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. , Schultz, T. Wayne
Current Investigators: Sayler, Gary S. , Layton, Alice C. , Schultz, T. Wayne , Sanseverino, John
Institution: University of Tennessee
EPA Project Officer: Klieforth, Barbara I
Project Period: October 1, 2003 through September 30, 2007
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: Environmental Justice , Endocrine Disruptors , Human Health , Safer Chemicals
The Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC) created by the Environmental Protection Agency 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 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 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 is to (i) validate the BLYES system and develop a standard operating procedure for routine chemical analysis; and (ii) develop an androgen bioluminescent reporter system analogous to the BLYES system.
The specific tasks of this research are:
1. Test the S. cerevisiae BLYES using the proposed 78 substances (see ICCVAM,
2002) listed for validation of estrogen receptors and correlate to the colorimetric
S. cerevisiae YES assay.
2. Develop the S. cerevisiae BLYES into a standard assay suitable for HTS of chemicals.
3. Modify the lux genes for optimum transcription/translation in S. cerevisiae thus increasing sensitivity of the assay.
4. Develop a yeast-based reporter for the detection of androgens.
In previous work, strain BLYES was exposed to the estrogenic compounds 17~-estradiol, 17~-estradiol, 17~-ethynyl estradiol, estrone, and 3,4~,5-trichloro-4-biphenylol and compared to the YES assay. The EC50 values correlated linearly (R2=0.97) between the two assays. Sensitivities of both assays decreased in the order 17~-estradiol > 17~-ethynyl - estradiol > estrone > 17~-estradiol, with no significant response generated from 3,4~,5-trichloro-4-biphenylol where the hydroxyl group is the stericly hindered by the paired ortho chlorines. The BLYES screen consistently detected estrogenic potencies at 5 to 10-fold lower levels than those attained in the YES assay. Moreover, bioluminescence was detectable in less than 4 hours as compared to 3 days for the colorimetric YES strain.
This proposal seeks to standardize the bioluminescent yeast assay for screening substances that potentially interact with the human estrogen receptor. Bioluminescence as a reporter has several advantages including:
· A reagentless reporter system eliminating the extra manipulation
and cost of adding exogenous reagents (compared to the colorimetric system).
· Speed – preliminary data using 17-estradiol indicated a bioluminescent response in 2-4 hours and a maximum response in 15 hours.
· A range of equipment can be used to detect bioluminescence.
· Dispensing of culture medium and cells into microtiter plates can be automated.
· Cells for the assay can be prepared fresh and used or stored at -80°C.
· Data collection and interpretation can be automated.
· No animals are used in this assay.
· Elimination of labor-intensive cell culture assays.
The expected results include two standardized, validated assays for rapidly screening chemicals for estrogenic, androgenic, anti-estrogenic, and anti-androgenic activity.