Biomedical subjects

I D Adler

Publications and source records attributed to I D Adler.

At least 19 recordsLinked to original sources

PubMed2001

Automated evaluation of frequencies of aneuploid sperm by laser-scanning cytometry (LSC).

BACKGROUND: Laser-scanning cytometry (LSC) allows fast automated scoring of fluorescence signals directly on microscopic slides. Frequencies of spontaneous aneuploidies in murine and human sperm were evaluated by using this new LSC technique. Rapid detection may be of great interest in reproductive toxicology, as certain chemicals act as aneugens during meiosis, increasing the production of aneuploid germ cells. Materials and Methods Selected chromosomes were detected by using fluorescence in situ hybridization (FISH) and fluorochrome-labeled DNA-probes. Sperm chromatin was counterstained with propidium iodide. By scanning across the slide, fluorescence signals within sperm nuclei were detected and counted. RESULTS: In murine sperm, the frequencies of disomies for chromosomes 8 and X were 0.019% and 0.021%, respectively. The automated assessment in human sperm resulted in disomy frequencies of 0.061% and 0.090% for chromosomes 13 and X, respectively. These results were comparable to data obtained from the same samples by manual microscopic scoring and to literature data. CONCLUSIONS: Frequencies of genotypically abnormal sperm were not significantly different between automated and manual scoring. In conclusion, sperm aneuploidy was reliably determined and disomic sperm were successfully relocated by LSC. By virtue of rapid and reliable analyses, LSC has the powerful potential to replace manual microscopic FISH analysis in molecular cytogenetics.

Aneuploidy↗

PubMed2001

Detection of aneuploidy in rodent and human sperm by multicolor FISH after chronic exposure to diazepam.

Aneuploidy induction in male germ cells of mice and men after chronic exposure to diazepam (DZ; CAS 439-14-5; Valium was assessed by multicolor fluorescence in situ hybridization (FISH). DZ, a widely administered sedative and muscle relaxant, was proposed to act as an aneugen by disturbing spindle function in various assay systems. Male mice were treated by oral intubation with 3mg/kg DZ once or daily for 14 consecutive days. At 22 days after the last treatment, epididymal sperm were collected from the caudae epididymes. Evaluation of aneuploid and diploid sperm (10,000 sperm per animal) was performed by multicolor FISH employing DNA probes specific for chromosomes X, Y, and 8 simultaneously. We found a significant increase in the frequency of disomy 8 in subchronically DZ-treated mice when compared to the concurrent solvent control group (2.4-fold; P<0.01), while no increase was detected for sex-chromosome hyperhaploidies. No effect was seen when mice were treated with a single dose (3mg/kg DZ). In a parallel human approach, two men were evaluated who chronically ingested >0.3mg/kg/d DZ for more than 6 months. Multicolor FISH was applied to human sperm probing for chromosomes X, Y, and 13. Frequencies for sperm with disomy 13, disomy X, and total sex-chromosomal disomies were found to be elevated among the two subjects after chronic DZ-exposure compared to control subjects. In conclusion, the results indicate that diazepam acts as an aneugen during meiosis in male spermatogenesis, both in mice and humans. The quantitative comparison indicates that humans may be at least 10 times more sensitive than mice for aneuploidy induction by DZ during male meiosis.

Adult↗

PubMed2001

Karyotyping mouse chromosomes by multiplex-FISH (M-FISH).

Karyotyping of mouse chromosomes is a skillful art, which is laborious work even for experienced cytogeneticists. With the growing number of mouse models for human diseases, there is an increasing demand for automated mouse karyotyping systems. Here, such a karyotyping system for mouse chromosomes based on the multiplex-fluorescence in-situ hybridization (M-FISH) technology is shown. The system was tested on a number of individual mice with numerical and structural aberrations and its reproducibility and robustness verified. Mouse M-FISH should be a valuable tool for the analysis of chromosomal rearrangements in mice.

Animals↗

PubMed2001

Effect of chemicals on the duration of male meiosis in mice detected with laser scanning cytometry.

Aneuploidy studies in sperm such as the sperm-FISH assay require a precise knowledge of the duration of spermatogenesis, especially of the meiotic stages. This is important in order to sample sperm from the epididymis at appropriate intervals after animal treatment. However, aneugens may delay the cell cycle. The progression from meiotic divisions to epididymal sperm was determined by labelling the last S-phase before meiosis with the thymidine analogue 5-bromo-2'-deoxyuridine (BrdU) and treating the animals 13 days later with the test chemicals. In a time frame of 20--24 days after treatment, BrdU-containing sperm were identified with a FITC-labelled anti-BrdU antibody and green fluorescent sperm were scored with a laser scanning cytometer (LSC). We studied the effects of the chemicals acrylamide, colchicine, diazepam, griseofulvin, taxol, thiobendazole, trichlorfon and vinblastine on the duration of meiotic divisions in male mice. Colchicine treatment prolonged the duration of meiotic divisions by about 48 h. On days 21 and 22, the frequencies of BrdU-labelled sperm in the colchicine group were 11.7 and 9.4%, respectively, while they were 28.4 and 30.6%, respectively, in the concurrent controls (P > 0.01). On day 24 after treatment, the frequency of labelled sperm in the colchicine group reached the control level. Etoposide treatment resulted in an elevation of BrdU-labelled sperm at 23 rather than 22 days. The other chemicals showed no significant effect of prolonging meiotic cell cycle progression. On the basis of the colchicine and etoposide data, it is suggested that the effect of a chemical on the meiotic cell cycle progression is determined first in order to chose the appropriate sperm sampling time to detect aneuploidy induction.

Animals↗

PubMed2000

Molecular cloning and functional characterization of the mouse organic-anion-transporting polypeptide 1 (Oatp1) and mapping of the gene to chromosome X.

We have cloned a murine member of the organic-anion-transporting polypeptide (Oatp) family of membrane-transport proteins from mouse liver. The cloned cDNA insert of 2783 bp with an open reading frame of 2011 bp codes for a 12-transmembrane 670-amino-acid protein with highest amino acid identity with the rat Oatp1. When expressed in Xenopus laevis oocytes, the mouse Oatp exhibited the same substrate specificity as the rat Oatp1. Besides the common Oatp substrates bromosulphophthalein, taurocholate, oestrone 3-sulphate and ouabain, the new mouse Oatp also mediates transport of the Oatp1-specific magnetic-resonance-imaging agent gadoxetate. The Oatp2-specific cardiac glycoside digoxin, however, is not transported. Kinetic analyses performed for taurocholate and oestrone 3-sulphate revealed apparent K(m) values of 12 microM and 5 microM respectively. Northern-blot analysis demonstrated a predominant expression in the liver with an additional moderate expression in the kidney. Taken together, the amino acid identity, the functional characteristics and the tissue distribution suggest that we have isolated the murine orthologue of the rat Oatp1, and consequently the identified protein will be called Oatp1. Using fluorescence in situ hybridization, the murine Oatp1 gene was mapped to chromosome XA3-A5.

Amino Acid Sequence↗

PubMed2000

Recommendations for the categorization of germ cell mutagens.

Germ cell mutagens are currently classified into three categories in the German List of MAK and BAT Values. These categories have been revised and extended by analogy with the new categories for carcinogenic chemicals. Germ cell mutagens produce heritable gene mutations, and heritable structural and numerical chromosome aberrations in germ cells. The original categories 1 and 2 for germ cell mutagens remain unchanged. Two new categories 3A and 3B are proposed for chemicals suspected to be germ cell mutagens. A new category 5 is proposed for germ cell mutagens with low potency that contribute negligibly to human genetic risk provided the MAK value is observed.

Germ Cells↗

PubMed2000

Incidence of mosaic cell lines in vivo and malsegregation of chromosome 21 in lymphocytes in vitro of trisomy 21 patients: detection by fluorescence in situ hybridization on binucleated lymphocytes.

In order to detect aneuploidy in interphase human lymphocytes, both in vivo and in vitro, fluorescence in situ hybridization (FISH) was carried out on binucleated cells cytokinesis-blocked by cytochalasin B at the first mitosis after phytohemagglutinin stimulation. A pericentric chromosome-21-specific DNA probe prepared from yeast artificial chromosome clone 881D2 by the polymerase chain reaction was employed. One thousand binucleated cells per individual were scored from cultures from twelve trisomy 21 patients aged 0.01-8.9 years (mean 4.3 years) and 20 normal children of similar age. Of trisomy 21 patients, increased frequencies of disomic cells in vivo (1.690+/-1.070%) and cells containing six signals with nondisjunction (0.822+/-0.554%) were found, compared with those of monosomic 21 cells in vivo (0.265+/-0.130%) and cells containing four signals with nondisjunction in normal children (0.369+/-0.250%; P=0.000 and P=0.000, respectively). These results show that malsegregation of chromosome 21 occurs more often in trisomic 21 cells than in disomic cells from normal children. The frequency of nondisjunction was significantly higher than the loss of chromosome 21 in both cultured trisomic (0.822+/-0.554% vs 0.043+/-0.049%, P=0.000) and disomic (0.369+/-0.250% vs 0.010+/-0.30%, P=0.000) cells. Comparisons of in vivo and in vitro data on aneuploidy indicate that a cell selection mechanism may exist in vivo. All these results show that FISH, with a chromosome-specific probe, on binucleated lymphocytes is a powerful tool for simultaneously detecting mosaic cell lines in vivo and malsegregation (loss and nondisjunction) of a corresponding chromosome in vitro in the same cell population.

Case-Control Studies↗

PubMed2000

Spermatogenesis and mutagenicity of environmental hazards: extrapolation of genetic risk from mouse to man.

To perform germ cell mutagenicity studies it is mandatory to know the duration of the different stages of spermatogenesis. The timing of male germ cell development determines the test protocols. Chemical mutagens are characterized by their differential spermatogenic responses, e.g. different chemicals induce mutations in different germ cell stages. Knowledge of the sensitive germ cell stages for a test agent is essential for the evaluation of the genetic hazard, i.e. stem cell effects present permanent genetic hazards and post-stem cell effects present transient hazards. A variety of assays are available to determine germ cell mutagenicity in treated animals or in the progeny of treated animals. Germ cell cytogenetics in differentiating spermatogonia and the dominant lethal assay are used for genetic hazard identification. Their results allow categorization of chemicals as germ cell mutagens (Maximale Arbeitsplatz Konzentration categories for germ cell mutagens). Gene mutations or reciprocal chromosome translocations induced in germ cells are assessed by observation of mutant offspring of treated males. These results are applicable to the quantification of genetic hazards for chemical exposures which cannot be avoided, i.e. for occupational exposures to chemicals such as butadiene.

Animals↗

PubMed2000

Trichlorfon induces spindle disturbances in V79 cells and aneuploidy in male mouse germ cells.

In order to assess the effects of trichlorfon on cell division and on aneuploidy induction, we conducted an in vitro assay for spindle disturbances using V79 cells and an in vivo assay for aneuploidy induction in meiosis of male mice using multicolour fluorescence in situ hybridization (FISH) with epididymal sperm. In the in vitro assay, the chemical caused a concentration-dependent increase in the incidence of initial and full c-mitoses in the dose range 40-120 microg/ml trichlorfon. The mitotic index (MI) was decreased between 40 and 100 microg/ml trichlorfon, whereas at 120 microg/ml the MI was back to the control level, coinciding with the dramatic increase in c-mitoses. The results confirm that trichlorfon is a potent spindle poison in V79 cells. In the in vivo multicolour FISH assay, administration of trichlorfon to male mice at single doses of 200, 300 and 405 mg/kg caused a dose-dependent increase of the frequencies of disomic sperm (0.068, 0.074 and 0.134%, respectively) compared with the corresponding controls (0.046, 0.042 and 0.056%, respectively). The prevalence of X-X-8 and Y-Y-8 sperm suggests that trichlorfon affected chromosome segregation predominantly during the second meiotic division. Diploid sperm were not induced by trichlorfon treatment, indicating that no meiotic block occurred. It is concluded that trichlorfon is a potent spindle poison in V79 cells and induces aneuploidy in mouse spermatocytes during meiosis.

Aneuploidy↗

PubMed2000

1-Aminobenzotriazole inhibits acrylamide-induced dominant lethal effects in spermatids of male mice.

Acrylamide (AA) is a germ cell mutagen and induces clastogenic effects predominantly in spermatids of mice. The mechanism of AA clastogenicity has been a matter of dispute. Since the reactivity of AA with DNA is low but is high with proteins containing SH groups, it was suggested that protamine alkylation could be the mechansim of clastogenicity by AA in spermatids. This was substantiated by the observation that the time course of protamine alkylation and dominant lethal effects in spermatids of mice induced by AA was strictly parallel. Another suggestion was that AA may be metabolized by cytochrome P-450 to the epoxide glycidamide (GA), which is then the ultimate DNA-reactive clastogen. This suggestion was based on the similarity of the stage specificity pattern for dominant lethality and heritable translocation induction by AA and GA. To test this latter assumption, 1-aminobenzotriazole (ABT), an inhibitor of P-450 metabolism, was used in the present experiments. Male mice were pretreated with ABT (3x50 mg/kg) on three consecutive days followed by AA treatment (125 mg/kg) on day 4. Parallel groups of animals were treated with AA (125 mg/kg), ABT (3x50 mg/kg) or with the solvent double-distilled water. The experiment was repeated once with slightly varied mating parameters. The results of both experiments showed that ABT inhibited or significantly reduced the AA-induced dominant lethal effects. Thus, the present data support the hypothesis that the AA metabolite GA is the ultimate clastogen in mouse spermatids.

Acrylamide↗

PubMed2000

In vivo rodent erythrocyte micronucleus assay. II. Some aspects of protocol design including repeated treatments, integration with toxicity testing, and automated scoring.

An expert working group on the in vivo micronucleus assay, formed as part of the International Workshop on Genotoxicity Test Procedures (IWGTP), discussed protocols for the conduct of established and proposed micronucleus assays at a meeting held March 25-26, 1999 in Washington, DC, in conjunction with the annual meeting of the Environmental Mutagen Society. The working group reached consensus on a number issues, including: (1) protocols using repeated dosing in mice and rats; (2) integration of the (rodent erythrocyte) micronucleus assay into general toxicology studies; (3) the possible omission of concurrently-treated positive control animals from the assay; (4) automation of micronucleus scoring by flow cytometry or image analysis; (5) criteria for regulatory acceptance; (6) detection of aneuploidy induction in the micronucleus assay; and (7) micronucleus assays in tissues (germ cells, other organs, neonatal tissue) other than bone marrow. This report summarizes the discussions and recommendations of this working group. In the classic rodent erythrocyte assay, treatment schedules using repeated dosing of mice or rats, and integration of assays using such schedules into short-term toxicology studies, were considered acceptable as long as certain study criteria were met. When the micronucleus assay is integrated into ongoing toxicology studies, relatively short-term repeated-dose studies should be used preferentially because there is not yet sufficient data to demonstrate that conservative dose selection in longer term studies (longer than 1 month) does not reduce the sensitivity of the assay. Additional validation data are needed to resolve this point. In studies with mice, either bone marrow or blood was considered acceptable as the tissue for assessing micronucleus induction, provided that the absence of spleen function has been verified in the animal strains used. In studies with rats, the principal endpoint should be the frequency of micronucleated immature erythrocytes in bone marrow, although scoring of peripheral blood samples gives important supplementary data about the time course of micronucleus induction. When dose concentration and stability are verified appropriately, concurrent treatment with a positive control agent is not necessary. Control of staining and scoring procedures can be obtained by including appropriate reference samples that have been obtained from a separate experiment. For studies in rats or mice, treatment/sampling regimens should include treatment at intervals of no more than 24 hr (unless the test article has a half-life of more than 24 hr) with sampling of bone marrow or blood, respectively, within 24 or 40 hr after the last treatment. The use of a DNA specific stain is recommended for the identification of micronuclei, especially for studies in the rat. In the case of a negative assay result with a non-toxic test article, it is desirable that systemic exposure to the test article is demonstrated. The group concluded that successful application of automated scoring by both flow cytometry and image analysis had been achieved, and defined criteria that should be met if automated scoring is employed. It was not felt appropriate to attempt to define specific recommended protocols for automated scoring at the present time. Other issues reviewed and discussed by the working group included micronucleus assays that have been developed in a number of tissues other than bone marrow. The group felt that these assays were useful research tools that could also be used to elucidate mechanisms in certain regulatory situations, but that these assays had not yet been standardized and validated for routine regulatory application.

Animals↗

PubMed1999

Numerical and structural chromosomal abnormalities detected in human sperm with a combination of multicolor FISH assays.

A pair of multicolor FISH assays (X-Y-21 and A-M-16) was developed for human sperm to simultaneously measure sex ratios; aneuploidies involving chromosomes 1, 16, 21, X, and Y; meiotic diploidies; and structural aberrations involving chromosome 1p. Sex ratios in sperm were not significantly different from unity among healthy men. Baseline frequencies of disomic sperm for chromosomes 1, 8, and 21 were similar (6.7 per 10(4) sperm, 95% CI of 5.6-8.1), suggesting that among these three chromosomes, chromosome 21 was not especially prone to nondisjunction. Frequencies of disomy 16 sperm were significantly lower, however (3.5 per 10(4) sperm, 95% CI of 2.0-6.2; P < 0.02). The baseline frequencies of sperm disomy by FISH for chromosomes 16 and 21 were validated against aneuploidy data obtained by the hamster-egg technique for human sperm cytogenetics. The frequencies of X-X, Y-Y, X-Y ("Klinefelter") sperm and sex-null ("Turner") sperm were 5.5, 5.1, 5.5, and 7.8 per 10(4) sperm, respectively. For chromosomes 16 and 21, the frequencies of nullisomic and disomic sperm were similar, suggesting that gain and loss events occurred symmetrically. However, more gain than loss was reported for chromosomes 1, X, and Y. The frequency of MI and MII diploid sperm (with flagella) was approximately 12 per 10(4) (range 8.3-16.7 per 10(4) sperm). Based on flagella data, the frequency of somatic cells in the semen was estimated to be approximately 1.8 per 10(4) sperm. Loss or gain of a portion of chromosome-arm 1p occurred in 5.5 per 10(4) sperm, and the percentage of sperm carrying structural aberrations within the haploid genome as calculated from FISH (1.4%), was similar to that obtained with the hamster-egg technique. These complementary sperm FISH assays have promising applications in studies of chromosomally abnormal sperm after exposure to occupational, medical, and environmental toxicants.

Adult↗

PubMed1999

Butadiene diolepoxide- and diepoxybutane-derived DNA adducts at N7-guanine: a high occurrence of diolepoxide-derived adducts in mouse lung after 1,3-butadiene exposure.

Butadiene (BD) is a high production volume chemical and is known to be tumorigenic in rodents. BD is metabolized to butadiene monoepoxide (BMO), diepoxybutane (DEB) and butadiene diolepoxide (BDE). These epoxides are genotoxic and alkylate DNA both in vitro and in vivo, mainly at the N7 position of guanine. In this study, a 32P-post-labeling/thin-layer chromatography (TLC)/high-pressure liquid chromatography (HPLC) assay for BDE and DEB adducts at the N7 of guanine was developed and was used in determining the enantiomeric composition of the adducts and the organ dose of BD exposure in lung. Exposure of 2'-deoxyguanosine (dGuo), 2'-deoxyguanosine-5'-phosphate (5'-dGMP) and 2'-deoxyguanosine-3'-phosphate (3'-dGMP) to racemic BDE followed by neutral thermal hydrolysis gave two products (products 1 and 2) that were identified by MS and UV and NMR spectroscopy as a diastereomeric pair of N7-(2,3,4-trihydroxybutan-1-yl)-guanines. Exposure of dGuo nucleotides to RR/SS DEB (also referred to as dl DEB) followed by thermal depurination resulted in a single product coeluting with the BDE product 1. If the reaction mixture of BDE and 5'-dGMP was analyzed by HPLC before hydrolysis of the glycosidic bond, four major nucleotide alkylation products (A, B, C and D) with identical UV sepectra were detected. The products were isolated and hydrolyzed, after which A and C coeluted with product 1 and B and D coeluted with the product 2. The major adduct of DEB-exposed 5'-dGMP was N7-(2-hydroxy-3,4-epoxy-1-yl)-dGMP (product E). A 32P-post-labeling assay was used to detect BDE- and DEB-derived N7-dGMP adducts in DNA. Levels of adducts increased with a dose of BDE and DEB and exhibited a half life of 30 +/- 3 (r = 0.98) and 31 +/- 4 h (r = 0.95), respectively. Incubation of DEB-modified DNA at 37 degrees C at neutral pH for up to 142 h did not lead to an increase of N7-(2,3,4-trihydroxybutan-1-yl)-dGMP in the DNA. These observations led to the conclusion that the N7-(2,3, 4-trihydroxybutan-1-yl)-dGMP adducts in DNA can be used as a marker of BDE exposure and that N7-(2-hydroxy-3,4-epoxy-1-yl)-dGMP adducts are related to DEB exposure. Dose-related levels of BDE- and DEB-derived adducts were detected in lungs of mice inhaling butadiene. Most of the N7-dGMP adducts (73%; product D) were derived from the 2R-diol-3S-epoxide of 1,3-butadiene. The data presented in this paper indicate that in vivo, 98% of N7-dGMP alkylation after BD exposure is derived from BDE, and approximately 2% of the adducts were derived from DEB and BMO.

Animals↗

PubMed1999

Isolation and embryonic expression of the novel mouse gene Hic1, the homologue of HIC1, a candidate gene for the Miller-Dieker syndrome.

The human gene HIC1 (hypermethylated in cancer) maps to chromosome 17p13.3 and is deleted in the contiguous gene disorder Miller-Dieker syndrome (MDS) [Makos-Wales et al. (1995) Nature Med., 1, 570-577; Chong et al. (1996) Genome Res., 6, 735-741]. We isolated the murine homologue Hic1, encoding a zinc-finger protein with a poxvirus and zinc-finger (POZ) domain and mapped it to mouse chromosome 11 in a region exhibiting conserved synteny to human chromosome 17. Comparison of genomic and cDNA sequences predicts two exons for the murine Hic1. The second exon exhibits 88% identity to the human HIC1 on DNA level. During embryonic development, Hic1 is expressed in mesenchymes of the sclerotomes, lateral body wall, limb and cranio-facial regions embedding the outgrowing peripheral nerves during their differentiation. During fetal development, Hic1 additionally is expressed in mesenchymes apposed to precartilaginous condensations, at many interfaces to budding epithelia of inner organs, and weakly in muscles. We observed activation of Hic1 expression in the embryonic anlagen of many tissues displaying anomalies in MDS patients. Besides lissencephaly, MDS patients exhibit facial dysmorphism and frequently additional birth defects, e.g. anomalies of the heart, kidney, gastrointestinal tract and the limbs (OMIM 247200). Thus, HIC1 activity may correlate with the defective development of the nose, jaws, extremities, gastrointestinal tract and kidney in MDS patients.

Abnormalities, Multiple↗

PubMed1999

Detection of aneuploidy by multicolor FISH in mouse sperm after in vivo treatment with acrylamide, colchicine, diazepam or thiabendazole.

Multicolor fluorescence in situ hybridization (FISH) was used to investigate the induction of aneuploidy during meiosis in young adult male mice treated with chemicals chosen for the EU sponsored aneuploidy project (acrylamide, colchicine, diazepam and thiabendazole). The aim of the present study was to evaluate the frequency of aneuploid sperm induced by each of these chemicals by sperm FISH. Male (102/ElxC3H/El)F1 mice were treated with acrylamide (120 and 60 mg/kg single dose i.p.), colchicine (1.5 and 3 mg/kg single dose, i.p.), diazepam (300, 150 and 75 mg/kg single dose by oral intubation) or thiabendazole (100 and 300 mg/kg daily for 11 days by oral intubation). At 22 days after the last treatment, sperm were collected from the cauda epididymis. Three chromosome FISH was applied to determine hyperhaploid and diploid sperm with DNA probes specific for the chromosomes X, Y and 8. Five animals were treated per dose group and sperm aneuploidy was evaluated in 10,000 sperm per animal. We found significant increases in the frequency of total hyperhaploidy for the males treated with 3.0 mg/kg colchicine (0.092 versus 0.056%, P < 0.05) and with 1.5 mg/kg colchicine (0.082 versus 0.050%, P < 0.05), as well for the males treated with 300 mg/kg diazepam (0.081 versus 0.050%, P < 0.05), indicating that colchicine and diazepam each induced germ cell aneuploidy. We also found significant increases in the frequency of total diploidy for the males treated with 300 mg/kg diazepam (P < 0.05) and with 300 mg/kg thiabendazole (P < 0.05). No significant effects were found for 120 and 60 mg/kg acrylamide or for the other doses of diazepam and thiabendazole. These first results indicate that the multicolor FISH method is useful to determine aneuploidy induction in sperm of mice.

Acrylamide↗

PubMed1999

[Aneuploidy detection by multi-color fluorescence in situ hybridization in mouse sperm].

Multi-color fluorescence in situ hybridization (FISH) was employed to evaluate the frequency of aneuploid sperm in young adult mice after treated by 2-(4'-thiazolyl) benzimidazole or thiabendazole (TB). The animals were given TB (100 200 300 mg/kg) by oral incubation daily for 11 days, and sacrificed 22 days after the last treatment of TB. Three-chromosome FISH was applied to determine the frequencies of diploid, disomic and nullisomic sperm with DNA probes specific for the chromosomes X, Y and 8. The aneuploid frequencies were not significantly different between the sperm in 100 mg/kg group and solvent control. The frequencies aneuploid and diploid sperm in middle dose group were significantly higher than the solvent control (P < 0.001-0.05). There were no significant aneuploid frequency increases were found in 300 mg/kg group excepting the frequency of diploid sperm. It is conclude that TB is a potential germinal aneugen in mammalian, which may block the cleavage during the meiosis. Epididymal sperm aneuploidy can be reliably determined by sperm FISH analysis in mice.

Aneuploidy↗

PubMed1999

[Detection of numerical chromosomal aberrations in epididymal sperm of mice using three-color FISH with chromosome-specific DNA probes].

To detect malsegregation of chromosomes during meiosis in male mice, three-color FISH using DNA probes specific for mouse chromosomes X, Y and 8 was performed on epididymal sperm decondensed for 30 min each in 10 mmol/L DTT and 4 mmol/L LIS, and conventional chromosome counting was also carried out on C-banded MMII cells. Comparisons between these two methods indicate as the following: (1) three-color FISH is a simple, rapid and sensitive approach; (2) The estimate of the frequency of aneuploidy obtained from three-color FISH analysis is more reliable since extremely large numbers of sperm (more than 10,000 sperm per animal) are scored; (3) The rates of cells arrested at meiosis I and meiosis II (representing diploid sperm) can be detected only by using the three-color FISH approach; (4) Malsegregation of chromosomes occurred during both meiosis I and meiosis II can be detected simultaneously by using this new approach. The probes and scoring criteria used in FISH on interphase sperm nuclei, and the necessity of using three or more chromosome-specific DNA probes to thoroughly evaluate aneugenic effects of factors tested on meiosis in male mice were discussed.

Animals↗

PubMed1998

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.

Animals↗