title: Synergistic induction of AHR regulated genes in developmental toxicity from co-exposure to two model PAHs in zebrafish.
abstract: 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.
authors.affiliation: Integrated Toxicology and Environmental Health Program, Nicholas School of the Environment and Earth Sciences, Duke University, Box 90328, Durham, NC 27708, USA.
authors.forename: Alicia R
authors.initials: AR
authors.lastname: Timme-Laragy
authors.forename: Crystal J
authors.initials: CJ
authors.lastname: Cockman
authors.forename: Cole W
authors.initials: CW
authors.lastname: Matson
authors.forename: Richard T
authors.initials: RT
authors.lastname: Di Giulio
mesh_terms: D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics; D001571:Benzoflavones / Q000633:toxicity*; D019363:Cytochrome P-450 CYP1A1 / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics; D065633:Cytochrome P-450 CYP1B1; D004305:Dose-Response Relationship, Drug; D004357:Drug Synergism; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D012097:Repressor Proteins / Q000819:agonists* / Q000037:antagonists & inhibitors*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015533:Transcriptional Activation; D014874:Water Pollutants, Chemical / Q000633:toxicity; D015027:Zebrafish; D029961:Zebrafish Proteins / Q000819:agonists* / Q000037:antagonists & inhibitors*; D019324:beta-Naphthoflavone / Q000633:toxicity*
chemical_list: D001571:Benzoflavones; D012333:RNA, Messenger; D012097:Repressor Proteins; D014874:Water Pollutants, Chemical; D029961:Zebrafish Proteins; C504745:ahrrb protein, zebrafish; C011512:alpha-naphthoflavone; D019324:beta-Naphthoflavone; D001189:Aryl Hydrocarbon Hydroxylases; D019363:Cytochrome P-450 CYP1A1; D065633:Cytochrome P-450 CYP1B1