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A Bioluminescent Yeast-Reporter System For A Bioluminescent Yeast-Reporter System for Screening Chemicals for Estrogenic Effects John Sanseverino, The Center for Environmental Biotechnology The University of Tennessee Abstract Saccharomyces cerevisiae BLYES The Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC) created by the Environmental Protection Agency was mandated with developing methods to screen PGK GPD ADH1 ERE luxA approximately 87,000 chemicals for biological effects on estrogen, androgen and thyroid luxC ERE luxE IRES hormone systems. As part of this mandate, EDSTAC proposed that EPA develop rapid, high IRES IRES pBLYES luxD pLCIRESDE- frp throughput screening systems to assess a compound’s effects on hormonal systems. The ~10kb luxB IRESfrp Center for Environmental Biotechnology at the University of Tennessee has re-engineered ~12.1kb the Saccharomyces cerevisiae YES colorimetric estrogen reporter system (Routledge and Sumpter, 1996) to produce bioluminescence in response to estrogen or environmental A B estrogens (S. cerevisiae BLYES; Figure 1; Gupta et al., 2003). Bioluminescence is a Luciferase FMNH reagentless system eliminating the need for expensive chromophores. Light detection is 2 C D E more sensitive than absorbance detection thus shortening the development time of the assay. Light FMN 490nm In previous work, strain BLYES was exposed to the estrogenic compounds 17β-estradiol, Myristyl aldehyde 17α-estradiol, 17α-ethynyl estradiol, estrone, and 3,4′,5-trichloro-4-biphenylol and 2 O compared to the YES assay (Fig. 2). The EC values correlated linearly (R =0.97) between 2 50 Myristic acid the 2 assays. Sensitivities of both assays decreased in the order 17β-estradiol > 17α-ethynyl H O - estradiol > estrone > 17α-estradiol, with no significant response generated from 3,4′,5- 2 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 Figure. 1. Construction of the estrogen inducible bioreporter S. cerevisiae BLYES. detectable in less than 4 hours as compared to 3 days for the colorimetric YES strain. Synthesis of luxA is regulated on plasmid pEREAB by upstream incorporation of two The primary objective of this research is to validate the BLYES system and develop a sequential estrogen response elements (ERE) coupled to a human phosphoglycerate kinase standard operating procedure for routine chemical analysis. Work is in progress to test the (PGK) promoter. The luxB component of the luciferase is supplied via independent S. cerevisiae BLYES using the proposed 78 substances (ICCVAM, 2002) listed for expression from a fused IRES. The luxC, luxD, luxE and frp genes are under control of validation of estrogen receptors and correlate to the colorimetric S. cerevisiae YES assay. constitutive promoters on the plasmid pLCDEfrp. The human estrogen receptor (hER-α) is Parallel research includes developing the S. cerevisiae BLYES into a standard assay suitable inserted in the S. cerevisiae chromosome. for HTS of chemicals and to modify the lux genes for optimum transcription/translation in S. cerevisiae thus increasing sensitivity of the assay. Goals The purpose of Tier 1 screening is to identify substances that have the potential to interact 200 ) 17β-estradiol (R2 = 0.98) with the endocrine system. The colorimetric-based YES assay has been widely used in the 600 180 17α-ethynyl estradiol (R2 = 0.99) literature and this laboratory and is a very useful tool for assessing estrogenicity of a estrone (R2 = 0.99) 160 17α-estradiol (R2 = 0.98) compound or environmental sample. Development of the bioluminescent version of the 140 YES system (S. cerevisiae BLYES) has the potential to enhance the utility of this system. 120 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 100 bioluminescent reporter system analogous to the BLYES system. 80 -7.6 -7.8 y = 0.78x - 1.03 2 The specific tasks of this research are: 60 R = 0.99 -8.0 40 -8.2 1. Test the S. cerevisiae BLYES using the proposed 78 substances (see ICCVAM, 2002) -8.4 20 log [BLYES] (M) listed for validation of estrogen receptors and correlate to the colorimetric S. cerevisiae -8.6 YES assay. 0 -8.8 -10.0 -9.8 -9.6 -9.4 -9.2 -9.0 -8.8 -8.6 -8.4 Normalized Bioluminescence (cps/OD Bioluminescence Normalized log [YES] (M) 2. Develop the S. cerevisiae BLYES into a standard assay suitable for HTS of chemicals. 10-11 10-10 10-9 10-8 10-7 10-6 10-5 Concentration (M) 3. Modify plasmid pEREAB construct for improved sensitivity. 4. Develop a yeast-based reporter for the detection of androgens. Figure 2. EC50 dose response profiles of the S. cerevisiae BLYES bioreporter to the estrogenic compounds 17β-estradiol (z), 17α-ethynyl estradiol (V), estrone (), and 17α-estradiol (); (n = 4). Inset: EC50 dose response correlations between the lacZ based YES and lux based BLYES estrogenic assays. 70,000 Impact 60,000 50,000 This EPA-sponsored research seeks to standardize a bioluminescent yeast assay for screening substances that potentially interact with the human estrogen receptor. The 40,000 bioluminescent reporter system has several advantages including: 30,000 • A low-cost, reagentless reporter system that eliminates the extra manipulation and cost of adding exogenous reagents. Bioluminescence 20,000 • Speed; preliminary data using 17β-estradiol indicated a bioluminescent response 10,000 in 2-4 hours and a maximum response in 15 hours (Fig. 3). 0 62.5 0 3 6 • Amenable to automated, high throughput analysis, data collection and 9 12 ppb interpretation. 15 18 21 24 0 27 Hours 30 o 33 • Cells for the assay can be prepared fresh or stored at –80 C. 36 • No animals are used in this assay. Figure 3. 3-Dimensional plot of bioluminescence vs. time for 0-62.5 ppb 17β-estradiol. Initial bioluminescence is observed in as little as 2 hours and peaks at approximately 15 h. • Elimination of labor-intensive cell culture assays. Acknowledgements Literature Cited Funds for this research are provided by EPA STAR Grant R-831302 and Gupta, R.K., S.S. Patterson, S. Ripp, M.L. Simpson, and G.S. Sayler. 2003. Expression of the The Center for Environmental Biotechnology at The University of Photorhabdus luminescens lux genes (luxA, B, C, D, and E) in Saccharomyces cerevisiae. FEMS Yeast Research. 4:305-313. Tennessee. Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM). 2002. Expert Principle Investigators/Contact Information Panel Evaluation on the Validation Status of In Vitro Test Methods for Detecting Endocrine Disruptors: Gary Sayler, T. Wayne Schultz, Alice Layton, John Sanseverino, Leslie Estrogen Receptor and Androgen Receptor Binding and Transcription Activation Assays. Expert Panel Final Report. Saidak, James Easter, Victoria Garrett. The Center for Environmental Biotechnology, The University of Tennessee 676 Dabney Hall, Routledge, E.J., and J.P. Sumpter. 1996. Estrogenic activity of surfactants and some of their degradation Knoxville, Tn 37996 products assessed using a recombinant yeast screen. Environ. Toxic. Chem. 15(3):241-248..
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