FDA Center for Devices and Radiological Health (HFZ-114), 9200 Corporate Boulevard, Rockville, MD 20850, USA
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Abstract |
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Abbreviations: ATCC, American Type Culture Collection; CUA, Catholic University of America; DCS, donor calf serum; DFMO, -difluoromethylornithine; EMEM, Eagle's minimal essential medium; EMF, electric and magnetic fields; FDA, Food and Drug Administration; FGF, fibroblast-derived growth factor; ODC, ornithine decarboxylase; PBS, phosphate-buffered saline; PDGF, platelet-derived growth factor; REEF, Regional Electromagnetic Exposure Facility.
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Introduction |
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Ornithine decarboxylase (ODC, EC 4.1.1.17) is essential for growth by virtue of its role in the synthesis of precursors of DNA and proteins. ODC is the first and rate-limiting enzyme in the pathway for biosynthesis of polyamines, which have been shown to be necessary for cell growth and proliferation. ODC activity is closely regulated and is stimulated by a number of growth factors and by all known tumor promoters. Thus, it is a sensitive molecular marker for tumor- and growth-promoting agents.
Several investigators have described induction of ODC in cells exposed to electromagnetic fields with an extremely low frequency component. Byus et al. found increased ODC activity in a number of cell lines exposed to 60 Hz electric fields (5) and to microwaves modulated at 16 Hz. Litovitz et al. also observed ODC induction in L929 murine fibroblasts exposed to 55, 60 and 65 Hz magnetic fields and to microwaves modulated at 60 Hz (6,7). Litovitz et al. have used the ODC enhancement effect to investigate the importance of modulation and other physical characteristics of the signal to try to identify the cellular receptor for interaction with the field and thus deduce a biophysical mechanism (811). Indeed, they found that shifting between 55 and 65 Hz fields at intervals of <10 s decreased or abolished ODC enhancement, as did the superposition of random noise upon a 60 Hz field.
Although magnetic field enhancement of ODC activity has been cited as having been replicated independently (3), the work of Azadniv et al. (12) failed to substantiate such claims. Their methodology differed in several ways from that of Litovitz and co-workers. We report here the results of a comprehensive effort to confirm the accuracy and reproducibility of the reported enhancement of ODC activity in a 60 Hz magnetic field. In addition, we extend the previously reported investigations by testing a number of exposure conditions and other associated variables.
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Materials and methods |
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Cells from four flasks were pooled and counted. Six 75 cm2 flasks were plated at a density of 3 000 000 cells/flask in 15 ml of EMEM and incubated for ~18 h before exposure. Three flasks were exposed and three were sham exposed. Caps were tightened immediately prior to exposure, as was done by Litovitz et al. (8).
Cell harvesting
Cells were removed from the field prior to deactivating the Helmholtz coils to preclude exposure to switching transients. Immediately after exposure, the medium was decanted from each flask and each monolayer was washed twice with 10 ml of cold (4°C) Ca2+/Mg2+-free phosphate-buffered saline (PBS). After the second wash, cells were suspended in 10 ml of cold PBS using a cell scraper and each cell suspension was transferred to a 15 ml centrifuge tube and centrifuged at 500 g for 5 min. After centrifugation the PBS was removed and each cell pellet was resuspended in 1 ml of cold PBS and transferred to a microcentrifuge tube. Cell suspensions were again centrifuged at 500 g for 5 min, the PBS was removed and the pellet was drained of excess PBS by inverting the tube onto absorbent paper. The cell pellet from each of the six flasks in an experiment was stored at 70°C until it was assayed in duplicate for ODC activity as described below.
Extremely low frequency magnetic field exposure systems
The various exposure systems used in this work are described below. Specifications of the respective systems are summarized in Table I.
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Regional ELF Exposure Facility (REEF). The REEF system consists of an incubator which supplies air of uniform temperature, humidity and CO2 content to two physically separate exposure chambers. Monitored data from the exposure chamber provide feedback to computer software which controls field variables. The chambers are mechanically and electrically isolated. Each chamber is equipped with a thermocouple and a three-axis AC magnetic field sensor. Two concentric sets of Merritt coils with a common vertical axis surround each exposure chamber. The coils are energized by a function generator driving a power amplifier which energizes the Merritt coils through a system of relays which randomly selects the exposure chamber to be activated under computer control. The inner coils provide exposure and the outer coils limit stray fields from reaching the sham exposure chamber. Each coil system also has two Helmholtz coils with horizontally directed orthogonal axes. These coils can be used to compensate for the ambient static field or generate a horizontal alternating field. All data such as time, flux density, temperature and incubator humidity and CO2 levels are saved to a computer file for inspection and plotting. The REEF system was designed by Pacific Northwest Laboratory (PNL) and manufactured by CMI (Kennewick, WA). Similar REEF facilities are located at PNL, Oak Ridge National Laboratories and the National Institute of Occupational Safety and Health (Cincinnati, OH).
CUA exposure system at FDA. The CUA exposure system consists of two similar E-field shielded Helmholtz coils (average radius 10.5 cm) enclosed with a mu-metal cover (24.9 cm long, 24.1 cm wide, 26.7 cm high). Each coil is placed in an identical water jacketed incubator (VWR 6000) at 37°C. The Helmholtz coils were connected to the 8 speaker output of a power amplifier. This system uses the same coils, amplifier and mu-metal shield boxes that Litovitz and co-workers used in their earlier ODC experiments. The Helmholtz coils were energized using a Krohn-Hite (model 5200A) function generator (FDA property) connected to a 35 W (Radioshack Realistic model MPA-46) audio amplifier. The current was determined by measuring the voltage drop across a 3
resistor connected in series with the coil, using a Hewlett Packard (model 34401A) multimeter. Calibration of the relationship between voltage drop across the coil and flux density were previously determined by CUA and was independently verified both by Dr Penafiel of CUA and by Dr Misakian of the National Institute of Standards and Technology (Gaithersburg, MD).
For experiments using the CUA coils and CUA mu-metal covers in the FDA system, the covers of the FDA mu-metal enclosures were removed. The CUA electronic equipment described above was used for all experiments conducted with the CUA coils. For experiments at CUA, cells were cultured at FDA, transported to CUA and exposed the next day.
ODC assay
ODC activity was determined by the method of Seely and Pegg (13) as modified for cell culture by Litovitz et al. (7). All reagents were from Sigma (St Louis, MO) except as noted. Briefly, cell pellets were lysed by adding 240 µl of lysis buffer and vortexing at maximum speed for 20 s. The lysis buffer contained 25 mM Tris, pH 7.4, 2.5 mM dithiothreitol, 100 µM EDTA, 0.10% Nonidet P-40, 50 µM pyridoxal 5'-phosphate and 50 µg/ml leupeptin. Cell lysates were centrifuged at 104 g for 30 min at 4°C. Duplicate 100 µl aliquots of supernatant were incubated for 1 h at 37°C with 2.77x105 d.p.m. 14C-labeled L-ornithine (Dupont NEN, Boston, MA), 0.4 mM unlabeled L-ornithine, 50 mM pyridoxal 5'-phosphate, 1.58 mM dithiothreitol, 50 µg/ml leupeptin and 0.2% Nonidet P-40 (250 µl final volume). Incubation was carried out in 15 ml tubes (Kimble 45160-15) in a shaking water bath. The 14CO2 generated by ODC was absorbed with 100 µl of 1.0 N sodium hydroxide contained in a center well (Kontes, Vineland, NJ) secured in a rubber septum-type stopper (Kontes). Following incubation, the reaction was terminated by the addition of 300 µl of 20% aqueous trichloroacetic acid through a 21 G needle inserted through the rubber septum and incubated for an additional 1 h. The sodium hydroxide in the center well was then transferred to a scintillation vial containing 8 µl acetic acid and 10 ml scintillation fluid (Packard Ultima Gold) and counted by liquid scintillation (Packard Tricarb 2000CA). The amount of protein in 10 µl of cell lysate was determined by the Bradford method (BioRad, Hercules, CA). Total ODC activity was calculated as pmol 14CO2/30 min/mg lysate protein multiplied by 41 (to account for the specific activity of the ornithine in the reaction).
Positive control experiments
Cells were serum starved for 24 h and then treated for 4 h with fresh EMEM containing one of the following: 5% calf serum, 100 ng/ml fibroblast growth factor (FGF), 125 ng/ml platelet-derived growth factor (PDGF) or 2.5 µM insulin-like growth factor. Non-serum-starved cells were kept in normal EMEM with 5% DCS to allow comparison between serum-starved cells and cells under normal growth conditions.
Data analysis
For each flask, the ODC activities calculated from duplicate assays were averaged. These independent values from the three flasks in each exposure condition were used to calculate both the average cellular ODC activity and the standard deviation of that value for a given exposure condition. For each experiment, the average and the standard deviation for each of the exposure conditions were used to calculate the ODC activity ratio. This ratio is expressed as exposed:control for experimental treatments and as chamber A:chamber B for sham versus sham control experiments. ODC activity ratio is used to account for variations between experiments in control ODC activity. This facilitates comparisons among experiments (8). For a series of 10 experiments conducted over several weeks, the variability of the baseline ODC activity was 36 and 42% (SD ÷ meanx100) for ATCC cells and CUA cells, respectively. This variability compares favorably with that reported by Litovitz et al. for experiments conducted under comparable conditions (9; Tables III).
Statistical comparisons were performed using a paired, two-tailed Student's t-test. Data were analyzed using the INSTAT computer program (GraphPad Software, San Diego, CA).
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Results |
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Positive control experiments
In order to demonstrate cell responsiveness as well as the capability of the assay to detect enhancement of ODC activity, an initial experiment was conducted in which L929 cells were exposed to various factors known to stimulate ODC (14). Serum-starved cells exposed to fresh serum showed a 19.4-fold enhancement in ODC activity over serum-starved cells, increasing from 20.9 to 406 pmol/30 min/mg. FGF-treated cells showed 4.6-fold enhancement, PDGF-treated cells showed 4.9-fold enhancement and insulin-like growth factor-treated cells showed a 4.5-fold enhancement over serum-starved cells. Thus, the cells responded to each of four positive control agents and our assay methods were sufficiently sensitive to detect the change in ODC activity. Furthermore, we were able to establish the serum concentration which caused an approximate doubling of ODC activity and included such positive controls in many of the experiments reported herein.
Specificity of ODC assay
In order to determine the specificity of the assay for ODC, a series of L929 cell lysates was assayed with and without the addition of 1.0 mM -difluoromethylornithine (DFMO; Hoechst Marion Roussel, Cincinnati, OH), a specific inhibitor of ODC (15). It has been shown that under some assay conditions, ODC assay results reflect non-specific cellular components (16). Addition of DFMO to the assay mixture resulted in a 96% inhibition of basal ODC activity and a 99% inhibition of the ODC activity of cells stimulated for 4 h with fresh medium. These results established that, under the conditions we employed, the enzyme assay primarily responded to ODC rather than to non-specific cellular components.
Exposures in the FDA exposure system
A series of 20 separate experiments was conducted in the FDA exposure system using 4 h exposures at 10 µT, 60 Hz and 37 ± 0.2°C. The magnetic field vector was oriented in the horizontal plane, as it was in the laboratory of Litovitz and co-workers. There was no superimposed DC magnetic field for these exposures. The active exposure chamber was arbitrarily alternated between the top (A) chamber and the bottom (B) chamber. The results of these experiments are shown in Figure 1 (upper, filled bars). The ODC activity ratio (exposed:control) was 0.99 ± 0.21 (A coils active) or 0.96 ± 0.17 (B coils active). In neither case was the result significantly different from unity, based on statistical analysis. A series of five separate shamsham exposures conducted in the FDA exposure system gave an ODC activity ratio of 1.02 ± 0.11 (chamber A:chamber B).
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Exposures conducted using the CUA Helmholtz coils
Exposures in neither of the systems at the FDA (above) demonstrated an effect of magnetic field exposure on ODC activity. Therefore, a series of three experiments (4 h, 10 µT) was conducted in the original investigators' exposure system at CUA. These experiments had an ODC activity ratio of 2.09 ± 0.35. However, the significance of these results was unclear because it was not possible to perform shamsham exposures for this series of experiments. Furthermore, we were unable to repeat this result at a later date (see below).
The positive results obtained with cells exposed at CUA suggested an effect that needed to be pursued. The CUA Helmholtz coils were installed in paired incubators (identical to each other, but not identical to those used at CUA) in our laboratory (Materials and methods). Exposures were alternated between the coil sets. The ODC activity ratio for the series was 1.07 ± 0.07 with coil set A active and 1.52 ± 0.26 with coil set B active (Figure 1, lower, filled bars). Shamsham exposures conducted with neither coil energized gave an ODC activity ratio of 0.96 ± 0.04 (coil set A:coil set B). To further evaluate the CUA coils sets and to take advantage of the lower ambient fields and more uniform temperatures provided by the FDA exposure system, the CUA coils were placed inside it. Magnetic field exposures in this experimental setup resulted in an ODC activity ratio of 1.23 ± 0.12 (coil A active) or 0.96 ± 0.07 (coil B active); under shamsham conditions the ODC activity ratio was 1.29 ± 0.25 (coil A:coil B; Figure 1
, lower, open bars). To address the possibility of temperature differences between exposed and control samples due to enclosure in the small metal boxes without air circulation, a series of experiments was performed without the CUA mu-metal covers (shielding from ambient fields was provided by the larger mu-metal shield boxes of the FDA system). The ODC activity ratio was 1.08 ± 0.25 (coil A active) or 1.21 ± 0.29 (coil B active); under shamsham conditions the ODC activity ratio was 0.94 ± 0.2 (coil A:coil B; Figure 1
, lower, hatched bars).
During the course of our studies, the exposure system used by Litovitz and co-workers at CUA was modified with new Helmholtz coils and mu-metal shields, using the same dimensions as the original equipment. Preliminary experiments conducted by Litovitz and co-workers at CUA using the modified system gave positive results for magnetic field induction of ODC activity (T.Litovitz, personal communication). These observations raised the possibility that ODC activity might respond to some exposure parameter unique to the CUA system. Thus, a new series of nine experiments was performed wherein cells were cultured at FDA and exposed at CUA. Three separate chambers were used for each experiment: (i) a sham chamber; (ii) a 10 µT chamber; (iii) a chamber at 10 µT with superimposed random noise. Exposure to 10 µT for 4 h at CUA gave an ODC ratio of 1.19 ± 0.33 (Table II). Cells exposed to 10 µT with random noise superimposed gave an ODC ratio of 1.04 ± 0.37. There was no statistically significant difference between magnetic field-exposed and sham-exposed cell ODC activity (P = 0.27). Neither was there a statistically significant difference between field-exposed and field-exposed with superimposed random noise (P = 0.51).
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To determine whether there was a change in ODC response in cells exposed for varying times, cells were exposed (60 Hz, 10 µT) in the FDA system for 1, 2, 4 and 6 h. There was no observed change in ODC activity for varying exposure duration in three separate experiments. ODC activity ratios were 1.04 ± 0.06, 1.03 ± 0.18, 1.00 ± 0.07 and 1.05 ± 0.05 for exposures of 1, 2, 4 and 6 h, respectively.
To characterize the importance of DC magnetic field for the response of ODC activity to an AC field, we conducted experiments using a 60 Hz field with a superimposed DC field. A 1.4 µT (1.2 µT vertical component, 0.7 µT horizontal component) DC field was generated in the FDA system to approximate the measured DC field within the mu-metal enclosures of the CUA exposure system. For six experiments performed with this superimposed DC field, the ODC activity ratio was 0.92 ± 0.15. In addition, six experiments (each) were performed with superimposed horizontal (only) DC fields of 1.2, 5 and 10 µT in the FDA system. These experimental series gave ODC ratios of 0.83 ± 0.05, 0.99 ± 0.28 and 1.20 ± 0.22, respectively. Thus, in no case did addition of DC fields to the AC exposure alter the ODC activity of cells. Similarly, a series of experiments performed in the REEF system with an applied DC field of 5.7 µT (vertical component 3.8 µT) resulted in an ODC activity ratio of 0.96 ± 0.16.
Effect of cell culture conditions
To address the possibility that different subcultures of L929 cells might differ with respect to ODC response to EMF exposure, we compared cells obtained from ATCC to those obtained from Litovitz and co-workers. For 17 experiments, EMF exposure of L929 cells gave ODC activity ratios of 0.91 ± 0.18 (CUA cells) and 1.00 ± 0.20 (ATCC cells). The results from the two subcultures are not significantly different (P = 0.34).
We also examined the possibility that the source of cell culture medium might influence the response of cellular ODC to EMF exposure by comparing medium obtained from CUA with that prepared in FDA laboratories. Cells grown in CUA medium gave an ODC activity ratio of 1.19 ± 0.29 and those grown in FDA medium gave a ratio of 1.01 ± 0.14. These results do not differ significantly (P = 0.34).
To test the possibility that the magnetic field exposure history of cell cultures was a critical parameter for ODC induction by 60 Hz magnetic fields, we performed a series of five experiments using cells which had been cultured in an incubator that we identified as having low ambient AC fields (<0.3 µT) with no other shielding. Thus, the magnetic field exposure history of the cells included exposure to the ambient geomagnetic field. Four hour, 60 Hz, 10 µT exposures of L929 cells grown in this manner gave an ODC activity ratio of 0.93 ± 0.20, which is not significantly different from similar exposures of cells that had been shielded from the geomagnetic field during culture.
We also evaluated whether the purity of CO2 could be important for the response of cellular ODC to EMF exposure. High purity CO2 was used for the work reported above. However, welding grade purity CO2 is used in many laboratories for cell culture. When we exposed L929 cells grown for several passages using welding grade CO2 to a 60 Hz, 10 µT field, the ODC activity ratio was 1.05 ± 0.15 for a series of five experiments. Thus, no difference in results was obtained by using CO2 of lower purity.
Finally, we examined the influence of cell culture density on ODC response to the magnetic field. In a series of three experiments, exposure of cells plated at two densities (3 000 000 and 6 000 000 cells/flask) to a 60 Hz, 10 µT field showed no significant difference from each other and in neither case did the ODC activity of the exposed cells differ significantly from the paired sham-exposed cells (data not shown).
Statistical evaluation indicated no effect of a 4 h, 10 µT, 60 Hz magnetic field exposure on ODC activity. Presentation of the data from such experiments as frequency distribution plots (Figure 2) further shows that while an occasional experiment might appear to indicate ODC enhancement by EMF exposure, there is no discernible difference in the distribution of data between sham versus sham experiments and exposed versus control experiments.
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Discussion |
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We report here the results of experiments to address the accuracy and reproducibility of the EMF effect on ODC using reagents, methods and exposure conditions that duplicated those of the original investigators as closely as was practical. In addition, we worked closely with the original investigators toward identifying and testing previously unexplored parameters that might be critical to the ability to observe EMF induction of ODC activity. We also extended the previously reported investigations by testing a number of exposure conditions and other associated variables.
Initial results of exposures of L929 cells conducted in the Regional EMF Exposure System at 60 Hz and 10 µT for 4 h did not indicate a significant increase in ODC activity, in contrast to the doubling of ODC activity reported by the Litovitz group (8). Similar results were obtained from exposures in the FDA Exposure Facility. An extensive series of experiments was then conducted to test the effects of varying magnetic field strength, superimposed static field strength, cell density, serum concentration and source, source of cell growth medium and purity of CO2 in the growth incubator. This experimental series did not identify the conditions sufficient to demonstrate an effect of magnetic field exposure on ODC activity. While three preliminary cell exposures in the original investigators' system at CUA indicated a doubling of ODC activity, neither experiments conducted at FDA with the major components of the CUA exposure system nor a subsequent repetition of the exposures in the exposure system at CUA showed any enhancement of enzyme activity.
Though it is impossible to prove the absence of an effect, statistical evaluation and presentation of data as frequency distribution plots demonstrated only the baseline variability of data from a particular biological assay system. Thus, our data provide no evidence supporting an effect of exposure in vitro on the activity of a cellular enzyme.
Litovitz et al. (611) reported a significant enhancement of ODC activity in response to either 60 Hz magnetic fields or modulated microwave exposures. The magnitude of this effect was dependent on signal coherence time and superimposed random noise. The reason why we have been unable to replicate the enhancement of ODC activity by 60 Hz fields is unknown. It is possible that this effect may be dependent on biological or exposure parameters unique to the conditions that once existed at CUA. It is tempting to speculate that insufficiently defined parameters have contributed to a decrease in the magnitude of the effect observed at CUA. Their earlier work reported a doubling of ODC activity (6), whereas more recently they have reported only a 70% increase (10).
Our studies raise questions regarding the relevance of magnetic field-induced ODC enhancement to any plausible health effects of magnetic fields in the intact organism. Sensitive dependence of an EMF-induced enhancement of ODC activity in vitro upon insufficiently defined parameters may be interpreted as indicating that the biological effect is too weak to overcome the homeostatic mechanisms in the intact organism and thus would not influence the development of cancer or other adverse effects.
We have conducted a large series of experiments using a methodology which is very similar to, and probably identical to, that employed by Litovitz et al. Our ability to duplicate the original conditions was significantly enhanced by the fact that one of us (A.B.D.) was a member of the Litovitz laboratory and performed many of the experiments published by that group. Through close work with Litovitz and co-workers, we were able to share their detailed original protocols, critical reagents and exposure system components. This relationship also helped us to characterize the importance of many variables for the effect of EMF exposure on cellular ODC activity. Despite this high level of cooperation, we have been unable to define conditions that are necessary and sufficient to observe induction of ODC activity by EMF exposure.
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Acknowledgments |
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Notes |
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References |
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