ABSTRACT: Production of PFAS has shifted from the most-studied PFAS, longer-chain perfluorinated alkyl acids, to shorter-chain or other alternative moieties including fluoroethers like HFPO-DA (“GenX chemicals”), Nafion Byproducts, and the PFOx homologous series that includes PFO4DA and PFO5DoA. In contaminated study areas, the fluoroethers PFO4DA and PFO5DoA, although detected at relatively low levels in surface water, have been detected in serum and/or tissues with high frequency in humans, wildlife including fish and alligators, and pets including horses. Although these compounds have been detected, there are extremely limited toxicity data available and no developmental toxicology studies that could be used to inform risk assessment or regulatory actions. To address these data gaps, 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/day and, in conjunction with apical measures of toxicity, hepatic transcriptomics in both dam and offspring and serum metabolomics in dams were performed. Differential expression analysis was performed for transcriptomics and metabolomics, and pathway analysis, including a multi-omic integrative analysis, was performed. For PFO5DoA, overt toxicity (substantial body weight loss, fetal mortality) was observed at the highest dose, so data only include up to 30 mg/kg/day. Dimensionality reduction measures including principal components analysis and heatmaps revealed strong separation of 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 differential expression analysis, PFO4DA exposure in dams resulted in a maximum of 367 differentially expressed genes (DEG) at the highest dose (62.5 mg/kg/d) whereas PFO5DoA resulted in 4,506 DEG at the highest dose (30 mg/kg/d). 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 62.5 mg/kg/d and PFO5DoA 3,653 DEG at 30 mg/kg/d. Several DEG were changed by every dose of PFO5DoA (≥ 0.3 mg/kg/d) in both maternal and fetal samples, including multiple acyl-coA thioesterases (Acot1, Acot2, Acot3, Acot4) and related enzymes that hydrolyze acyl-coA to free fatty acids, as well as Vnn1, a corticosteroid-responsive gene. Hallmark and Reactome pathway analyses of fetal and maternal livers revealed remarkable changes in metabolism of bile acids and salts, fatty acid metabolism, metabolism of lipids and lipoproteins, PPARα activation of gene expression, and phospholipid metabolism. In maternal serum metabolomics, PFO4DA did not result in significant changes at doses up to 62.5 mg/kg/d. In contrast, 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 10mg/kg, and 119 at 30 mg/kg/d. Multi-omics KEGG and Reactome pathway analyses 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. Notable multi-omics pathway level changes include fatty acid metabolism at all doses of PFO5DoA and at PFO4DA ≥ 10 mg/kg/d, with PFO4DA changes driven by the magnitude of transcriptomic changes and low/null detection of metabolites. Striking pathway-level changes identified for hepatic transcriptome and serum metabolome in exposed dams included metabolism, metabolism of lipids, hemostasis, signaling by G-protein coupled receptors (GPCRs), neuronal system, immune and innate immune system, PPARα activation of gene expression, and regulation of lipid metabolism by PPARα. The transcriptomic and metabolomic effects of PFO4DA and PFO5DoA appear consistent with other carboxylic acid PFAS, with primary changes related to lipid metabolism, bile acids, cholesterol, and cellular stress. Although PFO4DA and PFO5DoA are structurally similar, separated by only a CF2O, the potency of transcriptomic and metabolomic effects of developmental exposure to each was significantly different, with 5-day exposure to PFO5DoA causing changes in glucose and lipid metabolism and stress signals at much lower doses than PFO4DA in both dams (~ 35-fold more potent at inducing hepatic DEG) and fetuses (~ 11.6-fold more potent at inducing hepatic DEG). Although it is likely that some differences in toxicity between PFO4DA and PFO5DoA are caused by accumulation differences resulting in different dosimetry, PFO5DoA demonstrated much greater transcriptomic and metabolomic toxicity at equivalent oral doses.
