Introduction 1 2 3 4 5 6 7 8 9 10 11 13 14 16 17 19 20 22 β β β Materials and methods Chemicals 23 α β β α α β β β Yeast strains Saccharomyces cerevisiae URA3 − HIS3 − LEU − β Plasmids CYC1 Construction of the p403-GPD-hAR expression vector Xba Bam Xba Bam Xba Bam 2 CYC1 Sac Msc AGAACA TGTTCT AGAACA TGTTCT AGAACA TGTTCT AGAACA TGTTCT CYC1 24 Hind Sal Hind Sal 2 CYC1 Transformation of yeast cells − − − 23 2 CYC1 Stu URA3 l l Nsi HIS3 2 CYC1 PCR controls 2 CYC1 CYC1 Streamlined yEGFP assay with the yeast androgen bioassay β \documentclass[12pt]{minimal}
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$$\end{document} 50 lacZ β 4 β β o β d β d o o Results and discussion CYC1 URA3 Sac Msc CYC1 Xho Sph CYC1 23 HIS3 β lacZ PCR controls 1 1 2 2 CYC1 CYC1 1 CYC1 CYC1 CYC1 CYC1 CYC1 CYC 1 2 CYC1 Fig. 1 a 2 2 CYC1 CYC1 CYC1 CYC1 CYC1 b yEGFP  Dose–response curves obtained with the new yeast androgen bioassay 2 α β β α β β β β β 25 26 27 β Fig. 2 β α β β 17β-T β DHT α Prog Dex 17β-E2 β Bold β  1 50 50 β 50 α β β β β α α β Table 1 50 RAP Compound a b 50 c d β Positive (11/11) Strong AR agonist 76 1.0 α Positive (21/21) Strong AR agonist, weak ER agonist 33 2.3 β   510 0.15 β Positive Binds strongly to AR 52 1.5 Methyltrienolone Positive (8/8) AR agonist 54 1.4 Tetrahydrogestrinone  AR agonist 65 1.2 β Positive (10/11) AR agonist and antagonist, strong ER agonist 9,000 0.0084 Estrone Positive (2/2) AR agonist, strong ER agonist NR NR α Negative (1/1) ER agonist NR NR α Negative (1/1) Strong ER agonist NR NR Diethylstilbestrol Negative (2/2) Strong ER agonist NR NR e Negative (1/1) ER antagonist NR NR f Positive (7/9)  1,700 0.045 Medroxyprogesterone acetate  Positive (4/4) Weak AR agonist 1,500 0.051 Corticosterone Negative (1/1) Binds weakly to AR NR NR Dexamethasone Positive (3/4) AR agonist NR NR Flutamide Negative (5/5) AR antagonist NR NR 2,4,6-Tribromophenol    NR NR BDE-19   NR NR 4-OH-BDE-17   NR NR AR ER NR a 27 b 27 c 50 d 50 β 50 e f β 27 27 β β β 10 β 28 lacZ 3 lacZ 2 50 50 29 lacZ 50 α 50 lacZ α lacZ β lacZ lacZ lacZ 30 lacZ lacZ 50 15 β 50 31 Fig. 3 β α β β lacZ  Table 2 50 Compound 50 a 29 50 b RAP β 4.7 11.5 1.0 α 3.5 4.9 2.3 β ND 70 0.2 β ND 13 0.9 Methyltrienolone ND 3.7 3.1 Tetrahydrogestrinone ND 11.5 1.0 β 86.1 95 0.1 α ND NR NR Progesterone 89.3 170 0.068 Corticosterone ND NR NR Dexamethasone ND NR NR ND a 29 b lacZ Antiandrogenic activity 4 α 1 4 α 50 32 Fig. 4 α α α TBP  Conclusions A recombinant yeast cell was constructed that expresses the hAR and yEGFP as a reporter protein in response to androgens. Compared with other yeast androgen bioassays, this new biosassay showed a similar limit of detection and dynamic range. However, the measurement of the fluorescence (yEGFP) can be followed as a function of incubation time and is easier, quicker, and cheaper than the measurement of the β-galactosidase or the luciferase activity, which needs cell wall disruption and/or the addition of expensive substrates. Owing to the ease of the yEGFP measurement, standard deviations are generally less than 3%. Moreover, the assay seems to be more robust and more specific for detecting compounds with a pure androgenic mode of action. α α 16 33 34