Magnetic fields and cancer development in animal models

Transcription

1 Radio Science, Volume 30, Number 1, Pages , January-February 1995 Magnetic fields and cancer development in animal models Bo Holmberg and Agneta Rannug Department of Toxicology, National Institute of Occupational Health, Solna, Sweden Abstract. Severalaboratory animal studies have been up to July 1993 published with the aim to investigate possible carcinogenic or cocarcinogenic effects of magnetic fields. No large-scale study of complete carcinogenicity, considering tumor as an endpoint, has been performed. Nor has any study on tumor initiation only been performed. In studies designed for tumor promotion, small parallel series for complete carcinogenicity have also been included som experiments. These small series do not permit an evaluation of the possible complete carcinogenicity or noncarcinogenicity of magnetic fields. Studies designed to illuminate the possibility of a tumor-promotive effect of magnetic fields seem not to support the hypothesis that continuous magnetic fields act as promoters in rats or mice. Observations skin tumors in mice s m to indicate a possible copromotional effect of a 2-mT 60-Hz magnetic field and a simultaneous treatment with a known skin tumor promoter in the sensitive Sencar mouse strain. Introduction Epidemiological studies during the last two and a half decades have suggested [see review by Bates, 1991; Sagan, 1992] a link between extremely low frequency magnetic field (MF) exposure and an increased risk for leukemia or brain tumors among residents or workers. Recently, four epidemiological studies [London et al., 1991; Feychting and Ahlbom, 1993; Floderus et al., 1993; Savitz and Kaune, 1993] strengthened the suggestion by more precise exposure descriptions and/or measurements. In the two Swedish studies, a tendency toward a dose-response was obtained, although the absolute numbers of cancer cases were small. In an epidemiological paper [Wertheirner and Leeper, 1987], tumor promotion was mentioned as a possible mechanism. Carcinogenesis is a multistep and multifactorial phenomenon [Herrno, 1987]. The first animal experimental studies demonstrating the multistep character of tumorigenesis [Rous and Kidd, 1941; Berenblurn, 1941; Friedewald and Rous, 1944; Mottram, 1944; Berenblurn and Shubik, 1947] defined the two major steps as initiation and promotion, by observing the development of tumors in mouse skin after local treatment with two different types of carcinogens. The term promotion was an operational definition, associated with the particular experimental protocol used. Initiation has since been largely associated with genotoxic effects due to direct or indirect interactions between the carcinogen, or its metabolites, and DNA. The Copyright 1995 by thc Amcrican Geophysical Union. Paper number 94RS /95/94 RS promotion stage is responsible for the conversion of initiated cells to transformed cells. Promotion is associated with a number of subcellular events, largely nongenotoxic. Some animal studies also form the basis for a further division of the promotion stage into stage I (conversion) and stage II (propagation). A continuing cellular evolution into a fully invasive and metastasizing tumor cell tissue is termed progression. A chemical or physical factor capable of being effective in all stages is termed a complete carcinogen. It may be possible in a series of animal experiments to define the activity of a factor for one or more of these (often overlapping) biological steps; cellular studies, however, are most helpful in guiding the understanding of the mechanisms of a carcinogenic effect. A typical two-phase protocol for animal carcinogenicity studies does not differentiate between promotion and progression. The multistage phenomenon of cancer development has been extensively studied for skin tumors and also to some extent for tumors of the liver, urinary bladder, and respiratory tract. The phenomenon is, however, assumed to be general for the development of all cancer types. Data from long-term animal studies play a definite role in the characterization of a carcinogenic agent. An animal carcinogen, as established in a test for complete carcinogenicity, may be regarded as a human carcinogen an overall assessment of scientific data even in the absence of hum',,n data [Vainio and Wilbourn, 1992]. (Animal data, of course, also support epidemiological findings and can give at least an indication of the mechanism of action, which is far more difficult to demonstrate by epidemiology.) Studies with cellular systems using different exposure setups, exposure durations, amplitudes, and frequencies indicate that biological effects of magnetic fields are at 223

2 224 HOLMBERG AND RANNUG: ANIMAL CANCER STUDIES hand [Goldberg and Creasy, 1991; Szmigielski, 1993; Blask, 1990], which may be related to cell multiplication or even tumor promotion. On the other hand, studies on the possible genotoxicity of MFs suggest that MFs have no direct or indirect effects on DNA [Murphy et al., 1993]. A general assumption among scientists is that a tumor promotion mechanism is to be searched for, if any carcinogenic effect can be ascribed to MF exposure. The arguments for tumor promotion are, however, indirect, based upon the absence of genotoxic properties [Easterly, 1981; Doll et al., 1992]. No animal long-term studies, performed lege artes, on the carcinogenic effects of low-frequency electromagnetic fields have been published until recently. Such studies are needed in order to characterize the carcinogenic properties of MFs, if any. Animal studies are essential to define the exposure parameters responsible for an effect. The flux density, the frequency, the exposure duration, and the profile are among the important critical variables. Animal studies, as well as cell studies, are also essential in order to get an indication of the carcinogenic mechanism. The identification of the carcinogenic mechanism is important for a solid risk assessment and a quantitative risk extrapolation. Model Designs Long-term carcinogenicity studies can be performed according to different protocols depending on the underlying hypothesis of mechanisms. Testing the assumption that the factor is a complete carcinogen [Harem, 1985; Huff, 1992] requires months of exposure of mice or rats to the unknown factor at high dose levels, usually up to maximum tolerable dose, with no other exposure. The observations of the animals should be performed during the major part of the lifetime (2 to 2.5 years) of the animals, and the protocol involves among other things complete necropsy and histopatholoõy of all tissues. The occurrence of tumors is the critical endpoint. The requirements are also a reasonably high survival for most of the 2-year period and the use of at least 50 animals per group, two to three dose groups, both sexes and controls. If the animal groups in a lifelong complete carcinogenicity study are too small the sensitivity of the test system will be low. Assuming that the factor acts as an initiator or a promoter requires a two-phase protocol for testing [Slaga and Nesnow, 1985]. A typical protocol for an initiator implies one high-dose exposure (or high doses during a short time) to the unknown factor and a subsequent long-term repeated exposure to a model promoter. A protocol for an unknown promoter implies a single exposure to a known initiator and a subsequent long-term exposure to the factor. Two common models in beth cases are skin rumor formation in mice [Slaga and Nesnow, 1985] and the formation of preneoplasticell conglomerates in rat liver [Pereira, 1985]. In the case of the mouse skin tumor model, 30 animals are normally used in each group, and treatment and observations are made during at least 6 or 12 months, depending on strain and design. The appearance of skin tumors (papillomas and possible carcinomas) is recorded. The time of tumor appearance, number of tumors per animal, and number of rumor-bearing animals in the study are the important parameters. An effective initiator for skin tumorigenesis is 7,12-dimethylbenz[a]anthracene (DMBA), but other initiators may be selected. A model promoter must be used in parallel groups as a positive control. A common model promoter for mouse skin tumors is 12-0-tetradecanoylphorbol-13-acetate (TPA), which is a very potent chemical acting via mechanisms involving hyperplasia in the skin, thus enhancing the proliferation and development of initiated tumor cells in the skin to papillomas and eventually carcinomas. The enzyme-altered rat liver foci model is the second classical model system for a two-phase protocol [Pereira, 1985]. The endpoint of this model is the appearance of preneoplastic "cell islands"in the liver, which, in part, may eventually develop into liver tumors. There are several similar protocols available. In one protocol, the animals are subjected to a partial hepatectomy and subsequent initiation with a known carcinogen, e.g., diethylnitrosoamine (DENA), made in order to enhance cell replication in the regeneration process. Preneoplastic foci may be observed after 2-3 months by histochemical staining of enzyme markers. The foci frequency, foci area, and foci volume may be increased by liver promoters. As control promoter, phenobarbital (PB) in the drinking water is used. The rat liver model is usually performed with about 10 rats in each group. Both the mouse and the rat models should preferably include nonexposed controls, vehicle controls, and positive controls. A positive control (using a known carcinogen) is selected to assess the design, animal strain, and protocol. Both models reflect biological phenomena underlying tumor formation in general, phenomena which are supposed to be relevant for induction and development of most, if not all, types of tumors. The best-studied model with mouse skin tumor formation has been used for decades to demonstrate the carcinogenicity of e.g. supposed lung carcinogens. Data from such studies have been used recently [Nesnow, 1990] as the basis for quantitative human risk assessment of air pollutants. Two-phase protocols for other tumor types, such as leukemia and brain tumors, have been less commonly used in the past. However, a few such studies have already been performed with MFs and more are planned or ongoing with different initiator regimens. Papers [Batkin and Tabrah, 1977; Bellossi and Toujas, 1982; Thomson et al., 1988; Baumann et al., 1989] have been published on the effects of magnetic fields on the growth of tumor tissue transplanted into host animals. Such

3 HOLMBERG AND RANNUG: ANIMAL CANCER STUDIES 225 studies may be of value to study possible effects on cell growth and tumor growth enhancement (e.g., progression). The biology of rumor growth in transplants is, however, different from the biology of rumor induction and growth of the animal's own tissues. These papers are therefore not discussed in the present context. Complete Carcinogenicity Studies Well-performed lifelong animal studies assuming that MF act as a complete carcinogen are needed. Such studies are planned or already started in the United States (National Toxicology Program), Canada, and Italy. Data from these studies will appear within the next few years. In studies, mentioned below, designed mainly to reveal a possible rumor promotive effect, parallel groups of animals have also been included in order to give an indication of possible complete carcinogenic activity (Table 1). This is the case in a mouse skin tumor promotion study [Rannug et al., 1993a], a mammary tumor promotion study [Beniashvili et al., 1991] in rats, and a lymphoma study in mice [$vedenst&l and Holmberg, 1993]. Exposing femme NMRI mice to 0.S-roT and $0-gT continuous MF at $0-Hz for 103 weeks without previous initiation [Rannug et al., 1993a] did not produce skin tumors or exposure-related tumors in other organs. The small number (30 animals per group) of animals in these particular groups in the experiment makes this part of the study rather insensitive for evaluating complete carcinogenicity. A similar comment regarding group sizes is valid for a Georgian study [Beniashvili et al., 1991] with female rats, wherein four groups of $0-Hz ac fields and dc fields at 20-gT have been applied for 0.5 hour or 3.0 hours daily. These particular groups had 25 animals each. The design of the study was otherwise according to a tumor promotion protocol (see below) using nitrosomethyl urea (NMU) as an initiator. In the series of the experiment, where the MFs were applied daily during 3.0 hours without previous initiation, mammary gland tumors were found in 28% of the exposed animals. No mammary tumors developed in a control group of $0 unexposed rats. The report is lacking information on exposure details. Female CBA/S mice were exposed [$vedenst&l and Holmberg, 1993] to saw-tooth-shaped pulsed MF with a flux density of l$-gt (peak to peak) at 20-kHz for their lifetime in an experiment designed for tumor promotion. Two groups were not initiated but served as MF exposed controls and as nontreated controls. The design otherwise involved roentgen treatment and subsequent MF exposure as promoter. The spontaneous lymphoma frequency in this strain was not increased by MF treatment. In a series of papers [Bellossi and Toujas, 1982; Bellossi et al., 1988a, b; Bellossi, 1986, 1991], various types of fields (pulsed or static) at many different flux densities have been studied in mouse strains prone to develop a virusdependent leukemia or mammary tumor. In these studies, mortality has been the primary observation. In one study [Bellossi, 1991], no effect was seen over five generations of exposure to pulsed fields. In other studies, a longer survival was obtained under some exposure conditions to pulsed [Bellossi et al., 1988a] or static fields [Bellossi, Tumor Initiation Studies So far no such study has been performed. Tumor Promotion Studies In one 2-year tumor promotion study with continuous 50- Hz magnetic fields, using 0.$-mT and $0-gT exposure on femme NMRI mice [Rannug et al., 1993a], the appearance of skin tumors and other neoplastic lesions was observed. Also, skin hyperplasia analysis was performed on the animals as a short-term marker of possible skin tumor development. The animals were exposed during 19 hours or 21 hours a day with two exposure-free intervals, to permit observation, chemical treatment, and animal care. DMB A (100 nmol) was used as an initiator and TPA as a positive control for skin tumor promotion. In two series, MF exposure was used as the only treatment in order to get an indication of whether MFs could act as a complete carcinogen (see above). The results of these studies are listed in Table 2. The results /Ranhug et al., 1993a] did not show any difference in skin rumor development among DMBA- and MF-exposed animals compared to the DMBA-treated animals. This analysis was made with correction for survival and made for both cumulated rumor-bearing animals and a cumulated number of skin tumors. Other neoplasms than skin tumors were also analyzed, and no MF-related effect on systemic tumors could be seen. The skin hyperplasia analyses did not reveal any skin hyperplasia among DMBA- and MF-exposed mice related to magnetic field exposure. In three papers on skin tumor promotion in Sencar mice [$tuchly et al., 1991, 1992; McLean et al 1991], 2-mT and 60-Hz continuous MF exposure was used during 6 hours/day, 5 days/week up to weeks. In the first experiment [McLean et al., 1991] a rumor-promoting effect of MF was investigated in groups of 32 female mice. The initiation was performed with topical application of 10 nmol DMBA dissolved in 200 gl acetone, and starting 1 week after treatment with DMBA the animals were exposed to 2-rot MF or sham exposed from 0800 to 1400 LT for 21 weeks. None of the MF-exposed or sham-exposed mice developed papillomas. A study [$vedenst&l and Holmberg, 1993], using female CBA/S mice and pulsed magnetic fields at 20-kHz with a

4 226 HOLMBERG AND RANNUG: ANIMAL CANCER STUDIES o o o o o o o

5 HOLMBERG AND RANNUG: ANIMAL CANCER STUDIES 227 C C C

6 228 HOLMBERG AND RANNUG: ANIMAL CANCER STUDIES saw-tooth shape and a flux density of 15-1xT (peak to peak), was designed in order to observe a possible enhancing effect of X-ray-induced lymphomas or of spontaneous lymphomas (for the latter, see above) in this particular strain. Four X-ray doses of 1.31 Gy each were administered at 4-day intervals, and the subsequent MF exposure was performed 24 hours a day over the lifetime of the animals. In the X-ray-treated series a high lymphoma frequency was obtained. In the X-ray-irradiated group, 65.6% of the animals had lymphomas, and in the X-ray- and MF-exposed group, 71.4% of the animals had lymphomas. This difference was not statistically significant. In this study, hemoglobin analyses and blood counts also were made to get another indication of anemia and leukemogenic effects. A large German study [Mevissen et al., 1993] using Sprague-Dawley rats involved four different experiments and exposures to 15-mT dc fields or to 1-1xT, as well as 30-mT, continuous ac fields, after initiation of mammary ramors with repeated (4 times 5 mg) oral administrations of DMBA. The exposure was performed 24 hours/day during 91 days. MF did not alter the induced tumor frequency except in one experiment, where 30-mT increased the number of ramors per rumor-bearing animal. The 15-mT dc fields increased rumor weight but not rumor frequency in one experiment. The number of animals in the groups was small (18-36), limiting the sensitivity of the study. Also, the exposure and observation period was short. The experiment with 30-mT ac fields was repeated without showing an increase in the number of tumors per tumor-bearing animals. In the Georgian study [Beniashvili et al., 1991], female rats (strain not specified) were initiated with NMU at a dose of 50 mg/kg body weight administered into the caudal vein to induce mammary ramors, which could possibly be enhanced by 50-Hz MF exposure. Two types of MFs were used, variable (ac) fields and static (dc) fields with an intensity of 20-1xT. The MF exposure was 0.5 hour or 3 hours daily starting 2 days after NMU administration and possibly ongoing for lifetime (exposure details are not specified). Three hours of daily exposure to ac or dc fields increased the incidence of NMU-induced mammary ramors, while 0.5 hour of daily exposure did not. Also the time to rumor appearance was shorter among the animals exposed during 3 hours daily. In these groups, malignant ramors dominated among the histological types, compared to the control group. The results obtained in these experimental series need to be confirmed. The rat liver loci model has been used in a series of (partly, ongoing) experiments [Rannug et al., 1993b, c], where the animals were initiated with 30 mg/kg body weight DENA. The exposure lasts for 12 weeks, when animals are sacrificed and liver samples are taken for staining for enzyme markers, the placental form of glutathione transferase (GSTp) and T-glutamyl transpeptidase (GGT). In one study [Rannug et al., 1993b], 0.5-, 5-, and 50-1xT and.<.<.<

7 HOLMBERG AND RANNUG: ANIMAL CANCER STUDIES mT continuous MF at 50-Hz were used in a promotion protocol using Sprague-Dawley rats. This study did not show an enhancing effect of a continuous MF on the foci development. After initiation with methylcholanthrene, several hundred Swiss mice were exposed to static uniform MFs at a number of different exposure levels (flux densities between 25- and 800-mT) for different time periods [Bellossi, 1984] without any observed effects on survival, body weight, or spleen weights. In this paper the carcinogen was injected intramuscularly, and no data of tumor sites or frequencies were given. Interaction Studies The results of interaction studies are shown in Table 3. In the investigation by McLean et al. [1991], both promotion (see above) and copromotion of skin papillomas was studied in female Sencar mice exposed to 2-mT, 60-Hz MF. Using an initiating treatment of 2.56!xg of DMBA (10 nmol) and a subsequent exposure to 1!xg of TPA applied to the dorsal skin weekly, they tested whether a 21- week MF exposure could modify tumor development. They found a slightly earlier development of tumors in the MF-exposed animals; but the difference in time to appearance of tumors was however not statistically significant. In a follow-up study [Stuchly et al., 1992] the same strain and sex was exposed under the same field conditions, using a weekly dose of 0.3!xg of TPA. The rate of tumor development was found to be increased in the MF-exposed group compared to the sham-exposed TPA group. A difference in the cumulated number of mice with tumors was observed, but this difference did not reach statistical significance at the end of the observation period. The results suggest a copromotional activity of MF with a skin-specific tumor promoter. In one of the Canadian studies [McLean et al., 1991] the spleens were enlarged in the TPA+MF-exposed mice, and there was a greater number of mononuclear cells in the spleen of animals with combined exposure as well as a depressed natural killer (NK) cell activity. This is suggestive of a suppression of the immune system, possibly related to a development of leukemia/lymphomas. In one liver foci experiment [Rannug et al., 1993c], the 50-Hz continuous MF exposure with 0.5-lxT and 0.5-rot was applied both during the hepatectomy and initiation phase and during the promotion phase in combination with a phenobarbital (PB) treatment (300 ppm in drinking water). The foci formation was slightly inhibited in terms of foci area, frequency and volume for both enzyme markers. The results suggest either an interaction of MF with some process in preneoplastic cell development or a result of general stress. Conclusions The information on the possible effects of MFs on tumor development in experimental animals is slowly emerging. It is far too early to draw other than tentative conclusions from the present data set. The information so far on complete carcinogenicity is difficult to evaluate due to small animal groups or lack of critical information on exposures or on tumor observations. Large-scale complete carcinogenicity studies are needed. The tumor promotion studies performed up until now seem not to indicate an effect of continuous MFs per se at investigated flux densities. This seems to be established with different treatment protocols where exposures have been performed for the major part of the 24 hours each day. The finding in one study of a possible promotive effect of 3 hours of daily exposure to 20-lxT alternating MFs of NMUinduced mammary tumors is surprising and needs to be confirmed. Interaction studies with continuous MF exposure and treatments with known carcinogenic substances may suggest the presence of some sort of interaction. This information is, however, presently difficult to assess and to extrapolate to human risk. However, these observations may contribute to the future understanding of cancer development when well-performed, well-reported, and repeatable animal studies more clearly confirm the suspicion of carcinogenic or modulating effects of MF exposure. References Bates, M. N., Extremely low frequency electromagnetic fields and cancer: The epidemiological evidence, Environ. Health Perspect., 95, , Batkin, S., and F. L. Tabrah, Effects of alternating magnetic field (12 gauss) on transplanted neuroblastoma, Res. Commun. Chemical Pathol. Pharmacol., 16, , Baumann, S., R. Cooper, E. Berman, D. House, and W. loines, Lack of effects from 2000-Hz magnetic fields on mammary adenocarcinomand reproductive hormones in rats, Bioelectromagnetics, 10, , Bellossi, A., The effect of a static uniform magnetic field on mice. A study on methylcholanthrene carcinogenesis, Radiat. Environ. Biophys., 23, , Bellossi, A., Effect of static magnetic fields on survival of leukemia-prone AKR mice, Radiat. Environ. Biophys., 25, 75-80, Bellossi, A., Effect of pulsed magnetic fields on leukemiaprone AKR mice. No-effect on mortality through five generations, Leukemia Res., 15, , Bellossi, A., and L. Toujas, The effect of a static uniform

8 230 HOLMBERG AND RANNUG: ANIMAL CANCER STUDIES magnetic field on mice. A study of a Lewis tumour graft, Radiat. Environ. Biophys., 20, , Bellossi, A., I. Bernard-Griffiths, and M. Le Gall, Effect of ELF pulsed magnetic fields on survival of leukemiaprone AKR mice, In Vivo, 2, , 1988a. Bellossi, A., A. Desplaces, and R. Morin, Effect of a pulsed magnetic field on tumoral C3H/Bi female mice, Cancer Biochem. Biophys., 10, 59-66, 1988b. Huff, J., Design strategies, results and evaluations of longterm chemical carcinogenesistudies, Scand. J. Work Environ. Health, 18, Suppl. 1, 31-37, London, S. J., D.C. Thomas, J. D. Bowman, E. Sobel, T.- C. Cheng, and J. M. Peters, Exposure to residential electric and magnetic fields and risk of childhood leukemia, Am. J. Epidemiol., 134, , McLean, J. R. N., M. A. Stuchly, R. E. J. Mitchel, D. Beniashvili, D. Sh., V. G. Bilanishvili, and M. Z. Wilkinson, H. Yang, M. Goddard, D. W. Lecuyer, M. Menabde, Low-frequency electromagnetic radiation enhances the induction of rat mammary tumors by nitrosomethyl urea, Cancer Lett., 61, 75-79, Berenblum, I., The cocarcinogenic action of croton resin, Cancer Res., 1, 44-48, Berenblum, I., and P. Shubik, The role of croton oil applications, associated with a single painting of a Schunk, E. Callary, and S. D. Morrison, Cancer promotion in a mouse-skin model by a 60 Hz magnetic field: li. Tumor development and immune response, Bioelectromagnetics, 12, , Mevissen, M., A. Stamm, S. Buntenk6tter, R. Zwingelberg, U. W ahnschaffe, and W. L6scher, Effects of magnetic fields on mammary tumor development induced by 7,12- carcinogen, in turnour induction of the mouse's skin, Br. dimethylbenz(a) anthracene in rats, J. Cancer, 1, , Blask, D. E., The emerging role of the pineal gland and melatonin in oncogenesis, in Extremely Low Frequency Electromagnetic Fields: The Question of Cancer, edited by B. W. Wilson, R. G. Stevens, and L. E. Anderson, pp , Battelle, Columbus, Ohio, Bioelectromagnetics, 14, , Mottram, J. C., A developing factor in experimental blastogenesis, J. Pathol. Bacteriol., 56, , Murphy, J. C., D. A. Kaden, J. Warren, and A. Sivak, Power frequency electric and magnetic fields: A review of Doll, R., V. Beral, R. Cox, N. E. Day, M. J. Gardner, E. H. genetic toxicology, Mutation Res.,296, , Grant, A. F. McKinlay, C. R. Muirhead, R. D. Saunders, and J. W. Stather, Electromagnetic Fields and the Risk of Cancer, p. 138, National Radiological Protection Board, Chilton, United Kingdom, Easterly, C. E., Cancer link to magnetic field exposure: a hypothesis, Am. J. Epidemiol., 114, , Feychting, M., and A. Ahlbom, Magnetic fields and cancer Nesnow, S., Mouse skin tumours and human lung cancer: relationships with complex environmental emissions, Complex Mixtures and Cancer Risk, edited by H. Vainio, M. Sorsa, and A. J. McMichael, pp , IARC Sci. Publ. 104, Int. Agency for Res. on Cancer, Lyon, France, in children residing near Swedish high-voltage power Peteira, M., Rat liver foci assay, in Handbook of lines, Am. J. Epidemiol., 138, , Flodems, B., T. Petsson, C. Stenlund, A. Wennberg,,t,. Ost, and B. Knave, Occupational exposure to electromagnetic fields in relation to leukemia and brain rumours; a casecontrol study in Sweden, Cancer Causes Control, 4, , Friedewald, W. F., and P. Rous., The initiating and promoting elements in tumor production. An analysis of the effects of tar, benzpyrene, and methylcholanthrene on rabbit skin, J. Exp. Med., 80, , Goldberg, R. B., and W. A. Creasy, A review of cancer induction by extremely low frequency electromagnetic Carcinogenicity Testing, edited by H. A. Milman and E. K. Weisburger, pp , Noyes, Park Ridge, N.J Ranhug, A., T. Ekstr6m, K. Hansson Mild, B. Holmberg, I. Gimenez-Conti, and T. J. Slaga, A study on skin tumour formation in mice with 50 Hz magnetic field exposure, Carcinogenesis, 14, , 1993a. Ranhug, A., B. Holmberg, and K. Hansson Mild, A rat liver foci promotion study with 50 Hz alternating magnetic fields, Environ. Res., 62, , 1993b. Rannug, A., B. Holmberg, T. Ekstr6m, and K. Hansson Mild, Rat liver foci study on coexposure with 50 Hz fields. Is there a plausible mechanism?, M ed. magnetic fields and known carcinogens, Hypotheses, 35, , Hamm, T. E., Design of a long-term animal bioassay for carcinogenicity, in Handbook of Carcinogenicity Testing, edited by H. A. Milman and E. K. Weisburger, pp , Noyes, Park Ridge, N.J., Hertoo, H., Chemical carcinogenesis: tumor initiation and promotion, in Occupational Cancer and Carcinogenesis, Occup. Med.: State of the Art Rev., vol 2., no. 1, edited Bioelectromagnetics, 14, 17-27, 1993c. Rous, P., and J. G. Kidd, Conditional neoplasms and substhreshold neoplastic states. A study of tar tumors of rabbits, J. Exp. Med., 73, , Sagan, L., Epidemiological and laboratory studies of power frequency electric and magnetic fields, J. Am. Med. Assoc., 268, , Savitz, D. A., and W. T. Kaune, Childhood cancer in by P. W. Brandt-Rauf, pp. 1-25, Hanley & Belfus, relation to a modified residential wire code. Environ. Philadelphia, Health Perspect., 101, 76-80, 1993.

9 HOLMBERG AND RANNUG: ANIMAL CANCER STUDIES 231 Slaga, T. J., and S. Nesnow, SENCAR mouse tumorigenesis, in Handbook of Carcinogenicity Testing, edited by H. A. Milman and E. K. Weisburger, pp , Noyes, Park Ridge, N.J., $tuchly, M. A., D.W. Lecuyer, and J. McLean, Cancer promotion in a mouse-skin model by 60 Hz magnetic field: I. Experimental design and exposure system, Bioelectromagnetics, 12, , Stuchly, M. A., J. R. N. McLean, R. Burnett, M. Goddard, D. W. Lecuyer, and R. E. J. Mitchel, Modification of tumor promotion in the mouse skin by exposure to an alternating magnetic field, Cancer Lett, 65, 1-7, Svedenstttl, B. M., and B. Holmberg, Lymphoma development among mice exposed to X-rays and pulsed magnetic fields, Int. J. Radiation Biol., 64, , Szm.igielski, S., Electromagnetic fields and neoplasms with reference to extremely low frequencies, Bioelectrochem. Bioenerg., 30, , Thomson, R. A. E., S. M. Michaelson, and Q. A. Nguyen, Influence of 60-Hertz magnetic fields on leukemia, Bioelectromagnetics, 9, , Vainio, H., and J. Wilbourn, Identification of carcinogens within the IARC monograph program, Scan& J. Work Environ. Health, 18, Suppl. 1, 64-73, Wertheimer, N., and E. Leeper, Magnetic field exposure related to cancer subtypes, Ann. N.Y. Acad. $ci., 502, 45-54, i iii i i B. Holmberg and A. Rannug, Department of Toxicology, National Institute of Occupational Health, S Solna, Sweden. (Received July 8, 1993; revised January 4, 1994; accepted February 21, 1994.)