Dominant lethal study of ribavirin in male rats

Ribavirin, a new synthetic antiviral agent, was studied for dominant lethal effects in male CD rats. The drug was administered intraperitoneally at doses of 50, 100 and 200 mg/kg/day for 5 days. Males were mated weekly with 8 consecutive batches of female rats. Marginal increase in early foetal death detected in Assessment Weeks 3 and 8 in females mated with the low-dose and high-dose males were not dose-related and were most probably chance events caused by the particularly low vehicle control frequencies for these 2 weeks. Also, the slightly reduced pregnancy proportion among females mated with the high-dose treated males was to a substantial extent the effect of a single male rate which failed to fertilize any females. Ribavirin was, therefore, regarded as being devoid of any mutagenic potential demonstrable by a rat dominant lethal assay.     

Assessing overdispersion and dose-response in the male dominant lethal assay.

In dominant lethal studies the primary variables of interest are typically expressed as discrete counts or proportions (e.g., live implants, resorptions, percent pregnant). Simple statistical sampling models for discrete data such as binomial or Poisson generally do not fit this type of data because of extra-binomial or extra-Poisson departures from variability predicted under these simple models. Extra-variability in the fetal response may originate from parental contributions. These can lead to over- or under-dispersion seen as, e.g., extra-binomial variability in the proportion response. Utilizing a large control database, we investigated the relative impact of extra-variability from male or female contributions on the endpoints of interest. Male-related effects did not seem to contribute to overdispersion in our database; female-related effects were, however, evidenced. Various statistical methods were considered to test for significant treatment differences under these forms of sampling variability. Computer simulations were used to evaluate these methods and to determine which are most appropriate for practical use in the evaluation of dominant lethal data. Our results suggest that distribution-free statistical methods such as a nonparametric permutation test or rank-based tests for trend can be recommended for use.     

Dominant lethal effects of subchronic acrylamide administration in the male Long-Evans rat

Acrylamide, a widely used vinyl monomer, is well known as a neurotoxin but inactive as a mutagen in bacterial test systems. The experiments reported demonstrate that after subchronic oral dosing in the male rat, acrylamide induced significant elevations in both pre- and post-implantation loss following dominant lethal testing. These effects were seen at doses which failed to produce clinical or pathological evidence of neurotoxicity. In an accompanying cytogenetic study, no increase in chromosome aberrations was observed in spermatogonia or spermatocytes of treated animals. When spermatocytes from treated spermatogonial stem cells were analyzed, reciprocal translocations (4) were observed in the treated animals and not in the control, but the significance of this result still needs to be established.     

Dominant lethal studies in male mice after exposure to 2.45 GHz microwave radiation

Adult male mice had the lower halves of their bodies exposed in a waveguide system to 2.45 GHz microwave radiation for 30 min. The half body dose-rate of 43 W kg-1 had been shown in a previous study [7] to deplete severely the heat-sensitive stages of sperm production. The males were mated at intervals to adult hybrid females over the following 8-10 weeks. There was no significant reduction in post-implantation survival, suggesting that the microwave exposure did not have a mutagenic effect on the male germ cells. However, pregnancy rate was significantly reduced in weeks 3, 4, 5 and 6; reaching a minimum of about 10% of the control value in weeks 4 and 5. The occurrence of low values in weeks 4 and 5 correlated well with the expected reductions in sperm count due to the pattern of depletion of the spermatogenic epithelium of the testes. Thus it was concluded that the reduced pregnancy rate resulted from reduced male fertility. Pre-implantation survival can also be affected by reduced sperm count [8] and was significantly reduced in this study but it correlated less well with the anticipated heat response. A further study is in progress looking at the contribution of sperm count and sperm abnormality to the results.     

Dominant lethal studies in male mice after exposure to a 50-Hz electric field

Male C3H/He mice were sham-exposed or exposed continuously for 2 weeks to a vertical, 50-Hz, electric field at 20 kV/m rms. Densities of currents induced in the testes are estimated to be near 100 microA/m2. After the exposure, each male was mated with two different female mice each week during a period of 8 weeks. By this schedule, female mice were impregnated with sperm that had been exposed to the electric field at different stages of the spermatogenic cycle. No significant differences as a function of exposure condition were observed in pregnancy rates or in survival of embryos before or after implantation. The absence of effects was not due to insensitivity of assays; other mice that were exposed to X-rays (dose to testes = 1.5 Gy) presented reliable evidence of mutagenesis.     

Dominant lethal cell mutants detected by the autoradiographic assay for exotoxin A resistance

The autoradiographic assay (AR assay) for P. aeruginosa exotoxin A (PE) resistance in cultured mouse fibroblasts detects mutants able to synthesize proteins in the presence of the toxin, presumably due to mutations in the structural gene for elongation factor 2 (EF-2). Detection by the AR assay of PER cells is independent of their ability to divide. The frequencies of both spontaneous and mutagen-induced PER cells are higher than those detected by the conventional colony assay. Examination of phenotypic expression times in the PER cells, and of their in situ proliferation, reveals that this higher sensitivity of the AR assay is due to its ability to detect cells in which the PER mutation prevents proliferation, thus escaping detection by the colony assay. Expression of the mutant phenotype in the PER cells detected in the AR assay after mutagenesis with ethyl methanesulfonate (EMS) follows a pattern similar to that observed in the colony assay, reaching a maximum in 3 days, and then remaining constant for at least 4 more. After treatment with X rays (which fail to induce PER mutants in the colony assay), the frequency of PER cells detected in the AR assay also reaches a maximum on day 3, but then declines sharply, returning to the spontaneous level on day 7. In the absence of PE, the majority of the spontaneous or mutagen-induced PER cells detected in the AR assay are either incapable of dividing at all, or capable of undergoing a limited number of cell divisions to produce micro-colonies. Only few of them may continue to grow into 'full-size' colonies comparable to those detected in the colony assay. In the presence of the toxin, the proportion of PER cells which are able to divide is even smaller, and that of cells able to form full-sized PER colonies detectable in the AR assay is comparable to the results obtained in the conventional colony assay. We presume that the lethality of the PER mutations in the cells detected by the AR assay is due to abnormal protein synthesis resulting from the same mutational change that made these cells resistant to PE. While incapable of supporting colony formation, and hence detection by the colony assay, such abnormal protein synthesis still allows the detection of the mutant cells by the AR assay.     

Dominant lethal assay in female mice after oral dosing with dichlorvos or exposure to atmospheres containing dichlorovos.

The effect of a synthetic auxin-like substance (2,4-D) and a synthetic cell division factor (kinetin) on the induction of chromosome aberrations was studied on tissue cultures of Nicotiana glauca and the tumorous amphidiploid hybrid Nicotiana glauca × Nicotiana langsdorffii.The aberration frequencies in normal Nicotiana glauca tissue were proportional to the length of time of culture in the presence of 2,4-D. Moreover, both 2,4-D and kinetin increased chromosome breakage in the habiatouated Nicotiana glauca tissue but not in the amphidiploid hybrid tissue.The data are discussed in terms of genotype-hormone equilibria in long-term development of plant tissue culture.     

Dominant lethal effects of triethylenemelamine in the guppy Poecilia reticulata.

The need to screen potential chemical pollutants for mutagenicity has increased with the increasing volume of such materials being introduced into natural water. The present study demonstrates the utility of a fish, the guppy (Poecilia reticulata), as a model test system in which water-borne chemical mutagens may be assayed for dominant lethal effects. Mature male guppies were injected with three doses of triethylenemelamine (0.1, 0.2 and 0.4 mg/kg) in addition to a control sham treatment. Each male was subsequently mated to virgin females. In an alternative test, male fish were allowed to swim in triethylenemelamine solutions of known concentration for a period of 24 h prior to being mated to virgin female guppies. 10 days following matings, females were dissected, and numbers of live and dead embryos were recorded. Significant dose effects were demonstrated by analysis of variance techniques in both the injection and the emersion tests with the results showing higher percentages of dead embryos and lower total number of embryos with increasing doses of TEM.     

Dominant lethal effects of 2,4-D in Biomphalaria glabrata

Dominant lethal effects of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) were evaluated in the freshwater snail Biomphalaria glabrata. Wild-type snails were exposed during 10 days to 50, 75 and 100ppm of 2,4-D dimethylamine salt (2,4-D DMA) and paired with non-exposed albino snails 1, 11, 25 and 40 days after the exposure. The offspring of the non-exposed albino snails was scored for lethal malformations. One day after the exposure, a significant effect was observed at 75 and 100ppm without a dose-response relationship. After 11 days, the effect was observed only at the highest dose. After 25 days, significant increases in the dominant lethal effects occurred at 50 and 75ppm; effects were directly related to the doses. Background levels of lethal malformations were resumed after 40 days. Although the major and direct measure of dominant lethal mutations is the rate of lethal malformations in the heterozygous offspring of the albino snails, the sensitivity of the assay was substantially increased with the evaluation of all non-viable embryos, that are the sum of those with lethal malformations, identified or not as wild-type.     

Role of recessive lethal genes in spontaneous embryonal lethality in random-bred rat and mouse populations in Cuba

Spontaneous embryonic lethality and the recessive lethal gene frequency in the gene pool of noninbred mice and rats of the bioteria of the National Research Center of the Cuba Republic Academy of Sciences were investigated. It was established that the level of spontaneous embryonic lethality among mice increased greatly as compared to that in 1977 and was 40% (versus 20% in 1977). Of this number, 30% falls within the postimplantation period. In noninbred rats, these values constituted 21.4 and 8.1%, respectively. High embryonic lethality among noninbred mice may be determined by genetic causes and the influence of ecological factors. The frequency of recessive lethals among noninbred mice was 28%, that among rats 20.8%. It is characteristic that in both categories, recessive lethals became apparent only in the postimplantation period of embryogenesis.     

Dominant mutations in familial lethal and severe osteogenesis imperfecta

Summary Four families presenting with familial osteogenesis imperfecta (OI) have been studied: 2 with the lethal type II and 2 with the severe type III form. Fibroblasts of the patients, all issue from non-consanguineous parents, produced normal and abnormal (I) chains. These heterozygous mutations differentiate the recurrent forms from homozygous mutations characteristic of autosomal recessive forms. Although the identity of the mutations could not be determined, such recurrence of autosomal dominant OI is probably the result of germinal mosaicism in one of the parents. Biochemical results were consistent with a somatic mosaicism in the father's fibroblasts in one family. Moreover, our studies show that not only OI type II but also severe OI type III can arise from gonadal mosaicism. We discuss the importance of such a phenomenon for genetic counseling.     

Validation of the anthrax lethal toxin neutralization assay.

A validation of the performance characteristics of a toxin neutralization assay is presented. This in vitro assay measures the functional ability of antisera, containing antibodies to anthrax lethal toxin, to specifically protect J774A.1 cells against Bacillus anthracis lethal toxin cytotoxicity. This colormetric assay is based upon the reduction of MTT by living cells. Human and rabbit antisera produced against anthrax vaccine absorbed (AVA) were used to validate the assay. Results showed a high level of repeatability and reproducibility, particularly for a bio-assay. Inter-assay variability in absorbance values was the most prominent negative finding however, an acceptable level was demonstrated with a ratio [neutralization ratio (NR)] of the test serum 50% effective dose (ED(50)) to the reference standard ED(50). Accuracy was maintained, even in samples with minimal neutralizing capacity, and linearity was noted when sample dilutions resulted in accurate prediction of the Y(max)and Y(min). Specificity tests demonstrated that normal sera did not have an observable effect on the ability of the reference standard to neutralize toxin. The assay remained stable against time, temperature, and freeze/thaw effects on the reference standards, but not on the toxin. The assay also remained stable against media and solution storage effects. Cell passage number and cell plating density were two critical parameters identified during the robustness studies that may be responsible for inter-assay variability in absorbance values. The work was performed in accordance with the FDA's Bioanalytical Method Validation Guidance for Industry and the FDA's Good Laboratory Practice for Nonclinical Laboratory Studies (21 CFR Part 58).