2008 Progress Report: Development of Receptor- to Population-Level Analytical Tools for Assessing Endocrine Disruptor Exposure in Wastewater-Impacted Estuarine SystemsEPA Grant Number: R832737
Title: Development of Receptor- to Population-Level Analytical Tools for Assessing Endocrine Disruptor Exposure in Wastewater-Impacted Estuarine Systems
Investigators: Ferguson, P. Lee , Chandler, G. Thomas
Institution: University of South Carolina at Columbia
EPA Project Officer: McOliver, Cynthia
Project Period: January 1, 2006 through December 31, 2009 (Extended to December 31, 2010)
Project Period Covered by this Report: January 1, 2008 through December 31,2008
Project Amount: $526,028
RFA: Exposure Measurement Tools for Endocrine Disrupting Chemicals in Mixtures (2005) RFA Text | Recipients Lists
Research Category: Endocrine Disruptors , Health , Safer Chemicals
The general objectives of the proposed research are to:
- Develop nuclear hormone receptor-affinity extraction techniques as tools for isolating endocrine disrupting chemicals (EDCs) from complex wastewater mixtures.
- Apply these methods in combination with high performance mass spectrometry for activity-directed analysis of EDCs in wastewater and estuarine receiving waters on the SC coast.
- Utilize sensitive vertebrate (zebrafish) and invertebrate (copepod) EDC-exposure laboratory bioassays to link exposure measurements (above) to biological effects.
- Apply novel biomolecular endpoints to assess EDC exposure in field populations of sensitive meiobenthic invertebrates in wastewater-impacted estuarine environments.
We have developed and fully validated a method based on receptor-affinity extraction of xenoestrogenic contaminants from complex wastewater environments (specific objective 1). This was enabled by our ability to successfully express and purify active ER-LBD (in fusion with his6 and thioredoxin) in ~10 mg L-1 yield and > 95% purity from bacterial expression hosts. The receptor thus purified is active to bind [3H]-17β-estradiol in solution and has been used in subsequent receptor-affinity extraction experiments. This work represents the first application of nuclear hormone receptors as analytical tools for isolating endocrine disrupting chemicals from complex mixtures.
The method that we have developed was used to isolate and quantify xenoextrogenic contaminants from biologically-treated wastewater and wastewater-impacted surface waters on Kiawah Island, SC. Estrogen receptor-affinity extraction was used in combination with HPLC-MS/MS to assay several known xenoestrogenic contaminants in water and wastewater. These included the female sex hormones 17β-estradiol (E2), estrone (E1), and estriol (E3) as well as the synthetic contraceptive ethynylestradiol (EE2). In addition, we quantified the non-steroidal xenoestrogens 4-nonylphenol (NP), 4-tert-octylphenol (OP), and bisphenol A (BPA). As expected, concentrations for most xenoestrogen analytes were highest in the wastewater effluent with decreased levels in surface waters, consistent with a wastewater source for these contaminants. NP and BPA were the most concentrated xenoestrogens, consistent with previous reports and with the use of these compounds in many consumer products. OP was present at much lower concentration in effluent than was NP, but was measured above 10 ng·L-1 in surface waters, possibly due to generation of OP from degradation of octylphenol ethoxylate surfactants following wastewater discharge. Biogenic steroid estrogens (E1, E2, and E3) were present at low (< 10 ng·L-1) concentrations in all receptor-affinity extracts with the exception of E3 in effluent (20 – 30 ng·L-1). The contraceptive xenoestrogen EE2 was not detected in any sample.
In addition, we have successfully expressed and purified the ligand-binding domains of the ecdysone receptor (EcR) and ultraspiracle (USP) from the mysid shrimp Mysidopsis bahia. These proteins have been produced in milligram yield in fusion with glutathione s-transferase and will be utilized in developing a receptor-affinity extraction methodology for isolating invertebrate-relevant endocrine disruptors from wastewater and wastewater-impacted surface waters.
In parallel to the studies above, we have cloned and sequenced the ecdysone receptor (EcR) from the harpacticoid copepod Amphiascus tenuiremus, in support of objectives 3 and 4. We are currently in the process of developing a quantitative real-time PCR assay for this gene in the copepod, which will be used in concert with previously developed molecular endocrine assays (e.g. ecdysteroid titre, vitellin expression) in bioassays of endocrine disruption in this important invertebrate model organism. Preliminary results indicate that basal EcR expression in the copepod increases from the nauplius stage to the copepodite stage during development, but then decreases to approximately naupliar (larval) levels in adult and gravid adult copepods. This result indicates that the developmentally-active copepodite stage of A. tenuiremus may be an appropriate bioassay target for assessing the effects of EcR-active endocrine-disrupting contaminants on this organism.
Our future work on this project will focus on completing the application of estrogen receptor-affinity extraction coupled to HPLC-MS/MS for analyzing xenoestrogens in Kiawah Island wastewater and surface water. This work will be coupled with fish vitellogenesis and embryo development assays (Japanese medaka) of wastewater/water extracts to correlate measured xenoestrogen identities and concentrations with biological activity of these mixtures. Our purified mysid shrimp ecdysone receptor/ultraspiracle ligand binding domain heterodimer will be fully integrated into the receptor-affinity extraction workflow and utilized in concert with HPLC-MS/MS to isolate and identify invertebrate-relevant endocrine disruptors from Kiawah Island water and wastewater samples. In concert with these measurements, we will also utilize the newly sequenced ecdysone receptor/ultraspiracle genes from the copepod Amphiascus tenuiremus together with previously developed molecular endocrine disruption endpoints in this organism to assay the biological activity of these extracts in a sensitive invertebrate model. Although the human androgen receptor has proved refractory to expression and purification so far, we will continue efforts to produce a soluble and active receptor for use in receptor-affinity extraction and analysis of xenoandrogenic contaminants.
Journal Articles on this Report : 1 Displayed | Download in RIS Format
|Other project views:||All 10 publications||2 publications in selected types||All 2 journal articles|
||Kashiwada S, Tatsuta H, Kameshiro M, Sugaya Y, Sabo-Attwood T, Chandler GT, Ferguson PL, Goka K. Stage-dependent differences in effects of carbaryl on population growth rate in Japanese medaka (Oryzias latipes). Environmental Toxicology and Chemistry 2008;27(11):2397-2402.||