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

Summary of zebrafish AHR ligand binding, activity and expression.

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

<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

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

MMA enhances <i>P</i>. <i>aeruginosa</i> induced immune response.

<p>Cytokine secretion by HBEC exposed to MMA^III ± <i>P</i>. <i>aeruginosa</i>. n = 4 donors for each treatment. Different letters indicate statistically significant treatment means. Data labeled a are not statistically different from each other but are statistically different from data labeled b or c (p<0.05 as measured by one-way ANOVA). Data with the same letter are not significantly different. (A) IL-8 secretion (B) IL-6 secretion (C) CXCL1 secretion (D) CXCL2 secretion.</p

Arsenic exposure does not cause cytotoxicity.

<p>LDH release by HBEC was used to assess arsenic cytotoxicity and was measured using the Promega CytoTox 96 Non-Radioactive Cytotoxicity assay per manufacturers instructions. Data reported as optical density (OD, at 490 nm) of the cell culture medium bathing 500K cells. The first two bars represent LDH released by cells lysed by Triton-X (Lysed Cells). Data presented as mean ± SEM. LDH release was not significantly different from 0 in vehicle and arsenic treated cells, with and without <i>P</i>. <i>aeruginosa</i> exposure. n = 4 donors per treatment group.</p

DMA does not alter <i>P</i>. <i>aeruginosa</i> induced immune response.

<p>Cytokine secretion by HBEC exposed to DMA^V ± <i>P</i>. <i>aeruginosa</i>. n = 4 donors for each treatment. Different letters indicate statistically significant treatment means. Data labeled a are not statistically different from each other but are statistically different from data labeled b (p<0.05 as measured by one-way ANOVA). Data with the same letter are not significantly different. (A) IL-8 secretion (B) IL-6 secretion (C) CXCL1 secretion (D) CXCL2 secretion.</p