Moraes E C, Keyse S M, Pidoux M, Tyrrell R M
ISREC, Epalinges, Switzerland.
Nucleic Acids Res. 1989 Oct 25;17(20):8301-12. doi: 10.1093/nar/17.20.8301.
Treatment of a plasmid shuttle vector (pZ189) with a combination of hydrogen peroxide and a ferric iron/EDTA complex prior to transfection and passage in simian (CV-1) cells increases the frequency of mutations at the supF locus by up to 60-fold over the spontaneous background. This increase in mutation frequency is abolished when the inhibitors desferrioxamine, superoxide dismutase, catalase or dimethyl sulfoxide are included in the initial reaction or when the iron/EDTA complex is omitted, a strong indication that the premutagenic damage arises as a result of direct attack by hydroxyl radical generated in a superoxide driven Fenton reaction. DNA sequence analysis of the mutated plasmids shows that 1) Deletions occuring in combination with base-substitutions arise in 22.5 percent of the induced mutants compared with only 3 percent of spontaneous mutants 2) Sixty percent of all induced deletion mutations involve the loss of a single base and 77 percent of these (20 out of 26) occur at two adenine-containing sites 3) The base-change spectrum of mutants arising in the treated plasmid population is marked by the predominance of mutants containing a single base-change and by an increase in changes at AT base pairs. These results provide direct information concerning the nature of mutations arising in mammalian cells as a result of hydroxyl radical mediated DNA damage.
在转染和在猴（CV-1）细胞中传代之前，用过氧化氢和铁离子/乙二胺四乙酸（EDTA）复合物处理质粒穿梭载体（pZ189），与自发背景相比，可使supF位点的突变频率增加高达60倍。当在初始反应中加入去铁胺、超氧化物歧化酶、过氧化氢酶或二甲基亚砜等抑制剂，或省略铁/EDTA复合物时，突变频率的这种增加就会消失，这有力地表明，诱变前损伤是由超氧化物驱动的芬顿反应中产生的羟基自由基直接攻击所致。对突变质粒的DNA序列分析表明：1）与仅3%的自发突变体相比，22.5%的诱导突变体出现了与碱基替换相结合的缺失；2）所有诱导缺失突变的60%涉及单个碱基的丢失，其中77%（26个中的20个）发生在两个含腺嘌呤的位点；3）处理过的质粒群体中产生的突变体的碱基变化谱的特点是，含有单个碱基变化的突变体占主导，且AT碱基对处的变化增加。这些结果提供了有关哺乳动物细胞中因羟基自由基介导的DNA损伤而产生的突变性质的直接信息。
