Nomoto M, Yamaguchi R, Kawamura M, Kohno K, Kasai H
Department of Molecular Biology, School of Medicine, University of Occupational and Environmental Health Japan, Kitakyushu.
Carcinogenesis. 1999 May;20(5):837-41. doi: 10.1093/carcin/20.5.837.
The frequency of oxidative base damage, such as 8-hydroxyguanine (8-OH-Gua), was determined at the nucleotide level of resolution using the ligation-mediated PCR technique. Administration of a renal carcinogen, ferric nitrilotriacetate (Fe-NTA), is known to induce oxidative stress and subsequent formation of 8-OH-Gua in the rat kidney. Whole genomic DNA was isolated from the rat kidney after or without Fe-NTA treatment and then cleaved with hot piperidine. In order to assess the frequency of 8-OH-Gua formation, we chose three genes, the tumor suppressor gene p53, the heat shock protein 70 (HSP70-1) gene and the Na,K-ATPase alpha1 subunit gene. No alteration in the cleavage profile was observed in the p53 and HSP70 genes after Fe-NTA treatment. In the case of the p53 gene, a low incidence of point mutations has been observed in this carcinogenesis system. On the other hand, time-dependent alterations, corresponding to the time course of overall 8-OH-Gua formation and repair, were detected in the promoter region of the Na,K-ATPase alpha1 subunit gene. GpG and GpGpG in specific regions seem to be hotspots for the formation of 8-OH-Gua. These results were confirmed by formamidopyrimidine-DNA glycosylase-dependent DNA cleavage patterns. Thus, oxidative base damage, such as 8-OH-Gua, was not distributed uniformly along the whole genome, but seemed to be restricted to particular genes and regions.
使用连接介导的聚合酶链反应（PCR）技术，在核苷酸分辨率水平上测定了氧化碱基损伤的频率，如8-羟基鸟嘌呤（8-OH-Gua）。已知给予肾致癌物次氮基三乙酸铁（Fe-NTA）会诱导大鼠肾脏产生氧化应激并随后形成8-OH-Gua。在有或无Fe-NTA处理的情况下，从大鼠肾脏中分离出全基因组DNA，然后用热哌啶进行切割。为了评估8-OH-Gua的形成频率，我们选择了三个基因，即肿瘤抑制基因p53、热休克蛋白70（HSP70-1）基因和钠钾ATP酶α1亚基基因。Fe-NTA处理后，在p53和HSP70基因中未观察到切割图谱的改变。在这个致癌系统中，p53基因的点突变发生率较低。另一方面，在钠钾ATP酶α1亚基基因的启动子区域检测到与整体8-OH-Gua形成和修复的时间进程相对应的时间依赖性改变。特定区域的GpG和GpGpG似乎是8-OH-Gua形成的热点。这些结果通过甲酰胺嘧啶-DNA糖基化酶依赖性DNA切割模式得到证实。因此，氧化碱基损伤，如8-OH-Gua，并非均匀分布于整个基因组，而是似乎局限于特定的基因和区域。
