Activation, NADPH Oxidase

7440-61-1

JFALSRSLKYAFGM-UHFFFAOYSA-N

Uranium

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

DTXSID1042522

7440-43-9

BDOSMKKIYDKNTQ-UHFFFAOYSA-N

Cadmium

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

DTXSID1023940

D019255

MESH NADPH Oxidase

PR:000003106

PR map kinase p38

PR:G5EGD2

PR hypoxia-inducible factor 1 (Caenorhabditis elegans)

CHEBI:16991

CHEBI deoxyribonucleic acid

GO:0005739

GO mitochondrion

CHEBI:26523

CHEBI reactive oxygen species

GO:0023052

GO signaling

GO:0000003

GO reproduction

MP:0003674

MP oxidative stress

GO:0006281

GO DNA repair

GO:0006915

GO apoptotic process

GO:1903409

GO reactive oxygen species biosynthetic process

WIKI increased

WIKI decreased

WIKI functional change

Silver nanoparticles

2017-02-15T03:19:15

Uranium

2021-08-05T14:28:50

Nanoparticles and Micrometer Particles

2022-02-04T13:43:43

Cadmium

2017-10-25T08:33:12

9606

NCBI Homo sapiens

10090

NCBI Mus musculus

10116

NCBI Rattus norvegicus

6239

NCBI Caenorhabditis elegans

WCS_9606

common toxicological species human

10090

NCBI mouse

10116

NCBI rat

WCS_7227

common ecological species Drosophila melanogaster

WikiUser_28

Vertebrates

WikiUser_25

Wikiuser: Cyauk human and other cells in culture

WCS_35525

common ecological species crustaceans

WCS_4472

common ecological species Lemna minor

WCS_7955

common ecological species zebrafish

Activation, NADPH Oxidase

Molecular

CL:0000255

CL eukaryotic cell

AOPWiki 2016-11-29T18:41:30

2017-09-16T10:17:43

Reproductive failure

Individual

AOPWiki 2017-02-15T03:18:10

2017-02-15T03:18:10

Increase, Oxidative Stress / Activation, PMK-1 P38 MAPK

Cellular

CL:0000255

CL eukaryotic cell

AOPWiki 2017-02-15T03:24:58

2017-09-16T10:17:44

Activation, HIF-1

Cellular

CL:0000255

CL eukaryotic cell

AOPWiki 2017-02-15T03:25:30

2017-09-16T10:17:44

Increased, DNA Damage-Repair

Cellular

CL:0000255

CL eukaryotic cell

AOPWiki 2017-02-15T03:27:13

2017-09-16T10:17:44

Apoptosis

Cellular

Apoptosis, the process of programmed cell death, is characterized by distinct morphology with DNA fragmentation and energy dependency [Elmore, 2007]. Apoptosis, also called “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]. An AOP focuses existes on p21 pathway leading to apoptosis, however, alternative pathways such as NF-kappaB signaling pathways may be involved in the apoptosis of spermatocytes [Wang et al., 2017]. Apoptosis is defined as a programmed cell death.  A decrease in apoptosis or a resistance to cell death is noted is described as a hallmark of cancer by Hanahan et al. It is widely admitted as an essential step in tumor proliferation (Adams, Lowe).  Apoptosis occurs after activation of a number of intrinsic and extrinsic signals which activate the protease caspase system which in turn activates the destruction of the cell.    In mammals, the foetal ovary produces hundreds of thousands of oocytes. But most of them die before birth due to apoptosis (Kaur, S., & Kurokawa, M., 2023). The apoptotic process has a specific pattern at different stages: in foetal ovaries, the majority of apoptotic activity was found in germ cells, whereas in adult quiescent cortical follicles, apoptosis occurred from both granulosa and oocyte cells. The oocyte has been shown to be the one that triggers the apoptotic process and causes follicular atresia (Jin, X., et al. (2011). In humans, the primordial follicles' ovarian endowment is formed throughout foetal development. Apoptotic cell death, which is carried out with the assistance of multiple players and routes conserved from worms to humans, depletes this endowment by at least two-thirds prior to birth. As of right now, apoptosis has been linked to atresia, oocyte loss/selection, folliculogenesis, and oogenesis (Hussein MR, 2005) The Bcl-2 is a protein family suppressing apoptosis by binding and inhibiting two proapoptotic proteins (Bax and Bak) and transferring them to the mitochondrial outer membrane. In the absence of inhibition by Bcl2, Bax and Bak destroy the mitochondrial membrane and releases proapoptotic signaling proteins, such as cytochrome c which activated the caspase system. An increased expression of these antiapoptotic proteins (Bcl-2, Bcl-xL) occurs in cancer (Hanahan, Adams, Lowe). Several others pathways such as the loss of TP53 tumor suppressor function, or the increase of survival signals (Igf1/2), or decrease of proapoptotic factors (Bax, Bim, Puma) can also increase tumor growth (Hanahan, Juntilla). In breast cancer a decrease in apoptosis and a resistance to cell death has been described thoroughly, especially using a dysregulation of the Bcl2 system or TP53 (Parton, Williams, Shahbandi).

Apoptosis is characterized by many morphological and biochemical changes such as homogenous condensation of chromatin to one side or the periphery of the nuclei, membrane blebbing and formation of apoptotic bodies with fragmented nuclei, DNA fragmentation, enzymatic activation of pro-caspases, or phosphatidylserine translocation that can be measured using electron and cytochemical optical microscopy, proteomic and genomic methods, and spectroscopic techniques [Archana et al., 2013; Martinez et al., 2010; Taatjes et al., 2008; Yasuhara et al., 2003]. ・DNA fragmentation can be quantified with comet assay using electrophoresis, where the tail length, head size, tail intensity, and head intensity of the comet are measured [Yasuhara et al., 2003]. ・The apoptosis is detected with the expression alteration of procaspases 7 and 3 by Western blotting using antibodies [Parajuli et al., 2014]. ・The apoptosis is measured with down-regulation of anti-apoptotic gene baculoviral inhibitor of apoptosis protein repeat containing 2 (BIRC2, or cIAP1) [Parajuli et al., 2014]. ・Apoptotic nucleosomes are detected using Cell Death Detection ELISA kit, which was calculated as absorbance subtraction at 405 nm and 490 nm [Parajuli et al., 2014]. ・Cleavage of PARP is detected with Western blotting [Parajuli et al., 2014]. ・Caspase-3 and caspase-9 activity is measured with the enzyme-catalyzed release of p-nitroanilide (pNA) and quantified at 405 nm [Wu et al., 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]

・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 the nematode (Caenorhabditis elegans) [Elmore, 2007]. <ul> <li>Apoptosis occurs in breast cancer cells, human and mouse (Parton)</li> <li style="text-align:justify">Apoptosis applicable to fishes, hence be used to study as models (dos Santos, N. M., et al. (2008).</li> <li style="text-align:justify">Apoptosis in humans and baboon ovaries (Kugu, K., et al. (1998)</li> <li style="text-align:justify">Apoptosis in amphibians during metamorphosis (Ishizuya-Oka, A., et al. (2010).</li> <li style="text-align:justify">Apoptosis in Drosophila melanogaster (Steller, H. (2008)</li> <li style="text-align:justify">Apoptosis is a highly conserved and essential process across a broad taxonomic range, from unicellular eukaryotes to complex multicellular animals, it is also evident in metazoans (Suraweera, C. D., et al. (2022).</li> <li> Sex Applicability: Both sexes. Apoptosis occurs in male and female systems (e.g., oocyte and sperm cell turnover). </li> <li> Life Stage Applicability: All stages. Especially critical during embryonic development and in maintaining adult tissue homeostasis. </li> </ul>

UBERON:0000062

UBERON organ

CL:0000000

CL cell

High Unspecific

High

Not Otherwise Specified

High High High High

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 Yasuhara, S. et al. (2003), "Comparison of comet assay, electron microscopy, and flow cytometry for detection of apoptosis", J Histochem Cytochem 51:873-885 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   Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. 2011 Mar 4;144(5):646-74. doi: 10.1016/j.cell.2011.02.013. PMID: 21376230 Adams JM, Cory S. The Bcl-2 apoptotic switch in cancer development and therapy. Oncogene. 2007 Feb 26;26(9):1324-37. doi: 10.1038/sj.onc.1210220. PMID: 17322918; PMCID: PMC2930981. Lowe, S., Cepero, E. & Evan, G. Intrinsic tumour suppression. Nature 432, 307–315 (2004). <a href="https://doi.org/10.1038/nature03098" style="color:#467886; text-decoration:underline">https://doi.org/10.1038/nature03098</a> Parton M, Dowsett M, Smith I. Studies of apoptosis in breast cancer. BMJ. 2001 Jun 23;322(7301):1528-32. doi: 10.1136/bmj.322.7301.1528. PMID: 11420276; PMCID: PMC1120573. Junttila MR, Evan GI. p53--a Jack of all trades but master of none. Nat Rev Cancer. 2009 Nov;9(11):821-9. doi: 10.1038/nrc2728. Epub 2009 Sep 24. PMID: 19776747. Williams MM, Cook RS. Bcl-2 family proteins in breast development and cancer: could Mcl-1 targeting overcome therapeutic resistance? Oncotarget. 2015 Feb 28;6(6):3519-30. doi: 10.18632/oncotarget.2792. PMID: 25784482; PMCID: PMC4414133. Shahbandi A, Nguyen HD, Jackson JG. TP53 Mutations and Outcomes in Breast Cancer: Reading beyond the Headlines. Trends Cancer. 2020 Feb;6(2):98-110. doi: 10.1016/j.trecan.2020.01.007. Epub 2020 Feb 5. PMID: 32061310; PMCID: PMC7931175. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. 2011 Mar 4;144(5):646-74. doi: 10.1016/j.cell.2011.02.013. PMID: 21376230 Adams JM, Cory S. The Bcl-2 apoptotic switch in cancer development and therapy. Oncogene. 2007 Feb 26;26(9):1324-37. doi: 10.1038/sj.onc.1210220. PMID: 17322918; PMCID: PMC2930981. Lowe, S., Cepero, E. & Evan, G. Intrinsic tumour suppression. Nature 432, 307–315 (2004). <a href="https://doi.org/10.1038/nature03098" style="color:#467886; text-decoration:underline">https://doi.org/10.1038/nature03098</a> Parton M, Dowsett M, Smith I. Studies of apoptosis in breast cancer. BMJ. 2001 Jun 23;322(7301):1528-32. doi: 10.1136/bmj.322.7301.1528. PMID: 11420276; PMCID: PMC1120573. Junttila MR, Evan GI. p53--a Jack of all trades but master of none. Nat Rev Cancer. 2009 Nov;9(11):821-9. doi: 10.1038/nrc2728. Epub 2009 Sep 24. PMID: 19776747. Williams MM, Cook RS. Bcl-2 family proteins in breast development and cancer: could Mcl-1 targeting overcome therapeutic resistance? Oncotarget. 2015 Feb 28;6(6):3519-30. doi: 10.18632/oncotarget.2792. PMID: 25784482; PMCID: PMC4414133. Shahbandi A, Nguyen HD, Jackson JG. TP53 Mutations and Outcomes in Breast Cancer: Reading beyond the Headlines. Trends Cancer. 2020 Feb;6(2):98-110. doi: 10.1016/j.trecan.2020.01.007. Epub 2020 Feb 5. PMID: 32061310; PMCID: PMC7931175.   Parton M, Dowsett M, Smith I. Studies of apoptosis in breast cancer. BMJ. 2001 Jun 23;322(7301):1528-32. doi: 10.1136/bmj.322.7301.1528. PMID: 11420276; PMCID: PMC1120573.   <ul> <li>Kaur S, Kurokawa M. Regulation of Oocyte Apoptosis: A View from Gene Knockout Mice. Int J Mol Sci. 2023;24(2).</li> <li>Jin X, Xiao LJ, Zhang XS, Liu YX. Apotosis in ovary. Front Biosci (Schol Ed). 2011;3(2):680-97.</li> <li>Hussein MR. Apoptosis in the ovary: molecular mechanisms. Hum Reprod Update. 2005;11(2):162-77.</li> <li>dos Santos NM, do Vale A, Reis MI, Silva MT. Fish and apoptosis: molecules and pathways. Curr Pharm Des. 2008;14(2):148-69.</li> <li>Kugu K, Ratts VS, Piquette GN, Tilly KI, Tao XJ, Martimbeau S, et al. Analysis of apoptosis and expression of bcl-2 gene family members in the human and baboon ovary. Cell Death Differ. 1998;5(1):67-76.</li> <li>Ishizuya-Oka A, Hasebe T, Shi YB. Apoptosis in amphibian organs during metamorphosis. Apoptosis. 2010;15(3):350-64.</li> <li>Steller H. Regulation of apoptosis in Drosophila. Cell Death & Differentiation. 2008;15(7):1132-8.</li> <li>Suraweera CD, Banjara S, Hinds MG, Kvansakul M. Metazoans and Intrinsic Apoptosis: An Evolutionary Analysis of the Bcl-2 Family. International Journal of Molecular Sciences. 2022;23(7):3691.</li> </ul> AOPWiki 2017-02-07T13:21:50

2025-05-31T08:50:09

Increase, Mitochondrial dysfunction

Cellular

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

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

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

UBERON:0000062

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Measurements of mitochondrial calcium in vivo. Biochimica Et Biophysica Acta (BBA) - Bioenergetics, 1787(11), 1317-1323. doi:<a href="https://doi.org/10.1016/j.bbabio.2008.11.012" target="_blank">https://doi.org/10.1016/j.bbabio.2008.11.012</a> Promega GSH-Glo Glutathione Assay Technical Bulletin, TB369, Promega Corporation, Madison, WI. Pryor, W.A., J.P. Stanley, and E. Blair. (1976) Autoxidation of polyunsaturated fatty acids: II. A Suggested mechanism for the Formation of TBA-reactive materials from prostaglandin-like Endoperoxides. Lipids, 11:370-379. Radkowsky, A.E. and E.M. Kosower (1986) Bimanes 17. (Haloalkyl)-1,5-diazabicyclo[3.3.O]octadienediones (halo-9,10- dioxabimanes): reactivity toward the tripeptide thiol, glutathione, J. Am. Chem. Soc 108:4527-4531. Roos, D., Seeger, R., Puntel, R., & Vargas Barbosa, N. (2012). Role of calcium and mitochondria in MeHg-mediated cytotoxicity. Journal of Biomedicine and Biotechnology, 2012, 1-15. doi:10.1155/2012/248764 Ruch, W., P.H. 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Effects of lead and/or cadmium on the oxidative damage of rat kidney cortex mitochondria. Biol.Trace Elem.Res., 137, 69-78. doi:10.1007/s12011-009-8560-1 Wang Y., and Qin ZH., Molecular and cellular mechanisms of excitotoxic neuronal death, Apoptosis, 2010, 15:1382-1402. Wieloch T. (2001). Mitochondrial Involvement in Acute Neurodegeneration 52:247–254. Winklhofer, K. Haass,C (2010) Mitochondrial dysfunction in Parkinson's disease, Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease, 1802: 29-44. Wong A, Cortopassi GA. High-throughput measurement of mitochondrial membrane potential in a neural cell line using a fluorescence plate reader. Biochem Biophys Res Commun. 2002 Nov 15;298(5):750-4. doi: 10.1016/s0006-291x(02)02546-9. PMID: 12419317.  Yoo I, Ahn I, Lee J, Lee N. Extracellular flux assay (Seahorse assay): Diverse applications in metabolic research across biological disciplines. Mol Cells. 2024 Aug;47(8):100095. doi: 10.1016/j.mocell.2024.100095. Epub 2024 Jul 18. 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2026-02-11T07:06:25

Increase, Reactive oxygen species

Increase, ROS

Cellular

Biological State: increased reactive oxygen species (ROS) Biological compartment: an entire cell -- may be cytosolic, may also enter organelles. Reactive oxygen species (ROS) are O2- derived molecules that can be both free radicals (e.g. superoxide, hydroxyl, peroxyl, alcoxyl) and non-radicals (hypochlorous acid, ozone and singlet oxygen) (Bedard and Krause 2007; Ozcan and Ogun 2015). ROS production occurs naturally in all kinds of tissues inside various cellular compartments, such as mitochondria and peroxisomes (Drew and Leeuwenburgh 2002; Ozcan and Ogun 2015). Furthermore, these molecules have an important function in the regulation of several biological processes – they might act as antimicrobial agents or triggers of animal gamete activation and capacitation (Goud et al. 2008; Parrish 2010; Bisht et al. 2017).  However, in environmental stress situations (exposure to radiation, chemicals, high temperatures) these molecules have its levels drastically increased, and overly interact with macromolecules, namely nucleic acids, proteins, carbohydrates and lipids, causing cell and tissue damage (Brieger et al. 2012; Ozcan and Ogun 2015).  <div> Reactive oxygen species (ROS) refers to the chemical species superoxide, hydrogen peroxide, and their secondary reactive products. In the biological context, ROS are signaling molecules with important roles in cell energy metabolism, cell proliferation, and fate. Therefore, balancing ROS levels at the cellular and tissue level is an important part of many biological processes. Disbalance, mainly an increase in ROS levels, can cause cell dysfunction and irreversible cell damage. ROS are produced from both exogenous stressors and normal endogenous cellular processes, such as the mitochondrial electron transport chain (ETC). Inhibition of the ETC can result in the accumulation of ROS. Exposure to chemicals, heavy metal ions, or ionizing radiation can also result in increased production of ROS. Chemicals and heavy metal ions can deplete cellular antioxidants reducing the cell’s ability to control cellular ROS and resulting in the accumulation of ROS. Cellular antioxidants include glutathione (GSH), protein sulfhydryl groups, superoxide dismutase (SOD). ROS are radicals, ions, or molecules that have a single unpaired electron in their outermost shell of electrons, which can be categorized into two groups: free oxygen radicals and non-radical ROS [Liou et al., 2010]. <Free oxygen radicals> <div> <table cellspacing="0" class="MsoTableGrid" style="border-collapse:collapse; border:none"> <tbody> <tr> <td style="border-bottom:2px solid black; border-left:2px solid black; border-right:2px solid black; border-top:2px solid black; vertical-align:top; width:290px"> superoxide </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:2px solid black; vertical-align:top; width:290px"> O2·- </td> </tr> <tr> <td style="border-bottom:2px solid black; border-left:2px solid black; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> hydroxyl radical </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> ·OH </td> </tr> <tr> <td style="border-bottom:2px solid black; border-left:2px solid black; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> nitric oxide </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> NO· </td> </tr> <tr> <td style="border-bottom:2px solid black; border-left:2px solid black; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> organic radicals </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> R· </td> </tr> <tr> <td style="border-bottom:2px solid black; border-left:2px solid black; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> peroxyl radicals </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> ROO· </td> </tr> <tr> <td style="border-bottom:2px solid black; border-left:2px solid black; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> alkoxyl radicals </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> RO· </td> </tr> <tr> <td style="border-bottom:2px solid black; border-left:2px solid black; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> thiyl radicals </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> RS· </td> </tr> <tr> <td style="border-bottom:2px solid black; border-left:2px solid black; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> sulfonyl radicals </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> ROS· </td> </tr> <tr> <td style="border-bottom:2px solid black; border-left:2px solid black; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> thiyl peroxyl radicals </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> RSOO· </td> </tr> <tr> <td style="border-bottom:2px solid black; border-left:2px solid black; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> disulfides </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> RSSR </td> </tr> </tbody> </table> </div> <Non-radical ROS> <div> <table cellspacing="0" class="MsoTableGrid" style="border-collapse:collapse; border:none"> <tbody> <tr> <td style="border-bottom:2px solid black; border-left:2px solid black; border-right:2px solid black; border-top:2px solid black; vertical-align:top; width:290px"> hydrogen peroxide </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:2px solid black; vertical-align:top; width:290px"> H2O2 </td> </tr> <tr> <td style="border-bottom:2px solid black; border-left:2px solid black; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> singlet oxygen </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> 1O2 </td> </tr> <tr> <td style="border-bottom:2px solid black; border-left:2px solid black; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> ozone/trioxygen </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> O3 </td> </tr> <tr> <td style="border-bottom:2px solid black; border-left:2px solid black; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> organic hydroperoxides </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> ROOH </td> </tr> <tr> <td style="border-bottom:2px solid black; border-left:2px solid black; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> hypochlorite </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> ClO- </td> </tr> <tr> <td style="border-bottom:2px solid black; border-left:2px solid black; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> peroxynitrite </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> ONOO- </td> </tr> <tr> <td style="border-bottom:2px solid black; border-left:2px solid black; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> nitrosoperoxycarbonate anion </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> O=NOOCO2- </td> </tr> <tr> <td style="border-bottom:2px solid black; border-left:2px solid black; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> nitrocarbonate anion </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> O2NOCO2- </td> </tr> <tr> <td style="border-bottom:2px solid black; border-left:2px solid black; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> dinitrogen dioxide </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> N2O2 </td> </tr> <tr> <td style="border-bottom:2px solid black; border-left:2px solid black; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> nitronium </td> <td style="border-bottom:2px solid black; border-left:none; border-right:2px solid black; border-top:none; vertical-align:top; width:290px"> NO2+ </td> </tr> <tr> <td colspan="2" style="border-bottom:2px solid black; border-left:2px solid black; border-right:2px solid black; border-top:none; vertical-align:top; width:580px"> highly reactive lipid- or carbohydrate-derived carbonyl compounds </td> </tr> </tbody> </table> </div> Potential sources of ROS include NADPH oxidase, xanthine oxidase, mitochondria, nitric oxide synthase, cytochrome P450, lipoxygenase/cyclooxygenase, and monoamine oxidase [Granger et al., 2015]. ROS are generated through NADPH oxidases consisting of p47phox and p67phox. ROS are generated through xanthine oxidase activation in sepsis [Ramos et al., 2018]. Arsenic produces ROS [Zhang et al., 2011]. Mitochondria-targeted paraquat and metformin mediate ROS production [Chowdhury et al., 2020]. ROS are generated by bleomycin [Lu et al., 2010]. Radiation induces dose-dependent ROS production [Ji et al., 2019]. ROS are generated in the course of cellular respiration, metabolism, cell signaling, and inflammation [Dickinson and Chang 2011; Egea et al. 2017]. Hydrogen peroxide is also made by the endoplasmic reticulum in the course of protein folding. Nitric oxide (NO) is produced at the highest levels by nitric oxide synthase in endothelial cells and phagocytes. NO production is one of the main mechanisms by which phagocytes kill bacteria [Wang et al., 2017]. The other species are produced by reactions with superoxide or peroxide, or by other free radicals or enzymes. ROS activity is principally local. Most ROS have short half-lives, ranging from nano- to milliseconds, so diffusion is limited, while reactive nitrogen species (RNS) nitric oxide or peroxynitrite can survive long enough to diffuse across membranes [Calcerrada et al. 2011]. Consequently, local concentrations of ROS are much higher than average cellular concentrations, and signaling is typically controlled by colocalization with redox buffers [Dickinson and Chang 2011; Egea et al. 2017]. Although their existence is limited temporally and spatially, ROS interact with other ROS or with other nearby molecules to produce more ROS and participate in a feedback loop to amplify the ROS signal, which can increase RNS. Both ROS and RNS also move into neighboring cells, and ROS can increase intracellular ROS signaling in neighboring cells [Egea et al. 2017]. In the primary event, photoreactive chemicals are excited by the absorption of photon energy.  The energy of the photoactivated chemicals transfer to oxygen and then generates the reactive oxygen species (ROS), including superoxide (O2−) via type I reaction and singlet oxygen (1O2) via type II reaction, as principal intermediate species in phototoxic reaction (Foote, 1991, Onoue et al. , 2009). </div>

Photocolorimetric assays (Sharma et al. 2017; Griendling et al. 2016) or through commercial kits purchased from specialized companies. Yuan, Yan, et al., (2013) described ROS monitoring by using H2-DCF-DA, a redox-sensitive fluorescent dye. Briefly, the harvested cells were incubated with H2-DCF-DA (50 µmol/L final concentration) for 30 min in the dark at 37°C. After treatment, cells were immediately washed twice, re-suspended in PBS, and analyzed on a BD-FACS Aria flow cytometry. ROS generation was based on fluorescent intensity which was recorded by excitation at 504 nm and emission at 529 nm. Lipid peroxidation (LPO) can be measured as an indicator of oxidative stress damage Yen, Cheng Chien, et al., (2013). Chattopadhyay, Sukumar, et al. (2002) assayed the generation of free radicals within the cells and their extracellular release in the medium by addition of yellow NBT salt solution (Park et al., 1968). Extracellular release of ROS converted NBT to a purple colored formazan. The cells were incubated with 100 ml of 1 mg/ml NBT solution for 1 h at 37 °C and the product formed was assayed at 550 nm in an Anthos 2001 plate reader. The observations of the ‘cell-free system’ were confirmed by cytological examination of parallel set of explants stained with chromogenic reactions for NO and ROS. On the basis of the pathogenesis of drug-induced phototoxicity, a reactive oxygen species (ROS) assay was proposed to evaluate the phototoxic risk of chemicals. The ROS assay can monitor generation of ROS, such as singlet oxygen and superoxide, from photoirradiated chemicals, and the ROS data can be used to evaluate the photoreactivity of chemicals (Onoue et al. , 2014, Onoue et al. , 2013, Onoue and Tsuda, 2006).  The ROS assay is a recommended approach by guidelines to evaluate the phototoxic risk of chemicals (ICH, 2014, PCPC, 2014). <div> <Direct detection> Many fluorescent compounds can be used to detect ROS, some of which are specific, and others are less specific. ・ROS can be detected by fluorescent probes such as p-methoxy-phenol derivative [Ashoka et al., 2020]. ・Chemiluminescence analysis can detect the superoxide, where some probes have a wider range for detecting hydroxyl radical, hydrogen peroxide, and peroxynitrite [Fuloria et al., 2021]. ・ROS in the blood can be detected using superparamagnetic iron oxide nanoparticles (SPION)-based biosensor [Lee et al., 2020]. ・Hydrogen peroxide (H2O2) can be detected with a colorimetric probe, which reacts with H2O2 in a 1:1 stoichiometry to produce a bright pink colored product, followed by the detection with a standard colorimetric microplate reader with a filter in the 540-570 nm range. ・The levels of ROS can be quantified using multiple-step amperometry using a stainless steel counter electrode and non-leak Ag|AgCl reference node [Flaherty et al., 2017]. ・Singlet oxygen can be measured by monitoring the bleaching of p-nitrosodimethylaniline at 440 nm using a spectrophotometer with imidazole as a selective acceptor of singlet oxygen [Onoue et al., 2014]. <Indirect Detection> Alternative methods involve the detection of redox-dependent changes to cellular constituents such as proteins, DNA, lipids, or glutathione [Dickinson and Chang 2011; Wang et al. 2013; Griendling et al. 2016]. However, these methods cannot generally distinguish between the oxidative species behind the changes and cannot provide good resolution for the kinetics of oxidative activity. </div>

ROS is a normal constituent found in all organisms, lifestages, and sexes.

UBERON:0000062

UBERON organ

CL:0000000

CL cell

High Unspecific High Mixed

High

All life stages

High Moderate Moderate Moderate High High High

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"Reactive oxygen species mediate arsenic induced cell transformation and tumorigenesis through Wnt/β-catenin pathway in human colorectal adenocarcinoma DLD1 cells. " Toxicology and Applied Pharmacology, 256(2), 114-121. doi:10.1016/j.taap.2011.07.016 AOPWiki 2016-11-29T18:41:29

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AOPWiki 2025-04-03T17:19:15

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AOPWiki 2025-04-03T17:19:29

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AOPWiki 2017-02-15T03:32:01

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AOPWiki 2024-03-14T12:10:52

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AOPWiki 2017-02-15T03:32:27

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AOPWiki 2017-02-15T03:33:10

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AOPWiki 2024-03-13T14:59:55

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AOPWiki 2017-02-15T03:33:35

2017-02-15T03:33:35

NADPH oxidase and P38 MAPK activation leading to reproductive failure in Caenorhabditis elegans

NADPH oxidase activation leading to reproductive failure

Jinhee Choi

Jinhee Choi, University of Seoul, Republic of Korea Nivedita Chatterjee, University of Seoul, Republic of Korea Jaeseong Jeong, University of Seoul, Republic of Korea Elizabeth Dufourcq Sekatcheff, University of Seoul, Republic of Korea Ji-yeon Roh, Knoell Korea, Republic of Korea

1.0

NADPH oxidases are proteins that transfer electrons across biological membranes. The biological function of this enzymes is the generation of reactive oxygen species(ROS). ROS may be involved in mediation of apoptosis via variety cellular effects like oxidative stress, DNA damage and mitochondrial damage, and finally lead to reproductive failure. This AOP is constituted the MIE as NADPH oxidase activation, KEs as Reactive Oxygen Species (ROS) formation, oxidative stress, mitochondrial damage, DNA damage and apoptosis, and AO as reproductive failure. Silver nanoparticles (AgNPs) are used as stressor. To experimentally validate the link between MIE, KEs and AO, C. elegans functional genetic tools are used. C. elegans functional mutants involved in each MIE and KE, such as, bli-3  (NADPH Oxidase), pmk-1 (P38 MAPK), hif-1 (Transcription factor), fmo-2 (Effector), cyp35a2 (Effector), sod-3 (Effector), nth-1 (DNA damage/repair) and ndx-4 (DNA damage/repair).

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All life stages

High

<div> <table class="table table-bordered table-fullwidth"> <thead> <tr> <th>Modulating Factor (MF)</th> <th>Influence or Outcome</th> <th>KER(s) involved</th> </tr> </thead> <tbody> <tr> <td> </td> <td> </td> <td> </td> </tr> </tbody> </table> </div>

High

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