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

Miyamae, Youichi; Iwasaki, Kouichi; Kinae, Naohide; Tsuda, Shuji; Murakami, Michiko; Tanaka, Makiko; Sasaki, Yu

doi:10.1016/s1383-5718(97)00091-0

Cited by 88 publications

(42 citation statements)

References 13 publications

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“…An 8-OH-dG assay has been shown to determine the DNA damage oxidized by ROS and a comet assay shown to detect the double-strand breaks (DSBs) at pH 9, SSBs and DSBs at pH 12.1 and the alkali-labile sites, SSBs and DSBs at pH 13 (Anderson and Plewa, 1998;Fairbairn et al, 1995;Miyamae et al, 1997;Singh et al, 1988). In this study, 8-OH-dG showed age-dependent increases in the liver and kidney and this result was consistent with previous reports (Fraga et al, 1990;Hamilton et al, 2001;Izzotti et al, 1999;Kaneko et al, 1996;Schmerold and Niedermuller, 2001;Sohal et al, 1994).…”

Section: Discussionsupporting

confidence: 92%

“…However, the repair of 8-OH-dG generates AP sites and single-strand breaks (SSBs) in the course of BER and the detection of AP sites and SSBs is important to evaluate the ability of BER. A single-cell gel electrophoresis (comet) assay developed by Singh et al can detect SSBs and alkali-labile sites such as AP sites as well as double-strand breaks (DSBs) (Anderson and Plewa, 1998;Miyamae et al, 1997;Singh et al, 1988). Furthermore, Sasaki et al have applied a comet assay in vivo to detect various organs and tissues in animals (Sasaki et al, 1997a(Sasaki et al, , 1997b.…”

Section: Introductionmentioning

confidence: 99%

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DNA DAMAGE MEASURED BY COMET ASSAY AND 8-OH-dG FORMATION RELATED TO BLOOD CHEMICAL ANALYSES IN AGED RATS

et al. 2007

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-To evaluate the effects of aging on DNA damage, spontaneous and chemical-induced DNA damage and its repair were examined using comet assays at pH 9, 12.1 and 13, and an 8-OH-dG assay in the liver and kidney of young (9-week-old) and aged (20-month-old) rats. Additionally, blood chemistry was examined to investigate any correlation between vital functions and age-dependent DNA damage. DNA migration at pH 13 and 8-OH-dG levels increased in the liver and/or kidney of aged rats, but DNA migration did not increase at pH 9 or 12.1; that is, alkali-labile sites and 8-OH-dG were concomitantly accumulated in aged rats. These results suggest that 8-OH-dG production caused by reactive oxygen species exceeded glycosylation and that the glycosylation activity is far more than the AP endonucleation in aged rats. Methyl methanesulfonate (MMS, 80 mg/kg, i.p.) increased DNA migration at pH 12.1 and 13 in the liver and kidney at 3 and 24 hr after treatment in young and aged rats. The DNA damage in aged rats was less and decreased more slowly compared with young rats. The pictures of MMSinduced DNA migrations at pH 12.1 and 13 were very similar to each other. These results suggest that the adduct glycosylation and repair of the single-strand breaks (SSBs) of aged rats are less than those of young rats, although AP endonucleation is sufficient to remove the AP sites. N-nitrosodiethylamine (160 mg/kg, i.p.) increased DNA migration at pH 12.1 and 13 in the liver and kidney at 3 and 24 hr in young rats and at pH 12.1 and 13 in the kidney at 24 hr in aged rats. These results showed that SSBs were predominantly detected as chemical-induced DNA damage and DNA repairs such as N-glycosylase, DNA polymerase and DNA ligase, and that the metabolic activation declined in aged rats. Aspartate aminotransferase, alanine aminotransferase, total bilirubin, total cholesterol, total protein, globulin, creatinine and chloride age-dependently increased and alkaline phosphates, albumin/globulin ratio, inorganic phosphorus and potassium age-dependently decreased, and these changes were correlated with the DNA migration at pH 13 and/or 8-OH-dG. These results suggest that the activity of DNA repair and metabolic activation enzymes declines in aged rats and that the accumulation of spontaneous DNA damage may affect vital functions.

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Section: Discussionsupporting

confidence: 92%

Section: Introductionmentioning

confidence: 99%

DNA DAMAGE MEASURED BY COMET ASSAY AND 8-OH-dG FORMATION RELATED TO BLOOD CHEMICAL ANALYSES IN AGED RATS

et al. 2007

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“…After lysis of cells with detergent at high salt concentration, DNA unwinding and electrophoresis is carried out at a specific pH. Unwinding of the DNA and electrophoresis at neutral pH (7-8) predominantly facilitates the detection of double strand breaks and cross links; unwinding and electrophoresis at pH 12.1-12.4 facilitates the detection of single and double strand breaks, incomplete excision repair sites and cross-links; whereas unwinding and electrophoresis at a pH greater than 12.6 expresses alkali labile sites in addition to all types of lesions listed above (Miyamae et al 1997). When subjected to an electric field, the DNA migrates out of the cell, in the direction of the anode, appearing like a 'comet'.…”

Section: Introductionmentioning

confidence: 99%

Comet Assay measurements: a perspective

Kumaravel¹,

Vilhar

Faux³

et al. 2007

Cell Biol Toxicol

317

162

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“…The alkaline comet assay has been successfully performed using human mammary epithelial cells (17) and to detect alkylation damage (18)(19)(20)(21). Primary rat mammary epithelial cells were isolated from Fischer 344 rats as described for the treatment in culture limiting dilution in vivo transplantation assay as described in Aim 1.…”

Section: Methodsmentioning

confidence: 99%

Impulse Response of Alternative Synthetic Apertures for Subsurface Detection

Ralston¹

2001

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

Cited by 88 publications

References 13 publications

DNA DAMAGE MEASURED BY COMET ASSAY AND 8-OH-dG FORMATION RELATED TO BLOOD CHEMICAL ANALYSES IN AGED RATS

DNA DAMAGE MEASURED BY COMET ASSAY AND 8-OH-dG FORMATION RELATED TO BLOOD CHEMICAL ANALYSES IN AGED RATS

Comet Assay measurements: a perspective

Impulse Response of Alternative Synthetic Apertures for Subsurface Detection

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