Kohara Arihiro, Suzuki Takayoshi, Honma Masamitsu, Oomori Takashi, Ohwada Tomohiko, Hayashi Makoto
Division of Genetics and Mutagenesis, National Institute of Health Sciences, 1-18-1, Kamiyoga, Setagaya-ku, 158-8501, Tokyo, Japan.
Mutat Res. 2002 Mar 25;515(1-2):73-83. doi: 10.1016/s1383-5718(02)00007-4.
Dinitropyrenes (DNPs), 1,3-, 1,6- and 1,8-dinitropyrene, are carcinogenic compounds found in diesel engine exhaust. DNPs are strongly mutagenic in the bacterial mutation assay (Ames test), mainly inducing frameshift type mutations. To assess mutagenicity of DNPs in vivo is important in evaluating their possible involvement in diesel exhaust-induced carcinogenesis in human. For this purpose, we used the lambda/lacZ transgenic mouse (Muta Mouse) to examine induction of mutations in multiple organs. A commercially available mixture of DNPs (1,3-, 1,6-, 1,8-, and unidentified isomer (s) with a content of 20.2, 30.4, 35.2, and 14.2%, respectively) was injected intragastrically at 200 and 400mg/kg once each week for 4 weeks. Seven days after the final treatment, liver, lung, colon, stomach, and bone marrow were collected for mutation analysis. The target transgene was recovered by the lambda packaging method and mutation of lacZ gene was analyzed by a positive selection with galE(-) E. coli. In order to determine the sequence alterations by DNPs, the mutagenicity of the lambda cII gene was also examined by the positive selection with hfl(-) E. coli. Since cII gene (294bp) is much smaller than the lacZ (3024bp), it facilitated the sequence analysis. Strongest increases in mutant frequencies (MFs) were observed in colon for both lacZ (7.5x10(-5) to 43.3x10(-5)) and cII (2.7x10(-5) to 22.5x10(-5)) gene. Three-four-fold increases were observed in stomach for both genes. A statistically significant increase in MFs was also evident in liver and lung for the lacZ gene, and in lung and bone marrow for the cII gene. The sequence alterations of the cII gene recovered from 37 mutants in the colon were compared with 50 mutants from untreated mice. Base substitution mutations predominated for both untreated (91%) and DNP-treated (84%) groups. The DNPs treatment increased the incidence of G:C to T:A transversion (2-43%) and decreased G:C to A:T transitions (70-22%). The G:C to T:A transversions, characteristic to DNPs treatment, is probably caused by the guanine-C8 adduct, which is known as a major DNA-adduct induced by DNPs, through an incorporation of adenine opposite the adduct ("A"-rule). The present study showed a relevant use of the cII gene as an additional target for mutagenesis in the Muta Mouse and revealed a mutagenic specificity of DNPs in vivo.
二硝基芘（DNPs），即1,3 -、1,6 - 和1,8 - 二硝基芘，是在柴油机尾气中发现的致癌化合物。DNPs在细菌突变试验（艾姆斯试验）中具有很强的致突变性，主要诱导移码型突变。评估DNPs在体内的致突变性对于评估它们可能参与人类柴油机尾气诱导的致癌作用很重要。为此，我们使用λ/lacZ转基因小鼠（突变小鼠）来检测多个器官中的突变诱导情况。一种市售的DNPs混合物（1,3 -、1,6 -、1,8 - 以及含量分别为2 . 2%、30.4%、35.2%和14.2%的未鉴定异构体）以200和400mg/kg的剂量每周一次经胃内注射，共注射4周。在最后一次治疗后7天，收集肝脏、肺、结肠、胃和骨髓进行突变分析。通过λ包装方法回收目标转基因，并通过用galE(-)大肠杆菌进行阳性选择来分析lacZ基因的突变。为了确定DNPs引起的序列改变，还通过用hfl(-)大肠杆菌进行阳性选择来检测λ cII基因的致突变性。由于cII基因（294bp）比lacZ基因（3024bp）小得多，这便于进行序列分析。在结肠中观察到lacZ基因（从7.5×10(-5)到43.3×10(-5)）和cII基因（从2.7×10(-5)到22.5×10(-5)）突变频率（MFs）增加最为显著。在胃中两个基因的突变频率都增加了三到四倍。对于lacZ基因，在肝脏和肺中以及对于cII基因，在肺和骨髓中，MFs也有统计学上显著的增加。将从结肠中的37个突变体回收的cII基因的序列改变与未处理小鼠的50个突变体进行了比较。在未处理组（91%）和DNP处理组（84%）中，碱基取代突变都占主导。DNPs处理增加了G:C到T:A颠换的发生率（2% - 43%），并降低了G:C到A:T转换的发生率（70% - 22%）。G:C到T:A颠换是DNPs处理的特征，可能是由鸟嘌呤 - C8加合物引起的，已知该加合物是DNPs诱导的主要DNA加合物，通过在加合物对面掺入腺嘌呤（“A”规则）。本研究表明cII基因可作为突变小鼠中诱变的另一个靶点，并揭示了DNPs在体内的诱变特异性。
