Effects of Dose on the Induction of Dominant-Lethal Mutations and Heritable Translocations with Ethyl Methanesulfonate in Male Mice

Genetic damage by ethyl methanesulfonate (EMS) in male mice was measured at doses ranging from 50 to 300 mg/kg with dominant-lethal mutations and reciprocal translocations as endpoints. No appreciable increase in dominant-lethal mutations was detected following a dose of 100 mg/kg. Dominant lethals induced by EMS were convincingly detected only after a dose of 150 mg/kg, but in the translocation experiment an increase in the genetic effect was detectable at the 50 mg/kg dose. It is likely that dominant lethals had also been induced at the 50 and 100 mg/kg doses, but were not detected due to the relative insensitivity of the dominant..lethal procedure. Thus, for detection of low levels of EMS-induced chromosome breakage, translocations are a much more reliable endpoint than are dominant-lethal mutations. A procedure for large-scale screening of induced translocations is described.—The dominant-lethal dose-response curve, plotted on the basis of living embryos as a percentage of the control value, is clearly not linear as it is markedly concave downward. Similarly, the translocation dose-response curve showed a more rapid increase in the number of translocations with dose than would be expected on the basis of dose-square kinetics. It is clear for both of these endpoints that the effectiveness of EMS in inducing chromosome breakage is proportionately much lower at low doses.     

Heritable translocation test and dominant-lethal assay in mice with methyl methanesulfonate.

A dominant-lethal test and a heritable translocation test were performed with methyl methanesulphonate (MMS) at 40 mg/kg by treating the sensitive periods of post-meiotic spermatogenesis i.e. spermatozoa and spermatids. In the dominant-lethal test 25 to 60% dominant-lethal mutations were obtained depending on the mating intervals. In the heritable translocation test 11% sterile and partially sterile F1 males were observed in 250 offspring of the MMS group. All of the 14 partially sterile and 6 of the 14 sterile F1 males were demonstrated to be translocation carriers. Fertility of the partial steriles was about 40% of normal fertility. The translocation frequencies in the primary spermatocytes of the partially sterile F1 males varied between 2 and 99%. Transmission of partial sterility and translocations was confirmed in the F2 generation. There were no partially sterile or sterile males among the 245 controls.     

Studies on the dominant-lethal and fertility effects of the heavy metal compounds methylmercuric hydroxide, mercuric chloride, and cadmium chloride in male and female mice

Dominant-lethal effects of 10 mg/kg methylmercuric hydroxide were studied in male mice from two hybrid stocks and in females from one of these stocks. Two other compounds, mercuric chloride (2 mg/kg) and cadmium chloride (2 mg/kg), were studied only in females for dominant-lethal (in one hybrid stock) and reproductive capacity effects (in two hybrid and one mixed stocks). All compounds were administered in a single intraperitoneal injection. When males of one of the two stocks studied were treated with methylmercuric hydroxide, the females to which they were mated exhibited a slight reduction in the total number of implantations and in the number of living embryos. These reductions were accompanied by a very small increase in the incidence of dead implantations. In females, cadmium chloride had no detectable dominant-lethal or other fertility effects, except superovulation. On the other hand, the two mercury compounds slightly reduced the numbers of implants and living embryos in females subjected to dominant-lethal studies. The two mercury compounds also induced a slight reduction in the long-term reproductive performance of one stock of females. These results and those reported earlier by others, indicate that the mercury compounds studied so far are not potent inducers of dominant-lethal mutations in male and female mice. It is not clear whether the small effects on male or female fertility induced in some cases, particularly the increase in dead implantations and reductions in the number of living embryos, were attributable to dominant-lethal mutations or to nongenetic causes.     

The mutagenicity of azathioprine in mice, Drosophila melanogaster and Neurospora crassa.

The chemotherapeutic coumpound azathioprine was tested for possible mutagenicity in Swiss Albino mice, Drosophila melanogaster and Neurospora crassa. Utilizing the dominant-lethal assay it was found that acute oral doses of azathioprine (2 times 25 mg/kg body weight), induced dominant-lethal mutations in mouse spermatocytes. Chronic oral doses of azathioprine (2 times 25 mg/kg body weight/week for 10 weeks) resulted in a greater rate of dominant-lethality. This increase was not permanent, and by week 4 of gamete sampling there was no significant increase in dominant-lethal mutations. Histological sections showed that chronic treatment of male mice with azathioprine caused pyknosis of spermatocyte nuclei and depletion of the spermatid population. Both acute and chronic doses of azathioprine caused a temporary reduction in sperm viability. Oral treatment of male Canton-S, D. melanogaster with azathioprine caused an increase in dominant-lethality in broods assumed to correspond to spermatid and spermaotcyte stages. Azathioprine also increased the rate of non-disjunction of the X and Y chromosomes, loss of the long arm of the Y chromosome, and loss of the X or Y chromosome in treated male R(I)2, vf/BsYy+D. melanogaster. Since sex-ratio deviation did not occur in progeny from treated rod-X (yv/B2Yy+) male D. melanogaster, it was concluded that the observed sex-ration deviation in the treated ring-X stock was the result of induced ring-X lethality. Azathioprine induced recessive-lethal mutations in the ad-3 region of a N. crassa heterokaryon. In the host-mediated assay using this same heterokaryon and male Swiss Albino mice as host, the mutagenic activity of azathioprine did not appear to be potentiated or detoxified by the host. The results show that azathioprine has a deleterious effect on reproduction in mice and probably induces mutational events in mice, D. melanogaster and N. crassa.     

Lack of association between induction of dominant-lethal mutations and induction of heritable translocations with benzo[a]pyrene in postmeiotic germ cells of male mice

Benzo[a]pyrene was tested for induction of dominant-lethal mutations in germ cells of male mice. Clear-cut dominant-lethal effects were induced in middle and early spermatoza. In contrast to the dominant-lethal effects observed the study showed no detectable increase in hertiable translocations for these stages over the spontaneous level. Thus, the results provide another example of a chemical mutagen that is effective in inducing dominant-lethal mutations but relatively ineffective in inducing heritable translocations in male postmeiotic germ cells.     

Difference in the Ratio of Dominant-Lethal Mutations to Heritable Translocations Produced in Mouse Spermatids and Fully Mature Sperm after Treatment with Triethylenemelamine (Tem)

The relative induction of dominant-lethal mutations and heritable translocations in triethylenemelamine-treated male postmeiotic germ cells of mice was determined depending on the stage treated. Males were mated either 11.5–14.5 days after treatment (middle spermatids) or less than 2.5 hours after treatment (fully mature sperm). Results clearly showed that, even though similar levels of dominant-lethal mutations were induced in fully mature sperm and in middle spermatids, the frequency of heritable translocations induced in mature sperm was markedly lower than that induced in middle spermatids. This observation was used, together with earlier ones, to suggest a mechanism by which dominant-lethal mutations and heritable translocations are produced following chemical treatment of male postmeiotic germ cels.     

Tests for dominant-lethal effects of 1,2-dibromo-3-chloropropane (DBCP) in male and female mice

DBCP was studied for dominant-lethal effects in male and female mice and for total reproductive effects in females. In males it was administered either intraperitoneally or subcutaneously while in females it was given only by the former route. No DBCP-related response was observed in either males or females indicating its ineffectiveness in inducing chromosomal aberrations or cytotoxicity in mouse germ cells. These findings differ markedly from the observations made in rats by other investigators. Thus, the probable existence of a species difference in germ cell response to DBCP has been strengthened by the availability of the present results. It should be noted, however, that only two stocks of male mice have been studied so far for dominant-lethal and germ cell cytotoxicity effects.     

Effects of Dose on the Induction of Dominant-Lethal Mutations with Triethylenemelamine in Male Mice

Dose effects of triethylenemelamine (TEM) in the induction of dominant-lethal mutations were studied at the early spermatozoon, midspermatid and spermatocyte stages. The pattern of effects on spermatocytes, unlike midspermatids and early spermatozoa, indicated possible cytotoxic damage, so for the determination of TEM dose-response curves in the induction of genetic damage only the data for midspermatids and early spermatozoa were used. The TEM dose-effect curves for those two stages differ markedly from ethyl methanesulfonate (EMS) dose-effect curves. Beginning with the lowest doses at which significant effects are observed, there is a considerably more rapid increase in dominant-lethal effects with dose of EMS than TEM. Another marked difference between the two compounds is in the ratio of the genetically effective dose (as measured by dominant-lethal mutations) to the lethal dose. The ratio is 1:100 for TEM and only 1:3.5 for EMS; thus, TEM is mutagenic far below its toxic level. Obviously, these results have important implications not only for our understanding of the nature of chemical induction and recovery of chromosomal aberrations but also for the practical problems of evaluating the mutagenic effects of chemicals.     

Induction of specific-locus and dominant-lethal mutations by cyclophosphamide and combined cyclophosphamide-radiation treatment in male mice

Cyclophosphamide is the most widely used antineoplastic agent. It is also used to condition patients for bone-marrow transplantations. Because of the general interest of this compound we initiated a systematic study of the induction of dominant-lethal and specific-locus mutations in male mice. In addition, we investigated the induction of specific-locus mutations by the combined treatment of cyclophosphamide and ionizing radiation.A dose of 40 mg/kg bw of cyclophosphamide caused dominant-lethal mutations in male mice only in the 1st and 2nd week after treatment. A dose of 120 mg/kg induced dominant-lethal mutations in the mating intervals 1–21 days posttreatment. No dominant lethal mutations were observed after the 3rd week. The same differential spermatogenic response was observed for the induction of specific-locus mutations. Cyclophosphamide induced recessive mutations exclusively in spermatozoa and spermatids. No mutations were recovered from treated spermatocytes and spermatogonia. In contrast to cyclophosphamide, radiation induces specific-locus mutations in all germ-cell stages.The pretreatment with cyclophosphamide 24 h before radiation enhanced the frequency of specific-locus mutations in spermatogonia. The distribution of the observed mutations among the 7 loci and their viability supports the hypothesis that these mutations were induced by radiation rather than by cyclophosphamide. The compound causes an immediate inhibition of DNA and RNA synthesis in spermatogonia. The inhibition very likely interferes with the repair process. The disturbance of the repair process is probably the cause of the synergistic effect for the induction of specific-locus mutations in spermatogonia of mice after pretreatment with cyclophosphamide 24 h before irradiation.     

Mutagenic effects of dimethyl terephthalate on mouse somatic cells in vivo

The mutagenic activity of dimethyl terephthalate (DMtP) was evaluated in the micronucleus test in mice. A clear clastogenic effect was obtained at all concentrations studied (0.2-1.0 mmole/kg body weight). The maximum number of micronuclei occurred 24 h after a single intraperitoneal (i.p.) injection. The time-course for the DMtP-induced micronuclei was in agreement with the available data on the rapid excretion of phthalates from the mammalian body. The dose-effect response was best described by a linear equation with a logarithmic component. The emergence of the latter term was related to the toxic effects of DMtP at higher concentrations on bone marrow erythropoietic function. A comparison of the effects induced by DMtP and by methyl nitrosourea indicated that DMtP cannot be considered a strong mutagenic compound. We have compared the sensitivity of the mouse micronucleus test and that of Drosophila dominant-lethal test by contrasting the effects obtained at similar exposure doses. This comparison leads to the conclusion that the micronucleus test is capable of responding to far lower phthalate concentrations than the Drosophila dominant-lethal mutation test. Our results testify to the ability of dimethyl terephthalate to cause genotoxic damages in vivo in both somatic and germinal cells of higher organisms. Thus, the chemical in question may be of potential genetic hazard to man.     

The conditioned place preference paradigm in rats: effect of bupropion

The effect of bupropion in the conditioned place preference paradigm in rats is described. In doses between 10 and 50 mg/kg i.p. bupropion increased the time rats spent in a shuttle box compartment conditioned to this compound. This effect of bupropion was not blocked by pretreatment of the animals with 0.1 mg/kg i.p. haloperidol or 50 mg/kg i.p. sulpiride. The same doses of the neuroleptics did, however, attenuate locomotor hyperactivity induced by bupropion. Bupropion thus seems to belong to the group of CNS stimulants whose effect in the conditioned place preference paradigm is not blocked by neuroleptics.     

Clastogenic effects of acrylamide in mouse bone marrow cells

Acrylamide, known to induce dominant-lethal mutations (Shelby et al., 1986; Smith et al., 1986) and heritable translocations (Shelby et al., 1987) in rodent germ cells, was hitherto a questionable clastogen in rodent bone marrow (Shiraishi, 1978). Therefore, it was tested for chromosomal aberrations in mouse bone marrow cells, spermatogonia and by the micronucleus test. The intraperitoneally injected doses ranged from 50 to 150 mg/kg. In the chromosomal bone marrow test and the micronucleus assay positive results were obtained with acrylamide, and in the latter test the effect increased linearly with dose. Chromosomal aberrations were not induced in differentiating spermatogonia by the acute acrylamide treatment. Cisplatin was used as a positive control and gave the expected positive response in all 3 tests. The present results demonstrate that acrylamide is no exception among clastogens. It breaks chromosomes not only in mammalian germ cells but also in somatic cells.     

6-Mercaptopurine,an inducer of cytogenetic and dominant-lethal effects in premeiotic and early meiotic germ cells of male mice

Dominant-lethal effects of 6-mercaptopurine on male mice were studied using eight doses, ranging from 150 to 482 mg/kg. Effects of the 150-mg/kg dose were studied over the entire spermatogenic cycle, and those of the higher doses for matings made between days 28.5 and 41.5 after treatment. It was found that, with low doses, there was only one period in which clearcut increases in induced dominant-lethal mutations were detected, namely in matings that occurred 32.5 to 35.5 days after treatment. With higher doses, effects could be detected beyond that period through day 39.5. Spermatozoa utilized for matings during the period of greatest response were presumably derived from germ cells that were in late differentiating spermatogonial and early meiotic spermatocyte stages at the time of treatment. These results are similar to those of Ray and Hyneck. To date, 6-mercaptopurine is unique in inducing dominant lethality only at these particular stages.A study of chromatid aberration induction in the treated males themselves was carried out for 150 and 250 mg/kg doses of 6-mercaptopurine over the period of 9 to 16 days after treatment. A considerable increase in isochromatid and chromatid deletions was observed in diakinesis-metaphase-I spermatocytes on days 14 and 15 after treatment. For reasons discussed, the cells sampled at this time may be assumed to have been in early meiosis (preleptotene), with some in late differentiating spermatogonial stages, at the time of treatment. The rough agreement in sensitive cell type for dominant lethality and chromatid aberration induction suggests that chromatid deletions are the cause of dominant lethality in this study. Conservative estimates of the frequency of dominant lethality expected from the chromatid aberration frequencies tend to substantiate this suggestion.     

Influence of GABA-agonists and antagonists on neuroleptic-induced catalepsy in rats.

Direct (muscimol) or indirect (aminooxyacetic acid, diaminobutyric acid, pyrrolidinone) GABA-mimetic compounds significantly potentiate neuroleptic induced catalepsy in rats. In contrast at subconvulsant doses, direct (bicuculline, picrotoxinin and indirect (allylglycine) GABA antagonists antagonized haloperidol-induced catalepsy. The effect of bicuculline and picrotoxinin was biphasic with the lowest doses increasing catalepsy. These results indicate that GABA mechanisms are involved in the induction of catalepsy by neuroleptics.     

Induction of dominant-lethal mutations after administration of ethylenethiourea in combination with nitrite or of N-nitroso-ethylenethiourea in mice

Mutagenic potentials of ethylenethiourea (ETU) in combination with sodium nitrite or of N-nitroso-ETU, a nitrosttion product of ETU in vitro, were investigated in the mouse dominant-lethal test. Simultaneous 5-day p.o. administration of ETU (150 mg/kg) and sodium nitrite (50 mg/kg) caused a significant decrease in the percentage pregnancy and the number of implants in weeks 5 and 6 of testing, although no effects were obtained on these indices when the chemicals were applied separately. However, in the group treated with 30 mg ETU plus 10 mg sodium nitrite per kg no dominant-lethal mutations were induced. 5-day oral administration of 100 mg of N-nitroso-ETU per kg also exhibited similar effects to those observed after treatment with 150 mg ETU plus 50 mg sodium nitrite per kg.     

The mutagenicity of hydrazine and some of its derivatives.

The mutagenic effect of ethylenethiourea (ETU), a degradation product and metabolite of ethylenebisdithiocarbamates, which are widely used as fungicides, was studied in different test systems.ETU induced mutations of the base-pair substitution type in Salmonella typhimurium TA 1530 in vitro as well as in the host-mediated assay. In the host-mediated assay, a dose of 6000 mg/kg (LD₅₀ = 5400 mg/kg) resulted in a slight but significant increase of the reversion frequency by a factor of 2.37.The results of the micronucleus test were negative after two-fold oral applications of 700, 1850 and 6000 mg/kg to Swiss albino mice. Thus it is concluded that ETU hardly induces any chromosomal anomality in the bone marrow.No dominant-lethal effect was observed after single oral doses of 500, 1000 and 3500 mg/kg given to male mice.     

Bleomycin, unlike other male-mouse mutagens, is most effective in spermatogonia, inducing primarily deletions

Dominant-lethal tests [P.D. Sudman, J.C. Rutledge, J.B. Bishop, W.M. Generoso, Bleomycin: female-specific dominant lethal effects in mice, Mutat. Res. 296 (1992) 205-217] had suggested that Bleomycin sulfate (Blenoxane), BLM, might be a female-specific mutagen. While confirming that BLM is indeed a powerful inducer of dominant-lethal mutations in females that fails to induce such mutations in postspermatogonial stages of males, we have shown in a specific-locus test that BLM is, in fact, mutagenic in males. This mutagenicity, however, is restricted to spermatogonia (stem-cell and differentiating stages), for which the specific-locus mutation rate differed significantly (P<0.008) from the historical control rate. In treated groups, dominant mutations, also, originated only in spermatogonia. With regard to mutation frequencies, this germ-cell-stage pattern is different from that for radiation and for any other chemical studied to date, except ethylnitrosourea (ENU). However, the nature of the spermatogonial specific-locus mutations differentiates BLM from ENU as well, because BLM induced primarily (or, perhaps, exclusively) multilocus deletions. Heretofore, no chemical that induced specific-locus mutations in spermatogonia did not also induce specific-locus as well as dominant-lethal mutations in postspermatogonial stages, making the dominant lethal test, up till now, predictive of male mutagenicity in general. The BLM results now demonstrate that there are chemicals that can induce specific-locus mutations in spermatogonia without testing positive in postspermatogonial stages. Thus, BLM, while not female-specific, is unique, (a) in its germ-cell-stage specificity in males, and (b) in inducing a type of mutation (deletions) that is atypical for the responding germ-cell stages (spermatogonia).     

The genetic action of ammonium molybdate and cadmium chloride and iodide]

Ammonium molybdate cadmium iodide and cadmium chloride have been studied in test for their genotoxic effect on induction of DNA-cellular bonding, extrasynthesis of DNA in spermatozoa of mice as well as in test to estimate a fertility criterion of Drosophila males. Ammonium molybdate, cadmium iodide and cadmium chloride are stated to be able to induce injuries of native DNA in test on induction of DNA-cellular bonding and DNA-sex cells of mice and Drosophila melanogaster in dominant-lethal test and in experiments on estimation of a fertility coefficient of Drosophila males, respectively.     

Reproductive performance and mutagenic response of the wasp Bracon hebetor following treatment with the antibiotic bleomycin

Bleomycin (BM) induced dominant-lethal genetic lesions in the gametes of both sexes of adult Bracon hebetor wasps following ingestion. This effect was demonstrated by decreased fertility in the unfertilized eggs of treated females as well as in eggs fertilized by mature sperm derived from treated males. Death in the unhatched eggs occurred prior to blastoderm formation indicating BM caused chromosome breakage. Affected ovarian cells were limited to those exposed to BM between the developmental stages of vitellogenesis and early metaphase I. Sperm-associated fertility reductions were temporary in duration with substantial reductions being observed starting on the second day following the initiation of oviposition. BM lowered general egg-formation capacity in treated females but, unlike the fertility effects, decreased fecundity was caused by somatic debility. Although the BM-induced genetic damage observed in B. hebetor was radiomimetic in nature, the time of appearance and duration of the effects did not correspond with previous patterns found when B. hebetor adults were exposed to X-rays. While the differences could be due to the BM detoxification or the repair of BM-induced chromosome damage, further investigation would be required to demonstrate the presence of such systems in this hymemopteran.     

Induction of specific-locus and dominant-lethal mutations in male mice by diethyl sulfate (DES)

Diethyl sulfate (DES), a monofunctional alkylating agent, induces mutations and chromosomal aberrations in many different organisms and cell systems, including dominant-lethal mutations in male mice. However, until now it could not be demonstrated that DES induces specific-locus mutations in mice. This observation would contradict the close correlation observed between the induction of dominant-lethal mutations and specific-locus mutations in mice with other chemicals. DES induces dominant-lethal and specific-locus mutations in spermatozoa and late spermatids of mice. The mutation frequency for dominant-lethal mutations is dose-dependent, while for specific-locus mutations it is independent of the dose. In the mating interval 5-8 days post-treatment the mutation frequency for 200 mg/kg DES is 17.0 X 10(-5) and for 300 mg/kg 7.5 X 10(-5) mutations per locus. The dose-dependent increase of dominant-lethal mutations probably reduced the chance of recovering specific-locus mutations. The importance of these findings for mutagenicity testing is discussed.