Increase, 11β-Hydroxysteroid dehydrogenase type 1 activity

7440-61-1

JFALSRSLKYAFGM-UHFFFAOYSA-N

Uranium

Uranium, isotope of mass 238 238U Element UN 2979 (DOT) Uranium I

DTXSID1042522

7440-43-9

BDOSMKKIYDKNTQ-UHFFFAOYSA-N

Cadmium

Cadimium CADMIUM BLUE CADMIUM, IN PLATTEN, STANGEN, BROCKEN,KOERNER

DTXSID1023940

PR:000011406

PR glucocorticoid receptor

GO:0005739

GO mitochondrion

CL:0000091

CL Kupffer cell

CL:0000632

CL hepatic stellate cell

CHEBI:3815

CHEBI collagen

UBERON:0002107

UBERON liver

GO:0004883

GO glucocorticoid receptor activity

HP:0001397

HP Hepatic steatosis

GO:0008219

GO cell death

GO:0042116

GO macrophage activation

GO:0035733

GO hepatic stellate cell activation

GO:0032964

GO collagen biosynthetic process

MP:0003333

MP liver fibrosis

WIKI increased

WIKI functional change

WIKI occurrence

Uranium

2021-08-05T14:28:50

Nanoparticles and Micrometer Particles

2022-02-04T13:43:43

Cadmium

2017-10-25T08:33:12

WikiUser_28

Vertebrates

WCS_9606

common toxicological species human

10090

NCBI mouse

10116

NCBI rat

WCS_7227

common ecological species Drosophila melanogaster

6239

NCBI Caenorhabditis elegans

WikiUser_25

Wikiuser: Cyauk human and other cells in culture

10116

NCBI Rattus norvegicus

9823

NCBI pigs

9606

NCBI Homo sapiens

WikiUser_17

mammals

Increase, 11β-Hydroxysteroid dehydrogenase type 1 activity

Increase, 11β-HSD1 activity

Molecular

AOPWiki 2026-02-18T08:38:26

2026-02-18T08:38:26

Increase, Hepatic intracellular active glucocorticoids

Increase, Hepatic intracellular active GC

Cellular

AOPWiki 2026-02-18T08:43:05

2026-02-26T06:48:35

Increase, Glucocorticoid receptor activation

Increase, GR activation

Molecular

Site of action:  The molecular site of action is the glucocorticoid receptor (GR), nuclear receptor part of a superfamily of highly conserved which bind to steroids, sterols, thyroid hormones, retinoids, and orphan receptors (Weikum et al., 2017). In humans, the formal gene name of this receptor is nuclear receptor subfamily 3, group C, member 1 – NR3C1 (Oakley & Cidlowski, 2013). More specifically, the GR agonism occurs through the interaction of a chemical (endogenous compounds such as cortisol, or an external stressor) with the ligand binding domain. In the absence of a ligand, the GR is transcriptionally inactive in the cytoplasm (Barnes, 1998). Responses at the macromolecular level:  Once bound to a hormonal ligand, the GR is translocated from the cytoplasm to the nucleus where the activated GR interacts with genomic glucocorticoid-response elements (GRE) and regulates transcription of associated genes. Interactions with double stranded DNA and transcription factors can cause both activation and repression of downstream genes via directly binding to a consensus site, binding to other transcription factors to form a heterodimer, or homodimerization prior to DNA binding (Oakley & Cidlowski, 2013).

Glucocorticoid receptor activation can be measured via bioanalytical tools such as in vitro bioassays where results are typically reported in Dexamethasone-equivalents (DEX-EQ) . However it should be noted that these assays have differences in sensitivity (Cole & Brooks, 2023).             <table border="1" cellpadding="1" cellspacing="1" style="width:500px"> <caption>In Vitro Assays Employed in Glucocorticoid Receptor Agonism Detection</caption> <tbody> <tr> <td>Assay</td> <td>Receptor Organism</td> <td>Tissue</td> <td>Citation</td> </tr> <tr> <td> TOX21 GR BLA Agonist Ratio </td> <td>Human</td> <td>Cervix</td> <td>Huang et al., 2011</td> </tr> <tr> <td>GR CALUX</td> <td>Human</td> <td>Osteosarcoma</td> <td>Been et al., 2021; Macikova et al., 2014; Schriks et al., 2010; Suzuki et al., 2015</td> </tr> <tr> <td>Attagene GR TRANS</td> <td>Human</td> <td>Liver</td> <td>Martin et al., 2010; Medvedev et al., 2018; Romanov et al., 2008</td> </tr> <tr> <td>Attagene GRe CIS</td> <td>Human</td> <td>Liver</td> <td>Martin et al., 2010; Medvedev et al., 2018; Romanov et al., 2008</td> </tr> <tr> <td>CV1-hGR</td> <td>Human</td> <td>Kidney</td> <td>Medlock Kakaley et al., 2019</td> </tr> <tr> <td>GR-GeneBlazer</td> <td>Human</td> <td>Kidney</td> <td>Jia et al., 2016</td> </tr> <tr> <td>NovaScreen NR hGR</td> <td>Human</td> <td>N/A</td> <td>Knudsen et al., 2011; Sipes et al., 2013</td> </tr> <tr> <td>Trout GR1</td> <td>Trout</td> <td>Kidney</td> <td>Kugathas & Sumpter, 2011</td> </tr> <tr> <td>Trout GR2</td> <td>Trout</td> <td>Kidney</td> <td>Kugathas & Sumpter, 2011</td> </tr> <tr> <td>Indigo hGR</td> <td>Human</td> <td>N/A</td> <td>Cavaillin et al., 2021; Cole et al., 2025</td> </tr> <tr> <td>Indigo zfGR</td> <td>Zebrafish</td> <td>N/A</td> <td>Cole et al., 2025</td> </tr> </tbody> </table>   In addition to bioanalytical techniques, induction of GR-regulated genes are also indicative of GR agonism in vivo (Cavallin et al., 2021; Cole et al., 2025; Garland et al., 2019).

Taxonomic Applicability: The GR is present in almost every vertebrate cell (Weikum et al., 2017). The evolutionary conservation of GR activation across taxa was examined in silico through the employment of EPA’s Sequence Alignment to Predict Across Species Susceptibility (SeqAPASS) Tool, and 623 orthologs were identified confirming conservation in vertebrate species. Additionally, bioanalytical methods comparing zebrafish (Danio rerio) GR and human GR show conservation of ligand binding and receptor agonism when using dexamethasone and beclomethasone dipropionate. Lastly, the fathead minnow (Pimephales promelas) model has been employed to examine susceptibility to synthetic glucocorticoids in the following in vivo exposure to dexamethasone and beclomethasone dipropionate (Cole et al., 2025). Through the processes of gene duplication and divergence, the GR and mineralocorticoid receptor (MR) evolved from a corticoid receptor in jawless fish. While only possessing one isoform of MR, teleost fish possess two isoforms of the GR and all three have affinity for endogenous cortisol (Baker et al., 2013). Conservation of susceptibility does not infer similarities in sensitivity which varies based on species, receptor isoform, and tissue (Aedo et al., 2023; Baker et al., 2013; Bury & Sturm, 2007; Gilmour, 2005; Jerez-Cepa et al., 2019; Small & Quiniou, 2018; Stolte et al., 2006) <img alt="Results from (A) Level 1 Sequence Alignment to Predict Cross-Species Susceptibility (SeqAPASS) comparing 1,631 protein sequences to zebrafish glucocorticoid receptor (zfGR). Analysis resulted in 782 ortholog candidates at a susceptibility cut-off of 20.55%. (B) Level 2 SeqAPASS analysis examining the ligand binding domain (LDB) of zfGR which resulted in 784 orthologs at a susceptibility cut-off of 34.47%." src="https://aopwiki.org/system/dragonfly/production/2025/04/18/9oyje58ylm_image_5_.png" style="height:1745px; width:1505px" /> Figure: Results from (A) Level 1 Sequence Alignment to Predict Cross-Species Susceptibility (SeqAPASS) comparing 1,631 protein sequences to zebrafish glucocorticoid receptor (zfGR). Analysis resulted in 782 ortholog candidates at a susceptibility cut-off of 20.55%. (B) Level 2 SeqAPASS analysis examining the ligand binding domain (LDB) of zfGR which resulted in 784 orthologs at a susceptibility cut-off of 34.47%.   Life Stage Applicability: This MIE is not life stage specific. However, the downstream transcriptional effects of GR agonism may vary based on life stage. (LaLone et al., 2012; Watanabe et al., 2016).   Sex Applicability: This MIE is not sex specific.

CL:0000255

CL eukaryotic cell

High Unspecific

Moderate

All life stages

High

Aedo, J. E., Zuloaga, R., Aravena-Canales, D., Molina, A., & Valdés, J. A. (2023). Role of glucocorticoid and mineralocorticoid receptors in rainbow trout (Oncorhynchus mykiss) skeletal muscle: A transcriptomic perspective of cortisol action. Frontiers in Physiology, 13, 1048008. https://doi.org/10.3389/fphys.2022.1048008 Baker, M. E., Funder, J. W., & Kattoula, S. R. (2013). Evolution of hormone selectivity in glucocorticoid and mineralocorticoid receptors. The Journal of Steroid Biochemistry and Molecular Biology, 137, 57–70. Barnes, P. J. (1998). Anti-inflammatory actions of glucocorticoids: Molecular mechanisms. Clinical Science (London, England: 1979), 94(6), 557–572. https://doi.org/10.1042/cs0940557 Been, F., Pronk, T., Louisse, J., Houtman, C., van der Velden-Slootweg, T., van der Oost, R., & Dingemans, M. M. L. (2021). Development of a framework to derive effect-based trigger values to interpret CALUX data for drinking water quality. Water Research, 193. https://doi.org/10.1016/j.watres.2021.116859 Bury, N. R., & Sturm, A. (2007). Evolution of the corticosteroid receptor signalling pathway in fish. General and Comparative Endocrinology, 153(1), 47–56. https://doi.org/10.1016/j.ygcen.2007.03.009 Cavallin, J. E., Battaglin, W. A., Beihoffer, J., Blackwell, B. R., Bradley, P. M., Cole, A. R., Ekman, D. R., Hofer, R. N., Kinsey, J., Keteles, K., Weissinger, R., Winkelman, D. L., & Villeneuve, D. L. (2021). Effects-Based Monitoring of Bioactive Chemicals Discharged to the Colorado River before and after a Municipal Wastewater Treatment Plant Replacement. Environmental Science & Technology, 55(2), 974–984. https://doi.org/10.1021/acs.est.0c05269 Cole, A. R., & Brooks, B. W. (2023). Comparative Endpoint Sensitivity of Bioanalytical Tools for Glucocorticoid Receptor Agonism Surveillance in Aquatic Matrices. ACS ES&T Water, 3(9), 3082–3092. <a href="https://doi.org/10.1021/acsestwater.3c00253">https://doi.org/10.1021/acsestwater.3c00253</a> Cole, A. R., Blackwell, B. R., Cavallin, J. E., Collins, J. E., Kittelson, A. R., Shmaitelly, Y. M., Langan, L. M., Villenueve, D. L., & Brooks, B. W. (2025). Comparative Glucocorticoid Receptor Agonism: In Silico, In Vitro, and In Vivo and Identification of Potential Biomarkers for Synthetic Glucocorticoid Exposure. Environmental Toxicology and Chemistry, vgae041. https://doi.org/10.1093/etojnl/vgae041 Garland, M. A., Sengupta, S., Mathew, L. K., Truong, L., de Jong, E., Piersma, A. H., La Du, J., & Tanguay, R. L. (2019). Glucocorticoid receptor-dependent induction of cripto-1 (one-eyed pinhead) inhibits zebrafish caudal fin regeneration. Toxicology Reports, 6, 529–537. https://doi.org/10.1016/j.toxrep.2019.05.013 Gilmour, K. M. (2005). Mineralocorticoid receptors and hormones: Fishing for answers. Endocrinology, 146(1), 44–46. Huang, R., Xia, M., Cho, M.-H., Sakamuru, S., Shinn, P., Houck, K. A., Dix, D. J., Judson, R. S., Witt, K. L., Kavlock, R. J., Tice, R. R., & Austin, C. P. (2011). Chemical Genomics Profiling of Environmental Chemical Modulation of Human Nuclear Receptors. Environmental Health Perspectives, 119(8), 1142–1148. https://doi.org/10.1289/ehp.1002952 Jerez-Cepa, I., Gorissen, M., Mancera, J. M., & Ruiz-Jarabo, I. (2019). What can we learn from glucocorticoid administration in fish? Effects of cortisol and dexamethasone on intermediary metabolism of gilthead seabream (Sparus aurata L.). Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 231, 1–10. https://doi.org/10.1016/j.cbpa.2019.01.010 Jia, A., Wu, S., Daniels, K. D., & Snyder, S. A. (2016). Balancing the Budget: Accounting for Glucocorticoid Bioactivity and Fate during Water Treatment. Environmental Science & Technology, 50(6), 2870–2880. https://doi.org/10.1021/acs.est.5b04893 Knudsen, T. B., Houck, K. A., Sipes, N. S., Singh, A. V., Judson, R. S., Martin, M. T., Weissman, A., Kleinstreuer, N. C., Mortensen, H. M., Reif, D. M., Rabinowitz, J. R., Setzer, R. W., Richard, A. M., Dix, D. J., & Kavlock, R. J. (2011). Activity profiles of 309 ToxCastTM chemicals evaluated across 292 biochemical targets. Toxicology, 282(1), 1–15. https://doi.org/10.1016/j.tox.2010.12.010 Kugathas, S., & Sumpter, J. P. (2011). Synthetic Glucocorticoids in the Environment: First Results on Their Potential Impacts on Fish. Environmental Science & Technology, 45(6), 2377–2383. https://doi.org/10.1021/es104105e LaLone, C. A., Villeneuve, D. L., Olmstead, A. W., Medlock, E. K., Kahl, M. D., Jensen, K. M., Durhan, E. J., Makynen, E. A., Blanksma, C. A., Cavallin, J. E., Thomas, L. M., Seidl, S. M., Skolness, S. Y., Wehmas, L. C., Johnson, R. D., & Ankley, G. T. (2012). Effects of a glucocorticoid receptor agonist, dexamethasone, on fathead minnow reproduction, growth, and development. Environmental Toxicology and Chemistry, 31(3), 611–622. https://doi.org/10.1002/etc.1729 Macikova, P., Groh, K. J., Ammann, A. A., Schirmer, K., & Suter, M. J.-F. (2014). Endocrine Disrupting Compounds Affecting Corticosteroid Signaling Pathways in Czech and Swiss Waters: Potential Impact on Fish. Environmental Science & Technology, 48(21), 12902–12911. https://doi.org/10.1021/es502711c Martin, M. T., Dix, D. J., Judson, R. S., Kavlock, R. J., Reif, D. M., Richard, A. M., Rotroff, D. M., Romanov, S., Medvedev, A., Poltoratskaya, N., Gambarian, M., Moeser, M., Makarov, S. S., & Houck, K. A. (2010). Impact of Environmental Chemicals on Key Transcription Regulators and Correlation to Toxicity End Points within EPA’s ToxCast Program. Chemical Research in Toxicology, 23(3), 578–590. https://doi.org/10.1021/tx900325g Medlock Kakaley, E., Cardon, M. C., Gray, L. E., Hartig, P. C., & Wilson, V. S. (2019). Generalized Concentration Addition Model Predicts Glucocorticoid Activity Bioassay Responses to Environmentally Detected Receptor-Ligand Mixtures. Toxicological Sciences, 168(1), 252–263. https://doi.org/10.1093/toxsci/kfy290 Medvedev, A., Moeser, M., Medvedeva, L., Martsen, E., Granick, A., Raines, L., Zeng, M., Makarov, S., Houck, K. A., & Makarov, S. S. (2018). Evaluating biological activity of compounds by transcription factor activity profiling. Science Advances, 4(9), eaar4666. https://doi.org/10.1126/sciadv.aar4666 Oakley, R. H., & Cidlowski, J. A. (2013). The Biology of the Glucocorticoid Receptor: New Signaling Mechanisms in Health and Disease. The Journal of Allergy and Clinical Immunology, 132(5), 1033–1044. https://doi.org/10.1016/j.jaci.2013.09.007 Romanov, S., Medvedev, A., Gambarian, M., Poltoratskaya, N., Moeser, M., Medvedeva, L., Gambarian, M., Diatchenko, L., & Makarov, S. (2008). Homogeneous reporter system enables quantitative functional assessment of multiple transcription factors. Nature Methods, 5(3), 253–260. https://doi.org/10.1038/nmeth.1186 Schriks, M., van Leerdam, J. A., van der Linden, S. C., van der Burg, B., van Wezel, A. P., & de Voogt, P. (2010). High-Resolution Mass Spectrometric Identification and Quantification of Glucocorticoid Compounds in Various Wastewaters in The Netherlands. Environmental Science & Technology, 44(12), 4766–4774. https://doi.org/10.1021/es100013x Sipes, N. S., Martin, M. T., Kothiya, P., Reif, D. M., Judson, R. S., Richard, A. M., Houck, K. A., Dix, D. J., Kavlock, R. J., & Knudsen, T. B. (2013). Profiling 976 ToxCast Chemicals across 331 Enzymatic and Receptor Signaling Assays. Chemical Research in Toxicology, 26(6), 878–895. https://doi.org/10.1021/tx400021f Small, B. C., & Quiniou, S. M. A. (2018). Characterization of two channel catfish, Ictalurus punctatus, glucocorticoid receptors and expression following an acute stressor. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 216, 42–51. https://doi.org/10.1016/j.cbpa.2017.11.011 Stolte, E. H., Kemenade, B. M. L. V. van, Savelkoul, H. F. J., & Flik, G. (2006). Evolution of glucocorticoid receptors with different glucocorticoid sensitivity. Journal of Endocrinology, 190(1), 17–28. https://doi.org/10.1677/joe.1.06703 Suzuki, G., Sato, K., Isobe, T., Takigami, H., Brouwer, A., & Nakayama, K. (2015). Detection of glucocorticoid receptor agonists in effluents from sewage treatment plants in Japan. Science of The Total Environment, 527–528, 328–334. https://doi.org/10.1016/j.scitotenv.2015.05.008 Watanabe, Y., Grommen, S. V. H., & De Groef, B. (2016). Corticotropin-releasing hormone: Mediator of vertebrate life stage transitions? General and Comparative Endocrinology, 228, 60–68. https://doi.org/10.1016/j.ygcen.2016.02.012 Weikum, E. R., Knuesel, M. T., Ortlund, E. A., & Yamamoto, K. R. (2017). Glucocorticoid receptor control of transcription: Precision and plasticity via allostery. Nature Reviews Molecular Cell Biology, 18(3), 159–174. https://doi.org/10.1038/nrm.2016.152   AOPWiki 2016-11-29T18:41:22

2026-02-12T07:24:33

Increase, De novo lipogenesis

Cellular

UBERON:0002107

UBERON liver

CL:0000182

CL hepatocyte

AOPWiki 2017-04-12T14:46:28

2026-02-10T04:39:07

Increase, Liver steatosis

Organ

Biological state: liver steatosis is the inappropriate storage of fat in hepatocytes.   Four major pathways for triglyceride accumulation are: 1. Increased fatty acid uptake; 2. Increased De Novo FA and Lipid Synthesis; 3. Decreased FA Oxidation; 4. Decreased Lipid Efflux (Angrish et al. 2016).  Chemical stressors can increase gene expression of key genes involving these pathways, leading to increased accumulation of triglycerides (Aguayo-Orozco et al. 2018).  In addition, excessive dietary compounds of fatty compounds can also increase likelihood of accumulation of triglycerides (Nguyen et al. 2008).  Biological compartment: steatosis is generally an organ-level diagnosis; however, the pathology occurs within the hepatocytes. Role in biology: steatosis is an adverse endpoint.  Consequences: Liver steatosis, or fatty liver, serves as a pivotal factor in the development of liver fibrosis by triggering a cascade of pathological events. According to the two-strikes hypothesis (Day and James, 1998), liver damage progresses in two stages: the first strike involves the accumulation of lipids in hepatocytes, often due to metabolic disturbances such as insulin resistance, excess free fatty acids, or oxidative stress. This stage, though asymptomatic, increases liver vulnerability by inducing mild oxidative stress and inflammation. The second strike introduces additional insults, such as inflammatory mediators or cellular damage, exacerbating liver injury and promoting fibrogenesis. The accumulation of fat sensitizes the liver to oxidative stress and triggers mechanisms like the activation of hepatic stellate cells (HSCs) and hepatocyte apoptosis or necrosis, central to the fibrotic process. While early-stage steatosis is reversible, chronic steatosis perpetuates a cycle of inflammation and fibrosis, creating a feedback loop that amplifies liver damage (Pafili K et al, 2021). Consequently, liver steatosis is not only a precursor but also a critical driver of fibrosis progression. Day CP, James OF. Steatohepatitis: a tale of two "hits"? Gastroenterology. 1998 Apr;114(4):842-5. doi: 10.1016/s0016-5085(98)70599-2. PMID: 9547102. Pafili K, Roden M. Nonalcoholic fatty liver disease (NAFLD) from pathogenesis to treatment concepts in humans. Mol Metab. 2021 Aug;50:101122. doi: 10.1016/j.molmet.2020.101122. Epub 2020 Nov 19. PMID: 33220492; PMCID: PMC8324683. Description from EU-ToxRisk: Activation of stellate cells results in collagen accumulation and change in extracellular matrix composition in the liver causing fibrosis. (Landesmann, 2016; Koo et al 2016)

Steatosis is measured by lipidomics approaches (e.g. Yang and Han 2016) that measure lipid levels, or by histology.  Concentrations of triglycerides, cholesterols, fatty acids, and related compounds are measured biochemically include high throughput enzymatic analyses, analytical ultracentrifuging, gradient gel electrophoresis, Nuclear Magnetic Resonance, and other direct assessment techniques (Schaefer et al. 2016).

Steatosis is the result of perturbations in well-known metabolic pathways that are well-studied and well-known in many taxa. Life Stage: The life stage applicable to this key event is all life stages with a liver.  Older individuals are more likely to manifest this adverse outcome pathway (adults > juveniles) due to accumulation of triglycerides. Sex: This key event applies to both males and females. Taxonomic: This key event appears to be present broadly in vertebrates, with most representative studies in mammals (humans, lab mice, lab rats).

UBERON:0002107

UBERON liver

High Unspecific

High

All life stages

High

Aguayo-Orozco, A.A., Bois, F.Y., Brunak, S., and Taboureau, O.  2018.  Analysis of Time-Series Gene Expression Data to Explore Mechanisms of Chemical-Induced Hepatic Steatosis Toxicity.  Frontiers in Genetics 9(Article 396): 1-15. Angrish, M.M., Kaiser, J.P., McQueen, C.A., and Chorley, B.N.  2016.  Tipping the Balance: Hepatotoxicity and the 4 Apical Key Events of Hepatic Steatosis.  Toxicological Sciences 150(2): 261–268. Day CP, James OF. Steatohepatitis: a tale of two "hits"? Gastroenterology. 1998 Apr;114(4):842-5. doi: 10.1016/s0016-5085(98)70599-2. PMID: 9547102. Landesmann, B. (2016). Adverse Outcome Pathway on Protein Alkylation Leading to Liver Fibrosis, (2). https://doi.org/10.1016/j.molcel.2005.08.010   Koo, J. H., Lee, H. J., Kim, W., & Kim, S. G. (2016). Endoplasmic Reticulum Stress in Hepatic Stellate Cells Promotes Liver Fibrosis via PERK-Mediated Degradation of HNRNPA1 and Up-regulation of SMAD2. Gastroenterology, 150(1), 181–193.e8. https://doi.org/10.1053/j.gastro.2015.09.039 Nguyen, P., Leray, V., Diez, M., Serisier, S., Le Bloc’h, J., Siliart, B., and Dumon, H.  2008.  Liver lipid metabolism.  Journal of Animal Physiology and Animal Nutrition 92: 272–283.   Pafili K, Roden M. Nonalcoholic fatty liver disease (NAFLD) from pathogenesis to treatment concepts in humans. Mol Metab. 2021 Aug;50:101122. doi: 10.1016/j.molmet.2020.101122. Epub 2020 Nov 19. PMID: 33220492; PMCID: PMC8324683. Schaefer EJ, Tsunoda F, Diffenderfer M, Polisecki, E., Thai, N., and Astalos, B. The Measurement of Lipids, Lipoproteins, Apolipoproteins, Fatty Acids, and Sterols, and Next Generation Sequencing for the Diagnosis and Treatment of Lipid Disorders. [Updated 2016 Mar 29]. In: Feingold KR, Anawalt B, Blackman MR, et al., editors. Endotext [Internet]. South Dartmouth (MA): MDText.com, Inc.; 2000-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK355892/ Yang, K. and Han, X.  2016.  Lipidomics: Techniques, applications, and outcomes related to biomedical sciences.  Trends in Biochemical Sciences 2016 November ; 41(11): 954–969. NOTE: Italics symbolize edits from John Frisch AOPWiki 2016-11-29T18:41:24

2026-02-11T05:41:52

Increase, Hepatocellular lipotoxicity

Cellular

AOPWiki 2026-02-10T04:40:07

2026-02-10T04:40:07

Increase, Mitochondrial dysfunction

Cellular

Mitochondrial dysfunction is a consequence of inhibition of the respiratory chain leading to oxidative stress. Mitochondria can be found in all cells and are considered the most important cellular consumers of oxygen. Furthermore, mitochondria possess numerous redox enzymes capable of transferring single electrons to oxygen, generating the superoxide (O2-). Some mitochondrial enzymes that are involved in reactive oxygen species (ROS) generation include the electron-transport chain (ETC) complexes I, II and III; pyruvate dehydrogenase (PDH) and glycerol-3-phosphate dehydrogenase (GPDH). The transfer of electrons to oxygen, generating superoxide, happens mainly when these redox carriers are charged enough with electrons and the potential energy for transfer is elevated, like in the case of high mitochondrial membrane potential. In contrast, ROS generation is decreased if there are not enough electrons and the potential energy for the transfer is not sufficient (reviewed in Lin and Beal, 2006). Cells are also able to detoxify the generated ROS due to an extensive antioxidant defence system that includes superoxide dismutases, glutathione peroxidases, catalase, thioredoxins, and peroxiredoxins in various cell organelles (reviewed in Lin and Beal, 2006). It is worth mentioning that, as in the case of ROS generation, antioxidant defences are also closely related to the redox and energetic status of mitochondria. If mitochondria are structurally and functionally healthy, an antioxidant defence mechanism balances ROS generation, and there is not much available ROS production. However, in case of mitochondrial damage, the antioxidant defence capacity drops and ROS generation takes over. Once this happens, a vicious cycle starts and ROS can further damage mitochondria, leading to more free-radical generation and further loss of antioxidant capacity. During mitochondrial dysfunction the availability of ATP also decreases, which is considered necessary for repair mechanisms after ROS generation. A number of proteins bound to the mitochondria or endoplasmic reticulum (ER), especially in the mitochondria-associated ER membrane (MAM), are playing an important role of communicators between these two organelles (reviewed Mei et al., 2013). ER stress induces mitochondrial dysfunction through regulation of Ca2+ signaling and ROS production (reviewed Mei et al., 2013). Prolonged ER stress leads to release of Ca2+ at the MAM and increased Ca2+ uptake into the mitochondrial matrix, which induces Ca2+-dependent mitochondrial outer membrane permeabilization and apoptosis. At the same, ROS are produced by proteins in the ER oxidoreductin 1 (ERO1) family. ER stress activates ERO1 and leads to excessive production of ROS, which, in turn, inactivates SERCA and activates inositol-1,4,5- trisphosphate receptors (IP3R) via oxidation, resulting in elevated levels of cytosolic Ca2+, increased mitochondrial uptake of Ca2+, and ultimately mitochondrial dysfunction. Just as ER stress can lead to mitochondrial dysfunction, mitochondrial dysfunction also induces ER Stress (reviewed Mei et al., 2013). For example, nitric oxide disrupts the mitochondrial respiratory chain and causes changes in mitochondrial Ca2+ flux which induce ER stress. Increased Ca2+ flux triggers loss of mitochondrial membrane potential (MMP), opening of mitochondrial permeability transition pore (mPTP), release of cytochrome c and apoptosis inducing factor (AIF), decreasing ATP synthesis and rendering the cells more vulnerable to both apoptosis and necrosis (Wang and Qin, 2010). Metal-induced Mitochondrial Dysfunction Mitochondria are an important site of Ca2+ regulation and storage, taking up Ca2+ ions electrophoretically from the cytosol through a Ca2+ uniporter, which can then accumulate in the mitochondria (Roos et al., 2012; Orrenius et al., 2015). Similarities between calcium and metals, such as cadmium and lead, makes the entrance and accumulation of these metals into the mitochondria via calcium metals possible by mode of molecular mimicry (Mathews et al., 2013; Adiele et al., 2012). The outer mitochondrial membrane also contains the divalent metal transporter (DMT1), which allows for mitochondrial uptake of divalent metals such as Fe and Mn. When cells are under heavy metal-induced stress, DMT has been shown to be overexpressed in the mitochondrial membrane, making the mitochondria targets of metal toxicity and accumulation. Heavy metal exposure in aerobic organisms increases ROS formation through redox cycling, where metals with different valence states (Fe, Cu, Cr, etc.) directly produce ROS as they are reduced by cellular antioxidants and then react with oxygen (Shaki et al., 2012; Shaki et al., 2013; Pourahmad et al., 2006; Santos et al., 2007). The production of highly reactive hydroxyl radicals under mitochondrial oxidative stress and in the presence of transition metals occurs via the Fenton reaction or Haber-Weiss reaction (Hancock et al., 2001; Valko et al., 2005; Adam-Vizi et al., 2010). Metals and ROS are capable of damaging mitochondrial DNA as well as mechanisms of DNA repair and proliferation arrest (Valko et al., 2005). Metals and ROS have the potential to directly damage mitochondrial membranes and structure by binding to and oxidizing membrane lipids and proteins. This structural damage can collapse the MMP and lead to the opening of the MPTP (Orrenius et al., 2015; Roos et al., 2012; Pourahmad et al., 2006). Uranium and mercury, for example, have both been shown to directly inhibit the mitochondrial electron transport chain and interfere with ATP production (Shaki et al., 2012; Roos et al., 2012). Furthermore, as previously mentioned, metals have been shown to inhibit ROS-detoxifying enzymes. By binding to these enzymes, metals can inhibit their antioxidant functions, and cause an accumulation of ROS and increased synthesis of more antioxidant enzymes in order to combat the oxidative stress (Blajszczak and Bonini, 2017). Summing up: Mitochondria play a pivotal role in cell survival and cell death because they are regulators of both energy metabolism and apoptotic/necrotic pathways (Fiskum, 2000; Wieloch, 2001; Friberg and Wieloch, 2002). The production of ATP via oxidative phosphorylation is a vital mitochondrial function (Kann and Kovács, 2007; Nunnari and Suomalainen, 2012). The ATP is continuously required for signalling processes (e.g. Ca2+ signalling), maintenance of ionic gradients across membranes, and biosynthetic processes (e.g. protein synthesis, heme synthesis or lipid and phospholipid metabolism) (Kang and Pervaiz, 2012), and (Green, 1998; McBride et al., 2006). Inhibition of mitochondrial respiration contributes to various cellular stress responses, such as deregulation of cellular Ca2+ homeostasis (Graier et al., 2007) and ROS production (Nunnari and Suomalainen, 2012; reviewed Mei et al., 2013).). It is well established in the existing literature that mitochondrial dysfunction may result in: (a) an increased ROS production and a decreased ATP level, (b) the loss of mitochondrial protein import and protein biosynthesis, (c) the reduced activities of enzymes of the mitochondrial respiratory chain and the Krebs cycle, (d) the loss of the mitochondrial membrane potential, (e) the loss of mitochondrial motility, causing a failure to re-localize to the sites with increased energy demands (f) the destruction of the mitochondrial network, and (g) increased mitochondrial Ca2+ uptake, causing Ca2+ overload (reviewed in Lin and Beal, 2006; Graier et al., 2007), (h) the rupture of the mitochondrial inner and outer membranes, leading to (i) the release of mitochondrial pro-death factors, including cytochrome c (Cyt. c), apoptosis-inducing factor, or endonuclease G (Braun, 2012; Martin, 2011; Correia et al., 2012; Cozzolino et al., 2013), which eventually leads to apoptotic, necrotic or autophagic cell death (Wang and Qin, 2010). Due to their structural and functional complexity, mitochondria present multiple targets for various compounds.

Mitochondrial dysfunction can be detected using isolated mitochondria, intact cells or cells in culture as well as in vivo studies. Such assessment can be performed with a large range of methods (revised by Brand and Nicholls, 2011) for which some important examples are given. All approaches to assess mitochondrial dysfunction fall into two main categories: the first assesses the consequences of a loss-of-function, i.e. impaired functioning of the respiratory chain and processes linked to it. Some assay to assess this have been described for KE1, with the limitation that they are not specific for complex I. In the context of overall mitochondrial dysfunction, the same assays provide useful information, when performed under slightly different assay conditions (e.g. without addition of complex III and IV inhibitors). The second approach assesses a ‘non-desirable gain-of-function’, i.e. processes that are usually only present to a very small degree in healthy cells, and that are triggered in a cell, in which mitochondria fail. I. Mitochondrial dysfunction assays assessing a loss-of function. 1. Cellular oxygen consumption. See KE1 for details of oxygen consumption assays. The oxygen consumption parameter can be combined with other endpoints to derive more specific information on the efficacy of mitochondrial function. One approach measures the ADP-to-O ratio (the number of ADP molecules phosphorylated per oxygen atom reduced (Hinkle, 1995 and Hafner et al., 1990). The related P/O ratio is calculated from the amount of ADP added, divided by the amount of O2 consumed while phosphorylating the added ADP (Ciapaite et al., 2005; Diepart et al., 2010; Hynes et al., 2006; James et al., 1995; von Heimburg et al., 2005). 2. Mitochondrial membrane potential (Δψm ). - Revision of AOP3 (Project: <a href="https://www.efsa.europa.eu/en/call/npefsaprev202402-development-aop-network-parkinsonian-motor-symptoms" rel="noreferrer noopener" target="_blank">NP/EFSA/PREV/2024/02</a>): The mitochondrial membrane potential (Δψm) is the electric potential difference across the inner mitochondrial membrane. It requires a functioning respiratory chain in the absence of mechanisms that dissipate the proton gradient without coupling it to ATP production. Quantitative assessment of ΔΨm in living cells is most commonly achieved through the use of cationic, lipophilic fluorescent probes that accumulate within the mitochondrial matrix in proportion to the electrochemical gradient (Leonard et al., 2014). Among these, tetramethylrhodamine derivatives such as TMRE (tetramethylrhodamine ethyl ester) and TMRM (tetramethylrhodamine methyl ester) are widely employed due to their reversible, potential-dependent distribution across the inner mitochondrial membrane (Scaduto and Grotyohann, 1999; Creed and McKenzie, 2019). When applied at non-quenching, nanomolar concentrations, these dyes allow linear and quantitative detection of ΔΨm, as fluorescence intensity directly correlates with mitochondrial polarization. Detection can be performed by flow cytometry for population-level quantification, by high-content microscopy for spatially resolved analysis, or by fluorescence plate readers for higher throughput (Wong and Cortopassi, 2002; Valdebenito and Dunchen, 2022). Quantitative interpretation requires the use of appropriate controls, typically involving treatment with protonophores such as FCCP or CCCP, which fully dissipate ΔΨm and thereby establish baseline fluorescence, and inhibitors such as oligomycin or antimycin A to reveal different components of mitochondrial respiration. In parallel, dyes such as JC-1 are also used, though their ratiometric readout is less sensitive at low potentials and more prone to artifacts compared with TMRE or TMRM (Leonard et al., 2022). For accurate normalization, measurements are often corrected for cell number, mitochondrial content, or total protein, and fluorescence changes are expressed relative to maximal depolarization. In addition to chemical probes, genetically encoded sensors, such as mitochondria-targeted fluorescent proteins fused to potential-sensitive domains, provide complementary tools for ΔΨm monitoring in live-cell and in vivo contexts (Leonard et al., 2022). - Not endorsed  3. Enzymatic activity of the electron transport system (ETS). Determination of ETS activity can be dene following Owens and King's assay (1975). The technique is based on a cell-free homogenate that is incubated with NADH to saturate the mitochondrial ETS and an artificial electron acceptor [l - (4 -iodophenyl) -3 - (4 -nitrophenyl) -5-phenylte trazolium chloride (INT)] to register the electron transmission rate. The oxygen consumption rate is calculated from the molar production rate of INT-formazan which is determined spectrophotometrically (Cammen et al., 1990). 4. ATP content. For the evaluation of ATP levels, various commercially-available ATP assay kits are offered  based on luciferin and luciferase activity. For isolated mitochondria various methods are available to continuously measure ATP with electrodes (Laudet 2005), with luminometric methods, or for obtaining more information on different nucleotide phosphate pools (e.g. Ciapaite et al., (2005). <div> - Revision of AOP3 (Project: <a href="https://www.efsa.europa.eu/en/call/npefsaprev202402-development-aop-network-parkinsonian-motor-symptoms">NP/EFSA/PREV/2024/02</a>): Determination of mitochondrial ATP production based on extracellular flux analysis   The method is based on the detection of OCR (Oxygen Consumption Rate) that represents mitochondrial respiration as well as on the detection of ECAR (extracellular acidification rate) / proton efflux rate (PER): reflects extracellular acidification, a proxy for glycolysis (lactate release) plus contributions from CO₂/HCO₃⁻. PER is preferred over raw ECAR since it corrects for CO₂-derived acidification (Desousa et al., 2023; Espinosa et al., 2022). Application of inhibitors of individual complexes of the respiratory chain allows the detection of ATP-linked OCR: portion of oxygen consumption directly driving ATP synthesis (lost after ATP synthase inhibition) (Yoo et al., 2024). The proton leak & non-mitochondrial OCR represents remaining oxygen consumption after ATP synthase and electron transport chain inhibitor addition. The difference yields the ATP-coupled respiration component.   Calculation of mitochondrial ATP production  Mito ATP production rate (pmol ATP/min) = OCRATP (pmol O2/min) × 2 × P/O    OCR_ATP: ATP-coupled portion of OCR.   Factor 2: each O₂ molecule contains two oxygen atoms.   P/O ratio: number of ATP molecules synthesized per oxygen atom reduced. A mean P/O ≈ 2.75 is typically assumed (validated across many cell types but substrate- and condition-dependent) (Plitzko and Loesgen, 2018; Mookerjee et al., 2017; Motawe et al., 2024).    Limitations   P/O ratio varies by substrate (glucose vs. fatty acids), cell type, and conditions. Fixed values are approximations.   Non-mitochondrial oxygen consumption (oxidases, peroxidases, etc.) can confound OCR, hence use of ETC inhibitors.   PER vs. ECAR: CO₂-driven acidification must be corrected to avoid overestimating glycolytic ATP.   Normalization: results are usually expressed per cell, protein content, DNA, or mitochondrial mass — interpretation depends on normalization method.  - Not endorsed </div> II. Mitochondrial dysfunction assays assessing a gain-of function. 1. Mitochondrial permeability transition pore opening (PTP). The opening of the PTP is associated with a permeabilization of mitochondrial membranes, so that different compounds and cellular constituents can change intracellular localization. This can be measured by assessment of the translocation of cytochrome c, adenylate kinase or AIF from mitochondria to the cytosol or nucleus. The translocation can be assessed biochemically in cell fractions, by imaging approaches in fixed cells or tissues or by life-cell imaging of GFP fusion proteins (Single 1998; Modjtahedi 2006). An alternative approach is to measure the accessibility of cobalt to the mitochondrial matrix in a calcein fluorescence quenching assay in live permeabilized cells (Petronilli et al., 1999). 2. mtDNA damage as a biomarker of mitochondrial dysfunction. Various quantitative polymerase chain reaction (QPCR)-based assays have been developed to detect changes of DNA structure and sequence in the mitochondrial genome. mtDNA damage can be detected in blood after low-level rotenone exposure, and the damage persists even after CI activity has returned to normal. With a more sustained rotenone exposure, mtDNA damage is also detected in skeletal muscle. These data support the idea that mtDNA damage in peripheral tissues in the rotenone model may provide a biomarker of past or ongoing mitochondrial toxin exposure (Sanders et al., 2014a and 2014b). 3. Generation of ROS and resultant oxidative stress. a. General approach. Electrons from the mitochondrial ETS may be transferred ‘erroneously’ to molecular oxygen to form superoxide anions. This type of side reaction can be strongly enhanced upon mitochondrial damage. As superoxide may form hydrogen peroxide, hydroxyl radicals or other reactive oxygen species, a large number of direct ROS assays and assays assessing the effects of ROS (indirect ROS assays) are available (Adam-Vizi, 2005; Fan and Li 2014). Direct assays are based on the chemical modification of fluorescent or luminescent reporters by ROS species. Indirect assays assess cellular metabolites, the concentration of which is changed in the presence of ROS (e.g. glutathione, malonaldehyde, isoprostanes,etc.) At the animal level the effects of oxidative stress are measured from biomarkers in the blood or urine. b. Measurement of the cellular glutathione (GSH) status. GSH is regenerated from its oxidized form (GSSH) by the action of an NADPH dependent reductase (GSSH + NADPH + H+ à 2 GSH + NADP+). The ratio of GSH/GSSG is therefore a good indicator for the cellular NADH+/NADPH ratio (i.e. the redox potential). GSH and GSSH levels can be determined by HPLC, capillary electrophoresis, or biochemically with DTNB (Ellman’s reagent). As excess GSSG is rapidly exported from most cells to maintain a constant GSH/GSSG ratio, a reduction of total glutathione (GSH/GSSG) is often a good surrogate measure for oxidative stress. c. Quantification of lipid peroxidation. Measurement of lipid peroxidation has historically relied on the detection of thiobarbituric acid (TBA)-reactive compounds such as malondialdehyde generated from the decomposition of cellular membrane lipid under oxidative stress (Pryor et al., 1976). This method is quite sensitive, but not highly specific. A number of commercial assay kits are available for this assay using absorbance or fluorescence detection technologies. The formation of F2-like prostanoid derivatives of arachidonic acid, termed F2-isoprostanes (IsoP) has been shown to be more specific for lipid peroxidation. A number of commercial ELISA kits have been developed for IsoPs, but interfering agents in samples requires partial purification before analysis. Alternatively, GC/MS may be used, as robust (specific) and sensitive method. d. Detection of superoxide production. Generation of superoxide by inhibition of complex I and the methods for its detection are described by Grivennikova and Vinogradov (2014). A range of different methods is also described by BioTek (<a class="external free" href="http://www.biotek.com/resources/articles/reactive-oxygen-species.html" rel="nofollow" target="_blank">http://www.biotek.com/resources/articles/reactive-oxygen-species.html</a>). The reduction of ferricytochrome c to ferrocytochrome c may be used to assess the rate of superoxide formation (McCord, 1968). Like in other superoxide assays, specificity can only be obtained by measurements in the absence and presence of superoxide dismutase. Chemiluminescent reactions have been used for their increased sensitivity. The most widely used chemiluminescent substrate is lucigenin. Coelenterazine has also been used as a chemiluminescent substrate. Hydrocyanine dyes are fluorogenic sensors for superoxide and hydroxyl radical, and they become membrane impermeable after oxidation (trapping at site of formation). The best characterized of these probes are Hydro-Cy3 and Hydro-Cy5. generation of superoxide in mitochondria can be visualized using fluorescence microscopy with MitoSOX™ Red reagent (Life Technologies). MitoSOX™ Red reagent is a cationic derivative of dihydroethidium that permeates live cells and accumulates in mitochondria. e. Detection of hydrogen peroxide (H2O2) production. There are a number of fluorogenic substrates, which serve as hydrogen donors that have been used in conjunction with horseradish peroxidase (HRP) enzyme to produce intensely fluorescent products in the presence of hydrogen peroxide (Zhou et al., 1997: Ruch et al., 1983). The more commonly used substrates include diacetyldichloro-fluorescein, homovanillic acid, and Amplex® Red. In these examples, increasing amounts of H2O2 form increasing amounts of fluorescent product (Tarpley et al., 2004). Summing up, mitochondrial dysfunction can be measured by: • ROS production: superoxide (O2-), and hydroxyl radicals (OH−) • Nitrosative radical formation such as ONOO− or directly by: • Loss of mitochondrial membrane potential (MMP) • Opening of mitochondrial permeability transition pores (mPTP) • ATP synthesis • Increase in mitochondrial Ca2+ • Cytochrome c release • AIF (apoptosis inducing factor) release from mitochondria • Mitochondrial Complexes enzyme activity • Measurements of mitochondrial oxygen consumption • Ultrastructure of mitochondria using electron microscope and mitochondrial fragmentation measured by labelling with DsRed-Mito expression (Knott et al, 2008) Mitochondrial dysfunction-induced oxidative stress can be measured by: • Reactive carbonyls formations (proteins oxidation) • Increased 8-oxo-dG immunoreactivity (DNA oxidation) • Lipid peroxidation (formation of malondialdehyde (MDA) and 4- hydroxynonenal (HNE) • 3-nitrotyrosine (3-NT) formation, marker of protein nitration • Translocation of Bid and Bax to mitochondria • Measurement of intracellular free calcium concentration ([Ca2+]i): Cells are loaded with 4 μM fura-2/AM). • Ratio between reduced and oxidized form of glutathione (GSH depletion) (Promega assay, TB369; Radkowsky et al., 1986) • Neuronal nitric oxide synthase (nNOS) activation that is Ca2+-dependent. All above measurements can be performed as the assays for each readout are well established in the existing literature (e.g. Bal-Price and Brown, 2000; Bal-Price et al., 2002; Fujikawa, 2015; Walker et al., 1995). See also KE <a href="/wiki/index.php/Event:209" title="Event:209"> Oxidative Stress, Increase</a> <table border="1" cellpadding="1" cellspacing="1"> <tbody> <tr> <td> Assay Type & Measured Content </td> <td>Description</td> <td>Dose Range Studied</td> <td> Assay Characteristics (Length/Ease of use/Accuracy) </td> </tr> <tr> <td> Rhodamine 123 Assay Measuring Mitochondrial membrane potential (MMP) and its collapse  (Shaki et al., 2012) </td> <td> Mitochondrial uptake of cationic fluorescent dye, rhodamine 123, is used for estimation of mitochondrial membrane potential. The fluorescence was monitored using Schimadzou RF-5000U fluorescence spectrophotometer at the excitation and emission wavelength of 490 nm and 535 nm, respectively. </td> <td>50, 100 and 500 μM of uranyl acetate</td> <td> Short / easy Medium accurancy </td> </tr> <tr> <td> TMRE fluorescence Assay Measuring Mitochondrial permeability transition pore (mPTP) opening (Huser et al., 1998) </td> <td>Laser scanning confocal microscopy in combination with the potentiometric fluorescence dye tetramethylrhodamine ethyl ester to monitor relative changes in membrane potential in single isolated cardiac mitochondria. The cationic dye distributes across the membrane in a voltage-dependent manner. Therefore, the large potential gradient across the inner mitochondrial membrane results in the accumulation of the fluorescent dye within the matrix compartment. Rapid depolarizations are caused by the opening of the transition pore.</td> <td>1 µM cyclosporin A</td> <td> Short / easy Low accurancy </td> </tr> <tr> <td> GSH / GSSG Determination Assay Measuring  cellular glutathione (GSH) status; ratio of GSH/GSSG (Owen & Butterfield, 2010; Shaki et al., 2013) </td> <td>GSH and GSSG levels are determinted biochemically with DTNB (Ellman’s reagent). The developed yellow color was read at 412 nm on a spectrophotometer.</td> <td>100 µM uranyl acetate</td> <td> Short / easy Low accurancy </td> </tr> <tr> <td> TBARS Assay Quantification of lipid peroxidation (Yuan et al., 2016) </td> <td>MDA content, a product of lipid peroxidation, was measured using a thiobarbituric acid reactive substances (TBARS) assay. Briefly, the kidney cells were collected in 1 ml PBS buffer solution (pH 7.4) and sonicated. MDA reacts with thiobarbituric acid forming a colored product which can be measured at an absorbance of 532 nm.</td> <td>200, 400, 800 µM uranyl acetate</td> <td> Medium / medium High accurancy </td> </tr> <tr> <td> Aequorin-based bioluminescence assay Increase in mitochondrial Ca2+ influx (Pozzan & Rudolf, 2009) </td> <td>Together with GFP, the aequorin moiety acts as Ca2+ sensor in vivo, which delivers emission energy to the GFP acceptor molecule in a BRET (Bioluminescence Resonance Energy Transfer) process; the Ca2+ can then be visualized with fluorescence microscopy.</td> <td> </td> <td> Short / easy Low accurancy </td> </tr> <tr> <td> Western blot & immunostaining analyses Measuring cytochrome c release (Chen et al., 2000)</td> <td>Examining the redistribution of Cyto c in cytosolic and mitochondrial cellular fractions. Cells are homogenized and centrifuged, then prepared for immunoblots. Cellular fractions were washed in PBS and lysed in 1% NP-40 buffer. Cellular proteins were separated by SDS–PAGE, transferred onto nitrocellulose membranes, probed using immunoblot analyses with antibodies specific to cyto c (6581A for Western and 65971A for immunostaining; Pharmingen)</td> <td> </td> <td> Short / easy Medium accurancy </td> </tr> <tr> <td> Quantikine Rat/Mouse Cytochrome c Immunoassay Measuring cytochrome c release (Shaki et al., 2012) </td> <td>Cytochrome C release was measured a monoclonal antibody specific for rat/mouse cytochrome c was precoated onto the microplate. Seventy-five microliter of conjugate (containing mono- clonal antibody specific for cytochrome c conjugated to horseradish peroxidase). After 2 h of incubation, the substrate solution (100 μl) was added to each well and incubated for 30 min. After 100 μl of the stop solution was added to each well; the optical density of each well was determined by the aforementioned microplate spectrophotometer set to 450 nm.</td> <td> </td> <td> Short / easy Low accurancy </td> </tr> <tr> <td> Membrane potential and cell viability – Flow Cytometry Measuring cytochrome c release (Kruidering et al., 1997) </td> <td>“Dc and viability were determined by analyzing the R123 and propidium iodide fluorescence intensity with a FACScan flow cytometer (Becton Dickinson, San Jose, CA) equipped with an argon laser, with the Lysis software program (Becton Dickinson). R123 is a cationic dye that accumulates in the negatively charged inner side of the mitochondria. When the potential drops, less R123 accumulates in the mitochondria, which results in a lower fluorescence signal. The potential was measured as follows: at the indicated times, a 500-ml sample of the cell suspension was taken and transferred to an Eppendorf minivial. To this sample, 100 ml of 6 mM R123 in buffer D was added. After incubation for 10 min at 37°C, the cell suspension was centrifuged for 5 min at 80 3 g. The cell pellet was resuspended in 200 ml of buffer D, containing 0.2 mM R123 and 10 mM propidium iodide, to prevent loss of R123 and to stain nonviable cells, respectively. The samples were transferred to FACScan tubes and analyzed immediately. Analysis was performed at a flow rate of 60 ml/min. R123 fluorescence was detected by the FL1 detector with an emission detection limit below 560 nm. Propidium iodide fluorescence was detected by the FL3 detector, with emission detection above 620 nm. Per sample 3,000 to 5,000 cells were counted (Van de Water et al., 1993)”</td> <td> </td> <td> Short / easy Medium accurancy </td> </tr> </tbody> </table>

Mitochondrial dysfunction is a universal event occurring in cells of any species (Farooqui and Farooqui, 2012). Many invertebrate species (drosophila, C, elegans) are considered as potential models to study mitochondrial function. New data on marine invertebrates, such as molluscs and crustaceans and non-Drosophila species, are emerging (Martinez-Cruz et al., 2012). Mitochondrial dysfunction can be measured in animal models used for toxicity testing (Winklhofer and Haass, 2010; Waerzeggers et al., 2010) as well as in humans (Winklhofer and Haass, 2010). - Revision of AOP3 (Project: <a href="https://www.efsa.europa.eu/en/call/npefsaprev202402-development-aop-network-parkinsonian-motor-symptoms" rel="noreferrer noopener" target="_blank">NP/EFSA/PREV/2024/02</a>): Endogenous ROS formation by complex I: In mammals, complex I is a dominant site of mitochondrial ROS, especially via RET. In plants (Senkler et al. 2017; Maldonado), mitochondria contain alternative NAD(P)H dehydrogenases and an alternative oxidase (AOX) that bypass Complex I and III These pathways reduce ROS formation by preventing over-reduction of the ETC. Complex I still produces ROS, but generally less damaging due to AOX. Yeast: S. cerevisiae lacks a canonical Complex I entirely, relying instead on alternative NADH dehydrogenases. Consequently, mitochondrial ROS production from a Complex I-like source is absent. Other fungi with true Complex I (e.g., Neurospora crassa) do generate ROS similar to animals. - Not endorsed

UBERON:0000062

UBERON organ

CL:0000255

CL eukaryotic cell

High Male High Female

Not Specified

All life stages

High High High High High

Adam-Vizi V. Production of reactive oxygen species in brain mitochondria: contribution by electron transport chain and non-electron transport chain sources. Antioxid Redox Signal. 2005, 7(9-10):1140-1149. Adam-Vizi, V., & Starkov, A. A. (2010). Calcium and mitochondrial reactive oxygen species generation: How to read the facts. Journal of Alzheimer's Disease : JAD, 20 Suppl 2, S413-S426. doi:10.3233/JAD-2010-100465 Adiele, R. C., Stevens, D., & Kamunde, C. (2012). Differential inhibition of electron transport chain enzyme complexes by cadmium and calcium in isolated rainbow trout (oncorhynchus mykiss) hepatic mitochondria. Toxicological Sciences, 127(1), 110-119. doi:10.1093/toxsci/kfs091 Bal-Price A. and Guy C. Brown. Nitric-oxide-induced necrosis and apoptosis in PC12 cells mediated by mitochondria. J. Neurochemistry, 2000, 75: 1455-1464. Bal-Price A, Matthias A, Brown GC., Stimulation of the NADPH oxidase in activated rat microglia removes nitric oxide but induces peroxynitrite production. J. Neurochem. 2002, 80: 73-80. Belyaeva, E. A., Sokolova, T. V., Emelyanova, L. V., & Zakharova, I. O. (2012). Mitochondrial electron transport chain in heavy metal-induced neurotoxicity : Effects of cadmium , mercury , and copper. Thescientificworld, 2012, 1-14. doi:10.1100/2012/136063 Blajszczak, C., & Bonini, M. G. (2017). Mitochondria targeting by environmental stressors: Implications for redox cellular signaling. Toxicology, 391, 84-89. doi:10.1016/j.tox.2017.07.013 Brand MD, Nicholls DG. Assessing mitochondrial dysfunction in cells. Biochem J. 2011 Apr 15;435(2):297-312. Braun RJ. (2012). Mitochondrion-mediated cell death: dissecting yeast apoptosis for a better understanding of neurodegeneration. Front Oncol 2:182. Cammen M. Corwin, Susannah Christensen. John P. (1990) Electron transport system (ETS) activity as a measure of benthic macrofaunal metabolism MARINE ECOLOGY PROGRESS SERIES- (65) : 171-182. Chen, Q., Gong, B., & Almasan, A. (2000). Distinct stages of cytochrome c release from mitochondria: Evidence for a feedback amplification loop linking caspase activation to mitochondrial dysfunction in genotoxic stress induced apoptosis. Cell Death and Differentiation, 7(2), 227-233. doi:10.1038/sj.cdd.4400629 Ciapaite, Lolita Van Eikenhorst, Gerco Bakker, Stephan J.L. Diamant, Michaela. Heine, Robert J Wagner, Marijke J. V. Westerhoff, Hans and Klaas Krab (2005) Modular Kinetic Analysis of the Adenine Nucleotide Translocator–Mediated Effects of Palmitoyl-CoA on the Oxidative Phosphorylation in Isolated Rat Liver Mitochondria Diabetes 54:4 944-951. Correia SC, Santos RX, Perry G, Zhu X, Moreira PI, Smith MA. (2012). Mitochondrial importance in Alzheimer’s, Huntington’s and Parkinson’s diseases. Adv Exp Med Biol 724:205 – 221. Cozzolino M, Ferri A, Valle C, Carri MT. (2013). Mitochondria and ALS: implications from novel genes and pathways. Mol Cell Neurosci 55:44 – 49. Creed S, McKenzie M. Measurement of Mitochondrial Membrane Potential with the Fluorescent Dye Tetramethylrhodamine Methyl Ester (TMRM). Methods Mol Biol. 2019;1928:69-76. doi: 10.1007/978-1-4939-9027-6_5. PMID: 30725451.  Desousa BR, Kim KK, Jones AE, Ball AB, Hsieh WY, Swain P, Morrow DH, Brownstein AJ, Ferrick DA, Shirihai OS, Neilson A, Nathanson DA, Rogers GW, Dranka BP, Murphy AN, Affourtit C, Bensinger SJ, Stiles L, Romero N, Divakaruni AS. Calculation of ATP production rates using the Seahorse XF Analyzer. EMBO Rep. 2023 Oct 9;24(10):e56380. doi: 10.15252/embr.202256380. Epub 2023 Aug 7. PMID: 37548091; PMCID: PMC10561364.  Diepart, C, Verrax, J Calderon, PU, Feron, O., Jordan, BF, Gallez, B (2010) Comparison of methods for measuring oxygen consumption in tumor cells in vitroAnalytical Biochemistry 396 (2010) 250–256. Espinosa JA, Pohan G, Arkin MR, Markossian S. Real-Time Assessment of Mitochondrial Toxicity in HepG2 Cells Using the Seahorse Extracellular Flux Analyzer. Curr Protoc. 2021 Mar;1(3):e75. doi: 10.1002/cpz1.75. Erratum in: Curr Protoc. 2022 Aug;2(8):e551. doi: 10.1002/cpz1.551. PMID: 33735523.  Farooqui T. and . Farooqui, A. A (2012) Oxidative stress in Vertebrates and Invertebrate: molecular aspects of cell signalling. Wiley-Blackwell,Chapter 27, pp:377- 385. Fan LM, Li JM. Evaluation of methods of detecting cell reactive oxygen species production for drug screening and cell cycle studies. J Pharmacol Toxicol Methods. 2014 Jul-Aug;70(1):40-7. Fiskum G. Mitochondrial participation in ischemic and traumatic neural cell death. J Neurotrauma. 2000 Oct;17(10):843-55. Friberg H, Wieloch T. (2002). Mitochondrial permeability transition in acute neurodegeneration. Biochimie 84:241–250. Fujikawa DG, The Role of Excitotoxic Programmed Necrosis in Acute Brain Injury. Computational and Structural Biotechnology Journal, 2015, 13: 212–221. Graier WF, Frieden M, Malli R. (2007). Mitochondria and Ca2+ signaling: old guests, new functions. Pflugers Arch 455:375–396. Green DR. (1998). Apoptotic pathways: the roads to ruin. Cell 94:695-698. Grivennikova VG, Vinogradov AD. Generation of superoxide by the mitochondrial Complex I. Biochim Biophys Acta. 2006 May-Jun;1757(5-6):553-61. doi: 10.1016/j.bbabio.2006.03.013. Epub 2006 Apr 17. PMID: 16678117.   Hafner RP, Brown GC, Brand MD: Analysis of the control of respiration rate, phosphorylation rate, proton leak rate and protonmotive force in isolated mitochondria using the ‘top-down’ approach of metabolic control theory. Eur J Biochem188 :313 –319,1990. Hancock, J. T., Desikan, R., & Neill, S. J. (2001). Role of reactive oxygen species in cell signalling pathways. Biochemical Society Transactions, 29(Pt 2), 345-350. doi:10.1042/0300-5127:0290345 [doi] Hao, Y., Huang, J., Liu, C., Li, H., Liu, J., Zeng, Y., . . . Li, R. (2016). Differential protein expression in metallothionein protection from depleted uranium-induced nephrotoxicity. Scientific Reports, doi:10.1038/srep38942 Hao, Y., Ren, J., Liu, C., Li, H., Liu, J., Yang, Z., . . . Su, Y. (2014). Zinc protects human kidney cells from depleted uranium induced apoptosis. Basic & Clinical Pharmacology & Toxicology, 114, 271-280. doi:10.1111/bcpt.12167 Hinkle PC (1995) Measurement of ADP/O ratios. In Bioenergetics: A Practical Approach. Brown GC, Cooper CE, Eds. Oxford, U.K., IRL Press, p.5 –6. Huerta-García, E., Perez-Arizti, J. A., Marquez-Ramirez, S. G., Delgado-Buenrostro, N. L., Chirino, Y. I., Iglesias, G. G., & Lopez-Marure, R. (2014). Titanium dioxide nanoparticles induce strong oxidative stress and mitochondrial damage in glial cells. Free Radical Biology and Medicine, 73, 84-94. doi:10.1016/j.freeradbiomed.2014.04.026 Hüser, J., Rechenmacher, C. E., & Blatter, L. A. (1998). Imaging the permeability pore transition in single mitochondria. Biophysical Journal, 74(4), 2129-2137. doi:10.1016/S0006-3495(98)77920-2 Hynes, J.. Marroquin, L.D Ogurtsov, V.I. Christiansen, K.N. Stevens, G.J. Papkovsky, D.B. Will, Y. (2006)) Investigation of drug-induced mitochondrial toxicity using fluorescence-based oxygen-sensitive probes, Toxicol. Sci. 92 186–200. James, P.E. Jackson, S.K.. Grinberg, O.Y Swartz, H.M. (1995) The effects of endotoxin on oxygen consumption of various cell types in vitro: an EPR oximetry study, Free Radic. Biol. Med. 18 (1995) 641–647. Kang J, Pervaiz S. (2012). Mitochondria: Redox Metabolism and Dysfunction. Biochem Res Int 2012:896751. Kann O, Kovács R. (2007). Mitochondria and neuronal activity. Am J Physiol Cell Physiol 292:C641-576. Karlsson, H. L., Gustafsson, J., Cronholm, P., & Möller, L. (2009). Size-dependent toxicity of metal oxide particles—A comparison between nano- and micrometer size. Toxicology Letters, 188(2), 112-118. doi:<a href="https://doi.org/10.1016/j.toxlet.2009.03.014" target="_blank">10.1016/j.toxlet.2009.03.014</a> Knott Andrew B., Guy Perkins, Robert Schwarzenbacher & Ella Bossy-Wetzel. Mitochondrial fragmentation in neurodegeneration. Nature Reviews Neuroscience, 2008, 229: 505-518. Kruidering, M., Van De Water, B., De Heer, E., Mulder, G. J., & Nagelkerke, J. F. (1997). Cisplatin-induced nephrotoxicity in porcine proximal tubular cells: Mitochondrial dysfunction by inhibition of complexes I to IV of the respiratory chain. The Journal of Pharmacology and Experimental Therapeutics, 280(2), 638-649. Llaudet E, Hatz S, Droniou M, Dale N. Microelectrode biosensor for real-time measurement of ATP in biological tissue. Anal Chem. 2005, 77(10):3267-73. Lee HC, Wei YH. (2012). Mitochondria and aging. Adv Exp Med Biol 942:311-327. Leonard AP, Cameron RB, Speiser JL, Wolf BJ, Peterson YK, Schnellmann RG, Beeson CC, Rohrer B. Quantitative analysis of mitochondrial morphology and membrane potential in living cells using high-content imaging, machine learning, and morphological binning. Biochim Biophys Acta. 2015 Feb;1853(2):348-60. doi: 10.1016/j.bbamcr.2014.11.002. Epub 2014 Nov 13. PMID: 25447550; PMCID: PMC4289477.  Li N, Ragheb K, Lawler G, Sturgis J, Rajwa B, et al. Mitochondrial complex I inhibitor rotenone induces apoptosis through enhancing mitochondrial reactive oxygen species production. J Biol Chem.2003;278:8516–8525. Lin MT, Beal MF. Mitochondrial dysfunction and oxidative stress in neurodegenerative diseases. Nature 2006. 443:787-795. Maldonado M, Padavannil A, Zhou L, Guo F, Letts JA. Atomic structure of a mitochondrial complex I intermediate from vascular plants. Elife. 2020 Aug 25;9:e56664. doi: 10.7554/eLife.56664. PMID: 32840211; PMCID: PMC7447434.  Martin LJ. (2011). Mitochondrial pathobiology in ALS. J Bioenerg Biomembr 43:569 – 579. Martinez-Cruz, Oliviert Sanchez-Paz, Arturo Garcia-Carreño, Fernando Jimenez-Gutierrez, Laura Ma. de los Angeles Navarrete del Toro and Adriana Muhlia-Almazan. Invertebrates Mitochondrial Function and Energetic Challenges (www.intechopen.com), Bioenergetics, Edited by Dr Kevin Clark, <a class="internal mw-magiclink-isbn" href="/wiki/index.php/Special:BookSources/9789535100904">ISBN 978-953-51-0090-4</a>, Publisher InTech, 2012, 181-218. Mathews, C. K., Holde, K. E. van, Appling, D. R., & Anthony-Cahill, S. J. (2013). Biochemistry (4th ed.). Toronto: Pearson. McBride HM, Neuspiel M, Wasiak S. (2006). Mitochondria: more than just a powerhouse. Curr Biol 16:R551–560. McCord, J.M. and I. Fidovich (1968) The Reduction of Cytochrome C by Milk Xanthine Oxidase. J. Biol. Chem. 243:5733-5760. Mei Y, Thompson MD, Cohen RA, Tong X. (2013) Endoplasmic Reticulum Stress and Related Pathological Processes. J Pharmacol Biomed Anal.. 1:100-107. Miccadei, S., & Floridi, A. (1993). Sites of inhibition of mitochondrial electron transport by cadmium. Elsevier Scientific Publishers Ireland Ltd., 89, 159-167.Xu, X. M., & Møller, S. G. (2010). ROS removal by DJ-1: Arabidopsis as a new model to understand Parkinson's Disease. Plant signaling & behavior, 5(8), 1034–1036. doi:10.4161/psb.5.8.12298 Modjtahedi N, Giordanetto F, Madeo F, Kroemer G. Apoptosis-inducing factor: vital and lethal. Trends Cell Biol. 2006 May;16(5):264-72. Mookerjee SA, Gerencser AA, Nicholls DG, Brand MD. Quantifying intracellular rates of glycolytic and oxidative ATP production and consumption using extracellular flux measurements. J Biol Chem. 2017 Apr 28;292(17):7189-7207. doi: 10.1074/jbc.M116.774471. Epub 2017 Mar 7. Erratum in: J Biol Chem. 2018 Aug 10;293(32):12649-12652. doi: 10.1074/jbc.AAC118.004855. PMID: 28270511; PMCID: PMC5409486.  Motawe ZY, Abdelmaboud SS, Breslin JW. Evaluation of Glycolysis and Mitochondrial Function in Endothelial Cells Using the Seahorse Analyzer. Methods Mol Biol. 2024;2711:241-256. doi: 10.1007/978-1-0716-3429-5_20. PMID: 37776463; PMCID: PMC11368073. Nunnari J, Suomalainen A. (2012). Mitochondria: in sickness and in health. Cell 148:1145–1159. Hajnóczky G, Csordás G, Das S, Garcia-Perez C, Saotome M, Sinha Roy S, Yi M. (2006). Mitochondrial calcium signalling and cell death: approaches for assessing the role of mitochondrial Ca2+ uptake in apoptosis. Cell Calcium 40:553-560. Oliviert Martinez-Cruz, Arturo Sanchez-Paz, Fernando Garcia-Carreño, Laura Jimenez-Gutierrez, Ma. de los Angeles Navarrete del Toro and Adriana Muhlia-Almazan. Invertebrates Mitochondrial Function and Energetic Challenges (www.intechopen.com), Bioenergetics, Edited by Dr Kevin Clark, <a href="/wiki/index.php/Special:BookSources/9789535100904">ISBN 978-953-51-0090-4</a>, Publisher InTech, 2012, 181-218. Orrenius, S., Gogvadze, V., & Zhivotovsky, B. (2015). Calcium and mitochondria in the regulation of cell death. Biochemical and Biophysical Research Communications, 460(1), 72-81. doi:10.1016/j.bbrc.2015.01.137 Owen, J. B., & Butterfield, D. A. (2010). Measurement of oxidized/reduced glutathione ratio. Methods in Molecular Biology (Clifton, N.J.), 648, 269-277. doi:10.1007/978-1-60761-756-3_18 [doi] Owens R.G. and King F.D. The measurement of respiratory lectron-transport system activity in marine zooplankton. Mar. Biol. 1975, 30:27-36. Pan, Y., Leifer, A., Ruau, D., Neuss, S., Bonrnemann, J., Schmid, G., . . . Jahnen-Dechent, W. (2009). Gold nanoparticles of diameter 1.4 nm trigger necrosis by oxidative stress and mitochondrial damage. Small, 5(8), 2067-2076. doi:10.1002/smll.200900466 Petronilli V, Miotto G, Canton M, Brini M, Colonna R, Bernardi P, Di Lisa F: Transient and long-lasting openings of the mitochondrial permeability transition pore can be monitored directly in intact cells by changes in mitochondrial calcein fluorescence. Biophys J 1999, 76:725-734. Plitzko B, Loesgen S. Measurement of Oxygen Consumption Rate (OCR) and Extracellular Acidification Rate (ECAR) in Culture Cells for Assessment of the Energy Metabolism. Bio Protoc. 2018 May 20;8(10):e2850. doi: 10.21769/BioProtoc.2850. PMID: 34285967; PMCID: PMC8275291.  Pourahmad, J., Ghashang, M., Ettehadi, H. A., & Ghalandari, R. (2006). A search for cellular and molecular mechanisms involved in depleted uranium (DU) toxicity. Environmental Toxicology, 21(4), 349-354. doi:10.1002/tox.20196 Pozzan, T., & Rudolf, R. (2009). Measurements of mitochondrial calcium in vivo. Biochimica Et Biophysica Acta (BBA) - Bioenergetics, 1787(11), 1317-1323. doi:<a href="https://doi.org/10.1016/j.bbabio.2008.11.012" target="_blank">https://doi.org/10.1016/j.bbabio.2008.11.012</a> Promega GSH-Glo Glutathione Assay Technical Bulletin, TB369, Promega Corporation, Madison, WI. Pryor, W.A., J.P. Stanley, and E. Blair. (1976) Autoxidation of polyunsaturated fatty acids: II. A Suggested mechanism for the Formation of TBA-reactive materials from prostaglandin-like Endoperoxides. Lipids, 11:370-379. Radkowsky, A.E. and E.M. Kosower (1986) Bimanes 17. (Haloalkyl)-1,5-diazabicyclo[3.3.O]octadienediones (halo-9,10- dioxabimanes): reactivity toward the tripeptide thiol, glutathione, J. Am. Chem. Soc 108:4527-4531. Roos, D., Seeger, R., Puntel, R., & Vargas Barbosa, N. (2012). Role of calcium and mitochondria in MeHg-mediated cytotoxicity. Journal of Biomedicine and Biotechnology, 2012, 1-15. doi:10.1155/2012/248764 Ruch, W., P.H. Cooper, and M. Baggiollini (1983) Assay of H2O2 production by macrophages and neutrophils with Homovanillic acid and horseradish peroxidase. J. Immunol Methods 63:347-357. Sanders LH, McCoy J, Hu X, Mastroberardino PG, Dickinson BC, Chang CJ, Chu CT, Van Houten B, Greenamyre JT. (2014a). Mitochondrial DNA damage: molecular marker of vulnerable nigral neurons in Parkinson's disease. Neurobiol Dis. 70:214-23. Sanders LH, Howlett EH2, McCoy J, Greenamyre JT. (2014b) Mitochondrial DNA damage as a peripheral biomarker for mitochondrial toxin exposure in rats. Toxicol Sci. Dec;142(2):395-402. Santos, N. A. G., Catão, C. S., Martins, N. M., Curti, C., Bianchi, M. L. P., & Santos, A. C. (2007). Cisplatin-induced nephrotoxicity is associated with oxidative stress, redox state unbalance, impairment of energetic metabolism and apoptosis in rat kidney mitochondria. Archives of Toxicology, 81(7), 495-504. doi:10.1007/s00204-006-0173-2 Scaduto RC Jr, Grotyohann LW. Measurement of mitochondrial membrane potential using fluorescent rhodamine derivatives. Biophys J. 1999 Jan;76(1 Pt 1):469-77. doi: 10.1016/S0006-3495(99)77214-0. PMID: 9876159; PMCID: PMC1302536.  Senkler J, Senkler M, Braun HP. Structure and function of complex I in animals and plants - a comparative view. Physiol Plant. 2017 Sep;161(1):6-15. doi: 10.1111/ppl.12561. Epub 2017 Apr 26. Erratum in: Physiol Plant. 2018 Nov;164(3):364-365. doi: 10.1111/ppl.12844. PMID: 28261805.   Shaki, F., Hosseini, M. J., Ghazi-Khansari, M., & Pourahmad, J. (2012). Toxicity of depleted uranium on isolated rat kidney mitochondria. Biochimica Et Biophysica Acta - General Subjects, 1820(12), 1940-1950. doi:10.1016/j.bbagen.2012.08.015 Shaki, F., Hosseini, M., Ghazi-Khansari, M., & Pourahmad, J. (2013). Depleted uranium induces disruption of energy homeostasis and oxidative stress in isolated rat brain mitochondria. Metallomics, 5(6), 736-744. doi:10.1039/c3mt00019b Single B, Leist M, Nicotera P. Simultaneous release of adenylate kinase and cytochrome c in cell death. Cell Death Differ. 1998 Dec;5(12):1001-3. Tahira Farooqui and Akhlaq A. Farooqui. (2012) Oxidative stress in Vertebrates and Invertebrate: molecular aspects of cell signalling. Wiley-Blackwell,Chapter 27, pp:377- 385. Tarpley, M.M., D.A. Wink, and M.B. Grisham (2004) Methods for detection of reactive Metabolites of Oxygen and Nitrogen: in vitro and in vivo considerations. Am . J. Physiol Regul Integr Comp Physiol. 286:R431-R444. Valdebenito GE, Duchen MR. Monitoring Mitochondrial Membrane Potential in Live Cells Using Time-Lapse Fluorescence Imaging. Methods Mol Biol. 2022;2497:319-324. doi: 10.1007/978-1-0716-2309-1_22. PMID: 35771453.  Valko, M., Morris, H., & Cronin, M. T. (2005). Metals, toxicity and oxidative stress. Current Medicinal Chemistry, 12(10), 1161-1208. doi:10.2174/0929867053764635 [doi] von Heimburg, D. Hemmrich, K. Zachariah S.,. Staiger, H Pallua, N.(2005) Oxygen consumption in undifferentiated versus differentiated adipogenic mesenchymal precursor cells, Respir. Physiol. Neurobiol. 146 (2005) 107–116. Waerzeggers, Yannic Monfared, Parisa Viel, Thomas Winkeler, Alexandra Jacobs, Andreas H. (2010) Mouse models in neurological disorders: Applications of non-invasive imaging, Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease, Volume 1802, Issue 10, Pages 819-839. Walker JE, Skehel JM, Buchanan SK. (1995) Structural analysis of NADH: ubiquinone oxidoreductase from bovine heart mitochondria. Methods Enzymol.;260:14–34. Wang A, Costello S, Cockburn M, Zhang X, Bronstein J, Ritz B. (2011). Parkinson’s disease risk from ambient exposure to pesticides. Eur J Epidemiol 26:547-555. Wang, L., Li, J., Li, J., & Liu, Z. (2009). Effects of lead and/or cadmium on the oxidative damage of rat kidney cortex mitochondria. Biol.Trace Elem.Res., 137, 69-78. doi:10.1007/s12011-009-8560-1 Wang Y., and Qin ZH., Molecular and cellular mechanisms of excitotoxic neuronal death, Apoptosis, 2010, 15:1382-1402. Wieloch T. (2001). Mitochondrial Involvement in Acute Neurodegeneration 52:247–254. Winklhofer, K. Haass,C (2010) Mitochondrial dysfunction in Parkinson's disease, Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease, 1802: 29-44. Wong A, Cortopassi GA. High-throughput measurement of mitochondrial membrane potential in a neural cell line using a fluorescence plate reader. Biochem Biophys Res Commun. 2002 Nov 15;298(5):750-4. doi: 10.1016/s0006-291x(02)02546-9. PMID: 12419317.  Yoo I, Ahn I, Lee J, Lee N. Extracellular flux assay (Seahorse assay): Diverse applications in metabolic research across biological disciplines. Mol Cells. 2024 Aug;47(8):100095. doi: 10.1016/j.mocell.2024.100095. Epub 2024 Jul 18. PMID: 39032561; PMCID: PMC11374971.  Yuan, Y., Zheng, J., Zhao, T., Tang, X., & Hu, N. (2016). Uranium-induced rat kidney cell cytotoxicity is mediated by decreased endogenous hydrogen sulfide (H2S) generation involved in reduced Nrf2 levels. Toxicology Research, 5(2), 660-673. doi:10.1039/C5TX00432B Zhang, H., Chang, Z., Mehmood, K., Abbas, R. Z., Nabi, F., Rehman, M. U., . . . Zhou, D. (2018). Nano copper induces apoptosis in PK-15 cells via a mitochondria-mediated pathway. Biological Trace Element Research, 181(1), 62-70. doi:10.1007/s12011-017-1024-0 Zhou, M., Z.Diwu, Panchuk-Voloshina, N. and R.P. Haughland (1997), A Stable nonfluorescent derivative of resorufin for the fluorometric determination of trace hydrogen peroxide: application in detecting the activity of phagocyte NADPH oxidase and other oxidases. Anal. Biochem 253:162-168. AOPWiki 2016-11-29T18:41:23

2026-02-11T07:06:25

Increase, Cell injury/death

Cell injury/death

Cellular

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. 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 (see explanation below). Conversely, when apoptosis is massive, it can exceed the capacity for rapid phagocytosis, resulting in the eventual appearance of secondary necrosis. 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). 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).

Necrosis: 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).  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). 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) 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). 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). Moreover, quantification of ATP, signaling the presence of metabolically active cells, can be performed (CellTiter-Glo; Promega). ATP assay: Quantification of ATP, signaling the presence of metabolically active cells (CellTiter-Glo; Promega). Apoptosis: 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. 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). 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).  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.

Cell death is an universal event occurring in cells of any species (Fink and Cookson,2005).

CL:0000255

CL eukaryotic cell

Not Specified Unspecific

Not Specified

All life stages

High High High High

<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, <a class="external free" href="http://www.medscape.com/viewarticle/433631" rel="nofollow" target="_blank">http://www.medscape.com/viewarticle/433631</a> (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> AOPWiki 2016-11-29T18:41:22

2024-05-27T07:23:38

Increase, Kupffer cell activation

Cellular

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). <a href="#cite_note-1">[1]</a> KCs constitute 80%-90% of the tissue macrophages in the reticuloendothelial system and account for approximately 15% of the total liver cell population <a href="#cite_note-2">[2]</a> 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.<a href="#cite_note-Roberts2007-3">[3]</a> 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). <a href="#cite_note-4">[4]</a> <a href="#cite_note-Luckey_2001-5">[5]</a> 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 ceells (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<a href="#cite_note-6">[6]</a> <a href="#cite_note-7">[7]</a><a href="#cite_note-Roberts2007-3">[3]</a> 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. <a href="#cite_note-8">[8]</a> <a href="#cite_note-9">[9]</a> 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 <a href="#cite_note-10">[10]</a> <a href="#cite_note-11">[11]</a> 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. 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.<a href="#cite_note-12">[12]</a>

Kupffer cell activation can be measured by means of expressed cytokines, e.g. tissue levels of TNF-a <a href="#cite_note-13">[13]</a>, IL-6 expression, measured by immunoassays or Elisa (offered by various companies), soluble CD163 <a href="#cite_note-14">[14]</a> <a href="#cite_note-15">[15]</a> or increase in expression of Kupffer cell marker genes such as Lyz, Gzmb, and Il1b, (Genome U34A Array, Affymetrix); <a href="#cite_note-16">[16]</a>

Human: <a href="#cite_note-17">[17]</a><a href="#cite_note-18">[18]</a><a href="#cite_note-19">[19]</a> Rat: <a href="#cite_note-Luckey_2001-5">[5]</a> Mouse: <a href="#cite_note-20">[20]</a>

UBERON:0002107

UBERON liver

CL:0000091

CL Kupffer cell

High High High High

<ol> <li><a href="#cite_ref-1">↑</a> Haubrich, W.S. (2004), Kupffer of Kupffer cells, Gastroenterology, vol. 127, no. 1, p. 16.</li> <li><a href="#cite_ref-2">↑</a> 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.</li> <li>↑ <a href="#cite_ref-Roberts2007_3-0">3.0</a> <a href="#cite_ref-Roberts2007_3-1">3.1</a> 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><a href="#cite_ref-4">↑</a> 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> <li>↑ <a href="#cite_ref-Luckey_2001_5-0">5.0</a> <a href="#cite_ref-Luckey_2001_5-1">5.1</a> 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><a href="#cite_ref-6">↑</a> Guo, J. and S.L. Friedman (2007), Hepatic Fibrogenesis, Semin Liver Dis, vol. 27, no. 4, pp. 413-426.</li> <li><a href="#cite_ref-7">↑</a> Friedman, S.L. (2002), Hepatic Fibrosis-Role of Hepatic Stellate Cell Activation, MedGenMed, vol. 4, no. 3, pp. 27.</li> <li><a href="#cite_ref-8">↑</a> 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><a href="#cite_ref-9">↑</a> 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><a href="#cite_ref-10">↑</a> 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.</li> <li><a href="#cite_ref-11">↑</a> Tacke, F. and H.W. Zimmermann (2014), Macrophage heterogeneity in liver injury and fibrosis, J Hepatol, vol. 60, no. 5, pp. 1090-1096.</li> <li><a href="#cite_ref-12">↑</a> 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.</li> <li><a href="#cite_ref-13">↑</a> 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.</li> <li><a href="#cite_ref-14">↑</a> 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.</li> <li><a href="#cite_ref-15">↑</a> Møller, H.J. (2012), Soluble CD163.Scand J Clin Lab Invest, vol. 72, no. 1, pp. 1-13.</li> <li><a href="#cite_ref-16">↑</a> 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.</li> <li><a href="#cite_ref-17">↑</a> 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.</li> <li><a href="#cite_ref-18">↑</a> 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, <a class="external free" href="http://doi.org/10.1155/2015/640631" rel="nofollow" target="_blank">http://doi.org/10.1155/2015/640631</a>.</li> <li><a href="#cite_ref-19">↑</a> 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.</li> <li><a href="#cite_ref-20">↑</a> 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> </ol> AOPWiki 2016-11-29T18:41:22

2026-02-11T05:16:36

Increase, Steatohepatitis

Organ

Steatohepatitis is characterized by hepatic steatosis accompanied by hepatocellular injury and lobular inflammation. It represents a pathological progression beyond simple steatosis and is a defining feature of metabolic dysfunction–associated steatohepatitis (MASH). Histologically, steatohepatitis includes: <ul> <li> Macrovesicular steatosis </li> <li> Hepatocyte ballooning degeneration </li> <li> Lobular inflammatory cell infiltration </li> <li> Mallory–Denk bodies (in some cases) </li> <li> Variable degrees of perisinusoidal fibrosis </li> </ul>

<h3>1. Histopathology (Primary Method)</h3> <ul> <li> Hematoxylin and eosin (H&E) staining </li> <li> NAFLD Activity Score (NAS) </li> <li> Steatosis–Activity–Fibrosis (SAF) scoring system </li> <li> Assessment of: <ul> <li> Steatosis grade </li> <li> Ballooning degeneration </li> <li> Lobular inflammation </li> </ul> </li> </ul> Histological scoring systems are considered the gold standard for detection. <h3>2. Serum Biomarkers</h3> <ul> <li> Elevated ALT (alanine aminotransferase) </li> <li> Elevated AST (aspartate aminotransferase) </li> <li> Inflammatory cytokines (TNF-α, IL-6) </li> </ul> Biochemical markers provide supportive but not definitive evidence. <h3>3. Molecular Markers</h3> <ul> <li> Increased expression of inflammatory genes (e.g., TNF-α, MCP-1) </li> <li> Markers of hepatocyte injury (e.g., cytokeratin-18 fragments) </li> </ul> <h3>4. Imaging (Clinical Context)</h3> <ul> <li> MRI-PDFF (steatosis quantification) </li> <li> Elastography (for associated fibrosis) </li> </ul> However, histology remains required for definitive diagnosis of steatohepatitis.

Steatohepatitis is a well-defined pathological entity in humans and is reproducibly induced in rodent models of metabolic dysfunction and lipotoxic stress. The KE is most applicable to: <ul> <li> Mammalian species with comparable hepatic architecture </li> <li> Conditions involving chronic metabolic stress </li> <li> Adult or metabolically mature organisms </li> </ul> The weight of evidence for this KE is strong due to consistent clinical and experimental characterization.

UBERON:0002107

UBERON liver

High Unspecific

High

All life stages

High AOPWiki 2017-11-13T12:47:00

2026-02-24T09:13:44

Increase, Transforming growth factor-beta signaling

Activation of TGF-β signaling

Molecular

AOPWiki 2017-02-15T02:45:16

2026-02-11T05:39:19

Increase, Hepatic stellate cell activation

Increase, HSC activation

Cellular

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. 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).

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. 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).    <pre>  </pre>

Human: Friedman, 2008 Rat: George et al.,1999 Mouse: Chang et al., 2014 Pig: Costa et al., 2001

UBERON:0002107

UBERON liver

CL:0000632

CL hepatic stellate cell

Not Specified Unspecific

Not Specified

All life stages

High High High High High

<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> AOPWiki 2016-11-29T18:41:23

2026-02-11T07:04:19

Increase, Collagen accumulation

Tissue

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. 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). 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). 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. 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).

Determination of the amount of collagen produced in vitro can be done in a variety of ways ranging from simple colorimetric assays to elaborate chromatographic procedures using radioactive and non-radioactive material. What most of these procedures have in common is the need to destroy the cell layer to obtain solubilized collagen from the pericellular matrix. Rishikof et al. describe several methods to assess the in vitro production of type I collagen: Western immunoblotting of intact alpha1(I) collagen using antibodies directed to alpha1(I) collagen amino and carboxyl propeptides, the measurement of alpha1(I) collagen mRNA levels using real-time polymerase chain reaction, and methods to determine the transcriptional regulation of alpha1(I) collagen using a nuclear run-on assay (Rishikof et al., 2005).  Histological staining with stains such as Masson Trichrome, Picro-sirius red are used to identify the tissue/cellular distribution of collagen, which can be quantified using morphometric analysis both in vivo and in vitro. The assays are routinely used and are quantitative. Sircol Collagen Assay for collagen quantification: 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). Hydroxyproline assay: 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). Ex vivo precision cut tissue slices 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 1/4th of what will be used in whole animal exposure studies (Rahman et al., 2020).    <pre>  </pre>

Humans: Bataller and  Brenner, 2005; Decaris et al., 2015.   Mice: Dalton et al., 2009; Leung et al., 2008; Nan et al., 2013. Rats: Hamdy and El-Demerdash, 2012; Li et al., 2012; Luckey and Petersen, 2001; Natajaran et al., 2006.

UBERON:0002384

UBERON connective tissue

Not Specified Unspecific

Not Specified

All life stages

High High High

<ol> <li> Bataller R, Brenner DA. Liver fibrosis. J Clin Invest. 2005 Feb;115(2):209-18. doi: 10.1172/JCI24282.  </li> <li> Chen CZ, Raghunath M. Focus on collagen: in vitro systems to study fibrogenesis and antifibrosis state of the art. Fibrogenesis Tissue Repair. 2009 Dec 15;2:7. doi: 10.1186/1755-1536-2-7.  </li> <li> Dalton SR, Lee SM, King RN, Nanji AA, Kharbanda KK, Casey CA, McVicker BL. Carbon tetrachloride-induced liver damage in asialoglycoprotein receptor-deficient mice. Biochem Pharmacol. 2009 Apr 1;77(7):1283-90. doi: 10.1016/j.bcp.2008.12.023.  </li> <li> Decaris ML, Emson CL, Li K, Gatmaitan M, Luo F, Cattin J, Nakamura C, Holmes WE, Angel TE, Peters MG, Turner SM, Hellerstein MK. Turnover rates of hepatic collagen and circulating collagen-associated proteins in humans with chronic liver disease. PLoS One. 2015 Apr 24;10(4):e0123311. doi: 10.1371/journal.pone.0123311. </li> <li> Di Lullo GA, Sweeney SM, Korkko J, Ala-Kokko L, San Antonio JD. Mapping the ligand-binding sites and disease-associated mutations on the most abundant protein in the human, type I collagen. J Biol Chem. 2002 Feb 8;277(6):4223-31. doi: 10.1074/jbc.M110709200. </li> <li> Diegelmann R. Collagen Metabolism. Wounds. 2001;13:177-82. Available at www.medscape.com/viewarticle/423231 (accessed on 20 January 2016). </li> <li> 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.  </li> <li> 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. </li> <li> Kivirikko KI, Risteli L. Biosynthesis of collagen and its alterations in pathological states. Med Biol. 1976 Jun;54(3):159-86. </li> <li> 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.  </li> <li> 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.  </li> <li> 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. </li> <li> Miller EJ, Gay S. The collagens: an overview and update. Methods Enzymol. 1987;144:3-41. doi: 10.1016/0076-6879(87)44170-0.  </li> <li> 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. </li> <li> 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. </li> <li> 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.  </li> <li> 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.  </li> <li> 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. </li> <li> 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.  </li> </ol> AOPWiki 2016-11-29T18:41:22

2026-02-11T06:58:07

Increase, Liver fibrosis

Organ

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) 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).

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) .

Human: Bataller and Brenner, 2005;Merck Manual, 2015; Blachier et al., 2013. Rat, mouse: Liedtke et al., 2013

UBERON:0002107

UBERON liver

Not Specified Unspecific

Not Specified

All life stages

High High High

<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> AOPWiki 2016-11-29T18:41:24

2026-02-11T05:35:21

Increase, Regenerative nodule formation

Tissue

UBERON:0002107

UBERON liver

AOPWiki 2026-02-10T04:47:33

2026-02-10T06:47:33

Increase, Cirrhosis

Organ

Cirrhosis represents the end-stage consequence of chronic liver injury and sustained fibrogenesis. It is characterized by: <ul> <li> Extensive deposition of extracellular matrix (primarily type I collagen) </li> <li> Bridging fibrosis connecting portal and central regions </li> <li> Formation of regenerative nodules </li> <li> Distortion of normal hepatic architecture </li> <li> Altered vascular structure and portal hypertension </li> </ul>

<h3>1. Histopathology (Gold Standard)</h3> <ul> <li> Masson's trichrome staining </li> <li> Sirius Red staining for collagen deposition </li> <li> Assessment of: <ul> <li> Bridging fibrosis </li> <li> Nodular regeneration </li> <li> Architectural distortion </li> </ul> </li> </ul> Common scoring systems: <ul> <li> METAVIR (F4 stage indicates cirrhosis) </li> <li> Ishak fibrosis score </li> <li> SAF scoring system </li> </ul> <h3>2. Imaging (Clinical Context)</h3> <ul> <li> Transient elastography (FibroScan) </li> <li> Magnetic resonance elastography </li> <li> Ultrasound imaging (nodular surface, splenomegaly) </li> </ul> Imaging provides non-invasive assessment but histology confirms diagnosis. <h3>3. Serum Biomarkers (Supportive)</h3> <ul> <li> Decreased albumin </li> <li> Elevated bilirubin </li> <li> Prolonged prothrombin time </li> <li> Fibrosis biomarkers (e.g., hyaluronic acid, procollagen peptides) </li> </ul> These support functional impairment but do not independently confirm cirrhosis.

Cirrhosis is a clinically recognized end-stage liver disease in humans and is reproducible in mammalian models of chronic hepatic injury. The mechanisms of fibrosis progression and architectural remodeling are highly conserved in mammals. This KE is most applicable under: <ul> <li> Chronic exposure conditions </li> <li> Sustained inflammatory and fibrogenic signaling </li> <li> Adult or metabolically mature organisms </li> </ul> The biological plausibility is strong due to well-established fibrogenic pathways (e.g., TGF-β–mediated stellate cell activation) and consistent cross-species pathology.

UBERON:0002107

UBERON liver

Not Specified Unspecific

Not Specified

Adult

Not Specified Not Specified Not Specified Not Specified AOPWiki 2026-02-10T04:48:48

2026-02-11T07:34:30

<upstream-id>861e4d37-b7ac-45ee-87e4-7add8e597c0e</upstream-id> <downstream-id>34346224-6872-4ee1-aca2-4787039b629c</downstream-id>

AOPWiki 2026-02-24T08:32:30

2026-02-24T08:32:30

<upstream-id>34346224-6872-4ee1-aca2-4787039b629c</upstream-id> <downstream-id>094ea521-430e-40ac-bb7b-b3c60d898475</downstream-id>

AOPWiki 2026-02-24T08:32:45

2026-02-24T08:32:45

<upstream-id>094ea521-430e-40ac-bb7b-b3c60d898475</upstream-id> <downstream-id>13bf2bd9-c840-4212-8412-19530ae045d8</downstream-id>

AOPWiki 2026-02-12T07:41:40

2026-02-12T07:41:40

<upstream-id>13bf2bd9-c840-4212-8412-19530ae045d8</upstream-id> <downstream-id>845bad55-f1a7-4c4e-aaaf-36717d1d2fc1</downstream-id>

AOPWiki 2026-02-11T05:41:20

2026-02-11T05:41:20

<upstream-id>845bad55-f1a7-4c4e-aaaf-36717d1d2fc1</upstream-id> <downstream-id>0189ab59-d7df-4621-bef8-f222e9117e39</downstream-id>

AOPWiki 2026-02-10T08:59:43

2026-02-10T08:59:43

<upstream-id>0189ab59-d7df-4621-bef8-f222e9117e39</upstream-id> <downstream-id>54a555ce-3342-4bea-a51e-3fb9546c1403</downstream-id>

AOPWiki 2026-02-10T08:59:52

2026-02-10T08:59:52

<upstream-id>9daa396f-0caf-4995-9c72-a4642a8cb408</upstream-id> <downstream-id>1c6f22a8-44f1-462b-bd2f-505b88e4ef24</downstream-id> 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 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. <a href="#cite_note-Winwood_1993-1">[1]</a> <a href="#cite_note-Luckey_2001-2">[2]</a> <a href="#cite_note-Roberts_2007-3">[3]</a> <a href="#cite_note-Malhi_2010-4">[4]</a> <a href="#cite_note-Canbay_2004-5">[5]</a> <a href="#cite_note-Orrenius_2011-6">[6]</a> <a href="#cite_note-Kolios_2006-7">[7]</a> <a href="#cite_note-Kisseleva-8">[8]</a> <a href="#cite_note-Jaeschke_2011-9">[9]</a> <a href="#cite_note-Li_2008-10">[10]</a> <a href="#cite_note-Poli_2000-11">[11]</a>

There is a functional relationship between cell injury/death and KC activation, consistent with established biological knowledge. <a href="#cite_note-Winwood_1993-1">[1]</a> <a href="#cite_note-Luckey_2001-2">[2]</a> <a href="#cite_note-Roberts_2007-3">[3]</a> <a href="#cite_note-Malhi_2010-4">[4]</a> <a href="#cite_note-Canbay_2004-5">[5]</a> <a href="#cite_note-Orrenius_2011-6">[6]</a> <a href="#cite_note-Kolios_2006-7">[7]</a> <a href="#cite_note-Kisseleva-8">[8]</a> <a href="#cite_note-Jaeschke_2011-9">[9]</a> <a href="#cite_note-Li_2008-10">[10]</a> <a href="#cite_note-Poli_2000-11">[11]</a>

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.<a href="#cite_note-12">[12]</a><a href="#cite_note-13">[13]</a> 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 in vivo 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.<a href="#cite_note-Tukov_2006-14">[14]</a> Canbay et al could prove that engulfment of hepatocyte apoptotic bodies stimulated KC generation of cytokines. <a href="#cite_note-15">[15]</a>

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. <a href="#cite_note-Roberts_2007-3">[3]</a>

no quantitative data

High High

Human: <a href="#cite_note-Winwood_1993-1">[1]</a><a href="#cite_note-Roberts_2007-3">[3]</a><a href="#cite_note-Kolios_2006-7">[7]</a> Rat: <a href="#cite_note-Tukov_2006-14">[14]</a><a href="#cite_note-Roberts_2007-3">[3]</a>

<ol class="references"> <li id="cite_note-Winwood_1993-1">↑ <a href="#cite_ref-Winwood_1993_1-0">1.0</a> <a href="#cite_ref-Winwood_1993_1-1">1.1</a> <a href="#cite_ref-Winwood_1993_1-2">1.2</a> 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> <li id="cite_note-Luckey_2001-2">↑ <a href="#cite_ref-Luckey_2001_2-0">2.0</a> <a href="#cite_ref-Luckey_2001_2-1">2.1</a> 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 id="cite_note-Roberts_2007-3">↑ <a href="#cite_ref-Roberts_2007_3-0">3.0</a> <a href="#cite_ref-Roberts_2007_3-1">3.1</a> <a href="#cite_ref-Roberts_2007_3-2">3.2</a> <a href="#cite_ref-Roberts_2007_3-3">3.3</a> <a href="#cite_ref-Roberts_2007_3-4">3.4</a> 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 id="cite_note-Malhi_2010-4">↑ <a href="#cite_ref-Malhi_2010_4-0">4.0</a> <a href="#cite_ref-Malhi_2010_4-1">4.1</a> Malhi, H. et al. (2010), Hepatocyte death: a clear and present danger, Physiol Rev, vol. 90, no. 3, pp. 1165-1194. </li> <li id="cite_note-Canbay_2004-5">↑ <a href="#cite_ref-Canbay_2004_5-0">5.0</a> <a href="#cite_ref-Canbay_2004_5-1">5.1</a> 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 id="cite_note-Orrenius_2011-6">↑ <a href="#cite_ref-Orrenius_2011_6-0">6.0</a> <a href="#cite_ref-Orrenius_2011_6-1">6.1</a> 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 id="cite_note-Kolios_2006-7">↑ <a href="#cite_ref-Kolios_2006_7-0">7.0</a> <a href="#cite_ref-Kolios_2006_7-1">7.1</a> <a href="#cite_ref-Kolios_2006_7-2">7.2</a> 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 id="cite_note-Kisseleva-8">↑ <a href="#cite_ref-Kisseleva_8-0">8.0</a> <a href="#cite_ref-Kisseleva_8-1">8.1</a> Kisseleva T and Brenner DA, (2008), Mechanisms of Fibrogenesis, Exp Biol Med, vol. 233, no. 2, pp. 109-122. </li> <li id="cite_note-Jaeschke_2011-9">↑ <a href="#cite_ref-Jaeschke_2011_9-0">9.0</a> <a href="#cite_ref-Jaeschke_2011_9-1">9.1</a> 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 id="cite_note-Li_2008-10">↑ <a href="#cite_ref-Li_2008_10-0">10.0</a> <a href="#cite_ref-Li_2008_10-1">10.1</a> 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 id="cite_note-Poli_2000-11">↑ <a href="#cite_ref-Poli_2000_11-0">11.0</a> <a href="#cite_ref-Poli_2000_11-1">11.1</a> Poli, G. (2000), Pathogenesis of liver fibrosis: role of oxidative stress, Mol Aspects Med, vol. 21, no. 3, pp. 49 – 98. </li> <li id="cite_note-12"><a href="#cite_ref-12">↑</a> 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 id="cite_note-13"><a href="#cite_ref-13">↑</a> Soldatow, V.Y. et al. (2013), In vitro models for liver toxicity testing, Toxicol Res, vol. 2, no.1, pp. 23–39. </li> <li id="cite_note-Tukov_2006-14">↑ <a href="#cite_ref-Tukov_2006_14-0">14.0</a> <a href="#cite_ref-Tukov_2006_14-1">14.1</a> 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 id="cite_note-15"><a href="#cite_ref-15">↑</a> 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> </ol> AOPWiki 2016-11-29T18:41:33

2016-11-29T19:54:53

<upstream-id>1c6f22a8-44f1-462b-bd2f-505b88e4ef24</upstream-id> <downstream-id>a4861196-926d-41a0-bfaf-3b2e41aacacf</downstream-id>

AOPWiki 2026-02-10T09:00:33

2026-02-10T09:00:33

<upstream-id>a4861196-926d-41a0-bfaf-3b2e41aacacf</upstream-id> <downstream-id>681c59d7-40a7-485d-be1b-f86f07e3c2fa</downstream-id>

AOPWiki 2026-02-10T09:00:43

2026-02-10T09:00:43

<upstream-id>681c59d7-40a7-485d-be1b-f86f07e3c2fa</upstream-id> <downstream-id>f043f2b9-ec39-4bb5-946b-15211bc55d7e</downstream-id>

AOPWiki 2026-02-10T09:01:01

2026-02-10T09:01:01

<upstream-id>f043f2b9-ec39-4bb5-946b-15211bc55d7e</upstream-id> <downstream-id>46314f8b-4f00-4d6c-8017-30a84a6d26c1</downstream-id> 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. 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. 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).

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).

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).

no inconsistencies

no quantitative data

Not Specified Unspecific

Not Specified

All life stages

High High

Human: Safadi and Friedman, 2002; Bataller and Brenner, 2005; Lee und Friedman 2011. Rat: Li, Li et al., 2012; Luckey and Petersen, 2001;  Rockey et al., 1992

<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> AOPWiki 2016-11-29T18:41:33

2018-12-05T08:51:54

<upstream-id>46314f8b-4f00-4d6c-8017-30a84a6d26c1</upstream-id> <downstream-id>2439d8b3-0b5f-4baa-91ef-349d8221bb13</downstream-id> 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).

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).

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).

no inconsistencies

no quantitative data

Not Specified Unspecific

Not Specified

All life stages

High High

Human: Lee and Friedman, 2011; Bataller and Brenner, 2005; Brancatelli et al., 2009; Rockey and Friedman, 2006; Poynard et al., 1997.   Rat :Liedtke et al., 2013.

<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> AOPWiki 2016-11-29T18:41:33

2018-12-05T08:52:45

<upstream-id>2439d8b3-0b5f-4baa-91ef-349d8221bb13</upstream-id> <downstream-id>0cb786cb-185c-42a0-a4e4-aa045ff44e8d</downstream-id>

AOPWiki 2026-02-10T09:02:04

2026-02-10T09:02:04

<upstream-id>0cb786cb-185c-42a0-a4e4-aa045ff44e8d</upstream-id> <downstream-id>51af6d21-4309-49ca-a3ab-fec80fc6b4c7</downstream-id>

AOPWiki 2026-02-10T09:02:13

2026-02-10T09:02:13

Increased 11β-Hydroxysteroid dehydrogenase type 1 activity leading to MASLD progression via lipogenesis-associated mitochondrial dysfunction

Increased 11β-HSD1 leading to MASLD via DNL-associated mitochondrial dysfunction

Cataia Ives

You Song1, Jorke H. Kamstra2, Matej Oresic3,4 1 Norwegian Institute for Water Research, Økernveien 94, Oslo, Norway 2 Utrecht University, Institute for Risk Assessment Sciences (IRAS), Utrecht, the Netherlands 3 Örebro University, School of Medical Sciences, Örebro, Sweden 4 University of Turku, Turku Bioscience Centre, Turku, Finland   Acknowledgement: This project was supported by the “Investigation of endocrine-disrupting chemicals as contributors to progression of metabolic dysfunction-associated steatotic liver disease” (EDC-MASLD) consortium funded by the Horizon Europe Program of the European Union (Grant Agreement 101136259).  Shihori Tanabe

BY-SA

Under Development

2.7

his adverse outcome pathway (AOP) describes a mechanistic sequence linking altered glucocorticoid receptor (GR) signaling to the progression of metabolic dysfunction–associated steatotic liver disease (MASLD) through insulin resistance–associated endoplasmic reticulum (ER) stress. Disruption of GR signaling promotes systemic insulin resistance, enhanced adipose lipolysis, and increased hepatic free fatty acid (FFA) influx, resulting in hepatocellular lipotoxicity. Lipid overload and impaired insulin signaling induce ER stress, triggering unfolded protein response activation, hepatocyte injury, and inflammatory signaling. Subsequent activation of Kupffer cells, hepatic stellate cells, and TGF-β–mediated profibrotic pathways drives collagen deposition, fibrosis, and progression to cirrhosis. This AOP provides a biologically plausible and regulatory-relevant framework for identifying endocrine-disrupting chemicals (EDCs) that promote MASLD progression through GR-mediated insulin resistance and ER stress pathways. Endoplasmic reticulum (ER) stress is a central cellular stress response implicated in the progression from simple steatosis to steatohepatitis and fibrosis. In hepatocytes, insulin resistance, lipid overload, and disrupted metabolic signaling converge on the ER, overwhelming protein folding capacity and activating maladaptive unfolded protein response pathways. Chronic ER stress promotes hepatocyte injury, inflammatory signaling, and fibrogenesis. Glucocorticoid receptor (GR) signaling plays a key role in metabolic homeostasis, insulin sensitivity, and lipid flux regulation. Altered GR signaling—whether due to dysregulation or chemical interference—can induce systemic insulin resistance, increased adipose lipolysis, and hepatic lipid influx, thereby promoting ER stress. This AOP was developed to explicitly capture ER stress as a mechanistically distinct and biologically important mediator linking GR dysregulation to MASLD progression.

The AOP was developed using an expert-driven conceptual framework supported by targeted literature evaluation across endocrinology, metabolism, ER stress biology, and chronic liver disease. Initial scoping identified insulin resistance–associated ER stress as a recurring mechanistic feature in experimental and clinical MASLD progression. Focused literature searches were conducted to identify evidence supporting: <ul> <li> GR signaling perturbation and systemic insulin resistance </li> <li> Adipose lipolysis and hepatic FFA influx </li> <li> Induction of ER stress by lipid overload and insulin resistance </li> <li> ER stress–mediated hepatocyte injury and inflammatory activation </li> <li> Fibrogenic signaling driven by TGF-β and hepatic stellate cell activation </li> </ul> Evidence from human studies, animal models, and mechanistic in vitro systems was prioritized, with emphasis on chronic and low-dose perturbations relevant to endocrine disruption.

Steatosis is a regulatory endpoint and has been used as an endpoint in many US EPA assessments, including IRIS assessments.

Steatohepatitis represents a clinically recognized and pathologically defined stage of metabolic dysfunction–associated steatotic liver disease (MASLD), characterized by hepatic steatosis accompanied by hepatocellular injury and inflammation. It is a critical transition point between reversible metabolic steatosis and progressive, potentially irreversible liver pathology, including fibrosis, cirrhosis, and hepatocellular carcinoma. As such, an increase in steatohepatitis severity constitutes a biologically meaningful and adverse health outcome. <h2>Human Health Relevance</h2> Steatohepatitis (formerly NASH; now MASH under MASLD nomenclature) is associated with: <ul> <li> Increased risk of liver fibrosis progression </li> <li> Elevated liver-related morbidity and mortality </li> <li> Increased risk of hepatocellular carcinoma </li> <li> Higher overall cardiometabolic mortality </li> </ul> Histologically confirmed steatohepatitis is a strong predictor of disease progression compared to simple steatosis. Therefore, regulatory concern is substantially higher once inflammatory and hepatocellular injury components are present. <h2>Scientific Basis for Domain of Applicability</h2> <h3>Taxonomic Applicability</h3> The adverse outcome is highly relevant to mammals, particularly humans, due to conserved hepatic architecture, lipid metabolism, inflammatory signaling, and fibrogenic pathways. Rodent models (e.g., high-fat diet, Western diet, glucocorticoid exposure models) reliably reproduce key histopathological features of steatohepatitis, including: <ul> <li> Steatosis with hepatocyte ballooning </li> <li> Lobular inflammation </li> <li> Early perisinusoidal fibrosis </li> </ul> This cross-species concordance supports high biological plausibility within Mammalia. <h3>Life Stage Applicability</h3> The adverse outcome is most relevant in adolescent and adult life stages, where metabolic systems are fully developed and chronic exposure conditions can lead to progressive disease. While pediatric MASLD exists, the majority of mechanistic and regulatory evidence derives from adult populations and adult rodent models. <h3>Sex Applicability</h3> Steatohepatitis occurs in both males and females. Sex differences in susceptibility and progression rate have been reported, likely reflecting hormonal influences on lipid metabolism and inflammation. However, the pathological entity and its progression mechanisms are conserved across sexes. <h2>Weight of Evidence for Adversity</h2> The weight of evidence supporting steatohepatitis as an adverse outcome is strong based on: <ol> <li> Clinical Evidence <ul> <li> Histologically confirmed steatohepatitis predicts fibrosis progression and mortality. </li> <li> Longitudinal human studies demonstrate increased liver-related outcomes compared to simple steatosis. </li> </ul> </li> <li> Pathophysiological Evidence <ul> <li> Hepatocyte ballooning reflects cellular injury and cytoskeletal disruption. </li> <li> Inflammatory infiltration drives sustained tissue damage and fibrogenesis. </li> <li> Cytokine and TGF-β signaling link inflammation directly to fibrosis progression. </li> </ul> </li> <li> Consistency Across Models <ul> <li> Reproducible induction in multiple rodent models. </li> <li> Mechanistic concordance between experimental systems and human disease. </li> </ul> </li> <li> Irreversibility Consideration <ul> <li> While early steatohepatitis may be partially reversible, sustained inflammation significantly increases the probability of irreversible fibrosis. </li> </ul> </li> </ol> <h2>Regulatory Relevance</h2> An increase in steatohepatitis severity represents: <ul> <li> A clear adverse effect at the organ level </li> <li> A progression beyond adaptive metabolic perturbation </li> <li> A disease-defining pathological state recognized in clinical practice </li> </ul> From a regulatory perspective, this adverse outcome is relevant for: <ul> <li> Hazard identification </li> <li> Chronic toxicity assessment </li> <li> Endocrine and metabolic disruptor evaluation </li> <li> Integration into adverse outcome pathways supporting chemical prioritization </li> </ul> Because steatohepatitis is a well-defined diagnostic and pathological entity with established clinical consequences, it provides a robust anchor for AOP-based risk assessment frameworks.

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)"

An increase in cirrhosis represents: <ul> <li> A severe, adverse organ-level outcome </li> <li> Largely irreversible structural liver damage </li> <li> A major determinant of liver-related morbidity and mortality </li> </ul> From a regulatory perspective, cirrhosis constitutes a high-concern adverse outcome suitable for: <ul> <li> Chronic toxicity hazard identification </li> <li> Chemical prioritization </li> <li> Risk assessment frameworks </li> </ul> Because cirrhosis reflects irreversible architectural and functional liver impairment, it anchors the most severe end of MASLD progression within the AOP network.

adjacent

Not Specified

Not Specified adjacent

Not Specified

Not Specified adjacent

Not Specified

Not Specified adjacent

Not Specified

Not Specified adjacent

Not Specified

Not Specified adjacent

Not Specified

Not Specified adjacent

Not Specified

Not Specified adjacent

Not Specified

Not Specified adjacent

Not Specified

Not Specified adjacent

Not Specified

Not Specified adjacent

Not Specified

Not Specified adjacent

Not Specified

Not Specified adjacent

Not Specified

Not Specified adjacent

Not Specified

Not Specified Not Specified Not Specified

This AOP is biologically plausible and supported by moderate to strong empirical evidence linking insulin resistance, ER stress, and progressive liver injury. The sequence of key events reflects conserved cellular stress and inflammatory mechanisms observed across mammalian species and aligns with established pathological features of MASLD progression. The AOP is particularly relevant for hazard identification and prioritization of chemicals that interfere with GR signaling and metabolic regulation but may not directly target mitochondrial pathways. It is well suited for integration into AOP networks describing multiple converging routes to MASLD. <ul> <li> Taxa: Mammals (humans and laboratory rodents) </li> <li> Life stage: Primarily adolescents and adults </li> <li> Sex: Applicable to both sexes; sex-specific differences may occur due to hormonal and metabolic modulation </li> <li> Biological context: Chronic metabolic stress, insulin resistance, and endocrine perturbation </li> </ul> This AOP is not intended to represent acute hepatotoxicity and is most applicable to chronic exposure scenarios.

Evidence supporting the essentiality of the key events includes: <ul> <li> Altered GR signaling: Experimental modulation of GR activity alters insulin sensitivity and lipid flux. </li> <li> Insulin resistance: Genetic and pharmacological induction or attenuation of insulin resistance directly influences hepatic lipid accumulation and ER stress. </li> <li> ER stress: Inhibition of ER stress pathways reduces hepatocyte injury, inflammation, and disease severity in experimental MASLD models. </li> <li> Inflammatory and fibrogenic activation: Suppression of Kupffer cell activation, hepatic stellate cell activation, or TGF-β signaling attenuates fibrosis progression. </li> </ul> Together, these findings support the essential role of each KE in driving downstream MASLD outcomes.

Across the KERs in this AOP: <ul> <li> Biological plausibility is strong, supported by well-characterized links between insulin resistance, lipid overload, ER stress, and liver injury. </li> <li> Empirical support is moderate to strong, with consistent directional evidence across in vivo and in vitro models. </li> <li> Quantitative understanding is limited, particularly regarding thresholds for ER stress–induced transition from adaptive to maladaptive responses. </li> </ul> Overall, the weight of evidence supports confidence in the pathway for regulatory-relevant applications.

<table> <thead> <tr> <th>Modulating Factor (MF)</th> <th>Influence or Outcome</th> <th>KER(s) involved</th> </tr> </thead> <tbody> <tr> <td>Nutrient excess</td> <td>Exacerbates insulin resistance and ER stress</td> <td>Insulin resistance → ER stress</td> </tr> <tr> <td>Lipid species composition</td> <td>Modulates ER membrane stress and toxicity</td> <td>Lipotoxicity → ER stress</td> </tr> <tr> <td>Chaperone capacity</td> <td>Influences resilience to ER stress</td> <td>ER stress → cell injury</td> </tr> <tr> <td>Inflammatory milieu</td> <td>Amplifies hepatocyte injury and fibrosis</td> <td>Cell injury → fibrosis</td> </tr> </tbody> </table>

Quantitative relationships have been described for individual links between insulin resistance, lipid overload, and ER stress markers. However, integration across downstream inflammatory and fibrotic events remains limited. Accordingly, this AOP is best applied qualitatively or semi-quantitatively.

This AOP may support: <ul> <li> Identification of GR-modulating chemicals that induce ER stress–mediated liver injury </li> <li> Integration of ER stress biomarkers into MASLD-relevant testing strategies </li> <li> Complementary assessment of non-mitochondrial stress pathways in MASLD </li> <li> AOP network development capturing converging intracellular stress responses </li> </ul>

AOPWiki 2026-02-10T08:25:42

2026-06-26T16:12:16