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Aop: 296

AOP Title

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Oxidative DNA damage leading to chromosomal aberrations and mutations

Short name:

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Oxidative DNA damage, chromosomal aberrations and mutations

Graphical Representation

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Authors

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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, INRA, 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

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Carole Yauk   (email point of contact)

Contributors

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  • Carole Yauk

Status

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Author status OECD status OECD project SAAOP status
Under development: Not open for comment. Do not cite


This AOP was last modified on October 11, 2019 16:47

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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 11, 2019 16:42
Increase, DNA strand breaks October 18, 2019 13:20
Increase, Mutations October 18, 2019 15:41
Increase, Chromosomal aberrations October 18, 2019 15:47
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 September 04, 2019 14:08
Increase, DNA strand breaks leads to Increase, Mutations September 05, 2019 18:58
N/A, Inadequate DNA repair leads to Increase, Mutations September 30, 2019 11:51
N/A, Inadequate DNA repair leads to Increase, Chromosomal aberrations September 04, 2019 15:06
Increase, DNA strand breaks leads to Increase, Chromosomal aberrations July 03, 2019 15:16
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

Abstract

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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)

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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; 8-oxodG is an accepted biomarker of oxidative stress and oxidative damage to DNA (Roszkowski et al., 2011; Guo et al., 2017).

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 replicative or translesion polymerases), 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 an intermediate. With increasing oxidative lesions and more lesions in close proximity to each other, the quality and efficiency of repair may be compromised, resulting in retention of unrepaired lesions and repair intermediates. Accumulated intermediate SSBs, along with unrepaired oxidative lesions and other intermediates like abasic sites, can interfere with repair at other damaged sites nearby and/or with the replication fork, and lead to double strand breaks (DSBs) which are more toxic and laborious 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 during excision repair, these SSBs may be converted to DSBs. 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. Importantly, this work highlights notable gaps in the empirical evidence despite the fact that this is a long-studied area in genetic toxicology. Quantifying the extent to which levels of oxidative DNA damage must increase before DNA repair processes are overwhelmed and the AOs result is required to improve our ability to predict whether this pathway is relevant to a chemical’s toxicological effects.


Summary of the AOP

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Events: Molecular Initiating Events (MIE)

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Key Events (KE)

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Adverse Outcomes (AO)

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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)

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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

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Stressors

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Life Stage Applicability

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Life stage Evidence
All life stages

Taxonomic Applicability

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

Sex Applicability

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Sex Evidence
Unspecific

Overall Assessment of the AOP

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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 and leveraged decades of experience and research on DNA repair and genetic toxicology.

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 adducts on nucleotides leads to the formation of incorrect base pairs with incoming nucleotides without causing structural disturbance and, thus, evading mismatch repair (Cooke et al., 2003). It is well-understood that both 8-oxodG and FapydG readily form base pairs with adenine, giving rise to G to T transversions, which are predominant base substitutions caused by oxidative stress (Cadet and Wagner, 2013; Poetsch et al., 2018). 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.

The biology behind 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.

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 site, and APE1, which removes the AP site by cleaving 5’ to the AP site. A spike in BER substrates could lead to an imbalance in the initiating steps of BER, causing an accumulation of abasic sites and single strand break (SSB) intermediates (Coquerelle et al., 1995; Yang et al., 2006; Nemec et al., 2010). It is known that BER glycosylases are constitutively expressed and that APE1 is an abundant enzyme (Tell et al., 2009). Another biologically plausible way in 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, continuing the cycle of BER at the site and potentially causing an accumulation of SSBs. SSBs can turn into DSBs if they occur in close proximity to each other on opposite strands, or cause replication fork 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 can shift away from their original position and result in two incorrect ends being joined or loss of DNA segments, leading to structural aberrations (Obe et al., 2010; Durante et al., 2013)

Misrepair of DSBs (KE1 Description section 3) can also lead to mutations, providing an alternate pathway to AO1, 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 homologous chromosome or sister chromatid as a template to ensure fidelity of the reconstructed strands (Mao et al., 2008a; Mao et al., 2008b). 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 salavage 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).

Time- and dose-response concordance:

The WOE supporting the time- and dose-response concordance of the KEs of these AOPs and the overall network 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 in vitro models, 8-oxodG has been quantified as early as 15 minutes following chemical exposure (Ballmaier and Epe, 2006). Time and concentration-response concordance in oxidative lesion formation and induction of strand breaks have been demonstrated by in vitro time course experiments, where increases in oxidative lesions was 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 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 mutation frequencies are also very limited.

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 indicates 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).

Domain of Applicability

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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 and induction of oxidative stress due to exposure and/or diseases [e.g., urinary 8-oxodG (Hanchi et al., 2017); 8-oxodG in tumour samples (Mazlumoglu et al., 2017)]. Micronucleus is also regularly quantified as a biomarker of genotoxic exposure in humans. Numerous examples of MN detection 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

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A large number of studies exploring the effects of modulating different events in this network and measuring downstream effects have been published. These studies broadly provide strong support to the essentiality of the events to the pathway and AOs. Below we provide examples to demonstrate the effect of modulating each KE on the downstream KEs/AOs.

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 reduction in GSH was observed and the reduction was significant at all time points compared to 0h. The level of 8-oxo-dG lesions was measured at 6 and 24 hours; at both time points, there was a significant increase in oxidative DNA lesions, with a larger amount of lesions present at 24 hours. Strand breaks were also measured concurrently. While there was no observable increase in strand breaks at 6 hours, the increase at 24 hours was significant compared to control (p<0.01) (Beddowes et al., 2003).
  • Antioxidant treatment reduces oxidative lesions and downstream strand breaks and MN (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 micronucleus (MN) assay at 24 hours indicated a 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 - FapyG and FapyA lesions were measured in the liver nuclear extracts from wild type and Nth1-/- mice. A significant increase in FapyG and FapyA was 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 comet assay, compared to Ogg1+/+ MEFs. Time series (5 – 90 minutes) immunoblotting of the genomic DNA using anti-8-oxo-dG antibody indicated a larger magnitude of increase in oxidative lesions in Ogg1-/- cells compared to wild type.  Overall, the results demonstrate the role of Ogg1 in the generation of strand breaks as an intermediate in base excision repair 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 was observed on day 7 of the exposure and following 7 days of recovery. The magnitude of increase in DSBs after the 7-day recovery was smaller in the 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, immediately following exposure; however, after 7 and 14 days of recovery, the increase was no longer significant. In Ogg1-/- mice, the increase in MN was significant 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 (Tatsumi-Miyajima et al., 1993).
  • Reduction in strand breaks leads to decreases in MN frequency (AO2)
    • PCCL3 normal differentiated rat thyroid cells 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 prevented the ROS spike 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 spike in 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 at 24 and 48 hours following irradiation was significantly lower in NAC-treated cells compared to non-treated cells (Kurashige et al., 2017).

Evidence Assessment

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1. Support for biological plausibility

Defining Question

High (Strong)

Moderate

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

STRONG

The repair mechanisms for oxidative DNA damage have been extensively studied and well-understood. It is generally accepted that there exist limits 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

STRONG

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

KE2 →KE1:

Increase, DNA strand breaks leads to Inadequate repair

STRONG

It is well recognized that almost all types of DNA lesions will result in recruitment of repair enzymes and factors to the site of damage, and the pathway involved in the repair of DSBs has been well-documented in a number of reviews, many of which also discuss the error-prone nature of DNA repair.

KE1 → AO1: Inadequate repair leads to Increase, mutations

STRONG

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

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

STRONG

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 types of chromosomal aberrations can occur, depending on the timing and type of erroneous repair. Examples of chromosomal aberrations 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.

Non-adjacent:

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

STRONG

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

Non-adjacent

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

MODERATE

Increase in strand breaks due to failed attempted 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 are limited.

Non-adjacent

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

STRONG

Strong empirical evidence exist in literature demonstrating increase in mutations 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.

Non-adjacent

KE2→AO2:

Increase, DNA strand breaks leads to Increase, chromosomal aberrations

STRONG

DNA strands breaks must occur for chromosomal aberrations to occur.

2. Support for Essentiality of KEs

Defining Question

High (Strong)

Moderate

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

MODERATE

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

KE1: Inadequate repair

STRONG

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

KE2: DNA strand breaks

MODERATE

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

3. Empirical Support for KERs

Defining Question

High (Strong)

Moderate

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?

Inconsistencies?

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

MODERATE

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

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

MODERATE

Limited in vivo data are available. A few In vitro studies have demonstrated a larger increase in DNA strand breaks in DNA repair-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 following a hydrogen peroxide exposure - 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

MODERATE

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 organisms, 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 how error-prone NHEJ truly is, differences in responses depending on the level of exposure of a genotoxic substance, and confounding factors (such as smoking) that affect double-strand break repair fidelity.

KE1 → AO1: Inadequate repair leads to Increase, mutations

STRONG

Repair deficiency causing increase in mutations has been extensively demonstrated in both in vitro and in vivo models. Overexpression of repair enzymes has been shown to reduce mutation frequency following chemical exposure in vitro; these data further support the causal relationship between these two KEs.

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

MODERATE

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.

Non-adjacent:

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

MODERATE

Evidence demonstrating dose and temporal concordance in the two KEs are available in both in vitro and in vivo studies. These studies used a few different chemicals and ionizing radiation as stressors.

Non-adjacent

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

MODERATE

Both in vitro and in vivo data are available that demonstrate dose-response concordance in oxidative DNA lesions formation and strand breaks following exposure to various stressors. However, the temporal concordance between the KEs in these results 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). 

Non-adjacent

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

STRONG

This KER was demonstrated both in vitro and in vivo via knock-down of oxidative DNA damage repair protein (OGG1) and exposure to different ROS-inducing chemicals. Increase in oxidative DNA lesions followed by an increase in mutant frequency or G to T transversions was clearly shown in these studies. 

Non-adjacent

KE2→AO2:

Increase, DNA strand breaks leads to Increase, chromosomal aberrations

MODERATE

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.  


Quantitative Understanding

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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 may vary by cell type.

 


Considerations for Potential Applications of the AOP (optional)

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Genotoxicity testing is a fundamental requirement of all chemical and pharmaceutical assessments. Mutations and chromosome damage are inarguably tied to the induction of many genetic diseases including cancer. Oxidative DNA damage is a well-known genotoxic hazard and, thus, a critical endpoint in genotoxicity hazard assessment.  Indeed, when the mode of genotoxic action of a chemical is uncertain, oxidative DNA damage is frequently suspected to be the cause. 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. Moreover, the field of applied genetic toxicology is in the midst of a paradigm shift (Dearfield et al., 2017; White and Johnson, 2016), transitioning from a strictly qualitative hazard identification approach to applications in quantitative risk assessment. AOP networks such as this one are a critical element of this paradigm change, informing how different test methods align with the measurement of adverse genotoxic outcomes. Quantitative understanding is necessary in order to be able to determine the extent of oxidative DNA lesions, and single and double strand breaks, necessary to lead to mutations and chromosomal aberrations. This AOP network documents clear gaps in quantitative understanding that must be defined in order to enhance risk assessment and predict toxicology for chemicals that induce oxidative DNA lesions. Overall, the AOP serves a variety of potential regulatory purposes including: (a) facilitating mode of action analysis for chemicals hypothesized to operate through this pathway; (b) identifying test methods and strategies that can be used to test these hypothetical AOPs for new chemicals; (c) facilitating the development of new testing strategies; and (c) highlighting gaps and uncertainties in genotoxicity modes of action.


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