PNNL

Abstract

Polycyclic aromatic hydrocarbons (PAHs) are a class of both natural and anthropogenic organic compounds of concern due to their frequent detection in the environment and known and suspected toxicities. Many PAHs activate the aryl hydrocarbon receptor (AHR), inducing the expression of a battery of target genes, including xenobiotic metabolizing enzymes like Cytochrome P450s (CYPs). While this molecular initiating event is common across many PAHs, it is increasingly clear that PAHs do not all act via the same mechanisms. To further classify PAHs by their unique activity, we screened several parent and substituted PAHs in zebrafish and human in vitro AHR activation assays to definitively identify AHR ligands. Retene, a teratogenic 3-ring alkylated PAH, did not activate human AHR or zebrafish AHR2 even though it appears to act like a direct AHR ligand in whole-animal models including the zebrafish. We hypothesize that a retene metabolite, rather than the parent compound, activates AHR2 in zebrafish to induce developmental toxicity. To further investigate the role of metabolism in retene toxicity, studies were performed to determine the functional role of cyp1a, cyp1b1, and the microbiome in retene toxicity, identify the zebrafish window of susceptibility, and measure retene uptake, loss, and metabolite formation in vivo. cyp1a-null fish were generated for this study using CRISPR-Cas9 and a functional knockout was validated. Cyp1a and cyp1b1 knockout fish displayed increased sensitivity to retene toxicity while the lack of microbiome did not alter retene toxicity. Zebrafish were most susceptible to retene toxicity from 24 to 48 hours post-fertilization (hpf). After static exposure, retene concentration increased in zebrafish embryos from 6 to 24 hpf, peaked before 36 hpf and declined rapidly to the final measurement at 48 hpf, suggesting 36-48 hpf as a key window of time for metabolite formation. This observation was confirmed by the absence of retene metabolites at 12 and 24 hpf, and the detection and increase of retene metabolites from 36 to 48 hpf. This study highlights the value of combining molecular and systems biology approaches to mechanistic and predictive toxicology and challenges assumptions of AHR activating chemicals, emphasizing the need for further interrogation of PAH-AHR interactions.