• Title/Summary/Keyword: single cell gel electrophoresis

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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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Smoking Related DNA Damage in Human Lymphocytes Assessed by the Comet Assay (단세포전기영동법으로 평가한 흡연자의 백혈구 DNA손상)

  • 선수진;정해원;한정호
    • Environmental Mutagens and Carcinogens
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    • v.22 no.2
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    • pp.83-89
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    • 2002
  • The single cell gel electrophoresis (comet) assay is one of the useful tools for the study of genetic damage in humans exposed to environmental mutagens and carcinogens. This study was undertaken to evaluate the status of DNA damage in peripheral lymphocytes depending on their sex, age, smoking habits, and other factors in normal healthy Korean population. The 99 volunteers included in the study and out of these, 36 volunteers were smoker and 63 volunteers were non-smoker aged between 20-59 years. All individual answered a questionnaire that assessed their general information including smoking habits and the extent of the environmental tobacco smoke (ETS) exposure, and blood samples were obtained. There was a statistically significant difference in the extent of DNA damage between smoker and non-smoker (p<0.001). A significant difference was also observed between male and female (p<0.001) and amongst the different group of age (p<0.005), however, correlation analysis showed that only smoking habit was a significant factor for DNA damage. No significant effect of smoking duration, number of cigarettes smoking a day, SPY (smoke pack years) in smokers and environmental tobacco smoke exposure in non-smokers on the status of DNA damage was observed.

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Genotoxicity of Di-2-Ethylhexyl phthalate, 2-EthylHexanoic Acid and Di-2-Ethyl Phthalate in Human Lymphocytes in vitro (Di-2-EthylHexyl Phthalate, 2-EthylHexanoic Acid 및 Di-2-Ethyl Phthalate의 유전독성 평가)

  • Song, Joo-Young;Cho, Yoon-Hee;Kim, Yang-Jee;Chung, Hai-Won
    • Environmental Mutagens and Carcinogens
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    • v.25 no.3
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    • pp.110-117
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    • 2005
  • DEHP is one of well known endocrine disrupter and it is used as additives for the production of PVC. There has been contradictional result on the genotoxicity of DEHP. In order to examine genotoxicity of a endocrine disruptors, DEHP (Di-2-EthylHexyl Phthalate) and it's metabolites, EHA (2-EthylHexanoic Acid) and DEP (Di-2-Ethyl Phthalate), chromosome aberration (CA), sister chromatid exchange (SCE), micronuclei (MN) and single cell gel electrophoresis were analysised. No increase of the frequency of CA was observed by DEHP and its two metabolites. DEHPincreased the frequency of SCE and MN whereas EHA only increased the frequency of SCE. DEP increased the frequency of SCE but the increase was not statistically significant. DEHP and DEP, also induced DNA damage. It is suggested that combination of different methods were recomended to find the genotoxicity of DEHP and its metabolites.

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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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Purification of Internal Invertase in Rhodosporidium toruloides Mating Type A Cells (Rhodosporidium toruloides의 접합형 A 세포내 Invertase의 정제)

  • Jeong, Yong-Kee;Ryu, Beung-Ho
    • Journal of the Korean Society of Food Science and Nutrition
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    • v.21 no.6
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    • pp.725-730
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    • 1992
  • The internal invertase was purified from cell free extract of Rhodosporidium toruloides IFO 0559-M-919 by acid precipitation, ion-exchange chromatography and gel filtration to the unique enzyme protein on disc electrophoresis. We have found out that molecular weight of purified internal invertase was 90,000 by gel filtration and the purified enzyme was protein with 4 homogeneous subunits appearing as single band of 22,000daltons on SDS-polyacrylamide gel electrophoresis.

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Proteomic Analysis of Circadian Clock Mutant Mice

  • Lee Joon-Woo;Kim Han-Gyu;Bae Kiho
    • Biomedical Science Letters
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    • v.11 no.4
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    • pp.493-501
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    • 2005
  • Circadian rhythms, time on a scale of about 24 hours, are present in a number of organisms including animals, plants, and bacteria. The control of the biochemical, physiological and behavioral processes is regulated by endogenous clocks in the suprachiasmatic nucleus (SCN). At the core of this timing mechanism is molecular machinery that are present both in the brain and in the peripheral tissues throughout the body, and even in a single cultured cell. In this study, we performed two-dimensional gel electrophoresis to figure out any correlation between protein expression patterns and the requirement of two canonical clock proteins, either mPER1 or mPER2, by comparing global protein expression profiles in livers from wildtype or mPer1/mPer2 double mutant mice. We could identify several differentially expressed protein candidates with respect to time and genotypes. Further analysis of these candidate proteins in detail in vivo will lead us to the better understanding of how circadian clock functions in mammals.

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Assessment of DNA Damage using an Alkaline Single Cell Gel Electrophoresis (SCGE) Comet Assay and Toxic Effects in Chickens by T-2 Toxin Treatment (T-2 toxin을 투여한 닭에서 Comet assay 방법을 이용한 DNA 손상 평가와 독성)

  • Hah Dae-Sik;Heo Jung-Ho;Lee Kuk-Cheon;Cho Myung-Heui;Kim Kuk-Hun;Kim Chung-Hui;Lue Jae-Du;Lee Seung-Hwan;Kim Gon-Sup;Kim Eui-Gyung;Kim Jong-Shu
    • Toxicological Research
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    • v.22 no.2
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    • pp.75-85
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    • 2006
  • This study was designed to evaluate the possible DNA damaging effects of T-2 toxin using an alkaline single cell gel electrophoresis (SCGE) comet assay and also to investigate toxic effects in chickens. A total of 20 chickens were used in these experiments. Graded concentrations of dietary T-2 toxin (0, 4, 8, and $16{\mu}g/g$ of diet) were given to groups of 5 broiler chickens. In comet assay, The DNA damage was analysed by the tail extent moment (TEM) and tail length (TL), which were used as markers of DNA strand breaks in SCGE. A significant dose-dependent increase in the extent of DNA migration as well as in the percentage of cells with tails was observed after treatment with T-2 toxin (P<0.05). Treatment with the low T-2 toxin ($4{\mu}/g$ of diet) induced a relatively low level of DNA damage in comparison with the high T-2 toxin ($16{\mu}/g$ of diet) group. The growth rate was significantly reduced by concentrations of 8, and $16{\mu}/g$ of diet (P < 0.05). The feed conversion ratio were significantly affected by any concentrations (P < 0.05). The relative weight of the spleen, and lung was decreased by the growth inhibitory concentrations. The bursa of Fabricius, thymus, and kid- ney were decreased in relative weight by concentrations of $16{\mu}/g$ of diet. The relative weight of the liver and heart were unaffected. The hemoglobin (Hb), hematocrit (HCT), and mean corpuscular hemoglobin (MCH) were decreased at concentration of $16{\mu}/g$ of diet. As compared with control chickens, there was no marked change in serum components except uric acid in T-2 treated chickens. All lymphoid tissues retained atrophic and lymphoid cell depletion throughout the three weeks trial.

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.

Protective Effects of Ginkgo Biloba Leaf Extract(GBE) against 1,2,4-benzenetriol Induced Toxicity in Vitro (Ginkgo biloga 잎 추출물의 1,2,4-benzenetriol에 대한 항산화 효과에 대한 연구)

  • 이영준;김태연;정해원
    • Journal of Environmental Health Sciences
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    • v.27 no.1
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    • pp.124-130
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    • 2001
  • Ginkgo biliba has been used for bronchitis and asthma in oriental countries and its leaf extract(GBE) contains 24% ginkgoflavone glycoside and 6% terpenoid. Flavonoids and terpenoids are known to have various antioxidant effects such as scavenging of free radicals and chelation of transtional metals. Antioxidant effect of GBE against 1,2,4-benzenetriol(BT), one of toxic metabolites of benzene, was demonstrated throughbsister chromatid exchange(SCE) analysis, single cell gel electrophoresis(SCGE) analysis, DNA cleavage assay and lipid peroxidation production analysis. The means of SCE frequencies at 10, 25 and 50$\mu$M concentration of BT were 7.72, 8.02, 9.22 respectively. In addition of GBE with concentration of 50, 200 and 500$\mu\textrm{g}$/$m\ell$, SCE frequencies were decreased significantly.(p<0.05) According to SCGE analysis, BT induced DNA damage in a dose-dependent manner at concentration of 10 and 50 $\mu$m and the DNA damage induced by BT was significantly protected by GBE(p<0.001). No genotoxicity was observed by GBE treatment alone on DNA cleavage. The effect of BT on lipid peroxidation product, Malondiadehyde(MDA), was increased with concentration of BT(10 and 50 $\mu$M) and reduction in MDA was noted when GBE was added. From above results it is suggested that GBE could protect the cell and DNA from pro-oxidant effect by reactive oxigen species induced by BT.

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