Pfau W, Lecoq S, Hughes N C, Seidel A, Platt K L, Grover P L, Phillips D H
Department of Toxicology, University of Hamburg, Germany.
IARC Sci Publ. 1993(124):233-42.
Relatively few reported attempts have been made to substitute HPLC for the thin-layer ion-exchange chromatography (TLC) conventionally used in the 32P-postlabelling assay. Using a reverse-phase phenyl-modified silica gel column and a gradient of methanol in 0.5 M sodium phosphate buffer (pH 2.0), we were able to improve the resolution of very similar adducts. Combined with on-line detection of Cerenkov radiation, this method allows separation of sub-femtomole quantities of 32P-labelled nucleoside 3',5'-bisphosphates modified by bulky carcinogens. Using this method, we were able to separate nine of the ten major adducts formed by reaction of the diol-epoxides of ten polycyclic aromatic hydrocarbons with DNA, and resolve different adducts formed by a single carcinogen. The major adducts formed by benzo[b]fluoranthene (BbF) or dibenz[a,h]anthracene in mouse skin in vivo have been shown to be distinct from the adducts formed directly by the bay-region diol-epoxides. The heterocyclic amines IQ and MeIQ have each been shown to form one major DNA adduct in several in vitro and in vivo systems; using HPLC we were able to resolve the two adducts formed by these food mutagens. HPLC is especially useful for the identification of adducts by means of chromatographic comparisons and in the analysis of the multiple adducts formed by complex mixtures of environmental carcinogens. The major adducts formed by benzo[a]pyrene (BaP) and BbF in mouse skin in vivo that were not resolved on TLC were well separated by HPLC and thus a major DNA adduct formed in the skin of mice treated topically with coal tar was found to be derived from BaP rather than BbF.
相对而言，将高效液相色谱法（HPLC）替代32P后标记分析中传统使用的薄层离子交换色谱法（TLC）的报道尝试较少。使用反相苯基改性硅胶柱和在0.5M磷酸钠缓冲液（pH 2.0）中的甲醇梯度，我们能够提高非常相似加合物的分辨率。结合切伦科夫辐射的在线检测，该方法可分离亚飞摩尔量的由大分子致癌物修饰的32P标记的核苷3',5'-二磷酸。使用该方法，我们能够分离十种多环芳烃的二醇环氧化物与DNA反应形成的十种主要加合物中的九种，并解析由单一致癌物形成的不同加合物。苯并[b]荧蒽（BbF）或二苯并[a,h]蒽在小鼠皮肤中体内形成的主要加合物已被证明与湾区二醇环氧化物直接形成的加合物不同。杂环胺IQ和MeIQ在几个体外和体内系统中均已显示各自形成一种主要的DNA加合物；使用HPLC我们能够解析由这些食物诱变剂形成的两种加合物。HPLC对于通过色谱比较鉴定加合物以及分析环境致癌物复杂混合物形成的多种加合物特别有用。苯并[a]芘（BaP）和BbF在小鼠皮肤中体内形成的在TLC上未分离的主要加合物通过HPLC得到了很好的分离，因此发现在局部用煤焦油处理的小鼠皮肤中形成的一种主要DNA加合物源自BaP而非BbF。
