Activation of the aryl hydrocarbon receptor by TCDD inhibits mammary tumor metastasis in a syngeneic mouse model of breast cancer

Treatment with aryl hydrocarbon receptor (AhR) agonists can slow or reverse the growth of primary mammary tumors in rodents, which has fostered interest in developing selective AhR modulators for treatment of breast cancer. However, the major goal of breast cancer therapy is to inhibit metastasis, the primary cause of mortality in women with this disease. Studies conducted using breast cancer cell lines have demonstrated that AhR agonists suppress proliferation, invasiveness, and colony formation in vitro; however, further exploration using in vivo models of metastasis is warranted. To test the effect of AhR activation on metastasis, 4T1.2 mammary tumor cells were injected into the mammary gland fat pad of syngeneic Balb/c mice treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Primary tumor growth was monitored for 4 weeks, at which time metastasis was determined. TCDD treatment suppressed metastasis by approximately 50%, as measured both in the lung and in mammary glands at sites distant from the primary tumor. Primary tumor growth was not suppressed by TCDD exposure nor was proliferation of 4T1.2 cells affected by TCDD treatment in vitro. Taken together, these results suggest that the protective effect of AhR activation was selective for the metastatic process and not simply the result of a direct decrease in tumor cell proliferation or survival at the primary site. These observations in immunologically intact animals warrant further investigation into the mechanism of the protective effects of AhR activation and support the promise for use of AhR modulators to treat breast cancer.     

The aryl hydrocarbon receptor affects distinct tissue compartments during ontogeny of the immune system.

There is growing evidence that prenatal and early postnatal environmental factors influence the development and programming of the immune system, causing long-lasting negative health consequences. The aryl hydrocarbon receptor (AhR) is an important modulator of the development and function of the immune system; however, the mechanism is poorly understood. Exposure to the AhR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin throughout gestation and during lactation yields adult offspring with persistent defects in their immune response to influenza virus. These functional alterations include suppressed lymphocyte responses and increased inflammation in the infected lung despite normal cellularity and anatomical development of lymphoid organs. The studies presented here were conducted to determine the critical period during immune ontogeny that is particularly sensitive to inappropriate AhR activation. We also investigated the contribution of AhR-mediated events within and extrinsic to hematopoietic cells. Our findings show that AhR activation alters different elements of the immune system at different times during development by affecting different tissue targets. In particular, diminished T-cell responses arise due to deregulated events within bone marrow-derived cells. In contrast, increased interferon gamma levels in the infected lung result from AhR-regulated events extrinsic to bone marrow-derived cells, and require AhR agonist exposure during early gestation. The persistence of AhR activation induced immune modulation was also compared, revealing that AhR activation causes long-lasting functional alterations in the developing immune system, whereas the impact on the mature immune system is transient.     

Exposure to low doses of oxybenzone during perinatal development alters mammary gland morphology in male and female mice

Oxybenzone (benzophenone-3) is an ultraviolet radiation filter commonly used in personal care products including sunscreens, textiles and inks, and food and beverage containers, among others. Due to its widespread use, human exposures to oxybenzone are widespread. Oxybenzone is considered an endocrine disrupting chemical due to its antiestrogenic and antiandrogenic properties. We evaluated the effects of oral exposures to oxybenzone on the growth and morphology of the mammary gland, body weight and anogenital distance in BALB/c mice exposed to 30, 212 or 3000 μg/kg/day in utero and during lactation. Developmental exposures to oxybenzone reduced the size and growth of mammary gland in males prior to and during puberty. In exposed females, oxybenzone reduced mammary cell proliferation, decreased the number of cells expressing estrogen receptor α, and altered mammary gland morphology in adulthood. These results suggest that even low doses of oxybenzone can disrupt hormone sensitive organs during critical windows of development.     

Cause of pregnancy-dependent mammary tumour-induced suppression of lactation in GR/A mice.

To clarify the cause of pregnancy-dependent mammary tumour (PDMT)-induced suppression of lactation due to the retardation of mammary gland growth, we studied the following: (I) mammary gland growth at the end of pregnancy in mice with PDMT [PDMT(+)] in comparison with mice without PDMT [PDMT(-)] and (II) effects of PDMT removal at the end of pregnancy on mammary gland growth and function. In Experiment I, little difference was observed between PDMT(-) and PDMT(+) groups in mammary DNA content and plasma level of lactogenic hormone or progesterone at the end of pregnancy, indicating that the retardation of mammary gland growth by PDMT does not occur during pregnancy. In Experiment II, the surgical removal of PDMT 1 or 2 days before parturition resulted in a complete restoration of mammary gland growth and lactation. All results strongly suggest that PDMT has a deleterious effect on mammary gland growth during their abrupt regression after parturition, perhaps by secreting some mammary growth inhibitory factor(s).     

Fetal and neonatal exposure to the mycotoxin zearalenone induces phenotypic alterations in adult rat mammary gland

Zearalenone is a mycotoxin that is widespread in cereal food. We questioned whether this mycotoxin, administered during known critical exposure periods such as the fetal period and the first days of life, at doses compatible with mean daily intake in humans, could have an effect on mammary gland development in rodents. Wistar female rats were exposed to zearalenone (0.2 μg/kg to 5 mg/kg) during the last 14 days of fetal life and the first 5 post-natal days (PND). The mammary tissue was examined for development and maturation by morphologic analyses and immunochemistry. At PND 30, the mean length of terminal buds was significantly enhanced in all of the zearalenone-exposed females (p < 0.05). The mammary tissue, as evaluated by scoring of tissue slides, was significantly more differentiated in the 1 mg/kg treated group than in controls (p < 0.05). At PND 180, mammary tissue was more differentiated in all of the zearalenone treated groups (p < 0.05). At six months, 4 of 18 females exposed to 5 mg/kg of zearalenone presented mammary hyperplasia lesions. The induction of phenotypic alterations by zearalenone administered in utero and in the neonatal period at doses as low as 0.2 μg/kg suggests that zearalenone could contribute to the induction of breast endocrine disorders.     

Exposure parameters necessary for delayed puberty and mammary gland development in Long-Evans rats exposed in utero to atrazine

Our studies suggested that prenatal exposure to the herbicide atrazine (ATR) could delay vaginal opening (VO) and mammary development in the offspring of Long-Evans (LE) rats. To evaluate ATR exposure parameters required for pubertal delays, including mammary gland development, we used cross-fostering to determine if effects were strictly dam-mediated (via milk) or a direct effect (transplacental) on the pups. Timed-pregnant LE rats (N = 20/treatment group) were gavaged on gestational days (GD) 15-19 with 100 mg ATR/kg body weight (BW) or vehicle (controls, C). On PND1, half of all litters were cross-fostered, creating four treatment groups: C-C, ATR-C, C-ATR, and ATR-ATR (dam-milk source, respectively). A significant delay in VO and increase in VO BW was seen only in the litters receiving milk from ATR-exposed dams. However, mammary glands of female offspring (two per dam) in all groups exposed to ATR (ATR-C, C-ATR, and ATR-ATR) displayed significant delays in epithelial development. These changes were detected as early as PND4 and stunted development was evident through PND40. Further, at all developmental stages examined, offspring in the ATR-ATR group exhibited the least developed glands. These delays in pubertal endpoints do not appear to be related to body weight or endocrine hormone concentrations. Our data suggest that the delay in VO of ATR-exposed offspring (C-ATR lactationally, ATR-ATR lactationally and in utero) is mediated via the dam [milk], whereas brief direct exposure to ATR in utero can cause delays in mammary gland development. Our data suggest that milk-derived factors (growth factors or hormones), in addition to transplacental exposure during mammary bud outgrowth, may be involved in ATR mode of action on delayed mammary gland development.     

Mammary gland differentiation by early life exposure to enantiomers of the soy isoflavone metabolite equol

The role of soy in reducing breast cancer risk has been suggested to be associated with early exposure to isoflavones, which alter mammary gland morphology. The objective of the study was to determine the effect of dietary exposure to the enantiomers of a key soy isoflavone metabolite, equol, on mammary gland development and later chemoprotection using the DMBA-induced animal model of breast cancer. Animals were exposed to S-(−)equol or R-(+)equol (250 mg/kg diet) during the neonatal (0–21 days) or prepubertal (21–35 days) periods only. Histological evaluation of the mammary glands showed that both enantiomers fed neonatally via the dam led to significant precocial mammary gland differentiation. By day 50, early S-(−)equol or R-(+)equol exposure resulted in a decrease in immature terminal end structures and an increase in mature lobules, suggesting an early ‘imprinting’ effect. Despite these morphological changes to the mammary gland, neonatal and prepubertal exposure to equol had no long-term chemoprevention against mammary tumors induced by DMBA, although for R-(+)equol there was a trend to delaying tumor formation. In summary, early exposure to equol was not chemopreventive, but neither did it increase tumor formation in response to DMBA, suggesting exposure in early life does not influence breast cancer risk.     

Modulation of mammary gland development in prepubertal male rats exposed to genistein and methoxychlor.

The estrogenic isoflavone genistein is a common dietary component that has been shown to affect reproductive development in experimental animals at high doses. The objective of the present study was to examine interactions of genistein and the hormonally active pesticide methoxychlor on mammary gland development in juvenile rats. Timed-pregnant Sprague-Dawley rats were fed a soy- and alfalfa-free diet containing different combinations of genistein (300 and 800 ppm) and methoxychlor (800 ppm). Rats were fed these diets starting on gestation day (GD)1 and continuing through pregnancy and lactation until postnatal day (PND) 22, when the pups were killed. Inguinal mammary glands from both female and male pups were processed as whole-mount preparations for morphometric analysis. The total glandular area and the numbers of branch points, lateral buds, and terminal end buds in the male rats were found to be significantly greater in the groups exposed to methoxychlor than those exposed to genistein only. These effects were not observed in the female rats. In the male rats, methoxychlor had the most prominent effect on elongating the glandular ducts, while genistein enhanced the ductile branching. The 2 compounds in combination promoted the development of alveolar-lobular structure, an effect not observed with either compound alone. Immunostaining for proliferating cell nuclear antigen revealed a high percentage of immunopositive cells in the mammary epithelia of the males exposed to methoxychlor and genistein (800 ppm) compared to the controls. While no significant changes in serum levels of mammotrophic hormones were detected, increased immunostaining for insulin-like growth factor-1 receptor, estrogen receptor alpha, and progesterone receptor in the genistein + methoxychlor group suggested that local factors involved in regulating mammary growth may have played a role in propagating the endocrine effects of these two compounds. These results indicated that the mammary glands of juvenile male rather than juvenile female rats may be more sensitive to certain endocrine-active compounds and that high levels of phytoestrogens have the potential to alter the toxicological behaviors of other hormone mimics.     

The early embryo loss caused by 2,3,7,8-tetrachlorodibenzo-p-dioxin may be related to the accumulation of this compound in the uterus

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a reproductive and developmental toxicant that can alter endocrine status, leading to decreased fertility and altered embryonic development; however, there are limited reports on TCDD toxicity during early pregnancy. In the present study, pregnant and pseudopregnant NIH mice were exposed to TCDD orally (2, 50 and 100 ng/kg body weight) during early gestation (days 1-8), pre-implantation stages (days 1-3), and peri-implantation to early post-implantation stages (days 4-8). TCDD concentration in uterus, liver, kidney, brain and fat on day 9 of pregnancy was monitored by an aryl hydrocarbon receptor (Ah receptor, AhR)-mediated LacZ reporter system in yeast. Results showed that the number of implanted embryos was significantly reduced on day 9 of gestation by 50 and 100 ng/kg TCDD exposure. The number of implantation sites was lower for animals exposed to TCDD on days 1-3 versus those exposed during days 4-8. Decidualization in pseudopregnant mice was also inhibited by TCDD exposure. TCDD concentrations as low as 2 ng/kg significantly decreased serum progesterone levels but had no effect on serum estradiol. TCDD level in the uterus was equal to levels in the liver, but lower than the fat tissue. These results suggest that TCDD sensitivity might be attributed its local accumulation in the uterus.     

Alterations in c-Src/HER1 and estrogen receptor α signaling pathways in mammary gland and tumors of hexachlorobenzene-treated rats

Hexachlorobenzene (HCB) is an organochlorine pesticide that acts as an endocrine disruptor in humans and rodents. The development of breast cancer strongly depends on endocrine conditions modulated by environmental factors. We have demonstrated that HCB is a tumor co-carcinogen in rats and an inducer of proliferation in MCF-7 cells, in an estrogen receptor α (ERα)-dependent manner, and of migration in MDA-MB-231 breast cancer cell line. In the present study, we examined HCB effect on c-Src/human epidermal growth factor receptor (HER1) and ERα signaling pathways in mammary glands and in N-nitroso-N-methylurea (NMU)-induced mammary tumors in rats. Furthermore, we evaluated histopathological changes and serum hormone levels. Rats were separated into four groups: control, HCB (100 mg/kg b.w.), NMU (50 mg/kg b.w.) and NMU-HCB. Our data show that HCB increases c-Src and HER1 activation, c-Src/HER1 association, and Y699-STAT5b and ERK1/2 phosphorylation in mammary glands. HCB also enhances Y537-ERα phosphorylation and ERα/c-Src physical interaction. In tumors, HCB also induces c-Src and HER1 activation, c-Src/HER1 association, as well as T308-Akt and Y699-STAT5b phosphorylation. In addition, the pesticide increases ERα protein content and decreases p-Y537-ERα levels and ERα/c-Src association in tumors. HCB increases serum 17-beta estradiol and prolactin contents and decreases progesterone, FSH and LH levels in rats without tumors, while the opposite effect was observed in rats with tumors. Taken together, our results indicate that HCB induces an estrogenic effect in mammary gland, increasing c-Src/HER1 and ERα signaling pathways. HCB stimulates c-Src/HER1 pathway, but decreases ERα activity in tumors, appearing to shift them towards a higher malignancy phenotype.     

Prenatal TCDD exposure predisposes for mammary cancer in rats

Epidemiological data are conflicting in the link between 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure and breast cancer causation. We have hypothesized that timing of exposure to endocrine disruptors, such as TCDD, will alter breast cancer susceptibility. Using a carcinogen induced rat mammary cancer model, we have shown that prenatal exposure to TCDD alters mammary gland differentiation and increases susceptibility for mammary cancer. Investigations into imprinting via DNA methylation mechanisms showed that there were no changes in protein expression in DNA methyltransferases, ER-alpha, ER-beta, GST-pi, or MDGI. Using 2D gels and mass spectrometry, we have found seven proteins to be differentially regulated, including a decrease in superoxide dismutase 1 (SOD1). Down-regulation of SOD1 could provide an environment ill equipped to deal with subsequent free radical exposure. We conclude that prenatal TCDD can predispose for mammary cancer susceptibility in the adult offspring by altering the mammary proteome.     

Equine estrogen-induced mammary tumors in rats

Long-term hormone replacement therapy is associated with an increased risk of breast, ovarian and endometrial cancers in women. Equine estrogens are a principal component of hormone replacement therapy; however, their tumorigenic potential toward mammary tissue and reproductive organs has not been extensively explored. A pellet containing equilin was inserted under the skin of female ACI rats and the development of mammary tumors was monitored. Histological examination revealed premalignant lesions such as apocrine metaplasia in whole-mount preparations of mammary gland from the equilin-treated rats. ACI rats given 10 mg equilin developed palpable mammary tumors at 13 weeks of treatment, and 37.5% of the rats developed mammary tumors within 15 weeks. For 2.5 mg equilin, palpable tumors were observed in 8.3% of the rats after 8 weeks’ treatment; the frequency was lower than that (42.9%) observed with 2.5 mg E₂. No tumors were observed in the untreated rats. Evidently, equilin is a mammary carcinogen, and this potential may be associated with development of breast and reproductive cancers in women receiving hormone replacement therapy.     

The role of metalloproteinases and their inhibitors in regulating mammary epithelial morphology and function in vivo

Summary An intact basement membrane (BM) is essential for the proper function, differentiation and morphology of many epithelial cells. The disruption or loss of this BM occurs during normal development as well as in the disease state. To examine the importance of the BM during mammary gland development in vivo, we generated transgenic mice which inappropriately express autoactivating isoforms of the matrix metalloproteinase, stromelysin-1. The mammary glands from these mice are both functionally and morphologically altered throughout development. The mammary glands from virgin transgenic mice had supernumerary branches and showed precocious development of alveoli that expressed -casein. During midpregnancy, the alveolar structures were collapsed and mouse mammary epithelial cells underwent apoptosis. We have now documented a dramatic incidence of breast tumors in several independent lines of these mice. These data suggest that overexpression of SL-1 and disruption of the BM may play a key role in mammary gland branching morphogenesis, apoptosis and breast cancer induction and progression.     

HCG hastens both the development of mammary carcinoma and the metastatization of HCG/LH and ERBB-2 receptor-positive cells in mice

Breast cancer is more frequent in human nulliparae, whereas its incidence is reduced by early fullterm pregnancy. Rodent studies suggest that chorionic gonadotropin secretion during pregnancy affords protection by inducing breast structure differentiation. Opposite effects, however, have been observed in cancer prone transgenic mice overexpressing the β subunit of chorionic gonadotropin or pituitary luteinic hormone (LH). Here we assessed the effect of administration of human chorionic gonadotropin (hCG) for 21 days (corresponding to the duration of a mouse pregnancy) in virgin female mice transgenic for the activated rat (r-) ERBB-2 oncogene (BALB-neuT). In these mice, the onset of atypical mammary duct hyperplasia and its progression towards multiple mammary carcinomas is accelerated by hCG. hCG enhances the in vitro proliferation and in vivo metastatization of tumor cells from a BALB-neuT mammary tumor expressing the hCG/LH as well as the ERBB-2 receptors. These findings suggest that hCG favours the growth and progression of hCG/LH and ERBB-2 receptor-positive breast tumors.