Comparison of the duration of spermatogenesis between male rodents and humans.
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Animals↗
Biomedical subjects
I D Adler
Publications and source records attributed to I D Adler.
Detection of aneuploidy in human and rodent sperm using FISH and applications of sperm assays of genetic damage in heritable risk evaluation.
Efficient molecular methods are being developed for detecting various types of cytogenetic genetic damage in sperm, especially numerical aneuploidy for chromosomes involved in trisomies that survive at birth. These methods provide new approaches for identifying potentially detrimental environmental exposures, genetic predisposition, chromosomal rearrangements, and physiologic factors which may increase a man's risk of fathering a genetically defective offspring. Corollary methods are also being developed for detecting sperm aneuploidy in laboratory rodents and these will be used to make inter-species comparisons of mutagen sensitivities and for investigating mechanisms of induction and persistence of aneuploidy. Validated assays for detecting genetic alterations in human and rodent sperm (of which sperm aneuploidy is a first example) permit comparisons of somatic and germinal response to mutagens within individuals, comparisons of human and rodent germinal sensitivity to mutagens, and can be applied in an extended parallelogram model to sperm for assessing heritable risk resulting from paternal mutagen exposures.
Aneuploidy↗
Induction of hypoploidy and cell cycle delay by acrylamide in somatic and germinal cells of male mice.
Monomeric acrylamide was tested for its potential to induce aneuploidy in spermatocytes and bone marrow cells of mice. For this purpose, chromosomes from metaphase spreads were counted semi-automatically. In both test systems, cell proliferation was monitored, determining the meiotic index of spermatocytes and the average generation time of bone marrow cells after BrdU incorporation, respectively. No indications could be seen for different sensitivity of somatic and germinal cells towards acrylamide. With a dose of 120 mg/kg, the chemical caused cell cycle delay in both germ line and somatic cells. There was diverging response with respect to the balance of hypo- and hyperploidy. While the percentage of chromosome loss was significantly elevated in both test systems, acrylamide treatment did not increase the frequency of hyperploid cells. Interpreting these results on the basis of conventional test protocols, acrylamide should not be considered as an aneugen. The conservative approach, however, may be inadequate for the detection of aneugenic mechanisms different from non-disjunction.
Acrylamide↗
Activity of iPMS and nPMS in mouse bone marrow micronucleus assays: comparison with mouse dominant lethal assay data.
isoPropyl methanesulphonate (iPMS) and its n-propyl analogue (nPMS) are shown to be active in mouse bone marrow micronucleus assays using male CBA, male and female (C3H/El X 102/E1)Fl and male and female Muta Mouse mice. iPMS was significantly more active than nPMS. No significant strain or gender differences were observed. These findings reflect the differences reported earlier for these two chemicals in mouse dominant lethal mutation assays. The earlier described dominant lethal assay data are represented schematically and discussed.
Alkylating Agents↗
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.
Aneuploidy↗
Future research directions to study genetic damage in germ cells and estimate genetic risk.
The late Frits Sobels developed a parallelogram model to estimate genetic risk to humans based on experimental data in somatic cells (peripheral blood) of exposed animals and humans and on data from progeny studies of exposed animals (mice). Recently, an extension to the original parallelogram model was proposed to bridge the gap of extrapolation between rodent and human germ cells by studying sperm samples. The comparison in the parallelogram of rodent/human sperm data with data from rodent progeny tests to derive at an estimate of human progeny at risk is more promising. Therefore, data on all possible end points, DNA adducts, mutations, chromosomal aberrations, and aneuploidy, should be obtained in sperm of exposed rodents and humans. The technology from somatic cell studies is available or adaptable to sperm studies. Sperm samples lend themselves to automated analyses because they are a homogeneous cell population. By flow cytometry or image analysis, large cell samples can be studied per individual. Animal experiments could be conducted in the actual range of chronic human exposure to low doses. The acceptability of extrapolation from the high acute doses so far used in animal experiments to low chronic doses of human exposure could be assessed. Proof could be obtained in human germ cells for the assumption that data from animal experiments can be extrapolated to humans. Data from transgenic rodent systems may play an important role in the extension of the parallelogram approach to genetic risk estimation by providing a link between cancer and genetic risk estimates.
Aneuploidy↗
Inhalation exposure of rats and mice to 1,3-butadiene induces N6-adenine adducts of epoxybutene detected by 32P-postlabeling and HPLC.
In this paper we report DNA binding of butadiene monoepoxide, a first metabolite of 1,3-butadiene catalyzed by monooxygenases. We prepared alkylated purines as marker compounds for 32-P-postlabeling and electrochemical analysis and developed methods to measure the corresponding products. The traditional postlabeling assay was modified by incorporating a solid phase extraction column and high-performance liquid chromatography (HPLC) enrichment steps to the assay prior to labeling. The final analysis of adducted N6 adenines is based on two dimensional thin-layer chromatography (TLC) and an on-line HPLC/radioactivity analysis. The qualitative and quantitative results are based on positively identified marker compounds. Alkylated N7 guanines were released from DNA by neutral thermal hydrolysis, prepurified by HPLC, and analyzed by HPLC with a sensitive electrochemical detection procedure. By using these methods, we found alkylation of calf thymus DNA exposed to butadiene monoepoxide in vitro at adenine N6 and guanine N7 sites. Analysis of lung DNA samples from mice and rats exposed to butadiene through inhalation showed that adenine N6 adducts were formed in vivo in a dose responsive manner.
Adenine↗
1,3-Butadiene working group report.
During the Workshop in North Carolina, the in vivo metabolism, adduct formation and genotoxicity data available from rodent and human exposure to 1,3-butadiente (BD) were reviewed and they are summarized in the present report. BD is metabolized by cytochrome P-450-dependent monoxygenases to the primary metabolite 1,2-epoxybutene-3 (epoxybutene, EB). EB is subjected to further metabolism: oxidation to 1,2:3,4-diepoxybutane (DEB), hydrolysis to 3-butene-1,2-diol and conjugation to glutathione. The first pathway seems to prevail in mice while the latter is characteristic for rats and possibly for humans. Species differences exist in adduct formation of the monoepoxide to hemoglobin, for which the following pattern has been found: mice > rats > humans. Genotoxity of BD was found in mice with all applied tests; however, negative results were obtained in rats. In exposed humans, the cytogenetic studies in peripheral blood lymphocytes did not show genotoxic effects, although one report described elevated hprt variant levels in peripheral blood lymphocytes of exposed workers. It was concluded that the presently available data are insufficient for the application of the parallelogram model to estimate genetic risk for humans. As an alternative approach, a tentative estimate of the doubling dose for induction of hprt mutations in somatic cells of mice and men was performed and the calculated values were surprisingly similar, i.e. 9000 ppmh. However, this estimate is burdened with a number of caveats which were discussed in detail. The working group identified a series of urgent research needs to provide the appropriate data for the application of the parallelogram model, such as identification of metabolic pathways in different rodent species and humans, metabolic studies in mice, rats and humans considering metabolic polymorphisms, studies of adducts to DNA and hemoglobin especially of DEB and other butadiene metabolites in rodents and humans, studies of mutational spectra (mutational fingerprinting) in somatic and germinal cells, confirmation of the human hprt mutation data, conformation of the rodent malformation data, dose-response studies in rodent germ cell tests and studies on repair kinetics of mono-adducts induced by EB as opposed to repair of cross-links produced by DEB. Finally, it was suggested that the original parallelogram consisting of data from somatic cell studies in rodents and humans plus studies of heritable effects in rodents to extrapolate to germ cell risk for humans should be supplemented with studies in sperm of experimental animals and exposed men.
Animals↗
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.
Administration, Inhalation↗
Analysis of chemically induced spindle aberrations in male mouse germ cells: comparison of differential and immunofluorescent staining procedures.
Differential staining of spindle and chromatin was adapted to meiotic cells of male mice. Immunostaining with antitubulin antibodies was developed. Both methods were used to test acrylamide, diazepam and vinblastine for their potential to cause spindle disturbances in male mouse germ cells. The analyses were performed after in vivo treatment, comparing the classical safranin/brilliant blue spindle staining to an immunofluorescent assay. The two staining methods complemented each other. Differential staining seems to be more sensitive to the detection of misplaced chromatin whereas the immunostaining is superior when scoring for spindle structure aberrations. All three chemicals showed spindle activity. Acrylamide mainly caused multipolar spindles which possibly develop from a separation of mother and daughter centrioles during an unspecific meiotic block. Diazepam elevated the level of monopolar spindles and induced the loss of single chromosomes, suggesting an effect on motor proteins. Vinblastine was the strongest spindle poison by far. It produced high numbers of shortened and monopolar spindles as well as multiple chromosome loss.
Acrylamide↗
Clastogenicity of diepoxybutane in bone marrow cells and male germ cells of mice.
The bifunctional metabolite of 1,3-butadiene, 1,2:3,4-diepoxybutane (DEB), was tested in the mouse bone marrow micronucleus assay and in male mouse germ cell tests, namely the analysis of first cleavage divisions and the dominant lethal assay. All experiments were performed with single intraperitoneal treatment of the animals. In the micronucleus test, DEB doses of 4.5, 9.0, 18.0 and 36.0 mg/kg body weight were tested at a sampling interval of 24 h for bone marrow. The dose response for the induction of micronuclei in polychromatic erythrocytes was linear with the lowest effective dose of 9.0 mg/kg body weight. No sensitivity difference was observed between male and female mice. the cytogenetic analysis of first cleavage division chromosomes was performed after treatment of male mice with 17, 26, 34, 43 and 52 mg/kg body weight of DEB and mating the males to hormonally stimulated females on days 7, 14, 21 and 28 after treatment. The two higher doses caused general toxicity evidenced by the poor mating behavior of the males. Only 13 and 20% of the mated females were fertilized on day 7 after treatment of the males with 43 and 52 mg/kg body weight of DEB, respectively. An increased number of unfertilized oocytes was obtained from fertilized females on day 7 after treatment of the males with 34 mg/kg body weight of DEB. With a dose of 26 mg/kg body weight, it was demonstrated that chromosomal aberrations were only induced in spermatozoa (mating on day 7 after treatment) while spermatids (mating on days 14 and 21) and spermatocytes (mating on day 28) were not susceptible to the clastogenic effect of DEB. The response in spermatozoa in the dose range 17-34 mg/kg body weight was linear up to 26 mg/kg body weight and reached a plateau thereafter. The results of the dominant lethal experiments performed in the dose range 18-54 mg/kg body weight gave results similar to the cytogenetic study. With the highest dose tested, the toxicity and cytotoxicity during the first 8 mating days after treatment dramatically reduced the number of pregnant females and, consequently, the total implantations, so that no significant dominant lethal effect could be assessed. During mating days 9-12 (treated late spermatids), a significant dominant lethal effect was observed. With the two lower doses (18 and 36 mg/kg body weight), the dominant lethal effect was restricted to spermatozoa. The good correlation of the chromosomal aberrations with dominant lethal mutations confirms the chromosomal origin of dominant lethal effects. The clastogenic effect of DEB in somatic cells and in germ cells of mice was of the same order of magnitude.
Animals↗
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.
Acrylamide↗
Dominant lethal effects after inhalation exposure to 1,3-butadiene.
Two independent dominant lethal experiments were performed using different protocols with respect to strains of mice, inhalation exposure duration of 1,3-butadiene, and mating regimen. The short communication summarizes the results of the experiments and compares the induced dominant lethality according to the formula published by Ehling in 1978. Despite the differences in methodology the results are in close agreement. The sum of the dominant lethal effects observed during the first three mating weeks after 1 week of butadiene exposure in one experiment (23.1%) is surprisingly similar to the dominant lethal effect observed during 1 week of mating after 10 weeks of butadiene exposure (28.1%) in the other experiment. The results of the two independent experiments strengthen the conclusion that butadiene is a germ cell mutagen. Furthermore, the results indicate that the effect observed after 10 weeks of exposure is representative of the last three treatment weeks, i.e. treated spermatozoa and spermatids.
Administration, Inhalation↗
Mutagenicity of 1,3-butadiene inhalation in somatic and germinal cells of mice.
Inhalation exposure of mice to 50, 200, 500 or 1300 ppm of 1,3-butadiene for 6 h per day for 5 consecutive days caused micronuclei in mouse bone marrow and peripheral blood erythrocytes. The dose response was non-linear. The slope of the curve flattened with increasing exposure concentration. Coat color spots were found in the mouse spot test after exposure of pregnant females on pregnancy days 8-12 to 500 ppm of 1,3-butadiene. Dominant lethal mutations were induced in spermatozoa and late spermatids after exposure of male mice to 1300 ppm with the 5-day exposure regimen. Thus, in the mouse 1,3-butadiene is a somatic and germ cell mutagen.
Administration, Inhalation↗
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.
Animals↗
Summary report of the Working Group on Mammalian Germ Cell Tests.
The two tests considered by the Working Group were the mammalian germ cell cytogenetic assay and the rodent dominant lethal test. It was agreed that both tests were mainly used for identification of germ cell hazards, however, that the commonly applied protocol of the dominant lethal assay often supplied information for hazard characterization such as sensitivity of particular developmental stages of male germ cells. No particular species or strains were indicated. Concurrent solvent controls were regarded as indispensable for both tests. In the discussion of the mammalian germ cell cytogenetic assay, harmonization was obtained to a large extent with the cytogenetic bone marrow assay regarding the number of animals (5), the number of cells analyzed per animal (200), the highest exposure dose (MTD) and sampling times (twice within 24 and 48 h after dosing). However, it was pointed out that only the single acute exposure was adequate for the mammalian germ cell cytogenetic assay. Furthermore, it was stated that only structural chromosome aberrations could be analyzed and that it was not informative to score polyploidies or aneuploidies. In the discussion of the rodent dominant lethal test, it was stated that the assay was generally performed with treated males, however, increasing concern about female specific effects required that a protocol for female dominant lethal testing should be developed and validated. Acute and subacute treatment schedules were considered equally acceptable. It was regarded as highly important that the entire male germ cell development from meiosis to mature sperm was covered in the test protocol either by the appropriate mating schedules after single dosing or by subchronic dosing during the respective period. Postimplantation loss, preimplantation loss and fertility rate were the main parameters to be assessed in the rodent dominant lethal tests. It was agreed that the size of the experiment depended on the spontaneous frequency of dead implants, the mating scheme and the statistical design of the experiment.
Aneuploidy↗
The performance of short-term tests in identifying potential germ cell mutagens: a qualitative and quantitative analysis.
A retrospective analysis was undertaken to assess the performance of selected short-term tests in the discrimination of mammalian germ cell mutagens and nonmutagens using data derived from the U.S. Environmental Protection Agency/International Agency for Research on Cancer Genetic Activity Profile (EPA/IARC GAP) and EPA GENE-TOX databases. The short-term tests selected were gene mutation in Salmonella (S. typhimurium), cultured mammalian cell gene mutation and chromosomal aberrations, and mammalian bone marrow cytogenetics (micronucleus and chromosomal aberrations). These are the first level tests used in the EPA mutagenicity testing guidelines. The results of this analysis showed good sensitivity of short-term in vitro tests for mammalian cell gene mutation (96%) or chromosomal aberrations (92%) in identifying germ cell mutagens, while the sensitivity of tests for gene mutation in S. typhimurium was lower (79%). Bone marrow micronucleus or chromosomal aberration assays in vivo each displayed a sensitivity of 96%. Thus, both the in vitro and in vivo tests may be used effectively to screen chemicals for potential germ cell mutagenicity. In contrast, the in vitro tests mentioned above performed poorly in discriminating putative germ cell nonmutagens, giving results for specificity at or below what is expected due to chance alone (50-11%). The bone marrow assays were more efficient in this regard, the micronucleus test yielding a specificity of 63% and the chromosomal aberrations assay 64%. The mouse bone marrow micronucleus test also performed well on a quantitative basis, responding at or below the lowest effective doses tested in the mouse dominant lethal assay. Regression analysis of the mean lowest effective doses of chemicals evaluated in vivo showed approximately 1:1 linear correlations for mouse germ cell assays (heritable translocation vs dominant lethal or specific locus tests) as well as for mouse bone marrow assays (micronucleus vs chromosomal aberration). The results suggest the value of the bone marrow micronucleus test as an assay for potential germ cell mutagenicity and the dominant lethal test as a relatively inexpensive choice for confirmation of germ cell damage. The sensitivity of the in vitro assays investigated and the discriminatory capability of the in vivo bone marrow assay affirmed the utility of these tests within the framework of the EPA mutagenicity testing guidelines.
Animals↗
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)
Animals↗