• 한국환경성돌연변이발암원학회지
• /
• 제15권2호
• /
• 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.

• Molecules and Cells
• /
• 제39권3호
• /
• pp.204-210
• /
• 2016

DNA damage responses are important for the maintenance of genome stability and the survival of organisms. Such responses are activated in the presence of DNA damage and lead to cell cycle arrest, apoptosis, and DNA repair. In Caenorhabditis elegans, double-strand breaks induced by DNA damaging agents have been detected indirectly by antibodies against DSB recognizing proteins. In this study we used a comet assay to detect DNA strand breaks and to measure the elimination of DNA strand breaks in mitotic germline nuclei of C. elegans. We found that C. elegans brc-1 mutants were more sensitive to ionizing radiation and camptothecin than the N2 wild-type strain and repaired DNA strand breaks less efficiently than N2. This study is the first demonstration of direct measurement of DNA strand breaks in mitotic germline nuclei of C. elegans. This newly developed assay can be applied to detect DNA strand breaks in different C. elegans mutants that are sensitive to DNA damaging agents.

• Toxicological Research
• /
• 제2권1호
• /
• 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.

• Journal of Nutrition and Health
• /
• 제32권6호
• /
• pp.654-660
• /
• 1999

Folate, a precursor of the coenzyme tetrahydrofolate, plays an important role in DNA replication and cell proliferation, and thus could influence rapidly proliferating immune cells such as leukocytes and splenocytes. The effects of dietary folate on folate concentrations of plasma, thymus, spleen and leukocytes were investigated in rats. The animals were raised for 6 weeks on semipurifed experimental diets containing 0mg, 2mg, 4mg, 8mg folate/kg diet. Folate concentrations were determined microbiologically using Lactobacillus casei(ATCC 7469), and DNA strand breaks produced by alkaline treatment were analyzed fluorometrically. When compared to folate adequate diet, the folate deficient diet(0mg folate/kg diet) resulted in lowest folate levels in plasma, thymus, spleen and leukocytes, and the highest DNA strand breaks in spleen cells and leukocytes. Dietary folate levels significantly increased folate concentrations of immune tissues, leukocytes, and the plasma in a dose dependent manner, folate concentrations being highest with a diet providing 8mg folate/kg diet. The percentages of the double strand DNA remaining in the splenocytes and leukocytes after alkaline treatment were significantly increased with higher amounts of dietary folate in a dose dependent manner. Folate intakes of 8mg than 4mg/kg diet was found to be more effective in the prevention of DNA strand breaks. The results of this study suggest that increased folate above the requirement level could improve DNA stabilities in immune cells.

• Journal of Ginseng Research
• /
• 제16권3호
• /
• pp.210-216
• /
• 1992

The effect of saponin extracted from Panax grneng CA Meyer on DNA repair and replicative DNA synthesis were examined in CHO-Kl cells cotreated with benzo(a)pyrene and rat liver S-15 fraction. The DNA strand breaks inititated by benzo(a)pyrene metabolites were measured by alkaline election technique. The addition of ginseng saponin to the culture media resulted in decrease of benzo(a)pyrene-induced DNA strand breaks, and restored the suppressed-semiconservative-DNA-synthesis by the carcinogen. DNA repair synthesis in the damaged cells was also elevated by the ginseng treatment when the repairing activites were measured for the (3H)-thymidine incorporation into the carcinogen damaged cellular DNk Comparative analysis of DNA-adduces of benzo(a)pyrene metabortes in microsomes suggested that ginseng saponin treatment in rats reduced the formation of electrophilic metabolites of benzo (a)-pyrene in the rat liver microsomes.

• 대한수의학회지
• /
• 제31권3호
• /
• 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.

• Radiation Oncology Journal
• /
• 제11권2호
• /
• pp.219-225
• /
• 1993

각 방사선량의 조사에 따른 사람 정상임파구, 마우스 정상임파구, 그리고 EL-4마우스 백혈병 세포의 DNA double strand breaks 발생율에 대하여 비교분석 하였던 바 다음과 같은 결과를 얻었다. LPS와 PHA를 첨가하여 각각 배양한 마우스 정상임파구의 DNA double strand breaks의발생율을 strand scission factor의 기울기로 비교평가해 본 바 LPS를 첨가배양한 군이 DNA DSB가 더 많이 형성되었다(P<0.005). 이것으로 볼 때 DNA double strand breaks발생에 있어서는 B cells이 T cells보다 더 민감하였다. 또한 EL-4 백혈병 세포는 정상 임파구보다 유의하게 DNA DSB가 더 많이 형성된 것이 관찰되었다(p<0.005). 한편 사람 정상임파구와 마우스 정상임파구 사이의 intrinsic radiosensitivity는 시험관내 실험에서 비교적 유사한 kinetics를 나타냈으나, 마우스 정상임파구가 사람 정상임파구보다 DNA DSB수율이 더 낮았다. 그래서 방사선을 조사하고 3.5시간이 지난후에 $10\%$ DNA DSB발생에 필요한 선량을 두 군간에 비교평가해 본 바 마우스 정상임파구의 선량이 더 높게 나타났다. 이상의 실험결과를 정확하게 설명하기는 어 렵지만 아마도 환경적인 요인과 유전적인 요인 때문인 것으로 사료되었다. 그리고 본 연구결과는 방사선조사에 따른 임상반응의 이해와 cytometric assessment의 방사선 생물학적 parameter로 이용될 수 있을 것으로 판단되었다.

• Radiation Oncology Journal
• /
• 제8권2호
• /
• pp.137-144
• /
• 1990

Filter elution 방법으로 EL 4백서 백혈병 세포 및 C57BL/6백서 유래의 비장 임파구에 대한 Co-60 $\gamma$ 선의 DNA single-strand breaks (SSB) 효과를 정량적으로 측정하였다. 임파구는 [${^3}H$]thymidine을 표지하기 위하여lipopolysaccharide (LPS, 20 $\mug/ml$)를 첨가하여 자극하고 부유 상태의 EL 4 세포 및 임파구를 $0^{\circ}C$에서 0 Gy, 1 Gy, 5 Gy, 10 Gy 또는 15 Gy조사하였으며, elution용액의 pH는 12.1로 하였다. $\gamma$ 선 조사에 따른 single-strand breaks의 수는 방사선 조사량에 따라 증가 하였으며 21 ml elution 양을 기준으로 한 strand scission factor (SSF)는 EL 4 세포에서 $0.01301\pm0.00096\;Gy^{-1}(n=5)$이었고, 임파구는 $0.01097\pm0.00091\;Gy^{-1}(n=5)$를 나타내므로 본 실험에서는 EL 4 세포가 정상 임파구에 비하여 방사선에 의한 DNA SSB가 민감함을 알 수 있었다(p<0.005). 본 연구 결과 DNA strand breaks의 측정법을 이용하여 방사선의 특성 및 생물학적 효과의 파악은 물론 나아가 기존의 방호제 및 새로운 약제의 DNA에 대한 효과를 판별할 수 있을 것이다.

• Radiation Oncology Journal
• /
• 제13권2호
• /
• pp.113-120
• /
• 1995

Purpose : To investigate the effect of alkaloid fraction from Korean ginseng on radiation-induced DNA double strand breaks (dsb) formation and repair in murine lymphocytes Materials and Methods : We used the neutral filter elution technique to assay $^{60}Co\;{\gamma}$ ray-induced DNA double strand breaks formation and repair in C57BL/6 mouse spleen lymphocytes for evaluating the dose-response relationship in the presence of alkaloid fraction as a radioprotective agent. The lymphocytes were stimulated with Phytohemagglutinin (PHA, 2 u g/ml) to label $^3[H]-thymidine.$ Isotope-labelled lymphocytes in suspension were exposed to 100 Gy at $0^{\cdot}C$ in the alkaloid fraction-treated group and elution procedure was performed at PH 9.6. The extents of formation of radiation-induced DNA double strand breaks and repair were compared respectively via strand scission factor (SSF) and relative strand scission factor (RSSF). Results: Alkaloid fraction reduced the formation of double strand breaks with dose modification factor of 2 15, compared to control group Rejoining of DNA dsb appeared to take place via two components. The first fast component was completed within 20.4 minutes, but the second slow component was not completed until 220.2 minutes after irradiation. About $30\%$ of dsb formed by irradiation was ultimately unrejoined despite the administration of alkaloid fraction. The administration of alkaloid fraction had a great effect on the second slow component of repair; the half-time of fast component repair was not changed, but that of slow component was 621.8 minutes. Conclusion: Neutral filter elution assay Proved to be a very effective method to quantitate the extents of DNA dsb formation and its repair. By using this technique, we were able to evaluate the efficiency of alkaloid fraction from Korean ginseng as a valuable radioprotector. Alkaloid fraction can be used prophylactically to prevent or ameliorate the severe radiation damages in workers and neighbors around the atomic power plants. For more refined study, however, more advanced purification of alkaloid fraction wil be needed in the near future.

• Molecular & Cellular Toxicology
• /
• 제1권2호
• /
• 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.