<td>Under development: Not open for comment. Do not cite</td>
<td>Open for adoption</td>
<td></td>
<td></td>
<td>Under Development</td>
</tr>
</tbody>
</table>
</div>
</div>
<!-- Abstract Section, text as generated by author -->
<div id="abstract">
<h2>Abstract</h2>
<hr>
<p>Endocrine disrupting chemicals (EDC), particularly estrogen receptor (ER) agonists, are thought to contribute to the incidence of breast cancer. The majority (approximately 75 percent) of breast cancer cases express the estrogen receptor. Both animal and human studies strongly support that activation of the estrogen receptor stimulates breast cancer development and progression. We created the ER-mediated breast cancer AOP to frame how ER activation (the MIE) leads to breast cancer (the AO). For more information regarding the AOP, refer to the Morgan & Johnson et al. (2015) citation.</p>
<h2>Abstract</h2>
<p>Endocrine disrupting chemicals (EDC), particularly estrogen receptor (ER) agonists, are thought to contribute to the incidence of breast cancer. The majority (approximately 75 percent) of breast cancer cases express the estrogen receptor. Both animal and human studies strongly support that activation of the estrogen receptor stimulates breast cancer development and progression. We created the ER-mediated breast cancer AOP to frame how ER activation (the MIE) leads to breast cancer (the AO). For more information regarding the AOP, refer to the Morgan & Johnson et al. (2015) citation.</p>
<p>Activation of the estrogen receptor in breast epithelial cells stimulates genomic and non-genomic changes, which alters epithelial gene expression and subsequent protein production. Consequently, breast epithelial cells experience increased proliferation, decreased apoptosis, dysfunction of mitochondrial dynamics, increased DNA damage, increased cell motility, and increased oxidative stress. These cellular changes translate to a tissue level where ductal hyperplasia and cell invasion is increased.</p>
<p>While breast epithelial cells are the cancer cell type in ER+ adenocarcinomas, other cell types of the microenvironment interact with the AOP. For example, endothelial cells express ER and upon ER activation, undergo gene expression and protein production changes. Consequently, endothelial cell proliferation and migration is increased, leading to increased angiogenesis, which supports the proliferation of breast cancer epithelial cells. While estrogens do not target fibroblasts, adipocytes, or macrophages directly, they become activated as breast cancer progresses. It is not well understood if there is a direct relationship between estrogen signaling and stromal cell activation, however, activated cells stimulate cancer cell proliferation, influence chemical response, increase cell motility, and rearrange the extracellular matrix. Moreover, adipocytes contribute to the AOP through metabolism of testosterone to estrogen, and fibroblasts have been shown to regulate estrogen receptor regulated genes in epithelial cells. Therefore, due to how the breast microenvironment interacts with and stimulates the AOP, we have included activation of these cell types into our framework.</p>
<p>Overall, the ER-mediated breast cancer AOP is a useful framework that can identify both readouts and components of the breast microenvironment that are important in disease progression.</p>
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<p><strong>Sex.</strong> While females have a higher incidence of breast cancer, estrogen-receptor mediated breast cancer can occur in males and females.</p>
<p><strong>Sex.</strong> While females have a higher incidence of breast cancer, estrogen-receptor mediated breast cancer can occur in males and females.</p>
<p><strong>Life stages.</strong> Breast cancer affects adult women and men. Older adult women have a higher probability of having an ER+ breast cancer (vs. ER-) than younger adult women.</p>
<p><strong>Taxonomic applicability.</strong> Breast cancer occurs naturally in humans, cats, and dogs. <em>In vivo</em> studies primarily study breast cancer in mice.</p>
<h3>Weight of Evidence Summary</h3>
<p>The weight of evidence for the KERs related to epithelial cells is mostly strong. The KERs between ER activation, motility, and invasion were labeled as a moderate weight of evidence due to discrepancies in the literature regarding whether ER activation decreases motility/invasion, vs. increases motility/invasion. ER activation leading to non-genomic signaling was labeled as moderate due to the limited evidence supporting this KER. For non-epithelial cell types, we labeled the KERs relationship as mostly weak. ER activation has direct effects on endothelial cells as they express ER and several studies have correlated ER activation with increased proliferation, migration, and angiogenesis. Macrophages, fibroblasts, and adipocytes are influenced by and stimulate breast cancer progression, however, the exact correlation between ER activation and these events is still unclear.</p>
<h3>Weight of Evidence Summary</h3>
<p>The weight of evidence for the KERs related to epithelial cells is mostly strong. The KERs between ER activation, motility, and invasion were labeled as a moderate weight of evidence due to discrepancies in the literature regarding whether ER activation decreases motility/invasion, vs. increases motility/invasion. ER activation leading to non-genomic signaling was labeled as moderate due to the limited evidence supporting this KER. For non-epithelial cell types, we labeled the KERs relationship as mostly weak. ER activation has direct effects on endothelial cells as they express ER and several studies have correlated ER activation with increased proliferation, migration, and angiogenesis. Macrophages, fibroblasts, and adipocytes are influenced by and stimulate breast cancer progression, however, the exact correlation between ER activation and these events is still unclear.</p>
<h3> </h3>
</div>
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<div id="references">
<h2>References</h2>
<hr>
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Zheng, S., Huang, J., Zhou, K., Zhang, C., Xiang, Q., Tan, Z., et al. (2011). 17β-Estradiol Enhances Breast Cancer Cell Motility and Invasion via Extra-Nuclear Activation of Actin-Binding Protein Ezrin PLoS One (Vol. 6). Zivadinovic, D., Gametchu, B., & Watson, C. S. (2004). Membrane estrogen receptor-α levels in MCF-7 breast cancer cells predict cAMP and proliferation responses. [Research article]. Breast Cancer Research, 7(1).</p>
<td><a href="/aops/29">Aop:29 - Estrogen receptor agonism leading to reproductive dysfunction</a></td>
<td>MolecularInitiatingEvent</td>
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<td><a href="/aops/52">Aop:52 - ER agonism leading to skewed sex ratios due to altered sexual differentiation in males</a></td>
<td>MolecularInitiatingEvent</td>
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<td><a href="/aops/53">Aop:53 - ER agonism leading to reduced survival due to renal failure</a></td>
<td>MolecularInitiatingEvent</td>
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<td><a href="/aops/536">Aop:536 - Estrogen receptor agonism leading to reduced survival and population growth due to renal failure</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/537">Aop:537 - Estrogen receptor agonism leads to reduced fecundity via increased vitellogenin in the liver</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
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<td><a href="/aops/112">Aop:112 - Increased dopaminergic activity leading to endometrial adenocarcinomas (in Wistar rat)</a></td>
<td>KeyEvent</td>
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<td><a href="/aops/200">Aop:200 - Estrogen receptor activation leading to breast cancer </a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/167">Aop:167 - Early-life estrogen receptor agonism leading to endometrial adenosquamous carcinoma via promotion of sine oculis homeobox 1 progenitor cells</a></td>
<p><a name="_Hlk165971069">Taxonomic applicability:</a> In mammals there are two ER subtypes, ER alpha (ERα) and ER beta (ERβ), which are located on chromosome 6 and 14 and encoded by two different genes (ESR1 and ESR2) <a name="_Hlk162433655"></a>(Ascenzi et al., 2006). ERs were conventionally identified as mammal specific, but most vertebrates contain functional ERs. However, although teleost fish have receptors homologous to mammilian ERα, ERβ is divided into ERβ1 and ERβ2 resulting in three distinct ERs (Asnake et al., 2019; Menuet et al., 2004; Menuet et al., 2002). The majority of invertebrates (i.e. mollusks) possess a gene that is the orthologue of the vertebrate ER but in many species it has been demonstrated to only have constitutive transcriptional activity, and is not activated by ligand binding (Balbi et al., 2019). However, ERs in annelids share functional characteristics with vertebrate ERs and its transcriptional activity can be disrupted by known endocrine-disrupting substances (Keay & Thornton, 2009).</p>
<p>This event would generally be viewed as relevant to vertebrates, but not invertebrates.</p>
<p><a name="_Hlk165905099">Life stage:</a><a name="_Hlk165899451"> </a>This event is applicable to all life stages.</p>
<p>Sex: This event is applicable to both sexes.</p>
<h4>Key Event Description</h4>
<p>Site of action: The molecular site of action is the estrogen receptor (ER). ERs <a name="_Hlk162335835">are members of the steroid hormone receptor family which belongs to a group of nuclear receptors </a>that are transcriptionally activated by ligands leading to downstream activation of many cellular processes. ERs are composed of three principal domains – N-terminal domain (NTD), DNA binding domain (DBD), and the ligand binding domain (LBD). ER binds to specific DNA sequences known as estrogen response elements (EREs); EREs are generally short sequences located in the promoter region but can also exist in introns or exons (Klinge, 2001). ER-mediated gene transcription is initiated by binding of the DBD to an ERE with two distinct transcriptional activation domains, AF1 and AF2, located on the NTD and LBD respectively (Kumar et al., 2011).</p>
<p>Responses at the macromolecular level: ER’s bind to endogenous and exogenous compounds and are activated by endogenous ligands such as estrone (E1), estradiol (E2) and estriol (E3) (Ng et al., 2014). There are numerous compounds (e.g., natural or pharmaceutical estrogens, alkylphenols, organochlorine pesticides, phthalates, etc.) that can act as estrogen agonists or antagonists, and effectively mimic or block the natural effects of estrogens on the ER (Pillon et al., 2005; Schmieder et al., 2014).</p>
<p>ER is part of a multi-protein complex consisting of HSP 90, HSP 70, and immunophilins (Stice & Knowlton, 2008). In this multi-protein complex HSP 90 is the dominant protein and its binding to ER is essential for ER conformational binding of 17β-estradiol (Segnitz & Gehring, 1997). When binding on the LBD receptor occurs ER dissociates from HSP 90 and leads to receptor dimerization which can either be homodimers from the same isoform (ERα-Erα) or heterodimers containing one unit from both isoforms (ERα-Erβ) (Fliss et al., 2000). The translocation of these dimers into the nucleus modulates gene transcription (Aranda & Pascual, 2001).</p>
<h4>How it is Measured or Detected</h4>
<ul>
<li>OECD Test No. 455: Performance-based test guideline for stably transfected transactivation in vitro assays to detect estrogen receptor agonists and antagonists (OECD 2021).</li>
<li>OECD Test No. 457: BG1Luc Estrogen Receptor Transactivation Test Method for Identifying Estrogen Receptor Agonists and Antagonists (OECD 2012).</li>
<li>Standard Evaluation Procedure (SEP) for estrogen receptor transcriptional activation (Human Cell Line HeLa-9903) assay was developed by the U.S. Environmental Protection Agency (EPA).</li>
<li>ER-based transactivation assays that have been used to detect ER agonists and antagonist using cell lines include T47D-Kbluc assay (Wehmas et al., 2011), the ERα CALUX assay (Van et al.); MELN assay (Berckmans et al., 2007); and the yeast estrogen screen (YES; (De Boever et al., 2001)). The T47D-Kbluc assay responds to both ERα and ERß agonists but support the assumption that ERα is inducing more reporter expression than ERß. Each of these assays have undergone some level of validation.</li>
<li>Browne et al. (2015) integrated 18 ER ToxCast high-throughput screening (HTS) assays, measuring ER binding, dimerization, chromatin binding, transcriptional activation and ER-dependent cell proliferation, into the ToxCast ER pathway model. This mathematical model that in vitro assays to predict whether a chemical is an ER agonist or antagonist.</li>
<li>OECD Test No. 440: Uterotrophic Bioassay in Rodents: A Short-Term Screenign Test for Oestrogenic Properties. OCED Publishing. 2018. has been used to detect in vivo estrogenic activity.</li>
</ul>
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</div>
<h4>References</h4>
<p>Aranda, A., & Pascual, A. (2001). Nuclear hormone receptors and gene expression. Physiological reviews, 81(3), 1269-1304.</p>
<p>Ascenzi, P., Bocedi, A., & Marino, M. (2006). Structure–function relationship of estrogen receptor α and β: Impact on human health. Molecular aspects of medicine, 27(4), 299-402.</p>
<p>Asnake, S., Modig, C., & Olsson, P.-E. (2019). Species differences in ligand interaction and activation of estrogen receptors in fish and human. The Journal of steroid biochemistry and molecular biology, 195, 105450.</p>
<p>Balbi, T., Ciacci, C., & Canesi, L. (2019). Estrogenic compounds as exogenous modulators of physiological functions in molluscs: Signaling pathways and biological responses. Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology, 222, 135-144.</p>
<p>Berckmans, P., Leppens, H., Vangenechten, C., & Witters, H. (2007). Screening of endocrine disrupting chemicals with MELN cells, an ER-transactivation assay combined with cytotoxicity assessment. Toxicology in vitro, 21(7), 1262-1267.</p>
<p>Browne, P., Judson, R. S., Casey, W. M., Kleinstreuer, N. C., & Thomas, R. S. (2015). Screening Chemicals for Estrogen Receptor Bioactivity Using a Computational Model. Environmental Science & Technology, 49(14), 8804-8814. <a href="https://doi.org/10.1021/acs.est.5b02641">https://doi.org/10.1021/acs.est.5b02641</a></p>
<p>De Boever, P., Demaré, W., Vanderperren, E., Cooreman, K., Bossier, P., & Verstraete, W. (2001). Optimization of a yeast estrogen screen and its applicability to study the release of estrogenic isoflavones from a soygerm powder. Environmental Health Perspectives, 109(7), 691-697.</p>
<p>Fliss, A. E., Benzeno, S., Rao, J., & Caplan, A. J. (2000). Control of estrogen receptor ligand binding by Hsp90. The Journal of steroid biochemistry and molecular biology, 72(5), 223-230.</p>
<p>Keay, J., & Thornton, J. W. (2009). Hormone-activated estrogen receptors in annelid invertebrates: implications for evolution and endocrine disruption. Endocrinology, 150(4), 1731-1738.</p>
<p>Klinge, C. M. (2001). Estrogen receptor interaction with estrogen response elements. Nucleic Acids Res, 29(14), 2905-2919. <a href="https://doi.org/10.1093/nar/29.14.2905">https://doi.org/10.1093/nar/29.14.2905</a></p>
<p>Kumar, R., Zakharov, M. N., Khan, S. H., Miki, R., Jang, H., Toraldo, G., Singh, R., Bhasin, S., & Jasuja, R. (2011). The dynamic structure of the estrogen receptor. Journal of amino acids, 2011.</p>
<p>Menuet, A., Le Page, Y., Torres, O., Kern, L., Kah, O., & Pakdel, F. (2004). Analysis of the estrogen regulation of the zebrafish estrogen receptor (ER) reveals distinct effects of ERalpha, ERbeta1 and ERbeta2. Journal of Molecular Endocrinology, 32(3), 975-986.</p>
<p>Menuet, A., Pellegrini, E., Anglade, I., Blaise, O., Laudet, V., Kah, O., & Pakdel, F. (2002). Molecular characterization of three estrogen receptor forms in zebrafish: binding characteristics, transactivation properties, and tissue distributions. Biology of reproduction, 66(6), 1881-1892.</p>
<p>Ng, H. W., Perkins, R., Tong, W., & Hong, H. (2014). Versatility or Promiscuity: The Estrogen Receptors, Control of Ligand Selectivity and an Update on Subtype Selective Ligands. International Journal of Environmental Research and Public Health, 11(9), 8709-8742. <a href="https://www.mdpi.com/1660-4601/11/9/8709">https://www.mdpi.com/1660-4601/11/9/8709</a></p>
<p>Pillon, A., Boussioux, A.-M., Escande, A., Aït-Aïssa, S., Gomez, E., Fenet, H., Ruff, M., Moras, D., Vignon, F., & Duchesne, M.-J. (2005). Binding of estrogenic compounds to recombinant estrogen receptor-α: application to environmental analysis. Environmental Health Perspectives, 113(3), 278-284.</p>
<p>Schmieder, P. K., Kolanczyk, R. C., Hornung, M. W., Tapper, M. A., Denny, J. S., Sheedy, B. R., & Aladjov, H. (2014). A rule-based expert system for chemical prioritization using effects-based chemical categories. SAR and QSAR in Environmental Research, 25(4), 253-287. <a href="https://doi.org/10.1080/1062936X.2014.898691">https://doi.org/10.1080/1062936X.2014.898691</a></p>
<p>Segnitz, B., & Gehring, U. (1997). The function of steroid hormone receptors is inhibited by the hsp90-specific compound geldanamycin. Journal of Biological Chemistry, 272(30), 18694-18701.</p>
<p>Stice, J. P., & Knowlton, A. A. (2008). Estrogen, NFκB, and the heat shock response. Molecular Medicine, 14, 517-527.</p>
<p>Van, d., Winter, R., Weimer, M., Beckmanns, P., Suzuki, G., Gijsberg, L., Jonas, A., Van, d. W., Hilda, & Aarts, J. Optimization and Prevalidation of the in Vitro ER CALUX Method to Test Estrogenic and Antiestrogenic Activity of Compounds.</p>
<p>Wehmas, L. C., Cavallin, J. E., Durhan, E. J., Kahl, M. D., Martinovic, D., Mayasich, J., Tuominen, T., Villeneuve, D. L., & Ankley, G. T. (2011). Screening complex effluents for estrogenic activity with the T47D‐KBluc cell bioassay: Assay optimization and comparison with in vivo responses in fish. Environmental toxicology and chemistry, 30(2), 439-445.</p>
<p>Proliferation occurs when changes in external signals release inhibitory controls limiting entry into the cell cycle, and oncogenic mutations act via these same pathways to generate abnormal proliferation (Hanahan and Weinberg 2011; Weber, Desai et al. 2017). Inhibitory signals such as contact inhibition or TGF-β (Polyak, Kato et al. 1994; Francis, Bergsied et al. 2009) stabilize the mechanisms limiting entry into the cell cycle. Proliferative signals such as those following progesterone or estrogen (Croce 2008; Weber, Desai et al. 2017) or compensatory <a name="Prolif_KE2_Howthisworks">proliferation </a>after apoptosis (Fogarty and Bergmann 2017) relieve inhibition and enable cells to enter the cell cycle. Mutations that inactivate inhibitory signals (tumor suppressors) or activate proliferative signals (oncogenes) promote proliferation outside of the normal biological context (Gustin, Karakas et al. 2009; Francis, Chakrabarti et al. 2011; Hanahan and Weinberg 2011; Weber, Desai et al. 2017). Abnormal proliferation is typically met with apoptosis or senescence, so additional mutations or other mechanisms are required to escape these additional levels of control for proliferation to continue indefinitely (Garbe, Bhattacharya et al. 2009; Shay and Wright 2011; Fernald and Kurokawa 2013).</p>
<p>Proliferation increases mutations as DNA damage and replication errors become integrated into the genome (Kiraly, Gong et al. 2015). Proliferation can also promote the expansion of existing cells with proliferative mutations. Genomic mutations favoring further proliferation are positively selected from among the expanded cells, resulting in the accumulation of mutational errors and moving the organism further towards cancer. Different clonal populations can also collaborate to promote growth (Marusyk, Tabassum et al. 2014; Franco, Tyson et al. 2016).</p>
<p>Past cellular proliferation can be measured directly using labels that are incorporated into cells upon cell division (BRDU or cytoplasmic proliferation dyes) or indirectly by measuring a change in population size. Ongoing current proliferation can be quantified by labeling a protein associated with the cell cycle (e.g. Ki67). Methods for measuring proliferation were reviewed in (Romar, Kupper et al. 2016) and summarized in Table 1.</p>
<p>Table 1. Common methods for detecting proliferation</p>
<p>Stable, so can see proliferation from a specific time point onward. Can be used in vivo. BRDU must be labeled with a secondary fluorescent or other label for visualization, so it cannot be measured in living cells.</p>
<p>Enables quantification of successive cell divisions and differentiation between slowly and rapidly cycling cells. Cells survive analysis, so these dyes can be used as part of ongoing experiments. The dyes are better suited to in vitro experiments.</p>
</td>
</tr>
<tr>
<td style="width:153px">
<p><strong>Past proliferation</strong></p>
</td>
<td style="width:139px">
<p>Cell counting</p>
</td>
<td style="width:157px">
<p>Microscopy</p>
</td>
<td style="width:190px">
<p>An increase in cell numbers over time could represent proliferation or a decrease in apoptosis. Better suited to in vitro experiments, unless a label can be used to uniquely label a population of cells.</p>
<p>Labels all non-G0 phase proliferating cells. Labeling requires permeabilization so examination terminates the experiment.</p>
<p> </p>
</td>
</tr>
</tbody>
</table>
<h4>References</h4>
<p><a name="_ENREF_1">Croce, C. M. (2008). "Oncogenes and cancer." The New England journal of medicine 358(5): 502-511.</a></p>
<p><a name="_ENREF_2">Fernald, K. and M. Kurokawa (2013). "Evading apoptosis in cancer." Trends in cell biology 23(12): 620-633.</a></p>
<p><a name="_ENREF_3">Fogarty, C. E. and A. Bergmann (2017). "Killers creating new life: caspases drive apoptosis-induced proliferation in tissue repair and disease." Cell death and differentiation 24(8): 1390-1400.</a></p>
<p><a name="_ENREF_4">Francis, S. M., J. Bergsied, et al. (2009). "A functional connection between pRB and transforming growth factor beta in growth inhibition and mammary gland development." Molecular and cellular biology 29(16): 4455-4466.</a></p>
<p><a name="_ENREF_5">Francis, S. M., S. Chakrabarti, et al. (2011). "A context-specific role for retinoblastoma protein-dependent negative growth control in suppressing mammary tumorigenesis." PLoS One 6(2): e16434.</a></p>
<p><a name="_ENREF_6">Franco, O. E., D. R. Tyson, et al. (2016). "Altered TGF-alpha/beta signaling drives cooperation between breast cancer cell populations." FASEB journal : official publication of the Federation of American Societies for Experimental Biology 30(10): 3441-3452.</a></p>
<p><a name="_ENREF_7">Garbe, J. C., S. Bhattacharya, et al. (2009). "Molecular distinctions between stasis and telomere attrition senescence barriers shown by long-term culture of normal human mammary epithelial cells." Cancer research 69(19): 7557-7568.</a></p>
<p><a name="_ENREF_8">Gustin, J. P., B. Karakas, et al. (2009). "Knockin of mutant PIK3CA activates multiple oncogenic pathways." Proceedings of the National Academy of Sciences of the United States of America 106(8): 2835-2840.</a></p>
<p><a name="_ENREF_9">Hanahan, D. and R. A. Weinberg (2011). "Hallmarks of cancer: the next generation." Cell 144(5): 646-674.</a></p>
<p><a name="_ENREF_10">Kiraly, O., G. Gong, et al. (2015). "Inflammation-induced cell proliferation potentiates DNA damage-induced mutations in vivo." PLoS Genet 11(2): e1004901.</a></p>
<p><a name="_ENREF_11">Marusyk, A., D. P. Tabassum, et al. (2014). "Non-cell-autonomous driving of tumour growth supports sub-clonal heterogeneity." Nature 514(7520): 54-58.</a></p>
<p><a name="_ENREF_12">Polyak, K., J. Y. Kato, et al. (1994). "p27Kip1, a cyclin-Cdk inhibitor, links transforming growth factor-beta and contact inhibition to cell cycle arrest." Genes & development 8(1): 9-22.</a></p>
<p><a name="_ENREF_13">Romar, G. A., T. S. Kupper, et al. (2016). "Research Techniques Made Simple: Techniques to Assess Cell Proliferation." The Journal of investigative dermatology 136(1): e1-7.</a></p>
<p><a name="_ENREF_14">Shay, J. W. and W. E. Wright (2011). "Role of telomeres and telomerase in cancer." Seminars in cancer biology 21(6): 349-353.</a></p>
<p><a name="_ENREF_15">Weber, R. J., T. A. Desai, et al. (2017). "Non-autonomous cell proliferation in the mammary gland and cancer." Current opinion in cell biology 45: 55-61.</a></p>
<td><a href="/aops/48">Aop:48 - Binding of agonists to ionotropic glutamate receptors in adult brain causes excitotoxicity that mediates neuronal cell death, contributing to learning and memory impairment.</a></td>
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<td><a href="/aops/77">Aop:77 - Nicotinic acetylcholine receptor activation contributes to abnormal foraging and leads to colony death/failure 1</a></td>
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<td><a href="/aops/78">Aop:78 - Nicotinic acetylcholine receptor activation contributes to abnormal role change within the worker bee caste leading to colony death failure 1</a></td>
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<td><a href="/aops/79">Aop:79 - Nicotinic acetylcholine receptor activation contributes to impaired hive thermoregulation and leads to colony loss/failure</a></td>
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<td><a href="/aops/80">Aop:80 - Nicotinic acetylcholine receptor activation contributes to accumulation of damaged mitochondrial DNA and leads to colony loss/failure</a></td>
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<td><a href="/aops/87">Aop:87 - Nicotinic acetylcholine receptor activation contributes to abnormal foraging and leads to colony loss/failure </a></td>
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<td><a href="/aops/3">Aop:3 - Inhibition of the mitochondrial complex I of nigro-striatal neurons leads to parkinsonian motor deficits</a></td>
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<td><a href="/aops/144">Aop:144 - Endocytic lysosomal uptake leading to liver fibrosis</a></td>
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<td><a href="/aops/178">Aop:178 - Nicotinic acetylcholine receptor activation contributes to mitochondrial dysfunction and leads to colony loss/failure</a></td>
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<td><a href="/aops/200">Aop:200 - Estrogen receptor activation leading to breast cancer </a></td>
<td><a href="/aops/48">Aop:48 - Binding of agonists to ionotropic glutamate receptors in adult brain causes excitotoxicity that mediates neuronal cell death, contributing to learning and memory impairment.</a></td>
<td>KeyEvent</td>
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<td><a href="/aops/77">Aop:77 - Nicotinic acetylcholine receptor activation contributes to abnormal foraging and leads to colony death/failure 1</a></td>
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<td><a href="/aops/78">Aop:78 - Nicotinic acetylcholine receptor activation contributes to abnormal role change within the worker bee caste leading to colony death failure 1</a></td>
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<td><a href="/aops/79">Aop:79 - Nicotinic acetylcholine receptor activation contributes to impaired hive thermoregulation and leads to colony loss/failure</a></td>
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<td><a href="/aops/80">Aop:80 - Nicotinic acetylcholine receptor activation contributes to accumulation of damaged mitochondrial DNA and leads to colony loss/failure</a></td>
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<td><a href="/aops/87">Aop:87 - Nicotinic acetylcholine receptor activation contributes to abnormal foraging and leads to colony loss/failure </a></td>
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<td><a href="/aops/3">Aop:3 - Inhibition of the mitochondrial complex I of nigro-striatal neurons leads to parkinsonian motor deficits</a></td>
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<td><a href="/aops/144">Aop:144 - Endocytic lysosomal uptake leading to liver fibrosis</a></td>
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<td><a href="/aops/178">Aop:178 - Nicotinic acetylcholine receptor activation contributes to mitochondrial dysfunction and leads to colony loss/failure</a></td>
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<td><a href="/aops/200">Aop:200 - Estrogen receptor activation leading to breast cancer </a></td>
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<td><a href="/aops/273">Aop:273 - Mitochondrial complex inhibition leading to liver injury</a></td>
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<td><a href="/aops/377">Aop:377 - Dysregulated prolonged Toll Like Receptor 9 (TLR9) activation leading to Multi Organ Failure involving Acute Respiratory Distress Syndrome (ARDS)</a></td>
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<td><a href="/aops/437">Aop:437 - Inhibition of mitochondrial electron transport chain (ETC) complexes leading to kidney toxicity</a></td>
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<td><a href="/aops/423">Aop:423 - Toxicological mechanisms of hepatocyte apoptosis through the PARP1 dependent cell death pathway </a></td>
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<td><a href="/aops/479">Aop:479 - Mitochondrial complexes inhibition leading to left ventricular function decrease via increased myocardial oxidative stress</a></td>
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<td><a href="/aops/480">Aop:480 - Mitochondrial complexes inhibition leading to heart failure via decreased ATP production</a></td>
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<td><a href="/aops/481">Aop:481 - AOPs of amorphous silica nanoparticles: ROS-mediated oxidative stress increased respiratory dysfunction and diseases.</a></td>
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<td><a href="/aops/509">Aop:509 - Nrf2 inhibition leading to vascular disrupting effects through activating apoptosis signal pathway and mitochondrial dysfunction</a></td>
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<td><a href="/aops/511">Aop:511 - The AOP framework on ROS-mediated oxidative stress induced vascular disrupting effects </a></td>
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<td><a href="/aops/256">Aop:256 - Inhibition of mitochondrial DNA polymerase gamma leading to kidney toxicity</a></td>
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<td><a href="/aops/258">Aop:258 - Renal protein alkylation leading to kidney toxicity</a></td>
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<td><a href="/aops/464">Aop:464 - Calcium overload in dopaminergic neurons of the substantia nigra leading to parkinsonian motor deficits</a></td>
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<td><a href="/aops/500">Aop:500 - Activation of MEK-ERK1/2 leads to deficits in learning and cognition via ROS and apoptosis</a></td>
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<td><a href="/aops/205">Aop:205 - AOP from chemical insult to cell death</a></td>
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<td><a href="/aops/335">Aop:335 - AOP for urothelial carcinogenesis due to chemical cytotoxicity by mitochondrial impairment </a></td>
<td><a href="/aops/130">Aop:130 - Phospholipase A2 (LPLA2) inhibitors leading to hepatotoxicity</a></td>
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<td><a href="/aops/497">Aop:497 - ERa inactivation alters mitochondrial functions and insulin signalling in skeletal muscle and leads to insulin resistance and metabolic syndrome</a></td>
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<td><a href="/aops/34">Aop:34 - LXR activation leading to hepatic steatosis</a></td>
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<td><a href="/aops/447">Aop:447 - Kidney failure induced by inhibition of mitochondrial electron transfer chain through apoptosis, inflammation and oxidative stress pathways</a></td>
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<td><a href="/aops/207">Aop:207 - NADPH oxidase and P38 MAPK activation leading to reproductive failure in Caenorhabditis elegans</a></td>
<td>KeyEvent</td>
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<td><a href="/aops/476">Aop:476 - Adverse Outcome Pathways diagram related to PBDEs associated male reproductive toxicity</a></td>
<td><a href="/aops/622">Aop:622 - Calcineurin inhibitor induced nephrotoxicity leading to kidney failure</a></td>
<td>KeyEvent</td>
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<td><a href="/aops/624">Aop:624 - Increased 11β-Hydroxysteroid dehydrogenase type 1 activity leading to MASLD progression via insulin resistance-associated mitochondrial dysfunction</a></td>
<td>KeyEvent</td>
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<td><a href="/aops/627">Aop:627 - Increased 11β-Hydroxysteroid dehydrogenase type 1 activity leading to MASLD progression via lipogenesis-associated mitochondrial dysfunction</a></td>
<p>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).</p>
<br>
</div>
<p>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).</p>
<p><strong>- Revision of AOP3 (Project: </strong><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><strong>)</strong>: 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. <strong>- Not endorsed</strong></p>
<!-- event text -->
<h4>Key Event Description</h4>
<p>Mitochondrial dysfunction is a consequence of inhibition of the respiratory chain leading to oxidative stress.</p>
<h4>Key Event Description</h4>
<p>Mitochondrial dysfunction is a consequence of inhibition of the respiratory chain leading to oxidative stress.</p>
<p>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).</p>
<p>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.</p>
<p>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).</p>
<p>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).</p>
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.</p>
<p>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).</p>
<p><strong>Summing up:</strong> 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.</p>
<br>
<h4>How it is Measured or Detected</h4>
<p>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.</p>
<h4>How it is Measured or Detected</h4>
<p>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.</p>
<p>I. Mitochondrial dysfunction assays assessing a loss-of function.</p>
<p>1. Cellular oxygen consumption.</p>
<p>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 O<sub>2</sub> 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).</p>
<p>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. The classical, and still most quantitative method uses a tetraphenylphosphonium ion (TPP+)-sensitive electrode on suspensions of isolated mitochondria. The Δψm can also be measured in live cells by fluorimetric methods. These are based on dyes which accumulate in mitochochondria because of Δψm. Frequently used are tetramethylrhodamineethylester (TMRE), tetramethylrhodaminemethyl ester (TMRM) (Petronilli et al., 1999) or 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazole carbocyanide iodide (JC-1). Mitochondria with intact membrane potential concentrate JC-1, so that it forms red fluorescent aggregates, whereas de-energized mitochondria cannot concentrate JC-1 and the dilute dye fluoresces green (Barrientos et al., 1999). Assays using TMRE or TMRM measure only at one wavelength (red fluorescence), and depending on the assay setup, de-energized mitochondria become either less fluorescent (loss of the dye) or more fluorescent (attenuated dye quenching).</p>
<p><strong>- Revision of AOP3 (Project: </strong><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><strong>):</strong> 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 (tetramethylrhodamineethyl 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). <strong>- Not endorsed</strong> </p>
<p>3. Enzymatic activity of the electron transport system (ETS).</p>
<p>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).</p>
<p>4. ATP content.</p>
<p>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).</p>
<p><strong>Determination of mitochondrial ATP production based on extracellular flux analysis </strong></p>
<p>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. </p>
<p><strong>Calculation of mitochondrial ATP production </strong></p>
<p>Mito ATP production rate (pmol ATP/min) = OCRATP (pmol O2/min) × 2 × P/O </p>
<p>OCR_ATP: ATP-coupled portion of OCR. </p>
<p>Factor 2: each O₂ molecule contains two oxygen atoms. </p>
<p>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). </p>
<p><strong>Limitations</strong> </p>
<p>P/O ratio varies by substrate (glucose vs. fatty acids), cell type, and conditions. Fixed values are approximations. </p>
<p>Non-mitochondrial oxygen consumption (oxidases, peroxidases, etc.) can confound OCR, hence use of ETC inhibitors. </p>
<p>PER vs. ECAR: CO₂-driven acidification must be corrected to avoid overestimating glycolytic ATP. </p>
<p>Normalization: results are usually expressed per cell, protein content, DNA, or mitochondrial mass — interpretation depends on normalization method. </p>
<p><span style="font-size:12.0pt"><span style="font-family:Arial"><span style="color:#212529"><span style="background-color:white"><strong>- Not endorsed</strong></span></span></span></span></p>
</div>
<p><br />
II. Mitochondrial dysfunction assays assessing a gain-of function.</p>
<p>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).</p>
<p>2. mtDNA damage as a biomarker of mitochondrial dysfunction.</p>
<p>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).</p>
<p>3. Generation of ROS and resultant oxidative stress.</p>
<p>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.</p>
<p>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.</p>
<p>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.</p>
<p><br />
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.</p>
<p>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.</p>
<p>e. Detection of hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>) 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 H<sub>2</sub>O<sub>2</sub> form increasing amounts of fluorescent product (Tarpley et al., 2004).</p>
<p>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></p>
<br>
<p>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></p>
<p><strong>Assay Type & Measured Content</strong></p>
</td>
<td><strong>Description</strong></td>
<td><strong>Dose Range Studied</strong></td>
<td>
<p><strong>Assay Characteristics</strong></p>
<p><strong>(Length/Ease of use/Accuracy)</strong></p>
</td>
</tr>
<tr>
<td>
<p><strong>Rhodamine 123 Assay</strong></p>
<p>Measuring Mitochondrial membrane potential (MMP) and its collapse </p>
<p>(Shaki et al., 2012)</p>
</td>
<td>
<p>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.</p>
<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>
<p>Measuring cellular glutathione (GSH) status; ratio of GSH/GSSG</p>
<p>(Owen & Butterfield, 2010; Shaki et al., 2013)</p>
</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>
<p>Short / easy</p>
<p>Low accurancy</p>
</td>
</tr>
<tr>
<td>
<p><strong>TBARS Assay</strong></p>
<p>Quantification of lipid peroxidation</p>
<p>(Yuan et al., 2016)</p>
</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>
<p>Increase in mitochondrial Ca<sup>2+</sup> influx</p>
<p>(Pozzan & Rudolf, 2009)</p>
</td>
<td>Together with GFP, the aequorin moiety acts as Ca<sup>2+</sup> sensor <em>in vivo</em>, 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>
<p>Short / easy</p>
<p>Low accurancy</p>
</td>
</tr>
<tr>
<td>
<p><strong>Western blot & immunostaining analyses</strong></p>
<p>Measuring cytochrome c release</p>
(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>
<p>Short / easy</p>
<p>Medium accurancy</p>
</td>
</tr>
<tr>
<td>
<p><strong>Quantikine Rat/Mouse Cytochrome c Immunoassay</strong></p>
<p>Measuring cytochrome c release</p>
<p>(Shaki et al., 2012)</p>
</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>
<p>Short / easy</p>
<p>Low accurancy</p>
</td>
</tr>
<tr>
<td>
<p><strong>Membrane potential and cell viability – Flow Cytometry</strong></p>
<p>Measuring cytochrome c release</p>
<p>(Kruidering et al., 1997)</p>
</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 <em>g</em>. 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<br />
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 <em>et al.</em>, 1993)”</td>
<td> </td>
<td>
<p>Short / easy</p>
<p>Medium accurancy</p>
</td>
</tr>
</tbody>
</table>
<p> </p>
<p> </p>
<h4>References</h4>
<p> </p>
<h4>References</h4>
<p>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.</p>
<p>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.</p>
<p>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</p>
<p>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</p>
<p>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.</p>
<p>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.</p>
<p>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</p>
<p>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</p>
<p>Brand MD, Nicholls DG. Assessing mitochondrial dysfunction in cells. Biochem J. 2011 Apr 15;435(2):297-312.</p>
<p>Braun RJ. (2012). Mitochondrion-mediated cell death: dissecting yeast apoptosis for a better understanding of neurodegeneration. Front Oncol 2:182.</p>
<p>Barrientos A., and Moraes C.T. (1999) Titrating the Effects of Mitochondrial Complex I Impairment in the Cell Physiology. Vol. 274, No. 23, pp. 16188–16197.</p>
<p>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.</p>
<p>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</p>
<p>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.</p>
<p>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.</p>
<p>Cozzolino M, Ferri A, Valle C, Carri MT. (2013). Mitochondria and ALS: implications from novel genes and pathways. Mol Cell Neurosci 55:44 – 49.</p>
<p>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. </p>
<p>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. </p>
<p>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.</p>
<p>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. </p>
<p>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.</p>
<p>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.</p>
<p>Fiskum G. Mitochondrial participation in ischemic and traumatic neural cell death. J Neurotrauma. 2000 Oct;17(10):843-55.</p>
<p>Friberg H, Wieloch T. (2002). Mitochondrial permeability transition in acute neurodegeneration. Biochimie 84:241–250.</p>
<p>Fujikawa DG, The Role of Excitotoxic Programmed Necrosis in Acute Brain Injury. Computational and Structural Biotechnology Journal, 2015, 13: 212–221.</p>
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<p>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.</p>
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<td><a href="/aops/220">Aop:220 - Cyp2E1 Activation Leading to Liver Cancer</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/17">Aop:17 - Binding of electrophilic chemicals to SH(thiol)-group of proteins and /or to seleno-proteins involved in protection against oxidative stress during brain development leads to impairment of learning and memory</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/284">Aop:284 - Binding of electrophilic chemicals to SH(thiol)-group of proteins and /or to seleno-proteins involved in protection against oxidative stress leads to chronic kidney disease</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/377">Aop:377 - Dysregulated prolonged Toll Like Receptor 9 (TLR9) activation leading to Multi Organ Failure involving Acute Respiratory Distress Syndrome (ARDS)</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/411">Aop:411 - Oxidative stress Leading to Decreased Lung Function </a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/424">Aop:424 - Oxidative stress Leading to Decreased Lung Function via CFTR dysfunction</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/425">Aop:425 - Oxidative Stress Leading to Decreased Lung Function via Decreased FOXJ1</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/429">Aop:429 - A cholesterol/glucose dysmetabolism initiated Tau-driven AOP toward memory loss (AO) in sporadic Alzheimer's Disease with plausible MIE's plug-ins for environmental neurotoxicants</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/452">Aop:452 - Adverse outcome pathway of PM-induced respiratory toxicity</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/464">Aop:464 - Calcium overload in dopaminergic neurons of the substantia nigra leading to parkinsonian motor deficits</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/470">Aop:470 - Deposition of energy leads to abnormal vascular remodeling</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/478">Aop:478 - Deposition of energy leading to occurrence of cataracts</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/479">Aop:479 - Mitochondrial complexes inhibition leading to left ventricular function decrease via increased myocardial oxidative stress</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/481">Aop:481 - AOPs of amorphous silica nanoparticles: ROS-mediated oxidative stress increased respiratory dysfunction and diseases.</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/482">Aop:482 - Deposition of energy leading to occurrence of bone loss</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/483">Aop:483 - Deposition of Energy Leading to Learning and Memory Impairment</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/505">Aop:505 - Reactive Oxygen Species (ROS) formation leads to cancer via inflammation pathway</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/521">Aop:521 - Essential element imbalance leads to reproductive failure via oxidative stress</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/26">Aop:26 - Calcium-mediated neuronal ROS production and energy imbalance</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/488">Aop:488 - Increased reactive oxygen species production leading to decreased cognitive function</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/396">Aop:396 - Deposition of ionizing energy leads to population decline via impaired meiosis</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/437">Aop:437 - Inhibition of mitochondrial electron transport chain (ETC) complexes leading to kidney toxicity</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/535">Aop:535 - Binding and activation of GPER leading to learning and memory impairments</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/171">Aop:171 - Chronic cytotoxicity of the serous membrane leading to pleural/peritoneal mesotheliomas in the rat.</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/138">Aop:138 - Organic anion transporter (OAT1) inhibition leading to renal failure and mortality</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/177">Aop:177 - Cyclooxygenase 1 (COX1) inhibition leading to renal failure and mortality</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/186">Aop:186 - unknown MIE leading to renal failure and mortality</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/200">Aop:200 - Estrogen receptor activation leading to breast cancer </a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/444">Aop:444 - Ionizing radiation leads to reduced reproduction in Eisenia fetida via reduced spermatogenesis and cocoon hatchability</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/447">Aop:447 - Kidney failure induced by inhibition of mitochondrial electron transfer chain through apoptosis, inflammation and oxidative stress pathways</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/476">Aop:476 - Adverse Outcome Pathways diagram related to PBDEs associated male reproductive toxicity</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/497">Aop:497 - ERa inactivation alters mitochondrial functions and insulin signalling in skeletal muscle and leads to insulin resistance and metabolic syndrome</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/457">Aop:457 - Succinate dehydrogenase inhibition leading to increased insulin resistance through reduction in circulating thyroxine</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/459">Aop:459 - AhR activation in the thyroid leading to Subsequent Adverse Neurodevelopmental Outcomes in Mammals</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/507">Aop:507 - Nrf2 inhibition leading to vascular disrupting effects via inflammation pathway</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/509">Aop:509 - Nrf2 inhibition leading to vascular disrupting effects through activating apoptosis signal pathway and mitochondrial dysfunction</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/510">Aop:510 - Demethylation of PPAR promotor leading to vascular disrupting effects</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/511">Aop:511 - The AOP framework on ROS-mediated oxidative stress induced vascular disrupting effects </a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/538">Aop:538 - Adverse outcome pathway of PFAS-induced vascular disrupting effects via activating oxidative stress related pathways </a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/260">Aop:260 - CYP2E1 activation and formation of protein adducts leading to neurodegeneration</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/450">Aop:450 - Inhibition of AChE and activation of CYP2E1 leading to sensory axonal peripheral neuropathy and mortality</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/501">Aop:501 - Excessive iron accumulation leading to neurological disorders</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/540">Aop:540 - Oxidative Stress in the Fish Ovary Leads to Reproductive Impairment via Reduced Vitellogenin Production</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/471">Aop:471 - Neuron defect induced early behavioral change</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/31">Aop:31 - Oxidation of iron in hemoglobin leading to hematotoxicity</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/534">Aop:534 - Succinate dehydrogenase (SDH) inhibition leads to cancer through oxidative stress</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/462">Aop:462 - Activation of reactive oxygen species leading the atherosclerosis</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/324">Aop:324 - Excessive reactive oxygen species leading to growth inhibition via uncoupling of oxidative phosphorylation and cell death</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/325">Aop:325 - Excessive reactive oxygen species leading to growth inhibition via uncoupling of oxidative phosphorylation and reduced cell growth</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/326">Aop:326 - Excessive reactive oxygen species leading to growth inhibition via uncoupling of oxidative phosphorylation and reduced cell proliferation</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/331">Aop:331 - Excessive reactive oxygen species leading to growth inhibition via lipid peroxidation and cell death</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/332">Aop:332 - Excessive reactive oxygen species leading to growth inhibition via lipid peroxidation and reduced cell growth</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/333">Aop:333 - Excessive reactive oxygen species leading to growth inhibition via lipid peroxidation and reduced cell proliferation</a></td>
<td><a href="/aops/596">Aop:596 - Excessive reactive oxygen species leading to growth inhibition via protein oxidation and cell injury/death</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/598">Aop:598 - Excessive reactive oxygen species leading to growth inhibition via protein oxidation and reduced cell proliferation</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/599">Aop:599 - Excessive reactive oxygen species leading to growth inhibition via fatty acid oxidation and cell injury/death</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/600">Aop:600 - Excessive reactive oxygen species leading to growth inhibition via fatty acid oxidation and reduced cell growth</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/601">Aop:601 - Excessive reactive oxygen species leading to growth inhibition via fatty acid oxidation and reduced cell proliferation</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/602">Aop:602 - Excessive reactive oxygen species leading to growth inhibition via oxidative DNA damage</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/603">Aop:603 - Excessive reactive oxygen species leading to growth inhibition via protein oxidation and cell cycle disruption</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/608">Aop:608 - Thyroid Hormone Excess Leading to Reduced, Swimming Performance via Hypomyelination</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/616">Aop:616 - organic UV filter and its Photoproducts reproductive toxicity pathways </a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/622">Aop:622 - Calcineurin inhibitor induced nephrotoxicity leading to kidney failure</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/625">Aop:625 - Increased 11β-Hydroxysteroid dehydrogenase type 1 activity leading to MASLD progression via insulin resistance-associated oxidative stress</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/628">Aop:628 - Increased 11β-Hydroxysteroid dehydrogenase type 1 activity leading to MASLD progression via lipogenesis-associated oxidative stress</a></td>
<p><span style="color:#27ae60"><strong>Taxonomic applicability: </strong>Occurrence of oxidative stress is not species specific. </span></p>
<p><span style="color:#27ae60"><strong>Life stage applicability:</strong> Occurrence of oxidative stress is not life stage specific. </span></p>
<p><span style="color:#27ae60"><strong>Sex applicability: </strong>Occurrence of oxidative stress is not sex specific. </span></p>
<p><span style="color:#27ae60"><strong>Evidence for perturbation by prototypic stressor:</strong> There is evidence of the increase of oxidative stress following perturbation from a variety of stressors including exposure to ionizing radiation and altered gravity (Bai et al., 2020; Ungvari et al., 2013; Zhang et al., 2009). </span></p>
<div>
<div>
<h4><a href="/events/1187">Event: 1187: Increased, ER binding to DNA (classical pathway) </a><br></h4>
<h5>Short Name: Increased, ER binding to DNA (classical pathway) </h5>
<p>Oxidative stress is defined as an imbalance in the production of reactive oxygen species (ROS) and antioxidant defenses. High levels of oxidizing free radicals can be very damaging to cells and molecules within the cell. As a result, the cell has important defense mechanisms to protect itself from ROS. For example, Nrf2 is a transcription factor and master regulator of the oxidative stress response. During periods of oxidative stress, Nrf2-dependent changes in gene expression are important in regaining cellular homeostasis (Nguyen, et al., 2009) and can be used as indicators of the presence of oxidative stress in the cell. </p>
<p>In addition to the directly damaging actions of ROS, cellular oxidative stress also changes cellular activities on a molecular level. Redox sensitive proteins have altered physiology in the presence and absence of ROS, which is caused by the oxidation of sulfhydryls to disulfides on neighboring amino acids (Antelmann & Helmann 2011). Importantly Keap1, the negative regulator of Nrf2, is regulated in this manner (Itoh, et al. 2010). </p>
<p>ROS also undermine the mitochondrial defense system from oxidative damage. The antioxidant systems consist of superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase, as well as antioxidants such as α-tocopherol and ubiquinol, or antioxidant vitamins and minerals including vitamin E, C, carotene, lutein, zeaxanthin, selenium, and zinc (Fletcher, 2010). The enzymes, vitamins and minerals catalyze the conversion of ROS to non-toxic molecules such as water and O2. However, these antioxidant systems are not perfect and endogenous metabolic processes and/or exogenous oxidative influences can trigger cumulative oxidative injuries to the mitochondria, causing a decline in their functionality and efficiency, which further promotes cellular oxidative stress (Balasubramanian, 2000; Ganea & Harding, 2006; Guo et al., 2013; Karimi et al., 2017). </p>
<p>However, an emerging viewpoint suggests that ROS-induced modifications may not be as detrimental as previously thought, but rather contribute to signaling processes (Foyer et al., 2017). </p>
<p> </p>
<p><strong>Sources of ROS Production </strong></p>
<p><strong>Direct Sources: </strong>Direct sources involve the deposition of energy onto water molecules, breaking them into active radical species. When ionizing radiation hits water, it breaks it into hydrogen (H*) and hydroxyl (OH*) radicals by destroying its bonds. The hydrogen will create hydroxyperoxyl free radicals (HO2*) if oxygen is available, which can then react with another of itself to form hydrogen peroxide (H2O2) and more O2 (Elgazzar and Kazem, 2015). Antioxidant mechanisms are also affected by radiation, with catalase (CAT) and peroxidase (POD) levels rising as a result of exposure (Seen et al. 2018; Ahmad et al. 2021). </p>
<p><strong>Indirect Sources</strong>: An indirect source of ROS is the mitochondria, which is one of the primary producers in eukaryotic cells (Powers et al., 2008). As much as 2% of the electrons that should be going through the electron transport chain in the mitochondria escape, allowing them an opportunity to interact with surrounding structures. Electron-oxygen reactions result in free radical production, including the formation of hydrogen peroxide (H2O2) (Zhao et al., 2019). The electron transport chain, which also creates ROS, is activated by free adenosine diphosphate (ADP), O2, and inorganic phosphate (Pi) (Hargreaves et al. 2020; Raimondi et al. 2020; Vargas-Mendoza et al. 2021). The first and third complexes of the transport chain are the most relevant to mammalian ROS production (Raimondi et al., 2020). The mitochondria has its own set of DNA and it is a prime target of oxidative damage (Guo et al., 2013). ROS is also produced through nicotinamide adenine dinucleotide phosphate oxidase (Nox) stimulation, an event commenced by angiotensin II, a product/effector of the renin-angiotensin system (Nguyen Dinh Cat et al. 2013; Forrester et al. 2018). Other ROS producers include xanthine oxidase, immune cells (macrophage, neutrophils, monocytes, and eosinophils), phospholipase A2 (PLA2), monoamine oxidase (MAO), and carbon-based nanomaterials (Powers et al. 2008; Jacobsen et al. 2008; Vargas-Mendoza et al. 2021). </p>
<h4>How it is Measured or Detected</h4>
<p><strong>Oxidative Stress:</strong> Direct measurement of ROS is difficult because ROS are unstable. The presence of ROS can be assayed indirectly by measurement of cellular antioxidants, or by ROS-dependent cellular damage. Listed below are common methods for detecting the KE, however there may be other comparable methods that are not listed </p>
<ul>
<li>Detection of ROS by chemiluminescence (https://www.sciencedirect.com/science/article/abs/pii/S0165993606001683) </li>
<li>Detection of ROS by chemiluminescence is also described in OECD TG 495 to assess phototoxic potential. </li>
<li>Glutathione (GSH) depletion. GSH can be measured by assaying the ratio of reduced to oxidized glutathione (GSH:GSSG) using a commercially available kit (e.g., http://www.abcam.com/gshgssg-ratio-detection-assay-kit-fluorometric-green- ab138881.html). </li>
<li>TBARS. Oxidative damage to lipids can be measured by assaying for lipid peroxidation using TBARS (thiobarbituric acid reactive substances) using a commercially available kit. </li>
<li>8-oxo-dG. Oxidative damage to nucleic acids can be assayed by measuring 8-oxo-dG adducts (for which there are a number of ELISA based commercially available kits),or HPLC, described in Chepelev et al. (Chepelev, et al. 2015). </li>
</ul>
<p> </p>
<p><strong>Molecular Biology:</strong> Nrf2. Nrf2’s transcriptional activity is controlled post-translationally by oxidation of Keap1. Assay for Nrf2 activity include: </p>
<ul>
<li>Immunohistochemistry for increases in Nrf2 protein levels and translocation into the nucleus Western blot for increased Nrf2 protein levels </li>
<li>Western blot of cytoplasmic and nuclear fractions to observe translocation of Nrf2 protein from the cytoplasm to the nucleus qPCR of Nrf2 target genes (e.g., Nqo1, Hmox-1, Gcl, Gst, Prx, TrxR, Srxn), or by commercially available pathway-based qPCR array (e.g., oxidative stress array from SABiosciences) </li>
<li>Whole transcriptome profiling by microarray or RNA-seq followed by pathway analysis (in IPA, DAVID, metacore, etc.) for enrichment of the Nrf2 oxidative stress response pathway (e.g., Jackson et al. 2014) </li>
<li>OECD TG422D describes an ARE-Nrf2 Luciferase test method </li>
</ul>
<p>In general, there are a variety of commercially available colorimetric or fluorescent kits for detecting Nrf2 activation.</p>
<table border="1">
<tbody>
<tr>
<td>
<p><strong>Assay Type & Measured Content </strong></p>
</td>
<td>
<p><strong>Description </strong></p>
</td>
<td>
<p><strong>Dose Range Studied </strong></p>
</td>
<td>
<p><strong>Assay Characteristics (Length/Ease of use/Accuracy) </strong></p>
</td>
</tr>
<tr>
<td>
<p>ROS </p>
<p>Formation in the Mitochondria assay (Shaki et al., 2012) </p>
</td>
<td>
<p>“The mitochondrial ROS measurement was performed flow cytometry using DCFH-DA. Briefly, isolated kidney mitochondria were incubated with UA (0, 50, 100 and 200 µM) in respiration buffer containing (0.32 mM sucrose, 10mM Tris, 20 mM Mops, 50 µM EGTA, 0.5 mM MgCl2, 0.1 mM KH2PO4 and 5 mM sodium succinate) [32]. In the interval times of 5, 30 and 60 min following the UA addition, a sample was taken and DCFH-DA was added (final concentration, 10 µM) to mitochondria and was then incubated for 10 min.Uranyl acetate-induced ROS generation in isolated kidney mitochondria were determined through the flow cytometry (Partec, Deutschland) equipped with a 488-nm argon ion laser and supplied with the Flomax software and the signals were obtained using a 530-nm bandpass filter (FL-1 channel). Each determination is based on the mean fluorescence intensity of 15,000 counts.” </p>
<p> </p>
</td>
<td>
<p>0, 50,100 and 200 µM of Uranyl Acetate </p>
<p> </p>
</td>
<td>
<p> Long/ Easy High accuracy </p>
<p> </p>
</td>
</tr>
<tr>
<td>
<p>Mitochondrial Antioxidant Content Assay Measuring GSH content (Shaki et al., 2012) </p>
<p> </p>
</td>
<td>
<p>“GSH content was determined using DTNB as the indicator and spectrophotometer method for the isolated mitochondria. The mitochondrial fractions (0.5 mg protein/ml) were incubated with various concentrations of uranyl acetate for 1 h at 30 °C and then 0.1 ml of mitochondrial fractions was added into 0.1 mol/l of phosphate buffers and 0.04% DTNB in a total volume of 3.0 ml (pH 7.4). The developed yellow color was read at 412 nm on a spectrophotometer (UV-1601 PC, Shimadzu, Japan). GSH content was expressed as µg/mg protein.” </p>
</td>
<td>
<p>0, 50, </p>
<p>100, or </p>
<p>200 µM </p>
<p>Uranyl Acetate </p>
</td>
<td>
<p> </p>
</td>
</tr>
<tr>
<td>
<p>H2O2 Production Assay Measuring H2O2 Production in isolated mitochondria (Heyno et al., 2008) </p>
<p> </p>
</td>
<td>
<p>“Effect of CdCl2 and antimycin A (AA) on H2O2 production in isolated mitochondria from potato. H2O2 production was measured as scopoletin oxidation. Mitochondria were incubated for 30 min in the measuring buffer </p>
<p>(see the Materials and Methods) containing 0.5 mM succinate as an electron donor and 0.2 µM mesoxalonitrile 3‐chlorophenylhydrazone (CCCP) as an uncoupler, 10 U horseradish peroxidase and 5 µM scopoletin.” </p>
</td>
<td>
<p>0, 10, 30 </p>
<p>µM Cd2+ </p>
<p> </p>
<p>2 µM antimycin A </p>
</td>
<td>
<p> </p>
</td>
</tr>
<tr>
<td>
<p>Flow Cytometry ROS & Cell Viability (Kruiderig et al., 1997) </p>
<p> </p>
</td>
<td>
<p>“For determination of ROS, samples taken at the indicated time points were directly transferred to FACScan tubes. Dih123 (10 mM, final concentration) was added and cells were incubated at 37°C in a humidified atmosphere (95% air/5% CO2) for 10 min. At t 5 9, propidium iodide (10 mM, final concentration) was added, and cells were analyzed by flow cytometry at 60 ml/min. Nonfluorescent Dih123 is cleaved by ROS to fluorescent R123 and detected by the FL1 detector as described above for Dc (Van de Water 1995)”“For determination of ROS, samples taken at the indicated time points were directly transferred to FACScan tubes. Dih123 (10 mM, final concentration) was added and cells were incubated at 37°C in a humidified atmosphere (95% air/5% CO2) for 10 min. At t 5 9, propidium iodide (10 mM, final concentration) was added, and cells were analyzed by flow cytometry at 60 ml/min. Nonfluorescent Dih123 is cleaved by ROS to fluorescent R123 and detected by the FL1 detector as described above for Dc (Van de Water 1995)” </p>
</td>
<td>
<p> </p>
</td>
<td>
<p> </p>
<p> </p>
<p> </p>
<p> </p>
<p> </p>
<p>Strong/easy medium </p>
</td>
</tr>
<tr>
<td>
<p>DCFH-DA </p>
<p>Assay Detection of hydrogen peroxide production (Yuan et al., </p>
<p>2016) </p>
</td>
<td>
<p>Intracellular ROS production was measured using DCFH-DA as a probe. Hydrogen peroxide oxidizes DCFH to DCF. The probe is hydrolyzed intracellularly to DCFH carboxylate anion. No direct reaction with H2O2 to form fluorescent production. </p>
<p> </p>
</td>
<td>
<p>0-400 </p>
<p>µM </p>
</td>
<td>
<p>Long/ Easy High accuracy </p>
</td>
</tr>
<tr>
<td>
<p>H2-DCF-DAAssay Detection of superoxide production (Thiebault etal., 2007) </p>
<p> </p>
</td>
<td>
<p>This dye is a stable nonpolar compound which diffuses readily into the cells and yields H2-DCF. Intracellular OH or ONOO- react with H2-DCF when cells contain peroxides, to form the highly fluorescent compound DCF, which effluxes the cell. Fluorescence intensity of DCF is measured using a fluorescence spectrophotometer. </p>
<p>The dye (CM-H2DCFDA) diffuses into the cell and is cleaved by esterases, the thiol reactive chlormethyl group reacts with intracellular glutathione which can be detected using flow cytometry. </p>
</td>
<td>
<p> </p>
</td>
<td>
<p>Long/Easy/ High Accuracy </p>
</td>
</tr>
</tbody>
</table>
<p> </p>
<table border="1">
<tbody>
<tr>
<td>
<p><strong>Method of Measurement </strong></p>
</td>
<td>
<p><strong>References </strong></p>
</td>
<td>
<p><strong>Description </strong></p>
</td>
<td colspan="2">
<p><strong>OECD-Approved Assay </strong></p>
</td>
</tr>
<tr>
<td>
<p>Chemiluminescence </p>
</td>
<td>
<p>(Lu, C. et al., 2006; </p>
<p>Griendling, K. K., et al., 2016) </p>
</td>
<td>
<p>ROS can induce electron transitions in molecules, leading to electronically excited products. When the electrons transition back to ground state, chemiluminescence is emitted and can be measured. Reagents such as luminol and lucigenin are commonly used to amplify the signal. </p>
</td>
<td colspan="2">
<p>No </p>
<p> </p>
</td>
</tr>
<tr>
<td>
<p>Spectrophotometry </p>
</td>
<td>
<p>(Griendling, K. K., et al., 2016) </p>
</td>
<td>
<p>NO has a short half-life. However, if it has been reduced to nitrite (NO2-), stable azocompounds can be formed via the Griess Reaction, and further measured by spectrophotometry. </p>
</td>
<td colspan="2">
<p>No </p>
</td>
</tr>
<tr>
<td>
<p>Direct or Spin Trapping-Based electron paramagnetic resonance (EPR) Spectroscopy </p>
</td>
<td>
<p>(Griendling, K. K., et al., 2016) </p>
</td>
<td>
<p>The unpaired electrons (free radicals) found in ROS can be detected with EPR and is known as electron paramagnetic resonance. A variety of spin traps can be used. </p>
</td>
<td colspan="2">
<p>No </p>
</td>
</tr>
<tr>
<td>
<p>Nitroblue Tetrazolium Assay </p>
</td>
<td>
<p>(Griendling, K. K., et al., 2016) </p>
</td>
<td>
<p>The Nitroblue Tetrazolium assay is used to measure O2.− levels. O2.− reduces nitroblue tetrazolium (a yellow dye) to formazan (a blue dye), and can be measured at 620 nm. </p>
</td>
<td colspan="2">
<p>No </p>
</td>
</tr>
<tr>
<td>
<p>Fluorescence analysis of dihydroethidium (DHE) or Hydrocyans </p>
</td>
<td>
<p>(Griendling, K. K., et al., 2016) </p>
</td>
<td>
<p>Fluorescence analysis of DHE is used to measure O2.− levels. O2.− is reduced to O2 as DHE is oxidized to 2-hydroxyethidium, and this reaction can be measured by fluorescence. Similarly, hydrocyans can be oxidized by any ROS, and measured via fluorescence. </p>
</td>
<td colspan="2">
<p>No </p>
</td>
</tr>
<tr>
<td>
<p>Amplex Red Assay </p>
</td>
<td>
<p>(Griendling, K. K., et al., 2016) </p>
</td>
<td>
<p>Fluorescence analysis to measure extramitochondrial or extracellular H2O2 levels. In the presence of horseradish peroxidase and H2O2, Amplex Red is oxidized to resorufin, a fluorescent molecule measurable by plate reader. </p>
<p>An indirect fluorescence analysis to measure intracellular H2O2 levels. H2O2 interacts with peroxidase or heme proteins, which further react with DCFH, oxidizing it to dichlorofluorescein (DCF), a fluorescent product. </p>
</td>
<td colspan="2">
<p>No </p>
</td>
</tr>
<tr>
<td>
<p>HyPer Probe </p>
</td>
<td>
<p>(Griendling, K. K., et al., 2016) </p>
</td>
<td>
<p>Fluorescent measurement of intracellular H2O2 levels. HyPer is a genetically encoded fluorescent sensor that can be used for in vivo and in situ imaging. </p>
</td>
<td colspan="2">
<p>No </p>
</td>
</tr>
<tr>
<td>
<p>Cytochrome c Reduction Assay </p>
</td>
<td>
<p>(Griendling, K. K., et al., 2016) </p>
</td>
<td>
<p>The cytochrome c reduction assay is used to measure O2.− levels. O O2.− is reduced to O2 as ferricytochrome c is oxidized to ferrocytochrome c, and this reaction can be measured by an absorbance increase at 550 nm. </p>
<p>The redox state of tissue is detected through nuclear magnetic resonance/magnetic resonance imaging, with the use of a nitroxide spin probe or biradical molecule. </p>
</td>
<td colspan="2">
<p>No </p>
<p> </p>
<p> </p>
<p> </p>
<p> </p>
<p> </p>
<p> </p>
</td>
</tr>
<tr>
<td>
<p>Glutathione (GSH) depletion </p>
</td>
<td>
<p>(Biesemann, N. et al., 2018) </p>
</td>
<td>
<p>A downstream target of the Nrf2 pathway is involved in GSH synthesis. As an indication of oxidation status, GSH can be measured by assaying the ratio of reduced to oxidized glutathione (GSH:GSSG) using a commercially available kit (e.g., <a href="http://www.abcam.com/gshgssg-ratio-detection-assay-kit-fluorometric-green-ab138881.html" rel="noreferrer noopener" target="_blank">http://www.abcam.com/gshgssg-ratio-detection-assay-kit-fluorometric-green-ab138881.html</a>). </p>
<p>Oxidative damage to lipids can be measured by assaying for lipid peroxidation with TBARS using a commercially available kit. </p>
</td>
<td colspan="2">
<p>No </p>
</td>
</tr>
<tr>
<td>
<p>Protein oxidation (carbonylation) </p>
</td>
<td>
<p>(Azimzadeh et al., 2017; Azimzadeh et al., 2015; Ping et al., 2020) </p>
</td>
<td>
<p>Can be determined with ELISA or a commercial assay kit. Protein oxidation can indicate the level of oxidative stress. </p>
</td>
<td colspan="2">
<p>No </p>
</td>
</tr>
<tr>
<td>
<p>Seahorse XFp Analyzer </p>
</td>
<td>
<p>Leung et al. 2018 </p>
</td>
<td>
<p>The Seahorse XFp Analyzer provides information on mitochondrial function, oxidative stress, and metabolic dysfunction of viable cells by measuring respiration (oxygen consumption rate; OCR) and extracellular pH (extracellular acidification rate; ECAR). </p>
</td>
<td>
<p>No </p>
</td>
</tr>
</tbody>
</table>
<p> </p>
<p>Molecular Biology: Nrf2. Nrf2’s transcriptional activity is controlled post-translationally by oxidation of Keap1. Assays for Nrf2 activity include: </p>
<table border="1">
<tbody>
<tr>
<td>
<p>Method of Measurement </p>
</td>
<td>
<p>References </p>
</td>
<td>
<p>Description </p>
</td>
<td>
<p>OECD-Approved Assay </p>
</td>
</tr>
<tr>
<td>
<p>Immunohistochemistry </p>
</td>
<td>
<p>(Amsen, D., de Visser, K. E., and Town, T., 2009) </p>
</td>
<td>
<p>Immunohistochemistry for increases in Nrf2 protein levels and translocation into the nucleus </p>
</td>
<td>
<p>No </p>
</td>
</tr>
<tr>
<td>
<p>qPCR </p>
</td>
<td>
<p>(Forlenza et al., 2012) </p>
</td>
<td>
<p>qPCR of Nrf2 target genes (e.g., Nqo1, Hmox-1, Gcl, Gst, Prx, TrxR, Srxn), or by commercially available pathway-based qPCR array (e.g., oxidative stress array from SABiosciences) </p>
</td>
<td>
<p>No </p>
</td>
</tr>
<tr>
<td>
<p>Whole transcriptome profiling via microarray or via RNA-seq followed by a pathway analysis </p>
</td>
<td>
<p>(Jackson, A. F. et al., 2014) </p>
</td>
<td>
<p>Whole transcriptome profiling by microarray or RNA-seq followed by pathway analysis (in IPA, DAVID, metacore, etc.) for enrichment of the Nrf2 oxidative stress response pathway </p>
</td>
<td>
<p>No </p>
</td>
</tr>
</tbody>
</table>
<p> </p>
<h4>References</h4>
<p>Ahmad, S. et al. (2021), “60Co-γ Radiation Alters Developmental Stages of Zeugodacus cucurbitae (Diptera: Tephritidae) Through Apoptosis Pathways Gene Expression”, Journal Insect Science, Vol. 21/5, Oxford University Press, Oxford, <a href="https://doi.org/10.1093/jisesa/ieab080" rel="noreferrer noopener" target="_blank">https://doi.org/10.1093/jisesa/ieab080</a> </p>
<p>Antelmann, H. and J. D. Helmann (2011), “Thiol-based redox switches and gene regulation.”, Antioxidants & Redox Signaling, Vol. 14/6, Mary Ann Leibert Inc., Larchmont, <a href="https://doi.org/10.1089/ars.2010.3400" rel="noreferrer noopener" target="_blank">https://doi.org/10.1089/ars.2010.3400</a> </p>
<p>Amsen, D., de Visser, K. E., and Town, T. (2009), “Approaches to determine expression of inflammatory cytokines”, in Inflammation and Cancer, Humana Press, Totowa, <a href="https://doi.org/10.1007/978-1-59745-447-6_5" rel="noreferrer noopener" target="_blank">https://doi.org/10.1007/978-1-59745-447-6_5</a> </p>
<p>Azimzadeh, O. et al. (2015), “Integrative Proteomics and Targeted Transcriptomics Analyses in Cardiac Endothelial Cells Unravel Mechanisms of Long-Term Radiation-Induced Vascular Dysfunction”, Journal of Proteome Research, Vol. 14/2, American Chemical Society, Washington, <a href="https://doi.org/10.1021/pr501141b" rel="noreferrer noopener" target="_blank">https://doi.org/10.1021/pr501141b</a> </p>
<p>Azimzadeh, O. et al. (2017), “Proteome analysis of irradiated endothelial cells reveals persistent alteration in protein degradation and the RhoGDI and NO signalling pathways”, International Journal of Radiation Biology, Vol. 93/9, Informa, London, <a href="https://doi.org/10.1080/09553002.2017.1339332" rel="noreferrer noopener" target="_blank">https://doi.org/10.1080/09553002.2017.1339332</a> </p>
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<p>Bai, J. et al. (2020), “Irradiation-induced senescence of bone marrow mesenchymal stem cells aggravates osteogenic differentiation dysfunction via paracrine signaling”, American Journal of Physiology - Cell Physiology, Vol. 318/5, American Physiological Society, Rockville, <a href="https://doi.org/10.1152/ajpcell.00520.2019." rel="noreferrer noopener" target="_blank">https://doi.org/10.1152/ajpcell.00520.2019.</a> </p>
<p>Balasubramanian, D (2000), “Ultraviolet radiation and cataract”, Journal of ocular pharmacology and therapeutics, Vol. 16/3, Mary Ann Liebert Inc., Larchmont, <a href="https://doi.org/10.1089/jop.2000.16.285.%22%20/t%20%22_blank" rel="noreferrer noopener" target="_blank">https://doi.org/10.1089/jop.2000.16.285.</a> </p>
<p>Biesemann, N. et al., (2018), “High Throughput Screening of Mitochondrial Bioenergetics in Human Differentiated Myotubes Identifies Novel Enhancers of Muscle Performance in Aged Mice”, Scientific Reports, Vol. 8/1, Nature Portfolio, London, <a href="https://doi.org/10.1038/s41598-018-27614-8" rel="noreferrer noopener" target="_blank">https://doi.org/10.1038/s41598-018-27614-8</a>. </p>
<p>Elgazzar, A. and N. Kazem. (2015), “Chapter 23: Biological effects of ionizing radiation” in The Pathophysiologic Basis of Nuclear Medicine, Springer, New York, pp. 540-548 </p>
<p>Eruslanov, E., & Kusmartsev, S. (2010). Identification of ROS using oxidized DCFDA and flow-cytometry. Methods in molecular biology ,N.J., Vol. 594, https://doi.org/10.1007/978-1-60761-411-1_4 </p>
<p>Fletcher, A. E (2010), “Free radicals, antioxidants and eye diseases: evidence from epidemiological studies on cataract and age-related macular degeneration”, Ophthalmic Research, Vol. 44, Karger International, Basel, <a href="https://doi.org/10.1159/000316476.%22%20/t%20%22_blank" rel="noreferrer noopener" target="_blank">https://doi.org/10.1159/000316476.</a> </p>
<p>Forlenza, M. et al. (2012), “The use of real-time quantitative PCR for the analysis of cytokine mRNA levels” in Cytokine Protocols, Springer, New York, https://doi.org/10.1007/978-1-61779-439-1_2 </p>
<p>Forrester, S.J. et al. (2018), “Angiotensin II Signal Transduction: An Update on Mechanisms of Physiology and Pathophysiology”, Physiological Reviews, Vol. 98/3, American Physiological Society, Rockville, <a href="https://doi.org/10.1152/physrev.00038.201" rel="noreferrer noopener" target="_blank">https://doi.org/10.1152/physrev.00038.201</a> </p>
<p>Foyer, C. H., A. V. Ruban, and G. Noctor (2017), “Viewing oxidative stress through the lens of oxidative signalling rather than damage”, Biochemical Journal, Vol. 474/6, Portland Press, England, https://doi.org/10.1042/BCJ20160814 </p>
<p>Ganea, E. and J. J. Harding (2006), “Glutathione-related enzymes and the eye”, Current eye research, Vol. 31/1, Informa, London, <a href="https://doi.org/10.1080/02713680500477347.%22%20/t%20%22_blank" rel="noreferrer noopener" target="_blank">https://doi.org/10.1080/02713680500477347.</a> </p>
<p>Griendling, K. K. et al. (2016), “Measurement of reactive oxygen species, reactive nitrogen species, and redox-dependent signaling in the cardiovascular system: a scientific statement from the American Heart Association”, Circulation research, Vol. 119/5, Lippincott Williams & Wilkins, Philadelphia, <a href="https://doi.org/10.1161/RES.0000000000000110" rel="noreferrer noopener" target="_blank">https://doi.org/10.1161/RES.0000000000000110</a> </p>
<p>Guo, C. et al. (2013), “Oxidative stress, mitochondrial damage and neurodegenerative diseases”, Neural regeneration research, Vol. 8/21, Publishing House of Neural Regeneration Research, China, <a href="https://doi.org/10.3969/j.issn.1673-5374.2013.21.009" rel="noreferrer noopener" target="_blank">https://doi.org/10.3969/j.issn.1673-5374.2013.21.009</a> </p>
<p>Hargreaves, M., and L. L. Spriet (2020), “Skeletal muscle energy metabolism during exercise.”, Nature Metabolism, Vol. 2, Nature Portfolio, London, <a href="https://doi.org/10.1038/s42255-020-0251-4" rel="noreferrer noopener" target="_blank">https://doi.org/10.1038/s42255-020-0251-4</a> </p>
<p>Hladik, D. and S. Tapio (2016), “Effects of ionizing radiation on the mammalian brain”, Mutation Research/Reviews in Mutation Research, Vol. 770, Elsevier, Amsterdam, <a href="https://doi.org/10.1016/j.mrrev.2016.08.003" rel="noreferrer noopener" target="_blank">https://doi.org/10.1016/j.mrrev.2016.08.003</a> </p>
<p>Itoh, K., J. Mimura and M. Yamamoto (2010), “Discovery of the negative regulator of Nrf2, Keap1: a historical overview”, Antioxidants & Redox Signaling, Vol. 13/11, Mary Ann Leibert Inc., Larchmont, <a href="https://doi.org/10.1089/ars.2010.3222" rel="noreferrer noopener" target="_blank">https://doi.org/10.1089/ars.2010.3222</a> </p>
<p>Jackson, A.F. et al. (2014), “Case study on the utility of hepatic global gene expression profiling in the risk assessment of the carcinogen furan.”, Toxicology and Applied Pharmacology, Vol. 274/11, Elsevier, Amsterdam, <a href="https://doi.org/10.1016/j.taap.2013.10.019" rel="noreferrer noopener" target="_blank">https://doi.org/10.1016/j.taap.2013.10.019</a> </p>
<p>Jacobsen, N.R. et al. (2008), “Genotoxicity, cytotoxicity, and reactive oxygen species induced by single-walled carbon nanotubes and C60 fullerenes in the FE1-MutaTM Mouse lung epithelial cells”, Environmental and Molecular Mutagenesis, Vol. 49/6, John Wiley & Sons, Inc., Hoboken, <a href="https://doi.org/10.1002/em.20406" rel="noreferrer noopener" target="_blank">https://doi.org/10.1002/em.20406</a> </p>
<p>Karimi, N. et al. (2017), “Radioprotective effect of hesperidin on reducing oxidative stress in the lens tissue of rats”, International Journal of Pharmaceutical Investigation, Vol. 7/3, Phcog Net, Bengaluru, <a href="https://doi.org/10.4103/jphi.JPHI_60_17.%E2%80%AF" rel="noreferrer noopener" target="_blank">https://doi.org/10.4103/jphi.JPHI_60_17. </a> </p>
<p>Leung, D.T.H., and Chu, S. (2018), “Measurement of Oxidative Stress: Mitochondrial Function Using the Seahorse System” In: Murthi, P., Vaillancourt, C. (eds) Preeclampsia. Methods in Molecular Biology, vol 1710. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7498-6_22 </p>
<p>Lu, C., G. Song, and J. Lin (2006), “Reactive oxygen species and their chemiluminescence-detection methods”, TrAC Trends in Analytical Chemistry, Vol. 25/10, Elsevier, Amsterdam, <a href="https://doi.org/10.1016/j.trac.2006.07.007" rel="noreferrer noopener" target="_blank">https://doi.org/10.1016/j.trac.2006.07.007</a> </p>
<p>Nguyen Dinh Cat, A. et al. (2013), “Angiotensin II, NADPH oxidase, and redox signaling in the vasculature”, Antioxidants & redox signaling, Vol. 19/10, Mary Ann Liebert, Larchmont, <a href="https://doi.org/10.1089/ars.2012.4641" rel="noreferrer noopener" target="_blank">https://doi.org/10.1089/ars.2012.4641</a> </p>
<p>Ping, Z. et al. (2020), “Oxidative Stress in Radiation-Induced Cardiotoxicity”, Oxidative Medicine and Cellular Longevity, Vol. 2020, Hindawi, <a href="https://doi.org/10.1155/2020/3579143" rel="noreferrer noopener" target="_blank">https://doi.org/10.1155/2020/3579143</a> </p>
<p>Powers, S.K. and M.J. Jackson. (2008), “Exercise-Induced Oxidative Stress: Cellular Mechanisms and Impact on Muscle Force Production”, Physiological Reviews, Vol. 88/4, American Physiological Society, Rockville, <a href="https://doi.org/10.1152/physrev.00031.2007" rel="noreferrer noopener" target="_blank">https://doi.org/10.1152/physrev.00031.2007</a> </p>
<p>Raimondi, V., F. Ciccarese and V. Ciminale. (2020), “Oncogenic pathways and the electron transport chain: a dangeROS liason”, British Journal of Cancer, Vol. 122/2, Nature Portfolio, London, <a href="https://doi.org/10.1038/s41416-019-0651-y" rel="noreferrer noopener" target="_blank">https://doi.org/10.1038/s41416-019-0651-y</a> </p>
<p>Seen, S. and L. Tong. (2018), “Dry eye disease and oxidative stress”, Acta Ophthalmologica, Vol. 96/4, John Wiley & Sons, Inc., Hoboken, <a href="https://doi.org/10.1111/aos.13526" rel="noreferrer noopener" target="_blank">https://doi.org/10.1111/aos.13526</a> </p>
<p>Ungvari, Z. et al. (2013), “Ionizing Radiation Promotes the Acquisition of a Senescence-Associated Secretory Phenotype and Impairs Angiogenic Capacity in Cerebromicrovascular Endothelial Cells: Role of Increased DNA Damage and Decreased DNA Repair Capacity in Microvascular Radiosensitivity”, The Journals of Gerontology Series A: Biological Sciences and Medical Sciences, Vol. 68/12, Oxford University Press, Oxford, <a href="https://doi.org/10.1093/gerona/glt057." rel="noreferrer noopener" target="_blank">https://doi.org/10.1093/gerona/glt057.</a> </p>
<p> </p>
<p>Vargas-Mendoza, N. et al. (2021), “Oxidative Stress, Mitochondrial Function and Adaptation to Exercise: New Perspectives in Nutrition”, Life, Vol. 11/11, Multidisciplinary Digital Publishing Institute, Basel, <a href="https://doi.org/10.3390/life11111269" rel="noreferrer noopener" target="_blank">https://doi.org/10.3390/life11111269</a> </p>
<p>Wang, H. et al. (2019), “Radiation-induced heart disease: a review of classification, mechanism and prevention”, International Journal of Biological Sciences, Vol. 15/10, Ivyspring International Publisher, Sydney, <a href="https://doi.org/10.7150/ijbs.35460" rel="noreferrer noopener" target="_blank">https://doi.org/10.7150/ijbs.35460</a> </p>
<p>Zhang, R. et al. (2009), “Blockade of AT1 receptor partially restores vasoreactivity, NOS expression, and superoxide levels in cerebral and carotid arteries of hindlimb unweighting rats”, Journal of applied physiology, Vol. 106/1, American Physiological Society, Rockville, <a href="https://doi.org/10.1152/japplphysiol.01278.2007" rel="noreferrer noopener" target="_blank">https://doi.org/10.1152/japplphysiol.01278.2007</a>. </p>
<p>Zhao, R. Z. et al. (2019), “Mitochondrial electron transport chain, ROS generation and uncoupling”, International journal of molecular medicine, Vol. 44/1, Spandidos Publishing Ltd., Athens, <a href="https://doi.org/10.3892/ijmm.2019.4188" rel="noreferrer noopener" target="_blank">https://doi.org/10.3892/ijmm.2019.4188</a> </p>
<h4><a href="/events/1187">Event: 1187: Increased, ER binding to DNA (classical pathway) </a></h4>
<h5>Short Name: Increased, ER binding to DNA (classical pathway) </h5>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">Endothelial cell migration refers to the movement of endothelial cells, which are the cells lining the inner surface of blood vessels, across tissues. This dynamic process is essential for various physiological functions, including vascular development, tissue repair, and angiogenesis</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529"> (</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212121">Michaelis</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212121">, </span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">Fonseca).</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">.</span></span></span></span></span></p>
<p style="text-align:justify"> </p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">During migration, endothelial cells undergo a series of coordinated steps, including sensing chemotactic signals, altering their cytoskeleton to form protrusions</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529"> (Extension of finger-like projections (filopodia) at the leading edge of the cell to sense the environment)</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">, adhering to the extracellular matrix</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529"> through molecules such as integrins</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">,</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">contraction (pulling the cell forward using actin) and finally detachment for movement (</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212121">Michaelis</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212121">, </span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">Fonseca)</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">. These movements are crucial for the remodeling and maintenance of blood vessels.</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529"> This is regulated by chemical signs (VEGG, integrins) and physical cues (</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212121">Michaelis</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212121">, </span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">Fonseca, Norton).</span></span></span></span></span></p>
<p style="text-align:justify"> </p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">The role of endothelial cell migration is in (</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212121">Michaelis</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212121">, </span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">Fonseca).: </span></span></span></span></span></p>
<ul>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">Angiogenesis:</span></span></span> <span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">One of the primary roles of endothelial cell migration is in angiogenesis, the formation of new blood vessels. In response to signals from growth factors like vascular endothelial growth factor (VEGF), endothelial cells migrate towards the site of angiogenesis, contributing to the expansion of the vascular network</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529"> (Michaelis, Lamalis)</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">.</span></span></span></span></span></li>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">Tissue Repair:</span></span></span> <span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">Endothelial cell migration is crucial for repairing damaged blood vessels. In response to injury or inflammation, endothelial cells migrate to the site of damage, facilitating the restoration of vascular integrity</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529"> (Michaelis)</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">.</span></span></span></span></span></li>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">Vascular Development:</span></span></span> <span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">During embryonic development, endothelial cell migration is essential for the formation and remodeling of blood vessels</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529"> (Scarpa)</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">. This process helps establish the intricate vascular network required for organ development.</span></span></span></span></span></li>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">Immune Response:</span></span></span> <span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">Endothelial cells play a role in immune responses by facilitating the migration of immune cells across blood vessel walls to sites of infection or injury</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529"> (Sturtzel)</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">.</span></span></span></span></span></li>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">Lymphangiogenesis:</span></span></span> <span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">Endothelial cell migration is involved in lymphangiogenesis, the formation of new lymphatic vessels. This process is crucial for fluid drainage, immune surveillance, and can also play a role in cancer metastasis</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529"> (Pengchung)</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">.</span></span></span></span></span></li>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">Wound Healing:Endothelial cells contribute to wound healing by migrating to the site of injury and participating in the formation of new blood vessels, a process known as neovascularization</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529"> (Lamalis, Amersfoort)</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">.</span></span></span></span></span></li>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">Cancer Metastasis:</span></span></span> <span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">In cancer, endothelial cell migration is hijacked by tumors to support their growth and metastasis. Tumor cells release angiogenic factors, inducing the migration of endothelial cells to form new blood vessels that supply nutrients to the growing tumor</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529"> (Lamalis)</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">.</span></span></span></span></span></li>
</ul>
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</div>
<h4>How it is Measured or Detected</h4>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">Assays used to study endothelial cell migration (Guo): </span></span></span></span></span></p>
<ul>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">Boyden chamber: evaluates the ability of cells to migrate through a porous membrane towards a chemoattractant (substance that attracts cells) placed in the lower chamber.</span></span></span></span></span></li>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">Scratch wound assay: collective movement of endothelial cells to close a "wound" created by scratching a confluent monolayer of cells.</span></span></span></span></span></li>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">Microfluidic assay: microfluidic channels to create controlled environments that mimic the physiological flow conditions experienced by endothelial cells in vivo (Shih)</span></span></span></span></span></li>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">Tube formation: assays evaluate the ability of endothelial cells to form tube-like structures, mimicking the process of blood vessel formation (angiogenesis) (Guo)</span></span></span></span></span></li>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">Collagen Invasion Assay: Assess the invasive capacity of endothelial cells through a three-dimensional collagen matrix</span></span></span></span></span></li>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">Time-lapse microscopy: using live-cell imaging</span></span></span></span></span></li>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">In vivo: vessel density in fat pads</span></span></span></span></span></li>
<p>Mierke CT. Role of the endothelium during tumor cell metastasis: is the endothelium a barrier or a promoter for cell invasion and metastasis? J Biophys. 2008;2008:183516. doi: 10.1155/2008/183516. Epub 2009 Mar 5. PMID: 20107573; PMCID: PMC2809021.</p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212121">Michaelis UR. Mechanisms of endothelial cell migration. Cell Mol Life Sci. 2014 Nov;71(21):4131-48. doi: 10.1007/s00018-014-1678-0. Epub 2014 Jul 20. PMID: 25038776.</span></span></span></span></span></p>
<p style="text-align:justify"> </p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212121">Guo S, Lok J, Liu Y, Hayakawa K, Leung W, Xing C, Ji X, Lo EH. Assays to examine endothelial cell migration, tube formation, and gene expression profiles. Methods Mol Biol. 2014;1135:393-402. doi: 10.1007/978-1-4939-0320-7_32. PMID: 24510881; PMCID: PMC4035906.</span></span></span></span></span></p>
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<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#222222">Shih, HC., Lee, TA., Wu, HM. </span></span></span><em>et al.</em> Microfluidic Collective Cell Migration Assay for Study of Endothelial Cell Proliferation and Migration under Combinations of Oxygen Gradients, Tensions, and Drug Treatments. <em>Sci Rep</em> <strong>9</strong>, 8234 (2019). https://doi.org/10.1038/s41598-019-44594-5</span></span></p>
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<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">Fonseca CG, Barbacena P, Franco CA. Endothelial cells on the move: dynamics in vascular morphogenesis and disease. Vasc Biol. 2020 Jul 2;2(1):H29-H43. doi: 10.1530/VB-20-0007. PMID: 32935077; PMCID: PMC7487603.</span></span></span></span></span></p>
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<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212121">Yu P, Wu G, Lee HW, Simons M. Endothelial Metabolic Control of Lymphangiogenesis. Bioessays. 2018 Jun;40(6):e1700245. doi: 10.1002/bies.201700245. Epub 2018 May 11. PMID: 29750374; PMCID: PMC6237195.</span></span></span></span></span></p>
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<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#222222">Amersfoort, J., Eelen, G. & Carmeliet, P. Immunomodulation by endothelial cells — partnering up with the immune system?. </span></span></span><em>Nat Rev Immunol</em> <strong>22</strong>, 576–588 (2022). <a href="https://doi.org/10.1038/s41577-022-00694-4" style="color:#467886; text-decoration:underline">https://doi.org/10.1038/s41577-022-00694-4</a></span></span></p>
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<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212121">Sturtzel C. Endothelial Cells. Adv Exp Med Biol. 2017;1003:71-91. doi: 10.1007/978-3-319-57613-8_4. PMID: 28667554.</span></span></span></span></span></p>
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<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212121">Lamalice L, Le Boeuf F, Huot J. Endothelial cell migration during angiogenesis. Circ Res. 2007 Mar 30;100(6):782-94. doi: 10.1161/01.RES.0000259593.07661.1e. PMID: 17395884.</span></span></span></span></span></p>
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<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212121">Scarpa E, Mayor R. Collective cell migration in development. J Cell Biol. 2016 Jan 18;212(2):143-55. doi: 10.1083/jcb.201508047. PMID: 26783298; PMCID: PMC4738384.</span></span></span> </span></span></p>
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<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212121">Michaelis UR. Mechanisms of endothelial cell migration. Cell Mol Life Sci. 2014 Nov;71(21):4131-48. doi: 10.1007/s00018-014-1678-0. Epub 2014 Jul 20. PMID: 25038776.</span></span></span></span></span></p>
<p style="text-align:justify"> </p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212121">Norton KA, Popel AS. Effects of endothelial cell proliferation and migration rates in a computational model of sprouting angiogenesis. Sci Rep. 2016 Nov 14;6:36992. doi: 10.1038/srep36992. PMID: 27841344; PMCID: PMC5107954.</span></span></span></span></span></p>
<td><a href="/aops/200">Aop:200 - Estrogen receptor activation leading to breast cancer </a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/293">Aop:293 - Increased DNA damage leading to increased risk of breast cancer</a></td>
<td>MolecularInitiatingEvent</td>
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<tr>
<td><a href="/aops/294">Aop:294 - Increased reactive oxygen and nitrogen species (RONS) leading to increased risk of breast cancer</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/396">Aop:396 - Deposition of ionizing energy leads to population decline via impaired meiosis</a></td>
<td>KeyEvent</td>
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<td><a href="/aops/432">Aop:432 - Deposition of Energy by Ionizing Radiation leading to Acute Myeloid Leukemia</a></td>
<td>KeyEvent</td>
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<td><a href="/aops/388">Aop:388 - Deposition of ionising energy leading to population decline via programmed cell death</a></td>
<td>KeyEvent</td>
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<td><a href="/aops/435">Aop:435 - Deposition of ionising energy leads to population decline via pollen abnormal</a></td>
<td>KeyEvent</td>
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<tr>
<td><a href="/aops/441">Aop:441 - Ionizing radiation-induced DNA damage leads to microcephaly via apoptosis and premature cell differentiation</a></td>
<td>KeyEvent</td>
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<td><a href="/aops/444">Aop:444 - Ionizing radiation leads to reduced reproduction in Eisenia fetida via reduced spermatogenesis and cocoon hatchability</a></td>
<td>MolecularInitiatingEvent</td>
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<tr>
<td><a href="/aops/423">Aop:423 - Toxicological mechanisms of hepatocyte apoptosis through the PARP1 dependent cell death pathway </a></td>
<td>KeyEvent</td>
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<td><a href="/aops/476">Aop:476 - Adverse Outcome Pathways diagram related to PBDEs associated male reproductive toxicity</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/591">Aop:591 - DBDPE-induced DNA damage increase in liver leading to Non-alcoholic fatty liver disease via liver steatosis and inhibition of regeneration</a></td>
<p>DNA nucleotide damage, single, and double strand breaks occur in the course of cellular operations such as DNA repair and replication and can be induced directly and in neighboring “bystander” cells by internal or external stressors like reactive oxygen species, chemicals, and radiation. Ionizing radiation and RONS such as hydroxyl radicals or peroxide can create a range of lesions (a change in molecular structure) in the base of the nucleotide, with guanine particularly vulnerable because of its low redox potential (David, O'Shea et al. 2007). The same stressors can also break the sugar (deoxyribose)-phosphate backbone creating a single strand break. Simultaneous proximal breaks in both strands of DNA form double strand breaks, which are considered to be more destructive and mutagenic than lesions or single strand breaks. Double strand breaks can generate chromosomal abnormalities including changes in chromosomal number, breaks and gaps, translocations, inversions, and deletions (Yang, Craise et al. 1992; Haag, Hsu et al. 1996; Ponnaiya, Cornforth et al. 1997; Yang, Georgy et al. 1997; Unger, Wienberg et al. 2010; Behjati, Gundem et al. 2016; Morishita, Muramatsu et al. 2016).</p>
<p>However, DNA lesions and single strand breaks can also be destructive and mutagenic. Lesions can lead to point mutations (David, O'Shea et al. 2007) or single strand breaks (Regulus, Duroux et al. 2007). Lesions and single strand breaks can also promote the formation of double strand breaks: replication fork collapse and double strand breaks sometimes occur during mitosis when the replisome encounters an unrepaired single strand break (Kuzminov 2001), and clustered lesions and closely opposed single strand breaks can also form double strand breaks (Chaudhry and Weinfeld 1997; Vispe and Satoh 2000; Shiraishi, Shikazono et al. 2017). Complex damage consists of any combination of closely opposed DNA lesions, abasic sites, crosslinks, single, or double strand breaks in proximity. While classically induced by ionizing radiation, there is also evidence that it can be induced by oxidative activity (Sharma, Collins et al. 2016) or even by a single oxidizing particle (Ravanat, Breton et al. 2014). Complex damage is more difficult to repair (Kuhne, Rothkamm et al. 2000; Stenerlow, Hoglund et al. 2000; Pinto, Prise et al. 2005; Rydberg, Cooper et al. 2005).</p>
<p>DNA damage and resulting repair activity can trigger a halt in the cell cycle, cell death (apoptosis), and cause permanent changes to DNA including deletions, translocations, and sequence changes. DNA damage is also associated with an increase in genomic instability - the new appearance of DNA damage including double strand breaks, mutations, and chromosomal damage following repair of initial damage in affected cells or in clonal descendants or neighbors of DNA damaged cells. The mechanism behind this long term DNA damage is not clear, but telomere erosion appears to play a major role (Murnane 2012; Sishc, Nelson et al. 2015). Genomic instability is more common and longer lasting following complex damage (Ponnaiya, Cornforth et al. 1997), and is influenced by multiple factors including variants in DNA repair genes (Ponnaiya, Cornforth et al. 1997; Yu, Okayasu et al. 2001; Yin, Menendez et al. 2012), RONS (Dayal, Martin et al. 2008), estrogen (Kutanzi and Kovalchuk 2013), caspases (Liu, He et al. 2015), and telomeres (Sishc, Nelson et al. 2015).</p>
<p>DNA damage can be studied in isolated DNA, fixed cells, or living cells. Types of damage that can be detected include single and double strand breaks, nucleotide damage, complex damage, and chromosomal or telomere damage. The OECD test guideline for DNA synthesis Test No. 486 (OECD 1997) detects nucleotide excision repair, so it will reflect the formation of bulky DNA adducts but not the majority of oxidative damage to nucleotides, which is typically repaired via the Base Excision Repair pathway. The OECD test guideline alkaline comet assay Test No. 489 (OECD 2016) detects single and double strand breaks, including those arising from repair as well as some (alkali sensitive) nucleotide lesions including some lesions from oxidative damage. OECD tests for chromosomal damage and micronuclei Test No. 473, 475, 483, and 487 measure longer term effects of DNA damage but these tests require the damaged cell to subsequently undergo replication (OECD 2016; OECD 2016; OECD 2016; OECD 2016). They can therefore reflect a wider range of sources of DNA damage including changes in mitosis. Finally, tests for mutations reveal past DNA damage that resulted in a heritable change, and these are described in the key event ‘Increase in Mutation’.</p>
<p>Many other (non-test guideline) techniques have been used to examine specific forms of DNA damage (Table 1). Double strand breaks are commonly reported because of the significant risk attributed to breaks and the relative ease of detecting and quantifying them. Historically, single and double strand breaks were measured using gel electrophoresis, but are now commonly visualized microscopically using fluorescent or other labeled probes for double and single strand break repair such as H2AX and XRCC2. Base lesions can also be detected using labeled probes for base excision repair enzymes, or by chemical methods such as mass spectroscopy. Refinements on these methods can be used to characterize complex or clustered damage, in which various forms of damage occur in close proximity on a DNA molecule (Lorat, Timm et al. 2016; Nikitaki, Nikolov et al. 2016).</p>
<p>Certain challenges are common to all methods of detecting DNA damage. In the time required to initiate the detection method, some DNA may already be repaired, leading to undercounting of damage. On the other hand, apoptotic DSBs may be incorrectly included in a measurement of direct (non-apoptotic) induction of DSB damage unless controlled. All methods have difficulty distinguishing individual components of clustered lesions, and microscopic methods may undercount disparate breaks that are processed together in repair centers (Barnard, Bouffler et al. 2013). Methods that use isolated DNA (gel electrophoresis, analytical chemistry) are vulnerable to artifacts and must ensure that the DNA sample is protected from oxidative damage during extraction (Pernot, Hall et al. 2012; Barnard, Bouffler et al. 2013; Ravanat, Breton et al. 2014).</p>
<p>Table 1. Common methods of detecting DNA damage</p>
<p>Single cell gel electrophoresis (comet assay) with restriction enzymes (Collins 2004)</p>
</td>
<td style="height:22px; width:133px">
<p>Gel electrophoresis</p>
<p> </p>
</td>
<td style="height:22px; width:211px">
<p>A variant of the comet assay in which restriction enzymes allow the identification of different types of nucleotide damage.</p>
<p>The comet assay is more sensitive than PFGE, detecting damage from 0.1 Gy ionizing radiation (Pernot, Hall et al. 2012). A reproducible high-throughput application of the assay is available (Ge, Prasongtanakij et al. 2014; Sykora, Witt et al. 2018), and the test requires only a small (single cell) sample. Requires destruction of the cell.</p>
</td>
</tr>
<tr>
<td style="height:22px; width:127px">
<p><strong>Nucleotide damage</strong></p>
</td>
<td style="height:22px; width:167px">
<p>Labeled probes including Biotrin OxyDNA and anti- 8-oxoguanine-DNA glycosylase (OGG1) for oxidative damage and AP</p>
<p>endonuclease (APE1) for Base Excision Repair of less bulky lesions such as oxidative damage.</p>
</td>
<td style="height:22px; width:133px">
<p>Microscopy, FACS</p>
</td>
<td style="height:22px; width:211px">
<p>Most useful with FACS or other measures of average or relative intensity, as locations and numbers of damaged nucleotides can be difficult to distinguish using fluorescence microscopy. (Ogawa, Kobayashi et al. 2003; Nikitaki, Nikolov et al. 2016).</p>
</td>
</tr>
<tr>
<td style="height:22px; width:127px">
<p><strong>Nucleotide damage</strong></p>
</td>
<td style="height:22px; width:167px">
<p>High performance liquid chromatography (HPLC), tandem mass spectrometry (MS/MS)</p>
</td>
<td style="height:22px; width:133px">
<p>Analytical chemistry</p>
</td>
<td style="height:22px; width:211px">
<p>Capable of quantifying low levels of specific nucleotide lesions (Madugundu, Cadet et al. 2014; Ravanat, Breton et al. 2014). Requires destruction of the cell.</p>
</td>
</tr>
<tr>
<td style="height:22px; width:127px">
<p><strong>Nucleotide damage</strong></p>
</td>
<td style="height:22px; width:167px">
<p>Unscheduled DNA synthesis test OECD Test Guideline 486 (OECD 1997)</p>
</td>
<td style="height:22px; width:133px">
<p>Autoradiography</p>
</td>
<td style="height:22px; width:211px">
<p>Measures DNA damage that is repaired using Nucleotide Excision Repair - mostly bulky adducts (OECD (Organisation for Economic Co-operation and Development) 2016).</p>
</td>
</tr>
<tr>
<td style="height:22px; width:127px">
<p><strong>Non-specific DNA strand breaks</strong></p>
</td>
<td style="height:22px; width:167px">
<p>Single cell gel electrophoresis (comet assay), alkali conditions</p>
<p>OECD Test Guideline 489 (OECD 2016)</p>
</td>
<td style="height:22px; width:133px">
<p>Gel electrophoresis</p>
</td>
<td style="height:22px; width:211px">
<p>When used in alkali conditions, the comet assay reveals single and double strand breaks and alkali-sensitive nucleotide lesions. See single cell gel electrophoresis (comet assay) with restriction enzymes above for further comments. </p>
<p> </p>
</td>
</tr>
<tr>
<td style="height:22px; width:127px">
<p><strong>Single strand breaks</strong></p>
</td>
<td style="height:22px; width:167px">
<p>Labeled probe pXRCC1 (Lorat, Brunner et al. 2015)</p>
</td>
<td style="height:22px; width:133px">
<p>Microscopy</p>
</td>
<td style="height:22px; width:211px">
<p>Fluorescent probes can label single strand breaks in cells, while immunogold labeling is able to distinguish multiple single strand breaks in clusters (Lorat, Timm et al. 2016; Nikitaki, Nikolov et al. 2016).</p>
</td>
</tr>
<tr>
<td style="height:22px; width:127px">
<p><strong>Double strand breaks</strong></p>
</td>
<td style="height:22px; width:167px">
<p>Single cell gel electrophoresis (comet assay), neutral conditions</p>
</td>
<td style="height:22px; width:133px">
<p>Gel electrophoresis</p>
</td>
<td style="height:22px; width:211px">
<p>Neutral conditions help minimize the release of single strand breaks coiled DNA and alkali lesions, allowing the measurement of double strand breaks. Since single strand breaks can still appear, assay is not very sensitive or specific to double strand breaks (Pernot, Hall et al. 2012). See single cell gel electrophoresis (comet assay) with restriction enzymes above for further comments.</p>
</td>
</tr>
<tr>
<td style="height:22px; width:127px">
<p><strong>Double strand breaks</strong></p>
</td>
<td style="height:22px; width:167px">
<p>Pulsed field gel electrophoresis (PFGE)</p>
</td>
<td style="height:22px; width:133px">
<p>Gel electrophoresis</p>
</td>
<td style="height:22px; width:211px">
<p>Permits the quantitative measurement of double strand breaks, and can be combined with immunoblotting to detect DNA-associated proteins (Lobrich, Rydberg et al. 1995; Kawashima, Yamaguchi et al. 2017). Considered less sensitive than comet assay, but detected damage from 0.25 Gy ionizing radiation (Gradzka and Iwanenko 2005). Requires destruction of the cell.</p>
</td>
</tr>
<tr>
<td style="height:22px; width:127px">
<p><strong>Double strand breaks</strong></p>
</td>
<td style="height:22px; width:167px">
<p>Labeled probes including phosphorylated H2AX, 53BP1, Ku70, ATM (Lorat, Brunner et al. 2015)</p>
</td>
<td style="height:22px; width:133px">
<p>Microscopy</p>
</td>
<td style="height:22px; width:211px">
<p>Fluorescent probes can label individual double breaks in cells allowing for quantification, with immunogold labeling resolving breaks in clusters (Lorat, Timm et al. 2016; Nikitaki, Nikolov et al. 2016). Sensitive: detects damage from 0.001 Gy ionizing radiation (Rothkamm and Lobrich 2003; Ojima, Ban et al. 2008).</p>
</td>
</tr>
<tr>
<td style="height:22px; width:127px">
<p><strong>Chromosomal damage</strong></p>
</td>
<td style="height:22px; width:167px">
<p>Chromosomal aberrations and micronuclei</p>
<p>OECD Test Guidelines 473, 475, 483, and 487 (OECD 2016; OECD 2016; OECD 2016; OECD 2016)</p>
</td>
<td style="height:22px; width:133px">
<p>Microscopy</p>
</td>
<td style="height:22px; width:211px">
<p>Detects major DNA damage resulting from large breaks and rearrangements, or mitotic failures. Damage does not appear until DNA undergoes mitosis, so slower and limited to damage in replicating cells. Insensitive tosmall deletions and substitutions.</p>
</td>
</tr>
</tbody>
</table>
<h4>Regulatory Significance of the AO</h4>
<p>DNA damage increases the susceptibility to and probability of subsequent mutations, described in the key event ‘Increase in Mutation’. Mutations can impair the functional capacity of the cell and are an endpoint of regulator significance in their own right.</p>
<p>Multiple guideline toxicity tests exist for DNA damage. The OECD test guideline for DNA synthesis Test No. 486 (OECD 1997) detects nucleotide excision repair, so it will reflect the formation of bulky DNA adducts but not the majority of oxidative damage to nucleotides, which is typically repaired via the Base Excision Repair pathway. The OECD test guideline alkaline comet assay Test No. 489 (OECD 2016) detects single and double strand breaks, including those arising from repair as well as some (alkali sensitive) nucleotide lesions including some lesions from oxidative damage. OECD tests for chromosomal damage and micronuclei Test No. 473, 475, 483, and 487 measure longer term effects of DNA damage but these tests require the damaged cell to subsequently undergo replication (OECD 2016; OECD 2016; OECD 2016; OECD 2016). They can therefore reflect a wider range of sources of DNA damage including changes in mitosis.</p>
<h4>References</h4>
<p><a name="_ENREF_1">Barnard, S., S. Bouffler, et al. (2013). "The shape of the radiation dose response for DNA double-strand break induction and repair." Genome integrity 4(1): 1.</a></p>
<p><a name="_ENREF_2">Behjati, S., G. Gundem, et al. (2016). "Mutational signatures of ionizing radiation in second malignancies." Nat Commun 7: 12605.</a></p>
<p><a name="_ENREF_3">Chaudhry, M. A. and M. Weinfeld (1997). "Reactivity of human apurinic/apyrimidinic endonuclease and Escherichia coli exonuclease III with bistranded abasic sites in DNA." The Journal of biological chemistry 272(25): 15650-15655.</a></p>
<p><a name="_ENREF_4">Collins, A. R. (2004). "The comet assay for DNA damage and repair: principles, applications, and limitations." Molecular biotechnology 26(3): 249-261.</a></p>
<p><a name="_ENREF_5">David, S. S., V. L. O'Shea, et al. (2007). "Base-excision repair of oxidative DNA damage." Nature 447(7147): 941-950.</a></p>
<p><a name="_ENREF_6">Dayal, D., S. M. Martin, et al. (2008). "Hydrogen peroxide mediates the radiation-induced mutator phenotype in mammalian cells." Biochem J 413(1): 185-191.</a></p>
<p><a name="_ENREF_7">Ge, J., S. Prasongtanakij, et al. (2014). "CometChip: a high-throughput 96-well platform for measuring DNA damage in microarrayed human cells." Journal of visualized experiments : JoVE(92): e50607.</a></p>
<p><a name="_ENREF_8">Gradzka, I. and T. Iwanenko (2005). "A non-radioactive, PFGE-based assay for low levels of DNA double-strand breaks in mammalian cells." DNA repair 4(10): 1129-1139.</a></p>
<p><a name="_ENREF_9">Haag, J. D., L. C. Hsu, et al. (1996). "Allelic imbalance in mammary carcinomas induced by either 7,12-dimethylbenz[a]anthracene or ionizing radiation in rats carrying genes conferring differential susceptibilities to mammary carcinogenesis." Mol Carcinog 17(3): 134-143.</a></p>
<p><a name="_ENREF_10">Kawashima, Y., N. Yamaguchi, et al. (2017). "Detection of DNA double-strand breaks by pulsed-field gel electrophoresis." Genes to cells : devoted to molecular & cellular mechanisms 22(1): 84-93.</a></p>
<p><a name="_ENREF_11">Kuhne, M., K. Rothkamm, et al. (2000). "No dose-dependence of DNA double-strand break misrejoining following alpha-particle irradiation." International journal of radiation biology 76(7): 891-900.</a></p>
<p><a name="_ENREF_12">Kutanzi, K. and O. Kovalchuk (2013). "Exposure to estrogen and ionizing radiation causes epigenetic dysregulation, activation of mitogen-activated protein kinase pathways, and genome instability in the mammary gland of ACI rats." Cancer Biol Ther 14(7): 564-573.</a></p>
<p><a name="_ENREF_13">Kuzminov, A. (2001). "Single-strand interruptions in replicating chromosomes cause double-strand breaks." Proceedings of the National Academy of Sciences of the United States of America 98(15): 8241-8246.</a></p>
<p><a name="_ENREF_14">Liu, X., Y. He, et al. (2015). "Caspase-3 promotes genetic instability and carcinogenesis." Mol Cell 58(2): 284-296.</a></p>
<p><a name="_ENREF_15">Lobrich, M., B. Rydberg, et al. (1995). "Repair of x-ray-induced DNA double-strand breaks in specific Not I restriction fragments in human fibroblasts: joining of correct and incorrect ends." Proceedings of the National Academy of Sciences of the United States of America 92(26): 12050-12054.</a></p>
<p><a name="_ENREF_16">Lorat, Y., C. U. Brunner, et al. (2015). "Nanoscale analysis of clustered DNA damage after high-LET irradiation by quantitative electron microscopy--the heavy burden to repair." DNA repair 28: 93-106.</a></p>
<p><a name="_ENREF_17">Lorat, Y., S. Timm, et al. (2016). "Clustered double-strand breaks in heterochromatin perturb DNA repair after high linear energy transfer irradiation." Radiotherapy and oncology : journal of the European Society for Therapeutic Radiology and Oncology 121(1): 154-161.</a></p>
<p><a name="_ENREF_18">Madugundu, G. S., J. Cadet, et al. (2014). "Hydroxyl-radical-induced oxidation of 5-methylcytosine in isolated and cellular DNA." Nucleic acids research 42(11): 7450-7460.</a></p>
<p><a name="_ENREF_19">Morishita, M., T. Muramatsu, et al. (2016). "Chromothripsis-like chromosomal rearrangements induced by ionizing radiation using proton microbeam irradiation system." Oncotarget 7(9): 10182-10192.</a></p>
<p><a name="_ENREF_20">Murnane, J. P. (2012). "Telomere dysfunction and chromosome instability." Mutation research 730(1-2): 28-36.</a></p>
<p><a name="_ENREF_21">Nikitaki, Z., V. Nikolov, et al. (2016). "Measurement of complex DNA damage induction and repair in human cellular systems after exposure to ionizing radiations of varying linear energy transfer (LET)." Free radical research 50(sup1): S64-S78.</a></p>
<p><a name="_ENREF_22">OECD (1997). Test No. 486: Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo.</a></p>
<p><a name="_ENREF_23">OECD (2016). Test No. 473: In Vitro Mammalian Chromosomal Aberration Test.</a></p>
<p><a name="_ENREF_24">OECD (2016). Test No. 475: Mammalian Bone Marrow Chromosomal Aberration Test.</a></p>
<p><a name="_ENREF_26">OECD (2016). Test No. 487: In Vitro Mammalian Cell Micronucleus Test.</a></p>
<p><a name="_ENREF_27">OECD (2016). Test No. 489: In Vivo Mammalian Alkaline Comet Assay.</a></p>
<p><a name="_ENREF_28">OECD (Organisation for Economic Co-operation and Development) (2016). Overview of the set of OECD Genetic Toxicology Test Guidelines and updates performed in 2014–2015. No. 238.</a></p>
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<p><a name="_ENREF_49">Yu, Y., R. Okayasu, et al. (2001). "Elevated breast cancer risk in irradiated BALB/c mice associates with unique functional polymorphism of the Prkdc (DNA-dependent protein kinase catalytic subunit) gene." Cancer Res 61(5): 1820-1824.</a></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">Cell invasion refers to the active movement of cells into and through tissues, barriers, or extracellular matrices (ECM)</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529"> (Friedl)</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">. It involves a series of coordinated processes by which cells penetrate physical barriers, navigate through the extracellular environment, and potentially reach distant locations</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529"> (Hynes)</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">.</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529"> It is regulated by growth factors (VEGF), signaling pathways and cell-cell interactions.</span></span></span></span></span></p>
<p style="text-align:justify"> </p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">Key Steps in Cell Invasion:</span></span></span></span></span></p>
<ul>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">Detachment</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">: </span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">Detachment</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529"> of cells to the extracellular matrix (ECM) or neighboring cells through interactions with adhesion molecules, including integrins and cadherins.</span></span></span></span></span></li>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">Proteolysis: Degradation of ECM components by proteolytic enzymes, such as matrix metalloproteinases (MMPs), secreted by invasive cells. This process creates pathways for cell movement.</span></span></span></span></span></li>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">Motility:</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529"> Dynamic changes in the cell's cytoskeleton, involving the formation of actin-rich structures like lamellipodia and filopodia, which facilitate cell movement.</span></span></span></span></span></li>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">Intravasation: Invasion of cells into blood vessels or lymphatic vessels, allowing them to enter the circulatory system and potentially spread to distant sites.</span></span></span></span></span></li>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">Extravasation: Exit of invasive cells from the bloodstream or lymphatic vessels at a secondary site, facilitating colonization and the formation of secondary tumors.</span></span></span></span></span></li>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">Adhesion: Cells form new attachments to the ECM at the leading edge, allowing for continued movement.</span></span></span></span></span></li>
</ul>
<p style="text-align:justify"> </p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">There are many roles for cell invasion: </span></span></span></span></span></p>
<ul>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">Development and Tissue Repair: Cell invasion is crucial during embryonic development for processes such as tissue patterning and organ formation. In adults, invasion is essential for tissue repair and regeneration.</span></span></span></span></span></li>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">Embryonic development: </span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#1f1f1f">During development, cells migrate to form different organs and tissues, shaping the intricate structure of the organism</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#1f1f1f"> (</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">Heisenberg)</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#1f1f1f">.</span></span></span></span></span></li>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">Immune Response: Immune cells use invasion to migrate to sites of infection or injury, where they participate in immune responses.</span></span></span></span></span></li>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">Angiogenesis: Endothelial cells migrate to form new blood vessels, delivering oxygen and nutrients to growing tissues or healing wounds (Carmeliet, Lamalice).</span></span></span></span></span></li>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">Wound Healing: Invasive migration of cells is essential for wound healing, allowing cells to move into the wounded area and contribute to tissue repair</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529"> (Grinnell)</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">.</span></span></span></span></span></li>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">Cancer Metastasis: In cancer, invasion is a hallmark of malignancy and a critical step in metastasis. Cancer cells acquire the ability to invade surrounding tissues, enter blood or lymphatic vessels, and establish secondary tumors at distant sites</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529"> (Krakhmal)</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">.</span></span></span></span></span></li>
</ul>
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</div>
<h4>How it is Measured or Detected</h4>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">Several assays can be used to study cell invasion (Justus): </span></span></span></span></span></p>
<ul>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">Transwell Invasion Assay:</span></span></span> <span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">Cells migrate through a porous membrane coated with ECM proteins toward a chemoattractant</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529"> (Hulkower)</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">.</span></span></span></span></span></li>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">Boyden Chamber Assay:</span></span></span> <span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">cell migration and invasion through a porous membrane in response to a gradient of chemoattractants.</span></span></span></span></span></li>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">3D Spheroid Invasion Assay:</span></span></span> <span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">spheroids embedded in a 3D matrix, and invasion is assessed as cells migrate out from the spheroid into the surrounding matrix</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529"> (Pijuan)</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">.</span></span></span></span></span></li>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">Collagen Invasion Assay:</span></span></span> <span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">Cells invade through a collagen matrix, simulating the extracellular environment.</span></span></span></span></span></li>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">Matrigel Invasion Assay:</span></span></span> <span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">Cells invade through Matrigel, a basement membrane matrix rich in ECM proteins.</span></span></span></span></span></li>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">Zymography:</span></span></span> <span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">Assess the activity of matrix metalloproteinases (MMPs), enzymes involved in ECM degradation and cell invasion.</span></span></span></span></span></li>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">Electric Cell-Substrate Impedance Sensing (ECIS):</span></span></span> <span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">Measure changes in electrical impedance as cells invade and interact with a substrate.</span></span></span></span></span></li>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">Microfluidic Invasion Assays:</span></span></span> <span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">Use microfluidic devices to create controlled environments for studying cell invasion</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529"> (Fonseca)</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">.</span></span></span></span></span></li>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">In Vivo Invasion Assays:</span></span></span> <span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">Intravital imaging or xenograft models to study cell invasion in vivo.</span></span></span></span></span></li>
</ul>
<div>
<div>
<h4><a href="/events/1194">Event: 1194: Increase, DNA damage</a><br></h4>
<h5>Short Name: Increase, DNA Damage</h5>
</div>
<h4>References</h4>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212121">Justus CR, Leffler N, Ruiz-Echevarria M, Yang LV. In vitro cell migration and invasion assays. J Vis Exp. 2014 Jun 1;(88):51046. doi: 10.3791/51046. PMID: 24962652; PMCID: PMC4186330.</span></span></span></span></span></p>
<p style="text-align:justify"> </p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212121">Fonseca CG, Barbacena P, Franco CA. Endothelial cells on the move: dynamics in vascular morphogenesis and disease. Vasc Biol. 2020 Jul 2;2(1):H29-H43. doi: 10.1530/VB-20-0007. PMID: 32935077; PMCID: PMC7487603.</span></span></span></span></span></p>
<p style="text-align:justify"> </p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212121">Pijuan J, Barceló C, Moreno DF, Maiques O, Sisó P, Marti RM, Macià A, Panosa A. </span></span></span><em>In vitro</em> Cell Migration, Invasion, and Adhesion Assays: From Cell Imaging to Data Analysis. Front Cell Dev Biol. 2019 Jun 14;7:107. doi: 10.3389/fcell.2019.00107. PMID: 31259172; PMCID: PMC6587234.</span></span></p>
<p style="text-align:justify"> </p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212121">Hulkower KI, Herber RL. Cell migration and invasion assays as tools for drug discovery. Pharmaceutics. 2011 Mar 11;3(1):107-24. doi: 10.3390/pharmaceutics3010107. PMID: 24310428; PMCID: PMC3857040.</span></span></span></span></span></p>
<p style="text-align:justify"> </p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">Friedl, P., & Weigelin, B. (2008). Interstitial cell migration and invasion in tumorous environments: Past, present and future. Cell adhesion & migration, 2(1), 115-125. <a href="https://pathsocjournals.onlinelibrary.wiley.com/doi/full/10.1002/path.3031" style="color:#467886; text-decoration:underline">https://pathsocjournals.onlinelibrary.wiley.com/doi/full/10.1002/path.3031</a></span></span></span></span></span></p>
<p style="text-align:justify"> </p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">Hynes, R. O. (2009). The extracellular matrix in action. Cell, 137(5), 910-921. <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4185430/" style="color:#467886; text-decoration:underline">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4185430/</a></span></span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212121">Lamalice L, Le Boeuf F, Huot J. Endothelial cell migration during angiogenesis. Circ Res. 2007 Mar 30;100(6):782-94. doi: 10.1161/01.RES.0000259593.07661.1e. PMID: 17395884.</span></span></span></span></span></p>
<p style="text-align:justify"> </p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">Heisenberg, C. P., & Bellairs, R. (2013). </span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">Cell migration in development and disease. Nature reviews. Molecular cell biology, 14(7), 481-494. <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4457291/" style="color:#467886; text-decoration:underline">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4457291/</a></span></span></span></span></span></p>
<p style="text-align:justify"> </p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">Grinnell, F. (2003). Fibroblast biology: From contraction to proliferation. Journal of cell physiology, 197(1), 301-303. <a href="https://pubmed.ncbi.nlm.nih.gov/8106541/" style="color:#467886; text-decoration:underline">https://pubmed.ncbi.nlm.nih.gov/8106541/</a></span></span></span></span></span></p>
<p style="text-align:justify"> </p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">Carmeliet, P., & Jain, R. K. (2011). Angiogenesis in disease and the angiogenic switch. Nature medicine, 17(7), 755-763</span></span></span></span></span></p>
<p>It is well documented that alterations of histone acetylation have an impact on gene expression. Therefore if the acetylation status of the epigenetic set-up at the regulatory sequences of genes is altered, this leads to changes in gene expression.</p>
<!-- organ term -->
<div>
<h4>Organ term</h4>
<h4>How it is Measured or Detected</h4>
<p>Gene specific alterations in histone acetylation at gene regulatory seqences can be measured by chromatin immunoprecipitation (ChIPs) and gene expression analysis by RT-qPCR or whole transcriptomics (RNAseq, gene chips).</p>
<p><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-family:"Times New Roman",serif">Cell motility has been lar</span><span style="font-family:"Times New Roman",serif">gely described in h</span><span style="font-family:"Times New Roman",serif">uman breast cancer cell lines</span><span style="font-family:"Times New Roman",serif">, m</span><span style="font-family:"Times New Roman",serif">ice</span><span style="font-family:"Times New Roman",serif"> and f</span><span style="font-family:"Times New Roman",serif">ish</span><span style="font-family:"Times New Roman",serif"> (Stuelten)</span></span></span></p>
<h4>Key Event Description</h4>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">Cell motility is the capacity of cells to translocate onto a solid substratum. </span></span></span></span></span></p>
<p style="text-align:justify"> </p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">In order to move several actions such as : </span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#222222">cell–substrate adhesion, cell–cell adhesion, cell cortex rigidity (membrane and cytoskeleton), actin polymerization-mediated protrusion and actomyosin contractilit</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#222222">y (</span></span></span><span style="font-family:"Times New Roman",serif">Stuelten</span><span style="font-family:"Times New Roman",serif">, </span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212121">Lauffenburger</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212121">, </span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212121">Montell</span></span></span><span style="font-family:"Times New Roman",serif">).</span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">Several key factors contribute to cell motility in cancer</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529"> (</span></span></span><span style="font-family:"Times New Roman",serif">Friedl</span><span style="font-family:"Times New Roman",serif">, </span><span style="font-family:"Times New Roman",serif">Lamouille</span><span style="font-family:"Times New Roman",serif">, Sahail)</span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">:</span></span></span></span></span></p>
<ul>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">Actin Cytoskeleton Dynamics:</span></span></span> <span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">The actin cytoskeleton plays a crucial role in cell motility. Remodeling of the actin cytoskeleton is essential for cell shape changes, protrusion formation, and cell migration. This process is tightly regulated by proteins such as actin polymerization factors, focal adhesion proteins, and myosin motors.</span></span></span></span></span></li>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">Cell Adhesion and Extracellular Matrix (ECM) Interactions:</span></span></span> <span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">Integrins and other cell adhesion molecules mediate the interaction between cancer cells and the ECM. These interactions activate signaling pathways that influence cell motility. Changes in adhesion molecules can enhance or inhibit the migratory potential of breast cancer cells.</span></span></span></span></span></li>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">Epithelial-Mesenchymal Transition (EMT):</span></span></span> <span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">EMT is a biological process in which epithelial cells acquire mesenchymal characteristics, including increased motility. EMT is associated with the invasive behavior of cancer cells, allowing them to detach from the primary tumor and migrate to distant sites.</span></span></span></span></span></li>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">Chemotaxis and Gradients:</span></span></span> <span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">Cancer cells can respond to chemical gradients, a process known as chemotaxis. Growth factors and cytokines in the tumor microenvironment can attract or repel cancer cells, influencing their direction of movement.</span></span></span></span></span></li>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">Proteolytic Enzymes and Matrix Metalloproteinases (MMPs):</span></span></span> <span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">Proteolytic enzymes, especially MMPs, are involved in degrading the ECM, facilitating cancer cell invasion. The degradation of the surrounding matrix creates space for cell movement and allows cancer cells to penetrate adjacent tissues.</span></span></span></span></span></li>
</ul>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">In breast cancer, cell motility can favor metastasis through different steps:</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#0d0d0d"> loss of epithelial polarity, breakdown of tissue architecture, breach of the basement membrane, intravasation, extravasation, migration into new tissues, and expansion of metastatic colonies</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#0d0d0d"> (Stuelten)</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#0d0d0d">.</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#0d0d0d"> For instance, </span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">an increase in invasion of the </span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">surrounding tissues and blood vessels. Once cancer cells have invaded the local tissue, they may enter the bloodstream through a process called intravasation. Subsequently, they must migrate through the vasculature to reach distant organs, a process known as extravasation</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529"> (</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">Chambers</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">)</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">.</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529"> Once in the circulation, </span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">cells utilize chemotaxis, responding to chemokines and other signals in the microenvironment, to navigate through the bloodstream and reach specific distant organs. The ability of cancer cells to home in on specific organs depends on their motility and the interactions with the target tissue</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529"> (</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">Psaila</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">, Labelle). </span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">Once cancer cells reach a distant organ, they need to extravasate and establish micrometastases. Motility enables cancer cells to navigate through the tissue, invade the local environment, and form secondary tumor foci</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529"> (Nguyen)</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">.</span></span></span></span></span></p>
<h4>How it is Measured or Detected</h4>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-family:"Times New Roman",serif">Several assays can be used to measure cell motility, and the choice depends on the specific requirements and characteristics of the cells being studied. Here are some commonly used assays for measuring cell motility</span> <span style="font-family:"Times New Roman",serif">(Justus)</span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-family:"Times New Roman",serif">Principle: Create a controlled "wound" or scratch in a cell monolayer and monitor the closure of the gap over time.</span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-family:"Times New Roman",serif">Measurement: Quantify the rate of cell migration by measuring the reduction in the wound area.</span></span></span></p>
<ul>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-family:"Times New Roman",serif">Transwell Migration Assay:</span></span></span></li>
</ul>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-family:"Times New Roman",serif">Principle: Cells migrate through a porous membrane from one side to the other in response to a chemoattractant.</span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-family:"Times New Roman",serif">Measurement: Count the number of cells that have migrated through the membrane or quantify fluorescence if cells are labeled.</span></span></span></p>
<ul>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-family:"Times New Roman",serif">Boyden Chamber Assay:</span></span></span></li>
</ul>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-family:"Times New Roman",serif">Principle: Similar to the Transwell assay, cells migrate through a membrane towards a chemoattractant.</span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-family:"Times New Roman",serif">Measurement: Assess the migrated cells on the lower surface of the membrane.</span></span></span></p>
<ul>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-family:"Times New Roman",serif">Time-Lapse Microscopy:</span></span></span></li>
</ul>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-family:"Times New Roman",serif">Principle: Track the movement of individual cells over time using live-cell imaging.</span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-family:"Times New Roman",serif">Measurement: Analyze cell trajectories, speed, and directionality.</span></span></span></p>
<ul>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-family:"Times New Roman",serif">Collagen Invasion Assay:</span></span></span></li>
</ul>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-family:"Times New Roman",serif">Principle: Assess cell invasion through a three-dimensional collagen matrix.</span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-family:"Times New Roman",serif">Measurement: Quantify the extent of cell invasion into the matrix</span></span></span></p>
<ul>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-family:"Times New Roman",serif">Fluorescence Recovery After Photobleaching (FRAP):</span></span></span></li>
</ul>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-family:"Times New Roman",serif">Principle: Measure the mobility of fluorescently labeled molecules or proteins within cells.</span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-family:"Times New Roman",serif">Measurement: Assess the recovery of fluorescence in a photobleached region over time.</span></span></span></p>
<ul>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-family:"Times New Roman",serif">Single-Cell Tracking:</span></span></span></li>
</ul>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-family:"Times New Roman",serif">Principle: Monitor individual cell movements using time-lapse microscopy.</span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-family:"Times New Roman",serif">Measurement: Analyze parameters such as speed, persistence, and directionality for each tracked cell.</span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-family:"Times New Roman",serif">Principle: Measure changes in electrical impedance as cells migrate and interact with a substrate.</span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-family:"Times New Roman",serif">Measurement: Quantify impedance-based parameters to assess cell motility.</span></span></span></p>
<ul>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-family:"Times New Roman",serif">Bead-Based Motility Assay:</span></span></span></li>
</ul>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-family:"Times New Roman",serif">Principle: Attach beads to cells and track their movement using microscopy.</span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-family:"Times New Roman",serif">Measurement: Analyze the displacement of beads to determine cell motility.</span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-family:"Times New Roman",serif">Selecting the most appropriate assay depends on factors such as the nature of the cells, the desired readout, and the specific aspects of cell motility being investigated. Researchers often use a combination of these assays to gain a comprehensive understanding of cell motility in different contexts</span></span></span></p>
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<h4>References</h4>
<p>Stuelten, C., Parent, C. & Montell, D. Cell motility in cancer invasion and metastasis: insights from simple model organisms. <em>Nat Rev Cancer</em> <strong>18</strong>, 296–312 (2018). https://doi.org/10.1038/nrc.2018.15</p>
<p>Justus CR, Leffler N, Ruiz-Echevarria M, Yang LV. In vitro cell migration and invasion assays. J Vis Exp. 2014 Jun 1;(88):51046. doi: 10.3791/51046. PMID: 24962652; PMCID: PMC4186330.</p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212121">Lauffenburger DA, Horwitz AF. Cell migration: a physically integrated molecular process. Cell. 1996 Feb 9;84(3):359-69. doi: 10.1016/s0092-8674(00)81280-5. PMID: 8608589.</span></span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">Chambers, A. F., Groom, A. C., & MacDonald, I. C. (2002). Dissemination and growth of cancer cells in metastatic sites. Nature Reviews Cancer, 2(8), 563–572. doi:10.1038/nrc865</span></span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212121">Montell DJ. Morphogenetic cell movements: diversity from modular mechanical properties. Science. 2008 Dec 5;322(5907):1502-5. doi: 10.1126/science.1164073. PMID: 19056976.</span></span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-family:"Times New Roman",serif">Friedl, P., & Wolf, K. (2003). Tumour-cell invasion and migration: diversity and escape mechanisms. Nature Reviews Cancer, 3(5), 362–374. doi:10.1038/nrc1075</span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-family:"Times New Roman",serif">Lamouille, S., Xu, J., & Derynck, R. (2014). Molecular mechanisms of epithelial-mesenchymal transition. Nature Reviews Molecular Cell Biology, 15(3), 178–196. doi:10.1038/nrm3758</span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-family:"Times New Roman",serif">Sahai, E. (2005). Mechanisms of cancer cell invasion. Current Opinion in Genetics & Development, 15(1), 87–96. doi:10.1016/j.gde.2004.12.002</span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">Psaila, B., & Lyden, D. (2009). The metastatic niche: adapting the foreign soil. Nature Reviews Cancer, 9(4), 285–293. doi:10.1038/nrc2621</span></span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">Labelle, M., & Hynes, R. O. (2012). The initial hours of metastasis: the importance of cooperative host-tumor cell interactions during hematogenous dissemination. Cancer Discovery, 2(12), 1091–1099. doi:10.1158/2159-8290.CD-12-0329</span></span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">Nguyen, D. X., & Bos, P. D. (2009). Massagué, J. (2009). Metastasis: from dissemination to organ-specific colonization. Nature Reviews Cancer, 9(4), 274–284. doi:10.1038/nrc2622</span></span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">Quail, D. F., & Joyce, J. A. (2013). Microenvironmental regulation of tumor progression and metastasis. Nature Medicine, 19(11), 1423–1437. doi:10.1038/nm.3394</span></span></span></span></span></p>
<h4><a href="/events/1242">Event: 1242: Increased, Second Messenger Production</a></h4>
<h5>Short Name: Increased, Second Messenger Production</h5>
<p>This can be applied to adult women and men and mice.</p>
<h4>Key Event Description</h4>
<p>Cancers are thought to arise from a collection of permissive factors which interact within and between different cells of a tissue or tumor to promote tumor growth and invasive characteristics (Sonnenschein and Soto 1999; Hanahan and Weinberg 2011; Floor, Dumont et al. 2012; Goodson, Lowe et al. 2015; Schwarzman, Ackerman et al. 2015; Smith, Guyton et al. 2016; Grashow, De La Rosa et al. 2018). Permissive factors or hallmarks include changes to the cell’s dependence on growth signals, proliferation, metabolism, apoptosis, senescence, angiogenesis, and invasion and metastasis. These hallmarks are modified by other factors including growth factors, inflammation, oxidative stress, changes to the microenvironment, DNA damage, and changes in gene expression.</p>
<p>The mammary gland is a hormone responsive organ with multiple phases of development from embryogenesis into adulthood. Consequently, certain hallmarks and contributing factors including proliferative response to growth signals, growth factors, changes to the microenvironment, and changes in gene expression play a larger role in this organ, and the importance of various factors shifts depending on developmental stage (Rudel, Fenton et al. 2011). Established risk factors of breast cancer extend beyond genetic contributors (principally alterations in DNA damage response genes) and DNA damaging environmental agents to include exposure to pharmaceutical hormones, timing of puberty and first birth, and lifetime exposure to estrogen and progesterone ((IOM) Institute of Medicine 2012). </p>
<p>Hormonal and other environmental influences during proliferation and differentiation alter the pace and structure of cellular or mammary gland development to leave tissue in the adult gland more susceptible to cancer. In addition, the elevated hormone concentrations associated with the menstrual cycle and pregnancy provide a regular proliferative stimulus to any pre-cancerous cells present in the breast (Rudel, Fenton et al. 2011). A substantial majority of breast cancers express hormone receptors, and these cancers are particularly responsive to hormones (Badowska-Kozakiewicz, Patera et al. 2015).</p>
<p>Consistent with the importance of growth factors and DNA damage in the development of cancer, driver mutations (mutations that favor the success of the nascent cancer cells and are therefore selected) commonly appear in the growth factor related signaling pathways (BRAF, EGRF, RAS, PI3K, STK11) and in DNA damage response and cell cycle checkpoint signal pathways (ATM, TP53, CHEK2, CDKN2B (P15), CDK4) (Greenman, Stephens et al. 2007; Croce 2008; Kaufmann, Nevis et al. 2008; Stratton, Campbell et al. 2009; Vandin, Upfal et al. 2012). These and other mutations are acquired over the development of a cancer and contribute to the evolution of the cancer (Wang, Waters et al. 2014; Yates, Gerstung et al. 2015; Begg, Ostrovnaya et al. 2016).</p>
<p>In breast cancer, TP53, PI3K and GATA3 are each mutated in more than 10% of cancers, amplification or mutation of the RB1 pathway are common, and HER2 (an EGFR receptor) is amplified in HER2 type cancers (CGAN 2012). EGFR, HER2, BRAF, RAS, and PI3K participate in the EGFR (growth factor) signaling pathway. Activating mutations in PI3K generate growth factor independent proliferation of mammary epithelial cells, possibly via the RB1 pathway (Gustin, Karakas et al. 2009). GATA is a transcription factor that maintains luminal epithelial cell differentiation and suppresses proliferation, and mutation results in the proliferation of undifferentiated cells (Kouros-Mehr, Slorach et al. 2006; Shahi, Wang et al. 2017).</p>
<p>Environmental factors contribute significantly to the total number of breast cancers. Women exposed to the synthetic hormone DES or the pesticide DDT in utero are up to two to four times more likely to be diagnosed with breast cancer in their fifties (Palmer, Wise et al. 2006; Cohn, La Merrill et al. 2015). A study in 2002 found that recipients of hormone replacement therapy (HRT) around menopause are 26% more likely to be diagnosed with breast cancer (Narod 2011). When prescriptions of HRT began to fall in response to the study, so did cancer diagnoses. Over the next few years, approximately 5% fewer cancers were diagnosed in women over 45 (Glass, Lacey et al. 2007) with an estimated 126,000 fewer cases of breast cancer over the next ten years (Roth, Etzioni et al. 2014).</p>
<h4>How it is Measured or Detected</h4>
<p>In rodent bioassays, tumors can be detected via visual observation or palpation of live animals, necropsy of dead animals, and via microscopic examination of tissue. Malignant tumors including carcinomas in situ are distinguishable from benign tumors on the basis of the thickness or shape of the epithelial cell layer, regularity of the lumen or the presence of cribiform luminae, inflammation or desmoplastic reaction of the stroma, dominance of a less differentiated cell type, and larger nuclei, while diagnosis of invasiveness depends on the identification of metastases or invasion of neoplastic cells into surrounding tissue (Russo and Russo 2000).</p>
<p>In humans, lumps are commonly detected by palpation or mammogram. Further imaging, biopsy, and/or surgical excision of the affected tissue are used to differentiate benign, cancerous, and invasive tumors (McDonald, Clark et al. 2016).</p>
<p> </p>
<p style="text-align:justify"> </p>
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<h4>Regulatory Significance of the AO</h4>
<p>Because of the long latency of mammary tumors, the two-year rodent carcinogenicity bioassay is the primary assay for this adverse outcome. The assay is included in the OECD Test No. 451 and 453 for carcinogenicity and combined toxicity and carcinogenicity (OECD 2009; OECD 2009), and is also used by the US National Toxicology program (Chhabra, Huff et al. 1990), and the FDA (FDA (Food and Drug Administration) 2007), and referenced by the EPA (EPA (Environmental Protection Agency) 2005) in guidelines for risk assessments. Other assays from short term (2-4 weeks) and subchronic (90 day) to chronic (1 year) toxicity also call for the documentation of mammary tumors (FDA (Food and Drug Administration) 2007; OECD (Organisation for Economic Cooperation and Development) 2018), so these assays could capture the early onset of tumors, and could be modified to report earlier key events like proliferation and inflammation.</p>
<p>Several characteristics of classic cancer bioassays limit the sensitivity of these assays to mammary gland carcinogens. First, no assays require prenatal or early post-natal exposures for carcinogenicity testing. The US NIH’s National Toxicology Program assays start exposures at five to six weeks of age and OECD regulatory assay exposures suggest (but do not require) exposures beginning after weaning and before eight weeks of age. Assays initiating exposures at later ages have diminished sensitivity to agents that affect breast development and increase future susceptibility to cancer, such as estrogenic hormones, DDT and dioxins (EPA (Environmental Protection Agency) 2005; Rudel, Fenton et al. 2011). Agents with similar activity to ionizing radiation and DNA damaging chemicals may not be fully captured in some of these assays, since sensitivity appears to peak around or before week seven for these agents (around puberty) (Imaoka, Nishimura et al. 2013). Second, carcinogenicity assay guidelines do not require the best methods for detecting tumors in mammary gland: whole mount preparations of mammary gland coupled with longitudinal sections (dorsoventral sections parallel to the body) of mammary gland for histology (Tucker, Foley et al. 2017). Palpation and transverse sections of mammary gland can easily miss tumors or lesions of interest. Interestingly the NTP reproductive toxicity guidelines do specify these preferable methods for mammary gland analysis.</p>
<p>Two additional factors affect the sensitivity of standard carcinogenicity assays. First, benign tumors are not always considered to be an indicator of carcinogenicity, leading to a possible underestimation of risk. NTP and EPA guidance suggest that benign tumors provide additional weight of evidence if malignant tumors are also present or if studies suggest benign tumors can progress to carcinogenicity. In a short-term study, benign tumors may indicate a need for a longer-term study. However, benign mammary tumors (fibroadenomas) almost always coincide with carcinogenic tumors in mammary gland or other organs, and carcinomas sometimes grow from fibroadnomas (Rudel, Attfield et al. 2007; Russo 2015) suggesting that benign tumors may be an underutilized indicator of carcinogenicity.</p>
<p>Finally, the dose selection guidance in carcinogenicity testing typically calls for a high dose that is sufficiently toxic to suppress body weight (OECD 2009). However, body weight interacts with risk of breast cancer (Haseman, Young et al. 1997; Rudel, Attfield et al. 2007), reducing the sensitivity of the upper end of the dose range and the likelihood of a positive dose-response.</p>
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</div>
<h2>Appendix 2</h2>
<h2>List of Key Event Relationships in the AOP</h2>
<h4><a href="/relationships/3529">Relationship: 3529: Agonism, Estrogen receptor leads to Increased, ER binding to T.F. to DNA (non-classical pathway)</a></h4>
<h4><a href="/relationships/1250">Relationship: 1250: Increased, ER binding to DNA (classical pathway) leads to Increase, Cell Proliferation (Epithelial Cells)</a></h4>
<h4><a href="/relationships/1251">Relationship: 1251: Increased, ER binding to T.F. to DNA (non-classical pathway) leads to Increase, Cell Proliferation (Epithelial Cells)</a></h4>
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</div>
<h2>Appendix 2</h2>
<h2>List of Key Event Relationships in the AOP</h2>
<!-- Evidence for relationship links section, this lists the relationships and then supports them -->
<div id="evidence_supporting_links">
<hr>
<h3>List of Adjacent Key Event Relationships</h3>
<div>
<h4><a href="/relationships/1246">Relationship: 1246: Activation, Estrogen receptor leads to Increased, ER binding to DNA (classical pathway) </a></h4>
<h4><a href="/relationships/1249">Relationship: 1249: Activation, Estrogen receptor leads to Increased, ER binding to T.F. to DNA (non-classical pathway)</a></h4>
<td><a href="/aops/294">Increased reactive oxygen and nitrogen species (RONS) leading to increased risk of breast cancer</a></td>
<td>adjacent</td>
<td>High</td>
<td>Not Specified</td>
</tr>
</thead>
<tbody>
<tr>
<th><a href="/aops/200">Estrogen receptor activation leading to breast cancer </a></th>
<th>adjacent</th>
<th>High </th>
<th>High</th>
</tr>
</tbody>
</table>
</div>
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</tbody>
</table>
</div>
<h4>Key Event Relationship Description</h4>
<p>Proliferative lesions are believed to evolve over time and with successive cell divisions to take on the hallmarks of carcinogenesis, either directly or via other cell types recruited to the site such as fibroblasts and macrophages.</p>
<h4>Evidence Supporting this KER</h4>
<p><strong><em> Biological Plausibility is High.</em></strong> <em>It is generally accepted that proliferation contributes to cancer. Proliferation increases mutations, which can further promote proliferation and/or changes to the local microenvironment.</em></p>
<p><strong><em>Empirical support is High.</em></strong><em> Carcinogenic agents increase proliferation and hyperplasia as well as tumors. Proliferation and hyperplasia appears prior to or at the same time as tumors, grow into carcinomas, and are more effective at forming mammary tumors than non-proliferating tissue. Disruption of proliferation is associated with decreased tumor growth, and tumor resistant rats do not show proliferation. </em><em>However, the discrepancy between the non-linear proliferative and linear mammary tumor response to carcinogen dose coupled with evidence of independent occurrences of proliferation and tumorigenesis suggests that while proliferation and hyperplasia likely promote carcinogenesis, additional factors also contribute.</em></p>
<strong>Biological Plausibility</strong>
<div><strong><em> Biological Plausibility is High.</em></strong> <em>It is generally accepted that proliferation contributes to cancer. Proliferation increases mutations, which can further promote proliferation and/or changes to the local microenvironment.</em> For example, cells that become insensitive to certain TGF-β signaling pathways would be resistant to contact or TGF-β inhibition (Polyak, Kato et al. 1994) or apoptosis (Chapman, Lourenco et al. 1999), and cells that release or promote the stromal release of MMPs remodel the stroma and promote tumorigenesis and invasiveness (Sternlicht, Lochter et al. 1999; Ha, Moon et al. 2001).</div>
<strong>Empirical Evidence</strong>
<p><strong><em>High.</em></strong><em> Carcinogenic agents increase proliferation and hyperplasia as well as tumors. Proliferation and hyperplasia appears prior to or at the same time as tumors, grow into carcinomas, and are more effective at forming mammary tumors than non-proliferating tissue. Disruption of proliferation is associated with decreased tumor growth, and tumor resistant rats do not show proliferation. </em><em>However, the discrepancy between the non-linear proliferative and linear mammary tumor response to carcinogen dose coupled with evidence of independent occurrences of proliferation and tumorigenesis suggests that while proliferation and hyperplasia likely promote carcinogenesis, additional factors also contribute.</em></p>
<p>Factors that increase proliferation or hyperplasia also increase tumors. Proliferative epithelial cells, nodules and hyperplasia appear in mammary gland of rats and mice after exposure to chemical carcinogens (Beuving, Bern et al. 1967; Beuving, Faulkin et al. 1967; Russo, Saby et al. 1977; Purnell 1980) and ionizing radiation (Faulkin, Shellabarger et al. 1967; Ullrich and Preston 1991; Imaoka, Nishimura et al. 2006; Nguyen, Oketch-Rabah et al. 2011; Snijders, Marchetti et al. 2012; Suman, Johnson et al. 2012; Tang, Fernandez-Garcia et al. 2014). A subpopulation of post-senescent epithelial cells also proliferate following IR in vitro (Mukhopadhyay, Costes et al. 2010).</p>
<p>Proliferation and hyperplasia precede or are detected at the same time as tumors (Beuving, Bern et al. 1967; Beuving, Faulkin et al. 1967; Faulkin, Shellabarger et al. 1967; Haslam and Bern 1977; Russo, Saby et al. 1977; Purnell 1980; Imaoka, Nishimura et al. 2005; Imaoka, Nishimura et al. 2006) and form tumors more effectively than non-proliferating tissue (Deome, Faulkin et al. 1959; Beuving 1968; Rivera, Hill et al. 1981). Adenocarcinomas appear to form from terminal end bud hyperplasia in rats (Haslam and Bern 1977; Russo, Saby et al. 1977; Purnell 1980), similar to the origin of many breast cancers for humans and for some mice after IR (Medina and Thompson 2000).</p>
<p>Interrupting or preventing proliferation or hyperplasia reduces the incidence (or growth) of tumors. Disruption of proliferation or hyperplasia formation disrupts tumor growth (Luo, Fan et al. 2009; Connelly, Barham et al. 2011; Tang, Fernandez-Garcia et al. 2014). Similarly, ACI rats exhibit no proliferation or hyperplasia following IR and are resistant to tumors following IR (Shellabarger, Stone et al. 1976; Kutanzi, Koturbash et al. 2010).</p>
<strong>Uncertainties and Inconsistencies</strong>
<p>In the relatively small number of studies that examine the dose-dependence of proliferation and hyperplasia in models of carcinogenesis, proliferation does not appear to increase linearly with dose (Han, Chen et al. 2010; Mukhopadhyay, Costes et al. 2010; Nguyen, Oketch-Rabah et al. 2011; Tang, Fernandez-Garcia et al. 2014) while tumor formation and carcinogenesis does increase linearly with dose.</p>
<p>Some studies report carcinogenesis in the absence of hyperplasia (Middleton 1965; Sinha and Dao 1974) and others do not find increased tumorigenesis from transplanted hyperplasia (Haslam and Bern 1977; Sinha and Dao 1977). In Copenhagen rats resistant to tumors from MNU treatment, hyperplasia appear after MNU treatment but do not progress into carcinomas in situ, instead disappearing over time (Korkola and Archer 1999). Similarly, Fisher rats are less sensitive to tumor induction by DMBA, and hyperplasia from these rats do not go on to form tumors when transplanted (Beuving, Bern et al. 1967).</p>
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<p><a name="_ENREF_2">Beuving, L. J., H. A. Bern, et al. (1967). "Occurrence and Transplantation of Carcinogen-Induced Hyperplastic Nodules in Fischer Rats2." JNCI: Journal of the National Cancer Institute 39(3): 431-447.</a></p>
<p><a name="_ENREF_3">Beuving, L. J., J. L. J. Faulkin, et al. (1967). "Hyperplastic Lesions in the Mammary Glands of Sprague-Dawley Rats After 7,12-Dimethylbenz[a]anthracene Treatment2." JNCI: Journal of the National Cancer Institute 39(3): 423-429.</a></p>
<p><a name="_ENREF_4">Chapman, R. S., P. C. Lourenco, et al. (1999). "Suppression of epithelial apoptosis and delayed mammary gland involution in mice with a conditional knockout of Stat3." Genes & development 13(19): 2604-2616.</a></p>
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<p><a name="_ENREF_6">Deome, K. B., L. J. Faulkin, Jr., et al. (1959). "Development of mammary tumors from hyperplastic alveolar nodules transplanted into gland-free mammary fat pads of female C3H mice." Cancer Res 19(5): 515-520.</a></p>
<p><a name="_ENREF_7">Faulkin, J. L. J., C. J. Shellabarger, et al. (1967). "Hyperplastic Lesions of Sprague-Dawley Rat Mammary Glands After X Irradiation2." JNCI: Journal of the National Cancer Institute 39(3): 449-459.</a></p>
<p><a name="_ENREF_8">Ha, H. Y., H. B. Moon, et al. (2001). "Overexpression of membrane-type matrix metalloproteinase-1 gene induces mammary gland abnormalities and adenocarcinoma in transgenic mice." Cancer research 61(3): 984-990.</a></p>
<p><a name="_ENREF_9">Han, W., S. Chen, et al. (2010). "Nitric oxide mediated DNA double strand breaks induced in proliferating bystander cells after alpha-particle irradiation." Mutation research 684(1-2): 81-89.</a></p>
<p><a name="_ENREF_10">Haslam, S. Z. and H. A. Bern (1977). "Histopathogenesis of 7,12-diemthylbenz(a)anthracene-induced rat mammary tumors." Proceedings of the National Academy of Sciences of the United States of America 74(9): 4020-4024.</a></p>
<p><a name="_ENREF_11">Imaoka, T., M. Nishimura, et al. (2006). "Persistent cell proliferation of terminal end buds precedes radiation-induced rat mammary carcinogenesis." In Vivo 20(3): 353-358.</a></p>
<p><a name="_ENREF_12">Imaoka, T., M. Nishimura, et al. (2005). "Cooperative induction of rat mammary cancer by radiation and 1-methyl-1-nitrosourea via the oncogenic pathways involving c-Myc activation and H-ras mutation." Int J Cancer 115(2): 187-193.</a></p>
<p><a name="_ENREF_13">Korkola, J. E. and M. C. Archer (1999). "Resistance to mammary tumorigenesis in Copenhagen rats is associated with the loss of preneoplastic lesions." Carcinogenesis 20(2): 221-227.</a></p>
<p><a name="_ENREF_14">Kutanzi, K. R., I. Koturbash, et al. (2010). "Imbalance between apoptosis and cell proliferation during early stages of mammary gland carcinogenesis in ACI rats." Mutation research 694(1-2): 1-6.</a></p>
<p><a name="_ENREF_15">Luo, M., H. Fan, et al. (2009). "Mammary epithelial-specific ablation of the focal adhesion kinase suppresses mammary tumorigenesis by affecting mammary cancer stem/progenitor cells." Cancer research 69(2): 466-474.</a></p>
<p><a name="_ENREF_16">Medina, D. and H. J. Thompson (2000). A Comparison of the Salient Features of Mouse, Rat, and Human Mammary Tumorigenesis. Methods in Mammary Gland Biology and Breast Cancer Research. M. M. Ip and B. B. Asch. Boston, MA, Springer US: 31-36.</a></p>
<p><a name="_ENREF_17">Middleton, P. J. (1965). "The histogenesis of mammary tumours induced in the rat by chemical carcinogens." British journal of cancer 19(4): 830-839.</a></p>
<p><a name="_ENREF_18">Mukhopadhyay, R., S. V. Costes, et al. (2010). "Promotion of variant human mammary epithelial cell outgrowth by ionizing radiation: an agent-based model supported by in vitro studies." Breast cancer research : BCR 12(1): R11.</a></p>
<p><a name="_ENREF_19">Nguyen, D. H., H. A. Oketch-Rabah, et al. (2011). "Radiation acts on the microenvironment to affect breast carcinogenesis by distinct mechanisms that decrease cancer latency and affect tumor type." Cancer Cell 19(5): 640-651.</a></p>
<p><a name="_ENREF_20">Polyak, K., J. Y. Kato, et al. (1994). "p27Kip1, a cyclin-Cdk inhibitor, links transforming growth factor-beta and contact inhibition to cell cycle arrest." Genes & development 8(1): 9-22.</a></p>
<p><a name="_ENREF_21">Purnell, D. M. (1980). "The relationship of terminal duct hyperplasia to mammary carcinoma in 7,12-dimethylbenz(alpha)anthracene-treated LEW/Mai rats." The American journal of pathology 98(2): 311-324.</a></p>
<p><a name="_ENREF_22">Rivera, E. M., S. D. Hill, et al. (1981). "Organ culture passage enhances the oncogenicity of carcinogen-induced hyperplastic mammary nodules." In vitro 17(2): 159-166.</a></p>
<p><a name="_ENREF_23">Russo, J., J. Saby, et al. (1977). "Pathogenesis of Mammary Carcinomas Induced in Rats by 7, 12-Dimethylbenz[a]anthracene2." JNCI: Journal of the National Cancer Institute 59(2): 435-445.</a></p>
<p><a name="_ENREF_24">Shellabarger, C. J., J. P. Stone, et al. (1976). "Synergism between neutron radiation and diethylstilbestrol in the production of mammary adenocarcinomas in the rat." Cancer research 36(3): 1019-1022.</a></p>
<p><a name="_ENREF_25">Sinha, D. and T. L. Dao (1974). "A Direct Mechanism of Mammary Carcinogenesis Induced by 7,12-Dimethylbenz[a]anthracene2." JNCI: Journal of the National Cancer Institute 53(3): 841-846.</a></p>
<p><a name="_ENREF_26">Sinha, D. and T. L. Dao (1977). "Hyperplastic alveolar nodules of the rat mammary gland: tumor-producing capability in vivo and in vitro." Cancer letters 2(3): 153-160.</a></p>
<p><a name="_ENREF_27">Snijders, A. M., F. Marchetti, et al. (2012). "Genetic differences in transcript responses to low-dose ionizing radiation identify tissue functions associated with breast cancer susceptibility." PLoS One 7(10): e45394.</a></p>
<p><a name="_ENREF_28">Sternlicht, M. D., A. Lochter, et al. (1999). "The stromal proteinase MMP3/stromelysin-1 promotes mammary carcinogenesis." Cell 98(2): 137-146.</a></p>
<p><a name="_ENREF_29">Suman, S., M. D. Johnson, et al. (2012). "Exposure to ionizing radiation causes long-term increase in serum estradiol and activation of PI3K-Akt signaling pathway in mouse mammary gland." International journal of radiation oncology, biology, physics 84(2): 500-507.</a></p>
<p><a name="_ENREF_30">Tang, J., I. Fernandez-Garcia, et al. (2014). "Irradiation of juvenile, but not adult, mammary gland increases stem cell self-renewal and estrogen receptor negative tumors." Stem Cells 32(3): 649-661.</a></p>
<p><a name="_ENREF_31">Ullrich, R. L. and R. J. Preston (1991). "Radiation induced mammary cancer." Journal of radiation research 32 Suppl 2: 104-109.</a></p>
</div>
<br>
<div>
<h4><a href="/relationships/1250">Relationship: 1250: Increased, ER binding to DNA (classical pathway) leads to Increase, Cell Proliferation (Epithelial Cells)</a></h4>
<h4><a href="/relationships/1251">Relationship: 1251: Increased, ER binding to T.F. to DNA (non-classical pathway) leads to Increase, Cell Proliferation (Epithelial Cells)</a></h4>
<p>The activation of the AhR can lead to an increased endothelial cell migration. This was found when HMEC-1 or EA.hy926 cells were co-cultured with ER-positive MCF-7 cells and triple negative MDA-MB-231 cells (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0500" name="bb0500">Pontillo et al., 2015 Nov 19</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0705" name="bb0705">Zárate et al., 2020 Aug</a>). The assay mainly used was the Matrigel® / tube formation assay. Only one study found an increase in endothelial cell proliferation and not migration, therefore it was not kept as a KE (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0500" name="bb0500">Pontillo et al., 2015 Nov 19</a>). The main pathway explaining this relationship was again related to the activation of COX2 and subsequently to the increase in VEGF. The association between the activation of the AhR and endothelial cell migration was classified as “weak” since only 2 studies explored this feature, and both used hexachlorobenzene as a stressor. However, these works were robust with strong evidence, and both found a reversed association after AhR blockage. No contradicting results were found in the scientific literature.</p>
<p>As opposed to our work, another AOP displayed a link between AhR activation and angiogenesis (AOP 150) and found that activation of the receptor could decrease VEGF production with moderate evidence and quantitative understanding. It must be noted that these AOPs applied only to chicken, zebrafish, and certain rodents whereas our AOP concerns humans. As detailed further, the AhR presents a variability between species which must be considered.</p>
<p>Pontillo et al. treated mice with increasing doses of hexachlorobenzene and then calculated the vessel density in mammary fat pads (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0500" name="bb0500">Pontillo et al., 2015 Nov 19</a>). They found that mice treated with hexachlorobenzene had a higher vessel density with a dose–response concordance. Treatment by AhR antagonists completely reversed this association (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0500" name="bb0500">Pontillo et al., 2015 Nov 19</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0705" name="bb0705">Zárate et al., 2020 Aug</a>). The relationship between endothelial migration and angiogenesis was not detailed here since there is existing extensive knowledge (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0325" name="bb0325">Lamalice et al., 2007 Mar 30</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0440" name="bb0440">Norton and Popel, 2016 Nov 14</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0015" name="bb0015">Ausprunk and Folkman, 1977 Jul 1</a>). The KER 12 was considered as “strong”.</p>
<h4><a href="/relationships/1295">Relationship: 1295: Increased, Non-genomic signaling leads to Increased, ER binding to T.F. to DNA (non-classical pathway)</a></h4>
<h4><a href="/relationships/1297">Relationship: 1297: Increased, ER binding to T.F. to DNA (non-classical pathway) leads to Altered, Gene Expression</a></h4>
<td><a href="/aops/444">Ionizing radiation leads to reduced reproduction in Eisenia fetida via reduced spermatogenesis and cocoon hatchability</a></td>
<td>adjacent</td>
<td>Moderate</td>
<td>Not Specified</td>
</tr>
</tbody>
</table>
</div>
</div>
<br>
<div>
<div>
<h4><a href="/relationships/1301">Relationship: 1301: Increase, DNA Damage leads to Altered, Gene Expression</a></h4>
<p><span style="font-size:12.0pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#212529">Increased cell motility is a crucial factor contributing to increased invasion in various biological processes, including cancer metastasis. Cell motility refers to the ability of cells to move from one location to another, and when this ability is enhanced, it can facilitate the invasion of cells into surrounding tissues. </span></span></span></span></p>
<p>The relation between cell migration and organ invasion has already been shown (KER-1306, <a href="https://aopwiki.org/relationships/1306" rel="noreferrer noopener" target="_blank">https://aopwiki.org/relationships/1306</a>). Since the 2 are closely linked, most articles studied both cell migration (chemo-tactic) and the capacity to invade the extra-cellular matrix. Cell invasion is indeed defined as the capacity of a cell to migrate and degrade/invade the extracellular matrix. <em>In vitro</em>, this process was evaluated mostly using transwell chamber with Matrigel® and the presence of matrix metalloproteinases (MMP). This effect was found in ER-positive cells, triple negative cell lines and cells overexpressing the Her2.</p>
<h4>Evidence Supporting this KER</h4>
<p style="text-align:justify"> </p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="background-color:white"><span style="color:#212529">Increased cell motility is a crucial factor contributing to increased invasion in various biological processes, including cancer metastasis. Cell motility refers to the ability of cells to move from one location to another, and when this ability is enhanced, it can facilitate the invasion of cells into surrounding tissues. Here's how increased cell motility leads to increased invasion:</span></span></span></span></p>
<p style="text-align:justify"> </p>
<ul>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="background-color:white"><span style="color:#212529">Chemotaxis and Chemoattractants: Cells with increased motility are more responsive to chemoattractants (Signaling molecules released by tissues thatt attract and guide cells toward specific locations), allowing them to efficiently navigate through tissue barriers.</span></span></span></span></li>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="background-color:white"><span style="color:#212529">Enhanced Migration Through Extracellular Matrix (ECM): Increased cell motility is often associated with enhanced secretion of proteolytic enzymes, such as matrix metalloproteinases (MMPs), that degrade the ECM (Egeblad). Cells with higher motility can efficiently squeeze through ECM spaces created by their own proteolytic activity, facilitating invasion into surrounding tissues.</span></span></span></span></li>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="background-color:white"><span style="color:#212529">Formation of Cellular Protrusions: Highly motile cells often form dynamic structures such as lamellipodia (sheet-like protrusions) and filopodia (finger-like protrusions). These structures increase the surface area of contact between the cell and the surrounding environment, promoting effective movement through tissues.</span></span></span></span></li>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="background-color:white"><span style="color:#212529">Adhesion to Extracellular Matrix: Motile cells form focal adhesions, dynamic connections between the cell and ECM components. Increased motility enhances the ability of cells to dynamically form and disassemble these adhesions, promoting efficient movement through the ECM (Friedl). Increased motility allows cancer cells to detach from neighboring cells through mechanisms like downregulation of E-cadherin, a key cell adhesion molecule (</span></span><span style="color:#1f1f1f">Friedl</span><span style="color:#1f1f1f">)</span><span style="background-color:white"><span style="color:#212529">. This disrupts the tight junctions holding them together, creating space for individual cells to move.</span></span></span></span></li>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="background-color:white"><span style="color:#212529">Cytoskeletal Rearrangement: Increased cell motility is often accompanied by dynamic rearrangements of the actin cytoskeleton. Cells with enhanced motility can rapidly change shape, allowing them to navigate through complex tissue environments.</span></span></span></span></li>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="background-color:white"><span style="color:#212529">Cell-Cell and Cell-ECM Interactions: Motile cells can interact dynamically with neighboring cells, forming transient contacts. Enhanced motility allows cells to engage with ECM components more efficiently, promoting effective migration and invasion.</span></span></span></span></li>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="background-color:white"><span style="color:#212529">Epithelial to mesenchymal transition (EMT): cell motility increases EMT (Sahai)</span></span></span></span></li>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="background-color:white"><span style="color:#212529">Intravasion and extravasation: cell with increased motility can enter the bloodstream and then exit the main circulation thus promoting invasion (Kumar).</span></span></span></span></li>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="background-color:white"><span style="color:#212529">Involvement in Collective Migration: Groups of motile cells can move collectively, promoting invasion as a coordinated front. Enhanced motility of individual cells within the group contributes to the overall invasive potential of the collective migration.</span></span></span></span></li>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="background-color:white"><span style="color:#212529">Adaptation to Microenvironmental Challenges: Cells with increased motility can better navigate physical barriers within tissues, overcoming challenges posed by the surrounding microenvironment.</span></span></span></span></li>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="background-color:white"><span style="color:#212529">Increased cell motility is a multifaceted process involving various molecular and cellular mechanisms. In the context of cancer, understanding and targeting these mechanisms are crucial for developing strategies to inhibit tumor invasion and metastasis.</span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="background-color:white"><span style="color:#212529">Extracellular Matrix (ECM) Interaction: Cancer cells with enhanced motility often exhibit changes in surface receptors and cytoskeletal dynamics, allowing them to adhere to and move through the ECM more effectively. This is crucial for breaching physical barriers and invading neighboring tissues.</span></span></span></span></p>
<p style="text-align:justify"> </p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="background-color:white"><span style="color:#212529">Proteolytic Enzyme Secretion: Highly motile cancer cells often secrete proteolytic enzymes, such as matrix metalloproteinases (MMPs), that break down ECM proteins. This proteolytic activity facilitates the remodeling of the ECM, enabling cancer cells to invade surrounding tissues and enter blood or lymphatic vessels.</span></span></span></span></p>
<p style="text-align:justify"> </p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="background-color:white"><span style="color:#212529">Dynamic Cell-Cell and Cell-ECM Interactions: Increased cell motility allows cancer cells to form and disassemble focal adhesions with neighboring cells and the ECM. This dynamic interaction promotes efficient migration and invasion, enabling cancer cells to adapt to the changing microenvironment during invasion.</span></span></span></span></p>
<p style="text-align:justify"> </p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="background-color:white"><span style="color:#212529">Chemotaxis and Chemoattractants: Cancer cells with increased motility are more responsive to chemoattractants, signaling molecules that guide cell movement. Chemotaxis allows cancer cells to navigate towards blood vessels, lymphatic vessels, or specific tissues, facilitating invasion into distant organs.</span></span></span></span></p>
<p style="text-align:justify"> </p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="background-color:white"><span style="color:#212529">Formation of Cellular Protrusions: Highly motile cancer cells often extend dynamic protrusions like lamellipodia and filopodia. These structures increase the surface area of contact with the surrounding environment, allowing cancer cells to explore and invade tissues efficiently.</span></span></span></span></p>
<p style="text-align:justify"> </p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="background-color:white"><span style="color:#212529">Adaptation to Microenvironmental Challenges: Increased motility enables cancer cells to navigate through physical barriers, adapt to varying oxygen levels, and respond to microenvironmental cues. This adaptability is crucial for successful invasion into different organ environments.</span></span></span></span></p>
<p style="text-align:justify"> </p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="background-color:white"><span style="color:#212529">Involvement in Collective Migration: Collective migration, where multiple motile cells move as a coordinated front, enhances the invasive potential of a group of cancer cells. Increased motility of individual cells contributes to the overall success of collective invasion.</span></span></span></span></p>
<p style="text-align:justify"> </p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="background-color:white"><span style="color:#212529">Survival and Escape from Immune Surveillance: Increased motility allows cancer cells to escape immune surveillance by quickly moving through tissues and avoiding immune responses. This is crucial for the survival of circulating tumor cells during metastasis.</span></span></span></span></p>
<p style="text-align:justify"> </p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="background-color:white"><span style="color:#212529">Angiogenesis and Intravasation: Increased motility contributes to the ability of cancer cells to intravasate into vessels. It is also associated with angiogenesis, the formation of new blood vessels, providing a route for cancer cells to enter the bloodstream.</span></span></span></span></p>
<strong>Empirical Evidence</strong>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="background-color:white"><span style="color:#212529">Empirical evidence supporting the association between increased cell motility and organ invasion comes from various experimental studies, both in vitro and in vivo. Here are examples of empirical evidence demonstrating how enhanced cell motility contributes to organ invasion:</span></span></span></span></p>
<p style="text-align:justify"> </p>
<ul>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="background-color:white"><span style="color:#212529">In Vitro Cell Migration and Invasion Assays (Transwell or Boyden chamber assays): Cells with increased motility demonstrate higher migration through porous membranes coated with extracellular matrix (ECM) components (Friedl, Yurchenco). In invasive assays, these cells penetrate ECM barriers more efficiently than less motile cells, providing direct evidence of a correlation between motility and invasion. In a wound healing assay, cancer cells with enhanced motility exhibit faster wound closure, indicating increased invasive potential (Agarwal)</span></span></span></span></li>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="background-color:white"><span style="color:#212529">Three-Dimensional (3D) Invasion Models: Cells with enhanced motility exhibit increased invasion into 3D matrices. These models better represent the complexity of tissue architecture, highlighting the relevance of motility in navigating through a three-dimensional environment.</span></span></span></span></li>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="background-color:white"><span style="color:#212529">In Vivo Orthotopic Xenograft Models: High motility correlates with increased invasion into surrounding tissues and metastasis to distant organs (wiseman, valastyan). Imaging techniques, such as bioluminescence or positron emission tomography (PET), allow tracking of tumor cells in vivo, providing evidence of invasion into organs.</span></span></span></span></li>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="background-color:white"><span style="color:#212529">Intravital Imaging: Intravital imaging studies reveal that cells with increased motility exhibit enhanced invasion into organs. Researchers can directly observe the movement of cells within the complex microenvironment of living tissues.</span></span></span></span></li>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="background-color:white"><span style="color:#212529">Genetic Manipulation of Cell Motility (e.g., overexpression or knockdown of motility-related genes) : Cells with increased motility, due to genetic modifications, consistently demonstrate higher invasive potential. Conversely, inhibiting motility-related genes results in decreased invasion.</span></span></span></span></li>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="background-color:white"><span style="color:#212529">Pharmacological Modulation of Motility: Treatment with motility-inhibiting compounds results in reduced invasion both in vitro and in vivo. Conversely, stimulation of motility-related pathways increases invasive behavior.</span></span></span></span></li>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="background-color:white"><span style="color:#212529">Correlation Studies in Patient Samples: Increased expression of motility-related markers in patient tumors correlates with a higher likelihood of organ invasion and metastasis. This clinical evidence supports the association between cell motility and invasive behavior. Moreover, patients with tumors exhibiting higher motility markers often have a poorer prognosis, increased likelihood of metastasis, and higher rates of organ invasion compared to those with less motile tumors (Gupta, Sahao).</span></span></span></span></li>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="background-color:white"><span style="color:#212529">In Vitro Microfluidic Models: Cells with increased motility demonstrate enhanced invasion through microfluidic channels, simulating the conditions encountered in the circulatory system or within organs.</span></span></span></span></li>
<strong>Uncertainties and Inconsistencies</strong>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="background-color:white"><span style="color:#212529">While there is substantial empirical evidence supporting the notion that increased cell motility contributes to increased organ invasion, it is important to acknowledge uncertainties and inconsistencies in the literature. These uncertainties stem from the complexity of biological systems, the heterogeneity of tumors, and variations in experimental methodologies. Here are some potential uncertainties and inconsistencies:</span></span></span></span></p>
<p style="text-align:justify"> </p>
<ul>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="background-color:white"><span style="color:#212529">Tumor Heterogeneity:</span></span> <span style="background-color:white"><span style="color:#212529">Variability in motility within a tumor may lead to inconsistent observations, with some regions demonstrating enhanced invasion while others do not</span></span><span style="background-color:white"><span style="color:#212529"> (Friedl, Valastyan)</span></span><span style="background-color:white"><span style="color:#212529">.</span></span></span></span></li>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="background-color:white"><span style="color:#212529">Context Dependency:</span></span> <span style="background-color:white"><span style="color:#212529">The role of increased cell motility in invasion may vary depending on the tumor type, microenvironment, and organ of interest</span></span><span style="background-color:white"><span style="color:#212529"> (Friedl, Valastyan)</span></span><span style="background-color:white"><span style="color:#212529">.</span></span><span style="background-color:white"><span style="color:#212529"> For example, breast and pancreatic cancers exhibit a strong dependence on motility for invasion, while gliomas (brain tumors) infiltrate surrounding tissues through a different mechanism known as amoeboid movement, potentially minimizing the role of classical motility (Friedl, Valastyan)</span></span><span style="background-color:white"><span style="color:#212529">.</span></span></span></span></li>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="background-color:white"><span style="color:#212529">In Vitro vs. In Vivo Discrepancies:</span></span> <span style="background-color:white"><span style="color:#212529">Increased motility observed in cell culture may not translate to enhanced invasion in complex in vivo settings, possibly due to differences in the microenvironment and additional factors influencing invasion.</span></span><span style="background-color:white"><span style="color:#212529"> For example, The ECM composition in model systems often doesn't fully capture the diverse components and architecture found in real tumors, potentially influencing cell motility and invasion differently (Hodgkinson). </span></span></span></span></li>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="background-color:white"><span style="color:#212529">Divergent Experimental Models:</span></span> <span style="background-color:white"><span style="color:#212529">Varied experimental models, including xenografts, organoids, and in vitro cultures, may produce different outcomes.</span></span><span style="background-color:white"><span style="color:#212529"> For instance, Most preclinical models lack a functional immune system, neglecting the role of immune cells in either aiding or hindering invasion (Gentezl).</span></span></span></span></li>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="background-color:white"><span style="color:#212529">Dynamic Tumor Microenvironment:</span></span> <span style="background-color:white"><span style="color:#212529">The tumor microenvironment is dynamic, and factors such as hypoxia, inflammation, and immune responses can influence invasion.</span></span> <span style="background-color:white"><span style="color:#212529">Interactions with the dynamic microenvironment may modulate the relationship between cell motility and invasion, introducing complexities and uncertainties.</span></span></span></span></li>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="background-color:white"><span style="color:#212529">Adaptation and Compensation:</span></span> <span style="background-color:white"><span style="color:#212529">Over time, compensatory mechanisms may mask the direct impact of increased motility on invasion, leading to inconsistencies in experimental outcomes.</span></span></span></span></li>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="background-color:white"><span style="color:#212529">Genetic and Epigenetic Variations:</span></span> <span style="background-color:white"><span style="color:#212529">The contribution of increased motility to invasion may be influenced by other genetic or epigenetic alterations, introducing variability in experimental outcomes.</span></span></span></span></li>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="background-color:white"><span style="color:#212529">Timing of Measurements:</span></span> <span style="background-color:white"><span style="color:#212529">Assessments conducted at different stages of tumor progression may yield varied results, and the temporal dynamics of increased motility and invasion need to be considered.</span></span></span></span></li>
<li style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="background-color:white"><span style="color:#212529">Artifactual Observations:</span></span> <span style="background-color:white"><span style="color:#212529">Inconsistencies may arise from variations in the accuracy and sensitivity of experimental methods used to assess motility and invasion.</span></span></span></span></li>
</ul>
<h4>References</h4>
<p><span style="font-size:12pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#1f1f1f">Hodgkinson, P., et al. (2012). Tumor microenvironment and the efficacy of anticancer treatment. <strong>Cancer Treatment Reviews</strong>, 38(3), 231-239. <a href="https://pubmed.ncbi.nlm.nih.gov/21727232/" style="color:var(--bard-color-primary); text-decoration:underline" target="_blank">https://pubmed.ncbi.nlm.nih.gov/21727232/</a></span></span></span></span></p>
<p><span style="font-size:12pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#1f1f1f">Gentzel, D. B., et al. (2016). Transforming the cancer research paradigm: investigating the role of the tumor microenvironment in cancer progression. <strong>The Journal of Clinical Investigation</strong>, 126(8),</span></span></span></span></p>
<p><span style="font-size:12pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="font-family:"Helvetica Neue""><span style="color:#1f1f1f">Egeblad, M., & Werb, Z. (2002). New functions for the matrix metalloproteinases in cancer progression. <strong>Trends in Cell Biology</strong>, 12(3), 104-110. <a href="https://pubmed.ncbi.nlm.nih.gov/11850277/" target="_blank"><span style="color:blue">https://pubmed.ncbi.nlm.nih.gov/11850277/</span></a></span></span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#1f1f1f">Y</span><span style="color:#1f1f1f">urchenco, P. D., & Patton, B. T. (2009). Basement membrane assembly and function. <strong>Biochimica et Biophysica Acta (BBA) - Molecular Cell Research</strong>, 1790(10), 473-489. <a href="https://pubmed.ncbi.nlm.nih.gov/19436234/" style="color:var(--bard-color-primary); text-decoration:underline" target="_blank">https://pubmed.ncbi.nlm.nih.gov/19436234/</a></span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#1f1f1f">Agarwal, R., et al. (2013). EMT and Cancer Cell Migration: Roles of TGF-β and Rho Kinase Signaling. <strong>Cancers (Basel) 5(2),</strong> 820-830. [invalid URL removed]</span></span></span></span></p>
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