2004 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, 2003 through August 31, 2004
Project Amount: $391,598
RFA: Development of High-Throughput Screening Approaches for Prioritizing Chemicals for the Endocrine Disruptors Screening Program (2003) RFA Text |  Recipients Lists
Research Category: Endocrine Disruptors , Economics and Decision Sciences , Health , Safer Chemicals


The objective of this research project is to provide a scientific foundation for the development of a mechanism-based high-throughput screening assay for evaluating estrogen, androgen, and thyroid-like activities in an invertebrate species that also can be used to evaluate the interactive effects of endocrine-active compounds. The currently proposed Tier 1 screening battery to detect endocrine activity of chemicals suffers from two major deficiencies: (1) no invertebrate screen is included despite the proposed use of a Tier 2 multigenerational test with a crustacean, and (2) the battery is not equipped to assess combined effects of diverse endocrine toxicants.

Progress Summary:

Aim 1. Develop a High-Throughput Screening Format To Assess Endocrine Activity of Chemicals Towards an Invertebrate

Efforts during Year 1 of the project focused largely upon developing a screening approach to detect juvenoid/antijuvenoid activity of chemicals. Juvenoid hormones regulate many processes in crustaceans that are associated with estrogens and androgens in vertebrates. We had demonstrated that the juvenoid hormone methyl farnesoate is responsible for male sex determination in the crustacean Daphnia magna. During the past year, we demonstrated that this regulatory activity of methyl farnesoate is not unique to D. magna but is also operative in other cladoceran species (D. pulex and D. pulicaria). We also demonstrated that methyl farnesoate is capable of elevating hemoglobin levels in daphnids through induction of the hemoglobin hb2 gene. This activity holds promise as an endpoint for the detection of juvenoid/antijuvenoid activity as the induction of hemoglobin causes a readily measurable change in the color of the organisms.

A screening protocol was developed that utilizes male sex determination of offspring as a means of screening chemicals for juvenoid/antijuvenoid activity. Maternal daphnids were exposed to chemicals being screened and sex of individuals in the third brood of offspring produced was determined (see Figure 1). The third brood of offspring was evaluated because this is the first brood in which exposure during the critical period of sex determination was guaranteed to have occurred. Assay conditions were such that, in the absence of exogenous juvenoid, only female offspring were produced. Assay duration was approximately 9 days. Seventeen chemicals were evaluated for juvenoid activity as indicated by the production of male offspring. Among these 17 chemicals were juvenoid hormones (positive controls), insect growth regulating (IGR) insecticides, compounds of animal and plant origin that structurally resemble juvenoids, agricultural antimicrobials/fungicides, cyclodiene insecticides, and industrial chemicals. The assay detected juvenoid activity with the positive controls, the IGR insecticides, and the cyclodiene insecticide dieldrin. The positive response to the IGR insecticides is consistent with their known juvenoid activity in insects. The positive response with dieldrin was unprecedented but highly reproducible.

Schematic Representation of the Screening Assay Experimental Design Used To Detect Juvenoid-Related Activity of Chemicals

Figure 1. Schematic Representation of the Screening Assay Experimental Design Used To Detect Juvenoid-Related Activity of Chemicals

Seven of the chemicals were also evaluated for antijuvenoid activity using the same experimental design as for detecting juvenoid activity; however, a juvenoid hormone was included in the assay and chemicals were evaluated for their ability to interfere with juvenoid-stimulated male offspring production. None of the chemicals exhibited antijuvenoid activity.

Finally, six chemicals were evaluated for juvenoid potentiation ability using the same experimental design as used for detecting juvenoid activity; however, the juvenoid hormone methyl farnesoate was included in the assay at a concentration that stimulated a low incidence of male offspring and the chemicals were evaluated for their ability to increase the incidence of male offspring. Five of the six chemicals that were evaluated exhibited juvenoid potentiation activity. We propose that juvenoid potentiation occurs when a chemical binds to and inhibits the enzyme (probably an esterase) that is responsible for inactivating methyl farnesoate. As a consequence, endogenous methyl farnesoate levels increase and juvenoid activity is elevated. These initial results suggest that juvenoid potentiation may be a major endocrine modulating activity of environmental chemicals.

Experiments also were performed to identify endpoints of ecdysteroid signal disruption that could be used to screen chemicals for ecydsteroid/antiecdysteroidal activity. Ecdysteroids regulate many processes in crustaceans that are related to development, growth, and reproduction. Embryonic abnormalities associated with disruption of ecdysteroid-dependent developmental processes have been investigated and have been judged to have potential as endpoints in screening assays for antiecdysteroidal, but not ecdysteroidal, activity. We also have partially cloned several putative ecdysteroid-responsive genes (EcR, USP, HR38, and HR3) from D. magna. Experiments currently are underway to determine whether any of these genes could be used in real time RT-PCR based assays as indicators of either ecdysteroid or antiecdysteroidal activity of chemicals.

Aim 2. Investigate Receptor Crosstalk and Interactive Effects of Endocrine Toxicants as a Consequence of Receptor Crosstalk

Studies in insects provide precedence that ecdysteroid and juvenoid signaling pathways have the ability to modulate each other. Accordingly, chemicals that interact with one pathway may have the ability to modulate activity associated with the other. Experiments were conducted to test the hypothesis that juvenoids, as identified under Aim 1, also can interfere with ecdysteroid-regulated processes through signaling crosstalk. The juvenoids methyl farnesoate, pyriproxyfen, and fenoxycarb all disrupted ecdysteroid-regulated aspects of embryo development in daphnids. The juvenoid pyriproxyfen was shown to attenuate ecdysteroid-mediated arrest of cell proliferation and induction of EcR and USP mRNA levels in an ecdysteroid-responsive cell line. These results indicate that juvenoids can function as antiecdysteroids by negatively regulating ecdysteroid signaling activity.

Although juvenoids were shown to be capable of modulating ecdysteroids signaling, we found no evidence that ecdysteroids modulated juvenoid signaling activity. The combined effects of pyriproxyfen with the ecdysteroid synthesis inhibitor fenarimol or the ecdysteroid receptor antagonist testosterone were evaluated in an effort to discern whether the action of the juvenoids were additive with those of known antiecdysteroids. The antiecdysteroid effects of pyriproxyfen were nonadditive with those of either antiecdysteroid. Rather, joint effects conformed to a model of synergy. These results demonstrated that juvenoids elicit antiecdysteroidal activity through a unique mechanism of action. We have hypothesized that both the ecdysteroid receptor and the juvenoid receptor share components required of activation, and the crosstalk between signaling pathways occurs via competition for these components.

Experiments also were performed to determine whether a chemical that does not have intrinsic juvenoid activity but functions as a juvenoid potentiator as determined under Aim 1 could similarly interfere with ecdysteroid signaling. These experiments were performed with the ubiquitous environmental chemical bisphenol A. Bisphenol A was found to exhibit antiecdysteroidal activity as indicated by the prolongation of the intermolt duration and interference with embryo development. This antiecdysteroidal activity was neither due to reduced availability of endogenous ecdysteroid nor due to ecdysteroid-receptor antagonism. Rather, as demonstrated in the screening assay in Aim 1, bisphenol A potentiated the juvenoid activity of methyl farnesoate. We surmised that bisphenol A enhanced the activity of endogenous methyl farnesoate resulting in its interference with ecdysteroid-regulated processes. A definitive assessment of the effects of bisphenol A on the reproductive capacity of daphnids revealed a concentration-response relationship that extended at least one order of magnitude below exposure levels that were overtly toxic to the maternal organisms. These results demonstrated that bisphenol A is chronically toxic to daphnids, probably through its ability to interfere with ecdysteroid/juvenoid regulated processes. Effects are elicited at levels, however, that are not likely to pose environmental concern.

Aim 3. Evaluate the Relationships Between Effects Observed in the High-Throughput Mechanistic Screen and the Proposed Tier 2 Multigenerational Mysid Test

Experiments have not yet been initiated under Aim 3.

Future Activities:

As part of Aim 1, experiments will continue during Year 2 of this project to identify and evaluate endpoints that would be of value in detecting ecdysteroid/antiecdysteroid activity of chemicals. The receptors thus far identified (EcR, USP, HR38, and HR3) will be assessed for induction/suppression in response to ecdysteroids and juvenoids. Candidate endpoints then will be used in method development for the incorporation of these endpoints in a screening assay format.

We also anticipate that these gene products will be of value to further assess crosstalk between the ecdysteroid and juvenoid signaling pathways. The role of these two signaling pathways in regulating the expression of these receptors will be evaluated in an effort to decipher such interaction between these pathways.

Screening approaches will continue to be used to establish their use in detecting juvenoid/antijuvenoid and/or ecdysteroid/antiecdysteroid activity of chemicals.

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 Mu XY, LeBlanc GA. Cross communication between signaling pathways: juvenoid hormones modulate ecdysteroid activity in a crustacean. Journal of Experimental Zoology Part A–Comparative Experimental Biology 2004;301A(10):793-801. R831300 (2004)
R826129 (Final)
R829358 (2004)
R829358 (Final)
  • Abstract from PubMed
  • Abstract: Wiley-Abstract
  • Journal Article Mu XY, Rider CV, Hwang GS, Hoy H, LeBlanc GA. Covert signal disruption: anti-ecdysteroidal activity of bisphenol A involves cross talk between signaling pathways. Environmental Toxicology and Chemistry 2005;24(1):146-152. R831300 (2004)
    R831300 (Final)
    R829358 (2004)
    R829358 (Final)
    R832739 (2008)
  • Abstract from PubMed
  • Full-text: ResearchGate-PDF
  • Abstract: Wiley-Abstract
  • 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. R831300 (2004)
    R831300 (2006)
    R831300 (Final)
    R829358 (Final)
    R832739 (2008)
  • Abstract from PubMed
  • Full-text: JEB-Full Text HTML
  • Abstract: JEB-Abstract
  • Other: JEB-PDF
  • Supplemental Keywords:

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

    Relevant Websites:

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

    Progress and Final Reports:

    Original Abstract
    2005 Progress Report
    2006 Progress Report
    Final Report