Identification of sex pheromone of red swamp crayfish Procambarus clarkii and exploration of the chemosensory mechanism of their antennae.

Red swamp crayfish, Procambarus clarkii, is a globally invasive species, which has caused great damage to biodiversity, agriculture, and fishing. Therefore, the development of effective management methods, such as pheromone control, is necessary for biological control and biodiversity protection. However, the components of P. clarkii sex pheromones have not yet been explored, and the chemosensory mechanism of the P. clarkii antennae after stimulation by sex pheromone also remains unknown. In this study, we isolated and identified the candidate bioactive component of the female P. clarkii sex pheromone using ultrafiltration centrifugation, semi-preparative liquid phase separation and omics technologies and conducted bioassays to determine its attraction ability. Meanwhile, RNA-Seq technology was used to analyze the potential chemosensory mechanism of antennae. Our results indicated that the male P. clarkii were uniaxially attracted to the female crude conditioned water (FCW), medium fraction (MF, isolated by ultrafiltration centrifugation), and preparative fragment 6 of females (PFF6, isolated by semi-preparative liquid phase separation). Metabolomic analysis revealed the presence of 18 differential metabolites between the PFF6 and PFM6 samples, among which 15 were significantly upregulated in the PFF6 sample. Bioassay test also showed that mestranol, especially at concentrations of 10-5-10-2 mol∙l-1, could significantly attract P. clarkii males; therefore, mestranol was identified as the candidate sex pheromone component of P. clarkii females. Furthermore, RNA-Seq results showed that most differentially expressed genes (DEGs) enriched in lipid metabolism and signal transduction pathways were up-regulated in P. clarkii males. In addition, high expressions of Ca2+-binding protein and ion transporting ATPases may enhance the sensitivity of the antennae of P. clarkii males towards sex pheromones. Our study provides data on P. clarkii sex pheromone composition and reveals the molecular mechanism of sex pheromone response in P. clarkii. Moreover, our study provides a referable method for the isolation of candidate bioactive molecules from the P. clarkii sex pheromone.

Effects of low-dose bisphenol AF on mammal testis development via complex mechanisms: alterations are detectable in both infancy and adulthood.

Despite growing concern about adverse effects of bisphenol AF (BPAF) due to its endocrine disrupting properties, there is a lack of toxicity data from low-dose studies and direct evidence linking its adverse effects to endocrine disrupting properties. Here, we investigated the effects of gestational and postnatal exposure to BPAF through drinking water (0.15-15 µg/mL, equivalent to the daily intake of ~ 50 and 5 mg/kg/day) on testis development in mice. We found that like mestranol, 5 mg/kg/day BPAF resulted in remarkable decreases in multiple male reproductive parameters in adulthood, such as the sperm number and serum testosterone level. Notably, 50 µg/kg/day BPAF also caused significant decreases in anogenital distance (AGD), the luteinizing hormone level and spermatocyte number, along with declining trends in sperm number and the serum levels of testosterone and follicle-stimulating hormone. In line with the adverse outcomes observed in adulthood, on postnatal day (PND) 9, we also observed BPAF-caused dose-dependent alterations, including reduced AGD, seminiferous tubule area and numbers of total germ cells, spermatocytes and Leydig cells, coupled with down-regulated expression of male-biased genes in testes. Even when exposure to 5 mg/kg/day BPAF as well as MES was initiated from PND 0, similar alterations in male reproductive parameters were also found on PND 9, along with a decrease in the GnRH content in the hypothalamus; moreover, testicular alterations and the reduction in AGD were partly antagonized by the estrogen receptor (ER) antagonist ICI 182,780, but the reduction of GnRH production was not done, showing that the effects of BPAF on testis development may be partially mediated by ER signaling. In conclusion, all the findings demonstrate that low-dose BPAF can partly disrupt mammal testis development and cause adverse testicular outcomes in adulthood, indicating a potential reproductive risk to mammals including humans. Importantly, our finding that developmental alterations elicited by BPAF have been detectable on PND 9 provides important motivation for the development of effective methods for early detection of adverse effects of estrogenic chemicals on testis development.

Oral contraceptive use by formulation and endometrial cancer risk among women born in 1947-1964: The Nurses' Health Study II, a prospective cohort study.

Oral contraceptives (OCs) have been associated with long-term lower endometrial cancer risk; relatively little is known about associations with more recent OC formulations and associations with longer-term risk. A total of 107,069 women from the Nurses' Health Study II recalled OC use from age 13 to baseline (1989); biennial questionnaires updated data on OC use until 2009. OCs were classified by estrogen and progestin type, dose, and potency based on reported brand. 864 incident endometrial cancer cases were identified through 2017. Multivariable Cox proportional hazards models estimated hazard ratios (HR) and 95% confidence intervals [95% CI] for the association of OC use with endometrial cancer risk. OC use was associated with lower endometrial cancer risk (ever use, HR 0.77 [95% CI 0.65-0.91]; >10 years of use, 0.43 [0.32-0.58] vs. never OC use). Inverse associations for duration were evident regardless of time since last use. Longer durations (> 5 years) of ethinyl estradiol (0.52 [0.41-0.67]) and second-generation progestins (0.43 [0.30-0.61]), both versus never use, were more strongly associated with lower risk than mestranol (0.66 [0.50-0.88], p-het = 0.01) and first-generation progestins (0.62 [0.49-0.78], p-het = 0.03). Inverse associations were generally observed for cross-classified cumulative average estrogen and progestin dose and potency (< vs. ≥ median; ever use vs. never OC use), with the exception of high estrogen and low progestin dose. OCs were associated with lower endometrial cancer risk, independent of time since last use. Use of ethinyl estradiol and second-generation progestins were more strongly inversely associated with risk compared with older formulations.

A survey of 17α-ethinylestradiol and mestranol residues in Hawkesbury River, Australia, using a highly specific enzyme-linked immunosorbent assay (ELISA) demonstrates the levels of potential biological significance.

This study reports on the potential status of 17α-ethinylestradiol (EE2) and mestranol (MeEE2) residues in aquatic environments in New South Wales (NSW), Australia, based on the analysis by a specific ELISA we developed. Polyclonal antibodies were raised against the EE2 hapten with a linker attached at the C3-position to direct the antibody binding towards the ring D of EE2/MeEE2. Using this approach, an ELISA highly specific to EE2 and MeEE2 was successfully developed, showing less than 3.1% cross-reactivity (% CR) with other major steroidal sex hormones and their derivatives. The assay performed with the limit of detection (LOD) of 0.04 ± 0.01µg/L for both EE2 and MeEE2, and the limit of quantitation (LOQ) of 0.05 ± 0.01ng/L when it was coupled with the SM2-Biobeads solid phase extraction. Prior to conducting the survey study, it was validated against the gas chromatography-mass spectrophotometry (GC-MS) method, which showed high correlation with R2 of 0.934. Fresh surface water samples collected at different sites along Hawkesbury River in New South Wales (NSW) were analyzed for the EE2/ MeEE2 residues using the developed ELISA. The EE2/MeEE2 levels were found to range between 4.1 and 8.3ng/L in Emigrant Creek, NSW, where the primary activity was macadamia plantation, and higher levels between 15 and 29ng/L in South Creek, NSW, Greater Western Sydney at sites upstream and downstream of the municipal sewage treatment plants.

Effect of CYP2C9 Genetic Polymorphism in a Chinese Population on the Metabolism of Mestranol in vitro.

BACKGROUND: Mestranol is a widely used estrogen, which is converted into its active metabolite ethinyl estradiol by cytochrome P450 (CYP) 2C9. To comprehensively examine the enzymatic activity of reported CYP2C9 variants in Chinese individuals in response to mestranol, wild-type CYP2C9*1 and 35 allelic variants were highly expressed in Sf21 insect cell microsomes and used for the detection of their enzymatic values in vitro. These results showed that the majority of tested variants exhibited decreased clearance values compared to wild type, except for CYP2C9*40 and *36. METHOD: Insect microsomes expressing the 36 CYP2C9 variants were incubated with 0.25-8 µmol/l mestranol for 30 min at 37°C. Then, the production of the metabolite of mestranol, ethinyl estradiol, was analyzed using high-performance liquid chromatography. RESULTS: Most CYP-catalyzed reactions were sufficiently described by classical Michaelis-Menten kinetic parameters (e.g., Km and Vmax), while 9 variants exhibited atypical or non-Michaelis-Menten kinetic values, which were largely due to the self-inhibitory effect in response to mestranol. CONCLUSION: This is the first report of these rare alleles for mestranol metabolism, which provides fundamental data for further clinical studies on CYP2C9 alleles for mestranol metabolism.

Amphidynamic crystals of a steroidal bicyclo[2.2.2]octane rotor: a high symmetry group that rotates faster than smaller methyl and methoxy groups.

The synthesis, crystallization, single crystal X-ray structure, and solid state dynamics of molecular rotor 3 provided with a high symmetry order and relatively cylindrical bicyclo[2.2.2]octane (BCO) rotator linked to mestranol fragments were investigated in this work. By use of solid state (13)C NMR, three rotating fragments were identified within the molecule: the BCO, the C19 methoxy and the C18 methyl groups. To determine the dynamics of the BCO group in crystals of 3 by variable temperature (1)H spin-lattice relaxation (VT (1)H T1), we determined the (1)H T1 contributions from the methoxy group C19 by carrying out measurements with the methoxy-deuterated isotopologue rotor 3-d6. The contributions from the quaternary methyl group C18 were estimated by considering the differences between the VT (1)H T1 of mestranol 8 and methoxy-deuterated mestranol 8-d3. From these studies it was determined that the BCO rotator in 3 has an activation energy of only 1.15 kcal mol(-1), with a barrier for site exchange that is smaller than those of methyl (E(a) = 1.35 kcal mol(-1)) and methoxy groups (E(a) = 1.92 kcal mol(-1)), despite their smaller moments of inertia and surface areas.

Effect-directed analysis of a hospital effluent sample using A-YES for the identification of endocrine disrupting compounds.

An effect-directed analysis (EDA) approach was used to identify the compounds responsible for endocrine disruption in a hospital effluent (Basque Country). In order to facilitate the identification of the potentially toxic substances, a sample was collected using an automated onsite large volume solid phase extraction (LV-SPE) system. Then, it was fractionated with a two-step orthogonal chromatographic separation and tested for estrogenic effects with a recombinant yeast (A-YES) in-vitro bioassay. The fractionation method was optimized and validated for 184 compounds, and its application to the hospital effluent sample allowed reducing the number of unknowns from 292 in the raw sample to 35 after suspect analysis of the bioactive fractions. Among those, 7 of them were confirmed with chemical standards. In addition, target analysis of the raw sample confirmed the presence of mestranol, estrone and dodemorph in the fractions showing estrogenic activity. Predictive estrogenic activity modelling using quantitative structure-activity relationships indicated that the hormones mestranol (5840 ng/L) and estrone (128 ng/L), the plasticiser bisphenol A (9219 ng/L) and the preservative butylparaben (1224 ng/L) were the main contributors of the potential toxicity. Derived bioanalytical equivalents (BEQs) pointed mestranol and estrone as the main contributors (56 % and 43 %, respectively) of the 50 % of the sample's explained total estrogenic activity.

Ultrasonic-assisted derivatization of estrogenic compounds in a cup horn booster and determination by GC-MS.

Cup horn boosters are miniaturized ultrasound baths that maximize efficiency and precision. The optimization of an ultrasonic-assisted derivatization step by means of a cup horn booster and the determination of estrone, 17beta-estradiol, estriol, 17alpha-ethynyl estradiol and mestranol was developed by GC-MS. Different derivatization reagents and solvents were studied for maximizing the di-derivatization of 17alpha-ethynyl estradiol under ultrasound energy. Only N,O-bis(trimethylsilyl)trifluoroacetamide with 1% of trimethylchlorosilane in pyridine gave satisfactory results and this mixture was further used in the optimization of the ultrasound assisted derivatization. The experiment designs included sonication time (1-10 min), sonication power (20-80%), sonication cycles (1-9), derivatization reagent volume (25-125 microL) and solvent volume (25-125 microL). Once the optimum conditions were fixed, the effect of organic matter and the frequency of the water bath change were studied. Finally, the validation of the analytical method was carried out using spiked natural and synthetic waters. Recoveries (natural (138-70%) and synthetic (112-89%)), the LODs (0.35-1.66 ng/L), and LOQs (1.16-5.52 ng/L) and the precision (0.2-5.3%) of the method were studied. This is the first work in the literature where a cup horn booster is used with the aim of minimizing derivatization time during the determination of estrogenic compounds.

The effect of etoricoxib on the pharmacokinetics of oral contraceptives in healthy participants.

The pharmacokinetics of oral contraceptive (OC) components, ethinyl estradiol (EE) and norethindrone (NET), were evaluated after coadministration with etoricoxib in 3 double-blind, randomized, 2-period crossover studies of healthy women. There were 16, 39, and 24 participants enrolled in studies 1 (part I, part II), and 2, respectively. Each participant received triphasic OC (EE 35 microg/NET 0.5 mgx7 days, 0.75 mgx7 days, 1.0 mgx7 days) throughout each 28-day period. OC was coadministered with 21 days of etoricoxib daily followed by placebo for 7 days; the alternate period followed the reverse regimen (placebo to etoricoxib). Study 1 (part I) examined concurrent (morning) administration of OC/etoricoxib 120 mg, study 1 (part II) examined staggered (morning/night) administration of OC/etoricoxib 120 mg, and study 2 examined concurrent (morning) administration of OC/etoricoxib 60 mg. Coadministration of OC and etoricoxib 120 mg once daily was associated with a approximately 50% to 60% increase in EE concentrations, whereas etoricoxib 60 mg once daily was associated with a approximately 37% increase in EE concentrations. Coadministration of OC and etoricoxib was generally well tolerated. A clinically important change in NET AUC0-24 h was not observed. Adverse events included dyspepsia, diarrhea, headache, nausea, fatigue, loss of appetite, and taste disturbance.

Serum copper alteration after ingestion of an oral contraceptive.

Changes in the concentration of copper in the serum after administration of an oral contraceptive were determined with atomic absorption spectrophotometry. Statistically significant (P = .001) increases were observed in all volunteers.

Acute phase protein in serum of women using hormonal contraceptives.

Acute phase protein precipitating sonatic C-polysaccharide of pneumococci appears in serum of women under treatment with hormonal contraceptives in a significantly higher number of cases when compared with control groups. The summarized results of three preliminary studies show that in 80 control serums there were four positive specimens (5 percent) and in 80 serums from women using hormonal contraceptives there were 58 positive specimens (72.5 percent).

Pigeon control by chemosterilization: population model from laboratory results.

Reproductivity of pigeons is inhibited with mestranol incorporated in a synthetic grit; continual erosion releases daily doses. Young squabs may be permanently sterilized when fed crop milk by treated birds. A theoretical model of pigeon population dynamics using laboratory-obtained data shows the advantages of chemosterilization over killing as a means of pigeon control.

Improved synthesis of mestranol and ethinyl estradiol (EE) related degradation products as authentic references.

Preparative chemical methods for the synthesis of 10 degradation or photodecomposition products of mestranol and ethinyl estradiol (EE) are described. The synthesized compounds are useful as reference materials and standards for pharmaceutical analysis of mestranol and EE as bulk chemical or in formulated product. New synthetic methods were presented and the known synthetic procedures were improved. Detailed structural characterization of the degradation or photodecomposition products of mestranol and EE and related compounds was reported.

Parallel Solid-Phase Synthesis using a New Diethylsilylacetylenic Linker and Leading to Mestranol Derivatives with Potent Antiproliferative Activities on Multiple Cancer Cell Lines.

BACKGROUND: RM-133 belongs to a new family of aminosteroid derivatives demonstrating interesting anticancer properties, as confirmed in vivo in four mouse cancer xenograft models. However, the metabolic stability of RM-133 needs to be improved. After investigation, the replacement of its androstane scaffold by a more stable estrane scaffold led to the development of the mestranol derivative RM-581. METHODS: Using solid-phase strategy involving five steps, we quickly synthesized a series of RM-581 analogs using the recently-developed diethylsilylacetylenic linker. To establish structure-activity relationships, we then investigated their antiproliferative potency on a panel of cancer cell lines from various cancers (breast, prostate, ovarian and pancreatic). RESULTS: Some of the mestranol derivatives have shown in vitro anticancer activities that are close to, or better than, those observed for RM-581. Compound 23, a mestranol derivative having a ((3,5-dimethylbenzoyl)- L-prolyl)piperazine side chain at position C2, was found to be active as an antiproliferative agent (IC50 = 0.38 ± 0.34 to 3.17 ± 0.10 µM) and to be twice as active as RM-581 on LNCaP, PC-3, MCF-7, PANC-1 and OVCAR-3 cancer cells (IC50 = 0.56 ± 0.30, 0.89 ± 0.63, 1.36 ± 0.31, 2.47 ± 0.91 and 3.17 ± 0.10 µM, respectively). CONCLUSION: Easily synthesized in good yields by both solid-phase organic synthesis and classic solution-phase chemistry, promising compound 23 could be used as an antiproliferative agent on a variety of cancers, notably pancreatic and ovarian cancers, both having very bad prognoses.

Evaluation of antimicrobial efficacy of Trachyspermum ammi (Ajwain) oil and chlorhexidine against oral bacteria: An in vitro study.

INTRODUCTION: Plaque removal is of utmost importance for control of dental caries and other associated diseases of oral cavity. However, various natural agents have proven their efficacy over chemotherapeutic agents in terms of antibacterial activity against various microorganisms. The effect is mainly due to polyphenol as its major constituent. AIM: In this in vitro study, we aimed to determine the antibacterial efficacy of Trachyspermum ammi oil at different concentrations against five oral bacteria. HYPOTHESIS: Herbal compound, T. ammi oil is effective in reducing five oral plaque-forming bacteria. MATERIALS AND METHODS: We determined the antimicrobial activity of T. ammi oil (test material) against chlorhexidine (gold standard). Pure cultures of Streptococcus mutans MTCC No 497, Streptococcus oralis MTCC No. 2696, Lactobacillus acidophilus MTCC No. 10307, Lactobacillus fermentum MTCC No. 903, and Candida albicans MTCC No. 183 were obtained and grown in selective culture media. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of both materials were evaluated by serial dilution and disc diffusion method, respectively. RESULTS: Our results revealed that T. ammi oil moderately inhibits bacterial growth with mean MIC of 250, 125, 250, 125, and 250 µg/ml, respectively. Mean MBC for T. ammi oil obtained was 18.60 ± 0.65, 11.60 ± 1.14, 14.10 ± 0.55, 11.50 ± 0.61, and 15.10 ± 0.74 mm. The MIC and MBC values were higher as compared to chlorhexidine gluconate and it was statistically significant. CONCLUSION: T. ammi (ajwain) can serve as a potential, natural, nontoxic, and economical therapeutic antiplaque agent.

Characterization of precursor and secreted forms of human angiotensinogen.

To define the basis of the heterogeneity of angiotensinogen, we have characterized the immunoreactivity of high molecular weight (HMW) and low molecular weight (LMW) plasma angiotensinogen, the angiotensinogen precursor synthesized by cell-free translation, and angiotensinogen secreted by human hepatoma (Hep G2) cells. Angiotensinogen precursor synthesized by rabbit reticulocyte lysate primed with RNA prepared from liver or Hep G2 cells was compared with angiotensinogen secreted by Hep G2 cells by using immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). So as to assess the contribution of N-glycosylation of angiotensinogen, Hep G2 cells were incubated in the presence of tunicamycin. Glycosylation of secreted angiotensinogen was further characterized by using chromatography on concanavalin A-Sepharose, digestion with neuraminidase, and treatment with trifluoromethane sulfonic acid. In Sephadex G-200 column chromatography, HMW plasma angiotensinogen eluted just after the column void volume and was clearly separated from LMW angiotensinogen which eluted just before bovine serum albumin. Both HMW and LMW plasma angiotensinogen were shown to bind to monoclonal and polyclonal antibodies raised against pure LMW angiotensinogen. Only one angiotensinogen precursor (mol wt 50,000) was identified by cell-free translation which, after cleavage by renin, was reduced to mol wt 45,600. Angiotensinogen secreted by Hep G2 cells showed electrophoretic heterogeneity (mol wt 53,100-65,400). Tunicamycin-treated Hep G2 cells secreted five discrete forms of angiotensinogen, a predominant form of mol wt 46,200, with other forms (mol wt 46,800, 48,100, 49,200, and 49,600) representing 10% of secreted angiotensinogen. All five forms showed a similar reduction in molecular weight after cleavage by renin. The predominant 46,200-mol wt protein represented nonglycosylated angiotensinogen in that, after cleavage by renin, it had an electrophoretic mobility (mol wt 45,600) identical to the desangiotensin I-angiotensinogen resulting from renin cleavage of the angiotensinogen precursor. The other higher molecular weight forms of angiotensinogen secreted by tunicamycin-treated Hep G2 cells were shown to represent O-glycosylated angiotensinogen in that they were reduced to 46,200 mol wt by treatment with trifluoromethane sulfonic acid. Dexamethasone (10(-7) and 10(-6)M) stimulated angiotensinogen secretion by Hep G2 cells two- to fourfold, both in the absence and presence of tunicamycin. However, a small stimulatory effect of mestranol (10(-7) M) was evident only in the presence of tunicamycin. Neither dexamethasone nor mestranol influenced the electrophoretic pattern (SDS-PAGE) of angiotensinogen secreted by Hep G2 cells. However, when incubation media were chromatographed on Sephadex G-200 with subsequent immunoprecipitation of the column fractions, both dexamethasone and mestranol were shown to stimulate the secretion of HMW angiotensinogen (eluting just after the column void volume) which, on SDS-PAGE, migrated in a position identical to LMW angiotensinogen. From these studies, we conclude that all forms of human angiotensinogen are derived from a single precursor. The heterogeneity of secreted angiotensinogen represents differences in posttranslational processing of angiotensinogen. This processing includes both N- and O-glycosylation, and also the formation of HMW complexes (HMW angiotensinogen) through association either with other angiotensinogen molecules or with some other protein(s) whose secretion by hepatocytes is stimulated by glucocorticoids and estrogens.

Measurement of human luteinizing hormone in plasma by radioimmunoassay.

The recent isolation of highly purified human pituitary luteinizing hormone (LH) has permitted the development of a sensitive and specific radioimmunoassay for this hormone in plasma. Results of this immunoassay system employing anti-LH serum agree closely with previous reports for the measurement of plasma LH in which immunoassays employing cross-reactive antisera to human chorionic gonadotropin were used. The immunoassay and bioassay of LH in several crude and partially purified pituitary and urinary extracts show acceptable agreement. The sensitivity of the LH immunoassay (0.2 mmug/ml) is adequate to measure LH levels in almost half of all prepuberal children and in all but a few normal adults. A small, but significant, rise in plasma LH level occurs at pubescence in both boys and girls. In women, plasma LH level varies with both age and the phase of the menstrual cycle. The mean LH concentration in nine normal women during the follicular phase (1.2 mmug/ml was found to be significantly higher than during the luteal phase (1.0 mmug/ml). At midcycle, the mean peak LH level was 10.2 mmug/ml. In a large group of normal women, the mean plasma LH concentration rose significantly at menopause to a level of 5.8 mmug/ml during the fifth decade and 10.5 mmug/ml during the seventh decade. A small, but significant, rise in plasma LH concentration also occurred in men from the third and fourth decades (0.7 mmug/ml to the seventh and eighth decades (1.7 mmug/ml). Both estrogen and testosterone suppress plasma LH levels, but marked variation in response exists. The immunoassay serves as a useful diagnostic tool in evaluating men with gonadal failure, amenorrheic women of reproductive age, and postmenopausal women suspected of hypopituitarism. From the half-time disappearance of LH-(131)I in plasma (mean 69 min) and the calculated volume of distribution (2.5-2.8 liters) it has been determined that approximately 30 mug of LH is secreted per day in men, and in women except at midcycle, at which time the release of LH is estimated to be 10-15 times this basal rate.

Effects of exogenous estrogen and progestin on pituitary responsiveness to synthetic luteinizing hormone-releasing factor.

The effect of estrogen and progestin on pituitary responsiveness to 150 mug synthetic luteinizing hormone-releasing factor (LRF) was assessed in premenopausal women receiving sequential (n=12) and combination (n=7) contraceptive steroids. A marked contrast in the time-course and maximal response to LRF was found; a prompt but quantitatively smaller luteinizing hormone (LH) response was seen during cyclic combination therapy, while a delayed (five times) but enhanced (fivefold) LH response was observed during estrogen segments of cyclic sequential therapy. For follicle-stimulating hormone (FSH), the maximum rise was also higher, and the peak response was similarly delayed in the latter group. The quantitative secretion in response to LRF for LH (area under the curve), but not for FSH, was significantly greater (P < 0.01) in subjects receiving sequential, as compared to subjects receiving combination treatment. In both groups, characteristic gonadotropin responses to LRF were reproducible and were independent of the duration of treatment. Since LRF studies were performed during the estrogen segment of treatment cycle in subjects receiving sequential steroids, our data suggest that estrogen exerts a direct feedback action at the pituitary level and that pituitary responsiveness to LRF is augmented by estrogen.

Microwave-accelerated derivatization for the simultaneous gas chromatographic-mass spectrometric analysis of natural and synthetic estrogenic steroids.

A rapid microwave-accelerated derivatization process for the GC-MS analysis of steroid estrogens, estrone (E1), 17beta-estradiol (E2), estriol (E3), 17alpha-ethynylestradiol (EE2) and mestranol (MeEE2), was developed. Under microwave irradiation, the five estrogenic hormones studied were simultaneously derivatized with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA)+trimethylchlorosilane (TMCS) in pyridine solution. Effects of irradiation time (15-120 s) and power level (240-800 W) on the yield of the derivatization were investigated. The derivatization under the irradiation of 800 W microwave for 60s produced comparable results when compared with the conventional heating process in a sand bath for 30 min at 80 degrees C in terms of derivatization yield, linearity and precision for all steroid hormones tested. The calibration curves are linear between 3.00 and 3.00 x 10(2) microg mL(-1). The square of the regression coefficients (R(2)) range from 0.979 to 1.000. The applicability of the method was evaluated on spiked river and distilled water samples at two concentrations, 25.0 and 2.00 x 10(2) ng mL(-1). The recoveries obtained by using microwave heating (60s, 800 W) were similar to those by conventional heating. When combined solid-phase extraction (SPE) with the application of the microwave-accelerated derivatization proposed here, the detection limits of 0.02-0.1 ng L(-1) for the steroid hormones have been achieved. The results demonstrated that microwave-accelerated derivatization is an efficient and suitable sample preparation method for the GC-MS analysis of estrogenic steroids.

Immunomodulation of Mytilus hemocytes by individual estrogenic chemicals and environmentally relevant mixtures of estrogens: in vitro and in vivo studies.

Endocrine disrupting compounds (EDCs) are almost ubiquitous in the aquatic environment. In the marine bivalve Mytilus the natural estrogen 17beta-estradiol (E2) and different EDCs have been recently demonstrated to affect the function of the immune cells, the hemocytes. The effects were Tamoxifen-sensitive and were mediated by rapid modulation of kinase-mediated transduction pathways. In this work we compared the in vitro effects of individual estrogenic chemicals (E2, EE: 17alpha-ethynyl estradiol; MES: mestranol; NP: nonylphenol; NP1EC: nonylphenol monoethoxylate carboxylate; BPA: bisphenol A; BP: benzophenone) on hemocyte parameters: lysosomal membrane stability (LMS), phagocytosis, lysozyme release. LMS was the most sensitive effect parameter, showing a decreasing trend at increasing concentrations of estrogens. EC50 values obtained from LMS data were utilized to calculate the estradiol equivalency factor (EEF) for each compound; these EEFs allowed for an estimation of the estrogenic potential of a synthetic mixture with a composition very similar to that previously found in waters of the Venice lagoon. Concentrated mixtures significantly affected hemocyte parameters in vitro and the effects were prevented by Tamoxifen. Significant effects of the mixture were also observed in vivo, at longer exposure times and at concentrations comparable with environmental exposure levels. The results indicate that Mytilus immune parameters can be suitably utilized to evaluate the estrogenic potential of environmental samples.