<p><strong>Xavier Coumoul<sup>*</sup>, Min Ji Kim<sup>$</sup>, Karine Audouze*, Etienne Blanc<sup>*</sup>, Jean-Pascal de Bandt<sup>*</sup></strong></p>
<td>Under development: Not open for comment. Do not cite</td>
<td></td>
<td></td>
<td></td>
</tr>
</tbody>
</table>
</div>
</div>
<div id="coaches">
<h2>Coaches</h2>
<ul>
<li class="contributor" id="coach_95">
Tanja Burgdorf
</li>
</ul>
</div>
<div id="abstract">
<h2>Abstract</h2>
<p>Liver fibrosis, characterized by excessive accumulation of extracellular matrix proteins, represents a significant health burden worldwide. The Ah receptor (AhR), a ligand-activated transcription factor primarily known for its involvement in xenobiotic metabolism, has emerged as a key player in various physiological processes, including liver homeostasis and inflammation. Recent studies have implicated the AhR signaling pathway in the development and progression of liver fibrosis. This AOP provides a comprehensive overview of the molecular mechanisms underlying the association between AhR activation and liver fibrogenesis. AhR activation by endogenous ligands, such as tryptophan metabolites and environmental toxins, triggers a cascade of events leading to hepatic stellate cell activation, inflammation, and fibrogenesis. Understanding the intricate interplay between AhR and liver fibrosis offers novel insights into the pathogenesis of chronic liver diseasesand highlights AhR as a potential therapeutic target for the management of liver fibrosis.</p>
<p style="text-align:justify"><span style="font-size:medium"><span style="font-family:"Times New Roman",serif"><span style="color:#000000"><strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">“Liver fibrosis, characterized by excessive accumulation of extracellular matrix proteins, represents a significant health burden worldwide. </span></span></span></strong><strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:#00b050">It is a hallmark of chronic hepatotoxicity, which is characterized by excessive deposition of extracellular matrix (ECM) proteins, primarily collagen, leading to disrupted liver architecture and function. It is a precursor to cirrhosis and affects millions globally, with over 1.5 million deaths annually attributed to advanced liver diseases such as cirrhosis or liver cancer. At the cellular level, hepatic fibrosis involves the activation of hepatic stellate cells (HSCs) into myofibroblasts, driven by pro-fibrotic cytokines like TGF-β1. Molecularly, it is defined by increased expression of fibrotic markers such as α-SMA and COL1A1, and dysregulation of pathways like Wnt/β-catenin and NF-κB. These processes often result from chronic exposure to hepatotoxic agents, including alcohol, drugs, or viral infections, underscoring the critical link between hepatotoxicity and fibrosis. The chemical exposome is therefore suspected to influence the occurence of liver fibrosis.</span></span></span></strong><strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red"> The Ah receptor (AhR), a ligand-activated transcription factor primarily known for its involvement in xenobiotic metabolism, has emerged as a key player in various physiological processes, including liver homeostasis and inflammation. </span></span></span></strong><strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:#00b050">The AhR is now considered as an exposome receptor. </span></span></span></strong><strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">Recent studies have implicated the AhR signaling pathway in the development and progression of liver fibrosis. </span></span></span></strong><strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:#00b050">Importantly, predictive assays are missing and a single predictive <em>in vitro </em>assay appears difficult to be setup with current technical knowledge. AOPs can be used to define a framework linking cellular and molecular events up to an AO, starting with AhR activation (MIE). We therefore propose an</span></span></span></strong><strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">AOP provid</span></span></span></strong><strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:#00b050">ing</span></span></span></strong><strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red"> a comprehensive overview of the molecular mechanisms underlying the association between AhR activation and liver fibrogenesis. AhR activation by endogenous ligands, such as tryptophan metabolites and environmental toxins, triggers a cascade of events leading to hepatic stellate cell activation, inflammation, and fibrogenesis. </span></span></span></strong><strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:#00b050">The 5 key events presented here can serve as a knowledge repository for identifying and developing new in vitro methodological approaches, integrated into the AOP in the form of IATA (Integrated Approaches to Testing and Assessment). These 5 KEs link hepatic steatosis induced by AhR activation to cell death, which leads to the recruitment and activation of stellate cells that are responsible for the production of extracellular matrix components, and hence to hepatic fibrosis.</span></span></span></strong><strong> </strong><strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:#00b050">If tests are lacking, the AOP can help guide their development to assess the hazard associated with new substances or substances under development. </span></span></span></strong><strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">Understanding the intricate interplay between AhR and liver fibrosis offers novel insights into the pathogenesis of chronic liver diseases and highlights AhR as a potential therapeutic target for the management of liver fibrosis. “</span></span></span></strong></span></span></span></p>
<p style="text-align:justify"><span style="font-size:medium"><span style="font-family:"Times New Roman",serif"><span style="color:#000000"><span style="font-size:8pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">Pierre S, Chevallier A, Aryl hydrocarbon receptor-dependent induction of liver fibrosis by dioxin. </span></span></span><span style="font-size:8pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">Toxicol Sci. 2014 Jan;137(1):114-24. doi: 10.1093/toxsci/kft236. Epub 2013 Oct 23. PMID: 24154488.</span></span></span></span></span></span></p>
<p><span style="font-size:8pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">Larigot L, Benoit L, et al. </span></span></span><span style="font-size:8pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">Aryl Hydrocarbon Receptor and Its Diverse Ligands and Functions: An Exposome Receptor. Annu Rev Pharmacol Toxicol. 2022 Jan 6;62:383-404. doi: 10.1146/annurev-pharmtox-052220-115707. Epub 2021 Sep 9. PMID: 34499523.</span></span></span></p>
</div>
<div id="background">
<h3>Background</h3>
<p>Understanding the biological link between Ah receptor (AhR) activation and liver fibrosis holds significant relevance due to its implications in the pathogenesis of various liver diseases. Liver fibrosis represents a common pathological process underlying the progression of chronic liver disorders, including hepatitis, alcoholic liver disease, and non-alcoholic fatty liver disease (NAFLD). Notably, NAFLD, characterized by hepatic steatosis, inflammation, and fibrosis, has become a global health concern, closely associated with obesity, metabolic syndrome, and insulin resistance. Given that AhR activation has been implicated in the regulation of lipid metabolism and inflammation, elucidating its role in liver fibrosis provides valuable insights into the molecular mechanisms driving NAFLD progression. Moreover, the interconnected nature of liver diseases underscores the importance of investigating AhR-mediated pathways as potential therapeutic targets for the management of liver fibrosis and its comorbidities, including hepatic steatosis. Therefore, establishing a biological link between AhR activation and liver fibrosis not only enhances our understanding of disease pathogenesis but also offers promising avenues for the development of targeted therapies for liver-related disorders.</p>
<h2>AOP Development Strategy</h2>
<div id="context">
<h3>Context</h3>
<p>Understanding the biological link between Ah receptor (AhR) activation and liver fibrosis holds significant relevance due to its implications in the pathogenesis of various liver diseases. Liver fibrosis represents a common pathological process underlying the progression of chronic liver disorders, including hepatitis, alcoholic liver disease, and non-alcoholic fatty liver disease (NAFLD). Notably, NAFLD, characterized by hepatic steatosis, inflammation, and fibrosis, has become a global health concern, closely associated with obesity, metabolic syndrome, and insulin resistance. Given that AhR activation has been implicated in the regulation of lipid metabolism and inflammation, elucidating its role in liver fibrosis provides valuable insights into the molecular mechanisms driving NAFLD progression. Moreover, the interconnected nature of liver diseases underscores the importance of investigating AhR-mediated pathways as potential therapeutic targets for the management of liver fibrosis and its comorbidities, including hepatic steatosis. Therefore, establishing a biological link between AhR activation and liver fibrosis not only enhances our understanding of disease pathogenesis but also offers promising avenues for the development of targeted therapies for liver-related disorders.</p>
</div>
</div>
<div id="development_strategy">
<h3>Strategy</h3>
<p>We have been working for many years on chronic liver diseases and AhR. We had written a review on the subject and, following on from experimental work showing the links between AhR and fibrosis (two publications), we decided as part of the EU PARC project to take advantage of this expertise to propose this AOP. </p>
<p>The <em>biological plausibility</em> of KERs is defined by the OECD as the « understanding of the fundamental biological processes involved and whether they are consistent with the causal relationship being proposed in the AOP ». The biological plausibility is strong due to the presence of overwhelming evidence present in different studies. A minor setback would be the difficulty to dismiss alternative mechanisms caused by the ligands used for AhR activation. </p>
<p>The <em>essentiality of KEs</em> refers to « experimental data for whether or not downstream KEs or the AO are prevented or modified if an upstream event is blocked ». The essentiality of KEs is strong: most works converge to imply the AhR in fibrotic processes. One setback would be that AhR knockout mice also develop a specific liver fibrosis. We propose that exogenous ligands alter the activity of endogenous ligands and therefore contribute just like the knockout to the occurence of liver fibrosis. The AhR activation needs to be considered then as the binding of exogenous ligands (xenobiotics) counteracting on the physiological processes which regulate the physiological functions.</p>
<p>Finally, the <em>empirical support</em> of KERs, is often « based on toxicological data derived by one or more reference chemicals where dose–response and temporal concordance for the KE pair can be assessed ». The overall assessment of the empirical support of our KERs is also strong. There is evidence in human cell lines and mice showing a dose–response and temporal concordance for severity of our KEs and the adverse outcomes (for example, a dose-dependant effect of TCDD on the development of liver fibrosis in mice).</p>
<p style="text-align:justify"><span style="font-size:11pt"><span style="color:#000000"><strong><span style="font-size:10pt"><span style="color:red">The AOP described is valid for both sexes. Epidemiological studies carried out on chronic hepatitis patients living around the Da Nang air base in Vietnam have shown that increased levels of TCDD in the blood were associated, among other things, with elevated stages of liver fibrosis (classified using the METAVIR fibrosis staging system); this is also true for PCDF. Dioxins and furans are specific ligands of the AhR (whose activation represents MIE and hepatic fibrosis AO) (doi: 10.3390/toxics10060315). These results suggest that exposure to TCDD can influence an evolution of the hepatic environment towards fibrosis, which also increases the risk of liver cancer. They suggest that all subjects living in dioxin-contaminated areas in Vietnam should undergo regular check-ups, in particular liver function tests and imaging examinations. These results are consistent with experimental studies, which are consistent with the conservation of the AhR pathway in vertebrates. Thus, animal models (mainly rodents) have been used to study this fibrogenesis, demonstrating that the co-occurrence of external factors (high-fat diet, for example) contributes to this fibrosis at low doses of AhR ligands.</span></span></strong></span></span></p>
<p style="text-align:justify"><span style="font-size:11pt"><span style="color:#000000"><strong><span style="font-size:10pt"><span style="color:red">Pham PQ, Nguyen VB, Pham TT, Duong NX, Nguyen HT, Ha QV, Nguyen TD, Hoang TM, Dinh DT, Tran QTN, Bui LK, Vu TT, Phan MV, Luong TM, Nguyen K, Vu DA, Pham TN. Histopathological Alterations in the Livers of Chronic Hepatitis Patients Exposed to Agent Orange/Dioxin in Vietnam. Toxics. 2022 Jun 10;10(6):315. doi: 10.3390/toxics10060315. PMID: 35736923; PMCID: PMC9229980.</span></span></strong></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">STRONG</span></span></span></strong></span></span></p>
</td>
<td>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">Liver steatosis or fatty liver, is essential in promoting liver fibrosis because it sets off a cascade of pathological mechanisms that drive liver damage and the fibrotic response (and all other key events) and subsequently It is considered as a risk factor for fibrosis, according to the two-strikes hypothesis (Day and James, 1998): this hypothesis is a conceptual framework describing how liver damage progresses, often culminating in fibrosis, cirrhosis, or even liver failure. This hypothesis highlights the sequential and synergistic nature of liver injury and involves two key stages: the first strike involves an initial liver insult, such as accumulation of fat in liver cells (KE 459); this stage is often asymptomatic and involves increased liver vulnerability due to mild oxidative stress, fat accumulation, or mild inflammation. The second strike involves additional insults that exacerbate the damage, leading to inflammation and fibrogenesis (see other KEs below).</span></span></span></strong></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">Liver steatosis is characterized by the accumulation of lipids in hepatocytes. This can result from metabolic disturbances, such as excess free fatty acids, insulin resistance, or oxidative stress (Pafili K et al, 2021). Subsequently, steatosis sensitizes the liver to further injury by increasing oxidative stress, ultimately leading to activation of Hepatic Stellate Cells (HSCs), apoptosis or necrosis of hepatocytes.</span></span></span></strong></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">While liver steatosis is reversible in early stages, chronic steatosis exacerbates damage as it perpetuates a feedback loop of inflammation and fibrosis. This sequence makes steatosis an essential precursor and contributor to liver fibrosis.</span></span></span></strong></span></span></p>
<p style="text-align:justify"> </p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><strong><em><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">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.</span></span></span></em></strong></span></span></p>
<p style="text-align:justify"> </p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><strong><em><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">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.</span></span></span></em></strong></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">STRONG</span></span></span></strong></span></span></p>
</td>
<td>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><a name="bb0210"><strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">Cell injury and death are crucial initiating events in this AOP leading to liver fibrosis. Indeed, damaged hepatocytes release damage-associated molecular patterns (DAMPs), cytokines, and reactive oxygen species (ROS), which stimulate inflammation and HSC activation, a key event in liver fibrosis (see below). Hepatocyte cell death in the liver also leads to Kupffer cell activation (liver-resident macrophages), which secrete pro-inflammatory cytokines like TNF-</span></span></span></strong></a><strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">α</span></span></span></strong><strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red"> and IL-1</span></span></span></strong><strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">β</span></span></span></strong><strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">.</span></span></span></strong></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">The essentiality of this KE regarding liver fibrosis is demonstrated by inhibition of hepatocyte apoptosis or necrosis which reduces fibrosis in animal models (Guo R et al, 2022; Jiang M et al, 2023). Moreover, chronic liver diseases associated with fibrosis, such as non-alcoholic steatohepatitis (NASH), viral hepatitis, or alcoholic liver disease, show marked hepatocyte injury/death (Gaul S et al, 2021).</span></span></span></strong></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">Such observations make increased cell injury/death, an essential precursor and contributor to liver fibrosis.</span></span></span></strong></span></span></p>
<p style="text-align:justify"> </p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><strong><em><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">Guo R, Jia X, Ding Z, Wang G, Jiang M, Li B, Chen S, Xia B, Zhang Q, Liu J, Zheng R, Gao Z, Xie X. Loss of MLKL ameliorates liver fibrosis by inhibiting hepatocyte necroptosis and hepatic stellate cell activation. </span></span></span></em></strong><strong><em><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">Theranostics. 2022 Jul 4;12(11):5220-5236. doi: 10.7150/thno.71400. PMID: 35836819; PMCID: PMC9274737.</span></span></span></em></strong></span></span></p>
<p style="text-align:justify"> </p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><strong><em><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">Jiang M, Huang C, Wu Q, Su Y, Wang X, Xuan Z, Wang Y, Xu F, Ge C. Sini San ameliorates CCl4-induced liver fibrosis in mice by inhibiting AKT-mediated hepatocyte apoptosis. </span></span></span></em></strong><strong><em><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">J Ethnopharmacol. 2023 Mar 1;303:115965. doi: 10.1016/j.jep.2022.115965. Epub 2022 Nov 29. PMID: 36460296.</span></span></span></em></strong></span></span></p>
<p style="text-align:justify"> </p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><strong><em><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">Gaul S, Leszczynska A, Alegre F, Kaufmann B, Johnson CD, Adams LA, Wree A, Damm G, Seehofer D, Calvente CJ, Povero D, Kisseleva T, Eguchi A, McGeough MD, Hoffman HM, Pelegrin P, Laufs U, Feldstein AE. </span></span></span></em></strong><strong><em><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">Hepatocyte pyroptosis and release of inflammasome particles induce stellate cell activation and liver fibrosis. J Hepatol. 2021 Jan;74(1):156-167. doi: 10.1016/j.jhep.2020.07.041. Epub 2020 Aug 4. PMID: 32763266; PMCID: PMC7749849.</span></span></span></em></strong></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">STRONG</span></span></span></strong></span></span></p>
</td>
<td>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">Activation of hepatic stellate cells (HSCs) in liver fibrosis is considered as a critical and central event in the pathogenesis of fibrosis. Indeed, activation of HSCs leads to HSCs transition from a quiescent, vitamin A-storing phenotype to a myofibroblast-like phenotype that actively produces extracellular matrix (ECM) components such as collagen type I (Kamm DR et al, 2022), but also to the secretion of pro-fibrotic mediators (e.g., transforming growth factor-beta, TGF-β) and cytokines, amplifying the fibrotic response and creating a feed-forward loop of chronic liver damage (Dewidar B et al, 2019). </span></span></span></strong></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">A link of causality can be drawn as inhibition of HSC activation (e.g., through targeting TGF-β signaling, oxidative stress, or specific HSC markers like Hexokinase 2) significantly reduces fibrosis in animal models (Rho H et al, 2023).</span></span></span></strong></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">As a conclusion, the activation of HSC is essential for liver fibrosis, with strong experimental and mechanistic evidence supporting its central role. It serves as a critical target in developing anti-fibrotic therapies.</span></span></span></strong></span></span></p>
<p style="text-align:justify"> </p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><strong><em><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">Kamm DR, McCommis KS. Hepatic stellate cells in physiology and pathology. J Physiol. 2022 Apr;600(8):1825-1837. doi: 10.1113/JP281061. Epub 2022 Mar 30. PMID: 35307840; PMCID: PMC9012702.</span></span></span></em></strong></span></span></p>
<p style="text-align:justify"> </p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><strong><em><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">Dewidar B, Meyer C, Dooley S, Meindl-Beinker AN. TGF-</span></span></span></em></strong><strong><em><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">β</span></span></span></em></strong><strong><em><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red"> in Hepatic Stellate Cell Activation and Liver Fibrogenesis-Updated 2019. </span></span></span></em></strong><strong><em><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">Cells. 2019 Nov 11;8(11):1419. doi: 10.3390/cells8111419. PMID: 31718044; PMCID: PMC6912224.</span></span></span></em></strong></span></span></p>
<p style="text-align:justify"> </p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><strong><em><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">Rho H, Terry AR, Chronis C, Hay N. Hexokinase 2-mediated gene expression via histone lactylation is required for hepatic stellate cell activation and liver fibrosis. Cell Metab. 2023 Aug 8;35(8):1406-1423.e8. doi: 10.1016/j.cmet.2023.06.013. Epub 2023 Jul 17. PMID: 37463576; PMCID: PMC11748916.</span></span></span></em></strong></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">HIGH</span></span></span></strong></span></span></p>
</td>
<td>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">Extracellular matrix (ECM) deposition is a critical hallmark of liver fibrosis. It represents the accumulation of fibrotic scar tissue due to excessive synthesis and reduced degradation of ECM components (e.g., collagen types I and III) (Caligiuri A et al, 2021). This deposition disrupts liver architecture and impairs function.</span></span></span></strong></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">ECM deposition is considered a highly essential event for liver fibrosis progression because it marks the transition from reversible inflammation to irreversible fibrosis, it correlates with disease severity and progression to cirrhosis (Caligiuri A et al, 2021).</span></span></span></strong></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">Experimental evidence often supports the essentiality, such as fibrosis models showing reduced ECM deposition when specific pathways (e.g., TGF-β or integrin signaling) are inhibited (Fan W et al, 2019). ECM deposition is a measurable KE using histological, biochemical, and imaging methods (e.g., Masson’s trichrome staining, hydroxyproline content, or MRI elastography, Red sirius).</span></span></span></strong></span></span></p>
<p style="text-align:justify"> </p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><strong><em><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">Caligiuri A, Gentilini A, Pastore M, Gitto S, Marra F. Cellular and Molecular Mechanisms Underlying Liver Fibrosis Regression. </span></span></span></em></strong><strong><em><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">Cells. 2021 Oct 15;10(10):2759. doi: 10.3390/cells10102759. PMID: 34685739; PMCID: PMC8534788.</span></span></span></em></strong></span></span></p>
<p style="text-align:justify"> </p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><strong><em><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">Fan W, Liu T, Chen W, Hammad S, Longerich T, Hausser I, Fu Y, Li N, He Y, Liu C, Zhang Y, Lian Q, Zhao X, Yan C, Li L, Yi C, Ling Z, Ma L, Zhao X, Xu H, Wang P, Cong M, You H, Liu Z, Wang Y, Chen J, Li D, Hui L, Dooley S, Hou J, Jia J, Sun B. ECM1 Prevents Activation of Transforming Growth Factor </span></span></span></em></strong><strong><em><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">β</span></span></span></em></strong><strong><em><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">, Hepatic Stellate Cells, and Fibrogenesis in Mice. </span></span></span></em></strong><strong><em><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">Gastroenterology. 2019 Nov;157(5):1352-1367.e13. doi: 10.1053/j.gastro.2019.07.036. Epub 2019 Jul 27. PMID: 31362006.</span></span></span></em></strong></span></span></p>
<td><a href="/aops/21">Aop:21 - Aryl hydrocarbon receptor activation leading to early life stage mortality, via increased COX-2</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/57">Aop:57 - AhR activation leading to hepatic steatosis</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/131">Aop:131 - Aryl hydrocarbon receptor activation leading to uroporphyria</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/150">Aop:150 - Aryl hydrocarbon receptor activation leading to early life stage mortality, via reduced VEGF</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/310">Aop:310 - Embryonic Activation of the AHR leading to Reproductive failure, via epigenetic down-regulation of GnRHR</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/151">Aop:151 - AhR activation leading to preeclampsia</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/414">Aop:414 - Aryl hydrocarbon receptor activation leading to lung fibrosis through TGF-β dependent fibrosis toxicity pathway</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/415">Aop:415 - Aryl hydrocarbon receptor activation leading to lung fibrosis through IL-6 toxicity pathway</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/416">Aop:416 - Aryl hydrocarbon receptor activation leading to lung cancer through IL-6 toxicity pathway</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/417">Aop:417 - Aryl hydrocarbon receptor activation leading to lung cancer through AHR-ARNT toxicity pathway</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/418">Aop:418 - Aryl hydrocarbon receptor activation leading to impaired lung function through AHR-ARNT toxicity pathway</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/419">Aop:419 - Aryl hydrocarbon receptor activation leading to impaired lung function through P53 toxicity pathway</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/420">Aop:420 - Aryl hydrocarbon receptor activation leading to lung cancer through sustained NRF2 toxicity pathway</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/439">Aop:439 - Activation of the AhR leading to metastatic breast cancer </a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/455">Aop:455 - Aryl hydrocarbon receptor activation leading to early life stage mortality via sox9 repression induced impeded craniofacial development</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/456">Aop:456 - Aryl hydrocarbon receptor activation leading to early life stage mortality via sox9 repression induced cardiovascular toxicity</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/458">Aop:458 - AhR activation in the liver leading to Subsequent Adverse Neurodevelopmental Outcomes in Mammals</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/494">Aop:494 - AhR activation leading to liver fibrosis </a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/459">Aop:459 - AhR activation in the thyroid leading to Subsequent Adverse Neurodevelopmental Outcomes in Mammals</a></td>
<p>The AHR structure has been shown to contribute to differences in species sensitivity to DLCs in several animal models. In 1976, a 10-fold difference was reported between two strains of mice (non-responsive DBA/2 mouse, and responsive C57BL/6 14 mouse) in CYP1A induction, lethality and teratogenicity following TCDD exposure<sup><a href="#cite_note-Poland1976-3">[3]</a></sup>. This difference in dioxin sensitivity was later attributed to a single nucleotide polymorphism at position 375 (the equivalent position of amino acid residue 380 in chicken) in the AHR LBD<sup><a href="#cite_note-Ema1994-30">[30]</a></sup><sup><a href="#cite_note-Poland1982-19">[19]</a></sup><sup><a href="#cite_note-Poland1994-31">[31]</a></sup>. Several other studies reported the importance of this amino acid in birds and mammals<sup><a href="#cite_note-Backlund2004-32">[32]</a></sup><sup><a href="#cite_note-Ema1994-30">[30]</a></sup><sup><a href="#cite_note-Karchner2006-22">[22]</a></sup><sup><a href="#cite_note-Murray2005-33">[33]</a></sup><sup><a href="#cite_note-Pandini2007-34">[34]</a></sup><sup><a href="#cite_note-Pandini2009-35">[35]</a></sup><sup><a href="#cite_note-Poland1994-31">[31]</a></sup><sup><a href="#cite_note-Ramadoss2004-36">[36]</a></sup>. It has also been shown that the amino acid at position 319 (equivalent to 324 in chicken) plays an important role in ligand-binding affinity to the AHR and transactivation ability of the AHR, due to its involvement in LBD cavity volume and its steric effect<sup><a href="#cite_note-Pandini2009-35">[35]</a></sup>. Mutation at position 319 in the mouse eliminated AHR DNA binding<sup><a href="#cite_note-Pandini2009-35">[35]</a></sup>.</p>
<p>The first study that attempted to elucidate the role of avian AHR1 domains and key amino acids within avian AHR1 in avian differential sensitivity was performed by Karchner <em>et al.</em><sup><a href="#cite_note-Karchner2006-22">[22]</a></sup>. Using chimeric AHR1 constructs combining three AHR1 domains (DBD, LBD and TAD) from the chicken (highly sensitive to DLC toxicity) and common tern (resistant to DLC toxicity), Karchner and colleagues<sup><a href="#cite_note-Karchner2006-22">[22]</a></sup>, showed that amino acid differences within the LBD were responsible for differences in TCDD sensitivity between the chicken and common tern. More specifically, the amino acid residues found at positions 324 and 380 in the AHR1 LBD were associated with differences in TCDD binding affinity and transactivation between the chicken (Ile324_Ser380) and common tern (Val324_Ala380) receptors<sup><a href="#cite_note-Karchner2006-22">[22]</a></sup>. Since the Karchner et al. (2006) study was conducted, the predicted AHR1 LBD amino acid sequences were been obtained for over 85 species of birds and 6 amino acid residues differed among species<sup><a href="#cite_note-Farmahin2013b-14">[14]</a></sup><sup><a href="#cite_note-Head2008-37">[37]</a></sup> . However, only the amino acids at positions 324 and 380 in the AHR1 LBD were associated with differences in DLC toxicity in ovo and AHR1-mediated gene expression in vitro<sup><a href="#cite_note-Farmahin2013b-14">[14]</a></sup><sup><a href="#cite_note-Head2008-37">[37]</a></sup><sup><a href="#cite_note-Manning2012-16">[16]</a></sup>. These results indicate that avian species can be divided into one of three AHR1 types based on the amino acids found at positions 324 and 380 of the AHR1 LBD: type 1 (Ile324_Ser380), type 2 (Ile324_Ala380) and type 3 (Val324_Ala380)<sup><a href="#cite_note-Farmahin2013b-14">[14]</a></sup><sup><a href="#cite_note-Head2008-37">[37]</a></sup><sup><a href="#cite_note-Manning2012-16">[16]</a></sup>.</p>
<ul>
<li>Little is known about differences in binding affinity of AhRs and how this relates to sensitivity in non-avian taxa.</li>
<li>Low binding affinity for DLCs of AhR1s of African clawed frog (<em>Xenopus laevis</em>) and axolotl (<em>Ambystoma mexicanum</em>) has been suggested as a mechanism for tolerance of these amphibians to DLCs (Lavine et al 2005; Shoots et al 2015).</li>
<li>Among reptiles, only AhRs of American alligator (<em>Alligator mississippiensis</em>) have been investigated and little is known about the sensitivity of American alligator or other reptiles to DLCs (Oka et al 2016).</li>
<li>Among fishes, great differences in sensitivity to DLCs are known both for AhRs and for embryos among species that have been tested (Doering et al 2013; 2014).</li>
<li>Differences in binding affinity of the AhR2 have been demonstrated to explain differences in sensitivity to DLCs between sensitive and tolerant populations of Atlantic Tomcod (<em>Microgadus tomcod</em>) (Wirgin et al 2011).
<ul>
<li>This was attributed to the rapid evolution of populations in highly contaminated areas of the Hudson River, resulting in a 6-base pair deletion in the AHR sequence (outside the LBD) and reduced ligand binding affinity, due to reduces AHR protein stability.</li>
</ul>
</li>
<li>Information is not yet available regarding whether differences in binding affinity of AhRs of fishes are predictive of differences in sensitivity of embryos, juveniles, or adults (Doering et al 2013).</li>
</ul>
<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif">The AhR is a very conserved and ancient protein (95) and the AhR is present in human and mice (96–98). </span></span><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-size:10.0pt"><span style="font-family:"Times New Roman",serif">The AhR is present in human physiology and pathology. T</span></span><span style="font-size:10.0pt"><span style="font-family:"Times New Roman",serif">he AhR is highly expressed at several important physiological barriers such as the placenta, lung, gastrointestinal system, and liver in human (Wakx, Marinelli, Watanabe). In these tissues, the AhR is involved in both detoxication processes involving xenobiotic metabolizing enzymes such as cytochromes P450, and in immune functions translating chemical signals into immune defence pathways (Marinelli, Stobbe). Moreover, it has a regulatory role in human dendritic cells and myelination (Kado, Shackleford).</span></span> <span style="font-size:10.0pt"><span style="font-family:"Times New Roman",serif">The lung constitutes another barrier exposed to components of air pollution such as particles and hydrocarbons (air pollution, cigarette smoke). The AhR detects such hydrocarbons and protects the pulmonary cells from their deleterious effects through metabolization.</span></span> <span style="font-size:10.0pt"><span style="font-family:"Times New Roman",serif">The regulatory effect on blood cells of the AhR, balancing different related cell types, can be extended to the megakaryocytes and their precursors; indeed, StemRegenin 1 (SR1), an antagonist of the AhR increases the human population of CD34+CD41low cells, a fraction of very efficient precursors of proplatelets (Bock).</span></span> <span style="font-size:10.0pt"><span style="font-family:"Times New Roman",serif">The occurrence of a nystagmus has been subsequently diagnosed in humans bearing a AhR mutation (Borovok).</span></span></span></span></p>
<p><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-size:10.0pt"><span style="font-family:"Times New Roman",serif">In human cancer, the AhR has either a pro or con tumor effect depending on the tissue, the ligand, and the duration of the activation (Zudaire, Chang, Litzenburg, Gramatzki, Lin, Wang). In human breast cancer, the AhR is thoughts to be responsible of its progression (Goode, Kanno, Optiz, Novikov, Hall, </span></span><span style="font-size:10.0pt"><span style="font-family:"Times New Roman",serif">Subramaniam, Barhoover</span></span><span style="font-size:10.0pt"><span style="font-family:"Times New Roman",serif">). </span></span><span style="font-size:10.0pt"><span style="font-family:"Times New Roman",serif">In human mammary benign cells, Brooks et al. noted that a high level of AhR was associated with a modified cell cycle (with a 50% increase in population doubling time in cells expressing the AhR by more than 3-fold) and EMT including increased cell migration. Narasimnhan et al. found that suppression of the AhR pathway had a pro-tumorigenic effect in vitro (EMT, tumor migration) in triple negative breast cancer.</span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-size:10.0pt"><span style="font-family:"Times New Roman",serif">Many endogenous and exogenous ligands are present for the AhR in human (Optiz, Adachi, Schroeder, </span></span><span style="font-size:10.0pt"><span style="font-family:"Times New Roman",serif">Rothhammer</span></span><span style="font-size:10.0pt"><span style="font-family:"Times New Roman",serif">). </span></span><span style="font-size:10.0pt"><span style="font-family:"Times New Roman",serif">Indoles, such as indole-3-carbinol or one of its secondary metabolites, 3-3'- Diindolylmethane, are degradation products found in cruciferous vegetables and characterized as AhR ligands (Ema, Kall, Miller) they are also inducers of the human and rat CYP1A1 (Optiz). FICZ is the most potent AhR ligand known to date: it has a stronger affinity than TCDD for the human AhR (TCDD Kd=0.48 nM/FICZ Kd=0.07 nM) (Coumoul).</span></span></span></span></p>
<p> </p>
<p> </p>
<h4>Key Event Description</h4>
<h3>The AHR Receptor</h3>
<p>The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that belongs to the basic helix-loop-helix Per-ARNT-Sim (bHLH-PAS) superfamily and consists of three domains: the DNA-binding domain (DBD), ligand binding domain (LBD) and transactivation domain (TAD)<sup><a href="#cite_note-Okey2007-1">[1]</a></sup>. Other members of this superfamily include the AHR nuclear translocator (ARNT), which acts as a dimerization partner of the AHR <sup><a href="#cite_note-Hoffman1991-2">[2]</a></sup><sup><a href="#cite_note-Poland1976-3">[3]</a></sup>; Per, a circadian transcription factor; and Sim, the “single-minded” protein involved in neuronal development <sup><a href="#cite_note-Gu2000-4">[4]</a></sup><sup><a href="#cite_note-Kewley2004-5">[5]</a></sup>. This group of proteins shares a highly conserved PAS domain and is involved in the detection of and adaptation to environmental change<sup><a href="#cite_note-Gu2000-4">[4]</a></sup>.</p>
<p>Investigations of invertebrates possessing early homologs of the AhR suggest that the AhR evolutionarily functioned in regulation of the cell cycle, cellular proliferation and differentiation, and cell-to-cell communications (Hahn et al 2002). However, critical functions in angiogenesis, regulation of the immune system, neuronal processes, metabolism, development of the heart and other organ systems, and detoxification have emerged sometime in early vertebrate evolution (Duncan et al., 1998; Emmons et al., 1999; Lahvis and Bradfield, 1998).</p>
<div>Figure 1: The molecular mechanism of activation of gene expression by AHR.</div>
<div> </div>
</div>
</div>
<p>The molecular mechanism for AHR-mediated activation of gene expression is presented in Figure 1. In its unliganded form, the AHR is part of a cytosolic complex containing heat shock protein 90 (HSP90), the HSP90 co-chaperone p23 and AHR-interacting protein (AIP)<sup><a href="#cite_note-Fujii2010-6">[6]</a></sup>. Upon ligand binding, the AHR migrates to the nucleus where it dissociates from the cytosolic complex and forms a heterodimer with ARNT<sup><a href="#cite_note-Mimura2003-7">[7]</a></sup>. The AHR-ARNT complex then binds to a xenobiotic response element (XRE) found in the promoter of an AHR-regulated gene and recruits co-regulators such as CREB binding protein/p300, steroid receptor co-activator (SRC) 1, SRC-2, SRC-3 and nuclear receptor interacting protein 1, leading to induction or repression of gene expression<sup><a href="#cite_note-Fujii2010-6">[6]</a></sup>. Expression levels of several genes, including phase I (e.g. cytochrome P450 (CYP) 1A, CYP1B, CYP2A) and phase II enzymes (e.g. uridine diphosphate glucuronosyl transferase (UDP-GT), glutathione S-transferases (GSTs)), as well as genes involved in cell proliferation (transforming growth factor-beta, interleukin-1 beta), cell cycle regulation (p27, jun-B) and apoptosis (Bax), are regulated through this mechanism <sup><a href="#cite_note-Fujii2010-6">[6]</a></sup><sup><a href="#cite_note-Giesy2006-8">[8]</a></sup><sup><a href="#cite_note-Mimura2003-7">[7]</a></sup><sup><a href="#cite_note-Safe1994-9">[9]</a></sup>.</p>
<h3>AHR Isoforms</h3>
<ul>
<li>Over time the AhR has undergone gene duplication and diversification in vertebrates, which has resulted in multiple clades of AhR, namely AhR1, AhR2, and AhR3 (Hahn 2002).</li>
<li>Fishes and birds express AhR1s and AhR2s, while mammals express a single AhR that is homologous to the AhR1 (Hahn 2002; Hahn et al 2006).</li>
<li>The AhR3 is poorly understood and known only from some cartilaginous fishes (Hahn 2002).</li>
<li>Little is known about diversity of AhRs in reptiles and amphibians (Hahn et al 2002).</li>
<li>In some taxa, subsequent genome duplication events have further led to multiple isoforms of AhRs in some species, with up to four isoforms of the AhR (α, β, δ, γ) having been identified in Atlantic salmon (<em>Salmo salar</em>) (Hansson et al 2004).</li>
<li>Although homologs of the AhR have been identified in some invertebrates, compared to vertebrates these AhRs have differences in binding of ligands in the species investigated to date (Hahn 2002; Hahn et al 1994).</li>
</ul>
<p> </p>
<p>Roles of isoforms in birds:</p>
<p>Two AHR isoforms (AHR1 and AHR2) have been identified in the black-footed albatross (<em>Phoebastria nigripes</em>), great cormorant (<em>Phalacrocorax carbo</em>) and domestic chicken (<em>Gallus gallus domesticus</em>)<sup><a href="#cite_note-Yasui2007-10">[10]</a></sup>. AHR1 mRNA levels were similar in the kidney, heart, lung, spleen, brain, gonad and intestine from the great cormorant but were lower in muscle and pancreas. AHR2 expression was mainly observed in the liver, but was also detected in gonad, brain and intestine. AHR1 levels represented a greater proportion (80%) of total AHR levels than AHR2 in the cormorant liver<sup><a href="#cite_note-Yasui2007-10">[10]</a></sup>, and while both AHR isoforms bound to TCDD, AHR2 was less effective at inducing TCDD-dependent transactivation compared to AHR1 in black-footed albatross, great cormorant and domestic chicken<sup><a href="#cite_note-Lee2009-11">[11]</a></sup><sup><a href="#cite_note-Yasui2007-10">[10]</a></sup>.</p>
<ul>
<li>AhR1 and AhR2 both bind and are activated by TCDD <em>in vitro</em> (Yasui et al 2007).</li>
<li>AhR1 has greater binding affinity and sensitivity to activation by TCDD relative to AhR2 (Yasui et al 2007).</li>
<li>AhR1 is believed to mediate toxicities of DLCs, while AhR2 has no known role in toxicities (Farmahin et al 2012; Farmahin et al 2013; Manning et al 2012).</li>
</ul>
<p>Roles of isoforms in fishes:</p>
<ul>
<li>AhR1 and AhR2 both bind and are activated by TCDD <em>in vitro</em> (Bak et al 2013; Doering et al 2014; 2015; Karchner et al 1999; 2005).</li>
<li>AhR1 has greater sensitivity to activation by TCDD than AhR2 in red seabream (<em>Pagrus major</em>), white sturgeon (<em>Acipenser transmontanus</em>), and lake sturgeon (<em>Acipenser fulvescens</em>) (Bak et al 2013; Doering et al 2014; 2015)</li>
<li>AhR2 has greater binding affinity or activation by TCDD than AhR1 in zebrafish (<em>Danio rerio</em>) and mummichog (<em>Fundulus heteroclitus</em>) (Karchner et al 1999; 2005).</li>
<li>AhR2 is believed to mediate toxicities in fishes, while AhR1 has no known role in toxicities. Specifically, knockdown of AhR2 protects against toxicities of dioxin-like compounds (DLCs) and polycyclic aromatic hydrocarbons (PAHs) in zebrafish (<em>Danio rerio</em>) and mummichog (<em>Fundulus heteroclitus</em>), while knockdown of AhR1 offers no protection (Clark et al 2010; Prasch et al 2003; Van Tiem & Di Giulio 2011).</li>
</ul>
<p>Roles of isoforms in amphibians and reptiles:</p>
<ul>
<li>Less is known about AhRs of amphibians or reptiles.</li>
<li>AhR1 is believed to mediate toxicities in amphibians (Hahn 2002; Lavine et al 2005; Oka et al 2016; Shoots et al 2015). However, all AhRs of amphibians that have been investigated have very low affinity for TCDD (Hahn 2002; Lavine et al 2005; Oka et al 2016; Shoots et al 2015).</li>
<li>Both AhR1s and AhR2 of American alligator (<em>Alligator mississippiensis</em>) are activated by agonists with comparable sensitivities (Oka et al 2016). AhRs of no other reptiles have been investigated.</li>
</ul>
<p><em><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:14px">Role in mammals</span></span></em></p>
<p><em><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:14px">AhR expression is essentially ubiquitous in mammals consistent with a broad-spectrum homeostatic role, however expression levels varying widely across tissues with the liver, thymus, lung, kidney, spleen, and placenta exhibiting greatest expression (Harper PA). Additionally, AhR expression is developmentally regulated, and more recent evidence indicates a role for the AhR in developmental process affecting hematopoiesis, immune system biology, neural differentiation, and liver architecture (Wright E J) . AHR is involved in regulating the rate of apoptosis of oocytes in germ cell nests during embryonic life and in regulating survival of oocytes in the fetal and neonatal ovary. Specifically, studies have shown that ovaries obtained from AHRKO mice on ED13.5 and cultured for 72 h in the absence of hormonal support with the aim of inducing apoptosis, contained higher numbers of non-apoptotic germ cells compared to wild-type (WT) ovaries cultured in the same conditions (Hernández-Ochoa)</span></span></em></p>
<p> </p>
<h4>How it is Measured or Detected</h4>
<p><em>Methods that have been previously reviewed and approved by a recognized authority should be included in the Overview section above. All other methods, including those well established in the published literature, should be described here. Consider the following criteria when describing each method: 1. Is the assay fit for purpose? 2. Is the assay directly or indirectly (i.e. a surrogate) related to a key event relevant to the final adverse effect in question? 3. Is the assay repeatable? 4. Is the assay reproducible? </em></p>
<p>Transient transfection transactivation is the most common method for evaluating nuclear receptor activation<sup><a href="#cite_note-Raucy2010-12">[12]</a></sup>. Full-length AHR cDNAs are cloned into an expression vector along with a reporter gene construct (chimeric luciferase, P-lactamase or CAT reporter vectors containing the appropriate response elements for the gene of interest). There are a number of commercially available cell lines that can serve as recipients for these vectors (CV-1, HuH7, FLC-7, LS174T, LS180 MCF-7, HEC1, LLC-PK1, HEK293, HepG2, and Caco-2 cells)<sup><a href="#cite_note-Raucy2010-12">[12]</a></sup>. The greatest advantage of using transfected cells, rather than primary cell cultures, is the assurance that the nuclear receptor of interest is responsible for the observed induction. This would not be possible in a primary cell culture due to the co-regulation of different receptors for the same target genes. This model makes it easy to compare the responsiveness of the AHR across multiple species under the same conditions simply by switching out the AHR clone. One disadvantage to the transient transfection assay is the inherent variability associated with transfection efficiency, leading to a movement towards the use of stable cell lines containing the nuclear receptor and reporter gene linked to the appropriate response elements<sup><a href="#cite_note-Raucy2010-12">[12]</a></sup>.</p>
<h5>Luciferase reporter gene (LRG) assay</h5>
<p>The described luciferase reporter gene (LRG) assays have been used to investigate activation of AhRs of:</p>
<ul>
<li>Humans (<em>Homo sapiens</em>) (Abnet et al 1999) </li>
<li>Species of birds, namely chicken (<em>Gallus gallus</em>), ring-necked pheasant (<em>Phasianus colchicus</em>), Japanese quail (<em>Coturnix japonica</em>), and common tern (<em>Sterna hirundo</em>) (Farmahin et al 2012; Manning et al 2013), Mutant AhR1s with ligand binding domains resembling those of at least 86 avian species have also been investigated (Farmahin et al 2013). AhR2s of birds have only been investigated in black-footed albatross (<em>Phoebastria nigripes</em>) and common cormorant (<em>Phalacrocorax carbo</em>) (Yasio et al 2007).</li>
<li>American alligator (<em>Alligator mississippiensis</em>) is the only reptile for which AhR activation has been investigated (Oka et al 2016), AhR1A, AhR1B, and AhR2 of American alligator were assayed (Oka et al 2016).</li>
<li>AhR1 of two amphibians have been investigated, namely African clawed frog (<em>Xenopus laevis</em>) and salamander (<em>Ambystoma mexicanum</em>) (Lavine et al 2005; Shoots et al 2015; Ohi et al 2003),</li>
<li>AhR1s and AhR2s of several species of fish have been investigated, namely Atlantic salmon (<em>Salmo salar</em>), Atlantic tomcod (<em>Microgadus tomcod</em>), white sturgeon (<em>Acipenser transmontanus</em>), rainbow trout (<em>Onchorhynchys mykiss</em>), red seabream (<em>Pagrus major</em>), lake sturgeon (<em>Acipenser fulvescens</em>), and zebrafish (<em>Danio rerio</em>) (Andreasen et al 2002; Abnet et al 1999; Bak et al 2013; Doering et al 2014; 2015; Evans et al 2005; Hansson & Hahn 2008; Karchner et al 1999; Tanguay et al 1999; Wirgin et al 2011).</li>
</ul>
<p>For demonstrative purposes, a luciferase reporter gene assay used to measure AHR1-mediated transactivation for avian species is described here. However, comparable assays are utilized for investigating AHR1s and AHR2s of all taxa. A monkey kidney cell line (Cos-7) that has low endogenous AHR1 expression was transfected with the appropriate avian AHR1 clone, cormorant ARNT1, a CYP1A5 firefly luciferase reporter construct and a <em>Renilla</em> luciferase vector to control for transfection efficiency. After seeding, the cells were exposed to DLC and luciferase activity was measured using a luminometer. Luminescence, which is proportional to the extent of AHR activation, is expressed as the ratio of firefly luciferase units to <em>Renilla</em> luciferase units <sup><a href="#cite_note-Farmahin2012-13">[13]</a></sup>. This particular assay was modified from its original version to increase throughput efficiency; (a) cells were seeded in 96-well plates rather than Petri dishes or 48- well plates, (b) DLCs were added directly to the wells without changing the cell culture medium, and (c) the same 96-well plates were used to measure luminescence without lysing the cells and transferring to another plate. Similar reporter gene assays have been used to measure AHR1 activation in domestic and wild species of birds, including the chicken, ring-necked pheasant (Phasianus colchicus), Japanese quail (Coturnix japonica), great cormorant, black-footed albatross and peregrine falcon (Falco peregrinus).<sup><a href="#cite_note-Farmahin2013b-14">[14]</a></sup><sup><a href="#cite_note-Farmahin2012-13">[13]</a></sup><sup><a href="#cite_note-Fujisawa2012-15">[15]</a></sup><sup><a href="#cite_note-Lee2009-11">[11]</a></sup><sup><a href="#cite_note-Manning2012-16">[16]</a></sup><sup><a href="#cite_note-Mol2012-17">[17]</a></sup></p>
<h4>Transactivation in stable cell lines</h4>
<p>Stable cell lines have been developed and purified to the extent that each cell contains both the nuclear receptor and appropriate reporter vector, eliminating the variability associated with transfection <sup><a href="#cite_note-Raucy2010-12">[12]</a></sup>. A stable human cell line containing a luciferase reporter driven by multiple dioxin response elements has been developed that is useful in identifying AhR agonists and antagonists<sup><a href="#cite_note-Yueh2005-18">[18]</a></sup>. An added benefit of this model is the potential to multiplex 3 assays in a single well: receptor activation, cell viability and enzyme activity<sup><a href="#cite_note-Raucy2010-12">[12]</a></sup>. Such assays are used extensively in drug discovery due to their high throughput efficiency, and may serve just as useful for risk assessment purposes.</p>
<h3>Ligand-Binding Assays</h3>
<p>Ligand binding assays measure the ability of a test compound to compete with a labeled, high-affinity reference ligand for the LBD of a nuclear receptor. It is important to note that ligand binding does not necessitate receptor activation and therefore cannot distinguish between agonists and antagonists; however, binding affinities of AHR ligands are highly correlated with chemical potencies<sup><a href="#cite_note-Poland1982-19">[19]</a></sup> and can explain differences in species sensitivities to DLCs<sup><a href="#cite_note-Hesterman2000-20">[20]</a></sup><sup><a href="#cite_note-Farmahin2014-21">[21]</a></sup><sup><a href="#cite_note-Karchner2006-22">[22]</a></sup>; they are therefore worth mentioning. Binding affinity and efficacy have been used to develop structure-activity relationships for AHR disruption<sup><a href="#cite_note-Hesterman2000-20">[20]</a></sup><sup><a href="#cite_note-Lee2015-23">[23]</a></sup> that are potentially useful in risk-assessment. There has been tremendous progress in the development of ligand-binding assays for nuclear receptors that use homogenous assay formats (no wash steps) allowing for the detection of low-affinity ligands, many of which do not require a radiolabel and are amenable to high throughput screening<sup><a href="#cite_note-Jones2003-24">[24]</a></sup><sup><a href="#cite_note-Raucy2010-12">[12]</a></sup>. This author however was unable to find specific examples of such assays in the context of AHR binding and therefore some classic radioligand assays are described instead.</p>
<h4>Hydroxyapatite (HAP) binding assay</h4>
<p>The HAP binding assay makes use of an <em>in vitro</em> transcription/translation method to synthesize the AHR protein, which is then incubated with radiolabeled TDCPP and a HAP pellet. The occupied protein adsorbs to the HAP and the radioactivity is measured to determine saturation binding. An additional ligand can also be included in the mixture in order to determine its binding affinity relative to TCDD (competitive binding)<sup><a href="#cite_note-Gasiewicz1982-25">[25]</a></sup><sup><a href="#cite_note-Karchner2006-22">[22]</a></sup>. This assay is simple, repeatable and reproducible; however, it is insensitive to weak ligand-receptor interactions<sup><a href="#cite_note-Karchner2006-22">[22]</a></sup><sup><a href="#cite_note-Farmahin2014-21">[21]</a></sup><sup><a href="#cite_note-Nakai1995-26">[26]</a></sup>.</p>
<h4>Whole cell filtration binding assay</h4>
<p>Dold and Greenlee<sup><a href="#cite_note-Dold1990-27">[27]</a></sup> developed a method to detect specific binding of TCDD to whole mammalian cells in culture and was later modified by Farmahin et al.<sup><a href="#cite_note-Farmahin2014-21">[21]</a></sup> for avian species. The cultured cells are incubated with radiolabeled TCDD with or without the presence of a competing ligand and filtered. The occupied protein adsorbs onto the filter and the radioactivity is measured to determine saturation binging and/or competitive binding. This assay is able to detect weak ligand-receptor interactions that are below the detection limit of the HAP assay<sup><a href="#cite_note-Farmahin2014-21">[21]</a></sup>.</p>
<h3>Protein-DNA Interaction Assays</h3>
<p>The active AHR complexed with ARNT can be measured using protein-DNA interaction assays. Two methods are described in detail by Perez-Romero and Imperiale<sup><a href="#cite_note-Perez2007-28">[28]</a></sup>. Chromatin immunoprecipitation measures the interaction of proteins with specific genomic regions <em>in vivo</em>. It involves the treatment of cells with formaldehyde to crosslink neighboring protein-protein and protein-DNA molecules. Nuclear fractions are isolated, the genomic DNA is sheared, and nuclear lysates are used in immunoprecipitations with an antibody against the protein of interest. After reversal of the crosslinking, the associated DNA fragments are sequenced. Enrichment of specific DNA sequences represents regions on the genome that the protein of interest is associated with <em>in vivo</em>. Electrophoretic mobility shift assay (EMSA) provides a rapid method to study DNA-binding protein interactions in vitro. This relies on the fact that complexes of protein and DNA migrate through a nondenaturing polyacrylamide gel more slowly than free DNA fragments. The protein-DNA complex components are then identified with appropriate antibodies. The EMSA assay was found to be consistent with the LRG assay in chicken hepatoma cells dosed with dioxin-like compounds<sup><a href="#cite_note-Heid2001-29">[29]</a></sup>.</p>
<h3>In silico Approaches</h3>
<p>In silico homology modeling of the ligand binding domain of the AHR in combination with molecular docking simulations can provide valuable insight into the transactivation-potential of a diverse array of AHR ligands. Such models have been developed for multiple AHR isoforms and ligands (high/low affinity, endogenous and synthetic, agonists and antagonists), and can accurately predict ligand potency based on their structure and physicochemical properties (Bonati et al 2017; Hirano et al 2015; Sovadinova et al 2006).</p>
<h4>References</h4>
<ol>
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<li><a href="#cite_ref-Hoffman1991_2-0">↑</a> Hoffman, E. C., Reyes, H., Chu, F. F., Sander, F., Conley, L. H., Brooks, B. A., and Hankinson, O. (1991). Cloning of a factor required for activity of the Ah (dioxin) receptor. <em>Science</em> <strong>252</strong>, 954-958.</li>
<li>↑ <sup><a href="#cite_ref-Poland1976_3-0">3.0</a></sup> <sup><a href="#cite_ref-Poland1976_3-1">3.1</a></sup> Poland, A., Glover, E., and Kende, A. S. (1976). Stereospecific, high affinity binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin by hepatic cytosol. Evidence that the binding species is receptor for induction of aryl hydrocarbon hydroxylase. <em>J.Biol.Chem.</em> <strong>251</strong>, 4936-4946.</li>
<li>↑ <sup><a href="#cite_ref-Gu2000_4-0">4.0</a></sup> <sup><a href="#cite_ref-Gu2000_4-1">4.1</a></sup> Gu, Y. Z., Hogenesch, J. B., and Bradfield, C. A. (2000). The PAS superfamily: sensors of environmental and developmental signals. <em>Annu.Rev.Pharmacol.Toxicol.</em> <strong>40</strong>, 519-561.</li>
<li><a href="#cite_ref-Kewley2004_5-0">↑</a> Kewley, R. J., Whitelaw, M. L., and Chapman-Smith, A. (2004). The mammalian basic helix-loop-helix/PAS family of transcriptional regulators. <em>Int.J.Biochem.Cell Biol.</em> <strong>36</strong>, 189-204.</li>
<li>↑ <sup><a href="#cite_ref-Fujii2010_6-0">6.0</a></sup> <sup><a href="#cite_ref-Fujii2010_6-1">6.1</a></sup> <sup><a href="#cite_ref-Fujii2010_6-2">6.2</a></sup> <sup><a href="#cite_ref-Fujii2010_6-3">6.3</a></sup> Fujii-Kuriyama, Y., and Kawajiri, K. (2010). Molecular mechanisms of the physiological functions of the aryl hydrocarbon (dioxin) receptor, a multifunctional regulator that senses and responds to environmental stimuli. <em>Proc.Jpn.Acad.Ser.B Phys.Biol.Sci.</em> <strong>86</strong>, 40-53.</li>
<li>↑ <sup><a href="#cite_ref-Mimura2003_7-0">7.0</a></sup> <sup><a href="#cite_ref-Mimura2003_7-1">7.1</a></sup> Mimura, J., and Fujii-Kuriyama, Y. (2003). Functional role of AhR in the expression of toxic effects by TCDD. <em>Biochimica et Biophysica Acta - General Subjects</em> <strong>1619</strong>, 263-268.</li>
<li><a href="#cite_ref-Giesy2006_8-0">↑</a> Giesy, J. P., Kannan, K., Blankenship, A. L., Jones, P. D., and Newsted, J. L. (2006). Toxicology of PCBs and related compounds. In Endocrine Disruption Biological Bases for Health Effects in Wildlife and Humans (D. O. Norris, and J. A. Carr, Eds.), pp. 245-331. Oxford University Press, New York.</li>
<li>↑ <sup><a href="#cite_ref-Safe1994_9-0">9.0</a></sup> <sup><a href="#cite_ref-Safe1994_9-1">9.1</a></sup> <sup><a href="#cite_ref-Safe1994_9-2">9.2</a></sup> Safe, S. (1994). Polychlorinated biphenyls (PCBs): Environmental impact, biochemical and toxic responses, and implications for risk assessment. <em>Critical Reviews in Toxicology</em> <strong>24</strong>, 87-149.</li>
<li>↑ <sup><a href="#cite_ref-Yasui2007_10-0">10.0</a></sup> <sup><a href="#cite_ref-Yasui2007_10-1">10.1</a></sup> <sup><a href="#cite_ref-Yasui2007_10-2">10.2</a></sup> Yasui, T., Kim, E. Y., Iwata, H., Franks, D. G., Karchner, S. I., Hahn, M. E., and Tanabe, S. (2007). Functional characterization and evolutionary history of two aryl hydrocarbon receptor isoforms (AhR1 and AhR2) from avian species. <em>Toxicol.Sci</em>. <strong>99</strong>, 101-117.</li>
<li>↑ <sup><a href="#cite_ref-Lee2009_11-0">11.0</a></sup> <sup><a href="#cite_ref-Lee2009_11-1">11.1</a></sup> Lee, J. S., Kim, E. Y., and Iwata, H. (2009). Dioxin activation of CYP1A5 promoter/enhancer regions from two avian species, common cormorant (Phalacrocorax carbo) and chicken (Gallus gallus): association with aryl hydrocarbon receptor 1 and 2 isoforms. <em>Toxicol.Appl.Pharmacol</em>. <strong>234</strong>, 1-13.</li>
<li>↑ <sup><a href="#cite_ref-Raucy2010_12-0">12.0</a></sup> <sup><a href="#cite_ref-Raucy2010_12-1">12.1</a></sup> <sup><a href="#cite_ref-Raucy2010_12-2">12.2</a></sup> <sup><a href="#cite_ref-Raucy2010_12-3">12.3</a></sup> <sup><a href="#cite_ref-Raucy2010_12-4">12.4</a></sup> <sup><a href="#cite_ref-Raucy2010_12-5">12.5</a></sup> Raucy, J. L., and Lasker, J. M. (2010). Current in vitro high throughput screening approaches to assess nuclear receptor activation. <em>Curr. Drug Metab</em> <strong>11</strong> (9), 806-814.</li>
<li>↑ <sup><a href="#cite_ref-Farmahin2012_13-0">13.0</a></sup> <sup><a href="#cite_ref-Farmahin2012_13-1">13.1</a></sup> <sup><a href="#cite_ref-Farmahin2012_13-2">13.2</a></sup> Farmahin, R., Wu, D., Crump, D., Hervé, J. C., Jones, S. P., Hahn, M. E., Karchner, S. I., Giesy, J. P., Bursian, S. J., Zwiernik, M. J., and Kennedy, S. W. (2012). Sequence and in vitro function of chicken, ring-necked pheasant, and Japanese quail AHR1 predict in vivo sensitivity to dioxins. Environ.Sci.Technol. 46, 2967-2975.</li>
<li>↑ <sup><a href="#cite_ref-Farmahin2013b_14-0">14.0</a></sup> <sup><a href="#cite_ref-Farmahin2013b_14-1">14.1</a></sup> <sup><a href="#cite_ref-Farmahin2013b_14-2">14.2</a></sup> <sup><a href="#cite_ref-Farmahin2013b_14-3">14.3</a></sup> Farmahin, R., Manning, G. E., Crump, D., Wu, D., Mundy, L. J., Jones, S. P., Hahn, M. E., Karchner, S. I., Giesy, J. P., Bursian, S. J., Zwiernik, M. J., Fredricks, T. B., and Kennedy, S. W. (2013b). Amino acid sequence of the ligand binding domain of the aryl hydrocarbon receptor 1 (AHR1) predicts sensitivity of wild birds to effects of dioxin-like compounds. Toxicol.Sci. 131, 139-152.</li>
<li><a href="#cite_ref-Fujisawa2012_15-0">↑</a> Fujisawa, N., Ikenaka, Y., Kim, E. Y., Lee, J. S., Iwata, H., and Ishizuka, M. (2012). Molecular evidence predicts aryl hydrocarbon receptor ligand insensitivity in the peregrine falcon (Falco peregrines). European Journal of Wildlife Research 58, 167-175.</li>
<li>↑ <sup><a href="#cite_ref-Manning2012_16-0">16.0</a></sup> <sup><a href="#cite_ref-Manning2012_16-1">16.1</a></sup> <sup><a href="#cite_ref-Manning2012_16-2">16.2</a></sup> Manning, G. E., Farmahin, R., Crump, D., Jones, S. P., Klein, J., Konstantinov, A., Potter, D., and Kennedy, S. W. (2012). A luciferase reporter gene assay and aryl hydrocarbon receptor 1 genotype predict the embryolethality of polychlorinated biphenyls in avian species. Toxicol.Appl.Pharmacol. 263, 390-399.</li>
<li><a href="#cite_ref-Mol2012_17-0">↑</a> Mol, T. L., Kim, E. Y., Ishibashi, H., and Iwata, H. (2012). In vitro transactivation potencies of black-footed albatross (Phoebastria nigripes) AHR1 and AHR2 by dioxins to predict CYP1A expression in the wild population. Environ.Sci.Technol. 46, 525-533.</li>
<li><a href="#cite_ref-Yueh2005_18-0">↑</a> Yueh, M. F., Kawahara, M., and Raucy, J. (2005). Cell-based high-throughput bioassays to assess induction and inhibition of CYP1A enzymes. <em>Toxicol. In Vitro</em> <strong>19</strong> (2), 275-287.</li>
<li>↑ <sup><a href="#cite_ref-Poland1982_19-0">19.0</a></sup> <sup><a href="#cite_ref-Poland1982_19-1">19.1</a></sup> Poland, A., and Knutson, J. C. (1982). 2,3,7,8-tetrachlorodibenzo-p-dioxin and related halogenated aromatic hydrocarbons: examination of the mechanism of toxicity. <em>Annu. Rev. Pharmacol. Toxicol. </em> <strong>22</strong>, 517-554.</li>
<li>↑ <sup><a href="#cite_ref-Hesterman2000_20-0">20.0</a></sup> <sup><a href="#cite_ref-Hesterman2000_20-1">20.1</a></sup> Hestermann, E. V., Stegeman, J. J., and Hahn, M. E. (2000). Relative contributions of affinity and intrinsic efficacy to aryl hydrocarbon receptor ligand potency. <em>Toxicol. Appl. Pharmacol </em> <strong>168</strong> (2), 160-172.</li>
<li>↑ <sup><a href="#cite_ref-Farmahin2014_21-0">21.0</a></sup> <sup><a href="#cite_ref-Farmahin2014_21-1">21.1</a></sup> <sup><a href="#cite_ref-Farmahin2014_21-2">21.2</a></sup> <sup><a href="#cite_ref-Farmahin2014_21-3">21.3</a></sup> <sup><a href="#cite_ref-Farmahin2014_21-4">21.4</a></sup> Farmahin, R., Jones, S. P., Crump, D., Hahn, M. E., Giesy, J. P., Zwiernik, M. J., Bursian, S. J., and Kennedy, S. W. (2014). Species-specific relative AHR1 binding affinities of 2,3,4,7,8-pentachlorodibenzofuran explain avian species differences in its relative potency. <em>Comp Biochem. Physiol C. Toxicol. Pharmacol.</em> <strong>161C</strong>, 21-25.</li>
<li>↑ <sup><a href="#cite_ref-Karchner2006_22-0">22.0</a></sup> <sup><a href="#cite_ref-Karchner2006_22-1">22.1</a></sup> <sup><a href="#cite_ref-Karchner2006_22-2">22.2</a></sup> <sup><a href="#cite_ref-Karchner2006_22-3">22.3</a></sup> <sup><a href="#cite_ref-Karchner2006_22-4">22.4</a></sup> <sup><a href="#cite_ref-Karchner2006_22-5">22.5</a></sup> <sup><a href="#cite_ref-Karchner2006_22-6">22.6</a></sup> Karchner, S. I., Franks, D. G., Kennedy, S. W., and Hahn, M. E. (2006). The molecular basis for differential dioxin sensitivity in birds: Role of the aryl hydrocarbon receptor. <em>Proc. Natl. Acad. Sci. U. S. A</em> <strong>103</strong> (16), 6252-6257.</li>
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<li><a href="#cite_ref-Gasiewicz1982_25-0">↑</a> Gasiewicz, T. A., and Neal, R. A. (1982). The examination and quantitation of tissue cytosolic receptors for 2,3,7,8-tetrachlorodibenzo-p-dioxin using hydroxylapatite. <em>Anal. Biochem. </em> <strong>124</strong> (1), 1-11.</li>
<li><a href="#cite_ref-Nakai1995_26-0">↑</a> Nakai, J. S., and Bunce, N. J. (1995). Characterization of the Ah receptor from human placental tissue. <em>J Biochem. Toxicol. </em> <strong>10</strong> (3), 151-159.</li>
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<li>↑ <sup><a href="#cite_ref-Poland1994_31-0">31.0</a></sup> <sup><a href="#cite_ref-Poland1994_31-1">31.1</a></sup> Poland, A., Palen, D., and Glover, E. (1994). Analysis of the four alleles of the murine aryl hydrocarbon receptor. Mol.Pharmacol. 46, 915-921.</li>
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<li><a href="#cite_ref-Murray2005_33-0">↑</a> Murray, I. A., Reen, R. K., Leathery, N., Ramadoss, P., Bonati, L., Gonzalez, F. J., Peters, J. M., and Perdew, G. H. (2005). Evidence that ligand binding is a key determinant of Ah receptor-mediated transcriptional activity. Arch.Biochem.Biophys. 442, 59-71.</li>
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<li>↑ <sup><a href="#cite_ref-Pandini2009_35-0">35.0</a></sup> <sup><a href="#cite_ref-Pandini2009_35-1">35.1</a></sup> <sup><a href="#cite_ref-Pandini2009_35-2">35.2</a></sup> Pandini, A., Soshilov, A. A., Song, Y., Zhao, J., Bonati, L., and Denison, M. S. (2009). Detection of the TCDD binding-fingerprint within the Ah receptor ligand binding domain by structurally driven mutagenesis and functional analysis. Biochemistry 48, 5972-5983.</li>
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<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-size:10.0pt"><span style="font-family:"Times New Roman",serif">Shackleford G, Sampathkumar NK, Hichor M, Weill L, Meffre D, et al. 2018. Involvement of Aryl hydrocarbon receptor in myelination and in human nerve sheath tumorigenesis. Proc Natl Acad Sci U S 115(6):E1319–28</span></span></span></span></p>
<p style="text-align:justify"> </p>
<p style="text-align:justify"> </p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-size:10.0pt"><span style="font-family:"Times New Roman",serif">Zudaire E, Cuesta N, Murty V, Woodson K, Adams L, et al. 2008. The aryl hydrocarbon receptor repressor is a putative tumor suppressor gene in multiple human cancers. J Clin Invest. 118(2):640–50</span></span></span></span></p>
<p style="text-align:justify"> </p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-size:10.0pt"><span style="font-family:"Times New Roman",serif">Goode GD, Ballard BR, Manning HC, Freeman ML, Kang Y, Eltom SE. 2013. Knockdown of aberrantly upregulated aryl hydrocarbon receptor reduces tumor growth and metastasis of MDA-MB- 231 human breast cancer cell line. Int J Cancer. 133(12):2769–80</span></span></span></span></p>
<p style="text-align:justify"> </p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-size:10.0pt"><span style="font-family:"Times New Roman",serif">Chang JT, Chang H, Chen P-H, Lin S-L, Lin P. 2007. Requirement of aryl hydrocarbon receptor overexpression for CYP1B1 up-regulation and cell growth in human lung adenocarcinomas. Clin Cancer</span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-size:10.0pt"><span style="font-family:"Times New Roman",serif">Res. 13(1):38–45</span></span></span></span></p>
<p style="text-align:justify"> </p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-size:10.0pt"><span style="font-family:"Times New Roman",serif">Kanno Y, Takane Y, Izawa T, Nakahama T, Inouye Y. 2006. </span></span><span style="font-size:10.0pt"><span style="font-family:"Times New Roman",serif">The inhibitory effect of aryl hydrocarbon receptor repressor (AhRR) on the growth of human breast cancer MCF-7 cells. Biol Pharm Bull. 29(6):1254–57</span></span></span></span></p>
<p style="text-align:justify"> </p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-size:10.0pt"><span style="font-family:"Times New Roman",serif">Goode G, Pratap S, Eltom SE. 2014. Depletion of the aryl hydrocarbon receptor in MDA-MB- 231 human breast cancer cells altered the expression of genes in key regulatory pathways of cancer. PLoS One. 9(6):e100103</span></span></span></span></p>
<p style="text-align:justify"> </p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-size:10.0pt"><span style="font-family:"Times New Roman",serif">Opitz CA, Litzenburger UM, Sahm F, Ott M, Tritschler I, et al. 2011. An endogenous tumour promoting ligand of the human aryl hydrocarbon receptor. Nature. 478(7368):197–203</span></span></span></span></p>
<p style="text-align:justify"> </p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-size:10.0pt"><span style="font-family:"Times New Roman",serif">Novikov O, Wang Z, Stanford EA, Parks AJ, Ramirez-Cardenas A, et al. 2016. An Aryl Hydrocarbon Receptor-Mediated Amplification Loop That Enforces Cell Migration in ER-/PR-/Her2- Human Breast Cancer Cells. Mol Pharmacol. 90(5):674–88</span></span></span></span></p>
<p style="text-align:justify"> </p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-size:10.0pt"><span style="font-family:"Times New Roman",serif">Litzenburger UM, Opitz CA, Sahm F, Rauschenbach KJ, Trump S, et al. 2014. Constitutive IDO expression in human cancer is sustained by an autocrine signaling loop involving IL-6, STAT3 and the AHR. Oncotarget. 5(4):1038–51</span></span></span></span></p>
<p style="text-align:justify"> </p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-size:10.0pt"><span style="font-family:"Times New Roman",serif">Hall JM, Barhoover MA, Kazmin D, McDonnell DP, Greenlee WF, Thomas RS. 2010. Activation of the aryl-hydrocarbon receptor inhibits invasive and metastatic features of human breast cancer cells and promotes breast cancer cell differentiation. Mol Endocrinol. 24(2):359–69</span></span></span></span></p>
<p style="text-align:justify"> </p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-size:10.0pt"><span style="font-family:"Times New Roman",serif">Gramatzki D, Pantazis G, Schittenhelm J, Tabatabai G, K.hle C, et al. 2009. </span></span><span style="font-size:10.0pt"><span style="font-family:"Times New Roman",serif">Aryl hydrocarbon receptor inhibition downregulates the TGF-beta/Smad pathway in human glioblastoma cells. Oncogene. 28(28):2593–2605</span></span></span></span></p>
<p style="text-align:justify"> </p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-size:10.0pt"><span style="font-family:"Times New Roman",serif">Subramaniam V, Ace O, Prud’homme GJ, Jothy S. 2011. Tranilast treatment decreases cell growth, migration and inhibits colony formation of human breast cancer cells. Exp Mol Pathol. 90(1):116–22</span></span></span></span></p>
<p style="text-align:justify"> </p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-size:10.0pt"><span style="font-family:"Times New Roman",serif">Rothhammer V, Borucki DM, Kenison JE, Hewson P, Wang Z, et al. 2018. Detection of aryl hydrocarbon receptor agonists in human samples. Sci Rep. 8(1):4970</span></span></span></span></p>
<p style="text-align:justify"> </p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-size:10.0pt"><span style="font-family:"Times New Roman",serif">Lin P, Chang H, Tsai W-T, Wu M-H, Liao Y-S, et al. 2003. </span></span><span style="font-size:10.0pt"><span style="font-family:"Times New Roman",serif">Overexpression of aryl hydrocarbon receptor in human lung carcinomas. Toxicol Pathol. 31(1):22–30</span></span></span></span></p>
<p style="text-align:justify"> </p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-size:10.0pt"><span style="font-family:"Times New Roman",serif">Barhoover MA, Hall JM, Greenlee WF, Thomas RS. 2010. Aryl hydrocarbon receptor regulates cell cycle progression in human breast cancer cells via a functional interaction with cyclin-dependent kinase 4. Mol Pharmacol. 77(2):195–201</span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-size:10.0pt"><span style="font-family:"Times New Roman",serif">Wang K, Li Y, Jiang Y-Z, Dai C-F, Patankar MS, et al. 2013. </span></span><span style="font-size:10.0pt"><span style="font-family:"Times New Roman",serif">An endogenous aryl hydrocarbon receptor ligand inhibits proliferation and migration of human ovarian cancer cells. Cancer Lett. 340(1):63–71</span></span></span></span></p>
<p style="text-align:justify"> </p>
<p><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-size:10.0pt"><span style="font-family:"Times New Roman",serif">Borovok N, Weiss C, Sharkia R, Reichenstein M, Wissinger B, et al. 2020. Gene and Protein Expression in Subjects With a Nystagmus-Associated AHR Mutation. Front Genet. 11:582796</span></span></span></span></p>
<p><em><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:14px">Harper PA, Riddick DS, Okey AB. Regulating the regulator: factors that control levels and activity of the aryl hydrocarbon receptor. Biochem Pharmacol. 2006;72(3):267-79.</span></span></em></p>
<p><em><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:14px">Hernández-Ochoa I, Karman BN, Flaws JA. The role of the aryl hydrocarbon receptor in the female reproductive system. Biochem Pharmacol. 2009;77(4):547-59.</span></span></em></p>
<p> </p>
<p><br />
</p>
<h3>List of Key Events in the AOP</h3>
<h4><a href="/events/80">Event: 80: Up Regulation, CYP1A1</a></h4>
<td><a href="/aops/57">Aop:57 - AhR activation leading to hepatic steatosis</a></td>
<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 - Increased 11β-Hydroxysteroid dehydrogenase type 1 activity leading to MASLD progression via insulin resistance-associated mitochondrial dysfunction</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/625">Aop:625 - Increased 11β-Hydroxysteroid dehydrogenase type 1 activity leading to MASLD progression via insulin resistance-associated oxidative stress</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/626">Aop:626 - Increased 11β-Hydroxysteroid dehydrogenase type 1 activity leading to MASLD progression via insulin resistance-associated endoplasmic reticulum stress</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/627">Aop:627 - Increased 11β-Hydroxysteroid dehydrogenase type 1 activity leading to MASLD progression via lipogenesis-associated mitochondrial dysfunction</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/628">Aop:628 - Increased 11β-Hydroxysteroid dehydrogenase type 1 activity leading to MASLD progression via lipogenesis-associated oxidative stress</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/629">Aop:629 - Increased 11β-Hydroxysteroid dehydrogenase type 1 activity leading to MASLD progression via lipogenesis-associated endoplasmic reticulum stress</a></td>
<td><a href="/aops/325">Aop:325 - Thermal stress leading to population decline (2)</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/396">Aop:396 - Deposition of ionizing energy leads to population decline via impaired meiosis</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/481">Aop:481 - AOPs of amorphous silica nanoparticles: ROS-mediated oxidative stress increased respiratory dysfunction and diseases.</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/494">Aop:494 - AhR activation leading to liver fibrosis </a></td>
<td>KeyEvent</td>
<td>Unspecific</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
<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. <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><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>
<h4>How it is Measured or Detected</h4>
<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><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>
<td><a href="/aops/48">Aop:48 - Binding of agonists to ionotropic glutamate receptors in adult brain causes excitotoxicity that mediates neuronal cell death, contributing to learning and memory impairment.</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/13">Aop:13 - Chronic binding of antagonist to N-methyl-D-aspartate receptors (NMDARs) during brain development induces impairment of learning and memory abilities</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/38">Aop:38 - Protein Alkylation leading to Liver Fibrosis</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/12">Aop:12 - Chronic binding of antagonist to N-methyl-D-aspartate receptors (NMDARs) during brain development leads to neurodegeneration with impairment in learning and memory in aging</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/144">Aop:144 - Endocytic lysosomal uptake leading to liver fibrosis</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/17">Aop:17 - Binding of electrophilic chemicals to SH(thiol)-group of proteins and /or to seleno-proteins involved in protection against oxidative stress during brain development leads to impairment of learning and memory</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/278">Aop:278 - IKK complex inhibition leading to liver injury</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/281">Aop:281 - Acetylcholinesterase Inhibition Leading to Neurodegeneration</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/273">Aop:273 - Mitochondrial complex inhibition leading to liver injury</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/377">Aop:377 - Dysregulated prolonged Toll Like Receptor 9 (TLR9) activation leading to Multi Organ Failure involving Acute Respiratory Distress Syndrome (ARDS)</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/265">Aop:265 - Uncoupling of oxidative phosphorylation leading to growth inhibition via increased cytosolic calcium</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/264">Aop:264 - Uncoupling of oxidative phosphorylation leading to growth inhibition via ATP depletion associated cell death</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/266">Aop:266 - Uncoupling of oxidative phosphorylation leading to growth inhibition via decreased Na-K ATPase activity</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/268">Aop:268 - Uncoupling of oxidative phosphorylation leading to growth inhibition via mitochondrial swelling</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/479">Aop:479 - Mitochondrial complexes inhibition leading to left ventricular function decrease via increased myocardial oxidative stress</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/490">Aop:490 - Co-activation of IP3R and RyR leads to socio-economic burden through reduced IQ and non-cholinergic mechanisms</a></td>
<td><a href="/aops/490">Aop:490 - Co-activation of IP3R and RyR leads to reduced IQ and increased socio-economic burden through non-cholinergic mechanisms</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/494">Aop:494 - AhR activation leading to liver fibrosis </a></td>
<td><a href="/aops/324">Aop:324 - Excessive reactive oxygen species leading to growth inhibition via uncoupling of oxidative phosphorylation and cell death</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/331">Aop:331 - Excessive reactive oxygen species leading to growth inhibition via lipid peroxidation and cell death</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/596">Aop:596 - Excessive reactive oxygen species leading to growth inhibition via protein oxidation and cell injury/death</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/599">Aop:599 - Excessive reactive oxygen species leading to growth inhibition via fatty acid oxidation and cell injury/death</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/624">Aop:624 - Increased 11β-Hydroxysteroid dehydrogenase type 1 activity leading to MASLD progression via insulin resistance-associated mitochondrial dysfunction</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/625">Aop:625 - Increased 11β-Hydroxysteroid dehydrogenase type 1 activity leading to MASLD progression via insulin resistance-associated oxidative stress</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/626">Aop:626 - Increased 11β-Hydroxysteroid dehydrogenase type 1 activity leading to MASLD progression via insulin resistance-associated endoplasmic reticulum stress</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/627">Aop:627 - Increased 11β-Hydroxysteroid dehydrogenase type 1 activity leading to MASLD progression via lipogenesis-associated mitochondrial dysfunction</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/628">Aop:628 - Increased 11β-Hydroxysteroid dehydrogenase type 1 activity leading to MASLD progression via lipogenesis-associated oxidative stress</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/629">Aop:629 - Increased 11β-Hydroxysteroid dehydrogenase type 1 activity leading to MASLD progression via lipogenesis-associated endoplasmic reticulum stress</a></td>
<p>Cell death is an universal event occurring in cells of any species (Fink and Cookson,2005).<sup> </sup></p>
<h4>Key Event Description</h4>
<p style="text-align:justify">Two types of cell death can be distinguished by morphological features, although it is likely that these are two ends of a spectrum with possible intermediate forms. Apoptosis involves shrinkage, nuclear disassembly, and fragmentation of the cell into discrete bodies with intact plasma membranes. These are rapidly phagocytosed by neighbouring cells. An important feature of apoptosis is the requirement for adenosine triphosphate (ATP) to initiate the execution phase. In contrast, necrotic cell death is characterized by cell swelling and lysis. This is usually a consequence of profound loss of mitochondrial function and resultant ATP depletion, leading to loss of ion homeostasis, including volume regulation, and increased intracellular Ca2+. The latter activates a number of nonspecific hydrolases (i.e., proteases, nucleases, and phospholipases) as well as calcium dependent kinases. Activation of calpain I, the Ca2+-dependent cysteine protease cleaves the death-promoting Bcl-2 family members Bid and Bax which translocate to mitochondrial membranes, resulting in release of truncated apoptosis-inducing factor (tAIF), cytochrome c and endonuclease in the case of Bid and cytocrome c in the case of Bax. tAIF translocates to cell nuclei, and together with cyclophilin A and phosphorylated histone H2AX (γH2AX) is responsible for DNA cleavage, a feature of programmed necrosis. Activated calpain I has also been shown to cleave the plasma membrane Na+–Ca2+ exchanger, which leads to build-up of intracellular Ca2+, which is the source of additional increased intracellular Ca2+. Cytochrome c in cellular apoptosis is a component of the apoptosome.</p>
<p style="text-align:justify">DNA damage activates nuclear poly(ADP-ribose) polymerase-1(PARP-1), a DNA repair enzyme. PARP-1 forms poly(ADP-ribose) polymers, to repair DNA, but when DNA damage is extensive, PAR accumulates, exits cell nuclei and travels to mitochondrial membranes, where it, like calpain I, is involved in AIF release from mitochondria. A fundamental distinction between necrosis and apoptosis is the loss of plasma membrane integrity; this is integral to the former but not the latter. As a consequence, lytic release of cellular constituents promotes a local inflammatory reaction, whereas the rapid removal of apoptotic bodies minimizes such a reaction. The distinction between the two modes of death is easily accomplished in vitro but not in vivo. Thus, although claims that certain drugs induce apoptosis have been made, these are relatively unconvincing. DNA fragmentation can occur in necrosis, leading to positive TUNEL staining <span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11.0pt">(<span style="font-size:16px">see explanation below</span>)</span></span>. Conversely, when apoptosis is massive, it can exceed the capacity for rapid phagocytosis, resulting in the eventual appearance of secondary necrosis.</p>
<p style="text-align:justify">Two alternative pathways - either extrinsic (receptor-mediated) or intrinsic (mitochondria-mediated) - lead to apoptotic cell death. The initiation of cell death begins either at the plasma membrane with the binding of TNF or FasL to their cognate receptors or within the cell. The latter is due to the occurrence of intracellular stress in the form of biochemical events such as oxidative stress, redox changes, covalent binding, lipid peroxidation, and consequent functional effects on mitochondria, endoplasmic reticulum, microtubules, cytoskeleton, or DNA. The intrinsic mitochondrial pathway involves the initiator, caspase-9, which, when activated, forms an “apoptosome” in the cytosol, together with cytochrome c, which translocates from mitochondria, Apaf-1 and dATP. The apoptosome activates caspase-3, the central effector caspase, which in turn activates downstream factors that are responsible for the apoptotic death of a cell (Fujikawa, 2015). Intracellular stress either directly affects mitochondria or can lead to effects on other organelles, which then send signals to the mitochondria to recruit participation in the death process (Fujikawa, 2015; Malhi et al., 2010).<sup> </sup>Constitutively expressed nitric oxide synthase (nNOS) is a Ca2+-dependent cytosolic enzyme that forms nitric oxide (NO) from L-arginine, and NO reacts with the free radical such as superoxide (O2−) to form the very toxic free radical peroxynitrite (ONOO−). Free radicals such as ONOO−, O2 − and hydroxyl radical (OH−) damage cellular membranes and intracellular proteins, enzymes and DNA (Fujikawa, 2015; Malhi et al., 2010; Kaplowitz, 2002; Kroemer et al., 2009). </p>
<h4>How it is Measured or Detected</h4>
<p> </p>
<p><strong>Necrosis:</strong></p>
<p style="text-align:justify">Lactate dehydrogenase (LDH) is a soluble cytoplasmic enzyme that is present in almost all cells and is released into extracellular space when the plasma membrane is damaged. To detect the leakage of LDH into cell culture medium, a tetrazolium salt is used in this assay. In the first step, LDH produces reduced nicotinamide adenine dinucleotide (NADH) when it catalyzes the oxidation of lactate to pyruvate. In the second step, a tetrazolium salt is converted to a colored formazan product using newly synthesized NADH in the presence of an electron acceptor. The amount of formazan product can be colorimetrically quantified by standard spectroscopy. Because of the linearity of the assay, it can be used to enumerate the percentage of necrotic cells in a sample (Chan et al., 2013). </p>
<p style="text-align:justify">The MTT assay is a colorimetric assay for assessing cell viability. NAD(P)H-dependent cellular oxidoreductase enzymes may reflect the number of viable cells present. These enzymes are capable of reducing the tetrazolium dye MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to its insoluble formazan, which has a purple color. Other closely related tetrazolium dyes include XTT, MTS and the WSTs. Tetrazolium dye assays can also be used to measure cytotoxicity (loss of viable cells) or cytostatic activity (shift from proliferation to quiescence) of potential medicinal agents and toxic materials. MTT assays are usually done in the dark since the MTT reagent is sensitive to light (Berridgeet al.,2005).</p>
<p style="text-align:justify">Propidium iodide (PI) is an intercalating agent and a fluorescent molecule used to stain necrotic cells. It is cell membrane impermeant so it stains only those cells where the cell membrane is destroyed. When PI is bound to nucleic acids, the fluorescence excitation maximum is 535 nm and the emission maximum is 617 nm (Moore et al.,1998)</p>
<p style="text-align:justify">Alamar Blue (resazurin) is a fluorescent dye. The oxidized blue non fluorescent Alamar blue is reduced to a pink fluorescent dye in the medium by cell activity (O'Brien et al., 2000) (12).</p>
<p style="text-align:justify">Neutral red uptake, which is based on the ability of viable cells to incorporate and bind the supravital dye neutral red in lysosomes (Repetto et al., 2008)(13). <span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Moreover, quantification of ATP, signaling the presence of metabolically active cells, can be performed (CellTiter-Glo; Promega).</span></span></p>
<p style="text-align:justify">ATP assay: Quantification of ATP, signaling the presence of metabolically active cells (CellTiter-Glo; Promega).</p>
<p style="text-align:justify"><br />
<strong>Apoptosis:</strong></p>
<p style="text-align:justify">TUNEL is a common method for detecting DNA fragmentation that results from apoptotic signalling cascades. The assay relies on the presence of nicks in the DNA which can be identified by terminal deoxynucleotidyl transferase or TdT, an enzyme that will catalyze the addition of dUTPs that are secondarily labeled with a marker. It may also label cells that have suffered severe DNA damage.</p>
<p style="text-align:justify">Caspase activity assays measured by fluorescence. During apoptosis, mainly caspase-3 and -7 cleave PARP to yield an 85 kDa and a 25 kDa fragment. PARP cleavage is considered to be one of the classical characteristics of apoptosis. Antibodies to the 85 kDa fragment of cleaved PARP or to caspase-3 both serve as markers for apoptotic cells that can be monitored using immunofluorescence (Li, Peng et al., 2004).</p>
<p style="text-align:justify">Hoechst 33342 staining: Hoechst dyes are cell-permeable and bind to DNA in live or fixed cells. Therefore, these stains are often called supravital, which means that cells survive a treatment with these compounds. The stained, condensed or fragmented DNA is a marker of apoptosis (Loo, 2002; Kubbies and Rabinovitch, 1983). </p>
<p style="text-align:justify">Acridine Orange/Ethidium Bromide staining is used to visualize nuclear changes and apoptotic body formation that are characteristic of apoptosis. Cells are viewed under a fluorescence microscope and counted to quantify apoptosis.</p>
<h4>References</h4>
<ul>
<li>Fujikawa, D.G. (2015), The role of excitotoxic programmed necrosis in acute brain injury, Comput Struct Biotechnol J, vol. 13, pp. 212-221.</li>
<li>Malhi, H. et al. (2010), Hepatocyte death: a clear and present danger, Physiol Rev, vol. 90, no. 3, pp. 1165-1194.</li>
<li>Kaplowitz, N. (2002), Biochemical and Cellular Mechanisms of Toxic Liver Injury, Semin Liver Dis, vol. 22, no. 2,<span style="color:#000000"> </span><a class="external free" href="http://www.medscape.com/viewarticle/433631" rel="nofollow" target="_blank"><span style="color:#000000">http://www.medscape.com/viewarticle/433631</span></a><span style="color:#000000"> </span>(accessed on 20 January 2016).</li>
<li>Kroemer, G. et al., (2009), Classification of cell death: recommendations of the Nomenclature Committee on Cell Death, Cell Death Differ, vol. 16, no. 1, pp. 3-11.</li>
<li>Chan, F.K., K. Moriwaki and M.J. De Rosa (2013), Detection of necrosis by release of lactate dehydrogenase (LDH) activity, Methods Mol Biol, vol. 979, pp. 65–70.</li>
<li>Berridge, M.V., P.M. Herst and A.S. Tan (2005), Tetrazolium dyes as tools in cell biology: new insights into their cellular reduction. Biotechnology Annual Review, vol. 11, pp 127-152.</li>
<li>Moore, A, et al.(1998), Simultaneous measurement of cell cycle and apoptotic cell death,Methods Cell Biol, vol. 57, pp. 265–278.</li>
<li>Li, Peng et al. (2004), Mitochondrial activation of apoptosis, Cell, vol. 116, no. 2 Suppl,pp. S57-59, 2 p following S59.</li>
<li>Loo, D.T. (2002), TUNEL Assay an overview of techniques, Methods in Molecular Biology, vol. 203: In Situ Detection of DNA Damage, chapter 2, Didenko VV (ed.), Humana Press Inc.</li>
<li>Kubbies, M. and P.S. Rabinovitch (1983), Flow cytometric analysis of factors which influence the BrdUrd-Hoechst quenching effect in cultivated human fibroblasts and lymphocytes, Cytometry, vol. 3, no. 4, pp. 276–281.</li>
<li>Fink, S.L. and B.T. Cookson (2005), Apoptosis, pyroptosis, and necrosis: mechanistic description of dead and dying eukaryotic cells, Infect Immun, vol. 73, no. 4, pp.1907-1916.</li>
<li>O'Brien J, Wilson I, Orton T, Pognan F. 2000. Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity. European journal of biochemistry / FEBS 267(17): 5421-5426.</li>
<li>Repetto G, del Peso A, Zurita JL. 2008. Neutral red uptake assay for the estimation of cell viability/cytotoxicity. Nature protocols 3(7): 1125-1131.</li>
<td><a href="/aops/624">Aop:624 - Increased 11β-Hydroxysteroid dehydrogenase type 1 activity leading to MASLD progression via insulin resistance-associated mitochondrial dysfunction</a></td>
<td><a href="/aops/625">Aop:625 - Increased 11β-Hydroxysteroid dehydrogenase type 1 activity leading to MASLD progression via insulin resistance-associated oxidative 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. <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>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>
<h4>How it is Measured or Detected</h4>
<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><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>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>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/626">Aop:626 - Increased 11β-Hydroxysteroid dehydrogenase type 1 activity leading to MASLD progression via insulin resistance-associated endoplasmic reticulum stress</a></td>
<td><a href="/aops/38">Aop:38 - Protein Alkylation leading to Liver Fibrosis</a></td>
<td><a href="/aops/627">Aop:627 - Increased 11β-Hydroxysteroid dehydrogenase type 1 activity leading to MASLD progression via lipogenesis-associated mitochondrial dysfunction</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/144">Aop:144 - Endocytic lysosomal uptake leading to liver fibrosis</a></td>
<td><a href="/aops/628">Aop:628 - Increased 11β-Hydroxysteroid dehydrogenase type 1 activity leading to MASLD progression via lipogenesis-associated oxidative stress</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/494">Aop:494 - AhR activation leading to liver fibrosis </a></td>
<td><a href="/aops/629">Aop:629 - Increased 11β-Hydroxysteroid dehydrogenase type 1 activity leading to MASLD progression via lipogenesis-associated endoplasmic reticulum stress</a></td>
<p>Stellate cell activation means a transdifferentiation from a quiescent vitamin A–storing cell to a proliferative and contractile myofibroblast. Multiple cells and cytokines play a part in the regulation of hepatic stellate cell (HSC) activation that consists of discrete phenotype responses, mainly proliferation, contractility, fibrogenesis, matrix degradation, chemotaxis, and retinoid loss.</p>
<p>HSCs undergo activation through a two-phase process. The first step, the initiation phase, is triggered by injured hepatocytes, reactive oxygen speecies (ROS) and paracrine stimulation from neighbouring cell types (Kupffer cells (KCs), Liver sinusoidal endothelial cells (LSECs), and platelets) and make HSCs sensitized to activation by up-regulating various receptors. The perpetuation phase refers to the maintenance of HSC activation, which is a dynamic process including the secretion of autocrine and paracrine growth factors (such as TGF-β1), chemokines, and the up-regulation of collagen synthesis (mainly type I collagen). In response to growth factors (including Platelet-derived Growth Factor (PDGF) and Vascular Endothelial Growth Factor (VEGF)) HSCs proliferate. Increased contractility (Endothelin-1 and NO are the key opposing counter-regulators that control HSC contractility, in addition to angiotensinogen II, and others) leads to increased portal resistance. Driven by chemoattractants their accumulation in areas of injury is enhanced. TGF-β1 synthesis promotes activation of neighbouring quiescent hepatic stellate cells, whereas the release of HGF (hepatocyte growth factor) stimulates regeneration of adjacent hepatocytes. The release of chemoattractants (monocyte chemoattractant protein-1(MCP-1) and colony-stimulating factors (CSFs)) amplifies inflammation (Lee and Friedman; 2011; Friedman, 2010; 2008; 2000; Bataller and Brenner, 2005; ↑ Lotersztain et al., 2005; Poli, 2000). Activated HSCs (myofibroblasts) are the primary collagen producing cell, the key cellular mediators of fibrosis and a nexus for converging inflammatory pathways leading to fibrosis. Experimental inhibition of stellate cell activation prevents fibrosis (Li, Jing-Ting et al.,2008; George et al. (1999).<sup> </sup></p>
<h4>How it is Measured or Detected</h4>
<p>Alpha-smooth muscle actin (α-SMA) is a well-known marker of hepatic stellate cells activation. Anti-alpha smooth muscle Actin [1A4] monoclonal antibody reacts with the alpha smooth muscle isoform of actin.</p>
<p>Gene expression profiling confirmed early changes for known genes related to HSC activation such as alpha smooth muscle actin (Acta2), lysyl oxidase (Lox) and collagen, type I, alpha 1 (Col1a1). Insulin-like growth factor binding protein 3 (Igfbp3) was identified as a gene strongly affected and as marker for culture-activated HSCs and plays a role in HSC migration (Morini et al., 2005; Mannaerts et al., 2013). </p>
<pre>
</pre>
<h4>References</h4>
<ul>
<li>Lee, U.E. and S.L. Friedman (2011), Mechanisms of Hepatic Fibrogenesis, Best Pract Res Clin Gastroenterol, vol. 25, no. 2, pp. 195-206.</li>
<li>Friedman, S.L (2010), Evolving challenges in hepatic fibrosis, Nat. Rev. Gastroenterol. Hepatol, vol. 7, no. 8, pp. 425–436.</li>
<li>Friedman, S.L. (2008), Mechanisms of Hepatic Fibrogenesis, Gastroenterology, vol. 134, no. 6, pp. 1655–1669.</li>
<li>Friedman, S.L (2000), Molecular regulation of hepatic fibrosis, an integrated cellular response to tissue injury, J. Biol. Chem, vol. 275, no. 4, pp. 2247-2250.</li>
<li>Bataller, R. and D.A. Brenner (2005), Liver Fibrosis, J.Clin. Invest, vol. 115, no. 2, pp. 209-218.</li>
<li>Lotersztain, S. et al. (2005), Hepatic fibrosis: molecular mechanisms and drug targets, Annu. Rev. Pharmacol. Toxicol, vol. 45, pp. 605–628.</li>
<li>Poli, G. (2000), Pathogenesis of liver fibrosis: role of oxidative stress, Mol Aspects Med, vol. 21, no. 3, pp. 49 – 98.</li>
<li>Li, Jing-Ting et al. (2008), Molecular mechanism of hepatic stellate cell activation and antifibrotic therapeutic strategies, J Gastroenterol, vol. 43, no. 6, pp. 419–428.</li>
<li>George, J. et al. (1999), In vivo inhibition of rat stellate cell activation by soluble transforming growth factor beta type II receptor: a potential new therapy for hepatic fibrosis. Proc Natl Acad Sci, vol. 96, no. 22, pp. 12719-12724.</li>
<li>Morini, S. et al. (2005), GFAP expression in the liver as an early marker of stellate cells activation, Ital J Anat Embryol, vol. 110, no. 4, pp. 193-207.</li>
<li>Mannaerts, I. et al. (2013), Gene expression profiling of early hepatic stellate cell activation reveals a role for Igfbp3 in cell migration, PLoS One, vol. 8, no.12, e84071.</li>
<li>Chang et al., 2014, Isolation and culture of hepatic stellate cells from mouse liver. Acta Biochim Biophys Sin (Shanghai).;46(4):291-8.</li>
<li>Costa et al., 2001, Early activation of hepatic stellate cells and perisinusoidal extracellular matrix changes during ex vivo pig liver perfusion. J Submicrosc Cytol Pathol.;33(3):231-40.</li>
<p>The inflammatory response is the cornerstone of the body’s defense mechanism against bacterial and viral pathogens, as well as physical-, chemical- and environmental-mediated tissue and organ damage. Leucocyte recruitment at the site of pathogen evasion or sterile tissue injury is a critical adaptation for the preservation of tissue integrity. Neutrophils are the cell population that acutely responds to the alterations of inflammatory micro-environment. Neutrophil infiltration takes place within 6-8 hours from the initiation of the inflammatory process and is followed by the recruitment of other cell populations, like monocytes, lymphocytes, and eosinophils, which either promote or drive the resolution of inflammation. Leukocyte infiltration into sites of infection or sterile inflammation is a tightly regulated process that follows a sequence of adhesive events, termed as leukocyte adhesion cascade. One can broadly generalize that most leukocytes follow a similar multi-step cascade in the peripheral (non-lymphoid) vasculature with some exceptions. Accordingly, an updated adhesion cascade in postcapillary venules involves free-flowing leukocytes initial attachment or tethering and slow velocity rolling (step 1),stable adhesion (arrest) on endothelial cells (step 2), leukocyte flattening (step 3), and subsequent crawling on the vascular endothelium, transendothelial cell migration (TEM) between (paracellular route) or through (transcellular) the vascular endothelium (step 4), and uropod elongation to complete transmigration of postcapillary venules (step 5). The initial attachment and rolling steps are initiated by interactions of endothelial E- and P-selectins and their counterreceptors on leukocytes L-selectin (Leick et al., 2014).</p>
<p>Each of these steps is necessary for effective leukocyte recruitment; these steps are not phases of inflammation, but represent the sequence of events from the perspective of each leukocyte. At any given moment they all happen in parallel, involving different leukocytes in the same microvessels.</p>
<p>From the initial selectin-dependent leukocyte tethering to endothelial cells to the final migration of leukocytes into the sub-endothelium, this process depends on the interplay between leukocyte receptors and endothelial cell counter-receptors, as well as on the presence of endogenous inhibitors of leukocyte adhesion enabling the targeted recruitment of leukocytes to inflamed tissues.</p>
<p>To enable the infiltration of leukocytes at the site of inflammation, a series of alterations in endothelial cells and leukocytes takes place:</p>
<ul style="list-style-type:circle">
<li>regulation of the expression of adhesion molecules in leukocytes</li>
<li>increased secretion of chemokines by endothelial cells</li>
<li>increased expression of adhesion molecules in the luminal surface of endothelial cells</li>
</ul>
<p>(Kourtzelis and Mitroulis, 2015) (Robbins and Cotran: Pathologic Basis of Disease 2010).</p>
<p>After recruitment, activation includes phenotype modification with morphologic alterations, changes in marker proteins (MHC, adhesion molecules, co-stimulatory signal), expression of mediators, enzymes, and pro-inflammatory proteins/lipids. Recruited monocytes recruited mature into macrophages with phagocytic activity and elaboration of a myriad of mediators of inflammation. The macrophage can replicate within tissues or die, including by apoptosis.</p>
<p> </p>
<p> </p>
<p> </p>
<h4>How it is Measured or Detected</h4>
<p><u>in vivo imaging: </u></p>
<ul style="list-style-type:circle">
<li>Flow cytometry (FC/FACS),</li>
<li>immunhistochemistry</li>
<li>two photon-intravital microscopy (TP-IVM) (van Grinsven et al., 2017)</li>
<li>Spinning Disk Confocal Microscopy-IVM (Jenne et al., 2011)</li>
<li>Histology, increased cell numbers and altered composition</li>
</ul>
<p><u>In vitro </u></p>
<ul style="list-style-type:circle">
<li>transwell Migration Assay (Justus et al., 2014)</li>
<li>T-Lymphocyte & Innate Immune Cell Activation Assays</li>
<li>Leukocyte Surface Markers (Monoclonal Antibodies to Leukocyte Surface Markers)</li>
<li>Markers of leukocyte activation – protease release, ROS/RNS, NADPH oxidase (NOX), defense response - expression of anti-oxidants.</li>
<li>organs-on-a-chip (Bnam et al., 2016; Ribas et al., 2017; Wufuer et al. 2016)</li>
</ul>
<p>REFERENCES:</p>
<p>Benam KH, Villenave R, Lucchesi C, Varone 1, Hubeau C, Lee HH, Alves SE, Salmon M, Ferrante TC, Weaver JC, Bahinski A, Hamilton GA, Ingber DE., Small airway-on-a-chip enables analysis of human lung inflammation and drug responses in vitro, Nat Methods. 2016 Feb;13(2):151-7.</p>
<p>Ribas, J., Zhang, Y. S., Pitrez, P. R., Leijten, J., Miscuglio, M., Rouwkema, J., Dokmeci, M. R., Nissan, X., Ferreira, L. and Khademhosseini, A. (2017), Organ-On-A-Chip: Biomechanical Strain Exacerbates Inflammation on a Progeria-on-a-Chip Model doi:10.1002/smll.201770087</p>
<p>Wufuer M, Lee G, Hur W, Jeon B, Kim BJ, Choi TH, Lee SH, Skin-on-a-chip model simulating inflammation, edema and drug-based treatment, Nature Scientific Reports 6, Article number: 37471 (2016) doi:10.1038/srep37471</p>
<h4>References</h4>
<p>Kourtzelis I and Mitroulis I, Encyclopedia of Inflammatory Diseases, Leukocyte Recruitment, pp 1-9, Compendium of Inflammatory Diseases, Editors: Michael J. Parnham , Springer Basel, 2015, DOI 10.1007/978-3-0348-0620-6_177-1</p>
<p>Kumar, V.; Abbas, AK.; Fausto, N.; Aster, J. Robbins and Cotran: Pathologic Basis of Disease. 8. Elsevier; Philadelphia: 2010.</p>
<p>Leick M, Azcutia V, Newton G, Luscinskas FW., Leukocyte recruitment in inflammation: basic concepts and new mechanistic insights based on new models and microscopic imaging technologies, Cell Tissue Res. 2014 Mar;355(3):647-56</p>
<p>Nourshargh S, Alon R., Leukocyte migration into inflamed tissues., Immunity. 2014 Nov 20;41(5):694-707</p>
<tr><th scope="col">Level of Biological Organization</th></tr>
</thead>
<tbody class="tbody-striped">
<tr><td>Tissue</td></tr>
</tbody>
</table>
</div>
<h4>Key Event Description</h4>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">ECM is a macromolecular structure that provides physical support to tissues and is essential for organ function. The composition of ECM is tissue specific and consists mainly of fibrous proteins, glycoproteins, and proteoglycans. The ECM in lung is compartmentalised to basement membrane and the interstitial space. Fibroblasts found in the interstitial space are the main sources of ECM in lung (White, 2015). Altered composition of ECM is observed in several lung diseases of inflammatory origin in humans including chronic obstructive pulmonary disease, asthma and idiopathic lung fibrosis. The composition and architecture of the ECM determines 1) the open sites of attachment that are available to cells, 2) the mechanical properties of the ECM and 3) the mechanical loading (breathing) experienced by the cells. Thus, changes in the ECM composition during the exaggerated wound healing process determines if an organism commits to fibrotic process or completes the wound healing (<span style="color:red">Blaauboer et al., 2014</span>).</span></span></p>
<p><strong><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><em><span style="color:red">Evidence for its perturbation in the context of pulmonary fibrosis:</span></em></span></span></strong></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">In lung fibrosis, an exaggerated amount of ECM is distributed in the alveolar parenchyma in a non-heterogenous manner, leading to lower spirometry readings implying occlusion of alveolar regions and reduced gas exchange. Collagen 1 and Collagen III are suggested to be the main components of the ECM in the thickened alveolar septa in fibrosis with other constituents such as fibronectin, elastin and tenacin C (Zhang et al., 1994; Hinz, 2006; Kuhn & McDonald, 1991; Crabb et al., 2006; Bensadoun et al., 1996; Klingberg et al., 2012; McKleroy et al., 2013). It is suggested that ECM composition dramatically changes during the fibrotic process. The early fibrotic process is characterised by collagen III deposition and collagen 1 predominates the later stages of the fibrosis. Excessive collagen production by myofibroblasts is necessary for the development of fibrosis (scarred tissue), with established areas of scar formation containing almost exclusively Type I collagen (Bateman et al., 1981; McKleroy et al., 2013; Zhang et al., 1994). Studies have demonstrated that while total collagen increases in IPF, there is also a shift toward the less elastic type I collagen, which contributes to the stiffness of the scar tissue within the lung (Nimni, 1983; Rozin et al., 2005; McKleroy et al., 2013).</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><u><strong>In lung fibrosis</strong></u>, an exaggerated amount of ECM is distributed in the alveolar parenchyma in a non-heterogenous manner, leading to lower spirometry readings implying occlusion of alveolar regions and reduced gas exchange. Collagen 1 and Collagen III are suggested to be the main components of the ECM in the thickened alveolar septa in fibrosis with other constituents such as fibronectin, elastin and tenacin C (Zhang et al., 1994; Hinz, 2006; Kuhn & McDonald, 1991; Crabb et al., 2006; Bensadoun et al., 1996; Klingberg et al., 2012; McKleroy et al., 2013). It is suggested that ECM composition dramatically changes during the fibrotic process. The early fibrotic process is characterised by collagen III deposition and collagen 1 predominates the later stages of the fibrosis. Excessive collagen production by myofibroblasts is necessary for the development of fibrosis (scarred tissue), with established areas of scar formation containing almost exclusively Type I collagen (Bateman et al., 1981; McKleroy et al., 2013; Zhang et al., 1994). Studies have demonstrated that while total collagen increases in IPF, there is also a shift toward the less elastic type I collagen, which contributes to the stiffness of the scar tissue within the lung (Nimni, 1983; Rozin et al., 2005; McKleroy et al., 2013).</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">The fibrotic ECM contains characteristic accumulation of fibroblasts and myofibroblasts, which are the major contributors of ECM synthesised. The proliferation of fibroblasts and their differentiation into myofibroblasts is, in turn, guided by the composition and structure of the ECM. For example, <span style="color:red">s</span>tudies have demonstrated that cytokines secreted in response to inflammation are capable of activating fibroblasts, and that these changes could cause alterations in the fibroblasts that lead to excessive proliferation and ECM deposition (Sivakumar et al., 2012; Wynn, 2011).</span></span></p>
<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><strong><span style="font-size:10pt"><span style="color:red"><u>In liver fibrosis,</u> extracellular matrix (ECM) deposition is a critical hallmark. It represents the accumulation of fibrotic scar tissue due to excessive synthesis and reduced degradation of ECM components (e.g., collagen types I and III) (</span></span></strong><strong><span style="font-size:10pt"><span style="background-color:yellow"><span style="color:red">PMID: 34685739</span></span></span></strong><strong><span style="font-size:10pt"><span style="color:red">). This deposition disrupts liver architecture and impairs function. </span></span></strong></span></span></span><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><strong><span style="font-size:10pt"><span style="color:red">ECM deposition is considered a highly essential event for liver fibrosis progression because it marks the transition from reversible inflammation to irreversible fibrosis, it correlates with disease severity and progression to cirrhosis (</span></span></strong><strong><span style="font-size:10pt"><span style="background-color:yellow"><span style="color:red">PMID: 34685739</span></span></span></strong><strong><span style="font-size:10pt"><span style="color:red">). </span></span></strong></span></span></span><strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">Experimental evidence often supports the essentiality, such as fibrosis models showing reduced ECM deposition when specific pathways (e.g., TGF-β or integrin signaling) are inhibited (<span style="background-color:yellow">PMID: 31362006</span>). ECM deposition is a measurable KE using histological, biochemical, and imaging methods (e.g., Masson’s trichrome staining, hydroxyproline content, or MRI elastography, Red sirius).</span></span></span></strong></p>
<p style="text-align:justify"> </p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:"Times New Roman",serif"><span style="color:#000000"><em><strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">Caligiuri A, Gentilini A, Pastore M, Gitto S, Marra F. Cellular and Molecular Mechanisms Underlying Liver Fibrosis Regression.</span></span></span></strong></em><strong><em> </em></strong><em><strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">Cells. 2021 Oct 15;10(10):2759. doi: 10.3390/cells10102759. PMID: 34685739; PMCID: PMC8534788.</span></span></span></strong></em></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:"Times New Roman",serif"><span style="color:#000000"><em><strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">Fan W, Liu T, Chen W, Hammad S, Longerich T, Hausser I, Fu Y, Li N, He Y, Liu C, Zhang Y, Lian Q, Zhao X, Yan C, Li L, Yi C, Ling Z, Ma L, Zhao X, Xu H, Wang P, Cong M, You H, Liu Z, Wang Y, Chen J, Li D, Hui L, Dooley S, Hou J, Jia J, Sun B. ECM1 Prevents Activation of Transforming Growth Factor</span></span></span></strong></em><strong><em> </em></strong><em><strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">β</span></span></span></strong></em><em><strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">, Hepatic Stellate Cells, and Fibrogenesis in Mice.</span></span></span></strong></em><strong><em> </em></strong><em><strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">Gastroenterology. 2019 Nov;157(5):1352-1367.e13. doi: 10.1053/j.gastro.2019.07.036. Epub 2019 Jul 27. PMID: 31362006.</span></span></span></strong></em></span></span></span></p>
<h4>How it is Measured or Detected</h4>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><strong><em><span style="color:red">qRT-PCR, Immunosorbant assays, and immunohistochemistry:</span></em></strong></span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">The qRT-PCR, ELISA, and immunohistochemistry are routinely used to assess the levels of protein and mRNA levels. The various genes and proteins that are assessed include, collagen I, collagen III, elastin and tenacin C. Histological staining with stains such as Masson Trichrome, Picro-sirius red are used to identify the tissue/cellular distribution of collagen, which can be quantified using morphometric analysis both in vivo and in vitro. The assays are routinely used and are quantitative.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><strong><em>Sircol Collagen Assay for collagen quantification:</em></strong></span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">The Serius dye has been used for many decades to detect collagen in histology samples. The Serius Red F3BA selectively binds to collagen and the signal can be read at 540 nm (Chen & Raghunath, 2009; Nikota et al., 2017).</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Hydroxyproline is a non-proteinogenic amino acid formed by the prolyl-4-hydroxylase. Hydroxyproline is only found in collagen and thus, it serves as a direct measure of the amount of collagen present in cells or tissues. Colorimetric methods are readily available and have been extensively used to quantify collagen using this assay (Chen & Raghunath, 2009; Nikota et al., 2017).</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><strong><em><span style="color:red">Ex vivo and in vitro models of ECM deposition:</span></em></strong></span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="color:red">No models currently exist which allow for in vitro assessment of ECM deposition. Using single, or co-cultures containing fibroblasts, the production of soluble ECM components can be assessed after exposure to a stressor of interest using either ELISA or qRT-PCR experiments as a proxy. </span></span></span></p>
<h4>References</h4>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">1. Bateman, E., Turner-Warwick, M. and Adelmann-Grill, B. (1981). Immunohistochemical study of collagen types in human foetal lung and fibrotic lung disease. Thorax, 36(9), pp.645-653.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">2. Bensadoun, E., Burke, A., Hogg, J. and Roberts, C. (1996). Proteoglycan deposition in pulmonary fibrosis. American Journal of Respiratory and Critical Care Medicine, 154(6), pp.1819-1828.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">3. Blaauboer M et al. Extracellular matrix proteins: A positive feedback loop in lung fibrosis. Matrix Biology, 2014, 34, 170-178</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">4. Chen, C. and Raghunath, M. (2009). Focus on collagen: in vitro systems to study fibrogenesis and antifibrosis _ state of the art. Fibrogenesis & Tissue Repair, 2(1).</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">5. Crabb, R., Chau, E., Decoteau, D. and Hubel, A. (2006). Microstructural Characteristics of Extracellular Matrix Produced by Stromal Fibroblasts. Annals of Biomedical Engineering, 34(10), pp.1615-1627.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">6. HINZ, B. (2006). Masters and servants of the force: The role of matrix adhesions in myofibroblast force perception and transmission. European Journal of Cell Biology, 85(3-4), pp.175-181.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">7. Kuhn C, McDonald JA. The roles of the myofibroblast in idiopathic pulmonary fibrosis. Ultrastructural and immunohistochemical features of sites of active extracellular matrix synthesis. Am J Pathol. 1991;138(5):1257–1265.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">8. Klingberg, F., Hinz, B. and White, E. (2012). The myofibroblast matrix: implications for tissue repair and fibrosis. The Journal of Pathology, 229(2), pp.298-309.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">9. McKleroy, W., Lee, T. and Atabai, K. (2013). Always cleave up your mess: targeting collagen degradation to treat tissue fibrosis. American Journal of Physiology-Lung Cellular and Molecular Physiology, 304(11), pp.L709-L721.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">10. Nikota, J., Banville, A., Goodwin, L., Wu, D., Williams, A., Yauk, C., Wallin, H., Vogel, U. and Halappanavar, S. (2017). Stat-6 signaling pathway and not Interleukin-1 mediates multi-walled carbon nanotube-induced lung fibrosis in mice: insights from an adverse outcome pathway framework. Particle and Fibre Toxicology, 14(1).</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">11. Nimni, M. (1983). Collagen: Structure, function, and metabolism in normal and fibrotic tissues. Seminars in Arthritis and Rheumatism, 13(1), pp.1-86.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">12. Rozin, G., Gomes, M., Parra, E., Kairalla, R., de Carvalho, C. and Capelozzi, V. (2005). Collagen and elastic system in the remodelling process of major types of idiopathic interstitial pneumonias (IIP). Histopathology, 46(4), pp.413-421.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">13. Sivakumar, P., Ntolios, P., Jenkins, G. and Laurent, G. (2012). Into the matrix. Current Opinion in Pulmonary Medicine, 18(5), pp.462-469.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">14. White, E. (2015). Lung Extracellular Matrix and Fibroblast Function. Annals of the American Thoracic Society, 12(Supplement 1), pp.S30- S33. </span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">15. Wynn, T. (2011). Integrating mechanisms of pulmonary fibrosis. The Journal of Experimental Medicine, 208(7), pp.1339-1350.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">16. Zhang K, Rekhter MD, Gordon D, Phan SH. Myofibroblasts and their role in lung collagen gene expression during pulmonary fibrosis. A combined immunohistochemical and in situ hybridization study. Am J Pathol. 1994;145(1):114–125</span></span></p>
<td><a href="/aops/38">Aop:38 - Protein Alkylation leading to Liver Fibrosis</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/144">Aop:144 - Endocytic lysosomal uptake leading to liver fibrosis</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/383">Aop:383 - Inhibition of Angiotensin-converting enzyme 2 leading to liver fibrosis</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/494">Aop:494 - AhR activation leading to liver fibrosis </a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/624">Aop:624 - Increased 11β-Hydroxysteroid dehydrogenase type 1 activity leading to MASLD progression via insulin resistance-associated mitochondrial dysfunction</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/625">Aop:625 - Increased 11β-Hydroxysteroid dehydrogenase type 1 activity leading to MASLD progression via insulin resistance-associated oxidative stress</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/626">Aop:626 - Increased 11β-Hydroxysteroid dehydrogenase type 1 activity leading to MASLD progression via insulin resistance-associated endoplasmic reticulum stress</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/627">Aop:627 - Increased 11β-Hydroxysteroid dehydrogenase type 1 activity leading to MASLD progression via lipogenesis-associated mitochondrial dysfunction</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/628">Aop:628 - Increased 11β-Hydroxysteroid dehydrogenase type 1 activity leading to MASLD progression via lipogenesis-associated oxidative stress</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/629">Aop:629 - Increased 11β-Hydroxysteroid dehydrogenase type 1 activity leading to MASLD progression via lipogenesis-associated endoplasmic reticulum stress</a></td>
<p>Human: Bataller and Brenner, 2005;Merck Manual, 2015; Blachier et al., 2013.</p>
<p>Rat, mouse: Liedtke et al., 2013</p>
<h4>Key Event Description</h4>
<p>Liver fibrosis results from perpetuation of the normal wound healing response, as a result of repeated cycles of hepatocyte injury and repair and is a dynamic process, characterised by an excessive deposition of ECM (extracellular matrix) proteins including glycoproteins, collagens, and proteoglycans. It is usually secondary to hepatic injury and inflammation, and progresses at different rates depending on the aetiology of liver disease and is also influenced by environmental and genetic factors. If fibrosis continues, it disrupts the normal architecture of the liver, altering the normal function of the organ and ultimately leading to liver damage. Cirrhosis represents the final stage of fibrosis. It is characterised by fibrous septa which divide the parenchyma into regenerative nodules which leads to vascular modifications and portal hypertension with its complications of variceal bleeding, hepatic encephalopathy, ascites, and hepatorenal syndrome. In addition, this condition is largely associated with hepatocellular carcinoma with a further increase in the relative mortality rate (Bataller and Brenner, 2005; Merck Manual,2015)<sup> </sup></p>
<p>Liver fibrosis is an important health issue with clear regulatory relevance. The burden of disease attributable to liver fibrosis is quite high; progressive hepatic fibrosis, ultimately leading to cirrhosis, is a significant contributor to global health burden (Lim and Kim, 2008). In the European Union, 0.1 % of the population is affected by cirrhosis, the most advanced stage of liver fibrosis with full architectural disturbances (Blachier et al., 2013). Besides the epidemiological relevance, liver fibrosis also imposes a considerable economic burden on society. Indeed, the only curative therapy for chronic liver failure is liver transplantation. More than 5.500 orthotopic liver transplantations are currently performed in Europe on a yearly basis, costing up to €100.000 the first year and €10.000 yearly thereafter (Van Agthoven et al., 2001). </p>
<h4>How it is Measured or Detected</h4>
<p>Liver biopsy is an important part of the evaluation of patients with a variety of liver diseases. Besides establishing the diagnosis, the biopsy is often used to assess the severity of the disease. Until recently it has been assumed that fibrosis is an irreversible process, so most grading and staging systems have relatively few stages and are not very sensitive for describing changes in fibrosis. In all systems, the stages are determined by both the quantity and location of the fibrosis, with the formation of septa and nodules as major factors in the transition from one stage to the next. The absolute amount of fibrous tissue is variable within each stage, and there is considerable overlap between stages. Commonly used systems are the Knodell score with 4 stages - no fibrosis (score 0) to fibrous portal expansion (score 2) to bridging fibrosis (score 3) and Cirrhosis (score 4) – and the more sensitive Ishak fibrosis score with six stages - from no fibrosis (stage 0) over increasing fibrous expansion on portal areas (stages 1-2), bridging fibrosis (stages 3-4), and nodules (stage 5) to cirrhosis (stage 6) (Goodman, 2007). Liver biopsy is an invasive test with many possible complications and the potential for sampling error. Noninvasive tests become increasingly precise in identifying the amount of liver fibrosis through computer-assisted image analysis. Standard liver tests are of limited value in assessing the degree of fibrosis. Direct serologic markers of fibrosis include those associated with matrix deposition — e.g.procollagen type III amino-terminal peptide (P3NP), type I and IV collagens, laminin, hyaluronic acid, and chondrex. P3NP is the most widely studied marker of hepatic fibrosis. Other direct markers of fibrosis are those associated with matrix degradation, ie, matrix metalloproteinases 2 and 3 (MMP-2, MMP- 3) and tissue inhibitors of metalloproteinases 1 and 2 (TIMP-1, TIMP-2).These tests are not commercially available, and the components are not readily available in most clinical laboratories. Some indirect markers that combine several parameters are available but not very reliable. Conventional imaging studies (ultrasonography and computed tomography) are not sensitive for fibrosis. Hepatic elastography, a method for estimating liver stiffness, is a recent development in the noninvasive measurement of hepatic fibrosis. Currently, elastography can be accomplished by ultrasound or magnetic resonance. Liver biopsy is still needed if laboratory testing and imaging studies are inconclusive (Carey, 2010; Germani et al., 2011) .</p>
<h4>Regulatory Significance of the AO</h4>
<p>From the OECD - GUIDANCE DOCUMENT ON DEVELOPING AND ASSESSING ADVERSE OUTCOME PATHWAYS - Series on Testing and Assessment 18: "...an adverse effect that is of regulatory interest (e.g. repeated dose liver fibrosis)"</p>
<h4>References</h4>
<ul>
<li>Bataller, R. and D.A. Brenner (2005), Liver Fibrosis, J.Clin. Invest, vol. 115, no. 2, pp. 209-218.</li>
<li>Merck Manual available at: <a class="external free" href="http://www.merckmanuals.com/professional/hepatic_and_biliary_disorders/fibrosis_and_cirrhosis/hepatic_fibrosis.html,(accessed" rel="nofollow" target="_blank">http://www.merckmanuals.com/professional/hepatic_and_biliary_disorders/fibrosis_and_cirrhosis/hepatic_fibrosis.html,(accessed</a> 10 February 2015).</li>
<li>Lim, Y. and W. Kim (2008), The global impact of hepatic fibrosis and end-stage liver disease, Clin Liver Dis, vol. 12, no. 4, pp. 733-746.</li>
<li>Blachier, M. et al. (2013), The burden of liver disease in Europe: a review of available epidemiological data, J Hepatol, vol. 58, no. 3, pp. 593-608.</li>
<li>Van Agthoven, M. et al. (2001), A comparison of the costs and effects of liver transplantation for acute and for chronic liver failure. Transpl Int, vol. 14, no. 2, pp. 87-94.</li>
<li>Goodman, Z.D. (2007), Grading and staging systems for inflammation and fibrosis in chronic liver diseases, Journal of Hepatology, vol. 47, no. 4, pp. 598-607.</li>
<li>Carey, E. (2010), Noninvasive tests for liver disease, fibrosis, and cirrhosis: Is liver biopsy obsolete? Cleveland Clinic Journal of Medicine, vol. 77, no. 8, pp. 519-527.</li>
<li>Germani, G. et al. (2011), Assessment of Fibrosis and Cirrhosis in Liver Biopsies, Semin Liver Dis, vol. 31, no. 1, pp. 82-90. available at <a class="external free" href="http://www.medscape.com/viewarticle/743946_2,(accessed" rel="nofollow" target="_blank">http://www.medscape.com/viewarticle/743946_2,(accessed</a> 10 February 2015).</li>
<li>Liedtke, C. et al. (2013), Experimental liver fibrosis research: update on animal models, legal issues and translational aspects, Fibrogenesis Tissue Repair, vol. 6, no. 1, p. 19.</li>
</ul>
<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/19">Relationship: 19: Activation, AhR leads to Up Regulation, CYP1A1</a></h4>
<h4><a href="/relationships/3219">Relationship: 3219: Activation, AhR leads to Increase, Liver steatosis</a></h4>
<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="font-size:10pt"><span style="color:red">The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates cellular responses to environmental toxins, dietary components, and endogenous metabolites. Upon ligand binding, AhR translocates to the nucleus, dimerizes with the aryl hydrocarbon receptor nuclear translocator (ARNT), and binds to xenobiotic response elements (XREs) in the promoter regions of target genes. While its primary function involves detoxification through the regulation of cytochrome P450 enzymes (e.g., CYP1A1, CYP1B1), chronic or excessive AhR activation is implicated in metabolic disorders, including liver steatosis (</span></span><span style="font-size:10pt"><span style="background-color:yellow"><span style="color:red">PMID: 34830313</span></span></span><span style="font-size:10pt"><span style="color:red">, <span style="background-color:yellow">PMID: 37284280</span>)</span></span><span style="font-size:10pt"><span style="color:red">. </span></span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="font-size:10pt"><span style="color:red">AhR activation modulates lipid homeostasis through the upregulation of key lipogenic pathways. It regulates the expression of sterol regulatory element-binding protein 1c (SREBP-1c), a master regulator of fatty acid and triglyceride synthesis (<span style="background-color:yellow">PMID: 29694888</span>). Increased SREBP-1c expression enhances the transcription of lipogenic enzymes, such as acetyl-CoA carboxylase (ACC) and fatty acid synthase (FASN), leading to de novo lipogenesis (</span></span><span style="font-size:10pt"><span style="background-color:yellow"><span style="color:red">PMID: 21029304</span></span></span><span style="font-size:10pt"><span style="color:red">)</span></span><span style="font-size:10pt"><span style="color:red">. Concurrently, AhR increases the expression of CD36, a key transporter of fatty acids, which contributes to the import of these molecules in the cytoplasm and subsequently to de novo lipogenesis (</span></span><span style="font-size:10pt"><span style="background-color:yellow"><span style="color:red">PMID: 20303349</span></span></span><span style="font-size:10pt"><span style="color:red">). </span></span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="font-size:10pt"><span style="color:red">AhR activation also drives liver steatosis by inducing inflammatory responses. It promotes the production of pro-inflammatory cytokines, such as interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) (</span></span><span style="font-size:10pt"><span style="background-color:yellow"><span style="color:red">PMID: 27713108</span></span></span><span style="font-size:10pt"><span style="color:red">). </span></span><span style="font-size:10pt"><span style="color:red">This inflammatory milieu exacerbates hepatic lipid accumulation by impairing insulin signaling, which further upregulates SREBP-1c activity. Additionally, AhR activation increases the production of reactive oxygen species (ROS), leading to oxidative stress, lipid peroxidation, and hepatocyte injury, all of which contribute to the pathogenesis of steatosis (</span></span><span style="font-size:10pt"><span style="background-color:yellow"><span style="color:red">PMID: 34830313</span></span></span><span style="font-size:10pt"><span style="color:red">)</span></span><span style="font-size:10pt"><span style="color:red">. Furthermore, AhR-mediated repression of fibroblast growth factor 21 (FGF21), a hormone critical for lipid metabolism and insulin sensitivity, has been implicated in exacerbating steatosis (<span style="background-color:yellow">PMID: 27226639</span>). </span></span></span></span></span></p>
<h4>Evidence Supporting this KER</h4>
<strong>Biological Plausibility</strong>
<p style="margin-left:48px; text-align:start"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="font-size:10pt"><span style="color:red">There is a mechanistical relationship between both KEs (see ‘<u>Key Event Relationship Description’)</u></span></span></span></span></span></p>
<strong>Empirical Evidence</strong>
<p style="margin-left:48px; text-align:start"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="font-size:10pt"><span style="color:red">There is temporal concordance as AhR activation leads to adaptative responses in the liver including metabolic switch. In tumor cells, AhR activation is associated with a Warburg effect and increased glycolytic activity; subsequently, mitochondrial metabolism is decreased which is consistent with liver steatosis that depends on lipid catabolism mainly observed in the mitochondrial matrix with beta-oxidation (PMID: 26582802).</span></span></span></span></span></p>
<strong>Uncertainties and Inconsistencies</strong>
<p style="margin-left:48px; text-align:start"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="font-size:10pt"><span style="color:red">There are no inconsistencies</span></span></span></span></span></p>
<h4>References</h4>
<p style="text-align:justify"><span style="font-size:medium"><span style="font-family:"Times New Roman",serif"><span style="color:#000000"><strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">Fling RR, Zacharewski TR. Aryl Hydrocarbon Receptor (AhR) Activation by 2,3,7,8-Tetrachlorodibenzo-<em>p</em>-Dioxin (TCDD) Dose-Dependently Shifts the Gut Microbiome Consistent with the Progression of Non-Alcoholic Fatty Liver Disease. Int J Mol Sci. 2021 Nov 18;22(22):12431. doi: 10.3390/ijms222212431. PMID: 34830313; PMCID: PMC8625315.</span></span></span></strong></span></span></span></p>
<p style="text-align:justify"><span style="font-size:medium"><span style="font-family:"Times New Roman",serif"><span style="color:#000000"><strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">Patil NY, Friedman JE, Joshi AD. Role of Hepatic Aryl Hydrocarbon Receptor in Non-Alcoholic Fatty Liver Disease. Receptors (Basel). 2023 Mar;2(1):1-15. doi: 10.3390/receptors2010001. Epub 2023 Jan 4. PMID: 37284280; PMCID: PMC10240927.</span></span></span></strong></span></span></span></p>
<p style="text-align:justify"><span style="font-size:medium"><span style="font-family:"Times New Roman",serif"><span style="color:#000000"><strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">Krishnan S, Ding Y, Saedi N, Choi M, Sridharan GV, Sherr DH, Yarmush ML, Alaniz RC, Jayaraman A, Lee K. Gut Microbiota-Derived Tryptophan Metabolites Modulate Inflammatory Response in Hepatocytes and Macrophages. Cell Rep. 2018 Apr 24;23(4):1099-1111. doi: 10.1016/j.celrep.2018.03.109. Erratum in: Cell Rep. 2019 Sep 17;28(12):3285. doi: 10.1016/j.celrep.2019.08.080. PMID: 29694888; PMCID: PMC6392449.</span></span></span></strong></span></span></span></p>
<p style="text-align:justify"><span style="font-size:medium"><span style="font-family:"Times New Roman",serif"><span style="color:#000000"><strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">Ferré P, Foufelle F. Hepatic steatosis: a role for de novo lipogenesis and the transcription factor SREBP-1c. Diabetes Obes Metab. 2010 Oct;12 Suppl 2:83-92. doi: 10.1111/j.1463-1326.2010.01275.x. PMID: 21029304.</span></span></span></strong></span></span></span></p>
<p style="text-align:justify"><span style="font-size:medium"><span style="font-family:"Times New Roman",serif"><span style="color:#000000"><strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">Lee JH, Wada T, Febbraio M, He J, Matsubara T, Lee MJ, Gonzalez FJ, Xie W. A novel role for the dioxin receptor in fatty acid metabolism and hepatic steatosis. Gastroenterology. 2010 Aug;139(2):653-63. doi: 10.1053/j.gastro.2010.03.033. Epub 2010 Mar 17. PMID: 20303349; PMCID: PMC2910786.</span></span></span></strong></span></span></span></p>
<p style="text-align:justify"><span style="font-size:medium"><span style="font-family:"Times New Roman",serif"><span style="color:#000000"><strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">Duval C, Teixeira-Clerc F, Leblanc AF, Touch S, Emond C, Guerre-Millo M, Lotersztajn S, Barouki R, Aggerbeck M, Coumoul X. Chronic Exposure to Low Doses of Dioxin Promotes Liver Fibrosis Development in the C57BL/6J Diet-Induced Obesity Mouse Model. Environ Health Perspect. 2017 Mar;125(3):428-436. doi: 10.1289/EHP316. Epub 2016 Oct 7. PMID: 27713108; PMCID: PMC5332187.</span></span></span></strong></span></span></span></p>
<p style="text-align:justify"><span style="font-size:medium"><span style="font-family:"Times New Roman",serif"><span style="color:#000000"><strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">Xia H, Zhu X, Zhang X, Jiang H, Li B, Wang Z, Li D, Jin Y. Alpha-naphthoflavone attenuates non-alcoholic fatty liver disease in oleic acid-treated HepG2 hepatocytes and in high fat diet-fed mice. Biomed Pharmacother. 2019 Oct;118:109287. doi: 10.1016/j.biopha.2019.109287. Epub 2019 Aug 8. PMID: 31401392.</span></span></span></strong></span></span></span></p>
<p><strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">Eti NA, Flor S, Iqbal K, Scott RL, Klenov VE, Gibson-Corley KN, Soares MJ, Ludewig G, Robertson LW. PCB126 induced toxic actions on liver energy metabolism is mediated by AhR in rats. Toxicology. 2022 Jan 30;466:153054. doi: 10.1016/j.tox.2021.153054. Epub 2021 Nov 27. PMID: 34848246; PMCID: PMC8748418.</span></span></span></strong></p>
</div>
<div>
<h4><a href="/relationships/3218">Relationship: 3218: Increased, Liver Steatosis leads to Cell injury/death</a></h4>
<h4><a href="/relationships/3218">Relationship: 3218: Increase, Liver steatosis leads to Cell injury/death</a></h4>
<td><a href="/aops/494">AhR activation leading to liver fibrosis </a></td>
<td>adjacent</td>
<td>Moderate</td>
<td>Moderate</td>
</tr>
</tbody>
</table>
</div>
<h4>Evidence Supporting Applicability of this Relationship</h4>
<div>
</div>
<div>
</div>
<div>
</div>
<ul>
<li style="text-align:justify"><span style="font-size:12pt"><span style="color:red"><span style="font-family:"Times New Roman",serif"><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif">Human : PMID: 32044315</span></span></span></span></span></li>
<li style="text-align:justify"><span style="font-size:12pt"><span style="color:red"><span style="font-family:"Times New Roman",serif"><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif">Rat: PMID: 26674288</span></span></span></span></span></li>
<li style="text-align:justify"><span style="font-size:12pt"><span style="color:red"><span style="font-family:"Times New Roman",serif"><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif">Mouse : PMID: 36690638</span></span></span></span></span></li>
</ul>
<p style="text-align:justify"><span style="font-size:medium"><span style="font-family:"Times New Roman",serif"><span style="color:#000000"><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">Understanding this KER is critical for the development of interventions targeting early lipid accumulation to prevent downstream liver damage. It also aids in the establishment of biomarkers predictive of NAFLD/NASH progression.</span></span></span></span></span></span></p>
<h4>Key Event Relationship Description</h4>
<p style="text-align:justify"><span style="font-size:medium"><span style="font-family:"Times New Roman",serif"><span style="color:#000000"><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">Liver steatosis involves the abnormal accumulation of lipids within hepatocytes, which, when excessive, induces cellular stress through mechanisms such as oxidative stress, mitochondrial dysfunction, and endoplasmic reticulum stress. These disruptions compromise cell integrity and function, leading to cell death through apoptosis or necrosis. Indeed, empirical evidence supports this relationship (see below also) across various models, including <em>in vitro</em> studies where lipid accumulation triggers reactive oxygen species (ROS) production and cell death, as well as <em>in vivo</em> models of diet-induced steatosis that demonstrate progressive liver damage. Clinically, in patients with non-alcoholic fatty liver disease (NAFLD), a transition from simple steatosis to non-alcoholic steatohepatitis (NASH) involves hepatocyte injury and death, evidenced by histopathological changes and increased biomarkers of liver damage (PMID: 34848246).</span></span></span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:medium"><span style="font-family:"Times New Roman",serif"><span style="color:#000000"><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">The quantitative link between lipid accumulation and cell injury/death shows a dose-dependent relationship, although modulating factors such as the type of lipids, genetic predispositions, and environmental influences can affect the severity and onset of cell damage. Understanding this KER is essential for identifying interventions aimed at mitigating the progression of liver steatosis to irreversible liver injury.</span></span></span></span></span></span></p>
<h4>Evidence Supporting this KER</h4>
<strong>Biological Plausibility</strong>
<ul>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">Liver steatosis, characterized by excessive accumulation of lipids in hepatocytes, predisposes cells to injury and death due to lipotoxicity. Excessive lipid accumulation leads to oxidative stress, mitochondrial dysfunction, and endoplasmic reticulum stress. These cellular disturbances compromise membrane integrity, impair energy homeostasis, and activate apoptotic and necrotic pathways, resulting in cell injury and death. Chronic steatosis amplifies inflammatory signaling, creating a self-perpetuating cycle of liver damage (PMID: 37400694).</span></span></span></span></span></li>
</ul>
<strong>Empirical Evidence</strong>
<ul>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">I<strong>n vitro studies:</strong> Hepatocytes exposed to elevated free fatty acid levels (e.g., palmitate) exhibit lipid accumulation followed by markers of oxidative stress (e.g., ROS production) and cell death (apoptosis or necrosis).</span></span></span></span></span></li>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">Animal models:</span></span></span></strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red"> Rodents fed a high-fat diet show progression from liver steatosis to hepatocyte injury, evidenced by elevated serum alanine aminotransferase (ALT) levels and histological detection of necrotic foci.</span></span></span></span></span></li>
<li style="text-align:justify"><strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">Clinical observations:</span></span></span></strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red"> In patients with non-alcoholic fatty liver disease (NAFLD), progression from simple steatosis to non-alcoholic steatohepatitis (NASH) involves significant hepatocyte ballooning (a marker of injury) and cell death</span></span></span></li>
</ul>
<strong>Uncertainties and Inconsistencies</strong>
<ul>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">Heterogeneity of lipid species:</span></span></span></strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red"> Not all lipid accumulations are equally toxic. For example, triglycerides may serve as inert storage, whereas free fatty acids and ceramides are directly cytotoxic. Same remark regarding the type of fatty acids (saturated ones vs omega3)</span></span></span></span></span></li>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">Species differences:</span></span></span></strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red"> Rodents and humans may exhibit differences in susceptibility to lipid-induced liver damage.</span></span></span></span></span></li>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">Temporal dynamics:</span></span></span></strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red"> The time lag between lipid accumulation and detectable cell injury varies across models, complicating predictions.</span></span></span></span></span></li>
</ul>
<h4>Quantitative Understanding of the Linkage</h4>
<p style="text-align:justify"><span style="font-size:medium"><span style="font-family:"Times New Roman",serif"><span style="color:#000000"><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">The relationship between liver steatosis and cell injury/death exhibits a dose-dependent response. Higher levels of lipid accumulation correlate with increased markers of hepatocyte damage, such as ALT, AST, and lactate dehydrogenase (LDH) release. Thresholds for lipid overload beyond which cellular injury becomes inevitable have been proposed in experimental studies but remain context-dependent, influenced by factors like lipid species and duration of exposure.</span></span></span></span></span></span></p>
<td><span style="font-size:medium"><span style="font-family:"Times New Roman",serif"><span style="color:#000000"><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red"> Variants in genes such as PNPLA3 influence susceptibility to lipid-induced hepatocyte injury.</span></span></span></span></span></span></td>
<td><span style="font-size:medium"><span style="font-family:"Times New Roman",serif"><span style="color:#000000"><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">Antioxidants or omega-3 fatty acids may mitigate the progression from steatosis to cell death.</span></span></span></span></span></span></td>
<p style="text-align:justify"><span style="font-size:medium"><span style="font-family:"Times New Roman",serif"><span style="color:#000000"><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">Schwabe RF, Tabas I, Pajvani UB. Mechanisms of Fibrosis Development in Nonalcoholic Steatohepatitis. Gastroenterology. 2020 May;158(7):1913-1928. doi: 10.1053/j.gastro.2019.11.311. Epub 2020 Feb 8. PMID: 32044315; PMCID: PMC7682538.</span></span></span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:medium"><span style="font-family:"Times New Roman",serif"><span style="color:#000000"><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">Moravcová A, Červinková Z, Kučera O, Mezera V, Rychtrmoc D, Lotková H. The effect of oleic and palmitic acid on induction of steatosis and cytotoxicity on rat hepatocytes in primary culture. Physiol Res. 2015;64(Suppl 5):S627-36. doi: 10.33549/physiolres.933224. Epub 2015 Dec 15. PMID: 26674288.</span></span></span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:medium"><span style="font-family:"Times New Roman",serif"><span style="color:#000000"><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">Inaba Y, Hashiuchi E, Watanabe H, Kimura K, Oshima Y, Tsuchiya K, Murai S, Takahashi C, Matsumoto M, Kitajima S, Yamamoto Y, Honda M, Asahara SI, Ravnskjaer K, Horike SI, Kaneko S, Kasuga M, Nakano H, Harada K, Inoue H. The transcription factor ATF3 switches cell death from apoptosis to necroptosis in hepatic steatosis in male mice. </span></span></span><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">Nat Commun. 2023 Jan 23;14(1):167. doi: 10.1038/s41467-023-35804-w. PMID: 36690638; PMCID: PMC9871012.</span></span></span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:medium"><span style="font-family:"Times New Roman",serif"><span style="color:#000000"><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">Eti NA, Flor S, Iqbal K, Scott RL, Klenov VE, Gibson-Corley KN, Soares MJ, Ludewig G, Robertson LW. PCB126 induced toxic actions on liver energy metabolism is mediated by AhR in rats. Toxicology. 2022 Jan 30;466:153054. doi: 10.1016/j.tox.2021.153054. Epub 2021 Nov 27. PMID: 34848246; PMCID: PMC8748418.</span></span></span></span></span></span></p>
<p>Damaged hepatocytes can lead to activation of hepatic stellate cells (HSCs) through the release of ROS, cytokines and chemokines. Engulfment of apoptotic bodies from hepatocytes results in HSC activation and induces NOX (NADPH oxidases) expression in HSCs. DNA from apoptotic hepatocytes induces toll-like receptor 9 (TLR9)-dependent changes of HSCs that are consistent with late stages of HSC differentiation (activation), with up-regulation of collagen production and inhibition of platelet derived growth factor (PDGF)-mediated chemotaxis to retain HSCs at sites of cellular apoptosis.
The release of latent TGF-beta complex into the micro-environment by damaged hepatocytes is likely to be one of the first signals for adjacent HSCs leading to their activation.
</p><p>Damaged hepatocytes also influence liver sinusoidal endothelial cell (LSECs), which make an integral part of the hepatic reticulo-endothelial system and have a role in HSC activation. LSECs are morphologically identified by their fenestrations, which are transcytoplasmic canals arranged in sieve plates. In healthy liver, hepatocytes and HSCs maintain this phenotype of LSECs through release of vascular endothelial growth factor (VEGF). Differentiated (i.e. fenestrated) LSECs prevent HSC activation and promote reversal of activated HSC to quiescence, but LSEC lose this effect when they are de-differentiated due to liver injury. Preclinical studies have demonstrated that LSECs undergo defenestration as an early event that not only precedes liver fibrosis, but may also be permissive for it. Changes in LSEC differentiation might be an integral part of the development of fibrosis. Furthermore, in fibrosis LSECs become highly pro-inflammatory and secrete an array of cytokines and chemokines
</p><p>There is temporal concordance as HSC activation follows hepatic injury and there is experimental evidence for this KER.
Canbay et al. could show that Fas-mediated hepatocyte injury is mechanistically linked to liver fibrogenesis. Markers of HSC activation were significantly reduced when apoptosis was prevented in Fas-deficient bile duct ligated mice. These findings (reduction of inflammation, markers of HSC activation, and collagen I expression) could be repeated by pharmacological inhibition of liver cell apoptosis using a pan-caspase inhibitor.
Coulouarn et al found in a co-culture model that hepatocyte - HSC crosstalk engenders a permissive inflammatory microenvironment.
<strong>Uncertainties and Inconsistencies</strong>
<p>There are no inconsistencies
</p>
<h4>References</h4>
<ol class="references">
<li id="cite_note-Roth_1998-1"><span class="mw-cite-backlink">↑ <sup><a href="#cite_ref-Roth_1998_1-0">1.0</a></sup> <sup><a href="#cite_ref-Roth_1998_1-1">1.1</a></sup></span> <span class="reference-text">Roth, S., K. Michel and A.M. Gressner (1998), (Latent) transforming growth factor beta in liver parenchymal cells, its injury-dependent release, and paracrine effects on rat HSCs, Hepatology, vol. 27, no. 4, pp. 1003-1012.</span>
</li>
<li id="cite_note-Gressner_2002-2"><span class="mw-cite-backlink">↑ <sup><a href="#cite_ref-Gressner_2002_2-0">2.0</a></sup> <sup><a href="#cite_ref-Gressner_2002_2-1">2.1</a></sup></span> <span class="reference-text">Gressner , A.M. et al. (2002), Roles of TGF-β in hepatic fibrosis. Front Biosci, vol. 7, pp. 793-807.</span>
</li>
<li id="cite_note-Malhi_2010-3"><span class="mw-cite-backlink">↑ <sup><a href="#cite_ref-Malhi_2010_3-0">3.0</a></sup> <sup><a href="#cite_ref-Malhi_2010_3-1">3.1</a></sup></span> <span class="reference-text">Malhi, H. et al. (2010), Hepatocyte death: a clear and present danger, Physiol Rev, vol. 90, no. 3, pp. 1165-1194.</span>
</li>
<li id="cite_note-Canbay_2004-4"><span class="mw-cite-backlink">↑ <sup><a href="#cite_ref-Canbay_2004_4-0">4.0</a></sup> <sup><a href="#cite_ref-Canbay_2004_4-1">4.1</a></sup> <sup><a href="#cite_ref-Canbay_2004_4-2">4.2</a></sup></span> <span class="reference-text">Canbay, A., S.L. Friedman and G.J. Gores (2004), Apoptosis: the nexus of liver injury and
fibrosis, Hepatology, vol. 39, no. 2, pp. 273-278.</span>
</li>
<li id="cite_note-Orrenius_2011-5"><span class="mw-cite-backlink">↑ <sup><a href="#cite_ref-Orrenius_2011_5-0">5.0</a></sup> <sup><a href="#cite_ref-Orrenius_2011_5-1">5.1</a></sup></span> <span class="reference-text">Orrenius, S., P. Nicotera and B. Zhivotovsky (2011), Cell death mechanisms and their
implications in toxicology, Toxicol. Sci, vol. 119, no. 1, pp. 3-19.</span>
</li>
<li id="cite_note-Kolios_2006-6"><span class="mw-cite-backlink">↑ <sup><a href="#cite_ref-Kolios_2006_6-0">6.0</a></sup> <sup><a href="#cite_ref-Kolios_2006_6-1">6.1</a></sup></span> <span class="reference-text">Kolios, G., V. Valatas and E. Kouroumalis (2006), Role of Kupffer cells in the pathogenesis of liver disease, World J.Gastroenterol, vol. 12, no. 46, pp. 7413-7420.</span>
</li>
<li id="cite_note-Kisseleva_2008-7"><span class="mw-cite-backlink">↑ <sup><a href="#cite_ref-Kisseleva_2008_7-0">7.0</a></sup> <sup><a href="#cite_ref-Kisseleva_2008_7-1">7.1</a></sup> <sup><a href="#cite_ref-Kisseleva_2008_7-2">7.2</a></sup></span> <span class="reference-text">Kisseleva T and Brenner DA, (2008), Mechanisms of Fibrogenesis, Exp Biol Med, vol. 233, no. 2, pp. 109-122.</span>
</li>
<li id="cite_note-Li_2008-8"><span class="mw-cite-backlink">↑ <sup><a href="#cite_ref-Li_2008_8-0">8.0</a></sup> <sup><a href="#cite_ref-Li_2008_8-1">8.1</a></sup></span> <span class="reference-text">Li, Jing-Ting et al. (2008), Molecular mechanism of hepatic stellate cell activation and
<li id="cite_note-Xie_2012-12"><span class="mw-cite-backlink">↑ <sup><a href="#cite_ref-Xie_2012_12-0">12.0</a></sup> <sup><a href="#cite_ref-Xie_2012_12-1">12.1</a></sup></span> <span class="reference-text">Xie, G. et al. (2012), Role of differentiation of liver sinusoidal endothelial cells in progression and regression of hepatic fibrosis in rats, Gastroenterology, vol. 142, no. 4, pp. 918–927.</span>
</li>
<li id="cite_note-13"><span class="mw-cite-backlink"><a href="#cite_ref-13">↑</a></span> <span class="reference-text">Xie, G. et al. (2013), Hedgehog signalling regulates liver sinusoidal endothelial cell capillarisation, Gut, vol. 62, no. 2, pp. 299–309.</span>
</li>
<li id="cite_note-14"><span class="mw-cite-backlink"><a href="#cite_ref-14">↑</a></span> <span class="reference-text">Ding, B.S. et al. (2014), Divergent angiocrine signals from vascular niche balance liver regeneration and fibrosis, Nature, vol. 505, no. 7481, pp. 97–102.</span>
</li>
<li id="cite_note-15"><span class="mw-cite-backlink"><a href="#cite_ref-15">↑</a></span> <span class="reference-text">Connolly, M.K. et al. (2010), In hepatic fibrosis, liver sinusoidal endothelial cells acquire enhanced immunogenicity, J Immunol, vol. 185, no. 4, pp. 2200-2208.</span>
</li>
<li id="cite_note-16"><span class="mw-cite-backlink"><a href="#cite_ref-16">↑</a></span> <span class="reference-text">Canbay, A. et al. (2002), Fas enhances fibrogenesis in the bile duct ligated mouse: a link between apoptosis and fibrosis, Gastroenterology, vol. 123, no. 4, pp. 1323-1330.</span>
</li>
<li id="cite_note-17"><span class="mw-cite-backlink"><a href="#cite_ref-17">↑</a></span> <span class="reference-text">Canbay, A. et al. (2004), The caspase inhibitor IDN-6556 attenuates hepatic injury and fibrosis in the bile duct ligated mouse, J Pharmacol Exp Ther, vol. 308, no. 3, pp. 1191-1196.</span>
</li>
<li id="cite_note-Coulouarn_2012-18"><span class="mw-cite-backlink">↑ <sup><a href="#cite_ref-Coulouarn_2012_18-0">18.0</a></sup> <sup><a href="#cite_ref-Coulouarn_2012_18-1">18.1</a></sup></span> <span class="reference-text">Coulouarn, C. et al. (2012), Hepatocyte-stellate cell cross-talk in the liver engenders a permissive inflammatory micro-environment that drives progression in hepatocellular carcinoma, Cancer Res, vol. 72, no. 10, pp. 2533–2542.</span>
</li>
</ol>
</div>
<div>
<h4><a href="/relationships/2890">Relationship: 2890: Cell injury/death leads to Leukocyte recruitment/activation</a></h4>
<td><a href="/aops/494">AhR activation leading to liver fibrosis </a></td>
<td>adjacent</td>
<td>High</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
<h4>Evidence Supporting Applicability of this Relationship</h4>
<div>
</div>
<div>
</div>
<div>
</div>
<ul>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">This KER is most relevant to hepatic tissues, where stellate cells play a crucial role in fibrosis development.</span></span></span></span></span></li>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">It applies primarily to chronic fibrotic diseases such as liver fibrosis, but also potentially to chronic pancreatitis, and retinal fibrosis.</span></span></span></span></span></li>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">This relationship has been observed across multiple mammalian species, although species-specific differences in regulatory pathways may influence the quantitative aspects of the response.</span></span></span></span></span></li>
</ul>
<h4>Key Event Relationship Description</h4>
<p style="text-align:justify"><span style="font-size:medium"><span style="font-family:"Times New Roman",serif"><span style="color:#000000"><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">Upon activation, <strong>hepatic stellate cells</strong> undergo a transition into a myofibroblast-like phenotype, leading to excessive extracellular matrix deposition, primarily consisting of <strong>collagen type I and III</strong>, fibronectin, and other fibrillar proteins (PMID: 37152902). This activation is triggered by <strong>oxidative stress</strong>, <strong>pro-inflammatory cytokines</strong> (e.g., <strong>TGF-</strong></span></span></span><strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">β</span></span></span></strong><strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">, PDGF</span></span></span></strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">), and <strong>chronic tissue injury</strong>. Activated stellate cells express </span></span></span><strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">α</span></span></span></strong><strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">-SMA (alpha-smooth muscle actin)</span></span></span></strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red"> and exhibit increased secretion of ECM components. This process is central to the development of fibrosis in the liver, pancreas, and retina, contributing to tissue stiffness and organ dysfunction.</span></span></span></span></span></span></p>
<h4>Evidence Supporting this KER</h4>
<strong>Biological Plausibility</strong>
<ul>
<li style="list-style-type:none">
<ul style="list-style-type:circle">
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">The relationship between stellate cell activation and ECM accumulation is well-established. <strong>TGF-</strong></span></span></span><strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">β</span></span></span></strong><strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">1</span></span></span></strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red"> is a key cytokine that directly stimulates the expression of ECM-related genes (PMID: 24265236).</span></span></span></span></span></li>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">Inhibition of <strong>TGF-</strong></span></span></span><strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">β</span></span></span></strong><strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">1</span></span></span></strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red"> has been shown to prevent stellate cell activation and reduce collagen production (PMID: 37923895).</span></span></span></span></span></li>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">Transcriptomic analyses reveal a strong correlation between stellate cell activation markers (<strong>ACTA2, COL1A1, TIMP-1</strong>) and ECM deposition in fibrotic tissues (PMID: 39062514).</span></span></span></span></span></li>
</ul>
</li>
</ul>
<strong>Empirical Evidence</strong>
<ul style="list-style-type:circle">
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">In vitro studies</span></span></span></strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">: When cultured with <strong>TGF-</strong></span></span></span><strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">β</span></span></span></strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">, stellate cells exhibit increased collagen and fibronectin production (PMID: 30584275).</span></span></span></span></span></li>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">Animal models</span></span></span></strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">: In <strong>TGF-</strong></span></span></span><strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">β</span></span></span></strong><strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red"> or </span></span></span></strong><strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">α</span></span></span></strong><strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">-SMA knockout mice</span></span></span></strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">, chemically induced liver fibrosis (e.g., <strong>CCl₄ exposure</strong>) is significantly attenuated, indicating a direct causal role (PMID: 30584275).</span></span></span></span></span></li>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">Clinical data</span></span></span></strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">: In patients with liver fibrosis, activated stellate cells (</span></span></span><strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">α</span></span></span></strong><strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">-SMA+, Desmin+</span></span></span></strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">) are strongly correlated with the degree of ECM deposition (PMID: 38056058).</span></span></span></span></span></li>
</ul>
<strong>Uncertainties and Inconsistencies</strong>
<ul>
<li style="list-style-type:none">
<ul style="list-style-type:circle">
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">Suppressing stellate cell activation may not completely prevent ECM deposition; <strong>other cell types</strong> (e.g., portal fibroblasts, macrophages) may also contribute.</span></span></span></span></span></li>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">Species-specific differences in the regulation of stellate cell activation and ECM production may affect the translatability of animal model findings to humans.</span></span></span></span></span></li>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">Certain pro-inflammatory cytokines (<strong>IL-6, TNF-</strong></span></span></span><strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">α</span></span></span></strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">) may have <strong>context-dependent effects</strong> that modulate stellate cell activation and fibrogenesis differently.</span></span></span></span></span></li>
</ul>
</li>
</ul>
<h4>References</h4>
<p style="text-align:justify"><span style="font-size:medium"><span style="font-family:"Times New Roman",serif"><span style="color:#000000"><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">Zhao YQ, Deng XW, Xu GQ, Lin J, Lu HZ, Chen J. Mechanical homeostasis imbalance in hepatic stellate cells activation and hepatic fibrosis. </span></span></span><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">Front Mol Biosci. 2023 Apr 20;10:1183808. doi: 10.3389/fmolb.2023.1183808. PMID: 37152902; PMCID: PMC10157180.</span></span></span></span></span></span></p>
<p style="text-align:justify"> </p>
<p style="text-align:justify"><span style="font-size:medium"><span style="font-family:"Times New Roman",serif"><span style="color:#000000"><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">Kasahara N, Imi Y, Amano R, Shinohara M, Okada K, Hosokawa Y, Imamori M, Tomimoto C, Kunisawa J, Kishino S, Ogawa J, Ogawa W, Hosooka T. A gut microbial metabolite of linoleic acid ameliorates liver fibrosis by inhibiting TGF-</span></span></span><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">β</span></span></span><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red"> signaling in hepatic stellate cells. </span></span></span><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">Sci Rep. 2023 Nov 3;13(1):18983. doi: 10.1038/s41598-023-46404-5. PMID: 37923895; PMCID: PMC10624680.</span></span></span></span></span></span></p>
<p style="text-align:justify"> </p>
<p style="text-align:justify"><span style="font-size:medium"><span style="font-family:"Times New Roman",serif"><span style="color:#000000"><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">Puche JE, Saiman Y, Friedman SL. </span></span></span><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">Hepatic stellate cells and liver fibrosis. </span></span></span><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">Compr Physiol. 2013 Oct;3(4):1473-92. doi: 10.1002/cphy.c120035. PMID: 24265236.</span></span></span></span></span></span></p>
<p style="text-align:justify"> </p>
<p style="text-align:justify"><span style="font-size:medium"><span style="font-family:"Times New Roman",serif"><span style="color:#000000"><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">Buakaew W, Krobthong S, Yingchutrakul Y, Potup P, Thongsri Y, Daowtak K, Ferrante A, Usuwanthim K. Investigating the Antifibrotic Effects of </span></span></span><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">β</span></span></span><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">-Citronellol on a TGF-</span></span></span><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">β</span></span></span><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">1-Stimulated LX-2 Hepatic Stellate Cell Model. </span></span></span><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">Biomolecules. 2024 Jul 5;14(7):800. doi: 10.3390/biom14070800. PMID: 39062514; PMCID: PMC11274813.</span></span></span></span></span></span></p>
<p style="text-align:justify"> </p>
<p style="text-align:justify"><span style="font-size:medium"><span style="font-family:"Times New Roman",serif"><span style="color:#000000"><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">Mu M, Zuo S, Wu RM, Deng KS, Lu S, Zhu JJ, Zou GL, Yang J, Cheng ML, Zhao XK. </span></span></span><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">Ferulic acid attenuates liver fibrosis and hepatic stellate cell activation via inhibition of TGF-</span></span></span><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">β</span></span></span><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">/Smad signaling pathway. Drug Des Devel Ther. 2018 Dec 3;12:4107-4115. doi: 10.2147/DDDT.S186726. Erratum in: Drug Des Devel Ther. 2019 May 24;13:1819. doi: 10.2147/DDDT.S215949. </span></span></span><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">PMID: 30584275; PMCID: PMC6284527.</span></span></span></span></span></span></p>
<p style="text-align:justify"> </p>
<p style="text-align:justify"><span style="font-size:medium"><span style="font-family:"Times New Roman",serif"><span style="color:#000000"><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">Parola M, Pinzani M. Liver fibrosis in NAFLD/NASH: from pathophysiology towards diagnostic and therapeutic strategies. Mol Aspects Med. 2024 Feb;95:101231. doi: 10.1016/j.mam.2023.101231. Epub 2023 Dec 5. PMID: 38056058.</span></span></span></span></span></span></p>
<p style="text-align:justify"> </p>
<p style="text-align:justify"><span style="font-size:medium"><span style="font-family:"Times New Roman",serif"><span style="color:#000000"><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">Hirabaru M, Mochizuki K, Takatsuki M, Soyama A, Kosaka T, Kuroki T, Shimokawa I, Eguchi S. Expression of alpha smooth muscle actin in living donor liver transplant recipients. World J Gastroenterol. 2014 Jun 14;20(22):7067-74. doi: 10.3748/wjg.v20.i22.7067. PMID: 24966580; PMCID: PMC4051953.</span></span></span></span></span></span></p>
<td><a href="/aops/383">Inhibition of Angiotensin-converting enzyme 2 leading to liver fibrosis</a></td>
<td>adjacent</td>
<td>Moderate</td>
<td>Not Specified</td>
</tr>
<tr>
<td><a href="/aops/494">AhR activation leading to liver fibrosis </a></td>
<td>adjacent</td>
<td>High</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
<h4>Evidence Supporting Applicability of this Relationship</h4>
<div>
</div>
<div>
</div>
<div>
</div>
<ul style="list-style-type:disc">
<li style="text-align:justify"><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif">ECM overproduction is a hallmark of chronic liver diseases’ progression. Even if specific to the liver, this KER could have been highly relevant for fibrosis in other tissues (lungs, kidneys, heart, and skin).</span></span></li>
<li style="text-align:justify"><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif">The relationship has been observed across mammalian species, though the time course of fibrosis development varies.</span></span></li>
</ul>
<h4>Key Event Relationship Description</h4>
<p style="text-align:justify"><span style="font-size:medium"><span style="font-family:"Times New Roman",serif"><span style="color:#000000"><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">Fibrosis is the <strong>pathological consequence</strong> of excessive ECM deposition, primarily composed of <strong>collagen type I and III</strong>, fibronectin, and glycosaminoglycans. When ECM production exceeds its degradation, tissues undergo <strong>progressive stiffening and scarring</strong>, leading to functional impairment of affected organs. This process occurs in response to <strong>chronic inflammation, mechanical stress, and prolonged injury</strong>, where ECM-producing cells (e.g., <strong>myofibroblasts, activated stellate cells, fibroblasts</strong>) become <strong>overactive</strong> and produce an excessive matrix. The excessive ECM deposition disrupts normal tissue architecture, interferes with cellular signaling, and promotes further <strong>inflammation, hypoxia, and cell death</strong>, establishing a <strong>self-perpetuating cycle</strong> of fibrosis progression (</span></span></span><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">PMID: 24265236)</span></span></span><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">.</span></span></span></span></span></span></p>
<h4>Evidence Supporting this KER</h4>
<strong>Biological Plausibility</strong>
<ul>
<li style="list-style-type:none">
<ul style="list-style-type:circle">
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">Fibrosis is characterized by a dysregulated balance between ECM synthesis and degradation. Excess ECM, especially cross-linked collagen, alters tissue elasticity, leading to organ dysfunction (PMID: 30213667)</span></span></span></span></span></li>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">Studies show that fibrosis is largely irreversible when cross-linked ECM accumulates and inhibits normal tissue remodeling (PMID: 39377183).</span></span></span></span></span></li>
</ul>
</li>
</ul>
<strong>Empirical Evidence</strong>
<ul>
<li style="list-style-type:none">
<ul style="list-style-type:circle">
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><strong><em><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">In vitro studies</span></span></span></em></strong><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">: Overexpression of collagen in fibroblasts leads to increased matrix stiffness and activation of fibrotic signaling pathways (e.g., TGF-</span></span></span><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">β</span></span></span><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif"><span style="color:red">) (PMID: 39656528)</span></span></span></span></span></li>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:"Times New Roman",serif"><span style="color:#000000"><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif">Puche JE, Saiman Y, Friedman SL. Hepatic stellate cells and liver fibrosis. Compr Physiol. 2013 Oct;3(4):1473-92. doi: 10.1002/cphy.c120035. PMID: 24265236.</span></span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:"Times New Roman",serif"><span style="color:#000000"><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif">Parola M, Pinzani M. Liver fibrosis: Pathophysiology, pathogenetic targets and clinical issues. Mol Aspects Med. 2019 Feb;65:37-55. doi: 10.1016/j.mam.2018.09.002. Epub 2018 Sep 13. PMID: 30213667.</span></span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:"Times New Roman",serif"><span style="color:#000000"><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif">Pei Z, Fan J, Tang M, Li Y. Ferroptosis: A New Strategy for the Treatment of Fibrotic Diseases. Adv Biol (Weinh). 2024 Oct 8:e2400383. doi: 10.1002/adbi.202400383. Epub ahead of print. </span></span><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif">PMID: 39377183.</span></span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:"Times New Roman",serif"><span style="color:#000000"><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif">Guan Y, Fang Z, Hu A, Roberts S, Wang M, Ren W, Johansson PK, Heilshorn SC, Enejder A, Peltz G. Live-cell imaging of human liver fibrosis using hepatic micro-organoids. </span></span><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif">JCI Insight. 2024 Dec 10;10(2):e187099. doi: 10.1172/jci.insight.187099. PMID: 39656528.</span></span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:"Times New Roman",serif"><span style="color:#000000"><span style="font-size:10pt"><span style="font-family:Calibri,sans-serif">Xia S, Huang Y, Zhang Y, Zhang M, Zhao K, Han P, Tian D, Liao J, Liu J. Role of macrophage-to-myofibroblast transition in chronic liver injury and liver fibrosis. Eur J Med Res. 2023 Nov 8;28(1):502. doi: 10.1186/s40001-023-01488-7. PMID: 37941043; PMCID: PMC10631085.</span></span></span></span></span></p>