Our general objective is to test the hypothesis that 60-Hz magnetic fields (MF) have effects on genetic material leading to cancer. Accordingly, our project has two aims: (1) to investigate messenger RNA (mRNA) expression of c-myc, c-fos, p53 and ornithine decarboxylase (ODC) genes using several patterns of exposure and post-exposure times; (2) to identify and study other known and novel differentially expressed genes after the exposure conditions similar to those in Aim 1. Our Aim-1 studies include cells, 60-Hz magnetic field-exposure conditions and mRNA assays previously investigated and published by others. All the studies include additional non-MF treatments known to affect the proposed genes (i.e., X-irradiation, hyperthermia or a chemical tumor promoter).

Our first-year studies investigate the effects of power frequency (60 Hz) magnetic fields on transcription of several cellular tumor suppressor genes and protooncogenes. Carcinogen-induced deregulation of these genes creates the biochemical framework for aberrant cell proliferation, loss of differentiation, and development of metastasis. The c-myc, c-fos and p53 gene products (proteins) regulate expression of other genes, including ODC, and are involved in the signaling mechanisms within the cell that lead to an increase in de novo DNA synthesis and cell division; in addition, the ODC, c-myc and c-fos genes are important markers and mediators of tumor promoting agents. According to a recent hypothesis, power-frequency magnetic fields may act as a tumor promoter.

Variable exposure periods of 10 min to 24 h of human breast cancer (MCF7 WT) or pre-leukemic (HL-60) cells are carried out to investigate whether there is a critical exposure duration. It is still unknown which field parameter is the biologically most important MF exposure parameter. "Window" effects have been suggested for field intensity and time for c-myc, -actin and c-fos gene transcripts in HL-60 cells exposed to 60-Hz magnetic fields. Identification of an exposure metric (magnetic field intensity and/or exposure duration) that correlates with any cancer-related effect is one of the important goals of our ongoing studies. Field- or sham-exposed cells are lysed for RNA extraction at variable times ranging from 0 to 24 h after the cessation of a treatment, as suggested by the literature results. A typical experiment consists of 15 MF (test and matching control) groups and up to 20 non-MF (positive control) groups. Up to 20 RNA samples can be analyzed on a single Northern blot.

In Aim-1 studies, we have assessed alterations in mRNA levels by Northern blot analysis. Radiographic results are quantified by densitometry. Optical densities of experimental gene transcripts are compared to those of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 18S rRNA, 28S rRNA transcripts in exposed and corresponding control cells. The rRNA species approximate the total amount of RNA; the amounts of GAPDH and -actin are considered constant and proportional to the total amount of extracted mRNA under most circumstances (except for - actin following 60 Hz ELF exposures in one study); therefore, they are customarily used as control transcripts in messenger RNA (mRNA) analyses. RT-PCR method will be used to detect, when needed, low-level message alterations; The basal c-fos and ODC transcripts are undetectable in untreated MCF-7 and HL-60 cells. In Aim-2 studies, planned for years 2 and 3, HL-60 or MCF7 cDNA libraries will be screened with cDNA probes constructed from the total RNAs extracted from control and test cells. This novel method permits studying of gene expression regardless their basal level.

Field levels in hood areas have been measured to be less than 0.1 µT. Cell cultures are grown in commercial CO2-incubators. AC/DC fields inside the incubators' chambers were mapped; we use the incubators and/or incubator shelves with the lowest fields (less than 1 µT). Fields mapped in other work spaces and areas are less than 0.2 µT.

Our 60-Hz magnetic field exposure facility is consistent with the characterizations suggested by the NIEHS RAPID Program. Temperature and exposure parameters are monitored and recorded during experiments. Cells are exposed in a stringently controlled environment and assays are performed under blind conditions. RNA samples are coded by individuals not involved in performing molecular assays. In addition, in some of the AIM-1 experiments, the coded RNA samples obtained are divided between two teams and analyzed independently in two laboratories [in the Department of Radiation Oncology (EKBK) or Medicine (SJM)]. The autoradiographic data are analyzed statistically to avoid subjective interpretation. Positive (non-MFs) controls are included.

Fig. 1: Induction of c-fos mRNA following several different treatments. 60-Hz MF exposures at 5.7 or 570 µT lasting 10 to 40 min produced no positive response. RNA was extracted immediately after MF exposure or, in the case of non-MF treatments, at times indicated on the abscissa. Our results for TPA and hyperthermia are in a detailed agreement with those previously reported for HL-60 cells and c-fos by others (e.g, PNAS 87, 5663, 1990).

Our results do not support the original conclusions: we did not see MF-inducible gene expression for field intensities and exposure durations investigated previously. In contrast, we found that the variation of c-myc, -actin and c-fos in MF-exposed or control HL-60 cells was non-specific, i.e., significantly shared with the GAPDH transcript, 18S rRNA and with the total RNA (represented by 28S rRNA). In all the cases, the fluctuations of mRNA in exposed cells was similar to those in sham-exposed cells, i.e. ± 2.5 standard deviations. Such variations are expected for c-myc and - actin, whose basal levels are high in HL-60 cells. As shown in Fig. 1, the c-fos transcripts are not elevated by MF-exposure, in contrast to the previous report. Similar MF-exposures also did not induce the ODC transcription; this result confirms the previous observations published in the literature on MF effects.

Our lack of confirmation of MF effects on c-myc, c-fos, ODC and -actin certainly does not preclude the possibility of observing positive molecular responses for other genes and/or MF exposure conditions. Given the complexity of the emerging picture of the molecular basis of cell biology, we are confident that our remaining studies will shed light on possible connections between MF and cancer.

E.K. Balcer-Kubiczek, G.H. Harrison, X. Zhang, L. Ampey, Z.M. Shi, J.M. Abraham, W.A. McCready, S.J. Meltzer and C.C. Davis, Rodent cell transformation and gene expression following 60-Hz magnetic field exposure (Abstract)*. The Bioelectromagnetics Society Meeting, June 18-22, 1995, Boston, MA.

* This material will be included in the paper to be submitted for publication in Environmental Health Perspectives (manuscript in preparation).