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 wildlife and epidemiological studies supports the “endocrine disrupter hypothesis,” which postulates that environmental chemicals (even at low doses and transient exposures) can mimic or block natural hormones and the biological processes that they control. This project focuses on “xenoestrogens,” including PCBs, pesticides, and dioxins found at many Superfund sites, which have the potential to interfere with estrogen-regulated nervous system development.
Zebrafish (Danio rerio) are used as a model for conducting controlled experiments in the laboratory to establish clear cause-and-effect relationships between specific xenoestrogens and disrupted neural genes and cellular processes (mechanisms). A second, wild caught fish species, the killifish (Fundulus heteroclitus), is collected at a Superfund site (New Bedford Harbor, also studied in Project 5) in order to determine the significance of laboratory findings for animals living long term in a highly polluted environment. P450 aromatase B (a brain specific gene) has been developed as a molecular marker for screening chemicals with estrogen-like effects in the embryonic nervous system. The assay is applicable to routine, high-throughput screening of individual known or previously untested endocrine disrupting chemicals. The same assay can be used for detecting estrogen-like bioactivity in ground water and sediment samples collected from a second Superfund site (Massachusetts Military Reservation) but the specific contaminants at this site remain to be identified.
This year additional molecular markers for monitoring embryonic development, estrogen signaling, and/or neural processes were cloned and characterized. These include (a) P450 aromatase A (an ovary/testis specific form); (b) three distinct subtypes of the ER (a, ba, bb); and (c) a family of nuclear receptors expressed at high levels in the nervous system (Nur77, Nor1, Nurr1). Together with previously cloned zebrafish genes (GAP-43, actin, alpha-tubulin, vitellogenin, cyp1A1), this panel of gene markers reveals that hormonally active environmental chemicals can disrupt estrogen signaling in embryos and adults by altering (a) genes that are directly responsive to estrogen; (b) genes that control production of ER and mediate estrogen effects (alpha form); (c) genes that control endogenous estrogen biosynthesis (P450aromatase B and –A) and metabolism (P4501A1); and (d) mechanism required for correct splicing of gene products (P450aromatase B). Results indicate that, in addition to xenoestrogen actions via ER, dioxin-like chemicals acting through arylhydrocarbon receptors (AhR) affect estrogen signaling in the embryonic and adult nervous system through one or more of the above mechanisms. Comparison of adult killifish from polluted (New Bedford Harbor) and clean environments shows that the identified gene markers are capable of identifying estrogenic chemicals (most likely PCBs) and endocrine disruption in wild fish after multigenerational exposures.
Finally, microscopic analyses have shown abnormalities of neurological development after high-dose estrogen/xenoestrogen exposure, but molecular markers of neurological functions (specifically, neurogenesis, synaptogenesis, and neurotrophin action) are more sensitive indicators of estrogenic effects. This work focuses on the molecular mechanisms of xenoestrogen actions (transcription, splicing). Using microarrays, PCR cloning and bioinformatics, current research thus focuses on (a) the organization and sequence of these genetic markers to identify conserved features (for extrapolation across species: e.g., fish to humans); and (b) the global estrogen signaling network to clarify mechanism.