W.M. Generoso

Exposure of female mice to ethylene oxide within hours after mating leads to fetal malformation and death

When previously mated female mice were exposed to inhaled ethylene oxide at the time of fertilization of their eggs or during early pronuclear stage of the zygote (before DNA synthesis), a high incidence of mortality among conceptuses and of congenital abnormalities among both the dead and the surviving fetuses was observed. The developmental stage at which death occurred ranged from near the time of implantation to day 17 of gestation when examination of the uterine contents was performed. In comparison, midgestation and late fetal deaths were absent or minimal when the females were exposed either before mating or when conceptuses were in later zygotic stages (pronuclear DNA synthesis) or had reached the early two-cell stage. The random types of congenital abnormality observed and the remarkable stage-dependent sensitivity suggest a genetic basis for the response. The effects differ, both from genetic damages induced in premating germ cells, which lead only to death near the time of implantation, and from teratogenic damage, which leads to malformations only when exposure of embryos occurs during the period of major organogenesis.

Etoposide induces heritable chromosomal aberrations and aneuploidy during male meiosis in the mouse

Etoposide, a topoisomerase II inhibitor widely used in cancer therapy, is suspected of inducing secondary tumors and affecting the genetic constitution of germ cells. A better understanding of the potential heritable risk of etoposide is needed to provide sound genetic counseling to cancer patients treated with this drug in their reproductive years. We used a mouse model to investigate the effects of clinical doses of etoposide on the induction of chromosomal abnormalities in spermatocytes and their transmission to zygotes by using a combination of chromosome painting and 4′,6-diamidino-2-phenylindole staining. High frequencies of chromosomal aberrations were detected in spermatocytes within 64 h after treatment when over 30% of the metaphases analyzed had structural aberrations (P < 0.01). Significant increases in the percentages of zygotic metaphases with structural aberrations were found only for matings that sampled treated pachytene (28-fold, P < 0.0001) and preleptotene spermatocytes (13-fold, P < 0.001). Etoposide induced mostly acentric fragments and deletions, types of aberrations expected to result in embryonic lethality, because they represent loss of genetic material. Chromosomal exchanges were rare. Etoposide treatment of pachytene cells induced aneuploidy in both spermatocytes (18-fold, P < 0.01) and zygotes (8-fold, P < 0.05). We know of no other report of an agent for which paternal exposure leads to an increased incidence of aneuploidy in the offspring. Thus, we found that therapeutic doses of etoposide affect primarily meiotic germ cells, producing unstable structural aberrations and aneuploidy, effects that are transmitted to the progeny. This finding suggests that individuals who undergo chemotherapy with etoposide may be at a higher risk for abnormal reproductive outcomes especially within the 2 months after chemotherapy.

Heritable translocation and dominant-lethal mutation induction with ethylene oxide in mice

Ethylene oxide was studied for induction of dominant-lethal mutations and heritable translocations in male mice. The chemical was prepared in water and injected intraperitoneally. The dominant-lethal study was conducted using a single injection of 150 mg/kg (maximum tolerated dose); in the heritable translocation study males were injected daily on weekdays for 5 weeks with 60 or 30 mg/kg dose per day. Results clearly showed that ethylene oxide is effective in inducing dominant-lethal mutations and that the 4 stocks of untreated females used do not differ or may differ only slightly in the ability of their eggs to repair ethylene oxide-induced lesions in male germ cells. Increases in the frequencies of heritable translocations were also observed at the 2-dose levels. These frequencies did not deviate significantly from those expected on the basis of dose-square kinetics.

Strain and sex variations in the sensitivity of mice to dominant-lethal induction with ethyl methanesulfonate.

Abstract Male and female mice from two hybrid strains and a random-bred one were subjected to dominant-lethal induction with ethyl methanesulfonate. In female mice, 1 strain revealed a strong mutagenic action of ethyl methanesulfonate while the other 2 strains did not. In male mice, all 3 strains exhibited high sensitivity to the mutagenic action of ethyl methanesulfonate with only minor strain differences. The results thus revealed the existence of large sex differences in 2 of the 3 strains and large strain differences in the response of females.

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.

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 ischromatid 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 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.

Developmental anomalies derived from exposure of zygotes and first-cleavage embryos to mutagens

Results of continuing studies indicate that the mouse zygote and two-cell embryo stages are a window of susceptibility in the experimental induction of congenital anomalies with certain mutagenic agents. The mechanisms by which the mutagens initiate the pathogenesis of these developmental defects are not known. However, in certain cases there is evidence that a nonconventional, perhaps epigenetic, mechanism is involved. Detailed characterization of the spectrum of anomalies induced and comparison of responses at the various stages exposed allowed classification of the mutagens generally into two groups. One group is characterized by being effective only in the early stages of zygote development and capable of producing a relatively high incidence of fetal death and hydrops. The other group affects all of the zygote stages studied as well as the two cell-embryo, but does not increase the incidence of fetal death and hydrops. Except for hydrops, chemicals in the two groups do not differ in terms of the types of anomalies present among malformed live fetuses, which bear a resemblance to a subset of common, sporadic human developmental anomalies that are of unknown etiology. This similarity raises the possibility that certain human developmental defects may have their origins in events that happen in the zygote and early pre-implantation stages.

Female-specific dominant lethal effects in mice

For some chemicals, induction of presumed dominant lethal mutations has been observed only in female mice and not in males. In those cases, questions arise as to (1) whether the increased embryonic mortality is due to genetic effects of the chemicals in the oocyte or, (2) is caused indirectly through maternal toxicity, and, if genetic, (3) the basis for the sex difference. These questions were studied using the compounds adriamycin and platinol. Neither compound induces dominant lethals in male germ cells, but both increased early embryonic mortality when females were treated prior to mating (treatment of maturing oocytes). Reciprocal zygote transfer experiments ruled out, either entirely or for the large part, maternal toxicity as the cause, and cytogenetic analysis of first-cleavage metaphases revealed high incidences of chromosomal aberrations. The results of both of these experiments thus provide evidence that the early embryonic mortality resulted from genetic effects induced in oocytes. Most interestingly, each compound produced unexpected types of chromosomal aberrations. Adriamycin produced deletions, rings, and presumed chromosome-type rearrangements. Platinol, on the other hand, produced a few chromatid-type aberrations, but the bulk of aberrations were characterized by disorganization of the chromatin, minute fragments, and thread-like chromatin bridges between fragments and chromosomes or between two or more chromosomes. The latter type of cytogenetic damage was observed primarily in the centromeric region. It is hypothesized that the female-specific dominant lethal effects of the two compounds are associated with the diffused state of the maturing oocyte chromosomes.

Effects of alkylating chemicals on reproductive capacity of adult female mice

Abstract Adult female mice were subjected to the total reproductive capacity test after a single intraperitoneal injection with one of the following compounds: ethyl methanesulfonate (EMS), methyl methanesulfonatte (MMS), n-propyl methanesulfonate (PMS), isopropyl methanesulfonate (IMS), 1,4-di(methanesulfonoxy) butane (Myleran), triethylenemelamine (TEM), N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), 2-methoxy-6-chloro-9-[3-(ethyl-2-chloroethyl)aminopropylamino]acridine dihydrochloride (ICR-170). The results demonstrate clearly a wide range of fertility effects of alkylating chemicals on female mice. Genetics studies and histological examination of the ovaries were used to analyze fertility effects. For example, the early effects of IMS and Myleran were attributable mainly to dominant-lethal mutations. The drastic stoppage of reproduction with Myleran and with TEM was due to killing of oocytes in the early stages of follicular development. The late effects of IMS were also found to be due to oocyte killing, but the similar, marked late effects of EMS or MNNG were presumably an indirect manifestation of chemical damages outside the ovary. Thus it was shown that a reduction in the fertility of female mice after chemical treatment can be attributed to genetic or cell-killing effects on oocytes or, presumably, to non-ovarian toxic effects. Because of its ability to detect a wide range of unfavorable effects, the total reproductive capacity procedure is recommended for incorporation into the practical screening of chemicals.

Dominant lethal mutations, heritable translocations, and unscheduled DNA synthesis induced in male mouse germ cells by glycidamide, a metabolite of acrylamide

The hypothesis that acrylamide induces dominant lethal mutations and heritable translocations in male mice, not through direct adduction, but by conversion to the reactive epoxide, glycidamide, was investigated. Three studies, namely, induction of dominant lethal mutations, heritable translocations, and unscheduled DNA synthesis in spermatids, which were conducted earlier in this laboratory for acrylamide, were also performed for glycidamide to determine its mutagenic properties and to compare responses. Results of these studies are consistent with the proposal that in vivo conversion to glycidamide is responsible for the mutagenicity of acrylamide in male mice.