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Articles

Postpubertal Development of the Rat Mammary Gland Is Preserved during Iron Deficiency¹

Constance J. Grill^*, Wendie S. Cohick² and Adria R. Sherman^*

Departments of ^* Nutritional Sciences and Animal Sciences, Rutgers, the State University of New Jersey, New Brunswick, New Jersey 08901

²To whom correspondence should be addressed at Rutgers, The State University of New Jersey, Department of Animal Sciences, 59 Dudley Road, 108 Foran Hall, New Brunswick, NJ 08901-8502. E-mail: cohick{at}aesop.rutgers.edu

	ABSTRACT

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We previously showed that moderate iron deficiency is associated with increased susceptibility to chemically induced breast carcinogenesis. Epidemiological and experimental data suggest that breast cancer risk may be modulated by the developmental and proliferative state of the mammary epithelium. The adverse effects of iron deficiency on organ growth are well documented. However, the role of iron in mammary gland development has not been examined. Therefore, we studied the effect of iron deficiency on mammary gland development and epithelial cell kinetics in female Sprague-Dawley rats. Weanling rats were fed experimental diets that provide 6 (severe), 12 (moderate) or 35 (control) mg Fe/kg diet. After 6 wk of treatment, hematocrit and blood hemoglobin were lower in iron-restricted rats than in controls, with significant differences from controls observed in rats receiving 6 mg Fe/kg diet (P < 0.05). Liver iron was reduced 90 and 80% in severe and moderate groups, respectively, compared with controls. Puberty onset and 17-ß-estradiol levels were unaltered by iron status, but plasma progesterone was significantly lower in iron-restricted groups (P < 0.05). Microscopic examination of mammary gland whole mounts revealed an increased density of terminal end buds in thoracic glands from iron-restricted rats, indicative of decreased differentiation, although the differences were not statistically significant compared with controls (P = 0.21). Mammary epithelial cell proliferation, determined in contralateral glands by measuring 5-bromo-2'-deoxyuridine incorporation, did not differ between rats receiving 12 and 35 mg Fe/kg diet. In conclusion, these results suggest that alveolar development of the mammary gland and the proliferative capacity of the mammary epithelium are refractory to iron deficiency during early postpubertal growth of the rat.

KEY WORDS: • iron deficiency • mammary gland • rats

	INTRODUCTION

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An important concept emerging from experimental and epidemiological breast cancer research is that the developmental stage of the mammary gland at the time of insult may be an important factor in determining breast cancer risk. Although clinical disease is often not evident until midlife or later, it has been hypothesized that breast cancer is initiated early in a woman’s reproductive life. This coincides with the time that mammary epithelia are actively proliferating and most likely to incur genetic damage (1 ,2) . The terminal end bud (TEB),³ the least differentiated of the glandular structures, has been shown to be the site of cancer initiation in rat models of carcinogenesis (2 ,3) . Therefore, factors that modify the developmental profile of the gland may also modify breast cancer risk.

Postnatal mammary gland development is largely controlled by steroid and peptide hormones, as well as by peptide growth factors. In addition, dietary constituents influence glandular development in the rat. Maternal diets low in protein (4) or high in fat (5 ,6) increase the number of TEB and decrease the number of alveolobules (LOB) of female offspring. In contrast, dietary genisten, the major isoflavone found in soy, is associated with enhanced glandular development in the rat (7) .

An association between iron deficiency and breast cancer has not been reported in epidemiological studies, but we find the association both plausible and intriguing. Anemia, which often goes undetected and untreated, is a common clinical finding that is coincident during the breast cancer susceptibility timeframe. In addition, iron is critical for many aspects of cellular physiology (8 9 10 11) , and alterations in several of these processes have been implicated in cancer etiology (1) . Finally, many of the classic clinical manifestations of iron deficiency anemia involve impairments of epithelial cell growth and development.

We have previously shown that moderate iron deficiency increases 7,12-dimethylbenz[a]anthracene (DMBA)-induced breast tumor incidence and burden in virgin female Sprague-Dawley rats (12) . The carcinogenic potential of DMBA is dependent on the developmental stage of the mammary gland. The least differentiated TEB has been shown to be the target structure of DMBA (13) , and tumor incidence is directly related to the number of TEB (5 ,7 ,14) . Therefore, we hypothesized that the induction of moderate iron deficiency in prepubertal female rats would result in impaired alveolobular development of the gland as characterized by an increase in the number of TEB and a decrease in the number of LOB relative to controls. This hypothesis was tested in a rat model of iron deficiency by examining the morphological development of the mammary gland during moderate and severe iron deficiency.

	MATERIALS AND METHODS

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Animals and diets.

Female weanling Sprague-Dawley rats (Harlan Sprague Dawley, Indianapolis, IN) were randomly assigned to either control or treatment groups (n = 5). Rats were individually housed in wire mesh cages and maintained on a 12-h light/dark cycle. Rats were fed a pelleted semipurified AIN-76 diet (15) (Science Diets, Piscataway, NJ) formulated to provide all recommended nutrients as previously described (12) , with the exception that iron sulfate was added at 35 mg Fe/kg diet (control), 12 mg Fe/kg diet (moderate) or 6 mg Fe/kg diet (severe). Rats were allowed free access to diets and distilled deionized water. Food intake was recorded daily, and rats were weighed each week. Estrous phase was determined by microscopic examination of vaginal smears. Rats were fed the experimental diets for 6 wk and killed at 63–65 d of age while in estrus. At 2 h before killing, rats received an intraperitoneal injection of 5-bromo-2'-deoxyuridine (BrdU) (100 mg/kg body; Sigma Chemical Co., St. Louis, MO) dissolved in dimethylsulfoxide. Rats were exposed to ether, and closed cardiac puncture was performed. Cardiac blood was used to measure hematocrit and hemoglobin at the time of killing. Plasma was collected from the remaining blood sample and stored at -80°C for hormone analyses. Livers were excised, weighed, frozen in liquid nitrogen and stored at -80°C. Liver iron levels were measured on a flame atomic absorption spectrophotometer (Thermo Jarrell Ash, Franklin, MA) after dry ashing at 500°C and digestion in concentrated nitric acid. All conditions and handling of rats met National Institutes of Health guidelines for the care and use of laboratory animals (16) .

Mammary tissue.

Mammary tissue was obtained from rats by removing the intact pelt containing all six pairs of mammary glands. The pelt was stretched and fixed in 10% neutral buffered formalin (Fisher Scientific, Pittsburgh, PA) for 48 h. The left third thoracic and fourth abdominal mammary glands were excised and processed for whole mounting. The entire fat pad containing the intact mammary gland was dissected from the pelt, defatted in acetone for 24–48 h, hydrated through a graded series of ethanol to distilled water, stained with toluidine blue, dehydrated through a graded series of ethanol and cleared in xylene. Stained glands were microdissected to achieve maximum resolution of the gland and mounted on glass slides using Permount (Fisher Scientific, Pittsburgh, PA).

The contralateral glands were removed from the pelt and processed for paraffin embedding. Briefly, tissues were dehydrated through a graded series of ethanol to xylene, embedded in paraffin blocks, sectioned at 5 µm and mounted on Superfrost slides (Fisher Scientific) for detection of BrdU incorporation by immunohistochemistry.

Mammary gland morphological analysis

The effect of iron deficiency on mammary gland development was determined by microscopic examination of toluidine blue–stained mammary gland whole mounts. The classification and quantification of the different mammary gland structures were performed using a stereomicroscope fitted with an ocular micrometer. The microscope was calibrated using a 1-mm stage micrometer. The type and number of mammary gland structures were determined within a defined area that encompassed the periphery of the gland. Structures were classified as TEB, terminal ducts or LOB based on the criteria developed by Russo and Russo (13) . TEB, which are the growth point of the mammary epithelial tree, are abundant at the periphery of the gland, so this area was chosen for study. TEB are identified by their club-shaped morphology and exist as solitary structures or in clusters. In whole mount preparations, classification is confirmed by a minimum diameter of 100 µm when measured at the widest point. In longitudinal sections, TEB appear round or oval shaped with multiple layers of epithelial cells enclosing a central duct. The terminal duct, which represents a TEB in the process of regression (13) , appears as slender TEB in whole mount preparations. Classification is confirmed by a diameter of <100 µm. In longitudinal sections, terminal ducts appear as ducts with a single layer of epithelial cells and a central lumen. Alveoli consist of two to five alveolar buds clustered around a central duct. In the present study, TEB undergoing the process of cleavage were classified as alveoli. Lobules arise from the further cleavage of alveolar structures to form clusters of six or more alveolar buds. For this study, these two related structures were grouped under the single classification of LOB. In longitudinal sections, LOB structures are identified as clusters of acini composed of a single layer of myoepithelial cells and a single layer of epithelial cells enclosing a central lumen.

The densities of the different mammary structures within the third and fourth glands were determined by first dividing the gland into grids of 0.5 x 0.5 cm. Structures within each grid were then classified and quantified. The data were expressed as the number of each structure present within 1 cm² of gland. Coded whole mounts from each treatment group were counted in duplicate with a minimum of 1 wk between determinations (r² = 0.812).

Puberty onset and reproductive hormone levels.

Sexual maturation was established by monitoring the day of vaginal opening. Plasma levels of 17-ß-estradiol and progesterone at the time of killing were determined using standard radioimmunoassay kits according to the manufacturer’s directions (Coat-A-Count; Diagnostic Products Corp., Los Angeles, CA).

DNA-labeling index.

The effect of iron deficiency on mammary epithelial cell (MEC) DNA synthesis was determined in rats fed 12 and 35 mg Fe/kg diet. Mammary tissue sections of 5 µm thickness were heat immobilized overnight at 60°C, deparaffinized in xylene and rehydrated through a graded series of ethanol to distilled water. Endogenous peroxidase activity was quenched by a 30-min incubation in 3% H₂O₂. Antigen unmasking was accomplished by incubation in 2 mol HCl/L at 40°C for 1 h. Nonspecific binding of IgG was blocked by incubation in 5% horse serum (Vector Labs, Burlingame, CA). Anti-BrdU antibody (Amersham, Piscataway, NJ) was diluted 1:100 in Tris-buffered saline (TBS). After an overnight incubation at 4°C in a humidified chamber, slides were incubated with horse anti-mouse biotinylated secondary antibody (Vector Labs) followed by treatment with horseradish peroxidase–conjugated streptavidin (Vector Labs). 3,3'-Diaminobenzidine tetrahydrochloride (Vector Labs) was used as the chromogen. Slides were lightly counterstained with hematoxylin. Sections incubated with nonimmune serum were used as negative controls. Immunostaining was evaluated using a bright field microscope. The number of nuclei staining brown was expressed as a percentage of the total number of nuclei counted.

Statistical analysis.

Data were analyzed by one-way ANOVA and Fisher’s protected least significant difference test using StatView software for Macintosh. Differences were considered significant when P < 0.05.

	RESULTS

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Iron status feeding.

Iron-restricted diets providing 6 or 12 mg Fe/kg diet resulted in the development of severe or moderate iron deficiency, respectively, as determined by hematological indices and body iron stores (Table 1 ). Hematocrit and hemoglobin were significantly lower (32 and 45%, respectively) in rats fed 6 mg Fe/kg diet than in controls (P < 0.05). Rats fed 12 mg Fe/kg diet tended to have lower hematocrit and hemoglobin values compared to controls (16% and 13%, respectively), but these differences were not significant (P = .12). In contrast, liver iron stores were significantly depleted in moderately as well as severely deficient rats compared with controls (P < 0.05). Final body weights did not differ among groups (data not shown).

Mammary gland development and DMBA-induced carcinogenesis are hormone-dependent processes. In addition, mitotic activity of the MEC varies with phase of the estrous cycle, increasing during the luteal phase and decreasing in the follicular phase (13 ,17) . Therefore, in addition to measuring estrogen and progesterone levels, puberty onset was also established. Feeding 6 or 12 mg Fe/kg diet beginning at 21 d of age did not affect the onset of sexual maturity; control and treated rats reached sexual maturity at 35 d. Plasma estrogen levels did not differ among groups (P = 0.54), but plasma progesterone levels were significantly lower in both iron-restricted groups than in controls (P < 0.05, Table 1 ).

Mammary gland morphogenesis.

The effect of iron deficiency on mammary gland morphogenesis was evaluated in both thoracic and abdominal mammary glands after 6 wk of iron restriction. The overall size and gross morphology of the abdominal glands were similar in iron-deficient and control rats (Fig. 1 ). Similar results were obtained for the thoracic glands (data not shown). Because the leading edge of the gland is the area richest in TEB, this region was chosen for study. The area (in cm²) of thoracic (P = 0.82) and abdominal (P = 0.57) glands included for study did not differ among groups. TEB and LOB were present in the glands of all rats regardless of dietary treatment. The density of thoracic TEB in severely and moderately iron-deficient rats did not differ from controls (P = 0.20) (Table 2 ). The degree of iron deficiency also had no effect on the density of TEB in the fourth abdominal gland (P = 0.16). Similarly, LOB development in thoracic (P = 0.82) and abdominal glands (P = 0.57) was unaffected by dietary iron.

View larger version (176K): [in this window] [in a new window]   Figure 1. Morphological development of the fourth abdominal mammary gland in 63-d female iron-deficient and control rats. Whole mounts were prepared as described in the text. Glands are from rats fed 35 mg Fe/kg diet (Control) or 6 mg Fe/kg diet (Iron-deficient) beginning at 21 d of age for 6 wk. Alveolobules (LOB) and terminal end buds (TEB) and their predominant locations within the gland are depicted by the insets (100x).

We have found that only moderate, and not severe, iron deficiency leads to increased tumor incidence and burden in response to chemical carcinogen exposure. This is the condition that most closely reflects the average premenopausal woman living in industrialized societies. Therefore, the DNA-labeling index was studied in only the moderately iron-deficient and control groups. In addition, only the thoracic gland was studied because we have found that this site accounts for the majority of between-group differences in mammary gland tumor burden (number of tumors per rat). BrdU incorporation was used to determine the percentage of epithelial cells in S-phase of the cell cycle at the time of killing. Within the treatment and control groups, the DNA-labeling index was highest in the TEB and lowest in the LOB (Fig. 2 ). This is in agreement with the results of others (18) . DNA synthesis in the TEB of iron-deficient rats did not differ from controls (P = 0.29). We found no effect of moderate iron deficiency on MEC proliferative activity in any of the other ductal structures studied.

View larger version (27K): [in this window] [in a new window]   Figure 2. 5-Bromo-2'-deoxyuridine (BrdU) incorporation in mammary gland structures of moderately iron-deficient and control rats. Rats were fed experimental diets containing 12 or 35 mg Fe/kg diet for 6 wk. At 2 h before killing, rats received an intraperitoneal injection of 100 mg BrdU/kg body. Glands were excised and processed for immunohistochemistry as described in the text. The results are expressed as the percent BrdU incorporation, which represent the proportion of the total cells counted that were in the S-phase of the cell cycle. One thousand nuclei per glandular structure were counted per rat for each treatment group. Values are the mean ± SEM, n = 5.

	DISCUSSION

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Many factors have been shown to influence the carcinogenic potential of DMBA, including age, reproductive hormone levels and the rate of target cell proliferation (14 ,22) . The goal of the present study was to identify potential mechanisms mediating the increase in tumor outcome observed during moderate iron deficiency in rats (12) . We investigated the effect of iron restriction on mammary gland development, MEC proliferation and ovarian function.

Feeding iron-restricted diets that provide 6 and 12 mg Fe/kg diet to female weanling rats resulted in the development of severe and moderate iron deficiency as indicated by hematological indices and liver iron concentration. Moderate iron deficiency was characterized by a significant depletion of body iron stores, which was reflected in the decreased liver iron content. In contrast, the hematocrit and hemoglobin levels, although decreased, were not significantly different from those of control rats. Clinically, iron deficiency anemia is defined as hemoglobin values that are 2 SD below the population mean (23) . However, the determination of moderate iron deficiency, or iron deficiency without anemia, is more ambiguous. In the National Health and Nutrition Education Survey III study, the designation of iron deficiency without anemia was based on an individual having a normal hemoglobin with two or more other indicators of iron status that fell below a predetermined cutoff value (20) . In our studies, moderate iron deficiency was defined as having nonsignificant reductions in hemoglobin and hematocrit but a significant reduction in body iron stores. These criteria are consistent with the clinical picture of moderate iron deficiency seen in premenopausal women in the United States, a condition that was our intention to mimic.

Normal postnatal mammary gland development is dependent in part on the action of the reproductive hormones (24) . In addition, the mitotic activity of the MEC in humans and rats is influenced by the phase of estrus (13 ,17) . Therefore, it was important to establish the effect of dietary treatment on these variables. Iron status had no effect on the onset of sexual maturity. Circulating levels of 17-ß-estradiol were not affected by either severe or moderate iron deficiency. In contrast, iron restriction was associated with a significant reduction in plasma progesterone levels relative to control rats. Our results indicate that the lower progesterone levels did not affect mammary gland growth or development. Therefore, the physiological relevance of this finding is unclear at the present time. Progesterone has been shown to regulate, either directly or indirectly, the iron-containing proteins lactoferrin (25 ,26) and uteroferrin (27) . Whether these two findings are related remains to be determined. In contrast, the effect of iron deficiency on circulating progesterone levels has not been reported. One possibility would be that the metabolism of progesterone is altered in iron deficiency. Clearly, this observation warrants further investigation.

In rats, pubertal development of the mammary gland is characterized by a rapid expansion of the mammary parenchyma largely through the mitotic activity of the TEB. It has been estimated that mammary gland growth proceeds at a rate four times that of body growth between 23 to 40 d of age in rats (28) . The ubiquitous role of iron in cellular physiology would be expected to place a large demand on iron stores during this increase in cellular growth. We found no significant effects of iron status on postpubertal mammary gland morphology when iron deficiency was initiated at 21 d of age in the female rat. Although the density of TEB in the thoracic gland was numerically higher in both iron-restricted groups compared with control rats, the differences were not statistically significant. Thoracic mammary gland development was studied in three to five rats per group. Therefore, the lack of significant treatment effects may be due to the combination of subtle changes in gland development and the small sample size studied. It is possible that evaluation of a greater number of glands would have reduced the within-group variability, resulting in significant differences among groups.

Results in BrdU labeling confirm the morphological findings. The incorporation of BrdU was similar between moderately deficient and control rats in all structures examined. In contrast to the present observations in the mammary epithelium, thymic and splenic atrophy has been observed in rats with similar degrees of iron deficiency as achieved in the present study (29 ,30) . The apparent differences in the proliferative or growth capabilities of these two cell types during similar conditions of iron depletion raise interesting questions regarding the prioritization of iron utilization among different tissues during the depletion of body iron stores. Little information is available concerning the regulation of iron homeostasis in the MEC. However, because cellular iron homeostasis is regulated at the level of iron uptake, one mechanism governing such a prioritization is the regulation of transferrin receptor (TfR) expression (31) . TfR expression has been shown to be increased in proliferating MEC and T-lymphocytes (32 33 34) . However, in contrast to the present results, which indicate no effect of iron deficiency on MEC proliferation, iron deficiency does significantly impair T-lymphocyte proliferation. This suggests that factors other than TfR expression may be mediating iron uptake and utilization in the mammary gland during iron restriction.

We had hypothesized that iron deficiency would impair the morphological development of the mammary gland and that this would be manifested as an increase in the numbers of precursor structures, the TEB. An increase in TEB density could then account for our observation that moderate iron deficiency is a risk factor for DMBA-induced tumor formation in the rat. The data presented here do not support this hypothesis. The possibility that moderate iron deficiency may lead to subtle metabolic abnormalities at the cellular level or impair defense mechanisms directed toward elimination of genetic damage warrants further examination.

	ACKNOWLEDGMENTS

 
The authors are grateful to Irma and Jose Russo of the Breast Cancer Research Laboratory (Fox Chase Cancer Center, Philadelphia, PA) for their expert guidance in the technique of mammary gland whole mounting, as well as their assistance with the histological studies presented. In addition, the authors thank Deborah Hrabinski for her technical assistance

	FOOTNOTES

 
¹ Supported by a grant from the American Institute for Cancer Research.

³ Abbreviations used: BrdU, 5-bromo-2'-deoxyuridine; DMBA, 7,12-dimethylbenz[a]anthracene; LOB, alveolobule; MEC, mammary epithelial cell; TEB, terminal end bud; TfR, transferrin receptor.

Manuscript received November 30, 2000. Initial review completed January 3, 2001. Revision accepted February 24, 2001.

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This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Grill, C. J. Articles by Sherman, A. R. Search for Related Content PubMed PubMed Citation Articles by Grill, C. J. Articles by Sherman, A. R.