Zimmer D M, Zhang X B, Harbach P R, Mayo J K, Aaron C S
Pharmacia & Upjohn, Inc., Kalamazoo, Michigan 49007, USA.
Environ Mol Mutagen. 1996;28(4):325-33. doi: 10.1002/(SICI)1098-2280(1996)28:4<325::AID-EM5>3.0.CO;2-A.
Big Blue Rat-2 cells were evaluated for mutagenesis and mutational spectra (spontaneous and ethylnitrosourea [ENU]-induced). Survival, mutant frequency, population doubling time, and kinetics of mutant increase (to 120 hr) were determined. Exposures were 100, 200, 400, 600, and 1,000 micrograms ENU/ml. The spontaneous mutant frequency was similar to that previously reported in vivo, i.e., 5 X 10(5). Dose-related increases in mutant frequency were observed following ENU treatment. Kinetics (time course) of mutant frequency increase, population doubling, and mutational spectra were investigated following treatment at 1,000 micrograms ENU/ml. Among 39 spontaneous mutants, 26 independent mutations were found as follows: nine (34.6%) G:C-->A:T transitions (five at CpG sites), six (23%) G:C-->T:A transversions, three (11.5%) G:C-->C:G transversions (two at CpG sites), two (7.7%) frameshifts, five (19%) deletions or insertions, and one (3.8%) complex (deletion+insertion) mutation. Among 46 ENU-induced mutants, 37 independent mutations (all base substitutions) were found as follows: 15 (40.5%) G:C-->A:T transitions (four at CpG sites), five (13.5%) A:T-->G:C transitions, four (10.8%) G:C-->T:A transversions, 11 (30%) A:T-->T:A transversions, and two (5.4%) A:T-->C:G transversions. Nearly 50% of the base substitutions in the ENU-treated cells were at A:T base pairs, in contrast to the spontaneous mutants where none was found. Both the spontaneous and the ENU-induced mutational spectra were similar to that reported in vivo and for other cells. An important aspect of the experiment is that all mutations sequenced following ENU treatment (1,000 micrograms/ml) occurred under conditions which our experiments show corresponded to very little mitotic activity.
对大蓝鼠 - 2细胞进行了诱变和突变谱分析（自发突变和乙基亚硝基脲[ENU]诱导的突变）。测定了细胞存活率、突变频率、群体倍增时间以及突变增加的动力学（至120小时）。ENU暴露浓度为100、200、400、600和1000微克/毫升。自发突变频率与先前体内报道的相似，即5×10⁻⁵。ENU处理后观察到突变频率呈剂量相关增加。在1000微克/毫升的ENU处理后，研究了突变频率增加、群体倍增和突变谱的动力学（时间进程）。在39个自发突变体中，发现了26个独立突变，如下所示：9个（34.6%）G:C→A:T转换（5个发生在CpG位点），6个（23%）G:C→T:A颠换，3个（11.5%）G:C→C:G颠换（2个发生在CpG位点），2个（7.7%）移码突变，5个（19%）缺失或插入突变，以及1个（3.8%）复合（缺失 + 插入）突变。在46个ENU诱导的突变体中，发现了37个独立突变（均为碱基替换），如下所示：15个（40.5%）G:C→A:T转换（4个发生在CpG位点），5个（13.5%）A:T→G:C转换，4个（10.8%）G:C→T:A颠换，11个（30%）A:T→T:A颠换，以及2个（5.4%）A:T→C:G颠换。与未发现A:T碱基对发生自发突变的情况相反，ENU处理细胞中近50%的碱基替换发生在A:T碱基对处。自发突变谱和ENU诱导的突变谱均与体内及其他细胞报道的相似。该实验的一个重要方面是，在ENU处理（1000微克/毫升）后测序的所有突变均发生在我们的实验显示有极少有丝分裂活性的条件下。
