Electromagnetic Fields and Cancer: Laboratory Studies - American

Downloaded by COLUMBIA UNIV on July 25, 2012 | http://pubs.acs.org Publication Date: May 5, 1995 | doi: 10.1021/ba-1995-0250.ch012

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Electromagnetic Fields and Cancer: Laboratory Studies Larry E. Anderson and Lyle B. Sasser Biology and Chemistry Department, Battelle, Pacific Northwest Laboratories, P.O. Box 999, Richland, WA 99352

Convincing evidencefroma large number of laboratories indicates that exposure to extremely lowfrequency(ELF) magnetic and elec tricfieldsproduces biological responses in animals. However, no animal studies clearly demonstrate deleterious effects of ELF fields, although several suggest potential health impacts. A major current emphasis in laboratory research is to determine whether the re ported epidemiological studies that suggest an association between electromagneticfield(EMF) exposure and risk of cancer are sup ported in studies using animal models. Several approaches are out linedin the experimental approach to this question. From the per spective of laboratory animal studies, this chapter will discuss studies investigating the potential relationship between ELF mag netic and electricfieldexposure and the risk of cancer. 'HE INTERACTION OF A N AGENT WITH HUMANS is o f prime importance and

A concern, but many areas of biological investigation are more efficiently and appropriately conducted with various animal species. Animal studies provide an integrated system that can be used in studies in which experimental variables can be controlled, specific hypotheses can be explored, and exposure can be precisely assessed. Given the uncertainty and the relatively low power of electro-

0065-2393/95/0250-0225$12.00/0 ©1995 American Chemical Society

In Electromagnetic Fields; Blank, M.; Advances in Chemistry; American Chemical Society: Washington, DC, 1995.


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magnetic field (EMF) epidemiologic studies to ascertain the relationship between E M F exposure and possible adverse health effects, animal studies are especially important in evaluating any potential association between E M F and cancer. However, animal studies for risk assessment purposes have limitations that must be addressed. Extrapolation of experimental results from laboratory animals to humans remains somewhat tentative, both because of important biological differences between humans and animals and because the mechanisms (and hence the biologically relevant exposure parameters) by which the effects may arise are often unknown. Furthermore, studies using laboratory animals are typically carried out with high doses throughout the lifetime of the animals. The relevance of these exposure conditions to those generally experienced by humans is uncertain.

Animal Models Several different approaches and animal models can be used in laboratory cancer studies. The selection of a specific model depends largely upon the hypothesis chosen to evaluate a particular underlying mechanism. For example, i f one desires to test an agent (e.g., E M F ) for its potential to be a complete carcinogen (an agent that by its application alone causes a cancer to develop), 1/4 to 2 years of exposure of mice or rats to the agent is necessary. During that time it is important to keep exposure to other possibly confounding agents to a minimum. In this regimen the animals are observed during the major portion of their lifetime, and the number, type, and time of development of tumors are the critical end points. This type of study should include several dose groups and requires a relatively large number of animals, particularly i f the natural incidence of a tumor type is low. A s one would surmise, studies evaluating complete carcinogenicity are expensive because of both the length of time and the number of animals involved. It is also an open question as to whether such studies can yield meaningful results when studying E M F . This problem arises because of the inadequacy of present knowledge on what aspects of the E M F signal may be biologically active. Because carcinogenesis is a multistep process, another approach is to assume that the agent of interest acts either as an initiator or a promoter for which a two-phase protocol is required for testing. Initiation is defined as a genotoxic event in which the carcinogen interacts with the organism to directly affect the D N A . Promotion is operationally defined, associated with the experimental protocol, and occurs when the promoting agent is applied subsequently to initiation and generally over a protracted period of time. Promotion is associated with a number of subcellular events that are generally nongenotoxic and is responsible for the conversion of initiated cells to cancerous cells. To evaluate E M F as


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an initiator, one high dose of E M F would be given followed by repeated exposure to a model promoter (e.g., 12-0-tetradecanoylphorbol-13-acetate, T P A ) over a long term. If E M F were to be investigated for possible promotional effects, the animals would be treated with a known initiator (e.g., 7,12dimethylbenz[a]anthracene, D M B A ) , and subsequently exposed to E M F over a long term (months). These initiation-promotion approaches have the advantage of using fewer animals, a shorter time, and less cost. However, a given model is usually limited to evaluating a specific type of cancer and may provide only general information on possible biological mechanisms of the agent of interest (EMF) and cancer development.

Spontaneous Tumor Development and Complete Carcinogen Studies Few truly life-long animal studies examining E M F as a complete carcinogen have yet been fully reported, although several are under way (in the United States, Italy, Japan, and Canada). Japanese workers reported (/) preliminary results of a life-span study to determine the spontaneous carcinogenic effect of 50-Hz sinusoidal A C (alternating current) magnetic fields in rats. Groups of 48 Fischer-344 rats of each gender were exposed to flux densities of 5 mT, 0.5 mT, or sham fields for 2 years. Exposure to the fields did not increase the incidence of total tumors, nor was the incidence of leukemia or brain tumors altered. In a multigeneration study (2-4% C F W mice were exposed to 60-Hz A C magnetic fields at 25 mT (250 G) for three generations. Animals from the second and third generations were conceived and raised in the fields. Preliminary results from two sets of animals suggest a significant relationship between exposures to the magnetic field as a function of time and malignant lymphoma. O f 55 exposed animals, 45% developed malignant lymphoma by the age of 140-273 days; none were found in the controls (3% In another group of animals, 78% (32) of the exposed animals had premalignant changes or malignant lymphoma compared to 6% (2) of the controls when evaluated at 363-418 days of age (4% This work has appeared only in abstract form, and the exposure intensities were extremely high. However, i f the results are indeed related to the fields rather than artifacts from the high fields (i.e., heat, noise, vibration, etc.), then these data could provide a starting point for understanding dose-response relationships for magnetic field exposure and health-related end points. Additionally, several studies that were designed to evaluate E M F as a promoter of cancer contained control groups that were exposed to E M F without being initiated with a carcinogen (e.g., chemicals or radiation). Three such studies contained parallel groups of animals to investigate E M F as a possible complete carcinogen. These studies include an investigation of lymphoma development in mice (5% a mammary tumor-promotion study in rats (6), and a



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mouse skin tumor-promotion study (7). A major deficiency of using such "add on" studies to evaluate complete carcinogenicity is the small group sizes in volved. In the lymphoma study, Svedenstal and Holmberg (J) investigated the in fluence of pulsed magneticfieldson the lymphoma development in the radia tion-induced lymphoma mouse model and on spontaneous lymphoma develop ment in a life-time study. Mice (227 total) were divided into four groups and exposed to (1) 5.24-Gy X-ray, (2) pulsed 20-kHz magneticfields(vertical, sawtooth shaped with afluxdensity of 15 μΤ), (3) both X-rays and magnetic fields, or (4) no treatment. No significant differences were found in the induced or spontaneous lymphoma incidence as a result of magneticfieldexposure. The Beniashvili et al. study (6) found an increase in mammary gland tu mors in rats exposed to 20 μΤ for 3 h/day (7 of 25 animals) compared to no tumors in 50 unexposed animals. However, the number of animals studied is extremely small for this type of study, and no conclusions can be drawn from these results. The skin tumor study reported no increase in tumors with longterm exposure to magneticfields(500 or 50 μΤ). Both studies are discussed in more detail in the following section.

Initiation-Promotion Studies Leukemia Studies. Clinical progression of leukemia was investigated in 8-week-old female mice (10 per group) that were implanted with P388 leu kemia cells and subsequently exposed to 60-Hz sinusoidal magneticfieldsin Helmholtz coils for 6 h/day, 5 days/week until death at flux densities of 1.4,200, or 500 μΤ (8). Neither incidence nor clinical progression of the leukemia in the implanted animals was altered by the magnetic fields at any intensity studied. Mammary Cancer Studies. Beniashvili et al. (6) induced mam mary tumors in rats with nitrosomethylurea (NMU) at age 55 days (five groups of 50 animals). Following administration of the NMU, two groups of the rats were exposed to a 50-Hz magneticfieldfor 30 or 180 min each day. Two other groups were exposed to a static magneticfieldfor either 30 or 180 min/day. The results of this experiment are striking, as animals exposed to either the variable or the staticfieldfor 180 min/day had a higher incidence of mammary tumors as well as a greater number of tumors. In addition, the latent period was decreased in the exposed animals. No tumors developed in 50 control rats over the 2-year study period, whereas seven appeared in 25 rats exposed for 3 h/day to the 50Hz magnetic field (without NMU). Such results suggest a possible promoter effect of EMF in rat mammary carcinogenesis. In two preliminary studies using 60-Hz electricfields(40 kV/m), Leung et al. (9) reported that rats initiated with DMBA by gavage at 54 days of age had slightly more mammary tumors per tumor-bearing animal at either 18 or 23



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weeks of exposure than did similarly initiated animals that were not subse quently exposed to the field. A statistically significant difference could not be demonstrated in either individual study for this response, nor was there evidence of an increase in tumor incidence in either study. However, by pooling the data of the two studies, the authors found a statistically significant increase in the number of tumors per tumor-bearing animal. Even though the two studies were not identical replicates, the results suggest a weak promotional effect of electric fields. Subsequently, a series of mammary initiation-promotion studies were conducted by Mevissen et al. (10). Female rats were given 5 mg of D M B A by gastric intubation and then sham-exposed; exposed to a 50-Hz, 30-mT field; or exposed to a 15-mT D C (direct current) field. A l l animals received three addi tional doses of D M B A at weekly intervals. Reference controls and shamexposed controls had an average mammary tumor incidence of 66% and 61%, respectively. Exposure to the 15-mT D C field did not alter either the incidence or number of tumors per animal. The tumor weight, however, was significantly increased in the exposed animals. Animals that were exposed to the 50-Hz, 30mT field did not exhibit a significant difference in tumor incidence, but the exposed animals had twice as many tumors as the controls. When this experi ment was repeated using a smaller number of animals, no differences were found between exposed and controls. A third experiment in which DMBA-treated rats were exposed to a gradient (0.3-1.0 μΤ) 50-Hz field showed no differences between exposed and control animals. This study is handicapped by a small number of animals in each group. Loscher et al. (11) performed an experiment using D M B A to initiate mammary tumors in rats and reported a significant increase in mammary tumor induction in the rats exposed to a magnetic field. A l l rats received four weekly doses of 5 mg of D M B A beginning at 52 days of age. A group of 99 rats was exposed, after D M B A treatment, to 50-Hz magnetic fields at a flux density of 0.1 mT for 24 h/day for 3 months. Another group of 99 was sham-exposed. After 3 months of exposure, mammary tumor incidence was about 50% higher in the exposed group (51 tumors) compared to the sham-exposed (34 tumors). This difference was statistically significant (P < 0.05). The tumors were also larger in the exposed group (P = 0.0134), but a difference was not found in the number of tumors per tumor-bearing rat. Loscher and colleagues followed up their initial studies with two further mammary carcinogenesis studies. In one study, groups of 36 rats were treated with D M B A and subsequently exposed or sham-exposed to magnetic fields for 13 weeks (12). The gradient magnetic fields in this study, of 0.3-1 μΤ, were considerably lower in intensity than in the other study reported (11). The authors reported that although a detailed evaluation of mammary lesions showed a slight increase in the incidence of hyperplasias and tumors in the magnetic field ex posed animals, the changes were not statistically significant and therefore not supportive of the hypothesis that E M F exposure may be associated with promo-


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tion of mammary cancer in animals. In the second study, large groups of animals (99 animals per group) were exposed to 10-μΤ fields for 13 weeks (13). A non significant tendency to decreased tumor latency was observed in the exposed animals; however, no differences in overall tumor incidence were reported. These apparent differences in results from the same research group (i.e., from the D M B A mammary cancer study described) may reflect differences in animal response at the different field intensities used or may simply be a reflection of the differences in group size between the experiments. Skin Tumor Models. Skin tumor promotion, after initiation with D M B A , was examined in mice exposed to a 2-mT, 60-Hz continuous magnetic field, 6 h/day, 5 days/ week for up to 21 weeks (14). Groups of 32 female mice were initiated with D M B A (10 nmol) on the dorsal skin. The mice were then exposed to the magnetic field with or without T P A promotion (1 μg/week for 21 weeks). Because none of the exposed or sham-exposed mice developed papillo mas, the authors concluded that the fields did not act as a classical tumor pro moter. In a second study, two groups of 48 mice were similarly initiated with D M B A , and promoted with 0.3 μg (4.9 nmol) of T P A for 23 weeks with or without magnetic field exposure to investigate the copromotional effect of the fields. The onset of tumor development was slightly earlier in mice that were treated with T P A plus magnetic fields, compared to sham-exposed. However, at the end of the study, neither the number of animals affected nor the number of tumors per animal was different between the two groups (15). A life-span skin carcinogenicity study in N M R I / H A N mice exposed to A C sinusoidal magnetic fields found no evidence that magnetic fields promoted the formation of skin tumors (7). Groups of 36 mice were initiated with D M B A (25 μg applied to the dorsal skin of each mouse) and exposed to 50-Hz sinusoi dal magnetic fields with flux densities of 50 μΤ or 50 mT for 103 weeks, 19-21 h/day, or sham-exposed. These results do not support the findings that continu ous magnetic fields promote skin carcinogenicity in S E N C A R mice (15); how ever, the flux densities used in the S E N C A R mice were much greater than that used by Rannug et al. (7). The tumor-promoting effects of continuous and intermittent magnetic fields were compared in a long-term skin carcinogenesis study of female S E N C A R mice after initiation with D M B A (16). Groups of 40 mice were treated with D M B A (10 nmol) 1 week before being exposed to the magnetic fields. Continuous and intermittent (15 s οη-ofï) 50-Hz horizontal A C fields with flux densities of 50 μΤ and 0.5 mT were applied for 19 (weekdays) or 21 (weekends) h/day for 105 weeks. Appropriate untreated, DMBA-treated, and TPA-promoted control groups were included in the study design. N o tumors were found in animals treated with D M B A and exposed to continuous fields of 50 μΤ or 0.5 mT. Totals of four tumors in four animals and 13 tumors in five animals were found in the 50-μΤ and 0.5-mT intermittent groups, respectively. Statistically



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significant differences were not found when the tumor-bearing animals and cumulated skin tumors of intermittent groups were compared to the control group. However, when the two intermittent groups were pooled and compared to the continuous exposed groups, a statistically significant difference (P = 0.014) effect was found. No hyperplastic response was associated with the magnetic field exposures. The results did not support the hypothesis that continuous mag netic field exposure promotes skin carcinogenesis in S E N C A R mice at flux densities of 50 μΤ and 0.5 mT. However, a weak promotional effect may be associated with intermittent exposures. The different results from the continuous and intermittent exposures are rather intriguing, but not well understood. Further laboratory experiments will provide replication of experiments and better understanding of possible mecha nisms underlying the potential health effects associated with E M F . Liver Cancer Studies. In a series of experiments, Rannug and co workers (77, 18) investigated the possibility that magnetic fields interact with known initiators or promoters of cancer to induce preneoplastic lesions in rats. As a model, they used partially hepatectomized male Sprague-Dawley rats treated with diethylnitrosamine (DENA) to initiate tumor development. They then treated the animals with phénobarbital for 12 weeks, beginning 1 week after initiation, to promote growth of enzymatically altered foci of the liver cells. In two studies, rats were initiated with D E N A , then promoted with phénobarbital or with 50-Hz horizontal, magnetic fields having flux densities of 0.5 and 50 μΤ (Experiment 1) or 5 and 500 μΤ (Experiment 2). A slight increase in γ-glutamyl transpeptidase (GGT)-staining foci was reported in Experiment 1, but not in Experiment 2. In a third study, animals were exposed to magnetic fields having flux densities of 0.5 μΤ or 0.5 mT, both during D E N A initiation and also throughout phénobarbital treatment (copromotion; 18). The magnetic field inhibited, rather than increased, the size and number of focal lesions. These results may suggest a weak interaction of the fields on the process of carcinogenesis, but evidence of a promotion or copromotional role of magnetic fields was not demonstrated.

Conclusions Many studies on possible effects of E M F on tumor development in animals are ongoing in several countries. However, relatively little information is yet available, and any conclusions drawn at this point would be premature. E M F as a complete carcinogen or an initiator of cancer has not been demonstrated in laboratory studies. Only one set of experiments has suggested such a link; however, those studies used extremely high field intensities and therefore results may have been confounded by problems associated with such extreme exposures.



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In the tumor-promotion studies that have been reported, results seem to be mixed. Some studies reported increased tumor development with exposure, and others showed no association. The strongest laboratory evidence for an E M F related cancer association is in the area of mammary carcinogenesis. Several studies have been conducted, most of which suggest some increase in mammary cancer due to exposure. In these experiments, tumors must be initiated with a chemical carcinogen for the E M F to have its apparent effect. Some data support the idea that E M F may act in a possible copromotional manner, and E M F expo sure alone may not be as effective in promoting cancer development. Further more, the association between E M F and biological effect may be greater for intermittent exposures than for continuous exposure. Laboratory work has not yet confirmed the results of the epidemiological studies that there may be an association between E M F exposure and increased risk of cancer, and further carefully controlled and designed experimental re search in the laboratory will be required to determine whether such an associa tion is real. If further evidence suggests a relationship between exposure and potential health end points, studies in animal models will be necessary to con firm the evidence and to understand the possible mechanisms by which those effects occur. In particular, laboratory experiments allow specific hypotheses to be employed in addressing the issue of whether adverse health outcomes can result from E M F exposure.

References 1. Yasui, M . ; Kikuchi, T.; Ogawa, M . ; Otaka, Y.; Tsuchitani, M . ; Iwata, H. In Ab -stracts of Papers, Fifteenth Annual Meeting of the Bioelectromagnetics Society, June 13-17, 1993, Los Angeles, CA, pp 153-154. 2. Fam, W. Z.; Mikhail, E. L. In Abstracts of Papers, Twelfth Annual Meeting of the Bioelectromagnetics Society, June 10-14, 1990, San Antonio, TX, pp 17-18. 3. Mikhail, E. L.; Fam, W. Z. In Abstracts of Papers, Thirteenth Annual Meeting of the Bioelectromagnetics Society, June 23-27, 1991, Salt Lake City, UT, p 24. 4. Mikhail, E. L.; Fam, W. Z. In Abstracts of Papers, The First World Congress for Electricity and Magnetism in Biology and Medicine, June 14-19, 1992, Lake Buena Vista, FL, p 28. 5. Svedenstal, B. M . ; Holmberg, B. Int. J. Radiat.Biol.1993, 64, 119-125. 6. Beniashvili, D. Sh.; Bilanishvili, V. G.; Menabde, M . Z. Cancer Lett. 1991, 61, 7579. 7. Rannug, Α.; Ekstrom, T.; Hansson Mild, K.; Holmberg, B.; Gimenez-Conti, I.; Slaga, T. J. Carcinogenesis 1993, 14, 573-578. 8. Thomson, R. A. E.; Michaelson, S. M . ; Ngugen, Q. A. Bioelectromagnetics 1988, 9, 149-158. 9. Leung, F. C.; Rommereim, D. N.; Stevens, R. G.; Wilson, B. W.; Buschbom, R. L.;


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Anderson, L. E. In Abstracts of Papers, Tenth Annual Meeting of the Bioelectro -magnetics Society, Gaithersburg, MD, p 2. Mevissen, M . ; Stamm, Α.; Buntenkotter, S.; Zwingelberg, R.; Wahnschaffe, U . ; Loscher, W. Bioelectromagnetics 1993, 14, 131-143. Loscher, W.; Mevissen, M . ; Lehmacher, W.; Stamm, A. CancerLett.1993, 71, 7581. Loscher, W.; Wahnschaffe, U.; Mevissen, M . ; Lerchl, Α.; Stamm, A . Oncology 1994, 51, 288-295. Mevissen, M . ; Loscher, W.; Lerchl, Α.; Stamm, A . In Abstracts ofPapers, Sixteenth Annual Meeting of the Bioelectromagnetics Society, June 12-17, 1994, Copenha -gen, Denmark, p 73. McLean, J. R. N . ; Stuchly, Μ. Α.; Mitchel, R. E. J.; Wilkinson, D.; Yang, H.; Goddard, M . ; Lecuyer, D. W.; Schunk, M . ; Callary, E.; Morrison, D. Bioelectro -magnetics 1991, 12, 273-287. Stuchly, Μ. Α.; McLean, J. R. N . ; Burnett, R.; Goddard, M . ; Lecuyer, D. W.; Mitchel, R. E. J. Cancer Lett. 1992, 65, 1-7. Rannug, Α.; Holmberg, B.; Ekstrom, T.; Hansson Mild, K.; Gimenez-Conti, I.; Slaga, T. J. Carcinogenesis 1994, 15, 153-157. Rannug, Α.; Holmberg, B.; Hansson Mild, K. Environ. Res. 1993, 62, 223-229. Rannug, Α.; Holmberg, B.; Ekstrom, T.; Hansson Mild, K. Bioelectromagnetics 1993, 14, 17-21.

RECEIVED for review April 6, 1994. ACCEPTED revised manuscript September 26, 1994.


Electromagnetic Fields and Cancer: Laboratory Studies - American

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