Superfund Research Program
Estrogen Receptor-Arylhydrocarbon Receptor Interactions in the CNS
Project Leader: Gloria V. Callard
Grant Number: P42ES007381
Funding Period: 2000-2012
Epidemiological, wildlife, and laboratory studies indicate that disorders of development and reproduction can be ascribed to exposure to “hormonally active agents” (HAA) in the environment. Termed “endocrine disrupting chemicals” (EDC), 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 objective of this project is to understand how HAA, such as PCBs and dioxins (which are commonly found at Superfund sites), impact estrogen receptor (ER) and arylhydrocarbon receptor (AhR) regulated processes of neurodevelopment, neuroplasticity, and neural repair.
To develop a panel of biomarkers that can be used in a laboratory setting to screen for neuroactive HAA among the ~80,000 chemicals added to the environment, Dr. Gloria Callard and her team of researchers are using zebrafish embryos as a whole animal bioassay to measure expressed levels of known neural and non-neural, estrogen- and dioxin-responsive genes. The assay has been optimized, standardized, and characterized for authentic ER and AhR ligands stage-by-stage through development in detailed dose-response and time-course studies. The assay is currently being used to understand the complexity in the interrelationship between ER- and AhR pathways by testing mixtures of the two ligand classes, and by screening samples collected from polluted and reference environments. To expand the utility of the zebrafish embryo assay for detecting developmental toxicants, they are using a global gene finding approach to identify additional targets of estrogen and dioxin action and to define overlapping regulatory domains.
To detect perturbations in the developing and adult brain in response to any class or mixture of neurotoxicants, including estrogen- and dioxin-like chemicals, the researchers are developing biomarkers of specific processes of neurodevelopment, plasticity, and repair. To date, they have isolated and characterized the NR4 family of orphan nuclear receptors in zebrafish (Nor1, Nur77, Nurr1), and have shown that interference with Nurr1 expression in embryos blocks development of midbrain dopaminergic (DA) neurons, a population known to be impacted in Parkinson’s. Consistent with studies in mice, these results signify that Nurr1 is structurally and functionally conserved through evolution, and provide a sound scientific rationale for using zebrafish as genetic models in neurotoxicology research.
To obtain a panel of markers for assessing chemical effects on processes of neural repair (essential for maintaining homeostasis in response to damage) in the adult nervous system, they are using the zebrafish ONX (optic nerve section) model. The fish nervous system retains a remarkable potential for neuroregeneration throughout life (e.g., restoration of vision ~3 wk after ONX); thus, they predict that markers of damage-induced repair and perturbations by neurotoxicants are exaggerated and more readily identified than in mammals. The team is targeting known biomarkers of estrogen and dioxin action and effect (e.g., P450 aromatase B and –A; ERα, -βa, βb; cyp1A), known markers of axonogenesis and synaptogenesis (e.g., GAP43, βtubulin), and a global gene finding approach (microarray analysis) to profile changes in the retina after ONX in time-course and treatment studies (e.g., +/- estrogen antagonist, dioxin). Comparison of more than 14,900 transcripts in sham and ONX retina reveals 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 have been validated by quantitative polymerase chain reaction (qPCR) analysis and independent microarrays, and demonstrate that blockade of estrogen signaling compromises expression of neural repair genes. An unexpected but potentially valuable outcome of this work is the observation that no one “housekeeping gene” commonly used for normalizing qPCR is adequate for assessing neural processes or toxicant effects. The reserachers are systematically analyzing 9 housekeeping genes (β-actin, glyceraldehyde-3-phosphate dehydrogenase, α-tubulin, glucose-6-phosphate dehydrogenase, eukaryotic elongation factor α, 18S ribosomal protein, TATA-box binding protein, and beta-2-microglobulin) under different developmental stages and conditions. These data can be expected to provide the zebrafish community and toxicologists with important needed information for experimental design and interpretation. The work to date using a laboratory fish (zebrafish) will provide a foundation for their proposed field studies with killifish in the natural environment.