2006 Progress Report: Systems Approach to Assessing Cumulative Exposure to Endocrine Disrupting Chemicals

EPA Grant Number: R832739
Title: Systems Approach to Assessing Cumulative Exposure to Endocrine Disrupting Chemicals
Investigators: LeBlanc, Gerald A.
Institution: North Carolina State University
EPA Project Officer: Hahn, Intaek
Project Period: October 1, 2005 through September 30, 2008 (Extended to September 30, 2009)
Project Period Covered by this Report: October 1, 2005 through September 30, 2006
Project Amount: $585,206
RFA: Exposure Measurement Tools for Endocrine Disrupting Chemicals in Mixtures (2005) RFA Text |  Recipients Lists
Research Category: Economics and Decision Sciences , Health , Safer Chemicals , Endocrine Disruptors


The effective assessment of risk associated with endocrine-disrupting chemicals (EDCs) requires the development of tools with which exposure to the chemicals can be assessed. The overall objective of this research program is to develop a gene-expression based, whole-organism approach to evaluating cumulative exposure to EDCs.

Progress Summary:

Aim 1: Identify a Suite of Gene-Expression, Whole-Organism Based Biological Markers That Specifically Respond to Modulators of Hormone-Signaling Pathways

The whole-organism model selected for use in this program is the water flea Daphnia magna. This species is small and easily cultured. It is conducive to lab exposures and field deployment, and members of this genus are ubiquitous in freshwater environments, allowing for field sampling and evaluation. Finally, the endocrinology of daphnids has been well described. We selected two endocrine signaling pathways that function autonomously and in combination to regulate various activities related to development, growth, and reproduction in daphnids: the ecdysteroid signaling pathway and the terpenoid signaling pathway. These pathways are representative of steroid and retinoid signaling pathways, respectively, in vertebrates.

Using targeted PCR with consensus-degenerate hybrid oligonucleotide primers, we identified 19 gene products that will comprise our holistic, systems approach to measuring alterations in endocrine signaling following exposure to EDCs. These gene products are: hormone receptors (EcR and RXR); putative intermediate signaling factors (HR38, HR78, HR96, E75, E78, HSP90, and FTZ); hormone-regulated genes (HR3, Hb1, Hb2, Hb3, Vtg1, and Vtg2); and house-keeping genes (actin, cyclophilin, GAPDH, and ubiquitin conjugating enzyme [UCE]).


  • Generate gene-specific, functional primer sets. Percentage complete: 100.
  • Optimize conditions for high efficiency quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) of the gene products. Percentage complete: 50.

Aim 2: Evaluate the Performance and Versatility of the Biomarkers of Exposure

Biomarker sensitivity experiments are underway to establish: (1) the minimum number of daphnids necessary to measure changes in expression of the targeted genes; (2) variability in the level of expression of the individual genes among control animals; (3) variability in the expression of the individual genes as related to exposure conditions; and (4) responsiveness of the individual genes to EDC exposure.

Results to Date:

  • Groups of five daphnids provide sufficient biomass for the extraction of RNA and its conversion to ample cDNA for use in PCR analyses. Percentage complete: 100.
  • Experiments completed with EcR, RXR, HR38, Vtg1, Vtg2, Hb1, Hb2, Hb3, Actin, Cyclophilin, GAPDH, UCP, and HSP90 indicate that variability among independent control samples is low and these gene products will provide high-sensitivity assessments of induction or suppression in response to EDC exposure. HR3 expression is significantly elevated during the molt cycle, which may confound attempts to measure changes in the expression of this gene in groups of animals in which the molt cycle is not synchronized. Percentage complete: 74.
  • Several biomarkers have been evaluated for responsiveness to terpenoid hormone agonists (pyriproxyfen, fenoxycarb, and methoprene). The three Hb genes were induced by terpenoid hormone agonists, while RXR was suppressed by terpenoid agonist exposure, and HR38 expression was not affected by these treatments. HR3 gene expression was significantly induced by ecdysteroid treatment, which explains its induction during the molt cycle. HR3 may provide a good biomarker of anti-ecdysteroid activity by measuring decreases in gene expression in daphnids pre-treated with ecdysteroid. Percentage complete: 5.
  • Daphnids have been exposed to various chemicals, including: pyriproxyfen, fenoxycarb, methoprene, kinoprene, dieldrin, cis-chlordane, 4-nonylphenol, fenarimol, piperonyl butoxide, 20-hydroxyecdysone, bisphenol A, cyperoterone acetate, testosterone, diethyl stilbestrol, acetone, chloroform, methylene chloride, naphthalene, pentachlorophenol, and zinc sulfate. Tissue samples are currently being processed for the preparation of total cDNA. Once development of the biomarkers is complete, these samples will be evaluated for changes in expression of the targeted genes. Percentage complete: 25.

Aim 3: Evaluate the Ability of the Sensor to Detect EDC Exposure Associated With Chemical Mixtures

Work has not begun on the study of chemical mixtures. Percentage complete: 0.

Future Activities:

During the next funding period (2007), we will complete our optimization of real-time RT-PCR quantification of the biomarker gene products (Aim 1). Subsequent to optimization, the sensitivity of the individual biomarkers will be assessed and optimized where necessary (Aims 1 and 2). The majority of the 2007 funding period will be used to characterize the responsiveness of the sensor to various chemicals having both known and unknown endocrine disrupting properties. We intend to evaluate approximately 20 chemicals (listed above) during 2007. Results from these analyses will be assimilated to determine the systems-level responses to the chemicals and to identify patterns of responses that are diagnostic of the type of EDC to which the sensor was exposed.

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

Other project views: All 15 publications 15 publications in selected types All 15 journal articles
Type Citation Project Document Sources
Journal Article Olmstead AW, LeBlanc GA. The environmental-endocrine basis of gynandromorphism (intersex) in a crustacean. International Journal of Biological Sciences 2006;3(2):77-84. R832739 (2006)
R832739 (2007)
R832739 (2008)
R832739 (Final)
R831300 (Final)
  • Full-text from PubMed
  • Abstract from PubMed
  • Associated PubMed link
  • Full-text: International Journal of Biological Sciences-Full Text HTML
  • Abstract: International Journal of Biological Sciences-Abstract
  • Other: International Journal of Biological Sciences-PDF
  • 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. R832739 (2006)
    R832739 (2007)
    R832739 (Final)
    R831300 (2006)
    R831300 (Final)
  • Abstract from PubMed
  • Full-text: ScienceDirect-Full Text HTML
  • Other: ScienceDirect-PDF
  • Supplemental Keywords:

    exposure assessment, endocrinology, computational toxicology,, Health, Scientific Discipline, POLLUTANTS/TOXICS, Health Risk Assessment, Chemicals, Risk Assessments, Environmental Monitoring, chemical exposure, cumulative exposure, endocrine disrupting chemicals, rapid detection, genetic analysis, chemical mixtures, endocrine disruptors, human exposure, deconvolution technique, biomarker based exposure inference, sensor, biochemical research, human health risk

    Relevant Websites:

    http://www.tox.ncsu.edu/faculty/leblanc/ Exit
    http://www.ncsu.edu/project/toxresearch/projects/ Exit

    Progress and Final Reports:

    Original Abstract
  • 2007 Progress Report
  • 2008 Progress Report
  • Final Report