Tohru Shibuya

Other transgenic mutation assays: A new transgenic mouse mutagenesis test system using Spi− and 6-thioguanine selections

A new transgenic mouse mutagenesis test system has been developed for the efficient detection of point mutations and deletion mutations in vivo. The mice carry lambda EG10 DNA as a transgene. When the rescued phages are infected into Escherichia coli YG6020‐expressing Cre recombinase, the phage DNA is converted into plasmid pYG142 carrying the chloramphenicol‐resistance gene and the gpt gene of E. coli. The gpt mutants can be positively detected as colonies arising on plates containing chlaramphenicol and 6‐thioguanine. The EG10 DNA carries a chi site along with the red and gam genes so that the wild‐type phages display Spi+ (sensitive to P2 interference) phenotype. Mutant phages lacking both red and gam genes can be positively detected as plaques that grow in P2 lysogens of E. coli. These mutant phages are called lambda Spi−. The spontaneous gpt mutation frequencies of five independent transgenic lines were 1.7 to 3.3 × 10−5 in bone marrow. When the mice were treated with ethylnitrosourea (single i.p. treatments with 150 mg/kg body weight; killed 7 days after the treatments), mutation frequencies were increased four‐ to sevenfold over the background in bone marrow. The average rescue efficiencies were more than 200,000 chloramphenicol‐resistant colonies per 7.5 μg bone marrow DNA per packaging reaction. In contrast to gpt mutation frequencies, spontaneous Spi− mutation frequencies were 1.4 × 10−6 and 1.1 × 10−6 in bone marrow and sperm, respectively. No spontaneous Spi− mutants have been detected so far in spleen, although 930,000 phages rescued from untreated mice were screened. In gamma‐ray‐treated animals, however, induction of Spi− mutations was clearly observed in spleen, at frequencies of 1.4 × 10−5 (5 Gy), 1.2 × 10−5 (10 Gy), and 2.0 × 10−5 (50 Gy). These results suggest that the new transgenic mouse “gpt delta” could be useful for the efficient detection of point mutations and deletion mutations in vivo.

The induction of recessive mutations in mouse primordial germ cells with N-ethyl-N-nitrosourea

A specific-locus test was carried out to examine the mutagenic activity of N-ethyl-N-nitrosourea (ENU) on mouse primordial germ cells (PGC). Embryos of C3H/He mice were treated transplacentally with 30 or 50 mg ENU per kg of maternal body weight on day 8.5, 10.5, or 13.5 of gestation (G8.5 day, G10.5 day, or G13.5 day). Male and female mice that had been treated with ENU in embryonic stages were mated with female or male tester PW mice to detect recessive mutations induced in PGC. ENU induced recessive mutations at a relatively high rate in PGC at these developmental stages. The most sensitive stage was G10.5 day. On G8.5 day, the induced mutation rate in males and females was not significantly different. Cluster mutations, which originate from the limited number of PGC and cell killing, were more frequently induced at an earlier developmental stage. The induced mutation rate per unit dose of ENU (1 mg/kg) was higher in G8.5 and G10.5 day PGC than in stem-cell spermatogonia. It can be concluded that mouse PGC are more sensitive than stem-cell spermatogonia to the induction of recessive mutations by ENU.

Partial hepatectomy strongly increased the mutagenicity of N-ethyl-N-nitrosourea in Muta™Mouse liver

The relationship between cell proliferation and mutagenesis can be investigated in vivo due to the advent of transgenic animal mutation assays. In these assays, slowly proliferating tissues, such as mammary gland and liver, that are exposed to mutagens generally have longer manifestation times for mutations and lower mutant frequencies. This may be because the cells have enough time prior to cell division to repair DNA damage. We carried out this study using the Muta™Mouse positive selection system to investigate the effect of a high rate of cell proliferation induced by partial hepatectomy (PH) on mutation induction. We used a 2 × 2 experimental design for PH (or no PH) and 50 mg/kg N‐ethyl‐N‐nitrosourea (ENU) (or phosphate buffer), with the chemical injected 16–19 hr after PH. In the non‐ENU groups, the mean MF was slightly but not significantly higher in the PH group than in the non‐PH group. In the ENU non‐PH group, the MF was also slightly but not significantly increased. In the ENU PH group, in contrast, the mean MF was 7 times the mean MF of the group that received either treatment alone. These results strongly support the hypothesis that ENU induced pre‐mutational DNA lesions in liver are completely repaired prior to cell division, and PH increases the mutagen‐induced MF by reducing the amount of time available for such repair. Environ. Mol. Mutagen. 34:121–123, 1999

Mouse spot tests with alkylnitrosoureas.

Spot tests with various alkylnitrosoureas were carried out using the newly established PW strain of male and female C57BL/6 mice. Chemicals tested were N-methyl-N-nitrosourea (MNU), N-ethyl-N-nitrosourea (ENU), N-propyl-N-nitrosourea (PNU) and N-butyl-N-nitrosourea (BNU). Although MNU had a severe teratogenic effect, no clearly positive color spot was obtained, while apparent induction of their color spots was observed with ENU, PNU and BNU. The frequency of appearance of these color spots was dose-dependent. In F1 males from the treated group, the weight of the testes was significantly lower in mice about 30 days old. It is suggested that these chemicals may have mutagenic effects on both developing somatic and germ cells of mice.

Mutation induction by N-propyl-N-nitrosourea in eight Muta™Mouse organs

As a part of the 2nd Collaborative Study for the Transgenic Mouse Mutation Assay, we studied the organ specificity and the temporal changes in mutant frequency (MF) of the lacZ gene following intraperitoneal injection of 250 mg/kg N-propyl-N-nitrosourea into male MutaMouse. We used a positive selection system and examined eight organs, i.e., bone marrow, liver, kidney, lung, spleen, brain, heart, and testis. The chemical caused a significant increase in MF in all organs except for brain, and the bone marrow was the most sensitive organ, exhibiting a MF on day 7 that was 10 times that of the control. The MF increased from day 7 to day 28 in liver, kidney, and testis, while it decreased in bone marrow. The relationship between the results of this study and the target organs of carcinogenesis, and the cause of the temporal changes in MF, are discussed.

Effects of O (6)-alkylguanine-DNA alkyltransferase deficiency in Escherichia coli as the host for the detection of mutations in lacI transgenic mice.

Transgenic mice are widely used to detect gene mutations in vivo induced by a variety of chemicals. It is known, however, that no mutagenicity of methyl methanesulfonate (MMS) is detected in epididymal sperm in various transgenic mice assays, although MMS induces the dominant lethal and specific locus mutations in male mice. To investigate the issue of whether unrepaired lesions in DNA of mature sperm can be transformed into mutations during replication of the lambda phage in Escherichia coli cells, we developed an E. coli strain YG5152, which is a derivative of strain SCS‐8 but is deficient in the genes encoding O 6‐alkylguanine‐DNA alkyltransferases. When lambda LIZα phages were treated with MMS or N‐ethyl‐N‐nitrosourea (ENU) in vitro and infected to the E. coli strains, the mutant frequencies of lacI were markedly higher in strain YG5152 than in strain SCS‐8. When Big Blue™ mice were treated with MMS (160 mg/kg) or ENU (125 or 250 mg/kg) and the phages rescued from mature sperm were infected to the strains, the mutation frequency (MF) of phages from ENU‐treated mice at a dose of 250 mg/kg in strain YG5152 was about two times higher than that in strain SCS‐8. However, no increase in the MF was observed in the MMS‐treated mice even in strain YG5152. These results suggest that, although strain YG5152 efficiently detects ex vivo mutations caused by mutagenic alkyl adducts formed by MMS in lambda phage DNA, no detectable levels of mutagenic methyl adducts are present in mature sperm of MMS‐treated mice. Possible reasons for this lack of mutagenicity of MMS in mature sperm using transgenic mice assays are discussed. Environ. Mol. Mutagen. 34:221–226, 1999

Dose-dependent induction of recessive mutations with N-ethyl-N-nitrosourea in primordial germ cells of male mice

Using a specific locus test, we previously found that N-ethyl-N-nitrosourea (ENU) induces recessive mutations at a relatively high rate in male mouse primordial germ cells (PGC) at 8.5, 10.5 and 13.5 days of development (G8.5, G10.5 and G13.5). A large difference was observed on the induced mutation rate between 30 and 50 mg/kg ENU in 10.5-day PGC. We therefore carried out specific locus tests to ascertain whether ENU induces recessive mutations in a dose-dependent manner in G8.5 and G10.5 PGC. We also gave multiple doses of 25 mg/kg ENU using an 18-h interval, the approximate doubling time of PGC at these developmental stages, to test for an additive effect on the induced mutations rate. A dose-dependent induction of recessive mutations by ENU was observed in both G8.5 and G10.5 PGC, and multiple dosing of 25 mg/kg ENU showed an additive effect. Comparing these results to data on spermatogonial stem cells, we conclude the capacity to repair ENU-induced premutagenic damages is less effective in male mouse PGC at these developmental stages than in spermatogonial stem cells.

The induction of specific-locus mutations with N-propyl-N-nitrosourea in stem-cell spermatogonia of mice

A specific-locus test to determine the effect of N-propyl-N-nitrosourea (PNU) on the stem-cell spermatogonia of mice has been performed. Male wild-type mice (C3H/He) were treated with an intraperitoneal injection of 200 mg/kg [corrected] of PNU. Eight weeks after the injections, the males were mated with tester stock females (PW), homozygous for 6 visible recessive genes. Twelve mutants among 8605 offspring were observed. The mutation frequency with PNU was calculated to be 23.2 x 10(-5)/locus/gamete, showing a significant difference from that of the non-treated control. The mutations were all heritable and half of them were viable in homozygous condition. The mutation frequency with PNU was about one-third of that with N-ethyl-N-nitrosourea, a highly potent mutagen for mouse stem-cell spermatogonia.

Mouse spot tests with dimethylbenz[a]anthracene with and without phenobarbital pretreatment

Mouse spot tests using dimethylbenz[a]anthracene (DMBA) were carried out on PW strain male mice and female C57BL/6 mice. DMBA induced somatic gene mutations in developing mouse embryonic cells. Pretreatment of pregnant females with phenobarbital (PB) reduced the incidence of somatic mutation by DMBA. The testes of males treated with DMBA in utero, whether treated with PB or not, showed severe retardation of development. The amount of cytochrome p-450 in the liver of C57BL/6 females increased about 2-fold by the pretreatment schedule, carried out on days 10-12 of pregnancy.

Modifying effects of the enzyme inducers, phenobarbital and 3-methylcholanthrene, on dominant lethal events induced by 7,12-dimethylbenz[a]anthracene in mice

The effects of pretreatment with either phenobarbital (PB) or 3-methylcholanthrene (MC) on the induction of dominant lethal events by 7, 12-dimethylbenz[a]anthracene (DMBA) in male mice were studied. DMBA induced dominant lethals in both post- and pre-meiotic germ cells of mice. The incidences of DMBA-induced dominant lethals were markedly reduced in pre-meiotic germ cells by the pretreatment with PB, whereas a significant reduction of the lethal events in post-meiotic germ cells was observed with MC. No significant reduction of living implants was detected in pre-meiotic germ cells on pretreatment with PB. The contents of liver microsomal cytochrome p-450 of mice pretreated with PB or MC were about twice those of non-treated mice.