Fluorescent ligand for human progesterone receptor imaging in live cells.

We employed molecular modeling to design and then synthesize fluorescent ligands for the human progesterone receptor. Boron dipyrromethene (BODIPY) or tetramethylrhodamine were conjugated to the progesterone receptor antagonist RU486 (Mifepristone) through an extended hydrophilic linker. The fluorescent ligands demonstrated comparable bioactivity to the parent antagonist in live cells and triggered nuclear translocation of the receptor in a specific manner. The BODIPY labeled ligand was applied to investigate the dependency of progesterone receptor nuclear translocation on partner proteins and to show that functional heat shock protein 90 but not immunophilin FKBP52 activity is essential. A tissue distribution study indicated that the fluorescent ligand preferentially accumulates in tissues that express high levels of the receptor in vivo. The design and properties of the BODIPY-labeled RU486 make it a potential candidate for in vivo imaging of PR by positron emission tomography through incorporation of (18)F into the BODIPY core.

[Glyphosate and its formulations--toxicity, occupational and environmental exposure]. / Glifosat i jego preparaty--toksycznosc, narazenie zawodowe i srodowiskowe.

Glyphosate (N-(phosphonomethyl)glycine) is an active ingredient of the most widely used herbicide formulations in protecting agricultural and horticultural crops. Numerous results (mostly published in the years 2010-2013) concerning the action of glyphosate and its formulations in the recent decade were analyzed. Initial reports about alleged biodegradability of glyphosate in the environment turned out to be wrong. It has been shown that glyphosate remains in the soil and can reach people by spreading along with groundwater. Recent publications have shown that glyphosate is detected at low concentrations in the human blood. Publications cited in this article, which indicate a possible induction of neoplastic changes by glyphosate formulation, have raised great concern and controversy in the scientific world. Presenting adverse effects of glyphosate and its formulations we focused on the role of glyphosate formulations in hormonal disorders by impeding the expression of steroidogenic acute regulatory protein and the inhibition of aromatase activity. The impact of glyphosate on oxygen reactive species formation, changes in redox system and the effect on necrosis and apoptosis in various types of cells was shown. We also revealed that glyphosate as a phosphonate herbicide does not inhibit directly the activity of acetylcholinesterase. Based on numerous studies it was noted that commercial formulations of glyphosate exhibit higher toxicity than that of the active substance itself. The discussed problems clearly show the need to evaluate the toxicity of glyphosate and its formulations and related potential threat to humans.

Analysis of hormone antagonists in clinical and municipal wastewater by isotopic dilution liquid chromatography tandem mass spectrometry.

A comprehensive method was developed for the simultaneous trace analysis of ten hormone antagonist pharmaceuticals (raloxifene, exemestane, letrozole, anastrozole, mifepristone, finastride, tamoxifen, N-desmethyltamoxifen, clomiphene, and toremifene) in municipal sewage and hospital wastewater samples. The target compounds were firstly extracted using an Oasis HLB cartridge, followed by purification by an aminopropyl cartridge, and were then analyzed by liquid chromatography electrospray ionization tandem mass spectrometry in positive ion mode. The recoveries for the analytes based on internal standard calibration in different test matrices ranged from 67.6 to 118.6% (with the exception of mifepristone in clinical wastewater samples), with relative standard deviations less than 20%. The method quantification limits of the ten pharmaceuticals were in the range 0.10-2.0 ng/L. Excluding exemestane and N-desmethyltamoxifen, eight drugs were detected at 0.20-195.0 ng/L in hospital wastewater and municipal wastewater samples from Beijing.

Specific immunoassays for endocrine disruptor monitoring using recombinant antigens cloned by degenerated primer PCR.

Vitellogenin (VTG) and choriogenin (CHO) are valuable biomarkers of endocrine-disrupting compound (EDC) exposure in fish. Existing immunoassays are limited to a few species, which restricts their use for the analysis of local wildlife sentinels. Using C. facetum as a relevant South American model fish, this work presents a new strategy for the preparation of antibodies to VTG and CHO, with zero cross-reactivity with fish serum components. Recombinant fragments of Cichlasoma facetum VTG (280-mer) and CHO (223-mer) were prepared by degenerate primer RT-PCR and expression in E. coli. Polyclonal and monoclonal antibodies prepared with these antigens were used to develop rapid dotblot assays for VTG and CHO. Both the polyclonal and monoclonal antibodies prepared with the recombinant antigens reacted against the native proteins adsorbed on to nitrocellulose allowing the set up of sensitive dotblot assays. The VTG assay was further validated with spiked samples and purified native VTG. Exposure experiments with several estrogenic compounds revealed the potential of C. facetum as a sensitive biomonitor that produced measurable responses at concentrations of 100 ng L(-1) of 17-beta-estradiol, 100 ng L(-1) of ethynylestradiol, and 6.6 microg L(-1) of nonylphenol. The approach described here may be applied to other native species to produce highly specific and sensitive rapid tests. It may be particularly advantageous for species that cannot be kept in captivity or when homogeneous purification of the immunizing proteins is particularly challenging. In conclusion, we present a novel approach to develop a strategy for the generation of immunoassay reagents for vitellogenin (VTG) and choriogenin (CHO), which will facilitate regional studies on the impact of endocrine-disrupting chemicals on local wildlife.

Identification of new gonadotrophin-releasing hormone partial agonists.

GnRH agonists or antagonists are currently utilized as therapeutic agents in a number of diseases. A side-effect of prolonged treatment with GnRH analogues is hypoestrogenism. In this study, we tested the in vitro potency of different GnRH analogues originally found to be partial agonists (i.e. analogues with decreased efficacy for activating or stimulating their cognate receptor) as well as novel analogues, to identify compounds that might potentially be useful for partial blockade of gonadotrophin release. Cultured COS-7 cells transiently expressing the rat or human GnRH receptor (GnRHR) were exposed to increasing concentrations (10(-8) to 10(-5) M) of GnRH analogues (c(4-10)[Asp4,DNal6,Dpr10]-GnRH; c(4-10) [Dpr4,DNal6,Asp10]-GnRH; c(4-10)[Cys(4,10),DNal6]-GnRH; c[Eaca1,DNal6]-GnRH; c[Gly1,DNal6]-GnRH; c[betaAla1,DTrp6]-GnRH; c[Dava1,DNal6]-GnRH; c[Gaba1, DNal6]-GnRH), and the ability of these analogues to provoke or antagonize GnRH-stimulated inositol phosphate production was assessed. With both human and rat GnRHRs, c[Eaca1,DNal6]-GnRH, c[Gly1,DNal6]-GnRH, c[betaAla1,DTrp6]-GnRH and c[Dava1,DNal6]-GnRH exhibited partial agonist activity (35-87% of the maximal efficacy shown by 10(-6) M GnRH), whereas c[Gaba1,DNal6]-GnRH behaved as a partial agonist with the human GnRHR and as full agonist with the rat GnRHR. c(4-10)[Asp4, DNal6,Dpr10]-GnRH and c(4-10)[Dpr4,DNal6,Asp10]-GnRH exhibited full antagonist activity with both GnRHRs, and c(4-10) [Cys(4,10),DNal6]-GnRH was a weak, partial agonist with the human GnRHR and a full antagonist with the rat GnRHR. With the exception of c[Gaba1,DNal6]-GnRH stimulation of the human GnRHR, and c[Dava1,DNal6]-GnRH and c[Gaba1, DNal6]-GnRH stimulation of the rat GnRHR, all partial agonists also exhibited antagonist activity in the presence of the exogenous full agonist. The results demonstrate that structurally similar analogues display variable potencies and efficacies in vitro for a specific GnRHR as well as for the human versus the rat GnRHR. Their ultimate in vivo usefulness to treat clinical conditions in which complete suppression of gonadotroph activity is not required remains to be investigated.

An enzyme-linked immunosorbent assay for rare minnow (Gobiocypris rarus) vitellogenin and comparison of vitellogenin responses in rare minnow and zebrafish (Danio rerio).

A competitive enzyme-linked immunosorbent assay (ELISA) was developed to determine vitellogenin (Vtg) in rare minnow (Gobiocypris rarus) based on the separation and purification of rare minnow Vtg (r-Vtg) as well as the production of polyclonal antibody against r-Vtg in rabbits. Three different ELISAs for measuring r-Vtg were then compared: (1) indirect ELISA with the antibody against carp (Cyprinus carpio) Vtg (c-Vtg) (IC-ELISA); (2) competitive ELISA with the antibody against c-Vtg, and using r-Vtg for coating the plates and preparing standard curve (CC-ELISA); (3) competitive ELISA with the antibody against r-Vtg, and using r-Vtg for coating the plates and preparing standard curve (CR-ELISA). The result showed that the homologous CR-ELISA was the most sensitive among the three assays for quantifying r-Vtg. The sensitivities to 17alpha-ethinylestradiol (EE(2)) of rare minnow and zebrafish (Danio rerio) were compared upon the establishment of homologous competitive ELISA. The lowest observed effect concentrations (LOECs) to induce Vtg were found to be 0.8 ng EE(2) l(-1) for rare minnow and 4 ng EE(2) l(-1) for zebrafish respectively. Afterwards, CR-ELISA was applied to measure Vtg concentration in whole body homogenate (WBH) of juvenile rare minnow fed by three diets (tubifex from wastewater treatment plant, Artemia nauplii and commercial pellet food), and the agreements between bioassay and GC-MS analysis demonstrated that rare minnow was a sensitive fish model for assessing estrogenic effects of endocrine disrupting compounds in aquatic environment.

Distribution of Dip-allatostatin I-like immunoreactivity in the brain of the locust Schistocerca gregaria with detailed analysis of immunostaining in the central complex.

The distribution and morphology of neurons containing allatostatin-related substances in the brain of the locust Schistocerca gregaria was investigated using an antiserum against Diploptera punctata allatostatin I (Dip-allatostatin I, APSGAQRLYGFGL-amide). In each brain hemisphere, about 550 neurons in the midbrain and 500 neurons in the optic lobe exhibit Dip-allatostatin I-like immunoreactivity, including about eight lateral neurosecretory cells with processes to the retrocerebral complex. All major brain areas except the antennal lobe, the mushroom body, and large parts of the lamina, are innervated by Dip-allatostatin I-immunoreactive processes. Immunostaining in the central complex was studied in detail. The central complex is innervated by more than 260 Dip-allatostatin I-immunoreactive neurons belonging to six different cell types, four sets of tangential neurons and two sets of columnar neurons. These neurons give rise to intense immunostaining in the protocerebral bridge, in several layers of the upper division of the central body, and in the dorsalmost layer of the lower division of the central body. Double-label experiments show colocalization of Dip-allatostatin I- and serotonin-like immunoreactivities in one type of columnar and one type of tangential neurons of the central complex. The similar patterns of Dip-allatostatin I- and galanin message-associated peptide-like immunoreactivities result from cross-reactivity of the anti-galanin message-associated peptide antiserum with Dip-allatostatin I. The results provide further insight into the anatomical and neurochemical organization of the locust central complex and suggest a prominent neuroactive role for Dip-allatostatin I-related peptides in this brain area.

Immunocytochemistry of GABA in the central complex of the locust Schistocerca gregaria: identification of immunoreactive neurons and colocalization with neuropeptides.

The central complex is a highly organized neuropil structure in the insect brain and plays a role in motor control and visual orientation. We describe the distribution of gamma-aminobutyric acid (GABA) immunostaining in the central complex of the locust Schistocerca gregaria in an effort to analyze inhibitory neural circuits within this brain area. Antisera against GABA and the GABA-synthesizing enzyme glutamic acid decarboxylase resulted in identical patterns of immunostaining. Cell counts revealed about 100 bilateral pairs of GABA-immunoreactive neurons with arborizations in the central complex. Five types of immunostained neurons could be identified through reconstruction of the staining pattern, comparison with individually stained neurons, and double labeling experiments with Neurobiotin-injected neurons. All of these GABA-immunostained neurons are tangential neurons that connect the lateral accessory lobes to distinct layers of the central body. Three types of immunostained neurons (TL2, TL3, TL4) invade the lower division of the central body, and two additional types of neurons (TU1, TU2) have ramifications in layers I and II of the upper division of the central body. Double-labeling experiments with peptide antisera suggest that peptides related to Phe-Met-Arg-Phe-NH2/bovine pancreatic polypeptide and Dip-allatostatin might act as cotransmitters with GABA in TL4 neurons of the lower division and (Dip-allatostatin only) in TU2 neurons of the upper division of the central body. The high conservation in the pattern of GABA immunostaining in all insect species investigated so far suggests that GABA plays an essential role in the basic neural circuitry of the central complex in insects.

Placental transfer of SR49059 in the human dually perfused cotyledon in vitro.

SR49059 is an antagonist of vasopressin V(1a)receptors currently developed as a tocolytic drug. We investigated the transplacental transfer of SR49059 in vitro using the single pass dually perfused human cotyledon model. Thirteen placentae were collected from normal term pregnancies immediately after delivery. The placental transfer of SR49059 was tested at steady state at three different concentrations (100 ng/ml, 200 ng/ml and 500 ng/ml) along with that of antipyrine 20 mg/l as a reference substance. Concentrations were assayed by liquid chromatography with UV (antipyrine) or mass spectrometry (SR49059) detection. At steady state, the mean+/-s.d. fetal transfer rate of SR49059 was 10.80+/-4.33 per cent, 9.34+/-4.41 per cent, and 11.78+/-3.26 per cent at 100 ng/ml, 200 ng/ml and 500 ng/ml, respectively. The corresponding clearance indices were 0.29+/-0.14, 0.25+/-0.08, and 0.31+/-0.06, respectively. The absence of saturation kinetics is consistent with a passive mechanism of transfer. Moderate amounts of SR49059 are transferred from the maternal to the fetal circulation. The clearance index of SR49059 appeared to be very similar to that of ritodrine, which is currently used as a tocolytic.

The postembryonic development of somatostatin immunoreactivity in the central posterior/prepacemaker nucleus of weakly electric fish, Apteronotus leptorhynchus: a double-labelling study.

The neuropeptide somatostatin (SS) is widely distributed in both the central and peripheral nervous system of vertebrates. Its widespread distribution is paralleled by a large variety of diverse functions. While embryonic and perinatal development of SS-like immunoreactivity have been well examined, little is known about the postnatal development of this neuropeptide. Since, in teleosts, neurogenesis persists in many brain regions during adulthood, these vertebrates are well suited to investigate this phenomenon. In the present study, we have, therefore, examined the development of somatostatinergic cells born during adulthood in the central posterior/prepacemaker nucleus (CP/PPn) of Apteronotus leptorhynchus, a weakly electric gymnotiform fish. This was achieved by labelling proliferating cells with the thymidine analogue 5-bromo-2'-deoxyuridine (BrdU) and by simultaneous immunocytochemical detection of SS-like immunoreactivity. SS-like immunoreactivity is adopted in a period between 2 days and 3.5 days after birth. While the number of BrdU-labelled cells in the CP/PPn decreases 10 days after birth, the percentage of double-labelled cells among the BrdU-labelled cells remains with 1.0-7.6% in the period between 3.5 days and 100 days after birth rather constant. This percentage matches well the fraction of SS-positive cells in the total population of cells present in the CP/PPn.

Determination of SR 49059 in human plasma and urine by LC-APCI/MS/MS.

SR 49059 ((2S 1-[(2R 3S)-5-chloro-3-(2-chlorophenyl)-1-(3, 4-dimethoxybenzene-sulfonyl)-3-hydroxy-2,3-dihydro-1 H-indole-2-carbonyl]-pyrrolidine-2-carboxamide) is an orally active non-peptide vasopressin V1a antagonist. A sensitive, selective, and robust LC-MS/MS method was developed to determine the plasma and urine concentrations of SR 49059 in support of clinical studies. Plasma samples were prepared based on a rapid extraction procedure using Chem Elut cartridges. The extracted samples were analyzed on a C18 HPLC column interfaced with a Finnigan TSQ 700 mass spectrometer. Positive atmospheric chemical ionization (APCI) was employed as the ionization source. The analyte and its internal standard (2H6-SR 49059) were detected by use of multiple reaction monitoring (MRM) mode. The plasma matrix had a calibration range 0.2-20 ng ml-1, with within and between run accuracy and precision both less than 10%. The chromatographic run time was approximately 3 min. Urine samples were prepared based on a simple dilution with water, followed by analysis under the same conditions as plasma. The calibration range for urine matrix was 20-5000 ng ml-1, with within and between run accuracy and precision less than 11%. The method has been successfully applied to the clinical sample analysis. The plasma assay was also evaluated on a Finnigan TSQ 7000 mass spectrometer. The performance based on precision and accuracy was virtually identical to that on the TSQ 700, with the exception of linearity in calibration curve (the TSQ 700 was linear, the TSQ 7000 was quadratic).

Endocrine and neuroendocrine effects of Azadirachtin in adult females of the earwig Labidura riparia.

In previous studies we have shown that injection of the insect growth regulator Azadirachtin (AZA) into young vitellogenic females induces inhibition of vitellogenesis in a dose-dependent manner. Juvenile hormone treatment rescues vitellogenin synthesis and ovarian growth. The cytopathological effects on ovaries and fat body are not linked to an inhibition of feeding. In this work we investigated the effects of AZA on the endocrine and neuroendocrine system. Enzyme immunoassay reveals that ovarian ecdysteroid levels are drastically reduced, in a dose-dependent fashion, by AZA. Ultrastructural study indicates that corpus allatum cells exhibit signs of inactivity and degenerative changes after AZA exposure. Using an antibody against allastostatin-3 of Blatella germanica (BLAST-3), we show the appearance of strong immunoreactivity of numerous cells and axons in the brain of AZA-injected females. We conclude that vitellogenesis inhibition by AZA consists of a direct cytotoxic effect as well as a generalized disruption of endocrine and neuroendocrine functions.

Assessment of environmental endocrine disruptors in bald eagles of the Great Lakes.

Environmental endocrine disruption in wildlife has primarily focused on estrogenic/androgenic end points and their antagonists. We describe here the work that has occurred within the Great Lakes of North America that has used the bald eagle (Haliaeetus leucocephalus) as a sentinel species of the effects of environmental toxicants, including endocrine disruption. Our data suggests that population level effects of hormone disrupting chemicals, not necessarily estrogen/androgen mimics and their antagonists, have been associated with reproductive and teratogenic effects observed in the bald eagle population within the Great Lakes Basin. Additional laboratory and field studies are necessary to further clarify the role of environmental endocrine disruptors on reproduction in avian populations. The use of sea eagles (Haliaeetus spp.) as biosentinels of pollution in other regions of the world is also discussed.

Analysis of selective androgen receptor modulators by gas chromatography-microchip atmospheric pressure photoionization-mass spectrometry.

A gas chromatography-microchip atmospheric pressure photoionization-mass spectrometric (GC-microAPPI-MS) method was developed and used for the analysis of three 2-quinolinone-derived selective androgen receptor modulators (SARMs). SARMs were analyzed from spiked urine samples, which were hydrolyzed and derivatized with N-methyl-N-(trimethylsilyl)trifluoroacetamide before analysis. Trimethylsilyl derivatives of SARMs formed both radical cations (M(+*)) and protonated molecules ([M + H](+)) in photoionization. Better signal-to-noise ratios (S/N) were obtained in MS/MS analysis using the M(+*) ions as precursor ions than using the [M + H](+) ions, and therefore the M(+*) ions were selected for the precursor ions in selected reaction monitoring (SRM) analysis. Limits of detection (LODs) with the method ranged from 0.01 to 1 ng/mL, which correspond to instrumental LODs of 0.2-20 pg. Limits of quantitation ranged from 0.03 to 3 ng/mL. The mass spectrometric response to the analytes was linear (R > or = 0.995) from the LOQ concentration level up to 100 ng/mL concentration, and intra-day repeatabilities were 5%-9%. In addition to the GC-microAPPI-MS study, the proof-of-principle of gas chromatography-microchip atmospheric pressure chemical ionization-Orbitrap MS (GC-microAPCI-Orbitrap MS) was demonstrated.

Development and validation of a capillary high-performance liquid chromatography/electrospray tandem mass spectrometric method for the quantification of bisphenol A in air samples.

Bisphenol A (BPA) is a toxic industrial chemical that affects the endocrine system even at low concentrations. A new method, based on capillary high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS) analysis, has been developed to determine BPA in atmospheric samples. The method involves collection of air samples (typically 2 m(3)) on glass fiber filters, with ultrasonic extraction and sample concentration under vacuum before analysis. HPLC analysis was performed isocratically at a flow rate of 10 microL min(-1) using a capillary reversed-phase column and MS/MS analysis in negative ion multiple reaction monitoring (MRM) mode, using BPA-d(16) as internal standard. The present method provides linear response in the range 0.007-3.5 microg/filter (R(2) > 0.999) and is characterized by high accuracy (mean bias 2%) and good reproducibility (mean RSD 5%). High sensitivity (LOD = 2 ng/m(3) based on 2 m(3) of air collected), specificity, and speed of the analysis make the present method suitable for routine determination of BPA in the atmosphere, both for ambient and personnel monitoring.

Allatostatin-like and AKH/HrTH-like peptides in the aphid Megoura viciae.

Knowledge of the structures of neuropeptides that regulate development, metabolism, and behaviour in insects is extensive, but nothing is known of the identity of regulatory peptides in the aphid neuroendocrine system. The present study applies a radioimmunoassay to reveal the existence of at least two allatostatin-like peptides in the aphid, Megoura viciae. Immunocytochemistry using antibodies recognising cockroach and dipteran allatostatins (Dip-AST-7 and Cav-AST-1) revealed the presence of allatostatin-like peptides in the protocerebrum of the brain, in the supraoesophageal ganglion, and in the fused thoracic ganglia. Both the corpora cardiaca and the corpus allatum, as well as the nervi corporis cardiaci I, stained strongly with the allatostatin antibodies. AKH/ HrTH-like peptides were detected in extracts of M. viciae using conspecific bioassays for hypertrehalosaemic and hyperlipaemic activity. Endocrine cells of the corpora cardiaca contained AKH-like material that reacted to antibodies directed to the N- and C-terminus of Lom-AKH-I. Antibodies specific for the C-terminus of Lom-AKH-I gave extensive staining in the brain and immunoreactive fibres were also found in the suboesophageal and fused thoracic ganglia. In contrast, staining with antibodies recognising the N-terminus of Lom-AKH-I was restricted to the corpora cardiaca and a region of the pars intercerebralis. There was no difference between apterous and alate morphs of M. viciae in the distribution of both AKH-like and allatostatin-like peptides. These results suggest an endocrine role for AKH/HrTH and allatostatin-like peptides in aphids.

Sonic spray ionization applied to liquid chromatography/mass spectrometry analysis of endocrine-disrupting chemicals in environmental water samples.

A sonic spray ionization liquid chromatography/mass spectrometry (LC/SSI-MS) procedure combined with off-line solid-phase extraction was optimized for the analysis of 20 endocrine-disrupting chemicals (EDCs) in water samples. Method development included a comparison of the novel sonic spray ionization (SSI) with more traditional ion sources, i.e. pneumatically assisted electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI). It was demonstrated that SSI and ESI spectra were very similar, but were more prone to the formation of solvent cluster ions as compared with APCI spectra. This phenomenon was most prominent for SSI and resulted in an increased chemical background in full-scan mass spectra. However, this chemical noise did not affect the overall sensitivity of SSI and ESI. After optimization of LC and MS parameters, the LC/SSI-MS method was validated. Recoveries ranged from 76.3 up to 113.4% for all compounds. Limits of detection (LOD) and quantitation (LOQ) were established between 3.0 and 11.5 ng/L and 9.9 and 38.0 ng/L, respectively. Within-day (n = 5) and between-day (n = 5) reproducibility were investigated at three levels and ranged from 3.3-16.5% and 7.6-19.2%, respectively. Eight-point calibration curves were established and showed linearity for all compounds (r(2) > 0.987) over a linear dynamic range of 10-10 000 ng/L.