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| TITLE: | EMF Induced Signal Transduction and Gene Expression | ||
| Principal Investigator |
Steven C. Miller, Ph.D. | SRI International | |
| Health Relevance |
Cancer | ||
| Research Categories |
Cellular Function | Gene Expression | Signal Transduction |
| FY95 Funds | R01ES07127 $ 198,742 | Start Date 09/28/94 | End Date 08/31/98 |
| Rationale and Summary |
Evidence from epidemiological studies has suggested a possible association between
exposure to power frequency electric and magnetic fields (EMF) and increased incidence of
cancers. Despite numerous studies on the biological effects of EMF, no clear consensus has
been reached by the scientific community concerning the health effects of EMF. Well
characterized EMF exposure systems and reproducible biological model systems are
necessary to effectively investigate this problem.
Our aim is to evaluate the hypothesis that 60 Hz EMF may amplify the signal transduction pathway induced by the chemical tumor-promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Because the EMF exposure parameters and the fundamental principles by which the energy in EMF may be coupled to a biophysical process are unclear, our first goal was to use a sensitive model system that would allow both positive and negative controls to be included in the experimental design to systematically evaluate the effect of the EMF. This goal has been achieved by focusing our initial studies on the regulation of NF-kB or AP-1 dependent reporter gene expression in the human promonocytic cell line U937. Gene expression refers to the process of stimulating the production of messenger RNAs, which code for specific proteins. These unique proteins coded for by specific genes, then take part in various cell activities, from growth and metabolism to cell division Our model system measures gene expression using a model reporter gene with an assay system that detects the amount of reporter protein produced in cells. Thus, this system evaluates the ability of a 60 Hz EMF to modulate a TPA-induced signal transduction pathway by acting through the well-defined transcription factors: NF-kB; the human immune deficiency virus type 1 long terminal repeat (HIV-1 LTR), which contains two copies of the kB sequence, was used as the biological sensor for the detection of signal transduction pathways acting on the HIV-1 LTR directed chloramphenicol acetyltransferase (CAT) reporter gene. In addition, another DNA construct containing four copies of the kB sequence (4XkBCAT) has been developed for use in this model system to demonstrate that effects are mediated through the NF-kB binding site. AP-1; the CAT gene is placed downstream of a human metallothionein IIA minimal promoter and four AP-1 recognition sequences (4XAP-1CAT). |
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| Experimental Design and Exposure Conditions |
The reporter gene is introduced into U937 cells by electroporation, 24 h later, replicate
cultures are treated with TPA to induce CAT expression. Replicate culture flasks are placed
into a control, sham, or EMF exposure incubator and 24 h later harvested. Protein values are
determined for cell extracts and the amount of CAT protein is determined by CAT ELISA.
Because the CAT protein is relatively stable, measuring the amount of CAT protein
measures the sum of gene expression from the time of electroporation, treatment with TPA,
to the time of harvest. Because this methodology is very sensitive and reproducible it is
particularly well suited for EMF studies.
Our experiments are performed in T-25 Corning flasks (25- cm2) containing 5 ml of medium. Flasks are oriented in the magnetic field in the parallel configuration (the magnetic field is oriented parallel to the base of the flask). Culture flasks are placed into defined positions marked by a grid within the sham and exposure chambers which are temperature controlled (37 ± 0.1 oC) by circulating water through the jacket of the walls and doors of the chambers. A set of Helmholtz coils produce spatially uniform magnetic fields with a maximum field of about 13 gauss (1.3 mT). The field is generated from an unregulated commercial 60 Hz source using a variable autotransformer to control the field intensity. Experiments are done in replicates under blinded conditions to systematically evaluate the EMF exposure parameters. |
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| Quality Assurance Measures |
A site visit and project review coordinated by William Wisecup, Frederick Dietrich, and Paul Gailey of DOE was held on December 20, 1994 at SRI at the request of the Principal Investigator. The purpose of this site visit was to impartially investigate and document possible causes of repeatable results which seemed to reflect an effect related to position within the exposure system. Their review suggested a temperature gradient or a nonuniform field. A series of temperature and field measurements made by the site visit team with their equipment documented the presence of a temperature gradient that could account for the differences observed between exposed and non-exposed cells. This site visit greatly assisted the Principal Investigator in obtaining internal funds for designing a new exposure facility for these studies. The new system has two identical exposure units temperature controlled by water circulation to allow for the simultaneous incubation for sham and EMF exposed cells. | ||
| Results and Discussion |
The HIV-1 LTR or 4XkB directed CAT reporter gene is strongly responsive to TPA (>100-
fold induction at >4 ng/ml TPA). A series of experiments with the HIV-1 LTR construct
demonstrated that the calcium ionophore ionomycin stimulated the effect of suboptimal
concentrations of TPA in a dose-dependent manner. Similar results were found with
thapsigargin which has been shown to increase intracellular calcium from internal stores.
Thus, this model system is appropriate to evaluate the hypothesis, based on observations by
others, that EMF may alter gene expression by increasing intracellular calcium levels.
Because any observed effects of EMF on signal transduction pathways are likely to be weak, a series of experiments were done using TPA and the antioxidant pyrrolidinedithiocarbamate (PDTC). These studies established a framework using well defined chemicals for demonstrating the ability to detect stimulatory and inhibitory activity on the signal transduction pathways controlling reporter gene expression. The DOE site visit revealed that a repeatable effect was related to position within the exposure system because of a temperature gradient. That our preliminary studies have detected an artifact related to a temperature gradient in the exposure system attests to the sensitivity of the assay system. Our new sham and exposure system has water jacketed doors; initial studies have demonstrated temperature control between chambers at 37 ± 0.1 oC. A blinded experimental protocol has been developed to assess possible field effects on TPA-induced signal transduction. |
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| Recent Publications |
J. Haberer and S. C. Miller, A Model System for Evaluating the Biological and Physical
Exposure Parameters Responsible for the Effect of 60 Hz Electromagnetic Field on Gene
Expression. Cell and Molecular Biology Laboratory, Life Sciences Division, SRI
International, Menlo Park, CA 94025. Abstract presented at the 43rd annual meeting of the
Radiation Research Society, 1995.
S. C. Miller and J. Haberer, Development and Use of a Model System for Evaluating the Biological and Physical Exposure Parameters Responsible for the Effect of 60 Hz Electromagnetic Field on Gene Expression. Cell and Molecular Biology Laboratory, Life Sciences Division, SRI International, Menlo Park, CA 94025. Abstract presented at the 17th annual meeting of the Bioelectromagnetics Society, 1995. |
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