Gyula Ficsor

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.

A report of the U.S. environmental protection agency gene-tox program: Part I Host-mediated assay☆

Abstract 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 the 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 by strict, in vitro assays.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.

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.

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.

Germ cell-specific decrease of acrosomal proteolytic activity, sperm motility, and number in mitomycin C-treated mice.

Assessment of mammalian sperm acrosomal proteolytic activity, sperm motility, and sperm count may be useful for detecting mutagens, carcinogens, developmentally active agents, and antifertility effects. Groups of six albino mice were given a single i.p. injection of 5 mg/kg mitomycin C (MC) or saline. One treated and one control group of mice were killed 1, 3, 5, 7, or 10 weeks later. Sperm extracted from the vasa deferentia at these killing times were derived from cells treated as spermatozoa, spermatids, preleptotene-late spermatogonial cells, spermatogonial cells, and spermatogonial stem cells. In sperm derived from treated preleptotene or spermatogonial cells, the sperm count, sperm motility, and acrosomal proteolytic activity were decreased significantly. Acrosomal proteolytic activity was also decreased in sperm from spermatogonial stem cells. None of these sperm phenotypes were decreased in treated spermatozoa and spermatids. We propose the hypothesis that induced loss of sperm motility and acrosomal proteolytic activity in single spermatozoa derived from MC-treated spermatogonial cells is caused by mutational or developmental effects, whereas in preleptotene-derived and late-spermatogonium-derived sperm similar dysfunction results from developmental effects. Our data support the hypothesis indirectly. Since a low sperm count is correlated with decreased fertility and acrosomal proteolytic activity is essential for penetration of the zona pellucida by the sperm, the presence of these sperm phenotypes may help to detect chemicals with antifertility effects.

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.

Differential mutagenicity of streptozotocin analogs of the carbohydrate moiety

The mutagenicity of streptozotocin (SZN), 8 of its analogs and N-msthyl-N-nitrosourea (MNU) were compared in Salmonella typhimurium. SZN and its analogs carry MNU attached to the carbohydrate moiety at the C-2 position. The C-1 analogs tested were α- and β-methyl-SZN, α-ethyl-SZN, β-propyl-SZN, α- and β-butyl-SZN; in 2 analogs glucose was replaced by α- or β-inositol. When the ability of these compounds to revert the hisG46 auxotroph was compared, they fell into 4 groups which differed by about 10-fold in mutagenicity from one another. The most mutagenic was (i) SZN, followed by (ii) β-methyl-SZN; (iii) α-methyl-SZN, α-ethyl-SZN, β-propyl-SZN, α- and β-butyl-SZN; (iv) α and β-inositol-MNU. These results suggest that the presence of the glucose moiety is conducive to a high level of mutagenicity of SZN. Alterations of the glucose moiety by addition of larger alkyl groups, especially in the α position lead to decreased mutagenicity. The least mutagenic analogs are those in which the glucose moiety is replaced by inositols. The mutagenicity of SZN, β-methyl-SZN and of β-butyl-SZN was also compared in a mouse tissue-mediated assay. SZN was about 500-fold more mutagenic than its β-methyl analog, while the β-butyl analog was not mutagenic. Depletion of SZN and 4 of its analogs from the medium in presence of bacteria was determined spectrophotometrically. The more mutagenic compounds were depleted more rapidly but the quantitative differences in mutagenicity between these compounds could not be accounted for by depletion alone.

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.

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.

Preparation of mouse-sperm DNA for PCR.

The polymerase chain reaction (PCR) can be used to amplify a mutation in a single sperm to a detectable level (Li et al., 1988). With modification, the PCR may allow for the development of a direct germ-cell mutation assay (Ficsor, 1988). We have encountered two problems with using mousesperm samples for PCR. The first is that sperm extracted from the vas deferens contains non-germ cells. The DNA from these cells will also show PCR and interfere with the interpretation o f the germcell mutation data. The second problem is that DNA from mouse-sperm samples does not show PCR as well as DNA from somatic cells. We report on methods to solve these problems.