The Effect of Electromagnetic Fields on Rat Mammary Carcinoma

 

Douglas M. Evans, M.D., et al.

The processes of cancer cell invasion involve the many faces of the fibrinolytic system. What was initially thought to be a mechanism to control the effects of intravascular thrombus formation has been shown to be far more complex. The fibrinolytic cascade is nearly as complex as the coagulation cascade. A major role in the capability of cells to migrate through tissue has been recognized to belong to this biologic system. The aspects of this system that are important from the aspect of tumor invasive and metastatic potential are the collagenolytic activities indicated.
Incorporated into this system are several levels of checks and balances in the form of inhibitors and stimulators. Not surprisingly, this same system facilitates the migration of inflammatory cells and fibroblasts, making it a vital part of the host defense mechanism. Ironically, this vital biologic asset becomes a significant liability when coded into tumor cells. This is not unlike the transformation of rational adults into congressmen, thereby converting protection into invasion. The key to the invasive potential imparted by this system is that the activated plasmin produced by the
cascade is bound to the cell surface at the receptor site uPAR. Even more unusual is the finding that most tumor systems studied to date show cellular capability of producing the uPA which then binds to its own surface
receptors and initiates the cascade. It is also worthy of note that the resultant bound plasmin is some how protected from the normal inactivation mechanism of alpha 2 antiplasmin. In some tumor systems there can be as many as several hundred such sites per cell, allowing for matrix dissolution in all directions. In contrast, inflammatory cells have
their receptors grouped in only one locale, providing a unidirectional migratory pattern, in addition to the direct collagenolytic effect of surface bound plasmin, this uPA/uPAR/plasmin complex causes activation of other tissue components, particularly the metalloproteases, which intensify the matrix degradation required for invasion andmetastasis. In essence then, as illustrated in this slide, we have an aberration of a normal mechanism which
allows a lethal process to perpetuate itself. That same system which promotes healing, wound repair, infection control, ova fertilization and implantation and the spontaneous resolution of intravascular thrombosis can be parasitized by the malignant cell and utilized for the invasion and spread of the malignant process. The purpose of the exercise I am here to describe is to investigate the effect of electromagnetic fields on the capability of the cancer cell to pervert the fibrinolytic system. 

The Fisher rat MATB tumor cell line was chosen for this study because it is of mammary origin, and has been shown previously in the laboratory to produce uPA. The degree of tumor production can be readily reproduced from a given size of the inoculum. In order to establish that uPA was indeed essential to the care and feeding of these cells, a preliminary study was done to establish that the inhibition of uPA would impair the growth of this tumor. PAI 2, the uPA inhibitor, was graciously donated by (name withheld) and was utilized as the inhibitory agent of uPA. The tumor model used was the creation of lung metastases by injecting the tumor cells into the external iuqular vein of the Fisher rat and counting the number of metastases produced in standardized lung sections. The inhibitor was given
separately via the osmotic pump in the first half of the experiment, and by mixing the inhibitor with the tumor cells in the pump in the second stage. 

The results confirm that this tumor system is susceptible to uPA suppression and that the model is productive in creating metastases in predictable numbers.

An in vitro assay of uPA output of the cells in tissue culture was carried out under varying levels of exposure to electrical, magnetic and electromagnetic fields. uPA was assayed from the culture media. The levels obtained from
field exposure produced by 300uA of current and a 106 gauss magnetic field are shown here. Exposure times were 1 hour q 12 hrs x 48. These exposures were not selected with the view of mimicking physiologic situations, but rather to establish the presence or absence of a gross effect.

The methods of exposure are shown in these slides. Anticipating that increased amounts of uPA production would produce bigger and better tumors the pilot study was set up as follows. Forty Fisher rats were divided into four groups of ten each. The` control group received 10 MATB cells injected into the gluteus muscle by direct vision. Ten rats received cells exposed to the electrical fields as described, ten received cells exposed to the magnetic fields and the final ten received cells exposed simultaneously to both electrical and magnetic fields. The study was terminated at ten days post tumor inoculation. The tumors were harvested, measured, weighed and preserved for electron microscopy. To my surprise the tumors produced by the exposed cells were uniformly and significantly smaller than
the controls. (slides) When viewed under the electron microscope the cell membranes were grossly different, but this needs be interpreted with some
caution.The cell membrane of cultured cells, represented by the controls, tend to show more echinosis than those taken from a confluent mass. The appearance of microtubules did not seem to show morphologic change.

Being forced to reconcile the evidence that the treated cells consistently produced smaller tumors in the face of what was deemed to be an increased production of uPA it was recalled that the action in terms of tissue invasion was via surface bound uPA/plasmin complexes. The assay was repeated using both media and cellular homogenate and using plasmin as the assay target rather than free uPA. These results suggest that significantly less surface bound plasmin is produced by the treated cells than that produced by the control cells. Those levels of plasmin correlate roughly with the tumor sizes generated by the corresponding treated cell groups. Referring back to the electron microscopic findings, the appearance of the cell membranes of the treated cells could suggest that the receptor sites for uPA may be either reduced in number or otherwise defunctionalized by the field exposures, thereby resulting in measurably less
bound plasmin and consequently reduced penetrating power.

The apparent increase in free uPA demonstrated by the media assay would then be accounted for by having notably fewer binding sites for the uPA produced by the tumor cells with a larger fraction available in the free state while a proportionally smaller fraction is converted to the surface bound plasmin required for tissue matrix dissolution. This latter hypothesis is currently in the process of verification by monoclonal antibody labeling of the receptor sites for uPA.

This line of inquiry is obviously barely in its initial stages. At this point we have only a suspicion of an iceberg. if indeed there is an iceberg here, it is entirely submerged and a great amount of exploration will be required to
determine its dimensions.

Abstract:

EMF exposure and MATB Effects of Exposure of MATB Rat Mammary Cancer
Cells to Electric, Magnetic and Electromagnetic Fields: Increased Cell Growth and
Proliferation, Decreased Tumor Growth and Cell Invasiveness

Douglas M. Evans, M.D. , et al.

EMF Effect on Induced Ca

The implication of environmental exposure of electromagnetic fields on the incidence of certain malignancies is of continued interest. This study shows inhibitory effects of exposure of rat mammary cancer cells to electrical, magnetic and combined electromagnetic fields on tumor growth and cell invasive capacity, while cell proliferation was enhanced. Tumor growth was measured in Fisher 344 rats by direct intramuscular inoculation of EMF exposed/unexposed MATB rat mammary cancer cells. Invasive capacity was measured by incubating exposed/unexposed cells in Matrigel invasion chambers. Cell proliferation was measured by thymidine uptake combined with numerical counts of exposed/unexposed cells. Cell exposure in each aspect of this study was identical in field strength and exposure time to unexposed controls. While each field caused significant suppression of tumor growth and invasive capacity, as well as cell proliferation enhancement, the responses to the combined fields were somewhat greater. The determinations demonstrated strong dose related inhibitory response. Key words: Cancer Inhibition; Tumor Inhibition Environmental and occupational electromagnetic field (EMF) exposure have been implicated as a causese of certain malignancies. Leukemia and the lymphomas have been the primary neoplasms in question [1,2,3,4,5,6,7] although there are also reports of an increase in the incidence of breast cancer in employees exposed to electrical fields in the workplace [8,9,10,11], and questions of a link between brain cancer and occupational exposure to EMF [12]. The basis for these reports in each instance has been retrospective epidemiological data. Accurate retrospective measurement of the intensity of field exposures in these studies is not possible. While an approximation of field strength can be made in some instances, i.e. proximity to power lines, the use of household appliances, electric blankets etc., the extent of exposure to these field sources is uncertain. There have been recent
studies on the impact of EMF exposure on gene expression in exposed human leukemic, HL60 and Daudi cells [13,14,15], the results of which are in conflict. To date there have been no reports of prospective approaches to permit comparisons of quantified exposures over defined periods of time to biologic effects. Moreover, while there have been numerous reports of the possible linkage between EMF exposure and the etiology of specific malignancies, there have been no studies aimed at the impact of such exposures on the invasive and growth characteristics of cancer cells. To this end, this study was designed to investigate the effect of measured EMF exposures on cancer cells with respect to the capacity to invade, to proliferate and to the growth of tumors induced by exposed vs. unexposed cells.
Since the actual biologic effects of field exposures are ill defined, the field components were assessed separately. Electrical fields (E) were created through the conversion of petri dishes to capacitors in order to deliver pure voltage to the cultures without the effect of electrolysis on the culture media. The application of a
defined voltage to the capacitors using a current-limited transformer at a static strength of 235.4 milliVolts (mV) produced an electric potential of 300 milliAmps (mA)/meter2. A variable voltage filtered power supply with the addition of an inline transformer was used to provide either direct current or alternating current. A 11.5cm diameter coil (10 turns/cm) with an internal nonconducting platform onto which the culture dishes were placed provided magnetic field exposures (M) to the cells. 60Hz alternating current at 1.47 Amps created a 10 Gauss field within the coil. Combined exposures (EM) were provided by connecting the DC power supply to the capacitors, during exposure to a 10G field within the interior of the coil. All exposures were accomplished within the confines of an incubator at 37 degrees
centigrade and 5% C02. MATB 13762 rat mammary cancer cells (ATCC, Rockville, Md.) were maintained in culture with McCoy's 5A(90%) Bovine Serum (FBS) (10%) and penicillin-streptomycin 1%. MATB cells centrifuged and counted for viability with Trypan blue stain on a hemacytometer. Millicells (30mm,3um; Millipore Corp., Bedford MA) were used inside the petri dishes to provide an elevated surface for
growth so the cells would not contact the capacitor surface. The pore size of the Millicells obstructed passage of the MATB cells but permitted diffusion of the media. For cellular field exposures 106 MATB cells were distributed into the Millicells and the outer petri dish was filled with 30 ml of media. In each phase of the experiment control (unexposed) cells were prepared in an identical paired fashion but
conducted in an incubator separate from that in which the fields were generated.

Determination of the effect of field exposure on tumor growth utilized cells exposed to each field (E, M, and EM) for one hour periods twice daily for 48 hours. Ten Fisher 344 rats per exposure type were subsequently inoculated by intramuscular injection with l0u viable exposed cells. An identical inoculum of 10u unexposed cells was carried out on 10 Fisher 344 rats for controlled comparison. The central gluteal musculature was selected as the inoculation site under direct vision by virtue of a 3mm skin incision. The same inoculation schedule was followed for two groups of
forty rats each. At 10 days post inoculation the tumors were harvested, measured and weighed. Data was analysed by t-test with comparison of each exposure group to unexposed controls.

The effect of field exposure on the capability of MATB cells to invade was determined using the synthetic basement membrane Matrigel (Collaborative Biomedical Products, Bradford, MA), and standard invasion chambers. Matrigel
(100mg/cm2) was layered into Millicells (12 mm, 12 um) in 24- well plates. The size of the invasion chamber inoculation remained at l0u exposed viable cells. Field exposures were again imposed within the confines of an incubator at 37 degrees C. and 5% CO2 incubators. Exposure lengths were expanded to 24, 48 and 72 hours with one and two hour exposure times. A separate set of 12 individual chambers was used for each combination of field exposures. The cells were separately retrieved from each layer and counted by Trypan blue stain. The percentage of invasion was calculated by numbers of cells in the outer well of the invasion chamber divided by the total numbers of cells retrieved. Statistical analysis was by t-test for individual comparison of the exposure group to the unexposed controls. ANOVA was added for comparison of invasion values with respect to length and duration of exposure.

The effect of field exposures on cellular growth and proliferation was measured by both H-thymidine incorporation and by cell counts following incubation during field exposures. MATB cells were prepared, placed in Millicells and intermittently exposed to E, M, and EM fields as with the tumor growth and invasion experiments. The exposure length for this section of the study was Z hours twice daily over a duration of 72 hours. The media was removed from the outer portion of the petri dish and replaced with serum-free media to quiesce the cells for 48 hours, prior to exposure. The cells were removed from the exposure and control chambers and counted with Trypan blue stain in ahemocytometer. MATB cells were resuspended in media containing 10% fetal bovine serum and 0.25 Ci/ml of H-thymidine (NEN Dupont, Boston MA). 10 viable cells per well were aliquotted into 6 wells per exposure group and incubated for 48 hours. The suspended cells from each well were transferred to microcentrufuge tubes, centrifuged and media aspirated. The wells and pellets were washed with media to remove non-specific binding. MATB pellets and
attached cells were lysed with 0.5ml of 0.25 NaOH and incubated for 30 minutes at room temperature. The lysates were diluted with 5ml of Ecolite scintillation cocktail and counted on a Packard PLD beta counter. Growth and proliferation analysis was compared by t-test for each exposure group.

The results of the tumor growth aspect at the study are depicted graphically in Figure 1. Comparison of tumor weights showed significant reduction of tumor mass (all comparisons p<0.02) in lesions produced by exposed cells over those by controls. Tumors produced by unexposed MATB cells weighed 0.9766 -+10.16 grams, those receiving electrical exposure weighed 0.5334+/-0.10 gms., those receiving magnetic exposure weighed 0.43424+/- 0.07 gms. and those receiving combined exposure weighed 0.3597 -+0.06gms. EMF significantly decreased the growth and local
invasio0n of the MATB cells.
The invasive capacity of MATB cells showed variable results with the exposure durations of 24 and 48 hours, but more consistent effects were observed with 72 hour exposure periods (Figs 2 & 3). Compared to unexposed cells, those exposed to electrical fields showed invasiveness of 60% of controls (p=<O.OO2), those exposed to magnetic fields showed 64% invasiveness of controls (p=<0.05) and cells exposed to electromagnetic fields revealed 38$ invasiveness of controls (p=<0.001). Further analysis indicated a dose response from the effect on exposed cells that correlated to the effect on cell invasiveness. Exposure of the cells for 2 hours showed no more
pronounced effect than that for 1 hour, but a direct effect was observed with respect to the duration of exposure. Consistent with the decreased tumor growth of EMF exposed cells noted a significant decrease was noted in the invasion of Matrigel by when field exposure was extended to 72 hours. The more pronounced effect was produced by the combined fields.

EMF effects on MATB cellular proliferation by incorporation of 3H-thymidine and growth by cell counts were compared to the indices of unexposed cells (100%). Exposure to electrical fields increased cell proliferation to 150% of control, exposure to magnetic fields increased proliferation to 160% of control and combined field exposure produced increases of 228%. All field exposures increased the cellular proliferation (P<0.001) of the MATB cells. Furthermore, cell counts for the E, M and EM exposed cells showed increased cell growth (Table 1) to 105% of control, 116% of control and 137% of control for respective p values of 0.32, 0.053 and 0.005. The results confirm that the MATB cells remain
viable through EMF exposure. In contrast, the increased growth and proliferative capacities of the exposed cells did not correlate with their resultant tumor size and invasive activity.
The potential for biologic effects of exposure to electromagnetic fields was raised initially in the mid 1960s by Asanova and Rakov [16] reporting from the Soviet
Union. A complex of symptoms including malaise, headache, insomnia, fatigue, loss of libido and cardiovascular disorders was indentified among railroad switchyard workers. Subsequent studies of workers with similar levels of exposure in western countries were nonconfirmatory [17,108,19). The report of Wertheimer and
Leeper [1] in 1979 first raised the question of possible linkage between field exposures and cancer. A two- to three-fold increase in cancer was reported among children living in proximity to electrical lines of high-current configuration. Leukemia. lymphoma and central nervous system tumors were the prominent lesions found in this study. Subsequently at least 50 epidemiologic studies have been reported, focusing on either occupational or residential exposures. The results and appropriate abstracts of these studies have been presented in extensive literature reviews by Creasey and Goldberg [20] and Ahlbom [21] These may be summarized as showing a plausible but weak and unproven relationship between specific neoplasms and electromagnetic exposures. Some of the data suggest a possible role of EMF exposures serving a promotional rather than causative role with respect to the incidence of certain cancers.

In support of the concept of a promotional role for EMF exposures, Leung et al [22] reported an increased number of rat mammary tumors produced in animals exposed to both dimethylbenzanthracene (DMBA) and 60Hz electrical fields. Bemiashvili [23] likewise showed promotional effects of both static and variable magnetic fields imposed on rats previously injected with nitrosomethyl urea (NMU). The results reported by Rannug [24, 25] in two separate studies of promotional effects of magnetic fields on the production of liver tumors were inconclusive. Loscher [26] studied the promotional influence of magnetic fields in DMBA treated female rats. Varying ranges of magnetic field exposures were used with equivocal results. Conversely, inhibition of expected growth of spontaneous tumors In animals exposed
to pulsed magnetic fields has been reported by Bellossi [27], Rius [28] and Iur'ev [29]. It should be noted that the levels of energy of the induced fields in these studies were at considerable variance.
Of the several studies focused on the influence of EMF exposures on tumor growth, only the study reported here investigated both the effects of EMF exposure on tumor cells of the same type and at the same levels of exposure. Rius [28] reported inhibition of growth of a mouse mammary tumor cell line, demonstrated. Conversely, Phillips and associates [30] reported the increased growth of two separate cell lines of human colon cancer exposed to magnetic fields of 1G and electrical fields or 3O0mA/m2. West et al [31] showed enhanced growth of mouse JB6 cells exposed to magnetic fields of 10G. The results of the cell proliferation aspect of the study reported here, measured by 3H-thymidine uptake of exposed cells, showed similar increases in growth rates. These findings suggest that the cellular mechanisms controlling cell proliferation and those determining invasive capacity and tumor size may be mediated in separate fashions. There is support for this concept. Mahnensmith and Aronsan [] identified the role of the Na+/H+ plasma membrane antiporter in mammalian cells. Grinstein and Rothstein [] as well as the work of Rozengurt and Mendoza [] and that of Moolenaar et al [] lucidated the regulation of that exchanger. L'Alleman et al [] demonstrated the blockade of the antiport will
shut dawn DNA synthesis and cell proliferation in fibroblasts. Cell invasion and metastasis, on the other hand, have been clearly shown to be controlled by the activation of plasminogen to surface bound plasmin by the autocrine binding of urokinase plasminogen activator to its receptor (uPA/uPAR). These actions initiate the proteolytic cascade required for tumor cell invasion and metastasis. Separate effects of EMF exposure on the Na+/H+ antiport from those on the uPA/uPAR ligand may well explain the apparent paradox of reduced cellular invasiveness and smaller tumor production in the face of increased cell proliferation and growth resultant from the same stimulus.Studies designed to demonstrate effects on growth, embryonic development, organ system function as well as cytogenetics have been carried out on avian, mammalian and bacterial species have again yielded variable results [32.33.34.35.36.37.38.39.40.41.42]. Through the comparisons of these similar studies which have produced conflicting or even contradictory results there emerges a strong suggestion of specificity of exposure effect. The variations of length, intensity and frequency of exposures have been offered as explanations of conflicting results.

The results of the present study, showing variable levels of response to changes in the length and duration of exposures, are supportive of this concept.

Both electrical and magnetic fields singly imposed on MATB rat mammary cancer cells produced significant inhibition of tumor growth and cell invasiveness. Combined electric and magnetic field exposures produced the most profound change. EMF exposure of identical cells at identical energy levels which demonstrated decreased cell invasiveness and tumor growth provoked seemingly incongruent increase in cell growth and proliferation. These results are consistent with the current understanding of the regulatory pathways of cell proliferation vs those of invasion and metastasis.
The disparate findings of increased cell growth in the face of diminished tumor mass and cell invasiveness may be the result of the response of separate regulatory devices to the same stimulus. Further inquiry into the mechanisms involved in the responses found in this report are currently the subject of further inquiry. Comparison of the range of response to the varying exposure levels utilized in this study to the existing literature supports the concept of specificity of field strength and exposure duration on the effects produced.

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