Analytical approach for monitoring endocrine-disrupting compounds in urban waste water treatment plants

The presence of endocrine-disrupting compounds in influent and effluent water samples from four waste water treatment plants located in Italy was studied. The estrogen-like activity of the water samples was measured using a chemiluminescent recombinant yeast assay which is based on genetically engineered yeast cells that express the human estrogen receptor. This receptor, once activated, elicits the expression of the reporter gene lac-Z and, consequently, the production of β-galactosidase, which is then measured by chemiluminescence. To control and minimize sample matrix effects, an external control based on a modified yeast strain stably expressing β-galactosidase was developed and also used in the assay. Rapid and sensitive chemiluminescent enzyme immunoassays were also developed and validated for the quantification of 17β-estradiol, estrone, and estriol in waste water samples. Results from both methods were compared with a reference high-performance liquid chromatography and electrospray ionization tandem mass spectrometry (HPLC ESI-MS-MS) method developed for the quantification of natural estrogens. The recombinant yeast assay revealed a significant estrogenic activity in the influent samples, ranging from 80 to 400 pmol/L 17β-estradiol equivalents (EEQ), which was reduced by 70–95 % in the effluent samples. The yeast assay also showed a systematic 20–30 % overestimation of estrogenic activity relative to the HPLC ESI-MS-MS method, suggesting the presence of other compounds in the samples with estrogenic activity. The chemiluminescent enzyme immunoassays showed the presence of estrogens in the influent samples (mean concentrations: 350–450 pmol/L for estrone, 5–100 pmol/L for 17β-estradiol, 25–300 pmol/L for estriol), with significantly lower concentrations detected in the respective effluent samples. The waste water treatment was able to reduce natural estrogen concentrations by 40–95 %, although a high variability was observed. The enzyme immunoassay data correlated well with data obtained by the HPLC ESI-MS-MS method. Although the recombinant yeast assay represents a useful tool for a first-level screening of estrogenic activity due to its simplicity and high analytical throughput, sample matrix effects observed in waste water of industrial origin were found to strongly affect the yeast cells response, even when properly corrected for using the external control, thereby limiting its use to urban waste water. Its integration with chemiluminescent enzyme immunoassays would improve its performance by reducing false negative results, thereby enabling its use in extensive studies monitoring for the presence of endocrine-disrupting compounds in urban treatment plant effluents.     

Agonistic and antagonistic estrogens in licorice root (Glycyrrhiza glabra)

The roots of licorice (Glycyrrhiza glabra) are a rich source of flavonoids, in particular, prenylated flavonoids, such as the isoflavan glabridin and the isoflavene glabrene. Fractionation of an ethyl acetate extract from licorice root by centrifugal partitioning chromatography yielded 51 fractions, which were characterized by liquid chromatography–mass spectrometry and screened for activity in yeast estrogen bioassays. One third of the fractions displayed estrogenic activity towards either one or both estrogen receptors (ERs; ERα and ERβ). Glabrene-rich fractions displayed an estrogenic response, predominantly to the ERα. Surprisingly, glabridin did not exert agonistic activity to both ER subtypes. Several fractions displayed higher responses than the maximum response obtained with the reference compound, the natural hormone 17β-estradiol (E₂). The estrogenic activities of all fractions, including this so-called superinduction, were clearly ER-mediated, as the estrogenic response was inhibited by 20–60% by known ER antagonists, and no activity was found in yeast cells that did not express the ERα or ERβ subtype. Prolonged exposure of the yeast to the estrogenic fractions that showed superinduction did, contrary to E₂, not result in a decrease of the fluorescent response. Therefore, the superinduction was most likely the result of stabilization of the ER, yeast-enhanced green fluorescent protein, or a combination of both. Most fractions displaying superinduction were rich in flavonoids with single prenylation. Glabridin displayed ERα-selective antagonism, similar to the ERα-selective antagonist RU 58668. Whereas glabridin was able to reduce the estrogenic response of E₂ by approximately 80% at 6 × 10⁻⁶ M, glabrene-rich fractions only exhibited agonistic responses, preferentially on ERα.     

Continuous Production of Ethanol from Starch Using Glucoamylase and Yeast Co-Immobilized in Pectin Gel

This work presents a continuous simultaneous saccharification and fermentation (SSF) process to produce ethanol from starch using glucoamylase and Saccharomyces cerevisiae co-immobilized in pectin gel. The enzyme was immobilized on macroporous silica, after silanization and activation of the support with glutaraldehyde. The silica–enzyme derivative was co-immobilized with yeast in pectin gel. This biocatalyst was used to produce ethanol from liquefied manioc root flour syrup, in three fixed bed reactors. The initial reactor yeast load was 0.05 g wet yeast/ml of reactor (0.1 g wet yeast/g gel), used in all SSF experiments. The enzyme concentration in the reactor was defined by running SSF batch assays, using different amount of silica–enzyme derivative, co-immobilized with yeast in pectin gel. The chosen reactor enzyme concentration, 3.77 U/ml, allowed fermentation to be the rate-limiting step in the batch experiment. In this condition, using initial substrate concentration of 166.0 g/l of total reducing sugars (TRS), 1 ml gel/1 ml of medium, ethanol productivity of 8.3 g/l/h was achieved, for total conversion of starch to ethanol and 91% of the theoretical yield. In the continuous runs, feeding 163.0 g/l of TRS and using the same enzyme and yeast concentrations used in the batch run, ethanol productivity was 5.9 g ethanol/l/h, with 97% of substrate conversion and 81% of the ethanol theoretical yield. Diffusion effects in the extra-biocatalyst film seemed to be reduced when operating at superficial velocities above 3.7 × 10⁻⁴ cm/s.     

Screening of synthetic and plant-derived compounds for (anti)estrogenic and (anti)androgenic activities

Recently we constructed yeast cells that either express the human estrogen receptor α or the human androgen receptor in combination with a consensus ERE or ARE repeat in the promoter region of a green fluorescent protein (yEGFP) read-out system. These bioassays were proven to be highly specific for their cognate agonistic compounds. In this study the value of these yeast bioassays was assessed for analysis of compounds with antagonistic properties. Several pure antagonists, selective estrogen receptor modulators (SERMs) and plant-derived compounds were tested. The pure antiestrogens ICI 182,780 and RU 58668 were also classified as pure ER antagonists in the yeast estrogen bioassay and the pure antiandrogen flutamide was also a pure AR antagonist in the yeast androgen bioassay. The plant-derived compounds flavone and guggulsterone displayed both antiestrogenic and antiandrogenic activities, while 3,3′-diindolylmethane (DIM) and equol combined an estrogenic mode of action with an antiandrogenic activity. Indol-3-carbinol (I3C) only showed an antiandrogenic activity. Coumestrol, genistein, naringenin and 8-prenylnaringenin were estrogenic and acted additively, while the plant sterols failed to show any effect. Although hormonally inactive, in vitro and in vivo metabolism of the aforementioned plant sterols may still lead to the formation of active metabolites in other test systems.     

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.     

Xenoestrogens: mechanisms of action and detection methods

Estrogenic compounds exert pleiotropic effects in wildlife and humans, and endogenous estrogens, like 17-estradiol, regulate growth and development of their target tissues. Environmental, industrial, or naturally occurring chemicals that possess estrogenic and/or antiestrogenic activities are termed xenoestrogens and may interfere with endocrine systems. These xenoestrogens are therefore defined as endocrine-active or endocrine-disrupting compounds. The estrogen receptor (ER) is the major regulatory unit within the estrogen-signaling pathway and the molecular mechanisms of estrogen and ER actions are described briefly. Based on the mechanism of ER action, in vitro test systems are described that can be employed for screening but also for the elucidation of mechanisms of action of (anti)estrogenic compounds. How screening assays and mechanistic studies can aid in human risk assessment for potential endocrine-active compounds is discussed also.     

Reversed-phase liquid chromatography coupled on-line to estrogen receptor bioaffinity detection based on fluorescence polarization

We describe the development and validation of a high-resolution screening (HRS) platform which couples gradient reversed-phase high-performance liquid chromatography (RP-HPLC) on-line to estrogen receptor α (ERα) affinity detection using fluorescence polarization (FP). FP, which allows detection at high wavelengths, limits the occurrence of interference from the autofluorescence of test compounds in the bioassay. A fluorescein-labeled estradiol derivative (E2-F) was synthesized and a binding assay was optimized in platereader format. After subsequent optimization in flow-injection analysis (FIA) mode, the optimized parameters were translated to the on-line HRS bioassay. Proof of principle was demonstrated by separating a mixture of five compounds known to be estrogenic (17β-estradiol, 17α-ethinylestradiol and the phytoestrogens coumestrol, coumarol and zearalenone), followed by post-column bioaffinity screening of the individual affinities for ERα. Using the HRS-based FP setup, we were able to screen affinities of off-line-generated metabolites of zearalenone for ERα. It is concluded that the on-line FP-based bioassay can be used to screen for the affinity of compounds without the disturbing occurrence of autofluorescence.     

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.     

Combined biological and chemical assessment of estrogenic activities in wastewater treatment plant effluents

Five wastewater treatment plant effluents were analyzed for known endocrine disrupters and estrogenicity. Estrogenicity was determined by using the yeast estrogen screen (YES) and by measuring the blood plasma vitellogenin (VTG) concentrations in exposed male rainbow trout (Oncorhynchus mykiss). While all wastewater treatment plant effluents contained measurable concentrations of estrogens and gave a positive response with the YES, only at two sites did the male fish have significantly increased VTG blood plasma concentrations after the exposure, compared to pre-exposure concentrations. Estrone (E1) concentrations ranged up to 51 ng L^–1, estradiol (E2) up to 6 ng L^–1, and ethinylestradiol (EE2) up to 2 ng L^–1 in the 90 samples analyzed. Alkylphenols, alkylphenolmonoethoxylates and alkylphenoldiethoxylates, even though found at µg L^–1 concentrations in effluents from wastewater treatment plants with a significant industrial content, did not contribute much to the overall estrogenicity of the samples taken due to their low relative potency. Expected estrogenicities were calculated from the chemical data for each sample by using the principle of concentration additivity and relative potencies of the various chemicals as determined with the yeast estrogen screen. Measured and calculated estradiol equivalents gave the same order of magnitude and correlated rather well (R ²=0.6).An erratum to this article can be found at     

Development of an online p38α mitogen-activated protein kinase binding assay and integration of LC–HR-MS

A high-resolution screening method was developed for the p38α mitogen-activated protein kinase to detect and identify small-molecule binders. Its central role in inflammatory diseases makes this enzyme a very important drug target. The setup integrates separation by high-performance liquid chromatography with two parallel detection techniques. High-resolution mass spectrometry gives structural information to identify small molecules while an online enzyme binding detection method provides data on p38α binding. The separation step allows the individual assessment of compounds in a mixture and links affinity and structure information via the retention time. Enzyme binding detection was achieved with a competitive binding assay based on fluorescence enhancement which has a simple principle, is inexpensive, and is easy to interpret. The concentrations of p38α and the fluorescence tracer SK&F86002 were optimized as well as incubation temperature, formic acid content of the LC eluents, and the material of the incubation tubing. The latter notably improved the screening of highly lipophilic compounds. For optimization and validation purposes, the known kinase inhibitors BIRB796, TAK715, and MAPKI1 were used among others. The result is a high-quality assay with Z′ factors around 0.8, which is suitable for semi-quantitative affinity measurements and applicable to various binding modes. Furthermore, the integrated approach gives affinity data on individual compounds instead of averaged ones for mixtures.     

The Effect of Naphthalene-Acetic Acid on Biomass Productivity and Chlorophyll Content of Green Algae,Coccolithophore, Diatom,and Cyanobacterium Cultures

The application of biochemical stimulants to enhance biomass and metabolite productivity is being investigated here and may be a simpler approach to achieve our goals of higher productivity and lower costs than methods such as genetic modification. The research builds on prior work of screening various biochemical stimulants representing different types of plant growth regulators with the green alga, Chlorella sorokiniana. Here, we report the impact on biomass and chlorophyll productivity by comparing the delivery method of a previously identified superior stimulant, the synthetic auxin naphthalene-acetic acid (NAA), solubilized in ethanol or methanol. Algae evaluated included the green alga, C. sorokiniana, as well as a mixed consortium that includes C. sorokiniana along with two other wild-isolated green algae, Scenedesmus bijuga and Chlorella minutissima. It was found that NAA dissolved in ethanol was more effective in enhancing biomass productivity of C. sorokiniana. However, no differences were observed with the mixed consortia. The most effective treatment from this step, EtOH_500ppm + NAA_5ppm, along with two other NAA concentrations (EtOH_500ppm + NAA_2.5ppm and EtOH_500ppm + NAA_10ppm), was then applied to six diverse species of microalgae to determine if the treatment dosage was effective for other freshwater and marine green algae, cyanobacteria, coccolithophore, and diatoms. It was found that three of the species bioassayed, Pleurochrysis carterae, C. sorokiniana, and Haematococcus pluvialis exhibited a substantial boost in biomass productivity over the 10-day growth period. The use of ethanol and NAA at a combined dosage of EtOH_500ppm + NAA_5ppm was found to generate the highest biomass productivity for each of the species that responded positively to the treatments. If scalable, NAA and ethanol may have the potential to lower production costs by increasing biomass yields for commercial microalgae cultivation.     

Kinetics and mechanism of oxidation of dimethyl sulphoxide by sodium bromate-sodium bisulphite reagent in aqueous medium

Rates of oxidation of dimethyl sulphoxide (DMSO) by HOBr producedin situ from sodium bromate-sodium bisulphite reagent have been studied iodometrically in aqueous medium. The order in [DMSO] is one when [DMSO] < 001 mol dm^-3, fractional when [DMSO] is between 0.01 and 0.5mol dm^-3 and zero when (DMSO) > 0.5 mol dm^-3. Different rate laws are operative under these three conditions though HOBr is the effective oxidizing species in all the cases. A mechanism involving an intermediate four-membered cyclic transition state between DMSO and HOBr (formation constantK), which decomposes in a slow step with a rate constant(k) has been proposed. Thermodynamic parameters for the adduct formation step and activation parameters for the first-order decomposition of the adduct step have been evaluated in the temperature range 308–323 K. Activation parameters have also been determined while the orders in [DMSO] are unity and zero. The reaction product has been identified as dimethyl sulphone (DMSO₂).     

Molecular recognition of endocrine disruptors by synthetic and natural 17β-estradiol receptors: a comparative study

A β-estradiol receptor binding mimic was synthesised using molecular imprinting. Bulk polymers and spherical polymer nanoparticles based on methacrylic acid and ethylene glycol dimethacrylate as the functional monomer and crosslinker, respectively, were prepared in acetonitrile. The selectivity was evaluated by radioligand binding assays. The imprinted polymers were very specific to β-estradiol since the control polymers bound virtually none of the radioligand. The bulk polymer was then employed to screen endocrine disrupting chemicals. Structurally related steroids like α-estradiol, estrone and ethynylestradiol showed, respectively, 14.0, 5.0 and 0.7% of relative binding to the β-estradiol polymer, whereas most unrelated chemicals did not bind at all. These results are compared to those obtained with a bioassay using stably transfected yeast cells in culture bearing the human estrogen receptor. The receptor was activated by several estrogen-like chemicals and to a lesser extent by some structurally related chemicals. Figure A molecularly imprinted polymer that was a synthetic receptor for beta-estradiol was used for the screening of endocrine disrupting chemicals that are structurally related or unrelated to beta-estradiol. The results were compared with the recognition of the compounds by the biological estrogen receptor expressed in yeast cells. Related steroids like alpha-estradiol, estrone and ethynylestradiol showed significant binding to the beta-estradiol imprinted polymer, whereas most unrelated chemicals did not bind. The biological receptor was activated by several estrogen-like chemicals, and to a lesser extent by some structurally related chemicals     

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.     

A deuterium-labelling mass spectrometry–tandem diode-array detector screening method for rapid discovery of naturally occurring electrophiles

Because electrophiles regulate many signalling pathways in cells, by modifying cysteine residues in proteins, they have a wide range of biological activity. In this study, a deuterium-labelling mass spectrometry–tandem diode-array detector (MS–DAD) screening method was established for rapid discovery of naturally occurring electrophiles. Glutathione (GSH) was used as a probe and incubated with natural product extracts. To distinguish different types of electrophile, incubation was performed in two reaction solvents, H₂O and D₂O. Ten types of naturally occurring electrophile were chosen, on the basis of their properties, to undergo the screening assay. By using this screening method, we successfully discovered the bioactive electrophile 4-hydroxyderricin in an ethanol extract of Angelica keiskei. This electrophile had potent NAD(P)H:quinone oxidoreductase 1 (NQO1)-inducing activity at a concentration of 20 μmol L⁻¹.     

A new approach to improving the performance ofZymomonas in continuous ethanol fermentations

Although most fermentation ethanol is currently produced in traditional batch processes with yeast, the ethanologenic bacteriumZymomonas mobilis is recognized as an alternative process organism for fuel alcohol production. Different strategies for improving the productivity of ethanol fermentations are reviewed. In batch and open-type continuous fermentations the advantage of replacing yeast byZymomonas relates principally to the 10% higher fermentation efficiency (product yield), whereas in high cell density, closed-type continuous systems (operating with cell recycle or retention) the superior kinetic properties ofZymomonas can be exploited to affect about a five-fold improvement in volumetric productivity. Unlike yeast, the rate of energy supply (conversion of glucose to ethanol) inZymomonas is not strictly regulated by the energy demand and a nongrowing culture exhibits a maintenance energy coefficient that is at least 25 times higher than yeast. As an alternative to process improvement through genetic engineering of the process organism this investigation has taken a biochemical and physiological approach to increasing the kinetic performance ofZ. mobilis through manipulation and control of the chemical environment. Energetically “uncoupled” phenotypes with markedly increased specific rates of ethanol production were generated under conditions of nutritional limitation (nitrogen, phosphate, or potassium) in steady-state continuous culture. The pH was shown to influence energy coupling inZymomonas affecting the maintenance coefficient (m _e ) rather than the max growth yield coefficient (Y _x ^sάx ). Whereas the pH for optimal growth ofZ. mobilis (ATCC 29191) in a complex medium was 6.0–6.5, the specific rate of ethanol production in continuous fermentations was maximal in the range 4.0–4.5. Fermentation conditions are specified for maximizing the specific productivity of aZymomonas-based continuous ethanol fermentation where the potential exists for improving the volumetric productivity in dense culture fermentations with an associated 35–40% reduction in capital costs of fermentation equipment and an estimated savings of 10–15% on cost of product recovery (distillation), and 3–7% on overall production costs based on the projected use of inexpensive feedstocks.     

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.     

Validation of the HS-GC-FID method for the determination of ethanol residue in tablets

This paper describes the validation of a HS-GC-FID method (based on the Pharmacopeia’s method) for the determination of ethanol content in tablets. A general view of the procedure development/optimization process is presented. The main point of this study is the calculation of validation parameters. Selectivity of the method was determined. Linearity (r > 0.997) was observed in the range from 9.0 to 3,040 μg of ethanol per sample (because the mass of the tablets used was around 200 mg, this corresponds to 45–15,200 μg g⁻¹). The method showed good recoveries (average 99.0%), and a relative standard deviation for repeatability and intermediate precision of 4.5% and 5.5% respectively. The limit of detection was calculated to be 3.0 μg of ethanol per sample (15 μg g⁻¹). The uncertainty budget was done according to the "Guide to the Expression of Uncertainty in Measurement" (GUM)[1], and a relative expanded uncertainty was estimated as 4.8%.     

A novel microplate reader-based high-throughput assay for estrogen receptor binding

Coumestrol is a well-known ligand for the estrogen receptor (ER). The compound itself is fluorescent, and its fluorescence intensity at 408?nm increases upon binding to the ER. Here we describe a novel binding assay in 96-well plate format for estrogenic compounds, based on the competition between fluorescent coumestrol and estrogenic compounds for binding to the ligand binding domain (LBD) of the ER-alpha. Displacement of coumestrol was measured as a decrease in fluorescence intensity using a Victor² 1420 multilabel reader. Competitive binding curves for the well-known estrogenic compounds, 17β-estradiol (E₂), ethinylestradiol, 4-nonylphenol, 4-octylphenol, genistein, bisphenol A, tamoxifen and diethylstilbestrol were constructed by using 7–10 different concentrations of the compounds and a fixed concentration of ER-α-LBD (14?nmol) and coumestrol (100?nmol). IC₅₀ values and relative potencies (compared to E₂) of the estrogenic compounds were determined. The assay was validated by comparing the relative potencies to those from standard radioligand binding assays in the literature. Within day and between day variations were determined and the performance of the assay was assessed by determining the coefficients of variation and Z′ values. The present fluorescent binding assay has proven to be fast and easy, and allows accurately quantifying the binding affinity of estrogenic ligands. The method is also suitable as a high-throughput screening assay for ER ligands.