Natural epiestriol-16 act as potential lead molecule against prospective molecular targets of multidrug resistant Acinetobacter baumannii-Insight from in silico modelling and in vitro investigations.

The current study aimed to identify putative drug targets of multidrug resistant Acinetobacter baumannii (MDRAb) and study the therapeutic potential of natural epiestriol-16 by computer aided virtual screening and in vitro studies. The clinical isolates (n = 5) showed extreme dug resistance to carbapenems and colistins (p ≤ .05). Computational screening suggested that out of 236 natural molecules selected, 06 leads were qualified for drug likeliness, pharmacokinetic features and one potential molecule namely natural epiestriol-16 (16b-Hydroxy-17a-estradiol) exhibited significant binding potential towards four prioritised drug targets in comparison with the binding of faropenem to their usual target. Natural epiestriol demonstrated profound binding to the outer membrane protein (Omp38), protein RecA (RecA), orotate phosphoribosyltransferase (PyrE) and orotidine 5'-phosphate decarboxylase (PyrF) with binding energy of -6.0, -7.3, -7.3 and -8.0 kcal/mol respectively. MD simulations suggested that 16-epiestriol-receptor complexes demonstrated stability throughout the simulation. The growth curve and time kill assays revealed that MDRAb showed resistance to faropenem and polymyxin-B and the pure epiestriol-16 showed significant inhibitory properties at a concentration of 200 µg/mL (p ≤ .5). Thus, natural epiestriol-16 can be used as potential inhibitor against the prioritised targets of MDRAb and this study provide insight for drug development against carbapenem and colistin resistant A. baumannii.

Ionic liquid-supported magnetite nanoparticles as electrode modifier materials for estrogens sensing.

This paper reports the application of a carbon paste electrode modified with magnetite nanoparticles and the ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate in the electroanalytical determination of 17ß-estradiol and estriol. These estrogens are potential endocrine disruptors and thus it is relevant the development of devices for their monitoring. Transmission electron microscopy, scanning electron microscopy and zeta potential techniques were applied to characterization of the modifier materials. In cyclic voltammetry experiments, irreversible oxidation peaks were observed for 17ß-estradiol and estriol at +0.320 V and +0.400 V, respectively. The anodic currents obtained were approximately three times greater than those provided by the unmodified electrode due to the presence of magnetic nanoparticles and the ionic liquid, which improved the sensitivity of modified electrode. For the analysis, the parameters of the square-wave voltammetry (scan increment, amplitude and frequency) were optimized by Box-Behnken factorial design for each estrogen. For 17ß-estradiol in B-R buffer pH 12.0, the calibration plot was linear from 0.10 to 1.0 µmol L-1, with a detection limit of 50.0 nmol L-1. For estriol in B-R buffer pH 11.0, the linear range was 1.0 to 10.0 µmol L-1, with a detection limit of 300.0 nmol L-1. The modified electrode was applied in the determination of 17ß-estradiol and estriol in pharmaceutical formulations and the results were comparable to those obtained using UV/VIS spectrometry. Statistical tests were applied to evaluate the results and it was concluded that there was no significant difference regarding the precision and accuracy of the data provided by the two methods.

Quantitation of estradiol by competitive light-initiated chemiluminescent assay using estriol as competitive antigen.

BACKGROUND: Light-initiated chemiluminescent assays (LICA) are homogeneous assays that are sensitive, specific, and free of separation and washing steps and have high throughput and high precision. METHODS: In this research, we developed a competitive method by LICA to achieve accurate quantification of estradiol (E2) in human serum. E2 competed with estriol (E3) for binding to anti-human E2 antibodies. E3 was linked to biotin via bovine serum albumin as a linker. As this assay used competition between the labeled tracer and the analyte, an increase in E2 concentration will cause a signal decrease. RESULTS: The expected detection range of E2 was 20-5000 pg/mL. The analytical and functional sensitivities were 7.16 and 13.7 pg/mL, respectively. The intra- and inter-assay coefficients of variation were both below 15%, and the recovery rate ranged from 97.5% to 106.8%. The interference rates ranged from -3.6% to 5.4% and met detection requirements for E2 in hyperbilirubinemia, hemolysis, and lipemia in clinical samples. In addition, the cross-reactivity rates between E2 and structural analogs and some reproductive hormones varied from 1.9% to 10.6% which showed that LICA is highly specific for E2. Moreover, our results showed high accordance with the IMMULITE 2000 (y = 0.6695x + 47.92, r2  = .843) and VIDAS systems (y = 1.099x - 821.5, r2  = .9392). CONCLUSION: Our data show that the LICA, which is easy to automate, is a promising technique for quantification of E2 in human serum and could be used for clinical detection.

Towards the Importance of a Roller Mill in Compounding Practice: An Experimental Approach.

There are a substantial amount of suppliers for roller mills in the market, but there is a lack of scientific evidence of a roller mill's capacity to improve particle size reduction/distribution or homogenization. In this concise paper, we evaluate the use a roller mill in the final steps of compounding semisolid dosage forms. We performed three simple tests to verify these claims: 1) particle size evaluation through dynamic light scattering and scanning electron microscopy techniques, 2) content uniformity through high-performance liquid chromatography technique, and 3) cross contamination through a cleaning validation method. Dynamic light scattering and scanning electron microscopy techniques of benzoyl peroxide 5% (gel) and testosterone 1% (cream) showed a significant reduction on particle diameter. Content uniformity testing of creams containing progesterone 5%, estradiol 0.1%, and estriol 0.4% showed better homogeneity when using the roller mill. Finally, the proposed cleaning procedure decreased the presence of the compounded preparation to a "none-detection" level after the procedure. This suggests that the roller mill used does, in fact, play a role in the final aspect and quality of pharmaceutical semisolid dosage forms.

A novel dehydrogenase 17ß-HSDx from Rhodococcus sp. P14 with potential application in bioremediation of steroids contaminated environment.

Steroids are endocrine disrupting compounds in human and are distributed in various environments. Our previous study showed that a marine bacterium Rhodococcus sp. P14 was able to efficiently degrade one typical steroid estradiol. In this study, we showed that P14 could also use other steroids, including estriol and testosterone, as sole carbon source for growth. Two dehydrogenation products, 16-hydroxestrone and androst-4-ene-3, 17-dione, were detected during estriol and testosterone degradation, respectively. By screening the genome, a short chain dehydrogenase gene was identified and named as 17ß-HSDx. Expression of 17ß-HSDx was induced in P14 when estriol, estradiol or testosterone was used as single carbon source. In addition, 17ß-HSDx was shown to have dehydrogenation ability of transforming estriol to 16-hydroxestrone, estradiol to estrone and testosterone to androst-4-ene-3, 17-dione. This is the first short chain dehydrogenase identified in bacteria with dehydrogenation ability on various steroids substrates. Overall, this study reveals that 17ß-HSDx has potential application in the bioremediation of steroids contaminated environment.

Alternative adsorbents applied to the removal of natural hormones from pig farming effluents and characterization of the biofertilizer.

Pig farming has a very strong economic importance in Brazil. The residues from this activity are applied to the soil because of their excellent characteristics as biofertilizers. The present study aimed at studying the estrone, 17ß-estradiol, and estriol natural hormones, emerging contaminants present in this type of residue that are not mentioned in the current legislation. The characterization of the pig farming effluent presented high concentrations of hormones (mg L-1). The objective was to apply the biosorbents to the removal of the hormones in batch systems directly in the manure heaps without affecting the potential of the effluent as a fertilizer. It was verified that the adsorption of hormones using the rice husk biomass in natura and soybean hull in natura, abundant alternative adsorbents, presented a good capacity of removal of hormones. The presence of the organic materials (rice husk and soybean hull) caused few alterations in the biofertilizer characteristics, demonstrating that these adsorbents present a potential of application in batch treatment systems, with possible applications related to pig farming effluents containing natural hormones.

Efficient extraction of estrogen receptor-active compounds from environmental surface water via a receptor-mimic adsorbent, a hydrophilic PEG-based molecularly imprinted polymer.

We report an efficient screening procedure for the selective detection of compounds that are actively bound to estrogen receptor (ER) from environmental water samples using a receptor-mimic adsorbent prepared by a molecularly imprinted polymer (MIP). To mimic the recognition ability of ER, we improved the typical MIP preparation procedure using a hydrophilic matrix with a polyethylene glycol (PEG)-based crosslinker and a hydrophobic monomer to imitate the hydrophobic pocket of ER. An optimized MIP prepared with methacrylic acid as an additional functional monomer and estriol (E3), an analogue of 17ß-estradiol (E2), exhibited highly selective adsorption for ER-active compounds such as E2 and E3, with significant suppression of non-specific hydrophobic adsorption. The prepared MIP was then applied to the screening of ER-active compounds in sewage samples. The fraction concentrated by the MIP was evaluated by in vitro bioassay using the yeast two-hybrid (Y2H) method and liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-Q-TOFMS). Compared to an authentic adsorbent, styrene-divinylbenzene (SDB)-based resin, the fraction concentrated by the MIP had 120% ER activity in the Y2H assay, and only 25% peak volume was detected in LC-Q-TOFMS. Furthermore, a few ER-active compounds were identified only from the fraction concentrated by the MIP, although they could not be determined in the fraction concentrated by the SDB-based resin due to ion suppression along with high levels of hydrophobic compounds. These results indicated that the newly developed MIP effectively captured ER-active compounds and while allowing most non-ER-active compounds to pass through.

Physical and Chemical Stability of Estriol 0.025% to 1% Vaginal Creams (VersaBase).

Estrogen replacement therapy is often recommended when female patients present with lower than normal physiologic levels, such as patients going through menopause. The physical and chemical stability of estriol 0.25 mg/g and 10 mg/g vaginal creams (VersaBase) was tested over a period of 182 days, at room temperature and refrigerated conditions, in order to determine the corresponding beyond-use date. The physical characterization consisted in observing all samples for color/appearance and odor, and testing for pH, whereas the chemical characterization consisted in ultra-performance liquid chromatography assay testing. Both vaginal creams were proven physically and chemically stable, and the ultra-performance liquid chromatography method was proven stability indicating. As a result, the beyond-use date of the estriol 0.025% to 1% vaginal creams (VersaBase), in electronic mortar and pestle plastic jars, is six months at both room temperature and refrigerated conditions.

Combined Biophysical Chemistry Reveals a New Covalent Inhibitor with a Low-Reactivity Alkyl Halide.

17ß-Hydroxysteroid dehydrogenase type 1 (17ß-HSD1) plays a pivotal role in the progression of estrogen-related diseases because of its involvement in the biosynthesis of estradiol (E2), constituting a valuable therapeutic target for endocrine treatment. In the present study, we successfully cocrystallized the enzyme with the reversible inhibitor 2-methoxy-16ß-( m-carbamoylbenzyl)-E2 (2-MeO-CC-156) as well as the enzyme with the irreversible inhibitor 3-(2-bromoethyl)-16ß-( m-carbamoylbenzyl)-17ß-hydroxy-1,3,5(10)-estratriene (PBRM). The structures of ternary complexes of 17ß-HSD1-2-MeO-CC-156-NADP+ and 17ß-HSD1-PBRM-NADP+ comparatively show the formation of a covalent bond between His221 and the bromoethyl side chain of the inhibitor in the PBRM structure. A dynamic process including beneficial molecular interactions that favor the specific binding of a low-reactivity inhibitor and subsequent N-alkylation event through the participation of His221 in the enzyme catalytic site clearly demonstrates the covalent bond formation. This finding opens the door to a new design of alkyl halide-based specific covalent inhibitors as potential therapeutic agents for different enzymes, contributing to the development of highly efficient inhibitors.

Detection and Identification of Estrogen Based on Surface-Enhanced Resonance Raman Scattering (SERRS).

Many studies have shown that it is important to consider the harmful effects of phenolic hormones on the human body. Traditional UV detection has many limitations, so there is a need to develop new detection methods. We demonstrated a simple and rapid surface-enhanced resonance Raman scattering (SERRS) based detection method of trace amounts of phenolic estrogen. As a result of the coupling reaction, there is the formation of strong SERRS activity of azo compound. Therefore, the detection limits are as low as 0.2 × 10-4 for estrone (E1), estriol (E3), and bisphenol A (BPA). This method is universal because each SERRS fingerprint of the azo dyes a specific hormone. The use of this method is applicable for the testing of phenolic hormones through coupling reactions, and the investigation of other phenolic molecules. Therefore, this new method can be used for efficient detection.

Comparative study of substituted poly(4-vinylbenzyl chloride/ethylene glycol dimethacrylate) sorbents for enrichment of selected pharmaceuticals and estrogens from aqueous samples.

This study reports the syntheses of four polymeric sorbents based on nucleophilic substitution of Poly(4-vinylbenzylchloride/ethylene glycol dimethacrylate). Polymerization was executed by a simple thermal initiated bulk polymerization procedure. Ground polymer particles were functionalized through reaction with the nucleophiles triethylamine, imidazole, piperidine and pyrrolidine. Mixed-mode phases were characterized by infrared spectroscopy, nitrogen sorption porosimetry and potentiometric titration for determination of chloride content. Furthermore, materials were tested and evaluated for enrichment of seven pharmaceutical and endocrine-disrupting compounds at low ng mL-1 levels. Results demonstrate that the imidazole modified sorbent led to high and constant recovery rates for nearly all tested compounds. Therefore, this polymer was further tested for applicability with two environmental samples. Spiked tap and river water showed similar results as in evaluation experiments. Moreover, the developed method was validated regarding linearity, repeatability, instrumental limits and stability of analytes according to international guidelines.

Resource efficiency analysis for photocatalytic degradation and mineralization of estriol using TiO2 nanoparticles.

A resource efficiency analysis was developed that evaluated photocatalyst loading and temperature inputs, and assessed hydroxyl radical (OH) production. Catalyst loading (Aeroxide® TiO2 P25) between 1 and 1500 mg L-1 and temperatures between 5 and 50 °C were analyzed as input resources for OH production. After, the best experimental conditions were used to degrade and mineralize estriol (E3). The analysis showed that a low catalyst concentration lead to poor absorption of radiation and a slow reaction. When high catalyst concentrations were tested, most of the radiation was absorbed, which produced results near the top of the slowing rate of OH generation. Temperature was found a relevant resource for increasing interfacial transfer to facilitate OH production following the Arrhenius model. Two indices to measure resource efficiency were proposed: 1) the OH generation index (OHI) and 2) the initial degradation efficiency (IDE). OHI was used to measure the efficiency of a catalyst using photonic flux to generate OH production. IDE evaluated the relationship between the photocatalytic reactor set-up, catalyst, and E3 degradation. It was observed that 1.18 OH was produced when a photon interacts with a photocatalyst particle when a load of 5 mg L-1 of photocatalyst is used at 20 °C. It was found that at initial time, 2.4 OH was generated in the systems to produce a degradation of one E3 molecule when using a photocatalyst load of 20 mg L-1 at 20 °C. Additionally, it was demonstrated that E3 mineralization was feasible under different catalyst loading scenarios.

A novel mechanism of non-feminizing estrogens in neuroprotection.

Estrogens are potent and efficacious neuroprotectants both in vitro and in vivo in a variety of models of neurotoxicity. We determined the structural requirements for neuroprotection in an in vitro assay using a panel of >70 novel estratrienes, synthesized to reduce or eliminate estrogen receptor (ER) binding. We observed that neuroprotection could be enhanced by as much as 200-fold through modifications that positioned a large bulky group at the C2 or C4 position of the phenolic A ring of the estratriene. Further, substitutions on the B, C or D rings either reduced or did not markedly change neuroprotection. Collectively, there was a negative correlation between binding to ERs and neuroprotection with the more potent compounds showing no ER binding. In an in vivo model for neuroprotection, transient cerebral ischemia, efficacious compounds were active in protection of brain tissue from this pro-oxidant insult. We demonstrated that these non-feminizing estrogens engage in a redox cycle with glutathione, using the hexose monophosphate shunt to apply cytosolic reducing potential to cellular membranes. Together, these results demonstrate that non-feminizing estrogens are neuroprotective and protect brain from the induction of ischemic- and Alzheimer's disease (AD)-like neuropathology in an animal model. These features of non-feminizing estrogens make them attractive compounds for assessment of efficacy in AD and stroke, as they are not expected to show the side effects of chronic estrogen therapy that are mediated by ER actions in the liver, uterus and breast.

Immunoanalytical characteristics of unconjugated estriol: indications and analytical performances.

After a short description of the structure and the physiological roles of unconjugated estriol, this paper points out pre-analytical conditions and performances of the serum unconjugated estriol immunoassay, essentially used for Down syndrome screening.

Using Fenton Oxidation to Simultaneously Remove Different Estrogens from Cow Manure.

The presence of estrogens in livestock excrement has raised concerns about their potential negative influence on animals and the overall food cycle. This is the first investigation to simultaneously remove estrogens, including estriol (E3), bisphenol A (BPA), diethylstilbestrol (DES), estradiol (E2), and ethinyl estradiol (EE2), from cow manure using a Fenton oxidation technique. Based on the residual concentrations and removal efficiency of estrogens, the Fenton oxidation reaction conditions were optimized as follows: a H2O2 dosage of 2.56 mmol/g, a Fe(II) to H2O2 molar ratio of 0.125 M/M, a solid to water mass ratio of 2 g/mL, an initial pH of 3, and a reaction time of 24 h. Under these conditions, the simultaneous removal efficiencies of E3, BPA, DES, E2, and EE2, with initial concentrations in cow manure of 97.40, 96.54, 100.22, 95.01, and 72.49 mg/kg, were 84.9%, 99.5%, 99.1%, 97.8%, and 84.5%, respectively. We clarified the possible Fenton oxidation reaction mechanisms that governed the degradation of estrogens. We concluded that Fenton oxidation technique could be effective for efficient removal of estrogens in livestock excrement. Results are of great importance for cow manure reuse in agricultural management, and can be used to reduce the threat of environmental estrogens to human health and ecological safety.

Charge Density and Electrostatic Potential Study of 16α,17ß-Estriol and the Binding of Estrogen Molecules to the Estrogen Receptors ERα and ERß.

An accurate X-ray diffraction study at 20 K combined with DFT theoretical calculations has been performed for the estriol crystal with two conformationally different molecules in the asymmetric unit. The electron density has been modeled via a multipole expansion, using both experimental and theoretical structure factors, and a topological analysis has been performed. The experimental molecular geometry, hydrogen bonding, atomic charges, dipole moments, and other topological characteristics are compared with those calculated theoretically. In particular, the molecular electrostatic potential has been extracted and compared with those reported for other estrogen molecules exhibiting different binding affinities to the estrogen receptors (ERα and ERß).

Photodegradation behaviour of estriol: An insight on natural aquatic organic matter influence.

Estriol (E3) is one of the steroidal estrogens ubiquitously found in the aquatic environment, photodegradation being an important pathway for the elimination of such endocrine disrupting compounds. However, it is important to understand how environmentally important components present in aquatic matrices, such as organic matter, may affect their photodegradation. The main objective of this work was to investigate the photodegradation of E3 in water, under simulated solar radiation, as well as the effect of humic substances (HS - humic acids (HA), fulvic acids (FA) and XAD-4 fraction) in E3 photodegradation. Moreover, the photodegradation behaviour of E3 when present in different environmental aquatic matrices (fresh, estuarine and waste water samples) was also assessed. Results showed a completely different E3 degradation rate depending on the aquatic matrix. In ultrapure water the half-life obtained was about 50 h, while in presence of HS it varied between 5 and 10 h. Then, half-life times between 1.6 and 9.5 h were determined in environmental samples, in which it was observed that the matrix composition contributed up to 97% for the overall E3 photodegradation. Therefore, E3 photodegradation in the considered aquatic matrices was mostly caused by photosensitizing reactions (indirect photodegradation).

Palladium-Catalyzed Ortho-Arylation of Carbamate-Protected Estrogens.

The palladium-catalyzed ortho-arylation of diethyl carbamate-protected estrone and estriol with aryl iodides gives the 2-arylated analogues. Subsequent removal of the carbamate directing group furnishes 2-arylated estrone, estradiol, or estriol depending on the method used.

Photodegradation of estrogenic endocrine disrupting steroidal hormones in aqueous systems: Progress and future challenges.

This article reviews different photodegradation technologies used for the removal of four endocrine disrupting chemicals (EDCs): estrone (E1), 17ß-estradiol (E2), estriol (E3) and 17α-ethinylestradiol (EE2). The degradation efficiency is greater under UV than visible light; and increases with light intensity up to when mass transfer becomes the rate limiting step. Substantial rates are observed in the environmentally relevant range of pH7-8, though higher rates are obtained for pH above the pKa (~10.4) of the EDCs. The effects of dissolved organic matter (DOM) on EDC photodegradation are complex with both positive and negative impacts being reported. TiO2 remains the best catalyst due to its superior activity, chemical and photo stability, cheap commercial availability, capacity to function at ambient conditions and low toxicity. The optimum TiO2 loading is 0.05-1gl(-1), while higher loadings have negative impact on EDC removal. The suspended catalysts prove to be more efficient in photocatalysis compared to the immobilised catalysts, while the latter are considered more suitable for commercial scale applications. Photodegradation mostly follows 1st or pseudo 1st order kinetics. Photodegradation typically eradicates or moderates estrogenic activity, though some intermediates are found to exhibit higher estrogenicity than the parent EDCs; the persistence of estrogenic activity is mainly attributed to the presence of the phenolic moiety in intermediates.

Performance of metal-organic framework MIL-101 after surfactant modification in the extraction of endocrine disrupting chemicals from environmental water samples.

The research presented in this paper explored the modification and application of a metal-organic framework, MIL-101, with nonionic surfactant-Triton X-114 in dispersive solid-phase extraction for the preconcentration of four endocrine disrupting chemicals (estrone, 17α-ethynylestradiol, estriol and diethylstilbestrol) from environmental water samples. Triton X-114 molecules could be adsorbed by the hydrophobic surface of the MIL-101 crystals, and thus improved the dispersibility of MIL-101 in aqueous solution by serving as a hydrophilic coating. Cloud point phase separation from Triton X-114 accelerated the separation of extracts from the aqueous matrix. The proposed method combines the favorable attributes of strong adsorption capacity resulting from the porous structure of MIL-101 and self-assembly of Triton X-114 molecules. Post-extraction derivatization using N-methyl-N-(trimethylsilyl)trifluoroacetamide was employed to facilitate the quantitative determination of the extracts by gas chromatography-mass spectrometry. The main factors affecting the preparation of modified MIL-101, and extraction of the analytes, such as the amount of surfactant, the ultrasonic and vortex durations, solution pH and desorption conditions, were investigated in detail. Under the optimized conditions, the present method yielded low limits of detection (0.006-0.023 ng/mL), good linearity from 0.09 to 45 ng/mL (coefficients of determination higher than 0.9980) and acceptable precision (relative standard deviations of 2.2-13%). The surface modified MIL-101 was demonstrated to be effective for the extraction of the selected estrogens from aqueous samples, giving rise to markedly improved extraction performance compared to the unmodified MIL-101.