Final Report: Development of Receptor- to Population-Level Analytical Tools for Assessing Endocrine Disruptor Exposure in Wastewater-Impacted Estuarine Systems

EPA 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 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

Objective:

The research supported by this STAR grant was aimed primarily at developing and validating novel analytical tools for identifying and quantifying receptor-active endocrine disrupting chemicals in complex environmental samples. In addition, we have linked these new bioanalytical tools to sensitive in-vivo bioassays for assessing the potential for exposure of aquatic organisms to wastewater-derived endocrine disrupting chemicals. Specific objectives of the project were to:
 
1.  Develop nuclear hormone receptor-affinity extraction techniques as tools for isolating endocrine 
     disrupting chemicals (EDCs) from complex wastewater mixtures.
2.  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.
3.  Utilize sensitive invertebrate (copepod) EDC-exposure laboratory bioassays to link exposure
     measurements (above) to biological effects.
4.  Apply novel biomolecular endpoints to assess EDC exposure in field populations of sensitive
     meiobenthic invertebrates in wastewater-impacted estuarine environments.

Summary/Accomplishments (Outputs/Outcomes):

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. We have applied this method successfully to isolate, identify, and quantify estrogenic contaminants in wastewater-impacted surface waters on the coast of SC (specific objective 2). The details of the nuclear hormone receptor affinity method for isolating and identifying estrogenic contaminants in environmental samples are described in Section I of the detailed project report submitted to EPA; however, we briefly summarize the results of the method development and its application to a wastewater-impacted coastal golf course environment in this summary report.
 
We have developed a method, hormone receptor-affinity extraction, for isolating xenoestrogenic environmental contaminants from complex water and wastewater samples prior to analysis by HPLC-MS/MS and HPLC-QTOF MS. This technique utilizes the specificity of hexahistidine-tagged, recombinant human estrogen receptor (alpha isoform) ligand binding domain (ERα-LBD) to bind trace estrogenic compounds in solution prior to co-purification of the ERα-LBD-xenoestrogen complex using immobilized metal-affinity chromatography. This method reduces sample complexity and enriches for estrogen receptor-relevant endocrine disruptors. Xenoestrogens in municipal wastewater effluent and in surface waters impacted by irrigation runoff from land-application of wastewater effluent were isolated by receptor-affinity extraction and quantified by HPLC coupled to triple-quadrupole mass spectrometry using stable-isotope dilution. Synthetic xenoestrogens (nonylphenol, bisphenol A, and octylphenol) were detected in water and wastewater at concentrations up to approximately 350 ng·L-1, while biogenic estrogens (17β-estradiol, estrone, and estriol) were present in these samples at lower concentrations (typically below 10 ng·L-1). The pharmaceutical contraceptive estrogen 17α-ethynylestradiol was not measured in water or wastewater samples above detection limits (<1 ng·L-1). Qualitative analysis of non-target xenoestrogens in wastewater receptor-affinity extracts was performed by HPLC-QTOF MS/MS, and the weak xenoestrogen 2-(2-(4-nonylphenoxy)ethoxy)acetic acid (NP2EC) was identified by accurate mass measurement and high resolution MS/MS. Identification of this compound (an oxidative degradation product of nonylphenol polyethoxylate surfactants) in estrogen-receptor affinity isolates of wastewater effluent demonstrates the utility of this method for characterizing trace xenoestrogens in complex mixtures without prior knowledge of their identities.
 
In parallel to the studies described above, we have successfully cloned and sequenced the ecdysone receptor (EcR) from the harpacticoid copepod Amphiascus tenuiremus, in support of objectives 3 and 4. We have developed a quantitative real-time PCR assay for this gene in the copepod, which can 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. 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 indicated that the developmentally-active copepodite stage of A. tenuiremus was an appropriate bioassay target for assessing the effects of EcR-active endocrine-disrupting contaminants on this organism. The objective of this research was to obtain and characterize sequence information for the A. tenuiremis ecdysone receptor and investigate modulation of expression levels by fipronil, an insecticide that causes infertility in males and reduced egg extrusion in female copepods, and ponasterone, a natural ecdysone receptor agonist. Results show successful cloning and phylogenetic analysis of the ecdysone receptor for A. tenuiremis, providing the first genetic information for a hormone receptor in this species. Exposure of copepodites to fipronil for 1, 2, 4, 18 and 30 hours caused a significant increase in ecdysone receptor transcriptional expression at 30 hours compared to control unexposed animals. This work illustrates a potential mechanism whereby exposure to fipronil, and potentially other endocrine disrupting compounds, results in impacted reproduction. Furthermore, this exemplifies the potential utility of ecdysone receptor transcriptional measurement as a sensitive and rapid biomarker of ecological relevance when linked to traditional A. tenuiremis bioassays.

Conclusions:

Receptor-affinity extraction using the ERα-LBD has been shown to be a highly effective method for isolating environmental xenoestrogens from trace aqueous samples prior to HPLC-MS/MS or HPLC-QTOF-MS/MS analysis. This technique enables both quantitative (with appropriate stable-isotope labeled surrogate standards) and qualitative determination of a wide range of xenoestrogen compounds at trace levels. Application of the ERα-LBD-based receptor affinity extraction technique to wastewater-impacted aquatic environments provides a new tool for assessing which compounds in a complex mixture are of most concern for estrogen-mediated endocrine disruption in wildlife and human populations. In addition, the general analytical scheme can be applied to other nuclear hormone receptor systems (e.g., the androgen receptor or ecdysteroid receptor) to create unique and highly specific endocrine-activity directed tools for assessing exposure of vulnerable populations to additional endocrine disruptor classes. While the sensitivity of the current method is acceptable for analyzing wastewater and wastewater-impacted surface waters (enabling compound detection at low ng·L-1 concentrations), additional sensitivity for analysis of trace xenoestrogens in less polluted aqueous samples may be attained by extraction of larger volume samples (e.g., > 1 L) during the SPE step prior to receptor-affinity extraction.
 
Previously observed effects of the phenylpyrazole insecticide fipronil on A. tenuiremis including infertility in males, reduced egg extrusion in females, and increased vitellin levels in females and males may be related to newly reported fipronil-induced transcriptional expression of EcR. We have presented the first phylogenetic information for A. tenuiremis showing that this species is aligned most closely genetically with the related tidepool copepod Tigriopus japonicas. A. tenuiremis is presently undergoing validation as a USEPA and OECD-approved test species for the screening and evaluation of endocrine and reproductive toxicants in marine environments. This work illustrates the utility of EcR transcriptional measurement as a sensitive and rapid endocrine disruption biomarker of ecological relevance when linked to traditional A. tenuiremis bioassay and toxicity testing [e.g., ASTM E2317-0490]. The small tissue mass obtainable from individual copepods has restricted similar molecular biomarker developments in the past. However, new micro-extraction techniques for RNA in microgram tissue quantities coupled with PCR have brought the former impossibility of copepod-based biomarker development to reality.


Journal Articles on this Report : 2 Displayed | Download in RIS Format

Other project views: All 10 publications 2 publications in selected types All 2 journal articles
Type Citation Project Document Sources
Journal Article Gaertner K, Chandler GT, Quattro J, Ferguson PL, Sabo-Attwood T. Identification and expression of the ecdysone receptor in the harpacticoid copepod, Amphiascus tenuiremis, in response to fipronil. Ecotoxicology and Environmental Safety 2012;76(2):39-45. R832737 (Final)
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  • Journal Article 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. R832737 (2008)
    R832737 (Final)
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  • Supplemental Keywords:

    environmental chemistry, emerging contaminants, estuary, marine, mass spectrometry, estradiol, Health, Scientific Discipline, POLLUTANTS/TOXICS, Health Risk Assessment, Chemicals, Risk Assessments, Environmental Monitoring, chemical exposure, coastal ecosystem, wastewater, endocrine disrupting chemicals, rapid detection, bioavailability, chemical mixtures, endocrine disruptors, human exposure, deconvolution technique, biomarker based exposure inference, human health risk

    Progress and Final Reports:

    Original Abstract
  • 2006 Progress Report
  • 2007 Progress Report
  • 2008 Progress Report
  • 2009