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Relationship: 1913

Title

A descriptive phrase which clearly defines the two KEs being considered and the sequential relationship between them (i.e., which is upstream, and which is downstream). More help

Increase, Oxidative DNA damage leads to Increase, DNA strand breaks

Upstream event

The causing Key Event (KE) in a Key Event Relationship (KER). More help

Increase, Oxidative DNA damage

Downstream event

The responding Key Event (KE) in a Key Event Relationship (KER). More help

Increase, DNA strand breaks

Key Event Relationship Overview

The utility of AOPs for regulatory application is defined, to a large extent, by the confidence and precision with which they facilitate extrapolation of data measured at low levels of biological organisation to predicted outcomes at higher levels of organisation and the extent to which they can link biological effect measurements to their specific causes.Within the AOP framework, the predictive relationships that facilitate extrapolation are represented by the KERs. Consequently, the overall WoE for an AOP is a reflection in part, of the level of confidence in the underlying series of KERs it encompasses. Therefore, describing the KERs in an AOP involves assembling and organising the types of information and evidence that defines the scientific basis for inferring the probable change in, or state of, a downstream KE from the known or measured state of an upstream KE. More help

AOPs Referencing Relationship

AOP Name	Adjacency	Weight of Evidence	Quantitative Understanding	Point of Contact	Author Status	OECD Status
Oxidative DNA damage leading to chromosomal aberrations and mutations	non-adjacent	Moderate	Low	Carole Yauk (send email)	Open for comment. Do not cite	WPHA/WNT Endorsed
Deposition of energy leading to occurrence of cataracts	adjacent	Low	Low	Vinita Chauhan (send email)	Open for citation & comment

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) that help to define the biological applicability domain of the KER.In general, this will be dictated by the more restrictive of the two KEs being linked together by the KER. More help

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

Sex Applicability

An indication of the the relevant sex for this KER. More help

Sex	Evidence
Unspecific	Moderate
Male	Low

Life Stage Applicability

An indication of the the relevant life stage(s) for this KER. More help

Term	Evidence
All life stages	Moderate

Key Event Relationship Description

Provides a concise overview of the information given below as well as addressing details that aren’t inherent in the description of the KEs themselves. More help

The repair of oxidative DNA lesions produced by exposure to reactive oxygen species (ROS) involves excision repair, where damaged base is removed by glycosylases, a strand break is generated 5’ to the apurinic/apyrimidinic (AP) site by lyases and endonucleases, and finally, a new strand is synthesized across the break. Although these strand breaks are mostly transient under normal conditions, elevated levels of oxidative DNA lesions can increase the early AP lyase activities generating a higher number of SSBs that can be more persistent (Yang et al., 2004; Yang et al., 2006). These SSBs can exacerbate the DNA damage by interfering with the replication fork causing it to collapse, and ultimately becoming double strand breaks (DSBs). Additionally, SSBs in close proximity can become complex lesions to form DSBs (Caldecott, 2024).

Evidence Collection Strategy

Include a description of the approach for identification and assembly of the evidence base for the KER. For evidence identification, include, for example, a description of the sources and dates of information consulted including expert knowledge, databases searched and associated search terms/strings. Include also a description of study screening criteria and methodology, study quality assessment considerations, the data extraction strategy and links to any repositories/databases of relevant references.Tabular summaries and links to relevant supporting documentation are encouraged, wherever possible. More help

The strategy for collating the evidence to support the relationship is described in Kozbenko et al 2022. Briefly, a scoping review methodology was used to prioritize studies based on a population, exposure, outcome, endpoint statement.

Evidence Supporting this KER

Addresses the scientific evidence supporting KERs in an AOP setting the stage for overall assessment of the AOP. More help

Overall Weight of Evidence: Low

Biological Plausibility

Addresses the biological rationale for a connection between KEupstream and KEdownstream. This field can also incorporate additional mechanistic details that help inform the relationship between KEs, this is useful when it is not practical/pragmatic to represent these details as separate KEs due to the difficulty or relative infrequency with which it is likely to be measured. More help

The mechanism of repair of oxidative DNA damage in humans is well-established and numerous literature reviews are available on this topic (Berquist and Wilson III, 2012; Cadet and Wagner, 2013). Oxidative DNA damage is mostly repaired via base excision repair (BER) and via nucleotide excision repair (NER) to a lesser extent. With an increase in oxidative DNA lesions, the more glycosylase and lyase activities occur, introducing SSBs at a higher rate than at homeostasis. It is highly plausible that an increase in SSBs also increases the risk for DSBs, which are more difficult to repair accurately. Previous studies have reported thresholded dose-response curves in oxidative DNA damage and attributed these observations to failed repair at the inflection point on the curve, thus allowing strand breaks to accumulate (Gagne et al., 2012; Seager et al., 2012). When DNA bases sustain oxidative damage via ROS through base oxidation or deletion, this creates small nicks in the DNA strand (Cannan & Pederson, 2016). The bases guanine and adenine are most vulnerable to oxidative damage due to their low oxidation potentials (Fong, 2016). The mechanism of repair, BER, will work to fix these SSBs. If there are multiple SSBs close together in space and time, there will be many sites of BER occurring close together that can cause strain on the strand and result in the conversion of the SSBs to DSBs prior to completion of repair (Cannan & Pederson, 2016).

Empirical Evidence

Provides specific (citable) evidence that supports the idea of a change in the upstream KE (KEupstream) leading to, or being associated with, a subsequent change in the downstream KE (KEdownstream), assuming the perturbation of KEupstream is sufficient. More help

The studies collected frequently address both dose and temporal concordance within a single study. Thus, we have not split out these types of empirical data by sub-headings. Instead, we indicate what evidence is available both in vitro and in vivo.

In vitro studies

Concentration concordance in the formation of oxidative DNA lesions and strand breaks in HepG2 cells treated with nodularin (ROS-inducing substance (Bouaicha and Maatouk, 2004)) (Lankoff et al., 2006):
- A concentration-dependent increase in oxidative lesions and strand breaks was observed after 6, 12, and 24h of treatment using Fpg-modified and regular comet assays, respectively.
  - At 6h, the increase in oxidative lesions was significant at 2.5, 5, and 10 µg/mL, while the increase strand breaks was significant at 5 and 10 µg/mL.
  - At 12 and 24 h, the increase in lesions was significant from 1 µg/mL and above, while significant increase in strand breaks occurred from 2.5 µg/mL and above.
- At all time points, significant increase in oxidative DNA lesions occurred at a lower concentration than DNA strand breaks.
- These results demonstrate the concentration concordance in the formation of oxidative DNA lesions and DNA strand breaks.
Concentration and temporal concordance in human glioblastoma LN-229 cells treated with artesunate, a ROS inducing agent (Berdelle et al., 2011).
- Concentration and time dependent increases in oxidative lesions were observed using the +Fpg comet test and immunofluorescence staining of 8-oxo-dG.
  - Significant increases in oxidative lesions were observed in cells treated with 25 µg/ml after 6 and 24 hours of treatment, but not 2 and 4 hours, using the + Fpg comet. No increases were observed using -Fpg comet.
  - Concentration-dependent increases in oxidative lesions were observed at the 24 hour timepoint using the +Fpg comet (50 and 75 µg/ml).
  - Oxidative lesions were also measured using immunofluorescence staining of 8-OxodG. Significant increases in oxidative lesions were observed at 6 and 8 hours of continuous treatment with 15 ug/ml artesunate, but not 1 and 4 hours.
    - Upon removal of test chemical, 8-OxodG levels decreased, returning to negative control level after 6 hours.
- Significant increases in strand breaks as measured by ɣH2AX were observed 2 and 10 hours after treatment (15 µg/ml).
Deferme et al. (2013) exposed HepG2 cells to 100 µM menadione, 200 µM tert butylhydroperoxide, and 50 µM hydrogen peroxide for increasing durations (30 min, 1, 2, 4, 6, 8, 24 h). The temporal profiles of strand breaks and oxidative lesions were analyzed. The results shown below demonstrate incidence and temporal concordance in oxidative lesion formation and strand breaks (Deferme et al., 2013).
- Strand breaks were measured by alkaline comet assay.
- Oxidative DNA lesions were measured by Fpg-modified comet assay
- Menadione: strand breaks and oxidative lesions increased in a time-dependent manner from 30 min to 4h, when both reached their maximum. The tail moment values of fpg-digested comets were significantly higher than those of no-fpg comets at 1, 2, and 4h, indicating that the induction of oxidative lesions was significant at these time points. After 4h, both strand breaks and oxidative lesions gradually decreased.
- Tert butylhydroperoxide: From 30 min to 1h, both strand breaks and oxidative lesions increased and gradually decreased from 2 to 24h. Oxidative lesion induction was significant at both 30min and 1h.
- Hydrogen peroxide: The highest amount of strand breaks and oxidative lesions occurred at 30 min. From 1h onward, the levels of both decreased. Notably, the induction of oxidative lesions was significant at 30min and also at 1h, despite the decrease from 30min.
Rat alveolar epithelial type II cells (AECII) were isolated from neonatal Wistar rats within 24h of birth and cultured. Cells were then incubated under either normorxic conditions (21% O₂and 5% CO₂) or hyperoxic conditions (90% O₂and 5% CO₂) for 12, 24, 48 or 72h (Jin et al., 2015).
- Time-dependent increases in 8-oxodG were detected by ELISA under hyperoxic conditions; the level of 8-oxodG at 24h was significantly higher than at 12h (p-value <0.05), and the level had further increased significantly when measured at 48h (p-value <0.05) and remained constant until 72h.
- At all time points, the level of 8-oxodG in hyperoxic cells was signficantly higher than in normoxic cells.
- Time-dependent increases in DNA strand breaks were also observed in hyperoxic cells in the alkaline comet assay. The Olive tail moment in hyperoxic cells was significantly higher than in normoxic cells at all time points. However, the time-dependent increase in strands breaks in hyperoxic cells was statistically significant only at 72h (p-value <0.01).
- No change was observed in the level of DNA strands breaks or 8-oxodG in normoxic cells across all time points.

In vivo studies

Concentration concordance in Wistar rats orally exposed to ochratoxin A (OTA) and fumonisin B1 (FB1), ROS inducing agents (Domijan et al., 2006).
- Kidney cells of male Wistar rats were examined using the comet assay +/- Fpg after oral exposure to OTA for 15 days (5ng, 0.05 mg, 0.5 mg/kg b.w.) or FB1 for 5 days (200 ng, 0.05 mg, 0.5 mg/kg b.w.).
  - Significant increases in oxidative lesions were observed using +Fpg comet at all concentrations tested of both OTA and FB1
  - Significant increases were observed in strand breaks using the standard comet assay at all concentrations of both OTA and FB1.

Uncertainties and Inconsistencies

Addresses inconsistencies or uncertainties in the relationship including the identification of experimental details that may explain apparent deviations from the expected patterns of concordance. More help

As demonstrated by the Domijan et al paper, results can be complicated by mixed MOA’s. The comet results were positive with and without Fpg suggesting oxidative stress is not the only mechanism.

Known modulating factors

This table captures specific information on the MF, its properties, how it affects the KER and respective references.1.) What is the modulating factor? Name the factor for which solid evidence exists that it influences this KER. Examples: age, sex, genotype, diet 2.) Details of this modulating factor. Specify which features of this MF are relevant for this KER. Examples: a specific age range or a specific biological age (defined by...); a specific gene mutation or variant, a specific nutrient (deficit or surplus); a sex-specific homone; a certain threshold value (e.g. serum levels of a chemical above...) 3.) Description of how this modulating factor affects this KER. Describe the provable modification of the KER (also quantitatively, if known). Examples: increase or decrease of the magnitude of effect (by a factor of...); change of the time-course of the effect (onset delay by...); alteration of the probability of the effect; increase or decrease of the sensitivity of the downstream effect (by a factor of...) 4.) Provision of supporting scientific evidence for an effect of this MF on this KER. Give a list of references. More help

Quantitative Understanding of the Linkage

Captures information that helps to define how much change in the upstream KE, and/or for how long, is needed to elicit a detectable and defined change in the downstream KE. More help

A limited number of studies explored the quantitative correlation between oxidative DNA lesions and DNA strand breaks. There are computational models availabe that describe this relationship. Spassova et al. (2015) developed a simulated kinetic model of KBrO₃-induced oxidative DNA damage based on Michaelis-Menten enzyme kinetics to study the effect of BER on the shape of the dose-response curve of 8-oxo-dG lesions and strand breaks (Spassova et al., 2015).

Both time and concentration dependence of the responses were explored.
The time course simulation of a sustained exposure at various concentrations produced a sharp increase in 8-oxo-dG immediately following exposure.
- The authors attributed this accumulation to lagged, inefficient repair.
This increase was later followed by a steep decrease in 8-oxo-dG lesions, accompanied by a linear increase in SSBs.
- The repair of adducts by BER, both successful and failed, are responsible for the decrease of 8-oxo-dG; the SSBs are generated as a result of repair failure.
Moreover, the concentration-response model of 8-oxo-dG showed a thresholded curve, where no DNA damage was observed at low concentrations due to effective repair up to a certain concentration of KBrO₃ indicating insufficient repair at the inflection point.

Response-response Relationship

Provides sources of data that define the response-response relationships between the KEs. More help

Time-scale

Information regarding the approximate time-scale of the changes in KEdownstream relative to changes in KEupstream (i.e., do effects on KEdownstream lag those on KEupstream by seconds, minutes, hours, or days?). More help

Known Feedforward/Feedback loops influencing this KER

Define whether there are known positive or negative feedback mechanisms involved and what is understood about their time-course and homeostatic limits. More help

N/A

Domain of Applicability

A free-text section of the KER description that the developers can use to explain their rationale for the taxonomic, life stage, or sex applicability structured terms. More help

This KER is plausible in all life stages, sexes, and organisms with DNA. The majority of the evidence is from in vivo male rats and human male adolescent in vitro models.

References

List of the literature that was cited for this KER description. More help

Berdelle, N., Nikolova, T., Quiros, S., Efferth, T., Kaina, B. (2011), Artesunate Induces Oxidative DNA Damage, Sustained DNA Double-Strand Breaks, and the ATM/ATR Damage Response in Cancer Cells, Mol Cancer Ther, 10:2224-2233.

Berquist, B., Wilson III, D. (2012), Pathways for Repairing and Tolerating the Spectrum of Oxidative DNA Lesions, Cancer Lett, 327:61-72.

Bouaicha, N., Maatouk, I. (2004), Microcystin-LR and nodularin induce intracellular glutathione alteration, reactive oxygene species production and lipid peroxidation in primary cultured rat hepatocytes, Toxicol Lett, 148:53-63.

Cadet, J., Wagner, J.R. (2013), DNA Base Damage by Reactive Oxygen Species, Oxidizing Agents, and UV Radiation, Cold Spring Harb Perspect Biol, 5:a012559.

Cannan, W. and D. Pederson. (2016), “Mechanisms and consequences of double-strand DNA break formation in chromatin”, Journal of Cell Physiology, Vol.231/1, Wiley, Hoboken, https://doi.org/10.1002/jcp.25048.

Deferme, L., Briede, J.J., Claessen, S.M., Jennen, D.G., Cavill, R., Kleinjans, J.C. (2013), Time series analysis of oxidative stress response patterns in HepG2: A toxicogenomics approach , Toxicol, 306:24-34.

Domijan, A., Zeljezic, D., Kopjar, D., Peraica, M. (2006), Standard and Fpg-modified comet assay in kidney cells of ochratoxin A- and fumonisin B(1)-treated rats, Toxciol, 222:53-59.

Fong, C.W. (2016), “Platinum anti-cancer drugs: Free radical mechanism of Pt-DNA adduct formation and anti-neoplastic effect”, Free Radical Biology and Medicine, Vol.95/June 2016, Elsevier, Amsterdam, https://doi.org/10.1016/j.freeradbiomed.2016.03.006.

Gagne, J., Rouleau, M., Poirier, G. (2012), PARP-1 Activation— Bringing the Pieces Together, Science, 336:678-279.

Jin, L., Yang, H., Fu, J., Xue, X., Yao, L., Qiao, L. (2015), Association between oxidative DNA damage and the expression of 8-oxoguanine DNA glycosylase 1 in lung epithelial cells of neonatal rats exposed to hyperoxia, Mol Med Rep, 11: 4079-4086.

Kozbenko, T. et al. (2022), “Deploying elements of scoping review methods for adverse outcome pathway development: a space travel case example”, International Journal of Radiation Biology, 1–12. https://doi.org/10.1080/09553002.2022.2110306

Lankoff, A., Wojcik, A., Fessard, V., Meriluoto, J. (2006), Nodularin-induced genotoxicity following oxidative DNA damage and aneuploidy in HepG2 cells, Toxicol Lett, 164:239-248.

Seager, A., Shah, U., Mikhail, J., Nelson, B., Marquis, B., Doak, S., Johnson, G., Griffiths, S., Carmichael, P., Scott, S., Scott, A., Jenkins, G. (2012), Pro-oxidant Induced DNA Damage in Human Lymphoblastoid Cells: Homeostatic Mechanisms of Genotoxic Tolerance, Toxicol Sci, 128:387-397.

Spassova, M., Miller, D., Nikolov, A. (2015), Kinetic Modeling Reveals the Roles of Reactive Oxygen Species Scavenging and DNA Repair Processes in Shaping the Dose-Response Curve of KBrO3-Induced DNA Damage, Oxid Med Cell Longev, 2015:764375.

Yang, N., Chaudry, A., Wallace, S. (2006), Base excision repair by hNTH1 and hOGG1: A two edged sword in the processing of DNA damage in gamma-irradiated human cells, DNA Repair, 5:43-51.

Yang, N., Galick, H., Wallace, S. (2004), Attempted base excision repair of ionizing radiation damage in human lymphoblastoid cells produces lethal and mutagenic double strand breaks, DNA Repair, 3:1323-1334.