A sequential screening of the cytogenetic damage induced by triaziquone.

A sequence is described of test procedures for a screening in vivo of the clastogenic potential of the alkylating agent triaziquone (Trenimon). Two intraperitoneal injections of 0.125 mg/kg body weight caused a considerable increase in the number of aberrations in both the micronucleus test and the bone-marrow metaphase test, but not in the spermatocyte translocation test or the spermatogonial metaphase test. With the latter test a severe cell-killing effect was detected. An analysis of whole mounts of seminiferous tubules showed that 0.125 mg/kg was a lethal dose for all B- and intermediate-type spermatogonia and partly killed A-type spermatogonia. A single administration of the same dose caused stable chromosomal rearrangements in spermatids that could be demonstrated with the F1 translocation test, and gave rise to dominant lethality of fetuses originating from post-meiotic sperm. The comparative triaziquone study has provided arguments in favor of the micronucleus test as a reliable screening method for chromosomal aberrations. The analysis of seminiferous tubules is a recommendable method for studying lethal effects of a compound on germ cells, whereas the F1 translocation test gives important information about viable aberrations and their effect on the fertility of the progeny.     

Mechanism of induction of micronuclei and chromosome aberrations in mouse bone marrow by multiple treatments of methotrexate.

Methotrexate (MTX), an inhibitor of dihydrofolate reductase (DHFR), slightly induced micronuclei and this induction of micronuclei was enhanced by multiple treatments with the drug (Yamamoto et al., 1981; Hayashi et al., 1984; CSGMT/JEM.MMS, 1990). More micronuclei and chromosomal aberrations in mouse bone marrow cells were induced by multiple than by single treatment. The MTX level in mouse plasma and bone marrow showed little (or no) differences between single and quadruple treatments several hours after the injection(s). On the other hand, the DHFR activity in bone marrow cells 3 h after one and four injections was decreased to approximately 38 and 0%, respectively, of that in non-treated mice. Furthermore, the intracellular MTX level in the bone marrow cells (but not in total bone marrow) after four injections was about 10-fold higher than that after one injection. The amount of MTX bound to protein 3 h after four injections, as assayed by gel filtration (Sephadex G-25), was approximately 8-fold greater than after one injection. Therefore, the multiple-dose effects of MTX on the induction of micronuclei and chromosomal aberrations may be explained by the intracellular accumulation of MTX resulting in an enhancement of enzyme inhibition.     

1-Methyl-2-pyrrolidinone (NMP) does not induce structural and numerical chromosomal aberrations in vivo

1-Methyl-2-pyrrolidinone induces aneuploidy in yeast, but only under special treatment conditions. Other genotoxic effects have not been found in vitro, and in vivo no data are available in the literature. Therefore, NMP was investigated in the mouse micronucleus test and the Chinese hamster bone marrow test for structural and numerical chromosomal aberrations. These tests can detect both types of alterations as demonstrated by appropriate positive control substances (cyclophosphamide, vincristine sulfate and benomyl). NMP at single oral doses up to 3800 mg/kg body weight (∼ 80% of the LD₅₀) did not lead to an increase either in micronucleated erythrocytes or in structural or numerical chromosomal aberrations when bone marrow was sampled 16, 24 and 48 h after treatment in the micronucleus test or after 24 and 48 h for karyotype analysis.     

三种有丝分裂抑制剂诱发小鼠骨髓细胞非整倍体的比较研究

以秋水仙素有丝分裂(CM)效应、微核(MN)及染色体畸变(CA)三种体内细胞遗传学指标综合评估了有丝分裂抑制剂(秋水仙素、益康唑及对苯二酚)诱发小鼠骨髓细胞非整倍体的效应。结果表明:秋水仙素是典型的多倍体及非整倍体诱发剂。益康唑对细胞有丝分裂有与秋水仙素相类似的效应,进一步分析表明其在哺乳动物体细胞内无非整倍体诱发效应。对苯二酚在哺乳动物活体实验系统中,可能具有诱发非整倍体及染色体结构畸变的多种遗传毒性。结果提示三种细胞遗传学指标能为非整倍体诱发剂的检出提供依据。     

Lack of clastogenic effects of irradiated glucose in somatic and germ cells of mice

The clastogenic ability of irradiated glucose was evaluated using the micronucleus test in bone marrow cells and the chromosomal aberration assay in the spermatogonial cells of mice. Glucose was irradiated with 0.2, 2.0, 20 and 50 kGy of gamma-rays and administered to mice for various periods. No evidence of mutagenic effects was observed in somatic or germ cells.     

Mutagenicity of polycyclic hydrocarbons III. Monitoring genetic hazards of benz(a)anthracene

Mutagenicity tests (micronucleus test and chromosome aberrations) have been performed with benz (a) anthracene in spermatogonia and bond marrow cells of Chinese hamsters and in NMRI mice oocytes. Mutagenic effects of the polycyclic hydrocarbon could be demonstrated.     

Technical aspects of automatic micronucleus analysis in rodent bone marrow assays

The mouse bone marrow micronucleus assay is anin vivo test commonly used in the pharmaceutical industry to evaluate the genotoxic potential of new compounds. The test detects agent-induced chromosomal damage or damage of the mitotic spindle apparatus. In this paper the state-of-the-art in automated rodent micronucleus evaluation using computerized image analyis in combination with high-quality slides obtained by the cellulose column fractionation technique is reviewed. The latter allows the effective removal of nucleated cells from rodent bone marrow. It has been found that automatic micronucleus scoring with the Leitz MIAC image analyzer is substantially faster than labor-intensive manual analysis. Automatic scoring can be performed overnight for up to 16 slides. We have been successfully using automatic micronucleus analysis for the testing of new pharmaceutical drugs for more than 3 years.Abbreviations MNE NCE containing micronuclei - MPE PCE containing micronuclei - NCE normochromatic erythrocyte - PCE polychromatic erythrocyte deceased on 25 May 1994     

FSH-initiated differentiation of newt spermatogonia to primary spermatocytes in germ-somatic cell reaggregates cultured within a collagen matrix

We previously cultured fragments of newt testes in chemically defined media and showed that mammalian follicle-stimulating hormone (FSH) stimulates proliferation of spermatogonia as well as their differentiation into primary spermatocytes (Ji et al., 1992; Abe and Ji, 1994). Next, we indicated in cultures composed of spermatogonia and somatic cells (mainly Sertoli cells) that FSH stimulates germ cell proliferation via Sertoli cells (Maekawa et al., 1995). However, the spermatogonia did not differentiate into primary spermatocytes, but instead died. In the present study, we embedded large reaggregates of spermatogonia and somatic cells (mainly Sertoli cells) within a collagen matrix and cultured the reaggregates on a filter that floated on chemically defined media containing FSH; in this revised culture system, spermatogonia proliferated and differentiated into primary spermatocytes. The viability and percentage of germ cells differentiating into primary spermatocytes were proportional to the percentage of somatic cells in the culture, indicating that differentiation of spermatogonia into primary spermatocytes is mediated by Sertoli 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.     

Nicotine is not clastogenic at doses of 1 or 2 mg/kg body weight given orally to male mice

Nicotine has been tested in the conventional mouse bone marrow assay. Single doses of 1mg/kg bw or 2mg/kg bw were given by oral intubations and bone marrow was sampled at 24h (1mg/kg) or at 6, 12 and 18 h after treatment (2mg/kg). Nicotine treatment did not increase the micronucleus frequencies in polychromatic erythrocytes while the positive control compound mitomycin C yielded the expected result. These data contradict the only published in vivo study of nicotine in which 1.1mg/kg bw was called positive for the induction of chromosomal aberrations in mouse bone marrow cells at all sampling intervals, even as early as 6h after treatment. It is discussed that aberration scoring is a matter of subjectivity and depends on strict discrimination criteria between gaps and true DNA discontinuities, i.e. breaks. International collaboration has shown that micronucleus scoring is less subjective, hence more reliable. Therefore it is concluded that nicotine is not clastogenic at the doses and time intervals tested in the present experiments.     

Kinetics of micronucleus formation in relation to chromosomal aberrations in mouse bone marrow

A simulation analysis of the kinetics of micronucleus formation in polychromatic erythrocytes in mouse bone marrow was performed after a single administration of 3 chemicals--mitomycin C (MMC), 6-mercaptopurine (6-MP) and 1-beta-D-arabinofuranosylcytosine (Ara-C)--with different modes of action. The time-response patterns in the incidence of chromosomal aberrations and micronuclei after treatment with each chemical were compared and subjected to the simulation study with 3 parameters. Two of them, the time between the final mitotic metaphase of the erythroid series and nucleus expulsion (T1), and the duration of the polychromatic erythrocyte (PCE) stage in the bone marrow (T2), were almost identical for the 3 chemicals. However, the coefficients of formation rate of micronucleated cells resulting from cells with chromosomal aberration(s) (k) differed: Ara-C differed from the other two. These results indicate that chromosomal aberrations, especially chromatid breaks and probably gaps, induced by this chemical, effectively contribute to micronucleus formation. The DNA content of micronuclei was also compared to the length of acentric fragments induced by Ara-C and it was found that their distributions were comparable. These findings strongly suggest that chromosomal aberrations induced by chemicals are essential events for the induction of micronuclei in the PCE of bone marrow.     

Genotoxicity of two anticancer drugs,gemcitabine and topotecan,in mouse bone marrow in vivo

In this study, the genotoxic effects of gemcitabine and topotecan were investigated in mouse bone marrow cells using the micronucleus and chromosomal aberration test systems. Gemcitabine increased the frequency of micronuclei, particularly at the median dose for the 24-, 36-, and 48-h sampling intervals. It had cytotoxic effects on the bone marrow and decreased the polychromatic/normochromatic erythrocyte ratio dose-dependently for all sampling intervals. Gemcitabine significantly decreased the mitotic index at the 24-h time point. It increased the number of abnormal cells and induced a significant increase in total chromosomal aberrations. For the 6-h sampling time, gemcitabine neither induced chromosomal aberrations nor reduced the mitotic index. Topotecan also induced high levels of micronuclei, particularly for the 24- and 36-h sampling times and it decreased the polychromatic/normochromatic erythrocyte ratio for all sampling intervals, which is indicative of bone marrow cytotoxicity. The bone marrow metaphase analysis showed that topotecan significantly elevated the number of abnormal metaphases and total chromosomal aberrations at 6 and 24h, in a dose-dependent manner. It also decreased the mitotic index for both sampling intervals. In conclusion, the results of this study indicate that the two chemotherapeutics gemcitabine and topotecan have cytotoxic and genotoxic effects in mouse bone marrow.     

Interrelation of the dynamics of antigen intake into bone marrow and the proliferation of hematopoietic stem cells

As it is previously shown (Tsyrlova et al., 1986), the level of humoral immune response is not only determined by the reaction of peripheral lymphoid system on antigenic effect, but also is bound up with the observed stem blood cell (SBC) proliferation in bone marrow (Frindel et al., 1976, Kozlov et al., 1982). Dynamics of label accumulation in bone marrow was examined when injecting antigen--sheep red blood cells labeled by radioisotope 51Cr, 125I. The peak of label accumulation in bone marrow, accompanied by the increase in proliferative SBC, was observed on the 3rd day after antigen injection. Furthermore in the course of immunization 51Cr labeled macrophage assortment was changed in time in such a way that a greater number of macrophages was accumulated in bone marrow on the 3rd and 4th days in the immunized animals in comparison with the intact ones. The macrophages in bone marrow are likely to take part in antigen uptake and to mediate its effect on SBC proliferation.     

Action of mitomycin C on mouse spermatogonia

The induction of chromosome aberrations in mouse spermatogonia and bone marrow cells by treatment with Mitomycin (MC) was tested. The following dosages were used: 3.5; 1.75; 0.35; and 0.035 mg/kg body weight. Chromatid interchanges and terminal deletions were induced in both tissues. Regarding the chromosome damage, spermatogonia seemed to be more sensitive to the test substance than bone marrow cells.The aberrations observed were considered to represent the cause of dominant lethals induced in spermatocytes after treatment with MC by others. The squash technique adapted for examination of mitoses of mouse spermatogonia proved to be a useful tool in testing potential chemical mutagens.     

Nicotine is not clastogenic at doses of 1 or 2 mg/kg body weight given orally to male mice

Nicotne has been tested in the conventional mouse bone marrow assay. Single doses of 1 mg/kg bw or 2 mg/kg bw were given by oral intubations and bone marrow was sampled at 24 h (1 mg/kg) or at 6, 12 and 18 h after treatment (2 mg/kg). Nicotine treatment did not increase the micronucleus frequencies in polychromatic erythrocytes while the positive control compound mitomycin C yielded the expected result. These data contradict the only published in vivo study of nicotine in which 1.1 mg/kg bw was called positive for the induction of chromosomal aberrations in mouse bone marrow cells at all sampling intervals, even as early as 6 h after treatment. It is discussed that aberration scoring is a matter of subjectivity and depends on strict discrimination criteria between gaps and true DNA discontinuities, i.e. breaks. International collaboration has shown that micronucleus scoring is less subjective, hence more reliable. Therefore it is concluded that nocotine is not clastogenic at the doses and time intervals tested in the present experiments.     

Serial analysis of chromosomal aberrations and proliferation kinetics of mouse spermatogonia after single or fractionated X-ray exposures

Dose-fractionation studies on translocation induction in stem-cell spermatogonia of mice, as measured by spermatocyte analysis many cell generations after irradiation, revealed that a small conditioning dose of X-rays sensitizes the stem cells to the induction of translocations by a second dose 24 h later (Van Buul and Léonard, 1974, 1980). To find out whether such sensitization effects also occur at other spermatogonial stages, a comparison was made of the effects of single (50, 100 and 150 rad) and fractionated (100 + 50 rad, with 24 h in between) doses of X-rays on the induction of chromosomal aberrations in spermatogonia by analysing spermatogonial metaphases shortly after irradiation at multiple sampling times (0–48 h; every 4 h). In addition, the kinetics of spermatogonial proliferation was studied by using, in vivo, a BrdU chromosome-labelling procedure. The recorded frequencies of chromosomal aberrations did not indicate any sensitization effect of dose fractionation. It is concluded that the sensitization effects, as observed for chromosomal aberrations in male premeiotic germ cells, are characteristic for the stem-cell spermatogonia and do not occur in the more differentiated spermatogonia.     

Genetic toxicology of 1,2-dibromo-3-chloropropane (DBCP)

1,2-Dibromo-3-chloropropane (DBCP) is a nematocide, which has been used extensively as a soil fumigant in agriculture. Since sterility was found among male workers involved in the manufacture of DBCP, great concern has been focused on the genetic hazards of DBCP. DBCP gave positive results in many tests such as microbial, in vitro cytogenetics, and Drosophila studies. In mammalian test systems, DBCP caused chromosomal aberrations in the bone marrow cells and dominant-lethal mutations in germ cells in rats. In mice, there were no signs of DBCP-induced heritable mutation in germ cells, although point mutations were detected in somatic cells. The occurrence of Y-chromosomal non-disjunction was indicated in DBCP-exposed male workers by an increased number of sperm containing 2 Y-chromosomes.     

Mutagenic and cytotoxic effects of acrylates

Increasing amount of fructose-1-phosphate aldolase activity in rat blood serum and induction of chromosomal aberrations in rat bone marrow cells were observed after injection of acrylamide. The cytotoxic activity of acrylamide in rat depended on the dose. Methylmethacrylate and butyl methacrylate demonstrated no such activity.     

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.     

Absence of correlation between the chromosomal radiosensitivity of peripheral blood lymphocytes and stem-cell spermatogonia in mammals

To evaluate the reliability of quantitative extrapolation of radiation-induced chromosomal damage from somatic cells to germ cells, data on the effects of several biological and physical factors on the chromosomal radiosensitivity of blood lymphocytes and stem-cell spermatogonia have been collected from the literature. The results show that most of the factors considered, such as chromosomal constitution, age, genetic constitution, species, sampling time and dose fractionation, had differential effects on the induction of chromosomal aberrations in both systems. These differential effects can easily be explained in terms of the biological differences between in-vitro-stimulated peripheral blood lymphocytes and stem-cell spermatogonia. It is concluded that only direct experiments on germ cells of higher primates and man can be used for a quantitative estimation of human genetic radiation risks arising from structural chromosomal aberrations.