AOP ID and Title:
Graphical Representation
Status
| Author status | OECD status | OECD project | SAAOP status |
|---|---|---|---|
| Under development: Not open for comment. Do not cite |
Summary of the AOP
Events
Molecular Initiating Events (MIE), Key Events (KE), Adverse Outcomes (AO)
| Sequence | Type | Event ID | Title | Short name |
|---|---|---|---|---|
| MIE | 1115 | Increase, Reactive oxygen species | Increase, ROS | |
| KE | 1445 | Increase, Lipid peroxidation | Increase, LPO | |
| KE | 1505 | Cell cycle, disrupted | Cell cycle, disrupted | |
| KE | 1821 | Decrease, Cell proliferation | Decrease, Cell proliferation | |
| AO | 1521 | Decrease, Growth | Decrease, Growth |
Key Event Relationships
| Upstream Event | Relationship Type | Downstream Event | Evidence | Quantitative Understanding |
|---|---|---|---|---|
| Increase, Reactive oxygen species | adjacent | Increase, Lipid peroxidation | ||
| Increase, Lipid peroxidation | adjacent | Cell cycle, disrupted | ||
| Cell cycle, disrupted | adjacent | Decrease, Cell proliferation | ||
| Decrease, Cell proliferation | adjacent | Decrease, Growth | ||
Overall Assessment of the AOP
References
Appendix 1
List of MIEs in this AOP
Event: 1115: Increase, Reactive oxygen species
Short Name: Increase, ROS
Key Event Component
| Process | Object | Action |
|---|---|---|
| reactive oxygen species biosynthetic process | reactive oxygen species | increased |
AOPs Including This Key Event
Biological Context
| Level of Biological Organization |
|---|
| Cellular |
Cell term
| Cell term |
|---|
| cell |
Organ term
| Organ term |
|---|
| organ |
Domain of Applicability
Taxonomic Applicability| Term | Scientific Term | Evidence | Links |
|---|---|---|---|
| Vertebrates | Vertebrates | High | NCBI |
| human | Homo sapiens | Moderate | NCBI |
| human and other cells in culture | human and other cells in culture | Moderate | NCBI |
| mouse | Mus musculus | Moderate | NCBI |
| crustaceans | Daphnia magna | High | NCBI |
| Lemna minor | Lemna minor | High | NCBI |
| zebrafish | Danio rerio | High | NCBI |
| Life Stage | Evidence |
|---|---|
| All life stages | High |
| Sex | Evidence |
|---|---|
| Unspecific | High |
| Mixed | High |
ROS is a normal constituent found in all organisms, lifestages, and sexes.
Key Event Description
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).
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>
|
superoxide |
O2·- |
|
hydroxyl radical |
·OH |
|
nitric oxide |
NO· |
|
organic radicals |
R· |
|
peroxyl radicals |
ROO· |
|
alkoxyl radicals |
RO· |
|
thiyl radicals |
RS· |
|
sulfonyl radicals |
ROS· |
|
thiyl peroxyl radicals |
RSOO· |
|
disulfides |
RSSR |
<Non-radical ROS>
|
hydrogen peroxide |
H2O2 |
|
singlet oxygen |
1O2 |
|
ozone/trioxygen |
O3 |
|
organic hydroperoxides |
ROOH |
|
hypochlorite |
ClO- |
|
peroxynitrite |
ONOO- |
|
nitrosoperoxycarbonate anion |
O=NOOCO2- |
|
nitrocarbonate anion |
O2NOCO2- |
|
dinitrogen dioxide |
N2O2 |
|
nitronium |
NO2+ |
|
highly reactive lipid- or carbohydrate-derived carbonyl compounds |
|
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).
How it is Measured or Detected
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).
<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.
References
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Ashoka, A. H., et al. (2020). "Recent Advances in Fluorescent Probes for Detection of HOCl and HNO." ACS omega, 5(4), 1730-1742. doi:10.1021/acsomega.9b03420
B.H. Park, S.M. Fikrig, E.M. Smithwick Infection and nitroblue tetrazolium reduction by neutrophils: a diagnostic aid Lancet, 2 (1968), pp. 532-534
Bedard, Karen, and Karl-Heinz Krause. 2007. “The NOX Family of ROS-Generating NADPH Oxidases: Physiology and Pathophysiology.” Physiological Reviews 87 (1): 245–313.
Bisht, Shilpa, Muneeb Faiq, Madhuri Tolahunase, and Rima Dada. 2017. “Oxidative Stress and Male Infertility.” Nature Reviews. Urology 14 (8): 470–85.
Brieger, K., S. Schiavone, F. J. Miller Jr, and K-H Krause. 2012. “Reactive Oxygen Species: From Health to Disease.” Swiss Medical Weekly 142 (August): w13659.
Calcerrada, P., et al. (2011). "Nitric oxide-derived oxidants with a focus on peroxynitrite: molecular targets, cellular responses and therapeutic implications." Curr Pharm Des 17(35): 3905-3932.
Chattopadhyay, Sukumar, et al. "Apoptosis and necrosis in developing brain cells due to arsenic toxicity and protection with antioxidants." Toxicology letters 136.1 (2002): 65-76.
Chowdhury, A. R., et al. (2020). "Mitochondria-targeted paraquat and metformin mediate ROS production to induce multiple pathways of retrograde signaling: A dose-dependent phenomenon." Redox Biol. doi: 10.1016/j.redox.2020.101606. PMID: 32604037; PMCID: PMC7327929.
Dickinson, B. C. and Chang C. J. (2011). "Chemistry and biology of reactive oxygen species in signaling or stress responses." Nature chemical biology 7(8): 504-511.
Drew, Barry, and Christiaan Leeuwenburgh. 2002. “Aging and the Role of Reactive Nitrogen Species.” Annals of the New York Academy of Sciences 959 (April): 66–81.
Egea, J., et al. (2017). "European contribution to the study of ROS: A summary of the findings and prospects for the future from the COST action BM1203 (EU-ROS)." Redox biology 13: 94-162.
Flaherty, R. L., et al. (2017). "Glucocorticoids induce production of reactive oxygen species/reactive nitrogen species and DNA damage through an iNOS mediated pathway in breast cancer." Breast Cancer Research, 19(1), 1–13. https://doi.org/10.1186/s13058-017-0823-8
Foote CS. Definition of type I and type II photosensitized oxidation. Photochem Photobiol. 1991;54:659.
Fuloria, S., et al. (2021). "Comprehensive Review of Methodology to Detect Reactive Oxygen Species (ROS) in Mammalian Species and Establish Its Relationship with Antioxidants and Cancer." Antioxidants (Basel, Switzerland) 10(1) 128. doi:10.3390/antiox10010128
Go, Y. M. and Jones, D. P. (2013). "The redox proteome." J Biol Chem 288(37): 26512-26520.
Goud, Anuradha P., Pravin T. Goud, Michael P. Diamond, Bernard Gonik, and Husam M. Abu-Soud. 2008. “Reactive Oxygen Species and Oocyte Aging: Role of Superoxide, Hydrogen Peroxide, and Hypochlorous Acid.” Free Radical Biology & Medicine 44 (7): 1295–1304.
Granger, D. N. and Kvietys, P. R. (2015). "Reperfusion injury and reactive oxygen species: The evolution of a concept" Redox Biol. doi: 10.1016/j.redox.2015.08.020. PMID: 26484802; PMCID: PMC4625011.
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 119(5): e39-75.
Griendling, Kathy K., Rhian M. Touyz, Jay L. Zweier, Sergey Dikalov, William Chilian, Yeong-Renn Chen, David G. Harrison, Aruni Bhatnagar, and American Heart Association Council on Basic Cardiovascular Sciences. 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 119 (5): e39–75.
ICH. ICH Guideline S10 Guidance on Photosafety Evaluation of Pharmaceuticals.: International Council on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use; 2014.
Itziou, A., et al. (2011). "In vivo and in vitro effects of metals in reactive oxygen species production, protein carbonylation, and DNA damage in land snails Eobania vermiculata." Archives of Environmental Contamination and Toxicology, 60(4), 697–707. https://doi.org/10.1007/s00244-010-9583-5
Ji, W. O., et al. "Quantitation of the ROS production in plasma and radiation treatments of biotargets." Sci Rep. 2019 Dec 27;9(1):19837. doi: 10.1038/s41598-019-56160-0. PMID: 31882663; PMCID: PMC6934759.
Kruk, J. and Aboul-Enein, H. Y. (2017). "Reactive Oxygen and Nitrogen Species in Carcinogenesis: Implications of Oxidative Stress on the Progression and Development of Several Cancer Types." Mini-Reviews in Medicinal Chemistry, 17:11. doi:10.2174/1389557517666170228115324
Lee, D. Y., et al. (2020). "PEGylated Bilirubin-coated Iron Oxide Nanoparticles as a Biosensor for Magnetic Relaxation Switching-based ROS Detection in Whole Blood." Theranostics, 10(5), 1997-2007. doi:10.7150/thno.39662
Li, Z., et al. (2020). "Inhibition of MiR-25 attenuates doxorubicin-induced apoptosis, reactive oxygen species production and DNA damage by targeting pten." International Journal of Medical Sciences, 17(10), 1415–1427. https://doi.org/10.7150/ijms.41980
Liou, G. Y. and Storz, P. "Reactive oxygen species in cancer." Free Radic Res. 2010 May;44(5):479-96. doi:10.3109/10715761003667554. PMID: 20370557; PMCID: PMC3880197.
Lu, Y., et al. (2010). "Phosphatidylinositol-3-kinase/akt regulates bleomycin-induced fibroblast proliferation and collagen production." American journal of respiratory cell and molecular biology, 42(4), 432–441. https://doi.org/10.1165/rcmb.2009-0002OC
Onoue, S., et al. (2013). "Establishment and intra-/inter-laboratory validation of a standard protocol of reactive oxygen species assay for chemical photosafety evaluation." J Appl Toxicol. 33(11):1241-50. doi: 10.1002/jat.2776. Epub 2012 Jun 13. PMID: 22696462.
Onoue S, Hosoi K, Toda T, Takagi H, Osaki N, Matsumoto Y, et al. Intra-/inter-laboratory validation study on reactive oxygen species assay for chemical photosafety evaluation using two different solar simulators. Toxicology in vitro : an international journal published in association with BIBRA. 2014;28:515-23.
Onoue S, Hosoi K, Wakuri S, Iwase Y, Yamamoto T, Matsuoka N, et al. Establishment and intra-/inter-laboratory validation of a standard protocol of reactive oxygen species assay for chemical photosafety evaluation. Journal of applied toxicology : JAT. 2013;33:1241-50.
Onoue S, Kawamura K, Igarashi N, Zhou Y, Fujikawa M, Yamada H, et al. Reactive oxygen species assay-based risk assessment of drug-induced phototoxicity: classification criteria and application to drug candidates. J Pharm Biomed Anal. 2008;47:967-72.
Onoue S, Seto Y, Gandy G, Yamada S. Drug-induced phototoxicity; an early in vitro identification of phototoxic potential of new drug entities in drug discovery and development. Current drug safety. 2009;4:123-36.
Onoue S, Tsuda Y. Analytical studies on the prediction of photosensitive/phototoxic potential of pharmaceutical substances. Pharmaceutical research. 2006;23:156-64.
Ozcan, Ayla, and Metin Ogun. 2015. “Biochemistry of Reactive Oxygen and Nitrogen Species.” In Basic Principles and Clinical Significance of Oxidative Stress, edited by Sivakumar Joghi Thatha Gowder. Rijeka: IntechOpen.
Parrish, A. R. 2010. “2.27 - Hypoxia/Ischemia Signaling.” In Comprehensive Toxicology (Second Edition), edited by Charlene A. McQueen, 529–42. Oxford: Elsevier.
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Ramos, M. F. P., et al. (2018). "Xanthine oxidase inhibitors and sepsis." Int J Immunopathol Pharmacol. 32:2058738418772210. doi:10.1177/2058738418772210
Ravanat, J. L., et al. (2014). "Radiation-mediated formation of complex damage to DNA: a chemical aspect overview." Br J Radiol 87(1035): 20130715.
Schutzendubel, A. and Polle, A. (2002). "Plant responses to abiotic stresses: heavy metal-induced oxidative stress and protection by mycorrhization." Journal of Experimental Botany, 53(372), 1351–1365. https://doi.org/10.1093/jexbot/53.372.1351
Seto Y, Kato M, Yamada S, Onoue S. Development of micellar reactive oxygen species assay for photosafety evaluation of poorly water-soluble chemicals. Toxicology in vitro : an international journal published in association with BIBRA. 2013;27:1838-46.
Sharma, Gunjan, Nishant Kumar Rana, Priya Singh, Pradeep Dubey, Daya Shankar Pandey, and Biplob Koch. 2017. “p53 Dependent Apoptosis and Cell Cycle Delay Induced by Heteroleptic Complexes in Human Cervical Cancer Cells.” Biomedicine & Pharmacotherapy = Biomedecine & Pharmacotherapie 88 (April): 218–31.
Silva, R., et al. (2019). "Light exposure during growth increases riboflavin production, reactive oxygen species accumulation and DNA damage in Ashbya gossypii riboflavin-overproducing strains." FEMS Yeast Research, 19(1), 1–7. https://doi.org/10.1093/femsyr/foy114
Tsuchiya K, et al. (2005). "Oxygen radicals photo-induced by ferric nitrilotriacetate complex." Biochim Biophys Acta. 1725(1):111-9. doi:10.1016/j.bbagen.2005.05.001
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Wang, X., et al. (2013). "Imaging ROS signaling in cells and animals." Journal of molecular medicine 91(8): 917-927.
Yen, Cheng Chien, et al. "Inorganic arsenic causes cell apoptosis in mouse cerebrum through an oxidative stress-regulated signaling pathway." Archives of toxicology 85 (2011): 565-575.
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List of Key Events in the AOP
Event: 1445: Increase, Lipid peroxidation
Short Name: Increase, LPO
Key Event Component
| Process | Object | Action |
|---|---|---|
| lipid oxidation | polyunsaturated fatty acid | increased |
AOPs Including This Key Event
Biological Context
| Level of Biological Organization |
|---|
| Molecular |
Domain of Applicability
Taxonomic Applicability Life Stage Applicability| Life Stage | Evidence |
|---|---|
| All life stages | High |
| Sex | Evidence |
|---|---|
| Unspecific | High |
ROS is a normal constituent found in all organisms, therefore, all organisms containing lipid membranes may be affected by lipid peroxidation.
Structure: Regardless of sex or life stage, when exposed to free radicals, there is potential for lipid peroxidation as a auxiliary response where there are lipid membranes.
Key Event Description
Lipid peroxidation is the direct damage to lipids in the membrane of the cell or the membranes of the organelles inside the cells. Ultimately the membranes will break due to the build-up damage in the lipids. This is mainly caused by oxidants which attack lipids specifically, since these contain carbon-carbon double bonds. During lipid peroxidation several lipid radicals are formed in a chain reaction. These reactions can interfere and stimulate each other. Antioxidants, such as vitamin E, can react with lipid peroxy radicals to prevent further damage in the cell (Cooley et al. 2000).
How it is Measured or Detected
The main product of lipid peroxidation, malondialdehyde and 4-hydroxyalkenals, is used to measure the degree of this process. This is measured by photocolorimetric assays, quantification of fatty acids by gaseous liquid chromatography (GLC) or high performance (HPLC) (L. Li et al. 2019; Jin et al. 2010a) or through commercial kits purchased from specialized companies.
References
Cooley HM, Evans RE, Klaverkamp JF. 2000. Toxicology of dietary uranium in lake whitefish (Coregonus clupeaformis). Aquatic Toxicology. 48(4):495–515. https://doi.org/10.1016/S0166-445X(99)00057-0
Jin, Yuanxiang, Xiangxiang Zhang, Linjun Shu, Lifang Chen, Liwei Sun, Haifeng Qian, Weiping Liu, and Zhengwei Fu. 2010a. “Oxidative Stress Response and Gene Expression with Atrazine Exposure in Adult Female Zebrafish (Danio Rerio).” Chemosphere 78 (7): 846–52.
Li, Luxiao, Shanshan Zhong, Xia Shen, Qiujing Li, Wenxin Xu, Yongzhen Tao, and Huiyong Yin. 2019. “Recent Development on Liquid Chromatography-Mass Spectrometry Analysis of Oxidized Lipids.” Free Radical Biology & Medicine 144 (November): 16–34.
Event: 1505: Cell cycle, disrupted
Short Name: Cell cycle, disrupted
Key Event Component
| Process | Object | Action |
|---|---|---|
| regulation of cell cycle | cell cycle-related cyclin | disrupted |
AOPs Including This Key Event
Biological Context
| Level of Biological Organization |
|---|
| Cellular |
Cell term
| Cell term |
|---|
| cell |
Organ term
| Organ term |
|---|
| organ |
Domain of Applicability
Taxonomic Applicability| Term | Scientific Term | Evidence | Links |
|---|---|---|---|
| Homo sapiens | Homo sapiens | High | NCBI |
| Mus musculus | Mus musculus | High | NCBI |
| Life Stage | Evidence |
|---|---|
| Not Otherwise Specified | Moderate |
| Sex | Evidence |
|---|---|
| Unspecific | High |
The histone gene expression alters in each phase of the cell cycle in human HeLa cells (Homo sapiens) [Heintz et al., 1982].
Key Event Description
The disruption of the cell cycle leads to a decrease in cell number. The cell cycle consists of G1, S, G2, M, and G0 phases. The cell cycle regulation is disrupted by the cell cycle arrest in certain cell cycle phases. The histone gene expression is regulated in cell cycle phases [Heintz et al., 1983].
How it is Measured or Detected
The percentage of cells at G1, G0, S, and G2/M phases can be detected by flow cytometry [Li et al., 2013]. Cell cycle distribution was analyzed by fluorescence-activated cell sorter (FACS) analysis with a Partec PAS-II sorter [Zupkovitz et al., 2010]. The four cell-cycle phases in living cells can be measured with four-color fluorescent proteins using live-cell imaging [Bajar et al., 2016]. The incorporation of [3H]deoxycytidine or [3H]thymidine into cell DNA during the S phase can be monitored as DNA synthesis [Heintz et al., 1982].
References
Bajar, B.T. et al. (2016), "Fluorescent indicators for simultaneous reporting of all four cell cycle phases", Nat Methods 13:993-996
Heintz, N. et al. (1983), "Regulation of human histone gene expression: Kinetics of accumulation and changes in the rate of synthesis and in the half-lives of individual histone mRNAs during the HeLa cell cycle", Molecular and Cellular Biology 3:539-550
Li, Q. et al. (2013), "Glyphosate and AMPA inhibit cancer cell growth through inhibiting intracellular glycine synthesis", Drug Des Devel Ther 7:635-643
Event: 1821: Decrease, Cell proliferation
Short Name: Decrease, Cell proliferation
Key Event Component
| Process | Object | Action |
|---|---|---|
| cell proliferation | cell | decreased |
AOPs Including This Key Event
Stressors
| Name |
|---|
| 2,4-Dinitrophenol |
| Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone |
| Carbonyl cyanide m-chlorophenyl hydrazone |
| Pentachlorophenol |
| Triclosan |
| Emodin |
| Malonoben |
Biological Context
| Level of Biological Organization |
|---|
| Cellular |
Cell term
| Cell term |
|---|
| cell |
Domain of Applicability
Taxonomic Applicability| Term | Scientific Term | Evidence | Links |
|---|---|---|---|
| zebrafish | Danio rerio | High | NCBI |
| human | Homo sapiens | High | NCBI |
| rat | Rattus norvegicus | High | NCBI |
| mouse | Mus musculus | High | NCBI |
| Life Stage | Evidence |
|---|---|
| Embryo | High |
| Juvenile | High |
| Sex | Evidence |
|---|---|
| Unspecific | High |
Taxonomic applicability domain
This key event is in general applicable to all eukaryotes, as most organisms are known to use cell proliferation to achieve growth.
Life stage applicability domain
This key event is in general applicable to all life stages. As cell proliferation not only occurs in developing organisms, but also in adults.
Sex applicability domain
This key event is sex-unspecific, as both genders use the same cell proliferation mechanisms.
Key Event Description
Decreased cell proliferation describes the outcome of reduced cell division and cell growth. Cell proliferation is considered the main mechanism of tissue and organismal growth (Conlon 1999). Decreased cell proliferation has been associated with abnormal growth-factor signaling and cellular energy depletion (DeBerardinis 2008).
How it is Measured or Detected
Multiple types of in vitro bioassays can be used to measure this key event:
- ToxCast high-throughput screening bioassays such as “BSK_3C_Proliferation”, “BSK_CASM3C_Proliferation” and “BSK_SAg_Proliferation” can be used to measure cell proliferation status.
- Commercially available methods such as the well-established 5-bromo-2’-deoxyuridine (BrdU) (Raza 1985; Muir 1990) or 5-ethynyl-2’-deoxyuridine (EdU) assay. Both assays measure DNA synthesis in dividing cells to indicate proliferation status.
References
Conlon I, Raff M. 1999. Size control in animal development. Cell 96:235-244. DOI: 10.1016/s0092-8674(00)80563-2.
DeBerardinis RJ, Lum JJ, Hatzivassiliou G, Thompson CB. 2008. The biology of cancer: metabolic reprogramming fuels cell growth and proliferation. Cell Metabolism 7:11-20. DOI: https://doi.org/10.1016/j.cmet.2007.10.002.
Muir D, Varon S, Manthorpe M. 1990. An enzyme-linked immunosorbent assay for bromodeoxyuridine incorporation using fixed microcultures. Analytical Biochemistry 185:377-382. DOI: https://doi.org/10.1016/0003-2697(90)90310-6.
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List of Adverse Outcomes in this AOP
Event: 1521: Decrease, Growth
Short Name: Decrease, Growth
Key Event Component
| Process | Object | Action |
|---|---|---|
| growth | multicellular organism | decreased |
AOPs Including This Key Event
Stressors
| Name |
|---|
| 2,4-Dinitrophenol |
| Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone |
| Carbonyl cyanide m-chlorophenyl hydrazone |
| Pentachlorophenol |
| Triclosan |
| Emodin |
| Malonoben |
Biological Context
| Level of Biological Organization |
|---|
| Individual |
Domain of Applicability
Taxonomic Applicability| Term | Scientific Term | Evidence | Links |
|---|---|---|---|
| human | Homo sapiens | Moderate | NCBI |
| rat | Rattus norvegicus | Moderate | NCBI |
| mouse | Mus musculus | Moderate | NCBI |
| zebrafish | Danio rerio | High | NCBI |
| fathead minnow | Pimephales promelas | High | NCBI |
| Lemna minor | Lemna minor | High | NCBI |
| Daphnia magna | Daphnia magna | Moderate | NCBI |
| Life Stage | Evidence |
|---|---|
| Embryo | High |
| Juvenile | High |
| Sex | Evidence |
|---|---|
| Unspecific | High |
Taxonomic applicability domain
This key event is in general applicable to all eukaryotes.
Life stage applicability domain
This key event is applicable to early life stages such as embryo and juvenile.
Sex applicability domain
This key event is sex-unspecific.
Key Event Description
Decreased growth refers to a reduction in size and/or weight of a tissue, organ or individual organism. Growth is normally controlled by growth factors and mainly achieved through cell proliferation (Conlon 1999).
How it is Measured or Detected
Growth can be indicated by measuring weight, length, total volume, and/or total area of a tissue, organ or individual organism.
Regulatory Significance of the AO
Growth is a regulatory relevant chronic toxicity endpoint for almost all organisms. Multiple OECD test guidelines have included growth either as a main endpoint of concern, or as an additional endpoint to be considered in the toxicity assessments. Relevant test guidelines include, but not only limited to:
-Test No. 201: Freshwater Alga and Cyanobacteria, Growth Inhibition Test
-Test No. 208: Terrestrial Plant Test: Seedling Emergence and Seedling Growth Test
-Test No. 211: Daphnia magna Reproduction Test
-Test No. 212: Fish, Short-term Toxicity Test on Embryo and Sac-Fry Stages
-Test No. 215: Fish, Juvenile Growth Test
-Test No. 221: Lemna sp. Growth Inhibition Test
-Test No. 228: Determination of Developmental Toxicity to Dipteran Dung Flies (Scathophaga stercoraria L. (Scathophagidae), Musca autumnalis De Geer (Muscidae))
-Test No. 241: The Larval Amphibian Growth and Development Assay (LAGDA)
-Test No. 407: Repeated Dose 28-day Oral Toxicity Study in Rodents
-Test No. 408: Repeated Dose 90-Day Oral Toxicity Study in Rodents
-Test No. 416: Two-Generation Reproduction Toxicity
-Test No. 422: Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test
-Test No. 443: Extended One-Generation Reproductive Toxicity Study
-Test No. 453: Combined Chronic Toxicity/Carcinogenicity Studies
References
Conlon I, Raff M. 1999. Size control in animal development. Cell 96:235-244. DOI: 10.1016/s0092-8674(00)80563-2.
Appendix 2
List of Key Event Relationships in the AOP
List of Adjacent Key Event Relationships
Relationship: 2460: Increase, ROS leads to Increase, LPO
AOPs Referencing Relationship
| AOP Name | Adjacency | Weight of Evidence | Quantitative Understanding |
|---|---|---|---|
| Oxidation and antagonism of reduced glutathione leading to mortality via acute renal failure | adjacent | High | Moderate |
| Glutathione conjugation leading to reproductive dysfunction via oxidative stress | adjacent | High | High |
| Essential element imbalance leads to reproductive failure via oxidative stress | non-adjacent | ||
| Excessive reactive oxygen species leading to growth inhibition via lipid peroxidation and cell death | adjacent | ||
| Excessive reactive oxygen species leading to growth inhibition via lipid peroxidation and reduced cell proliferation | adjacent | ||
| Excessive reactive oxygen species production leading to mortality (3) | adjacent |
Evidence Supporting Applicability of this Relationship
| Life Stage | Evidence |
|---|---|
| All life stages | High |
| Sex | Evidence |
|---|---|
| Unspecific | High |
Considering the empirical domain of the evidence, the increased, reactive oxygen species leading to increased, lipid peroxidation is known to occur in fish and mammals, but, based on scientific reasoning, the biologically plausible domain of applicability can be eukaryotic organisms in general. It can be measured at any stage of life and in both male and female species.
Evidence Supporting this KER
Biological PlausibilityBiological plausibility of this KER lies in the fact that reactive species, in excess, react and change macromolecules such as proteins, nucleic acids and lipids. Membrane lipids are particularly susceptible to damage by free radicals, as they are composed by unsaturated fatty acids (Su et al. 2019). Hence, increase in ROS production beyond antioxidant system defense capability of cells enables free circulation of molecules such as O2·−, HO·, H2O2, which removes electrons from membrane lipids and then triggers lipid peroxidation (Auten and Davis 2009; Su et al. 2019).
Empirical EvidenceAnalyses performed to support this relation show that KER3 is unchained by the three previously selected xenobiotics, as well as it takes place in a conserved way among species. Connection among the KEs is observed in both in vitro experimental models and in vivo systems, including fishes, birds and mammals.
In cultures of rat hepatocytes, progressive ROS increase during 4 hours of treatment, triggered by DEM (5 mM), is followed by a continuous growth in levels of thiobarbituric acid reactive substances (TBARS), lipid peroxidation markers (Tirmenstein et al. 2000). This chemical depletes GSH content, leading to an augmentation of ROS levels and, consequently, to lipid peroxidation. In an in vivo model, 52 μM of DEM intraperitoneally injected in male Balb/c mice for two weeks caused a significant decrease in the GSH, increase in GSSG, ROS generation and increase in lipid peroxidation in testicles (Kalia and Bansal 2008).
ATZ (46.4 µM) causes an increase of 48.97% of ROS and of 12.5% in MDA content in cultures of Sertoli-Germ cells from Wistar rats (25–28 days old), after, respectively, 3 and 24 h post-exposure. At a higher concentration (232 µM), these cells reach a maximum peak of ROS production after 6h of exposure, while MDA generation gets to the peak only after 24 h of treatment (Abarikwu, Pant, and Farombi 2012). In in vivo model, ATZ (38.5, 77 e 154 mg/Kg bw/day) led to a decrease in total antioxidant capacity (TAC) in a dose-dependent manner in male Sprague-Dawley rats of Specific Pathogen Free (SPF) ATZ-treated for 30 days. Which indirectly suggests increase in ROS levels – and increased malondialdehyde (MDA) content in 154 mg/Kg (Song et al. 2014).
In relation to Hg, it was found that male young Wistar rats exposed to an initial dose of 4.6 μg/Kg of this metal (with following doses of 0.07 μg/Kg/day) displayed an increase in ROS levels, followed by an elevation of MDA content in testicles and epididymis of these rats 60 days post-exposure (Rizzetti et al. 2017). Other assays still carried out with male rats showed that the heavy metal induces oxidative stress with a single subcutaneous dose of 5 mg/Kg, by a substantial diminishment of activity of the main testicle antioxidant enzymes: SOD, CAT and GPX. Consequently, blood hydroperoxide and testicle MDA levels rose in a relevant way (El-Desoky et al. 2013).
Furthermore, Hy-Line Brown laying hens fed with 4 experimental diets containing graded levels of Hg at 0.280, 3.325, 9.415, and 27.240 mg/Kg, respectively, for 10 weeks had GSH content significantly decreased in all Hg-treatment groups in ovaries, whilst SOD, CAT, GPX and glutathione reductase (GR) enzyme activities were significantly reduced, pointing to ROS accumulation. MDA content strongly increased in the 27.240-mg/Kg Hg group (Ma et al. 2018).
Hence, it can be deduced that, as in other adjacent relations evaluated, there is also evidence here that upstream KE is initially required in order to downstream KE take place, which reaffirms time concordance. Besides this, data enhance dose and incidence concordances for this KER.
Quantitative Understanding of the Linkage
Mechanisms involving lipid peroxidation, such as that one caused by ROS accumulation in cells, have been investigated for decades (Tirmenstein et al. 2000; Yin, Xu, and Porter 2011; Su et al. 2019). For this reason, there is much experimental data about response-response relationships or a growth of upstream KE in relation to downstream KE.
Response-response relationshipThis mechanism can be better understood through a process chain that consists of initiation, propagation and termination, as discussed by (Yin, Xu, and Porter 2011). In their review, these authors summarized a series of chemical reactions that develop during all this self-oxidation process and represent them in a schematic manner, as displayed in figure below.
Furthermore, although phospholipid oxidizability is lower, once their rate of diffusion in membranes is slower, the kinetics for this kind of reaction shown in figure follows the same law of velocity (steady-state rate) of homogeneous systems (equation below) (Yin, Xu, and Porter 2011). Oxygen consumption of the equation represents the rate of steady state, while rate of radical generation is defined by Ri, the constant of propagation rate is expressed as kp and the termination rate constant for the reaction is called kt.
-d[O] / dt = kp / (2kt)1/2. [L-H] . Ri1/2
Time-scaleFor instance, empirical evidences show that rat hepatocytes begin ROS production after the first 30 minutes of DEM exposition (5 mM), growing linearly for all the remaining time, whereas the increase in products of lipid peroxidation (TBARS) starts only from the first hour of exposure (Tirmenstein et al. 2000).
Known modulating factors
| Modulating Factor (MF) | MF Specification | Effect(s) on the KER | Reference(s) |
|---|---|---|---|
| antioxidant | vitamin E | prevents lipid peroxidation | Auten and Davis 2009 |
| antioxidant | vitamin C | prevents lipid peroxidation | Auten and Davis 2009 |
References
Su, Lian-Jiu, Jia-Hao Zhang, Hernando Gomez, Raghavan Murugan, Xing Hong, Dongxue Xu, Fan Jiang, and Zhi-Yong Peng. 2019. “Reactive Oxygen Species-Induced Lipid Peroxidation in Apoptosis, Autophagy, and Ferroptosis.” Oxidative Medicine and Cellular Longevity 2019 (October): 5080843.
Auten, Richard L., and Jonathan M. Davis. 2009. “Oxygen Toxicity and Reactive Oxygen Species: The Devil Is in the Details.” Pediatric Research 66 (2): 121–27.
Tirmenstein, M. A., F. A. Nicholls-Grzemski, J. G. Zhang, and M. W. Fariss. 2000. “Glutathione Depletion and the Production of Reactive Oxygen Species in Isolated Hepatocyte Suspensions.” Chemico-Biological Interactions 127 (3): 201–17.
Kalia, Sumiti, and M. P. Bansal. 2008. “Diethyl Maleate-Induced Oxidative Stress Leads to Testicular Germ Cell Apoptosis Involving Bax and Bcl-2.” Journal of Biochemical and Molecular Toxicology 22 (6): 371–81.
Abarikwu, S. O., E. O. Farombi, and A. B. Pant. 2011. “Biflavanone-Kolaviron Protects Human Dopaminergic SH-SY5Y Cells against Atrazine Induced Toxic Insult.” Toxicology in Vitro: An International Journal Published in Association with BIBRA 25 (4): 848–58.
Rizzetti, Danize Aparecida, Caroline Silveira Martinez, Alyne Goulart Escobar, Taiz Martins da Silva, José Antonio Uranga-Ocio, Franck Maciel Peçanha, Dalton Valentim Vassallo, Marta Miguel Castro, and Giulia Alessandra Wiggers. 2017. “Egg White-Derived Peptides Prevent Male Reproductive Dysfunction Induced by Mercury in Rats.” Food and Chemical Toxicology: An International Journal Published for the British Industrial Biological Research Association 100 (February): 253–64.
El-Desoky, Gaber E., Samir A. Bashandy, Ibrahim M. Alhazza, Zeid A. Al-Othman, Mourad A. M. Aboul-Soud, and Kareem Yusuf. 2013. “Improvement of Mercuric Chloride-Induced Testis Injuries and Sperm Quality Deteriorations by Spirulina Platensis in Rats.” PloS One 8 (3): e59177.
Ma, Yan, Mingkun Zhu, Liping Miao, Xiaoyun Zhang, Xinyang Dong, and Xiaoting Zou. 2018. “Mercuric Chloride Induced Ovarian Oxidative Stress by Suppressing Nrf2-Keap1 Signal Pathway and Its Downstream Genes in Laying Hens.” Biological Trace Element Research 185 (1): 185–96.
Yin, Huiyong, Libin Xu, and Ned A. Porter. 2011. “Free Radical Lipid Peroxidation: Mechanisms and Analysis.” Chemical Reviews 111 (10): 5944–72.
Auten, Richard L., and Jonathan M. Davis. 2009. “Oxygen Toxicity and Reactive Oxygen Species: The Devil Is in the Details.” Pediatric Research 66 (2): 121–27.
Relationship: 3364: Increase, LPO leads to Cell cycle, disrupted
AOPs Referencing Relationship
| AOP Name | Adjacency | Weight of Evidence | Quantitative Understanding |
|---|---|---|---|
| Excessive reactive oxygen species leading to growth inhibition via lipid peroxidation and reduced cell proliferation | adjacent |
Relationship: 3363: Cell cycle, disrupted leads to Decrease, Cell proliferation
AOPs Referencing Relationship
| AOP Name | Adjacency | Weight of Evidence | Quantitative Understanding |
|---|---|---|---|
| Excessive reactive oxygen species leading to growth inhibition via lipid peroxidation and reduced cell proliferation | adjacent | ||
| Excessive reactive oxygen species leading to growth inhibition via oxidative DNA damage and reduced cell proliferation | adjacent |
Relationship: 2205: Decrease, Cell proliferation leads to Decrease, Growth
AOPs Referencing Relationship
Evidence Supporting Applicability of this Relationship
| Term | Scientific Term | Evidence | Links |
|---|---|---|---|
| zebrafish | Danio rerio | High | NCBI |
| Life Stage | Evidence |
|---|---|
| Embryo | High |
| Sex | Evidence |
|---|---|
| Unspecific | High |
Taxonomic applicability
Relationship 2205 is considered applicable to all eukaryotes (both unicellular and multicellular), as growth (or population growth of alga) is well known to be achieved through cell proliferation in animals, plants and some microorganisms.
Sex applicability
Relationship 2205 is considered applicable to both all sexes, as cell proliferation leading to growth is a fundamental process and not sex-specific.
Life-stage applicability
Relationship 2205 is considered applicable to all life stages, as cell proliferation leading to growth is essential for maintaining basic biological processes throughout an organism’s life.
Key Event Relationship Description
This key event relationship describes reduced cell proliferation (cell growth, division or a combination of these) leading to reduced tissue, organ or individual growth.
Evidence Supporting this KER
The overall evidence supporting Relationship 2205 is considered moderate.
Biological PlausibilityThe biological plausibility of Relationship 2205 is considered high.
Rationale: The biological structural and functional relationship between cell proliferation and growth is well established. It is commonly accepted that the size of an organism, organ or tissue is dependent on the total number and volume of the cells it contains, and the amount of extracellular matrix and fluids (Conlon 1999). Impairment to cell proliferation can logically affect tissue and organismal growth.
Empirical EvidenceThe empirical support of Relationship 2205 is considered low.
Rationale: Because cell proliferation is typically measured in vitro, while growth of an organism is measured in vivo, few studies have measured both in the same experiment. There is one zebrafish study reporting concordant relationship between reduced cell proliferation and embryo growth with some inconsistencies (Bestman 2015).
Uncertainties and Inconsistencies- In zebrafish embryos exposed to 2,4-DNP, significant growth inhibition (AO), as indicated by whole embryo length, caudal primary (CaP) motor neuron axons and otic vesicle length (OVL) ratio after 21h, somite width and eye diameter after 45h exposure was identified, after 21h, whereas a non- significant reduction in cell proliferation was observed (Bestman 2015).
References
Bestman JE, Stackley KD, Rahn JJ, Williamson TJ, Chan SS. 2015. The cellular and molecular progression of mitochondrial dysfunction induced by 2,4-dinitrophenol in developing zebrafish embryos. Differentiation 89:51-69. DOI: 10.1016/j.diff.2015.01.001.
Binder BJ, Landman KA, Simpson MJ, Mariani M, Newgreen DF. 2008. Modeling proliferative tissue growth: a general approach and an avian case study. Phys Rev E Stat Nonlin Soft Matter Phys 78:031912. DOI: 10.1103/PhysRevE.78.031912.
Conlon I, Raff M. 1999. Size control in animal development. Cell 96:235-244. DOI: 10.1016/s0092-8674(00)80563-2.
Jarrett AM, Lima EABF, Hormuth DA, McKenna MT, Feng X, Ekrut DA, Resende ACM, Brock A, Yankeelov TE. 2018. Mathematical models of tumor cell proliferation: A review of the literature. Expert Review of Anticancer Therapy 18:1271-1286. DOI: 10.1080/14737140.2018.1527689.
Mosca G, Adibi, M., Strauss, S., Runions, A., Sapala, A., Smith, R.S. 2018. Modeling Plant Tissue Growth and Cell Division. In Morris R., ed, Mathematical Modelling in Plant Biology. Springer, Cham.