Analysis of 8-hydroxyguanine in rat kidney genomic DNA after administration of a renal carcinogen, ferric nitrilotriacetate.

Abstract

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.