Yang J L, Yeh S C, Chang C Y
Department of Life Sciences, National Tsing Hua University, Hsinchu, Taiwan, Republic of China.
Mol Carcinog. 1996 Dec;17(4):181-91. doi: 10.1002/1098-2744(199612)17:4<181::aid-mc2940170402>3.0.co;2-f.
The molecular nature of lead-induced mutations was examined in this study to more thoroughly understand lead mutagenesis. Chinese hamster ovary K1 cells were exposed to 0.5-3 mM lead acetate for 24 h. The median lethal dose (LD50) value was 1.5 mM, and the hypoxanthine guanine phosphoribosyltransferase (HPRT) mutant frequency increased linearly as lead concentrations were raised from 0.5 to 1.5 mM. We also amplified the HPRT cDNAs of 56 independent lead-induced mutants by reverse transcriptase-polymerase chain reaction (PCR). Forty-two mutant cDNAs were successfully amplified: 36 mutants had transcripts of normal or slightly smaller than normal size, and six mutants had large deletions. The other 14 mutants whose HPRT cDNA could not be amplified were subjected to genomic-DNA PCR analysis. All of those mutants had one or more exons missing from their genomic HPRT DNA. DNA sequencing of mutant cDNAs showed that 22 had single-base substitutions, four had small alterations, 10 had single-exon deletions, and six were missing two or three exons. Furthermore, DNA sequencing of the HPRT intron-exon boundaries in eight splice mutants revealed that all of them had single-base substitutions in their genomic DNA. G.C base substitutions occurred 3.3-fold more frequently than A.T base substitutions. Similar frequencies were observed for G.C-->A.T, G.C-->T.A, and G.C-->C.G mutations. These results suggest that G.C base pairs may be the primary target sites for lead mutagenesis.
本研究检测了铅诱导突变的分子本质，以更全面地了解铅的诱变作用。将中国仓鼠卵巢K1细胞暴露于0.5 - 3 mM醋酸铅中24小时。半数致死剂量（LD50）值为1.5 mM，随着铅浓度从0.5 mM升高至1.5 mM，次黄嘌呤鸟嘌呤磷酸核糖转移酶（HPRT）突变频率呈线性增加。我们还通过逆转录聚合酶链反应（PCR）扩增了56个独立的铅诱导突变体的HPRT cDNA。成功扩增出42个突变体cDNA：36个突变体的转录本大小正常或略小于正常，6个突变体有大片段缺失。另外14个无法扩增HPRT cDNA的突变体进行了基因组DNA PCR分析。所有这些突变体的基因组HPRT DNA中都有一个或多个外显子缺失。突变体cDNA的DNA测序显示，22个有单碱基替换，4个有小的改变，10个有单外显子缺失，6个缺失两个或三个外显子。此外，对8个剪接突变体的HPRT内含子 - 外显子边界进行DNA测序发现，它们的基因组DNA中均有单碱基替换。G.C碱基替换的发生频率比A.T碱基替换高3.3倍。G.C→A.T、G.C→T.A和G.C→C.G突变的频率相似。这些结果表明，G.C碱基对可能是铅诱变的主要靶位点。
