Potassium diazoacetate-induced p53 mutations in vitro in relation to formation of O6-carboxymethyl- and O6-methyl-2′-deoxyguanosine DNA adducts:: relevance for gastrointestinal cancer

Nitrosated glycine derivatives react with DNA to form O-6-carboxymethyl-2'-deoxyguanosine (O-6-CMdG) and O-6-methyl-2'-deoxyguanosine (O-6-MedG) adducts concurrently. O-6-CMdG is not repaired by O-6-alkylguanine alkyltransferases and might be expected to lead to mutations via a similar mechanism to O-6-MedG. Potassium diazoacetate (KDA) is a stable form of nitrosated glycine and its ability to induce mutations in the p53 gene in a functional yeast assay was studied. Treatment of a plasmid containing the human p53 cDNA sequence with KDA afforded readily detectable levels of O-6-CMdG and O-6-MedG. The treated plasmid was used to transform yeast cells and coloured colonies harbouring a p53 sequence with functional mutations were detected. Recovery of the mutated plasmids followed by DNA sequencing enabled the mutation spectrum of KDA to be characterised. The most common mutations induced by KDA were substitutions with > 50% occurring at GC base pairs. In contrast to the methylating agent methylnitrosourea which gives predominantly (> 80%) GC -> AT transitions, KDA produced almost equal amounts of transitions (GC -> AT) and transversions (GC -> TA and AT -> TA). This difference is probably due to a different mode of base mispairing for O-6-CMdG compared with O-6-MedG. The pattern of mutations induced by KDA was very similar to the patterns observed in mutated p53 in human gastrointestinal tract tumours. These results are consistent with the hypothesis that nitrosation of glycine (or glycine derivatives) may contribute to characteristic human p53 mutation profiles. This conclusion is borne out by recent observations that O-6-CMdG is present in human DNA both from blood and exfoliated colorectal cells and is consistent with recent epidemiological studies that have concluded that endogenous nitrosation arising from red meat consumption is related to an increased risk of colorectal cancer.