A correlative study on the genetic damage induced by chemical mutagens in bone marrow and spermatogonia of mice : I. CNU-ethanol

Cytogenetic damage induced by a wide range of concentrations of CNU-ethanol in mice was evaluated by determining the frequencies of (a) micronuclei nuclei in polychromatic erythrocytes of the bone marrow, (b) chromatid aberrations in bone marrow, (c) chromatid aberrations in spermatogonia, and (d) reciprocal translocations induced in spermatogonia and scored in spermatocytes.For CNU-ethanol the following order of sensitivity was found between the tests performed: micronuclei > aberrations in bone marrow > aberrations in spermatogonia > translocations in spermatocytes.Correlation coefficients were calculated for the first three parameters. Positive correlations existed (a) between micronuclei in polychromatic erythrocytes and chromatid aberrations in bone marrow on the first day after treatment, and (b) between chromatid aberrations in bone marrow and spermatogonia at the first day after treatment.Three reciprocal translocations were induced in spermatogonia and recorded in primary spermatocytes; all were of a rare type, namely between an X chromosome and an autosome.     

Clastogenic effect of thiophosphamide in spermatogonia of inbred strains of mice

Sensitivity of spermatogonia of 11 mouse inbred strains to induction of chromosome damages by thiophosphamide (thioTEPA) was studied. Metaphase chromosome preparations were made 24 h after treatment with thioTEPA (at 2.25 mg/kg, i/p). With respect to frequency of cells with chromosome damages, strains were ranked as follows: A/Sn (17.5 + 4.4%) greater than 101/H greater than TPS greater than WR = C57BL/6 = AKR = NZB greater than CBA/Lac greater than C3H/Sn greater than MRL greater than BALB/c (5.0 + 2.2%). This distribution does not coincide with that for sensitivity of bone marrow cells, though the data support, in general, the estimations obtained earlier for strains' mutability. Comparison of the data presented with those from literature demonstrates that the sensitivity to clastogenic effect of thioTEPA (and other alkylating agents) correlates neither with spontaneous level of SCE, nor with unscheduled DNA synthesis, nor with radiosensitivity of inbred mice. The frequency of induced chromosome aberrations in spermatogonia is relatively low and spermatogonia cannot substitute bone marrow cells as a test system when assaying chemical mutagens.     

Clastogenic effects of acrylamide in mouse bone marrow cells

Acrylamide, known to induce dominant-lethal mutations (Shelby et al., 1986; Smith et al., 1986) and heritable translocations (Shelby et al., 1987) in rodent germ cells, was hitherto a questionable clastogen in rodent bone marrow (Shiraishi, 1978). Therefore, it was tested for chromosomal aberrations in mouse bone marrow cells, spermatogonia and by the micronucleus test. The intraperitoneally injected doses ranged from 50 to 150 mg/kg. In the chromosomal bone marrow test and the micronucleus assay positive results were obtained with acrylamide, and in the latter test the effect increased linearly with dose. Chromosomal aberrations were not induced in differentiating spermatogonia by the acute acrylamide treatment. Cisplatin was used as a positive control and gave the expected positive response in all 3 tests. The present results demonstrate that acrylamide is no exception among clastogens. It breaks chromosomes not only in mammalian germ cells but also in somatic cells.     

Clastogenic effects of hydroquinone: induction of chromosomal aberrations in mouse germ cells

The clastogenic activity of hydroquinone (HQ) in germ cells of male mice was evaluated by analysis of chromosomal aberrations in primary spermatocytes and differentiating spermatogonia. In the first experiment with treated spermatocytes the most sensitive stage of meiotic prophase to aberration induction by HQ was determined. Testicular material was sampled for microscopic analysis of cells in diakinesis-metaphase I at 1, 5, 9, 11, and 12 days after treatment with 80 mg/kg of HQ, corresponding to treated diplotene, pachytene, zygotene, leptotene and preleptotene. The frequencies of cells with structural chromosome aberrations peaked at 12 days after treatment (p less than 0.01). This indicates that the preleptotene when DNA synthesis occurred was the most sensitive stage of meiotic prophase. In the second experiment the dose response was determined 12 days post treatment by applying 2 additional doses of 40 mg/kg and 120 mg/kg. The clastogenic effects induced by 40 and 80 mg/kg were significantly different from the controls (p less than or equal to 0.01) and higher than the results obtained with 120 mg/kg of HQ. A humped dose-effect relationship was observed. In a third experiment the same doses were used to analyse chromosomal aberrations in dividing spermatogonia of mice 24 h after treatment with HQ. All the administered doses gave results statistically different from the control values (p less than or equal to 0.01) and the data were fitted to a linear equation. HQ was found to be clastogenic in male mouse germ cells. It is concluded that the clastogenic effect in male germ cells is of the same order of magnitude as in mouse bone marrow cells.     

Cytogenetic effect of mitomycin C and cytosine arabinoside on mice of different genotypes]

Cytogenetic effect of mitomycin C (MC) and cytosine arabinoside (CA) on bone marrow cells of male mice of the strains 101/HY, C57BL/6Y C,3H/SnY and of the (C3HX101) F1 hybrids was studied. The frequencies of cells with chromosome aberrations after the treatment with MC at a 5 mg/kg dose were 54,4%; 41,8%; 40,4% and 26,8% in 101H, B6, C3H/Sn mice and in the F1 hybrids (C3HX101) respectively. The frequencies of cells with chromosome aberrations after the treatment with CA at a 500 mg/kg dose were 25,2%; 17,8%; 10,8% and the 101/H, B6, C3H/Sn mice and in the F1 hybrids (C3HX101) respectively. Both mutagens induced the greatest number of chromosome aberrations in the 101/H strain and the smallest number in the F1 hybrid (C3HX101). A positive correlation was established between the levels of induced and spontaneous chromosome lesions.     

Comparative cytogenetic study after treatment of mouse spermatogonia with mitomycin C

Male (101 × C₃H)F₁ hybrid mice, 10–12 weeks old, were injected i.p. with single doses of 2.5, 3.75 or 5.0 mg/kg of mitomycin C (MC). Spermatogonia were sample for mitotic chromosome analyses 6, 18 or 24 h after treatment. Spermatocytes were sample for meiotic chromosome analyses 50 or 60 days after treatment.The maximal yield of chromatid-type aberrations induced in spermatogonia was found 24 h after treatment with 5.0 mg/kg of MC. More than 50% of the cells carried at least one chromatid exchange. The majority (90%) of these were whole-arm exchanges derived from breaks in the centromeric heterochromatin.No translocation multivalents were found in spermatocytes analysed 50 or 60 days after treatment. The discrepancy between the presence of many symmetrical exchanges in spermatogonia and the absence of translocation multivalents in primary spermatocytes may be result of insensitivity of the stem cell spermatogonia against exchange induction by MC or of complete germinal selection against induced translocations before and/or during early meiosis. However, the possibility of missing translocations due to whole-arm exchanges in acrocentric chromosomes during the analysis of diakineses-metaphases I is also discussed.It is emphasized that comparisons of chromatid exchange frequencies in spermatogonia with the yield of translocation multivalents in spermatocytes descended from these spermatogonia as opposed to those from stem cells might provide an estimate of pre-diakinesis germinal selection against chromatid exchanges or the resulting translocations. This estimate is important for the quantitative evaluation of the genetic risk from environmental mutagens.     

Cytogenetic effects of malathion insecticide on somatic and germ cells of mice

Male mice dermally exposed to single or multiple treatment (5 days/2 weeks) showed that the ability of malathion to induce chromosome aberrations in somatic (bone marrow) and germ cells (primary spermatocytes) was related to the type of treatment and dose used. Statistically significant increases of chromosome aberrations in bone marrow cells occurred after single treatment (500 and 2000 mg/kg body wt) when chromatid gaps were included and after multiple treatment (250 and 500 mg/kg) when they were excluded. No dose-response relationships were observed for either treatment. In germ cells, malathion induced a significant increase of univalents in both types of treatment but structural chromosome aberrations were induced only by multiple treatment. Malathion induced a significant decrease of the mitotic indices in the bone marrow.     

Clastogenic effects of cis-diamminedichloroplatinum. I. Induction of chromosomal aberrations in somatic and germinal cells of mice

The clastogenicity of cisplatin, cis-diamminedichloroplatinum(II), an extensively used antitumor drug, has been studied employing (101/E1 X C3H/E1)F1 mice, aged 12-14 weeks. Chromosomal aberrations were assessed in mitotic divisions of bone marrow cells and differentiating spermatogonia. The drug was tested at 3 doses, 0.5, 1.0 and 2.5 mg/kg and 1.0, 2.5 and 5.0 mg/kg, respectively, for bone marrow and spermatogonia. Cisplatin had a clastogenic effect which was dose-dependent in both cell types. The frequencies of aberrant cells increased non-linearly in bone marrow and the dose-response relationship could be best described by a linear-quadratic equation. At the highest dose the affected cells carried multiple aberrations. An average of 2.7 aberrations per aberrant cell was observed 12 h after treatment of the mice with 2.5 mg/kg of cisplatin. In differentiating spermatogonia the dose response for aberrant cells could be described by a linear equation. The damage to the individual affected cell was less dramatic than in bone marrow, averaging 1.4 aberrations per damaged cell at the highest dose tested. Gaps were excluded from these considerations but they generally also showed a dose-related increase. A quantitative comparison of the clastogenic response to cisplatin was based on the dose-response relationships using 2 criteria, the doubling dose and the dose of unit increase (DUI). For both comparisons the general conclusion was that bone marrow cells were twice as sensitive as differentiating spermatogonia to the clastogenic action of cisplatin.     

The Production of Chromosome Aberrations in Various Mammalian Cells by Triethylenemelamine

The cytogenetic effects of triethylenemelamine (TEM) were studied using five different mammalian tissues. Treatments of 0.1 and 0.2 mg/kg TEM on differentiating mouse spermatogonia and bone marrow cells showed no significant differences in the frequency of chromosomal aberrations produced in these two tissues. At higher doses, however, the sensitivites of the two tissues appear to be different. The frequency of aberrations varies with time after treatment, with the greatest amount occurring at the latter fixation times. Results of an experiment on primary spermatocytes indicated a correlation between the frequency of chromosome aberrations and DNA replication. Human peripheral leukocytes were utilized in an attempt to clarify the cell-stage specificity of TEM-induced chromosome aberrations. Cultures were treated with TEM prior to PHA stimulation (G0), as well as various time intervals after stimulation (late G,1 S, and G2). The most sensitive stages of the cell cycle to aberration induction were later G1 and S, with chromatid aberrations the predominant type. A very low yield of chromosome damage was observed with the G0 and G1 treated stages. The experiments described tend to support the view that TEM is most effective at inducing aberrations when an intervening round of DNA replication has occurred.     

Comparison of the levels of chromosome aberration and sister chromatid exchange induced by chemical mutagens in vitro

Dose dependencies of the induction of sister chromatid exchanges (SCEs) and chromosome aberrations were studied under in vivo exposure of mouse bone marrow cells to 5 alkylating agents. The efficacy of the induction of SCEs for all the substances was 20 to 60 times higher than that of the induction of chromosome aberrations. It was demonstrated that SCEs induced by chemical mutagens in vivo and in vitro are more sensitive tests than chromosome aberrations.     

小鼠体内脾脏,骨髓及精原细胞姐妹染色单体交换的比较研究

本文对几种化学诱变剂诱发小鼠体内脾脏、骨髓和精原细胞的SCE进行了比较研究,同时分析了几类常见化合物在小鼠脾脏细胞中诱发SCE的活力。结果显示诱变剂在脾脏细胞中诱发SCE比骨髓和精原细胞敏感。几类化合物都能显著地诱发小鼠脾脏SCE的增加,与对照相比差异显著(P<0.05)或极显著(P<0.01),说明利用小鼠脾脏细胞检测环境诱变物是相当灵敏的。     

A search for chromosome aberrations induced in mouse spermatogonia by chemical mutagens

Two established chemical mutagens—ethylmethanesulphonate (EMS) and triethylenemelamine (TEM)—were tested for the ability to induce chromosome aberrations in mouse spermatogonia. While not a single aberration was detected following the EMS treatment, a low frequency of translocations and fragments was found in the TEM groups. These findings are in agreement with the data obtained with the specific locus mutation test as applied to male mouse premeiotic germ cells but contrast with the effectiveness of these chemicals in breaking chromosomes in male mouse postmeiotic germ cells. A differential sensitivity of post- and premeiotic germ cells to any kind of genetic damage by these chemical mutagens is most likely to be the correct interpretation of all the data. However, it is also suggested that a high proportion of translocations induced in spermatogonia by chemical mutagens may not be detectable by present methods.     

Spontaneous and induced chromosome aberrations in the bone marrow cells of different strains of mice during aging

Sensitivity of bone marrow cell chromosomes to alkylating agent thiophosphamide and to gamma-irradiation has been studied in the course of ageing in 101/H, A/He, CBA, BALB/c and C57BL/6 mice. The effects of both the kinds of mutagenic treatment and of the genotype of the animals on the age-dependent changes in sensitivity of bone marrow cell chromosomes were found. Following gamma-irradiation under our experimental conditions, no variation in the output of chromosomal aberrations was observed between the strains studied. Following thiophosphamide treatment, aged mice of strains 101/H, A/He and CBA showed an increased chromosome instability as compared to young ones. In C57BL/6 mice the level of induced chromosome aberrations was found to be age-independent. Following thiophosphamide treatment, cells with multiple chromosome lesions were found in the bone marrow. The higher instability of aged animals in some strains was mainly due to a sharp increase in the number of such cells. In the intact mice of all the strains studied no age-dependent increase in the number of cells showing structural chromosome aberrations was observed, while accumulation of aneuploid cells varied with genotype.     

DNA-repair kinetics and the sensitivity of cells to X-ray-induced chromosome aberrations: a mouse myeloid leukemia cell line and normal mouse bone marrow cells

The frequency of X-ray-induced chromosome aberrations in G1 ML-1 mouse myeloid leukemia cells and normal mouse bone marrow cells increased with post-irradiation incubation with the DNA-repair resynthesis inhibitor 1-beta-D-arabinofuranosylcytosine (araC), but the frequency of aberrations in the leukemic cells increased with quite a different time response compared to the normal cells. Irradiated normal mouse bone marrow cells had a rapid increase in the frequency of chromosome exchanges and deletions with increasing araC incubation time, for example, an increase was observed with 0.5 h araC incubation. In contrast, the ML-1 cells did not have a significant increase in aberrations until 1-2 h post-irradiation incubation with araC. These results suggest that the ML-1 cells, per unit time, initially undergo less repair of the X-ray-induced DNA damage that can be converted into chromosome aberrations. We previously showed that the ML-1 cells have a higher frequency of X-ray-induced chromosome aberrations compared to normal cells and the results presented here indicate that a slower rate of repair resynthesis is contributing to the increased sensitivity of the ML-1 cells.     

A comparative study on the genotoxic effect of pyrimethamine in bone marrow and spermatogonial mice cells

Pyrimethamine is an antimalarial agent widely used in clinical therapy. We aimed to compare its mutagenic potential in mammalian spermatogonial and bone marrow cells. For studying chromosomal aberrations mice were treated acutely (single treatment) with 4 dose levels of pyrimethamine (5, 10, 20 and 40 mg/kg). Pyrimethamine was found to produce a significant increase in structural chromosomal aberrations after acute treatment in bone marrow cells of mice (p < 0.001). It also induced chromosome abnormalities in spermatogonial cells (p < 0.05) at the highest dose.     

A correlative study of the genetic damage induced by chemical mutagens in bone marrow and spermatogonia of mice. III. 1,3-Bis (2-chloroethyl)-3-nitrosourea (BCNU)

Cytogenetic damage induced by various concentrations of BCNU was evaluated by determining the frequencies of (a) micronuclei in polychromatic erythrocytes of bone marrow, (b) chromatid aberrations in bone marrow, (c) chromatid aberrations in spermatogonia, and (d) reciprocal translocations induced in spermatogonia and scored in spermatocytes. The order of sensitivity for the four parameters tested was: micronuclei greater than chromatid aberrations in bone marrow greater than aberrations in spermatogonia greater than translocations in spermatocytes, a situation similar to that found in an earlier study with CNU-ethanol. When the effect of concentration of the chemical was taken into consideration there was no correlation among the four parameters tested, so that information on induced frequencies of one parameter does not have predictive value for the frequencies of the others. A comparison of the results obtained with the bifunctional BCNU and the mono-functional CNU-ethanol at equal concentrations indicated that BCNU had a similar or a lesser clastogenic effect than did CNU-ethanol. In an experiment in vitro the situation was different in that BCNU was more effective than CNU-ethanol.     

Radiation- and chemically-induced chromosome aberrations in mouse oocytes: a comparison with effects in males.

Data from studies on radiation- and chemically-induced chromosome aberrations in mouse oocytes have been summarized. An attempt has been made to assess the relative sensitivity to mutagenic agents of female and male germ cells through comparison of observations from mutation studies of female and male mice. No unequivocal evidence of a mutagenic effect limited to a single sex could be found in the cytogenetic data, although differences in relative germ cell sensitivity could be inferred for ionizing radiation and some chemicals. However, the pattern of inter-sex variations was not consistent: for example, irradiation of dictyate oocytes yielded a lower rate of heritable chromosome translocations than the same dose to spermatogonia; in contrast, some chemicals, such as mitomycin C, yielded a larger incidence of chromosome anomalies after treatment of dictyate oocytes than spermatogonia. Overall, the limitations in quality and quantity of cytogenetic data, and the uncertainties associated with comparing information obtained in disparate assays, place severe constraints on the use of observations on induced chromosome aberrations to assess the relative sensitivities of female and male germ cells to environmental mutagens.     

Mutagenic effect of human adenovirus type I on the somatic and sex cells of male mice

Human adenovirus 1 was studied for its effect on the chromosomal apparatus both in bone marrow cells and male sex cells of mice. Chromosome aberrations were most early detected in spermatocytes of the 1st order mice infected with human adenovirus 1. In bone marrow cells of mice the highest level of chromosome aberrations was observed 30, 60, 90 days after the inoculation, which corresponds to a more frequent detection of the adenoviral antigen. The UV-irradiated-virus caused chromosome aberrations in the later periods after the inoculation which might be induced by the virus reactivation in a cell.     

Induction of chromosomal aberrations in male mouse germ cells by uranyl fluoride containing enriched uranium

Cytogenetic damage induced by a wide range of concentrations of uranyl fluoride injected into mouse testes was evaluated by determining the frequencies of chromosomal aberrations in spermatogonia and primary spermatocytes. Breaks, gaps and polyploids were observed in spermatogonia. The frequencies of the significant type of aberration, breaks, were induced according to the injected doses of uranyl fluoride. Primary spermatocytes were examined for fragments, univalents and multivalents. The multivalents observed in this study resulted either from chromatid interchanges or from reciprocal translocations. The reciprocal translocations were induced in spermatogonia and recorded in primary spermatocytes. For primary spermatocytes the incidence of aberrant cells largely depended on the administered dose. Sampling time after treatment could affect the frequencies of chromosomal aberrations in male mouse germ cells.     

A 39,X/40,XY/41,XYY mosaic male mouse

Cytogenetic analyses of bone marrow and gonadal cells in a male mouse, which appeared to be normal, revealed mosaicism in both tissues. Three chromosome complements, 39,X, 40,XY, and 41,XYY, were found in both bone marrow and spermatogonia, while only the last two complements were found in spermatocytes. In this mouse, unlike in the human, the XYY cells showed a proliferative advantage over the XY cells. In XYY cells at diakinesis/metaphase I the gonosomes showed all possible types of association, and a pairing advantage of the X chromosome was clearly demonstrated. The fertility of the mouse was not determined. However, since the epididymal sperm count was reduced by only 55% and the incidence of sperm head abnormality was near normal, it is not evident that the mouse was sterile.