Studies for evaluation and protection of DNA damage due to reactive oxygen species

活性氧造成的 DNA 损伤的评估和保护研究

• 批准号: 04670308
• 负责人: TAKEUCHI Toru
• 金额: $ 1.41万
• 依托单位: Osaka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1992
• 资助国家: 日本
• 起止时间: 1992 至 1993
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-04670308/
• 关键词: Reactive oxygen species; 8-hydroxydeoxy-guanosine; DNA damage; Fanconi's anemia; Asbestos; カタラーゼ

Reactive oxygen species (ROS) is one of the most effective species to cause disease. Among ROS induced damages, DNA damage is important in relation to cancer and aging. We established systems to determine typical mutagenic oxidative DNA damages, 8-hydroxydeoxyguanosine (8OHDG) and DNA strand breaks (SB). We could determine 8OHDG in cellular DNA quantitatively by HPLC with electrochemical detection. We could detect SB by pulsed-field gel electrophoresis, however, the method was not suitable for quantitative assay. Lymphoblasts from Fanconi's anemia patients (whose cells are hypersensitive to ROS) induced 8OHDG more than those from controls with H202 at 20 mM.The defference was indicated to be derived from decreased catalase activity in patients' cells. To study the effect of asbestos, we used human promyelocytic leukemia cell line, HL60, which defferentiates to neutrophils and macrophages. Opsonized asbestos induced O2- release from neutrophil-like HL60, however, it did not induce 80HdG-increase in the cellular DNA.On the other hand, asbestos did not increase ROS release from macrophage-like HL60, it was phagocytozed by the cell and increased 80HdG.The 80HdG-increase was not inhibited by SOD or catalase, however, it was inhibited by cytochalasin B, a inhibitor for phagocytosis. For the evaluation of oxidative DNA damage in populations, we determined 8OHdG in white blood cells (WBC) from factory workers. Polymorphonuclear cells which release ROS upon various stimuli, contained similar 8OHdG levels to mononuclear cells. Contaminated red blood cells in the cell preparation increased 8OHdG artificially. We could not find correlations between 8OHdG levels and age or lifestyle factors, probably because the distribution range of 8OHdG levels in WBC was too small against the experimental error of 8OHdG determination.

活性氧（Reactive oxygen species，ROS）是引起疾病的最有效物质之一。在ROS诱导的损伤中，DNA损伤与癌症和衰老有关。建立了8-羟基脱氧鸟苷（8 OHDG）和DNA链断裂（SB）两种典型致突变性DNA氧化损伤的检测体系。采用高效液相色谱电化学检测法可定量测定细胞DNA中的8-羟基脱氧葡萄糖。脉冲场凝胶电泳法可以检测到SB，但不适合定量分析。来自范可尼贫血患者（其细胞对ROS超敏感）的淋巴母细胞在20 mM H2 O2下诱导的8 OHDG比来自对照的淋巴母细胞多。为了研究石棉的作用，我们使用人早幼粒细胞白血病细胞系HL 60，它不同于中性粒细胞和巨噬细胞。石棉可诱导HL 60细胞释放O2-，但不能诱导细胞DNA中80 HdG-的增加;石棉不能诱导HL 60细胞释放ROS，但能被细胞吞噬并增加80 HdG-，而80 HdG-的增加不受SOD或过氧化氢酶的抑制，但能被吞噬抑制剂细胞松弛素B抑制。为了评价人群中氧化性DNA损伤，我们测定了工厂工人白色血细胞（WBC）中的8 OHdG。在各种刺激下释放ROS的多形核细胞含有与单核细胞相似的8 OHdG水平。细胞制剂中污染的红细胞人为增加了8 OHdG。我们没有发现8 OHdG水平与年龄或生活方式因素之间的相关性，可能是因为8 OHdG水平在WBC中的分布范围太小，而8 OHdG测定的实验误差太大。

项目成果

Takeuchi,T.他: "Crocidolite asbestos increased 8-hydroxydeoxyguanosine levels in cellular DNA of a human promyelocytic leukemia cell line,HL60" Carcinogenesis. (in press).

Takeuchi, T. 等人：“青石棉增加了人类早幼粒细胞白血病细胞系 HL60 细胞 DNA 中的 8-羟基脱氧鸟苷水平”致癌作用（正在出版）。

Takeuchi, T.et al.: "Spin trapping of super-oxide released by opsonized asbestos from human promyelocytic leukemia cell line, HL60" Biochem. Biophys. Res. Commun. 194. 57-64 (1993)

Takeuchi, T.等人：“自旋捕获人早幼粒细胞白血病细胞系 HL60 中调理石棉释放的超氧化物”Biochem。

森本 兼曩 他: "ライフスタイルと免疫学的防御機構" Therapeutic Research. 13. 2486-2492 (1992)

Kanehiro Morimoto 等人：“生活方式和免疫防御机制”治疗研究 13. 2486-2492 (1992)。

Takeshita, T.et al.: "Elevated chromosome aberration frequency after X-ray exposure of cutured fibroblasts derived from patients with porokeratosis" Cancer Genet. Cytogenet. in press.

Takeshita, T.et al.：“来自汗孔角化症患者的切割成纤维细胞经 X 射线照射后染色体畸变频率升高”Cancer Genet。

Takeuchi,T.他: "Increased formation of 8-hydroxy deoxy guanosine,an oxidative DNA damage,in lymphoblasts from Fanconi´s anemia patients due to possible catalase deficiency" Carcinogenesis(in press).

Takeuchi, T. 等人：“由于可能存在过氧化氢酶缺陷，范可尼贫血患者的淋巴母细胞中 8-羟基脱氧鸟苷（一种氧化性 DNA 损伤）的形成增加”致癌作用（正在出版）。