Etoposide and merbarone are clastogenic and aneugenic in the mouse bone marrow micronucleus test complemented by fluorescence in situ hybridization with the mouse minor satellite DNA probe.

The topoisomerase II (topo II) inhibitors etoposide (VP-16) and merbarone (MER) were investigated with the in vivo micronucleus test (MN test) combined with fluorescence in situ hybridization (FISH) using the mouse minor satellite DNA probe to discriminate MN of clastogenic and aneugenic origin. All experiments were performed with male (102/ElxC3H/El) F1 mice bred in the mouse colony of the GSF Research Center. The sample size per experimental group was five animals and 2,000 polychromatic erythrocytes (PCE) were scored per animal from coded slides in the conventional MN test. A separate set of coded slides was used for the FISH analysis. All treatments consisted of single intraperitoneal injections. Colchicine (COL, 3 mg/kg) and mitomycin (MMC, 1 mg/kg) were used as a positive control aneugen and clastogen, respectively, and these compounds produced the expected responses. A dose of 1 mg/kg VP-16 induced 3.44% MNPCE (compared to the concurrent solvent control of 0.37%, P < 0.001) and of these 39.9% (1.4% MNPCE) showed one or more fluorescent signals. MER (7.5-60 mg/kg) increased the MNPCE frequencies in a dose-dependent manner, with 15 mg/kg being the lowest positive dose. At the highest dose of 60 mg/kg of MER, a total of 4.26% MNPCE were found (compared to 0.31% in the concurrent solvent control, P < 0.001) and of these 46.2% (2.0% MNPCE) contained one or more fluorescent signals. The data demonstrate that VP-16 and MER induced both clastogenic and aneugenic events despite their different modes of topo II inhibition.

Recommendations for statistical designs of in vivo mutagenicity tests with regard to subsequent statistical analysis.

A workshop was held on September 13 and 14, 1993, at the GSF, Neuherberg, Germany, to start a discussion of experimental design and statistical analysis issues for three in vivo mutagenicity test systems, the micronucleus test in mouse bone marrow/peripheral blood, the chromosomal aberration tests in mouse bone marrow/differentiating spermatogonia, and the mouse dominant lethal test. The discussion has now come to conclusions which we would like to make generally known. Rather than dwell upon specific statistical tests which could be used for data analysis, serious consideration was given to test design. However, the test design, its power of detecting a given increase of adverse effects and the test statistics are interrelated. Detailed analyses of historical negative control data led to important recommendations for each test system. Concerning the statistical sensitivity parameters, a type I error of 0.05 (one tailed), a type II error of 0.20 and a dose related increase of twice the background (negative control) frequencies were generally adopted. It was recommended that sufficient observations (cells, implants) be planned for each analysis unit (animal) so that at least one adverse outcome (micronucleus, aberrant cell, dead implant) would likely be observed. The treated animal was the smallest unit of analysis allowed. On the basis of these general consideration the sample size was determined for each of the three assays. A minimum of 2000 immature erythrocytes/animal should be scored for micronuclei from each of at least 4 animals in each comparison group in the micronucleus assays. A minimum of 200 cells should be scored for chromosomal aberrations from each of at least 5 animals in each comparison group in the aberration assays. In the dominant lethal test, a minimum of 400 implants (40-50 pregnant females) are required per dose group for each mating period. The analysis unit for the dominant lethal test would be the treated male unless the background frequency of dead implants (DI) is so low that multiple males would need to be integrated to meet the minimum observation of one adverse outcome (DI) per analysis unit. A three-step strategy of data analysis was proposed for the cytogenetic assays. Use of negative historical controls was allowed in certain circumstances for interpretation of results from micronucleus tests and chromosomal aberration tests.

Dose response study for 1,3-butadiene-induced dominant lethal mutations and heritable translocations in germs cells of male mice.

Butadiene (BD) and its metabolites have extensively been studied in the EU sponsored research project "Multi-endpoint analysis of genetic damage induced by 1,3-butadiene and its major metabolites". Within this project a dominant lethal test and a heritable translocation test were performed with male mice to study the dose-response relationships for the respective endpoints. BD concentrations of 130 and 500 ppm were tested in the dominant lethal assay by exposing male mice on 6 h/day for five consecutive days resulting in doses of 3900 and 15,000 ppmh, respectively. Males were mated for four consecutive weeks at a ratio of 1:2 to untreated females. A positive dominant lethal effect was observed in the first mating week in the experiment with 15,000 ppmh but no dominant lethality was found with the lower dose of 3900 ppmh. The present dominant lethal data have to be viewed together with the data already published for a BD dose of 39,000 ppmh (1300 ppm at 6 h/day on 5 consecutive days) [1]. The main difference between results with the highest and the middle dose is that mating weeks one and two (sperm and late spermatids) showed an effect at 39,000 ppmh while only mating week one (sperm) showed an effect at 15,000 ppmh. In the heritable translocation assay, males mice were exposed with a BD dose of 15,000 ppmh and mated for one week to untreated females. Among 434 F1 offspring, we found 5 translocation carriers (1.15% vs. 0.05% in the historical control, p < 0.001). In the previous heritable translocation experiment with a BD dose of 39,000 ppmh of DB exposure, 2.7% of the offspring carried a reciprocal translocation [2]. These data can be used for quantification of genetic risk. The dose response for BD-induced heritable translocations in sperm and late spermatids of mice was linear (Y = 0.05 + 6.9 x 10(-5)X) and a doubling dose of 725 ppmh could be calculated.

Extruded micronuclei induced by colchicine or acrylamide contain mostly lagging chromosomes identified in paintbrush smears by minor and major mouse DNA probes.

In the mouse bone marrow micronucleus assay, it was studied whether micronuclei (MN) could be expelled from polychromatic erythrocytes (PCE) in a similar way to the main nucleus. To avoid the disrupting centrifugation step of the conventional bone marrow preparation procedure, the paintbrush technique was used in the present experiments. With May-Grunwald-Giemsa staining of paintbrush slides, 5 % of the colchicine (COL)-induced MN were found attached to the outside membranes of PCE and were regarded as extruded. Of the acrylamide (AA)-induced MN, 22% were extruded. After fluorescence in situ hybridization (FISH) of a total of 300 MN per chemical treatment with the mouse minor and major satellite DNA probes, 9.7% MN were extruded in the COL group and 8.3% MN were extruded in the AA group. FISH showed that 76% of the retained COL-induced MN were signal-positive, indicating that they contained entire chromosomes. With AA, 29% minor-positive and 28.3 % major-positive retained MN were found, confirming its known clastogenicity. However, the observed frequency of signal-positive MN (1.7 MNPCE(pos)/ 1000 PCE) in the AA group was about three times higher than in the control (0.5 MNPCE(pos)/1000 PCE) which indicates that AA has aneugenic potential. FISH analysis of the extruded MN showed 72-100% major as well as minor signals. It is concluded that expelled MN contain mostly entire chromosomes.

Spontaneous rates of sex chromosomal aneuploidies in sperm and offspring of mice: a validation of the detection of aneuploid sperm by fluorescence in situ hybridization.

This study was designed to evaluate the frequency of aneuploid sperm in young adult mice of the genotype (102/E1 x C3H/E1)F1 determined by the fluorescence in situ hybridization (FISH) procedure and to evaluate the frequencies of aneuploid sperm observed by FISH compared with the frequencies of aneuploid offspring. Three-chromosome FISH was applied to determine the fractions of hyperhaploid and diploid sperm with DNA probes specific for chromosomes X, Y and 8. The animals were treated with three common solvents. Sperm smears were prepared for FISH by two similar protocols and were scored by different persons and in two different laboratories. There were no significant differences between scorers or laboratories. The frequencies of the sex chromosome aneuploidies in sperm (Y-Y and X-Y) were compared to the frequencies of mice carrying sex chromosome aneuploidy among controls of the heritable translocation assay in studies conducted from 1975-1995. To identify aneuploid individuals, untreated males and females of the genotype (102/E1 x C3H/E1)F1 were mated to assess their fertility by observing three consecutive litters. Semisterile and sterile animals were further analysed by meiotic cytogenetics and by karyotyping to determine the incidence of reciprocal translocations and sex chromosome aneuploidies (XXY and XYY). Based on the analysis of 175247 sperm and 9840 progeny, the frequency of Y-Y sperm was 0.01% while 0.03% of the offspring were XYY. The frequency of X-Y sperm was 0.005% while 0.02% of the offspring were XXY. The frequencies of aneuploid sex chromosomes were not significantly different between sperm and offspring. This allows two conclusions. First, there was no detectable prenatal selection against these sex-chromosomal aneuploid offspring, and second, germ cell aneuploidy can be reliably determined in mice by sperm FISH analyses.

Analysis of micronuclei induced by 1,3-butadiene and its metabolites using fluorescence in situ hybridization.

In our previous study, micronuclei (MN) were induced in bone marrow cells of mice following inhalation exposure to 1300 ppm of 1,3-butadiene (BD) for 6 h per day on 5 consecutive days, and in splenocytes of mice and rats treated intraperitoneally with 80 mg/kg 1,2-epoxybutene (EB) and 30 mg/kg 1,2,3,4-diepoxybutane (DEB), respectively. In the present study, the nature of MN induced by BD, EB and DEB was analyzed by means of fluorescence in situ hybridization (FISH) using mouse minor satellite DNA and rat satellite I DNA as probes. Percentages of MN with centromere signals (MN+) measured following exposures to BD, EB and DEB indicate that these agents are predominantly clastogens. Frequencies of MN+ per 1000 cells suggest that BD, EB and DEB are not only strong clastogens, but also weak aneugens in mice. The weak aneugenic effect of EB and DEB was not observed in rats. Analysis of the number of centromere signals in individual MN, and the size distribution of MN with centromere signals in EB- and DEB-treated animals, and in animals exposed to the positive controls diethylstilbestrol (DES) and mitomycin C (MMC) led to the following conclusions: (1) analysis of MN for the number of centromere signals may be a useful indicator for identifying chemicals with aneugenic properties; (2) there is no correlation between the size of MN and their origin (i.e., chromosome loss/gain or fragment).

Derivation and characterization of a somatic cell hybrid containing the portion of mouse chromosome 11 (MMU11) homologous to human chromosome 17q.

To contribute to the physical gene map of mouse chromosome 11 (MMU11) and to extend the mapping resources available for this chromosome, we have produced mouse x rat somatic cell hybrids containing only bands B5 to E of MMU11. Characterization of the hybrids by polymerase chain reaction (PCR) amplification and Southern blot analyses of MMU11 markers revealed two hybrids, T16Ad14B and T16Ad19A, that had selectively retained the 3(11) translocation product containing distal MMU11 (bands B5-E). Cytogenetic analysis of the hybrid T16Ad14B by fluorescence in situ hybridization (FISH) and conventional G-banding confirmed the presence of the 3(11) translocation chromosome. Mapping of markers in both the T16Ad14B and T16Ad19A hybrids localized the T16Ad translocation breakpoint between the proximal markers Atplb2 and Acrb and the more distal markers Scya2 and Mpo. Loci for D11Mit5, Rpo2-1, Trp53, Glut4, Acrb, and Atplb2 could all be localized proximal to the T16Ad breakpoint in band B5, between bands B1 and B5 on MMU11.

Heritable translocations induced by inhalation exposure of male mice to 1,3-butadiene.

Previously, we reported that dominant lethal mutations were induced in spermatids after inhalation exposure of male (102/El x C3H/El)F1 mice to 1300 ppm of 1,3-butadiene on 5 days for 6 h per day (exposure dose 39,000 ppm h). The same inhalation exposure was given to male C3H/El inbred mice which were mated to inbred line 102/El females 8-14 d after the end of exposure. Male and female F1 hybrid progeny were tested for the presence of heritable translocations by observation of litter sizes and by cytogenetic analyses in meiotic and somatic cells. 1,3-Butadiene induced heritable translocations in late spermatids. The translocation frequency after 1,3-butadiene exposure to 39,000 ppm h was 2.7% (16 translocation heterozygotes among 559 F1 offspring). This frequency is 54 times higher than the historical control frequency (0.05%; 5 translocation heterozygotes among 9500 F1 offspring). Thus, 1,3-butadiene causes heritable germ cell effects in mice.

Dose response for heritable translocations induced by acrylamide in spermatids of mice.

A heritable translocation test was carried out with acrylamide (AA) to obtain a dose-response relationship for induction of reciprocal translocations in late spermatids of the mouse. Male C3H/E1 mice were treated with single i.p. doses of 50 and 100 mg/kg of acrylamide and mated 7-16 days after treatment to untreated female 102/E1 mice. Translocation carriers among the F1 progeny were selected by a sequential procedure of fertility testing and cytogenetic analysis including G-band karyotyping to determine the chromosomes involved in the respective translocations. The translocation frequencies observed with 50 mg/kg and 100 mg/kg of AA were 0.6% and 2.7%, respectively. The historical control translocation frequency was 0.04%. Doubling dose estimates based on these and previous data are discussed.

Differentiation of micronuclei in mouse bone marrow cells: a comparison between CREST staining and fluorescent in situ hybridization with centromeric and telomeric DNA probes.

Immunofluorescent staining (CREST) of kinetochore proteins and in situ hybridization (FISH) with centromeric DNA probes are able to distinguish between micronuclei (MN) containing lagging chromosomes or acentric fragments. Different frequencies of signal-positive MN induced by mitomycin C (MMC) were obtained by Miller et al. (Mutagenesis, 6, 297-302, 1991) between CREST labelling and FISH with the mouse major-gamma-satellite DNA probe (major probe). Both modes of identifying the presence of an entire chromosome in a MN are theoretically prone to misclassification. Breaks induced in pericentric heterochromatin can produce fragment-containing MN with a major signal. Alternatively, alterations of kinetochore proteins can produce CREST-negative MN containing lagging chromosomes. To improve the reliability of MN differentiation two additional DNA probes, the mouse minor satellite DNA probe (minor probe) and the telomere repeat (5'-TTAGGG-3')7, have been used and double labelling has been employed with minor/major and minor/telomere probes. At 1 mg/kg of MMC the labelling frequencies of MN with CREST and the minor probe were identical (18.5 and 19%, respectively) and the major probe showed a higher labelling rate (30.5%). Using double-labelling the difference between minor and major probe responses was confirmed (17 and 31.5%, respectively). At 5 mg/kg of MMC, CREST labelling gave the lowest (6%), the minor probe gave intermediate (10 and 11.5% after single- and double-labelling, respectively) and the major probe gave the highest signal frequencies (16.5 and 15% after single- and double-labelling, respectively). The CREST and the minor signal frequencies were not significantly different at either dose of MMC whereas the minor and the major signal frequencies were significantly different.(ABSTRACT TRUNCATED AT 250 WORDS)

Clastogenicity of trophosphamide in somatic and germinal cells of mice.

Trophosphamide, a chemotherapeutic agent structurally related to cyclophosphamide, was tested in the micronucleus and heritable translocation assays in mice. It induced a linear increase of micronuclei in polychromatic erythrocytes 24 h after treatment with 1, 5, 25 or 50 mg/kg. In spermatids and spermatozoa of mice heritable translocations were induced by 150 mg/kg with an average frequency of 6%. The doubling doses calculated for micronucleus induction and heritable translocation induction were 5.0 and 1.3 mg/kg, respectively. These values are in the same order of magnitude and suggest that somatic and germinal cells are similarly sensitive to the clastogenic action of trophosphamide.

A mouse stock with 38 chromosomes derived from the reciprocal translocation T(7;15)33Ad.

A reciprocal translocation, T(7;15)33Ad, with presumed breakpoints in bands 7A1 and 15F3 was induced in late spermatids by injecting male (102/E1 x C3H/E1)F1 mice five times with acrylamide (50 mg/kg body weight). Outcrosses of the original semisterile T(7;15) female generated three males monosomic for the short marker 7(15) [Ms(7(15))] among a total of 15 males. The Ms(7(15)) males sired small litters and had reduced testes weights. From inter se matings of Ms(7(15)) animals, nullisomic progeny for chromosome 7(15) were obtained and mated to produce a breeding stock of mice with 38 chromosomes. For comparison, mice carrying the reciprocal translocation T(4;8), with similarly located breakpoints, were also analyzed. Fluorescent in situ hybridization (FISH) with major and minor satellite DNA probes and a telomeric DNA probe was utilized. The observed FISH signals suggest that in chromosomes 7 and 8 the breaks occurred within the pericentric heterochromatic block immediately below the centromere and in chromosomes 15 and 4 at a point near the distal telomeres. The long markers 15(7)and 4(8) are tandem fusion chromosomes. The short markers 7(15) and 8(4) also showed all appropriate FISH signals for intact chromosomes. The loss of the small chromosome 7(15) was compatible with survival, suggesting that no essential genes are located on the small reciprocal translocation product. The development of this tandem fusion stock is described as a laboratory example of one possible step in karyotypic evolution.

Telomeric signals in robertsonian fusion and fission chromosomes: implications for the origin of pseudoaneuploidy.

In situ hybridization with synthetic plant telomeric sequences resulted in labeling of all broad bean (Vicia faba) chromosomes at their ends only. Telocentric chromosomes derived by fission of the metacentric satellite chromosome of V. faba also showed signals at both of their ends, whereas the ancestral metacentric did not display signals at its primary constriction, the point of fission. As in V. faba, all acrocentric mouse chromosomes were labeled by in situ hybridization with a vertebrate telomeric probe at both ends of each chromatid exclusively. However, different metacentric Robertsonian chromosomes derived by fusion of defined acrocentrics did not show signals at their primary constrictions. The mechanism of Robertsonian rearrangement leading to a pseudoaneuploid increase or decrease in chromosome number therefore cannot consist solely of a simple fission or fusion of chromosomes without a concomitant gain or loss of chromatin material. The additional assumption of a subdetectable deletion of telomeric sequences after fusion and amplification of these sequences following fission is necessary to explain the present observations.