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.     

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.     

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.     

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.     

Chemical induction of presumed dominant-lethal mutations in postcopulation germ cells and zygotes of mice. II. Sensitivity of different postcopulation-precleavage stages to three alkylating chemicals.

The relative sensitivities of various postcopulation-precleabage and pronuclear stages to dominant-lethal effects of isopropyl methanesulfonate (IMS), ethyl methanesulfonate (EMS), and triethylenemelamine (TEM) were investigated. The pattern of sensitivity differed with the chemical. IMS was most effective when pronuclear formation was already completed and the majority of the zygotes were presumably undergoing DNA synthesis. EMS, on the other hand, induced its most pronounced effects when eggs in the course of second meiotic division and zygotes in early pronuclear stages were treated. The greatest effect of TEM was observed when zygotes were treated at the early pronuclear stage. EMS and TEM, in contrast to IMS, are similar to radiations in that zygotes undergoing DNA synthesis are more resistant to them than are the early pronuclear stages. In the case of IMS, effects induced in the most sensitive postcopulation-precleavage stage were 6 to 9 times greater than in the most sensitive precopulatory dictyate oocytes or male germ cells. On the other hand, in the case of EMS and TEM, the most sensitive precopulatory male germ cells, but not the dictyate oocytes, were more sensitive than the most sensitive postcopulation stages.     

Induction of dominant lethals with ethyl methane-sulfonate in male germ cells of mulberry silkwork, Bombyx mori l.

Sensitivity of male germ cells in the mulberry silkworm, Bombyx mori L., to ethyl methanesulfonate (EMS) was determined by treating newly emerged 5th- instar larvae, and 2-day- and 7-day-old pupae with 3 concentrations, 0.05, 0.1 and 0.15%, of the mutagen. The frequency of dominant-lethal mutations induced by EMS treatment was used as the parameter for the study. Spermatids and spermatozoa were markedly sensitive to EMS. Statistical analysis confirmed that differences in respect of percentage of egg hatch among the 3 different treatments as well as the interactions between the 3 factors, e.g. stages, hatchability and EMS treatment, were highly significant.     

DNA adduct formation in mouse testis by ethylating agents: a comparison with germ-cell mutagenesis

DNA adduct formation in various organs of mice was determined after i.p. injection with the ethylating agents N-ethyl-N-nitrosourea (ENU), ethyl methanesulfonate (EMS), and diethyl sulfate (DES). The potency of the 3 chemicals to react either at the O6 position of guanine or at the N-7 position of guanine was related to their potency to induce mutations in the specific-locus assay of the mouse. ENU, which produces relatively high levels of O-alkylations (O6-ethylguanine), is primarily mutagenic in spermatogonia of the mouse, whereas EMS and DES, which produce relatively high levels of N-alkylations (7-ethylguanine) in DNA, are much more mutagenic in post-meiotic stages of male germ cells. The relationship between exposure to ENU and the dose, determined as O6-ethylguanine per nucleotide in testicular DNA, is non-linear. However, the relationship between dose and mutation induction in spermatogonia by ENU appears to be linear, which is expected if O6-ethylguanine is the major mutagenic lesion. The relatively high mutagenic potency of EMS and DES in the late stages of spermatogenesis is probably due to the accumulation of apurinic sites which generate mutations after fertilization. A comparison of mutation induction by ENU in spermatogonia and mutation induction in cultured mammalian cells indicates that about 10 O6-ethylguanine residues were necessary in the coding region of a gene to generate a mutation.     

Induction of specific-locus mutations in male mice by ethyl methanesulfonate (EMS)

Using a sequential mating procedure, the induction of specific-locus mutations by ethyl methanesulfonate (EMS) was reinvestigated in male mice. Doses of 175 mg/kg b.w. and 250 mg/kg b.w. of EMS induce gene mutations in the mating intervals 5-8 and 9-12 days post treatment. However, only the frequency of dominant lethal mutations increases with the dose, not the frequency of specific-locus mutations. This observation implies that with a higher dose of EMS a larger fraction of mutagenized spermatozoa and spermatids are selectively eliminated, leading to underestimation of the specific-locus mutation yield at high doses. EMS does not induce specific-locus mutations in spermatogonia.     

Inhalation exposure-rate of ethylene oxide affects the level of DNA breakage and unscheduled DNA synthesis in spermiogenic stages of the mouse

The effect of ethylene oxide (EtO) inhalation-exposure rate on the induction of DNA breakage in late spermatids and on unscheduled DNA synthesis (UDS) in early spermatids was studied. The exposures were 450 parts per million (ppm) for 4 h, 900 ppm for 2 h, and 1800 ppm for 1 h. Thus, the total exposure was always 1800 ppm-h. Both DNA breakage and UDS were found to increase by a factor of approximately 3 in going from the low to high EtO concentration, suggesting that the molecular dose of EtO to the testis had increased by a similar factor. Our results are consistent with the EtO exposure-rate effect found by Generoso et al. (1986) for induction of dominant-lethal mutations in late spermatids and early spermatozoa.     

Dose-rate effects of ethyl methanesulfonate on survival and mutation induction in cultured Chinese hamster cells

The dose-rate effects of ethyl methanesulfonate (EMS) on the survival and induction of mutations in Chinese hamster Don cells were investigated. The most effective time of exposure to EMS for reducing the surviving fraction of cells was 4 h, shorter and longer exposure times being less effective. The threshold or minimal concentration of EMS giving a surviving fraction of 0.5 was 0.05 mg/ml. The minimal effective time of exposure to EMS for cell death was 1 h. Corrected survival curves showed that longer exposure times at lower dose rates of EMS had less cytotoxic effect than shorter exposure times at higher dose rates.After exposure of Don cells to various doses of EMS for various times, the frequencies of mutations resistant to 6-thioguanine (6TG) were measured. An exposure time of 4 h produced a lower mutation frequency than shorter or longer exposure times that resulted in the same surviving fraction of cells. An exposure time of 20 h produced the highest induced mutation frequency.This system using cultured Chinese hamster cells should be useful as a sensitive procedure for detecting the mutagenic actions of chemicals.     

A comparison of the effects of physical and chemical mutagens in sesame (Sesamum indicum L.)

Three sesame genotypes (Rama, SI 1666 and IC 21706) were treated with physical (γ-rays: 200 Gy, 400 Gy or 600 Gy) or chemical (ethyl methane sulphonate, EMS: 0.5%, 1.0%, 1.5% or 2.0%) mutagens and their mutagenic effectiveness and efficiency were estimated in the M (2) generation. The M (3) generation was used to identify the most effective mutagen and dose for induction of mutations. The average effectiveness of EMS was much higher than γ-rays. The lowest dose of γ-rays (200 Gy) and the lowest concentration of EMS (0.5%) showed the highest mutagenic efficiency in all genotypes. Analysis of the M (3) generation data based on parameters such as the variance ratio and the difference in residual variances derived from the model of Montalván and Ando indicated that 0.5% concentration of EMS was the most effective treatment for inducing mutations.     

Evidence that induction and suppression of mutations and recombinations by chemical mutagens in S. cerevisiae during mitosis are jointly correlated.

Mutagen-induced intergenic and interallelic recombination as well as forward mutation were studied in one and the same strain of S. cerevisiae. In nontoxic dose ranges, the induction of mutants and recombinants was parallel after treatment with ethyl methanesulfonate (EMS), methyl methanesulfonate (MMS), N-methyl-N'-nitro-M-nitrosoguanidine (MNNG), triethylene melamine (TEM), 4-nitroquinoline 1-oxide (4-NQO), sodium nitrite (NaNO2), and 1-fluoro-2,4-dinitrobenzene (2,4-DNFB). Acridine orange (AO) after treatment without light induced recombinants, but reduced the frequency of spontaneous mutations. In combination with TEM, AO exerted the same effect, i.e., reduced its mutagenic effect and enhanced its recombinogenic effect. 4,5,6-Trichloro-2-(2,4-dichlorophenoxy) phenol (Cl5-predioxin) induced mutants and intergenic recombinants, but specifically reduced the spontaneous frequency of interallelic recombinants. In combination with TEM, it enhanced its mutagenic and intergenic recombinogenic effects but reduced its interallelic recombinogenic effect. The main conclusions of the present study, that is 1. Essentially similar lesions can lead to different genetic consequences, and 2. Induction of mutation and recombination are jointly correlated, i.e., suppression of mutations leads to an enhancement of recombinations, while suppression of recombinations leads to an enhancement of mutations, are used to set up a speculative concept for mutation and recombination induction in the diploid yeast cell during mitosis.     

Mutagen-induced fetal anomalies and death following treatment of females within hours after mating

In an earlier study (Generoso et al., 1987), it was observed that the mutagen, ethylene oxide (EtO), produced remarkable increases in the incidence of developmental abnormalities and death of fetuses when early zygotic stages were exposed. This is a major finding in experimental induction of embryopathy, implicating genetic damage to the zygotes as the likely cause. In the subsequent study reported here, 3 other mutagens--ethyl methanesulfonate (EMS), ethyl nitrosourea (ENU), and triethylene melamine (TEM), were studied for embryopathic effects following exposure of dictyate oocytes, prefertilization oviducal eggs and sperm, early pronuclear zygotes, zygotes undergoing pronuclear DNA synthesis, and two-cell embryos. All 4 mutagens produced developmental abnormalities among living fetuses following exposure of early pronuclear zygotes (the only stage studied for this endpoint in this report). With respect to stage specificity and gestational timing of death of conceptuses, EMS and EtO on one hand and ENU and TEM on the other, are very similar to one another. EMS, like EtO, produced a high incidence of midgestation and late fetal deaths only in prefertilization oviducal eggs and sperm and in early pronuclear eggs. In contrast, ENU and TEM produced high losses of conceptuses in all postmating stages studied but death occurred primarily prior to or around the time of implantation. Thus, the frequency of induction and the expression of embryopathy, which ranged from early embryonic preimplantation and late fetal deaths to subtle fetal anomalies, are dependent upon the stage exposed and the mutagen used.     

Induction and expression of mutations in mammalian cells in the absence of DNA synthesis and cell division

The induction of mutations by the alkylating agent ethyl methanesulfonate (EMS) was determined with Chinese hamster ovary cells maintained in serum-free medium to arrest DNA synthesis and cell division. The arrested cultures were treated with EMS and maintained in serum-free medium for various time intervals post-treatment before serum containing medium was added to initiate DNA synthesis and cell division. The concentration-dependent increase in 6-thioguanine-resistant mutants in the arrested cultures was similar to that found with exponentially dividing cultures when serum was added to the arrested cultures immediately after the EMS treatment; the time course of phenotypic expression was also similar with both cultures. In addition, maintenance of the arrested cultures in serum-free medium for up to 18 days post-treatment resulted in no change in the mutant frequency. This suggests that the mutagenic damage is not removed in these arrested cultures. Furthermore, maintenance of the arrested state for increasing time intervals before serum addition results in decreases in the time necessary for maximum phenotypic expression. Cultures maintained in serum-free medium for 16 days after mutation treatment show complete expression of the mutations with no need for subculture. This last result suggests that the mutagenic damage induced by EMS in Chinese hamster ovary cells is not removed and that this damage results in both the induction and expression of mutation in the absence of DNA replication.     

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).     

Mechanisms of mutation induction in germ cells of the mouse as assessed by the specific locus test.

Mouse germ cell specific locus mutagenesis data and a molecular characterization of mutant alleles have been reviewed to arrive at an understanding of the mechanism of mutation induction in mammals. (a) The spermatogenic stage specificity for the sensitivity to mutation induction by 20 chemical mutagens is considered. (b) The effects of a saturable repair process and its recovery over time are examined for the mutagenic efficiency of ethylnitrosourea. (c) The mutagenic events following methylnitrosourea and chlorambucil are shown to be mainly deletions. In contrast the mutations recovered after ethylnitrosourea treatment are almost exclusively base pair substitutions. (d) It is emphasized that to date very few specific locus experiments have been designed to test for mutagenic events outside the interval stem cell spermatogonia-mature spermatozoa. A specific locus mutation has recently been shown to be due to loss of heterozygosity via mitotic recombination in an early zygote stage and suggests a broader range of possible mechanisms of mutation when these stages are considered. (e) With the cloning of all 7 marker loci mutation analysis at the molecular level will allow a more direct assessment of the mutation process in future studies.     

Induction of dominant lethal mutations by combined X-ray-acrylamide treatment in male mice

The induction of mutations following combined treatment with acrylamide (AA) plus X-rays has been determined using the dominant lethal mutations test in Pzh:SFISS male mice. Combinations of a mutagenic dose of both agents (1.00 Gy, 125 mg/kg b.w.) and a non-mutagenic dose, i.e., a dose that alone does not produce dominant lethals (0.25 Gy, 25 mg/kg b.w.), were used. For the discussion of the effects of combined action of X-rays and acrylamide the term 'enhancement in risk' was used whenever the effects observed after combined exposure significantly exceeded the sum of the effects produced separately by the agents. Such an enhanced risk has been observed in late spermatids after combined action of X-rays and AA at non-mutagenic doses, and in spermatozoa, spermatids and late spermatocytes after exposure to mutagenic doses.     

Concepts of threshold in mutagenesis and carcinogenesis

Although the existence of a threshold in the dose effect relationship is well documented for many, if not most, types of toxicological effects the existence of a threshold for the mutagenic effects of ionising radiation and of certain chemicals has been questioned since the middle of the century and only recently the question of thresholds for radiation and chemical carcinogenesis has been addressed. The essential facts for the interpretation of threshold dose–response curves are common to all type of effects and are: (i) the number and the identity of the target; (ii) the type and sensitivity of the endpoint used to quantify the effect. We therefore will first try to model the type of interactions which may be expected between a mutagen and its target and define from this whether a threshold dose-effect can be expected; in a second step the concept will be extended to heritable mutations and carcinogenesis.     

Unscheduled DNA synthesis in spermatogenic cells of mice treated in vivo with the indirect alkylating agents cyclophosphamide and mitomen

Cyclophosphamide (CPA) and mitomen (DMO) are chemical mutagens that require metabolic activation to produce their biological effect. We have used an in vivo UDS assay in various meiotic and postmeiotic germ-cell stages of male mice to study DNA repair after treatment with these chemicals. EMS, a compound requiring no metabolic activation, was also used for comparative purposes.CPA and DMO induced UDS in meiotic through early-to-midspermatid stages, but no UDS was detected in late spermatids and mature sperm. While EMS produced a maximum UDS response in the germ cells immediately after treatment, CPA and DMO did not produce a maximum response until ～0.5 to 1 h after injection. This delay is attributed to the time required for CPA and DMO to be enzymatically vonverted active alkylating metabolites.Unlike the results found with EMS, mutation frequencies (dominant lethals, translocations, specific-locus mutations) following CPA treatment are not noticeably reduced in germ-cell stages in which UDS occurred. In the case of DMO, mutations are induced only in mature spermatozoa, and these germ-cell stages represent only a fraction of those in which no UDS is detected. The results with CPA and DMO thus still leave unclear the relationship between DNA repair and the differential spermatogenic response of mice to genetic damage.