Effect of magnetic field exposure on anchorage-independent growth of a promoter-sensitive mouse epidermal cell line (JB6).

The anchorage-independent growth of mouse epidermal cells (JB6) exposed to 60-Hz magnetic fields (MF) was investigated. Promotion-responsive JB6 cells were suspended in agar (10(4)cells/plate) and exposed continuously to 0.10 or 0.96 mT, 60-Hz magnetic fields for 10-14 days, with or without concurrent treatment with the tumor promoter tetradecanoylphorbol acetate (TPA). Exposures to MF were conducted in a manner such that the experimenter was blind to the treatment group of the cells. At the end of the exposure period, the anchorage-independent growth of JB6 cells on soft agar was examined by counting the number of colonies larger than 60 microm (minimum of 60 cells). The use of a combined treatment of the cells with both MF and TPA was to provide an internal positive control to estimate the success of the assay and to allow evaluation of co-promotion. Statistical analysis was performed by a randomized block design analysis of variance to examine both the effect of TPA treatment (alone and in combination with MF exposure) and the effect of intra-assay variability. Transformation frequency of JB6 cells displayed a dose-dependent response to increasing concentrations of TPA. Coexposure of cells to both TPA and 0.10 or 0.96 mT, 60-Hz MF did not result in any differences in transformation frequency for any TPA concentrations tested (0-1 ng/ml). These data indicate that exposure to a 0.10 or 0.96 mT, 60-Hz MF does not act as a promoter or co-promoter in promotion-sensitive JB6 cell anchorage-independent growth. ImagesFigure 1

The Electric and Magnetic Fields Research and Public Information Dissemination (EMF-RAPID) program established under the Energy Policy Act (Section 7118) of 1992 has the central goal of determining if electric and magnetic fields associated with the generation, transmission, and use of electricity pose a risk to human health. The National Institute for Occupational Safety and Health (NIOSH), as part of an interagency agreement, maintains one of four Regional extremely low frequency magnetic field (MF) exposure systems. In addition to providing access and support for researchers outside of NIOSH desiring to use the regional exposure system, NIOSH is also responsible for performing replication studies to investigate the effect of 60-Hz MF on biological processes. Epidemiologic evidence has demonstrated a putative link between exposure to MF and an increased risk for certain types of cancer (1,2). Because epidemiologic studies in the literature are often contradictory and at best can only demonstrate associations between cause and effect (3), in vivo and in vitro studies are often used to examine whether MF encountered in homes or the work place may exert biological effects (4-A. Promotion-sensitive subclones of the mouse epidermal cell line JB6 (clone 41) respond to treatment with tumor-promoting agents by increasing colony growth under anchorage-independent conditions. Increased colony growth has been observed in JB6 cells treated with phorbol esters, epidermal growth factor, lanthanides, and phthalate esters ($). West et al. (9) reported that 60-Hz sinusoidal MF exposure altered the growth of promotion-sensitive JB6 cells under anchorageindependent conditions. In their research, exposure of cells to a continuous 1.1 -mT MF (11 G) led to significant increases in the soft agar colony-forming efficiency, i.e., 40-70% greater numbers compared with unexposed cells (>95% confidence level), at 10-14 days of MF exposure. A second study by this group investigated anchorage-independent growth of JB6 cells exposed to MF of 100, 10, and 1 jiT (1, 0.1, and 0.01 G, respectively). Although increases in anchorage-independent growth were observed in nearly all cell cultures exposed to MFs, there appeared to be no consistent response correlating increasing field strength with increased colony numbers (10). The present report describes efforts by NIOSH to investigate the effect of 0.10 and 0.96 mT MFs (1 and 9.6 G) on the growth of JB6 cells on soft agar. Promotion-responsive JB6 cells, obtained from Robert West at the National Center for Toxicological Research (NCTR; clone 41 passage 83), and also from the American Tissue Culture Collection (Rockville, MD), were suspended in agar and exposed continuously to a 0.10 or 0.96 mT, 60-Hz MF for 10-14 days, with or without concurrent treatment with the promoter tetradecanoylphorbol acetate (TPA). The exposures were conducted in the RAPID program's regional MF exposure facility at NIOSH. The faciity consists of twin exposure chambers in which the MF is produced by a stacked Merritt coil design, and controlled by a computer system that randomizes which chamber serves as the exposure chamber.

Materials and Methods
Plasticware, media, serum, and all other reagents were obtained from identical commercial sources as used previously by West et al. (9). Prior to beginning experiments at NIOSH, one of the authors J.E.S.) visited NCTR, and 50 vials of JB6 cells (passage 83) were cryopreserved to provide sufficient quantities of similar passage cells to last the entire experiment. Because serum source and lot may affect in vitro transformation experiments with JB6 cells (11), the original serum lot (3M) from JRH Biosciences (Lenexa, KS), as used by West, was used in these experiments. Experiments with another lot of JRH serum (4M) that met specific requirements for JB6 cell growth were also performed to examine the effect of serum on JB6 anchorage-independent transformation. In addition, promotion-sensitive cells (clone 41) were also obtained from ATCC to examine differences in transformation frequency between cells from different sources.
Cell culture. Cells were maintained in minimum essential medium with Earles salts and supplemented with 5% heat-inactivated (30 min at 56°C) fetal bovine serum, 2 mM glutamine, and penicillin-streptomycin (50 U/nil, 50 pg/nil). Previously frozen cells were maintained in logarithmic growth for 1 week before use. Incubator conditions were maintained at 37°C, 5% CO2 + 95% air, and 90% humidity. Temperature and CO2 were monitored by the incubator's internal controls. In addition, daily measurements were made with a recording thermometer and Fyrite analyzer for CO2 (Bacharach Inc., Pittsburgh, PA).
with appropriate concentrations of TPA. For these assays, three sets (A, B, C) of three or more replications per treatment were used in each assay. The cell-containing layer was applied over a similar layer of 0.5% agar in 60-mm dishes. Dishes were sealed with Parafilm (American National Can, Greenwich, CN) and the plates from group A and B were placed in their respective exposure chambers of the Department of Energy (DOE) regional MF exposure facility at NIOSH. The exposure conditions of both chambers were randomized by the system. Cell plates marked "C" were maintained in the incubator used for normal cell culture. At days 10 or 14, plates were examined and scored for colony growth. Using an inverted microscope, colonies >60 pm (>60 cells) were counted and results were expressed as mean number of colonies + standard deviation/treatment.
Double-blind trials were conducted to examine the effect of 60-Hz magnetic fields on JB6 cells with or without TPA. This was a slight deviation from the work of West et al. (9) in that study cells were exposed without TPA. The combined treatment of the cells (TPA and/or MF) resulted in an internal positive control that provided an estimate for the success of the assay and allowed the evaluation of co-promotion.
DOE regional MF exposure facility at NIOSH. The DOE regional MF exposure facility is located at the Taft Laboratories and is under the custodial administration of the Division for Biomedical and Behavioral Sciences, NIOSH. The facility at NIOSH is one of four nationally that were manufactured by Columbia Magnetics Inc. of Kennewick, WA, for the DOE and provided to NIOSH, the U.S. Food and Drug Administration, Oak Ridge National Laboratory, and Battelle Pacific Northwest Laboratory. The exposure device, designated the Model 2xc Exposure System, consists of two coil systems energized by a function generator/power amplifier combination under computer control. The software is custom programmed in LabVIEW (National Instruments, Austin, TX) to control field configuration and monitor temperature and MF flux density. The available MF flux density range is 0.1 uT-1 mT. Inside each of the two coil systems are double-walled, Abbreviations: DOE, Department of Energy; NIOSH, National Institute of Safety and Health. Cells were treated as described in "Methods" and allowed to grow in the incubator used for normal cell culture or the DOE regional exposure facility. The number of colonies per plate was determined at day 14. Data are mean number of colonies/plate + standard deviation: n = 3 or 4 plates/treatment. amn sham/sham exposure, double-wound coils were energized with opposing current flow so that the net applied magnetic field was 0 bincubator used for normal cell culture.
cChamber A of the DOE regional exposure facility at NIOSH. dChamber B of the DOE regional exposure facility at NIOSH.  (12). Statistical analysis. Statistical analysis was performed by a randomized block design analysis of variance to examine the effect of TPA treatment (aloune and in combination with MF exposure) as well as the effect of intra-assay variability.

Results
The DOE regional MF exposure facility at NIOSH maintained identical physical conditions in either chamber. Temperature, atmospheric CO2, and relative humidity never differed by more than 1% between chambers; only the presence or absence of an applied MF differed between the two chambers (data not shown). Although our goal was to expose cells to magnetic fields of 0, 0.1, and 1.0 mT, a MF strength of 0.96 ± 0.01 mT was the greatest field that was consistently maintained by the facility over the duration of the exposure period. The background static (DC) field for the chambers was determined with a MultiWave II magnetic field exposure monitor (ERM, Inc., State College, PA). The magnitude of the static field in chamber A was 40.0 pT; field inclination (elevation) was 760 above horizontal, and field declination (azimuthal angle) was 900 (rotated clockwise from north). For chamber B, static field magnitude was 40.5 pT; field inclination (elevation) was 580 above horizontal, and field declination (azimuthal angle) was 1220 (rotated clockwise from north). Magnetic fields measured in inactive chambers and during sham exposures were below 2 mG in Cells were treated with TPA as described in "Methods" and exposed to either no applied MF or 0.10-mT MF in chamber A or chamber B of the Department of Energy (DOE) regional exposure system. Transformation frequency of JB6 cells displayed a similar dose-dependent response to increasing concentrations of TPA, whether grown in the incubator used for cell culture or the DOE regional MF exposure facility (Table 1). Exposure to MF of 0.10 mT (Table 2) or 0.96 mT (Table 3) did not alter colony formation of JB6 cells. Co-exposure of cells to both TPA and the 0.10 mT (Table 4) or 0.96 mT (Table 5), 60-Hz field also did not result in any differences in transformation frequency for any TPA concentrations tested (0-1 ng/ml). In tests comparing cells obtained from ATCC or West and the same lot of serum, cells obtained from West produced significantly greater numbers of colonies without TPA treatment or the presence of MF (Fig. 1).
In tests to determine reasons for the discrepancy between our results and those reported by West et al. (9,10), factors such as serum lot, cell-plating technique, the effect of increasing or decreasing the time the cells are held at 42°C prior to plating, and other experimenter-dependent factors were examined. Of the factors above, serum lot and cell plating order had the greatest effect on colony formation. Two serum lots obtained from the same supplier (JRH Biosciences) were examined. Initial growth tests determined that the two serum lots were both capable of supporting the JB6 cell line URH 3M and JRH 4M). JRH 3M was provided by West as a lot that gave consistent positive results of MF exposure in his laboratory. Samples ofJRH 4M were provided to West and failed to show a positive effect of MF exposure on JB6 cell-proliferation in his laboratory (personal communication). It was observed in our laboratory that JRH 3M caused a noticeable aggregation of cells despite repeated tituration. Because the aggregated cells tended to settle to the bottom of the pipet during the plating procedure, and based on West's technique of plating six plates from a single 10-ml pipette, in our normal replication studies every effort was made to ensure that each treatment group had at least one dish from the first 4.5 ml of suspended cells. In tests comparing the effect of plating order on cell colony formation, significant differences were observed between JRH serum lots 3M and 4M. As seen in Table 6, with JRH 3M serum there was a significant difference between colony counts of plates that were plated with the initial 4.5 ml of cell 25 Cells were treated with TPA as described in "Methods" and exposed to either no applied MF or 0.96-mT MF in chamber A or chamber B of the Department of Energy regional exposure system.  suspension versus the second 4.5 ml. This was not observed with JRH serum lot 4M (Table 6).

Discussion
The data obtained using the NIOSH regional exposure facility indicate that exposure of JB6 cells to a 0.10 or 0.96 mT, 60-Hz MF, does not increase the anchorage-independent proliferation of promotion-sensitive JB6 cells. In addition, the data support the conclusion that exposure to 0.10 or 0.96 mT MF does not enhance the effect of TPA on cellular transformation frequency.
Extensive efforts have been made to decrease intra-assay variability while duplicating the experimental design of West et al. (9); cells, serum, and other cell culture supplies were either from the same commercial source or supplied by West. The methods used in the present study were identical to those of West et al. (9,10) except for the use of the DOE regional facility for cell exposures. Despite these efforts, our findings were different than those previously reported (9,10). Although we had hoped the NIOSH regional exposure facility could consistently maintain MF of 1.1 mT, this was not possible. Experiments by West et al. (10) and Saffer et al. (11) demonstrated there was no correlation between MF strength and JB6 transformation frequency when MF differed as much as threefold. In light of those findings, failure to maintain 1.1 mT in the present study should not have been a factor in the absence of an MF-dependent effect on anchorage-independent cell proliferation. Because of equipment malfunctions, two experiments were ended after 10 days. However, we chose to indude these experiments as valid replications for two reasons. First, the TPA-treated cells showed a typical dose-response curve compared to 14day exposed cells (with predictably lower cell numbers). Second, previous experiments by West et al. (9) reported that although MF-dependent differences were not evident at day 7 of exposure, differences were discernable after 10 days.
The possibility that serum lot contributed to differences between the data reported here and those reported by West et al. (9,10) cannot be discounted. As indicated in Table 6, plating order with JRH serum lot 3M (a serum lot previously reported to yield an increase in JB6 anchorage-independent growth) produced apparent increases in cell proliferation similar in magnitude to those reported by previous researchers (9,10). Our experimental design always induded three groups (EMFexposed, sham, and control) of five TPA concentrations and exposure assignment of these was random, decreasing the likelihood of plating order contributing to erroneous interpretation. In previous studies (9,10) only two groups of cells were included with no internal control (no TPA). Although not discounting the effect of MF exposure, if only two groups of cells are plated (as in the previous publications), the differences that could result from plating order may be inadvertently attributed to MF exposure.
The NIOSH JB6 replication effort closely mirrors that of Saffer et al. (11). These researchers reported that 60-Hz MF of 0.01, 0.1, 1.0, or 1.1 mT flux density did not induce anchorage-independent growth. In addition, these authors reported that MF did not enhance TPA-induced transformation.
In conclusion, although we observed that JB6 cells displayed a dose-dependent response to increasing concentrations of TPA, co-exposure of cells to both TPA and 0.10 or 0.96 mT, 60-Hz MF did not result in any differences in transformation frequency for any TPA concentrations tested (0-1 ng/ml). These data indicate that exposure to a 0.10 or 0.96 mT, 60-Hz MF does not act as a promoter or co-promoter in promotion-sensitive JB6 cell anchorageindependent growth.