Significance of specific-locus germ-cell mutations in mice.

On the basis of a historical-control incidence of 39 mutants in 688 921 progeny (5.7 mutants/10(5) animals or 0.82 mutations/locus/10(5) gametes) proposals are made for the numbers of test progeny required when screening for possible mutagens using the specific-locus test in mice (7 loci). It is recommended that 25,000 control progeny should be included in each test, to establish homogeneity with the historical controls. This would also be the number of progeny required from treated males, unless significantly positive results had been obtained with smaller numbers. It would appear that the greater sensitivity of post-spermatogonial stages could more than compensate for practical difficulties in sampling these stages, rather than spermatogonia, in screening tests.     

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 and dominant lethal mutations in male mice by chlormethine

Chlormethine (WHO), a nitrogen mustard (2,2'-dichloro-N-methyldiethylamine), induces dominant lethal and specific-locus mutations in spermatozoa and spermatids of mice.     

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.     

Genomic analysis of gamma-ray-induced germ-cell mutations at the b locus recovered from the medaka specific-locus test.

To study how gamma-ray-induced germ-cell mutations are fixed at the early embryonic stage of the next generation, genomic alterations in the b locus mutants (colorless melanophores) detected during development in the medaka specific-locus test (SLT) were analyzed. First, nine anonymous DNA markers linked to the b locus were cloned and mapped into the region extending about 47cM surrounding the b locus. Next, losses of paternal alleles of these DNA markers were examined in each of the 51 gamma-ray-induced b locus mutants obtained after irradiation of sperm or spermatids. In these mutants, 47 were dominant lethals, three were semi-viable and one was viable. All the mutants examined had large deletions surrounding the b locus. One viable mutant had an interstitial deletion, while all the semi-viable and dominant lethal ones appeared to have terminal deletions. Deletions extending about 20-35cM were the most frequently observed in 18 of the 51 mutants examined. The largest one extended more than 40cM. These results suggest that most of the gamma-ray induced germ cell mutations recovered as total specific-locus mutants were accompanied by large genomic deletions, which eventually led the mutant embryos to dominant lethality.     

The induction of specific-locus mutations with N-propyl-N-nitrosourea in stem-cell spermatogonia of mice.

A specific-locus test to determine the effect of N-propyl-N-nitrosourea (PNU) on the stem-cell spermatogonia of mice has been performed. Male wild-type mice (C3H/He) were treated with an intraperitoneal injection of 200 mg/kg [corrected] of PNU. Eight weeks after the injections, the males were mated with tester stock females (PW), homozygous for 6 visible recessive genes. Twelve mutants among 8605 offspring were observed. The mutation frequency with PNU was calculated to be 23.2 x 10(-5)/locus/gamete, showing a significant difference from that of the non-treated control. The mutations were all heritable and half of them were viable in homozygous condition. The mutation frequency with PNU was about one-third of that with N-ethyl-N-nitrosourea, a highly potent mutagen for mouse stem-cell spermatogonia.     

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.     

Dominant cataract and recessive specific-locus mutations detected in offspring of procarbazine-treated male mice

The induction of dominant cataract mutations by procarbazine was studied concomitantly with the induction of specific-locus mutations in treated male mice. The most effective dose in the specific-locus test, 600 mg/kg of procarbazine, and a fractionated dose of 5 X 200 mg/kg were used. The frequencies of dominant cataract mutations were higher, but not significantly different from the historical control. The ratio between the number of recovered specific-locus and dominant cataract mutations was in accordance with that found in our experiments with gamma-rays (Ehling et al., 1982; Kratochvilova, 1981) or in experiments with ethylnitrosourea (Favor, 1986). A total of 3 dominant cataract mutations were recovered in the offspring of procarbazine-treated spermatogonial stem cells. Two mutations had complete penetrance while the third exhibited a reduced penetrance of approximately 70%. The viability and fertility of the heterozygotes of all 3 mutations were not affected. Only 1 mutation was shown to be viable as a homozygote.     

Induction of specific-locus mutations in female mice by 1-ethyl-1-nitrosourea and procarbazine

Using the specific-locus method, the ability of 1-ethyl-1-nitrosourea (ENU) and procarbazine hydrochloride (procarbazine) to induce gene mutations in mouse oocytes was tested and confirmed. The sensitive stage for the induction of mutations in oocytes with 160 mg/kg of ENU is 2-4 weeks post treatment. The induced mutation frequency in this mating interval was 5.1 X 10(-7) mutations/locus/gamete/mg/kg. The induction of mutations by procarbazine occurred 8-33 weeks after treatment. The induced mutation frequency in this mating interval for the 400 mg/kg group was 0.4 X 10(-7) mutations/locus/gamete/mg/kg. One third of the induced mutations was lethal in homozygous condition in both experiments. ENU and procarbazine have a lower mutational response in oocytes than in stem-cell spermatogonia.     

Induction of specific-locus and dominant lethal mutations in male mice by ifosfamide (Holoxan)

Ifosfamide induced dominant lethal mutations in spermatozoa of mice at doses of 200 and 300 mg/kg and in spermatids and spermatocytes at 600 mg/kg. The highest dose also induced specific-locus mutations in post-spermatogonial germ-cell stages of mice but not in spermatogonial stem cells. The nature of the induced mutations suggests they are intergenic. The spermatogenic specificity of ifosfamide in mouse germ cells is similar to that of the structurally related cytostatic drugs cyclophosphamide and trofosfamide. Due to the post-spermatogonial germ cell specificity of ifosfamide, the genetic risk is limited to a few weeks after exposure.     

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.     

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.     

Tests for urethane induction of germ-cell mutations and germ-cell killing in the mouse

Urethane, a chemical that has given varied results in mutagenesis assays, was tested in the mouse specific-locus test, and its effect on germ-cell survival was explored. Altogether 32,828 offspring were observed from successive weekly matings of males exposed to the maximum tolerated i.p. dose of 1750 mg urethane/kg. The combined data rule out (at the 5% significance level) an induced mutation rate greater than 1.7 times the historical control rate. For spermatogonial stem cells alone, the multiple ruled out is 3.2, and for poststem-cell stages, 3.5. Litter sizes from successive conceptions made in any of the first 7 weeks give no indication of induced dominant lethality, confirming results of past dominant-lethal assays. That urethane (or an active metabolite) reaches germ cells is indicated by SCE induction in spermatogonia demonstrated by other investigators. Cytotoxic effects in spermatogonia are suggested by our finding of a slight reduction in numbers of certain types of spermatogonia in seminiferous tubule cross-sections and of a borderline decrease in the number of litters conceived during the 8th and 9th posttreatment weeks. The negative results for induction of gene mutations as well as clastogenic damage are at variance with Nomura's reports of dominant effects (F1 cancers and malformations) produced by urethane.     

DNA crosslinking, sister-chromatid exchange and specific-locus mutations

Chinese hamster ovary cells were treated with the DNA-crosslinking chemicals, mitomycin C (MMC) and porfiromycin (POR), and their monofunctional derivative decarbamoyl mitomycin C (DCMMC). After exposure, the cells were studied for the induction of sister-chromatid exchanges (SCEs) and mutations at the hypoxanthine phosphoribosyltransferase and adenine phosphoribosyltransferase loci. The frequency of SCEs varied significantly in successive sampling intervals, requiring the weighting of each interval by the percentage of second-division mitosis in that interval to obtain the mean SCE frequency for each dose. All 3 compounds were potent inducers of SCEs but weakly mutagenic. All 3 chemicals by concentration were approximately equally effective in inducing SCEs or mutations. When the induced SCEs and mutations were compared at equal levels of survival, DCMMC was slightly more effective than MMC or POR in inducing SCEs and somewhat less mutagenic. These results indicate that the DNA interstrand crosslink is not the major lesion responsible for the induction of SCE or mutation by these compounds.     

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.     

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.     

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.