Toxicol Sci. 2006 Oct;93(2):357-68.
Estrogenic effect of soy isoflavones on mammary gland morphogenesis and gene expression profile.
Thomsen AR, Almstrup K, Nielsen JE, Sørensen IK, Petersen OW, Leffers H, Breinholt VM.
Department of Toxicology and Risk Assessment, Danish Institute for Food and Veterinary Research, Mørkhøj Bygade 19, Søborg, Denmark.
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We examined the effect of 17beta-estradiol (E2) and soy isoflavones' exposure on morphogenesis and global gene expression in the murine mammary gland. Three exposure regimens were applied: isoflavones added to the diet throughout either the lactational period (via the dams) or the postweaning period and E2 administered orally during the lactational period. Whole mounts of mammary glands were evaluated both in juvenile and adult animals with respect to branching morphogenesis and terminal end bud (TEB) formation. At postnatal day (PND) 28, we observed a significant increase in branching morphogenesis in all treated groups with the most pronounced effect after E2 exposure. For the E2-treated animals there was also a significant increase in TEB formation. At PNDs 42-43 the postweaning isoflavone and the E2 groups showed a transient reduction in the number of TEBs. A similar response after isoflavone and E2 exposure was further substantiated by changes in gene expression, since the same groups of genes were up- and downregulated, particularly in the E2 and postweaning isoflavone regimen. All changes in gene expression correlated with changes in the cellular composition of the gland, i.e., more and larger TEBs and ducts. The results suggest an estrogenic response of physiological doses of isoflavones on mammary gland development at both the morphological and molecular level, which resembled that induced by puberty.

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... Although phytoestrogens may contribute to the prevention of hormone-dependent cancers, compounds that possess estrogenic activity or can disrupt steroidogenesis may also cause endocrine disruption, particularly when exposure occurs prior to puberty (Newbold et al., 1990; Palmer et al., 2005). Genistein, an isoflavone abundantly present in soybeans, induces an estrogenic response at physiological levels in the uterus and in ER–positive cancer cells in vivo (Jefferson et al., 2002; Matsumura et al., 2005). In addition, isoflavones are reported to mediate estrogen-like adverse effects on the formation of mammary and uterine adenomas (Allred et al., 2004; Day et al., 2001; Luijten et al., 2004).

Animal experiments suggest that exposure to estrogens, including genistein, has an influence on breast cancer risk, conceivably by altering mammary gland development (Cabanes et al., 2004; Hilakivi-Clarke et al., 1998, 1999a; Murrill et al., 1996). At birth, the mammary gland of most mammalian species, including humans, is not completely formed but consists of a small primitive anlage consisting of a few ducts extending from the nipple and growing isometrically in relation to the rest of the body. With the onset of puberty, estrogen level rises and, together with other hormones and growth factors, initiates the development of the mammary gland. In mice, this process begins around postnatal day (PND) 28 and is characterized by increased proliferation at the duct termini, resulting in the formation of the terminal end bud (TEB) (Hovey et al., 2002). These structures are the major sites of proliferation from which ducts elongate and branch dichotomously and sympodially and thereby form the mammary tree. Estrogen signaling is essential for TEB formation and ductal elongation (Korach et al., 1996). Thus, it is likely that the differentiation of the TEB is responsive to environmental estrogens including isoflavones. The TEB may contain immature cells that are potential precursor cells of mammary tumors (Russo and Russo, 1978). Indeed, in rodents the majority of tumors seem to originate from the immature TEB, and the elimination of these structures by differentiation into alveolar structures may reduce the subsequent risk of breast cancer (Russo and Russo, 1978). Animal experiments suggest that estrogenic compounds can alter the course of TEB differentiation; however, the results are contradicting as to whether the differentiation is enhanced or inhibited (Hilakivi-Clarke et al., 1998, 1999b; Murrill et al., 1996). Although the results are controversial, they nevertheless suggest the existence of a "window" in early life where temporal exposure to estrogenic compounds causes alterations in the subsequent mammary gland development.

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The results suggest that isoflavones given at physiological concentrations (resulting in serum levels of approximately 500nM) cause analogous morphological and gene expression changes, as treatment with E2 at doses known to induce an estrogenic response in vivo (2.5 mg/kg body weight). Early postnatal exposure to 17beta-estradiol acetate (E2) and isoflavones affected the pattern of mammary gland morphogenesis in the juvenile gland by inducing an increased branching of the ductal tree in the juvenile gland, suggesting an early growth stimulatory effect. Also, when mice were exposed to E2 during PNDs 10–20, the number of proliferative TEBs was increased at PND 28. An increase in TEBs was found following postweaning isoflavone exposure (PNDs 21–28) as well, although it did not reach statistical significance. At PNDs 42–43, the number of the proliferative TEB was reduced by all treatments, with statistical significance after postweaning isoflavone treatment and E2 treatment, supporting the notion of accelerated or induced onset of pubertal maturation, which in theory may result in a reduced cancer risk (Murrill et al., 1996). However, this effect was transient, as no reduction in the TEB number was observed at adulthood (PNDs 70–73). This could suggest that the treatment did not as such induce the formation of additional TEBs but rather enhanced the normal development. If the number of TEBs, i.e., the number of immature proliferating cells, is an important aspect in the etiology of mammary tumorigenesis, then the effect of environmental estrogens may depend on the specific timing of tumor initiation: a protective effect may be expected if the tumor transformation is initiated late in puberty but the opposite may be true if the tumor transformation is initiated at an earlier point in life.

To complement the findings of morphological alterations in the juvenile gland (PND 28) and to clarify whether exposure to isoflavones and E2 induced distinct pathways, we compared the global gene expression profiles in the different exposure groups. We found that the gene expression profiles of the isoflavone exposures were remarkably similar to those induced by E2. The result is in accordance with a previous study, where gene expression after exposure to genistein and synthetic and physiologic estrogens was investigated in immature mouse uterus. The authors showed that the different types of estrogens induced similar genetic responses and that the differences in transcriptional responses probably were caused by dose-dependent variations in magnitude and kinetics of gene expression rather than induction of distinct pathways (Moggs et al., 2004). Moreover, ER seemed downregulated (~50%) after exposure to both isoflavone and E2 during the lactational period. This effect was previously observed in the mammary gland after exposure to exogenous E2, probably as a consequence of tissue-specific autoregulation (Hatsumi and Yamamuro, 2006).

Having identified a set of differentially expressed genes, we sought to clarify if the effects of isoflavones and E2 on gene expression were caused by the induction of genes in existing cell types or by an altered number of the cells expressing the genes, i.e., changes in cellularity. Based on their relevance to mammary gland development, three of the upregulated genes (AP-2, Crk, and Clusterin) were selected for investigation of the cell types expressing the genes. AP-2 was expressed in ductal epithelia cells and myofibroblasts in the adult mammary gland in a pattern similar to what has previously been described (Zhang et al., 2003). The AP-2 family of transcription factors might participate in direct activation of ER-, Insulin-like Growth Factor Receptor, and ErbB-mediated proliferative signaling and may also participate in regulating mammary gland morphogenesis (Hoei-Hansen et al., 2004; Turner et al., 1998; Zhang et al., 2003); AP-2 is significantly upregulated in early-stage breast tumors and in testicular carcinomas (Hoei-Hansen et al., 2004; Turner et al., 1998). The highest expression of Crk was observed in cap/transition cells in the TEBs, but it was also expressed in body cells and luminal epithelial cells. Crk is involved in epithelial invasion and morphogenesis and is a mediator of ErbB signaling, and its expression pattern is thus in accordance with its putative function in cell migration (Lamorte et al., 2002). For both Crk and AP-2, we could not detect a change in staining intensity between mammary glands from treated and control animals. However, the TEBs from the treated glands (groups III and IV) were significantly larger in size and seemed further developed compared to the controls (Fig. 5). Finally, we used ISH to determine the precise expression pattern of Clusterin (Apo J), which, in the microarray data showed a significantly higher expression in all treatment groups as compared to controls. ISH showed that the Clusterin mRNA was localized to cap/transition cells of the TEBs and to the leading edge of lateral branches. As for Ap-2 and Crk, the staining intensity among controls and treated animals was similar, but there were more and larger TEBs in the treated glands. Clusterin is involved in tissue remodeling and in promoting and preventing apoptosis (Trougakos and Gonos, 2002), and Clusterin is highly expressed in bladder transitional cell carcinoma (Miyake et al., 2002) and in human breast carcinoma, whereas it is barely detectable in normal adult breast tissue (Redondo et al., 2000).

Together, the observations suggest that the increased expression of these genes probably reflects (1) an increase in the number of cells in the TEBs, (2) an increase in lateral branches, and (3) the presence of more luminal epithelial cells in the mammary gland. That changes in cellularity is the cause of the changes in gene expression is supported by the increased expression in all experiments of four cytokeratins (CK7, CK8, CK18, and CK19). The cytokeratins are markers for luminal epithelial cells (Petersen et al., 2003; Stingl et al., 2005), and an increased total ductal volume and, thus, a higher percentage of ductal epithelia cells can explain their increased expression.

Interestingly, three of the identified genes, CK19, Crk, and AP-2, are referred to as stem cell markers and are also highly expressed in mammary cancer cells. In the human mammary gland, CK19 is expressed in a subpopulation of cells with stem cell characteristics located within the luminal epithelial compartment and is also expressed in the majority of breast cancer cells arising from these compartments (Petersen et al., 2003). It has been proposed that exposure to hormones in utero or during the immediate early postnatal growth may modulate the number of stem cells and their proliferative potential (Baik et al., 2005; Trichopoulos, 1990). The increase in the number of cells expressing CK19, Crk, and AP-2 may imply an increase in mammary stem cells introduced by early exposure to E2 and isoflavones. Since mammary stem cells/progenitor cells are suggested to be the cellular origin of at least a subset of breast cancers (Petersen et al., 2003), an increase in their number may result in an increased breast cancer risk.

In conclusion, the changes in gene expression after E2 and isoflavone exposure most likely reflect altered cellularity, whereas an increase in ductal epithelium or in the number and volume of TEBs would result in an apparent upregulation of all genes that are highly expressed in these specific structures. Hence, the working hypothesis that estrogenic compounds could enhance mammary gland development by promoting an earlier proliferation in the mammary gland is supported by the results herein, as evident by increased branching and increased number of TEBs. In addition, the notion that this early event should lead to increased glandular differentiation was also supported, as we observed a significant transient reduction in TEB number at midpuberty (PNDs 42–43). This reduction in TEB number implies that the further differentiation of the immature cells of the TEB into luminal epithelial cells is enhanced by both isoflavone and E2 treatment, which in theory could reduce the subsequent breast cancer risk. However, as a consequence of the initial increase in the number of proliferating cells (at PND 28), the potential effect of these compounds on breast cancer risk may depend on the specific timing of tumor initiation.

The appearance of TEBs and initiation of ductal branching observed in the treated groups are also indicators of the onset of puberty. Thus, the observed effects could be caused by treatment-induced puberty, resulting in a more developed stage of the gland. Thus, it remains to be elucidated whether the alterations in morphology and gene expression pattern are caused directly by the E2 and isoflavone exposure or whether these compounds induced early puberty.

Categories: 2006, Soy, Phytoestrogens, Endocrine, Estrogen, Gene expression, Sexual development, Puberty, Premature puberty, Hormone disruptors, Cancer, Nutrition and diet