Genetic effects of low-dose chronic irradiation of developing seeds of Arabidopsis thaliana Heynh

A six-year study, starting in 1987, focused on the frequency of embryonic lethals and chlorophyll mutations that arose in developing seeds of Arabidopsis thaliana growing at sites varying in the level of radioactive contamination in the 30-km control region of the Chernobyl Atomic Power Plant. The dose rate of chronic irradiation varied from 200 microR/h to 2.4 R/h. To study the genetic effects of various levels of radioactive contamination, the frequency of mutations arising in a particular generation was determined and the irradiation dose was estimated for the given generation. The dose dependence of the mutation frequency proved to follow a power function with a power less than unity, suggesting a relatively high effect for low-dose irradiation. Possible explanations of this phenomenon are discussed.     

Genetic effects in plant populations in the zone of the Chernobyl accident

During 6 years, starting from 1986, the monitoring of the dynamics of the frequency of embryo lethal and of chlorophyll mutations was carried out in arabidopsis populations in areas with different levels of radioactive contamination by the Muller embryo-test in the 30 km of ChNPP. The dose rate of chronic irradiation in the examined areas varied from 0.014 to 17 nA/Kg. Monitoring of the dynamics of the mutation process in natural arabidopsis populations showed the correlation between the level of the mutation process and the dose rate of chronic irradiation. The genetic effects of different levels of radioactive contamination were estimated by determining the frequency of mutations occurred in this generation and by calculating the dose of irradiation of one was found. That the dependence of the mutation frequency on the dose of irradiation presents a power function with a power index less 1, which suggests a higher efficiency of low radiation doses per unit dose. Possible explanations of this phenomenon are considered in the work. The studies of cytogenetic effects in chronically exposed Crepis tectorum populations in the zones of the Chernobyl accident showed that starting from the second year after the Chernobyl disaster there appeared plants with an altered karyotype and their frequency of chromosome aberrations correlates in root meristem cells.     

Genetic damage in domestic mice inhabiting in the areas with elevated background radiation

Genetic effects of irradiation in males of wild house mice which were caught in the region of Chernobyl Nuclear Power Station were studied. The dose rate on the ground surface varied from 0.04 to 200 mR/h of gamma-irradiation. The increasing yield of dominant lethal mutations was only observed in males from the most contaminated sector. Reciprocal translocations were observed in spermatocytes of mice at all the levels of contamination. The rate of reciprocal translocations was relatively low and increased linearly with the elevation of the dose rate. The extent of testis damages increased also, as the dose rate grew. The frequency of abnormal sperm heads, the yield of recessive lethal mutations, litter size and radiosensitivity of the first progeny were not changed, depending on the dose rate.     

Comparison of radiation-and chemically-induced dominant lethal mutations in male mice

Ionizing radiation-induced dominant lethal mutations in all spermatogenic stages. After irradiation of male mice with 200 R the yield of induced mutations in early spermatids was twice the yield in spermatozoa, late spermatids, and spermatocytes. After irradiation with 400 R or 800 R the spermatocytes were the most sensitive stage for the induction of dominant lethal mutations. The frequency of radiation-induced dominant lethal mutations in postspermatogonial stages was dose-dependent. The yield of dominant lethal mutations in spermatogonia was independent of the dose.     

Mutation induction by different dose rates of gamma rays in radiation-sensitive mutants of mouse leukemia cells

Induction of cell killing and mutation to 6-thioguanine resistance was examined in a radiation-sensitive mutant strain LX830 of mouse leukemia cells following gamma irradiation at dose rates of 30 Gy/h (acute), 20 cGy/h (low dose rate), and 6.2 mGy/h (very low dose rate). LX830 cells were hypersensitive to killing by acute gamma rays. A slight but significant increase was observed in cell survival with decreasing dose rate down to 6.2 mGy/h, where the survival leveled off above certain total doses. The cells were also hypersensitive to mutation induction compared to the wild type. The mutation frequency increased linearly with increasing dose for all dose rates. No significant difference was observed in the frequency of induced mutations versus total dose at the three different dose rates so that the mutation frequency in LX830 cells at 6.2 mGy/h was not significantly different from that for moderate or acute irradiation.     

Chromosomal instability in offspring of voles in unfavorable radiation zones

A high frequency of cells having chromosomal aberrations was shown to be preserved in the first generation of laboratory offspring of common voles, whose ancestors were captured near the East Ural radioactive track (EURT). In F2 and F3, a decrease in frequency was observed. Most likely, the cytogenetic effects observed were caused by environmental contamination with plutonium in the locality of capture. An increased chromosomal instability observed in the first laboratory generation of nonirradiated common voles was caused by reversible modifications in the expression of mutator genes inherited from parents exposed to alpha-irradiation rather than by classic mutations occurred in specific loci.     

Mitochondrial DNA mutation frequencies in experimentally irradiated compost worms, Eisenia fetida

The compost worm Eisenia fetida is routinely used in ecotoxicological studies. A standard assay to assess genetic damage in this species would be extremely valuable. Since mitochondrial DNA (mtDNA) is known to exhibit an increased mutation rate following exposure to ionising radiation we assessed the validity of a mtDNA-based assay for measuring increases in mutation rate in laboratory-irradiated compost worms. To this end the mutation frequency in the mtDNA of the compost worm E. fetida was quantified following in vivo gamma-irradiation of adult worms in three dose groups. Five adult worms exposed to 1.4 mGy/h for 55 days (total dose 1.85 Gy), five adult worms exposed to 8.5 mGy/h for 55 days (total dose 11.22 Gy) and five adult control worms were used to assess the effect of irradiation on mtDNA mutation induction. DNA samples extracted from irradiated adult worms were used in high-fidelity PCR of a 486 bp region of mtDNA spanning the ATPase 8 gene, chosen for its high spontaneous mutation rate. PCR products were cloned and sequenced to identify mutations, with 89-102 clones successfully sequenced per individual. A significant elevation in mtDNA mutation frequency (p=0.032) was seen in worms exposed at the higher dose rate (8.5 mGy/h, total dose 11.22 Gy; mutation frequency 27.98+/-4.85 x 10(-5)mutations/bp) in comparison to controls (mutation frequency 12.68+/-3.06 x 10(-5)mutations/bp), but no elevation in mutation frequency (p=0.764) was seen for the lower dose rate (1.4 mGy/h, total dose 1.85 Gy; mutation frequency 13.74+/-1.29 x 10(-5)mutations/bp) compared with controls. This indicates that although the technique has the potential to detect an elevation in mutation frequency, it does not have sufficient sensitivity at the doses likely to be encountered in environmental monitoring scenarios.     

Is there a proportionality between the spontaneous and the X-ray induction rates of mutations?: Experiments with mutations at 13 X-chromosome loci in Drosophila melanogaster

The X-ray induction of recessive visible specific locus mutations at 14 X-chromsome loci was studied in Drosophila melanogaster using the "Maxy" technique. The X-ray exposure was 3000 R to 5-day-old males and the sampling of germ cells was restricted to mature spermatozoa. Presumptive mutant females recovered in the F1 generation were tested for transmission, allelism, fertility and viability in males. A total of 128 mutations (115 completes and 13 mosaics including those that were male viable as well as male-lethal) recovered among 38 898 female progeny were found to be transmitted. On the basis of the above frequency, the average mutation rate can be estimated as 7.8 X 10(-8)/locus/R; for mutations that were viable and fertile in males, the rate is 3.0 X 10(-5)/locus/R (49 mutations among 38 898 progeny). The frequency of mutations at the different loci encompassed a wide range: while no mutations were recovered at the raspberry and carnation loci, at others, the numbers ranged from 1 at echinus to 31 at garnet; in addition, the proportion of mutations that was male-viable was also different, depending on the locus. Schalet's extensive data on spontaneous mutations at 13 (of the 14 loci employed in the present study) loci permit an estimate of the spontaneous rate which is 6.1 X 10(-6)/locus (a total of39 mutations among 490 000 progeny); for mutations that were viable and fertile in males, the rate is 3.0 X 10(-6)/locus (19 mutations among 490 000 progeny). The mutability of the different loci varied over a 9-fold range. When the different loci are ranked depending on their relative mutability (for spontaneous and induced mutations) it is found that in general, loci that mutate spontaneously relatively more frequently are also those at which more mutations have been recovered in the radiation experiments and likewise, those that are less mutable spontaneously are also those that mutate less after irradiation. Since the data are limited, it is concluded that the above finding is not inconsistent with the assumption of proportionality between spontaneous and induction rates of mutations. On the basis of the above results, a doubling dose of 100 R can be calculated for the X-ray induction of specific-locus mutations in Drosophila spermatozoa.     

Radioactive contamination of aquatic organisms of the Yenisei River in the area affected by the activity of the Mining-and-Chemical Combine

The study was done to investigate the content of manmade radionuclides in aquatic organisms of the Yenisei River near the Mining-and-Chemical Combine (MCC) and to estimate the exposure dose rates to organisms from various sources. The results of the investigation and calculations suggest that the main source of radioactive contamination of aquatic organisms is the coolant of the third MCC reactor, which is still being released into the Yenisei. Gamma-spectrometric analysis revealed 23 manmade radionuclides in the biomass of aquatic plants. The aquatic animal Phylolimnogammarus viridis and diatoms also contain manmade radionuclides. Among aquatic organisms, the highest dose rate is received by aquatic plants (up to 39 microGy/day). For most aquatic organisms under study, the dose received from the technogenic irradiation is an order of magnitude higher than the dose received from natural irradiation. The water moss (Fontinalis antipyretica) features the highest capacity to accumulate manmade radionuclides; hence, it accumulates the largest technogenic exposure dose among the study aquatic organisms.     

Induction of lethal mutations in female mice by 9 generations of gamma-irradiation during foetal development.

Female CBA mice were chronically gamma-irradiated in utero during either of two periods, the 10th to 14th days or the 14th to 18th days of gestation. The doses administered were 34 rad/generation in the earlier group and 160 rad/generation in the latter with dose rates of 0.3 rad/h and 1.7 rad/h, respectively. The doses were given through 9 generations. The effect of the irradiation was expressed as an increased frequency in the rate of recessive lethal equivalents by just above 4%. This corresponds to a mutation rate of 1.5 X 10(-4) mutation/rad/genome in the animals irradiated during the 10th to 14th gestational days and 0.3 X 10(-4) mutation/rad/genome in the 14th to 18th day group. As in earlier investigations, neither dominant mutations nor dominance effects of induced recessive lethal equivalents were found.     

Specific-locus experiments show that female mice exposed near the time of birth to low-LET ionizing radiation exhibit both a low mutational response and a dose-rate effect

Female mice were exposed to 300 R of 73-93 R/min X-radiation either as fetuses at 18.5 d post conception (p.c.) or within 9 h after birth. Combining the similar results from these two groups yielded a specific-locus mutation frequency of 9.4 X 10(-8) mutation/locus/R, which is statistically significantly higher than the historical-control mutation frequency, but much lower than the rate obtained by irradiating mature and maturing oocytes in adults. Other females, exposed at 18.5 days p.c. to 300 R of 0.79 R/min gamma-radiation, yielded a mutation frequency that was statistically significantly lower than the frequency at high dose rates. The low-dose-rate group also had markedly higher fertility. It appears that the dose-rate effect for mutations induced near the time of birth may be more pronounced than that reported for mature and maturing oocytes of adults. A hypothesis sometimes advanced to explain low mutation frequencies recovered from cell populations that experience considerable radiation-induced cell killing is that there is selection against mutant cells. The reason for the relatively low mutational response following acute irradiation in our experiments is unknown; however, the finding of a dose-rate effect in these oocytes in the presence of only minor radiation-induced cell killing (as judged from fertility) makes it seem unlikely that selection was responsible for the low mutational response following acute exposure. Had selection been an important factor, the mutation frequency should have increased when oocyte killing was markedly reduced.     

Genetic action of 131I and 125I on the germ cells of male mice

A study was made of the genetic effects of iodine radioactive isotopes in male germ cells of (CBA X C57Bl)F1 hybrid mice. After a single intraperitoneal administration of Na131I (1.48 to 740 kBq/g) or Na125I (148 to 7400 kBq/g) to males the occurrence of dominant lethal mutations (DLM), reciprocal translocations (RT), and abnormal sperm heads (ASH) was studied. The radioactive iodine isotopes induced DLM at the postmeiotic spermatogenesis stages only. After the effect of the isotopes, the frequency of RT increased insignificantly with dose. The frequency of ASH was only increased with the highest 131I dose. Relative biological effectiveness of 131I and 125I was less than 1 with a reference to the indices under study.     

Differential spermatogonial stem-cell survival and mutation frequency

One group of adult C3H×101 hybrid male mice was given 3 injections of 12.5 μCi of [³H]thymidine at 9-h intervals and irradiated 24 h after the last injection with X-ray doses of 100, 300, 500, 600, 1000 R or the first fraction of a split 1000-R dose given as two 500-R exposures 24 h apart. Mice were killed 207 and 414 h after irradiation. A second group of mice was given a single injection of 12.5 μCi of [³H]thymidine 1 h before irradiation with single exposures of 300, 500, 600, 1000 R, or the first fraction of a 1000-R exposure given as two 500-R fractions 24 h apart. Mice were killed 120 and 207 h after irradiation. In both experiments, parallel groups of mice were given X-ray only as a control for the effect of [³H]thymidine. Two sets of slides were prepared for each mouse receiving [³H]thymidine: one set was not autoradiographed and was used for scoring cell survival; the second set was coated with emulsion and used for scoring percentage of labeled cells. The dose-response curves for survival at 120 and 207 h were curvilinear, with no evidence of discontinuity over the 100–1000-R range. After multiple injections of [³H]thymidine and irradiation 24 h later, percentage of labeled cells at 207 h was comparable for controls, 100, 300, and 600 R; significantly lower than controls for 1000 R; and significantly above controls after 500 + 500 R. Thus the surviving stem-cell population was qualitatively the same for that portion of the dose-response curve giving a linear increase in mutation rate but was different for both 1000-R and 500 + 500-R exposures, and the single and fractionated 1000-R exposures differed from each other. This parallelism between survival of labeled cells and mutation frequency in spermatogonial stem cells suggests that a stage in the cell cycle 24–42 h after DNA synthesis is resistant to cell killing but sensitive to mutation induction. The mutation rate after a single 1000-R exposure is low because labeled, mutation-sensitive cells have been selectively killed. Mutation frequency after the 500 + 500-R dose is increased because of synchronization induced by the first dose combined with selective killing of unlabeled cells by the second fraction. Irradiation 1 h after labeling with [³H]-thymidine demonstrated that the S phase of the spermatogonial stem-cell cycle is sensitive to radiation-induced cell killing.     

Genetic variability in Scotch pine populations of the Bryansk Region radioactively contaminated in the Chernobyl accident

The method of isozyme analysis of megagametophytes is used to estimate the genetic variability in Scotch pine populations (Pinus sylvestris L.) of the Bryansk Region sites with contrasting levels of radioactive contamination (soil ¹³⁷Cs, 60 to 17 800 Bq/kg) resulting from the Chernobyl accident. All indices of genetic variability (heterozygosity, frequency of polymorphic loci, Zhivotovskii index) and frequencies of loss-of-function enzyme mutations increase with the dose absorbed by plant generative organs. The data show that high mutability is intrinsic for seeds of these pine trees, and genetic diversity in the populations is essentially conditioned by radiation exposure.     

Targeted exome sequencing of unselected heavy‐ion beam‐irradiated populations reveals less‐biased mutation characteristics in the rice genome

Heavy‐ion beams have been widely utilized as a novel and effective mutagen for mutation breeding in diverse plant species, but the induced mutation spectrum is not fully understood at the genome scale. We describe the development of a multiplexed and cost‐efficient whole‐exome sequencing procedure in rice, and its application to characterize an unselected population of heavy‐ion beam‐induced mutations. The bioinformatics pipeline identified single‐nucleotide mutations as well as small and large (>63 kb) insertions and deletions, and showed good agreement with the results obtained with conventional polymerase chain reaction (PCR) and sequencing analyses. We applied the procedure to analyze the mutation spectrum induced by heavy‐ion beams at the population level. In total, 165 individual M₂ lines derived from six irradiation conditions as well as eight pools from non‐irradiated ‘Nipponbare’ controls were sequenced using the newly established target exome sequencing procedure. The characteristics and distribution of carbon‐ion beam‐induced mutations were analyzed in the absence of bias introduced by visual mutant selections. The average (±SE) number of mutations within the target exon regions was 9.06 ± 0.37 induced by 150 Gy irradiation of dry seeds. The mutation frequency changed in parallel to the irradiation dose when dry seeds were irradiated. The total number of mutations detected by sequencing unselected M₂ lines was correlated with the conventional mutation frequency determined by the occurrence of morphological mutants. Therefore, mutation frequency may be a good indicator for sequencing‐based determination of the optimal irradiation condition for induction of mutations.     

Mutation induction by very low dose rate gamma rays in cultured mouse leukemia cells L5178Y

Induction of cell killing and mutation to 6-thioguanine resistance was studied in growing mouse leukemia cells in culture following gamma rays at dose rates of 30 Gy/h, 20 cGy/h, and 6.3 mGy/h, i.e., acute, low dose rate, and very low dose rate irradiation. A marked increase was observed in the cell survival with decreasing dose rate; no reduction in the surviving fraction was detected after irradiation at 6.3 mGy/h until a total dose of 4 Gy. Similarly, the induced mutation frequency decreased after low dose rate irradiation compared to acute irradiation. However, the frequency after irradiation at 6.3 mGy/h was unexpectedly high and remained at a level which was intermediate between acute and low dose rate irradiation. No appreciable changes were observed in the responses to acute gamma rays (in terms of cell killing and mutation induction) in the cells which had experienced very low dose rate irradiation.     

The effect of dose rate on the frequency of specific-locus mutations induced in mouse spermatogonia is restricted to larger lesions; a retrospective analysis of historical data

A series of 19 large-scale germ-cell mutagenesis experiments conducted several decades ago led to the conclusion that low-LET radiation delivered to mouse spermatogonia at dose rates of 0.8 R/min and below induced only about one-third as many specific-locus mutations as did single, acute exposures at 24 R/min and above. A two-hit origin of the mutations was deemed unlikely in view of the then prevailing evidence for the small size of genetic lesions in spermatogonia. Instead, the dose-rate effect was hypothesized to be the result of a repair system that exists in spermatogonia, but not in more mature male reproductive cells. More recent genetic and molecular studies on the marker genes have identified the phenotypes associated with specific states of the mutant chromosomes, and it is now possible retrospectively to classify individual past mutations as "large lesions" or "other lesions". The mutation-frequency difference between high and low dose rates is restricted to the large lesion mutations, for which the dose-curve slopes differ by a factor exceeding 3.4. For other lesion mutations, there is essentially no difference between the slopes for protracted and acute irradiations; induced other lesions frequencies per unit dose remain similar for dose rates ranging over more than 7 orders of magnitude. For large lesions, these values rise sharply at dose rates >0.8 R/min, though they remain similar within the whole range of protracted doses, failing to provide evidence for a threshold dose rate. The downward bend at high doses that had been noted for X-ray-induced specific-locus mutations as a whole and ascribed to a positive correlation between spermatogonial death and mutation load is now found to be restricted to large lesion mutations. There is a marked difference between the mutation spectra (distributions among the seven loci) for large lesions and other lesions. Within each class, however, the spectra are similar for acute and protracted irradiation.     

The effect of prolonged gamma irradiation at a decreasing dose rate on survivability and gene mutation induction in cultured Chinese hamster cells

Continuous gamma irradiation at decreasing dose rate was shown to be less effective than acute exposure with regard to the lethal effect and frequency of mutations of resistance to 6-thioguanine in cultured Chinese hamster cells. The cell population subjected to continuous irradiation was more radioresistant than the intact one. Lethal and genetic effects of continuous irradiation at decreasing dose rate were mainly determined by the contribution of the radiation dose received during the first 24 h of exposure.     

The relative frequency of induced mutations recovered from Drosophila melanogaster gametogenic stages irradiated at different dose rates

Inseminated Drosophila females were exposed to radiation doses of approx. 2000 or 4000 R at dose rates ranging from 8 to 240 000 R/h. The observed frequencies of sex-linked lethal mutations induced in spermatozoa and oogonia suggest that dose-rate effects were absent in spermatozoa over the whole range and in oogonia up to 3000 R/h. There was a consistent increase in oogonial mutation frequency from 3000 to 240 000 R/h. These results are discussed in relation to the well-established dose-rate effects observed in the mouse.     

X-ray-induced specific-locus mutation rate in newborn male mice

The specific-locus mutation frequency resulting from 300 R of acute X-irradiation has been determined for the germ cells present in newborn male mice. The frequency is 13.7·10^?8 mutations/locus/R, which is statistically significantly lower than that of 29.1·10^?8 mutations/locus/R found earlier for the same loci in spermatogonia of the adult male by W. L. Russell. The mutation rate for newborn males does not differ significantly from the induced specific-locus frequency reported for fetal males by T. C. Carteret al.The incidence of clusters of specific-locus mutations found following the irradiation of the newborn males was statistically significantly higher than the cluster incidence reported by W. L. Russell for similar irradiation of adult males. This presumably indicates the survival of relatively fewer reproductive cells following irradiation of the day-o testis.Although there are suggestions that the distribution of mutations among the loci following irradiation of the newborn males may be different from that of the irradiated adults, no statistically significant differences are demonstrated.It is quite possible that the testis of the newborn mouse may be comparable to the relatively undifferentiated human testis which persists for approx. 10 years. Until the present research was undertaken, no attempt had been made to determine the specific-locus mutation frequency resulting from X-irradiation of newborn male mice. Although some important questions still remain concerning the explanation for the lower mutational response of the newborn mouse testis, from the hazard standpoint it is reassuring that the mutation frequency of the newborn male is statistically significantly lower than that of the adult.