Bonfanti M, Magagnotti C, Galli A, Bagnati R, Moret M, Gariboldi P, Fanelli R, Airoldi L
Laboratorio di Farmacologia e Tossicologia Ambientali, Istituto di Ricerche Farmacologiche, Mario Negri, Milan, Italy.
Cancer Res. 1990 Nov 1;50(21):6870-5.
A sensitive, specific, and rapid method for quantitating the minor adduct O6-butylguanine (O6BuG) in hydrolyzed DNA has been developed by combining immunoaffinity chromatography and high resolution gas chromatography-negative ion chemical ionization-mass spectrometry. Polyclonal antibodies raised against O6BuG were coupled to CNBr-activated Sepharose 4B and used for sample clean-up and extraction of the specific O6-alkylguanine. After addition of O6BuG and its deuterium labeled analogue (O6BuG-D7), used as internal standard, hydrolyzed DNA was applied on the immunoaffinity column and washed with water, and the immunoadsorbed butylated guanines were eluted with acetone/water cetome/water (95/5) before gas chromatographic derivatization. O6BuG and O6BuG-D7 were analyzed and quantitated by high resolution gas chromatography-negative ion chemical ionization-mass spectrometry as their pentafluorobenzyl-trimethylsilyl derivatives. Immunoaffinity column capacity and O6BuG recovery from this column were 1.53 nmol O6BuG/column and 62 +/- 5%, respectively. The method was applied to evaluate O6BuG levels in DNA butylated in vitro with 10 mM N-nitroso-Nr-butylurea or isolated from rats given an i.p. dose of 185 mg/kg N-nitroso-N-butylurea or N-nitrosodibutylamine. In the first case the level of modifications present in calf thymus DNA was 104 mumol O6BuG/mol guanine, and in the second case O6BuG in liver DNA was about 6 times higher after N-nitroso-N-butylurea (2.11 mumol O6BuG/mol guanine) than after N-nitrosodibutylamine (0.34 mumol O6BuG/mol guanine) treatment. These results indicate that O6BuG formed in vivo can be isolated and quantitated by this method, which may also be useful for studying DNA damage and repair mechanisms.
通过结合免疫亲和色谱法和高分辨率气相色谱 - 负离子化学电离质谱法，已开发出一种灵敏、特异且快速的方法，用于定量水解DNA中的微量加合物O6 - 丁基鸟嘌呤（O6BuG）。针对O6BuG产生的多克隆抗体与溴化氰活化的琼脂糖4B偶联，用于样品净化和特异性O6 - 烷基鸟嘌呤的提取。加入O6BuG及其氘代类似物（O6BuG - D7）作为内标后，将水解的DNA应用于免疫亲和柱，用水洗涤，然后用丙酮/水（95/5）洗脱免疫吸附的丁基化鸟嘌呤，再进行气相色谱衍生化。O6BuG和O6BuG - D7作为其五氟苄基 - 三甲基硅烷基衍生物，通过高分辨率气相色谱 - 负离子化学电离质谱法进行分析和定量。免疫亲和柱容量和从该柱中回收的O6BuG分别为1.53 nmol O6BuG/柱和62±5%。该方法用于评估体外经10 mM N - 亚硝基 - N - 丁基脲丁基化的DNA中O6BuG的水平，或从腹腔注射剂量为185 mg/kg N - 亚硝基 - N - 丁基脲或N - 亚硝基二丁胺的大鼠中分离出的DNA中O6BuG的水平。在第一种情况下，小牛胸腺DNA中存在的修饰水平为104 μmol O6BuG/mol鸟嘌呤，在第二种情况下，肝脏DNA中的O6BuG在N - 亚硝基 - N - 丁基脲处理后（2.11 μmol O6BuG/mol鸟嘌呤）比N - 亚硝基二丁胺处理后（0.34 μmol O6BuG/mol鸟嘌呤）高约6倍。这些结果表明，体内形成的O6BuG可以通过该方法分离和定量，该方法也可能有助于研究DNA损伤和修复机制。
