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Key words: EMF, molecular biology, biological effects, oncogenes, biochemistry, ODC, cell biology, glioblastoma, microwaves, radiofrequency
Health issues concerning exposure to electric and magnetic fields (EMF) are discussed extensively in the news. There are a number of products under the regulatory authority of FDA which emit or utilize EMF radiation. These include electric blankets, video display terminals, and medical devices such as those used to treat non-unions (bone fractures), as well as wireless communications devices such as cellular telephones. Concern stems, in part, from epidemiological reports suggesting that EMF may play a role in cancer promotion. The program described below is part of a national research effort with contributions from NIH, CDC, DOE, and FDA. The data from this research will be used in an NIH risk analysis process leading to a Congressional report. This will place the FDA in a better position to evaluate safety claims of products emitting EMF. The principal objectives of the program are (1) to establish and maintain a regional in vitro magnetic field exposure facility and (2) to perform studies which address the issue of reproducibility of published EMF biological effects. This program was supported by NIH, FDA, and the Office of Naval Research. This program resulted in 10 published abstracts and 3 invited seminars at international scientific meetings during FY 96.
A state-of-the-art regional EMF exposure facility has been developed. The two systems that comprise the core of this facility are capable of exposing samples to environmentally relevant, extremely low frequency magnetic fields. They have been used for the work described below, as well as for research performed in collaboration with non-FDA investigators.
1. EMF and Gene Expression
This project (see program description above) has addressed the issue of cancer promotion through investigation of the effects of 60 Hz EMF exposure on gene expression. Specifically, the effect of EMF exposure on gene expression in human leukemia cells has been analyzed by screening for changes in steady state levels of specific messenger RNAs. This study also addresses the issues of repeatability and accuracy of published research. Specifically, it includes a replication of the published experiments that were performed to conclude that the expression of the MYC protooncogene is increased when cells are exposed to 60 Hz EMF.
A detailed protocol for the gene expression replication study was developed in collaboration with the original investigators and peer-reviewed by a panel of experts. The protocol was endorsed by the original investigators. Reciprocal site visits were made with members of the laboratories of original investigators to discover differences that existed between the original methods and OST methods, and protocols were revised accordingly. HL60 human leukemia cells were expanded to a large stock and their growth characteristics were carefully examined. Control exposures and RNA isolations were performed to acquire proficiency in procedures unique to the replication.
Control baseline studies were performed to determine the precision and accuracy of the assay methods. These experiments demonstrated that the methods used were capable of detecting reproducible changes in gene expression of greater than 10%. Positive control studies using chemical agents known to cause gene induction were initiated to provide a frame of reference for the magnitude of signals observed in the presence of EMF. These experiments demonstrated that the cells responded to a known tumor promoter and that the assay system was capable of making quantitative measurements of the resulting changes in gene expression.
Replication experiments were performed with cells obtained from the American Type Culture Collection as well as with cells obtained from the original investigators. Cells were exposed to the conditions previously reported to give maximal enhancement of MYC expression: 63 milligauss for 20 minutes. The 60-Hz magnetic field was generated by Helmholtz coils located inside a mu metal shield box in an incubator. Control cells were sham-exposed in an identical mu metal box in the same incubator. Temperatures and magnetic fields were continuously monitored and recorded during all experiments. Following exposure, cells were assayed for specific gene expression by Northern blot analysis and quantitative storage phosphor imaging autoradiography, using probes for MYC and for beta-2-microglobulin. MYC gene expression in EMF exposed cells was not distinguishable from that in sham-exposed cells, suggesting that the exposure conditions defined by the original investigators are not sufficient to cause an EMF exposure-associated change in MYC gene expression. Experiments designed to analyze possible confounding variables have been initiated, as have experiments designed to assess the effect of EMF exposure on the expression of other genes. This project contributed to four published abstracts and three invited seminars at international scientific meetings during FY 96.
2. EMF and Enzyme Activity
This project (see program description above) has addressed the issue of cancer promotion through investigation of the effects of 60 Hz EMF exposure on enzyme activity. Specifically, the effect of EMF exposure on enzyme activity in mammalian cells has been analyzed by screening for changes in the activity of ornithine decarboxylase (ODC), an important marker for cell proliferation and tumor promotion. Specifically, it includes a replication of the published experiments that were performed to prove that a weak, extremely low frequency (ELF) magnetic field could enhance the activity of ornithine decarboxylase (ODC) in mouse L929 cells.
Cell lines were obtained from the original investigators as well as from the American Type Culture Collection (ATCC). They were exposed to the conditions previously reported to give the maximal (two-fold) enhancement: 100 milligauss for 4 hours. The 60-Hz magnetic field was generated by Helmholtz coils located inside a mu-metal shield box in an incubator. Control cells were shamexposed in an identical mumetal box in the same incubator. Temperatures and magnetic fields were continuously monitored and recorded during all experiments. Following exposure, cells were assayed for ODC activity. ODC activity in exposed cells was indistinguishable from that in shamexposed cells. This was the case using either of two state-of-the-art exposure systems. This lack of a magnetic field effect was also observed using an exposure system constructed from components supplied by the original investigators. Interestingly, three experiments previously performed at the original investigators' facility for exposure gave a doubling of ODC activity, consistent with the published results. This suggests that the inability to replicate the ODC enhancement in other exposure systems might depend upon some unknown characteristic of the magnetic field, in addition to field strength, and is critical for this particular biochemical endpoint. Continuing efforts to replicate the ODC effect involve other cellular systems that are believed to respond to magnetic field exposure. This project contributed to four published abstracts and three invited seminars at international scientific meetings during FY 96.
3. EMF and Nerve Cell Biology
This project (see program description above) has addressed the issues of cancer promotion and developmental effects through investigation of the effects of 60-Hz EMF exposure on cell differentiation. Specifically, the effect of EMF exposure on nerve growth factor stimulated neurite outgrowth in rat pheochromocytoma (PC-12) cells has been analyzed by screening for changes in the number and size of neurites. In particular, it includes a replication of the published experiments that were performed to conclude that a weak, extremely low frequency (ELF) magnetic field could inhibit the formation of nerve growth factor stimulated neurite outgrowth in PC-12 cells
PC-12 cells from three different sources, including the original investigators, were used. Growth characteristics and responsiveness to nerve growth factor (NGF) was established with these cells. Under conditions of good cell growth and approximately half maximal neurite outgrowth response to NGF, no change in neurite outgrowth or proliferation in any of the PC-12 cell lines could be attributed to exposure to the magnetic field conditions specified by the original investigators to yield the maximum neurite outgrowth suppression. Collaborative attempts to identify confounding factors included the participation of the original investigators, but no confounding factor was identified to explain the disparate results.
In related experiments, a video imaging system was developed for quantifying neurite extension by normal cells as they were being exposed to electric fields. Electric fields similar to those present in developing tissue have been proposed as components of some nerve-bridging devices for stimulating or directing the regeneration of nerves in spinal cord or peripheral nerve injury. Outgrowth of neurites from explanted chick embryo dorsal root ganglia was monitored for up to 5 days as they were exposed to substantial electric fields of 0.1 to 1.0 V/cm. Effects on various aspects of neurite outgrowth were observed. Some of these effects were due to pH or heating artifacts from the fields rather than to the electric current itself. These experiments suggest that pH and heating artifacts need to be carefully documented and reviewed in evaluating safety and efficacy claims for the use of electric fields from electrodes implanted in nerve bridging medical devices.
This project contributed to two published abstracts and two invited seminars at international scientific meetings during FY 96.
4. Radiofrequency EMF and Cell Cycle Control
Concern has been raised over the possibility that the microwave radiation from cellular phones might in some way promote the occurrence of brain cancer. While these reports are more anecdotal than substantiated epidemiological studies, OST has begun independent studies looking at issues related to in vitro tumor promotion of brain cancer cells in cellular phone microwave fields. Specifically, human glioblastoma T98G cells were exposed to radiofrequency fields generated by a cell phone and flow cytometry was used to study the parameters of the cell growth cycle.
Proliferation of T98G cells ceases when the cells touch each other, a feature of normal cells that is generally lost in carcinogenesis. Tumor-promoting agents are known to interfere with the cell communication that results in contact inhibition. Thus, this cell line exhibits preneoplastic features important to tumor promotion. Cells in T25 culture flasks were exposed to a 835-MHz, simulated digital telephone-modulated electromagnetic field. The proportion of cells in the various phases of the cell cycle was measured using Cell-Fit software analysis of flow cytometric data. [PostMS, ProA]
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Key words: ultraviolet radiation, photosensitizing drugs, epidemiology, HIV
Skin diseases are extremely common among HIV-infected persons, and in many cases, being refractory to drug therapy, require treatment with ultraviolet radiation (UV)-alone or in combination with photosensitizing drugs. Treatments with UV-emitting devices are highly effective; however the safety of such devices in HIV infection has not been established. At the same time, it is known that UV activates HIV and is immunosuppressive. To address these issues, CDRH conducts epidemiological, clinical, and laboratory studies, and performs risk analyses.
1. Epidemiology
Final analysis of the data shows that the prevalence of known HIV-positive patients among all UV patients is substantial (8.4%). Probably, the proportion of HIV-positive UV therapy patients is even higher (the positive HIV status of some patients is unknown to them or to the treatment facility). We estimate that 2,000 to 8,000 new HIV-positive patients enter UV therapy each year in the U.S., and they are treated predominantly (80%) with UVB radiation. Only about 10% of HIVinfected UV therapy patients receive PUVA, a combined treatment with a photosensitizing drug (8methoxypsoralen) and UVA radiation. At the same time, PUVA is used to treat at least 40% of uninfected UV therapy patients.
2. Clinical Studies
The Center conducts studies on changes in the blood of uninfected and HIV-infected UV therapy patients to support analyses of possible adverse effects of UV therapy on HIV disease. In FY 96, in collaboration with the National Institutes of Health, Bethesda, Maryland, and The University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, studies have been completed on the effects of UVB therapy on cytokine profiles in the control group of uninfected patients with psoriasis. These patients improved clinically after 6 weeks of therapy, while their cytokine profiles (including interferon-gamma, interleukin (IL)-2, IL-4, IL-6, and IL-10) remained unchanged at the systemic level. The data obtained indicate also that cytokine profiles of untreated psoriatic patients might be considered to be Th-1 dominant with plasma levels of IL-10 higher than in normal volunteers.
3. Risk Analysis
Risk analysis of HIV activation by direct interaction with UV delivered during dermatological therapy has been completed for the two basic modalities, UVB and PUVA. It was found that the probability of HIV activation in the skin is low, since HIV is localized sufficiently far from the skin surface to allow attenuation of therapeutic UV to relatively small doses. Since HIV load in the skin is relatively low, direct HIV activation in the skin is considered a phenomenon of potentially small impact on HIV disease. Such small impact may be, however, significant when novel antiviral treatments are used to reduce the total viral load to undetectable levels, and complete eradication of the virus becomes the therapeutic goal. Our analysis indicates also that the probability of HIV activation in the skin is substantially higher in the case of UVB therapy as compared to PUVA. [PostMS]
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Key words: UV, UVA1 phototherapy, autoimmune, HIV, AIDS
Different phototherapy regimens are being used investigationally in the United States to treat a number of diseases, such as UVA1 phototherapy for systemic lupus erythematosus (SLE). In pilot studies, UVA1 radiation (>340 nm) has been reported to alleviate autoimmune symptoms associated with SLE and some other autoimmune diseases. The major cause of mortality in SLE is renal (kidney) failure. To investigate potential health risks of phototherapy, OST scientists used a mouse model of SLE and irradiation with UVA1 or unshielded fluorescent light (low UV) and compared the results with the known exacerbating effects of UVB exposure (primarily 311 nm) on SLE. SLE mice were shaved and exposed to UVA1 (at levels comparable to low dose therapy), UVB radiation, or to fluorescent lights (approximating a work environment) over a period of two months. Immunological analysis of lymphocytes demonstrated that a significant increase in the cytolytic (CD8+) T-cell population occurred in the UVA1-exposed mice (p<0.05) but not in the UVB- or fluorescent light-exposed mice. This may be a beneficial effect since the cytolytic T cells help to rid the body of defective cells, such as autoimmune cells. The UVB- and fluorescent lightexposed mice had significant decreases in the helper (CD4+) T-cell population (p<0.05), while the UVA1-exposed mice showed no significant difference. Thus, UVA1 may not result in immunosuppression at these dose levels. As determined by pathology, neither UVA1 nor fluorescent light exposure resulted in any significant detrimental effects to the kidneys, where as UVB caused significant adverse effects (p<0.05). Collectively, the results suggest that low doses of UVA1 radiation, unlike UVB, may beneficially alter the immune status without adversely affecting the kidneys of SLE mice. [ProA]