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AOP: 529
Title
Perfluorooctanesulfonic acid (PFOS) binding to peroxisome proliferator-activated receptors (PPARs) causes dysregulation of lipid metabolism and subsequent liver steatosis
Short name
Graphical Representation
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Point of Contact
Contributors
- Erik Mylroie
- Kurt A. Gust
- Evgeniia Kazymova
Coaches
OECD Information Table
| OECD Project # | OECD Status | Reviewer's Reports | Journal-format Article | OECD iLibrary Published Version |
|---|---|---|---|---|
This AOP was last modified on May 26, 2024 20:40
Revision dates for related pages
| Page | Revision Date/Time |
|---|---|
| Accumulation, Fatty acid | September 16, 2017 10:14 |
| Increased, Liver Steatosis | May 21, 2024 10:00 |
| Decreased, Mitochondrial fatty acid beta-oxidation | September 16, 2017 10:14 |
| Accumulation, Triglyceride | March 26, 2024 13:09 |
| Dysregulation of transcriptional expression within PPAR signaling network | May 23, 2024 08:55 |
| Disrupted Lipid Storage | May 23, 2024 13:39 |
| Stressor binding PPAR isoforms | May 22, 2024 12:52 |
| Disrupted PPAR isoform nuclear signaling | May 22, 2024 15:59 |
| Binding PPAR isoforms leads to Disrupted PPAR isoform nuclear signaling | May 23, 2024 15:55 |
| Disrupted Lipid Storage leads to Accumulation, Triglyceride | May 08, 2024 15:11 |
| Disrupted PPAR isoform nuclear signaling leads to Dysregulation of transcriptional expression within PPAR signaling network | May 23, 2024 16:17 |
| Dysregulation of transcriptional expression within PPAR signaling network leads to Disrupted Lipid Storage | May 23, 2024 17:01 |
| Dysregulation of transcriptional expression within PPAR signaling network leads to Decreased, Mitochondrial fatty acid beta-oxidation | May 24, 2024 15:12 |
| Decreased, Mitochondrial fatty acid beta-oxidation leads to Disrupted Lipid Storage | May 24, 2024 16:39 |
| Disrupted Lipid Storage leads to Accumulation, Fatty acid | May 08, 2024 15:10 |
| Accumulation, Fatty acid leads to Accumulation, Triglyceride | December 03, 2016 16:37 |
| Accumulation, Triglyceride leads to Increased, Liver Steatosis | March 27, 2024 10:09 |
| Perfluorooctanesulfonic acid | May 08, 2024 10:21 |
| PPARalpha antagonists | June 02, 2017 14:46 |
| PPAR agonist | May 08, 2024 10:22 |
| Per- and Polyfluorinated Substances (PFAS) | May 08, 2024 10:23 |
Abstract
This AOP describes the chain of events where the molecular initiating event (MIE) of perfluorooctanesulfonic acid (PFOS) binding to the ligand-binding domain of the peroxisome proliferator-activated receptor (PPAR) causes a cascade of key events (KEs) including altered transcriptional expression of genes involved in lipid metabolism leading to impacted lipid transport, metabolism, and storage, ultimately leading to lipid accumulation in the liver and the adverse outcome (AO) of liver steatosis. Specifically, ligand binding analyses and molecular modeling studies have indicated the potential for PFOS to bind to the lipid-binding domain of various PPAR isoforms (the MIE) resulting in disruption of PPAR nuclear signaling (KE1). Disruption of PPAR nuclear signaling leads to KE2 in which the activity of PPAR as a transcriptional regulator is altered affecting transcriptional expression of a suite of genes within the PPAR signaling network. Transcriptional studies have shown that exposure to PFOS results in broad dysregulation of gene expression for a suite of genes involved in lipid metabolism which ultimately result in decreased β-oxidation (KE3) and disrupted lipid storage (KE4). Altered expression of β-oxidation related genes (acox1, acadm, cpt1a, cyp4a1) have been observed in conjunction with inhibition of β-oxidation in PFOS exposures. Also, transcriptional expression of genes involved in both lipogenesis and lipid transport including, apoa, apoe, acacb, CD36, fabp isoforms, Plin isoforms and lpl, have been observed to be affected by PFOS exposure in conjunction with disrupted of lipid storage (KE4). Alterations in fatty acids, triglycerides (TG), and total cholesterol (TC) accumulation and profiles have been observed in the livers of PFOS-exposed vertebrates including fish, reptiles, birds, and mammals and serve as evidence of KE5 (accumulation of fatty acids) and KE6 (accumulation of TG/TC) in liver tissue. KE5 and KE6 thus contribute to hepatocellular vacuolation as seen in multiple histopathological assessments performed on livers of vertebrate species exposed to PFOS, including work funded under SERDP project ER20-1542 (Mylroie et al, manuscript in development). Finally, KE5 and KE6 ultimately drive the adverse outcome (AO) of liver steatosis. Additional, more systemic AOs may also be affected by this MIE and the cascade of KEs that can ultimately alter global energy metabolism, such as AOs of impacted growth and reproduction.
AOP Development Strategy
Context
Poly- and perfluoroalkyl substances (PFAS) are a large group of fluorinated compounds that have a wide variety of commercial and industrial applications ranging from use in firefighting foams to non-stick coatings to fishing lines (DeWitt, et al. 2019; Annunziato et al. 2020; Glüge et al. 2020). PFAS exposure can have negative effects on development, growth, reproduction, hepatic function, immune function, neurological function, and lipid metabolism in humans and other vertebrates (Sunderland et al. 2019; Lee et al. 2020; Agency for Toxic Substances and Disease Registry (ATSDR), 2021; Ankley et al. 2021; Bell et al. 2021; Fragki et al. 2021; Ho et al. 2021; Boyd et al. 2022). Research in terrestrial and aquatic vertebrates has shown the liver to be a target organ of PFAS accumulation and resulting hepatoxicity (Lee et al. 2020; Costello et al. 2022; Ducatman and Fenton 2022; Huang et al. 2022a; Wang et al. 2022b). Here we propose an adverse outcome pathway (AOP) linking the binding of a specific PFAS, perfluorooctanesulfonic acid (PFOS), to peroxisome proliferator-activated receptors (PPARs) as the molecular initiating event (MIE) causing perturbation of PPAR-linked lipid metabolism which ultimately results in the adverse outcome (AO) of liver steatosis in PFOS-exposed vertebrates.
PPARs are a family of nuclear receptors in vertebrates that bind lipids as signaling molecules resulting in a cascade of transcriptional regulatory events that maintain energy homeostasis (Grygiel-Gorniak 2014). Specifically, PPARα is integral in regulating fatty acid catabolism and energy production through beta-oxidation; PPARγ regulates fatty acid synthesis and storage; and PPARβ/δ plays a key role in glucose homeostasis and beta-oxidation (Varga et al. 2011; Grygiel-Gorniak 2014; Lamas-Bervejillo and Ferreira 2019; Gust et al. 2019). Despite their more discrete roles, the crosstalk between all PPAR isoforms is essential to maintaining energy homeostasis; and therefore, any over-activation or repression of the PPAR signaling network can have deleterious outcomes for the organism.
Strategy
Summary of the AOP
Events:
Molecular Initiating Events (MIE)
Key Events (KE)
Adverse Outcomes (AO)
| Type | Event ID | Title | Short name |
|---|
| MIE | 2226 | Stressor binding PPAR isoforms | Binding PPAR isoforms |
| KE | 2227 | Disrupted PPAR isoform nuclear signaling | Disrupted PPAR isoform nuclear signaling |
| KE | 2224 | Dysregulation of transcriptional expression within PPAR signaling network | Dysregulation of transcriptional expression within PPAR signaling network |
| KE | 179 | Decreased, Mitochondrial fatty acid beta-oxidation | Decreased, Mitochondrial fatty acid beta-oxidation |
| KE | 2225 | Disrupted Lipid Storage | Disrupted Lipid Storage |
| KE | 327 | Accumulation, Fatty acid | Accumulation, Fatty acid |
| KE | 291 | Accumulation, Triglyceride | Accumulation, Triglyceride |
| AO | 459 | Increased, Liver Steatosis | Increased, Liver Steatosis |
Relationships Between Two Key Events (Including MIEs and AOs)
| Title | Adjacency | Evidence | Quantitative Understanding |
|---|
| Binding PPAR isoforms leads to Disrupted PPAR isoform nuclear signaling | adjacent | High | Moderate |
| Disrupted PPAR isoform nuclear signaling leads to Dysregulation of transcriptional expression within PPAR signaling network | adjacent | High | Moderate |
| Dysregulation of transcriptional expression within PPAR signaling network leads to Disrupted Lipid Storage | adjacent | High | Moderate |
| Dysregulation of transcriptional expression within PPAR signaling network leads to Decreased, Mitochondrial fatty acid beta-oxidation | adjacent | High | Moderate |
| Decreased, Mitochondrial fatty acid beta-oxidation leads to Disrupted Lipid Storage | adjacent | Moderate | Moderate |
| Disrupted Lipid Storage leads to Accumulation, Fatty acid | adjacent | Moderate | Moderate |
| Accumulation, Fatty acid leads to Accumulation, Triglyceride | adjacent | Moderate | Moderate |
| Accumulation, Triglyceride leads to Increased, Liver Steatosis | adjacent | High | Moderate |
| Disrupted Lipid Storage leads to Accumulation, Triglyceride | non-adjacent | Moderate | Moderate |
Network View
Prototypical Stressors
Life Stage Applicability
| Life stage | Evidence |
|---|---|
| Embryo | Moderate |
| Juvenile | Moderate |
| Adult, reproductively mature | High |
Taxonomic Applicability
Sex Applicability
| Sex | Evidence |
|---|---|
| Male | High |
| Female | Moderate |
Overall Assessment of the AOP
The weight of evidence from the literature indicates the potential for the molecular initiating even (MIE) of PFOS binding to the lipid-binding site of PPAR isoforms resulting in the key event of dysregulation of PPAR nuclear signaling (KE1). This KE results in the downstream KE of impacted regulation of diverse transcriptional expression pathways (KE2) that subsequently control KEs of altered lipid metabolism and transport. The effects of these KEs thus affect systemic lipid profiles resulting in the KEs of lipid accumulation in livers and hepatocellular vacuolation. Finally, these key events drive the adverse outcome (AO) of liver steatosis. Additional, more systemic AOs may also be affected by this MIE and cascade of KEs that can ultimately alter global energy metabolism, such as AOs of impacted growth and reproduction.
Domain of Applicability
The AOP is likely to be relevant for the majority of vertebrate species as an overall phylogenetic group across various lifestages and for both sexes.
Life Stage Applicability
There is evidence of disruption of PPAR isoforms in all life stages and evidence of perturbed lipid accumulation has also been seen at all lifestages across multiple vertebrate species. However, the liver (or proto-liver) is only formed and characterized in a subset of the organisms used for generating experimental data (e.g. zebrafish), and therefore evidence of the AO is limited across all potential vertebrates at the embyo stage. MIE, KE, and AO has been characterized in adults across mutliple vertebrate species types.
| Life Stage | Evidence |
| Embryo | Moderate |
| Juvenile | Moderate |
| Adult | High |
Taxanomic Applicability
The conservation of PPAR molecular structure and function among vertebrates (Gust et al 2020) indicates the MIE is likely to be conserved among this broad phylogenetic group. Further, evidence for the various KEs and the AO were assembled from investigations in various vertebrate species including mammals, birds, reptiles, amphibians, and fish where responses were largely congruent among the species tested. These observations indicate that the overall AOP is likely be relevant across the majority of vertebrate species. Further, these observations indicate the potential to use non-animal models, such as zebrafish embryo tests, in the context of this AOP to provide screening-level assessments that have relevance for human health, especially when rapid toxicity screening of diverse PFAS structures remains a critical need.
Sex Applicability
AOP is expected to be applicable across both sexes. However, it is important to note that in many of the fish studies in adults where sex differences were examined, lipid accumulation in liver was more severe in males than in females.
| Sex | Evidence |
| Male | High |
| Female | Moderate |
Essentiality of the Key Events
Essentiality of Key Events
MIE: Stressor binding PPAR isoforms: Numerous studies have shown the ability of synthetic ligands to bind the ligand binding domains of the PPAR isoforms (α, β/δ, γ). Specifically, the prototypical stressor, PFOS, has been shown to bind the three PPAR isoforms with varying degrees of affinity through in vitro ligand binding assays (Vanden Heuvel et al. 2006; Takacs and Abbot 2007; Wolf et al. 2008; Behr et al. 2020; Evans et al. 2022; Sun et al. 2023) as well as through computational binding/docking analyses (Li et al. 2018; Yi et al. 2019; Almedia et al. 2021; Garoche et al. 2021; Khazee et al. 2021; Huang et al. 2022b; Wang et al. 2022a, Wang et al. 2022b; Kowalska et al. 2023).
Key Event 1: Disruption of PPAR Isoform Nuclear Signaling: Studies have demonstrated that exposure to the prototypical stressor, PFOS, can have a direct effect on the transcriptional expression of the PPAR isoforms in vertebrates (Lee et al. 2020; Beale et al. 2022) with these studies showing expression changes occurring primarily in the PPARα and PPARγ isoforms. Furthermore, activation of one PPAR isoform can have effects on the expression of other PPAR isoforms. For example, agonism of PPARβ/δ can cause reduced expression of PPARα and PPARγ isoforms (Shi et al. 2002; Kim et al. 2020), and certain coregulators can have effects (sometimes opposite) on different PPAR isoforms (Tahri-Joutey et al. 2021). Finally, omics studies have shown that agonist and antagonist of PPAR isoforms alter PPAR signaling transcripts (Louisse et al. 2020; Heintz et al. 2024). Overall, this evidence displays that disruption of PPAR isoforms stressor chemicals can effect other PPAR isoforms and impact PPAR nuclear signaling.
Key Event 2: Dysregulation of Transcriptional Expression within PPAR Signaling Network: There is abundant evidence of showing how stressors can affect transcriptional expression in the PPAR signaling network and key genes involved in lipid homeostasis. Specifically, pathway and gene ontology (GO) enrichment analyses have identified lipid metabolism, lipid transport, fatty acid degradation, PPAR signaling pathway, and lipid homeostasis as being transcriptionally altered in response to PFOS exposure (Chen et al. 2014; Jacobsen et al. 2018; Rodríguez-Jorquera et al. 2018; Martinez et al. 2019; Christou et al. 2020; Dong et al. 2021; Lee et al. 2021; Mylroie et al. 2021; Beale et al. 2022; Davidsen et al. 2022; Haimbuagh et al. 2022; Wang et al. 2022a; Mylroie et al. IN PREP).
Key Event 3: Decreased β-oxidation: Decreased β-oxidation has been linked to liver steatosis and the PPAR isoforms play a key role in regulating β-oxidation (Cherkaoui-Malki et al. 2012). PPARα knockouts have shown decreased β-oxidation and subsequent lipid accumulation in the liver (Hashimoto et al. 2000; Reddy 2001; Badmann et al. 2007) whereas activation of PPARα has been shown to increase β-oxidation (Tahri-Joutey et al. 2021). PPARβ/δ has also been shown to have a critical role in the regulation β-oxidation (Roberts et al. 2011).
Key Event 4: Disrupted Lipid Storage: Disruption of the PPAR isoforms can have effects on lipid storage and transport (Dixon et al. 2021). PPARγ over expression results in promotes storage of lipids in the liver and thus exacerbates hepatic steatosis (Yu et al. 2003; Patsouris et al. 2006). Conversely, deletion of PPARα resulted in an increased liver lipid (Ptsouris et al. 2006). Wang et al. (2003) demonstrated that PPARβ/δ deficient mice had increased obesity which, while potentially not a function of improper lipid storage, underpins the importance of all PPAR isoforms in proper lipid homeostasis. Evidence of disruption of lipogenesis at the transcriptional level has also been observed across multiple studies using PFAS as the stressor (Tse et al. 2016; Cui et al. 2017; Huck et al. 2018; Liu et al. 2019; Martinez 2019; Yi et al. 2019; Louisse et al. 2020; Wang et al. 2022a).
Key Event 5: Accumulation of Fatty Acids in Liver Tissues: A Pparα-null strain in mice exhibited signs of increased fatty acid accumulation during fasting and over time under normal dietary conditions as Pparα-null strain mie cannot properly catabolize fatty acids (Montager et al. 2016). Under exposure to a stressor, Sant et al. (2021) observed increased accumulation of fatty acids and changes in fatty acid ratios when PFOS exposed zebrafish embryos were compared to control fish and Yang et al. (2022) observed differing lipid profiles between PFOS and PFOA exposed zebrafish embryos.
Key Event 6: Accumulation of Triglycerides (TG) and Total Cholesterol (TC) in the Liver Tissue: Disruption of the PPAR isoforms can be linked to accumulation of TG and TC in the liver tissue. In a review by Wang et al. (2020), it is explained how increased PPARγ expression can alter triacylglycerol levels. As examples of exposure to a stressor, studies using human cell cultures demonstrated increases in TG levels after exposure to PFOS (Liu et al. 2019; Louisse et al. 2020), and a metadata analysis performed on the blood lipid profiles of adult and juvenile humans showed that PFOS exposure was significantly correlated with an increase in TC levels in the blood and showed a trend of decreased TG levels in the blood (Ho et al. 2021).
Adverse Outcome: Liver Steatosis: The PPAR isoforms are essential for regulation of energy metabolism and specifically lipid metabolism (Wang et al. 2010). There is significant evidence in the literature demonstrating that repression, overexpression, or complete knock-out (KO) of the various PPAR isoforms can lead to disruptions in lipid metabolism and the adverse outcome of liver steatosis. An extensive review by Wang et al. (2020) presented evidence of how differential repression or activation of the various PPAR isoforms can affect metabolic regulation in mice livers and could lead to lipid accumulation and steatosis in the liver. A Pparα-null strain in mice exhibited signs of increased fatty acid accumulation and steatosis during fasting and over time under normal dietary conditions (Montager et al. 2016). Conversely, overexpression of PPARγ in mice increased the rate of hepatosteatosis (Yu et al. 2003; Wang et al. 2020). In fish, Li et al. (2020) demonstrated that a pparα knockout zebrafish, resulted in altered fatty acid oxidation enzymes and an increase in lipid accumulation in zebrafish livers. Conversely as to what was observed in mice, PPARγ KO male zebrafish showed indicators of hepatic steatosis under standard diet conditions (Zhao et al. 2022). Overall, there is evidence in multiple species of vertebrates that repression, overexpression, or complete knock-out of the PPAR isoforms can disrupt lipid metabolism and lead to the AO of liver steatosis even in the absence of a stressor such as PFOS.
Evidence Assessment
Evidence of PFOS/PPAR Interaction as the Molecular Initiating Event (MIE)
Perflouroalkyl substances like PFOS have structural similarities to fatty acids which are natural ligands of PPARs. Binding analyses and molecular docking models have shown that PFOS and other PFAS can bind to the ligand binding site of PPARs in both the agonist and antagonistic confirmations of the PPARs (Li et al. 2018; Yi et al. 2019; Almedia et al. 2021; Garoche et al. 2021; Khazee et al. 2021; Huang et al. 2022b; Wang et al. 2022a, Wang et al. 2022b; Kowalska et al. 2023) representing the molecular initiating event (MIE) of the present AOP. Activity assays in in vitro cell assay studies involving expressed PPAR receptors from mammals have also shown activation of PPARs by PFOS (Vanden Heuvel et al. 2006; Takacs and Abbot 2007; Wolf et al. 2008; Behr et al. 2020; Evans et al. 2022; Sun et al. 2023). An omics-based metadata study examining the response to PFAS exposure across multiple terrestrial and aquatic organisms (Beale et al. 2022) identified PPAR receptors as one of the key molecular targets of PFAS after exposure. Investigation of PPARα molecular structure and function indicated a high degree of conservation among vertebrate species including mammals, birds, reptiles, amphibians, and fish, whereas there was little conservation across invertebrates (Gust et al. 2020), which indicates that the MIE is likely to be conserved for majority of vertebrate species as an overall phylogenetic group.
Evidence of Disruption of PPAR Nuclear Signaling (KE1)
Evidence of disruption of PPAR nuclear signaling (KE1) following biding of PFOS to PPAR isoforms can be evidenced by numerous studies demonstrating that exposure to PFOS can have a direct effect on the transcriptional expression of the PPAR isoforms in vertebrates (Lee et al. 2020; Beale et al. 2022) with these studies showing expression changes occurring primarily in the PPARα and PPARγ isoforms. Investigations in human cells (Liu et al. 2019), mice [Mus musculus] (Das et al. 2018; Huck et al. 2018), Atlantic salmon [Salmo salar] (Arukwe and Mortensen 2011), and zebrafish [Danio rerio] (Olivares-Rubio and Vega Lopez 2016; Christou et al. 2020; Mylroie et al. 2021; Sant et al. 2021; Wang et al. 2022a) have shown both up- and down-regulation of PPAR transcriptional expression. In some cases, the expression of different PPAR isoforms can be regulated in opposite directions in the same exposure as was observed in Rodríguez-Jorquera et al. (2018) after fathead minnows [Pimephales promelas] were exposed to PFOS. Finally, studies in zebrafish have indicated that modulation of PPAR isoform signaling by PPAR agonist and antagonist results in apical toxicity outcomes similar to those seen as a result of PFOS and other PFAS exposures (Venezia et al. 2021). Given the sum of these observations, it is reasonable to hypothesize that PFASs, such as PFOS, can directly interact with PPARs through receptor binding and thus affect the downstream transcriptional signaling cascade and resultant enzymatic expression events that control lipid homeostasis with implications for all vertebrate species, with the best described outcomes associated with mammals.
Evidence of Disruption in PPAR Pathway Causing Early Key Events (KE2, KE3, & KE4)
Evidence of the overall dysregulation of transcriptional expression within the PPAR signaling network (KE2) can been observed in global and pathway-centered gene expression analyses in vertebrate embryos, larvae, and adult tissues which have shown that exposure to PFOS and other PFAS disrupts gene expression in multiple PPAR pathway-associated genes. Specifically, pathway and gene ontology (GO) enrichment analyses have identified lipid metabolism, lipid transport, fatty acid degradation, PPAR signaling pathway, and lipid homeostasis as being transcriptionally altered in response to PFOS exposure (Chen et al. 2014; Jacobsen et al. 2018; Rodríguez-Jorquera et al. 2018; Martinez et al. 2019; Christou et al. 2020; Dong et al. 2021; Lee et al. 2021; Mylroie et al. 2021; Beale et al. 2022; Davidsen et al. 2022; Haimbuagh et al. 2022; Wang et al. 2022a; Mylroie et al. IN PREP).
In addition to observations of dysregulation in transcriptional expression of the PPAR receptors, there is ample evidence that PFOS exposure results in transcriptional expression changes in downstream genes involved in the specific process of fatty acid metabolism (KE3), lipid storage (KE4), and lipid transport. For example, in mammal models, up-regulation of β -oxidation related genes Thiolase B and cyp4a1 have been observed in rats [Rattus norvegicus] (Davidsen et al. 2022) and with cyp4a14 and acadm observed as upregulated in mice (Rossen et al. 2010). At a cellular level, Wan et al. (2012) and Geng et al. (2019) demonstrated decreases in overall mitochondrial β -oxidation rates in liver tissue from PFOS exposed mice and chicken [Gallus gallus] embryos. In zebrafish, Cheng et al. (2016) observed increased transcriptional expression for genes related to β-oxidation (acox1, acadm, cpt1a) which is suggestive of a compensatory response to β-oxidation inhibition caused by PFOS exposure. Similarly, Wang et al. (2022a) also observed trends of increased transcriptional expression of genes in the β -oxidation pathway in zebrafish after PFOS exposure, and Yi et al. (2019) observed increased transcriptional expression of genes within the β -oxidation pathway including acox1 and acadm in response to PFOS. However, other investigations using zebrafish have observed genes in the β -oxidation pathway having decreased expression or mixed profiles of both increased and decreased expression (Tu et al. 2019; Mylroie et al. 2021).
Disruption of lipid storage (KE4) can occur when the genes involved in lipogenesis and/or lipid transport experience dysregulation and can be exacerbated by simultaneous effects on lipid metabolism such as altered β-oxidation (KE3). Evidence of disruption of lipogenesis at the transcriptional level has also been observed across multiple studies (Tse et al. 2016; Cui et al. 2017; Huck et al. 2018; Liu et al. 2019; Martinez 2019; Yi et al. 2019; Louisse et al. 2020; Wang et al. 2022a). Changes in lipogenesis could result in an accumulation of lipids in liver cells if lipogenesis is increased or transport is perturbed. Huck et al. (2018) saw a decrease expression in apoa1 and apoa2 in mice which has been associated with increased risk of liver steatosis (Karavia et al. 2012). Liu et al. (2019) and Louisse et al. (2020) saw an increase in expression in perilipin (Plin) family genes in human liver and stem cells exposed to PFOS, but Rodríguez-Jorquera et al. (2018) saw a decrease in Plin expression in livers from exposed fathead minnows. Plin family genes are involved in the formation and degradation of lipid droplets and thus dysregulation of these genes may impact proper lipid storage in the liver (Carr and Ahima 2016). Tse et al. (2016) saw an increase in apoe expression in zebrafish, which can signal a shift towards accumulation of lipids in hepatocytes. Furthermore, Wang et al. (2022a) saw a trend of decreased transcriptional expression of genes involved in lipid synthesis in zebrafish in response to PFOS; whereas Yi et al. (2109) saw PFOS exposure result in an increase in acacb transcriptional expression, a gene involved in fatty acid synthesis.
Disruption in lipid transport in and out of liver cells can result in excess lipid accumulation in cells which can ultimately lead to liver steatosis. Specifically, previous work has shown that along with disruptions to β-oxidation and lipogenesis, PFOS exposure can result in transcriptional changes to lipid transport genes in terrestrial vertebrates and fish (Cheng et al. 2016; Tse et al. 2016; Cui et al. 2017; Rodríguez-Jorquera et al. 2018; Sant et al. 2018; Martinez 2019; Christou et al. 2020; Mylroie et al. 2021; Davidsen et al. 2022; Wang et al. 2022a). Studies in mice (Huck et al. 2018; Liu et al. 2019), rats (Davidsen et al. 2022), and human cells (Wan et al. 2012), showed increases in CD36 expression in response to PFOS exposure. CD36 is responsible for transport of lipids in liver cells and an increase in CD36 expression due to PFOS exposure has been linked in increased TG levels in the liver (Jai et al. 2023). Dysregulation in fabp isoforms, which are responsible for the transport of fatty acids for fates such as β-oxidation and lipogenesis, was observed in mammals and fish exposed to PFOS (Rossen et al. 2010; Jacobsen et al. 2018; Sant et al. 2018; Mylroie et al. 2021; Wang et al. 2022a). Furthermore, lpl, which is involved in the proper transport of triglycerides was shown to be upregulated in studies in human cells (Wan et al. 2012) and mice (Liu et al. 2019); conversely Cheng et al. (2016) and Tse et al. (2016) showed lpl to be downregulated in response to PFOS exposure in zebrafish. Finally, Rodríguez-Jorquera et al. (2018) saw an overall decrease in lipid transport related genes in livers from PFOS exposed fathead minnow.
Overall, the results from these transcriptional studies show that PFOS exposure caused various disruptions of gene-transcript expression within the PPAR nuclear signaling network which are involved in fundamental processes that control lipid homeostasis and lipid profiles in liver tissue. Further, evidence for these KEs span multiple vertebrate species suggesting conservation of responses throughout vertebrates as a phylogenetic group.
Evidence of Changes in Lipid Profiles Indicative of Downstream Key Events (KE5 & KE6)
The observed dysregulation in β-oxidation and lipid storage should ultimately result in observable alterations in fatty acid, triglyceride, and total cholesterol profiles and accumulation as evidence of KE5 and KE6. Studies examining, whole body, serum, and liver lipid profiles have shown that PFOS exposure results in disrupted lipid profiles and accumulation in vertebrates, including humans. For example, Geng et al. (2019) saw increases in multiple types of lipid classes, including TGs, in developing chicken embryo livers after exposure to PFOS. Wan et al. (2012) and Huck et al. (2018) observed that mice had increased levels of TG in hepatic tissues after exposure to PFOS. Two studies using human cell cultures demonstrated increases in TG levels after exposure to PFOS (Liu et al. 2019; Louisse et al. 2020), and a metadata analysis performed on the blood lipid profiles of adult and juvenile humans showed that PFOS exposure was significantly correlated with an increase in TC levels in the blood and showed a trend of decreased TG levels in the blood (Ho et al. 2021).
Similar patterns of lipid alterations have been observed in fish. Sant et al. (2021) observed increased accumulation of fatty acids and changes in fatty acid ratios when PFOS exposed zebrafish embryos were compared to control fish and Yang et al. (2022) observed differing lipid profiles between PFOS and PFOA exposed zebrafish embryos. Cheng et al. (2016) observed a decrease in serum triglyceride (TG) and total cholesterol (TC) levels in the serum of male fish and an accumulation of TG in male and female livers (with males have significant increased TC levels as well). Cui et al. (2017) also observed a decrease in serum TG and TC levels in male fish and observed an increase in TC levels in female fish exposed to the highest PFOS concentration. Wang et al. (2022a) observed a significant increase in TC levels in adult zebrafish livers. The decrease in serum TC and TG levels combined with the increase in those same parameters in the liver tissue suggest a dysregulation of lipid homeostasis and preferential deposition of TC and TG in liver tissues.
These measured observations in fatty acid, TG, and TC profiles and accumulation provide further evidence of PPAR pathway dysregulation and are the downstream Key Events of the disruption of lipid metabolism and storage, a response conserved across the vertebrate species that were investigated.
Evidence of Lipid Accumulation in the Liver and the Adverse Outcome (AO) of Liver Steatosis
The contribution of KE5 and KE6 to lipid accumulation and lipid-induced hepatocellular vacuolation has been observed in various vertebrate species exposed to PFOS and other PFASs (Lee et al. 2020; Beale et al. 2022). In mice and rats, multiple studies have observed that PFOS exposure resulted in lipid-style hepatocellular vacuolation, lipid accumulation, or liver steatosis (Cui et al. 2009; Rosen et al. 2010, Zhang et al. 2016; Huck et al. 2018; Salter et al. 2021; Davidsen et al. 2022). Meanwhile in amphibians, results from Lin et al. (2022) showed that black-spotted frogs [Rana nigromaculata] exposed to PFOS had increased levels of hepatocellular vacuolation when compared to control frogs. Numerous studies have shown that increased lipid accumulation and/or hepatocellular vacuolation occurs in the developing liver of zebrafish beginning at the embryo/larval stage (Tse et al. 2016; Yi et al. 2019; Sant et al. 2021). At the adult stage, Mylroie et al. (IN PREP) found significant incidences of hepatocellular vacuolation in male zebrafish after exposure to 100 µg/L PFOS with other studies reporting similar outcomes at differing concentrations of PFOS exposure (Du et al. 2009; Keiter et al. 2012; Cheng et al. 2016; Cui et al. 2017; Huang et al. 2022a; Wang et al. 2022a). It is important to note that in many of the studies in adults where sex differences were examined, lipid accumulation in liver was more severe in males than in females. Excess accumulation of lipids in the liver, as seen by the evidence here, is a key factor in the ultimate adverse outcome (AO) of liver steatosis.
Known Modulating Factors
| Modulating Factor (MF) | Influence or Outcome | KER(s) involved |
|---|---|---|
Quantitative Understanding
Considerations for Potential Applications of the AOP (optional)
This AOP is likely to be applicable to chemicals, such as PFAS, that have been shown to interact with and disrupt the signaling of more than one PPAR isoform. The risk for this AOP is likely dependent on the concentrations of the chemical stressor and the duration of the exposure. It is possible that co-factors such as diet, genetic predisposition, and lack of physical activity could exacererbate or hasten the onset of the adverse outcome.
References
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