1. Department of Pharmaceutical Sciences, Ritsumeikan University, Shiga, Japan
2. Department of Biochemistry and Molecular Biology, Faculty of Medicine, Osaka University, Osaka, Japan
3. Faculty of Pharmacy, Meijo University, Nagoya, Japan
4. Graduate School of Science, Nagoya University, Nagoya, Japan
5. Department of Genomic Drug Discovery Science, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan.
Abstract Text: Plastic pollution and exposure to synthetic chemicals are becoming a serious problem. There has been accumulating evidence that exposure to endocrine disrupting chemicals (EDCs) during development can lead to various adverse effects in humans. Brain and neuronal development are highly dependent on a combination of multiple signaling molecules and considered to be susceptible to EDCs. In fact, there are an increasing number of incidents of neurodevelopmental impairments such as autism spectrum disorder (ASD) and attention deficit hyperactivity disorder (ADHD). However, the causal link between these neurodevelopmental disorders and EDCs is not clear. One of the reasons for this is due to the lack of a suitable testing method.
Here, we used human induced pluripotent stem cells (iPSC) and zebrafish embryos to investigate the effect of Bisphenol A (BPA) on brain and neuronal development. BPA is the most highly studied EDC and has been widely used in manufacturers of polycarbonate plastics and epoxy resins including food and drink packages, baby bottles, dental sealants, and thermal receipt paper. We found that BPA enhanced HOX gene expression in the presence of exogenous retinoic acid (RA), but not when BPA was used alone in iPSC. This effect was impeded with retinoic acid receptor (RAR) antagonists, but not with estrogen receptor (ER) antagonists, suggesting that ER is not involved in this signaling. To verify their physiological relevance, we also applied BPA to hindbrain formation in zebrafish. Spatial expression analysis of hoxb1a, fgf8, and otx2 revealed that BPA enhanced RA-induced rostral shift of these brain markers, implying posteriorization of brain region, but not when BPA was used by itself. This rostral shift was successfully simulated with mathematical modeling. Co-exposure to BPA and RA also resulted in the duplication of Mauthner cells and the abnormal craniofacial cartilage formation. Transcriptome analysis revealed that 3’HOX genes and genes related to brain formation exhibited a similar expression profile between iPSC and zebrafish under these experimental conditions. We compared these transcriptomes using GO-based gene set enrichment analysis and found characteristic clusters, which consists of GO terms related to anterior/posterior pattern specification, brain formation, and skeletal system development. These GO terms were highly related to the adverse outcomes we observed. All the data we obtained are consistent with the notion that BPA potentiates RA signaling. Therefore, we are able to link the chemical treatment and detrimental outcomes with potentiation of RA signaling. As RA signaling plays a key role in many fundamental biological processes, pleiotropic effect of BPA showed good agreement with the interference of RA signaling. This result also implies that iPSC can be a useful model for predicting adverse effects in zebrafish and provide a new in vitro method in toxicology and environmental sciences.
