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Developmental toxicity of polycyclic aromatic hydrocarbons : defining mechanisms with systems-based transcriptional profiling Public Deposited

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  • Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous in the environment as components of fossil fuels and by-products of combustion. Defining toxicity mechanisms for this large family of multi-ring structures and substituted derivatives is a substantial challenge. Several PAHs, such as benzo(a)pyrene (BaP), are mutagenic, toxic to wildlife, and classified as probable carcinogens to humans. PAHs are present in the environment both in the gaseous phase as well as associated with particulates, and exposures occur via complex mixtures; combustion emissions contain PAHs along with many other contaminants. Cardiac dysfunction and adverse birth outcomes associated with exposure to airborne PAHs suggest that this family of compounds may have non-mutagenic biological activities that affect human health. Some PAHs exert toxic effects via binding the aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor that mediates transcription of many downstream target genes, including cytochrome P450 metabolizing enzymes. Unlike planar halogenated hydrocarbons, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), PAHs are readily metabolized by CYP1A, CYP1B1 and other enzymes, which create reactive intermediates and/or facilitate excretion. Mechanisms of PAH toxicity therefore include canonical AHR signaling, induction of oxidative stress, and other lesser-understood activities that do not require the AHR. We employed zebrafish as a model to rapidly assess developmental toxicity, global transcriptional responses and AHR activation in embryos exposed to parent and oxygenated PAHs (OPAHs). Using comparative analysis of mRNA expression profiles from microarrays with embryos exposed to benz(a)anthracene (BAA), dibenzothiophene (DBT) and pyrene (PYR), we identified expression biomarkers and disrupted biological processes that precede developmental abnormalities. These transcriptional responses were associated with PAH body burdens in the embryos detected by GC-MS. We found that uptake data were essential for discerning molecular pathways from dose-related differences, and identified two primary toxicity profiles. While BAA disrupted transcripts involved in vasculogenesis, DBT and PYR misregulated ion homeostasis and muscle-related genes. NfKB signaling was predicted to be involved in both responses, but canonical AHR signaling was only activated by BAA. In order to study the role of the AHR in mediating toxicity of PAHs, we developed an AHR2 mutant zebrafish line, which has a mutation in the transactivation domain of AHR2. We used AHR agonists TCDD and leflunomide as toxicological probes to characterize AHR activity in the mutant line, and determined that the mutants were functionally null. Finally, we used AHR2 deficient zebrafish embryos to investigate mechanisms by which two four-ring OPAHs induced developmental effects. 1,9 benz-10-anthrone (BEZO) and benz(a)anthracene-7,12-dione (7,12-B[a]AQ) both caused malformations in developing embryos, but they differentially induced CYP1A expression. Despite this difference, the toxicity produced from both compounds was AHR2-dependent. We used mRNA-seq to compare the transcriptional profiles of BEZO and 7,12-B[a]AQ, and identified transcriptional networks that will be investigated further to determine how ligands differentially modulate AHR activity. We also discovered novel transcripts that are potentially important mediators of AHR toxic effects. Comparison across all five parent and OPAHs highlighted clusters of genes that, surprisingly, were similarly expressed in response to the OPAHs, DBT and PYR. These commonly-regulated transcripts may be important to consider when investigating toxicity of PAH mixtures. Together, these studies show that PAHs act via different transcriptional mechanisms, but can be categorized based on transcriptional profiles and differential AHR activation. The clusters of transcripts identified may be involved in common pathways; further investigation of transcription factors and coactivators that interact with mixexpressed genes is a promising area of research for elucidating diverse functions of the AHR.
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