Position-Specific Methylation of Benzo[a]pyrene Drives AHR-Dependent Fin Duplication in Zebrafish

Polycyclic aromatic hydrocarbons (PAHs) are an environmental contaminant class characterized by fused aromatic rings. PAHs are released through the incomplete combustion of organic materials as well as petrogenic sources like oil and gas. Benzo[a]pyrene (BaP) is one of the most well-studied PAHs and is known to cause cancer through the formation of DNA adducts and strand breaks. In larval zebrafish exposed from 6 hpf, BaP caused behavioral effects by 4 µM but no morphological effects up to 50 µM by 120 hours post-fertilization (hpf). In contrast, 8-methylbenzo[a]pyrene caused a distinct fin duplication phenotype by 0.26 µM and severe edema and mortality 1 µM. Alkyl substitutions in different positions (7-, 6-, 9-, and 10-MBaP) did not elicit morphological effects at similar concentrations. Utilizing knock-out lines, we determined that the effects of 8-MBaP were Ahr2 dependent and that Cyp1a served a protective role. To identify transcriptomic changes underlying the observed phenotypic effects, larvae were exposed to three concentrations of BaP, 6-MBaP, and 8-MBaP. Whole larvae were collected at 48 and 72 hpf which was before and during phenotype onset, respectively. A systematic approach allowed us to identify the concentration-dependent transcriptional responses linked to the downstream morphological phenotypes unique to BaP methylation at the eight position. This study improves environmental and human health hazard assessment by identifying critical structural features and mechanisms of action contributing to the toxicity of PAH mixtures in the environment. Overall design: Poly-A tail bulk RNA sequencing was performed for three chemicals (8-MBaP, 6-MBaP, and BaP) at three concentrations and two timepoints to elucidate transcriptomic changes unique to 8-MBaP exposure and development of the x-fin phenotype. Embryonic zebrafish were exposed to three concentrations (0.133, 1.33, and 13.3 µM) of 3 chemicals (8-methylbenzo[a]pyrene, 6-methylbenzo[a]pyrene, and benzo[a]pyrene) and a DMSO control at 6 hpf. Embryos were exposed to chemical in 100µL of embryonic medium in 96-well plates. Pools of 6 embryos were collected at 48 hpf and 72 hpf. RNA sequencing was performed by Alithia Genomics (Epalignes, Switzerland) using their Mercurius™ BRB-SEQ service, which is a multiplexed 3' RNA sequencing library preparation technique.