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Relationship: 1939

Title

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Increase, DNA strand breaks leads to Increase, Chromosomal aberrations

Upstream event

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Increase, DNA strand breaks

Downstream event

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Increase, Chromosomal aberrations

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
rat Rattus norvegicus High NCBI
mouse Mus musculus 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 strand breaks (single and double) can arise from endogenous processes (e.g., topoisomerase reaction, excision repair, and VDJ recombination) and exogenous insults (e.g., replications stressors, ionizing radiation, and reactive oxygen species). Single strand breaks (SSBs) are generally repaired rapidly without error. However, multiple SSBs in close proximity to each other and interference of replication by unrepaired SSBs can lead to double strand breaks (DSB). DSB are more difficult to repair and are more toxic than SSB (Kuzminov, 2001). DSBs may lead to chromosomal breakages that may permanently alter the structure of chromosomes (i.e., chromosomal aberrations) and cause loss of DNA segments.

Evidence Supporting this KER

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Biological Plausibility

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DNA strand breaks are a necessity for chromosomal aberrations to occur. However, not all strand breaks lead to clastogenic events as most of them is repaired rapidly by a variety of different repair mechanisms. DNA DSBs are the critical damage because they lead to chromosome breakage. It is well-understood that unrepaired DSBs can lead to chromosomal aberrations. Studies have demonstrated DSBs leading to irreversible structural damage; for example, treatment of cultured cells with replication stress-inducing agents such as hydroxyurea induced micronuclei that are positive for gamma-H2AX, a marker of DSBs (Xu et al., 2010). The link between DSBs and the importance of DSB repair in preventing chromosomal aberrations/genomic instability is extensively discussed in literature and many reviews are available (van Gent et al., 2001; Ferguson and Alt, 2001; Hoeijmakers, 2001; Iliakis et al., 2004; Povirk, 2006; Weinstock et al., 2006; Natarajan and Palitti, 2008; Lieber et al., 2010; Mehta and Haber, 2014; Ceccaldi et al., 2016; Chang et al., 2017; Sishc and Davis, 2017; Brunet and Jasin, 2018).

In addition, attempted repair of DSBs can lead to chromosomal aberrations such as translocations; NHEJ is a recognized source of oncogenic translocations in human cancers (Ferguson and Alt, 2001; Weinstock et al., 2006; Byrne et al., 2014; Brunet and Jasin, 2018), and a contributor to the carcinogenic process (Hoeijmakers, 2001; Sishc and Davis, 2017).

Empirical Evidence

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In vitro studies demonstrating dose and temporal concordance

  • In the 2009 and 2011 studies by Platel et al. TK6 cells were exposed to bleomycin and glucose oxidase (H2O2-generating enzyme) for 1 hour at increasing concentrations (Platel et al., 2009; Platel et al., 2011).
    • Concentration-dependent increase in DNA strand breaks was measured using the alkaline comet assay 1 hr post-exposure
      • First statistically significant concentration: bleomycin: 0.5 µM; GOx: 1.08x10-5 units/mL
      • NOEL could not be defined, indicating that there was response at every tested concentration.
    • MN frequency was measured 23 hours post exposure; concentration-dependent increase in MN frequency was observed and NOEL was identified.
      • NOEL: bleomycin: 0.023 µM; GOx: 1.78x10-5 units/mL
      • All concentrations above the NOEL induced significant increases in MN frequency.
    • Thus, the data demonstrate temporal concordance for both stressors; lack of concordance in the concentration at which response for bleomycin occurs is likely due to differences in detection sensitivities between these assays.


 

  • Strand breaks and chromosomal breakage were measured in V79 cells with the comet assay and the MN test after exposure to hyperbaric oxygen at 3 bar for different periods of time (Rothfuss et al., 1999).
    • Stand breaks were observed in the comet assay after treatment of 3 bar hyperbaric oxygen starting at treatment times of 30 mins. The effect increased constantly up to 180 min.
    • The MN frequency was measured 20 h post treatment and showed increasing numbers of MN starting at treatment times of 30 mins, being clearly increased at treatment times of 60 min up to 180 min. 
    • These data demonstrate both dose- and temporal concordance in DNA strand breaks observed by comet assay and MN frequency.

 

  • Lymphoblastoid cell lines were investigated with the comet assay and the MN test using gamma irradiation of 1 and 2 Gy (Trenz et al., 2003). Pulsed field gel electrophoresis was used additionally to investigate the occurrence of strand breaks (Trenz et al., 2005).
    • Strand breaks were shown in the comet assay in all cell lines tested, immediately after treatment with 1 and 2 Gy.  
    • 40 h post treatment the cell lines were prepared for MN analysis: an increase in MN frequency was shown in all cell lines after treatment with 1 and 2 Gy.
    • Thus, the data demonstrate both temporal and dose concordance.

 

  •  Watters et al. (2009) treated mouse embryonic fibroblasts (MEFs) with bleomycin for 4 hours and conducted comparative investigations using the H2AX assay, the comet assay and the MN test (Watters et al., 2009).
    • The occurrence of DNA DSB was shown with the gamma-H2AX assay immediately following exposure. The number of foci increased up to 0.1 µg/ml; however, it was not statistically significant until 1 µg/ml and above.
    • The comet assay showed a continuous increase in tail moment immediately following exposure, showing more than 2-fold increase at 10 µg/ml, but did not reach statistical significance.
    • Significant increases in MN frequency was observed 26h post exposure (~1.5 cycles) at concentrations of 0.1µg/ml and above.
    • These data support temporal concordance; lack of concordance in the dose at which the endpoints reach statistical significance is likely the rest of different sensitivities of these assays.

 

  • Using bleomycin as a stressor, Kawaguchi et al. monitored DNA strand breaks in TK6 human lymphoblastoid cells with the comet assay/modified comet assay using DNA repair inhibitors and monitored clastogenic events with the MN test after a treatment period of 2h (Kawaguchi et al., 2010).
    • In the regular alkaline comet assay an increase in DNA strand breaks was observed immediately following the 2h exposure, reaching significance at 12.5 µg/mL, and in the modified AraC/HU version at 6.25 µg/ml.
    • A statistically significant increase in MN frequency was observed 24 h after treatment at 5 µg/mL.
    • This provides support for temporal-concordance and the lack of dose-concordance is consistent with the increased sensitivity of the MN assay relative to the comet assay.

 

  • Wild type and N-methylpurine DNA glycosylase (MPG)-deficient (Mpg-/-) Mouse embryonic fibroblasts (MEFs) were treated with increasing concentrations of methyl methane sulfonate (MMS) (0.5, 1, 1.5, 2.5 mM) for 1 hour (Ensminger et al., 2014).
    • DSBs were measured as the number of γH2AX foci immediately following the exposure.
    • There was a concentration-dependent increase in DSBs in wild type MEFs, and the increase was significantly larger in wild type compared to Mpg-/- cells at every concentration.
    • Chromosomal aberrations (breaks and translocations) were monitored in metaphase spreads 24h following 1h 1 mM MMS treatment.
    • At 1 mM MMS, the amount of chromatid breaks and translocations was significantly larger in wild type cells, compared to Mpg-/- cells, concordant with the observations in DSBs.
    •  The results support that increases in DSBs lead to increases in chromosomal aberrations.

 

  • Dertinger et al. (2019) exposed TK6 cells to 34 diverse genotoxic chemicals over a range of concentrations for 24 hrs (Dertinger et al., 2019). At 4 and 24 hr time points cell aliquots were evaluated with the MultiFlow assay, which includes the gH2AX biomarker. At the 24 hr time point, remaining cells were evaluated with the in vitro MicroFlow assay, which includes %MN measurements.
    • Benchmark dose analyses were conducted to estimate Point of Departure values for MN and gamma-H2AX responses.   
    • In vitro MN and gamma-H2AX BMD confidence intervals for 18 clastogens were graphed on cross system plots. Good correlations were observed for 24 hr MN and 24 hr gamma-H2AX (shown), as well as 24 hr MN and 4 hr gamma-H2AX (not shown).
    • Thus, the data demonstrate both temporal and dose concordance for these endpoints.

 

  • Isolated lymphocytes and whole blood samples taken from four healthy, adult males were exposed to gamma-ray radiation at 20 cGy/minute at doses ranging from 0 – 50 cGy. Immediately following irradiation, DNA strand breaks were assessed using the comet assay and chromosomal aberrations were examined by cytogenetic analysis (Sudprasert et al., 2006).
    • In irradiated lymphocytes, there were dose-dependent increases in the number of DNA strand breaks, with significant increases in strand breaks evident from 5 – 50 cGy doses.    
    • Irradiated whole blood samples showed significantly increased strand breaks by 10 cGy, but this level stayed relatively stable from 10 - 50 cGy.
    • Analysis of chromosomal aberrations in irradiated whole blood samples indicated dose-dependent increases in deletions and dicentric chromosomes across 50 cGy, with more deletions detected than dicentrics. All doses (5 – 50 cGy) showed significantly more aberrations than unirradiated controls.
    • The results of this study support dose concordance and are suggestive of time concordance.

 

  • In a study by Chernikova et al. 1999, PL61 cells were exposed to radiation sensitizer/DNA repair inhibitor wortmannin prior to gamma-ray irradiation, and then analyzed for DSBs and micronuclei (indicative of chromosomal aberrations) (Chernikova et al., 1999).
    • DSB experiments were performed with cells treated with 25 µM of wortmannin + radiation, and with cells exposed only to radiation. In both cases, there was a linear, dose-dependent increase in the number of DSBs across radiation doses ranging from 0 – 60 Gy, as measured by the FAR assay. Wortmannin treatment did not affect the number of DSBs that were formed.
    • In terms of DNA repair, however, cells irradiated with 45 Gy of gamma-rays showed a dose-dependent decline in the percentage of DNA repair with increasing wortmannin concentrations from 0 – 25 µM.
    • Furthermore, cells treated with wortmannin + 2 Gy of radiation demonstrated a dose-dependent increase in the number of micronuclei from 0 – 25 µM of wortmannin.
    • Overall, the results of this study suggest that as the number of DSBs increase and repair processes are inhibited, there is a corresponding increase in the number of chromosomal aberrations. Thus the data demonstrate dose concordance and essentiality.

 

  • Iliakis, et al. (2019) studied the relationship between DSB damage and chromosomal aberrations using an experimental model that mimics the clustered DNA DSB damage induced by high linear energy transfer (LET) radiation (Iliakis et al., 2019). Chinese hamster ovary cells and human retinal epithelial cells were engineered to carry I-SceI meganuclease recognition sites at specific locations in order to generate specific DSB clustered damage. Cells were then transfected with plasmids expressing I-SceI to induce the DNA breakages. Twelve hours or 24 hours post-transfection, cells were analyzed by immunofluorescence microscopy for DSBs, and by cytogenetic analysis for chromosome translocations.    
    • DSBs were increased in all cells transfected with the endonuclease relative to cells from the same cell lines that underwent a mock transfection.
    • Chromosomal translocations were also elevated in cell lines transfected with an endonuclease, with increasing chromosomal translocations found in cells with increasing DSB cluster damage.
    • This study shows an association between DSB cluster damage and chromosomal translocation incidence.

 

In vivo studies

  • Sprague-Dawley rats were dosed with different genotoxic compounds at select concentrations (methotrexate, cisplatin, chlorambucil, and cyclophosphamide) and blood samples were collected at different time points following the dosing (6, 12, 24, 36, 48, 72, and 96 hours post dosing) (Mughal et al., 2010).
    • Peripheral blood lymphocytes were isolated for comet assay and peripheral blood erythrocytes were used to measure MN at each time point.
      • Different comet assay parameters such as tail length, moment, olive tail moment, and % tail DNA were compared to MN frequency
      • All comet assay parameters had a positive correlation to MN frequency demonstrated in all chemical treatments.
      • DNA tail length and % tail DNA showed visible increases in strand breaks at early time points (6 and 12h), while the increase in MN frequency was not observed until after 12-24 h.
      • This early response at 6 h was not observed in tail moment or olive tail moment; these two paramenters did not show as strong of a response as tail length and % tail DNA to all four chemical treatments.
    • The results suggest temporal concordance in strand breaks measured by comet assay and induction of MN, where strand breaks are observed earlier than MN.

 

  • C57BL/6 mice were irradiated with increasing doses of X-rays (1.1, 2.2, 4.4 Gy) at rate of 1.03 Gy/min (acute high dose) and 0.31 cGy/min (low dose rate). Lymphocytes were isolated and collected 24h and 7 days from the start of irradiation (different mice were used for each time point) (Turner et al., 2015).
    • γH2AX measured at 24h showed a dose-dependent increase in DSBs in both acute and low dose rate exposed mice.
      • The level of DSBs due to the acute dose treatment was significantly higher than due to the low dose rate treatment at 1.1 and 2.2 Gy.
    • MN frequency was also measured 24h and 7 days post exposure;
      • At both time points and in both treatment groups, MN frequency increased with dose from 1.1 and 2.2 Gy. However, there was no further increase at 4.4 Gy
      • There was no statistical difference in the two treatment groups

Overall, the above data demonstrate that when strand breaks occur there is an increase in MN frequency, which is indicative of chromosomal aberrations. There is a clear temporal-concordance but dose-concordance is not always consistent due to differences in assay sensitivity.

Uncertainties and Inconsistencies

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As described above, statistically significant increases in MN occur, in some cases, at lower concentrations than strand breaks measured with the comet assay (Platel et al., 2001; Watters et al., 2009; Kawaguchi et al., 2010). The two assays measure different endpoints at different time points; the MN test may appear to be more sensitive than the comet assay but it is difficult to directly compare these two assays.

Mughal et al. (2010) study compared different parameters of comet assay (tail moment, length, and % tail DNA) to MN frequency. Depending on the parameter, the observation of increase in strand breaks varied. For example, % tail DNA would show a visible increase in strand breaks at one concentration; however, no change would be observed in the tail moment calculated using the same data. Use of different parameters in presenting comet assay data may add subjectivity to the results that are reported in certain papers.

Rossner Jr. et al. exposed human embryonic lung fibroblasts (HEL12469) to 1, 10, and 25 µM of benzo[a]pyrene (B[a]P) for 24 hours and measured DSB (γH2AX immunodetection by Western blotting) and translocations (by fluorescence in situ hybridization of chromosomes 1, 2, 4, 5, 7, 17) (Rossner Jr. et al., 2014).

  • Increases in γH2AX were observed only at 25 µM B[a]P (~2.5 fold increase) after the 24h exposure.
  • Translocations were quantified and expressed as the genomic frequency of translocations per 100 cells (FG/100)
    • All concentrations of B[a]P induced an elevated frequency of translocations compared to the DMSO control (DMSO: ~0.19/100; 1 µM: ~0.53/100 cells; 10 µM: ~0.33/100; 25 µM: ~0.39/100)

In this study, the increase in translocations was detected at concentrations that did not induce an increase in γH2AX signal. This observation of the discordant relationship between γH2AX and translocations may be due to the differences in assay sensitivity. In addition, immunodetection by Western blotting cannot precisely measure small changes in protein content.

Quantitative Understanding of the Linkage

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As with the regularly used alkaline comet assay a variety of DNA damage is detected – SSBs, DSBs, alkaline labile sites, as well as sites of DNA repair; thus, a quantitative understanding for specific types of damage is rather difficult. There exists the possibility to quantify the amount of DNA breaks by comparing the induced damage with Gy equivalents (Collins et al., 2008), however, this is not the standard. DSBs can be measured more specific with the neutral version of the Comet assay, however, this version is not that regularly used. As reviewed in Takahashi et al, 2005 the efficiency of DSB detection measured with the PFGE, the neutral Comet assay and the DNA elution assay has a lower detection limit of 100 DSB per cell (Collins et al., 2008). In contrast to this, each Gamma-H2AX focus seems to represent a DNA DSB in vivo (Rogakou et al., 1999)

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 chromosomal aberrations can occur in any eukaryotic and prokaryotic cell.

References

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Brunet, E. & M. Jasin (2018), "Induction of chromosomal translocations with CRISPR-Cas9 and other nucleases: Understanding the repair mechanisms that give rise to translocations.", Adv. Exp. Med. Biol. 1044:15-25.

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

Ceccaldi, R., B. Rondinelli & A.D. D'Andrea (2016), "Repair Pathway Choices and Consequences at the Double-Strand Break.", Trends Cell Biol. 26(1):52-64.

Chang, H. et al. (2017), "Non-homologous DNA end joining and alternative pathways to double‑strand break repair.", Nature Rev. Mol. Cell. Biol., 18:495-506.

Chernikova, S.B., R.L. Wells & M. Elkind (1999), "Wortmannin Sensitizes Mammalian Cells to Radiation by Inhibiting the DNA-Dependent Protein Kinase-Mediated Rejoining of Double-Strand Breaks.", Radiat. Res., 151:159-166.

Collins, A.R. et al. (2008), "The comet assay: topical issues.", Mutagenesis, 23:143-151.

Dertinger, S.D. et al. (2019), "Predictions of genotoxic potential, mode of action, molecular targets, and potency via a tiered multiflow® assay data analysis strategy.", Environ. Mol. Mutagen., 60(6):513-533

Ensminger, M. et al. (2014), "DNA breaks and chromosomal aberrations arise when replication meets base excision repair.", J. Cell Biol.,  206:29.

Ferguson, D.O. & F.W. Alt (2001), "DNA double strand break repair and chromosomal translocation: Lessons from animal models.", Oncogene 20(40):5572–5579.

Hoeijmakers, J.H. (2001), "Genome maintenance mechanisms for preventing cancer.", Nature, 411:366-374.

Iliakis, G. et al. (2019), "Defined Biological Models of High-LET Radiation Lesions.", Radiat. Protect Dosimet., 183:60-68.

Iliakis, G. et al. (2004), "Mechanisms of DNA double strand break repair and chromosome aberration formation.", Cytogenet. Genome Res. 104:14-20.

Kawaguchi, S. et al. (2010), "Is the comet assay a sensitive procedure for detecting genotoxicity?.", J. Nucleic Acids, 2010:541050.

Kuzminov, A. (2001), "Single-strand interruptions in replicating chromosomes cause double-strand breaks.", Proc. Natl. Acad. Sci. USA 95:8241-8246.

Lieber, M. et al. (2010), "Nonhomologous DNA End Joining (NHEJ) and Chromosomal Translocations in Humans.", Subcell Biochem., 50:279-296.

Mehta, A. & J. Haber (2014), "Sources of DNA Double-Strand Breaks and Models of Recombinational DNA Repair.", Cold Spring Harb. Perspect Biol., 6:a016428.

Mughal, A. et al. (2010), "Micronucleus and comet assay in the peripheral blood of juvenile rat: Establishment of assay feasibility, time of sampling and the induction of DNA damage.", Mutat. Res. Gen. Tox. En., 700:86-94.

Natarajan, A.T & F. Palitti (2008), "DNA repair and chromosomal alterations.", Mutat. Res., 657:3-7.

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.

Platel, A. et al. (2009), "Study of oxidative DNA damage in TK6 human lymphoblastoid cells by use of the in vitro micronucleus test: Determination of No-Observed-Effect Levels.", Mutat. Res., 678:30-37.

Povirk, L. (2006), "Biochemical mechanisms of chromosomal translocations resulting from DNA double-strand breaks.", DNA Repair 5:1199-1212.

Rogakou, E.P. et al. (1999), "Megabase chromatin domains involved in DNA double-strand breaks in vivo.", J. Cell Biol., 146:905-916.

Rossner, Jr. P et al. (2014), "Nonhomologous DNA end joining and chromosome aberrations in human embryonic lung fibroblasts treated with environmental pollutants.", Mutat. Res., 763-764:28-38.

Rothfuss, A. et al. (1999), "Evaluation of mutagenic effects of hyperbaric oxygen (HBO) in vitro.", Environ. Mol. Mutagen., 34:291-296.

Sishc, B.J. & A.J. Davis (2017), "The Role of the Core Non-Homologous End Joining Factors in Carcinogenesis and Cancer.", Cancers (Basel), 9(7): pii E82.

Sudprasert, W., P. Navasumrit & M. Ruchirawat (2006), "Effects of low-dose gamma radiation on DNA damage, chromosomal aberration and expression of repair genes in human blood cells.", Int. J. Hyg. Environ.-Health, 206:503-511.

Trenz, K., J. Landgraf & G. Speit (2003), "Mutagen sensitivity of human lymphoblastoid cells with a BRCA1 mutation.", Breast Cancer Res. Treat., 78:69-79.

Trenz, K., P. Schutz & G. Speit (2005), "Radiosensitivity of lymphoblastoid cell lines with a heterozygous BRCA1 mutation is not detected by the comet assay and pulsed field gel electrophoresis.", Mutagenesis, 20:131-137.

Turner, H.C. et al. (2015), "Effect of Dose Rate on Residual c-H2AX Levels and Frequency of Micronuclei in X-Irradiated Mouse Lymphocytes.", Radiat. Res., 183:315-324.

van Gent, D., J.H. Hoeijmakers & R. Kanaar (2001), "Chromosomal Stability and the DNA Double-Stranded Break Connection.", Nature 2:196-206.

Watters, G.P. et al. (2009), "H2AX phosphorylation as a genotoxicity endpoint.", Mutat. Res., 670:50-58.

Weinstock, D. et al. (2006), "Modeling oncogenic translocations: Distinct roles for double-strand break repair pathways in translocation formation in mammalian cells.", DNA Repair 5:1065-1074.

Xu, B. et al. (2010), "Replication Stress Induces Micronuclei Comprising of Aggregated DNA Double-Strand Breaks.", PLoS One, 6:e18618.