Cytochemical detection of hyaluronidase activity in single human and mouse sperm by an improved substrate-film technique.

A substrate-film method is described that allows the detection of hyaluronidase activity in nearly 100% of single human and mouse sperm. The level of hyaluronidase activity as determined by halo diameters was greater in mouse than in human sperm. This simple method may have use as a screening method for identifying compounds that cause developmental or genetic defects in male germ cells, or for the diagnosis of infertility due to decreased hyaluronidase activity.

Cytogenetic and teratogenic test of polybrominated biphenyls in rodents.

Previously we reported negative terata and c-mitosis synergism of FireMaster (polybrominated biphenyls) with colchicine in subacutely treated rats. Now we report absence of chromosome aberrations from FireMaster and absence of c-mitosis synergism of FireMaster and colchicine in male mice. For the study of chromosome aberrations groups of three mice received 0, 50, or 500 mg/kg FireMaster or 4.5 mg/kg triethylenemelamine (TEM) dissolved in dimethyl sulfoxide through a single stomach gavage administration. Five hours before killing the animals were injected with 5 mg colchicine/kg. Groups of 3 mice from each treatment killed 12, 24, and 48 hr after treatment. From the bone marrow of each of 36 mice 100 metaphases were scored for gaps, chromatid and chromosome breaks, rearrangements and pulverized chromosomes. Only TEM induced chromosome damage. For detection of synergism between FireMaster and colchicine, slides prepared for chromosome analysis were also scored for metaphase and mitotic indeces. Control mice for detection of synergism were treated as for the chromosome study but were not injected with colchicine. Approximately 1000 cells were scored from each of 72 animals for determination of metaphase and mitotic indeces. FireMaster did not show c-mitosis synergism with colchicine in mice. Treatment with FireMaster did not cause visually recognizable toxicity.

Detection of low copy number inversions in mouse genomic DNA with unidirectional PCR primers.

The recessive brachypodism (bp) mutation, located in the growth/differentiation factor 5 (GDF5) gene, causes highly specific skeletal changes in the limbs of brachypod mice. Although Southern blot analysis does not distinguish sequence disruptions in the GDF5 sequence of brachypod mice, sequencing and mapping GDF5 mRNA reveals the bp mechanism to be an inversion preceded by a small deletion. We report here a simple and sensitive method of bp detection from mouse genomic DNA. Previous bp detection used degenerative PCR sequencing. However, without automation, sequencing is a laborious effort for GDF5 inversion detection. The method developed utilizes two unidirectional primers in PCR (UP-PCR), which allow for quick and sensitive analysis of gel electrophoresed PCR products. UP-PCR of the GDF5 gene in wildtype mouse genomic DNA cannot amplify a fragment due to the unidirectional primers. However, UP-PCR of the GDF5 gene in bp mouse genomic DNA does amplify a fragment from the GDF5 gene. Amplification occurs because of the inverted fragment in bp GDF5. This fragment changes the direction of the second forward primer 180 degrees to the position of a reverse primer. UP-PCR detection of the bp inverted fragment is highly sensitive. Amplified fragments were obtained from the bp genomic DNA in the presence of wildtype genomic DNA in ratios up to 1:10(6), respectively. The sensitivity and simplicity of this method allow for quick, inexpensive, and reliable detection of the bp inversion.

Micronuclei in mice treated with monocrotaline with and without phenobarbital pretreatment.

Monocrotaline is a very potent toxin, producing significant effects of pneumotoxicity, hepatotoxicity, and teratogenicity, as well as carcinogenicity. In addition, the compound has been clearly shown to be mutagenic after metabolic activation. The goal of the experiments reported here was to confirm the reported clastogenesis induced by this agent in vivo and to evaluate the impact of modulation of metabolic activity by phenobarbital, a potent P-450 inducer (both Phase I and Phase II enzymes). The method used in addressing this problem relied on a new technique for monitoring clastogenesis in vivo, i.e., the acridine orange micronucleus assay method originally exploited by Hayashi et al. [1990]. The result of our experiments confirmed monocrotaline to be an effective clastogen in vivo, using the acridine orange method of assessment. The peak in induction of micronuclei occurred on the second day following intraperitoneal administration of the drug. Administration of phenobarbital prior to monocrotaline did appear to modulate the micronucleus induction. At 30 mg/kg bw monocrotaline, the pretreatment with phenobarbital appears to increase the intensity of monocrotaline clastogenesis, while the effect at higher doses (60 and 125 mg/kg bw) is a reduction in potency, presumably reflecting increased importance of Phase II metabolism for monocrotaline at these doses. Thus the study reported here confirms the potent in vivo clastogenesis of monocrotaline, and provides evidence for a dose-related shift in mechanism for the phenomenon.

Gelatin-substrate film technique for detection of acrosin in single mammalian sperm.

Sperm acrosin proteolytic activity in single sperm can be detected by a protein-free halo on a gelatin-substrate film. With current techniques, halos have variable sizes and are often absent because of unevenness of the hand-spread gelatin-substrate film. We prepared gelatin-substrate films with a coating machine. Using these films, halos were formed uniformly throughout the gelatin-substrate films in the vicinity of single mammalian sperm. The level of acrosin activity as determined by halo diameters was human greater than dog greater than squirrel monkey greater than mouse greater than rat. This simple and reproducible technique may be used to diagnose infertility due to decreased acrosin activity, as a screening method for identifying compounds with male sterility effects, and for identifying agents with developmental and/or genetic effects.

Ethylnitrosourea treatment increases lectin binding to mouse germ cells.

Germ cell toxicity was assessed by investigating the binding of FITC-labeled lectins to mouse testis cells before and 18 days after treatment with ethylnitrosourea (ENU). Flow cytometry of testis cells dual-labeled with FITC-lectin plus the DNA stain, propidium iodide, allowed analysis of haploid (1C), diploid (2C), and dividing (4C) cell populations. Soybean agglutinin, wheat germ agglutinin, concanavalin A and Limax flavus agglutinin bound to normal mouse testis cells containing 1C, 2C or 4C DNA. Asparagus pea lectin and Bandeireae simplicifolia I isolectin B4 did not. ENU treatment reduced the number of testis cells and increased lectin binding, particularly of those lectins which bound to untreated cells.

Testes weight reflect ethylnitrosourea induced histopathology in mice.

The powerful mutagen/carcinogen ethylnitrosourea (ENU) decreases testis weight in mice. A histopathological cause was determined for this effect. Groups of 3 mice were injected with 0, 50, 100 or 200 mg ENU/kg b.w. and were killed 1, 2, 4, 5, 7, 9, 13 or 15 weeks later. Microscopic examination of PAS-hematoxylin-stained sections showed a dose-and-time-dependent loss of germ cells from the seminiferous tubules 1-7 weeks after treatment followed by recovery from the damage. Testis weight decrease and recovery followed a similar course.

The effect of hydroxyurea and mitomycin C on sperm motility in mice.

The mutagen, mitomycin C, and the teratogen, hydroxyurea, were found to decrease sperm motility in mice in a dose-dependent manner. Positive results with these compounds suggest that sperm motility may have been decreased through either mutations or developmental disturbances. Sperm motility can be determined quickly and may be done in conjunction with a sperm-morphology assay.

Mitomycin C- and streptozotocin-induced motility and numerical sperm variants in mice.

Groups of HA (ICR) albino male mice were injected once i.p. with o, 2.5 or 5.0 mg/kg mitomycin C (MC), or 2.65 or 26.5 mg/kg streptozotocin (SZ). 4 weeks after treatment each male was mated with 2 untreated CF1 albino females. 5 weeks after treatment the males were killed for determination of sperm motility and count from the cauda and vas. Both agents decreased sperm motility and count in a dose-dependent manner in the treated males. The effect is believed to be caused primarily by interference with gene expression involved in spermatogenesis. The females mated to treated males showed decreased percentage pregnancy and had few progeny. Mean percentage motility and sperm count was significantly lower in the F1 male progeny of MC- or SZ-treated males compared to control males. 50-60% of F1 males form treated male parents had percentage sperm motility below control range. Decreased motility and count in the F1 progeny of treated males may have been caused by gene mutations and/or chromosomal aberrations. The method which is presented may be useful for detecting mutations and/or developmental effects.

Growth of Chinese hamster ovary (CHO) cells in the presence of MMC in low-serum medium.

Chinese hamster ovary (CHO-WBLT) cells growing in McCoy's 5a with 10% fetal bovine serum (FBS) were adapted to 0.5% FBS in CHO-1 Complete Media System, a serum-free medium from Ventrex. Cells in these two media were exposed to 10(-7) M and 10(-8) M mitomycin C (MMC) for 24 h. Comparison of cell growth over 10 days showed that cells in 0.5% serum proliferate, though at a slower rate than cells in 10% serum. Treatment with MMC revealed that at 10(-7) M, MMC is cytotoxic to cells to both the media; at 10(-8) M, MMC is non-cytotoxic to cells in both media.

Mutagenicity testing of benomyl, methyl-2-benzimidazole carbamate, streptozotocin and N-methyl-N'-nitro-N-nitrosoguanidine in Salmonella typhimurium in vitro and in rodent host-mediated assays.

The fungicide benomyl and its commercial preparations Fundazol 50WP and Benlate 50WP and the benomyl metabolite methyl-2-benzimidazole carbamate and its commercial preparation MBC 50WP were tested for mutagenicity in in vitro spot tests, in microsomal plate assay, in liquid-culture treatments, or in rodent host-mediated assay. The base-pair substitution Salmonella typhimurium mutant hisG46 and the hisG46-bearing uvrB excision-repair-deficient mutants TA100, TA1530, TA1535 or TA1950 were used as test organisms. Complete genotypic information of these mutants is given in Ames et al. [2]. Captain 50WP, streptozotocin (SZN), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 2-aminopurine and N-acetylaminofluorene were used as positive control compounds. In nonoverlay spot tests Benlate 50WP was not mutagenic over a dose range of 50-5000 microgram/spot in hisG46 and TA1535. In overlay spot tests 50 or 100 microgram/spot Benomyl, MBC, Fundazol 50WP, Benlate 50WP and MBC 50WP were tested in hisG46, TA1530 or TA1950. Only a non-commercial MBC sample at 100 microgram/spot showed weak mutagenic activity in hisG46. In microsomal activation plate assay MBC, benomyl, Fundazol 50WP and Benlate 50WP were tested in TA100 over a dose range of 50-2000 microgram/plate. None of the compounds showed mutagenicity. In a 20-h liquid-culture treatment 10, 100, 1000 and 10 000 microgram/ml Fundazol 50WP were not mutagenic in TA 30. In 1-h liquid-culture treatments benomyl, Benlate 50WP or Fundazol 50WP failed to induce mutations in hisG46, TA100 or TA1950 over a dose range of 0.25-1000 microgram/ml. Appropriate positive controls were mutagenic in each experiment. The consistently negative results in this study with commercial MBC and benomyl preparations are contrary to positive results reported earlier with similar methods and similar commercial preparations. Possible reasons to explain the different results are presented. The alkylating agents SZN and MNNG induced fewer mutations in TA1530 and TA1950 uvrB excision-repair-deficient strains than in the hisG46 excision-proficient strain, indicating that with these mutagens excision-repair is also a mutation-prone process. In rodent host-mediated assays with Fundazol 50WP in mice 3 consecutive subcutaneous hourly doses of 500 mg/kg in hisG46 and TA1950 and in rats or mice an oral dose of 4000 mg/kg in TA1950 were not mutagenic. The positive control SZN was mutagenic.

Bone marrow and lymphocyte cytogenetics of rhesus monkeys (Macaca mulatta) treated with the clastogen Mitomycin C.

Rhesus monkeys (Macaca mulatta) were used to determine their effectiveness as experimental animals for different cytogenetic tests with mitomycin C (MC). The micronucleus test (MNT and/or chromosome analysis of blood and bone marrow were made before and/or after the treatment with mitomycin C. Thus, the controls data and treated data were obtained from the same animals. With the employed methology, the micronucleus test could not be performed on living animals. Less chromosomal damage was detected in the micronucleus test of post-mortem samples than in the chromosome analysis of bone marrow. No influence by the mutagen could be observed in lymphocyte chromosomes at any of the different times of analysis. In contrast to this, bone-marrow chromosomes seemed to be highly affected by mitomycin C at day 1, 2 and 3 after injection. However, before treatment and at day 14, 16 and 17 after treatment there was no visible increase in chromosomal aberration in bone marrow.

Rhesus monkey (Macaca mulatta) model in genetic toxicology mitomycin C clastogenicity in germ cells.

The value of rhesus monkeys (Macaca mulatta) as a genetic toxicology model is limited by their scarcity, expense, and impracticality of progeny testing. However, in some special circumstances, e.g., accidental exposure of humans to potential mutagens, rhesus monkeys or other primates may provide a superior animal model to help to cope with a difficult public health situation. Using the testis as a target organ we found that when primary spermatocytes were treated in pre-leptotene stage with 1 mg mitomycin C/kg body weight, the frequency of exchanges, fragments, sex-chromosome and autosomal univalents increased significantly at diakinesis-metaphase I. This response was absent in cells treated during diplotene, late pachytene or during spermatogonial stages. We suggested that animals should be evaluated not only for genetic toxicology parameters, but also toxicologically, histologically, behaviorally, for carcinogenesis and seminal cytology. Whenever possible, the animals should be recycled.

Mammalian host- and fluid-mediated mutagenicity assays of captan and streptozotocin in Salmonella typhimurium.

The mutagenicity of captan and of streptozotocin was tested in vivo by reversion of hisG46 base-pair substitution histidine auxotrophs of Salmonella typhimurium in the peritoneal cavity or in blood, plasma or urine of rats or mice. Genetic response was determined by the frequency of revertants (quantitative test) or by the number of revertants per plate (semiquantitative test). In quantitative HMA captan gave negative results following 3 hourly 500 mg/kg s.c. doses or 1000 mg/kg oral dose in mice with the hisG46 mutant or 2000 mg/kg oral dose in rats with the hisG46, uvrB (TA1950) mutant. The positive control SZN induced many reversions at 0.5 mg/kg i.p. or 10 or 100 mg/kg oral doses. In semiquantitative in vivo blood or urine assays captan gave negative results after a 250 mg/kg oral dose with hisG46. SZN in the same experiment gave positive results in both semiquantitative and quantitative in vivo blood assays following 1000 mg/kg i.p. or 2000 mg/kg oral doses in the rat with TA1950. Rat blood mixed with captan for 45 min before adding TA1950 cells inactivated 1000 mug captan/ml but not 5000 mg/ml in the semiquantitative test. Corresponding figures in the quantitative test were 500 mu/ml and 1000 mug/ml. Rat plasma inactivated the mutagenicity of about 10 times less captan than rat blood. Human blood inactivated about as much captan as rat blood. The mutagenicity of captan was inactivated more efficiently than of SZN by blood. The results of the experiments suggested that captan's mutagenicity is probably inactivated by glutathione of the erythrocytes. Rat S-9 liver microsomal fraction also strongly decreased captan's mutagenicity in a semiquantitative test with the R factor, uvrB, hisG46 (TA100) mutant.

An evaluation of the host-mediated assay and body fluid analysis. A report of the U.S. Environmental Protection Agency Gene-Tox Program.

The methodologies and status of the Host-Mediated Assay were reviewed using the published literature available up to June 1980. The Working Group reviewed 274 documents, including abstracts, research articles, review articles, and publicly available contracts and grant final reports. From this group, abstracts and reviews were rejected from critical evaluation. 77 documents were accepted and reviewed by the Working Group and the test results summarized. These selected documents yielded 208 chemicals that were evaluated in th host-mediated assay. Of these chemicals, 133 were mutagenic in this assay with one or more indicators. 76 chemicals, several of which are not considered to be carcinogenic, were not detected by any of the indicators. Of the 208 chemicals, 125 had been tested in carcinogenicity assay in rodents. 90, or 71%, of the carcinogens were detected as mutagens in the Host-Mediated Assay. In several cases, those carcinogens not detected may have been negative because of improper selection of the indicator. The Working Group concluded that the Host-Mediated Assay is an important test in mutagenicity/carcinogenicity research and that, by proper selection of protocols and indicators, valuable information can be gained that otherwise would be overlooked strict, in vitro assays.

Enhancing cervical cancer detection using nucleic acid hybridization and acetic acid tests.

Acetowhitening of abnormal cervical epithelium has been suggested as an indicator of increased cervical cancer risk. The presence of human papillomavirus (HPV) types 16, 18, 31, 33 and 35 may also indicate increased cervical cancer risk. Hence, tests that detect these two abnormal conditions may augment that Papanicolaou smear (Pap test) as predictors of cervical cancer risk. The cohort consisted of 145 women aged 14 to 47 (mean 21 years) attending health clinics. Thirty women (20.6 percent) showed acetowhitening of the cervical epithelium following exposure to vinegar of 4-percent acetic acid content. Fourteen (9.6 percent) had a positive Pap test and 13 (9 percent) carried a cervical HPV infection as determined by the commercially available ViraPap and ViraType nucleic acid tests. Statistical analysis of the data showed a positive correlation between Pap, ViraPap and acetic acid tests results. The acetic acid test and the nucleic acid tests were the sole positive tests for 21 (14.5 percent) and nine (6.2 percent) women, respectively. Four women with negative Pap results were infected with HPV types previously shown to have an association with cervical intraepithelial neoplasias, carcinoma in situ and cervical cancer. The authors have concluded that the acetic acid and nucleic acid tests detect women at risk for cervical cancer who would not have been detected by the Pap test alone.