DNA Repair Measured by the Comet Assay

Azqueta, Amaya; Shaposhnikov, Sergey; Collins, Andrew

doi:10.5772/22504

Cited by 13 publications

(10 citation statements)

References 49 publications

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“…We used two approaches to measure repair of DNA damage [11]. If cells are subjected to damage (SBs or base oxidation), and then incubated to allow cellular repair, the residual lesions can be measured at intervals to show the kinetics of damage removal.…”

Section: Introductionmentioning

confidence: 99%

Vitamin C in Cultured Human (HeLa) Cells: Lack of Effect on DNA Protection and Repair

et al. 2013

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Aims: Dietary antioxidants, including vitamin C, may be in part responsible for the cancer-preventive effects of fruits and vegetables. Human intervention trials with clinical endpoints have failed to confirm their protective effects, and mechanistic studies have given inconsistent results. Our aim was to investigate antioxidant/ pro-oxidant effects of vitamin C at the cellular level. Experimental approach: We have used the comet assay to investigate effects of vitamin C on DNA damage, antioxidant status, and DNA repair, in HeLa (human tumor) cells, and HPLC to measure uptake of vitamin C into cells. Results: Even at concentrations in the medium as high as 200 μM, vitamin C did not increase the background level of strand breaks or of oxidized purines in nuclear DNA. Vitamin C is taken up by HeLa cells and accumulates to mM levels. Preincubation of cells with vitamin C did not render them resistant to strand breakage induced by H2O2 or to purine oxidation by photosensitizer plus light. Vitamin C had no effect on the rate of repair of strand breaks or oxidized bases by HeLa cells. However, vitamin C at a concentration of less than 1 μM, or extract from cells preincubated for 6 h with vitamin C, was able to induce damage (strand breaks) in lysed, histone-depleted nuclei (nucleoids). Conclusion: In these cultured human cells, vitamin C displays neither antioxidant nor pro-oxidant properties; nor does it affect DNA strand break or base excision repair.

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Section: Introductionmentioning

confidence: 99%

Vitamin C in Cultured Human (HeLa) Cells: Lack of Effect on DNA Protection and Repair

et al. 2013

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“…The alkaline COMET assay was used to detect DNA damage in the form of single-strand breaks and alkali-labile sites after exposure to 5 μM of thia/isothiacalothrixin B analogues for 48 hours to HCT116 cells. The alkaline comet assay is simple and known for its sensitivity to detect DNA strand breaks [ 27 ]. The single cell suspension obtained from drug-treated culture plates were embedded in agarose on a microscope slide, lysed and electrophoresed at a high pH (>13).…”

Section: Resultsmentioning

confidence: 99%

Novel isothiacalothrixin B analogues exhibit cytotoxic activity on human colon cancer cells in vitro by inducing irreversible DNA damage

et al. 2018

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Preliminary cytotoxic analysis of sulphur containing isosteric analogues of calothrixin B identified the useful anti-tumour activity of thia/isothiacalothrixin B which necessitated it’s biological evaluation in colon and lung cancer cell lines. The isothia analogues induced cytotoxicity of HCT116 in a time-dependent manner and inhibited the clonogenic survival of HCT116 and NCI-H460 cells in a dose-dependent manner comparable to the standard anti-cancer drug camptothecin. Herein employing flow cytometry, we demonstrate that isothiacalothrixin B analogues inhibited proliferation of colon cancer cells by the arrest of cells in S and G2/M phases over a period of 48 hours at a concentration of 5 μM. Our results also suggest that the cytotoxicity of thia analogues of calothrixin B is partially mediated by induction of cellular DNA strand breaks. The UV-Vis spectroscopic studies with CT-DNA revealed groove binding for calothrixin B and its thia analogues wherein subsequent in silico molecular modelling studies indicated preferential binding to the AT-rich regions of minor groove of DNA. Furthermore, thiacalothrixin B caused transcriptional activation of p21waf1/cip1 promoter and upregulation of its protein levels independent of p53. The induction of DNA damage response pathway leads to apoptosis in isothiacalothrixin B but not in thiacalothrixin B treated cells. The isothia analogues SCAB 4 induced DNA strand breaks and cell cycle arrest even after treatment for a short period (i.e., 4 hours) and the cell cycle effects were irreversible. For the first time, this study provides detailed cellular effects on the potential use of isothiacalothrixin B analogues as cytotoxic agents.

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“…Comet assays traditionally use cell suspensions, which are embedded in agarose on a microscope slide, and exposed to lysis by exposure to detergent and high salt solutions (for review Collins et al, 2008 ; Azqueta et al, 2009 ). Lysis allows removing membranes and soluble cell components, leaving a supercoiled DNA nucleoid (Azqueta et al, 2011b ). When submitted to electrophoretic conditions, DNA fragments will migrate toward the anode, forming a typical “comet tail.” The amount of strand breaks is overall proportional to the amount of DNA in the tail respectively to the DNA remaining in the head (Hovhannisyan, 2010 ).…”

Section: Basic Principles and Methodologiesmentioning

confidence: 99%

The use of comet assay in plant toxicology: recent advances

2015

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The systematic study of genotoxicity in plants induced by contaminants and other stress agents has been hindered to date by the lack of reliable and robust biomarkers. The comet assay is a versatile and sensitive method for the evaluation of DNA damages and DNA repair capacity at single-cell level. Due to its simplicity and sensitivity, and the small number of cells required to obtain robust results, the use of plant comet assay has drastically increased in the last decade. For years its use was restricted to a few model species, e.g., Allium cepa, Nicotiana tabacum, Vicia faba, or Arabidopsis thaliana but this number largely increased in the last years. Plant comet assay has been used to study the genotoxic impact of radiation, chemicals including pesticides, phytocompounds, heavy metals, nanoparticles or contaminated complex matrices. Here we will review the most recent data on the use of this technique as a standard approach for studying the genotoxic effects of different stress conditions on plants. Also, we will discuss the integration of information provided by the comet assay with other DNA-damage indicators, and with cellular responses including oxidative stress, cell division or cell death. Finally, we will focus on putative relations between transcripts related with DNA damage pathways, DNA replication and repair, oxidative stress and cell cycle progression that have been identified in plant cells with comet assays demonstrating DNA damage.

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DNA Repair Measured by the Comet Assay

Cited by 13 publications

References 49 publications

Vitamin C in Cultured Human (HeLa) Cells: Lack of Effect on DNA Protection and Repair

Vitamin C in Cultured Human (HeLa) Cells: Lack of Effect on DNA Protection and Repair

Novel isothiacalothrixin B analogues exhibit cytotoxic activity on human colon cancer cells in vitro by inducing irreversible DNA damage

The use of comet assay in plant toxicology: recent advances

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