Superfund Research Program
Estrogen Receptor-Arylhydrocarbon Receptor Interactions in the CNS
Project Leader: Gloria V. Callard
Grant Number: P42ES007381
Funding Period: 2000-2012
Evidence from epidemiological, wildlife, and laboratory studies increasingly indicates that disorders of development and reproduction can be ascribed to exposure to "hormonally active agents" (HAA) in the environment. Often termed "endocrine disrupting chemicals," HAA are rarely toxic but, even at low doses and transient exposures, have the potential to induce lifelong anatomical and functional defects by mimicking or blocking essential hormone actions during critical periods of development. In laboratory and field studies, the goal of this project is to understand how HAA-like PCBs and dioxins (which are commonly found at Superfund sites) impact estrogen receptor (ER)-regulated processes of neurodevelopment, neuroplasticity, and neural repair. One approach is to develop a panel of biomarkers that can be used to identify neuroactive HAA among the approximately 80,000 chemicals that have been added to the environment.
Dr. Callard and her team of investigators are using zebrafish embryos as a whole animal bioassay to measure expressed levels of known neural, non-neural, estrogen- and dioxin-responsive genes. The assay has been optimized, standardized, and characterized in detailed dose-response and time-course studies, and it is currently being validated through tests of diverse suspected HAA and samples from polluted and reference environments. To expand the zebrafish biomarker array to genes known to mediate neural processes and disease (e.g. Parkinson's), the investigators have used a homology cloning approach to isolate and characterize the NR4 family of orphan nuclear receptors in zebrafish: Nor1, Nur77, Nurr1. In rodent models, this gene family has been linked to critical steps in neurodevelopment, although many questions about their normal function and dysfunction remain.
Initial experiments to selectively knock-down Nurr1 functions in zebrafish embryos show that this gene is essential for dopaminergic neuron development, which agrees with studies in mice and is indicative of evolutionarily ancient and conserved structure and function. To obtain a global perspective on genes involved in neuroplasticity and repair, project investigators applied microarray analysis of the zebrafish retina 24 hours after severing the optic nerve (ONX). In marked contrast to mammals, zebrafish retain a potential for functional neuroregeneration of the optic nerve throughout life, such that vision is restored within three weeks of damage. Comparison of more than 14,900 transcripts in sham and ONX retina revealed 484 up-regulated sequences, including structural components (GAP43, tubulin, fibronectins) and cell adhesion factors (connexins, integrins). Among the 433 down-regulated sequences are calcium ion binding proteins (parvalbumin and calreticulin families). Results of this array are currently being validated by quantitative polymerase chain reaction analysis and cross-referenced with an earlier microarray identifying estrogen- and dioxin-responsive genes.
To establish environmental relevance of laboratory studies in zebrafish and investigate long-term, multi-generational effects and adaptations in an "estrogenic" environment, project investigators are using the same biomarker approach to study killifish at the New Bedford Harbor (MA) Superfund site. In comparison with reference site fish, the investigators are evaluating functions of the estrogen signal transduction pathway and impacts on functions of neural, neuroendocrine, and reproductive processes. Expanded knowledge of neuroactive environmental chemicals in laboratory and sentinel fish species will help reduce the uncertainty of risk assessment in polluted environments and provide a foundation for predicting adverse neural effects and possible adaptations.