Detection of DNA lesions induced by chemical mutagens by the single cell gel electrophoresis (Comet) assay.

Miyamae, Youichi; Zaizen, Kazuyo; Ohara, Kaname; Mine, Yasuhiro; Sasaki, Yu

doi:10.1016/s1383-5718(97)00090-9

Cited by 39 publications

(8 citation statements)

References 11 publications

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“…To further evaluate the capacity of ␤-pol null cells to repair MMSinduced lesions, DNA damage in genomic DNA was measured with the comet assay (Fig. 1b); this assay detects single-strand breaks plus alkali-labile sites (e.g., AP sites) (36). MMS induced an increase in comet assay tail moment, indicating an increase in DNA lesions in both cell lines.…”

Section: Resultsmentioning

confidence: 99%

Mutations associated with base excision repair deficiency and methylation-induced genotoxic stress

Sobol

Watson

Nakamura

et al. 2002

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

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The long-term effect of exposure to DNA alkylating agents is entwined with the cell's genetic capacity for DNA repair and appropriate DNA damage responses. A unique combination of environmental exposure and deficiency in these responses can lead to genomic instability; this ''gene-environment interaction'' paradigm is a theme for research on chronic disease etiology. In the present study, we used mouse embryonic fibroblasts with a gene deletion in the base excision repair (BER) enzymes DNA ␤-polymerase (␤-pol) and alkyladenine DNA glycosylase (AAG), along with exposure to methyl methanesulfonate (MMS) to study mutagenesis as a function of a particular gene-environment interaction. The ␤-pol null cells, defective in BER, exhibit a modest increase in spontaneous mutagenesis compared with wild-type cells. MMS exposure increases mutant frequency in ␤-pol null cells, but not in isogenic wild-type cells; UV light exposure or N-methyl-N -nitro-N-nitrosoguanidine exposure increases mutant frequency similarly in both cell lines. The MMS-induced increase in mutant frequency in ␤-pol null cells appears to be caused by DNA lesions that are AAG substrates, because overexpression of AAG in ␤-pol null cells eliminates the effect. In contrast, ␤-pol͞AAG double null cells are slightly more mutable than the ␤-pol null cells after MMS exposure. These results illustrate that BER plays a role in protecting mouse embryonic fibroblast cells against methylation-induced mutations and characterize the effect of a particular combination of BER gene defect and environmental exposure. B ase excision repair (BER) (1) is considered the predominant DNA repair system in mammalian cells for eliminating small DNA lesions generated either exogenously or endogenously at DNA bases (1-3). Such DNA damage can be caused by exposure to environmental agents or by normal cellular metabolic processes that produce reactive oxygen species, alkylating molecules, and other reactive metabolites capable of modifying DNA. The first two steps of the mammalian BER pathway are removal of a damaged base residue by a DNA glycosylase and subsequent action of apurinic͞apyrimidinic (AP) endonuclease. The product of these reactions is single-nucleotide gapped DNA with 5Ј deoxyribosephosphate (dRP) and 3Ј OH at the margins of the gap. A DNA ␤-polymerase (␤-pol)-mediated DNA synthesis step fills the single-nucleotide gap (4-6), and the 5Ј dRP group is removed by the dRP lyase activity of ␤-pol (7-11). DNA ligase I or III conducts the final, nick-sealing step in the pathway. Many laboratories have presented evidence in support of the roles of the enzymes noted here in single-nucleotide BER, mediated either by crude cell extracts or purified enzymes (3, 4, 12-16). Another important feature of single-nucleotide BER is that some of the enzymes in the pathway can interact with each other, forming a series of ''subassemblies'' that may pass along the substrates and products like a baton in a relay (for review, see ref. 17).It is well known that ␤-pol null mouse cells are hypersen...

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Section: Resultsmentioning

confidence: 99%

Mutations associated with base excision repair deficiency and methylation-induced genotoxic stress

Sobol

Watson

Nakamura

et al. 2002

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

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“…By increasing the effective molecular weight of the DNA, alkylating cross-linking agents reduce the ability of DNA to migrate in an electric field [59]. For this reason, cells with DNA cross-linking lesions show no increased or even decreased DNA migration in the alkaline comet assay [60]. It is well known that the relationship between the onset of DNA damage and the time after exposure depend on the mechanism of DNA damage and that the measured damage level is a result of equilibrium between damage infliction and repair [61].…”

Section: Discussionmentioning

confidence: 99%

Alkaline comet assay study with breast cancer patients: evaluation of baseline and chemotherapy-induced DNA damage in non-target cells

et al. 2006

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The sensitivity of the alkaline comet assay for the evaluation of baseline and treatment-induced DNA damage in white blood cells of breast cancer patients receiving adjuvant chemotherapy according to three conventional anthracycline- and cyclophosphamide-containing protocols was investigated. Additionally, baseline DNA damage in cancer patients was compared with the levels of DNA damage recorded in healthy women. Altogether 30 patients with diagnosed breast cancer and 30 female blood donors with no known familial history of breast cancer participated in the study. Alkaline comet assay was performed according to standard protocol and DNA migration in peripheral blood leukocytes was measured by a computer-based image analysis system. For each subject the frequency of "damaged" cells, i.e., long-tailed nuclei (LTN) with tail length exceeding 95th percentile for the considered parameter among controls, is also reported. Breast cancer patients had significantly increased background levels of DNA damage in their peripheral blood leukocytes as compared to healthy women. Prior to the chemotherapy, a majority of patients showed a statistically significant increase in the number of LTN compared to healthy blood donors. Marked interindividual variations in baseline DNA damage among patients were recorded, some of them related to the disease stage and status. The present study confirmed the alkaline comet assay as a sensitive technique able to detect significantly elevated DNA migration in blood cells of patients already one hour after completion of the first cycle of chemotherapy. Administration of antineoplastic drugs in three chemotherapy protocols studied induced a similar increase of primary DNA damage in nontarget cells. The evaluation of the LTN frequencies indicates the best response to the protocol containing cyclophosphamide, methotrexate and 5-fluorouracil (CMF). Our results point to the significance of simultaneous evaluation of DNA migration and frequency of LTN in the same subject and approved the use of alkaline comet assay as a suitable method for the routine detection of critical DNA lesions produced after administration of antineoplastic drugs in the clinical settings.

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“…Oxidized pyrimidines are detected with use of endonuclease III, while oxidized purines with formamidopyrimidine DNA glycosylase (FPG). Modifications have been made in the protocol to specifically detect double-strand breaks (neutral Comet assay ;Singh 2000), single-strand breaks (at pH 12.1; Miyamae et al 1997), DNA cross-links (decrease in DNA migration due to cross-links, Singh 2000), and apoptosis (Singh 2000). Neutral comet assay also helps to distinguish apoptosis from necrosis as evidenced by the increased Comet score in apoptotic cells and the almost zero Comet score in necrotic cells (Yasuhara et al 2003).…”

Section: Specificity Sensitivity and Limitations Of The Comet Assaymentioning

confidence: 99%

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

2008

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New chemicals are being added each year to the existing burden of toxic substances in the environment. This has led to increased pollution of ecosystems as well as deterioration of the air, water, and soil quality. Excessive agricultural and industrial activities adversely affect biodiversity, threatening the survival of species in a particular habitat as well as posing disease risks to humans. Some of the chemicals, e.g., pesticides and heavy metals, may be genotoxic to the sentinel species and/or to non-target species, causing deleterious effects in somatic or germ cells. Test systems which help in hazard prediction and risk assessment are important to assess the genotoxic potential of chemicals before their release into the environment or commercial use as well as DNA damage in flora and fauna affected by contaminated/polluted habitats. The Comet assay has been widely accepted as a simple, sensitive, and rapid tool for assessing DNA damage and repair in individual eukaryotic as well as some prokaryotic cells, and has increasingly found application in diverse fields ranging from genetic toxicology to human epidemiology. This review is an attempt to comprehensively encase the use of Comet assay in different models from bacteria to man, employing diverse cell types to assess the DNA-damaging potential of chemicals and/or environmental conditions. Sentinel species are the first to be affected by adverse changes in their environment. Determination of DNA damage using the Comet assay in these indicator organisms would thus provide information about the genotoxic potential of their habitat at an early stage. This would allow for intervention strategies to be implemented for prevention or reduction of deleterious health effects in the sentinel species as well as in humans.

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Detection of DNA lesions induced by chemical mutagens by the single cell gel electrophoresis (Comet) assay.

Cited by 39 publications

References 11 publications

Mutations associated with base excision repair deficiency and methylation-induced genotoxic stress

Mutations associated with base excision repair deficiency and methylation-induced genotoxic stress

Alkaline comet assay study with breast cancer patients: evaluation of baseline and chemotherapy-induced DNA damage in non-target cells

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

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