A new highly specific and robust yeast androgen bioassay for the detection of agonists and antagonists

Abstract

Public concern about the presence of natural and anthropogenic compounds which affect human health by modulating normal endocrine functions is continuously growing. Fast and simple high-throughput screening methods for the detection of hormone activities are thus indispensable. During the last two decades, a panel of different in vitro assays has been developed, mainly for compounds with an estrogenic mode of action. Here we describe the development of an androgen transcription activation assay that is easy to use in routine screening. Recombinant yeast cells were constructed that express the human androgen receptor and yeast enhanced green fluorescent protein (yEGFP), the latter in response to androgens. Compared with other reporters, the yEGFP reporter protein is very convenient because it is directly measurable in intact living cells, i.e., cell wall disruption and the addition of a substrate are not needed. When yeast was exposed to 17β-testosterone, the concentration where half-maximal activation is reached (EC 50 ) was 50 nM. The relative androgenic potencies, defined as the ratio between the EC 50 of 17β-testosterone and the EC 50 of the compound, of 5α-dihydrotestosterone, methyltrienolone, and 17β-boldenone are 2.3, 1.4, and 0.15 respectively. The results presented in this paper demonstrate that this new yeast androgen bioassay is fast, sensitive, and very specific and also suited to detect compounds that have an antiandrogenic mode of action.

Evidence of the indirect hormonal activity of prohormones using liver S9 metabolic bioactivation and an androgen bioassay

“…Transformants of a Saccharomyces cerevisiae strain that express the human androgen receptor (hAR) and an yEGFP reporter system [10] were grown on selective minimal medium plates supplemented with L-leucine. Minimal medium consisted of yeast nitrogen base without ammonium sulphate or amino acids (1.7 g/l), dextrose (20 g/l), ammonium sulphate (5 g/l) and was supplemented with Lleucine (6 mg/l).…”

“…Biological transcription activation assays, however, have the advantage of detecting compounds based on bioactivity. For screening of hormones a wide range of mammalian or yeast cell-based bioassays have been developed [7][8][9][10]. These assays focus mainly on ligandreceptor interactions in which activation of a specific receptor is linked to a transcription reporter mechanism.…”

Analysis of Environmental Samples with Yeast-Based Bioluminescent Bioreporters

“…These assays have been adapted to environmental matrices including environmental waterways (Thomas et al, 2002), aquifers (Conroy et al, 2005), wastewater treatment systems (Layton et al, 2000) and dairy manure (Raman et al, 2004). Additional yeast-based bioreporters have been developed using either a colorimetric detection (Bovee et al, 2004;Gaido et al, 1997;Le Guevel & Pakdel, 2001;Rehmann et al, 1999), green 7 fluorescent protein (Bovee et al, 2007;Bovee et al, 2004) or the firefly luciferase bioreporter (Bovee et al, 2004;Leskinen et al, 2005;Michelini et al, 2005). While the YES and YAS assays were highly specific for their target compounds, the colorimetric assays have disadvantages including addition of the chromophore for color development and a 3-5 day reaction time.…”

A Yeast Bioassay for Androgenic and Anti-Androgenic Compounds Based on the NH2- and COOH-Terminal Interaction of Androgen Receptor

“…12) On the other hand, in the previous yeast-based yEGFP reporter bioassay, 7) the EC 50 values of DHT and testosterone are estimated at 33 nM and 76 nM respectively.…”

“…The limit of detection of DHT in the N-C/ -catenin system (2.5 nM) was comparable to that in a previous yeast-based yEGFP reporter bioassay (3 nM). 7) The dose-responses for various steroids were determined in the N-C/ -catenin system. As shown in Fig.…”