Detection of O6-carboxymethyl-2′-deoxyguanosine in DNA following reaction of nitric oxide with glycine and in human blood DNA Using a quantitative immunoslot blot assay

Previous research has shown that a range of nitrosated glycine derivatives react with DNA to form O-6-carboxymethylguanine and O-6-methylguanine DNA adducts [Harrison et al. (1999) Chem. Res. Toxicol. 12, 106-111). Nitrosated glycine derivatives may be formed in the gastrointestinal tract from the reaction of dietary glycine with nitrosating agents. The aim of this study was to further investigate the role of dietary glycine in the formation of O-6-guanine adducts at physiologically relevant concentrations. In vitro studies were performed by reacting 10 muM to 50 mM glycine with nitric oxide in the presence of oxygen. An HPLC assay was developed to measure the resulting nitrosated glycine derivative, diazoacetate anion. The amount of nitrosating agent present in the reaction mixture was determined by colorimetric measurement of nitrite, the hydrolysis product of N2O3. Diazoacetate anion formation depended linearly on glycine concentration. Solutions of nitrosated glycine reacted with 2'-deoxyguanosine and calf thymus DNA to give O-6-carboxymethyl-2'-deoxyguanosine and, at high concentrations of glycine and nitric oxide, O-6-methyl-2'-deoxyguanosine. At physiological concentrations of glycine and nitric oxide, diazoacetate anion was not detectable. Studies with synthetic diazoacetate anion showed that concentrations < 14 muM did not give detectable O-6-carboxyethylguanine in DNA, even when a sensitive immunoslot blot assay was used. However, O-6-carboxymethylguanine was detected in human blood DNA samples obtained from three volunteers consuming a standardized high meat diet, using the immunoslot blot assay. O-6-Carboxymethylguanine levels ranged from 35 to 80 (detection limit = 15) O-6-carboxymethylguanine per 10(8) bases. These studies provide further evidence that nitrosated amino acids may be risk factors for gastrointestinal tract cancers.