Polycyclic Aromatic Hydrocarbons (PAHs) are diverse environmental pollutants associated with adverse human health effects. Many studies focus on the carcinogenic effects of a limited number of PAHs and there is an increasing need to understand mechanisms of developmental toxicity of more varied yet environmentally relevant PAHs. A previous study characterized the developmental toxicity of 123 PAHs in zebrafish. Based on phenotypic responses ranging from complete inactivity to acute mortality, we classified these PAHs into eight bins, selected 16 representative PAHs, and exposed developing zebrafish to the concentration of each PAH that induced 80% phenotypic effect. We conducted RNA sequencing at 48 h post fertilization to identify gene expression changes as a result of PAH exposure. Using the Context Likelihood of Relatedness algorithm, we inferred a network that links the PAHs based on coordinated gene responses to PAH exposure. The 16 PAHs formed two broad clusters: Cluster A was transcriptionally more similar to the controls, while Cluster B consisted of PAHs that were generally more developmentally toxic, significantly elevated cyp1a transcript levels, and induced Ahr2-dependent Cyp1a protein expression in the skin confirmed by gene-silencing studies. We found that cyp1a transcript levels were associated with transcriptomic response, but not with PAH developmental toxicity. While all cluster B PAHs predominantly activated Ahr2, they also each enriched unique pathways like ion transport signaling, which likely points to differing molecular events between the PAHs downstream of Ahr2. Thus, using a systems biology approach, we have begun to evaluate, classify, and define mechanisms of PAH toxicity.

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Background: A structurally diverse group of chemicals, including dioxins [e.g., 2,3,7,8-tetrachlorodibenzo- p -dioxin (TCDD)] and polycyclic aromatic hydrocarbons (PAHs), can xenobiotically activate the aryl hydrocarbon receptor (AHR) and contribute to adverse health effects in humans and wildlife. In the zebrafish model, repression of sox9b has a causal role in several AHR-mediated toxic responses, including craniofacial cartilage malformations; however, the mechanism of sox9b repression remains unknown. We previously identified a long noncoding RNA, sox9b long intergenic noncoding RNA ( slincR ), which is increased (in an AHR-dependent manner) by multiple AHR ligands and is required for the AHR-activated repression of sox9b . Objective: Using the zebrafish model, we aimed to enhance our understanding of the signaling events downstream of AHR activation that contribute to toxic responses by identifying: a ) whether slincR is enriched on the sox9b locus, b ) slincR ’s functional contributions to TCDD-induced toxicity, c ) PAHs that increase slincR expression, and d ) mammalian orthologs of slincR . Methods: We used capture hybridization analysis of RNA targets (CHART), qRT-PCR, RNA sequencing, morphometric analysis of cartilage structures, and hemorrhaging screens. Results: The slincR transcript was enriched at the 5′ untranslated region (UTR) of the sox9b locus. Transcriptome profiling and human ortholog analyses identified processes related to skeletal and cartilage development unique to TCDD-exposed controls, and angiogenesis and vasculature development unique to TCDD-exposed zebrafish that were injected with a splice-blocking morpholino targeting slincR . In comparison to TCDD exposed control morphants, slincR morphants exposed to TCDD resulted in abnormal cartilage structures and a smaller percentage of animals displaying the hemorrhaging phenotype. In addition, slincR expression was significantly increased in six out of the sixteen PAHs we screened. Conclusion: Our study establishes that in zebrafish, slincR is recruited to the sox9b 5′ UTR to repress transcription, can regulate cartilage development, has a causal role in the TCDD-induced hemorrhaging phenotype, and is up-regulated by multiple environmentally relevant PAHs. These findings have important implications for understanding the ligand...

Over the last two decades, the zebrafish (Danio rerio) has emerged as a stellar model for unraveling molecular signaling events mediated by the Aryl Hydrocarbon Receptor (AHR), an important ligand-activated receptor found in all eumetazoan animals. Zebrafish have three AHRs – AHR1a, AHR1b, and AHR2, and studies have demonstrated the diversity of both the endogenous and toxicological functions of the zebrafish AHRs. In this contemporary review, we first highlight the evolution of the zebrafish ahr genes, and the characteristics of the receptors including developmental and adult expression, their endogenous and inducible roles, and the predicted ligands from homology modeling studies. We then review the toxicity of a broad spectrum of AHR ligands across multiple life stages (early stage, and adult), discuss their transcriptomic and epigenetic mechanisms of action, and report on any known interactions between the AHRs and other signaling pathways. Through this article, we summarize the promising research that furthers our understanding of the complex AHR pathway through the extensive use of zebrafish as a model, coupled with a large array of molecular techniques. Since much of the research has focused on the functions of AHR2 during development and the mechanism of TCDD toxicity, we illustrate the need to address the considerable knowledge gap in our understanding of both the mechanistic roles of AHR1a and AHR1b, and the diverse modes of toxicity of the various AHR ligands.

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