Bleomycin: female-specific dominant lethal effects in mice.

Limited comparative data in mice indicate that chemical mutagens that induce dominant lethal mutations in males are not necessarily effective in females, but those which are effective in females are generally equally or more effective in males. Recently, however, a few chemicals have been identified that are female-specific with respect to induction of dominant lethal mutations. The antitumor antibiotic adriamycin is among them. Another antitumor antibiotic, bleomycin was examined for its ability to induce dominant lethal mutations in the reproductive cells of male and female mice. No dominant lethal or cytotoxic effects were observed in males treated with bleomycin, even at a maximum tolerated dose. In females, on the other hand, a dose nearly 1/4 of that used in males induced not only a high level of dominant lethal mutations but also killed oocytes in certain stages of follicular development. The effectiveness of bleomycin in inducing dominant lethal mutations in mouse oocytes makes it a valuable tool for investigating whether gonadal transport, inherent differences in the configuration of chromatin in the germ cells of the two sexes or other factors are responsible for the differential susceptibility to bleomycin, which implies potential gender-specific genetic risk in cancer chemotherapy.     

The dominant lethal effect of dietary triethylenemelamine.

Triethylenemelamine (TEM) was administered in the diet to adult male mice at doses of 0.1, 0.3, 1, 10 or 50 mg/kg body weight for 45 days or at doses of 0.1 or 0.3 mg/kg b.w. for 10 days. As a comparison, male mice were treated intraperitoneally with 5 daily doses of 0.25 or 0.5 mg TEM/kg b.w. At the end of the treatment period, males were mated sequentially with 2 untreated virgin females each for 2 or 3 weeks. Near mid-pregnancy the number of implantation sites and fetal deaths were determined. TEM, administered in the diet at 10 or 50 mg/kg b.w. for 45 dyas, was lethal to male mice. Surviving males from the 1 mg/kg level failed to impregnate any females during the two matings. TEM, given in the diet at 0.1 or 0.3 mg/kg for 10 or 45 dyas, decreased fertility and increased dominant lethal mutations in a dose and time dependent manner. These results were comparable to those obtained from males treated i.p. with TEM at 0.25 or 0.5 mg/kg b.w.     

Induction of specific-locus and dominant lethal mutations in male mice in the low dose range by methyl methanesulfonate (MMS)

Methyl methanesulfonate (MMS) induces specific-locus and dominant lethal mutations in spermatozoa and spermatids of mice. A dose of 15 mg/kg b.w. of MMS induces 9% dominant lethal mutations in the most sensitive germ-cell stages, corresponding to the mating intervals 5-8 and 9-12 days post treatment. A dose of 150 mg/kg b.w. of MMS in the same mating intervals induces 100% dominant lethal mutations. The sensitivity pattern for the induction of dominant lethal and specific-locus mutations is the same. In the mating interval 5-8 days a dose of 20 mg/kg b.w. of MMS induced 3.8 x 10(-5) mutations per locus per gamete. The yield of specific-locus and dominant lethal mutations in the low dose range increases proportionally with the dose. A dose given in 2, 4 or 5 fractions yields the same frequency of mutations as a single injection of the total dose. The additivity of small doses proves that the pre-mutational lesions are not or only partially repaired in these stages and that MMS is not or only partially detoxified. In addition, the frequency of dominant lethal and specific-locus mutations depends on the germ-cell stage.     

Mechanism of inactivation and mutagenesis induced by ethyleneimine in Drosophila germ cells. V. Relation of complete and mosaic mutations during the supplementary action of high temperature]

Supplementary effect of high temperature (37 degrees C) an hour after the treatment of mature sperms of Drosophila with ethylene imine resulted in an increased level of the inactivation (the frequency of dominant lethal mutation). The frequency of complete mutations (recessive sex-linked lethal mutations) increased by the supplementary effect of high temperature at low doses of E1, and it did not change under a comparatively high dose of the mutagen. The frequency of mosaic mutations decreased under the effect of high temperature at both doses of E1. No effect of high temperature was observed in 4 and 24 h after the E1 treatment. The results obtained are discussed in connection with the proportion of inactivation and mutagenesis under the effect of chemical mutagens on Drosophila germ cells.     

Comparison of radiation-and chemically-induced dominant lethal mutations in male mice

Ionizing radiation-induced dominant lethal mutations in all spermatogenic stages. After irradiation of male mice with 200 R the yield of induced mutations in early spermatids was twice the yield in spermatozoa, late spermatids, and spermatocytes. After irradiation with 400 R or 800 R the spermatocytes were the most sensitive stage for the induction of dominant lethal mutations. The frequency of radiation-induced dominant lethal mutations in postspermatogonial stages was dose-dependent. The yield of dominant lethal mutations in spermatogonia was independent of the dose.     

Induction of specific-locus and dominant lethal mutations in male mice by busulfan

The chemotherapeutic agent busulfan was tested for the induction of dominant lethal and specific-locus mutations in male mice. A dose of 5 mg/kg b.w. of busulfan induces dominant lethal mutations in spermatozoa. A dose of 20 mg/kg b.w. induces dominant lethal mutations in spermatozoa and spermatids. A total of 83,196 offspring were scored in the specific-locus experiments. Busulfan-induced specific-locus mutations were recovered in spermatozoa and spermatids, but not in spermatogonia. The sensitivity patterns for the induction of dominant lethal and specific-locus mutations by busulfan in germ cells of male mice are similar but not identical.     

Genetic damage in domestic mice inhabiting in the areas with elevated background radiation

Genetic effects of irradiation in males of wild house mice which were caught in the region of Chernobyl Nuclear Power Station were studied. The dose rate on the ground surface varied from 0.04 to 200 mR/h of gamma-irradiation. The increasing yield of dominant lethal mutations was only observed in males from the most contaminated sector. Reciprocal translocations were observed in spermatocytes of mice at all the levels of contamination. The rate of reciprocal translocations was relatively low and increased linearly with the elevation of the dose rate. The extent of testis damages increased also, as the dose rate grew. The frequency of abnormal sperm heads, the yield of recessive lethal mutations, litter size and radiosensitivity of the first progeny were not changed, depending on the dose rate.     

Genetic effects induced by nickel sulfate in germline and somatic cells of WR mice

We examined the effects of nickel sulfate at doses 0.5 to 5.0 mg/kg (LD50) on the frequency of dominant lethal mutations and two-strand DNA breaks (TSBs) in germline cells and on an increase in frequency in gene mutations W(y) in pigment cells of first-generation mice. The results indicated that spermatogenesis stages most sensitive to nickel sulfate (at a dose of 1.0 mg/kg) are spermatozoids, early spermatids, late spermatocytes, and stem spermatogonia. No statistically significant increase in the total TSB level was detected in spermatozoids 4 weeks after exposure. At the same time, a significant (P < 0.05) increase in percentage of cells with an extremely high level of DNA fragmentation (supposedly apoptotic cells) was observed upon exposure at a dose of 0.5 mg/kg. Nickel sulfate at doses of 5.0 and 1.0 mg/kg induced a marked increase in the c-kit gene expression in pigment cells of heterozygous first-generation WR mice as compared to control (P < 0.001). It was shown that the nonobservable adverse effect level (NOAEL) of nickel sulfate on the dominant lethal mutation frequency and gene mutations was 1/200 LD50, while the lowest observable adverse effect level (LOAEL) was 1/100 LD50.     

Dominant-lethal test results with known mutagens in two laboratories

During investigations of the potential dominant lethal activity of various chemicals, concurrent controls were run to check the sensitivity and the reproducibility of the test systems. These experiments were carried out over an 18-month period in two laboratories using similar protocols.Negative control substances used were maize oil, dispersol, tween 80 and water. Positive control substance used were cyclophosphamide, ethyl methane-sulphonate and mechlorethamine hydrochloride (nitrogen mustard). The substances were administered either intraperitoneally or by gavage.The results were analysed using, principally, a hierarchical analysis of variance of the total implants per pregnancy and the transformed early deaths per pregnancy data.Pregnancy frequency generally did not fall below 80%. The total number of implantations per pregnancy was usually about 11.8 in negative controls and was variably reduced by the mutagens used. With cyclophosphamide or ethyl methanesulphonate (EMS), there were some quantitative variations in the results obtained but qualitative agreement was good in the results both between experiments in the same laboratory and between the two laboratories. It was demonstrated that EMS is a useful positive control substance in experiments with orally administered materials. Sensitivity of the system was indicated by positive effects with a relatively low dose of EMS and consistent positive results with mechlorethamine for which a dominant lethal effect has not been demonstrated previously.It is concluded that the dominant lethal test gives reproducible data and it is, thus, possible to have confidence in the results and to compare findings in different laboratories using similar protocols.     

Current status of bioassays in genetic toxicology--the dominant lethal assay. A report of the U.S. Environmental Protection Agency Gene-Tox Program

The term dominant lethal may be defined as death of the heterozygote arising through multiple chromosomal breaks. The assay is generally conducted by treating male animals, usually mice or rats, acutely (1 dose), subacutely (5 doses), or over the entire period of spermatogenesis. Animals treated acutely or subacutely are mated at weekly intervals to females for a sufficient number of weeks to cover the period of spermatogenesis. Those treated for the entire spermatogenic cycle are mated for 1 or 2 successive weeks at the termination of treatment. Females usually are killed at 14 days of pregnancy and examined for the number of total implantations in the uterus, the number of implantations classified as early deaths, and, in some cases, the number of corpora lutea. The category of early death is the most significant index of dominant lethality. A total of 249 papers were reviewed and 140 chemicals were evaluated. Of the 140 chemicals, 65 were positive by the criteria used by the Work Group in evaluating each publication. The category of "positive" includes those responses of a borderline nature. 99 chemicals were declared negative. There is considerable overlap of chemicals in both categories, which accounts for the incongruity in the total number of chemicals tested and the number considered positive and negative. A total of 44 animal carcinogens have been tested in the dominant lethal assay, 26 of which were positive and 18 negative for a correlation of 59%. The role of the assay should be that of confirming positive results from lower tier chromosomal aberration-detecting systems (confirming in the sense of indicating the ability of the chemical to penetrate gonadal tissue and to produce cytogenetic damage). The dominant lethal assay should not be used as a risk assessment method.     

Heritable translocation study in male mice with trimethyl phosphate

Dominant lethal and heritable translocation studies were performed in male mice receiving a single intraperitoneal injection of trimethyl phosphate (TMP). The germ cell stage investigated was the spermatid. Methyl methanesulfonate (MMS) was used as a positive control in the latter study. A dominant lethal assay gave marked dose-dependent increases in early fetal deaths. Heritable translocations were detected at 1000 or 1500 mg of TMP/kg in F1 male progeny when screening for semi-sterility and cytogenetically analyzing the meiotic or mitotic chromosomes. Translocation induction was higher at the higher TMP dose (14.3%) than at the lower dose (5.3%) and the yield from the higher dose was similar to that induced by 50 mg of MMS/kg (11.0%). Most of the translocation carriers were semi-sterile or sterile. The data confirm conclusions from other dominant lethal studies showing TMP to be capable of causing chromosomal damage in mouse spermatids and show that certain types of damage result in heritable translocations.     

Isolation of Chinese hamster cells hyposensitive to ethyl methanesulfonate and their characterization in induction and antagonization of sister-chromatid exchanges

Dominant lethal tests were performed on female mice injected intraperitoneally with cyclophosphamide (200 mg/kg) or with mitomycin C (0.2 or 5 mg/kg) at the preovulatory stage of oogenesis. Complementary experiments were undertaken to clarify the results obtained. Embryo culture showed that sterility found after treatment with cyclophosphamide or with the high dose of mitomycin C was the reflection of true dominant lethal effects. Mortality after cyclophosphamide treatment occurred predominantly at the 2- and 3-cell stages, while it was reported in all preimplantation stages after treatment with the high dose of mitomycin C. Embryos treated with the low dose of mitomycin C developed normally to the blastocyst stage, confirming the absence of preimplantation effects found with this dose in the dominant lethal test. Cytogenetic analysis of female pronuclei at the first cleavage division were performed after mating treated females with males homozygous for one Robertsonian translocation. This method allowed one to distinguish easily the female pronuclei from the male ones, which exhibited one translocated 'marker' chromosome. After treatment with cyclophosphamide, most female pronuclei showed multiple chromatid exchanges or shattering of the entire genome. After treatment with the high dose of mitomycin C, various types of premature chromosome condensation were found, and they were often accompanied by important interchromosome associations. After treatment with the low dose of mitomycin C, no structural chromosome aberrations were found, and the number of numerical anomalies was not significantly different from that found in control embryos. These last results suggest that the increase in rate of postimplantation loss obtained in the dominant lethal test with the low dose of mitomycin C was not due to clastogenic effects of this compound in the female germ cells, but rather to indirect effects on the maternal organism.     

Genetically determined resistance to lethal murine cytomegalovirus infection is mediated by interferon-dependent and -independent restriction of virus replication.

Susceptibility of 4-week-old mice of different strains to lethal murine cytomegalovirus (MCMV) infection was studied. Strains homozygous for H-2k and C57BL strains were resistant to greater than or equal to 10(5.5) PFU. B10.BR mice congenic for C57BL background genes and H-2k were about 10-fold more resistant than either C3H/HeN or C57BL strains. BALB/c mice (H-2d) were susceptible (50% lethal dose, 10(5.05) PFU). This susceptibility was dominant over resistance associated with H-2k but not that associated with C57BL background genes. The dominant susceptibility trait segregated in backcross mice as if carried by a single gene. Virus replication in spleen cells in vivo correlated with susceptibility to lethal infection. A similar trend was found in tests of salivary glands. Replication of MCMV in vitro in cultures of adherent spleen cells and primary mouse embryo cells correlated with replication in vivo. Neutralization of interferon (IFN) in cultures of adherent spleen cells reversed H-2k-linked restriction of viral replication but had minor effects on cells of other strains. Natural killer cell responses to infection were often higher in more resistant strains, but B10.BR mice developed minimal natural killer cell responses. Specific antibody and cytotoxic T cell responses in B10.BR mice were similar or lower than in other strains. Thus, resistance to lethal MCMV infection was not immunologically mediated, was dependent on and reflected by the capacity of cells from a given mouse strain to support replication in vivo and in vitro, and was IFN dependent and recessive if linked to H-2k but IFN independent when associated with C57BL background genes.     

Increment kinetics of recessive lethal mutations and dominant lethals in offspring of Drosophila melanogaster on storage of methyl-methanesulfonate-treated sperm in females

The frequencies of sex-linked recessive lethal mutations in F1 males after feeding adult male Drosophila melanogaster with 0.25 and 0.5 mM methyl methanesulfonate (MMS) orally for 24 h increased approximately linearly with storage of the treated spermatozoa in females, whereas the number of hits of dominant lethals in the sperm after feeding 0.3 and 0.5 mM MMS increased approximately with the square of the storage time. Chromosome losses and mosaics in F1 males also increased with the dose of MMS to males, but their yields were too low to be analyzed quantitatively, only indicating a slight increase of chromosome loses and a slight decrease of mosaics with the time of storage of sperm. Maternal non-disjunctions (or chromosome losses), detected in F1 males, decreased with the dose of MMS to spermatozoa and their yield decreased with the time of storage of sperm of both MMS-treated and the control groups. A unitary model is proposed to explain the effect of storage on the dominant lethals and recessive lethal mutations.     

Mutagenicity of Ascaris chymotrypsin inhibitor in germ cells of mice

Chymotrypsin inhibitor isolated from Ascaris suum (ACHI) was tested for the induction of dominant lethal mutations in male mice. Dominant lethal effects of ACHI for the main stages of germ cell development were analyzed by mating at specific time points after dosing. Two groups of adult BALB/c males received 24 or 40 mg per kilogram body weight (BW) per day intraperitoneal (IP) injection of ACHI in sterile phosphate-buffered saline (PBS) for five consecutive days (subacute exposure). Males from a third group were administered single IP injections of ACHI—60 mg/kg BW (acute exposure). The control group received concurrent injections of PBS for five successive days. After the last dose, each male was mated with two untreated females. For fractionated examination with regard to successive germ cell stages (spermatozoa, spermatids, spermatocytes, spermatogonia), every second week, two other untreated virgin females were placed with each male for mating. The uteri of the females were inspected on the 15th day of gestation, and preimplantation loss and postimplantation loss determined from dominant lethal parameters. Exposure of mice germ cells to ACHI did not impair mating activity of males. Fertility index was reduced (P < 0.05) only for females mated at the third week with males exposed to the highest dose of ACHI. In the females bred to ACHI-treated males, significant (P < 0.05) increase in preimplantation loss was observed at postinjection weeks 1 (reflecting exposure to spermatozoa after single treatment and to spermatozoa or late spermatids after subacute dosing) and 3 (reflecting exposure to mid and early spermatids for acute dosing and to mid and early spermatids or late spermatocytes following acute treatment), regardless of dose and length of exposure to the inhibitor. At the 60-mg/kg-BW group, a significant increase of this parameter was also noted at week 5 (reflecting exposure to early spermatocytes). During mating days 15–21, a significant (P < 0.05) increase in postimplantation loss and dominant lethal effects were observed for all doses of ACHI. Acute ACHI exposure 5 weeks prior to mating resulted in dominant lethal effects in early spermatocytes. These preliminary data suggest that ACHI induces dominant lethal mutations at postmeiotic and meiotic stages of spermatogenesis, but spermatids are the most sensitive cell stage to the effect of ACHI. These results show that ACHI may be one of the factors causing disturbances in spermatogenesis leading to a reduction of host reproductive success.     

Mutations and their use in insect control

Traditional chemically based methods for insect control have been shown to have serious limitations, and many alternative approaches have been developed and evaluated, including those based on the use of different types of mutation. The mutagenic action of ionizing radiation was well known in the field of genetics long before it was realized by entomologists that it might be used to induce dominant lethal mutations in insects, which, when released, could sterilize wild female insects. The use of radiation to induce dominant lethal mutations in the sterile insect technique (SIT) is now a major component of many large and successful programs for pest suppression and eradication. Adult insects, and their different developmental stages, differ in their sensitivity to the induction of dominant lethal mutations, and care has to be taken to identify the appropriate dose of radiation that produces the required level of sterility without impairing the overall fitness of the released insect. Sterility can also be introduced into populations through genetic mechanisms, including translocations, hybrid incompatibility, and inherited sterility in Lepidoptera. The latter phenomenon is due to the fact that this group of insects has holokinetic chromosomes. Specific types of mutations can also be used to make improvements to the SIT, especially for the development of strains for the production of only male insects for sterilization and release. These strains utilize male translocations and a variety of selectable mutations, either conditional or visible, so that at some stage of development, the males can be separated from the females. In one major insect pest, Ceratitis capitata, these strains are used routinely in large operational programs. This review summarizes these developments, including the possible future use of transgenic technology in pest control.     

On the mutagenicity of methadone hydrochloride. Induced dominant lethal mutation and spermatocyte chromosomal aberrations in treated males

The mutagenicity of methadone hydrochloride was tested in male mice using the dominant lethal mutation technique and the spermatocyte test of treated mice. Male mice of C3H inbred strain received one of the following doses, 1, 2, 4 or 6 mg/kg body weight once a day for 3 consecutive days. Another group of mice served as control and received saline instead. Treated males were then mated to virgin females at 3-day intervals for a period of 45 days. Pregnant females were dissected at mid-term and the corpora lutea and intrauterine contents were recorded. The spermatocytes of treated males were examined 45-50 d after treatments with methadone and abnormal pairing configurations were scored. The methadone treatment was found to increase the rate of preimplantation deaths consistently in all post-meiotic stages with all doses used. In addition, the higher doses, 4 and 6 mg, affected spermatogonia stages. Quantitatively, the dose-response relationship cannot be demonstrated though the spectrum of effect increased with higher doses as more spermatogenesis stages became more sensitive to the treatment. In many cases the frequency of live implants showed a positive correlation with preimplantation deaths in contrast with the frequency of early deaths which showed only sporadic variation. The mutation indices based on total embryonic death indicate that methadone hydrochloride affected several stages of germ-cell maturation namely, spermatozoa (M.I. 14-35), late spermatids (M.I. 15-48), early spermatids (M.I. 14-50), late spermatocytes (M.I. 15-43) and spermatogonial stages (M.I. 12-63). Chromosome analysis at diakinesis-metaphase 1 revealed significant increase in the frequency of sex chromosome and autosome univalents with different doses of methadone. The smallest dose applied was quite effective and the data represent direct dose-response relationship. Of the multivalent configuration, the most frequent type was chain quadrivalents. The frequencies of total translocations per cell were estimated as 0.1, 0.16 and 0.2 for the 4 applied doses illustrating a dose-response relationship for the doses: 1, 2 and 4 mg, whereas with the higher dose, 6 mg, an abrupt decrease was apparent (0.05). This study calls for concern regarding the possible genetic hazards this drug may impose upon human populations.     

Acute toxoplasmosis leads to lethal overproduction of Th1 cytokines

Virulence in Toxoplasma gondii is strongly influenced by the genotype of the parasite. Type I strains uniformly cause rapid death in mice regardless of the host genotype or the challenge dose. In contrast, the outcome of infections with type II strains is highly dependent on the challenge dose and the genotype of the host. To understand the basis of acute virulence in toxoplasmosis, we compared low and high doses of the RH strain (type I) and the ME49/PTG strain (type II) of T. gondii in outbred mice. Differences in virulence were reflected in only modestly different growth rates in vivo, and both strains disseminated widely to different tissues. The key difference in the virulent RH strain was the ability to reach high tissue burdens rapidly following a low dose challenge. Lethal infections caused by type I (RH) or type II (PTG) strain infections were accompanied by extremely elevated levels of Th1 cytokines in the serum, including IFN-gamma, TNF-alpha, IL-12, and IL-18. Extensive liver damage and lymphoid degeneration accompanied the elevated levels of cytokines produced during lethal infection. Increased time of survival following lethal infection with the RH strain was provided by neutralization of IL-18, but not TNF-alpha or IFN-gamma. Nonlethal infections with a low dose of type II PTG strain parasites were characterized by a modest induction of Th1 cytokines that led to control of infection and minimal damage to host tissues. Our findings establish that overstimulation of immune responses that are normally necessary for protection is an important feature of acute toxoplasmosis.     

Irradiated laboratory animal diets: Dominant lethal studies in the mouse

In 4 separate dominant lethal experiments groups of mice of either Charles River CD1 or Alderley Park strains were fed laboratory diets (Oakes, 41B, PRD, BP nutrition rat and mouse maintenance diet No. 1). The diets were either untreated (negative control diets) or irradiated at 1, 2.5 and 5 megarad and were freshly irradiated, or stored. The animals were fed their test diets for a period of 3 weeks prior to mating. Groups of mice given a single intraperitoneal injection of 200 mg cyclophosphamide per kg body weight served as the positive controls.

Freshly irradiated PRD diet fed to male mice of both strains caused an increase in early deaths in females mated to the males in week 7 and to a lesser extent in week 4. The increase due to irradiation was small by comparison with that produced by the positive control compound. The responses for the other irradiated diets showed no significant increases in early deaths although some values for Oakes diet were high. The effect of storage was examined with PRD and BPN diet on one occasion and produced conflicting results.

Thus there was some evidence that irradiated PRD diet has weak mutagenic activity in the meiotic and/or pre-meiotic phase of the spermatogenic cycle which appeared to be lessened on storage; the inclusion of such a diet in toxicological studies would therefore need to be carefully considered.