Upstream eventIncrease, Oxidative DNA damage
N/A, Inadequate DNA repair
Key Event Relationship Overview
AOPs Referencing Relationship
|AOP Name||Adjacency||Weight of Evidence||Quantitative Understanding|
|Oxidative DNA damage leading to chromosomal aberrations and mutations||adjacent||High||Low|
Life Stage Applicability
|All life stages|
Key Event Relationship Description
Oxidative DNA lesions are present in the cell at steady state due to low levels of reactive oxygen species (ROS) and other free radicals generated by endogenous processes involving redox reactions. The most prominent examples of oxidative DNA lesions include 7, 8-dihydro-8oxo-deoxyGuanine (8-oxo-dG), 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FaPydG), and thymidine glycol (Tg). Under homeostatic conditions, cells are able to regulate the level of free radicals and readily repair oxidized DNA bases using basal repair mechanisms to prevent irreversible damage (Swenberg et al., 2011). Oxidative DNA lesions are mainly repaired by base excision repair (BER) initiated by DNA glycosylases such as oxoguanine glycosylase 1 (OGG1), endonuclease III homologue 1 (NTH1), and Nei-like DNA glycosylases (NEIL 1/2), which detect and remove damaged bases. Abasic sites are then cleaved by endonucleases or lyases, resulting in transient single-strand breaks (SSB) that enter either short-patch or long-patch repair. Nucleotide excision repair (NER) is involved in repairing oxidized bases to a lesser extent (Shafirovich et al., 2016). Increase in free radicals or exposure to oxidizing agents can increase the level of oxidative DNA lesions and overwhelm the repair pathways, compromising the quality of repair. If the repair mechanisms are compromised, oxidative lesions may accumulate (insufficient repair) and cause incorrect base pairing during replication or incomplete repair (indicated by accumulation of repair intermediates) (Markkanen, 2017).
Evidence Supporting this KER
Inadequate repair of oxidative lesions is indicated by an increase in oxidative lesions above background, activation of repair enzymes, increase in repair intermediates (abasic sites and single strand breaks), and incorrect base insertion opposite lesion during replication (lesion bypass by translesion DNA synthesis).
The mechanism of repair of oxidative DNA lesions in humans is well-established and numerous literature reviews are available on this topic (Berquist and Wilson III, 2012; Cadet and Wagner, 2013). As described above, oxidative DNA lesions are mostly repaired via BER and, to a lesser extent, NER. Previous studies have reported thresholded dose-response curves in oxidative DNA damage and attributed these observations to exceeded repair capacity at the inflection point on the curve (Gagne et al., 2012; Seager et al., 2012). In vivo, increase and accumulation of oxidative DNA lesions despite the activation of BER have been observed following chemical exposures, demonstrating insufficient repair of oxidative DNA lesions past a certain level (Ma et al., 2008).
OGG1 and NTH1, the glycosylases that initiate the BER of 8-oxo-dG and thymine glycol (Tg) lesions, respectively, are bifunctional, containing both glycosylase and lyase activities. The glycosylase removes the oxidized guanine by cleaving the glycosidic bond, giving rise to an apurinic site. The lyase then cleaves the phosphodiester bond 5’ to the AP site; a transient SSB is created for further processing in BER (Delaney et al., 2012). Abasic sites created by OGG1 and other glycosylases are also processed by apuric/apyrimidinic endonucleases (APE1) to create the 5’ nick (Allgayer et al., 2016).
Previous studies have demonstrated that an imbalance in any one of the multiple steps of BER can lead to an accumulation of repair intermediates and failed repair. Given that OGG1 is relatively slower in releasing its catalytic product than other glycosylases, it is highly likely that a disproportionate increase in oxidative DNA lesions compared to the level of available OGG1 would lead to an imbalance between lesions and the initiating step of BER (Brenerman et al., 2014). Accumulation of oxidative lesions would be observed as a result. Moreover, studies have reported accumulation of SSB due to OGG1 and NTH1 overexpression, demonstrating that the imbalanced lyase activity generates excessive SSB intermediates (Yang et al., 2004; Yoshikawa et al., 2015; Wang et al., 2018).
Increases in oxidative lesions may produce more lesions and repair intermediates in close proximity to each other. Previous studies in mammalian cell extracts have reported reduction in repair efficiency when oxidative lesions are in tandem or opposite each other. For example, OGG1 showed reduced binding to 8-oxo-dG near an AP site incision. Furthermore, the OGG1-8-oxo-dG complex has been observed to hinder the repair of neighbouring AP site incision, delaying the completion of BER; this interaction between BER enzymes has been suggested to cause an accumulation of oxidative lesions and repair intermediates (Pearson et al., 2004; Budworth et al., 2005; Bellon et al., 2009; Yoshikawa et al., 2015; Sharma et al., 2016).
If oxidative lesions persist in the genome due to insufficient repair, incorrect base insertion opposite unrepaired oxidative DNA lesions may occur during replication. This is a well-established event. For example, 8-oxo-dG and FaPydG, the two most prominent oxidative DNA lesions, are able to form base pairs with dATP, giving rise to G:C→T:A transversions after subsequent DNA synthesis (Freudenthal et al., 2013; Gehrke et al., 2013; Markkanen, 2017). Replicative DNA polymerases such as DNA polymerase α, δ, and ε (pol α, δ, ε) have a poor ability to extend the DNA strand past 8-oxo-dG:dCTP base pairs and may cause replication to stall or incorrectly insert dATP opposite 8-oxo-dG (Hashimoto et al., 2004; Markkanen et al., 2012). In stalled replication forks, repair polymerases may be recruited to perform translesion DNA synthesis (TLS). Human Y-family DNA polymerases (Rev 1, pol κ, ι, and η) are DNA repair polymerases mainly involved in TLS in stalled replication forks. However, TLS is not free of error and its accuracy differs for each repair polymerase. For example, it is known that pol κ and η perform TLS across 8-oxo-dG and preferentially insert dATP opposite the lesion, generating G:C→T:A transversions. The error-prone nature of bypassing unrepaired oxidative lesions has been described in many previous studies and reviews (Greenberg, 2012; Maddukuri et al., 2014; Taggart et al., 2014; Shah et al., 2018).
Repair by OGG1 requires 8-oxo-dG:dC base pairing, thus, it is unable to repair 8-oxo-dG:dA mispairing in newly synthesized strands. The repair of 8-oxo-dG:dA base pairs post-replication is performed by MUT Y homologue, MYH, an adenine DNA glycosylase. However, the removal of dA instead of the damaged guanine may lead to futile cycles of BER because: 1) another dA is often inserted opposite the lesion, or 2) BER ligases have a poor ability of ligating the 3’end of dC opposite 8-oxo-dG (Hashimoto et al., 2004; Caglayan and Wilson, 2015). Accumulated 8-oxo-dG may be more resistant to repair post-replication due to this futile BER.
Example in vitro studies demonstrating dose and temporal concordance, or essentiality
- Human normal hepatocytes (HL-7702) were exposed to N,N-dimethylformamide for 24 hours at increasing concentrations (C. Wang et al., 2016)
- Concentration-dependent increase in ROS was observed; the increase was statistically significant compared to control at all concentrations (6.4, 16, 40, 100 mM)
- No significant increase in 8-oxodG was observed until the highest two concentrations (40 and 100 mM) indicating insufficient repair at these concentrations
- Significant up-regulation of excision repair genes (XRCC2 and XRCC3) occurred at 6.4 and 16 mM, below the concentrations that significantly induced 8-oxodG, supporting sufficient DNA repair at these low concentrations.
- These results demonstrate that repair is sufficient at low concentrations (rapidly removing 8-oxodG) and not until higher concentrations is repair overwhelmed (i.e., insufficient), where 8-oxo-dG significantly increases.
- AS52 Chinese hamster ovary cells (wild type and OGG1-overexpressing) were exposed to varying doses of ultraviolet A (UVA) radiation (Dahle et al., 2008)
- Formamidopyrimidine glycosylase (Fpg)-sensitive sites were quantified using alkaline elution after increasing repair times (0, 1, 2, 3, 4 h) following 100 kJ/m2 UVA irradiation
- OGG1-overexpressing AS52 cells (OGG1+): Fpg-sensitive sites reduced to 71% within half an hour and down to background levels at 4h
- Wild type AS52 cells: at 4h, 70% of the Fpg-sensitive sites remained, indicating accumulation of oxidative lesions
- The above results demonstrated that excess OGG1 was able to prevent the accumulation of oxidative lesions, while the amount of OGG1 in wild type was insufficient to handle the amount of lesions induced by the same magnitude of UVA irradiation.
- Mutations in the Gpt gene was quantified in both wild type and OGG1+ cells by sequencing after 13-15 days following 400 kJ/m2 UVA irradiation
- G:C→T:A mutations in UVA-irradiated OGG1+ cells were completely eliminated (thus, repair was sufficient when repair overexpressed).
- G:C→T:A mutation frequency in wild type cells increased from 1.8 mutants/million cells to 3.8 mutants/million cells following irradiation – indicating incorrect repair or lack of repair of accumulated 8-oxo-dG.
- The above result also demonstrates the essentiality of 8-oxo-dG formation in the oxidative DNA damage-induced G to T transversion mutations.
- HL-60 human leukemia cells were irradiated with X-rays at a rate of 0.5 Gy/min for increasing durations (i.e., increasing doses). 8-OHdG levels were quantified by HPLC as number of 8-OHdG per 106 deoxyguanosine (Li et al., 2013)
- No increase in 8-OHdG was observed up to 2 Gy (sufficient repair at low doses), above which the level of lesions increased linearly up to 20 Gy (insufficient repair)
- This thresholded dose-response curve, indicative of overwhelmed repair processes, was also observed in mouse liver in the same study described below.
In vivo studies demonstrating dose concordance
- Two groups of 5-week-old C57BL/6J mice were exposed to increasing doses of X-rays at a rate of 0.5 Gy/min (200 kV, 12 mA). The livers were collected from one group immediately after exposure and urine samples were collected over 24 hours following irradiation in the second group of mice (Li et al., 2013).
- 8-OHdG in the mouse liver DNA were quantified by HPLC and expressed as 8-OHdG per 106 deoxyguanosine
- Between 0 and 0.5 Gy, no increase in lesions was observed
- Between 0.5 and 30 Gy, a linear dose-response in 8-OHdG was observed
- The thresholded dose-response curve was concordant in the urine samples; no increase in urinary 8-OHdG (8-OHdG/creatinine (ng/mg)) was observed between 0 and 0.1 Gy but between 0.1 and 5 Gy, the number of lesions increased linearly with dose
- Male Sprague-Dawley rats were fed 0.5 mmol aniline/kg/day for 30 days. Genomic DNA, nuclear extracts, and mitochondrial extracts were collected from spleen tissues (Ma et al., 2008).
- 8-OHdG was quantified using enzyme-linked immunosorbent assay (ELISA) on digested genomic DNA. There was a significant 2.8-fold increase in lesions in aniline-fed rats than in control rats.
- Both the nuclear extracts and mitochondrial extracts were tested for OGG1 activity, where 1.32-fold and 1.15-fold increase in enzyme activity (both significant; p<0.05) were observed in the respective extracts of aniline-treated rats.
- The OGG1 enzyme content in the extracts was detected using Western blotting; the increase in OGG1 content in aniline-treated rats was consistent with the OGG1 activity assay.
- Despite the increase in OGG1 enzyme content and activity, the quantity of 8-OHdG increased.
- Together, these results demonstrate that the level of OGG1 capacity was exceeded for repair of 8-oxo-dG, leading to accumulation of these lesions during this sub-chronic exposure.
Uncertainties and Inconsistencies
Although the dual functionality of OGG1 as a glycosylase and lyase has been widely accepted and demonstrated experimentally, there are studies showing that the cleavage of phosphodiester bond 5’ to the lesion is mainly performed by apurinic endonuclease 1 (APE1) (Allgayer et al., 2016; R. Wang et al., 2018). In some cases, APE1 may be the main factor driving the accumulation of BER intermediates. Some studies suggest that OGG1 is involved in the repair of non-transcribed strands and is not required for transcription-coupled repair of 8-oxo-dG; Le Page et al. reported efficient repair of 8-oxo-dG in the transcribed sequence in Ogg1 knockout mouse cells (Le Page et al., 2000). Moreover, the repair of 8-oxo-dG is also affected by the neighbouring sequence; the position of the lesions may have a negative effect on repair efficiency (Pastoriza-Gallego et al., 2007). We note that the study by Allgayer et al. was investigating the fate and effect of 8-oxo-dG during transcription; repair mechanism may vary by situation and availability of repair enzymes at the time.
Quantitative Understanding of the Linkage
The precise relationship between levels of oxidative DNA lesions and when repair can be considered inadequate have not been fully defined; this relationship will very likely differ between cell types and tissues and, thus, difficult to define. There are computational models of repair kinetics of 8-oxo-dG.
Sokhansanj and Wilson III  applied a quantitative model of BER and the literature value for the rate of formation of endogenous 8-oxo-dG to investigate the rate of clearance of BER repair intermediates (Sokhansanj and Wilson III, 2004).
- The BER model used Michaelis-Menten enzyme kinetics and included the activities of OGG1, AP lyases, polymerases, and ligases.
- The model assumed the formation rate of endogenous oxidative lesions to be 500 8-oxo-dG/day
- Based on the above, it was estimated that following a sudden spike in 8-oxo-dG up to 20,000 8-oxo-dG/cell, the total level of repair intermediates would return to baseline within 4000 seconds (less than 1 hour)
- This model also assumed that OGG1 was available in excess
- When APE1 (AP site endonuclease) is present, glycosylase reaction kinetics of OGG1(a bifunctional glycosylase/lyase) was observed to increase
- Suggested to be due to the coordinated action of the two enzymes
- A 10-fold reduction in OGG1 kinetics led to 10-fold increase in 8-oxo-dG, while no other repair intermediates increased.
Known modulating factors
Known Feedforward/Feedback loops influencing this KER
Domain of Applicability
Oxidative DNA damage can occur and overwhelm the repair mechanisms in any eukaryotic and prokaryotic cell. Observation of this KER has been described in mammalian cells, yeast, and bacteria.
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