Grantee Research Project Results
Final 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 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 primary objectives of this research were to (i) validate the Saccharomyces cerevisiae 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 project activities performed to meet these objectives were:
- Redesigning and constructing S. cerevisiae BLYES and S. cerevisiae BLYR for the detection and measuring estrogenic and chemical toxicity, respectively,
- Designing and constructing S. cerevisiae BLYAS for the detection of androgenic compounds and measuring androgenic activity,
- Developing the assay as a high throughput method for screening chemicals,
- Testing each assay using a suite of test substances as outlined by the ICCVAM report (2002).
Summary/Accomplishments (Outputs/Outcomes):
Three S. cerevisiae strains were developed to produce a measurable bioluminescent signal in response to chemicals with estrogenic, androgenic, or toxic activities (Sanseverino et al., 2005; Eldridge et al., 2007). Assays with strains BLYES and BLYAS were characterized using 17β-estradiol and dihydroxytestosterone (DHT) as standards. In addition, these strains were tested using a suite of chemicals with known hormonal responses (ICCVAM, 2002). When used as a Tier I screening tool, four outcomes were determined to be:
- Chemical is hormonally active. These chemicals induce bioluminescence in BLYES and BLYAS, produce a sigmoidal curve, and exhibit no toxicity. Chemicals tested that fall into this category include17β-Estradiol, butyl benzyl phthalate, diethylstilbestrol, p-nonylphenol, 4-OH-tamoxifen, and DHT, androstenedione, clomiphene citrate, and 17β-trenbolone.
- Chemical is toxic. These chemicals result in a decrease in bioluminescence in the constitutive strain BLYR. Chemicals that fall into this category include atrazine, haloperidol, kepone, methoxychlor, and sodium azide. Generally, an IC50 cannot be determined from this data, but an IC20 can be calculated. The IC20 is defined as the concentration at which bioluminescence is reduced by 20 percent.
- Chemical is not hormonally active and not toxic. There is no increase in bioluminescence in the BLYES and BLYAS strains and no decrease in bioluminescence in the BLYR strain. An example is phenolphthalein.
- Chemical has hormonal activity but an EC50 cannot be calculated. This group produced limited bioluminescence (no sigmoidal curve) in BLYES and BLYAS. Bioluminescence may be hampered due to a chemical’s toxicity, uptake by the cells, or the concentration range tested was not broad enough to capture the full sigmoidal curve. Examples of this type of response include bisphenol A, fenarimol, flavone, ketoconaide, phenobarbitol, bicalutamide and sodium azide. A minimum concentration to induce bioluminescence can be calculated from this data.
The reproducibility of the standard curves and the range of responses for each test chemical, allowed development of quantitative rules for data interpretation. The proposed rules define if the data obtained from each assay is acceptable or not and if an EC50 can be determined. These rules are:
- Bioluminescence produced in the methanol control must be less than 150% of the bioluminescence produced in the assay blank.
- Minimum bioluminescence in the 17β-estradiol and DHT standard curve must be approximately 29,600 ± 9,400 and 23,500 ± 6,000 counts per second, respectively.
- Maximum bioluminescence in the 17β-estradiol and DHT standard curve must be approximately 190,200 ± 14,000 and 379,000 ± 88,500 counts per second, respectively.
- 17β-Estradiol and DHT standard curve plots and test chemical plots should be sigmoidal.
- Values for the upper and lower limbs for each test chemical must be within the standard deviation of the respective standard curves.
- Chemical toxicity, measured by the constitutive bioluminescent strain S. cerevisiae BLYR, must be absent in the sigmoidal part of the standard curve.
Conclusions:
These bioreporter strains can be used in high throughput Tier I screening to determine if a chemical has endocrine disrupting activity or toxicity. Uses of these assays offer the following improvements over other methods of detection of hormonally active compounds:
- Automation of chemical, medium, and cell distribution to microtiter plates and, with the proper robotics, transfer to a plate reader.
- Speed of data collection with data collected in 3-4 hours.
- Ease of data collection by downloading to a spreadsheet and analysis and interpretation by computer algorithm.
These bioluminescent bioreporter strains may also be used for monitoring of waterways, wastewater treatment plant influents and effluents, runoff from farms, etc. It is well-documented that pharmaceuticals and personal care products, as well as other organic pollutants, that cause endocrine-disrupting activity are present in our nation’s water ways (e.g. Kolpin et al., 2002). These assays may provide a rapid means of assessing if a water sample has activity before conducting expensive analytical methodology. This activity can be conducted in the laboratory via water collection and spotting of the microtiter plates as described. An alternative approach would be to conduct real-time online monitoring by integrating these bioluminescent bioreporters with integrated circuitry equipped with photodetectors.
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, Eldridge ML, Layton AC, Easter JP, Yarbrough J, Schultz TW, Sayler GS. Screening of potentially hormonally active chemicals using bioluminescent yeast bioreporters. Toxicological Sciences 2009;107(1):122-134. |
R831302 (Final) R832740 (2008) R832740 (Final) |
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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.