AHR2-Mediated Transcriptomic Responses Underlying the Synergistic Cardiac Developmental Toxicity of PAHs

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants and are known to produce a wide range of toxic effects including carcinogenesis, mutagenesis, disruption of immune and endocrine function, and developmental defects. Considering the increasing introduction of complex PAH mixtures particularly into the aquatic environment due to increased industrialization and urban run-off, investigating mechanisms mediating toxicity of PAH-mixtures is critical for adequate protection of environmental and human health (Nebert et al., 2004; Neilson, 1998). The heart is a target organ of PAH-induced developmental toxicity in teleost fish. For example, embryonic exposure to weathered crude oil containing PAHs lead to impaired heart looping, atrioventricular (AV) conduction block, and cardiac arrhythmia in zebrafish (Incardona et al., 2005) and benzo[k]fluoranthene (BkF) and beta-naphthoflavone (BNF) induced pericardial edema and elongated heart in killifish (Fundulus heteroclitus) (Clark et al., 2010). The aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor, is thought to play a role in mediating cardiotoxicity of some PAHs (Billiard et al., 2006; Van Tiem and Di Giulio, 2011; Wassenberg and Di Giulio, 2004) and dioxins (Prasch et al., 2003; Dong et al., 2004). However, there is some evidence that cardiotoxicity of certain PAHs may also occur through AHR-independent mechanisms (Incardona et al. 2005, 2011). Upon ligand binding, the aryl hydrocarbon receptor (AHR) undergoes a conformational change, translocates from the cytoplasm to the nucleus and forms a heterodimer with the aryl hydrocarbon receptor nuclear translocator (ARNT). The heterodimer then activates xenobiotic response elements inducing expression of genes involved in metabolism and transport of xenobiotic compounds. In mammals, the AHR gene battery includes cytochrome P450 1A (CYP1A), aldehyde dehydrogenase 3, glutathione S-transferases (GSTs), NAD(P)H:quinone oxidoreductase 1, and UDP-glucuronosyltransferase 6 (Nebert et al., 2004). Cytochrome P450s, AHRR (AHR repressor), certain GSTs, and redox-responsive genes are also induced by AHR activation in fish (Garner and Di Giulio, 2012; Timme-Laragy et al., 2007, 2009) and are involved in the detoxification of numerous lipophilic compounds, including some AHR ligands. Based on evidence from TCDD studies, it was originally hypothesized that inhibition of CYP1A would protect from the toxicity induced by an AHR-agonist-PAH (Cantrell et al., 1996; Uno et al., 2004). However, exposure to a simple binary mixture of PAHs that contains an AHR agonist and a CYP1 inhibitor results in pericardial edema and an elongated heart (the “stringy heart” phenotype) during development (Billiard et al., 2006; Van Tiem and Di Giulio, 2011; Wassenberg and Di Giulio, 2004). Furthermore, rather than rescuing toxicity, CYP1A knockdown exacerbates toxicity of BNF in zebrafish and killifish embryos (Billiard et al., 2006; Matson et al., 2008). On the other hand, knockdown of AHR2 in zebrafish prevents the toxicity of these AHR agonist + CYP1 inhibitor mixtures (Billiard et al., 2006; Van Tiem and Di Giulio, 2011). AHR2 knockdown also prevents PAH- and PCB-126-induced cardiac toxicity in killifish (Clark et al., 2010). In addition, AHR2 mediates TCDD toxicity in zebrafish (Prasch et al., 2003), and expression of a constitutively active AHR2 in zebrafish cardiomyocytes has been recently shown to be associated with the same deformities as those mediated by TCDD: cardiac and craniofacial deformities, decreased circulation, and pericardial edema (Lanham et al., 2014). Despite a plethora of data characterizing the phenotypic effects of PAH toxicity and mediation of this toxicity via the AHR, the precise changes at the molecular level immediately following exposure to an AHR agonist and a CYP1 inhibitor in a developing heart remain unknown. In this study, we sought to identify novel genes involved in the cardiotoxicity induced by a simple PAH mixture and to determine whether these genes are controlled in some part by the AHR2. Using a microarray approach, we investigated global gene expression changes in hearts extracted from zebrafish embryos injected with an AHR2 morpholino or control morpholino and exposed to the AHR agonist BaP and the CYP1 inhibitor FL. To determine AHR2-dependent gene expression changes immediately following PAH exposure and to explore how those changes might potentially underlie cardiac morphological deformities, we extracted hearts across several timepoints (2, 6, 12, and 18 hpe) preceding the appearance of detectable deformities. We also characterized heart rates (HRs) at each timepoints across all treatment groups to examine effects of these exposures on overall cardiac function. We hypothesized that while exposure to BaP, FL, and the combination of the two would activate transcription of a large number of genes, knockdown of AHR2 that prevents the BaP + FL toxicity would differentially modulate a smaller number of genes, permitting a more focused analysis of the mechanism of AHR2-dependent cardiotoxicity of this simple PAH mixture.