The adsorption, kinetics, and interaction mechanisms of various types of estrogen on electrospun polymeric nanofiber membranes.

This study focuses on the adsorption kinetics of four highly potent sex hormones (estrone (E1), 17ß-estradiol (E2), 17α-ethinylestradiol (EE2), and estriol (E3)), present in water reservoirs, which are considered a major cause of fish feminization, low sperm count in males, breast and ovarian cancer in females induced by hormonal imbalance. Herein, electrospun polymeric nanostructures were produced from cellulose acetate, polyamide, polyethersulfone, polyurethanes (918 and elastollan), and polyacrylonitrile (PAN) to simultaneously adsorbing these estrogenic hormones in a single step process and to compare their performance. These nanofibers possessed an average fiber diameter in the range 174-330 nm and their specific surface area ranged between 10.2 and 20.9 m2g-1. The adsorption-desorption process was investigated in four cycles to determine the effective reusability of the adsorption systems. A one-step high-performance liquid chromatography technique was developed to detect and quantify concurrently each hormone present in the solution. Experimental data were obtained to determine the adsorption kinetics by applying pseudo-first-order, pseudo-second-order and intraparticle diffusion models. Findings showed that E1, E2 and EE2 best fitted pseudo-second-order kinetics, while E3 followed pseudo-first-order kinetics. It was found that polyurethane Elastollan nanofibers had maximum adsorption capacities of 0.801, 0.590, 0.736 and 0.382 mg g-1for E1, E2, EE2 and E3, respectively. In addition, the results revealed that polyurethane Elastollan nanofibers had the highest percentage efficiency of estrogens removal at ∼58.9% due to its strong hydrogen bonding with estrogenic hormones, while the least removal efficiency for PAN at ∼35.1%. Consecutive adsorption-desorption cycles demonstrated that polyurethane maintained the best efficiency, even after being repeatedly used four times compared to the other polymers. Overall, the findings indicate that all the studied nanostructures have the potential to be effective adsorbents for concurrently eradicating such estrogens from the environment.

The Effect of a Synthetic Estrogen, Ethinylestradiol, on the hERG Block by E-4031.

Inhibition of K+-conductance through the human ether-a-go-go related gene (hERG) channel leads to QT prolongation and is associated with cardiac arrhythmias. We previously reported that physiological concentrations of some estrogens partially suppress the hERG channel currents by interacting with the S6 residue F656 and increase the sensitivity of hERG blockade by E-4031. Although these studies suggested that clinically used synthetic estrogens with similar structures have the marked potential to alter hERG functions, the hERG interactions with synthetic estrogens have not been assessed. We therefore examined whether ethinylestradiol (EE2), a synthetic estrogen used in oral contraceptives, affects hERG function and blockade by drugs. Supratherapeutic concentrations of EE2 did not alter amplitudes or kinetics of the hERG currents elicited by train pulses at 20 mV (0.1 Hz). On the other hand, EE2 at therapeutic concentrations reduced the degree of hERG current suppression by E-4031. The administration of EE2 followed by E-4031 blockade reversed the current suppression, suggesting that the interaction of EE2 and E-4031 alters hERG at the drug-binding site. The effects of EE2 on hERG blockade raised the possibility that other estrogens, including synthetic estrogens, can alter hERG blockade by drugs that cause QT prolongation and ventricular arrhythmias.

ERRγ ligand HPB2 upregulates BDNF-TrkB and enhances dopaminergic neuronal phenotype.

Brain derived neurotrophic factor (BDNF) promotes maturation of dopaminergic (DAergic) neurons in the midbrain and positively regulates their maintenance and outgrowth. Therefore, understanding the mechanisms regulating the BDNF signaling pathway in DAergic neurons may help discover potential therapeutic strategies for neuropsychological disorders associated with dysregulation of DAergic neurotransmission. Because estrogen-related receptor gamma (ERRγ) is highly expressed in both the fetal nervous system and adult brains during DAergic neuronal differentiation, and it is involved in regulating the DAergic neuronal phenotype, we asked in this study whether ERRγ ligand regulates BDNF signaling and subsequent DAergic neuronal phenotype. Based on the X-ray crystal structures of the ligand binding domain of ERRγ, we designed and synthesized the ERRγ agonist, (E)-4-hydroxy-N'-(4-(phenylethynyl)benzylidene)benzohydrazide (HPB2) (Kd value, 8.35 µmol/L). HPB2 increased BDNF mRNA and protein levels, and enhanced the expression of the BDNF receptor tropomyosin receptor kinase B (TrkB) in human neuroblastoma SH-SY5Y, differentiated Lund human mesencephalic (LUHMES) cells, and primary ventral mesencephalic (VM) neurons. HPB2-induced upregulation of BDNF was attenuated by GSK5182, an antagonist of ERRγ, and siRNA-mediated ERRγ silencing. HPB2-induced activation of extracellular-signal-regulated kinase (ERK) and phosphorylation of cAMP-response element binding protein (CREB) was responsible for BDNF upregulation in SH-SY5Y cells. HPB2 enhanced the DAergic neuronal phenotype, namely upregulation of tyrosine hydroxylase (TH) and DA transporter (DAT) with neurite outgrowth, both in SH-SY5Y and primary VM neurons, which was interfered by the inhibition of BDNF-TrkB signaling, ERRγ knockdown, or blockade of ERK activation. HPB2 also upregulated BDNF and TH in the striatum and induced neurite elongation in the substantia nigra of mice brain. In conclusion, ERRγ activation regulated BDNF expression and the subsequent DAergic neuronal phenotype in neuronal cells. Our results might provide new insights into the mechanism underlying the regulation of BDNF expression, leading to novel therapeutic strategies for neuropsychological disorders associated with DAergic dysregulation.

UPLC-MS/MS determination of steroid hormones via a novel reaction based on derivatisation by a cyclic-organophosphate.

A cyclic-organophosphate, specifically 2-chloro-5,5-dimethyl-1,3,2-dioxaphosphorinane-2-oxide, was used to derivatise the hydroxyl group at the C3 position of selected steroid hormones to analyse the derivatives using UPLC-MS/MS (ultra-performance liquid chromatography-tandem mass spectrometry). Reactions were performed in an anhydrous pyridine environment in the presence of AlCl3 at 50 °C. The developed reaction is suitable for analytical chemistry applications and was validated by analysis of selected contraceptive drugs. The sensitivity of the method depends on hormone tested and the limit of detection ranges from 130 pg/mL for ß-estradiol to 240 pg/mL for estriol. The estimated efficiency of derivatisation reactions varies in the range from 77.5 to 95.7%, and depends upon the hormone undergoing derivatisation. The method's recovery rate for the lowest concentration tested (800 pg/mL) is 88.1-96.3%. The method exhibits linearity in the 390 pg/mL to 2.5 µg/mL range, with R2 = 0.997. The developed steroid hormone derivatisation reaction was validated experimentally using UHPLC-QTOF-MS (ultra-high performance liquid chromatography quadrupole time of flight mass spectrometry) and NMR (nuclear magnetic resonance) spectroscopy. These studies show that the developed derivatisation reaction provides a precise and repeatable determination of selected steroid hormones in contraceptive drugs. At n = 10, CV (Coefficient of Variation) did not exceed 7%, which is a very good result compared with other analytical methods.

Adsorption of Trace Estrogens in Ultrapure and Wastewater Treatment Plant Effluent by Magnetic Graphene Oxide.

In the current study, graphene oxide, Fe3+, and Fe2+ were used for the synthesis of magnetic graphene oxide (MGO) by an in situ chemical coprecipitation method. Scanning electron microscopy, transmission electron microscopy, Fourier transform infrared spectroscopy, and X-ray diffraction were used to characterize the well-prepared MGO. The prepared MGO was used as an adsorbent to remove five typical estrogens (estrone (E1), 17β-estradiol (E2), 17α-ethinylestradiol (17α-E2), estriol (E3), and synthetic estrogen (EE2)) at the ppb level from spiked ultrapure water and wastewater treatment plant effluent. The results indicated that the MGO can efficiently remove estrogens from both spiked ultrapure water and wastewater treatment plant effluent in 30 min at wide pH ranges from 3 to 11. The temperature could significantly affect removal performance. A removal efficiency of more than 90% was obtained at 35 °C in just 5 min, but at least 60 min was needed to get the same removal efficiency at 5 °C. In addition, an average of almost 80% of the estrogens can still be removed after 5 cycles of MGO regeneration but less than 40% can be reached after 10 cycles. These results indicate that MGO has potential for practical applications to remove lower levels of estrogens from real water matrixes and merits further evaluation.

Oxidation of steroid estrogens by peroxymonosulfate (PMS) and effect of bromide and chloride ions: Kinetics, products, and modeling.

Recently, in situ chemical oxidation (ISCO) using peroxymonosulfate (PMS) for environmental decontamination has received increasing interest. In this study, oxidation kinetics and products of four steroid estrogens (i.e., estrone, 17ß-estradiol, estriol, and 17α-ethinylestradiol) by PMS under various conditions were investigated. PMS could fairly degrade steroid estrogens over the pH range of 7-10, and the degradation rate increased with the increase of solution pH. This pH-dependence was well described by parallel reactions between individual acid-base species of steroid estrogens (E and E-) and PMS (HSO5- and SO52-), where specific second-order rate constants for E- with HSO5- and SO52- were in the range of 2.11-5.58 M-1s-1 and 0.77-1.25 M-1s-1, respectively. Identification of oxidation products by liquid chromatography/electrospray ionization quadrupole time-of-flight mass spectrometer showed that PMS readily oxidized the phenolic group of steroid estrogens, leading to the generation of hydroxylated and ring-opening products. The presence of bromide and chloride ions (Br- and Cl-) at environmentally relevant levels could greatly accelerate the degradation of steroid estrogens by PMS with the formation of halogenated aromatic products. This effect was quantitatively estimated by a kinetic model, where the formation of free bromine and chorine and their rapid electrophilic substitution with steroid estrogens were taken into consideration. Eco-toxicity of transformation products of 17α-ethinylestradiol by PMS treatment in the absence and presence of bromide and chloride was estimated by quantitative structure-activity relationship analysis using ECOSAR. These findings advance the understanding of ISCO using PMS.

High-Performance Prediction of Human Estrogen Receptor Agonists Based on Chemical Structures.

Many agonists for the estrogen receptor are known to disrupt endocrine functioning. We have developed a computational model that predicts agonists for the estrogen receptor ligand-binding domain in an assay system. Our model was entered into the Tox21 Data Challenge 2014, a computational toxicology competition organized by the National Center for Advancing Translational Sciences. This competition aims to find high-performance predictive models for various adverse-outcome pathways, including the estrogen receptor. Our predictive model, which is based on the random forest method, delivered the best performance in its competition category. In the current study, the predictive performance of the random forest models was improved by strictly adjusting the hyperparameters to avoid overfitting. The random forest models were optimized from 4000 descriptors simultaneously applied to 10,000 activity assay results for the estrogen receptor ligand-binding domain, which have been measured and compiled by Tox21. Owing to the correlation between our model's and the challenge's results, we consider that our model currently possesses the highest predictive power on agonist activity of the estrogen receptor ligand-binding domain. Furthermore, analysis of the optimized model revealed some important features of the agonists, such as the number of hydroxyl groups in the molecules.

Advanced oxidative processes and membrane separation for micropollutant removal from biotreated domestic wastewater.

The presence of micropollutants in sewage is already widely known, as well as the effects caused by natural and synthetic hormones. Thus, it is necessary to apply treatments to remove them from water systems, such as advanced oxidation processes (AOPs) and membrane separation processes, which can oxidize and remove high concentrations of organic compounds. This work investigated the removal of 17ß-estradiol (E2), 17α-ethinylestradiol (EE2), and estriol (E3) from biotreated sewage. Reverse osmosis processes were conducted at three recoveries (50, 60, and 70 %). For E2 and EE2, the removals were affected by the recovery. The best results for RO were as follows: the E2 compound removal was 89 % for 60 % recovery and the EE2 compound removal was 57 % for 50 % recovery. The RO recovery did not impact the E3 removal. It was concluded that the interaction between the evaluated estrogens, and the membrane was the major factor for the hormone separation. The AOP treatment using H2O2/UV was carried out in two sampling campaigns. First, we evaluated the variation of UV doses (24.48, 73.44, 122.4, and 244.8 kJ m-2) with 18.8 mg L-1 of H2O2 in the reaction. EE2 showed considerable removals (around 70 %). In order to optimize the results, an experimental design was applied. The best result was obtained with higher UV dose (122.4 kJ m-2) and lower H2O2 concentration (4 mg L-1), achieving removal of 91 % for E3 and 100 % for E2 and EE2.

The role and impact of estrogens and xenoestrogen on the development of cervical cancer.

Throughout an individual's lifetime, the human body is exposed to many different chemical compounds, including xenoestrogens (XEs) that can be found in the environment, food, air, cosmetics and other substances, which have a positive or negative impact on their health and lifestyle. Whereas high-risk human papillomavirus (HR-HPV) is necessary but not sufficient for full malignant cervical cell transformation, other compounds such as estrogens and XEs may be risk factors for cervical cancer (CC) development. The causes and effects of some diseases such as cancer, cardiovascular, metabolic or immune system disorders are partly due to signaling pathways in response to estrogens. XEs are a vast group of natural and synthetic compounds, behaving like estrogens, that have been studied over the recent years and which may interact with estrogen receptors. The major problem with XEs is the difficulty in studying the mechanism of such complex substances as well as investigating the influences of some of the compounds (dose-dependent) over time. The impact of XEs on CC is variable, with no direct comparison between in vitro studies and in vivo XEs action.

Biomagnification of endocrine disrupting chemicals (EDCs) by Pleuronectes yokohamae: Does P. yokohamae accumulate dietary EDCs?

We evaluated the potential for biomagnification of endocrine disrupting chemicals (EDCs) such as nonylphenol (NP), octylphenol (OP), bisphenol A (BP), and natural estrogens such as estrone (E1) and 17ß-estradiol (E2) in a benthic fish, Pleuronectes yokohamae. The assimilation efficiencies (AE) of most EDCs ranged from 88 to 96% suggesting that they were efficiently incorporated and assimilated into P. yokohamae, except for NP (50%). However, the biomagnification factor (BMF) values were <1.0 suggesting that the compounds were not biomagnifying. Additionally, three of the target EDCs were not detected (BP, E1 and E2). Glucuronidation activity towards BP (11.44 ± 2.5 nmol/mg protein/min) and E2 (12.41 ± 3.2 nmol/mg protein/min) was high in the intestine suggesting that EDCs were glucuronidated prior to excretion into bile. Thus, we conclude that biomagnification of dietary EDCs is reduced in P. yokohamae because of effective glucuronidation.

[Study of osteoprotective and hypolipidemic effects of estrogen 8α-analogues].

The aim of this work was to study the ability of some estrogen 8α-analogues, that have CH3-group in the C-3 position, exhibit osteoprotective and cholesterolemic effects. The properties of these analogues was comparisoned with effects of native estradiol and 17α-ethynylestradiol (EE). We showed that compounds 3 ((d,l)-17ß-acethoxy-3-methoxy-8α-estra-1,3,5(10)-triene) and 4 ((d,l)-3-methoxy-8α-estra-1,3,5(10)-triene-17-one) had the same osteoprotective and cholesterolemic effects as EE. The utherotropic effects of compound 3 and EE were the same, while the utherotropic activity of 17-keto derivative (compound 4) was higher than effect of EE. The osteoprotective and cholesterolemic effects of compounds 5 and 6 (d- or l-17ß-acethoxy-3-methoxy-13-ethyl-8α-gone-1,3,5(10)-triene) were approximately the same, however the utherotropic action of these compounds was different: the compound 5 had significantly lower activity, but the compound 6 had the same effect in comparison with EE. Thus, all studied estrogen 8α-analogues may be used as basic constructions for structural modifications which is necessary as medications with while spectrum of biological properties.

Simultaneous Determination of Naturally Occurring Estrogens and Mycoestrogens in Milk by Ultrahigh-Performance Liquid Chromatography-Tandem Mass Spectrometry Analysis.

A simple, fast, and reproducible method for the simultaneous determination of natural estrogens and mycoestrogens (resorcylic acid lactones) in milk by ultrahigh-performance liquid chromatography combined with electrospray ionization triple quadrupole tandem mass spectrometry (UHPLC/ESI-MS/MS) is described. The extraction was carried out by solid-phase extraction (SPE) using graphitized carbon black as solid sorbent. The use of carbon black allowed us to avoid any type of sample pretreatment, and the extraction was performed simply by diluting milk samples in water. Correlation coefficient values were obtained in the range between 0.9991 and 1, with good recoveries (67-107% at the lowest spiked level), repeatability (4.8-16.8%), and reproducibility (3.2-16.3%). Moreover, a very low matrix effect was observed for both estrogens and mycoestrogens. With respect to a previous method based on SPE with Oasis MAX cartridges, the one here described allowed us to detect all the analytes under investigation, at the lowest tested concentration level, including free estrogens (in particular estriol). Finally, the developed UHPLC/ESI-MS/MS method was applied to the analysis of some whole milk samples from different lactating animals (cow, goat, and donkey) as well as ultrahigh-temperature-treated cow milk and powder milk samples.

Mass spectrometry evidence for formation of estrogen-homocysteine conjugates: estrogens can regulate homocysteine levels.

Homocysteine (HCys), a sulfur-containing amino acid, is formed during the metabolism of methionine. An imbalance between the rate of production and the use of HCys during methionine metabolism can result in an increase in the plasma and urinary levels of HCys. HCys has been shown to be toxic to vascular endothelial cells through several pathways. Many earlier clinical studies have revealed an association between plasma HCys and cardiovascular and other diseases. In contrast, estrogens are suggested to lower the risk of cardiovascular disease. Several studies indicate that estrogen metabolites could be responsible for cardiovascular protection. It has been demonstrated that electrophilic estrogen quinones, E1(E2)-2,3-Q and E1(E2)-3,4-Q, can alkylate DNA as well as form conjugates with glutathione. I hypothesize that estrogen quinones generated in situ by oxidative enzymes, metal ions, or molecular oxygen can interact with HCys to form conjugates. This in turn could lower the levels of toxic HCys as well as quenching the reactive estrogen quinones, resulting in cardiovascular protective effects. To test the feasibility of a protective estrogen-HCys pathway, estrogen quinones were treated with HCys. Tandem mass spectrometry analysis of the assay mixture shows the formation of estrogen-HCys conjugates. Furthermore, incubation of catechol estrogens with myeloperoxidase (MPO) in the presence of HCys resulted in the formation of respective estrogen-HCys conjugates. The identities of estrogen-HCys conjugates in MPO assay extracts were confirmed by comparing them to pure synthesized estrogen-HCys standards. I propose that through conjugation estrogens could chemically regulate HCys levels; moreover these conjugates could be used as potential biomarkers in determining health.

Hollow-fiber liquid-phase microextraction for the determination of natural and synthetic estrogens in milk samples.

In this work, a group of nine estrogens, four of them being natural (estriol, 17ß-estradiol, 17α-estradiol and estrone), four being synthetic (17α-ethynylestradiol, diethylstibestrol, dienestrol and hexestrol) and one metabolite (2-hydroxyestradiol) have been extracted and preconcentrated from milk samples with different fat content (whole, semi-skimmed and skimmed). After protein precipitation with acetonitrile containing acetic acid, evaporation of the supernatant and reconstitution of the residue in water, hollow-fiber liquid-phase microextraction (HF-LPME) using 1-octanol as extraction solvent was applied to further preconcentrate the analytes. Separation, determination and quantification were achieved by high-performance liquid chromatography coupled to a diode array detector and a fluorescence detector set in series. Deproteinization conditions, as well as parameters affecting the extraction efficiency in HF-LPME (pH of the sample, ionic strength, extraction time, stirring speed, temperature and desorption conditions) were investigated and optimized. Calibration, precision and accuracy studies were carried out to validate the methodology in different types of milk providing LODs in the low µg/L range.

Carbon isotopic (13C and 14C) composition of synthetic estrogens and progestogens.

RATIONALE: Steroids are potent hormones that are found in many environments. Yet, contributions from synthetic and endogenous sources are largely uncharacterized. The goal of this study was to evaluate whether carbon isotopes could be used to distinguish between synthetic and endogenous steroids in wastewater and other environmental matrices. METHODS: Estrogens and progestogens were isolated from oral contraceptive pills using semi-preparative liquid chromatography/diode array detection (LC/DAD). Compound purity was confirmed by gas chromatography/flame ionization detection (GC/FID), gas chromatography/time-of-flight mass spectrometry (GC/TOF-MS) and liquid chromatography/mass spectrometry using negative electrospray ionization (LC/ESI-MS). The (13)C content was determined by gas chromatography/isotope ratio mass spectrometry (GC/IRMS) and (14)C was measured by accelerator mass spectrometry (AMS). RESULTS: Synthetic estrogens and progestogens are (13)C-depleted (δ(13)C(estrogen) = -30.0 ± 0.9 ‰; δ(13)C(progestogen) = -30.3 ± 2.6 ‰) compared with endogenous hormones (δ(13)C ~ -16 to -26 ‰). The (14)C content of the majority of synthetic hormones is consistent with synthesis from C(3) plant-based precursors, amended with 'fossil' carbon in the case of EE(2) and norethindrone acetate. Exceptions are progestogens that contain an ethyl group at carbon position 13 and have entirely 'fossil' (14)C signatures. CONCLUSIONS: Carbon isotope measurements have the potential to distinguish between synthetic and endogenous hormones in the environment. Our results suggest that (13)C could be used to discriminate endogenous from synthetic estrogens in animal waste, wastewater effluent, and natural waters. In contrast, (13)C and (14)C together may prove useful for tracking synthetic progestogens.

A quantitative method evaluating the selective adsorption of molecularly imprinted polymer.

Adsorption isotherms of 4 estrogenic compounds, estrone, 17ß-estradiol, 17α-ethinylestradiol and Bisphenol A, using molecularly imprinted polymer were studied. The isotherms can be simulated by Langmuir model. According to the adsorption isotherms and the template's mass balance, an experimental concept, selective adsorption ratio, SAR, was proposed to assess how many template molecules extracted out of MIP could create selective binding sites. The SAR of the molecularly imprinted polymer was 74.3% for E2. This concept could be used to evaluate quantitatively the selective adsorption.

A multiplexed screening method for agonists and antagonists of the estrogen receptor protein.

The estrogen receptor (ER) is regarded as a significant drug target because of its important physical and pathological function. In this article, we describe a novel screening method to obtain agonists and antagonists of ER. ER was immobilized onto an aldehyde-modified glass slide. The affinity of Cy3-labeled estradiol for ER protein microarrays was then determined. Two libraries, one containing 29 synthetic compounds and the other with 384 natural products that served as a model, were screened to find new ligands for ER. The IC(50) values obtained for tamoxifen and raloxifene were consistent with those found in the literature (4.85 × 10(-7) M versus 1.74~4.23 × 10(-7) M and 7.58 × 10(-8) M versus 0.89~5.84 × 10(-8) M, respectively). Finally, 65 active ligands (5 synthetic compounds and 60 natural products) of ER were identified. This novel method gave identical results to a conventional fluorescence polarization assay, thus verifying the accuracy of this simultaneous multireceptor screening method based on protein microarrays. The presented method is sensitive, accurate, and reliable, and shows great potential for use in high-throughput drug-screening research.

The structures and inhibitory effects of Buame [N-(3-hydroxy-1,3,5(10)-estratrien-17ß-yl)-butylamine] and Diebud [N,N'-bis-(3-hydroxy-1,3,5(10)-estratrien-17ß-yl)-1,4-butanediamine] on platelet aggregation.

Compounds with estrogenic effects that also inhibit platelet aggregation might be useful in reducing thrombotic events associated with estrogenic therapy. In this study, two aminoestrogens, Buame [N-(3-hydroxy-1,3,5(10)-estratrien-17ß-yl)-butylamine] and Diebud [N,N'-bis-(3-hydroxy-1,3,5(10)-estratrien-17ß-yl)-1,4-butanediamine], were synthesized and characterized using common analytical methods and spectrophotometric analyses. The location and orientation of these molecules on the estrogenic receptor α (ERα) were also evaluated. Platelet inhibitory effects were elucidated ADP-induced platelet aggregation and ADP- and collagen-induced ATP release. Molecular docking demonstrated that Buame can reach and bind to the ERα in the ligand binding domain (LBD) similar to 17ß-estradiol (co-crystallized ligand). On the other hand, Diebud binds only to the surface of ERα due to its high molecular volume compared to 17ß-estradiol and Buame.

Chemical kinetics and interactions involved in horseradish peroxidase-mediated oxidative polymerization of phenolic compounds.

The primary objective of this research was to evaluate various factors that affect the reaction rate of oxidative coupling (OXC) reaction of phenolic estrogens catalyzed by horseradish peroxidase (HRP). Kinetic parameters were obtained for the conversion of phenol as well as natural and synthetic estrogens estrone (E(1)), 17ß-estradiol (E(2)), estriol (E(3)), and 17α-ethinylestradiol (EE(2)). Molecular orbital theory and Autodock software were employed to analyze chemical properties and substrate binding characteristics. Reactions were first order with respect to phenolic concentration and reaction rate constants (k(r)) were determined for phenol, E(3), E(1), E(2) and EE(2) (in increasing order). Oxidative coupling was controlled by enzyme-substrate interactions, not collision frequency. Docking simulations show that higher binding energy and a shorter binding distance both promote more favorable kinetics. This research is the first to show that the OXC of phenolics is an entropy-driven and enthalpy-retarded process.

Degradation of 17α-ethinylestradiol by ozonation--identification of the by-products and assessment of their estrogenicity and toxicity.

The presence of the synthetic estrogen 17α-ethinylestradiol (EE2) in waters at low levels is a concern due to its ability to act as an endocrine disruptor. Ozone (O(3)) is commonly used in water treatment and reacts with EE2 to form by-products having characteristics that are mostly unknown. The aim of this study was to identify the by-products of E2 and EE2 ozonation and determine their estrogenicity and toxicity relative to the parent compound. Ozonation by-products were identified via LC-MS analysis. The estrogenicity was measured using the YES assay, and toxicity was determined by monitoring effects on histology of fetal rat testes and testosterone secretion by these tissues. Two EE2 by-products were identified with open phenolic ring structures (masses 302 and 344 u). The Yeast Estrogen Screening (YES) assay showed a decreased but incomplete removal of estrogenicity after ozonation of EE2. Histological analysis of fetal testes revealed that neither E2 nor EE2, with or without ozonation, had any effect on seminiferous cord formation; however, a remarkable negative effect on testosterone secretion was observed, with EE2 by-products after ozonation showing the most rapid and extensive inhibition. These results show that the removal of EE2 via reaction with O(3) resulted in the formation of by-products that are less estrogenic (as demonstrated by the YES assay), but have a greater negative impact on testosterone secretion. Thus, the disappearance of the parent compound is not a sufficient endpoint, as the by-products created may be more toxic. Care should be taken when implementing oxidation applications such as ozone during waste water treatment.