Effect-directed analysis of endocrine-disrupting compounds in multi-contaminated sediment: identification of novel ligands of estrogen and pregnane X receptors

Effect-directed analysis (EDA)-based strategies have been increasingly used in order to identify the causative link between adverse (eco-)toxic effects and chemical contaminants. In this study, we report the development and use of an EDA approach to identify endocrine-disrupting chemicals (EDCs) in a multi-contaminated river sediment. The battery of in vitro reporter cell-based bioassays, measuring estrogenic, (anti)androgenic, dioxin-like, and pregnane X receptor (PXR)-like activities, revealed multi-contamination profiles. To isolate active compounds of a wide polarity range, we established a multi-step fractionation procedure combining: (1) a primary fractionation step using normal phase-based solid-phase extraction (SPE), validated with a mixture of 12 non-polar to polar standard EDCs; (2) a secondary fractionation using reversed-phase-based high-performance liquid chromatography (RP-HPLC) calibrated with 33 standard EDCs; and (3) a purification step using a recombinant estrogen receptor (ER) affinity column. In vitro SPE and HPLC profiles revealed that ER and PXR activities were mainly due to polar to mid-polar compounds, while dioxin-like and anti-androgenic activities were in the less polar fractions. The overall procedure allowed final isolation and identification of new environmental PXR (e.g., di-iso-octylphthalate) and ER (e.g., 2,4-di-tert-butylphenol and 2,6-di-tert-butyl-α-methoxy-p-cresol) ligands by using gas chromatography coupled with mass spectrometry with full-scan mode acquisition in mid-polar fractions. In vitro biological activity of these chemicals was further confirmed using commercial standards, with di-iso-octylphthalate identified for the first time as a potent hPXR environmental agonist.     

Validation and application of reporter gene assays for the determination of estrogenic and androgenic endocrine disruptor activity in sport supplements

Previously developed estrogen and androgen mammalian reporter gene assays (RGAs) were assessed for their potential use as a quantitative screening method in the detection of estrogenic and androgenic endocrine disruptors (EDs) in sport supplements. The validation of both RGAs coupled with dispersive solid phase extraction (dSPE) was performed in accordance with European Commission Decision EC/2002/6579 for biological screening methods. Decision limits (CCα) and detection capabilities (CCβ) were established for both the estrogen and androgen RGAs. All samples were compliant with CCα and CCβ in both bioassays. Recovery rates were 96 % for 17β-estradiol and 115 % for dihydrotestosterone as obtained in their corresponding RGA. Both estrogens and androgens were stable in samples for more than 3 weeks, when stored at -20 °C. Specificity, good repeatability (coefficients of variation (CV), 12-25 %), reproducibility and robustness of both bioassays were also observed. Four different ED modes of action were determined for estrogens and androgens in 53 sport supplements, using the validated RGAs. This study revealed that 89 % of the investigated sport supplements contained estrogenic EDs and 51 % contained androgenic compounds. In conclusion, both bioassays are suitable for sport supplement screening of estrogenic and androgenic EDs.     

Application of chemometric methods to the investigation of main microcontaminant sources of endocrine disruptors in coastal and harbour waters and sediments

Identification, resolution and distribution of main microcontaminant sources of endocrine disruptors in Spanish harbours, coastal waters and sediments are investigated using chemometric methods. We investigated eighteen different endocrine disruptor chemical compounds, including non-ionic surfactants, their degradation products and linear alkylbenzene sulfonates, found in a total number of 74 samples (35 water samples and 39 sediment samples) over a period of 16 months from March 1999 to July 2000, and in 32 different geographical sites along the Spanish Mediterranean Coast (e.g. Barcelona, Tarragona, Almeria Harbour, Malaga and the Bay of Cadiz). Main environmental contamination sources of these endocrine disruptor compounds were investigated and interpreted according to their chemical composition and according to their resolved geographical distribution profiles.An erratum to this article can be found at     

长江沿岸某化工园区土壤、底泥中酚类化合物的污染现状

建立了同时测定土壤（底泥）样品中12种酚类化合物的检测方法。采用加速溶剂萃取（ASE）与凝胶渗透色谱（GPC）协同净化进行前处理,气相色谱和质谱联用技术（GC-MS）进行定性定量分析。方法检出限为0.410~13.1 μg/kg（干重）,回收率在70.7%~122%之间,相对标准偏差（RSD）为1.2%~16%。基于上述分析方法研究了长江沿岸某化工园区土壤及长江底泥中12种酚类化合物的污染水平。17个土壤样品和7个底泥样品中除对苯二酚外的11种酚类化合物均有检出。土壤和底泥中酚类污染物总含量范围分别为10.16~30.66 mg/kg和18.00~29.83 mg/kg,平均含量分别为18.26 mg/kg和22.51 mg/kg。土壤和底泥中最主要的酚类污染物为4-硝基苯酚和4-氯-3-甲酚,其次为邻氯对苯二酚、4,6-二硝基邻甲基苯酚和2,4,6-三氯酚。该化工园区周边土壤及长江底泥中12种酚类污染物污染水平较低、环境风险较小。     

Resolution and identification of co‐eluting alkylphenols in comprehensive two‐dimensional gas chromatography–mass spectrometry by multivariate curve resolution‐alternating least squares

The interest in the analysis of alkylphenols (APs) has widely increased in the last decades because of the endocrine disrupting features of these phenol derivatives. However, the isolation and identification of many of the multiple chemical structures of all APs is a very challenging task because of their similar physicochemical properties. In this work, the co‐elution of the isomers present in technical mixtures and using comprehensive two‐dimensional gas chromatography coupled to quadrupole mass spectrometry was resolved using multivariate curve resolution‐alternating least squares algorithm. The mass spectrum of each resolved compound was compared with the theoretical mass spectrum obtained from the literature, in order to assign the appropriate identification of each isomer. Two commercial mixtures were studied; in one of them, 34 compounds were resolved, and in the second mixture, 40 compounds were resolved. The relative abundances of the compounds were also calculated in both mixtures. Copyright © 2015 John Wiley & Sons, Ltd.     

The application of reporter gene assays for the detection of endocrine disruptors in sport supplements

The increasing availability and use of sports supplements is of concern as highlighted by a number of studies reporting endocrine disruptor contamination in such products. The health food supplement market, including sport supplements, is growing across the Developed World. Therefore, the need to ensure the quality and safety of sport supplements for the consumer is essential. The development and validation of two reporter gene assays coupled with solid phase sample preparation enabling the detection of estrogenic and androgenic constituents in sport supplements is reported. Both assays were shown to be of high sensitivity with the estrogen and androgen reporter gene assays having an EC(50) of 0.01 ng mL(-1) and 0.16 ng mL(-1) respectively. The developed assays were applied in a survey of 63 sport supplements samples obtained across the Island of Ireland with an additional seven reference samples previously investigated using LC-MS/MS. Androgen and estrogen bio-activity was found in 71% of the investigated samples. Bio-activity profiling was further broken down into agonists, partial agonists and antagonists. Supplements (13) with the strongest estrogenic bio-activity were chosen for further investigation. LC-MS/MS analysis of these samples determined the presence of phytoestrogens in seven of them. Supplements (38) with androgen bio-activity were also selected for further investigation. Androgen agonist bio-activity was detected in 12 supplements, antagonistic bio-activity was detected in 16 and partial antagonistic bio-activity was detected in 10. A further group of supplements (7) did not present androgenic bio-activity when tested alone but enhanced the androgenic agonist bio-activity of dihydrotestosterone when combined. The developed assays offer advantages in detection of known, unknown and low-level mixtures of endocrine disruptors over existing analytical screening techniques. For the detection and identification of constituent hormonally active compounds the combination of biological and physio-chemical techniques is optimal.     

景洪哥纳香化学成分研究

景洪哥纳香为蕃荔枝科哥纳香属植物,主要分布在我国云南省。为了从该属植物中寻长新类型的抗肿瘤化合物,我们通过药理筛选,发现哥纳香根的醇提取物对人肿瘤细胞培养系有较强的素毒性。以生物活性跟踪为手段,指导分离景洪哥纳香根的醇提取物,得到38个化合物,通过光谱学及化学方法,确定其中两个化合物是新化合物,分别命名为哥纳香醌及哥纳香呋喃并呋喃酮丙亚基化物。     

毛细管电泳结合高效液相色谱-质谱用于天然产物活性成分的筛选

发展了毛细管电泳(CE)和高效液相色谱-质谱(HPLC-MS)相结合的用于天然产物中活性成分筛选和鉴定的方法。该方法中,用HPLC半制备柱对天然产物粗提物进行分离纯化,再用CE对HPLC纯化后的组分进行活性测试。根据HPLC-MS/MS提供的二级质谱数据,即可确定活性成分的化学结构。以乙酰胆碱酯酶为实验模型,对我们发展的筛选方法进行了验证。从黄连粗提物中确定了药根碱、巴马汀等7种活性成分,并通过CE测定了它们的半抑制率(IC₅₀)值。与传统的天然产物分离纯化和活性筛选方法相比,该方法具有简单、微量、快速、准确的优点。本文建立的方法为天然产物粗提物中活性成分的筛选提供了新技术。     

Optimization of Maceration Conditions for Improving the Extraction of Phenolic Compounds and Antioxidant Effects of Momordica Charantia L. Leaves Through Response Surface Methodology (RSM) and Artificial Neural Networks (ANNs)

The main goals of this research were the chemical and biological characterization of the bitter melon (Momordica charantia) isolate obtained by traditional (maceration) extraction, as well as optimization of this process using response surface methodology (RSM) and artificial neural networks (ANNs). Experiments were performed using Box–Behnken experimental design on three levels and three variables: extraction temperature (20?°C, 40?°C, and 60?°C), solvent concentration (30%, 50%, and 70%) and extraction time (30, 60, and 90?min). The measurements consisted of 15 randomized runs with 3 replicates in a central point. The antioxidant activity of obtained extracts was determined by the 1,1-diphenyl-2-picrylhydrazyl (DPPH), cupric ion reducing antioxidant capacity (CUPRAC) and ferric reducing antioxidant power (FRAP) assays while chemical characterization was done in terms of the total phenolic content (TPC). The methodology shows positive influence of solvent concentration on all four observed outputs, while temperature showed a negative impact. RSM showed that the optimal extraction conditions were 20?°C, 70% methanol, and an extraction time of 52.2?min. Under these conditions, the TPCs were 20.66 milligrams of gallic acid equivalents (mg GAE/g extract), DPPH 30.22 milligrams of trolox equivalents (mg TE/g extract), CUPRAC 67.78 milligrams of trolox equivalents (mg TE/g extract), and FRAP 45.48 milligrams of trolox equivalents (mg TE/g extract). The neural network coupled with genetic algorithms (ANN-GA) was also used to optimize the conditions for each of the outputs separately. It is anticipated that results reported herein will establish baseline data and also demonstrate that that the present model can be applied in the food and pharmaceutical industries.     

Medicinal Use of Testosterone and Related Steroids Revisited

Testosterone derivatives and related compounds (such as anabolic-androgenic steroids—AAS) are frequently misused by athletes (both professional and amateur) wishing to promote muscle development and strength or to cover AAS misuse. Even though these agents are vastly regarded as abusive material, they have important pharmacological activities that cannot be easily replaced by other drugs and have therapeutic potential in a range of conditions (e.g., wasting syndromes, severe burns, muscle and bone injuries, anemia, hereditary angioedema). Testosterone and related steroids have been in some countries treated as controlled substances, which may affect the availability of these agents for patients who need them for therapeutic reasons in a given country. Although these agents are currently regarded as rather older generation drugs and their use may lead to serious side-effects, they still have medicinal value as androgenic, anabolic, and even anti-androgenic agents. This review summarizes and revisits the medicinal use of compounds based on the structure and biological activity of testosterone, with examples of specific compounds. Additionally, some of the newer androgenic-anabolic compounds are discussed such as selective androgen receptor modulators, the efficacy/adverse-effect profiles of which have not been sufficiently established and which may pose a greater risk than conventional androgenic-anabolic agents.     

Novel modes of capillary electrophoresis for the determination of endocrine disrupting chemicals

The synthesis and usage of a wide range of organic chemicals has increased dramatically over the last five decades. These compounds sometimes termed endocrine disrupting chemicals include agricultural pesticides, industrial solvents, dyes, plasticisers, detergents and heat exchangers. Concerns have been raised about the potential adverse effects of these compounds on humans and wildlife species. Our objectives are to develop a method to identify, using novel capillary electrophoretic techniques, the endocrine disrupting compounds that are reported to be present in environmental samples. The CE modes, capillary zone electrophoresis, micellar electrokinetic chromatography (MEKC), cyclodextrin-modified MEKC (CD-MEKC) and electroosmotic flow-suppressed CD-MEKC were investigated for the determination of a range of endocrine disrupting chemical compounds. This paper shows some initial results obtained.     

卵黄蛋白原的分离测定及其在环境内分泌干扰物质筛选中的应用

环境内分泌干扰物的存在直接威胁野生动物的生存和人类的健康，对其作用机制及筛选方法的研究，已经成为环境科学研究的热点领域。近年来，卵黄蛋白原作为环境内分泌干扰物的“生物标志物”，得到了较深入的研究。本文讨论了卵黄蛋白原的分离测定方法及其在内分泌干扰物筛选中应用的最新进展，为建立更有效的卵黄蛋白分离测定方法及发展新的环境内分泌干扰物筛选技术提供参考。     

Screening and analysis of potentially active components in Shenxiong glucose injection using UHPLC coupled with photodiode array detection and MS/MS

Shenxiong glucose injection, a pharmaceutical preparation containing a water extract of the roots of Salvia miltiorrhizae and ligustrazine hydrochloride, is widely used in clinical to treat cardiovascular diseases in China. The chemical components of the water extract have been reported and the cardioprotective effects of the injection have been evaluated. However, the chemical constituents of the injection and their correlations with its pharmacological effects have not been established. In this study, 13 chemical constituents of the injection have been identified or characterized by ultra‐high performance liquid chromatography with diode array detection and electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry. Besides, the potentially active compounds of this preparation that directly act on cardiac cells have been screened by cell extraction and ultra high performance liquid chromatography targeted multiple reaction monitoring. As a result, eight potentially active compounds, danshensu ( 1 ), ligustrazine hydrochloride ( 4 ), salvianolic acid I/H ( 7 ), lithospermic acid ( 8 ), salvianolic acid D ( 9 ), rosmarinic acid ( 10 ), salvianolic acid B ( 12 ), and salvianolic acid C ( 13 ), were obtained and structurally characterized from the 11 target compounds used for screening. The liquid chromatography with quadrupole time‐of‐flight mass spectrometry and liquid chromatography with multiple reaction monitoring tandem mass spectrometry combination method has demonstrated its potency for the screening, detection, and structural identification of bioactive compounds in a complex matrix.     

Discovery and SAR exploration of <Emphasis Type="Italic">N</Emphasis>-aryl-<Emphasis Type="Italic">N</Emphasis>-(3-aryl-1,2,4-oxadiazol-5-yl)amines as potential therapeutic agents for prostate cancer

A new chemical series of antiproliferative compounds was identified via high-throughput screening on DU-145 human prostate carcinoma cell line (hit compound potency - 5.7 μM). Exploration of the two peripheral diversity vectors of the hit molecule in a hit-targeted library and testing of the resulting compounds led to SAR generalizations and identification of the 'best' pharmacophoric moieties. The latter were merged in a single compound that exhibited a 200-fold better potency than the original hit compound. Specific cancer cell cytotoxicity was confirmed for the most potent compounds.     

Monitoring of environmental phenolic endocrine disrupting compounds in treatment effluents and river waters, Korea

The last two decades have witnessed growing scientific and public concerns over endocrine disrupting compounds (EDCs) that have the potential to alter the normal structure or functions of the endocrine system in wildlife and humans. In this study, the phenolic EDCs such as alkylphenol, chlorinated phenol and bisphenol A were considered. They are commonly found in wastewater discharges and in sewage treatment plant. In order to monitor the levels and seasonal variations of phenolic EDCs in various aquatic environments, a total of 15 water samples from the discharged effluent from sewage and wastewater treatment plants and river water were collected for 3 years. Ten environmental phenolic EDCs were determined by GC-MS and laser-induced fluorescence (LIF). GC-MS analysis revealed that most abundant phenolic EDCs were 4-n-heptylphenol, followed by nonlyphenol and bisphenol A during 2002-2003, while 4-t-butylphenol and 4-t-octylphenol were newly detected in aquatic environments in 2004.The category of phenolic EDCs showed similar fluorescence spectra and nearly equal fluorescence decay time. This makes it hard to distinguish each phenolic EDC from the EDCs mixture by LIF. Therefore, the results obtained from LIF analysis were expressed in terms of the fluorescence intensity of the total phenolic EDCs rather than that of the individual EDC. However, LIF monitoring and GC-MS analysis showed consistent result in that the river water samples had lower phenolic EDCs concentration compared to the effluent sample. This revealed a lower fluorescence intensity and the phenolic EDCs concentration in summer was lower than that in winter. For the validation of LIF monitoring for the phenolic EDCs, the correlation between EDCs concentration acquired from GC-MS and fluorescence intensity from LIF was obtained (R = 0.7379). This study supports the feasibility of the application of LIF into EDCs monitoring in aquatic systems.     

Screening of endocrine disrupting chemicals using a surface plasmon resonance sensor.

Because concern over endocrine disrupting reactions caused by chemicals to humans and animals is growing, a rapid and reliable screening assay for endocrine disrupting chemicals is required. We have developed an in vitro screening assay based on a hormone receptor mechanism using a surface plasmon resonance (SPR) sensor. The interaction between an estrogen receptor alpha (ER) and an estrogen response element (ERE) is monitored in real time, when ER is injected over the SPR sensor chip on which a DNA fragment containing ERE is immobilized. In the presence of a chemical with estrogenic activity, the ER-ERE interaction is enhanced and the kinetic parameters are altered. We have validated the assay in terms of its specificity, dose dependency, optimal reaction conditions and reproducibility. It has been shown that the assay is very reliable as a rapid and quantitative screening method to judge the estrogenic activities of chemicals.