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

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-β- d-galactoside. Spontaneous mutant frequencies (MF) in vehicle-treated control mice were approximately 1×10 −5 and 3×10 −5 in testicular germ cells and sperm, respectively, at both sampling times. ENU treatment increased the MF in the testicular germ cells to 5×10 −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.