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.     

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.     

Frequency and nature of specific-locus mutations induced in female mice by radiations and chemicals: a review.

The inducibility of heritable mutations in female mammals has been measured in the mouse specific-locus test (SLT). For radiation-induced mutations, a large body of data has been accumulated that includes information about biological and physical factors that influence mutation yields. However, relatively few SLT studies in females have been conducted with chemicals to date. A single estimate of the spontaneous mutation rate in oocytes, 6/536,207, has been derived as the most appropriate one to subtract from experimental rates. This rate is highly significantly below the spontaneous mutation rate in males. Mutations recovered from females mutagenized at any time after about the 12th day post-conception are induced in non-dividing cells. In adult females, most oocytes are arrested in small follicles; maturation from this stage to ovulation takes several weeks. High-dose-rate radiations are more mutagenic in mature and maturing oocytes than in spermatogonia of the male; on the other hand, no clearly induced mutations have been recovered from irradiated arrested oocytes. Efficient repair processes have been invoked to explain the latter finding as well as the upward-curving dose-effect relation for acute irradiation, and the fact that dose protraction drastically reduces mutation yield from mature and maturing oocytes. The dose-protraction effect is much greater than that found in spermatogonia. Radiation-induced mutation rates in embryonic, fetal, and newborn females are overall lower than those in the mature and maturing oocytes of adults. A dose-protraction effect has also been demonstrated at an early developmental stage when the nuclear morphology of mouse oocytes most resembles that of the human. Of only 5 chemicals so far explored for their effect in oocytes, 2 (ethylnitrosourea, ENU, and triethylenemelamine, TEM), and possibly a third (procarbazine hydrochloride, PRC), are mutagenic--with at least one of these (ENU) mutagenic in arrested as well as maturing oocytes. However, the mutation rate is, in each case, lower than for treated male germ cells. By contrast, ENU-induced mutation yield for the maternal genome of the zygote is an order of magnitude higher than that for the zygote's paternal genome or for spermatogonia. A high proportion of mutants derived from chemical treatment of oocytes (including the oocyte genome in zygotes) are mosaics, probably owing to lesions affecting only 1 strand of the DNA. A characteristic of specific-locus mutations induced in oocytes is that they include a considerably higher percentage of large (multi-locus) lesions (LLs) than do mutations induced in spermatogonia.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

X-ray-induced specific-locus mutation rates in young male mice

The specific-locus mutation frequency resulting from 300 R of acute X-irradiation has been determined for the germ cells present in male mice at 2, 4, 6, 8, 10, 14, 21, 28, and 35 days of age. The sample size was large enough for each of these nine age groups to ensure that a high mutation rate would be noticed. The testis of the mouse undergoes many developmental changes between birth, when most or all germ cells are gonocytes, and 35 days, when the cell population has come to resemble that of the adult. It was important to know if the germ cells present in these developmental stages of immature male mice yield the same mutation frequency as that found earlier for spermatogonia in the adult by W. L. Russell.None of the nine age groups has a mutation rate statistically significantly higher than that of the adult. Taken together, the nine groups of males have an average mutation frequency quite to that of the adult. This does not rule out the possibility that individual age groups may have a mutation frequency somewhat different from that of the adult.The distribution of mutations among the loci seems to be similar to that found for mutations induced in spermatogonia of the adult. Clusters of specific-locus mutations were found only on day 21.This paper and that presented earlier on the newborn report the first specific-locus mutation-rate studies on male mice irradiated between birth and adulthood. If the results can be carried over to man, it can be concluded that irradiation of the immature testis, from birth to puberty, will not present any greatly increased genetic hazard over that from irradiation of the adult testis. In fact, as the data stand in the mouse, they indicate a mutation rate similar to the adult for all but the earlier stages tested and, for these stages, a probably lower rate, representing a transition from the significantly lower rate reported earlier for newborns.     

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).     

Tests of induction in mice by acute and chronic ionizing radiation and ethylnitrosourea of dominant mutations that cause the more common skeletal anomalies

Assessment-of-Dominant-Damage (ADD) experiments explored induction by proven specific-locus mutagens of dominant mutations that cause skeletal anomalies, cataracts, and stunted growth in offspring of mutagenized male mice. The data set reported here includes 6134 offspring. Mutagenic treatments included 600 R (i.e., approximately 6 Gy) of X-rays delivered in about 7 min, 600 R of gamma rays delivered over about 110 days, and four weekly intraperitoneal (i.p.) injections of 77.5 mg/kg of ethylnitrosourea (ENU). The results reported in this paper are restricted to mutations induced in stem-cell spermatogonia and to the 34 more common skeletal anomalies (i.e., those found in 0.5% or more of the control offspring). Mutation induction was demonstrated for eight anomalies in the acute X-ray experiment and for 17 anomalies (including those same eight anomalies) in the ENU experiment. In spite of the surprisingly high mutation rates found for these treatments, there was no hint of any induction of such dominant mutations by 600 R of chronic gamma radiation. Our results suggest that several anomalies related to variation in the sacralization pattern may be particularly useful for revealing induction of dominant mutations.     

X-ray-induced specific-locus mutation rate in newborn male mice

The specific-locus mutation frequency resulting from 300 R of acute X-irradiation has been determined for the germ cells present in newborn male mice. The frequency is 13.7·10^?8 mutations/locus/R, which is statistically significantly lower than that of 29.1·10^?8 mutations/locus/R found earlier for the same loci in spermatogonia of the adult male by W. L. Russell. The mutation rate for newborn males does not differ significantly from the induced specific-locus frequency reported for fetal males by T. C. Carteret al.The incidence of clusters of specific-locus mutations found following the irradiation of the newborn males was statistically significantly higher than the cluster incidence reported by W. L. Russell for similar irradiation of adult males. This presumably indicates the survival of relatively fewer reproductive cells following irradiation of the day-o testis.Although there are suggestions that the distribution of mutations among the loci following irradiation of the newborn males may be different from that of the irradiated adults, no statistically significant differences are demonstrated.It is quite possible that the testis of the newborn mouse may be comparable to the relatively undifferentiated human testis which persists for approx. 10 years. Until the present research was undertaken, no attempt had been made to determine the specific-locus mutation frequency resulting from X-irradiation of newborn male mice. Although some important questions still remain concerning the explanation for the lower mutational response of the newborn mouse testis, from the hazard standpoint it is reassuring that the mutation frequency of the newborn male is statistically significantly lower than that of the adult.     

Analysis of the Albino-Locus Region of the Mouse. I. Origin and Viability

Numerous specific-locus experiments designed to test the mutagenic effect of external radiation have yielded, in over 3,600,000 animals observed, altogether 119 presumed mutations involving the c locus. Of these, 55 were viable and albino (cav), 13 were viable and of various intermediate pigment types (cxv), four were subvital (cas and cxs), seven were neonatally lethal albinos (cal), 28 prenatally lethal albinos (cal); 12 died untested. All of the prenatally lethal and at least one of the neonatally lethal c-locus mutations (cal classes) are probably deficiencies that we have analyzed extensively in other experiments. Since absence of the locus mimics albino in phenotype, the intermediates (cxv and cxs groups) probably resulted from intragenic changes. The class of viable albino mutants (cav) might include, in addition to intragenic changes, some extremely small deficiencies. --The effects on viability of c-locus lethals (cal's) in heterozygous condition are not drastic enough to be perceived in stocks of mixed genetic background except in the case of the two longest known deficiencies and a few others. --Analysis of the relation between radiation treatment and type of c-locus mutants obtained shows that the relative frequency of viable mutations, for each germ-cell type, is greater for low-LET than for neutron irradiation; however, the difference for any individual cell type is not significant. The majority (66.7%) of mutations derived from X- or gamma-ray irradiated spermatogonia are viable, and the proportion of "intermediates" among these viables is similar to that among presumed spontaneous c-locus mutations. No significant dose-rate effect on the proportion of lethals could be demonstrated within the set of mutants induced by low-LET irradiation of spermatogonia. Although sets from other germ-cell stages are too small for statistical tests, the results for oocytes are similar, as far as they go. Furthermore, most of the c-locus mutations induced in spermatogonia, even by high-dose-rate X-ray or gamma irradiation, are of a type most likely to result from single-tract events (62% cxv, cxs, and cav; plus 16% presumed deficiencies not involving the closest marker). These results support the view that most of the reduction in mutation frequency at low dose rates is not due to a change in relative proportion of two-track and one-track ionizing events.     

Induction of specific-locus and dominant lethal mutations in male mice by 1-methyl-1-nitrosourea (MNU)

1-Methyl-1-nitrosourea (MNU) induced specific-locus mutations in mice in all spermatogenic stages except spermatozoa. After intraperitoneal injection of 70 mg/kg body weight of MNU a high yield of specific-locus mutations was observed in spermatids (21.8 × 10⁻⁵ mutations per locus per gamete). The highest mutational yield was induced in differentiating spermatogonia. In 1954 offspring we observed 5 specific-locus mutants (44.8 × 10⁻ mutations per locus per gamete). In addition, 2 mosaics were recovered, which gave a combined mutation rate of 62.7 × 10⁻⁵. In A_s spermatogonia the mutation rate was 3.9 × 10⁻⁵. The same dose of 70 mg/kg of MNU induced dominant lethal mutations 5–48 days post treatment, mainly due to post-implantation loss in spermatids and spermatocytes. It is interesting to compare the induction pattern of mutations by MNU with methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS) and ethylnitrosourea (ENU). Based on the different spermatogenic response of the induction of specific-locus mutations we can characterize the 4 mutagens in the following way: EMS = MMS ≠ MNU ≠ ENU.     

Dose-rate effect on transgenerational mutation frequencies in spermatogonial stem cells of the medaka fish

The estimation of transgenerational genetic risk of radiation exposure to non-human species is crucial for the protection of ecosystems. Here we determined the frequency of specific-locus mutations at the five pigmentation loci in medaka spermatogonial stem cells after gamma irradiation at 0.03 cGy/min and 95 cGy/min. At each total dose, the mutation frequency was significantly lower in the 0.03-cGy/min group than in the 95-cGy/min group, suggesting a dose-rate effect. The ratio of the induced mutation frequency at 0.03 cGy/min to that at 95 cGy/min was approximately 0.42 from 0 to 1.9 Gy and approximately 0.33 from 1.9 to 4.75 cGy. In the mouse, this ratio is estimated to be 0.33 (Russell and Kelly, Proc. Natl. Acad. Sci. USA 79, 542-544, 1982). It is thus possible that the magnitude of the dose-rate effect on transgenerational mutation frequencies is comparable between mouse and medaka spermatogonia, suggesting similar dose-rate effects among vertebrates.     

Specific-locus experiments show that female mice exposed near the time of birth to low-LET ionizing radiation exhibit both a low mutational response and a dose-rate effect

Female mice were exposed to 300 R of 73-93 R/min X-radiation either as fetuses at 18.5 d post conception (p.c.) or within 9 h after birth. Combining the similar results from these two groups yielded a specific-locus mutation frequency of 9.4 X 10(-8) mutation/locus/R, which is statistically significantly higher than the historical-control mutation frequency, but much lower than the rate obtained by irradiating mature and maturing oocytes in adults. Other females, exposed at 18.5 days p.c. to 300 R of 0.79 R/min gamma-radiation, yielded a mutation frequency that was statistically significantly lower than the frequency at high dose rates. The low-dose-rate group also had markedly higher fertility. It appears that the dose-rate effect for mutations induced near the time of birth may be more pronounced than that reported for mature and maturing oocytes of adults. A hypothesis sometimes advanced to explain low mutation frequencies recovered from cell populations that experience considerable radiation-induced cell killing is that there is selection against mutant cells. The reason for the relatively low mutational response following acute irradiation in our experiments is unknown; however, the finding of a dose-rate effect in these oocytes in the presence of only minor radiation-induced cell killing (as judged from fertility) makes it seem unlikely that selection was responsible for the low mutational response following acute exposure. Had selection been an important factor, the mutation frequency should have increased when oocyte killing was markedly reduced.     

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.     

Procarbazine-induced specific-locus mutations in male mice.

Procarbazine is used in drug-combination treatment of Hodgkin's disease. The specific locus method was used to test and confirm the ability of procarbazine to induce gene mutations in pre- and post-meiotic germ cells of male mice. The lowest dose of procarbazine that significantly increased the mutation frequency in As spermatogonia over the control frequency was 400 mg/kg (P = 0.003). The corresponding dose for the post-spermatogonial germ-cell stages was 600 mg/kg (P = 0.009). The dose--response was linear for the point estimates of the mutation frequencies after treatment of As spermatogonia with 0, 200, 400 and 600 mg/kg. The point estimate of the mutation frequency at the 800 mg/kg level was one-third of that expected from a linear extrapolation. Variation in mutation rates among the 7 loci between the lowest (a locus) and the highest (p locus) was 12-fold. Only 24% of procarbazine-induced specific locus mutations in As spermatogonia were lethal in the homozygous condition. From the mutation spectra and the viability tests, it is concluded that procarbazine-induced mutations may be mainly due to base-pair changes. Procarbazine-induced specific-locus mutations fulfilled the criteria for the estimation of the doubling dose, the dose necessary to induce as many mutations as occur spontaneously. The doubling dose of procarbazine in As spermatogonia of mice was 114 mg/kg. The therapeutic dose for procarbazine is about 215 mg/kg. If man and mouse were equally sensitive, this dose would induce 1.9 times as many mutations as arise spontaneously. From the incidence of patients with Hodgkin's disease (1 : 42 000) the calculated population dose of procarbazine is 5.12 micrograms/kg. Assuming equal sensitivity between the sexes we can calculate, for an estimated number of 30 000 genes, the induction of about 22 mutations per million children due to procarbazine treatment. The same number of induced mutations can be calculated if the risk of patients is used for the estimation of the genetic hazard.     

Induction of specific-locus mutations in male germ cells of the mouse by acrylamide monomer

Acrylamide monomer (AA), injected into male mice at the maximum tolerated dose of 5 x 50 mg/kg (24-h intervals), significantly increased the specific-locus mutation rate in certain poststem-cell stages of spermatogenesis, but not in spermatogonial stem cells. Germ-cell stages in which the treatment induced dominant lethals--namely, exposed spermatozoa and late spermatids (number of surviving offspring only 3% and 27%, respectively, of those in concurrent controls)--jointly yielded the highest frequency of specific-locus mutations. AA thus conforms to Pattern 1 in our earlier classification of chemicals according to the spermatogenic stage at which they elicit maximum response (Russell et al., 1990). No specific-locus mutations were observed among 17,112 offspring derived from exposed spermatogonial stem cells, a result which rules out (at the 5% significance level) an induced mutation rate greater than 2.3 times the historical control rate. A sustained high productivity in matings made for several months following week 3 indicates that there is no significant spermatogonial killing and that cell selection is presumably not the explanation for the negative result. On the basis of genetic and/or cytogenetic evidence, the mutations induced postmeiotically by AA were 'large lesions' (multi-locus), while one of 2 recovered from exposure of differentiating spermatogonia is probably a small lesion. An earlier survey of mammalian mutagenesis results led us to conclude that, regardless of the classification of a chemical according to the stage at which it elicits its maximum response, the nature of mutations is determined by the germ-cell stage in which they are induced (Russell et al., 1990). The AA results on lesion size and on distribution of mutations among the loci fit the general pattern.     

A dose-response analysis of ethylnitrosourea-induced recessive specific-locus mutations in treated spermatogonia of the mouse

A dose-response analysis was carried out with 2 independent data sets available for ethylnitrosourea-induced specific-locus mutations in spermatogonia of the mouse. It was assumed that the occurrence of mutation is binomially distributed and maximum-likelihood procedures were employed to determine the appropriateness of 4 alternative models, Linear, Linear-Quadratic, Power, and Threshold, in describing the dependence of the binomial parameter on dose. For both data sets, the Threshold model yielded a far superior fit and the threshold dose was estimated to be between 34 and 39 mg/kg. These results are supported by the relatively inefficient response of ethylnitrosourea at lower doses in inducing DNA adducts. Relevant specific-locus mutation results in the mouse for low-dose fractionated treatment as well as the recovery of mutation mosaics indicate the threshold model to be an oversimplification. Rather than a threshold dose below which 100% of the induced DNA adducts are repaired, we propose that some DNA adducts which may eventually be fixed as a mutation persist through a number of repair-competent cell divisions and do not interfere with normal cell function nor do they induce a repair response before being eventually fixed as a mutation. We interpret the thresholded response for ethylnitrosourea-induced specific-locus mutations to be due to a saturable repair process which at lower doses results in ethylnitrosourea being less efficient in inducing mutation. Once this repair process is saturated, a clear dose-related increase in the mutation rate is observed.     

Effects of Male Germ-Cell Stage on the Frequency,Nature, and Spectrum of Induced Specific-Locus Mutations in the Mouse

By means of the mouse specific-locus test (SLT) with visible markers, which is capable of detecting intragenic mutations as well as larger lesions, about 20 mutagens have been studied comparatively across arrays of male germ-cell stages. In addition, a very large historical control, accumulated over decades, provides data on spontaneous mutations in males. Each mutagen has a characteristic germ-cell-stage sensitivity pattern. Although most chemicals yield their maximum numbers of mutations following exposure of spermatozoa and late spermatids, mutagens have now been identified that peak in each of the major stages of spermatogenesis and spermiogenesis, including those in which effects on recombination can also be induced. Stem-cell spermatogonia have yielded positive results with only five of 15 mutagenic chemicals. In postspermatogonial stages, all chemicals, as well as radiations, induce primarily large lesions (LL). By contrast, in spermatogonia (either stem-cell or differentiating) all chemicals except one (bleomycin) produce very few such lesions. The spectrum of relative mutation frequencies at the seven loci of the SLT is characteristic for treated germ-cell stage and mutagen. Treatments that induce primarily LL are characterized by a great preponderance of s (Ednrb)-locus mutations (possibly due to a paucity of haplo-insufficient genes in the surrounding region); and those that induce very few, if any, LL by a great preponderance of p-locus mutations. Spontaneous locus-spectra differ from both types of treatment-induced spectra; moreover, there are two distinct types of spontaneous spectra, depending on whether mutations occurred in mitotic cells or during the perigametic interval.     

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.     

Comparison of the genetic effects of equimolar doses of ENU and MNU: while the chemicals differ dramatically in their mutagenicity in stem-cell spermatogonia, both elicit very high mutation rates in differentiating spermatogonia

Mutagenic, reproductive, and toxicity effects of two closely related chemicals, ethylnitrosourea (ENU) and methylnitrosourea (MNU), were compared at equimolar and near-equimolar doses in the mouse specific-locus test in a screen of all stages of spermatogenesis and spermiogenesis. In stem-cell spermatogonia (SG), ENU is more than an order of magnitude more mutagenic than MNU. During post-SG stages, both chemicals exhibit high peaks in mutation yield when differentiating spermatogonia (DG) and preleptotene spermatocytes are exposed. The mutation frequency induced by 75mgMNU/kg during this peak interval is, to date, the highest induced by any single-exposure mutagenic treatment - chemical or radiation - that allows survival of the exposed animal and its germ cells, producing an estimated 10 new mutations per genome. There is thus a vast difference between stem cell and differentiating spermatogonia in their sensitivity to MNU, but little difference between these stages in their sensitivity to ENU. During stages following meiotic metaphase, the highest mutation yield is obtained from exposed spermatids, but for both chemicals, that yield is less than one-quarter that obtained from the peak interval. Large-lesion (LL) mutations were induced only in spermatids. Although only a few of the remaining mutations were analyzed molecularly, there is considerable evidence from recent molecular characterizations of the marker genes and their flanking chromosomal regions that most, if not all, mutations induced during the peak-sensitive period did not involve lesions outside the marked loci. Both ENU and MNU treatments of post-SG stages yielded significant numbers of mutants that were recovered as mosaics, with the proportion being higher for ENU than for MNU. Comparing the chemicals for the endpoints studied and additional ones (e.g., chromosome aberrations, toxicity to germ cells and to animals, teratogenicity) revealed that while MNU is generally more effective, the opposite is true when the target cells are SG.     

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.