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.     

A genome scanning approach to assess the genetic effects of radiation in mice and humans

We used Restriction Landmark Genome Scanning (RLGS) to assess, on a genome-wide basis, the mutation induction rate in mouse germ cells after radiation exposure. Analyses of 1,115 autosomal NotI DNA fragments per mouse for reduced spot intensity, indicative of loss of one copy, in 506 progeny derived from X-irradiated spermatogonia (190, 237 and 79 mice in 0-, 3-, and 5-Gy groups, respectively), permitted us to identify 16 mutations affecting 23 fragments in 20 mice. The 16 mutations were composed of eight small changes (1-9 bp) at microsatellite sequences, five large deletions (more than 25 kb), and three insertions of SINE B2 or LINE1 transposable elements. The maximum induction rate of deletion mutations was estimated as (0.17 +/- 0.09) x 10(-5)/locus Gy(-1). The estimate is considerably lower than 1 x 10(-5)/locus Gy(-1), the mean induction rate of deletion mutations at Russell's 7 loci, which assumed that deletion mutations comprise 50% of all mutations. We interpret the results as indicating that the mean induction rate of mutations in the whole genome may be substantially lower than that at the 7 loci. We also demonstrate the applicability of RLGS for detection of human mutations, which allows direct comparisons between the two species.     

Cellular and molecular effects for mutation induction in normal human cells irradiated with accelerated neon ions

We investigated the linear energy transfer (LET) dependence of mutation induction on the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in normal human fibroblast-like cells irradiated with accelerated neon-ion beams. The cells were irradiated with neon-ion beams at various LETs ranging from 63 to 335 keV/microm. Neon-ion beams were accelerated by the Riken Ring Cyclotron at the Institute of Physical and Chemical Research in Japan. Mutation induction at the HPRT locus was detected to measure 6-thioguanine-resistant clones. The mutation spectrum of the deletion pattern of exons of mutants was analyzed using the multiplex polymerase chain reaction (PCR). The dose-response curves increased steeply up to 0.5 Gy and leveled off or decreased between 0.5 and 1.0 Gy, compared to the response to (137)Cs gamma-rays. The mutation frequency increased up to 105 keV/microm and then there was a downward trend with increasing LET values. The deletion pattern of exons was non-specific. About 75-100% of the mutants produced using LETs ranging from 63 to 335 keV/mum showed all or partial deletions of exons, while among gamma-ray-induced mutants 30% showed no deletions, 30% partial deletions and 40% complete deletions. These results suggested that the dose-response curves of neon-ion-induced mutations were dependent upon LET values, but the deletion pattern of DNA was not.     

Large deletions are tolerated at the hprt locus of in vivo derived human T-lymphocytes.

A cloning assay was used to recover hprt- T-lymphocytes from adult human males. Analysis of crude cellular extracts by polymerase chain reactions (PCRs) demonstrated that 7% (16/218) of the hprt mutations were due to total deletion of the hprt gene. 14 of the 16 mutants were examined by PCR for the presence of flanking DNA to determine the extent of the deletions. The deletion mutation in 13 mutants was at least 350 kb with 5 of these deletions being at least 700 kb. The largest deletions were greater than 15 times the size of the hprt gene. Therefore, large deletions are tolerated at the hprt locus of human T-lymphocytes.     

Biochemical and recessive visible specific locus responses of C3H/HeH to fractionated,acute radiation

The recessive visible specific locus test has been widely used for many years to investigate the genetic effects of radiation in mice. We devised an electrophoretic-specific locus test so that biochemical mutations leading to alterations in the activity or amount of four enzymes and proteins, as well as charge changes could be detected. We measured the yield of recessive visible and electrophoretic mutations in the same experiment so that a direct comparison of mutation incidence could be made. Dominant visible mutations were also scored. The recessive visible specific locus response of male C3H/HeH to a fractionated dose of 3 + 3 Gy X-irradiation separated by 24 h was similar to that previously reported for the F1 hybrid widely used in mutagenesis studies, and other strains. The response of C3H/HeH was significantly greater for the recessive visible mutations than for the biochemical mutations, supporting the contention that the recessive visible loci are more mutable than others. Mutational analysis of some of the mutants showed that the lesions ranged from a very deletion (30% of chromosome 14 deleted) to a point mutation. The number of loci scored in the electrophoretic test has been reassessed, and it is now considered that six, not four were scored, and this has implications for the calculation of the doubling dose.     

Sequence specificity of Hprt lymphocyte mutation in rats fed the hepatocarcinogen 2-acetylaminofluorene

Rats fed the hepatocarcinogen 2-acetylaminofluorene (2-AAF) have a low, but significantly increased, frequency of lymphocyte Hprt mutants. In this study, mutants from 2-AAF-fed and control F344 rats were examined for mutations in the Hprt gene in order to determine if the 2-AAF treatment resulted in an agent-specific mutation profile. The most common mutation from 2-AAF-treated rats was G:C-->T:A transversion (32% of all mutations) followed by 1-basepair (bp) deletion (19%); there were very few (5%) G:C-->A:T transitions. Among mutations from control rats, G:C-->A:T transition was the most common (43%), and there were very few G:C-->T:A transversions (5%) and no 1-bp deletions. The profile of mutations from 2-AAF-fed rats was significantly different from control rats (P = 0.003) and was consistent with the types of mutations produced by 2-AAF in vitro. The results of this study indicate that even weak mutational responses in the lymphocyte Hprt assay are capable of producing mutation profiles that reflect the DNA damage inducing them.     

Ionizing radiation and genetic risks. III. Nature of spontaneous and radiation-induced mutations in mammalian in vitro systems and mechanisms of induction of mutations by radiation

This paper (1) presents an analysis of published data on the molecular nature of spontaneously arising and radiation-induced mutations in mammalian somatic cell systems and (2) examines whether the molecular nature and mechanisms of origin of radiation-induced mutations, in mammalian in vivo and in vitro systems, as currently understood, are consistent with expectations based on the biophysical and microdosimetric properties of ionizing radiation. Depending on the test system (CHO cells, human T lymphocytes and human lymphoid cell line TK6), 80-97% of spontaneous HPRT mutations show normal Southern patterns; the remainder is due to gross changes, predominantly partial (intragenic) deletions. Total gene deletions at the HPRT locus are rare except in the TK6 cell line. At the APRT locus in CHO cells, 80-97% of spontaneous mutations are due to base-pair changes, the remainder being, mostly, partial deletions. The latter can extend upstream in the 5' direction but not beyond the APRT gene in the 3' direction. At the human HLA-A locus (T lymphocytes), the percentage of mutations with normal Southern patterns is lower than that for HPRT, and in the range of 50-60%. At the HLA-A locus, mitotic recombination contributes substantially to the mutation spectrum (approximately 30% of mutations recovered) and this is likely to be true of the TK locus in the TK6 cell line as well. With a few exceptions, most of the radiation-induced mutations show altered Southern patterns and are consistent with their being deletions and/or other gross changes (HPRT, 70-90% (CHO); 50-85% (TK6); 50-75% (T lymphocytes); TK, 60-80% (TK6); HLA-A, 80% (T lymphocytes); DHFR, 100% (CHO]. The exceptions are APRT mutations in CHO cells (16-20% of mutants with deletions or other changes) and HPRT mutations in T lymphocytes from A-bomb survivors (15-25%); the latter finding is consistent with the occurrence of in vivo selection against HPRT mutant cells. In cases of HPRT intragenic deletions analyzed (CHO cells and V79 Chinese hamster cells), there is evidence for a non-random distribution of breakpoints. The spontaneous mutation frequencies vary widely, from about 0.04/10(6) cells (sickle cell mutations at the human HBB locus) to 30.8/10(6) cells (HLA-A mutations in T lymphocytes) and are dependent on the locus, the system employed and a number of other factors. Those for the other loci fall between these limits.(ABSTRACT TRUNCATED AT 400 WORDS)     

Radiation-induced mutation at tandem repeat DNA Loci in the mouse germline: spectra and doubling doses

The spectra and dose response for mutations at expanded simple tandem repeat (ESTR) loci in the germline of male mice acutely exposed to low-LET X or gamma rays at pre-meiotic stages of spermatogenesis were compared in five strains of laboratory mice. Most mutation events involved the gain or loss of a relatively small number of repeat units, and the distributions of length changes were indistinguishable between the exposed and control males. Overall, a significant bias toward gains of repeats was detected, with approximately 60% of mutants showing gains. The values for ESTR mutation induction did not differ substantially between strains. The highest values of doubling dose were obtained for two genetically related strains, BALB/c and C.B17 (mean value 0.98 Gy). The estimates of doubling dose for three other strains (CBA/H, C57BL/6 x CBA/H F1 and 129SVJ x C57BL/6) were lower, with a mean value of 0.44 Gy. The dose response for ESTR mutation across all five strains was very close to that for the specific loci (Russell 7-locus test). The mechanisms of ESTR mutation induction and applications of this system for monitoring radiation-induced mutation in the mouse germline are discussed.     

Molecular characterization of methyl methanesulphonate (MMS)-induced HPRT mutations in V79 cells

Spontaneous and methyl methanesulphonate-induced HPRT-deficient mutants were analysed for changes in the hprt gene structure using multiplex polymerase chain reaction. The PCR amplification pattern of 21 MMS-induced mutations revealed one total deletion of the hprt coding exons and one small deletion within exon 5, while 19 mutants showed the V79 wild-type pattern. Molecular analysis of 30 spontaneous mutations revealed no mutants with amplification patterns which differed from those of wild-type cells. We further analysed MMS-induced mutants in a different V79 cell line with a high (40%) spontaneous deletion frequency. MMS caused a dose-dependent increase in the mutant frequency but the incidence of deletions was reduced to 6% at 2 × 10⁻⁴ M and to 13% at 5 × 10⁻⁴ M indicating that mainly point mutations were induced. The repair inhibitor cytosine arabinoside (araC) enhanced mutation induction by MMS but did not change the proportion of deletions in the mutation spectrum. The results indicate that different V79 cell lines spontaneously produce different amounts of deletion mutations. The frequency of MMS-induced deletions does not depend on the frequency of spontaneous deletions in a given cell line. The MMS-induced mutation spectrum seems to be unchanged even at high concentrations with a strong cytotoxic effect. Deletions are not increased as a consequence of araC-inhibited repair of MMS-inducd lesions.     

Radiation-induced genetic instability in vivo depends on p53 status

In response to ionizing radiation and other agents that damage DNA, the p53 tumor suppressor protein activates multiple cellular processes including cell cycle checkpoints and programmed cell death. Although loss of p53 function is associated with radiation-induced genetic instability in cell lines, it is not clear if this relationship exists in vivo. To study the role of p53 in maintenance of genetic stability in normal tissues following irradiation, we have measured mutant frequencies at the adenine phosphoribosyltransferase (Aprt) and hypothanine-guanine phosphoribosyltransferase (Hprt) loci and examined mechanisms of loss of heterozygosity (LOH) in normal T cells of p53-deficient, Aprt heterozygous mice that were subjected to whole-body irradiation with a single dose of 4Gy X-rays. The radiation-induced mutant frequency at both the Aprt and Hprt loci was elevated in cells from mice with different p53 genotypes. The radiation-induced elevation of p53-/- mice was significantly greater than that of p53+/- or p53+/+ mice and was caused by several different kinds of mutational events at the both chromosomal and intragenic levels. Most significantly, interstitial deletion, which occurs rarely in unirradiated mice, became the most common mechanism leading to LOH in irradiated p53 null mice. These observations support the idea that absence or reduction of p53 expression enhances radiation-induced tumorigenesis by increasing genetic instability at various loci, such as those for tumor suppressor genes.     

Adaptive response to mutagenesis and its molecular basis in a human T-cell leukemia line primed with a low dose ofγ-rays

The effect was studied of a low dose of-ray preexposure on the frequency and molecular spectrum of radiation-induced mutations at the hprt locus in a human T-cell leukemia line. When the cells were preexposed to 0.01 Gy of-rays, the yield of mutations induced by a subsequent 2-Gy challenge dose was reduced by 60%, compared with the 2 Gy of irradiation alone. The data of Southern blot analysis showed that 47% of the mutants induced by 2 Gy in the cells without low-dose preexposure were of the deletion or rearranged mutations type. In contrast, in the low-dose radioadapted cells the proportion of this type of 2-Gy-induced mutations decreased to 28%. This is close to the control level (22%) of spontaneous mutations. Our results confirm that a low dose of-ray preexposure leads to a decreased susceptibility to gene deletions and rearrangements after high-dose irradiation.     

Induction of mutations by bismuth-212 alpha particles at two genetic loci in human B-lymphoblasts.

The human lymphoblast cell line TK6 was exposed to the alpha-particle-emitting radon daughter 212Bi by adding DTPA-chelated 212Bi directly to the cell suspension. Cytotoxicity and mutagenicity at two genetic loci were measured, and the molecular nature of mutant clones was studied by Southern blot analysis. Induced mutant fractions were 2.5 x 10(-5)/Gy at the hprt locus and 3.75 x 10(-5)/Gy at the tk locus. Molecular analysis of HPRT- mutant DNAs showed a high frequency (69%) of clones with partial or full deletions of the hprt gene among radiation-induced mutants compared with spontaneous mutants (31%). Chi-squared analyses of mutational spectra show a significant difference (P < or = 0.005) between spontaneous mutants and alpha-particle-induced mutants. Comparison with published studies of accelerator-produced heavy-ion exposures of TK6 cells indicates that the induction of mutations at the hprt locus, and perhaps a subset of mutations at the tk locus, is a simple linear function of particle fluence regardless of the ion species or its LET.     

Molecular changes in UV-induced and γ-ray-induced mutations in human lymphoblastoid cells

We have characterized the structural changes in the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene of 14 UV-induced, 15 γ-ray-induced and 17 spontaneous mutants of human lymphoblastoid cells selected for 6-thioguanine (6TG) resistance. Southern blot analysis using the full-length HPRT cDNA as a probe revealed that 29% (5/17) of the spontaneous mutants contained detectable alterations in their restriction fragment patterns. Among the 15 mutants induced by γ rays, 7 (47%) had such alterations indicative of large deletions in the HPRT gene. In contrast, all 14 UV-induced mutants exhibited hybridization patterns indistinguishable from those of the wild-type cells. These results suggest that UV is likely to induce point mutations at the HPRT locus on the human chromosome and that the molecular mechanism of UV-induced mutation is quite different from that of ionizing radiation-induced mutation or spontaneous mutation in human cells.     

Molecular analysis of spontaneous mutations at the gpt locus in Chinese hamster ovary (AS52) cells

AS52 cells are Chinese hamster ovary (CHO) cells that carry a single functional copy of the bacterial gpt gene and allow the isolation of 6-thioguanine-resistant (6TGr)mutants arising from mutation at the chromosally integrated gpt locus. The gpt locus in AS52 cells is extremely stable, giving rise to 6TGr mutants at frequencies comparable to the endogenous CHO hprt locus. In this study, we describe the spectrum of spontaneous mutations observed in AS52 cells by Southern blot and DNA sequence analyses. Using the polymerase chain reaction (PCR) and the Thermus aquaticus (Taq) polymerase, we have enzymatically amplified 6TGr mutant gpt sequences in vitro. The PCR product was then sequenced without further cloning manipulations to directly identify gpt structural gene mutations. Deletions predominant among the 62 spontaneous 6TGr-AS52 mutant clones analyzed in this study. Of these, 79% (49/62) of the mutations were identified as deletions either by Southern blotting, PCR amplification or DNA sequence analysis. Among these deletions is a predominant 3-base deletion that was observed in 31% (19/62) of the mutants. These data provide a basis for future comparisons of induced point mutational spectra derived in the AS52 cell line, and demonstrate the utility of PCR in the generation of DNA sequence spectra derived from chromosomally integrated mammalian loci.     

Characterization of trimethylpsoralen as a mutagen for mouse embryonic stem cells

Given a large number of genes with unknown functions in model organisms, collections of mutants are valuable resources for studying gene function. For the mouse, embryonic stem cell technology offers the possibility to manipulate the genome and select for mutations in vitro. Mutant mice can then be generated from clones of interest to study the phenotype of these animals. We manipulate the genome of mouse embryonic stem (ES) cells chemically using the mutagen trimethylpsoralen (TMP). TMP predominantly causes deletions in the genome of Caenorhabditis elegans and Escherichia coli, but has not been established as a mutagen in mammalian systems yet. We have characterized TMP as a mutagen for mouse ES cells regarding death rates, mutation frequencies, and mutation spectrum. Allowing for 12.5% of cell survival, the mutation frequency at the mouse hypoxanthine-guanine phosphoribosyltransferase (Hprt) locus was 3.5 x 10(-5) on average. The characterization of a non-redundant set of 17 Hprt-deficient ES clones revealed that only 12% of clones contained genomic deletions and almost 50% were point mutations. Base substitutions were mostly transversions and all affected AT base pairs. We conclude that the mutation spectrum of TMP in mouse ES cells is different from that observed in C. elegans and E. coli.     

Molecular analysis of mutation at the hprt locus of Chinese hamster V79 cells induced by ethyl methanesulphonate and mitomycin C

Mutation at the hprt locus of Chinese hamster V79 cells were induced by treatment with ethyl methanesulphonate (EMS), considered primarily a point mutagen and mitomycin C (MMC), a potent clastogen. EMS gave a dose-dependent induction of mutants while MMC induced a poor mutagenic response. Mutations were analysed using Southern and Northern blotting.Analysis of 9 EMS-induced and 4 spontaneous mutants yielded no detectable alterations in the hprt locus after digestion of DNA with 6 restriction enzymes. Mutants without detectable changes carried presumptive point mutations. In contrast, 4 out of 12 MMC-induced mutants had detectable alterations. 2 of these appeared to have lost the entire hprt gene while the other 2had prodable partial deletions. For these 4 deletion mutants no hprt mRNA was detected. 3 MMC-induced and 1 EMS-induced mutants had reduced levels of hprt mRNA. All the other mutants showed normal levels of hprt mRNA and the message detected was always of the correct size.It is suggested that the poor mutagenic response induced by MMC may be due to the lethal nature of large deletions involving both the hemizygous hprt locus and adjacent essential genes. This may lead to an underestimate of the mutagenicity of clastogenic agents such as MMC in the V79 HPRT mutation assay.     

Molecular cloning and mapping of the ecotropic leukemia provirus Emv-23 provides molecular access to the albino-deletion complex in mouse chromosome 7

Genetic analysis of radiation-induced deletion mutations involving the chromosome 7 albino (c) locus has expanded the functional map of this 6 to 11-cM region of the mouse genome. To generate one of many points of molecular access necessary for intensifying the analysis of the genes and phenotypes associated with this particular complex of deletions, we have cloned an endogenous ecotropic leukemia provirus (Emv-23), known to be closely linked to c, along with its flanking chromosome 7 sequences. A unique-sequence probe (23.3), derived from a region immediately 5' to the proviral integration site, was found to map less than 0.5 cM from c in a standard backcross analysis. Southern blot analysis of DNAs from animals carrying homozygous or overlapping albino deletions demonstrated that the 23.3 probe was deleted in several relatively small c-region deletions. The deletion mapping of the 23.3 probe places the Emv-23 locus between c and Mod-2, just proximal to a region important for male fertility and juvenile fitness. Mapping of this locus also provides a refinement of the genetic/deletion map for several mutations within this deletion complex.     

Frequency of CD59 mutations induced in human-hamster hybrid A(L) cells by low-dose X-irradiation

Determination of the genotoxic effects of ionizing radiation, especially at low-doses, is of great importance for risk assessment, e.g. in radiological diagnostics. The human-hamster hybrid A(L) cell line has been shown previously to be a well-suited in vitro model for the study of mutations induced by various mutagens. The A(L) cells contain a standard set of hamster chromosomes and a single human chromosome 11, which confers the expression of the human cell surface protein CD59. Using CD59 specific antibodies, cells mutated in the CD59 gene can be detected and quantified by the loss of the cell surface marker. In contrast to previous studies, prior to irradiation we removed spontaneous mutants by magnetic cell separation (MACS) which allows analysis of radiation-induced mutation events only. We exposed A(L) cells to 100kV X-rays at 0.1 to 5Gy. The proportions of X-irradiation-induced CD59(-) mutants were quantified by flow cytometry after immunofluorescence labeling. Between 0.2 and 5Gy the yield of CD59 mutants was a linear function of dose. The molecular analysis of individual CD59-negative clones induced after exposure of 1, 3 and 5Gy of X-ray revealed a dose-dependent linear increase of large deletions (>6Mbp), whereas, point mutations could be seen only in spontaneous CD59 mutants or after low-dose exposure (< or =1Gy). We conclude that the modified A(L) assay presented here is appropriate for detection and quantification of non-lethal DNA lesions induced by low-dose ionizing radiation.     

Molecular analysis of two mouse dilute locus deletion mutations: spontaneous dilute lethal20J and radiation-induced dilute prenatal lethal Aa2 alleles.

The dilute (d) coat color locus of mouse chromosome 9 has been identified by more than 200 spontaneous and mutagen-induced recessive mutations. With the advent of molecular probes for this locus, the molecular lesion associated with different dilute alleles can be recognized and precisely defined. In this study, two dilute mutations, dilute-lethal20J (dl20J) and dilute prenatal lethal Aa2, have been examined. Using a dilute locus genomic probe in Southern blot analysis, we detected unique restriction fragments in dl20J and Aa2 DNA. Subsequent analysis of these fragments showed that they represented deletion breakpoint fusion fragments. DNA sequence analysis of each mutation-associated deletion breakpoint fusion fragment suggests that both genomic deletions were generated by nonhomologous recombination events. The spontaneous dl20J mutation is caused by an interstitial deletion that removes a single coding exon of the dilute gene. The correlation between this discrete deletion and the expression of all dilute-associated phenotypes in dl20J homozygotes defines the dl20J mutation as a functional null allele of the dilute gene. The radiation-induced Aa2 allele is a multilocus deletion that, by complementation analysis, affects both the dilute locus and the proximal prenatal lethal-3 (pl-3) functional unit. Molecular analysis of the Aa2 deletion breakpoint fusion fragment has provided access to a previously undefined gene proximal to d. Initial characterization of this new gene suggests that it may represent the genetically defined pl-3 functional unit.