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.