Heinrich V. Malling

An evaluation of the mouse sperm morphology test and other sperm tests in nonhuman mammals. A report of the U.S. Environmental Protection Agency Gene-Tox Program.

The literature on the mouse sperm morphology test and on other sperm tests in nonhuman mammals was reviewed (a) to evaluate the relationship of these tests to chemically induced spermatogenic dysfunction, germ-cell mutagenicity, and carcinogenicity, and (b) to make an interspecies comparison to chemicals. A total of 71 papers were reviewed. The mouse sperm morphology test was used to assess the effects of 154 of the 182 chemical agents covered. 4 other murine sperm tests were also used: the induction of acrosomal abnormalities (4 agents), reduction in sperm counts, (6 agents), motility (5 agents), and F1 sperm morphology (7 agents)). In addition, sperm tests for the spermatogenic effects of 35 agents were done in 9 nonmurine mammalian species; these included analyses for sperm count, motility, and morphology, using a large variety of study designs. For the mouse sperm morphology test, 41 agents were judged by the reviewing committee to be positive inducers of sperm-head shape abnormalities, 103 were negative, and 10 were inconclusive. To evaluate the relationship between changes in sperm morphology and germ cell mutagenicity, the effects of 41 agents on mouse sperm shape were compared to available data from 3 different mammalian germ-cell mutational tests (specific locus, heritable translocation, and dominant lethal). The mouse sperm morphology test was found to be highly sensitive to germ-cell mutagens; 100% of the known mutagens were correctly identified as positives in the sperm morphology test. Data are insufficient at present to access the rate of false positives. Although it is biologically unclear why one might expect changes in sperm morphology to be related to carcinogenesis, we found that (a) a positive response in the mouse sperm morphology test is highly specific for carcinogenic potential (100% for the agents surveyed), and (b) overall, only 50% of carcinogens were positive in the test (i.e., sensitivity approximately equal to 50%). Since many carcinogens do not produce abnormally shaped sperm even at lethal doses, negative findings with the sperm test cannot be used to classify agents as noncarcinogens. We conclude that the mouse sperm morphology test has potential use for identifying chemicals that induce spermatogenic dysfunction and perhaps heritable mutations. Insufficient numbers of chemicals agents have been studied by the other sperm tests to permit similar comparisons. A comparison of 25 chemicals tested with sperm counts, motility, and morphology in at least 2 species (including man, mouse and 9 other mammals) demonstrated good agreement in response among species. With further study, interspecies comparisons of chemically induced sperm changes may be useful for predicting and evaluating human effects.

An evaluation of human sperm as indicators of chemically induced alterations of spermatogenic function: A report of the U.S. environmental protection agency Gene-Tox program

To evaluate the utility of sperm tests as indicators of chemical effects on human spermatogenesis, the literature on 4 sperm tests used to assess chemically induced testicular dysfunction was reviewed. The tests surveyed included sperm count, motility, morphology (seminal cytology), and double Y-body (a fluorescence-based test thought to detect Y-chromosomal nondisjunction). There were 132 papers that provided sufficient data for evaluation. These reports encompassed 89 different chemical exposures: 53 were to single agents; 14 to complex mixtures; and 22 to combinations of 2 or more identified agents. Approximately 85% of the exposures were to experimental or therapeutic drugs, 10% were to occupational or environmental agents, and 5% were to drugs for personal use. The most common sperm parameter studied was sperm count (for 87 of the 89 exposures reviewed). Sperm motility was evaluated for 59 exposures, morphology for 44, and double Y-bodies for only 4. The 89 exposures reviewed were grouped into 4 classes: those which adversely effected spermatogenesis, as measured by one or more of the sperm tests (52); those suggestive of improving semen quality (11); those showing inconclusive evidence of adverse effects from exposure (14); and those showing no significant changes (12). Since the reviewed reports had a large variety of study designs, and since every attempt was made to include all reports with interpretable data, these classifications were based on reviewing committee decisions rather than on uniform statistical criteria. This review gives strong evidence that human sperm tests can be used to identify chemicals that affect sperm production, but because of our limited understanding of underlying mechanisms, the extent to which they can detect mutagens, carcinogens or agents that affect fertility remains uncertain. For the very few agents studied with both human and mouse sperm tests, similar test-responses were seen; thus sperm tests in mice and other laboratory mammals may have a potential role in hazard identification. An overall comparison of the 4 human sperm tests suggests that no one test is biologically more responsive than another; all of them may thus be needed when testing for chemically induced changes from agents of unknown activity. This review also gives evidence that sperm tests can be used to assess the extent and the potential reversibility of induced spermatogenic damage. The reviewing committee recommends further studies to determine (a) the dose-response characteristics of the human sperm tests, (b) details of the reversibility of induced changes with time after exposure, (c) the relative responses in the 4 sperm tests in exposed individuals, (d) the mechanism of action, (e) the biological and genetic implications of chemically induced effects, and (f) the comparison of responses among different species for risk assessment. The reviewing committee outlines specific considerations for planning new sperm studies on chemically exposed men.

Genetic effects of crocidolite asbestos in Chinese hamster lung cells

Chinese hamster lung cells cultured in the presence of crocidolite asbestos displayed inhibition of cell growth. Cell death was directly associated with the phagocytosis of larger fibres as observed with the aid of trypan blue. Water soluble components of crocidolite did not appear to inhibit cell growth. Chromosomal aberrations were induced by the asbestos. The aberrations were confined mainly to structural aberrations--breaks and fragments. Electron-microscopic preparation indicated that asbestos was readily contained in phagosomes. Phagocytosed asbestos appeared to be a weak mutagen in its ability to induce gene mutation at the hypoxanthine-guanine phosphoribosyl transferase locus.

Mutagenic activation or dimethylnitrosamine and diethylnitrosamine in the host-mediated assay and the microsomal system

Summary Dimethylnitrosamine (DMN) and diethylnitrosamine (DEN) were tested for the formation of mutagenic metabolites in vitro using liver microsomal preparations from mice or rats or in vivo in the host-mediated assay using mice or rats as hosts. The indicator organism for mutagenesis was strain G-46 of Salmonella typhimurium (mutations were scored as histidine reversions). In the host-mediated assay, the bacteria were injected into the peritoneal cavity. DMN gave approximately the same maximum reversion frequencies under host-mediated assay condition whether mice or rats were used as hosts. In comparison, the mouse liver microsome preparation was more than ten times more active than the rat liver microsome preparation. DEN showed almost no mutagenic effect in the host-mediated assay. DENs mutagenic action was clearly shown in the mouse liver microsome system, although its activity was much lower than DMN in the same system.

Metabolic activation of dimethylnitrosamine and diethylnitrosamine to mutagens

Abstract Dimethylnitrosamine (DMN) and diethylnitrosamine (DEN) are not mutagenic by themselves, but they can be converted by mammalian enzymes to highly mutagenic products. As indicators for mutagenic activity, Neurospora crassa and Salmonella typhimurium were used. The ad -3 forward-mutation system was used to detect specific locus mutations; mutants in this system can range from multi-locus deletions to leaky mutations. The induction of mutations in S. typhimurium is detected as induction of histidine revertants of the histidine-requiring strain G 4 6. The activation of DMN is microsomal, inhibited by SKF 525-A, and requires co-factors. The activating enzyme is induced in mice by pretreatment with phenobarbital, 3-methylcholanthrene and butylated hydroxytoluene. The mutagenic activity of the reaction products is directly correlated with the metabolic formation of formaldehyde with and without induction by 3-methylcholanthrene and across strains of mine. Formaldehyde does not contribute to the mutagenic activity of the reaction products. It is clear from the data that the reversion sites in G 4 6 are more sensitive than the ad -3 loci of Neurospora crassa to the mutagenic action of DMN metabolites formed by mammalian liver. The microsomal assay is a few orders of magnitude more sensitive than the intraperitoneal host-mediated assay, and the intrahepatic host-mediated assay is a few orders of magnitude more sensitive than the in vitro microsomal system.

Use of ΦX174 as a shuttle vector for the study of in vivo mammalian mutagenesis

The most promising new techniques for the study of in vivo mammalian mutagenesis make use of transgenic mice carrying a recoverable vector. Mutation systems in mammals can be based on the selection of altered phenotypes among cells sampled from the whole animal, but they are then limited to the very few cell types in which the marker gene is expressed. Such systems require both in vivo and in vitro cell proliferation for expression and verification of the mutations. To avoid these complications, the study of mutations in most tissues must be based on the detection of genetic alterations in a vector that is independent of the phenotype of the mammalian cell. The vector is only a small portion of the mammalian genome, and many of the procedures for recovering the vector are inhibited by the host DNA. For this reason, partial purification is necessary. The purification is made possible by using vectors which are not cut by restriction enzymes that cut the host DNA to pieces of an average size considerably smaller than the vector. The efficiency for measuring mutation frequencies depends on the number of vectors which can be recovered from a certain amount of DNA and is affected by the number of vectors per mammalian genome and the transfection efficiency of the partially-purified vector. In order to avoid selection against or for the spontaneous or induced mutations, the transfection efficiency of the vector from the transformed DNA and of the pure vector DNA should be of the same order of magnitude. Differences in the response to mutagens between the mammalian genome and the procaryotic vector may be expected due to the lack of unique mammalian topographical features in the vectors. Any mutation induction which depends preferentially on these unique features of the mammalian genome may not be detected in a shuttle vector system unless the vector has been engineered or specifically designed to include such topographical characters. The shortcoming of short-term tests that use mutagenicity for predicting human carcinogenicity is usually lack of correlation between mutagenesis in the short-term tests and the corresponding results in carcinogenesis bioassays in mammals. One factor which could contribute to the lack of correlation between the short-term test systems and the bioassays is that we are comparing mutations in totally different genes in different organisms. By using the phi X174 shuttle system, one of the variables may be eliminated.(ABSTRACT TRUNCATED AT 400 WORDS)

Mutagenesis of φX174 am3 cs70 incorporated into the genome of mouse L-cells

Abstract The objective of our work with φX174 has been to develop a shuttle vector that can be used comparatively in bacterial cells, different types of mammalian cells, and possibly in the various tissues of transfenic mice, with a constant mechanism for detection and analysis of mutations independent of any host-cell type. Toward that end, we have efficiently rescued ΦX174 am3 cs70 that is host-silent and stably integrated into the genome of mouse L-cells. The particular mouse L-cell line contains tandem arrays, single copies, and fragments of ΦX that, upon restriction enzyme excision, can result in 5 potentially active copies per diploid genome. The excised ΦX DNA is recovered by column chromatography, ligated, and transfected into higly competent spheroplasts. The Rescue Efficiency, defined as the number of viable phages produced out of the total number of potentially recoverable copies, is approx. 10−3. The Recovery Ratio, defined as the Rescue Efficiency for chromosomally-integrated phage DNA divided by the Rescue Efficiency for ΦX am3 cs70, is close to one. Mouse L-cels containing the integrated ΦX174 am3 cs70 were treated with 20 mM ethyl methanesulfonate. The reversion frequency of am3 among progeny phages rescued from treated cells was 1.4 × 10−5 (193 revertants in 1.4 × 107 phages). This is significantly higher than the 5.8 × 10−7 reversion frequency of am3 (7 revertants in 1.2 × 10−7 phages) among progeny phages rescued from untreated cells.

A double antibody radioimmunoassay for mouse hemoglobins: Use of polyethylene glycol in conjunction with the second antibody

Abstract A double antibody radioimmunoassay (RIA) system is described for detection of small quantities of hemoglobins. Mouse (C57BL/6) hemoglobin and horse anti-mouse hemoglobin antiserum were used to develop the system. The first phase of the RIA, i.e., the initial reaction between the antigen and the antibody, was found to be complete within 24 h. The reaction proceeded better at 4°C than at 25°C. The second phase, i.e., separation of bound from unbound antigen, was achieved by precipitation with a second antibody (goat anti-horse IgG) and polyethylene glycol (PEG). A 50 g/l concentration of PEG was found to be best suited for the assay. Mixing of all the reagents together was found to decrease the binding as compared to the system in which second antibody and PEG were added after completion of the first phase. Maximum precipitation of the complex took place within 30 min after the addition of the second antibody and 1 h after the addition of 50 g/l PEG. The RIA system described here combines the conventional double antibody RIA with the PEG method. This method has decreased the amount of time necessary for precipitation from 24 h (or longer) to 1 h. Large molecular weight antigens could not be estimated in the conventional PEG method because of their insolubility in 200 g/l PEG utilized in the assay. The use of a low concentration of PEG along with the second antibody in the method described here allows the estimation of large molecular weight antigens. This double antibody-PEG method has a general applicability for small as well as large molecular weight antigens.

Effect of procarbazine treatment of mice on α-glycerolphosphate dehydrogenase activity and frequency of selected abnormalities in sperm

The in situ activity of mitochondrial alpha-glycerol phosphate dehydrogenase (alpha-GPD) as well as 2 specific sperm abnormalities [headless and disorganization of the mitochondria assembly (PL-type)] have been studied in sperm from mice treated with single doses of procarbazine (100, 200, 400, 600 and 800 mg/kg) at intervals up to 55 days after treatment. The frequency of sperm without active alpha-GPD and the frequency of the morphology variants increased with increasing dose of procarbazine. The variance in the level of alpha-GPD among sperm with active enzyme and the variance between animals also increased with increasing dose. The lowest effective dose was 200 mg/kg and there were no observable effects 85 days after treatment.

Mutagenesis of a single AT basepair in mice transgenic for PhiX174 am3 cs70: I. Spleen and testis

Mutations induced in a single AT base pair were studied in spleen and testis by using mice transgenic for PhiX174 am3, cs70 and ethylnitrosourea (ENU) as the mutagen. The transgenic mice were produced on the C57BL6/J background. The line (am54), which carries 50 copies of PhiX per haploid genome integrated in a tandem array, was selected for experimental use and was maintained by random breeding. The animals for mutagenesis studies were produced by mating homozygous am54 males to wildtype C57BL6/J females. Hemizygous male offspring (8 to 10 weeks old) from this cross were injected i.p. with 150 mg ENU per kg and were euthanized 3, 10 or 110 days after treatment. The spontaneous revertant frequency in the spleen was 1.42 x 10(-6) per plaque forming unit (pfu) and in the testis it was 1.41 x 10(-6) per pfu. There was no significant difference between the two tissues. In spleen, it was not until 110 days after ENU treatment that the average revertant frequency among treated animals was significantly higher than the revertant frequency among the control animals. In spleen, the induced frequency of basepair substitutions in the center AT basepair in the am3 nonsense codon was 2 x 10(-6). Also at this post-injection interval the variance of revertant frequencies in the spleen was not different from control variance. In testis, the average revertant frequency 110 days post ENU injection was not significantly different from the control. However, two important observations were made regarding the testis data. First, one animal had a significantly increased revertant frequency 110 days after ENU treatment in comparison to the other four animals in the group that had revertant frequencies equal to or lower than the average control frequency. Second, the variance of revertant frequencies in the testis among the treated animals increased as the post injection period increased. Taken together, these observations may indicate that the revertants formed large clusters in one testis sample.