• Title, Summary, Keyword: comet assay

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Evaluation of the Genetic Toxicity of Synthetic Chemicals (XIII) - Single Cell Gel Electrophoresis of Benzoyl Chloride, 2-Propyn-1-ol, and 2-Phenoxyethanol in Chinese Hamster lung Fibroblast -

  • Ryu, Jae-Chun;Kim, Youn-Jung
    • Environmental Mutagens and Carcinogens
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    • v.24 no.2
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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.

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Antimutagenic Effects of Ginsenoside Rb$_1$, Rg$_1$ in the CHO-K1 Cells by Benzo[a]pyrene with Chromosomal Aberration Test and Comet Assay

  • Kim, Jong-Kyu;Kim, Soo-Jin;Rim, Kyung-Taek;Cho, Hae-Won;Kim, Hyeon-Yeong;Yang, Jeong-Sun
    • Molecular & Cellular Toxicology
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    • v.5 no.2
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    • pp.126-132
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    • 2009
  • The usage and types of chemicals are advancing, specializing, large-scaled increasing, and new chemical exposed workers are concerning to occupational disease. The generation of reactive oxygen in the body from carcinogen, mutation and DNA damage in cancer is protected by natural antioxidants (phytochemicals) with antimutagenic effect. There were many reports of ginsenoside Rb$_1$, Rg$_1$ grievances of the genetic mutation to suppress the effect confirm the genetic toxicity test with chromosomal aberration test and the Comet (SCGE) assay confirmed the suppression effect occurring chromosomal DNA damage. We had wanted to evaluate the compatibility and sensitivity between the chromosomal aberration (CA) test and the Comet assay. We used the CA test and Comet assay to evaluate the anti-genotoxicity of ginsenoside Rb$_1$ and Rg$_1$, in CHO-K1 (Chinese hamster ovary fibroblast) cell in vitro, composed negative control (solvent), positive control (benzo[a]pyrene), test group (carcinogen+variety concentration of ginsenoside) group. The positive control was benzo[a]pyrene (50 $\mu$M), well-known carcinogen, and the negative control was the 1 % DMSO solvent. The test group was a variety concentration of ginsenoside Rb$_1$, Rg$_1$ with 10$^{-8}$%, 10$^{-6}$%, 10$^{-4}$%, 10$^{-2}$%, 1%, 10%. In chromo-somal aberration test, we measured the number of cells with abnormally structured chromosome. In Comet assay, the Olive tail moment (OTM) and Tail length (TL) values were measured. The ratio of cell proliferation was increased 8.3% in 10$^{-8}$%, 10$^{-6}$%, 10$^{-4}$%, 10$^{-2}$%, 1%, 10% Rb$_1$ treated groups, and increased 10.4% in 10$^{-10}$%, 10$^{-8}$%, 10$^{-6}$%, 10$^{-4}$%, 10$^{-2}$%, 1% Rg$_1$ treated groups. In the CA test, the number of chromosomal aberration was decreased all the Rb$_1$ and Rg$_1$ treated groups. In the Comet assay, the OTM values were decreased in all the Rb$_1$ and Rg$_1$ treated groups. To evaluate the compatibility between CA and Comet assay, we compared the reducing ratio of chromosomal abnormalities with its OTM values, it was identified the antimutagenicity of ginsenoside, but it was more sensitive the CA test than the Comet assay. Ginsenoside Rb$_1$ and Rg$_1$ significantly decrease the number of cells with chromosomal aberration, and decrease the extent of DNA migration. Therefore, ginsenoside Rb$_1$, Rg$_1$ are thought as an antioxidant phytochemicals to protect mutagenicity. The in vitro Comet assay seems to be less sensitive than the in vitro chromosomal aberration test.

Single Cell Gel Electrophoresis (comet assay) to Detect DNA Damage and Apoptosis in Cell Level (DNA damage와 Apoptosis를 정량화하는 단세포전기영동법)

  • 류재천;김현주;서영록;김경란
    • Environmental Mutagens and Carcinogens
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    • v.17 no.2
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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.

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Detection of Irradiated Beef and Pork by DNA Comet Assay (DNA Comet Assay를 이용한 방사선 조사 쇠고기와 돼지고기의 검지 기술)

  • 박준영;오경남;김경은;양재승
    • Journal of the Korean Society of Food Science and Nutrition
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    • v.29 no.6
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    • pp.1025-1029
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    • 2000
  • This study was conducted to investigate whether a DNA comet assay could be applied for identifying irradiated pork and beef. Pork and beef were irradiated with Co-60 gamma rays at 0.1, 0.3, 0.5, 0.7 and 1.0 kGy, and stored in a freezer Cells separated from the samples were embedded in agarose gel on a slide, dissolved in a lysis solution, and electrophoresed at 2 V/cm for 2.0 min by horizontal electrophoesis. The cells were then stained with a silver staining in order to visualize the DNA using a micro-scope. The DNA fragments of the irradiated cells stretched or migrated out of the cells and formed tails towards the anode, giving the appearance of comets, while unirradiated cells formed very short or no tails. The distance of DNA migration increased with irradiation dose. Since the statistical analysis showed a significant correlation between tail length and irradiation dose, a DNA comet assay could provide not only identification but also estimation of the irradiation dose for irradiated beef and pork.

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Identification Characteristics of Irradiated Dried Red Pepper during Storage by Analysis of Thermoluminescence, DNA Comet, and DEFT/APC (Thermoluminescence, DNA Comet 및 DEFT/APC 분석에 의한 방사선처리 건고추의 저장 중 검지 특성)

  • Kim, Byeong-Keun;Kwon, Joong-Ho
    • Korean Journal of Food Science and Technology
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    • v.36 no.6
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    • pp.851-856
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    • 2004
  • Minerals separated from irradiated dried red pepper (whole) at 2.5 kGy or higher showed typical thermoluminescence (TL) glow curves ($TL_1$) at around $150^{\circ}C$, which increased with irradiation dose. The TL ratio ($TL_1/TL_2$) through re-irradiation step at 1 kGy enhanced reliability of TL identification results. DNA comet assay indicated that the intact cell was observed in non-irradiated pepper (seed), while some long tails were found in irradiated ones, showing relationship between irradiation dose and tail length. Log DEPT/APC values increased in proportion to irradiation doses in powdered and whole peppers. Based on overall results, irradiated dried red peppers could be screened using DNA comet assay or log DEFT/APC, and moreover the identification results were verified by TL analysis.

Evaluation of sperm DNA fragmentation using multiple methods: a comparison of their predictive power for male infertility

  • Javed, Aamir;Talkad, Muralidhar Srinivasaih;Ramaiah, Manjula Kannasandra
    • 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.

Study on Mutagenicity of DehydroevodiamineㆍHCl(DHED) (치료제 DehydroevodiamineㆍHCl(DHED)의 변이원성 연구)

  • 성이숙;정성윤;정주연;채규영;진미령;최봉웅;장병모;김대경
    • YAKHAK HOEJI
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    • v.46 no.3
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    • pp.208-212
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    • 2002
  • Dehydroevodiamine HCl (DHED), which is a component separated from Evodia rutaecarpa Bentham, has novel anticholinesterase and antiamnesic activities in the scopolamine-induced amnesia model. Several studies suggest that DHED might be an effective drug for the Alzheimer's disease and the vascular type of dementia. In order to evaluate the mutagenic potential of DHED, Salmonella typhimurium reversion assay, chromosomal aberration test on Chinese hamster lung cells, in vivo micronucleus assay using mouse bone marrow cells, and comet assay were performed. DHED did not increase the number of revertant in the reverse mutation test using Salmonella typhimurium TA1535, TA1537, TA98, TA100. DHED HCl, at concentration of 5 and 10 $\mu\textrm{g}$/mι, increased the number of chromosome aberrated Chinese hamster lung cells with 5 and 10%, respectively. In mouse micronucleus test, no significant increase in the occurrence of micronucleated polychromatic erythrocyte was observed in ICR mice orally administered with DHED. DHED was tested for ability to induce genotoxic effect in L5178Y cells (mouse lymphoma cells) using the single cell gel electrophoresis assay (comet assay). In comet assay, tail moment did not increase in L5178Y cells treated with 10, 100, 300 $\mu$M DHED.

Use of the Comet Assay to Assess DNA Damage in Hemocytes and Gill of Oyster(Crassostrea gigas) Exposed to Pyrene and Benzo(a)pyrene (Pyrene과 Benzo(a)pyrene에 노출된 굴의 혈구세포과 아가미 세포에서의 DNA손상 측정을 위한 Comet assay의 이용)

  • 김기범;배세진
    • Journal of Aquaculture
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    • v.16 no.3
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    • pp.196-201
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    • 2003
  • Sessile organisms such as the oyster Crassostrea gigas have been given much attention as a potential biomonitoring indicator to assess the impact of toxicants on aquatic organism. In this study, we exposed cells isolated from gill of oyster (Crassostrea gigas) to hydrogen peroxide in vitro. In addition oysters were in vivo exposed to pyrene and benzo(a)pyrene at various concentrations for 2 weeks. Comet assay was used to detect DNA single strand breaks and to investigate the application of this technique as a tool for aquatic biomonitoring. Hydrogen peroxide increased DNA single strand break with increasing concentration after 30 minutes exposure in vitro. Pyrene and benzo(a)pyrene caused DNA damage only at very high concentration (100 $\mu\textrm{g}$/L or 1000 $\mu\textrm{g}$/L) at two week exposure in vivo. DNA damage was relatively higher at hemocyte than at gill. It suggested that metabolized PAHs are transferred to hemolymph from digestive gland which have a relatively high enzyme activity, and attacked the DNA of hemocyte, while gill accumulated PAHs without degrading them to their metabolites due to low enzyme activity at gill. Both in vitro and in vivo exposure experiments showed that the comet assay is an effective tool on screening whether the organism are exposed to genotoxic contaminants.

Comparison of the Protective Effect of Antioxidant Vitamins and Fruits or Vegetable Juices on DNA Damage in Human Lymphocyte Cells Using the Comet Assay (Comet Assay를 이용한 항산화 비타민과 과일.야채즙의 인체 임파구 세포 DNA 손상 감소 효과 비교)

  • 전은재;박유경;김정신;강명희
    • Journal of Nutrition and Health
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    • v.37 no.6
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    • pp.440-447
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    • 2004
  • In this study the in vitro protective effects of several antioxidant vitamins (vitamin C, $\alpha$-tocopherol, $\beta$-carotene), fruits and vegetables (strawberry, tangerine, orange and 100% orange juice, carrot juice), on the levels of isolated human lymphocyte DNA damage was measured using Comet assay. Comet assay has been used widely to assess the level of the DNA damage in the individual cells. Lymphocytes were pre-treated for 30 minutes with antioxidant vitamins (10, 50, 100, 500 $\mu$M) or fruits$.$vegetables (10, 100, 500, 1000 $\mu$g/ml), an4 then oxidatively challenged with 100 $\mu$M $H_2O$$_2$ for 5 min at 4$^{\circ}C$. The protective effect of antioxidant vitamins against DNA damage at a concentration of 50 $\mu$M were 50% in vitamin C, 32% in $\alpha$-tocopherol, whereas, fJ-carotene showed a 55% protection at a dose as low as 10 $\mu$M. The inhibitory effects of DNA damage by strawberry, tangerine, orange, orange juices, carrot juices were 50 - 60% with wide ranges of doses. The results of the present study indicate that most the antioxidant vitamins and fruits$.$vegetables juices produced a significant reduction in oxidative DNA damage.

The Antigenotoxic Effects of Korean Native Fermented Food, Baechu Kimchi Using Comet Assay (Comet Assay를 이용한 전통발효식품인 배추김치의 항유전 독성효과)

  • 지승택;박종흠;현창기;신현길
    • Journal of the Korean Society of Food Science and Nutrition
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    • v.29 no.2
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    • pp.316-321
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    • 2000
  • This study carried out to eluciate the cancer chemoprevention of Korean native fermented food, baechu kimchi using Comet assay (in other words, single cell microgel electrophoresis). For this purpose, baechu kimchi was fractionated by water, n-hexane, chloroform and ethyl acetate. 5 strains of dominant fermented bacteria were isolated from baechu kimchi. The water fraction, n-hexane fraction, chloroform fraction, ethyl acetate fraction and water insoluble fractions showed no antigenotoxicitie in non-tumoral normal 3T3 cells. Among 5 bacteria isolates from baechu kimchi, two isolates bacteria 1 and 2 strongly inhibited genotoxicity of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in non-tumoral normal 3T3 cells (p<0.05). Bacteria 3, 4 and 5 were also not antigenotoxic.

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