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

Primer sequences for PCR experiments.

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

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

<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

Primer sequences for RT-PCR experiments.

<p><b>H: Human M: Mouse, FP: Forward Primer, RP: Reverse Primer.</b></p><p><b>¹</b><b>Sequences described previously.</b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013128#pone.0013128-Bisson1" target="_blank">[<b>33</b>]</a></p

Leflunomide, but not its active metabolite, A771726, activates the AhR.

<p>(A) Structures of leflunomide (left) and its metabolite A771726 (right). (B–C) Reporter gene assays were conducted in Hepa1.1 cells or in HepG2 cells transiently transfected with XRE-Luc reporter gene. Results are the mean ± SEM of at least three independent experiments, each of which consisted of at least three biological replicates. ***: p<0.001 compared with vehicle treatment and p<0.05 compared with corresponding dose of A771726. (D) To confirm the observations of the reporter gene assays, we performed semi quantitative RT-PCR in WT Hepa1 cells for CYP1A1 with total RNA isolated from cells treated with vehicle (0.1% DMSO), leflunomide (L, 20 µM) or A771726 (M, 20 µM) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013128#pone-0013128-g002" target="_blank">Figure 2</a>. GAPDH was included as a control. Consistent with reporter gene assays, A771726 failed to activate CYP1A1 beyond that of vehicle treatment, while leflunomide induced strong CYP1A1 expression. (E) Cellular localization of AhR was analyzed by immunofluorescence of Hepa1 cells treated with TCDD, leflunomide, or A771726 for 1 or 3 hours. The FITC (green) channel represents AhR staining, while DAPI (blue) represents the nucleus. The AhR translocated to the nucleus following treatment with both TCDD and leflunomide, while it remained in the cytosol following treatment with A771726. (F) M2 is the major tautomeric form of A771726. Molecular docking of M2 and leflunomide in the homology models of mouse (G) and human (H) AhR ligand binding domain reveal favorable energetic and docking for leflunomide but not M2.</p

Activation of AhR target genes by leflunomide requires Arnt.

<p>(A) Induction of the AhR target genes CYP1A1, UGT1A1, and NQO1 was performed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013128#pone-0013128-g002" target="_blank">figure 2</a>. Vehicle (V, 0.1% DMSO), TCDD (T, 1 nM), leflunomide (L20, 20 µM; L10, 10 µM). GAPDH expression was used as a control. PCR cycle numbers are indicated. Target gene induction in Hepa1 vT{2} cells expressing a functional Arnt protein was similar as to that seen with WT Hepa1 cells. CYP1A1 and NQO1 were not induced by leflunomide in Hepa1 C4 cells that do not express a functional Arnt protein. (B–C) XRE-Luc reporter gene assays in Hepa1 vT{2} and C4 cells. Cells were transfected and treated with leflunomide or controls (VEH, Vehicle, 0.1% v/v DMSO; TCDD, 1 nM; and LEF, leflunomide,10 µM) as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013128#s2" target="_blank">methods</a> section. (B) Consistent with semi quantitative RT-PCR analysis, TCDD and leflunomide induced expression of the XRE-Luc reporter gene in Hepa1 vT{2} cells. (C) Treatment with TCDD or leflunomide failed to activate the XRE-Luc reporter in the Hepa1 C4 cells. However, transient co-expression of Arnt rescued XRE-Luc reporter gene induction. Reporter gene assays are the mean ± SEM of three independent experiments.</p