Dose-dependent changes in the RLWs 24 h post dose for TCDD, PCB126, and TCDF. Results are displayed as mean ± SE of at least four independent replicates. Data were analyzed by ANOVA followed by Dunnett’s post hoc test: *p < 0.05 for vehicle versus treated samples. No additional significant treatment-related alterations in liver or organ weights were noted in the dose-response study.

Hepatic (A) TCDD, (B) PCB126, and (C) TCDF levels per g liver wet weight measured 24 h post dose using HRGC/HRMS. The data are displayed on a log scale to visualize tissue concentrations at all doses. The results are displayed as mean ± SE of at least three independent samples. Data were analyzed by ANOVA followed by Dunnett’s post hoc test: *p< 0.05 for vehicle versus treated samples.

Representative hematoxylin and eosin–stained liver sections 224 h postexposure to PCB126, TCDF, or TCDD. Selected doses resulted in vacuolization, single cell necrosis, and/or immune cell infiltration. (A) Vehicle treatment resulted in no visible hepatic alterations. (B) 300 μg/kg PCB126 elicited minimal vacuolization compared with (C) 1000 μg/kg TCDF and (D) 300 μg/kg TCDD, which exhibited more pronounced vacuolization. (E) 3000 μg/kg TCDF resulted in immune cell infiltration as well as (F) instances of necrosis (arrow). Bars = 50 μm.

Dose-dependent induction of EROD activity following exposure to TEF-equivalent doses of TCDD, PCB126, and TCDF. The ToxResponse Modeler calculated ED₅₀ values of 1.1, 53.6, and 54.2 μg/kg for TCDD, PCB126, and TCDF, respectively, yielding PCB126 and TCDF EROD REPs of 0.02. Symbols represent the mean ± SE of four independent samples. Sigmoidal dose-response curve fitting was done using GraphPad Prism 5.0.

(A) Comprehensive TCDD, TCDF, and PCB126 dose-response analysis—Part 1/3. Differentially regulated features were identified using a relaxed P1(t) cutoff of 0.90 to maximize the number of differentially expressed genes under consideration. TCDD elicited ∼1.6 times more differentially expressed features compared with TCDF (13,476 vs. 8655) and ∼1.3 times more differentially expressed features compared with PCB126 (13,476 vs. 10,983). Unions were taken to identify all differentially regulated features. Features missing data at any dose, as well as Agilent controls, were removed and not considered further. Features were mapped to specific genes for further analysis. (B) Comprehensive TCDD, TCDF, and PCB126 dose-response analysis—Part 2/3. Differentially expressed genes were analyzed further if the change in expression was greater than 1.5-fold for at least one dose. Genes exhibiting a sigmoidal dose-response were identified and intersected to identify genes responsive to both TCDD and TCDF and to TCDD and PCB126 at 24 h. Genes were examined further if the model-based ED₅₀ value, calculated by ToxResponse Modeler, was within the experimental dose range. Genes exhibiting a sigmoidal dose-response curve for both TCDD and TCDF were intersected to identify 1506 genes that exhibited an expression change greater than 1.5-fold for at least one dose, a P1(t) > 0.90, a sigmoidal profile, and an ED₅₀ value within the experimental range. Identical analysis yielded only 388 sigmoidal genes for TCDD and PCB126. PCB126 lost a majority of differentially expressed dose-responsive genes from further consideration because the ED_50s were not within the experimental dose range. (C) Comprehensive TCDD, TCDF, and PCB126 dose-response analysis—Part 3/3. In the final analysis, assumptions regarding similarities in the slopes and shapes of corresponding TCDD and TCDF as well as TCDD and PCB126 sigmoidal dose-response curves were assessed by calculating the correlation coefficients of the elicited dose-response curves. The correlation analysis identified 210 genes from TCDD versus TCDF comparisons, and only 40 genes from TCDD and PCB126 comparisons with correlation coefficients greater than 0.60. The distribution of individual gene expression REPs is provided. The median REP for hepatic gene expression in the immature ovariectomized C57BL/6 mouse at 24 h was calculated to be 0.06 and 0.02 for TCDF and PCB126, respectively.

Three-way Venn analysis of TCDD, PCB126, and TCDF elicited differentially expressed genes at (A) stringent (|fold change| > 1.5, P1(t) > 0.99) and (B) relaxed statistical criteria (|fold change| > 1.5, P1(t) > 0.90).

QRT-PCR verification of selected AhR-regulated genes: Cyp1a1, Cyp1a2, Nqo1, Tiparp, and Notch1 for (A) TCDD, (B) PCB126, and (C) TCDF at 24 h. The same RNA samples used in the dose-response microarray studies were also used for QRT-PCR analysis. All fold changes were calculated relative to vehicle controls. Bars (left y-axis) and lines (right y-axis) represent QRT-PCR and microarray data, respectively. The genes are represented by their official gene symbols. Bars represent the mean ± SE of four independent QRT-PCR samples. QRT-PCR data were analyzed by ANOVA followed by Dunnett’s post hoc test: *p < 0.05 for vehicle versus treated samples. ED₅₀ values for microarray and QRT-PCR mRNA levels are provided in Supplementary table 7.