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Polybrominated diphenyl ethers (PBDEs) have been used as flame retardants in household materials. Their environmental persistence has led to continuous human exposure and significant tissue levels. Three PBDE congeners (BDE-47, BDE-100, and BDE-153) have been frequently detected in human serum. Although these compounds appear to possess endocrine disrupting activity, studies are largely missing to determine the biological mechanisms of PBDEs in breast cancer cells. Here, we assessed PBDE bioactivities with three complementary strategies: receptor binding/activity assays; nonbiased RNA-sequencing analysis using an estrogen-dependent breast cancer cell line MCF-7aroERE; and in vivo assessments using patient-derived xenograft (PDX) models of human breast cancer. According to the results from in vitro experiments, the PBDE congeners regulate distinct nuclear receptor signaling pathways. BDE-47 acts as a weak agonist of both estrogen receptor α (ERα) and estrogen-related receptor α (ERRα); it could stimulate proliferation of MCF-7aroERE and induced expression of ER-regulated genes (including cell cycle genes). BDE-153 was found to act as a weak antagonist of ERα. BDE-100 could act as (1) an agonist of aryl hydrocarbon receptor (AhR), inducing expression of CYP1A1 and CYP1B1 and (2) as a very weak agonist/antagonist of ERα. In vivo, a mixture of the three congeners with ratios detected in human serum was tested in an ER+ PDX model. The mixture exhibited estrogenic activity through apoptosis/cell cycle regulation and increased the expression of a proliferation marker, Ki-67. These results advance our understanding of the mechanisms of PBDE exposure in breast cancer cells., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Polycyclic aromatic hydrocarbons (PAHs) are a diverse group of widespread environmental pollutants, some of which have been found to be estrogenic or antiestrogenic. Recent data have shown that hydroxylated PAH metabolites may be responsible for the estrogenic effects of some PAHs. The purpose of this study was to investigate the effects of several PAHs, as well as their monohydroxylated metabolites, on estrogen receptors (ERs), ERα and ERβ. Three parent PAHs and their monohydroxylated metabolites were each evaluated using transcriptional reporter assays in isogenic stable cell lines to measure receptor activation, competitive binding assays to determine ligand binding, and bioluminescence resonance energy transfer assays to assess dimerization. Finally, the estrogenic effects of the hydroxylated metabolites were confirmed by quantitative real-time PCR of estrogen-responsive target genes. Although the parent PAHs did not induce ERα or ERβ transcriptional activity, all of the monohydroxylated PAHs (1-OH naphthanol, 9-OH phenanthrene, 1-OH pyrene) selectively induced ERβ transcriptional activity at the concentrations tested, while not activating ERα. Additionally, the monohydroxylated PAHs were able to competively bind ERβ, induce ERβ homodimers, and regulate ERβ target genes. Although monohydroxylated PAHs appeared to have weak agonist activity to ERβ, our results showed that they can elicit a biologically active response from ERβ in human breast cancer cells and potentially interfere with ERβ signaling pathways.

It has been demonstrated in our previous studies that Calbindin-D9k (CaBP-9k) is a potent biomarker for screening estrogen-like chemicals in the rat model. Although treatments with 17beta-estradiol (E2) and endocrine disrupting compounds resulted in the up-regulation of uterine CaBP-9k, the mechanism of CaBP-9k induction by these compounds through two subtypes of estrogen receptors (ERalpha and ERbeta) is unclear. Thus, in the present study, immature rats were treated with propyl pyrazole triol (PPT, an ERalpha-selective ligand), diarylpropionitrile (DPN, an ERbeta-selective ligand), E2, or dimethyl sulfoxide (DMSO, a vehicle control) for three days in order to clarify which subtype of ER is involved in the uterine CaBP-9k induction. Following injection with these ER ligands, uterine CaBP-9k expression was analyzed by Northern blot and immunoblot assays. Uterine CaBP-9k expression is mainly mediated by PPT in a dose- and time-dependent manner in immature rats, whereas no significant alteration of the uterine CaBP-9k gene was observed after DPN treatment. In addition, an estrogenicity of PPT in inducing CaBP-9k expression was completely blocked by the anti-estrogen ICI 182,780, implying that uterine CaBP-9k is solely induced by ERalpha. A single treatment with PPT rapidly increased the protein levels of ERalpha and PR, an E2-mediated gene, in these tissues. Taken together, these results indicate that uterine CaBP-9k is induced by E2 and endocrine disrupting chemicals via the ERalpha pathway, but not ERbeta, in the uterus of immature rats.

Estrogenic and thyroid activities of 91 monohydroxylated PCBs were measured with two-hybrid assays using yeast cells containing the human estrogen receptor ERalpha or human thyroid receptor TRalpha. Estrogenic activity of 30 of the 91 compounds, including all compounds active in the yeast two-hybrid assay, were also measured by a reporter gene assay employing Chinese hamster ovary cells. The mammalian cell assay was more sensitive than the yeast assay but the rank order of estrogenicities of the compounds were in broad agreement for the two assays. Results for estrogenicity and thyroid activity were analyzed by inspection and those for estrogenicity by a theoretical treatment. Inspection indicated para-hydroxyl was more likely to be estrogenically active than meta-, which was more likely to be active than ortho-; one ortho-chlorine was important for activity but additional ortho-chlorines did not increase activity; and 2 lateral chlorines or 2,4,6-chloro- substitution of the non-phenol ring were favorable. In contrast, thyroid activity appeared not to depend strongly on the position of the hydroxyl group although ortho-hydroxyls occurred in the most active compounds. Activity was usually associated with at least one ortho-chlorine, with 2 chlorines in the phenolic ring and, importantly, two chlorines in the non-phenolic ring, and with 1 or 2 chlorines ortho to the hydroxyl group. Examination of the torsion angle between the rings, in the theoretical examination of estrogenicity, suggested that perpendicular orientation (i.e., rigidity) was not essential for activity. Intramolecular hydrogen bonding of the phenolic groups to adjacent chlorines or to the pi-electron cloud of the non-phenol ring possibly decreased activity--the hydroxyl should be free of intramolecular interactions for maximum activity. It was difficult to predict the estrogenic activity of a congener from its obtained potential energy curve (PEC). In general, estrogenically active congeners had large values for the sum of the atomic charges on the carbon atoms of the hydroxylated ring, and on the oxygen atom. Hydroxyl substitution at the para-position allowed the compounds to become more polarizable in the x-axis (molecular axis), whereas OH substitution at the ortho-position made the congeners less polarizable in the same direction. However, no general statement about polarizability and estrogenic activity was possible.

While the pesticide methoxychlor (MXC) has a variety of adverse effects on the female reproductive system, the effects of MXC on the ovarian surface epithelium (OSE) are unknown. Thus, this study tested the hypothesis that MXC alters the growth of the OSE. Mouse OSE cells were isolated by enzymatic digestion and cultured with vehicle, 3 microM of MXC, or 3 microM of 2,2-bis[p-hydroxyphenyl]-1,1,1,-trichloroethane (HPTE) for 14 days. After culture, proliferation and apoptosis were assessed by measurement of cell density, immunohistochemistry, and real-time polymerase chain reaction. Cell density was 66% greater for MXC-treated cells and 95% greater for HPTE-treated cells than controls (p < or = 0.05). The estrogen receptor blocker ICI 182,780 abolished MXC- and HPTE-induced increases in cell density. Proliferating cell nuclear antigen (PCNA) staining was positive in only 22 +/- 2.3% of controls, compared to 35 +/- 2.4% of MXC-treated cells and 40 +/- 2.4% of HPTE-treated cells (p < or = 0.05). The cell cycle regulators, cyclinD2 and cdk4, were significantly increased in MXC- and HPTE-treated cells compared to controls. The ApopTag assay demonstrated apoptotic cells in 4.8 +/- 0.45% of controls, 2.2 +/- 0.56% of MXC-treated cells, and 2.1 +/- 0.33% of HPTE-treated cells (p < or = 0.005). Expression of bcl-2 was significantly increased in MXC- and HPTE-treated cells, while bax was decreased in MXC- and HPTE-treated cells compared to controls. Collectively, these data indicate that MXC and HPTE stimulate OSE cell growth by increasing proliferation and inhibiting apoptosis. Further, since ICI 182,780 blocked MXC- and HPTE-induced OSE growth, these data suggest that the effects of MXC and HPTE on the OSE are mediated by estrogen receptors.

Genistein, a phytoestrogen possessing a high affinity for estrogen receptor beta (ERbeta), is of increasing interest because of its possible influence on the physiology of mammalian reproductive tracts. Although estrogen has been demonstrated to regulate Calbindin-D9k (CaBP-9k) in the rat uterus as with other calcium binding proteins, the role of ERbeta on the modulation of CaBP-9k remains to be elucidated. To elucidate the effect of genistein as a selective ERbeta agonist on uterine expression of CaBP-9k mRNA and protein, immature female rats were injected with genistein daily for three consecutive days in a dose-dependent (0.4, 4, and 40 mg/kg/day) and time-dependent (40 mg/kg/day; 3, 6, 12, 24, 48, and 72 h) manner. Then, the expression of CaBP-9k mRNA and protein was analyzed by Northern hybridization and Western blot, respectively, in the absence or presence of ICI 182,780 (ICI), an estrogen antagonist. In addition, the protein levels of ERalpha and ERbeta and mRNA level of progesterone receptor (PR) were further measured following genistein treatment to elucidate which of ERs is involved in CaBP-9k modulation. In a dose-dependent experiment, the highest dose of genistein (40 mg/kg/day) for 3 days significantly induced uterine CaBP-9k protein as 17beta-estradiol (E2) did. In addition, its maximal mRNA expression was observed at 3 and 6 h, and it returned to control level at 24 h in a time-dependent experiment. In parallel with its mRNA level, the protein level of CaBP-9k was significantly induced by genistein at 3 h and sustained up to 48 h. The pretreatment with ICI, followed by genistein or E2, completely blocked genistein- and E2-induced CaBP-9k protein in the uterus of immature rats. Interestingly, genistein was demonstrated to induce ERalpha protein, but not ERbeta and PR mRNA, an E2-responsive gene, in this tissue. These results imply that genistein, an ERbeta ligand, may regulate CaBP-9k gene through ERalpha pathway. Taken together, the present study demonstrated that genistein enhanced CaBP-9k gene via ERalpha in the uterus of immature rats, suggesting that ERalpha may be a key mediator in uterine CaBP-9k gene induction in immature rats.

Recently several advisory committees (EDSTAC, ICCVAM) have recommended that stable estrogen-dependent gene expression assays be developed for screening chemicals for estrogenic activity because of the high degree of specificity of the response and potential for use in a high-throughput mode. In this paper we describe a specific, sensitive assay developed for screening chemicals for estrogenic and antiestrogenic activities. T47D human breast cancer cells, which naturally express estrogen receptor (ER) alpha and beta, were stably transfected with a triplet ERE (estrogen-responsive elements)-promoter-luciferase reporter gene construct. The transformed cells were named T47D-KBluc. These cells are sensitive to the potent estrogens, 17beta-estradiol, ethynyl estradiol, and diethylstibesterol, and well-characterized weaker environmental estrogens like genistein, HPTE (an estrogenic pesticide metabolite), and 4-nonylphenol. The EC50 for estradiol was about 0.01 nM, reaching maximal induction at 0.1 nM. The antiestrogen, ICI 182,780, was able to completely inhibit the induction of luciferase expression by 0.1 nM estradiol at 10 nM, with an IC50 of 1 nM. In addition, we were able to replicate, in this in vitro assay, the observation that low concentrations of cadmium were able to induce estrogen-dependent gene expression, an effect that was completely inhibited by the potent antiestrogen ICI 182,780. The potent glucocorticoid receptor agonist, dexamethasone, was without effect as an ER agonist at concentrations up to 10 nM, whereas the potent androgen, dihydrotestosterone (DHT), showed no induction at concentration of 50 microM, but was a partial agonist at high concentrations of 0.2 mM and above. In summary, we have developed a specific, sensitive estrogen-responsive gene expression assay in a stable cell line that could possibly be adapted for high throughput screening of large numbers of chemicals for estrogenic and antiestrogenic activity. In addition, herein we also provide key protocol recommendations necessary to identify and eliminate common problems encountered in in vitro screening for estrogenicity.

High oral doses of atrazine (ATRA) disrupt normal neuroendocrine function, resulting in suppression of the luteinizing hormone (LH) surge in adult, ovariectomized (OVX) estrogen-primed female rats. While the mechanism by which ATRA inhibits LH secretion is not known, current data indicate that ATRA does have anti-estrogenic properties in vitro and in vivo. In the body, ATRA is rapidly converted to diaminochlorotriazine (DACT). The present study was conducted to investigate the effects of ATRA and DACT on the estradiol benzoate (EB)/progesterone (P) induced LH surge and to determine if such changes correlate with impaired estrogen receptor (ER) function. ATRA, administered by gavage for five consecutive days to adult OVX, female Sprague-Dawley rats, caused a dose-dependent suppression of the EB/P induced LH surge. Although to a lesser degree than ATRA, DACT significantly suppressed total plasma LH and peak LH surge levels in EB/P primed animals by 60 and 58%, respectively. DACT treatment also decreased release of LH from the pituitary in response to exogenous gonadotropin releasing hormone (GnRH) by 47% compared to control. Total plasma LH secretion was reduced by 37% compared to control, suggesting that in addition to potential hypothalamic dysfunction, pituitary function is altered. To further investigate the mechanism by which hypothalamic function might be altered, potential anti-estrogenicity of ATRA and DACT were assessed by evaluating ER function treated rats. Using an in vitro receptor binding assay, ATRA, but not DACT, inhibited binding of [(3)H]-estradiol to ER. In contrast, ATRA, administered to female rats under dosing conditions which suppressed the LH surge, neither changed the levels of unoccupied ER nor altered the estrogen induced up-regulation of progesterone receptor mRNA. Collectively, these results indicate that although ATRA is capable of binding ER in vitro, the suppression of LH after treatment with high doses of ATRA is not due to alterations of hypothalamic ER function.