Misra R R, Chiang S Y, Swenberg J A
Department of Environmental Science and Engineering, University of North Carolina at Chapel Hill 27599.
Carcinogenesis. 1994 Aug;15(8):1647-52. doi: 10.1093/carcin/15.8.1647.
1,N6-Ethenodeoxyadenosine (edA) and 3,N4-ethenodeoxycytidine (edC) are two mutagenic adducts associated with exposure to ethyl carbamate (urethane) and vinyl chloride. We have recently developed two ultrasensitive methods for determining the molecular dose of these adducts in cellular DNA. In both methods, purified DNA was first enzymatically digested to 2c-deoxyribonucleotide 3c-monophosphates. Etheno-modified nucleotides were then separated from normal nucleotides in one of two ways: either by reverse phase, ion-pair HPLC coupled with 260 nm UV detection, or by immunoaffinity chromatography using reusable microcolumns containing specific monoclonal antibodies coupled to Protein A-Sepharose. Fractions enriched for the adducted nucleotides were labeled using T4 poly-nucleotide kinase and [32P]ATP, and individual nucleotides were subsequently resolved by two-dimensional TLC, visualized by autoradiography, and quantified by liquid scintillation counting. When used to analyze the same sample of etheno-modified calf thymus DNA, both assays produced similar results. However, when both methods were used to analyze rat liver DNA 'spiked' with known amounts of etheno nucleotide standards, the immuno-affinity/32P TLC procedure proved to be more sensitive and more reproducible than the HPLC/32P TLC method: while the detection limit of the immunoaffinity/32P TLC technique was < 4 etheno adducts/10(9) parent deoxynucleotides, the HPLC/32P TLC method often failed to detect adducts at concentrations < 2/10(8). In other experiments, the immunoaffinity/32P TLC method was used to demonstrate formation of edA and edC in cells treated with vinyl chloride monomer. Because of its exquisite sensitivity, the immunoaffinity/32P TLC method promises to be extremely useful for measuring both background and induced levels of etheno adducts, making it possible to examine the role of these adducts in inducing mutations and/or carcinogenesis.
1,N6-乙烯基脱氧腺苷（edA）和3,N4-乙烯基脱氧胞苷（edC）是两种与接触氨基甲酸乙酯（尿烷）和氯乙烯相关的诱变加合物。我们最近开发了两种超灵敏方法来测定细胞DNA中这些加合物的分子剂量。在这两种方法中，首先将纯化的DNA酶解为2'-脱氧核糖核苷酸3'-单磷酸。然后通过以下两种方式之一将乙烯基修饰的核苷酸与正常核苷酸分离：要么通过反相离子对高效液相色谱（HPLC）结合260 nm紫外检测，要么通过免疫亲和色谱，使用含有与蛋白A-琼脂糖偶联的特异性单克隆抗体的可重复使用微柱。富含加合核苷酸的馏分用T4多核苷酸激酶和[32P]ATP进行标记，随后通过二维薄层层析（TLC）分离单个核苷酸，通过放射自显影进行可视化，并通过液体闪烁计数进行定量。当用于分析相同的乙烯基修饰小牛胸腺DNA样品时，两种测定方法产生了相似的结果。然而，当两种方法都用于分析添加了已知量乙烯基核苷酸标准品的大鼠肝脏DNA时，免疫亲和/32P TLC方法被证明比HPLC/32P TLC方法更灵敏、更可重复：免疫亲和/32P TLC技术的检测限<4个乙烯基加合物/10(9)个亲本脱氧核苷酸，而HPLC/32P TLC方法在浓度<2/10(8)时常常无法检测到加合物。在其他实验中，免疫亲和/32P TLC方法被用于证明在用氯乙烯单体处理的细胞中edA和edC的形成。由于其极高的灵敏度，免疫亲和/32P TLC方法有望在测量乙烯基加合物的背景水平和诱导水平方面极其有用，从而有可能研究这些加合物在诱导突变和/或致癌作用中的作用。
