Key Event Title
|Level of Biological Organization|
Key Event Components
Key Event Overview
AOPs Including This Key Event
|AOP Name||Role of event in AOP|
|AOP on basal cytotoxicity||AdverseOutcome|
|NADPH oxidase activation leading to reproductive failure||KeyEvent|
|Histone deacetylase inhibition leading to testicular atrophy||KeyEvent|
|Inhibition of N-linked glycosylation leads to liver injury||KeyEvent|
|Not Otherwise Specified||High|
Key Event Description
Apoptosis, the process of programmed cell death, is characterized by distinct morphology with DNA fragmentation and energy dependency [Elmore, 2007]. Apoptosis, also called as “physiological cell death”, is involved in cell turnover, physiological involution and atrophy of various tissues and organs [Kerr et al., 1972]. The formation of apoptotic bodies involves marked condensation of both nucleus and cytoplasm, nuclear fragmentation, and separation of protuberances [Kerr et al., 1972]. Apoptosis is characterized by DNA ladder and chromatin condensation. Several stimuli such as hypoxia, nucleotides deprivation, chemotherapeutical drugs, DNA damage, and mitotic spindle damage induce p53 activation, leading to p21 activation and cell cycle arrest [Pucci et al., 2000]. The SAHA or TSA treatment on neonatal human dermal fibroblasts (NHDFs) for 24 or 72 hrs inhibited proliferation of the NHDF cells [Glaser et al., 2003]. Considering that the acetylation of histone H4 was increased by the treatment of SAHA for 4 hrs, histone deacetylase inhibition may be involved in the inhibition of the cell proliferation [Glaser et al., 2003]. The impaired proliferation was observed in HDAC1-/- ES cells, which was rescued with the reintroduction of HDAC1 [Zupkovitz et al., 2010]. The present AOP focuses on p21 pathway leading to apoptosis, however, the alternative pathway such as NF-kB signaling pathways may be involved in apoptosis of spermatocytes [Wang et al., 2017].
How It Is Measured or Detected
Apoptosis is characterized by many morphological and biochemical changes such as cell shrinkage and pyknosis, cell fragments into apoptotic bodies, DNA fragmentation, enzymatic activation of pro-caspases or phosphatidylserine translocation that can be measured using electron and cytochemical optical microscopy, proteoic and genomic methods, spectroscopic techniques [Martines, 2010, Taatjes, 2008, Archana, 2013]. Some of the methods are described as follows:
・The apoptosis is detected with the expression alteration of procaspases 7 and 3 by Western blotting using antibodies [Parajuli, 2014].
・The apoptosis is measured with down-regulation of anti-apoptotic gene baculoviral inhibitor of apoptosis protein repeat containing 2 (BIRC2, or cIAP1) [Parajuli, 2014].
・Apoptotic nucleosomes are detected using Cell Death Detection ELISA kit, which were calculated as absorbance subtraction at 405 nm and 490 nm [Parajuli, 2014].
・Cleavage of PARP is detected with Western blotting [Parajuli, 2014].
・Caspase-3 and caspase-9 activity is measured with the enzyme-catalyzed release of p-nitroanilide (pNA) and quantified at 405 nm [Wu, 2016].
・Apoptosis is measured with Annexin V-FITC probes, and the relative percentage of Annexin V-FITC-positive/PI-negative cells is analyzed by flow cytometry [Wu et al., 2016].
・Apoptosis is detected with the Terminal dUTP Nick End-Labeling (TUNEL) method to assay the endonuclease cleavage products by enzymatically end-labeling the DNA strand breaks [Kressel and Groscurth, 1994].
・For the detection of apoptosis, the testes are fixed in neutral buffered formalin, and embedded in paraffin. Germ cell death is visualized in testis sections by Terminal dUTP Nick End-Labeling (TUNEL) staining method [Wade et al., 2008]. The incidence of TUNEL-positive cells is expressed as the number of positive cells per tubule examined for one entire testis section per animal [Wade et al., 2008].
Domain of Applicability
・Apoptosis is induced in human prostate cancer cell lines (Homo sapiens) [Parajuli et al., 2014].
・Apoptosis occurs in B6C3F1 mouse (Mus musculus) [Elmore, 2007].
・Apoptosis occurs in Sprague-Dawley rat (Rattus norvegicus) [Elmore, 2007].
・Apoptosis occurs in nematode (Caenorhabditis elegans) [Elmore, 2007].
Regulatory Significance of the Adverse Outcome
Archana, M. et al. (2013), "Various methods available for detection of apoptotic cells", Indian J Cancer 50:274-283
Elmore, S. (2007), "Apoptosis: a review of programmed cell death", Toxicol Pathol 35:495-516
Glaser, K.B. et al. (2003), "Gene expression profiling of multiple histone deacetylase (HDAC) inhibitors: defining a common gene set produced by HDAC inhibition in T24 and MDA carcinoma cell lines", Mol Cancer Ther 2:151-163
Kerr, J.F.R. et al. (1972), "Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics", Br J Cancer 26:239-257
Kressel, M. and Groscurth, P. (1994), "Distinction of apoptotic and necrotic cell death by in situ labelling of fragmented DNA", Cell Tissue Res 278:549-556
Martinez, M.M. et al. (2010), "Detection of apoptosis: A review of conventioinal and novel techniques", Anal Methods 2:996-1004
Parajuli, K.R. et al. (2014), "Methoxyacetic acid suppresses prostate cancer cell growth by inducing growth arrest and apoptosis", Am J Clin Exp Urol 2:300-313
Pucci, B. et al. (2000), "Cell cycle and apoptosis", Neoplasia 2:291-299
Taatjes, D.J. et al. (2008), "Morphological and cytochemical determination of cell death by apoptosis", Histochem Cell Biol 129:33-43
Wade, M.G. et al. (2008), "Methoxyacetic acid-induced spermatocyte death is associated with histone hyperacetylation in rats", Biol Reprod 78:822-831
Wang, C. et al. (2017), "CD147 regulates extrinsic apoptosis in spermatocytes by modulating NFkB signaling pathways", Oncotarget 8:3132-3143
Wu, R. et al. (2016), "microRNA-497 induces apoptosis and suppressed proliferation via the Bcl-2/Bax-caspase9-caspase 3 pathway and cyclin D2 protein in HUVECs", PLoS One 11:e0167052
Zupkovitz, G. et al. (2010), "The cyclin-dependent kinase inhibitor p21 is a crucial target for histone deacetylase 1 as a regulator of cellular proliferation", Mol Cell Biol 30:1171-1181