Detection of concomitant formation of O6-carboxymethyl- and O6-methyl-2′-deoxyguanosine in DNA exposed to nitrosated glycine derivatives using a combined immunoaffinity/HPLC method

A previous observation that an N-nitroso-N-carboxymethyl derivative reacts with DNA to give both O-6-carboxymethyl-2'-deoxyguanosine (O-6-CMdGuo) and O-6-methyl-2'-deoxyguanosine (O-6-MedGuo) [Shuker, D. E. G., and Margison, G. P. (1997) Cancer Res. 57, 366-369] has been confirmed using a range of nitrosated glycine derivatives [N-acetyl-N'-nitroso-N'-prolylglycine (APNG), azaserine (AS), and potassium diazoacetate (KDA)I. In addition, mesyloxyacetic acid (MAA) was also found to give both O-6-adducts in DNA. O-6-CMdGuo and O-6-MedGuo were assessed in enzymatic hydrolysates of treated calf thymus DNA using a combined immunoaffinity/HPLC/fluorescence procedure. The ratio of O-6-CMdGuo to O-6-MedGuo varied somewhat between the different compounds with APNG giving the most methylation (O-6-CM:O-6-Me ratio of 10) and AS the least (39), with KDA and MAA giving intermediate amounts (16 and 18, respectively). The formation of O-6-MedGuo by the four compounds probably arises through decarboxylation at various stages in the decomposition pathways, but the exact mechanisms remain to be clarified. The formation of O-6-MedGuo from reactions of nitrosated glycine derivatives with DNA in vitro may explain the frequent detection of this adduct in human gastrointestinal DNA, as nitrosation of dietary glycine may occur. O-6-CMdGuo is likely to be a useful biomarker of this pathway in vivo and has been detected in human tissues.