Jennifer J. Schlezinger

Per- and polyfluoroalkyl substances (PFAS) are significant environmental contaminants that cause a number of adverse human health effects. Studies increasingly link exposure to PFAS with disruptions in osteoblast and osteoclast differentiation and function, reduced bone mineral density (BMD), and negative impacts on skeletal structure. In vivo studies on the effects of PFAS on bone health remain limited, with none investigating the impacts in mice expressing human peroxisome proliferator activated receptor α (PPARα). Given PFAS’s ability to interact with the peroxisome proliferator activated receptor family and their importance in bone health, we present work investigating the effects of perfluorooctanoic acid (PFOA) exposure and PPARα genotype (mouse, human or null) on bone quality across two exposure periods (6 and 14 weeks) in mice fed a human relevant diet. In both exposure scenarios, the serum PFOA concentrations were ≈ 50 μg/mL, similar to a highly exposed fluorochemical worker. We began by establishing the liver phenotype across exposures and genotypes. Across genotypes and exposure timing, PFOA increased liver to body weight ratios, but the contribution of increased triglycerides was reduced at 14 weeks. Exposure to PFOA increased hepatic PPARα-dependent gene expression ( Acox ) in both sexes and exposure periods, but only in mPPARα mice. The effect of PFOA on hepatic CAR- and PXR-dependent gene expression ( Cyp2b10 and Cyp3a11 , respectively) was more complex; with sex-, genotype-, and exposure timing-dependent differences. Trabecular and cortical bone parameters were not impacted by PFOA treatment, but genotype was a significant factor across sexes and exposure periods. mPPARα mice had higher trabecular tissue mineral density at 9 and 20 weeks of age than hPPARα mice, but the lower trabecular bone volume evident at 9 weeks of age resolved at 20 weeks of age. mPPARα mice had higher cortical tissue mineral density and bone volume than hPPARα and PPARα null mice. Analysis of serum markers of osteoblast and osteoclast activity following 14 weeks of exposure to PFOA revealed a PPARα-dependent decrease in PINP but no effect on CTX. Our results reveal the importance of PPARα genotype for bone quality parameters and emerging evidence of the effect of PFOA on osteoblast activity. Given the unusual mouse bone quality model (sv/129 background), this work underscores the need for future in vivo research shifts to investigate the effects of PFAS on in bone quality.

Exposure to pollutants, including the ubiquitous “forever chemical,” perfluorooctane sulfonate (PFOS) has increasingly been associated with metabolic dysfunction-associated steatotic liver disease. Recent epidemiological evidence has identified associations between per- and polyfluoroalkyl substances (PFAS) exposure and increased liver injury in alcohol consumers, suggesting potential interactions between these exposures. However, the intersection of pollutant exposures and alcohol-associated liver disease (ALD) is not well studied. We hypothesize that pollutants may disrupt hepatic metabolism to modify ALD severity. Recently, we developed a two-hit (ethanol [EtOH] plus pollutant) mouse model, enabling testing of this hypothesis. Here, we elucidate the metabolic and disease-modifying effects of PFOS in this model. Male C57BL/6J mice were fed isocaloric control or 5% EtOH Lieber-DeCarli diet for 15 days. From day 6 of feeding, mice were concurrently gavaged with 1 mg/kg PFOS or 2% tween-80 vehicle for 10 days, followed by a 5 g/kg EtOH binge dose and euthanized 5 to 6 h later. Approximately 60% of the administered PFOS dose accumulated in the liver. PFOS exacerbated EtOH-induced hepatic steatosis and was associated by higher levels of plasma very low-density lipoprotein and alanine aminotransferase. PFOS upregulated hepatic EtOH-metabolizing enzymes and lowered blood alcohol levels. Ingenuity Pathway Analysis (IPA) Top Toxicity Functions/Lists associated with hepatic gene expression following PFOS co-exposure in EtOH-fed mice included: Fatty acid metabolism and liver steatosis; nuclear receptor activation, cytochrome P450, and reactive oxygen species; apoptosis; liver fibrosis; and hepatocellular carcinoma (HCC). Gene Ontology/Kyoto Encyclopedia of Genes and Genomes analyses similarly revealed enrichment in fatty acid, xenobiotic, alcohol, or glutathione metabolic processes; and peroxisome proliferator-activated receptor (PPAR) signaling. PFOS upregulated hepatic expression of several nuclear receptors (e.g. Pparα, Car, and Pxr) and their P450 target genes (e.g. Cyp4a10, Cyp2b10, and Cyp3a11) by real-time-PCR or Western blot, confirming key IPA predictions. PFOS is a metabolism-disrupting chemical that worsens ALD severity. PFOS activated hepatic nuclear receptors and enriched hepatic transcriptional pathways associated with steatosis, xenobiotic metabolism, oxidative stress, cell death, fibrosis, and HCC. These data demonstrate a novel mechanism whereby PFOS exacerbates ALD through coordinated dysregulation of lipid homeostasis and liver injury, potentially mediated by nuclear receptor activation. The identification of PFOS as an ALD risk modifier highlights the critical need to evaluate environmental pollutants as potential contributors to liver disease progression. More data are required on environmental pollution as a disease-modifying factor in ALD. Impact Statement: The present study demonstrates that PFOS exacerbates alcohol-induced liver injury through nuclear receptor activation and metabolic disruption. These findings provide novel insights into how environmental pollutants can act as significant risk modifiers in ALD, how PFAS exposures may contribute to the growing public health burden of liver disease, and underscore the importance of considering combined risk factors in developing targeted interventions.

Extensive use of per- and polyfluoroalkyl substances (PFAS) has resulted in their ubiquitous presence in human blood. PFAS exposures have been associated with multiple adverse human health effects. Biomonitoring studies have focused on long-chain PFASs, but these are being replaced by short-chain PFASs or with alternate PFAS chemistries (or replacement chemistries such as GenX), resulting in changes in human exposures with time. Here, we take advantage of serum samples collected as part of a clinical trial testing the efficacy of a dietary fiber intervention to reduce serum cholesterol to investigate exposure to PFASs in Canadian participants. Serum samples were collected from 72 participants (adult males with elevated cholesterol) in 2019–2020 at baseline and after 4 weeks of the intervention and were analyzed for 17 PFASs. The highest geometric mean serum concentrations of PFAS measured at baseline corresponded to PFOSA (7.1 ng/ml), PFOS (4.2, ng/ml), PFOA (1.8 ng/ml) and PFHxS (1.3 ng/ml). Four long-chain PFASs (PFOA, PFOS, PFOSA and PFHxS) and two short-chain PFASs (PFBA, PFHxA) were detected in 100% of participants. GenX was detected in 71% of participants. Analyses of associations between serum-PFAS concentrations and biomarkers of adverse health outcomes showed the PFBA, PFHxA, PFDA and PFOSA were associated with higher serum gamma-glutamyl transferase concentrations but not with measures of serum-total or low-density lipoprotein cholesterol. Comparison of PFAS concentrations at baseline and after a 4-week follow-up showed that the total PFAS detected decreased in both the control and cholesterol intervention groups. However, the suite of long-chain PFASs of concern identified by the United States National Academies of Sciences, Engineering, and Medicine, significantly decreased only in the cholesterol intervention group. This observation suggests that a sustained dietary fiber intervention may reduce long-chain PFAS body burden, but future intervention studies need to control for PFAS exposure sources and extend the dietary supplement intake beyond 4 weeks. Overall, the results show that exposures to short-chain and replacement chemistry PFASs are common in this Canadian population.

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.

Per-and polyfluorinated substances (PFAS) are synthetic chemicals that are used to make fluoropolymer coatings found in many products, such as non-stick pans, clothing, cosmetics, and food packaging. These highly persistent molecules are known as “forever chemicals” since they neither degrade environmentally nor break down enzymatically within biological systems. PFAS compounds readily contaminate water sources, and as a result, certain PFAS molecules have bioaccumulated in exposed species including humans. The purpose of this study was to define the effect of two PFAS molecules, the ostensibly more toxic perfluorooctanoic acid (PFOA) and the more recent, reportedly safer chemical hexafluoropropylene oxide dimer acid (Gen X), on the development of Lytechinus variegatus sea urchin embryos. We examined the effects of PFOA and Gen X on development and patterning using morphological analysis, immunostaining, HCR-FISH, and Particle Image Velocimetry (PIV). The results show that both PFAS compounds are teratogenic to sea urchin embryos. PFOA and Gen X each function at different intervals during development and provoke distinct phenotypic and gene expression outcomes. Despite beliefs that Gen X would be a safer alternative, our findings indicate that Gen X has earlier and more severe effects on endomesoderm and dorsal-ventral axis specification, neural development and function, and pattern formation compared to PFOA. These results illustrate the dangerous teratogenic potential of environmentally accumulating PFAS like Gen X, underscoring the negative ecological implications that accompany continuing commercial and industrial use of PFAS in the absence of remediation strategies. HIGHLIGHTS: Gen X is more teratogenic in sea urchins than PFOA Gen X acts earlier than PFOA and perturbs axial and germ layer specification Each PFAS reduces neural numbers and perturbs ciliary behavior and swimming

Extensive use of per- and polyfluoroalkyl substances (PFAS) has resulted in their ubiquitous presence in human blood. PFAS exposures have been associated with multiple adverse human health effects including increased risk of liver damage, elevated serum lipids, impaired vaccine response, adverse birth outcomes and cancer. Biomonitoring studies have focused on measuring long-chain PFAS, but these are being replaced by shorter chain PFAS and PFAS with alternative structures, resulting in incomplete understanding of human exposures. Here, we take advantage of serum samples collected as part of a clinical trial testing the efficacy of a dietary fiber intervention to reduce serum cholesterol to investigate exposure to legacy and replacement PFAS chemicals in Canadian participants. Serum samples were collected from 72 participants in 2019-2020 at baseline and after 4 weeks of the intervention and were analyzed for 17 PFAS species. The highest geometric mean concentrations of PFAS measured at baseline corresponded to PFOSA (7.1 ng/ml), PFOS (4.2, ng/ml), PFOA (1.8 ng/ml) and PFHxS (1.3 ng/ml). Short chain PFAS including PFBuA, PFHxS and/or PFHpA were detected in 100% of participants and GenX was detected in 70% of participants. Analyses of associations between PFAS serum concentrations and biomarkers of adverse health outcomes showed the PFBuA, PFHxA, PFDA and PFOSA were associated with higher serum gamma-glutamyl transferase concentrations but not with measures of serum total or low-density lipoprotein cholesterol. Comparison of PFAS concentrations at baseline and after a 4-week follow-up showed that total PFAS concentrations decreased in both the control and cholesterol intervention groups. However, the suite of PFAS of concern identified by the United States National Academies of Sciences, Engineering, and Medicine, significantly decreased only in the cholesterol intervention group. This observation suggests that a dietary fiber intervention may reduce PFAS body burden, but future intervention studies need to control for PFAS exposure sources and extend beyond 4 weeks. Overall, the results show that exposures to short-chain and alternative PFAS are common in this Canadian population.

Conservative estimates by the World Health Organization suggest that at least a quarter of global cardiovascular diseases are attributable to environmental exposures. Associations between air pollution and cardiovascular risk have garnered the most headlines and are strong, but less attention has been paid to other omnipresent toxicants in our ecosystem. Perfluoroalkyl and polyfluoroalkyl substances (PFASs) are man-made chemicals that are extensively used in industrial and consumer products worldwide and in aqueous film-forming foam utilized in firefighting. As such, our exposure to PFAS is essentially ubiquitous. Given the long half-lives of these degradation-resistant chemicals, virtually, all people are carrying a body burden of PFAS. Health concerns related to PFAS are growing such that the National Academies of Sciences, Engineering and Medicine has recommended standards for clinical follow-up of individuals with high PFAS blood levels, including prioritizing screening for dyslipidemia. The link between PFAS and dyslipidemia has been extensively investigated, and evidence for associations is compelling. However, dyslipidemia is not the only cardiovascular risk factor with which PFAS is associated. Here, we review the epidemiological evidence for links between PFAS of concern identified by the National Academies of Sciences, Engineering and Medicine and risk factors for cardiovascular disease, including overweight/obesity, glucose intolerance, hypertension, dyslipidemia, and hyperuricemia. Moreover, we review the potential connections of PFAS with vascular disease and atherosclerosis. While observational data support associations between the National Academies of Sciences, Engineering and Medicine PFAS and selected cardiac risk factors, additional research is needed to establish causation and better understand how exposure to PFAS leads to the development of these conditions.

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.