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Extramural Research

2006 Progress Report: Mechanistic Approach to Screening Chemicals and Mixtures for Endocrine Activity Using an Invertebrate Model

EPA Grant Number: R831300
Title: Mechanistic Approach to Screening Chemicals and Mixtures for Endocrine Activity Using an Invertebrate Model
Investigators: LeBlanc, Gerald A.
Institution: North Carolina State University
EPA Project Officer: Pascual, Pasky
Project Period: September 1, 2003 through August 31, 2006 (Extended to May 31, 2007)
Project Period Covered by this Report: September 1, 2005 through August 31, 2006
Project Amount: $391,598
RFA: Development of High-Throughput Screening Approaches for Prioritizing Chemicals for the Endocrine Disruptors Screening Program (2003)
Research Category: Endocrine Disruptors , Economics and Decision Sciences

Description:

Objective:

The overall objective of this research project is to provide a scientific foundation for the development a mechanism-based high-throughput screening assay for evaluating estrogen, androgen, thyroid (known as EAT)-like activities in an invertebrate species that also can be used to evaluate interactive effects of endocrine-active compounds.

Progress Summary:

Develop a High-Throughput Screening Format to Assess Endocrine Activity of Chemicals Towards an Invertebrate

This aim was completed during the previous grant period. See 2005 summary report.

Investigate Receptor Cross-Talk and Interactive Effects of Endocrine Toxicants as a Consequence of Receptor Cross-Talk

Significant effort was directed towards the identification of the methyl farnesoate receptor of crustaceans. Methyl farnesoate is a crustacean hormone that regulates many of the processes regulated by androgens and estrogens in vertebrate species. Identification of the methyl farnesoate receptor will provide the foundation for the development of receptor-based high-throughput screening assays. Cloning of the receptor and expression of recombinant protein also will allow for studies of cross-talk between this and other hormone signaling pathways. During the previous grant cycle, we identified a putative response element on the hemoglobin 2 gene to which the methyl farnesoate receptor binds. This response element was used as bait to capture and affinity purify the methyl farnesoate receptor from daphnids treated with the hormone. A protein was precipitated that has a molecular mass of 52 kd. Commensurate with these studies, we used targeted PCR to isolate a cDNA candidate for the methyl farnesoate. The gene targeted was based on presumed high conservation between the DNA-binding domain of the putative receptor and the DNA-binding domain of hormone receptors of other species that bind response elements similar to that which we had identified. We successfully identified and fully cloned a cDNA for a NR2B receptor family member that shares a high level of homology to the retinoid X-receptors (RXR). We expressed this cDNA in vitro and characterized the recombinant protein with respect to molecular mass. The mass of this protein was identical to that of the candidate receptor that was affinity purified with the methyl farnesoate response element (52 kD). Experiments are underway to establish whether the affinity purified protein and the recombinant protein from the cloned cDNA are the same. Additionally, we are currently producing the large amounts of the recombinant protein necessary for ligand-binding experiments.

The complete daphnid cDNA for EcR, the major steroid receptor of crustaceans, has been cloned and sequenced during this grant cycle. This receptor is related to the thyroid hormone receptor of vertebrates and, in conjunction with its ecdysteroid ligands, mediates many of the activities associated with thyroid hormones. The cloned receptor will be used to express EcR protein, which then will be used, in conjunction with the cloned methyl farnesoate receptor, to assess cross-talk between these receptors and the ability of endocrine toxicants to interfere with endocrine signaling by interfering with this crosstalk.

In the last progress report, we described efforts to clone the receptor HR3 and HR38, two putative contributors to ecdysteroid and methyl farnesoate signaling in crustaceans. We have abandoned efforts to clone HR38 because of time and cost retrains. Efforts to clone and characterize HR3 continue. To date, we have sequenced 279 base pairs, which translates to 92 amino acids in the open reading frame. This sequence encompasses the entire DNA-binding domain of the receptor and an additional 27 amino acids. We estimate that we have cloned approximately 10 percent of the cDNA. Using real-time PCR with primers derived from the sequenced portion of the cDNA, we have demonstrated that HR3 is highly induced (~30-fold) during the molt cycle. HR3 induction occurs following the surge in ecdysteroid levels, suggesting that this protein functions in the ecdysteroid signaling cascade. Ecdysteroid responsiveness was confirmed when HR3 mRNA levels were induced with exogenous 20-hydroxyecdysone exposure.

Future Activities:

Express RXR Protein

  • Assess ligand-binding characteristics with respect to hormones and environmental chemicals.
  • Assess ligand-activated binding to the putative methyl farnesoate response element.
  • Develop a rapid, high-throughput framework to assess interaction of xenobiotics with the receptor.

Express EcR Protein

  • Assess interactions with RXR with respect to ligand binding and gene activation.
  • Develop a rapid, high-throughput framework to assess cross-talk between the EcR and RXR receptors and xenobiotics interference with cross-talk.

Further advance the cloning and characterization of the role of HR3 in hormone signaling.


Journal Articles on this Report : 3 Displayed | Download in RIS Format

Other project views: All 22 publications 9 publications in selected types All 9 journal articles

Type Citation Project Document Sources
Journal Article Gorr TA, Rider CV, Wang HY, Olmstead AW, LeBlanc GA. A candidate juvenoid hormone receptor cis-element in the Daphnia magna hb2 hemoglobin gene promoter. Molecular and Cellular Endocrinology 2006;247(1-2):91-102.
abstract available   full text available
R831300 (2005)
R831300 (2006)
R831300 (Final)
R829358 (Final)
R832739 (2008)
  • Abstract from PubMed
  • Full-text: ScienceDirect-FullText HTML
    Exit
  • Other: ScienceDirect-PDF
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  • Journal Article Rider CV, Gorr TA, Olmstead AW, Wasilak BA, LeBlanc GA. Stress signaling: coregulation of hemoglobin and male sex determination through a terpenoid signaling pathway in a crustacean. Journal of Experimental Biology 2005;208(Pt 1):15-23.
    abstract available   full text available
    R831300 (2004)
    R831300 (2006)
    R831300 (Final)
    R829358 (Final)
    R832739 (2008)
  • Abstract from PubMed
  • Full-text: JEB-Full Text HTML
    Exit
  • Abstract: JEB-Abstract
    Exit
  • Other: JEB-PDF
    Exit
  • Journal Article Wang YH, Wang G, LeBlanc GA. Cloning and characterization of the retinoid X receptor from a primitive crustacean Daphnia magna. General and Comparative Endocrinology 2007;150(2):309-318.
    abstract available   full text available
    R831300 (2006)
    R831300 (Final)
    R832739 (2006)
    R832739 (2007)
    R832739 (Final)
  • Abstract from PubMed
  • Full-text: ScienceDirect-Full Text HTML
    Exit
  • Other: ScienceDirect-PDF
    Exit
  • Supplemental Keywords:

    hazard assessment, endocrinology, computational toxicology, estrogen, androgen, thyroid, compounds, hormone activities, genes, endocrine screening, bioindicator, biomarkers, ecological risk assessment model, endocrine disrupting chemicals, endocrine disrupting chemicals, estrogen receptors, estrogen response, estuarine crustaceans, exposure, exposure assessment, exposure studies, hormone production, human growth and development, human health risk, invertebrate model, invertebrates, mechanistic screening, sexual development,, RFA, Health, Scientific Discipline, PHYSICAL ASPECTS, ENVIRONMENTAL MANAGEMENT, POLLUTANTS/TOXICS, Environmental Chemistry, Health Risk Assessment, Chemicals, Endocrine Disruptors - Environmental Exposure & Risk, endocrine disruptors, Risk Assessments, Biochemistry, Physical Processes, Biology, Endocrine Disruptors - Human Health, Risk Assessment, bioindicator, assays, biomarkers, EDCs, endocrine disrupting chemicals, exposure, exposure studies, animal model, sexual development, 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, estuarine crustaceans, exposure assessment, human health risk

    Relevant Websites:

    http://www.tox.ncsu.edu/faculty/leblanc/ Exit

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

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    The 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.

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