AOP-Wiki

AOP ID and Title:

AOP 529: Perfluorooctanesulfonic acid (PFOS) binding to peroxisome proliferator-activated receptors (PPARs) causes dysregulation of lipid metabolism and subsequent liver steatosis

Short Title: PFOS binding to PPARs leads to liver steatosis

Graphical Representation

Authors

J. Erik Mylroie¹, Kurt A. Gust¹, David W. Moore¹

¹US Army, Engineer Research and Development Center, Environmental Laboratory 3909 Halls Ferry Rd. Vicksburg, MS

Status

Author status	OECD status	OECD project	SAAOP status
Under development: Not open for comment. Do not cite

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.

Background

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.

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	Decrease, Fatty acid beta-oxidation	Decrease, Fatty acid β-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

Key Event Relationships

Upstream Event	Relationship Type	Downstream Event	Evidence	Quantitative Understanding
Stressor binding PPAR isoforms	adjacent	Disrupted PPAR isoform nuclear signaling	High	Moderate
Disrupted PPAR isoform nuclear signaling	adjacent	Dysregulation of transcriptional expression within PPAR signaling network	High	Moderate
Dysregulation of transcriptional expression within PPAR signaling network	adjacent	Disrupted Lipid Storage	High	Moderate
Dysregulation of transcriptional expression within PPAR signaling network	adjacent	Decrease, Fatty acid beta-oxidation	High	Moderate
Decrease, Fatty acid beta-oxidation	adjacent	Disrupted Lipid Storage	Moderate	Moderate
Disrupted Lipid Storage	adjacent	Accumulation, Fatty acid	Moderate	Moderate
Accumulation, Fatty acid	adjacent	Accumulation, Triglyceride	Moderate	Moderate
Accumulation, Triglyceride	adjacent	Increased, Liver Steatosis	High	Moderate

Disrupted Lipid Storage	non-adjacent	Accumulation, Triglyceride	Moderate	Moderate

Stressors

Name	Evidence
Perfluorooctanesulfonic acid
PPARalpha antagonists
PPAR agonist
Per- and Polyfluorinated Substances (PFAS)

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

Life Stage Applicability

Life Stage	Evidence
Embryo	Moderate
Juvenile	Moderate
Adult, reproductively mature	High

Taxonomic Applicability

Term	Scientific Term	Evidence	Links
Vertebrates	Vertebrates	High	NCBI
mouse	Mus musculus	High	NCBI
rat	Rattus norvegicus	High	NCBI
zebrafish	Danio rerio	High	NCBI

Sex Applicability

Sex	Evidence
Male	High
Female	Moderate

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 mice 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.

Weight of Evidence Summary

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.

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

Agency for Toxic Substances and Disease Registry (ATSDR), 2021. Toxicological profile for Perfluoroalkyls. Atlanta, GA: U.S. Department of Health and Human Services, Public Health Service

Almeida, N.M., Eken, Y. and Wilson, A.K., 2021. Binding of per-and polyfluoro-alkyl substances to peroxisome proliferator-activated receptor gamma. ACS omega, 6(23), pp.15103-15114.

Ankley, G.T., Cureton, P., Hoke, R.A., Houde, M., Kumar, A., Kurias, J., Lanno, R., McCarthy, C., Newsted, J., Salice, C.J. and Sample, B.E., 2021. Assessing the ecological risks of per‐and polyfluoroalkyl substances: Current state‐of‐the science and a proposed path forward. Environmental toxicology and chemistry, 40(3), pp.564-605.

Annunziato, K.M., Doherty, J., Lee, J., Clark, J.M., Liang, W., Clark, C.W., Nguyen, M., Roy, M.A. and Timme-Laragy, A.R., 2020. Chemical characterization of a legacy aqueous film-forming foam sample and developmental toxicity in zebrafish (Danio rerio). Environmental Health Perspectives, 128(9), p.097006.

Arukwe, A. and Mortensen, A.S., 2011. Lipid peroxidation and oxidative stress responses of salmon fed a diet containing perfluorooctane sulfonic-or perfluorooctane carboxylic acids. Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology, 154(4), pp.288-295.

Badman, M.K., Pissios, P., Kennedy, A.R., Koukos, G., Flier, J.S. and Maratos-Flier, E., 2007. Hepatic fibroblast growth factor 21 is regulated by PPARα and is a key mediator of hepatic lipid metabolism in ketotic states. Cell metabolism, 5(6), pp.426-437.

Beale, D.J., Sinclair, G., Shah, R., Paten, A., Kumar, A., Long, S.M., Vardy, S. and Jones, O.A., 2022. A review of omics-based PFAS exposure studies reveals common biochemical response pathways. Science of The Total Environment, p.157255.

Behr, A.C., Plinsch, C., Braeuning, A. and Buhrke, T., 2020. Activation of human nuclear receptors by perfluoroalkylated substances (PFAS). Toxicology in Vitro, 62, p.104700.

Bell, E.M., De Guise, S., McCutcheon, J.R., Lei, Y., Levin, M., Li, B., Rusling, J.F., Lawrence, D.A., Cavallari, J.M., O'Connell, C. and Javidi, B., 2021. Exposure, health effects, sensing, and remediation of the emerging PFAS contaminants–Scientific challenges and potential research directions. Science of the Total Environment, 780, p.146399.

Boyd, R.I., Ahmad, S., Singh, R., Fazal, Z., Prins, G.S., Madak Erdogan, Z., Irudayaraj, J. and Spinella, M.J., 2022. Toward a Mechanistic Understanding of Poly-and Perfluoroalkylated Substances and Cancer. Cancers, 14(12), p.2919.

Carr, R.M. and Ahima, R.S., 2016. Pathophysiology of lipid droplet proteins in liver diseases. Experimental cell research, 340(2), pp.187-192.

Chen, J., Tanguay, R.L., Tal, T.L., Gai, Z., Ma, X., Bai, C., Tilton, S.C., Jin, D., Yang, D., Huang, C. and Dong, Q., 2014. Early life perfluorooctanesulphonic acid (PFOS) exposure impairs zebrafish organogenesis. Aquatic toxicology, 150, pp.124-132.

Cheng, J., Lv, S., Nie, S., Liu, J., Tong, S., Kang, N., Xiao, Y., Dong, Q., Huang, C. and Yang, D., 2016. Chronic perfluorooctane sulfonate (PFOS) exposure induces hepatic steatosis in zebrafish. Aquatic toxicology, 176, pp.45-52.

Cherkaoui-Malki, M., Surapureddi, S., I El Hajj, H., Vamecq, J. and Andreoletti, P., 2012. Hepatic steatosis and peroxisomal fatty acid beta-oxidation. Current Drug Metabolism, 13(10), pp.1412-1421.

Christou, M., Fraser, T.W., Berg, V., Ropstad, E. and Kamstra, J.H., 2020. Calcium signaling as a possible mechanism behind increased locomotor response in zebrafish larvae exposed to a human relevant persistent organic pollutant mixture or PFOS. Environmental Research, 187, p.109702.

Costello, E., Rock, S., Stratakis, N., Eckel, S.P., Walker, D.I., Valvi, D., Cserbik, D., Jenkins, T., Xanthakos, S.A., Kohli, R. and Sisley, S., 2022. Exposure to per-and polyfluoroalkyl substances and markers of liver injury: a systematic review and meta-analysis. Environmental Health Perspectives, 130(4), p.046001.

Cui, L., Zhou, Q.F., Liao, C.Y., Fu, J.J. and Jiang, G.B., 2009. Studies on the toxicological effects of PFOA and PFOS on rats using histological observation and chemical analysis. Archives of environmental contamination and toxicology, 56, pp.338-349.

Cui, Y., Lv, S., Liu, J., Nie, S., Chen, J., Dong, Q., Huang, C. and Yang, D., 2017. Chronic perfluorooctanesulfonic acid exposure disrupts lipid metabolism in zebrafish. Human & experimental toxicology, 36(3), pp.207-217.

Das, K.P., Wood, C.R., Lin, M.T., Starkov, A.A., Lau, C., Wallace, K.B., Corton, J.C. and Abbott, B.D., 2017. Perfluoroalkyl acids-induced liver steatosis: Effects on genes controlling lipid homeostasis. Toxicology, 378, pp.37-52.

Davidsen, N., Ramhøj, L., Lykkebo, C.A., Kugathas, I., Poulsen, R., Rosenmai, A.K., Evrard, B., Darde, T.A., Axelstad, M., Bahl, M.I. and Hansen, M., 2022. PFOS-induced thyroid hormone system disrupted rats display organ-specific changes in their transcriptomes. Environmental Pollution, 305, p.119340.

DeWitt, J.C., Blossom, S.J. and Schaider, L.A., 2019. Exposure to per-fluoroalkyl and polyfluoroalkyl substances leads to immunotoxicity: epidemiological and toxicological evidence. Journal of exposure science & environmental epidemiology, 29(2), pp.148-156.

Dixon, E.D., Nardo, A.D., Claudel, T. and Trauner, M., 2021. The role of lipid sensing nuclear receptors (PPARs and LXR) and metabolic lipases in obesity, diabetes and NAFLD. Genes, 12(5), p.645.

Dong, G., Zhang, R., Huang, H., Lu, C., Xia, Y., Wang, X. and Du, G., 2021. Exploration of the developmental toxicity of TCS and PFOS to zebrafish embryos by whole-genome gene expression analyses. Environmental Science and Pollution Research, 28(40), pp.56032-56042.

Du, Y., Shi, X., Liu, C., Yu, K. and Zhou, B., 2009. Chronic effects of water-borne PFOS exposure on growth, survival and hepatotoxicity in zebrafish: a partial life-cycle test. Chemosphere, 74(5), pp.723-729.

Ducatman, A. and Fenton, S.E., 2022. Invited Perspective: PFAS and Liver Disease: Bringing All the Evidence Together. Environmental health perspectives, 130(4), p.041303.

Evans, N., Conley, J.M., Cardon, M., Hartig, P., Medlock-Kakaley, E. and Gray Jr, L.E., 2022. In vitro activity of a panel of per-and polyfluoroalkyl substances (PFAS), fatty acids, and pharmaceuticals in peroxisome proliferator-activated receptor (PPAR) alpha, PPAR gamma, and estrogen receptor assays. Toxicology and Applied Pharmacology, 449, p.116136.

Fragki, S., Dirven, H., Fletcher, T., Grasl-Kraupp, B., Bjerve Gützkow, K., Hoogenboom, R., Kersten, S., Lindeman, B., Louisse, J., Peijnenburg, A. and Piersma, A.H., 2021. Systemic PFOS and PFOA exposure and disturbed lipid homeostasis in humans: what do we know and what not?. Critical reviews in toxicology, 51(2), pp.141-164.

Garoche, C., Boulahtouf, A., Grimaldi, M., Chiavarina, B., Toporova, L., den Broeder, M.J., Legler, J., Bourguet, W. and Balaguer, P., 2021. Interspecies Differences in Activation of Peroxisome Proliferator-Activated Receptor γ by Pharmaceutical and Environmental Chemicals. Environmental Science & Technology, 55(24), pp.16489-16501.

Geng, D., Musse, A.A., Wigh, V., Carlsson, C., Engwall, M., Orešič, M., Scherbak, N. and Hyötyläinen, T., 2019. Effect of perfluorooctanesulfonic acid (PFOS) on the liver lipid metabolism of the developing chicken embryo. Ecotoxicology and environmental safety, 170, pp.691-698.

Glüge, J., Scheringer, M., Cousins, I.T., DeWitt, J.C., Goldenman, G., Herzke, D., Lohmann, R., Ng, C.A., Trier, X. and Wang, Z., 2020. An overview of the uses of per-and polyfluoroalkyl substances (PFAS). Environmental Science: Processes & Impacts, 22(12), pp.2345-2373.

Grygiel-Górniak, B., 2014. Peroxisome proliferator-activated receptors and their ligands: nutritional and clinical implications-a review. Nutrition journal, 13, pp.1-10.

Gust, K.A., Ji, Q., Luo, X., 2020. Example of Adverse Outcome Pathway Concept Enabling Genome-to-Phenome Discovery in Toxicology. Integr. Comp. Biol. 60, 375-384.

Gust KA, Wilbanks MS, Collier ZA, Burgoon LD, Perkins EJ (2019). Adverse Outcome Pathway on antagonist binding to PPARα leading to body-weight loss. OECD Series on Adverse Outcome Pathways, No. 10, OECD Publishing, Paris, https://doi.org/10.1787/29d4e08d-en.

Haimbaugh, A., Wu, C.C., Akemann, C., Meyer, D.N., Connell, M., Abdi, M., Khalaf, A., Johnson, D. and Baker, T.R., 2022. Multi-and transgenerational effects of developmental exposure to environmental levels of PFAS and PFAS mixture in zebrafish (Danio rerio). Toxics, 10(6), p.334.

Hashimoto, T., Cook, W.S., Qi, C., Yeldandi, A.V., Reddy, J.K. and Rao, M.S., 2000. Defect in peroxisome proliferator-activated receptor α-inducible fatty acid oxidation determines the severity of hepatic steatosis in response to fasting. Journal of Biological Chemistry, 275(37), pp.28918-28928.

Heintz, M.M., Klaren, W.D., East, A.W., Haws, L.C., McGreal, S.R., Campbell, R.R. and Thompson, C.M., 2024. Comparison of transcriptomic profiles between HFPO-DA and prototypical PPARα, PPARγ, and cytotoxic agents in mouse, rat, and pooled human hepatocytes. Toxicological Sciences, p.kfae044.

Ho, S.H., Soh, S.X.H., Wang, M.X., Ong, J., Seah, A., Wong, Y., Fang, Z., Sim, S. and Lim, J.T., 2022. Perfluoroalkyl substances and lipid concentrations in the blood: A systematic review of epidemiological studies. Science of The Total Environment, p.158036.

Huang, J., Liu, Y., Wang, Q., Yi, J., Lai, H., Sun, L., Mennigen, J.A. and Tu, W., 2022a. Concentration-dependent toxicokinetics of novel PFOS alternatives and their chronic combined toxicity in adult zebrafish. Science of The Total Environment, 839, p.156388.

Huang, J., Wang, Q., Liu, S., Lai, H. and Tu, W., 2022b. Comparative chronic toxicities of PFOS and its novel alternatives on the immune system associated with intestinal microbiota dysbiosis in adult zebrafish. Journal of Hazardous Materials, 425, p.127950.

Huck, I., Beggs, K. and Apte, U., 2018. Paradoxical Protective Effect of Perfluorooctanesulfonic Acid Against High-Fat Diet–Induced Hepatic Steatosis in Mice. International journal of toxicology, 37(5), pp.383-392.

Jacobsen, A.V., Nordén, M., Engwall, M. and Scherbak, N., 2018. Effects of perfluorooctane sulfonate on genes controlling hepatic fatty acid metabolism in livers of chicken embryos. Environmental Science and Pollution Research, 25, pp.23074-23081.

Jia, Y., Zhu, Y., Wang, R., Ye, Q., Xu, D., Zhang, W., Zhang, Y., Shan, G. and Zhu, L., 2023. Novel insights into the mediating roles of cluster of differentiation 36 in transmembrane transport and tissue partition of per-and polyfluoroalkyl substances in mice. Journal of Hazardous Materials, 442, p.130129.

Karavia, E.A., Papachristou, D.J., Liopeta, K., Triantaphyllidou, I.E., Dimitrakopoulos, O. and Kypreos, K.E., 2012. Apolipoprotein AI modulates processes associated with diet-induced nonalcoholic fatty liver disease in mice. Molecular Medicine, 18(6), pp.901-912.

Keiter, S., Baumann, L., Färber, H., Holbech, H., Skutlarek, D., Engwall, M. and Braunbeck, T., 2012. Long-term effects of a binary mixture of perfluorooctane sulfonate (PFOS) and bisphenol A (BPA) in zebrafish (Danio rerio). Aquatic toxicology, 118, pp.116-129.

Khazaee, M., Christie, E., Cheng, W., Michalsen, M., Field, J. and Ng, C., 2021. Perfluoroalkyl acid binding with peroxisome proliferator-activated receptors α, γ, and δ, and fatty acid binding proteins by equilibrium dialysis with a comparison of methods. Toxics, 9(3), p.45.

Kim, D.H., Kim, D.H., Heck, B.E., Shaffer, M., Yoo, K.H. and Hur, J., 2020. PPAR-δ agonist affects adipo-chondrogenic differentiation of human mesenchymal stem cells through the expression of PPAR-γ. Regenerative Therapy, 15, pp.103-111.

Kowalska, D., Sosnowska, A., Bulawska, N., Stępnik, M., Besselink, H., Behnisch, P. and Puzyn, T., 2023. How the Structure of Per-and Polyfluoroalkyl Substances (PFAS) Influences Their Binding Potency to the Peroxisome Proliferator-Activated and Thyroid Hormone Receptors—An In Silico Screening Study. Molecules, 28(2), p.479.

Lamas Bervejillo, M. and Ferreira, A.M., 2019. Understanding peroxisome proliferator-activated receptors: from the structure to the regulatory actions on metabolism. Bioactive Lipids in Health and Disease, pp.39-57.

Lee, J.W., Choi, K., Park, K., Seong, C., Do Yu, S. and Kim, P., 2020. Adverse effects of perfluoroalkyl acids on fish and other aquatic organisms: A review. Science of the Total Environment, 707, p.135334.

Lee, H., Sung, E.J., Seo, S., Min, E.K., Lee, J.Y., Shim, I., Kim, P., Kim, T.Y., Lee, S. and Kim, K.T., 2021. Integrated multi-omics analysis reveals the underlying molecular mechanism for developmental neurotoxicity of perfluorooctanesulfonic acid in zebrafish. Environment International, 157, p.106802.

Li, C.H., Ren, X.M., Ruan, T., Cao, L.Y., Xin, Y., Guo, L.H. and Jiang, G., 2018. Chlorinated polyfluorinated ether sulfonates exhibit higher activity toward peroxisome proliferator-activated receptors signaling pathways than perfluorooctanesulfonate. Environmental science & technology, 52(5), pp.3232-3239.

Li, L.Y., Lv, H.B., Jiang, Z.Y., Qiao, F., Chen, L.Q., Zhang, M.L. and Du, Z.Y., 2020. Peroxisomal proliferator‐activated receptor α‐b deficiency induces the reprogramming of nutrient metabolism in zebrafish. The Journal of Physiology, 598(20), pp.4537-4553.

Lin, H., Wu, H., Liu, F., Yang, H., Shen, L., Chen, J., Zhang, X., Zhong, Y., Zhang, H. and Liu, Z., 2022. Assessing the hepatotoxicity of PFOA, PFOS, and 6: 2 Cl-PFESA in black-spotted frogs (Rana nigromaculata) and elucidating potential association with gut microbiota. Environmental Pollution, 312, p.120029.

Liu, S., Yang, R., Yin, N., Wang, Y.L. and Faiola, F., 2019. Environmental and human relevant PFOS and PFOA doses alter human mesenchymal stem cell self-renewal, adipogenesis and osteogenesis. Ecotoxicology and environmental safety, 169, pp.564-572.

Louisse, J., Rijkers, D., Stoopen, G., Janssen, A., Staats, M., Hoogenboom, R., Kersten, S. and Peijnenburg, A., 2020. Perfluorooctanoic acid (PFOA), perfluorooctane sulfonic acid (PFOS), and perfluorononanoic acid (PFNA) increase triglyceride levels and decrease cholesterogenic gene expression in human HepaRG liver cells. Archives of toxicology, 94(9), pp.3137-3155.

Martínez, R., Navarro-Martín, L., Luccarelli, C., Codina, A.E., Raldúa, D., Barata, C., Tauler, R. and Piña, B., 2019. Unravelling the mechanisms of PFOS toxicity by combining morphological and transcriptomic analyses in zebrafish embryos. Science of the Total Environment, 674, pp.462-471.

Montagner, A., Polizzi, A., Fouché, E., Ducheix, S., Lippi, Y., Lasserre, F., Barquissau, V., Régnier, M., Lukowicz, C., Benhamed, F. and Iroz, A., 2016. Liver PPARα is crucial for whole-body fatty acid homeostasis and is protective against NAFLD. Gut, 65(7), pp.1202-1214.

Mylroie, J.E., Wilbanks, M.S., Kimble, A.N., To, K.T., Cox, C.S., McLeod, S.J., Gust, K.A., Moore, D.W., Perkins, E.J. and Garcia‐Reyero, N., 2021. Perfluorooctanesulfonic acid–induced toxicity on zebrafish embryos in the presence or absence of the chorion. Environmental toxicology and chemistry, 40(3), pp.780-791.

Mylroie, J.E., Gust, K.A., Kimble, A.N., Wilbanks, M.W., Steward, C., Chapman, K.A., Kennedy, A.L., Jensen, K., Erickson, R., Ankley G.T, Conder, J., Vinas, N.G., Moore, D.W., Histological and Transcriptomic Evidence of Disrupted Lipid Metabolism in a Three-Generation Exposure of the Zebrafish (Danio rerio) to Perfluorooctane Sulfonate (PFOS). IN PREP.

Olivares-Rubio, H.F. and Vega-López, A., 2016. Fatty acid metabolism in fish species as a biomarker for environmental monitoring. Environmental Pollution, 218, pp.297-312.

Patsouris, D., Reddy, J.K., Müller, M. and Kersten, S., 2006. Peroxisome proliferator-activated receptor α mediates the effects of high-fat diet on hepatic gene expression. Endocrinology, 147(3), pp.1508-1516.

Reddy, J.K., 2001. III. Peroxisomal β-oxidation, PPARα, and steatohepatitis. American Journal of Physiology-Gastrointestinal and Liver Physiology, 281(6), pp.G1333-G1339.

Roberts, L.D., Murray, A.J., Menassa, D., Ashmore, T., Nicholls, A.W. and Griffin, J.L., 2011. The contrasting roles of PPARδ and PPARγ in regulating the metabolic switch between oxidation and storage of fats in white adipose tissue. Genome biology, 12, pp.1-19.

Rodríguez-Jorquera, I.A., Colli-Dula, R.C., Kroll, K., Jayasinghe, B.S., Parachu Marco, M.V., Silva-Sanchez, C., Toor, G.S. and Denslow, N.D., 2018. Blood transcriptomics analysis of fish exposed to perfluoro alkyls substances: assessment of a non-lethal sampling technique for advancing aquatic toxicology research. Environmental science & technology, 53(3), pp.1441-1452.

Rosen, M.B., Schmid, J.R., Corton, J.C., Zehr, R.D., Das, K.P., Abbott, B.D. and Lau, C., 2010. Gene expression profiling in wild-type and PPARα-null mice exposed to perfluorooctane sulfonate reveals PPARα-independent effects. PPAR research, 2010.

Salter, D.M., Wei, W., Nahar, P.P., Marques, E. and Slitt, A.L., 2021. Perfluorooctanesulfonic acid (PFOS) thwarts the beneficial effects of calorie restriction and metformin. Toxicological Sciences, 182(1), pp.82-95.

Sant, K.E., Sinno, P.P., Jacobs, H.M. and Timme-Laragy, A.R., 2018. Nrf2a modulates the embryonic antioxidant response to perfluorooctanesulfonic acid (PFOS) in the zebrafish, Danio rerio. Aquatic toxicology, 198, pp.92-102.

Sant, K.E., Annunziato, K., Conlin, S., Teicher, G., Chen, P., Venezia, O., Downes, G.B., Park, Y. and Timme-Laragy, A.R., 2021. Developmental exposures to perfluorooctanesulfonic acid (PFOS) impact embryonic nutrition, pancreatic morphology, and adiposity in the zebrafish, Danio rerio. Environmental Pollution, 275, p.116644.

Shi, Y., Hon, M. and Evans, R.M., 2002. The peroxisome proliferator-activated receptor δ, an integrator of transcriptional repression and nuclear receptor signaling. Proceedings of the National Academy of Sciences, 99(5), pp.2613-2618.

Sun, X., Xie, Y., Zhang, X., Song, J. and Wu, Y., 2023. Estimation of Per-and Polyfluorinated Alkyl Substance Induction Equivalency Factors for Humpback Dolphins by Transactivation Potencies of Peroxisome Proliferator-Activated Receptors. Environmental Science & Technology, 57(9), pp.3713-3721.

Sunderland, E.M., Hu, X.C., Dassuncao, C., Tokranov, A.K., Wagner, C.C. and Allen, J.G., 2019. A review of the pathways of human exposure to poly-and perfluoroalkyl substances (PFASs) and present understanding of health effects. Journal of exposure science & environmental epidemiology, 29(2), pp.131-147.

Tahri-Joutey, M., Andreoletti, P., Surapureddi, S., Nasser, B., Cherkaoui-Malki, M. and Latruffe, N., 2021. Mechanisms mediating the regulation of peroxisomal fatty acid beta-oxidation by PPARα. International journal of molecular sciences, 22(16), p.8969.

Takacs, M.L. and Abbott, B.D., 2007. Activation of mouse and human peroxisome proliferator–activated receptors (α, β/δ, γ) by perfluorooctanoic acid and perfluorooctane sulfonate. Toxicological Sciences, 95(1), pp.108-117.

Tse, W.K.F., Li, J.W., Tse, A.C.K., Chan, T.F., Ho, J.C.H., Wu, R.S.S., Wong, C.K.C. and Lai, K.P., 2016. Fatty liver disease induced by perfluorooctane sulfonate: Novel insight from transcriptome analysis. Chemosphere, 159, pp.166-177.

Tu, W., Martinez, R., Navarro-Martin, L., Kostyniuk, D.J., Hum, C., Huang, J., Deng, M., Jin, Y., Chan, H.M. and Mennigen, J.A., 2019. Bioconcentration and metabolic effects of emerging PFOS alternatives in developing zebrafish. Environmental Science & Technology, 53(22), pp.13427-13439.

Vanden Heuvel, J.P., Thompson, J.T., Frame, S.R. and Gillies, P.J., 2006. Differential activation of nuclear receptors by perfluorinated fatty acid analogs and natural fatty acids: a comparison of human, mouse, and rat peroxisome proliferator-activated receptor-α,-β, and-γ, liver X receptor-β, and retinoid X receptor-α. Toxicological Sciences, 92(2), pp.476-489.

Varga, T., Czimmerer, Z. and Nagy, L., 2011. PPARs are a unique set of fatty acid regulated transcription factors controlling both lipid metabolism and inflammation. Biochimica et Biophysica Acta (BBA)-Molecular Basis of Disease, 1812(8), pp.1007-1022.

Venezia, O., Islam, S., Cho, C., Timme-Laragy, A.R. and Sant, K.E., 2021. Modulation of PPAR signaling disrupts pancreas development in the zebrafish, Danio rerio. Toxicology and applied pharmacology, 426, p.115653.

Wan, H.T., Zhao, Y.G., Wei, X., Hui, K.Y., Giesy, J.P. and Wong, C.K., 2012. PFOS-induced hepatic steatosis, the mechanistic actions on β-oxidation and lipid transport. Biochimica et Biophysica Acta (BBA)-General Subjects, 1820(7), pp.1092-1101.

Wang, Y.X., Lee, C.H., Tiep, S., Ruth, T.Y., Ham, J., Kang, H. and Evans, R.M., 2003. Peroxisome-proliferator-activated receptor δ activates fat metabolism to prevent obesity. Cell, 113(2), pp.159-170.

Wang, Y.X., 2010. PPARs: diverse regulators in energy metabolism and metabolic diseases. Cell research, 20(2), pp.124-137.

Wang, Y., Nakajima, T., Gonzalez, F.J. and Tanaka, N., 2020. PPARs as metabolic regulators in the liver: lessons from liver-specific PPAR-null mice. International journal of molecular sciences, 21(6), p.2061.

Wang, Q., Huang, J., Liu, S., Wang, C., Jin, Y., Lai, H. and Tu, W., 2022a. Aberrant hepatic lipid metabolism associated with gut microbiota dysbiosis triggers hepatotoxicity of novel PFOS alternatives in adult zebrafish. Environment International, 166, p.107351.

Wang, P., Liu, D., Yan, S., Cui, J., Liang, Y. and Ren, S., 2022b. Adverse effects of perfluorooctane sulfonate on the liver and relevant mechanisms. Toxics, 10(5), p.265.

Wolf, C.J., Takacs, M.L., Schmid, J.E., Lau, C. and Abbott, B.D., 2008. Activation of mouse and human peroxisome proliferator− activated receptor alpha by perfluoroalkyl acids of different functional groups and chain lengths. Toxicological Sciences, 106(1), pp.162-171.

Yang, Z., Fu, L., Cao, M., Li, F., Li, J., Chen, Z., Guo, A., Zhong, H., Li, W., Liang, Y. and Luo, Q., 2022. PFAS-induced lipidomic dysregulations and their associations with developmental toxicity in zebrafish embryos. Science of The Total Environment, p.160691.

Yi, S., Chen, P., Yang, L. and Zhu, L., 2019. Probing the hepatotoxicity mechanisms of novel chlorinated polyfluoroalkyl sulfonates to zebrafish larvae: Implication of structural specificity. Environment international, 133, p.105262.

Yu, S., Matsusue, K., Kashireddy, P., Cao, W.Q., Yeldandi, V., Yeldandi, A.V., Rao, M.S., Gonzalez, F.J. and Reddy, J.K., 2003. Adipocyte-specific gene expression and adipogenic steatosis in the mouse liver due to peroxisome proliferator-activated receptor γ1 (PPARγ1) overexpression. Journal of Biological Chemistry, 278(1), pp.498-505.

Zhang, L., Krishnan, P., Ehresman, D.J., Smith, P.B., Dutta, M., Bagley, B.D., Chang, S.C., Butenhoff, J.L., Patterson, A.D. and Peters, J.M., 2016. Editor’s highlight: Perfluorooctane sulfonate-choline ion pair formation: A potential mechanism modulating hepatic steatosis and oxidative stress in mice. Toxicological Sciences, 153(1), pp.186-197.

Zhao, Y., Castro, L.F.C., Monroig, Ó., Cao, X., Sun, Y. and Gao, J., 2022. A zebrafish pparγ gene deletion reveals a protein kinase network associated with defective lipid metabolism. Functional & Integrative Genomics, 22(4), pp.435-450.

Appendix 1

List of MIEs in this AOP

Event: 2226: Stressor binding PPAR isoforms

Short Name: Binding PPAR isoforms

Key Event Component

Process	Object	Action
receptor binding		occurrence

AOPs Including This Key Event

AOP ID and Name	Event Type
Aop:529 - Perfluorooctanesulfonic acid (PFOS) binding to peroxisome proliferator-activated receptors (PPARs) causes dysregulation of lipid metabolism and subsequent liver steatosis	MolecularInitiatingEvent

Biological Context

Level of Biological Organization
Molecular

Cell term

Cell term
eukaryotic cell

Organ term

Organ term
liver

Domain of Applicability

Taxonomic Applicability

Term	Scientific Term	Evidence	Links
Vertebrates	Vertebrates	High	NCBI

Life Stage Applicability

Life Stage	Evidence
Embryo	Moderate
Juvenile	High
Adult, reproductively mature	High

Sex Applicability

Sex	Evidence
Male	High
Female	Moderate

The conservation of PPAR molecular structure and function among vertebrates (Gust et al 2020) indicates this key event is likely to be conserved among this broad phylogenetic group. Furthermore, PPAR isoforms play a crucial role in lipid metabolism across representative vertebrate species. However, given that species to species variation does exist in structure and specific function, it is important to exercise care when looking to extrapolate across species.

Key Event Description

Both natural and synthetic ligands can interact with all 3 main PPAR isoforms with unsaturated fatty acids and other lipid-derived molecules being the primary natural ligands the PPAR isoforms (Ferré 2004). This Key Event describes the binding of stressor ligands to the PPAR isoforms with either agonist or antagonist interactions. Numerous studies have shown the ability of synthetic ligands to bind the ligand binding domains of the PPAR isoforms (α, β/δ, γ). Some of these synthetic ligands can be PPAR isoform specific whereas others, like bezafibrate, can bind and activate all 3 main PPAR isoforms (Grygiel-Górniak 2014). 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).

How it is Measured or Detected

Nuclear signaling assays, affinity assays, x-ray crystallography, and in silico analyses can all be used to assess the affinity and location of binding by known or potential ligands to the PPAR isoforms (Vanden Heuvel et al. 2006; Takacs and Abbot 2007; Capelli et al. 2016; Rajapaksha et al. 2017; Li et al. 2018; Behr et al. 2020; Almedia et al. 2021; Garoche et al. 2021; Evans et al. 2022; Sun et al. 2023). In silico analyses are a powerful screening tool to determine if a molecule of interest may be able to bind to one or more of the PPAR isoforms; however, confirmation of binding location should be done via x-ray crystallography. Nuclear signaling assays can be used to determine if a potential ligand of interest acts as an agonists or antagonists. A comprehensive example of in silico primary analyses coupled with confirmation steps using cell-based report assays and x-ray crystallography for PPAR isoforms can be found in Capelli et al. (2016).

References

Almeida, N.M., Eken, Y. and Wilson, A.K., 2021. Binding of per-and polyfluoro-alkyl substances to peroxisome proliferator-activated receptor gamma. ACS omega, 6(23), pp.15103-15114.

Behr, A.C., Plinsch, C., Braeuning, A. and Buhrke, T., 2020. Activation of human nuclear receptors by perfluoroalkylated substances (PFAS). Toxicology in Vitro, 62, p.104700.

Capelli, D., Cerchia, C., Montanari, R., Loiodice, F., Tortorella, P., Laghezza, A., Cervoni, L., Pochetti, G. and Lavecchia, A., 2016. Structural basis for PPAR partial or full activation revealed by a novel ligand binding mode. Scientific reports, 6(1), p.34792.

Ferré, P., 2004. The biology of peroxisome proliferator-activated receptors: relationship with lipid metabolism and insulin sensitivity. Diabetes, 53(suppl_1), pp.S43-S50.

Grygiel-Górniak, B., 2014. Peroxisome proliferator-activated receptors and their ligands: nutritional and clinical implications-a review. Nutrition journal, 13, pp.1-10.

Gust, K.A., Ji, Q., Luo, X., 2020. Example of Adverse Outcome Pathway Concept Enabling Genome-to-Phenome Discovery in Toxicology. Integr. Comp. Biol. 60, 375-384.

Huang, J., Wang, Q., Liu, S., Lai, H. and Tu, W., 2022. Comparative chronic toxicities of PFOS and its novel alternatives on the immune system associated with intestinal microbiota dysbiosis in adult zebrafish. Journal of Hazardous Materials, 425, p.127950.

Rajapaksha, H., Bhatia, H., Wegener, K., Petrovsky, N. and Bruning, J.B., 2017. X-ray crystal structure of rivoglitazone bound to PPARγ and PPAR subtype selectivity of TZDs. Biochimica et Biophysica Acta (BBA)-General Subjects, 1861(8), pp.1981-1991.

Wang, P., Liu, D., Yan, S., Cui, J., Liang, Y. and Ren, S., 2022b. Adverse effects of perfluorooctane sulfonate on the liver and relevant mechanisms. Toxics, 10(5), p.265.

List of Key Events in the AOP

Event: 2227: Disrupted PPAR isoform nuclear signaling

Short Name: Disrupted PPAR isoform nuclear signaling

Key Event Component

Process	Object	Action
peroxisome proliferator activated receptor signaling pathway		disrupted

AOPs Including This Key Event

AOP ID and Name	Event Type
Aop:529 - Perfluorooctanesulfonic acid (PFOS) binding to peroxisome proliferator-activated receptors (PPARs) causes dysregulation of lipid metabolism and subsequent liver steatosis	KeyEvent

Biological Context

Level of Biological Organization
Molecular

Cell term

Cell term
eukaryotic cell

Organ term

Organ term
liver

Domain of Applicability

Taxonomic Applicability

Term	Scientific Term	Evidence	Links
Vertebrates	Vertebrates	High	NCBI

Life Stage Applicability

Life Stage	Evidence
Embryo	Moderate
Juvenile	High
Adult, reproductively mature	High

Sex Applicability

Sex	Evidence
Male	High
Female	Moderate

Key Event Description

This Key Event describes disruption of PPAR isoform nuclear signaling following the binding of stressor ligands to the PPAR isoforms with either agonist or antagonist interactions. Following binding with an activating ligand, PPAR isoforms heterodimerize with the retinoid X receptor (RXR) with this complex then recognizing the peroxisome proliferator response elements (PPRE) of the PPAR isoform target genes promoting gene expression (Capelli et al. 2016). Therefore, non-native ligands that bind the PPAR isoforms either agonistically or antagonistically can disrupt proper PPAR activity and signaling of either expression or repression of target genes. Results from activity assays, nuclear signaling assays, and transcriptomic analyses using PPAR isoform agonist and antagonist have demonstrate that PPAR ligands directly affect PPAR activity, nuclear signaling, and the transcription of PPAR mediated target genes (Kojo et al. 2003; Behr et al. 2020; Gao et al. 2020; Evans et al. 2022; Murase et al. 2023; Ardenkjær-Skinnerup et al. 2024). Moreover, 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.

Beyond the direct effects of stressor ligands on PPAR isoforms, 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 via stressor chemicals can affect other PPAR isoforms and impact PPAR nuclear signaling.

How it is Measured or Detected

Activity assays, nuclear signaling assays, and transcriptomic or proteomic analyses can identify disrupted nuclear signaling as the result of ligand binding to PPAR isoforms (Kojo et al. 2003; Li et al. 2017; Gao et al. 2020; Murase et al. 2023; Ardenkjær-Skinnerup et al. 2024). These assays can be used to determine if a potential ligand of interest acts as an agonists or antagonists either via direct activity assays or by analysis of gene targets in the PPAR isoform pathways.

References

Ardenkjær-Skinnerup, J., Nissen, A.C.V.E., Nikolov, N.G., Hadrup, N., Ravn-Haren, G., Wedebye, E.B. and Vogel, U., 2024. Orthogonal assay and QSAR modelling of Tox21 PPARγ antagonist in vitro high-throughput screening assay. Environmental Toxicology and Pharmacology, 105, p.104347.

Behr, A.C., Plinsch, C., Braeuning, A. and Buhrke, T., 2020. Activation of human nuclear receptors by perfluoroalkylated substances (PFAS). Toxicology in Vitro, 62, p.104700.

Gao, P., Wang, L., Yang, N., Wen, J., Zhao, M., Su, G., Zhang, J. and Weng, D., 2020. Peroxisome proliferator-activated receptor gamma (PPARγ) activation and metabolism disturbance induced by bisphenol A and its replacement analog bisphenol S using in vitro macrophages and in vivo mouse models. Environment international, 134, p.105328.

Gust, K.A., Ji, Q., Luo, X., 2020. Example of Adverse Outcome Pathway Concept Enabling Genome-to-Phenome Discovery in Toxicology. Integr. Comp. Biol. 60, 375-384.

Kojo, H., Fukagawa, M., Tajima, K., Suzuki, A., Fujimura, T., Aramori, I., Hayashi, K.I. and Nishimura, S., 2003. Evaluation of human peroxisome proliferator-activated receptor (PPAR) subtype selectivity of a variety of anti-inflammatory drugs based on a novel assay for PPARδ (β). Journal of pharmacological sciences, 93(3), pp.347-355.

Li, Y., Liu, X., Niu, L. and Li, Q., 2017. Proteomics analysis reveals an important role for the PPAR signaling pathway in DBDCT-induced hepatotoxicity mechanisms. Molecules, 22(7), p.1113.

Murase, W., Kubota, A., Ikeda-Araki, A., Terasaki, M., Nakagawa, K., Shizu, R., Yoshinari, K. and Kojima, H., 2023. Effects of perfluorooctanoic acid (PFOA) on gene expression profiles via nuclear receptors in HepaRG cells: Comparative study with in vitro transactivation assays. Toxicology, 494, p.153577.

Event: 2224: Dysregulation of transcriptional expression within PPAR signaling network

Short Name: Dysregulation of transcriptional expression within PPAR signaling network

Key Event Component

Process	Object	Action
regulation of gene expression		disrupted

AOPs Including This Key Event

AOP ID and Name	Event Type
Aop:529 - Perfluorooctanesulfonic acid (PFOS) binding to peroxisome proliferator-activated receptors (PPARs) causes dysregulation of lipid metabolism and subsequent liver steatosis	KeyEvent

Biological Context

Level of Biological Organization
Molecular

Cell term

Cell term
eukaryotic cell

Organ term

Organ term
liver

Domain of Applicability

Taxonomic Applicability

Term	Scientific Term	Evidence	Links
Vertebrates	Vertebrates	High	NCBI

Life Stage Applicability

Life Stage	Evidence
Embryo	High
Juvenile	High
Adult, reproductively mature	High

Sex Applicability

Sex	Evidence
Male	High
Female	Moderate

Key Event Description

This Key Event describes dysregulation of PPAR mediated transcriptional expression within the PPAR signaling network following the binding of stressor ligands to the PPAR isoforms with either agonist or antagonist interactions. There is abundant evidence of showing how synthetic ligands can affect transcriptional expression in the PPAR signaling network and of key genes involved in lipid homeostasis (Meierhofer et al. 2014; Li et al. 2020; Cariello et al. 2021; Heintz et al. 2022; Eide et al. 2023; Heintz et al. 2024). 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. 2022; Mylroie et al. IN PREP).

How it is Measured or Detected

Targeted gene expression assays along with “omic” tools such as transcriptomics or proteomics can be used to determine if known or suspected ligands of the PPAR isoforms disrupt gene expression in the PPAR pathway. There are abundant resources available describing methodologies to assess disruption of 1 or more of the PPAR isoform pathways (Meierhofer et al. 2014; Li et al. 2020; Cariello et al. 2021; Mylroie et al. 2021; Heintz et al. 2022; Eide et al. 2023; Heintz et al. 2024).

References

Cariello, M., Piccinin, E. and Moschetta, A., 2021. Transcriptional regulation of metabolic pathways via lipid-sensing nuclear receptors PPARs, FXR, and LXR in NASH. Cellular and molecular gastroenterology and hepatology, 11(5), pp.1519-1539.

de la Rosa Rodriguez, M.A., Sugahara, G., Hooiveld, G.J., Ishida, Y., Tateno, C. and Kersten, S., 2018. The whole transcriptome effects of the PPARα agonist fenofibrate on livers of hepatocyte humanized mice. BMC genomics, 19, pp.1-16.

Eide, M., Goksøyr, A., Yadetie, F., Gilabert, A., Bartosova, Z., Frøysa, H.G., Fallahi, S., Zhang, X., Blaser, N., Jonassen, I. and Bruheim, P., 2023. Integrative omics-analysis of lipid metabolism regulation by peroxisome proliferator-activated receptor a and b agonists in male Atlantic cod. Frontiers in physiology, 14, p.1129089.

Heintz MM, Chappell GA, Thompson CM, Haws LC. Evaluation of transcriptomic responses in livers of mice exposed to the short-chain PFAS compound HFPO-DA. Frontiers in Toxicology. 2022 Jun 27;4:937168.

Li, Y., Zhang, Q., Fang, J., Ma, N., Geng, X., Xu, M., Yang, H. and Jia, X., 2020. Hepatotoxicity study of combined exposure of DEHP and ethanol: A comprehensive analysis of transcriptomics and metabolomics. Food and chemical toxicology, 141, p.111370.

Meierhofer, D., Weidner, C. and Sauer, S., 2014. Integrative analysis of transcriptomics, proteomics, and metabolomics data of white adipose and liver tissue of high-fat diet and rosiglitazone-treated insulin-resistant mice identified pathway alterations and molecular hubs. Journal of proteome research, 13(12), pp.5592-5602.

Wang, Q., Huang, J., Liu, S., Wang, C., Jin, Y., Lai, H. and Tu, W., 2022. Aberrant hepatic lipid metabolism associated with gut microbiota dysbiosis triggers hepatotoxicity of novel PFOS alternatives in adult zebrafish. Environment International, 166, p.107351.

Event: 179: Decrease, Fatty acid beta-oxidation

Short Name: Decrease, Fatty acid β-oxidation

Key Event Component

Process	Object	Action
fatty acid beta-oxidation	fatty acid	decreased

AOPs Including This Key Event

AOP ID and Name	Event Type
Aop:36 - Peroxisomal Fatty Acid Beta-Oxidation Inhibition Leading to Steatosis	KeyEvent
Aop:60 - NR1I2 (Pregnane X Receptor, PXR) activation leading to hepatic steatosis	KeyEvent
Aop:497 - ERa inactivation alters mitochondrial functions and insulin signalling in skeletal muscle and leads to insulin resistance and metabolic syndrome	KeyEvent
Aop:529 - Perfluorooctanesulfonic acid (PFOS) binding to peroxisome proliferator-activated receptors (PPARs) causes dysregulation of lipid metabolism and subsequent liver steatosis	KeyEvent

Biological Context

Level of Biological Organization
Cellular

Domain of Applicability

Taxonomic Applicability

Term	Scientific Term	Evidence	Links
Homo sapiens	Homo sapiens	High	NCBI

See review for Human PPARalpha signaling in (Evans et al 2004).

Key Event Description

Fatty acid oxidation in liver tissue is controlled by PPARalpha signaling networks (Evans et al 2004). The PPARalpha signaling network controls expression of the genes within metabolic pathways that catalyze fatty acid oxidation reactions (Desvergne and Wahli 1999).

How it is Measured or Detected

A variety of approaches establishing the effects of PPARalpha signaling on fatty acid oxidation are reviewed in Evans et al (2004).

References

Desvergne B, Wahli W (1999) Peroxisome proliferator-activated receptors: nuclear control of metabolism. Endocrine Reviews 20(5): 649-688.

Evans RM, Barish GD, Wang YX: PPARs and the complex journey to obesity. Nat Med 2004, 10(4):355-361.

Event: 2225: Disrupted Lipid Storage

Short Name: Disrupted Lipid Storage

Key Event Component

Process	Object	Action
lipid storage		disrupted

AOPs Including This Key Event

AOP ID and Name	Event Type
Aop:529 - Perfluorooctanesulfonic acid (PFOS) binding to peroxisome proliferator-activated receptors (PPARs) causes dysregulation of lipid metabolism and subsequent liver steatosis	KeyEvent

Biological Context

Level of Biological Organization
Cellular

Cell term

Cell term
eukaryotic cell

Organ term

Organ term
liver

Domain of Applicability

Taxonomic Applicability

Term	Scientific Term	Evidence	Links
Vertebrates	Vertebrates	High	NCBI

Life Stage Applicability

Life Stage	Evidence
Embryo	Moderate
Juvenile	High
Adult, reproductively mature	High

Sex Applicability

Sex	Evidence
Male	High
Female	Moderate

Key Event Description

This Key Event describes the disruption of normal lipid storage in liver cells. Disruption of lipid storage and transport can be identified by excess accumulation of fatty acids or other lipids in the liver or altered ratios of expected lipid species which can ultimately lead to liver steatosis (Ipsen et al. 2018). An example of an event that can cause disrupted lipid storage is the binding of stressor ligands to the PPAR isoforms with either agonist or antagonist interactions which can lead to 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 (Patsouris 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).

How it is Measured or Detected

There are numerous methodologies available for measuring disrupted lipid storage in the liver cells. Fatty acids and other lipid species can be measure directly or measured globally using lipidomic methodologies (Wang et al. 2022; Albers et al. 2024), and histopathology can confirm lipid deposits in liver sections (Huck et al. 2018; Wang et al. 2022). Also, targeted or global gene expression analyses can reveal disruptions in key genes responsible for proper lipid storage and transport (Tse et al. 2016; Yi et al. 2019; Louisse et al. 2020).

References

Albers, J., Mylroie, J., Kimble, A., Steward, C., Chapman, K., Wilbanks, M., Perkins, E. and Garcia-Reyero, N., 2024. Per-and Polyfluoroalkyl Substances: Impacts on Morphology, Behavior and Lipid Levels in Zebrafish Embryos. Toxics, 12(3), p.192.

Dixon, E.D., Nardo, A.D., Claudel, T. and Trauner, M., 2021. The role of lipid sensing nuclear receptors (PPARs and LXR) and metabolic lipases in obesity, diabetes and NAFLD. Genes, 12(5), p.645.

Ipsen, D.H., Lykkesfeldt, J. and Tveden-Nyborg, P., 2018. Molecular mechanisms of hepatic lipid accumulation in non-alcoholic fatty liver disease. Cellular and molecular life sciences, 75, pp.3313-3327.

Event: 327: Accumulation, Fatty acid

Short Name: Accumulation, Fatty acid

Key Event Component

Process	Object	Action
	fatty acid	increased

AOPs Including This Key Event

AOP ID and Name	Event Type
Aop:36 - Peroxisomal Fatty Acid Beta-Oxidation Inhibition Leading to Steatosis	KeyEvent
Aop:57 - AhR activation leading to hepatic steatosis	KeyEvent
Aop:58 - NR1I3 (CAR) suppression leading to hepatic steatosis	KeyEvent
Aop:60 - NR1I2 (Pregnane X Receptor, PXR) activation leading to hepatic steatosis	KeyEvent
Aop:529 - Perfluorooctanesulfonic acid (PFOS) binding to peroxisome proliferator-activated receptors (PPARs) causes dysregulation of lipid metabolism and subsequent liver steatosis	KeyEvent

Biological Context

Level of Biological Organization
Organ

Organ term

Organ term
liver

Event: 291: Accumulation, Triglyceride

Short Name: Accumulation, Triglyceride

Key Event Component

Process	Object	Action
	triglyceride	increased

AOPs Including This Key Event

AOP ID and Name	Event Type
Aop:34 - LXR activation leading to hepatic steatosis	KeyEvent
Aop:57 - AhR activation leading to hepatic steatosis	KeyEvent
Aop:318 - Glucocorticoid Receptor activation leading to hepatic steatosis	KeyEvent
Aop:517 - Pregnane X Receptor (PXR) activation leads to liver steatosis	KeyEvent
Aop:518 - Liver X Receptor (LXR) activation leads to liver steatosis	KeyEvent
Aop:529 - Perfluorooctanesulfonic acid (PFOS) binding to peroxisome proliferator-activated receptors (PPARs) causes dysregulation of lipid metabolism and subsequent liver steatosis	KeyEvent
Aop:580 - Mineralocorticoid Receptor Activation Leading to Increased Body Mass Index	KeyEvent

Biological Context

Level of Biological Organization
Cellular

Cell term

Cell term
hepatocyte

Domain of Applicability

Taxonomic Applicability

Term	Scientific Term	Evidence	Links
Vertebrates	Vertebrates	High	NCBI

Life Stage Applicability

Life Stage	Evidence
Adult	High
Juvenile	Moderate

Sex Applicability

Sex	Evidence
Unspecific	High

Life Stage: Older individuals are more likely to manifest this key event (adults > juveniles) due to accumulation of triglycerides.

Sex: Applies to both males and females.

Taxonomic: Appears to be present broadly in vertebrates, with most representative studies in mammals (humans, lab mice, lab rats). Likely pervasive in many animal taxa.

Key Event Description

Triglycerides are important building blocks for a wide variety of compounds found in organisms, with cellular concentrations reflecting the relative rate of influx and efflux, as well as the relative rate of synthesis and breakdown. However, excess accumulation leads to Fatty Liver Cells and steatosis.

In this key event we focus on excessive accumulation of triglycerides in mammalian systems. Four major pathways for triglyceride accumulation are: 1. Increased fatty acid uptake; 2. Increased De Novo FA and Lipid Synthesis; 3. Decreased FA Oxidation; 4. Decreased Lipid Efflux (Angrish et al. 2016). Chemical stressors can increase gene expression of key genes involving these pathways, leading to increased accumulation of triglycerides (Aguayo-Orozco et al. 2018). In addition, excessive dietary compounds of fatty compounds can also increase likelihood of accumulation of triglycerides (Nguyen et al. 2008). Nuclear receptors that have been implicated in causing excessive accumulation of triglycerides leading to steatosis, when overexpressed, include (Mellor et al. 2016): Aryl hydrocarbon receptor (AHR), Constitutive androstane receptor (CAR), Oestrogen receptor (ER), Farnesoid X receptor (FXR), Glucocorticoid receptor (GXR), Liver X receptor (LXR), Peroxisome proliferator-activated receptor (PPAR), Pregnane X receptor (PXR), and Retinoic acid receptor (RAR or RXR).

How it is Measured or Detected

Concentrations of triglycerides, cholesterols, fatty acids, and related compounds are measured biochemically to assess levels in control versus potentially affected individuals; common techniques include high throughput enzymatic analyses, analytical ultracentrifuging, gradient gel electrophoresis, Nuclear Magnetic Resonance, lipidomics, and other direct assessment techniques (Schaefer et al. 2016; Yang and Han 2016). Analysis is often performed to look at gene expression levels to see which pathway(s) have increased expression levels, to attribute plausibility to changes in influx, eflux, synthesis, and/or breakdown pathways (Nguyen et al. 2008; Mellor et al. 2016, Aguayo-Orozco et al. 2018). Assessment of cellular components including mitochondria and membrane integrity can also be used as evidence of alteration of normal function within cells.

References

Aguayo-Orozco, A.A., Bois, F.Y., Brunak, S., and Taboureau, O. 2018. Analysis of Time-Series Gene Expression Data to Explore Mechanisms of Chemical-Induced Hepatic Steatosis Toxicity. Frontiers in Genetics 9(Article 396): 1-15.

Angrish, M.M., Kaiser, J.P., McQueen, C.A., and Chorley, B.N. 2016. Tipping the Balance: Hepatotoxicity and the 4 Apical Key Events of Hepatic Steatosis. Toxicological Sciences 150(2): 261–268.

Mellor, C.L., Steinmetz, F.P., and Cronin, T.D. 2016. The identification of nuclear receptors associated with hepatic steatosis to develop and extend adverse outcome pathways. Critical Reviews in Toxicology, 46(2): 138-152.

Nguyen, P., Leray, V., Diez, M., Serisier, S., Le Bloc’h, J., Siliart, B., and Dumon, H. 2008. Liver lipid metabolism. Journal of Animal Physiology and Animal Nutrition 92: 272–283.

Schaefer EJ, Tsunoda F, Diffenderfer M, Polisecki, E., Thai, N., and Astalos, B. The Measurement of Lipids, Lipoproteins, Apolipoproteins, Fatty Acids, and Sterols, and Next Generation Sequencing for the Diagnosis and Treatment of Lipid Disorders. [Updated 2016 Mar 29]. In: Feingold KR, Anawalt B, Blackman MR, et al., editors. Endotext [Internet]. South Dartmouth (MA): MDText.com, Inc.; 2000-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK355892/

Yang, K. and Han, X. 2016. Lipidomics: Techniques, applications, and outcomes related to biomedical sciences. Trends in Biochemical Sciences 2016 November ; 41(11): 954–969.

NOTE: Italics symbolize edits from John Frisch

List of Adverse Outcomes in this AOP

Event: 459: Increased, Liver Steatosis

Short Name: Increased, Liver Steatosis

Key Event Component

Process	Object	Action
Hepatic steatosis		increased

AOPs Including This Key Event

AOP ID and Name	Event Type
Aop:58 - NR1I3 (CAR) suppression leading to hepatic steatosis	AdverseOutcome
Aop:60 - NR1I2 (Pregnane X Receptor, PXR) activation leading to hepatic steatosis	AdverseOutcome
Aop:61 - NFE2L2/FXR activation leading to hepatic steatosis	AdverseOutcome
Aop:62 - AKT2 activation leading to hepatic steatosis	AdverseOutcome
Aop:36 - Peroxisomal Fatty Acid Beta-Oxidation Inhibition Leading to Steatosis	AdverseOutcome
Aop:213 - Inhibition of fatty acid beta oxidation leading to nonalcoholic steatohepatitis (NASH)	KeyEvent
Aop:285 - Inhibition of N-linked glycosylation leads to liver injury	KeyEvent
Aop:318 - Glucocorticoid Receptor activation leading to hepatic steatosis	AdverseOutcome
Aop:517 - Pregnane X Receptor (PXR) activation leads to liver steatosis	AdverseOutcome
Aop:518 - Liver X Receptor (LXR) activation leads to liver steatosis	AdverseOutcome
Aop:529 - Perfluorooctanesulfonic acid (PFOS) binding to peroxisome proliferator-activated receptors (PPARs) causes dysregulation of lipid metabolism and subsequent liver steatosis	AdverseOutcome
Aop:232 - NFE2/Nrf2 repression to steatosis	AdverseOutcome
Aop:57 - AhR activation leading to hepatic steatosis	AdverseOutcome
Aop:494 - AhR activation leading to liver fibrosis	KeyEvent

Biological Context

Level of Biological Organization
Organ

Organ term

Organ term
liver

Domain of Applicability

Taxonomic Applicability

Term	Scientific Term	Evidence	Links
Vertebrates	Vertebrates	High	NCBI

Life Stage Applicability

Life Stage	Evidence
All life stages	High

Sex Applicability

Sex	Evidence
Unspecific	High

Steatosis is the result of perturbations in well-known metabolic pathways that are well-studied and well-known in many taxa.

Life Stage: The life stage applicable to this key event is all life stages with a liver. Older individuals are more likely to manifest this adverse outcome pathway (adults > juveniles) due to accumulation of triglycerides.

Sex: This key event applies to both males and females.

Taxonomic: This key event appears to be present broadly in vertebrates, with most representative studies in mammals (humans, lab mice, lab rats).

Key Event Description

Biological state: liver steatosis is the inappropriate storage of fat in hepatocytes. Four major pathways for triglyceride accumulation are: 1. Increased fatty acid uptake; 2. Increased De Novo FA and Lipid Synthesis; 3. Decreased FA Oxidation; 4. Decreased Lipid Efflux (Angrish et al. 2016). Chemical stressors can increase gene expression of key genes involving these pathways, leading to increased accumulation of triglycerides (Aguayo-Orozco et al. 2018). In addition, excessive dietary compounds of fatty compounds can also increase likelihood of accumulation of triglycerides (Nguyen et al. 2008).

Biological compartment: steatosis is generally an organ-level diagnosis; however, the pathology occurs within the hepatocytes.

Role in biology: steatosis is an adverse endpoint.

Consequences: Liver steatosis, or fatty liver, serves as a pivotal factor in the development of liver fibrosis by triggering a cascade of pathological events. According to the two-strikes hypothesis (Day and James, 1998), liver damage progresses in two stages: the first strike involves the accumulation of lipids in hepatocytes, often due to metabolic disturbances such as insulin resistance, excess free fatty acids, or oxidative stress. This stage, though asymptomatic, increases liver vulnerability by inducing mild oxidative stress and inflammation. The second strike introduces additional insults, such as inflammatory mediators or cellular damage, exacerbating liver injury and promoting fibrogenesis. The accumulation of fat sensitizes the liver to oxidative stress and triggers mechanisms like the activation of hepatic stellate cells (HSCs) and hepatocyte apoptosis or necrosis, central to the fibrotic process. While early-stage steatosis is reversible, chronic steatosis perpetuates a cycle of inflammation and fibrosis, creating a feedback loop that amplifies liver damage (Pafili K et al, 2021). Consequently, liver steatosis is not only a precursor but also a critical driver of fibrosis progression.

Day CP, James OF. Steatohepatitis: a tale of two "hits"? Gastroenterology. 1998 Apr;114(4):842-5. doi: 10.1016/s0016-5085(98)70599-2. PMID: 9547102.

Pafili K, Roden M. Nonalcoholic fatty liver disease (NAFLD) from pathogenesis to treatment concepts in humans. Mol Metab. 2021 Aug;50:101122. doi: 10.1016/j.molmet.2020.101122. Epub 2020 Nov 19. PMID: 33220492; PMCID: PMC8324683.

Description from EU-ToxRisk:

Activation of stellate cells results in collagen accumulation and change in extracellular matrix composition in the liver causing fibrosis. (Landesmann, 2016; Koo et al 2016)

How it is Measured or Detected

Steatosis is measured by lipidomics approaches (e.g. Yang and Han 2016) that measure lipid levels, or by histology. Concentrations of triglycerides, cholesterols, fatty acids, and related compounds are measured biochemically include high throughput enzymatic analyses, analytical ultracentrifuging, gradient gel electrophoresis, Nuclear Magnetic Resonance, and other direct assessment techniques (Schaefer et al. 2016).

Regulatory Significance of the AO

Steatosis is a regulatory endpoint and has been used as an endpoint in many US EPA assessments, including IRIS assessments.

References

Angrish, M.M., Kaiser, J.P., McQueen, C.A., and Chorley, B.N. 2016. Tipping the Balance: Hepatotoxicity and the 4 Apical Key Events of Hepatic Steatosis. Toxicological Sciences 150(2): 261–268.

Day CP, James OF. Steatohepatitis: a tale of two "hits"? Gastroenterology. 1998 Apr;114(4):842-5. doi: 10.1016/s0016-5085(98)70599-2. PMID: 9547102.

Landesmann, B. (2016). Adverse Outcome Pathway on Protein Alkylation Leading to Liver Fibrosis, (2).

https://doi.org/10.1016/j.molcel.2005.08.010

Koo, J. H., Lee, H. J., Kim, W., & Kim, S. G. (2016). Endoplasmic Reticulum Stress in Hepatic Stellate Cells Promotes Liver Fibrosis via PERK-Mediated Degradation of HNRNPA1 and Up-regulation of SMAD2. Gastroenterology, 150(1), 181–193.e8. https://doi.org/10.1053/j.gastro.2015.09.039

Nguyen, P., Leray, V., Diez, M., Serisier, S., Le Bloc’h, J., Siliart, B., and Dumon, H. 2008. Liver lipid metabolism. Journal of Animal Physiology and Animal Nutrition 92: 272–283.

Yang, K. and Han, X. 2016. Lipidomics: Techniques, applications, and outcomes related to biomedical sciences. Trends in Biochemical Sciences 2016 November ; 41(11): 954–969.

NOTE: Italics symbolize edits from John Frisch

Appendix 2 List of Key Event Relationships in the AOP

List of Adjacent Key Event Relationships

Relationship: 3220: Binding PPAR isoforms leads to Disrupted PPAR isoform nuclear signaling

AOPs Referencing Relationship

AOP Name	Adjacency	Weight of Evidence	Quantitative Understanding
Perfluorooctanesulfonic acid (PFOS) binding to peroxisome proliferator-activated receptors (PPARs) causes dysregulation of lipid metabolism and subsequent liver steatosis	adjacent	High	Moderate

Evidence Supporting Applicability of this Relationship

Taxonomic Applicability

Term	Scientific Term	Evidence	Links
Vertebrates	Vertebrates	High	NCBI

Life Stage Applicability

Life Stage	Evidence
Embryo	Moderate
Juvenile	High
Adult, reproductively mature	High

Sex Applicability

Sex	Evidence
Male	High
Female	Moderate

Key Event Relationship Description

Both natural and synthetic ligands can interact with all 3 main PPAR isoforms (α, β/δ, γ) with unsaturated fatty acids and other lipid-derived molecules being the primary natural ligands the PPAR isoforms (Ferré 2004). Numerous studies have shown the ability of synthetic ligands to bind the ligand binding domains of the PPAR isoforms (Ferré 2004; Grygiel-Górniak 2014). This Key Event Relationship describes the binding of stressor ligands to the PPAR isoforms with either agonist or antagonist interactions which then disrupts downstream PPAR isoform nuclear signaling. The ligands that bind the PPAR isoforms either agonistically or antagonistically can disrupt proper PPAR activity and nuclear signaling for the either expression or repression of target genes.

Evidence Supporting this KER

Biological Plausibility

Natural and synthetic ligands can interact with all 3 main PPAR isoforms (α, β/δ, γ) with unsaturated fatty acids and other lipid-derived molecules being the primary natural ligands the PPAR isoforms (Ferré 2004). Following binding with an activating ligand, PPAR isoforms heterodimerize with the retinoid X receptor (RXR) with this complex then recognizing the peroxisome proliferator response elements (PPRE) of the PPAR isoform target genes and promoting gene expression (Capelli et al. 2016). Therefore, ligands that act either agonistically or antagonistically beyond or more persistently than the normal biological range can disrupt proper nuclear signaling and subsequent gene expression.

Empirical Evidence

Synthetic ligands can be PPAR isoform specific whereas others, like bezafibrate, can bind and activate all 3 main PPAR isoforms (Grygiel-Górniak 2014). 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. 2022; Wang et al. 2022a, Wang et al. 2022b; Kowalska et al. 2023).

Results from activity assays, nuclear signaling assays, and transcriptomic analyses using PPAR isoform agonist and antagonist have demonstrate that PPAR ligands directly affect PPAR activity, nuclear signaling, and the transcription of PPAR mediated target genes (Kojo et al. 2003; Behr et al. 2020; Gao et al. 2020; Evans et al. 2022; Murase et al. 2023; Ardenkjær-Skinnerup et al. 2024). Moreover, 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.

Beyond the direct effects of stressor ligands on PPAR isoforms, 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; Kim et al. 2023), 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 via stressor chemicals can affect other PPAR isoforms and impact PPAR nuclear signaling.

Uncertainties and Inconsistencies

Quantitative Understanding of the Linkage

Response-response relationship

Unknown.

Time-scale

Rapid Molecular Interactions.

Known Feedforward/Feedback loops influencing this KER

As PPAR signaling is essential for maintaining energy homeostasis, there is a complex network of feedforward/feedback loops influencing PPAR nuclear signaling via ligands, products, and the PPAR isoforms acting on each other. Due to extensive detail needed to properly describe all potential feedforward/feedback loops that could influence this KER, the authors direct readers to reviews by Ament et al. (2012) and Lamichane et al. (2018).

References

Almeida, N.M., Eken, Y. and Wilson, A.K., 2021. Binding of per-and polyfluoro-alkyl substances to peroxisome proliferator-activated receptor gamma. ACS omega, 6(23), pp.15103-15114.

Ament, Z., Masoodi, M. and Griffin, J.L., 2012. Applications of metabolomics for understanding the action of peroxisome proliferator-activated receptors (PPARs) in diabetes, obesity and cancer. Genome Medicine, 4, pp.1-12.

Behr, A.C., Plinsch, C., Braeuning, A. and Buhrke, T., 2020. Activation of human nuclear receptors by perfluoroalkylated substances (PFAS). Toxicology in Vitro, 62, p.104700.

Ferré, P., 2004. The biology of peroxisome proliferator-activated receptors: relationship with lipid metabolism and insulin sensitivity. Diabetes, 53(suppl_1), pp.S43-S50.

Grygiel-Górniak, B., 2014. Peroxisome proliferator-activated receptors and their ligands: nutritional and clinical implications-a review. Nutrition journal, 13, pp.1-10.

Gust, K.A., Ji, Q., Luo, X., 2020. Example of Adverse Outcome Pathway Concept Enabling Genome-to-Phenome Discovery in Toxicology. Integr. Comp. Biol. 60, 375-384.

Kim, I.S., Silwal, P. and Jo, E.K., 2023. Peroxisome proliferator-activated receptor-targeted therapies: challenges upon infectious diseases. Cells, 12(4), p.650.

Lamichane, S., Dahal Lamichane, B. and Kwon, S.M., 2018. Pivotal roles of peroxisome proliferator-activated receptors (PPARs) and their signal cascade for cellular and whole-body energy homeostasis. International journal of molecular sciences, 19(4), p.949.

Li, Y., Liu, X., Niu, L. and Li, Q., 2017. Proteomics analysis reveals an important role for the PPAR signaling pathway in DBDCT-induced hepatotoxicity mechanisms. Molecules, 22(7), p.1113.

Wang, P., Liu, D., Yan, S., Cui, J., Liang, Y. and Ren, S., 2022b. Adverse effects of perfluorooctane sulfonate on the liver and relevant mechanisms. Toxics, 10(5), p.265.

Relationship: 3221: Disrupted PPAR isoform nuclear signaling leads to Dysregulation of transcriptional expression within PPAR signaling network

AOPs Referencing Relationship

AOP Name	Adjacency	Weight of Evidence	Quantitative Understanding
Perfluorooctanesulfonic acid (PFOS) binding to peroxisome proliferator-activated receptors (PPARs) causes dysregulation of lipid metabolism and subsequent liver steatosis	adjacent	High	Moderate

Evidence Supporting Applicability of this Relationship

Taxonomic Applicability

Term	Scientific Term	Evidence	Links
Vertebrates	Vertebrates	High	NCBI

Life Stage Applicability

Life Stage	Evidence
Embryo	Moderate
Juvenile	High
Adult, reproductively mature	High

Sex Applicability

Sex	Evidence
Male	High
Female	Moderate

Key Event Relationship Description

This Key Event Relationship describes how the disruption of PPAR isoform nuclear signaling affects transcriptional expression within the PPAR signaling network. The ligands that bind the PPAR isoforms either agonistically or antagonistically can disrupt proper PPAR activity and nuclear signaling for the either expression or repression of target genes in the PPAR signaling network.

Evidence Supporting this KER

Biological Plausibility

Following binding with an activating ligand, PPAR isoforms heterodimerize with the retinoid X receptor (RXR) with this complex then recognizing the peroxisome proliferator response elements (PPRE) of the PPAR isoform target genes and promoting gene expression (Capelli et al. 2016). Therefore, ligands that act either agonistically or antagonistically beyond or more persistently than the normal biological range can disrupt proper nuclear signaling and subsequent gene expression in the PPAR signaling pathway.

Empirical Evidence

Beyond the direct effects of stressor ligands on PPAR isoforms, 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; Kim et al. 2023), 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 via stressor chemicals can affect other PPAR isoforms and impact PPAR nuclear signaling.

Dysregulation of gene expression follows disrupted nuclear signaling as can be seen from abundant evidence of showing how synthetic ligands can affect transcriptional expression in the PPAR signaling network and of key genes involved in lipid homeostasis (Meierhofer et al. 2014; Li et al. 2020; Cariello et al. 2021; Heintz et al. 2022; Eide et al. 2023; Heintz et al. 2024). 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. 2022; Mylroie et al. IN PREP).

Uncertainties and Inconsistencies

While the PPAR molecular structure and function among vertebrates is largely conserved (Gust et al 2020), species to species variation does exist in structure and specific function; and therefore, it is important to exercise care when looking to extrapolate across species. The binding affinity of certain ligands and the magnitude of response in PPAR nuclear signaling may differ from species to species due to variations in PPAR molecular structure. Furthermore, the direction and magnitude of gene expression response may differ from species to species or even within species depending on the ligand assayed and the concentration used.

Quantitative Understanding of the Linkage

Response-response relationship

Unknown

Time-scale

Rapid Molecular Interactions

Known Feedforward/Feedback loops influencing this KER

As PPAR signaling is essential for maintaining energy homeostasis, there is a complex network of feedforward/feedback loops influencing PPAR nuclear signaling and gene expression via ligands, products, and the PPAR isoforms acting on each other. Due to the extensive detail needed to properly describe all potential feedforward/feedback loops that could influence this KER, the authors direct readers to reviews by Ament et al. (2012) and Lamichane et al. (2018).

References

Behr, A.C., Plinsch, C., Braeuning, A. and Buhrke, T., 2020. Activation of human nuclear receptors by perfluoroalkylated substances (PFAS). Toxicology in Vitro, 62, p.104700.

Gust, K.A., Ji, Q., Luo, X., 2020. Example of Adverse Outcome Pathway Concept Enabling Genome-to-Phenome Discovery in Toxicology. Integr. Comp. Biol. 60, 375-384.

Heintz MM, Chappell GA, Thompson CM, Haws LC. Evaluation of transcriptomic responses in livers of mice exposed to the short-chain PFAS compound HFPO-DA. Frontiers in Toxicology. 2022 Jun 27;4:937168.

Li, Y., Liu, X., Niu, L. and Li, Q., 2017. Proteomics analysis reveals an important role for the PPAR signaling pathway in DBDCT-induced hepatotoxicity mechanisms. Molecules, 22(7), p.1113.

Relationship: 3224: Dysregulation of transcriptional expression within PPAR signaling network leads to Disrupted Lipid Storage

AOPs Referencing Relationship

AOP Name	Adjacency	Weight of Evidence	Quantitative Understanding
Perfluorooctanesulfonic acid (PFOS) binding to peroxisome proliferator-activated receptors (PPARs) causes dysregulation of lipid metabolism and subsequent liver steatosis	adjacent	High	Moderate

Evidence Supporting Applicability of this Relationship

Taxonomic Applicability

Term	Scientific Term	Evidence	Links
Vertebrates	Vertebrates	High	NCBI

Life Stage Applicability

Life Stage	Evidence
Embryo	Moderate
Juvenile	High
Adult, reproductively mature	High

Sex Applicability

Sex	Evidence
Male	High
Female	Moderate

Key Event Relationship Description

This Key Event Relationship describes how the dysregulation of transcriptional expression within the PPAR signaling network results in disrupted lipid storage, specifically in liver cells. All 3 PPAR isoforms and the genes they regulate are essential for proper lipid storage and transport; and therefore, dysregulation in the expression profiles of any or all of the PPAR isoform controlled signaling networks can disrupt the proper storage of lipids in cells (Ament et al. 2012; Dixon et al. 2021; Xiao et al. 2021).

Evidence Supporting this KER

Biological Plausibility

Empirical Evidence

When a stressor ligand binds to the PPAR isoforms with either agonist or antagonist interactions which can lead to 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 (Patsouris 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. 2022). 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. (2022) saw a trend of decreased transcriptional expression of genes involved in lipid synthesis in zebrafish in response to PFOS; whereas Yi et al. (2019) 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. 2022). 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 (Rosen et al. 2010; Jacobsen et al. 2018; Sant et al. 2018; Mylroie et al. 2021; Wang et al. 2022). 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.

Uncertainties and Inconsistencies

Quantitative Understanding of the Linkage

Response-response relationship

Unknown

Time-scale

Rapid Molecular Interactions

Known Feedforward/Feedback loops influencing this KER

References

Carr, R.M. and Ahima, R.S., 2016. Pathophysiology of lipid droplet proteins in liver diseases. Experimental cell research, 340(2), pp.187-192.

Heintz MM, Chappell GA, Thompson CM, Haws LC. Evaluation of transcriptomic responses in livers of mice exposed to the short-chain PFAS compound HFPO-DA. Frontiers in Toxicology. 2022 Jun 27;4:937168.

Xiao, Y., Kim, M. and Lazar, M.A., 2021. Nuclear receptors and transcriptional regulation in non-alcoholic fatty liver disease. Molecular Metabolism, 50, p.101119.

Relationship: 3223: Dysregulation of transcriptional expression within PPAR signaling network leads to Decrease, Fatty acid β-oxidation

AOPs Referencing Relationship

AOP Name	Adjacency	Weight of Evidence	Quantitative Understanding
Perfluorooctanesulfonic acid (PFOS) binding to peroxisome proliferator-activated receptors (PPARs) causes dysregulation of lipid metabolism and subsequent liver steatosis	adjacent	High	Moderate

Evidence Supporting Applicability of this Relationship

Taxonomic Applicability

Term	Scientific Term	Evidence	Links
Vertebrates	Vertebrates	High	NCBI

Life Stage Applicability

Life Stage	Evidence
Embryo	Moderate
Juvenile	High
Adult, reproductively mature	High

Sex Applicability

Sex	Evidence
Male	High
Female	Moderate

The conservation of PPAR molecular structure and function among vertebrates (Gust et al 2020) indicates this key event is likely to be conserved among this broad phylogenetic group. Furthermore, PPAR isoforms play a crucial role in lipid metabolism and β-oxidation across representative vertebrate species. However, given that species to species variation does exist in structure and specific function, it is important to exercise care when looking to extrapolate across species.

Key Event Relationship Description

This Key Event Relationship describes how the dysregulation of transcriptional expression within the PPAR signaling network results in disrupted β-oxidation and specifically cause a decrease in β-oxidation. All 3 PPAR isoforms and the genes they regulate are essential for proper energy homeostasis of which β-oxidation is a key component; and therefore, dysregulation in the expression profiles of any or all of the PPAR isoform controlled signaling networks can disrupt fatty acid β-oxidation in cells (Ament et al. 2012; Liu et al. 2020; Dixon et al. 2021; Xiao et al. 2021).

Evidence Supporting this KER

Biological Plausibility

Ligands that act either agonistically or antagonistically beyond or more persistently than the normal biological range can disrupt proper nuclear signaling and subsequent gene expression in the PPAR signaling pathway. The complex control of lipid metabolism means dysregulation of gene expression in the PPAR signaling network can have a disruptive effect on β-oxidation (Dixon et al. 2021; Xiao et al. 2021) as 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; Badman 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 and PPARγ activation promotes lipid storage and decreases fatty acid β-oxidation (Reddy 2001; Roberts et al. 2011).

Empirical Evidence

Dysregulation of gene expression follows disrupted nuclear signaling as can be seen from abundant evidence of showing how synthetic ligands can affect transcriptional expression in the PPAR signaling network and of key genes involved in lipid homeostasis (Meierhofer et al. 2014; Li et al. 2020; Cariello et al. 2021; Heintz et al. 2022; Eide et al. 2023; Heintz et al. 2024). Specifically, pathway and gene ontology (GO) enrichment analyses have identified lipid metabolism, lipid transport, fatty acid β-oxidation, PPAR signaling pathway, and lipid homeostasis as being transcriptionally altered in response to PFAS exposure (Chen et al. 2014; Tse et al. 2016; Cui et al. 2017; Huck et al. 2018; Jacobsen et al. 2018; Rodríguez-Jorquera et al. 2018; Liu et al. 2019; Martinez et al. 2019; Christou et al. 2020; Louisse 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. 2022; Mylroie et al. IN PREP).

The proper control of mitochondrial β -oxidation is reliant on PPAR induced transcription of the enzymes integral to carrying out fatty acid oxidation (Fan and Evans 2015; Hong et al. 2019). Agonist of PPARα increase gene expression of genes involved in mitochondrial fatty acid β -oxidation (Bougarne et al. 2018) whereas PPARα null mice have a decreased expression of fatty acid oxidation genes with the same being seen in PPARβ/δ knockouts (Wang 2010).

Stressors can impact the expression of genes involved in β -oxidation. 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 (Rosen et al. 2010) after exposure to PFAS. 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. (2022) 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).

Uncertainties and Inconsistencies

Quantitative Understanding of the Linkage

Response-response relationship

Unknown

Time-scale

Rapid Molecular Interactions

Known Feedforward/Feedback loops influencing this KER

References

Bougarne, N., Weyers, B., Desmet, S.J., Deckers, J., Ray, D.W., Staels, B. and De Bosscher, K., 2018. Molecular actions of PPAR α in lipid metabolism and inflammation. Endocrine reviews, 39(5), pp.760-802.

Carr, R.M. and Ahima, R.S., 2016. Pathophysiology of lipid droplet proteins in liver diseases. Experimental cell research, 340(2), pp.187-192.

Dixon, E.D., Nardo, A.D., Claudel, T. and Trauner, M., 2021. The role of lipid sensing nuclear receptors (PPARs and LXR) and metabolic lipases in obesity, diabetes and NAFLD. Genes, 12(5), p.645.

Fan, W. and Evans, R., 2015. PPARs and ERRs: molecular mediators of mitochondrial metabolism. Current opinion in cell biology, 33, pp.49-54.

Heintz MM, Chappell GA, Thompson CM, Haws LC. Evaluation of transcriptomic responses in livers of mice exposed to the short-chain PFAS compound HFPO-DA. Frontiers in Toxicology. 2022 Jun 27;4:937168.

Hong, F., Pan, S., Guo, Y., Xu, P. and Zhai, Y., 2019. PPARs as nuclear receptors for nutrient and energy metabolism. Molecules, 24(14), p.2545.

Liu, Z., Ding, J., McMillen, T.S., Villet, O., Tian, R. and Shao, D., 2020. Enhancing fatty acid oxidation negatively regulates PPARs signaling in the heart. Journal of molecular and cellular cardiology, 146, pp.1-11.

Reddy, J.K., 2001. III. Peroxisomal β-oxidation, PPARα, and steatohepatitis. American Journal of Physiology-Gastrointestinal and Liver Physiology, 281(6), pp.G1333-G1339.

Wang, Y.X., 2010. PPARs: diverse regulators in energy metabolism and metabolic diseases. Cell research, 20(2), pp.124-137.

Xiao, Y., Kim, M. and Lazar, M.A., 2021. Nuclear receptors and transcriptional regulation in non-alcoholic fatty liver disease. Molecular Metabolism, 50, p.101119.

Relationship: 3209: Decrease, Fatty acid β-oxidation leads to Disrupted Lipid Storage

AOPs Referencing Relationship

AOP Name	Adjacency	Weight of Evidence	Quantitative Understanding
Perfluorooctanesulfonic acid (PFOS) binding to peroxisome proliferator-activated receptors (PPARs) causes dysregulation of lipid metabolism and subsequent liver steatosis	adjacent	Moderate	Moderate

Evidence Supporting Applicability of this Relationship

Taxonomic Applicability

Term	Scientific Term	Evidence	Links
Vertebrates	Vertebrates	High	NCBI

Life Stage Applicability

Life Stage	Evidence
Embryo	Moderate
Juvenile	High
Adult, reproductively mature	High

Sex Applicability

Sex	Evidence
Male	High
Female	Moderate

β-oxidation is a crucial biological function maintained across representative vertebrate species. However, given that species to species variation does exist in gene sequences and enzyme specific structures; therefore, it is important to exercise care when looking to extrapolate across species.

Key Event Relationship Description

This Key Event Relationship describes how a decrease in β-oxidation can disrupt proper lipid storage. Disruption of lipid storage and transport can be identified by excess accumulation of fatty acids or other lipids in the liver or altered ratios of expected lipid species which can ultimately lead to liver steatosis (Ipsen et al. 2018). Decreased or impaired mitochondrial β-oxidation has been linked to the accumulation of lipids and potentially liver steatosis (Cherkaoui-Malki et al. 2012; Fromenty 2019).

Evidence Supporting this KER

Biological Plausibility

Mitochondrial fatty acid β-oxidation is an important biochemical mechanism that is vital in maintaining energy homeostasis in the liver (Houten and Wanders 2010; Naguib et al. 2019). It is important in whole organism energy production during fasting but also serves as the main mechanism for fatty acid degradation and removal (Houten and Wanders 2010; Cherkaoui-Malki et al. 2012; Naguib et al. 2019). When fatty acid β-oxidation is decreased in the liver, lipids are not able to be eliminated as efficiently and can to begin to accumulate in the liver (Fromenty 2019; He et al. 2019; Naguib et al. 2019). Therefore, a decrease in or complete inhibition of mitochondrial fatty acid β-oxidation can result in disrupted lipid storage in the liver.

Empirical Evidence

There is ample evidence showing how the decrease or inhibition of mitochondrial fatty acid β-oxidation can cause disrupted lipid storage in the liver. Fromenty et al. (2019) present a comprehensive review of multiple examples of drug-induced inhibition of mitochondrial fatty acid β-oxidation disruptions in lipid liver storage resulting in steatosis. Specifically, drugs such as acetaminophen, linezolid, and traglitazone that decrease or inhibit fatty acid β-oxidation causes triglycerides to accumulate as small or large droplets in liver tissue. He et al. (2019) showed that cadmium (Cd) exposure in mice inhibited mitochondrial fatty acid oxidation via a suppression of SIRT1 and PPARα signaling resulting in excess lipid accumulation in the liver. Finally, Massart et al. (2019) presented multiple modes of actions for drug-induced inhibition of mitochondrial fatty acid oxidation and disrupted lipid storage in the liver with direct inhibition of mitochondrial fatty acid β-oxidation and disruptions of PPARα activity as two pathways for disruption of fatty acid β-oxidation.

Uncertainties and Inconsistencies

Energy homeostasis is a complex system in vertebrates and controlled via the cross-talk of numerous pathways. Therefore, it is important to understand that factors like age, sex, and the fed state of the organism could all have a direct effect on lipid storage and subsequent fatty acid accumulation in the liver of the target organism.

Quantitative Understanding of the Linkage

Response-response relationship

Unknown

Time-scale

Hours to days.

Known Feedforward/Feedback loops influencing this KER

Mitochondrial fatty acid β-oxidation is a well-studied biological process integral to energy homeostasis. The feedforward/feedback loops involved in regulating mitochondrial fatty acid β-oxidation are extensive and present a challenge to properly represent in this KER summary. The authors suggest reading the reviews by Houten and Wanders (2010) and Morris et al. (2011) for a comprehensive summary of feedforward/feedback loops influencing this KER.

References

Fromenty, B., 2019. Inhibition of mitochondrial fatty acid oxidation in drug-induced hepatic steatosis. Liver Research, 3(3-4), pp.157-169.

He, X., Gao, J., Hou, H., Qi, Z., Chen, H. and Zhang, X.X., 2019. Inhibition of mitochondrial fatty acid oxidation contributes to development of nonalcoholic fatty liver disease induced by environmental cadmium exposure. Environmental science & technology, 53(23), pp.13992-14000.

Houten, S.M. and Wanders, R.J., 2010. A general introduction to the biochemistry of mitochondrial fatty acid β-oxidation. Journal of inherited metabolic disease, 33, pp.469-477.

Massart, J., Begriche, K., Buron, N., Porceddu, M., Borgne-Sanchez, A. and Fromenty, B., 2013. Drug-induced inhibition of mitochondrial fatty acid oxidation and steatosis. Current Pathobiology Reports, 1, pp.147-157.

Morris, E.M., Rector, R.S., Thyfault, J.P. and Ibdah, J.A., 2011. Mitochondria and redox signaling in steatohepatitis.

Naguib, G., Morris, N., Yang, S., Fryzek, N., Haynes‐Williams, V., Huang, W.C.A., Norman‐Wheeler, J. and Rotman, Y., 2020. Dietary fatty acid oxidation is decreased in non‐alcoholic fatty liver disease: A palmitate breath test study. Liver International, 40(3), pp.590-597.

Relationship: 3210: Disrupted Lipid Storage leads to Accumulation, Fatty acid

AOPs Referencing Relationship

AOP Name	Adjacency	Weight of Evidence	Quantitative Understanding
Perfluorooctanesulfonic acid (PFOS) binding to peroxisome proliferator-activated receptors (PPARs) causes dysregulation of lipid metabolism and subsequent liver steatosis	adjacent	Moderate	Moderate

Evidence Supporting Applicability of this Relationship

Taxonomic Applicability

Term	Scientific Term	Evidence	Links
Vertebrates	Vertebrates	Moderate	NCBI

Life Stage Applicability

Life Stage	Evidence
Embryo	Moderate
Juvenile	Moderate
Adult, reproductively mature	Moderate

Sex Applicability

Sex	Evidence
Male	Moderate
Female	Moderate

Lipid storage and transport is a crucial biological function maintained across representative vertebrate species. However, given that species to species variation in genes and specific regulatory mechanisms do exist it is important to exercise care when looking to extrapolate across species.

Key Event Relationship Description

This Key Event Relationship describes how disrupted lipid storage in the liver results in the accumulation of fatty acids. Disruption of lipid storage and transport can be identified by excess accumulation of fatty acids or other lipids in the liver or altered ratios of expected lipid species which can ultimately lead to liver steatosis (Ipsen et al. 2018). Disruption of lipid metabolism through dysregulation of transcriptional control and/or decreased or impaired mitochondrial β-oxidation can result in improper lipid storage and an accumulation of fatty acids in liver cells (Ament et al. 2012; Cherkaoui-Malki et al. 2012; Fromenty 2019; Dixon et al. 2021; Xiao et al. 2021).

Evidence Supporting this KER

Biological Plausibility

Empirical Evidence

There is ample evidence outlining how improper lipid storage and transport can result in the accumulation of fatty acids in the liver (Ipsen et al. 2018). For example, overexpression of a fatty acid transport gene CD36 in mice increased fatty acid uptake and accumulation in livers (Koonen et al. 2007). The over expression of human hepatic lipase (hHL) in mice resulted in increased de novo synthesis of fatty acids and upregulation of fatty acid synthesis genes such as Srebf1, Fasn, Acaca, and Nr1h3 (Cedó et al. 2017). Finally, overexpression of sterol regulatory element–binding proteins (SREBP), which is one of the key regulatory elements in lipid synthesis, resulted in an increase in fatty acid synthesis and fatty acid synthase (Fas) gene expression in mouse livers (Horton et al. 2002).

Uncertainties and Inconsistencies

Quantitative Understanding of the Linkage

Response-response relationship

Unknown

Time-scale

Hours to Days

Known Feedforward/Feedback loops influencing this KER

Lipid homeostasis is a well-studied biological process integral to vertebrates and invertebrates. The feedforward/feedback loops involved in regulating lipid storage and transport are extensive and present a challenge to properly represent in this KER summary. The authors suggest reading the reviews by Ipsen et al. (2018) and Geng et al. (2021) for comprehensive summaries of feedforward/feedback loops influencing this KER.

References

Cedó, L., Santos, D., Roglans, N., Julve, J., Pallarès, V., Rivas-Urbina, A., Llorente-Cortes, V., Laguna, J.C., Blanco-Vaca, F. and Escola-Gil, J.C., 2017. Human hepatic lipase overexpression in mice induces hepatic steatosis and obesity through promoting hepatic lipogenesis and white adipose tissue lipolysis and fatty acid uptake. PLoS One, 12(12), p.e0189834.

Dixon, E.D., Nardo, A.D., Claudel, T. and Trauner, M., 2021. The role of lipid sensing nuclear receptors (PPARs and LXR) and metabolic lipases in obesity, diabetes and NAFLD. Genes, 12(5), p.645.

Fromenty, B., 2019. Inhibition of mitochondrial fatty acid oxidation in drug-induced hepatic steatosis. Liver Research, 3(3-4), pp.157-169.

Geng, Y., Faber, K.N., de Meijer, V.E., Blokzijl, H. and Moshage, H., 2021. How does hepatic lipid accumulation lead to lipotoxicity in non-alcoholic fatty liver disease?. Hepatology international, 15, pp.21-35.

Horton, J.D., Goldstein, J.L. and Brown, M.S., 2002. SREBPs: activators of the complete program of cholesterol and fatty acid synthesis in the liver. The Journal of clinical investigation, 109(9), pp.1125-1131.

Houten, S.M. and Wanders, R.J., 2010. A general introduction to the biochemistry of mitochondrial fatty acid β-oxidation. Journal of inherited metabolic disease, 33, pp.469-477.

Koonen, D.P., Jacobs, R.L., Febbraio, M., Young, M.E., Soltys, C.L.M., Ong, H., Vance, D.E. and Dyck, J.R., 2007. Increased hepatic CD36 expression contributes to dyslipidemia associated with diet-induced obesity. diabetes, 56(12), pp.2863-2871.

Morris, E.M., Rector, R.S., Thyfault, J.P. and Ibdah, J.A., 2011. Mitochondria and redox signaling in steatohepatitis.

Xiao, Y., Kim, M. and Lazar, M.A., 2021. Nuclear receptors and transcriptional regulation in non-alcoholic fatty liver disease. Molecular Metabolism, 50, p.101119.

Yoon, H., Shaw, J.L., Haigis, M.C. and Greka, A., 2021. Lipid metabolism in sickness and in health: Emerging regulators of lipotoxicity. Molecular cell, 81(18), pp.3708-3730.

Relationship: 472: Accumulation, Fatty acid leads to Accumulation, Triglyceride

AOPs Referencing Relationship

AOP Name	Adjacency	Weight of Evidence	Quantitative Understanding
Perfluorooctanesulfonic acid (PFOS) binding to peroxisome proliferator-activated receptors (PPARs) causes dysregulation of lipid metabolism and subsequent liver steatosis	adjacent	Moderate	Moderate

Evidence Supporting Applicability of this Relationship

Taxonomic Applicability

Term	Scientific Term	Evidence	Links
Vertebrates	Vertebrates	Moderate	NCBI

Life Stage Applicability

Life Stage	Evidence
Embryo	Moderate
Juvenile	Moderate
Adult, reproductively mature	Moderate

Sex Applicability

Sex	Evidence
Male	Moderate
Female	Moderate

Key Event Relationship Description

This Key Event Relationship describes how the accumulation of fatty acids in the liver results in an increase in and accumulation of triglycerides (TG) in the liver. Disruption of lipid storage and transport can be identified by excess accumulation of fatty acids followed by an accumulation of triglycerides and other lipids which can ultimately lead to liver steatosis (Ipsen et al. 2018).

Evidence Supporting this KER

Biological Plausibility

Proper lipid homeostasis is controlled by the balance of lipid influx and efflux as well as the balance between lipogenesis and lipid catabolism (Ipsen et al. 2018; Kloska et al. 2020; Geng et al. 2021; Yoon et al. 2021). Disruption of this balance via diet, disease, or environmental stressor can lead to the improper storage and transport of lipids in the liver and the subsequent accumulation of fatty acids (Ipsen et al. 2018). When an excess of accumulation of fatty acid occurs in the liver via increased import, de novo synthesis, and/or reduced β-oxidation TG synthesis increases for storage and export and to also protect cells from lipotoxicity under periods of extremely high free fatty acid accumulation (Listenberger 2003; Reddy and Rao 2006; Rada et al. 2020). Therefore, it is plausible to assume that an increase in fatty acid accumulation would lead to an increase in TG accumulation especially under conditions of greater lipid homeostasis perturbation due to a stressor.

Empirical Evidence

There is ample evidence outlining how accumulation of fatty acids in the liver results in an increased accumulation of triglycerides (Reddy and Rao 2006; Angrish et al. 2016; Ipsen et al. 2018). For example, overexpression of sterol regulatory element–binding proteins (SREBP), which is one of the key regulatory elements in lipid synthesis, resulted in an increase in fatty acid synthesis and an accumulation of TG species in the liver (Horton et al. 2002). Selen et al. (2021) demonstrate that mice with a KO in a key gene involved in β-oxidation showed increased fatty acid accumulation and increased TG content when fed a high-fate diet. Finally, Koonen et al. (2007) showed that overexpression of CD36 in mice resulted in an influx of fatty acids and increased triglyceride levels.

Uncertainties and Inconsistencies

Energy homeostasis is a complex system in vertebrates and controlled via the cross-talk of numerous pathways. Therefore, it is important to understand that factors like age, sex, and the fed state of the organism could all have a direct effect on fatty acid accumulation and subsequent triglyceride accumulation in the liver of the target organism/species.

Quantitative Understanding of the Linkage

Response-response relationship

Unknown

Time-scale

Hours to Days

Known Feedforward/Feedback loops influencing this KER

References

Angrish, M.M., Kaiser, J.P., McQueen, C.A. and Chorley, B.N., 2016. Tipping the balance: hepatotoxicity and the 4 apical key events of hepatic steatosis. Toxicological Sciences, 150(2), pp.261-268.

Kloska, A., Węsierska, M., Malinowska, M., Gabig-Cimińska, M. and Jakóbkiewicz-Banecka, J., 2020. Lipophagy and lipolysis status in lipid storage and lipid metabolism diseases. International journal of molecular sciences, 21(17), p.6113.

Listenberger, L.L., Han, X., Lewis, S.E., Cases, S., Farese Jr, R.V., Ory, D.S. and Schaffer, J.E., 2003. Triglyceride accumulation protects against fatty acid-induced lipotoxicity. Proceedings of the National Academy of Sciences, 100(6), pp.3077-3082.

Rada, P., González-Rodríguez, Á., García-Monzón, C. and Valverde, Á.M., 2020. Understanding lipotoxicity in NAFLD pathogenesis: is CD36 a key driver?. Cell death & disease, 11(9), p.802.

Reddy, J.K. and Sambasiva Rao, M., 2006. Lipid metabolism and liver inflammation. II. Fatty liver disease and fatty acid oxidation. American Journal of Physiology-Gastrointestinal and Liver Physiology, 290(5), pp.G852-G858.

Selen, E.S., Choi, J. and Wolfgang, M.J., 2021. Discordant hepatic fatty acid oxidation and triglyceride hydrolysis leads to liver disease. JCI insight, 6(2).

Yoon, H., Shaw, J.L., Haigis, M.C. and Greka, A., 2021. Lipid metabolism in sickness and in health: Emerging regulators of lipotoxicity. Molecular cell, 81(18), pp.3708-3730.

Relationship: 2265: Accumulation, Triglyceride leads to Increased, Liver Steatosis

AOPs Referencing Relationship

AOP Name	Adjacency	Weight of Evidence	Quantitative Understanding
Glucocorticoid Receptor activation leading to hepatic steatosis	adjacent
Pregnane X Receptor (PXR) activation leads to liver steatosis	adjacent	High	Not Specified
Liver X Receptor (LXR) activation leads to liver steatosis	adjacent	High	Not Specified
Perfluorooctanesulfonic acid (PFOS) binding to peroxisome proliferator-activated receptors (PPARs) causes dysregulation of lipid metabolism and subsequent liver steatosis	adjacent	High	Moderate
AhR activation leading to hepatic steatosis	adjacent	High	High

Evidence Supporting Applicability of this Relationship

Taxonomic Applicability

Term	Scientific Term	Evidence	Links
Homo sapiens	Homo sapiens	Moderate	NCBI
Mus musculus	Mus musculus	Moderate	NCBI

Life Stage Applicability

Life Stage	Evidence
Adult	High
Juvenile	Moderate

Sex Applicability

Sex	Evidence
Unspecific	Moderate

Life Stage: All life stages with a liver. Older individuals are more likely to manifest this adverse outcome pathway (adults > juveniles) due to accumulation of triglycerides.

Sex: Applies to both males and females.

Taxonomic: Appears to be present broadly in vertebrates, with most representative studies in mammals (humans, lab mice, lab rats).

Key Event Relationship Description

Steatosis is a key event representing increased accumulation of fat in liver cells. In this key event relationship we are focused on accumulation of triglycerides leading to steatosis. Increased accumulation of triglycerides in cells is evidence of imbalance in the influx and synthesis versus metabolism or breakdown of lipid compounds. Increased accumulation of triglycerides can be enhanced by chemical stressors, or alteration of regulation by gene expression.

Evidence Supporting this KER

Biological Plausibility

The biological plausibility linking accumulation of triglycerides to steatosis is strong. Increased accumulation of triglycerides represents an imbalanced influx and synthesis of compounds versus normal function, resulting in liver steatosis.

Empirical Evidence

Species	Duration	Dose	Accumulated triglycerides?	Liver steatosis	Summary	Citation
Human (Homo sapiens)	14 days	In vitro exposure of 20 mM amiodarone, 50 mM tetracycline.	yes	yes	HepG2 human cells showed correlated increases in triglycerides and other lipid compounds and steatosis oxidation after 14 days of tetracycline exposure and after both 1 and 14 days of amiodarone exposure.	Antherieu et al. (2011)
Human (Homo sapiens)	24 hours	In vitro exposure of at least 6 concentrations to 28 compounds selected for steatogenic potential.	yes	yes	HepG2 human cells exposed to fialuridine, sodium valproate, doxycycline, amiodarone, tetracycline showed corresponding increases in lipid accumulation, with higher doses exhibiting greater lipid accumulation and correlated steatosis.	Donato et al. (2009)
Human (Homo sapiens) and mouse (Mus musculus)	16 weeks	Transgenic and wild-type mice with normal and high cholesterol diet.	yes	yes	Human subjects with liver steatosis had increased RBP4 gene expression. Transgenic mice with human RBP4 gene had correlated increases in triglycerides associated with steatosis, in comparison to wild-type mice.	Liu et al. (2016)

References

References
Antherieu, S., Rogue, A., Fromenty, B., Guillouzo, A., and Robin, M.-A. 2011. Induction of Vesicular Steatosis by Amiodarone and Tetracycline Is Associated with Up-regulation of Lipogenic Genes in HepaRG Cells. Hepatology 53:1895-1905.

Donato, M.T., Martinez-Romero, A. Jimenez, N., Negro, A., Gerrerad, G., Castell, J.V., O’Connor, J.-E., and Gomez-Lechon, M.J. 2009. Cytometric analysis for drug-induced steatosis in HepG2 cells. Chemico-Biological Interactions 181: 417–423.

Landesmann, B., Goumenou, M., Munn, S., and Whelan, M. 2012. Description of Prototype Modes-of-Action Related to Repeated Dose Toxicity. European Commission Report EUR 25631, 49 pages. https://op.europa.eu/en/publication-detail/-/publication/d2b09726-8267-42de-8093-8c8981201d65/language-en

Liu, Y., Mu, D., Chen, H., Li, D., Song, J., Zhong, Y., and Xia, M. 2016. Retinol-Binding Protein 4 Induces Hepatic Mitochondrial Dysfunction and Promotes Hepatic Steatosis. The Journal of Clinical Endocrinology and Metabolism 101: 4338–4348.

Negi, C.K., Bajard, L., Kohoutek, J., and Blaha, L. 2021. An adverse outcome pathway based in vitro characterization of novel flame retardants-induced hepatic steatosis. Environmental Pollution 289: 117855.

List of Non Adjacent Key Event Relationships

Relationship: 3211: Disrupted Lipid Storage leads to Accumulation, Triglyceride

AOPs Referencing Relationship

AOP Name	Adjacency	Weight of Evidence	Quantitative Understanding
Perfluorooctanesulfonic acid (PFOS) binding to peroxisome proliferator-activated receptors (PPARs) causes dysregulation of lipid metabolism and subsequent liver steatosis	non-adjacent	Moderate	Moderate

Evidence Supporting Applicability of this Relationship

Taxonomic Applicability

Term	Scientific Term	Evidence	Links
Vertebrates	Vertebrates	Moderate	NCBI

Life Stage Applicability

Life Stage	Evidence
Embryo	Moderate
Juvenile	Moderate
Adult, reproductively mature	Moderate

Sex Applicability

Sex	Evidence
Male	Moderate
Female	Moderate

Key Event Relationship Description

This Key Event Relationship describes how disrupted lipid storage in the liver results in the accumulation of triglycerides. Disruption of lipid storage and transport can be identified by excess accumulation of triglycerides or other lipids in the liver or altered ratios of expected lipid species which can ultimately lead to liver steatosis (Alves-Bezerra and Cohen 2017; Ipsen et al. 2018; Dixon et al. 2021).

Evidence Supporting this KER

Biological Plausibility

Empirical Evidence

There is ample evidence outlining how improper lipid storage and transport can result in the accumulation of TG in the liver (Ipsen et al. 2018). For example, overexpression of diacylglycerol acyltransferases (DGAT2) in the liver resulted in increased levels of TG in mice livers (Monetti et al. 2007). Disruption of G3P acyltransferase (GPAT) enzymes in the liver, which is necessary for maintaining the balance between lipid storage and fatty acid oxidation, can result in increased TG levels in hepatocytes (Lewin et al. 2005; Alves-Bezerra and Cohen). Impaired secretion of TG as TG-enriched very low-density lipoprotein (VLDL) can result in increased TG accumulation in the liver. This connection has been demonstrated via inhibition of microsomal triglyceride transfer protein (MTP), which is critical for proper TG-VLDL packing and export, which was shown to increase TG content in liver in mice where expression was inhibited (Josekutty et al. 2013). Finally, lipid droplets (LD) are TG are stored temporarily in the liver for use in fatty acid oxidation; and thus, a disruption in regulation of the formation of LD can thus result in accumulation of TG in the liver (Alves-Bezerra and Cohen 2017). Perilipin proteins (PLIN) are critical for formation of LD and Trevino et al. (2015) demonstrated that overexpression of PLIN5 resulted an increase of TG and other lipids in mouse livers.

Uncertainties and Inconsistencies

Quantitative Understanding of the Linkage

Response-response relationship

Unknown

Time-scale

Hours to Days

Known Feedforward/Feedback loops influencing this KER

Lipid homeostasis is a well-studied biological process integral to vertebrates and invertebrates. The feedforward/feedback loops involved in regulating lipid storage and transport are extensive and present a challenge to properly represent in this KER summary. The authors suggest reading the reviews by Alves-Bezerra and Cohen (2017) and Ipsen et al. (2018) for comprehensive summaries of feedforward/feedback loops influencing this KER.

References

Alves-Bezerra, M. and Cohen, D.E., 2017. Triglyceride metabolism in the liver. Comprehensive physiology, 8(1), p.1.

Josekutty, J., Iqbal, J., Iwawaki, T., Kohno, K. and Hussain, M.M., 2013. Microsomal triglyceride transfer protein inhibition induces endoplasmic reticulum stress and increases gene transcription via Ire1α/cJun to enhance plasma ALT/AST. Journal of Biological Chemistry, 288(20), pp.14372-14383.

Lewin, T.M., Wang, S., Nagle, C.A., Van Horn, C.G. and Coleman, R.A., 2005. Mitochondrial glycerol-3-phosphate acyltransferase-1 directs the metabolic fate of exogenous fatty acids in hepatocytes. American Journal of Physiology-Endocrinology and Metabolism, 288(5), pp.E835-E844.

Monetti, M., Levin, M.C., Watt, M.J., Sajan, M.P., Marmor, S., Hubbard, B.K., Stevens, R.D., Bain, J.R., Newgard, C.B., Farese, R.V. and Hevener, A.L., 2007. Dissociation of hepatic steatosis and insulin resistance in mice overexpressing DGAT in the liver. Cell metabolism, 6(1), pp.69-78.

Trevino, M.B., Mazur-Hart, D., Machida, Y., King, T., Nadler, J., Galkina, E.V., Poddar, A., Dutta, S. and Imai, Y., 2015. Liver perilipin 5 expression worsens hepatosteatosis but not insulin resistance in high fat-fed mice. Molecular endocrinology, 29(10), pp.1414-1425.

Yoon, H., Shaw, J.L., Haigis, M.C. and Greka, A., 2021. Lipid metabolism in sickness and in health: Emerging regulators of lipotoxicity. Molecular cell, 81(18), pp.3708-3730.