• Title, Summary, Keyword: comet assay

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Genotoxicity Study on Khal, a Halocidin Derivative, in Bacterial and Mammalian Cells

  • Kim, Youn-Jung;Kim, Mi-Soon;Jeon, Hee-Kyoung;Ryu, Jae-Chun
    • Molecular & Cellular Toxicology
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    • v.2 no.3
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    • pp.151-158
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    • 2006
  • Khal was a synthetic congener of halocidin, a heterodimeric peptide consisting of 19 and 15 amino acid residues detected in Halocynthia aurantium. This compound was considered a candidate for the development of a novel peptide antibiotic. The genotoxicity of Khal was subjected to high throughput toxicity screening (HTTS) because they revealed strong antibacterial effects. Mouse lymphoma thymidine kinase ($tk^{+/-}$) gene assay (MOLY), single cell gel electrophoresis (Comet) assay and chromosomal aberration assay in mammalian cells and Ames reverse mutation assay in bacterial system were used as simplified, inexpensive, short-term in vitro screening tests in our laboratory. These compounds are not mutagenic in S. typhimurium TA98 and TA100 strains both in the presence and absence of metabolic activation. Before performing the comet assay, $IC_{20}$ of Khal was determined the concentration of $25.51\;{\mu}/mL\;and\;21.99\;{\mu}g/mL$ with and without S-9, respectively. In the comet assay, Khal was not induced DNA damage in mouse lymphoma cell line. Also, the mutation frequencies in the Khal-treated cultures were similar to the vehicle controls. It is suggests that Khal is non-mutagenic in MOLY assay. And no clastogenicity was observed in Khal-treated Chinese hamster lung cells. The results of this battery of assays indicate that Khal has no genotoxic potential in bacterial or mammalian cell systems. Therefore, we suggest that Khal, as the optimal candidates with both no genotoxic potential and antibacterial effects must be chosen.

DNA Damage and Micronuclei Induced by Di (2-ethylhexyl) phthalate in Human Breast Carcinoma MCF-7 cells (Di(2-ethylhexyl) phthalate에 의해 유도된 DNA손상과 소핵 형성)

  • 김종원;한의식;박미선;엄미옥;김인숙;전혜승;정해관;심웅섭;오혜영
    • Environmental Mutagens and Carcinogens
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    • v.21 no.1
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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.

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In Vitro Studies on the Genotoxic Effects of Wood Smoke Flavors

  • Chung, Young-Shin;Ahn, Jun-Ho; Eum, Ki-Hwan;Choi, Seon-A;Oh, Se-Wook;Kim, Yun-Ji;Park, Sue-Nie;Yum, Young-Na;Kim, Joo-Hwan;Lee, Michael
    • Toxicological Research
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    • v.24 no.4
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    • pp.321-328
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    • 2008
  • Smoke flavors based on the thermal decomposition of wood have been applied to a variety of food products as an alternative for traditional smoking. Despite its increasing use, the available genotoxicity data on wood smoke flavors (WSF) are still controversial. Thus, potential genotoxic effects of WSF in four short-term in vitro genotoxicity assays were investigated, which included the Ames assay, chromosomal aberration assay, micronucleus test and the alkaline comet assay. WSF did not cause any mutation in the Ames assay using five tester strains at six concentrations of 0.16, 0.31, 0.63, 1.25, 2.5 and 5 ${\mu}l/plate$. To assess clastogenic effect, the in vitro chromosomal aberration assay was performed using Chinese hamster lung cells. No statistically significant increase in the number of metaphases with structural aberrations was observed at the concentrations of 1.25, 2.5, and 5 ${\mu}l/ml$. The in vitro comet assay and micronucleus test results obtained on L5178Y cells also revealed that WSF has no genotoxicity potential, although there was a marginal increase in micronuclei frequencies and DNA damage in the respective micronucleus and comet assays. Taken together, based on the results obtained from these four in vitro studies, it is concluded that WSF is not a mutagenic agent in bacterial cells and causes no chromosomal and DNA damage in mammalian cells in vitro.

DNA Damage Effect of Botanical Insecticides Using Chinese Hamster Lung Cells

  • Kim, Areumnuri;Jeong, Mihye;Park, Kyung-Hun;Chon, Kyongmi;Cho, Namjun;Paik, Min Kyoung
    • Korean Journal of Environmental Agriculture
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    • v.34 no.4
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    • pp.350-354
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    • 2015
  • BACKGROUND: Botanical insecticides, especially Azadirachta Indica extract (AIE) and Sophorae radix extract (SRE) are widely used in Agriculture field. In our previous studies on genotoxicity test of AIE and SRE samples, a suspicious clastogenic properties was shown. Herein, we investigated the DNA damage effect of these botanical insecticide samples through the in vitro comet assay. METHODS AND RESULTS: Chinese hamster lung (CHL) fibroblast cell line was used, and methyl methanesulphonate was as positive control. Respective two samples of AIE and SRE were evaluated using Single Cell Gel Electrophoresis (Comet) assay and measured as the Olive tail moment (OTM). Results from this study indicated that all tested AIE and SRE samples did not show DNA damage in comet assay using CHL cells, compared with control. CONCLUSION: AIE and SRE samples used in this study were not cause genetic toxicity and are suitable for use as organic materials.

Application of Single Cell Gel Electrophoresis for Detection of DNA Single Strand Breaks in DNA of Fish Blood Cell (어류혈구세포에 있어서 Single Cell Gel Electrophoresis를 응용한 DNA Single Strand Breack의 측정)

  • KIM Gi Beum;LEE Richard F.;MARUYA Keith A.
    • Korean Journal of Fisheries and Aquatic Sciences
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    • v.36 no.4
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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.

Evaluation of protective effect of peach kernel extracts on radiation-induced DNA damage in human blood lymphocytes in the single cell gel electrophoresis assay (단세포 겔 전기영동법을 이용한 사람 림프구 DNA 손상에 대한 복숭아씨 추출물의 방사선 방어효과 평가)

  • Kim, Jin-Kyu;Park, Tae-Won;Lee, Chang-Joo;Chai, Young-Gyu
    • Journal of Radiation Protection and Research
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    • v.24 no.2
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    • pp.93-99
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    • 1999
  • The alkaline single-cell gel electrophoresis (SCGE) assay, called the comet assay, has been applied to the detection of DNA damage from a number of chemical and biological factors in vivo and in vitro. The comet assay is a novel method to assess DNA single-strand breaks, alkali-labile sites in individual cells. The effect of peach kernel extracts on radiation-induced DNA damage in human blood lymphocytes was evaluated by the SCGE assay. The lymphocytes, with or without pretreatment of the extracts, were exposed to 0, 0.1, 0.3, 0.5, 1.0 and 2.0 Gy of $^{60}Co$ gamma ray. Significantly increased tail moment, which was a marker of DNA strand breaks in the comet assay, showed an excellent dose-response relationship. The treatment of the peach kernel extracts reduced the DNA damage by 30 % in irradiated groups as compared to that in non-treated control groups. The result indicates that the extracts shows radioprotective effect on lymphocyte DNA when assessed by the comet assay.

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EVALUATION OF THE GENOTOXICITY OF FERRIC SULFATE BY COMET ASSAY (Comet assay를 이용한 Ferric Sulfate의 유전자 독성에 대한 연구)

  • Kang, Ho-Seung;Kim, Shin;Jeong, Tae-Sung;Park, Hae-Ryoun
    • JOURNAL OF THE KOREAN ACADEMY OF PEDTATRIC DENTISTRY
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    • v.27 no.1
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    • pp.77-84
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    • 2000
  • Although ferric sulfate has been proposed as an alternative to formocresol in pulpotomy treatment in primary teeth, it has been given little concern regarding its cytotoxicity and mutagenicity. In the present study, we assessed the in vitro genotoxic effect of a ferric sulfate on human gingival fibroblast cell line (HGF-1). DNA damage was evaluated using comet assay (single cell alkaline gel electrophoresis) and obtained the results as follows: 1. A dose-response relationship was found between ferric sulfate concentrations (0 to 5mM) and DNA damages. 2. Above the concentration of 0.1mM, DNA damage was significantly increased than those of the control (p<0.05). 2. At the fixed concentration of 0.05mM, no significant difference was found between exposure time and DNA damage. These findings suggest that ferric sulfate as a pulpotomy agent can induce DNA damage in human gingival fibroblasts.

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Effect of N-acetyl-L-cysteine on human chronic myeloid leukemia cells KCL22 treated with mitomycin C

  • Simonyan, Anna;Hovhannisyan, Galina;Aroutiounian, Rouben;Kim, Jin Kyu
    • Journal of Ecology and Environment
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    • v.37 no.1
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    • pp.31-34
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    • 2014
  • The effectiveness of N-acetyl-L-cysteine (NAC) to protect blood cells against Mitomycin C (MMC) induced genotoxicity was investigated in human chronic myeloid leukemia cells (KCL22) using the alkaline comet assay. The comet assay was selected as sensitive and rapid method for analysis of DNA damage and repair in individual cells. NAC treatment alone did not produce any damage in KCL22 cell. But NAC was found to be effective in reducing genotoxic damage in KCL22 cells exposed to MMC. These results confirm the literature data that, given the safety and ability to reduce DNA damage. NAC may be useful to prevent drug-mediated genotoxicity.

DNA Damage Induced by New Pophyrins of Different Chemical Structure

  • Galina Hovhannisyan;Samvel Haroutiunian;Kristina Margaryan;Robert Ghazaryan;Rouben Aroutiounian
    • Korean Journal of Environmental Biology
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    • v.23 no.4
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    • pp.379-382
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    • 2005
  • The new cationic meso-substituted N-quarternized 4-pyridylporphyrins and their metal derivatives were synthesized as novel chemotherapeutics. The level of DNA damage induced by porphyrins TOBut4PyP, TOBut4PyP, TOEt4PyPMn and TOBut4PyPMn and its dependence on the chemical structure of compounds were analyzed by the Comet-assay. On the base of data obtained, the investigated porphyrins may be arranged by their genotoxic activity in the following order: TOEt4PyP>TOEt4PyPMn>TOBut4PyP>TOBut4PyPMn. Thus, i) the genotoxicity of the Mn-derivatives of TOEt4PyP and TOBut4PyP is higher than the original porphyrins and ii) the genotoxicity of TOEt4PyP and TOEt4PyPMn is increased after substitution of a butyl radical for ethyl one. The applied Comet-assay permits to reveal the dependence of DNA damage induction on the chemical structure of porphyrins.