Yeast Estrogen Screen Assay Research Articles

Simultaneous removal of E1, E2, EE2 and levonorgestrel from water using TiO2 catalyst anchored on activated carbon: Processes optimization, materials characterization, and assessment of the estrogenicity reduction

Removal of estrogen hormones from water matrices is crucial owing to its adverse effects on aquatic ecosystems and human health. The present investigation applies an advanced approach to assess the effectiveness of combined processes (adsorption and visible-light-driven photo-degradation) for simultaneous removal of estrone (E1), 17β-estradiol (E2), 17α-ethinylestradiol (EE2), and levonorgestrel (LEVO) in water, applying TiO2-activated carbon composite selected through design of experiment (DoE) for process optimization. Based on a central composite rotatable design (CCRD), composites were synthesized with percentages of activated carbon (AC) ranging from 2.93% to 17.07% (wt.) and calcination temperatures between 259 and 541 °C. The composite with the best performance TiO2-AC15%-541 (15% wt. AC, calcined at 541 °C), achieved a total removal of 98.3 ± 1.15% for E1, 99.0 ± 1% for E2, 99.3 ± 1.15% for EE2, and 96.0 ± 2.65% for LEVO at the initial concentration of 100 μg L−1 under simulated solar irradiation. Further optimization using once more CCRD involved three independent variables: pH; hormone concentration/TiO2-AC15%-541 loading ratio; and the intensity of simulated solar irradiation. Under optimized conditions (pH 2.64, hormone concentration/TiO2-AC15%-541 loading ratio of 3.8 mg g−1, and irradiation intensity of 41 W m−2 UV-A), the TiO2-AC15%-541 composite removed 99.8% of E1, 99.8% of E2, 99.0% of EE2, and 92.1% of LEVO. Furthermore, the process achieved a 99.9% reduction in estrogenic activity, assessed with yeast estrogen screen (YES) assay. These results demonstrate that TiO2-AC15%-541 is an efficient and cost-effective remediation agent for treating mixed estrogen compounds in water, with significant potential for commercial applications.

The use of in vitro bioassays and chemical screening to assess the impact of a minimally processed vegetable facility on wastewater quality.

Fruit- and vegetable-processing facilities may contaminate wastewater via contaminants found in the produce and disinfecting chemicals used. These contaminants may include agrochemicals, pesticides, and disinfectants such as chlorine and quaternary ammonium compounds (QACs). Some compounds may exhibit harmful endocrine-disrupting activity. This study investigated the impact of a minimally processed vegetable facility on wastewater quality via in vitro bioassays and chemical screening. Estrogen activity was assessed via a yeast estrogen screen (YES), and (anti-)androgenic and glucocorticoid activities were evaluated via an MDA-kb2 reporter gene assay. The samples were screened via gas and liquid chromatography-tandem mass spectrometry (GC-MS/MS and LC-MS/MS) to identify target compounds, and GC coupled with time-of-flight mass spectrometry (GC-TOFMS) was used for non-targeted screening. Sample complexity and chemical profiles were assessed using GC-TOFMS. Estrogenic activity was detected in 16 samples (n = 24) with an upper limit of 595 ± 37ng/L estradiol equivalents (EEqs). The final wastewater before discharge had an EEq of 0.23ng/L, which is within the ecological effect-based trigger value range for the estrogenic activity of wastewater (0.2-0.4ng/L EEq). Androgenic activity was detected in one sample with a dihydrotestosterone equivalent (DHTEq) value of 10 ± 2.7ng/L. No antiandrogenic activity was detected. The GC-MS/MS and LC-MS/MS results indicated the presence of multiple pesticides, nonylphenols, triclocarban, and triclosan. Many of these compounds exhibit estrogenic activity, which may explain the positive YES assay findings. These findings showed that wastewater from the facility contained detergents, disinfectants, and pesticides and displayed hormonal activity. Food-processing facilities release large volumes of wastewater, which may affect the quality of the water eventually being discharged into the environment. We recommend expanding conventional water quality monitoring efforts to include additional factors like endocrine activity and disinfectant byproducts.

Estrogenic activity in wastewater treatment plants through in vitro effect-based assays: Insights into extraction phase

Effluents of wastewater treatment plants can abundantly spread endocrine disrupting chemicals in the environment. To improve water quality monitoring, the use of effect-based tools that measure estrogenic activity has been suggested, however their results could be influenced by different factors.This study compared the estrogenic activity of wastewater samples extracted with two stationary phases and tested with two in vitro effect-based assays to investigate whether and how stationary phases and assays could influence biomonitoring data.During four seasonal periods, the effluents of six WWTPs located in northern Italy were sampled. After the extraction using two different stationary phases (HLB, C18), the samples (n = 72) were tested using two effect-based assays: a gene reporter luciferase assay on mammalian cells (MELN) and yeast estrogen screen assay (YES).The results showed that estrogenic activity of HLB extracts was significantly different from the activity of C18 extracts, suggesting that extraction phase can influence biomonitoring data. Moreover, the estrogenic activity was overall higher using gene reporter MELN assay than using YES assay, suggesting that, due to difference in cell membrane permeability and metabolic activation, the applied cell model can affect the biomonitoring results. Finally, from the comparison between the activity of the final effluent and the environmentally safe estrogenic levels in surface waters, MELN data suggested that the activity of this effluent may pose an environmental risk, while YES data showed that it should not be considered a threat to the receiving surface waters.This study pointed out that a standardized approach is needed to assess the estrogenic activity of waters; it reported important data to select the most suitable stationary phase for samples extraction (samples extracted with C18 sorbent showed higher estradiol equivalent concentration values) and the most appropriate bioassay (gene reporter luciferase MELN assay was more sensitive than YES assay) to assess the environmental risk, thus protecting human health.

Microcarrier-based fluorescent yeast estrogen screen assay for fast determination of endocrine disrupting compounds

The presence of endocrine-disrupting compounds (EDCs) in water poses a significant threat to human and animal health, as recognized by regulatory agencies throughout the world. The Yeast Estrogen Screen (YES) assay is an excellent method to evaluate the presence of these compounds in water due to its simplicity and capacity to assess the bioaccessible forms/fractions of these compounds. In the presence of a compound with estrogenic activity, Saccharomyces cerevisiae cells, containing a lacZ reporter gene encoding the enzyme β-galactosidase, are induced, the enzyme is synthesised, and released to the extracellular medium. In this work, a YES-based approach encompassing the use of a lacZ reporter gene modified strain of S. cerevisiae, microcarriers as solid support, and a fluorescent substrate, fluorescein di-β-d-galactopyranoside, is proposed, allowing for the assessment of EDCs’ presence after only 2 h of incubation. The proposed method provided an EC50 of 0.17 ± 0.03 nM and an LLOQ of 0.03 nM, expressed as 17β-estradiol. The assessment of different EDCs provided EC50 values between 0.16 and 1.2 × 103 nM. After application to wastewaters, similar results were obtained for EDCs screening, much faster, compared to the conventional 45 h spectrophotometric procedure using a commercial kit, showing potential for onsite high-throughput screening of environmental contamination.

Yeast estrogen screen assay applied in the assessment of estrogenic activity removal from dairy cattle wastewater treated by anaerobic digestion

The presence of estrogen-like endocrine disrupting chemicals (EEDC) in surface waters, soils, and groundwater has been frequently reported in the literature. Since these compounds can be excreted by humans and animals, the release of domestic sewage and animal wastewaters may be an important source of EEDC in the environment. In this context, there is a growing concern regarding the presence of these substances in the environment and their potential adverse effects on human health. However, most studies have been focused on the presence of EEDC in domestic wastewaters and the available information regarding EEDC in livestock wastewater is still limited. Therefore, this work aimed to quantify the estrogenic activity (EA) in the liquid phase of dairy cattle wastewaters and evaluate the removal of EA during anaerobic digestion, applying an adapted methodology based on the yeast estrogen screen (YES) assay, which has been widely used to analyze EA in domestic sewage. Influent and effluent dairy cattle wastewater samples from a full-scale anaerobic digester placed in an experimental farm were collected during seven months. EA concentrations in the liquid phase of raw wastewater ranged from 228 to 2182 ng.L−1 E2-eq. Anaerobic digestion was not efficient in EA removal, achieving effluent concentrations ranging from 109 to 946 ng.L−1 E2-eq. Results also indicate that the application of an estradiol-based fixed time artificial insemination protocol did not statistically contribute to the increase of EA concentrations in dairy cattle wastewater which reuse in cleaning of animal feeding operation facilities and fertigation are beneficial and economically advantageous to dairy production systems, reducing water demand in parallel to promoting organic matter and nutrient recycling. However, the potential environmental and health risks associated with the presence of EEDC in dairy cattle wastewaters should be better elucidated and evaluated.

Estrogenic activity and acute toxicity assessments of sediments from a chronically polluted estuarine area in southeastern Brazil

AbstractCompounds displaying estrogenic potential can lead to serious negative aquatic ecosystem impacts, and exposure to mixtures of endocrine disruptors present in environmental matrices with very complex characteristics, including sediments, can cause serious risks to biota, animals, and humans. Within this perspective, the aim of the present study was to assess estrogenic activity and toxicity in sediments sampled from Guanabara Bay, the most important, although chronically polluted, estuary in southeastern Brazil. To this end, estrogenic activity determinations were performed by the yeast estrogen screen (YES) assay, and acute toxicity assays were carried out employing Vibrio fischeri. Estrogenic activity was quantified up for 8.7 ng g−1 in E2‐EQ, although high levels of cytotoxicity (up to 95%) were observed and might have hindered the determination of the real estrogenic potential in most samples. Acute toxicity to V. fischeri ranged from 0 to 45.21%. These results point to potential deleterious effects caused by micropollutants and underline the urgency of continuous monitoring of the degradation degree of the Guanabara Bay estuary.

Combining chemical, bioanalytical and predictive tools to assess persistence, seasonality, and sporadic releases of organic micropollutants within the urban water cycle

Using a novel liquid chromatography-tandem mass spectrometry method with large volume direct injection and quantitation via isotope dilution, we evaluated the presence of 55 organic micropollutants in wastewater effluents, and locations within the Bow River and Elbow River watersheds in and around the city of Calgary, Alberta, Canada. In addition to establishing baseline micropollutant data for water utility operations, our study aimed to enhance our understanding of micropollutant behavior in the urban water cycle, assess the contributions of three wastewater treatment plants (WWTPs) to downstream receiving waters, explain the potential causes of total estrogenicity measured using the yeast-estrogen screen assay (YES), and prioritize a subset of substances for continuous monitoring. With data spanning 48 months and 95 river km, our results indicate the extensive persistence of metformin (antidiabetic), seasonality of N,N‑diethyl-m-toluamide (DEET, insect repellant), O-desmethylvenlafaxine (antidepressant metabolite), and sulfamethoxazole (antibiotic) in source waters, and sporadic detections of a well-known perfluoroalkyl substance (PFOA). The seasonality of pharmaceuticals at the sentinel downstream monitoring site appeared to coincide with river dilution while that of DEET was likely attributable to peak usage during the warmer months. Steroidal estrogens were rarely detected in wastewater effluents although total estrogenicity via YES was evident, suggesting the presence of less potent but more abundant non-steroidal estrogens (e.g., flame retardants, bisphenols, and phthalates). A conservative mass balance analysis suggests that the largest WWTP (serving a population of >1 million) consistently contributed the highest load of micropollutants, with the exception of metformin, which appeared to be influenced by a smaller WWTP (serving 115,000) that operates a different activated sludge process. We consider metformin, sucralose, diclofenac, and venlafaxine as more effective conservative tracers of wastewater pollution due to their notably higher concentrations and persistence in the Bow River compared to carbamazepine and caffeine, respectively. Finally, hierarchical clustering revealed a close association between E. coli and caffeine, supporting the use of caffeine as an indicator of short-term, untreated anthropogenic inputs. Overall, this study yields valuable insights on the presence, behavior, and sources of organic micropollutants in the urban water cycle and identifies indicators of anthropogenic impacts that are useful for prioritizing future monitoring campaigns in Calgary and elsewhere.

Interferences in the yeast estrogen screen (YES) assay for evaluation of estrogenicity in environmental samples, chemical mixtures, and individual substances

The Yeast Estrogen Screen (YES) has a specific mechanism of action that allows for the analysis of estrogenic EDC at low concentrations, and it has been broadly used to estimate the estrogenic potential of environmental samples. However, the experimental parameters of this assay still demand an investigation, such as cell density, incubation time, wavelength on the experimental outcome, cytotoxicity, and estrogenic activity adsorbed on suspended solids. We studied these interferences and applied the assay to single substances, mixtures, and environmental matrices from different sources. The increase in cell density amplifies the assay sensitivity only to a limited extent, while the reduction in incubation time decreased assay sensitivity - although it was not significant for surface water, no differences were observed between estradiol-equivalents derived of 48 h and 72 h measurements. The particulate phase was of utmost importance for the total estrogenic activity of the landfill leachate and surface water. Surface waters, landfill leachates and sediments also showed antiestrogenic activity and the integration of both estrogenic and antiestrogenic endpoints provided deeper insights into the potential risk associated with EDC. This study elucidated experimental interferences that may arise during the implementation and use of this assay, bringing more understanding to experimental parameters during the application of the assay for estrogenicity screening.

(Anti)estrogenic activity impacted by complex environmental matrices: A DOM and multiphase distribution approach

The presence of estrogenic endocrine disruptors in aquatic environments has been a concern and bioassays are recommended tools for their monitoring. However, the physicochemical properties of contaminants and the environmental matrix features may influence the resultant response. This study aimed to assess this influence on the Yeast Estrogen Screen (YES) assay. Mixtures of 17β-estradiol (E2) and humic acid (HA) were evaluated through the Schild approach aiming to investigate the interactions between estrogens and dissolved organic matter (DOM). Moreover, environmental samples from municipal landfill leachate and wastewater treatment plant (WWTP) influents and effluents were screened for (anti)estrogenic activity at both dissolved and particulate phases. Finally, results were statistically confronted with physicochemical parameters through principal component analysis (PCA). The HA test concentrations strongly reduced the E2 response, even at low levels. Humic substances may not only reduce estrogen bioavailability, but also interfere with the assay mechanism through enzymatic inhibition thus masking the sample estrogenic potential. Landfill leachate had total E2-Eq in the range 1282–2591 ng L−1, while WWTP influent and effluent were in the range 12.1–41.4 and <DL–2.3 ng L−1, so estrogenicity was reduced 92% in average. Particulate phase was responsible for 33–100% of measured E2-Eq between matrices, though cytotoxicity occurred in some extracts. Antiestrogenic activity was observed in both phases and might also have masked the estrogenicity of samples. PCA did not resulted in positive correlations supporting a multiphase distribution pattern of estrogenic compounds. Nevertheless, the solids and organic matter characteristics supported the data interpretation. In conclusion, the in vitro YES assay is subjected to factors intrinsic to the environmental sample that can influence on the measured estrogenic response. Therefore, results interpretation should be performed together with organic matter characterization parameters, cytotoxicity and antiestrogenic activity evaluation.

4,4′-(9-Fluorenylidene)dianiline (BAFL) is antiestrogenic and has adverse effects on female development in CD-1 mice

Many phenolic compounds have been found to have endocrine disrupting activities, but their arylamine analogs, the phenolic hydroxyl groups substituted by aniline amino groups, have rarely been reported. 4,4′-(9-Fluorenylidene)dianiline (BAFL) is an arylamine analog of fluorene-9-bisphenol (BHPF) and BHPF has been reported to be a strong antiestrogen which could cause endometrial atrophy, ovarian damage and adverse pregnancy outcomes in animals. BAFL has been widely used as material to synthetize polymers, such as polyimides, polyamide, and polyamine, for various uses since the 1970s. Here, we assessed the antiestrogenicity of BAFL using a variety of methods and looked into its impacts on the development of females in CD-1 mice. With the aid of a yeast estrogen screen assay, we found BAFL possessed obviously antiestrogenic activity (IC50 = 8.15 × 10−6 M), which close to that of tamoxifen and BHPF. Using a 10-d mouse uterotrophic assay, we found that BAFL obviously decreased uterine weight in a dose-dependent way. Histological analyses of mouse uteri revealed that BAFL induced marked endometrial atrophy and inhibited the uterine development. Immunohistochemical analyses showed that Sprr2d, an estrogen-responsive gene encoding protein, was mainly expressed in endometrial epithelial cells and BAFL decreased the areas and levels of Sprr2d staining in mouse uteri. It was clear from uterine transcriptome investigations that BAFL significantly downregulated the expressions of multiple genes responding to estrogen. Molecular docking showed that BAFL could effectively occupy the antagonist-binding pocket of hERα, and one of the amino groups of BAFL formed hydrogen bonds with the side chains of Arg394 and Glu353 in the receptor. These results indicated that BAFL exhibited clearly antiestrogenic characteristics and could interfere with normal female development in mice, which should be avoided using in commodities that come into direct contact with humans. Moreover, this study indicated that the arylamine analogs of phenolic endocrine disrupting chemicals might also have endocrine disrupting activities.

Estrogenic Activity and Endocrine Disruptor Compounds Determined in Guanabara Bay (Brazil) by Yeast Estrogen Screen Assays and Chemical Analyses

Studies assessing the presence of endocrine disrupting compounds in marine environments have increased in the last decades. In Brazil, the combination of poor sanitation conditions and low investment in sewage treatment plants leads to significant contamination of receiving waters. The risks of these micropollutants in the aquatic biota include biochemical and histopathological alterations of the liver, gonads, and kidneys, as well as, reproductive process and development modifications, and behavioral changes, among others. The aim of this study was to evaluate the quality of the surface and deep waters of Guanabara Bay, southeastern Brazil, regarding the presence of estrogenic substances. Acute toxicity assays were also conducted employing Vibrio fischeri. The estrogenic activity of the water samples was determined by Yeast Estrogen Screen assay and the quantification of the Bisphenol A, estriol, 17β-estradiol, and 17α-ethinylestradiol by high-performance liquid chromatography, using fluorescence and diode array detectors. Estrogenic activity ranged from 9 to 77 ng L-1 of estradiol equivalents. The highest micropollutants concentrations were detected for bisphenol A (298.5 and 465.5 ng L-1), followed by 17α-ethinylestradiol (248 and 256.9 ng L-1), estriol (70.7 and 179.6 ng L-1), and 17β-estradiol (167 and 174.8 ng L-1) for surface and deep waters, respectively. The findings indicate significant risks for the Guanabara Bay ecosystem. No acute toxicity effects were observed in the V. fisheri assay. These data reflect the current environmental degradation situation of the bay’s waters and highlight the need for the systematic monitoring of this important estuary.

Novel hyphenation of DGT in-situ passive sampling with YES assay to ascertain the potency of emerging endocrine disruptors in water systems in New Zealand

This study represents the first attempt to investigate selected estrogenic compounds that include 17α-ethynylestradiol (EE2), 17β-estradiol (E2) bisphenol A (BPA), and bisphenol AF (BPAF) along the drinkable water, from river-to-tap, and wastewater, from effluent-to-treated wastewater, treatment processes of the Hamilton City Council and the monitoring of the freshwater, from source-to-outfall, of the Waikato River in New Zealand. This was accomplished by the adoption of a novel combination of diffusive gradients in thin films (DGTs) in-situ passive sampling coupled with high-performance liquid chromatography/mass spectrometry analysis (HPLC/MS) and the Yeast Estrogen Screen (YES). Estradiol equivalency quantities, integrated in time, were evaluated theoretically (cEEQ) by DGT-HPLC/MS and experimentally (EEQ) by DGT-YES assay. cEEQ and EEQ highlighted that primary treatments are not suitable for estrogens and bisphenolic plastics removal both at drinkable and wastewater treatment plants in Hamilton where they worsen the water quality in terms of estrogenicity making these pollutants more available in the water phase. All downstream sites monitored along the Waikato River showed higher cEEQ and EEQ, moreover the Waikato River water quality showed a moderate worsening moving from Taupo (source) to Tuakau (outfall). The most polluted sites were downstream of Hamilton city and Huntly township wastewater treatment plants that serve the main conurbations in the area. cEEQ and EEQ generally showed good agreement at low concentrations but differed substantially at more polluted sites where cEEQ consistently underestimated estrogenic potency, possibly due to DGT accumulation of estrogenic compounds not quantified by HPLC/MS.

New solvent systems to separate some estrogenically active compounds by high-performance thin-layer chromatography (HPTLC)

Two solvent mixtures for high-performance thin-layer chromatographic (HPTLC) separation of some compounds showing estrogenic activity in the yeast estrogen screen (YES) assay are presented. The new method, planar yeast estrogen screen (pYES) combines the YES assay and a chromatographic separation on silica gel HPTLC plates with the performance of the YES assay. For separation, the analytes were applied bandwise to HPTLC plates (10 × 20 cm) with fluorescent dye (Merck, Germany). The plates were developed in a vertical developing chamber after 30 min of chamber saturation over a separation distance of 70 mm, using cyclohexane‒methyl-ethyl ketone (2:1, V/V) or cyclohexane‒CPME (3:2, V/V) as solvents. Both solvents allow separation of estriol, daidzein, genistein, 17β-estradiol, 17α-ethinyl estradiol, estrone, 4-nonylphenol and bis(2-ethylhexyl) phthalate.

Combined reverse osmosis and UV/H2O2 treatment of aqueous solutions of bisphenol A and 17α-ethinylestradiol: assessment of estrogenic activity

ABSTRACT Bisphenol-A (BPA) and 17α-ethinylestradiol (EE2) are considered endocrine disrupting compounds (EDC) and they may be harmful to the normal functioning of endocrine systems of humans and animals. Moreover, the presence of these compounds in superficial and groundwater may represent serious risks, even in low concentrations like ng·L−1. The objectives of this study were to remove BPA and EE2 from solutions containing a mixture of these compounds in ultrapure water at low concentrations through reverse osmosis (RO) membrane combined with a UV/H2O2 process. Furthermore, to assess the estrogenic activity reduction after such treatments, in vitro recombinant yeast-estrogen screen (YES) assay was used. The removal efficiencies of target micropollutants increased with the increase of H2O2 dosage. For RO permeate stream, they enhanced from 91% to 96% for EE2 and from 76% to 90% for BPA while, for the concentrate stream, from 70% to 81% for EE2 and 41% to 84% for BPA as the H2O2 concentration were increased from 100 to 1000 µg·L−1. The OH radicals’ generation was the dominant factor in the degradation of EDC during the UV/H2O2 treatment since the photolysis itself was not enough to degrade BPA or EE2. The estrogenic activity reduction after UV/H2O2 treatment was high, ranging from 92% to 98% for the permeate stream and from 50% to 93% for the concentrate stream. The EE2 was responsible for the whole observed estrogenic activity since BPA does not present estrogenicity, by in vitro YES assay, in the concentrations observed.

The Fate of Atrazine in Tropical Environments: Photolysis, Acute Toxicity and Endocrine Disruptor Potential

Triazine herbicides are widely used both in Brazil and internationally and are frequently detected in natural environments and drinking water. This study assesses atrazine degradation through solar radiation under tropical conditions and determines the toxicity of the intermediates. Atrazine degradation is tested through ultrapure water, humic acid solution and natural water experiments, with exposure to sunlight to simulate a natural environment. A yeast estrogen screen (YES) assay and Artemia salina test are carried out during the abiotic degradation. The atrazine degradation depends on the radiation intensity, since the experiments conducted in the summer reached 50% after ca. 17 days. No significant variations in this herbicide concentration are observed after 90 days of exposure in the fall. Atrazine degradation is observed only in humic acid and is responsible for indirect photolysis. Intermediates, namely, desethylatrazine (DEA) and deisopropylatrazine (DIA), are identified and quantified at the μg L–1 level. Thus, with the degradation of atrazine in water, the medium toxicity may decrease, since DEA and DIA have mean effective concentrations that kill 50% of Artemia salina (EC50) similars to atrazine (13 mg L–1). No estrogenic activity in the YES is detected for atrazine and its metabolites. These findings evidence that radiation intensity and organic substances in tropical countries influence the half-life of pesticides in aquatic environments.

Manipulating Estrogenic/Anti-Estrogenic Activity of Triphenylethylenes towards Development of Novel Anti-Neoplastic SERMs.

Selective estrogen receptor modulators (SERMs) act as estrogen receptor (ERα) agonists or antagonists depending on the target issue. Tamoxifen (TAM) (a non-steroidal triphenylethylene derivative) was the first SERM approved as anti-estrogen for the treatment of metastatic breast cancer. On the hunt for novel SERMs with potential growth inhibitory activity on breast cancer cell lines yet no potential to induce endometrial carcinoma, we designed and synthesized 28 novel TAM analogs. The novel analogs bear a triphenylethylene scaffold. Modifications on rings A, B, and C aim to attenuate estrogenic/anti-estrogenic activities of the novel compounds so they can potentially inhibit breast cancer and provide positive, beneficial estrogenic effects on other tissues with no risk of developing endometrial hyperplasia. Compound 12 (E/Z-1-(2-{4-[1-(4-Chloro-phenyl)-2-(4-methoxy-phenyl)-propenyl]-phenoxy}-ethyl)-piperidine) showed an appreciable relative ERα agonistic activity in a yeast estrogen screen (YES) assay. It successfully inhibited the growth of the MCF-7 cell line with GI50 = 0.6 µM, and it was approximately three times more potent than TAM. It showed no potential estrogenicity on Ishikawa endometrial adenocarcinoma cell line via assaying alkaline phosphatase (AlkP) activity. Compound 12 was tested in vivo to assess its estrogenic properties in an uterotrophic assay in an ovariectomized rat model. Compared to TAM, it induced less increase in wet uterine wet weight and showed no uterotrophic effect. Compound 12 is a promising candidate for further development due to its inhibition activity on MCF-7 proliferation with moderate AlkP activity and no potential uterotrophic effects. The in vitro estrogenic activity encourages further investigations toward potential beneficial properties in cardiovascular, bone, and brain tissues.

Estrogenic activity in reused water: comparison of concentration methods

Determining the presence of endocrine disrupting substances in waters is a relevant aspect for monitoring environmental health. Given its relevance, it is important to use methods that can make the total concentration of substances with estrogenic activity (eg endocrine disruptors), being faster, and without the use of compounds that pollute the environment. The purpose of the study was to compare the effectiveness of the methods of concentration by lyophilization and by vacuum concentration of substances with estrogenic activity present in reused water, using the commonly used methodology, solid phase extraction. Three methods were compared: solid phase extraction, lyophilization, and vacuum centrifugation. Sample aliquots of reused water received 17β-estradiol at a final concentration of 2 μg L-1 and were concentrated by the three methods. The analysis of estrogenic activity was performed by the in vitro YES (Yeast Estrogen Screen) assay. The results showed that the vacuum centrifugation, solid phase extraction and lyophilization methods had different percentages in the recovery of substances with estrogenic activity, being 45%, 40%, and 31%, respectively. The study pointed out that the lyophilization and vacuum centrifugation methods were effective as alternative methods for concentrating samples containing substances with estrogenic activity.

Improved yeast-screening assay for phytoestrogens.

A yeast estrogen screening (YES) assay was improved to increase sensitivity for detection of phytoestrogens. New yeast strains minus one or the other of transporters Pdr5 or Snq2 and harboring yEGFP as a reporter gene were developed. The new strains showed 2-100-fold improvement in sensitivity for detection of standard estrogens and antiestrogens. In addition, the assay time (1 h) using the newly developed strains was shorter than that (4 h) previously reported. Furthermore, the snq2-minus strains were most effective for detection of estrogenic activity while the pdr5-minus strains were most effective for detection of antiestrogenic activity. The efficacy of the new methods was evaluated and confirmed by testing with 23 Thai medicinal plant species. The new strains were also tested for detection of xenoestrogens. The results revealed that the newly developed YES methods were specific and rapid and suitable for simple high-throughput screening or detection of estrogen-like compounds.

Effective removal of pharmaceutical compounds and estrogenic activity by a hybrid anaerobic osmotic membrane bioreactor – Membrane distillation system treating municipal sewage

Pharmaceutically active compounds (PhACs) may cause harmful effects in living beings, and advanced treatment is required to improve wastewater treatment plant efficiency. In this context, this study aimed to assess the performance of a hybrid anaerobic osmotic membrane bioreactor coupled with a membrane distillation system (AnOMBR-MD) for removing PhACs and estrogenic activity from municipal sewage. Human health and environmental risks of produced water were also assessed. The removal efficiency of dissolved organic carbon and P-PO43- reached 97.2% and 98.0%, respectively. N-NH4+ accumulated in the bioreactor since anaerobic treatment can not remove it. Salinity increase in the bioreactor caused a great change in the microbial community, with Chao 1 and Shannon indexes higher in the sludge after 50 days of operation than in the sludge used as inoculum. Estrogenic activity of municipal sewage spiked with PhACs was >3 times higher than the expected value calculated by the additive model (2 µg L−1E2-eq.), which indicates that some of the PhACs effects increased in the presence of others. Estrogenicity was not detected in distillate samples, which greatly reduced human health risks to acceptable values. Of the 7 PhACs selected in this study, only betamethasone, fluconazole, and prednisone were detected in the distillate. However, the overall removal of PhACs by the AnOMBR-MD system was higher than 96.4%. The chronic environmental risk considering the estrogenic activity was classified as high because of the detection limit in the yeast estrogen screen (YES), which supports the need for improving bioassay sensibility. The results demonstrated that the use of the YES assay combined with detection and identification of micropollutants allowed an effective assessment of the overall treatment performance.

Hydrogen peroxide and persulfate activation using UVA-UVB radiation: Degradation of estrogenic compounds and application in sewage treatment plant waters

In the present work, the degradation of three estrogens (17β-estradiol (E2), estrone (E1) and 17α-ethinylestradiol (EE2)) was investigated under photoactivation of hydrogen peroxide and persulfate. Lab-scale irradiation experiments showed that both UVA and UVB radiations are able to photoactivate the oxidant precursors, although UVB is more efficient to generate radicals and therefore to degrade the targets. The efficiency of both oxidant precursors was investigated showing higher efficiency in the system with persulfate. The pseudo-first order degradation rate constants and the second order rate constants between the hydroxyl or the sulfate radicals and estrogens were measured. In order to evaluate the process efficiency in real treatment conditions, the degradation of the estrogens spiked into sewage treatment plant effluent was studied. Measurements of second order rate constants between the radical and the effluent organic matter by laser flash photolysis allowed to understand the involved quenching mechanisms. A Yeast Estrogen Screen (YES) assay was used to follow the decrease in estrogenic activity during the estrogen degradation. This assay permitted to ensure that the studied processes are not only able to degrade the estrogens but also to remove their estrogenic activity.