Takeuchi T, Matsugo S, Morimoto K
Department of Hygiene and Preventive Medicine, Osaka University School of Medicine, Suita, Japan.
Carcinogenesis. 1997 Nov;18(11):2051-5. doi: 10.1093/carcin/18.11.2051.
We have investigated the mutagenicity of oxidative DNA damage induced in V79 Chinese hamster lung fibroblast, and measured 8-hydroxydeoxyguanosine (8OHdG) levels as an indicator of this damage. A hydroxyl radical generator, N,N'-bis(2-hydroxyperoxy-2-methoxyethyl)-1,4,5,8-naphthalene-tetra -carboxylic-diimide (NP-III), induced 8OHdG in V79 upon irradiation with 366 nm ultraviolet light (UV) for 15 min. 8OHdG was determined by HPLC with electrochemical detection after anaerobic sample processing. The 8OHdG level in the cells treated without NP-III was 0.49 per 10(5) dG, whereas levels in the cells treated with 5, 10 or 20 microM NP-III and UV irradiation were 1.84, 4.06 or 6.95 per 10(5) dG, respectively. The 8OHdG induced by 20 microM NP-III with UV irradiation decreased rapidly, and the half-life of the induced 8OHdG was approximately 6 h. NP-III with UV irradiation also induced DNA strand breaks in all cells uniformly, as determined by single cell gel assay. Mutant frequencies at the hypoxanthine-guanine phosphoribosyltransferase (hprt) locus in V79 were determined as the number of 6-thioguanine-resistant cells per 10(6) cells. Mutant frequency of the cells without NP-III was 8.0, and frequencies of the cells treated with 5, 10 or 20 microM NP-III and UV irradiation were 14.9, 20.6 or 24.7 respectively. Treatment with 20 microM NP-III and UV irradiation decreased the cell number, determined 3 days after the treatment, to 20.8%. These findings indicate that acutely induced oxidative DNA damage including mutagenic 8OHdG is only weakly mutagenic in V79.
我们研究了V79中国仓鼠肺成纤维细胞中诱导产生的氧化性DNA损伤的致突变性，并测量了8-羟基脱氧鸟苷（8OHdG）水平作为这种损伤的指标。一种羟基自由基生成剂，N,N'-双(2-羟基过氧-2-甲氧基乙基)-1,4,5,8-萘四羧酸二酰亚胺（NP-III），在366nm紫外光（UV）照射15分钟后在V79细胞中诱导产生8OHdG。厌氧样品处理后，通过高效液相色谱-电化学检测法测定8OHdG。未用NP-III处理的细胞中8OHdG水平为每10(5)个dG中有0.49个，而用5、10或20μM NP-III处理并经UV照射的细胞中8OHdG水平分别为每10(5)个dG中有1.84、4.06或6.95个。20μM NP-III与UV照射诱导产生的8OHdG迅速减少，诱导产生的8OHdG的半衰期约为6小时。单细胞凝胶试验表明，NP-III与UV照射在所有细胞中均一致地诱导DNA链断裂。V79细胞次黄嘌呤-鸟嘌呤磷酸核糖转移酶（hprt）位点的突变频率以每10(6)个细胞中6-硫代鸟嘌呤抗性细胞的数量来确定。未用NP-III处理的细胞的突变频率为8.0，用5、10或20μM NP-III处理并经UV照射的细胞的突变频率分别为14.9、20.6或24.7。用20μM NP-III处理并经UV照射后，处理3天后测定细胞数量减少至20.8%。这些发现表明，包括致突变性8OHdG在内的急性诱导的氧化性DNA损伤在V79细胞中仅具有弱致突变性。
