Maher V M, Yang J L, McCormick J J
Department of Microbiology, Michigan State University, East Lansing 48824-1316.
Acta Biol Hung. 1990;41(1-3):173-86.
An SV40-based shuttle vector, pZ189, carrying a bacterial suppressor tRNA target gene (supF), was treated with radiolabeled polycyclic aromatic carcinogens and the number of covalently bound residues (adducts) per plasmid was determined. The plasmids were transfected into human cell line 293, allowed to replicate, and the progeny plasmids rescued and assayed for the frequency of supF mutants. The agents tested were the 7,8-diol-9,10-epoxide of benzo(a)pyrene (BPDE) and 1-nitrosopyrene (1-NOP). With each agent there was a linear increase in the frequency of supF mutants as a function of the number of DNA adducts formed, reaching frequencies 15 to 25 times higher than the background frequency of 1.4 x 10(-4). When compared on the basis of adducts formed per plasmid BPDE, which forms its principal DNA adduct at the N2 position of guanine, was approximately four times more mutagenic than 1-NOP, which binds principally at the C8 position of guanine. This difference in mutagenic effectiveness may reflect intrinsic differences in the nature of the adducts and their location in the DNA molecule, but it could also reflect a difference in the rate of removal of particular adducts by nucleotide excision repair since the 293 host cell line excised BPDE-induced adducts from genomic DNA at least three times slower than 1-NOP-induced adducts. Agarose gel electrophoresis and DNA sequencing analysis of mutants derived from untreated plasmids showed that the majority (70%) involved deletions, insertions, or altered gel mobility (gross rearrangements). In contrast, the majority of those derived from carcinogen-treated plasmids were base substitutions. DNA sequencing of 86 unequivocally independent mutants derived from BPDE-treated plasmid and 60 from 1-NOP-treated plasmid indicated that 70% to 80% contained a single base substitution, 5%-10% had two base substitutions, and 4%-10% had small insertions or deletions (one or two basepairs). The majority (83%) of the base substitutions in mutants from BPDE- or 1-NOP-treated plasmid were transversions, mainly G.C----T.A. Each carcinogen produced its own spectrum of mutations.
一种基于SV40的穿梭载体pZ189，携带细菌抑制性tRNA靶基因（supF），用放射性标记的多环芳烃致癌物处理，并测定每个质粒上共价结合残基（加合物）的数量。将质粒转染到人细胞系293中，使其复制，然后拯救后代质粒并检测supF突变体的频率。所测试的试剂是苯并(a)芘的7,8-二醇-9,10-环氧化物（BPDE）和1-亚硝基芘（1-NOP）。对于每种试剂，supF突变体的频率随形成的DNA加合物数量呈线性增加，达到比1.4×10(-4)的背景频率高15至25倍的频率。当基于每个质粒形成的加合物进行比较时，在鸟嘌呤的N2位置形成其主要DNA加合物的BPDE的致突变性比主要结合在鸟嘌呤的C8位置的1-NOP高约四倍。这种致突变效力的差异可能反映了加合物性质及其在DNA分子中位置的内在差异，但也可能反映了核苷酸切除修复去除特定加合物速率的差异，因为293宿主细胞系从基因组DNA中切除BPDE诱导的加合物的速度比1-NOP诱导的加合物至少慢三倍。对未处理质粒产生的突变体进行琼脂糖凝胶电泳和DNA测序分析表明，大多数（70%）涉及缺失、插入或凝胶迁移率改变（大规模重排）。相比之下，致癌物处理质粒产生的突变体大多数是碱基替换。对86个明确独立的来自BPDE处理质粒的突变体和60个来自1-NOP处理质粒的突变体进行DNA测序表明，70%至80%含有单个碱基替换，5% - 10%有两个碱基替换，4% - 10%有小的插入或缺失（一或两个碱基对）。来自BPDE或1-NOP处理质粒的突变体中大多数（83%）的碱基替换是颠换，主要是G.C----T.A。每种致癌物都产生其自身的突变谱。
