title: Zebrafish cardiotoxicity: the effects of CYP1A inhibition and AHR2 knockdown following exposure to weak aryl hydrocarbon receptor agonists.
abstract: 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 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 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 (Wassenberg and Di Giulio Environ Health Perspect 112(17):1658-1664, 2004a; Wassenberg and Di Giulio Res 58(2-5):163-168, 2004b; Billiard et al. Toxicol Sci 92(2):526-536, 2006; Van Tiem and Di Giulio Toxicol Appl Pharmacol 254(3):280-287, 2011). The weak AHR agonists included the following: carbaryl, phenanthrene, 2-methylindole, 3-methylindole, indigo, and indirubin. 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 ethoxyresorufin-O-deethylase (EROD) assay, and cardiac deformities. Carbaryl, 2-methylindole, and 3-methylindole induced the highest CYP1A activity in zebrafish. Experiments were then conducted to determine the individual cardiotoxicity of each compound. Next, zebrafish were coexposed 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-methylindole, CYP1A knockdown caused a similar level of pericardial edema to that caused by exposure to 2-methylindole and FL. The results showed a complex pattern of cardiotoxic response to weak agonist inhibitor exposure and morpholino-knockdown. However, CYP1A knockdown in phenanthrene and 3-methylindole only moderately increased pericardial edema relative to coexposure 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 of cardiac toxicity among known AHR agonists.
authors.affiliation: Nicholas School of the Environment, Duke University, Durham, NC, USA, daniel.r.browndu@gmail.com.
authors.forename: Daniel R
authors.initials: DR
authors.lastname: Brown
authors.forename: Bryan W
authors.initials: BW
authors.lastname: Clark
authors.forename: Lindsey V T
authors.initials: LV
authors.lastname: Garner
authors.forename: Richard T
authors.initials: RT
authors.lastname: Di Giulio
mesh_terms: D000818:Animals; D001564:Benzo(a)pyrene / Q000378:metabolism; D066126:Cardiotoxicity; D019363:Cytochrome P-450 CYP1A1 / Q000235:genetics / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D004625:Embryo, Nonmammalian / Q000187:drug effects*; D005449:Fluorenes / Q000494:pharmacology; D018507:Gene Expression Regulation, Developmental; D055785:Gene Knockdown Techniques; D011078:Polychlorinated Biphenyls / Q000378:metabolism; D011084:Polycyclic Aromatic Hydrocarbons / Q000633:toxicity; D018336:Receptors, Aryl Hydrocarbon / Q000819:agonists* / Q000235:genetics / Q000378:metabolism; D015027:Zebrafish / Q000196:embryology*; D029961:Zebrafish Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: C474372:AHR2 protein, zebrafish; D005449:Fluorenes; D011084:Polycyclic Aromatic Hydrocarbons; D018336:Receptors, Aryl Hydrocarbon; D029961:Zebrafish Proteins; D001564:Benzo(a)pyrene; C007738:fluoranthene; D003577:Cytochrome P-450 Enzyme System; D011078:Polychlorinated Biphenyls; D019363:Cytochrome P-450 CYP1A1