Superfund Research Program
Estrogen Receptor-Arylhydrocarbon Receptor Interactions in the CNS
Project Leader: Gloria V. Callard
Grant Number: P42ES007381
Funding Period: 2000-2012
The problem of environmental chemicals which mimic estrogen ("xenoestrogens") is becoming more urgent as researchers find that many Superfund pollutants, including PCBs and dioxins, can bind to the "estrogen receptor" (ER) in the same way the body's own estrogen does, upsetting the normal hormone balance. This project uses the zebrafish (Danio rerio) as a model to investigate two possible impacts of exposure to xenoestrogens. First, effects on neurological development may occur if xenoestrogens disrupt normal ER pathways in the brain. Second, xenoestrogens upregulate a gene (P450aromB) involved in synthesis of estrogens in the brain -- potentially causing a feedback loop with detrimental effects on normal endocrine balance. Dr. G. Callard's team seeks to develop a screening system for identifying environmental chemicals with estrogen-like activity in the nervous system. They have focused on measuring the activity of the P450aromB gene in zebrafish embryos as a measure of xenoestrogenic activity. Additionally, they have cloned and characterized three distinct subtypes of the ER (a, ba, bb), each with a unique developmental program, tissue distribution, and hormone sensitivity. This screening system is doubly useful because it is also sensitive to a different set of environmental pollutants -- those which, like dioxin and some PCBs, act through the aryl hydrocarbon receptor (AhR), and which appear to decrease the activity of a related gene (P450aromA), predominantly found in the ovaries. Thus, this zebrafish embryo test system can be used for simultaneous detection of two main classes of chemical pollutants. The environmental relevance of this assay for unknown chemical mixtures was validated using samples from polluted and unpolluted sites near the Massachusetts Military Reservation Superfund site (MMR). The system is now being adapted to a high-throughput, quantitative screening test for environmental pollutants using all five genes of interest. Project investigators have documented the patterns of gene expression and tissue distribution of these five genes in all developmental stages (embryo through aged adult).
Additionally, the researchers are looking at neurodevelopment (in embryos) and neuroregeneration and neuroplasticity (in adult fish) to investigate the specific effects of these estrogenic environmental chemicals. Microscopic analyses have shown abnormalities of neurological development after high-dose estrogen/xenoestrogen exposure; however, it was found that molecular markers of neurological functions (specifically, neurogenesis, synaptogenesis, and neurotrophin action) are more sensitive indicators of estrogenic effects.
Project investigators are also focusing on the detailed molecular mechanisms of xenoestrogen actions, particularly by examining the differences between the two forms of the estrogen sythetase gene (P450aromA and P450aromB). They have shown that estrogenic chemicals from plants (like those in soybeans and fruits) bind more strongly to the aromB (brain) form than to the aromA form (found primarily in the ovaries), suggesting that this class of xenoestrogen can preferentially inhibit ovarian estrogen synthesis. Using bioinformatics techniques and PCR cloning, they are analyzing the specific sequence and organization of these genes. The researchers are also examining the interactions with other genes, like the three ER subtypes and the two AhRs from Project 5, which help mediate the xenoestrogenic activation of the P450aromA/B genes.
The researchers are also investigating a possible novel mechanism of endocrine disruption that was revealed by MMR sample testing. Although the active agent is still unknown, samples from polluted sites increased the proportion of a P450aromB variant. Whether this splicing mechanism has functional effects requires further study. Growing concern over the effects of even low levels of xenoestrogens (possibly acting synergistically) has made the investigation of the mechanisms of action a critical field of research. This fish model affords valuable data about these genes and mechanisms, and the screening test based on this model promises to provide a rapid and effective tool for investigations of contaminated sites.