* Institute of Human Virology, Division of Infectious Diseases, Department of Medicine,
Departments of Epidemiology and Preventive Medicine, and
Physiology, University of Maryland Medical School, Baltimore Maryland 21201; and
School of Pharmacy and Environmental Toxicology Center, University of Wisconsin, Madison, Wisconsin 53706
Received December 22, 2000; accepted March 19, 2001
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ABSTRACT |
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Key Words: TCDD; estrogen receptor ; mammary gland; in utero and lactational exposure; differentiation..
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INTRODUCTION |
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2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is an aryl hydrocarbon receptor (AhR) agonist with direct anti-estrogenic action in mammary epithelial cells (Safe et al., 1991, 1998
). AhR is expressed in the mammary gland and acute exposure to either 2,3,7,8-tetrachlorodibenzofuran (TCDF) (Hushka et al., 1998
) or TCDD (Brown and Lamartiniere, 1995
) suppresses lobule development. In utero exposure to TCDD results in altered development of hormonally responsive tissues including prostate (Roman and Peterson, 1998
; Roman et al., 1995
, 1998
), testes (Barthold et al. 1999
; Sommer et al., 1996
), epididymis (Barthold et al. 1999
), vagina (Flaws et al., 1997
; Gray and Ostby 1995
; Gray et al., 1997
), and mammary gland (Brown et al., 1998
; Youngblood et al., 2000
). Increased expression levels of ER
have been reported in testes (Barthold et al. 1999
), hypothalamus, uterus, and ovary (Chaffin et al. 1996
; Chaffin et al., 1997
).
In the mammary gland, the percentage of epithelial cells expressing ER changes with hormonally induced differentiation. Less-well differentiated structures such as terminal end buds (TEBs) exhibit a higher percentage of ER
-expressing cells (Fendrick et al., 1998
; Russo et al. 1999
; Russo and Russo, 1978
). The percentage of mammary epithelial cells expressing ER
decreases as terminal end buds differentiate into lobular structures.
The study demonstrates that expression levels of ER are increased in postpubertal mammary glands following in utero and lactational TCDD exposure, and that this is correlated with impaired mammary gland differentiation. Significantly, the response to exogenous estrogen was not impaired by in utero and lactational TCDD exposure.
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MATERIALS AND METHODS |
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On gestational day (GD) 15, 12 rats were administered 1 µg TCDD/kg (98% purity, Cambridge Isotope Laboratories, Woburn, MA) as a single oral gavage dose (2 ml/kg) and 9 control rats received an equivalent volume of vehicle (Wesson corn oil/acetone, 19/1, v/v). All dams were weighed daily and visually inspected until delivery. After parturition at postnatal day (PND) 0, the live pups were counted and sexed. On PND 1, pups were toe-clipped and numerically marked. The litters were adjusted to 5 males and 5 females to enable similar nutritional and lactational exposure to TCDD for all pups.
The offspring were weaned on PND 21 and housed with littermates, 2 to 3 animals per cage, in wire-bottomed stainless-steel cages. Male pups were terminated for experiments reported elsewhere. Female pups were transferred to the University of Maryland Animal Care Facility where they were housed on a 12-h light/dark cycle under temperature control (22 ± 1°C), 2 to 3 animals per clear plastic cage (41 x 20 x 20) containing aspen-chip bedding. Rat Chow 5012, Purina Mills (St. Louis, MO, USA) and tap water were provided ad libitum.
At nine weeks of age, the female offspring were ovariectomized under anesthesia (DePaolo et al. 1981; Hirshfield 1989
). At eleven weeks of age, both the TCDD-exposed and control non-exposed, ovariectomized female offspring were divided into 3 groups. Each group was composed of pups from different litters. A maximum of one pup per litter was represented in each group. Group 1 was treated with an implant containing 0.1 mg 17ß-estradiol (Innovative Research of America, Sarasota, FL) placed under the dorsal hind skin (TCDD exposure: n = 9; Control: n = 7). Group 2 were treated with an implant containing 0.025 mg 17ß-estradiol (Innovative Research) placed under the dorsal hind skin (TCDD exposure: n = 9; Control: n = 7). Group 3 were treated with a placebo implant (Innovative Research) placed under the dorsal hind skin (TCDD exposure: n = 11; Control: n = 6). The total number of rats analyzed was 37. After 48 h, the rats were euthanized by decapitation and one of the fourth mammary glands removed at the time of necropsy. The mammary tissue removed was divided into 3 portions. One portion was frozen at 70°C and stored for protein and RNA assays, one portion was fixed in 10% neutral formalin and embedded in paraffin for sectioning, and the third portion was taken for whole-mount analysis. Equivalent representative portions of each gland were taken from each animal for analysis of structural differentiation, RNA, and histology. Whole mounts were performed on the caudal portion of the gland.
Mammary gland whole mounts and structural differentiation.
Each mammary gland specimen was spread on a glass slide and fixed in Carnoy's solution (100% ethanol:chloroform:glacial acetic acid, 6:3:1) for 60 min at room temperature. Following fixation, the glands were washed with 70% ethanol for 15 min, followed by a wash with distilled H2O for 5 min. The staining of the glands was performed in carmine alum (1 g carmine; Sigma Chemical Co., St. Louis, MO) and 2.5 g aluminum potassium sulfate [Sigma] in 500 ml H2O) at 4°C overnight. The tissues were then dehydrated and mounted on glass slides. The whole mounts were examined at 10 x and the total number of terminal-end buds (TEB), terminal ducts (TD), and Lobules I and II were determined for each whole mount (Brown et al., 1998; Russo and Russo, 1978
). Structural differentiation was determined by calculating the percentage of ductal structures ending in terminal end buds (Fig. 1A
), terminal ducts (Fig. 1B
), lobules I (Fig. 1C
) and Lobules II (Fig. 1D
). Terminal end buds differentiate into Lobules I and II. A poorly differentiated gland demonstrates a higher percentage of terminal end buds as compared to Lobules I and II. A better-differentiated gland demonstrates a lower percentage of terminal end buds and terminal ducts as compared to Lobules I and II. Terminal end buds are composed of 36 layers of closely packed mammary epithelial cells and exhibit a rounded appearance at the end of a duct. Terminal ducts have a smaller diameter and are only 2 cell layers thick. Terminal ducts form from terminal end buds that do not differentiate into Lobules I and II and instead exhibit progressive hypoplasia. Lobules I and II are groups of alveoli around a main duct. Lobules II exhibit more extensive alveolar growth and are larger, fuller structures than Lobules I (Russo and Russo, 1978
). Means ± SE were calculated. Tests for normality were applied and the data was not normally distributed. Kruskal-Wallis tests were performed to determine the statistical significance of observed differences; p < 0.05 was considered significant.
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Analysis of ER and ß-actin protein expression levels.
Tissue was homogenized in lysis buffer using a Polytron and cellular proteins were extracted utilizing RIPA buffer (PBS, 1% NP40, 5% sodium deoxycholate, 0.1% SDS) with protease inhibitors PMSF, aprotinin, and sodium orthovanadate, and fractionated on an SDS-polyacrylamide gel as described previously (Li et al., 2000). Proteins were transferred onto NOVEX PVDF membranes using a NOVEX Western Transfer Apparatus. After transfer and blocking with buffer (5% non-fat milk, 10 mM Tris pH 7.5, 100 mM NaCl, 1% Tween 20) for 1 h at room temperature (RT), the membranes were exposed to a 1:400 dilution of an antibody directed against ER
(NCL-ER-6511, Novo Castra, Burlingame, CA) for 1 h. This was followed by incubation with the appropriate horseradish peroxidase-conjugated antibody at a dilution of 1:2000 (sc-2005, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for 1 h at RT. ER
was visualized using ECL (Amersham, Arlington Heights, IL). The membrane was then washed and reprobed with a 1:500 dilution of antibody directed against ß-actin (sc-1616, Santa Cruz Biotechnology, Inc.) for 1 h, followed by incubation with the appropriate horseradish peroxidase-conjugated antibody at a dilution of 1:5000 (sc-2020, Santa Cruz Biotechnology Inc.) for 1 h. ß-actin was visualized using ECL.
Ki67 immunohistochemistry and in situ detection of apoptosis.
Tissue was fixed in 10% neutral formalin solution and embedded in paraffin using routine methods (Li et al., 2000). Five-micron sections were prepared for hematoxylin and eosin (H&E) staining, Ki67 immunohistochemistry, and in situ detection of apoptosis. For Ki67 immunohistochemistry, sections were deparaffinized, rehydrated with decreasing concentrations of ethanol, quenched in 3% hydrogen peroxide for 5 min, digested with Auto/Zyme buffer (Biomeda, Foster City, CA) at 37°C for 30 min, equilibrated with buffer, incubated with anti-Ki67 antigen/horseradish peroxidase (HRP) (Dako, Carpinteria, CA) for 1 h at RT, and washed with 1x Tris buffered saline (TBS). Color was developed for 15 min using 3,3'-dimethylaminoazobenzene peroxidase substrate (Sigma) diluted in 5 ml sterile H2O. Sections were counterstained with methyl green. The percentage of cells with Ki67-stained nuclei was determined by counting labeled and unlabeled mammary epithelial cell nuclei. Sections from 4 different, randomly chosen mammary glands from each treatment group were analyzed. A minimum of 1000 cells was counted for each mammary gland. Means ± SE were determined. Apoptotic cell nuclei were identified using the ApopTag kit (Oncor, Gaithersburg, MD) as described previously (Li et al., 1996
). The same glands utilized for Ki67 staining were tested for the presence of apoptotic cells. The percentage of apoptotic cells was determined by counting labeled and unlabeled mammary epithelial cells. A minimum of 1000 cells was counted for each animal.
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RESULTS |
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DISCUSSION |
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TCDD is reported to exert anti-estrogenic effects on mammary epithelial cells (Caruso et al. 1999; Kharat and Saatcioglu 1996
; Safe 1995
; Tian et al. 1998
; Wormke et al., 2000
). The impaired differentiation observed in this experiment is consistent with an anti-estrogenic effect. In the mouse, expression of ER
in stroma cells is required for normal mammary gland development although expression in mammary epithelial cells is dispensable (Bocchinfuso and Korach, 1997
). Reciprocal mammary gland transplant experiments have demonstrated that mammary epithelial cells exposed to TCDD in utero develop normally when transplanted to the fat pad of a non-exposed rat, but that non-exposed mammary epithelial cells transplanted to a fat pad exposed to TCDD in utero grow more slowly. The resulting gland appears underdeveloped, suggesting that TCDD may be able to act on mammary epithelial cells through the stroma (Youngblood et al., 2000
). These experiments do not exclude possible systemic effects with secondary effects on the mammary gland. However, hypothalamic-pituitarygonadal hormonal cyclicity has been described as normal in gestationally exposed animals and normal serum FSH, LH, progesterone, androstenedione and estrogen levels are reported (Chaffin et al., 1996
, 1997
; Flaws et al., 1997
; Gray et al. 1997
; Heimler et al., 1998
; Wolf et al., 1999
).
The TCDD-exposed glands were capable of responding to estrogen stimulation with both increased expression levels of PR and glandular differentiation. The glands were not rendered permanently refractory to the effects of estrogen by TCDD exposure. This implies that it is possible for a gestationally exposed gland to recover at least some normal differentiation with hormonal stimulation. The increase in ER expression levels in the whole gland may simply be a reflection of the increased percentage of terminal end buds found in the less differentiated TCDD-exposed glands. Alternatively, the anti-estrogenic activity of TCDD may have increased expression levels of ER
in the stroma, as has been reported in ovariectomized, postmenopausal mice (Fendrick et al., 1998
). It is important to note that susceptibility to chemical carcinogenesis in rats is linked to the number of terminal end buds present in the gland (Brown et al., 1998
; Lamartiniere et al., 1995
; Murrill et al., 1996
; Russo and Russo, 1978
, 1996
). The increased numbers of terminal end buds found after in utero TCDD exposure appears to render the animals more susceptible to chemical carcinogenesis as shown by Brown et al. (1998).
It is not known if in utero or lactational TCDD exposure alters mammary gland differentiation in humans. Potential effects on susceptibility to carcinogenesis in humans are debated. However, the experiments presented here lay the foundation for a molecular understanding of the effect of TCDD exposure on the developing mammary gland. They identify the mammary gland as a potential target organ for TCDD exposure but illustrate that it still can respond normally to estrogen stimulation, at least in the short term. In the rat model, a next step would be to determine if hormonally induced differentiation would protect the TCDD-exposed gland from chemical carcinogenesis and normalize ER expression levels. For human studies, it is necessary to determine if mammary gland differentiation is impaired or ER
expression levels altered by TCDD exposure to the developing mammary gland. If any changes are found, it will be most important to establish if the alterations are fixed or if hormonal exposure through estrous cycling, pregnancy, or exogenous sources can normalize the differences.
In summary, mammary gland differentiation was impaired by in utero and lactational exposure to TCDD in the rat. ER expression levels were increased. Exposure to exogenous estrogen induced progesterone-receptor expression and -differentiation of the terminal ductal structures in the TCDD-exposed glands, demonstrating that the glands were not refractory to estrogen stimulation.
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ACKNOWLEDGMENTS |
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NOTES |
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2 Present address: Bates College, Department of Biology, Lewiston, ME 04240.
3 To whom correspondence should be addressed at the Institute of Human Virology, University of Maryland Medical School, 725 West Lombard Street, Room N545, Baltimore, MD 21201. Fax: (410) 706-1992. E-mail: furth{at}umbi.umd.edu.
Portions of this work were presented in preliminary form at the annual meetings of the Society of Toxicology in 1999, New Orleans, LA, and 2000, Philadelphia, PA.
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