Transcriptional pathways linked to fetal and maternal hepatic dysfunction caused by gestational exposure to perfluorooctanoic acid (PFOA) or hexafluoropropylene oxide-dimer acid (HFPO-DA or GenX) in CD-1 mice

Abstract

Per- and polyfluoroalkyl substances (PFAS) comprise a diverse class of chemicals used in industrial processes, consumer products, and fire-fighting foams which have become environmental pollutants of concern due to their persistence, ubiquity, and associations with adverse human health outcomes, including in pregnant persons and their offspring. Multiple PFAS are associated with adverse liver outcomes in adult humans and toxicological models, but effects on the developing liver are not fully described. Here we performed transcriptomic analyses in the mouse to investigate the molecular mechanisms of hepatic toxicity in the dam and its fetus after exposure to two different PFAS, perfluorooctanoic acid (PFOA) and its replacement, hexafluoropropylene oxide-dimer acid (HFPO-DA, known as GenX). Pregnant CD-1 mice were exposed via oral gavage from embryonic day (E) 1.5-17.5 to PFOA (0, 1, or 5 mg/kg-d) or GenX (0, 2, or 10 mg/kg-d). Maternal and fetal liver RNA was isolated (N = 5 per dose/group) and the transcriptome analyzed by Affymetrix Array. Differentially expressed genes (DEG) and differentially enriched pathways (DEP) were obtained. DEG patterns were similar in maternal liver for 5 mg/kg PFOA, 2 mg/kg GenX, and 10 mg/kg GenX (R²: 0.46-0.66). DEG patterns were similar across all 4 dose groups in fetal liver (R²: 0.59-0.81). There were more DEGs in fetal liver compared to maternal liver at the low doses for both PFOA (fetal = 69, maternal = 8) and GenX (fetal = 154, maternal = 93). Upregulated DEPs identified across all groups included Fatty Acid Metabolism, Peroxisome, Oxidative Phosphorylation, Adipogenesis, and Bile Acid Metabolism. Transcriptome-phenotype correlation analyses demonstrated > 1000 maternal liver DEGs were significantly correlated with maternal relative liver weight (R² >0.92). These findings show shared biological pathways of liver toxicity for PFOA and GenX in maternal and fetal livers in CD-1 mice. The limited overlap in specific DEGs between the dam and fetus suggests the developing liver responds differently than the adult liver to these chemical stressors. This work helps define mechanisms of hepatic toxicity of two structurally unique PFAS and may help predict latent consequences of developmental exposure.

Keywords: Animal models; Developmental exposure; Emerging contaminants; Liver disease; PFAS.

Fig. 1.

Matthews correlation coefficient plot demonstrating similarity between significant DEGs (shown in Table 1) across the four dose conditions for maternal and fetal liver. Gene expression changes tended to be more similar across dose groups in fetal liver (0.59–0.81) than across dose groups in maternal liver (0.15–0.66). Correlation coefficients between maternal and fetal liver across the dose conditions were least similar when comparing fetal dose groups to maternal 1 mg/kg PFOA (0.03–0.09) whereas all other correlation permutations were comparable (0.31–0.37).

Fig. 2.

Differentially expressed genes (DEGs) in maternal liver after gestational exposure to PFOA (1 and 5 mg/kg) or GenX (2 and 10 mg/kg). (A) Overlapping upregulated DEGs and (B) overlapping downregulated DEGs are shown in the Venn diagrams. (C) Heatmap showing row z-score of the normalized expression values for 80 genes significantly altered in the 5 mg/kg PFOA, 2 mg/kg GenX, and 10 mg/kg GenX exposure groups where each column represents an individual animal to highlight biologic variability within a given treatment group. Three non-coding genes were identified and include Gm11844, Gm15441, and Mir292b. DEGs were considered significant if absolute fold-change values were ≥ 2 and p ≤ 0.005.

Fig. 3.

Differentially expressed genes (DEGs) in fetal liver after in utero exposure to PFOA (1 and 5 mg/kg) or GenX (2 and 10 mg/kg). (A) Overlapping upregulated DEGs and (B) overlapping downregulated DEGs are shown in the Venn diagrams. (C) Heatmap showing row z-score of the normalized expression values for 61 genes significantly altered in altered in all four exposure groups where each column represents an individual animal to highlight biologic variability within a given treatment group. One non-coding gene, Gm15035, was identified as a DEG across all treatment groups and one additional non-coding gene, 1300002E11Rik, was identified as a DEG across all treatment groups except 1 mg/kg PFOA. DEGs were considered significant if absolute fold-change values were ≥ 2 and p ≤ 0.005.

Fig. 4.

Normalized enrichment scores (NES) for significantly enriched MSigDB Hallmark Pathways in maternal (top panels) and fetal liver (bottom panels). Size of the dot indicates the log10(p value) with larger dots corresponding to more significant p values. Dot color gradient corresponds to the number of groups (1–8, with lighter colors corresponding to higher values) for which the pathway was significantly enriched and blank regions on the plot indicate no statistical significance of the pathway NES for that specific group. P ≤ 0.05 for all pathways shown.

Fig. 5.

Gene networks for the Oxidative Phosphorylation Hallmark Pathway and related Hallmark Pathways constructed in Ingenuity Pathway Analysis using statistically significant DEGs in (A) maternal liver after gestational exposure to 1 mg/kg PFOA, (B) fetal liver after in utero exposure to 1 mg/kg PFOA, (C) maternal liver after gestational exposure to 2 mg/kg GenX, and (D) fetal liver after in utero exposure to 2 mg/kg GenX. DEGs were considered significant if absolute fold-change values were ≥ 2 and p ≤ 0.005.

Fig. 6.

Correlation between individual dam gene expression and phenotype for two example genes, Aco2 and F11, and relative liver weight. Box and whiskers plot showing dam relative liver weights (A) and normalized expression of (B) Aco2 and (C) F11 in the liver, with treatment groups significantly different from the Vehicle Control in solid blue. Transcriptome-phenotype correlation plots, r-squared values, and adjusted p-values for dam relative liver weight and (D) Aco2 and (E) F11 expression. N = 5 dams per treatment group.