Slamenová D, Gábelová A, Ruzeková L, Chalupa I, Horváthová E, Farkasová T, Bozsakyová E, Stĕtina R
Cancer Research Institute of Slovak Academy of Sciences, Bratislara, Slovak Republic.
Mutat Res. 1997 May 1;383(3):243-52. doi: 10.1016/s0921-8777(97)00007-4.
Human cells (VH10 or Hep G2) and hamster cells V79 were exposed to different concentrations of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and the level of DNA lesions was evaluated by the DNA unwinding technique, alkaline elution of DNA and the comet assay. All three methods were able to detect the effects of MNNG but with a clear difference in sensitivity. At low concentrations of MNNG the most sensitive method appeared to be the comet assay. After the short-term treatment the comet assay was able to detect the lesions induced by MNNG at approx. 0.1 microgram/ml, alkaline elution of DNA at 1 microgram/ml and DNA unwinding at 1-2 micrograms/ml. MNNG treated VH10 cells, human lymphocytes and V79 cells were also tested cytogenetically, confirming that MNNG induced chromosomal aberrations at concentrations > 1 microgram/ml in VH10 cells (short-term treatment): > 0.2 microgram/ml in V79 cells (long-term treatment) and > 8 micrograms/ml in human lymphocytes (long-term treatment). In some experiments we tried to increase the level of MNNG-induced DNA breaks with help of DNA repair inhibitors cytosine arabinoside (Ara C) and hydroxyurea (HU) which were applied either after or during MNNG treatment. Our results showed that the level of MNNG-induced lesions was increased by simultaneous treatment of cells with MNNG and Ara C and HU. 2 x 10(-5) M Ara C and 2 x 10(-3) MHU were as effective as 10-times higher concentrations of inhibitors. Ara C and HU increased the level of MNNG-induced DNA breaks mainly in combination with lower concentrations of MNNG (< 2 micrograms/ml). Rejoining of DNA breaks was observed in human cells VH10 and Hep G2 as well as in Chinese hamster cells V79 damaged by both lower and higher MNNG-concentrations. All methods showed that MNNG-induced DNA breaks had been gradually rejoined.
将人细胞（VH10或Hep G2）和仓鼠细胞V79暴露于不同浓度的N-甲基-N'-硝基-N-亚硝基胍（MNNG），并通过DNA解旋技术、DNA碱性洗脱和彗星试验评估DNA损伤水平。所有这三种方法都能够检测到MNNG的作用，但灵敏度存在明显差异。在低浓度的MNNG下，最敏感的方法似乎是彗星试验。短期处理后，彗星试验能够检测到约0.1微克/毫升MNNG诱导的损伤，DNA碱性洗脱可检测到1微克/毫升的损伤，DNA解旋可检测到1 - 2微克/毫升的损伤。对经MNNG处理的VH10细胞、人淋巴细胞和V79细胞也进行了细胞遗传学检测，证实MNNG在以下浓度时可诱导染色体畸变：VH10细胞中>1微克/毫升（短期处理）；V79细胞中>0.2微克/毫升（长期处理）；人淋巴细胞中>8微克/毫升（长期处理）。在一些实验中，我们试图借助DNA修复抑制剂阿糖胞苷（Ara C）和羟基脲（HU）来提高MNNG诱导的DNA断裂水平，这些抑制剂在MNNG处理之后或处理期间使用。我们的结果表明，细胞同时用MNNG与Ara C和HU处理时，MNNG诱导的损伤水平会增加。2×10⁻⁵M的Ara C和2×10⁻³M的HU与浓度高出10倍的抑制剂效果相同。Ara C和HU主要在与较低浓度的MNNG（<2微克/毫升）联合使用时增加MNNG诱导的DNA断裂水平。在受较低和较高MNNG浓度损伤的人细胞VH10和Hep G2以及中国仓鼠细胞V79中均观察到DNA断裂的重新连接。所有方法均表明MNNG诱导的DNA断裂已逐渐重新连接。
