107-18-6XXROGKLTLUQVRX-UHFFFAOYSA-NXXROGKLTLUQVRX-UHFFFAOYSA-N
Allyl alcohol2-Propen-1-ol
1-Hydroxy-2-propene
1-Propen-3-ol
2-Propenyl alcohol
3-Hydroxy-1-propene
3-Hydroxypropene
alcohol alilico
Alcool allylique
Allylalkohol
Allylic alcohol
NSC 6526
PROP-2-EN-1-OL
Propenyl alcohol
Shell unkrauttod A
UN 1098
Vinyl carbinol
Vinylcarbinol
DTXSID802004456-23-5VZGDMQKNWNREIO-UHFFFAOYSA-NVZGDMQKNWNREIO-UHFFFAOYSA-N
Carbon tetrachloride(CT
Benzinoform
Carbona
Flukoids
Kohlenstofftetrachlorid
Necatorina
NSC 97063
Perchloromethane
TETRACHLORKOHLENSTOFF
Tetrachlorocarbon
Tetrachlorure de carbone
tetracloruro de carbono
Tetrafinol
Tetraform
Tetrasol
UN 1846
Univerm
Vermoestricid
Tetrachloromethane
Methane, tetrachloro-
DTXSID802025068-26-8FPIPGXGPPPQFEQ-OVSJKPMPSA-NFPIPGXGPPPQFEQ-OVSJKPMPSA-N
RetinolAll-trans retinol
(all-E)-3,7-Dimethyl-9-(2,6,6-trimethyl-1-cyclohexen-1-yl)-2,4,6,8-nonatetraen-1-ol
(all-E)-3,7-Dimethyl-9-(2,6,6-trimethyl-1-cyclohexene-1-yl)-2,4,6,8-nonatetraen-1-ol
2,4,6,8-Nonatetraen-1-ol, 3,7-dimethyl-9-(2,6,6-trimethyl-1-cyclohexen-1-yl)-, (all-E)-
Agiolan
Agoncal
Alcovit A
all-trans-Retinol
all-trans-Retinyl alcohol
all-trans-Vitamin A
all-trans-Vitamin A alcohol
all-trans-Vitamin A1
Alphalin
Alphasterol
A-Mulsal
Anatola
Anatola A
Anti-Infective vitamin
Antixerophthalmic vitamin
Apostavit
Aquasol A Parenteral
Aquasynth
A-Vi-Pel
A-Vitan
Axerophthol
Bentavit A
Biosterol
Cylasphere
Disatabs Tabs
Dohyfral A
Epiteliol
Hi-A-Vita
Lard Factor
Myvpack
Nio-A-Let
NSC 122759
Oleovitamin a
Ophthalamin
Plivit A
Prepalin
Retinol 50C
Retinol, all-trans-
Retinyl A
Tegosphere VitA
Testavol
Thalasphere
trans-Retinol
trans-Vitamin A alcohol
Veroftal
Vi-Alpha
Vi-Dom-A
Vitamin A alcohol
Vitamin A alcohol, all-trans-
Vitamin A1 alcohol
Vitamin A1 alcohol, all-trans-
Vitamin A1, all-trans-
Vitavel A
Vogan-Neu
β-Retinol
Vitamin A
DTXSID302355662-75-9UMFJAHHVKNCGLG-UHFFFAOYSA-NUMFJAHHVKNCGLG-UHFFFAOYSA-N
N-NitrosodimethylamineDMN
Methanamine, N-methyl-N-nitroso-
DIMETHYLAMINE, N-NITROSO-
Dimethylnitrosoamin
Dimethylnitrosoamine
dimetilnitrosoamina
Nitrosodimethylamine
Nitrosodimetilamina
N-Methyl-N-nitrosomethanamine
N-NITROSODIMETHYLAMIN
N-Nitroso-N,N-dimethylamine
NSC 23226
Dimethylnitrosamine
DTXSID702102962-55-5YUKQRDCYNOVPGJ-UHFFFAOYSA-NYUKQRDCYNOVPGJ-UHFFFAOYSA-N
ThioacetamideEthanethioamide
Acetamide, thio-
Acetimidic acid, thio-
Acetothioamide
NSC 2120
Thiacetamide
Thioacetamid
tioacetamida
DTXSID9021340UBERON:0002107liverCL:0000632hepatic stellate cellCHEBI:3815collagenMP:0003333liver fibrosisGO:0008213protein alkylationGO:0008219cell deathGO:0035733hepatic stellate cell activationGO:0032964collagen biosynthetic processGO:0002263cell activation involved in immune responseGO:0002526acute inflammatory response3occurrence1increasedAllyl Alcohol2016-11-29T18:42:082016-11-29T21:18:43Carbon tetrachloride2016-11-29T18:42:082016-11-29T21:18:52Retinol2016-11-29T18:42:262016-11-29T21:20:38Dimethyl nitrosamine2016-11-29T18:42:112016-11-29T21:19:02Thioacetamide2016-11-29T18:42:262016-11-29T21:20:46WCS_9606human10116Rattus norvegicus10090mouseWikiUser_25human and other cells in culture9823pigs9541Macaca fascicularis10116ratWCS_7955zebrafishWikiUser_28VertebratesWikiUser_14Monkey10090Mus musculusN/A, Liver fibrosisN/A, Liver fibrosisOrgan<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>
<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>
<p>Human: Bataller and Brenner, 2005;Merck Manual, 2015; Blachier et al., 2013.</p>
<p>Rat, mouse: Liedtke et al., 2013</p>
UBERON:0002107liverNot SpecifiedUnspecificNot SpecifiedAll life stagesHighHighHigh<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>
2016-11-29T18:41:242018-12-05T08:29:26Alkylation, ProteinAlkylation, ProteinMolecular<p>Alkylation is the transfer of an alkyl group from one molecule to another. The alkyl group may be transferred as an alkyl carbocation, a free radical, a carbanion or a carbene (or their equivalents). Protein alkylation is the addition of an alkyl group to a protein amino acid. An alkyl group is any group derived from an alkane by removal of one hydrogen atom.
Alkylating agents are highly reactive chemicals that introduce alkyl groups into biologically active molecules and thereby prevent their proper functioning. Alkylating agents are classified according to their nucleophilic or electrophilic character. Nucleophilic alkylating agents deliver the equivalent of an alkyl anion (carbanion). These compounds typically can add to an electron-deficient carbon atom such as at a carbonyl group. Electrophilic alkylating agents deliver the equivalent of an alkyl cation. Alkyl halides can also react directly with amines to form C-N bonds; the same holds true for other nucleophiles such as alcohols, carboxylic acids, thiols, etc. Alkylation with only one carbon is termed methylation. <sup id="cite_ref-1" class="reference"><a href="#cite_note-1">[1]</a></sup> <sup id="cite_ref-2" class="reference"><a href="#cite_note-2">[2]</a></sup>
</p><p>Covalent protein alkylation by reactive electrophiles was identified as a key triggering event in chemical toxicity over 40 years ago and these reactions remain a major cause of chemical-induced toxicity. Interestingly, some chemical molecules produce significant protein covalent binding without causing toxicity, which suggests that only a critical subset of protein alkylation events contributes to injury.
The study by Codreanu et al. (2014) describes an inventory of electrophile- mediated protein damage in intact cells and suggests that non-toxic covalent binding may largely be survivable damage to cytoskeletal components, whereas toxic covalent binding produces lethal injury by targeting protein synthesis and catabolism and possibly mitochondrial electron transport. <sup id="cite_ref-Codreanu_2014_3-0" class="reference"><a href="#cite_note-Codreanu_2014-3">[3]</a></sup>
<sup id="cite_ref-4" class="reference"><a href="#cite_note-4">[4]</a></sup> <sup id="cite_ref-5" class="reference"><a href="#cite_note-5">[5]</a></sup>
<sup id="cite_ref-Kehrer2000_6-0" class="reference"><a href="#cite_note-Kehrer2000-6">[6]</a></sup>
<sup id="cite_ref-7" class="reference"><a href="#cite_note-7">[7]</a></sup>
</p><p><em>
</p><p><br />
HPLC-ESI-MS/MS analysis
</p><p>High Performance Liquid Chromatography – electrospray tandem mass spectrometry (HPLC-ESI-MS/MS) is the most popular MS technique. It combines the separation ability of HPLC along with the sensitivity and specificity of detection from MS. One of the advantages of HPLC-MS is that it allows samples to be rapidly desalted online, so no sample preparation is required unlike samples for GC-MS. Electrospray ionisation can produce singly or multiply charged ions. Typically high molecular weight compounds have multiple charges i.e. peptides and proteins. This technique is particularly suited to analysing polar molecules of mass <2000 Dalton and requires no prior derivatisation in most applications.
<sup id="cite_ref-8" class="reference"><a href="#cite_note-8">[8]</a></sup> <sup id="cite_ref-Codreanu_2014_3-1" class="reference"><a href="#cite_note-Codreanu_2014-3">[3]</a></sup> <sup id="cite_ref-9" class="reference"><a href="#cite_note-9">[9]</a></sup>
</p><p>MALDI-TOF/MS (Matrix Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry)
</p><p>Matrix-assisted laser desorption/ionization (MALDI) is a soft ionization technique used in mass spectrometry, allowing the analysis of biomolecules (biopolymers such as DNA, proteins, peptides and sugars) and large organic molecules (such as polymers, dendrimers and other macromolecules), which tend to be fragile and fragment when ionized by more conventional ionization methods. MALDI methodology is a three-step process. First, the sample is mixed with a suitable matrix material and applied to a metal plate. Second, a pulsed laser irradiates the sample, triggering ablation and desorption of the sample and matrix material. Finally, the analyte molecules are ionized by being protonated or deprotonated in the hot plume of ablated gases, and can then be accelerated into whichever mass spectrometer is used to analyse them. <sup id="cite_ref-10" class="reference"><a href="#cite_note-10">[10]</a></sup>
</p><p><br />
</p><p></em>
</p><p>Human, rat and mouse <sup id="cite_ref-EPA_2010_11-0" class="reference"><a href="#cite_note-EPA_2010-11">[11]</a></sup>
</p>CL:0000255eukaryotic cellHighHighHigh<ol class="references">
<li id="cite_note-1"><span class="mw-cite-backlink"><a href="#cite_ref-1">↑</a></span> <span class="reference-text">The European Bioinformatics Institute <a rel="nofollow" target="_blank" class="external free" href="http://www.ebi.ac.uk/QuickGO/GTerm?id=GO:0008213">http://www.ebi.ac.uk/QuickGO/GTerm?id=GO:0008213</a> (accessed on 20 January 2016).</span>
</li>
<li id="cite_note-2"><span class="mw-cite-backlink"><a href="#cite_ref-2">↑</a></span> <span class="reference-text">NLM, Medical Subject Headings, National Library of Medicine, <a rel="nofollow" target="_blank" class="external free" href="http://www.nlm.nih.gov/cgi/mesh/2011/MB_cgi?mode=&term=Alkylating+agents">http://www.nlm.nih.gov/cgi/mesh/2011/MB_cgi?mode=&term=Alkylating+agents</a> (accessed on 20 January 2016).</span>
</li>
<li id="cite_note-Codreanu_2014-3"><span class="mw-cite-backlink">↑ <sup><a href="#cite_ref-Codreanu_2014_3-0">3.0</a></sup> <sup><a href="#cite_ref-Codreanu_2014_3-1">3.1</a></sup> <sup><a href="#cite_ref-Codreanu_2014_3-2">3.2</a></sup></span> <span class="reference-text">Codreanu, S.G. et al. (2014), Alkylation damage by lipid electrophiles targets functional protein systems, Molecular & Cellular Proteomics, vol. 13, no. 3, pp.849–859.</span>
</li>
<li id="cite_note-4"><span class="mw-cite-backlink"><a href="#cite_ref-4">↑</a></span> <span class="reference-text">Grattagliano, I. et al. (2009), Biochemical mechanisms in drug-induced liver injury: certainties and doubts, World J Gastroenterol, vol. 15, no. 39, pp. 4865-4876.</span>
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<li id="cite_note-5"><span class="mw-cite-backlink"><a href="#cite_ref-5">↑</a></span> <span class="reference-text">Livertox <a rel="nofollow" target="_blank" class="external free" href="http://livertox.nlm.nih.gov/AlkylatingAgents.htm">http://livertox.nlm.nih.gov/AlkylatingAgents.htm</a></span>
</li>
<li id="cite_note-Kehrer2000-6"><span class="mw-cite-backlink">↑ <sup><a href="#cite_ref-Kehrer2000_6-0">6.0</a></sup> <sup><a href="#cite_ref-Kehrer2000_6-1">6.1</a></sup></span> <span class="reference-text">Kehrer, J.P. and S. Biswal (2000), The Molecular Effects of Acrolein, Toxicol. Sciences,vol.57,pp.6-15.</span>
</li>
<li id="cite_note-7"><span class="mw-cite-backlink"><a href="#cite_ref-7">↑</a></span> <span class="reference-text">Schopfer, F.J., C. Cipollina and B.A. Freeman (2011), Formation and Signaling Actions of Electrophilic Lipids, Chem Rev, vol. 111, no. 10,pp.5997–6021.</span>
</li>
<li id="cite_note-8"><span class="mw-cite-backlink"><a href="#cite_ref-8">↑</a></span> <span class="reference-text">Zhang F et al. (2005), Differential adduction of proteins vs. deoxynucleosides by methyl methanesulfonate and 1-methyl-1-nitrosourea in vitro, Mass Spectrom, vol 19, no. 4, pp. 438–448.</span>
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<li id="cite_note-9"><span class="mw-cite-backlink"><a href="#cite_ref-9">↑</a></span> <span class="reference-text">Gundry, R.L. et al. (2009), Preparation of proteins and peptides for mass spectrometry analysis in a bottom-up proteomics workflow, Curr Protoc Mol Biol, chapter 10, section VI, unit 10.25, pp. 1-23.</span>
</li>
<li id="cite_note-10"><span class="mw-cite-backlink"><a href="#cite_ref-10">↑</a></span> <span class="reference-text">Kislinger, T. et al. (2005), Analysis of protein glycation products by MALDI-TOF/MS, Ann N Y Acad Sci, vol. 1043, pp. 249-259.</span>
</li>
<li id="cite_note-EPA_2010-11"><span class="mw-cite-backlink">↑ <sup><a href="#cite_ref-EPA_2010_11-0">11.0</a></sup> <sup><a href="#cite_ref-EPA_2010_11-1">11.1</a></sup></span> <span class="reference-text">EPA Toxicological review of Carbon Tetrachloride (CAS No. 56-23-5). March 2010 EPA/635/R-08/005F available at: <a rel="nofollow" target="_blank" class="external free" href="http://cfpub.epa.gov/ncea/iris/iris_documents/documents/toxreviews/0020tr.pdf">http://cfpub.epa.gov/ncea/iris/iris_documents/documents/toxreviews/0020tr.pdf</a> (accessed 24.10.2015)</span>
</li>
<li id="cite_note-12"><span class="mw-cite-backlink"><a href="#cite_ref-12">↑</a></span> <span class="reference-text">Auerbach, S.S. et al. (2008), A comparative 90 day toxicity study of allyl acetate, allyl alcohol and acrolein, Toxicology, Vol. 253, No.1-3, pp.79–88.</span>
</li>
<li id="cite_note-13"><span class="mw-cite-backlink"><a href="#cite_ref-13">↑</a></span> <span class="reference-text">Huang, L. et al. (2008), Genes related to apoptosis predict necrosis of the liver as a phenotype observed in rats exposed to a compendium of hepatotoxicants, BMC Genomics, vol. 9: 288.</span>
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<li id="cite_note-14"><span class="mw-cite-backlink"><a href="#cite_ref-14">↑</a></span> <span class="reference-text">Mohammad, M.K. et al. (2012), Acrolein cytotoxicity in hepatocytes involves endoplasmic reticulum stress, mitochondrial dysfunction and oxidative stress, Toxicol Appl Pharmacol, vol. 265, no. 1, pp. 73-82.</span>
</li>
<li id="cite_note-15"><span class="mw-cite-backlink"><a href="#cite_ref-15">↑</a></span> <span class="reference-text">Yamada T et al., (2013), A category approach to predicting the repeated-dose hepatotoxicity of allyl esters, Regulatory Toxicology and Pharmacology, vol. 65, no. 2, pp. 189–195.</span>
</li>
<li id="cite_note-16"><span class="mw-cite-backlink"><a href="#cite_ref-16">↑</a></span> <span class="reference-text">Basu, S. (2003), Carbon tetrachloride-induced lipid peroxidation: eicosanoid formation and their regulation by antioxidant nutrients, Toxicology,vol.189, no.1-2, pp. 113-127.</span>
</li>
<li id="cite_note-17"><span class="mw-cite-backlink"><a href="#cite_ref-17">↑</a></span> <span class="reference-text">Calabrese, E.J., L.A. Baldwin and H.M. Mehendale (1993), G2 subpopulation in rat liver induced into mitosis by low-level exposure to carbon tetrachloride: an adaptive response, Toxicol Appl Pharmacol, vol. 121. no. 1, pp. 1-7.</span>
</li>
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</li>
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</li>
<li id="cite_note-20"><span class="mw-cite-backlink"><a href="#cite_ref-20">↑</a></span> <span class="reference-text">Lee Kwang-Jong et al. (2004), Induction of molecular chaperones in carbon tetrachloride-treated rat liver: implications in protection against liver damage, Cell Stress Chaperones, vol. 9, no. 1, pp. 58-68.</span>
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</li>
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no. 2, pp. 105-136.</span>
</li>
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hepatocytes and liver S-9 fraction, Chem Res Toxicol, vol. 22, no. 2, pp. 332-340.</span>
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</li>
<li id="cite_note-30"><span class="mw-cite-backlink"><a href="#cite_ref-30">↑</a></span> <span class="reference-text">Shin, N.Y. et al. (2007), Protein targets of reactive electrophiles in human liver microsomes, Chem Res Toxicol, vol. 20, no. 6, pp. 859-867.</span>
</li>
</ol>2016-11-29T18:41:232017-09-16T10:14:57Cell injury/deathCell injury/deathCellular<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>
<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>
<p>Cell death is an universal event occurring in cells of any species (Fink and Cookson,2005).<sup> </sup></p>
CL:0000255eukaryotic cellNot SpecifiedUnspecificNot SpecifiedAll life stagesHighHighHighHigh<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>
</ul>
2016-11-29T18:41:222022-07-15T09:46:25Activation, Stellate cellsActivation, Stellate cellsCellular<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>
<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>
<p>Human: Friedman, 2008</p>
<p>Rat: George et al.,1999</p>
<p>Mouse: Chang et al., 2014</p>
<p>Pig: Costa et al., 2001</p>
CL:0000632hepatic stellate cellNot SpecifiedUnspecificNot SpecifiedAll life stagesHighHighHighHighHigh<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>
</ul>
2016-11-29T18:41:232019-11-10T05:25:03Accumulation, CollagenAccumulation, CollagenTissue<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">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.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">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></span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">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.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">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).</span></span></p>
<p> </p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Determination of the amount of collagen produced <em>in vitro</em> 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 <em>in vitro</em> 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). </span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,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 <em>in vivo</em> and <em>in vitro</em>. 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"><em><strong>Sircol Collagen Assay for collagen quantification:</strong></em></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 and Raghunath, 2009; Nikota et al., 2017).</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><em><strong>Hydroxyproline assay:</strong></em></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 and 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>Ex vivo precision cut tissue slices</em></strong></span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,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>
<span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">
</span></span></pre>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Humans: Bataller and Brenner, 2005; Decaris et al., 2015. </span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Mice: Dalton et al., 2009; Leung et al., 2008; Nan et al., 2013.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Rats: Hamdy and El-Demerdash, 2012; Li et al., 2012; Luckey and Petersen, 2001; Natajaran et al., 2006.</span></span></p>
<p> </p>
UBERON:0002384connective tissueNot SpecifiedUnspecificNot SpecifiedAll life stagesHighHighHigh<ol>
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<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Bataller R, Brenner DA. Liver fibrosis. J Clin Invest. 2005 Feb;115(2):209-18. doi: 10.1172/JCI24282. </span></span></p>
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<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">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. </span></span></p>
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<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">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. </span></span></p>
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<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">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.</span></span></p>
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<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">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.</span></span></p>
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<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Diegelmann R. Collagen Metabolism. Wounds. 2001;13:177-82. Available at www.medscape.com/viewarticle/423231 (accessed on 20 January 2016).</span></span></p>
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<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Hamdy N, El-Demerdash E. New therapeutic aspect for carvedilol: antifibrotic effects of carvedilol in chronic carbon tetrachloride-induced liver damage. Toxicol Appl Pharmacol. 2012 Jun 15;261(3):292-9. doi: 10.1016/j.taap.2012.04.012. </span></span></p>
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<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Henderson NC, Iredale JP. Liver fibrosis: cellular mechanisms of progression and resolution. Clin Sci (Lond). 2007 Mar;112(5):265-80. doi: 10.1042/CS20060242.</span></span></p>
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<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Kivirikko KI, Risteli L. Biosynthesis of collagen and its alterations in pathological states. Med Biol. 1976 Jun;54(3):159-86.</span></span></p>
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<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Leung TM, Tipoe GL, Liong EC, Lau TY, Fung ML, Nanji AA. Endothelial nitric oxide synthase is a critical factor in experimental liver fibrosis. Int J Exp Pathol. 2008 Aug;89(4):241-50. doi: 10.1111/j.1365-2613.2008.00590.x. </span></span></p>
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<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Li L, Hu Z, Li W, Hu M, Ran J, Chen P, Sun Q. Establishment of a standardized liver fibrosis model with different pathological stages in rats. Gastroenterol Res Pract. 2012;2012:560345. doi: 10.1155/2012/560345. </span></span></p>
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<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Luckey SW, Petersen DR. Activation of Kupffer cells during the course of carbon tetrachloride-induced liver injury and fibrosis in rats. Exp Mol Pathol. 2001 Dec;71(3):226-40. doi: 10.1006/exmp.2001.2399.</span></span></p>
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<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Miller EJ, Gay S. The collagens: an overview and update. Methods Enzymol. 1987;144:3-41. doi: 10.1016/0076-6879(87)44170-0. </span></span></p>
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<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Nan YM, Kong LB, Ren WG, Wang RQ, Du JH, Li WC, Zhao SX, Zhang YG, Wu WJ, Di HL, Li Y, Yu J. Activation of peroxisome proliferator activated receptor alpha ameliorates ethanol mediated liver fibrosis in mice. Lipids Health Dis. 2013 Feb 6;12:11. doi: 10.1186/1476-511X-12-11.</span></span></p>
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<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Natarajan SK, Thomas S, Ramamoorthy P, Basivireddy J, Pulimood AB, Ramachandran A, Balasubramanian KA. Oxidative stress in the development of liver cirrhosis: a comparison of two different experimental models. J Gastroenterol Hepatol. 2006 Jun;21(6):947-57. doi: 10.1111/j.1440-1746.2006.04231.x.</span></span></p>
</li>
<li>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Nikota J, Banville A, Goodwin LR, Wu D, Williams A, Yauk CL, Wallin H, Vogel U, Halappanavar S. 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. Part Fibre Toxicol. 2017 Sep 13;14(1):37. doi: 10.1186/s12989-017-0218-0. </span></span></p>
</li>
<li>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Prockop DJ, Kivirikko KI. Collagens: molecular biology, diseases, and potentials for therapy. Annu Rev Biochem. 1995;64:403-34. doi: 10.1146/annurev.bi.64.070195.002155. </span></span></p>
</li>
<li>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Rahman L, Williams A, Gelda K, Nikota J, Wu D, Vogel U, Halappanavar S. 21st Century Tools for Nanotoxicology: Transcriptomic Biomarker Panel and Precision-Cut Lung Slice Organ Mimic System for the Assessment of Nanomaterial-Induced Lung Fibrosis. Small. 2020 Sep;16(36):e2000272. doi: 10.1002/smll.202000272.</span></span></p>
</li>
<li>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Rishikof DC, Kuang PP, Subramanian M, Goldstein RH. Methods for measuring type I collagen synthesis in vitro. Methods Mol Med. 2005;117:129-40. doi: 10.1385/1-59259-940-0:129. </span></span></p>
</li>
</ol>
2016-11-29T18:41:222023-05-17T15:55:30Tissue resident cell activationTissue resident cell activationCellular<p>Tissue resident cell activation is considered as a hallmark of inflammation irrespective of the tissue type. Strategically placed cells within tissues respond to noxious stimuli, thus regulating the recruitment of neutrophil and the initiation and resolution of inflammation (Kim and Luster, 2015). Examples for these cells are resident immune cells, parenchymal cells, vascular cells, stromal cells, or smooth muscle cells. These cells may be specific for a certain tissue, but they have a common tissue-independent role.</p>
<p>Under healthy conditions there is a homeostatic state, characterized as a generally quiescent cellular milieu. Various danger signals or alarmins that are involved in induction of inflammation like pathogen-associated molecular pattern molecules (PAMPs) and damage-associated molecular pattern molecules (DAMPs) activate these resident cells in affected tissues. </p>
<p>Examples of well-characterized DAMPs (danger signals or alarmins) (Saïd-Sadier and Ojcius, 2012)</p>
<table border="1" cellpadding="0" cellspacing="3">
<thead>
<tr>
<th>
<p>DAMPs</p>
</th>
<th style="width:193px">
<p>Receptors</p>
</th>
<th style="width:299px">
<p>Outcome of receptor ligation</p>
</th>
</tr>
</thead>
<tbody>
<tr>
<td>
<p>Extracellular nucleotides<br />
(ATP, ADP, adenosine)</p>
</td>
<td style="width:193px">
<p>PI, P2X and P2Y receptors (ATP, ADP); Al, A2A, A2B and A3 receptors (adenosine)</p>
</td>
<td style="width:299px">
<p>Dendritic cell (DC) maturation, chemotaxis, secretion of cytokines (IL-1β, IL-18), inflammation</p>
</td>
</tr>
<tr>
<td>
<p>Extracellular heat shock<br />
proteins</p>
</td>
<td style="width:193px">
<p>CD14, CD91, scavenger<br />
receptors, TLR4, TLR2, CD40</p>
</td>
<td style="width:299px">
<p>DC maturation, cytokine induction, DC, migration to lymph nodes</p>
</td>
</tr>
<tr>
<td>
<p>Extracellular HMGB1</p>
</td>
<td style="width:193px">
<p>RAGE, TLR2, TLR4</p>
</td>
<td style="width:299px">
<p>Chemotaxis, cytokine induction, DC activation, neutrophil recruitment, inflammation, activation of immune cells</p>
</td>
</tr>
<tr>
<td>
<p>Uric acid crystals</p>
</td>
<td style="width:193px">
<p>CD14, TLR2, TLR4</p>
</td>
<td style="width:299px">
<p>DC activation, cytokine induction, neutrophil recruitment, gout induction</p>
</td>
</tr>
<tr>
<td>
<p>Oxidative stress</p>
</td>
<td style="width:193px">
<p>Intracellular redox-sensitive proteins</p>
</td>
<td style="width:299px">
<p>Cell death, release of endogenous DAMPs, inflammation</p>
</td>
</tr>
<tr>
<td>
<p>Laminin</p>
</td>
<td style="width:193px">
<p>Integrins</p>
</td>
<td style="width:299px">
<p>Neutrophil recruitment, chemotaxis</p>
</td>
</tr>
<tr>
<td>
<p>S100 proteins or<br />
calgranulins</p>
</td>
<td style="width:193px">
<p>RAGE</p>
</td>
<td style="width:299px">
<p>Neutrophil recruitment, chemotaxis, cytokine secretion, apoptosis</p>
</td>
</tr>
<tr>
<td>
<p>Hyaluronan</p>
</td>
<td style="width:193px">
<p>TLR2, TLR4, CD44</p>
</td>
<td style="width:299px">
<p>DC maturation, cytokine production, adjuvant activity</p>
</td>
</tr>
</tbody>
</table>
<p>Activation refers to a phenotypic modification of the resident cells that includes alterations in their secretions, activation of biosynthetic pathways, production of pro-inflammatory proteins and lipids, and morphological changes. While these represent a pleiotropic range of responses that can vary with the tissue, there are a number of common markers or signs of activation that are measurable.</p>
<p>Examples of Common markers are</p>
<ul>
<li><span style="color:#3498db">CD11b</span></li>
<li><span style="color:#3498db">Iba1</span></li>
<li><span style="color:#3498db">GFAP</span></li>
<li><span style="color:#3498db">CD68</span></li>
<li><span style="color:#3498db">CD86</span></li>
<li><span style="color:#3498db">Mac-1</span></li>
<li>NF-kB</li>
<li>AP-1</li>
<li>Jnk</li>
<li>P38/mapk</li>
</ul>
<p>These described commonalities allow the use of this KE as a hub KE in the AOP network. However, despite the similarities in the inflammatory process, the type of reactive cells and the molecules triggering their reactivity may be tissue-specific. Therefore, for practical reasons, a tissue specific description of the reactive cells and of the triggering factors is necessary in order to specify in a tissue-specific manner, which cell should be considered and what should be measured.</p>
<p><strong>BRAIN </strong></p>
<p>The most easily detectable feature of brain inflammation or neuroinflammation is activation of microglial cells and astrocytes. It is evidenced by changes in shape, increased expression of certain antigens, and accumulation and proliferation of the glial cells in affected regions (Aschner, 1998; Graeber & Streit, 1990; Monnet-Tschudi et al, 2007; Streit et al, 1999; Kraft and Harry, 2011; Claycomb et al., 2013). Upon stimulation by cytokines, chemokines or inflammogens (e.g. from pathogens or from damaged neurons), both glial cell types activate inflammatory signaling pathways, which result in increased expression and/or release of inflammatory mediators such as cytokines, eicosanoids, and metalloproteinases (Dong & Benveniste, 2001) (cf KE: pro-inflammatory mediators, increased), as well as in the production of reactive oxygen species (ROS) and nitrogen species (RNS) (Brown & Bal-Price, 2003). Different types of activation states are possible for microglia and astrocytes, resulting in pro-inflammatory or anti-inflammatory signaling, and other cellular functions (such as phagocytosis) (Streit et al., 1999; Nakajima and Kohsaka, 2004). Therefore, neuroinflammation can have both neuroprotective/neuroreparative and neurodegenerative consequences (Carson et al., 2006; Monnet-Tschudi et al, 2007; Aguzzi et al., 2013 ; Glass et al., 2010). Under normal physiological conditions, microglial cells survey the nervous system for neuronal integrity (Nimmerjahn et al, 2005) and for invading pathogens (Aloisi, 2001; Kreutzberg, 1995; Kreutzberg, 1996; Rivest, 2009). They are the first type of cell activated (first line of defense), and can subsequently induce astrocyte activation (Falsig, 2008). Two distinct states of microglial activation have been described (Gordon, 2003; Kigerl et al, 2009; Maresz et al, 2008; Mosser & Edwards, 2008; Perego et al; Ponomarev et al, 2005): The M1 state is classically triggered by interferon-gamma and/or other pro-inflammatory cytokines, and this state is characterized by increased expression of integrin alpha M (Itgam) and CD86, as well as the release of pro-inflammatory cytokines (TNF-alpha, IL-1beta, IL-6), and it is mostly associated with neurodegeneration. The M2 state is triggered by IL-4 and IL-13 (Maresz et al, 2008; Perego et al, 2011; Ponomarev et al, 2007) and induces the expression of mannose receptor 1 (MRC1), arginase1 (Arg 1) and Ym1/2; it is involved in repair processes. The activation of astrocytes by microglia-derived cytokines or TLR agonists resembles the microglial M1 state (Falsig 2006). Although classification of the M1/M2 polarization of microglial cells may be considered as a simplification of authentic microglial reaction states (Ransohoff, 2016), a similar polarization of reactive astrocytes has been described recently Liddlelow et al., 2017): Interleukin-1 alpha (IL-1a), TNF and subcomponent q (C1q) released by activated microglial cells induce A1-reactive astrocytes, which lose the ability to promote neuronal survival, outgrowth, synaptogenesis and phagocytosis and induce the death of neurons and oligodendrocytes.</p>
<p><strong>Regulatory examples using the KE</strong></p>
<p>Measurement of glial fibrillary acidic protein (GFAP) in brain tissue, whose increase is a marker of astrocyte reactivity, is required by the US EPA in rodent toxicity studies for fuel additives (40 CFR 79.67). It has been used on rare occasions for other toxicant evaluations.</p>
<p><span style="font-size:14px"><strong>LIVER:</strong></span></p>
<p>Kupffer cells (KCs) are a specialized population of macrophages that reside in the liver; they were first described by Carl Wilhelm von Kupffer (1829–1902) [Haubrich 2004]. KCs constitute 80%-90% of the tissue macrophages in the reticuloendothelial system and account for approximately 15% of the total liver cell population [Bouwens et al., 1986]. They play an important role in normal physiology and homeostasis as well as participating in the acute and chronic responses of the liver to toxic compounds. Activation of KCs results in the release of an array of inflammatory mediators, growth factors, and reactive oxygen species. This activation appears to modulate acute hepatocyte injury as well as chronic liver responses including hepatic cancer. Understanding the role KCs play in these diverse responses is key to understanding mechanisms of liver injury [Roberts et al.,2007]. Besides the release of inflammatory mediators including cytokines, chemokines, lysosomal and proteolytic enzymes KCs are a main source of TGF-β1 (transforming growth factor-beta 1, the most potent profibrogenic cytokine). In addition latent TGF-β1 can be activated by KC-secreted matrix metalloproteinase 9 (MMP-9)[Winwood and Arthur, 1993; Luckey and Peeterson, 2001] through the release of biologically active substances that promote the pathogenic process. Activated KCs also release ROS like superoxide generated by NOX (NADPH oxidase), thus contributing to oxidative stress. Oxidative stress also activates a variety of transcription factors like NF-κB, PPAR-γ leading to an increased gene expression for the production of growth factors, inflammatory cytokines and chemokines. KCs express TNF-α (Tumor Necrosis Factor-alpha), IL-1 (Interleukin-1) and MCP-1 (monocyte-chemoattractant protein-1), all being mitogens and chemoattractants for hepatic stellate cells (HSCs) and induce the expression of PDGF receptors on HSCs which enhances cell proliferation. Expressed TNF-α, TRAIL (TNF-related apoptosis-inducing ligand), and FasL (Fas Ligand) are not only pro-inflammatory active but also capable of inducing death receptor-mediated apoptosis in hepatocytes [Guo and Friedman, 2007; Friedman 2002; Roberts et al., 2007]. Under conditions of oxidative stress macrophages are further activated which leads to a more enhanced inflammatory response that again further activates KCs though cytokines (Interferon gamma (IFNγ), granulocyte macrophage colony-stimulating factor (GM-CSF), TNF-α), bacterial lipopolysaccharides, extracellular matrix proteins, and other chemical mediators [Kolios et al., 2006; Kershenobich Stalnikowitz and Weissbrod 2003].</p>
<p>Besides KCs, the resident hepatic macrophages, infiltrating bone marrow-derived macrophages, originating from circulating monocytes are recruited to the injured liver via chemokine signals. KCs appear essential for sensing tissue injury and initiating inflammatory responses, while infiltrating Ly-6C+ monocyte-derived macrophages are linked to chronic inflammation and fibrogenesis. The profibrotic functions of KCs (HSC activation via paracrine mechanisms) during chronic hepatic injury remain functionally relevant, even if the infiltration of additional inflammatory monocytes is blocked via pharmacological inhibition of the chemokine CCL2 [Baeck et al., 2012; Tacke and Zimmermann, 2014].</p>
<p>KC activation and macrophage recruitment are two separate events and both are necessary for fibrogenesis, but as they occur in parallel, they can be summarised as one KE.</p>
<p>Probably there is a threshold of KC activation and release above which liver damage is induced. Pre-treatment with gadolinium chloride (GdCl), which inhibits KC function, reduced both hepatocyte and sinusoidal epithelial cell injury, as well as decreased the numbers of macrophages appearing in hepatic lesions and inhibited TGF-β1 mRNA expression in macrophages. Experimental inhibition of KC function or depletion of KCs appeared to protect against chemical-induced liver injury [Ide et al.,2005]. </p>
<p><strong><span style="font-size:18px">In General:</span></strong></p>
<p>Measurement <u>targets</u> are cell surface and intracellular markers; the specific markers may be cell and species-specific. </p>
<p>Available <u>methods</u> include cytometry, immunohistochemistry, gene expression sequencing; western blotting, ELISA, and functional assays.</p>
<h2><strong><span style="font-size:18px">BRAIN </span></strong></h2>
<p>Neuroinflammation, i.e. the activation of glial cells can be measured by quantification of cellular markers (most commonly), or of released mediators (less common). As multiple activation states exist for the two main cell types involved, it is necessary to measure several markers of neuroinflammation:</p>
<ol>
<li>Microglial activation can be detected based on the increased numbers of labeled microglia per volume element of brain tissue (due to increase of binding sites, proliferation, and immigration of cells) or on morphological changes. A specific microglial marker, used across different species, is CD11b. Alternatively various specific carbohydrate structures can be stained by lectins (e.g. IB4). Beyond that, various well-established antibodies are available to detect microglia in mouse tissue (F4/80), phagocytic microglia in rat tissue (ED1) or more generally microglia across species (Iba1). Transgenic mice are available with fluorescent proteins under the control of the CD11b promoter to easily quantify microglia without the need for specific stains.</li>
<li>The most frequently used astrocyte marker is glial fibrillary acidic protein, GFAP (99% of all studies) (Eng et al., 2000). This protein is highly specific for astrocytes in the brain, and antibodies are available for immunocytochemical detection. In neuroinflamatory brain regions, the stain becomes more prominent, due to an upregulation of the protein, a shape change/proliferation of the cells, and/or better accessibility of the antibody. Various histological quantification approaches can be used. Occasionally, alternative astrocytic markers, such as vimentin of the S100beta protein, have been used for astrocyte staining (Struzynska et al., 2007). Antibodies for complement component 3 (C3), the most characteristic and highly upregulated marker of A1 neurotoxic reactive astrocytes are commercially available.</li>
<li>All immunocytochemical methods can also be applied to cell culture models.</li>
<li>In patients, microglial accumulation can be monitored by PET imaging, using [11C]-PK 11195 as a microglial marker (Banati et al., 2002).</li>
<li>Activation of glial cells can be assessed in tissue or cell culture models also by quantification of sets of M1/M2 phenotype markers. This can for instance be done by PCR quantification, immunocytochemistry, immunoblotting.</li>
</ol>
<ul>
<li>Itgam, CD86 expression as markers of M1 microglial phenotype</li>
<li>Arg1, MRC1, as markers of M2 microglial phenotype</li>
</ul>
<p style="margin-left:21.3pt">(for descriptions of techniques, see Falsig 2004; Lund 2006 ; Kuegler 2010; Monnet-Tschudi et al., 2011; Sandström et al., 2014; von Tobel et al., 2014)</p>
<p style="margin-left:21.3pt"><span style="font-size:14px"><strong>LIVER:</strong></span></p>
<p style="margin-left:21.3pt">Kupffer cell activation can be measured by means of expressed cytokines, e.g. tissue levels of TNF-a [Vajdova et al,2004], IL-6 expression, measured by immunoassays or Elisa (offered by various companies), soluble CD163 [Grønbaek etal., 2012; Møller etal.,2012] or increase in expression of Kupffer cell marker genes such as Lyz, Gzmb, and Il1b, (Genome U34A Array, Affymetrix); [Takahara et al.,2006]</p>
<p>Extend to at least invertebrates</p>
<p>Not to plants and not to single-celled organisms</p>
<p><span style="font-size:14px"><strong>BRAIN:</strong></span></p>
<p>Tissue resident activation is observed in human, monkey, rat, mouse, and zebrafish, in association with neurodegeneration or following toxicant exposure. Some references (non-exhaustive list) are given below for illustration:</p>
<p>Human: Vennetti et al., 2006</p>
<p>Monkey (Macaca fascicularis): Charleston et al., 1994, 1996</p>
<p>Rat: Little et al., 2012; Zurich et al., 2002; Eskes et al., 2002</p>
<p>Mouse: Liu et al., 2012</p>
<p>Zebrafish: Xu et al., 2014.</p>
<p><span style="font-size:14px"><strong>LIVER:</strong></span></p>
<p>Human: Su et al., 2002; Kegel et al., 2015; Boltjes et al.,2014</p>
<p>Rat: Luckey and Peterson,2001</p>
<p>Mouse: Dalton t al., 2009</p>
<p><span style="color:#3498db"><strong>Life stage applicability:</strong> This key event is mainly applicable to all life stages most evidence is derived from adult models (Betlazar et al., 2016; Paladini et al., 2021). </span></p>
<p><span style="color:#3498db"><strong>Sex applicability:</strong> This key event is not sex specific (Betlazar et al., 2016; Paladini et al., 2021). </span></p>
<p><span style="color:#3498db"><strong>Evidence for perturbation by a prototypic stressor:</strong> Current literature provides ample evidence of tissue resident cell activation being induced by ionizing radiation (Allen et al., 2020; Krukowski et al., 2018; Parihar et al., 2020; Parihar et al., 2018; Parihar et al., 2016; Poulose et al., 2011; Raber et al., 2019; Sumam et al., 2013). </span></p>
Not SpecifiedAll life stagesNot SpecifiedNot SpecifiedNot SpecifiedNot SpecifiedNot Specified<p><span style="color:#3498db">Allen, B. D. et al. (2020), "Mitigation of helium irradiation-induced brain injury by microglia depletion", Journal of Neuroinflammation, Vol. 17/1, Nature, https://doi.org/10.1186/s12974-020-01790-9. </span></p>
<p><span style="color:#3498db">Betlazar, C. et al. (2016), "The impact of high and low dose ionising radiation on the central nervous system", Redox Biology, Vol. 9, Elsevier, Amsterdam, https://doi.org/10.1016/j.redox.2016.08.002. </span></p>
<p>Chan JK, Roth J, Oppenheim JJ, Tracey KJ, Vogl T, Feldmann M, Horwood N, Nanchahal J., Alarmins: awaiting a clinical response. J Clin Invest. 2012 Aug;122(8):2711-9.</p>
<p>Davies LC, Jenkins SJ, Allen JE, Taylor PR, Tissue-resident macrophages, Nat Immunol. 2013 Oct;14(10):986-95. </p>
<p>Escamilla-Tilch M, Filio-Rodríguez G, García-Rocha R, Mancilla-Herrera I, Mitchison NA, Ruiz-Pacheco JA, Sánchez-García FJ, Sandoval-Borrego D, Vázquez-Sánchez EA, The interplay between pathogen-associated and danger-associated molecular patterns: an inflammatory code in cancer? Immunol Cell Biol. 2013 Nov-Dec;91(10):601-10.</p>
<p>Hussell T, Bell TJ, Alveolar macrophages: plasticity in a tissue-specific context, Nat Rev Immunol. 2014 Feb;14(2):81-93.</p>
<p>Kim ND, Luster AD. The role of tissue resident cells in neutrophil recruitment ,Trends Immunol. 2015 Sep;36(9):547-55.</p>
<p><span style="color:#3498db">Krukowski, K. et al. (2018), "Female mice are protected from space radiation-induced maladaptive responses", Brain, Behavior, and Immunity, Vol. 74, Academic Press Inc., https://doi.org/10.1016/j.bbi.2018.08.008. </span></p>
<p><span style="color:#3498db">Paladini, M. S. et al. (2021), "Microglia depletion and cognitive functions after brain injury: From trauma to galactic cosmic ray", Neuroscience Letters, Vol. 741, Elsevier, Amsterdam, https://doi.org/10.1016/j.neulet.2020.135462. </span></p>
<p><span style="color:#3498db">Parihar, V. K. et al. (2016), "Cosmic radiation exposure and persistent cognitive dysfunction", Scientific Reports, Vol. 6/June, Nature Publishing Group, https://doi.org/10.1038/srep34774. </span></p>
<p><span style="color:#3498db">Parihar, V. K. et al. (2018), "Persistent nature of alterations in cognition and neuronal circuit excitability after exposure to simulated cosmic radiation in mice", Experimental Neurology, Vol. 305, Academic Press Inc., https://doi.org/10.1016/j.expneurol.2018.03.009. </span></p>
<p><span style="color:#3498db">Parihar, V. K. et al. (2020), "Sex-Specific Cognitive Deficits Following Space Radiation Exposure", Frontiers in behavioral neuroscience, Vol. 14, Frontiers, https://doi.org/10.3389/fnbeh.2020.535885. </span></p>
<p><span style="color:#3498db">Poulose, S. M. et al. (2011), "Exposure to 16O-particle radiation causes aging-like decrements in rats through increased oxidative stress, inflammation and loss of autophagy", Radiation Research, Vol. 176/6, BioOne, https://doi.org/10.1667/RR2605.1. </span></p>
<p><span style="color:#3498db">Raber, J. et al. (2019), "Combined Effects of Three High-Energy Charged Particle Beams Important for Space Flight on Brain, Behavioral and Cognitive Endpoints in B6D2F1 Female and Male Mice", Frontiers in physiology, Vol. 10, Frontiers, https://doi.org/10.3389/fphys.2019.00179. </span></p>
<p>Saïd-Sadier N, Ojcius DM., Alarmins, inflammasomes and immunity. Biomed J. 2012 Nov-Dec;35(6):437-49.</p>
<p>Schaefer L, Complexity of danger: the diverse nature of damage-associated molecular patterns, J Biol Chem. 2014 Dec 19;289(51):35237-45.</p>
<p><span style="color:#3498db">Suman, S. et al. (2013), "Therapeutic and space radiation exposure of mouse brain causes impaired dna repair response and premature senescence by chronic oxidant production", Aging, Vol. 5/8, https://doi.org/10.18632/aging.100587. </span></p>
<p><span style="font-size:14px"><strong>BRAIN:</strong></span></p>
<p>Aschner M (1998) Immune and inflammatory responses in the CNS: modulation by astrocytes. ToxicolLett 103: 283-287</p>
<p>Banati, R. B. (2002). "Visualising microglial activation in vivo." Glia 40: 206-217.</p>
<p>Brown GC, Bal-Price A (2003) Inflammatory neurodegeneration mediated by nitric oxide, glutamate, and mitochondria. Mol Neurobiol 27: 325-355</p>
<p>Charleston JS, Body RL, Bolender RP, Mottet NK, Vahter ME, Burbacher TM. 1996. Changes in the number of astrocytes and microglia in the thalamus of the monkey Macaca fascicularis following long-term subclinical methylmercury exposure. NeuroToxicology 17: 127-138.</p>
<p>Charleston JS, Bolender RP, Mottet NK, Body RL, Vahter ME, Burbacher TM. 1994. Increases in the number of reactive glia in the visual cortex of Macaca fascicularis following subclinical long-term methyl mercury exposure. ToxicolApplPharmacol 129: 196-206.</p>
<p>Dong Y, Benveniste EN (2001) Immune Function of Astrocytes. Glia 36: 180-190</p>
<p>Eng LF, Ghirnikar RS, Lee YL (2000) Glial Fibrillary Acidic Protein: GFAP-Thirty-One Years (1969-2000). NeurochemRes 25: 1439-1451</p>
<p>Eskes C, Honegger P, Juillerat-Jeanneret L, Monnet-Tschudi F. 2002. Microglial reaction induced by noncytotoxic methylmercury treatment leads to neuroprotection via interactions with astrocytes and IL-6 release. Glia 37(1): 43-52.</p>
<p>Falsig J, Latta M, Leist M. Defined inflammatory states in astrocyte cultures correlation with susceptibility towards CD95-driven apoptosis. J Neurochem. 2004 Jan;88(1):181-93.</p>
<p>Falsig J, Pörzgen P, Lund S, Schrattenholz A, Leist M. The inflammatory transcriptome of reactive murine astrocytes and implications for their innate immune function. J Neurochem. 2006 Feb;96(3):893-907.</p>
<p>Falsig J, van Beek J, Hermann C, Leist M. Molecular basis for detection of invading pathogens in the brain. J Neurosci Res. 2008 May 15;86(7):1434-47.</p>
<p>Glass CK, Saijo K, Winner B, Marchetto MC, Gage FH (2010). Mechanisms underlying inflammation in neurodegeneration. Cell. 2010 Mar 19;140(6):918-34.</p>
<p>Gordon S (2003) Alternative activation of macrophages. Nat Rev Immunol 3: 23-35</p>
<p>Graeber MB, Streit WJ (1990) Microglia: immune network in the CNS. Brain Pathol 1: 2-5</p>
<p>Kigerl KA, Gensel JC, Ankeny DP, Alexander JK, Donnelly DJ, Popovich PG (2009) Identification of two distinct macrophage subsets with divergent effects causing either neurotoxicity or regeneration in the injured mouse spinal cord. J Neurosci 29: 13435-13444</p>
<p>Kuegler PB, Zimmer B, Waldmann T, Baudis B, Ilmjärv S, Hescheler J, Gaughwin P, Brundin P, Mundy W, Bal-Price AK, Schrattenholz A, Krause KH, van Thriel C, Rao MS, Kadereit S, Leist M. Markers of murine embryonic and neural stem cells, neurons and astrocytes: reference points for developmental neurotoxicity testing. ALTEX. 2010;27(1):17-42</p>
<p>Kreutzberg GW (1995) Microglia, the first line of defence in brain pathologies. Arzneimttelforsch 45: 357-360</p>
<p>Kreutzberg GW (1996) Microglia : a sensor for pathological events in the CNS. Trends Neurosci 19: 312-318</p>
<p>Liddelow SA, Guttenplan KA, Clarke LE, Bennett FC, Bohlen CJ, Schirmer L, et al. 2017. Neurotoxic reactive astrocytes are induced by activated microglia. Nature 541(7638): 481-487.</p>
<p>Little AR, Miller DB, Li S, Kashon ML, O'Callaghan JP. 2012. Trimethyltin-induced neurotoxicity: gene expression pathway analysis, q-RT-PCR and immunoblotting reveal early effects associated with hippocampal damage and gliosis. Neurotoxicol Teratol 34(1): 72-82.</p>
<p>Liu Y, Hu J, Wu J, Zhu C, Hui Y, Han Y, et al. 2012. alpha7 nicotinic acetylcholine receptor-mediated neuroprotection against dopaminergic neuron loss in an MPTP mouse model via inhibition of astrocyte activation. J Neuroinflammation 9: 98.</p>
<p>Lund S, Christensen KV, Hedtjärn M, Mortensen AL, Hagberg H, Falsig J, Hasseldam H, Schrattenholz A, Pörzgen P, Leist M. The dynamics of the LPS triggered inflammatory response of murine microglia under different culture and in vivo conditions. J Neuroimmunol. 2006 Nov;180(1-2):71-87.</p>
<p>Maresz K, Ponomarev ED, Barteneva N, Tan Y, Mann MK, Dittel BN (2008) IL-13 induces the expression of the alternative activation marker Ym1 in a subset of testicular macrophages. J Reprod Immunol 78: 140-148</p>
<p>Monnet-Tschudi F, Zurich MG, Honegger P (2007) Neurotoxicant-induced inflammatory response in three-dimensional brain cell cultures. Hum Exp Toxicol 26: 339-346</p>
<p>Monnet-Tschudi, F., A. Defaux, et al. (2011). "Methods to assess neuroinflammation." Curr Protoc Toxicol Chapter 12: Unit12 19. </p>
<p>Mosser DM, Edwards JP (2008) Exploring the full spectrum of macrophage activation. Nat Rev Immunol 8: 958-969</p>
<p>Nakajima K, Kohsaka S. 2004. Microglia: Neuroprotective and neurotrophic cells in the central nervous system. Current Drug Targets-Cardiovasc & Haematol Disorders 4: 65-84.</p>
<p>Perego C, Fumagalli S, De Simoni MG (2011) Temporal pattern of expression and colocalization of microglia/macrophage phenotype markers following brain ischemic injury in mice. J Neuroinflammation 8: 174</p>
<p>Ponomarev ED, Maresz K, Tan Y, Dittel BN (2007) CNS-derived interleukin-4 is essential for the regulation of autoimmune inflammation and induces a state of alternative activation in microglial cells. J Neurosci 27: 10714-10721</p>
<p>Ponomarev ED, Shriver LP, Maresz K, Dittel BN (2005) Microglial cell activation and proliferation precedes the onset of CNS autoimmunity. J Neurosci Res 81: 374-389</p>
<p>Ransohoff RM. 2016. A polarizing question: do M1 and M2 microglia exist? Nat Neurosci 19(8): 987-991.</p>
<p>Sandstrom von Tobel, J., D. Zoia, et al. (2014). "Immediate and delayed effects of subchronic Paraquat exposure during an early differentiation stage in 3D-rat brain cell cultures." Toxicol Lett. DOI : 10.1016/j.toxlet.2014.02.001</p>
<p>Struzynska L, Dabrowska-Bouta B, Koza K, Sulkowski G (2007) Inflammation-Like Glial Response in Lead-Exposed Immature Rat Brain. Toxicol Sc 95:156-162</p>
<p>von Tobel, J. S., P. Antinori, et al. (2014). "Repeated exposure to Ochratoxin A generates a neuroinflammatory response, characterized by neurodegenerative M1 microglial phenotype." Neurotoxicology 44C: 61-70.</p>
<p>Venneti S, Lopresti BJ, Wiley CA. 2006. The peripheral benzodiazepine receptor (Translocator protein 18kDa) in microglia: from pathology to imaging. Prog Neurobiol 80(6): 308-322.</p>
<p>Xu DP, Zhang K, Zhang ZJ, Sun YW, Guo BJ, Wang YQ, et al. 2014. A novel tetramethylpyrazine bis-nitrone (TN-2) protects against 6-hydroxyldopamine-induced neurotoxicity via modulation of the NF-kappaB and the PKCalpha/PI3-K/Akt pathways. Neurochem Int 78: 76-85.</p>
<p>Zurich M-G, Eskes C, Honegger P, Bérode M, Monnet-Tschudi F. 2002. Maturation-dependent neurotoxicity of lead aceate in vitro: Implication of glial reactions. J Neurosc Res 70: 108-116.</p>
<p><span style="font-size:14px"><strong>LIVER:</strong></span></p>
<p><span style="color:#000000">Baeck, C. et al. (2012), Pharmacological inhibition of the chemokine CCL2 (MCP-1) diminishes liver macrophage infiltration and steatohepatitis in chronic hepatic injury, Gut, vol. 61, no. 3, pp.416–426.</span></p>
<p><span style="color:#000000">Boltjes, A. et al. (2014), The role of Kupffer cells in hepatitis B and hepatitis C virus infections, J Hepatol, vol. 61, no. 3, pp. 660-671.</span></p>
<p><span style="color:#000000">Bouwens, L. et al. (1986), Quantitation, tissue distribution and proliferation kinetics of Kupffer cells in normal rat liver, Hepatology, vol. 6, no. 6, pp. 718-722.</span></p>
<p><span style="color:#000000">Dalton, S.R. et al. (2009), Carbon tetrachloride-induced liver damage in asialoglycoprotein receptor-deficient mice, Biochem Pharmacol, vol. 77, no. 7, pp. 1283-1290.</span></p>
<p><span style="color:#000000">Friedman, S.L. (2002), Hepatic Fibrosis-Role of Hepatic Stellate Cell Activation, MedGenMed, vol. 4, no. 3, pp. 27.</span></p>
<p><span style="color:#000000">Grønbaek, H. et al. (2012), Soluble CD163, a marker of Kupffer cell activation, is related to portal hypertension in patients with liver cirrhosis, Aliment Pharmacol Ther, vol 36, no. 2, pp. 173-180.</span></p>
<p><span style="color:#000000">Guo, J. and S.L. Friedman (2007), Hepatic Fibrogenesis, Semin Liver Dis, vol. 27, no. 4, pp. 413-426.</span></p>
<p><span style="color:#000000">Haubrich, W.S. (2004), Kupffer of Kupffer cells, Gastroenterology, vol. 127, no. 1, p. 16</span></p>
<p><span style="color:#000000">Ide, M. et al. (2005), Effects of gadolinium chloride (GdCl(3)) on the appearance of macrophage populations and fibrogenesis in thioacetamide-induced rat hepatic lesions, J. Comp. Path, vol. 133, no. 2-3, pp. 92–102.</span></p>
<p><span style="color:#000000">Kegel, V. et al. (2015), Subtoxic concentrations of hepatotoxic drugs lead to Kupffer cell activation in a human in vitro liver model: an approach to study DILI, Mediators Inflamm, 2015:640631, </span><a href="http://doi.org/10.1155/2015/640631"><span style="color:#000000">http://doi.org/10.1155/2015/640631</span></a><span style="color:#000000">.</span></p>
<p><span style="color:#000000">Kershenobich Stalnikowitz, D. and A.B. Weissbrod (2003), Liver Fibrosis and Inflammation. A Review, Annals of Hepatology, vol. 2, no. 4, pp.159-163.</span></p>
<p><span style="color:#000000">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></p>
<p><span style="color:#000000">Luckey, S.W., and D.R. Petersen (2001), Activation of Kupffer cells during the course of carbon tetrachloride-induced liver injury and fibrosis in rats, Exp Mol Pathol, vol. 71, no. 3, pp. 226-240.</span></p>
<p><span style="color:#000000">Møller, H.J. (2012), Soluble CD163.Scand J Clin Lab Invest, vol. 72, no. 1, pp. 1-13.</span></p>
<p><span style="color:#000000">Roberts, R.A. et al. (2007), Role of the Kupffer cell in mediating hepatic toxicity and carcinogenesis, Toxicol Sci, vol. 96, no. 1, pp. 2-15.</span></p>
<p><span style="color:#000000">Su, G.L. et al. (2002), Activation of human and mouse Kupffer cells by lipopolysaccharide is mediated by CD14, Am J Physiol Gastrointest Liver Physiol, vol. 283, no. 3, pp. G640-645.</span></p>
<p><span style="color:#000000">Tacke, F. and H.W. Zimmermann (2014), Macrophage heterogeneity in liver injury and fibrosis, J Hepatol, vol. 60, no. 5, pp. 1090-1096.</span></p>
<p><span style="color:#000000">Takahara, T et al. (2006), Gene expression profiles of hepatic cell-type specific marker genes in progression of liver fibrosis, World J Gastroenterol, vol. 12, no. 40, pp. 6473-6499.</span></p>
<p><span style="color:#000000">Vajdova, K. et al. (2004), Ischemic preconditioning and intermittent clamping improve murine hepatic microcirculation and Kupffer cell function after ischemic injury, Liver Transpl, vol. 10, no. 4, pp. 520–528</span></p>
<p><span style="color:#000000">Winwood, P.J., and M.J. Arthur (1993), Kupffer cells: their activation and role in animal models of liver injury and human liver disease, Semin Liver Dis, vol. 13, no. 1, pp. 50-59.</span></p>
<p> </p>
2017-11-28T08:59:272023-03-22T16:03:39Increased Pro-inflammatory mediatorsIncreased pro-inflammatory mediatorsTissue<p>Inflammatory mediators are soluble, diffusible molecules that act locally at the site of tissue damage and infection, and at more distant sites. They can be divided into exogenous and endogenous mediators.</p>
<p>Exogenous mediators of inflammation are bacterial products or toxins like endotoxin or LPS. Endogenous mediators of inflammation are produced from within the (innate and adaptive) immune system itself, as well as other systems. They can be derived from molecules that are normally present in the plasma in an inactive form, such as peptide fragments of some components of complement, coagulation, and kinin systems. Or they can be released at the site of injury by a number of cell types that either contain them as preformed molecules within storage granules, e.g. histamine, or which can rapidly switch on the machinery required to synthesize the mediators.</p>
<p>Table1: a non-exhaustive list of examples for pro-inflammatory mediators</p>
<table border="1" cellpadding="0" cellspacing="0">
<tbody>
<tr>
<td style="width:253px">
<p><strong>Classes of inflammatory mediators</strong></p>
</td>
<td style="width:361px">
<p><strong>Examples</strong></p>
</td>
</tr>
<tr>
<td style="width:253px">
<p>Pro-inflammatory cytokines</p>
</td>
<td style="width:361px">
<p>TNF-a, Interleukins (IL-1, IL-6, IL-8), Interferons (IFN-g), chemokines (CXCL, CCL, GRO-α, MCP-1), GM-CSF</p>
</td>
</tr>
<tr>
<td style="width:253px">
<p>Prostaglandins</p>
</td>
<td style="width:361px">
<p>PGE2</p>
</td>
</tr>
<tr>
<td style="width:253px">
<p>Bradykinin</p>
</td>
<td style="width:361px">
<p> </p>
</td>
</tr>
<tr>
<td style="width:253px">
<p>Vasoactive amines</p>
</td>
<td style="width:361px">
<p>histamine, serotonin</p>
</td>
</tr>
<tr>
<td style="width:253px">
<p>Reactive oxygen species (ROS)</p>
</td>
<td style="width:361px">
<p>O<sup>2-</sup>, H<sub>2</sub>O<sub>2</sub></p>
</td>
</tr>
<tr>
<td style="width:253px">
<p>Reactive nitrogen species (RNS)</p>
</td>
<td style="width:361px">
<p>NO, iNOS</p>
</td>
</tr>
</tbody>
</table>
<p>The increased production of pro-inflammatory mediators can have negative consequences on the parenchymal cells leading even to cell death, as described for TNF-a or peroxynitrite on neurons (Chao et al., 1995; Brown and Bal-Price, 2003). <span style="color:#2980b9">Along with TNF-α, IL-1β and IL-6 have been shown to exhibit negative consequences on neurogenesis and neuronal precursor cell proliferation when overexpressed. IFN-γ is also associated with neuronal damage, although it is not as extensively studied compared to TNF-α, IL-1β and IL-6.</span> In addition, via a feedback loop, they can act on the reactive resident cells thus maintaining or exacerbating their reactive state; and by modifying elements of their signalling pathways, they can favour the M1 phenotypic polarization and the chronicity of the inflammatory process (Taetzsch et al., 2015).</p>
<p>Basically, this event occurs equally in various tissues and does not require tissue-specific descriptions. Nevertheless, there are some specificities such as the release of glutamate by brain reactive glial cells (Brown and Bal-Price, 2003; Vesce et al., 2007).The differences may rather reside in the type of insult favouring the increased expression and/or release of a specific class of inflammatory mediators, as well the time after the insult reflecting different stages of the inflammatory process. For these reasons, the analyses of the changes of a battery of inflammatory mediators rather than of a single one is a more adequate measurement of this KE.</p>
<p style="margin-right:13px; text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif"><strong><span style="font-size:7pt"><span style="font-size:11.0pt">Regulatory examples using the KE</span></span></strong></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:7pt"><span style="font-size:11.0pt">CD54 and CD 86 as well as IL-8 expression is used to assess skin sensitization potential (OECD TG 442E). IL-2 expression is used to assess immunotoxicity (and will become an OECD test guideline); for the latter see also doi: 10.1007/s00204-018-2199-7. </span></span></span></p>
<p> </p>
<p><span style="font-size:14px"><strong>LIVER:</strong></span></p>
<p>When activated, resident macrophages (Kupffer cells) release inflammatory mediators including cytokines, chemokines, lysosomal, and proteolytic enzymes and are a main source of TGF-β1 - the most potent pro-fibrogenic cytokine. Following the role of TGF-β is described in more detail.</p>
<p>Transforming growth factor β (TGF-β) is a pleiotropic cytokine with potent regulatory and</p>
<p>inflammatory activity [Sanjabi et al., 2009; Li and Flavell, 2008a;2008b]. The multi-faceted effects of TGF-β on numerous immune functions are cellular and environmental context dependent [Li et al., 2006]. TGF-β binds to TGF-β receptor II (TGF-βRII) triggering the kinase activity of the cytoplasmic domain that in turn activates TGF-βRI. The activated receptor complex leads to nuclear translocation of Smad molecules,</p>
<p>and transcription of target genes [Li et al., 2006a]. The role of TGF-β as an immune modulator of T cell activity is best exemplified by the similarities between TGF-β1 knockout and T cell specific</p>
<p>TGF-β receptor II knockout mice [Li et al., 2006b; Marie et al., 2006;Shull et al., 1992]. The animals in both of these models develop severe multi-organ autoimmunity and succumb to death within a few weeks after birth [Li et al., 2006b; Marie et al., 2006; Shull et al., 1992]. In addition, in mice where TGF-β signaling is blocked specifically in T cells, the development of natural killer T (NKT) cells, natural regulatory T (nTreg) cells, and CD8+ T cells was shown to be dependent on TGF-β signaling in the thymus [Li et al., 2006b; Marie et al., 2006].</p>
<p>TGF-β plays a major role under inflammatory conditions. TGF-β in the presence of IL-6 drives the differentiation of T helper 17 (Th17) cells, which can promote further inflammation and augment autoimmune conditions [Korn et al., 2009]. TGF-β orchestrates the differentiation of both Treg and Th17 cells in a concentration-dependent manner [Korn et al., 2008]. In addition, TGF-β in combination with IL-4, promotes the differentiation of IL-9- and IL-10-producing T cells, which lack</p>
<p>suppressive function and also promote tissue inflammation [Dardalhon et al., 2008; Veldhoen et al., 2008]. The biological effects of TGF-β under inflammatory conditions on effector and memory CD8+ T cells are much less understood. In a recent study, it was shown that TGF-β has a drastically opposing role on naïve compared to antigen-experienced/memory CD8+ T cells [Filippi et al., 2008]. When cultured <em>in vitro</em>, TGF-β suppressed naïve CD8+ T cell activation and IFN-γ production, whereas TGF-β enhanced survival of memory CD8+ T cells and increased the production of IL-17 and IFN-γ [Filippi et al., 2008]. TGF-β also plays an important role in suppressing the cells of the innate immune system.</p>
<p>The transforming growth factor beta (TGF-β) family of cytokines are ubiquitous, multifunctional, and essential to survival. They play important roles in growth and development, inflammation and repair, and host immunity. The mammalian TGF-β isoforms (TGF-β1, β2 and β3) are secreted as latent precursors and have multiple cell surface receptors of which at least two mediate signal transduction. Autocrine and paracrine effects of TGF-βs can be modified by extracellular matrix, neighbouring cells and other cytokines. The vital role of the TGF-β family is illustrated by the fact that approximately 50% of TGF-1 gene knockout mice die in utero and the remainder succumb to uncontrolled inflammation after birth. The role of TGF-β in homeostatic and pathogenic processes suggests numerous applications in the diagnosis and treatment of various diseases characterised by inflammation and fibrosis. [Clark and Coker, 1998; Santibañez et al., 2011; Pohlers et al., 2009] Abnormal TGF-β regulation and function are implicated in a growing number of fibrotic and inflammatory pathologies, including pulmonary fibrosis, liver cirrhosis, glomerulonephritis and diabetic nephropathy, congestive heart failure, rheumatoid arthritis, Marfan syndrome, hypertrophic scars, systemic sclerosis, myocarditis, and Crohn’s disease. [Gordon and Globe,2008] TGF-β1 is a polypeptide member of the TGF-β superfamily of cytokines. TGF-β is synthesized as a non-active pro-form, forms a complex with two latent associated proteins latency-associated protein (LAP) and latent TGF- β binding protein (LTBP) and undergoes protolithic cleavage by the endopeptidase furin to generate the mature TGF-β dimer. Among the TGF-βs, six distinct isoforms have been discovered although only the TGF-β1, TGF-β2 and TGF-β3 isoforms are expressed in mammals, and their human genes are located on chromosomes 19q13, 1q41 and 14q24, respectively. Out of the three TGF-β isoforms (β1, β2 and β3) only TGF-β1 was linked to fibrogenesis and is the most potent fibrogenic factor for hepatic stellate cells. [Roberts, 1998; Govinden and Bhoola, 2003]. During fibrogenesis, tissue and blood levels of active TGF-β are elevated and overexpression of TGF-β1 in transgenic mice can induce fibrosis. Additionally, experimental fibrosis can be inhibited by anti-TGF-β treatments with neutralizing antibodies or soluble TGF-β receptors [Qi et al.; 1999; Shek and Benyon , 2004; De Gouville et al., 2005; Chen et al., 2009]. TGF-β1 induces its own mRNA to sustain high levels in local sites of injury. The effects of TGF-β1 are classically mediated by intracellular signalling via Smad proteins. Smads 2 and 3 are stimulatory whereas Smad 7 is inhibitory. [Parsons et al., 2013; Friedman, 2008; Kubiczkova et al., 2012] Smad1/5/8, MAP kinase (mitogen-activated protein) and PI3 kinase are further signalling pathways in different cell types for TGF-β1 effects.</p>
<p>TGF-β is found in all tissues, but is particularly abundant in bone, lung, kidney and placental tissue. TGF-β is produced by many, but not all parenchymal cell types, and is also produced or released by infiltrating cells such as lymphocytes, monocytes/macrophages, and platelets. Following wounding or inflammation, all these cells are potential sources of TGF-β. In general, the release and activation of TGF-β stimulates the production of various extracellular matrix proteins and inhibits the degradation of these matrix proteins. [Branton and Kopp, 1999]</p>
<p>TGF-β 1 is produced by every leukocyte lineage, including lymphocytes, macrophages, and dendritic cells, and its expression serves in both autocrine and paracrine modes to control the differentiation, proliferation, and state of activation of these immune cells. [Letterio and Roberts; 1998]</p>
<p>In the liver TGF-β1 is released by activated Kupffer cells, liver sinusoidal endothelial cells, and platelets; in the further course of events also activated hepatic stellate cells express TGF-β1. Hepatocytes do not produce TGF-β1 but are implicated in intracellular activation of latent TGF-β1. [Roth et al., 1998; Kisseleva and Brenner, 2007; Kisseleva and Brenner, 2008; Poli, 2000; Liu et al., 2006]</p>
<p>TGF-β1 is the most established mediator and regulator of epithelial-mesenchymal-transition (EMT) which further contributes to the production of extracellular matrix. It has been shown that TGF-β1 mediates EMT by inducing snail-1 transcription factor and tyrosine phosphorylation of Smad2/3 with subsequent recruitment of Smad4. [Kolios et al., 2006; Bataller and Brenner, 2005; Guo and Friedman,2007; Brenner,2009; Kaimori et al., 2007; Gressner et al., 2002; Kershenobich Stalnikowitz and Weisssbrod, 2003; Li et al., 2008; Matsuoka and Tsukamoto, 1990; Kisseleva and Brenner, 2008; Poli, 200; Parsons et al., 2007; Friedman 2008; Liu et al., 2006]</p>
<p>TGF-β1 induces apoptosis and angiogenesis in vitro and in vivo through the activation of vascular endothelial growth factor (VEGF) High levels of VEGF and TGF-β1 are present in many tumors. Crosstalk between the signalling pathways activated by these growth factors controls endothelial cell apoptosis and angiogenesis. [Clark and Coker; 1998]</p>
<p> </p>
<p><span style="font-family:arial,helvetica,sans-serif"><span style="font-size:11pt">T<span style="font-size:14px">he specific type of measurement(s) might vary with tissue, environment and context and will need to be described for different tissue contexts as used within different AOP descriptions</span></span><span style="font-size:14px">.</span></span></p>
<p><span style="font-size:14px">In general, quantification of inflammatory markers can be done by:</span></p>
<ul>
<li><span style="font-size:14px">qRT-PCR (mRNA expression)</span></li>
<li><span style="font-size:14px">ELISA</span></li>
<li><span style="font-size:14px">Immunocytochemistry</span></li>
<li><span style="font-size:14px">Immunoblotting</span></li>
</ul>
<p><span style="font-size:14px">For descriptions of techniques, see Falsig 2004; Lund 2006 ; Kuegler 2010; Monnet-Tschudi et al., 2011; Sandström et al., 2014; von Tobel et al., 2014</span><br />
</p>
<p><span style="font-size:14px"><strong>LIVER:</strong></span></p>
<p>There are several assays for TGB-β1 measurement available.</p>
<p>e.g. Human TGF-β1 ELISA Kit. The Human TGF-β 1 ELISA (Enzyme –Linked Immunosorbent Assay) kit is an in vitro enzyme-linked immunosorbent assay for the quantitative measurement of human TGF-β1 in serum, plasma, cell culture supernatants, and urine. This assay employs an antibody specific for human TGF-β1 coated on a 96-well plate. Standards and samples are pipetted into the wells and TGF-β1 present in a sample is bound to the wells by the immobilized antibody. The wells are washed and biotinylated anti-human TGF-β1 antibody is added. After washing away unbound biotinylated antibody, HRP- conjugated streptavidin is pipetted to the wells. The wells are again washed, a TMB substrate solution is added to the wells and colour develops in proportion to the amount of TGF-β1 bound. The StopSolution changes the colour from blue to yellow, and the intensity of the colour is measured at 450 nm [Mazzieri et al., 2000]</p>
<p><span style="color:#2980b9">Listed below are common methods for detecting the KE, however there may be other comparable methods that are not listed.</span></p>
<table border="1">
<tbody>
<tr>
<td>
<p><span style="color:#2980b9">Assay </span></p>
</td>
<td>
<p><span style="color:#2980b9">Reference </span></p>
</td>
<td>
<p><span style="color:#2980b9">Description </span></p>
</td>
<td>
<p><span style="color:#2980b9">OECD Approved Assay </span></p>
</td>
</tr>
<tr>
<td>
<ul>
<li>
<p><span style="color:#2980b9">RT-qPCR </span></p>
</li>
<li>
<p><span style="color:#2980b9">Q-PCR </span></p>
</li>
</ul>
</td>
<td>
<p><span style="color:#2980b9">(Veremeyko et al., 2012; Alwine et al, 1977; Forlenza et al., 2012) </span></p>
</td>
<td>
<p><span style="color:#2980b9">Measures mRNA expression of cytokines, chemokines and inflammatory markers </span></p>
</td>
<td>
<p><span style="color:#2980b9">No </span></p>
</td>
</tr>
<tr>
<td>
<p><span style="color:#2980b9">Immunoblotting (western blotting) </span></p>
</td>
<td>
<p><span style="color:#2980b9">(Lee et al., 2008) </span></p>
</td>
<td>
<p><span style="color:#2980b9">Uses antibodies specific to proteins of interest, can used to detect presence of pro-inflammatory mediators in samples of cell or tissue lysate </span></p>
</td>
<td>
<p><span style="color:#2980b9">No </span></p>
</td>
</tr>
<tr>
<td>
<p><span style="color:#2980b9">Whole blood stimulation assay </span></p>
</td>
<td>
<p><span style="color:#2980b9">(Thurm & Halsey, 2005) </span></p>
</td>
<td>
<p><span style="color:#2980b9"> Detects inflammatory cytokines in blood </span></p>
</td>
<td>
<p><span style="color:#2980b9">No </span></p>
</td>
</tr>
<tr>
<td>
<p><span style="color:#2980b9">Imaging tests </span></p>
</td>
<td>
<p><span style="color:#2980b9">(Rollins & Miskolci, 2014) </span></p>
</td>
<td>
<p><span style="color:#2980b9">A qualitative technique using a cytokine specific antibodies and fluorophores can be used to visualize expression patterns, subcellular location of the target and protein-protein interactions. </span></p>
<p><span style="color:#2980b9">Common examples include double immunofluorescence confocal microscopy or other molecular imaging modalities. </span></p>
</td>
<td>
<p><span style="color:#2980b9">No </span></p>
</td>
</tr>
<tr>
<td>
<p><span style="color:#2980b9">Flow-cytometry </span></p>
</td>
<td>
<p><span style="color:#2980b9">(Karanikas et al., 2000) </span></p>
</td>
<td>
<p><span style="color:#2980b9">Detects the intracellular cytokines with stimulation. </span></p>
</td>
<td>
<p><span style="color:#2980b9">No </span></p>
</td>
</tr>
<tr>
<td>
<p><span style="color:#2980b9">Immunoassays (ex. enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunospot (ELISpot), radioimmunoassay) </span></p>
</td>
<td>
<p><span style="color:#2980b9">(Amsen et al., 2009; Engvall & Perlmann, 1972; Ji & Forsthuber, 2016; Goldsmith, 1975) </span></p>
</td>
<td>
<p><span style="color:#2980b9">Plate based assay technique using antibodies to detect presence of a protein in a liquid sample. </span></p>
<p><span style="color:#2980b9">Can be used to identify presence of an inflammatory cytokine of interest especially when in low concentrations. </span></p>
</td>
<td>
<p><span style="color:#2980b9">No </span></p>
</td>
</tr>
<tr>
<td>
<p><span style="color:#2980b9">Inflammatory cytokine arrays </span></p>
<p> </p>
</td>
<td>
<p><span style="color:#2980b9">(Amsen et al., 2009) </span></p>
<p> </p>
</td>
<td>
<p><span style="color:#2980b9">Similar to the ELISA, except using a membrane-based rather than plate-based approach. Can be used to measure multiple cytokine targets concurrently. </span></p>
</td>
<td>
<p><span style="color:#2980b9">No </span></p>
</td>
</tr>
<tr>
<td>
<p><span style="color:#2980b9">Immunohistochemistry (IHC) </span></p>
</td>
<td>
<p><span style="color:#2980b9">(Amsen et al., 2009; Coons et al., 1942) </span></p>
</td>
<td>
<p><span style="color:#2980b9">Immobilized tissue or cell cultures are stained using antibodies for specificity of ligands of interest. Versions of the assays can be used to visualize localization of inflammatory cytokines. </span></p>
</td>
<td>
<p><span style="color:#2980b9">No </span></p>
</td>
</tr>
</tbody>
</table>
<p><span style="font-size:14px"><strong>LIVER:</strong></span></p>
<p>Human [Santibañez et al., 2011]</p>
<p>Rat [Luckey and Petersen, 2001]</p>
<p>Mouse [Nan et al., 2013]</p>
<p><strong>BRAIN:</strong></p>
<p><span style="font-size:14px">Falsig 2004; Lund 2006 ; Kuegler 2010; Monnet-Tschudi et al., 2011; Sandström et al., 2014; von Tobel et al., 2014</span></p>
<p> </p>
<p><span style="color:#2980b9"><strong>Taxonomic applicability</strong>: The inflammatory response and increase of the pro-inflammatory mediators has been observed across species from simple invertebrates such as Daphnia to higher order vertebrates (Weavers & Martin, 2020).</span></p>
<p><span style="color:#2980b9"><strong>Life stage applicability</strong>: This key event is not life stage specific (Kalm et al., 2013; Veeraraghan et al., 2011; Hladik & Tapio, 2016). </span></p>
<p><span style="color:#2980b9"><strong>Sex applicability</strong>: Most studies conducted were on male models, although sex-dependent differences in pro-inflammatory markers have been previously reported (Cekanaviciute et al., 2018; Parihar et al., 2020). </span></p>
<p><span style="color:#2980b9"><strong>Evidence for perturbation by a prototypic stressor</strong>: There is evidence of the increase of pro-inflammatory mediators following perturbation from a variety of stressors including exposure to ionizing radiation. (Abdel-Magied et al., 2019; Cho et al., 2017; Gaber et al., 2003; Ismail et al., 2016; Kim et al. 2002; Lee et al., 2010; Parihar et al., 2018).</span></p>
Not SpecifiedUnspecificNot SpecifiedAll life stagesNot SpecifiedNot Specified<p> <span style="color:windowtext">Brown GC, Bal-Price A (2003) Inflammatory neurodegeneration mediated by nitric oxide, glutamate, and mitochondria. Mol Neurobiol 27: 325-355</span></p>
<p><span style="font-size:14px"><span style="color:windowtext">Dong Y, Benveniste EN (2001) Immune Function of Astrocytes. Glia 36: 180-190</span></span></p>
<p><span style="font-size:14px"><span style="color:windowtext">Falsig J, Latta M, Leist M. Defined inflammatory states in astrocyte cultures correlation with susceptibility towards CD95-driven apoptosis. J Neurochem. 2004 Jan;88(1):181-93. </span></span></p>
<p><span style="font-size:14px"><span style="color:windowtext">Falsig J, Pörzgen P, Lund S, Schrattenholz A, Leist M. The inflammatory transcriptome of reactive murine astrocytes and implications for their innate immune function. J Neurochem. 2006 Feb;96(3):893-907. </span></span></p>
<p><span style="font-size:14px"><span style="color:windowtext">Falsig J, van Beek J, Hermann C, Leist M. Molecular basis for detection of invading pathogens in the brain. J Neurosci Res. 2008 May 15;86(7):1434-47.</span></span></p>
<p><span style="font-size:14px"><span style="color:windowtext">Hamadi N, Sheikh A, Madjid N, Lubbad L, Amir N, Shehab SA, Khelifi-Touhami F, Adem A: Increased pro-inflammatory cytokines, glial activation and oxidative stress in the hippocampus after short-term bilateral adrenalectomy. BMC Neurosci 2016, <strong>17:</strong>61.</span></span></p>
<p><span style="font-size:14px"><span style="color:windowtext">Kuegler PB, Zimmer B, Waldmann T, Baudis B, Ilmjärv S, Hescheler J, Gaughwin P, Brundin P, Mundy W, Bal-Price AK, Schrattenholz A, Krause KH, van Thriel C, Rao MS, Kadereit S, Leist M. Markers of murine embryonic and neural stem cells, neurons and astrocytes: reference points for developmental neurotoxicity testing. ALTEX. 2010;27(1):17-42</span></span></p>
<p><span style="font-size:14px"><span style="color:windowtext">Lund S, Christensen KV, Hedtjärn M, Mortensen AL, Hagberg H, Falsig J, Hasseldam H, Schrattenholz A, Pörzgen P, Leist M. The dynamics of the LPS triggered inflammatory response of murine microglia under different culture and in vivo conditions. J Neuroimmunol. 2006 Nov;180(1-2):71-87. </span></span></p>
<p><span style="font-size:14px"><span style="color:windowtext">Monnet-Tschudi, F., A. Defaux, et al. (2011). "Methods to assess neuroinflammation." Curr Protoc Toxicol Chapter 12: Unit12 19.</span></span></p>
<p><span style="font-size:14px"><span style="color:windowtext">Sandstrom von Tobel, J., D. Zoia, et al. (2014). "Immediate and delayed effects of subchronic Paraquat exposure during an early differentiation stage in 3D-rat brain cell cultures." Toxicol Lett. DOI : 10.1016/j.toxlet.2014.02.001</span></span></p>
<p><span style="font-size:14px"><span style="color:windowtext">Taetzsch T, Levesque S, McGraw C, Brookins S, Luqa R, Bonini MG, Mason RP, Oh U, Block ML (2015) Redox regulation of NF-kappaB p50 and M1 polarization in microglia. Glia 5, <strong>63:</strong>423-440.</span></span></p>
<p><span style="font-size:14px"><span style="color:windowtext">Vesce S, Rossi D, Brambilla L, Volterra A (2007) Glutamate release from astrocytes in physiological conditions and in neurodegenerative disorders characterized by neuroinflammation. Int Rev Neurobiol. 82 :57-71.</span></span></p>
<p><span style="font-size:14px"><span style="color:windowtext"> <strong>LIVER:</strong></span></span></p>
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<p><span style="font-size:14px"><span style="color:windowtext"> </span></span> </p>
<p><span style="font-size:14px"><span style="color:#2980b9">Abdel-Magied, N., S. M., Shedid and Ahmed, A. G. (2019), “Mitigating effect of biotin against irradiation-induced cerebral cortical and hippocampal damage in the rat brain tissue”, Environmental Science and Pollution Research, Vol. 26/13, Springer, London, </span><a href="https://doi.org/10.1007/S11356-019-04806-X" rel="noreferrer noopener" target="_blank"><span style="color:#2980b9">https://doi.org/10.1007/S11356-019-04806-X</span></a><span style="color:#2980b9">. </span></span></p>
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<p><span style="font-size:14px"><span style="color:#2980b9">Weavers, H. and P. Martin (2020), “The cell biology of inflammation: From common traits to remarkable immunological adaptations”, Journal of Cell Biology, Vol. 219, Rockefeller University Press, New York, </span><a href="https://doi.org/10.1083/jcb.202004003" rel="noreferrer noopener" target="_blank"><span style="color:#2980b9">https://doi.org/10.1083/jcb.202004003</span></a><span style="color:#2980b9"> </span></span></p>
2017-11-28T09:00:542023-03-21T15:50:49579cc09b-5709-4639-b519-3785d1be3a4a4a8acd45-6b90-44cf-bd65-d8b3023484f6<p>Alkylating agents are highly reactive chemicals that may produce cellular damage by covalently binding to cellular macromolecules to form adducts and thereby preventing their proper functioning. Covalent protein alkylation by reactive electrophiles was identified as a key triggering event in chemical toxicity; it disturbs the cellular redox balance - contributing also to the development of oxidative stress - through interaction with glutathione, which leads to disruption of multiple biochemical pathways in exposed cells and is associated with mitochondrial dysfunction, which in turn, can trigger the death of exposed cells via either apoptosis and/or necrosis. <sup id="cite_ref-1" class="reference"><a href="#cite_note-1">[1]</a></sup><sup id="cite_ref-Boll2001_2-0" class="reference"><a href="#cite_note-Boll2001-2">[2]</a></sup><sup id="cite_ref-3" class="reference"><a href="#cite_note-3">[3]</a></sup><sup id="cite_ref-4" class="reference"><a href="#cite_note-4">[4]</a></sup><sup id="cite_ref-5" class="reference"><a href="#cite_note-5">[5]</a></sup>
</p><p>For example, Acrolein, the metabolite of Allyl Alcohol is a highly reactive electrophilic aldehyde and rapidly binds to cellular nucleophiles like glutathione. Thiol redox balance is critical for numerous cell functions Acrolein has been identified as both a product and initiator of lipid peroxidation.
<sup id="cite_ref-Kehrer2000_6-0" class="reference"><a href="#cite_note-Kehrer2000-6">[6]</a></sup>
The high toxic potential of Acrolein reflects its possession of two strongly electrophilic centres which ensure it readily reacts with nucleophilic groups on biological molecules including glutathione and proteins. These reactions typically proceed via Michael addition of nucleophiles to the a,b-unsaturated bond of Acrolein, generating carbonyl-retaining adducts with the ability to undergo
further crosslinking. Reaction of the carbonyl group in the first instance to form Schiff base adducts is typically much less preferred. Adduction of a diverse range of targets, in addition to disruption of the cellular redox balance, appears to underlie the disruption of multiple biochemical pathways in Acrolein-exposed cells. Such events can trigger the death of exposed cells via either apoptosis and/or necrosis. <sup id="cite_ref-Thompson2008_7-0" class="reference"><a href="#cite_note-Thompson2008-7">[7]</a></sup>
</p><p>It has been suggested that the alkylation of nucleophilic groups of cellular macromolecules effected by Acrolein after glutathione depletion is the event actually leading to cell injury.<sup id="cite_ref-8" class="reference"><a href="#cite_note-8">[8]</a></sup>
</p><p>Another example for an alkylating agent is Carbon Tetrachloride (CCl4), for which consensus has emerged that its toxicity is a mutifactorial process involving the generation of CCl4-derived free radicals, lipid peroxidation, covalent binding to macromolecules, loss of calcium homeostasis, nucleic acid hypomethylation and inflammatory cytokines. CCl4-derived free radicals are highly reactive species that are able to alkylate proteins and nucleic acids to generate CCl4-derived adducts. <sup id="cite_ref-9" class="reference"><a href="#cite_note-9">[9]</a></sup>
</p><p>Cell injury caused by covalent binding is biologically plausible. The mechanistic relationship between MIE and KE 1 consistent with established biological knowledge.
<sup id="cite_ref-Codreanu_2014_10-0" class="reference"><a href="#cite_note-Codreanu_2014-10">[10]</a></sup>
<sup id="cite_ref-11" class="reference"><a href="#cite_note-11">[11]</a></sup><sup id="cite_ref-Kehrer2000_6-1" class="reference"><a href="#cite_note-Kehrer2000-6">[6]</a></sup>
</p><p><em>
</p><p></em>
</p><p>Even though protein alkylation is a generic process having an impact on multiple physiological processes in the cell, certain connections related to which alkylated proteins lead and/or contribute to cell injury have been described with high biological plausibility. Better understanding of the effects of alkylating agents at the molecular level is aided by recent application of new toxicogenomics technologies. <sup id="cite_ref-12" class="reference"><a href="#cite_note-12">[12]</a></sup>
However, further efforts are certainly needed. Here we list literature-based evidence on how protein alkylation induced by allyl alcohol and carbon tetrachloride (CCl4) could be leading to cell injury (apoptosis/necrosis).
</p><p>Allyl alcohol/Acrolein-induced apoptosis of human cells is associated with depletion of cellular GSH and intracellular generation of oxidants <sup id="cite_ref-13" class="reference"><a href="#cite_note-13">[13]</a></sup>, achieved by alkylation of various proteins involved in the process.<sup id="cite_ref-14" class="reference"><a href="#cite_note-14">[14]</a></sup><sup id="cite_ref-15" class="reference"><a href="#cite_note-15">[15]</a></sup> More specifically, allyl alcohol/acrolein is considered a mitochondrial toxin that leads to cell death.<sup id="cite_ref-16" class="reference"><a href="#cite_note-16">[16]</a></sup> Whether apoptosis or necrosis ensues after acrolein exposure appears to be related to dose and cell type. In regards to activation of caspases as part of the mitochondrial death pathway it was shown that apoptosis could be both caspase-dependent: in human neuroblastoma cells <sup id="cite_ref-17" class="reference"><a href="#cite_note-17">[17]</a></sup> and in A549 lung cells <sup id="cite_ref-18" class="reference"><a href="#cite_note-18">[18]</a></sup>, as well as caspase-independent: in CHO cells. <sup id="cite_ref-19" class="reference"><a href="#cite_note-19">[19]</a></sup> It was suggested that the activation of certain caspases may arise from a partial inhibition of their active site cysteine residue through direct alkylation by acrolein.<sup id="cite_ref-20" class="reference"><a href="#cite_note-20">[20]</a></sup>
</p><p>Furthermore, using biotin hydrazide labeling, it was shown that NF-κB RelA and p50, as well as JNK2, were revealed as direct targets for alkylation by acrolein, affecting the GSH depletion. Mass spectrometry analysis of acrolein-modified recombinant JNK2 indicated adduction to Cys(41) and Cys(177), putative important sites involved in mitogen-activated protein kinase (MAPK) kinase (MEK) binding and JNK2 phosphorylation.<sup id="cite_ref-21" class="reference"><a href="#cite_note-21">[21]</a></sup> In complimentary work, exposure of cultured hepatocytes to acrolein led to a sustained activation of ERK1/2, JNK, and p38, which was associated with ER and mitochondrial stress and apoptosis. The cytotoxic effects of acrolein were decreased by JNK inhibitor, suggesting that kinase activation may be linked to cell death and liver injury. <sup id="cite_ref-22" class="reference"><a href="#cite_note-22">[22]</a></sup>
</p><p>Schwend et al. (2008) tried to identify new proteins that undergo alkylation by acrylamide by treating three human cell lines (Jurkat, Caco-2 and HepG2 cells) with acrylamide and analyzing extracted proteins by MALDI-TOF for potentially alkylated candidates. They could identify two novel acrylamide target proteins that may contribute to the toxicity of acrylamide in cell cultures. Acrylamide showed dose-dependent cytotoxic effects in all three tested cultures (IC50 2-4 mM for the three cell lines). Protein alkylation could be observed already at lower, sub-cytotoxic doses (10uM). Their data confirmed that acrylamide causes cytotoxicity effects in cell cultures and this cytotoxicity is most likely mediated by protein alkylation. <sup id="cite_ref-Schwend2008_23-0" class="reference"><a href="#cite_note-Schwend2008-23">[23]</a></sup>
</p><p>Thompson and Burcham (2008) studied the impact of culture media composition on the extent of damage occurring at protein targets within acrolein-exposed cells (A549 cells), and saw that acrolein induced concentration- dependent damage to cell proteins and increased cytochrome c release as marker of apoptotic cell death. <sup id="cite_ref-Thompson2008_7-1" class="reference"><a href="#cite_note-Thompson2008-7">[7]</a></sup>
</p><p>Cai et al. (2005) investigated the mitochondria-initiated apoptosis pathway involved in CCl4 hepatotoxicity in vitro and observed a time-and dose-dependent decrease in cellular glutathione content, along with a concomitant increase in malondialdehyde levels following the application of CCl4. Caspase 3 activity was stimulated at all doses of CCl4, with the most significant activation at 3 mmol/L. Cytochrome c was released obviously after CCl4 treatment. A time-dependent decrease in Bcl-XL expression was observed. DNA fragmentation results revealed apoptosis and necrosis following CCl4 treatment. They concluded that oxidative damage is one of the essential mechanisms of CCl4 hepatotoxicity, which triggers apoptosis via the mitochondria-initiated pathway.<sup id="cite_ref-24" class="reference"><a href="#cite_note-24">[24]</a></sup>
</p><p>Perrissoud et al. (1980) investigated the effect of CCl4 on isolated rat hepatocytes. The ultrastructural alterations and release of lactate dehydrogenase (LDH) and glutamate-oxaloacetate transaminase (GOT), were recorded after different periods of incubation. After 5 min incubation with CC14, morphological changes observed by electron microscopy, involved the plasma membrane. The endoplasmic reticulum and mitochondria were altered later. These morphological alterations were accompanied by an early release of LDH and GOT into the incubation medium.<sup id="cite_ref-25" class="reference"><a href="#cite_note-25">[25]</a></sup> .
</p><p>Johnston and Kroening (1998) investigated mechanisms of early carbon tetrachloride toxicity in cultured rat hepatocytes and found that primary rat hepatocytes in culture were killed after a 2 hr incubation with CCl4 gas at partial pressures above a threshold between 45 and 54 mmHg. They concluded that early hepatocyte death in cell culture is independent of metabolism of CCl4, and may be related to direct effects of CCl4 on intracellular membranes.<sup id="cite_ref-26" class="reference"><a href="#cite_note-26">[26]</a></sup>
</p><p>Boll et al. (2001) modelled CCl4-induced liver damage in monolayer cultures of rat primary hepatocytes with a focus on involvement of covalent binding of CC14 metabolites to cell components and/ or peroxidative damage as the cause of injury. They observed that covalent binding of 1 4 C-labelled metabolites was detected in hepatocytes immediately after exposure to CC14. Electrophoresis of microsomal proteins from [14C]-CCl4-treated hepatocytes revealed that, aside of the start and the front of the gel, radioactive label was found primarily between 70 and 80 kDa.<sup id="cite_ref-Boll2001_2-1" class="reference"><a href="#cite_note-Boll2001-2">[2]</a></sup><sup id="cite_ref-27" class="reference"><a href="#cite_note-27">[27]</a></sup>
</p><p>In a study performed on isolated hepatocytes it was demonstrated that direct alkylation of critical sulfhydryl groups in proteins leads to a sustained increase in free Ca++ concentrations which, via Ca++ - dependent protease activates the enzyme xantine oxidase. This activation generates a substantial amount of superoxide anion free radical and other ROS that oxidize other protein sulfhydryl groups. Thiol depletion in the cytoplasm is primarily linked to the alkylation by the reactive metabolite acrolein.<sup id="cite_ref-28" class="reference"><a href="#cite_note-28">[28]</a></sup>
</p><p>Boot (1996) described toxicological data of organic mercury compounds (alkylating agents) in rat hepatocytes, primary human hepatocytes, and in situ perfused total rat livers. Significant effects like induction of glutathione depletion, inhibition of cellular glucose and amino acid uptake with blocked albumin synthesis were observed in almost all tested physiological parameters. <sup id="cite_ref-Boot_1996_29-0" class="reference"><a href="#cite_note-Boot_1996-29">[29]</a></sup>
</p><p>Codreanu et al. (2014) intended to profile the accumulation of proteome damage in human cells (RKO and THP-1 cells) treated with lipid electrophile probes. Damage occurred selectively across functional protein interaction networks, with the most highly alkylation- susceptible proteins mapping to networks involved in cytoskeletal regulation. <sup id="cite_ref-Codreanu_2014_10-1" class="reference"><a href="#cite_note-Codreanu_2014-10">[10]</a></sup>
</p><p>Though covalent protein alkylation by reactive electrophiles was identified as a key triggering event in chemical toxicity already over 40 years ago and despite the intense effort expended over the past few years, our understanding of the mechanism and consequences of protein modification by reactive intermediates – both oxidizing and alkylating agents - is still quite limited. Covalent protein alkylation is a feature of many hepatotoxic drugs and the overall extent of binding does not adequately distinguish toxic from non-toxic binding. Directly relating covalent binding to hepatotoxicity is likely an oversimplification of the process whereby adduct formation ultimately leads to toxicity. Understanding underlying complexities (e.g., which macromolecules are important covalent binding targets) will be essential to any understanding of the problem of metabolism-dependent hepatotoxicity and predicting toxicity from in vitro experiments. <sup id="cite_ref-30" class="reference"><a href="#cite_note-30">[30]</a></sup><sup id="cite_ref-31" class="reference"><a href="#cite_note-31">[31]</a></sup>
Data from Codreanu et al. suggest that non-toxic covalent binding may largely be survivable damage to cytoskeletal components and other highly reactive protein targets, whereas toxic covalent binding produces lethal injury by targeting protein synthesis and catabolism and possibly mitochondrial electron transport. Future studies with appropriate probe molecules for toxic and non-toxic drugs could test these hypotheses and provide a better mechanistic basis for interpreting protein alkylation in drugsafety evaluation <sup id="cite_ref-Codreanu_2014_10-2" class="reference"><a href="#cite_note-Codreanu_2014-10">[10]</a></sup>
</p><p>For this AOP it is not known whether protein alkylation to certain proteins is required and whether particular proteins and various binding sites influence the further downstream process. Further we do not know whether there is a threshold and if this threshold would refer to the number of alkylation of a single protein or of a threshold number of proteins.
</p><p><em>
Quantitative data are hardly available.
</p><p>Schwend et al found that Acrylamide concentrations causing serious cytotoxicity were 2 – 4 mM. Acrylamide toxicity <i>in vivo</i> and <i>in vitro</i> is most likely the result of protein alkylation.Protein alkylation could be observed already at lower, sub-cytotoxic doses (10uM). The effects were dose-dependent and these IC 50 values were found for the three treated cell types: Jurkat cells: 2mM, HepG2 cells: 2mM, Caco-2 cells: 4mM.
Cells were grown in 96-well plates and treated with acrylamide for 48 h. Cell viability was measured by the MTT assay (0.05 mg/mL MTT). IC50 values were calculated from dose-response curves 48 h after acrylamide treatment.<sup id="cite_ref-Schwend2008_23-1" class="reference"><a href="#cite_note-Schwend2008-23">[23]</a></sup>
</p><p>Codreanu et al. performed adduct profiling experiments with alkynyl analogs of the prototypical lipid electrophiles 4-hydroxy-2-nonenal (HNE) and 4-oxo-2-nonenal (ONE) in human colorectal carcinoma (RKO) cells and human monocytic leukemia (THP-1) cells. Treatment with aHNE and aONE produced widespread protein alkylation in both cell types. IC50 concentrations for HNE and ONE and their alkynyl analogs in both cell types were 20 uM. Protein alkylation could be observed already at nontoxic concentrations (5 and 10uM).<sup id="cite_ref-Codreanu_2014_10-3" class="reference"><a href="#cite_note-Codreanu_2014-10">[10]</a></sup>
</em>
</p>HighHigh<p>human:<sup id="cite_ref-Schwend2008_23-2" class="reference"><a href="#cite_note-Schwend2008-23">[23]</a></sup>
rat:<sup id="cite_ref-Boot_1996_29-1" class="reference"><a href="#cite_note-Boot_1996-29">[29]</a></sup>
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<li id="cite_note-30"><span class="mw-cite-backlink"><a href="#cite_ref-30">↑</a></span> <span class="reference-text">Bauman, J.N. et al. (2008), Comparison of the bioactivation potential of the antidepressant and hepatotoxin nefazodone with aripiprazole, a structural analog and marketed drug, Drug Metab. Dispos, vol. 36, no. 6, pp. 1016–1029.</span>
</li>
<li id="cite_note-31"><span class="mw-cite-backlink"><a href="#cite_ref-31">↑</a></span> <span class="reference-text">Bauman, J.N. et al. (2009), Can in vitro metabolism-dependent covalent binding data distinguish hepatotoxic from nonhepatotoxic drugs? An analysis using human hepatocytes and liver S-9 fraction, Chem Res Toxicol, vol. 22, no. 2, pp. 332-340.</span>
</li>
</ol>2016-11-29T18:41:332016-11-29T20:02:374a8acd45-6b90-44cf-bd65-d8b3023484f625836309-52ad-49bd-a86a-8de73055cea8<p>The pioneering work of Kreutzberg and coworkers (1995, 1996) has shown that neuronal injury leads to neuroinflammation, with microglia and astrocyte reactivity. Several chemokines and chemokines receptors (fraktalkine, CD200) control the neuron-microglia interactions, and a loss of this control can trigger microglial reactivity (Blank and Prinz, 2013; Chapman et al., 2000; Streit et al., 2001). Upon injury causing neuronal death (mainly necrotic), signals termed Damage-Associated Molecular Patterns (DAMPs) are released by damaged neurons and promote microglial reactivity (Marin-Teva et al., 2011; Katsumoto et al., 2014). Toll-like receptors (TLRs) are pattern-recognition receptors that recognize specific pathogen- and danger-associated molecular signatures (PAMPs and DAMPs) and subsequently initiate inflammatory and immune responses. Microglial cells express TLRs, mainly TLR-2, which can detect neuronal cell death (for review, see Hayward and Lee, 2014). TLR-2 functions as a master sentry receptor to detect neuronal death and tissue damage in many different neurological conditions including nerve trans-section injury, traumatic brain injury and hippocampal excitotoxicity (Hayward and Lee, 2014). Astrocytes, the other cellular mediator of neuroinflammation (Ranshoff and Brown, 2012) are also able to sense tissue injury via TLR-3 (Farina et al., 2007; Rossi, 2015).</p>
<p><strong><span style="font-size:14px">LIVER:</span></strong></p>
<p>Damaged hepatocytes release reactive oxygen species (ROS), cytokines such as TGF-β1 and TNF-α, and chemokines which lead to oxidative stress, inflammatory signalling and finally activation of the resident macrophages in the liver, Kupffer cells (KCs). ROS generation in hepatocytes results from oxidative metabolism by NADH oxidase (NOX) and cytochrome 2E1 activation as well as through lipid peroxidation. Damaged liver cells trigger a sterile inflammatory response with activation of innate immune cells through release of damage-associated molecular patterns (DAMPs), which activate KCs through toll-like receptors and recruit activated neutrophils and monocytes into the liver. Central to this inflammatory response is the promotion of ROS formation by these phagocytes. Upon initiation of apoptosis hepatocytes undergo genomic DNA fragmentation and formation of apoptotic bodies; these apoptotic bodies are consecutively engulfed by KCs and cause their activation. This increased phagocytic activity strongly up-regulates NOX expression in KCs, a superoxide producing enzyme of phagocytes with profibrogenic activity, as well as nitric oxide synthase (iNOS) mRNA transcriptional levels with consequent harmful reaction between ROS and nitricoxide (NO), like the generation of cytotoxic peroxinitrite (N2O3). ROS and/or diffusible aldehydes also derive from liver sinusoidal endothelial cells (LSECs) which are additional initial triggers of KC activation. [Winwood and Arthur,1993; Luckey and Petersen, 2001; Roberts et al., 2007; Malhi, H. et al., 2010; Canbay et al., 2004; Orrenius et al., 2012; Kisseleva and Brenner, 2008; Jaeschke, 2011; Li et al., 2008; Poli, 2000]</p>
<p><span style="font-size:14px"><strong>LIVER:</strong></span></p>
<p>There is convincing theoretical evidence that hepatocyte injury and apoptosis causes KC activation, as well as inflammation and oxidative stress. But there are only limited experimental studies which could show that there is a direct relationship between these two events with temporal concordance. Specific markers for activated KCs have not been identified yet. KC activation cannot be detected morphologically by staining techniques since cell morphology does not change, but cytokines release can be measured (with the caveat that KCs activate spontaneously in vitro) and used as marker for KC activation. [Canbay et al., 2003; Soldatow et al., 2013] Tukov et al. examined the effects of KCs cultured in contact with rat hepatocytes. They found that by adding KCs to the cultures they could mimic <em>in vivo</em> drug-induced inflammatory responses. Experiments on cells of the macrophage lineage showed significant aldehyde-induced stimulation of the activity of protein kinase C, an enzyme involved in several signal transduction pathways. Further, 4-Hydroxynonenal (HNE) was demonstrated to up-regulate TGF-β1 expression and synthesis in isolated rat KCs. [Tukov et al., 2006] Canbay et al could prove that engulfment of hepatocyte apoptotic bodies stimulated KC generation of cytokines. [LeCluyse et al., 2012] </p>
<p>It is widely accepted that cell/neuronal injury and death lead to neuroinflammation (microglial and astrocyte reactivities) in adult brain. In the developing brain, neuroinflammation was observed after neurodegeneration induced by excitotoxic lesions (Acarin et al., 1997; Dommergues et al., 2003) or after ethanol exposure (Tiwari et al., 2012; Ahmad et al., 2016). It is important to note that physiological activation of microglial cells is observed during normal brain development for removal of apoptotic debris (Ashwell 1990, 1991). But exposure to toxicant (ethanol), excitotoxic insults (kainic acid) or traumatic brain injury during development can also induce apoptosis in hippocampus and cerebral cortex, as measured either by TUNEL, BID or caspase 3 upregulation associated to an inflammatory response, as evidenced by increased level of pro- inflammatory cytokines IL-1b, TNF-a, of NO, of p65 NF-kB or of the marker of astrogliosis, glial fibrillary acidic protein (GFAP), suggesting that, during brain development, neuroinflammation can also be triggerred by apoptosis induced by several types of insult (Tiwari and Chopra, 2012; Baratz et al., 2015; Mesuret et al., 2014).</p>
<p><strong>Mercury</strong></p>
<p>Young mice receiving a fish diet (MeHgCl) for 3 months exhibited in cortex a decrease of the chemokine Ccl<sub>2</sub> and neuronal death, as measured by a decrease in cell density, as well as microglial reactivity (increase in Iba1-labelled cells) (Godefroy et al., 2012)</p>
<p>Perinatal exposure to MeHgCl (GD7-PD21, 0.5 mg/kg bw/day in drinking water) lead to a delayed decrease (PD 36) of cholinergic muscarinic receptors in cerebellum accompanied by astrogliosis (Roda et al., 2008).</p>
<p>Immature rat brain cell cultures maintained in 3D conditions were exposed to either MeHgCl or HgCl<sub>2</sub> (10<sup>-9</sup> – 10<sup>-6</sup> M, for 10 days). This treatment caused microglial and astrocyte activation without neuronal death, but a reversible decrease of the expression of the neuronal marker MAP2 (Monnet-Tschudi et al., 1996 ; Eskes et al., 2002).</p>
<p>Adult marmoset exposed acutely to 5 mg Hg/kg/day p.o. exhibited apoptosis in occipital cortex, as well as glial reactivity (GFAP and Iba1 increased). Mercury content in occipital cortex was 31 mg/g (Yamamoto et al., 2012).</p>
<p>Monkeys exposed to MeHgCl (50 mg/bw for 6,12,18 months) showed microglial and astrocyte activation without any change in neuronal number. Both astrocyte and microglia accumulated elevated levels of inorganic mercury, suggesting a direct effect of mercury on glial cells (Charleston et al., 1996).</p>
<p>Human LUHMES cells as model of dopaminergic neurons and the human astrocyte cell line CFF-STTG1 were exposed to MeHgCl (0.25 -5 mM), thiomersal (0.25 – 5 mM) or HgCl2 (5-35 mM), what affected their cell viability. Neurons were much more sensitive than astrocytes (Lohner et al., 2015).</p>
<p>A direct activation of rat primary microglial cells and astrocytes was observed after exposure to MeHgCl (10<sup>-10</sup>-10<sup>-6</sup> M, for 5 days). (Eskes et aé., 2002).</p>
<p>Astrocyte + microglia in co-cultures exposed to mercury (1-5 mM for 30 min to 6 days) showed lower levels of GSH in microglia than in astrocytes (Ni et al., 2011 ; 2012).</p>
<p>Human primary astrocyte cell line exposed to MeHgCl (1.125 mM) for 24h and 72h did not exhibit an increase of GFAP, but of NfkB after the 72h (Malfa et al., 2014).</p>
<p>Human mast cells (leukemic LAD2, derived from umbilical cord blood) showed an increase of IL-6 release when exposed to HgCl<sub>2</sub> (0.1-10 mM, for 10 min to 24h). It is hypothesized that mast cell activation could lead to BBB disruption and to neuroinflammation. (Kempurai et al., 2010).</p>
<p> </p>
<p><em>Sex dependency</em></p>
<p>In prairie voles 10 weeks exposure to 600 ppm HgCl<sub>2</sub> in drinking water lead to an increase of TNF-a in hippocampus of male, but not in female (Curtis et al., 2011).</p>
<p> </p>
<p><strong>Acrylamide</strong> (acrylamide is a common food contaminant generated by heat processing)</p>
<p>Adult mice received 10, 20, 30 mg/kg bw for 4 weeks. The dose of 20 mg/kg bw caused neurological symptoms (ex. cognitive impairment) associated to an increased oxidative stress, a decrease of GSH and glial reactivity (GFAP and Iba1 increased) in cortex, hippocampus and striatum. An increase in TNF-a, IL-1b and i-NOS expression in all 3 brain regions was also observed. (Santhanasabepathy et al., 2015)</p>
<p>Isolated and/or co-cultures of microglial cells or astrocytes treated with acrylamide 0-1mM for 24-96h exhibited an increased release of TNF-a, IL-1b, IL-6 and G-CSF, suggesting a direct effect of acrylamide on glial cells (Zhao et al., 2017a,b).</p>
<p>Neonatal rat astrocytes treated with acrylamide (0.1-1mM) for 7, 11, 15, or 20 days increased their proliferation rate as measured by PCNA staining. Astrocyte proliferation is also a sign of reactivity. (Aschner et al., 2005).</p>
<p> </p>
<p><strong>Acrolein</strong></p>
<p>Adult rat received an infusion of acrolein (15, 50, 150 nmoles/0.5 ml) directly in substantia nigra which caused a decrease of Tyrosine hydroxylase immunostaining, an increase in caspase 1 and an activation of microglial cells and astrocytes (Wang et al., 2017).</p>
<p>Similar treatment as above induced an increase in lipid peroxidation, of hsp32 and of caspase 1 with an increase in GFAP and in ED1 (marker of macrophagic microglial cells) as well as of IL-1b (Zhao et al., 2017).</p>
<p> </p>
<p><strong>Mercury</strong></p>
<p>Mouse developmental exposure to 50 mM of HgCl<sub>2</sub> in maternal drinking water from GD8 to PD21 did not induce any change in GM-CSF, IFN-g, IL-1b, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70. IL-13, IL-17, MCP1, MIP2 and TNF-a measured by Luminex in brain slices of PD21 and PD70. No sex differences, but brain increase of IgG and increased sociability in females (Zhang et al., 2012).</p>
<p>3D rat brain cell cultures treated for 10 days with HgCl2 or MeHgCl (10-10 - 10-6 M) exhibited increased apotosis measured by TUNEL, but exclusively in immature cultures. The proportion of cells undergoing apoptotis was highest for astrocytes than for neurons. But the apoptotic nuclei were not associated with reactive microglial cells as evidenced by double staining (Monnet-Tschudi, 1998).</p>
<p><strong>Acrylamide</strong></p>
<p>A 2 weeks exposure to acrylamide in drinking water (44mg/kg/day) induced behavioral effects, such a decreased in locomotor activity, but with no effect at gene level on neuronal and inflammatory markers analyzed in somatosensory and motor cortex (Bowyer et al., 2009).</p>
<p><span style="font-size:14px"><strong>LIVER:</strong></span></p>
<p>The detailed mechanisms of the KC - hepatocyte interaction and its consequences for both normal and toxicant-driven liver responses remain to be determined. KC activation followed by cytokine release is associated in some cases with evident liver damage, whereas in others this event is unrelated to liver damage or may be even protective; apparently this impact is dependent on the quantity of KC activation; excessive or prolonged release of KC mediators can switch an initially protective mechanism to a damaging inflammatory response. Evidence suggests that low levels of cytokine release from KCs constitute a survival signal that protects hepatocytes from cell death and in some cases, stimulates proliferation. [Roberts et al., 2007] </p>
HighUnspecificHighDuring brain development, adulthood and agingHighAll life stagesHighHighHighHighHigh<p><span style="font-size:14px"><strong>Liver:</strong></span></p>
<p>Human [Winwood and Arthur,1993; Roberts et al., 2007; Kolios et al., 2006] </p>
<p>Rat [Tukov et al., 2006; Roberts et al., 2007]</p>
<p>Acarin L, González B, Castellano B, Castro AJ. 1997. Quantitative analysis of microglial reaction to a cortical excitotoxic lesion in the early postnatal brain. ExpNeurol 147: 410-417.</p>
<p>Ahmad A, Shah SA, Badshah H, Kim MJ, Ali T, Yoon GH, et al. 2016. Neuroprotection by Vitamin C Against Ethanol-Induced Neuroinflammation Associated Neurodegeneration in the Developing Rat Brain. CNS Neurol Disord Drug Targets 15(3): 360-370.</p>
<p>Aschner, M., Wu, Q., Friedman, M.A., 2005. Effects of acrylamide on primary neonatal rat astrocyte functions. Ann N Y Acad Sci. 1053<strong>,</strong> 444-54.</p>
<p>Ashwell K. 1990. Microglia and cell death in the developing mouse cerebellum. DevBrain Res 55: 219-230.</p>
<p>Ashwell K. 1991. The distribution of microglia and cell death in the fetal rat forebrain. DevBrain Res 58: 1-12.</p>
<p>Baratz R, Tweedie D, Wang JY, Rubovitch V, Luo W, Hoffer BJ, et al. 2015. Transiently lowering tumor necrosis factor-alpha synthesis ameliorates neuronal cell loss and cognitive impairments induced by minimal traumatic brain injury in mice. J Neuroinflammation 12: 45.</p>
<p>Blank T, Prinz M. Microglia as modulators of cognition and neuropsychiatric disorders. Glia, 2013, 61: 62-70.</p>
<p>Bowyer, J.F., et al., 2009. The mRNA expression and histological integrity in rat forebrain motor and sensory regions are minimally affected by acrylamide exposure through drinking water. Toxicol Appl Pharmacol. 240<strong>,</strong> 401-11.</p>
<p>Chapman GA, Moores K, Harrison D, Campbell CA, Stewart BR, Strijbos PJLM. Fractalkine Cleavage from Neuronal Membrans Represents an Acute Event in Inflammatory Response to Excitotoxic Brain Damage. J Neurosc., 2000, 20 RC87: 1-5.</p>
<p>Charleston JS, Body RL, Bolender RP, Mottet NK, Vahter ME, Burbacher TM: Changes in the number of astrocytes and microglia in the thalamus of the monkey Macaca fascicularis following long-term subclinical methylmercury exposure. <em>NeuroToxicology </em>1996, <strong>17:</strong>127-138.</p>
<p>Thomas Curtis, J., et al., 2011. Chronic inorganic mercury exposure induces sex-specific changes in central TNFalpha expression: importance in autism? Neurosci Lett. 504<strong>,</strong> 40-4.</p>
<p>Dommergues MA, Plaisant F, Verney C, Gressens P. 2003. Early microglial activation following neonatal excitotoxic brain damage in mice: a potential target for neuroprotection. Neuroscience 121(3): 619-628.</p>
<p>Eskes C, Honegger P, Juillerat-Jeanneret L, Monnet-Tschudi F. 2002. Microglial reaction induced by noncytotoxic methylmercury treatment leads to neuroprotection via interactions with astrocytes and IL-6 release. Glia 37(1): 43-52.</p>
<p>Farina C, Aloisi F, Meinl E. Astrocytes are active players in cerebral innate immunity. Trends Immunol, 2007, 28(3): 138-145.</p>
<p>Godefroy, D., et al., 2012. The chemokine CCL2 protects against methylmercury neurotoxicity. Toxicol Sci. 125<strong>,</strong> 209-18.</p>
<p>Hayward JH, Lee SJ. A Decade of Research on TLR2 Discovering Its Pivotal Role in Glial Activation and Neuroinflammation in Neurodegenerative Diseases. Experimental Neurobiology, 2014, 23(2): 138-147.</p>
<p>Katsumoto A, Lu H, Miranda AS, Ransohoff RM. Ontogeny and functions of central nervous system macrophages. J Immunol., 2014, 193(6): 2615-2621.</p>
<p>Kempuraj, D., et al., 2010. Mercury induces inflammatory mediator release from human mast cells. J Neuroinflammation. 7<strong>,</strong> 20.</p>
<p>Kreutzberg GW. Microglia, the first line of defence in brain pathologies. Arzneimttelforsch, 1995, 45: 357-360.</p>
<p>Kreutzberg GW. Microglia : a sensor for pathological events in the CNS. Trends Neurosci., 2006, 19: 312-318.</p>
<p>Lohren, H., et al., 2015. Toxicity of organic and inorganic mercury species in differentiated human neurons and human astrocytes. J Trace Elem Med Biol. 32<strong>,</strong> 200-8.</p>
<p>Malfa, G.A., et al., 2014. "Reactive" response evaluation of primary human astrocytes after methylmercury exposure. J Neurosci Res. 92<strong>,</strong> 95-103.</p>
<p>Marin-Teva JL, Cuadros MA, Martin-Oliva D, Navascues J., Microglia and neuronal cell death. Neuron glia biology, 2011, 7(1): 25-40.</p>
<p>Mesuret G, Engel T, Hessel EV, Sanz-Rodriguez A, Jimenez-Pacheco A, Miras-Portugal MT, et al. 2014. P2X7 receptor inhibition interrupts the progression of seizures in immature rats and reduces hippocampal damage. CNS neuroscience & therapeutics 20(6): 556-564.</p>
<p>Ni, M., et al., 2011. Comparative study on the response of rat primary astrocytes and microglia to methylmercury toxicity. Glia. 59<strong>,</strong> 810-20.</p>
<p>Ni, M., et al., 2012. Glia and methylmercury neurotoxicity. J Toxicol Environ Health A. 75<strong>,</strong> 1091-101.</p>
<p>Ransohoff RM, Brown MA. Innate immunity in the central nervous system. J Clin Invest., 2012, 122(4): 1164-1171.</p>
<p>Roda, E., et al., 2008. Cerebellum cholinergic muscarinic receptor (subtype-2 and -3) and cytoarchitecture after developmental exposure to methylmercury: an immunohistochemical study in rat. J Chem Neuroanat. 35<strong>,</strong> 285-94.</p>
<p>Rossi D. Astrocyte physiopathology: At the crossroads of intercellular networking, inflammation and cell death. Prog Neurobiol., 2015, 130: 86-120.</p>
<p>Santhanasabapathy, R., et al., 2015. Farnesol quells oxidative stress, reactive gliosis and inflammation during acrylamide-induced neurotoxicity: Behavioral and biochemical evidence. Neuroscience. 308<strong>,</strong> 212-27.</p>
<p>Streit WJ, Conde J, Harrison JK. Chemokines and Alzheimer's disease. Neurobiol Aging., 2001, 22: 909-913.</p>
<p>Tiwari V, Chopra K. 2012. Attenuation of oxidative stress, neuroinflammation, and apoptosis by curcumin prevents cognitive deficits in rats postnatally exposed to ethanol. Psychopharmacology (Berl) 224(4): 519-535</p>
<p>Wang, Y.T., et al., 2017. Acrolein acts as a neurotoxin in the nigrostriatal dopaminergic system of rat: involvement of alpha-synuclein aggregation and programmed cell death. Sci Rep. 7<strong>,</strong> 45741.</p>
<p>Yamamoto, M., et al., 2012. Increased expression of aquaporin-4 with methylmercury exposure in the brain of the common marmoset. J Toxicol Sci. 37<strong>,</strong> 749-63.</p>
<p>Zhang, Y., Bolivar, V.J., Lawrence, D.A., 2012. Developmental exposure to mercury chloride does not impair social behavior of C57BL/6 x BTBR F(1) mice. J Immunotoxicol. 9<strong>,</strong> 401-10.</p>
<p>Zhao, M., et al., 2017. Effect of acrylamide-induced neurotoxicity in a primary astrocytes/microglial co-culture model. Toxicol In Vitro. 39<strong>,</strong> 119-125.</p>
<p>Zhao, M., et al., 2017. Acrylamide-induced neurotoxicity in primary astrocytes and microglia: Roles of the Nrf2-ARE and NF-kappaB pathways. Food Chem Toxicol. 106<strong>,</strong> 25-35.</p>
<p>Zhao, W.Z., et al., 2017. Neuroprotective Effects of Baicalein on Acrolein-induced Neurotoxicity in the Nigrostriatal Dopaminergic System of Rat Brain. Mol Neurobiol.</p>
<p><span style="font-size:14px"><strong>LIVER:</strong></span></p>
<ul style="list-style-type:circle">
<li>Canbay, A. et al. (2003), Kupffer cell engulfment of apoptotic bodies stimulates death ligand and cytokine expression, Hepatology, vol. 38, no. 5, pp. 1188-1198.</li>
<li>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.</li>
<li>Jaeschke, H. (2011), Reactive oxygen and mechanisms of inflammatory liver injury: Present concepts, J Gastroenterol Hepatol. vol. 26, suppl. 1, pp. 173-179.</li>
<li>Kisseleva T and Brenner DA, (2008), Mechanisms of Fibrogenesis, Exp Biol Med, vol. 233, no. 2, pp. 109-122.</li>
<li>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.</li>
<li>LeCluyse, E.L. et al. (2012), Organotypic liver culture models: meeting current challenges in toxicity testing, Crit Rev Toxicol, vol. 42, no. 6, 501-548.</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>Luckey, S.W., and D.R. Petersen (2001), Activation of Kupffer cells during the course of carbon tetrachloride-induced liver injury and fibrosis in rats, Exp Mol Pathol, vol. 71, no. 3, pp. 226-240.</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>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.</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>Roberts, R.A. et al. (2007), Role of the Kupffer cell in mediating hepatic toxicity and carcinogenesis, Toxicol Sci, vol. 96, no. 1, pp. 2-15.</li>
<li>Soldatow, V.Y. et al. (2013), In vitro models for liver toxicity testing, Toxicol Res, vol. 2, no.1, pp. 23–39.</li>
<li>Tukov, F.F. et al. (2006), Modeling inflammation-drug interactions in vitro: a rat Kupffer cell-hepatocyte co-culture system, Toxicol In Vitro, vol. 20, no. 8, pp. 1488-1499.</li>
<li>Winwood, P.J., and M.J. Arthur (1993), Kupffer cells: their activation and role in animal models of liver injury and human liver disease, Semin Liver Dis, vol. 13, no. 1, pp. 50-59.</li>
</ul>
<p> </p>
<p> </p>
<p> </p>
<p> </p>
<p> </p>
<p> </p>
2018-02-01T05:53:152018-08-02T03:02:0525836309-52ad-49bd-a86a-8de73055cea867d8d2cd-f4a7-4b55-9a5e-acf1b82ffbe2<p><span style="font-size:14px"><strong>LIVER:</strong></span></p>
<p>Following activation the liver resident macrophages, Kupffer cells (KCs), become a major source for inflammatory mediators including cytokines, chemokines, lysosomal, and proteolytic enzymes and for reactive oxygen species (ROS) and also the main source for TGF-β1, the most potent profibrogenic cytokine. [Luckey and Petersen 2001; Winwood and Arthur 1993]</p>
<p>Expressed TNF-α (Tumor Necrosis Factor -alpha), TRAIL (TNF-related apoptosis-inducing ligand), and FasL (Fas Ligand) are pro-inflammatory active and also capable of inducing death receptor-mediated apoptosis in hepatocytes.</p>
<p>Activated KCs are an important source of ROS like superoxide (generated by NADPH oxidase (NOX). KCs express TNF-α, IL-1 (Interleukin-1) and MCP-1 (monocyte-chemoattractant protein-1), all being mitogens and chemoattractants for HSCs and induce the expression of platelet-derived growth factor (PDGF) receptors on hepatic stellate cells (HSCs) which further enhances HSCs proliferation. [Kamimura and Tsukamoto, 1995; Li et al.,2008; Kolios et al., 2006; Bataller and Brenner, 2005; Lee and Friedman,2011; Brenner,2009, Fujiwara and Kobayashi, 2005; Kirkham, 2007; Reuter et al., 2010</p>
<p><span style="font-size:14px"><strong>LIVER:</strong></span></p>
<p>The functional relationship between these KEs is consistent with biological knowledge. [Kamimura and Tsukamoto, 1995; Li et al.,2008; Kolios et al., 2006; Bataller and Brenner, 2005; Lee and Friedman,2011; Guo and Friedman, 2007; Brenner,2009, Fujiwara and Kobayashi, 2005; Kirkham, 2007; Reuter et al., 2010]</p>
<p><strong><span style="font-size:14px">LIVER:</span></strong></p>
<p>Cytokine release is one of the features that define KC activation and there is sound empirical evidence for this KER. Experimental studies have shown enhanced cytokine gene expression by KCs in evolution of experimental liver injury. Northern blot analysis of freshly isolated KCs showed enhanced mRNA expression of three acute phase cytokines by the hepatic resident macrophages, TNF-α, IL-6 and TGF-β. [Kamimura and Tsukamoto, 1995; De Bleser et al., 1997; Chu et al., 2013] </p>
<p>Experiments by Matsuoka and Tsukamoto already 1990 showed that KCs isolated from rat liver with alcoholic fibrosis express and release TGF-β1 and that this cytokine is largely responsible for the KC-conditioned medium-induced stimulation of collagen formation by HSCs. [Matsuoka and Tsukamoto, 1990]</p>
<p> </p>
HighUnspecificHighAll life stagesHighHigh<p><span style="font-size:14px"><strong>LIVER:</strong></span></p>
<p>Human <a href="https://aopwiki.org/relationships/147#cite_note-Bataller_2005-4">[Bataller and Brenner, 2005; Kolios et al., 2006] </a> </p>
<p>Rat <a href="https://aopwiki.org/relationships/147#cite_note-De_Blaser_1997-11">[De Bleser et al., 1997]</a></p>
<p><span style="font-size:14px"><strong>LIVER:</strong></span></p>
<ul style="list-style-type:circle">
<li>Bataller, R. and D.A. Brenner (2005), Liver Fibrosis, J.Clin. Invest, vol. 115, no. 2, pp. 209-218.</li>
<li>Brenner, D.A. (2009), Molecular Pathogenesis of Liver Fibrosis, Trans Am Clin Climatol Assoc, vol. 120, pp. 361–368.</li>
<li>Chu, P.S. et al. (2013), C-C motif chemokine receptor 9 positive macrophages activate hepatic stellate cells and promote liver fibrosis in mice, Hepatology, vol. 58, no. 1, pp. 337-350.</li>
<li>De Bleser, P.J. et al. (1997), Transforming growth factor-beta gene expression in normal and fibrotic rat liver, J Hepatol, vol. 26, no. 4, pp. 886-893.</li>
<li>Fujiwara, N. and K. Kobayashi (2005), Macrophages in inflammation, Curr Drug Targets Inflamm Allergy, vol. 4, no. 3, pp. 281-286.</li>
<li>Guo, J. and S. L. Friedman (2007), Hepatic fibrogenesis, Semin Liver Dis, vol. 27, no. 4, pp. 413-426.</li>
<li>Kamimura, S. and H. Tsukamoto (1995), Cytokine gene expression by Kupffer cells in experimental alcoholic liver disease, Hepatology, vol. 22, no. 4, pp. 1304-1309.</li>
<li>Kirkham, P. (2007), Oxidative stress and macrophage function: a failure to resolve the inflammatory response, Biochem Soc Trans, vol. 35, no. 2, pp. 284-287.</li>
<li>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.</li>
<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>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>Luckey, S.W., and D.R. Petersen (2001), Activation of Kupffer cells during the course of carbon tetrachloride-induced liver injury and fibrosis in rats, Exp Mol Pathol, vol. 71, no. 3, pp. 226-240</li>
<li>Matsuoka, M. and H. Tsukamoto, (1990), Stimulation of hepatic lipocyte collagen production by Kupffer cell-derived transforming growth factor beta: implication for a pathogenetic role in alcoholic liver fibrogenesis, Hepatology, vol. 11, no. 4, pp. 599-605.</li>
<li>Reuter, S. et al. (2010), Oxidative stress, inflammation, and cancer: how are they linked? Free Radic Biol Med, vol. 49, no. 11, pp. 1603-1616.</li>
<li>Winwood, P.J., and M.J. Arthur (1993), Kupffer cells: their activation and role in animal models of liver injury and human liver disease, Semin Liver Dis, vol. 13, no. 1, pp. 50-59.</li>
</ul>
2018-08-02T03:05:102018-08-02T03:37:5867d8d2cd-f4a7-4b55-9a5e-acf1b82ffbe238fc8b7c-df66-4ba4-98a1-e8cf17183abf<p>HSC Initiation is associated with rapid gene induction resulting from paracrine stimulation by inflammatory cells and injured hepatocytes . Also Kupffer cell infiltration and activation play a prominent role in HSC activation.[Li et al., 2008]</p>
<p>Lymphocytes, especially CD4 T-helper (Th) lymphocytes, help orchestrate the host response via cytokine production and can differentiate into Th1 and Th2 subsets. In general, Th1 cells produce cytokines promoting cell-mediated immunity, including interferon (IFN)-γ, TNF, and interleukin (IL)-2. Th2 cells produce IL-4, IL-5, IL-6, and IL-13 and promote humoral immunity. Results from previous experimental models imply that Th2 lymphocytes favor fibrogenesis in liver injury over Th1 lymphocytes. [Shi et al., 1997] However, recent studies of Wynn [Wynn,2004] suggest that more than two T-cell subsets underlying a highly complex, orchestrated response are involved, and they also provide us a more important paradigm for how these intersecting pathways may regulate fibrosis. In animal models, IL-13 has emerged as a key mediator because it increases TGF-β1 and MMP expression by macrophages, whereas IL-4 has a limited role. One study examined the activity of IL-13 in cultured HSCs and suggested that IL-4 and IL-13 directly affect HSCs by increasing collagen production and suppressing HSC proliferation. [Sugimoto et al., 2005] </p>
<p>Leukocytes recruited to the liver during injury join with Kupffer cells in producing compounds that modulate HSC behavior</p>
<p>Transforming growth factor beta 1 (TGF-β1) is the most potent fibrogenic factor for hepatic stellate cells (HSCs). In response to TGF-β1, HSCs activate into myofibroblast-like cells, producing type I, III and IV collagen, proteoglycans like biglycan and decorin, glycoproteins like laminin, fibronectin, tenascin and glycosaminoglycan. [Kisseleva and Brenner, 2007] In the further course of events activated HSCs themselves express TGF-β1. TGF-β1 induces its own mRNA to sustain high levels in local sites of liver injury. The effects of TGF-β1 are mediated by intracellular signalling via Smad proteins. Smads 2 and 3 are stimulatory whereas Smad 7 is inhibitory. Smad1/5/8, MAP kinase and PI3 kinase are further signalling pathways in different cell types for TGF-β1 effects. [Parsons et al., 2007] Concomitant with increased TGF-β production, HSC increase production of collagen. Connective tissue growth factor (CTGF) is a profibrogenic peptide induced by TGF-β, that stimulates the synthesis of collagen type I and fibronectin and may mediate some of the downstream effects of TGF-β. It is upregulated during activation of HSC, suggesting that its expression is another determinant of a fibrogenic response to TGF-β. [Williams et al.,2000] During fibrogenesis, tissue and blood levels of active TGF-β are elevated and overexpression of TGF-β1 in transgenic mice can induce fibrosis. Additionally, experimental fibrosis can be inhibited by anti-TGF-β treatments with neutralizing antibodies or soluble TbRs (TGF-β receptors). [Qi et al., 1999] </p>
<p>There is good understanding and broad acceptance of this KER. [Kisseleva and Brenner, 2007; Williams et al., 2000; Qi et al., 1999; Gressner et al., 2002; Kolios et al., 2006; Bataller and Brenner, 2005; Guo and Friedman, 2007; Brenner, 2009; Kaimori et al., 2007; Kershenobich Stalnikowitz and Weissbrod, 2003; Li et al., 2008; Matsuoka and Tsukamoto, 1990; Kisseleva and Brenner, 2008; Poli, 2000; Parsons et al., 2007; Friedman, 2008; Liu et al., 2006]</p>
<p>It is difficult to get experimental evidence in vitro for TGF-β1-induced HSC activation because HSCs undergo spontaneous activation when cultured on plastic; nevertheless qualitative empirical evidence for temporal and incidence concordance for this KER exists. Czaja et al could prove that treatment of cultured hepatic cells with TGF-β1 increased type I pro-collagen mRNA levels 13-fold due to post-transcriptional gene regulation. Tan et al. discovered that short TGF-β1 pulses can exert long-lasting effects on fibroblasts. HSCs activated in culture do not fully reproduce the changes in gene expression observed in vivo. De Minicis et al investigated gene expression changes in 3 different models of HSC activation and compared gene expression profiles in culture (mice HSCs in co-culture with KCs) and in vivo and did not find a proper correlation. [Czaja et al., 1989; Tan et al., 2013; Yin et al., 2013; De Minicis et al., 2007]</p>
HighUnspecificHighAll life stagesHighHigh<p>Human [Kolios et al., 2006; Guo and Friedman, 2007] </p>
<p>Rat [Dooley et al., 2000] </p>
<ul style="list-style-type:circle">
<li>Bataller, R. and D.A. Brenner (2005), Liver Fibrosis, J.Clin. Invest, vol. 115, no. 2, pp. 209-218.</li>
<li>Brenner, D.A. (2009), Molecular Pathogenesis of Liver Fibrosis, Trans Am Clin Climatol Assoc, vol. 120, pp. 361–368.</li>
<li>Czaja, M.J. et al. (1989), In vitro and in vivo association of transforming growth factor-beta 1 with hepatic fibrosis, J Cell Biol, vol. 108, no. 6, pp. 2477-2482.</li>
<li>De Minicis, S. et al. (2007), Gene expression profiles during hepatic stellate cell activation in culture and in vivo, Gastroenterology, vol. 132, no. 5, pp. 1937-1946.</li>
<li>Dooley, S. et al. (2000), Modulation of transforming growth factor b response and signaling during transdifferentiation of rat hepatic stellate cells to myofibroblasts,Hepatology, vol. 31, no. 5, pp. 1094-1106.</li>
<li>Friedman, S.L. (2008), Mechanisms of Hepatic Fibrogenesis, Gastroenterology, vol. 134, no. 6, pp. 1655–1669.</li>
<li>Gressner , A.M. et al. (2002), Roles of TGF-β in hepatic fibrosis. Front Biosci, vol. 7, pp. 793-807.</li>
<li>Guo, J. and S. L. Friedman (2007), Hepatic fibrogenesis, Semin Liver Dis, vol. 27, no. 4, pp. 413-426.</li>
<li>Jing-Ting Li, Zhang-Xiu Liao, Jie Ping, Dan Xu, and Hui Wang, Molecular mechanism of hepatic stellate cell activation and antifi brotic therapeutic strategies, J Gastroenterol 2008; 43:419–428</li>
<li>Kaimori, A. et al. (2007), Transforming growth factor-beta1 induces an epithelial-to-mesenchymal transition state in mouse hepatocytes in vitro, J Biol Chem, vol. 282, no. 30, pp. 22089-22101.</li>
<li>Kershenobich Stalnikowitz, D. and A.B. Weissbrod (2003), Liver Fibrosis and Inflammation. A Review, Annals of Hepatology, vol. 2, no. 4, pp.159-163.</li>
<li>Kisseleva T and Brenner DA, (2008), Mechanisms of Fibrogenesis, Exp Biol Med, vol. 233, no. 2, pp. 109-122.</li>
<li>Kisseleva, T. and Brenner, D.A. (2007), Role of hepatic stellate cells in fibrogenesis and the reversal of fibrosis, Journal of Gastroenterology and Hepatology, vol. 22, Suppl. 1; pp. S73–S78.</li>
<li>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.</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>Liu, Xingjun et al. (2006), Therapeutic strategies against TGF-beta signaling pathway in hepatic fibrosis. Liver Int, vol.26, no.1, pp. 8-22.</li>
<li>Matsuoka, M. and H. Tsukamoto, (1990), Stimulation of hepatic lipocyte collagen production by Kupffer cell-derived transforming growth factor beta: implication for a pathogenetic role in alcoholic liver fibrogenesis, Hepatology, vol. 11, no. 4, pp. 599-605.</li>
<li>Parsons, C.J., M.Takashima and R.A. Rippe (2007), Molecular mechanisms of hepatic fibrogenesis. J Gastroenterol Hepatol, vol. 22, Suppl.1, pp. S79-S84.</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>Qi Z et al. (1999), Blockade of type beta transforming growth factor signaling prevents liver fibrosis and dysfunction in the rat, Proc Natl Acad Sci USA, vol. 96, no. 5, pp. 2345-2349.</li>
<li>Shi Z, Wakil AE, Rockey DC. Strain-specifi c differences in mouse hepatic wound healing are mediated by divergent T helper cytokine responses. Proc Natl Acad Sci USA 1997;94:10663–8.</li>
<li>Sugimoto R, Enjoji M, Nakamuta M, Ohta S, Kohjima M, Fukushima M, et al. Effect of IL-4 and IL-13 on collagen production in cultured LI90 human hepatic stellate cells. Liver Int 2005;25:420–8.</li>
<li>Tan, A.B. et al. (2013), Cellular re- and de-programming by microenvironmental memory: why short TGF-β1 pulses can have long effects, Fibrogenesis Tissue Repair, vol. 6, no. 1, p. 12.</li>
<li>Williams, E.J. et al. (2000), Increased expression of connective tissue growth factor in fibrotic human liver and in activated hepatic stellate cells, J Hepatol, vol. 32, no. 5, pp. 754-761.</li>
<li>Wynn TA. Fibrotic disease and the T(H)1/T(H)2 paradigm. Nat Rev Immunol 2004;4:583–94.</li>
<li>Yin, C. et al. (2013), Hepatic stellate cells in liver development, regeneration, and cancer, J Clin Invest, vol. 123, no. 5, pp. 1902–1910.</li>
</ul>
2018-08-02T03:39:392018-08-02T03:43:2238fc8b7c-df66-4ba4-98a1-e8cf17183abf7f46e0c8-d5ed-4978-9904-4101d6b9d6b1<p>Up-regulation of collagen synthesis following hepatic stellate cell (HSC) activation is among the most striking molecular responses of HSCs to injury and is mediated by both transcriptional and post-transcriptional mechanisms. Activated HSCs do not only proliferate and increase cell number, but also increase collagen production per cell. Synthesis of type I collagen is initiated by expression of the col1a1 and col1a2 genes, giving rise to α 1(I) and α 2(I) procollagen mRNAs in a 2:1 ratio. Upon activation of HSCs and other myofibroblast precursors, there is a > 50-fold increase in α 1(I) procollagen mRNA levels. The half-life of collagen α1(I) mRNA increases 20-fold in activated HSCs compared with quiescent HSCs. Monocytes and macrophages are involved in inflammatory actions by producing large amounts of Nitric oxide (NO) and inflammatory cytokines such as TNF-α which have a direct stimulatory effect on HSC collagen synthesis. Synthesis of TGF-α and TGF-β promotes activation of neighbouring quiescent HSCs, whereas the release of HGF (hepatocyte growth factor) stimulates regeneration of adjacent hepatocytes.</p>
<p>The basement membrane-like matrix is normally comprised of collagens IV and VI, which is progressively replaced by collagens I and III and cellular fibronectin during fibrogenesis. Although multiple extracellular matrix (ECM) components are up-regulated, type I collagen is the most abundant protein. These changes in ECM composition initiate several positive feedback pathways that further amplify collagen production. Increasing matrix stiffness is a stimulus for HSC activation and matrix-provoked signals link to other growth factor receptors through integrin-linked kinase and transduce via membrane-bound guanosine triphosphate binding proteins, in particular Rho67 and Rac, signals to the actin cytoskeleton that promote migration and contraction.</p>
<p>The overall amount of collagen deposited by fibroblasts is a regulated balance between collagen synthesis and collagen catabolism. Down-regulated expression of degrading Matrix metalloproteinases (MMPs) and up-regulation of tissue inhibitors of metalloproteinases (TIMPs), MMP- inhibitors, lead to a net decrease in protease activity, and therefore, matrix accumulation. Chronic inflammation, hypoxia and oxidative stress reactivate epithelial-mesenchymal transition (EMT) developmental programmes that converge in the activation of NF-kB. Cells that may transdifferentiate into fibrogenic myofibroblasts are hepatocytes and cholangiocytes. Additional sources of ECM include bone marrow (which probably gives rise to circulating fibrocytes) and portal fibroblasts (Benyon and Arthur; 2001; Milani et al., 1994; Safadi and Friedman, 2002; Kolios et al.,2006; Bataller and Brenner, 2005; Lee und Friedman 2011; Guo and Friedman, 2007; Li, Jing-Ting et al., 2008; Kershenobich Stalnikowitz and Weisssbrod , 2003; López-Novoa and Nieto, 2009; Friedman, 2010; 2008; Dalton et al., 2009; Leung, et al., 2008; Nan et al., 2013; Hamdy and El-Demerdash, 2012;Li, Li et al., 2012; Natajaran et al., 2006; Luckey and Petersen, 2001; Chen and Raghunath, 2009;Thompson et al., 2011; Henderson and Iredale, 2007).</p>
<p> </p>
<p> </p>
<p>There is general acceptance that HSCs are collagen producing cells and key actors in fibrogenesis. The functional relationship between these KEs is consistent with biological knowledge (Benyon and Arthur; 2001; Milani et al., 1994; Safadi and Friedman, 2002; Kolios et al.,2006; Bataller and Brenner, 2005; Lee und Friedman 2011; Guo and Friedman, 2007; Li, Jing-Ting et al., 2008; Kershenobich Stalnikowitz and Weisssbrod , 2003; López-Novoa and Nieto, 2009).</p>
<p> </p>
<p>It is difficult to stimulate sufficient collagen production and its subsequent incorporation into a pericellular matrix in vitro; therefore analytical methods have focused on measurement of pro-collagen secreted into culture medium or measurement of α-smooth muscle actin (α-SMA) expression, a marker of fibroblast activation. In primary culture, HSCs from normal liver begin to express α-SMA coincident with culture-induced activation ( Chen and Raghunath, 2009; Rockey et al.,1992).<sup> </sup></p>
<p>no inconsistencies</p>
<p> </p>
<p>no quantitative data</p>
Not SpecifiedUnspecificNot SpecifiedAll life stagesHighHigh<p>Human: Safadi and Friedman, 2002; Bataller and Brenner, 2005; Lee und Friedman 2011.</p>
<p>Rat: Li, Li et al., 2012; Luckey and Petersen, 2001; Rockey et al., 1992</p>
<p> </p>
<ul>
<li>Benyon, R.C. and M.J. Arthur (2001), Extracellular matrix degradation and the role of stellate cells, Semin Liver Dis, vol. 21, no. 3, pp. 373-384.</li>
<li>Milani, S. et al. (1994), Differential expression of matrix-metalloproteinase-1 and -2 genes in normal and fibrotic human liver, Am J Pathol, vol. 144, no. 3, pp. 528-537.</li>
<li>↑Safadi, R. and S.L. Friedman (2002), Hepatic fibrosis--role of hepatic stellate cell activation, MedGenMed, vol 4, no. 3, p. 27.</li>
<li>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.</li>
<li>Bataller, R. and D.A. Brenner (2005), Liver Fibrosis, J.Clin. Invest, vol. 115, no. 2, pp. 209-218.</li>
<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>Guo, J. and S. L. Friedman (2007), Hepatic fibrogenesis, Semin Liver Dis, vol. 27, no. 4, pp. 413-426.</li>
<li>Brenner, D.A. (2009), Molecular Pathogenesis of Liver Fibrosis, Trans Am Clin Climatol Assoc, vol. 120, pp. 361–368.</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>Kershenobich Stalnikowitz, D. and A.B. Weisssbrod (2003), Liver Fibrosis and Inflammation. A Review, Annals of Hepatology, vol. 2, no. 4, pp.159-163.</li>
<li>López-Novoa, J.M. and M.A. Nieto (2009), Inflammation and EMT: an alliance towards organ fibrosis and cancer progression, EMBO Mol Med, vol. 1. no. 6-7, pp. 303–314.</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>Dalton, S.R. et al. (2009), Carbon tetrachloride-induced liver damage in asialoglycoprotein receptor-deficient mice, Biochem Pharmacol, vol. 77, no. 7, pp. 1283-1290.</li>
<li>Leung, T.M. et al. (2008), Endothelial nitric oxide synthase is a critical factor in experimental liver fibrosis, Int J Exp Pathol, vol. 89, no. 4, pp. 241-250.</li>
<li>Nan, Y.M. et al. (2013), Activation of peroxisome proliferator activated receptor alpha ameliorates ethanol mediated liver fibrosis in mice, Lipids in Health and Disease, vol. 12, p. 11.</li>
<li>Hamdy, N. and E. El-Demerdash. (2012), New therapeutic aspect for carvedilol: antifibrotic effects of carvedilol in chronic carbon tetrachloride-induced liver damage, Toxicol Appl Pharmacol, vol. 261, no. 3, pp. 292-299.</li>
<li>Li, Li et al. (2012), Establishment of a standardized liver fibrosis model with different pathological stages in rats, Gastroenterol Res Pract; vol. 2012, Article ID 560345.</li>
<li>Natajaran, S.K. et al. (2006), Oxidative stress in the development of liver cirrhosis: a comparison of two different experimental models, J Gastroenterol Hepatol, vol. 21, no. 6, pp. 947-957.</li>
<li>Luckey, S.W., and D.R. Petersen (2001), Activation of Kupffer cells during the course of carbon tetrachloride-induced liver injury and fibrosis in rats, Exp Mol Pathol, vol. 71, no. 3, pp. 226-240.</li>
<li>Chen, C. and M. Raghunath (2009), Focus on collagen: in vitro systems to study fibrogenesis and antifibrosis state of the art, Fibrogenesis Tissue Repair, vol. 15, no. 2, p. 7.</li>
<li>Thompson, K.J., I.H. McKillop and L.W. Schrum (2011), Targeting collagen expression in alcoholic liver disease, World J Gastroenterol, vol. 17, no. 20, pp. 2473-2481.</li>
<li>Henderson, N.C. and J.P. Iredale (2007), Liver fibrosis: cellular mechanisms of progression and resolution, Clin Sci (Lond), vol. 112, no. 5, pp. 265-280.</li>
<li>Rockey, D.C. et al. (1992), Rat hepatic lipocytes express smooth muscle actin upon activation in vivo and in culture, J Submicrosc Cytol Pathol, vol. 24, no. 2, pp. 193-203.</li>
</ul>
2016-11-29T18:41:332018-12-05T08:51:547f46e0c8-d5ed-4978-9904-4101d6b9d6b144b95250-171c-47e2-a791-30653ca666c9<p>Liver fibrosis is the excessive accumulation of extracellular matrix (ECM) proteins including collagen. Liver fibrosis results from an imbalance between the deposition and degradation of ECM and a change of ECM composition; the latter initiates several positive feedback pathways that further amplify fibrosis. With chronic injury, there is progressive substitution of the liver parenchyma by scar tissue. Deposition of collagen in the liver progressively disrupts the normal hepatic architecture so that the normal relationship between vascular inflow and outflow is destroyed and the normal collagen content around hepatic sinusoids in regenerating nodules becomes modified.Advanced liver fibrosis results in cirrhosis (Lee and Friedman, 2011; Bataller and Brenner, 2005; Pellicoro et al., 2014;Brancatelli et al., 2009; Rockey and Friedman, 2006; Poynard et al., 1997). </p>
<p><sup> </sup></p>
<p>By definition, liver fibrosis is the excessive accumulation of ECM proteins that are produced by HSCs. The KER between this KE and the AO is undisputed (Lee and Friedman, 2011; Bataller and Brenner, 2005;Brancatelli et al., 2009; Rockey and Friedman, 2006; Poynard et al., 1997). </p>
<p><sup> </sup></p>
<p> </p>
<p>There is a smooth transition from ECM accumulation to liver fibrosis without a definite threshold and plenty in vivo evidence exists that ECM accumulation is a pre-stage of liver fibrosis (Lee and Friedman, 2011; Bataller and Brenner, 2005;Brancatelli et al., 2009; Rockey and Friedman, 2006; Poynard et al., 1997). </p>
<p><sup> </sup></p>
<p>no inconsistencies</p>
<p><em>no quantitative data </em></p>
Not SpecifiedUnspecificNot SpecifiedAll life stagesHighHigh<p>Human: Lee and Friedman, 2011; Bataller and Brenner, 2005; Brancatelli et al., 2009; Rockey and Friedman, 2006; Poynard et al., 1997. </p>
<p>Rat :Liedtke et al., 2013.</p>
<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>Bataller, R. and D.A. Brenner (2005), Liver Fibrosis, J.Clin. Invest, vol. 115, no. 2, pp. 209-218.</li>
<li>Pellicoro, A. et al. (2014), Liver fibrosis and repair: immune regulation of wound healing in a solid organ, Nat Rev Immunol, vol. 14, no. 3, pp. 181-194.</li>
<li>Brancatelli, G. et al. (2009), Focal confluent fibrosis in cirrhotic liver: natural history studied with serial CT, AJR Am J Roentgenol, vol. 192, no. 5, pp. 1341-1347.</li>
<li>Rockey, D.C. and S.L. Friedman (2006), Hepatic fibrosis and cirrhosis, Zakim and Boyer's Hepatology, 5th edition, section 1, chapter 6, pp. 87-109.</li>
<li>Poynard, T., P. Bedossa and P. Opolon (1997), Natural history of liver fibrosis progression in patients with chronic hepatitis C. The OBSVIRC, METAVIR, CLINIVIR, and DOSVIRC groups, Lancet, vol. 349, no. 9055, pp. 825-832.</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>
2016-11-29T18:41:332018-12-05T08:52:454a8acd45-6b90-44cf-bd65-d8b3023484f638fc8b7c-df66-4ba4-98a1-e8cf17183abf<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><sup id="cite_ref-Roth_1998_1-0" class="reference"><a href="#cite_note-Roth_1998-1">[1]</a></sup>
<sup id="cite_ref-Gressner_2002_2-0" class="reference"><a href="#cite_note-Gressner_2002-2">[2]</a></sup>
<sup id="cite_ref-Malhi_2010_3-0" class="reference"><a href="#cite_note-Malhi_2010-3">[3]</a></sup>
<sup id="cite_ref-Canbay_2004_4-0" class="reference"><a href="#cite_note-Canbay_2004-4">[4]</a></sup>
<sup id="cite_ref-Orrenius_2011_5-0" class="reference"><a href="#cite_note-Orrenius_2011-5">[5]</a></sup>
<sup id="cite_ref-Kolios_2006_6-0" class="reference"><a href="#cite_note-Kolios_2006-6">[6]</a></sup>
<sup id="cite_ref-Kisseleva_2008_7-0" class="reference"><a href="#cite_note-Kisseleva_2008-7">[7]</a></sup>
<sup id="cite_ref-Li_2008_8-0" class="reference"><a href="#cite_note-Li_2008-8">[8]</a></sup>
<sup id="cite_ref-Friedman_2008_9-0" class="reference"><a href="#cite_note-Friedman_2008-9">[9]</a></sup>
<sup id="cite_ref-Lee_2011_10-0" class="reference"><a href="#cite_note-Lee_2011-10">[10]</a></sup>
</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
<sup id="cite_ref-11" class="reference"><a href="#cite_note-11">[11]</a></sup>
<sup id="cite_ref-Xie_2012_12-0" class="reference"><a href="#cite_note-Xie_2012-12">[12]</a></sup> <sup id="cite_ref-13" class="reference"><a href="#cite_note-13">[13]</a></sup> <sup id="cite_ref-14" class="reference"><a href="#cite_note-14">[14]</a></sup> <sup id="cite_ref-15" class="reference"><a href="#cite_note-15">[15]</a></sup>
</p><p>This relationship is classified as indirect as HSCs activation is partly mediated by TGF-β1 and LSECs.
</p><p>There is a functional relationship between KE 1 and KE 4 consistent with established biological knowledge.
<sup id="cite_ref-Roth_1998_1-1" class="reference"><a href="#cite_note-Roth_1998-1">[1]</a></sup>
<sup id="cite_ref-Gressner_2002_2-1" class="reference"><a href="#cite_note-Gressner_2002-2">[2]</a></sup>
<sup id="cite_ref-Malhi_2010_3-1" class="reference"><a href="#cite_note-Malhi_2010-3">[3]</a></sup>
<sup id="cite_ref-Canbay_2004_4-1" class="reference"><a href="#cite_note-Canbay_2004-4">[4]</a></sup>
<sup id="cite_ref-Orrenius_2011_5-1" class="reference"><a href="#cite_note-Orrenius_2011-5">[5]</a></sup>
<sup id="cite_ref-Kolios_2006_6-1" class="reference"><a href="#cite_note-Kolios_2006-6">[6]</a></sup>
<sup id="cite_ref-Kisseleva_2008_7-1" class="reference"><a href="#cite_note-Kisseleva_2008-7">[7]</a></sup>
<sup id="cite_ref-Li_2008_8-1" class="reference"><a href="#cite_note-Li_2008-8">[8]</a></sup>
<sup id="cite_ref-Friedman_2008_9-1" class="reference"><a href="#cite_note-Friedman_2008-9">[9]</a></sup>
<sup id="cite_ref-Lee_2011_10-1" class="reference"><a href="#cite_note-Lee_2011-10">[10]</a></sup>
</p><p><em>
</p><p></em>
</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.
<sup id="cite_ref-16" class="reference"><a href="#cite_note-16">[16]</a></sup>
<sup id="cite_ref-17" class="reference"><a href="#cite_note-17">[17]</a></sup>
<sup id="cite_ref-Coulouarn_2012_18-0" class="reference"><a href="#cite_note-Coulouarn_2012-18">[18]</a></sup>
</p><p>There are no inconsistencies
</p><p><em>
</p><p></em>
</p><p>There are no quantitative data
</p>HighHighHigh<p>Human: <sup id="cite_ref-Kisseleva_2008_7-2" class="reference"><a href="#cite_note-Kisseleva_2008-7">[7]</a></sup><sup id="cite_ref-Coulouarn_2012_18-1" class="reference"><a href="#cite_note-Coulouarn_2012-18">[18]</a></sup>
Rat: <sup id="cite_ref-Xie_2012_12-1" class="reference"><a href="#cite_note-Xie_2012-12">[12]</a></sup>
Mouse: <sup id="cite_ref-Canbay_2004_4-2" class="reference"><a href="#cite_note-Canbay_2004-4">[4]</a></sup>
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<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>
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<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>
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<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>
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<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>2016-11-29T18:41:332016-11-29T19:54:57Protein Alkylation leading to Liver FibrosisProtein Alkylation to Liver Fibrosis<p>Brigitte Landesmann</p>
<p>F3 Chemical Safety and Alternative Methods Unit incorporating EURL ECVAM</p>
<p>Directorate F – Health, Consumers and Reference Materials</p>
<p>Joint Research Centre, European Commission</p>
<p>brigittelandesmann (at) gmail.com</p>
Open for citation & commentWPHA/WNT EndorsedIncluded in OECD Work Plan1.14<p>Hepatotoxicity in general is of special interest for human health risk assessment. Liver fibrosis in particular is an important human health issue associated with chemical exposure and predictive assays are lacking; it is a typical result of chronic or repeated-dose toxic injury and one of the considered endpoints for regulatory purposes. It is a long-term process in which inflammation, tissue destruction, and repair occur simultaneously, together with sustained production of growth factors and fibrogenic cytokines due to a complex interplay between various hepatic cell types, various receptors and signalling pathways which lead to an imbalance between the deposition and degradation of extracellular matrix (ECM) and a change of ECM composition. Due to this complex situation an adequate cell model is not available and an <em>in vitro</em> evaluation of fibrogenic potential is therefore not feasible. A sufficiently detailed description of the AOP to liver fibrosis might support chemical risk assessment by indicating early (upstream) markers for downstream events and facilitate a testing strategy without the need for a sophisticated cell model. This systematic and coherent display of currently available mechanistic-toxicological information can serve as a knowledge-based repository for identification/selection/development of <em>in vitro</em> methods suitable for measuring key events (KEs) and their relationships along the AOP and to facilitate the use of alternative data for regulatory purposes. Identified uncertainties and knowledge gaps can direct future research by priority setting and targeted testing. The KE descriptions can be used for hazard identification and read-across to assess the toxic potential of an untested substance.</p>
<p>This AOP describes the linkage between hepatic injury caused by protein alkylation and the formation of liver fibrosis. The molecular initiating event (MIE) is protein alkylation, leading to structural and functional cell injury and further to cell death, the first KE. Apoptotic hepatocytes undergo genomic DNA fragmentation and formation of apoptotic bodies. Upon engulfment of apoptotic bodies Kupffer cells (KCs) are activated, the next KE along the pathway. Activated KCs are the main source of TGF-β1, the most potent profibrogenic cytokine. TGF-β1 expression therefore is considered a KE that causes the next KE, hepatic stellate cell (HSCs) activation, meaning the transdifferentiation from a quiescent vitamin A–storing cell to a proliferative and contractile myofibroblast, the central effector in hepatic fibrosis. Activated HSCs cause progressive collagen accumulation, which together with changes in ECM composition signifies the KE on tissue level. The excessive accumulation of extracellular matrix proteins progressively affects the whole organ and alters its normal functioning, which corresponds to liver fibrosis, the adverse outcome (AO).</p>
<p>There are two further events that play an important role in driving fibrogenesis, namely oxidative stress and chronic inflammation. Both are on-going processes being present throughout the pathway and interconnected with most of the KEs. Hence, they are not classified as KEs themselves and described in the individual KE and key event relationship (KER) descriptions. The inflammatory response plays an important role in driving fibrogenesis, since persistent inflammation precedes fibrosis. Inflammatory signalling stems from injured hepatocytes, activated KCs and HSCs. Inflammatory and fibrogenic cells stimulate each other in amplifying fibrosis. Chemokines and their receptors provoke further fibrogenesis, as well as interacting with inflammatory cells to modify the immune response during injury. Oxidative stress, as well, plays a crucial role in liver fibrogenesis by inducing hepatocyte apoptosis, activation of KCs and HSCs and fuelling inflammation. ROS contributing to oxidative stress are generated by hepatocytes, KCs, HSCs and inflammatory cells.</p>
<p>This purely qualitative AOP description is plausible, the scientific data supporting the AOP are logic, coherent and consistent and there is temporal agreement between the individual KEs. Quantitative data on dose-response-relationships and temporal sequences between KEs are still lacking; the provision of quantitative data will further strengthen the weight of evidence and make the AOP applicable for a wide range of purposes.</p>
<p>Two prototypical chemicals acting via protein alkylation are Allyl Alcohol <sup id="cite_ref-12" class="reference"><a href="#cite_note-12">[12]</a></sup><sup id="cite_ref-13" class="reference"><a href="#cite_note-13">[13]</a></sup><sup id="cite_ref-Kehrer2000_6-1" class="reference"><a href="#cite_note-Kehrer2000-6">[6]</a></sup><sup id="cite_ref-14" class="reference"><a href="#cite_note-14">[14]</a></sup><sup id="cite_ref-15" class="reference"><a href="#cite_note-15">[15]</a></sup>
and Carbon Tetrachloride (CCl4)<sup id="cite_ref-EPA_2010_11-1" class="reference"><a href="#cite_note-EPA_2010-11">[11]</a></sup><sup id="cite_ref-16" class="reference"><a href="#cite_note-16">[16]</a></sup> <sup id="cite_ref-17" class="reference"><a href="#cite_note-17">[17]</a></sup> <sup id="cite_ref-18" class="reference"><a href="#cite_note-18">[18]</a></sup>
<sup id="cite_ref-19" class="reference"><a href="#cite_note-19">[19]</a></sup><sup id="cite_ref-20" class="reference"><a href="#cite_note-20">[20]</a></sup><sup id="cite_ref-21" class="reference"><a href="#cite_note-21">[21]</a></sup><sup id="cite_ref-22" class="reference"><a href="#cite_note-22">[22]</a></sup> . <sup id="cite_ref-23" class="reference"><a href="#cite_note-23">[23]</a></sup>
<sup id="cite_ref-24" class="reference"><a href="#cite_note-24">[24]</a></sup> .<sup id="cite_ref-25" class="reference"><a href="#cite_note-25">[25]</a></sup>
<sup id="cite_ref-26" class="reference"><a href="#cite_note-26">[26]</a></sup>
</p><p>Covalent protein alkylation is a feature of many cytotoxic drugs but the overall extent of binding does not adequately distinguish toxic from non-toxic binding. <sup id="cite_ref-27" class="reference"><a href="#cite_note-27">[27]</a></sup> Interestingly, some chemicals significantly alkylate proteins without causing toxicity, which suggests that only alkylation of a specific protein subset critical subset contributes to injury. Indeed, Codreanu presented an inventory of proteins affected by electrophile-mediated alkylation in intact cells and suggested that non-toxic covalent binding largely affects cytoskeletal protein components, whereas toxic covalent binding induces lethal injury by targeting factors involved in protein synthesis and catabolism and possibly mitochondrial electron transport. <sup id="cite_ref-Codreanu_2014_3-2" class="reference"><a href="#cite_note-Codreanu_2014-3">[3]</a></sup>
In vitro covalent binding studies to macromolecules have been used to elucidate the biochemical mechanisms of chemical-induced toxicity. Experimental work with kidney epithelial cells by Chen et al suggested that following alkylation of cellular macromolecules as initial cytotoxic event both sulfhydryl depletion and lipid peroxidation are components of the cytotoxic mechanism <sup id="cite_ref-28" class="reference"><a href="#cite_note-28">[28]</a></sup> Dennehy et al have analyzed the protein targets in nuclear and cytoplasmic proteomes from human embryonic kidney cells (HEK293) treated in vitro with two biotin-tagged, thiol-reactive electrophiles and mapped the adducts. Certain protein families appeared particularly susceptible to alkylation. <sup id="cite_ref-29" class="reference"><a href="#cite_note-29">[29]</a></sup> Shin et al have identified protein targets of two biotin-tagged model electrophiles in human liver microsomes through LC-MS-MS and showed that different target selectivities of the two electrophile probes correlated with different biological outcomes and that alkylation reactions of specific targets could be quantified. <sup id="cite_ref-30" class="reference"><a href="#cite_note-30">[30]</a></sup>
</p><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>
adjacentNot SpecifiedModerateadjacentNot SpecifiedHighadjacentNot SpecifiedHighadjacentNot SpecifiedHighadjacentNot SpecifiedHighadjacentNot SpecifiedHighnon-adjacentNot SpecifiedHigh<p>The essentiality of each of the KEs for this AOP was rated high as there is much experimental evidence that the blocking of one KE prevents (or attenuates where complete blocking is not possible) the next downstream KE and therefore the whole AOP. Much evidence arises from preclinical research for antifibrotic agents, which is mainly based on the interference with or blockade of a key event. For details see the table above</p>
Not SpecifiedUnspecificNot SpecifiedNot Otherwise SpecifiedHighHigh<p> </p>
<p> </p>
<h3><strong>Assessment of the Weight-of-Evidence supporting the AOP</strong></h3>
<h4><strong>Concordance of dose-response relationships</strong></h4>
<p>This is a qualitative description of the pathway; the currently available literature does not provide quantitative information on dose-response relationships. But there is empirical evidence to support that a change in KEup leads to an appropriate change in the respective KEdown.</p>
<h4><strong>Temporal concordance among the key events and adverse outcome</strong></h4>
<p>Empirical evidence shows temporal concordance between the individual KEs leading to the AO.</p>
<h4><strong>Strength, consistency, and specificity of association of adverse outcome and initiating event</strong></h4>
<p>The scientific evidence on the linkage between MIE and AO has been described. The ample literature is consistent in describing this association between AO and MIE</p>
<h4><strong>Biological plausibility, coherence, and consistency of the experimental evidence</strong></h4>
<p>The available data supporting the AOP are logic, coherent and consistent with established biological knowledge.</p>
<h4><strong>Alternative mechanism(s) that logically present themselves and the extent to which they may distract from the postulated AOP</strong></h4>
<p>There are some other important fibrogenic signalling pathways that influence HSC activation and fibrogenesis without constituting another AOP:</p>
<h5><strong>Adipokine pathways</strong></h5>
<p>Adipokines are secreted mainly by adipose tissue, but also by resident and infiltrating macrophages and are increasingly recognised as mediators of fibrogenesis.</p>
<p>Leptin promotes HSC fibrogenesis and enhances TIMP-1 expression and further acts as a pro-fibrotic through suppression of peroxisome proliferator-activated receptor-gamma (PPARg), an anti-fibrogenic nuclear receptor that can reverse HSC activation. The expression of leptin receptor is up-regulated during HSC activation and leptin activity is therefore increased through enhanced signaling. Downstream effects include increased release of TGF-b1 from KCs. The counter-regulatory hormone adiponectin is reduced in hepatic fibrosis. <sup><a href="#cite_note-Lee_and_Friedman_2011-1">[1]</a></sup><sup><a href="#cite_note-Friedmann2010-2">[2]</a></sup></p>
<h5><strong>Neuroendocrine pathways</strong></h5>
<p>The fibrogenic function of HSCs is also influenced by neurochemical and neurotrophic factors. Upon chronic liver injury, the local neuroendocrine system is up-regulated, and activated HSCs express specific receptors, most prominently those regulating cannabinoid signaling. Activated HSCs are additionally a key source of the endogenous cannabinoid,2-Arachidonylglycerol (2-AG), which drives increased (cannabinoid-receptor) CB 1 signalling. Stimulation of the CB1 receptor is profibrogenic, whereas the CB2 receptor is anti-fibrotic and hepatoprotective. Opioid signaling increases proliferation and collagen production in HSCs. Serotonin has a pro-fibrotic effect that synergizes with PDGF signaling. Also thyroid hormones enhance activation of HSCs (through increased p75 neurotrophin receptor (p75NTR) and activation of Rho), thereby accelerating the development of liver fibrosis. <sup><a href="#cite_note-Friedman_2008-3">[3]</a></sup><sup><a href="#cite_note-Friedmann2010-2">[2]</a></sup><sup><a href="#cite_note-Lee_and_Friedman_2011-1">[1]</a></sup></p>
<h5><strong>Renin–angiotensin pathway</strong></h5>
<p>Angiotensin II (Ang II) is a pro-oxidant and fibrogenic cytokine that stimulates DNA synthesis, cell migration, procollagen α1(I) mRNA expression, and secretion of TGF-β1 and inflammatory cytokines. These fibrogenic actions are mediated by NOX. <sup><a href="#cite_note-Kisseleva_and_Brenner_2008-4">[4]</a></sup> <sup><a href="#cite_note-Friedmann2010-2">[2]</a></sup> <sup><a href="#cite_note-Lee_and_Friedman_2011-1">[1]</a></sup> <sup><a href="#cite_note-5">[5]</a></sup></p>
<h4><strong>Uncertainties, inconsistencies and data gaps</strong></h4>
<p>Ths AOP description is plausible, though purely qualitative; the addition of quantitative data on dose response-relationships and temporal sequences is needed and would substantially improve its applicability.</p>
<p>Protein alkylation is a broad, non-specific MIE. Covalent protein alkylation is a feature of many hepatotoxic drugs but the overall extent of binding does not adequately distinguish toxic from non-toxic binding. For this AOP it is unclear whether protein alkylation per se is sufficient to start the pathway or whether alkylation to specific proteins or families of proteins needs to be affected and whether various binding sites influence the further downstream process. The identification and specification of the targeted biomolecules is needed for the structural definition of chemical initiators and consecutively for profiling and categorising of chemicals related to the initiation of this AOP. Likewise it is necessary for the establishment of a distinct relationship with the next downstream event. Further it is unknown whether there is a threshold and if this threshold would refer to the number of alkylation of a single protein or of a threshold number of proteins. Future studies could provide a better mechanistic basis for interpreting protein alkylation in chemical safety evaluation.</p>
<p>By definition, an AOP has only one MIE and one final AO, the two anchor points of the AOP that have to be clearly defined. Any other MIE that leads to cell injury and further to liver fibrosis via the same downstream KEs would constitute another AOP. There are various types of liver injury that are caused by different agents, initiated by various MIEs and finally lead to fibrosis via the same described pathway; therefore, the question arises whether hepatocyte injury itself, independently from the cause of injury, might be the initiating event for this pathway to fibrosis. Obviously hepatocyte injury does not inevitably lead to fibrosis in all cases and there is a wide range of hepatotoxic chemicals (like Acetaminophen, Aflatoxin or Chlorpromazine) for which liver fibrosis cannot be observed. Apoptosis, necrosis, transdifferentiation/transition and repair/regeneration, all these might occur in response to cellular stressors and the difference in progression to liver fibrosis might lie in these various cellular responses. There is increasing evidence for apoptosis being the main fibrogenic trigger. Yet, both necrosis and apoptosis are often present simultaneously and necrosis may only represent the more severe cellular response to stronger damaging stimuli. It also might well be that hepatocyte insult/injury, rather than death is sufficient to trigger fibrosis and the key question would then be whether there are fibrosis-specific features of cell injury. It could be rather the amount (quantitative difference) than the kind (qualitative difference) of cell injury that matters. The rate of cell injury/death, i.e. the amount of injury within a certain time frame could be another plausible initiating parameter, as fibrosis is resulting from chronic injury. Assuming hepatocyte injury being the crucial KE without which fibrosis could not occur via this AOP, then simple investigation of <em>in vitro</em> hepatotoxicity could provide relevant information for potential fibrosis prediction without the need of highly elaborated cell models.</p>
<p>The initial AOP case study was based on data of two prototypic fibrogenic chemicals, namely Carbon Tetrachloride (CCl4)<sup><a href="#cite_note-6">[6]</a></sup><sup><a href="#cite_note-7">[7]</a></sup><sup><a href="#cite_note-8">[8]</a></sup><sup><a href="#cite_note-9">[9]</a></sup><sup><a href="#cite_note-10">[10]</a></sup><sup><a href="#cite_note-11">[11]</a></sup><sup><a href="#cite_note-12">[12]</a></sup><sup><a href="#cite_note-13">[13]</a></sup><sup><a href="#cite_note-14">[14]</a></sup><sup><a href="#cite_note-15">[15]</a></sup><sup><a href="#cite_note-16">[16]</a></sup><sup><a href="#cite_note-17">[17]</a></sup> <sup><a href="#cite_note-18">[18]</a></sup><sup><a href="#cite_note-19">[19]</a></sup> <sup><a href="#cite_note-20">[20]</a></sup><sup><a href="#cite_note-21">[21]</a></sup><sup><a href="#cite_note-22">[22]</a></sup><sup><a href="#cite_note-luckey_2001-23">[23]</a></sup><sup><a href="#cite_note-24">[24]</a></sup><sup><a href="#cite_note-25">[25]</a></sup><sup><a href="#cite_note-26">[26]</a></sup><sup><a href="#cite_note-27">[27]</a></sup><sup><a href="#cite_note-Manibusan_2007-28">[28]</a></sup><sup><a href="#cite_note-29">[29]</a></sup><sup><a href="#cite_note-30">[30]</a></sup><sup><a href="#cite_note-Nan_2013-31">[31]</a></sup><sup><a href="#cite_note-Natajaran2006-32">[32]</a></sup><sup><a href="#cite_note-33">[33]</a></sup><sup><a href="#cite_note-34">[34]</a></sup><sup><a href="#cite_note-35">[35]</a></sup><sup><a href="#cite_note-36">[36]</a></sup><sup><a href="#cite_note-37">[37]</a></sup><sup><a href="#cite_note-38">[38]</a></sup><sup><a href="#cite_note-39">[39]</a></sup> <sup><a href="#cite_note-40">[40]</a></sup><sup><a href="#cite_note-41">[41]</a></sup><sup><a href="#cite_note-42">[42]</a></sup> and Allyl Alcohol <sup><a href="#cite_note-43">[43]</a></sup><sup><a href="#cite_note-44">[44]</a></sup><sup><a href="#cite_note-45">[45]</a></sup><sup><a href="#cite_note-Kehrer2000-46">[46]</a></sup><sup><a href="#cite_note-47">[47]</a></sup><sup><a href="#cite_note-48">[48]</a></sup>. Further knowledge was gathered by looking for mechanistic data of other chemicals which are known inducers of liver fibrosis, namely Thioacetamide <sup><a href="#cite_note-49">[49]</a></sup><sup><a href="#cite_note-50">[50]</a></sup><sup><a href="#cite_note-51">[51]</a></sup><sup><a href="#cite_note-52">[52]</a></sup><sup><a href="#cite_note-53">[53]</a></sup><sup><a href="#cite_note-54">[54]</a></sup><sup><a href="#cite_note-Ide_2005-55">[55]</a></sup><sup><a href="#cite_note-56">[56]</a></sup><sup><a href="#cite_note-57">[57]</a></sup><sup><a href="#cite_note-58">[58]</a></sup><sup><a href="#cite_note-59">[59]</a></sup><sup><a href="#cite_note-60">[60]</a></sup> <sup><a href="#cite_note-Natajaran2006-32">[32]</a></sup><sup><a href="#cite_note-61">[61]</a></sup><sup><a href="#cite_note-62">[62]</a></sup><sup><a href="#cite_note-63">[63]</a></sup><sup><a href="#cite_note-64">[64]</a></sup><sup><a href="#cite_note-65">[65]</a></sup>, Amiodarone <sup><a href="#cite_note-66">[66]</a></sup><sup><a href="#cite_note-67">[67]</a></sup><sup><a href="#cite_note-68">[68]</a></sup> <sup><a href="#cite_note-69">[69]</a></sup><sup><a href="#cite_note-70">[70]</a></sup><sup><a href="#cite_note-71">[71]</a></sup><sup><a href="#cite_note-72">[72]</a></sup>, Methotrexate <sup><a href="#cite_note-73">[73]</a></sup><sup><a href="#cite_note-74">[74]</a></sup><sup><a href="#cite_note-75">[75]</a></sup><sup><a href="#cite_note-76">[76]</a></sup><sup><a href="#cite_note-77">[77]</a></sup><sup><a href="#cite_note-78">[78]</a></sup><sup><a href="#cite_note-79">[79]</a></sup>, Isoniazid <sup><a href="#cite_note-80">[80]</a></sup><sup><a href="#cite_note-81">[81]</a></sup><sup><a href="#cite_note-82">[82]</a></sup><sup><a href="#cite_note-83">[83]</a></sup>, Dimethyl Nitrosamine <sup><a href="#cite_note-84">[84]</a></sup><sup><a href="#cite_note-85">[85]</a></sup><sup><a href="#cite_note-86">[86]</a></sup><sup><a href="#cite_note-87">[87]</a></sup>, Ethanol <sup><a href="#cite_note-88">[88]</a></sup><sup><a href="#cite_note-89">[89]</a></sup><sup><a href="#cite_note-Nan_2013-31">[31]</a></sup><sup><a href="#cite_note-90">[90]</a></sup><sup><a href="#cite_note-91">[91]</a></sup><sup><a href="#cite_note-92">[92]</a></sup><sup><a href="#cite_note-93">[93]</a></sup>, Retinol<sup><a href="#cite_note-94">[94]</a></sup><sup><a href="#cite_note-95">[95]</a></sup><sup><a href="#cite_note-96">[96]</a></sup>, Ethinyl Estradiol <sup><a href="#cite_note-97">[97]</a></sup><sup><a href="#cite_note-98">[98]</a></sup><sup><a href="#cite_note-99">[99]</a></sup>, and Chlopromazine <sup><a href="#cite_note-100">[100]</a></sup><sup><a href="#cite_note-101">[101]</a></sup> <sup><a href="#cite_note-102">[102]</a></sup>. Mechanistic data related to these additional chemicals are rather scarce, because classical <em>in vivo</em> studies were mainly looking at the AO than at intermediate (key) events and <em>in vitro</em> studies investigating liver fibrosis tend to use always the same reference chemicals. The overall gathered information was summarised in a data matrix that displays how many (if any) individual studies have observed the same findings at the MIE, KE and AO levels. Blue boxes refer to the KEs described in this AOP to liver fibrosis and green boxes indicate the observation of the event (the number within the box showing how many individual publications reported this specific event). It must be noted that these studies have not intended to investigate KEs on various levels of biological information; therefore, absence of a KE description does not necessarily mean that this KE did not occur, but rather that it has not been investigated or described. This matrix shows that protein binding was indicated as MIE only for three more chemicals Thioacetamide, Retinol and Dimethylnitrosamine) and therefore only these were added to the list of chemical initiators of this AOP. This matrix also demonstrates that hepatocyte injury/death is an early convergent KE that is valid for all described fibrogenic chemicals.</p>
<p><a class="image" href="/wiki/index.php/File:Mechanistic_data_matrix.jpg"><img alt="Mechanistic data matrix.jpg" src="/wiki/images/thumb/9/99/Mechanistic_data_matrix.jpg/600px-Mechanistic_data_matrix.jpg" style="height:450px; width:600px" /></a></p>
<h3><strong>Assessment of the quantitative understanding of the AOP</strong></h3>
<p>See above</p>
<p>The described AOP is valid for both sexes and any life stage. This pathway description is also based on studies of formation and progression of fibrosis in human patients. Findings suggest common conserved pathways across different species which initiate and promote liver fibrosis. Animal models are used to study fibrogenesis and CCl4 intoxication in rats and mice is probably the most widely studied and therefore best characterised model with respect to histological, biochemical, cell, and molecular changes associated with the development of fibrosis</p>
<p>The essentiality of each of the KEs for this AOP was rated high as there is much experimental evidence that the blocking of one KE prevents (or attenuates where complete blocking is not possible) the next downstream KE and therefore the whole AOP. Much evidence arises from preclinical research for antifibrotic agents, which is mainly based on the interference with or blockade of a key event. For details see the table above</p>
<table border="1" style="border-collapse:collapse">
<tbody>
<tr>
<td colspan="3">
<p><strong>Support for Essentiality of KEs</strong></p>
<p> </p>
</td>
</tr>
<tr>
<td>
<p>MIE</p>
<p>Protein Alkylation</p>
</td>
<td>
<p>Alkylating agents are highly reactive chemicals that introduce alkyl groups into biologically active molecules and thereby prevent their proper functioning.</p>
</td>
<td>
<p>Essentiality of the MIE is high.</p>
<p>Covalent protein alkylation by reactive electrophiles was identified as a key triggering event in chemical toxicity over 40 years ago. These reactions remain a major cause of chemical-induced toxicity. <sup><a href="#cite_note-Codreanu_2014-103">[103]</a></sup><sup><a href="#cite_note-Liebler_2008-104">[104]</a></sup></p>
</td>
</tr>
<tr>
<td>
<p>KE 1</p>
<p>cell injury/death</p>
</td>
<td>
<p>Covalent binding to liver proteins and oxidative stress can directly affect cells or influence signalling pathways, finally leading to necrotic or apoptotic cell death.</p>
</td>
<td>
<p>Essentiality of KE 1 is high.</p>
<p>Up-regulated apoptosis of hepatocytes is increasingly viewed as a nexus between liver injury and fibrosis. Pharmacological inhibition of liver cell apoptosis attenuates liver injury and fibrosis, suggesting a critical role for hepatocyte apoptosis in the initiation of HSC activation and hepatic fibrogenesis. <sup><a href="#cite_note-Malhi_2010-105">[105]</a></sup><sup><a href="#cite_note-Canbay_2004apoptosis-106">[106]</a></sup> <sup><a href="#cite_note-Orrenius_2011-107">[107]</a></sup><sup><a href="#cite_note-108">[108]</a></sup><sup><a href="#cite_note-Canbay_2002-109">[109]</a></sup><sup><a href="#cite_note-Canbay_2003-110">[110]</a></sup><sup><a href="#cite_note-111">[111]</a></sup><sup><a href="#cite_note-112">[112]</a></sup><sup><a href="#cite_note-Canbay_2004caspaseinhibitor-113">[113]</a></sup></p>
</td>
</tr>
<tr>
<td>
<p>KE 2</p>
<p>Kupffer cell (KC) activation and macrophage recruitment</p>
</td>
<td>
<p>Activated KCs are a major source of inflammatory mediators, including cytokines, chemokines, lysosomal and proteolytic enzymes and a main source of TGF-β, as well as a major source of reactive oxygen species (ROS).</p>
</td>
<td>
<p>Essentiality of KE 2 is high.</p>
<p>Probably there is a threshold of KC activation and release above which liver damage is induced. Pre-treatment with gadolinium chloride (GdCl), which inhibits KC function, reduced both hepatocyte and sinusoidal epithelial cell injury, as well as decreased the numbers of macrophages appearing in hepatic lesions and inhibited TGF-b1 mRNA expression in macrophages. Experimental inhibition of KC function or depletion of KCs appeared to protect against liver injury from the alkylating agent melphalan, the chemical thioacetamide and the immunostimulants concanavalin A and Pseudomonas exotoxin. <sup><a href="#cite_note-Kolios_2006-114">[114]</a></sup><sup><a href="#cite_note-Kisseleva_and_Brenner_2008-4">[4]</a></sup><sup><a href="#cite_note-Roberts_2007-115">[115]</a></sup><sup><a href="#cite_note-Ide_2005-55">[55]</a></sup><sup><a href="#cite_note-Canbay_2004caspaseinhibitor-113">[113]</a></sup><sup><a href="#cite_note-116">[116]</a></sup><sup><a href="#cite_note-117">[117]</a></sup><sup><a href="#cite_note-118">[118]</a></sup></p>
</td>
</tr>
<tr>
<td>
<p>KE 3</p>
<p>TGF-β1 expression</p>
</td>
<td>
<p>TGF-β1 is the most potent profibrogenic cytokine and plays a central role in fibrogenesis, mediating a cross-talk between parenchymal, inflammatory and collagen expressing cells.</p>
</td>
<td>
<p>Essentiality of KE 3 is high.</p>
<p>TGF-β1 is considered the most potent pro-fibrogenic cytokine and several reviews assign this cytokine a central role in fibrogenesis, especially in HSC activation. Strategies aimed at disrupting TGF-β1 expression or signalling pathways are extensively being investigated because blocking this cytokine may not only inhibit matrix production, but also accelerate its degradation. Animal experiments using different strategies to block TGF-β1 have demonstrated significant anti-fibrotic effect for liver fibrosis. Experimental fibrosis can be inhibited by anti-TGF-β treatments with neutralizing antibodies or soluble TbRs (TGF-β receptors). <sup><a href="#cite_note-Liu_2006-119">[119]</a></sup><sup><a href="#cite_note-Gressner_2002-120">[120]</a></sup><sup><a href="#cite_note-121">[121]</a></sup><sup><a href="#cite_note-122">[122]</a></sup><sup><a href="#cite_note-Qi_1999-123">[123]</a></sup></p>
</td>
</tr>
<tr>
<td>
<p>KE 4</p>
<p>hepatic stellate cell (HSC) activation</p>
</td>
<td>
<p>HSC activation (in response to TGF-β1) means a transdifferentiation from a quiescent vitamin A–storing cell to a proliferative and contractile myofibroblast and is the dominant event in liver fibrosis. Activated HSCs (myofibroblasts) are the primary collagen producing cells, the key cellular mediators of fibrosis and a nexus for converging inflammatory pathways leading to fibrosis.</p>
</td>
<td>
<p>Essentiality of KE 4 is high.</p>
<p>Experimental inhibition of HSC activation prevents fibrosis. <sup><a href="#cite_note-Kisseleva_and_Brenner_2008-4">[4]</a></sup><sup><a href="#cite_note-Friedman_2002-124">[124]</a></sup><sup><a href="#cite_note-125">[125]</a></sup><sup><a href="#cite_note-126">[126]</a></sup><sup><a href="#cite_note-127">[127]</a></sup><sup><a href="#cite_note-128">[128]</a></sup></p>
</td>
</tr>
<tr>
<td>
<p>KE 5</p>
<p>collagen accumulation</p>
</td>
<td>
<p>Excess ECM (extracellular matrix) deposition and changes in ECM composition.</p>
</td>
<td>
<p>Essentiality of KE 5 is high.</p>
<p>Continuing imbalance between the deposition and degradation of ECM is a pre-requisite of liver fibrosis; therefore this KE is essential for the AO. <sup><a href="#cite_note-Lee_and_Friedman_2011-1">[1]</a></sup></p>
</td>
</tr>
<tr>
<td>
<p>Adverse Outcome</p>
<p>liver fibrosis</p>
</td>
<td>
<p>Excessive deposition of ECM proteins occurs as a result of repeated cycles of hepatocytes injury and repair and results in liver fibrosis.</p>
</td>
<td>
<p>It is generally accepted that any chronic form of liver damage, including any chemical causing sub-massive hepatocellular injury, can result in myofibroblast activation, leading to hepatic fibrosis and cirrhosis in humans <sup><a href="#cite_note-129">[129]</a></sup><sup><a href="#cite_note-130">[130]</a></sup><sup><a href="#cite_note-131">[131]</a></sup><sup><a href="#cite_note-132">[132]</a></sup><sup><a href="#cite_note-133">[133]</a></sup></p>
</td>
</tr>
<tr>
<td>
<p> </p>
</td>
<td>
<p>There are two further events that play an important role in driving fibrogenesis, namely chronic inflammation and oxidative stress. Both are on-going processes being present throughout the pathway and interconnected with most of the KEs. Therefore they are not classified as KEs themselves and described in the individual KE and KER descriptions. Nevertheless a short overview is given below.</p>
</td>
<td>
<p> </p>
</td>
</tr>
<tr>
<td>
<p>Associated Event</p>
<p>chronic inflammation</p>
</td>
<td>
<p>Hepatic fibrosis is commonly preceded by chronic inflammation and persistent inflammation has been associated with progressive hepatic fibrosis. Hepatic inflammation is a driver of hepatic fibrosis as the whole fibrinogenic cascade is initiated and maintained by inflammatory mediators and inflammatory and fibrogenic cells stimulate each other in amplifying fibrosis. Damaged hepatocytes release inflammatory cytokines that activate KCs and stimulate the recruitment of inflammatory cells, which produce profibrotic cytokines and chemokines that further activate fibroblastic cells. Activated HSCs secrete various cytokines (like macrophage colony-stimulating factor (M-CSF), MCP-1 and IL-6) and inflammatory chemokines, they interact directly with immune cells through expression of adhesion molecules (mediated by TNF-α and facilitating the recruitment of inflammatory cells), and they modulate the immune system through antigen presentation. Signaling of HSCs in response to either lipopolysaccharides (LPS) or endogenous TLR4 ligands down-regulates the protein activin membrane-bound inhibitor (BAMBI), a transmembrane suppressor of TGF-β1. Other inflammatory cells regulating progression and resolution of fibrosis include T-cells, dendritic cells, liver sinusoidal endothelial cells (LSECs) and natural killer cells (NKs), which exert an anti-fibrotic activity by inducing HSC apoptosis through production of IFN γ. Chronic inflammatory response is often accompanied simultaneously by tissue destruction and repair. Activated inflammatory cells represent a major source of oxidative stress-related molecules.</p>
<p> </p>
</td>
<td>
<p>Essentiality of inflammation is high.</p>
<p>Suppression of inflammatory activity by eliminating the etiological agent (e.g. a virus) or dampening the immune response (lymphocytic proliferation and infiltration) can halt and even reverse the fibrotic process. <sup><a href="#cite_note-Stalnikowitz_2003-134">[134]</a></sup><sup><a href="#cite_note-135">[135]</a></sup><sup><a href="#cite_note-136">[136]</a></sup><sup><a href="#cite_note-Sivakumar_and_Das_2008-137">[137]</a></sup><sup><a href="#cite_note-138">[138]</a></sup><sup><a href="#cite_note-Lee_and_Friedman_2011-1">[1]</a></sup><sup><a href="#cite_note-Parola_and_Robino_2001-139">[139]</a></sup><sup><a href="#cite_note-Bataller_2005-140">[140]</a></sup><sup><a href="#cite_note-141">[141]</a></sup></p>
<p> </p>
</td>
</tr>
<tr>
<td>
<p>Associated Event</p>
<p>oxidative stress</p>
</td>
<td>
<p>Oxidative stress corresponds to an imbalance between the rate of oxidant production and that of degradation and plays a crucial role in liver fibrogenesis by inducing hepatocyte apoptosis and activation of KCs and HSCs. Oxidative stress-related molecules act as mediators to modulate tissue and cellular events responsible for the progression of liver fibrosis. ROS, including superoxide, hydrogen peroxide, hydroxyl radicals and aldehydic end products, may be derived from hepatocytes (generated through cytochrome P450, lipid peroxidation), as well as from activated KCs, other inflammatory cells and HSCs (by NOX). Excessive levels of ROS can lead to hepatocellular injury and death. Under conditions of oxidative stress macrophages are activated, which leads to a more enhanced inflammatory response. Oxidative stress can activate a variety of transcription factors like NF-κB, PPAR-γ which may further lead to increased gene expression for the production of growth factors, inflammatory cytokines and chemokines.</p>
</td>
<td>
<p>Essentiality of oxidative stress is moderate.</p>
<p>Oxidative stress-related molecules act as mediators to modulate tissue and cellular events responsible for the progression of liver fibrosis. Hence ROS likely contribute to both onset and progression of fibrosis, being simultaneously cause and consequence of the observed condition. <sup><a href="#cite_note-Parsons_2007-142">[142]</a></sup><sup><a href="#cite_note-Poli_2000-143">[143]</a></sup> <sup><a href="#cite_note-Parola_and_Robino_2001-139">[139]</a></sup><sup><a href="#cite_note-144">[144]</a></sup><sup><a href="#cite_note-145">[145]</a></sup><sup><a href="#cite_note-146">[146]</a></sup><sup><a href="#cite_note-Kisseleva_and_Brenner_2007-147">[147]</a></sup><sup><a href="#cite_note-Kirkham_2007-148">[148]</a></sup></p>
</td>
</tr>
<tr>
<td colspan="3">
<p><strong>Support for Biological Plausibility of KERs</strong></p>
</td>
</tr>
<tr>
<td>
<p>MIE => KE 1</p>
</td>
<td>
<p>Hepatocytes are damaged by alkylating agents via both covalent binding to liver proteins and lipid peroxidation accompanied by oxidative stress and collapse of mitochondrial membrane potential, which triggers apoptotic cell death.</p>
</td>
<td>
<p>Biological Plausibility of the MIE => KE1 is high.</p>
<p>There is a mechanistic relationship between MIE and KE 1 consistent with established biological knowledge. <sup><a href="#cite_note-Codreanu_2014-103">[103]</a></sup><sup><a href="#cite_note-Kehrer2000-46">[46]</a></sup><sup><a href="#cite_note-149">[149]</a></sup><sup><a href="#cite_note-Manibusan_2007-28">[28]</a></sup></p>
</td>
</tr>
<tr>
<td>
<p>KE 1 => KE 2</p>
</td>
<td>
<p>Damaged hepatocytes release ROS, cytokines and chemokines which lead to oxidative stress, inflammatory signalling and activation of KCs. Apoptotic hepatocytes undergo genomic DNA fragmentation and formation of apoptotic bodies. Upon engulfment of apoptotic bodies KCs are activated. Liver cells trigger a sterile inflammatory response with activation of innate immune cells through release of damage-associated molecular patterns (DAMPs). Through toll-like receptors KCs are additionally activated.</p>
</td>
<td>
<p>Biological Plausibility of KE1 => KE2 is high.</p>
<p>There is a functional relationship between KE 1 and KE 2 consistent with established biological knowledge. <sup><a href="#cite_note-150">[150]</a></sup><sup><a href="#cite_note-luckey_2001-23">[23]</a></sup> <sup><a href="#cite_note-Roberts_2007-115">[115]</a></sup><sup><a href="#cite_note-Malhi_2010-105">[105]</a></sup><sup><a href="#cite_note-Canbay_2004apoptosis-106">[106]</a></sup> <sup><a href="#cite_note-Orrenius_2011-107">[107]</a></sup><sup><a href="#cite_note-Kolios_2006-114">[114]</a></sup><sup><a href="#cite_note-Kisseleva_and_Brenner_2008-4">[4]</a></sup> <sup><a href="#cite_note-151">[151]</a></sup><sup><a href="#cite_note-Li_2008-152">[152]</a></sup><sup><a href="#cite_note-Poli_2000-143">[143]</a></sup></p>
</td>
</tr>
<tr>
<td>
<p>KE 1 => KE 4</p>
</td>
<td>
<p>Like KCs, also HSCs are activated by damaged hepatocytes through the release of ROS, cytokines and chemokines and upon engulfment of apoptotic bodies from hepatocytes. 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-β 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>
</td>
<td>
<p>Biological Plausibility of KE1 => KE4 is high.</p>
<p>HSCs activation by hepatocytes is only a contributing factor and not the main route; partly it is mediated by TGF-β1; therefore this relationship is classified as indirect. Nevertheless, there is a functional relationship between KE 1 and KE 4 consistent with established biological knowledge. <sup><a href="#cite_note-153">[153]</a></sup><sup><a href="#cite_note-Gressner_2002-120">[120]</a></sup><sup><a href="#cite_note-Malhi_2010-105">[105]</a></sup><sup><a href="#cite_note-Canbay_2004apoptosis-106">[106]</a></sup> <sup><a href="#cite_note-Orrenius_2011-107">[107]</a></sup><sup><a href="#cite_note-Kolios_2006-114">[114]</a></sup><sup><a href="#cite_note-Kisseleva_and_Brenner_2008-4">[4]</a></sup> <sup><a href="#cite_note-Li_2008-152">[152]</a></sup><sup><a href="#cite_note-Friedman_2008-3">[3]</a></sup><sup><a href="#cite_note-Lee_and_Friedman_2011-1">[1]</a></sup></p>
</td>
</tr>
<tr>
<td>
<p>KE 2 => KE 3</p>
</td>
<td>
<p>Following activation KCs become a main source of TGF-β1, the most potent profibrogenic cytokine, as well as a major source of inflammatory mediators, chemokines, and ROS.</p>
</td>
<td>
<p>Biological Plausibility of KE2 => KE3 is high.</p>
<p>The functional relationship between KE 2 and KE 3 is consistent with biological knowledge. <sup><a href="#cite_note-154">[154]</a></sup><sup><a href="#cite_note-Li_2008-152">[152]</a></sup><sup><a href="#cite_note-Kolios_2006-114">[114]</a></sup><sup><a href="#cite_note-Bataller_2005-140">[140]</a></sup><sup><a href="#cite_note-Lee_and_Friedman_2011-1">[1]</a></sup><sup><a href="#cite_note-Guo_and_Friedman_2007-155">[155]</a></sup><sup><a href="#cite_note-Brenner_2009-156">[156]</a></sup><sup><a href="#cite_note-157">[157]</a></sup><sup><a href="#cite_note-Kirkham_2007-148">[148]</a></sup> <sup><a href="#cite_note-158">[158]</a></sup></p>
</td>
</tr>
<tr>
<td>
<p>KE 3 => KE 4</p>
</td>
<td>
<p>TGF-β1 activates HSCs, i.e. stimulates cell proliferation, matrix synthesis, and release of retinoids by HSCs and is the most potent fibrogenic factor for HSCs.</p>
</td>
<td>
<p>Biological Plausibility of KE3 => KE4 is high.</p>
<p>There is good understanding and broad acceptance of the KER between KE 3 and KE 4.</p>
<p><sup><a href="#cite_note-Kisseleva_and_Brenner_2007-147">[147]</a></sup><sup><a href="#cite_note-159">[159]</a></sup><sup><a href="#cite_note-Qi_1999-123">[123]</a></sup><sup><a href="#cite_note-Gressner_2002-120">[120]</a></sup><sup><a href="#cite_note-Kolios_2006-114">[114]</a></sup><sup><a href="#cite_note-Bataller_2005-140">[140]</a></sup><sup><a href="#cite_note-Guo_and_Friedman_2007-155">[155]</a></sup><sup><a href="#cite_note-Brenner_2009-156">[156]</a></sup> <sup><a href="#cite_note-160">[160]</a></sup> <sup><a href="#cite_note-Stalnikowitz_2003-134">[134]</a></sup><sup><a href="#cite_note-Li_2008-152">[152]</a></sup><sup><a href="#cite_note-Matsuoka_1990-161">[161]</a></sup><sup><a href="#cite_note-Kisseleva_and_Brenner_2008-4">[4]</a></sup><sup><a href="#cite_note-Poli_2000-143">[143]</a></sup> <sup><a href="#cite_note-Parsons_2007-142">[142]</a></sup><sup><a href="#cite_note-Friedman_2008-3">[3]</a></sup><sup><a href="#cite_note-Liu_2006-119">[119]</a></sup></p>
<p> </p>
</td>
</tr>
<tr>
<td>
<p>KE 4 => KE 5</p>
</td>
<td>
<p>In response to TGF-β1 activated HSCs up-regulate collagen synthesis. Together with decreased matrix degradation ECM composition changes and further stimulates HSC activation and production of TGF-β1, which further promotes activation of neighbouring quiescent HSCs.</p>
</td>
<td>
<p>Biological Plausibility of KE4 => KE5 is high.</p>
<p>The functional relationship between KE 4 and KE 5 is consistent with biological knowledge and generally accepted. <sup><a href="#cite_note-162">[162]</a></sup><sup><a href="#cite_note-163">[163]</a></sup><sup><a href="#cite_note-164">[164]</a></sup><sup><a href="#cite_note-Kolios_2006-114">[114]</a></sup><sup><a href="#cite_note-Bataller_2005-140">[140]</a></sup><sup><a href="#cite_note-Lee_and_Friedman_2011-1">[1]</a></sup><sup><a href="#cite_note-Guo_and_Friedman_2007-155">[155]</a></sup><sup><a href="#cite_note-Brenner_2009-156">[156]</a></sup><sup><a href="#cite_note-Li_2008-152">[152]</a></sup><sup><a href="#cite_note-Stalnikowitz_2003-134">[134]</a></sup><sup><a href="#cite_note-165">[165]</a></sup></p>
</td>
</tr>
<tr>
<td>
<p>KE 5 => AO</p>
</td>
<td>
<p>Excessive accumulation of ECM proteins leads to disruption of normal hepatic architecture.</p>
</td>
<td>
<p>Biological Plausibility of KE5 => AO is high.</p>
<p>By definition, liver fibrosis is the excessive accumulation of ECM proteins that are produced by HSCs. The KER between KE 5 and the AO is undisputed. <sup><a href="#cite_note-Lee_and_Friedman_2011-1">[1]</a></sup><sup><a href="#cite_note-Bataller_2005-140">[140]</a></sup><sup><a href="#cite_note-Pellicoro_2014-166">[166]</a></sup><sup><a href="#cite_note-Brancatelli_2009-167">[167]</a></sup><sup><a href="#cite_note-Rockey_2006-168">[168]</a></sup><sup><a href="#cite_note-Ponard_1997-169">[169]</a></sup></p>
<p> </p>
<p> </p>
</td>
</tr>
<tr>
<td colspan="3">
<p><strong>Empirical Support for KERs</strong></p>
</td>
</tr>
<tr>
<td colspan="3">
<p>There is a need for more advanced <em>in vitro</em> models systems for chemical-induced hepatotoxicity to study intercellular signalling and dose-response data on KERs. Nevertheless, some empirical evidence exists to support that a change in KEup leads to an appropriate change in the respective KEdown.</p>
</td>
</tr>
<tr>
<td>
<p>MIE => KE 1</p>
</td>
<td>
<p>It is general accepted knowledge that alkylating chemicals damage cells. Although covalent protein alkylation is a feature of many hepatotoxic drugs the overall extent of binding does not adequately distinguish toxic from non-toxic binding. It is not known whether protein alkylation to certain proteins is required and whether particular proteins and various binding sites influence the further downstream process. Further, we do not know whether there is a threshold and if this threshold would refer to the number of alkylation of a single protein or of a threshold number of proteins.</p>
</td>
<td>
<p><br />
Empirical Support of the MIE => KE 1 is moderate.</p>
<p>There is exposure-dependent change in both events following exposure with temporal concordance. <sup><a href="#cite_note-170">[170]</a></sup><sup><a href="#cite_note-171">[171]</a></sup><sup><a href="#cite_note-172">[172]</a></sup><sup><a href="#cite_note-173">[173]</a></sup><sup><a href="#cite_note-Codreanu_2014-103">[103]</a></sup></p>
</td>
</tr>
<tr>
<td>
<p>KE 1 => KE 2</p>
<p> </p>
<p> </p>
</td>
<td>
<p>Specific markers for activated KCs have not been identified yet. KC activation cannot be detected by staining techniques since cell morphology does not change, but cytokines release can be measured (with the caveat that KCs activate spontaneously <em>in vitro</em>). Tukov et al. examined the effects of KCs cultured in contact with rat hepatocytes. They found that by adding KCs to the cultures they could mimic <em>in vivo</em> drug-induced inflammatory responses. Canbay et al could prove that engulfment of hepatocyte apoptotic bodies stimulated cytokine expression by KCs.</p>
</td>
<td>
<p>Empirical Support of the KE 1 => KE 2 is moderate.</p>
<p>There are limited experimental studies which could show that there is a direct relationship between these two events with temporal concordance. <sup><a href="#cite_note-174">[174]</a></sup><sup><a href="#cite_note-175">[175]</a></sup><sup><a href="#cite_note-176">[176]</a></sup><sup><a href="#cite_note-Canbay_2003-110">[110]</a></sup></p>
<p> </p>
</td>
</tr>
<tr>
<td>
<p>KE 1 => KE 4</p>
<p> </p>
<p> </p>
</td>
<td>
<p>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. Watanabe et al. could demonstrate <em>in vitro</em> that DNA from apoptotic hepatocytes acts as an important mediator of HSC differentiation by providing a stop signal to mobile HSCs when they have reached an area of apoptosing hepatocytes and inducing a stationary phenotype- associated up-regulation of collagen production. Coulouarn et al found in a co-culture model that hepatocyte - HSC crosstalk engenders a permissive inflammatory micro-environment.</p>
</td>
<td>
<p>Empirical Support of the KE 1 => KE 4 is moderate.</p>
<p>There is experimental evidence for this KER. <sup><a href="#cite_note-Canbay_2002-109">[109]</a></sup><sup><a href="#cite_note-Canbay_2004caspaseinhibitor-113">[113]</a></sup><sup><a href="#cite_note-177">[177]</a></sup><sup><a href="#cite_note-178">[178]</a></sup></p>
</td>
</tr>
<tr>
<td>
<p>KE 2 => KE 3</p>
<p> </p>
<p> </p>
</td>
<td>
<p>Experiments by Matsuoka and Tsukamoto already 1990 showed that KCs isolated from rat liver with alcoholic fibrosis express and release TGF-β1 and that this cytokine is largely responsible for the KC-conditioned medium-induced stimulation of collagen formation by HSCs. Accumulated CD11b1 macrophages are critical for activating HSCs (via expression of TGF-β1) (Chu et al, 2013)</p>
</td>
<td>
<p>Empirical Support of the KE 2 => KE 3 is moderate.</p>
<p>Cytokine release is one of the features that define KC activation and there is sound empirical evidence for this KER. <sup><a href="#cite_note-Matsuoka_1990-161">[161]</a></sup><sup><a href="#cite_note-179">[179]</a></sup></p>
<p> </p>
</td>
</tr>
<tr>
<td>
<p>KE 3 => KE 4</p>
<p> </p>
<p> </p>
</td>
<td>
<p>Czaja et al could prove that treatment of cultured hepatic cells with TGF-β1 increased type I pro-collagen mRNA levels 13-fold due to post-transcriptional gene regulation. Tan et al. discovered that short TGF-β1 pulses can exert long-lasting effects on fibroblasts. Difficulties are that HSCs cultured on plastic, undergo spontaneous activation and HSCs activated in culture do not fully reproduce the changes in gene expression observed <em>in vivo</em>. De Minicis et al investigated gene expression changes in 3 different models of HSC activation and compared gene expression profiles in culture (mice HSCs in co-culture with KCs) and <em>in vivo</em> and did not find a proper correlation.</p>
<p> </p>
</td>
<td>
<p>Empirical Support of the KE 3 => KE 4 is moderate.</p>
<p>Qualitative empirical evidence with temporal and incidence concordance for this KER exists. <sup><a href="#cite_note-180">[180]</a></sup><sup><a href="#cite_note-181">[181]</a></sup><sup><a href="#cite_note-182">[182]</a></sup><sup><a href="#cite_note-183">[183]</a></sup></p>
<p> </p>
</td>
</tr>
<tr>
<td>
<p>KE 4 => KE 5</p>
<p> </p>
<p> </p>
</td>
<td>
<p>It is difficult to stimulate sufficient production of collagen and its subsequent incorporation into a pericellular matrix <em>in vitro</em>; therefore analytical methods have focused on measurement of pro-collagen secreted into culture medium or measurement of α-smooth muscle actin (α-SMA) expression, a marker of fibroblast activation. In primary culture, HSCs from normal liver began to express α-SMA coincident with culture-induced activation.</p>
</td>
<td>
<p>Empirical Support of the KE 4 => KE 5 is moderate.</p>
<p>It is general accepted knowledge that activated HSCs (=myofibroblasts) are collagen-producing cells. <sup><a href="#cite_note-184">[184]</a></sup><sup><a href="#cite_note-185">[185]</a></sup></p>
<p> </p>
<p> </p>
<p> </p>
</td>
</tr>
<tr>
<td>
<p>KE 5 => AO</p>
<p> </p>
</td>
<td>
<p>Liver fibrosis results from chronic damage in conjunction with the accumulation of ECM proteins, which distorts the hepatic architecture by forming a fibrous scar. The onset of liver fibrosis is usually insidious and progression to cirrhosis occurs after an interval of 15–20 years.</p>
</td>
<td>
<p>Empirical Support of the KE 5 => AO is high.</p>
<p>There is a smooth transition from ECM accumulation to liver fibrosis without a definite threshold and plenty <em>in vivo</em> evidence exists that ECM accumulation is a pre-stage of liver fibrosis <sup><a href="#cite_note-Bataller_2005-140">[140]</a></sup></p>
<p> </p>
</td>
</tr>
</tbody>
</table>
<p>More advanced <em>in vitro</em> models systems are needed to study chemical-induced hepatotoxicity. Modulations of hepatotoxicity by intercellular signalling cannot be addressed in primary cultures of hepatocytes alone but require co-cultures of different liver cell types. Various co-cultures systems with two or more different liver cell types are currently being developed, but quantitative data on KERs are not available yet.</p>
<p>This systematic and coherent display of currently available mechanistic-toxicological information can serve as a knowledge-based repository for identification/selection/development of <em>in vitro</em> methods suitable for measuring KEs and their relationships along the AOP and to facilitate the use of alternative data for regulatory purposes. Identified uncertainties and knowledge gaps can direct future research by priority setting and targeted testing. The KE descriptions can be used for hazard identification and read-across to assess the toxic potential of an untested substance. A sufficiently detailed description of the AOP to liver fibrosis might support chemical risk assessment by indicating early (upstream) markers for downstream events and facilitate a testing strategy without the need for an elaborated cell model.</p>
<h2>Confidence in the AOP</h2>
<p><em>Elaborate on the domains of applicability listed in the summary section above. Specifically, provide the literature supporting, or excluding, certain domains. </em></p>
<p>The biological plausibility, i.e the mechanistic relationship between of each of the KERs in this AOP was rated high, because there is good scientific understanding of these relationships and they are consistent with established biological knowledge. The empirical support for the KERs is considered moderate because there are only limited (nevertheless consistent) data available. For the KER between collagen accumulation and liver fibrosis exists a lot of empirical and clinical evidence and therefore empirical support is rated high. Modulations of hepatotoxicity by intercellular signalling cannot be addressed in primary cultures of hepatocytes alone but require co-cultures of different liver cell types. Due to the limited availability of adequate cell models dose-response data on KERs are not available yet. But there is some empirical evidence to support that a change in KEup leads to an appropriate change in the respective KEdown; some experimental studies could demonstrate a dependent relationship between two consecutive KEs with temporal concordance following exposure to a toxicant.</p>
<h3><strong>How well characterised is the AOP?</strong></h3>
<p>The adverse outcome is well understood qualitatively, but quantitative data are lacking.</p>
<h4><strong>How well are the initiating and other key events causally linked to the outcome?</strong></h4>
<p>The relationships between each key event and adverse outcome are well established.</p>
<h4><strong>What are the limitations in the evidence in support of the AOP?</strong></h4>
<p>This AOP description is plausible and consistent with existing literature in describing the association between AO and MIE across different levels of biological organisation. Animal studies are mainly focused on the AO and do not describe mechanistic sequences in detail. Due to the pathogenic complexity of liver fibrosis involving many different cells there is currently no suitable cell model available to mimic and further explore the sequence of events, especially in quantitative terms.</p>
<p><a name="cite_ref-194"></a></p>
<p><span style="font-size:14px">Prestigiacomo et al.<sup><a href="#ref-194">[194]</a></sup> generated a cell system containing the three key players of liver fibrosis (hepatocytes, Kupffer cells and hepatic stellate cells) to assess the response to fibrogenic compounds and they could recapitulate <em>in vitro</em> the KEs leading to liver fibrosis, as described in this AOP.</span></p>
<p> </p>
<h4><strong>Is the AOP specific to certain tissues, life stages / age classes?</strong></h4>
<p>The complex mechanism of fibrogenesis does not only affect a single organ, but causes a systemic response which equally damages other organs and tissues. The described findings in liver fibrosis parallel those in studies of fibrogenesis in other organs; everywhere are the same kind of cells and soluble factors involved <sup><a href="#cite_note-Pellicoro_2014-166">[166]</a></sup><sup><a href="#cite_note-Friedman_2002-124">[124]</a></sup><sup><a href="#cite_note-Friedmann2010-2">[2]</a></sup>. For example the reference compound CCl4 equally affects lymphoid organs, lungs and kidneys <sup><a href="#cite_note-Kisseleva_and_Brenner_2008-4">[4]</a></sup>. Fibrosis may affect lung, kidney, heart and blood vessels, eye, skin, pancreas, intestine, brain and bone marrow. Multi-organ fibrosis occurs due to mechanical injury or can be drug- or radiation-induced <sup><a href="#cite_note-186">[186]</a></sup><sup><a href="#cite_note-Sivakumar_and_Das_2008-137">[137]</a></sup><sup><a href="#cite_note-187">[187]</a></sup><sup><a href="#cite_note-188">[188]</a></sup>. As many fibrogenic pathways are conserved across tissues, recent findings in the liver could be extended to studies of fibrosis in the lungs, the kidneys, the heart and other organs.</p>
<h4><strong>Are the initiating and key events expected to be conserved across taxa?</strong></h4>
<p>Findings also suggest common conserved pathways across different species which initiate and significantly modulate the progression of liver fibrosis <sup><a href="#cite_note-189">[189]</a></sup> <sup><a href="#cite_note-190">[190]</a></sup><sup><a href="#cite_note-191">[191]</a></sup><sup><a href="#cite_note-192">[192]</a></sup><sup><a href="#cite_note-193">[193]</a></sup>.</p>
<h2>Acknowledgements</h2>
<p>I want to thank Clemens Wittwehr for his repeated and patient editing assistance, as well as Steve Edwards for his prompt availability whenever a technical problem occurred.</p>
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