The detection of mutations induced in vitro in the human p53 gene by hydrogen peroxide with the restriction site mutation (RSM) assay

We have analysed five mutation hotspots within the p53 gene (codons 175, 21 3, 248, 249, and 282) for mutations induced by hydrogen peroxide (H2O2), em ploying the restriction site mutation (RSM) assay. In addition, four other restriction sites covering non-hotspot codons of exons 5-9 of the p53 gene (codons 126, 153/54, 189 and the 3 ' splice site of exon 9) were analysed b y the RSM assay for H2O2-induced mutations. Two cell types were concurrentl y analysed in this study, i.e. primary fibroblast cells and a gastric cance r cell line. Using the RSM assay, H2O2-induced mutations were only detected in exon 7 of the p53 gene. This was true for both cell types. These mutati ons were mainly induced in the Msp I restriction site (codon 247/248) and w ere predominantly GC to AT transitions (71%). Hence these GC to AT mutation s were presumably due to H2O2 exposure, possibly implicating the 50HdC addu ct, which is known to induce C to T mutations upon misreplication. Importan tly, this study demonstrates that the RSM methodology is capable of detecti ng rare oxidative mutations within the hotspot codons of the p53 tumour sup pressor gene. Hence, this methodology may allow the detection of early p53 mutations in pre-malignant tissues. (C) 2001 Elsevier Science B.V. All righ ts reserved.