<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Of the originating work: Chander Negi, Lola Bajard, Jiri Kohoutek, and Ludek Blaha, Faculty of Science, Masaryk University, Brno, Czech Republic</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 Do not cite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">Pregnane X receptor (PXR) 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), Liver X receptor (LXR), Peroxisome proliferator-activated receptor (PPAR), 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). Pregnenolone and progesterone are ligands in normal molecular activation of PXR (Mellor <em>et al</em>. 1996), while an increasing number of chemical stressors have been shown to increase PXR expression (Bajard <em>et al.</em> 2019; Moya <em>et al</em>. 2020). Activation of PXR has been linked to increased gene expression of CD36 (Zhou <em>et al</em>. 2006). The transmembrane protein CD36 has been shown to have a central role in fatty acid influx (Glatz <em>et al</em>. 2010), with fatty acid influx one of the main pathways for increase 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">Pregnane X receptor (PXR) belongs to a class of nuclear receptors [Aryl hydrocarbon receptor (AHR), Constitutive androstane receptor (CAR), Oestrogen receptor (ER), Farnesoid X receptor (FXR), Glucocorticoid receptor (GR), Liver X receptor (LXR), Peroxisome proliferator-activated receptor (PPAR), 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). Pregnenolone and progesterone are ligands in normal molecular activation of PXR (Mellor <em>et al</em>. 1996), while an increasing number of chemical stressors have been shown to increase PXR expression (Bajard <em>et al.</em> 2019; Moya <em>et al</em>. 2020). Activation of PXR has been linked to increased gene expression of CD36 (Zhou <em>et al</em>. 2006). The transmembrane protein CD36 has been shown to have a central role in fatty acid influx (Glatz <em>et al</em>. 2010), with fatty acid influx one of the main pathways for increase 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</span></span><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">, ultimately resulting in steatosis as a primary adverse outcome (Angrish <em>et al.</em> 2016; Mellor <em>et al</em>. 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 Pregnane X Receptor (PXR) leads to liver steatosis through increased fatty acid influx. 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 PXR gene and coordinated molecular responses leading to increased fatty acid influx. This pathway has been particular well studied in mammals (humans, lab mice, lab rats).</span></span></p>
<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>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</em>. 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><br />
<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 Pregnane X Receptor (PXR) leads to liver steatosis through increased fatty acid influx. 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 PXR gene and coordinated molecular responses leading to increased fatty acid influx. 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). Negi et al. (2021) provided initial network analysis based on empirical study of gene expression, lipid levels, and cell viability of human hepatocellular carcinoma cells (HepG2) exposed to 9 flame retardants (TDCIPP, TPHP, TMPP, TBBPA, EHDPP, TCEP, TNBP, TCIPP, and TBOEP) in order to obtain dose-response data. ToxCast database analysis and in silico molecular modeling supplemented cell viability and lipid accumulation empirical data, and led to the proposed Adverse Outcome Pathway.</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>Strong support.</strong> The relationship between activation of Pregnane-X receptor and Up Regulation of CD36 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 66: Up Regulation, CD36 leads to Increase, FA Influx</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>Strong support. </strong>The relationship between Up Regulation of CD36 and Increase, FA Influx 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>Increase, FA Influx 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><span style="color:#212529">Strong support.</span></strong><span style="color:#212529"> Activation of Pregnane-X receptor is a primary activator for increases in CD36 gene expression. Evidence is available from toxicant and gene-knockout studies.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong><span style="color:#212529">Strong support. </span></strong><span style="color:#212529">Up Regulation of CD36 expression is one gene linked to increases in fatty acid influx. Evidence is available from toxicant, gene-knockout, and high lipid diet studies.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong><span style="color:#212529">Moderate support. </span></strong><span style="color:#212529">Increase in fatty acid influx is a primary factor in increased triglyceride levels in cells. Evidence is available from toxicant and gene-knockout studies.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong><span style="color:#212529">Strong support. </span></strong><span style="color:#212529">Accumulation of triglyceride is linked to liver steatosis. Evidence is available from toxicant, gene-knockout, and high lipid diet studies.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong><span style="color:#212529">Strong support. </span></strong><span style="color:#212529">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></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong><span style="color:#212529">Moderate to strong support. </span></strong><span style="color:#212529">Direct evidence from empirical studies from laboratory mammals for most key events, with more inferential evidence for fatty acid influx.</span></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>Strong support. </strong>Increases in Pregnane X-receptor expression lead to increases in upregulation of CD36 expression, 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 66: Up Regulation, CD36 leads to Increase, FA Influx</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>Strong support.</strong> Increases in upregulation of CD36 expression lead to increases in fatty acid influx, primarily through measured increases in CD36 gene expression and increased triglyceride levels. Increased fatty influx 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 fatty acid influx 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">Bajard, L., Melymuk, L., and Blaha, L. 2019. Prioritization of hazards of novel flame retardants using the mechanistic toxicology information from ToxCast and Adverse Outcome Pathways. Environmental Sciences Europe 31:14.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:#444444">Glatz, J.F.C., Luiken, J.J.F.P., and Bonen, A. 2010. Membrane Fatty Acid Transporters as Regulators of Lipid Metabolism: Implications for Metabolic Disease. Physiological Reviews 90: 367–417.</span></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">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></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:#444444">Zhou, J., Zhai, Y., Mu, Y., Gong, H., Uppal, H., Toma, D., Ren, S., Evans, R.M., and Xie, W. 2006. A Novel Pregnane X Receptor-mediated and Sterol Regulatory Element-binding Protein-independent Lipogenic Pathway. Journal of Biological Chemistry 281(21): 15013-15020.</span></span></span></p>
<td><a href="/aops/8">Aop:8 - Upregulation of Thyroid Hormone Catabolism via Activation of Hepatic Nuclear Receptors, and Subsequent Adverse Neurodevelopmental Outcomes in Mammals</a></td>
<td>MolecularInitiatingEvent</td>
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<td><a href="/aops/517">Aop:517 - Pregnane X Receptor (PXR) activation leads to liver steatosis</a></td>
<td>MolecularInitiatingEvent</td>
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<tr>
<td><a href="/aops/545">Aop:545 - Activation, Pregnane-X receptor, NR1l2 leads to increased plasma low-density lipoprotein (LDL) cholesterol via increased cholesterol synthesis</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/548">Aop:548 - Activation, Pregnane-X receptor, NR1l2 leads to increased plasma low-density lipoprotein (LDL) cholesterol via increased PCSK9 protein expression</a></td>
<p><em>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.</em></p>
<p><em>Life Stage: All life stages.</em></p>
<p><em>Sex: This AOP applies to both males and females.</em></p>
<p><em>Sex: Applies to both males and females.</em></p>
<p><em>Taxonomic: This AOP appears to be present broadly in vertebrates, with most representative studies in mammals (humans, lab mice, lab rats).</em></p>
<p><em>Taxonomic: Appears to be present broadly in vertebrates, with most representative studies in mammals (humans, lab mice, lab rats). According to the evaluation of the plausible taxonomic domain of applicability (tDOA) by Haigis et al., 2023, PXR was found only to be conserved across mammalian species compared to the human (Homo sapiens) protein target using the U.S. Environmental Protection Agency's Sequence Alignment to Predict Across Species Susceptibility (SeqAPASS v6.0; seqapass.epa.gov/seqapass/) tool, while acknowledging the potential existence of interspecies differences in conservation. No structural protein conservation of amphibian, fish, reptilian or avian PXR was found.</em></p>
<h4>Key Event Description</h4>
<p><em>Pregnane X receptor (PXR), also known as steroid NR subfamily 1, group I, member 2 (NR1I2), is a nuclear receptor that can be activated by pregnenolone and progesterone and many chemicals due to low specificity; once activated it binds to Retinoid X receptor (RXR) and increases upregulation of target genes (Mellor et al. 2016). PXR has an important role in lipid homeostasis by regulating the rate of lipid intake particularly through activation of cluster of differentiation 36, also known as platelet glycoprotein 4 [(CD36) (Zhou et al. 2006; Gwag et al. 2019)].</em></p>
<h4>How it is Measured or Detected</h4>
<p><em>Effects of Pregnane X receptor (PXR) activation on expression of downstream genes can be investigating using transcriptomics and/or RT-qPCR approaches. The following ToxCast assays measure PXR activation: ATG_PXRE_CIS; ATG_PXR_TRANS; TOX21_PXR_agonist; ATG_mPXR_XSP2; NVS_NR_hPXR (U.S. EPA 2024). Indigo Biosciences sells a commercial assay for human PXR activation (INDIGO Biosciences 2024).</em></p>
<h4>References</h4>
<p><em>Gwag, T., Meng, Z., Sui, Y., Helsley, R.N., Park, S.-H., Wang, S., Greenberg, R.N., and Zhou, C. 2019. Non-nucleoside reverse transcriptase inhibitor efavirenz activates PXR to induce hypercholesterolemia and hepatic steatosis. Journal of Hepatology 70: 930–940.</em></p>
<p><em>Haigis A-C., Vergauwen L., Lalone C.A., Villeneuve D.L., O'Brien J.M., Knapen D. (2023). Cross-species applicability of an adverse outcome pathway network for thyroid hormone system disruption. Toxicol Sci. 195, 1-27.</em></p>
<p><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></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. DOI:10.3109/10408444.2015.1089471.</em></p>
<p><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></p>
<p><em>Zhou, J., Zhai, Y., Mu, Y., Gong, H., Uppal, H., Toma, D., Ren, S., Evans, R.M., and Xie, W. 2006. A Novel Pregnane X Receptor-mediated and Sterol Regulatory Element-binding Protein-independent lipogenic pathway. The Journal of Biological Chemistry 281(21): 15013–15020.</em></p>
<p><em>NOTE: Italics indicate edits from John Frisch June 2024</em><br />
</p>
<h3>List of Key Events in the AOP</h3>
<h4><a href="/events/54">Event: 54: Up Regulation, CD36</a></h4>
<p><em>Life Stage: Older individuals are more likely to manifest this key event (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>
<p> </p>
<h4>Key Event Description</h4>
<p>Fatty acid translocase CD36 (FAT/CD36) is a scavenger protein mediating uptake and intracellular transport of long-chain fatty acids (FA) in diverse cell types <sup><a href="#cite_note-1">[1]</a></sup>, <sup><a href="#cite_note-2">[2]</a></sup>. In addition, CD36 can bind a variety of molecules including acetylated low density lipoproteins (LDL), collagen and phospholipids <sup><a href="#cite_note-3">[3]</a></sup>. CD36 has been shown to be expressed in liver tissue <sup><a href="#cite_note-4">[4]</a></sup>, <sup><a href="#cite_note-5">[5]</a></sup>. It is located in lipid rafts and non-raft domains of the cellular plasma membrane and most likely facilitates LCFA transport by accumulating LCFA on the outer surface <sup><a href="#cite_note-6">[6]</a></sup>, <sup><a href="#cite_note-7">[7]</a></sup>, <sup><a href="#cite_note-8">[8]</a></sup>.</p>
<p>FAT/CD36 gene is a liver specific target of LXR activation <sup><a href="#cite_note-9">[9]</a></sup>. Studies have confirmed that the lipogenic effect of LXR and activation of FAT/CD36 was not a simple association, since the effect of LXR agonists on increasing hepatic and circulating levels of triglycerides and free fatty acids (FFAs) was largely abolished in FAT/CD36 knockout mice suggesting that intact expression and/or activation of FAT/CD36 is required for the steatotic effect of LXR agonists <sup><a href="#cite_note-10">[10]</a></sup>, <sup><a href="#cite_note-11">[11]</a></sup>. In addition to the well-defined pathogenic role of FAT/CD36 in hepatic steatosis in rodents the human up-regulation of the FAT/CD36 in NASH patients is confirmed <sup><a href="#cite_note-12">[12]</a></sup>. There are now findings that can accelerate the translation of FAT/CD36 metabolic functions determined in rodents to humans <sup><a href="#cite_note-13">[13]</a></sup> and suggest that the translocation of this fatty acid transporter to the plasma membrane of hepatocytes may contribute to liver fat accumulation in patients with NAFLD and HCV <sup><a href="#cite_note-14">[14]</a></sup>. In addition, hepatic FAT/CD36 up-regulation is significantly associated with insulin resistance, hyperinsulinaemia and increased steatosis in patients with NASH and HCV G1 (Hepatitis C Virus Genotype1) with fatty liver. Recent data show that CD36 is also increased in the liver of morbidly obese patients and correlated to free FA levels <sup><a href="#cite_note-15">[15]</a></sup>.</p>
<h4>How it is Measured or Detected</h4>
<p><em>CD36 is measured by changes in gene expression and protein levels. </em></p>
<h4>References</h4>
<ol>
<li><a href="#cite_ref-1">↑</a> Su & Abumrad 2009 - Su X., Abumrad N.A., Cellular fatty acid uptake: a pathway under construction. Trends<br />
Endocrinol. Metab., 20 (No 2), 72-77, 2009</li>
<li><a href="#cite_ref-2">↑</a> He et al. 2011 - He J. et al, The emerging roles of fatty acid translocase/CD36 and the aryl hydrocarbon<br />
receptor in fatty liver disease, Exp. Med. And Biology, 236, 1116-1121, 2011</li>
<li><a href="#cite_ref-3">↑</a> Krammer 2011 - Krammer J. et al, Overexpression of CD36 and Acyl-CoA Synthetases FATP2, FATP4<br />
and ACSL1 Increases Fatty Acid Uptake in Human Hepatoma Cells, Int. J. Med. Sci.,<br />
8(7), 599-614, 2011</li>
<li><a href="#cite_ref-4">↑</a> Pohl et al. 2005 - Pohl J., et al, FAT/CD36-mediated long-chain fatty acid uptake in adipocytes requires<br />
<li><a href="#cite_ref-5">↑</a> Cheung et al. 2007 - Cheung L., et al, Hormonal and nutritional regulation of alternative CD36 transcripts<br />
in rat liver--a role for growth hormone in alternative exon usage, BMC Mol. Biol., 8, 60,<br />
2007</li>
<li><a href="#cite_ref-6">↑</a> Ehehalt et al. 2008 - Ehehalt R., et al, Uptake of long chain fatty acids is regulated by dynamic interaction<br />
of FAT/CD36 with cholesterol/sphingolipid enriched microdomains (lipid rafts). BMC<br />
Cell. Biol., 9, 45, 2008</li>
<li><a href="#cite_ref-7">↑</a> Pohl et al. 2005 - Pohl J., et al, FAT/CD36-mediated long-chain fatty acid uptake in adipocytes requires<br />
<li><a href="#cite_ref-8">↑</a> Krammer 2011 - Krammer J. et al, Overexpression of CD36 and Acyl-CoA Synthetases FATP2, FATP4<br />
and ACSL1 Increases Fatty Acid Uptake in Human Hepatoma Cells, Int. J. Med. Sci.,<br />
8(7), 599-614, 2011</li>
<li><a href="#cite_ref-9">↑</a> Zhou 2008 - Zhou J., Hepatic fatty acid transporter Cd36 is a common target of LXR, PXR, and<br />
PPAR gamma in promoting steatosis, Gastroenterology, 134 (No 2),556-567, 2008</li>
<li><a href="#cite_ref-10">↑</a> Febbraio et al. 1999 - Febbraio M., et al, A null mutation in murine CD36 reveals an important role in fatty<br />
acid and lipoprotein metabolism, J Biol Chem, 274, 19055–19062, 1999</li>
<li><a href="#cite_ref-11">↑</a> Lee et al. 2008 - Febbraio M., et al, A null mutation in murine CD36 reveals an important role in fatty<br />
acid and lipoprotein metabolism, J Biol Chem, 274, 19055–19062, 1999</li>
<li><a href="#cite_ref-11">↑</a> Lee et al. 2008 - Lee J.H., et al, PRX and LXR in hepatic Steatosis: a new dog and an old dog with new<br />
tricks, Mol. Pharm., 5(No 1),60-66, 2008</li>
<li><a href="#cite_ref-12">↑</a> Zhu et al. 2011 - Zhu L., et al, Lipid in the livers of adolescents with non-alcoholic steatohepatitis:<br />
combined effects of pathways on steatosis, Metabolism Clinical and experimental, 30,<br />
1001-1011, 2011</li>
<li><a href="#cite_ref-13">↑</a> Love-Gregory et al. 2011 - Love-Gregory L., Abumrad N.A., CD36 genetics and the metabolic complications of<br />
obesity, Current Opinions in Clinical Nutition and Metabolic Care, 14 (No 6), 527-534,<br />
2011</li>
<li><a href="#cite_ref-14">↑</a> Miquilena-Colina et al. 2011 - Miquilena-Colina M.E., et al, Hepatic fatty acid translocase CD36 upregulation is<br />
associated with insulin resistance, hyperinsulinaemia and increased steatosis in nonalcoholic<br />
steatohepatitis and chronic hepatitis C, Gut., 60 (No 10), 1394-1402 , 2011</li>
<li><a href="#cite_ref-15">↑</a> Bechmann et al. 2010 - Bechmann L.P., et al, Apoptosis is associated with CD36/fatty acid translocase<br />
upregulation in non-alcoholic steatohepatitis, Liver Int., 30 (No 6), 850-859, 2010</li>
upregulation in non-alcoholic steatohepatitis, Liver Int., 30 (No 6), 850-859, 2010 </li>
</ol>
<p><em>NOTE: Italics symbolize edits from John Frisch</em></p>
<h4><a href="/events/115">Event: 115: Increase, FA Influx</a></h4>
<p><em>Life Stage: Older individuals are more likely to manifest this key event (adults > juveniles) due to increased opportunity to increase fatty acid influx.</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>Fat influx to the liver is usually increased under condition like obesity. Free fatty acids (FFA) increase in blood leads to an increase of FFA uptake in the liver. Especially the long chain fatty acids (LCFAs) are translocated across the plasma membrane, reassembled to triglycerides and stored in lipid droplets causing hepatic steatosis <sup><a href="#cite_note-1">[1]</a></sup>.</p>
<p>As mentioned above CD36 has consistently been shown to be expressed at the plasma membrane and to enhance LCFA uptake upon over-expression <sup><a href="#cite_note-2">[2]</a></sup>, <sup><a href="#cite_note-3">[3]</a></sup>.</p>
<p>CD36 has consistently been shown to be expressed at the plasma membrane and to enhance LCFA uptake upon over-expression <sup><a href="#cite_note-2">[2]</a></sup>, <sup><a href="#cite_note-3">[3]</a></sup>.</p>
<h4>How it is Measured or Detected</h4>
<p><em>Increases in fatty acid influx are generally measured by increases in triglycerides, fatty acids, cholesterols, and similar compounds in cells. In addition, assessment is generally made for plasma membrane stability and/or gene expression increases with genes associated with influx, to associate the increase in fatty acid compounds with influx rather than other pathways (ex. synthesis).</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><a href="#cite_ref-1">↑</a> Amacher 2011 - Amacher D.E., The mechanistic basis for the induction of hepatic steatosis by<br />
xenobiotics, Expert Opinion on Drug Metabolism and Toxicology, 7 (No 8), 949-965,<br />
2011</li>
<li><a href="#cite_ref-2">↑</a> Baranowski 2008 - Baranowski, Biological role of liver X receptors, Journal of Physiology and<br />
<li><a href="#cite_ref-3">↑</a> Su & Abumrad 2009 - Su X., Abumrad N.A., Cellular fatty acid uptake: a pathway under construction. Trends<br />
Endocrinol. Metab., 20 (No 2), 72-77, 2009</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 - DBDPE-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 - DBDPE-induced DNA damage increase in liver leading to Non-alcoholic fatty liver disease via liver steatosis and inhibition of regeneration</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/624">Aop:624 - Altered glucocorticoid receptor signaling leading to MASLD progression via insulin resistance-associated mitochondrial dysfunction</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/625">Aop:625 - Altered glucocorticoid receptor signaling leading to MASLD progression via reduced very low-density lipoprotein export-associated mitochondrial dysfunction</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/626">Aop:626 - Altered glucocorticoid receptor signaling leading to MASLD progression via reduced VLDL export-associated mitochondrial dysfunction</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/627">Aop:627 - Altered glucocorticoid receptor signaling leading to MASLD progression via insulin resistance-associated endoplasmic reticulum stress</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/628">Aop:628 - Altered glucocorticoid receptor signaling leading to MASLD progression via reduced very low-density lipoprotein export-associated endoplasmic reticulum stress</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/629">Aop:629 - Altered glucocorticoid receptor signaling leading to MASLD progression via reduced lipogenesis-associated endoplasmic reticulum stress</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/3100">Relationship: 3100: Activation, Pregnane-X receptor, NR1l2 leads to Up Regulation, CD36</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>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 Pregnane-X receptor (PXR) gene expression has been shown to lead to increased gene expression and protein levels of CD36. CD36 is a transmembrane protein, and is of scientific interest because of the role of CD36 in fatty acid influx into cells.</p>
<p>Activation of Pregnane-X receptor (PXR) gene expression has been shown to lead to increased gene expression and protein levels of CD36 (Zhou et al. 2006; Gwag et al. 2009). CD36 is a transmembrane protein, and is of scientific interest because of the role of CD36 in fatty acid influx into cells.</p>
<h4>Evidence Supporting this KER</h4>
<strong>Biological Plausibility</strong>
<p>The biological plausibility linking increased PXR expression to CD36 expression is moderate. Gene expression studies in mammalian systems have linked activation of PXR to increased gene expression and protein levels of CD36.</p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Increased PXR gene expression vs control in hepatocytes exposed to 20 um efavirenz for 24 hours and increased CD36 gene expression in hepatocytes exposed to 20 um efavirenz for 24 hours.</span></span></p>
<p>Gwag, T., Meng, Z., Sui, Y., Helsley, R.N., Park, S.-H., Wang, S., Greenberg, R.N., and Zhou, C. 2019. Non-nucleoside reverse transcriptase inhibitor efavirenz activates PXR to induce hypercholesterolemia and hepatic steatosis Journal of Hepatology 70: 930–940.</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>Zhou, J., Zhai, Y., Mu, Y., Gong, H., Uppal, H., Toma, D., Ren, S., Evans, R.M., and Xie, W. 2006. A Novel Pregnane X Receptor-mediated and Sterol Regulatory Element-binding Protein-independent lipogenic pathway. The Journal of Biological Chemistry 281(21): 15013–15020.</p>
</div>
<div>
<h4><a href="/relationships/66">Relationship: 66: Up Regulation, CD36 leads to Increase, FA Influx</a></h4>
<h4><a href="/relationships/66">Relationship: 66: Up Regulation, CD36 leads to Increase, FA influx</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>Life Stage: Older individuals are more likely to manifest this adverse outcome pathway (adults > juveniles) due to accumulation of triglycerides.</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>CD36 gene expression has been shown to be a key regulator of fatty acid influx, primarily in mammal studies. CD36 is a transmembrane protein, and increased CD36 gene expression can result in increased fatty acid influx. Chemical stressors or high fat diets can help trigger fatty acid influx.</p>
<h4>Evidence Supporting this KER</h4>
<p> </p>
<p> </p>
<strong>Biological Plausibility</strong>
<p>The biological plausibility linking increased CD36 expression to increased fatty acid uptake is moderate. CD36 is a transmembrane protein, and upregulation of CD36 has been linked to increased fatty acid uptake, primarily in mammalian systems.</p>
<strong>Empirical Evidence</strong>
<p>Since the link between upregulation of CD36 and increased fatty acid influx has been established, empirical studies often measure increased CD36 gene expression and increased lipid content in cells and infer that the mechanism was increased fatty acid influx (Moya et al. 2010). </p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Transgenic-human PXR mice showed increased expression of CD36 genes in livers and increased lipid accumulation versus wild-type mice. FA influx was inferred as there was no increase in gene expression of SREBP, which would be expected to be upregulated if de novo fatty acid synthesis was the mechanism for increased triglycerides.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Children and adolescents exhibiting steatosis versus children and adolescents without steatosis</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">CD36, FABPpm, SLC27A2, SLC27A5 gene expression were upregulated and CD36 and CPT-1 protein expression was upregulated in subjects exhibiting steatosis linking increased triglyceride levels to fatty acid influx; FASN, SCD1, and acyl-COA gene expression were also upregulated in subjects exhibiting steatosis linking increased triglyceride levels to de novo fatty acid synthesis; both pathways appear to be responsible for increased triglycerides.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Mouse fed high fat diet had higher CD36 expression and triglyceride accumulation than mice fed low fat diet; transgenic mice and hepatocytes with CD36 gene had higher fatty acid influx than null mice and hepatocytes measured by the fluorescent fatty acid analog BODIPY.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Increased CD36 gene expression vs control in hepatocytes exposed to 20 um efavirenz and correlated higher fatty acid transport as measured by palmitic acid uptake.</span></span></p>
<p>Gwag, T., Meng, Z., Sui, Y., Helsley, R.N., Park, S.-H., Wang, S., Greenberg, R.N., and Zhou, C. 2019. Non-nucleoside reverse transcriptase inhibitor efavirenz activates PXR to induce hypercholesterolemia and hepatic steatosis Journal of Hepatology 70: 930–940.</p>
<p>Koonen, D.P.Y., Jacobs, R.L., Febbraio, M. Young, M.E., Soltys, C.-L.M., Ong, H., Vance, D.E., and Dyck, J.R.B. 2007. Increased hepatic CD36 expression contributes to dyslipidemia associated with diet-induced obesity. Diabetes 56: 2863-2871.</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>Moya, M., Gomez-Lechon, M.J., Castell, J.V., and Jover, R. 2010. Enhanced steatosis by nuclear receptor ligands: A study in cultured human hepatocytes and hepatoma cells with a characterized nuclear receptor expression profile. 184: 376–387.</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>Zhu, L., Baker, S.S., Liu, W., Tao, M.-H., Patel, R., Nowak, N.J., and Baker, R.D. 2011. Lipid in the livers of adolescents with nonalcoholic steatohepatitis: combined effects of pathways on steatosis. Metabolism Clinical and Experimental 60: 1001-1011.</p>
<p>Zhou, J., Zhai, Y., Mu, Y., Gong, H., Uppal, H., Toma, D., Ren, S., Evans, R.M., and Xie, W. 2006. A Novel Pregnane X Receptor-mediated and Sterol Regulatory Element-binding Protein-independent lipogenic pathway. The Journal of Biological Chemistry 281(21): 15013–15020.</p>
</div>
<div>
<h4><a href="/relationships/132">Relationship: 132: Increase, FA Influx leads to Accumulation, Triglyceride</a></h4>
<h4><a href="/relationships/132">Relationship: 132: Increase, FA influx 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>Life Stage: Older individuals are more likely to manifest this adverse outcome pathway (adults > juveniles) due to accumulation of triglycerides.</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>Increased fatty acid influx 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>The biological plausibility linking increased fatty acid influx to accumulation of triglycerides is strong, as a main pathway conserved across taxa. </p>
<p>The biological plausibility linking increased fatty acid influx to accumulation of triglycerides is strong, as a main pathway conserved across taxa. Stressors can disrupt normal rates of fatty acid influx, increasing the accumulation of trigylcerides.</p>
<strong>Empirical Evidence</strong>
<p>In empirical studies, the link between increased fatty acid influx and accumulation of triglycerides is generally inferred. Zhou et al. (2006) link accumulation of triglycerides to increased fatty acid influx in the livers of transgenic mice with increased Pregnane X Receptor expression compared to wild-type mice. <br />
Increased expression of genes and/or signaling molecules known to facilitate fatty acid influx, and corresponding increases in triglyceride content in cells, are correlated to show evidence that increases are due to increased influx rather than alternative pathways. Angrish et al. (2016) review genes, signaling molecules, and chemical stressors linked to increased fatty acid influx, as well as other pathways leading to accumulation of triglycerides in cells. For a review of membrane proteins facilitating fatty acid influx, see Glatz et al. (2010).</p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Heptatocytes from transgenic-CD36 mice showed increased fatty acid influx than null mice and measured by the fluorescent fatty acid analog BODIPY and correlated increased triglycerides.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Hepatocytes exposed to 20 um efavirenz for 24 hours had increased fatty acid influx as measured by palmitic acid uptake and correlated increased triglycerides and cholesterol to mice exposed to 100 mg/kg/day efavirenz for 1 week.</span></span></p>
<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>Glatz, J.F.C., Joost, J.F., Luiken, P., and Bonen, A. 2010. Membrane fatty acid transporters as regulators of lipid metabolism: Implications for metabolic disease. Physiological Reviews 90: 367–417.</p>
<p>Gwag, T., Meng, Z., Sui, Y., Helsley, R.N., Park, S.-H., Wang, S., Greenberg, R.N., and Zhou, C. 2019. Non-nucleoside reverse transcriptase inhibitor efavirenz activates PXR to induce hypercholesterolemia and hepatic steatosis Journal of Hepatology 70: 930–940.</p>
<p>Koonen, D.P.Y., Jacobs, R.L., Febbraio, M. Young, M.E., Soltys, C.-L.M., Ong, H., Vance, D.E., and Dyck, J.R.B. 2007. Increased hepatic CD36 expression contributes to dyslipidemia associated with diet-induced obesity. Diabetes 56: 2863-2871.</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>Zhou, J., Zhai, Y., Mu, Y., Gong, H., Uppal, H., Toma, D., Ren, S., Evans, R.M., and Xie, W. 2006. A Novel Pregnane X Receptor-mediated and Sterol Regulatory Element-binding Protein-independent Lipogenic Pathway. The Journal of biological chemistry 281(21): 15013–15020.</p>
</div>
<div>
<h4><a href="/relationships/2265">Relationship: 2265: Accumulation, Triglyceride leads to Increased, Liver Steatosis</a></h4>
<h4><a href="/relationships/2265">Relationship: 2265: Accumulation, Triglyceride leads to Increase, 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">DBDPE-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>