Exploring maternal and developmental toxicity of PFO4DA and PFO5DoA using hepatic transcriptomics and serum metabolomics
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Sprague-Dawley rat dams were exposed via oral gavage from GD18-22 to PFO4DA or PFO5DoA across a series of doses ranging from 0.1 to 62.5 mg/kg/d. Fetal livers and maternal livers and serum were collected on GD22 and hepatic transcriptomics in both dam and offspring and serum metabolomics in dams were performed with significance cutoffs of p < 0.05 and absolute fold change > 20%. Four-parameter logistic regression was used to predict the lowest maternal oral dose at which >=1000 differentially expressed genes (DEGs) would occur, with calculations performed separately for each chemical in dams and fetuses. Because doses for PFO4DA are below the linear portion of the dose-response curve, slope of the regression was set using the PFO5DoA regression slope.
For PFO5DoA, overt toxicity (substantial body weight loss, fetal mortality) was observed at the highest dose, so transcriptomic and metabolomic data only include up to 30 mg/kg/d. Dimensionality reduction measures including principal components analysis and heatmaps revealed strong clustering between maternal and fetal samples and between chemicals (PFO4DA vs PFO5DoA). Both dams and fetuses exhibited dose-responsive patterns of hepatic gene expression in response to PFO4DA or PFO5DoA. Of the 14,928 genes included in analysis, PFO4DA exposure in dams resulted in a maximum of 367 DEG at the highest dose, whereas PFO5DoA resulted in 4,506 DEG at the highest dose. Even at 10 mg/kg/d, PFO5DoA resulted in 1,915 DEG in dam liver. Fetuses were more affected at lower doses, but the pattern was similar, with PFO4DA resulting in 1,378 DEG at the highest dose and PFO5DoA 3,653 DEG at the highest dose. The transcriptomic dose causing >= 1000 DEG was estimated at 120 for Maternal PFO4, 5.3 for Maternal PFO5, 39 for Fetal PFO4DA, and 3.1 mg/kg/d for Fetal PFO5DoA. Several genes were changed by all doses of PFO5DoA in both maternal and fetal samples, including multiple acyl-coA thioesterases and related enzymes that hydrolyze acyl-coA to free fatty acids, as well as Vnn1, a corticosteroid-responsive gene. Hallmark and Reactome pathway analysis of fetal and maternal liver metabolome revealed remarkable changes in metabolism of bile acids and salts, fatty acid metabolism, metabolism of lipids and lipoproteins, PPARa activation of gene expression, and phospholipid metabolism. In maternal serum metabolomics, PFO4DA exposure did not result in meaningful changes at doses up to 62.5 mg/kg/d, but PFO5DoA exposure resulted in differential metabolite abundance for 149 unique metabolites, with 4 significantly altered at 0.3 mg/kg, 31 metabolites at 1 mg/kg, 51 at 3 mg/kg, 58 at 10 mg/kg, and 119 at 30 mg/kg/d. Multi-omics KEGG and Reactome pathway analysis of integrated maternal liver transcriptomics and serum metabolomics revealed significant convergent pathway-level changes as low as 3 mg/kg/d PFO4DA and 0.3 mg/kg/d PFO5DoA exposure, including fatty acid metabolism at all doses of PFO5DoA and all doses of PFO4DA above 10 mg/kg/d, with all PFO4DA changes driven entirely by the magnitude of transcriptomic changes. Striking pathway-level changes shared between hepatic transcriptome and serum metabolome in exposed dams included metabolism of lipids, hemostasis, signaling by G-protein coupled receptors (GPCRs), neuronal system, immune and innate immune system, PPARa activation of gene expression, and regulation of lipid metabolism by PPARa.