Genomic profiling reveals an alternate mechanism for hepatic tumor promotion by perfluorooctanoic acid in rainbow trout

Abstract

Background: Perfluorooctanoic acid (PFOA) is a potent hepatocarcinogen and peroxisome proliferator (PP) in rodents. Humans are not susceptible to peroxisome proliferation and are considered refractory to carcinogenesis by PPs. Previous studies with rainbow trout indicate they are also insensitive to peroxisome proliferation by the PP dehydroepiandrosterone (DHEA), but are still susceptible to enhanced hepatocarcinogenesis after chronic exposure.

Objectives: In this study, we used trout as a unique in vivo tumor model to study the potential for PFOA carcinogenesis in the absence of peroxisome proliferation compared with the structurally diverse PPs clofibrate (CLOF) and DHEA. Mechanisms of carcinogenesis were identified from hepatic gene expression profiles phenotypically anchored to tumor outcome.

Methods: We fed aflatoxin B(1) or sham-initiated animals 200-1,800 ppm PFOA in the diet for 30 weeks for tumor analysis. We subsequently examined gene expression by cDNA array in animals fed PFOA, DHEA, CLOF, or 5 ppm 17beta-estradiol (E(2), a known tumor promoter) in the diet for 14 days.

Results: PFOA (1,800 ppm or 50 mg/kg/day) and DHEA treatments resulted in enhanced liver tumor incidence and multiplicity (p < 0.0001), whereas CLOF showed no effect. Carcinogenesis was independent of peroxisome proliferation, measured by lack of peroxisomal beta-oxidation and catalase activity. Alternately, both tumor promoters, PFOA and DHEA, resulted in estrogenic gene signatures with strong correlation to E(2) by Pearson correlation (R = 0.81 and 0.78, respectively), whereas CLOF regulated no genes in common with E(2).

Conclusions: These data suggest that the tumor-promoting activities of PFOA in trout are due to novel mechanisms involving estrogenic signaling and are independent of peroxisome proliferation.

Keywords: clofibrate; dehydroepiandrosterone; estradiol; hepatocarcinogenesis; microarray; perfluorooctanoic acid; peroxisome proliferation; rainbow trout.

Figure 1

Effects of test compounds on tumor incidence, serum VTG, and serum E₂ compared with DMSO vehicle control. (A) Tumor incidence and multiplicity after exposure to CLOF, DHEA, or PFOA in the diet for 6 months compared with control (postinitiation by immersion in 10 ppb AFB₁ (each treatment consisted of a single tank of 68–100 individuals). (B) Serum VTG in trout after exposure to E₂, DHEA, and PFOA in the diet for 5 days as determined by Western blot (representative images shown) and ELISA (n = 4). (C) Serum E₂ in trout after exposure to E₂, DHEA, and PFOA in the diet for 14 days, as determined by enzyme immunosorbent assay. For (B) and (C), pools of blood plasma were obtained from five individual males in each replicate tank (n = 3). **p < 0.01 compared with control by one-way ANOVA with Dunnett’s multiple comparison test. ^#p < 0.0001 for tumor incidence and ^##p < 0.0001 for multiplicity compared with control, calculated by logistic regression analysis and Kruskal-Wallis test, respectively.

Figure 2

Hepatic gene expression analysis after dietary exposure to 0.1% DMSO (control), 500 or 1,800 ppm PFOA, 750 ppm DHEA, 1,800 ppm CLOF, or 5 ppm E₂ for 14 days. PCA, principal components analysis. (A) Bidirectional hierarchical clustering of hepatic gene expression in trout by Euclidean distance. Results are shown as fold change (log₂) of control of dye-swapped slides for biological replicates (n = three per treatment). Heatmap reflects expression profiles for genes differentially regulated 1.8-fold up or down (p < 0.05) in at least one treatment group. Red indicates up-regulation; green, down-regulation; black, unchanged expression; and grey, missing values. (B) Pairwise correlations of hepatic gene profiles; values are fold change (log₂) compared with control and were plotted to generate Pearson correlation coefficients (R) among the treatments (p < 0.0001). Lines indicate least-squares linear regression. (C) PCA on condition. Symbols represent biological replicates (n = 3), and ovals indicate overlap among E₂, PFOA, and DHEA treatments.

Figure 3

Comparison of gene expression of CYP2K5, VTG, CTSD, CYP1A, and ESR1 measured by microarray and real-time qRT-PCR. Values are expressed as fold change (log₂; mean ± SD) compared with vehicle control. Dashed lines indicate 1.8-fold change (± 0.847 log₂).