High-performance liquid chromatographic determination of 17 beta-estradiol and 17 beta-estradiol-3-acetate solubilities and diffusion coefficients in silicone elastomeric intravaginal rings

A rapid, sensitive reversed-phase high-performance liquid chromatographic method has been developed for the determination of in vitro release of 17 beta-estradiol and its ester prodrug, 17 beta-estradiol-3-acetate, from silicone intravaginal rings. Partial hydrolysis of the acetate under the aqueous conditions provided by the 1% benzalkonium chloride release medium necessitates its conversion to 17 beta-estradiol prior to HPLC analysis. Both steroid peaks have been fully resolved from the benzalkonium chloride peaks by the reported chromatographic method,which employs a C-18 bonded reversed-phase column, an acetonitrile-water (50:50, v/v) mobile phase and a UV detection wavelength of 281 nm. The peak area versus 17 beta-estradiol concentration was found to be linear over the range of 0.0137-1347 mu g ml(-1) The HPLC method has also been used to determine the silicone solubilities and diffusion coefficients of the two related steroids. The almost 100-fold increase in 17 beta-estradiol-3-acetate release from the silicone core-type intravaginal rings compared to 17 beta-estradiol is shown to be due to a 60-fold increase in silicone solubility and a one and a half-fold increase in diffusitivity. The results demonstrate that an effective estrogen replacement therapy dose of 17 beta-estradiol may be administered from a silicone intravaginal reservoir device containing the labile 17 beta-estradiol-3-acetate prodrug. (C) 2000 Elsevier Science B.V. All rights reserved.

Elimination kinetic of 17 beta-estradiol 3-benzoate and 17 beta-nandrolone laureate ester metabolites in calves' urine

Efficient control of the illegal use of anabolic steroids must both take into account metabolic patterns and associated kinetics of elimination; in this context, an extensive animal experiment involving 24 calves and consisting of three administrations of 17 beta-estradiol 3-benzoate and 17 beta-nandrolone laureate esters was carried out over 50 days. Urine samples were regularly collected during the experiment from all treated and non-treated calves. For sample preparation, a single step high throughput protocol based on 96-well C-18 SPE was developed and validated according to the European Decision 2002/657/EC requirements. Decision limits (CC alpha) for steroids were below 0.1 mu g L-1, except for 19-norandrosterone (CC alpha = 0.7 mu g L-1) and estrone (CC alpha = 0.3 mu g L-1). Kinetics of elimination of the administered 17 beta-estradiol 3-benzoate and 17 beta-nandrolone laureate were established by monitoring 17 beta-estradiol, 17 alpha-estradiol, estrone and 17 beta-nandrolone, 17 alpha-nandrolone, 19-noretiocholanolone, 19-norandrostenedione, respectively. All animals demonstrated homogeneous patterns of elimination both from a qualitative (metabolite profile) and quantitative point of view (elimination kinetics in urine). Most abundant metabolites were 17 alpha-estradiol and 17 alpha-nandrolone (> 20 and 2 mg L-1, respectively after 17 beta-estradiol 3-benzoate and 17 beta-nandrolone laureate administration) whereas 17 beta-estradiol, estrone, 17 beta-nandrolone, 19-noretiocholanolone and 19-norandrostenedione were found as secondary metabolites at concentration values up to the mu g L-1 level. No significant difference was observed between male and female animals. The effect of several consecutive injections on elimination profiles was studied and revealed a tendency toward a decrease in the biotransformation of administered steroid 17 beta form. (c) 2008 Elsevier Ltd. All rights reserved.

Estrogen Receptor-beta Affects the Prognosis of Human Malignant Mesothelioma

Malignant pleural mesothelioma is an asbestos-related neoplasm with poor prognosis, refractory to current therapies, the incidence of which is expected to increase in the next decades. Female gender was identified as a positive prognostic factor among other clinical and biological prognostic markers for malignant mesothelioma, yet a role of estrogen receptors (ERs) has not been studied. Our goal was to investigate ERs expression in malignant mesothelioma and to assess whether their expression correlates with prognosis. Immunohistochemical analysis revealed intense nuclear ER beta staining in normal pleura that was reduced in tumor tissues. Conversely, neither tumors nor normal pleura stained positive for ER alpha. Multivariate analysis of 78 malignant mesothelioma patients with pathologic stage, histologic type, therapy, sex, and age at diagnosis indicated that FRO expression is an independent prognostic factor of better survival. Moreover, studies in vitro confirmed that treatment with 17 beta-estradiol led to an ER beta-mediated inhibition of malignant mesothelioma cell proliferation as well as p21(CIP1) and p27(KIP1) up-regulation. Consistently cell growth was suppressed by ER beta overexpression, causing a G(2)-M-phase cell cycle arrest, paralleled by cyclin B1 and survivin down-regulation. Our data support the notion that ER beta acting as a tumor suppressor is of high potential relevance to prediction of disease progression and to therapeutic response of malignant mesothelioma patients. [Cancer Res 2009;69(11):4598-604]

Endocrine disruptor activity in bottled mineral and flavoured water

A panel of reporter gene assays (RGAs) coupled with a single solid phase extraction (SPE) step was developed and used to screen bottled mineral water for the presence of four classes of endocrine disruptors (EDs), oestrogens, androgens, progestagens and glucocorticoids.

Fourteen brands of bottled mineral water in triplicate (42 samples) were analysed. Overall, hormonal activity was found in 78% of the samples. Oestrogenic, androgenic, progestagenic and glucocorticoid activity was found in 38%, 38%, 36% and 55% of the samples, respectively at an average concentration of 10 ng/l 17 beta-estradiol equivalent (EEQ), 26 ng/l testosterone equivalent (TEQ), 123 ng/l progesterone equivalent (PEQ) and 13.5 ng/l hydrocortisone equivalent (HEQ).

The level of oestrogenic, androgenic and progestagenic activity observed is not considered a matter of concern for the consumers' health. It is unknown whether the glucocorticoid levels observed are safe. The ED source, long term exposure and mixture effects remain to be investigated. (C) 2012 Elsevier Ltd. All rights reserved.

Plasma biomarker profiling in the detection of growth promoter use in calves

The detection of illicit growth promoter use during meat production within the European Union is reliant on residue testing which is a limiting factor on the number of animals which can be tested and consequently compromises the efficacy of testing procedures. The present study examined a novel detection strategy based on the profiling of plasma component concentrations in response to growth promoter administrations. Calves subjected to nortestosterone decanoate, 17 beta-oestradiol benzoate and dexamethasone were found to have altered urea, aminoterminal propeptide of type III procollagen and sex hormone binding globulin profiles in response to treatments. These findings demonstrate the potential of using the identification of perturbed profiles within a panel of biomarkers which cover a spectrum of biological activity to reveal growth promoter abuse.

Effect of phytoestrogen and antioxidant supplementation on oxidative DNA damage assessed using the comet assay

Antioxidant species may act in vivo to decrease oxidative damage to DNA, protein and lipids thus reducing the risk of coronary heart disease and cancer. Phytoestrogens are plant compounds which are a major component of traditional Asian diets and which may be protective against certain hormone-dependent cancers (breast and prostate) and against coronary heart disease. They may also be able to function as antioxidants, scavenging potentially harmful free radicals. In this study, the effects of the isoflavonoids (a class of phytoestrogen) genistein and equol on hydrogen peroxide-mediated DNA damage in human lymphocytes were determined using alkaline single-cell gel electrophoresis (the comet assay). Treatment with hydrogen peroxide significantly increased the levels of DNA strand breaks. Pre-treatment of the cells with both genistein and equol offered protection against this damage at concentrations within the physiological range. This protection was greater than that offered by addition of the known antioxidant vitamins ascorbic acid and alpha -tocopherol, or the compounds 17 beta -oestradiol and Tamoxifen which have similar structures to isoflavonoids and are known to have weak antioxidant properties. These findings are consistent with the hypothesis that phytoestrogens can, under certain conditions, function as antioxidants and protect against oxidatively-induced DNA damage. (C) 2001 Elsevier Science B.V. All rights reserved.

Effect-based proteomic detection of growth promoter abuse

Unregulated growth promoter use in food-producing animals is an issue of concern both from food safety and animal welfare perspectives. However, the monitoring of such practices is analytically challenging due to the concerted actions of users to evade detection. Techniques based on the monitoring of biological responses to exogenous administrations have been proposed as more sensitive methods to identify treated animals. This study has, for the first time, profiled plasma proteome responses in bovine animals to treatment with nortestosterone decanoate and 17 beta-oestradiol benzoate, followed by dexamethasone administration. Two-dimensional fluorescence differential in-gel electrophoresis analysis revealed a series of hepatic and acute-phase proteins within plasma whose levels were up- or down-regulated within phases of the treatment regime. Surface plasmon resonance (SPR) immuno-assays were developed to quantify responses of identified protein markers during the experimental treatment study with a view to developing methods which can be used as screening tools for growth promoter abuse detection. SPR analysis demonstrated the potential for plasma proteins to be used as indicative measures of growth promoter administrations and concludes that the sensitivity and robustness of any detection approach based on plasma proteome analysis would benefit from examination of a range of proteins representative of diverse biological processes rather being reliant on specific individual markers.

Identification of RBCK1 as a novel regulator of FKBPL: implications for tumor growth and response to tamoxifen

FKBPL has been implicated in processes associated with cancer, including regulation of tumor growth and angiogenesis with high levels of FKBPL prognosticating for improved patient survival. Understanding how FKBPL levels are controlled within the cell is therefore critical. We have identifed a novel role for RBCK1 as an FKBPL-interacting protein, which regulates FKBPL stability at the post-translational level via ubiquitination. Both RBCK1 and FKBPL are upregulated by 17-b-estradiol and interact within heat shock protein 90 chaperone complexes, together with estrogen receptor-a (ERa). Furthermore, FKBPL and RBCK1 associate with ERa at the promoter of the estrogen responsive gene, pS2, and regulate pS2 levels. MCF-7 clones stably overexpressing RBCK1 were shown to have reduced proliferation and increased levels of FKBPL and p21. Furthermore, these clones were resistant to tamoxifen therapy, suggesting that RBCK1 could be a predictive marker of response to endocrine therapy. RBCK1 knockdown using targeted small interfering RNA resulted in increased proliferation and increased sensitivity to tamoxifen treatment. Moreover, in support of our in vitro data, analysis of mRNA microarray data sets demonstrated that high levels of FKBPL and RBCK1 correlated with increased patient survival, whereas high RBCK1 predicted for a poor response to tamoxifen. Our findings support a role for RBCK1 in the regulation of FKBPL with important implications for estrogen receptor signaling, cell proliferation and response to endocrine therapy.

Effect of growth-promoting 17ß estradiol, 19 nortestosterone and dexamethasone on circulating levels of nine potential biomarker candidates in veal calves

The use of screening methods based on the detection of biological effects of growth promoters is a promising approach to assist residue monitoring. To reveal useful effects on protein metabolism, male and female veal calves at 10 weeks of age were treated thrice with a combination of 25 mg 17ß-estradiol 3-benzoate and 150 mg 19-nortestosterone decanoate with 2 weeks intervals and finally once with 4 mg dexamethasone. Hormone-treated calves showed a significant accelerated growth rate over 6 weeks. Plasma samples of treated and control calves were analysed for immunoreactive inhibin (ir-inhibin), osteocalcin, insulin-like growth factor 1 (IGF-1), insulin-like growth factor-binding protein 2 (IGFBP-2), IGFBP-3, luteinzing hormone (LH), follicle-stimulating hormone (FSH) and prolactin using immunoaffinity assays. Hormone treatment did not affect levels of IGF-1, IGFBP-2, IGFBP-3, LH, FSH and prolactin. The concentration of circulating ir-inhibin decreased, however, significantly (P < 0.05) in bull calves upon administration of the sex steroids, whereas it remained unchanged in the female animals. Dexamethasone treatment decreased significantly (P < 0.05) circulating levels of osteocalcin in both female and male animals. Ir-inhibin and osteocalcin were, therefore, considered as candidates for a protein biomarker-based screening assay for detection of abuse of estrogens, androgens and/or glucocorticoids in cattle fattening, which is being developed in the framework of EU research project BioCop

Endocrine disrupting effects of zearalenone, alpha- and beta-zearalenol at the level of nuclear receptor binding and steroidogenesis.

The mycotoxin zearalenone (ZEN) is a secondary metabolite of fungi which is produced by certain species of the genus Fusarium and can occur in cereals and other plant products. Reporter gene assays incorporating natural steroid receptors and the H295R steroidogenesis assay have been implemented to assess the endocrine disrupting activity of ZEN and its metabolites -zearalenol (-ZOL) and -zearalenol (-ZOL). -ZOL exhibited the strongest estrogenic potency (EC50 0.022 ± 0.001 nM), slightly less potent than 17- estradiol (EC50 0.015 ± 0.002 nM). ZEN was ~70 times less potent than -ZOL and twice as potent as -ZOL. Binding of progesterone to the progestagen receptor was shown to be synergistically increased in the presence of ZEN, -ZOL or -ZOL. ZEN, -ZOL or -ZOL increased production of progesterone, estradiol, testosterone and cortisol hormones in the H295R steroidogenesis assay, with peak productions at 10 M. At 100 M, cell viability decreased and levels of hormones were significantly reduced except for progesterone. -ZOL increased estradiol concentrations more than -ZOL or ZEN, with a maximum effect at 10 M, with -ZOL (562 ± 59 pg/ml) > -ZOL (494 ± 60 pg/ml) > ZEN (375 ± 43 pg/ml). The results indicate that ZEN and its metabolites can act as potential endocrine disruptors at the level of nuclear receptor signalling and by altering hormone production.

Nitric Oxide Produced in Response to Engagement of beta 2 Integrins on Human Neutrophils Activates the Monomeric GTPases Rap1 and Rap2 and Promotes Adhesion

We found that engagement of beta 2 integrins on human neutrophils increased the levels of GTP-bound Rap1 and Rap2. Also, the activation of Rap1 was blocked by PP1, SU6656, LY294002, GF109203X, or BAPTA-AM, which indicates that the downstream signaling events in Rap1 activation involve Src tyrosine kinases, phosphoinositide 3-kinase, protein kinase C, and release of calcium. Surprisingly, the integrin-induced activation of Rap2 was not regulated by any of the signaling pathways mentioned above. However, we identified nitric oxide as the signaling molecule involved in beta 2 integrin-induced activation of Rap1 and Rap2. This was illustrated by the fact that engagement of beta 2 integrins increased the production of nitrite, a stable end-product of nitric oxide. Furthermore, pretreatment of neutrophils with N-monomethyl-L-arginine, or 1400W, which are inhibitors of inducible nitric-oxide synthase, blocked integrin-induced activation of Rap1 and Rap2. Similarly, Rp-8pCPT-cGMPS, an inhibitor of cGMP-dependent serine/threonine kinases, also blunted the integrin-induced activation of Rap GTPases. Also nitric oxide production and its downstream activation of cGMP-dependent serine/threonine kinases were essential for proper neutrophil adhesion by beta 2 integrins. Thus, we made the novel findings that beta 2 integrin engagement on human neutrophils triggers production of nitric oxide and its downstream signaling is essential for activation of Rap GTPases and neutrophil adhesion.

Non-thiazolidinedione antihyperglycemic agents. 2: alpha-Carbon substituted-beta-phenylpropanoic acids.

Buckle, D. R.; Cantello, B. C. C.; Cawthorne, M. A.; Coyle, P. J.; Dean, D. K.; Faller, A.; Haigh, D.; Hindley, R. M.; Lefcott, L. J.; et al. Dep. Medicinal Chem., Smithkline Beecham Pharmaceuticals, Surrey, UK. Bioorganic & Medicinal Chemistry Letters (1996), 6(17), 2127-2130. Publisher: Elsevier, CODEN: BMCLE8 ISSN: 0960-894X. Journal written in English. CAN 125:238227 AN 1996:573179 CAPLUS (Copyright (C) 2009 ACS on SciFinder (R)) Abstract The thiazolidine-2,4-dione ring of insulin-sensitizing antidiabetic agents can be replaced by ?-acyl-, ?-alkyl- and ?-(aralkyl)-carboxylic acids. The inclusion of an addnl. lipophilic moiety affords compds., equipotent to BRL 48482.