• Title/Summary/Keyword: DNA single-strand breaks

Search Result 65, Processing Time 0.026 seconds

Sensitization Effects of Hyperthermia on Bleomycininduced DNA Strand Breaks and Replication Inhibition in CHO-$K_1$ Cells in Vitro

  • Kim, Chan-Gil;Kim, Hyeon-Suk;Park, Sang-Dai
    • Environmental Mutagens and Carcinogens
    • /
    • v.15 no.2
    • /
    • pp.88-93
    • /
    • 1995
  • Effects of hyperthermia on the induction of DNA single strand breaks and replication inhibition were studied in bleomycin-treated CHO-K$_1$ cells by alkaline elution and alkaline sucrose gradient sedimentation. Bleomycin-induced DNA single strand breaks of DNA were dose-and time-dependently increased, and these strand breaks of DNA were gradually rejoined as post-incubation time passed. Treatment with hyperthermia alone did not affect the induction of DNA single strand breaks. However, pre-exposure of cells to hyperthermia followed by bleomycin treatment greatly increased the single strand breaks, and also reduced the rejoining processes of bleomycin-induced DNA single strand breaks. Bleomycin selectively inhibited the replicon initiation. The combined treatment with hyperthermia and bleomycin markedly potentiated the nonspecific inhibition of replication.

  • PDF

DETECTION OF DNA SINGLE-STRAND BREAKS AND UNSCHEDULED DNA SYNTHESIS INDUCED BY PROCARCINOGENS IN PRIMARY CULTURES OF RAT HEPATOCYTES

  • Kim, D.H.;Kim, Bok-Ryang;K. H. Yang
    • Toxicological Research
    • /
    • v.2 no.1
    • /
    • pp.1-7
    • /
    • 1986
  • Procarcinogen induced DNA single-strand breaks and unschduled DNA synthesis were measured in primary rat hepatocytes culture. For DNA single-strand breaks assay, rat liver DNA was prelabeled by injection 3H-thymidine during the peak of DNA synthesis following partial hepatectomy. Hepatocytes were isolated from the rat 2 weeks after surgery by a collagenase perfusion techinique and maintained as monolayers in serum free medium on collagen-coated culture dishes. DNA sigle-strand breaks were measured by the alkaline elution techinique.

  • PDF

Quantitative Analysis of DNA Single-strand Breaks in EL 4 cells and Mouse Spleen Lymphocytes after Irradiation (방사선에 의한 EL 4 백서 백혈병 세포 및 정상 백서 비장 임파구 DNA Single-Strand Breaks의 정량적 분석과 측정)

  • Yoo Seong Yul;Cho Chul Koo;Koh Kyung Hwan;Park Woo Yoon;Park Young Hwan;Kim Sung Ho;Kim Tae Hwan;Chung In Yong
    • Radiation Oncology Journal
    • /
    • v.8 no.2
    • /
    • pp.137-144
    • /
    • 1990
  • The filter elution technique was used to assay Co-60 $\gamma$ ray-induced DNA single-strand breaks(SSB) in EL 4 mouse leukemia cell and mouse spleen lymphocyte. The lymphocytes were stimulated with lipopolysaccharide (LPS, 20 $\mug/ml$) to label [${^3}H$] thymidine. EL 4 cells and lymphocytes in suspension were exposed at $0^{\circ}C$ to 0 Gy, 1 Gy, 5 Gy,10 Gy of Co-60 radiation and elution procedure was performed at PH 12.1. The number of DNA single-strand breaks increased with increasing doses of $\gamma$ rays. The strand scission factor (SSF) was estimated in each experiment (eluted volume 21 ml). The slope for EL 4 cells was $0.01301\pm0.00096\;Gy^{-1}(n=5)$ and the slope for lymphocytes was $0.01097\pm0.00091\;Gy^{-1}(n=5)$. The slopes were significantly different (P<0.005). Thus EL 4 cells were more sensitive to induction of DNA SSB by ionizing radiation than lymphocytes.

  • PDF

Radiation-induced DNA strand breaks in EL4 cells and mouse spleen lymphocytes (방사선에 의한 EL4 마우스 백혈병세포 및 정상 마우스 비장 임파구 DNA strand breaks의 측정)

  • Kim, Sung-ho;Kim, Tae-hwan;Chung, In-yong;Yoo, Seong-yul;Cho, Chul-koo;Chin, Soo-yil
    • Korean Journal of Veterinary Research
    • /
    • v.31 no.3
    • /
    • pp.329-335
    • /
    • 1991
  • The filter elution technique was used to assay $^{60}Co$ $\gamma$ ray-induced DNA strand breaks(SB) in EL4 mouse leukemia cell and mouse spleen lymphocyte. The lymphocytes were stimulated with lipopolysaccharide (LPS, $20{\mu}g/ml$) to label $[^3H]$ thymidine. EL4 cells and lymphocytes in suspension were exposed at $0^{\circ}C$ to 0Gy, 1Gy, 5Gy, 10Gy or l5Gy for DNA single strand breaks(SSB) assay and 0Gy, 25Gy, 50Gy, 75Gy or 100Gy for DNA double strand breaks(DSB) assay of $^{60}Co$ radiation and elution procedure was performed at pH12.1 and 9.6. The number of DNA strand breaks increased with increasing doses of r rays. The strand scission factor(SSF) was estimated in each experiment (eluted volume 21ml). The slope of SSB EL4 cells was $0.01301{\pm}0.00096Gy^{-1}$ (n=5), the slope of SSB for lymphocytes was $0.01097{\pm}0.00091Gy^{-1}$ (n=5) and the slope of DSB for lymphocytes was $0.001707{\pm}0.0000573Gy^{-1}$ (n=5). Thus EL4 cells were more sensitive to induction of DNA SSB by ionizing radiation than lymphocytes (p<0.005). The ratio of slope of dose-response relationship (SSF versus dose) of lymphocytes DNA SSB as compared with the slope of DNA DSB was 6.4.

  • PDF

DNA Single Strand Breaks of Perchloroethylene and Its Bio-degradation Products by Single Cell Gel Electrophoresis Assay in Mammalian Cell System

  • Jeon, Hee-Kyoung;Kim, Young-Seok;Sarma, Sailendra Nlath;Kim, Youn-Jung;Sang, Byoung-In;Ryu, Jae-Chun
    • Molecular & Cellular Toxicology
    • /
    • v.1 no.2
    • /
    • pp.99-105
    • /
    • 2005
  • Perchloroethylene (tetrachloroethylene, PCE), a dry cleaning and degreasing solvent, can enter ground-water through accidental leak or spills. PCE can be degraded to trichloroethylene (TCE), 1, 1-dichloroethylene (DCE) and vinyl chloride (VC) as potential bio-product. These compounds have been reported that they can cause clinical diseases and cytotoxicity. However, only a little genotoxic information of these compounds has been known. In this study, we investigated DNA single strand breaks of PCE, TCE, DCE and VC by single cell gel electrophoresis assay, (comet assay) which is a sensitive, reliable and rapid method for DNA single strand breaks with mouse lymphoma L5178Y cells. From these results, $37.5\;{\mu}g/ml$ of PCE, $189\;{\mu}g/ml$ of TCE and $56.4\;{\mu}g/ml$ of DCE were revealed significant DNA damages in the absence of S-9 metabolic activation system meaning direct-acting mutagen. And in the presence of S-9 metabolic activation system, $41.5\;{\mu}g/ml$ of PCE, $328.7\;{\mu}g/ml$ of TCE and $949\;{\mu}g/ml$ of DCE were induced significant DNA damage. In the case of VC, it was revealed a significant DNA damage in the presence of S-9 metabolic activation system. Therefore, we suggest that chloroethylene compounds (PCE, TCE, DCE and VC) may be induced the DNA damage in a mammalian cell.

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
    • /
    • v.36 no.4
    • /
    • pp.346-351
    • /
    • 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.

Optimal Conditions of Single Cell Gel Electrophoresis (Comet) Assay to detect DNA single strand breaks in Mouse Lymphoma L5178Y cells

  • Ryu, Jae-Chun;Kwon, Oh-Seung;Kim, Hyung-Tae
    • Environmental Mutagens and Carcinogens
    • /
    • v.21 no.2
    • /
    • pp.89-94
    • /
    • 2001
  • Recently, single cell gel electrophoresis, also known as comet assay, is widely used for the detection and measurement of DNA strand breaks in vitro and in vivo in many toxicological fields such as radiation exposure, human monitoring and toxicity evaluation. As well defined, comet assay is a sensitive, rapid and visual method for the detection of DNA strand breaks in individual cells. Briefly, a small number of damaged cells suspended in a thin agarose gel on a microscope slide were lysed, unwinded, electrophoresed, and stained with a fluorescent DNA binding dye. The electric current pulled the charged DNA from the nucleus such that relaxed and broken DNA fragments migrated further. The resulting images which were subsequently named for their appearance as comets, were measured to determine the extent of DNA damages. However, some variations could be occurred in procedures, laboratories's conditions and kind of cells used. Hence, to overcome and to harmonize these matters in comet assay, International Workshop on Genotoxicity Test Procedure (IWGTP) was held with several topics including comet assay at Washington D.C. on March, 1999. In spite of some consensus in procedures and conditions in IWGTP, there are some problems still remained to be solved. In this respect, we attempted to set the practical optimal conditions in the experimental procedures such as lysis, unwinding, electrophoresis and neutralization conditions and so on. First of all, we determined optimal lysis and unwinding time by using 150 $\mu$M methyl methanesulfonate (MMS) which is usually used concentration. And then, we determined optimal positive control concentrations of benzo(a)pyrene (BaP) and MMS in the presence and absence of S9 metabolic activation system, respectively.

  • PDF

Suppressive Effect of Galangin on the Formation of 8-OH2'dG and DNA Single Strand Breaks by Hydrogen Peroxide ($H_2O_2$ 유도 8-OH2'dG 생성 및 DNA Single Strand Break에 미치는 Galangin의 억제효과)

  • Kim, Soo-Hee;Heo, Moon-Young
    • YAKHAK HOEJI
    • /
    • v.54 no.1
    • /
    • pp.32-38
    • /
    • 2010
  • The aim of this study was to evaluate the effect of galangin towards hydrogen peroxide-induced DNA damage. The calf thymus DNA and Chinese Hamster Lung (CHL) cells were used to measure 8-hydroxy-2'-deoxyguanosine(8-OH2'dG) as an indicator of DNA oxidative damage using high performance liquid chromatography with electrochemical detection. Hydrogen peroxide in the presence of Fe(II) ion induced the formation of 8-OH2'dG in both calf thymus DNA and CHL cells. The DNA damage effects were enhanced by increasing the concentration of Fe(II) ion and inhibited by galangin. In the single cell gel electrophoresis (Comet assay), galangin and dl-a-tocopherol showed an inhibitory effect in CHL on hydrogen peroxide induced DNA single strand breaks. Galangin showed more potent activity than dl-$\alpha$-tocopherol under our experimental conditions. These results indicate that galangin can modify the action mechanisms of the oxidative DNA damage and may act as chemopreventive agents against oxidative stress.

Effect of fisetin on UVB-induced apoptosis and DNA single strand breaks in NIH3T3 cells (NIH3T3 세포에서 UVB에 의한 세포고사와 DNA 단사절단에 미치는 fisetin의 효과)

  • Jeong, Se-Jin;Kim, Don-Young;Han, Seol-Hee;Shin, Sang-Min;Cha, Jae-Young;Park, Nou-Bog;Lee, Jung-Sup;Park, Jong-Kun
    • Journal of Life Science
    • /
    • v.17 no.1 s.81
    • /
    • pp.64-69
    • /
    • 2007
  • In the present study, we have investigated the effect of fisetin on the apoptosis and DNA single strand breaks in ultraviolet light B (UVB)-exposed NIH3T3 cells. Exposure of cells to UVB light $(200J/m^2)$ and post-incubation in growth medium for 48 hr resulted in about 50% of cells with apoptotic nuclear fragmentation. Addition of various concentrations of fisetin in the postincubation medium, however, significantly reduced the apoptotic nuclear fragmentation as compared with the values expected when the effects are additive and independent. DNA single strand breaks induced by UVB exposure were also significantly decreased by postincubation with fisetin. By Western blot analysis, fisetin post-incubation was shown to attenuate the p53 upregulation upon UVB exposure. Furthermore, the decrease of proliferating cell nuclear antigen (PCNA) level upon UVB exposure was alleviated by fisetin postincubation. These results suggest that fisetin decrease the apoptosis and increae DNA repair in a possible association with alteration of p53 and PCNA levels in UVB-exposed cells.

Effects of Aronia melanocarpa and Korean Red Ginseng Ethanol Extracts Combination on Cytotoxicity induced by Fludarabine, a DNA Chain Terminating Anti-Cancer Drug (DNA 사슬 종결형 항암제인 플루다라빈에 의해 유도된 세포독성에 대한 아로니아-홍삼 에탄올 혼합 추출물의 효과)

  • Kim, Min Seob;Chung, You Heon;Oh, Hong Geun;Park, Jong Kun
    • The Korean Journal of Food And Nutrition
    • /
    • v.30 no.4
    • /
    • pp.673-680
    • /
    • 2017
  • Fludarabine, a chain terminating anti-cancer drug, is a purine analogue that causes DNA strand breaks in normal cells. In this study, we determined if A. melanocarpa and Korean red ginseng extract mixture reduce cytotoxicity of fludarabine. Treatment of HaCaT cells with $10{\mu}M$ of fludarabine for 24 hours decreased cell viability and increased DNA strand breaks. Treatment of A. melanocarpa and Korean red ginseng extract mixture for 24 hours increased cell viability as compared with single extract treatment. The protective effect of these extracts on cell activity increased in a concentration-dependent manner. DNA strand breaks induced by fludarabine decreased as concentration of extract mixture increased. p-H2AX level, a marker of DNA strand breakage, decreased depending on the concentration of extract mixture. The effect of mixed extract of A. melanocarpa and Korean red ginseng on DNA damage is due to the anti-oxidative effect of A. melanocarpa and signal transmission through glucocorticoid receptor upon binding of saponin of Korean red ginseng.