CIIT Centers for Health Research, Research Triangle Park, North Carolina 27709-2137
Received July 24, 2001; accepted December 20, 2001
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ABSTRACT |
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Key Words: genistein; methoxychlor; endocrine disruptor; mammary development; male mammary gland; male endocrine effects.
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INTRODUCTION |
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The endocrine effects of one toxicant may be influenced by the presence of additional endocrine-active compounds (EACs). The likelihood of such influence is high, considering the degree of human and animal exposure to various EACs in the environment. One principal source of such EACs is diet, which often contains substantial levels of phytoestrogens. A prototypical example of phytoestrogens is the isoflavone genistein, which is present in soybeans and other legumes. Genistein has a well documented ability to bind to the ER and exert estrogenic effects on reproductive development in animals (Casanova et al., 1999; Cheng et al., 1955
; Lamartiniere et al., 1998
). Genistein is commonly found in commercial diets for rodents used in toxicological studies, but the effects of phytoestrogens on the outcome of reproductive studies in endocrine toxicology have not been adequately investigated. Human populations are also exposed to diet-based phytoestrogens. In addition, an increasing number of people are taking extracted isoflavones as a dietary supplement in an attempt to benefit from reported chemopreventive action and for postmenopausal estrogenic maintenance (Anderson et al., 1999
).
The mammary gland is a highly endocrine-sensitive organ that relies on ovarian steroids and other hormonal signals for its proper growth and differentiation. Among the health issues believed to be related to estrogenic exposures at early developmental stages are earlier commencement of puberty in young girls and breast cancer in the adult female population (MacMahon et al., 1982). In order to understand the responsiveness to EACs of the mammary glands in prepubertal rats and to evaluate the potential of genistein to influence the toxicity of a second toxicant, we exposed pregnant rats to combinations of genistein and methoxychlor through diet during pregnancy and the lactational period. Two levels of genistein were used in this study. Genistein at a 300-ppm level was intended to represent roughly the total phytoestogens found in the commonly used NIH-07 rodent diet, and an 800-ppm level was included to approximate a level that was demonstrated to be estrogenic in vivo (Casanova et al., 1999
). The 800-ppm concentration of methoxychlor used in this study was to represent a level that was endocrine-active (Harris et al., 1974
) so that potential influence of genistein on the toxicological behavior of methoxychlor could be evaluated. Growth and differentiation of the mammary glands in rat offspring were evaluated in conjunction with a larger study on the effects of such exposure on sexual and reproductive development. Our results indicated that, at the 800-ppm dose level, these two estrogenic compounds might have a complex interplay in their effects on prepubertal mammary gland development.
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MATERIALS AND METHODS |
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The pregnant dams were housed individually in plastic cages with alpha dry cellulose bedding and provided with deionized water ad libitum. The animal room was maintained within a temperature range of 2225°C and relative humidity of 50 ± 10% with 12-h (7:0019:00) light cycles. Following parturition, the offspring rats were housed with their respective dams until postnatal day (PND) 22, at which time they were killed for necropsy. Starting on the day of arrival at the animal facility (GD 0), the pregnant dams were assigned to treatment groups and provided with the appropriate treatment diet. The same diets were maintained throughout the study.
To produce the treatment diets, appropriate amounts of genistein (>98% purity, Indofine, Somerville, NJ) and methoxychlor (M) (95% purity; Sigma, St. Louis, MO) were blended with a base diet, a custom-prepared soy- and alfalfa-free diet (SAFD) (Zeigler Bros., Gardners, PA, formula code: 54120000) of previously described composition (Casanova et al., 1999
).
Three levels of genistein (nominal 0, 300, and 800, for either mg/kg or parts per million) and two levels of methoxychlor (nominal and 800 ppm) were used in 6 different combinations. The treatment diets with test chemical contents were control: without either genistein or methoxychlor (base SAFD diet); 800 M: 800 ppm methoxychlor; 300 G: 300 ppm genistein; 300 G + 800 M: 300 ppm genistein plus 800 ppm ethoxychlor. To produce the treatment diets, SAFD was mixed with appropriate amounts of genistein to result in diets containing 0, 300, and 800 ppm genistein (for diets 2 and 3). The diet at each genistein level was then apportioned into two parts, one of which was mixed with appropriate amounts of methoxychlor to achieve a dietary concentration of 800 ppm methoxychlor. The uniformity of blending was verified through chromatographic analyses. Each batch of diets was prepared to last for approximately 4 weeks, and the prepared diets were stored at 4050°F.
Mammary gland whole mounts and quantification.
Both sides of the inguinal mammary glands from one male and one female pup in each litter were removed at necropsy on PND 22 for evaluation of glandular development. One gland in each animal (4 animals per treatment group) was used for whole-mount preparation, and the other was used for tissue section preparation. The whole-mount preparation protocol was a modification of a procedure described by Murrill et al (1996). To prepare whole-mount samples, freshly dissected tissue pieces were flatly placed between 2 glass slides and fixed in 70% formalin for 24 h before being dehydrated in ethanol solutions with graded concentrations of 70, 95, and 100% (about 3 h in each concentration). The samples were then defatted in acetone for approximately 12 h and rehydrated for 3 h at each concentration of increasingly diluted ethanol solutions ranging from 100 to 40%. The samples were stained with 0.005% toluidine blue for 30 min and then dehydrated again in ethanol solutions. The tissue pieces were finally treated in xylene for approximately 6 h. The glandular tissue, along with the associated lymph node and fat pad, was mounted on a glass slide with Permount and cover slip. The area of each mammary gland was calculated using the Image-I image analysis software (Universal Imaging Corp., West Chester, PA). The number of generations of branching in the mammary duct structure as well as the total number of terminal end buds (TEB) and lateral buds (LB) were counted based on the images of the whole-mount mammary gland, according the procedures described by Thompson et al., 1995.
Serum hormones.
Truck blood samples were collected at necropsy following decapitation. Serum samples were prepared by centrifuging coagulated blood samples at approximately 1000 g and 4°C for 30 min and stored below 20°C until the hormone assay. Serum level of prolactin was measured using a radioimmunoassay kit from Amersham (Buskinghamshire, UK). The measurements of rat-specific serum insulin-like growth factor-1 (IGF-1) were performed using radioimmunoassay kits from Diagnostic Systems Laboratories (Webster, TX).
Immunohistochemistry.
The inguinal mammary glands from control and treated male rats, contralateral to the ones used for whole mounts, were fixed in 10% neutral buffered formalin for 24 h. Fixed tissue samples were embedded in paraffin, and 5-mm sections were cut and processed for immunohistochemistry, using a procedure previously described (You and Sar, 1998). Briefly, the tissue sections were deparaffinized and treated with 3% H2O2 in PBS (pH 7.4) for 10 min, followed by antigen retrieval for 36 min in a microwave oven, using citrate buffer (pH 5.55.7, BioGenex, Dublin, CA). Appropriate primary antibodies, either the anti-IGF-1 receptor-ß antibody (1:500 dilution, Santa Cruz Biotechnology, Santa Cruz, CA), a monoclonal anti-ER
antibody (1:500 dilution, Dako Corp., Carpinteria, CA), a mouse monoclonal antiproliferating cell nuclear antigen (anti-PCNA) antibody (1:1000 dilution, Signet Pathology Systems, Dedham, MA), or a polyclonal rabbit antirat progesterone receptor antibody (1:400 dilution, Santa Cruz Biotechnology) was used on paraffin sections. After incubating at 4°C overnight, the tissue sections were stained using the avidin-biotin peroxidase method, in which the secondary antibodies (a biotinylated goat antirabbit IgG for polyclonal primary antibodies or biotinated horse antimouse IgG for monoclonal antibodies) and the avidin-biotin peroxidase complex (Vector Labs, Burlingame, CA) were used at a dilution of 1:200 PBS. Liquid DAB (BioGenex) was used as a chromagen for polyclonal antibody and AEC (Xymed Laboratories, South San Francisco, CA) as chromagen for monoclonal antibody. Immunostained sections were counterstained with hematoxylin and mounted with Permount. Slides were evaluated and photographed on a Vanox-S photomicroscope (Melville, NY). The PCNA-stained slides were evaluated for labeling index (LI), which is the ratio of actively dividing cells to the total number of cells in a mammary gland section. This evaluation was done by projecting the magnified image of a mammary gland section onto a computer screen, randomly selecting areas of the image using a grid (the number of gridded fields used for each sample section ranged from 8 to 32), and counting the total number of cells in these areas and the number of cells stained positive for PCNA.
Statistics.
Mammary gland morphometric measurements and serum hormone data were expressed as means ± SD. Sex difference in the morphometric measurements was probed with a three-way analysis of variance (ANOVA) test using genistein, methoxychlor, and sex as the main effects. The differences between the male and female pups in the morphometric measurements in the control group were probed by a paired t-test for each parameter. Homogeneity of variances for the treatment groups were tested with Levene's test, and all statistical analyses used untransformed data. The morphometric parameters of the male whole-mount mammary glands, the PCNA staining scores, and the serum hormone data were analyzed using a two-way analysis of variance procedure (two-way ANOVA), with genistein and methoxychlor as the treatment factors along with a term of interaction. When the methoxychlor term was significant in an analysis, a treatment effect due to methoxychlor was concluded. However, when the genistein term became positive, a t-test was used to contrast individual pairs between the zero and 300-ppm, and zero and 800-ppm dose levels, and the Bonferroni correction was used to adjust the significance values with respect to multiple pair-wise comparisons. The level of significance for all tests was set at p < 0.05, and all procedures were performed using the JMP statistical software (SAS Institute, Cary, NC).
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RESULTS |
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In the tissue sections, the amounts of glandular epithelium in the various treatment groups corresponded well with the degree of glandular growth observed in the whole mounts. The magnitude of glandular proliferation was also indicated by the PCNA staining in situ. In the control group, a modest amount of mammary epithelium was observed in the surrounding fat pad (Fig. 4A). The epithelial tissue in this group displayed the characteristics of extending ducts with a limited amount of budding activities. The sizes of the ducts were small, not reaching the lymph nodes, and their density was low, occupying a small proportion of the fat pad area. Only a small number of the epithelial cells were PCNA-positive in the control group, indicating a relatively inactive state of cell proliferation. The proliferative status of the mammary glandular ducts was drastically different in the 800 M group compared to the controls (Fig. 4B
). The glands contained a large number of ducts with high density in the mammary pad. Many of these ducts had numerous PCNA-positive epithelial cells lining the luminal wall of the ducts, and the epithelium frequently contained multilayer cells. In the high-dose genistein-treated rats 800 G (Fig. 4C
), the degree of epithelial proliferation was greater than the control group. However, the characteristics of glandular proliferation in this group were different from those in group 800 M. The genistein treatment resulted mainly in bud formation (portions of these budding structures were PCNA-positive) in contrast to mainly ductal growth seen in the methoxychlor-only group. These buds were predominately in the LB category, with a limited number of alveolar buds seen in the terminal portion of the glandular tree. The microscopic morphology of the 800 G + 800 M group was dramatically different from the other groups (Fig. 4D
). In these animals, not only were the bud structures numerous in the tissue sections but distinctive lobular structures could also be seen throughout the glandular area. In this group, most of the glandular epithelium consisted of structures of multilayer cells and a high degree of PCNA staining, indicative of active proliferation. The mammary epithelium in various groups was presented in higher magnification in Figure 5
, showing that the genistein- and methoxychlor-exposed groups demonstrated a higher level of glandular differentiation and proliferation, which was evident with the multilayer structure of the epithelium and numerous budding structures. Morphometric analysis of the PCNA immunostaining supported the general observation of glandular proliferation through the tissue sections and the whole-mount preparations. Both genistein and methoxychlor significantly increased the PCNA labeling of the mammary gland epithelium (p < 0.05, two-way ANOVA) without significant interaction. Genistein was only effective at the 800-ppm dose level (Fig. 6
, p < 0.05, t-test, with Bonferroni correction).
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DISCUSSION |
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Cardy (1991) described the sexual dimorphism of mammary gland morphology in the rat. The histological features of mammary glands were essentially identical between the sexes at the time of birth. This similarity lasts throughout the prepubertal stages until PND 35 or so, around the beginning of male pubertal development and before the rate of ductal development in the female mammary gland outpaces that in the male. The present study also indicated a lack of sexual dimorphism in the prepubertal rats in that we observed similar, although not identical, mammary gland gross morphology for both sexes at PND 22. We do not know, however, why the female juvenile pups have similar morphology to the male pups, even though the females have higher endogenous estrogen levels than the males (the serum level of 17ß-estradiol in PND-21 female Sprague-Dawley rats is approximately 50 pg/ml, about 10-fold that of males at the same stage; O'Connor et al., 1999; Ronis et al., 1996
). Nor do we know what was responsible for the difference between the two sexes in their responses to genistein and methoxychlor, even though they have similar developmental characteristics at the prepubertal stage.
In the present study, the effects of genistein on the male prepubertal mammary glands was mainly expressed as enhanced glandular differentiation at the 800-ppm level, which is believed to be an estrogenic dose (Casanova et al., 1999). The 300-ppm level, which for the most part did not produce a mammary effect in the present study, was thought to be roughly equivalent to the total phytoestrogen level found in the commonly used NIH-07 rodent diet (Casanova et al., 1999
). Genistein is known to affect mammary gland development in exposed rodents. Murrill et al. (1996) reported that genistein injection (sc) to female rats on PND 16, 18, and 20 at the dose of 50 µg/g significantly increased mammary gland size and the number of TEBs. Our observation in the present study was somewhat different, in that we detected no significant change in gland size in either the male or female rats on PND 22 in response to the genistein treatment. This difference might be due to the different treatment regimens involved. The genistein in our study was given to the rats in their diet during gestation and lactation. The offspring were exposed to the compound mostly through transplacental and lactational transfer, and the exposure was direct only during the last phase of nursing when gradual increases in the offspring feed intake occurred. Genistein is highly conjugated in vivo (Piskula, 2000
), and factors affecting its biotransformation status could alter the estrogenic potency of the compound. In fact, the treatment of dietary genistein in our study did not result in a significant increase in the uterine weights of the exposed animals (data not shown), contrary to the outcome following high-dose genistein injections as done in the Murill et al. study (1996).
The dose level of methoxychlor used in this study was not intended to represent likely environmental exposure; rather, the 800-ppm level was chosen because it approximates a toxicologically effective dose (Harris et al., 1974). Only limited information is available on the effects of methoxychlor exposure on the mammary gland. Female rats exposed to methoxychlor lacked alveolar development (Chapin et al., 1997
). Meanwhile, mammary glandular proliferation was noted in methoxychlor-treated adult male swine after exposure to the compound at 1000 ppm in the diet for several months (Reuber, 1980
). Since no sex comparison was made in those studies, we do not know whether the male and female in these respective species would have responded differently under their experimental conditions.
Both genistein and methoxychlor have the ability to activate the ER in vitro. However, our observations of developmental effects on the mammary gland were very different with respect to the two compounds. Genistein mainly enhanced the differentiation of the glands, expressing as moderately increased number of lateral buds and a limited degree of alveolar formation, while methoxychlor by itself caused extensive ductal proliferation. Such differences indicate that estrogenicity alone does not explain the observed effects in prepubertal male rats following treatment. While signaling mediated by the ER is important in the normal development of mammary glands, the effects of estrogens may also be due to their regulation of other mammotrophic signals, including the ones involved with the progesterone and prolactin pathways (Bocchinfuso et al., 2000).
In addition, although both genistein and methoxychlor are regarded as estrogenic compounds, their receptor reactivities have important differences. Genistein is an agonist at both ER and ERß, but methoxychlor, through its metabolism to HPTE, can be an ER
receptor agonist and an ERß antagonist (Gaido et al., 1999
). ERß is expressed in the mammary tissue, although its physiological roles are not yet well understood (Saji et al., 2000
). In addition, the effect of methoxychlor might have an androgen component due to the androgen receptor antagonism of HPTE. The effects of androgens on mammary gland growth are complex and may be species-specific. While testosterone is thought to virilize the female rat mammary gland during development (Goldman et al., 1976
), this androgen can also increase mammary epithelium density when given to female rats (Xie et al., 1999
). We have little information at present regarding the nature of involvement for the antiandrogenicity of HPTE in the mammary phenotypes associated with methoxychlor exposure.
A central issue in the health effect of chemicals is the potential of one exogenous compound to affect the toxicological behavior of another when simultaneous exposures occur. The likelihood of biological interactions among compounds has particular importance for endocrine-active agents because of the background exposure to and supplemental usage of phytoestrogens. Such a case of interaction was clearly seen in the present study. Methoxychlor alone promoted proliferation of the mammary ductal tissue, while genistein mainly enhanced the glandular differentiation and moderated the ductal elongating effect of methoxychlor when administered together. The combined administration of genistein and methoxychlor in the 800 G + 800 M group also promoted lobular growth in the mammary epithelium of prepubertal male rats. Such lobular structures are normally seen in postpubertal or early pregnant female rats (Russo et al., 1989a,b
). Similar features were not seen in the genistein-only and methoxychlor-only groups. Namely, combined exposure to the two compounds resulted in phenotypic effects there were distinctively different from the two types of effects associated with each compound alone.
One may suspect that mammary effects observed in the 800 G + 800 M group may be due to the additive estrogenic action of the two compounds. This is likely to be true in regard to the estrogenicity component of the combined treatment but unlikely to account for the observed lobular development. We do not know exactly what the endocrine conditions are that promote lobular development in male mammary glands. In the female, however, lobular proliferation requires the presence of progesterone (Plaut et al., 1999; Russo et al., 1989a
); this requirement for progesterone fits the role of this hormone in preparing the mammary gland during pregnancy for impending milk production.
IGF-1, a mediator for the mammotrophoic growth hormone, is essential for mammogenesis (Ruan and Kleinberg, 1999), and its effects on mammary tissue are believed to depend on local circuits (Akers et al., 2000
). In the present study, serum IGF-1, most of which is produced in the liver, was not affected by either genistein or methoxychlor. The immunostaining of IGF-1 receptor, while not a quantitative measure, suggests that the IGF-1/IGF-I receptor-mediated signal was enhanced in the hyperproliferative mammary glands of the treated animals.
While it has been demonstrated that both ER and ERß are presented in prepubertal female rats (Saji et al., 2000
; Zeps et al., 1999
), there is little information available regarding their expression in the mammary glands of prepubertal male rats. In the present study, we were not able to demonstrate immunoreactive ERß. However, the level of ER
seems to be inducible by the combined treatment of genistein and methoxychlor. Estrogen effects on female mammary gland growth are well documented in many standard texts. However, a possible role of ER
in male mammary growth has not been established, and we do not know whether upregulation in the expression of ER
is indeed a mediator of the treatment effects or simply a consequence of it. The level of progesterone receptor (PR) expression in cellular systems or at tissue sites has been frequently associated with estrogenic influence. In the mammary glands, upregulation of PR in the nuclei of the mammary epithelial cells, as observed in the 800 G + 800 M group of the present study, may have pathophysiological consequences, since the signals mediated by this receptor are a key regulator of mammary epithelial differentiation, particularly in regard to ductal formation in the mammary glands (Atwood et al., 2000
; Humphreys et al., 1997
). Indeed, mammary morphology in the 800 G + 800 M group was similar to that of male mice exposed to both estradiol and progesterone (Freeman and Topper, 1978
). Due to limited sample availability, our evaluation of both ER and PR did not include all treatment groups. Thus, we do not know if upregulation in the expression of these receptors might have been caused by either genistein or methoxychlor alone. Based on the physiological role of PR in promoting branch formation in mammary ductal trees, we speculate that methoxychlor exposure, which caused a high degree of ductal growth, could be the major factor responsible for the elevated PR presence in the nuclei of the mammary epithelium.
In conclusion, we found that mammary glands of prepubertal male rats were sensitive to genistein and methoxychlor. When these two compounds were given in combination at high doses, the mammary glands displayed lobular growth that did not present in animals treated with either compound alone. In addition to the hormone-like actions of the two compounds, due to their ER reactivities, the observed effects might have also involved pathways mediating the signals of other steroid hormones and some local growth factor pathways regulating mammary growth. Our future studies will explore what the primary responses are in the mammary glands following treatment, and what, if any, long-term consequence in development may be associated with the effects seen at prepubertal stages. Equally as important, research should also be directed at understanding the relevance of the effects described in the present study and their associated mechanisms to the levels of exposure that are likely to occur in real-life scenario for humans and animals.
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ACKNOWLEDGMENTS |
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NOTES |
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