In Vitro Assays and Zebrafish as an in Vivo Model to Study Nuclear Receptor Signaling

Humans are exposed daily to cocktails of chemicals from different sources. Some of these chemicals can interfere with hormonal signaling, and are referred to as endocrine disrupting chemicals (EDCs). A mechanism through which EDCs may act is by binding and modulating the activity of nuclear receptors (NRs), which could lead to adverse health outcomes, especially during critical developmental windows, such as fetal development and perinatal life. As we are exposed to chemical cocktails, there is an increased concern regarding adverse effects resulting from exposures to combinations of EDCs. Mixture effects of bisphenol A and phytoestrogens, well-known xenoestrogens, were assessed in transactivation assays mediated by the estrogen receptors (ERs) and in MCF-7 cell proliferation assays. We observed additive effects with a combination of nanomolar concentrations of these chemicals, levels that are relevant to human exposure. Another major concern is the need to refine, reduce and replace animal models, and zebrafish have been used as an alternative in vivo model. However, to support zebrafish for risk assessment of EDCs, it is crucial to evaluate the transcriptional activity/selectivity of mammalian NR ligands towards their zebrafish homologues. By using stable reporter cell lines expressing human or zebrafish NRs, we observed clear differences in the selectivity/activity of ER and peroxisome-proliferator activated receptor gamma (PPARg) ligands between human and their zebrafish counterparts, demonstrating the importance of assessing the transcriptional profile of EDCs prior to using them in vivo. Liver X receptors (LXRs), also NRs, are key regulators of lipid and cholesterol metabolism in mammals. Transcriptomic analysis of zebrafish larvae treated with the mammalian LXR agonists T0901317 and GW3965, also activators of zebrafish LXR, revealed a primary role of LXR in regulating lipid metabolism and transport during zebrafish development. Tissue enrichment analysis predicted the liver, the yolk syncytial layer and the eye to be affected by LXR activation in zebrafish, which correlated with increased lipid uptake from the yolk, and with morphological abnormalities in the retina and lens of developing zebrafish. The expression of important genes for eye development and maintenance of photoreceptors, was modulated in zebrafish exposed to the ligands as well as in LXR knockout mice, revealing, for the first time, a new potential physiological role of LXRs in regulating expression of gene networks important for visual function in vertebrates.