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.     

A new luciferase reporter gene assay for the detection of androgenic and antiandrogenic effects based on a human prostate specific antigen promoter and PC3/AR human prostate cancer cells.

We developed a new mammalian cell-based luciferase reporter gene assay for androgenic and antiandrogenic activities of chemicals and environmental samples. Environmental samples usually have a complex matrix that may contain the constituents acting as androgen receptor (AR) agonists, AR antagonists or aryl hydrocarbon receptor (AhR) agonists. AhR agonists are known to elicit the antiandrogenic effect through cross-talk between AR and AhR signal transduction pathways. In this study, PC3/AR human prostate carcinoma cells were transiently transfected with a prostate-specific antigen (PSA) promoter-driven luciferase expression plasmid. The cells were treated with a test compound or an environmental sample for 24 h at 37 degrees C and then measured for luciferase activity. The luciferase activity was induced by dihydrotestosterone (DHT) in a concentration-dependent manner in a concentration range from 10 fM to 1 nM. R1881, a synthetic androgen receptor agonist, induced luciferase activity and its inductive effects was additive to that of DHT. The luciferase activity was not induced by cortisol, a glucocorticoid, progesterone, a progestin, and 17beta-estradiol, an estrogen in a concentration range of up to 1 microM. DHT-induced luciferase activity was reduced by bicalutamide and cyproterone acetate, AR antagonists, and also by benzo[a]pyrene, an aryl hydrocarbon receptor agonist, through AhR-mediated pathways. All of these findings indicate that the present assay system correctly responds to AR agonists, AR antagonists and AhR agonist and, therefore, it is a powerful tool for the sensitive and selective screening of chemicals and environmental samples for their androgenic and antiandrogenic activities. We developed the first assay system, in which the expression of luciferase was driven by the promoter of a prostate-specific antigen gene, a typical human androgen-regulated gene.     

Retinol-binding protein as a biomarker to assess endocrine-disrupting compounds in the environment

Endocrine-disrupting compounds (EDC) are predominantly investigated with respect to their ability to mimic or block estrogenic actions. However, it is well-known that EDC can act as agonists or antagonists of androgen- and estrogen-response systems. For that reason, there is an obvious need for bioassays providing the possibility of detecting (anti-)estrogenic and (anti-)androgenic effects. The retinol-binding protein (RBP) seems to be a useful molecular biomarker for assessing all modes of action of EDC, because it is regulated by sex steroid hormones. This study was conducted to establish RBP as a biomarker for determination of (anti-)estrogenic and (anti-)androgenic effects of EDC using a Xenopus laevis primary hepatocyte culture system. It could be shown that RBP mRNA expression in X. laevis hepatocytes was stimulated by estrogens in a dose-dependant manner whereas a combination of estrogen and androgen or estrogen and anti-estrogen treatment suppressed estrogenic stimulating effects. Androgens testosterone and dihydrotestosterone were able to reduce RBP mRNA expression and the anti-androgen vinclozolin could abolish the mRNA synthesis-suppressing activity of the androgen dihydrotestosterone. These results clearly demonstrated that RBP mRNA expression patterns in Xenopus laevis hepatocytes have different modes of (anti-)estrogenic and (anti-)androgenic action and can be used for examination of suspected EDC. Moreover, water samples from sewage-treatment plant effluents were applied to liver cells and expression levels of RBP and estrogen receptor mRNA (a known estrogenic biomarker) were detected. These samples had high estrogenicity but caused low to moderate induction of RBP mRNA synthesis, leading to the conclusion that RBP levels represent the sum of all possible effects (estrogenic and other effects) of EDC in environmental samples.Abbreviations EDC Endocrine-disrupting compounds - RBP Retinol-binding protein - STP Sewage-treatment plants - ER Estrogen receptor - AR Androgen receptor - E2 17-Estradiol - EE Ethynylestradiol - T Testosterone - DHT Dihydrotestosterone - MT Methyltestosterone - TAM Tamoxifen - VC Vinclozolin - CMF Calcium-magnesium free - ME Minimal essential     

Miniaturization of a panel of high-throughput yeast-cell-based nuclear receptor assays in 384- and 1536-well microplates

A panel of luminescent Saccharomyces cerevisiae cell-based nuclear receptor assays, consisting of human estrogen receptors α and β, androgen receptor, and aryl hydrocarbon receptor, was miniaturized from the standard 96-well microplate format to high-throughput 384- and 1536-well microplate formats. In these assays, firefly luciferase lacking the peroxisome targeting sequence was used as a reporter and D-luciferin substrate was pre-mixed with the yeast cells before the incubation step, eliminating cell lysis and substrate addition steps, and allowing multiple readings at any desired time point. All of the assays were highly functional in the 384-well format, and most functioned well in the 1536-well format. The detection limit of the estrogen receptor α assay was even lower in the miniaturized microplate formats than in the original 96-well format. The panel of yeast-cell-based nuclear receptor assays can be used for high-throughput chemical testing and environmental monitoring of potential endocrine-disrupting activity of compounds and samples.     

Use of reporter cell lines for detection of endocrine-disrupter activity

We have studied stable transformed human mammary cell lines with highly inducible steroid receptor-mediated luciferase reporter gene expression. Cells responding specifically to glucocorticoids, progestagens, androgens, or estrogens are described and characterized. The use of this high-throughput, cell-based assay for analysis of steroid (ant)agonists is reported. Systematic characterization of endocrine-disrupting activity on human receptors and in a human-cell system is interpreted for a selection of xenobiotics. We show that the phytoestrogens apigenin and genistin have progestagenic and androgenic activity, respectively. Finally, application of cell-based assays to the analysis of environmental samples is discussed.     

Applicability of a yeast bioassay in the detection of steroid esters in hair

The aim of the present study was to demonstrate the applicability of a yeast androgen and estrogen bioassay in the detection of steroid esters in hair samples of animals treated with a hormone ester cocktail. The outcome of the activity screenings was critically compared with the results previously obtained with LC-MS/MS analysis. Hair samples of one pour-on treated animal, 10 ml DMSO containing 25 mg estradiol benzoate (EB), 60 mg testosterone decanoate (TD) and 60 mg testosterone cypionate (TC), were selected and analyzed with the androgen and estrogen yeast bioassay. Results showed that by the introduction of a hydrolysis step, bioassays can be used to screen for the presence of hormone esters in hair samples. Based on the difference in fluorescence responses between the non-hydrolyzed and the hydrolyzed hair samples, it was possible to detect the presence of EB up to at least 56 days after a single pour-on treatment and to detect the presence of TC and TD up to at least 14 days after the treatment. Although the LC-MS/MS analysis could detect TC and TD up to 49 days after treatment, bioassays have the advantage that they can also detect any (un)known steroid ester.     

Identification of anabolic steroids and derivatives using bioassay-guided fractionation, UHPLC/TOFMS analysis and accurate mass database searching

Biological tests can be used to screen samples for large groups of compounds having a particular effect, but it is often difficult to identify a specific compound when a positive effect is observed. The identification of an unknown compound is a challenge for analytical chemistry in environmental analysis, food analysis, as well as in clinical and forensic toxicology. In this study bioassay-guided fractionation, ultra high performance liquid chromatography combined with time-of-flight mass spectrometry (UHPLC/TOFMS) and accurate mass database searching was tested to detect and identify unknown androgens. Herbal mixtures and sport supplements were tested using an androgen bioassay and modifications in sample preparations were carried out in order to activate inactive pro-androgens, androgen esters and conjugated androgens to enable their detection in the bioassay. Two of the four herbal mixtures tested positive and bioassay-guided fractionation followed by UHPLC/TOFMS of positive fractions resulted in the identification of nortestosterone phenylpropionate, testosterone cyclohexanecarboxylate and methyltestosterone. Three of the four sport supplements reacted toxic in the bioassay or gave inconclusive results and were further investigated using UHPLC/TOFMS in combination with data processing software and an accurate mass database having approximately 40,000 entries. This accurate mass database was derived from the PubChem database on the internet and coupled to the TOFMS software. This resulted in the tentative identification of several androgens, including methylboldenone, testosterone and the androgen esters methyltestosterone propionate or testosterone isobutyrate, testosterone buciclate and methylenetestosterone acetate. The study showed that bioassay-guided fractionation in combination with UHPLC/TOFMS analysis is a useful procedure to detect, isolate and identify unknown androgens in suspected samples. As an alternative, the use of data processing software in combination with an accurate mass database and coupled on-line with the TOFMS instrument software enabled the identification of androgens and androgen esters in the chromatogram even without bioassay-guided fractionation.     

Novel method for evaluation of chemicals based on ligand-dependent recruitment of GFP labeled coactivator to estrogen receptor displayed on bacterial magnetic particles

We established a novel method to evaluate endocrine disrupting chemicals (EDCs) by assembling the estrogen receptor-ligand binding domain (ERLBD) and GFP labeled coactivator on magnetic nanoparticles. EDC can promote or inhibit coactivator recruitment to the ligand-ERLBD complex. ERLBD was displayed on the surface of nano-sized bacterial magnetic particles (BacMPs) produced by the magnetic bacterium, Magnetospirillum magneticum AMB-1. Our method resulted in 38 molecules of ERLBD molecules on a BacMPs with diameter of 75 nm. Furthermore, ligand-dependent recruitment assays of GFP labeled coactivator to ERLBD-BacMPs was performed by measuring the fluorescence intensity. 17β-estradiol (E2), estriol, diethylstilbestrol, zeralenone (full agonist), octylphenol (partial agonist) and ICI 182,780 (antagonist) were evaluated by this method. Full agonists tested showed increased fluorescence with increasing agonist concentration. Octylphenol had lower fluorescence intensity than E2. ICI 182,780 did not produce any fluorescence. The method developed in this study can evaluate the estrogenic potential of chemicals by discriminating whether they are an ER full agonist, partial agonist, or antagonist. Finally, this method is amenable adaptation into a high throughput format by using automated magnetic separation.     

Phytoestrogens Inhibiting Androgen Receptor Signal and Prostate Cancer Cell Proliferation

The androgen receptor(AR) signaling activated by dihydrotestosterone(DHT) plays critical roles in prostate cancer development and progression. Phytoestrogens, which are diphenolic compounds with estrogen and anti-estrogen effects, can bind to estrogen receptors. However, their function on AR signaling has not been fully elucidated. In this study, dual-luciferase reporter assay, immunobloting, docking system test, MTT assay, immunofluorescence and chromatin immunoprecipitation(ChIP) assays were employed to examine the potential effects of three phytoestrogens(genistein, daidzein, flavone) on DHT-activated prostate specific antigen(PSA) activation, cell proliferation and AR transactivation in lymph node carcinoma of prostate(LNCaP) cells. Phytoestrogens were detected to down-regulate DHT-activated AR-mediated PSA promoter transactivation by dual-luciferase reporter system. Furthermore, three phytoestrogens, especially genistein, were demonstrated to significantly decrease AR-activated PSA protein expression by Western blotting analysis. MTT experiment proves that phytoestrogens, especially genistein, remarkably inhibits the DHT-induced cell proliferation in LNCaP cells. To provide reasonable explanations for experimental phenomena mentioned above, we did docking system test and detected phytoestrogens to share the same AR-binding site with DHT. To further prove the competition between phytoestrogen and DHT on AR binding, we examined the effects of phytoestrogens on DHT-activated AR nuclear translocation and immunofluorescence analysis which confirms that phytoestrogens, especially genistein, inhibit DHT-activated androgen receptor nuclear translocation. Results from ChIP show that phytoestrogens down-regulate DHT-induces AR binding to the androgen response elements(AREs, including AREI, AREII, and AREIII) in PSA promoter. Genistein remarkably down-regulates AR, binding to the AREI located in -250― -39 bp and AREIII in -4170― -3978 bp in the presence of DHT. In general, three phytoestrogens were identified to inhibit DHT-AR binding by competitively binding to AR and inhibit AR-mediated transactivation. And genistein shows the strongest effects among three phytoestrogens. Our findings confirm that phytoestrogens are AR antagonist in the regulation of AR-related PSA activation and cell proliferation, which provides valuable insights into the treatment of prostate cancer.     

Application of chromatography–mass spectrometry methods to the control of sport nutrition and medicines marketed via internet

Several sport nutrition products and doping drugs sold in the period from 2014 to 2016 were studied using gas and liquid chromatography coupled with mass-spectrometry. In the study, WADA-banned substances were detected in the composition of pre-workout supplements, fat burners, and prohormones. A series of selective androgen receptor modulators and peptide doping drugs were also studied. It was shown that, in some cases, preparations can be adulterated.     

An LC-MS screening method with library identification for the detection of steroids in dietary supplements

For many years anabolic-androgenic steroids (AAS) are by far the most frequently detected pharmacological substances in doping control. In order to improve their performances, professional sportsmen are often tempted to take dietary supplements. However, due to the frequent and widespread occurrence of contaminated supplements, the use of such products is not without risk for the athletes involved. In order to minimize the chances of an unattended positive doping test or serious health problems, fast and reliable screening methods for the detection of anabolic steroids in dietary supplements are needed. A general screening procedure requires the fast and unambiguous detection of a large range of steroids. Gas chromatography-mass spectrometry (GC-MS) has been used intensively in the detection of doping substances for the past 40 years. Over time, many laboratories have delivered spectra to be included in standard reference databases, one of which is maintained by the National Institute of Standards and Technology (NIST) (Gaithersburg, MD, USA). In recent years, however, liquid chromatography coupled to mass spectrometry (LC-MS) has gained popularity. Unfortunately, existing GC-MS libraries are not applicable to LC-MS analysis. In the present study, a new mass spectral library of 88 steroids was developed, along with a fast UPLC-MS method. For the construction of this mass spectral library, three different mass spectra were measured for each steroid, with a sample cone voltage of 30, 60 and 100 V, respectively. This method was then successfully tested on contaminated dietary supplements which had previously been tested by means of a targeted LC-MS/MS method. Overall, the library search was shown to identify the same compounds as the MRM method.     

A reactivity pattern for discrimination of ER agonism and antagonism based on 3-D molecular attributes

Various models have been developed to predict the relative binding affinity (RBA) of chemicals to estrogen receptors (ER). These models can be used to prioritize chemicals for further tiered biological testing to assess the potential for endocrine disruption. One shortcoming of models predicting RBA has been the inability to distinguish potential receptor antagonism from agonism, and hence in vivo response. It has been suggested that steroid receptor antagonists are less compact than agonists; thus, ER binding of antagonists may prohibit proper alignment of receptor protein helices preventing subsequent transactivation. The current study tests the theory of chemical bulk as a defining parameter of antagonism by employing a 3-D structural approach for development of reactivity patterns for ER antagonists and agonists. Using a dataset of 23 potent ER ligands (16 agonists, 7 antagonists), molecular parameters previously found to be associated with ER binding affinity, namely global ( E HOMO ) and local (donor delocalizabilities and charges) electron donating ability of electronegative sites and steric distances between those sites, were found insufficient to discriminate ER antagonists from agonists. However, parameters related to molecular bulk, including solvent accessible surface and negatively charged Van der Waal's surface, provided reactivity patterns that were 100% successful in discriminating antagonists from agonists in the limited data set tested. The model also shows potential to discriminate pure antagonists from partial agonist/antagonist structures. Using this exploratory model it is possible to predict additional chemicals for their ability to bind but inactivate the ER, providing a further tool for hypothesis testing to elucidate chemical structural characteristics associated with estrogenicity and anti-estrogenicity.     

A reactivity pattern for discrimination of ER agonism and antagonism based on 3-D molecular attributes

Various models have been developed to predict the relative binding affinity (RBA) of chemicals to estrogen receptors (ER). These models can be used to prioritize chemicals for further tiered biological testing to assess the potential for endocrine disruption. One shortcoming of models predicting RBA has been the inability to distinguish potential receptor antagonism from agonism, and hence in vivo response. It has been suggested that steroid receptor antagonists are less compact than agonists; thus, ER binding of antagonists may prohibit proper alignment of receptor protein helices preventing subsequent transactivation. The current study tests the theory of chemical bulk as a defining parameter of antagonism by employing a 3-D structural approach for development of reactivity patterns for ER antagonists and agonists. Using a dataset of 23 potent ER ligands (16 agonists, 7 antagonists), molecular parameters previously found to be associated with ER binding affinity, namely global (E(HOMO)) and local (donor delocalizabilities and charges) electron donating ability of electronegative sites and steric distances between those sites, were found insufficient to discriminate ER antagonists from agonists. However, parameters related to molecular bulk, including solvent accessible surface and negatively charged Van der Waal's surface, provided reactivity patterns that were 100% successful in discriminating antagonists from agonists in the limited data set tested. The model also shows potential to discriminate pure antagonists from partial agonist/antagonist structures. Using this exploratory model it is possible to predict additional chemicals for their ability to bind but inactivate the ER, providing a further tool for hypothesis testing to elucidate chemical structural characteristics associated with estrogenicity and anti-estrogenicity.     

Rapid surface plasmon resonance immunobiosensor assay for microcystin toxins in blue-green algae food supplements

A surface plasmon resonance (SPR) immunobiosensor assay was developed and validated to detect microcystin toxins in Spirulina and Aphanizomenon flos-aquae blue-green algae (BGA) food supplements. A competitive inhibition SPR-biosensor was developed using a monoclonal antibody to detect microcystin (MC) toxins. Powdered BGA samples were extracted with an aqueous methanolic solution, centrifuged and diluted in HBS-EP buffer prior to analysis. The assay was validated in accordance with the performance criteria outlined in EU legislation 2002/657/EC. The limit of detection (LOD) of the assay was calculated from the analysis of 20 known negative BGA samples to be 0.561 mg kg⁻¹. The detection capability (CCβ) of the assay was determined to be ≤0.85 mg kg⁻¹ for MC-LR. The biosensor assay was successfully applied to detect MC-LR toxins in BGA samples purchased on the Irish retail market. MC-LR was detected in samples at levels ranging from <0.5 to 2.21 mg kg⁻¹. The biosensor results were in good agreement with an established LC-MS/MS assay. The assay is advantageous because it employs a simple clean-up procedure compared to chemical assays and allows automated unattended analysis of samples unlike ELISA.     

Masking effect of anti-androgens on androgenic activity in European river sediment unveiled by effect-directed analysis

This study shows that the androgen receptor agonistic potency is clearly concealed by the effects of androgen receptor antagonists in a total sediment extract, demonstrating that toxicity screening of total extracts is not enough to evaluate the full in vitro endocrine disrupting potential of a complex chemical mixture, as encountered in the environment. The anti-androgenic compounds were masking the activity of androgenic compounds in the extract with relatively high anti-androgenic potency, equivalent to 200 nmol flutamide equivalents/g dry weight. A two-step serial liquid chromatography fractionation of the extract successfully separated anti-androgenic compounds from androgenic compounds, resulting in a total androgenic potency of 3,820 pmol dihydrotestosterone equivalents/g dry weight. The fractionation simplified the chemical identification analysis of the original complex sample matrix. Seventeen chemical structures were tentatively identified. Polyaromatic hydrocarbons, a technical mixture of nonylphenol and dibutyl phthalate were identified to contribute to the anti-androgenic potency observed in the river sediment sample. With the GC/MS screening method applied here, no compounds with AR agonistic disrupting potencies could be identified. Seventy-one unidentified peaks, which represent potentially new endocrine disrupters, have been added to a database for future investigation.     

Evaluation of an hPXR reporter gene assay for the detection of aquatic emerging pollutants: screening of chemicals and application to water samples

Many environmental endocrine-disrupting compounds act as ligands for nuclear receptors. Among these receptors, the human pregnane X receptor (hPXR) is well described as a xenobiotic sensor to various classes of chemicals, including pharmaceuticals, pesticides, and steroids. To assess the potential use of PXR as a sensor for aquatic emerging pollutants, we employed an in vitro reporter gene assay (HG5LN-hPXR cells) to screen a panel of environmental chemicals and to assess PXR-active chemicals in (waste) water samples. Of the 57 compounds tested, 37 were active in the bioassay and 10 were identified as new PXR agonists: triazin pesticides (promethryn, terbuthryn, terbutylazine), pharmaceuticals (fenofibrate, bezafibrate, clonazepam, medazepam) and non co-planar polychlorobiphenyls (PCBs; PCB101, 138, 180). Furthermore, we detected potent PXR activity in two types of water samples: passive polar organic compounds integrative sampler (POCIS) extracts from a river moderately impacted by agricultural and urban inputs and three effluents from sewage treatment works (STW). Fractionation of POCIS samples showed the highest PXR activity in the less polar fraction, while in the effluents, PXR activity was mainly associated with the dissolved water phase. Chemical analyses quantified several PXR-active substances (i.e., alkylphenols, hormones, pharmaceuticals, pesticides, PCBs, bisphenol A) in POCIS fractions and effluent extracts. However, mass-balance calculations showed that the analyzed compounds explained only 0.03% and 1.4% of biological activity measured in POCIS and STW samples, respectively. In effluents, bisphenol A and 4-tert-octylphenol were identified as main contributors of instrumentally derived PXR activities. Finally, the PXR bioassay provided complementary information as compared to estrogenic, androgenic, and dioxin-like activity measured in these samples. This study shows the usefulness of HG5LN-hPXR cells to detect PXR-active compounds in water samples, and further investigation will be necessary to identify the detected active compounds.