We propose to characterize this MF interaction at the cellular level and to assess several plausible mechanisms of action. In biophysical studies we will a) determine the dose- threshold for the magnetic field strength, b) define the role of the induced electric vs. magnetic field, c) examine the frequency-dependence, d) test for an exposure-time dependence, and e) assess the reversibility of the MF blocking effect. A biological basis for the field interaction will be investigated in several ways. MF are reported to trigger early signal transduction events such as [Ca+2]i and it is possible that stimulated MCF-7 cells could overcome growth inhibition of MLT and TAX. We will follow changes in [Ca+2]i in real time at the single-cell level using digital imaging technology. Alternatively, MF may directly alter entry and/or distribution of MLT and TAX in MCF-7 cells, and immunohistological studies will assess this interaction. It is possible that MF may specifically influence estrogen receptor expression which could explain MF blocking TAX action, and this will be assessed. The above studies will significantly help define how a environmental MF act to influence hormone/drug regulation of proliferating human breast cancer cells.

Exposure System: We have developed over the last several years a special exposure system for long-term exposure of cells to very uniform magnetic fields. This system includes a Merritt four-coil exposure apparatus that is placed inside of a mu metal chamber, and this assembly is then placed inside of a commercial cell culture incubator. We have taken great lengths to ensure that our cells are only exposed to the waveforms and magnetic fields generated by the four-coil Merritt device. The use of a mu metal chamber is critical since it acts as a shield against both electric and magnetic fields that are generated during the operation of the cell incubator. These magnetic fields can be considerable (many times greater than the "environmental" level field exposures of 12mG(rms) which we employ in our studies). We routinely map the magnetic fields before and after experiments, and we monitor temperature and CO2 continuously and record values daily In addition, we have nine (9) identical exposure systems that are comprised of identical components, including incubators from the same manufacturer. We have found that ambient magnetic field levels inside of operating incubators can vary significantly both in spatial distribution and in time depending on the manufacturer. Thus, our use of nine identical exposure systems ensure that exposures will be identical. We design experiments to use multiple incubators simultaneously so as to conduct experiments with the same passage cells. This is a factor in reproducibility of our results.

Exposure Regime: Exposures to MCF-7 cells will be at levels that are considered "environmental": 2 - 12 mG(sinusoidal waveform). The DC magnetic field will be nulled to values near zero mG. We plan to vary field intensity (2- 12mG) to determine a dose- threshold. We will also vary frequency over a 5-fold range of 60Hz harmonics (15-300 Hz). Exposures will be continuous over the entire length of the 7-8 day growth period, however, we will also investigate if shorter exposure times are also effective in altering growth.

Blinding: We blind the experiments at three levels in the follow manner. First the experimenter is not aware of the treatment group for melatonin or for tamoxifen. Second the experimenter does not know which magnetic fields are generated in the incubator(s) they are using for experiments. Third, the data can be worked-up in a blinded manner and then the codes are broken.

Experimental Design: We design experiments to use multiple incubators simultaneously so as to conduct experiments with the same passage cells. We feel that this is a critical factor in reproducibility of our results. We are able to do this because we have nine (9) identical exposure systems that are comprised of identical components, including incubators from the same manufacturer. We have found that ambient magnetic field levels inside of operating incubators can vary significantly both in spatial distribution and in time depending on the manufacturer. Thus, our use of nine identical exposure systems ensure that exposures will be identical and that same passage cells can be used in experiments. In addition, we will be rotating experiments so as to use different incubators for testing in a random manner; this will rule-out any confounding effect due to the use of specific incubators.

Positive Controls: We design experiments so that we always culture several plates of cells in every incubator in the absence of melatonin or tamoxifen. This permits us to compare growth characteristics of MCF-7 cells in each incubator; magnetic fields have not been observed to alter cell growth of MCF-7 cells unless melatonin or tamoxifen is present. Thus, the growth curves of non-treated cells should be nearly identical across all incubators - which can be used as a QA check.

In addition, a positive biological control we use in all of our studies is the inhibition of MCF-7 cell growth by melatonin or tamoxifen in a 2mG(60Hz) magnetic field. This response should be within standard prescribed limits defined by our historical data and this establishes that the cells have the proper biological response to drug treatment - cells that are unresponsive to drug/hormone treatment fail this biological check and are not used in the magnetic field experiments.

1. The follow-on studies we have conducted confirm our original findings as presented in the original proposal.

2. The oncostatic action of melatonin and Tamoxifen are blocked or inhibited by a 12 mG environmental-level magnetic field.

3. This interaction involves the magnetic field itself and is not associated with the induced electric field.

4. The interaction clearly requires the presence of the hormone melatonin or the drug Tamoxifen, but the dose response data suggest that the interactions are mediated in a different manner. Perhaps this reflects a difference in the way the two compounds are processed in the cell. We plan to investigate the role of the estrogen receptor in the future. This may reveal a mode of interaction for Tamoxifen since it is known to act through the ER.

5. We observe that a 12 mG magnetic field inhibits Tamoxifen action at the pharmacological dose of Tamoxifen. This in vitro data suggests that environmental level magnetic fields may interfere with the drug s efficacy in humans. It is important to note that this is speculation and represents an extrapolation to the in vivo case for which we have no data.

6. The inhibition of Tamoxifen s action by 12 mG magnetic fields can be overcome by increasing Tamoxifen s dose several log units. This suggests that raising Tamoxifen s dose in vivo may be an appropriate strategy to avoid magnetic field effects. This raises issues of drug toxicity and side-effects that are more properly addressed in a clinical setting.

2. Inhibition of Melatonin s and Tamoxifen s Action in MCF-7 Cells by Magnetic Fields. J.D. Harland and R.P. Liburdy. Presented at the 34th Meeting of the American Society for Cell Biology. San Francisco, California. Abstract 107. Published in Molecular Biology of the Cell 5: 19a (1994).

3. 60Hz ELF Inhibition of Melatonin and Tamoxifen Action on MCF-7 Cells; E vs. B Field Components. J.D. Harland & R.P. Liburdy. Presented at the 17th Bioelectromagnetics Society meeting held on June 18-22 in Boston, MA. Abstract 3-3

Publications.
1. Environmental Magnetic Field Inhibition of Effects of Melatonin and Tamoxifen on Breast Cancer Cell Growth, J.D. Harland & R.P. Liburdy. Journal of the National Cancer Institute, submitted.