Enhanced Detection of Estrogen-like Compounds by Genetically Engineered Yeast Sensor Strains.

The release of endocrine-disrupting compounds (EDCs) to the environment poses a health hazard to both humans and wildlife. EDCs can activate or inhibit endogenous endocrine functions by binding hormone receptors, leading to potentially adverse effects. Conventional analytical methods can detect EDCs at a high sensitivity and precision, but are blind to the biological activity of the detected compounds. To overcome this limitation, yeast-based bioassays have previously been developed as a pre-screening method, providing an effect-based overview of hormonal-disruptive activity within the sample prior to the application of analytical methods. These yeast biosensors express human endocrine-specific receptors, co-transfected with the relevant response element fused to the specific fluorescent protein reporter gene. We describe several molecular manipulations of the sensor/reporter circuit in a Saccharomyces cerevisiae bioreporter strain that have yielded an enhanced detection of estrogenic-like compounds. Improved responses were displayed both in liquid culture (96-well plate format) as well as in conjunction with sample separation using high-performance thin-layer chromatography (HPTLC). The latter approach allows for an assessment of the biological effect of individual sample components without the need for their chemical identification at the screening stage.

Estrogenicity of chemical mixtures revealed by a panel of bioassays.

Estrogenic compounds are widely released to surface waters and may cause adverse effects to sensitive aquatic species. Three hormones, estrone, 17ß-estradiol and 17α-ethinylestradiol, are of particular concern as they are bioactive at very low concentrations. Current analytical methods are not all sensitive enough for monitoring these substances in water and do not cover mixture effects. Bioassays could complement chemical analysis since they detect the overall effect of complex mixtures. Here, four chemical mixtures and two hormone mixtures were prepared and tested as reference materials together with two environmental water samples by eight laboratories employing nine in vitro and in vivo bioassays covering different steps involved in the estrogenic response. The reference materials included priority substances under the European Water Framework Directive, hormones and other emerging pollutants. Each substance in the mixture was present at its proposed safety limit concentration (EQS) in the European legislation. The in vitro bioassays detected the estrogenic effect of chemical mixtures even when 17ß-estradiol was not present but differences in responsiveness were observed. LiBERA was the most responsive, followed by LYES. The additive effect of the hormones was captured by ERα-CALUX, MELN, LYES and LiBERA. Particularly, all in vitro bioassays detected the estrogenic effects in environmental water samples (EEQ values in the range of 0.75-304 × EQS), although the concentrations of hormones were below the limit of quantification in analytical measurements. The present study confirms the applicability of reference materials for estrogenic effects' detection through bioassays and indicates possible methodological drawbacks of some of them that may lead to false negative/positive outcomes. The observed difference in responsiveness among bioassays - based on mixture composition - is probably due to biological differences between them, suggesting that panels of bioassays with different characteristics should be applied according to specific environmental pollution conditions.

Coupling high-performance thin-layer chromatography with a battery of cell-based assays reveals bioactive components in wastewater and landfill leachates.

Over the last two decades, effect-directed analysis (EDA) gained importance as a seminal screening tool for tracking biological effects of environmental organic micro-pollutants (MPs). As EDA using high-performance liquid chromatography and bioassays is costly and time consuming, recent implementations of this approach have combined high-performance thin-layer chromatography (HPTLC) with effect-based methods (EBMs) using cell-based bioassays, enabling the detection of estrogenic, androgenic, genotoxic, photosystem II (PSII)- inhibiting, and dioxin-like sample components on a HPTLC plate. In the present study, the developed methodologies were applied as a HPTLC-based bioassay battery, to investigate toxicant elimination efficiency of wastewater treatment plants (WWTPs), and to characterize the toxic potential of landfill leachates. Activity levels detected in untreated landfill leachates, expressed as reference compound equivalence (EQ) concentration, were up to 16.8 µg ß-naphthoflavone-EQ L-1 (indicating the degree of dioxin-like activity), 1.9 µg estradiol-EQ L-1 (estrogenicity) and 8.3 µg diuron-EQ L­1 (PSII-inhibition), dropping to maximal concentrations of 47 ng ß-naphthoflavone-EQ L-1, 0.7 µg estradiol-EQ L-1 and 53.1 ng diuron-EQ L-1 following treatment. Bisphenol A (BPA) is suggested to be the main contributor to estrogenic activity, with concentrations determined by the planar yeast estrogen screen corresponding well to results from chemical analysis. In the investigated WWTP samples, a decrease of estrogenic activity of 6-100% was observed following treatment for most of the active fractions, except of a 20% increase in one fraction (Rf = 0.568). In contrast, androgenicity with concentrations up to 640 ng dihydrotestosterone-EQ L-1 was completely removed by treatment. Interestingly, genotoxic activity increased over the WWTP processes, releasing genotoxic fractions into receiving waters. We propose this combined HPTLC and EBM battery to contribute to an efficient, cheap, fast and robust screening of environmental samples; such an assay panel would allow to gain an estimate of potential biological effects for prioritization prior to substance identification, and its routine application will support an inexpensive identification of the toxicity drivers as a first tier in an EDA strategy.

Yeast-Based Fluorescent Sensors for the Simultaneous Detection of Estrogenic and Androgenic Compounds, Coupled with High-Performance Thin Layer Chromatography.

The persistence of endocrine disrupting compounds (EDCs) throughout wastewater treatment processes poses a significant health threat to humans and to the environment. The analysis of EDCs in wastewater remains a challenge for several reasons, including (a) the multitude of bioactive but partially unknown compounds, (b) the complexity of the wastewater matrix, and (c) the required analytical sensitivity. By coupling biological assays with high-performance thin-layer chromatography (HPTLC), different samples can be screened simultaneously, highlighting their active components; these may then be identified by chemical analysis. To allow the multiparallel detection of diverse endocrine disruption activities, we have constructed Saccharomyces cerevisiae-based bioreporter strains, responding to compounds with either estrogenic or androgenic activity, by the expression of green (EGFP), red (mRuby), or blue (mTagBFP2) fluorescent proteins. We demonstrate the analytical potential inherent in combining chromatographic compound separation with a direct fluorescent signal detection of EDC activities. The applicability of the system is further demonstrated by separating influent samples of wastewater treatment plants, and simultaneously quantifying estrogenic and androgenic activities of their components. The combination of a chemical separation technique with an optical yeast-based bioassay presents a potentially valuable addition to our arsenal of environmental pollution monitoring tools.

Combination of yeast-based in vitro screens with high-performance thin-layer chromatography as a novel tool for the detection of hormonal and dioxin-like compounds.

The combination of classic in vitro bioassays with high-performance thin-layer chromatography (HPTLC) is a promising technique to directly link chemical analysis of contaminants to their potential adverse biological effects. With respect to endocrine disruption, much work is focused on estrogenicity. While a direct combination of HPTLC and the yeast estrogen screen is already developed, it is well accepted that further endocrine effects are relevant for monitoring environmental wellbeing. Here we show that non-estrogenic specific biological endpoints, (partly) related to the endocrine system, can also be addressed by combining respective yeast reporter gene assays with HPTLC to support effect-directed analysis (EDA). These are: androgenicity (YAS), thyroidogenicity (YTS), dioxin-like effects (YDS), effects on the vitamin D (YVS) and the retinoic acid receptor (YRaS). A proof of principle is demonstrated within this study by the characterization of dose-dependent responses to different model compounds for the respective receptors with and without chromatographic development of the HPTLC-plate. Limits of quantification (LOQ) for several model compounds were determined, e.g. 37 pg for testosterone (p-YAS), 0.476 ng for ß-naphthoflavone (p-YDS) and 1.02 ng for calcipotriol hydrate (p-YVS) with chromatographic development. The LOQ for p-YTS and p-YRaS were 10.16 pg for 3,3',5-triiodothyroacetic acid (p-YTS) and 0.41 pg for tamibarotene (p-YRaS), without chromatographic separation. Furthermore, we challenged the developed methodology using environmental samples, demonstrating an elimination efficiency of androgenic activity from municipal wastewater by a wastewater treatment plant between 99.4 and 100%. We anticipate our methodology to substantially broaden the spectrum of specific endpoints combined with HPTLC for an efficient and robust screening of environmental samples to guide a subsequent in-depth EDA.

Coupling High-Performance Thin-Layer Chromatography with Bacterial Genotoxicity Bioreporters.

We present an innovative technological platform for monitoring the direct genotoxicity of individual components in complex environmental samples, based on bioluminescent Escherichia coli genotoxicity bioreporters, sprayed onto the surface of a high-performance thin-layer chromatography (HPTLC) plate. These sensor strains harbor plasmid-borne fusions of selected gene promoters of the E. coli SOS DNA repair system to the Photorhabdus luminescens luxABCDE gene cassette, and mark by increased luminescence the presence of potentially DNA-damaging sample components separated on the plate. We demonstrate an "on plate" quantifiable dose-dependent response to several model genotoxicants (without metabolic activation). We further demonstrate the applicability of the system by identifying as genotoxic specific components of HPTLC-separated influent and effluent samples of wastewater treatment plants, thereby alleviating the need for a comprehensive chemical analysis of the sample.