Prenatal death and malformations after irradiation of mouse zygotes with neutrons or X-rays

Female mice (strain: "Heiligenberger Stamm") were irradiated with neutrons (7 MeV) or X-rays when embryos were at the early zygote stage; uterine contents were examined on gestation day 19 for prenatal mortality and malformed fetuses. For both radiation qualities, the dose-dependent survival curve fitted well to a simple exponential equation; the neutron relative biological efficiency (RBE) value was 2.3. The major fraction of deaths induced by exposure to neutrons or X-rays occurred before implantation. Aside from dead embryos, malformed fetuses were observed 19 days p.c. (postconception). The number of malformed fetuses increased with a linear-quadratic function of neutron or X-ray dose. Malformations were mainly gastroschisis, although omphaloceles and anencephalies were also observed. The neutron RBE value for the induction of malformations varied from 2.0 to 2.8 in the dose range tested. Except after 75-cGy neutrons, no significant increase in the proportion of stunted or skeletally malformed fetuses was noted. Our results indicated that the reaction of preimplantation embryos to irradiation could be more complex than the simple "all-or-none" response considered so far.     

Cytogenetic effects of X-rays and fission neutrons in female mice

The induction by X-rays of chromosomal damage in oocytes was studied, while the genetic consequences of X- and neutron-induced damage in female mice were looked for by testing offspring for dominant lethality and semi-sterility. None out of 386 sons of hybrid females given 300 rad X-rays showed evidence of semi-sterility or translocation heterozygosity, but 9 out of 294 daughters were diagnosed as semi-sterile. At least 3 and probably 4 of these (1.4%) carried reciprocal translocations, 2 of which caused male sterility. Complete or partial loss of the X-chromosome may have been responsible for some of the other sermi-steriles. Examination of oocytes at metaphase-I during the first and third weeks after X-irradiation with 100 or 400 rad revealed both multivalents (some of the ring quadrivalent type) and fragments (mainly double). These were thought to arise mainly from chromatid intercchanges (both symmetrical and asymmetrical) and isochromatid intrachanges respectively. Since neither the proportion of asymmetrical interchanges nor the amount of hidden damage was known it was not thought possible to predict the magnitude of F₁ effects from metaphase-I findings. The aberration frequency in oocytes rose with dose and (at the 400 rad level only) with time after irradiation, reaching a maximum of 10% multivalents and 22% fragments in the third week after 400 rad. The frequency of univalents showed no consistent trend, but chiasma counts decreased in the first week after 400 rad. The increase in levels of chromosomal damage with dose and time after irradiation was reflected in dominant lethal frequencies after the same radiation-conception intervals and doses of 0–400 rad. Induced post-implantation lethality was over twice as high in the third week after 200–400 rad than in the first. Pre-implantation loss also greatly increased in the third week after 300 or 400 rad; this was associated with increased non-fertilization of ova. No evidence for the induction of translocations in oogonia or resting oocytes by fast neutron irradiation was obtained, although there was evidence for X-chromosomal loss after 200 rad to oocytes. The relative biological effectiveness (RBE) for fission neutrons vs. X-rays with respect to dominant lethal induction in oocytes was found to vary with dose, but seamed to be around 1 at lower levels.     

The relationship between radiation-induced DNA double-strand breaks and cell kill in hamster V79 fibroblasts irradiated with 250 kVp X-rays, 2.3 MeV neutrons or 238Pu alpha-particles

Using the neutral filter elution technique, the induction of DNA double-strand breaks (dsb) has been measured in 250 kVp X-irradiated V79-379A Chinese hamster cells irradiated under air or nitrogen. The dose-effect curves for induced dsb were curvilinear, mirroring cell survival curves, such that there was an approximately linear relationship between induced dsb and lethal lesions (-In (cell survival)) which was independent of oxygen. With cells irradiated with 2.3 MeV neutrons or 238Pu alpha-particles the correlations between lethal events and dsb, although also approximately linear, do not match those for X-rays. With neutrons there is approximately a 2.5-fold reduction in the level of dsb induction per lethal event. Thus either the apparently linear relationships found are spurious, and there is no general correlation between induced dsb and lethal effect, or there are qualitative differences between neutron, alpha-particle and X-ray induced dsb that give them differing probabilities of cell kill.     

The mouse splenocyte assay, an in vivo/in vitro system for biological monitoring: studies with X-rays, fission neutrons and bleomycin.

A modified mouse splenocyte culture system was standardized after testing different mitogens (i.e., phytohemagglutinin (PHA), concanavalin A (Con A)). The mitotic index was determined for comparison between different mitogens. Following selection of appropriate mitogen (PHA 16, Flow), a series of experiments were conducted to evaluate the application of a cytokinesis-block for scoring micronuclei and assays for chromosomal aberrations produced by treatment in G0 and G2 for the purposes of biological dosimetry following in vivo and/or in vitro exposure to X-rays, fission neutrons and bleomycin. In the X-irradiation studies, the frequencies of micronuclei and chromosomal aberrations (i.e., dicentrics and rings) increased in a dose-dependent manner. These data could be fitted to a linear-quadratic model. No difference was observed between irradiation in vivo and in vitro, suggesting that measurement of dicentrics and micronuclei in vitro after X-irradiation can be used as an in vivo dosimeter. Following in vivo irradiation with 1 MeV fission neutrons and in vitro culturing of mouse splenocytes, linear dose-response curves were obtained for induction of micronuclei and chromosomal aberrations. The lethal effects of neutrons were shown to be significantly greater than for a similar dose of X-rays. The relative biological effectiveness (RBE) was 6-8 in a dose range of 0.25-3 Gy for radiation-induced asymmetrical exchanges (dicentrics and rings), and about 8 for micronuclei in a dose range of 0.25-2 Gy. Furthermore, the induction of chromosomal aberrations by bleomycin was investigated in mouse G0 splenocytes (in vitro) and compared with X-ray data. Following bleomycin treatment (2 h) a similar pattern of dose-response curve was obtained as with X-rays. In this context a bleomycin rad equivalent of 20 micrograms/ml = 0.50 Gy was estimated.     

Meiotic non-disjunction induced by fission neutrons relative to X-rays observed in mouse secondary spermatocytes. II. Dose-effect relationships after treatment of pachytene cells

(C57B1/Cne X C3H/Cne)F1 male mice were irradiated with single acute doses of 0.4 MeV neutrons (from 0.11 to 0.72 Gy) or 250 kV X-rays (from 0.25 to 3 Gy) and sacrificed 5 days later. Chromosome preparations of secondary spermatocytes, irradiated at the stage of pachytene, were analysed and the incidence of hyper-haploidies and chromosome fragments was recorded. Data on numerical aberrations were fitted by highly significant linear relationships for both types of radiation. A relative biological effectiveness (RBE) value of 5.65 was estimated by the ratio between the slopes of the two regression lines. The same linear fitting was applied to frequencies of cells with fragments, even if in this case other types of functions could not be excluded. An RBE value was estimated in the same way as for numerical aberrations and yielded a comparable figure of 5.23. A significant correlation was also found between the incidence of numerical and structural aberrations, which points to the chromosome itself as the prevalent target for radiation-induced non-disjunction (ND). In addition, the highly significant linearity of the dose-effect relationship observed for the induction of aneuploidies suggests, as the simplest hypothesis, a single-hit mechanism of radiation action, possibly through pre-non-disjunctional damage to the centromeric region, rather than an indirect induction of segregational difficulties after primarily induced chromatid interchanges.     

Genetic background damage accompanying reciprocal translocations induced by X-rays and fission neutrons in Arabidopsis and Secale

Translocations were induced in Arabidopsis and rye by fission neutrons and X-rays using doses with equal effects. Segregations of these translocations were studied in M₃, M₄ and S₁ of backcrosses. When there was a deficit of non-translocation homozygotes it was concluded that genetic damage had occurred in chromosomes homologous with the translocation chromosomes. The frequency of families with shortage (including absence) of translocation homozygotes was much larger than families with background damage. This demonstrates that reduced viability in the former was due to true breakpoint damage and not to linked damage. The frequency of translocation breakpoint damage was the same for X-rays and fission neutrons, but the latter probably induced more serious damage. Background damage was also the same for the two types of radiation. Therefore, neutrons should not be considered a ‘cleaner’ inducer of translocation than X-rays.     

Combined treatment of preimplantation mouse embryos in vitro with sodium nitrite and X-rays

Summary Man takes up nitrite in a considerable amount. Effects of nitrite on DNA have been reported; therefore, interaction between nitrite and radiation might be possible.Preimplantation mouse embryos in vitro were treated with a combination of sodium nitrite (1 mM or 2.5 mM) and X-rays (0.94 Gy) in order to obtain some information whether radiation risk is influenced by the presence of nitrite. The microscopic visible development up to 144 h post conceptionem (h p.c.), the number of cell nuclei, and the number of micronuclei were determined.None of the experimental results gives any indication that radiation risk is influenced by nitrite. All effects after combined treatment correspond to the sum of the single effects.     

Survival of V79 cells following simultaneous irradiation with X-rays and neutrons in air or hypoxia

V79 Chinese hamster cells have been irradiated with X-rays and neutrons given simultaneously. The oxygen enhancement ratio and r.b.e. were measured as a function of the proportion of the dose due to the neutrons, which varied from 0 to 100 per cent. These were compared with the values calculated assuming the two types of radiation act independently, following an approach suggested by Curtis. The o.e.r. was less than the predicted value when the neutrons contributed less than about 40 per cent of the total dose. The r.b.e. also did not vary as predicted on the basis of independent action. The 'oxygen gain factor' reached half its maximum value when the proportion of the dose due to neutrons was only about 27 per cent. The results imply that there may be interaction between the damage caused by X-rays and neutrons and that beams having only 20 to 30 per cent of their dose due to high l.e.t. radiation, could be of therapeutic benefit.     

The effects of lead and X-rays, alone or in combination, on blastocyst formation and cell kinetics of preimplantation mouse embryos in vitro

Two-cell embryos in late G2-phase (cytofluorometrically determined) at 32 hours post-conception were treated in vitro with PbC12 (0 . 1 or 1 . 0 micrograms per ml). An additional group was X-irradiated 1 hour later (0 . 94 Gy) with or without lead treatment. Lead alone impaired formation and hatching of blastocysts. The combined treatment increased this deleterious effect on preimplantation development but synergism was not observed. Cell proliferation was disturbed by lead alone but to a higher degree by lead plus X-rays. A pronounced reduction of cell number per embryo was found during the period after reaching the morula stage although the labelling index increased. Apparently cell death occurred. Unlabelled S-phase cell nuclei (comparison between autoradiographic results and cytofluorometric DNA-determinations on the same isolated cell nuclei) and cell nuclei with a hyperploid (0 . 1 microgram PbC12 + 0 . 94 Gy) or hypoploid (1 . 0 microgram PcC12 + 0 . 94 Gy) DNA-content may have contributed to this cell loss. Labelling of cell nuclei which according to their DNA-content were not in S-phase was only observed when 0 . 1 microgram PbC12 alone or in combination with X-rays was used. This effect may be due to unscheduled DNA-repair synthesis or to the induction of chromosome aberrations (acentric fragments or non-disjunction).