Greylin Nielsen

Per- and polyfluoroalkyl substances (PFAS) are a large group of persistent chemicals that are pervasive in the environment leading to widespread exposure for humans. Perfluorooctanoic acid (PFOA), one of the most commonly measured PFAS in people, disrupts liver and serum lipid homeostasis as shown in animal toxicity and human epidemiological studies. We tested the hypothesis that the effects of PFOA exposure in mice expressing mouse PPARα (mPPARα) are driven largely through PPARα-dependent mechanisms while non-PPARα dependent mechanisms will be more apparent in mice expressing human PPARα (hPPARα). Female and male mPPARα, hPPARα, and PPARα null mice were exposed to PFOA (0.5, 1.4 or 6.2 mg PFOA/L) via drinking water for 14 weeks. Concurrently, mice consumed an American diet containing human diet-relevant amounts of fat and cholesterol. Here, we focused on the effects in female mice, given the dearth of data reported on PFAS-induced effects in females. Increasing the duration of PFOA exposure reduced weight gain in all genotypes of female mice while end-of-study body fat was lower in PFOA exposed hPPARα and PPARα null mice. Serum cholesterol, but not triacylglyceride, concentrations were increased by PFOA exposure in a PPARα-dependent manner. Hepatic triacylglycerides were higher in vehicle-exposed mPPARα and PPARα null mice than hPPARα mice, and PFOA significantly reduced concentrations in mPPARα and PPARα null mice only. In contrast, PFOA increased hepatic cholesterol content in a PPARα-dependent manner. Changes in liver and serum cholesterol may be explained by a strong, PPARα-dependent downregulation of Cyp7a1 expression. PFOA significantly increased PPARα target gene expression in mPPARα mice. Other nuclear receptors were examined: CAR target gene expression was only induced by PFOA in hPPARα and PPARα null mice. PXR target gene expression was induced by PFOA in all genotypes. Results were similar in male mice with two exceptions: (1) vehicle-exposed male mice of all genotypes were equally susceptible to diet-induced hepatic steatosis; (2) male mice drank less water, resulting in lower serum PFOA levels, which may explain the less significant changes in lipid endpoints. Overall, our results show that PFOA modifies triacylglyceride and cholesterol homeostasis independently and that PPARα plays an important role in PFOA-induced increases in liver and serum cholesterol.

Adverse lung outcomes from exposure to per-and polyfluoroalkyl substances (PFAS) are known; however, the mechanism of action is poorly understood. To explore this, human bronchial epithelial cells were grown and exposed to varied concentrations of short-chain (perfluorobutanoic acid, perflurobutane sulfonic acid and GenX) or long-chain (PFOA and perfluorooctane sulfonic acid (PFOS)) PFAS, alone or in a mixture to identify cytotoxic concentrations. Non-cytotoxic concentrations of PFAS from this experiment were selected to assess NLRP3 inflammasome activation and priming. We found that PFOA and PFOS alone or in a mixture primed and activated the inflammasome compared with vehicle control. Atomic force microscopy showed that PFOA but not PFOS significantly altered the membrane properties of cells. RNA sequencing was performed on the lungs of mice that had consumed PFOA in drinking water for 14 weeks. Wild type (WT), PPARα knock-out (KO) and humanized PPARα (KI) were exposed to PFOA. We found that multiple inflammation- and immune-related genes were affected. Taken together, our study demonstrated that PFAS exposure could alter lung biology in a significant manner and may contribute to asthma/airway hyper-responsiveness.

Human exposure to per- and polyfluoroalkyl substances (PFAS) is ubiquitous, with mixtures of PFAS detected in drinking water, food, household dust, and other exposure sources. Animal toxicity studies and human epidemiology indicate that PFAS may act through shared mechanisms including activation of peroxisome proliferator activated receptor α (PPARα). However, the effect of PFAS mixtures on human relevant molecular initiating events remains an important data gap in the PFAS literature. Here, we tested the ability of modeling approaches to predict the effect of diverse PPARα ligands on receptor activity using Cos7 cells transiently transfected with a full length human PPARα (hPPARα) expression construct and a peroxisome proliferator response element-driven luciferase reporter. Cells were treated for 24 hours with two full hPPARα agonists (pemafibrate and GW7647), a full and a partial hPPARα agonist (pemafibrate and mono(2-ethylhexyl) phthalate), or a full hPPARα agonist and a competitive antagonist (pemafibrate and GW6471). Receptor activity was modeled with three additive approaches: effect summation, relative potency factors (RPF), and generalized concentration addition (GCA). While RPF and GCA accurately predicted activity for mixtures of full hPPARα agonists, only GCA predicted activity for full and partial hPPARα agonists and a full agonist and antagonist. We then generated concentration response curves for seven PFAS, which were well-fit with three-parameter Hill functions. The four perfluorinated carboxylic acids (PFCA) tended to act as full hPPARα agonists while the three perfluorinated sulfonic acids (PFSA) tended to act as partial agonists that varied in efficacy between 28-67% of the full agonist, positive control level. GCA and RPF performed equally well at predicting the effects of mixtures with three PFCAs, but only GCA predicted experimental activity with mixtures of PFSAs and a mixture of PFCAs and PFSAs at ratios found in the general population. We conclude that of the three approaches, GCA most accurately models the effect of PFAS mixtures on hPPARα activity in vitro . Highlights: Perfluorinated carboxylic acids are full human PPARα agonists Perfluorinated sulfonic acids are partial human PPARα agonists GCA predicts human PPARα activity for mixtures of full and partial agonists GCA predicts human PPARα activity for mixtures of agonists and competitive antagonists GCA accurately predicts human PPARα activity in response to PFAS mixtures

Humans are exposed to per- and polyfluoroalkyl substances (PFAS) in their drinking water, food, air, dust in their homes, and by direct use of consumer products. Increased concentrations of serum total cholesterol and low density lipoprotein cholesterol are among the endpoints best supported by epidemiology. The objectives of this study were to generate a new model for examining PFAS-induced dyslipidemia and to conduct molecular studies to better define mechanism(s) of action. We tested the hypothesis that PFOA exposure at a human-relevant level dysregulates expression of genes controlling cholesterol homeostasis in livers of mice expressing human PPARα (hPPARα). Female and male hPPARα and PPARα null mice were fed a diet based on the “What we eat in America” analysis and exposed to perfluorooctanoic acid (PFOA) in drinking water (8 µM) for 6 weeks. This resulted in a serum PFOA concentration of 48 μg/ml. PFOA increased liver mass, which was associated with histologically-evident lipid accumulation. PFOA induced PPARα and constitutive androstane receptor target gene expression in liver. Expression of genes in four pathways regulating cholesterol homeostasis were also measured. PFOA decreased expression ofHmgcrin a PPARα-dependent manner. PFOA decreased expression ofLdlrandCyp7a1in a PPARα-independent manner.Apobexpression was not changed. Gene expression in females appeared to be more sensitive to PFOA exposure than in males. This novel study design (hPPARα mice, American diet, long term exposure) generated new insight on the effects of PFOA on cholesterol regulation in the liver and the role of hPPARα.