Cytogenetic analysis of malformed mouse fetuses derived from balanced translocation heterozygotes.

Reciprocal translocations are readily induced by various physical and chemical mutagens in certain germ-cell stages. Carriers of balanced reciprocal translocations generally exhibit no abnormal phenotypes, except for occasional male sterility, but about half (on average) of their progeny carry grossly unbalanced chromosome complements and die prenatally, so that the carriers are said to be 'semisterile'. Since death of such progeny generally occurs in very early embryonic stages, it would be of minor importance in an analogous human situation. Several types of unbalanced segregants, however, survive to late gestational or even to postnatal stages and are often malformed. Recently, it was determined in this laboratory that over one half of the male carriers of methylene-bisacrylamide-induced translocations, sired litters that had late-dying and/or malformed fetuses (Rutledge et al., 1990). Five high-anomaly translocation stocks derived from that study and four derived from studies with other mutagens were analyzed cytogenetically to determine (1) the chromosomes and breakpoints involved, (2) the nature of chromosome imbalance in malformed fetuses, and (3) the types of meiotic segregation that produce late-surviving unbalanced segregants. Cytogenetic analysis of the 9 translocation stocks revealed 18 breakpoints located in 12 chromosomes. Each translocation had at least one breakpoint located near the centromere or the telomere. All translocations produced abnormal fetuses that were partially monosomic for a very short terminal chromosome segment, and partially trisomic for a segment that can be of various lengths, 2-10 times as long as the monosomic segment. In 6 stocks, these abnormal fetuses arose by adjacent-1 or alternate segregation; in the other three they arose by adjacent-2 segregation. In addition, tertiary trisomy by 3-1 missegregation was also observed in two of the stocks.

Comparison of two stocks of mice in spermatogonial response to different conditions of radiation exposure.

In a previous report (Generoso et al., 1985) it was shown that the two hybrid stocks of mice, (C3H/R1 x 101/R1)F1 and (SEC/R1 x C57BL/6)F1, differed in their responses to induction of chromosomal aberrations following exposure of the stem-cell spermatogonia to 500 R x 4 (4-week intervals) acute X-rays. The levels of response in the two stocks were paralleled by the effects on the length of the sterile period, which presumably results from stem-cell killing and repopulation. The present study was conducted in order to determine whether the differences between the two stocks in these parameters hold true also for other conditions of radiation exposure. Thus, comparative experiments were conducted using the following acute exposure regimens: 500 R single dose, 500 R + 500 R (24-h interval), 100 R + 900 R (24-h interval), and 500 R x 4 (8-week intervals). The endpoints measured were chromosome rearrangements in diakinesis/metaphase-I meiocytes, embryonic lethality in conceptuses, length of sterile period and testis weight. Trend analysis indicated that higher frequencies of chromosome rearrangements and embryonic lethality were recovered from (C3H/R1 x 101/R1)F1 than from (SEC/R1 x C57BL/6)F1 males, that there were no significant differences between stocks in testis weight reductions, and that there was no consistency in the direction of the significant differences that occurred in the length of the sterile period. A definitive conclusion regarding the possible association between induction of chromosomal aberrations and induction of cell killing awaits direct histological analysis of the stem-cell population.

Increased incidence of developmental anomalies among descendants of carriers of methylenebisacrylamide-induced balanced reciprocal translocations.

N,N'-Methylenebisacrylamide (MBA), a dimer of the monomeric acrylamide, was studied for induction of clastogenic effects in germ cells of male mice. It was found to be effective in inducing dominant-lethal mutations and heritable translocations in maturing sperm. The semisterile translocation carriers and their normal counterparts were used to determine the health impact of transmitted chromosomal rearrangements through anatomical analysis of their immediate descendants in utero. As expected, semisterility resulted primarily from embryonic death during the periimplantation stages presumably caused by sperm segregants with unbalanced chromosome complement fertilizing some of the eggs. Among conceptuses that survived to mid- and late-gestation stages, there was an increased incidence of developmental anomalies including fetal death and phenotypic defects. These anomalies are assumed to be caused by certain types of unbalanced segregants that are compatible with survival beyond the periimplantation period. This class of unbalanced segregants represent in humans a major health problem to the mother and her conceptus.

Concentration-response curves for ethylene-oxide-induced heritable translocations and dominant lethal mutations.

Male mice were subjected to repeated inhalation exposures to different concentrations (165, 204, 250, or 300 ppm) of ethylene oxide (EtO) during an 8.5-week period. Transmitted clastogenic effects of these exposures were measured in terms of induction of dominant lethal mutations and heritable translocations. The concentration-response curves for both endpoints are not linear but are markedly concave upward. Significant increases in dominant lethals were detected at all concentrations, except the lowest one. In comparison, the incidences of heritable translocations were significantly increased at all concentrations.

Fetal anomalies produced subsequent to treatment of zygotes with ethylene oxide or ethyl methanesulfonate are not likely due to the usual genetic causes.

Earlier studies in this laboratory revealed that ethylene oxide (EtO) or ethyl methanesulfonate (EMS) induced high frequencies of midgestation and late fetal deaths, and of malformations among some of the surviving fetuses, when female mice were exposed at the time of fertilization of their eggs or during the early pronuclear stage of the zygote. Effects of the two mutagens are virtually identical. Thus, in investigating the mechanisms responsible for the dramatic effects in the early pronuclear zygotes, the two compounds were used interchangeably in the experiments. First, a reciprocal zygote-transfer study was conducted in order to determine whether the effect is directly on the zygotes or indirectly through maternal toxicity. And second, cytogenetic analyses of pronuclear metaphases, early cleavage embryos, and midgestation fetuses were carried out. The zygote transplantation experiment rules out maternal toxicity as a factor in the fetal maldevelopment. Together with the strict stage specificity observed in the earlier studies, this result points to a genetic cause for the abnormalities. However, the cytogenetic studies failed to show structural or numerical chromosome aberrations. Since intragenic base changes and deletions may also be ruled out, it appears that the lesions in question induced in zygotes by the two mutagens are different from conventional ones and, therefore, could be a novel one in experimental mammalian mutagenesis. Alternatively, the mechanism could involve a non-mutational 'imprinting' process that caused changes in gene expression.

No evidence found for induction of dominant lethal mutations and heritable translocations in male mice by calcium cyclamate.

Calcium cyclamate, an artificial sweetener, was studied for its effectiveness in inducing transmissible chromosomal aberrations in germ cells of male mice. Both the dominant-lethal and the heritable translocation tests were carried out following daily treatment (on weekdays) of males by oral intubation with the maximum tolerated dose for 6 weeks. Calcium cyclamate is negative in both tests; therefore, there is no evidence of induced chromosome breakage and exchange.

Acrylamide: induction of heritable translocation in male mice.

Acrylamide (AA), known to induce dominant lethals in male rodents, was studied in the mouse heritable translocation test by using intraperitoneal injections on 5 consecutive days. Matings on days 7-10 following the last injection yielded a high frequency of translocation carriers in the F1 male population, which demonstrated that acrylamide is an effective inducer of translocations in postmeiotic germ cells. As an inducer of both dominant lethals and heritable translocations in late spermatids and early spermatozoa, AA is similar to alkylating agents such as ethylmethanesulfonate and ethylene oxide. However, AA's chemical structure, the nature of adducts formed with DNA, and it lack of mutagenicity in bacteria suggest a different mechanism as the basis for AA's germ cell mutagenicity.

A balanced translocation in mice with a neurological defect.

A semisterile male translocation heterozygote [t(2; 14) 1Gso] that exhibited neurological symptoms and an inability to swim (diver) was found among the offspring of male mice treated with triethylenemelamine. All breeding and cytogenetic data showed a complete concordance between translocation heterozygosity and the neurological disorders. Homozygosity for the translocation seemed to be lethal at an early embryonic stage. Despite the distinctive neurologic symptoms, no anatomic or histological defects in either the ear or in the central nervous system were observed. Thus, a balanced chromosomal translocation can produce disease with an inheritance pattern that mimics a single dominant gene defect.

Difference in the response of two hybrid stocks of mice to X-ray induction of chromosome aberrations in spermatogonial stem cells.

Ionizing radiation induces balanced reciprocal translocations in spermatogonial stem cells of mice. From cells carrying these rearrangements, which can be scored cytologically in the diakinesis-metaphase I stage, balanced normal, balanced translocated and unbalanced (duplication/deficiency) sperm can be produced. The relationship between expected (calculated from cytological data) and observed frequencies of embryonic lethality (presumably as a result of unbalanced sperm fertilizing the egg) following exposure of spermatogonial stem cells to X-rays was studied in two hybrid stocks. A marked difference in the incidence of induced embryonic lethality was found between the two stocks. Similarly, a difference in the cytological frequencies of translocations was also found, although smaller than that observed for embryonic lethality. Thus, it appears that the difference between the two stocks in the frequencies of embryonic lethality may be attributable both to processes occurring prior to metaphase I and to a difference in the rate of transmission of unbalanced chromosome constitutions.

239Plutonium-induced heritable translocations in male mice.

This study was conducted to estimate the frequency of transmitted reciprocal translocations per rad of exposure to alpha particles from [239Pu]citrate. Data indicate that the rate of induction of heritable translocations is related linearly to the duration of spermatogonia stem cell exposure. The estimated increase in heritable translocations per rad of exposure of the stem cell to alpha particles is in the range of 1.45-2.91 X 10(-5)/gamete.

Response of mouse spermatogonial stem cells to X-ray induction of heritable reciprocal translocations.

Although heritable translocations are an important endpoint for the assessment of genetic risk from radiation, there has been a serious information gap with regard to their induction in spermatogonial stem cells, the most important cell stage in males for risk considerations. This led to uncertainty in estimating the magnitude of risk per unit exposure. Further, the relationship between the frequency of reciprocal exchanges scored by cytological analysis of the exposed male's meiocytes and the frequency of those transmitted to first-generation offspring needed to be re-examined. In order to fill in these gaps, two radiation studies, i.e., dose response and dose fractionation, were conducted on spermatogonial stem cells in which heritable and cytologically detected translocations were scored. The present data are by far the most extensive, to date, for heritable translocation induction in spermatogonial stem cells. The linearity of the rising portion of the dose-effect curve and the additivity of effects observed in the fractionation study allow a direct estimation of the number of transmissible translocations expected per unit exposure. Thus, the expected increase in heritable translocations per rad of acute X-rays is 3.89 X 10(-5) per gamete. The data also show a lack of consistency between cytologically and genetically scored translocations.

Effect of dose fractionation on the ethylnitrosourea induction of specific-locus mutations in mouse spermatogonia.

As measured by specific-locus mutations in mouse spermatogonia, fractionating a dose of 100 mg of ethylnitrosourea per kg of body weight into doses of 10 mg/kg injected intraperitoneally at weekly intervals greatly reduces the mutation frequency compared with that from a single dose of 100 mg/kg. Because there is independent evidence that the doses of 10 and 100 mg/kg reach the germ cells in amounts directly proportional to the injected dose, the lower mutational response with the fractionated dose is attributed to repair. The induced mutation rate expected from a single 10-mg/kg dose (on the assumption that this would be 1/10th the rate observed after 10 such doses) would be only 75% of the spontaneous mutation rate. Mouse spermatogonia apparently have an efficient repair system that is effective even against a potent mutagen.

Difference in the ratio of dominant-lethal mutations to heritable translocations produced in mouse spermatids and fully mature sperm after treatment with triethylenemelamine (TEM).

The relative induction of dominant-lethal mutations and heritable translocations in triethylenemelamine-treated postmeiotic germ cells of mice was determined depending on the stage treated. Males were mated either 11.5-14.5 days after treatment (middle spermatids) or less than 2.5 hours after treatment (fully mature sperm). Results clearly showed that, even through similar levels of dominant-lethal mutations were induced in fully mature sperm and in middle spermatids, the frequency of heritable translocations induced in mature sperm was markedly lower than that induced in middle spermatids. This observation was used, together with earlier ones, to suggest a mechanism by which dominant-lethal mutations and heritable translocations are produced following chemical treatment of male postmeiotic germ cels.