A simple mesoporous silica nanoparticle-based fluorescence aptasensor for the detection of zearalenone in grain and cereal products.

Zearalenone (ZEN) is a type of estrogenic mycotoxin commonly occurring in cereals. The aim of this study was to design a simple, rapid, inexpensive and ultrasensitive fluorescence assay for the determination of ZEN. Here, amino-modified mesoporous silica nanoparticles (MSNs-NH2) were synthesized to be the positive charge-rich reactor. A 6-carboxy-fluorescein-labeled aptamer (aptamer-FAM) was designed as the signal probe, ZEN-capture probe and negative charge reactor. In the absence of ZEN, the negatively charged aptamer-FAM combined with the positively charged MSNs-NH2 in an electrostatic manner. In the presence of ZEN, the fluorescence intensity in the supernatant increased significantly because the aptamer-FAM could bind to ZEN instead of MSNs-NH2. Under the optimal experimental conditions, this assay exhibited excellent specificity, repeatability and a wide linearity range of 0.005-150 ng/mL, with a detection limit of 0.012 ng/mL. Additionally, it showed high recovery (83.3-101.5%) for the spiked samples. There was no statistically significant difference in the ZEN concentrations detected by the proposed assay and HPLC in naturally contaminated samples. Overall, this design provides a new strategy for the rapid, inexpensive and sensitive detection of ZEN, and it could be applied to develop fluorometric assays for different targets by the selection of appropriate aptamers. Graphical abstract.

Novel hapten design, antibody recognition mechanism study, and a highly sensitive immunoassay for diethylstilbestrol in shrimp.

Over the past few years, there has been a lack of progress in the quality of diethylstilbestrol (DES) antibodies used in immunoassay. In this study, a new immunizing hapten was designed for remarkably sensitive and specific antibody generation against diethylstilbestrol. By introducing a benzene ring instead of the traditional linear chain alkane as the hapten spacer, a more specific immune reaction was induced in the process of immunization. The developed polyclonal antibodies were characterized using the indirect competitive enzyme-linked immunosorbent assay (icELISA). Under optimized conditions, the half maximal inhibitory concentration (IC50) of the best polyclonal antibody was 0.14 ng/mL and it displayed low cross-reactions (CRs) with the structural analogs such as hexestrol (HEX) and dienestrol (DI). The molecular modeling and quantum chemical computation revealed that the lowest CR of the DES antibody to DI was mainly due to the huge three-dimensional conformational difference between DES and DI. Finally, a highly sensitive icELISA method based on the polyclonal antibody was developed for the determination of DES in shrimp tissue. The limit of detection (LOD) was as low as 0.2 µg/kg in shrimp and the recoveries in the spiked samples ranged from 83.4 to 90.8% with the coefficient of variation less than 13.8%. These results indicated that the use of an aromatic ring as the immunizing hapten spacer arm could be a potential strategy for the enhancement of anti-DES antibody sensitivity, and the established icELISA was applicable to the trace detection of DES in shrimp. Graphical abstract.

MECHANISMS IN ENDOCRINOLOGY: Estrogens in consumer milk: is there a risk to human reproductive health?

Possible effects of xenoestrogens on human health, in particular on male reproductive health, have attracted considerable attention in recent years. Cow's milk was suggested in numerous publications as one of possible sources of xenoestrogens that could affect human health. Although milk has undoubtedly many beneficial health effects and could even have a role in reducing incidence of some cancers, concerns were raised about presumably high levels of estrogens in cow's milk. In intensive farming, concentrations of estrogens in milk are higher due to long milking periods that today extend long into the pregnancy, when concentrations of estrogens in the cow's body rise. Numerous studies examined potential effects of milk on reproductive health and endocrine-related cancers in both experimental studies with laboratory animals, and in human epidemiological studies. In the present review article, we compiled a review of recently published literature about the content of estrogens in cow's milk and potential health effects, in particular on reproductive system, in humans. Although results of published studies are not unequivocal, it seems that there is stronger evidence suggesting that amounts of estrogens in cow's milk are too low to cause health effects in humans.

Analysis of Diethylstilbestrol Residues in Chicken Using Surface-Enhanced Raman Spectroscopy (SERS) Coupled with Multivariate Analysis.

Estrogen residues, including diethylstilbestrol in chicken, are one of the main food safety concerns all over the world owing to a series of negative effects on the human body. Surface-enhanced Raman spectroscopy (SERS) coupled with multivariate analysis was applied to detect rapidly diethylstilbestrol residues in chicken. The detection conditions, including the sizes of colloidal gold nanoparticles (Au NPs) and the additional amounts of Au NPs, chicken extract containing diethylstilbestrol, and magnesium sulfate solution, as well as the adsorption time, were optimized by a single factor experiment to obtain a better detection effect of diethylstilbestrol residues in chicken. Partial least squares regression (PLSR) was the best quantitative model for the detection of diethylstilbestrol residues in chicken by comparing four chemometric models. Diethylstilbestrol residues in chicken could be predicted by PLSR with the low root mean square error (RMSE = 0.4128 mg/L), and the high determination coefficient (R2 = 0.9811) and ratio of prediction to deviation (RPD = 7.2566) for the test set. A novel approach, which has the potential for the analysis of other estrogen residues in meat, was developed to detect rapidly the diethylstilbestrol residues in chicken by using SERS coupled with multivariate analysis.

Ultrasensitive competitive method-based electrochemiluminescence immunosensor for diethylstilbestrol detection based on Ru(bpy)32+ as luminophor encapsulated in metal-organic frameworks UiO-67.

In this work, Ru(bpy)32+ encapsulated in metal-organic frameworks (MOFs) UiO-67 (Ru(bpy)32+/UiO-67) as luminophor was easily prepared and firstly applied in constructing an electrochemiluminescence (ECL) immunosensor to efficiently estimate diethylstilbestrol (DES). The competitive method-based ECL immunosensor platform was fabricated by amino-silicon dioxide which possesses large surface area. The poriness of UiO-67 was splendid so that Ru(bpy)32+ could be easily encapsulated. Ru(bpy)32+/UiO-67 with excellent ECL luminescence signal existed large specific surface area for easily labeled with antibodies. DES competed with bovine serum albumin-diethylstilbestrol (BSA-DES) for binding to antibody-specific sites in the constructed immunosensor. However DES was micromolecule, which was easier to bond to antibodies than BSA-DES. The ECL signal was gradually decreases with the increase of the concentration of DES. Under optimal conditions, the proposed immunosensor exhibited a wide linear range from 0.01 pg mL-1 to 50 ng mL-1 with a low detetion limit of 3.27 fg mL-1 (S/N = 3). The novel fabricated immunosensor with interference immunity and high stability may cause an attractive approach for the other targets determination.

Molecularly imprinted vs. reversed-phase extraction for the determination of zearalenone: a method development and critical comparison of sample clean-up efficiency achieved in an on-line coupled SPE chromatography system.

Sample preparation prior to chromatographic separation plays an important role in the analytical process. To avoid time-consuming and manual handling sample-prep, automated on-line techniques such as on-line SPE-HPLC are therefore preferred. In this study, two different on-line extraction approaches for mycotoxin/endocrine disruptor zearalenone (ZEA) determination using either molecularly imprinted polymer (MIP) with selective cavities and binding sites for extraction or a reversed-phase sorbent C18 providing non-selective interactions have been developed, validated, and compared. The validation characteristics were compared and the two methods were evaluated as being almost equal in terms of linearity, repeatability, precision, and recovery. Recoveries were in the range of 99.0-100.1% and limits of detection were found the same for both methods (1.5 µg L-1). Method precision calculated for spiked beer samples was better for C18 sorbent (2.5 vs. 5.4% RSD). No significant differences in the selectivity of either extraction method were observed. The possible reasons and further details associated with this finding are discussed. Finally, both validated methods were applied for the determination of ZEA contamination in beer samples. Due to ZEA's native fluorescence, chromatographic separation with fluorimetric detection (λex = 270 nm and λem, = 458 nm) was selected. Graphical abstract Determination of zearalenone in beer using an on-line extraction chromatography system.

The mycoestrogen zearalenone in Portuguese flowing waters and its potential environmental impact.

This study provides the first environmental risk assessment (ERA) for zearalenone (ZEN), the only known mycoestrogen, based on a broad-scale investigation on its occurrence in rivers and creeks from Portugal. Water sample filtration and immunoaffinity columns (IAC) clean-up followed by liquid chromatograph with tandem mass spectrometry (LC/MS n ) provided an analytical method with good analytical performance. ZEN levels were determined for seven Portuguese rivers and one creek, during two different seasons, in a total of 38 samples collected upstream wastewater treatment plants (WWTPs). Overall, 23.7% were contaminated with ZEN at levels ranging between 5.6 and 82.6 ng/L. The highest concentration was observed during spring, although no statistically significant differences were observed between spring and autumn sampling campaigns. The potential ecotoxicological risk from ZEN to different trophic levels of aquatic organisms was evaluated by means of risk quotients (RQs) calculation. Although all the RQs obtained were lower than 1, our results confirm that ZEN is a relatively frequent contaminant in flowing waters in Portugal and might contribute to the overall estrogenic activity in the environment.

Determination of bisphenols with estrogenic activity in plastic packaged baby food samples using solid-liquid extraction and clean-up with dispersive sorbents followed by gas chromatography tandem mass spectrometry analysis.

Bisphenols (BPs) are a family of chemicals with known endocrine disrupting activity. Bisphenol A (BPA) is the most representative prototype of this group of chemicals. Recently, the use of BPA, a prototype of endocrine disruptors, has been reduced and replaced with structural analogs due to its negative effects on both the environment and consumers. In this work, a new method is presented for the determination of seven BPs, with estrogenic activity in ready-to-eat plastic packaged baby foods. The procedure involves the isolation of the analytes using solid-liquid phase extraction with acetonitrile followed by a clean-up step with a mixture of dispersive-SPE sorbents (C18 and PSA) and magnesium sulphate, to reduce matrix effect from proteins, sugars and lipids. Extraction parameters were optimized using multivariate optimization methods. The compounds were detected and quantified by gas chromatography tandem mass spectrometry (GC-MS/MS). The limits of quantification were between 0.1 and 1.2ngg-1 for the studied analytes. The method was validated using matrix-matched calibration and recovery assays with spiked samples. Recovery rates were between 91% and 110% and % RSD was lower than 13% in all cases. The method has been successfully applied for the determination of these endocrine disrupting chemicals (EDCs) in samples of a novel type of food consumed by pre-schoolers. This is the first study to analyze EDCs in plastic packaged foods consumed by this target group.

A smartphone colorimetric reader integrated with an ambient light sensor and a 3D printed attachment for on-site detection of zearalenone.

Smartphone biosensors could be cost-effective, portable instruments to be used for the readout of liquid colorimetric assays. However, current reported smartphone colorimetric readers have relied on photos of liquid assays captured using a camera, and then analyzed using software programs. This approach results in a relatively low accuracy and low generality. In this work, we reported a novel smartphone colorimetric reader that has been integrated with an ambient light sensor and a 3D printed attachment for the readout of liquid colorimetric assays. The portable and low-cost ($0.15) reader utilized a simplified electronic and light path design. Furthermore, our reported smartphone colorimetric reader can be compatible with different smartphones. As a proof of principle, the utility of this device was demonstrated using it in conjunction with an enzyme-linked immunosorbent assay to detect zearalenone. Results were consistent with those obtained using a professional microplate reader. The developed smartphone colorimetric reader was capable of providing scalable, cost-effective, and accurate results for liquid colorimetric assays that related to clinical diagnoses, environment pollution, and food testing. Graphical abstract A novel smartphone colorimetric reader that has been integrated with an ambient light sensor and a 3D printed attachment for the readout of liquid colorimetric assays.

Rapid and ultrasensitive detection of endocrine disrupting chemicals using a nanosensor-enabled cell-based platform.

Endocrine disrupting chemicals (EDCs) interact with estrogen receptors (ERs), causing a broad range of adverse health effects. Current assays for EDC activity are slow and often lack sensitivity. We report here an ultra-sensitive nanosensor that can detect estrogenic cellular changes in ER(+) MCF-7 cells rapidly (minutes) at several orders of magnitude lower than the generally used assays. Notably, the sensor responses at these ultra-low EDC levels correlated with an increased synthesis phase (S-phase) cell population of EDC-treated cells. The nanosensor was also able to detect binary EDC mixture effects, with synergism observed for bisphenol A (BPA) - 17ß-estradiol (E2), and antagonism for dicyclohexylphthalate (DCHP) - E2 and benzo(a)pyrene (BaP) - E2.

Synthesis and characterization of Fullerene modified ZnAlTi-LDO in photo-degradation of Bisphenol A under simulated visible light irradiation.

In this study, ZnAlTi layered double hydroxide (ZnAlTi-LDH) combined with fullerene (C60) was fabricated by the urea method, and calcined under vacuum atmosphere to obtain nanocomposites of C60-modified ZnAlTi layered double oxide (ZnAlTi-LDO). The morphology, structure and composition of the nanocomposites were analyzed by Scanning Electron Microscopy, High-resolution transmission electron microscopy, X-ray diffraction patterns, Fourier transform infrared and specific surface area. The UV-vis diffuse reflectance spectra indicated that the incorporation of C60 expanded the absorption of ZnAlTi-LDO to visible-light region. The photo-degradation experiment was conducted by using a series of C60 modified ZnAlTi-LDO with different C60 weight percentage to degrade Bisphenol A (BPA) under simulated visible light irradiation. In this experiment, the degradation rate of C60 modified ZnAlTi-LDO in photo-degradation of BPA under simulated visible light irradiation was over 80%. The intermediates formed in the degradation of BPA process by using LDO/C60-5% were 4-hydroxyphenyl-2-propanol, 4-isopropenylphenol and Phenol. Photogenerated holes, superoxide radical species, ·OH and singlet oxygen were considered to be responsible for the photodegradation process, among which superoxide radical species and ·OH played a predominant role in the photocatalytic reaction system. C60 modified ZnAlTi-LDO catalysts for photocatalytic reduction shows great potential in degradation of organic pollutants and environmental remediation.

Development of a new colorimetric assay for detection of bisphenol-A in aqueous media using green synthesized silver chloride nanoparticles: experimental and theoretical study.

In the present study, a cost-effective, green and simple synthesis method was applied for preparation of stable silver chloride nanoparticles (AgCl-NPs). The method was done by forming AgCl-NPs from Ag+ ions using aqueous extract of brown algae (Sargassum boveanum) obtained from the Persian Gulf Sea. This extract served as capping agent during the formation of AgCl-NPs. Creation of AgCl-NPs was confirmed by UV-visible spectroscopy, powder X-ray diffraction, energy-dispersive X-ray spectroscopy, and high-resolution transmission electron microscopy, while the morphology and size analyses were characterized using high-resolution transmission electron microscopy and dynamic light scattering. After optimization of some experimental conditions, particularly pH, a simple and facile system was developed for the naked-eye detection of bisphenol-A. Moreover, a theoretical study of AgCl interaction with bisphenol-A was performed at the density functional level of theory in both gas and solvent phases. Theoretical results showed that electrostatic and van der Waal interactions play important roles in complexation of bisphenol-A with AgCl-NPs, which can lead to aggregation of the as-prepared AgCl-NPs and results in color change from specific yellow to dark purple, where a new aggregation band induced at 542 nm appears. The absorbance at 542 nm was found to be linearly dependent on the bisphenol-A concentration in the range of 1 × 10-6-1 × 10-4 M, with limit of detection of 45 nM. In conclusion, obtained results from the present study can open up an innovative application of the green synthesis of AgCl-NPs using brown algae extract as colorimetric sensors.

Estrogenic compound profiles in an urbanized industry-impacted coastal bay and potential risk assessment by pollution indices and multivariative statistical methods.

The occurrence and distribution of target estrogenic compounds in a highly urbanized industry-impacted coastal bay were investigated, and contamination profiles were evaluated by estimating total estradiol equivalents (∑EEQs) and risk quotients (RQs). Phenolic compounds were the most abundant xenoestrogens, but seldom showed contribution to the ∑EEQs. The diethylstilbestrol (DES) and 17α-ethinylestradiol (EE2) were the major contributors followed by 17ß-estradiol (E2) in comparison with a slight contribution from estrone (E1) and estriol (E3). Both ∑EEQs and RQs indicated likely adverse effects posed on resident organisms. Further, multivariate statistical method comprehensively revealed pollution status by visualized factor scores and identified multiple "hotspots" of estrogenic sources, demonstrating the presence of complex pollution risk gradients inside and particularly outside of bay area. Overall, this study favors the integrative utilization of pollution indices and factor analysis as powerful tool to scientifically diagnose the pollution characterization of human-derived chemicals for better management decisions in aquatic environments.

Local effect of bisphenol A on the estradiol synthesis of ovarian granulosa cells from PCOS.

Close relationship between polycystic ovary syndrome (PCOS) and bisphenol A (BPA) has drawn much attention in recent years, while the underlying mechanisms are poorly understood. In our study, we aim to detect BPA concentration in the follicular fluid and investigate its effect on estradiol synthesis in human granulosa cells from PCOS and non-PCOS patients. Follicular fluid and granulosa cells were collected from women who underwent controlled ovarian stimulation for in vitro fertilization or intracytoplasmic sperm injection. BPA concentration in the follicular fluid from PCOS patients (440.50 ± 63.70 pg/ml) was significantly higher than that from non-PCOS patients (338.00 ± 57.88 pg/ml). Expression of aromatase and estradiol synthesis in cultured granulosa cells was examined after treatment with BPA from 0.01 to 1 µM for 24 h. Expression of aromatase and estradiol synthesis was downregulated by BPA in a dose-dependent manner in PCOS, but no effect was observed in granulosa cells from non-PCOS patients. These findings provide evidence that increased BPA concentration in the follicular fluid of PCOS patients may play an important role in its pathogenesis by attenuating the expression of aromatase in granulosa cells.

Differential BPA levels in sewage wastewater effluents from metro Detroit communities.

The endocrine disruptor Bisphenol A (BPA) is ubiquitous in both aquatic and surface sediment environments because it is continuously released into sewage wastewater effluent. The measurement of BPA at wastewater treatment plants is rarely performed even though the United States Environmental Protection Agency (EPA) states that current levels of environmental BPA could be a threat to aquatic organisms. Therefore, the aims of this study were to measure BPA levels in sewage wastewater at different collection points over a 1-year period and to compare the levels of BPA to 8-isoprostane, a human derived fatty acid, found in sewage wastewater. We analyzed pre-treated sewage samples collected from three source points located in different communities in the metropolitan Detroit area provided by the Detroit Water and Sewerage Department. Human urine samples were also used in the study. BPA and 8-isoprostane were measured using ELISA kits from Detroit R&D, Inc. BPA levels from the same collection point oscillated more than 10-fold over 1 year. Also, BPA levels fluctuated differentially at each collection point. Highly fluctuating BPA values were confirmed by LC/MS/MS. The concentration of BPA in sewage wastewater was ~100-fold higher than the concentration of 8-isoprostane, while urinary concentration was ~20-fold higher. Thus, BPA levels discharged into the sewage network vary among communities, and differences are also observed within communities over time. The difference in BPA and 8-isoprostane levels suggest that most of the BPA discharged to sewage wastewater might be derived from industries rather than from human urine. Therefore, the continuous monitoring of BPA could account for a better regulation of BPA release into a sewage network.

Natural incidence of zearalenone in Croatian pig feed, urine and meat in 2014.

The aim of this study was to determine the levels of zearalenone (ZEN) in different feed materials and feedstuffs for pigs, as well as in pig urine and pig meat following contaminated feed consumption. In total, 253 feed material and feedstuff samples were collected from Croatian pig farms. The results revealed the presence of ZEN in significant concentrations, the maximal being found in maize (5522 µg/kg), wheat (3366 µg/kg) and pig fattening feed (1949 µg/kg). In farms in which high feed contamination and pig hyperestrogenism were observed, samples of pig urine (n=30) and meat (n=30) were retrieved as well. The mean ZEN concentrations in pig urine and pig meat were 206±20.6 µg/L and 0.62±0.14 µg/kg, respectively. Despite high contamination of feedstuffs responsible for farmed pigs' intoxication, ZEN levels determined in pig meat were shown to be of little significance for human safety.

Invited review: Diagnosis of zearalenone (ZEN) exposure of farm animals and transfer of its residues into edible tissues (carry over).

The aim of the review was to evaluate the opportunities for diagnosing the zearalenone (ZEN) exposure and intoxication of farm animals by analyzing biological specimens for ZEN residue levels. Metabolism is discussed to be important when evaluating species-specific consequences for the overall toxicity of ZEN. Besides these toxicological facts, analytics of ZEN residues in various animal-derived matrices requires sensitive, matrix-adapted multi-methods with low limits of quantification, which is more challenging than the ZEN analysis in feed. Based on dose-response experiments with farm animals, the principle usability of various specimens as bio-indicators for ZEN exposure is discussed with regard to individual variation and practicability for the veterinary practitioner. ZEN residue analysis in biological samples does not only enable evaluation of ZEN exposure but also allows the risk for the consumer arising from contaminated foodstuffs of animal origin to be assessed. It was compiled from literature that the tolerable daily intake of 0.25 µg ZEN/kg body weight and day is exploited to approximately 8%, when a daily basket of animal foodstuffs and associated carry over factors are assumed at reported ZEN contamination levels of complete feed.

Analysis of estrogenic activity in environmental waters in Rio de Janeiro state (Brazil) using the yeast estrogen screen.

The estrogenicity of waters collected from an important hydrological system in Brazil (Paraiba do Sul and Guandu Rivers) was assessed using the yeast estrogen screen (YES) assay. Sampling was performed in rivers and at the outlets of conventional water treatment plants (WTP). The removal of estrogenic activity by ozonation and chlorination after conventional water treatment (clarification and sand filtration) was investigated employing samples of the Guandu River spiked with estrogens and bisphenol A (BPA). The results revealed a preoccupying incidence of estrogenic activity at levels higher than 1ngL(-1) along some points of the rivers. Another matter of concern was the number of samples from WTPs presenting estrogenicity surpassing 1ngL(-1). The oxidation techniques (ozonation and chlorination) were effective for the removal of estrogenic activity and the combination of both techniques led to good results using less amounts of oxidants.

Determination of estrogenic mycotoxins in environmental water samples by low-toxicity dispersive liquid-liquid microextraction and liquid chromatography-tandem mass spectrometry.

A novel, simple, rapid and eco-friendly method based on dispersive liquid-liquid microextraction using a bromosolvent was developed to determine six estrogenic mycotoxins (zearalenone, zearalanone, α-zearalanol, ß-zearalanol, α-zearalenol and ß-zearalenol) in water samples by liquid chromatography-electrospray ionization tandem mass spectrometry in the negative mode (LC-ESI-MS/MS). The optimal conditions for this method include the use of 100 µL bromocyclohexane as an extraction solvent (using a non-dispersion solvent), 10 mL of aqueous sample (adjusted to pH 4), a vortex extraction time of 2 min, centrifugation for 10 min at 3500 rpm and no ionic strength adjustment. The calibration function was linear and was verified by applying the Mandel fitting test with a 95% confidence level. No matrix effect was observed. According to the relative standard deviations (RSDs), the precision was better than 13% for the repeatability and intermediate precision. The average recoveries of the spiked compounds ranged from 81 to 118%. The method limits of detection (LOD) and quantification (LOQ) considering a 125-fold pre-concentration step were 4-20 and 8-40 ng L(-1), respectively. Next, the method was applied to the analysis of the environmental aqueous samples, demonstrating the presence of ß-zearalanol and zearalanone in the river water samples.