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Extramural Research

2006 Progress Report: Development and Application of a Bioluminescent Yeast-Reporter System for Screening Chemicals for Estrogenic and Androgenic Effects

EPA 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: Mustra, David
Project Period: October 1, 2003 through September 30, 2007
Project Period Covered by this Report: October 1, 2005 through September 30, 2006
Project Amount: $391,505
RFA: Development of High-Throughput Screening Approaches for Prioritizing Chemicals for the Endocrine Disruptors Screening Program (2003)
Research Category: Economics and Decision Sciences , Endocrine Disruptors

Description:

Objective:

The specific objectives of this research project are to: (1) test the Saccharomyces cerevisiae BLYES using the proposed 78 substances (see Interagency Coordinating Committee on the Validation of Alternative Methods [ICCVAM], 2002) listed for validation of estrogen receptors; (2) use the S. cerevisiae BLYES reporter for assessing antiestrogenic activity of known antiestrogens; and (3) develop a yeast-based reporter for the detection of androgens.

Progress Summary:

Test the S. cerevisiae BLYES Using the Proposed 78 Substances (see ICCVAM, 2002) Listed for Validation of Estrogen Receptors

Sixty-four substances listed in Appendix A have been tested (ICCVAM, 2002). Three chemicals (dibenzo[a,h]anthracence, reserpine, L-Thyroxine ) were not tested because of hydrophobicity issues. This portion of the project has moved into the data analysis phase. Data analysis includes determining the reproducibility of the assay and calculating the EC50 value for each test chemical and comparison to known literature values.

Use the S. cerevisiae BLYES Reporter for Assessing Antiestrogenic Activity of Known Antiestrogens

The bioluminescent assay was assessed for its utility in measuring antagonistic activity. The assay followed the procedure of Conroy, et al. (2005). In essence, 17β-estradiol dilution curves were assayed in a background of 4-OH tamoxifen present at different concentrations. As shown in Figure 1, the 17β-estradiol sigmoidal curves in the presence of increasing concentrations of 4-OH tamoxifen shifted to the right. EC50 values for 17β-estradiol increased in the presence of increasing concentrations of 4-OH tamoxifen (Table 1). Interestingly, a plot of these EC50 values versus the concentration of 4-OH tamoxifen showed a sigmoidal shaped curve (Figure 2). However, these data need to be reproduced.

Antagonist assays are being conducted with 4-OH tamoxifen, tamoxifen, flavone, and n-nonylphenol.

Sigmoidal Response Curves of 17β-Estradiol in the Presence of Increasing Concentrations of 4-OH Tamoxifen. Curve fitting was performed by SigmaPlot 9.0 using a sigmoidal 3 parameter function

Figure 1. Sigmoidal Response Curves of 17β-Estradiol in the Presence of Increasing Concentrations of 4-OH Tamoxifen. Curve fitting was performed by SigmaPlot 9.0 using a sigmoidal 3 parameter function.

Table 1. Calculated EC50 Values for 17β-Estradiol in the Presence of Increasing Concentrations of 4-OH Tamoxifen

4-OH Tamoxifen (μM)

Calculated EC50 Value

0

2.93 x 10-10

0.10

2.65 x 10-10

0.25

6.21 x 10-10

0.50

1.57 x 10-9

0.75

2.74 x 10-9

1.00

3.04 x 10-9

17beta-Estradiol EC<sub>50</sub> Values Plotted Against Increasing Concentrations of 4-OH Tamoxifen. Curve fitting was performed by SigmaPlot 9.0 using a sigmoidal 3 parameter function

Figure 2. 17β-Estradiol EC50 Values Plotted Against Increasing Concentrations of 4-OH Tamoxifen. Curve fitting was performed by SigmaPlot 9.0 using a sigmoidal 3 parameter function.

Develop a Yeast-Based Reporter for the Detection of Androgens Four variations of an androgen bioreporter have been developed and preliminary testing was conducted. Sequences for each variation are shown in Figure 3. Maximal expression was demonstrated when quadruple androgen response elements (AREs) were used (data not shown). This strain is called Saccharomyces cerevisiae BLYAS4. A constitutive bioluminescent strain to measure chemical toxicity has also been developed (S. cerevisiae BLYRa).

Sequence of Four Androgen Response Elements Based on Information From Horie-Inoue, et al. (2004) and Haendler, et al. (2001)

Figure 3. Sequence of Four Androgen Response Elements Based on Information From Horie-Inoue, et al. (2004) and Haendler, et al. (2001).

Currently, six known androgens have been tested with BLYAS4 in addition to the positive control 5α-DHT. Response curves and toxicity controls for three chemicals and 5α-DHT are shown in Figure 4A and 4B, respectively.

Figure 4. Androgen and Toxicity Response Curves for Selected Chemicals Using S. cerevisiae BLYAS4 (A) and S. cerevisiae BLYASa (B)

EC50 values calculated from these response curves are compared against literature values in Table 2. All chemicals tested except medroxyprogesterone were close to literature values. Medroxyprogesterone differed from the literature by three orders of magnitude. This may be because of differences in the assay conducted by Sonneveld, et al. (2006). Sonneveld, et al. used mammalian cell lines as host cells. Mammalian cell lines may be hydroxylating the medroxyprogesterone, causing the increase in androgenic activity. The yeast host does not hydroxylate the compound thereby giving a muted androgenic response.

Fluoxymestrone demonstrated toxicity at concentrations higher than 1 x 10-6 M, and Mifepristone had slight toxicity at concentrations higher than 1 x 10-6 M.

Table 2. Calculated EC50 Values for Selected Androgens Using S. cerevisiae BLYAS4

Chemicals

n

BLYAS EC50

Reported

Source

DHT

6

9.77 x 10-9

3.50 x 10-9 2.23 x 10-9 1.26 x 10-10

Gaido et al. 1997 Fang et al. 2003 Sonneveld et al. 2006

Mifepristone (RU486)

3

1.02 x 10-6

>1.0 x 10-5

Sonneveld et al. 2006

Fluoxymestrone

3

1.15 x 10-7

   

Medroxyprogesterone

3

2.36 x 10-6

6.31 x 10-9

Sonneveld et al. 2006

4-Androstenedione

3

9.97 x 10-8

5E-07 1.28 x 10-6 3.98 x 10-9

Michelini et al. 2005 Fang et al. 2003 Sonneveld et al. 2006

Testosterone

3

5.99 x 10-9

1 x 10-8 4.73 x 10-9 1.59 x 10-8 6.31 x 10-10

Michelini et al. 2005 Gaido et al. 1997 Fang et al. 2003 Sonneveld et al. 2006

17β-Trenbolone

3

1.96 x 10-8

2.75 x 10-9

Fang et al. 2003

Calculated EC50 values for androgens that have been both reported in the literature and tested with the BLYAS4 strain. Each EC50 value obtained with the BLYAS4 strain is the average of at least three independent replicates (n). Gaido, et al. (1997) performed androgen testing with a yeast-based colorimetric reporter strain. The study by Fang, et al. (2003) was a competitive binding assay (examined androgen receptor binding to chemicals, rather than using a reporter strain). Michelini, et al. (2005) performed a yeast-based bioluminescent assay utilizing the luc-skl system, thereby adding an exogenous substrate D-luciferin. Sonneveld, et al. (2006) used a human cell line (U2-OS)-based AR CALUX assay. Strain BLYAS is a self-contained yeast-based bioluminescent assay without requiring exogenous substrate addition.

Future Activities:

Future Activities: During Year 4, we will:

  • Perform statistical analysis of data gathered using the S. cerevisiae BLYES strain (Task 1) and manuscript preparation (Task 1).
  • Complete testing of the S. cerevisiae BLYAR strain (Task 4).
  • Test the androgen bioreporter using the same 78 substances (Task 4).
  • Prepare and submit two manuscripts for peer-review.
  • Prepare and submit the final report.

References:

Conroy O, Quanrud DM, Wendell PE, Wicke D, Lansey KE, Arnold RG. Fate of wastewater effluent hER-agonists and hER-antagonists during soil aquifer treatment. Environmental Science & Technology 2005;39:2287-2293.

Fang H, Tong W, Branham WS, Moland CL, Dial SL, Hong H, Xie Q, Perkins R, Owens W, Sheehan DM. Study of 202 natural, synthetic and environmental chemicals for binding to the androgen receptor. Chemical Research in Toxicology 2003;16:1338-1358.

Gaido KW, Leonard LS, Lovell S, Gould JC, Babai D, Portier CJ, McDonnell DP. Evaluation of chemicals with endocrine modulating activity in a yeast-based steroid hormone receptor gene transcription assay. Toxicology and Applied Pharmacology 1997;143:205-212.

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, 2002.

Michelini E, Leskinen P, Virta M, Karp M, Roda A. A new recombinant cell-based bioluminescent assay for sensitive androgen-like compound detection. Biosensors and Bioelectronics 2005;20:2261-2267.

Sonneveld E, Riteco JAC, Jansen HJ, Pieterse B, Brouwer A, Schoonen WG, van der Burg B. Comparison of in vitro and in vivo screening models for androgenic and estrogenic activities. Toxicological Sciences 2006;89(1):173-187.


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

Type Citation Project Document Sources
Journal Article 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.
abstract available   full text available
R831302 (2004)
R831302 (2005)
R831302 (2006)
R831302 (Final)
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  • Supplemental Keywords:

    automation, Photorhabdus luminescens, bioreporter, detection, endocrine disruption, androgen bioreporter, estrogen bioreporter, Saccharomyces cerevisiae,, RFA, Health, Scientific Discipline, POLLUTANTS/TOXICS, Environmental Chemistry, Health Risk Assessment, Chemicals, Endocrine Disruptors - Environmental Exposure & Risk, endocrine disruptors, Risk Assessments, Ecological Risk Assessment, Endocrine Disruptors - Human Health, bioindicator, assays, biomarkers, EDCs, endocrine disrupting chemicals, exposure studies, animal model, sexual development, bioluminescent testing, mechanistic screening, animal models, human growth and development, toxicity, endocrine disrupting chemcials, estrogen response, invertebrates, invertebrate model, hormone production, androgen, estrogen receptors, ecological risk assessment model, assessment technology

    Progress and Final Reports:
    Original Abstract
    2004 Progress Report
    2005 Progress Report
    Final Report

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