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Carcinogenesis 2006 27(12):2555-2564; doi:10.1093/carcin/bgl195
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Dietary effects of soy isoflavones daidzein and genistein on 7,12-dimethylbenz[a]anthracene-induced mammary mutagenesis and carcinogenesis in ovariectomized Big Blue® transgenic rats

Mugimane G. Manjanatha*, Sharon Shelton, Michelle E. Bishop, Lascelles E. Lyn-Cook and Anane Aidoo

Division of Genetic and Reproductive Toxicology, National Center for Toxicological Research, U.S. FDA Jefferson Laboratories Jefferson, AR, USA

*To whom correspondence should be addressed at: Division of Genetic and Reproductive Toxicology, 3900 NCTR Road, Jefferson, AR 72079, USA; Tel: +1 870 543 7098; Fax: +1 870 543 7393; Email: mmanjanatha{at}nctr.fda.gov

Abstract

The major constituents of isoflavones, daidzein (DZ) and genistein (GE) are known to interact with the {alpha} and ß estrogen receptors (ER{alpha}/ß) in several tissues including mammary. In this study, we used ovariectomy (OVX) to model menopause and determined the effects of DZ, GE or 17ß-estradiol (E2) exposures on chemically induced mutagenesis and carcinogenesis in the mammary glands of female Big Blue® (BB) transgenic rats. The rats were fed control diet containing the isoflavones and E2 and treated with a single oral dose of 7,12-dimethylbenz[a]anthracene (DMBA) at PND 50. Animals were sacrificed at 16 or 20 weeks post-carcinogen treatment to assess mutant frequencies (MFs) and histopathological parameters, respectively. The isoflavones or E2 supplementation alone resulted in modest increases in the lacI MF that were not significantly different from the MFs measured in rats fed the control diet alone. DMBA exposure, however, induced significant increases in the lacI MFs in the mammary of both OVX and ovary intact (INT) rats and Hprt MFs in spleen lymphocytes (P ≤ 0.01). In general, feeding the isoflavones or E2 separately did not cause any significant changes in DMBA-induced mutagenicity in the mammary. However, feeding the isoflavone mixture (DZG) resulted in a significant reduction in the DMBA-induced lacI MFs (P ≤ 0.05). Cell proliferation as measured by PCNA immunohistochemistry was increased in both OVX and INT rats exposed to DMBA as compared with rats fed control diet (P ≤ 0.05). Mammary histology indicated that hyperplasia was induced in most of the treatment groups including control. Although DMBA treatment did not induce mammary tumors in the OVX rats, adenoma and adenocarcinoma were detected in the mammary glands of INT rats.

Abbreviations: DZ, daidzein; GE, genistein; DMBA, 7,12-dimethylbenz[a]anthracene; BB, Big Blue; OVX, ovariectomized; INT, ovary intact.

Introduction

The major components of soybean-derived isoflavones daidzein (DZ) and genistein (GE) have been suggested as chemopreventive agents for certain types of cancer, particularly breast and prostate (1–3). Besides DZ and GE, soy products contain other constituents like saponins or antioxidants that also have health-promoting properties (4). Consequently, there is a growing interest in the possibility that dietary phytoestrogens may be an alternative to post-menopausal hormone replacement therapy, partly due to concerns about side effects and long-term health consequences of hormone therapy. However, studies conducted using in vitro systems and animal models indicate that these substances bind to estrogen receptors (ERs) and exert hormonal and anti-hormonal effects (1,5) that may influence cancer development.

In addition to their estrogenic action, soy isoflavones are known to exhibit certain biological activities including inhibition of angiogenesis, topoisomerase II and tyrosine kinases (5–7) which may be detrimental to the host. The potential for such activities of the isoflavones to invoke cytotoxicity in the host cells has been demonstrated for topoisomerase II inhibitors (8). Inhibition of DNA topoisomerase enzymes which function in DNA cleavage and religation during DNA replication and repair by GE has been shown to cause DNA strand breaks (9). Accumulated evidence indicates that isoflavones are not only clastogenic (10–12), but also they induce gene mutations (12,13). Moreover, GE has been shown to enhance chemically induced tumors in the colon (14) and in the mammary gland (15,16). Thus, it is conceivable that the biological effects of DZ or GE may be deleterious and possibly cause DNA damage that can potentiate the carcinogenesis process (17). Although, the intake of soy isoflavones in western cultures is much less than that consumed by Asian countries (18,19), dietary supplements of isoflavones are on the rise. Thus, the need for more studies to elucidate the potential genotoxicity or carcinogenicity of these compounds is warranted.

Previous studies in our laboratory suggest that chemically induced mutations in both target and non-target tissues using both the Big Blue® (BB) transgenic lacI and the Hprt assays (20–25) can serve as an indicator of carcinogen exposure. The uniqueness of BB rats stems from a transgenic mutational target that facilitates the measurement of in vivo mutations in any tissue with sufficient DNA for analysis. The strength of the Hprt assay is that it is one of the few genes suitable as a marker for mutation induction in animal systems and in humans. In rodents, the frequency of Hprt mutations is determined in populations of T-lymphocytes isolated from the spleen or the peripheral blood. This assay is limited only to circulating blood lymphocytes and was used in this study as an adjunct to the lacI assay.

7,12-Dimethylbenz[a]anthracene (DMBA) has been widely used to conduct hormone-dependent carcinogenesis experiments and has tremendously increased our understanding of chemically induced tumors in the mammary gland (26–28). The main target sites for the potent carcinogenicity of this chemical in rodents are the skin and the mammary gland; however, our previous experience has shown that spleen lymphocytes are capable of responding to DNA damage caused by environmental carcinogens in vivo (21,22,25). Thus, to evaluate the potential genotoxic effects of DZ or GE either alone or combined with a carcinogen, we have treated transgenic BB rats with a single dose of DMBA, and have conducted a mutagenesis assay in the target tissue, mammary, and a surrogate tissue, lymphocytes. Additionally, histopathology and immunohistochemistry were performed on the mammary tissues 20 weeks post-carcinogen treatment. Although, menopausal women are not entirely devoid of estrogen, BB rats with or without ovaries were used as models for pre-menopausal and post-menopausal conditions, respectively. Further, 17 ß-estradiol (E2) was used as a positive control for both treatment groups.

Materials and methods

Chemicals
DZ (lot 1-FSS-31-1) and GE (lot 6-ECGW-83-2) were purchased from Toronto Research Chemicals (Toronto, Canada). Purity as determined by nuclear magnetic resonance, desorption or direct electron ionization and gas chromatography/mass spectrometry and liquid chromatography-ultra-violet analyses was >99%. DMBA and E2 were from Sigma Chemicals (St. Louis, MO). TranspackTM in vitro {lambda} phage packaging extract and 5-bromo-4chloro-3-indolyl-b-D-glactopyranoside (X-gal) were obtained from Stratagene (La Jolla, CA). Mouse monoclonal anti-PCNA antibody was from Clone PC10, Dako Corporation (Carpinteria, CA). Biotinylated goat anti-mouse Immunoglobulin G was purchased from Boehringer-Mannheim (Indianapolis, IN), while streptavidin-conjugated horseradish peroxidase was supplied by Jackson Immuno-Research Laboratories (West Grove, PA).

Animals and diets
During the course of this experiment, we followed the recommendations set forth by the NCTR Institutional Animal Care and Use committee for the handling, maintenance, treatment, and euthanasia of the animals. Female Big Blue® (BB) rats were obtained from Taconic Farm (Germantown, NY) as weanlings and housed three per cage. BB rats were fed isoflavone-free, NIH-31C diet (29). NIH-31C diet has the same basic formulation as standard NIH-31 rat chow (Purina 5K96, Purina Mills Inc., St. Louis, MO) except that the protein contributed by soy meal and alfalfa was replaced by casein and the soy oil by corn oil. Beginning at 5 weeks of age, groups of rats were fed either the control diet or a diet containing DZ or GE at the following dose levels: 0.25 or 1.0 g/kg diet separately, or combined (1 g/kg diet of each DZ and GE) until the end of the study. The rationale for selecting 0.25 and 1 g/kg doses was based on a preliminary study in rats exposed for 16 weeks with 0.1 and 0.5 g/kg of GE that indicated that at a dose of 0.5 g/kg DMBA mutagenic response was enhanced while 0.1 g/kg was ineffective (A, Aidoo, personal communication). The high dose chosen for the present study is equivalent to a daily intake of 20 mg per rat. This amount falls within the range of 20–50 mg/day, consumed in Asian countries where soy-containing diets have been shown to reduce the risk for certain cancers and cardiovascular disease (18,19,30). Equally important, at the dose levels used in the present study, the isoflavones were detected in relevant concentrations in the serum as determined by HPLC analysis. Rats fed low and high doses of DZ or GE had values ranging from 15–100 nM and 170–570 nM, respectively. Equol, a metabolite of DZ was detected in the range of 500–1500 nM for rats fed low dose and 3000–7500 nM for the high dose, while no isoflavone was detected in the blood samples of animals fed control diet.

Additional animals in the study were fed a diet containing 0.005 g/kg E2 as a positive control (Figure 1). At postnatal day 50 (PND50), rats were gavaged with a single dose of 80 mg/kg DMBA or sesame oil (Figure 1). This dose of DMBA has produced high mutant frequencies between 16 and 18 weeks following treatment in BB and Fischer 344 rats (24). The PND50 treatment was based on carcinogenesis studies that indicate that rats at this age have high density of terminal end buds; ductal structures that are more sensitive to DMBA-induced mammary tumors (31).


Figure 1
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  		Fig. 1 Experimental design used to study the effects of isoflavones DZ, GE, DZG and E2 on DMBA-induced mutagenicity and carcinogenicity in ovariectomized (OVX) and intact (INT) female Big Blue (BB) rats 16 and 20 weeks, respectively following a single DMBA oral gavage. Plus indicates (+) rats were placed on diet containing isoflavone supplements and E2 2 weeks prior to DMBA treatment. Hash (#) indicates rats were gavaged with DMBA at PND 50. Asterias (*), rats were ovariectomized (OVX) 2 weeks post-carcinogen treatment.

 
After 2 weeks post-carcinogen treatment, rats were divided into two groups, and were either bilaterally ovariectomized (OVX; ovariectomy) under ketamine/xylazine (100 and 15 mg/kg, respectively), anesthesia, or the ovaries remained intact (INT). The rationale for treating the animals with the carcinogen before OVX is based on the fact that sensitivity of the rat to mammary tumor induction by DMBA is in part dependent on the hormonal state of the animal (32,33). Rats continued to have free access to food and water. Food consumption and body weight were recorded weekly.

Hprt mutagenesis assay
The Hprt experiments were conducted according to previously published methods (20,21) with minor changes. Briefly, the cloning efficiency (CE) of non-selected cells was measured by adding four target cells (lymphocytes isolated from the spleen and cultured for 2 days) per well to 96-well microtiter plates (round bottom), while for 6-thioguanine (6-TG)-selected cells, CE was measured by adding 5 x 104 cells/well. Following 10 day-incubation, the plates were scored for clone formation and the CE was calculated from the number of negative wells assuming a Poisson distribution of colonies. The mutant frequency (MF) was determined as the ratio of the CE in the selective plates to the CE in the non-selective plates.

DNA extraction from mammary epithelial cells and mammary gland
Six mammary glands and surrounding fat pads were removed from the inguinal region of female rats and stromal epithelial cells were isolated from freshly processed mammary gland tissue by collagenase digestion as described by Allaben et al. (34). The mammary epithelial cells were further processed to isolate nuclei and the genomic DNA was extracted from the nuclei using the BB RecoveraseTM DNA isolation kit according to the manufacturer's instruction manual. The DNA was then resuspended in TE buffer (10 mM Tris–HCl and 1 mM EDTA, pH 7.5) and quantitated by UV absorption.

Mammary lacI mutation assay
DNA extraction, lambda packaging, and plating for lacI mutant plaques were carried out in ‘blocked’ manner so as to minimize bias from day-to-day variations in experimental procedures. The lacI containing lambda shuttle vector was recovered by mixing the genomic DNA extracted from mammary gland, with TranspackTM in vitro {lambda} phage packaging extract as described previously (24). The resulting phages were pre-adsorbed to Escherichia coli SCS-8 cells for 20 min at 37°C, mixed with pre-warmed NZY top agar containing 1.5 mg/ml of X-gal, and poured into 250-mm assay trays containing Big Blue media. The plates were incubated overnight at 37°C and scored for mutant blue plaques. Color control mutants were included in all plating, and the results were accepted only if mutant CMI could be detected. Packaging and plating were repeated for the DNA samples until at least 2 x 105 plaques were scored for each data point. The mutant blue plaques were picked into individual tubes containing 0.5 ml of SM buffer and 50 µl of chloroform. To confirm the mutant phenotypes, all recovered putative mutant phages from the 250-mm assay plates were diluted 1:100 and re-plated on 100-mm plates with 3.5 ml of top agarose containing 1.5 mg/ml of X-gal. The sectored plaques were also verified for their phenotype as specified in previous experiments (24), and confirmed sectored plaques were separately scored. The lacI mutant frequency (MF) was calculated by dividing the number of verified mutant plaques by the total number of plaques analyzed.

PCNA/apoptosis analysis in mammary glands
The animals were euthanized at 27 weeks of age (20 weeks following DMBA treatment). The mammary glands were excised, fixed in 10% buffered formalin for 48–72 h, and processed for 8 h on a Shandon Pathcenter Tissue Processor (Shandon Inc. Pittsburg, PA). They were embedded in paraffin, sectioned at 4 µm, and mounted on positive (+) charged slides. Cell proliferation indices were determined for all the tissue samples by immunohistochemical localization of proliferating cell nuclear antigen (PCNA), slightly modified from Foley et al. (35). The slides were placed in xylene to remove paraffin, re-hydrated in a series of alcohol with decreasing water content and immersed in phosphate buffered saline. Endogenous peroxidase was quenched with 3% H2O2 containing 0.1% sodium azide. The sections were placed in antigen-retrieval solution consisting of 1% zinc sulfate in de-ionized water and heated for 7.5 min in a 700-watt microwave oven on full power. Routine streptavidin procedures were then performed, beginning with 0.5% casein to block non-specific binding of subsequent antibody and sequential incubation of sections in a mouse monoclonal anti-PCNA antibody, biotinylated goat anti-mouse IgG (Boehringer-Mannheim, Indianapolis, IN), and streptavidin-conjugated horseradish peroxidase. The PCNA-positive cells were visualized by incubating the sections in 3,3'-diaminobenzidine hydrochloride (DAB) chromogen, followed by counterstaining with Mayer's hematoxylin. The nuclei in the G0- phase cells were stained blue while the nuclei in the S-phase cells were stained dark brown and the nuclei in the G1-phase cells were stained light brown. The PCNA-positive cells were counted per a total of 1000 cells. For the enumeration of apoptotic cells, another set of slides was stained with the terminal deoxynucleotide transferase mediated dUTP nick-end labeling (TUNEL) as described by Gavrieli et al. (36).

Histopathological analysis
At necropsy, the mammary glands were examined grossly, removed and preserved in 10% neutral buffered formalin. Lesion descriptions were recorded on the IANR (Individual Animal Necropsy Record Form). Tissues were trimmed, processed and embedded in Tissue Prep II, sectioned at 4–6 µm, and stained with hematoxylin and eosin. In addition, select tissues were harvested for PCNA, apoptosis assay and in situ hybridization as described above. They were microscopically examined, and when applicable, non-neoplastic lesions were graded for severity.

Statistical analysis
MFs for the lacI, and Hprt genes were analyzed as a function of dose by one-way ANOVA. Since the MFs tend not to be normally distributed, a logarithmic transformation was performed before conducting the analyses. A paired t-test was used to compare the MFs between OVX and INT groups. One-way ANOVA followed by Holm-Sidak tests were used to analyze the PCNA and gross mammary tumor size data. Chi-squared tests were used to analyze histopathology data.

Results

Mortality, body weight, organ weight and food intake
In the OVX group, two animals receiving DZ 0.25 g/kg and treated with DMBA died early in the study. In INT animals, one rat in DMBA/E2 group also died early in the study but the cause of death was undetermined. Six other animals in various groups treated with DMBA were found to be moribund and therefore euthanized. All of these animals had a gross observation of a mammary gland mass that was diagnosed as an adenocarcinoma and included in the mammary tumor data. Body weight and food intake were measured weekly during the course of the study. DZ or GE feeding had no effect on body weight gain. In both OVX and INT rats receiving E2 with or without DMBA treatment, there was an apparent increase in body weight; however, as shown in a parallel experiment in the uterus, differences between means did not reach statistical significance (37). Total food consumption was essentially similar for all the treatment groups (data not shown). At 20 weeks sacrifice, organs/tissues were excised, examined and weighed. DZ or GE diet alone or combined with DMBA and fed to both OVX and INT rats had no effect on organ weights determined for selected tissues including mammary gland (data not shown).

Mammary lacI mutagenesis
Mammary lacI MFs for both OVX and INT rats fed isoflavones and estradiol with or without DMBA treatment are listed in Table I. The isoflavones and E2 intake alone generally produced a modest increase in the lacI MFs in both OVX and INT BB rats but none of the responses was significantly higher than the control. INT BB rats treated with DMBA alone or treated with DMBA+ isoflavones and E2 showed a significant increase in the lacI MFs as compared with their respective controls (P < 0.01), but none of the supplements or estradiol significantly altered the DMBA-induced lacI MF. In the OVX group, BB rats treated with DMBA alone or treated with DMBA+ isoflavones and E2 showed a significant increase in the lacI MFs as compared with their respective controls (P < 0.01). Unlike the responses found for the INT group, BB OVX rats fed the isoflavone mixture (DZG: 1 g/kg GE + 1 g/kg DZ) displayed a significant reduction in the DMBA-induced lacI MF (P < 0.05). To examine the effect of OVX on the mutagenicity of DMBA or the isoflavones and E2 in the mammary, a comparison of the MFs in the INT and OVX groups was made. The lacI MFs in the DMBA-treated groups with or without isoflavone supplements were significantly higher for INT rats compared with OVX rats (P < 0.05); whereas the control MFs with or without isoflavone supplements or E2 in the INT rats were not significantly different from those in the comparable OVX groups.


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  		Table I Mammary lacI MFs measured 16 weeks post-DMBA treatment in OVX and INT rats

 
Lymphocyte Hprt mutagenesis
Lymphocyte Hprt MFs measured in OVX and INT BB rats fed the isoflavone supplements with or without DMBA treatment are reported in Table II. The isoflavones and E2 intake alone generally produced a modest increase in the Hprt MFs in both OVX and INT BB rats, but none of the responses was significantly higher than the control. In the OVX group, BB rats treated with DMBA alone or treated with DMBA+ isoflavones and estradiol showed a significant increase in the Hprt MFs as compared with their respective controls (P < 0.01). None of the supplements or E2 significantly altered the DMBA-induced Hprt MF. INT BB rats treated with DMBA alone or treated with DMBA+ isoflavones or E2 showed a significant increase in the Hprt MFs as compared with their respective controls (P < 0.01). Compared with OVX group, INT BB rats fed the high dose DZ and isoflavone mixture showed a significant reduction in the DMBA-induced Hprt MF (P < 0.05). In contrast to the lacI MFs, the Hprt MFs measured in the DMBA-treated rats with or without isoflavone supplements or in the control rats with or without the isoflavones were not significantly different between INT and OVX rats (Table II).


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  		Table II Lymphocyte Hprt MFs measured 16 weeks post-DMBA treatment in OVX and INT rats

 
Mammary gland histopathology in OVX and INT rats
Both neoplastic and non-neoplastic lesions (%) in the mammary glands of OVX and INT rats fed isoflavones with DMBA are reported in Figure 2A and B, respectively. In the OVX group, although non-neoplastic changes such as atrophy and ductal hyperplasia predominated in most of the treatment groups, 20% of the BB rats fed E2 and exposed to DMBA also had adenomas (Figure 2A). The incidence of adenomas found only in E2 fed rats exposed to DMBA is probably associated with feeding exogenous E2 to the OVX rats treated with the carcinogen. The incidence of atrophy observed in most of the treatments including control is probably attributable to OVX and not treatment-related effects. However, the absence of atrophy and the preponderance of ductal hyperplasia seen in BB rats fed high dose DZ or the isoflavone mixture with or without DMBA exposure is likely due to an estrogenic effect of isoflavone DZ or its metabolite equol in OVX rats. On the contrary, BB rats fed both low and high doses of GE with or without DMBA treatment had mammary gland atrophy without hyperplasia (Figure 2A).


Figure 2
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  		Fig. 2 (A) Neoplastic and non-neoplastic lesions in the mammary glands in the OVX BB rats fed isoflavones, DZ, GE, DZG, and 0.005 g/kg diet of E2 and sacrificed 20 weeks post-DMBA (80 mg/kg) treatment. Values are mean ± SEM of 10 rats per treatment. *Significant increase in mammary adenoma tumors compared with DMBA treatment (P < 0.01). (B) Neoplastic and non-neoplastic lesions in the mammary glands in the INT BB rats fed isoflavones, DZ, GE, DZG and 0.005 g/kg diet of E2 and sacrificed 20 weeks post-DMBA (80 mg/kg) treatment. Values are mean ± SEM of 10 rats per treatment. No significant difference in adenoma/ adenocarcinoma between DMBA and DMBA+isoflavones.

 
In contrast to the mammary gland of the OVX rats with minimal neoplastic lesions, in the INT group, adenoma and adenocarcinoma were detected in most of the isoflavone-fed rats treated with DMBA (Figure 2B). Although DMBA alone induced adenoma/adenocarcinomas in 40% of BB rats, the incidence of adenoma/adenocarcinoma was increased (60%) in rats receiving 0.25 g/kg DZ, and 1.0 g/kg GE and to a lesser extent in animals given 0.25 g/kg GE, 1.0 g/kg DZ and 0.005 g/kg E2. The incidence of adenoma/adenocarcinoma was slightly lower (30%) relative to DMBA in rats fed the isoflavone mixture, 1.0 g/kg DZG but none of these changes was significantly different from DMBA alone.

Gross examination of mammary lesions in the INT animals revealed that the mammary tumors in rats treated with DMBA alone were larger in size with an average size approximating ≥28 mm (data not shown). There was a slight reduction in the average tumor size among rats treated with low doses of DZ (17 mm) and GE (23 mm). The maximum reduction in tumor size although non-significant, was seen among rats treated with high dose GE (11 mm) and DZG (8 mm), no such reduction was realized in rats fed either high dose DZ (25 mm) or E2 (21 mm).

PCNA/apoptotic index in mammary
The effect of dietary DZ, GE or E2 either separately or combined with DMBA treatment on cell proliferation and apoptosis was assessed by PCNA Immunohistochemistry and TUNEL, respectively (Figure 3). In general, rats fed isoflavone diets in the INT group (Figure 3A) had a slight increase in cell proliferation compared with OVX group (Figure 3B). On the other hand, all of the treatment groups with DMBA exposure produced markedly higher response in cell proliferation than the respective controls. In the OVX group, although there was no difference in the number of apoptotic cells, low dose GE and high dose DZ with DMBA treatment, produced significant increases in cell proliferation compared with DMBA alone (P < 0.05). In the INT group, rats fed with high dose GE showed significantly higher number of PCNA-positive cells in S-phase and G1-phase compared with the number of PCNA-positive cells in S-phase and G1-phase in rats fed control diets (P < 0.05). In addition, animals fed E2 diet or high dose DZ, with DMBA exhibited significantly higher cell proliferation rates than animals exposed to DMBA alone (P < 0.05).


Figure 3
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  		Fig. 3 Effect of DZ and GE on cell proliferation and apoptosis in the mammary of INT (A) and OVX (B) rats. Values are mean ± SEM of ten rats per treatment. *Significantly different from DMBA alone at P < 0.05. **Significantly different from control diet at P < 0.05.

 
Discussion

The main focus of this study was to determine whether or not the intake of GE or DZ, phytoestrogens currently used by women for menopausal health (38,39), modulates DMBA-induced mutagenicity and carcinogenicity in the mammary glands of BB rats. The results indicate that DMBA treatment significantly increased lacI MFs compared with the animals fed control diet without DMBA. In addition, feeding BB rats with low and high doses of DZ, GE or E2 separately did not alter either spontaneous or chemically induced lacI MFs in rat mammary (Table I). However, feeding the animals a diet containing the mixture (DZG, 1g/kg GE + 1 g/kg DZ) resulted in a significant reduction in the DMBA-induced lacI MF in the mammary of the OVX rats (P < 0.05, Table I). The lacI MFs in the DMBA-treated groups with or without isoflavone supplements were significantly higher in the INT rats compared with the OVX group (P < 0.05), indicating the potency of endogenous estrogens in augmenting DMBA mutagenicity. In contrast, the control MFs with or without isoflavone supplements or estradiol in the INT rats were not significantly different from the relative MFs in the OVX groups.

In addition to a target tissue, we examined a surrogate tissue, lymphocytes for DMBA-induced mutagenic activity in rats with and without ovaries. Unlike lacI MFs in mammary tissues, the Hprt MFs in lymphocytes showed no significant difference between INT and OVX rats (Table II) suggesting that the endogenous estrogen status did not influence DMBA-induced mutagenesis in lymphocytes. Irrespective of the condition of the animals (OVX or INT), the DMBA-induced mutagenic response in the surrogate (lymphocytes) appears to be lower than that observed in the target tissue (mammary); a response that may be partially attributable to the use of two fundamentally different genes with respect to the kinetics of mutant induction, the frequency of background mutations, and the sensitivity for measuring mutant induction by DMBA. In addition, INT rats fed with high doses of GE and DZG showed significant reductions in DMBA-induced MFs (P < 0.05; Table II) in lymphocytes, whereas similar reduction in the DMBA mutagencity to DZG exposure was observed only in the mammary of OVX rats (P < 0.05; Table I). This effect of isoflavones on DMBA-induced mutagenicity in two different tissues suggests that the isoflavones have pleotrophic properties and therefore can manifest different effects on different tissues depending on the presence or absence of estrogen receptors. Also, the effect of the phytoestrogens on DMBA metabolites which could affect the mutagenic outcome cannot be overlooked. Furthermore, recognizing that long tem effects of isoflavone intake in humans is not well established, the finding that DZ and GE modulated DMBA-induced mutagenicity in the target tissue, mammary and in the surrogate tissue, lymphocytes suggests that lymphocyte mutagenesis assay can be conducted with cells isolated from human peripheral blood (40) to predict breast cancer risk in individuals taking isoflavone supplements.

With the exception of rats fed the DZ (high dose), DZG, and E2 diets, mild atrophy was detected in the mammary glands of most of the treatment groups in the OVX animals (Figure 2A); however this condition was non-neoplastic and it was attributable to OVX and not a reflection of treatment-related effects. Animals exposed to a high dose of DZ or DZG and treated with DMBA had a mildly reduced severity in mammary gland atrophy. Animals fed the mixture had a marked increase in normally developed mammary gland compared with those treated with DMBA alone. Since atrophy was absent in animals fed the E2 diet, the reduction seen with the phytoestrogens clearly suggests an enhanced estrogenic action of DZ or GE when administered as a mixture. Also, the percentage of animals with mammary gland ductal hyperplasia (defined here as a relative increase per unit area of hypodermis of branching intralobular and/or interlobular ducts), was higher (100%) in OVX rats fed 1.0 g/kg DZ or the mixture alone (Figure 2A). The incidence was low, but relatively marked in isoflavone-fed rats exposed to DMBA. A similar response was seen with E2 either alone or combined with DMBA exposure. Interestingly, ductal hyperplasia was absent in OVX rats fed only the control diet or treated with DMBA alone (Figure 2A). Hyperplasia is generally initiated by hormonal stimuli or other factors and even though it reflects a non-neoplastic event such as cellular proliferation with neoplastic transformation potential, it also can serve as a physiologic and adaptive response useful to organisms. Thus, the high incidence of ductal hyperplasia seen in OVX rats fed the isoflavone alone appears to relate to a physiologic or adaptive response to the estrogenic action of DZ or GE. Although the effects of supplemental estrogens were nominal on PCNA-based cell proliferation (Figure 3A), this does not eliminate a causative effect on ductal hyperplasia. The PCNA assay provides a ‘snap shot’ of cell proliferation rates and altered proliferation rates may have occurred before the time-point analysis.

The combined administration of GE and DZ significantly reduced the DMBA-mutagenicity in the mammary gland in OVX rats, while this treatment was ineffective in INT rats. Although this appears to relate to the influence of estrogen dependency associated with DMBA initiated cells (26,41–44), the response is also suggestive of the anti-cancer potential of the isoflavones in conditions of estrogen deficiency as occurs in menopause. However, another possible explanation for the difference in response is that isoflavones possess selective estrogen-receptor modulator (SERM)-like properties and therefore can exert selective agonist or antagonist effects on various estrogen target tissues such as mammary (45). Thus, in the presence of endogenous estrogens in the INT rats, one would expect isoflavones, as agonist and antagonist to be comparatively ineffective; whereas in the absence of estrogen in the OVX rats, the isoflavones might effectively reduce DMBA-mutagenicity in the mammary.

Despite the significant DMBA mutagenic response seen in the OVX rats (Table I), histopathological examination of the mammary tissues in this group revealed that DMBA exposure was not associated with mammary tumor induction (Figure 2A). In the INT BB rats, the DMBA-mediated mutagenic response in the mammary was associated with ~40% of the tumors. This response was strikingly significant and may be specifically important given the fact that the sensitivity of BB rats (a derivative of Fischer 344 strain) to DMBA may be lower compared with the commonly used strain, Sprague–Dawley rats. Although, in the OVX rats, DMBA was effective at inducing the mutagenic response in the mammary comparable to that of INT rats, no tumors were formed. This is not unexpected since DMBA-mediated tumor formation in INT rats regresses in response to OVX (28). It also has been shown that administration of exogenous estrogen is accompanied by mammary tumor development in OVX rats exposed to DMBA (41). Further, in a companion study, it was observed that DMBA-mutagenicity was not associated with the occurrence of pathologic lesion in the uterus of OVX rats (37). These results are concordant with the lack of tumor formation in the mammary of DMBA-treated OVX rats, and suggest estrogen dependency in the transformation of DMBA initiated cells into malignancy.

Consistent with these findings is the observation that DMBA-treated rats fed with E2 in the present study resulted in a 20% incidence of adenomas (benign tumors) in the mammary of OVX rats. This response was associated with a 3-fold increase in PCNA/apoptosis ratio (cell proliferation: cell death), suggesting increased cell proliferation (Figure 3B). In the DMBA-treated rats fed DZ or GE no tumors were developed (Figure 2A). Although, the combination of isoflavones (DZG) significantly reduced DMBA-mutagenicity in the OVX rats, the absence of neoplasia in the face of DMBA exposure in DZ- and GE-fed OVX rats indicates, in part, that isoflavones have weak estrogenic activity, and may be less toxic in a menopausal condition when estrogen levels are low (46).

A high incidence of ductal hyperplasia similar to that observed in the OVX rats treated with isoflavones was also found in INT rats given isoflavones with or without DMBA treatment (Figure 2A and B). However, ductal hyperplasia also occurred in rats treated with the carcinogen alone, and the majority of INT rats developed mammary tumors by 20 weeks following carcinogen treatment (Figure 2B). These pathologic lesions in the mammary, classified as either adenoma or adenocarcinoma (benign or malignant tumors) were combined and their percentages are presented in Figure 2B. Of the DMBA-treated rats ~40% developed adenoma/adenocarcinoma and 60% had ductal hyperplasia (Figure 2B). The carcinogenic potency of DMBA in the INT rats appears to be due to the presence of endogenous ovarian hormones including estrogens (47–49); however, addition of exogenous E2 did not augment the carcinogenic outcome of DMBA.

In the rats fed DZ or GE, there was a slight increase in the percentage of animals with DMBA-induced mammary tumors, but the responses were not statistically significant. Although GE and DZ slightly increased the number of tumor bearing animals, the high dose GE substantially reduced the size of mammary tumors compared with tumor size seen in DMBA-treated rats alone, while this response was not observed in rats fed high dose DZ where cell proliferation was significantly increased relative to GE (Figure 3A). Feeding DMBA-treated rats with the mixture resulted in a reduced mammary tumor incidence and tumor size. Since in DMBA-treated rats receiving DZ alone tumor size was increased, it appears that GE was more effective compared with DZ.

Although the observed increase in DMBA carcinogenicity by the isoflavones was not significant, it clearly demonstrates the intrinsic estrogenic activity of phytoestrogens. The fact that isoflavones can increase DMBA-induced carcinogenicity suggests that like estrogens, these compounds can be co-carcinogens or tumor promoters in certain tissues if administered in the presence of a carcinogen or a pre-existing DNA damage. An increase in DMBA-induced mammary adenocarcinoma by 1.0 g/kg GE in wild-type, but not in ER-{alpha}-knockout mice has been reported (15). Chronic intake of 0.75 g/kg GE administered 6 weeks following carcinogen treatment or OVX (when tumors had already developed) increased mammary gland tumors in Sprague–Dawley rats (16). In addition, dietary GE 0.25 g/kg fed to female Fischer 344 rats exposed two times a week for 2 weeks with azoxymethane enhanced colon carcinogenesis (14). By contrast, in the present study rats fed diets containing high dose, 1.0 g/kg DZ or GE starting 2 weeks before DMBA treatment did not cause significant changes in mammary gland carcinogenesis in the INT rats (Figure 2B). Our results suggest that compared with E2, the isoflavones did not significantly alter DMBA-induced tumor incidence in the mammary. It should be noted, however, that BB rats were exposed to the carcinogen for up to 20 weeks compared with ~30 weeks in previous studies (14,16); this factor and the allocation of a limited number of rats for carcinogenicity study (10 rats per treatment) may have contributed to a lack of significant effects seen with the isoflavones. Thus, the smaller sample size and perhaps the short duration utilized in the present study, limit our ability to attribute these findings solely to a weak estrogenic nature of the isoflavones.

In summary, our results indicate that the intake of DZ or GE commencing 2 weeks prior to carcinogen treatment in both OVX and INT BB rats did not result in significant mutagenic or carcinogenic changes in the mammary tissue. However, administering DZ and GE in a mixture reduced DMBA-mediated mutagenicity and carcinogenicity in the mammary, with a significant reduction observed for the mutagenic response, suggesting the beneficial effect of isoflavone mixture.

Acknowledgements and notes

The authors wish to thank the Bionetics personnel for their expert technical assistance, and Dr Greg Olson and Alan Warbritton, Pathology Associates, Jackson Laboratories for their help in performing the histopathological and immunohistochemical analyses of the mammary tissues. Our gratitude also goes to Paul H. Siitonen, Division of Biochemical Toxicology for determining the purity of DZ and GE, and Drs William Witt and Jeff Carraway for veterinary services provided. This study was funded in part by the FDA Office of Women's Health, Rockville, MD.

Conflict of Interest Statement: The views presented in this article do not necessarily reflect those of the Food and Drug Administration.

Footnotes

This paper was previously published in Carcinogenesis 27(10) doi:10.1093/carcin/bgl195. This was not the final accepted paper and contained many errors. The publisher would like to apologize for this error.

References

  1. Messina M.J., Persky V., Setchell K.D., Barnes S. (1994) Soy intake and cancer risk: a review of the in vitro and in vivo data. Nutr. Cancer 21:113–131.[ISI][Medline]
  2. Kennedy A.R. (1995) The evidence for soybean products as cancer preventive agents. J. Nutr. 125:733s–743s.[Medline]
  3. Barnes S., Peterson T.G., Coward L. (1995) Rationale for the use of genistein-containing soy matrices in chemoprevention trials for breast and prostate cancer. J. Cell Biochem. Suppl. 22:181–187.[Medline]
  4. Knight D.C. and Eden J.A. (1996) A review of the clinical effects of phytoestrogen. Obstet. Gynecol. 87:897–904.[Abstract]
  5. Kurzer M.S. and Xu X. (1997) Dietary phytoestrogens. Annu. Rev. Nutr. 17:353–381.[CrossRef][ISI][Medline]
  6. Peterson G. and Barnes S. (1993) Genistein and biochanin A inhibit the growth of human prostate cancer cells but not epidermal growth factor receptor tyrosine autophosphorylation. Prostate 22:335–345.[ISI][Medline]
  7. Constantinou A. and Huberman E. (1995) Genistein as an inducer of tumor cell differentiation: possible mechanisms of action. Proc. Soc. Exp. Biol. Med. 208:109–115.[Abstract]
  8. Pommier Y., Zwelling L.A., Kao-Shan C.S., Whang-Peng J., Bradly M.O. (1985) Correlations between intercalator-induced DNA strand breaks and sister chromatid exchanges, mutations and cytotoxicity in Chinese hamster cells. Cancer Res. 45:3143–3149.[Abstract/Free Full Text]
  9. Kaufmann W.K. (1998) Human topoisomerase II function, tyrosine phosphorylation and cell cycle checkpoints. Proc. Soc. Exp. Biol. Med. 217:327–334.[Abstract]
  10. Kulling S.E. and Metzler M. (1997) Induction of micronuclei, DNA strand breaks and HPRT mutations in cultured Chinese Hamster V79 cells by the phytoestrogen cummoestrol. Food Chem. Toxicol. 35:605–601.[CrossRef][ISI][Medline]
  11. Morris S.M., Chen J.J., Domon O.E., McGarrity L.J., Bishop M.E., Manjanatha M.G., Casciano DA. (1998) P53, mutations, and apoptosis in genistein-exposed human lymphoblastoid cells. Mutat. Res. 405:41–56.[ISI][Medline]
  12. Misra R.R., Hursting S.D., Perkins S.N., Sathyamoorthy N., Mirsalis J.C., Riccio E.S., Crowell J.A. (2002) Genotoxicity and carcinogeniciy studies of soy isoflavones. Int. J. Toxicol. 21:277–285.[CrossRef][ISI][Medline]
  13. Giri A.K. and Lu L.J.W. (1995) Genetic damage and inhibition of 7,12-dimethylbenz(a)anthracene-induced genetic damage by the phytoestrogens, genistein and daidzein. Cancer Lett. 95:125–133.[CrossRef][ISI][Medline]
  14. Rao C.V., Wang C.X., Siml B., Lubet R., Kelloff G., Steele V., Reddy B.S. (1997) Enhancement of experimental colon cancer by genistein. Cancer Res. 57:3717–3722.[Abstract/Free Full Text]
  15. Day J.K., Besch-Williford C., McMann T.R., Hufford M.G., Lubahn D.B., MacDonald R.S. (2001) Dietary genistein increased DMBA-induced mammary adenocarcinoma in wild-type, but not ER alpha KO, mice. Nutr. Cancer 39:226–232.[CrossRef][ISI][Medline]
  16. Allred C.D., Allred K.F., Ju Y.H., Clausen L.M., Doerge D.R., Schantz S.L., Korol D.L., Wallig M.A., Helferich W.G. (2004) Dietary genistein results in larger MNU-induced, estrogen-dependent mammary tumors following ovariectomy of Sprague–Dawley rats. Carcinogenesis 25:211–218.[Abstract/Free Full Text]
  17. Abe T. (1999) Infantile leukemia and soybean- a hypothesis. Leukemia 13:317–320.[CrossRef][ISI][Medline]
  18. Chen Z., Zheng W., Custer L.J., et al. (1999) Usual dietary consumption of soy foods and its correlation with the excretion rate of isoflavonoids in overnight urine samples among Chinese women in Shanghai. Nutr. Cancer 33:82–87.[ISI][Medline]
  19. Arai Y., Uehara M., Sato Y., et al. (2000) Comparison of isoflavones among dietary intake, plasma concentration and urinary excretion for accurate estimation of phytoestrogen intake. J Epidemiol. 10:127–135.[Medline]
  20. Aidoo A., Lyn-Cook L.E., Mittelstaedt R.A., Heflich R.H., Casciano D.A. (1991) Induction of 6-thioguanine-resistant lymphocytes in Fischer 344 rats following in vivo exposure to N-ethyl-N-nitrosourea and cyclophosphamide. Environ. Mol. Mutagen. 17:141–151.[ISI][Medline]
  21. Azidoo A., Morris S.M., Casciano D.A. (1997) Development and utilization of the rat lymphocyte hprt mutation assay. Mutat. Res. 387:69–88.[CrossRef][ISI][Medline]
  22. Manjanatha M.G., Lyn-Cook L.E., Culp S.J., Beland F.A., Heflich R.H., Aidoo A. (1996) Lymphocyte mutant frequency in relation to DNA adduct formation in rats treated with tumorigenic doses of the mammary gland carcinogen 7,12-dimethylbenz[a]anthracene. Mutat. Res. 357:89–96.[ISI][Medline]
  23. Manjanatha M.G., Shelton S.D., Aidoo A., Lyn-Cook L.E., Casciano D.A. (1998) Comparison of in vivo mutagenesis in the endogenous Hprt gene and the lacI transgene of Big Blue rats treated with 7,12-dimethylbenz[a]anthracene. Mutat. Res. 401:165–178.[ISI][Medline]
  24. Manjanatha M.G., Shelton S.D., Culp S.J., Blankenship L.R., Casciano D.A. (2000) DNA adduct formation and molecular analysis of in vivo lacI mutations in the mammary tissue of Big Blue rats treated with 7, 12- dimethylbenz(a)anthracene. Carcinogenesis 21:265–273.[Abstract/Free Full Text]
  25. Chen T., Aidoo A., Manjanatha M.G., Mittelstaedt R.A., Shelton S.D., Lyn-Cook L.E., Casciano D.A., Heflich R.H. (1998) Comparison of mutant frequencies and types of mutations induced by thiotepa in the endogenous Hprt gene and transgenic LacI gene of Big Blue rats. Mutat. Res. 403:199–214.[ISI][Medline]
  26. Leung B.S., Sasaki G.H., Leung J.S. (1975) Estrogen-prolactin dependency in 7,12-dimethylbenz(a)anthracene-induced tumors. Cancer Res. 35:621–627.[Abstract/Free Full Text]
  27. Bradley C.J., Kledzik G.S., Meites J. (1976) Prolactin and estrogen dependency of rat mammary cancers at early and late stages of development. Cancer Res. 36:319–324.[ISI]
  28. Ip C. and Ip M.M. (1981) Serum estrogen and estrogen responsiveness in 7,12-dimethylbenz[a]anthracene-induced mammary tumors as influenced by dietary fat. J Natl Cancer Inst. 66:291–295.[ISI][Medline]
  29. Fu X., Blaydes B.S., Weis C.C., Latendresse J.R., Muskhelishvili L., Sutter T.R., Delclos K.B. (2003) Effects of dietary soy and estrous cycle on adrenal cytochrome p450 1B1 expression and DMBA metabolism in adrenal glands and livers in female Sprague–Dawley rats. Chem. Biol. Interact. 146:273–284.[CrossRef][ISI][Medline]
  30. Nagata C., Takatsuka N., Kurisu Y., Shimizu H. (1998) Decreased serum total cholesterol concentration is associated with high intake of soy products in Japanese men and women. J. Nutr. 128:209–214.[Abstract/Free Full Text]
  31. Russo J., Gusterson B.A., Rogers A.E., Russo I.H., Wellings S.R., van Zwieten M.J. (1990) Comparative study of human and rat mammary tumorigenesis. Lab. Invest. 62:244–278.[ISI][Medline]
  32. Dao T.L. (1962) The role of ovarian hormones in initiating the induction of mammary cancer in rats by polynuclear hydrocarbons. Cancer Res. 22:973–981.[Abstract/Free Full Text]
  33. Welsch C.W. (1985) Host factors affecting the growth of carcinogen-induced rat mammary carcinoma: a review and tribute to Charles Brenton Huggins. Cancer Res. 45:3415–3443.[Abstract/Free Full Text]
  34. Allaben W.T., Weis C.C., Fullerton N.F., Beland F.A. (1983) Formation and persistence of DNA adducts from the carcinogen N-hydroxy-2-acetylaminofluorene in rat mammary gland in vivo. Carcinogenesis 4:1067–1070.[Abstract/Free Full Text]
  35. Foley J.F., Dietrich D.R., Swenberg J.A., Maronpot R.R. (1991) Detection and evaluation of proliferating cell nuclear antigen (PCNA) in rat tissue by an improved immunohistochemical procedure. J. Histotechnol. 14:237–241.[Medline]
  36. Gavrieli Y., Sherman Y., Ben-Sasson S.A. (1992) Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J. Cell. Biol. 119:493–501.[Abstract/Free Full Text]
  37. Aidoo A., Bishop M.E., Shelton S.D., Lyn-Cook L.E., Chen T., Manjanatha M.G. (2005) Effects of Daidzein, Genistein, and 17 ß-estradiol on DMBA-induced mutagenicity and uterine dysplasia in ovariectomized rats. Nutr. Cancer 53:82–90.[CrossRef][ISI][Medline]
  38. Albertazzi P., Pansini F., Bonaccorsi G., Zanotti L., Forini E., de Aloysio D. (1998) The effect of dietary soy supplementation on hot flushes. Obstet. Gynecol. 91:6–11.[Abstract]
  39. Faure E.D., Chantre P., Mares P. (2002) Effects of a standardized soy extract on hot flushes: a multicenter, double-blind, randomized, placebo-controlled study. Menopause 9:329–334.[CrossRef][ISI][Medline]
  40. Cole J., Waugh A.P.W., Beare D.M., Sala-Trepat M., Stephens G., Green M.H.L. (1991) Hprt Mutant frequencies in circulating lymphocytes: population studies using normal donors, exposed groups and cancer-prone syndromes. Prog. Clin. Biol. Res. 372:319–328.[Medline]
  41. Teller M.N., Kaufman R.J., Bowie M., Stock C.C. (1969) Influence of estrogens and endocrine ablation on duration of remission produced by ovariectomy or androgen treatment of 7,12-dimethylbenz(a)anthracene-induced rat mammary tumors. Cancer Res. 29:349–352.[Abstract/Free Full Text]
  42. Bradley C.J., Kledzik G.S., Meites J. (1976) Prolactin and estrogen dependency in rat mammary cancer at later stages of development. Cancer Res. 36:319–324.[ISI]
  43. McCormick D.L., Mehta R.G., Thompson C.A., Dinger N., Caldwell J.A., Moon R.C. (1982) Enhanced inhibition of mammary carcinogenesis by combined treatment with N-(4-hydroxyphenyl) retinamide and ovariectomy. Cancer Res. 42:508–512.[Abstract/Free Full Text]
  44. Ohi Y. and Yoshida H. (1992) Influence of estrogen and progesterone on the induction of mammary carcinomas by DMBA in ovariectomized rats. Virchows. Arch. B Cell. Pathol. Incl. Mol. Pathol. 62:365–370.
  45. Riggs B.L. and Hartmann L.C. (2003) Selective estrogen-receptor modulators-mechanisms of action and application to clinical practice. N. Engl. J. Med. 348:8618–629.
  46. Adlercreutz H. and Mazur W. (1997) Phytoestrogens and western diseases. Ann. Med. 29:95–120.[ISI][Medline]
  47. Liehr J.G. (2000) Is estradiol a genotoxic mutagenic carcinogen? Endocr. Rev. 21:40–54.[Abstract/Free Full Text]
  48. Liehr J.G. and Roy D. (1990) Free radical generation by redox cycling of estrogens. Free Radic. Biol. Med. 8:415–423.[CrossRef][ISI][Medline]
  49. Liehr J.G., Roy D., Ari-Ulubelen A., Bui Q.D., Weisz J., Strobel H.W. (1990) Effect of chronic estrogen treatment of Syrian hamsters on microsomal enzymes mediating formation of catecholestrogens and their redox cycling: implications for carcinogenesis. J. Steroid Biochem. 35:555–560.[CrossRef][ISI][Medline]

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Decreased 7,12-dimethylbenz[a]anthracene-induced carcinogenesis coincides with the induction of antitumor immunities in adult female B6C3F1 mice pretreated with genistein
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