Measurement of oxidative damage at individual gene levels by quantitative PCR using 8-hydroxyguanine glycosylase (OGG1)

In this study, an attempt was made to develop a method to estimate oxidative damage of individual genes for assessing chemopreventive potential of dietary or medicinal plants. Oxidative damage was investigated on the two genes in gastric mucosal tissue infected with Helicobacter pylori, which were genes of glyceralclehydes-3-phosphate dehydrogenase (GAPDH), a house-keeping gene, and gene of insulin-like growth factor II receptor (IGFIIR), a gene known to be mutated frequently in gastric carcinoma. The oxidative damage in genomic DNA in the above tissue was confirmed by immunohistochemical study using monoclonal antibody to 8-hydroxyguanine (oh(8)G), which showed much higher degree of staining in their nuclei. Using the method we developed, it was demonstrated that the number of oh(8)G (indicated by 8-hydroxyguanine glycosylase (OGG1) sensitive sites) in GAPDH was almost not changed in H. pylori-infected tissue but in IGFIIR, it increased significantly. These results indicate that this method is valid for the estimate of oxidative damage of individual genes and also showed that the susceptibility of genomic DNA to attack of reactive oxygen species is not homogeneous but different depending upon the region of DNA. We expect to use this method in studies of carcinogenic mechanism and chemoprevention since it can provide more specific information pertaining to individual genes we are interested in. (C) 2003 Elsevier Science B.V. All rights reserved.