[Comparative analysis of ionizing radiation and xenobiotics influence on spermatogenic epithelium and dominant lethal mutations output in laboratory animals].

The study covered state of spermatogenic epithelium and dominant lethal mutations output in mice of BALB/c and CBA lines, subjected to total gamma-irradiation and in Wistar rats after intraperitoneal injection of potassium bichromate (K2Cr2,O7) in small and sublethal doses. The BALB/c line mice under low irradiation dose (0.25 Gy) demonstrated stimulation effect on spermatogenic epithelium, but in the CBA line mice no such effect was seen. Both mice lines under irradiation of 0.25 Gy and 1.0 Gy demonstrated increase in pathologic sperm counts and in percentage ofpreimplantation embryonal death. In rats, injection of potassium bichromate in doses of 0.028 mg/kg and 2.8 mg/kg increased number of micronuclear spermatids, larger pathologic sperm counts and percentage of postimplantation deaths. Thus, lower general embryonal deaths under radiation exposure is due to preimplantation embryonal deaths, under exposure to 6-valent chromium--is due to postimplantation losses.

Cellular radiosensitivity: expression of an MS susceptibility gene?

As a group, of 40 MS patients exhibited significantly more cellular sensitivity to ionizing (gamma) radiation than 30 age- and sex-matched controls (p less than 0.0001), as measured by radiation-induced chromosome aberrations. Studies of phytohemagglutinin-stimulated T lymphocytes, B lymphoblastoid cell lines, and fibroblasts indicated that the cellular radiosensitivity was a general property of the cells of an individual. Patterns of cellular radiosensitivity among the unaffected first-degree relatives of some MS patients suggested autosomal dominant inheritance. Cellular radiosensitivity may be due to mutations of DNA-processing that predispose to MS.

X-ray-induced dominant lethal mutations in mouse oocytes detected by an in vitro assay.

Female mice were X-irradiated with 0.5-4.5 Gy 2 h before mating to unirradiated males of the same strain. The dominant lethal frequencies (DLF) were determined by growing the embryos in vitro from the two-cell stage and determining the relative rates of successful embryogenesis to the blastocyst stage and to the trophectoderm outgrowth with proliferated inner cell mass stage. The DLF increased with increasing dose, the two linear aspects having a breakpoint at about 1.5 Gy. The nature of embryo failure was also dose dependent. At doses less than 2.0 Gy embryos failed predominantly after blastocyst formation, but at higher doses the embryos failed both before and after blastocyst formation. Over the dose range tested, the frequency with which lesions leading to dominant lethality were induced [i.e., -ln(1 - DLF)] increased linearly with increasing dose.

Mechanisms for dominance: Adh heterodimer formation in heterozygotes between ENU or X-ray induced null alleles and normal alleles in Drosophila melanogaster.

To study mechanisms for dominance of phenotype, eight ENU- and four X-ray-induced mutations at the alcohol dehydrogenase (Adh) locus were analyzed for partial dominance in their interaction with normal alleles. All ENU and one of the X-ray mutations were single base substitutions; the other three X-ray mutations were 9-21 base deletions. All but one of the 12 mutant alleles were selected for this study because they produced detectable mutant polypeptides, but seven of the 11 producing a peptide could not form dimers with the normal peptide and the enzyme activity of heterozygotes was about half that of normal homozygotes. Four mutations formed dimers with a decreased catalytic efficiency and two of these were near the limit of detectability; these two also inhibited the formation of normal homodimers. The mutant alleles therefore show multiple mechanisms leading to partial enzyme expression in heterozygotes and a wide range of dominance ranging from almost complete recessive to nearly dominant. All amino acid changes in mutant peptides that form dimers are located between amino acids 182 and 194, so this region is not critical for dimerization. It may, however, be an important surface domain for catalyzation.

Mutagenesis in murine spermatogonia by MOPP therapy.

Male mice were given mechlorethamine (2.0 mg/kg) followed immediately or 1, 2, 4, or 8 h later by procarbazine (100 mg/kg) and vincristine (0.67 mg/kg) in a protocol that stimulated the drugs and dosages used in a single cycle of clinical MOPP therapy. Seven weeks later and continuing for the next five weeks, the mice were mated to females and the dominant lethal mutant frequencies determined in their offspring by an in vitro assay. Significant (Student's t-test, p less than 0.05) mutant frequencies were detected when the interval between drug administration was between 2 and 8 h. In another series of experiments, mice were injected with these drugs using a 4-h interval between mechlorethamine and the vincristine and procarbazine (MOPP), two cycles of MOPP separated by one week, one cycle of MOPP and 2.0 Gy x-radiation separated by one week, or 2.0 Gy x-radiation alone. When each group was assayed for its dominant lethal mutant frequency, all were found to have similar, highly significant (0.001 less than p less than 0.005) induction. Together these data suggest that stable mutagenic lesions are induced in spermatagonial stem cells by both x-radiation and chemotherapeutic drugs and the frequency with which they are produced can be approximated by a single treatment. Clinical strategies to minimize genetic effects should concentrate on the sequence and timing of drug administration on those days when the drugs are administered in combination.

Induction of congenital malformation in mice by parental irradiation: transmission to later generations.

In order to investigate the genetic basis of the increased incidence of congenital malformations in the offspring of irradiated mice, the frequency of malformations among the offspring of individual F1 sons of irradiated females was studied in detail. Among 90 fully tested F1 sons of females which had been mated 15-21 days after receiving 360 cGy X-rays 4 were definite or probable carriers of dominant genes giving a low penetrance of malformations. This confirms that the malformations seen in the first generation are of genetic origin and can be transmitted to later generations. However, the incidence and penetrance of the mutant genes detected were too low to account for all the anomalies found in the first generation. It was concluded that the genetic basis of the original anomalies was heterogeneous, with some due to genetic changes of high penetrance and rapidly eliminated, and others due to genes of low penetrance like those found in this work. Other malformations, in both the irradiated and control series, were probably of non-genetic origin.

The relationship between radiation-induced and transposon-induced genetic damage during Drosophila oogenesis.

The combined effect of X-irradiation and transposon mobility on the frequencies of X-linked recessive lethals and dominant lethals was investigated in female hybrids in the P-M system of hybrid dysgenesis. X-linked lethals were measured in G2 hybrid dysgenic females whose X chromosome was derived from the M X P cross. To test for additivity or synergism, the mutation rate in irradiated dysgenic females was compared to that of unirradiated females as well as to irradiated nondysgenic hybrid females derived from M X M crosses. Eggs collected for 2 days after irradiation, were represented by the more radiation-sensitive A and B oocytes (about 75%) and the least sensitive C oocytes (about 25%). The production of X-linked lethal events in X-irradiated dysgenic females was 8.1%, as compared to 4.5% in dysgenic controls and 3.4% in irradiated, nondysgenic controls, demonstrating an additive effect of radiation and dysgenesis-induced genetic damage. The effect of irradiation on sterility of dysgenic hybrid females was a negative one, resulting in 20% less sterility than expected from an additive effect. The combined effect of radiation and dysgenesis on dominant lethality tested in A, B and C oocytes of the same hybrid females was synergistic. Egg broods collected for 3.5 days after irradiation showed that significantly more damage was induced in the presence of ionizing radiation in dysgenic females than in their nondysgenic counterparts. This effect was most obvious in B and C oocytes. The synergism observed may be related to the inability of cells to repair the increased number of chromosome breaks induced both by radiation and transposon mobility.

Genetic injury in hybrid male mice exposed to low doses of 60Co gamma-rays or fission neutrons. II. Dominant lethal mutation response to long-term weekly exposures.

Male B6CF1 mice were exposed to once-weekly doses of either fission neutrons or 60Co gamma-rays for periods up to one year and mated periodically to screen for the induction of dominant lethal mutations. Two independent experiments were performed, each involving a control and three dose levels of both neutrons and gamma-rays. Neutron doses were between 0.125 and 2.67 rad/week and gamma-ray levels were between 5 and 32 rad/week. Data on both pre- and post-implantation fetal deaths were obtained. Analyses that were intended to identify the potential contribution from age- or time-dependent factors, which could include changes in radiosensitivity and in spontaneous rates plus any cumulative damage to the stem cell population did not reveal a consistent significant contribution to the mutation rate/rad/week. Direct comparisons of these data with data from males exposed to single doses confirm that weekly neutron irradiation is significantly more effective than single doses for the induction of postimplant fetal losses, whereas single doses of gamma-rays are more effective than the same dose divided into weekly fractions. Neutron-induced augmentation appears limited in these data to lethal mutations induced in meiotic and postmeiotic cell stages. The relative biological effectiveness (RBE) of neutrons rises from 5 +/- 1 to 12 +/- 1 for single vs. weekly doses. Rates of preimplant loss, although significant, are not a sensitive measure of genetic injury at the low doses used here. They are extremely sensitive to litter size and best estimated in litters of seven or more implants along with appropriate statistical control of concurrent variation in the number of corpora lutea.

Innate metabolic differences and mutagen sensitivity of Drosophila melanogaster. I. Radiosensitivity of hyperkinetic mutant.

The sensitivity to gamma-rays of a hyperkinetic mutant (HK1), characterized by high metabolic activity, has been studied and compared with gamma-ray sensitivity of the wild-type Oregon-K. Radiation damage, as measured by the frequency of induced dominant lethals (DL) and sex-linked recessive lethals (SLRL), was more in HK1 as compared with Or-K. The maximal sensitivity differences for these parameters were observed in the first brood. Translocations, on the contrary, were in general fewer in the HK1 compared with Or-K. These results have been interpreted in terms of reduced repair of mutational lesions in the energy-stressed cells of KH1.

Investigations on radiosensitive and radioresistant populations of Drosophila melanogaster. XVI. Adaptation to the mutagenic effects of X-rays in several experimental populations with irradiation histories.

Selection responses of the laboratory stock Berlin wild (+60, +K) of Drosophila melanogaster to the mutagenic effects of high, accumulated exposures of X-rays were studied in several sub-populations with long irradiation histories. Interest was focussed on adaptive adjustments of mutation rates. In samples from the populations, radiosensitivities of immature oocytes were tested, by using dominant lethals (A), X-chromosome losses (B) and sex-linked recessive lethals (C) as end-points of genetic radiation damage. Populations RO II and RO V are similar to the previously studied population RO I and were exposed to 2100 R/generation, delivered to oocyte stages 6-14, mature sperm, and spermatids. RO II (first tests after 160 generations) is radioresistant relative to +60 (control). The resistance was characterized by dose-reduction factors (DRFs) of 1.72 (with respect to end-points A, B) and 1.53 (C), and these were similar to those previously obtained for RO I. The resistance of RO II was inherited semidominantly as was that of RO I, and the radiosensitivity of the hybrids RO I/RO II was similar to that of RO I and RO II with respect to end-points A and B. RO V did not become resistant within 25 generations of irradiation history (A). Populations RO III (6000 R/generation) and RO IV (7000 R/generation) have histories of irradiations given to oogonia and spermatogonia. Radiosensitivities of immature oocytes of RO III did not differ from those of +K after 55 generations, but after 105 and 135 generations of irradiation history, DRFs of 1.2 (A) and 1.44 (B) were observed. RO IV was characterized in generations 55-135 by DRFs of 1.31 (A) and 1.72 (B). Selection for relative radioresistance of immature oocytes was found (1) to be reproducible (RO II-RO IV), (2) not to require genetic pre-adaptation (RO V), and (3) to be, in part, also achieved by 'indirect' selection (RO III, RO IV). It is concluded that mutation rates in populations are selectively adjusted to evolutionary requirements.

The influence of mating status and age on the induction of chromosome aberrations and dominant lethals in irradiated female mice.

Young and old hybrid female mice were given 0.5 Gy or 2 Gy acute x-irradiation, followed by (i) in utero examination for dominant lethal mutations, or (ii) examination of metaphase I oocytes for chromosome aberrations 2-3 weeks after the irradiation. Some of the old females had been mated when young to males of a specific locus stock. Others were left unmated until after the irradiation when they, and the young females, were mated to the same specific locus stock and allowed to have 1 (if given 2 Gy) or 2 (if given 0.5 Gy) litters before the dominant lethal test. In both the 0.5-Gy and 2-Gy series, mean sizes of first litters in the old late-mated group were markedly lower than in the old early-mated or young groups, the differences being significant at the 2-Gy level. The intrauterine examinations showed that this difference was largely the result of a reduced ovulation rate in the old late-mated females. Preimplantation loss tended to be higher in all the old females than in the young ones, but differences between the groups in postimplantation lethality were less pronounced. In the chromosome studies, only about half as many oocytes were recovered from the ovaries of old females than from young ones. At both the 0.5-Gy and 2-Gy dose levels interchange frequencies were non-significantly higher in old than in young females (with no clear-cut effect of mating status), while the overall frequency of aberrations (interchanges + fragments) was significantly higher in oocytes of old than young females after 2 Gy X-rays (35.5% against 12.5%). No specific locus mutations were found in 5616 offspring of unirradiated females.

An enhancement of the yield of X-ray-induced Minute mutations in the c3G female-ywmf-2 male system of Drosophila melanogaster.

The possible enhancement of the yield of X-ray-induced Minute mutations in the c3G female-ywmf-2 male system which is proposed to be responsible for the high production of spontaneous Minute mutations was investigated. To determine and compare the yield of X-ray-induced Minute mutations exactly, four series of crosses were made: (a) ywmf-2 male x Oregon-R (OrR) female crosses, spontaneous Minute mutations were scored; (b) ywmf-2 male x c3G female crosses, spontaneous Minute mutations produced in the c3G female-ywmf-2 male system were evaluated; (c) X-irradiated ywmf-2 male x OrR female crosses, the yield of Minutes induced by X-rays in the different stages of male germ cells was evaluated; and (d) X-irradiated ywmf-2 male x c3G female crosses, the yield of X-ray-induced Minutes in the c3G female-ywmf-2 male system was evaluated. The results show that the yield of X-ray-induced Minutes recorded in the c3G female-ywmf-2 male system is 2.37-16.55 times more than that in the ywmf-2 male x OrR female crosses. This finding clearly indicates that the yield of these mutations is greatly enhanced in the c3G female-ywmf-2 male system. This possibly suggests that the c3G female-ywmf-2 male system may be responsible not only for the high production of spontaneous Minute mutations but also for the high formation of radiation-induced Minutes.

Response of mutagen-sensitive Drosophila melanogaster to hyperthermia and radiation.

In an attempt to understand the mechanism of hyperthermia sensitization of radiation-induced damage, males of 5 mutant mutagen-sensitive strains: mei-41A1, w mus(1)101D1, w mus(1)104D1, y mei-9a, and w mus(1)102D2, representing at least 3 different defects in the DNA-repair pathways were exposed to hyperthermia (38 degrees C for 1 h) and to 1000 R of X-rays. In all of the mutagen-sensitive strains, the radiation-induced dominant lethals were enhanced by the hyperthermia treatment in the 6--8 day brood, which included the meiotic division at the time of radiation, and in the postmeiotic broods. The radiation-induced recessive lethals were also significantly increased by hyperthermia treatment in all the mutagen-sensitive mutants except mei-41A1 in 4--6 day brood. However, mei-41A1 males produced a significant increase of lethals in earlier broods, 0--2 and 2--4 day. The hyperthermia which apparently sensitizes radiation-induced damage by inhibiting DNA and probably chromosome-repair systems, is not specific, at least for 3 different Drosophila repair pathways.

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 mutation rate after gamma-irradiation of the fish, Oryzias latipes.

When laying females or males of the small fish Oryzias latipes were irradiated with gamma-rays and then mated with a non-irradiated partner, the fertility and hatchability of the embryos were reduced as the doses increased. In respect to hatchability (the induction of dominant lethality), the male was more sensitive than the female, and mature sperm were most sensitive among the various stages of spermatogenetic cells. The dose-rate effects on the production of the dominant lethality were observed in spermatogonia and spermatocytes. The inbred strain of the fish, HB-1, was sensitive to gamma-rays. Since the relationship between dose and the decrease in hatchability was almost linear, at least within a limited range, we think that this system would be useful for monitoring mutagenic factors in an aquatic environment.

Genetic effects of radiocarbon in reproductive cells of male mice.

The genetic effect of incorporated radiocarbon was studied after single, long-term (33 days) and chronic (6 and 12 months) treatment of male mice (CBA X C57B1) F1 with [14C]glucose. The genetic effect in male germ cells was estimated by 3 tests: DLM frequency in post- and pre-meiotic cells, RT frequency in stem spermatogonia and frequency of abnormal sperm heads. Absorbed doses in the gonads were: 0.22, 0.50 and 1.01 Gy, after a single exposure; 0.74 and 1.47 Gy, after long-term exposures; and 0.006 and 0.031 Gy, after chronic exposure for 6 months; and 0.013 and 0.066 Gy, for 12 months. The results suggest that DLM frequency in post-meiotic cells increased linearly with increasing the dose of 14C single and long-term exposures at a dose of 1.47 Gy only. A chronic treatment with [14C]glucose induced no increase in DLM frequency. RT frequency in stem spermatogonia was statistically significantly higher than the control level after the single and long-term exposure to 14C. A comparison of the results with the results of external single and chronic gamma-irradiation allows the conclusion that the relative genetic efficiency of radiocarbon as compared with that of gamma-rays is about 1.

Induction of congenital malformations in the offspring of male mice treated with X-rays at pre-meiotic and post-meiotic stages.

The induction of congenital malformations among the offspring of male mice treated with X-rays at pre-meiotic and post-meiotic stages has been studied in two experiments. Firstly, animals were exposed to varying doses (108-504 cGy) of X-rays and mated at various time intervals (1-7, 8-14, 15-21 and 64-80 days post-irradiation), so as to sample spermatozoa, spermatids and spermatogonial stem cells. In the second experiment, only treated spermatogonial stem cells were sampled. One group of males was given a single 500-cGy dose, a second group a fractionated dose (500 + 500 cGy, 24 h apart) and a third group was left unexposed. In the first experiment, induced post-implantation dominant lethality increased with dose, and was highest in week 3, in line with the known greater radiosensitivity of the early spermatid stage. Preimplantation loss also increased with dose and was highest in week 3. There was no clear induction of either pre-implantation or post-implantation loss at spermatogonial stem cell stages. There was a clear induction of congenital malformations at post-meiotic stages, the overall incidence being 2.0 +/- 0.32% in the irradiated series and 0.24 +/- 0.17% among the controls. The induction was statistically significant at each dose. At the two highest doses the early spermatids (15-21 days) appeared more sensitive than spermatozoa, and at this stage the incidence of malformations increased with dose. The data from Expt. 1 on the induction of malformations by irradiation of spermatogonial stages were equivocal. In contrast, Expt. 2 showed a statistically significant induction of malformations at both dose levels (2.2 +/- 0.46% after 500 cGy and 3.1 +/- 0.57% after 500 + 500 cGy). The relative sensitivities of male stem cells, post-meiotic stages and mature oocytes to the induction of congenital malformations were reasonably similar to their sensitivities for specific-locus mutations, except that the expected enhancing effect of the fractionation regime used was not seen. Dwarfism and exencephaly were the two most commonly observed malformations in all series.

X-ray induced dominant lethal mutations in mature and immature oocytes of guinea-pigs and golden hamsters.

The induction of dominant lethal mutations by doses of 100-400 rad X-rays in oocytes of the guinea-pig and golden hamster was studied using criteria of embryonic mortality. For both species higher yields were obtained from mature than from immature oocytes, in contrast to results for the mouse. Data on fertility indicated that in the golden hamster, as in the mouse, immature oocytes were more sensitive to killing by X-rays than mature oocytes but that the converse was true in the guinea-pig. The dose-response relationship for mutation to dominant lethals in pre-ovulatory oocytes of guinea-pig and golden hamsters was linear, both when based on pre- and post-implantation loss and when on post-implantation loss only. The rate per unit dose was higher for the golden hamster, and the old golden hamsters were possibly slightly more sensitive than young ones. The mutation rate data for mature oocytes of the mouse, using post-implantation loss alone, also fitted a linear dose-response relationship, except that the rate per unit dose was lower than for the other two species.

Induction of dominant lethal mutations by combined X-ray-acrylamide treatment in male mice.

The induction of mutations following combined treatment with acrylamide (AA) plus X-rays has been determined using the dominant lethal mutations test in Pzh:SFISS male mice. Combinations of a mutagenic dose of both agents (1.00 Gy, 125 mg/kg b.w.) and a non-mutagenic dose, i.e., a dose that alone does not produce dominant lethals (0.25 Gy, 25 mg/kg b.w.), were used. For the discussion of the effects of combined action of X-rays and acrylamide the term 'enhancement in risk' was used whenever the effects observed after combined exposure significantly exceeded the sum of the effects produced separately by the agents. Such an enhanced risk has been observed in late spermatids after combined action of X-rays and AA at non-mutagenic doses, and in spermatozoa, spermatids and late spermatocytes after exposure to mutagenic doses.

Detection of gamma-ray-induced DNA damages in malformed dominant lethal embryos of the Japanese medaka (Oryzias latipes) using AP-PCR fingerprinting.

Adult male fish of the medaka HNI strain exposed to 9.5 Gy or 19 Gy (0.95 Gy/min) of gamma-rays were mated with non-irradiated female fish of the Hd-rR strain. Genomic DNA was prepared from malformed individual embryos which were expected to be dominant lethal and used for AP-PCR fingerprinting. By the use of a part of the T3 promoter sequence (20 mer), which, to our knowledge, is not found in the medaka genome as an arbitrary primer, we found polymorphisms in genomic fingerprints which could distinguish the parental strains. On the other hand, we found that the fingerprints of F1 hybrids were the sum of those of their parents. Based on these findings, we analyzed the fingerprints of genomic DNA of each severely malformed embryo, because we expect that radiation-induced genomic damages resulting in severe malformation and eventually in dominant lethals should be detected as changes in paternal fingerprints of F1 hybrids. Indeed, we succeeded in detecting changes in genomic DNA as loss of some paternal bands in fingerprints of malformed embryos. One of 10 malformed embryos obtained from 9.5 Gy gamma-irradiated males had lost one band of the paternal origin and 4 of 12 malformed embryos obtained from 19 Gy gamma-irradiated males had lost 5 bands. These results indicated a possibility that quantitative as well as qualitative estimation of gamma-ray-induced DNA damages can be made by this method which does not require the functional selection based on a specific target gene.