Discovery of Fluorotelomer Sulfones in the Blubberof Greenland Killer Whales (Orcinus orca)

<p dir="ltr">Per- and polyfluoroalkyl substances (PFAS) are a group of synthetic chemicals, abundantly produced and with a wide range of applications. The partial or full fluorination of alkyl substances with varying functional groups, leads to physicochemical properties that are mainly characterised by persistence and amphiphilicity. While these properties are highly valuable for industrial- and consumer products, they pose major challenges in environmental pollution and health implications. Especially influences on the lipid metabolism have been frequently connected to PFAS exposures, yet lack deeper mechanistic understanding, and were, hence, the main investigated experimental subject of this thesis.</p><p dir="ltr">Regulatory efforts for PFAS have been made in the past and are currently underway. A harmonised approach for the assessment of human health effects, following PFAS exposure is, however, still needed. Dose-response assessment, through benchmark dose (BMD) modelling is the state of the science for hazard characterisation for risk assessment purposes. Specifically, the selection of a critical effect size (CES) - a threshold for adversity - of the assessed effect (response), is important in this context.</p><p dir="ltr">This thesis set out to tackle the outlined challenges in toxicological assessments of PFAS in three main approaches.</p><p dir="ltr">1) Testing contrasting ways to select the CESs in different BMD modelling approaches and assess the outcome metrics for select PFAS case-studies (Study I).</p><p dir="ltr">2) Utilise human cell models to gain insights into PFAS-effects on the human lipid metabolism. This was done by investigating PFAS (single and mixture) exposures and PPARa receptor activation, lipid metabolism-related gene expression and lipid accumulation in Study III. Further, PFAS (single) exposure was investigated with regards to affecting hepatocyte lipidomic profiles in Study II.</p><p dir="ltr">3) Assessing the meaningfulness of comparative relative potency factors (RPFs) for PFAS, through characterisation of RPFs, based on the endpoints in Study III.</p><p dir="ltr">The findings of Study I reveal that the CES choice alters the results of a dose- response assessment significantly for both, frequentist and Bayesian BMD modelling. Further, it became apparent that for the selected case-studies, the Bayesian BMD modelling - paired with flexible, effect-specific CES selection - led to more stable and biologically relevant results, supporting their use in regulatory decision-making.</p><p dir="ltr">Study II unveiled wide-range influences of PFAS exposures on the intracellular (hepatocyte) lipid profile. Legacy PFAS (e.g., PFOS and PFOA), as well as substitute PFAS (e.g., HFPO-DA and ADONA), were shown to have lipid profile-altering properties, with PFOS and PFOA having displayed the largest effects.</p><p dir="ltr">The cell-based assays in Study III confirmed PFAS influences in multiple mechanistic steps of the lipid metabolism. This further underscores the body of evidence for the investigated PFAS and PFAS mixtures to be involved in alterations of important lipid metabolic pathways with likely relevance to cardiovascular diseases and other metabolic conditions. With regards to their relative potencies, PFAS appeared to be endpoint-specific, with no unambiguous pattern of potency.</p><p dir="ltr">This thesis offers an assessment of the BMD methodology in chemical hazard characterisation within the context of assessing the risks from PFAS. Further, multi-endpoint assessment and exposures to single PFAS and PFAS mixtures of human relevance, highlighted the importance of the lipid metabolism as a major target system for PFAS toxicity. The use of RPFs to compare PFAS effects in an endpoint-specific manner, however, needs to be further investigated and a universal approach across endpoints appears to be very challenging.</p><h3>List of scientific papers</h3><p dir="ltr">I. BRUNKEN, L., Vieira Silva, A., & Öberg, M. (2025). Selection of the critical effect size alters hazard characterization - a retrospective analysis of key studies used for risk assessments of PFAS. Frontiers in Toxicology. 7. <a href="https://doi.org/10.3389/ftox.2025.1525089" rel="noreferrer" target="_blank">https://doi.org/10.3389/ftox.2025.1525089</a></p><p dir="ltr">II. Kashobwe, L.#, Sadrabadi, F.#, BRUNKEN, L.#, Coelho, A. C. M. F., Sandanger, T. M., Braeuning, A., Buhrke, T., Öberg, M., Hamers, T., & Leonards, P. E. G. (2024). Legacy and alternative per- and polyfluoroalkyl substances (PFAS) alter the lipid profile of HepaRG cells. Toxicology. 506, 153862. # Equally contributed. <a href="https://doi.org/10.1016/j.tox.2024.153862" rel="noreferrer" target="_blank">https://doi.org/10.1016/j.tox.2024.153862</a></p><p dir="ltr">III. BRUNKEN, L., Vieira Silva, A., & Öberg, M. Relative Potency of PFAS in Human Cell Models: Linking PPARa Activation, Gene Regulation, and Lipid Accumulation. [Manuscript]</p>

<p dir="ltr">Per- and polyfluoroalkyl substances (PFAS) are a group of synthetic chemicals, abundantly produced and with a wide range of applications. The partial or full fluorination of alkyl substances with varying functional groups, leads to physicochemical properties that are mainly characterised by persistence and amphiphilicity. While these properties are highly valuable for industrial- and consumer products, they pose major challenges in environmental pollution and health implications. Especially influences on the lipid metabolism have been frequently connected to PFAS exposures, yet lack deeper mechanistic understanding, and were, hence, the main investigated experimental subject of this thesis.</p><p dir="ltr">Regulatory efforts for PFAS have been made in the past and are currently underway. A harmonised approach for the assessment of human health effects, following PFAS exposure is, however, still needed. Dose-response assessment, through benchmark dose (BMD) modelling is the state of the science for hazard characterisation for risk assessment purposes. Specifically, the selection of a critical effect size (CES) - a threshold for adversity - of the assessed effect (response), is important in this context.</p><p dir="ltr">This thesis set out to tackle the outlined challenges in toxicological assessments of PFAS in three main approaches.</p><p dir="ltr">1) Testing contrasting ways to select the CESs in different BMD modelling approaches and assess the outcome metrics for select PFAS case-studies (Study I).</p><p dir="ltr">2) Utilise human cell models to gain insights into PFAS-effects on the human lipid metabolism. This was done by investigating PFAS (single and mixture) exposures and PPARa receptor activation, lipid metabolism-related gene expression and lipid accumulation in Study III. Further, PFAS (single) exposure was investigated with regards to affecting hepatocyte lipidomic profiles in Study II.</p><p dir="ltr">3) Assessing the meaningfulness of comparative relative potency factors (RPFs) for PFAS, through characterisation of RPFs, based on the endpoints in Study III.</p><p dir="ltr">The findings of Study I reveal that the CES choice alters the results of a dose- response assessment significantly for both, frequentist and Bayesian BMD modelling. Further, it became apparent that for the selected case-studies, the Bayesian BMD modelling - paired with flexible, effect-specific CES selection - led to more stable and biologically relevant results, supporting their use in regulatory decision-making.</p><p dir="ltr">Study II unveiled wide-range influences of PFAS exposures on the intracellular (hepatocyte) lipid profile. Legacy PFAS (e.g., PFOS and PFOA), as well as substitute PFAS (e.g., HFPO-DA and ADONA), were shown to have lipid profile-altering properties, with PFOS and PFOA having displayed the largest effects.</p><p dir="ltr">The cell-based assays in Study III confirmed PFAS influences in multiple mechanistic steps of the lipid metabolism. This further underscores the body of evidence for the investigated PFAS and PFAS mixtures to be involved in alterations of important lipid metabolic pathways with likely relevance to cardiovascular diseases and other metabolic conditions. With regards to their relative potencies, PFAS appeared to be endpoint-specific, with no unambiguous pattern of potency.</p><p dir="ltr">This thesis offers an assessment of the BMD methodology in chemical hazard characterisation within the context of assessing the risks from PFAS. Further, multi-endpoint assessment and exposures to single PFAS and PFAS mixtures of human relevance, highlighted the importance of the lipid metabolism as a major target system for PFAS toxicity. The use of RPFs to compare PFAS effects in an endpoint-specific manner, however, needs to be further investigated and a universal approach across endpoints appears to be very challenging.</p><h3>List of scientific papers</h3><p dir="ltr">I. BRUNKEN, L., Vieira Silva, A., & Öberg, M. (2025). Selection of the critical effect size alters hazard characterization - a retrospective analysis of key studies used for risk assessments of PFAS. Frontiers in Toxicology. 7. <a href="https://doi.org/10.3389/ftox.2025.1525089" rel="noreferrer" target="_blank">https://doi.org/10.3389/ftox.2025.1525089</a></p><p dir="ltr">II. Kashobwe, L.#, Sadrabadi, F.#, BRUNKEN, L.#, Coelho, A. C. M. F., Sandanger, T. M., Braeuning, A., Buhrke, T., Öberg, M., Hamers, T., & Leonards, P. E. G. (2024). Legacy and alternative per- and polyfluoroalkyl substances (PFAS) alter the lipid profile of HepaRG cells. Toxicology. 506, 153862. # Equally contributed. <a href="https://doi.org/10.1016/j.tox.2024.153862" rel="noreferrer" target="_blank">https://doi.org/10.1016/j.tox.2024.153862</a></p><p dir="ltr">III. BRUNKEN, L., Vieira Silva, A., & Öberg, M. Relative Potency of PFAS in Human Cell Models: Linking PPARa Activation, Gene Regulation, and Lipid Accumulation. [Manuscript]</p>

It is generally accepted that per- and polyfluoroalkyl substances (PFASs) occur primarily in protein-rich tissues such as blood and liver, but few studies have examined the occurrence of PFASs (in particular emerging PFASs), in lipid-rich tissues such as blubber. Here we report the distribution of 24 PFASs, total fluorine (TF) and extractable organic fluorine (EOF) in eight different tissues of a killer whale (Orcinus orca) from East Greenland. The sum of target PFAS concentrations was highest in liver (352 ng/g ww) and decreased in the order blood > kidney ≈ lung ≈ ovary > skin ≈ muscle ≈ blubber. Most of the EOF was made up of known PFASs in all tissues except blubber, which displayed the highest concentration of EOF, almost none of which was attributed to targeted PFASs. Suspect screening using high-resolution mass spectrometry revealed the presence of additional PFASs but the magnitude of peak areas could not explain the high concentrations of EOF in blubber. While the identity of this unknown organofluorine and its pervasiveness in marine mammals requires further investigation, this work suggests that exposure of killer whales to organofluorine substances may be underestimated by determination of legacy PFASs exclusively in liver tissues.

Observation of emerging per- and polyfluoroalkyl substances (PFASs) in Greenland marine mammals.

Per- and polyfluoroalkyl substances (PFAS) are a group of commonly used compounds, known particularly for their hydrophobic, nonstick properties. Their unique chemistry has also led to their use in ski waxes. While competition rules and some regions have recently banned the use of fluorinated waxes, the persistence of PFAS means they could still be detected for years. Given the hazards and concern about PFAS contamination, this study investigated if PFAS could be detected at a ski area that supports a high-level race program, where these waxes would have been in use for many years. Samples were collected from a variety of locations within a ski area in New Hampshire, USA, to investigate the levels and trends of PFAS in this type of environment. While previous studies have focused on targeted analysis for known PFAS, this study utilized both targeted and nontargeted analysis with high-resolution mass spectrometry (HRMS) and ion mobility to look for new and unexpected PFAS. In the nontargeted analysis, detected peaks were first compared to an internal HRMS PFAS library for identification, and unknown peaks were selected for further scrutiny based on their detected drift time in the ion mobility dimension. An ion mobility filter was created to look for PFAS based on the unique trendlines of collisional cross section (CCS) vs m/z exhibited by halogenated molecules and applied to the list of detected peaks. Using this filter, a number of homologous series of PFAS were tentatively identified, in addition to those found with suspect screening. Two of the series included dioic perfluorinated acids and monohydrogen-substituted perfluoroalkyl carboxylic acids (H-PFCAs). While authentic standards were not available for many of the tentative identifications, two standards were purchased and compared with experimental data to confirm the proposed structures of shorter chain compounds in these series, thus increasing the evidence that identification of the homologous series in these cases was correct. This preliminary study, based on a limited number of water samples, indicated that PFAS contamination could be detected at the ski area. The inclusion of nontargeted analysis provided a more thorough understanding of the contamination's extent by identifying new species that would be overlooked using targeted methodologies.

Killer whales (Orcinus orca) have been deemed one of the most contaminated cetacean species in the world. However, concentrations and potential health implications of selected 'contaminants of emerging concern' (CECs) and new persistent organic pollutants (POPs) in endangered Southern Resident and threatened Bigg's (Transient) killer whales in the Northeastern Pacific (NEP) have not yet been documented. Here, we quantify CECs [alkylphenols (APs), triclosan, methyl triclosan, and per- and polyfluoroalkyl substances (PFAS)] and new POPs [hexabromocyclododecane (HBCCD), PFOS, PFOA, and PFHxS] in skeletal muscle and liver samples of these sentinel species and investigate in utero transfer of these contaminants. Samples were collected from necropsied individuals from 2006 to 2018 and analyzed by LC-MS/MS or HRBC/HRMS. AP and PFAS contaminants were the most prevalent compounds; 4-nonylphenol (4NP) was the predominant AP (median 40.84 ng/g ww), and interestingly, 7:3-fluorotelomer carboxylic acid (7:3 FTCA) was the primary PFAS (median 66.35 ng/g ww). Maternal transfer ratios indicated 4NP as the most transferred contaminant from the dam to the fetus, with maternal transfer rates as high as 95.1%. Although too few killer whales have been screened for CECs and new POPs to infer the magnitude of contamination impact, these results raise concerns regarding pathological implications and potential impacts on fetal development and production of a viable neonate. This study outlines CEC and new POP concentrations in killer whales of the NEP and provides scientifically derived evidence to support and inform regulation to mitigate pollutant sources and contamination of Southern Resident killer whale critical habitat and other marine ecosystems.

When Chemicals Go to the Dark Side

Per- and polyfluoroalkyl substances (PFASs) have been widely used since the 1940s in industry and everyday household products. They also persist in the environment and bioaccumulate in humans and wildlife. Despite these concerns, the identities of most PFASs in environmental and biological samples are unknown. Herein, we describe a novel cyclic ion mobility mass spectrometer (cIMS), hyphenated with gas chromatography (GC) atmospheric pressure chemical ionization, that can reveal the presence of unknown PFASs on the basis of the ratio of their mass and collision cross section (CCS). Prediction of the CCS of ca. 20,000 chemicals used in industry and commerce indicates that most compounds characterized by CCS values that are less than the sum of 100 Å2 and one-fifth of their mass are either PFASs or polybrominated flame retardants. When this filter is applied to GC-cIMS data collected from a set of 20 indoor dust samples, PFAS compounds are revealed without prior knowledge of their occurrence. Validation of this approach was performed using SRM 2585, a standard reference material of household dust, by comparing the PFASs detected with those (tentatively) identified in previous studies. Chlorofluoro phthalimides tentatively identified previously were confirmed with a synthesized standard. The method also reveals the presence of chlorofluoro n-alkanes as an emerging class of "forever chemicals" that contaminate the indoor environment.

Per- and polyfluoroalkyl substances (PFAS) are an ensemble of persistent organic pollutants of global interest because of their associations with adverse health outcomes. Currently, environmental PFAS pollution is prolific as a result of the widespread manufacturing of these compounds and their chemical persistence. In this work, we demonstrate the advantages of adding ion mobility spectrometry (IMS) separation to existing LC-MS workflows for PFAS analysis. Using a commercially available drift tube IMS-MS, we characterized PFAS species and isomeric content in both analytical standards and environmental water samples. Molecular trendlines based on intrinsic mass and structural relationships were also explored for individual PFAS subclasses (e.g. PFSA, PFCA, etc.). Results from rapid IMS-MS analyses provided a link between mass and collision cross sections (CCS) for specific PFAS families and are linked to compositional differences in molecular structure. In addition, CCS values provide additional confidence of annotating prioritized features in untargeted screening studies for potential environmental pollutants. Results from this study show that the IMS separation provides novel information to support traditional LC-MS PFAS analyses and will greatly benefit the evaluation of unknown pollutants in future environmental studies.

Per- and poly-fluoroalkyl substances (PFAS) have gained much attention recently due to their high persistence and potential health risks. PFAS are often used in the fabrication of various consumer products, such as firefighting foam, non-stick utensils, water-repellent clothes, stain-resistant materials, and food packaging products. PFAS are highly stable and persistent due to their strong carbon-fluorine bonds and have been found to be absorbed in humans and animals for significant amount of time. Studies have shown that drinking water may act as a reservoir of trace level PFAS and contribute to the accumulation of PFAS in human bodies, which may lead to adverse health issues in liver, kidney, and reproductive systems. However, limited studies have been done to remove trace level PFAS in point-of-use (POU) water treatment systems, including reverse osmosis (RO) and activated carbon (AC) systems. The objective of this study was to evaluate PFAS removal in POU systems and factors that may impact PFAS removal efficiencies. Mixtures of perfluorooctane sulfonic acid (PFOS), perfluorohexane sulfonic acid (PFHxS), and perfluorobutane sulfonic acid (PFBS) under two different concentrations (1 μg/l and 10 μg/l) were spiked into tap water and PFAS removal efficiencies in three RO and three AC systems were evaluated. Solid phase extraction coupled with liquid chromatography tandem mass spectrometry (SPE-LC/MS/MS) was used to quantify trace level PFAS concentrations. The results showed that PFAS were effectively removed in both RO and AC systems with average removal efficiencies greater than 90% in all tested POU systems. Among the three evaluated PFAS with different chain-lengths (PFOS: 8 carbons, PFHxS: 6 carbons, PFBS: 4 carbons), higher removal efficiencies were observed in long-chain perfluoroalkyl sulfonic acids (carbon chain-length ≥ 6), while relatively low removal efficiency and high variability was observed in PFBS removal. These results suggest that POU systems are generally effective to remove PFAS, but short-chain PFBS may not be consistently removed, even though they have been frequently used as alternative PFAS to replace conventional long-chain PFAS. The results from this study may improve the understanding of PFAS removal in POU systems and provide useful information for the design, operation, and maintenance of POU systems to minimize health risks of PFAS in drinking water.

As the occurrence of human diseases and conditions increase, questions continue to arise about their linkages to chemical exposure, especially for per-and polyfluoroalkyl substances (PFAS). Currently, many chemicals of concern have limited experimental information available for their use in analytical assessments. Here, we aim to increase this knowledge by providing the scientific community with multidimensional characteristics for 175 PFAS and their resulting 281 ion types. Using a platform coupling reversed-phase liquid chromatography (RPLC), electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI), drift tube ion mobility spectrometry (IMS), and mass spectrometry (MS), the retention times, collision cross section (CCS) values, and m/z ratios were determined for all analytes and assembled into an openly available multidimensional dataset. This information will provide the scientific community with essential characteristics to expand analytical assessments of PFAS and augment machine learning training sets for discovering new PFAS.

Toxicants with the potential to bioaccumulate in humans and animals have long been a cause for concern, particularly due to their association with multiple diseases and organ injuries. Per- and polyfluoro alkyl substances (PFAS) and polycyclic aromatic hydrocarbons (PAH) are two such classes of chemicals that bioaccumulate and have been associated with steatosis in the liver. Although PFAS and PAH are classified as chemicals of concern, their molecular mechanisms of toxicity remain to be explored in detail. In this study, we aimed to identify potential mechanisms by which an acute exposure to PFAS and PAH chemicals can induce lipid accumulation and whether the responses depend on chemical class, dose, and sex. To this end, we analyzed mechanisms beginning with the binding of the chemical to a molecular initiating event (MIE) and the consequent transcriptomic alterations. We collated potential MIEs using predictions from our previously developed ToxProfiler tool and from published steatosis adverse outcome pathways. Most of the MIEs are transcription factors, and we collected their target genes by mining the TRRUST database. To analyze the effects of PFAS and PAH on the steatosis mechanisms, we performed a computational MIE-target gene analysis on high-throughput transcriptomic measurements of liver tissue from male and female rats exposed to either a PFAS or PAH. The results showed peroxisome proliferator-activated receptor (PPAR)-α targets to be the most dysregulated, with most of the genes being upregulated. Furthermore, PFAS exposure disrupted several lipid metabolism genes, including upregulation of fatty acid oxidation genes (Acadm, Acox1, Cpt2, Cyp4a1-3) and downregulation of lipid transport genes (Apoa1, Apoa5, Pltp). We also identified multiple genes with sex-specific behavior. Notably, the rate-limiting genes of gluconeogenesis (Pck1) and bile acid synthesis (Cyp7a1) were specifically downregulated in male rats compared to female rats, while the rate-limiting gene of lipid synthesis (Scd) showed a PFAS-specific upregulation. The results suggest that the PPAR signaling pathway plays a major role in PFAS-induced lipid accumulation in rats. Together, these results show that PFAS exposure induces a sex-specific multi-factorial mechanism involving rate-limiting genes of gluconeogenesis and bile acid synthesis that could lead to activation of an adverse outcome pathway for steatosis.

Ion mobility spectrometry(IMS) is a rapid gas-phase separationtechnique, which can distinguish ions on the basis of their size,shape, and charge. The IMS-derived collision cross section (CCS) canserve as additional identification evidence for the screening of environmentalorganic micropollutants (OMPs). In this work, we summarize the publishedexperimental CCS values of environmental OMPs, introduce the currentCCS prediction tools, summarize the use of IMS and CCS in the analysisof environmental OMPs, and finally discussed the benefits of IMS andCCS in environmental analysis. An up-to-date CCS compendium for environmentalcontaminants was produced by combining CCS databases and data setsof particular types of environmental OMPs, including pesticides, drugs,mycotoxins, steroids, plastic additives, per- and polyfluoroalkylsubstances (PFAS), polycyclic aromatic hydrocarbons (PAHs), polychlorinatedbiphenyls (PCBs), and polybrominated diphenyl ethers (PBDEs), as wellas their well-known transformation products. A total of 9407 experimentalCCS values from 4170 OMPs were retrieved from 23 publications, whichcontain both drift tube CCS in nitrogen (DTCCSN2) and traveling wave CCS in nitrogen (TWCCSN2). A selection of publicly accessible and in-houseCCS prediction tools were also investigated; the chemical space coveredby the training set and the quality of CCS measurements seem to bevital factors affecting the CCS prediction accuracy. Then, the applicationsof IMS and the derived CCS in the screening of various OMPs were summarized,and the benefits of IMS and CCS, including increased peak capacity,the elimination of interfering ions, the separation of isomers, andthe reduction of false positives and false negatives, were discussedin detail. With the improvement of the resolving power of IMS andenhancements of experimental CCS databases, the practicability ofIMS in the analysis of environmental OMPs will continue to improve.

Per- and polyfluoroalkyl substances (PFAS) have garnered increasing attention in recent years and non-targeted analysis (NTA) has become essential for elucidating novel PFAS structures. NTA and PFAS research have been dominated by liquid chromatography - mass spectrometry (LC-MS) with gas chromatography - mass spectrometry (GC-MS) used less often as evidenced by bibliometrics. However, the performance of GC-MS in NTA studies (GC-NTA) rivals that of LC-ESI-MS and GC-MS is shown to cover a complimentary chemical space. An LC-ESI-MS amenability model applied to a list of approximately 12,000 PFAS revealed that less than 10% of known PFAS chemistry is predicted to be amenable to typical LC-MS analysis. Therefore, there is strong potential for applying GC-MS methods to more fully assess the PFAS environmental contamination landscape, uniquely shedding light on both known and novel PFAS, especially within the chemical space realm of volatile and semi-volatile PFAS. Waste streams from fluorochemical manufacturing facilities have been heavily studied using LC-MS and targeted GC-MS; however, GC-NTA is needed to discover novel PFAS that are not amenable to LC-MS emitted from facilities. Studies on the incineration of PFAS-containing materials, such as aqueous film forming foam, have focused on the destruction of parent compounds and little is known about the transformation products formed during such processes. GC-NTA holds the potential to elucidate transformation products formed when PFAS are incinerated. Wastewater treatment plants and landfills are known sources of PFAS to the environment, yet GC-NTA is needed to understand air emissions of PFAS and PFAS transformation products from these sources. Consumer products are known to lead to indoor exposures to PFAS via emissions to air and dust but research in this area has either used LC-MS or targeted GC-MS. Despite the challenges with advancing GC-NTA, we call on NTA researchers, grantors, managers, and other stakeholders to recognize the potential and necessity of GC-NTA in PFAS research so that we may face these challenges together.

Per- and poly-fluoroalkyl substances (PFAS) are a class of synthetic chemicals that have been released into the environment over the past six decades. Some perfluoroalkyl acids (PFAA), such as perfluorooctanesulfonic acid (PFOS) and perfluorooctanoic acid (PFOA), have been associated with hepatic steatosis in rodents and monkeys, however it is unclear if lipid accumulation occurs in humans. This thesis explores the liver effects of PFAS on non-alcoholic fatty liver disease, or NAFLD, using mouse and primary human hepatocytes. The central hypotheses are that diet and liver lipid composition alter PFAS-induced outcomes, and PFAS structure (i.e. functional groups and chain length) will also dictate outcomes in hepatocytes regarding lipid metabolism, gene activation, and lipid accumulation. In manuscript 1, PFOS was evaluated for augmentation of HFD-induced hepatic steatosis and the potential to interfere with lipid loss after switching from a high fat diet (HFD) to a standard diet (SD). 5-week-old male C57BL/6J mice were fed HFD for 4 weeks to increase body weight and induce liver lipid accumulation. Then, some HFD mice were switched to SD to mimic weight loss induced improvement of NAFLD. At that time, mice were also given diet only or diet containing 0.0003% PFOS. After 10 weeks on study, hepatic lipids, serum and liver PFOS levels, and gene and protein expression were evaluated. In manuscript 2, critical members of the PFAS family were screened for gene expression changes and lipid accumulation in a five-donor pool of cryopreserved human hepatocytes. Hepatocytes were cultured and treated with vehicle or various PFAS at concentrations of 0.25 to 25 μM in media. After 48-hr or 72-hr treatments, mRNA transcripts related to lipid transport, metabolism, and synthesis were measured or cells were stained with Nile Red dye to quantify intracellular lipids. In manuscript 3, to complement manuscript 1, the impact of maternal diet and PFAS exposure on the risk to mouse pup liver and diet related PFAS partitioning to pups was evaluated. Timed-pregnant dams received at gestation day 1 (GD1) were fed a standard chow diet (SD) or 60% Kcal high fat diet (HFD) and dosed with either vehicle (0.5% Tween 20 in water), 1 mg/kg PFOS, 1 mg/kg PFOA, 1mg/kg PFHxS, or PFAS mix (1 mg/kg of each PFOS, PFOA, and PFHxS) via oral gavage (10 mL/kg). At PND 21, dam and pup liver were collected, weighed, and liver lipids were isolated in dams and pups. Pup serum was also evaluated for PFAS