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1 The role of the aryl hydrocarbon receptor pathway in mediating synergistic developmental toxicity of polycyclic aromatic hydrocarbons to zebrafish. 2 ABSTRACT Planar halogenated aromatic hydrocarbons (pHAH), such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin), show strong binding affinity for the aryl hydrocarbon receptor (AHR) and are potent inducers of cytochrome P4501A (CYP1A). It is widely accepted that dioxin toxicity is largely AHR-mediated; however the role of CYP1A activity in causing that toxicity is less clear. Another class of AHR agonists of increasing concern because of their known toxicity and ubiquity in the environment is the polycyclic aromatic hydrocarbons (PAH). Like dioxin, some PAH also cause toxicity to early life stages of vertebrates. Symptoms include increased cardiovascular dysfunction, pericardial and yolk sac edemas, subcutaneous hemorrhages, craniofacial deformities, reduced growth and increased mortality rates. Although developmental effects are comparable between these two types of AHR agonists, the roles of both the AHR and CYP1A activity in PAH toxicity are unknown. As observed in previous studies with killifish (Fundulus heteroclitus), we demonstrate here that co-exposure of zebrafish (Danio rerio) embryos to the PAH-type AHR agonist-naphthoflavone (BNF), and the CYP1A inhibitor-naphthoflavone (ANF), significantly enhanced toxicity above that observed for single compound exposures. In order to elucidate the role of the AHR pathway in mediating synergistic toxicity of PAH mixtures to early life stages, we used a morpholino approach to knock down expression of zebrafish AHR2 and CYP1A proteins during development. We observed that while knock down of AHR2 reduces cardiac toxicity of BNF combined with ANF to zebrafish embryos, CYP1A knock down markedly enhanced toxicity of BNF alone and BNF + ANF co-exposures. These data support earlier chemical inducer/inhibitor studies and also suggest that mechanisms underlying developmental toxicity of PAH-type AHR agonists are different from that of pHAH. 3 Identifying the pathways involved in PAH toxicity will provide for more robust, mechanistic-based tools for risk assessment of single compounds and complex environmental mixtures.