnetic fields. In this configuration,
the solenoids produce a 1.8 milliTesla rms magnetic flux density for a 106 milliamp rms current.
The flux density varies less than 3% over the central 10 cm in the interior of the coil due to the use
of additional windings on the ends of the solenoids, permitting up to six eight-well Nunc tissue-tek
plates (48 samples) to be simultaneously exposed to a uniform field. In the sham exposure
configuration, the two windings of the solenoid coil are connected so as to produce opposing
magnetic fields. In this configuration, the measured magnetic flux in the interior of the coil is less
than 0.01 mT for a 16 milliamperes current. A single current source (Krohn-Hite Model 75) is used
to provide the current for both the sham and treatment solenoids so that any resistive heating in the
two coils will be similar. Each coil presents a total resistance of 40 ohms, so that power dissipation
in each coil is approximately 0.5 Watts. The resulting power dissipation results in a temperature
rise of the coils of 1.5 degrees C in both sham and exposure solenoids for a 30 Hz exposure
condition. The control, sham, and exposure solenoids are placed on the same shelf of an incubator
to ensure minimal variation in other environmental factors. The magnetically induced electric field
and current density distributions within the individual square wells have been calculated with 20
micrometer resolution using a 2-D impedance network solved via the method of successive over
relaxation. The solution at the monolayer surface demonstrates a peak tangential (horizontal)
electric field intensity of 6 microvolts/cm rms at the center of the well which decreases to zero at the
edges of the well. Correspondingly, the normal (vertical) electric field is maximal (1 microvolt/cm)
at the edge of the well, decaying to zero at the center of the well.
Pure electric fields are applied to cells during mechanical measurements within the atomic force microscope using platinum electrodes connected to the cell media via agar bridges. A current density is established within the media using a current source, and the induced electric field intensity is calculated based on knowing the geometry of the exposure chamber, the media depth, and the conductivity of the cell medium (65 ohm-cm for culture media with 10% serum). |
||
| Quality Assurance Measures |
Characterization of exposure system: Magnetic field intensity - up to 2 mT rms Duration of EMF exposure - 4 to 64 hours Field Frequencies - 15 - 150 Hz Static magnetic field - 507 milligauss at 85 degrees below horizontal Orientation of a.c. to d.c. magnetic field - 85 degrees Associated electric field - 8.4 microvolts/cm peak at 1.8 mT, 30 Hz Transient characteristics of exposure system: 74 mH, 40 ohms, implying 1.9 ms time constant Spatial homogeneity - 3% over exposure range of solenoid Non-EMF exposures - Max temp rise in solenoids - 1.5 degrees C rise in first 2 hours, sham and exposure coils similar within 0.1 degrees. Incidental exposures - Fan in incubator produces ambient 60 Hz flux of 14.5 microTesla oriented horizontally, in line with solenoids. |
||
| Results and Discussion |
This work is entering the third year of a three year grant period, and so substantial progress has
already been made on the specific aims. First, we have been able to clearly demonstrate that in our
EMF exposure system, cell response corresponds to the magnitude of the induced electric field,
rather than to the applied magnetic field. Using a histologic staining procedure for alkaline
phosphatase, a distinct spatial mapping of the cells responding to the pattern of the induced electric
field. This work provides a clear dose-response, at least for bone cells (MC-3T3-E1, a non-
transformed osteoblastic cell line). Moreover, these studies suggest a threshold for cellular
responses may be as low as 1 microvolt/cm, a field intensity threshold level consistent with that
which has been shown to be capable of influencing bone adaptation in vivo, and which can be
induced in the humans exposed to commonly encountered magnetic flux density levels.
Second, we have shown that the decrease in alkaline phosphatase activity seen in bone cells is consistent with a decrease in the number of cells entering the cell cycle, and flow cytometry studies have confirmed that field exposure results in a delay in progression through the G2/M phase of the cell cycle. Third, we have used flow cytometry to demonstrate that exposed cells tend to be larger than non- exposed cells, consistent with the delay in G2M, but also suggesting an interaction similar to the control effects caused by changes in nutrient level (i.e. richer nutrients result in growth delay while cells achieve larger size. Fourth, we have been able to show that while induced surface charge and alterations in fixed surface charge both affect cell growth control, only induced surface charge perturbations (either a.c. or d.c.) result in cellular changes similar to those found in field exposed cells. These data support the contention that not only is it the electric field which is critical to ensure cellular responses, but it is the influence of the electric field with charges and dipoles at the outer surface of the cell which is critical in this transduction process. Fifth, we have completed our numerical modeling of the electromechanical interaction of the field with the cell, incorporating the concepts of the development of an electric polarization force at the surface of the cell due to the low conductivity of the plasma membrane, the enhancement of this volume force by the dielectric enhancement occurring non-uniformly around the cell due to the high charge characteristics of the plasma membrane and cell coat, and the transference of this volume force to the cell surface via the glycocalyx. This model predicts specific spatial and temporal patterns that should arise during field exposure and which can be tested using atomic force microscopy. Finally, we have installed atomic force microscopy capability which will permit us to specifically measure the electro-mechanical coupling of the field to the cell. The combination of instantaneous force measurements and long duration recordings will permit isolation of any mechanical oscillators within the cell as well as the ability of the cell to temporally integrate the applied signal. |
||
| Recent Publications |
1. McLeod, K.J., Fontaine, M.A., Donahue, H.J. Rubin, C.T. (1993) Electric fields modulate bone
cell function in a density dependent manner. J. Bone & Mineral Res. 8(8):977-983.
2. McLeod, K.J., Fritton, C. & Brink, P. (1994) Spectral energy distribution of the resting membrane potential in osteoblastic cells obtained through patch recording, Annual Review of Research on Biological Effects of Electric and Magnetic Fields from the Generation, Delivery & Use of Electricity. 3. McLeod, K.J. & Rubin, C.T. (1994) Regulation of cell growth rates in vitro by alteration of induced charge density, Annual Review of Research on Biological Effects of Electric and Magnetic Fields from the Generation, Delivery & Use of Electricity. 4. McLeod, K.J. (1994) Exposure frequency and duration response characteristics for cell ensembles exposed to low level, ELF electric fields, Annual Review of Research on Biological Effects of Electric and Magnetic Fields from the Generation, Delivery & Use of Electricity. 5. Rubin, J., McLeod, K.J., Titus, L., Nanes, M.S., Catherwood, B.D. & Rubin, C.T. (1995) Extremely low frequency, low intensity electric fields attenuate formation of osteoclast-like cells in murine marrow culture. J. Ortho. Res. (In press). 6. Shim, J., Rubin, C.T. & McLeod, K.J. (1995) The interrelationship of alkaline phosphatase activity cell adhesion, and cell proliferation in osteoblastic cell lines. J. Cell Physiol. (Submitted) 7. Vander Molen, M. & McLeod, K.J. (1995) Surface Charge and Electric Field Effects on Cell Cycle Kinetics. Bioelectromagnetics Society, 17th Annual Meeting, 18-22 June, Boston, MA, p. 18. 8. McLeod, K.J. & Porres, L. (1995) Field intensity response characteristics for cell ensembles exposed to low level, ELF electric fields. Bioelectromagnetics Society, 17th Annual Meeting, 18-22 June, Boston, MA, p. 186-187. 9. McLeod, K.J. (1995) The role of calcium in transducing the effect of low frequency electromagnetic field exposure on cells and tissues, in Biological Effects of Environmental Electromagnetic Fields, M. Blank, ed., Advances in Chemistry Series 250:349-365. |
||