Detection of vincristine-induced hyperploidy in meiotic II metaphases of male Chinese hamsters.

Induction of hyperploidy in germ cells of male Chinese hamsters treated with vincristine at dose levels of 0.25, 0.50 or 0.75 mg/kg of body weight was investigated. Animals were killed at 6, 24, 48, 72 and 96 h after administration of the chemical by a single intraperitoneal injection. The testes were removed and processed for spermatogonial, meiotic I, and meiotic II metaphases. Significantly increased frequencies of hyperploidy were obtained in meiotic II cells harvested 6, 24 and 48 h but not 72 and 96 h after treatment, indicating the importance of multiple sampling times. Analysis of spermatogonial cells shows that the frequencies of hyperploidy in the treated samples were comparable to those of controls. Limited sampling times used in the present study as well as small sample size or possible loss of hyperploid cells may be responsible for the negative findings for spermatogonial cells. Examination of meiotic I cells from 53 animals reveals the presence of one animal with an elevated level of hyperploidy unrelated to the vincristine treatment.

The feasibility of using Chinese hamsters as an animal model for aneuploidy.

The utility of Chinese hamsters as a test species for aneuploidy analysis was studied using four chemicals--vincristine, methyl 2-benzimidazole carbamate (MBC), nocodazole, and cyclophosphamide. Ten or more male Chinese hamsters were used per dose and bone marrow was removed at intervals of 6-96 hr. Slides were coded and 50-100 metaphases were analyzed per animal. A metaphase with more than 22 chromosomes was classified as a hyperploid cell, and the data were evaluated by using a one-tailed Fisher's exact test. In experiments using vincristine, MBC, and nocodazole, the frequencies of hyperploid cells were 0.43, 1.14, and 0.91%, respectively, for the control groups. In the experiment using cyclophosphamide, the control value frequency was 3.75%. The treated groups showed no significant increase in hyperploid frequencies when compared to concurrent controls at each of the treated times, except the value at 24 hr for the group that had been treated with vincristine at 0.75 mg/kg. However, this increase was not significant when compared to the overall value for pooled controls, with or without the cyclophosphamide control. Therefore, no significant effects due to chemical treatment were obtained in the present study. The results illustrate the extent of animal-to-animal as well as experiment-to-experiment variability in hyperploid frequencies and the importance of incorporating concurrent controls in assays for aneuploidy.

A guide for mutagenicity testing using the dominant lethal assay.

The dominant lethal assay has been used and continues to be used to provide information about the effects of chemicals on the gonadal cells of male animals. Guidelines for conducting this test are useful but as with any guideline scientists should avoid interpreting them as protocols. Thus this document is a general approach to dominant lethal testing and should be used in conjunction with other available protocols and procedures.

Enhancement of concurrent yield of spermatogonial, meiotic I, and meiotic II metaphase chromosomes from Chinese hamster testes.

A procedure to enhance the concurrent yield of large numbers of spermatogonial, meiotic I, and meiotic II metaphases from Chinese hamster testes has been developed. Animals were injected intraperitoneally with 5, 10, 20, or 40 mg of colchicine/kg and killed 3 hr after the treatment. Both testes from each animal were excised; one testis was minced, and germ cells were isolated by using 0.1% trypsin solution; the other testis was minced, and germ cells were isolated by using a conventional procedure. Chromosomal preparations were made with a standard air-drying technique. Examination of slides revealed that with the trypsin-isolation procedure the concurrent yields of spermatogonial, meiotic I, and meiotic II metaphases were significantly higher (P less than .05) than the yields obtained with the conventional procedure. Furthermore, 40 mg of colchicine/kg produced maximum numbers of all three types of germ cell metaphases. At this maximum concentration, metaphases were stable with no evidence of structural aberrations, and the frequency of hyperploidy was not significantly different (P greater than .05) from hypoploidy.

Mammalian in vivo assays for aneuploidy in female germ cells.

This paper presents an evaluation of and offers recommendations for assays to detect chemically induced aneuploidy in mammalian female germ cells. 72 papers on female germ cell aneuploidy, published from 1970 to 1984, were reviewed. 28 papers were selected for critical evaluation; the other 44 papers were rejected according to pre-established criteria. Salient points emerging from the information reviewed allow an assessment of the current status of mammalian female germ cell tests for aneuploidy. The majority of data have been obtained by analyzing metaphase II mouse oocyte chromosomes following superovulation. Various classes of chemicals were administered usually around the time of ovulation. Dose-response relationships have not been obtained for the majority of chemicals evaluated. The method of data reporting and analysis usually was not conducive to comparisons among different studies. Few of the 16 chemicals studied can be regarded as negative for their ability to induce aneuploidy, whereas an even smaller number should be considered as positive. Certainly, a need exists to identify the chemicals and the dosages that could increase the incidence of aneuploidy in mammalian female germ cells. Obtaining such data definitely is feasible in cytogenetic laboratories. However, the mammalian female germ cell aneuploid assay should not be perceived as a rapid, inexpensive, routine procedure. The assay is capable of detecting aneuploidy following anaphase I when metaphase II oocytes are studied and following anaphases I and II when first-cleavage zygotes are studied.

Cultured cell lines for research on pulmonary physiology available through the American type culture collection.

Procedures are described by which cell lines of potential use for studies on pulmonary physiology were developed or otherwise obtained, characterized, and banked for distribution to the scientific community. Seventy-seven cell lines were submitted under this program. Forty-eight of these exhibited adequate doubling potential and were of sufficient interest and purity to permit accessioning and banking for distribution. An additional 12 lung cell lines had been added earlier during development of the Cell Repository. In all, 4 presumptive type II alveolar lines, 2 nonspecified epithelial-like lines, 2 endothelial lies, 1 mesothelial line, and 51 fibroblastlike lines are available. Eighteen species including humans, monkeys, and common laboratory animals are represented. An additional 19 lines were retained as token holdings because they either exhibited insufficient doubling potential or represented duplicate samples of differing passage levels. Ten lines were rejected because of poor viability, presence of contaminant microorganisms, or inappropriate species identification by the donor. The lines will be retained at the ATCC for distribution on request to the scientific community at large. As newly developed strains are identified and made available these may also be added to the existing collection. Appropriate cooperation from the scientific community and support from governmental funding agencies are required and acknowledge.

Morphological, growth, and chromosomal changes in bovine pancreatic duct epithelial cells exposed to N-methyl-N'-nitro-N-nitrosoguanidine.

Epithelial cells cultured from bovine pancreatic ducts were given a single treatment of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Multinucleated cells and giant cells were observed more frequently in carcinogen-treated cultures than in controls. The MNNG-treated cultures also contained a sizeable population of small, dense cells that were not observed in control cultures. At the concentration of 1.0 microgram/ml, MNNG caused an initial depression in the growth rate of the cells followed by growth stimulation for several weeks. The MNNG produced chromosomal damage in the cells as indicated by the observation that a substantial proportion of carcinogen-treated cells were heteroploid and contained a high frequency of metacentric and submetacentric chromosomes and a dicentric marker chromosome. The MNNG treated and control cultures did not acquire the ability to grow in soft agar or to produce tumors after transplantation into athymic, nude mice.

Survey of ATCC stocks of human cell lines for HeLa contamination.

Seed stocks of human cell lines deposited at the American Type Culture Collection (ATCC) have been examined for cross-contamination with HeLa cells using Giemsabanded marker chromosomes. Sixteen additional cell lines investigated have been found to exhibit marker chromosomes typical of HeLa cells. Quinacrine fluorescence studies further revealed the absence of Y chromosome in these lines. These observations indicate that the lines are HeLa derivatives.

Characterization of human heteroploid cell line J-111: Reverse banding patterns of marker chromosomes.

The karyotype of the human cell line, J-111, has been studied employing R-banding by fluorescence using acridine orange technique (RFA). The model chromosome number of this line was 112. All human chromosomes except the Y were present in each metaphase. Twenty-one marker chromosomes were distinguished and their possible origins were investigated. Of these, twelve were consistently present in all cells. Nine markers were highly variable. Four typical marker chromosomes of HeLa cells were found and their origins were identified, indicating that the line is a HeLa contaminant. The reverse banding patterns of all marker chromosomes are presented and the value of the RFA technique is discussed.

Latent meiotic anomalies related to an ancestral exposure to a mutagenic agent.

When urethan is administered to cytologically normal F(2) progeny descended from grandsires exposed to ethyl methanesulfonate, meiotic anomalies in the forms of site-specific X-chromosome deletions and bivalent associations are noted in spermatocytes of male Armenian hamsters examined 6 and 8 days after treatment, respectively. These latent anomalies are initiated and retained in a premutated state for two generations after the ancestral exposure to ethyl methanesulfonate, the additional impetus required to complete the mutational processes being supplied by urethan.