- Billions of pounds of bisphenol A (BPA) are produced annually around the globe for the manufacture of numerous consumer products, including polycarbonate food and water containers, the protective resin linings of food cans, thermal printing paper, and dental fillings. BPA exposure during nervous system development has been associated with learning and behavioral impairments in animal models. The mode of action for these effects is not clearly defined. While BPA is a weak estrogen receptor (ER) agonist, it is also an estrogen-related receptor gamma (ERRγ) agonist. ERRγ binds BPA with 100 times greater affinity than ERs. This study was designed to test the hypothesis that exposure to human-relevant BPA concentrations impacts nervous system development and behavior through ERRγ activation.
To examine whether BPA behaves more like an ER or ERRγ ligand, two positive control compounds were used throughout the study: 17β-estradiol (E2) and GSK4716, ER and ERRγ agonists, respectively. Initial behavior testing results included the observation that neurodevelopmental exposure to 0.01 or 0.1 μM BPA led to hyperactivity in larvae, while exposure to 0.1 or 1 μM BPA led to learning deficits in adult zebrafish. Exposure to 0.1 μM E2 or GSK4716 also led to larval hyperactivity. To identify early molecular signaling events that lead to the observed neurobehavioral phenotypes, a global gene expression analysis using a 135K zebrafish microarray was conducted. The concentrations of compounds tested were anchored to the common larval hyperactivity phenotype they elicited. Gross abnormalities, in the case of higher concentrations of BPA and E2, were also anchored phenotypes included
in the analysis. Functional pathway analysis of the BPA versus E2 results predicted an impact on prothrombin signaling from the two lower concentrations of BPA and E2. Both BPA and GSK4716 were also predicted to impact nervous system development, potentially involving inhibition of the upstream regulator, SIM1. Additionally, GSK4716 exposure was predicted to inhibit neuron migration. There were fewer similarities in transcriptional responses between BPA and E2 when the lower versus higher concentrations were compared, suggesting different mechanisms operated at the higher concentrations. Subsequent experiments were focused on the role of ERRγ in the larval hyperactivity phenotype. Transient ERRγ knockdown by antisense oligonucleotide morpholino during the first 24 hours of development abrogated the hyperactive phenotype induced by 0.1 μM BPA exposure. Transient ERRγ knockdown during the first 48 hours of development resulted in developmental delays, craniofacial defects, pericardial edema, and severe body axis curvature.
This work is the first to identify behavioral effects in a fish from developmental BPA exposure. It is also the first study to confirm a role for ERRγ in mediating BPA's neurobehavioral effects in any animal model. The global gene expression analysis identified similarities between BPA, E2, and GSK4716, suggesting that BPA's mode of action may involve crosstalk between ERRγ and other ERs. These results from human-relevant BPA exposures help explain the widely documented in vivo effects of BPA, despite low binding affinity exhibited by nuclear ERs. ERRγ is an evolutionarily conserved vertebrate receptor and the developmental impacts of BPA in the zebrafish are an indication of hazard potential to vertebrates. They are also an important translational step toward knowing the hazard potential from human developmental exposure to BPA and yet unknown environmental ligands of ERRγ.