Simultaneous determination of natural and synthetic steroid estrogens and their conjugates in aqueous matrices by liquid chromatography / mass spectrometry

An analytical method for the simultaneous determination of nine free and conjugated steroid estrogens was developed with application to environmental aqueous matrices. Solid phase extraction (SPE) was employed for isolation and concentration, with detection by liquid chromatography/mass spectrometry (LC/MS) using electrospray ionisation (ESI) in the negative mode. Method recoveries for various aqueous matrices (wastewater, lake and drinking water) were determined, recoveries proving to be sample dependent. When spiked at 50 ng/l concentrations in sewage influent, recoveries ranged from 62-89 % with relative standard deviations (RSD) < 8.1 %. In comparison, drinking water spiked at the same concentrations had recoveries between 82-100 % with an RSD < 5%. Ion suppression is a known phenomenon when using ESI; hence its impact on method recovery was elucidated for raw sewage. Both ion suppression from matrix interferences and the extraction procedure has bearing on the overall method recovery. Analysis of municipal raw sewage identified several of the analytes of interest at ng/l concentrations, estriol (E3) being the most abundant. Only one conjugate, estrone 3-sulphate (E1-3S) was observe

Fate of conjugated natural and synthetic steroid estrogens in crude sewage and activated sludge batch studies

This document is the unedited author's version of a Submitted Work that was subsequently accepted for publication in Environmental Science & Technology, copyright © American Chemical Society after peer review. To access the final edited and published work see http://pubs.acs.org/doi/abs/10.1021/es801952h.Steroids are excreted from the human body in the conjugated form but are present in sewage influent and effluent as the free steroid, the major source of estrogenic activity observed in water courses. The fate of sulfate and glucuronide conjugated steroid estrogens was investigated in batch studies using activated sludge grown on synthetic sewage in a laboratory-scale Husmann simulation and crude sewage from the field. A clear distinction between the fate of sulfate and glucuronide conjugates was observed in both matrices, with sulfated conjugates proving more recalcitrant and glucuronide deconjugation preferential in crude sewage. For each conjugate, the free steroid was observed in the biotic samples. The degree of free steroid formation was dependent on the conjugate moiety, favoring the glucuronide. Subsequent degradation of the free steroid (and sorption to the activated sludge solid phase) was evaluated. Deconjugation followed the first order reaction rate with rate constants for 17α-ethinylestradiol 3-glucuronide, estriol 16α-glucuronide, and estrone 3-glucuronide determined as 0.32, 0.24, and 0.35 h respectively. The activated sludge solid retention time over the range of 3−9 days had 74 to 94% of sulfate conjugates remaining after 8 h. In contrast, a correlation between increasing temperature and decreasing 17α-ethinylestradiol 3-glucuronide concentrations in the activated sludge observed no conjugate present in the AS following 8 h at 22 °C Based on these batch studies and literature excretion profiles, a hypothesis is presented on which steroids and what form (glucuronide, sulfate, or free) will likely enter the sewage treatment plant.EPSR

Progression in MCF-7 Breast Cancer Cell Tumorigenicity: Compared Effect of FGF-3 and FGF-4.

The transforming properties of fibroblast growth factor 3 (FGF-3) were investigated in MCF7 breast cancer cells and compared to those of FGF-4, a known oncogenic product. The short form of fgf-3 and the fgf-4 sequences were each introduced with retroviral vectors and the proteins were only detected in the cytoplasm of the infected cells, as expected. In vitro, cells producing FGF-3 (MCF7.fgf-3) and FGF-4 (MCF7.fgf-4) displayed an amount of estrogen receptors decreased to around 45% of the control value. However, MCF7.fgf-3 cell proliferation remained responsive to estradiol supply. The sensitivity of the MCF7.fgf-4 cells, if existant, was masked by the important mitogenic action exerted by FGF-4. In vivo, the MCF7.fgf-3 and MCF7.fgf-4 cells gave rise to tumors under conditions in which the control cells were not tumorigenic. Supplementing the mice with estrogen had the paradoxical effect of totally suppressing the start of the FGF-3 as well as the FGF-4 tumors. Tumorigenicity in the presence of matrigel was similar for MCF7.fgf-3 and control cells and was increased by estrogen supplementation. Once started, the MCF7.fgf-4 tumors grew with a characteristic high rate. Remarkably, FGF-4 but not FGF-3, stimulated the secretion of vascular endothelial growth factor (VEGF165) without altering the steady-state level of its mRNA, suggesting a possible regulation of VEGF synthesis at the translational level in MCF7 cells. The increased VEGF secretion is probably involved in the more aggressive phenotype of the MCF7.fgf-4 cells while a decreased dependence upon micro-environmental factors might be part of the increased tumorigenic potential of the MCF7.fgf-3 cells.Peer reviewe

Supporting data on combined transcriptomic and phosphoproteomic analysis of BMP4 signaling in human embryonic stem cells.

Human embryonic stem cells exhibit great potential as a therapeutic tool in regenerative medicine due to their self-renewal and trilineage differentiation capacity. Maintaining this unique cellular state has been shown to rely primarily on the Activin A / TGFβ signaling pathway. While most conventional culture media are supplemented with TGFβ, in the current study we utilize a modified version of the commercially available mTeSR1, substituting TGFβ for Activin A in order to preserve pluripotency. (1) Cells cultured in ActA-mTesR express pluripotency factors NANOG, OCT4 and SOX2 at comparable levels with cells cultured in TGFβ-mTeSR. (2) ActA-mTeSR cultured cells retain a physiological karyotype. (3) Cells in ActA-mTeSR maintain their trilineage differentiation capacity as shown in the teratoma formation assay. This system can be used to dissect the role of Activin A, downstream effectors and signaling cascades in human embryonic stem cell responses

Phytoestrogens and inhibition of angiogenesis

Peer reviewe

A Comparative Survey of Leguminous Plants as Sources of the Isoflavones, Genistein and Daidzein: Implications for Human Nutrition and Health

Over 80 taxa of mostly agriculturally important legumes were surveyed as sources of the metabolites, genistein and daidzein. Remarkably high concentrations (over 2 g · kg–1 dry weight) of the anticancer metabolite, genistein, were found in the leaves of Psoralea corylifolia (Indian bread root). All other legumes, with the exception of fermented soybean miso, had genistein levels <400 mg · kg–1 dry weight. Concentrations of over 1 g · kg–1 dry weight and 0.95 g · kg–1 dry weight of the anticancer metabolite, daidzein, were found in the stems of the fava bean (Vicia faba) and roots of kudzu vine (Pueraria lobata)' respectively. From this survey, our results indicate that the legumes, lupine (Lupinus spp.), fava bean, (Vicia faha), soybeans (Glycine max), kudzu (Pueraria lobata), and psoralea (Psoralea corylifolia), are excellent food sources for both genistein and daidzein. Miso, a fermented soybean product, is also a rich source of both isoflavones.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/63131/1/acm.1997.3.7.pd

Modification of Cantor High Entropy Alloy by the Addition of Mo and Nb: Microstructure Evaluation, Nanoindentation-Based Mechanical Properties, and Sliding Wear Response Assessment

The classic Cantor (FeCoCrMnNi) isoatomic high entropy alloy was modified by separate additions of Mo and Nb in an effort to optimize its mechanical properties and sliding wear response. It was found that the introduction of Mo and Nb modified the single phase FCC solid solution structure of the original alloy and led to the formation of new phases such as the BCC solid solution, σ-phase, and Laves, along with the possible existence of intermetallic phases. The overall phase formation sequence was approached by parametric model assessment and solidification considerations. Nanoindentation-based mechanical property evaluation showed that due to the introduction of Mo and Nb; the modulus of elasticity and microhardness were increased. Creep nanoindentation assessment revealed the beneficial action of Mo and Nb in increasing the creep resistance based on the stress sensitivity exponent, strain rate sensitivity, and critical volume for the dislocation nucleation considerations. The power law and power law breakdown were identified as the main creep deformation mechanisms. Finally, the sliding wear response was increased by the addition of Mo and Nb with this behavior obeying Archard’s law. A correlation between microstructure, wear track morphologies, and debris characteristics was also attempted

Genistein, a dietary-derived inhibitor of in vitro angiogenesis

Consumption of a plant-based diet can prevent the development and progression of chronic diseases that are associated with extensive neovascularization; however, little is known about the mechanisms. To determine whether prevention might be associated with dietary-derived angiogenesis inhibitors, we have fractionated urine of healthy human subjects consuming a plant-based diet and examined the fractions for their abilities to inhibit the proliferation of vascular endothelial cells. Using gas chromatography-mass spectrometry, we showed that one of the most potent fractions contained several isoflavonoids, which we subsequently synthesized. Of all synthetic compounds, the isoflavonoid genistein was the most potent and inhibited endothelial cell proliferation and in vitro angiogenesis at concentrations giving half-maximal inhibition of 5 and 150 microM, respectively. As we have previously demonstrated, genistein concentrations in urine of subjects consuming a plant-based diet are in the micromolar range, while those of subjects consuming a traditional Western diet are lower by a factor of > 30. The high excretion of genistein in urine of vegetarians and our present results suggest that genistein may contribute to the preventive effect of a plant-based diet on chronic diseases, including solid tumors, by inhibiting neovascularization. Thus, genistein may represent a member of a new class of dietary-derived anti-angiogenic compounds.Proc Natl Acad Sci U S

Luteolin reduces lipopolysaccharide-induced lethal toxicity and expression of proinflammatory molecules in mice

Luteolin is a flavonoid that has been shown to reduce proinflammatory molecule expression in vitro. In the present study, we have tested the ability of luteolin to inhibit lipopolysaccharide (LPS)- induced lethal toxicity and proinflammatory molecule expression in vivo. Mice receiving LPS (Salmonella enteriditis LPS, 32 mg/kg, intraperitoneally) exhibited high mortality with only 4.1% of the animals surviving seven days after the LPS challenge. On the contrary, mice that had received luteolin (0.2 mg/kg, intraperitoneally) before LPS showed an increased survival rate with 48% remaining alive on Day 7. To investigate the mechanism by which luteolin affords protection against LPS toxicity we measured intercellular adhesion molecule-1 (ICAM-1) and tumor necrosis factor-alpha (TNF-alpha) production in response to LPS in the presence or absence of luteolin pretreatment. Treatment of animals with LPS increased serum TNF-alpha levels in a time-dependent manner. The increase in peak serum TNF-alpha levels was sensitive to luteolin pretreatment. Luteolin pretreatment also reduced LPS-stimulated ICAM-1 expression in the liver and abolished leukocyte infiltration in the liver and lung. We conclude that luteolin protects against LPS-induced lethal toxicity, possibly by inhibiting proinflammatory molecule (TNF-alpha, ICAM-1) expression in vivo and reducing leukocyte infiltration in tissues.Am J Respir Crit Care Me