Validation and application of reporter gene assays for the determination of estrogenic and androgenic endocrine disruptor activity in sport supplements

Previously developed estrogen and androgen mammalian reporter gene assays (RGAs) were assessed for their potential use as a quantitative screening method in the detection of estrogenic and androgenic endocrine disruptors (EDs) in sport supplements. The validation of both RGAs coupled with dispersive solid phase extraction (dSPE) was performed in accordance with European Commission Decision EC/2002/6579 for biological screening methods. Decision limits (CCα) and detection capabilities (CCβ) were established for both the estrogen and androgen RGAs. All samples were compliant with CCα and CCβ in both bioassays. Recovery rates were 96 % for 17β-estradiol and 115 % for dihydrotestosterone as obtained in their corresponding RGA. Both estrogens and androgens were stable in samples for more than 3 weeks, when stored at -20 °C. Specificity, good repeatability (coefficients of variation (CV), 12-25 %), reproducibility and robustness of both bioassays were also observed. Four different ED modes of action were determined for estrogens and androgens in 53 sport supplements, using the validated RGAs. This study revealed that 89 % of the investigated sport supplements contained estrogenic EDs and 51 % contained androgenic compounds. In conclusion, both bioassays are suitable for sport supplement screening of estrogenic and androgenic EDs.     

The application of reporter gene assays for the detection of endocrine disruptors in sport supplements

The increasing availability and use of sports supplements is of concern as highlighted by a number of studies reporting endocrine disruptor contamination in such products. The health food supplement market, including sport supplements, is growing across the Developed World. Therefore, the need to ensure the quality and safety of sport supplements for the consumer is essential. The development and validation of two reporter gene assays coupled with solid phase sample preparation enabling the detection of estrogenic and androgenic constituents in sport supplements is reported. Both assays were shown to be of high sensitivity with the estrogen and androgen reporter gene assays having an EC(50) of 0.01 ng mL(-1) and 0.16 ng mL(-1) respectively. The developed assays were applied in a survey of 63 sport supplements samples obtained across the Island of Ireland with an additional seven reference samples previously investigated using LC-MS/MS. Androgen and estrogen bio-activity was found in 71% of the investigated samples. Bio-activity profiling was further broken down into agonists, partial agonists and antagonists. Supplements (13) with the strongest estrogenic bio-activity were chosen for further investigation. LC-MS/MS analysis of these samples determined the presence of phytoestrogens in seven of them. Supplements (38) with androgen bio-activity were also selected for further investigation. Androgen agonist bio-activity was detected in 12 supplements, antagonistic bio-activity was detected in 16 and partial antagonistic bio-activity was detected in 10. A further group of supplements (7) did not present androgenic bio-activity when tested alone but enhanced the androgenic agonist bio-activity of dihydrotestosterone when combined. The developed assays offer advantages in detection of known, unknown and low-level mixtures of endocrine disruptors over existing analytical screening techniques. For the detection and identification of constituent hormonally active compounds the combination of biological and physio-chemical techniques is optimal.     

Laccase-Mediated Transformations of Endocrine Disrupting Chemicals Abolish Binding Affinities to Estrogen Receptors and Their Estrogenic Activity in Zebrafish

Endocrine disrupting chemicals (EDCs) are known to mainly affect aquatic organisms, producing negative effects in aquaculture. Transformation of the estrogenic compounds 17??-estradiol (E2), bisphenol-A (BPA), nonylphenol (NP), and triclosan (TCS) by laccase of Coriolopsis gallica was studied. Laccase is able to efficiently transform them into polymers. The estrogenic activity of the EDCs and their laccase transformation products was evaluated in vitro as their affinity for the human estrogen receptor alpha (hER??) and for the ligand binding domain of zebrafish (Danio rerio) estrogen receptor alpha (zfER??LBD). E2, BPA, NP, and TCS showed higher affinity for the zfER??LBD than for hER??. After laccase treatment, no affinity was found, except a marginal affinity of E2 products for the zfER??LBD. Endocrine disruption studies in vivo on zebrafish were performed using the induction of vitellogenin 1 as a biomarker (VTG1 mRNA levels). The use of enzymatic bioreactors, containing immobilized laccase, efficiently eliminates the endocrine activity of BPA and TCS, and significantly reduces the effects of E2. The potential use of enzymatic reactors to eliminate the endocrine activity of EDCs in supply water for aquaculture is discussed.     

Xenoestrogens: mechanisms of action and detection methods

Estrogenic compounds exert pleiotropic effects in wildlife and humans, and endogenous estrogens, like 17-estradiol, regulate growth and development of their target tissues. Environmental, industrial, or naturally occurring chemicals that possess estrogenic and/or antiestrogenic activities are termed xenoestrogens and may interfere with endocrine systems. These xenoestrogens are therefore defined as endocrine-active or endocrine-disrupting compounds. The estrogen receptor (ER) is the major regulatory unit within the estrogen-signaling pathway and the molecular mechanisms of estrogen and ER actions are described briefly. Based on the mechanism of ER action, in vitro test systems are described that can be employed for screening but also for the elucidation of mechanisms of action of (anti)estrogenic compounds. How screening assays and mechanistic studies can aid in human risk assessment for potential endocrine-active compounds is discussed also.     

Effects of 17β-trenbolone in male eelpout Zoarces viviparus exposed to ethinylestradiol

To evaluate the interaction between 17β-trenbolone (TB) and 17α-ethinylestradiol (EE2), male eelpout, Zoarces viviparus, was exposed for 21 days (April to May 2008) to 5 ng l⁻¹ EE2 and 5 or 20 ng l⁻¹ TB, separately or in combination in a flow-through SW system. The effects on hepatosomatic (HSI) and gonadosomatic index (GSI), plasma vitellogenin (Vtg) concentration, gonadal histology, hepatic and testicular Vtg mRNA and estrogen receptor (ERα) mRNA expression were investigated. No effects on HSI were observed. A significant decrease was observed in the GSI of all males exposed to EE2 (<0.7%) when compared to controls (1.4%). Histological alterations and immature stages were observed in the testis of all exposed males; however, males exposed to EE2 were the most affected. Increased tubule number and proportionally decreased tubule diameter were observed in the testis of all EE2 groups. No effects in Vtg mRNA expression were observed in the testis; however, a significant decrease in testis ERα mRNA was observed in males exposed to 20 ng l⁻¹ TB. The groups exposed to EE2 showed a significant increase in plasma Vtg (>300-fold), hepatic Vtg mRNA (>450-fold), and ERα mRNA (>100-fold) when compared to controls. This study shows that lower concentrations of 17β-trenbolone are unable to counteract the EE2 estrogenic effects when the exposure is simultaneous.      

SPE-HPLC purification of endocrine-disrupting compounds from human serum for assessment of xenoestrogenic activity

Assessment of xenoestrogenic activity in human serum samples requires the removal of endogenous sex hormones to assure that the activity measured originates from xenobiotic compounds only. Serum samples representing high, medium and lower accumulation of persistent organic pollutants (POPs) were extracted using solid-phase extraction (SPE) followed by normal-phase high-performance liquid chromatography (NP-HPLC) for separation of POPs from endogenous hormones. The recovery of polychlorinated biphenyl (PCB) congeners in spiked serum samples was up to 86 %, making the extraction method suitable for the study. MVLN cells, stably transfected with an estrogen receptor (ER) luciferase reporter vector (estrogen response element chemically activated luciferase expression, ERE-CALUX), were exposed to the reconstituted SPE-HPLC extracts for determination of the integrated estrogenic activity. The effects of PCBs were analyzed by direct in vitro exposure of PCBs (nos. 138, 153, 180) and by ex vivo analysis of SPE-HPLC extracts from serum spiked with the PCBs. Similar effects on ER transactivation were observed for the direct in vitro and the ex vivo analysis experiments. The ER transactivation responses determined for actual serum samples were in the linear range of the dose-response curve. 17β-Estradiol titrations showed that the xenoestrogenic effects were mediated via ER. Moreover, our SPE-HPLC-ERE-CALUX assay was demonstrated to elicit high interlaboratory correlation. In the present study the combination of SPE-HPLC purification and the ex vivo estrogenic responses measured by ERE-CALUX was validated and considered to be a valuable tool to assess the combined ER effect of lipophilic serum POPs where additive/synergistic and agonistic/antagonistic effects are integrated giving an overall estimate of exposure and bioactivity.      

Development,standardization and refinement of procedures for evaluating effects of endocrine active compounds on development and sexual differentiation of <Emphasis Type="Italic">Xenopus laevis</Emphasis>

Xenopus laevis has been introduced as a model to study effects of endocrine-active compounds (EAC) on development and sexual differentiation. However, variable and inconsistent data have raised questions about the reliability of the test methods applied. The current study was conducted in two laboratories to develop, refine, and standardize procedures and protocols. Larvae were exposed in flow-through systems to 17β-estradiol (E2), at concentrations from 0.2 to 6.0 μg E2 L⁻¹ in Experiment 1A, and 0.015 to 2.0 μg E2 L⁻¹ in Experiment 1B. In both studies survival (92%, 99%) and percentage of animals that completed metamorphosis (97%, 99%) indicated reproducible biological performance. Furthermore, minor variations in husbandry led to significant differences in snout-to-vent length, weight, and gonad size. In Experiment 1A, almost complete feminization occurred in all E2 treatment groups whereas a concentration response was observed in Experiment 1B resulting in an EC₅₀ of 0.12 μg E2 L⁻¹. The final verified protocol is suitable for determining effects of EAC on development and sexual differentiation in X. laevis.     

Simultaneous determination of estrogenic and androgenic hormones in water by isotope dilution gas chromatography-tandem mass spectrometry

A rapid gas chromatography-tandem mass spectrometry (GC-MS/MS) analytical method was developed for the simultaneous analysis of 7 estrogenic hormones (17α-estradiol, 17β-estradiol, estrone, mestranol, 17α-ethynylestradiol, levonorgestrel, estriol) and 5 androgenic hormones (testosterone, androsterone, etiocholanolone, dihydrotestosterone, androstenedione) in aqueous matrices. This method is unique in its inclusion of all 12 of these estrogens and androgens and is of particular value due to its very short chromatographic run time of 15 min. The use of isotope dilution for all analytes ensures the accurate quantification, accounting for analytical variabilities that may be introduced during sample processing and instrumental analysis. Direct isotopically labelled analogues were used for 8 of the 12 hormones and satisfactory isotope standards were identified for the remaining 4 hormones. Method detection levels (MDLs) were determined to describe analyte concentrations sufficient to provide a signal with 99% certainty of detection. The established MDLs for most analytes were 1-5 ngL(-1) in a variety of aqueous matrices. However, slightly higher MDLs were observed for etiocholanolone, androstenedione, testosterone, levonorgestrel and dihydrotestosterone in some aqueous matrices. Sample matrices were observed to have only a minor impact on MDLs and the method validation confirmed satisfactory method stability over intra-day and inter-day analyses of surface water and tertiary treated effluent samples.     

Development of a nanomechanical biosensor for analysis of endocrine disrupting chemicals

A nanomechanical transducer is developed to detect and screen endocrine disrupting chemicals (EDCs) combining fluidic sample injection and delivery with bioreceptor protein functionalized microcantilevers (MCs). The adverse affects of EDCs on the endocrine system of humans, livestock, and wildlife provides strong motivation for advances in analytical detection and monitoring techniques. The combination of protein receptors, which include estrogen receptor alpha (ER-alpha) and estrogen receptor beta (ER-beta), as well as monoclonal antibodies (Ab), with MC systems employing modified nanostructured surfaces provides for excellent nanomechanical response sensitivity and the inherent selectivity of biospecific receptor-EDC interactions. The observed ranking of binding interaction of the tested EDCs with ER-beta is diethylstilbestrol (DES) > 17-beta-estradiol > 17-alpha-estradiol > 2-OH-estrone > bisphenol A > p,p'-dichlorodiphenyldichloroethylene (p,p'-DDE) with measurements exhibiting intra-day RSDs of about 3%. A comparison of responses of three EDCs, which include 17-beta-estradiol, 17-alpha-estradiol, and 2-OH-estrone, with ER-beta and ER-alpha illustrates which estrogen receptor subtype provides the greatest sensitivity. Antibodies specific to a particular EDC can also be used for analyte specific screening. Calibration plots for a MC functionalized with anti-17-beta-estradiol Ab show responses in the range of 1 x 10(-11) through 1 x 10(-7) M for 17-beta-estradiol with a linear portion extending over two orders of magnitude in concentration.     

Phosphorus macrocycles and cryptands

The reviews covers authors" studies dealing with the synthesis of P^III- and P^V-containing macrocycles and cryptands. The separation, structural characterization, and the chemical properties of a number of homeomorphic compounds with in,out bridgehead phosphorus atoms are described. Modification of the in-positions in macrobicyclic compounds with bulky groups is described for the first time.     

π-d-Interaction and the temperature dependent magnetic circular dichroism spectra of low spin Fe(III)-heme compounds

The magnetic circular dichroism (MCD) of metmyoglobin cyanide, ferricytochromec and horseradish peroxidase cyanide were measured in the region 340–800 nm over a range of temperatures from 293 to 15 K. All three species show the temperature dependent MCD (TheC-type effects ∼1/T) in both visibleQ and near UVB bands. While the MCD and absorption inB- region as well as the absorption inQ region are quite similar for all three species the MCD inQ- bands reveal the marked differences, especially at low temperatures. To explain these observations, the theoretical treatment based on our previous model (A. P. Mineyev and Yu. A. Sharonov, 1978, Theoret. Chim. Acta (Berl.)49, 295–307) is proposed. The key point of this consideration is the configuration π-d- interaction which in addition to our previous analysis involves the first excited Fe(III)-ion Kramers doublet and theB-Q-mixing effects. The simultaneous least square fit of MCD and absorption data allows to evaluate the π —d- parameters which appear to be of the order of 10²−10³ cm⁻¹. The role of the π -d- interaction in the forming of hemoprotein spectra are discussed.     

Melanopsins: Localization and Phototransduction in Xenopus laevis Melanophores

Xenopus laevis melanophores express two melanopsins, Opn4x and Opn4m. We identified Opn4x immunoreactivity throughout the melanophore cytoplasm and in the cell membrane. The strongest immunopositivity for Opn4m was observed in the nuclear region, and no labeling was seen in the cell membrane. This immunodistribution suggests Opn4x as the functional photopigment. In X. laevis melanophores, light triggers pigment dispersion and clock gene induction at blue wavelength, which maximally activates melanopsins. Although light stimulation activates phospholipase C and increases intracellular calcium and cGMP, this nucleotide does not participate in photo‐induced melanin dispersion. Nevertheless, the guanylyl cyclase activator YC‐1 stimulates Per1 expression, similar to blue light pulse, and the use of pharmacological inhibitors indicates the participation of the phosphoinositide cascade. Since cAMP levels does not change after blue light stimulation, the cAMP/PKA pathway most probably is not involved in blue light induction of Per in X. laevis melanophores. Given the localization of melanopsins and our pharmacological data, the light‐induced clock gene expression seems to be mediated by Opn4x through phosphoinositide cascade and rise in cGMP, thus leading to the reset of the biological clock in our model.     

A new luciferase reporter gene assay for the detection of androgenic and antiandrogenic effects based on a human prostate specific antigen promoter and PC3/AR human prostate cancer cells.

We developed a new mammalian cell-based luciferase reporter gene assay for androgenic and antiandrogenic activities of chemicals and environmental samples. Environmental samples usually have a complex matrix that may contain the constituents acting as androgen receptor (AR) agonists, AR antagonists or aryl hydrocarbon receptor (AhR) agonists. AhR agonists are known to elicit the antiandrogenic effect through cross-talk between AR and AhR signal transduction pathways. In this study, PC3/AR human prostate carcinoma cells were transiently transfected with a prostate-specific antigen (PSA) promoter-driven luciferase expression plasmid. The cells were treated with a test compound or an environmental sample for 24 h at 37 degrees C and then measured for luciferase activity. The luciferase activity was induced by dihydrotestosterone (DHT) in a concentration-dependent manner in a concentration range from 10 fM to 1 nM. R1881, a synthetic androgen receptor agonist, induced luciferase activity and its inductive effects was additive to that of DHT. The luciferase activity was not induced by cortisol, a glucocorticoid, progesterone, a progestin, and 17beta-estradiol, an estrogen in a concentration range of up to 1 microM. DHT-induced luciferase activity was reduced by bicalutamide and cyproterone acetate, AR antagonists, and also by benzo[a]pyrene, an aryl hydrocarbon receptor agonist, through AhR-mediated pathways. All of these findings indicate that the present assay system correctly responds to AR agonists, AR antagonists and AhR agonist and, therefore, it is a powerful tool for the sensitive and selective screening of chemicals and environmental samples for their androgenic and antiandrogenic activities. We developed the first assay system, in which the expression of luciferase was driven by the promoter of a prostate-specific antigen gene, a typical human androgen-regulated gene.     

Beiträge zur Chemie der Pyrrolpigmente, 42. Mitt. Kraftfeldrechnungen an Gallenfarbstoffen: Die Energiehyperfläche rubinoider Pigmente

The energy hypersurface of rubinoid bile pigments is calculated using a force field model previously described. Two cases are observed. I: The pigment is substituted symmetrically or unsymmetrically by apolar groups. This results in a very shallow energy valley containing several enantiomeric conformers of approximately equal energies. II: Substitution by polar groups, especially in position 8 and 12 of the rubin skeleton, (e.g.–CH₂–CH₂–COOH) is followed by a lock in bonding between such groups and the lactam ring functions. Thereby only two enantiomeric conformers, which are energetically stabilized, are possible. The barrier between these species amounts to about 40 kJ/mol. These results are compared with the experimental facts available so far. An analysis of the corresponding energy hypersurfaces of the diastereomeric forms of (Z,Z)-, (E,Z)-, (Z,E)- and (E,E)-configurations is given as well.

41. Mitt.:Falk H., Müller N., Mh. Chem.112, 791 (1981).     

Kinetic asymmetric protonation as a stereochemistry determining step in theMichael addition of acetic acid derivatives to α-substituted cinnamic acid derivatives

Summary The reaction between acetic acid derivatives and -substituted cinnamic acid derivatives has been studied inTHF andTHF:HMPT (80:20) as an alternative pathway of theMichael addition of phenylacetic and cinnamic acid derivatives. The regioselectivity observed is found to depend on the acceptor functional group and its geometry but not on the solvent used. The diastereoselectivity of the conjugate addition results from kinetic protonation of diastereotopic enolates (1,2-asymmetric induction). It varies from low in the presence ofHMPT to considerable or even high in pureTHF. The favouredanti orsyn configuration inTHF depends on the nature of the enolate. The results obtained are rationalized in terms of protonationvia transition structures different in type (openvs. chelated) and geometry.

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Kinetische asymmetrische Protonierung als bestimmender Schritt für die Stereochemie bei derMichael-Addition von Essigsäurederivaten an -substitutierte Zimtsäurederivate

Zusammenfassung Die Reaktion zwischen Essigsäurederivaten und -substitutierten Zimtsäurederivaten wurde inTHF undTHF:HMPT=80:20 als ein alternative Weg derMichael-Addition von Phenylessigsäure und Zimtsäurederivaten untersucht. Es wurde gefunden, daß die beobachtete Stereoselektivität von der Akzeptorgruppe und ihrer Geometrie, nicht jedoch vom Lösungsmittel abhängig ist. Die Diastereoselektivität der konjugierten Addition folgt aus der kinetischen Protonierung der diastereotopen Enolate (1,2-asymmetrische Induktion). Sie variiert von klein (bei Anwesenheit vonHMPT) bis bedeutend oder sogar groß (in reinemTHF). Die bevorzugteanti odersyn Konfiguration inTHF hängt von der Natur der Enolate ab. Die Ergebnisse werden durch eine Protonierungvia bezüglich Typ (offen oder chelatiert) und Geometrie unterschiedliche Strukturen des Übergangszustands erklärt.

     

Crystal and molecular structure ofN-phenyl-4-nitrobenzamidoxime

N-Phenyl-4-nitrobenzamidoxime has been studied by X-ray structural analysis. In two crystallographically independent molecules1A and1B, amidoxime groups adopt a planarZ configuration, and these fragments are in ans-trans conformation with respect to the =N-O and C-N(H) bonds; an intramolecular NH...O bond occurs. The nitrophenyl and phenyl groups are rotated with respect to the amidoxime plane [ONCN] by –57 and –32° in1A and –38 and –22° in1B, respectively. The rotation of the fragments about the C(1)-N(2) bond is –28 (1A) and –35° (1B). In crystals, molecules1A and1B are linked in oxime dimers through two intermolecular =N...(HO) hydrogen bonds; dimers form double chains through two NH...(O₂N) hydrogen bonds.Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 8, pp. 1516–1519, August, 1995.The work was supported by the Russian Foundation for Basic Research (Project No. 93-03-05069).     

化学修饰方法对聚乙二醇功能化碳纳米管的影响

通过酰氯化法与碳二亚胺缩合法(EDC/NHS)制备氨基化聚乙二醇(PEG1500N)修饰的多壁碳纳米管(MWNTs)并采用FTIR、Raman、TEM、原子力显微镜(AFM)、TGA-DTA-DSC、UV-Vis进行表征与分析。实验结果发现：两种方法PEG1500N都能很好地修饰MWNTs,但EDC/NHS缩合法采用更短的反应时间(反应1 d),达到了更好的接枝效果。EDC/NHS缩合法提高了碳管上羧基的利用率,接枝率大大提高。TGA-DTA分析表明缩合法接枝率为30%,而酰氯化法(反应4 d)为15%。UV-Vis分析表明EDC/NHS缩合法得到的产物溶解性也更好,溶解度由1.19 mg·mL^-1(酰氯化法得到的产物的溶解度)提高到2 mg·mL^-1以上。