Clive D, Glover P, Applegate M, Hozier J
Genetic Toxicology Laboratory, Burroughs Wellcome Co., Research Triangle Park, NC 27709.
Mutagenesis. 1990 Mar;5(2):191-7. doi: 10.1093/mutage/5.2.191.
Southern blot analyses were performed on DNA from at least 10 large and 10 small colony thymidine kinase-deficient (tk -/-) mutants induced by each of 10 mutagens [2-amino-N6-hydroxyadenine (AHA), ethyl methanesulfonate (EMS), methyl methanesulfonate, 2-acetylaminofluorene, methotrexate, caffeine, methapyrilene, 4-(9-acridinylamino)-methanesulfo-m-anisidide, hycanthone methanesulfonate and procarbazine]. Two molecular mutant genotypes were recognized upon digestion with NcoI and subsequent probing with a 1.1 kb cDNA insert from plasmid pMtk 4: (i) no detectable alteration, and (ii) the absence of the functional tkb allele as indicated by the absence of the 6.3 kb fragment. In combination with the previously established chromosomal nature of most small colony tk -/- mutants, this permitted the classification of these 10 mutagens according to the relative proportions of each of four classes of genetic damage they induced. AHA and EMS gave mutational spectra consistent with their point mutational effects in other systems. The other eight mutagens induced mostly small colony mutants, most of which had lost the entire original tkb allele. Methotrexate induced high frequencies of large colony mutants at the tk locus, most of which lacked the tkb allele, although it is weakly or non-mutagenic at the hemizygous hprt locus in these same cells. At least three of these mutagens-methotrexate, caffeine, methapyrilene (and possibly procarbazine)--lack structural alerts for DNA reactivity, implying a major class of non-DNA primary targets for mutagenicity in mammalian cells that interact secondarily with the chromosome. These results are discussed in relation to the known differences in sensitivity among various short-term tests for genotoxicity.
对由10种诱变剂[2-氨基-N6-羟基腺嘌呤（AHA）、甲磺酸乙酯（EMS）、甲磺酸甲酯、2-乙酰氨基芴、甲氨蝶呤、咖啡因、甲氧苄氨嘧啶、4-（9-吖啶基氨基）-甲磺基间茴香胺、甲磺基海恩酮和丙卡巴肼]分别诱导产生的至少10个大菌落和10个小菌落胸苷激酶缺陷型（tk-/-）突变体的DNA进行了Southern印迹分析。用NcoI消化并随后用质粒pMtk 4的1.1 kb cDNA插入片段进行探针杂交后，识别出两种分子突变基因型：（i）未检测到改变，（ii）如6.3 kb片段缺失所示，功能性tkb等位基因缺失。结合先前确定的大多数小菌落tk-/-突变体的染色体性质，这使得可以根据这10种诱变剂诱导的四类遗传损伤中每一类的相对比例对它们进行分类。AHA和EMS产生的突变谱与其在其他系统中的点突变效应一致。其他八种诱变剂主要诱导小菌落突变体，其中大多数失去了整个原始的tkb等位基因。甲氨蝶呤在tk位点诱导出高频率的大菌落突变体，其中大多数缺乏tkb等位基因，尽管它在这些相同细胞的半合子hprt位点是弱诱变剂或非诱变剂。这些诱变剂中至少有三种——甲氨蝶呤、咖啡因、甲氧苄氨嘧啶（可能还有丙卡巴肼）——缺乏对DNA反应性的结构警示，这意味着在哺乳动物细胞中存在一类主要的非DNA初级诱变靶点，它们与染色体发生二次相互作用。结合已知的各种遗传毒性短期试验之间的敏感性差异对这些结果进行了讨论。
