3D-QSAR and docking studies of estrogen compounds based on estrogen receptor β

Close attention has been paid to estrogen compounds because these chemicals may pose a serious threat to the health of humans and wildlife. Estrogen receptor (ER) exists as two subtypes, ERα and ERβ. The difference in amino acids sequence of the binding sites of ERα and ERβ might lead to a result that some synthetic estrogens and naturally occurring steroidal ligands have different relative affinities and binding modes for ERα and ERβ. In this investigation, comparative molecular similarity indices analysis...     

Screening of synthetic and plant-derived compounds for (anti)estrogenic and (anti)androgenic activities

Recently we constructed yeast cells that either express the human estrogen receptor α or the human androgen receptor in combination with a consensus ERE or ARE repeat in the promoter region of a green fluorescent protein (yEGFP) read-out system. These bioassays were proven to be highly specific for their cognate agonistic compounds. In this study the value of these yeast bioassays was assessed for analysis of compounds with antagonistic properties. Several pure antagonists, selective estrogen receptor modulators (SERMs) and plant-derived compounds were tested. The pure antiestrogens ICI 182,780 and RU 58668 were also classified as pure ER antagonists in the yeast estrogen bioassay and the pure antiandrogen flutamide was also a pure AR antagonist in the yeast androgen bioassay. The plant-derived compounds flavone and guggulsterone displayed both antiestrogenic and antiandrogenic activities, while 3,3′-diindolylmethane (DIM) and equol combined an estrogenic mode of action with an antiandrogenic activity. Indol-3-carbinol (I3C) only showed an antiandrogenic activity. Coumestrol, genistein, naringenin and 8-prenylnaringenin were estrogenic and acted additively, while the plant sterols failed to show any effect. Although hormonally inactive, in vitro and in vivo metabolism of the aforementioned plant sterols may still lead to the formation of active metabolites in other test systems.     

New polyhydroxysteroids and steroid glycosides from the far east starfishCeramaster patagonicus

Two new polyhydroxysteroids and five new glycosides were isolated from the starfishCeramaster patagonicus and their structures were elucidated: 5α-cholestane-3β,6α,15β,16β,26-pentol, (22E)-5α-cholest-22-ene-3β,6α,8,15α,24-pentol, (22E)-28-O-[O-(2-O-methyl-β-d-xylopyranosyl)-(1→2)-β-d-galactofuranosyl]-24-hydroxymethyl-5α-cholest-22-ene-3β,4β, 6α,8,15β,16β,28-heptol (ceramasteroside C₁), (22E)-28-O-[O-(2,4-di-O-methyl-β-d-xylopyranosyl)-(1→2)-β-d-galactofuranosyl]-24-hydroxymethyl-5α-cholest-22-ene-3β, 6α,8,15β,16β,28-hexol (ceramasteroside C₂), (22E)-28-O-[O-methyl-β-d-xylopyranosyl)-(1→2)-β-d-galactofuranosyl]-24-hydroxymethyl-5α-cholest-22-ene-3β,6α,8,15β,16β 28-hexol (eramasteroside C₃), (22E)-28-O-[O-(2-O-methyl-β-d-xylopyranosyl)-(1→2)-β-d-galactofuranosyl]-24-methyl-5α-cholest-22-ene-3β,4β,6α,8, 15β, 26-hexol (ceramasteroside C₄), and (22E)-28-O-[O-(2-O-methyl-β-d-xylopyranosyl)-(1→2)-β-d-xylopyranosyl]-5α-cholest-22-ene-3β,6α,8,15β,24-pentol (ceramasteroside C₅)). Three known polyhydroxysteroids (24-methylene-5α-cholestane-3β,6α,8,15β,16β,26-hexol, 5α-cholestane-3β,6α,8,15β,16β,26-hexol, and 5α-cholestane-3β,6β,15α,16β,26-pentol) were also isolated. Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 1, pp. 190–195, January, 1997.     

Dehydration Reaction of Manganese(II) Oxalate Dihydrate in the Solid State New Method in the Study of Non-isothermal Kinetics

A new method was proposed for determining the most probable mechanism function of a solid phase reaction. According to Coats-Redfern's integral equation E_β→0 was calculated by extrapolating β to zero using a series of TG curves with different heating rates. Similarly, E_α→0 was calculated according to Ozawa's equation. The most probable mechanism function of the solid phase dehydration of manganese(II) oxalate dihydrate was confirmed to be G(α)=(1-α)^1/2 by comparing E_α→0 with E_β→0. This revised version was published online in July 2006 with corrections to the Cover Date.     

Reversed-phase liquid chromatography coupled on-line to estrogen receptor bioaffinity detection based on fluorescence polarization

We describe the development and validation of a high-resolution screening (HRS) platform which couples gradient reversed-phase high-performance liquid chromatography (RP-HPLC) on-line to estrogen receptor α (ERα) affinity detection using fluorescence polarization (FP). FP, which allows detection at high wavelengths, limits the occurrence of interference from the autofluorescence of test compounds in the bioassay. A fluorescein-labeled estradiol derivative (E2-F) was synthesized and a binding assay was optimized in platereader format. After subsequent optimization in flow-injection analysis (FIA) mode, the optimized parameters were translated to the on-line HRS bioassay. Proof of principle was demonstrated by separating a mixture of five compounds known to be estrogenic (17β-estradiol, 17α-ethinylestradiol and the phytoestrogens coumestrol, coumarol and zearalenone), followed by post-column bioaffinity screening of the individual affinities for ERα. Using the HRS-based FP setup, we were able to screen affinities of off-line-generated metabolites of zearalenone for ERα. It is concluded that the on-line FP-based bioassay can be used to screen for the affinity of compounds without the disturbing occurrence of autofluorescence.     

The L Spectrum of Fe and Fe3O4

The intensities of the Fe L lines/bands l = L₃M₁, η = L₂M₁, α_1,2 = L₃M_4,5, β₁ = L₂M₄, and β_3,4 = L₁M_2,3 were measured for pure Fe and Fe₃O₄ using a TAP crystal as the dispersing element. The energy of the exciting electrons, E₀, was varied in the range 5 ≤ E₀ ≤ 25 keV. For pure Fe the following results were obtained. The net peak height ratio Ll/Lα remains relatively constant with varying E₀ at approximately 14%. The E₀ dependence of Lη is similar to that of Ll, although Lη is less intense than Ll by a factor of 7. Lβ₁/Lα decreases from 20% for E₀ = 5 keV to about 5% for 25 keV. Lβ_3,4 behaves like Lβ₁ but is weaker by a factor of 15. For Fe₃O₄ a much weaker intensity of Lα was observed which can be partially explained by its stronger absorption. Again, the E₀ dependence of Ll and Lη is similar with Ll/Lα = 19% and Lη/Lα = 4%. Lβ₁ and Lβ_3,4 show a comparable E₀ dependence. Lβ₁/Lα decreases from 50% for E₀ = 5 keV to 34% for 25 keV. Lβ_3,4 is weaker than Lβ₁ by a factor of about 25. The observed E₀ dependence of the different lines was used to estimate a set of mass absorption coefficients. Our value for Lα in Fe agrees well with other data which were deduced from variable E₀ measurements but differs considerably from data given by Heinrich and Henke.     

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.      

Analytical approach for monitoring endocrine-disrupting compounds in urban waste water treatment plants

The presence of endocrine-disrupting compounds in influent and effluent water samples from four waste water treatment plants located in Italy was studied. The estrogen-like activity of the water samples was measured using a chemiluminescent recombinant yeast assay which is based on genetically engineered yeast cells that express the human estrogen receptor. This receptor, once activated, elicits the expression of the reporter gene lac-Z and, consequently, the production of β-galactosidase, which is then measured by chemiluminescence. To control and minimize sample matrix effects, an external control based on a modified yeast strain stably expressing β-galactosidase was developed and also used in the assay. Rapid and sensitive chemiluminescent enzyme immunoassays were also developed and validated for the quantification of 17β-estradiol, estrone, and estriol in waste water samples. Results from both methods were compared with a reference high-performance liquid chromatography and electrospray ionization tandem mass spectrometry (HPLC ESI-MS-MS) method developed for the quantification of natural estrogens. The recombinant yeast assay revealed a significant estrogenic activity in the influent samples, ranging from 80 to 400 pmol/L 17β-estradiol equivalents (EEQ), which was reduced by 70–95 % in the effluent samples. The yeast assay also showed a systematic 20–30 % overestimation of estrogenic activity relative to the HPLC ESI-MS-MS method, suggesting the presence of other compounds in the samples with estrogenic activity. The chemiluminescent enzyme immunoassays showed the presence of estrogens in the influent samples (mean concentrations: 350–450 pmol/L for estrone, 5–100 pmol/L for 17β-estradiol, 25–300 pmol/L for estriol), with significantly lower concentrations detected in the respective effluent samples. The waste water treatment was able to reduce natural estrogen concentrations by 40–95 %, although a high variability was observed. The enzyme immunoassay data correlated well with data obtained by the HPLC ESI-MS-MS method. Although the recombinant yeast assay represents a useful tool for a first-level screening of estrogenic activity due to its simplicity and high analytical throughput, sample matrix effects observed in waste water of industrial origin were found to strongly affect the yeast cells response, even when properly corrected for using the external control, thereby limiting its use to urban waste water. Its integration with chemiluminescent enzyme immunoassays would improve its performance by reducing false negative results, thereby enabling its use in extensive studies monitoring for the presence of endocrine-disrupting compounds in urban treatment plant effluents.     

Electron Excited L X-Ray Spectra of the Elements 14 ≤ Z ≤ 22

The L spectra of the elements 14 ≤ Z ≤ 22 were reinvestigated. Because of the limited energy resolution of the multilayer reflectors neither Ll and Lη nor Lα and Lβ₁ could be resolved. Nevertheless, the line Lβ_3,4 = L₁M_2,3 was observed for all elements Z ≥ 16, but not for ¹⁴Si and ¹⁵P. Exceptions to this are ¹⁸Ar and ²²Ti. For ¹⁸Ar no suitable sample is available. In the case of ²²Ti the Lβ_3,4 line is overlapped by the O Kα line which has its origin in the oxide surface layer of the Ti standard. In all cases the Lβ_3,4 line was observed near to the expected position which was determined by means of the known electron binding energies. The L spectrum of ²¹Sc was studied for various energies of the incident electrons, E₀. These measurements show that Lβ_3,4 is much more strongly absorbed in Sc than Ll,η and Lα,β₁. From these measurements approximate mass absorption coefficients (m.a.c.) were deduced. The relative net peak height I(Lβ_3,4)/I(Lα,β₁) decreases from 15% at E₀ = 4 keV to 4% at E₀ = 20 keV, whereas I(Ll,η)/I(Lα,β₁) remains nearly constant at 90%. The peak to background ratio of Ll,η is greater than that of Lα,β₁.     

Polar steroidal compounds from the Far-Eastern starfish <Emphasis Type="Italic">Lethasterias fusca</Emphasis>

Nine steroidal compounds including three new steroidal glycosides, viz., sodium (24S)-3,24-di-O-(β-D-xylopyranosyl)-5α-cholestane-3β,6β,8,15α,24-pentol 15-sulfate (fuscaside A), (24S)-3,24-di-O-(β-D-xylopyranosyl)-5α-cholestane-3β,6β,8,15α,24-pentol (fuscaside B), and (22E,24R)-24-O-(β-D-xylopyranosyl)-5α-cholest-22-ene-3β,6α,8,15β,24-pentol (desulfated minutoside A); three previously known glycosides, viz., distolasterosides D₁ and D₂ and pycno-podioside A; two previously known polyhydroxysteroids, viz., 5α-cholestane-3β,6α,8,15β,16β,26-hexaol and 5α-cholestan-3β,4β,6α,7⇇8,15β,16β,26-octol; and the known sodium 24,25-dihydro-marthasterone 3-sulfate were isolated from the Far-Eastern starfish Lethasterias fusca. The structures of these compounds were elucidated by NMR spectroscopy and mass spectrometry. Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 1, pp. 196–200, January, 2008.     

Stereochemical effects during [M-H]− dissociations of epimeric 11-OH-17β-estradiols and distant electronic effects of substituents at C(11) position on gas phase acidity

The affinity of estradiol derivatives for the estrogen receptor (ER) depends strongly on nature and stereochemistry of substituents in C₍₁₁₎ position of the 17β-estradiol (I). In this work, the stereochemistry effects of the 11α-OH-17β-estradiol (III_α) and 11β-OH-17β-estradiol (III_β) were investigated using CID experiments and gas-phase acidity (ΔH_acid^∘) determination. The CID experiments showed that the steroids decompose via different pathways involving competitive dissociations with rate constants depending upon the α/β C₍₁₁₎ stereochemistry. It was shown that the fragmentations of both deprotonated [III_α-H]⁻ and [III_β-H]⁻ epimers were initiated by the deprotonation of the most acidic site, i.e. the phenolic hydroxyl at C₍₃₎. This view was confirmed by H/D exchange and double resonance experiments. Furthermore, the ΔH_acid^∘ of both epimers (III_α and III_β), 17β-estradiol (I), and 17-desoxyestradiol (II) was determined using the extended Cooks’ kinetic method. The resulting values allowed us to classify steroids as a function of their gas-phase acidity as follows: (III_β)≫(II)>(I)>(III_α). Interestingly, the α/β C₍₁₁₎ stereochemistry appeared to influence strongly the gas-phase acidity. This phenomenon could be explained through stereospecific proton interaction with π-orbital cloud of A ring, which was confirmed by theoretical calculation.     

Recombinant cell bioassays for the detection of (gluco)corticosteroids and endocrine-disrupting potencies of several environmental PCB contaminants

Sensitive and robust bioassays for glucocorticoids are very useful for the pharmaceutical industry, environmental scientists and veterinary control. Here, a recombinant yeast cell was constructed that expresses the human glucocorticoid receptor alpha and a green fluorescent reporter protein in response to glucocorticoids. Both the receptor construct and the reporter construct were stably integrated into the yeast genome. The correct and specific functioning of this yeast glucocorticoid bioassay was studied by exposures to cortisol and other related compounds and critically compared to a GR-CALUX bioassay based on a human bone cell. Although less sensitive, the new yeast glucocorticoid bioassay showed sensitivity towards all (gluco)corticoids tested, with the following order in relative potencies: budesonide >> corticosterone > dexamethasone > cortisol = betamethasone > prednisolone > aldosterone. Hormone representatives for other hormone nuclear receptors, like 17β-estradiol for the oestrogen receptor, 5α-dihydrotestosterone for the androgen receptor and progesterone for the progesterone receptor, showed no clear agonistic responses, whilst some polychlorinated biphenyls were clearly able to interfere with the GR activity.     

Detection of environmental chemicals by SPR assay using branched cyclodextrin as sensor ligand

We obtained the association constants Ka of estrogen (E2) and environmental chemicals by the surface plasmon resonance (SPR) assay using the immobilized mono-6-O-α-maltosyl-β-CD (G₂βCD) compared with the immobilized β-CD and the immobilized estrogen receptor (ER). The association behavior of G₂βCD was shown as a ER model compound. The calibration curve was determined by the initial rate of association depending on the various concentrations, and the minimum detectable concentrations in the order of parts per billion were calculated. The SPR assay has advantages that the pre-treatment of the sample is not necessary and the immobilized ligand is stable and useful for the repeated measurement.     

On the Apparent Compensation Effect Observed for Two Consecutive Reactions

A critical analysis is presented of the use of an overall single rate reaction equation instead of the true rate equation corresponding to a complex process consisting of two consecutive reactions. In accordance with this approximation, which is often used in the kinetic analysis of systems in which several reactions take place, the overall process is described by apparent activation parameters (the apparent activation energy E_ap and the apparent pre-exponential factor A_ap) and an apparent conversion function. The theoretical isotherms (α=α(t), where α is the conversion degree and t is time) were simulated for a system in which two consecutive reactions occur. In this case, the apparent activation parameters depend on (a) the considered range of temperature; and (b) the temperature for a given conversion degree. It is shown that the apparent activation parameters are correlated by the compensation effect relationship: ln A_ap = α^* + β^*E_ap where α^* and β^* are the linear regression parameters. The possibility of using the apparent kinetic parameters to predict the isotherms α=α(t) for temperatures lower than those for which these parameters were evaluated is discussed. This revised version was published online in August 2006 with corrections to the Cover Date.     

Factors affecting vitamin degradation in oil-in-water nano-emulsions

Fat fractions composed by different proportions of low (LMT) or high (HMT) melting temperature triacylglycerols were used, alone or in mixture with α-tocopherol for the preparation of oil-in-water protein stabilised nano-emulsions. Addition of α-tocopherol to the LMT or HMT fat fractions was accompanied by different changes in the emulsion characteristics such as fat droplet size distributions, under-cooling and polymorphic transitions, in parallel with different extent of α-tocopherol degradation reactions. Our results showed higher immobilisation pattern of α-tocopherol molecules and higher protection against degradation when incorporated in higher size fat droplets, which presented 2L_α → 2L_β′ polymorphic transitions under cooling and re-heating cycles.     

On the Apparent Compensation Effect Found for Two Consecutive Reactions

A critical analysis of the use of an overall single rate reaction equation instead of the true rate equation corresponding to a complex process consisting in two consecutive reactions is presented. In accordance with this approximation, often used in the kinetic analysis of the system in which several reactions take place, the overall process is described by the apparent activation parameters (the apparent activation energy, E _ap, and the apparent pre-exponential factor, A _ap) and the apparent conversion function. The theoretical isotherms (α=α(t), where a is the conversion degree and t is the time) have been simulated for a system in which two consecutive reactions occur. In this case, the apparent activation parameters depends on: (a) the considered range of the temperature; (b) the temperature, for a given conversion degree. It is shown that the apparent activation parameters are corrrelated by the compensation effect relationship: lnA _ap=α*+β*E _ap where α* and β* are the parameters of the linear regression. The possibility of using the apparent kinetic parameters to predict the isotherms α=α(t) for temperatures lower than those for which these parameters were evaluated, is discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Steroids from the marine bryozoan <Emphasis Type="Italic">Bugula neritina</Emphasis>

One new compound, 3β-hydroxy-25-methoxy-(23E)-cholesta-5,23-diene (1), together with five known steroids, cholesteryl myristate (2), cholest-4-en-3-one (3), cholesterol (4), 3β,5α,9α-trihydroxy-(22E,24R)-ergosta7,22-dien-6-one (5), and 3β,5α,6β-trihydroxy-(22E,24R)-ergosta-7,22-diene (6), were isolated and identified from the marine bryozoan Bugula neritina.     

Condensations of Ethyl 3-Ethoxy-4-(triphenylphosphoranylidene)-2-butenoate with α,β-Unsaturated Carbonyl Compounds

 Wittig condensations of α,β-unsaturated carbonyl compounds with ethyl 3-ethoxy-4-(triphenylphosphoranylidene)-2-butenoate gave good to high yields of (2E,4E,6E)-ethyl 3-ethoxy-2,4,6-alkatrienoates. Some of last mentioned compounds were almost quatitatively hydrolysed to (4E,6E)-ethyl 3-oxo-4,6-alkadienoates. This method can therefore be used as an attractive alternative for the preparation of unsaturated conjugated β-keto esters previously prepared in very low yields from α,β-unsaturated carbonyl compounds and ethyl 3-oxo-4-(triphenylphosphoranylidene)butanoate.     

Synthesis of amino-silane modified magnetic silica adsorbents and application for adsorption of flavonoids from <Emphasis Type="Italic">Glycyrrhiza uralensis</Emphasis> Fisch

Magnetic separation technology was applied in the separation of flavonoids from the licorice root in this work. Licorice flavonoids (LF) displayed a remarkable array of biological and pharmacological activities. The magnetic adsorbents with functional —NH₂ groups were synthesized by immobilization of amino-silane on the surface of the magnetic silica supports, which were prepared by co-precipitation method. The adsorption and desorption characteristics of the magnetic adsorbents for the separation of LF have been evaluated. The purity of an enriched extract with this method was 16.7% while the crude extract only had about 6.8% purity. Therefore, it can be concluded that these kinds of magnetic adsorbents have selectivity to the flavonoids to some extent. The affinity selectivity of the adsorbents is based on the formation of hydrogen bonding between the —NH₂ on the magnetic adsorbents and —OH, —CO on the flavonoids. Supported by the National High Technology Research and Development Program of China (Grant No. 2002AA302211) and the National Science Fund for Creative Research Groups of China (Grant No. 20221603)