Synergistic induction of AHR regulated genes in developmental toxicity from co-exposure to two model PAHs in zebrafish.

Polycyclic aromatic hydrocarbons (PAHs) are pollutants created by the incomplete combustion of carbon, and are increasing in the environment largely due to the burning of fossil fuels. PAHs occur as complex mixtures, and some combinations have been shown to cause synergistic developmental toxicity in fish embryos, characterized by pericardial edema and craniofacial malformations. Previous studies have indicated that in the zebrafish model, this toxicity is mediated by the aryl hydrocarbon receptor 2 (AHR2), and enhanced by inhibition of CYP1A activity. In this study, we further examined this interaction of the model PAH and AHR agonist beta-naphthoflavone (BNF) with and without the AHR partial agonist/antagonist and CYP1A inhibitor alpha-naphthoflavone (ANF) to determine (1) whether ANF was acting as an AHR antagonist, (2) what alterations BNF and ANF both alone and in combination had on mRNA expression of the AHR regulated genes cytochrome P450 (cyp) 1a, 1 b 1, and 1 c 1, and the AHR repressor (ahrr2) prior to versus during deformity onset, and (3) compare CYP1A enzyme activity with mRNA induction. Zebrafish embryos were exposed from 24-48 or 24-96 hpf to BNF, 1-100 microg/L, ANF, 1-150 microg/L, a BNF+ANF co-exposure (1 microg/L+100 microg/L), or a DMSO solvent control. RNA was extracted and examined by quantitative real-time PCR. Both BNF and ANF each individually resulted in a dose dependent increase CYP1A, CYP1B1, CYP1C1, and AHRR2 mRNA, confirming their activities as AHR agonists. In the BNF+ANF co-exposures prior to deformity onset, expression of these genes was synergistic, and expression levels of the AHR regulated genes resembled the higher doses of BNF alone. Gene induction during deformities was also significantly increased in the co-exposure, but to a lesser magnitude than prior to deformity onset. EROD measurements of CYP1A activity showed ANF inhibited activity induction by BNF in the co-exposure group; this finding is not predicted by mRNA expression, which is synergistically induced in this treatment. This suggests that inhibition of CYP1A activity may alter metabolism and/or increase the half-life of the AHR agonist(s), allowing for increased AHR activation. This study furthers a mechanistic understanding of interactions underlying PAH synergistic toxicity.

The role of the aryl hydrocarbon receptor pathway in mediating synergistic developmental toxicity of polycyclic aromatic hydrocarbons to zebrafish.

Planar halogenated aromatic hydrocarbons (pHAHs), 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 (PAHs). Like dioxin, some PAHs 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 coexposure of zebrafish (Danio rerio) embryos to the PAH-type AHR agonist beta-naphthoflavone (BNF) and the CYP1A inhibitor alpha-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 knockdown markedly enhanced toxicity of BNF alone and BNF + ANF coexposures. These data support earlier chemical inducer/inhibitor studies and also suggest that mechanisms underlying developmental toxicity of PAH-type AHR agonists are different from those of pHAHs. 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.