Glick James, Xiong Wennan, Lin Yiqing, Noronha Anne M, Wilds Christopher J, Vouros Paul
Barnett Institute and Department of Chemistry, Northeastern University, Boston, MA 02115, USA.
J Mass Spectrom. 2009 Aug;44(8):1241-8. doi: 10.1002/jms.1605.
Benzo[a]pyrene is a major carcinogen implicated in human lung cancer. Almost 60% of human lung cancers have a mutation in the p53 tumor suppressor gene at several specific codons. An on-line nanoLC/MS/MS method using a monolithic nanocolumn was applied to investigate the chemoselectivity of the carcinogenic diol epoxide metabolite, (+/-)-(7R,8S,9S,10R)-benzo[a]pyrene 7,8-diol 9,10-epoxide [(+/-)-anti-benzo[a]pyrene diol epoxide (BPDE)], which was reacted in vitro with a synthesized 14-mer double stranded oligonucleotide (5'-ACCCG5CG7TCCG11CG13C-3'/5'-GCGCGGGCGCGGGT-3') derived from the p53 gene. This sequence contained codons 157 and 158, which are considered mutational 'hot spots' and have also been reported as chemical 'hot spots' for the formation of BPDE-DNA adducts. In evaluating the effect of cytosine methylation on BPDE-DNA adduct binding, it was found that codon 156, containing the nucleobase G5 instead of the mutational hot spot codons 157 (G7) and 158 (G11), was the preferential chemoselective binding site for BPDE. In all permethylated cases studied, the relative ratio for adduction was found to be G5 >> G11 > G13 > G7. Permethylation of CpG dinucleotide sites on either the nontranscribed or complementary strand did not change the order of sequence preference but did enhance the relative adduction level of the G11 CpG site (codon 158) approximately two-fold versus the unmethylated oligomer. Permethylation of all CpG dinucleotide sites on the duplex changed the order of relative adduction to G5 >> G7 > G11 > G13. The three- to four-fold increase in adduction at the mutational hot spot codon 157 (G(7)) relative to the unmethylated or single-stranded permethylated cases suggests a possible relationship between the state of methylation and adduct formation for a particular mutation site in the p53 gene. Using this method, only 125 ng (30 pmol) of adducted oligonucleotide was analyzed with minimal sample cleanup and high chromatographic resolution of positional isomers in a single chromatographic run.
苯并[a]芘是一种与人类肺癌相关的主要致癌物。近60%的人类肺癌在p53肿瘤抑制基因的几个特定密码子处发生突变。采用在线纳米液相色谱/串联质谱法，使用整体式纳米柱研究致癌二醇环氧化物代谢物（±）-（7R，8S，9S，10R）-苯并[a]芘7,8-二醇9,10-环氧化物[（±）-反式苯并[a]芘二醇环氧化物（BPDE）]的化学选择性，该代谢物在体外与源自p53基因的合成14聚体双链寡核苷酸（5'-ACCCG5CG7TCCG11CG13C-3'/5'-GCGCGGGCGCGGGT-3'）反应。该序列包含密码子157和158，它们被认为是突变“热点”，也被报道为形成BPDE-DNA加合物的化学“热点”。在评估胞嘧啶甲基化对BPDE-DNA加合物结合的影响时，发现密码子156含有碱基G5，而不是突变热点密码子157（G7）和158（G11），是BPDE的优先化学选择性结合位点。在所有研究的全甲基化情况下，加合的相对比例为G5 >> G11 > G13 > G7。非转录链或互补链上CpG二核苷酸位点的全甲基化不会改变序列偏好顺序，但相对于未甲基化的寡聚物，确实使G11 CpG位点（密码子158）的相对加合水平提高了约两倍。双链体上所有CpG二核苷酸位点的全甲基化将相对加合顺序改变为G5 >> G7 > G11 > G13。相对于未甲基化或单链全甲基化情况，突变热点密码子157（G(7)）处的加合增加了三到四倍，这表明p53基因中特定突变位点的甲基化状态与加合物形成之间可能存在关系。使用这种方法，仅分析了125 ng（30 pmol）的加合寡核苷酸，样品净化最少，并且在一次色谱运行中对位置异构体具有高色谱分辨率。
