In order to maximize the mutagenic effectiveness of N-ethyl-N-nitrosourea in mouse stem-cell spermatogonia, advantage was taken of the fact that these cells can accumulate mutations from repeated doses given over relatively long time periods. Repeated doses (100 mg/kg) of ethylnitrosourea injected intraperitoneally into male mice at weekly intervals were found to allow adequate survival and fertility with total dosages of 300 and 400 mg/kg. The specificlocus mutation frequencies at these dosages were, respectively, 1.8 and 2.2 times that obtained with the maximal practicable single dose of 250 mg/kg. The mutation frequency induced by a 400 mg/kg dosage of ethylnitrosourea is 12 times the maximal mutation frequency achievable with a single exposure to x-rays and 36 times that reported for procarbazine, the most effective chemical mutagen previously known for mouse stem-cell spermatogonia. Ethylnitrosourea is already the mutagen of choice in deliberate attempts to create mouse models for human disease and in any experiments in which a maximal mutation rate is desired. Repeated-dose regimens similar to the ones reported here should increase the efficiency of such studies.One advantage of determining induced mutation rates in stem-cell spermatogonia is that these cells can accumulate mutational lesions throughout the reproductive life span of the animal; therefore, repeated doses of mutagens can be given over relatively long time periods to amass a total dosage higher than that tolerated in a single exposure. Ethylnitrosourea (EtNU) has been shown to be the most effective mutagen for induction of specific-locus mutations in mouse spermatogonia (1,2). This has led rapidly to its being the mutagen of choice by many investigators (3,4). Therefore, it seemed useful to test whether repeated doses could be given to yield higher specific-locus mutation rates than those obtained by single exposures. This paper presents data from specific-locus tests with repeated doses of EtNU and from survival and fertility tests that were conducted prior to the specific-locus tests in order to choose feasible dosage regimens. A brief abstract of the preliminary results on survival and fertility has appeared (5). MATERIALS AND METHODSSurvival and Fertility Tests. EtNU, synthesized in our laboratory by D. G. Doherty, was dissolved in phosphate buffer (6) adjusted to pH 6. The dose injected intraperitoneally was matched to the body weight of the animals by adjusting the volume of solution injected, which never exceeded 1 ml. Male (101 x C3H)F1 mice were injected with dosages of 300 or 400 mg of EtNU/kg, given in 100 mg/kg doses. In four separate experiments, the individual doses were spaced 1 day, 2 days, 4 days, or 7 days apart, respectively. Six mice, 12 weeks old at the first injection, were in each dosage group in each experiment. All injections were completed within 1 hr after the EtNU was dissolved. Seven weeks after the final injection, these males were mated to females of our standard specific-locus test strain, T, which is h...

No abstract

In the following is described a recently developed blood culture technique for the study of fish chromosomes.Whole blood culture : Whole blood about 0.2 ml in volume, was derived from ventriculus cordis of a young fish and suspended in a medium consisting of 2 ml calf serum and 0.1 ml PHA-M in 8 ml TC-199 (Dif co) to which penicillin and streptomycin were added.Separation of leucocytes : Heparinized blood about 1 ml in volume, was obtained from ventriculus cordis and allowed to stand in an ice bath for 1-2 hours. Then the sedimentation of erythrocytes was accomplished following the cetrifugation at 300-350 rpm for 10 minutes at 5°C. The supernatant plasma containing leucocytes, 0.2-0.3 ml in volume, was suspended in a culture bottle cotaining a mixture of 2 ml of calf serum, 8 ml of TC-199 (Difco), 0.1 ml of PHA-M, penicillin and streptomycin.The initial concentration of cells was 1 to 2>< 106/ml of the culture medium. The pH was adjusted to 7.0-7.2 initially.Harvesting: Examination of growing cells in vitro showed that an optimal mitotic activity was obtained on the 3 rd day of culture at 25-30°C. For chromosome slides, the culture was terminated at this limit. To accumulate a large number of cells at metaphase, colchicine was added at a 1 x 10-g M concentration (approximately one drop of 100 r Jml colchicine for culture bottle) at an interval of 5-6 hours prior to termination. Cells were harvested from the culture bottle by shaking or pipetting and then centrifuged at 800 rpm for 5-10 minutes. The most part of the supernatant medium was removed and the sedimentary cells were treated with hypotonic solution consisting of 1 part of Hanks' BSS and 3 parts of distilled water. The centrifuge bottle was cooled in ice water. Three ml of ice cooled Carnoy's fluid were added very gently to the cell suspension with a fine pipette. While the centrifuge bottle was still in ice water, the cells were mixed by bubbling of a fine stream of air with the aid of a fine pipette. After centrifugation at 800 rpm for 10 minutes the cells were suspended in cold Carnoy's solution for 1 hour. The cells were resuspended in the fixative and the chromosome slides were prepared by means of the air-drying method by Sandberg et al. (1962) and Ojima et al. (1964).The process described above achieved a fairly uniform and considerably flattening metaphase configuration in cells which is available for a precise karyotype analysis

Malignant cell transformation and its reversion, in cells transformed

Because of the unusually high number of chromosomes coupled with the extreme smallness of cell size in these cyprinid fish, the counting and the study of the morphology of the chromosomes are a very difficult task, when older techniques of fixing and staining are used. Recent surprising advances in cytological techniques have rendered possible precise and reliable observation of the chromosomes in germ cells as well as somatic cells of the fish, and noticeable information has increasingly been obtained on the chromosome morphology of lower vertebrates.In the senior author's laboratory, a cytogenetical survey of lower vertebrates including Pisces, Amphibia and Reptilia has been in progress, with a hope to contribute to advances of cytogenetic knowledge in these groups of animals. MATERIALS AND METHODSIn the present study the chromosomes were investigated mostly in the common carp (Cyprinus carpio). Both males and females were used as materials. Adult fishes were injected intraperitoneally with colchicine solution four hours before sampling. Then the testes and kidnies were dissected out and minced with scissors in a balanced salt solution. The dispersed cells of testis and kidney cells were collected in a centrifuge bottle, treated with a hypotonic solution, and fixed with acetic alcohol. Chromosome spreading was achieved by means of the air-drying method. RESULTSChromosome observations were exclusively made on excellent and reliable metaphasic cells. The results of chromosome counting showed that both germ cells and somatic cells derived from males contained 100 chromosomes at metaphase. The somatic cells from females also had 100 chromosomes in a diploid complement.The morphology of chromosomes was analyzed with special regard to their size, shape and the position of the centromere. The karyotype analysis was done on approximately 100 well-delineated metaphasic chromosomes in which no element showed