Because the body of epidemiological studies are inconclusive, the scientific community has looked to laboratory studies for clarification of the biological effects of EMF. The results of some of these laboratory studies indicate that biochemical changes can occur at the cellular level when cells are exposed to EMF. Despite the number of studies on the biological effects of EMF, there remains considerable controversy concerning whether observed bioeffects would lead to health effects. Among the reasons for the controversy are reports of both positive and negative effects in different laboratories doing similar studies. Another reason is that many of the bioeffects reported in the peer-reviewed literature have not been replicated. Also, there are scientists who believe that power frequency fields contain too little energy to cause any biological effects. These scientists argue first, the energy of a 60 Hz electromagnetic wave is too weak to break chemical bonds, and second, natural electric fields in the body are greater than those that can be induced by common EMF exposure. Thus, there is not as yet a consensus on the biological effects of EMF on the human health consequences for any such bioeffects.

One of the highly cited and studied areas for EMF bioeffects is on the behavior of cells exposed to electric and/or magnetic fields. Studies have examined a wide range of endpoints. For example, in vitro studies report effects on cell membranes, RNA transcription, ornithine decarboxylase activity, calcium-ion efflux, and cellular response to hormones. These responses to EMF at the cellular level display a considerably complexity. There have been a number of studies initiated to replicate these earlier results that have not obtained similar results; therefore, there continues to be controversy. However, in most instances, investigators have not completely duplicated exposure conditions and assays, and therefore, the lack of response reported by other investigators may be due to differences in the protocols. Thus, there is a need for additional research, particularly careful replication studies, on the biological effects of EMF exposure, especially at power line frequency (60 Hz).

The main objective of this research projects will be to validate or refute important findings of cellular responses of exposure to extremely low frequency (ELF) magnetic fields. Emphasis will be placed on attempted replication or validation of published work. Specifically, the cell environment (media, nutrients, temperature, humidity, etc.) As well as the specified field exposure conditions will be duplicated. Initial validation will be followed by confirmatory studies which will supplement prior findings and provide more insight into possible underlying mechanisms of bioelectromagnetic interactions. Three replication projects have been initiated: (1) analysis of the expression of proto-oncogenes in promyelogenous leukemia (HL-60) cells, (2) analysis of the activity of ornithine decarboxylase (ODC) in mouse L929 cells, and (3) analysis of rat PC-12 cell differentiation.

A number of exposure systems have been used. An exposure system was designed and built using a pair of identical mu metal enclosures to block out ambient fields (Davis et al, BEMS 17th Annual Meeting, 1995). This system has been used for each project. In addition, experiments have been conducted in exposure systems provided by the original investigators and/or in the laboratories of the original investigators. A regional ELF-EMF exposure facility (to be described at the 1995 DOE Contractors’ Review) has been installed and used for some of the experiments. Cell exposures were performed at 37 ± 0.3 C. Control cells were sham exposed in the same incubator. Temperatures and magnetic fields were continuously monitored and recorded during the experiments.

(1) Gene Expression in HL-60 Cells: Cell exposures are conducted at 60 Hz, 0.06 G for a 20 minute period of incubation. Gene expression relative to differing conditions of ELF-EMF is assayed as follows: Extraction of RNA from cells is performed by the method of Henderson and Goodman and analyzed by blot hybridization. Quantitation of radioactive hybridization data is achieved using a storage phosphor image analysis system.

(2) ODC activity in mouse L929 cells: Cell exposures were performed using the conditions previously reported to give the maximal (two-fold) enhancement: 60 Hz, 0.1 G for four hours. Following exposure cells were lysed in buffer and assayed for ODC activity by incubation with 14C- Ornithine with capture and quantitation of released 14CO2.

(3) Rat PC-12 cell Differentiation: Cell exposures use an AC/DC magnetic field combination reported as the most effective field in inhibiting neurite outgrowth (45 Hz, 233 mG AC; with colinear 366 mG DC). In close communication with the original investigators, we established a protocol and criteria for assaying neurite outgrowth in response to NGF treatment.

Reciprocal visits with the original investigators’ laboratories have been made for each of the projects.

Experimental samples have been exchanged with the original investigators’ laboratories for analysis.

Preliminary progress is presented at open scientific meetings. All work will be submitted for publication in the peer-reviewed scientific literature upon completion.

The gene expression studies incorporate the following types of controls: Control baseline studies to determine the precision and accuracy of the assay methods. Positive control studies to provide a frame of reference for the magnitude of signals observed in the presence of ELF-EMF. Internal controls are used to assure that data are comparable both across different samples within an experiment as well as between separate experiments.

All exposures are conducted in a blinded fashion, such that the respective identities of exposed and control samples remain unknown until all data are obtained.

(2) ODC activity in mouse L929 cells: When L929 cells are exposed in our facility, we do not observe an enhancement of ODC activity. This is true whether the cells are obtained from the original investigators or ATCC. This led to efforts to more closely duplicate the exposure conditions of Litovitz et. Al. Measurements at their facility revealed a small residual DC magnetic field which we attempted to stimulate using a coil energized with DC current. These efforts did not improve the agreement with the prior results. Interestingly, however, when we transported cells to the original investigators’ facility for exposure, and repeated the ODC assay here, we were always able to observe a doubling of ODC activity. Our inability to replicate the ODC enhancement in our exposure facility suggests that some unknown characteristic of the exposure conditions, in addition to magnetic field strength, is critical for this particular biochemical endpoint. We have attempted to rule out DC field as the missing factor. Continuing efforts to replicate the ODC effect will involve construction of an exposure system that is more nearly identical to that of Litovitz et. al.

(3) Rat PC-12 cell Differentiation: In the neurite-extension assay, sham exposures in the FDA double mu metal incubator produced no significant difference for a 5 ng/ml NGF exposure. In a series of 5 field experiments, a 50 Hz, 233 mG AC field plus colinear 366 mG AC field plus colinear 366 mG DC also produced no change from the controls. Sham exposure of ATCC cells in the mu metal box loaned by the original investigators showed no significant difference in the neurite-extension assay from the controls, as did field exposure at both 5 ng/ml and 2.5 ng/ml NGF. PC-12 cells obtained from the original investigators were compared to PC-12 cells obtained directly from ATCC. The PC-12 cell strain from the original investigators was much less responsive to NGF, and was morphologically different from the ACC P12 cell. In close communication with the original investigators, factors that might be preventing a field effect from being seen were investigated: presence of fungizone in the media, presence of greater than 5 mG non-colinear vectors, stress of disaggregation of clumps of cells, potential silicone sealing vapor in the FDA incubator, and the concentration of NGF used in the neurite extension assay. None of these factors seemed to be preventing the field effect from being seen. Our present work is concentrating on gearing up to doing the assays using the strain of PC-12 cells provided by the original investigators. It is possible that weaker responsiveness to NGF may be responsible for allowing field effects to be seen.