Mini-and microsatellite mutations in children from Chernobyl accident cleanup workers

Knowledge about possible genotoxic effects of low-dose radiation on the human germline is limited and relies primarily on extrapolations from high-dose exposures. To test whether ionizing radiation can cause paternal genetic mutations that are transmitted to offspring, we enrolled families of 88 Chernobyl cleanup workers exposed to ionizing radiation. We analyzed DNA isolated from lymphocytes for mutations via DNA blotting with the multi-locus minisatellite probes 33.6 and 33.15 and via PCR in a panel of six tetranucleotide repeats. Children conceived before and children conceived after their father's exposure showed no statistically significant differences in mutation frequencies. We saw an increase in germline microsatellite mutations after radiation exposure that was not statistically significant. We found no dependence of mutation rate on increasing exposure. A novel finding was that the tetranucleotide marker D7S1482 demonstrated germline hypermutability. In conclusion, our results do not support an increased level of germline minisatellite mutations but suggest a modest increase in germline mutations in tetranucleotide repeats. Small sample size, however, limited statistical power.     

Differential genetic responses to ionizing irradiation in individual families of Japanese medaka, Oryzias latipes

Although no statistically significant hereditary effects have yet been detected in the children of survivors from the atomic bombings in Hiroshima and Nagasaki, recent animal studies have found that exposure to ionizing radiation can cause genomic and epigenomic instability in the exposed individuals, as well as their offspring, and therefore, may have much larger genetic effects than predicted by earlier studies. When individuals are exposed to various environmental insults, including radiation, individual sensitivity to the insults often varies. Variance in germ-line response to radiation among individuals has been widely recognized, but it is difficult to address due to the use of inbred strains and the limited number of offspring that can be produced by a pair of mice, the common model used to study genetic effects of radiation. Herein is the first study to examine individual family responses to ionizing radiation using a parent-pedigree approach in an outbred strain of a vertebrate model, the Japanese medaka fish. Changes in frequencies of radiation-induced germline mutations at nine microsatellite loci were examined in the same families before and after exposure to one of four acute doses of ionizing radiation (0.1, 0.5, 2.5, 5Gy, plus sham-exposed controls). Families varied significantly in pre-exposure mutation frequencies and responses to irradiation, but germline mutations were elevated in at least one family after 0.1, 0.5, and 5Gy exposures. Variance among individuals in sensitivity to radiation is well documented for many endpoints, and our work now extends these endpoints to include germ-line mutations. Further studies are needed to elucidate dose response, effects at varying stages of spermatogenesis, and the mechanisms underlying the variance in these individual responses to radiation.     

Microsatellite mutations show no increases in the children of the Chernobyl liquidators

We performed a study on Belarusian "liquidators", exploring whether increase in the frequencies of germline mutations at microsatellite loci could be found in their progeny. The liquidators, mostly young males, were those involved (during 1986 and 1987) in clean-up operations in the radioactively contaminated area following the Chernobyl nuclear power plant accident in 1986. Many liquidators fathered children during the clean-up period and after the work had been terminated. The numbers of families studied were 64 (liquidators) and 66 (controls). A total of 72 loci (31 autosomal, one X-linked and 40 Y-linked) were used. DNA was isolated from peripheral blood lymphocytes and the microsatellite loci were amplified by the polymerase chain reaction with fluorescence-labelled primers. Mutations were detected as variations in the length of the loci. At the Y-linked loci, the mutation rates (expressed as number of mutations among the total number of loci for the individuals included) are 2.9 x 10(-3) (4/1392) and 2.1 x 10(-3) (3/1458) in the children of exposed and control parents, respectively. This difference is not statistically significant. At the autosomal loci, the corresponding estimates are 5.9 x 10(-3) (11/1862; exposed group) and 8.5 x 10(-3) (18/2108; control). Again, the difference is not significant. The possibility that the Belarusian population might have been unexpectedly exposed to some chemical contaminants in the environment appears unlikely in view of the finding that the spontaneous mutation rates at the same set of loci in several non-Belarusian populations were similar to those in Belarus. The estimated mean radiation dose to the liquidators was small, being about 39 mSv, and this might be one reason why no increases in mutation rates due to radiation could be found.     

Mutation in microsatellite repeats of DNA and embryonal death in humans

In the analysis of tetranucleotide DNA repeats inheritance carried out in 55 families with a history of spontaneous miscarriages and normal karyotypes in respect to 21 loci located on seven autosomes, 8 embryos (14.5%) demonstrating 12 cases of the presence of alleles absent in both parents were described. The study of chromosome segregation using other DNA markers permitted highly probable exclusion of false paternity as well as uniparental disomy as the reasons for parent/child allele mismatches. The high probability of paternity together with the presence of a "new" allele at any offspring locus points to the mutation having occurred during game-togenesis in one of the parents. Examination of mutation in spontaneous abortuses revealed an increased number of tandem repeat units at microsatellite loci in three cases and an decreased number of these repeats in six cases. In two abortuses, a third allele absent in both parents, which resulted from a somatic mutation that occurred during embryonic development, was observed. The prevalence of the male germline mutations, revealed during investigation of the mutation origin, was probably associated with an increased number of DNA replication cycles in sperm compared to the oocytes. In spontaneous abortuses, the mean mutation rate of the tetranucleotide repeat complexes analyzed was 9.8 x 10(-3) per locus per gamete per generation. This was about five times higher than the spontaneous mutation rate of these STR loci. It can be suggested that genome instability detected at the level of repeated DNA sequences can involve not only genetically neutral loci but also active genomic regions crucial for embryonic viability. This results in cell death and termination of embryonic development. Our findings indicate that the death of embryos with normal karyotypes in most cases is associated with an increased frequency of germline and somatic microsatellite mutations. The data of the present study also provide a practical tool for the quantitative evaluation of this phenomenon and for the analysis of the reasons for miscarriages and embryonic death in certain families.     

Expanded simple tandem repeat (ESTR) mutation induction in the male germline: lessons learned from lab mice

Expanded simple tandem repeat (ESTR) DNA loci that are unstable in the germline have provided the most sensitive tool ever developed for investigating low-dose heritable mutation induction in laboratory mice. Ionizing radiation exposures have shown that ESTR mutations occur mainly in pre-meiotic spermatogonia and stem cells. The average spermatogonial doubling dose is 0.62-0.69 Gy for low LET, and 0.18-0.34 Gy for high LET radiation. Chemical alkylating agents also cause significant ESTR mutation induction in pre-meiotic spermatogonia and stem cells, but are much less effective per unit dose than radiation. ESTR mutation induction efficiency is maximal at low doses of radiation or chemical mutagens, and may decrease at higher dose ranges. DNA repair deficient mice (SCID and PARP-1) with elevated levels of single and double-strand DNA breaks have spontaneously elevated ESTR mutation frequencies, and surprisingly do not show additional ESTR mutation induction following irradiation. In contrast, ESTR mutation induction in p53 knock-outs is indistinguishable from that of wild-type mice. Studies of sentinel mice exposed in situ to ambient air pollution showed elevated ESTR mutation frequencies in males exposed to high levels of particulate matter. These studies highlight the application of the ESTR assay for assessing environmental hazards under real-world conditions. All ESTR studies to date have shown untargeted mutations that occur at much higher frequencies than predicted. The mechanism of this untargeted mutation induction is unknown, and must be elucidated before we can fully understand the biological significance of ESTR mutations, or use these markers for formal risk assessment. Future studies should focus on the mechanism of ESTR mutation induction, refining dose responses, and developing ESTR markers for other animal species.     

Germline mutations of tetranucleotide DNA repeats in families with normal children and reproductive pathology

We have previously reported a high rate of tetranucleotide DNA repeat mutations, including mutations of both germline and somatic origin, in spontaneous human abortuses. To analyze in more detail mutational microsatellite (MS) variability in meiosis and its possible association with disturbed embryonic development, we have conducted a comparative study of mutation rates of a complex of 15 autosomal tetranucleotide MSs in 55 families with healthy children and in 103 families that have had spontaneous abortuses with normal karyotypes. In the families with miscarriage, the gametic MS mutation rate was higher than in the families with normal reproductive function (4.36 x 10(-3) versus 2.32 x 10(-3) per locus per gamete per generation), but this difference was statistically nonsignificant (P = 0.25). No association of MS mutations with familiar miscarriage was found. Mutations at the MS loci studied were recorded almost 3 times as often in spermatogenesis as in oogenesis, which is likely to result from a greater number of DNA replication cycles in male germline cell precursors than in female ones. Mutations increasing and reducing the MS sequence length appeared at virtually the same rate. Changes in MS DNA sequence length per one repeated element, i.e., single-step mutations (93% of cases) exceeded all other events of allele length change. The highest number of mutations (81.2%) was found in longer alleles. This distribution of mutations by size, direction, and parental origin corresponds to the multistep mutation model of their emergence via mechanism of DNA strand slippage during replication.     

Microsatellite mutations in the offspring of irradiated parents 19 years after the Cesium-137 accident

In September of 1987, a radiotherapy unit containing 50.9 TBq of Cs(137)Cl was removed from an abandoned radiotherapy clinic. This unit was subsequently disassembled leading to the most serious radiological accident yet to occur in the Western hemisphere. This event provides an opportunity to assess the genetic effects of ionizing radiation. We surveyed genetic variation of 12 microsatellite loci in 10 families of exposed individuals and their offspring and also in non-exposed families from the same area of Goias state. We found an increase in the number of new alleles in the offspring of the exposed individuals. The mutation rate was found to be higher in the exposed families compared to the control group. These results indicated that exposure to ionizing radiation can be detected in offspring of exposed individuals and also suggest that the elevated microsatellite mutation rate can be attributed to radioactive exposure.     

Instability of (GATA)<Subscript> n</Subscript> microsatellite loci in the parthenogenetic Caucasian rock lizard<Emphasis Type="Italic"> Darevskia unisexualis</Emphasis> (Lacertidae)

Mini- and microsatellites, comprising tandemly repeated short nucleotide sequences, are abundant dispersed repetitive elements that are ubiquitous in eukaryotic genomes. In humans and other bisexual species hypervariable mini- and microsatellite loci provide highly informative systems for monitoring of germline and somatic instability. However, little is known about the mechanisms by which these loci mutate in species that lack effective genetic recombination. Here, multilocus DNA fingerprinting was used to study M13 minisatellite and (GATA) _n microsatellite instability in the parthenogenetic Caucasian rock lizard Darevskia unisexualis (Lacertidae). DNA fingerprinting of 25 parthenogenetic families, from six isolated populations in Armenia (comprising a total of 84 siblings), using the oligonucleotide (GATA)₄ as a hybridization probe, revealed mutant fingerprinting phenotypes in 13 siblings that differed from their mothers in several restriction DNA fragments. In three families (8 siblings), the mutations were present in the germline. Moreover, the mutant fingerprint phenotypes detected in siblings were also present in population DNA samples. No intrafamily variations in DNA fingerprint patterns were observed with the M13 minisatellite probe. Estimates of the mutation rate for (GATA) _n microsatellite loci in D. unisexualis showed that it was as high as that seen in some bisexual species, reaching 15% per sibling or 0.95% per microsatellite band. Furthermore, in one case, a somatic (GATA) _n microsatellite mutation was observed in an adult lizard. These findings directly demonstrate that mutations in (GATA) _n microsatellite loci comprise an important source of genetic variation in parthenogenetic populations of D. unisexualis.Communicated by G. P. Georgiev     

No correlation between germline mutation at repeat DNA and meiotic crossover in male mice exposed to X-rays or cisplatin

To test the hypothesis that mouse germline expanded simple tandem repeat (ESTR) mutations are associated with recombination events during spermatogenesis, crossover frequencies were compared with germline mutation rates at ESTR loci in male mice acutely exposed to 1 Gy of X-rays or to 10 mg/kg of the anticancer drug cisplatin. Ionising radiation resulted in a highly significant 2.7–3.6-fold increase in ESTR mutation rate in males mated 4, 5 and 6 weeks after exposure, but not 3 weeks after exposure. In contrast, irradiation had no effect on meiotic crossover frequencies assayed on six chromosomes using 25 polymorphic microsatellite loci spaced at approximately 20 cM intervals and covering 421 cM of the mouse genome. Paternal exposure to cisplatin did not affect either ESTR mutation rates or crossover frequencies, despite a report that cisplatin can increase crossover frequency in mice.

Correlation analysis did not reveal any associations between the paternal ESTR mutation rate and crossover frequency in unexposed males and in those exposed to X-rays or cisplatin. This study does not, therefore, support the hypothesis that mutation induction at mouse ESTR loci results from a general genome-wide increase in meiotic recombination rate.     

No evidence of radiation effect on mutation rates at hypervariable minisatellite loci in the germ cells of atomic bomb survivors

Human minisatellites consist of tandem arrays of short repeat sequences, and some are highly polymorphic in numbers of repeats among individuals. Since these loci mutate much more frequently than coding sequences, they make attractive markers for screening populations for genetic effects of mutagenic agents. Here we report the results of our analysis of mutations at eight hypervariable minisatellite loci in the offspring (61 from exposed families in 60 of which only one parent was exposed, and 58 from unexposed parents) of atomic bomb survivors with mean doses of >1 Sv. We found 44 mutations in paternal alleles and eight mutations in maternal alleles with no indication that the high doses of acutely applied radiation had caused significant genetic effects. Our finding contrasts with those of some other studies in which much lower radiation doses, applied chronically, caused significantly increased mutation rates. Possible reasons for this discrepancy are discussed.     

Individual variation in microsatellite mutation rate in barn swallows

The hypermutable nature of some microsatellite loci implies realistic possibilities for the large-scale detection of germline mutations by pedigree analysis. We have developed a model system for mutation analysis by the characterisation of patterns of mutation at three hypervariable microsatellites (two tetranucleotide and one pentanucleotide repeat loci) in barn swallows, all three markers having mutation rates at the percentage level. Here, we study how the mutation rate varies between individual birds of a Spanish population of barn swallows. A total of 53 mutations were identified from 2920 germline transmissions in 90 families with a total of 694 offspring. Mutations were not randomly distributed among individuals (P = 0.020). Attempts to correlate mutation rate with allele size, degree of inbreeding, immunocompetence and male age only revealed a strong effect of allele size. The mean mutation rate differed between colonies of breeding swallows which was probably due to a corresponding variation in allele size between colonies. There was no difference in the mean mutation rate between the Spanish and an Italian population. These results corroborate earlier findings, at the population level, of an allele size effect on the microsatellite mutation rate.     

Microsatellite mutations in the offspring of irradiated parents 19 years after the Cesium-137 accident

In September of 1987, a radiotherapy unit containing 50.9 TBq of Cs¹³⁷Cl was removed from an abandoned radiotherapy clinic. This unit was subsequently disassembled leading to the most serious radiological accident yet to occur in the Western hemisphere. This event provides an opportunity to assess the genetic effects of ionizing radiation. We surveyed genetic variation of 12 microsatellite loci in 10 families of exposed individuals and their offspring and also in non-exposed families from the same area of Goias state. We found an increase in the number of new alleles in the offspring of the exposed individuals. The mutation rate was found to be higher in the exposed families compared to the control group. These results indicated that exposure to ionizing radiation can be detected in offspring of exposed individuals and also suggest that the elevated microsatellite mutation rate can be attributed to radioactive exposure.     

A high incidence of clustered microsatellite mutations revealed by parent-offspring analysis in the African freshwater snail, <Emphasis Type="BoldItalic">Bulinus forskalii</Emphasis> (Gastropoda,Pulmonata)

Genotyping of 11 microsatellites in 432 offspring from 28 families of the hermaphroditic, freshwater snail Bulinus forskalii detected 10 de novo mutant alleles. This gave an estimated mutation rate of 1.1 × 10^–3 per locus per gamete per generation. There was a trend towards repeat length expansion and, unlike most studies, multi-step mutations predominated, suggesting that the microsatellite mutation process does not conform to a strict stepwise mutation model. Interestingly, the ten mutant alleles appear to have arisen from only six independent germline mutation events within the microsatellite array, with seven of them residing in three mutational clusters. Our results extend observations of clustered microsatellite mutations to another taxonomic group and type of mating system, self-fertile gastropods, and provide compelling evidence of premeiotic germline mutations, a phenomenon that could greatly impact upon our understanding of mutation dynamics but which has received little attention.     

Extremely complex pattern of microsatellite mutation in the germline of wheat exposed to the post-Chernobyl radioactive contamination

The molecular structure of rare variants at 13 microsatellite loci found in a population of wheat plants grown for one generation in the heavily contaminated 30 km exclusion zone around the Chernobyl Nuclear Power Plant and in a control population was compared. Evidence for rare alterations (variants) was obtained for all 13 loci, including gain and loss of repeats, as well as the complete loss of microsatellite bands. The ratio between gains and losses among variants in the control group was similar to that in the exposed group. Sequencing of variants at six microsatellite loci found in the exposed population revealed extremely complex pattern of germline mutations, including complete deletions of loci, a bias towards mutations with gains and losses of multiple repeat units, and relatively frequent insertions of DNA of unknown origin. The occurrence of large deletions at two loci may be attributed to direct and inverted repeats sequences located just upstream and downstream of the array. The results of our study also suggest that the majority of mutations within the studied wheat microsatellite loci are represented by gains and losses of multiple repeat units, implying that a simple model of replication slippage cannot account for mutation events at these loci. Our data also support the conclusion that the spectra of spontaneous and radiation-induced mutation in wheat may be similar.     

Germline Mutations of Tetranucleotide DNA Repeats in Families with Normal Children and Reproductive Pathology

We have previously reported a high rate of tetranucleotide DNA repeat mutations, including mutations of both germline and somatic origin, in spontaneous human abortions. To analyze in more detail mutational microsatellite (MS) variability in meiosis and its possible association with disturbed embryonic development, we have conducted a comparative study of mutation rates of a panel of 15 autosomal tetranucleotide MSs in 55 families with healthy children and in 103 families that have had spontaneous abortions with normal karyotypes. In the families with miscarriage, the gametic MS mutation rate was higher than in the families with normal reproductive function (4.36 × 10⁻³ versus 2.32 × 10⁻³ per locus per gamete per generation), but this difference was statistically nonsignificant (P = 0.25). No association of MS mutations with familiar miscarriage was found. Mutations at the MS loci studied were recorded almost 3 times as often in spermatogenesis as in oogenesis, which is likely to result from a greater number of DNA replication cycles in male germline cell precursors than in female ones. Mutations increasing and reducing the MS sequence length appeared at virtually the same rate. Changes in MS DNA sequence length per one repeated element, i.e., single-step mutations (93% of cases) exceeded all other events of allele length change. The highest number of mutations (81.2%) was found in longer alleles. This distribution of mutations by size, direction, and parental origin corresponds to the multistep mutation model of their emergence via mechanism of DNA strand slippage during replication.__________Translated from Genetika, Vol. 41, No. 7, 2005, pp. 943–953.Original Russian Text Copyright © 2005 by Nikitina, Lebedev, Sukhanova, Nazarenko.     

Germline mutation rates in mice following in utero exposure to diesel exhaust particles by maternal inhalation

The induction of inherited DNA sequence mutations arising in the germline (i.e., sperm or egg) of mice exposed in utero to diesel exhaust particles (DEPs) via maternal inhalation compared to unexposed controls was investigated in this study. Previous work has shown that particulate air pollutants (PAPs) from industrial environments cause DNA damage and mutations in the sperm of adult male mice. Effects on the female and male germline during critical stages of development (in utero) are unknown. In mice, previous studies have shown that expanded simple tandem repeat (ESTR) loci exhibit high rates of spontaneous mutation, making this endpoint a valuable tool for studying inherited mutation and genomic instability. In the present study, pregnant C57Bl/6 mice were exposed to 19mg/m(3) DEP from gestational day 7 through 19, alongside air exposed controls. Male and female F1 offspring were raised to maturity and mated with control CBA mice. The F2 descendents were collected and ESTR germline mutation rates were derived from full pedigrees (mother, father, offspring) of F1 male and female mice. We found no evidence for increased ESTR mutation rates in females exposed in utero to DEP relative to control females. In contrast, a statistically significant increase in the mutation frequency of male mice exposed in utero to DEP was observed (2-fold; Fisher's exact p<0.05). Thus, maternal exposure to DEP results in increased mutation in sperm during development.     

The effects of maternal irradiation during adulthood on mutation induction and transgenerational instability in mice

The long-term genetic effects of maternal irradiation remain poorly understood. To establish the effects of radiation exposure on mutation induction in the germline of directly exposed females and the possibility of transgenerational effects in their non-exposed offspring, adult female BALB/c and CBA/Ca mice were given 1Gy of acute X-rays and mated with control males. The frequency of mutation at expanded simple tandem repeat (ESTR) loci in the germline of directly exposed females did not differ from that of controls. Using a single-molecule PCR approach, ESTR mutation frequency was also established for both germline and somatic tissues in the first-generation offspring of irradiated parents. While the frequency of ESTR mutation in the offspring of irradiated males was significantly elevated, maternal irradiation did not affect stability in their F(1) offspring. Considering these data and the results of our previous study, we propose that, in sharp contrast to paternal exposure to ionising radiation, the transgenerational effects of maternal high-dose acute irradiation are likely to be negligible.     

Genetic variation and de novo mutations in the parthenogenetic Caucasian rock lizard Darevskia unisexualis

Unisexual all-female lizards of the genus Darevskia that are well adapted to various habitats are known to reproduce normally by true parthenogenesis. Although they consist of unisexual lineages and lack effective genetic recombination, they are characterized by some level of genetic polymorphism. To reveal the mutational contribution to overall genetic variability, the most straightforward and conclusive way is the direct detection of mutation events in pedigree genotyping. Earlier we selected from genomic library of D. unisexualis two polymorphic microsatellite containing loci Du281 and Du215. In this study, these two loci were analyzed to detect possible de novo mutations in 168 parthenogenetic offspring of 49 D. unisexualis mothers and in 147 offspring of 50 D. armeniaca mothers. No mutant alleles were detected in D. armeniaca offspring at both loci, and in D. unisexualis offspring at the Du215 locus. There were a total of seven mutational events in the germ lines of four of the 49 D. unisexualis mothers at the Du281 locus, yielding the mutation rate of 0.1428 events per germ line tissue. Sequencing of the mutant alleles has shown that most mutations occur via deletion or insertion of single microsatellite repeat being identical in all offspring of the family. This indicates that such mutations emerge at the early stages of embryogenesis. In this study we characterized single highly unstable (GATA)(n) containing locus in parthenogenetic lizard species D. unisexualis. Besides, we characterized various types of mutant alleles of this locus found in the D. unisexualis offspring of the first generation. Our data has shown that microsatellite mutations at highly unstable loci can make a significant contribution to population variability of parthenogenetic lizards.     

Lack of effects of atomic bomb radiation on genetic instability of tandem-repetitive elements in human germ cells.

In a pilot study to detect the potential effects of atomic bomb radiation on germ-line instability, we screened 64 children from 50 exposed families and 60 from 50 control families for mutations at six minisatellite loci by using Southern blot analysis with Pc-1, lambda TM-18, ChdTC-15, p lambda 3, lambda MS-1, and CEB-1 probes. In the exposed families, one or both parents received a radiation dose > 0.01 Sv. Among the 64 children, only one child had parents who were both exposed. Thus, of a total of 128 gametes that produced the 64 children, 65 gametes were derived from exposed parents and 63 were from unexposed parents, the latter being included in a group of 183 unexposed gametes used for calculating mutation rates. The average parental gonadal dose for the 65 gametes was 1.9 Sv. We detected a total of 28 mutations at the p lambda g3, lambda MS-1, and CEB-1 loci, but no mutations at the Pc-1, lambda TM-18, and ChdTC-15 loci. We detected 6 mutations in 390 alleles of the 65 exposed gametes and 22 mutations in 1098 alleles of the 183 gametes from the unexposed parents. The mean mutation rate per locus per gamete in these six minisatellite loci was 1.5% in the exposed parents and 2.0% in the unexposed parents. We observed no significant difference in mutation rates in the children of the exposed and the unexposed parents (P = .37, Fisher's exact probability test).     

Radiation-induced germline mutations detected by a direct comparison of parents and first-generation offspring DNA sequences containing SNPs

Germline mutation induction has been detected in mice but not in humans. To estimate the genetic risk of germline mutation induction in humans, new techniques for extrapolating from animal data to humans or directly detecting radiation-induced mutations in man are expected to be developed. We have developed a new method to detect germline mutations by directly comparing the DNA sequences of parents and first-generation offspring. C3H male mice were irradiated with gamma-rays of 3, 2 and 1 Gy and 3 weeks later were mated with C57BL female mice of the same age. The nucleotide sequences of 160 UniSTS markers containing 300-900 bp and SNPs of the DNA of parent and offspring mice were determined by direct sequencing. At each dose of radiation, a total of 5 Mb DNA sequences were examined for radiation-induced mutations. We found 7 deletions in 3 Gy-irradiated mice, 1 deletion in 2 Gy-irradiated mice, 1 deletion in 1 Gy-irradiated mice and no mutations in control mice. The maximum mutation frequency was 2.0 x 10(-4)/locus/Gy at 3 Gy, and these results suggested that a non-linear increase of mutations with dose.