In the present study, effect-directed analysis was used to identify teratogenic compounds in porewater collected from a Superfund site along the Elizabeth River estuary (VA, USA). Zebrafish (Danio rerio) exposed to the porewater displayed acute developmental toxicity and cardiac teratogenesis, presumably because of elevated sediment levels of polycyclic aromatic hydrocarbons (PAHs) from historical creosote use. Pretreatment of porewater with several physical and chemical particle removal methods revealed that colloid-bound chemicals constituted the bulk of the observed toxicity. Size-exclusive chromatography and normal-phase high-performance liquid chromatography were used to fractionate Elizabeth River porewater. Acute toxicity of porewater extracts and extract fractions was assessed as the pericardial area in embryonic zebrafish. The most toxic fraction contained several known aryl hydrocarbon receptor (AhR) agonists (e.g., 1,2-benzofluorene and 1,2-benzanthracene) and cytochrome P450 A1 (CPY1A) inhibitors (e.g., dibenzothiophene and fluoranthene). The second most toxic fraction contained known AhR agonists (e.g., benzo[a]pyrene and indeno[1,2,3-cd]pyrene). Addition of a CYP1A inhibitor, fluoranthene, increased toxicity in all active porewater fractions, suggesting synergism between several contaminants present in porewaters. The results indicate that the observed acute toxicity associated with Elizabeth River porewater results from high concentrations of AhR agonistic PAHs and mixture effects related to interactions between compounds co-occurring at the Elizabeth River site. However, even after extensive fractionation and chemical characterization, it remains plausible that some active compounds in Elizabeth River porewater remain unidentified.

Polycyclic aromatic hydrocarbons (PAHs) induce developmental defects including cardiac deformities in fish. The aryl hydrocarbon receptor (AHR) mediates the toxicity of some PAHs. Exposure to a simple PAH mixture during embryo development consisting of an AHR agonist (benzo(a)pyrene-BaP) with fluoranthene (FL), an inhibitor of cytochrome p450 1(CYP1)--a gene induced by AHR activation--results in cardiac deformities. Exposure to BaP or FL alone at similar concentrations alters heart rates, but does not induce morphological deformities. Furthermore, AHR2 knockdown prevents the toxicity of BaP + FL mixture. Here, we used a zebrafish microarray analysis to identify heart-specific transcriptomic changes during early development that might underlie cardiotoxicity of BaP + FL. We used AHR2 morphant embryos to determine the role of this receptor in mediating toxicity. Control and knockdown embryos at 36 h post-fertilization were exposed to DMSO, 100 μg/l BaP, 500 μg/l FL, or 100 μg/l BaP + 500 μg/l FL, and heart tissues for RNA were extracted at 2, 6, 12, and 18 h-post-exposure (hpe), prior to the appearance of cardiac deformities. Data show AHR2-dependent BaP + FL effects on expression of genes involved in protein biosynthesis and neuronal development in addition to signaling molecules and their associated molecular pathways. Ca(2+)-cycling and muscle contraction genes were the most significantly differentially expressed category of transcripts when comparing BaP + FL-treated AHR2 morphant and control embryos. These differences were most prominent at 2 and 6 hpe. Therefore, we postulate that BaP + FL may affect cellular Ca(2+) levels and subsequently cardiac muscle function, potentially underlying BaP + FL cardiotoxicity.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that mediates many of the toxic effects of dioxin-like compounds (DLCs) and some polycyclic aromatic hydrocarbons (PAHs). Strong AHR agonists, such as certain polychlorinated biphenyls and 2, 3,7,, cause severe cardiac teratogenesis in fish embryos. Moderately strong AHR agonists, such as benzo [a]pyrene and β-naphthoflavone, have been shown to cause similar cardiotoxic effects when coupled with a cytochrome P450 1A (CYP1A) inhibitor, such as fluoranthene (FL). We sought to determine if weak AHR agonists, when combined with a CYP1A inhibitor (FL) or CYP1A morpholino gene knockdown, are capable of causing cardiac deformities similar to moderately strong AHR agonists Billiard, Timme-Laragy et al. 2006;Van Tiem and Di Giulio 2011). The weak AHR agonists included the following: carbaryl, phenanthrene, 2-methylindole, 3-methylindole, indigo, and indirubin. The results showed a complex pattern of cardiotoxic response to weak agonist inhibitor exposure and morpholino-knockdown. Danio rerio (zebrafish) embryos were first exposed to weak AHR agonists at equimolar concentrations. The agonists were assessed for their relative potency as inducers of CYP1 enzyme activity, measured by the ethoxyresorufino-deethylase (EROD) assay, and cardiac deformities. Carbaryl, 2-methylindole, and 3-methylindole induced the highest CYP1A activity in zebrafish. Experiments were then conducted Correspondence to: Daniel Ross Brown, daniel.r.browndu@gmail.com. to determine the individual cardiotoxicity of each compound. Next, zebrafish were co-exposed to each agonist (at concentrations below those determined to be cardiotoxic) and FL in combination to assess if CYP1A inhibition could induce cardiac deformities. Carbaryl, 2-methylindole, 3-methylindole, and phenanthrene significantly increased pericardial edema relative to controls when combined with FL. To further evaluate the interaction of the weak AHR agonists and CYP1A inhibition, a morpholino was used to knockdown CYP1A expression, and embryos were then exposed to each agonist individually. In embryos exposed to 2-MI, CYP1A knockdown caused a similar level of pericardial edema to that caused by exposure to 2-MI and FL. However, CYP1A knockdown in phenanthrene and 3-methylindole only moderately increased pericardial edema relative to co-exposure to FL. AHR2 expression was also knocked down using a morpholino to determine its role in mediating the observed cardiac teratogenesis. Knockdown of AHR2 did not rescue the pericardial edema as previously observed with strong AHR agonists. While some of the cardiotoxicity observed may be attributed to the combination of weak AHR agonism and CYP1A inhibition, other weak AHR agonists appear to be causing cardiotoxicity through an AHR2-independent mechanism. The data show that CYP1A is protective of the cardiac toxicity associated with weak AHR agonists and that knockdown can generate pericardial edema, but these findings are also suggestive of differing mechanisms o...

Various environmental contaminants are known agonists for the aryl hydrocarbon receptor (AHR), which is highly conserved across vertebrate species. Due to gene duplication events before and after the divergence of ray- and lobe-finned fishes, many teleosts have multiple AHR isoforms. The zebrafish (Danio rerio) has three identified AHRs: AHR1A and AHR1B, the roles of which are not yet well elucidated, and AHR2, which has been shown to mediate the toxicity of various anthropogenic compounds including dioxins, polychlorinated biphenyls (PCBs), and polycyclic aromatic hydrocarbons (PAHs). In this study, we sought to explore the role of the two AHR1 isoforms in PAH- and PCB-induced toxicity in zebrafish embryos utilizing morpholino gene knockdown of the AHR isoforms. Knockdown of AHR1B did not affect the toxicity of PAH mixtures or PCB-126, whereas knockdown of AHR1A exacerbated the cardiac toxicity caused by PAH mixtures and PCB-126. Knockdown of AHR1A did not impact the mRNA expression of CYP1A, CYP1B1, and CYP1C1 in exposed embryos, but it did result in increased CYP1 activity in exposed embryos. As has been shown previously, knockdown of AHR2 resulted in protection from PAH- and PCB-induced cardiac deformities and prevented CYP1 enzyme activity in exposed embryos. Co-knockdown of AHR1A and AHR2 resulted in an intermediate response compared to knockdown of AHR1A and AHR2 individually; co-knockdown did not exacerbate nor protect from PAH-induced deformities and embryos exhibited an intermediate CYP1 enzyme activity response. In contrast, co-knockdown of AHR1A and AHR2 did protect from PCB-126-induced deformities. These results suggest that AHR1A is not a nonfunctional receptor as previously thought and may play a role in the normal physiology of zebrafish during development and/or the toxicity of environmental contaminants in early life stages.