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,, 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...

Biological effects of 6-formylindolo[3,2-b]carbazole (FICZ) in vivo are enhanced by loss of CYP1A function in an Ahr2-dependent manner

“…In the cyp1a1 −/− mice continuous exposure to FICZ provoked a response similar to that caused by TCDD, whereas continuous FICZ exposure had no effect in wild type mice. Together with the results from the FICZ replenishment- and CYP1A-knockdown experiments present in our study, these data suggest that a functioning CYP1 biotransformation capacity is critical for controlling the toxic effects of FICZ and possibly other labile AHR agonists [41]. …”

“…However, other reports show no protective role of CYP1A against systemic toxicity of TCDD [37, 46, 47]. With PAH substrates, on the other hand, there are several reports demonstrating synergistic AHR-mediated toxicity with CYP1A knockdown or exposure to a CYP1-inhibiting PAH in combination with an AHR agonist [28, 41, 47-52]. …”

When evolution is the solution to pollution: Key principles, and lessons from rapid repeated adaptation of killifish (Fundulus heteroclitus) populations

“…Similarly, the AHR is not the only factor that influences sensitivity to effects of DLCs; the expression and function of other members of the AHR pathway, including ARNT (Nukaya, Walisser, et al., 2010; Tomita, Sinal, Yim, & Gonzalez, 2000; Walisser et al., 2004) and AIP (Nukaya, Lin, et al., 2010), also are important. In addition, the expression and inducibility of biotransformation enzymes can influence sensitivity, especially for labile AHR ligands such as PAHs (Billiard et al., 2008; Brown, Clark, Garner, & Di Giulio, 2015). Thus, there are multiple possible paths to reduced sensitivity to AHR ligands.…”

“…Embryo‐larval cardiotoxicity of some low molecular weight PAHs is independent of AHR signaling (Brown et al., 2015; Incardona, Linbo, & Scholz, 2011; Incardona et al., 2005, 2006, 2014), and may be mediated through disrupted regulation of intracellular potassium and calcium (Brette et al., 2014). Within top‐ranked selection signature regions specific to the ER population, we find genes for two proteins that make up the conductance pore of the voltage‐gated potassium channel ( KCNB2 , KCNC3 ) in cardiomyocytes and RYR3 that regulates intracellular calcium.…”

BDE-99, but not BDE-47, is a transient aryl hydrocarbon receptor agonist in zebrafish liver cells

“…The system's 3XRE activity was much more dose-dependent, with a higher induction fold, at 4 h than at 24 h. CYP1A expression has been well characterized as a consequence of AHR mediated XRE activation. Its enzymatic function formed a negative feedback loop, reducing the substrate's capacity to bind to the AHR and thus protecting the organism against the toxicity of AHR agonist (Brown et al, 2015). The results of previous in vitro reporter gene assays have also shown that in contrast with dioxin-like compounds-which are resistant to CYP1A catalysis and exhibit poor substrate properties that enable them to sustain the AHR signaling pathway, with deleterious effects-most AHR agonists, such as BaP, are metabolically liable and are only transient inducers (Jones et al, 2000;Postlind et al, 1993;Machala et al, 2001).…”