Comet assay: a reliable tool for the assessment of DNA damage in different models

Dhawan, Alok; Bajpayee, Mahima; Parmar, Devendra

doi:10.1007/s10565-008-9072-z

Cited by 334 publications

(193 citation statements)

References 276 publications

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“…It has been used to study a variety of cell types and species from all three biological kingdoms (17). The microwell arrays further increase the versatility of this assay by enabling unique capabilities, including cell multiplexing and analysis of cell aggregates.…”

Section: Discussionmentioning

confidence: 99%

“…Although most base lesions are not directly detected, base adducts can be detected when converted to abasic sites or single strand breaks with the addition of purified DNA repair enzymes (7)(8)(9)(10)(11). The comet assay has been used in a variety of applications, including genotoxicity testing, human biomonitoring and epidemiology, environmental health, and basic research on DNA damage and repair (7,8,(12)(13)(14)(15)(16)(17)(18)(19). Compared with other DNA damage assays (20)(21)(22), the comet assay is relatively inexpensive, is very sensitive, and can assess the integrated cellular response to many kinds of DNA lesions simultaneously (7,8,19).…”

mentioning

confidence: 99%

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Single cell trapping and DNA damage analysis using microwell arrays

Wood

Weingeist

Bhatia

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

240

284

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With a direct link to cancer, aging, and heritable diseases as well as a critical role in cancer treatment, the importance of DNA damage is well-established. The intense interest in DNA damage in applications ranging from epidemiology to drug development drives an urgent need for robust, high throughput, and inexpensive tools for objective, quantitative DNA damage analysis. We have developed a simple method for high throughput DNA damage measurements that provides information on multiple lesions and pathways. Our method utilizes single cells captured by gravity into a microwell array with DNA damage revealed morphologically by gel electrophoresis. Spatial encoding enables simultaneous assays of multiple experimental conditions performed in parallel with fully automated analysis. This method also enables novel functionalities, including multiplexed labeling for parallel single cell assays, as well as DNA damage measurement in cell aggregates. We have also developed 24-and 96-well versions, which are applicable to high throughput screening. Using this platform, we have quantified DNA repair capacities of individuals with different genetic backgrounds, and compared the efficacy of potential cancer chemotherapeutics as inhibitors of a critical DNA repair enzyme, human AP endonuclease. This platform enables high throughput assessment of multiple DNA repair pathways and subpathways in parallel, thus enabling new strategies for drug discovery, genotoxicity testing, and environmental health.base excision repair | comet assay | DNA repair D NA damage is a critical risk factor for cancer, aging, and heritable diseases (1-3), and it is the underlying basis for most frontline cancer therapies (4). Increased knowledge about DNA damage and repair would facilitate disease prevention, identification of individuals at increased risk of cancer, discovery of novel therapeutics, and better drug safety testing. Despite their obvious translational impact, most DNA damage assays have not changed significantly in more than two decades, and thus are not compatible with modern high throughput screening technologies. To better assess the biological impact of damage, we need data that reflect the integrated effect of multiple DNA repair pathways and subpathways, in response to a range of DNA lesions, measured in an accurate and high throughput fashion.The single cell gel electrophoresis or "comet" assay is based on the principle that relaxed loops (induced by single strand breaks) and DNA fragments migrate farther in an agarose gel than undamaged DNA (5-8). The alkali comet assay sensitively detects a range of DNA lesions, including strand breaks (single and double), as well as alkali sensitive sites. Although most base lesions are not directly detected, base adducts can be detected when converted to abasic sites or single strand breaks with the addition of purified DNA repair enzymes (7-11). The comet assay has been used in a variety of applications, including genotoxicity testing, human biomonitoring and epidemiology, environmental healt...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Single cell trapping and DNA damage analysis using microwell arrays

Wood

Weingeist

Bhatia

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

240

284

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“…The genome-protective eff ect of xanthohumol against oxidative DNA damage induced by H 2 O 2 was evaluated using the yeast comet assay. The yeast comet assay is a well-established, simple, versatile, sensitive and inexpensive technique used to assess DNA oxidative damage quantitatively and qualitatively in eukaryotic cell populations (30)(31)(32)(33). Yeast spheroplasts were incubated with diff erent xanthohumol concentrations and 10 mM H 2 O 2 in S buff er (containing 1 M sorbitol) in order to maintain osmotic protection.…”

Section: Xanthohumol Protects Yeast Cells From Dna Damage Induced By mentioning

confidence: 99%

Dose-Dependent Protective and Inductive Effects of Xanthohumol on Oxidative DNA Damage in Saccharomyces cerevisiae

Carvalho

Oliveira

Johansson

et al. 2016

Food Technol. Biotechnol.

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SummaryThe eff ect of xanthohumol, a prenylfl avonoid isolated from the hop plant (Humulus lupulus L.), on Saccharomyces cerevisiae DNA oxidative damage and viability was evaluated. Yeast cultures under oxidative stress, induced by H 2 O 2 , displayed stronger growth in the presence of 5 mg/L of xanthohumol than cultures with only H 2 O 2 . Likewise, DNA damage assessed by the comet assay was signifi cantly lower in cells co-incubated with xanthohumol and H 2 O 2 . Accordingly, fl uorescence of dichlorofl uorescein in cells treated with H 2 O 2 and xanthohumol was considerably lower than in cells exclusively treated with H 2 O 2 , indicative of a reactive oxygen species scavenging mechanism and consequent formation of oxidation products, as detected by mass spectrometry. However, at concentrations above 5 mg/L, xanthohumol elicited an opposite eff ect, leading to a slower growth rate and significant increase in DNA damage. A yeast yap1 deletion mutant strain sensitive to oxidative stress grew more slowly in the presence of at least 5 mg/L of xanthohumol than cultures of the wild type, suggesting that xanthohumol toxicity is mediated by oxidative stress. This evidence provides further insight into the impact of xanthohumol on yeast cells, supporting dose-dependent antioxidant/antigenotoxic and prooxidant/genotoxic eff ects.

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“…Compared to other tests for genotoxic damage, the principal advantages of the comet assay include its demonstrated sensitivity for detecting DNA damage at a very low degree, the requirement for a small number of cells per sample, its flexibility, low cost, and the relatively short time needed to complete a study. [43][44][45] The data collected in the present study suggest that the comet assay could contribute to treatment decisions by considering the patient's basal DNA damage before therapy. At present, therapy decisions are largely based on prognostic factors, such as the presence of node metastases, among others.…”

Section: A U T H O R P R O O Fmentioning

confidence: 81%

Relationship between Genotoxic Effects of Breast Cancer Treatments and Patient Basal DNA Integrity

Ceballos

Funes

Massa

et al. 2014

J Environ Pathol Toxicol Oncol

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Radiotherapy and chemotherapy cause genotoxic side effects that are highly variable among patients. In this study, we evaluated DNA integrity using the comet assay in peripheral blood lymphocytes from breast cancer patients before ("pre-treatment patients"; n = 47) and after ("post-treatment patients"; n = 24) radiotherapy and/or chemotherapy treatment and from healthy donors (n = 15). Comet evaluation was made by visual (types 0-4) and digital (percentage of DNA remaining in the comet head = % head DNA) analysis. The association between the level of DNA damage and cancer prognostic factors was assessed. The treatments caused a significant increase in DNA damage registered by both visual (p < 0.001) and digital (p < 0.001) analyses. No significant associations between the level of DNA damage in pre-treatment patients and cancer prognostic factors were found. A significant correlation between the comet results from each patient before and after treatment (r = 0.64, p = 0.001) was observed. The % head DNA in post-treatment samples from patients with a high level of DNA damage before treatment (30.3 ± 3.1%, p < 0.01) was lower than in post-treatment samples from patients with a low-to-medium level of DNA damage before therapy (49.2 ± 4.4%). These results support the usefulness of the comet assay as a sensitive technique to evaluate basal DNA status and DNA damage caused by cancer treatments. The comet assay could contribute to treatment decisions, especially by taking into account the patient's basal DNA damage before therapy.

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Comet assay: a reliable tool for the assessment of DNA damage in different models

Cited by 334 publications

References 276 publications

Single cell trapping and DNA damage analysis using microwell arrays

Single cell trapping and DNA damage analysis using microwell arrays

Dose-Dependent Protective and Inductive Effects of Xanthohumol on Oxidative DNA Damage in Saccharomyces cerevisiae

Relationship between Genotoxic Effects of Breast Cancer Treatments and Patient Basal DNA Integrity

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