• Korean Journal of Ecology and Environment
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• v.41 no.4
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• pp.466-471
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• 2008

Little is known about DNA-level and physiological levels for health assessments of stream or river environments. Recently, comet assay, so called Single Cell Gel Electrophoresis (SCGE) is introduced for assessments of DNA damage in the medical science, food science and mammal toxicology. The comet assay is known as a biomarker which is one of the best barometers in assessing the DNA damage by oxidative stress. In this study, we conducted the comet assay using sentinel species, Zacco platypus, as one of the pre-warning alarm systems for the aquatic ecosystem health assessments and also applied it to Gap Stream as a model system. Tail extent moments in the S1 and S2 were 5.20 and 9.90 respectively and the moment was 19.89 in the S3. Statistical ANOVA in the tail moments showed a significant difference (n=75, p<0.05) between S1 and S3. Also, the proportions of DNA in the tail were 14.47, 23.64, and $30.04{\mu}m$ in the upstream (control site), midstream, downstream sites, respectively. Our results in the downstream were accord with previous studies of individual-level, population-level, and community-level in Gap Stream. Our results suggest that the comet assay may be used as an important tool for diagnosing ecological health of aquatic ecosystems in the level of DNA.

• Journal of Plant Biotechnology
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• v.31 no.3
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• pp.225-230
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• 2004

We employed single cell gel electrophoresis method (comet assay) to analyze the degree of nucleus-DNA damage in the leaves of red pepper (Capsicum annuum L.) seedlings exposed to $^{60}$ CO v-radiation stress. Nucleus-DNA damage was measured as the ratio of tail length (T) to head length (H) in individual comet image isolated from pepper leaf cell. The T/H ratio of control-cells and treated-cells at 50 or 100 Gy were 1.28 and 3.54 or 3.39, respectively, suggesting that nuclei of pepper cells were severely damaged in the integrity of DNA strand by the treatment of enhanced v-radiation. The percentage of head-DNA in control-cells was 76.8%, whereas those of 50 and 100 Gy treated-cells were 55.9% and 59.9%, respectively. Pretreatment of low dose (4 to 20 Gy) radiation to seeds decreased DNA-damage in the leaves of seedlings treated with high dose radiation at 50 or 100 Gy. In this experiment, we developed a sensitive, reliable and rapid method for evaluating genotoxic effect in the nuclei of plant cells by employing comet assay.

• Journal of the Korean Society of Food Science and Nutrition
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• v.30 no.6
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• pp.1076-1081
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• 2001

Gamma-irradiated peanuts, Korean and Chinese origins, were investigated on detection properties by thermoluminescence (TL), electron spin resonance (ESR), and DNA comet assay (single cell gel electrophoresis). TL measurement showed that the non-irradiated sample revealed a glow curve with low intensity at about 25$0^{\circ}C$, while the irradiated samples showed higher intensity around at 18$0^{\circ}C$. TL ratio (TL$_1$/TL$_2$) of area for TL$_1$ glow curve to TL$_2$ was below 0.05 for the non-irradiated sample and 0.2 or more for the irradiated ones, thus identifying each other. ESR spectroscopy for the irradiated peanuts using outer skin showed negligible signals induced by irradiation, indicating ESR is little applicable to the detection of irradiated peanuts. In DNA comet assay, the non-sample had no or very short tails, whereas the irradiated samples revealed the cells with long tails. Significance in the increase of their lengths depending on irradiation dose (r=0.761/Korean, r=0.768/Chinese) was also found. There was no remarkable difference in detection properties by origins of samples in all determinations, It is concluded that TL analysis or DNA comet assay is suitable for detection of irradiated peanuts and a combined method is recommendable for enhancing the reliability of detection results.

• Clinical and Experimental Reproductive Medicine
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• v.46 no.1
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• pp.14-21
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• 2019

Objective: The usual seminal profile has been customarily used for diagnosing male infertility based on an examination of semen samples. However, sperm DNA fragmentation has also been causally linked to reproductive failure, suggesting that it should be evaluated as part of male infertility assessments. To compare the ability of the five most widely utilized methodologies of measuring DNA fragmentation to predict male infertility and reactive oxygen species by Oxisperm kit assay. Methods: In this case-control study, which received ethical committee approval, the participants were divided into fertile and infertile groups (50 patients in each group). Results: The alkaline comet test showed the best ability to predict male infertility, followed by the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay, the sperm chromatin dispersion (SCD) test, and the sperm chromatin structure assay (SCSA), while the neutral comet test had no predictive power. For our patient population, the projected cut-off point for the DNA fragmentation index was 22.08% using the TUNEL assay, 19.90% using SCSA, 24.74% using the SCD test, 48.47% using the alkaline comet test, and 36.37% using the neutral comet test. Significant correlations were found between the results of the SCD test and those obtained using SCSA and TUNEL (r = 0.70 and r = 0.68, respectively; p< 0.001), and a statistically significant correlation was also found between the results of SCSA and the TUNEL assay (r = 0.77, p< 0.001). Likewise, the results of the alkaline comet test showed significant correlations with those of the SCD, SCSA, and TUNEL tests (r = 0.59, r = 0.57, and r = 0.72, respectively; p< 0.001). Conclusion: The TUNEL assay, SCSA, SCD, and the alkaline comet test were effective for distinguishing between fertile and infertile patients, and the alkaline comet test was the best predictor of male infertility.

• Environmental Mutagens and Carcinogens
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• v.21 no.2
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• pp.89-94
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• 2001

Recently, single cell gel electrophoresis, also known as comet assay, is widely used for the detection and measurement of DNA strand breaks in vitro and in vivo in many toxicological fields such as radiation exposure, human monitoring and toxicity evaluation. As well defined, comet assay is a sensitive, rapid and visual method for the detection of DNA strand breaks in individual cells. Briefly, a small number of damaged cells suspended in a thin agarose gel on a microscope slide were lysed, unwinded, electrophoresed, and stained with a fluorescent DNA binding dye. The electric current pulled the charged DNA from the nucleus such that relaxed and broken DNA fragments migrated further. The resulting images which were subsequently named for their appearance as comets, were measured to determine the extent of DNA damages. However, some variations could be occurred in procedures, laboratories's conditions and kind of cells used. Hence, to overcome and to harmonize these matters in comet assay, International Workshop on Genotoxicity Test Procedure (IWGTP) was held with several topics including comet assay at Washington D.C. on March, 1999. In spite of some consensus in procedures and conditions in IWGTP, there are some problems still remained to be solved. In this respect, we attempted to set the practical optimal conditions in the experimental procedures such as lysis, unwinding, electrophoresis and neutralization conditions and so on. First of all, we determined optimal lysis and unwinding time by using 150 $\mu$M methyl methanesulfonate (MMS) which is usually used concentration. And then, we determined optimal positive control concentrations of benzo(a)pyrene (BaP) and MMS in the presence and absence of S9 metabolic activation system, respectively.

• Toxicological Research
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• v.20 no.2
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• pp.89-100
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• 2004

The comet assay (also called the single-cell gel electrophoresis assay) has been widely used for detecting DNA damage and repair in individual cells. Since the conventional methods of evaluating comet assay data using frequency statistics are unsatisfactory we developed a new quantitative measure of DNA damage/repair that is based on all information residing in the dose/time-response curves of a comet experiment. Blood samples were taken from 25 breast cancer patients before undergoing radiotherapy. The comet assay was performed under alkaline conditions using isolated lymphocytes. Tail DNA, tail length, tail moment and tail inertia of the comet were measured for each patient at four doses of $\gamma$-rays (0, 2, 4 and 8 Gy) and at four time points after irradiation (0, 10, 20 and 30 min) using 100 cells each. The resulting three-dimensional dose-time response surface was modeled by multiple regression, and the second derivative, termed 2D, on dose and time was determined. A software module was programmed in SAS/AF to compute 2D values. We applied the new method successfully to data obtained from cancer patients to be assessed for their radiation sensitivity. We computed the 2D values for the four damage measures, i.e., tail moment, tail length, tail DNA and tail inertia, and examined the pairwise correlation coefficients of 2D both on the log scale and the unlogged scale. 2D values based on tail moment and tail DNA showed a high correlation and, therefore, these two damage measures can be used interchangeably as far as DNA repair is concerned. 2D values based on tail inertia have a correlation profile different from the other 2D values which may reflect different facets of DNA damage/repair. Using the dose-time response surface, other statistical models, e.g., the proportional hazards model, become applicable for data analysis. The 2D approach can be applied to all DNA repair measures, Le., tail moment, tail length, tail DNA and tail inertia, and appears to be superior to conventional evaluation methods as it integrates all data of the dose/time-response curves of a comet assay.

• Journal of the Korean Society of Food Science and Nutrition
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• v.31 no.4
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• pp.594-598
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• 2002

The changes in DNA damage were investigated during storage after irradiation. Kiwi, orange and pear were irradiated at 0.1, 0.3, 0.5, 0.7 and 1.0 kGy and stored for 3 months at 4$^{\circ}C$. The comet assay was applied to the sample seeds alt the beginning of irradiation and at the end of storage. Seeds were isolated and crushed, and the suspended cells were embedded in an agarose layer. After lysis of the cells, they were electrophoresed for 2 min and then stained. DNA fragmentation in seeds caused by irradiation was quantified as tail length and tail moment (tail length $\times$ % DNA in tail) by comet image analyzing system. Immediately after irradiation, the differences in tail length between unirradiated and irradiated fruit seeds were significant (p<0.05) in kiwi, orange and pear seeds. With in-creasing the irradiation doses, statistically significant longer extension of the DNA from the nucleus toward anode was observed. The results represented as tail moment showed similar tendency to those of tail length, but tile latter parameter was more sensitive than the former. Similarly even 3 months after irradiation, all the irradiated fruit seeds significantly showed longer tail length than the unirradiated controls. These results indicate that the comet assay could be one of the simple methods of detecting irradiated fruit seeds. Moreover, the method could detect DNA damage even after 3 months after irradiation.

• Proceedings of the Korea Society of Environmental Biology Conference
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• 2002.11a
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• pp.145-153
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• 2002

유해 화학 물질류에 의해 오염된 해양 환경 시료의 환경독성 수준을 평가하기 위하여 다양한 화학물질에 대해 민감성이 우수한 생물학적 독성평가기법을 개발 하고자하였다. 지속성 유기오염 물질 중 다환 방향 족 탄화수소(PAHs)를 처리한 광어(Conger myriaster)와 개조개(Saxidomus pupurata)의 DNA 손상정도를 single cell gel electrophoresis assay(comet assay)를 통해 분석하였다. PAHs 중 광양만에서 높은 농도로 검출되는 benzo(a)pyrene을 농도별(0, 10, 50, 100 ppb)로 처리한 후 2일과 4일에 광어의 혈액세포와 개조개의 근육세포를 채취해 comet assay를 실시하였다. benso(a)pyrene에 대한 DNA 손상정도를 처리된 농도와 생물종에 따라 다르게 나타났는데 광어의 혈액세포는 2일에 가장 DNA 손상정도가 높았고, 4일에는 회복되는 경향을 나타냈다. 개조개의 근육세포는 시간이 지나면서 DNA 손상정도가 증가하는 경향을 보였다. 이와 같은 결과는 comet assay 기법이 유해 화학물질로 오염된 해양생물 종의 환경독성을 검색하는 유용한 수단이 될 수 있음을 보여준다.

• Proceedings of the Korea Society of Environmental Toocicology Conference
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• 2003.10a
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• pp.159-159
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• 2003

Comet assay is a potential monitoring tool because DNA strand breakage may be produced by a wide range of agents. The comet assay, also called the single-cell gell electrophoresis (SCGE) assay, is rapid and sensitive method for the detection of DNA damage in cells. This study was performed for the identification of DNA damage in the cells from flounders and clams exposed to PAHs. As a control experiments, flounder and clam cells were exposed to $H_2O$$_2$. The cells exposed to $H_2O$$_2$ were displayed a typical nuclei movement DNA damage of cells were significantly increased when the isolated cells from the blood of flounders and the tissue of clams were in vitro exposed to the different concentrations (5, 10, 50, 100 ppb) of five kinds of PAHs (benzo[a]pyrene, pyrene, fluoranthene, anthrancene, and phenanthrene). For the in vivo test, flounders and clams were exposed to the different concentrations of BaP for 4 days. The results showed that DNA strand breakage was effected by the concentration of BaP and the duration of exposure. In high concentration of BaP, the mean tail lengths of nuclei was longer than it In low concentration, while the mean size of head DNA decreased. In this research, both in vitro and in vivo genotoxicity of PAHs could be biomonitored by the comet assay. Especially, clams and flounders seem to be useful as materials for monitoring genotoxic damage by comet assay.

• Molecules and Cells
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• v.39 no.3
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• pp.204-210
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• 2016

DNA damage responses are important for the maintenance of genome stability and the survival of organisms. Such responses are activated in the presence of DNA damage and lead to cell cycle arrest, apoptosis, and DNA repair. In Caenorhabditis elegans, double-strand breaks induced by DNA damaging agents have been detected indirectly by antibodies against DSB recognizing proteins. In this study we used a comet assay to detect DNA strand breaks and to measure the elimination of DNA strand breaks in mitotic germline nuclei of C. elegans. We found that C. elegans brc-1 mutants were more sensitive to ionizing radiation and camptothecin than the N2 wild-type strain and repaired DNA strand breaks less efficiently than N2. This study is the first demonstration of direct measurement of DNA strand breaks in mitotic germline nuclei of C. elegans. This newly developed assay can be applied to detect DNA strand breaks in different C. elegans mutants that are sensitive to DNA damaging agents.