乙基亚硝基脲和甲磺酸甲酯对lacZ转基因小鼠（突变小鼠）精子和睾丸生殖细胞的致突变性。

Ethyl nitrosourea and methyl methanesulfonate mutagenicity in sperm and testicular germ cells of lacZ transgenic mice (Muta Mouse).

作者信息

Suzuki T, Itoh S, Takemoto N, Yajima N, Miura M, Hayashi M, Shimada H, Sofuni T

机构信息

Division of Genetics and Mutagenesis, National Institute of Health Sciences, Tokyo, Japan.

出版信息

Mutat Res. 1997 Feb 14;388(2-3):155-63. doi: 10.1016/s1383-5718(96)00112-x.

DOI:10.1016/s1383-5718(96)00112-x

PMID:9057876

Abstract

The germ cell mutagens ethyl nitrosourea (ENU) and methyl methanesulfonate (MMS), were tested for their genotoxicity in sperm cells and testicular germ cells using lacZ transgenic mice (Muta Mouse). Eight- to 10-week-old Muta mice were treated with ENU (150 mg/kg) or MMS (40 mg/kg) by intraperitoneal injection. Three and 14 days after treatment, testes and sperm were collected for lacZ mutation analysis. Sperm were isolated from the epididymis and vas deferens by washing out the minced tissue. Germ cell DNA was isolated from testicular germ cells and sperm with the help of 2-mercaptoethanol, and the target lacZ gene, which is integrated into a lambda shuttle vector, was recovered by in vitro packaging. The resultant phages were allowed to infect to E. coli C (galE), and the lacZ mutant plaques were dominantly selected on a plate containing phenyl-beta-D-galactoside. Spontaneous mutant frequencies (MF) in vehicle-treated control mice were approximately 1 x 10(-5) and 3 x 10(-5) in testicular germ cells and sperm, respectively, at both sampling times. ENU treatment increased the MF in the testicular germ cells to 5 x 10(-5) on days 3 and 14, but did not affect sperm MF. MMS was not mutagenic in either tissue. The peripheral blood micronucleus assay was performed on the same animals 48 h after treatment, and strong inductions of micronucleated reticulocytes (MNRETs) were observed in both ENU- and MMS-treated mice. These data suggest that agents mutagenic to premeiotic germ cells, e.g., ENU, can be detected by transgenic mutation assay system using germ cells isolated from the testis. On the other hand, those mutagenic to postmeiotic cells, e.g., MMS, are insensitive in the assay system.

摘要

使用 lacZ 转基因小鼠（Muta 小鼠），对生殖细胞诱变剂亚硝酸乙酯（ENU）和甲磺酸甲酯（MMS）在精子细胞和睾丸生殖细胞中的遗传毒性进行了测试。8至10周龄的 Muta 小鼠通过腹腔注射给予 ENU（150 mg/kg）或 MMS（40 mg/kg）。处理后3天和14天，收集睾丸和精子进行 lacZ 突变分析。通过冲洗切碎的组织从附睾和输精管中分离精子。借助2-巯基乙醇从睾丸生殖细胞和精子中分离生殖细胞DNA，并通过体外包装回收整合到λ穿梭载体中的目标 lacZ 基因。将所得噬菌体用于感染大肠杆菌C（galE），并在含有苯基-β-D-半乳糖苷的平板上显性选择 lacZ 突变噬菌斑。在两个采样时间，溶剂处理的对照小鼠睾丸生殖细胞和精子中的自发突变频率（MF）分别约为1×10⁻⁵ 和3×10⁻⁵。ENU 处理在第3天和第14天将睾丸生殖细胞中的 MF 增加到5×10⁻⁵，但不影响精子 MF。MMS 在两种组织中均无致突变性。在处理后48小时对同一批动物进行外周血微核试验，在ENU和MMS处理的小鼠中均观察到微核网织红细胞（MNRET）的强烈诱导。这些数据表明，对减数分裂前生殖细胞具有诱变作用的试剂，如ENU，可以通过使用从睾丸分离的生殖细胞的转基因突变检测系统进行检测。另一方面，对减数分裂后细胞具有诱变作用的试剂，如MMS，在该检测系统中不敏感。