Carcinogenesis, Vol. 20, No. 5, 837-841,
May 1999
© 1999 Oxford University Press
Analysis of 8-hydroxyguanine in rat kidney genomic DNA after administration of a renal carcinogen, ferric nitrilotriacetate
Department of Molecular Biology and
1 Department of Biology, School of Medicine and
2 Department of Environmental Oncology, Institute of Industrial Ecological Sciences, University of Occupational and Environmental Health Japan, Kitakyushu 807, Japan
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 
1 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 1 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.
Abbreviations: DMS, dimethylsulfate; Fe-NTA, ferric nitriloacetate; Fpg, formamidopyrimidine-DNA glycosylase; LM-PCR, ligation-mediated polymerase chain reaction; 8-OH-Gua, 8-hydroxyguanine; ROS, reactive oxygen species.
3 To whom correspondence should be addressed Email: m-nomoto{at}mail.med.uoeh-u.ac.jp
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