Glycine nitrosation and signature mutations in the p53 tumour suppressor gene : a molecular link between diet and cancers of the gastro-intestinal tract

Genotoxic agents derived from diet may contribute to the total human burden of potentially carcinogenic DNA damage. For example, glycine is a simple primary amino acid and a commonly found dietary constituent in the red meat. The nitrosation of glycine would probably constitute a major source of alkylating agents, as glycine is one of the most abundant amino acids found in nature. It has been shown that O6- carboxymethyl-2'-deoxyguanosine, the major 06-guanine adduct of nitrosated glycine derivatives, is not repaired by 06-alkylguanine-DNA-alkyltransferase so could accumulate in the DNA of GI tract tissues as a promutagenic lesion. In DNA extracted from human gastric biopsies and white blood cells, using a sensitive Immunoblot assay, 06-CMdG adduct has been detected suggesting that the formation of carboxymethylating agents, possibly via nitrosation of glycine, does occur in man. Therefore genotoxic properties of the 06-carboxymethyl-2'-deoxyguanosine adduct were studied. 06-carboxymethyl-2'-deoxyguanosine adduct has been successfully synthesised and incorporated site specifically in to oligonucleotides. Nucleoside composition analysis of site specifically modified sequences confirmed the presence of O6- carboxymethylguanine adduct. The melting curves studies showed qualitative differences between base pair stabilities formed by 06-CMdG adduct with respect to 06-MedG adduct and normal 2’-dG. 06-CMdG:T base pair was stable than O6- CMdG:C base pair suggesting that 06-CMdG adduct like 06-MedG might preferentially base pair with thymidine. However, quantitative studies on the melting temperatures of 06-CMdG adduct were not fully conclusive. Clearly further effort and experimentation is needed to substantiate the qualitative data. The mutagenic potential of 06-carboxymethyl-2'-guanosine adduct was studied using Amplification Refractory Mutation System (ARMS) PCR technique. A shuttle vector was constructed by incorporating a single 06-CMdG adduct site specifically in to p53 cDNA of plasmid, pLS76. The mutagenesis studies have indicated that O6- carboxymethyl-2'-deoxyguanosine adduct can induce both GC—>AT transition mutations and GC—>TA transversion mutations. Further studies are needed to focus on mutation specificity and frequency of 06-CMdG adduct. The mutagenesis studies on potassium diazoacetate, model compound of nitrosoglycine derivatives did not induce any functional mutations in the p53gene of embryonic fibroblast cells of Human p53 knock in (HUPKI) mouse. However, this is mainly due to problems associated with KDA stability rather than the toxicity of the compound. The results presented in this thesis highlight some key aspects of methodology as well as the significance of mutagenecity of 06-CMdG adduct with respect to diet-related cancer risk.