Evaluation of the suitability of recombinant yeast-based estrogenicity assays as a pre-screening tool in environmental samples.

This paper presents a study evaluating the suitability of recombinant yeast-based estrogenicity assays as a pre-screening tool for monitoring of the chemical status of water bodies in support of the Water Framework Directive (WFD). Three different recombinant yeast-based assays were evaluated; the Yeast Estrogen Screen (YES), the Recombinant Yeast Assay (RYA) and the Rikilt Estrogen bioAssay (REA), of which the YES assay was employed by two different laboratories. No significant difference between the performance of neither the different laboratories, nor the different yeast-assays was observed. Six batches of eleven samples each were analysed one week apart by the four participating laboratories and the robustness, repeatability and reproducibility of the participating yeast-based assays were evaluated. The setup included a correlation between bioassay results and results from chemical target analysis, which gave valuable information in the evaluation of the assays' performance. A good agreement was found between chemical and bioassay results, showing that the yeast-based assays can give valuable information in WFD work. However, the low sensitivity of the assays towards alkylphenols needs to be significantly improved if they are to be used for monitoring of these compounds. The study further led to suggestions on ways to improve traceability and quality assurance of the yeast-based assays.

Toxicity identification fractionation of environmental estrogens in waste water and sludge using gas and liquid chromatography coupled to mass spectrometry and recombinant yeast assay.

We developed a toxicity identification fractionation (TIF) procedure to determine estrogenic compounds in wastewaters and sludge. The procedure consisted in fractionation of samples through a C(18) solid-phase extraction cartridge, in which Fraction I contained nonylphenol (NP) and its mono (NPEO(1)) and diethoxylate (NPEO(2)) and the markers of faecal exposure, Fraction II contained bisphenol A (BPA) and synthetic and natural hormones, and Fraction III contained the hormone conjugates. These three fractions were analyzed in parallel using gas or liquid chromatography coupled to mass spectrometry and recombinant yeast assay (RYA). Water samples collected daily throughout a whole week contained from 0.45 to 7.22 microg L(-1) of NP > NPEO(1) > NPEO(2) and were responsible for the estrogenicity of these samples. Fractions II and III were not estrogenic and that was due to the low ng L(-1) level of hormones and hormone conjugates found, respectively. The biological treatment sewage treatment plant (STP) was capable to eliminate from 52 to 100% of the compounds, with bisphenol A being the least removed. Only alkylphenols were accumulated in sludge with concentrations from 8.69 to 26.3 mg kg(-1) dw of NPEO(1) > NPEO(2) > NP. The integrated procedure herein proposed can be used as a screening method to evaluate estrogenic compounds in STPs and to survey faecal elimination.

Removal of estrogenic activity of natural and synthetic hormones from a municipal wastewater: efficiency of horseradish peroxidase and laccase from Trametes versicolor.

Some researches studied the removal of steroid estrogens by enzymatic treatment, however none verified the residual estrogenicity after the enzymatic treatment at environmental conditions. In this study, the residual estrogenic activities of the key natural and synthetic steroid estrogens were investigated following enzymatic treatment with horseradish peroxidase (HRP) and laccase from Trametes versicolor. Synthetic water and municipal wastewater containing environmental concentrations of estrone, 17beta-estradiol, estriol, and 17alpha-ethinylestradiol were treated. Liquid chromatography-mass spectrometry analysis demonstrated that the studied steroid estrogens were completely oxidized in the wastewater reaction mixture after a 1-h treatment with either HRP (8-10 U ml(-1)) or laccase (20 U ml(-1)). Using the recombinant yeast assay, it was also confirmed that both enzymatic treatments were very efficient in removing the estrogenic activity of the studied steroid estrogens. The laccase-catalyzed process seemed to present great advantages over the HRP-catalyzed system for up-scale applications for the treatment of municipal wastewater.

Evaluating the interactions of vertebrate receptors with persistent pollutants and antifouling pesticides using recombinant yeast assays.

The development of in vitro methods for screening potentially harmful biological activities of new compounds is an extremely important way to increase not only their intrinsic environmental safety, but also the public perception of the safety standards associated with them. In this work we use two yeast systems to test the ability of different chemicals to bind and activate two vertebrate receptors which are intimately related to adverse biological effects of pollution in exposed fauna: the estrogen receptor (ER) and the aryl hydrocarbon receptor (AhR). The panel of compounds analysed here includes well-known pollutants, like PCBs, pp'-DDT and hexachlorobenzene, together with the less-known, emerging putative pollutants, such as Sea-Nine, Irgarol and diuron. Results show the ability of some of these compounds to interact with one or both receptors, provide hints about the relationship between structure and activity, and suggest mechanistic explanations for the biological activities already described in whole-animal experiments. In addition, we show that AhR may have an intrinsic ligand promiscuity comparable to that of ER, a feature not fully appreciated in the past due to the technical difficulties involved with testing highly lipophilic substances in yeast-based assays.

Detection of hormone receptor ligands in yeast by fluorogenic methods.

Yeast-based bioassays are becoming widespread tools for detection and quantification of ligands for vertebrate nuclear hormone receptors, including estrogens, progestans, androgens and dioxin-like compounds, both for agonistic and for antagonistic effects. These systems rely on the monitoring of transcription rates of reporter genes whose expression in yeast depends on binding of receptors to their natural or xenobiotic ligands. Among the different methods to quantify reporter gene transcription, those based on fluorescence detection are fast, very sensitive and reproducible. We propose a fast method for ligand detection for different vertebrate receptors in yeast, based on the use of fluorogenic substrates for the widely used reporter beta-galactosidase gene. In this method, beta-galactosidase activity is calculated from kinetic data, rather than from end-point measurements, which increases accuracy and facilitates the statistical analysis of the data. It also provides statistically rigorous procedures to distinguish between active and inactive compounds and to evaluate the fitness of the data to alternative models of dose/response mechanisms. All these features combined configure a flexible, fast (less than three hours for some systems) and reproducible method to evaluate the presence of potential endocrine disruptors in the environment.