Evaluation of an imaging-based in vitro screening platform for estrogenic activity with OECD reference chemicals.

Estrogen receptor alpha (ERα) is often a primary target of endocrine disrupting chemicals (EDCs) and therefore several biochemical and cell-based assays for the detection of chemicals with estrogenic properties have been developed in the past. However, the current approaches are not suitable for the monitoring of pathway activation dynamics, and they are mostly based on expression constructs that lack physiological promoter regulation. We recently developed MCF7 fluorescent reporter cell lines of 3 different green fluorescent protein (GFP)-tagged ERα target genes: GREB1, PGR and TFF1. These reporters are under control of the full physiological promoter region and allow the monitoring of dynamic pro-proliferative pathway activation on a single cell level using a live-cell imaging set-up. In this study, we systematically characterized the response of these reporters to a full reference compound set of known estrogenic and non-estrogenic chemicals as defined by the Organization for Economic Co-Operation and Development (OECD). We linked activation of the pro-proliferative ERα pathway to a potential adverse outcome by additionally monitoring cell cycle progression and proliferation. The correct classification of the OECD reference compounds showed that our reporter platform has the same sensitivity and specificity as other validated artificial ERα pathway reporters, such as the ERα CALUX and VM7 Luc ER TA assay. By monitoring several key events (i.e. ER target activation, cell cycle progression and proliferation), and subsequently determining Point-of-Departure (POD) values, our reporter panel can be used in high-throughput testing for a physiologically more relevant, quantitative temporal endocrine modulation analysis to improve human carcinogen risk assessment.

Comparative study of estrogenic activities of phytoestrogens using OECD in vitro and in vivo testing methods.

With growing scientific interest in phytoestrogens, a number of studies have investigated the estrogenic potential of phytoestrogens in a wide variety of assay systems. However, evaluations of individual phytoestrogens with different assay systems make it difficult for predicting their relative estrogenic potency. The objective of this study was to compare estrogenic properties of fifteen known phytoestrogens using an estrogen receptor-α (ER-α) dimerization assay and Organization for Economic Cooperation and Development (OECD) standardized methods including in vitro estrogen receptor (ER) transactivation assay using VM7Luc4E2 cells and in vivo uterotrophic assay using an immature rat model. Human ER-α dimerization assay showed positive responses of eight test compounds and negative responses of seven compounds. These results were consistently found in luciferase reporter assay results for evaluating ER transactivation ability. Seven test compounds exhibiting relatively higher in vitro estrogenic activities were subjected to uterotrophic bioassays. Significant increases in uterine weights were only found after treatments with biochanin A, 8-prenylnaringenin, and coumestrol. Importantly, their uterotrophic effects were lost when animals were co-treated with antagonist of ER, indicating their ER-dependent effects in the uterus. In addition, analysis of estrogen responsive genes revealed that these phytoestrogens regulated uterine gene expressions differently compared to estrogens. Test methods used in this study provided a high consistency between in vitro and in vivo results. Thus, they could be used as effective screening tools for phytoestrogens, particularly focusing on their interactions with ER-α.

An In vitro dimerization assay for the adverse outcome pathway approach in risk assessment of human estrogen receptor α-mediated endocrine-disrupting chemicals.

The adverse outcome pathway (AOP) has been recently proposed as an effective framework for chemical risk assessment. The AOP framework offers the advantage of effectively integrating individual in vitro studies and in silico prediction models. Thus, the development of an effective testing method to measure key events caused by chemicals is essential for chemical risk assessment through a fully developed AOP framework. We developed a human cell-based estrogen receptor α (ERα) dimerization assay using the bioluminescence resonance energy transfer (BRET) technique and evaluated the ERα dimerization activities of 72 chemicals. Fifty-one chemicals were identified to mediate dimerization of ERα, and the BRET-based ERα dimerization assay could effectively measure the events that mediated dimerization of ERα by the estrogenic chemicals. These results were compared with the results of pre-existing assay to determine whether the BRET-based ERα dimerization assay could be employed as an in vitro test method to provide scientific information for explaining key events as a part of the AOP framework. Consequently, we propose that the BRET-based ERα dimerization assay is suitable for measuring the chemical-mediated dimerization of ERα, a key event in the AOP framework for cellular-level risk assessment of estrogenic chemicals.

Protein druggability assessment for natural products using in silico simulation: A case study with estrogen receptor and the flavonoid genistein.

Traditional herbal medicine (THM) comprises a vast number of natural compounds. Most of them are metabolized into different structures after administration, which makes the clarification of THM's mode of action more complicated. To evaluate the biological activities of those components and metabolites, in silico simulation technology is helpful. We focused on mixed-solvent molecular dynamics (MD) simulation for druggability assessment of natural products. Mixed-solvent MD is an in silico simulation method for the exploration of ligand-binding sites on target proteins, which uses water and an organic molecule mixture. The selection of organic small molecules is an important factor for predicting the characteristics of natural products. In this study, we used the known crystal structure of estrogen receptors with genistein as a test case and explored fragments reflecting the characteristics of natural products. We found that structures with a 4-pyrone structure are more often included in the natural products database compared with the DrugBank database, and we selectively detected the known-binding sites of estrogen receptor α and ß. The results indicate that the 4-pyrone structure might be promising for predicting the protein druggability of flavonoids. Additionally, mixed-solvent MD simulation discriminates the selectivity of genistein between estrogen receptor ß and α, indicating that the simulation can be evaluated using indices that differ from those of traditional ligand docking. Although this approach is still in its early stages, it has the potential to provide valuable information for understanding the diverse biological activities of natural products.

Cyclic depsipeptide mycotoxin exposure may cause human endocrine disruption: Evidence from OECD in vitro stably transfected transcriptional activation assays.

The presence of cyclic depsipeptide mycotoxins in foods and feedstuffs could potentially cause endocrine disrupting effects on humans and wildlife by their inhibition of active steroidogenesis. Therefore, we attempted to assess the human estrogen receptor (ER) and androgen receptor (AR) agonistic/antagonistic effects of representative cyclic depsipeptide mycotoxins, enniatin A1 (ENN A1), and enniatin B1 (ENN B1), by OECD Performand Based Test Guideline (PBTG) No.455, VM7Luc ER transcriptional activation (TA) assay and OECD TG No. 458, 22Rv1/MMTV_GR-KO AR TA assay. No tested cyclic depsipeptide mycotoxins were found to be ER and AR agonists in VM7Luc ER TA and 22Rv1/MMTV_GR-KO AR TA assays. On the other hand, ENN A1, and ENN B1 exhibited the ER and AR antagonistic effects with IC30 and IC50 values in both TA assays. These two cyclic depsipeptide mycotoxins, which were determined as ER and AR antagonists by two in vitro assays, bound to ERα, and AR. Then ENN A1, and ENN B1 inhibited the dimerization of ERα, and AR. These results, for the first time indicated that ENN A1, and ENN B1 could have potential endocrine disrupting effects mediated by interaction of ERα and AR using international standard testing methods to determine the potential endocrine disrupting chemical.

Race, Ethnicity, and Clinical Outcomes in Hormone Receptor-Positive, HER2-Negative, Node-Negative Breast Cancer in the Randomized TAILORx Trial.

BACKGROUND: Black race is associated with worse outcomes in early breast cancer. We evaluated clinicopathologic characteristics, the 21-gene recurrence score (RS), treatment delivered, and clinical outcomes by race and ethnicity among women who participated in the Trial Assigning Individualized Options for Treatment. METHODS: The association between clinical outcomes and race (White, Black, Asian, other or unknown) and ethnicity (Hispanic vs non-Hispanic) was examined using proportional hazards models. All P values are 2-sided. RESULTS: Of 9719 eligible women with hormone receptor-positive, HER2-negative, node-negative breast cancer, there were 8189 (84.3%) Whites, 693 (7.1%) Blacks, 405 (4.2%) Asians, and 432 (4.4%) with other or unknown race. Regarding ethnicity, 889 (9.1%) were Hispanic. There were no substantial differences in RS or ESR1, PGR, or HER2 RNA expression by race or ethnicity. After adjustment for other covariates, compared with White race, Black race was associated with higher distant recurrence rates (hazard ratio [HR] = 1.60, 95% confidence intervals [CI] = 1.07 to 2.41) and worse overall survival in the RS 11-25 cohort (HR = 1.51, 95% CI = 1.06 to 2.15) and entire population (HR = 1.41, 95% CI = 1.05 to 1.90). Hispanic ethnicity and Asian race were associated with better outcomes. There was no evidence of chemotherapy benefit for any racial or ethnic group in those with a RS of 11-25. CONCLUSIONS: Black women had worse clinical outcomes despite similar 21-gene assay RS results and comparable systemic therapy in the Trial Assigning Individualized Options for Treatment. Similar to Whites, Black women did not benefit from adjuvant chemotherapy if the 21-gene RS was 11-25. Further research is required to elucidate the basis for this racial disparity in prognosis.

Accurate assessment of estrogen receptor profiles in non-functioning pituitary adenomas using RT-digital PCR and immunohistochemistry.

BACKGROUND: Non-functioning pituitary adenomas (NFPAs) are common pituitary tumors, and surgery is generally the only treatment option. Few attempts have been made to explore target molecules for the development of NFPA pharmacological treatments. METHOD: We quantitatively assessed the expression profiles of estrogen receptor (ER) transcripts and proteins in NFPA samples, using reverse transcription-digital polymerase chain reaction (RT-dPCR) and immunohistochemistry, and further investigated the correlations between the expression levels of ER and those of downstream responsive genes. All patients had undergone surgery at the same high-volume hospital. A total of 20 patients with NFPAs were included. All patients were new-onset, and none were diagnosed with intratumoral hemorrhages or cysts. RESULTS: NFPA samples exhibited a bimodal ESR1 expression pattern and were categorized into significantly different high- and low-ESR1 expression level groups (P < 0.05). In contrast, expression levels of ESR1 variants and ESR2 could barely be detected. Similar results were obtained through the immunohistochemical staining of NFPAs, using well-validated antibodies against ERs. The expression levels of ESR1 positively correlated with those of GREB1, an estrogen-responsive gene [correlation coefficient (r) = 0.623, P = 0.003]. CONCLUSIONS: ESR1 expression levels in NFPAs exhibited a bimodal pattern and were positively correlated with GREB1 expression levels. The accurate assessment of ER expression levels may further advance future NFPA-related research.

Racial differences in the relationship between surgical choice and subsequent patient-reported satisfaction outcomes among women with early-stage hormone-positive breast cancer.

PURPOSE: The last fifteen years has seen a rising proportion of women who are eligible for breast conserving therapy (BCT) choosing mastectomy despite equivalent survival in early-stage breast cancer. We aim to explore potential racial differences in the association of surgical choice with subsequent patient-reported satisfaction outcomes. METHODS: Women who were within one year of diagnosis with hormone receptor (HR)-positive breast cancer were asked the Short Version of Patient Satisfaction Questionnaire (PSQ-18), which assesses their overall satisfaction with their medical care. We conducted bivariate analyses, including paired t-tests, to clarify differences in these patient-reported factors by surgical choice and race. Multivariable linear regression models were used to adjust for clinical and demographic control variables. RESULTS: For the sample of 279 women who underwent definitive surgery, women who received a mastectomy had lower levels of overall satisfaction, 71 vs. 75 (out of 90) (p = .001). In stratifying this relationship by race, the difference in total satisfaction score was largest among Black women (69 among mastectomy patients vs. 75 among BCT patients; p = 0.016). On multivariable linear regression, Black race and mastectomy status (together) exhibited a significantly large negative association with total satisfaction score, with negative associations across all domains of the PSQ-18. CONCLUSION: Despite the high prevalence of mastectomy among Black women with early-stage, HR-positive breast cancer, this population is more likely to report lower levels of patient satisfaction compared to patients receiving BCT. These findings suggest there may be potential racial differences in the psychosocial consequences of surgical choice.

Cost-effectiveness analyses demonstrate that observation is superior to sentinel lymph node biopsy for postmenopausal women with HR + breast cancer and negative axillary ultrasound.

PURPOSE: To evaluate the cost-effectiveness of axillary observation versus sentinel lymph node biopsy (SLNB) after negative axillary ultrasound (AUS). In patients with clinical T1-T2 N0 breast cancer and negative AUS, SLNB is the current standard of care for axillary staging. However, SLNB is costly, invasive, decreasing in importance for medical decision-making, and is not considered therapeutic. Observation alone is currently being evaluated in randomized clinical trials, and is thought to be non-inferior to SLNB for patients with negative AUS. METHODS: We performed cost-effectiveness analyses of observation versus SLNB after negative AUS in postmenopausal women with clinical T1-T2 N0, HR+/HER2- breast cancer. Costs at the 2016 price level were evaluated from a third-party commercial payer perspective using the MarketScan® Database. We compared cost, quality-adjusted life years (QALYs), and net monetary benefit (NMB). Multiple sensitivity analyses varying baseline probabilities, costs, utilities, and willingness-to-pay thresholds were performed. RESULTS: Observation was superior to SLNB for patients with N0 and N1 disease, and for the entire patient population (NMB in US$: $655,659 for observation versus $641,778 for SLNB for the entire patient population). In the N0 and N1 groups, observation incurred lower cost and was associated with greater QALYs. SLNB was superior for patients with > 3 positive lymph nodes, representing approximately 5% of the population. Sensitivity analyses consistently demonstrated that observation is the optimal strategy for AUS-negative patients. CONCLUSION: Considering both cost and effectiveness, observation is superior to SLNB in postmenopausal women with cT1-T2 N0, HR+/HER2- breast cancer and negative AUS.

Oncotype DX Predictive Nomogram for Recurrence Score Output: The Novel System ADAPTED01 Based on Quantitative Immunochemistry Analysis.

PURPOSE: Oncotype DX (ODX) predicts breast cancer recurrence risk, guiding the choice of adjuvant treatment. In many countries, access to the test is not always available. We used correlation between phenotypical tumor characteristics, quantitative classical immunohistochemistry (IHC), and recurrence score (RS) assessed by ODX to develop a decision supporting system for clinical use. PATIENTS AND METHODS: Breast cancer patients who underwent ODX testing between 2014 and 2018 were retrospectively included in the study. The data selected for analysis were age, menopausal status, and pathologic and IHC features. IHC was performed with standardized quantitative methods. The data set was split into two subsets: 70% for the training set and 30% for the internal validation set. Statistically significant features were included in logistic models to predict RS ≤ 25 or ≤ 20. Another set was used for external validation to test reproducibility of prediction models. RESULTS: The internal set included 407 patients. Mean (range) age was 53.7 (31-80) years, and 222 patients (54.55%) were > 50 years old. ODX results showed 67 patients (16.6%) had RS between 0 and 10, 272 patients between 11 and 25 (66.8%), and 68 patients > 26 (16.6%). Logistic regression analysis showed that RS score (for threshold ≤ 25) was significantly associated with estrogen receptor (P = .004), progesterone receptor (P < .0001), and Ki-67 (P < .0001). Generalized linear regression resulted in a model that had an area under the receiver operating characteristic curve (AUC) of 92.2 (sensitivity 84.2%, specificity 80.1%) and that was well calibrated. The external validation set (183 patients) analysis confirmed the model performance, with an AUC of 82.3 and a positive predictive value of 91%. A nomogram was generated for further prospective evaluation to predict RS ≤ 25. CONCLUSION: RS was related to quantitative IHC in patients with RS ≤ 25, with a good performance of the statistical model in both internal and external validation. A nomogram for enhancing clinical approach in a cost-effective manner was developed. Prospective studies must test this application in clinical practice.

Hypothalamic estrogen receptor alpha establishes a sexually dimorphic regulatory node of energy expenditure.

Estrogen receptor a (ERa) signaling in the ventromedial hypothalamus (VMH) contributes to energy homeostasis by modulating physical activity and thermogenesis. However, the precise neuronal populations involved remain undefined. Here, we describe six neuronal populations in the mouse VMH by using single-cell RNA transcriptomics and in situ hybridization. ERa is enriched in populations showing sex biased expression of reprimo (Rprm), tachykinin 1 (Tac1), and prodynorphin (Pdyn). Female biased expression of Tac1 and Rprm is patterned by ERa-dependent repression during male development, whereas male biased expression of Pdyn is maintained by circulating testicular hormone in adulthood. Chemogenetic activation of ERa positive VMH neurons stimulates heat generation and movement in both sexes. However, silencing Rprm gene function increases core temperature selectively in females and ectopic Rprm expression in males is associated with reduced core temperature. Together these findings reveal a role for Rprm in temperature regulation and ERa in the masculinization of neuron populations that underlie energy expenditure.

Male breast cancer: a closer look at patient and tumor characteristics and factors that affect survival using the National Cancer Database.

OBJECTIVE: To comprehensively describe the tumor and clinical characteristics of breast cancer in a cohort of male patients and to assess the factors that affect survival. BACKGROUND: Much of the standard care of male breast cancer is based on the diagnosis and treatment strategies of female breast cancer. However, important clinical differences between the two have been elucidated, which suggests the need for unique attention to male breast cancer. METHODS: We evaluated the records of male patients who were diagnosed with breast cancer between 2004 and 2015 using the National Cancer Database (NCDB). Data obtained were demographic characteristics, clinical and tumor data, type of therapy, as well as survival data. We used descriptive statistics to characterize our study population. We then performed a survival and Cox proportional hazards analysis. RESULTS: We identified 16,498 patients (median age: 63 years). Several treatment modalities were used, of which surgery was the most common (14,882 [90.4%]). The total follow-up time was 13 years (156 months). Five-year survival was 77.7% (95% CI 76.9-78.4) and 10-year survival was 60.7%. In a Cox proportional hazards model, mastectomy was associated with the greatest survival (hazard ratio [HR] 0.49; p < 0.001). CONCLUSION: We report what is to our knowledge the largest national population-based cohort of male breast cancer patients. Importantly, our data suggests that similar to female patients, several treatment modalities are significantly associated with improved survival in male patients, particularly surgery. Increasing age, black race, government insurance, more comorbidities, and higher tumor stages are associated with decreased survival.

Associations between breast cancer subtype and neighborhood socioeconomic and racial composition among Black and White women.

PURPOSE: Studies of Black-White differences in breast cancer subtype often emphasize potential ancestry-associated genetic or lifestyle risk factors without fully considering how the social or economic implications of race in the U.S. may influence risk. We assess whether neighborhood racial composition and/or socioeconomic status are associated with odds of triple-negative breast cancer (TNBC) diagnosis relative to the less-aggressive hormone receptor-positive/HER2-negative subtype (HR+ /HER-), and whether the observed relationships vary across women's race and age groups. METHODS: We use multilevel generalized estimating equation models to evaluate odds of TNBC vs. HR+ /HER2- subtypes in a population-based cohort of 7291 Black and 74,208 White women diagnosed with breast cancer from 2006 to 2014. Final models include both neighborhood-level variables, adjusting for individual demographics and tumor characteristics. RESULTS: Relative to the HR+ /HER- subtype, we found modestly lower odds of TNBC subtype among White women with higher neighborhood median household income (statistically significant within the 45-64 age group, OR = 0.981 per $10,000 increase). Among Black women, both higher neighborhood income and higher percentages of Black neighborhood residents were associated with lower odds of TNBC relative to HR+ /HER2-. The largest reduction was observed among Black women diagnosed at age ≥ 65 (OR = 0.938 per $10,000 increase; OR = 0.942 per 10% increase in Black residents). CONCLUSION: The relationships between neighborhood composition, neighborhood socioeconomic status, and odds of TNBC differ by race and age. Racially patterned social factors warrant further exploration in breast cancer subtype disparities research.

Racial Differences in the Association Between Luminal Master Regulator Gene Expression Levels and Breast Cancer Survival.

PURPOSE: Compared with their European American (EA) counterparts, African American (AA) women are more likely to die from breast cancer in the United States. This disparity is greatest in hormone receptor-positive subtypes. Here we uncover biological factors underlying this disparity by comparing functional expression and prognostic significance of master transcriptional regulators of luminal differentiation. EXPERIMENTAL DESIGN: Data and biospecimens from 262 AA and 293 EA patients diagnosed with breast cancer from 2001 to 2010 at a major medical center were analyzed by IHC for functional biomarkers of luminal differentiation, including estrogen receptor (ESR1) and its pioneer factors, FOXA1 and GATA3. Integrated comparison of protein levels with network-level gene expression analysis uncovered predictive correlations with race and survival. RESULTS: Univariate or multivariate HRs for overall survival, estimated from digital IHC scoring of nuclear antigen, show distinct differences in the magnitude and significance of these biomarkers to predict survival based on race: ESR1 [EA HR = 0.47; 95% confidence interval (CI), 0.31-0.72 and AA HR = 0.77; 95% CI, 0.48-1.18]; FOXA1 (EA HR = 0.38; 95% CI, 0.23-0.63 and AA HR = 0.53; 95% CI, 0.31-0.88), and GATA3 (EA HR = 0.36; 95% CI, 0.23-0.56; AA HR = 0.57; CI, 0.56-1.4). In addition, we identify genes in the downstream regulons of these biomarkers highly correlated with race and survival. CONCLUSIONS: Even within clinically homogeneous tumor groups, regulatory networks that drive mammary luminal differentiation reveal race-specific differences in their association with clinical outcome. Understanding these biomarkers and their downstream regulons will elucidate the intrinsic mechanisms that drive racial disparities in breast cancer survival.

Pharmacological, Mechanistic, and Pharmacokinetic Assessment of Novel Melatonin-Tamoxifen Drug Conjugates as Breast Cancer Drugs.

Tamoxifen is used to prevent and treat estrogen receptor-positive (ER+) breast cancer (BC); however, its chronic use can increase uterine cancer risk and induce tamoxifen resistance. Novel melatonin-tamoxifen drug conjugates may be promising to treat BC and may help offset the adverse effects of tamoxifen usage alone due to the presence of melatonin. We synthesized and screened five drug conjugates (C2, C4, C5, C9, and C15 linked) for their effects on BC cell (MCF-7, tamoxifen-resistant MCF-7, mouse mammary carcinoma, MDA-MB-231, and BT-549) viability, migration, and binding affinity to melatonin receptor 1 (MT1R) and estrogen receptor 1 (ESR1). C4 and C5 demonstrated the most favorable pharmacological characteristics with respect to binding profiles (affinity for ESR1 and MT1R) and their potency/efficacy to inhibit BC cell viability and migration in four phenotypically diverse invasive ductal BC cell lines. C4 and C5 were further assessed for their actions against tamoxifen-resistant MCF-7 cells and a patient-derived xenograft triple-negative BC cell line (TU-BcX-4IC) and for their mechanisms of action using selective mitogen-activated protein kinase kinase MEK1/2, MEK5, and phosphoinositide 3-kinase (PI3K) inhibitors. C4 and C5 inhibited tamoxifen-resistant MCF-7 cells with equal potency (IC50 = 4-8 µM) and efficacy (∼90% inhibition of viability and migration) but demonstrated increased potency (IC50 = 80-211 µM) and efficacy (∼140% inhibition) to inhibit migration versus cell viability (IC50 = 181-304 mM; efficacy ∼80% inhibition) in TU-BcX-4IC cells. Unique pharmacokinetic profiles were observed, with C4 having greater bioavailability than C5. Further assessment of C4 and C5 demonstrates that they create novel pharmacophores within each BC cell that is context specific and involves MEK1/2/pERK1/2, MEK5/pERK5, PI3K, and nuclear factor κB. These melatonin-tamoxifen drug conjugates show promise as novel anticancer drugs and further preclinical and clinical evaluation is warranted.

Use of in vitro bioassays to facilitate read-across assessment of nitrogen substituted heterocycle analogues of polycyclic aromatic hydrocarbons.

Nitrogen-containing polycyclic aromatic hydrocarbons (PANHs or azaarenes) are compounds structurally similar to PAHs (carbon substituted by a nitrogen) reported to occur at low levels in food. Although limited, literature may suggest possible higher toxicity than for PAHs. Using a battery of in vitro assays, the toxicological properties of uncharacterized PANHs of increasing ring number were compared to those of characterized structural PAH analogues. The parameters measured covered key events relevant to the AOP developed for Benzo(a)pyrene: AhR activation, mutagenicity and DNA-damage with and without metabolic activation and endocrine receptors activation/inhibition. There was a strong correlation between the chemical structure and the biological activities of the compounds. AhR activation was the most sensitive parameter with a direct correlation between potency and ring number. The most potent genotoxic chemicals were found amongst the ones with the highest number of ring, and under metabolic activation. Such an approach allowed designing sub-groups based on biological properties in addition to structural similarities. Within a sub-group, toxicological data of tested chemicals may be used to characterize hazard of biologically similar but toxicologically uncharacterized substances. This indicates that in addition to structural properties, in vitro biological data may be useful to conduct read-across.

Assessment of human estrogen receptor agonistic/antagonistic effects of veterinary drugs used for livestock and farmed fish by OECD in vitro stably transfected transcriptional activation assays.

The presence of veterinary drug residues in foods and the environment could potentially cause adverse effects on humans and wildlife. Several veterinary drugs were reported to exhibit endocrine disrupting effects via binding affinities to sexual hormone receptors such as estrogen and androgen receptors. Therefore, we confirmed the human estrogen receptor (ER) agonistic/antagonistic effects of 135 chemicals that were used as veterinary drugs in Korea by the official Organization for Economic Cooperation and Development (OECD) in vitro ER transcriptional activation (TA) assay using the VM7Luc4E2 cell line. In the case of ER agonist screening, 7 veterinary drugs (cefuroxime, cymiazole, trenbolone, zeranol, phoxim, altrenogest and nandrolone) were determined to be ER agonists. In addition, only zeranol was found to exhibit weak ER antagonistic activity. These 7 veterinary drugs, which were determined as ER agonists and/or antagonists by an OECD in vitro assay, were also found to have binding affinity to ERs. These results indicate that various veterinary drugs possess potential (anti-)estrogenic effects. However, further study is needed to determine the precise endocrine-disrupting effects of these compounds.

Activation of hepatic estrogen receptor-α increases energy expenditure by stimulating the production of fibroblast growth factor 21 in female mice.

OBJECTIVE: The endogenous estrogen 17ß-estradiol (E2) promotes metabolic homeostasis in premenopausal women. In a mouse model of post-menopausal metabolic syndrome, we reported that estrogens increased energy expenditure, thus preventing estrogen deficiency-induced adiposity. Estrogens' prevention of fat accumulation was associated with increased serum concentrations of fibroblast growth factor 21 (FGF21), suggesting that FGF21 participates in estrogens' promotion of energy expenditure. METHODS: We studied the effect of E2 on FGF21 production and the role of FGF21 in E2 stimulation of energy expenditure and prevention of adiposity, using female estrogen receptor (ER)- and FGF21-deficient mice fed a normal chow and a cohort of ovariectomized women from the French E3N prospective cohort study. RESULTS: E2 acting on the hepatocyte ERα increases hepatic expression and production of FGF21 in female mice. In vivo activation of ERα increases the transcription of Fgf21 via an estrogen response element outside the promoter of Fgf21. Treatment with E2 increases oxygen consumption and energy expenditure and prevents whole body fat accumulation in ovariectomized female WT mice. The effect of E2 on energy expenditure is not observed in FGF21-deficient mice. While E2 treatment still prevents fat accumulation in FGF21-deficient mice, this effect is decreased compared to WT mice. In an observational cohort of ovariectomized women, E2 treatment was associated with lower serum FGF21 concentrations, which may reflect a healthier metabolic profile. CONCLUSIONS: In female mice, E2 action on the hepatocyte ERα increases Fgf21 transcription and FGF21 production, thus promoting energy expenditure and partially decreasing fat accumulation.

Assessment of morphological changes and steroid receptors in the uteri of postmenopausal women.

INTRODUCTION: The morphology of the endometrium constantly changes in the reproductive period, depending on the levels of ovarian steroid hormones, and undergoes atrophic changes during menopause as a result of their insufficiency. The purpose of this study was to analyze morphological and morphometric changes in the mucous and muscle layers of the uterine wall in postmenopausal women, and to assess localization and number of cells showing the expression of steroid hormone receptors, namely estrogen receptor α (ER-α), progesterone receptor (PR), and androgen receptor (AR) in glandular epithelial cells and smooth muscle cells in particular groups of women. MATERIAL AND METHODS: The study material consisted of uterine specimens sectioned across the full thickness of the uterine wall, and embedded in 164 paraffin blocks. The specimens came from women without menopausal hormone therapy (MHT) operated due to reproductive organ prolapse or uterine myomas. The material was divided into four groups depending on the time interval from menopause to surgery: group I - from 1 to 5 years after menopause, group II - from 6 to 10 years after menopause, group III - more than 11 years after menopause, and group IV - women over 70 years of age. The sections were stained by standard HE, Masson's trichrome, and immunohistochemical methods (ERα, PR, AR). Quantitative assessment of the results was based on computer image analysis. RESULTS: Analysis of morphological changes in the endometrium and myometrium revealed the presence of increasing regressive changes, such as various types of atrophy, fibrosis, and calcification, augmented over time from the last menstruation. Furthermore, endometrial polyps, foci of endometriosis, and leiomyomas were observed. Based on the results of morphometric measurements, a constant decrease in the endometrial and myometrial thickness was noticed in the studied groups (I-IV). Significant differences between the groups were observed in the number of ER-α positive cells in the myometrium, but not in the endometrial glandular epithelium. Statistically significant differences in the number of AR positive cells were detected in the endometrial epithelium and in the uterine muscle. The analysis the number of PR positive cells demonstrated differences between the groups in the endometrial stroma and the myometrium. CONCLUSION: The uterus of postmenopausal woman undergo major morphological changes (mainly atrophic lesions in the endometrium and myometrium), leading to a decline in their morphometric parameters over time from the last menstruation. Localization and number of cells showing the expression of steroid receptors: ER-α, PR, and AR in the uterus of postmenopausal women, depending on the time interval from the last menstruation.

N-terminal transactivation function, AF-1, of estrogen receptor alpha controls obesity through enhancement of energy expenditure.

OBJECTIVE: Studies using the estrogen receptor alpha (ERα) knock-out (αERKO) mice have demonstrated that ERα plays a crucial role in various estrogen-mediated metabolic regulations. ERα is a ligand dependent transcription regulator and its activity is regulated by estrogenic compounds. ERα consists of two transcriptional activation domains, AF-1 and AF-2. The activities of these domains are regulated through different mechanisms; however, the specific physiological role in metabolic regulation by these domains is still unclear. METHODS: We utilized an ERα AF-2 mutant knock-in mouse (AF2ERKI) to evaluate the physiological functionality of ERα transactivation domains. Due to the estrogen insensitive AF-2 mutation, the phenotypes of AF2ERKI mice are seemingly identical to the global αERKO including obesity in the females. Distinct from the αERKO, the AF-1 function of AF2ERKI mice can be activated by tamoxifen (Tam). Ovariectomized (OVX) AF2ERKI and WT females were treated with Tam and fed a high-fat diet (HFD) for 10 weeks. Additionally, indirect calorimetric analysis was performed using metabolic chambers with food intake and locomotor activity recorded for Tam-treated AF2ERKI and αERKO females. RESULTS: Obesity in HFD-fed AF2ERKI females was prevented by Tam treatment; particularly, inguinal fat accumulation was strongly blocked by Tam treatment. Alterations in fat metabolism genes, however, were not found in either inguinal fat nor visceral fat to be Tam-regulated, even though fat accumulation was strongly reduced by Tam treatment. Indirect calorimetric analysis revealed that without alteration of food intake and locomotor activity Tam treatment increased energy expenditure in AF2ERKI but not αERKO females. CONCLUSIONS: These results suggest that the activation of ERα AF-1 prevents fat accumulation. The prevention of obesity through AF-1 is mediated by induction of energy expenditure rather than ERα AF-1 functionality of lipid metabolism gene regulation in fat tissues.