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 some disorders of development and reproduction can be ascribed to exposure to hormonally active agents (HAA), also known as endocrine disruptors, in the environment. 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 the Estrogen Receptor-Arylhydrocarbon Receptor Interactions in the CNS project is to understand how HAA, such as PCBs and dioxins, 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 roughly 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 quantify 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 (agonists, antagonists); validated by screening multiple classes of known or suspected HAA (e.g., steroids, pesticides, industrial chemicals, phytoestrogens, cosmetics) alone and as mixtures; and used to detect the presence and nature of HAA in samples collected from polluted environments. To extend the applicability of the screening assay to a wider range of HAA target genes/tissue types, a global gene finding approach (microarray analysis) has been applied to identify unknown gene targets of ER, AhR, and ER/AhR interactions. Results suggest that living zebrafish embryos have greater potential for in vivo risk assessment than currently available from in vitro binding or reporter assays. Also, findings provide “proof of concept” for modifying the assay for high throughput screening in order to detect genes/processes targeted by HAA that target any other members of the nuclear receptor superfamily.
A complementary objective is to identify known biomarkers of neural processes in zebrafish for use in the screening bioassay, for mechanistic studies of HAA action and, by extension, to fish living long term in polluted environments (e.g., killifish). To date, the researchers have cloned & characterized members of the nuclear receptor 4 family. As previously reported in mice, they found that morpholino mediated knock down of Nurr1 in zebrafish causes defective development of midbrain dopaminergic (DA) neurons. To expand the panel of markers to assess processes of neural repair in the adult nervous system, they are using the zebrafish ONX (optic nerve section) model. To assure accuracy of methods used to quantify zebrafish gene expression changes, they have invested effort to identify and validate reference genes that are stably expressed under different experimental conditions, and thus suitable as normalizers. These results, already widely accessed, provide zebrafish researchers and toxicologists with important needed information for experimental design and interpretation. The research results provide a sound scientific rationale for using zebrafish to identify neuroactive environmental chemicals, and as genetic models in neurotoxicology research. This work provides a foundation for ongoing and proposed field studies with killifish populations living long term in polluted environments.