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.

Figure 1

Dose response of BNF and ANF on mRNA induction of AHR2 and AHR2 regulated genes at 48 hpf after exposure for 24 hours. A) Induction of AHR2 by BNF and ANF dose response curves. A “*” indicates a significant difference from control levels. B) BNF induction of CYP1A, 1B1, 1C1, and AHRR2. To differentiate significance between overlapping data points, the letter “a” indicates a significant difference from control levels for CYP1A induction, letter “b” for CYP1B1, letter “c” for CYP1C1, and the letter “r” for AHRR2 expression. C) ANF induction of CYP1A, 1B1, and 1C1, and AHRR2. Significance is indicated by the letters described above. Data presented as mean ± S.E.; n ≥ 6 replicates of pooled embryos; significance defined as p ≤ 0.05.

Figure 2

Induction of AHR2 mRNA at 48 hpf and at 96 hpf. A “*” indicates a significant difference from time-matched controls (p ≤ 0.05). Data presented as mean ± S.E.; 48 hpf time-point data n ≥ 15 replicates of pooled embryos; 96 hpf time-point data n = 6.

Figure 3

Induction of AHR2 regulated genes in the BNF+ANF co-exposure experiments at 48 and 96 hpf. A “#” indicates a synergistic interaction (ANOVA interaction term p ≤ 0.05). A “*” indicates a significant difference from time-matched control (p ≤ 0.05). Data presented as mean ± S.E.; for 48 hpf data n ≥ 12 replicates of pooled embryos; for 96 hpf data n ≥ 6.

Figure 4

CYP1A EROD activity vs. CYP1A mRNA induction. BNF induction of EROD activity (96 hpf) is inhibited by ANF while mRNA induction (48 hpf) is synergistically enhanced. A “#” indicates a synergistic interaction (ANOVA interaction term p ≤ 0.05). A “*” indicates a significant difference from control among EROD data, and “^‡” indicates a significant difference from controls among mRNA data (p ≤ 0.05). Data are presented as mean ± S.E.; EROD n = 3 replicates of pooled embryos and is representative of 75 larvae. mRNA n ≥ 12 replicates of pooled embryos.