Grantee Research Project Results
2005 Progress Report: Development and Application of a Bioluminescent Yeast-Reporter System for Screening Chemicals for Estrogenic and Androgenic Effects
EPA Grant Number: R831302Title: Development and Application of a Bioluminescent Yeast-Reporter System for Screening Chemicals for Estrogenic and Androgenic Effects
Investigators: Sayler, Gary S. , Layton, Alice C. , Schultz, T. Wayne , Sanseverino, John
Institution: University of Tennessee
EPA Project Officer: Aja, Hayley
Project Period: October 1, 2003 through September 30, 2007
Project Period Covered by this Report: October 1, 2004 through September 30, 2005
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
Objective:
The specific objectives of this research project are to:
(1) test the Saccharomyces cerevisiae BLYES using the proposed 78 substances (ICCVAM, 2002) listed for validation of estrogen receptors; (2) develop the S. cerevisiae BLYES into a standard assay suitable for high throughput screening (HTS) of chemicals; and (3) develop a yeast-based reporter for the detection of androgens.
Progress Summary:
Tasks 1 and 2
Task 1 (Test the S. Cerevisiae BLYES Using the Proposed 78 Substances (see ICCVAM, 2002) Listed for Validation of Estrogen Receptors) and Task 2 (Correlate to the Colorimetric S. Cerevisiae YES Assay and Develop the S. Cerevisiae BLYES Into a Standard Assay Suitable for HTS of Chemicals) have been proceeding simultaneously. The HTS assay development is built around a Beckman F/X Automated Liquid Handling System (LHS). This instrument includes a Span-8 pipettor assembly and a 96 pipette tip head. The Span-8 is 8 individual pipettors that can be programmed individually to perform unique liquid transfers while the 96-tip head can replicate microtiter plates rapidly. Our strategy is to let the LHS dilute the chemical over the test concentration range and deliver the dilutions to each microtiter plate.
- Each microtiter plate will include dilutions of four test chemicals, a methanol control, and a blank. Dilutions will range from 10-3 M to 2.5 x 10-9 M.
- Every third plate will have a 17β-estradiol standard curve.
- The LHS will prepare the dilutions on a master microtiter plate using the Span-8 pipettor.
- The 96-well pipette head will deliver 20 μL of the diluted chemicals to replicate microtiter plates. The methanol solvent will be allowed to dry.
- The 96-well pipette head will deliver 200 μL of the bioreporter to each well.
- Microtiter plates will be transferred to a plate reader and bioluminescence will be monitored for 12 hours; the data will be downloaded to an Excel spreadsheet for analysis.
One technical issue was encountered in programming the LHS. Initially, nondisposable tips were used for chemical dilutions and ethanol was used to wash the tips in between transfers. This approach, however, affected the accuracy of the volume being transferred and thus the reproducibility of the assay. Switching to disposable pipette tips and eliminating the ethanol washing improved accuracy and reproducibility in the assay.
Organic Solvent. A second technical issue involved ethanol as the carrier solvent for each chemical. Ethanol was eliminated as a solvent carrier and replaced with methanol. Ethanol affected the baseline response of our assay resulting in increased background. We hypothesized that ethanol in the LHS solubilized plasticizers in the lines. Gas chromatography analysis of ethanol sampled from the stock bottles confirmed the presence of phthalates, which are known estrogenic compounds. In addition, there was lot-to-lot variability in the quality of ethanol received. Switching to methanol as the solvent eliminated these problems.
Comparison of Manual Versus LHS Assays. A comparison was made with chemicals diluted manually versus the LHS performing the dilutions. The chemicals used were 17β-estradiol, estrone, and 17α-ethynyl estradiol. Bioluminescence response data show nearly identical curves for each chemical (data not shown). EC50 values show strong agreement between assays and with previously published data (Table 1).
Table 1. Comparison of EC50 Values Between Assays Performed Manually and With the LHS
Chemical | Manual |
LHS |
Published1 |
17β-estradiol |
5 x 10-11 |
1.3 x 10-10 |
2.4 ± 1.0 x 10-10 |
Estrone |
1 x 10-10 |
5.0 x 10-10 |
5.5 x 10-10 |
17α-ethynyl estradiol |
6.5 x 10-11 |
2.5 x 10-11 |
2.5 x 10-11 |
1 Sanseverino, et al., 2005. |
Chemical Testing. To date, 26 of the 78 chemicals diluted in methanol have been tested (Table 2). Data analysis and calculation of EC50 values is in progress.
Task 3
In support of Task 3 (Modify the ERE-luxCDABE Construct for Improved Sensitivity), we reconstructed the estrogen bioreporter by inserting tandem estrogen response elements (EREs) between divergent yeast promoters GPD and ADH1 on pUTK401 (formerly pUA27B7; Gupta, et al., 2003). 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 estrogenic compounds. This work has been published (Sanseverino, et al., 2005).
Table 2. List of Chemicals Tested Using the Bioluminescence Assay and the LHS
Actinomycin D | o,p’-DDT |
Ammonium perchlorate |
Dexamethasone |
4-Androstenedione |
Dibenzo[a,h]anthracene |
Atrazine |
Di-n-butyl phthalate |
Butylbenzyl phthalate |
Diethylstilbestrol |
2-sec-butylphenol |
Diethylhexyl phthalate |
Clomiphene citrate |
17α-estradiol |
Corticosterone |
Estrone |
4-cumylphenol |
17α-Ethynyl estradiol |
Cycloheximide |
Ethyl-4-hydroxybenzoate |
Cyproterone acetate |
Fenarimol |
Daidzein |
Flavone |
p,p’-DDE |
Fluoranthene |
Recent efforts have focused on integrating the lux genes into the chromosome, which would alleviate the need for selective pressure. So far, the luxC-IRES-luxE and the luxD-IRES-frp genes have been integrated into S. cerevisiae hER (estrogen host strain) and S. cerevisiae hAR strains (androgen host strain). This has been accomplished by cloning the lux gene constructs into the S. cerevisiae integrative plasmid pRS305. Work is underway to insert the luxA and luxB under control of the EREs into the chromosome as well. All strains will be evaluated for their bioluminescent responses to 17β-estradiol and 5-DHT. The desired outcome is a bioreporter strain that is stable genetically and does not require continued selection.
Task 4
In support of Task 4 (Develop a Yeast-Based Reporter for the Detection of Androgens), we constructed the bioluminescent androgen bioreporter. The ERE sequences from plasmid pUTK407 were substituted with triple androgen response elements (ARE) to create pUTK410. This construct was generated by synthesis of the GPD promoter followed by the ARE sequences followed by the ADH1 promoter. The AREs used were originally described in Purvis, et al. (1991). The series of three AREs used were separated by one base pair.
After transformation of the S. cerevisiae hAR strain with plasmids pUTK404 and pUTK410, only constitutive bioluminescence was observed. Recovery of each plasmid and subsequent analysis demonstrated that the ARE elements were being excised from pUTK410 by the yeast host strain. Because these sequences can form stem and loop structures that may be amenable to excision, the triple AREs were replaced with double AREs analogous to the estrogen reporter plasmids. Unfortunately, the double AREs also were excised from the reporter plasmid.
Horie-Inoue, et al. (2004) published a paper that identified all the androgen response elements in human prostate cancer cells using an in silico analysis of the human genome. They identified the consensus androgen receptor binding site: a palindromic 15-base pair sequence, which consists of 2 hexameric half-sites (5’-AGAACA-3’) arranged as an inverted repeat with a 3 base pair spacer. Further, Haendler, et al. (2001) described the occurrence of 2 or 4 near consensus AREs for maximal induction separated by approximately 12 base pairs. Based on this information, the reporter plasmid is being redesigned using 2-4 perfect palindromic sequences (Horie-Inoue, et al., 2004) separated by a 12 base pair sequence (Figure 1).
A.
5’ - GGT ACA AAA TGT TCT A GGT ACA AAA TGT TCT C GGT ACA AAA TGT TCT- 3’
B.
5’- aga tct AGA ACA cta TGT TCT cta cta cta cta AGA ACA cta TGT TCT cta
cta cta cta AGA ACA cta TGT TCT ggc gcc -3’
Figure 1. Sequence of Androgen Response Elements From (A) Purvis, et al. (1991) and (B) Horie-Inoue, et al. (2004).
Future Activities:
In Year 3 of the grant, we will:
- Complete testing the S. cerevisiae BLYES strain using the proposed 78 substances (see ICCVAM, 2002) listed for validation of estrogen receptors. (Task 1).
- Continue to automate the assay using a Beckman F/X Automated Liquid Handling System (Task 2).
- Complete development of the bioluminescent bioreporter for the detection of androgens (Task 4).
- Test the androgen bioreporter using the same 78 substances (Task 4).
References:
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.
Haendler B, Schüttke I, Schleuning WD. Androgen receptor signalling: comparative analysis of androgen response elements and implication of heat-shock protein 90 and 14-3-3eta. Molecular and Cellular Endocrinology 2001;173(1-2):63-73.
Hori-Inoue K, Bono H, Okazaki Y, Inoue S. Identification and functional analysis of consensus androgen response elements in human prostate cancer cells. Biochemical and Biophysical Research Communications 2004;325:1312-1317.
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.
Purvis IJ, Chotai D, Dykes CW, Lubahn DB, French FS, Wilson EM, Hobden AN. An androgen-inducible expression system for Saccharomyces cerevisiae. Gene 1991;106:35-42.
Journal Articles on this Report : 1 Displayed | Download in RIS Format
Other project views: | All 7 publications | 2 publications in selected types | All 2 journal articles |
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Type | Citation | ||
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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. |
R831302 (2004) R831302 (2005) R831302 (2006) |
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Supplemental Keywords:
automation, Photorhabdus luminescens, bioreporter, detection, endocrine disruption, androgen bioreporter, estrogen bioreporter, Saccharomyces cerevisiae,, RFA, Scientific Discipline, Health, POLLUTANTS/TOXICS, Environmental Chemistry, Health Risk Assessment, Endocrine Disruptors - Environmental Exposure & Risk, Chemicals, Risk Assessments, endocrine disruptors, Ecological Risk Assessment, Endocrine Disruptors - Human Health, bioindicator, biomarkers, assays, animal model, EDCs, exposure studies, endocrine disrupting chemicals, sexual development, bioluminescent testing, mechanistic screening, animal models, human growth and development, toxicity, endocrine disrupting chemcials, estrogen response, invertebrates, invertebrate model, estrogen receptors, hormone production, androgen, assessment technology, ecological risk assessment modelProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.