Lau H H, Baird W M
Department of Medicinal Chemistry and Pharmacognosy, School of Pharmacy and Pharmacal Sciences, Purdue University, West Lafayette, IN 47907.
Carcinogenesis. 1991 May;12(5):885-93. doi: 10.1093/carcin/12.5.885.
Two generally applicable [35S]phosphorothioate postlabeling procedures for the HPLC analysis of polycyclic aromatic hydrocarbon (PAH)-DNA adducts have been developed based upon [32P]phosphate postlabeling assays described by Gupta and Randerath et al. In one procedure, benzo[a]pyrene (B[a]P)-modified DNA was digested to nucleoside 3'-phosphates by micrococcal nuclease and spleen phosphodiesterase and the adducted nucleotides were extracted with 1-butanol. The adducted nucleoside-3'-phosphates were 5'-thiophosphorylated by T4 polynucleotide kinase (T4PNK) and adenosine 5'-O-(3-[35S]thiotriphosphate) to yield [35S]B[a]P-nucleoside-5'-phosphorothioate-3'-phosphate adducts. Although thiophosphorylation of B[a]P-DNA adducts was slower than the corresponding phosphorylation reaction, similar recoveries of the postlabeled adducts were achieved with longer incubation times and higher concentrations of T4PNK. A major advantage of this procedure over the 32P-postlabeling procedure is that the resistance of phosphorothioates to degradation by phosphatases allows selective removal of the unlabeled 3'-phosphate from the [35S]B[a]P-nucleoside-5'-phosphorothioate-3'-phosphate adducts by brief treatment with alkaline phosphatase. [35S]B[a]P-nucleoside-5'-phosphorothioate adducts were also prepared using a nuclease P1/prostatic acid phosphatase DNA degradation method. For anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE)-modified DNA, overall adduct recoveries were substantially higher with the nuclease P1/prostatic acid phosphatase method (48-51%) than with the micrococcal nuclease/spleen phosphodiesterase/alkaline phosphatase method (22-29%). There were no significant differences in the HPLC profiles of the [35S]phosphorothioate-postlabeled adducts obtained from these two procedures. HPLC analysis of B[a]P-DNA adducts formed in B[a]P-treated hamster embryo cell cultures demonstrated the formation of two major adducts, (+)syn-BPDE-deoxyguanosine-5'-phosphorothioate and (+)anti-BPDE-deoxyguanosine-5'-phosphorothioate, along with other minor adducts. Based upon an overall adduct recovery of 20% and 0.5 mol as the detection limit of this 35S-postlabeling/HPLC assay, the sensitivity of this assay is 1 adduct/10(8) nucleotides for a 60 micrograms DNA sample. This method offers the advantages of using 35S which has a longer half-life and lower radioactive decay energy than 32P and the ability to prepare PAH-DNA adducts at the monophosphorothioate level which greatly facilitates separation of individual 35S-postlabeled PAH-DNA adducts by HPLC.
基于古普塔和兰德拉斯等人描述的[32P]磷酸盐后标记分析方法，已开发出两种普遍适用的[35S]硫代磷酸酯后标记程序，用于高效液相色谱（HPLC）分析多环芳烃（PAH）-DNA加合物。在一种程序中，苯并[a]芘（B[a]P）修饰的DNA被微球菌核酸酶和脾磷酸二酯酶消化为核苷3'-磷酸，加合的核苷酸用1-丁醇萃取。加合的核苷-3'-磷酸被T4多核苷酸激酶（T4PNK）和腺苷5'-O-(3-[35S]硫代三磷酸)进行5'-硫代磷酸化，生成[35S]B[a]P-核苷-5'-硫代磷酸酯-3'-磷酸加合物。虽然B[a]P-DNA加合物的硫代磷酸化比相应的磷酸化反应慢，但通过延长孵育时间和提高T4PNK浓度，可实现后标记加合物的类似回收率。该程序相对于32P后标记程序的一个主要优点是，硫代磷酸酯对磷酸酶降解的抗性使得通过用碱性磷酸酶短暂处理，可从[35S]B[a]P-核苷-5'-硫代磷酸酯-3'-磷酸加合物中选择性去除未标记的3'-磷酸。[35S]B[a]P-核苷-5'-硫代磷酸酯加合物也使用核酸酶P1/前列腺酸性磷酸酶DNA降解方法制备。对于反式-7,8-二羟基-9,10-环氧-7,8,9,10-四氢苯并[a]芘（BPDE）修饰的DNA，核酸酶P1/前列腺酸性磷酸酶方法的总加合物回收率（48 - 51%）比微球菌核酸酶/脾磷酸二酯酶/碱性磷酸酶方法（22 - 29%）高得多。从这两种程序获得的松代磷酸酯后标记加合物的HPLC图谱没有显著差异。对B[a]P处理的仓鼠胚胎细胞培养物中形成的B[a]P-DNA加合物进行HPLC分析，结果表明除了其他次要加合物外，还形成了两种主要加合物，即(+)顺式-BPDE-脱氧鸟苷-5'-硫代磷酸酯和(+)反式-BPDE-脱氧鸟苷-5'-硫代磷酸酯。基于20%的总加合物回收率和0.5摩尔作为此35S后标记/HPLC分析的检测限，对于60微克DNA样品，该分析方法的灵敏度为1个加合物/10(8)个核苷酸。此方法具有以下优点：使用半衰期比32P长且放射性衰变能量低的35S，以及能够在单硫代磷酸酯水平制备PAH-DNA加合物，这极大地便于通过HPLC分离各个35S后标记的PAH-DNA加合物。
