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 17beta-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 17beta-testosterone and the EC(50) of the compound, of 5alpha-dihydrotestosterone, methyltrienolone, and 17beta-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.

Fig. 1

PCR controls. The PCR controls were performed as described in “PCR controls.” a Lanes 1, 6 and 11 contain a 100-bp ladder. PCR I was performed with primers on the backbone of the p406 plasmid and on the ARE₂ sequence. Lanes 2–5 are PCR I on the p406-ARE₂-CYC1-yEGFP reporter vector, the DNA that was isolated from the yeast transformant, the empty p406-CYC1 plasmid, and the DNA that was isolated from the empty yeast host (the nontransfected yeast cells), respectively. PCR II was performed with primers on the CYC1 promoter and the CYC1 terminator. Lanes 7–11 are PCR II on the reporter vector, the DNA from the yeast transformant, the empty p406-CYC1 plasmid, and the DNA from the empty yeast host respectively. b Lane 1 contains a 1-kb ladder. PCR III was performed with the primers on the human androgen receptor gene. Lanes 2–4 are PCR III on the p403-GPD-hAR expression vector, the DNA from the yeast transformant, and the DNA from the empty yeast host, respectively. yEGFP yeast enhanced green fluorescent protein

Fig. 2

Response of the yeast androgen biosensor to different substances. Exposure to 17β-testosterone, 5α-dihydrotestosterone, progesterone, dexamethasone, 17β-estradiol, and 17β-boldenone was started by adding to 200 μL of a yeast culture a 2-μL aliquot of a stock solution of the compound in dimethyl sulfoxide (DMSO). Fluorescence was determined after 24 h as described in “Streamlined yEGFP assay with the yeast androgen bioassay.” Fluorescence signals are the mean of a triplicate with the standard deviation (SD). 17β-T 17β-testosterone, DHT 5α-dihydrotestosterone, Prog progesterone, Dex dexamethasone, 17β-E2 17β-estradiol, Bold 17β-boldenone

Fig. 3

Response of the McDonnell yeast androgen bioassay to different substances. Exposure to 17β-testosterone, 5α-dihydrotestosterone, progesterone, dexamethasone, 17β-estradiol, and 17β-boldenone was started by adding to 200 μL of a yeast culture a 2-μL aliquot of a stock solution of the compound in DMSO. The β-galactosidase activity was determined after 24 h and corrected for the optical density at 630 nm as described in “lacZ-based yeast androgen bioassay: β-galactosidase assay.” Signals are the mean of a triplicate with the SD

Fig. 4

Inhibition of a submaximal response obtained by 5α-dihydrotestosterone with flutamide and three brominated flame retardants (BFRs). Coexposure to a concentration of 5α-dihydrotestosterone that induced a submaximal response was started by adding to 200 μL of a yeast culture, 1 μL of a 5α-dihydrotestosterone and 1 μL of the BFR stock solution in DMSO. Fluorescence was determined after 24 h as described in “Streamlined yEGFP assay with the yeast androgen bioassay.” Fluorescence signals are the mean of a triplicate with the SD. TBP 2,4,6-tribromophenol