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G protein-coupled estrogen receptor (GPER) is a member of the GPCR family and a key mediator of the rapid, nongenomic actions of estrogens as membrane estrogen receptors. In this study, we established a procedure for the expression and purification of recombinant human membrane estrogen receptor (hGPER) protein via the expression system using the methylotrophic yeast Pichia pastoris . By optimizing codon usage, we successfully expressed hGPER at a level that can be purified by column chromatography. The recombinant protein was purified via three chromatography steps. Purified hGPER showed specific estrogen-binding activity (Kd = 9.9 nM and Bmax = 1.76 nM) in a radiolabeled steroid-binding assay. We subsequently established a homogeneous assay for hGPER ligands by conjugating semiconductor nanoparticles known as graphene quantum dots (GQDs) to hGPER. GQDs coupled with hGPER (GQD-hGPER) caused a decrease in fluorescence at 520 nm from E2-BSA-FITC, which was activated by 370 nm light upon the addition of free estradiol to the reaction mixture. Fluorescence was decreased by the administration of hGPER ligands but not by steroids that do not interact with hGPER. Thus, we successfully established a ligand-binding assay for hGPER that is suitable for screening potential compounds. hGPER is a promising candidate for drug discovery for nongenomic estrogen-stimulating effects. The homogeneous assay established in this study will be usable for that purpose.

Bisphenol A (BPA) is an industrial compound and a well known endocrine-disrupting chemical with estrogenic activity. The widespread exposure of individuals to BPA is suspected to affect a variety of physiological functions, including reproduction, development and metabolism. Here we report that the mechanisms by which BPA and two congeners, bisphenol AF and bisphenol C (BPC), bind to and activate estrogen receptors (ER) α and β differ from that used by 17β-estradiol. We show that bisphenols act as partial agonists of ERs by activating the N-terminal activation function 1 regardless of their effect on the C-terminal activation function 2, which ranges from weak agonism (with BPA) to antagonism (with BPC). Crystallographic analysis of the interaction between bisphenols and ERs reveals two discrete binding modes, reflecting the different activities of compounds on ERs. BPA and 17β-estradiol bind to ERs in a similar fashion, whereas, with a phenol ring pointing toward the activation helix H12, the orientation of BPC accounts for the marked antagonist character of this compound. Based on structural data, we developed a protocol for in silico evaluation of the interaction between bisphenols and ERs or other members of the nuclear hormone receptor family, such as estrogen-related receptor γ and androgen receptor, which are two known main targets of bisphenols. Overall, this study provides a wealth of tools and information that could be used for the development of BPA substitutes devoid of nuclear hormone receptor-mediated activity and more generally for environmental risk assessment.

Recently we constructed yeast cells that either express the human estrogen receptor α or the human androgen receptor in combination with a consensus ERE or ARE repeat in the promoter region of a green fluorescent protein (yEGFP) read-out system. These bioassays were proven to be highly specific for their cognate agonistic compounds. In this study the value of these yeast bioassays was assessed for analysis of compounds with antagonistic properties. Several pure antagonists, selective estrogen receptor modulators (SERMs) and plant-derived compounds were tested. The pure antiestrogens ICI 182,780 and RU 58668 were also classified as pure ER antagonists in the yeast estrogen bioassay and the pure antiandrogen flutamide was also a pure AR antagonist in the yeast androgen bioassay. The plant-derived compounds flavone and guggulsterone displayed both antiestrogenic and antiandrogenic activities, while 3,3'-diindolylmethane (DIM) and equol combined an estrogenic mode of action with an antiandrogenic activity. Indol-3-carbinol (I3C) only showed an antiandrogenic activity. Coumestrol, genistein, naringenin and 8-prenylnaringenin were estrogenic and acted additively, while the plant sterols failed to show any effect. Although hormonally inactive, in vitro and in vivo metabolism of the aforementioned plant sterols may still lead to the formation of active metabolites in other test systems.

A novel magnetic dispersive solid phase extraction (MDSPE) procedure based on the deep eutectic solvent (DES) modified magnetic graphene oxide/metal organic frameworks nanocomposites (MGO@ZIF-8@DES) was established and used for the efficient enrichment of estradiol, estrone, and diethylstilbestrol in cosmetics (toner, lotion, and cream) for the first time. Then, the three estrogens were separated and determined by UHPLC-UV analysis method. In order to study the features and morphology of the synthesized adsorbents, various techniques such as FT-IR, SEM, and VSM measurements were executed. The MGO@ZIF-8@DES nanocomposites combine the advantages of high adsorption capacity, adequate stability in aqueous solution, and convenient separation from the sample solution. To achieve high extraction recoveries, the Box-Behnken design and single factor experiment were applied in the experimental design. Under the optimum conditions, the method detection limits for three estrogens were 20–30 ng g −1 . This approach showed a good correlation coefficient ( r more than 0.9998) and reasonable linearity in the range 70–10000 ng g −1 . The relative standard deviations for intra-day and inter-day were beneath 7.5% and 8.9%, respectively. The developed MDSPE-UHPLC-UV method was successfully used to determine  three estrogens in cosmetics, and acceptable recoveries in the intervals of 83.5–95.9% were obtained. Finally, three estrogens were not detected in some cosmetic samples. In addition, the Complex GAPI tool was used to evaluate the greenness of the developed pretreatment method. The developed MDSPE-UHPLC-UV method is sensitive, accurate, rapid, and eco-friendly, which provides a promising strategy for determining hormones in different complex samples. Graphical abstract

A covalent organic framework (COF) based on imine was synthesized using 2,5-dihexoxyterephthalaldehyde (DHT) and 1,3,5-tris(4-aminophenyl) benzene (TAPB) as starting materials. The TAPB-DHT-COF exhibited satisfactory chemical stability, making it a promising adsorbing material for stir bar sorptive extraction (SBSE) of four estrogens, including estrone (E1), β-estradiol (E2), hexestrol (HES), and mestranol (MeEE2), in ambient water samples. The extracted analytes were subsequently analyzed using a high-performance liquid chromatography-diode array detector (HPLC-DAD). A series of parameters affecting the SBSE process, such as solution pH, ionic strength, extraction time, and desorption solvent, were investigated by the controlled variable method. Under optimal conditions, the limit of detection (LODs) for the four targeted estrogens ranged from 0.06 to 0.15 µg/L, with a linear range from 0.2 to 100 µg/L. The observed enrichment factor (EF) ranged from 39 to 49, while the theoretical EF was estimated to be 50-fold. This methodology can be applied to the identification of estrogens in three environmental water samples.

In this study, three magnetic ionic liquids (MILs) were investigated for extraction of four estrogens, i.e., estrone (E1), estradiol (E2), estriol (E3), and ethinylestradiol (EE2), from environmental water. The cation trihexyl(tetradecyl)phosphonium ([P66614]+), selected to confer hydrophobicity to the resulting MIL, was combined with tetrachloroferrate(III), ferricyanide, and dysprosium thiocyanate to yield ([P66614][FeCl4]), ([P66614]3[Fe(CN)6]), and ([P66614]5[Dy(SCN)8]), respectively. After evaluation of various strategies to develop a liquid–liquid microextraction technique based on synthesized MILs, we placed the MILs onto a magnetic stir bar and used them as extracting solvents. After extraction, the MIL-enriched phase was dissolved in methanol and injected into an HPLC–UV for qualitative and quantitative analysis. An experimental design was used to simultaneously evaluate the effect of select variables and optimization of extraction conditions to maximize the recovery of the analytes. Under optimum conditions, limits of detection were in the range of 0.2 (for E3 and E2) and 0.5 μg L−1 (for E1), and calibration curves exhibited linearity in the range of 1–1000 μg L−1 with correlation coefficients higher than 0.998. The percent relative standard deviation (RSD) was below 5.0%. Finally, this method was used to determine concentration of estrogens in real lake and sewage water samples.

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.

Ecotoxicological screening of surface waters can involve multiple analyses using multiple bioassay and chemical analytical methods that require enriched samples to reach low concentrations. Such broad screening of the same sample necessitates sufficient sample volume—typically several liters—to produce a sufficient amount of enriched sample. Often, this is achieved by performing parallel solid-phase extractions (SPE) where extracts are combined into a pool—this is a laborious process. In this study, we first validated our existing SPE method for the chemical recovery of an extended set of compounds. We spiked four estrogenic compounds and 11 herbicides to samples from independent rivers (1 L) and wastewater treatment plant effluents (0.5 L). Then, we investigated the effect of increased sample loading of the SPE cartridges on both chemical and biological recoveries by comparing the validated volumes with four times larger sample volumes (i.e., 4 L river water and 2 L effluent). Samples were analyzed by LC-MS/MS and three bioassays: an estrogen receptor transactivation assay (ERα-CALUX), the combined algae test, and a bacterial bioluminescence inhibition assay. Our existing SPE method was found to be suitable for enriching the extended set of estrogens and herbicides in river water and effluents with near to perfect chemical recoveries (~ 100%), except for the herbicide metribuzin (46 ± 19%). In the large volume river and effluent samples, the biological activities and concentrations of the spiked compounds were between 87 and 104% of those measured with the lower sample loading, which is adequate. In addition, the ratio between the large and original volume SPE method for the non-target endpoint (bacterial bioluminescence inhibition) was acceptable (on average 82 ± 9%). Results indicate that our current water extraction method can be applied to up to four times larger sample volumes, resulting in four times more extract volumes, without significant reductions in recoveries for the tested estrogens and herbicides.

Chemical contamination of wastewater is a problem of great environmental concern, as it poses a hazard to both the ecosystem and to human health. In this study, we have performed a bioanalytical evaluation of the presence and removal efficiency for bioactive chemicals in wastewater treatment plants (WWTPs), using in vitro assays for toxicity endpoints of high relevance for human health. Water samples were collected at the inlet and outlet of five Swedish WWTPs, all adopting a treatment technology including pretreatment, primary treatment (sedimenation), seconday treatment (biological processes), post-sedimentation, and sludge handling. The water samples were analyzed for cytotoxicity, estrogenicity, androgenicity, aryl hydrocarbon receptor (AhR) activity, oxidative stress response (Nrf2) and the ability to activate NFĸB (nuclear factor kappa-light-chain-enhancer of activated B cells) signaling. We observed clear androgenic and estrogenic activities in all inlet samples. Androgenic and estrogenic activities were also observed in all outlet samples, but the activities were lower than the respective inlet sample. AhR activity was observed in all samples, with higher activities in the inlet samples compared to the outlet samples. The removal efficiency was found to be high for androgenic (>99% for two plants and 50–60% for two plants) and estrogenic (>90% for most plants) compounds, while the removal efficiency for AhR-inducing compounds was 50–60% for most plants and 16% for one plant.

As part of our ongoing research on phytoestrogens, we investigated the phytochemical profile and estrogen-like activities of eight extracts from the aerial parts of four Genista species of Greek flora using estrogen-responsive cell lines. Ethyl acetate and methanolic extracts of G. acanthoclada, G. depressa,G. hassertiana, and G. millii were obtained with accelerated solvent extraction and their phytochemical profiles were compared using ultra-high-performance liquid chromatography-high-resolution mass spectrometry (uHPLC-HRMS). Fourteen isoflavonoids, previously isolated from G. halacsyi, were used as reference standards for their identification in the extracts. Thirteen isoflavonoids were detected in both extracts of G. acanthoclada and G. hassertiana, while fewer and far fewer were detected in G. millii and G. depressa, respectively. The ethyl acetate extracts of G. hassertiana and G. acanthoclada displayed 2.45- and 1.79-fold higher, respectively, estrogen-like agonist activity in Ishikawa cells compared to MCF-7 cells at pharmacologically relevant concentrations. Both these extracts, but not that of G. depressa, contained mono- and di-O-β-d-glucosides of genistein as well as the aglycone, all three of which are known to display full estrogen-like activity at lower-than-micromolar concentrations. The possibility of using preparations rich in G. hassertiana and/or G. acanthoclada extracts as a potentially safer substitute for low-dose vaginal estrogen for menopausal symptoms is discussed.