Our goal is to obtain specific, measurable experimental endpoints which will allow us to determine whether EMF exposure is an initiating factor in neoplastic transformation and/or if exposure can mimic characteristics of the "second or other-step" counterpart in the multiple independent events leading to neoplastic disease. The specific question is whether characteristics of neoplastic transformation and/or immortalization occur in cells exposed to extremely low frequency (elf) EMF at 60 Hz under environmentally significant conditions.

The specific goals of this research project are:

Aim 1. To determine in cultured human cells exposed to defined ELF EMF: (a) if the expression of cellular proto-oncogenes is altered; (b) if changes in transcription are present in all cell types; i.e., those derived from both neoplastic and "normal" cells, and the time course of the response and if it consistent with that observed for known promoting factors, such as TPA.

Aim 2. To investigate the role of signal transduction in cell response to ELF EMF; can a relation with signal transduction (and the cell membrane) be made as predicted on the basis of calcium flux studies?

The cultured cells are exposed to EMF using the Helmholtz coils constructed by EBI, Inc., and employed in earlier experiments. The coils were made using 164 turn rectangular windings of gauge 19 magnet wire measuring approximately 13 cm x 14 cm with 8 cm spacing. The coils are 1 cm in diameter wound around an approximate square form. They are positioned with their central axis horizontal; samples are placed on a plexiglass stand in the horizontal plane in an area shown to have a uniform magnetic field and maximum field strength. Exposures take place in a mu metal can. We are currently using a Double Blind Helmholtz Coil Exposure System designed by Electric Research and Management, Inc. All features of our previous system were incorporated into the new design, such as coil dimensions, wire size, number of turns, coil form dimensions, coil form material, shielding enclosure dimensions and shielding enclosure material. This provides two functional units for simultaneous sham exposures. There are dual switches for selecting module controls; one set is inside a locked cabinet.

Single continuous exposures ranging from 2 minutes to 4 hours are carried out at a magnetic field intensity of 60 mG at 60 Hz with usual conditions equaling 20 minutes exposure with no post- exposure time. Harmonic content is sinusoidal; polarization is linear. The DC magnetic field is 82 mG 12.7o [100 vertical; 0 horizontal. The X component of DC fields is parallel to the AC field. The cells are shielded in mu metal container during exposure. The horizontal DC magnetic field in the container is less than 20 x 10-7 tesla (measurement by M. Masakian). Fields are turned on 30 minutes prior to exposure; they are turned off and the cells removed rapidly. Square 5 x 5 cm (T- 25) flasks are used for exposures. The bottom of the flask is 2 cm below the axis level. The height from flask bottom to top surface of liquid is about 1.1 cm; the height of the liquid is 0.6 cm. The cells are in suspension culture, but are on the bottom of the flasks; the concentration is about 1 x 106 cells / ml. The cells are maintained in T-75 flasks. A magnetic field survey in all portions of the laboratory is routinely done. The area around the exposure apparatus and preparation bench are monitored before each experiment. (Control and experimental samples remain in the same flasks and are treated identically until exposure). The field inside the mu metal container is 0.8 mG. Ambient static magnetic fields inside the mu metal can at its normal position are: Horizontal(east- west): 3.2 mG; Horizontal (north-south): 4.7 mG; Vertical: ~ 10.5 mG; Total: 11-12.9 mG (measurements by F. Dietrich). No vibration or warming of coils is detectable.

Experiments are performed in a small laboratory which is removed from possible sources of extraneous signals (elevator, NMR, X-ray diffraction, heavy equipment, etc.). The laboratory and equipment are monitored routinely by outside consultants. A map has been made of all possible sources of extraneous fields in the area of the exposure apparatus. The field inside the incubator used for exposures has been relatively constant at about 2 mG over a 5 year period of monitoring. A water-jacketed incubator is used.

A research associate is in charge of maintaining quality control for biological experiments, i.e., that appropriate procedures are followed, that coding takes place, reagents are standardized, all buffers sterile, etc. The laboratory is monitored before each experiment using a METEX digital multimeter. Temperature is monitored using a Physitemp thermocouple probe that is sensitive to 0.1oC.

(2) The response to EMF is rapid. Four minutes is the earliest time point after exposure that an increase in c-myc or c-fos transcripts can be measured.

(3) At the field strengths used in many of our experiments (8 or 80 µT), the increase in c-myc transcripts peaks at 20 minutes. Following 20 minute exposures, removal of cells at various time points shows that the effect is diminished with time.

(4) There may be overlapping pathways between heat shock and exposure to EMF. Although hsp70 mRNA is increased (as possibly other heat shock proteins on the basis of 2-D gel electrophoresis) in the presence of EMF, the reaction is not heat shock per se.

(5) The cell membrane is important in the response to EMF. Treatment of cells with colchicine disturbs microtubules and decreases plasma membrane fluctuations. Cytochalasin treatment influences microfilament structure and increases local membrane fluctuations. C-fos transcript levels are decreased in the presence of colchicine (+EMF), but enhanced in cytochalasin B (+EMF). We conclude that there is a correlation between the amplitude of plasma membrane fluctuations and the response of the cell to EMF.

(6) Reduction or absence of extra-cellular calcium negates the effect of EMF on transcript levels. The effect of EMF on the steady state levels of c-fos and c-myc is suppressed when HL-60 cells are placed under conditions of calcium reduction or the absence of calcium (EGTA) .

(7) Calcium channel blockers negate the effect of EMF (unpublished data). Calcium flux through L- and T-type transmembrane calcium channels was blocked 10 minutes prior to EMF exposure by the use-dependent calcium channel blocker, Verapamil (100 µM).The steady state level of c-myc mRNA was increased after EMF exposure, but the effect was blocked by adding Verapamil 10 minutes before exposure. 2-microglobulin gene expression was used as an internal control.

(8) CAT expression is increased in HeLa cells exposed to EMF following transfection of portions of the c-myc upstream control elements linked to a CAT reporter gene . C-myc upstream regulatory regions were transfected into both mouse (with integration) and human (transient) cells as CAT constructs. The presence of the 2.3 kb domain upstream of the human gene results in increased expression of CAT following 20 minute exposure of HeLa cells to 60 Hz EMF (8 µT). The transfection of a similar region from mouse c-myc regulatory DNA showed increased expression in mouse myeloma cells (PX3) [the maximum increase was at a B field of 80 µT, consistent with an increased expression of endogenous c-myc in this cell line]. Specific portions of the promoter region were deleted from the human promoter construct and introduced into cells to determine the responsive region to EMF. The region is less than 800 bp and is located between -353 and -1257 relative to the P1 promoter. Experiments in progress are designed to identify a more precise region.

(9) Various portions of upstream regulatory regions of human c-fos (0 to -700 bp) were linked to CAT constructs as a reporter gene. Maximum expression (~+25%) is seen within 20 minutes of exposure using the complete construct, but the activity drops rapidly. Deletion analysis of the upstream regulatory DNA narrowed the responsive region to ~340 base pairs. Further experiments will determine if this region is sufficient or if it acts in conjunction with another region. Fos protein is phosphorylated at serine residues. Other studies have shown that phosphorylation causes the protein, in conjugation with Jun, to down regulate its own promoter. Preliminary results, using immunoprecipitation, show that phosphorylation of the c-fos protein is increased in cells exposed to EM fields for 20 minutes. Protein was measured one hour after exposure, the point at which the protein was shown to peak. The increase in phosphorylation was about 30%. These studies suggest that EMF can enhance and down regulate the c-fos gene in a manner which is analogous to other growth factors.

Broude, N., R. Karabakhtsian, N. Shalts, R. Goodman and A. Henderson, 1994. Correlation between the amplitude of plasma fluctuations and the response of cells to electric and magnetic (EM) fields. Bioelect. Bioenerget. 31: 19-23.

Lin, H., R. Goodman and A. Henderson, 1994. Specific region of the c-myc promoter is responsive to electric and magnetic fields. J. Cell. Biochemistry 55:1-8.

Goodman, R., M. Blank, H. Lin, O. Khorkova, L. Soo, D.Weisbrot and A. Henderson, 1994. Increased levels of hsp70 transcripts induced when cells are exposed to low frequency electromagnetic fields. Bioelectrochem. Bioenerget.33: 115-120.

Gold, S., R. Goodman and A. Henderson, 1994. Exposure of human cells to electric and magnetic fields results in increased levels of T-antigen mRNA and protein. Bioelectromagnetics 15: 329-336.

Karabakhtsian, R., N. Broude, N. Shalts, S. Kochlatyi, R. Goodman and A. S. Henderson, 1994. Calcium is necessary in the cell response to EM fields. FEBS LETT. 349: 1-6.

Book Chapters:

Goodman, R. and A. Henderson, 1994. Effects of electric and magnetic fields on gene expression. IN Biological effects of electromagnetic fields (D. Carpenter, ed.) Academic Press, Orlando, FLA, pp.155-176.

Abstracts:

Karabakhtsian, R., N. Broude, N. Shalts, R. Goodman and A. Henderson, 1994. The presence of calcium is important in the cell's response to EM fields. BEMS, Copenhagen.

Rao, S., H. Lin, R. Goodman and A. Henderson, 1994. Regulatory DNA upstream of the c-fos and c-myc genes is responsive to electric and magnetic fields. BEMS, Copenhagen.

Goodman, R., M. Blank, H. Lin, L. Soo, D. Weisbrot and A. Henderson, 1994.Increased levels of hsp70 transcripts are induced when cells are exposed to low frequency electromagnetic fields. BEMS, Copenhagen

Goodman, R. H. Lin and A. Henderson, 1994. Increased transcripts for hsp70 and c-myc in cells re- stimulated with different exposure conditions. BEMS, Copenhagen

Kochlatyi, S., N. Shalts, R. Karabakhtsian and A. S. Henderson, 1994. The role of transmembrane calcium flux on EMF effects on cells. DOE Contractor's Meeting, Albuquerque.

Goodman,R., H. Lin, S. Rao, R. Karabakhtsian, N. Shalts, S. Kochlatyi and A. S. Henderson, 1994. Tests of EMF results; effects can be blocked. DOE Contractor's Meeting, Albuquerque.

Goodman, R. and A. S. Henderson, 1994. What constitutes replication? DOE Contractor's Meeting, Albuquerque.

Rao, S. and A. Henderson, 1995 Regulatory DNA upstream of the c-fos gene is responsive to EMF exposure, BEMS, Boston.