• Title, Summary, Keyword: Comet assay

Search Result 319, Processing Time 0.04 seconds

Validation of Photo-comet Assay as a Model for the Prediction of Photocarcinogenicity

  • Kim, Ji-Young;Koh, Woo-Suk;Lee, Mi-Chael
    • Toxicological Research
    • /
    • v.22 no.4
    • /
    • pp.423-429
    • /
    • 2006
  • Recent reports on the photocarcinogenicity and photogerotoxicity of many compounds led to an increasing awareness for the need of a standard approach to test for photogenotoxicity. The comet assay has been recently validated as a sensitive and specific test system for the quantification of DNA damage. Thus, the objectives of this study are to investigate the utility of photo-comet assay for detecting photo-mutagens, and to evaluate its ability to predict rodent photo-carcinogenicity. Photo-comet assays were performed using L5178Y $Tk^{+/-}$ mouse lymphoma cells on five test substances (8-methoxypsoralen, chlorpromazine, lomefloxacin, anthracene and retinoic acid) that demonstrated positive results in photocarcinogenicity tests. For the best discrimination between the test substance-mediated DNA damage and the undesirable DNA damage caused by direct UV absorption, a UV dose-response of the cells in the absence of the test substances was firstly fnalized. Out of 5 test substances, positive comet results were obtained for chlorpromazine, lomefloxacin, anthracene and retinoic acid while 8-methoxypsoralen found negative. An investigation into the predictive value of this photo-comet assay for determining the photocarcinogenicity showed that photo-comet assay has relatively high sensitivity. Therefore, the photo-comet assay with mammalian cells seems to be a good and sensitive predictor of the photocarcinogenic potential of new substances.

In vivo Comet Assay on Flounder and Clam Exposed to BaP and TBT (BaP 및 TBT에 노출된 넙치와 개조개의 in vivo Comet assay)

  • Kim, So-Jung;Chung, Young-Jae;Lee, Taek-Kyun
    • Ocean and Polar Research
    • /
    • v.33 no.2
    • /
    • pp.127-133
    • /
    • 2011
  • The comet assay, also called single-cell electrophoresis (SCGE) assay, is a potential sensitive monitoring tool for DNA damage in cells. The primary objective of this study was to use comet assay to ascertain if the blood cells of flounder (Pleuronichthys olivaceus) and muscle cells of clam (Saxidomus purpurata) are suitable for genotoxicity screening. This was achieved by initially exposing blood and muscle cells under in vitro conditions to the reference genotoxin hydrogen peroxide ($H_2O_2$); strong correlation between $H_2O_2$ concentration and comet values were found. Subsequently, the identification of DNA damage in isolated cells from flounder and clam was performed under in vivo exposure to benzo(a)pyrene (BaP) and tributyltin (TBT). Flounder and clam were exposed to different concentrations (1, 10, 50, 100 ${\mu}g/L$) of BaP or TBT for 4 days. Regardless of treated chemicals, blood cells of flounder were more prone to DNA breakage compared to muscle cells of clam. In conclusion, in vivo genotoxicity of BaP and TBT can be biomonitored using the comet assay. This study suggests that flounder and clam do show potential as mediums for monitoring genotoxic damage by comet assay.

Study on the Characteristics of DNA Comet Assay for Irradiated Vegetables and Grains (방사선조사된 채소류 및 곡물류의 DNA Comet Assay 특성 연구)

  • Seo, Jung-Eun;Oh, Se-Wook;Kim, Yun-Ji;Lee, Nam-Hyouck;Hong, Sang-Pill;Kim, Young-Ho
    • Journal of the Korean Society of Food Science and Nutrition
    • /
    • v.37 no.4
    • /
    • pp.472-476
    • /
    • 2008
  • The possibility of DNA comet assay as a rapid method for screening the irradiated vegetables and grains was evaluated. Vegetables (spring onion, garlic, and tomato) irradiated at $0{\sim}3$ kGy and grains (rice flour and black soybean) irradiated at $0{\sim}9$ kGy were used as samples. Optimum DNA comet assay conditions, such as elution, sedimentation of suspension, and lysis time of cell, were established. The optimum conditions for vegetables were 10 min for the elution time, 0 min for the sedimentation time, and 5 min for the lysis time. The optimum conditions for grains were 15 min for the elution time, 60 min for the sedimentation time, and 30 min for the lysis time. For the food application of DNA comet assay, it was possible to screen various food samples irradiated at the following doses: spring onion at 2 kGy, garlic at 3 kGy, tomato at 1 kGy, rice flour at 9 kGy, and black soybean at 3 kGy. Each sample showed different forms and sizes in DNA comet. Therefore, further studies on various methods using comet shape, concentration, or area in DNA comet assay are necessary.

A Comparison of Hydrated versus Dehydrated Gels for Evaluation of Apoptosis in Comet Assay Slides (Comet assay slides 에서 나타난 apoptosis 평가에서 함수 및 탈수 겔의 비교)

  • 최민철;수즌엠러루;에드워드엘질럿
    • Journal of Veterinary Clinics
    • /
    • v.13 no.2
    • /
    • pp.158-162
    • /
    • 1996
  • Comet assay 는 포유류 세포에서 DNA의 파괴를 측정하는데 있어 신속하고 단순하며 시각적이고 민감한 방법이다. Apoptosis에서는 세포핵의 광범위한 DNA의 붕괴가 일어나므로 comet assay는 종양세포에서 apoptosis가 발생되었는가를 알아내는데 유용하다. 본 연구는 apoptosis 연구의 결과가 변화되지 않도록 comet assay slides를 좀 더 오래 보관할 수 있는 방법을 개발하고자 시행되었다. 개의 종양세포를 가지고 alkali comet assay를 끝낸 뒤 slides를 진공 건조기에서 꺼내서 증류수로 점적하여 10-20분간 침수시키고 현광현미경하에서 육안적으로 관찰하였다. 건조후 3-4일, 1주, 2주, 3주, 4주 및 7주의 slides에서 apoptosis 회복율(%)은 각각 98.1, 98.3, 99.4, 80.8 및 35.2%이었다. 3주 이내의 slides에서 대조군과 비교하여 apoptosis 회복율에서 차이가 없었으나 4주 이상의 slides를 건조후 침수시키는 방법을 이용하였을 때 apoptosis 평가에서 건조 후 3주간까지는 처음의 결과와 차이가 없으며, 이 방법을 이용하여 comet slide의 좀 더 긴 기간이 보관과 보관후의 재평가에서 이용될 수 있는 좋은 방법이 된다.

  • PDF

Detection of Irradiated Beans Using the DNA Comet Assay (DNA Comet Assay를 이용한 콩류의 방사선 조사 확인)

  • 오경남;김경은;양재승
    • Journal of the Korean Society of Food Science and Nutrition
    • /
    • v.29 no.5
    • /
    • pp.843-848
    • /
    • 2000
  • The single cell-gel electrophoresis assay (comet assay) was used to identify irradiated beans. Soy beans, kidney beans, and red beans were irradiated with $^{60}Co$ gamma rays at 0.1, 0.3, 0.5, 0.7, and 1.0 kGy. Beans were peeled out, crushed lightly, and treated with phosphate-buffered saline (PBS) to extract cells. The extracted cell suspension was mixed with agarose gel solution and spread on an agarose precoated slide. After lysis of the cells, they were subjected to microgel electrophoresis for 2 minutes, and then silver-stained. We found that the DNA fragments of the irradiated samples were stretched, migrated out of the cells, and formed tails towards the anode giving the appearance of comets, while the unirradiated or the undamaged cells formed very short or no tails. The tail lengths of irradiated samples were significantly increased as irradiation dose increased at the above 0.3 kGy.

  • PDF

Application of the Alkaline Comet Assay for Detecting Oxidative DNA Damage in Human Biomonitoring (인체 산화적 DNA손상에 대한 Human Biomonitoring도구로서 Alkaline Comet Assay의 활용 가능성 연구)

  • 박은주;강명희
    • Journal of Nutrition and Health
    • /
    • v.35 no.2
    • /
    • pp.213-222
    • /
    • 2002
  • The alkaline comet assay has been used with increasing popularity to investigate the level of DNA damage in biomonitoring studies within the last decade in Western countries. The purpose of this study was to evaluate the usefulness of the alkaline comet assay as a biomarker of oxidative DNA damage for monitoring in the Korean population, and also to evaluate the effect of nutritional status and lifestyle factors on H2O2 induced oxidative DNA damage measured by the alkaline comet assay in human lymphocytes. The study population consisted of 61 healthy Korean male volunteers, aged 20-28. Epidemiological background data including dietary habits, smoking habits and anthropometrical measurements were collected through personal interviews. After blood collection, the comet assay in peripheral lymphocytes and plasma lipids analysis was carried out and the results analyzed. Tail moment (TM) and tail length (TL) of the comet assay were use\ulcorner to measure DNA damage in the lymphocytes of the subjects. Statistically significant (p < 0.05) positive correlations were observed between DNA damage (TM or TL) and smoking habits expressed as cigarettes smoked per day and pack years (r = 0.311 and 0.382 for TM, r = 0.294 and 0.350 for TL, respectively). There were also significant positive correlations between DNA damage parameter and waist-hip ratio. Higher plasma triglyceride levels were associated with increased damage to DNA. There were no correlations between the consumption frequencies of vegetables and DNA damage to the subjects. However, consumption frequencies of fruit and fruit juice intake were inversely associated with the TM and TL. The results indicate that die comet assay is a simple, rapid and sensitive method for detecting lymphocyte DNA damage induced by cigarette smoking. Consumption of fruit or fruit juices could potentiall modify the damaged DNA in the human peripheral lymphocytes of young Korean men.

Mechanism study on DNA damage and Apoptosis induced by heak shock using Comet Assay

  • Seo, Young-Rok;Han, Sung-Sik;Kim, L. O′Neill;Ryu, Jae-Chun
    • Proceedings of the Korea Society of Environmental Toocicology Conference
    • /
    • /
    • pp.101-101
    • /
    • 1997
  • Comet assay, single cell gel electrophoresis has been known as useful, rapid, simple, visual, and sensitive technique for measuring the DNA breakage in mammalian ce1ls. For evaluation of DNA damage using comet assay, early studies reported a change in comet length and intensity with DNA damage using simple visual technique, such as fluorescence microscopy with eyespiece. In recent, some workers are observing and analyzing nucleotide of comets using quantitative fluorescence image analysis system to estimate 'tail moment', which is defined as the product of the tail length and the fraction of total DNA in tail. Our laboratory also adopted the image analysis software for qualification. In addition, many of the practical features of comet assay render it potentially attractive as useful tool for molecular toxicology and carcinogenesis, because the system is already showing considerable promise as rapid predictor in both in vitro and in vivo experimental designs. Recently, the comet assay becomes a attractive technique to study of apoptosis, because apoptotic fragmentation of nuclear DNA into nucleosomal sizes can be evaluated by the comet assay. So, we attempted to apply the comet assay to studying the effect of various stress on the apoptosis-sensitive cell lines. Particularly, focusing on the hyperthermic apoptosis, we could find that heat shock(44˚C for 60 minutes) was sufficient to induced apoptosis in these cell lines. But using the highly sensitive comet assay, we could not detect DNA breaks immediately after heat shock.

  • PDF

Detection of Irradiated Fruits Using the DNA Comet Assay (DNA Comet Assay를 이용한 과일의 방사선 조사 확인)

  • Oh, Kyong-Nam;Park, Jun-Young;Kim, Kyeung-Eun;Yang, Jae-Seung
    • Korean Journal of Food Science and Technology
    • /
    • v.32 no.3
    • /
    • pp.531-537
    • /
    • 2000
  • The simple microgel electrophoresis of single cells, a 'comet assay', on fruit seeds enabled the rapid identification of irradiated fruits by comparing the intact non-irradiated cells and the damaged cells of irradiated fruits. Grapes and plums were irradiated with 0.1, 0.5, 0.7, 1.0 kGy and strawberries, peaches, apples, and nectarines were irradiated with only 1.0 kGy. Seeds were isolated, crushed, and the suspended cells were embedded in an agarose layer. After lysis of the cells, they were subjected to microgel electrophoresis for 2 minutes, and then stained. The DNA radiation-induced fragmentation of all the fruits stretched and migrated out of the cells forming a tail toward the anode giving the appearance of a comet, while the undamaged cells appeared as intact nuclei without tails. Grape and plum seeds irradiated at 0.5 kGy and higher showed significant increases in tail length. With increasing the irradiation doses, longer extention of the DNA from the nucleus toward the anode was observed. Strawberry, peach, apple, and nectarine seeds irradiated with 1.0 kGy also showed the longer tails than non-irradiated ones. DNA comet assay as a rapid and inexpensive screening technique could be an officially validated method for the detection of irradiated fruits.

  • PDF

Studies on Toxicological Evaluation of Freshwater Sediment using a PLHC-1 Cell Comet Assay (PLHC-1세포주의 Comet assay를 이용한 하천 퇴적토의 생태독성평가)

  • Bak, Jeong-Ah;Hwang, In-Young;Baek, Seung-Hong;Kim, Young-Sug
    • Korean Journal of Environmental Biology
    • /
    • v.29 no.1
    • /
    • pp.23-30
    • /
    • 2011
  • In this study, the Comet assay (evaluation of DNA damage) used the fish hepatocellular carinoma cell, PLHC-1, was tried to the sediment extract obtained from freshwater to understand its applicability as a tool for monitoring sediment toxicity. In parallel, induced EROD (7-ethoxyresorufin- O-deethylase) activity and DNA damage (TEM values) in PLHC-1 cells were measured for establishing the tandem endpoints of the PLHC-1cell test to test the ecotoxicity of sediment. Among several study sites in a small river passed through downtown and industrial park area, one of them, site B, showed a higher level of EROD activity and DNA damage than other sites. It indicates that a tandem endpoints of PLHC-1 cells could be useful tools for assessing the toxicity of sediment. The sensitivity of Comet assay with PLHC-1 cells was a little higher than that with a blood cell of frog tadpoles to the solvent extract of sediment. According to the results, a PLHC-1 cell-Comet assay could be used as a useful tool for evaluating ecotoxicity of the freshwater sediment. In addition, more detailed studies are needed to the contaminated site.

Discrimination of Irradiated Beef Using Comet Assay (Comet assay를 이용한 방사선 조사육의 판별)

  • Jeong, Seok-Kyu;Park, Jong-Heum;Ji, Seung-Taek;Park, Kum-Ju;Kim, Hai-Hong;Hyun, Chang-Kee;Shin, Heuyn-Kil
    • Korean Journal of Food Science and Technology
    • /
    • v.32 no.4
    • /
    • pp.747-754
    • /
    • 2000
  • DNA damages in post-mortem bovine muscle samples caused by gamma irradiation at doses of 1 to 10 kGy were determined by Comet assay. When the cell extract was prepared in a physical method and followed by neutral lysis and neutral electrophoresis, the optimal comet images could be obtained. DNA damages were evaluated from the mean tail length, the distributions of comet images in 4 groups divided by tail length and the relative damage index (RDI) values calculated from the distribution pattern. The mean tail length and RDI value were increased by increasing the irradiation dose, and the RDI value was found to be useful as an index for discriminating of irradiation and measuring the irradiated dose. Blind tests using Korean domestic (Hanwoo) and imported beef samples showed a higher RDI value for the latter. However, the value was lower than those of irradiated samples indicating that the cause of DNA damages in the imported beef samples might be not irradiation but low-temperature treatments. It was concluded from the results of this study that the irradiated beef and its irradiated dose could be detected and predicted by Comet assay.

  • PDF