<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Of the originating work: Brigitte Landesmann, Marina Goumenou, Sharon Munn and Maurice Whelan, Joint Research Centre, European Commission, Ispra, Italy</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Of the content populated in the AOP-Wiki: John R. Frisch and Travis Karschnik, General Dynamics Information Technology, Duluth, Minnesota; Daniel L. Villeneuve, US Environmental Protection Agency, Great Lakes Toxicology and Ecology Division, Duluth, MN</span></span></p>
<td>Under development: Not open for comment. Do not cite</td>
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<div id="abstract">
<h2>Abstract</h2>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Liver X receptor (LXR) belongs to a class of nuclear receptors [Arhyl hydrocarbon receptor (AHR), Constitutive androstane receptor (CAR), Oestrogen receptor (ER), Farnesoid X receptor (FXR), Glucocorticoid receptor (GR), Peroxisome proliferator-activated receptor (PPAR), Pregnane X receptor (PXR), Retinoic acid receptor (RAR)] that are needed for normal liver function, but for which increased expression (i.e. activation by binding by chemical stressors) lead to liver injury, including steatosis (Mellor <em>et al</em>. 1996). An increasing number of chemical stressors have been shown to increase LXR expression (Moya <em>et al</em>. 2020). Activation of LXR has been linked to increased expression of a group of genes (ChREBP, SREBP-1c, FAS and SCD1) involved in increasing <em>de novo</em> fatty acid synthesis (Mellor <em>et al</em>. 1996, Schultz <em>et al</em>. 2000, Postic and Girard 2008). Increases in <em>de novo</em> fatty acid synthesis is one of the main pathways for increases in triglycerides in livers (Angrish <em>et al.</em> 2016). Increases in triglycerides can result in decreased mitochondrial biochemical function or histological changes in mitochondria structure, ultimately resulting in steatosis as a primary adverse outcome (Angrish <em>et al.</em> 2016; Mellor <em>et al</em>. 1996).</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Liver X receptor (LXR) belongs to a class of nuclear receptors [Arhyl hydrocarbon receptor (AHR), Constitutive androstane receptor (CAR), Oestrogen receptor (ER), Farnesoid X receptor (FXR), Glucocorticoid receptor (GR), Peroxisome proliferator-activated receptor (PPAR), Pregnane X receptor (PXR), Retinoic acid receptor (RAR)] that are needed for normal liver function, but for which increased activaton (i.e. activation by binding by chemical stressors) can lead to liver injury, including steatosis (Mellor <em>et al</em>. 1996). An increasing number of chemical stressors have been shown to increase LXR activation (Moya <em>et al</em>. 2020). Activation of LXR has been linked to increased expression of a group of genes (ChREBP, SREBP-1c, FAS and SCD1) involved in increasing <em>de novo</em> fatty acid synthesis (Mellor <em>et al</em>. 1996, Schultz <em>et al</em>. 2000, Postic and Girard 2008). Increases in <em>de novo</em> fatty acid synthesis is one of the main pathways for increases in triglycerides in livers (Angrish <em>et al.</em> 2016). Increases in triglycerides can result in histological changes to cell structure and disruption of normal biochemical function, ultimately resulting in steatosis as a primary adverse outcome (Angrish et al. 2016; Mellor et al. 1996).</span></span></p>
</div>
<div id="background">
<h3>Background</h3>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">This Adverse Outcome Pathway (AOP) focuses on the pathway in which activation of Liver X receptor (LXR) leads to liver steatosis through increased <em>de novo</em> fatty acid synthesis. Environmental stressors result in activation of nuclear receptors linked to increases in triglyceride accumulation through several pathways. One of the primary pathways linked to triglyceride accumulation, and focus of this AOP, is through activation of the LXR gene and coordinated molecular responses leading to increased fatty acid synthesis. This pathway has been particular well studied in mammals (humans, lab mice, lab rats).</span></span></p>
<h2>AOP Development Strategy</h2>
<div id="context">
<h3>Context</h3>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">This Adverse Outcome Pathway (AOP) was developed as part of an Environmental Protection Agency effort to represent putative AOPs from peer-reviewed literature which were heretofore unrepresented in the AOP-Wiki. The originating work for this AOP was: <em>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</em></span></span><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"> This publication, and the work cited within, were used create and support this AOP and its respective KE and KER pages.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Flame retardants are of environmental and human health concern because of increased use and ability to leach into the environment. Exposure concerns include effects on reproduction, development, neurology, and endocrine pathways (Negi et al. 2021). This AOP focuses on a subset of endocrine disruption related to loss of lipid homeostasis, specifically the pathway in which activation of Liver X Receptor (LXR) leads to liver steatosis through increased <em>de novo</em> fatty acid synthesis. Environmental stressors result in activation of nuclear receptors linked to increases in triglyceride accumulation through several pathways. One of the primary pathways linked to triglyceride accumulation, and focus of this AOP, is through activation of the LXR gene and coordinated molecular responses leading to increased<em> de novo</em> fatty acid synthesis. This pathway has been particularly well studied in mammals (humans, lab mice, lab rats).</span></span></p>
<p><br />
<span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">The focus of the originating work was to use an AOP framework to integrate lines of evidence from multiple disciplines based on evolving guidance developed by the Organization for Economic Cooperation and Development (OECD). Landesmann et al. (2012) provided initial network analysis based on literature review of empirical studies with focus on pathways leading to liver steatosis. The authors then used the AOP framework to identify a pathway: 1. originating with LXR activation; 2. intermediate steps increased gene expression of ChREBP, SREBP-1c, FAS, and SCD1; 3. increased de novo fatty acid synthesis; 4. liver triglyceride accumulation; 5. organelle, cellular, and tissue steps leading to steatosis.</span></span><br />
</p>
</div>
</div>
<div id="development_strategy">
<h3>Strategy</h3>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">The originating authors conducted a literature search to develop a database of publications categorized by discipline or field of study: toxicology, epidemiology, exposure, and gene-environment interaction. The literature search relied on standard search engines such as Web of Science and Google Scholar, and the search strategy focused on toxicants known to disrupt lipid pathways in organisms, and diet studies with elevated levels of lipids. The originating authors reviewed references from individual citations to identify additional studies not captured through the literature search itself. They then included all relevant publications through 2023. Only studies focused primarily on developmental or neurotoxic endpoints were included; those focused on carcinogenesis or other systemic effects were not included unless there was a particular relevance to a neurotoxic or developmental outcome.</span></span></p>
<p><br />
<span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">The scope of the aforementioned EPA project was limited to re-representing the AOP(s) as presented in the originating publication. The literature used to support this AOP and its constituent pages began with the originating publication and followed to the primary, secondary, and tertiary works cited therein. KE and KER page creation and re-use was determined using Handbook principles where page re-use was preferred. </span></span></p>
<span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">The authors of AOP 518 also referred to AOP 34: LXR activation leading to hepatic steatosis by Marina Goumenou, coauthor of Landesmann et al. (2012). In contrast to AOP 34, we have condensed the number of key events leading to de novo fatty acid synthesis. We recognize that there is a complex interaction of genes within organisms, and focus attention on the role of upregulation of genes linked to increased LXR expression, leading to increased de novo fatty acid synthesis.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">1. Support for Biological Plausibility of Key Event Relationships: Is there a mechanistic relationship between KEup and KEdown consistent with established biological knowledge?</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Level of Support </span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Strong = Extensive understanding of the KER based on extensive previous documentation and broad acceptance.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>Moderate support.</strong> The relationship between activation of Liver X receptor and genes linked to regulation of <em>de novo</em> fatty acid synthesis is broadly accepted and consistently supported across taxa.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>Strong support.</strong> The relationship between activation of Liver X receptor and genes linked to regulation of <em>de novo</em> fatty acid synthesis is broadly accepted and consistently supported across taxa.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Relationship 3104: Increased, Expression of LXR activated genes leads to Synthesis, De Novo FA</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Relationship 3104: Increased, Expression of LXR activated genes leads to Synthesis, De Novo Fatty Acid (FA)</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Relationship 110: Synthesis, De Novo FA leads to Accumulation, Triglyceride </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Relationship 110: Synthesis, De Novo Fatty Acid (FA) leads to Accumulation, Triglyceride </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>Strong support. </strong>Increased <em>de novo</em> fatty acid synthesis is broadly recognized as a major pathway leading to accumulation of triglycerides, and consistently supported across taxa. </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>Strong support.</strong> The relationship between accumulation of triglycerides and liver steatosis is broadly accepted and consistently supported across taxa.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>Strong support</strong>. Extensive understanding of the relationships between events from empirical studies from a variety of taxa, including frequent testing in lab mammals.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Life Stage: The life stage applicable to this AOP 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.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Sex: This AOP applies to both males and females.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Taxonomic: This AOP appears to be present broadly in vertebrates, with most representative studies in mammals (humans, lab mice, lab rats).</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">2. Essentiality of Key Events: Are downstream KEs and/or the AO prevented if an upstream KE is blocked?</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Level of Support</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Strong = Direct evidence from specifically designed experimental studies illustrating essentiality and direct relationship between key events.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Moderate = Indirect evidence from experimental studies inferring essentiality of relationship between key events due to difficulty in directly measuring at least one of key events.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>Moderate support.</strong> Activation of Liver X receptor is a primary activator for increases in genes linked to regulation of <em>de novo</em> fatty acid synthesis. However, expression of these genes can be elicited by other nuclear receptors and molecular processes.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>Strong support.</strong> Activation of Liver X receptor is a primary activator for increases in genes linked to regulation of <em>de novo</em> fatty acid synthesis. However, expression of these genes can be elicited by other nuclear receptors and molecular processes.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>Moderate support. </strong>Increased, expression of LXR activated genes is one pathway linked to increases in <em>de novo</em> fatty acid synthesis. However, a variety of molecular signals and corresponding cellular changes are required in order for <em>de novo</em> fatty acid synthesis to increase.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>Strong support. </strong>Increased, expression of LXR activated genes is one pathway linked to increases in <em>de novo</em> fatty acid synthesis. However, a variety of molecular signals and corresponding cellular changes are required in order for <em>de novo</em> fatty acid synthesis to increase.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>Moderate support. </strong>Increase in <em>de novo</em> fatty acid synthesis is a primary factor in increased triglyceride levels in cells. However, triglycerides increase in cells via a number of pathways, including increased triglyceride influx into cells.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>Strong support. </strong>Accumulation of triglyceride is linked to liver steatosis. Evidence is available from toxicant, gene-knockout, and high lipid diet studies.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>Strong support.</strong> Liver steatosis occurs due to a variety of stressors and breakdown of multiple biochemical pathways and physiological changes with resulting increases in triglyceride levels. Evidence is available from toxicant and high lipid diet studies.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">3.<span style="background-color:#d0cece"> Empirical Support for Key Event Relationship: Does empirical evidence support that a change in KEup leads to an appropriate change in KEdown?</span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Level of Support </span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Strong = Experimental evidence from exposure to toxicant shows consistent change in both events across taxa and study conditions. </span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>Moderate support. </strong>Increases in Liver X receptor expression lead to increases in genes linked to regulation of <em>de novo</em> fatty acid synthesis, primarily from studies examining TOXCAST data, as well as changes in gene expression levels after exposure to chemical stressors.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>Strong support. </strong>Increases in Liver X receptor expression lead to increases in genes linked to regulation of <em>de novo</em> fatty acid synthesis, primarily from studies examining TOXCAST data, as well as changes in gene expression levels after exposure to chemical stressors.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Relationship 3104: Increased, Expression of LXR activated genes leads to Synthesis, De Novo FA</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Relationship 3104: Increased, Expression of LXR activated genes leads to Synthesis, De Novo Fatty Acid (FA)</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>Weak support.</strong> Increases in expression of LXR activated genes lead to increases in <em>de novo</em> fatty acid synthesis, primarily through measured increases in gene expression and increased triglyceride levels. Increased <em>de novo</em> fatty acid synthesis is inferred from increased triglyceride levels rather than directly observed.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>Strong support.</strong> Increases in expression of LXR activated genes lead to increases in <em>de novo</em> fatty acid synthesis, primarily through measured increases in gene expression and increased triglyceride levels. Increased <em>de novo</em> fatty acid synthesis is inferred from increased triglyceride levels rather than directly observed.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Relationship 110: Synthesis, De Novo FA leads to Accumulation, Triglyceride </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Relationship 110: Synthesis, De Novo Fatty Acid (FA) leads to Accumulation, Triglyceride </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>Strong support.</strong> Increases in <em>de novo</em> fatty acid synthesis is recognized as a primary pathway to accumulation of triglycerides.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>Strong support</strong>. Increases in accumulation of triglyceride is recognized as a primary pathway to liver steatosis.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>Strong support</strong>. Exposure from empirical studies shows consistent change in both events from a variety of taxa, including frequent testing in lab mammals.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>Strong support</strong>. Evidence from empirical studies shows consistent change in both events from a variety of taxa, including frequent testing in lab mammals.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:#444444">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.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:#444444">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. </span><a href="https://op.europa.eu/en/publication-detail/-/publication/d2b09726-8267-42de-8093-8c8981201d65/language-en" style="color:blue; text-decoration:underline">https://op.europa.eu/en/publication-detail/-/publication/d2b09726-8267-42de-8093-8c8981201d65/language-en</a></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:#444444">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.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:#444444">Moya, M., Gomez-Lechon, M.J., Castell, J.V., and Jovera, R. 2010. Enhanced steatosis by nuclear receptor ligands: A study in cultured human hepatocytes and hepatoma cells with a characterized nuclear receptor expression profile. Chemico-Biological Interactions 184: 376–387.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:#444444">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. </span><a href="https://op.europa.eu/en/publication-detail/-/publication/d2b09726-8267-42de-8093-8c8981201d65/language-en" style="color:blue; text-decoration:underline">https://op.europa.eu/en/publication-detail/-/publication/d2b09726-8267-42de-8093-8c8981201d65/language-en</a></span></span></p>
<p><span style="font-size:14px">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.</span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:#444444">Postic, C. and Girard, J. 2008. Contribution of de novo fatty acid synthesis to hepatic steatosis and insulin resistance: lessons from genetically engineered mice. The Journal of Clinical Investigation 118(3): 829-838.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:#444444">Schultz, J.R., Tu, H., Luk, A., Repa, J.J., Media, J.C., Li, L., Schwendner, S., Wang, S., Thoolen, M., Mangelsdorf, D.J., Lustig, K.D., and Shan, B. 2000. Role of LXRs in control of lipogenesis. Genes and Development 14:2831–2838.</span></span></span></p>
<p><em>Life Stage: Older individuals are more likely to manifest this adverse outcome pathway (adults > juveniles) due to increased opportunity to upregulate gene expression.</em></p>
<p><em>Sex: Applies to both males and females.</em></p>
<p><em>Taxonomic: Appears to be present broadly in vertebrates, with most representative studies in mammals (humans, lab mice, lab rats).</em></p>
<h4>Key Event Description</h4>
<h3>The LXR receptor</h3>
<p>Liver X receptors are ligand-activated transcription factors of the nuclear receptor superfamily first identified in 1994 in rat liver (Apfel et al. 1994, Song 1994). There are two LXR isoforms termed a and ß (NR1H3 and NR1H2) which upon activation form heterodimers with retinoid X receptor (RXR) and bind to the LXR response element found in the promoter region of the target genes (Baranowski 2008). LXRs were shown to function as sterol sensors protecting the cells from cholesterol overload by stimulating reverse cholesterol transport and activating its conversion to bile acids in the liver (Baranowski 2008).</p>
<p>Liver X receptors (LXR) are ligand-activated transcription factors of the nuclear receptor superfamily first identified in 1994 in rat liver (Apfel et al. 1994, Song 1994). There are two LXR isoforms termed a and ß (NR1H3 and NR1H2) which upon activation form heterodimers with retinoid X receptor (RXR) and bind to the LXR response element found in the promoter region of the target genes (Baranowski 2008). LXRs were shown to function as sterol sensors protecting the cells from cholesterol overload by stimulating reverse cholesterol transport and activating its conversion to bile acids in the liver (Baranowski 2008).</p>
<p>LXRa expression is restricted to liver, kidney, intestine, fat tissue, macrophages, lung, and spleen and is highest in liver, hence the name liver X receptor a (LXRa). LXRβ is expressed in almost all tissues and organs, hence the early name UR (ubiquitous receptor) (Ory 2004). The different pattern of expression suggests that LXRa and LXRβ have different roles in regulating physiological function. This is also supported from the observation that LXRa deficient mice do not develop hepatic steatosis when treated with LXR agonist that activates both types (Lund et al. 2006) and consequently the role of the two isoforms in relation to adverse effects could be different.</p>
<p> </p>
<h3>The molecular initiating event</h3>
<p>Generally speaking chemicals that are able to act through NRs are usually specific ligands. These chemicals are mainly lipophilic and they mimic the action of natural hormones. However, in some cases hydrophilic chemicals (like phthalates) are also capable to act as ligands in NRs due to the molecular structure of the proteins and the pocket sites of the receptors.</p>
<p>The molecular initiating event in the presented MoA is the binding to the LXR or the permissive RXR of the LXR-RXR dimer leading to activation. LXR activation can be achieved via a wide range of endogenous neutral and acidic ligands as shown by crystallographic analysis (Williams et al. 2003). There are known endogenous but also synthetic ligands that can act as agonists. Endogenous agonists for this receptor are the oxysterols (oxidized cholesterol derivatives like 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, 27-hydroxycholesterol, and cholestenoic acid) mainly with similar affinity for the two isoforms (Baranowski 2008). Oxysterols bind directly to the typical hydrophobic pocket in the C-terminal domain (Williams et al. 2003). Other endogenous ligands are the D-glucose and D-Glucose-6-phosphate (Mitro 2007). However, the hydrophilic nature of glucose and its low affinity for LXR present a challenge to the central dogma about the nature of the NR-ligand interaction (Lazar & Wilson 2007). Unsaturated fatty acids have also been shown to bind and regulate LXRa activity in cells. However, in contrast to the role of oxysterols, the biological relevance of this observation has not been established in vivo (Pawar et al. 2003). The function of LXRs is also modulated by many currently used drugs such as statins, fibrates, and thazolidinedione derivatives (Jamroz-Wiśniewska et al. 2007). Some synthetic LXR agonists have been developed like the non-steroidal agonists T0901317 and GW3965 (Schultz et al 2000, Collins et al. 2002). LXR forms a permissive dimer with the RXR which means that chemicals that can activate this receptor can trigger the same pathway as the LXR agonists. The endogenous RXR agonist is 9-cis-retinoic acid (Heyman et al. 1992) while synthetic agonists include LGD1069 and LG100268 (Boehm et al. 1994 and 1995).</p>
<p>In addition to the agonist binding in the LXR there are other mechanisms for its control. LXRa gene promoter contains also functional peroxisome proliferator response element (PPRE) and peroxisome proliferator-activated receptor (PPAR) a and γ agonists were shown to stimulate LXRa expression in human and rodent (Baranowski 2008). Control of the LXRa expression is also dependent on insulin and post-translationally by protein kinase A that phosphorylates receptor protein at two sites thereby impairing its dimerization and DNA-binding (Baranowski 2008).</p>
<p> </p>
<h3>Identification of the site of action</h3>
<p>As already mentioned above LXR isoforms are expressed in various tissues but in relation to the presented MoA we refer to LXRs that are expressed in the hepatocytes.</p>
<p>Nuclear receptors may be classified into two broad classes according to their sub-cellular distribution in the absence of ligand. Type I NRs (like ER and AhR) are located in the cytosol (and they are translocated into the nucleus after ligand binding) while type II NRs like LXRs (but also PXR, PPARa and PPARγ) are located in the nucleus of the cell.</p>
<p>The specific site of binding and the affinity of a ligand for the LXRs depend on the structure of the ligand.</p>
<p> </p>
<h3>Binding in the LXREs and target genes transcription</h3>
<p>Upon ligand-induced activation both isoforms form obligate heterodimers with the retinoid X receptor (RXR) and regulate gene expression through binding to LXR response elements (LXREs) in the promoter regions of the target genes (Fig. 1). The LXRE consists of two idealized hexanucleotide sequences (AGGTCA) separated by four bases (DR-4 element).</p>
Figure 1. Mechanism of transcriptional regulation mediated by LXRs. RXR - retinoid X receptor, LXRE - LXR response element (Baranowski 2008)</p>
<p>Target genes of LXRs are involved in cholesterol and lipid metabolism regulation (<sup><a href="#cite_note-1">[1]</a></sup>, <sup><a href="#cite_note-2">[2]</a></sup>) including:</p>
<ul>
<li>ABC - ATP Binding Cassette transporter isoforms A1, G1, G5, and G8</li>
<p>Human specific auto-regulated expression specifically of the LXRa has been demonstrated from several studies (Laffitte et al. 2001, Whitney et al. 2001, Li et al. 2002, Kase et al. 2007). Human LXRa gene promoter has a functional LXRE activated by both LXRa and β. In addition human liver LXRa expression is induced by both natural and synthetic LXR agonists.</p>
<h4>How it is Measured or Detected</h4>
<p><em>Liver X receptor (LXR) activation is measured by changes in gene expression and protein levels. Effects of LXR on expression of downstream genes can be investigating using metabolomics and RT-qPCR approaches. In addition, targeted ToxCast assays using SeqAPASS evaluations can evaluate gene expression changes from chemical exposure for model species (e.g. Lalone et al. 2018). Relevent ToxCast assays are ATG_LXRa_TRANS; ATG_LXRb_TRANS; ATG_DR4_LXR_CIS (U.S. EPA 2024).</em></p>
<h4>References</h4>
<ol>
<li><a href="#cite_ref-1">↑</a> Peet 1998 - Peet D.J., Cholesterol and Bile Acid Metabolism Are Impaired in Mice Lacking the<br />
Nuclear Oxysterol Receptor LXRa in mammals, Cell, 93, 693–704, 1998</li>
<li><a href="#cite_ref-2">↑</a> Edwardsa et al. 2002 - Edwardsa P.A., et al, LXRs; Oxysterol-activated nuclear receptors that regulate genes<br />
controlling lipid homeostasis, (Oxidized Lipids as Potential Mediators of<br />
Atherosclerosis), Vascular Pharmacology, 38 (No 4), 249–256, 2002</li>
<li><em>LaLone, C.A., Villeneuve, D.L., Doering, J.A., Blackwell, B.R., Transue, T.R., Simmons, C.W., Swintek, J., Degitz, S.J., Williams, A.J., and Ankley, G.T. 2018. Evidence for Cross Species Extrapolation of Mammalian-Based High-Throughput Screening Assay Results. Environmental Science and Technology 52: 13960−13971.</em></li>
<li><em>U.S. EPA. 2024. ToxCast & Tox21 Summary Files from invitrodb_v4. Retrieved from https://www.epa.gov/chemical-research/toxicity-forecaster-toxcasttm-data. </em></li>
</ol>
<p><em>NOTE: Italics symbolize edits from John Frisch</em></p>
<h3>List of Key Events in the AOP</h3>
<h4><a href="/events/2199">Event: 2199: Increased, Expression of LXR activated genes</a></h4>
<h5>Short Name: Increased, Expression of LXR activated genes</h5>
<p>Life Stage: Older individuals are more likely to manifest this adverse outcome pathway (adults > juveniles) due to increased opportunity to upregulate gene expression.</p>
<p>Sex: Applies to both males and females.</p>
<p>Taxonomic: Appears to be present broadly in vertebrates, with most representative studies in mammals (humans, lab mice, lab rats).</p>
<h4><a href="/events/89">Event: 89: Synthesis, De Novo FA</a></h4>
<h5>Short Name: Synthesis, De Novo FA</h5>
<h4>Key Event Component</h4>
<h4>Key Event Description</h4>
<p>Liver X receptor (LXR) gene expression activate a suite of genes responsible for de novo fatty acid synthesis. These genes include: Fatty Acid Synthase (FAS); Sterol Response Element Binding Proteins (SREBP); Carbohydrate Response Element Binding Proteins (ChREBP); stearoyl-CoA desaturase 1 (SCD1) (Schultz <em>et al</em>. 2000, Grefhorst <em>et al</em>. 2002; Kotokorpi<em> et al</em>. 2007; Nguyen et al. 2008).</p>
<h4>How it is Measured or Detected</h4>
<p>Differences are measured by changes in gene expression and protein levels. Effects on expression of downstream genes can be investigating using metabolomics and RT-qPCR approaches.</p>
<h4>References</h4>
<p>Grefhorst, A., Elzinga, B.M., Voshol, P.J., Plösch, T., Kok, T., Bloks, V.W., van der Sluijs, F.H., Havekes, L.M., Romijn, J.A., Verkade, H.J., and Kuipers, F. 2002. Stimulation of Lipogenesis by Pharmacological Activation of the Liver X Receptor Leads to Production of Large, Triglyceride-rich Very Low Density Lipoprotein Particles. The Journal of Biological Chemistry 277(37): 34182–34190.</p>
<p>Kotokorpi, P., Ellis, E., Parini, P., Nilsson, L.-M., Strom, S., Steffensen, K.R., Gustafsson, J.-A., and Mode, A. 2007. Physiological Differences between Human and Rat Primary Hepatocytes in Response to Liver X Receptor Activation by 3-[3-[N-(2-Chloro-3-trifluoromethylbenzyl)-(2,2-diphenylethyl)amino]propyloxy]phenylacetic Acid Hydrochloride (GW3965). Molecular Pharmacology 72(4): 947-955.</p>
<p>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. </p>
<p>Schultz, J.R., Tu, H., Luk, A., Repa, J.J., Medina, J.C., Li, L., Schwendner, S., Wang, S., Thoolen, M., Mangelsdorf, D.J., Lustig, K.D., and Shan, B. 2000. Role of LXRs in control of lipogenesis. Genes and Development 14:2831–2838. </p>
<h4><a href="/events/89">Event: 89: Synthesis, De Novo Fatty Acid (FA)</a></h4>
<h5>Short Name: Synthesis, De Novo Fatty Acid (FA)</h5>
<p><em>Life Stage: Older individuals are more likely to manifest this adverse outcome pathway (adults > juveniles) due to accumulation of triglycerides.</em></p>
<p><em>Sex: Applies to both males and females.</em></p>
<p><em>Taxonomic: Appears to be present broadly in vertebrates, with most representative studies in mammals (humans, lab mice, lab rats).</em></p>
<h4>Key Event Description</h4>
<p>A number of pathways and a great number of enzymes like GK, L-PK, ACC, FAS and SCD-1 are involved in the de novo FA synthesis <sup><a href="#cite_note-Postic_.26_Girard_2008-1">[1]</a></sup>. As it is already discussed above these enzymes are induced by LXR agonists (FAS, SCD1), the SREBP-1c (GK, ACC, FAS) and the ChREBP (L-PK, ACC, FAS) leading to enhancement of the de novo FA synthesis.</p>
Figure 1. Metabolic pathway for de novo FA synthesis and TG formation <sup><a href="#cite_note-Postic_.26_Girard_2008-1">[1]</a></sup></p>
<p>As proposed from Diraison et al 1997 the de novo FA synthesis contributes maximum 5% to the synthesis of FA and TG under normal conditions. Conditions associated with high rates of lipogenesis, such as low fat - high carbohydrate (LF/HC) diet, hyperglycemia, and hyperinsulinemia are associated with a shift in cellular metabolism from lipid oxidation to TG esterification, thereby increasing the availability of TGs derived from VLDL synthesis and secretion.</p>
<h4>How it is Measured or Detected</h4>
<p><em>Increases in fatty acid synthesis are generally measured by increases in triglycerides, fatty acids, cholesterols, and similar compounds in cells. In addition, assessment is generally made for cellular components such as mitochondria and/or gene expression increases with genes associated with synthesis, to associate the increase in fatty acid compounds with synthesis rather than other pathways (ex. influx).</em></p>
<p><em>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.</em></p>
<h4>References</h4>
<ol>
<li>↑ <sup><a href="#cite_ref-Postic_.26_Girard_2008_1-0">1.0</a></sup> <sup><a href="#cite_ref-Postic_.26_Girard_2008_1-1">1.1</a></sup> Postic & Girard 2008 - Postic C., Girard J., Contribution of de novo fatty acid synthesis to hepatic steatosis and<br />
insulin resistance: lessons from genetically engineered mice, J. Clin. Invest. 118 (No 3),<br />
829–838, 2008</li>
<li>Diraison et al 1997 - Diraison F., et al, Role of human liver lipogenesis and re-esterification in triglycerides<br />
secretion and in FFA re-esterification. Am J Physiol., 274 (2 Pt 1), E321-327, 1998</li>
</ol>
<p><em>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.</em></p>
<p><em>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.</em></p>
<p><em>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.</em></p>
<p><em>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/</em></p>
<p><em>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.</em></p>
<p><em>NOTE: Italics symbolize edits from John Frisch</em></p>
<td><a href="/aops/34">Aop:34 - LXR activation leading to hepatic steatosis</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/57">Aop:57 - AhR activation leading to hepatic steatosis</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/318">Aop:318 - Glucocorticoid Receptor activation leading to hepatic steatosis</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/517">Aop:517 - Pregnane X Receptor (PXR) activation leads to liver steatosis</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/518">Aop:518 - Liver X Receptor (LXR) activation leads to liver steatosis</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/529">Aop:529 - Xenobiotic binding to peroxisome proliferator-activated receptors (PPARs) causes dysregulation of lipid metabolism leading to liver steatosis</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/580">Aop:580 - Mineralocorticoid Receptor Activation Leading to Increased Body Mass Index</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/591">Aop:591 - DBEPE-induced DNA damage increase in liver leading to Non-alcoholic fatty liver disease via liver steatosis and inhibition of regeneration</a></td>
<p><em>Life Stage: Older individuals are more likely to manifest this key event (adults > juveniles) due to accumulation of triglycerides.</em></p>
<p><em>Sex: Applies to both males and females.</em></p>
<p><em>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.</em></p>
<h4>Key Event Description</h4>
<p>Leads to Fatty Liver Cells.
</p>
<p><em>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 </em>leads to Fatty Liver Cells <em>and steatosis</em>.</p>
<p><br />
<em>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). </em><br />
</p>
<p> </p>
<p> </p>
<h4>How it is Measured or Detected</h4>
<p><em>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.</em></p>
<h4>References</h4>
<p><em>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.</em></p>
<p><em>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.</em></p>
<p><br />
<em>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.</em></p>
<p><br />
<em>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.</em></p>
<p><em>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/</em></p>
<p><em>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.</em></p>
<p><em>NOTE: Italics symbolize edits from John Frisch</em></p>
<td><a href="/aops/58">Aop:58 - NR1I3 (CAR) suppression leading to hepatic steatosis</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/60">Aop:60 - NR1I2 (Pregnane X Receptor, PXR) activation leading to hepatic steatosis</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/61">Aop:61 - NFE2L2/FXR activation leading to hepatic steatosis</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/62">Aop:62 - AKT2 activation leading to hepatic steatosis</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/36">Aop:36 - Peroxisomal Fatty Acid Beta-Oxidation Inhibition Leading to Steatosis</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/213">Aop:213 - Inhibition of fatty acid beta oxidation leading to nonalcoholic steatohepatitis (NASH)</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/285">Aop:285 - Inhibition of N-linked glycosylation leads to liver injury</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/318">Aop:318 - Glucocorticoid Receptor activation leading to hepatic steatosis</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/517">Aop:517 - Pregnane X Receptor (PXR) activation leads to liver steatosis</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/518">Aop:518 - Liver X Receptor (LXR) activation leads to liver steatosis</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/529">Aop:529 - Xenobiotic binding to peroxisome proliferator-activated receptors (PPARs) causes dysregulation of lipid metabolism leading to liver steatosis</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/232">Aop:232 - NFE2/Nrf2 repression to steatosis</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/57">Aop:57 - AhR activation leading to hepatic steatosis</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/494">Aop:494 - AhR activation leading to liver fibrosis </a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/591">Aop:591 - DBEPE-induced DNA damage increase in liver leading to Non-alcoholic fatty liver disease via liver steatosis and inhibition of regeneration</a></td>
<p>Steatosis is the result of perturbations in well-known metabolic pathways that are well-studied and well-known in many taxa.</p>
<p><em>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.</em></p>
<p><em>Sex: This key event applies to both males and females.</em></p>
<p><em>Taxonomic: This key event appears to be present broadly in vertebrates, with most representative studies in mammals (humans, lab mice, lab rats).</em></p>
<h4>Key Event Description</h4>
<p>Biological state: liver steatosis is the inappropriate storage of fat in hepatocytes.</p>
<p>Biological state: liver steatosis is the inappropriate storage of fat in hepatocytes. <em>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). </em></p>
<p>Biological compartment: steatosis is generally an organ-level diagnosis; however, the pathology occurs within the hepatocytes.</p>
<p>Role in biology: steatosis is an adverse endpoint. </p>
<p> </p>
<p><span style="color:#d35400"><strong>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.</strong></span></p>
<p><span style="font-size:12px"><span style="color:#d35400"><strong>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.</strong></span></span></p>
<p><span style="font-size:12px"><span style="color:#d35400"><strong>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.</strong></span></span></p>
<p>Description from EU-ToxRisk:</p>
<p>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)</p>
<p>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)</p>
<h4>How it is Measured or Detected</h4>
<p>Steatosis is measured by lipidomics approaches that measure lipid levels, or by histology.</p>
<p>Steatosis is measured by lipidomics approaches<em> (e.g. Yang and Han 2016)</em> that measure lipid levels, or by histology. <em>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).</em></p>
<h4>Regulatory Significance of the AO</h4>
<p>Steatosis is a regulatory endpoint and has been used as an endpoint in many US EPA assessments, including IRIS assessments.</p>
<h4>References</h4>
<p>Landesmann, B. (2016). Adverse Outcome Pathway on Protein Alkylation Leading to Liver Fibrosis, (2).</p>
<p><em>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.</em></p>
<p><em>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.</em></p>
<p>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.</p>
<p>Landesmann, B. (2016). Adverse Outcome Pathway on Protein Alkylation Leading to Liver Fibrosis, (2).</p>
<p>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. <em>Gastroenterology</em>, <em>150</em>(1), 181–193.e8. https://doi.org/10.1053/j.gastro.2015.09.039</p>
<p><em>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. </em></p>
<p><em>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.</em></p>
<p><em>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/</em></p>
<p><em>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.</em></p>
<p><em>NOTE: Italics symbolize edits from John Frisch</em></p>
<h2>Appendix 2</h2>
<h2>List of Key Event Relationships in the AOP</h2>
<div id="evidence_supporting_links">
<h3>List of Adjacent Key Event Relationships</h3>
<div>
<h4><a href="/relationships/3103">Relationship: 3103: Activation, LXR leads to Increased, Expression of LXR activated genes</a></h4>
<p>Life Stage: Older individuals are more likely to manifest this adverse outcome pathway (adults > juveniles) due to increased opportunity to upregulate gene expression.</p>
<p>Sex: Applies to both males and females.</p>
<p>Taxonomic: Appears to be present broadly in vertebrates, with most representative studies in mammals (humans, lab mice, lab rats).</p>
<h4>Key Event Relationship Description</h4>
<p>Activation of Liver X receptor (LXR) gene expression has been shown to lead to increased gene expression and protein levels of loci associated with fatty acid synthesis, including Sterol regulatory element-binding protein (SRBEP), Fas cell surface death receptor (FAS), stearoyl-CoA desaturase 1 (SCD1), and Carbohydrate response element binding protein (CHREBP). Elevation of these molecular components increase the rate of fatty acid synthesis.</p>
<h4>Evidence Supporting this KER</h4>
<strong>Biological Plausibility</strong>
<p>The biological plausibility linking increased LXR expression to expression of genes associated with fatty acid synthesis is moderate. Gene expression studies in mammalian systems have linked activation of LXR to increased gene expression and protein levels of Sterol regulatory element-binding protein (SRBEP), Fas cell surface death receptor (FAS), stearoyl-CoA desaturase 1 (SCD1), and Carbohydrate response element binding protein (CHREBP), associated with fatty acid synthesis.</p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Increased LXR gene expression vs control in HEK293 cells and C57BL/6 mice, with correlated increases in CYP7A1, SCD-1, and SREBP-1 gene expression in a dose-dependent manner.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Increased LXR gene expression vs control in human and rat cells, with correlated increases in SREBP-1c, FASN, SCD1 in a dose-dependent manner.</span></span></p>
<p>Kotokorpi, P., Ellis, E., Parini, P., Nilsson, L.-M., Strom, S., Steffensen, K.R., Gustafsson, J.-A., and Mode, A. 2007. Physiological Differences between Human and Rat Primary Hepatocytes in Response to Liver X Receptor Activation by 3-[3-[N-(2-Chloro-3-trifluoromethylbenzyl)-(2,2-diphenylethyl)amino]propyloxy]phenylacetic Acid Hydrochloride (GW3965). Molecular Pharmacology 72(4): 947-955.</p>
<p>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</p>
<p>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.</p>
<p>Schultz, J.R., Tu, H., Luk, A., Repa, J.J., Medina, J.C., Li, L., Schwendner, S., Wang, S., Thoolen, M., Mangelsdorf, D.J., Lustig, K.D., and Shan, B. 2000. Role of LXRs in control of lipogenesis. Genes and Development 14:2831–2838.<br />
</p>
<p>Schultz, J.R., Tu, H., Luk, A., Repa, J.J., Medina, J.C., Li, L., Schwendner, S., Wang, S., Thoolen, M., Mangelsdorf, D.J., Lustig, K.D., and Shan, B. 2000. Role of LXRs in control of lipogenesis. Genes and Development 14:2831–2838.</p>
<p> </p>
</div>
<div>
<h4><a href="/relationships/3104">Relationship: 3104: Increased, Expression of LXR activated genes leads to Synthesis, De Novo FA</a></h4>
<h4><a href="/relationships/3104">Relationship: 3104: Increased, Expression of LXR activated genes leads to Synthesis, De Novo Fatty Acid (FA)</a></h4>
<p>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.</p>
<p><br />
Sex: Applies to both males and females.</p>
<p><br />
Taxonomic: Appears to be present broadly in vertebrates, with most representative studies in mammals (humans, lab mice, lab rats).<br />
</p>
<h4>Key Event Relationship Description</h4>
<p>Increased expression of genes activated by Liver X receptor (LXR) gene expression has been shown to lead to increased fatty acid synthesis pathway activity. Activated loci include: Sterol regulatory element-binding protein (SRBEP), Fas cell surface death receptor (FAS), stearoyl-CoA desaturase 1 (SCD1), and Carbohydrate response element binding protein (CHREBP). Elevation of these molecular components increase the rate of fatty acid synthesis.</p>
<h4>Evidence Supporting this KER</h4>
<strong>Biological Plausibility</strong>
<p>The biological plausibility linking increased expression of LXR activated genes to fatty acid synthesis is moderate. Gene expression studies in mammalian systems have linked increased expression of LXR activated genes and protein levels of Sterol regulatory element-binding protein (SRBEP), Fas cell surface death receptor (FAS), stearoyl-CoA desaturase 1 (SCD1), and Carbohydrate response element binding protein (CHREBP) to increased fatty acid synthesis.</p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Increased LXR gene expression vs control in HEK293 cells and C57BL/6 mice, with correlated increases in CYP7A1, SCD-1, and SREBP-1 gene expression in a dose-dependent manner.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Increased LXR gene expression vs control in human and rat cells, with correlated increases in SREBP-1c, FASN, SCD1 in a dose-dependent manner.</span></span></p>
<p>Kotokorpi, P., Ellis, E., Parini, P., Nilsson, L.-M., Strom, S., Steffensen, K.R., Gustafsson, J.-A., and Mode, A. 2007. Physiological Differences between Human and Rat Primary Hepatocytes in Response to Liver X Receptor Activation by 3-[3-[N-(2-Chloro-3-trifluoromethylbenzyl)-(2,2-diphenylethyl)amino]propyloxy]phenylacetic Acid Hydrochloride (GW3965). Molecular Pharmacology 72(4): 947-955.</p>
<p>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</p>
<p>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.</p>
<p>Schultz, J.R., Tu, H., Luk, A., Repa, J.J., Medina, J.C., Li, L., Schwendner, S., Wang, S., Thoolen, M., Mangelsdorf, D.J., Lustig, K.D., and Shan, B. 2000. Role of LXRs in control of lipogenesis. Genes and Development 14:2831–2838.<br />
</p>
</div>
<div>
<h4><a href="/relationships/110">Relationship: 110: Synthesis, De Novo FA leads to Accumulation, Triglyceride</a></h4>
<h4><a href="/relationships/110">Relationship: 110: Synthesis, De Novo Fatty Acid (FA) leads to Accumulation, Triglyceride</a></h4>
<p>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.</p>
<p><br />
Sex: Applies to both males and females.</p>
<p><br />
Taxonomic: Appears to be present broadly in vertebrates, with most representative studies in mammals (humans, lab mice, lab rats).<br />
</p>
<h4>Key Event Relationship Description</h4>
<p><em>De novo</em> fatty acid synthesis is a main pathway broadly accepted as a mechanism for accumulation of triglycerides in cells. Chemical stressors or alteration of gene expression levels can trigger increased fatty acid influx, as well as changes to membrane permeability and membrane proteins that facilitate fatty acid transport.</p>
<h4>Evidence Supporting this KER</h4>
<strong>Biological Plausibility</strong>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">The biological plausibility linking increased fatty acid synthesis to accumulation of triglycerides is strong, as a main pathway conserved across taxa. </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Increased CYP7A1, SCD-1, and SREBP-1 gene expression vs control in HEK293 cells and C57BL/6 mice, genes linked with fatty acid synthesis, with correlated increases in triglycerides, phospholipids, and HDL cholesterol in a dose-dependent manner.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Increased SREBP-1c, FASN, SCD1 gene expression vs control in human and rat cells, with correlated increases in fatty acid synthesis, pointing to increased de novo lipogenesis, in a dose-dependent manner.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Kotokorpi et al. (2007)</span></span></p>
</td>
</tr>
</tbody>
</table>
<p>In empirical studies, the link between increased fatty acid synthesis and accumulation of triglycerides is generally inferred. <br />
Increased expression of genes and/or signaling molecules known to facilitate fatty acid synthesis, and corresponding increases in triglyceride content in cells, are correlated to show evidence that increases are due to increased synthesis rather than alternative pathways. Angrish et al. (2016) review genes, signaling molecules, and chemical stressors linked to increased fatty acid synthesis, as well as other pathways leading to accumulation of triglycerides in cells. </p>
<h4>References</h4>
<p>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.</p>
<p>Grefhorst, A., Elzinga, B.M., Voshol, P.J., Plösch, T., Kok, T., Bloks, V.W., van der Sluijs, F.H., Havekes, L.M., Romijn, J.A., Verkade, H.J., and Kuipers, F. 2002. Stimulation of Lipogenesis by Pharmacological Activation of the Liver X Receptor Leads to Production of Large, Triglyceride-rich Very Low Density Lipoprotein Particles. The Journal of Biological Chemistry 277(37): 34182–34190.</p>
<p>Kotokorpi, P., Ellis, E., Parini, P., Nilsson, L.-M., Strom, S., Steffensen, K.R., Gustafsson, J.-A., and Mode, A. 2007. Physiological Differences between Human and Rat Primary Hepatocytes in Response to Liver X Receptor Activation by 3-[3-[N-(2-Chloro-3-trifluoromethylbenzyl)-(2,2-diphenylethyl)amino]propyloxy]phenylacetic Acid Hydrochloride (GW3965). Molecular Pharmacology 72(4): 947-955.</p>
<p>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</p>
<p>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.</p>
<p>Schultz, J.R., Tu, H., Luk, A., Repa, J.J., Medina, J.C., Li, L., Schwendner, S., Wang, S., Thoolen, M., Mangelsdorf, D.J., Lustig, K.D., and Shan, B. 2000. Role of LXRs in control of lipogenesis. Genes and Development 14:2831–2838.<br />
</p>
</div>
<div>
<h4><a href="/relationships/2265">Relationship: 2265: Accumulation, Triglyceride leads to Increased, Liver Steatosis</a></h4>
<td><a href="/aops/318">Glucocorticoid Receptor activation leading to hepatic steatosis</a></td>
<td>adjacent</td>
<td></td>
<td></td>
</tr>
<tr>
<td><a href="/aops/517">Pregnane X Receptor (PXR) activation leads to liver steatosis</a></td>
<td>adjacent</td>
<td>Moderate</td>
<td>High</td>
<td>Not Specified</td>
</tr>
<tr>
<td><a href="/aops/518">Liver X Receptor (LXR) activation leads to liver steatosis</a></td>
<td>adjacent</td>
<td>Moderate</td>
<td>High</td>
<td>Not Specified</td>
</tr>
<tr>
<td><a href="/aops/529">Xenobiotic binding to peroxisome proliferator-activated receptors (PPARs) causes dysregulation of lipid metabolism leading to liver steatosis</a></td>
<td>adjacent</td>
<td>High</td>
<td>Moderate</td>
</tr>
<tr>
<td><a href="/aops/57">AhR activation leading to hepatic steatosis</a></td>
<td>adjacent</td>
<td>High</td>
<td>High</td>
</tr>
<tr>
<td><a href="/aops/591">DBEPE-induced DNA damage increase in liver leading to Non-alcoholic fatty liver disease via liver steatosis and inhibition of regeneration</a></td>
<td>adjacent</td>
<td>High</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
<h4>Evidence Supporting Applicability of this Relationship</h4>
<p>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.</p>
<p><br />
Sex: Applies to both males and females.</p>
<p><br />
Taxonomic: Appears to be present broadly in vertebrates, with most representative studies in mammals (humans, lab mice, lab rats).<br />
</p>
<h4>Key Event Relationship Description</h4>
<p>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. </p>
<h4>Evidence Supporting this KER</h4>
<strong>Biological Plausibility</strong>
<p>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.</p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">In vitro exposure of 20 mM amiodarone, 50 mM tetracycline.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">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.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">In vitro exposure of at least 6 concentrations to 28 compounds selected for steatogenic potential.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">HepG2 human cells exposed to fialuridine, sodium valproate, doxycycline, amiodarone, tetracycline showed changes in the mitochondrial membrane potential by analysis of TMRM fluorescence and corresponding increases in lipid accumulation, with higher doses exhibiting greater lipid accumulation and correlated steatosis. </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">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. </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Transgenic and wild-type mice with normal and high cholesterol diet.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Human subjects with liver steatosis had increased RBP4 gene expression. Transgenic mice with human RBP4 gene had disrupted membranes, increased mitochondria dysfunction assessed by decreased citrate synthase activity, and correlated increases in triglycerides associated with steatosis, in comparison to wild-type mice.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">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.</span></span></p>
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.</p>
<p><br />
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.</p>
<p><br />
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</p>
<p> </p>
<p>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.</p>
<p> </p>
<p>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.</p>