• 한국환경성돌연변이발암원학회지
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• 제17권2호
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• pp.71-77
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• 1997

The single cell gel electrophoressis(SCGE) assay, also known as the comet assay, is a rapid, simple, visual and sensitive technique for measuring and analysing DNA breakage in mammalian cells. The SCGE or comet assay is a promising test for the detection of DNA damage and repair in individnal cells. It has widespread potential applications in DNA damage and repair studies, genotoxicity testing and biomonitoring. In this microgel electrophoresis technique, cells are embedded in agarose gel on microscope slides, iysed and electrophoresed under alkaline conditions. Cells with increased DNA damage display increased migration of DNA from the nucleus towards the anode. The length of DNA migration indicates the amount of DNA breakage in the cell. The comet assay is also capable of identifying apoptotic cells which contain highly fragmented DNA. Here we review the development of the SCGE assay, existing protocols for the detection and analysis of comets, the relevant underlying principles determining the behaviour of DNA and the potential applications of the technique.

• 한국수산과학회지
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• 제36권4호
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• pp.346-351
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• 2003

Single-cell gel electrophoresis (comet assay) was used to detect DNA single strand break in blood cells from several marine fish species. Three fish species were collected from Georgia coastal area. Mummichog, Fundulus heteroclitus showed higher DNA damage than sea bass, Lateolabrax japonicus and trout, Oncorhynchus masou masou under the same experimental conditions. Mummichogs had more alkaline-labile sites on their DNA than other fish species. The comet assay with mummichog blood cells at pH 12.5 showed a dose-response curve with the increasing concentrations of hydrogen peroxide. While the isolated leucocytes showed no increase of DNA damage after in vitro exposure to 2-methyl-1,4-naphthoquinone (MNQ), erythrocytes showed dose-dependent DNA damage. These results indicate that the comet assay can be applied successfully as a bioassay using erythrocyte for environmental monitoring.

• 한국환경성돌연변이발암원학회지
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• 제21권2호
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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.

• 한국환경성돌연변이발암원학회지
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• 제24권2호
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• pp.79-84
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• 2004

Three synthetic chemicals, benzoyl chloride, 2-propyn-l-ol, and 2-phenoxy ethanol were selected for genotoxicity testing, based on production quantity and available genotoxic data. In our previous report, benzoyl chloride induced chromosomal aberrations in Chinese hamster lung (CHL) fibroblast in vitro with and without metabolic activation, while 2-propyn-l-ol and 2-phenoxy ethanol induced only with metabolic activation. To compare the genotoxicity of chromosome aberration assay, the single cell gel electrophoresis (comet) assay subjected using CHL cells. As a result, statistically significant differences of tail moment values of benzoyl chloride, 2-propyn-1-ol, and 2-phenoxy ethanol were observed compared with control values on almost all concentrations with S9 or without S9 metabolic activation system. This results suggest that genotoxic results of the comet assay and the chromosome aberration assay show correlationship of genotoxicity in the CHL fibroblast. In summary, the positive result of chromosome aberration of benzoyl chloride, 2-propyn-l-ol, and 2-phenoxy ethanol was also induced DNA damages in comet assay with same cell line. Consequently, comet assay will be useful and more accurate tool to detect and to confirm the genotoxicity especially DNA damages in CHL fibroblast.

• 한국환경성돌연변이발암원학회지
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• 제21권1호
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• pp.34-43
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• 2001

Di-2-ethylhexyl phthalate (DEHP) is the most commonly used phthalate ester in polyvinyl chloride formulations including food packing and storage of human blood. DEHP is a well known as non-genotoxic carcinogen and endocrine disrupting chemical (EDC). DEHP have shown all negative results in ICH-guildeline recommended standard genotoxicity test battery. In this study, to assess the clastogenic and DNA damaging effect in human-derived tissue specific cells, DEHP was treated in human derived MCE-7 cells, HepG2 cells, LNCap cells, BeWo cells, MCE-10A cells, and female peripheral blood cells using micronucleus assay and in human breast carcinoma MCF-7 cells up to $1.28$\times$10^{-2}$ M using Comet assay. The in vitro micronucleus assay is a mutagenicity test system for the detection of chemicals which induce the formation of small membrane bound DNA fragment i.e. micronuclei in the cytoplasm of interphase cells, originated from clastogenic and/or aneugenic mechanism. The single cell gel electrophoresis assay (Comet assay) is used to detect DNA strand-breaks and alkaline labile site. In our results, DEHP increased significantly and/or dose-depentently and time-dependently micronucleus frequency at the 6 and 24 hr without metabolic activation system only in MCE-7 cells. DEHP treated with 2 hrs in MCF-7 cells using Comet assay induced DNA damage dose-depentantly.

• 한국독성학회:학술대회논문집
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• 한국독성학회 2002년도 Molecular and Cellular Response to Toxic Substances
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• pp.146-146
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• 2002

The alkaline single cell gel electrophoresis (comet) assay was used to clarify in vitro and in vivo genotoxicity of 2-bromopropane (2-BP). For in vitro studies, fresh medium containing 2-BP (2.50, 1.00, 0.50, 0.25, 0.10, 0.05, 0.01 mM, and vehicle control) were added in human lymphocytes.(omitted)

• 한국환경성돌연변이발암원학회지
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• 제21권2호
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• pp.128-134
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• 2001

Single cell gel electrophoresis (SCGE) assay, also called comet assay, is a rapid and sensitive method to detect DNA damage in single cell level. To evaluate the DNA damage of lymphocytes of pesticides sprayers, SCGE assay was carried out for 50 pesticides sprayer and 58 control subjects. They were interviewed with structured questionnaire to get the information about the kinds and amount of pesticide. Insecticides and fungicides were predominant among pesticides. Major components of pesticides were organophosphorus, organosulfate, cartap, carbamates, and triazole. Sprayed pesticides were classified into two groups. Group I included organophosphorus, organoarsenic, organotin, tetrazine, triazole and gramoxone, which were known to cause DNA damages. Group II pesticide were carbamates, surfactants, organosulfates, etc., which were not found as DNA damaging agents in scientific documents. Olive tail moments of 100 lymphocytes were measured by KOMET 3.1 program for each person. The means of tail moments were compared between farmers exposed to pesticides and control subjects. Farmers showed higher tail moments than control subjects (2.07$\pm$1.40 vs 1.53$\pm$0.77, p<0.05). The means of tail moments also were compared among group I sprayers (n=36), group II sprayers (n=24) and, control subject, and the means or tail moments were 3.4s$\pm$3.2o, 2.66$\pm$2.20 and 1.53$\pm$0.77 respectively. The difference between means of group I sprayers and controls was statistically significant (p<0.05). In conclusion, this study showed higher DNA damage in farmers exposed to pesticides than control subjects, and comet assay could be useful as a biological monitoring method of genotoxic pesticides for farmers.

• Environmental Analysis Health and Toxicology
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• 제16권1호
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• pp.17-20
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• 2001

The alkaline comet assay, employing a single-cell gel electrophoresis(SCGE), is a rapid, simple and sensitive technique for visualizing and measuring DNA damage leading to strand breakage in individual mammalian cells. The protecting effect of pretreatment with vitamin C and cysteine on the DNA damage of gamma ray was investigated in mice splenic lymphocytes. Vitamin C and cysteine were administered orally for five consecutive days before irradiation. Four week old ICR male mice were irradiated wish 3.5Gy of γ-radiation and were sacrificed 3 days later. Spleens were taken for DNA damage examination by Comet assay and the tail moments of DNA single -strand breaks in tole splenic lymphocytes were evaluated. The results show that pretreatment with vitamin C and cysteine were effective in protecting against DNA damage by gamma ray. Administration of antioxidants like vitamin C and cysteine to mice before irradiation was effective in reducing the tail moment of splenic lymphocytes DNA.

• Molecular & Cellular Toxicology
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• 제1권2호
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• pp.99-105
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• 2005

Perchloroethylene (tetrachloroethylene, PCE), a dry cleaning and degreasing solvent, can enter ground-water through accidental leak or spills. PCE can be degraded to trichloroethylene (TCE), 1, 1-dichloroethylene (DCE) and vinyl chloride (VC) as potential bio-product. These compounds have been reported that they can cause clinical diseases and cytotoxicity. However, only a little genotoxic information of these compounds has been known. In this study, we investigated DNA single strand breaks of PCE, TCE, DCE and VC by single cell gel electrophoresis assay, (comet assay) which is a sensitive, reliable and rapid method for DNA single strand breaks with mouse lymphoma L5178Y cells. From these results, $37.5\;{\mu}g/ml$ of PCE, $189\;{\mu}g/ml$ of TCE and $56.4\;{\mu}g/ml$ of DCE were revealed significant DNA damages in the absence of S-9 metabolic activation system meaning direct-acting mutagen. And in the presence of S-9 metabolic activation system, $41.5\;{\mu}g/ml$ of PCE, $328.7\;{\mu}g/ml$ of TCE and $949\;{\mu}g/ml$ of DCE were induced significant DNA damage. In the case of VC, it was revealed a significant DNA damage in the presence of S-9 metabolic activation system. Therefore, we suggest that chloroethylene compounds (PCE, TCE, DCE and VC) may be induced the DNA damage in a mammalian cell.

• 한국식품영양과학회지
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• 제29권5호
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• pp.843-848
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• 2000

콩류의 방사선 조사 여부를 DNA comet assay로 확인하였다. 대두, 강남콩, 그리고 팥을 각각 0.1, 0.3, 0.5, 0.7, 그리고 1.0 kGy의 저선량으로 조사하고 비조사 시료와 조사시료간의 DNA 손상정도를 현미경으로 육안 검사 및 comet의 tail 길이를 측정하여 상호 비교하였다. 모든 시료에서 비조사 시료보다 조사시료의 tail 길이가 더 길었으며 조사 선량이 증가할수록 tail 길이가 길게 나타났다. 비조사 시료에서도 손상된 세포의 comet 모양의 핵이 관찰되기는 하였으나 대부분 비손상된 세포의 원형모양의 핵이 관찰되었으며 조사된 시료에서는 comet 모양의 핵이 주로 관찰되는 경향이어서 비조사 시료와 조사 시료간에 comet 양상을 비교할 수 있었다. 한편 100개의 세포를 무작위로 선택하여 comet tail 길이를 측정하고 통계분한 결과, 비조사 시료와 조사시료간의 유의적인 차이가 있었고 조사선량이 증가할수록 tail 길이가 유의적으로 증가함을 확인하였다. DNA comet assay로 간단하고 비교적 저렴한 비용을 짧은 시간 내에 콩류의 방사선 조사 여부를 확인할 수 있었다.