FMA:84050CytokineFMA:241981ChemokineFMA:86578InterleukinCHEBI:3815collagenCHEBI:26523reactive oxygen speciesGO:0002534cytokine production involved in inflammatory responseGO:0090195chemokine secretionGO:0006956complement activationGO:0032964collagen biosynthetic processGO:0043122regulation of I-kappaB kinase/NF-kappaB signalingGO:1903409reactive oxygen species biosynthetic process1increasedReactive oxygen species2017-06-16T08:32:102017-08-15T10:43:2710090mouse10116ratsWCS_9606human10116Rattus norvegicus9606Homo sapiensWikiUser_28VertebratesBinding of agonist, Angiotensin II receptor type 1 receptor (AT1R)Binding of agonist, Angiotensin II receptor type 1 receptor (AT1R)Molecular2021-03-30T12:38:502021-03-30T12:38:50Increased, secretion of proinflammatory mediatorsIncreased proinflammatory mediatorsCellular<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Pro-inflammatory mediators are the chemical and biological molecules that initiate and regulate inflammatory reactions. Pro-inflammatory mediators are secreted following exposure to an inflammogen in a gender/sex or developmental stage independent manner. They are secreted during inflammation in all species. Different types of pro-inflammatory mediators are secreted during innate or adaptive immune responses across various species (Mestas and Hughes, 2004). Cell-derived pro-inflammatory mediators include cytokines, chemokines, and growth factors. Blood derived pro-inflammatory mediators include vasoactive amines, complement activation products and others. These modulators can be grouped based on the cell type that secrete them, their cellular localisation and also based on the type of immune response they trigger. For example, members of the interleukin (IL) family including <a href="https://bioregistry.io/genecards:IL2">IL-2</a>, <a href="https://bioregistry.io/genecards:IL4">IL-4</a>, <a href="https://bioregistry.io/genecards:IL7">IL-7</a>, <a href="https://bioregistry.io/genecards:IL9">IL-9</a>, <a href="https://bioregistry.io/genecards:IL15">IL-15</a>, <a href="https://bioregistry.io/genecards:IL21">IL-21</a>, <a href="https://bioregistry.io/genecards:IL3">IL-3</a>, <a href="https://bioregistry.io/genecards:IL5">IL-5</a> and Granulocyte-macrophage colony stimulating factor (<a href="https://bioregistry.io/genecards:CSF2">GM-CSF</a>) are involved in the adaptive immune responses. The pro-inflammatory cytokines include IL-1 family (<a href="https://bioregistry.io/genecards:IL1a">IL-1</a></span></span><span style="font-size:11.0pt"><span style="font-family:"Arial",sans-serif">α</span></span><span style="color:blue"><u><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif"> </span></span></u></span><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">, <a href="https://bioregistry.io/genecards:IL1b">IL-1</a></span></span><span style="font-size:11.0pt"><span style="font-family:"Arial",sans-serif">β</span></span><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">, <a href="https://bioregistry.io/genecards:IL1ra">IL-1r</a></span></span><span style="font-size:11.0pt"><span style="font-family:"Arial",sans-serif">α</span></span><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">, <a href="https://bioregistry.io/genecards:IL18">IL-18</a>, <a href="https://bioregistry.io/genecards:IL36a">IL-36</a></span></span><span style="font-size:11.0pt"><span style="font-family:"Arial",sans-serif">α</span></span><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">, <a href="https://bioregistry.io/genecards:IL36b">IL-36</a></span></span><span style="font-size:11.0pt"><span style="font-family:"Arial",sans-serif">β</span></span><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">, <a href="https://bioregistry.io/genecards:IL36g">IL-36</a></span></span><span style="font-size:11.0pt"><span style="font-family:"Arial",sans-serif">γ</span></span><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">, <a href="https://bioregistry.io/genecards:IL36Ra">IL-36R</a></span></span><span style="font-size:11.0pt"><span style="font-family:"Arial",sans-serif">α</span></span><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">, <a href="https://bioregistry.io/genecards:IL37">IL-37</a>), <a href="https://bioregistry.io/genecards:IL6">IL-6 </a>family, Tumor necrosis factor (<a href="https://bioregistry.io/genecards:TNF">TNF</a>) family, <a href="https://bioregistry.io/genecards:IL17">IL-17</a>, and Interferon gamma (<a href="https://bioregistry.io/genecards:IFNg">IFN</a>-</span></span><span style="font-size:11.0pt"><span style="font-family:"Arial",sans-serif">γ</span></span><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">) (Turner et al., 2014). While <a href="https://bioregistry.io/genecards:IL4">IL-4</a> and <a href="https://bioregistry.io/genecards:IL5">IL-5</a> are considered T helper (Th) cell type 2 response, <a href="https://bioregistry.io/genecards:IFNg">IFN</a>-</span></span><span style="font-size:11.0pt"><span style="font-family:"Arial",sans-serif">γ</span></span><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"> is suggested to be Th1 type response.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Different types of pro-inflammatory mediators are secreted during innate or adaptive immune responses across various species (Mestas and Hughes, 2004). However, <a href="https://bioregistry.io/genecards:IL1">IL-1</a> family cytokines, <a href="https://bioregistry.io/genecards:IL4">IL-4</a>, <a href="https://bioregistry.io/genecards:IL5">IL-5</a>, <a href="https://bioregistry.io/genecards:IL6">IL-6</a>, <a href="https://bioregistry.io/genecards:TNFa">TNF</a>-</span></span><span style="font-size:11.0pt"><span style="font-family:"Arial",sans-serif">α</span></span><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">, IFN-</span></span><span style="font-size:11.0pt"><span style="font-family:"Arial",sans-serif">γ</span></span><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"> are the commonly measured mediators in experimental animals and in humans. Similar gene expression patterns involving inflammation and matrix remodelling are observed in human patients of pulmonary fibrosis and mouse lungs exposed to bleomycin (Kaminski, 2002). </span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><strong><em><span style="color:red">Literature evidence for its perturbation:</span></em></strong></span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="color:red">Several studies show increased proinflammatory mediators in rodent lungs and bronchoalveolar lavage fluid, and in cell culture supernatants following exposure to a variety of carbon nanotube (CNT) types and other materials</span>. Poland et al., 2008 showed that long and thin CNTs (>5 µm) can elicit asbestos-like pathogenicity through the continual release of pro-inflammatory cytokines and reactive oxygen species. Exposure to crystalline silica induces release of inflammatory cytokines (TNF-</span></span><span style="font-size:11.0pt"><span style="font-family:"Arial",sans-serif">α</span></span><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">, IL-1, IL-6), transcription factors (Nuclear factor kappa B [NF-</span></span><span style="font-size:11.0pt"><span style="font-family:"Arial",sans-serif">κ</span></span><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">B], Activator protein-1 [AP-1]) and kinase signalling pathways in mice that contain NF-</span></span><span style="font-size:11.0pt"><span style="font-family:"Arial",sans-serif">κ</span></span><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">B luciferase reporter (Hubbard et al., 2002). Boyles et al., 2015 found that lung responses to long multi-walled carbon nanotubes (MWCNTs) included high expression levels of pro-inflammatory mediators Monocyte Chemoattractant Protein 1 (MCP-1), Transforming growth factor beta 1 (TGF-β1), and TNF-α (Boyles et al., 2015). Bleomycin administration in rodents induces lung inflammation and increased expression of pro-inflammatory mediators (Park et al., 2019). Inflammation induced by bleomycin, paraquat and CNTs is characterised by the altered expression of pro-inflammatory mediators. A large number of nanomaterials induce expression of cytokines and chemokines in lungs of rodents exposed via inhalation (Halappanavar et al., 2011; Husain et al., 2015a). Similarities are observed in gene programs involving pro-inflammatory event is observed in both humans and experimental mice (Zuo et al., 2002).</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">The selection of pro-inflammatory mediators for investigation varies based on the expertise of the lab, cell types studied and the availability of the specific antibodies.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><u>Real-time reverse transcription-polymerase chain reaction (qRT-PCR)</u> – will measure the abundance of cytokine mRNA in a given sample. The method involves three steps: conversion of RNA into cDNA by reverse transcription method, amplification of cDNA using the PCR, and the real-time detection and quantification of amplified products (amplicons) (Nolan T et al., 2006). Amplicons are detected using fluorescence, increase in which is directly proportional to the amplified PCR product. The number of cycles required per sample to reach a certain threshold of fluorescence (set by the user – usually set in the linear phase of the amplification, and the observed difference in samples to cross the set threshold reflects the initial amount available for amplification) is used to quantify the relative amount in the samples. The amplified products are detected by the DNA intercalating minor groove-binding fluorophore SYBR green, which produces a signal when incorporated into double-stranded amplicons. Since the cDNA is single stranded, the dye does not bind enhancing the specificity of the results. There are other methods such as nested fluorescent probes for detection, but SYBR green is widely used. RT-PCR primers specific to several pro-inflammatory mediators in several species including mouse, rat and humans, are readily available commercially.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><u>Enzyme-linked immunosorbent assays (ELISA)</u> – permit quantitative measurement of antigens in biological samples. The method is the same as described for the MIE. Both ELISA and qRT-PCR assays are used in vivo and are readily applicable to in vitro cell culture models, where cell culture supernatants or whole cell homogenates are used for ELISA or mRNA assays. Both assays are straight forward, quantitative and require relatively a small amount of input sample. </span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Apart from assaying single protein or gene at a time, cytokine bead arrays or cytokine PCR arrays can also be used to detect a whole panel of inflammatory mediators in a multiplex method (Husain et al., 2015b). This method is quantitative and especially advantageous when the sample amount available for testing is scarce. Lastly, immunohistochemistry can also be used to detect specific immune cell types producing the pro-inflammatory mediators and its downstream effectors in any given tissue (Costa et al., 2017). Immunohistochemistry results can be used as weight of evidence; however, the technique is not quantitative and depending on the specific antibodies used, the assay sensitivity may also become an issue (Amsen and De Visser, 2009).</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="color:red"><u>Cell models</u> - of varying complexity have been used to assess the expression of pro-inflammatory mediators. Two dimensional submerged monocultures of the main fibrotic effector cells – lung epithelial cells, macrophages, and fibroblasts – have routinely been used <em>in vitro</em> due to the large literature base, and ease of use, but do not adequately mimic the <em>in vivo</em> condition (Sundarakrishnan <em>et al.,</em> 2018, Sharma <em>et al.,</em> 2016). Recently, the EpiAlveolar <em>in vitro</em> lung model (containing epithelial cells, endothelial cells, and fibroblasts) was used to predict the fibrotic potential of MWCNT, and researchers noted increases in the pro-inflammatory molecules TNF-</span></span></span><span style="font-size:11.0pt"><span style="font-family:"Arial",sans-serif">α</span></span><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="color:red">, IL-1</span></span></span><span style="font-size:11.0pt"><span style="font-family:"Arial",sans-serif">β</span></span><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="color:red">, and the pro-fibrotic TGF-</span></span></span><span style="font-size:11.0pt"><span style="font-family:"Arial",sans-serif">β</span></span><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="color:red"> using ELISA (Barasova <em>et al.,</em> 2020). A similar, but less complicated co-culture model of immortalized human alveolar epithelial cells and idiopathic pulmonary fibrosis patient derived fibroblasts was used to assess pro-fibrotic signalling, and noted enhanced secretion of Platelet derived growth factor (PDGF) and Basic fibroblast growth factor (bFGF), as well as evidence for epithelial to mesenchymal transition of epithelial cells in this system (Prasad et al., 2014). Models such as these better capitulate the <em>in vivo</em> pulmonary alveolar capillary, but have lower reproducibility as compared to traditional submerged mono-culture experiments. </span></span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="color:red">Human, mouse, rat</span></span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Cytokines are the common pro-inflammatory mediators secreted following inflammogenic stimuli. Cytokines can be defined as diverse group of signaling protein molecules. They are secreted by different cell types in different tissues and in all mammalian species, irrespective of gender, age or sex. A lot of literature is available to support cross species, gender and developmental stage application for this KE. The challenge is the specificity; most cytokines exhibit redundant functions and many are pleotropic.</span></span></p>
CL:0000255eukaryotic cellHighMaleHighFemaleHighAdultsHighHighHigh<p><span style="color:#000000"><span style="font-family:Calibri">1. </span></span>Amsen D, de Visser KE, Town T. Approaches to determine expression of inflammatory cytokines. Methods Mol Biol. 2009;511:107-42. doi: 10.1007/978-1-59745-447-6_5. </p>
<p><span style="color:red"><span style="font-family:Calibri">2. </span></span>Barosova H, Maione AG, Septiadi D, Sharma M, Haeni L, Balog S, O'Connell O, Jackson GR, Brown D, Clippinger AJ, Hayden P, Petri-Fink A, Stone V, Rothen-Rutishauser B. Use of EpiAlveolar Lung Model to Predict Fibrotic Potential of Multiwalled Carbon Nanotubes. ACS Nano. 2020 Apr 28;14(4):3941-3956. doi: 10.1021/acsnano.9b06860. </p>
<p><span style="color:#000000"><span style="font-family:Calibri">3. </span></span>Boyles MS, Young L, Brown DM, MacCalman L, Cowie H, Moisala A, Smail F, Smith PJ, Proudfoot L, Windle AH, Stone V. Multi-walled carbon nanotube induced frustrated phagocytosis, cytotoxicity and pro-inflammatory conditions in macrophages are length dependent and greater than that of asbestos. Toxicol In Vitro. 2015 Oct;29(7):1513-28. doi: 10.1016/j.tiv.2015.06.012. </p>
<p><span style="color:#000000"><span style="font-family:Calibri">4. </span></span>Costa PM, Gosens I, Williams A, Farcal L, Pantano D, Brown DM, Stone V, Cassee FR, Halappanavar S, Fadeel B. Transcriptional profiling reveals gene expression changes associated with inflammation and cell proliferation following short-term inhalation exposure to copper oxide nanoparticles. J Appl Toxicol. 2018 Mar;38(3):385-397. doi: 10.1002/jat.3548.</p>
<p><span style="color:#000000"><span style="font-family:Calibri">5. </span></span>Halappanavar S, Jackson P, Williams A, Jensen KA, Hougaard KS, Vogel U, Yauk CL, Wallin H. Pulmonary response to surface-coated nanotitanium dioxide particles includes induction of acute phase response genes, inflammatory cascades, and changes in microRNAs: a toxicogenomic study. Environ Mol Mutagen. 2011 Jul;52(6):425-39. doi: 10.1002/em.20639. </p>
<p><span style="color:#000000"><span style="font-family:Calibri">6. </span></span>Hubbard AK, Timblin CR, Shukla A, Rincón M, Mossman BT. Activation of NF-kappaB-dependent gene expression by silica in lungs of luciferase reporter mice. Am J Physiol Lung Cell Mol Physiol. 2002 May;282(5):L968-75. doi: 10.1152/ajplung.00327.2001.</p>
<p><span style="color:#000000"><span style="font-family:Calibri">7. </span></span>Husain M, Kyjovska ZO, Bourdon-Lacombe J, Saber AT, Jensen KA, Jacobsen NR, Williams A, Wallin H, Halappanavar S, Vogel U, Yauk CL. Carbon black nanoparticles induce biphasic gene expression changes associated with inflammatory responses in the lungs of C57BL/6 mice following a single intratracheal instillation. Toxicol Appl Pharmacol. 2015a Dec 15;289(3):573-88. doi: 10.1016/j.taap.2015.11.003.</p>
<p><span style="color:#000000"><span style="font-family:Calibri">8. </span></span>Husain M, Wu D, Saber AT, Decan N, Jacobsen NR, Williams A, Yauk CL, Wallin H, Vogel U, Halappanavar S. Intratracheally instilled titanium dioxide nanoparticles translocate to heart and liver and activate complement cascade in the heart of C57BL/6 mice. Nanotoxicology. 2015b;9(8):1013-22. doi: 10.3109/17435390.2014.996192.</p>
<p><span style="color:#000000"><span style="font-family:Calibri">9. </span></span>Kaminski N. Microarray analysis of idiopathic pulmonary fibrosis. Am J Respir Cell Mol Biol. 2003 Sep;29(3 Suppl):S32-6.</p>
<p><span style="color:#000000"><span style="font-family:Calibri">10. </span></span>Mestas J, Hughes CC. Of mice and not men: differences between mouse and human immunology. J Immunol. 2004 Mar 1;172(5):2731-8. doi: 10.4049/jimmunol.172.5.2731.</p>
<p><span style="color:#000000"><span style="font-family:Calibri">11. </span></span>Nolan T, Hands RE, Bustin SA. Quantification of mRNA using real-time RT-PCR. Nat Protoc. 2006;1(3):1559-82. doi: 10.1038/nprot.2006.236.</p>
<p><span style="color:#000000"><span style="font-family:Calibri">12. </span></span>Park SJ, Im DS. Deficiency of Sphingosine-1-Phosphate Receptor 2 (S1P<sub>2</sub>) Attenuates Bleomycin-Induced Pulmonary Fibrosis. Biomol Ther (Seoul). 2019 May 1;27(3):318-326. doi: 10.4062/biomolther.2018.131.</p>
<p><span style="color:#000000"><span style="font-family:Calibri">13. </span></span>Poland CA, Duffin R, Kinloch I, Maynard A, Wallace WA, Seaton A, Stone V, Brown S, Macnee W, Donaldson K. Carbon nanotubes introduced into the abdominal cavity of mice show asbestos-like pathogenicity in a pilot study. Nat Nanotechnol. 2008 Jul;3(7):423-8. doi: 10.1038/nnano.2008.111.</p>
<p><span style="color:red"><span style="font-family:Calibri">14. </span></span>Prasad S, Hogaboam CM, Jarai G. Deficient repair response of IPF fibroblasts in a co-culture model of epithelial injury and repair. Fibrogenesis Tissue Repair. 2014 Apr 29;7:7. doi: 10.1186/1755-1536-7-7. </p>
<p><span style="color:red"><span style="font-family:Calibri">15. </span></span>Sharma M, Nikota J, Halappanavar S, Castranova V, Rothen-Rutishauser B, Clippinger AJ. Predicting pulmonary fibrosis in humans after exposure to multi-walled carbon nanotubes (MWCNTs). Arch Toxicol. 2016 Jul;90(7):1605-22. doi: 10.1007/s00204-016-1742-7. </p>
<p><span style="color:red"><span style="font-family:Calibri">16. </span></span>Sundarakrishnan A, Chen Y, Black LD, Aldridge BB, Kaplan DL. Engineered cell and tissue models of pulmonary fibrosis. Adv Drug Deliv Rev. 2018 Apr;129:78-94. doi: 10.1016/j.addr.2017.12.013.</p>
<p><span style="color:#000000"><span style="font-family:Calibri">17. </span></span>Turner MD, Nedjai B, Hurst T, Pennington DJ. Cytokines and chemokines: At the crossroads of cell signalling and inflammatory disease. Biochim Biophys Acta. 2014 Nov;1843(11):2563-2582. doi: 10.1016/j.bbamcr.2014.05.014. </p>
<p><span style="color:#000000"><span style="font-family:Calibri">18. </span></span>Zuo F, Kaminski N, Eugui E, Allard J, Yakhini Z, Ben-Dor A, Lollini L, Morris D, Kim Y, DeLustro B, Sheppard D, Pardo A, Selman M, Heller RA. Gene expression analysis reveals matrilysin as a key regulator of pulmonary fibrosis in mice and humans. Proc Natl Acad Sci U S A. 2002 Apr 30;99(9):6292-7. doi: 10.1073/pnas.092134099.</p>
2018-01-02T13:12:112023-04-21T09:51:32Accumulation, CollagenAccumulation, CollagenTissue<p>Collagen is mostly found in fibrous tissues such as tendons, ligaments and skin. It is also abundant in corneas, cartilage, bones, blood vessels, the gut, intervertebral discs, and the dentin in teeth. In muscle tissue, it serves as a major component of the endomysium. Collagen is the main structural protein in the extracellular space in the various connective tissues, making up from 25% to 35% of the whole-body protein content. In normal tissues, collagen provides strength, integrity, and structure. When tissues are disrupted following injury, collagen is needed to repair the defect. If too much collagen is deposited, normal anatomical structure is lost, function is compromised, and fibrosis results.</p>
<p>The fibroblast is the most common collagen producing cell. Collagen-producing cells may also arise from the process of transition of differentiated epithelial cells into mesenchymal cells. This has been observed e.g. during renal fibrosis (transformation of tubular epithelial cells into fibroblasts) and in liver injury (transdifferentiation of hepatocytes and cholangiocytes into fibroblasts) (Henderson and Iredale, 2007)<sup>.</sup></p>
<p>There are close to 20 different types of collagen found with the predominant form being type I collagen. This fibrillar form of collagen represents over 90 percent of our total collagen and is composed of three very long protein chains which are wrapped around each other to form a triple helical structure called a collagen monomer. Collagen is produced initially as a larger precursor molecule called procollagen. As the procollagen is secreted from the cell, procollagen proteinases remove the extension peptides from the ends of the molecule. The processed molecule is referred to as collagen and is involved in fiber formation. In the extracellular spaces the triple helical collagen molecules line up and begin to form fibrils and then fibers. Formation of stable crosslinks within and between the molecules is promoted by the enzyme lysyl oxidase and gives the collagen fibers tremendous strength (Diegelmann,2001)<sup>.</sup> The overall amount of collagen deposited by fibroblasts is a regulated balance between collagen synthesis and collagen catabolism. Disturbance of this balance leads to changes in the amount and composition of collagen. Changes in the composition of the extracellular matrix initiate positive feedback pathways that increase collagen production.</p>
<p>Normally, collagen in connective tissues has a slow turn over; degradating enzymes are collagenases, belonging to the family of matrix metalloproteinases. Other cells that can synthesize and release collagenase are macrophages, neutrophils, osteoclasts, and tumor cells (Di Lullo et al., 2002; Kivirikko and Risteli, 1976; Miller and Gay, 1987; Prockop and Kivirikko, 1995).</p>
<p> </p>
<p> </p>
<p>Determination of the amount of collagen produced in vitro can be done in a variety of ways ranging from simple colorimetric assays to elaborate chromatographic procedures using radioactive and non-radioactive material. What most of these procedures have in common is the need to destroy the cell layer to obtain solubilized collagen from the pericellular matrix. Rishikof et al. describe several methods to assess the in vitro production of type I collagen: Western immunoblotting of intact alpha1(I) collagen using antibodies directed to alpha1(I) collagen amino and carboxyl propeptides, the measurement of alpha1(I) collagen mRNA levels using real-time polymerase chain reaction, and methods to determine the transcriptional regulation of alpha1(I) collagen using a nuclear run-on assay (Rishikof et al., 2005). </p>
<p><span style="color:red"><span style="font-family:"Arial",sans-serif">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><em><strong><span style="color:red"><span style="font-family:"Arial",sans-serif">Sircol Collagen Assay for collagen quantification:</span></span></strong></em></p>
<p><span style="color:red"><span style="font-family:"Arial",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 and Raghunath, 2009; Nikota et al., 2017).</span></span></p>
<p><em><strong><span style="color:red"><span style="font-family:"Arial",sans-serif">Hydroxyproline assay:</span></span></strong></em></p>
<p><span style="color:red"><span style="font-family:"Arial",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 and Raghunath, 2009; Nikota et al., 2017).</span></span></p>
<p><strong><em><span style="color:red"><span style="font-family:"Arial",sans-serif">Ex vivo precision cut tissue slices</span></span></em></strong></p>
<p><span style="color:red"><span style="font-family:"Arial",sans-serif">Precision cut tissue slices mimic the whole organ response and allow histological assessment, an endpoint of interest in regulatory decision making. While this technique uses animals, the number of animals required to conduct a dose-response study can be reduced to 1/4<sup>th</sup> of what will be used in whole animal exposure studies (Rahman et al., 2020). </span></span></p>
<p> </p>
<pre>
</pre>
<p>Humans: Bataller and Brenner, 2005; Decaris et al., 2015. </p>
<p>Mice: Dalton et al., 2009; Leung et al., 2008; Nan et al., 2013.</p>
<p>Rats: Hamdy and El-Demerdash, 2012; Li, Li et al., 2012; Luckey and Petersen, 2001; Natajaran et al., 2006.</p>
<p> </p>
UBERON:0002384connective tissueNot SpecifiedUnspecificNot SpecifiedAll life stagesHighHighHigh<ol>
<li>Bataller R, Brenner DA. Liver fibrosis. J Clin Invest. 2005 Feb;115(2):209-18. doi: 10.1172/JCI24282. </li>
<li>Chen CZ, Raghunath M. Focus on collagen: in vitro systems to study fibrogenesis and antifibrosis state of the art. Fibrogenesis Tissue Repair. 2009 Dec 15;2:7. doi: 10.1186/1755-1536-2-7. </li>
<li>Dalton SR, Lee SM, King RN, Nanji AA, Kharbanda KK, Casey CA, McVicker BL. Carbon tetrachloride-induced liver damage in asialoglycoprotein receptor-deficient mice. Biochem Pharmacol. 2009 Apr 1;77(7):1283-90. doi: 10.1016/j.bcp.2008.12.023. </li>
<li>Decaris ML, Emson CL, Li K, Gatmaitan M, Luo F, Cattin J, Nakamura C, Holmes WE, Angel TE, Peters MG, Turner SM, Hellerstein MK. Turnover rates of hepatic collagen and circulating collagen-associated proteins in humans with chronic liver disease. PLoS One. 2015 Apr 24;10(4):e0123311. doi: 10.1371/journal.pone.0123311.</li>
<li>Di Lullo GA, Sweeney SM, Korkko J, Ala-Kokko L, San Antonio JD. Mapping the ligand-binding sites and disease-associated mutations on the most abundant protein in the human, type I collagen. J Biol Chem. 2002 Feb 8;277(6):4223-31. doi: 10.1074/jbc.M110709200.</li>
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2016-11-29T18:41:222023-04-25T11:56:49Lung fibrosisLung fibrosisOrgan<p>This consecutive KE resulting in the acquisition of the accumulation of excess fibrous connective tissue, the adverse outcome on pulmonary fibrosis. Scar formation, the accumulation of excess fibrous connective tissue (the process called fibrosis), leads to thickening of the walls, and causes reduced oxygen supply in the blood. As a consequence patients suffer from perpetual shortness of breath.</p>
2017-02-15T02:55:562017-12-26T02:10:27Increase activation, Nuclear factor kappa B (NF-kB)Increase activation, Nuclear factor kappa B (NF-kB)Cellular<p>The NF-kB pathway consists of a series of events where the transcription factors of the NF-kB family play a key role. The proinflammatory cytokine (IL-1beta) can be activated by NF-kB , including Reactive Oxygen Species produced by NADPH oxidase (NOX). Upon pathway activation, the IKK complex will be phosphorylated, which in turn phosphorylates IkBa. There, this transcription factor can express pro-inflammatory and pro-fibrotic genes. This can be achieved by ROS, IKK enhancer or nuclear translocation enhancer. </p>
<p>NF-kB transcriptional activity: Beta lactamase reporter gene assay (Miller et al. 2010). NF-kB transcription: Lentiviral NF-kB GFP reporter with flow cytometry (Moujalled et al. 2012)</p>
<p>NF-κB translocation: RelA-GFP reporter assay (Frederiksson 2012) (Huppelschoten 2017)</p>
<p>IκBa phosphorylation: Western blotting (Miller et al. 2010)</p>
<p>NF-κB p65 (Total/Phospho) ELISA</p>
<p>ELISA for IL-6, IL-8, and Cox</p>
<p>The ROS directly influences NF-κB signalling, resulting in differential production of cytokines and chemokines (McKay and Cidlowski, 1999; Pernis, 2007). NF-κB regulates pro-inflammatory responses that are transcriptionally mediated by NF‑κB.</p>
UBERON:0000479tissueCL:0000066epithelial cellNot SpecifiedMixedModerateNot Otherwise SpecifiedHigh2016-11-29T18:41:302021-03-30T13:17:17Increased, Reactive oxygen speciesIncreased, Reactive oxygen speciesCellular<p>Biological State: increased reactive oxygen species (ROS)</p>
<p>Biological compartment: an entire cell -- may be cytosolic, may also enter organelles.</p>
<p>Reactive oxygen species (ROS) are O2- derived molecules that can be both free radicals (e.g. superoxide, hydroxyl, peroxyl, alcoxyl) and non-radicals (hypochlorous acid, ozone and singlet oxygen) (Bedard and Krause 2007; Ozcan and Ogun 2015). ROS production occurs naturally in all kinds of tissues inside various cellular compartments, such as mitochondria and peroxisomes (Drew and Leeuwenburgh 2002; Ozcan and Ogun 2015). Furthermore, these molecules have an important function in the regulation of several biological processes – they might act as antimicrobial agents or triggers of animal gamete activation and capacitation (Goud et al. 2008; Parrish 2010; Bisht et al. 2017). <br />
However, in environmental stress situations (exposure to radiation, chemicals, high temperatures) these molecules have its levels drastically increased, and overly interact with macromolecules, namely nucleic acids, proteins, carbohydrates and lipids, causing cell and tissue damage (Brieger et al. 2012; Ozcan and Ogun 2015). </p>
<p>Photocolorimetric assays (Sharma et al. 2017; Griendling et al. 2016) or through commercial kits purchased from specialized companies.</p>
<p>ROS is a normal constituent found in all organisms.</p>
HighUnspecificHighAll life stagesHigh<p>Bedard, Karen, and Karl-Heinz Krause. 2007. “The NOX Family of ROS-Generating NADPH Oxidases: Physiology and Pathophysiology.” Physiological Reviews 87 (1): 245–313.</p>
<p>Ozcan, Ayla, and Metin Ogun. 2015. “Biochemistry of Reactive Oxygen and Nitrogen Species.” In Basic Principles and Clinical Significance of Oxidative Stress, edited by Sivakumar Joghi Thatha Gowder. Rijeka: IntechOpen.</p>
<p>Drew, Barry, and Christiaan Leeuwenburgh. 2002. “Aging and the Role of Reactive Nitrogen Species.” Annals of the New York Academy of Sciences 959 (April): 66–81.</p>
<p>Goud, Anuradha P., Pravin T. Goud, Michael P. Diamond, Bernard Gonik, and Husam M. Abu-Soud. 2008. “Reactive Oxygen Species and Oocyte Aging: Role of Superoxide, Hydrogen Peroxide, and Hypochlorous Acid.” Free Radical Biology & Medicine 44 (7): 1295–1304.</p>
<p>Parrish, A. R. 2010. “2.27 - Hypoxia/Ischemia Signaling.” In Comprehensive Toxicology (Second Edition), edited by Charlene A. McQueen, 529–42. Oxford: Elsevier.</p>
<p>Bisht, Shilpa, Muneeb Faiq, Madhuri Tolahunase, and Rima Dada. 2017. “Oxidative Stress and Male Infertility.” Nature Reviews. Urology 14 (8): 470–85.</p>
<p>Brieger, K., S. Schiavone, F. J. Miller Jr, and K-H Krause. 2012. “Reactive Oxygen Species: From Health to Disease.” Swiss Medical Weekly 142 (August): w13659.</p>
<p>Sharma, Gunjan, Nishant Kumar Rana, Priya Singh, Pradeep Dubey, Daya Shankar Pandey, and Biplob Koch. 2017. “p53 Dependent Apoptosis and Cell Cycle Delay Induced by Heteroleptic Complexes in Human Cervical Cancer Cells.” Biomedicine & Pharmacotherapy = Biomedecine & Pharmacotherapie 88 (April): 218–31.</p>
<p>Griendling, Kathy K., Rhian M. Touyz, Jay L. Zweier, Sergey Dikalov, William Chilian, Yeong-Renn Chen, David G. Harrison, Aruni Bhatnagar, and American Heart Association Council on Basic Cardiovascular Sciences. 2016. “Measurement of Reactive Oxygen Species, Reactive Nitrogen Species, and Redox-Dependent Signaling in the Cardiovascular System: A Scientific Statement From the American Heart Association.” Circulation Research 119 (5): e39–75.</p>
2016-11-29T18:41:292023-04-10T14:01:30900449fd-c070-43ff-997b-3feabf32397c26269fe7-4694-4889-a2d2-ee76992a237b2021-04-10T11:45:462021-04-10T11:45:4626269fe7-4694-4889-a2d2-ee76992a237b8964dffc-71f9-4ec4-b098-5e900016ccc82021-04-10T11:46:102021-04-10T11:46:108964dffc-71f9-4ec4-b098-5e900016ccc8c571fef2-ebc4-4792-a8f3-2f3628a3bef02021-03-30T13:01:132021-03-30T13:01:13c571fef2-ebc4-4792-a8f3-2f3628a3bef00a817baf-f34d-4499-ac5e-60c3d42738f62021-03-30T12:50:092021-03-30T12:50:090a817baf-f34d-4499-ac5e-60c3d42738f6f419df48-3dd5-4962-848f-35de2ae966c02020-05-15T18:00:132020-05-15T18:00:13Angiotensin II type 1 receptor (AT1R) agonism leading to lung fibrosisAT1R, lung fibrosisUnder development: Not open for comment. Do not citeUnder DevelopmentIncluded in OECD Work Plan1.96adjacentModerateModerateadjacentModerateModerateadjacentModerateModerateadjacentModerateModerateadjacentNot SpecifiedLow2021-03-30T12:00:282023-04-29T13:02:19