K.T. Cain

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.

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.

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.

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.

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.

Efficient gene-driven germ-line point mutagenesis of C57BL/6J mice

BackgroundAnalysis of an allelic series of point mutations in a gene, generated by N-ethyl-N-nitrosourea (ENU) mutagenesis, is a valuable method for discovering the full scope of its biological function. Here we present an efficient gene-driven approach for identifying ENU-induced point mutations in any gene in C57BL/6J mice. The advantage of such an approach is that it allows one to select any gene of interest in the mouse genome and to go directly from DNA sequence to mutant mice.ResultsWe produced the Cryopreserved Mutant Mouse Bank (CMMB), which is an archive of DNA, cDNA, tissues, and sperm from 4,000 G1 male offspring of ENU-treated C57BL/6J males mated to untreated C57BL/6J females. Each mouse in the CMMB carries a large number of random heterozygous point mutations throughout the genome. High-throughput Temperature Gradient Capillary Electrophoresis (TGCE) was employed to perform a 32-Mbp sequence-driven screen for mutations in 38 PCR amplicons from 11 genes in DNA and/or cDNA from the CMMB mice. DNA sequence analysis of heteroduplex-forming amplicons identified by TGCE revealed 22 mutations in 10 genes for an overall mutation frequency of 1 in 1.45 Mbp. All 22 mutations are single base pair substitutions, and nine of them (41%) result in nonconservative amino acid substitutions. Intracytoplasmic sperm injection (ICSI) of cryopreserved spermatozoa into B6D2F1 or C57BL/6J ova was used to recover mutant mice for nine of the mutations to date.ConclusionsThe inbred C57BL/6J CMMB, together with TGCE mutation screening and ICSI for the recovery of mutant mice, represents a valuable gene-driven approach for the functional annotation of the mammalian genome and for the generation of mouse models of human genetic diseases. The ability of ENU to induce mutations that cause various types of changes in proteins will provide additional insights into the functions of mammalian proteins that may not be detectable by knockout mutations.

Chromosome malsegregation and embryonic lethality induced by treatment of normally ovulated mouse oocytes with nocodazole

The mouse egg is ovulated with its nucleus arrested at the metaphase-II stage of meiosis. Sperm entry triggers the completion of the second meiotic division. It has been speculated that damage to the meiotic spindle of normally ovulated eggs at around the time of sperm entry could result in chromosome malsegregation and the death of conceptuses with numerical chromosome anomalies. This hypothesis was tested using nocodazole, a microtubule inhibitor. Nocodazole was administered either to maturing preovulatory oocytes or to normally ovulated eggs at one of the following stages: (1) the time of sperm entry, (2) early pronuclear stage, (3) pronuclear DNA synthesis, (4) prior to first cleavage division, (5) early 2-cell stage, or (6) prior to the second cleavage division. Little or no effect was observed for treatment times other than the time of sperm entry, when the egg is being activated to complete the second meiotic division. Remarkably high frequencies of embryonic lethality, expressed at around the time of implantation, were induced at this stage. Cytogenetic analysis of first cleavage metaphases of zygotes treated at the time of sperm entry revealed a high incidence of varied numerical chromosome anomalies, with changes in ploidy being predominant.

Dominant lethal and heritable translocation tests with chlorambucil and melphalan in male mice

Chemicals used in the treatment of cancer include several that are potent mutagens in a range of in vitro and in vivo assays. For some, genetic effects have also been demonstrated in humans, detected as chromosomal aberrations in peripheral lymphocytes. Because (1) many of these agents are confirmed mutagens, (2) humans are exposed to them in relatively high doses, and (3) an increasing number of early cancer victims are surviving to reproductive age, it is important that information be available on the genetic and reproductive hazards associated with exposure to these agents. Chlorambucil and melphalan are structurally related chemicals that are included in our efforts to identify and assess such hazards among cancer chemotherapy agents. To date, both have been reported to induce specific locus mutations in germ cells of male mice (Russell et al., 1989; Russel et al., 1992b) and melphalan is one of very few chemicals shown to induce such mutations in spermatogonial stem cells. More recently, both chemicals were found to have strong reproductive effects in female mice (Bishop and Generoso, 1995, in preparation). In the present studies, these chemicals were tested for the induction of dominant lethal mutations and heritable translocations in male mice. Both chemicals were found to have reproductive effects attributable to cytotoxicity in specific male germ cell stages and to induce dominant lethal mutations and heritable translocations in postmeiotic germ cells, particularly in mid to early stage spermatids. Thus, relatively extensive data are now available for assessing the genetic and reproductive hazards that may result from therapeutic exposures to these chemicals.

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 spermatozoa. In contrast to the dominant-lethal observed the study showed no detectable increase in heritable 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.