Globally, biomonitoring data demonstrate virtually all humans carry residues of multiple per- and polyfluoroalkyl substances (PFAS). Despite pervasive co-exposure, limited mixtures-based in vivo PFAS toxicity research has been conducted. Perfluorooctanoic acid (PFOA) and perfluorooctane sulfonic acid (PFOS) are commonly detected PFAS in human and environmental samples and both produce adverse effects in laboratory animal studies, including maternal and offspring effects when orally administered during pregnancy and lactation. To evaluate the effects of combined exposure to PFOA and PFOS, we orally exposed pregnant Sprague-Dawley rats from gestation day 8 (GD8) to postnatal day 2 (PND2) to PFOA (10-250 mg/kg/d) or PFOS (0.1-5 mg/kg/d) individually to characterize effects and dose response curve parameters, followed by a variable-ratio mixture experiment with a constant dose of PFOS (2 mg/kg/d) mixed with increasing doses of PFOA (3-80 mg/kg/d). The mixture study design was intended to: 1) shift the PFOA dose response curves for endpoints shared with PFOS, 2) allow comparison of dose addition (DA) and response addition (RA) model predictions, 3) conduct relative potency factor (RPF) analysis for multiple endpoints, and 4) avoid overt maternal toxicity. Maternal serum and liver concentrations of PFOA and PFOS were consistent between the individual chemical and mixture experiments. Combined exposure with PFOS significantly shifted the PFOA dose response curves towards effects at lower doses compared to PFOA-only exposure for multiple endpoints and these effects were well predicted by dose addition. For endpoints amenable to mixture model analyses, DA produced equivalent or better estimates of observed data than RA. All endpoints evaluated were accurately predicted by RPF and DA approaches except for maternal gestational weight gain, which produced less-than-additive results in the mixture. Data support the hypothesis of cumulative effects on shared endpoints from PFOA and PFOS co-exposure and dose additive approaches for predictive estimates of mixture effects.

Simultaneous exposure to multiple per- and polyfluoroalkyl substances (PFAS) is common in humans across the globe. Individual PFAS are associated with adverse health effects, yet the nature of mixture effects after exposure to two or more PFAS remains unclear. Previously we reported that oral administration of hexafluoropropylene oxide-dimer acid (HFPO-DA, or GenX), Nafion byproduct 2 (NBP2), or perfluorooctane sulfonate (PFOS) individually during pregnancy produced maternal and F1 effects. Here, we hypothesized that responses to the combined exposure to these three PFAS would be dose additive. Pregnant Sprague-Dawley rats were exposed to a fixed-ratio equipotent mixture where the top dose contained each PFAS at their ED50 for neonatal mortality (100 % dose = PFOS 3 mg/kg; NBP2 10 mg/kg; HFPO-DA 110 mg/kg), followed by a dilution series (33.3, 10, 3.3, and 1 %) and vehicle controls (0 % dose). Consistent with the single chemical studies, dams were exposed from gestation day (GD)14-18 or from GD8-postnatal day (PND2). Fetal and maternal livers on GD18 displayed multiple significantly upregulated genes associated with lipid and carbohydrate metabolism at all dose levels, while dams displayed significantly increased liver weight (≥3.3 % dose) and reduced serum thyroid hormones (≥33.3 % dose). Maternal exposure from GD8-PND2 significantly reduced pup bodyweights at birth (≥33.3 % dose) and PND2 (all doses), increased neonatal liver weights (≥3.3 % dose), increased pup mortality (≥3.3 % dose), and reduced maternal bodyweights and weight gain at the top dose. Echocardiography of adult F1 males and females identified significantly increased left ventricular anterior wall thickness (~10 % increase), whereas other cardiac morphological, functional, and transcriptomic measures were unaffected. Mixture effects in maternal and neonatal animals conformed to dose addition using a relative potency factor (RPF) analysis. Results support dose addition-based cumulative assessment approaches for estimating combined effects of PFAS co-exposure.

Various PFAS have been identified as potential endocrine-disrupting chemicals due to estrogen receptor activation, impacts on puberty timing, or impacts on hormonally sensitive endpoints in fish. This study screened multiple PFAS in the rat uterotrophic assay to determine potential estrogenic effects on the uterus with PFAS exposure. This study also explored PFAS-dependent uterine signaling with an osmotic stress mRNA gene expression array. Briefly, Sprague–Dawley rats (26–39 days old) were ovariectomized, and uterine tissue was allowed to regress for a 3-week period of recovery. Animals were then exposed daily via oral gavage to PFAS for 4 days, and then uterine weight was determined. In contrast to the positive control estrogens, the PFAS tested (4:2, 6:2, and 8:2FTOH; perfluorooctanesulfonamide (PFOSA), perfluorononanoic acid (PFNA), perfluorohexane sulfonate (PFHxS), perfluorooctane sulfonate (PFOS), nafion byproduct 2 (NBP2), 1H,1H,8H,8H-perfluorooctane-1,8-diol (FC8-diol) and 1H,1H,10H,10H-perfluorodecane-1,10-diol (FC10-diol)) caused no significant changes in the uterine weight. Hormonally active compounds can act as carcinogens, and because earlier rodent work has demonstrated that chronic PFOA exposure is associated with increased risk of uterine cancer, uterine mRNA gene expression was explored with an osmotic stress RT-qPCR array. PFAS exposure significantly upregulated multiple genes across the array, with PFOSA being the compound most similar to the reference estrogens (estradiol benzoate and ethinyl estradiol) in its expression pattern. Also, across all PFAS, pathway analysis revealed that the paxillin pathway, a pathway important in tumor suppressor gene SHP-2 signaling, was significantly upregulated with PFAS exposure. These results demonstrate that in vitro estrogen screens or impacts in fish may show different responses from direct impacts on mammalian uterine weight as assessed with the uterotrophic assay. This study also builds out new mechanisms that may contribute to understanding of carcinogenic changes seen in the uterus after PFAS exposure.