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Using the Cre-loxP system, we generated the first mouse model in which estrogen receptor-α non-nuclear signaling was inactivated in endothelial cells. Estrogen protection against mechanical vascular injury was impaired in this model. This result indicates the pivotal role of endothelial estrogen receptor-α non-nuclear signaling in the vasculoprotective effects of estrogen., Competing Interests: This work was supported by the Japan Foundation for Applied Enzymology, Japan Heart Foundation Research Grant, SENSHIN Medical Research Foundation, Kobayashi Foundation (Dr Takimoto), Takeda Science Foundation (Drs Ueda and Takimoto), National Institutes of Health HL 052233, National Institutes of Health HL 136962, DK 062729 (Dr Liao), AHA Northeast Research Consortium Postdoctoral Fellowship, Uehara Research Fellowship (Dr Hiroi), and Tri-Service General Hospital Medical Research Foundation Grant TSGH-PH-E-111017 (Dr Liu). All other authors have reported that they have no relationships relevant to the contents of this paper to disclose., (© 2023 The Authors.)

Purpose: Intrauterine adhesion (IUA), often leading to gynecological complications including amenorrhea, abdominal pain and infertility, is frequently induced by injuries to the endometrium. Hence it would be of great benefit to take efforts to prevent adhesion after intrauterine operations. Orally administration of 17β-estradiol (E2) is commonly used to promote endometrium regeneration, but is limited by low concentrations at the injured sites. We aim at preparing an E2-releasing uterine stent, which could improve the efficiency of E2 therapy and be utilized for IUA prevention., Methods: We designed a silicone rubber stent, which could be implanted in the uterine cavity and continuously release E2 in long term. Stents were placed in rodent uterine, and removed at different time points. Remaining E2 in stent was measured by high performance liquid chromatography (HPLC), and organ E2 concentrations were detected by enzyme-linked immuno sorbent assay (ELISA). Endometrium morphology was examined by histological staining of paraffin sections., Results: Our stent showed a controlled release of E2 in rodent uterine for over 60 days, and significantly increased E2 concentration in serum and in situ uterine. After the stent was removed from uterine, E2 rapidly reverted to a normal level. Also, the stent did not induce pathological changes in endometrium., Conclusions: The uterine stent provided abundant local E2 in uterine cavity with satisfactory safety. The silicone rubber based E2-releasing uterine stent could be further advanced by adjusting its shape and E2 load for its clinical application, and might promisingly help lowering the incidence of IUA., Competing Interests: The authors declare that they have no known competing interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Japanese Society for Regenerative Medicine. Production and hosting by Elsevier B.V.)

Abstract: Estrogenic activities of cigarette smoke condensates obtained from the extraction of particulate matters from mainstream and sidestream cigarette smoke with benzene/ethanol were evaluated by using a yeast two-hybrid assay system expressing human estrogen receptor α (hERα). To identify the constituents of the cigarette smoke condensate which are responsible for the estrogenic activity, the condensate was fractionated into eleven fractions by liquid–liquid extractions. Among these fractions, the neutral fractions of mainstream and sidestream smoke showed the strongest estrogen receptor-mediated activity by the yeast two-hybrid assay. Then the neutral fractions were fractionated by medium-pressure liquid chromatography with silica gel column. In the fractions that showed strong estrogenic activity, 2-hydroxyfluorene (2-OHFle), 2- and 3-hydroxyphenanthrene (2- and 3-OHPhe), 1-hydroxypyrene (1-OHPyr) and n-propyl-p-hydroxybenzoate (n-PHB) were identified by LC- and GC-MS and HPLC with fluorescence detection. 2-OHFle, 2-OHPhe and n-PHB exhibited estrogenic activity, whereas weak activity was observed with 3-OHPhe and 1-OHPyr. Several other hydroxylated polycyclic aromatic hydrocarbons having no activity were also identified. This is a first study to identify estrogenic hydroxylated PAHs in cigarette smoke condensate. The present findings points out the necessity for detailed investigation of exposure to aerosols containing apparently estrogenic compounds. [Copyright &y& Elsevier]

Abstract: In this study, we investigated the effects of endocrine disrupters bisphenol A (BPA) and nonylphenol (NP) on insulin secretion from rat pancreatic islets. Following acute exposure to BPA and NP, neither BPA nor NP (0.1, 1, 10, 100 and 1000μg/l) affected insulin secretion in concentrations of 16.7mM glucose. However, insulin secretion following long-term exposure to BPA or NP for 24h in 16.7mM glucose was significantly higher than without exposure. To determine whether increased insulin secretion resulting from long-term exposure to BPA and NP is induced via intracellular estrogen receptors, we blocked the cytosolic/nuclear estrogen receptors, using actinomycin-D (Act-D), an inhibitor of RNA synthesis, and ICI 182,780 (ICI), an estrogen receptor inhibitor. Following long-term exposure to BPA (10μg/l) or NP (10μg/l), Act-D or ICI treatment eliminated the facilitation of insulin secretion. In conclusion, we have demonstrated for the first time that long-term exposure to endocrine disrupters, such as BPA and NP, promotes in vitro insulin secretion from the pancreatic islets, via cytosolic/nuclear estrogen receptors. [Copyright &y& Elsevier]

Abstract: In this study, we examined the effects of exposure to phytoestrogen (daidzein), 17β-estradiol (E2), diethylstilbestrol (DES) and staurosporin on the TM4 testicular cell line, using comprehensive analysis, such as cDNA microarray and two-dimension polyacrylamide gel electropholesis (2D-PAGE) analysis, and we demonstrated if these toxicogenomic analyses could classify the chemical compounds. First, RNA was extracted from TM4 cells that had been treated with daidzein (80μM), DES, E2 (40μM) and stauroporin (100nM) for 30min. We performed cDNA microarray analysis, and the expression ratio data thus obtained were then analyzed using hierarchical clustering. This hierarchical clustering showed that daidzein exposure induced a different effect on gene expression change from that of E2, DES and staurosporin. Next, protein extracted from TM4 cells also underwent cDNA microarray analysis for 3h. We performed 2D-PAGE analysis, and the spot intensity ratio data thus obtained were analyzed using hierarchical clustering. As with cDNA microarray, the hierarchical clustering of protein spot ratios showed that daidzein exposure induced a different effect on gene expression change from that of the other substances. In conclusion, we have demonstrated for the first time that classification of these chemicals can be performed by clustering analysis, using data from cDNA microarray and 2D-PAGE analyses, and that exposure to daidzein induces effects different from those of E2, DES and staurosporin. [Copyright &y& Elsevier]

Abstract: Metabolism of polycyclic aromatic hydrocarbons (PAHs) has been studied intensively, and potential metabolites with estrogenic activity have been identified previously. However, little attention has been paid to the metabolic pathways in mammalians and to the combined effect of individual metabolites. Several hydroxylated metabolites of benzo[a]pyrene (BaP) and chrysene (CHN) were formed by rat liver microsomal cytochrome P450 (CYP) activity, some of which possess estrogenic activity. All mono- and several dihydroxylated metabolites of BaP and CHN were tested for ER affinity and estrogenic activity in a proliferation assay (E-screen) and in a reporter-gene assay (ER-CALUX). Twelve estrogenic metabolites were identified with EC50 values ranging from 40nM to 0.15mM. The combined effect of a mixture of seven PAH-metabolites was also studied in the ER binding assay. At concentrations that show little activity themselves, their joint action clearly exhibited significant estrogenic activity. BaP itself exhibited estrogenicity in the ER-CALUX assay due to bio-activation into estrogenic metabolites, probably via aryl hydrocarbon receptor (AhR) induced CYP activity. Furthermore, 2-hydroxy-CHN (2-OHCHN) induced supra-maximal (400%) estrogenic effects in the ER-CALUX assay. This effect was entirely ER-mediated, since the response was completely blocked with the ER-antagonist ICI182,780. We showed that 2-OHCHN increased ER-concentration, using ELISA techniques, which may explain the observed supra-maximal effects. Co-treatment with the AhR-antagonist 3′,4′-dimethoxyflavone (DMF) enhanced ER-signaling, possibly via blockage of AhR-ER inhibitory cross-talk. [Copyright &y& Elsevier]

The effects on cell proliferation of arylhydrocarbon receptor (AhR) agonists in estrogen-responsive T47D and ZR-75-1 cells were investigated. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and the non-ortho-substituted polychlorinated biphenyl (PCB) congeners, PCB 77, PCB 81, PCB 126, and PCB 169 each inhibited 17β-estradiol (E2)-stimulated cell proliferation in a dose–responsive manner. In the absence of added E2, TCDD, PCB 77, PCB 81, and PCB 169 had no significant effect on cell proliferation, while PCB 126 at high concentrations caused slight elevations. The order of effective inhibition of E2-stimulated cell proliferation by the PCB congeners was: PCB 81>PCB 126≃PCB 169>PCB 77. In the comparative literature, mammalian TEFs for these congeners toxic potency are in the order: PCB 126>PCB 169>PCB 81≃PCB 77 [Organohalogen Compd. 34 (1997) 237]. Our results thus show an unexpected different pattern for the inhibitory effects of PCBs congeners on E2-mediated cell proliferation. [Copyright &y& Elsevier]

The estrogen receptors (ERs) are members of a super family of ligand-activated transcription factors mediating estrogenic responses. A close functional kinship was found for the structurally related estrogen receptor-related receptor1 (ERR1), a constitutively active transcription factor. The aryl hydrocarbon receptor (AhR) mediates the toxic and estrogenic effects of a wide variety of environmental contaminants and industrial pollutants. Both the ERR1 and the AhR are known to modulate the ER’s signalling pathways in multiple ways. Organic chemicals with a certain structural relationship to steroid hormones often induce a tissue- or cell-specific variety of responses distinct from estrogenic responses and this may involve ERR1 and AhR. The UV-screens benzophenone-2 and benzophenone-3 (BP2, BP3), structurally related to known steroid receptor ligands, are used in cosmetics and plastics to improve product stability and durability. Both BP2 and BP3 were shown to exert uterotrophic effects and BP2 was shown to bind to the estrogen receptors. Whether such effects are also exerted in other organs is unknown. Therefore, an approach to a multi-organic risk assessment for these substances was made by measuring the gene-expression of the four mentioned receptors in the pituitary, the uterus and the thyroid after a five-day treatment in comparison to estradiol. Though BP2 seems to exert an estrogen-like effect while BP3 does not, there are regulatory effects on receptor expression for both substances that indicate a kind of endocrine disruption that is not assessed by “classical” estrogenic markers. [Copyright &y& Elsevier]

Steroid estrogens have been identified in the solid matrices of unit treatment processes in sewage treatment works (STWs) and in sediments of watercourses that receive effluent. This article discusses the sample preparation and analytical considerations necessary for reliable determination and the need to evaluate for possible matrix interferences during method development. Complementing this is a case study highlighting the potential for analyte transformation during sample preparation and the phenomena of ion suppression when utilising LC/MS ESI with a comparison of method recoveries by GC/MS. We discuss the use of LC/MS/MS and TOF instruments; however, at present, their use in environmental analyses appears to be limited because of their capital costs. [Copyright &y& Elsevier]

17β-Estradiol (E2) regulates neuronal activity via genomic and rapid, non-genomic mechanisms. The rat serotonergic neuronal cell line (RN46A) was used to investigate the rapid effects of E2 on serotonin (5-HT) reuptake and on potential intracellular signaling pathways. RN46A cells express the serotonin transporter (SERT) and estrogen receptor (ER)β, but not ERα. Fifteen minute E2 treatment (10-9M) decreased 5-HT uptake. Intracellular cAMP levels were not increased by 15min E2 treatment; however, E2 caused an increase in intracellular Ca2+ levels, with a maximum response within the first minute. The response was E2 specific, since other steroids (17α-estradiol, testosterone, and progesterone) had no effect. The ER antagonist ICI 182,780 blocked the rapid E2 effects on intracellular Ca2+ levels as did the selective ER modulator tamoxifen. In summary, changes in intracellular Ca2+ levels caused by E2 and mediated through ERβ may be responsible for observed rapid effects of E2 on SERT activity. [Copyright &y& Elsevier]

The anti-17β-estradiol antibody 57-2 has been a subject for several protein engineering studies that have produced a number of mutants with improved binding properties. Here, we generated a set of 16 antibody 57-2 variants by systematically combining mutations previously identified from phage display-derived improved antibody mutants. These mutations included three point mutations in the variable domain of the light-chain and a heavy-chain variant containing a four-residue random insertion in complementarity determining region CDR-H2. The antibody variants were expressed as Fab fragments, and they were characterized for affinity toward estradiol, for cross-reactivity toward three related steroids, and for dissociation rate of the Fab/estradiol complex by using time-resolved fluorescence based immunoassays. The double-mutant cycle method was used to address the cooperativity effects between the mutations. The experimental data were correlated with structural information by using molecular modeling and visual analysis of the previously solved antibody 57-2 crystal structures. These analyses provided information about the steroid-binding mode of the antibody, the potential mechanisms of individual mutations, and their mutual interactions. Furthermore, several combinatorial mutants with improved affinity and specificity were obtained. The capacity of one of these mutants to detect estradiol concentrations at a clinically relevant range was proved by establishing a time-resolved fluorescence based immunoassay. [ABSTRACT FROM AUTHOR]

Our understanding of estrogen signaling has undergone a true paradigm shift over recent years, following the discovery in 1995 of a second estrogen receptor, estrogen receptor β (ERβ). In many contexts ERβ appears to antagonize the actions of ERα (yin/yang relationship) although there also exist genes that are specifically regulated by one of the two receptors. Studies of ERβ knockout mice have shown that ERβ exerts important functions in the ovary, central nervous system, mammary gland, prostate gland, hematopoiesis, immune system, vessels and bone. The use of ERβ-specific ligands against certain forms of cancer represents one of the many pharmaceutical possibilities that have been created thanks to the discovery of ERβ. [Copyright &y& Elsevier]

The in vitro release characteristics of eight low-molecular-weight drugs (clindamycin, 17β-estradiol, 17β-estradiol-3-acetate, 17β-estradiol diacetate, metronidazole, norethisterone, norethisterone acetate and oxybutynin) from silicone matrix-type intravaginal rings of various drug loadings have been evaluated under sink conditions. Through modelling of the release data using the Higuchi equation, and determination of the silicone solubility of the drugs, the apparent silicone elastomer diffusion coefficients of the drugs have been calculated. Furthermore, in an attempt to develop a quantitative model for predicting release rates of new drug substances from these vaginal ring devices, it has been observed that linear relationships exist between the log of the silicone solubility of the drug (mg ml−1) and the reciprocal of its melting point (K−1) (y=3.558x−9.620, R=0.77), and also between the log of the diffusion coefficient (cm2 s−1) and the molecular weight of the drug molecule (g mol−1) (y=−0.0068x−4.0738, R=0.95). Given that the silicone solubility and silicone diffusion coefficient are the major parameters influencing the permeation of drugs through silicone elastomers, it is now possible to predict through use of the appropriate mathematical equations both matrix-type and reservoir-type intravaginal ring release rates simply from a knowledge of drug melting temperature and molecular weight. [Copyright &y& Elsevier]

We describe a highly efficient and sensitive yeast-based screening method for isolating human estrogen receptor α (ERα) mutants with altered trans-activation activity. This method takes advantage of the fact that estrogen receptor is a ligand-activated transcription factor, and links the transactivation activity of estrogen receptor to the growth rate of yeast cells. We used this method to screen a library of human ERα mutants created by random mutagenesis of the ligand binding domain of human ERα in the presence of ligand 17β-estradiol (E2). We isolated several human ERα mutants with significantly altered trans-activation activity toward E2 in yeast cells. We also used this method to screen a library of chemical compounds and showed that it can be used to rapidly identify estrogenic compounds and the different cell growth rates for these estrogenic compounds correlated well with their relative binding affinities. Thus, this method is suitable for selecting novel estrogenic compounds and estrogen receptor mutants. In principle, this method might also be used to isolate mutants of any nuclear receptors with altered trans-activation activity, which may greatly facilitate their structural and functional studies. [Copyright &y& Elsevier]

Resveratrol (RES) is a natural polyphenol present in red wines and various human food items. The estrogenic activity of RES was demonstrated in two in vitro assay systems, i.e. binding to human estrogen receptor α and stimulation of MCF-7 cell proliferation. To investigate the inhibition of cell proliferation observed at high concentrations of RES, we analyzed the compound for genotoxic potential. RES induced cellular toxicity, micronuclei, and metaphase chromosome displacement in L5178Y mouse lymphoma cells. Likewise, the induction of micronuclei was observed in Chinese hamster V79 cells. Determination of kinetochore signals in micronuclei and cell cycle analysis suggested that RES did not cause a direct disturbance of mitosis. In support of this notion, cell-free tubulin polymerization studies indicated no direct effect of RES on microtubule assembly. According to an estimation of daily intake and bioavailability, concentrations that were found genotoxic in vitro might be reached in human exposure. On the other side, the estrogenic acitivity might be beneficial. Therefore, further investigations of mechanisms, possibly including animal models, would be desirable to clarifiy a potential risk for humans. [Copyright &y& Elsevier]

Estrogen induces rapid and extensive degeneration of rodent uterine myometrial sympathetic innervation. To clarify the underlying mechanisms, we used explant cultures to assess whether estrogen affects the myometrium’s ability to induce sympathetic neuritogenesis and the sympathetic neuron’s ability to respond. Superior cervical ganglion explants from ovariectomized adult donors extended neurites when cultured alone in serum-free medium, and their numbers increased 2.3-fold in the presence of myometrial explants from ovariectomized adult rats. The myometrium’s ability to induce neuritogenesis was abolished by injection of myometrium donors with 17β-estradiol 24 h prior to tissue harvest. Myometrial neurite-promoting effects were also abolished by adding 2×10−8M estradiol to the culture medium. Because outgrowth from ganglia of ovariectomized rats cultured alone was not affected by estrogen in the culture medium, this indicates that estrogen acts directly on myometrium to abrogate its neurite-promoting effects. However, estrogen injection of ganglion donor rats also inhibited neurite extension toward ovariectomized myometrium, suggesting that some factor in ovariectomized rats normally acts on the ganglion to prevent estrogen from inhibiting neurite outgrowth. When ganglia from hypophysectomized ovariectomized donors were cultured alone, neuritogenesis was normal but estrogen added to the culture medium now attenuated outgrowth. Prolactin but not other pituitary-derived hormones reversed the suppression of neuritogenesis induced by estrogen.We conclude that estrogen acts directly on myometrium to inhibit its neuritogenic effects on sympathetic neurons. Estrogen can also attenuate neurite formation by acting directly on the ganglion; this effect normally is not apparent at low estrogen levels because a pituitary hormone (possibly prolactin) prevents the ganglion from responding fully to estrogen. With high in vivo estrogen, this pituitary hormone’s effects are abated, possibly through diminished release, and estrogen directly reduces ganglion neuritogenesis. Thus, estrogen regulates uterine sympathetic nerve remodeling through actions on myometrium, ganglion, and intermediary pituitary factors. [Copyright &y& Elsevier]

Fatty acid amide hydrolase (FAAH) is a membrane-bound enzyme that inactivates a family of fatty acid amide molecules which are implicated in physiological processes such as pain and sleep. We cloned a 1.9 kb fragment of the 5′-untranslated region of the mouse FAAH gene into the pGL3 basic luciferase reporter vector and showed that this sequence has promoter activity in vitro. By primer extension analysis, we have determined the transcription start site to be 200 bases upstream of the ATG initiation codon and found that a TATA motif was absent. A number of putative response elements, including those for estrogen and glucocorticoids, were identified in this sequence. We have demonstrated that the estrogen and glucocorticoid receptors down-regulate transcriptional activity independent of their ligand. These data should help in understanding the mechanisms of FAAH gene transcription. [Copyright &y& Elsevier]

Estrogen treatment offers neuro-protection in animal experiments in which excitotoxic mechanisms destroy neurons. In a model of delayed neuronal degeneration that depends on excitotoxicity, we tested whether females had an altered susceptibility, and whether physiologic doses of estrogen administered after the brain insult would protect susceptible neurons. Females were ovariectomized, exposed to striatal-pallidal ibotenic acid injury that caused delayed degeneration of substantia nigra neurons, and treated with 17β-estradiol (30 μg, subcutaneously every other day, beginning 2 days after the striatal injury) or vehicle. At 6 and 8 days post lesion, the 17β-estradiol treatment group maintained over 87 and 70% of control nigral neuron number, respectively. Physiologic levels of estrogen delivered days after the excitotoxic stress completely protected neurons in the substantia nigra reticulata 6 days post lesion and slowed degeneration 8 days post lesion. [Copyright &y& Elsevier]

Increased vitellogenin (vtg) levels in the blood of male fish are frequently used as an indicator of estrogenic exposure. Similar responses are expected for mussels, where the concentration of vtg-like proteins has been reported to depend on estrogens. To verify the role of hemolymph during vitellogenesis of mussels, the saltwater mussel Mytilus edulis and the freshwater mussel Anodonta cygnea were exposed to 17β-estradiol (E2) and wastewater treatment plant effluents, known for their estrogenic potential. Gel electrophoresis did not reveal any significant induction (or repression) of plasma proteins compared to control plasma. Our results do not support the hypothesis that mussel hemolymph is a carrier of estrogen-dependent major egg-yolk precursors (vtg-like proteins). However, additional information on a 35±2-kDa hemolymph protein, previously reported to bind heavy metals, was obtained by high-resolution two-dimensional electrophoresis. It was resolved in a cluster of single proteins with properties that match the characteristics of a previously reported histidine-rich glycoprotein. [Copyright &y& Elsevier]

Raloxifene (Ral) has estrogenic activity in bone and cardiovascular tissues, but is antiestrogenic in breast and has limited uterotrophic activity in mice. Here we report that Ral stimulates the growth of human endometrial Ishikawa tumors implanted in the mammary fat pad of nude ovariectomized mice. In cultured Ishikawa cells, Ral has agonist effects on transcription mediated by the progesterone receptor, an endogenous estrogen target gene, and on expression of reporter genes containing estrogen response elements (EREs). Both Ral and tamoxifen (Tam), but not estradiol, stimulated transcription mediated by the activator protein 1 at micromolar concentrations. However, this effect correlated with induction of cellular death at high concentrations of Ral or Tam and was not observed at lower concentrations. Our results suggest that Ral has stimulatory effects in Ishikawa cells on both cellular growth and gene transcription, and that EREs can mediate some of these effects. [Copyright &y& Elsevier]

Estradiol exerts beneficial effects on neurodegenerative disorders associated with the decline of cognitive performance. The present study was designed to further investigate the effect of 17β-estradiol on learning and memory, and to evaluate its neuroprotective action on cholinergic cells of the nucleus basalis magnocellularis, a neural substrate of cognitive performance. Female rats were ovariectomized at an age of 6 months. Three weeks later they received injections of either a mid-physiological dose of 17β-estradiol or vehicle (oil), every other day for 2 weeks. The effect of estradiol on cognitive performance was tested in two associative learning paradigms. In the two-way active shock avoidance task estradiol-replaced animals learned significantly faster, while in the passive shock avoidance test no differences were observed between the experimental groups. Subsequent unilateral infusion of N-methyl-D-aspartate in the nucleus basalis magnocellularis resulted in a significant loss of cholinergic neurons concomitant with the loss of their fibers invading the somatosensory cortex. Estradiol treatment did not affect the total number of choline-acetyltransferase-immunoreactive neurons and their coexpression of the p75 low-affinity neurotrophin receptor either contralateral or ipsilateral to the lesion. In contrast, cholinergic fiber densities in estradiol-treated animals were greater both in the contralateral and ipsilateral somatosensory cortices as was detected by quantitative choline-acetyltransferase and vesicular acetylcholine transporter immunocytochemistry. However, estradiol treatment did not affect the lesion-induced relative percentage loss of cholinergic fibers. A significant decline of synaptophysin immunoreactivity paralleled the cholinergic damage in the somatosensory cortex of oil-treated animals, whereas an almost complete preservation of synaptic density was determined in estradiol-treated rats.Our results indicate that estradiol treatment enhances the cortical cholinergic innervation but has no rescuing effect on cholinergic nerve cells in the basal forebrain against excitotoxic damage. Nevertheless, estradiol may restore or maintain synaptic density in the cerebral cortex following cholinergic fiber loss. This estradiol effect may outweigh the lack of cellular protection on cholinergic cells at the functional level. [Copyright &y& Elsevier]

The effects of dehydroepiandrosterone, estradiol and testosterone on 1-methyl-4-phenylpyridium (MPP+)-induced neurotoxicity of the nigrostriatal dopaminergic system were examined in rat. They were subjected to a unilateral intrastriatal infusion of the following treatment conditions: MPP+ alone or co-injection of MPP+ plus each hormone. Four days after injection, concentrations of dopamine and their metabolites were determined from the corpus striatum. To corroborate the neurochemical data an immunohistochemical analysis of tyrosine hydroxylase-immunoreactive fibers and acetylcholinesterase histochemistry in the striatum was performed. Moreover, we performed a dose–response study of the three hormones on the high-affinity dopamine transport system in rat striatal synaptosomes.Rats co-injected within the striatum with MPP+ and either dehydroepiandrosterone or estradiol had significantly greater concentrations of dopamine and less tyrosine hydroxylase-immunoreactive fibers and acetylcholinesterase fiber density loss compared with their respective controls. In addition, 4 days after injection, the brain was fixed and cut into coronal sections, and was immunostained with major histocompatibility complex class II antigens for activated microglia, and glial fibrillary acidic protein for activated astrocytes. Dehydroepiandrosterone also attenuated microglial cell activation. In contrast, testosterone showed reductions in dopamine concentrations similar to those obtained by MPP+. The protective effect of dehydroepiandrosterone against the MPP+ neurotoxic dopaminergic system may be produced by its partial prevention of MPP+ inhibition of NADH oxidase activity, whereas the estradiol may function as a neuroprotectant by reducing the uptake of MPP+ into dopaminergic neurons.Our findings we suggest indicate that dehydroepiandrosterone and estradiol by a non-genomic effect may have an important modulatory action, capable of attenuating degeneration within the striatum, and in this way serve as neuroprotectants of the nigrostriatal dopaminergic system. [Copyright &y& Elsevier]

Arginine-140 and isoleucine-141 were identified as key determinants of 17β-estradiol (E2) binding affinity of the sex-steroid-binding protein (SBP, or SHBG) of human plasma. Amino acid residues that differ between human and rabbit SBP sequences were replaced in the human protein and the products tested for lowered E2binding activity as are seen in the rabbit protein. Only mutants containing either R140K or I141L replacements display an E2 equilibrium dissociation constant (Kd) higher than the wild type, reaching a value of 30 nM when both were present. The 5α-dihydrotestosterone (DHT) equilibrium dissociation constant of these mutants was unaffected. The quadruple mutant M107I/I138V/R140K/I141L yielded an E2 Kd of 65 nM, significantly closer to the 80 nM rabbit SBP E2 Kd value. Although mutants containing the M107I and I138V replacements in the absence of R140K and I141L had normal E2 Kds, the presence of the M107I replacement in the quadruple mutant was necessary to obtain an accurate E2 Kd value by competitive Scatchard analysis. Molecular modeling using coordinates for the recently determined N-terminal domain of human SBP revealed a significant shift of the F56 phenyl ring away from ring A of E2 in mutant models containing the R140K and I141L replacements. We conclude that R140 and I141 are required for sustaining the right proximity of the phenyl ring of F56 to ring A of 17β-estradiol, thus optimizing the E2-binding affinity of human SBP. [ABSTRACT FROM AUTHOR]

A high-performance liquid chromatography with diode array detector (HPLC-DAD) method was developed and validated for the simultaneous quantification of 4 xenoestrogens in water for monitoring their photocatalytic degradation in synthetic water. The analytical parameters evaluated were linearity, limits of detection, and quantification (LODs and LOQs), selectivity, and accuracy, according to the US Food and Drug Administration (FDA) and Eurachem guidelines. The developed method shows good linearity (R 2  > 0.995 for all compounds), and LODs ranged from 0.02 to 0.04 mg L -1 , while LOQs ranged from 0.05 to 0.11 mg L -1 . Moreover, accuracy expressed as recovery and precision were within the required limits. Therefore, the developed method was considered accurate, and reliable. In addition, it was successfully applied for monitoring a mixture of 4 xenoestrogens in water during the photocatalytic treatment.•An HPLC-DAD method was developed to quantify 4 xenoestrogens in water simultaneously.•The developed HPLC-DAD method shows excellent linearity, selectivity, and accuracy.•A mixture of 4 xenoestrogens was reliably monitored during their photocatalytic degradation., (© 2022 The Author(s). Published by Elsevier B.V.)

Objectives: Estradiol is known to exert a protective effect against atherosclerosis, but the mechanism(s) whereby this protection is mediated is/are unclear. However, estradiol-treated castrated animals exhibit increased activity of endothelium-derived relaxing factor (EDRF), which could contribute to vasculoprotection. In the present work, we investigated the molecular mechanism(s) of the enhancement of EDRF activity in the thoracic aorta of oophorectomized female rats given 17β-estradiol (E2, 2 or 40 μg/kg/day) compared to those given a placebo. Methods and Results: The abundance in the thoracic aorta of NO synthase I, II and III mRNA (using RT-PCR) and of NO synthase I, II and III immunoreactive protein (using Western blotting) was unaltered by E2. NO synthase activity (based on arginine/citrulline conversion) in thoracic aorta homogenates did not differ significantly among the three groups, suggesting that NO production was not enhanced by E2. In contrast, lucigenin-enhanced chemiluminescence of aorta from the E2 group was decreased compared to that of the placebo group. Desendothelialization and exogenously added superoxide dismutase suggested that this difference was due to a decrease in extracellular endothelium-derived production of superoxide anion (O2−⋅). Experiments in cultured bovine aortic endothelial cells confirmed a decreased extracellular production of O2−⋅ in response to ethinylestradiol (1 nM) using both lucigenin-enhanced chemiluminescence and ESR spectroscopy. Luminol-enhanced chemiluminescence revealed that ethinylestradiol-treated cultured endothelial cells generated less peroxynitrite (the byproduct of NO⋅ and O2−⋅ interaction) than control cells. Conclusion: Estradiol increases rat aorta EDRF activity in the absence of changes in endothelial NO synthase gene expression. The decreased endothelium-derived generation of O2−⋅ in response to estrogens could account for enhanced EDRF–NO bioactivity and decreased peroxynitrite release. All of these effects could contribute to the vascular protective properties of estrogens. [ABSTRACT FROM PUBLISHER]

Background and Aim: Trigonella foenum-graecum L. seeds (TFG) are used as spices in Indian cuisine. In Indian traditional medicine, TFG is used to treat diabetes, dyslipidemia, obesity, arthritis, cancer, digestive disorders, and postmenopausal conditions. Pathophysiology of postmenopausal diseases involves low-grade systemic inflammation. The purpose of this study is to investigate the prophylactic effect of petroleum ether fraction of TFG-extract (PE-TFG) on inflammatory markers, and histopathological changes in ovariectomized rats (OVX-rats) fed with a high-fat diet (HFD)., Experimental Procedure: OVX female Sprague Dawley rats were used for the study. Three weeks after ovariectomy, rats were randomized in different groups and administered PE-TFG, atorvastatin, diosgenin, 17β-estradiol for 12 weeks along with HFD. The sham-operated rats (S.OVX) were fed with a standard pellet diet. At the end of 12-weeks, rats were sacrificed, and blood samples were used to estimate lipid profile, glucose, hepatic markers, TNF-α, and leptin. Liver, kidney, and common carotid artery were isolated for testing oxidative stress markers, mRNA expression of adiponectin, PPAR-γ, and histopathological changes., Results: Administration of PE-TFG significantly decreased (P < 0.05) total cholesterol, LDL, hepatic markers, leptin, TNF-α and improved mRNA expression of adiponectin and PPAR-γ in HFD-fed OVX-rats. Further, micro and macro hepatic steatosis, inflammation, glomerular hypertrophy, degenerated tubules in kidney, increased tunica intima, and media thickness of common carotid artery and the pathological changes were not significant upon PE-TFG administration compared to S.OVX-rats., Conclusion: PE-TFG protects cellular inflammation and metabolic alternations in HFD-fed OVX-rats and thus can be explored further in postmenopausal diseases as a prophylactic agent., Competing Interests: The authors declare that there is no conflict of interest in publishing the data generated in this work., (© 2021 Center for Food and Biomolecules, National Taiwan University. Production and hosting by Elsevier Taiwan LLC.)

Coffee brewed from capsule machines may contain estrogenic chemicals migrated from plastic, but the estrogenic activity of capsule coffee has not been evaluated. This study evaluated the estrogenic activity of capsule coffee using the VM7Luc4E2 estrogen receptor transcriptional activation assay. Estrogenic potentials of six capsule coffee samples were calculated using relative maximum amplitude response of E2 (>15%RME2 indicative of estrogenic activity) and estradiol equivalent factor (EEF). Estrogenic chemical content was determined using ultra-performance liquid chromatography with tandem mass spectrometry. All capsule coffee samples possessed estrogenic activity (48-56%RME2). EEFs were 6-7 orders of magnitude lower than that of E2, (1.2 × 10 -7 -1.7 × 10 -6 ), indicating substantially weaker estrogenic potencies. Bisphenol A, bisphenol F, benzophenone, 4-nonylphenol, dibutyl phthalate, and dimethyl terephthalate were detected in capsule coffee. Capsule coffee exhibited estrogenic activity in vitro, and its estrogenic chemical content is likely driving its estrogenicity, warranting further investigations to fully understand the degree to which they are related and to predict the estrogenic potential based on the concentration of estrogenic chemicals., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Author(s).)

Benzyl salicylate (BS) is a natural ingredient of essential oils and a widely used fragrance chemical. A number of in vitro screening studies have evaluated the estrogenic potential of BS with ambiguous results. Lack of dose-response information for the positive control 17β-estradiol (E2) in most studies makes an assessment of the relative potency and efficacy challenging. Notwithstanding this difficulty, BS has been added as the only fragrance ingredient to the list of the first 14 substances to be screened as potential endocrine disruptors by the European Scientific Committee for Consumer Safety (SCCS) and it is included in the Community rolling action plan (CoRAP) of the European REACH regulation to be assessed for the same property. Here we review all literature evidence and present new data to quantify the in vitro potency and efficacy of BS vs. E2 with full dose response analysis in both an estrogen response element (ERE) depending reporter gene assay and in the MCF7 cell proliferation (E-screen) assay. In both assays, very similar results for BS were found. BS is a partial agonist exhibiting 35-47 % maximal efficacy and it is active only close to the cytotoxic concentration. The extrapolated concentration to achieve 50 % efficacy is 21'000'000 higher as compared to E2 in the reporter gene assay. A ca. 36'000'000 higher concentration of BS as compared to E2 is required to reach equivalent partial cell proliferation stimulation in the MCF7 proliferation assay. This potency is significantly below the agonistic activity of known chemicals which cause estrogenic effects in in vivo assays. Importantly, in this study the weak agonistic activity is for the first time directly related to the activity of E2 in a full quantitative comparison in human cell lines which may help ongoing evaluations of BS by regulatory bodies., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests. All authors are employees of Givaudan, a company manufacturing and using benzyl salicylate. The authors receive no benefits except their salary for conducting this research., (© 2021 The Authors.)

Cancer cell invasion into the surrounding extracellular matrix (ECM) takes place when cell-cell junctions are disrupted upon epithelial-to-mesenchymal transition (EMT). Both cancer cell-stroma and cell-cell crosstalk are essential to support the continuous tumor invasion. Cancer cells release microvesicles and exosomes containing bioactive molecules and signal peptides, which are recruited by neighboring cells or carried to distant sites, thus supporting intercellular communication and cargo transfer. Besides this indirect communication mode, cancer cells can develop cytoplasmic intercellular protrusions or tunneling nanotubes (TNTs), which allow the direct communication and molecular exchange between connected distinct cells. Using scanning electron microscopy (SEM) we show for the first time that MDA-MB-231 (high metastatic potential) and shERβ MDA-MB-231 (low metastatic potential) breast cancer cells cultured on fibronectin and collagen type I or 17β-estradiol (E2) develop TNTs and very long flexible filopodia. Interestingly, the less aggressive shERβ MDA-MB-231 cells treated with E2 in 3D collagen matrix showed the highest development of TNTs and filopodia. TNTs were often associated to adhering exosomes and microvesicles surfing from one cell to another, but no filopodia exhibited vesicle-like cytoplasmic structures on their surface. Moreover, E2 affected the expression of matrix macromolecules and cell effectors mostly in the presence of ERβ. Our novel data highlights the significance of matrix substrates and the presence of E2 and ERβ in the formation of cellular protrusion and the production of surface structures, defining novel phenotypes that unravel nodal reports for breast cancer progression., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2020 The Authors.)

Focusing on licorice, a highly used raw material in health foods, quantitative analysis of functional/medicinal components and a safety and functional evaluation was carried out for herbal medicines, health food ingredients, and so-called health foods. A functional component, glabridin, was detected in herbal medicines from Glycyrrhiza glabra and G. inflata , health food ingredients, and in commercially available health foods that contain licorice. Likewise, glycyrrhizin, a medicinal component, was detected in these sources, except in licorice oil extract. Estrogen activity in vitro was detected in some of the herbal medicines, health food ingredients, and in health foods containing licorice. In the in vivo study, liver weight in ovariectomized (OVX) mice treated with licorice oil extract was significantly higher than that in OVX and sham mice in a dose dependent manner. These results suggest that excessive intake of licorice oil extract from health foods should be avoided, even though these ingredients might be beneficial for medical use in order to maintain bone health in postmenopausal women. Measurement of hepatic cytochrome P-450 (CYP) activity, reproductive organ weight, and fat and bone mass in OVX mice was considered useful for evaluating the safety and efficacy of estrogenic health food ingredients derived from herbal medicines., Competing Interests: The authors declare no conflict of interest.

Background and Objective: The Pro12Ala (exon 2) and His447His (exon 6) polymorphisms of PPAR-γ, and Gly972Arg polymorphism of IRS-1 have been implicated in insulin resistance (IR) and adiposity. Our aim was to investigate the influence of these polymorphisms on metabolic features of polycystic ovary syndrome (PCOS)., Methods: Fifty-three PCOS women and 26 control women underwent a clinical and biochemical evaluation, including a 75-g oral glucose tolerance test. Insulin secretion and insulin sensitivity indices were calculated., Results: Frequencies of PPAR-γ polymorphisms did not differ from those predicted by the Hardy-Weinberg equilibrium. Instead, the IRS-1 Gly972Arg allele was significantly more frequent in the PCOS group compared to controls. The most frequent allelic combinations were IRS1+/exon2-/exon6- (which prevailed in PCOS) and IRS-1-/exon2-/exon6- (which prevailed in controls). Among PCOS women, compared with the wild type patients, carriers of the Gly972Arg IRS-1 allele had lower E2 levels, while carriers of the Pro12Ala PPAR-γ (exon 2) allele had lower free testosterone levels. No other significant relationships were noted. When compared with the wild type, in PCOS group IR and beta-cell function were: (i) trendwise greater in carriers of the variant IRS-1 allele; (ii) trendwise lower in carriers of the variant PPAR-γ exon 6 allele; (iii) significantly lower in carriers of the variant PPAR-γ exon 2 allele., Conclusions: Our data support the protective influence of PPAR-γ-exon 2 and exon 6 variants on IR and beta cell function, whereas IRS-1 polymorphism is associated with an unfavorable metabolic profile. However, these associations do not fully explain the high metabolic risk associated with PCOS.

Polycyclic Aromatic Hydrocarbons (PAHs) are potent carcinogens. Among these, dimethylbenz(a)anthracene (DMBA) is well known for its capacity to induce mammary carcinomas in female Sprague-Dawley (SD) rats. Ovariectomy suppresses the susceptibility of this model to DMBA, thus suggesting that the inducible action of the carcinogen depends on ovarian hormones. The promotion of DMBA-induced adenocarcinoma is accompanied by a series of neuroendocrine disruptions of both Hypothalamo-Pituitary-Gonadal (HPG) and Hypothalamo-Pituitary-Adrenal (HPA) axes and of the secretion of melatonin during the latency period of 2 months that precedes the occurrence of the first mammary tumor. The present review analyses the various neuroendocrine disruptions that occur along the HPG and the HPA axes, and the marked inhibitory effect of the carcinogen on melatonin secretion. The possible relationships between the neuroendocrine disruptions, which essentially consist in an increased pre-ovulatory secretion of 17β-estradiol and prolactin, associated with a marked reduction of melatonin secretion, and the decrease in gene expression of the receptors for aryl-hydrocarbons receptor (AhR) and 17β-estradiol (ERα; ERβ) are also discussed.

The prototype dioxin congener 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is known to exert anti-estrogenic effects via activation of the aryl hydrocarbon receptor (AhR) by interfering with the regulation of oestrogen homeostasis and the estrogen receptor α (ERα) signalling pathway. The AhR/ER cross-talk is considered to play a crucial role in TCDD- and E2-dependent mechanisms of carcinogenesis, though the concerted mechanism of action in the liver is not yet elucidated. The present study investigated TCDD's impact on the transcriptional cross-talk between AhR and ERα and its modulation by 17β-estradiol (E2) in the human hepatoma cell line HepG2, which is AhR-responsive but ERα-negative. Transient transfection assays with co-transfection of hERα and supplementation of receptor antagonists showed anti-estrogenic action of TCDD via down-regulation of E2-induced ERα signaling. In contrast, enhancement of AhR signaling dependent on ERα was observed providing evidence for increased cytochrome P450 (CYP) induction to promote E2 metabolism. However, relative mRNA levels of major E2-metabolizing CYP1A1 and 1B1 and the main E2-detoxifying catechol-O-methyltransferase were not affected by the co-treatments. This study provides new evidence of a TCDD-activated AhR-mediated molecular AhR/ERα cross-talk mechanism at transcriptional level via indirect inhibition of ERα and enhanced transcriptional activity of AhR in HepG2 cells.

Pairing the selective estrogen receptor modulator bazedoxifene (BZA) with estrogen as a tissue-selective estrogen complex (TSEC) is a novel menopausal therapy. We investigated estrogen, BZA and TSEC effects in preventing diabetisity in ovariectomized mice during high-fat feeding. Estrogen, BZA or TSEC prevented fat accumulation in adipose tissue, liver and skeletal muscle, and improved insulin resistance and glucose intolerance without stimulating uterine growth. Estrogen, BZA and TSEC improved energy homeostasis by increasing lipid oxidation and energy expenditure, and promoted insulin action by enhancing insulin-stimulated glucose disposal and suppressing hepatic glucose production. While estrogen improved metabolic homeostasis, at least partially, by increasing hepatic production of FGF21, BZA increased hepatic expression of Sirtuin1, PPARα and AMPK activity. The metabolic benefits of BZA were lost in estrogen receptor-α deficient mice. Thus, BZA alone or in TSEC produces metabolic signals of fasting and caloric restriction and improves energy and glucose homeostasis in female mice.