Superfund Research Program
Estrogen Receptor-Arylhydrocarbon Receptor Interactions in the CNS
Project Leader: Gloria V. Callard
Grant Number: P42ES007381
Funding Period: 2000-2012
This project is investigating the hypothesis that low dose, transient exposures of embryos to estrogen-like environmental chemicals can disrupt neurodevelopment and generate perturbations of lifelong processes of neuroplasticity and neuroregeneration by altering molecular mechanisms of estrogen biosynthesis and action in the nervous system. An overall objective of Project 7 is to demonstrate the utility of zebrafish (Danio rerio) as a laboratory model for identification and mechanistic analysis of developmental neurotoxicants, and to show direct applicability of laboratory observations to changes in fish species exposed to pollutants in the natural environment.
Extending preliminary studies, cDNAs of the zebrafish cytochrome P450 aromatases (P450arom, estrogen synthetase) expressed in brain (-B isoform) and ovary (-A isoform) and three estrogen receptor subtypes (ER alpha, beta, gamma) were cloned and characterized for use as probes of estrogen signaling. Exposure of embryos to estradiol-l7-beta between 2 and 48 hours post-fertilization (hpf) resulted in a robust (2-5 fold) increase in the predominant brain P450arom (-B) mRNA, but ovarian (-A) mRNA was unaffected. The P450aromB response was sensitive and ER-mediated. Screening for neural estrogen effect using semi-quantitative RT-PCR analysis of P450aromB mRNA as an end point was initiated.
To obtain baseline information about the normal physiological role of estrogen signaling during embryogenesis and in adult fish, and to determine how estrogen exposure perturbs these patterns, RT-PCR was used to analyze the P450arom and ER mRNAs of interest in staged embryos/larvae, juvenile and adult fish. Evidence was obtained for maternal transfer of P450arom and ER mRNAs (all isoforms) and the onset of zygotic transcription beginning 12-24 hpf. Estrogen advanced and upregulated P450aromB (but not other mRNAs) as early as 24 hpf, and the effect persisted up to 2 wk after washout. Following microinjection of fertilized eggs with a plasmid containing a goldfish cyp19b promoter fused upstream of a green fluoroescent protein (GFP) reporter, GFP expression was visualized by confocal microscopy in neuron like cells of forebrain (24 hpf) and midbrain (48 hpf). Even more prominently labeled at 48 hpf, however, were retinal ganglion cells and their projections to central visual processing areas (optic tectum). These results are consistent with immunolocalization of P450arom in adult fish brain and visual system, and imply a role in neural proliferation, differentiation, and path- or target-finding.
Gross and microscopic anatomy of embryos, larvae and juveniles is being studied using whole mounted and sectioned specimens, standard phase contrast and confocal microscopy, and acridine orange as a vital stain for observing patterns of apoptosis. Although early estrogen exposure induces a "curly tail down" phenotype characteristic of mutants with defects of early CNS development, the dose and critical period of estrogen effect are consistent with toxicity rather than endocrine disruption.
Sequence and functional analysis of previously isolated goldfish cyp19 gene promoters has been completed. Among potential cis regulatory motifs identified were as follows: cyp19b, two estrogen responsive elements (EREs) and a Nur77 binding site, implying a site of convergence of both estrogen and neurotrophin mediated pathways; cyp19a, two AhR/ARNT elements and an SF-1 binding motif. Both cyp19b and cyp19a promoters had multiple SRY/SOX binding sites, suggesting that aromatase genes are immediately downstream in the sex determining pathway, which could explain intersex fish in polluted environments. Feasibility of transcriptional analysis of cyp19 promoters by microinjection of reporter constructs into fertilized eggs was tested using goldfish gene promoters. Whereas the promoter of the predominant brain gene (cyp19b) was active in embryonic brain or retina only, the ovarian gene promoter (cyp19a) was inactive up to 48 hpf. By contrast, a luciferase reporter driven by the cyp19a promoter had transcriptional activity in mammalian pituitary (GH3) cell lines whereas the cyp19b promoter had little or no activity. Results suggest that zebrafish embryos are a suitable system for cyp19b promoter analysis.