2004 Progress Report: Development and Application of a Bioluminescent Yeast-Reporter System for Screening Chemicals for Estrogenic and Androgenic EffectsEPA Grant Number: R831302
Title: Development and Application of a Bioluminescent Yeast-Reporter System for Screening Chemicals for Estrogenic and Androgenic Effects
Investigators: Sayler, Gary S. , Layton, Alice C. , Sanseverino, John , Schultz, T. Wayne
Institution: University of Tennessee - Knoxville
EPA Project Officer: Hahn, Intaek
Project Period: October 1, 2003 through September 30, 2007
Project Period Covered by this Report: October 1, 2003 through September 30, 2004
Project Amount: $391,505
RFA: Development of High-Throughput Screening Approaches for Prioritizing Chemicals for the Endocrine Disruptors Screening Program (2003) RFA Text | Recipients Lists
Research Category: Economics and Decision Sciences , Endocrine Disruptors , Health , Safer Chemicals
The Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC), created by the U.S. Environmental Protection Agency (EPA), was mandated with developing methods to screen approximately 87,000 chemicals for biological effects on estrogen, androgen, and thyroid hormone systems. As part of this mandate, EDSTAC proposed that EPA develop rapid, high throughput screening (HTS) systems to assess a compound’s effects on hormonal systems. The Center for Environmental Biotechnology at the University of Tennessee has re-engineered the Saccharomyces cerevisiae yeast estrogen screen (YES) colorimetric estrogen reporter system to produce bioluminescence in response to estrogen or environmental estrogens (S. cerevisiae BLYES). Bioluminescence is a reagentless system eliminating the need for expensive chromophores. Light-detection is more sensitive than absorbance detection thus shortening the development time of the assay.
The purpose of Tier 1 screening is to identify substances that have the potential to interact with the endocrine system (O’Conner, et al., 2002). The colorimetric-based YES has been widely used in the literature and this laboratory and is a very useful tool for assessing estrogenicity of a compound or environmental sample. Development of the bioluminescent version of the YES system (S. cerevisiae BLYES) has the potential to enhance the utility of this system. The primary objective of this research project is to:
- validate the BLYES system and develop a standard operating procedure for routine chemical analysis;
- and develop an androgen bioluminescent reporter system analogous to the BLYES system.
The specific tasks of this project are to:
- test the S. cerevisiae BLYES using the proposed 78 substances (see Interagency Coordinating Committee on the Validation of Alternative Methods [ICCVAM], 2003) listed for validation of estrogen receptors and correlate the results to the colorimetric S. cerevisiae YES assay;
- develop the S. cerevisiae BLYES into a standard assay suitable for HTS of chemicals;
- modify the lux genes for optimum transcription/translation in S. cerevisiae thus increasing sensitivity of the assay;
- and develop a yeast-based reporter for the detection of androgens.
Year 1 activities focused on improving the bioluminescent response of S. cerevisiae BLYES (Task 3). The strain originally described in the proposal had tandem estrogen response elements (ERE) in front of the PGK promoter upstream of the luxA-IRES-luxB construct. This construct was eliminated in favor of inserting tandem ERE between divergent yeast promoters GPD and ADH1 on pUTK401 (formerly pUA27B7; Gupta et al., 2003) that constitutively expressed luxA and luxB. Co-transformation of this plasmid with a second plasmid (pUTK404; formerly pLCIRESDEIRESEfrp, Gupta, et al., 2003) containing the genes required for aldehyde synthesis (luxCDE) and FMN reduction (frp) yielded a bioluminescent bioreporter responsive to estrogen disrupting compounds. Strain BLYES was exposed to the estrogenic compound 17 β-estradiol over the concentration range of 1.2 x 10-8 M through 5.6 x 10-12 M. The calculated EC 50 values from the colorimetric and bioluminescence assays (n = 7) were 4.4 ± 1.1 x 10-10 M and 2.4 ± 1.0 x 10-10 M. The lower and upper limits of detection for each assay were approximately 4.5 x 10-11 M to 2.8 x 10-9 M. Bioluminescence was observed in as little as 1 hour and reached its maximum in 6 hours. In comparison, the YES assay requires a minimum of 3 days for results. Strain BLYES fills the niche for rapid, HTS of estrogenic compounds along with the ability for remote, near real-time monitoring of endocrine disrupting chemicals in the environment.
In Year 2 of the research project, we will focus on the following activities:
- Testing the S. cerevisiae BLYES strain using the proposed 78 substances (see ICCVAM, 2002) listed for validation of estrogen receptors and correlate those results to the colorimetric S. cerevisiae YES assay (task 1).
- Continuing to automate the assay using a Beckman F/X Automated Liquid Handling System (task 2).
- Initiating development of the bioluminescent bioreporter for the detection of androgens (task 4).
Gupta RK, Patterson SS, Ripp S, Sayler GS. Expression of the Photorhabdus luminescens lux genes (luxA, B, C, D, and E) in Saccharomyces cerevisiae. FEMS Yeast Research 2003;4:305-313.
Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM). Expert panel evaluation on the validation status of in vitro test methods for detecting endocrine disruptors: estrogen receptor and androgen receptor binding and transcription activation assays. Expert Panel Final Report, NIH Publication No. 03-4503, May 2003.
O’Connor JC, Cook JC, Marty MS, Davis LG, Kaplan AM, Carney EW. Evaluation of tier I screening approaches for detecting endocrine-active compounds (EACs). Critical Review in Toxicology 2002;32(6):521-549.
Journal Articles on this Report : 1 Displayed | Download in RIS Format
|Other project views:||All 11 publications||3 publications in selected types||All 3 journal articles|
||Sanseverino J, Gupta RK, Layton AC, Patterson SS, Ripp SA, Saidak L, Simpson ML, Schultz TW, Sayler GS. Use of Saccharomyces cerevisiae BLYES expressing bacterial bioluminescence for rapid, sensitive detection of estrogenic compounds. Applied and Environmental Microbiology 2005;71(8):4455-4460.||