• Title/Summary/Keyword: single cell gel electrophoresis (comet assay)

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Genotoxicity Study of Sophoricoside, a Constituent of Sophora japonica, in Bacterial and Mammalian Cell System

  • Kim, Youn-Jung;Park, Hyo-Joung;Kim, Young-Soo;Kim, Mi-Kyung;Lee, Seung-Ho;Jung, Sang-Hun;Ryu, Jae-Chun
    • Environmental Mutagens and Carcinogens
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    • v.21 no.2
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    • pp.99-105
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    • 2001
  • Sophoricoside was isolated as the inhibitor of IL-5 bioactivity from Sophora japonica (Leguminosae). It has been reported to has an anti-inflammatory effect on rat paw edema model. To develope as an anti-allergic drug, genotoxicity of sophoricoside was investigated in bacterial and mammalian cell system such as Ames bacterial reversion test, chromosomal aberration assay and single cell gel electrophoresis (Comet) assay. As results, in the range of 1,250~40 $\mu\textrm{g}$/plate sophoricoside concentrations was not shown significant mutagenic effects in Salmonella typhimurium TA 98, TA 100, TA 1535 and TA 1537 strains in Ames test. The 80% cell growth inhibition concentration (IC/SUB 80/) of sophoricoside was determined as above 5,000 $\mu\textrm{g}$/$m\ell$ in Chinese hamster lung (CHL) fibroblast cell and L5178Y mouse lymphoma cell line for the chromosomal aberration and comet assay, respectively. Sophoricoside was not induced chromosomal aberration in CHL fibroblast cell at concentrations of 700, 350 and 175 $\mu\textrm{g}$/$m\ell$ or 600, 300 and 150 $\mu\textrm{g}$/$m\ell$ in the absence or presence of S-9 metabolic activation system, respectively. Also, in the comet assay, the induction of DNA damage was not observed in L5178Y mouse lymphoma cell line both in the absence or presence of S-9 metabolic activation system. From these results, no genotoxic effects of sophoricoside were observed in bacterial and mammalian cell systems used in these experiments.

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Protective Effects of Paeonia japonica against Radiation-induced Damage (방사선 장해에 대한 백작약의 방호효과)

  • Oh, Heon;Park, Hae-Ran;Jeong, Ill-Yun;Kim, Sung-Ho;Jo, Sung-Kee
    • Journal of Radiation Protection and Research
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    • v.27 no.3
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    • pp.181-188
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    • 2002
  • We investigated the effect of Paeonia japonica (PJ) on radiation-induced oxidative damage to macromolecules in vitro and in vivo. The PJ reduced the tail moment (TM) which was a marker of DNA strand break in single-cell gel electrophoresis (SCGE; comet assay) in the human peripheral blood lymphocytes. Lipid peroxidation in the liver of the ICR mouse, measured as malondialdehyde (MDA), was also reduced by PJ administration. Ethanol fraction of PJ was more effective than polysaccharide fraction of that on reduction of TM in SCGE and lipid peroxidation. Also, Their activities to scavenge DPPH radicals and hydroxyl radicals were observed in vitro, and the activities were due to its ethanol fraction. It is plausible that scavenging of flee radicals by PJ extract may have played an important role in providing the protection against the radiation-induced damage. These results indicated that Paeonia japonica might be a useful radioprotector, especially since it is a relatively nontoxic natural product.

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.

PROTECTION OF DNA BY SCUTELLARlA BAICALENSIS IN HL-60 CELLS EXPOSED TO $\gamma$-RAYS; ANALYSED BY MICRONUCLEI FORMATION AND SINGLE CELL GELL ELECTROPHORESIS

  • Heon Oh;Park, Hae-Ran;Ham, Yeon-Ho;Kim, Sung-Ho;Jo, Sung-Kee
    • Proceedings of the Korean Society of Toxicology Conference
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    • 2001.10a
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    • pp.107-107
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    • 2001
  • In the present study, the protective effect of Scutellaria baicalensis against DNA damage in HL-60 cells exposed to $^{60}$ Co ${\gamma}$-rays was evaluated using micronuclei formation and alkaline single cell gel electrophoresis (SCGE, comet assay). The frequency of micronuclei was decreased in groups treated with water extract (P<0.01), polysaccaride fraction (P<0.01) and methanol fraction (P<0.01) before/after exposure to 200 cGy of ${\gamma}$-rays.(omitted)

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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
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    • v.37 no.4
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    • pp.472-476
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    • 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.

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 Pediatric 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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Protective Effect of Yellow-Green Vegetable Juices on DNA Damage in Chinese Hamster Lung Cell Using Comet Assay (Comet Assay를 이용한 케일, 명일엽, 당근, 돌미나리 녹즙의 Chinese Hamster Lung 세포 DNA 손상 보호 효과)

  • 전은재;김정신;박유경;김태석;강명희
    • Journal of Nutrition and Health
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    • v.36 no.1
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    • pp.24-31
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    • 2003
  • The present study was attempted to investigate the antioxidant capacity of popular yellow-green vegetable juices (kale, Angelica keishei, carrot, small water dropwort) and to investigate the effect of vegetable juices on protecting oxidative damage to DNA in cultured Chinese hamster lung (CHL) cells. Antioxidant capacity was analyzed by TRAP assay (Total radical-trapping antioxidant potential). Cellular DNA dmamage was measured by SCGE (single-cell gel electrophoresis, also known as comet assay. Cells incubated in medium with PBS (negative control) or with various concentration of the freeze dried green juices (25, 50, 100, 250 $\mu\textrm{g}$/$m\ell$) resuspended in PBS were treated with $H_2O_2$ (200 ${\mu}{\textrm}{m}$) as an oxidative stimulus for 5 min at 4$^{\circ}C$. The physiological function of each vegetable juice on oxidative DNA damage was analyzed and expressed as tail moment (tail length X percentage migrated DNA in tail) . Kale juice had the highest TRAP value suggesting that kale has the highest antioxidant capacity followed by Angelica keishei, small water dropwort and carrot. Cells treated with $H_2O_2$ had extensive DNA damage compared with cells treated with PBS or pre-treated with vegetable juice extracts. All green juices inhibited $H_2O_2$-induced DNA damage with kale being the most effective juice among the tested juices. These results indicate that green juice supplementation to CHL cells followed by oxidative stimulus inhibited damage to cellular DNA, supporting a protective effect against oxidative damage induced by reactive oxygen species. (Korean J Nutrition 36(1) : 24-31, 2003)

Protective Effect of Green Tea Extract and EGCG on Ethanol-induced Cytotoxicity and DNA Damage in NIH/3T3 and HepG2 Cells

  • Kim, Nam Yee;Kim, Hyun Pyo;Heo, Moon Young
    • Journal of Food Hygiene and Safety
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    • v.31 no.1
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    • pp.1-7
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    • 2016
  • In the present study, our aim was to determine whether green tea extract (GTE) and its major constituent, epigallocatechin-3-gallate (EGCG) have a protective effect on ethanol-induced cytotoxicity and DNA damage in NIH/3T3 and HepG2 cells. The cell viability and DNA single strand breaks were examined by MTT assay and alkaline single cell gel electrophoresis (Comet assay), respectively. Ethanol decreased the cell viability and also increased DNA single strand breaks in a concentration-dependent manner. On the other hand, GTE showed the protective effect of cytotoxicity and DNA damage induced by ethanol in both cell lines. GTE and EGCG, were found to possess the anti-oxidative and anti-genotoxic activities by evaluation with DPPH test, LDL oxidation assay, oxidative DNA damage assay and 8OH-2'dG generation test. These results were also verified by the experimental results demonstrating the lower cytotoxicity and genotoxicity of commercial green tea liqueur compared to pure ethanol in same concentration. Thus it is concluded that the supplementation of GTE or EGCG may mitigate the ethanol-induced cytotoxicity and DNA damage.