Development of a rapid yeast estrogen bioassay, based on the expression of green fluorescent protein.

The aim of this study was to develop an estrogen transcription activation assay that is sensitive, fast and easy to use in the routine screening of estrogen activity in complex matrices such as agricultural products. Recombinant yeast cells were constructed that express the human estrogen receptor alpha (ER alpha) and beta-Galactosidase (beta Gal), Luciferase (Luc) or yeast Enhanced Green Fluorescence Protein (yEGFP) as a reporter protein. Compared to other yeast assays, these new cells contain both the receptor construct as well as the reporter construct stably integrated in the genome with only one copy of the reporter construct. Dose-response curves for 17beta-estradiol (E2) obtained with the beta Gal assay were similar to those reported and the calculated EC(50) of 0.2 nM was even slightly better. However, 5 days of incubation were required before the chlorophenol red product could be measured. The Luc assay was as sensitive as the beta Gal assay and gave an EC(50) of 0.2 nM, but the signals were rather low and, although the assay can be performed within 1 day, the procedure is laborious and caused variability. The yEGFP revealed an EC(50) of 0.4 nM, but compared to the beta Gal and the Luc assay, the response was much better. This yEGFP assay can be performed completely in 96 well plates within 4 h and does not need cell wall disruption nor does it need the addition of a substrate. This makes the test sensitive, rapid and convenient with high reproducibility and small variation. These qualities make that this yEGFP assay is suited to be used as a high throughput system.

[1]  R. Müller,et al.  Yeast vectors for the controlled expression of heterologous proteins in different genetic backgrounds. , 1995, Gene.

[2]  F. Sherman,et al.  Two types of TATA elements for the CYC1 gene of the yeast Saccharomyces cerevisiae. , 1991, Molecular and cellular biology.

[3]  John P. Sumpter,et al.  Widespread Sexual Disruption in Wild Fish , 1998 .

[4]  L. Guarente,et al.  Distinctly regulated tandem upstream activation sites mediate catabolite repression of the CYC1 gene of S. cerevisiae , 1984, Cell.

[5]  C Sonnenschein,et al.  The E-SCREEN assay as a tool to identify estrogens: an update on estrogenic environmental pollutants. , 1995, Environmental health perspectives.

[6]  K. Korach,et al.  Estrogen receptor null mice: what have we learned and where will they lead us? , 1999, Endocrine reviews.

[7]  F. Pakdel,et al.  Streamlined beta-galactosidase assay for analysis of recombinant yeast response to estrogens. , 2001, BioTechniques.

[8]  T. Zacharewski.  In Vitro Bioassays for Assessing Estrogenic Substances , 1997 .

[9]  K. Korach,et al.  Techniques for detection of estrogenicity. , 1995, Environmental health perspectives.

[10]  W. Verstraete,et al.  Optimization of a yeast estrogen screen and its applicability to study the release of estrogenic isoflavones from a soygerm powder. , 2001, Environmental health perspectives.

[11]  M. Schalk,et al.  Species-specific antagonism of Ah receptor action by 2,2',5,5'-tetrachloro- and 2,2',3,3'4,4'-hexachlorobiphenyl. , 1995, European journal of pharmacology.

[12]  K. Korach,et al.  Printed in U.S.A. Copyright © 1997 by The Endocrine Society Estrogenic Activity of a Dieldrin/Toxaphene Mixture in the Mouse Uterus, MCF-7 Human Breast Cancer Cells, and Yeast-Based Estrogen Receptor Assays: No Apparent Synergism* , 2022 .

[13]  K. Korach,et al.  Targeted Disruption of the Estrogen Receptor-α Gene in Female Mice: Characterization of Ovarian Responses and Phenotype in the Adult. , 1999, Endocrinology.

[14]  J. Sumpter,et al.  Endocrine disruption in wildlife: a critical review of the evidence. , 1998, Critical reviews in toxicology.

[15]  J. Pike,et al.  Analysis of estrogen receptor function in vitro reveals three distinct classes of antiestrogens. , 1995, Molecular endocrinology.

[16]  R. M. Sharpe,et al.  Are oestrogens involved in falling sperm counts and disorders of the male reproductive tract? , 1993, The Lancet.

[17]  K. Melcher,et al.  Zero background yeast reporter plasmids. , 2000, Gene.

[18]  L. Guarente,et al.  Heme regulates transcription of the CYC1 gene of S. cerevisiae via an upstream activation site , 1983, Cell.

[19]  J. Shine,et al.  Sequence and expression of human estrogen receptor complementary DNA. , 1986, Science.

[20]  A. Murk,et al.  Estrogenic activity of estradiol and its metabolites in the ER‐CALUX assay with human T47D breast cells Note , 2001, APMIS : acta pathologica, microbiologica, et immunologica Scandinavica.

[21]  J. Gustafsson,et al.  Estrogen receptor (ER)β knockout mice reveal a role for ERβ in migration of cortical neurons in the developing brain , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[22]  S. Inoue,et al.  Interaction of Phytoestrogens with Estrogen Receptors α and β , 2001 .

[23]  A D Vethaak,et al.  Development of a stably transfected estrogen receptor-mediated luciferase reporter gene assay in the human T47D breast cancer cell line. , 1999, Toxicological sciences : an official journal of the Society of Toxicology.

[24]  H. Kuiper,et al.  Validation and use of the CALUX-bioassay for the determination of dioxins and PCBs in bovine milk. , 1998, Food additives and contaminants.

[25]  K. Schramm,et al.  Applicability of a yeast oestrogen screen for the detection of oestrogen-like activities in environmental samples. , 1999, Chemosphere.

[26]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[27]  J. Sumpter,et al.  Structural Features of Alkylphenolic Chemicals Associated with Estrogenic Activity* , 1997, The Journal of Biological Chemistry.

[28]  J. White,et al.  Synergistic activation of transcription by the human estrogen receptor bound to tandem responsive elements. , 1990, The EMBO journal.

[29]  A R Woodward,et al.  Developmental abnormalities of the gonad and abnormal sex hormone concentrations in juvenile alligators from contaminated and control lakes in Florida. , 1994, Environmental health perspectives.

[30]  S Falkow,et al.  Yeast-enhanced green fluorescent protein (yEGFP): a reporter of gene expression in Candida albicans. , 1997, Microbiology.

[31]  M. Pike,et al.  Estrogens, progestogens, normal breast cell proliferation, and breast cancer risk. , 1993, Epidemiologic reviews.

[32]  C. Portier,et al.  Evaluation of chemicals with endocrine modulating activity in a yeast-based steroid hormone receptor gene transcription assay. , 1997, Toxicology and applied pharmacology.