Superfund Research Program
Developmental Toxicity of non-Dioxin-like PCBs and Chemical Mixtures
Studies and Results
Dr. Stegeman and his team have used their finding that expression of PXR is self-regulated in zebrafish embryos to address other genes regulated via the PXR, and to assess chemicals for efficacy as PXR agonists. The studies have suggested further that the CYP2AAs (Kubota et al., 2013), which were identified in zebrafish, include PXR responsive genes. Likewise, CYP3 genes were confirmed to be PXR regulated. One PXR knockdown study suggested that ortho-PCBs do not activate PXR in vivo. This is consistent with the observation obtained with the PXR ligand-binding domain in COS cells, showing that PCB 153 is not a PXR agonist in zebrafish. However, the in vivo analyses need to be repeated. The analysis of transcriptomic effects of PCB 153, and of PXR-dependent gene expression (obtained comparing PXR morpholino knockdown with control morpholino), is underway at this time, using RNAseq. The research team also has established the complex developmental pattern of redox potential (Timme-Laragy et al, 2013), setting the stage for analysis in the proposed oxidative stress studies.
The researchers have continued studies on variant forms (alleles) of PXR in zebrafish (further leveraging in-kind support) (see Bainy et al, 2013). Additional PXR variants have been identified, and they now have data to show that responses to a PXR agonist in embryos are related to the SNP present. This is critically important information for chemical testing. While there was little response of PXR to PCB153, the researchers have identified a consistent response in induction of an orphan gene, CYP20, by the ortho-PCBs PCB153 and PCB52. This is an unanticipated result with important implications, as in humans the orphan CYP20 is expressed in brain areas critical for memory and learning. If PXR is not activated yet CYP20 is induced by ortho-PCBs, it would imply that there is a mechanism of ortho-PCB effects on transcription other than via the PXR.
Studies of AHR and PXR agonist effects showed that pxr expression is induced by both pregnenolone and by PCB126. Knocking down PXR and AHR expression and assessing effects of agonists for these receptors points to a possible interaction between AHR and PXR agonists. The knockdown of PXR suppressed the PN induction of pxr, and in like manner, knockdown of AHR suppressed PCB126 induction of pxr. There could be interactive effects of bona fide agonists for the two receptors on the levels and possible downstream effects mediated by the PXR.
Analysis of killifish from New Bedford Harbor, which have stunning levels of ortho-PCBs, has continued in collaboration with scientists at the UC Davis SRP. The effects of ortho-PCBs on expression and function of genes for the RyR calcium channels and a number of accessory proteins in killifish have been substantiated. Furthermore, compared to the reference site, NBH killifish embryos and larvae displayed altered RyR related mRNA transcription and these results were consistent across multiple age classes and multiple generations reared in the laboratory. Changes in expression of RyR1 and associated proteins could contribute to a long-term and potentially transgenerational adaptation to continual non-coplanar PCB exposure in impacted fish populations. Analysis of effects of ortho- and non-ortho-PCBs on PXR and CYP3A expression also continued in collaboration with a group from Sweden. The NBH fish showed a lower level of expression in some organs than in the reference fish. There may be multiple types of adaptation to generational exposure to ortho-PCBs.
Research Translation and Training Activities:
This project has continued and has contributed to training of two postdoctoral researchers, both of whom are currently engaged in the continuing studies of this project. There also have been undergraduate students who have been involved in the research.
This is a robust approach for analysis of the effects of ortho-PCBs on early vertebrate development, using the zebrafish and killifish models, and is a uniquely comprehensive study of possible mechanisms of ortho-PCB effects. Developmental abnormalities affecting many systems occur in vertebrates exposed to chemicals found at Superfund sites. This is a highly important and poorly understood concern in toxicology. Most studies are focused on single compounds, but there is clear evidence for complex developmental effects of mixtures, including PCB mixtures. There is a need to establish screening tools for mechanistic integration of effects over an entire organism. The studies will elucidate interactions of ligands with proteins that could determine the effects. This will be the first study of CYP enzymes induced by or involved in metabolism of ortho-PCB congeners in zebrafish. The study also will address effects of mixtures of chemicals prevalent at Superfund sites. Developmental effects of co-occurring mixtures have rarely been systematically addressed in ways that could reveal interactive mechanisms. Studies in killifish will test whether the population in New Bedford Harbor, which acquired resistance to toxicity of dioxin-like PCBs, is resistant also to effects of ortho-PCBs, expanding this team's understanding of resistance adaptation.