AHR2 Mutant Reveals Functional Diversity of Aryl Hydrocarbon Receptors in Zebrafish

The aryl hydrocarbon receptor (AHR) is well known for mediating the toxic effects of TCDD and has been a subject of intense research for over 30 years. Current investigations continue to uncover its endogenous and regulatory roles in a wide variety of cellular and molecular signaling processes. A zebrafish line with a mutation in ahr2 (ahr2hu3335), encoding the AHR paralogue responsible for mediating TCDD toxicity in zebrafish, was developed via Targeting Induced Local Lesions IN Genomes (TILLING) and predicted to express a non-functional AHR2 protein. We characterized AHR activity in the mutant line using TCDD and leflunomide as toxicological probes to investigate function, ligand binding and CYP1A induction patterns of paralogues AHR2, AHR1A and AHR1B. By evaluating TCDD-induced developmental toxicity, mRNA expression changes and CYP1A protein in the AHR2 mutant line, we determined that ahr2hu3335 zebrafish are functionally null. In silico modeling predicted differential binding of TCDD and leflunomide to the AHR paralogues. AHR1A is considered a non-functional pseudogene as it does not bind TCCD or mediate in vivo TCDD toxicity. Homology modeling, however, predicted a ligand binding conformation of AHR1A with leflunomide. AHR1A-dependent CYP1A immunohistochemical expression in the liver provided in vivo confirmation of the in silico docking studies. The ahr2hu3335 functional knockout line expands the experimental power of zebrafish to unravel the role of the AHR during development, as well as highlights potential activity of the other AHR paralogues in ligand-specific toxicological responses

Summary of zebrafish AHR ligand binding, activity and expression.

<p>Summary of zebrafish AHR ligand binding, activity and expression.</p

<i>ahr2</i>^hu3335 embryos are resistant to TCDD-induced developmental abnormalities.

<p><b>A</b>) Percent of embryos with axis malformations and <b>B</b>) percent incidence pericardial edema at 120 hpf in embryos treated with 0, 0.1, 1 or 10 nM TCDD from 6–24 hpf. Vehicle control groups (c, 0.1% DMSO) are displayed at 10⁻⁴ for graphing purposes. Data represent three independent experiments with 20 embryos per treatment group. <b>C</b>) Representative image of 120 hpf <i>ahr2</i>⁺ and (<b>D</b>) <i>ahr2</i>^hu3335 embryos developmentally exposed to 10 nM TCDD.</p

Schematic diagram of predicted AHR2 protein in <i>ahr2</i>^hu3335 zebrafish.

<p>The <i>ahr2</i>^hu3335 zebrafish line has a T to A point mutation in residue 534, resulting in a premature stop codon in the transactivation domain of the protein. The predicted truncated protein contains the ligand binding, DNA binding and ARNT binding domains, but lacks the transactivation domain previously shown to be essential for a functional AHR2 protein <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029346#pone.0029346-Andreasen1" target="_blank">[21]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029346#pone.0029346-Hahn3" target="_blank">[58]</a>. NLS: nuclear localization signal, NES1: nuclear export signal 1, NES2: nuclear export signal 2.</p

Molecular docking of TCDD and Leflunomide in zebrafish AHR isoforms.

<p><b>A</b>) TCDD docking orientation in zebrafish AHR2- and <b>B</b>) AHR1B-LBD homology model binding pocket (ICM v3.5-1n, Molsoft). <b>C</b>) Leflunomide docking orientation into AHR2-, <b>D</b>) AHR1B- and <b>E</b>) AHR1A homology model binding pockets. The residues are displayed as sticks and colored by atom type with the carbon atoms in green. The protein backbone is displayed as ribbon and colored by secondary structure. The ligand is displayed as sticks and colored by atom type with carbon atoms in orange (<b>A, C</b>), magenta (<b>B, D</b>) and yellow (<b>E</b>). H-bonds are represented by black dashed lines.</p

Fin and skeletal abnormalities observed in adult <i>ahr2</i>^hu3335 zebrafish.

<p><b>A–B</b>) Brightfield and (<b>C–D</b>) microCt imaging of adult <i>ahr2</i>⁺ and <i>ahr2</i>^hu3335zebrafish. Notable differences were observed in the dentate (d), premaxilla (pm), maxilla (mx), supraorbital (so), infraorbital 3(inf) and operculum (op).</p

Primer sequences for PCR experiments.

<p><b>mo</b>- morpholino mis-splice detection.</p><p><b>mut</b>- mutant point mutation detection.</p

<i>ahr2</i>^hu3335 embryos express reduced endogenous AHR2 mRNA and are resistant to TCDD-induced CYP induction.

<p><b>A</b>) Comparative analysis of AHR1A, AHR1B, CYP1A, CYP1B1, CYP1C1 and CYP1C2 mRNA expression in wild-type 5D and <i>ahr2</i>^hu3335 mutant embryos at 48hpf . ΔΔCt values were calculated by comparing sample ΔCt values (normalized to β-actin) to the mean <i>ahr2</i>⁺ ΔCt for each gene. Data were analyzed by paired student's t-test, * p<.05. <b>B</b>) Developmental exposure (6–24 hpf) to 1 nM TCDD induced significant CYP1A, CYP1C1 and CYP1C2 expression at 48 hpf in <i>ahr2</i>⁺ embryos. Data is shown normalized to vehicle-treated controls and was analyzed with paired student's t-test, *p<.05, ** p<.01. <b>C</b>) Developmental exposure to 1 nM TCDD did not induce significant mRNA expression changes in <i>ahr2</i>^hu3335 embryos. While CYP1A was elevated, the difference was not significant.</p