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Key Event Title
Increase, Oxidative damage to DNA
|Level of Biological Organization|
Key Event Components
|regulation of response to reactive oxygen species||reactive oxygen species||occurrence|
Key Event Overview
AOPs Including This Key Event
|AOP Name||Role of event in AOP||Point of Contact||Author Status||OECD Status|
|Oxidative DNA damage, chromosomal aberrations and mutations||MolecularInitiatingEvent||Brendan Ferreri-Hanberry (send email)||Open for comment. Do not cite||EAGMST Approved|
|Energy deposition leading to population decline via DNA oxidation and follicular atresia||KeyEvent||Allie Always (send email)||Under development: Not open for comment. Do not cite|
|Energy deposition leading to population decline via DNA oxidation and oocyte apoptosis||KeyEvent||Agnes Aggy (send email)||Under development: Not open for comment. Do not cite|
|All life stages|
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). 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 from specific compounds, 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
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
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.
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.