A Modified Alkaline Comet Assay for Measuring DNA Repair Capacity in Human Populations

Trzeciak, Andrzej; Barnes, Janice; Evans, Michele K.

doi:10.1667/rr1101.1

Cited by 40 publications

(33 citation statements)

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“…There is significant interest in determining the extent of variation in DNA damage sensitivity and repair capacity between populations and individuals (24,32). Although it is extremely sensitive, the traditional comet assay is impractical for most large-scale studies.…”

Section: Resultsmentioning

confidence: 99%

“…The need for improvement is widely recognized, and previous approaches include reducing space requirements for electrophoresis (CometAssay 96, Trevigen), using a multiwell format for treating samples (23), incorporating internal controls (24,25), or improving imaging (26). These methods address individual issues, but do not provide a comprehensive solution for DNA damage analysis.…”

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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: Resultsmentioning

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

View full text Add to dashboard Cite

show abstract

“…Also, the human peripheral blood lymphocyte culture constitutes an excellent system to test the capacity of physical or chemical agents to cause DNA damage and can be used to monitor individuals exposed to them [39]. All the methods employed in the primary evaluation of DNA damage include the comet assay, which presents a potential to detect cytotoxic and genotoxic agents [32,37,[40][41][42]. Nucleus classification according to the migration of the fragments using the software CometScore 15: class 0 (no damage); class 1 (little damage, tail length smaller than the diameter of the nucleus); class 2 (medium damage, tail length once or twice the diameter of the nucleus); class 3 (significant damage, tail length between two and a half to three times the diameter of the nucleus); class 4 (significant damage, tail length longer than three times the diameter of the nucleus)…”

Section: Discussionmentioning

confidence: 99%

Mutagenic and Genotoxic Effects of cis-(Dichloro)tetraammineruthenium(III) Chloride on Human Peripheral Blood Lymphocytes

Ribeiro

Silva

Pereira

et al. 2009

Biol Trace Elem Res

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Chemotherapeutic agents play an important role in cancer treatment mostly due their systemic action on human organism allowing access to liquid tumors and even metastases. Among these drugs, ruthenium compounds have been showing promising results to treat tumors and represent an important development of new antitumor therapy. This study presents the evaluation of cis-(dichloro)tetraammineruthenium(III) chloride, cis-[RuCl(2)(NH(3))(4)]Cl, genotoxic effects using human peripheral blood lymphocytes cultured in vitro. Mitotic index (MI), chromosome aberrations (CA), and DNA damage using the comet assay were analyzed. MI in human peripheral blood lymphocyte cultures treated with 1, 10, 100, and 1,000 microg mL(-1) cis-[RuCl(2)(NH(3))(4)]Cl were 5.9%, 4.6%, 3.9%, and 0%, respectively. Doxorubicin chloridate was used as the positive control. CA derived from 1, 10, and 100 microg mL(-1) concentrations were defined as spontaneous when compared with the negative control, and at the concentration of 1,000 microg mL(-1), the cell cycle was inhibited (IM = 0%). Results obtained for the comet assay using cis-[RuCl(2)(NH(3))(4)]Cl suggest that this compound has no genotoxic activity against cultured human peripheral blood lymphocytes.

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“…The progressive disappearance of DNA strand breaks over time (up to 90 min) was monitored as an indication of DNA repair (30) and quantified by determining the mean percentage of DNA in tail for the most heavily damaged cells (constituting 20% of the total population) at each time , and DNA damage and BrdUrd incorporation were assessed by a modified alkaline comet assay. Select analysis of only the cells undergoing active DNA synthesis (BrdUrd-positive cells; top) revealed extensive damage in this population in both cell lines, with nearly every comet containing >60% DNA in tail.…”

Section: Preexposure Of Human Tumor Cells To Hypoxia Decreases Etoposmentioning

confidence: 99%

Hypoxia prevents etoposide-induced DNA damage in cancer cells through a mechanism involving hypoxia-inducible factor 1

Sullivan

Graham

2009

Molecular Cancer Therapeutics

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Intratumoral hypoxia is associated with resistance to therapy in many human cancers, and preexposure of tumor cells to hypoxia confers multidrug resistance. Whereas most anticancer drugs kill proliferating tumor cells by causing DNA damage, a role for hypoxia in the prevention and/or repair of drug-induced DNA damage has not been clear. Using the alkaline comet assay, we provide direct evidence that hypoxia-induced resistance to etoposide in human tumor cells (MDA-MB-231 breast carcinoma and DU-145 prostatic adenocarcinoma) is mainly due to prevention of drug-induced DNA damage (i.e., strand breaks) and that the amount of DNA damage present immediately after etoposide exposure is a good independent predictor of clonogenic survival. Our results also revealed that preexposure to hypoxia did not affect the apparent DNA repair capacity of cells. These findings indicate that the extent of DNA damage resulting from etoposide exposure is a more important determinant of survival than subsequent events after DNA damage. Furthermore, immunofluorescence analysis showed that, in a subpopulation of cells, preexposure to hypoxia decreased the levels of topoisomerase IIα, an enzyme that generates DNA strand breaks when poisoned with etoposide. Treatment of cells with small interfering RNA targeting hypoxia-inducible factor 1 prevented the hypoxia-induced decreases in topoisomerase IIα levels, abolished the protective effect of hypoxia against etoposide-induced DNA damage, and inhibited hypoxia-induced etoposide resistance. These findings support a model of hypoxia-induced drug resistance in which etoposide-induced DNA damage is prevented by HIF-1-dependent adaptations to hypoxia.

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A Modified Alkaline Comet Assay for Measuring DNA Repair Capacity in Human Populations

Cited by 40 publications

References 56 publications

Single cell trapping and DNA damage analysis using microwell arrays

Single cell trapping and DNA damage analysis using microwell arrays

Mutagenic and Genotoxic Effects of cis-(Dichloro)tetraammineruthenium(III) Chloride on Human Peripheral Blood Lymphocytes

Hypoxia prevents etoposide-induced DNA damage in cancer cells through a mechanism involving hypoxia-inducible factor 1

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