E-screen Assay Research Articles

In Vitro Evaluation of Endocrine-Related Adverse Effects of 5-Fluoroindole Derived Melatonin Analogues with Antioxidant Activity.

Melatonin (MLT) is a natural indolic hormone with well documented antioxidant properties, but it can also modulate the estrogen signaling pathway by inhibiting the aromatase enzyme and estrogen receptor modulating activity. This dual activity raises concerns about potential endocrine-related adverse effects when using MLT and its analogues as therapeutic agents in the prevention and treatment of oxidative stress related diseases. In this study, 34 novel 5-fluoroindole derivatives of MLT were synthesized and evaluated for their antioxidant, estrogen receptor modulatory, and aromatase inhibitory activities.Three compounds (4c, 5c, and 6c) demonstrated significant antioxidant activity, with compound 4c showing the highest efficacy in reducing intracellular reactive oxygen species (ROS) by 65 % in CHO-K1 cells and displaying DPPH radical scavenging comparable to the standard antioxidant, BHT. However, these same compounds also exhibited antiestrogenic effects in the E-Screen assay, with IC50 values of 3.36×10-5 M, 1.31×10-7 M, and 1.9×10-7 M, respectively, and inhibited aromatase activity by up to 29 % in a direct enzymatic assay. These findings indicate that, while the compounds have potent antioxidant properties, their significant antiestrogenic and aromatase inhibitory activities may pose risks for unintended endocrine related effects. Further studies are needed to better understand the implications of these activities in vivo and to balance the benefits and risks of such compounds in therapeutic applications.

Estrogen-like Cell Proliferation Abilities of Korea Forest Plant Resources on MCF-7 Cells and Analysis of Active Compounds

This study conducted a comprehensive analysis of functional substances and explored their biological activities using colorimetric and chromatographic techniques to identify high-value materials from 14 species of forest plants native to Korea. Comparative analysis between plant species included calculating the total polyphenol and flavonoid contents and qualitative and quantitative analysis of the phytochemical compounds caffeic acid (1), p-coumaric acid (2), and quercetin (3) using HPLC, and antioxidant activity tests (DPPH and ABTS+) and menopausal symptom relief tests (E-screen assay) to investigate their biological activities. The results highlighted Cercidiphyllum japonicum (FR 4), Aruncus dioicus (FR 6), and Pseudocydonia sinensis (FR 8) as excellent forest plant resource materials among the 14 forest plant resources. Notably, FR 4 exhibited significant activity in the E-screen assay. On the basis of these findings, we propose the use of FR 4 as a valuable material. Further analysis identified the phytochemical compounds maltol (4), chlorogenic acid (5), ellagic acid (6), and quercitrin (7) in FR 4 and analyzed their biological activities. Compounds 2–6 found in FR 4 were confirmed to possess strong antioxidant activity, and an E-screen assay revealed an excellent cell proliferation rate for quercitrin (7). This suggests that quercitrin (7) in FR 4 may be a significant indicator of the alleviation of menopausal symptoms.

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.

Combined Effects of Polystyrene Nanosphere and Homosolate Exposures on Estrogenic End Points in MCF-7 Cells and Zebrafish.

Micro- and nanoplastics (MNPs) and homosalate (HMS) are ubiquitous emerging environmental contaminants detected in human samples. Despite the well-established endocrine-disrupting effects (EDEs) of HMS, the interaction between MNPs and HMS and its impact on HMS-induced EDEs remain unclear. This study aimed to investigate the influence of MNPs on HMS-induced estrogenic effects and elucidate the underlying mechanisms in vitro and invivo. We assessed the impact of polystyrene nanospheres (PNSs; , ) on HMS-induced MCF-7 cell proliferation (HMS: , equivalent to ) using the E-SCREEN assay and explored potential mechanisms through transcriptomics. Adult zebrafish were exposed to HMS () with or without PNSs (, ) for 21 d. EDEs were evaluated through gonadal histopathology, fertility tests, steroid hormone synthesis, and gene expression changes in the hypothalamus-pituitary-gonad-liver (HPGL) axis. Coexposure of HMS and PNSs resulted in higher expression of estrogen receptor (ESR1) and the mRNAs of target genes (pS2, AREG, and PGR), a greater estrogen-responsive element transactivation activity, and synergistic stimulation on MCF-7 cell proliferation. Knockdown of serum and glucocorticoid-regulated kinase 1 (SGK1) rescued the MCF-7 cell proliferation induced by PNSs alone or in combination with HMS. In zebrafish, coexposure showed higher expression of SGK1 and promoted ovary development but inhibited spermatogenesis. In addition, coexposure led to lower egg hatchability, higher embryonic mortality, and greater larval malformation. Coexposure also modulated steroid hormone synthesis genes (cyp17a2, hsd17[Formula: see text]1, esr2b, vtg1, and vtg2), and resulted in higher () release in females. Conversely, males showed lower testosterone, , and gene expressions of cyp11a1, cyp11a2, cyp17a1, cyp17a2, and hsd17[Formula: see text]1. PNS exposure exacerbated HMS-induced estrogenic effects via SGK1 up-regulation in MCF-7 cells and disrupting the HPGL axis in zebrafish, with gender-specific patterns. This offers new mechanistic insights and health implications of MNP and contaminant coexposure. https://doi.org/10.1289/EHP13696.

Evaluation of Endocrine Related Adverse Effects of Non-Endocrine Targeted Pharmaceuticals in Cellular Systems.

Prenatal period is a critical developmental phase that is sensitive to hormonal disruption by natural and/or exogenous hormones. Some pharmaceuticals frequently prescribed and used safely during pregnancy are shown to interact with the developmental programming of fetus, resulting in endocrine-related adverse effects. In this research, we aimed to determine the endocrine disrupting potential of paracetamol, indomethacin, alpha-methyldopa and pantoprazole which are frequently prescribed pharmaceuticals during pregnancy. In vitro aromatase inhibitory, estrogen receptor (ER) agonist/antagonist (E-Screen assay) and hormone biosynthesis modulatory effects (H295R steroidogenesis assay) of the selected pharmaceuticals were evaluated. Furthermore, their effects on viability of MCF-7/BUS and H295R cells were also evaluated by MTT assay. None of the pharmaceuticals affected H295R cell viability. Only indomethacin reduced MCF-7/BUS cell viability at 100µM and 300µM. Among the tested pharmaceuticals, only paracetamol and indomethacin showed aromatase inhibitory activity with IC 50 values of 14.7 x 10 -5 M and 57.6 x 10 -5 M, respectively. Moreover, indomethacin displayed a biphasic ER agonist effect. ER antagonist effects of indomethacin and pantoprazole were confirmed by performing two stepped E-Screen assay. After the partial validation of the H295R steroidogenesis assay with forskolin and prochloraz, the effects of pharmaceuticals on synthesis of testosterone (T) and estradiol (E2) levels were tested. Alpha-methyldopa increased E2 at all tested concentrations and T at 1.48 and 4.4µM. Contrarily other tested pharmaceuticals did not affect steroidogenesis. Present data suggest that all tested pharmaceuticals may have potential endocrine disrupting effect, which should be considered when used in pregnancy.

Global Natural Products Social (GNPS)-Based Molecular-Networking-Guided Isolation of Phenolic Compounds from Ginkgo biloba Fruits and the Identification of Estrogenic Phenolic Glycosides

Ginkgo biloba L. stands as one of the oldest living tree species, exhibiting a diverse range of biological activities, including antioxidant, neuroprotective, anti-inflammatory, and cardiovascular activities. As part of our ongoing discovery of novel bioactive components from natural sources, we directed our focus toward the investigation of potential bioactive compounds from G. biloba fruit. The profiles of its chemical compounds were examined using a Global Natural Products Social (GNPS)-based molecular networking analysis. Guided by this, we successfully isolated and characterized 11 compounds from G. biloba fruit, including (E)-coniferin (1), syringin (2), 4-hydroxybenzoic acid 4-O-β-D-glucopyranoside (3), vanillic acid 4-O-β-D-glucopyranoside (4), syringic acid 4-O-β-D-glucopyranoside (5), (E)-ferulic acid 4-O-β-D-glucoside (6), (E)-sinapic acid 4-O-β-D-glucopyranoside (7), (1′R,2′S,5′R,8′S,2′Z,4′E)-dihydrophaseic acid 3′-O-β-D-glucopyranoside (8), eucomic acid (9), rutin (10), and laricitrin 3-rutinoside (11). The structural identification was validated through a comprehensive analysis involving nuclear magnetic resonance (NMR) spectroscopic data and LC/MS analyses. All isolated compounds were evaluated using an E-screen assay for their estrogen-like effects in MCF-7 cells. As a result, compounds 2, 3, 4, 8, and 9 promoted cell proliferation in MCF-7 cells, and these effects were mitigated by the ER antagonist, ICI 182,780. In particular, cell proliferation increased most significantly to 140.9 ± 6.5% after treatment with 100 µM of compound 2. The mechanism underlying the estrogen-like effect of syringin (2) was evaluated using a Western blot analysis to determine the expression of estrogen receptor α (ERα). We found that syringin (2) induced an increase in the phosphorylation of ERα. Overall, these experimental results suggest that syringin (2) can potentially aid the control of estrogenic activity during menopause.

Hormone effects of eighteen bisphenol analogues and their effects on cellular homeostasis and the typical signal pathways

Through the transfer chain of surroundings from feed to the farmed-animals and ultimately the corresponding livestock and poultry products, people are exposed to large amounts of bisphenol analogues (BPs), such as rational emissions from manufacturing plants, feed packaging bags and food packaging contact. Some BPs have been reported to show certain toxicological effects, especially, estrogen and endocrine disrupting effect. With the increasing application of BPs, the problem is becoming more and more serious. We systematically studied the hormonal effects of 18 BPs and their effects on cell homeostasis and classical signaling pathways by using classical E-SCREEN assay, fluorescent probes and western blotting. The results confirmed the estrogen-like effect of 13 BPs and 6 BPs obtained high docking scores (Scores < −9.0) for the three receptors simultaneously with the main interactions of hydrophobic, hydrogen and π-stacking of T-type bonds. BPAP regulates cells via apoptosis and steroid signaling pathway by intracellular ROS and mitochondrial followed the caspase pathway. BPE and BPS were involved in the classical NF-κB and Hippo signaling pathways. All data provides scientific basis for the safety risk assessment of endocrine disrupting and cellular homeostasis evaluation of BPs as chronic environmental pollution.

Xeno-estrogenic activity of real-life mixtures of perfluoroalkylated substances in human placenta homogenates

Humans are simultaneously exposed to complex chemical mixtures, and its combined effect can affect human health. As part of the HBM4EU project, the actual mixture of perfluoroalkylated substances (PFAS) in 25 human placenta samples was extracted by chromatographic methods and assessed for xeno-estrogenic activity using two in-vitro bioassays: the estrogen receptor transactivity and the E-Screen assay. Most of the PFAS extracts displayed xeno-estrogenic activity, in one or both assays. The xeno-estrogenic activities in the two bioassays were not correlated, but both assays showed an overall negative correlation with placenta concentrations of single PFAS. Xeno-estrogenic activities were significantly related to maternal characteristics; being higher in young, smokers and primiparous women, but not with fetal growth (birth weight, birth length, head circumference, gestational age, placenta weight). The presented extraction method can be used to study the combined effect of real-life mixtures of PFAS in relation to health outcomes in large-scale human biomonitoring studies.

Estrogenic and anti-amnesic potential of Millettia griffoniana Baill. (Fabaceae) ethanolic extract on scopolamine-induced memory impairment in ovariectomized Wistar rats.

Dementias including Alzheimer disease (AD) are three times higher in menopausal women than in men. Phytoestrogens, a group of plant-derived compounds are known to alleviate menopausal complaints including dementia. Millettia griffoniana Baill is a phytoestrogen-rich plant used to treat menopausal complaints and dementia. Evaluating the estrogenic and neuroprotective potential of Millettia griffoniana on ovariectomized (OVX) rats. The in vitro safety of M. griffoniana ethanolic extract was assayed by MTT in human mammary epithelial (HMEC) and mouse neuronal (HT-22) cells and its lethal dose 50 (LD50) was estimated following OECD 423 guidelines. For estrogenicity, in vitro the well known E-screen assay on MCF-7cells was performed and in vivo four groups of OVX rats were treated either with 75, 150 and 300mg/kg M. griffoniana extract doses or estradiol (1mg/kg BW) for three days; and changes in uterine and vagina were analyzed. Then, for neuroprotective effect, Alzheimer-type dementia induction was achieved by scopolamine (1.5mg/kg B.W., i.p.) injection four days/week and M. griffoniana extract as well as piracetam (standard) were administered daily for 2 weeks to evaluate the extract's neuroprotective potential. The endpoints were the assessment of learning and working memory, oxidative stress state (SOD, CAT, and MDA) in brain, acetylcholine esterase (AChE) activity and the histopathological changes in hippocampus. No toxic effect was observed when incubating mammary (HMEC) and neuronal (HT-22) cells with M. griffoniana ethanol extract for 24h and its LD50 was found >2000mg/kg. The extract also exhibited both in vitro and in vivo estrogenic activities, displayed by a significant (p<0.01) increment in MCF-7cells population in vitro and an increase in the epithelium height of the vagina and the wet weight of the uterus mainly with the 150mg/kg BW extract dose compared to untreated OVX rats. The extract also reversed scopolamine-induced memory impairment in rat by improving learning, working and reference memory. This was associated with an increment in CAT and SOD expression, alongside a decrement in MDA content and AChE activity in hippocampus. Further, the extract reduced neuronal cell loss in hippocampal structures (CA1, CA3 and dentate gyrus). High Performance Liquid Chromatography coupled with Mass Spectrometry (HPLC-MS) spectra, revealed the presence of numerous phytoestrogens in M. griffoniana extract. M. griffoniana ethanolic extract has estrogenic, anticholinesterase and antioxidant activities that could account for its anti-amnesic effects. These findings therefore sheds light on why this plant is commonly used in the therapy of menopausal complaints and dementia.

Estrogenic Activity of 4-Hydroxy-Benzoic Acid from Acer tegmentosum via Estrogen Receptor α-Dependent Signaling Pathways.

Acer tegmentosum, a deciduous tree belonging to Aceraceae, has been used in traditional oriental medicine for treating hepatic disorders, such as hepatitis, cirrhosis, and liver cancer. We evaluated the estrogen-like effects of A. tegmentosum using an estrogen receptor (ER)-positive breast cancer cell line, namely MCF-7, to identify potential phytoestrogens and found that an aqueous extract of A. tegmentosum promoted cell proliferation in MCF-7 cells. Five phenolic compounds (1-5) were separated and identified from the active fraction using bioassay-guided fractionation of crude A. tegmentosum extract and phytochemical analysis. The chemical structures of the compounds were characterized as vanillic acid (1), 4-hydroxy-benzoic acid (2), syringic acid (3), isoscopoletin (4), and (E)-ferulic acid (5) based on the analysis of their nuclear magnetic resonance spectra and liquid chromatography-mass spectrometry data. All five compounds were evaluated using an E-screen assay for their estrogen-like effects on MCF-7 cells. Among the tested compounds, only 4-hydroxy-benzoic acid (2) promoted the proliferation of MCF-7 cells, which was mitigated by the ER antagonist, ICI 182,780. The mechanism underlying the estrogen-like effect of 4-hydroxy-benzoic acid (2) was evaluated via western blotting analysis to determine the expression levels of extracellular signal-regulated kinase (ERK), phosphoinositide 3-kinase (PI3K), serine/threonine kinase (AKT), and ERα. Our results demonstrated that 4-hydroxy-benzoic acid (2) induced the increase in the protein expression levels of p-ERK, p-AKT, p-PI3K, and p-Erα, concentration dependently. Collectively, these experimental results suggest that 4-hydroxy-benzoic acid (2) is responsible for the estrogen-like effects of A. tegmentosum and may potentially aid in the control of estrogenic effects during menopause.

Cytotoxicity and estrogenicity of a novel 3-dimensional printed orthodontic aligner

Orthodontic aligners printed with in-office 3-dimensional (3D) procedures have been described, but no data on their biocompatibility exist. This study investigates the cytotoxicity and estrogenicity of a 3D-printed orthodontic aligner by assessing its biological and behavioral effects. Ten sets of 1 type of aligner were immersed in sterile deionized water for 14 days, and the cytotoxicity and estrogenicity of released factors were assessed via MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assays on human gingival fibroblasts and the estrogen-sensitive MCF-7 and the estrogen-insensitive MDA-MB-231 breast cancer cell lines. 17β-Estradiol and bisphenol-A were used as positive controls. The statistical analysis of data was performed with generalized linear models at a 0.05 level of significance. No signs of cytotoxicity were seen for the aligner samples for concentrations (v/v) of 20% (P= 0.32), 10% (P=0.79), or 5% (P=0.76). The antioxidant activity expressed as the capacity to reduce intracellular levels of reactive oxygen species was not affected in the aligner samples (P= 0.08). No significant estrogenicity was induced by thealigner samples compared with eluents from the negative control for both MCF-7 (P= 0.65) and MDA-MB-231 (P= 0.78). As expected, 17β-Estradiol and bisphenol-A stimulated MCF-7 cell proliferation, whereas no effect was observed on MDA-MB-231 cells. In conclusion, if any factors were released during the 14-day aging of 3D-printed aligners in water, these were not found to be cytotoxic for human gingival fibroblasts and did not affect their intracellular reactive oxygen species levels. Moreover, no estrogenic effects of these putative eluates were observed based on an E-screen assay.

Impedance-Based E-Screen Cell Biosensor for the Real-Time Screening of Xenoestrogenic Compounds

The E-screen assay is the gold standard method to screen for xenoestrogens. The method is based on the binding of estrogenic compounds to the human estrogen receptor of MCF-7 cells, resulting in increased cell proliferation. The conventional method is easy to perform but involves multiple manual steps including cell labeling and requires up to 6 days to evaluate the estrogenic activity. Here, we present an impedance-based E-screen cell biosensor in which the MCF-7 cells are cultured on electrodes. Upon estrogen treatment, the increased cell proliferation is detected as an increase in the recorded impedance with time. The estrogenic activity of test compounds is measured by comparing the increased impedance during the exponential growth phase with the impedance recorded in a hormone-free medium, which demonstrates that the impedance increase is directly proportional to the 17β-estradiol concentration. Two different compounds were tested for their estrogenic activities, confirming the known xenoestrogen, bisphenol-A, and the absence of xenoestrogenic activity of the antifouling agent, Irgarol 1051. All impedance-based results are compared to results obtained with the conventional assay. The impedance E-screen cell biosensor is label-free, easier to use, enables real-time analysis, which provides growth kinetic information upon estrogen exposure, and results in a shorter analysis time.

Estrogenic Activity of Mycoestrogen (3β,5α,22E)-Ergost-22-en-3-ol via Estrogen Receptor α-Dependent Signaling Pathways in MCF-7 Cells.

Armillariella tabescens (Scop.) Sing., a mushroom of the family Tricholomataceae, has been used in traditional oriental medicine to treat cholecystitis, improve bile secretion, and regulate bile-duct pressure. The present study evaluated the estrogen-like effects of A. tabescens using a cell-proliferation assay in an estrogen-receptor-positive breast cancer cell line (MCF-7). We found that the methanol extract of A. tabescens fruiting bodies promoted cell proliferation in MCF-7 cells. Using bioassay-guided fractionation of the methanol extract and chemical investigation, we isolated and identified four steroids and four fatty acids from the active fraction. All eight compounds were evaluated by E-screen assay for their estrogen-like effects in MCF-7 cells. Among the tested isolates, only (3β,5α,22E)-ergost-22-en-3-ol promoted cell proliferation in MCF-7 cells; this effect was mitigated by the ER antagonist, ICI 182,780. The mechanism underlying the estrogen-like effect of (3β,5α,22E)-ergost-22-en-3-ol was evaluated using Western blot analysis to detect the expression of extracellular signal-regulated kinase (ERK), phosphatidylinositol 3-kinase (PI3K), Akt, and estrogen receptor α (ERα). We found that (3β,5α,22E)-ergost-22-en-3-ol induced an increase in phosphorylation of ERK, PI3K, Akt, and ERα. Together, these experimental results suggest that (3β,5α,22E)-ergost-22-en-3-ol is responsible for the estrogen-like effects of A. tabescens and may potentially aid control of estrogenic activity in menopause.

In vitro and in vivo estrogenic activity of triclosan

ABSTRACT Triclosan (TCS) is an antibacterial and antifungal agent used in many consumer products and exhibits a chemical structure similar to non-steroidal estrogen, which is known to induce endocrine disruption. Triclosan has been found in human plasma, urine, and breast milk, and the safety of TCS-containing products has been disputed. Although studies attempted to determine the estrogenic activity of TCS, no clear results have emerged. The aim of the present study was to examine estrogenic activity of TCS using an in vitro E-screen assay and an in vivo uterotrophic assay. The in vitro E-screen assay demonstrated that TCS significantly enhanced proliferation of MCF-7 breast cancer cells, although not in a concentration-dependent manner. The in vivo uterotrophic results showed no significant change in the weight of uteri obtained from TCS-administered Sprague-Dawley rats. Further, to understand the estrogenic activity attributed to TCS at the molecular level, gene-expression profiling of uterus samples was performed from both TCS- or estrogen-treated rats and the genes and cellular processes affected by TCS or estrogen were compared. Data demonstrated that both the genes and cellular processes affected by TCS or estrogen were significantly similar, indicating the possibility that TCS-mediated estrogenic activity occurred at the global transcriptome level. In conclusion, in vitro and gene-profiling results suggested that TCS exhibited estrogenic activity.

Efficacy and reusability of mixed-phase TiO2–ZnO nanocomposites for the removal of estrogenic effects of 17β-Estradiol and 17α-Ethinylestradiol from water

Photocatalytic removal of estrogenic compounds (ECs), 17β-estradiol (E2), and 17α-ethinylestradiol (EE2) were assessed using a TiO2–ZnO nanocomposite (NC) over a range of initial EC concentration (Co; 10 mg/L – 0.05 mg/L). Photocatalytic removal was evaluated under UV and visible irradiation using 10 mg/L NC over 240 min duration. After 240 min, analysis using GCxGC TOF MS revealed 100% transformation at Co ≤ 1 mg/L and ≥25% transformation at Co ≤ 10 mg/L under visible irradiation. Degradation was accompanied by breakdown of the fused ring structure of E2, generating smaller molecular weight by-products which were subsequently mineralized as revealed through TOC removal. With UV photocatalysis, ~30% and ~20% mineralization was attained for E2 and EE2, respectively, for Co of 10 mg/L. Under visible irradiation, ~25% and ~10% mineralization was achieved for E2 and EE2, respectively. Estrogenicity variation was estimated using the E-screen assay conducted with estrogen receptor-positive MCF-7 breast cancer cells. Complete removal of estrogenicity of ECs was confirmed after 240 min of photocatalysis under UV and visible irradiation. FTIR spectroscopy-based analysis of the NC after E2 photocatalysis revealed the presence of sorbed organics. Desorption, followed by GC × GC TOF-MS analysis revealed these organics as by-products of photocatalysis. Desorption of sorbed organics followed by recalcination at 600 °C for 1 h regenerated the active sites on the NC, enabling its efficient reuse for 3 cycles under visible irradiation without loss in activity.

Estrogenic activity of ethyl gallate and its potential use in hormone replacement therapy

We aimed to compare the estrogenic activities of compounds isolated from Moutan Cortex Radicis (MRC, Paeonia suffruticosa Andrews) and identify their potential use in hormone replacement therapy. We quantified seven marker components (gallic acid, oxypaeoniflorin, paeoniflorin, ethyl gallate, benzoic acid, benzoylpaeoniflorin, and paeonol) in MRC using a high-performance liquid chromatography simultaneous analysis assay. To investigate the estrogenic activity of MRC and the seven marker components, an E-screen assay was conducted using the estrogen receptor (ER)-positive MCF-7 human breast cancer cell line. Among them, ethyl gallate caused cell proliferation in a concentration-dependent manner at concentrations above 25 µM and was clearly suppressed by combination treatment with the ER antagonist ICI 182,780. Therefore, ethyl gallate may be a compound of MRC that can increase the estrogenic effect in ER-positive MCF-7 cells.

Polarity Difference and the Presence of Phytoestrogen Compounds Affecting Estrogenic Activity of Peperomia pellucida Extracts

Peperomia pellucida (L.) Kunth has been studied as an anti-osteoporotic agent. However, there is no report about its estrogenic activity, which is important for its anti-osteoporotic activity. Thus, the aim of this research was to study the estrogenic potency of P. pellucida extracts. The estrogenic activity of P. pellucida extracts (n-hexane, ethyl acetate, ethanol, and water extracts) was studied using E-screen assay and confirmed with a molecular docking simulations. Further, the presence of phytoestrogen compounds was identified using thin layer chromatography (TLC), TLC densitometry, and high performance liquid chromatography. The n-hexane, ethyl acetate, and ethanol extracts at a concentration of 0.1 μg mL-1 exhibited a partial agonist effect, whereas the water extract showed full agonisteffect at the similar concentration. This activity was produced through a classical ligand-dependent mechanism similar to estradiol. N-hexane and ethyl acetate extracts showed antiestrogenic activity. The TLC chromatogram evidently depicted the presence of quercetin and stigmasterol in the n-hexane and ethyl acetate extracts. Apigenin and apigetrin at concentrations of 0.239±0.076 and 1.063±0.156 μg mg-1 extract, respectively, were present in the water extract. A docking study on estrogen receptors confirmed that apigetrin prefer to produce estrogenic activity, whereas the other compounds can produce both estrogenic and antiestrogenic activity. Hence, we suggest that the bioactive compounds in the water extract are flavonoids, such as apigenin and apigetrin. In summary, the water extract is recommended to be used as an estrogenic agent.

Comparison of In Vitro Endocrine Activity of Phthalates and Alternative Plasticizers.

Because of the deleterious effects of phthalates, regulations have been taken to decrease their use, and the needs for alternatives are increasing. Due to the concerns about the endocrine-disrupting properties of phthalates, it was deemed necessary to particularly investigate these effects for potential substitutes. In this study, we compared the in vitro endocrine activity of several already used potential alternative plasticizers (DEHT, DINCH, and TOTM) or new substitutes (POLYSORB® isosorbide and POLYSORB® ID 46) to one of 2 phthalates, DEHP and DINP. Effects of these chemicals on 3 common mechanisms of endocrine disruption, i.e., interaction with estrogen receptors (ER), androgen receptors (AR), or steroidogenesis, were studied using extensively used in vitro methods. In the E-Screen assay, only DEHP moderately induced MCF-7 cell proliferation; none of the other tested substances were estrogenic or antiestrogenic. No androgenic or antiandrogenic activity in MDA-kb2 cells was shown for any of the tested phthalates or alternatives. On the other hand, both DEHP and DINP, as well as DEHT, DINCH, and TOTM, disrupted steroidogenesis in the H295R assay, mainly by inducing an increase in estradiol synthesis; no such effect was observed for POLYSORB® isosorbide and POLYSORB® ID 46.

Isolation and Characterization of Phenylpropanoid and Lignan Compounds from Peperomia pellucida [L.] Kunth with Estrogenic Activities.

Extracts of Peperomia pellucida [L.] Kunth have previously been demonstrated to have in vivo estrogenic-like effects, thereby functioning as an anti-osteoporotic agent. However, the compounds responsible for these effects have not yet been determined. Therefore, the aim of this study is to isolate and elucidate potential compounds with estrogenic activity. The structures of the isolated compounds were identified using 1D 1H and 13C-NMR and confirmed by 2D FT-NMR. The estrogenic activity was evaluated using the E-SCREEN assay, and a molecular docking study was performed to predict the binding affinity of the isolated compounds to estrogen receptors. In this experiment, we successfully isolated three phenylpropanoids and two lignan derivatives, namely, 6-allyl-5-methoxy-1,3-benzodioxol-4-ol (1), pachypostaudin B (2), pellucidin A (3), dillapiole (4), and apiol (5). Among these compounds, the isolation of 1 and 2 from P. pellucida is reported for the first time in this study. Activity assays clearly showed that the ethyl acetate extract and its fractions, subfractions, and isolated compounds exerted estrogenic activity. Methanol fraction of the ethyl acetate extract produced the highest estrogenic activity, while 1 and 2 had partial agonist activity. Some compounds (derivates of dillapiole and pellucidin A) also had, in addition, anti-estrogenic activity. In the docking study, the estrogenic activities of 1–5 appeared to be mediated by a classical ligand-dependent mechanism as suggested by the binding interaction between the compounds and estrogen receptors; binding occurred on Arg 394 and His 524 of the alpha receptor and Arg 346 and His 475 of the beta receptor. In summary, we reveal that P. pellucida is a promising anti-osteoporotic agent due to its estrogenic activity, and the compounds responsible for this activity were found to be lignan and phenylpropanoid derivatives. The presence of other compounds in either the extract or fraction may contribute to a synergistic effect, as suggested by the higher estrogenic activity of the methanol fraction. Hence, we suggest further research on the osteoporotic activity and safety of the identified compounds, especially regarding their effects on estrogen-responsive organs.

Estrogens Determine Adherens Junction Organization and E-Cadherin Clustering in Breast Cancer Cells via Amphiregulin.

SummaryEstrogens play an important role in the development and progression of human cancers, particularly in breast cancer. Breast cancer progression depends on the malignant destabilization of adherens junctions (AJs) and disruption of tissue integrity. We found that estrogen receptor alpha (ERα) inhibition led to a striking spatial reorganization of AJs and microclustering of E-Cadherin (E-Cad) in the cell membrane of breast cancer cells. This resulted in increased stability of AJs and cell stiffness and a reduction of cell motility. These effects were actomyosin-dependent and reversible by estrogens. Detailed investigations showed that the ERα target gene and epidermal growth factor receptor (EGFR) ligand Amphiregulin (AREG) essentially regulates AJ reorganization and E-Cad microclustering. Our results not only describe a biological mechanism for the organization of AJs and the modulation of mechanical properties of cells but also provide a new perspective on how estrogens and anti-estrogens might influence the formation of breast tumors.