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Water Extract Screening in Yeast Receptor-Reporter Mediated Assay Functional Assay in Yeast Figure 1: Functional Yeast Assay. Yeast carrying a hormone responsive beta-galactosidase reporter plasmid and a steroid hormone receptor expression. The level of beta- galactosidase expression serves as a direct quantitative measure of steroid hormone receptor function. Abstract Endocrine disrupting chemicals (EDCs) are chemicals that interfere with the endocrine system in humans and animals. These nonsteroidial substances have the ability to bind to the human estrogen receptor, and have been found to mimic the activity of Estrodial (Estrogen 17 β-Estrodial), the natural form of estrogen. They are widely distributed within our environment, are associated with certain cancers, reproductive disorders, and developmental disorders, and have been previously detected in agriculturally intense watersheds. This study aims to use yeast bioassays to test for estrogenic activity in water and sediment sample extracts from experiments designed to measure 17 β-Estrodial and estrogen degradation in water and sediment . Synthetic Complete Media (SC-UW) and DSY- 219 yeast strains were used to test twenty-four water samples using receptor-mediated β- galactosidase reporter assays. Throughout a three-month period results were examined from a number of four- hour yeast experiments, and samples displayed significant estrogenic activity. These results will be correlated with additional data from chemical analysis in the future. These studies will ultimately aid in the understanding of the environmental fate, transport and degradation of these specific endocrine-disrupting chemicals. Introduction Nonsteroidal substances have the ability to bind to human estrogen receptor α (ERα) and mimic estrogen 17β-estradiol. These Xenoestrogens are thought to disrupt the normal endocrine function in humans and animals. The Cox lab studies steroid hormone receptor function, and it’s relation to human disease, specifically hormonal disorders. Saccharomyces cerevisiae is the model system used in the Functional Yeast Bioassay. Containing the appropriate estrogen receptor expression cassette, and reporter construct, it served as the model system for the detection of environmental estrogens in water and sediment samples. Conclusions/ Future Directions Estrogenic activity was found in environmental water extracts Results from this study correlated with further chemical analysis will be performed Testing for EDCs in our environment can only aid in finding a resolution to this growing problem of increased levels of environmental estrogens and human exposure The Yeast Receptor Mediated Assay can also be used to detect androgenic and steroidal environmental contaminants Acknowledgements This project was supported in part by grants to the Border Biomedical Research Center from the National Center for Research Resources (5G12RR008124) and the National Institute on Minority Health and Health Disparities (8G12MD007592) from the National Institutes of Health. We thank the Border Biomedical Research Center’s Biomolecule Analysis Core Facility (BACF), Tissue Culture Core Facility (TCF) , and the DNA Analysis Core Facility (DACF) for the use of the instruments. This project was also supported in part by American Recovery and Reinvestment Act (ARRA) funds through grant number SC1GM084863 to M.B.C. from the National Institute of General Medical Sciences, NIH and the Cancer Prevention and Research Institute of General Medical Sciences, NIH and the Cancer Prevention and Research Institute of Texas (CPRIT) References “A Four-Hour Yeast Bioassay for the Direct Measure of Estrogenic Activity in Wastewater without Sample Extraction, Concentration, or Sterilization” Heather A. Balsiger, Roberto de la Torre, Wen-Yee Lee, and Marc B. Cox “A Role for αSGT in Steroid Hormone Receptor Function” Atama Paul, Heather A. Balsinger, and Marc B. Cox Reina Hernandez 1 ; Jeffrey C. Sivils 1 , Ph.D. ; Yenni A. Garcia 1 ; Shannon Bartelt-Hunt 2 , Ph.D. ; Marc B. Cox 1 , Ph.D. 1: University of Texas at El Paso, Border Biomedical Research Center El Paso, TX. 2: University of Nebraska- Lincoln, Department of Civil Engineering, Lincoln, Nebraska. Yeast Bioassay is a Viable Tool to Determine Estrogenic Activity in Environmental Samples Figure 2: Functional Yeast Asssay. Yeast carrying a hormone responsive beta-galactosidase reporter plasmid and a steroid hormone receptor expression vector are transformed with αSGT and SGT2 expression vectors. The level of beta-galactosidase expression serves as a direct quantitative measure of steroid hormone receptor function. Figure 3. Yeast Bioassay is a Viable Tool to Determine Estrogenic Activity in Environmental Samples A. A dose response curve showing the reporter expression of S. cerevisiae in a yeast bioassay. The effective concentration of Estradiol is 17.0 nM against reporter expression. This data was used to determine relative estrogenic active of soil samples in B and C. Figure B and C demonstrate estrogenic activity over time in two different soil compositions. Relating to presume breakdown of the estrogenic compound over time.

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Water Extract Screening in Yeast Receptor-Reporter Mediated AssayFunctional Assay in Yeast

Figure 1: Functional Yeast Assay. Yeast carrying a hormone responsive beta-galactosidase reporter plasmid and a steroid hormone receptor expression. The level of beta-galactosidase expression serves as a direct quantitative measure of steroid hormone receptor function.

AbstractEndocrine disrupting chemicals (EDCs) are chemicals that interfere with the endocrine system in humans and animals. These nonsteroidial substances have the ability to bind to the human estrogen receptor, and have been found to mimic the activity of Estrodial (Estrogen 17 β-Estrodial), the natural form of estrogen. They are widely distributed within our environment, are associated with certain cancers, reproductive disorders, and developmental disorders, and have been previously detected in agriculturally intense watersheds. This study aims to use yeast bioassays to test for estrogenic activity in water and sediment sample extracts from experiments designed to measure 17 β-Estrodial and estrogen degradation in water and sediment . Synthetic Complete Media (SC-UW) and DSY-219 yeast strains were used to test twenty-four water samples using receptor-mediated β-galactosidase reporter assays. Throughout a three-month period results were examined from a number of four- hour yeast experiments, and samples displayed significant estrogenic activity. These results will be correlated with additional data from chemical analysis in the future. These studies will ultimately aid in the understanding of the environmental fate, transport and degradation of these specific endocrine-disrupting chemicals.

Introduction

Nonsteroidal substances have the ability to bind to human estrogen receptor α (ERα) and mimic estrogen 17β-estradiol. These Xenoestrogens are thought to disrupt the normal endocrine function in humans and animals. The Cox lab studies steroid hormone receptor function, and it’s relation to human disease, specifically hormonal disorders. Saccharomyces cerevisiae is the model system used in the Functional Yeast Bioassay. Containing the appropriate estrogen receptor expression cassette, and reporter construct, it served as the model system for the detection of environmental estrogens in water and sediment samples.

Conclusions/ Future Directions Estrogenic activity was found in

environmental water extracts Results from this study correlated with

further chemical analysis will be performed

Testing for EDCs in our environment can only aid in finding a resolution to this growing problem of increased levels of environmental estrogens and human exposure

The Yeast Receptor Mediated Assay can also be used to detect androgenic and steroidal environmental contaminants

AcknowledgementsThis project was supported in part by grants to the Border Biomedical Research Center from the National Center for Research Resources (5G12RR008124) and the National Institute on Minority Health and Health Disparities (8G12MD007592) from the National Institutes of Health. We thank the Border Biomedical Research Center’s Biomolecule Analysis Core Facility (BACF), Tissue Culture Core Facility (TCF) , and the DNA Analysis Core Facility (DACF) for the use of the instruments. This project was also supported in part by American Recovery and Reinvestment Act (ARRA) funds through grant number SC1GM084863 to M.B.C. from the National Institute of General Medical Sciences, NIH and the Cancer Prevention and Research Institute of General Medical Sciences, NIH and the Cancer Prevention and Research Institute of Texas (CPRIT) through grant number RP110444-P2. NIH 2R25GM069621-10 (RISE)

References “A Four-Hour Yeast Bioassay for the Direct

Measure of Estrogenic Activity in Wastewater without Sample Extraction, Concentration, or Sterilization” Heather A. Balsiger, Roberto de la Torre, Wen-Yee Lee, and Marc B. Cox

“A Role for αSGT in Steroid Hormone Receptor Function” Atama Paul, Heather A. Balsinger, and Marc B. Cox

Use of Yeast Bioassays in Testing for Estrogenic Compounds in Environmental SamplesReina Hernandez1 ; Jeffrey C. Sivils1, Ph.D. ; Yenni A. Garcia1 ; Shannon Bartelt-Hunt2, Ph.D. ; Marc B. Cox1, Ph.D.

1: University of Texas at El Paso, Border Biomedical Research Center El Paso, TX. 2: University of Nebraska- Lincoln, Department of Civil Engineering, Lincoln, Nebraska.

Yeast Bioassay is a Viable Tool to Determine Estrogenic Activity in Environmental Samples

Figure 2: Functional Yeast Asssay. Yeast carrying a hormone responsive beta-galactosidase reporter plasmid and a steroid hormone receptor expression vector are transformed with αSGT and SGT2 expression vectors. The level of beta-galactosidase expression serves as a direct quantitative measure of steroid hormone receptor function.

Figure 3. Yeast Bioassay is a Viable Tool to Determine Estrogenic Activity in Environmental SamplesA. A dose response curve showing the reporter expression of S. cerevisiae in a yeast bioassay. The effective concentration of Estradiol is 17.0 nM against reporter expression. This data was used to determine relative estrogenic active of soil samples in B and C. Figure B and C demonstrate estrogenic activity over time in two different soil compositions. Relating to presume breakdown of the estrogenic compound over time.