A comparative study of human and zebrafish glucocorticoid receptor activities of natural and pharmaceutical steroids.

Introduction: The action of environmental steroids on the human glucocorticoid receptor (hGR) has been pointed out with the risk to impair physiological immune and metabolic processes regulated by this nuclear receptor. However, there is still a lack of mechanistic information regarding their ability to interact with GR in aquatic species. Methods: To investigate ligand activation differences between hGR and zebrafish GR (zfGR), we tested several natural and synthetic steroids using reporter cell lines expressing hGR or zfGR. Results and discussion: Almost all the glucocorticoids tested (dexamethasone, cortisol, bimedrazol, medrol, cortivazol and fluticasone) are agonists of the two receptors with similar potencies. The dissociated glucocorticoids, RU24782 and RU24858 are agonists of both zfGR and hGR but with a better potency for the latter. On the other hand, the synthetic glucocorticoid forbimenol and the mineralocorticoid aldosterone are agonist on hGR but antagonist on zfGR. The other steroids tested, androgens and progestins, are all antagonists of both GRs with equal or lower potency on zfGR than on hGR. Surprisingly, the lower efficacy and potency on zfGR of aldosterone, forbimenol and the dissociated glucocorticoids is not related to their affinity for the receptors which would suggest that it could be related to less efficacious recruitment of coactivators by zfGR compared to hGR.

Vegetable oil-based hybrid microparticles as a green and biocompatible system for subcutaneous drug delivery.

The aim of this study was to evidence the ability of vegetable oil-based hybrid microparticles (HMP) to be an efficient and safe drug delivery system after subcutaneous administration. The HMP resulted from combination of a thermostabilized emulsification process and a sol-gel chemistry. First of all, castor oil was successfully silylated by means of (3-Isocyanatopropyl)trimethoxysilane in solvent-free and catalyst-free conditions. Estradiol, as a model drug, was dissolved in silylated castor oil (ICOm) prior to emulsification, and then an optimal sol-gel crosslinking was achieved inside the ICOm microdroplets. The resulting estradiol-loaded microparticles were around 80 µm in size and allowed to entrap 4 wt% estradiol. Their release kinetics in a PBS/octanol biphasic system exhibited a one-week release profile, and the released estradiol was fully active on HeLa ERE-luciferase ERα cells. The hybrid microparticles were cytocompatible during preliminary tests on NIH 3T3 fibroblasts (ISO 10993-5 standard) and they were fully biocompatible after subcutaneous injection on mice (ISO 10993-6 standard) underlining their high potential as a safe and long-acting subcutaneous drug delivery system.

Long-Term Exposure of Early-Transformed Human Mammary Cells to Low Doses of Benzo[a]pyrene and/or Bisphenol A Enhances Their Cancerous Phenotype via an AhR/GPR30 Interplay.

It is of utmost importance to decipher the role of chronic exposure to low doses of environmental carcinogens on breast cancer progression. The early-transformed triple-negative human mammary MCF10AT1 cells were chronically (60 days) exposed to low doses (10-10 M) of Benzo[a]pyrene (B[a]P), a genotoxic agent, and/or Bisphenol A (BPA), an endocrine disruptor. Our study revealed that exposed MCF10AT1 cells developed, in a time-dependent manner, an acquired phenotype characterized by an increase in cancerous properties (anchorage independent growth and stem-like phenotype). Co-exposure of MCF10AT1 cells to B[a]P and BPA led to a significantly greater aggressive phenotype compared to B[a]P or BPA alone. This study provided new insights into the existence of a functional interplay between the aryl hydrocarbon receptor (AhR) and the G protein-coupled receptor 30 (GPR30) by which chronic and low-dose exposure of B[a]P and/or BPA fosters the progression of MCF10AT1 cells into a more aggressive substage. Experiments using AhR or GPR30 antagonists, siRNA strategies, and RNAseq analysis led us to propose a model in which AhR signaling plays a "driver role" in the AhR/GPR30 cross-talk in mediating long-term and low-dose exposure of B[a]P and/or BPA. Retrospective analysis of two independent breast cancer cohorts revealed that the AhR/GPR30 mRNA expression signature resulted in poor breast cancer prognosis, in particular in the ER-negative and the triple-negative subtypes. Finally, the study identified targeting AhR and/or GPR30 with specific antagonists as a strategy capable of inhibiting carcinogenesis associated with chronic exposure to low doses of B[a]P and BPA in MCF10AT1 cells. Altogether, our results indicate that the engagement of both AhR and GPR30 functions, in particular in an ER-negative/triple-negative context of breast cells, favors tumor progression and leads to poor prognosis.

Diclofenac in the marine environment: A review of its occurrence and effects.

Interest in the presence and effects of diclofenac (DCF) and other pharmaceutical products (PPs) in the aquatic environment has been growing over the last 20 years. DCF has been included in the First Watch List of the EU Water Framework Directive in order to gather monitoring data in surface waters. Despite PP input in water bodies, few studies have been conducted to determine the extent of DCF occurrence and effects on marine ecosystems, which is usually the final recipient of surface waters. The present article reviews available published data on DCF occurrence in marine water, sediment and organisms, and its effects on marine organisms. The findings highlight the scarcity of available data on the occurrence and effects of DCF in marine ecosystems, and the need for further data acquisition to assess the risks associated with the presence of this compound in the environment.

In vitro biomonitoring of contamination by estrogenic compounds in coastal environments: comments on the use of M. galloprovincialis.

The use of mussel extracts in in vitro bioassays which express the estrogen receptor could provide valuable information on the bioavailability of endocrine disruptors in coastal environments. The aim of this study was to assess the temporal variability of the estrogenic responses in bioassays in Mytilus galloprovincialis. A 6-month in situ experiment was conducted in order to follow the estrogenic activity on MELN cell line during the reproduction stages of mussels. Estradiol equivalents (EEQ) determined in mussels using the MELN cell lines ranged from 0.79 to 3.72 ng/g dry weight (d.w.) in males, from 0.42 to 2.33 ng/g d.w. in females and from 3.41 to 4.2 d.w. in undifferentiated bivalves. We observed an increase in EEQ values during the spawning stage for males, not for female. The maximal EEQ values were observed at the indifferent stage. We discuss these results in regards to the actual knowledge on mussels' reproductive cycle and to the possible impact of xeno-estrogens. Variations of E2 levels in mussels must be taken into account for further studies on xeno-estrogens monitoring using hER reporter cell-lines bioassays.

Biological analysis of endocrine-disrupting compounds in Tunisian sewage treatment plants.

Endocrin-disrupting compounds (EDCs) are frequently found in wastewater treatment plants (WWTPs). So far, research has been mainly focused on the detection of estrogenic compounds and very little work has been carried out on other receptors activators. In this study, we used reporter cell lines, which allow detecting the activity of estrogen (ERalpha), androgen (AR), pregnane X (PXR), glucocorticoid (GR), progesterone (PR), mineralocorticoid (MR), and aryl hydrocarbon (AhR) receptors, to characterise the endocrine-disrupting profile of the aqueous, suspended particulate matter, and sludge fractions from three Tunisian WWTPs. The aqueous fraction exhibited estrogenic and androgenic activities. Suspended particulate matter and sludge extracts showed estrogenic, aryl hydrocarbon and pregnane X receptor activities. No GR, MR, or PR (ant) agonistic activity was detected in the samples, suggesting that environmental compounds present in sewage might have a limited spectrum of activity. By performing competition experiments with recombinant ERalpha, we demonstrated that the estrogenic activity detected in the aqueous fraction was due to EDCs with a strong affinity for ERalpha. Conversely, in the sludge fraction, it was linked to the presence of EDCs with weak affinity. Moreover, by using different incubation times, we determined that the EDCs present in suspended particulate matter and sludge, which can activate AhR, are metabolically labile compounds. Finally, we showed in this study that environmental compounds are mainly ER, AR, PXR, and AhR activators. Concerning AR and PXR ligands, we do not to know the nature of the molecules. Concerning ER and AhR compounds, competition experiments with recombinant receptor and analysis at different times of exposure of the AhR activation gave some indications of the compound's nature that need to be confirmed by chemical analysis.

New stably transfected bioluminescent cells expressing FLAG epitope-tagged estrogen receptors to study their chromatin recruitment.

BACKGROUND: Biological actions of estrogens are mediated by the two specific estrogen receptors ERalpha and ERbeta. However, due to the absence of adequate cellular models, their respective transcriptional activities are still poorly understood. For instance, the evaluation of such differing properties on the transcription of responsive genes using ChIP experiments was hindered by the deficiency of cells exhibiting the same genotypic background and properties but expressing only one of the ERs. We describe here the generation of such cells, using an estrogen receptor negative HELN cell line that was derived from HeLa cells stably transfected with an ERE-driven luciferase plasmid. These HELN-Falpha and HELN-Fbeta cell lines stably express either the alpha or beta (full length) estrogen receptor tagged with the FLAG epitope. The use of antibodies directed against the FLAG epitope allowed a direct comparative evaluation of the respective actions of both ERs using ChIP. RESULTS: HELN-Falpha and HELN-Fbeta cell lines were found to express comparable levels of their corresponding tagged receptors with a Kd for estradiol binding of 0.03 and 0.27 nM respectively. The presence of a stably transfected ERE-driven luciferase plasmid in these cells allowed the direct evaluation of the transcriptional activity of both tagged receptors, using natural or synthetic estrogens. FLAG-ERalpha and FLAG-ERbeta were found to exhibit similar transcriptional activity, as indicated by a kinetic evaluation of the transcriptional activation of the luciferase gene during 10 hrs of treatment with estradiol. The validity of these model cells was further confirmed by the predictable transcriptional regulations measured upon treatments with ERalpha or ERbeta specific ligands. The similar immunoprecipitation efficiency of both tagged receptors by an anti-FLAG antibody allowed the assessment of their kinetic recruitment on the synthetic luciferase promoter (containing an estrogen response element) by ChIP assays during 8 hours. A biphasic curve was obtained for both FLAG-ERalpha and FLAG-ERbeta, with a peak occurring either at 2 hr or at 1 hr, respectively, and a second one following 4 hr of E2 stimulation in both cases. In MCF-7 cells, the recruitment of ERalpha also exhibited a biphasic behaviour; with the second peak however not so important than in the HeLa cell lines. CONCLUSION: In HELN derived cell lines, no fundamental differences between kinetics were observed during 8 hours for FLAG-ERalpha and FLAG-ERbeta, as well as for polymerase II recruitment. However, the relative importance of recruitment between 1 hr and 4 hr was found to be different in HeLa cell line expressing exogenous tagged ERalpha and in MCF-7 cell line expressing endogenous ER.

Regulation of activities of steroid hormone receptors by tibolone and its primary metabolites.

This work was undertaken (i) to study deeply the estrogen, androgen and progestative activities of tibolone and its metabolites (ii) to determine whether tibolone and its metabolites present glucocorticoid or mineralocorticoid activity. For this purpose, we used human cell lines bearing a luciferase gene with a responsive element under the control of human estrogen receptor alpha (ERalpha) or estrogen receptor beta (ERbeta) or androgen receptor (AR) or chimeric Gal4 fusion with progesterone receptor (PR), glucocorticoid receptor (GR) or mineralocorticoid receptor (MR). The major tibolone metabolites, the two hydroxymetabolites, bind and activate ER with a preference for ERalpha. Tibolone and the Delta(4)-tibolone are agonists for AR and PR and surprisingly 3alpha- and 3beta-OH-tibolone are antagonists for them. Moreover we showed for the first time that tibolone and its primary metabolites are GR and MR antagonists with a stronger affinity for MR than for GR. In conclusion, tibolone by these actions on different receptors and by this capacity to transform in different metabolites, has more complex effects than initially supposed.

Profiling of benzophenone derivatives using fish and human estrogen receptor-specific in vitro bioassays.

Benzophenone (BP) derivatives, BP1 (2,4-dihydroxybenzophenone), BP2 (2,2',4,4'-tetrahydroxybenzophenone), BP3 (2-hydroxy-4-methoxybenzophenone), and THB (2,4,4'-trihydroxybenzophenone) are UV-absorbing chemicals widely used in pharmaceutical, cosmetics, and industrial applications, such as topical sunscreens in lotions and hair sprays to protect skin and hair from UV irradiation. Studies on their endocrine disrupting properties have mostly focused on their interaction with human estrogen receptor alpha (hERalpha), and there has been no comprehensive analysis of their potency in a system allowing comparison between hERalpha and hERbeta activities. The objective of this study was to provide a comprehensive ER activation profile of BP derivatives using ER from human and fish origin in a battery of in vitro tests, i.e., competitive binding, reporter gene based assays, vitellogenin (Vtg) induction in isolated rainbow trout hepatocytes, and proliferation based assays. The ability to induce human androgen receptor (hAR)-mediated reporter gene expression was also examined. All BP derivatives tested except BP3 were full hERalpha and hERbeta agonists (BP2>THB>BP1) and displayed a stronger activation of hERbeta compared with hERalpha, the opposite effect to that of estradiol (E2). Unlike E2, BPs were more active in rainbow trout ERalpha (rtERalpha) than in hERalpha assay. All four BP derivatives showed anti-androgenic activity (THB>BP2>BP1>BP3). Overall, the observed anti-androgenic potencies of BP derivatives, together with their proposed greater effect on ERbeta versus ERalpha activation, support further investigation of their role as endocrine disrupters in humans and wildlife.

Temporal study of estrogenic responses of mussel (Mytilus galloprovinciallis) extracts applied to reporter cell lines.

In vitro bioassays would facilitate monitoring of estrogen-like compounds in mussels (Mytilus galloprovincialis) since they tend to accumulate lipophilic compounds in their fat and muscle tissue. However, estradiol (E2) steroid (already identified in mussels) could induce false positive responses. This study focuses on temporal variability in estrogenic responses of mussel extracts and the possible relation of this response with E2 levels. The reproductive cycle and growth were thus monitored for six months in sexed mussels. E2 levels were similar between genders and these levels varied with reproductive stages. human estrogen receptor (hER) activities were similar at all stages except in February. E2 present in male and female extracts accounted for part of the hER activation observed, but no correlation was found between E2 levels and hER activities.

The transcription factor aryl hydrocarbon receptor nuclear translocator functions as an estrogen receptor beta-selective coactivator, and its recruitment to alternative pathways mediates antiestrogenic effects of dioxin.

The biological effects of dioxins are mediated by the aryl hydrocarbon receptor (AhR) and its dimerization partner, the AhR nuclear translocator (ARNT), and include interference with hormonal signaling pathways like the response to estrogens. The effects of estrogens are mediated by two estrogen receptor (ER) isoforms, ERalpha and ERbeta, which belong to the family of nuclear receptors. We have previously shown that ARNT can act as coactivator of the ERs. In this study, we show that recruitment of ARNT to AhR or hypoxia-inducible factor-1alpha signaling pathways as well as small interfering RNA-mediated down-regulation of ARNT levels lead to a reduction in ER transcriptional activity. Using chromatin immunoprecipitation assays, we demonstrate that this decrease coincides with reduced recruitment of ARNT to estradiol-regulated promoters. We show further that coactivation by ARNT as well as inhibition by dioxin acts stronger on ERbeta than on ERalpha activity. Additionally, we demonstrate that the effects of ARNT are dependent on the A/B domain of the ERs with the A/B domain of ERbeta being considerably stronger in mediating the coactivating effects of ARNT. Taken together, our studies show that recruitment of ARNT to the AhR after dioxin treatment can account for the antiestrogenic effect of dioxins. Moreover, we show for the first time that the inhibitory effects of dioxin are more pronounced on ERbeta than on ERalpha.

Estrogens and antiestrogens activate hPXR.

The pregnane X receptor (PXR, NR1I2) and the estrogen receptors (ERalpha, NR3A1 and ERbeta, NR3A2) bind a large number of compounds, including environmental pollutants and drugs, which exhibit remarkably diverse structural features. This prompted us to investigate if ER ligands could be PXR activators. We focused our attention on known estrogens from various chemical classes: physiological and synthetic estrogens and antiestrogens, plant and fungus estrogens, and other man-made chemicals belonging to phthalate plasticizers, surfactant-derived alkylphenols and cosmetics. Altogether, nearly 50 compounds were thus analyzed for their ability to activate human PXR in stably transfected cells, HGPXR cells, derived from HeLa cells and expressing luciferase under the control of a chimeric hPXR. Some of the newly identified hPXR activators were also checked for their ability to induce cytochrome P450 3A4 and 2B6 expressions in a primary culture of human hepatocytes. A significant proportion (54%) of compounds with estrogenic activity or able to bind ER were found to be hPXR activators: in particular, antiestrogens, mycoestrogens and phthalates. An even greater proportion is observed if estrogenic pesticides are included. Altogether, these results raise the question of the meaning and consequences of compounds with double PXR/ER activation ability.

Estrogen receptor alpha and the activating protein-1 complex cooperate during insulin-like growth factor-I-induced transcriptional activation of the pS2/TFF1 gene.

Insulin like growth factor I (IGF-I) displays estrogenic activity in breast cancer cells. This activity is strictly dependent on the presence of estrogen receptor alpha (ERalpha). However the precise molecular mechanisms involved in this process are still unclear. IGF-I treatment induces phosphorylation of the AF1 domain of ERalpha and activation of estrogen regulated genes. These genes are characterized by important differences in promoter architecture and response element composition. We show that promoter structure is crucial for IGF-I-induced transcription activation. We demonstrate that on a complex promoter such as the pS2/TFF1 promoter, which contains binding sites for ERalpha and for the activating protein-1 (AP1) complex, transcriptional activation by IGF-I requires both ERalpha and the AP1 complex. IGF-I is unable to stimulate transcription of an estrogen-regulated gene under the control of a minimal promoter containing only a binding site for ERalpha. We propose a molecular mechanism with stepwise assembly of the AP1 complex and ERalpha during transcription activation of pS2/TFF1 by IGF-I. IGF-I stimulation induces rapid phosphorylation and an increase in protein levels of the AP1 complex. Binding of the phosphorylated AP1 complex to the pS2/TFF1 promoter allows recruitment of the chromatin remodeling factor Brg1 followed by binding of ERalpha via its interaction with c-Jun.

Evaluation of ligand selectivity using reporter cell lines stably expressing estrogen receptor alpha or beta.

Estrogens control transcriptional responses through binding to two different nuclear receptors, estrogen receptor alpha (ERalpha) and beta (ERbeta). Since these two ER subtypes are thought to mediate different biological effects, there is intense interest in designing subtype-selective ER ligands. In this study, we evaluated the ERalpha and ERbeta selectivity of 19 known estrogens and antiestrogens using reporter cell lines previously developed in our laboratory. The HELN-ERalpha and HELN-ERbeta cells stably express full-length ERalpha and ERbeta, respectively, and are derived from HELN cells (HeLa cells stably transfected with an ERE-driven luciferase plasmid). We report that 16alpha-LE2, PPT and 3beta,5alpha-GSD have a high ERalpha-selective agonist potency while 8beta-VE2, DPN, genistein and biochanin A show ERbeta selectivity with 8beta-VE2 being the most potent and selective ERbeta agonist. We also tested ER antagonists and we showed that raloxifene and RU486 are ERalpha and ERbeta-selective antiestrogens, respectively. In all cases, selectivity is due to differences in binding affinities as indicated by whole-cell ligand-binding assays. Very interestingly, we demonstrate that a combination of genistein and raloxifene produces a full-ERbeta specific response. Together these results demonstrate the usefulness of our stably transfected cell lines to characterize ER ligands and indicate that treatments combining agonist/antagonist ligands produce full-ERbeta selectivity.

Binding of estrogenic compounds to recombinant estrogen receptor-alpha: application to environmental analysis.

Estrogenic activity in environmental samples could be mediated through a wide variety of compounds and by various mechanisms. High-affinity compounds for estrogen receptors (ERs), such as natural or synthetic estrogens, as well as low-affinity compounds such as alkylphenols, phthalates, and polychlorinated biphenyls are present in water and sediment samples. Furthermore, compounds such as polycyclic aromatic hydrocarbons, which do not bind ERs, modulate estrogen activity by means of the aryl hydrocarbon receptor (AhR). In order to characterize compounds that mediate estrogenic activity in river water and sediment samples, we developed a tool based on the ER-alphaligand-binding domain, which permitted us to estimate contaminating estrogenic compound affinities. We designed a simple transactivation assay in which compounds of high affinity were captured by limited amounts of recombinant ER-alpha and whose capture led to a selective inhibition of transactivation. This approach allowed us to bring to light that water samples contain estrogenic compounds that display a high affinity for ERs but are present at low concentrations. In sediment samples, on the contrary, we showed that estrogenic compounds possess a low affinity and are present at high concentration. Finally, we used immobilized recombinant ER-alpha to separate ligands for ER and AhR that are present in river sediments. Immobilized ER-alpha, which does not retain dioxin-like compounds, enabled us to isolate and concentrate ER ligands to facilitate their further analysis.