The present study demonstrates that the deleterious consequences of TCDD on pregnancy-associated mammary development and lactogenesis occur through local effects on the mammary gland and that AHR activation in both the epithelial parenchyma and in the associated stromal tissue are required for this impairment. Understanding how environmental pollutants, such as dioxins, influence pregnancy-associated mammary gland development provides greater insight into two important processes: pregnancy-associated mammary gland differentiation and AHR-regulated development. Synchronized and controlled development of mammary glands during pregnancy is necessary for appropriate milk production. Reasons for lactation insufficiency are not well understood; however, decreased milk production as a consequence of exposure to pollutants has been proposed as a contributing factor leading to difficulties in initiating or maintaining breastfeeding [8
]. Yet few studies have been undertaken to determine precisely how environmental toxicants affect mammary gland differentiation during pregnancy. In addition to helping understand how chemicals from our environment disrupt this process, studies of mammary gland development during pregnancy provide insight into how AHR regulates complex developmental processes. Mammary gland development during pregnancy follows a pattern of proliferation and differentiation that is quite similar to events that occur in other organs during prenatal development [4
]. Thus, processes that occur in other organs during fetal development can be studied in an adult animal simply by impregnating the female. Herein we used several different experimental models to better understand how AHR activation during pregnancy deregulates mammary development and impairs lactogenesis.
In addition to responding to xenobiotics, the AHR has been implicated as an important regulator of growth and development; however, the specific nature of AHR's physiological functions remains enigmatic [13
]. AHR activation by exogenous ligands disrupts proliferation and tissue differentiation in many experimental systems, and AHR-null mice have defects in hepatic growth, decreased fertility, ovarian changes, and abnormalities with aging [18
]. However, the role of AHR in normal mammary gland development remains controversial. We show here that lack of AHR did not alter normal mammary gland development, and homozygous AHR-deficient dams were able to nutritionally support their offspring. This is consistent with another report wherein AHR-null and ARNT-null mice were able to nurse their litters normally [24
]. Collectively, these data suggest AHR and its dimerization partner are not required for mammary gland development and lactation. However, others have reported that mammary glands from AHR-null mice showed impairment in branching and morphogenesis in an ex vivo culture system [23
]. These differences may arise because of distinctions in experimental procedures, physiological setting (in vivo vs. in vitro), endpoints measured, or mouse strain. For example, the report by Hushka et al. [23
] used AHR-null mice that were created by one group, while in our experiments we used Ahr
KO mice created in a different manner. Also, we examined this process in vivo, whereas they used an ex vivo system for differentiating the tissue. Despite such differences in studies with Ahr
KO mice, it remains clear that AHR activation by exogenous ligands has a profound impact on mammary gland differentiation.
Mammary gland development during pregnancy is controlled by many factors that are produced systemically and locally [4
]. Using several experimental approaches, we endeavored to tease apart which tissues and cell types are directly affected by TCDD. Ex vivo culture of whole organs revealed that TCDD acts directly on mammary glands, which is consistent with a prior report that mammary explants treated with 2,3,7,8-tetrachlorodibenzofuran (another potent AHR ligand) displayed decreased lobule development [23
]. Likewise, direct treatment of cultured mammary epithelial cells with TCDD impairs differentiation and β-casein induction [39
]. Collectively, these findings in mammary tissues and cells are consistent with reports showing that AHR ligands directly influence the differentiation of other organs [29
]. Yet, organs are made up of different cell types, making it somewhat difficult in many systems to delineate the specific subsets of cell types within an organ that are responsible for AHR-mediated changes in differentiation. It is possible to distinguish AHR-mediated events in mammary epithelium and stromal tissues by reciprocal transplantation, which is an approach that has been used to delineate the contribution of receptors and signaling molecules in other studies [49
]. Reciprocal transplantation of AHR+/+
mammary epithelium revealed that local factors produced by the mammary epithelium and stromal tissues (fat pad) both contain direct AHR targets, which are involved in disrupting pregnancy-associated differentiation. Future experiments using transgenic systems to conditionally eliminate AHR in specific mammary cell types would ultimately clarify whether epithelial or stromal AHR in the mammary gland explains the observed changes in pregnancy-associated differentiation.
In addition to demonstrating that AHR has targets within and extrinsic to the mammary epithelium, we present here the novel finding that whatever signaling events are triggered by TCDD exposure requires the AHR's DNA-binding domain. This further supports the idea that AHR regulates mammary gland differentiation at the level of transcriptional control. One potential target gene is cyclin D1, which showed reduced expression in whole mammary glands and sorted MECs from TCDD-treated mice. Given that AHR activation decreases MEC proliferation in early pregnancy, decreased cyclin D1 is consistent with a defect in proliferation [9
]. Of course, one possible explanation for reduced cyclin D1 is that that there are simply fewer MECs. However, many genes were not changed by TCDD treatment (data not shown), and other genes, such as Cyp1a1,
were markedly elevated by TCDD exposure. There are two additional pieces of evidence to suggest a relationship between AHR signaling and cyclin D1. First, the work of others has shown that activated AHR inhibits proliferation and induces cell cycle arrest in other types of cells and tissues; thus, observing this in mammary glands is consistent with these studies (reviewed in [17
]). In particular, AHR activation by TCDD decreased cyclin D1 in prostate cancer cells and breast cancer cell lines [53
] as well as in mouse liver [45
] and zebrafish caudal fin regeneration models [55
]. The second piece of evidence stems from reports that cyclin D1 is essential for mammary gland development during pregnancy [56
]. Loss of cyclin D1 leads to a paucity of alveolar cells, which fail to functionally differentiate. In fact, mammary gland development during pregnancy is stunted in cyclin D1-deficient mice with a phenotype that looks strikingly similar to TCDD-treated mice. Thus, an AHR-mediated diminution in cyclin D1 provides a possible mechanism for reduced pregnancy-associated MEC proliferation and may explain in part the decreased number of proliferating mammary epithelial cells in TCDD-treated pregnant mice. However, an impact on cyclin D1 does not rule out additional affects of TCDD on growth factors or other regulatory molecules that are locally produced in the mammary gland.
Indeed, we also noted that mammary glands from TCDD-treated mice had reduced expression of E-cadherin, which is a cell-adhesion molecule directly involved in and crucial for the development and differentiation of epithelial cells in various tissues, including the mammary gland [58
]. Other reports have shown that AHR activation reduces E-cadherin protein levels in whole mammary glands, MCF-7 cells, and SCp2 cells, suggesting a direct effect of TCDD on MEC and on this gene in particular [40
]. However, when we isolated MECs from TCDD-treated pregnant mice, we did not observe decreased E-cadherin expression levels. These contradictory results may be due to the fact that the cell lines were cultured with other factors and/or on Matrigel, which provides an exogenous extracellular matrix. Timing may also be a factor in sensitivity of E-cadherin to perturbation by AHR activation. In our prior work, we examined E-cadherin in late pregnancy (DP 17) and in SCp2 mammary epithelial cells, which were clonally derived from mice at midpregnancy [59
]. In contrast, in our present study, MECs were isolated from animals exposed to TCDD in vivo during early pregnancy (DP 0–6). Thus, E-cadherin expressed by MECs during early pregnancy may not be altered by TCDD exposure, whereas AHR may alter E-cadherin expression in later stages of pregnancy. Alternatively, AHR-mediated changes in E-cadherin levels may be downstream of a direct impact of AHR on other gene targets and signaling pathways, including gene targets that are within mammary tissue but not that are extrinsic to the epithelial compartment.
The new findings presented here suggest that impairment of pregnancy-associated mammary gland development after exposure to TCDD occurs due to AHR-mediated alterations in the normal function of the mammary epithelium and mammary fat pad (stroma). In addition to providing new information regarding how AHR ligands alter this orchestrated developmental process, these findings demonstrate that mammary tissues are directly targeted by a common environmental toxicant in a manner that impedes lactogenesis. When considering public health, the implications of this are profound. Decreased milk production as a consequence of exposure to environmental pollutants may contribute to poor nutrition, especially in places where breast milk is the only food available for the neonate and where exposures to pollution remain poorly controlled. In addition to human health, wild animal populations are exposed to dioxins and related chemicals, which may result in decreased milk production in these species. Another possible implication of this work is a potential connection to breast cancer. Although not directly examined in the work described herein, several molecules involved in normal mammary gland development and differentiation have been implicated in the formation of mammary tumors [60
]. This possible relationship is further suggested by the fact that TCDD is a known human carcinogen, and a relationship between exposure to TCDD and related AHR-binding pollutants and breast cancer has been reported [61
]. Thus, in addition to improving our understanding of how AHR signaling and exogenous AHR-binding pollutants influence lactogenesis, the results from the present study suggest possible involvement of AHR ligands in the development of breast cancer.