Optimised graphite/carbon black loading of recycled PLA for the production of low-cost conductive filament and its application to the detection of ß-estradiol in environmental samples.

The production, optimisation, physicochemical, and electroanalytical characterisation of a low-cost electrically conductive additive manufacturing filament made with recycled poly(lactic acid) (rPLA), castor oil, carbon black, and graphite (CB-G/PLA) is reported. Through optimising the carbon black and graphite loading, the best ratio for conductivity, low material cost, and printability was found to be 60% carbon black to 40% graphite. The maximum composition within the rPLA with 10 wt% castor oil was found to be an overall nanocarbon loading of 35 wt% which produced a price of less than £0.01 per electrode whilst still offering excellent low-temperature flexibility and reproducible printing. The additive manufactured electrodes produced from this filament offered excellent electrochemical performance, with a heterogeneous electron (charge) transfer rate constant, k0 calculated to be (2.6 ± 0.1) × 10-3 cm s-1 compared to (0.46 ± 0.03) × 10-3 cm s-1 for the commercial PLA benchmark. The additive manufactured electrodes were applied to the determination of ß-estradiol, achieving a sensitivity of 400 nA µM-1, a limit of quantification of 70 nM, and a limit of detection of 21 nM, which compared excellently to other reports in the literature. The system was then applied to the detection of ß-estradiol within four real water samples, including tap, bottled, river, and lake water, where recoveries between 95 and 109% were obtained. Due to the ability to create high-performance filament at a low material cost (£0.06 per gram) and through the use of more sustainable materials such as recycled polymers, bio-based plasticisers, and naturally occurring graphite, additive manufacturing will have a permanent place within the electroanalysis arsenal in the future.

Novel Indoline Spiropyrans Based on Human Hormones ß-Estradiol and Estrone: Synthesis, Structure, Chromogenic and Cytotoxic Properties.

The introduction of a switchable function into the structure of a bioactive compound can endow it with unique capabilities for regulating biological activity under the influence of various types of external stimuli, which makes such hybrid compounds promising objects for photopharmacology, targeted drug delivery and bio-imaging. This work is devoted to the synthesis and study of new spirocyclic derivatives of important human hormones-ß-estradiol and estrone-possessing a wide range of biological activities. The obtained hybrid compounds represent an indoline spiropyrans family, a widely known class of organic photochromic compounds. The structure of the compounds was confirmed by 1H and 13C NMR, IR, HRMS and single-crystal X-ray analysis. The intermolecular interactions in the crystals of spiropyran (3) were defined by Hirshfeld surfaces and 2D fingerprint plots, which were successfully acquired from CrystalExplorer (v21.5). All target hybrids demonstrated pronounced activity in the visible region of the spectrum. The mechanisms of thermal isomerization processes of spiropyrans and their protonated merocyanine forms were studied by DFT methods, which revealed the energetic advantage of the protonation process with the formation of a ß-cisoid CCCH conformer at the first stage and its further isomerization to more stable ß-transoid forms. The proposed mechanism of acidochromic transformation was confirmed by the additional NMR study data that allowed for the detecting of the intermediate CCCH isomer. The study of the short-term cytotoxicity of new spirocyclic derivatives of estrogens and their 2-formyl-precursors was performed on the HeLa cell model. The precursors and spiropyrans differed in toxicity, suggesting their variable applicability in novel anti-cancer technologies.

Efficient removal of endocrine disrupting compounds 17 α-ethynyl estradiol and 17 ß-estradiol by Enterobacter sp. strain BHUBP7 and elucidation of the degradation pathway by HRAMS analysis.

Owing to the increased population and their overuse, estrogens are being detected in the environment at alarming levels. They act as endocrine disrupting compounds (EDC's) posing adverse effects on animals and humans. In this study, a strain belonging to Enterobacter sp. strain BHUBP7 was recovered from a Sewage Treatment Plant (STP) situated in Varanasi city, U.P., India, and was capable of metabolizing both 17 α-Ethynylestradiol (EE2) and 17 ß-Estradiol (E2) separately as a sole carbon source. The strain BHUBP7 exhibited high rates of E2 degradation as compared to EE2 degradation. The degradation of E2 (10 mg/L) was 94.3% after four days of incubation, whereas the degradation of EE2 (10 mg/L) under similar conditions was 98% after seven days of incubation. The kinetics of EE2 and E2 degradation fitted well with the first-order reaction rate. FTIR analysis revealed the involvement of functional groups like C = O, C-C, C-OH during the degradation process. The metabolites generated during degradation of EE2 and E2 were identified using HRAMS and a plausible pathway was elucidated. It was observed that metabolism of both E2 and EE2 proceeded with the formation of estrone, which was then hydroxylated to 4-hydroxy estrone, followed by ring opening at the C4-C5 position, and was further metabolized by the 4,5 seco pathway leading to the formation of 3-(7a-methyl-1,5-dioxooctahydro-1H-inden-4-yl) propanoic acid (HIP). It is the first report on the complete pathway of EE2 and E2 degradation in Enterobacter sp. strain BHUBP7. Moreover, the formation of Reactive Oxygen Species (ROS) during the degradation of EE2 and E2 was observed. It was concluded that both hormones elicited the generation of oxidative stress in the bacterium during the degradation process.

Upregulated Talin1 synergistically boosts ß-estradiol-induced proliferation and pro-angiogenesis of eutopic and ectopic endometrial stromal cells in adenomyosis.

Adenomyosis (ADS) is an estrogen-dependent gynecological disease with unspecified etiopathogenesis. Local hyperestrogenism may serve a key role in contributing to the origin of ADS. Talin1 is mostly identified to be overexpressed and involved in the progression of numerous human carcinomas through mediating cell proliferation, adhesion and motility. Whether Talin1 exerts an oncogenic role in the pathogenesis of ADS and puts an extra impact on the efficacy of estrogen, no relevant data are available yet. Here we demonstrated that the adenomyotic eutopic and ectopic endometrial stromal cells (ADS_Eu_ESC and ADS_Ec_ESC) treated with ß-estradiol (ß-E2) presented stronger proliferative and pro-angiogenetic capacities, accompanied by increased expression of PCNA, Ki67, VEGFB and ANGPTL4 proteins. Meanwhile, these promoting effects were partially abrogated by Fulvestrant (ICI 182780, an estrogen-receptor antagonist). Aberrantly upregulation of Talin1 mRNA and protein level was observed in ADS endometrial specimens and stromal cells. Through performing functional experiments in vitro, we further determined that merely overexpression of Talin1 (OV-Talin1) also enhanced ADS stromal cell proliferation and pro-angiogenesis, while the most pronounced facilitating effects were found in the co-intervention group of OV-Talin1 plus ß-E2 treatment. Results from the xenograft nude mice model showed that the hypodermic endometrial lesions from co-intervention group had the highest mean weight and volume, compared with that of individual OV-Talin1 or ß-E2 treatment. The expression levels of PCNA, Ki67, VEGFB and ANGPTL4 in the lesions were correspondingly elevated the most in the co-intervention group. Our findings unveiled that overexpressed Talin1 might cooperate withß-E2 in stimulating ADS endometrial stromal cell proliferation and neovascularization, synergistically promoting the growth and survival of ectopic lesions. These results may be beneficial to provide a new insight for clarifying the pathogenesis of ADS.

Estradiol attenuates chronic restraint stress-induced dendrite and dendritic spine loss and cofilin1 activation in ovariectomized mice.

Ovarian hormone deprivation is associated with mood disorders, such as depression, and estradiol therapy is significantly more effective than placebos in treating major depression associated with menopause onset. However, the effect of estradiol on neuronal plasticity and its mechanisms remain to be further elucidated. In this study, behavioral assessments were used to examine the antidepressant effect of estradiol in ovariectomized (OVX) B6.Cg-TgN (Thy-YFP-H)-2Jrs transgenic mice on chronic restraint stress (CRS)-induced dendrite and dendritic spine loss; Yellow fluorescent protein (YFP) is characteristically expressed in excitatory neurons in transgenic mice, and its three-dimensional images were used to evaluate the effect of estradiol on the density of different types of dendritic spines. Quantification and distribution of cofilin1 and p-cofilin1 were determined by qPCR, Western blots, and immunohistochemistry, respectively. The results revealed that treatment with estradiol or clomipramine significantly improved depression-like behaviors. Estradiol treatment also significantly upregulated the dendritic density in all areas examined and increased the density of filopodia-type, thin-type and mushroom-type spines in the hippocampal CA1 and elevated the thin-type and mushroom-type spine density in the PFC. Consistent with these changes, estradiol treatment significantly increased the density of p-cofilin1 immunopositive dendritic spines. Thus, these data reveal a possible estradiol antidepressant mechanism, in that estradiol promoted the phosphorylation of cofilin1 and reduced the loss of dendrites and dendritic spines, which of these dendritic spines include not only immature spines such as filopodia-type, but also mature spines such as mushroom-type, and attenuated the depression-like behavior.

ß-estradiol promotes the growth of primary human fetal spermatogonial stem cells via the induction of stem cell factor in Sertoli cells.

BACKGROUND: Mammalian spermatogenesis is responsible for male fertility and is supported by the self-renewal and differentiation of spermatogonial stem cells (SSCs). Sertoli cells provide a supportive microenvironment for SSCs, in part by the production of stem cell factor (SCF), which is a potent regulator of spermatogonia proliferation and survival. METHODS: We investigated the novel role of ß-estradiol in modulating the proliferation and apoptosis of fetal SSCs via the regulation of SCF secretion in Sertoli cells isolated from human fetal testes. The proliferation of SSCs in the co-culture system was determined by colony formation and BrdU incorporation assays. TUNEL assay was used to measure SSC apoptosis in co-culture in response to treatment with control, ß-estradiol, or the combination of ß-estradiol and the estrogen receptor inhibitor ICI 182780. RESULTS: In the system with purified human fetal Sertoli cells (MIS+/c-Kit-/AP-), ß-estradiol upregulated the production of SCF in a dose- and time-dependent manner. In the co-culture system of primary human fetal SSCs (c-Kit+/SSEA-4+/Oct-4+/AP+) and Sertoli cells (MIS+), ß-estradiol markedly increased the proliferation of SSCs. Moreover, SSC apoptosis was significantly inhibited by ß-estradiol and was completely reversed by the combination of ß-estradiol and ICI 182780. CONCLUSION: Here we report, for the first time, that ß-estradiol can induce the increase of SCF expression in human fetal Sertoli cells and regulates the growth and survival of human fetal SSCs. These novel findings provide new perspectives on the current understanding of the role of estrogen in human spermatogenesis.

Beta-Cyclodextrin-Decorated Magnetic Activated Carbon as a Sorbent for Extraction and Enrichment of Steroid Hormones (Estrone, ß-Estradiol, Hydrocortisone and Progesterone) for Liquid Chromatographic Analysis.

A ß-cyclodextrin-decorated magnetic activated carbon adsorbent was prepared and characterized using various analytical techniques (X-ray diffraction (XRD), scanning electron microscopy-electron diffraction spectroscopy (SEM-EDS) and transmission electron microscopy (TEM)), and the adsorbent was used in the development of a magnetic solid-phase microextraction (MSPE) method for the preconcentration of estrone, ß-estradiol, hydrocortisone and progesterone in wastewater and river water samples. This method was optimized using the central composite design in order to determine the experimental parameters affecting the extraction procedure. The quantification of hormones was achieved using high-performance liquid chromatography equipped with a photodiode array detector (HPLC-DAD). Under optimum conditions, the linearity ranged from 0.04 to 300 µg L-1 with a correlation of determinations of 0.9969-0.9991. The limits of detection and quantification were between 0.01-0.03 and 0.033-0.1 µg L-1, with intraday and interday precisions at 1.1-3.4 and 3.2-4.2. The equilibrium data were best described by the Langmuir isotherm model, and high adsorption capacities (217-294 mg g-1) were obtained. The developed procedure demonstrated high potential as an effective technique for use in wastewater samples without significant interferences, and the adsorbent could be reused up to eight times.

Mouse strains and sexual divergence in corneal innervation and nerve regeneration.

A variety of mouse strains and sexes are used in studies of corneal wound healing and nerve regeneration. However, there is a gap of knowledge about corneal nerve density and its function in different mouse strains and sexes. In this study, we report a strain divergence of total and substance P (SP) sensory corneal nerves in uninjured mice. The BALB/c mouse showed the highest nerve density, corneal sensitivity, and tear volume followed by CFW and then C57BL/6. No differences were found in total nerves and SP-positive nerves between sexes. After injury damaged the corneal nerves, an important role for mouse strains, biologic sex, and their association to corneal nerve regeneration was identified. All female mice have a faster nerve regeneration rate than males. The molecular mechanism of this sexual divergence involves higher secretion neurotrophic factors in tears, which in turn modulate gene expression in trigeminal ganglion neurons. An important upstream signaling regulator was ß-estradiol, and topical treatment with ß-estradiol confirmed its function in corneal nerve regeneration. In conclusion, our study shows that the strain and sex of laboratory mice significantly affect the different indicators of corneal innervation and nerve regeneration. Researchers investigating corneal diseases should carefully consider these factors.-Pham, T. L., Kakazu, A., He, J., Bazan, H. E. P. Mouse strains and sexual divergence in corneal innervation and nerve regeneration.

Hormones modulate Candida vaginal isolates biofilm formation and decrease their susceptibility to azoles and hydrogen peroxide.

Vulvovaginal candidiasis (VVC) is an infection usually caused by Candida albicans and increasingly by Candida glabrata, which has an intrinsically high resistance to commonly used antifungals. Candida species possess virulence factors that contribute to VVC development, as the ability to form biofilms in vaginal walls and intrauterine devices. It is known that VVC is promoted by conditions that increase the hormones levels, during pregnancy, however, the effects of hormones on Candida cells are poorly studied, especially in C. glabrata. Thus, the influence of progesterone and ß-estradiol, at normal cycle and pregnancy concentrations, on biofilm formation and resistance of C. albicans and C. glabrata vaginal isolates, was analyzed using acidic conditions (pH 4). Biofilms of C. albicans developed in the presence of hormones presented reduced biomass (up to 65%) and impaired cells ability to produce filamentous forms. On the other hand, C. glabrata presented high adaptation to the presence of hormones, which did not affect its biofilm formation. Additionally, hormones impaired the susceptibility of C. albicans and C. glabrata cells to azoles, with potential clinical significance in the presence of pregnancy hormone levels. A similar result was obtained for the susceptibility to hydrogen peroxide, a biological vaginal barrier against Candida growth. Overall, the results of this study suggest that hormones may act as environmental cues promoting Candida protection from vaginal defenses and harmful conditions, what may have implications in Candida vaginal pathogenicity and treatment of VVC, especially in C. glabrata infections due to its high adaptability to vaginal conditions.

ß-Estradiol Enhanced Secretion of Lipoprotein Lipase from Mouse Mammary Tumor FM3A Cells.

The role of ß-estradiol (E2) in lipoprotein metabolism in mammary tumors is unclear, therefore, we investigated the effect of E2 on the secretion of lipoprotein lipase (LPL) from mouse mammary tumor FM3A cells. E2-treated cells increased the secretion of active LPL from FM3A cells in a time- and dose-dependent manner. Activity of mitogen-activated protein kinase (MAPK) was increased in the tumor cells treated with E2, and enhanced secretion of LPL was suppressed by MAPK kinase 1/2 inhibitor, PD98059, extracellular signal-regulated kinase (ERK) 1/2 inhibitor, FR180204, p38 MAPK inhibitor, SB202190, and phosphatidyl inositol 3-kinase (PI3K) inhibitor, LY294002. In addition, the effect of E2 on LPL secretion was markedly suppressed by an inhibitor of mammalian target of rapamycin complex (mTORC) 1 and 2, KU0063794, but were not by a mTORC1 inhibitor, rapamycin. Furthermore, a small interfering RNA (siRNA)-mediated decrease in the expression of rapamycin-insensitive companion of mTOR (Rictor), a pivotal component of mTORC2, suppressed secretion of LPL by E2. These results suggest that the stimulatory secretion of LPL by E2 from the tumor cells is closely associated with an activation of mTORC2 rather than mTORC1 possibly via the MAPK cascade.

Additive antidepressant-like effects of fasting with ß-estradiol in mice.

Our recent study has shown that acute fasting produces antidepressant-like effects in male mice. However, there is little evidence regarding the antidepressant-like effects of acute fasting in female mice. Moreover, it is not yet clear whether estrogen produces additive effects with acute fasting. Therefore, this study aims to investigate the antidepressant-like effects of acute fasting plus estrogen treatment. In this study, the acute fasting produced antidepressant-like effects in female mice and the antidepressant-like effects of 9 hours fasting with those of ß-estradiol (E2) were additive. Activity of the cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB)-brain-derived neurotrophic factor (BDNF) pathway in the prefrontal cortex (PFC) and hippocampus (HP) was increased, as well as neurogenesis in the subgranular zone of the hippocampus. Serum ghrelin and estrogen were also increased by fasting plus E2. Furthermore, RNA-seq analysis indicated that fasting and E2 co-regulate similar gene expression pathways, underlying similar neurological functions. Taken together, these data suggest that E2 produces additive antidepressant-like effects with fasting by activating the CREB-BDNF pathway in the PFC and HP. Genome-wide transcriptome mapping suggests that fasting may be used as an adjunct to estrogen replacement therapy for the treatment of depression associated with reduced estrogen function.

Role of ß-Estradiol in MCF-7 Breast Cancer Cell Line Based on the Bioinformatics Analysis.

BACKGROUND: This study aimed to investigate the action mechanism of ß-estradiol in MCF-7 breast cancer (BC) cells. METHODS: The cell samples were sequenced using Hiseq 2000, including 2 MCF-7 controls and 2 samples treated with ß-estradiol. Differentially expressed genes (DEGs) were screened using the NOISeq package in R, followed by the functions and pathways analyses using Database for Annotation, Visualization and Integrated Discovery. DEGs associated with ß-estradiol were selected using the WbeGestalt software, and the corresponding target miRNAs of these genes were analyzed from different miRNA databases. Additionally, protein-protein interaction network of the drug-associated genes was constructed using Cytoscape. RESULTS: A total of 1,835 DEGs in BC samples were screened. Thereinto, DEGs associated with BC (17 upregulated and 28 downregulated DEGs) were involved in the regulation of cell proliferation, response to endogenous stimulus, and response to hormone stimulus, while the genes participated in several significant pathways. Cyclin D1, estrogen receptor 1, catechol-O-methyltransferase, and cathepsin D (CTSD; hub genes) were the predicted new genes associated with ß-estradiol. Besides, hsa-miR-140-3p was the only target miRNA of CTSD. CONCLUSION: ß-Estradiol may play a key role in contributing to BC progression and metastasis by regulating the expression of the selected genes.

Lack of influence of sex hormones on Brugada syndrome-associated mutant Nav1.5 sodium channel.

Brugada syndrome (BS) is an autosomal dominant disease. The most common causes of BS are loss-of-function mutations occur in the SCN5A gene which encodes the sodium channel protein Nav1.5. BS has a higher incidence rate in males and the underlying mechanisms of the gender inequality are not yet fully understood. Considering sex hormones are among the most important factors behind gender differences and have previously been shown to regulate the activity of multiple cardiac ion channels, we hypothesized that sex hormones also affect Nav1.5 function which lead to BS predominantly affecting males. In this study, we investigate the protein expression level and current of Nav1.5 in the HEK293 cells cotransfected with SCN5A and sex hormone receptor plasmids using both wild-type SCN5A and BS-associated SCN5A channel mutants R878C and R104W. Our findings showed that sex hormones have no effects on the protein expression level and current of the wild-type Nav1.5, neither does it affect the protein expression level and current of BS-associated Nav1.5 mutants R878C and R104W, regardless of homozygous or heterozygous state. Our results suggest that the male preponderance of BS does not arise from the effects of the sex hormones on Nav1.5. Further studies are needed to explain the male preponderance of this disease.

Multidrug ABC transporter Cdr1 of Candida albicans harbors specific and overlapping binding sites for human steroid hormones transport.

The present study examines the kinetics of steroids efflux mediated by the Candida drug resistance protein 1 (Cdr1p) and evaluates their interaction with the protein. We exploited our in-house mutant library for targeting the 252 residues forming the twelve transmembrane helices (TMHs) of Cdr1p. The screening revealed 65 and 58 residues critical for ß-estradiol and corticosterone transport, respectively. Notably, up to 83% critical residues for corticosterone face the lipid interface compared to 54% for ß-estradiol. Molecular docking identified a possible peripheral corticosterone-binding site made of 8/14 critical/non-critical residues between TMHs 3, 4 and 6. ß-estradiol transport was severely hampered by alanine replacements of Cdr1p core residues involving TMHs 2, 5 and 8, in a binding site made of 10/14 critical residues mainly shared with rhodamine 6G with which it competes. By contrast, TMH11 was poorly impacted, although being part of the core domain. Finally, we observed the presence of several contiguous stretches of 3-5 critical residues in TMHs 2, 5 and 10 that points to a rotation motion of these helices during the substrate transport cycle. The selective structural arrangement of the steroid-binding pockets in the core region and at the lipid-TMD interface, which was never reported before, together with the possible rotation of some TMHs may be the structural basis of the drug-transport mechanism achieved by these type II ABC transporters.

Characterization of ß-Estradiol 3-Glucuronidation in Rat Brain.

ß-Estradiol is conjugated by uridine 5'-diphosphate-glucuronosyltransferase (UGT) 1A to 3-glucuronide in the human liver. UGT1A has been found in the brain; therefore, UGT1A may be involved in ß-estradiol 3-glucuronidation in the brain. In the present study, we aimed to characterize the ß-estradiol 3-glucuronidation reaction in the rat brain. ß-Estradiol 3-glucuronidation was detected in eight rat brain regions (cerebellum, frontal cortex, parietal cortex, piriform cortex, hippocampus, medulla oblongata, striatum, and thalamus). ß-Estradiol 3-glucuronidation in the cerebellum was fitted to the Hill equation (S50=8.0 µM, n=1.1). In inhibition experiments, ß-estradiol 3-glucuronidation was inhibited to 73.6% in the cerebellum by 50 µM bilirubin, whereas it was reduced to 20.5% with 5 µM bilirubin in the liver. Unlike in the liver, Ugt1a1 may not be the main isoform catalyzing this glucuronidation in the brain. Serotonin and acetaminophen at 10 mM inhibited glucuronidation to 1.17 and 25.5%, respectively, in the cerebellum. In induction experiments, the administration of ß-naphthoflavone, carbamazepine, and phenobarbital did not increase ß-estradiol 3-glucuronidation in the brain except for phenobarbital in the striatum. In addition, ß-estradiol 3-glucuronidation was not correlated with serotonin or acetaminophen glucuronidation in the brain, suggesting that Ugt1a6 and Ugt1a7 are not major isoforms of ß-estradiol 3-glucuronidation in the rat brain. In the present study, although we were unable to identify the isoform responsible for ß-estradiol 3-glucuronidation, we confirmed that ß-estradiol could be metabolized to glucuronide in the brain under a different metabolic profile from that in the liver.

Species and Tissue Differences in ß-Estradiol 17-Glucuronidation.

Uridine 5'-diphosphate-glucuronosyltransferase (UGT) is expressed in the liver and extrahepatic tissues. One of the major metabolic pathways of ß-estradiol (E2) is glucuronidation at the 17-hydroxy position by UGTs. This study was performed to determine E2 17-glucuronidation kinetics in human and rodent liver, small intestine, and kidney microsomes and to clarify the species and tissue differences. In the human liver and small intestine, Eadie-Hofstee plots exhibited biphasic kinetics, suggesting that E2 17-glucuronide (E17G) formation was catalyzed by more than two UGT isoforms in both tissues. The Km values for E17G formation by the high-affinity enzymes in the human liver and small intestine were 1.79 and 1.12 µM, respectively, and corresponding values for the low-affinity enzymes were 3.72 and 11.36 µM, respectively. Meanwhile, E17G formation in the human kidney was fitted to the Hill equation (S50=1.73 µM, n=1.63), implying that the UGT isoform catalyzing E17G formation in the kidney differed from that in the liver and small intestine. The maximum clearance for E17G formation in the human kidney was higher than the intrinsic clearance in the liver. E17G formation in the rat liver and kidney exhibited biphasic kinetics, whereas that in the small intestine was fitted to the Hill equation. In mice, all 3 tissues exhibited biphasic kinetics. In conclusion, we reported species and tissue differences in E2 17-glucuronidation, which occurred not only in the human liver but also in the extrahepatic tissues particularly the kidney.

17ß-estradiol differently affects osteogenic differentiation of mesenchymal stem/stromal cells from adipose tissue and bone marrow.

Adipose-derived and bone marrow stem/stromal cells (ASCs and BMSCs) have been often compared for their application in regenerative medicine, and several factors sustaining their differentiation and efficacy have been investigated. 17 ß-estradiol (E2) has been reported to influence some functions of progenitor cells. Here we studied the effects of 10 and 100nM E2 on ASC and BMSC vitality, proliferation and differentiation towards osteogenic and adipogenic lineages. E2 did not modulate ASC and BMSC vitality and growth rate, while the hormone produced a pro-adipogenic effect on both mesenchymal stem/stromal cells (MSCs). In particular, the synergy between 7-day pre-treatment and 100nM E2 led to the most evident result, increasing lipid vacuoles formation in ASCs and BMSCs of +44% and +82%, respectively. Despite the fact that E2 did not alter collagen deposition of osteo-induced MSCs, we observed a different modulation of ASC and BMSC alkaline phosphatase (ALP) activity. Indeed, this osteogenic marker was always enhanced by 17 ß-estradiol in BMSCs, and 7-day pre-treatment with 100nM E2 increased it of about 70%. In contrast, E2 weakened ASC osteogenic potential, reducing their ALP activity of about 20%, with the most evident effect on ASCs isolated from pre-menopausal women (-30%). Finally, we identified an estrogen receptor α (ERα) variant of about 37kDa expressed in both MSCs. Interestingly, adipogenic stimuli drastically reduced its expression, while osteogenic ones mildly increased this isoform in BMSCs only. In conclusion, E2 positively affected the adipogenic process of both MSCs while it favored osteogenic induction in BMSCs only, and both mesenchymal progenitors expressed a novel 37kDa ER-α variant whose expression was modulated during differentiation.

Effect of dioxin and 17ß-estradiol on the expression of cytochrome P450 1A1 gene via an estrogen receptor dependent pathway in cellular and xenografted models.

Cytochrome P450 (CYP) 1A1 plays a major role in the metabolic activation of procarcinogens to carcinogens via aryl hydrocarbon receptor (AhR) pathway. Especially, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is known as an agonist of AhR. In estrogen responsive cancers, 17ß-estradiol (E2) may influence on AhR dependent expression of CYP1 family via the interaction between estrogen receptor (ER) and AhR. In the present study, the effect of E2/ER on the expression of AhR and CYP1A1 genes was investigated for MCF-7 clonal variant (MCF-7 CV) breast cancer cells expressing ER. In reverse transcription-PCR and Western blot analysis, mRNA expression level of AhR was not altered, but its protein expression level was increased by TCDD or E2. The transcriptional and translational levels of CYP1A1 appeared to be increased by TCDD or E2. The increased expression of AhR and CYP1A1 induced by E2 was restored to the control level by the co-treatment of ICI 182,780, indicating that E2 induced the protein expression levels of AhR and CYP1A1 like TCDD via an ER dependent pathway. In an in vivo xenograft mouse model transplanted with MCF-7 CV cells, the protein expression levels of AhR and CYP1A1 of tumor masses were also increased by E2 or TCDD. Taken together, these results indicate that E2 may promote AhR dependent expression of CYP1A1 via ER dependent pathway in MCF-7 CV cells expressing ER in the absence of TCDD, an agonist of AhR. The relevance of E2 and ER in CYP1A1 activation of estrogen responsive cancers may be targeted for developing more effective cancer treatments.

17 ß-estradiol mineralization in human waste products and soil in the presence and the absence of antimicrobials.

Natural steroidal estrogens, such as 17 ß-estradiol (E2), as well as antimicrobials such as doxycycline and norfloxacin, are excreted by humans and hence detected in sewage sludge and biosolid. The disposal of human waste products on agricultural land results in estrogens and antibiotics being detected as mixtures in soils. The objective of this study was to examine microbial respiration and E2 mineralization in sewage sludge, biosolid, and soil in the presence and the absence of doxycycline and norfloxacin. The antimicrobials were applied to the media either alone or in combination at total rates of 4 and 40 mg kg-1, with the 4 mg kg-1 rate being an environmentally relevant concentration. The calculated time that half of the applied E2 was mineralized ranged from 294 to 418 days in sewage sludge, from 721 to 869 days in soil, and from 2,258 to 14,146 days in biosolid. E2 mineralization followed first-order and the presence of antimicrobials had no significant effect on mineralization half-lives, except for some antimicrobial applications to the human waste products. At 189 day, total E2 mineralization was significantly greater in sewage sludge (38 ±0.7%) > soil (23 ±0.7%) > biosolid (3 ±0.7%), while total respiration was significantly greater in biosolid (1,258 mg CO2) > sewage sludge (253 mg CO2) ≥ soil (131 mg CO2). Strong sorption of E2 to the organic fraction in biosolid may have resulted in reduced E2 mineralization despite the high microbial activity in this media. Total E2 mineralization at 189 day was not significantly influenced by the presence of doxycycline and/or norfloxacin in the media. Antimicrobial additions also did not significantly influence total respiration in media, except that total CO2 respiration at 189 day was significantly greater for biosolid with 40 mg kg-1 doxycycline added, relative to biosolid without antimicrobials. We conclude that it is unlikely for doxycycline and norfloxacin, or their mixtures, to have a significant effect on E2 mineralization in human waste products and soil. However, the potential for E2 to be persistent in biosolids, with and without the presence of antimicrobials, is posing a challenge for biosolid disposal to agricultural lands.

17 ß-estradiol and 17 α-ethinylestradiol mineralization in sewage sludge and biosolids.

Natural steroid estrogens (e.g., 17 ß-estradiol, E2), synthetic steroid estrogens (e.g., 17 α-ethinylestradiol, EE2) and pharmaceutical antibiotics (e.g., ciprofloxacin) are chemicals detected in biosolids and sewage sludges because they partition into the solids fraction during the wastewater treatment process. This research utilized a three-way factorial design (six media × two estrogens × three antibiotic treatments) to quantify cumulative E2 and EE2 mineralization over 133 d (MAX) in a range of sewage sludge and biosolid samples in the presence (4 and 40 mg kg(-1)) and absence of ciprofloxacin. The same three-way factorial design was utilized to quantify the impact of the six media, E2 or EE2, and ciprofloxacin on cumulative soil respiration over 133 d (RESP). Minimal ciprofloxacin mineralization was observed (<0.05% over 133 d), but despite its persistence, ciprofloxacin had no significant effect on MAX of E2 or EE2, and, in general, no significant effect on RESP. MAX ranged from 38.38% to 48.44% for E2 but from only 0.72% to 24.27% for EE2 although RESP was relatively similar, ranging from 101.00 to 866.54 mg CO2 in the presence of E2 and from 69.55 to 893.95 mg CO2 in the presence of EE2. The sorption-limited bioavailability of EE2, which is inherently resistant to biodegradation due to chemical structure, as MAX and Freundlich sorption coefficients (Kf) were negatively correlated. As such, the Kf values of EE2 were largest in composted biosolids in which EE2 was particularly resistant to microbial degradation as the MAX of EE2 was <3%. In contrast, the MAX of E2 showed a positive association with the Kf values of E2 because some steps in the E2 transformation process have been found to occur in the sorbed phase. The MAX of E2 was significantly greater in the biosolid and composted biosolid media than in any other media, whereas the MAX of E2 decreased in the following order: secondary sewage sludge > primary sewage sludge > biosolid = composted biosolid. This suggests that sewage sludges in municipal lagoons and pre-treatment holding lagoons are a more favorable media for mineralization of EE2, whereas biosolids in post-treatment storage lagoons are a more favorable media for the mineralization of E2. The presence of ciprofloxacin will have no impact on the potential E2 or EE2 mineralization rates in these cases.