Environmental estrogens (EEs) are associated with an increased prevalence of asthma. These epigenetic alter-ations of the immune cells may explain the multigenerational effects on asthma development. We hypothesized that exposure to immune cells enhances allergic sensitization by initiating signaling in these cells. Human T cell lines (TIB-152, CCL-119) were exposed to varying concentrations of estradiol, bisphenol A, bisphenol S, or bisphenol A + estradiol. H3K27me3, phosphorylations of EZH2 (pEZH2), AKT (pAKT), and phosphatidylinositide 3-kinase (pPI3K) were assessed. pAKT and pPI3K were decreased in response to some of the concentrations of these exposures in both cell lines. It is likely that EEs exposure to immune cells is one of the factors in the increase in the prevalence of asthma.

Ethinyl estradiol (EE2, the active component of many birth control formulations) persists in treated waste waters and it has become a concerning endocrine-disrupting contaminant throughout the world. Previous studies have not examined the behavior of EE2 in nongenomic signaling pathways and the subsequent functional responses (either alone or in mixtures) or conducted comparisons with the physiological estrogen estradiol (E2). In this study, mitogen-activated protein kinases (MAPKs), ERK, and JNK were activated in pituitary tumor cells by fM EE2, but p38 activation was insensitive to <nM doses. Both E2 and EE2 (10 fM to 10 nM) caused cell proliferation as well as triggering intracellular calcium increases and GTP charging of Gαi. E2 was more effective at causing prolactin release. Previously, we reported that the soy-based diadzein synthetic metabolite R-equol (R-eq) activated nongenomic responses in pituitary cells and impaired the actions of E2, thereby affecting both prolactin release and cell proliferation. In the present study, as expected, R-eq activated all MAPKs in a dose-dependent manner at concentrations ranging from fM to 100 nm, and it also modified the effects of environmentally and therapeutically relevant levels of EE2. The physiological/therapeutic doses of E2/EE2 that activated p38 were most effectively challenged by R-eq at ≥fM concentrations. R-eq did not alter the proliferative response to E2 but it elevated the cell numbers induced by EE2 at all concentrations of added R-eq. The more pronounced ability of R-eq to inhibit the cell-killing mechanisms associated with p38-induced inflammatory responses may explain its capacity to increase the numbers of EE2-stimulated pituitary tumor cells. Thus, widespread exposure to persistent pharmaceutical estrogens that imperfectly mimic endogenous estrogens may exacerbate cell proliferation in these responsive cells.

Rapid nongenomic signaling by estrogens (Es), initiated near the cell membrane, provides new explanations for the potent actions of environmental chemicals that imperfectly mimic physiological Es. These pathways can affect tumor growth, stabilization, or shrinkage via a number of signaling streams such as activation/inactivation of mitogen-activated protein kinases and caspases, generation of second messengers, and phospho-triggering of cyclin instability. Though prostate cancers are better known for their responsiveness to androgen deprivation, ∼17% of late stage tumors regress in response to high dose natural or pharmaceutical Es; however, the mechanisms at the cellular level are not understood. More accurate recent measurements show that estradiol (E2) levels decline in aging men, leading to the hypothesis that maintaining young male levels of E2 may prevent the growth of prostate cancers. Major contributions to reducing prostate cancer cell numbers included low E2 concentrations producing sustained ERK phospho-activation correlated with generation of reactive oxygen species causing cancer cell death, and phospho-activation of cyclin D1 triggering its rapid degradation by interrupting cell cycle progression. These therapeutic actions were stronger in early stage tumor cells (with higher membrane estrogen receptor levels), and E2 was far more effective compared to diethylstilbestrol (the most frequently prescribed E treatment). Xenoestrogens (XEs) exacerbated the growth of prostate cancer cells, and as we know from previous studies in pituitary cancer cells, can interfere with the nongenomic signaling actions of endogenous Es. Therefore, nongenomic actions of physiological levels of E2 may be important deterrents to the growth of prostate cancers, which could be undermined by the actions of XEs.

The 5-HT2A receptor (5-HT2AR) plays an important role in various neuropsychiatric disorders, including substance use disorder and schizophrenia. Homodimerization of this receptor has been suggested, but tools that allow direct assessment of the relevance of the 5-HT2AR:5-HT2AR homodimer in these disorders are necessary. We chemically modified the selective 5-HT2AR antagonist M100907 to synthesize a series of homobivalent ligands connected by ethylene glycol linkers of varying lengths that may be useful tools for probing 5-HT2AR:5-HT2AR homodimer function. We tested these molecules for 5-HT2AR antagonist activity in a cell line stably expressing the functional 5-HT2AR and quantified a downstream signaling target, activation (phosphorylation) of extracellular regulated kinases 1/2 (ERK1/2), in comparison to in vivo efficacy of altering spontaneous or cocaine-evoked locomotor activity in rats. All of the synthetic compounds inhibited 5-HT-mediated phosphorylation of ERK1/2 in the cellular signaling assay; the potency of the bivalent ligands varied as a function of linker length, with the intermediate linker lengths being the most potent. The Ki values for the binding of bivalent ligands to 5-HT2AR were only slightly lower than the values for the parent (+)-M100907 compound, but significant selectivity for 5-HT2AR over 5-HT2BR or 5-HT2CR binding was retained. In addition, the 11-atom-linked bivalent 5-HT2AR antagonist (2 mg/kg, intraperitoneally) demonstrated efficacy on par with that of (+)-M100907 in inhibiting cocaine-evoked hyperactivity. As we develop further strategies for ligand-evoked receptor assembly and analyses of diverse signaling and functional roles, these novel homobivalent 5-HT2AR antagonist ligands will serve as useful in vitro and in vivo probes of 5-HT2AR structure and function.

OBJECTIVE:The objective of this study is to determine if BPA exposure, as measured by maternal plasma (MP) and amniotic fluid (AF) BPA concentrations is associated with an increased risk of spontaneous preterm birth (PTB) and preterm premature rupture of membranes (pPROM). METHODS:In this nested case-control study, MP samples from women in term labor (n = 30), preterm labor that ended with preterm delivery (n = 25), or who had pPROM (n = 30) and amniotic fluid samples from term labor (n= 45), preterm labor (n = 60), and pPROM (n = 35) were assayed for BPA by enzyme immunoassay. RESULTS:BPA was detectible in 100% of MP and AF samples. Women with MP BPA concentrations in the fourth quartile were at increased risk of PTB (cOR = 4.12, 95% CI = 1.32-12.87; aOR = 4.78, 95% CI = 1.14-20) but not pPROM. High (fourth quartile) AF BPA values also tended to increase the risk of pPROM (cOR = 2.47, 95% CI = 0.96-6.37) but results were not statistically significant. CONCLUSIONS:Increased BPA concentration is associated with an increased risk for PTB or pPROM depending on the maternal-fetal compartment(s) affected. High MP plasma BPA concentrations are associated with PTB with intact membranes but high AF BPA concentrations may weakly be associated with pPROM.

ObjectiveActivation of the Mitogen-activated Protein Kinase (MAPK) pathway in amnion epithelial cells (AEC) has been documented in adverse pregnancy outcomes such as preterm premature rupture of membranes and preterm birth. Conventional approaches to evaluating MAPK signaling include ELISA and Western blot analysis, which are time consuming and expensive. The objective of this study was to develop a new methodology that would increase the throughput of MAPK signaling assay in AECS.Study DesignAECs from normal term not in labor parturients were plated at a density of 15,000 cells/well in poly-D-lysine coated 96 well plates. Serum deprived cells were treated with 12-O-tetradecanollyphorbol 13-acetate (TPA, 50 nM; positive control) or 0.01%ethanol (negative control). The cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 100% methanol for 20 minutes at -20°C, blocked with 0.1% fish gelatin in 0.1% Triton X-100 for 1 hour at room temperature, and then exposed to an antibody (to phospho ERK, phospho JNK, or phosphorp38) overnight at 4°C. Color was developed using biotinylated secondary antibody followed by incubation with ABC-AP solution and color development using alkaline phosphatase. Plates were read at A405. Cell number estimated by crystal violet staining, was used to normalize the data.ResultsAt a concentration of 50 nM, TPA caused a two fold increase in the phospho-ERK level after 10 min of treatment in human AECs compared to ethanol treatment (Figure 1).Conclusion ObjectiveActivation of the Mitogen-activated Protein Kinase (MAPK) pathway in amnion epithelial cells (AEC) has been documented in adverse pregnancy outcomes such as preterm premature rupture of membranes and preterm birth. Conventional approaches to evaluating MAPK signaling include ELISA and Western blot analysis, which are time consuming and expensive. The objective of this study was to develop a new methodology that would increase the throughput of MAPK signaling assay in AECS. Activation of the Mitogen-activated Protein Kinase (MAPK) pathway in amnion epithelial cells (AEC) has been documented in adverse pregnancy outcomes such as preterm premature rupture of membranes and preterm birth. Conventional approaches to evaluating MAPK signaling include ELISA and Western blot analysis, which are time consuming and expensive. The objective of this study was to develop a new methodology that would increase the throughput of MAPK signaling assay in AECS. Study DesignAECs from normal term not in labor parturients were plated at a density of 15,000 cells/well in poly-D-lysine coated 96 well plates. Serum deprived cells were treated with 12-O-tetradecanollyphorbol 13-acetate (TPA, 50 nM; positive control) or 0.01%ethanol (negative control). The cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 100% methanol for 20 minutes at -20°C, blocked with 0.1% fish gelatin in 0.1% Triton X-100 for 1 hour at room temperature, and then exposed to an antibody (to phospho ERK, phospho JNK, or phosphorp38) overnight at 4°C. Color was developed using biotinylated secondary antibody followed by incubation with ABC-AP solution and color development using alkaline phosphatase. Plates were read at A405. Cell number estimated by crystal violet staining, was used to normalize the data. AECs from normal term not in labor parturients were plated at a density of 15,000 cells/well in poly-D-lysine coated 96 well plates. Serum deprived cells were treated with 12-O-tetradecanollyphorbol 13-acetate (TPA, 50 nM; positive control) or 0.01%ethanol (negative control). The cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 100% methanol for 20 minutes at -20°C, blocked with 0.1% fish gelatin in 0.1% Triton X-100 for 1 hour at room temperature, and then exposed to an antibody (to phospho ERK, phospho JNK, or phosphorp38) overnight at 4°C. Color was developed using biotinylated secondary antibody followed by incubation with ABC-AP solution and color development using alkaline phosphatase. Plates were read at A405. Cell number estimated by crystal violet staining, was used to normalize the data. ResultsAt a concentration of 50 nM, TPA caused a two fold increase in the phospho-ERK level after 10 min of treatment in human AECs compared to ethanol treatment (Figure 1). At a concentration of 50 nM, TPA caused a two fold increase in the phospho-ERK level after 10 min of treatment in human AECs compared to ethanol treatment (Figure 1). Conclusion

We examined nongenomic signaling and functional effects (cell proliferation; prolactin release) of R-equol (R-eq), a synthetically produced metabolite of the phytoestrogen daidzein that preferentially binds to estrogen receptor-α, across a broad concentration range (10–16 to 10–7M). Membrane estrogen receptor-α, via which nongenomic signaling occurs, is enriched in the GH3/B6/F10 pituitary tumor cells used in these studies. We previously reported that other phytoestrogens, including daidzein, are potent inducers of nongenomic signaling acting via membrane receptors for estrogens, resulting in changes to these same functional responses. In the present studies, R-eq activated typical rapid signaling pathways normally evoked by estrogens, but with some differences in response timings and functional outcomes. Levels of R-eq (at 15 nM, a blood level typical for dietary phytoestrogens) were compared to a female physiological level of estradiol (E2; 1 nM). Both estrogens activated multiple mitogen-activated protein kinases (ERK, JNK and p38) by phosphorylation within 2.5–15 min, with subsequent oscillations, as observed previously for other estrogens. Like E2, R-eq also augmented intracellular calcium levels and caused prolactin release; in contrast to E2, it did not produce a dose-dependent increase in cell proliferation, as estrogens that activate ERK often do. R-eq and E2, both alone and in combination, activated Gαi by GTP-charging. However, R-eq suppressed 1 nM E2-activated ERK, JNK and p38, as well as cell proliferation (most pronounced at typical phytoestrogen blood levels of 10–10 to 10–7M). We conclude that R-eq, like E2, is a rapid activator of nongenomic signals, but when combined with E2, can interfere with E2-induced nongenomic estrogenic effects .

The goal of this project is to test the hypothesis that fetal exposure to environmental estrogens (EEs) enhances allergic sensitization initiating cell signaling in antigen-presenting and/or T cells, which leads to epigenetic alterations that promote the development of asthma. Cord blood mononuclear cells (CB-MNCs) were separated into CD4+T and CD8+T populations, incubated overnight in media with steroid-depleted serum, and then exposed to various concentrations (fM-pM) of estradiol, bisphenol A (BPA) and Bisphenol S (BPS). Supernatant from cell culture was collected for quantifying immunomodulatory cytokines. Intracellular signaling was assessed in a plate assay in cells that were fixed, permeabilized, blocked, and incubated with anti-phospho ERK (pARK), anti-phosphoAKT (pAKT) or anti-phospho EZH2 histone methyl transferase (pEZH2). Phosphatase activity was assessed using secondary antibody and colorimetrical measurement. The signals were normalize to the number of cells in each well. Current experiments suggest that estradiol, BPA and BPS significantly increase the phosphorylation of ERK, AKT and EZH2 in human MNCs, including CD8+T and CD4+T cells. The dose response to BPA and BPS indicate high sensitivity and typical non-monotonic responses. The concomitant increases in the exposure of EEs and prevalence of asthma, raise the possibility that EEs may be a factor in the increasing prevalence of childhood asthma. Understanding the molecular and cellular basis for EE's asthma promoting effects in animal models and human epidemiological studies will inform public policy concerning EE exposures, and may ultimately allow the design of future prevention measures, and potentially, new molecular-based treatments for childhood asthma.

Xenoestrogens (XEs) are exogenous mimics capable of binding to estrogen receptors (ERs), competing with/disrupting the actions of physiological estrogens, and promoting tumor growth in the prostate and other endocrine tissues. Humans are exposed to numerous XEs including environmental contaminants such as plastics monomer bisphenol A (BPA), and dietary phytoestrogens such as coumestrol and genistein from soy, and resveratrol, highest in red grapes. There is growing interest in the ability of phytoestrogens to prevent or treat tumors. We previously reported that multiple cellular mechanisms influence the number of prostate cancer cells after estradiol or diethylstilbestrol treatment. We now examine the effect of these XEs on signaling mechanisms that alter the number of LAPC-4 (androgen-dependent) and PC-3 (androgen-independent) cells at environment- and diet-relevant concentrations. Coumestrol and genistein both increased the number of LAPC-4 and PC-3 cells dramatically. Rapid alterations of phospho- and total-cyclin D1 levels most closely correlated with the XE-induced changes in cell numbers. Sustained activation (phosphorylation) of the extracellular signal-regulated kinases 1 and 2 as a prelude to generation of reactive oxygen species also partially contributed to the XE's effects on cell numbers. Early-stage cells expressed higher levels of all 3 ERs (including those in membranes) than did late-stage cells; ER subtypes were variably involved in the signaling responses. Taken together, these results show that each XE can elicit its own signature constellation of signaling responses, highlighting the importance of managing exposures to both environmental and dietary XEs for existing prostate tumors. These mechanisms may offer new cellular targets for therapy.

Evidence from the various sources indicates alterations in 5-HT2C receptor functions in anxiety, depression and suicide, and other stress-related disorders treated with antidepressant drugs. Although the notion of a 5-HT2C receptor desensitization following antidepressant treatments is rather well anchored in the literature, this concept is mainly based on in vitro assays and/or behavioral assays (hypolocomotion, hyperthermia) that have poor relevance to anxio-depressive disorders. Our objective herein is to provide a comprehensive overview of the studies that have assessed the effects of antidepressant drugs on 5-HT2C receptors. Relevant molecular (second messengers, editing), neurochemical (receptor binding and mRNA levels), physiological (5-HT2C receptor-induced hyperthermia and hormone release), behavioral (5-HT2C receptor-induced changes in feeding, anxiety, defense and motor activity) data are summarized and discussed. Setting the record straight about drug-induced changes in 5-HT2C receptor function in specific brain regions should help to determine which pharmacotherapeutic strategy is best for affective and anxiety disorders.

Some chemicals used in consumer products or manufacturing (e.g. plastics, surfactants, pesticides, resins) have estrogenic activities; these xenoestrogens (XEs) chemically resemble physiological estrogens and are one of the major categories of synthesized compounds that disrupt endocrine actions. Potent rapid actions of XEs via nongenomic mechanisms contribute significantly to their disruptive effects on functional endpoints (e.g. cell proliferation/death, transport, peptide release). Membrane-initiated hormonal signaling in our pituitary cell model is predominantly driven by mERα with mERβ and GPR30 participation. We visualized ERα on plasma membranes using many techniques in the past (impeded ligands, antibodies to ERα) and now add observations of epitope proximity with other membrane signaling proteins. We have demonstrated a range of rapid signals/protein activations by XEs including: calcium channels, cAMP/PKA, MAPKs, G proteins, caspases, and transcription factors. XEs can cause disruptions of the oscillating temporal patterns of nongenomic signaling elicited by endogenous estrogens. Concentration effects of XEs are nonmonotonic (a trait shared with natural hormones), making it difficult to design efficient (single concentration) toxicology tests to monitor their harmful effects. A plastics monomer, bisphenol A, modified by waste treatment (chlorination) and other processes causes dephosphorylation of extracellular-regulated kinases, in contrast to having no effects as it does in genomic signaling. Mixtures of XEs, commonly found in contaminated environments, disrupt the signaling actions of physiological estrogens even more severely than do single XEs. Understanding the features of XEs that drive these disruptive mechanisms will allow us to redesign useful chemicals that exclude estrogenic or anti-estrogenic activities.

We developed fixed-cell multi-well plate immunoassays that increase the throughput and ease of quantification for questions formerly assessed by immunoblot scanning. The assays make use of the now abundant antibodies designed to recognize receptor subtypes and posttranslationally modified signaling proteins. By optimizing permeabilization and fixation conditions, mainly based on specific cell types, the assay can be adapted to the study of many different antigens of importance to hormonal and neurotransmitter signaling scenarios.

Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Versions of published Taylor & Francis and Routledge Open articles and Taylor & Francis and Routledge Open Select articles posted to institutional or subject repositories or any other third-party website are without warranty from Taylor & Francis of any kind, either expressed or implied, including, but not limited to, warranties of merchantability, fitness for a particular purpose, or non-infringement. Any opinions and views expressed in this article are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor & Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content.

Diethylstilbestrol (DES) and other pharmaceutical estrogens have been used at ≥ µM concentrations to treat advanced prostate tumors, with successes primarily attributed to indirect hypothalamic‐pituitary‐testicular axis control mechanisms. However, estrogens also directly affect tumor cells, though the mechanisms involved are not well understood.

Background: Bisphenol A (BPA) is a well-known endocrine disruptor that imperfectly mimics the effects of physiologic estrogens via membrane-bound estrogen receptors (mERα, mERβ, and GPER/GPR30), thereby initiating nongenomic signaling. Bisphenol S (BPS) is an alternative to BPA in plastic consumer products and thermal paper. Objective: To characterize the nongenomic activities of BPS, we examined signaling pathways it evoked in GH3/B6/F10 rat pituitary cells alone and together with the physiologic estrogen estradiol (E2). Extracellular signal-regulated kinase (ERK)– and c-Jun-N-terminal kinase (JNK)–specific phosphorylations were examined for their correlation to three functional responses: proliferation, caspase activation, and prolactin (PRL) release. Methods: We detected ERK and JNK phosphorylations by fixed-cell immunoassays, identified the predominant mER initiating the signaling with selective inhibitors, estimated cell numbers by crystal violet assays, measured caspase activity by cleavage of fluorescent caspase substrates, and measured PRL release by radioimmunoassay. Results: BPS phosphoactivated ERK within 2.5 min in a nonmonotonic dose-dependent manner (10–15 to 10–7 M). When combined with 10–9 M E2, the physiologic estrogen’s ERK response was attenuated. BPS could not activate JNK, but it greatly enhanced E2-induced JNK activity. BPS induced cell proliferation at low concentrations (femtomolar to nanomolar), similar to E2. Combinations of both estrogens reduced cell numbers below those of the vehicle control and also activated caspases. Earlier activation of caspase 8 versus caspase 9 demonstrated that BPS initiates apoptosis via the extrinsic pathway, consistent with activation via a membrane receptor. BPS also inhibited rapid (≤ 1 min) E2-induced PRL release. Conclusion: BPS, once considered a safe substitute for BPA, disrupts membrane-initiated E2-induced cell signaling, leading to altered cell proliferation, cell death, and PRL release.