Replacement per- and polyfluoroalkyl substances (PFAS) are potent modulators of lipogenic and drug metabolizing gene expression signatures in primary human hepatocytes.

Per- and polyfluoroalkyl substances (PFAS) are a class of environmental toxicants, and some, such as perfluorooctanesulfonic acid (PFOS) and perfluorooctanoic acid (PFOA), have been associated with hepatic steatosis in rodents and monkeys. It was hypothesized that perfluorosulfonic acids (C4, 6, 8), perfluorocarboxylic acids (C4-14), perfluoro(2-methyl-3-oxahexanoic) acid (HFPO-DA), 1H, 1H, 2H, 2H-perfluorooctanesulfonic acid (6:2 FTS) along with 3 PFOS precursors could induce expression of lipid metabolism genes and lipid deposition in human hepatocytes. Five-donor pooled cryopreserved human hepatocytes were cultured and treated with 0.1% DMSO vehicle or various PFAS (0.25 to 25 µM) in media. After a 48-h treatment, mRNA transcripts related to lipid transport, metabolism, and synthesis were measured using a Quantigene Plex assay. After 72-h treatments, hepatocytes were stained with Nile Red dye to quantify intracellular lipids. Overall, PFAS were transcriptionally active at 25 µM. In this model, lipid accumulation was not observed with C8-C12 treatments. Shorter chain PFAS (C4-C5), 6:2 FTS, and PFOS precursor, metFOSA, induced significant liver lipid accumulation, and gene activation at lower concentrations than legacy PFAS. In summary short chain PFAS and other alternative PFAS were more potent gene inducers, and potential health effects of replacement PFAS should be critically evaluated in humans.

Evaluating risk, exposure, and detection capabilities for chemical threats in water.

The unexpected release of chemicals into the environment requires estimation of human health risks, followed by risk management decisions. When environmental concentrations of toxicants are associated with adverse health risks, the limit for analytical measurement needs to be at or below the risk threshold. The aim of this study was to assess chemical contaminants that have the potential to produce acute adverse human health impacts following oral consumption of contaminated drinking water. The U.S. Environmental Protection Agency's (EPA) Candidate Contaminant List, version 4 (CCL4) and EPA's Selected Analytical Methods (SAM) document were screened to identify 24 chemicals that exist as a solid or liquid at room temperature, with acute oral LD50 (lethal dose in 50% of the test population) values < 500 mg/kg-d and water solubility > 500 mg/L at ambient temperature. While these screening criteria were used to identify prioritized needs for targeted research, it does not imply that other chemicals on the CCL4 and SAM lists are not issues in acute and chronic exposures. Of these 24 most toxic and most soluble chemicals, this evaluation identified 6 chemicals (2-chlorovinylarsonous acid, lewisite, N-nitrosopyrrolidine, N-nitrosodiethylamine, 3-hydroxycarbofuran, and triethylamine) lacking either sufficient toxicity value information or analytical sensitivity required to detect at levels protective against adverse effects in adults for acute exposures. This assessment provides an approach for gap identification and highlights research needs related to water contamination incident involving these six priority chemicals.

Hepatoprotective and anti-inflammatory effects of a standardized pomegranate (Punica granatum) fruit extract in high fat diet-induced obese C57BL/6 mice.

Diets rich in fats are linked to elevated systemic inflammation, which augments the progression of inflammatory-related disorders including non-alcoholic fatty liver disease (NAFLD) and neurodegenerative diseases. A phenolic-enriched pomegranate fruit extract (PE) was investigated for its hepatoprotective and anti-inflammatory effects in male C57BL/6 mice fed either a high-fat diet or a standard rodent diet with or without 1% of PE for 12 weeks. Mouse livers and hippocampi were evaluated for the expression of genes associated with NAFLD and inflammation by multiplexed gene analysis. PE alleviated diet-induced fatty liver and suppressed hepatic lipid regulating genes including Cd36, Fas, Acot2 and Slc27a1. In addition, PE suppressed gene expression of pro-inflammatory cytokines including Il-1α, Il-7, Il-11, Ifnα, Tnfα and Lepr in the hippocampi. Our findings support the protective effects of PE against high-fat diet-induced hepatic and neurological disease.

Perfluorooctanesulfonic acid (PFOS) administration shifts the hepatic proteome and augments dietary outcomes related to hepatic steatosis in mice.

Hepatic steatosis increases risk of fatty liver and cardiovascular disease. Perfluorooctanesulfonic acid (PFOS) is a persistent, bio-accumulative pollutant that has been used in industrial and commercial applications. PFOS administration induces hepatic steatosis in rodents and increases lipogenic gene expression signatures in cultured hepatocytes. We hypothesized that PFOS treatment interferes with lipid loss when switching from a high fat diet (HFD) to a standard diet (SD), and augments HFD-induced hepatic steatosis. Male C57BL/6 N mice were fed standard chow diet or 60% kCal high-fat diet (HFD) for 4 weeks to increase body weight. Then, some HFD mice were switched to SD and mice were further divided to diet only or diet containing 0.0003% PFOS, for six treatment groups: SD, HFD to SD (H-SD), HFD, SD + PFOS, H-SD + PFOS, or HFD + PFOS. After 10 weeks on study, blood and livers were collected. HFD for 14 weeks increased body weight and hepatic steatosis, whereas H-SD mice returned to SD measures. PFOS administration reduced body weight in mice fed a SD, but not H-SD or HFD. PFOS administration increased liver weight in H-SD + PFOS and HFD + PFOS mice. PFOS increased hepatic steatosis in H-SD and HFD groups. Hepatic mRNA expression and SWATH-MS proteomic analysis revealed that PFOS induced lipid and xenobiotic transporters, as well as metabolism pathways. Overall, the findings herein suggest that PFOS treatment did interfere with lipid loss associated with switch to a SD and similarly augmented hepatic lipid accumulation in mice established on an HFD.

Legacy and Novel Per- and Polyfluoroalkyl Substances in Juvenile Seabirds from the U.S. Atlantic Coast.

Per- and polyfluoroalkyl substances (PFAS) are anthropogenic, globally distributed chemicals. Legacy PFAS, including perfluorooctane sulfonate (PFOS), have been regularly detected in marine fauna but little is known about their current levels or the presence of novel PFAS in seabirds. We measured 36 emerging and legacy PFAS in livers from 31 juvenile seabirds from Massachusetts Bay, Narragansett Bay, and the Cape Fear River Estuary (CFRE), United States. PFOS was the major legacy perfluoroalkyl acid present, making up 58% of concentrations observed across all habitats (range: 11-280 ng/g). Novel PFAS were confirmed in chicks hatched downstream of a fluoropolymer production site in the CFRE: a perfluorinated ether sulfonic acid (Nafion byproduct 2; range: 1-110 ng/g) and two perfluorinated ether carboxylic acids (PFO4DA and PFO5DoDA; PFO5DoDA range: 5-30 ng/g). PFOS was inversely associated with phospholipid content in livers from CFRE and Massachusetts Bay individuals, while δ 13C, an indicator of marine versus terrestrial foraging, was positively correlated with some long-chain PFAS in CFRE chick livers. There is also an indication that seabird phospholipid dynamics are negatively impacted by PFAS, which should be further explored given the importance of lipids for seabirds.

An 'Omics Approach to Unraveling the Paradoxical Effect of Diet on Perfluorooctanesulfonic Acid (PFOS) and Perfluorononanoic Acid (PFNA)-Induced Hepatic Steatosis.

Perfluoroalkyl substances (PFAS) are a family of toxicants universally detected in human serum and known to cause dyslipidemia in animals and humans. Hepatic steatosis, which is defined as lipid deposition in the liver, is known to be a consequence of poor diet. Similarly, PFAS are known to induce hepatic steatosis in animals on a low-fat chow. This study explored diet-PFAS interactions in the liver and their potential to modulate hepatic steatosis. Male C57BL/6J mice were fed with either a low-fat diet (10% kcal from fat, LFD) or a moderately high-fat diet (45% kcal from fat, HFD) with or without perfluorooctanesulfonic acid (3 ppm, PFOS) or perfluorononanoic acid (3 ppm, PFNA) in feed for 12 weeks. Livers were excised for histology and quantification of PFAS and lipids. The PFOS and PFNA coadministration with HFD reduced the hepatic accumulation of lipid and PFAS relative to the LFD treatment groups. Furthermore, transcriptomic analysis revealed that PFAS administration in the presence of an HFD significantly reduces expression of known hepatic PFAS uptake transporters, organic anion transporter proteins. Transcriptomics and proteomics further revealed several pathways related to lipid metabolism, synthesis, transport, and storage that were modulated by PFAS exposure and further impacted by the presence of dietary fat. Both dietary fat content and the chemical functional head group exerted significant influence on hepatic PFAS accumulation and the resulting biochemical signature, suggesting that diet and structure should be considered in the design and interpretation of research on PFAS induced hepatic steatosis.

Increased toxicity and retention of perflourooctane sulfonate (PFOS) in humanized CYP2B6-Transgenic mice compared to Cyp2b-null mice is relieved by a high-fat diet (HFD).

PFOS is a persistent, fluorosurfactant used in multiple products. Murine Cyp2b's are induced by PFOS and high-fat diets (HFD) and therefore we hypothesized that human CYP2B6 may alleviate PFOS-induced steatosis. Cyp2b-null and hCYP2B6-Tg mice were treated with 0, 1, or 10 mg/kg/day PFOS by oral gavage for 21-days while provided a chow diet (ND) or HFD. Similar to murine Cyp2b10, CYP2B6 is inducible by PFOS. Furthermore, three ND-fed hCYP2B6-Tg females treated with 10 mg/kg/day PFOS died during the exposure period; neither Cyp2b-null nor HFD-fed mice died. hCYP2B6-Tg mice retained more PFOS in serum and liver than Cyp2b-null mice presumably causing the observed toxicity. In contrast, serum PFOS retention was reduced in the HFD-fed hCYP2B6-Tg mice; the opposite trend observed in HFD-fed Cyp2b-null mice. Hepatotoxicity biomarkers, ALT and ALP, were higher in PFOS-treated mice and repressed by a HFD. However, PFOS combined with a HFD exacerbated steatosis in all mice, especially in the hCYP2B6-Tg mice with significant disruption of key lipid metabolism genes such as Srebp1, Pparg, and Hmgcr. In conclusion, CYP2B6 is induced by PFOS but does not alleviate PFOS toxicity presumably due to increased retention. CYP2B6 protects from PFOS-mediated steatosis in ND-fed mice, but increases steatosis when co-treated with a HFD.

The role of maternal high fat diet on mouse pup metabolic endpoints following perinatal PFAS and PFAS mixture exposure.

Per- and polyfluoroalkyl substances (PFAS) are a family of chemicals that are ubiquitous in the environment. Some of these chemicals, such as perfluorooctanesulfonic acid (PFOS), perfluorohexanesulfonate (PFHxS) and perfluorooctanoic acid (PFOA), are found in human sera and have been shown to cause liver steatosis and reduce postnatal survival and growth in rodents. The purpose of this work is to evaluate the impact of diet and PFAS exposure to mouse dam (mus musculus) on the risk to pup liver and metabolism endpoints later in life, as well as evaluate PFAS partitioning to pups. Timed-pregnant dams were fed a standard chow diet or 60 % kcal high fat diet (HFD). Dams were administered either vehicle, 1 mg/kg PFOA, 1 mg/kg PFOS, 1 mg/kg PFHxS, or a PFAS mixture (1 mg/kg of each PFOA, PFOS, and PFHxS) daily via oral gavage from gestation day 1 until postnatal day (PND) 20. At PND 21, livers of dams and 2 pups of each sex were evaluated for lipid changes while remaining pups were weaned to the same diet as the dam for an additional 10 weeks. Dam and pup serum at PND 21 and PND 90 were also evaluated for PFAS concentration, alanine aminotransferase (ALT), leptin and adiponectin, and glycosylated hemoglobin A1c. Perinatal exposure to a HFD, as expected, increased pup body weight, maternal liver weight, pup liver triglycerides, pup serum ALT, and pup serum leptin. PFOA and the PFAS mixture increased liver weights, and. treatment with all three compounds increased liver triglycerides. The maternal HFD increased dam and pup serum PFAS levels, however, was protective against PFOA-induced increase in serum ALT and observed increases in liver triglycerides. The PFAS mixture had very distinct effects when compared to single compound treatment, suggesting some cumulative effects, particularly when evaluating PFAS transfer from dam to pup. This data highlights the importance of diet and mixtures when evaluating liver effect of PFAS and PFAS partitioning.

Perfluorooctanesulfonic Acid and Perfluorohexanesulfonic Acid Alter the Blood Lipidome and the Hepatic Proteome in a Murine Model of Diet-Induced Obesity.

Perfluoroalkyl substances (PFAS) represent a family of environmental toxicants that have infiltrated the living world. This study explores diet-PFAS interactions and the impact of perfluorooctanesulfonic acid (PFOS) and perfluorohexanesulfonic (PFHxS) on the hepatic proteome and blood lipidomic profiles. Male C57BL/6J mice were fed with either a low-fat diet (10.5% kcal from fat) or a high fat (58% kcal from fat) high carbohydrate (42 g/l) diet with or without PFOS or PFHxS in feed (0.0003% wt/wt) for 29 weeks. Lipidomic, proteomic, and gene expression profiles were determined to explore lipid outcomes and hepatic mechanistic pathways. With administration of a high-fat high-carbohydrate diet, PFOS and PFHxS increased hepatic expression of targets involved in lipid metabolism and oxidative stress. In the blood, PFOS and PFHxS altered serum phosphatidylcholines, phosphatidylethanolamines, plasmogens, sphingomyelins, and triglycerides. Furthermore, oxidized lipid species were enriched in the blood lipidome of PFOS and PFHxS treated mice. These data support the hypothesis that PFOS and PFHxS increase the risk of metabolic and inflammatory disease induced by diet, possibly by inducing dysregulated lipid metabolism and oxidative stress.

Lack of Multidrug Resistance-associated Protein 4 Prolongs Partial Hepatectomy-induced Hepatic Steatosis.

Multidrug resistance-associated protein 4 (Mrp4) is an efflux transporter involved in the active transport of several endogenous and exogenous chemicals. Previously, we have shown that hepatic Mrp4 expression increases following acetaminophen overdose. In mice, these increases in Mrp4 expression are observed specifically in hepatocytes undergoing active proliferation. From this, we hypothesized that Mrp4 plays a key role in hepatocyte proliferation and that lack of Mrp4 impedes liver regeneration following liver injury and/or tissue loss. To evaluate the role of Mrp4 in these processes, we employed two-third partial hepatectomy (PH) as an experimental liver regeneration model. In this study, we performed PH-surgery on male wildtype (C57BL/6J) and Mrp4 knockout mice. Plasma and liver tissues were collected at 24, 48, and 72 h postsurgery and evaluated for liver injury and liver regeneration endpoints, and for PH-induced hepatic lipid accumulation. Our results show that lack of Mrp4 did not alter hepatocyte proliferation and liver injury following PH as evaluated by Ki-67 antigen staining and plasma alanine aminotransferase levels. To our surprise, Mrp4 knockout mice exhibited increased hepatic lipid content, in particular, di- and triglyceride levels. Gene expression analysis showed that lack of Mrp4 upregulated hepatic lipin1 and diacylglycerol O-acyltransferase 1 and 2 gene expression, which are involved in the synthesis of di- and triglycerides. Our observations indicate that lack of Mrp4 prolonged PH-induced hepatic steatosis in mice and suggest that Mrp4 may be a novel genetic factor in the development of hepatic steatosis.

Phospholipid Levels Predict the Tissue Distribution of Poly- and Perfluoroalkyl Substances in a Marine Mammal.

Exposure to poly- and perfluoroalkyl substances (PFASs) has been linked to many negative health impacts in humans and wildlife. Unlike neutral hydrophobic organic pollutants, many PFASs are ionic and have been hypothesized to accumulate in both phospholipids and protein-rich tissues. Here we investigate the role of phospholipids for PFAS accumulation by analyzing associations among concurrent measurements of phospholipid, total protein, total lipid and 24 PFASs in the heart, muscle, brain, kidney, liver, blubber, placenta and spleen of North Atlantic pilot whales (Globicephala melas). The sum of 24 PFASs ( ∑ 24 PFAS ) was highest in the liver (median 260 ng g-1; interquartile range (IQR) 216-295 ng g-1) and brain (86.0; IQR 54.5-91.3 ng g-1), while phospholipid levels were highest in brain. The relative abundance of PFASs in the brain greatly increases with carbon chain lengths of 10 or greater, suggesting shorter-chained compounds may cross the blood-brain barrier less efficiently. Phospholipids were significant predictors of the tissue distribution of the longest-chained PFASs: perfluorodecanesulfonate (PFDS), perfluorododecanoate (PFDoA), perfluorotridecanoate (PFTrA), and perfluorotetradecanoic acid (PFTA) (rs = 0.5-0.6). In all tissues except the brain, each 1 mg g-1 increase in phospholipids led to a 12%-25% increase in the concentration of each PFAS. We conclude that partitioning to phospholipids is an important mechanism of bioaccumulation for long-chained PFASs in marine mammals.