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Carcinogenesis Advance Access originally published online on October 4, 2007
Carcinogenesis 2008 29(1):186-193; doi:10.1093/carcin/bgm217
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Exposure of Fischer 344 rats to a weak power frequency magnetic field facilitates mammary tumorigenesis in the DMBA model of breast cancer

Maren Fedrowitz and Wolfgang Löscher*

Department of Pharmacology, Toxicology and Pharmacy, University of Veterinary Medicine, Bünteweg 17, D-30559 Hannover, Germany

* To whom correspondence should be addressed. Tel: +49 511 953 8720; Fax: +49 511 953 8581; Email: wolfgang.loescher{at}tiho-hannover.de


   Abstract
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 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 
The possibility that long-term exposure to relatively weak power frequency magnetic fields (MFs) emanating from the generation, transmission and use of electricity could increase the risk of breast cancer is a matter of ongoing debate. Laboratory studies using well-defined exposure conditions are useful to examine whether exposure to MF affects mammary tumorigenesis. Previous studies from different laboratories using the 7,12-dimethylbenz[a]anthracene (DMBA) model of breast cancer in female Sprague–Dawley (SD) rats have been inconclusive, which has been related to differences in MF sensitivity between SD substrains used in these studies. When we compared the effects of MF exposure on cell proliferation in the mammary gland of various outbred and inbred rat strains, Fischer 344 was the only inbred strain that exhibited a marked increase in cell proliferation. Based on these data, we suggested that MF exposure should significantly facilitate development and growth of mammary tumors in Fischer 344 rats, which was tested in the present study. Groups of 108 DMBA-treated rats were either MF exposed (100 µT, 50 Hz) or sham exposed for 26 weeks. MF exposure significantly facilitated mammary tumorigenesis. The incidence of rats with grossly recorded, histologically verified adenocarcinomas was increased by 45% (P = 0.0095). The most pronounced MF effect on tumor incidence was seen in the cranial inguinal complexes (L/R5). These data indicate that Fischer 344 rats are a suitable inbred strain to study the mechanisms underlying the effects of MF exposure on mammary tumorigenesis.

Abbreviations: AB, alveolar bud; BrdU, bromodeoxyuridine; DMBA, 7,12-dimethylbenz[a]anthracene; MF, magnetic field; SD, Sprague–Dawley; TEB, terminal end bud


   Introduction
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 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Funding
 References
 
Exposures to power frequency (50 or 60 Hz) electric and magnetic fields emanating from the generation, transmission and use of electricity are ubiquitous in modern life. The hypothesis that chronic exposure to electric and magnetic field may increase the risk of breast cancer, via a reduction in synthesis and secretion of the pineal hormone melatonin, was first made 20 years ago and has led to a great deal of research (1–5). On the basis of both experimental and epidemiologic findings, this issue remains highly controversial (3–6). Given the ubiquitous nature of electric and magnetic field exposure along with the high incidence of breast cancer, even a small risk would have a substantial public health impact, so that further study appears warranted.

We and others have used the 7,12-dimethyl-benz[a]anthracene (DMBA) model of breast cancer in rats to evaluate the effects of magnetic field (MF) exposure on mammary tumorigenesis (6,7). We found previously that prolonged exposure of female Sprague–Dawley (SD) rats to 50 Hz MFs at flux densities in the µTesla (µT) range increases cell proliferation in the mammary gland (8) and enhances mammary tumor development and growth in response to DMBA (9–12). However, attempts of other groups to replicate our findings failed (13–15) which led the involved researchers to suggest that genetic differences between substrains of SD rats used in our and other studies may be involved (16). Subsequent studies in different substrains of SD rats confirmed this suggestion (12), indicating that the genetic background plays a pivotal role in effects of MF exposure. Different strains or substrains of rats may thus serve to evaluate the genetic factors underlying sensitivity to co-carcinogenic or tumor-promoting effects of MF exposure. Such genetic factors or genetic predisposition could, of course, also play a role for adverse health effects in response to residential or occupational MF exposures in human populations.

To obtain more information about rat strain differences in sensitivity to MF exposure, we recently compared various outbred and inbred rat strains in respect to MF effects on cell proliferation in the mammary gland, using in vivo labeling of proliferating cells with bromodeoxyuridine (BrdU) (17). In addition to the MF-sensitive SD outbred substrain (SD1) previously used in our experiments, inbred Fischer 344 rats were the only strain in which MF exposure significantly enhanced BrdU labeling in the mammary epithelium, indicating a marked increase in cell proliferation (17). The MF-induced increase in BrdU labeling in Fischer 344 rats was similar to that seen after DMBA application. Furthermore, whole-mount analysis of mammary tissue from Fischer 344 rats demonstrated that MF exposure increased the number of terminal end buds (TEBs), i.e. the site of origin of mammary carcinomas (17). Based on these recent data on MF-induced increase in cell proliferation in the mammary gland, we hypothesized that MF exposure should significantly facilitate development and growth of mammary tumors in Fischer 344 rats (17). This hypothesis was tested in the present study.


   Materials and methods
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 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Funding
 References
 
Animals
Female Fischer 344 (F344) inbred rats were obtained from Charles River (Sulzfeld, Germany). Rats were allowed to acclimatize to the environmental conditions for at least 1 week, so that they were 50–54 days of age at onset of MF exposure. For comparison, some experiments were also performed with age-matched SD and Lewis rats also obtained from Charles River. The experimental protocols used in this study were in line with national and international ethical guidelines and were conducted in compliance with the German Animal Welfare Act and approved by the responsible governmental agency, including approval by an animal ethics committee. All efforts were made to minimize pain or discomfort of the animals used.

MF exposure of Fischer 344 rats
After acclimatization, the rats were brought into the room with the exposure chambers, placed in their home cage (nine rats per cage) in the exposure chambers (for details see ref. 18) and MF or sham exposure was started for 24 h/day (minus time for weighing, tumor palpation, cage cleaning, cage rotation) 7 days/week for a total duration of 26 weeks. Size of groups was 108 for MF exposure and 108 for sham exposure. Each rat received an administration of 10 mg DMBA (corresponding to ~90 mg/kg body wt) by gavage, dissolved in sesame oil (1 ml per rat), at onset of exposure. This dose of DMBA was chosen on the basis of preliminary dose-finding experiments in smaller groups of Fischer 344 rats (30 rats per dose), which indicated that at 10 mg DMBA ~50% of controls developed grossly recorded (macroscopically visible) mammary tumors thus allowing to evaluate whether MF exposure facilitates mammary tumorigenesis.

The exposure system and the protocol for MF exposure used for the present experiments have been described in detail elsewhere (18). In short, rats were exposed in exposure chambers to a horizontally polarized magnetic 50 Hz field with a flux density of 100 µT (i.e. 1 G) root mean square for 26 weeks. Identical but non-energized exposure chambers were used for sham exposure of control rats in the same room. Sham-exposed rats received a stray MF field from the energized coils, which was calculated (and measured) to be 0.1 µT in the volume of the sham exposure chambers.

Animals were weighed once per week; cage cleaning was done three times a week; cage rotation in the exposure and sham-exposure chambers was done once a week. The 50 Hz MF in the exposure chambers was measured twice per week with a µT-Vector2 meter (Physical Systems, Bradenton, Florida). In addition, the current generating the MF was continuously measured by a Clamp On Leak Hi Tester (Hioki E.E. Corp., Nagano, Japan) and recorded by a computer every 5 s. The mean current value of 1 min, the minimum, and the maximum values of the last 24 h were recalculated continuously, were visible at a monitor for direct control of stable exposure conditions during the experiment and were saved on a computer for retrospective analysis (for details see ref. 12). During the MF experiments, all of the field measurements were done by a person not involved in the animal experimentation.

During the 26 weeks of exposure, rats were housed within the exposure or sham exposure chambers under controlled conditions of temperature (23–24°C), humidity (~50%) and light (12 h dark/light cycle; light off at 6 p.m.); food (Altromin standard rat diet) and water were available ad libitum. Light intensity produced by artificial white light in the room with the exposure system varied between 16 and 35 lx (measured by a luxmeter in the exposure chambers). In the dark period, the room was weakly illuminated by dim red light, which led to a light intensity of <1 lx (measured in the exposure chambers).

Quantification of mammary tumors in Fischer 344 rats
During MF or sham exposure, rats were palpated once per week to assess the development of mammary tumors. The size of palpable tumors was estimated by a rating scale as recently described (19). Furthermore, the location of each tumor among the six mammary complexes of the rat was recorded. The investigator performing these experiments was not aware of the exposure status (sham or MF) of the rats.

After 26 weeks of MF or sham exposure, all rats were killed for necropsy. Blood was sampled for determining the hemogram. Furthermore, the weight of liver and spleen was recorded in all animals. Two rats died and 22 rats (10 sham, 12 MF exposed) had to be necropsied prior to the end of the exposure period because of large bleeding tumors. These rats were included in the pathological examination. For preparation of the mammary glands, the skin was opened by a midline incision to expose the six pairs of mammary glands extending from the salivary glands to the perianal region. Specific mammary glands were identified by site as L(left)1 through L6 and R(right)1 through R6, with 1 being the most cranial and 6 the most caudal gland. All grossly observed (i.e. macroscopically visible) mammary tumors were recorded, excised, trimmed and saved for further histopathological analysis. The size of macroscopically visible mammary tumors was measured by a calliper after dissection, and tumor volume was calculated from the length, width and depth of tumors on the basis of an ellipse. The mammary tumors were then fixed in 4% phosphate-buffered formalin (pH 7.3). The fixative was changed after 24 h. Small tumors were fixed in total or cut in two halves. For large tumors, 1–2 sections were cut vertical to the surface and to the midline. These tissue samples were embedded in Paraplast, sectioned at 3–4 µm and stained routinely with hematoxylin and eosin. Neoplastic lesions of the mammary glands were classified by microscopic examination according to Boorman et al. (20). The histopathological evaluation was done ‘blind’, i.e. the examiner was not aware of the group origin of sections. With respect to the tumors palpated before necropsy, only the neoplasms that were subsequently histologically verified as mammary tumors were used for group comparisons.

Whole-mount analysis in different rat strains
Because the developmental stage of the rat mammary gland determines its susceptibility to DMBA and, as indicated by our previous experiments in SD rats, also its susceptibility to MF-induced alterations in mammary tumorigenesis (11,12), whole-mount preparations were used to analyze the degree of mammary gland development in Fischer 344 rats at the age used for the DMBA experiments. In a first experiment, whole-mount preparations of different sham-exposed rat strains (Fischer 344, Lewis, SD) were compared. Rats were sham exposed for 2 weeks before whole-mount analysis. Age at onset of sham exposure was 52–54 days in all strains. In a second experiment, Fischer 344 rats were MF exposed for 2 weeks under the exposure conditions also used for the DMBA experiment (see above) in order to assess which structures in the mammary tissue were affected by MF exposure. Whole-mount preparation was performed for all mammary complexes (L/R1–6) as described recently (17). Because of disturbing muscle tissue at some positions, it was not always possible to evaluate the whole complex, so that areas with comparable size within the mammary gland complexes were evaluated in all glands and rat strains. These areas were located in the zone distal to the nipple (zone C) as described recently (17). In this zone, TEBs, alveolar buds (ABs) and lobules per square millimeter were counted. All analyses were performed in a blinded fashion.

Statistics
Differences between groups in tumor incidence were determined using Fisher's exact test and in the mean number, size and latency to onset of tumors by the Mann–Whitney U-test. Differences in the cumulative proportions of animals with tumors (incidence curves) were calculated by the log-rank test in which the two animals that died without tumors were included as censored. Differences between groups in body weight and organ weights were calculated by Student's t-test. Differences in data from whole-mount analysis in the three rat strains were determined by analysis of variance for non-parametric data (Kruskal–Wallis test), followed by post hoc testing with the Mann–Whitney U-test. The latter test was also used to calculate the significance of differences in whole-mount data between sham- and MF-exposed Fischer 344 rats. All statistical tests were used as two-sided tests and a P < 0.05 was considered significant.


   Results
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 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Funding
 References
 
Development and growth of mammary tumors in Fischer 344 rats
The cumulative proportion of DMBA-treated animals which developed mammary tumors during the period of MF or sham exposure is shown in Figure 1A. The first mammary tumors could be palpated in the MF-exposed group 6 weeks after DMBA application. During the subsequent weeks of exposure, tumor incidence in MF-exposed rats was always above that of sham-exposed rats. Individual differences in incidence of palpable tumors between the two groups were statistically significant at 15–25 weeks of exposure. In terms of the magnitude of differences between groups, the largest percent difference was seen after 16 weeks of exposure, at which tumor incidence in the MF group was 120% higher than that in the sham group (P = 0.0106). The percent differences became less marked during subsequent exposure. At time of necropsy, i.e. 26 weeks after DMBA application, 69 MF-exposed rats and 53 sham-exposed rats had developed macroscopically visible (and histologically verified) mammary tumors (P = 0.0393). Statistical evaluation of the cumulative proportions of animals with tumors in MF- and sham-exposed groups over the whole period of MF exposure by the log-rank test yielded a P value of 0.0055 (Figure 1A), indicating that the two groups differed significantly.


Figure 1
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  		Fig. 1. (A) Cumulative proportion of Fischer 344 rats with mammary tumors as a function of duration of MF exposure (incidence curves). DMBA was administered perorally at 10 mg per rat at onset of MF exposure. Group size was 108 rats per group. In addition to the data from palpation (weeks 5–26), the percentage of rats with macroscopically visible (and histologically verified) mammary tumors at necropsy (i.e. after 26 weeks of exposure) is shown (indicated by arrow). With respect to the tumors palpated before necropsy, only neoplasms which were subsequently histologically verified as mammary tumors are shown. Statistical evaluation of data from the palpation period by the log-rank test gave a P value of 0.0055, indicating that the two incidence curves differ significantly. Individual differences during exposure between groups are indicated by asterisk (P < 0.05). (B) Cumulative number of mammary tumors as a function of duration of MF exposure. For further details see above (A).

 
MF exposure affected mammary tumorigenesis not equally across the six pairs of mammary glands. In our previous studies in SD1 rats, the effect of MF exposure on mammary tumorigenesis in the DMBA model was predominantly due to an increased development and growth of mammary tumors in the cervical or cranial thoracic mammary complexes (L/R1 and L/R2) (11,12). In contrast, MF exposure of Fischer 344 rats did not affect mammary tumorigenesis in these complexes (Figure 2A), but the significant effect of MF exposure on the cumulative proportion of DMBA-treated animals that developed mammary tumors resulted from effects on mammary complexes in position 3–6 (Figure 2B), particularly the thoracic L/R3 and the cranial inguinal (L/R5) complexes. These complexes were also those glands in which in most MF-exposed rats the first tumor developed (Table I). When the incidence of the first palpable tumor was compared between groups (Table I), significantly more MF-exposed rats (17/108) developed their first adenocarcinoma in the cranial inguinal complexes (L/R5) than sham-exposed rats (7/108).


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  		Table I. Incidence of the first palpable mammary tumor in the six mammary complexes after the application of DMBA in sham- and MF-exposed Fischer 344 rats

 


Figure 2
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  		Fig. 2. (A) Cumulative proportion of Fischer 344 rats with mammary tumors in the cervical/cranial thoracic (L/R1 and 2) complexes as a function of duration of MF exposure (incidence curves). Statistical evaluation of data from the palpation period by the log-rank test gave a P value of 0.9518, indicating that the two incidence curves did not differ significantly. (B) Cumulative proportion of Fischer 344 rats with mammary tumors in the caudal thoracic (L/R3), abdominal (L/R4) and inguinal (L/R5 and 6) complexes as a function of duration of MF exposure (incidence curves). Statistical evaluation of data from the palpation period by the log-rank test gave a P value of 0.0158, indicating that the two incidence curves differ significantly. Individual differences during exposure between groups are indicated by asterisk (P < 0.05). (C and D) Cumulative number of mammary tumors in either L/R1 and 2 (C) or L/R3–6 (D) as a function of duration of MF exposure. For further details see legend to Figure 1A.

 
Figure 1B illustrates the cumulative number of mammary tumors in the two groups of rats during the 26 weeks of exposure. As could be expected from the incidence curves (Figure 1A), a higher number of mammary tumors was observed in the MF-exposed groups throughout the period of tumor development and growth. At time of necropsy, a total of 73 mammary tumors appeared in the group exposed to DMBA only, compared with 106 grossly recorded mammary tumors in the MF-exposed group. Again, the most marked inter-group difference in numbers of tumors during MF exposure was seen in the thoracic L/R3 and the cranial inguinal (L/R5) complexes (Figure 2D).

Both the data on incidence and cumulative number of mammary tumors (Figures 1 and 2) may suggest that MF exposure decreased the latency to tumor onset. Thus, after the delay of tumor appearance in the sham exposure controls, tumors developed at virtually the same rate as the MF-exposed group. Calculation of the mean latency to onset of the first palpable mammary tumor in each rat for the MF- and sham-exposure groups resulted in the following findings (Table II). When latency was calculated independently of the mammary complex in which the first tumor appeared, tumor latency was 125 days in MF-exposed rats versus 140 days in sham-exposed rats, which was close to statistical significance (P = 0.0581). When latency was calculated separately for each of the six mammary complexes in which the first tumor appeared, the tumor latency in rats with first tumor in the thoracic L/R3 complexes was significantly shorter in MF-exposed rats compared with controls (P = 0.0111). Because L/R3 was the complex in which most tumors developed in sham controls (Table I), the delay in tumor appearance in L/R3 in sham controls may be involved in the differences between incidence curves (Figures 1 and 2). We also calculated latencies to onset of first palpable mammary tumors for the groups of complexes shown in Figure 2, resulting in a significant reduction in latency in the L/R3–6 group of complexes (Table II).


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  		Table II. Latency to onset of the first palpable mammary tumor following application of DMBA in sham- and MF-exposed Fischer 344 rats

 
Tumor multiplicity, i.e. mean number of tumors per tumor-bearing rat, was not significantly different between sham- and MF-exposed groups over the duration of the experiment (not illustrated). Furthermore, the size of tumors as estimated by palpation did not differ between groups during exposure (not illustrated).

Histopathology in Fischer 344 rats
The incidence of histologically verified DMBA-induced mammary tumors was 49% in sham-exposed rats and 64% in MF-exposed rats, the difference being statistically significant (Table III). The predominant type of tumors was invasive adenocarcinomas, being observed in 40% of sham-exposed rats and 58% of MF-exposed rats (P = 0.0095). In both groups, comparable incidences of benign lesions (adenomas or fibroadenomas) were determined. In terms of total numbers of grossly recorded mammary tumors, again the predominant type of neoplasms was adenocarcinomas in both groups of rats (Table III). Except fibroadenomas, all types of mammary lesions occurred more frequently in MF-exposed rats (Table III). Hyperplasias were only found in sham-exposed rats; because we did not perform serial sections of the mammary glands, it is difficult to determine the reliability of this observation.


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  		Table III. Incidences of rats with grossly recorded, histologically verified mammary gland neoplasias induced by DMBA in sham- and MF-exposed Fischer 344 rats; furthermore, the absolute numbers of grossly recorded mammary gland neoplasias are shown

 
Incidence, number, multiplicity and volume of histologically verified tumors in the six mammary complexes are shown in Table IV. In both controls and MF-exposed rats, the highest number and incidence of tumors were determined in L/R3 and L/R5. Tumor multiplicity in mammary complexes was about the same in both groups of rats. Furthermore, tumor volume was not significantly different in sham- and MF-exposed rats. However, when only adenocarcinomas were used for comparison, tumor volume was significantly increased in L/R3 of MF-exposed rats versus sham-exposed controls [225 (25–7226) versus 94 (5–691) mm3; P = 0.0454; median and range of 20 adenocarcinomas in MF-exposed rats and 10 adenocarcinomas in sham-exposed rats].


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  		Table IV. Grossly recorded histologically verified mammary tumors in the six mammary glands of sham- and MF-exposed Fischer 344 rats

 
Other findings in the DMBA model in Fischer 344 rats
No differences between groups were seen in body weight gain or general behavior during the period of exposure. Average body weight (±standard deviation) in MF- and sham-exposed groups was 107 ± 8.8 g and 108 ± 6.8 g at onset of exposure and 222 ± 14.6 g and 223 ± 16.2 g after 26 weeks of exposure, respectively. Furthermore, weights of liver and spleen at time of necropsy did not differ significantly. Liver weights in MF- and sham-exposed rats (mean ± standard deviation) were 7.7 ± 1.1 g and 7.8 ± 1.5 g. Spleen weights in MF- and sham-exposed groups (mean ± standard deviation) were 0.52 ± 0.24 and 0.51 ± 0.18 g. No significant differences were seen between the hemograms of sham- and MF-exposed rats, and all differential counts were within the normal values known from Fischer 344 rats (not illustrated).

Whole-mount analysis in different rat strains
In our previous DMBA experiments in sham-exposed SD rats, tumor incidence was greater in thoracic glands, particularly L/R1 and/or L/R2, than glands in the abdomino-inguinal area (9–11,18), whereas this difference was less marked in the present experiments in Fischer 344 rats (Table IV), possibly indicating strain differences in the developmental stage of the mammary at time of DMBA exposure. This prompted us to directly compare the degree of mammy gland development in age-matched SD and Fischer 344 rats. Furthermore, Lewis rats were included in this comparison. As shown in Figure 3A, in both SD and Lewis rats the highest concentration of TEBs, i.e. the primary site of origin of mammary carcinomas, was determined in L/R2, whereas all other glands retained much fewer TEBs. In contrast, Fischer 344 rats exhibited significantly more TEBs in L/R3 and L/R4 than the other strains and also more TEBs in L/R6 than SD rats (Figure 3A). The density of ABs (Figure 3B) and of lobules (not illustrated) did not differ significantly between Fischer 344 rats and the two other strains.


Figure 3
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  		Fig. 3. Whole-mount analysis of mammary glands. (A) Number of terminal end buds (TEBs) per square millimeter in the six mammary complexes (averaged from left and right site) in three rat strains, SD, Lewis and Fischer 344 (F344). Data are shown as means ± SEM. All rats were sham exposed for 2 weeks, starting at 52–54 days of age, so that they were 66–68 days of age at time of whole-mount analysis. Group size was 3 (SD), 10 (Lewis) and 5 (F344) rats, except for L/R1 in Fischer 344 rats, for which n = 15, because data from 10 sham-exposed, age-matched rats of a previous experiment (17) were averaged with the data of the present experiment. Analysis of data by analysis of variance indicated significant differences between groups for L/R3 (P = 0.0316), L/R4 (P = 0.0078) and L/R6 (P = 0.0492). Significant differences between Fischer 344 rats and the other strains are indicated by asterisk (P < 0.05). (B) Number of ABs in the three strains. Analysis of data by analysis of variance indicated significant differences between groups only for L/R2 (P = 0.0311); asterisk indicates that Lewis rats differed significantly from SD rats in this position (P = 0.0485). For further details see (A). (C) Effect of MF exposure on TEB numbers in Fischer 344 rats. The rats were either sham or MF exposed for 2 weeks. Data are shown as means ± SEM of five rats per group except for L/R1, for which n = 15 per group, because data from a previous experiment (17) were averaged with the data of the present experiment (data from the two experiments did not differ significantly). Statistical analysis of data indicated that MF exposure increased TEBs significantly in L/R1 (P = 0.0210). (D) Effect of MF exposure on AB numbers in Fischer 344 rats. Statistical analysis of data indicated that MF exposure increased ABs significantly in L/R1 (P = 0.0151) and L/R5 (P = 0.0079).

 
Whole-mount analysis in MF-exposed Fischer 344 rats
We have reported previously that MF exposure significantly increases the number of TEBs in L/R1 of Fisher 344 rats (17). We now repeated the experiment to assess whether MF exposure also affects differentiation in other mammary complexes. As shown in Figure 3C, the concentration of TEBs was only increased in L/R1. The density of ABs was significantly increased by MF exposure in L/R1 and L/R5 (Figure 3C). No significant effects of MF exposure were observed for lobules (not illustrated).


   Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Funding
 References
 
To our knowledge, this is the first experimental study demonstrating that power frequency MF exposure facilitates mammary tumorigenesis in the DMBA model of breast cancer in Fischer 344 rats, one of the most widely used inbred strains in toxicology and oncology (21,22). We recently reported that MF exposure increases cell proliferation in the mammary gland of Fischer 344 rats, whereas several other inbred strains, including Lewis rats, were insensitive in this regard (17). The magnitude of MF's proliferative effects on the mammary epithelium in Fischer 344 rats was comparable with that of DMBA, so that we suggested that combined treatment of female Fischer 344 rats with DMBA and prolonged MF exposure will lead to enhanced mammary tumorigenesis (17). This suggestion was substantiated by the present experiments. Compared with sham controls, the incidence of histologically verified mammary gland tumors observed grossly in female Fischer 344 rats after 26 weeks of MF exposure was significantly increased by 31%, which is similar to the 28% increase that we observed previously with the same protocol in a MF-sensitive substrain (SD1) of SD rats (11). The incidence of adenocarcinomas was significantly increased by 45% in MF-exposed Fischer 344 rats, whereas a smaller (24%) increase was observed in our previous study in SD1 rats (11). Previous experiments with serial sections of mammary glands after MF exposure of DMBA-treated SD1 rats revealed that MF exposure does not increase the initiation of cancers by DMBA, but enhances the growth and progression of DMBA-induced lesions (18). The present data indicate that Fischer 344 rats are a suitable inbred strain to study the effect of MF exposure on mammary tumorigenesis and the mechanisms underlying this effect.

The Fischer 344 rats were originally generated by the Crocker Institute of Cancer Research and were selected as the standard rat for the Carcinogenesis Bioassay Program of the National Cancer Institute (21). Compared with other rat strains, the Fischer 344 rat shows qualitatively similar responses to toxic chemicals and potential carcinogens but is quantitatively neither the most sensitive nor the most resistant to cancer induction following exposure to a chemical in most cases (21). Fischer rats appear to exhibit an intermediate sensitivity to the carcinogen DMBA and carry neither mammary cancer suppressor genes nor susceptibility genes (12, 23, 24). The incidence of spontaneous tumors in aged Fischer 344 rats is moderate for most organs, except for testicular interstitial cell tumors in male animals that arise in 63–90% of aged rats (25–27). Another disease that appears in Fischer 344 rats is a mononuclear cell leukemia called the Fischer rat leukemia (26–28).

Several previous studies used Fischer 344 rats to assess whether exposure to power frequency (50 or 60 Hz) MF exerts effects on cancer development, including 2 year bioassays (29–34). Overall these studies were negative except increased incidences of thyroid gland C-cell adenomas and carcinomas in male rats exposed to 2 or 200 µT in one study, which was intepreted as equivocal evidence of carcinogenicity (31). The incidence of mammary adenocarcinoma in the lifelong bioassays in female Fischer 344 rats was very low (ranging between 0 and 4% in sham control groups), resulting in a low statistical power to detect effects of MF exposure on mammary carcinogenesis. This is illustrated by the 2 year MF exposure study of Boorman et al. (31), in which 2/100 female rats in the sham controls developed mammary adenocarcinoma, while 7/100 rats (P = 0.098 versus control) and 5/100 rats (P = 0.231) developed such cancers in the 2 and 200 µT MF-exposure groups, respectively. Thus, although these are increases in mammary cancer incidence of >100%, the differences to sham control were not statistically significant because of too low statistical power. Co-carcinogenicity models such as the DMBA model of breast cancer are more sensitive to detect whether MF acts as a promoter or co-promoter of tumorigenesis. The present experiments demonstrate that MF exposure facilitates mammary tumorigenesis in the DMBA model in Fischer 344 rats.

In the mammary gland, enhanced proliferation of epithelial stem cells as recently shown for MF exposure in Fischer 344 rats (17) is known to increase the risk of malignant transformation, e.g. in response to chemical carcinogens such as DMBA (35). The susceptibility of the mammary gland to a carcinogen depends upon its degree of differentiation at the time of exposure (35). The mostly undifferentiated gland of young virgin rats is highly susceptible due to the high proliferative rate of the terminal ductal structures called TEBs, which differentiate into ABs and lobules (35,36). TEBs, which are composed of an actively proliferating epithelium, are the most actively growing terminal ductal structures in the rat mammary gland, which explains the high susceptibility of the TEBs to neoplastic transformation in response to DMBA and other chemical carcinogens. Any factor at the time of DMBA treatment which enhances the proliferative state of the mammary epithelium appears important in determining the appearance of carcinomas.

Because of asynchrony in post-natal mammary gland development over the six mammary gland complexes of the female rat, these complexes exhibit a different susceptibility to neoplastic transformation (35). Three of the mammary complexes of the rat (L/R1, L/R2 and L/R3) are located along the thorax, extending cranially to the cervical region; one complex (L/R4) is located on the abdomen (L/R4) and two complexes (L/R5 and L/R6) in the inguinal region. Thoracic glands lag behind in post-natal development and retain a higher concentration of TEBs, i.e. the site of origin of mammary carcinomas, than abdominal glands, so that tumor incidence in rats treated with DMBA at the age of about 50 days is greater in thoracic glands than glands in the abdomino-inguinal area (35). This difference among mammary complexes is also relevant for MF exposure. Thus, in the MF-sensitive substrain of SD rats (SD1) that we used previously for studying effects of MF exposure in the DMBA model, MF exposure affected the development of mammary tumors not equally across the six mammary complexes, but the most pronounced effect was seen in the cervical and cranial thoracic complexes (L/R1 and L/R2) (11,12). These complexes of SD1 rats were also particularly sensitive to MF exposure in terms of increase of ornithine decarboxylase (37), indicating enhanced proliferation of breast stem cells at risk for malignant transformation.

In apparent contrast to SD1 rats, the present experiments with DMBA in Fischer 344 rats did not disclose any particular sensitivity of the cervical or cranial thoracic mammary complexes to MF exposure, but the most pronounced effect on mammary tumorigenesis was seen in the caudal thoracic (L/R3) and cranial inguinal (L/R5) complexes. Post-natal development of the six mammary gland complexes and differences in DMBA-induced tumorigenesis as a consequence of the topographic location of the complexes have almost exclusively been studied in the SD strain. This prompted us to evaluate the differentiation of the mammary gland by whole-mount analysis in Fischer 344 rats in comparison with SD rats. Furthermore, Lewis rats, i.e. an inbred strain in which we previously did not find any effect of MF exposure on stem cell proliferation in the mammary gland (17), were included in the comparison. Analysis of TEBs across the six mammary complexes of rats at about 6–7 weeks of age showed that both SD and Lewis rats exhibited the by far the highest density of TEBs in L/R2, confirming the asynchrony in post-natal mammary gland development reported previously for SD rats (35). In contrast, much less marked differences in TEB density across mammary complexes was observed in Fischer 344 rats. Furthermore, the strain comparison indicated that L/R3 and L/R4 of Fischer 344 rats lag behind in post-natal development and retain a higher concentration of TEBs compared with these complexes in SD and Lewis rats. This would be likely explanation for the present findings with DMBA in sham-exposed Fischer 433 rats in that differences in the incidence of DMBA-induced mammary tumors between thoracic and abdomino-inguinal glands were less marked than such differences reported previously for SD rats (9–11,18,35).

We also examined the effect of MF exposure on mammary gland differentiation in Fischer 344 rats, using whole-mount analysis. In a previous study on cell proliferation in the mammary gland of Fischer 344 rats, using in vivo labeling of proliferating cells with (BrdU) and whole-mount analysis, we found that MF exposure (50 Hz, 100 µT) for 2 weeks significantly increased the number of TEBs and BrdU labeling in the mammary epithelium in L/R1, but other mammary complexes were not evaluated in this study (17). The effect of MF exposure on TEBs in L/R1 was confirmed in the present study, but no increase of TEBs was observed in any of the other mammary gland complexes. However, an increased number of the more differentiated ABs was determined in L/R1 and L/R5 after MF exposure, which may indicate that MF exposure accelerated the differentiation from TEBs into ABs in these complexes. On the basis of these observations, it is difficult to explain why the most pronounced effects of MF exposure on mammary tumorigenesis were observed in L/R3 and L/R5. However, in contrast to the 26 weeks of MF exposure used in the DMBA experiments, MF exposure for the whole-mount analysis was only 2 weeks, so that definite conclusions have to await whole-mount analyses after different periods of MF exposure with and without DMBA treatment. In any event, our data strongly indicate that the site of origin of mammary carcinoma determine to what extent MF exposure increases mammary tumorigenesis in the DMBA model in different rat strains.

The cell proliferation and tumorigenesis-facilitating effects we have seen after MF exposure in mammary tissue of Fischer 344 rats may be local effects or mediated by a systemic effect. There is compelling evidence that static (earth-strength) MFs affect the pineal/melatonin system, with the retina considered as the site of magnetoreception (38–41). The resulting decrease in nocturnal production of the pineal hormone melatonin is not observed in acutely blinded rats (42). However, evidence involving pineal secretion of melatonin in biological effects of 50/60 Hz MF exposure is less consistent (41). Opposed to MF effects mediated by the visual system and the pineal gland, such effects may be locally mediated as demonstrated by in vitro studies using MCF-7 breast cancer cells (43–45). We plan to use primary cultures of mammary epithelial cells isolated from Fischer 344 rats to study the mechanisms involved in MF effects. Furthermore, we have started to use gene arrays to determine MF effects on gene expression in Fischer 344 rats and Lewis rats, i.e. an MF-insensitive rat strain (17). By such strain comparison, we hope to identify the genes that are involved in the effects of MF exposure observed in Fischer 344 rats in the present and previous studies (17).

Apart from effects of MF exposure on cell proliferation or differentiation in the mammary gland, MF exposure may alter mammary tumorigenesis by modifying the metabolism of DMBA, as DMBA is a procarcinogen, which has to be metabolized before being active. In this respect, experiments using the direct acting carcinogen, N-methyl-N-nitrosourea, might be of interest. To our knowledge, only one previous study used the N-methyl-N-nitrosourea model of breast cancer for evaluating the effects of MF exposure in female rats, indicating that MF exposure significantly decreased tumor latency and increased tumor incidence and the progression from benign to malignant tumors (46). Thus, these data do not support the notion that MF exposure facilitates mammary tumorigenesis only by modifying the metabolism of the carcinogen.

In conclusion, by comparison with MF-insensitive inbred strains, Fischer 344 rats can now be used to identify the genes involved in their MF response, e.g. by microarray gene expression studies and backcrossing. Because in contrast to outbred rat strains such as SD, genetic divergence among rats from different sources should not be a problem in an highly homozygous inbred strain such as Fischer 344; the use of this strain should minimize inter-laboratory variation in effects of MF exposure and thus help to clarify the role of MF exposure in mammary carcinogenesis.


   Funding
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
References
 
Deutsche Forschungsgemeinschaft (Lo 274/6-2); Forschungsverbund Elektromagnetische Verträglichkeit Biologischer Systeme (Department of High Voltage Engineering, Technical University, Braunschweig, Germany).


   Acknowledgments
 
We thank Prof. Kenji Kamino (Institute of Cell and Molecular Pathology, Hannover Medical School, Hannover, Germany) for help with the histological analysis of mammary tumors and Britta Sterzik for technical assistance. We appreciate the information on mammary gland development in rat strains provided by Prof. José Russo (Breast Cancer Research Laboratory, Fox Chase Cancer Center, Philadelphia, PA).

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 

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Received June 28, 2007; revised September 20, 2007; accepted September 23, 2007.


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