Aop: 296

AOP Title


Oxidative DNA damage leading to chromosomal aberrations and mutations

Short name:


Oxidative DNA damage, chromosomal aberrations and mutations

Graphical Representation


Click to download graphical representation template




Eunnara Cho1,2, Ashley Allemang3, Marc Audebert4, Vinita Chauhan5, Stephen Dertinger6, Giel Hendriks7, Mirjam Luijten8, Francesco Marchetti1,2, Sheroy Minocherhomji9, Stefan Pfuhler3, Daniel J. Roberts10, Kristina Trenz11, Carole L. Yauk1,2, *


1 Environmental Health Science and Research Bureau, Health Canada, Ottawa, ON, Canada

2 Department of Biology, Carleton University, Ottawa, ON, Canada

3 The Procter & Gamble Company, Mason, OH, United States

4 Toxalim, INRAE, Toulouse, France

Consumer and Clinical Radiation Protection Bureau, Health Canada, Ottawa, ON, Canada

Litron Laboratories, Rochester, NY, United States

7 Toxys, Leiden, The Netherlands

Centre for Health Protection, National Institute for Public Health and the Environment (RIVM), Bilthoven, the Netherlands

Amgen, Thousand Oaks, CA, United States

10 Charles River Laboratories, Skokie, IL, United States

11 Boehringer-Ingelheim, Ingelheim, Germany

*Corresponding author: Carole Yauk (carole.yauk@canada.ca)

Point of Contact


Carole Yauk   (email point of contact)



  • Carole Yauk



Author status OECD status OECD project SAAOP status
Open for comment. Do not cite

This AOP was last modified on September 22, 2020 15:38


Revision dates for related pages

Page Revision Date/Time
Increase, Oxidative damage to DNA June 04, 2019 05:16
N/A, Inadequate DNA repair October 30, 2019 10:07
Increase, DNA strand breaks May 29, 2020 14:45
Increase, Mutations October 25, 2019 13:12
Increase, Chromosomal aberrations October 30, 2019 10:11
Increase, Oxidative DNA damage leads to N/A, Inadequate DNA repair June 06, 2019 18:41
Increase, Oxidative DNA damage leads to Increase, DNA strand breaks June 01, 2019 18:10
N/A, Inadequate DNA repair leads to Increase, DNA strand breaks July 22, 2019 13:02
Increase, Oxidative DNA damage leads to Increase, Mutations September 11, 2019 15:23
Increase, DNA strand breaks leads to N/A, Inadequate DNA repair October 22, 2019 12:49
Increase, DNA strand breaks leads to Increase, Mutations October 29, 2019 14:41
N/A, Inadequate DNA repair leads to Increase, Mutations June 03, 2020 23:25
N/A, Inadequate DNA repair leads to Increase, Chromosomal aberrations October 29, 2019 08:28
Increase, DNA strand breaks leads to Increase, Chromosomal aberrations October 29, 2019 15:01
Hydrogen peroxide May 19, 2019 17:21
Potassium bromate May 19, 2019 17:21
Ionizing Radiation May 07, 2019 12:12
Cadmium chloride May 19, 2019 17:23
tert-Butyl hydroperoxide May 19, 2019 17:24
Reactive oxygen species August 15, 2017 10:43
Hydroquinone November 08, 2019 13:06
4-Nitroquinoline 1-oxide November 08, 2019 13:07



This adverse outcome pathway (AOP) network describes the linkage between oxidative DNA damage and irreversible genomic damage (chromosomal aberrations and mutations). Both endpoints are of regulatory interest because irreversible genomic damage is associated with various adverse health effects such as cancer and heritable disorders.

Mutagens are genotoxic substances that alter the DNA sequence and this includes single base substitutions, deletion or addition of a single base or multiple bases of DNA, and complex multi-site mutations. Mutations can occur in coding and non-coding regions of the genome and can be functional or silent. The site and type of mutation will determine its consequence. Clastogens are genotoxic substances that cause DNA single- and double-strand breaks that can result in deletion, addition, or rearrangement of sections in the chromosomes. As with mutagens, the type and extent of chromosome modification(s) determine cellular consequences.

The molecular initiating event (MIE) of this AOP is increase in oxidative DNA damage, indicated by increases in oxidative DNA lesions. DNA in any cell type is susceptible to oxidative damage due to endogenous (e.g., aerobic respiration) and exogenous (i.e., exposure to oxidants) oxidative insults. Although this is the MIE for this AOP network, we note that there are numerous upstream key events (KE) that can also lead to DNA oxidation. Thus, we expect this AOP to be expanded upstream, and to be incorporated into a variety of AOP networks. Generally, cells are able to tolerate and readily repair oxidative DNA lesions by basal repair mechanisms. However, excessive damage can override the basal repair capacity and lead to inadequate repair of oxidative damage (KE1). Mutations (AO1) can arise from incorrect repair following oxidative damage (KE1), where incorrect bases are inserted opposite lesions during DNA replication. Insufficiently or incompletely repaired oxidative DNA lesions can also lead to DNA strand breaks (KE2) that, if insufficiently repaired (KE1), may result in chromosome aberrations (AO2) and/or mutations (AO1) following DNA replication.

Support for this AOP is strong based on extensive understanding of the mechanisms involved in this pathway, evidence of essentiality of certain KE (i.e., studies using reactive oxidative species scavengers and modulating DNA repair enzymes), and a robust set of studies providing empirical support for many of the KERs.

We anticipate that this AOP will be of widespread use to the regulatory community as oxidative DNA damage is considered an important contributor to the adverse health effects of many environmental toxicants. Importantly, the AOP points to critical research gaps required to establish the quantitative associations and modulating factors that connect KEs across the AOP, and highlights the utility of novel test methods in understanding and evaluating the implications of oxidative DNA damage.

Background (optional)


This AOP network describes oxidative damage to DNA (MIE) leading to mutations (AO1) and chromosomal aberrations (AO2). The AOP summarizes the evidence supporting how increases in oxidative DNA lesions can overwhelm DNA repair mechanisms, causing an accumulation of unrepaired lesions and/or repair intermediates. Failure to resolve oxidative DNA damage can lead to permanent alterations to the genome. Increases in reactive oxygen and nitrogen species (RONS) that can lead to oxidative DNA lesions is a broad characteristic of many xenobiotics and indeed, is noted as one of the 'key characteristics of carcinogens' (Smith et al., 2016). Moreover, oxidative stress is often suspected to be the cause of DNA damage by substances whose mechanism of genotoxicity is uncertain [e.g., glyphosate (Kier and Kirkland, 2013; Benbrook, 2019), monosodium glutamate (Ataseven et al., 2016)]. Thus, this AOP network will serve as a key tool in mechanism-based genotoxic hazard identification and assessment.

Oxidative stress describes an imbalance of oxidants and antioxidants in the cell. Excess oxidants can occur following exposure to agents that: (a) generate free radicals and other RONS, (b) deplete cellular antioxidants, and/or (c) have oxidizing properties. The effects of oxidative stress in the cell are broad; all biomolecules are susceptible to damage by oxidizing agents. Oxidative stress and associated damage to cellular components have been implicated in various diseases, including neurodegenerative diseases, cardiovascular diseases, diabetes, and different cancers (Liguori et al., 2018).

Free radicals and other RONS are continuously generated as by-products of endogenous redox reactions (e.g., oxidative phosphorylation in the mitochondria, NADPH oxidation to NADP+ by NADPH oxidase) at steady state. The steady state concentration of oxidants is essential for cellular functions (e.g., as secondary signalling molecules) and is tightly regulated by endogenous antioxidants such as glutathione, superoxide dismutase, and catalase. However, exogenous sources such as ionizing radiation, ultraviolet (UV) radiation, and certain compounds can directly or indirectly generate reactive species, causing oxidative stress. Oxidizing compounds can also directly cause oxidative damage to cellular components (Liguori et al., 2018). The nitrogenous bases of the DNA are susceptible to oxidation by both endogenous and exogenous oxidants (Berquist and Wilson III, 2012).

Oxidizing agents cause a wide range of oxidative DNA lesions. In addition to strand breaks due to direct RONS attack on the phosphate backbone, the nitrogenous bases can be modified in various ways by free radicals and other reactive species. If these lesions are left unrepaired or the attempt at repair fails, mutations and strand breaks can occur, permanently altering the DNA sequence. All nitrogenous bases are susceptible to oxidative damage, however, to different extents. A variety of DNA lesions caused by RONS are described within this AOP (Cooke et al., 2003). Notably, guanine is most readily damaged by RONS and other oxidants due to its low reduction potential.

Indeed, 8-oxoG is the most abundant oxidative DNA lesion and has been extensively studied; within this AOP network, we mainly focus on 8-oxo-dG as oxidative DNA damage representing the MIE, for practicality. The fate of guanine lesions has been most extensively researched and well understood (Roszkowski et al., 2011; Whitaker et al., 2017; Cadet et al., 2017; Markkanen, 2017). Also, 8-oxodG is an accepted biomarker of oxidative stress and oxidative damage to DNA both in vitro and in vivo (Cooke et al., 2008; Roszkowski et al., 2011; P. Li et al., 2014; Guo et al., 2017). Several different detection methods for 8-oxo-dG are commercially available and, thus, are easy to access (e.g., immunodetection, comet assay). We note that 8-oxo-dG is not a terminal product of oxidative damage; 8-oxo-dG can be further oxidized to additional mutagenic lesions such as spiroiminodihydantoin and guanidinohydantoin (Jena and Mishra, 2012). However, as with many other oxidative lesions on pyrimidines and adenine, these guanine lesions are estimated to be small fractions compared to 8-oxo-dG (Yu et al., 2005; Cooke et al., 2008). 

The pathway to mutations (AO1) from oxidative DNA lesions can either proceed (a) directly to mutation through replication of unrepaired oxidized DNA bases (insertion of an incorrect nucleotide by a replicative or translesion polymerase), or (b) indirectly through the creation of strand breaks that can be misrepaired to introduce mutations (Taggart et al., 2014; Rodgers and McVey, 2016). Strand breaks can arise during attempted repair of oxidative DNA lesions. Oxidative base damage is predominantly repaired by base excision repair (BER), and by nucleotide excision repair (NER) to a lesser extent (Whitaker et al., 2017). In the excision repair pathways, single strand breaks (SSB) are transiently introduced as repair intermediates. With increasing oxidative lesions and more lesions in close proximity to each other, the quality and efficiency of repair may be compromised, resulting in persistent unrepaired lesions and repair intermediates. Accumulated repair intermediates such as SSBs, oxidatized bases, and abasic sites can interfere with proximal excision repair and/or impede replication fork elongation, leading to double strand breaks (DSBs), which are more toxic and difficult to repair (Yang et al., 2006; Sedletska et al., 2013; Ensminger et al., 2014). Furthermore, if a SSB is introduced nearby another SSB on the opposite strand prior to or during excision repair, these SSBs may be converted to DSBs. Some studies suggest that multiple DNA lesions within one or two helical turns can increase the rate of DSB formation (Cannan and Pederson, 2017). Insufficiently repaired DSBs (incorrect or lack of rejoining) can permanently alter the DNA sequence (e.g., insertion, deletion, translocations), and cause both mutations (AO1) and structural chromosomal aberrations (AO2) (Rodgers and McVey, 2016). These processes are described in more detail within the AOP.   

Overall, we anticipate that this AOP network will provide a key sub-network that will be relevant to many future AOPs. However, we note that the AOs herein, increased mutations and chromosomal aberrations, are regulatory endpoints of concern in and of themselves. This AOP also provides a template for designing testing strategies for RONS-induced genetic effects. Despite the fact that this is a long-studied area in genetic toxicology, this work highlights notable gaps in the empirical evidence linking adjacent KEs. For example, the extent to which the levels of oxidative DNA damage must increase before DNA repair processes are overwhelmed leading to an AO is currently poorly understood, and may vary based on the test system. Hence, further data are needed to improve our ability to predict whether this pathway is relevant to a chemical’s toxicological effects..

Summary of the AOP


Events: Molecular Initiating Events (MIE)


Key Events (KE)


Adverse Outcomes (AO)


Sequence Type Event ID Title Short name
1 MIE 1634 Increase, Oxidative damage to DNA Increase, Oxidative DNA damage
2 KE 155 N/A, Inadequate DNA repair N/A, Inadequate DNA repair
3 KE 1635 Increase, DNA strand breaks Increase, DNA strand breaks
4 AO 185 Increase, Mutations Increase, Mutations
5 AO 1636 Increase, Chromosomal aberrations Increase, Chromosomal aberrations

Relationships Between Two Key Events
(Including MIEs and AOs)


Title Adjacency Evidence Quantitative Understanding
Increase, Oxidative DNA damage leads to N/A, Inadequate DNA repair adjacent High Low
N/A, Inadequate DNA repair leads to Increase, DNA strand breaks adjacent High Low
Increase, DNA strand breaks leads to N/A, Inadequate DNA repair adjacent High Low
N/A, Inadequate DNA repair leads to Increase, Mutations adjacent High Low
N/A, Inadequate DNA repair leads to Increase, Chromosomal aberrations adjacent High Low
Increase, Oxidative DNA damage leads to Increase, DNA strand breaks non-adjacent Moderate Low
Increase, Oxidative DNA damage leads to Increase, Mutations non-adjacent High Low
Increase, DNA strand breaks leads to Increase, Mutations non-adjacent High Low
Increase, DNA strand breaks leads to Increase, Chromosomal aberrations non-adjacent High Low

Network View





Life Stage Applicability


Life stage Evidence
All life stages

Taxonomic Applicability


Term Scientific Term Evidence Link
human Homo sapiens NCBI
mice Mus sp. NCBI
rat Rattus norvegicus NCBI

Sex Applicability


Sex Evidence

Overall Assessment of the AOP


Biological plausibility:

Overall, the biological plausibility of this AOP network is strong. This network was developed by a team of experts within the Health and Environmental Sciences Institute’s Genetic Toxicology Technical Committee who have decades of experience in research on DNA repair and genetic toxicology.

Oxidative DNA lesions are primarily repaired by base excision repair (BER). BER is a multistep process that involves multiple enzymes including OGG1, which removes oxidized guanine bases and creates a nick 3’ to the damaged base, and APE1, which then removes the resulting abasic site by cleaving 5’ to the damaged base. It is known that BER glycosylases are constitutively expressed and that APE1 is an abundant enzyme (Tell et al., 2009). A spike in BER substrates could lead to an imbalance in the initiating steps of BER, causing an accumulation of abasic sites and other repair intermediates (e.g., SSBs) that can lead to the AOs described herein (Coquerelle et al., 1995; Yang et al., 2006; Nemec et al., 2010). Another biologically plausible mechanism by which oxidative DNA lesions can lead to clastogenic effects is through futile cycles of MUTY-initiated BER, which removes dA opposite 8-oxodG post-replication (Hashimoto et al., 2004). Replicative polymerases may repeatedly insert dA opposite 8-oxodG and cyclical rounds of BER may cause an accumulation of SSBs in S phase. SSBs can turn into DSBs if they occur in close proximity to each other on opposite strands, or cause replication forks to stall and collapse (Iliakis et al., 2004; Fujita et al., 2013; Mehta and Haber, 2014). If DSBs are not repaired in a timely manner, the broken ends may diffuse away from their original position and result in genetic translocation where incorrect ends are joined, or loss of DNA segments, leading to structural aberrations (AO2) (Obe et al., 2010; Durante et al., 2013)

Error-prone repair of DSBs (KE1 Description section 3) can also lead to mutations, providing an alternate pathway to AO1, an increase in mutations (Sedletska et al., 2013). Non-homologous end joining (NHEJ), the error-prone joining of two broken ends, is a faster process compared to homologous recombination (HR), which uses the homologous sequence in the sister chromatid or homologous chromosome as a template to ensure fidelity of the reconstructed strands (Mao et al., 2008a; Mao et al., 2008b). The preference for the use of sister chromatids versus homologous chromosomes in HR depends on the stage of cell cycle in which the DSB occurs. NHEJ may be preferred over HR in many instances, especially under stress, leading to altered sequences at the site of repair (Rodgers and McVey, 2016). The structure of the site of a DSB and end resection can determine the repair pathway and the repair outcome. Typically, breaks with single stranded overhangs are processed by end resection and proceed to HR or error-prone homology-based annealing (i.e, single strand annealing and alternative end-joining). DSBs with blunt ends are more likely to be rejoined by NHEJ (Ceccaldi et al., 2016). The error-prone nature of DSB repair by NHEJ has been extensively studied and widely accepted. Under stress by exogenous or endogenous sources (e.g., xenobiotics), DSBs can also lead to mutagenic salvage DNA repair pathways such as break-induced replication (BIR) and microhomology-mediated break-induced replication (MMBIR) which are linked to mutagenesis, chromosomal rearrangemnts, and genomic instability (Sakofsky et al., 2015; Kramara et al., 2018).

It is established and accepted that unrepaired oxidative DNA lesions, especially 8-oxodG and FapydG, are mutagenic (AO1). During DNA replication, the presence of these unrepaired adducts (KE1: Inadequate repair) on nucleotides leads to incorrect base pairing with incoming nucleosides. This occurs without causing structural disturbance leading to evasion of mismatch repair (Cooke et al., 2003). It is well-understood that both 8-oxodG and FapydG readily base pair with adenine, giving rise to G to T transversions, which are the predominant base substitutions caused by oxidative stress (Cadet and Wagner, 2013; Poetsch et al., 2018)

The underlying biology of the KERs leading to chromosomal aberrations (AO2) is more complex. There are a variety of biologically plausible mechanisms that link inadequate repair of oxidative DNA lesions (KE1; see section 2) of KE Description) to DNA strand breaks (KE2), which, if insufficiently repaired (KE1; see section 3) of KE Description), can cause chromosomal aberrations. Mechanistically, these pathways are well understood (Yang et al., 2006; Nemec et al., 2010; Markkanen, 2017). However, empirical evidence supporting the occurrence of these events is limited in the current literature.

Time- and dose-response concordance:

The WOE supporting the time- and dose-response concordance of KEs leading to the AOs is between moderate and strong.

The MIE (increase in oxidative DNA lesions) can be measured shortly following exposure to stressors. In cell-free systems and cell-based in vitro models, 8-oxodG has been quantified as early as 15 minutes following chemical exposure (Ballmaier and Epe, 2006). Oxidative lesion formation and induction of strand breaks have been demonstrated by time course experiments, where increases in oxidative lesions were detected at earlier time points and at lower concentrations than strand breaks following exposure to various oxidative stress-inducing chemicals [e.g., Ballmaier and Epe (2006), Deferme et al. (2013)]. Mutations (AO1) and chromosomal aberrations (AO2) must be measured after replication and cell division; therefore, these endpoints are only detected at much later time points than the MIE and KEs. Due to the vastly different sensitivities and dynamic ranges of the methodologies detecting the events in these AOPs, it is difficult to demonstrate concordance in concentration-response between the upstream events and AO.

Uncertainties, inconsistencies, and data gaps:

Currently, quantitative understanding of the amount of oxidative lesions that lead to the two AOs of this AOP network, mutations and chromosomal aberrations, is very limited. Very few studies have specifically investigated the extent of chromosomal aberrations induced by different levels of oxidative DNA lesions. Quantitative studies of different oxidative DNA lesions corresponding to mutation frequencies are also very limited. We note that the mutagenicity of 8-oxodG has been most extensively studied, while other oxidative DNA lesions have been studied to a lesser extent.

Quantitative understanding of the relationships comes primarily from studies that modulate levels of oxidative DNA damage through manipulation of repair enzyme activity. In these studies, conflicting observations have been made following modulation of OGG1, the primary repair enzyme for 8-oxodG lesions. While OGG1 protected against DSB formation and cytotoxicity of certain compounds (e.g., methyl mercury, bleomycin, hydrogen peroxide), DSBs were exacerbated by the presence of OGG1 in some other cases (e.g., ionizing radiation, conflicting results for hydrogen peroxide) (Ondovcik et al., 2012; Wang et al., 2018). Available literature indicate that the effect of inadequate repair of oxidative lesions manifests differently for different stressors; it has been suggested that these discrepancies may be due to the difference in proximity of lesions to each other (clustered lesions vs. single lesions) (Yang et al., 2004; Yang et al., 2006).

This AOP network primarily describes oxidative damage to the nuclear DNA (nDNA). However, we must acknowledge that oxidative damage occurs also in the deoxynucleotide triphosphate (dNTP) pool and mitochondrial DNA (mtDNA). Due to mtDNA's location, it is more susceptible to oxidative damage than nDNA. Indeed, crosstalk exists between the nucleus and mitochondria during oxidative stress (Cha et al., 2015; Saki and Prakash, 2017). BER maintains both mitochondrial and nuclear genomic integrity (Cha et al., 2015). Oxidized dNTPs, especially 8-oxodGMP, can be inserted into both genomes during replication and excision repair, resulting in mismatches and impediment of the repair of existing damage, respectively; both scenarios can directly lead to inadequate DNA repair, contributing to the progression of the AOP network (Colussi et al., 2002; Russo et al., 2004; Caglayan et al., 2017). Moving forward, KEs addressing oxidative damage to the dNTP pool and mtDNA are necessary to build a more complete map of oxidative stress-related genotoxicity and to expand the AOP network to other related AOs.  

Domain of Applicability


Theoretically, this AOP is relevant to any cell type in any organism at any life stage. Regardless of the type of cell or organism, DNA is susceptible to oxidative damage and repair mechanisms exist to protect the cell against permanent chromosomal damage. Generally, DNA repair pathways are highly conserved among eukaryotic organisms (Wirth et al., 2016). Base excision repair (BER), the primary repair mechanism for oxidative DNA lesions, and associated glycosylases are highly conserved across eukaryotes (Jacobs and Schar, 2012). DNA strand break repair pathways such as homologous recombination (HR) and non-homologous end joining (NHEJ) are shared among eukaryotes as well. Induction of chromosomal aberrations and mutations following oxidative DNA damage has been studied in both eukaryotic and prokaryotic cells. Notably, the KEs of this AOP have been measured in rodent models (i.e., rat and mouse) and mammalian cells in culture (e.g., TK6 human lymphoblastoid cells, HepG2 human hepatic cells, Chinese hamster ovary cells) (Klungland et al., 1999; Arai et al., 2002; Platel et al., 2009; Platel et al., 2011; Deferme et al., 2013).

The occurrence of oxidative DNA damage and chromosomal aberrations are well-established events in humans. Micronucleus and 8-oxodG have been quantified in various tissues and fluids as part of occupational health and biomonitoring studies. Detection of 8-oxodG is typically used as a measure of oxidiative DNA damage to link exposure and/or diseases to oxidative stress [e.g., urinary 8-oxodG (Hanchi et al., 2017); 8-oxodG in tumour samples (Mazlumoglu et al., 2017)]. Micronuclei (MN) are also regularly quantified as a biomarker of genotoxicant exposure or genotoxic stress in humans. Numerous examples of detecting MN in different human tissues (e.g., lymphocytes, buccal cells, urothelial cells) are available in the current literature (Li et al., 2014; Dong et al., 2019; Alpire et al., 2019). Mutations also have been measured in human samples of diverse cell types (Ojha et al., 2018; Zhu et al., 2019; Liljedahl et al., 2019). As such, observations of the MIE and the two AOs of this AOP have been extensively documented in humans.

Essentiality of the Key Events


A large number of studies have been published that explore the effects of KE modulation on downstream effects. These studies broadly provide strong support to the essentiality of the events within the AOP. Below are examples demonstrating the effects of KE modulation on downstream events.

Essentiality of Increase, oxidative DNA damage (MIE)

  • GSH depletion increases 8-oxo-dG (MIE), and DNA strand breaks (KE2)
    • HepG2 human hepatocytes were treated with 1 mM buthionine sulphoximine (BSO), a GSH-depleting agent, for 4, 8, and 24 hours. Time-dependent statistically significant reduction in GSH was observed at all time points when compared to baseline. The level of 8-oxo-dG lesions was measured 6 and 24 hours after BSO exposure and, at both time points, there was a statistically significant increase in oxidative DNA lesions. A higher magnitude of lesions were present at 24 hours and with statistically significant increases in strand breaks (measured via comet assay) as compared to control (p<0.01) (Beddowes et al., 2003).
  • Antioxidant treatment reduces oxidative lesions, downstream strand breaks, and MN induction (AO2)
    • A 3 hour exposure of HepG2 cells to increasing concentrations of tetrachlorohydroquinone (TCHQ) with N-acetylcysteine (NAC: a radical scanvenger and precursor to glutathione) pre-treatment reduced the amount of cellular ROS (measured by DCFH-DA assay), 8-oxodG, and strand breaks induced by TCHQ measured immediately following exposure. The MN assay at 24 hours indicated a statistically significant decrease in MN at the highest concentration (Dong et al., 2014).
    • Reduction of 8-oxo-dG levels following NAC treatment was also observed in embryos isolated from C57BL/6Jpun/pun mice treated with NAC via drinking water; NAC significantly reduced the number of 8-oxo-dG in the treatment group (Reliene et al., 2004). In human blood mononuclear cells collected in clinical studies, 72-hour NAC treatment significantly reduced the number of MN in the cells. Together, these data support the correlation between the levels of ROS, 8-oxo-dG, and MN frequency (Federici et al., 2015).

Essentiality of Inadequate DNA repair (KE1)

  • The effect of inadequate DNA repair on lesion accumulation and strand breaks (KE2)
    • Nth1 knock-out in vivo - FapyG and FapyA lesions were measured in liver nuclear extracts from wild type and Nth1-/- mice. Statistically significant increases in FapyG and FapyA were observed in Nth1-/- mice. These results demonstrate insufficient repair leading to accumulation of unrepaired oxidative lesions (Hu et al., 2005).
    • Ogg1 knock-out in vitro - In Ogg1-/- mouse embryonic fibroblasts (MEF) treated with 400 µM hydrogen peroxide for 30 minutes, there were significantly fewer strand breaks measured by alkaline comet assay, compared to Ogg1+/+ MEFs. Time series (5 – 90 minutes) immunoblotting of the genomic DNA using anti-8-oxo-dG antibodies indicated a larger magnitude of oxidative lesions in Ogg1-/- cells compared to wild type.  Overall, these results demonstrate the role of Ogg1 in the generation of strand breaks during BER following oxidative DNA damage (Wang et al., 2018).    
  • The effect of inadequate DNA repair on MN induction (AO2)
    • Ogg1 knock-out in vivo - In Ogg1-deficient mice exposed to silver nanoparticles (AgNPs) for seven days, a significant increase (compared to Ogg1+/+) in double strand breaks (indicated by % γ-H2AX positive cells) and 8-oxo-dG lesions were observed at the end of treatment and after 7 days of recovery. The magnitude of increase in DSBs after the 7-day recovery was smaller in wild type. Levels of MN were measured in erythrocytes at the same time points. Increases in MN frequency were significant in wild type (compared to untreated control) on day 7, but not after 7 and 14 days of recovery. In Ogg1-/- mice, the increase in MN was significantly higher on day 7 compared to Ogg1+/+ mice and untreated Ogg1-/- mice and remained significant 7 and 14 days after the exposure (Nallanthighal et al., 2017). Thus, the DNA damage was retained in repair deficient mice leading to persistent clastogenic effects.
  • The effect of inadequate DNA repair on mutations (AO1)
    • Suzuki et al. (2010) knocked-down BER-initiating glycosylases (OGG1, NEIL1, MYH, NTH1) in HEK293T human embryonic kidney cells transfected with plasmids that were either positive or negative for 8-oxodG. The resulting changes in mutant frequencies were measured. Compared to the negative control, all knock-downs caused the mutant frequency to increase in 8-oxodG plasmid-containing cells. Moreover, G:C to T:A transversion frequency increased in all analyzed cells. MYH knock-down decreased A:T to C:G transversion frequency of A paired to 8-oxo-dG; the latter result supports the futile MYH-initiated BER model for the repair of 8-oxo-dG opposite A (Suzuki et al., 2010). Overall, these findings support the essential role of DNA repair in mitigating the mutagenic effects of oxidative DNA lesions.

Essentiality of Increase, DNA strand breaks (KE2)

  • Double strand breaks leading to mutations (AO1)
    • Tatsumi-Miyajima et al. (1993) analyzed different mutations arising from the repair of DSBs induced by a restriction endonuclease, AvaI, in five different human fibroblast cell lines transfected with plasmids containing the AvaI restriction site in the supF gene. Cells containing non-digested plasmids (negative control) produced spontaneous supF mutation frequencies between 0.197 and 2.49 x10-3. In cells containing Ava1-digested plasmids, the number of supF mutants increased, indicated by the rejoining fidelity ((total colonies-supF mutants)/total colonies) between 0.50-0.86. Hence, up to 50% of the colonies were mutated at the AvaI restriction site due to erroneous repair of DSBs induced by the endonuclease.(Tatsumi-Miyajima et al., 1993).
  • Reduction in strand breaks leads to decreases in MN frequency (AO2)
    • Differentiated rat thyroid cells (PCCL3) were internally irradiated by 131I treatment and externally irradiated by 5 Gy X-rays, with or without NAC pre-treatment. Cellular ROS and strand breaks were measured at different time points after irradiation. NAC pre-treatment abrogated ROS induced by both internal and external irradiation at 30 min. The level of ROS was also significantly lower in the NAC-treated cells compared to the non-treated cells at later time points (2, 24, and 48 hours). Moreover, the induction of strand breaks at 30 min was also prevented by NAC pre-treatment and there was a reduction in strand breaks compared to the non-treated cells at later time points as well. Finally, the induction of MN measured 24 and 48 hours after irradiation was significantly lower in NAC-treated cells compared to non-treated cells (Kurashige et al., 2017).

Evidence Assessment


1. Support for biological plausibility

Defining Question

High (Strong)


Low (Weak)

Is there a mechanistic relationship between KEup and KEdown consistent with established biological knowledge?

Extensive understanding of the KER based on extensive previous documentation and broad acceptance.

KER is plausible based on analogy to accepted biological relationships, but scientific understanding is incomplete

Empirical support for association between

KEs, but the structural or functional relationship between them is not understood.

MIE → KE1: Increase, oxidative DNA damage leads to inadequate repair


The repair mechanisms for oxidative DNA damage have been extensively studied and are well-understood. It is generally accepted that limits exist on the amount of oxidative DNA damage that can be managed by these repair mechanisms.

KE1 → KE2: Inadequate repair leads to Increase, DNA strand breaks


It is well-established that failed attempts to repair of accumulated oxidative lesions and replication fork stalling by both unrepaired and incompletely repaired DNA lesions (e.g., repair intermediates such as abasic sites and SSBs) lead to increase in DNA strand breaks.

KE2 →KE1:

Increase, DNA strand breaks leads to Inadequate repair


It is well recognized that the pathways involved in the repair of DSBs is error-prone. In addition to errors induced by NHEJ, all repair mechanisms have a capacity limit; if the number of strand breaks exceed the repair capacity of the cell, unrepaired  SSBs and DSBs may accumulate.

KE1 → AO1: Inadequate repair leads to Increase, mutations


Numerous studies (cell-based and in vivo) have demonstrated increases in mutation due to unrepaired oxidative DNA lesions (insufficient repair) and incorrect repair (e.g., non-homologous end joining and error-prone lesion bypass). The mechanisms by which these events occur are well-understood.

KE1 → AO2: Inadequate repair leads to Increase, chromosomal aberrations


Chromosomal aberrations may result if DNA repair is inadequate, meaning that the double-strand breaks are misrepaired or not repaired at all. A multitude of different chromosomal aberrations can occur, depending on the timing (i.e., cell cycle) and type of inadequate repair. Examples include copy number variants, deletions, translocations, inversions, dicentric chromosomes, nucleoplasmic bridges, nuclear buds, micronuclei, centric rings, and acentric fragments. A multitude of publications are available that provide details on how these various chromosomal aberrations are formed in the context of inadequate repair.


KE2 →AO1: Increase, DNA strand breaks leads to Increase, mutations


Mechanisms of DNA strand break repair have been extensively studied. It is accepted that non-homologous end joining of DSBs can introduce deletions, insertions, translocations, or base substitution.   


MIE → KE2: Oxidative DNA lesions leads to Increase, DNA strand breaks


Increase in strand breaks due to failed repair of oxidative DNA lesions is an accepted mechanism for the clastogenic effects of oxidative damage. Concurrent increases in the two KEs have been observed in previous studies. However, data that demonstrate a causal relationship, in accordance with the Bradford-Hill criteria for causality, are limited.


MIE → AO1: Oxidative DNA lesions leads to Increase, mutations


Strong empirical evidence exists in literature demonstrating increases in mutation frequency due to increase in oxidative DNA lesions. Notably, mutagenicity of 8-oxodG, the most abundant oxidative DNA lesion, has been extensively studied and is well-known to cause G to T transversions.



Increase, DNA strand breaks leads to Increase, chromosomal aberrations


DNA strands breaks must occur for chromosomal aberrations to occur. Increase in strand breaks, especially DSBs, may increase the risk of inadequate repair (lack of repair or misrepair) of the damage, leading to translocations, inversions, insertions, and deletions.

2. Support for Essentiality of KEs

Defining Question

High (Strong)


Low (Weak)

Are downstream KEs and/or the AO prevented if an upstream KE is blocked?

Direct evidence from specifically designed experimental studies illustrating essentiality for at least one of the important KEs

Indirect evidence that sufficient modification of an expected modulating factor attenuates or augments a KE

No or contradictory experimental evidence of the essentiality of any of the KEs.

MIE: Increase, oxidative DNA damage


Studies have demonstrated that indirectly reducing or increasing the amount of oxidative DNA lesions by modulating cellular ROS levels (via antioxidant addition or depletion) causes concordant changes in the levels of strand breaks and MN.

KE1: Inadequate repair


Numerous studies have investigated inadequate BER of oxidative DNA lesions by disrupting BER through generating gene KO rodent or mammalian cell models. Modulation of the downstream KEs (i.e., DNA strand breaks, mutation, MN induction) by dysfunctional BER has been demonstrated in these studies.

KE2: DNA strand breaks


Theoretically, chromosomal aberrations (AO2) cannot occur unless DNA strand breaks occur. Predominantly, indirect evidence exists that support the essentiality of KE2 in leading to mutations (AO1).

3. Empirical Support for KERs

Defining Question

High (Strong)


Low (Weak)

Does empirical evidence support that a change in KEup leads to an appropriate change in KEdown?

Does KEup occur at lower doses and earlier time points than KE down and is the incidence of KEup> than that for KEdown?


Multiple studies showing dependent change in both events following exposure to a wide range of specific stressors.

No or few critical data gaps or conflicting data

Demonstrated dependent change in both events following exposure to a small number of stressors.

Some inconsistencies with expected pattern that can be explained by various factors.

Limited or no studies reporting dependent change in both events following exposure to a specific stressor; and/or significant inconsistencies in empirical support across taxa and species that don’t align with hypothesized AOP

MIE → KE1: Increase, oxidative DNA damage leads to inadequate repair


Empirical in vitro and in vivo data demonstrate that increases in oxidative DNA lesions leads to indications of inadequate repair (i.e., increases in mutation, retention of adducts, increases in lesions despite upregulation of repair enzymes).

KE1 → KE2: Inadequate repair leads to Increase, DNA strand breaks


Limited in vivo data are available. A few In vitro studies have demonstrated a larger increase in DNA strand breaks in BER-defective cells compared to wildtype cells, following various oxidative stresse-inducing chemical exposures. 

In certain cases, as demonstrated by Wang et al. (2018), knock-down of OGG1 (BER-initiating glycosylase) reduced the amount of  DNA strand breaks that formed after exposure to hydrogen peroxide - mostly likely due to the reduction in the incidences of incomplete repair. As such, deficiency in different DNA repair proteins can have varying effects on downstream strand breaks; inadequate repair may manifest differently for different stressors.

KE2 →KE1:

Increase, DNA strand breaks leads to Inadequate repair


Results from many studies indicate dose/incidence and temporal concordance between the frequency of double-strand breaks and the rate of inadequate repair. As DNA damage accumulates in cells, the incidence of inadequate DNA repair activity (in the form of non-repaired or misrepaired DSBs) also increases.

 Uncertainties in this KER include controversy surrounding the error rate of NHEJ, differences in responses depending on genotoxicant exposure levels and confounding clinical factors (such as smoking) that affect double-strand break repair fidelity.

KE1 → AO1: Inadequate repair leads to Increase, mutations


Repair deficiency causing increases in mutations has been extensively demonstrated in both in vitro and in vivo. Overexpression of repair enzymes has been shown to reduce mutation frequency following chemical exposure in vitro, which further supports the causal relationship between these two KEs.

KE1 → AO2: Inadequate repair leads to Increase, chromosomal aberrations


There is little empirical evidence available that directly examines the dose and incidence concordance between DNA repair and CAs within the same study. Similarly, there is not clear evidence of a temporal concordance between these two events. More research is required to establish empirical evidence for this KER.


KE2 →AO1: Increase, DNA strand breaks leads to Increase, mutations


Evidence from in vitro and in vivo studies demonstrating dose and temporal concordance of the two KEs are available. These investigations utilized various stressors such as chemicals and ionizing radiation.


MIE → KE2: Oxidative DNA lesions leads to Increase, DNA strand breaks


Both in vitro and in vivo data are available that the demonstrate dose-response concordance of oxidative DNA lesions formation and strand breakage following exposure to various stressors. However, the temporal concordance between the KEs is not strong; there are discrepancies in the temporal sequence of events that appear to be dependent on the endpoint used to measure the KE (i.e., Fpg comet assay vs. 8-oxodG immunodetection, comet assay vs. ɣ-H2AX immunodetection). 


MIE → AO1: Increase, oxidative DNA lesions leads to Increase, mutations


This KER was demonstrated by knocking out oxidative DNA damage repair protein (OGG1) and exposure to different ROS-inducing chemicals in vitro and in vivo. It is clear that an increase in oxidative DNA lesions is followed by an increase in mutant frequency or G to T transversions. 



Increase, DNA strand breaks leads to Increase, chromosomal aberrations


Temporal concordance is clear in both in vitro and in vivo data. However, due to the differences in the methods used to measure strand breaks and chromosomal aberrations, the concentration-response of these events often appear to be discordant.  


The text in blue were copied and pasted from AOP #272: Direct deposition of ionizing energy onto DNA leading to lung cancer.

Quantitative Understanding


The quantitative understanding of the KERs in this AOP is overall weak. Different cell types have different baseline levels of oxidative DNA lesion repair capacity; for example, Nishioka et al. (1999) demonstrated difference in the expression level of OGG1 mRNA across different human tissues (Nishioka et al., 1999). Thus, the quantity of oxidative DNA lesions required to overwhelm the repair mechanisms and lead to chromosomal damage or mutations by cell type.


Considerations for Potential Applications of the AOP (optional)


Genotoxicity testing is a fundamental requirement of all chemical and pharmaceutical assessments. Although there are established guidelines for in vitro tests, there is an urgent need for in vitro assays that can better predict in vivo genotoxicity. The current standard in vitro genotoxicity assays provide limited mechanistic information and suffer from high sensitivity and low specificity, leading to unnecessary in vivo studies. The field of applied genetic toxicology is in the midst of a paradigm shift to develop better in vitro testing strategies and to reduce the reliance on animal models. More biologically relevant in vitro models and mechanism-based test methods are required to transition away from the current strategies using bacteria and non-metabolically competent, transformed human cell lines (Whitwell et al., 2015). Mutations and chromosome damage are inarguably tied to the induction of many genetic diseases including cancer. However, there is also a movement away from the notion that genotoxicity testing is only valuable to inform potential hazards and mechanisms of carcinogenesis, and increasing understanding of the fact that genomic damage such as mutations, in and of themselves, are adverse (e.g., germ cell mutations) (Heflich et al., 2020). Indeed, there is increasing use of genotoxicity data in deriving points of departure to inform risk assessments (Klapacz and Gollapudi, 2020; Luijten et al., 2020; White et al., 2020). Overall, understanding the genotoxic mechanisms (i.e., the MIEs and KEs) that lead to genotoxic outcomes in vivo is critical to this paradigm change (Dearfield et al., 2017).

AOPs provide a framework for organizing and evaluating evidence for different mechanisms of genotoxicity and their ability to predict downstream genotoxic outcomes to support the paradigm changes that are underway. AOPs that describe the mechanisms leading to genotoxicity, such as the one herein, are useful for identifying relevant endpoints for mechanism-based testing. Oxidative DNA damage is a well-known genotoxic hazard and, thus, a critical endpoint in genotoxicity hazard assessment. This AOP network provides a framework for assembling information from different mechanism-based tests to determine the probability that an agent will cause genotoxicity through induction of oxidative DNA damage.

In addition to incorporating mechanistic information in assessments, there is an effort to transition from a strictly qualitative hazard identification approach to applications in quantitative risk assessment (White and Johnson, 2016; Dearfield et al., 2017; White et al., 2020). AOP networks that such as this one can inform how different testing methods, including mechanistic assays without established testing guidelines, quantitatively relate to the measurement of adverse genotoxic outcomes. This AOP network documents clear gaps in quantitative understanding of genomic damage induced by oxidative DNA lesions that are needed to enhance risk assessment and predictive toxicology for chemicals that induce oxidative DNA lesions.

Overall, AOPs that extend to adverse genotoxic outcomes can be applied in regulatory assessment of chemicals to (a) facilitate mode of action analysis of chemicals to hypothesize potential pathways of genotoxicity; (b) identify test methods and strategies that can be used to test hypothetical AOPs for untested chemicals; (c) highlight knowledge gaps and uncertainties in genotoxic MOAs; and (d) facilitate the development of new testing strategies.



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