Cupid Belinda C, Zeng Zuotao, Singh Rajinder, Shuker David E G
MRC Toxicology Unit, Hodgkin Building, University of Leicester, P.O. Box 138, Lancaster Road, Leicester LE1 9HN, United Kingdom.
Chem Res Toxicol. 2004 Mar;17(3):294-300. doi: 10.1021/tx0340706.
Previous research has shown that a range of nitrosated glycine derivatives react with DNA to form O6-carboxymethylguanine and O6-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 O6-guanine adducts at physiologically relevant concentrations. In vitro studies were performed by reacting 10 microM 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 O6-carboxymethyl-2'-deoxyguanosine and, at high concentrations of glycine and nitric oxide, O6-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 microM did not give detectable O6-carboxyethylguanine in DNA, even when a sensitive immunoslot blot assay was used. However, O6-carboxymethylguanine was detected in human blood DNA samples obtained from three volunteers consuming a standardized high meat diet, using the immunoslot blot assay. O6-Carboxymethylguanine levels ranged from 35 to 80 (detection limit = 15) O6-carboxymethylguanine per 10(8) bases. These studies provide further evidence that nitrosated amino acids may be risk factors for gastrointestinal tract cancers.
先前的研究表明，一系列亚硝化甘氨酸衍生物可与DNA反应，形成O6 - 羧甲基鸟嘌呤和O6 - 甲基鸟嘌呤DNA加合物[哈里森等人（1999年），《化学研究毒理学》12卷，第106 - 111页]。亚硝化甘氨酸衍生物可能在胃肠道中由膳食甘氨酸与亚硝化剂反应形成。本研究的目的是进一步研究膳食甘氨酸在生理相关浓度下对O6 - 鸟嘌呤加合物形成的作用。通过在有氧条件下使10微摩尔至50毫摩尔的甘氨酸与一氧化氮反应进行体外研究。开发了一种HPLC测定法来测量生成的亚硝化甘氨酸衍生物重氮乙酸根阴离子。反应混合物中存在的亚硝化剂的量通过比色法测量亚硝酸（N2O3的水解产物）来确定。重氮乙酸根阴离子的形成与甘氨酸浓度呈线性关系。亚硝化甘氨酸溶液与2'-脱氧鸟苷和小牛胸腺DNA反应，生成O6 - 羧甲基 - 2'-脱氧鸟苷，并且在高浓度的甘氨酸和一氧化氮条件下，生成O6 - 甲基 - 2'-脱氧鸟苷。在甘氨酸和一氧化氮的生理浓度下，未检测到重氮乙酸根阴离子。用合成重氮乙酸根阴离子进行的研究表明，即使使用灵敏的免疫斑点印迹测定法，浓度<14微摩尔时也不会在DNA中产生可检测到的O6 - 羧乙基鸟嘌呤。然而，使用免疫斑点印迹测定法，在三名食用标准化高肉饮食的志愿者的人血DNA样本中检测到了O6 - 羧甲基鸟嘌呤。O6 - 羧甲基鸟嘌呤水平范围为每10^8个碱基35至80（检测限 = 15）个O6 - 羧甲基鸟嘌呤。这些研究提供了进一步的证据，表明亚硝化氨基酸可能是胃肠道癌症的风险因素。
