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Endocrinology Vol. 147, No. 10 4871-4882
Copyright © 2006 by The Endocrine Society

Neonatal Exposure to the Phytoestrogen Genistein Alters Mammary Gland Growth and Developmental Programming of Hormone Receptor Levels

Elizabeth Padilla-Banks, Wendy N. Jefferson and Retha R. Newbold

Developmental Endocrinology and Endocrine Disruptor Section, Laboratory of Molecular Toxicology, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, North Carolina 27709

Address all correspondence and requests for reprints to: Retha R. Newbold, P.O. Box 12233, National Institute of Environmental Health Sciences, Mail Drop E4-02, Research Triangle Park, North Carolina 27709. E-mail: newbold1{at}niehs.nih.gov.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Developmental effects of genistein (Gen) on the mammary gland were investigated using outbred female CD-1 mice treated neonatally on d 1–5 by sc injections at doses of 0.5, 5, or 50 mg/kg·d. Examination of mammary gland whole mounts (no. 4) before puberty (4 wk) revealed no morphological differences in development after Gen treatment. However, mice treated with Gen-50 had stunted development characterized by less branching at 5 wk and decreased numbers of terminal end buds at 5 and 6 wk. Conversely, at 6 wk, Gen-0.5-treated mice exhibited advanced development with increased ductal elongation compared with controls. Measurements of hormone receptor levels showed increased levels of progesterone receptor protein and estrogen receptor-ß mRNA in Gen-0.5-treated mice compared with controls; ER{alpha} expression was decreased after all doses of Gen treatment. Lactation ability, measured by pup weight gain and survival, was not affected after neonatal Gen-0.5 and Gen-5. Mice treated with Gen-50 did not deliver live pups; therefore, lactation ability could not be determined. Evaluation of mammary glands in aged mice (9 months) showed no differences between Gen-0.5-treated mice and controls but mice treated with Gen-5 and Gen-50 exhibited altered morphology including reduced lobular alveolar development, dilated ducts, and focal areas of "beaded" ducts lined with hyperplastic ductal epithelium. In summary, neonatal Gen exposure altered mammary gland growth and development as well as hormone receptor levels at all doses examined; higher doses of Gen led to permanent long-lasting morphological changes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
EXPOSURE TO HIGH levels of estrogens during perinatal life has been suggested to be a risk factor for developing breast cancer later in life (1). For example, female twins developing in an elevated intrauterine estrogen environment have an increased incidence of breast cancer; this supports the idea that the fetal hormonal environment plays a role in predisposing women to breast cancer later in life (2, 3, 4). Also, increased levels of circulating estrogens usually found in older pregnant women have been associated with an increased breast cancer risk in their daughters (5, 6). Conversely, preeclampsia characterized by restricted estrogen production in the placenta and high maternal blood pressure correlates with a decreased incidence of breast cancer in daughters (4). Furthermore, epidemiological studies following a cohort of women exposed prenatally to the synthetic estrogen diethylstilbestrol (DES) have found an increased incidence of breast cancer (18%) as they age (7, 8). These studies provide evidence that early estrogen exposure is a risk factor for the subsequent development of breast cancer. These human data are supported by experimental animal data that demonstrate the proliferative and carcinogenic potential of estrogens in its target tissues including the mammary gland (9, 10, 11, 12).

Rodent models of developmental exposure to DES have been useful in reproducing and predicting abnormalities and tumors in humans prenatally exposed to DES (10, 11, 13, 14), and have shown that the mammary gland is extremely sensitive to DES (13, 15, 16). Prenatal or neonatal treatment of rats with DES increased the incidence of mammary lesions (hyperplastic alveolar nodules, dysplasia, and neoplasia) and decreased tumor latency (15). Neonatal treatment of mice with DES increased sensitivity to hormones and carcinogens later in life (17). Similar studies using BALB/c mice treated prenatally with DES showed that palpable mammary tumors developed in the presence of mouse mammary tumor virus (18), and in CD-1 mice mammary tumors developed even in the absence of this virus (19).

Over the last 25 yr, epidemiological studies have suggested the incidence of breast cancer in the United States is on the rise (20, 21, 22). Because only 50% of breast cancer can be explained by known risk factors such as age, nationality, family, and reproductive history, there is increasing interest in examining the role of developmental exposures to environmental toxicants and estrogen-mimicking chemicals in the etiology of this disease. Reports in animal models have suggested that exposure during development to environmental chemicals with estrogenic activity increases the risk of mammary cancer (23) as well as causes early puberty (24) and abnormal estrous cyclicity (25). Also, exposure to the estrogenic pesticide dieldrin has been shown to increase the risk of breast cancer in exposed women and decrease their survival rate in a dose-dependent manner (26, 27). Furthermore, in utero exposure of mice to low, environmentally relevant doses of bisphenol A caused abnormal histoarchitecture and altered DNA synthesis in mammary tissues, two changes that have been associated with carcinogenesis in both rodents and humans (28). Taken together, these data provide strong evidence that exposure to environmental chemicals with hormone activity during critical stages of development is associated with an increase risk of breast cancer later in life.

The isoflavone genistein (Gen) is a naturally occurring phytoestrogen found in soy products. Gen, generally in the glycosylated form in the diet, accounts for more than 65% of the isoflavones found in soy. Although Gen-rich foods and supplements have been proposed for use by menopausal women because of potential beneficial effects, adverse effects can occur if exposure occurs during development, particularly if exposure occurs during critical periods of differentiation of reproductive and breast tissues (29, 30, 31). Therefore, it is of concern that infants are exposed to Gen through soy-based formulas (32) and newly marketed soy-enriched products designed to appeal to children with the intention of providing them a healthy diet. Infants consuming soy-based formula are exposed to 6–11 mg/kg·d of isoflavones (4–7 mg/kg·d of total Gen) resulting in circulating levels of approximately 1–5 µM total Gen (33). In contrast, adults consuming a moderate to large amount of soy in the diet are exposed to approximately 1 mg/kg·d of total Gen resulting in circulating levels of approximately 0.5 µM total Gen (32). Although the form of Gen predominantly found in dietary sources is the glycosylated form, genistin, recent studies have shown that genistin is quickly hydrolyzed to the unconjugated form genistein in the gut and readily absorbed by infants because glucuronidated Gen metabolites and other metabolites were found in their urine (34). Although the typical route of exposure for humans is through the diet, a previous pharmacokinetic study from our laboratory using sc injections of Gen at a dose of 50 mg/kg·d produced serum circulating levels of 6.8 µM (in female pups), which is similar to human infants drinking soy-based infant formulas (1–5 µM). Furthermore, the percentage of circulating levels of the aglycone form of Gen (unconjugated, estrogenically active form) is much higher in neonates compared with adults (35, 36), making the developmental period especially sensitive to perturbation by estrogenic compounds like Gen. Thus, infants are exposed to much higher levels of Gen than adults, particularly because soy-based infant formulas can be their sole source of nutrition for many months. Unfortunately, there have been few epidemiology studies examining long-term effects of soy infant formula and soy-enriched diets for children.

Numerous experimental studies have examined developmental effects of Gen in estrogen target tissues (37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47). Several studies from our laboratory using outbred CD-1 mice have shown that neonatal exposure to Gen (0.5–50 mg/kg·d) by sc injection on d 1–5 caused deleterious effects on the female reproductive tract including altered ovarian differentiation, subfertility/infertility, and development of uterine cancer later in life (39, 45, 46, 47). Another study using rats exposed to Gen (40 mg/kg·d) neonatally by oral gavage showed similar adverse effects on the female reproductive system (48). Changes in sexually dimorphic areas of the brain, brain function, and altered reproductive behavior (44, 49, 50) have also been described. In humans, an epidemiological study showed an association with vegetarian diets during pregnancy and reproductive tract malformation (hypospadias) in the male offspring (51), and other studies indicate soy products have hormonal effects in adult women (52, 53, 54, 55, 56, 57).

The National Toxicology Program has evaluated the effects of dietary Gen in Sprague Dawley rats for multigenerational and chronic effects on reproductive tract tissues and other estrogen target tissues (16). Histopathological examination of the mammary gland of female pups from rat dams fed 250-1250 ppm Gen (~20–100 mg/kg·d) showed ductal/alveolar hyperplasia. In male pups, the most sensitive target tissue for endocrine disruptor effects was the mammary gland, which showed significant ductal/alveolar hyperplasia and hypertrophy at 25 ppm of Gen (~2 mg/kg·d). Chronic effects of Gen in the National Toxicology Program study suggested that Gen treatment decreased the incidence of benign mammary fibroadenomas but increased the incidence of mammary adenocarcinoma (Delclos, K. B., personal communication). A recent study showed similar effects on the male mammary gland after dietary Gen treatment (300 and 800 ppm) during pregnancy and lactation; furthermore, Gen increased the sensitivity of the mammary gland to the pesticide methoxychlor, a proestrogen (58). These data suggest that mammary gland development is particularly susceptible to disruption by estrogenic compounds if exposure occurs during critical stages of differentiation.

Other reports described the long-term effects on the mammary gland after developmental exposure to Gen (59, 60, 61, 62, 63). The timing of exposure, route, and dose of Gen appeared to be important factors in the overall effect on the mammary gland. For example, one study exposing rats to Gen (~1–10 mg/kg·d) through the diet from gestation d 0 through postnatal d 21 (weaning) showed resistance to dimethylbenz[a]anthracene (DMBA)-induced mammary tumors later in life (59). There was also reduced terminal end bud (TEB) development as well as immature terminal ductal structures that remained until adulthood. In another study from the same laboratory, exposure to high levels of Gen (~500 mg/kg·d) in the diet on d 16, 18, and 20 reduced mammary tumors after DMBA treatment in adulthood (62, 63). These studies show that Gen exposure during development of the mammary gland caused altered morphogenesis and abnormal mammary gland responses later in life to environmental insults. In addition, Hilakivi-Clarke’s laboratory reported similar findings when rats treated with Gen (~1 mg/kg·d) by sc injection on d 7–20 showed reduced DMBA-induced mammary gland tumors as well as reduced terminal duct formation (61). However, in another study from Hilakivi-Clarke’s laboratory, rats treated prenatally with Gen (~0.1, 0.5, or 1.5 mg/kg·d) on gestation d 15–20 had increased susceptibility of mammary gland tumors induced by DMBA (60). These data taken together showed that the timing of Gen exposure influences the development of the mammary gland and its subsequent response to carcinogens later in life such that prenatal exposure to Gen increased sensitivity to DMBA, a known mammary carcinogen.

Our current study focuses on the developmental effects of Gen on the mammary gland after neonatal exposure over a wide dose range; studies from other laboratories have primarily focused on prenatal or prepubertal exposure. The doses of Gen used in our study were chosen to span the range of human exposures with the highest dose (50 mg/kg·d) producing circulating serum levels of 6.8 µM total Gen in mice compared with 1–5 µM in human infants drinking soy-based formulas (36). Thus, the highest dose we used produced slightly higher circulating levels of total Gen than the levels reported in human infants, whereas our two lower doses cover the range of circulating levels 10- and 100-fold less. Because research in our laboratory and others have shown developmental exposure to estrogenic chemicals caused differential effects at low compared with high doses in ovary, uterus, and prostate tissues (46, 64, 65, 66, 67, 68), the study of a wide dose range including low doses was deemed particularly important.

Our study was conducted to gain more understanding of the processes involved in altered mammary gland growth and development after neonatal Gen treatment. The mammary gland was examined before puberty (4 wk) and during the time of active ductal morphogenesis (5 and 6 wk) after neonatal exposure to Gen for specific morphological and biochemical alterations including altered hormone receptor levels to elucidate potential mechanisms of Gen’s effects. We further compared morphological effects with subsequent functional alterations as determined by lactation and followed the long-lasting consequences by examining mammary gland morphology at 9 months of age.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Time pregnant CD-1 (Crl: CD-1 [ICR] BR) mice were obtained from the breeding colony at National Institute of Environmental Health Sciences (NIEHS), Research Triangle Park, NC (original supplier, Charles River Laboratories). Mice were housed in a temperature controlled room (21–22 C) under a 12-h light,12-h dark cycle and fed NIH-31 mouse diet and deionized water ad libitum. The diet was analyzed for estrogenic activity as previously described (69). All animal procedures complied with NIEHS/National Institutes of Health animal care guidelines. Dams delivered their young at 19 d of gestation; pups were culled together, separated into two groups by sex, and then randomly standardized to eight female pups per dam; male pups were used in another experiment. All female pups were treated by sc injections on neonatal d 1 through 5 with Gen (0.5, 5, and 50 mg/kg·d) suspended in corn oil or with corn oil alone as controls (Gen, 98% pure; Sigma Chemical Co., St. Louis, MO). There were six litters per treatment group. Pups were weaned on d 22 and housed four per cage. Mice were euthanized by carbon dioxide asphyxiation at various ages.

At 4 wk of age, the right abdominal mammary gland (no. 4) was removed from eight mice per treatment group for whole-mount preparation. At 5 and 6 wk of age, from three litters per treatment group, the right abdominal mammary gland (no. 4) was collected and fixed for whole-mount preparation (eight mice per treatment group per age) or frozen for Western blot analysis (four mice per treatment group per age), and the left abdominal mammary gland (no. 4) was removed and frozen for real-time RT-PCR (four mice per treatment group per age) or fixed in cold formalin for immunohistochemical analysis (eight mice per treatment group per age). Reproductive tract tissues and ovaries were also collected, fixed in 10% neutral buffered formalin, sectioned at 6 µm, and stained with hematoxylin and eosin to evaluate the stage of the estrous cycle and presence of ovarian corpora lutea (CL).

At 2 months of age, eight female mice per treatment group were housed with proven control males (two females to one male). When female mice were vaginal plug positive, they were individually housed, allowed to deliver their pups, and observed for lactation ability. Pups were counted on the day of birth (d 1) and remained with their mother. As a measure of mammary gland function, litter weights were determined on d 2 and 10, and survival rates were determined on d 22. Mice were killed and tissues collected but not further analyzed for this study.

At 9 months of age, mammary glands (no. 4) from eight mice per treatment group were removed and fixed for whole-mount preparation to observe long-term effects. Reproductive tract tissues and ovaries were collected, fixed in 10% neutral buffered formalin, sectioned at 6 µm, and stained with hematoxylin and eosin to evaluate the stage of the estrous cycle and presence of ovarian CLs in the ovaries. A minimum of five reproductive tract sections were evaluated per mouse. For CL counts, three sections from three different levels for a total of nine sections per ovary per mouse were evaluated.

Whole-mount preparation and analysis
Mammary glands were excised and processed as previously described (70). Briefly, the mammary gland was placed on a charged glass slide and fixed in Carnoy’s fixative for 1 h and then immersed in 70% alcohol for 15 min, rinsed for 5 min in distilled water, and stained overnight with carmine aluminum. Tissues were then dehydrated through a series of graded alcohols and xylenes and coverslipped.

Measurements were taken using an image analysis system interfaced with a Leica dissecting scope (Bannockburn, IL) using Image Pro software from SciMeasure (Decatur, GA). TEBs greater than or equal to 100 µm in diameter (at the widest point) were counted for each gland. At 4 wk of age, because the mammary gland has not expanded to the lymph node, ductal elongation was measured from the original point of bifurcation to the farthest expanded duct. At 5 and 6 wk of age, ductal elongation was measured from the proximal end of the lymph node to the TEB of the farthest elongated branch as previously described (28). Branch points per field were counted within an area of 1 x 107 µm2, between the nipple and the lymph node. The percent of the area occupied by mammary gland structures in the fat pad was also determined by digitizing and quantitating the images using a Canon EOS 1Ds camera (Canon USA, Inc., Lake Success, NY). Although eight mice per treatment group per age were analyzed for the number of TEBs and ductal elongation, four mice per treatment group per age were randomly selected and analyzed for the number of branch points because of the labor-intensive methods used to determine this parameter.

Immunohistochemistry
Mammary glands were fixed in formalin, embedded in paraffin, and cut at 6 µm; four mice per treatment group per age were evaluated by immunohistochemistry. Tissue sections were immunostained for estrogen receptor (ER){alpha}, ERß, and progesterone receptor (PR).

ER{alpha}.
Tissues were deparaffinized in xylene, hydrated in a series of alcohols, and rinsed with 1x automation buffer (AB) (Fisher Scientific, Norcross, GA). Sections were treated with 3% hydrogen peroxide to eliminate endogenous peroxidase, washed in AB, and placed in a Coplin jar with citrate buffer (pH 8.0) (Biocare Medical, Walnut Creek, CA). Sections were placed into a decloaker (Biocare Medical) for 5 min for antigen retrieval. Slides were then rinsed with distilled water and treated according to the instructions provided with the mouse-on-mouse kit (Vector Laboratories, Burlingame, CA). Briefly, sections were incubated in blocking solution for 1 h at room temperature, followed by a rinse with Tris-buffered saline (pH 7.4) and then incubated for 30 min with antimouse ER{alpha} (Oncogene Science, Manhasset, NY) diluted 1:100 in diluent provided in the kit. Negative controls without primary antibody were run for each experiment. Slides were then incubated with biotinylated goat antimouse and detection reagent for 5 min as described in the kit. Sections were rinsed with distilled water and stained with NOVA Red (Vector Laboratories) for 5–10 min until color reaction was observed. Sections were rinsed in distilled water, counterstained with hematoxylin, dehydrated in a graded series of alcohols and xylenes, and coverslipped for evaluation by light microscopy.

PR.
Adjacent mammary gland sections were immunostained using the same kit and concentrations described for ER{alpha} except the primary antibody was PR (Immunotech, Westbrook, ME) at a dilution of 1:25. This antibody recognized both PR-A and PR-B isoforms.

ERß. Mammary gland sections were processed as described for ER{alpha} immunohistochemistry until the antigen retrieval step. Nonspecific binding was blocked using 10% BSA in AB (pH 6.8) for 30 min. Sections were incubated with ERß antibody (Santa Cruz, La Jolla, CA) diluted 1:25 in 1% BSA and 1% milk in AB for 2 h. Biotinylated antirabbit (Vector) was applied for 1 h. followed by ExtrAvidin peroxidase (Sigma) for 30 min. Proteins were visualized using NOVA Red (Vector) as described for ER{alpha}.

PR Western blotting
Nuclear protein was isolated from approximately 40 mg of each mammary gland collected using the N-PER kit (Pierce, Rockford, IL). For Western blot analysis, 25 µg nuclear protein was run on a 10% Bis-Tris gel (Invitrogen, Carlsbad, CA) and transferred to nitrocellulose. The gel was then stained with Simply Blue (Invitrogen, Grand Island, NY) to ensure the efficiency of transfer. The blot was then washed in Tris-buffered saline with 1% Tween 20 at pH 7.4, blocked with 10% BSA for 30 min at room temperature, and then allowed to incubate with PR antibody (Immunotech) at a dilution of 1:50,000 for 1 h at room temperature. This antibody detects both PR-A and PR-B isoforms. Blots were then incubated with horseradish peroxidase antimouse (Amersham, Piscataway, NJ). Specific immunoreactivity was detected using the ECL Plus detection reagents (Amersham) according to the manufacturer’s protocol. Blots were exposed to film for 30 sec to 2 min. Image analysis was performed using Image Pro software (OPELCO, Dulles, VA); the area of each band was measured from three samples per treatment group per age.

ER{alpha} and ERß real-time RT-PCR
RNA was isolated using the RNeasy kit (QIAGEN, Valencia, CA) following the manufacturer’s instructions using approximately 20 mg frozen mammary gland tissue from individual mammary glands. Total RNA (1 µg) was reverse transcribed using the SuperScript First-Strand Synthesis System (Invitrogen) following the manufacturer’s instructions to assess ER{alpha} and ERß transcript levels. ER{alpha}, ERß, and18S primers were graciously provided by Vickie Walker, NIEHS, and the forward and reverse primers are as follows: ER{alpha}, forward 5'-GTCCAGCAGTAACGAGAAAGGAA-3' and reverse 5'-TCATTGCACACGGCACAGT-3'; ERß, forward 5'-CTGTTACTAGTCCAAGCGCCAA-3' and reverse 5'-CCCAGATGCATAATCACTGCA-3'; 18S, forward 5'-GAAACTGCGAATGGCTCATTAA-3' and reverse 5'-GAATCACCACAGTTATCCAAGTAGGA-3'. Control and Gen-treated mammary gland cDNA (20 ng) was added to a 96-well plate in duplicate with SYBR Green and primer sets for each gene. Amplification was carried out using the 7900 HT Sequence Detection System (Applied Biosystems, Foster City, CA). Expression levels were determined using the mathematical model previously described (71). Expression ratios were calculated by dividing the level of expression of the gene of interest by 18S (housekeeping gene) expression level for each sample. As a negative control, a sample containing RNA but no reverse transcriptase (minus RT) was also included.

Serum hormone analysis
To determine the circulating serum levels of progesterone and estradiol, blood was collected from the vena cava of mice treated with corn oil, Gen-0.5, Gen-5, and Gen-50 used for mammary gland collection at 5 and 6 wk of age (six to eight samples per treatment group per age were analyzed). All samples were centrifuged at 8000 rpm at 4 C for 10 min; serum was isolated and frozen at –70 C until further analysis. Serum progesterone and estradiol were measured using respective kits (Diagnostic Systems Laboratory, Webster, TX) according to the manufacturer’s instructions.

Statistical analysis
Statistical analysis was performed on the number of TEBs, ductal elongation, branching, density, body weights, percent weight gain of pups, percent survival, circulating levels of estradiol and progesterone, real-time RT-PCR, and area image analysis using ANOVA followed by Dunnett’s test; P < 0.05 was reported as significant. Data are presented as the mean ± SEM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Body weight and serum hormone levels
At 4, 5, and 6 wk of age, body weights in all Gen-treated groups (Gen 0.5, 5, and 50 mg/kg·d) were similar to age-matched controls (data not shown). At 5 and 6 wk, the stage of the estrous cycle was determined based on uterine and vaginal histology. All stages of the estrous cycle were represented in the controls and Gen-0.5- and Gen-5-treated mice. However, all of the mice in the Gen-50 treatment group at 5 wk (eight of eight) and 6 wk (eight of eight) were in estrus or metestrus at the time of collection similar to previously published data (47). At 5 and 6 wk, a comparison of circulating levels of estradiol and progesterone revealed no significant differences in hormone levels of the mice in similar stages of the cycle. However, progesterone levels in mice treated with the highest dose of Gen were generally lower than controls, most likely because of the stage of the estrous cycle (estrus) in these mice (Fig. 1Go).


Figure 1
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  		FIG. 1. Serum circulating levels of estradiol and progesterone at 5 and 6 wk of age after neonatal exposure to Gen. At 5 and 6 wk of age, serum was collected and assayed for estradiol (A) and progesterone (B). Individual levels are plotted on each graph (at least 14 mice were assayed from each treatment group). Co, Control.

 
Mammary gland morphogenesis after neonatal Gen exposure
Mammary gland growth before puberty.
Whole mounts of mammary glands collected before puberty (4 wk) also showed no differences between treatment groups (Fig. 2Go). The number of TEBs greater than 100 µm in diameter (control, 14.0 ± 1.6; Gen-0.5, 16.8 ± 1.5; Gen-5, 14.5 ± 1.7; Gen-50, 13.0 ± 2.1) and ductal elongation from the point of bifurcation to the farthest duct (control, 2.6 ± 0.3; Gen-0.5, 2.8 ± 0.2; Gen-5, 2.9 ± 0.4; Gen-50, 3.1 ± 0.2) was not significantly different across treatment groups at 4 wk of age, suggesting no morphological effects on mammary gland growth before puberty.


Figure 2
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  		FIG. 2. Representative mammary gland whole mounts from control and Gen-treated mice at 4 wk of age. At 4 wk of age, before puberty, mammary gland growth is just beginning, not reaching the lymph node. There are no apparent differences between treatment groups at this age. A, Control; B, Gen-0.5; C, Gen-5; D, Gen-50. Scale bar, 1000 µm.

 
Mammary gland growth during the time of active ductal morphogenesis.
Representative photographs demonstrating mammary gland ductal morphogenesis in control and Gen-treated mice at 5 and 6 wk of age are shown in Fig. 3Go. At 5 wk of age, mammary gland development was similar in Gen-0.5-treated mice compared with controls. However, by 6 wk of age, there was an apparent increase in elongation through the fat pad in the Gen-0.5-treated mice compared with their age-matched controls (Fig. 3Go, E and F). In contrast, ductal growth was limited in the highest Gen group compared with controls at both 5 and 6 wk of age (Fig. 3Go, D and H).


Figure 3
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  		FIG. 3. Representative mammary gland whole mounts from control and Gen-treated mice at 5 and 6 wk of age. The lymph node (LN) is used for orientation in each photo; ductal growth proceeds from the left to the right as shown by the direction of the arrow in A. At 5 wk of age (A–D), mammary gland development was similar in low-dose Gen-0.5 (B) compared with controls (A). However, expansion was slower in Gen-5 (C) and Gen-50 (D) groups as characterized by less branching points compared with controls. Gen-50 (D) also had less ductal elongation and fewer TEBs than controls. At 6 wk (E–H), mammary gland growth remained limited in the Gen-5 (G) and Gen-50 (H) mice with fewer TEBs. In contrast, the low dose of Gen-0.5 (F) showed a marked increase in growth with more ductal elongation and branching points compared with controls (E). The asterisk marks TEBs more than 100 µm in diameter (across the widest point) in B. Scale bar, 1000 µm.

 
The average number of mammary gland TEBs larger than 100 µm in diameter (at the widest point) is shown in Fig. 4Go. Mice treated with Gen-0.5 and Gen-5 had similar numbers of TEBs as controls at 5 wk of age; however, mice treated neonatally with Gen-50 showed a statistically significant decrease in the number of TEBs (>100 µm). By 6 wk, both Gen-5 and Gen-50 showed a statistically significant decrease in the number of TEBs compared with controls (Fig. 4BGo). The average extension of the mammary gland into the fat pad at 5 and 6 wk of age is also shown in Fig. 4Go. Although there is a decrease in ductal elongation after the highest dose of Gen at 5 and 6 wk compared with controls, this difference was not significant. However, there is a statistically significant increase in ductal elongation in the Gen-0.5 dose at 6 wk of age (Fig. 4DGo). These data suggest that mammary gland ductal morphogenesis was altered by neonatal exposure to Gen.


Figure 4
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  		FIG. 4. Number of TEBs and extent of ductal elongation at 5 and 6 wk of age after neonatal treatment with Gen. A and B, Average number of TEBs ± SEM at 5 wk (A) and 6 wk (B); C and D, average length (mm) ± SEM of ductal elongation from the lymph node to the tip of the furthest duct at 5 wk (C) and 6 wk (D). *, Significance at P < 0.05 using Dunnett’s test.

 
The average number of branch points at 5 and 6 wk is shown in Fig. 5Go. There is a decrease in the number of branch points after neonatal exposure to Gen at 5 wk of age that is statistically significant at the Gen-5 and -50 doses (Fig. 5AGo); however, this difference is resolved by 6 wk of age with no differences in Gen treatment compared with controls (Fig. 5BGo). The average area of the mammary gland at 5 and 6 wk of age is also shown in Fig. 5Go. Although there appears to be less mammary gland area in the highest Gen-dosed group compared with controls, this difference was not significant. These data suggest that ductal ramification is delayed after neonatal exposure to Gen.


Figure 5
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  		FIG. 5. Number of branch points and mammary gland area at 5 and 6 wk of age after neonatal treatment with Gen. A and B, Average number of branch points ± SEM at 5 wk (A) and 6 wk (B); C and D, average area (pixels) ± SEM of the mammary gland structure at 5 wk (C) and 6 wk (D). *, Significance at P < 0.05 using Dunnett’s test.

 
PR, ER{alpha}, and ERß expression after neonatal Gen treatment
PR.
Representative mammary gland sections immunostained for PR at 5 and 6 wk of age are shown in Fig. 6Go. PR protein is expressed in the nuclei of ductal epithelial cells as demonstrated by the dark brown immunostain in the mammary gland. Western blot analysis for PR (both A and B subunits) is shown in Fig. 6EGo. Quantification of the PR bands is as follows: at 5 wk, control was 331 ± 41, Gen-0.5 was 621 ± 56 (P < 0.05 by Dunnett’s test), Gen-5 was 637 ± 16 (P < 0.05 by Dunnett’s test), and Gen-50 was 309 ± 39; at 6 wk, control was 638 ± 28, Gen-0.5 was 831 ± 154, Gen-5 was 875 ± 47, and Gen-50 was 702 ± 28. Mammary gland PR protein expression is increased at 5 and 6 wk of age in the Gen-0.5 and Gen-5 treatment groups compared with controls, but it reaches statistical significance only at the 5-wk time point; no apparent difference in expression between control and Gen-50-treated mice is seen at either age. These data suggest that the mammary gland expresses higher levels of PR in mice treated with lower doses of Gen but no difference in PR levels at the highest dose.


Figure 6
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  		FIG. 6. PR expression in the mammary gland of mice treated neonatally with Gen. A–D, PR immunostaining in control (A), Gen-0.5 (B), Gen-5 (C), and Gen-50 (D) mammary glands at 6 wk of age. Scale bar, 100 µm . E, PR Western blot of mammary gland protein from control, Gen-0.5, Gen-5, and Gen-50 (three mice in each group) mice at 5 wk of age (top) and 6 wk of age (bottom). Note the significant increase in PR expression in the Gen-0.5- and Gen-5-treated mammary glands at 5 wk of age compared with controls (E).

 
ER{alpha}.
Representative mammary gland sections immunostained for ER{alpha} at 5 and 6 wk of age are shown in Fig. 7Go. ER{alpha} immunostaining is decreased in the nuclei of ductal epithelium after neonatal Gen treatment. Real-time RT-PCR for mammary gland ER{alpha} mRNA expression at 5 and 6 wk of age is also shown in Fig. 7Go, E and F. There is a decrease in ER{alpha} mRNA after neonatal Gen treatment at both 5 and 6 wk of age. When the data from the two age groups are combined (Fig. 7GGo), all three doses show a significant decrease in ER{alpha} compared with control levels by Dunnett’s test (P < 0.05). These data demonstrate that the mammary gland expresses lower levels of ER{alpha} after neonatal Gen treatment, particularly at the highest dose with a 50% reduction from control levels.


Figure 7
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  		FIG. 7. ER{alpha} expression in the mammary gland of mice treated neonatally with Gen. A–D, ER{alpha} immunostaining in control (A), Gen-0.5 (B), Gen-5 (C), and Gen-50 (D) mammary glands at 6 wk of age. Note the apparent decrease in ER{alpha} immunostaining in the Gen-treated mammary glands (B–D) compared with control (A). Scale bar, 100 µm. E–G, ER{alpha} real-time RT-PCR of mammary gland RNA from control, Gen-0.5, Gen-5, and Gen-50 (four mice each) at 5 wk (E), 6 wk (F), and 5 and 6 wk combined (G). *, Significance at P < 0.05 using Dunnett’s test.

 
ERß.
Representative mammary gland sections immunostained for ERß at 5 and 6 wk of age are shown in Fig. 8Go. ERß was expressed in the nuclei of ductal epithelial cells; no difference in expression levels could be detected across treatments by immunohistochemistry. Real-time RT-PCR for ERß mRNA is also shown in Fig. 8Go. There was an apparent increase in ERß mRNA expression in the mammary gland after neonatal Gen-0.5 treatment at 5 and 6 wk of age compared with controls. When the data from the two age groups are combined (Fig. 8GGo), the Gen-0.5 group showed a significant increase in ERß mRNA expression compared with controls using Dunnett’s test (P < 0.05).


Figure 8
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  		FIG. 8. ERß expression in the mammary gland of mice treated neonatally with Gen. A–D: ERß immunostaining in control (A), Gen-0.5 (B), Gen-5 (C), and Gen-50 (D) mammary glands at 6 wk of age. Scale bar, 100 µm. E–G, ERß real-time RT-PCR of mammary gland RNA from control, Gen-0.5, Gen-5, and Gen-50 (four mice each) at 5 wk (E), 6 wk (F), and 5 and 6 wk combined (G). *, Significance at P < 0.05 using Dunnett’s test.

 
Mammary gland function as determined by pup growth and survival during lactation
Mice treated neonatally with all three doses of Gen and controls were bred to proven control males and allowed to deliver their young; five of eight mice were vaginal plug positive in each of the four treatment groups. Female mice in the Gen-50 group did not deliver any live pups as previously described (47). For the other groups, the average number of pups per litter was not different between treatment groups (Table 1Go). One litter in the control group with only four pups was not included in the analysis because the number of pups in a litter affects weight gain and thus could not be compared with the other litters with 10–15 pups per litter. Litter weights and calculated average pup weights on d 2 and 10 were not different between treatment groups, and the percentage of mice surviving until weaning was also not different (Table 1Go). These data suggest that mice treated neonatally with Gen-0.5 or Gen-5 have the ability to lactate and sustain pup growth and survival until weaning similar to controls. The inability of the Gen-50-treated mice to deliver live pups did not allow assessment of lactation in this group.


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  		TABLE 1. Determination of mammary gland function (lactation) at 2 months of age after neonatal exposure to Gen

 
Mammary gland morphology of aged mice
Mammary glands were collected from mice at 9 months of age after neonatal treatment with Gen to evaluate permanent long-lasting effects as shown in Fig. 9Go. The stage of the estrous cycle was determined for each mouse, and comparisons were made across treatment groups based on stage of the cycle. Mice in the Gen-0.5 treatment group showed signs of estrous cyclicity with all stages of the cycle represented similar to controls. In addition, all of the mice in the control and Gen-0.5 treatment groups had similar numbers of CL (control, 28.1 ± 4.1; Gen-0.5, 34.2 ± 5.8). Mammary glands from mice treated with Gen-0.5 were similar to controls independent of the stage of the estrous cycle. This suggests that although these mice demonstrated advanced mammary gland growth during puberty, no apparent difference was observed at 9 months.


Figure 9
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  		FIG. 9. Mammary gland morphology at 9 months of age after neonatal Gen treatment. A, Control; B, Gen-0.5; C, Gen-5; D, Gen-50. Note that the two highest doses lack tertiary structures compared with controls [Gen-5 (C) and Gen-50 (D)]. E, Representative view of dilated beaded ducts from a Gen-50-treated mouse. F, Higher magnification of E. Scale bars, 2 mm (A), 20 µm (E), and 100 µm (F).

 
Interestingly, some of the mice in the Gen-5 treatment group were in persistent estrus (two of eight) with excessive vaginal cornification and lack of CL similar to previously published data (47). The remainder of the mice treated with Gen-5 had similar numbers of CL as controls (Gen-5, 33.2 ± 5.8; control, 28.1 ± 4.1). The mammary gland from all mice in the Gen-5 group exhibited signs of altered morphology with reduced lobular alveolar development (eight of eight), dilated ducts (four of eight), and focal areas of dilated beaded ducts lined with hyperplastic ductal epithelium with secretory material between the piling epithelial cells (three of eight).

All mice in the Gen-50 treatment group (eight of eight) exhibited persistent estrus and had no CL, similar to previously published data (47), suggesting they were not cycling. All Gen-50 (eight of eight) mice had marked decreased branching and alveolar development compared with age-matched controls in any stage of the cycle. The appearance of dilated beaded ducts was also apparent in three of eight mice in this group (Fig. 9Go, E and F). These data suggest that mice treated with the higher doses of Gen had permanently altered mammary gland development that persisted until much later in life.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study shows that neonatal exposure to the phytoestrogen Gen alters mammary gland morphogenesis long after the time of exposure, despite the lack of obvious effects before puberty. This suggests developmental programming of the mammary gland was altered such that at later time points the tissue responds differently to hormonal cues than would be expected. This was evidenced by altered hormone receptor levels as well as advanced mammary gland morphogenesis during puberty after neonatal exposure to the lower doses of Gen treatment (0.5 and 5 mg/kg) and delayed mammary gland morphogenesis in the highest dose of Gen (50 mg/kg). Although the development of the mammary gland was altered during the time of puberty in the Gen-0.5 and Gen-5 treatment groups, the ultimate function of the mammary gland appeared to be intact because mice from these treatment groups at 2 months of age were able to lactate and maintain pup growth. This would be expected because the morphological changes observed in the low dose of Gen treatment would have most likely led to expansion of the mammary gland through the fat pad at a faster rate but not necessarily affecting the function of the mammary gland. However, at a later time point (9 months), altered mammary gland morphology was apparent in the Gen-5 and Gen-50 treatment groups with less branching and less alveolar development compared with controls. In addition, some of the mice in the Gen-5 and Gen-50 groups exhibited the presence of dilated beaded ducts lined with hyperplastic ductal epithelium, which were not seen in controls at any age examined. The presence of these structures has been previously reported after prenatal exposure to the xenoestrogen zearalenone, supporting the idea that developmental exposure to an estrogenic compound caused this effect (72). Whether the lack of tertiary differentiation later in life is because of a direct effect on the mammary gland or to a lack of cyclicity and associated low levels of progesterone, is not known.

The differential effects on the mammary gland at low vs. high doses is not unique to this compound or this tissue. Several studies have shown an effect at a low dose and an opposite effect at a higher dose. For example, mice treated with the same low dose of Gen used in this study (Gen-0.5) have increased ovulation rates compared with control mice, whereas mice treated with the high dose (Gen-50) have decreased rates (46). Another study from our laboratory showed enhanced uterine responsiveness after neonatal exposure to low doses of DES and dampened uterine responsiveness after high doses (64). Others have also shown nonlinear effects on reproductive tissues such as the prostate after developmental exposure to endocrine-disrupting compounds including DES, bisphenol A, methoxychlor, arochlor, and ethynylestradiol (65, 66, 67, 68). Although the exact mechanisms controlling these observations remain unknown, there is clear evidence that biphasic effects occur. One possible explanation is that there are two separate mechanisms involved with one effect being mediated elsewhere in the endocrine system, perhaps the hypothalamus or pituitary, and the other effect being mediated in the tissue itself. One study supports this idea in that developmental exposure to low doses of Gen led to hyperresponsiveness of the pituitary, whereas higher doses of Gen led to lack of responsiveness (73). Additional study on differential effects at low vs. high doses is needed to determine the mechanism(s) responsible for each effect.

Mammary gland development has been studied for decades, but the exact mechanisms that control growth and development remain to be elucidated. The mammary gland is a rudimentary structure at birth in both rodents and humans (74). Extension and ramification of the mammary gland through the fat pad begins before puberty and continues throughout puberty. Additional differentiation of the mammary gland occurs in adulthood, responding to hormonal cues during the estrous cycle and pregnancy. Hormones such as estradiol and progesterone are necessary for proper mammary gland development and differentiation. Studies using transgenic mouse models of hormone receptors provide clues to the contributions of each receptor type on the development and subsequent differentiation of the mammary gland (75, 76).

Progesterone is important in mammary gland differentiation, and there are two forms of the PR, PR-A and PR-B; each one mediates differential effects in the mammary gland as well as other reproductive tissues (77). Transgenic mice overexpressing PR-A and PR-B showed defects in postpubertal differentiation of the mammary gland with increased ductal branching and hyperplasia in the PR-A-overexpressing mice (78) and reduced ductal branching in PR-B-overexpressing mice (79). Neither of these models showed differences in prepubertal development of the mammary gland, which suggest that PR and progesterone signaling is not the primary mechanism involved in early prepubertal development (77). In addition, transgenic mice lacking PR-A had normal mammary gland development (80) and mice lacking PR-B had reduced ductal branching associated with pregnancy (81). The fact that overexpression of PR-B or lack of PR-B caused decreased ductal branching suggests that the level of PR-B is carefully regulated and that differences in expression level can cause alterations in mammary gland morphogenesis.

Estrogens have also been implicated in mammary gland development and differentiation. There are two transgenic mouse models lacking either one or the other of the two receptor subtypes responsible for estrogen signaling. Mice lacking ER{alpha} ({alpha}ERKO) maintain a rudimentary duct even as adults (82). The lack of mammary development in these mice was found to result from a nonfunctioning pituitary with insufficient production of prolactin and subsequent reduction of progesterone in those mice (82). In fact, estrogen and progesterone treatment along with a transplanted functioning pituitary recovered mammary gland development in {alpha}ERKO mice. Interestingly, the formation of TEBs and ductal elongation occurred in {alpha}ERKO mice in the presence of estrogen and in absence of prolactin, suggesting that early mammary gland morphogenesis is estrogen dependent but not mediated by ER{alpha} (82). Mice lacking ERß (ßERKO) have a less dramatic effect on the mammary gland, but ERß may play a role in some aspects of mammary gland development (75). These mice have less ductal elongation as evidenced by incomplete penetration of the fat pad as well as less alveolar development, although specific studies during the time of development were not researched (75). In contrast, another laboratory showed that mammary gland development in ßERKO mice developed independently of ERß and that they have the ability to lactate and appear relatively normal during adulthood (82). However, ßERKO mice have much smaller litter sizes and may not need the full capacity of the mammary gland to sustain the litter. Early developmental studies on the mammary gland of the ßERKO mice were also not conducted to determine what role ERß might play in early mammary gland development.

The findings from the current study show differences in receptor expression levels after neonatal treatment with Gen long after exposure occurred. The most striking difference was the reduced levels of ER{alpha} mRNA after Gen treatment at both 5 and 6 wk of age. This is in agreement with previous studies that have also shown similar reductions in ER{alpha} expression after developmental exposure to Gen (61). The lower levels of ER{alpha} expression in the mammary gland after the highest dose of Gen are accompanied by reduced TEBs and reduced branching. This reduced or delayed expansion through the fat pad is reminiscent of the {alpha}ERKO mice, which lack TEBs and do not expand into the fat pad because of a nonfunctioning pituitary in these mice. In addition, these studies have shown that ductal elongation during prepuberty is dependent on estrogen, although its effects are not mediated by ER{alpha} (82). Therefore, the direct consequence of lower ER{alpha} expression in the mammary gland after developmental exposure to Gen remains unclear.

Another interesting finding is the increased expression of ERß mRNA in the lower dose of Gen treatment. However, this increase in ERß mRNA was not accompanied by increased ERß protein expression in the mammary ductal epithelium as determined by immunohistochemistry. The most likely explanation is that the immunohistochemical staining shows only that the ductal epithelial cells are positive for ERß and that the cells across treatments have similar intensities but not how many cells are actually staining positive throughout the entire mammary gland. The increase in mRNA may be more of a reflection of the higher percentage of mammary ductal epithelial cells present in the Gen-0.5-treated samples as opposed to increased ERß mRNA in individual cells. Because there is increased ductal elongation in the low dose, one would expect there to be more mammary gland ductal epithelium relative to the number of mammary stromal cells and fat cells. The increased proportion of mammary ductal epithelial cells could explain the increased levels of ERß mRNA. Therefore, the increased ERß mRNA in the mammary gland after the low dose of Gen treatment supports the finding of increased mammary gland structure in this treatment group during this period of development.

Another possible mechanism by which Gen could alter the development of the mammary gland is by disrupting the hypothalamic-pituitary-gonadal (HPG) axis. This would impact the secretory pathway of pituitary and ovarian hormones that are necessary for mammary gland morphogenesis and differentiation. This has been suggested previously by Faber and Hughes (73) showing that rats exposed neonatally to low doses of Gen (0.01 mg/kg) produced higher levels of LH in response to GnRH. Data from our laboratory support this idea because mice treated with lower doses of Gen exhibited enhanced ovulation rates after exogenous stimulation with gonadotropins, suggesting hyperresponsiveness of the pituitary (46). Faber and Hughes (73) also showed that rats exposed to higher doses of Gen during neonatal life were associated with decreased pituitary responsiveness. Because alterations in HPG signaling have been observed in similarly treated animals, the pituitary hormones involved in mammary gland morphogenesis may be disrupted as well. Additional studies in our laboratory are underway to further characterize the responsiveness of the hypothalamus and pituitary after developmental exposure to Gen.

The effects observed herein as a consequence of exposure to Gen is not unique because developmental exposure to other environmental chemicals also altered mammary gland development and differentiation. For example, exposure to dioxin prenatally inhibits mammary gland morphogenesis (83). Exposure to synthetic estrogenic or antiestrogenic chemicals such as DES, tamoxifen, or ICI during development also alter mammary gland development (12, 84). There are also several xenoestrogens that have been shown to alter mammary gland development including bisphenol A, zearalenone, and resveratrol (28, 72). In addition, developmental exposure to the growth factor TGF{alpha} has been shown to advance mammary gland growth during puberty (12), similar to our low-dose Gen-treated mice. Furthermore, IGF-I and GH are also known to play a role in mammary gland growth and differentiation and are thought to work in concert with estradiol to complete mammary gland morphogenesis (85). Future studies will be needed to determine possible effects of neonatal exposure to genistein on growth factors and growth factor signaling in these mice.

In summary, developmental exposure to Gen at environmentally relevant doses alters murine mammary gland morphogenesis during puberty, despite the lack of obvious effects before puberty. These data suggest that the brief exposure to estrogenic substances during development alters programming such that subsequent exposure to hormones results in altered growth and/or differentiation. This idea is supported by the fact that hormone receptor levels in the mammary gland are altered after neonatal Gen treatment; however, altered endocrine signaling from the HPG axis may also be involved in altered mammary gland development. There are also long-term effects on the mammary gland, including ductal epithelial hyperplasia in the higher doses of Gen treatment. This effect appears to coincide with lack of estrous cyclicity or persistent estrus in these mice, but a direct effect on the mammary gland epithelial cells during the time of treatment might also explain this effect. Whether these effects are observed in humans exposed developmentally to these compounds remains to be determined.


   Acknowledgments
 
We thank Drs. Suzanne Fenton, Richard DiAugustine, Diane Klotz, Silvia Hewitt, Suzanne Snedeker, and Gloria Jahnke for their comments and advice on this study. We also thank Drs. Bill Bullock and David Malarkey for pathological evaluations. Mr. Ryan J. Snyder and MRPath, Inc., are acknowledged for their technical support.


   Footnotes
 
This research was supported by the Intramural Research Program of the National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services.

Disclosure statement: The authors have nothing to disclose.

First Published Online July 20, 2006

Abbreviations: AB, Automation buffer; CL, corpora lutea; DES, diethylstilbestrol; DMBA, dimethylbenz[a]anthracene; ER, estrogen receptor; {alpha}ERKO, mice lacking ER{alpha}; Gen, genistein; HPG, hypothalamic-pituitary-gonadal; PR, progesterone receptor; TEB, terminal end bud.

Received March 27, 2006.

Accepted for publication July 10, 2006.


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 Introduction
 Materials and Methods
 Results
 Discussion
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