API

Relationship: 1931

Title

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Increase, DNA strand breaks leads to Increase, Mutations

Upstream event

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Increase, DNA strand breaks

Downstream event

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Increase, Mutations

Key Event Relationship Overview

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AOPs Referencing Relationship

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AOP Name Adjacency Weight of Evidence Quantitative Understanding
Oxidative DNA damage leading to chromosomal aberrations and mutations non-adjacent High Low
Direct deposition of ionizing energy onto DNA leading to lung cancer non-adjacent High Low

Taxonomic Applicability

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Term Scientific Term Evidence Link
human Homo sapiens High NCBI
mouse Mus musculus High NCBI
rat Rattus norvegicus High NCBI

Sex Applicability

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Sex Evidence
Unspecific High

Life Stage Applicability

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Term Evidence
All life stages High

Key Event Relationship Description

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DNA single strand breaks (SSB) are generally repaired rapidly and efficiently. However, if left unrepaired, SSBs can interfere with replication and cause the replication fork to collapse resulting in double strand breaks (DSB). Multiple SSBs in close proximity to each other can also give rise to DSBs. DSBs can be repaired virtually error-free by homologous recombination (HR), which uses DNA sequence in the homologous chromosome or sister chromatid as a template for new strand synthesis (Polo and Jackson, 2011). Alternatively, the broken ends may be joined to other sites in the genome regardless of homology via non-homologous end joining (NHEJ), irreversibly altering the DNA sequence (deletion, addition, rearrangement). Because HR is a more time-consuming and labour-intensive process, larger proportions of DSBs are repaired via NHEJ than via HR (Mao et al., 2008a; Mao et al., 2008b).

Alterations in DNA sequence can also occur from structural damage to the chromosomes; observations of micronucleus indicate chromosomal aberrations and that a permanent loss of DNA segments has occurred.

Evidence Supporting this KER

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The mechanisms by which strand breaks lead to mutations are very well studied and understood. Thus, we provide a small selection of empirical evidence below supporting this KER; i.e., we did not undertake and exhaustive literature search.

Biological Plausibility

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The error-prone nature of DSB repair in eukaryotes has been described in numerous reviews. In mammalian and yeast cells, both HR and NHEJ can lead to alteration in DNA sequence; insertions, deletions, and translocations can arise from NHEJ and base substitutions can occur during the repair synthesis of HR (Hicks and Haber, 2010; Bunting and Nussenzweig, 2013; Byrne et al., 2014; Rodgers and McVey, 2016; Dwivedi and Haber, 2018).

                                                                                                                            

Empirical Evidence

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The mechanisms by which strand breaks lead to mutations are very well studied and understood. Thus, we provide a small selection of empirical evidence below supporting this KER; i.e., we did not undertake and exhaustive literature search.

In vitro studies

  • Strand breaks and mutation frequencies were measured in TK6 cells after exposure to bleomycin and glucose oxidase (enzyme that generates H2O2) for 1 hour (Platel et al., 2011).
    • Concentration-dependent increases in strand breaks were measured using the alkaline comet assay.
    • At the same concentrations, mutation frequencies measured by TK gene mutation assay also showed a concentration-dependent increasing trend.
    • No Observed Genotoxic Effect Level was determined in TK assay (bleomycin: 0.6µM; GOx: 1.17x10-5 units/mL) while it couldn’t be identified in comet assay, indicating that every tested concentration induced an increase in strand breaks (First statistically significant concentration: bleomycin: 1.5 µM; GOx: 1.08x10-5 units/mL).

 

  • Spassova et al. (2013) combined the alkaline comet assay data from Luan et al. (2007) and Tk gene mutation assay data from Harrington-Brock et al. (2003) (Spassova et al., 2013).
    • Luan et al. treated TK6 cells with KBrO3 for 4 hours and performed alkaline comet assay to measure strand breaks.
    • Harrington-Brock et al. treated L5178Y/Tk+/- mouse lymphoma cells with KBrO3 for 4 hours and measured the Tk mutant frequency after a 13-day incubation.
    • Spassova et al. (2013) found no significant differences between the two experiments in regression analysis, thus, combined the datasets (same concentration range was used in both studies)
    • In both comet assay and Tk mutation assay, concentration-dependent increase in response was observed.
    • These results demonstrate the occurrence of DNA strand breaks followed by increase in mutations.

 

  • Indirect measurement of mutations by measuring misrejoined DSBs in vitro
    • Rydberg et al. (2005) exposed GM38 human primary dermal fibroblasts to increasing doses of X-rays and linear electron transfer (LET) by nitrogen, helium, and iron ions.
    • DSBs were measured by pulsed field gel electrophoresis (PFGE)
      • Dose-dependent increase in DSBs was observed immediately following irradiation.
    • Misrejoining of ends was monitored using the Hybridization assay:
      • DNA is digested using a restriction enzyme and fractionated by PFGE.
      • 32P-labeled probe for a 3.2-Mbp NotI restriction fragment is then used in Southern blotting to detect intact restriction fragments.
      • Failure to reconstitute the restriction fragment indicates incorrect joining of ends following DSBs and altered DNA sequence.
    • After 16 h of recovery following irradiation, Rydberg et al. observed a radiation dose-dependent increase in misrejoined DSBs in all four treatment groups.
    • A similar study by Kuhne et al. (2005) reported concordant results (Kuhne et al., 2005):
      • Subsequently, there was a dose-dependent increase in misrejoined DSBs 24h post irradiation.
      • Increasing doses of X-rays and γ rays immediately induced DSBs in primary human fibroblasts in a dose-dependent manner.
      • Alterations in the restriction fragment due to irradiation indicate changes in the DNA sequence (i.e., shorter fragments would suggest loss of DNA sequence), thus, induction of mutations (Rydberg et al., 2005; Kuhne et al., 2005).
      • These results demonstrate the concentration and temporal concordance in strand breaks leading to mutations.

 

  • In a study by Kuhne et al. (2000), irradiated normal human fibroblasts were examined for both DSBs and the percentage of misrejoined DSBs (Kuhne et al., 2000).
    • Increasing doses of alpha-particle radiation from 0 – 80 Gy resulted in a linear, dose-dependent increase in the number of DSBs per mega base pair, as measured by the FAR assay.
    • Using X-ray radiation, the percentage of misrejoined DSBs were found to increase approximately linearly from 0 – 40 Gy doses per fraction. By 80 Gy, the rate of misrejoining plateaued at approximately 50%, and this plateau was maintained at X-ray doses between 80 and 320 Gy.
    • Overall, these results provide indirect evidence suggesting that elevated numbers of DSBs may lead to the formation of increasingly more mutations, as indicated by the corresponding increased number of misrejoined DSBs.

 

  • Dikomey et al. (2000) performed a study using normal human skin fibroblasts that were irradiated with 200 kVp X-rays at doses ranging from 0 – 180 Gy, and then were examined for DSBs immediately following irradiation, and for non-repaired DSBs 24 hours after radiation exposure (Dikomey and Brammer, 2000).
    • As measured by constant field gel electrophoresis, there was a dose-dependent increase in the number of DSBs after exposure to X-rays doses of 0 – 80 Gy.
    • The number of non-repaired DSBs also increased with increasing radiation dose from 0 – 180 Gy. After 30 Gy, there were more non-repaired DSBs when cells were exposed to radiation with a high dose-rate (4 Gy/min) relative to those exposed to radiation with a low dose-rate (0.4 Gy/min).
    • These results suggest that there are increasing DSBs with increasing radiation dose, and that there are also an increasing number of DSBs that are not repaired with increasing radiation dose. This is important as non-repaired DSBs may result in mutations in the genome.

 

  • Both lung and dermal fibroblasts were irradiated with 80 kV X-rays at 23 Gy/min, and analyzed for the number of DSBs and the percentage of correctly rejoined DSBs in a study by (Lobrich et al., 2000).
    • Results from the FAR assay showed a linear increase in the number of DSBs in all cell lines for radiation doses ranging from 0 – 80 Gy.
    • After being irradiated with 80 Gy of X-rays, approximately 50% of the DSBs were correctly rejoined, as measured by the hybridization assay.
    • A dose-dependent increase in the number of rearrangements per mega base pair was found in cells irradiated with 0 – 80 Gy of X-rays.
    • The results of this study provide evidence of dose concordance, as the number of DSBs and the number of rearrangements both increase with increasing radiation dose.

In vivo studies

  • Strand breaks and mutation frequencies were measured in the leaves of Nicotiana tabacum var. xanthi after the seedling plants were irradiated with 0 – 10 Gy doses of gamma-ray radiation (Ptacek et al., 2001).
    • DNA strand breaks in the leaves were measured using the Comet assay immediately following irradiation. Results of this assay showed a linear, dose-dependent increase in strand breaks, which were resolved by 24 hour post-irradiation.
    • Mutations in the leaves were measured when the seedling plants put out their 6th or 7th true leaves following irradiation. Similar to results found for radiation-induced strand breaks, there was a corresponding dose-dependent increase in the number of mutations per radiation dose.
    • These results demonstrate a dose concordance between DNA strands breaks and mutation frequency, and suggest a time concordance.

Uncertainties and Inconsistencies

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In Kuhne et al. (2005) and Rydberg et al. (2005) studies provided above, mutation was not directly measured. The PFGE and hybridization assay detects a 3.2-Mbp restriction fragment from chromosome 21. Deviation of DNA restriction fragments from the 3.2-Mbp mark during electrophoresis suggests occurrence of breakage and failed reconstruction in this segment of chromosome 21; induction of mutations can be inferred from the change in the size of the restriction fragments. The remaining 22 chromosomes are not considered. This method may not be sensitive enough to detect small base changes.

Cell cycle can influence the repair pathway of DSBs and, thus, the risk of incorrect rejoining of broken ends. In G1 phase, NHEJ may be favoured, while in S, G2, or M phase, both HR and NHEJ have been observed to be active in repair (Mao et al., 2008b)

Quantitative Understanding of the Linkage

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McMahon et al. (2016) compiled the data from multiple studies spanning different human and mouse cell lines (including Lobrich et al. (2000) and Rydberg et al. (2005)) to model the IR dose-dependent increase in chromosomal aberrations, misrejoined DSBs, and mutation rate (per 104 cells). The data used to model the DSB misrepair rate were generated from the hybridization assay described in the Empirical evidence section. The mutation rate model was based on HPRT gene mutation assay in Chinese hamster cells (McMahon et al., 2016). Previously, in another study, the number of γH2AX foci formed following IR irradiation was quantified over increasing doses of radiation in human fibroblasts (Rothkamm and Lobrich, 2003). Further quantitative studies that exam the relationships between the quantities of strand breaks (e.g., quantity of γH2AX foci (DSBs) formed), DSB misrepair rate, and mutation rate would provide a better quantitative understanding of this KER.

Response-response Relationship

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Time-scale

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Known modulating factors

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Known Feedforward/Feedback loops influencing this KER

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Domain of Applicability

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DNA strand breaks and subsequent mutations can occur in any eukaryotic and prokaryotic cell. Any DNA strand break has potential to cause alterations in DNA sequence (e.g., deletions and insertions), whether it is due to insufficient or faulty repair.

References

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Bunting, S. & A. Nussenzweig (2013), "End-joining, Translocations and Cancer", Nat Rev Cancer, 13:443-454.

Byrne, M. et al. (2014), "Mechanisms of oncogenic chromosomal translocations", Ann. N.Y. Acad. Sci., 1310:89-97.

Dikomey, E. & I. Brammer (2000), "Relationship between cellular radiosensitivity and non-repaired double-strand breaks studied for different growth states, dose rates and plating conditions in a normal human Žfibroblast line.", Int. J. Radiat. Biol., 76:773-781.

Dwivedi, G. & J.E. Haber (2018), "Assaying Mutations Associated With Gene Conversion Repair of a Double-Strand Break", Methods Enzymiol., 601:145-160.

Hicks, W. & J.E. Haber (2010), "Increased Mutagenesis and Unique Mutation Signature Associated with Mitotic Gene Conversion", Nat. Rev. Cancer, 329:82-84.

Kuhne, M., K. Rothkamm & M. Lobrich (2000), "No dose-dependence of DNA double-strand break misrejoining following a -particle irradiation.", Int. J. Radiat. Biol. 76(7):891-900

Kuhne, M., G. Urban & M. Lo, (2005), "DNA Double-Strand Break Misrejoining after Exposure of Primary Human Fibroblasts to C K Characteristic X Rays, 29 kVp X Rays and Co g-Rays.", Radiation Research. 164(5):669-676. doi:10.1667/RR3461.1.

Lobrich, M. et al. (2000), "Joining of Correct and Incorrect DNA Double-Strand Break Ends in Normal Human and Ataxia Telangiectasia Fibroblasts.", 68(July 1999):59–68. doi: 10.1002/(SICI)1098-2264(200001)27:1<59::AID-GCC8>3.0.CO;2-9.

Mao, Z. et al. (2008a), "Comparison of nonhomologous end joining and homologous recombination in human cells.", DNA Repair, 7:1765-1771.

Mao, Z. et al. (2008b), "DNA repair by nonhomologous end joining and homologous recombination during cell cycle in human cells.", Cell Cycle, 7:2902-2906.

McMahon, S.J. et al. (2016), "Mechanistic Modelling of DNA Repair and Cellular Survival Following Radiation-Induced DNA Damage.", Nat. Publ. Gr.(April):1–14. doi:10.1038/srep33290.

Platel, A. et al. (2011), "Study of oxidative DNA damage in TK6 human lymphoblastoid cells by use of the thymidine kinase gene-mutation assay and the in vitro modified comet assay: Determination of No-Observed-Genotoxic-Effect-Levels.", Mutat. Res., 726:151-159.

Polo, S. & S. Jackson (2011), "Dynamics of DNA damage response proteins at DNA breaks: a focus on protein modifications.", Genes Dev., 25:409-433.

Ptacek, O. et al. (2001), "Induction and repair of DNA damage as measured by the Comet assay and the yield of somatic mutations in gamma-irradiated tobacco seedlings.", Mutat. Res., 491:17-23.

Rodgers, K. & M. McVey (2016), "Error-prone repair of DNA double-strand breaks.", J. Cell. Physiol., 231:15-24.

Rothkamm, K. & M. Lobrich (2003), "Evidence for a lack of DNA double-strand break repair in human cells exposed to very low x-ray doses.", PNAS, 100(9):5057-62. doi:10.1073/pnas.0830918100.

Rydberg, B. et al. (2005), "Dose-Dependent Misrejoining of Radiation-Induced DNA Double-Strand Breaks in Human Fibroblasts: Experimental and Theoretical Study for High- and Low-LET Radiation.", Radiat. Res. 163(5):526–534. doi:10.1667/RR3346.

Spassova, M. et al. (2013), "Dose-Response Analysis of Bromate-Induced DNA Damage andMutagenicity Is Consistent With Low-Dose Linear,Nonthreshold Processes", Environ. Mol. Mutagen., 54:19-35.