Evaluating the toxicity of estetrol, 17α-ethinylestradiol, and their combination with drospirenone on zebrafish larvae: A behavioural and proteomic study.

OBJECTIVE: To characterise and compare the toxicity of estetrol (E4) and 17α-ethinylestradiol (EE2), and their respective mixture with the progestin drospirenone (DRSP) in zebrafish (Danio rerio) embryos. METHODS: Zebrafish embryos were exposed to E4, EE2, DRSP, E4+DRSP, and EE2+DRSP in a fish embryo acute toxicity (FET) test. A second test examined behavioural responses and, using label-free proteomics, identified changes in protein expression in response to hormonal treatments, across a range of concentrations, including those that are considered to be environmentally relevant. RESULTS: In the FET test, no effects were found from E4 at concentrations ≤100 mg/L, while EE2 induced mortality and morphological abnormalities at concentrations of 1-2 mg/L. In the behavioural test, exposure to 30 ng/L EE2 (∼200 × predicted environmental concentration - PEC) resulted in hypoactivity in fish larvae and exposure to 0.3 ng/L EE2 (∼2 × PEC) led to quantitative changes in protein abundance, revealing potential impacts on RNA processing and protein synthesis machinery. Exposure to E4 did not alter behaviour, but several groups of proteins were modulated, mainly at 710 ng/L (∼200 × PEC), including proteins involved in oxidative phosphorylation. When combined with DRSP, EE2 induced reduced effects on behaviour and proteomic responses, suggesting an antagonistic effect of DRSP. E4+DRSP induced no significant effects on behaviour or proteomic profiles at tested concentrations. CONCLUSIONS: These findings suggest that E4-based combined oral contraceptives present a more favourable environmental profile than EE2-based contraceptives, particularly during the early developmental stages of fish.

Estetrol/GPER/SERPINB2 transduction signaling inhibits the motility of triple-negative breast cancer cells.

BACKGROUND: Estetrol (E4) is a natural estrogen produced by the fetal liver during pregnancy. Due to its favorable safety profile, E4 was recently approved as estrogenic component of a new combined oral contraceptive. E4 is a selective ligand of estrogen receptor (ER)α and ERß, but its binding to the G Protein-Coupled Estrogen Receptor (GPER) has not been described to date. Therefore, we aimed to explore E4 action in GPER-positive Triple-Negative Breast Cancer (TNBC) cells. METHODS: The potential interaction between E4 and GPER was investigated by molecular modeling and binding assays. The whole transcriptomic modulation triggered by E4 in TNBC cells via GPER was explored through high-throughput RNA sequencing analyses. Gene and protein expression evaluations as well as migration and invasion assays allowed us to explore the involvement of the GPER-mediated induction of the plasminogen activator inhibitor type 2 (SERPINB2) in the biological responses triggered by E4 in TNBC cells. Furthermore, bioinformatics analysis was aimed at recognizing the biological significance of SERPINB2 in ER-negative breast cancer patients. RESULTS: After the molecular characterization of the E4 binding capacity to GPER, RNA-seq analysis revealed that the plasminogen activator inhibitor type 2 (SERPINB2) is one of the most up-regulated genes by E4 in a GPER-dependent manner. Worthy, we demonstrated that the GPER-mediated increase of SERPINB2 is engaged in the anti-migratory and anti-invasive effects elicited by E4 in TNBC cells. In accordance with these findings, a correlation between SERPINB2 levels and a good clinical outcome was found in ER-negative breast cancer patients. CONCLUSIONS: Overall, our results provide new insights into the mechanisms through which E4 can halt migratory and invasive features of TNBC cells.

Estetrol/drospirenone versus 17α-ethinylestradiol/drospirenone: An extended one generation test to evaluate the endocrine disruption potential on zebrafish (Danio rerio).

Combined oral contraceptives, comprising of both an oestrogen and a progestin component, are released in aquatic environments and potentially pose a risk to aquatic wildlife by their capacity to disrupt physiological mechanisms. In this study, the endocrine disruptive potential of two mixtures, 17α-ethinylestradiol (EE2), a synthetic oestrogen, or estetrol (E4), a natural oestrogen, with the progestin drospirenone (DRSP) have been characterised in three generations of zebrafish, according to an adapted Medaka Extended One Generation Reproduction Test. Zebrafish (Danio rerio) were exposed to a range of concentrations of EE2/DRSP and E4/DRSP (∼1×, ∼3×, ∼10× and ∼30× predicted environmental concentration, PEC). Survival, growth, hatching success, fecundity, fertilisation success, vitellogenin (VTG), gonad histopathology, sex differentiation, and transcriptional analysis of genes related to gonadal sex steroid hormones synthesis were assessed. In the F0 generation, exposure to EE2/DRSP at ∼10 and ∼30× PEC decreased fecundity and increased male VTG concentrations. The highest concentration of EE2/DRSP also affected VTG concentrations in female zebrafish and the expression of genes implicated in steroid hormones synthesis. In the F1 generation, sex determination was impaired in fish exposed to EE2/DRSP at concentrations as low as ∼3× PEC. Decreased fecundity and fertility, and abnormal gonadal histopathology were also observed. No effects were observed in the F2 generation. In contrast, E4/DRSP induced only minor histopathological changes and an increase in the proportion of males, at the highest concentration tested (∼30× PEC) in the F1 generation and had no effect on hatching success of F2 generation. Overall, this study suggests that the combination E4/DRSP has a more favourable environmental profile than EE2/DRSP.

Estetrol: From Preclinical to Clinical Pharmacology and Advances in the Understanding of the Molecular Mechanism of Action.

Estetrol (E4) is the most recently described natural estrogen. It is produced by the human fetal liver during pregnancy and its physiological function remains unclear. E4 is the estrogenic component of a recently approved combined oral contraceptive. It is also in development for use as menopausal hormone therapy. In the context of these developments, the pharmacological activity of E4, alone or in combination with a progestin, has been extensively characterized in preclinical models as well as in clinical studies in women of reproductive age and postmenopausal women. Despite the clinical benefits, the use of oral estrogens for contraception or menopause is also associated with unwanted effects, such as an increased risk of breast cancer and thromboembolic events, due to their impact on non-target tissues. Preclinical and clinical data for E4 point to a tissue-specific activity and a more selective pharmacological profile compared with other estrogens, including a low impact on the liver and hemostasis balance. This review summarizes the characterization of the pharmacological properties of E4 as well as recent advances made in the understanding of the molecular mechanisms of action driving its activity. How the unique mode of action and the different metabolism of E4 might support its favorable benefit-risk ratio is also discussed.

Estetrol has a lower impact than 17α-ethinylestradiol on the reproductive capacity of zebrafish (Danio rerio).

Natural and synthetic oestrogens are commonly found in aquatic ecosystems. The synthetic oestrogen 17α-ethinylestradiol (EE2) is widely used in oral contraceptives and its ecotoxicological effects on aquatic organisms have been widely reported. The natural oestrogen estetrol (E4) was recently approved for use in a new combined oral contraceptive and, after therapeutic use, is likely to be found in the aquatic environment. However, its potential effects on non-target species such as fish is unknown. In order to characterize and compare the endocrine disruptive potential of E4 with EE2, zebrafish (Danio rerio) were exposed to E4 or EE2 in a fish short-term reproduction assay conducted according to OECD Test Guideline 229. Sexually mature male and female fish were exposed to a range of concentrations, including environmentally relevant concentrations of E4 and EE2, for 21 days. Endpoints included fecundity, fertilization success, gonad histopathology, head/tail vitellogenin concentrations, as well as transcriptional analysis of genes related to ovarian sex steroid hormones synthesis. Our data confirmed the strong impact of EE2 on several parameters including an inhibition of fecundity, an induction of vitellogenin both in male and female fish, an alteration of gonadal structures and the modulation of genes involved in sex steroid hormone synthesis in female fish. In contrast, only few significant effects were observed with E4 with no impact on fecundity. The results suggest that the natural oestrogen, E4, presents a more favorable environmental profile than EE2 and is less likely to affect fish reproductive capacity.

Microbe-mediated intestinal NOD2 stimulation improves linear growth of undernourished infant mice.

The intestinal microbiota is known to influence postnatal growth. We previously found that a strain of Lactiplantibacillus plantarum (strain LpWJL) buffers the adverse effects of chronic undernutrition on the growth of juvenile germ-free mice. Here, we report that LpWJL sustains the postnatal growth of malnourished conventional animals and supports both insulin-like growth factor-1 (IGF-1) and insulin production and activity. We have identified cell walls isolated from LpWJL, as well as muramyl dipeptide and mifamurtide, as sufficient cues to stimulate animal growth despite undernutrition. Further, we found that NOD2 is necessary in intestinal epithelial cells for LpWJL-mediated IGF-1 production and for postnatal growth promotion in malnourished conventional animals. These findings indicate that, coupled with renutrition, bacteria cell walls or purified NOD2 ligands have the potential to alleviate stunting.

GPR97 triggers inflammatory processes in human neutrophils via a macromolecular complex upstream of PAR2 activation.

Neutrophils play essential anti-microbial and inflammatory roles in host defense, however, their activities require tight regulation as dysfunction often leads to detrimental inflammatory and autoimmune diseases. Here we show that the adhesion molecule GPR97 allosterically activates CD177-associated membrane proteinase 3 (mPR3), and in conjugation with several protein interaction partners leads to neutrophil activation in humans. Crystallographic and deletion analysis of the GPR97 extracellular region identified two independent mPR3-binding domains. Mechanistically, the efficient binding and activation of mPR3 by GPR97 requires the macromolecular CD177/GPR97/PAR2/CD16b complex and induces the activation of PAR2, a G protein-coupled receptor known for its function in inflammation. Triggering PAR2 by the upstream complex leads to strong inflammatory activation, prompting anti-microbial activities and endothelial dysfunction. The role of the complex in pathologic inflammation is underscored by the finding that both GPR97 and mPR3 are upregulated on the surface of disease-associated neutrophils. In summary, we identify a PAR2 activation mechanism that directs neutrophil activation, and thus inflammation. The PR3/CD177/GPR97/PAR2/CD16b protein complex, therefore, represents a potential therapeutic target for neutrophil-mediated inflammatory diseases.

Profile of estetrol, a promising native estrogen for oral contraception and the relief of climacteric symptoms of menopause.

INTRODUCTION: Estrogens used in women's healthcare have been associated with increased risks of venous thromboembolism (VTE) and breast cancer. Estetrol (E4), an estrogen produced by the human fetal liver, has recently been approved for the first time as a new estrogenic component of a novel combined oral contraceptive (E4/drospirenone [DRSP]) for over a decade. In phase 3 studies, E4/DRSP showed good contraceptive efficacy, a predictable bleeding pattern, and a favorable safety and tolerability profile. AREAS COVERED: This narrative review discusses E4's pharmacological characteristics, mode of action, and the results of preclinical and clinical studies for contraception, as well as for menopause and oncology. EXPERT OPINION: Extensive studies have elucidated the properties of E4 that underlie its favorable safety profile. While classical estrogens (such as estradiol) exert their actions via both activation of nuclear and membrane estrogen receptor α (ERα), E4 presents a specific profile of ERα activation: E4 binds and activates nuclear ERα but does not induce the activation of membrane ERα signaling pathways in specific tissues. E4 has a small effect on normal breast tissue proliferation and minimally affects hepatic parameters. This distinct profile of ERα activation, uncoupling nuclear and membrane activation, is unique.

Metformin treatment for 8 days impacts multiple intestinal parameters in high-fat high-sucrose fed mice.

Although the mechanism of action of the antidiabetic drug metformin is still a matter of discussions, it is well accepted that the gut plays an important role. To gain more insights into the mechanisms occurring in the different regions of the intestine, adult male mice were fed a high-fat-high sucrose (HFS) diet for 8 days and treated with metformin by gavage (300 mg/day/kg body weight) during the HFS diet. Metformin counteracted HFS diet-induced overexpression of a network of genes involved in the transport of glucose and fatty acids in the different regions of the small intestine. It also induced beneficial modification of secondary bile acid profile in the caecum, with a reduction of deoxycholic acid and lithocholic acid levels and increased abundance of ursodeoxycholic acid and tauroursodeoxycholic acid, potentially leading to FRX inhibition. In parallel, metformin treatment was associated with specific changes of the microbiota composition in the lumen of the different regions of the intestine. Metformin induced a marked increase in the abundance of Akkermansia muciniphila in the lumen all along the gut and counteracted the effects of HFS diet on the abundances of some bacterial groups generally associated with metabolic disturbances (f-Lachnospiraceae, f-Petostreptococcaceae, g-Clostidium). Therefore, the present work clearly emphasises the role of all the regions of the intestinal tract in the beneficial action of the antidiabetic drug metformin in a prediabetic mouse model.

Estetrol Combined to Progestogen for Menopause or Contraception Indication Is Neutral on Breast Cancer.

Given the unequivocal benefits of menopause hormone therapies (MHT) and combined oral contraceptives (COC), there is a clinical need for new formulations devoid of any risk of breast cancer promotion. Accumulating data from preclinical and clinical studies support that estetrol (E4) is a promising natural estrogen for MHT and COC. Nevertheless, we report here that E4 remains active on the endometrium, even under a dose that is neutral on breast cancer growth and lung metastasis dissemination. This implies that a progestogen should be combined with E4 to protect the endometrium of non-hysterectomized women from hyperplasia and cancer. Through in vivo observations and transcriptomic analyses, our work provides evidence that combining a progestogen to E4 is neutral on breast cancer growth and dissemination, with very limited transcriptional impact. The assessment of breast cancer risk in patients during the development of new MHT or COC is not possible given the requirement of long-term studies in large populations. This translational preclinical research provides new evidence that a therapeutic dose of E4 for MHT or COC, combined with progesterone or drospirenone, may provide a better benefit/risk profile towards breast cancer risk compared to hormonal treatments currently available for patients.

Three-dimensional growth of breast cancer cells potentiates the anti-tumor effects of unacylated ghrelin and AZP-531.

Breast cancer is the most common type of cancer in women and notwithstanding important therapeutic advances, remains the second leading cause of cancer-related death. Despite extensive research relating to the hormone ghrelin, responsible for the stimulation of growth hormone release and appetite, little is known of the effects of its unacylated form, especially in cancer. The present study aimed to characterize effects of unacylated ghrelin on breast cancer cells, define its mechanism of action, and explore the therapeutic potential of unacylated ghrelin or analog AZP-531. We report potent anti-tumor effects of unacylated ghrelin, dependent on cells being cultured in 3D in a biologically-relevant extracellular matrix. The mechanism of unacylated ghrelin-mediated growth inhibition involves activation of Gαi and suppression of MAPK signaling. AZP-531 also suppresses the growth of breast cancer cells in vitro and in xenografts, and may be a novel approach for the safe and effective treatment of breast cancer.

Impact of Gut Microbiota on Host Glycemic Control.

Given that obesity and associated disorder type II diabetes mellitus have reached epidemic proportions worldwide, the development of efficient prevention and therapeutic interventions is a global public health interest. There is now a large body of evidence suggesting that the micro-organisms colonizing the human gut, known as gut microbiota, play a central role in human physiology and metabolism. Understanding how gut microbiota affects and regulates key metabolic functions such as glucose regulation and insulin resistance is an important health issue. The present review summarizes recent advances in our understanding of how gut bacterial species interfere with host metabolic phenotype. We will examine key biological molecular mechanisms underlying the impact of gut microbiota on host glycemic control including: incretin secretion, short-chain fatty acid production, bile acid metabolism, and adipose tissue regulation. We will highlight how prebiotic/probiotic interventions affect these bacterial processes and are now considered as promising approaches to treat obese and diabetic patients.

Revealing the role of microstructure architecture on strength and ductility of Ni microwires by in-situ synchrotron X-ray diffraction.

Deformation mechanisms of cold drawn and electropolished nickel microwires are studied by performing in-situ monotonous and cyclic tensile tests under synchrotron radiation. X-ray diffraction tests allow probing elastic strains in the different grain families and establishing a link with the deformation mechanisms taking place within the microwires. The measurements were carried out on several microwires with diameters ranging from as-drawn 100 µm down to 40 µm thinned down by electropolishing. The as-drawn wires exhibit a core-shell microstructure with <111> fiber texture dominant in core and heterogeneous dual fiber texture <111> and <100> in the shell. Reduction of specimen size by electropolishing results in a higher yield stress and tensile strength along with reduced ductility. In-situ XRD analysis revealed that these differences are linked to the global variation in microstructure induced by shell removal with electropolishing, which in turn affects the load sharing abilities of grain families. This study thus proposes a new way to increase ductility and retain strength in nickel microwires across different diameters by tuning the microstructure architecture.

Lymph/angiogenesis contributes to sex differences in lung cancer through oestrogen receptor alpha signalling.

Oestrogen signalling pathways are emerging targets for lung cancer therapy. Unravelling the contribution of oestrogens in lung cancer development is a pre-requisite to support the development of sex-based treatments and identify patients who could potentially benefit from anti-oestrogen treatments. In this study, we highlight the contribution of lymphatic and blood endothelia in the sex-dependent modulation of lung cancer. The orthotopic graft of syngeneic lung cancer cells into immunocompetent mice showed that lung tumours grow faster in female mice than in males. Moreover, oestradiol (E2) promoted tumour development, increased lymph/angiogenesis and VEGFA and bFGF levels in lung tumours of females through an oestrogen receptor (ER) alpha-dependent pathway. Furthermore, while treatment with ERb antagonist was inefficient, ERa antagonist (MPP) and tamoxifen decreased lung tumour volumes, altered blood and lymphatic vasculature and reduced VEGFA and bFGF levels in females, but not in males. Finally, the quantification of lymphatic and blood vasculature of lung adenocarcinoma biopsies from patients aged between 35 and 55 years revealed more extensive lymphangiogenesis and angiogenesis in tumour samples issued from women than from men. In conclusion, our findings highlight an E2/ERa-dependent modulation of lymphatic and blood vascular components of lung tumour microenvironment. Our study has potential clinical implication in a personalised medicine perspective by pointing to the importance of oestrogen status or supplementation on lung cancer development that should be considered to adapt therapeutic strategies.

Obesity and breast cancer - Role of estrogens and the molecular underpinnings of aromatase regulation in breast adipose tissue.

One in eight women will develop breast cancer over their lifetime making it the most common female cancer. The cause of breast cancer is multifactorial and includes hormonal, genetic and environmental cues. Obesity is now an accepted risk factor for breast cancer in postmenopausal women, particularly for the hormone-dependent subtype of breast cancer. Obesity, which is characterized by an excess accumulation of body fat, is at the origin of chronic inflammation of white adipose tissue and is associated with dramatic changes in the biology of adipocytes leading to their dysfunction. Inflammatory factors found in the breast of obese women considerably impact estrogen signaling, mainly by driving changes in aromatase expression the enzyme responsible for estrogen production, and therefore promote tumor formation and progression. There is thus a strong link between adipose inflammation and estrogen biosynthesis and their signaling pathways converge in obese patients. This review describes how obesity-related factors can affect the risk of hormone-dependent breast cancer, highlighting the different molecular mechanisms and metabolic pathways involved in aromatase regulation, estrogen production and breast malignancy in the context of obesity.

Accurate Control of 17ß-Estradiol Long-Term Release Increases Reliability and Reproducibility of Preclinical Animal Studies.

Estrogens are the subject of intensive researches aiming to elucidate their mechanism of action on the various tissues they target and especially on mammary gland and breast cancer. The use of ready-to-use slow releasing devices to administer steroids, especially estrogens, to small experimental animals remains the method of choice in terms of animal well-being and of safety for both the researcher and the animal. In this study, we evaluated and compared, in vitro and in vivo, the release kinetic of estradiol (E2) over sixty days from two different slow-releasing systems: the matrix pellet (MP) and the reservoir implant (RI). We compared the impact of these systems in three E2-sensitive mouse models : mammary gland development, human MCF7 adenocarcinoma xenograft and mouse melanoma progression. The real amount of E2 that is released from both types of devices could differ from manufacturer specifications due to inadequate release for MP and initial burst effect for RI. Compared to MP, the interindividual variability was reduced with RI thanks to a superior control of the E2 release. Depending on the dose-dependent sensitivity of the physiological or pathological readout studied, this could lead to an improvement of the statistical power of in vivo experiments and thus to a reduction of the required animal number. Altogether, our data draw attention on the importance to adequately select the slow-releasing device that is the most appropriated to a specific experiment to better fulfill the 3Rs rule (Replacement, Reduction, Refinement) related to animal welfare and protection.

Quantitative Assessment of Mouse Mammary Gland Morphology Using Automated Digital Image Processing and TEB Detection.

The assessment of rodent mammary gland morphology is largely used to study the molecular mechanisms driving breast development and to analyze the impact of various endocrine disruptors with putative pathological implications. In this work, we propose a methodology relying on fully automated digital image analysis methods including image processing and quantification of the whole ductal tree and of the terminal end buds as well. It allows to accurately and objectively measure both growth parameters and fine morphological glandular structures. Mammary gland elongation was characterized by 2 parameters: the length and the epithelial area of the ductal tree. Ductal tree fine structures were characterized by: 1) branch end-point density, 2) branching density, and 3) branch length distribution. The proposed methodology was compared with quantification methods classically used in the literature. This procedure can be transposed to several software and thus largely used by scientists studying rodent mammary gland morphology.

Combined estrogenic and anti-estrogenic properties of estetrol on breast cancer may provide a safe therapeutic window for the treatment of menopausal symptoms.

Increased risk of breast cancer is a critical side effect associated with the use of a menopausal hormone therapy (MHT). Estetrol (E4) is a natural estrogen produced by the human fetal liver and is a promising compound for clinical use in MHT. However, its impact on breast cancer is controversial and poorly defined. In this preclinical study, we show that E4 acts as a weak estrogen by stimulating the growth of hormone-dependent breast cancer only at concentrations exceeding menopausal therapeutic needs. E4 presents also an antitumor activity by decreasing the strong proliferative effect of estradiol (E2). While estrogen receptor alpha (ERα) is the predominant receptor mediating its effects, the dual weak-estrogenic/anti-estrogenic feature of E4 results from differential signaling pathways activation. Both nuclear and rapid extra-nuclear signaling pathway are necessary for a complete estrogenic effect of E4. However, the antitumor action of E4 is not due to a capacity to antagonize E2-induced nuclear activity. Altogether, our results highlight that E4 has a limited impact on breast cancer and may offer a safe therapeutic window for the treatment of menopausal symptoms.

The uterine and vascular actions of estetrol delineate a distinctive profile of estrogen receptor α modulation, uncoupling nuclear and membrane activation.

Estetrol (E4) is a natural estrogen with a long half-life produced only by the human fetal liver during pregnancy. The crystal structures of the estrogen receptor α (ERα) ligand-binding domain bound to 17ß-estradiol (E2) and E4 are very similar, as well as their capacity to activate the two activation functions AF-1 and AF-2 and to recruit the coactivator SRC3. In vivo administration of high doses of E4 stimulated uterine gene expression, epithelial proliferation, and prevented atheroma, three recognized nuclear ERα actions. However, E4 failed to promote endothelial NO synthase activation and acceleration of endothelial healing, two processes clearly dependent on membrane-initiated steroid signaling (MISS). Furthermore, E4 antagonized E2 MISS-dependent effects in endothelium but also in MCF-7 breast cancer cell line. This profile of ERα activation by E4, uncoupling nuclear and membrane activation, characterizes E4 as a selective ER modulator which could have medical applications that should now be considered further.