Radiation-induced Mutations Research Articles

Oligosyndactylism Mice Have an Inversion of Chromosome 8

The radiation-induced mutation Oligosyndactylism (Os) is associated with limb and kidney defects in heterozygotes and with mitotic arrest and embryonic lethality in homozygotes. We reported that the cell cycle block in Os and in the 94-A/K transgene-induced mutations is due to disruption of the Anapc10 (Apc10/Doc1) gene. To understand the genetic basis of the limb and kidney abnormalities in Os mice we characterized the structural changes of chromosome 8 associated with this mutation. We demonstrate that the Os chromosome 8 has suffered two breaks that are 5 cM ( approximately 10 Mb) apart and the internal fragment delineated by the breaks is in an inverted orientation on the mutant chromosome. While sequences in proximity to the distal break are present in an abnormal Os-specific Anapc10 hybrid transcript, transcription of these sequences in normal mice is low and difficult to detect. Transfer of the Os mutation onto an FVB/N background indicated that the absence of dominant effects in 94-A/K mice is not due to strain background effects on the mutation. Further analysis of this mutation will determine if a gene interrupted by the break or a long-range effect of the rearrangement on neighboring genes is responsible for the dominant effects of Os.

A role for long-lived radicals (LLR) in radiation-induced mutation and persistent chromosomal instability: counteraction by ascorbate and RibCys but not DMSO

Miazaki, Watanabe, Kumagai and their colleagues reported that induction of HPRT − mutants by X-rays in cultured human cells was prevented by ascorbate added 30 min after irradiation. They attributed extinction of induced mutation to neutralization by ascorbate of radiation-induced long-lived mutagenic radicals (LLR), found using spectroscopy to have half-lives of minutes or hours. We find that post-irradiation treatment with ascorbate reduces, but does not eliminate, induction of CD59 − mutants in human-hamster hybrid A L cells exposed to high-LET carbon-ions (LET of 100 KeV/μm). A L cells contain a standard set of Chinese hamster ovary (CHO) chromosomes and a single copy of human chromosome 11 containing the CD59 gene which encodes the CD59 cell surface antigen, a convenient marker for mutation. RibCys [2(R, S)-D-ribo-(1′,2′,3′,4′-tetrahydroxybutyl)thiazolidine-4(R)-carboxylic acid] a ‘prodrug’ of l-cysteine which also scavenges LLR, had a similar but lesser effect on induced mutation. DMSO, which scavenges classical radicals like H and OH but not LLR, also reduced mutation, but only when it was present during irradiation. The lethality of carbon-ions was not altered by ascorbate, RibCys no matter when added. Post-radiation addition of ascorbate and RibCys also affected the quality of CD59 − mutations induced by carbon-ions. The major change in mutant spectra was a reduction in the prevalence of small, intragenic mutations (mutations not detected by PCR) and in the prevalence of unstable, complicated mutants, which display high levels of persistent chromosomal instability. Thus, ascorbate and RibCys may suppress some kinds of mutations induced by ionizing radiation including those displaying aspects of radiation-induced genomic instability. Countering the effects of both classical radicals and LLR may be important in preventing genetic diseases.

The Dynamics of Radiation-Induced Mutations in Mammalian Differentiated Cells

The Dynamics of Radiation-Induced Mutations in Mammalian Differentiated Cells

Long-term genetic effects of radiation exposure.

To date, there has been little experimental knowledge on the genetic risks of human exposure to ionising radiation for humans. Recent data suggest that hypervariable tandem repeat minisatellite loci provide a useful and sensitive experimental approach for monitoring radiation-induced germline mutation in humans. Here, I review the results of studies on minisatellite mutation rates in human populations exposed to radioactive fallout after the Chernobyl accident and nuclear weapon tests in Kazakhstan.

Monitoring of radiation-induced germline mutation in humans.

Estimating the genetic hazards of radiation and other mutagens in humans depends on extrapolation from experimental systems. Recent data have shown that minisatellite loci provide a useful and sensitive experimental approach for monitoring radiation-induced mutation in humans. This review describes the progress made in validating this approach and presents the results of recent publications on the analysis of minisatellite mutation rates in the irradiated families.

Monitoring of radiation-induced germline mutation in humans.

Estimating the genetic hazards of radiation and other mutagens in humans depends on extrapolation from experimental systems. Recent data have shown that minisatellite loci provide a useful and sensitive experimental approach for monitoring radiation-induced mutation in humans. This review describes the progress made in validating this approach and presents the results of recent publications on the analysis of minisatellite mutation rates in the irradiated families.

The LNT model is the best we can do—today

The form of the dose–response curve for radiation-induced cancers, particularly atlow doses, is the subject of an ongoing and spirited debate. The present reviewdescribes the current database and basis for establishing a low dose, linear nothreshold (LNT) model. The requirement for a dose–response model to be usedfor risk assessment purposes is that it fits the great majority of data derived fromepidemiological and experimental tumour studies. Such is the case for the LNTmodel as opposed to other nonlinear models. This view is supported by datadeveloped for radiation-induced mutations and chromosome aberrations.Potential modifiers of low dose cellular responses to radiation (such asadaptive response, bystander effects and genomic instability) have not beenshown to be associated with tumour development. Such modifiers tend toinfluence the slope of the dose–response curve for cellular responses at lowdoses and not the shape—thereby resulting in a quantitative modificationrather than a qualitative one. Additional data pertinent to addressing theshape of the tumour dose–response relationship at low doses are needed.

Analysis of radiation-induced DNA double-strand breaks misrepair is not compromized by broken DNA in human fibroblasts.

It has been suggested that the technique for measuring repair fidelity of radiation-induced DNA double-strand breaks (DSBs) using Southern blotting and hybridization to defined regions of the genome could be compromised by broken or poorly-digested DNA. Since misrepair of DNA DSBs is an important aspect of radiation-induced chromosome aberrations, mutations, and cell killing, we checked for such a supposition in non-transformed human fibroblasts. DSB misrepair was assessed in a NotI-cleavable DNA fragment of 3.2 Mbp located on the long arm of chromosome 21 and detected by D21S1 probe. We hypothesized that the suggested DNA degradation, whether spurious in nature or the results of irradiation-induced phenomena such as apoptosis and/or necrosis, should be detectable with or without NotI restriction enzyme treatment. When the DNA embedded in agarose plugs was separated by electrophoresis without prior NotI restriction, no significant difference was observed in the relative amount of migrating DNA between the control (no irradiation) and 24 h of repair following 80 Gy irradiation. Furthermore, only about 10% of the total signal was located below the 3.2 Mbp band. This suggests that the amount of DNA fragmentation due to biological (apoptosis or necrosis) or technical processes was negligible. The Tunel assay supported these results, as there was little to no apoptosis detectable in these fibroblasts up to 24 h after irradiation. We conclude that in primary human fibroblasts, the NotI method for measuring radiation-induced misrepair is not compromised by DNA degradation.

Mutational analysis of CDKN2A genes in patients with squamous cell carcinoma of the skin.

Nonmelanoma skin cancers [squamous cell carcinomas (SCC) and basal cell carcinomas (BCC)] are the most common neoplasias of the Caucasian population. The purpose of our study was to determine the involvement of CDKN2A genes in the development of sporadic nonmelanoma skin cancer in Greek patients. Allelic imbalance analysis was performed in 22 SCC and five Bowen's disease specimens. Mutational analysis was performed on exons 1alpha, 1beta and 2 of the CDKN2A locus in 22 SCC, five Bowen's disease and 39 BCC specimens. Exon 1alpha was additionally screened in 28 BCC specimens to complete the mutational analysis of a previous study. Overall, 52% (14 of 27) of the SCC and Bowen's disease specimens exhibited loss of heterozygosity (LOH) in at least one microsatellite marker, whereas, only two of 27 (7%) exhibited microsatellite instability. LOH in 9p appears to be equally involved in both BCC and SCC tumours. Exons 1alpha, 1beta and 2 of the CDKN2A locus were screened for mutations. A Val28Gly substitution in exon 1alpha and a CCC-->TTT (Ala57Val and Arg58Ter) substitution in exon 2, resulting in a change in the amino acid sequence, are reported for the first time in two SCCs, the latter being indicative of a combination of an ultraviolet (UV) radiation-induced mutation and a point mutation. A previously described polymorphism of CDKN2A, the gene for p16INK4a, Ala148Thr, was also detected in an allelic frequency of 3.72%. No mutation was found in any of the five Bowen's disease specimens, or in exon 1beta of CDKN2A, also the gene for p14ARF. Mutations and the high incidence of 9p LOH detected in our SCC samples imply that inactivation of CDKN2A genes, via allelic loss and/or mutation (probably UV-induced) may play a significant role in nonmelanoma skin cancer development, particularly in the more aggressive SCC type.

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.

Increase of Radiation-induced Mutation Frequency in Regenerating Mouse Liver

Increase of Radiation-induced Mutation Frequency in Regenerating Mouse Liver

Circling, Deafness, and Yellow Coat Displayed by Yellow Submarine (Ysb) and Light Coat and Circling (Lcc) Mice with Mutations on Chromosome 3

We describe here two mouse mutants, yellow submarine (Ysb) and light coat and circling (Lcc). Ysb arose as the result of insertions of a transgene, pAA2, into the genome. Lcc is an independent, radiation-induced mutation. Both mutants are characterized by recessive circling behavior and deafness, associated with a non-segregating, semi-dominant yellow coat color. Complementation tests showed that Ysb and Lcc are allelic. We attribute the yellow coat in Ysb and Lcc mice to the absence of black awl overhairs, increased agouti zigzag underhairs, and the presence of agouti awls with long subapical yellow pigment. Chromosomal mapping and genomic characterization showed the Ysb and Lcc mutations involve complex chromosomal rearrangements in overlapping regions of mouse chromosome 3, A2/A3–B/C and B–E1, respectively. Ysb and Lcc show for the first time, to our knowledge, the presence of genes in the B–C region of chromosome 3 important for balance and hearing and the pigmentation and specification of coat hair.

Disruption of mesodermal enhancers forIgf2in the minute mutant

The radiation-induced mutation minute (Mnt) in the mouse leads to intrauterine growth retardation with paternal transmission and has been linked to the distal chromosome 7 cluster of imprinted genes. We show that the mutation is an inversion, whose breakpoint distal to H19 disrupts and thus identifies an enhancer for Igf2 expression in skeletal muscle and tongue, and separates the gene from other mesodermal and extra-embryonic enhancers. Paternal transmission of Mnt leads to drastic downregulation of Igf2 transcripts in all mesodermal tissues and the placenta. Maternal transmission leads to methylation of the H19 differentially methylated region (DMR) and silencing of H19, showing that elements 3' of H19 can modify the maternal imprint. Methylation of the maternal DMR leads to biallelic expression of Igf2 in endodermal tissues and foetal overgrowth, demonstrating that methylation in vivo can open the chromatin boundary upstream of H19. Our work shows that most known enhancers for Igf2 are located 3' of H19 and establishes an important genetic paradigm for the inheritance of complex regulatory mutations in imprinted gene clusters.

A novel single molecule analysis of spontaneous and radiation-induced mutation at a mouse tandem repeat locus

Expanded simple tandem repeat (ESTR) loci include some of the most unstable DNA in the mouse genome and have been extensively used in pedigree studies of germline mutation. We now show that repeat DNA instability at the mouse ESTR locus Ms6-hm can also be monitored by single molecule PCR analysis of genomic DNA. Unlike unstable human minisatellites which mutate almost exclusively in the germline by a meiotic recombination-based process, mouse Ms6-hm shows repeat instability both in germinal (sperm) DNA and in somatic (spleen, brain) DNA. There is no significant variation in mutation frequency between mice of the same inbred strain. However, significant variation occurs between tissues, with mice showing the highest mutation frequency in sperm. The size spectra of somatic and sperm mutants are indistinguishable and heavily biased towards gains and losses of only a few repeat units, suggesting repeat turnover by a mitotic replication slippage process operating both in the soma and in the germline. Analysis of male mice following acute pre-meiotic exposure to X-rays showed a significant increase in sperm but not somatic mutation frequency, though no change in the size spectrum of mutants. The level of radiation-induced mutation at Ms6-hm was indistinguishable from that established by conventional pedigree analysis following paternal irradiation. This confirms that mouse ESTR loci are very sensitive to ionizing radiation and establishes that induced germline mutation results from radiation-induced mutant alleles being present in sperm, rather than from unrepaired sperm DNA lesions that subsequently lead to the appearance of mutants in the early embryo. This single molecule monitoring system has the potential to substantially reduce the number of mice needed for germline mutation monitoring, and can be used to study not only germline mutation but also somatic mutation in vivo and in cell culture.

Induction and decline of HPRT mutants and deletions following a low dose radiation exposure at Chernobyl

This study was conducted to evaluate the ability of mutation in the hypoxanthine-phosphoribosyltransferase gene ( HPRT) to detect radiation-induced mutation in lymphocytes of Russian Chernobyl Clean-up workers, particularly as a function of time after exposure. It is part of a multi-endpoint study comparing HPRT mutation with chromosome translocation and glycophorin A mutation [Radiat. Res. 148 (1997) 463], and extends an earlier report on HPRT [Mutat. Res. 431 (1999) 233] by including data from all 9 years of our study (versus the first 6 years) and analysis of deletion size. Blood samples were collected from 1991 to 1999. HPRT mutant frequency (MF) as determined by the cloning assay was elevated 16% in Clean-up workers ( N=300, the entire group minus one outlier) compared to Russian Controls ( N=124) when adjusted for age and smoking status ( P=0.028). Since exposures occurred over a short relative to the long sampling period, the year of sampling corresponded roughly to the length of time since exposure (correlation coefficient=0.94). When date of blood sample was considered, Control MF was not time dependent. Clean-up worker MF was estimated to be 47% higher than Control MF in 1991 ( P=0.004) and to decline 4.4% per year thereafter ( P=0.03). A total of 1123 Control mutants and 2799 Clean-up worker mutants were analyzed for deletion type and size by PCR assay for retention of HPRT exons and flanking markers on the X chromosome. There was little difference between the overall deletion spectra of Clean-up workers and Controls. However, there was a decline in the average size of deletions of Clean-up workers as time after exposure at Chernobyl increased from 6 to 13 years ( P≤0.05). The results illustrate the sensitivity of HPRT somatic mutation as a biomarker for populations with low dose radiation exposure, and the dependence of this sensitivity on time elapsed since radiation exposure.

Cell cycle-dependent effects of wortmannin on radiation survival and mutation.

Wortmannin, a known radiation sensitizer, has been used in experiments with synchronized cells to compare its effect on radiation survival and mutation induction within the cell cycle. PL61 cells (CHO cells with an inactivated HPRT gene containing a single active copy of a bacterial gpt gene) were synchronized by mitotic selection. Wortmannin administered before gamma irradiation caused a greater sensitization in G(1)-phase cells relative to late S/G(2)-phase cells. Preferential radiosensitization of G(1)-phase cells by wortmannin sets a limit to the proposed use of wortmannin in radiation therapy, since, in contrast to normal tissues, tumors usually have high proportions of S-phase cells. Wortmannin increased mutation frequencies in both G(1)- and S/G(2)-phase cells. Interestingly, relative increases in radiation-induced mutations in G(1) and S/G(2) phases were comparable. The results are discussed in terms of the contributions of different repair modes in the production of mutations.

ESTIMATION OF THE HEREDITARY RISKS OF EXPOSURE TO IONIZING RADIATION

This paper provides a brief overview of the advances in the field of the estimation of the genetic risks of exposure of human populations to ionizing radiation from the early 1950's to the present and of the developments that are anticipated in the coming years. The latter are based on the view that the insights gained from human genetics, especially human molecular genetics, will be increasingly applied to address problems in risk estimation. Owing to the paucity of human data on radiation-induced mutations, mouse data on radiation-induced mutations are used to predict the risk of genetic diseases in humans using the doubling dose method. With this method, the risk per unit dose is expressed as a product of three quantities, i.e., P x 1/DD x MC where P is the baseline frequency of genetic diseases, 1/DD (the relative mutation risk per unit dose; DD refers to the doubling dose, i.e., the radiation dose required to produce as many mutations as those that occur spontaneously in a generation) and MC is the disease class-specific mutation component (a measure of the relative increase in disease frequency per unit relative increase in mutation rate). The five important changes that are now introduced in genetic risk estimation include (1) an upward revision of the baseline frequency of Mendelian diseases to 2.4% (from 1.25% used until the early 1990's); (2) a reversion to the conceptual basis for DD calculations used in the 1972 BEIR report of the U.S. National Academy of Sciences, namely, the use of human data on spontaneous mutation rates and mouse data on induced mutation rates (instead of the use of mouse data for both these rates as has been the case from mid-1970's until the early 1990's); (3) the fuller development and use of the MC concept for predicting the responsiveness of Mendelian and multifactorial diseases to increases in mutation rate; (4) the introduction of a new disease-class-specific quantity called the "potential recoverability correction factor" or PRCF in the risk equation to bridge the gap between the rates of induced mutations in mice and the risk of inducible genetic diseases in humans; and (5) the introduction of the concept that multisystem developmental abnormalities are likely to be among the principal phenotypes of radiation induced genetic damage in humans. All these advances now permit, for the first time in 40 y, the estimation of risks for all classes of genetic diseases. For a population exposed to low-LET, chronic or low-dose irradiation, the risks predicted for the first generation progeny are the following (all estimates are per million live born progeny per gray of parental irradiation): autosomal dominant and x-linked diseases, approximately 750 to 1,500 cases; autosomal recessive, nearly zero; chronic multifactorial diseases, approximately 250 to 1,200 cases; and congenital abnormalities, approximately 2000 cases. The total risk per gray is of the order of approximately 3,000 to 4,700 cases, which represent approximately 0.4 to 0.6% of the baseline frequency of these diseases (738,000 per million) in the population. The advances anticipated in the coming years are likely to permit the estimation of genetic risks of radiation with greater precision than is now possible.

Mouse paracentric inversion In(3)55Rk mutates the urate oxidase gene

The paracentric inversion In(3)55Rk on mouse Chromosome 3 (Chr 3) was induced by cesium irradiation. Genetic crosses indicate the proximal breakpoint cosegregates with D3Mit324 and D3Mit92; the distal breakpoint cosegregates with D3Mit127, D3Mit160, and D3Mit200. Giemsa-banded chromosomes show the inversion spans ∼80% of Chr 3. The proximal breakpoint occurs within band 3A2, not 3B as reported previously; the distal breakpoint occurs within band 3H3. Mice homozygous for the inversion exhibit nephropathy indicative of uricase deficiency. Southern blot analyses of urate oxidase, Uox, show two RFLPs of genomic mutant DNA: an EcoRI site between exons 4–8 and a BamHI site 3′ to exon 6. Mutant cDNA fails to amplify downstream of base 844 at the 3′ end of exon 7. FISH analysis of chromosomes from inversion heterozygotes, using a cosmid clone containing genomic wild-type DNA for Uox exons 2–4, shows that a 5′ segment of the mutated Uox allele on the inverted chromosome has been transposed from the distal breakpoint region to the proximal breakpoint region. Clinical, histopathological, and Northern analyses indicate that our radiation-induced mutation, uox^In, is a putative null.

A biophysical model for estimating the frequency of radiation-induced mutations resulting from chromosomal translocations.

Gene mutations can be induced by radiation as a result of chromosomal translocations. A biophysical model is developed to estimate the frequency of this type of mutation induced by low-LET radiation. Mutations resulting from translocations are assumed to be formed by misrejoining of two DNA double strand breaks (DSB), one within the gene and one on a different chromosome. The chromosome containing the gene is assumed to occupy a spherical territory and does not overlap spatially with other chromosomes. Misrejoining between two DSB can occur only if the two DSB are closer than an interaction distance at the time of their induction. Applying the model to mutations of the hprt gene induced in G0 human lymphocyte cells by low-LET radiation, it is calculated that mutations resulting from translocations account for about 14% of the total mutations. The value of the interaction distance is determined to be 0.6 micrometers by comparing with the observed frequency of translocations in the X-chromosome.

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 100 kV X-rays at 0.1 to 5 Gy. The proportions of X-irradiation-induced CD59 − mutants were quantified by flow cytometry after immunofluorescence labeling. Between 0.2 and 5 Gy 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 5 Gy of X-ray revealed a dose-dependent linear increase of large deletions (>6 Mbp), whereas, point mutations could be seen only in spontaneous CD59 mutants or after low-dose exposure (≤1 Gy). 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.