Androgen receptor transactivation assay using green fluorescent protein as a reporter

Beck, Verena; Reiter, Evelyne; Jungbauer, Alois

doi:10.1016/j.ab.2007.09.006

Cited by 27 publications

(19 citation statements)

References 45 publications

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“…The PRtransactivation assay was performed as the conventional assay with ␤-galactosidase as reporter. For the ER-and AR-transactivation assays we used our modified method using GFP as reporter [23,24].…”

Section: 21ˇ-gal -Transactivation Assaymentioning

confidence: 99%

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Receptor binding and transactivation activities of red clover isoflavones and their metabolites

Pfitscher

Reiter

Jungbauer

2008

The Journal of Steroid Biochemistry and Molecular Biology

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Section: 21ˇ-gal -Transactivation Assaymentioning

confidence: 99%

“…For the modified AR-transactivation assay [24], yeast cells were transformed with the expression plasmid YEpBUbiFLAG-AR coding for the AR and the reporter plasmid YRpE2-GFP-ARE carrying the GFP coding sequence.…”

Section: Plasmidsmentioning

confidence: 99%

Receptor binding and transactivation activities of red clover isoflavones and their metabolites

Pfitscher

Reiter

Jungbauer

2008

The Journal of Steroid Biochemistry and Molecular Biology

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“…All three are current-use pesticides [8,28] that range in affinity for the androgen receptor, M2 > procymidone > prochloraz [29,30]. Inhibition response curves were developed for each antagonist with three androgens also of varying potency: 17b-trenbolone > testosterone > androstenedione [31,32]. These data provide insights on how sensitive the MDA-kb2 cell line might be to different combinations of androgen receptor agonists and antagonists that could be present in environmental samples.…”

Section: Introductionmentioning

confidence: 99%

Characterization of the androgen‐sensitive MDA‐kb2 cell line for assessing complex environmental mixtures

Blake

Martinović

Gray

et al. 2010

Enviro Toxic and Chemistry

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Synthetic and natural steroidal androgens and estrogens and many other non-steroidal endocrine-active compounds commonly occur as complex mixtures in aquatic environments. It is important to understand the potential interactive effects of these mixtures to properly assess their risk. Estrogen receptor agonists exhibit additivity in mixtures when tested in vivo and in vitro. Little is known, however, concerning possible mixture interactions of androgen receptor agonists. In these studies we used the MDA-kb2 cell line, a human breast cancer cell line with endogenous androgen receptors and a stably transfected luciferase reporter gene construct to quantify the androgenic activity of seven natural and synthetic androgens: 17beta-trenbolone, dihydrotestosterone, methyltestosterone, testosterone, trendione, 17alpha-trenbolone, and androstenedione. We tested combinations of these androgens and compared the observed activity to expected androgenic activity based on a concentration addition model. Our analyses support the hypothesis that androgen receptor agonists cause additive responses in a mixture. Binary mixtures of 17beta-trenbolone with 17beta-estradiol or triclocarban (an anti-microbial found in the environment) were also tested. 17beta-Estradiol induced androgenic activity, but only at concentrations 600-fold greater than those found in the environment. Triclocarban enhanced the activity of 17beta-trenbolone. Additionally, three anti-androgens were each paired with three androgens of varying potencies. The relative potencies of the antagonists were a vinclozolin metabolite (M2) > procymidone > prochloraz regardless of the androgen used. The results of our studies demonstrate the potential utility of the androgen-responsive MDA-kb2 cell line for quantifying the activity of mixtures of endocrine-active chemicals in complex wastes such as municipal effluents and feedlot discharges.

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“…Some in vitro whole cell biosensors have focused on estrogen-or androgen-related compounds, for example the YES assay (using S. cerevisiae as the detection element) [24] or the A-YES assay (using A. adeninivorans) [25] and their counterparts such as YAS [26,27] and A-YAS assays for androgen binding substances [28]. For progesterone detection, similar assays have been developed using mammalian or yeast cells as host for the production of the progesterone receptor and an enzyme or a fluorescent protein as reporter [29][30][31][32][33][34].…”

Section: Introductionmentioning

confidence: 99%

Development of a recombinant Arxula adeninivorans cell bioassay for the detection of molecules with progesterone activity in wastewater

et al. 2015

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This study describes the development of a bioassay to detect the presence of progesterone and progesterone-like molecules in wastewater samples. The basis of the bioassay is the integration of the human progesterone receptor gene into the yeast Arxula adeninivorans for the constitutive synthesis of the receptor. After incubation, binding of the analyte to the receptor induces the production of a reporter protein. Two reporter proteins were compared for detection parameters such as half-maximal activity (EC50), limit of detection (LoD) and limit of quantification (LoQ). When the extracellular phytase K was used, an EC50 value of 155 ng L(-1) and a LoD of 27 ng L(-1) progesterone were obtained after 4 h incubation, while use of the fluorescent dsRED as the reporter protein, resulted in an EC50 of 320 ng L(-1) and a LoD of 65 ng L(-1) after 20 h incubation. Use of phytase K as the reporter protein offers decreased incubation time and increased sensitivity; however the dsRED reporter system is less labor-intensive. Additionally, the affinity of known agonists and antagonists of the human progesterone receptor was determined. The utility of this bioassay was confirmed by measuring total progesterone equivalent concentration of samples from a wastewater treatment plant. The A. adeninivorans-based transactivation assay was able to measure concentrations of about 311 ng L(-1) in the influent stream but could not detect progesterone activity in effluent. One key feature of the assay is the robustness of A. adeninivorans, which allows sample measurement without any sample preparation.

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Androgen receptor transactivation assay using green fluorescent protein as a reporter

Cited by 27 publications

References 45 publications

Receptor binding and transactivation activities of red clover isoflavones and their metabolites

Receptor binding and transactivation activities of red clover isoflavones and their metabolites

Characterization of the androgen‐sensitive MDA‐kb2 cell line for assessing complex environmental mixtures

Development of a recombinant Arxula adeninivorans cell bioassay for the detection of molecules with progesterone activity in wastewater

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