Jpn. J. Genet. (1983) 58, pp. 263-267

SHORT PAPER

Genetic analysis of mutants in mice induced by N-ethyl-N-

BY Tetsuro MUROTAand Tohru SHIBUYA Laboratory of Genetics, Hatano Research Institute, Food and Drug Safety Center, 729-5 Ochiai, Hadano, Kanagawa 257

(Received December 10, 1982) ABSTRACT Specific locus tests of the effects of N-ethyl-N-nitrosourea (ENU) on mouse spermatogonia using visible loci and hemoglobin beta-chain (Hbb) locus have been performed. Male wild-type (C3Hf/He) mice were injected intraperito- neally with ENU. Eight weeks after injection, the males were mated with tester strain females (PW) homozygous for six visible recessive . Ten visible and 2 hemoglobin mutants were obtained. The frequency was 60.3 x 10-5/locus/generation. These were all heritable, and nine out of ten mutations were viable under homozygous conditions. The results suggest that the mutations induced by ENU on spermatogonia of mice might have resulted from events at the genic rather than the chromosomal level.

1. INTRODUCTION The specific locus test in mice can be used to detect mutations caused by various . Analysis of germ cell mutations in mice can provide important information for estimation of the genetic risk of environmental mutagens to humans. Generally, the specific locus test in mice has been mainly applied for the detection of visible recessive mutations (Russell 1951). This test is advantageous in that (1) it can accurately determine the induced muta- tion frequency at a given locus in germ cells, (2) the differential stage sensi- tivity to chemical mutagens in in vivo germ cells can be determined and (3) the nature of the mutations induced can be characterized. Recently, the detection of mutations at enzyme and/or protein loci by means of electro- phoretic techniques has been developed. Most enzymes in mice are rather similar to those of humans, so that the induction of mutations at the enzyme level in mice could provide important information to estimate the genetic risk of environmental mutagens to humans. The nature of induced mutations at these loci was investigated in detail by biochemical and molecular methods. Russell et al. (1979) indicated that ENU has strong mutagenic activity for inducing visible recessive mutations in mouse spermatogonia. A study of 264 T. MUROTA and T. SHIBUYA mutagenicity at visible loci and hemoglobin beta-chain (Hbb) locus in sper- matogonia of mice after treatment with ENU was therefore carried out, and many mutants were obtained. Genetic analysis of these mutants should provide important information regarding the types of genetic damage induced by ENU. In this paper, the results of specific locus tests with ENU in mice and a genetic analysis of the mutants are reported.

2. MATERIALS AND METHODS Male 9-week-old C3Hf /He mice were given a single dose of N-ethyl-N- nitrosourea (ENU: Nakarai Chemical, Ltd., Japan) dissolved in 1/15M phosphate buffer adjusted to pH 6.0 at a concentration of 150 mg/kg. A volume of 0.5 ml per 25 g body weight was administered intraperitoneally. No treatment was given to control males. From 8 weeks after treatment, each male was mated with 12- to 16-week-old tester strain females (PW), homozygous for six visible recessive genes, a/a, b/b, cch/cch,d/d, se/se and p/p, as established by Tutikawa and Harada (1981). The pregnant females were separated and allowed to deliver offspring. The offspring were weaned at about 21 days of age and then scored for mutation at the six loci. The analysis of Hbb locus mutations was carried out by means of Titan III cellulose acetate (Helena Laboratories, U.S.A.) electrophoresis of fresh hemolysates and alkylated hemolysates treated with iodoacetate. Electrophoresis was performed according to the method described by Takahashi et al. (1978). The preparation of samples for electro- phoretic studies was described elsewhere (Murota et al., 1982). The genotypes of C3Hf /He and PW were Hbbd/Hbbd and Hbb'/Hbb', respectively. The allelism and viability tests on the visible mutants were performed according to the method described by Searle (1977). For the viability test of the mutant at the Hbb locus, they were backcrossed to the marker stock PW, and the result- ant backcross F1 offspring, classified as heterozygous mutants, were mated inter se. The homozygous viability of these mutations was judged on the basis of whether or not homozygous mutants were obtained by crossing of these heterozygous mutants inter se.

3. RESULTSAND DISCUSSION The results of specific locus tests with ENU are shown in Table 1. In the treated group, 2916 offspring were obtained, among which 2400 offspring were examined at the Hbb locus. Ten visible locus mutants and two Hbb locus mutants were obtained. The overall mutation frequency was calculated to be 60.3 X 10-5/locus/generation with 95% confidence intervals of 31.2 to 105.3 X 10-5. This value corresponds well with the results reported by Russell et al. (1982). In the control group, no mutation was detected among 1731 offspring. In Table 2, the results of allelism and viability tests on the detected mutants are listed. Specific locus tests in mice with ENU 265

Table 1. Mutation frequency at specific loci in the offspring of male mice treated withFlN-ethyl-N-nitrosourea

*: The numbers in parentheses indicate the numbers of offsping at the Hbb locus .

Table 2. Distribution among specific loci of mutations induced in spermatogonia in mice by N-ethyl-N-nitrosourea, and thier viability under homozygous conditions

* N . T.: Not tested for v iability.

The locus distribution of the mutants was as follows: b locus, 3; c locus, 2; d locus, 3; p locus, 2; and Hbb locus, 2. As shown in the Table, nine out of ten mutants were viable under homozygous conditions. Two female mutants died before the viability test could be performed, so that the viability could not be determined. Chemicals which are known to induce specific locus mutations on sper- matogonia in the mouse specific locus test include triethylenemelamine (Cattanach 1966), mitomycin C (Ehling 1974), (Ehling and Neu- hauser 1979) and ENU (Russell et al. 1979) . The induced mutation frequency of ENU was higher than that of the other chemical mutagens. The frequencies of homozygous viable mutations induced by mitomycin C (Ehling 1980) and procarbazine (Ehling and Neuhauser 1979) were reported to be 75% and 76%, respectively. The origin of mutations induced by these chemicals might have been mainly base-pair changes. ENU induces a high frequency of homozygous viable mutations (90%) in accord with the above view. In radiation muta- genesis, however, the frequency of X-ray-induced viable mutants in spermato- gonia was only 23% (Russell and Russell 1959), suggesting that mutations induced by X-rays in spermatogonia might include some chromosomal abnor- malities. Similar events were also observed in experiments using gamma-rays and fission neutrons (Batchelor et al. 1966). The frequency of homozygous 266 T. MUROTA and T. SHIBUYA viable mutations was clearly different between chemically-induced and radia- tion-induced mutations in spermatogonia. In the post-spermatogonial stage, procarbazine (Ehling and Neuhauser 1979) and methyl methanesulf onate (Ehling 1978) are known to induce specific locus mutations. The viable muta- tions amounted to 33% (Ehling and Neuhauser 1979) and 12% (Ehling 1980), respectively. The frequency of specific locus mutations which were viable under homozygous conditions was clearly dependent on the germ cell stage tested, i.e., post-spermatogonia and spermatogonia. It is likely that qualitative differences of specific locus mutations induced by some chemicals in post- spermatogonia and spermatogonia may exist. In mice, three alleles, Hbb', Hbbd and Hbbp have been reported at the Hbb locus which may code the structure of hemoglobin beta-chain polypeptides. It is well known that the amino acid composition of the beta-chain is different for each allele (Gilman 1974). Both Hbb' and Hbbd are found in most inbred strains, but Hbbp is rare (Staats 1980). Two mutants at Hbb locus were obtained in this experiment. These mutations were heritable and viable under homozygous conditions. One of the mutants was observed as a change in the mobility of the bands on electrophoresis (Murota et al. 1982) . The mobility of the bands was similar to that of Pon which carrier Hbb alleles under homo- zygous conditions (Watanabe et al. 1976). In the second mutant, new bands were observed. This observation suggested that the mutation had not origi- nated from Hbb locus, but from Hba locus. The other electrophoresic technique described by Chernoff and Pettit (1964) would account for this events. This may represent a gene mutations like the mutations observed at specific loci.

We are grateful to Dr. Akio Murakami, National Institute of Genetics, for a critical reading of the manuscript, and to Dr. Kiyoshi Tutikawa, National Institute of Genetics, for his valuable comments. We are also indebted to Drs. Koroku Hashimoto and Shigeo Iwahara of Hatano Research Institute for their constant encouragement during the present study.

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