Marsch Glenn A, Botta Sisir, Martin Martha V, McCormick W Andrew, Guengerich F Peter
Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
Chem Res Toxicol. 2004 Jan;17(1):45-54. doi: 10.1021/tx034156z.
The dihalomethane CH(2)Cl(2) is an industrial solvent of potential concern to humans because of its potential genotoxicity and carcinogenicity. To characterize DNA damage by dihalomethanes, a rapid DNA digestion under acidic conditions was developed to identify alkali labile DNA-dihalomethane nucleoside adducts using HPLC-electrospray mass spectrometry. DNA digestion worked best using pH 5.0 sodium acetate buffer, a 30 min incubation with DNase II and phosphodiesterase II, and a 2 h acid phosphatase digest. DNA was modified with S-(1-acetoxymethyl)glutathione (GSCH(2)OAc), a reagent modeling activated dihalomethanes. Adducts to G, A, and T were detected at high ratios of GSCH(2)OAc/DNA following digestion of the DNA with the procedure used here. The relative efficacy of adduct formation was G > T > A >> C. The four DNA nucleosides were also reacted with the dihalomethanes CH(2)Cl(2) and CH(2)Br(2) in the presence of glutathione (GSH) and GSH S-transferases from bacteria (DM11), rat (GST 5-5), and human (GST T1-1) under conditions that produce mutations in bacteria. All enzymes formed adducts to all four nucleosides, with dGuo being the most readily modified nucleoside. Thus, the pattern paralleled the results obtained with the model compounds GSCH(2)OAc and DNA. CH(2)Cl(2) and CH(2)Br(2) yielded similar amounts of adducts under these conditions. The relative efficiency of adduct formation by GSH transferases was rat 5-5 > human T1-1 > bacterial DM11, showing that human GSH transferase T1-1 can form dihalomethane adducts under the conditions used. Although the lability of DNA adducts has precluded more sophisticated experiments and in vivo studies have not yet been possible, the work collectively demonstrates the ability of several GSH transferases to generate DNA adducts from dihalomethanes, with G being the preferred site of adduction in both this and the GSCH(2)OAc model system.
二氯甲烷CH₂Cl₂是一种对人类具有潜在危害的工业溶剂，因其具有潜在的遗传毒性和致癌性。为了表征二卤甲烷对DNA的损伤，我们开发了一种在酸性条件下快速消化DNA的方法，以使用高效液相色谱 - 电喷雾质谱法鉴定对碱不稳定的DNA - 二卤甲烷核苷加合物。使用pH 5.0的醋酸钠缓冲液、与DNase II和磷酸二酯酶II孵育30分钟以及进行2小时的酸性磷酸酶消化时，DNA消化效果最佳。DNA用S-(1 - 乙酰氧基甲基)谷胱甘肽(GSCH₂OAc)进行修饰，GSCH₂OAc是一种模拟活化二卤甲烷的试剂。按照此处使用的方法消化DNA后，在高比例的GSCH₂OAc/DNA条件下检测到了与鸟嘌呤(G)、腺嘌呤(A)和胸腺嘧啶(T)的加合物。加合物形成的相对效率为G > T > A >> C。在细菌、大鼠和人类的谷胱甘肽(GSH)及GSH S - 转移酶存在的情况下，四种DNA核苷也与二卤甲烷CH₂Cl₂和CH₂Br₂发生反应，反应条件为能在细菌中产生突变的条件。所有酶都与所有四种核苷形成了加合物，其中脱氧鸟苷(dGuo)是最容易被修饰的核苷。因此，这种模式与使用模型化合物GSCH₂OAc和DNA获得的结果相似。在这些条件下，CH₂Cl₂和CH₂Br₂产生的加合物量相似。GSH转移酶形成加合物的相对效率为大鼠5 - 5 > 人类T1 - 1 > 细菌DM11，这表明人类GSH转移酶T1 - 1在所用条件下能够形成二卤甲烷加合物。尽管DNA加合物的不稳定性使得更复杂的实验受到限制，并且体内研究尚未可行，但这项工作共同证明了几种GSH转移酶能够从二卤甲烷生成DNA加合物，在本研究以及GSCH₂OAc模型系统中，鸟嘌呤(G)都是加合的首选位点。
