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Key Event Title
Increase, Cell death
|Level of Biological Organization|
Key Event Components
Key Event Overview
AOPs Including This Key Event
|AOP Name||Role of event in AOP||Point of Contact||Author Status||OECD Status|
|Uncoupling of OXPHOS leading to growth inhibition 2||KeyEvent||Evgeniia Kazymova (send email)||Open for citation & comment||Under Development|
|Uncoupling of OXPHOS leading to growth inhibition 4||KeyEvent||Brendan Ferreri-Hanberry (send email)||Under development: Not open for comment. Do not cite||Under Development|
|Uncoupling of OXPHOS leading to growth inhibition 5||KeyEvent||Arthur Author (send email)||Under development: Not open for comment. Do not cite||Under Development|
|Uncoupling of OXPHOS leading to growth inhibition 6||KeyEvent||Agnes Aggy (send email)||Under development: Not open for comment. Do not cite||Under Development|
|Mitochondrial ATP synthase antagonism leading to growth inhibition (2)||KeyEvent||Arthur Author (send email)||Under development: Not open for comment. Do not cite|
|Mitochondrial complex III antagonism leading to growth inhibition (2)||KeyEvent||Allie Always (send email)||Under development: Not open for comment. Do not cite|
|Cytochrome oxidase inhibition to nasal tissues outcomes||KeyEvent||Agnes Aggy (send email)||Under development: Not open for comment. Do not cite|
|TLR9 activation leading to Multi Organ Failure and ARDS||KeyEvent||Cataia Ives (send email)||Under development: Not open for comment. Do not cite|
|GSK3beta inactivation leads to increased mortality||KeyEvent||Cataia Ives (send email)||Open for citation & comment|
|AHR activation decreasing lung function via AHR-ARNT tox path||KeyEvent||Arthur Author (send email)||Under development: Not open for comment. Do not cite|
|All life stages||High|
Key Event Description
Cell death is part of normal development and maturation cycle, and is the component of many response patterns of living tissues to xenobiotic agents (i.e.. micro organisms and chemicals) and to endogenous modulations, such as inflammation and disturbed blood supply (Kanduc et al., 2002). Many physiological processes require cell death for their function (e.g.., embryonal development and immune selection of B and T cells) (Bertheloot et al., 2021). Defects in cells that result in their inappropriate survival or untimely death can negatively impact development or contribute to a variety of human pathologies, including cancer, AIDS, autoimmune disorders, and chronic infection. Cell death may also occur following exposure to environmental toxins or cytotoxic chemicals. Although this is often harmful, it can be beneficial in some cases, such as in the treatment of cancer (Crowley et al., 2016).
Cell death can be divided into: programmed cell death (cell death as a normal component of development) and non-programmed cell death (uncontrolled death of the cell). Although this simplistic view has blurred the intricate mechanisms separating these forms of cell death, studies have and will uncover new effectors, cell death pathways and reveal a more complex and intertwined landscape of processes involving cell death (Bertheloot et al., 2021).
Programmed cell death: is a form of cell death in which the dying cell plays an active part in its own demise (Cotter & Al-Rubeai, 1995).
Apoptosis At a morphological level, it is characterized by cell shrinkage rather than the swelling seen in necrotic cell death. It is characterized by a number of characteristic morphological changes in the structure of the cell, together with a number of enzyme‐dependent biochemical processes. The result of it being the clearance of cells from the body, with minimal damage to surrounding tissues. An essential feature of apoptosis is the release of cytochrome c from mitochondria, regulated by a balance between proapoptotic and antiapoptotic proteins of the BCL-2 family, initiator caspases (caspase-8, -9 and -10) and effector caspases (caspase-3, -6 and -7). Apoptosis culminates in the breakdown of the nuclear membrane by caspase-6, the cleavage of many intracellular proteins (e.g., PARP and lamin), membrane blebbing, and the breakdown of genomic DNA into nucleosomal structures (Bertheloot et al., 2021). Mechanistically, two main pathways contribute to the caspase activation cascade downstream of mitochondrial cytochrome c release:
- Intrinsic pathway is triggered by dysregulation of or imbalance in intracellular homeostasis by toxic agents or DNA damage. It is characterized by mitochondrial outer membrane permeabilization (MOMP), which results in the release of cytochrome c into the cytosol.
- Extrinsic pathway is initiated by activation of cell surface death receptors. Proapoptotic death receptors include TNFR1/2, Fas and the TNF-related apoptosis-inducing ligand (TRAIL) receptors DR4 and DR5.
Other pathways of programmed cell death are called »non-apoptotic programmed cell-death« or »caspase-independent programmed cell-death« (Blank & Shiloh, 2007).
Necroptosis: This type of regulated cell death, occurs following the activation of the tumor necrosis receptor (TNFR1) by TNFα. Activation of other cellular receptors triggers necroptosis. These receptors include death receptors (i.e., Fas/FasL), Toll-like receptors (TLR4 and TLR3) and cytosolic nucleic acid sensors such as RIG-I and STING, which induce type I interferon (IFN-I) and TNFα production and thus promote necroptosis in an autocrine feedback loop. Most of these pathways trigger NFκB- dependent proinflammatory and prosurvival signals.
Pyroptosis is a type of cell death culminating in the loss of plasma membrane integrity and induced by activation of so-called inflammasome sensors. These include the Nod-like receptor (NLR) family, the DNA receptor Absent in Melanoma 2 (AIM2) and the Pyrin receptor.
Autophagy: is a process where cellular components such as macro proteins or even whole organelles are sequestered into lysosomes for degradation (Mizushima et al., 2008; Shintani & Klionsky, 2004). The lysosomes are then able to digest these substrates, the components of which can either be recycled to create new cellular structures and/or organelles or alternatively can be further processed and used as a source of energy (D’Arcy, 2019).
Anoikis is apoptosis induced by loss of attachment to substrate or to other cells (anoikis). Anoikis overlaps with apoptosis in molecular terms, but is classified as a separate entity because of its specific form od induction (Blank & Shiloh, 2007). Induction of anoikis occurs when cells lose attachment to ECM, or adhere to an inappropriate type of ECM, the latter being the more relevant in vivo (Gilmore, 2005).
Cornification: is programmed cell death of keratinocytes. Cell death in the context of cornification involves distinct enzyme classes such as transglutaminases, proteases, DNases and others (Eckhart et al., 2013).
Non-programmed cell death: occurs accidentally in an unplanned manner.
Necrosis is generally characterized to be the uncontrolled death of the cell, usually following a severe insult, resulting in spillage of the contents of the cell into surrounding tissues and subsequent damage thereof (D’Arcy, 2019).
How It Is Measured or Detected
Assays for Quantitating Cell Death:
- Cell death can be measured by staining a sample of cells with trypan blue, assay is described in protocol: Measuring Cell Death by Trypan Blue Uptake and Light Microscopy (Crowley, Marfell, Christensen, et al., 2015d). Or with propidium Iodide, assay is described in protocol: Measuring Cell Death by Propidium Iodide (PI) Uptake and Flow Cytometry (Crowley & Waterhouse, 2015a)
- TUNEL technique: in situ terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling can be used to detect apoptotic cells (Bever & Fekete, 1999; Uribe et al., 2013).
Assays for Quantitating Cell Survival
Colony-forming assay can be used to define the number of cells in a population that are capable of proliferating and forming large groups of cells. Described in Protocol: Measuring Survival of Adherent Cells with the Colony-Forming Assay (Crowley, Christensen, & Waterhouse, 2015c); Measuring Survival of Hematopoietic Cancer Cells with the Colony-Forming Assay in Soft Agar (Crowley & Waterhouse, 2015b).
ASSAYS TO DISTINGUISH APOPTOSIS FROM NECROSIS AND OTHER DEATH MODALITIES
Detecting Nuclear Condensation: The nucleus is generally round in healthy cells but fragmented in apoptotic cells. Dyes such as Giemsa or hematoxylin, which are purple in color and therefore easily viewed using light microscopy, are commonly used to stain the nucleus. Other features of apoptosis and necrosis, such as plasma membrane blebbing or rupture, can be identified by staining the cytoplasm with eosin. Eosin is pinkish in color and can also be viewed using light microscopy. Hematoxylin and eosin are, therefore, commonly used together to stain cells. Assay is described in Protocol: Morphological Analysis of Cell Death by Cytospinning Followed by Rapid Staining (Crowley, Marfell, & Waterhouse, 2015c); Analyzing Cell Death by Nuclear Staining with Hoechst 33342 (Crowley, Marfell, & Waterhouse, 2015a).
Detection of DNA Fragmentation: Apoptotic cells with fragmented DNA can be identified and distinguished from live cells by staining with Propidium Iodide (PI) and measuring DNA content by flow cytometry. This assay is described in Protocol: Measuring the DNA Content of Cells in Apoptosis and at Different Cell-Cycle Stages by Propidium Iodide Staining and Flow Cytometry (Crowley, Chojnowski, & Waterhouse, 2015a). TUNEL technique can also be used: in situ terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling can be used to detect apoptotic cells (Bever & Fekete, 1999; Crowley, Marfell, & Waterhouse, 2015b; Uribe et al., 2013).
Detecting Phosphatidylserine Exposure: Apoptosis is also characterized by exposure of phosphatidylserine (PS) on the outside of apoptotic cells, which acts as a signal that triggers removal of the dying cell by phagocytosis. Annexin V, can selectively bind to PS to label apoptotic cells in which PS is exposed. Purified annexin V can be conjugated to various fluorochromes, which can then be visualized by fluorescence microscopy or detected by flow cytometry. This assay is described in protocol: Quantitation of Apoptosis and Necrosis by Annexin V Binding, Propidium Iodide Uptake, and Flow Cytometry (Crowley, Marfell, Scott, et al., 2015e).
Detecting Caspase Activity: antibodies that specifically recognize the cleaved fragments of caspases and their substrates can be used to specifically detect caspase activity in apoptotic cells by immunocytochemistry. Flow cytometry (using primary antibodies conjugated to fluorescent molecules, or by counter staining with fluorescently labeled antibodies against the primary antibody) can then be used to quantitate the number of apoptotic cells. This assay is described in protocol: Detecting Cleaved Caspase-3 in Apoptotic Cells by Flow Cytometry (Crowley & Waterhouse, 2015a).
Detecting Mitochondrial Damage: flow cytometry can be used to quantitate the number of cells that have reduced mitochondrial transmembrane potential, which is commonly associated with cytochrome c release during apoptosis. For this assay see protocol: Measuring Mitochondrial Transmembrane Potential by TMRE Staining (Crowley, Christensen, & Waterhouse, 2015b).
Domain of Applicability
The process of cell death is highly conserved within multi‐cellular organisms. (Lockshin & Zakeri, 2004).
Evidence for Perturbation by Stressor
Autophagy can be initiated by a variety of stressors, most notably by nutrient deprivation (caloric restriction) or can result from signals present during cellular differentiation and embryogenesis and on the surface of damaged organelles (Mizushima et al., 2008).
Gentamicin causes significant inner ear sensory hair cell death and auditory dysfunction in zebrafish (Uribe et al., 2013).
Bertheloot, D., Latz, E., & Franklin, B. S. (2021). Necroptosis, pyroptosis and apoptosis: an intricate game of cell death. Cellular & Molecular Immunology, 18, 1106–1121. https://doi.org/10.1038/s41423-020-00630-3
Bever, M. M., & Fekete, D. M. (1999). Ventromedial focus of cell death is absent during development of Xenopus and zebrafish inner ears. Journal of Neurocytology, 28(10–11), 781–793. https://doi.org/10.1023/a:1007005702187
Blank, M., & Shiloh, Y. (2007). Cell Cycle Programs for Cell Death: Apoptosis is Only One Way to Go. Cell Cycle, 6(6), 686–695. https://doi.org/10.4161/cc.6.6.3990
Cotter, T. G., & Al-Rubeai, M. (1995). Cell death (apoptosis) in cell culture systems. Trends in Biotechnology, 13(4), 150–155. https://doi.org/10.1016/S0167-7799(00)88926-X
Crowley, L. C., Chojnowski, G., & Waterhouse, N. J. (2015a). Measuring the DNA content of cells in apoptosis and at different cell-cycle stages by propidium iodide staining and flow cytometry. Cold Spring Harbor Protocols, 10, 905–910. https://doi.org/10.1101/pdb.prot087247
Crowley, L. C., Christensen, M. E., & Waterhouse, N. J. (2015b). Measuring mitochondrial transmembrane potential by TMRE staining. Cold Spring Harbor Protocols, 12, 1092–1096. https://doi.org/10.1101/pdb.prot087361
Crowley, L. C., Christensen, M. E., & Waterhouse, N. J. (2015c). Measuring survival of adherent cells with the Colony-forming assay. Cold Spring Harbor Protocols, 8, 721–724. https://doi.org/10.1101/pdb.prot087171
Crowley, L. C., Marfell, B. J., Christensen, M. E., & Waterhouse, N. J. (2015d). Measuring cell death by trypan blue uptake and light microscopy. Cold Spring Harbor Protocols, 7, 643–646. https://doi.org/10.1101/pdb.prot087155
Crowley, L. C., Marfell, B. J., Scott, A. P., Boughaba, J. A., Chojnowski, G., Christensen, M. E., & Waterhouse, N. J. (2016). Dead cert: Measuring cell death. Cold Spring Harbor Protocols, 2016(12), 1064–1072. https://doi.org/10.1101/pdb.top070318
Crowley, L. C., Marfell, B. J., Scott, A. P., & Waterhouse, N. J. (2015e). Quantitation of apoptosis and necrosis by annexin V binding, propidium iodide uptake, and flow cytometry. Cold Spring Harbor Protocols, 11, 953–957. https://doi.org/10.1101/pdb.prot087288
Crowley, L. C., Marfell, B. J., & Waterhouse, N. J. (2015a). Analyzing cell death by nuclear staining with Hoechst 33342. Cold Spring Harbor Protocols, 9, 778–781. https://doi.org/10.1101/pdb.prot087205
Crowley, L. C., Marfell, B. J., & Waterhouse, N. J. (2015b). Detection of DNA fragmentation in apoptotic cells by TUNEL. Cold Spring Harbor Protocols, 10, 900–905. https://doi.org/10.1101/pdb.prot087221
Crowley, L. C., Marfell, B. J., & Waterhouse, N. J. (2015c). Morphological analysis of cell death by cytospinning followed by rapid staining. Cold Spring Harbor Protocols, 9, 773–777. https://doi.org/10.1101/pdb.prot087197
Crowley, L. C., & Waterhouse, N. J. (2015a). Detecting cleaved caspase-3 in apoptotic cells by flow cytometry. Cold Spring Harbor Protocols, 11, 958–962. https://doi.org/10.1101/pdb.prot087312
Crowley, L. C., & Waterhouse, N. J. (2015b). Measuring survival of hematopoietic cancer cells with the Colony-forming assay in soft agar. Cold Spring Harbor Protocols, 8, 725. https://doi.org/10.1101/pdb.prot087189
D’Arcy, M. S. (2019). Cell death: a review of the major forms of apoptosis, necrosis and autophagy. Cell Biology International, 43(6), 582–592. https://doi.org/10.1002/cbin.11137
Eckhart, L., Lippens, S., Tschachler, E., & Declercq, W. (2013). Cell death by cornification. Biochimica et Biophysica Acta - Molecular Cell Research, 1833(12), 3471–3480. https://doi.org/10.1016/j.bbamcr.2013.06.010
Gilmore, A. P. (2005). Anoikis. Cell Death and Differentiation, 12, 1473–1477. https://doi.org/10.1038/sj.cdd.4401723
Kanduc, D., Mittelman, A., Serpico, R., Sinigaglia, E., Sinha, A. A., Natale, C., Santacroce, R., Di Corcia, M. G., Lucchese, A., Dini, L., Pani, P., Santacroce, S., Simone, S., Bucci, R., & Farber, E. (2002). Cell death: apoptosis versus necrosis (review). International Journal of Oncology, 21(1), 165–170. https://doi.org/10.3892/ijo.21.1.165
Lockshin, R. A., & Zakeri, Z. (2004). Apoptosis, autophagy, and more. International Journal of Biochemistry and Cell Biology, 36(12), 2405–2419. https://doi.org/10.1016/j.biocel.2004.04.011
Mizushima, N., Levine, B., Cuervo, A. M., & Klionsky, D. J. (2008). Autophagy fights disease through cellular self-digestion. Nature, 451(7182), 1069–1075. https://doi.org/10.1038/nature06639
Shintani, T., & Klionsky, D. J. (2004). Autophagy in health and disease: A double-edged sword. Science, 306(5698), 990–995. https://doi.org/10.1126/science.1099993
Uribe, P. M., Sun, H., Wang, K., Asuncion, J. D., & Wang, Q. (2013). Aminoglycoside-Induced Hair Cell Death of Inner Ear Organs Causes Functional Deficits in Adult Zebrafish (Danio rerio). PLoS ONE, 8(3), 58755. https://doi.org/10.1371/journal.pone.0058755