Juedes M J, Wogan G N
Massachusetts Institute of Technology, Division of Toxicology, Cambridge 02139, USA.
Mutat Res. 1996 Jan 17;349(1):51-61. doi: 10.1016/0027-5107(95)00152-2.
Peroxynitrite is a powerful oxidant formed through reaction of nitric oxide with superoxide. Because activated macrophages can produce both nitric oxide and superoxide, it has been proposed that peroxynitrite may contribute to cytotoxicity and increased cancer risks associated with the inflammatory response during chronic infections. We therefore investigated mutagenicity of peroxynitrite in the supF gene of the pSP189 shuttle vector as a mutation target. The plasmid was exposed to 2.5 mM peroxynitrite in vitro, then replicated in Eschericia coli MBL50 and in human AD293 cells. Mutation frequency increased 21-fold in pSP189 replicated in E. coli and 9-fold in plasmid replicated in human cells. Mutations were clustered within the 5' region of the supF gene in plasmids replicated in bacteria. The hot spots were located at positions 108, 113, 116, 124, 126 and 141; more than 25% of all mutations occurred at position 124. Following replication in human cells, mutations were more widely distributed over the gene, with hot spots at positions 113, 124, 133, 156 and 164; 15% occurred at position 124. In both systems, the majority of mutations occurred at G:C base pairs, predominantly involving G:C-->T:A transversions (65% when replication was in bacteria and 63% when in human cells). G:C-->C:G transversions were observed at lower frequency (28% in MBL50 and 11% in AD293 cells), and 11% of mutations found in vectors replicated in AD293 cells were G:C-->A:T transitions. A greater number of large deletions, insertions, tandem and multiple mutations occurred in plasmid replicated in AD293 cells. Differences in mutation spectra following replication in the two systems may be attributable to differences in recognition and repair of the lesions and/or properties of the replication apparatus.
过氧亚硝酸盐是一氧化氮与超氧化物反应形成的一种强氧化剂。由于活化的巨噬细胞可产生一氧化氮和超氧化物，因此有人提出过氧亚硝酸盐可能导致细胞毒性，并增加慢性感染期间炎症反应相关的癌症风险。因此，我们研究了以pSP189穿梭载体的supF基因为突变靶点时过氧亚硝酸盐的致突变性。将该质粒在体外暴露于2.5 mM过氧亚硝酸盐，然后在大肠杆菌MBL50和人AD293细胞中复制。在大肠杆菌中复制的pSP189中，突变频率增加了21倍，在人细胞中复制的质粒中增加了9倍。在细菌中复制的质粒中，突变聚集在supF基因的5'区域内。热点位于第108、113、116、124、126和141位；所有突变中超过25%发生在第124位。在人细胞中复制后，突变在基因上分布更广泛，热点位于第113、124、133、156和164位；15%发生在第124位。在这两个系统中，大多数突变发生在G:C碱基对处，主要涉及G:C→T:A颠换（在细菌中复制时为65%，在人细胞中复制时为63%）。G:C→C:G颠换的频率较低（在MBL50中为28%，在AD293细胞中为11%），在AD293细胞中复制的载体中发现的11%的突变是G:C→A:T转换。在AD293细胞中复制的质粒中发生了更多的大缺失、插入、串联和多重突变。在两个系统中复制后突变谱的差异可能归因于损伤识别和修复的差异和/或复制装置的特性。
