Screening bisphenols in complex samples via a planar Arxula adeninivorans bioluminescence bioassay.

The Arxula yeast bisphenol screen (A-YBS) utilizes the bioluminescent Arxula adeninivorans yeast-based reporter cells for tailored analysis of bisphenols, one of the major endocrine-disrupting compound groups. For the first time, this bioreporter has been applied on the high-performance thin-layer chromatography (HPTLC) adsorbent surface to develop a respective planar bioluminescence bioassay (pA-YBS). The goal was to combine the advantages of HPTLC with a more selective bioassay detection for bisphenols. The performance of this pA-YBS bioluminescence bioassay was demonstrated by calculating the half-maximal effective concentration (EC50) of bisphenols compared to references. The EC50 ranged from 267 pg/band for bisphenol Z and 322 pg/band for bisphenol A (BPA) to > 1 ng/band for other bisphenols (BPC, BPE, BPF, and BPS) and references (17ß-estradiol and 17α-ethinylestradiol). The EC50 value of BPA was three times more sensitive in signal detection than that of 17ß-estradiol. The visual or videodensitometric limit of detection of BPA was about 200 pg/zone. The higher signal intensity and sensitivity for BPA confirmed the tailored bioassay selectivity compared to the existing estrogen screen bioassay. It worked on different types of HPTLC silica gel plates. This HPTLC-UV/Vis/FLD-pA-YBS bioluminescence bioassay method was used to analyze complex mixtures such as six tin can migrates, five thermal papers, and eleven botanicals. The detected estrogenic compound zones in the tin can migrates were successfully verified via the duplex planar yeast antagonist estrogen screen (pYAES) bioassay. The two bisphenols A and S were identified in one out of five thermal papers and confirmed with high-resolution mass spectrometry. No bisphenols were detected in the botanicals investigated via the pA-YBS bioluminescence bioassay. However, the botanicals proved to contain phytoestrogens as detected via the pYAES bioassay, which confirmed the tailored bioassay selectivity. This HPTLC-UV/Vis/FLD-pA-YBS bioluminescence bioassay is suited for cost-efficient analysis of BPA in complex samples, with no need for sterile conditions due to the fast workflow.

Patterns of estrogenic activity in the Baltic Sea.

Compounds such as estradiol and ethinylestradiol belong to contaminants of emerging concern, as they can disrupt the endocrine system of an organism with a hormonal system. The determination of such compounds is still challenging due to required low detection and quantification limits. Bioassays have proved to be sensitive tools for investigating the full potential of all compounds that can elicit an estrogenic response. In this study, surface water samples from different sampling sites and seasons in the Baltic Sea were analyzed for estrogenic activity with the Arxula adeninivorans yeast estrogen screen. Observed estradiol equivalent concentrations were in the range of

Validation of Arxula Yeast Estrogen Screen assay for detection of estrogenic activity in water samples: Results of an international interlaboratory study.

Endocrine-active substances can adversely impact the aquatic ecosystems. A special emphasis is laid, among others, on the effects of estrogens and estrogen mimicking compounds. Effect-based screening methods like in vitro bioassays are suitable tools to detect and quantify endocrine activities of known and unknown mixtures. This study describes the validation of the Arxula-Yeast Estrogen Screen (A-YES®) assay, an effect-based method for the detection of the estrogenic potential of water and waste water. This reporter gene assay, provided in ready to use format, is based on the activation of the human estrogen receptor alpha. The user-friendly A-YES® enables inexperienced operators to rapidly become competent with the assay. Fourteen laboratories from four countries with different training levels analyzed 17ß-estradiol equivalent concentrations (EEQ) in spiked and unspiked waste water effluent and surface water samples, in waste water influent and spiked salt water samples and in a mixture of three bisphenols. The limit of detection (LOD) for untreated samples was 1.8ng/L 17ß-estradiol (E2). Relative repeatability and reproducibility standard deviation for samples with EEQ above the LOD (mean EEQ values between 6.3 and 20.4ng/L) ranged from 7.5 to 21.4% and 16.6 to 28.0%, respectively. Precision results are comparable to other frequently used analytical methods for estrogens. The A-YES® has been demonstrated to be an accurate, precise and robust bioassay. The results have been included in the ISO draft standard. The assay was shown to be applicable for testing of typical waste water influent, effluent and saline water. Other studies have shown that the assay can be used with enriched samples, which lower the LOD to the pg/L range. The validation of the A-YES® and the development of a corresponding international standard constitute a step further towards harmonized and reliable bioassays for the effect-based analysis of estrogens and estrogen-like compounds in water samples.

Separation and identification of hormone-active compounds using a combination of chromatographic separation and yeast-based reporter assay.

Arxula adeninivorans-based yeast cell assays for the detection of steroid hormones demonstrated their efficiency for the determination of total hormone activity in a variety of samples using a microtiter plate format. In this study, a preliminary chromatographic separation using thin-layer chromatography plates is introduced in order to allow a rapid identification of the compounds responsible for this hormonal activity. The yeast whole cell assay can then be performed on the plate, producing a detectable signal where a steroid hormone is present. Simultaneous detection of estrogens, progestogens and androgens on the same plate in the picogram range was achieved, while keeping the assay as simple and affordable as possible. The assay requires a single incubation of the thin-layer chromatography plate and the detection of reporter protein production can be performed by fluorescence scanning of the plate at different wavelengths. The chromatographic separation allows the separation of several estrogens, androgens and progestogens, thus making its application for 'real world' samples very useful. In this work, different water-based samples from environmental origins were used to demonstrate the capacity of this new bioassay. Trials showed that most samples, with the exception of complex samples such as wastewater influent, can be assayed.

Simultaneous detection of three sex steroid hormone classes using a novel yeast-based biosensor.

A biosensor detecting estrogens, progestogens, and androgens in complex samples and in a single step is described. Three Arxula adeninivorans yeast strains were created, each strain producing a different recombinant human hormone receptor and a different fluorescent reporter protein. These strains were then mixed to create G1212/YRC102-hHR-fluo, the biological component of the biosensor. During incubation with G1212/YRC102-hHR-fluo, hormones present in a sample bind to their target receptor, which leads to the production of a specific fluorescent protein. Three fluorescence scans of the yeast suspension determine which fluorescence protein has been produced, thus revealing which hormone receptor (estrogen, progesterone, and androgen) has been activated by the hormones or hormone mimics present in the sample. The biosensor has similar sensitivities to the existing A. adeninivorans cell-based assays. The detection of the three hormone classes in one single experiment reduces the labor and time required to assay for the three hormone classes. The biosensor was also trialed with animal serum samples for the detection of progestogens, androgens, and estrogens and gave results that correlated well with ELISA analysis in case of progestogens. These results highlight the potential usefulness of the biosensor for comprehensive determination of hormone status in samples from veterinary origin. Biotechnol. Bioeng. 2017;114: 1539-1549. © 2017 Wiley Periodicals, Inc.