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Event: 1634

Key Event Title

The KE title should describe a discrete biological change that can be measured. It should generally define the biological object or process being measured and whether it is increased, decreased, or otherwise definably altered relative to a control state. For example “enzyme activity, decreased”, “hormone concentration, increased”, or “growth rate, decreased”, where the specific enzyme or hormone being measured is defined. More help

Increase, Oxidative damage to DNA

Short name
The KE short name should be a reasonable abbreviation of the KE title and is used in labelling this object throughout the AOP-Wiki. The short name should be less than 80 characters in length. More help
Increase, Oxidative DNA damage

Biological Context

Structured terms, selected from a drop-down menu, are used to identify the level of biological organization for each KE. Note, KEs should be defined within a particular level of biological organization. Only KERs should be used to transition from one level of organization to another. Selection of the level of biological organization defines which structured terms will be available to select when defining the Event Components (below). More help
Level of Biological Organization
Molecular

Cell term

Further information on Event Components and Biological Context may be viewed on the attached pdf.The biological context describes the location/biological environment in which the event takes place.  For molecular/cellular events this would include the cellular context (if known), organ context, and species/life stage/sex for which the event is relevant. For tissue/organ events cellular context is not applicable.  For individual/population events, the organ context is not applicable. More help
Cell term
eukaryotic cell

Organ term

Further information on Event Components and Biological Context may be viewed on the attached pdf.The biological context describes the location/biological environment in which the event takes place.  For molecular/cellular events this would include the cellular context (if known), organ context, and species/life stage/sex for which the event is relevant. For tissue/organ events cellular context is not applicable.  For individual/population events, the organ context is not applicable. More help
Organ term
organ

Key Event Components

Further information on Event Components and Biological Context may be viewed on the attached pdf.Because one of the aims of the AOP-KB is to facilitate de facto construction of AOP networks through the use of shared KE and KER elements, authors are also asked to define their KEs using a set of structured ontology terms (Event Components). In the absence of structured terms, the same KE can readily be defined using a number of synonymous titles (read by a computer as character strings). In order to make these synonymous KEs more machine-readable, KEs should also be defined by one or more “event components” consisting of a biological process, object, and action with each term originating from one of 22 biological ontologies (Ives, et al., 2017; See List). Biological process describes dynamics of the underlying biological system (e.g., receptor signalling). The biological object is the subject of the perturbation (e.g., a specific biological receptor that is activated or inhibited). Action represents the direction of perturbation of this system (generally increased or decreased; e.g., ‘decreased’ in the case of a receptor that is inhibited to indicate a decrease in the signalling by that receptor).Note that when editing Event Components, clicking an existing Event Component from the Suggestions menu will autopopulate these fields, along with their source ID and description. To clear any fields before submitting the event component, use the 'Clear process,' 'Clear object,' or 'Clear action' buttons. If a desired term does not exist, a new term request may be made via Term Requests. Event components may not be edited; to edit an event component, remove the existing event component and create a new one using the terms that you wish to add. More help
Process Object Action
regulation of response to reactive oxygen species reactive oxygen species occurrence

Key Event Overview

AOPs Including This Key Event

All of the AOPs that are linked to this KE will automatically be listed in this subsection. This table can be particularly useful for derivation of AOP networks including the KE. Clicking on the name of the AOP will bring you to the individual page for that AOP. More help
AOP Name Role of event in AOP Point of Contact Author Status OECD Status
Oxidative DNA damage, chromosomal aberrations and mutations MolecularInitiatingEvent Carole Yauk (send email) Open for comment. Do not cite EAGMST Under Review

Stressors

This is a structured field used to identify specific agents (generally chemicals) that can trigger the KE. Stressors identified in this field will be linked to the KE in a machine-readable manner, such that, for example, a stressor search would identify this as an event the stressor can trigger. NOTE: intermediate or downstream KEs in one AOP may function as MIEs in other AOPs, meaning that stressor information may be added to the KE description, even if it is a downstream KE in the pathway currently under development.Information concerning the stressors that may trigger an MIE can be defined using a combination of structured and unstructured (free-text) fields. For example, structured fields may be used to indicate specific chemicals for which there is evidence of an interaction relevant to this MIE. By linking the KE description to a structured chemical name, it will be increasingly possible to link the MIE to other sources of chemical data and information, enhancing searchability and inter-operability among different data-sources and knowledgebases. The free-text section “Evidence for perturbation of this MIE by stressor” can be used both to identify the supporting evidence for specific stressors triggering the MIE as well as to define broad chemical categories or other properties that classify the stressors able to trigger the MIE for which specific structured terms may not exist. More help

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) can be selected from an ontology. In many cases, individual species identified in these structured fields will be those for which the strongest evidence used in constructing the AOP was available in relation to this KE. More help
Term Scientific Term Evidence Link
human and other cells in culture human and other cells in culture NCBI
yeast Saccharomyces cerevisiae NCBI
mouse Mus musculus NCBI
rat Rattus norvegicus NCBI
bovine Bos taurus NCBI
human Homo sapiens High NCBI

Life Stages

The structured ontology terms for life-stage are more comprehensive than those for taxa, but may still require further description/development and explanation in the free text section. More help
Life stage Evidence
All life stages

Sex Applicability

The authors must select from one of the following: Male, female, mixed, asexual, third gender, hermaphrodite, or unspecific. More help
Term Evidence
Unspecific

Key Event Description

A description of the biological state being observed or measured, the biological compartment in which it is measured, and its general role in the biology should be provided. For example, the biological state being measured could be the activity of an enzyme, the expression of a gene or abundance of an mRNA transcript, the concentration of a hormone or protein, neuronal activity, heart rate, etc. The biological compartment may be a particular cell type, tissue, organ, fluid (e.g., plasma, cerebrospinal fluid), etc. The role in the biology could describe the reaction that an enzyme catalyses and the role of that reaction within a given metabolic pathway; the protein that a gene or mRNA transcript codes for and the function of that protein; the function of a hormone in a given target tissue, physiological function of an organ, etc. Careful attention should be taken to avoid reference to other KEs, KERs or AOPs. Only describe this KE as a single isolated measurable event/state. This will ensure that the KE is modular and can be used by other AOPs, thereby facilitating construction of AOP networks. More help

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). 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. 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, 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.

This KE describes an increase in oxidative lesions in the nuclear DNA above the steady-state level. Oxidative DNA damage can occur in any cell type under oxidative stress.

How It Is Measured or Detected

One of the primary considerations in evaluating AOPs is the relevance and reliability of the methods with which the KEs can be measured. The aim of this section of the KE description is not to provide detailed protocols, but rather to capture, in a sentence or two, per method, the type(s) of measurements that can be employed to evaluate the KE and the relative level of scientific confidence in those measurements. Methods that can be used to detect or measure the biological state represented in the KE should be briefly described and/or cited. These can range from citation of specific validated test guidelines, citation of specific methods published in the peer reviewed literature, or outlines of a general protocol or approach (e.g., a protein may be measured by ELISA).Key considerations regarding scientific confidence in the measurement approach include whether the assay is fit for purpose, whether it provides a direct or indirect measure of the biological state in question, whether it is repeatable and reproducible, and the extent to which it is accepted in the scientific and/or regulatory community. Information can be obtained from the OECD Test Guidelines website and the EURL ECVAM Database Service on Alternative Methods to Animal Experimentation (DB-ALM). ?

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) (Breton et al., 2003; Zhao et al.)
    • 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 8-oxodG. Correlation between 8-oxodG measured by hOGG1-modified comet assay and LC-MS has been reported
  • We note that other types of oxidative lesions can be quantified using the methods described above.

Domain of Applicability

This free text section should be used to elaborate on the scientific basis for the indicated domains of applicability and the WoE calls (if provided). While structured terms may be selected to define the taxonomic, life stage and sex applicability (see structured applicability terms, above) of the KE, the structured terms may not adequately reflect or capture the overall biological applicability domain (particularly with regard to taxa). Likewise, the structured terms do not provide an explanation or rationale for the selection. The free-text section on evidence for taxonomic, life stage, and sex applicability can be used to elaborate on why the specific structured terms were selected, and provide supporting references and background information.  More help

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.

Evidence for Perturbation by Stressor

Overview for Molecular Initiating Event

When a specific MIE can be defined (i.e., the molecular target and nature of interaction is known), in addition to describing the biological state associated with the MIE, how it can be measured, and its taxonomic, life stage, and sex applicability, it is useful to list stressors known to trigger the MIE and provide evidence supporting that initiation. This will often be a list of prototypical compounds demonstrated to interact with the target molecule in the manner detailed in the MIE description to initiate a given pathway (e.g., 2,3,7,8-TCDD as a prototypical AhR agonist; 17α-ethynyl estradiol as a prototypical ER agonist). Depending on the information available, this could also refer to chemical categories (i.e., groups of chemicals with defined structural features known to trigger the MIE). Known stressors should be included in the MIE description, but it is not expected to include a comprehensive list. Rather initially, stressors identified will be exemplary and the stressor list will be expanded over time. For more information on MIE, please see pages 32-33 in the User Handbook.

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 hours (Platel et al., 2011).

References

List of the literature that was cited for this KE description. Ideally, the list of references, should conform, to the extent possible, with the OECD Style Guide (https://www.oecd.org/about/publishing/OECD-Style-Guide-Third-Edition.pdf) (OECD, 2015). More help

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.

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.

Jovanovic S, Simic M. (1986). One-electron redox potential of purines and pyrimidines. J Phys Chem 90:974-978.

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.

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.

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.

Swenberg J, Lu K, Moeller B, Gao L, Upton P, Nakamura J, Starr T. (2011). Endogenous versus Exogenous DNA Adducts: Their Role in Carcinogenesis, Epidemiology, and Risk Assessment. Toxicol Sci 120:S130-S145.

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.

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.