AOP ID and Title:

Short Title: ROS leading to growth inhibition via DNA damage and reduced proliferation

Authors

You Song 

Norwegian Institute for Water Research (NIVA), Økernveien 94, NO-0579 Oslo, Norway

Status

Summary of the AOP

Events

Molecular Initiating Events (MIE), Key Events (KE), Adverse Outcomes (AO)

Key Event Relationships

Stressors

Overall Assessment of the AOP

Domain of Applicability

Life Stage Applicability Taxonomic Applicability Sex Applicability

References

Appendix 1

List of MIEs in this AOP

Event: 1115: Increase, Reactive oxygen species

Short Name: Increase, ROS

Key Event Component

AOPs Including This Key Event

Biological Context

Cell term

Organ term

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

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). 

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).

References

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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

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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

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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.

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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.

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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.

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List of Key Events in the AOP

Event: 1634: Increase, Oxidative DNA damage

Short Name: Increase, Oxidative DNA damage

Key Event Component

AOPs Including This Key Event

Stressors

Biological Context

Cell term

Organ term

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

Taxonomic applicability: Theoretically, DNA oxidation can occur in any cell type, in any organism. Oxidative DNA lesions have been measured in mammalian cells (human, mouse, calf, rat) in vitro and in vivo, and in prokaryotes.

Life stage applicability: This key event is not life stage specific (Mesa & Bassnett, 2013; Suman et al., 2019). 

Sex applicability: This key event is not sex specific (Mesa & Bassnett, 2013). 

Evidence for Perturbation by Prototypic Stressor: H₂O₂ and KBrO₃ – A concentration-dependent increase in oxidative lesions was observed in both Fpg- and hOGG1-modified comet assays of TK6 cells treated with increasing concentrations of glucose oxidase (an enzyme that generates H₂O₂) and potassium bromate for 4 h (Platel et al., 2011).  

Evidence indicates that oxidative DNA damage is also induced by X-rays (Bahia et al., 2018), ⁶⁰Co γ-rays, ¹²C ions, α particles, electrons (Georgakilas, 2013), UVB (Mesa and Bassnett, 2013), γ-rays, ⁵⁶Fe ions (Datta et al., 2012), and protons (Suman et al., 2019).  

Key Event Description

The nitrogenous bases of DNA are susceptible to oxidation in the presence of oxidizing agents. Oxidative adducts form mainly on C5 and to a lesser degree on C6 of thymine and cytosine, and on C8 of guanine and adenine. Guanine is most prone to oxidation due to its low oxidation potential (Jovanovic and Simic, 1986). Indeed, 8-oxo-2’-deoxyguanosine (8-oxodG)/8-hydroxy-2’-deoxyguanosine (8-OHdG) is the most abundant and well-studied oxidative DNA lesion in the cell (Swenberg et al., 2011). It causes an A(anti):8-oxo-G(syn) mispair instead of the normal C(anti):8-oxo-G(syn) pair. This pairing does not cause large structural changes to the DNA backbone, and therefore remains undetected by the polymerase’s proofreading mechanism. Consequently, one of the daughter strands will have an AT pair instead of the correct GC pair after replication (Markkanen, 2017). 

Formamidopyrimidine lesions on guanine and adenine (FaPyG and FaPyA), 8-hydroxy-2'-deoxyadenine (8-oxodA), and thymidine glycol (Tg) are other common oxidative lesions. We refer the reader to reviews on this topic to see the full set of potential oxidative DNA lesions (Whitaker et al., 2017). Oxidative DNA lesions are present in the cell at a steady state due to endogenous redox processes (Swenberg et al., 2010). Under normal conditions, cells are able to withstand the baseline level of oxidized bases through efficient repair and regulation of free radicals in the cell. However, direct chemical insult from specific compounds, exposure to various forms of radiation, or induction of reactive oxygen species (ROS) from the reduction of endogenous molecules, as well as through the release of inflammatory cell-derived oxidants, can lead to increased DNA oxidation, a state known as oxidative stress (Turner et al., 2002; Schoenfeld et al., 2012; Tangvarasittichai and Tangvarasittichai, 2019). It is worth noting that ROS must be generated near the DNA to cause damage, otherwise, if ROS was produced more distantly, then it can be removed by the cell (Nilsson & Liu, 2020). Furthermore, although cells do possess repair mechanisms to deal with oxidative DNA damage, sometimes the repair intermediates can interfere with genome function or decrease stability of the genome. This creates a balancing act between when it is best to repair damage and when it is best to leave it (Poetsch, 2020a). 

This KE describes an increase in oxidative lesions of a broad spectrum (ie. superoxide radical (O2•−), hydroxyl radical (OH), peroxyl radical (RO22), single oxygen (1O2 ) in the nuclear DNA above the steady-state level. Oxidative DNA damage can occur in any cell type with nuclear DNA under oxidative stress.

How it is Measured or Detected

Relative Quantification of Oxidative DNA Lesions

• Comet assay (single cell gel electrophoresis) with Fpg and hOGG1 modifications (Smith et al., 2006; Platel et al., 2011)
  • Oxoguanine glycosylase (hOGG1) and formamidopyrimidine-DNA glycosylase (Fpg) are base excision repair (BER) enzymes in eukaryotic and prokaryotic cells, respectively
  • Both enzymes are bi-functional; the glycosylase function cleaves the glycosidic bond between the ribose and the oxidized base, giving rise to an abasic site, and the apurinic/apymidinic (AP) site lyase function cleaves the phosphodiester bond via β-elimination reaction and creates a single strand break
  • Treatment of DNA with either enzyme prior to performing the electrophoresis step of the comet assay allows detection of oxidative lesions by measuring the increase in comet tail length when compared against untreated samples.
• Enzyme-linked immunosorbant assay (ELISA) (Dizdaroglu et al., 2002; Breton et al., 2003; Xu et al., 2008; Zhao et al. 2017)
  • 8-oxodG can be detected using immunoassays, such as ELISA, that use antibodies against 8-oxodG lesions. It has been noted that immunodetection of 8-oxodG can be interfered by certain compounds in biological samples.

Absolute Quantification of Oxidative DNA Lesions

• Quantification of 8-oxodG using HPLC-EC  (Breton et al., 2003; Chepelev et al., 2015)
  • 8-oxodG can be separated from digested DNA and precisely quantified using high performance liquid chromatography (HPLC) with electrochemical detection
• Mass spectrometry LC-MRM/MS (Mangal et al., 2009)
  • Liquid chromatography can also be coupled with multiple reaction monitoring/ mass spectrometry to detect and quantify oxidative lesions. Correlation between lesions measured by hOGG1-modified comet assay and LC-MS has been reported

Gas chromatography-mass spectrometry (GC-MS) 

• DNA is hydrolyzed to release either free bases or nucleosides and then undergoes derivatization in order to increase their volatility. Finally, samples run through a gas chromatograph and then a mass spectrometer. The mass spectrometer results are used to determine oxidative DNA damage by identifying modified bases or nucleosides (Dizdaroglu, 1994). 

Sequencing assays 

• Various markers are used to detect and highlight sites of DNA damage; the result is then processed and sequenced. This category encompasses a wide range of assays such as snAP-seq, OGG1-AP-seq, oxiDIP-seq, OG-seq, and click-code-seq (Yun et al., 2017; Wu et al., 2018; Amente et al., 2019; Poetsch, 2020b). 
• We note that other types of oxidative lesions can be quantified using the methods described above.

References

Amente, S. et al. (2019), “Genome-wide mapping of 8-oxo-7,8-dihydro-2’-deoxyguanosine reveals accumulation of oxidatively-generated damage at DNA replication origins within transcribed long genes of mammalian cells”, Nucleic Acids Research 2019, Vol. 47/1, Oxford University Press, England, https://doi.org/10.1093/nar/gky1152 

Bahia, S. et al. (2018), “Oxidative and nitrative stress-related changes in human lens epithelial cells following exposure to X-rays”, International journal of radiation biology, Vol. 94/4, England, https://doi.org/10.1080/09553002.2018.1439194 

Breton J, Sichel F, Bainchini F, Prevost V. (2003). Measurement of 8-Hydroxy-2′-Deoxyguanosine by a Commercially Available ELISA Test: Comparison with HPLC/Electrochemical Detection in Calf Thymus DNA and Determination in Human Serum. Anal Lett 36:123-134.

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Chepelev N, Kennedy D, Gagne R, White T, Long A, Yauk C, White P. (2015). HPLC Measurement of the DNA Oxidation Biomarker, 8-oxo-7,8-dihydro-2'-deoxyguanosine, in Cultured Cells and Animal Tissues. Journal of Visualized Experiments 102:e52697.

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Lee, J. et al. (2004), “Reactive oxygen species, aging, and antioxidative nutraceuticals”, Comprehensive reviews in food science and food safety, Vol. 3/1, Blackwell Publishing Ltd, Oxford, https://doi.org/10.1111/j.1541-4337.2004.tb00058.x 

Mangal D, Vudathala D, Park J, Lee S, Penning T, Blair I. (2009). Analysis of 7,8-Dihydro-8-oxo-2′-deoxyguanosine in Cellular DNA during Oxidative Stress. Chem Res Toxicol 22:788-797.

Markkanen, E. (2017), “Not breathing is not an option: How to deal with oxidative DNA damage”, DNA repair, Vol. 59, Elsevier B.V., Netherlands, https://doi.org/10.1016/j.dnarep.2017.09.007 

Mesa, R. and S. Bassnett (2013), “UV-B induced DNA damage and repair in the mouse lens”, Investigative ophthalmology & visual science, Vol. 54/10, the Association for Research in Vision and Ophthalmology, United States, https://doi.org/10.1167/iovs.13-12644 

Nilsson R. and Liu N. (2020), “Nuclear DNA damages generated by reactive oxygen molecules (ROS) under oxidative stress and their relevance to human cancers, including ionizing radiation-induced neoplasia part I: Physical, chemical and molecular biology aspects”, Radiation Medicine and Protection, Vol. 1/3(3), https://doi.org/10.1016/j.radmp.2020.09.002 

Pendergrass, W. et al. (2010), “X-ray induced cataract is preceded by LEC loss, and coincident with accumulation of cortical DNA, and ROS; similarities with age-related cataracts”, Molecular vision, Vol. 16, Molecular Vision, United States, pp. 1496-1513 

Platel A, Nesslany F, Gervais V, Claude N, Marzin D. (2011). Study of oxidative DNA damage in TK6 human lymphoblastoid cells by use of the thymidine kinase gene-mutation assay and the in vitro modified comet assay: Determination of No-Observed-Genotoxic-Effect-Levels. Mutat Res 726:151-159.

Poetsch, Anna R. (2020a), “The genomics of oxidative DNA damage, repair, and resulting mutagenesis”, Computational and structural biotechnology journal 2020, Vol. 18, Elsevier B.V., Netherlands https://doi.org/10.1016/j.csbj.2019.12.013 

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Smith C, O'Donovan M, Martin E. (2006). hOGG1 recognizes oxidative damage using the comet assay with greater specificity than FPG or ENDOIII. Mutagenesis 21:185-190.

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Tangvarasittichai, O and S. Tangvarasittichai (2018), “Oxidative stress, ocular disease, and diabetes retinopathy”, Current Pharmaceutical Design, Vol. 24/40, Bentham Science Publishers, https://doi.org/10.2174/1381612825666190115121531 

Turner, N. D. et al. (2002), “Opportunities for nutritional amelioration of radiation-induced cellular damage”, Nutrition, Vol. 18/10, Elsevier Inc, New York, https://doi.org/10.1016/S0899-9007(02)00945-0 

Whitaker A, Schaich M, Smith MS, Flynn T, Freudenthal B. (2017). Base excision repair of oxidative DNA damage: from mechanism to disease. Front Biosci 22:1493-1522.

Wu, J. (2018), “Nucleotide-resolution genome-wide mapping of oxidative DNA damage by click-code-seq”, Journal of the American Chemical Society 2018, American Chemical Society, United States https://doi-org.proxy.bib.uottawa.ca/10.1021/jacs.8b03715 

Xu, X. et al. (2008). “Fluorescence recovery assay for the detection of protein-DNA binding”, Analytical Chemistry, Vol. 80/14, https://doi.org/10.1021/ac8007016 

Zhao M, Howard E, Guo Z, Parris A, Yang X. (2017). p53 pathway determines the cellular response to alcohol-induced DNA damage in MCF-7 breast cancer cells. PLoS One 12:e0175121.

Event: 1505: Cell cycle, disrupted

Short Name: Cell cycle, disrupted

Key Event Component

AOPs Including This Key Event

Biological Context

Cell term

Organ term

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

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

AOPs Including This Key Event

Stressors

Biological Context

Cell term

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

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.

Raza A, Spiridonidis C, Ucar K, Mayers G, Bankert R, Preisler HD. 1985. Double labeling of S-phase murine cells with bromodeoxyuridine and a second DNA-specific probe. Cancer Research 45:2283-2287.

Event: 1635: Increase, DNA strand breaks

Short Name: Increase, DNA strand breaks

Key Event Component

AOPs Including This Key Event

Stressors

Biological Context

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

Taxonomic applicability: DNA strand breaks are relevant to all species, including vertebrates such as humans, that contain DNA (Cannan & Pederson, 2016).  

Life stage applicability: This key event is not life stage specific as all life stages display strand breaks. However, there is an increase in baseline levels of DNA strand breaks seen in older individuals though it is unknown whether this change due to increased break induction or a greater retention of breaks due to poor repair (White & Vijg, 2016). 

Sex applicability: This key event is not sex specific as both sexes display evidence of strand breaks. In some cell types, such as peripheral blood mononuclear cells, males show higher levels of single strand breaks than females (Garm et al., 2012). 

Evidence for perturbation by a stressor: There are studies demonstrating that increased DNA strand breaks can result from exposure to multiple stressor types including ionizing & non-ionizing radiation, chemical agents, and oxidizing agents (EPRI, 2014; Hamada, 2014; Cencer et al., 2018; Cannan & Pederson, 2016; Yang et al., 1998).  

Key Event Description

DNA strand breaks are a type of damage resulting from the hydrolysis of phosphodiester groups in the backbone of DNA molecules (Gates, 2009) and can occur on a single strand (single strand breaks; SSBs) or both strands (double strand breaks; DSBs). SSBs arise when the sugar phosphate backbones connecting adjacent nucleotides in DNA are simultaneously hydrolyzed such that the hydrogen bonds between complementary bases are not able to hold the two strands together. DSBs are generated when both strands are simultaneously broken at sites that are sufficiently close to one another that base-pairing and chromatin structure are insufficient to keep the two DNA ends juxtaposed. As a consequence, the two DNA ends generated by a DSB can physically dissociate from one another, becoming difficult to repair and increasing the chance of inappropriate recombination with other sites in the genome (Jackson, 2002). SSB can turn into DSB if the replication fork stalls at the lesion leading to fork collapse. Strand breaks are intermediates in various biological events, including DNA repair (e.g., excision repair), as well as other normal cellular processes where DSBs act as genetic shufflers to generate genetic diversity for V(D)J recombination in lymphoid cells, and chromatin remodeling in both somatic cells and germ cells, and meiotic recombination in gametes. 

Strand breaks are intermediates in various biological events, including DNA repair (e.g., excision repair), V(D)J recombination in developing lymphoid cells and chromatin remodeling in both somatic cells and germ cells. The spectrum of damage can be complex, particularily if the stressor is from large amounts of deposited energy which can result in complex lesions and clustered damage defined as two or more oxidized bases, abasic sites or strand breaks on opposing DNA strands within a few helical turns. These lesions are more difficult to repair and have been studied in many types of models (Barbieri et al., 2019 and Asaithamby et al., 2011). DSBs and complex lesions are of particular concern, as they are considered the most lethal and deleterious type of DNA lesion. If misrepaired or left unrepaired, DSBs may drive the cell towards genomic instability, apoptosis or tumorigenesis (Beir, 1999). 

How it is Measured or Detected

Please refer to the table below for details regarding these and other methodologies for detecting DNA DSBs. 

Method of Measurement	References	Description	OECD Approved Method?
Comet Assay (Single Cell Gel Eletrophoresis - Alkaline)	Collins, 2004; Olive and Banath, 2006; Platel et al., 2011; Nikolova et al., 2017	To detect SSBs or DSBs, single cells are encapsulated in agarose on a slide, lysed, and subjected to gel electrophoresis at an alkaline pH (pH >13); DNA fragments are forced to move, forming a "comet"-like appearance	Yes
γ-H2AX Foci Quantification - Flow Cytometry	Rothkamm and Horn, 2009; Bryce et al., 2016	Measurement of γ-H2AX immunostaining in cells by flow cytometry, normalized to total levels of H2AX	No
γ-H2AX Foci Quantification - Western Blot	Burma et al., 2001; Revet et al., 2011	Measurement of γ-H2AX immunostaining in cells by Western blotting, normalized to total levels of H2AX	No
γ-H2AX Foci Quantification - Microscopy	Redon et al., 2010; Mah et al., 2010; Garcia-Canton et al., 2013	Quantification of γ-H2AX immunostaining by counting γ-H2AX foci visualized with a microscope	No
γ-H2AX Foci Quantification - ELISA	Ji et al., 2017	Measurement of γ-H2AX in cells by ELISA, normalized to total levels of H2AX	No
Pulsed Field Gel Electrophoresis (PFGE)	Ager et al., 1990; Gardiner et al., 1985; Herschleb et al., 2007; Kawashima et al., 2017	To detect DSBs, cells are embedded and lysed in agarose, and the released DNA undergoes gel electrophoresis in which the direction of the voltage is periodically alternated; Large DNA fragments are thus able to be separated by size	No
The TUNEL (Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling) Assay	Loo, 2011	To detect strand breaks, dUTPs added to the 3’OH end of a strand break by the DNA polymerase terminal deoxynucleotidyl transferase (TdT) are tagged with a fluorescent dye or a reporter enzyme to allow visualization	No
In Vitro DNA Cleavage Assays using Topoisomerase	Nitiss, 2012	Cleavage of DNA can be achieved using purified topoisomerase; DNA strand breaks can then be separated and quantified using gel electrophoresis	No
PCR assay	Figueroa‑González & Pérez‑Plasencia, 2017	Assay of strand breaks through the observation of DNA amplification prevention. Breaks block Taq polymerase, reducing the number of DNA templates, preventing amplification	No
Sucrose density gradient centrifuge	Raschke et al. 2009	Division of DNA pieces by density, increased fractionation leads to lower density pieces, with the use of a sucrose cushion	No
Alkaline Elution Assay	Kohn, 1991	Cells lysed with detergent-solution, filtered through membrane to remove all but intact DNA	No
Unwinding Assay	Nacci et al. 1992	DNA is stored in alkaline solutions with DNA-specific dye and allowed to unwind following removal from tissue, increased strand damage associated with increased unwinding	Yes
STRIDE assay	Zilio and Ulrich, 2021	STRIDE (SensiTive Recognition of Individual DNA Ends) combines in situ nick translation with the proximity ligation assay (PLA) to detect single-strand breaks (sSTRIDE) or double-strand breaks (dSTRIDE). In this process, lesions labeled through nick translation with biotinylated nucleotides are identified by a PLA signal, which arises from the interaction of two anti-biotin antibodies from different species.	No
sBLISS	Bouwmann et al. 2020	sBLISS (in-suspension breaks labeling in situ and sequencing)  labels double-strand breaks (DSBs) in cells immobilized on glass coverslips, using double-stranded oligonucleotide adaptors that facilitate selective linear amplification through T7-mediated in vitro transcription (IVT), followed by next-generation sequencing (NGS) library preparation	No

References

Ager, D. D., et al. (1990). Measurement of radiation-induced DNA double-strand breaks by pulsed-field gel electrophoresis. Radiation research, 122/(2), 181–187. 

Anderson, D. & Laubenthal J. (2013), “Analysis of DNA Damage via Single-Cell Electrophoresis. In: Makovets S, editor. DNA Electrophoresis. Totowa.”, NJ: Humana Press. p 209-218. 

Asaithamby, A., B. Hu and D.J. Chen. (2011) “Unrepaired clustered DNA lesions induce chromosome breakage in human cells.” Proc Natl Acad Sci U S A 108(20): 8293-8298 . 

Barbieri, S., G. Babini, J. Morini et a l (2019). . Predicting DNA damage foci and their experimental readout with 2D microscopy: a unified approach applied to photon and neutron exposures. Scientific Reports 9(1): 14019 

Bouwman, B. et al. (2020), “Genome-wide detection of DNA double-strand breaks by in-suspension BLISS”, Nature protocols,.15/12, Springer Nature, London, https://doi.org/10.1038/s41596-020-0397-2  

Bryce, S. et al. (2016), “Genotoxic mode of action predictions from a multiplexed flow cytometric assay and a machine learning approach.”, Environ Mol Mutagen. 57:171-189. Doi: 10.1002/em.21996. 

Burma, S. et al. (2001), “ATM phosphorylates histone H2AX in response to DNA double-strand breaks.”, J Biol Chem, 276(45): 42462-42467. doi:10.1074/jbc.C100466200 

Cannan, W.J. and D.S. Pederson (2016), "Mechanisms and Consequences of Double-Strand DNA Break Formation in Chromatin.", Journal of Cellular Physiology, Vol.231(/1), Wiley, New York, https://doi.org/10.1002/jcp.25048.  

Cencer, C. et al. (2018), “PARP-1/PAR Activity in Cultured Human Lens Epithelial Cells Exposed to Two Levels of UVB Light”, Photochemistry and Photobiology, Vol.(94/1), Wiley-Blackwell, Hoboken, https://doi.org/10.1111/php.12814.  

Charlton, E. D. et al. (1989), “Calculation of Initial Yields of Single and Double Stranded Breaks in Cell Nuclei from Electrons, Protons, and Alpha Particles.”, Int. J. Radiat. Biol. 56(1): 1-19. doi: 10.1080/09553008914551141. 

Collins, R. A. (2004), “The Comet Assay for DNA Damage and Repair. Molecular Biotechnology.”, Mol Biotechnol. 26(3): 249-61. doi:10.1385/MB:26:3:249 

EPRI (2014), Epidemiology and mechanistic effects of radiation on the lens of the eye: Review and scientific appraisal of the literature, EPRI, California. 

Figueroa‑González, G. and C. Pérez‑Plasencia. (2017), “Strategies for the evaluation of DNA damage and repair mechanisms in cancer”, Oncology Letters, Vol.133(/6), Spandidos Publications, Athens, https://doi.org/10.3892/ol.2017.6002. 

Garcia-Canton, C. et al. (2013), “Assessment of the in vitro p-H2AX assay by High Content Screening asa novel genotoxicity test.”, Mutat Res. 757:158-166. Doi: 10.1016/j.mrgentox.2013.08.002 

Gardiner, K. et al. (1986), “Fractionation of Large Mammalian DNA Restriction Fragments Using Vertical Pulsed-Field Gradient Gel Electrophoresis.”, Somatic Cell and Molecular Genetics. 12(2): 185-95.Doi: 10.1007/bf01560665. 

Garm, C. et al. (2012), “Age and gender effects on DNA strand break repair in peripheral blood mononuclear cells”, Aging Cell, Vol.12/1, Blackwell Publishing Ltd, Oxford, https://doi.org/10.1111/acel.12019. 

Hamada, N. (2014), “What are the intracellular targets and intratissue target cells for radiation effects?”, Radiation research, Vol. 181/1, The Radiation Research Society, Indianapolis, https://doi.org/10.1667/RR13505.1. 

Herschleb, J. et al. (2007), “Pulsed-field gel electrophoresis.”, Nat Protoc. 2(3): 677-684. doi:10.1038/nprot.2007.94 

Iliakis, G. et al. (2015), “Alternative End-Joining Repair Pathways Are the Ultimate Backup for Abrogated Classical Non-Homologous End-Joining and Homologous Recombination Repair: Implications for the Formation of Chromosome Translocations.”, Mutation Research/Genetic Toxicology and Environmental Mutagenesis. 2(3): 677-84. doi: 10.1038/nprot.2007.94 

Jackson, S. (2002). “Sensing and repairing DNA double-strand breaks.”, Carcinogenesis. 23:687-696. Doi:10.1093/carcin/23.5.687. 

Ji, J. et al. (2017), “Phosphorylated fraction of H2AX as a measurement for DNA damage in cancer cells and potential applications of a novel assay.”, PLoS One. 12(2): e0171582. doi:10.1371/journal.pone.0171582 

Kawashima, Y.(2017), “Detection of DNA double-strand breaks by pulsed-field gel electrophoresis.”, Genes Cells 22:84-93. Doi: 10.1111/gtc.12457. 

Khoury, L. et al. (2013), “Validation of high-throughput genotoxicity assay screening using cH2AX in-cell Western assay on HepG2 cells.”, Environ Mol Mutagen, 54:737-746. Doi: 10.1002/em.21817. 

Khoury, L. et al. (2016), “Evaluation of four human cell lines with distinct biotransformation properties for genotoxic screening.”, Mutagenesis, 31:83-96. Doi: 10.1093/mutage/gev058. 

Kohn, K.W. (1991), “Principles and practice of DNA filter elution”, Pharmacology & Therapeutics, Vol.49(/1), Elsevier, Amsterdam, https://doi.org/10.1016/0163-7258(91)90022-E. 

Loo, DT. (2011), “In Situ Detection of Apoptosis by the TUNEL Assay: An Overview of Techniques. In: Didenko V, editor. DNA Damage Detection In Situ, Ex Vivo, and In Vivo. Totowa.”, NJ: Humana Press. p 3-13.doi: 10.1007/978-1-60327-409-8_1. 

Mah, L. J. et al. (2010), “Quantification of gammaH2AX foci in response to ionising radiation.”, J Vis Exp(38). doi:10.3791/1957. 

Nacci, D. et al. (1992), “Application of the DNA alkaline unwinding assay to detect DNA strand breaks in marine bivalves”, Marine Environmental Research, Vol.33(/2), Elsevier BV, Amsterdam, https://doi.org/10.1016/0141-1136(92)90134-8. 

Nikolova, T., F. et al. (2017), “Genotoxicity testing: Comparison of the γH2AX focus assay with the alkaline and neutral comet assays.”, Mutat Res 822:10-18. Doi: 10.1016/j.mrgentox.2017.07.004. 

Nitiss, J. L. et al. (2012), “Topoisomerase assays. ”, Curr Protoc Pharmacol. Chapter 3: Unit 3 3. 

OECD. (2014). Test No. 489: “In vivo mammalian alkaline comet assay.” OECD Guideline for the Testing of Chemicals, Section 4 . 

Olive, P. L., & Banáth, J. P. (2006), “The comet assay: a method to measure DNA damage in individual cells.”, Nature Protocols. 1(1): 23-29. doi:10.1038/nprot.2006.5. 

Platel A. et al. (2011), “Study of oxidative DNA damage in TK6 human lymphoblastoid cells by use of the thymidine kinase gene-mutation assay and the in vitro modified comet assay: Determination of No-Observed-Genotoxic-Effect-Levels.”, Mutat Res 726:151-159. Doi: 10.1016/j.mrgentox.2011.09.003. 

Raschke, S., J. Guan and G. Iliakis. (2009), “Application of alkaline sucrose gradient centrifugation in the analysis of DNA replication after DNA damage”, Methods in Molecular Biology, Vol.521, Humana Press, Totowa, https://doi.org/10.1007/978-1-60327-815-7_18. 

Redon, C. et al. (2010), “The use of gamma-H2AX as a biodosimeter for total-body radiation exposure in non-human primates.”, PLoS One. 5(11): e15544. doi:10.1371/journal.pone.0015544 

Revet, I. et al. (2011), “Functional relevance of the histone γH2Ax in the response to DNA damaging agents.” Proc Natl Acad Sci USA.108:8663-8667. Doi: 10.1073/pnas.1105866108 

Rogakou, E.P. et al. (1998), “DNA Double-stranded Breaks Induce Histone H2AX Phosphorylation on Serine 139.” , J Biol Chem, 273:5858-5868. Doi: 10.1074/jbc.273.10.5858 

Rothkamm, K. & Horn, S. (2009), “γ-H2AX as protein biomarker for radiation exposure.”, Ann Ist Super Sanità, 45(3): 265-71. 

White, R.R. and J. Vijg. (2016), “Do DNA Double-Strand Breaks Drive Aging?”, Molecular Cell, Vol.63, Elsevier, Amsterdam, http://doi.org/10.1016/j.molcel.2016.08.004. 

Yang, Y. et al. (1998), “The effect of catalase amplification on immortal lens epithelial cell lines”, Experimental Eye Research, Vol.67(/6), Academic Press Inc, Cambridge, https://doi.org/10.1006/exer.1998.0560.  

Zilio, N. and H. D. Ulrich (2021), “Exploring the SSBreakome: genome-wide mapping of DNA single-strand breaks by next-generation sequencing”, The FEBS journal, 288(13), Wiley, Hoboken, https://doi.org/10.1111/febs.15568 

 

List of Adverse Outcomes in this AOP

Event: 1521: Decrease, Growth

Short Name: Decrease, Growth

Key Event Component

AOPs Including This Key Event

Stressors

Biological Context

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

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