Assessment of estrogenic potential from exudates of microcystin-producing and non-microcystin-producing Microcystis by metabolomics, machine learning and E-screen assay.

Cyanobacterial blooms, often dominated by Microcystis aeruginosa, are capable of producing estrogenic effects. It is important to identify specific estrogenic compounds produced by cyanobacteria, though this can prove challenging owing to the complexity of exudate mixtures. In this study, we used untargeted metabolomics to compare components of exudates from microcystin-producing and non-microcystin-producing M. aeruginosa strains that differed with respect to their ability to produce microcystins, and across two growth phases. We identified 416 chemicals and found that the two strains produced similar components, mainly organoheterocyclic compounds (20.2%), organic acids and derivatives (17.3%), phenylpropanoids and polyketides (12.7%), benzenoids (12.0%), lipids and lipid-like molecules (11.5%), and organic oxygen compounds (10.1%). We then predicted estrogenic compounds from this group using random forest machine learning. Six compounds (daidzin, biochanin A, phenylethylamine, rhein, o-Cresol, and arbutin) belonging to phenylpropanoids and polyketides (3), benzenoids (2), and organic oxygen compound (1) were tested and exhibited estrogenic potency based upon the E-screen assay. This study confirmed that both Microcystis strains produce exudates that contain compounds with estrogenic properties, a growing concern in cyanobacteria management.

Deciphering the synergistic effects of photolysis and biofiltration to actuate elimination of estrogens in natural water matrix.

The presence of estrogens in water environments has raised concerns for human health and ecosystems balance. These substances possess potent estrogenic properties, causing severe disruptions in endocrine systems and leading to reproductive and developmental problems. Unfortunately, conventional treatment methods struggle to effectively remove estrogens and mitigate their effects, necessitating technological innovation. This study investigates the effectiveness of a novel sequential photolysis-granular activated carbon (GAC) sandwich biofiltration (GSBF) system in removing estrogens (E1, E2, E3, and EE2) and improving general water quality parameters. The results indicate that combining photolysis pre-treatment with GSBF consistently achieved satisfactory performance in terms of turbidity, dissolved organic carbon (DOC), UV254, and microbial reduction, with over 77.5 %, 80.2 %, 89.7 %, and 92 % reduction, respectively. Furthermore, this approach effectively controlled the growth of microbial biomass under UV irradiation, preventing excessive head loss. To assess estrogen removal, liquid chromatography-tandem mass spectrometry (LC-MS) measured their concentrations, while bioassays determined estrogenicity. The findings demonstrate that GSBF systems, with and without photolysis installation, achieved over 96.2 % removal for estrogens when the spike concentration of each targeted compound was 10 µg L-1, successfully reducing estrogenicity (EA/EA0) to levels below 0.05. Additionally, the study evaluated the impact of different thicknesses of GAC layer filling (8 cm, 16 cm, and 24 cm) and found no significant difference (p>0.05) in estrogen and estrogenicity removal among them.

A 4-year study of bovine reproductive hormones that are induced by pharmaceuticals and appear as steroid estrogenic pollutants in the resulting slurry, using in vitro and instrumental analytical methods.

The main objective of the research was to study the environmental "price" of the large-scale, milk production from a rarely known perspective, from the mapping of the estrogenic footprint (the amount of oestrus-inducer hormonal products, and the generated endoestrogens) in the resulting slurry in a dairy cow farm. These micropollutants are endocrine-disrupting chemicals (EDCs) and can be dangerous to the normal reproductive functions even at ng/kg concentration. One of them, 17ß-estradiol, has a 20,000 times stronger estrogenic effect than bisphenol-A, a widely known EDC of industrial origin. While most studies on EDCs are short-term and/or laboratory based, this study is longitudinal and field-based. We sampled the slurry pool on a quarterly basis between 2017 and 2020. Our purpose was testing the estrogenic effects using a dual approach. As an effect-based, holistic method, we developed and used the YES (yeast estrogen screen) test employing the genetically modified Saccharomyces cerevisiae BJ3505 strain which contains human estrogenic receptor. For testing exact molecules, UHPLC-FLD was used. Our study points out that slurry contains a growing amount of EDCs with the risk of penetrating into the soil, crops and the food chain. Considering the Green Chemistry concept, the most benign ways to prevent of the pollution of the slurry is choosing appropriate oestrus-inducing veterinary pharmaceuticals (OIVPs) and the separation of the solid and liquid parts with adequate treatment methods. To our knowledge, this is the first paper on the adaptation of the YES test for medicine and slurry samples, extending its applicability. The adapted YES test turned out to be a sensitive, robust and reliable method for testing samples with potential estrogenic effect. Our dual approach was successful in evaluating the estrogenic effect of the slurry samples.

[Sorption Characteristics and Site Energy Distribution Theory of Typical Estrogens on Microplastics].

Microplastics (MPs) and estrogens are high-profile emerging contaminants at present, and MPs might become the carrier of estrogens in the environment and induce combined pollution. To study the adsorption behavior of polyethylene (PE) microplastics to typical estrogens, the adsorption isothermal properties of the six estrogens[estrone (E1), 17α-estradiol (17α-E2), 17ß-estradiol (17ß-E2), estriol (E3), diethylstilbestrol (DES), and ethinylestradiol (17α-EE2)] in single-solute and mixed-solute systems were studied through batch equilibrium adsorption experiments, in which the PE microplastics before and after adsorption were characterized by X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared spectroscopy (FTIR). Then, the site energy distribution theory of the adsorption of six estrogens on PE microplastics was further analyzed based on the Freundlich model. The results showed that the adsorption process of selected estrogens with two concentrations (100 µg·L-1 and 1000 µg·L-1) on PE were more consistent with the pseudo-second order kinetic model. The increase in initial concentration reduced the equilibrium time of adsorption and increased the adsorbing capacity of estrogens on PE. In the single system (one estrogen) or mixed system (six estrogens) with different concentrations (10 µg·L-1-2000 µg·L-1), the Freundlich model showed the best fitting effect for the adsorption isotherm data (R2>0.94). The results of isothermal adsorption experiments and XPS and FTIR spectra showed that the adsorption of estrogens on PE in the two systems was heterogeneous adsorption, and hydrophobic distribution and van der Waals forces were the principal factors in the process of adsorption. The occurrence of C-O-C (in only the DES and 17α-EE2 systems) and O-C[FY=,1]O (in only the 17α-EE2 system) indicated that the adsorption of synthetic estrogens on PE was affected slightly by chemical bonding function, but no obvious effects were observed for natural estrogens. The results of site energy distribution analysis showed that, compared with the single system, the adsorption site energy of each estrogen shifted to the high-energy region in its entirety in the mixed system, and the site energy increased by 2.15%-40.98%. The energy change in DES was the most significant among all of the estrogens, indicating its competitive advantage in the mixed system. The above results of this study can provide some reference for the study of adsorption behavior, mechanism of action, and environmental risks under the coexisting condition of organic pollutants and MPs.

Synthetic ERRα/ß/γ Agonist Induces an ERRα-Dependent Acute Aerobic Exercise Response and Enhances Exercise Capacity.

Repetitive physical exercise induces physiological adaptations in skeletal muscle that improves exercise performance and is effective for the prevention and treatment of several diseases. Genetic evidence indicates that the orphan nuclear receptors estrogen receptor-related receptors (ERRs) play an important role in skeletal muscle exercise capacity. Three ERR subtypes exist (ERRα, ß, and γ), and although ERRß/γ agonists have been designed, there have been significant difficulties in designing compounds with ERRα agonist activity. Additionally, there are limited synthetic agonists that can be used to target ERRs in vivo. Here, we report the identification of a synthetic ERR pan agonist, SLU-PP-332, that targets all three ERRs but has the highest potency for ERRα. Additionally, SLU-PP-332 has sufficient pharmacokinetic properties to be used as an in vivo chemical tool. SLU-PP-332 increases mitochondrial function and cellular respiration in a skeletal muscle cell line. When administered to mice, SLU-PP-332 increased the type IIa oxidative skeletal muscle fibers and enhanced exercise endurance. We also observed that SLU-PP-332 induced an ERRα-specific acute aerobic exercise genetic program, and the ERRα activation was critical for enhancing exercise endurance in mice. These data indicate the feasibility of targeting ERRα for the development of compounds that act as exercise mimetics that may be effective in the treatment of numerous metabolic disorders and to improve muscle function in the aging.

Rapid Detection of Estrogens in Cosmetics by Chemical Derivatization and Paper-Spray Ionization Mass-Spectrometry.

Estrogens in personal care products are harmful to customers. Conventional methods such as HPLC and LC-MS require tedious sample pretreatment and long analytical time. Paper-spray ionization mass spectrometry (PSI-MS) is a powerful tool for the determination of compounds with little time and minimal pretreatment procedures. Since most estrogens show poor responses in PSI-MS, we developed a chemical derivatization and PSI-MS method to determinate three estrogens: estradiol, estriol and ethinyloestradiol with estradiol valerate as the internal standard (I.S.). After derivatization with 2-fluoro-1-methyl-pyridinium-p-toluene-sulfonate, the three estrogens could be quantified in seconds. This method showed good linearity in the range of 0.1~30 µg·mL-1, with R2 > 0.999. Their recovery results were all between 85%~115%. The limits of detection (LOD) were 0.04 µg·mL-1, 0.02 µg·mL-1 and 0.02 µg·mL-1 for estradiol, estriol and ethinyloestradiol respectively, which improved around 200, 2000, and 900 times compared to non-derivative PSI-MS. The method could quantitatively determine estrogens in cosmetics.

Interferences in the yeast estrogen screen (YES) assay for evaluation of estrogenicity in environmental samples, chemical mixtures, and individual substances.

The Yeast Estrogen Screen (YES) has a specific mechanism of action that allows for the analysis of estrogenic EDC at low concentrations, and it has been broadly used to estimate the estrogenic potential of environmental samples. However, the experimental parameters of this assay still demand an investigation, such as cell density, incubation time, wavelength on the experimental outcome, cytotoxicity, and estrogenic activity adsorbed on suspended solids. We studied these interferences and applied the assay to single substances, mixtures, and environmental matrices from different sources. The increase in cell density amplifies the assay sensitivity only to a limited extent, while the reduction in incubation time decreased assay sensitivity - although it was not significant for surface water, no differences were observed between estradiol-equivalents derived of 48 h and 72 h measurements. The particulate phase was of utmost importance for the total estrogenic activity of the landfill leachate and surface water. Surface waters, landfill leachates and sediments also showed antiestrogenic activity and the integration of both estrogenic and antiestrogenic endpoints provided deeper insights into the potential risk associated with EDC. This study elucidated experimental interferences that may arise during the implementation and use of this assay, bringing more understanding to experimental parameters during the application of the assay for estrogenicity screening.

Establishment and validation of a sensitive LC-MS/MS method for quantification of urinary estrogens in women with gestational diabetes mellitus.

Gestational diabetes mellitus (GDM) is not only a threat to the health of pregnant women, but also has profound effects on the health of offspring. Studies have shown that the imbalance of estrogen metabolism is associated with an increased risk of GDM. In this study, an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was established and validated for simultaneous quantification of thirteen estrogens in the urine of GDM women, including estrone (E1), estradiol (E2), estriol (E3), and their hydroxylated and methylated metabolites. The method was achieved on a Waters CORTECS C18 column (2.1 mm × 150 mm, 1.6 µm) within 8.5 min. The linear range of thirteen estrogens in urine was 2-1000 pg·mL-1. Both intra- and inter-day precision for each analyte were less than 15%, with accuracies ranging from 8.3% to 7.3%. The extraction recoveries rate were between 86% and 111%, and stability verification results met the requirements for determination of biological samples. The results suggested that the concentrations of estrogens in all urine samples range from 0.08 to 134.06 (pg·mg-1 creatinine). The mean levels of E1, E2 and most estrogen metabolites in the urine of GDM women were higher than those in healthy pregnant women. Notably, the mean level of 2-hydroxyestrone (2-OHE1) in GDM women was 13.2-fold lower than that in healthy pregnant women. The types of estrogens with the highest mean levels in the urine of GDM and healthy pregnant women were obviously different, which are 2-methoxyestrone (2MeOE1) and E3, respectively. Our results demonstrated that this specific and sensitive method is suitable for quantifying estrogens in human urine and could provide support for further research on estrogen-related pathological mechanisms in GDM and other diseases.

Determination of estrogens in human serum using a novel chemical derivatization-assisted liquid chromatography-electrospray ionization-tandem mass spectrometry method.

RATIONALE: Assessing estrogen concentrations in biological systems can provide valuable information on physiological processes, which is crucial for the early diagnosis of many diseases. Because estrogens are present in the human body in low concentrations and in a wide dynamic range, analytical methods with high sensitivity and specificity are required for their determination in complex biological matrices. METHODS: To discover an appropriate derivatization reagent for estrogen mass spectrometry (MS) analysis, we compared five sulfonyl chloride derivatization reagents, namely 3-methyl-8-quinolinesulfonyl chloride (MQSCl) and 8-quinolinesulfonyl chloride (QSCl), 1-methyl-1H-pyrazole-4-sulfonyl chloride, 1,2-methyl-imidazole-5-sulfonyl chloride, and dansyl chloride. By selecting the derivatization reagent with the best performance, we developed and validated a novel chemical derivatization-assisted-liquid chromatography-electrospray ionization-tandem mass spectrometry (CD-LC-ESI-MS/MS) method to simultaneously determine the concentrations of estrone, estradiol, and estriol (E1, E2, and E3) in human serum. RESULTS: It was found that among the five investigated reagents, MQSCl-derivatized estrogens presented the highest sensitivity using LC-ESI-MS/MS. Based on this discovery, MQSCl was chosen to derivatize the analyzed estrogens to assist LC-ESI-MS/MS analysis. The limit of quantification of E1, E2, and E3 was measured as 2.7, 4.6, and 5.1 pg/mL, respectively. Inter- and intra-day precision, expressed as the coefficient of variation, was shown to be lower than 13.2% for all concentrations. The mean recovery was 72.4% overall, with good reproducibility at low, medium, and high concentrations in the calibration range. CONCLUSIONS: The developed method was successfully applied to the quantitative determination of estrogens in clinical human serum from pediatric and adult women, demonstrating the suitability of estrogen analysis in the biological matrix at low concentration (pg/mL).

Novel Evidence-Based Combination of Plant Extracts with Multitarget Mechanisms of Action for the Elimination of Hot Flashes during Menopause.

Hot flashes are considered the most bothersome complaint during menopause. Although hormone therapy is an effective option to relieve hot flashes, it has been associated with significant side effects. The aim of our study is to suggest a novel combination of different plant extracts with distinct mechanisms of action against hot flashes. We selected the rhizome of Glycyrrhiza glabra L. (Fabaceae), the rhizome of Actaea racemosa L. (Ranunculaceae), the aerial parts of Hypericum perforatum L. (Hypericaceae) to produce extracts rich in bioactive phytochemicals and the seed oil of Oenothera biennis L. (Onagraceae). We investigated their estrogenic and antioxidant potential and their inhibitory effect against prostaglandin D2 receptor 1 (DP1) as a novel mechanistic pathway for vasodilation in hot flashes, alone or in combination. The phytochemical footprint of the extracts was analyzed using HPLC-PDA and UPLC-HRMS. We observed that the tested extracts possess different mechanisms of action. A. racemosa exerts a beneficial activation of the estrogen receptor, H. perforatum possesses the highest antioxidant capacity and the seed oil of O. biennis inhibits the DP1 receptor. The triple combination in the optimal doses pertains to efficacy against all three mechanisms of action, serves as a multitarget plant-based therapy and could serve as a novel strategy for the alleviation of hot flashes in postmenopausal women.

A tissue-selective estrogen complex as treatment of osteoporosis in experimental lupus.

Osteoporosis is a common secondary complication in patients with systemic lupus erythematosus (SLE). Current osteoporosis treatment with bisphosphonates has some negative side effects and there is a lack of data regarding newer treatments options for SLE associated osteoporosis. The tissue-selective estrogen complex (TSEC) containing conjugated estrogens and the selective estrogen receptor modulator bazedoxifene (Bza) is approved for treatment of postmenopausal vasomotor symptoms and prevention of osteoporosis. However, it has not been evaluated for treatment of osteoporosis in postmenopausal SLE patients. Ovariectomized MRL/lpr mice constitute a model for postmenopausal lupus that can be used for osteoporosis studies. We used this model in a set of experiments where the mice were treated with different doses of 17ß-estradiol-3-benzoate (E2), Bza, or TSEC (E2 plus Bza), administered in the early or late phases of disease development. The skeleton was analyzed by dual-energy X-ray absorptiometry, peripheral quantitative computed tomography, and high-resolution microcomputed tomography. The lupus disease was assessed by determination of proteinuria, hematuria, and lupus disease markers in serum. Treatment with medium dose TSEC administered in early disease protected ovariectomized MRL/lpr mice from trabecular bone loss, while there were no differences in lupus disease parameters between treatments. This is the first experimental study to investigate TSEC as a potential new therapy for osteoporosis in postmenopausal SLE.

Estrogenic activity of Sage (Salvia officinalis L.) aerial parts and its isolated ferulic acid in immature ovariectomized female rats.

ETNOPHARMACOLOGICAL RELEVANCE: Common sage (Salvia officinalis L., Lamiaceae), a medicinal plant of Mediterranean origin, has been traditionally applied in cases of excessive sweating, and in menopausal complaints, including hot flushes. AIM OF THE STUDY: This study aims to study the possible estrogenic effect of the aerial parts of S. officinalis ethanolic extract in immature ovariectomized female rats. MATERIALS AND METHODS: The ethanolic extract was subjected to qualitative and quantitative HPLC analysis and phytochemical isolation. The estrogenic activity of S. officinalis ethanolic extract at oral doses of 50, 100 and 200 mg/kg b.wt. and its isolated ferulic acid at a dose of 50 mg/kg b.wt. for a week, was assessed on ovariectomized immature Wistar rats. The experiment was confirmed by luteinizing hormone (LH) and follicle stimulating hormone (FSH) serum levels determination, a histopathological examination and a histomorphometrical study. RESULTS: HPLC/PDA analysis revealed fourteen phenolic compounds the major constituents were methyl rosmarinate (24.86 mg/100 g) and ferulic acid (6.06 mg/100 g) together with five flavonoids where the major constituents were rutin, naringenin and quercetin. Two compounds were isolated from the polar fraction and identified as methyl rosmarinate (1) and ferulic acid (2). Oral administration of sage ethanolic extract and ferulic acid revealed a significant increase in the uterine weight compared to ovariectomized control rats. Moreover, S. officinalis and ferulic acid showed different phases of estrus cycle denoting estrogenic activity, and significantly decreased the serum levels of FSH and LH. CONCLUSION: From these results it could be concluded that S. officinalis ethanolic extract and its content of ferulic acid could be useful as a safe natural source for estrogenic activity, supporting its traditional use to improve postmenopausal symptoms.

Structural and Functional Analysis of Female Sex Hormones against SARS-CoV-2 Cell Entry.

Emerging evidence suggests that males are more susceptible to severe infection by the SARS-CoV-2 virus than females. A variety of mechanisms may underlie the observed gender-related disparities including differences in sex hormones. However, the precise mechanisms by which female sex hormones may provide protection against SARS-CoV-2 infectivity remains unknown. Here we report new insights into the molecular basis of the interactions between the SARS-CoV-2 spike (S) protein and the human ACE2 receptor. We further report that glycosylation of the ACE2 receptor enhances SARS-CoV-2 infectivity. Importantly, estrogens can disrupt glycan-glycan interactions and glycan-protein interactions between the human ACE2 and the SARS-CoV-2 thereby blocking its entry into cells. In a mouse model of COVID-19, estrogens reduced ACE2 glycosylation and thereby alveolar uptake of the SARS-CoV-2 spike protein. These results shed light on a putative mechanism whereby female sex hormones may provide protection from developing severe infection and could inform the development of future therapies against COVID-19.

Co-treatment of testosterone and estrogen mitigates heat-induced testicular dysfunctions in a rat model.

The two gonadal steroid hormones, testosterone and estrogen, regulate spermatogenesis by proliferation, differentiation, and apoptosis of testicular cells. It has been reported that heat stress or increased scrotal temperature impairs spermatogenesis in many mammals. Moreover, testicular heat stress has also been shown to suppress testosterone and estrogen biosynthesis. Furthermore, it is well known that testosterone and estrogen are important for testicular activity. Therefore, we hypothesised that exogenous testosterone and estrogen, alone or in combination, might alleviate the testicular activity in a heat-stressed rat model. To the best of our knowledge, this will be the first report of the exogenous treatment of both testosterone and estrogen in the heat-stressed rat. Our results showed that a combined testosterone and estrogen treatment significantly increased sperm concentration. The histopathological analysis also exhibited a normal histoarchitecture in the combined treatment group along with decreased oxidative stress. The improved spermatogenesis in the combined treatment group was also supported by the increase in PCNA, GCNA, tubule diameter, germinal epithelium height, and Johnsen score in the combined treatment group. Furthermore, the combined treatment also increased the expression of Bcl2, pStat3, and active caspase-3 and decreased expression of Bax. Thus, increased proliferation, apoptotic and anti-apoptotic markers, along with improved histology in the combined treatment group suggest that estrogen and testosterone synergistically act to stimulate spermatogenesis by increasing proliferation and differentiation of germ cells and may also remove the heat-induced damaged germ cells by apoptosis. Overall, the final mechanism of testosterone- and estrogen-mediated improvement of testicular activity could be attributed to amelioration of oxidative stress.

A new α-pyrone from Arthrinium pseudosinense culture medium and its estrogenic activity in MCF-7 cells.

A new α-pyrone analog, arthrifuranone A (1) was isolated from an EtOAc-extract of Arthrinium pseudosinense culture medium. The isolation workflow was guided by a Molecular Networking-based dereplication strategy. The chemical structure of the new compound was elucidated using MS and NMR spectroscopic techniques, and the absolute configuration was established by the Mosher's method and gauge-including atomic orbital NMR chemical shift calculations, followed by DP4 + analysis. The isolated compound was evaluated for its estrogenic activity using the MCF-7 estrogen responsive human breast cancer cells. Compound 1 showed estrogenic activity by increasing the proliferation of MCF-7 cells at the concentration of 3.125 µM via phosphorylation of estrogen receptor-α.

Iron accumulation deteriorated bone loss in estrogen-deficient rats.

BACKGROUND: Postmenopausal osteoporosis is characterized by an imbalance of bone resorption exceeding bone formation, resulting in a net loss of bone mass. Whether a menopause-related excess of iron contributes to the development of postmenopausal osteoporosis has remained unresolved due to a lack of an appropriate animal model. This study aimed to explore the effects of iron accumulation in bone mass in estrogen-deficient rats. METHODS: In the present study, ovariectomy (OVX) was performed in female rats and the changes of iron metabolism and some related modulated genes were detected. Ferric ammonium citrate (FAC) was used as a donor of iron for OVX rats. Moreover, micro-CT was performed to assess the bone microarchitecture in sham group, OVX, and FAC groups. Histological detection of iron in liver was assessed by Perl's staining. The expressions of ß-CTX and osteocalcin were assessed by ELISA. RESULTS: It was found that serum iron decreased after OVX. It was found that the expressions of Hepcidin in liver and Fpn, DMT-1 in duodenum significantly decreased at transcriptional level in OVX group than sham group. However, no difference existed in the expression of DMT-1. Then, ferric ammonium citrate (FAC) was used as a donor of iron for OVX rats. The FAC group manifested significant iron accumulation by increased serum iron and hepatic iron content. In addition, FAC treatment accelerated bone loss and decreased BMD and biomechanics in OVX rats. Moreover, bone biomarker ß-CTX rather than osteocalcin increased significantly in FAC groups than OVX group. CONCLUSIONS: In conclusion, no iron accumulation occurred in OVX rats. Furthermore, iron accumulation could further deteriorate osteopenia through enhanced bone resorption.

Limitations of currently available in vitro oestrogenicity bioassays for effect-based testing of whole foods as the basis for decision making.

The idea that previously unknown hazards can be readily revealed in complex mixtures such as foods is a seductive one, giving rise to the hope that data from effect-based assays of food products collected in market surveys is of suitable quality to be the basis for data-driven decision-making. To study this, we undertook a comparative study of the oestrogenicity of blinded cereal samples, both in a number of external testing laboratories and in our own facility. The results clearly showed little variance in the activities of 9 samples when using a single method, but great differences between the activities from each method. Further exploration of these findings suggest that the oestrogenic activity is likely an inherent part of the natural food matrix which the varying sample preparation methods are able to release and extract to differing degrees. These issues indicate the current poor suitability of these types of datasets to be used as the basis for consumer advice or food decision-making. Data quality must be improved before such testing is used in practice.

Structure-Activity Relationship of para-Carborane Selective Estrogen Receptor ß Agonists.

Selective agonism of the estrogen receptor (ER) subtypes, ERα and ERß, has historically been difficult to achieve due to the high degree of ligand-binding domain structural similarity. Multiple efforts have focused on the use of classical organic scaffolds to model 17ß-estradiol geometry in the design of ERß selective agonists, with several proceeding to various stages of clinical development. Carborane scaffolds offer many unique advantages including the potential for novel ligand/receptor interactions but remain relatively unexplored. We synthesized a series of para-carborane estrogen receptor agonists revealing an ERß selective structure-activity relationship. We report ERß agonists with low nanomolar potency, greater than 200-fold selectivity for ERß over ERα, limited off-target activity against other nuclear receptors, and only sparse CYP450 inhibition at very high micromolar concentrations. The pharmacological properties of our para-carborane ERß selective agonists measure favorably against clinically developed ERß agonists and support further evaluation of carborane-based selective estrogen receptor modulators.

Assessing the chemical-induced estrogenicity using in silico and in vitro methods.

Multiple substances are considered endocrine disrupting chemicals (EDCs). However, there is a significant gap in the early prioritization of EDC's effects. In this work, in silico and in vitro methods were used to model estrogenicity. Two Quantitative Structure-Activity Relationship (QSAR) models based on Logistic Regression and REPTree algorithms were built using a large and diverse database of estrogen receptor (ESR) agonism. A 10-fold external validation demonstrated their robustness and predictive capacity. Mechanistic interpretations of the molecular descriptors (C-026, nArOH,PW5, B06[Br-Br]) used for modelling suggested that the heteroatomic fragments, aromatic hydroxyls, and bromines, and the relative bond accessibility areas of molecules, are structural determinants in estrogenicity. As validation of the QSARs, ESR transactivity of thirteen persistent organic pollutants (POPs) and suspected EDCs was tested in vitro using the MMV-Luc cell line. A good correspondence between predictions and experimental bioassays demonstrated the value of the QSARs for prioritization of ESR agonist compounds.

ERα-agonist and ERß-antagonist bifunctional next-generation bisphenols with no halogens: BPAP, BPB, and BPZ.

As demonstrated for bisphenol AF (BPAF), the electrostatic halogen bond based on the London dispersion force of halogen atoms was found to be a major driving force of their bifunctional ERα-agonist and ERß-antagonist activities. Because similar electronic effects are anticipated for hydrocarbon groups (alkyl or aryl groups), we hypothesized that bisphenol compounds consisting of such groups also work bifunctionally. In the present study, we examined bisphenol AP (BPAP), B (BPB), and Z (BPZ). After recognizing their considerably strong receptor binding affinities, we evaluated the abilities of BPAP, BPB, and BPZ to activate ERα and ERß in a luciferase reporter gene assay. These bisphenols were fully active for ERα but completely inactive for ERß. When we examined their inhibitory activities for 17ß-estradiol in ERß by two different qualitative and quantitative analytical methods, we found that those bisphenols worked as definite antagonists. Consequently, they were established as bifunctional ERα-agonists and ERß-antagonists. The present structure-activity analyses revealed that the dispersion force works not only on the halogens but also on the hydrocarbon groups, and that it is a major driving force of bifunctional ERα-agonist and ERß-antagonist activities.