Frequency Of Radiation-induced Mutations Research Articles

Abstract PO-021: Single-cell DNA sequencing as a means to directly examine the size and frequency of radiation-induced mutations - An exploratory study

Abstract This study examines if single-cell DNA sequencing may be used to study the mutational effects of ionizing radiation. As the action of ionizing radiation is a stochastic process, each cell in an irradiated sample experiences its own unique radiation-induced DNA damage. As a result, conventional sequencing methods such as bulk cell sequencing cannot be used to identify individual mutations. In this work, Epstein-Barr virus (EBV) transformed B-lymphoblastoid cells were irradiated with 6 MV X-ray radiation using a medical linear accelerator. Four samples of cells from the same population (400,000 cells/ml) were exposed to sham irradiation (0 Gray [Gy]; control), 0.5 Gy, 1.5 Gy and 3.0 Gy respectively at a common dose rate of about 600 cGy/min. Irradiated samples were incubated for 24 hrs and subsequently underwent single-cell whole-genome DNA sequencing to characterize the radiation impact. Mutational profiles of approximately 500 cells, randomly selected from each sample, were individually analyzed and compared to identify the variation of several mutational parameters as a function of dose. We quantified the copy number variations (CNV) for each cell in our samples. Additionally, we segregated insertion CNVs and deletion CNVs and independently analyzed their dose dependences. We found that the total number of CNVs (insertion and deletion combined) increased with dose, and the number of deletion CNVs consistently increased most. We have repeated the experiment and a new round of single-cell DNA sequencing is underway. If confirmed, our results will demonstrate that the mutational effects of ionizing radiation may be examined directly using single-cell sequencing. Citation Format: Felix Mathew, Jonathan Yeo, Luc Galarneau, Norma Ybarra, Patricia Tonin, Yu Chang Wang, Ioannis Ragoussis, John Kildea. Single-cell DNA sequencing as a means to directly examine the size and frequency of radiation-induced mutations - An exploratory study [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-021.

Verification of a dose rate-responsive dynamic equilibrium model on radiation-induced mutation frequencies in mice

Purpose: We have proposed a mathematical model (WAM model) expressing increment of the dose-rate dependent mutation frequency caused by artificial radiations. In this model, it is defined that the pool of mutant cells in dynamic equilibrium in organisms. We verified the accuracy of the WAM prediction of mutation frequency in mice.Materials and methods: The theoretical values calculated by the WAM model were compared with the experimental values obtained from the large mouse genetics program at the Oak Ridge National Laboratory (ORNL).Results: Most of all the theoretical values in acute and chronic irradiation conditions nearly coincided with the experimental values. However, the theoretical value of the chronic conditions at the dose-rate of 0.8 R/min was significantly higher than its experimental value. This discordance was able to be minimized in the WAM assumption, when the period from the end of exposure to start mating was two weeks longer.Conclusions: As a result of comparison between experimental and theoretical data, the certainty of the WAM model was confirmed in mice and it was shown that the genetic influence varies depending on the dose-rate.

Deficiency of the DNA repair protein nibrin increases the basal but not the radiation induced mutation frequency in vivo

Nibrin (NBN) is a member of a DNA repair complex together with MRE11 and RAD50. The complex is associated particularly with the repair of DNA double strand breaks and with the regulation of cell cycle check points. Hypomorphic mutation of components of the complex leads to human disorders characterised by radiosensitivity and increased tumour occurrence, particularly of the lymphatic system. We have examined here the relationship between DNA damage, mutation frequency and mutation spectrum in vitro and in vivo in mouse models carrying NBN mutations and a lacZ reporter plasmid. We find that NBN mutation leads to increased spontaneous DNA damage in fibroblasts in vitro and high basal mutation rates in lymphatic tissue of mice in vivo. The characteristic mutation spectrum is dominated by single base transitions rather than the deletions and complex rearrangements expected after abortive repair of DNA double strand breaks. We conclude that in the absence of wild type nibrin, the repair of spontaneous errors, presumably arising during DNA replication, makes a major contribution to the basal mutation rate. This applies also to cells heterozygous for an NBN null mutation. Mutation frequencies after irradiation in vivo were not increased in mice with nibrin mutations as might have been expected considering the radiosensitivity of NBS patient cells in vitro. Evidently apoptosis is efficient, even in the absence of wild type nibrin.

P53-Dependent suppression of genome instability in germ cells

Radiation increases mutation frequencies at tandem repeat loci. Germline mutations in γ-ray-irradiated medaka fish (Oryzias latipes) were studied, focusing on the microsatellite loci. Mismatch-repair genes suppress microsatellite mutation by directly removing altered sequences at the nucleotide level, whereas the p53 gene suppresses genetic alterations by eliminating damaged cells. The contribution of these two defense mechanisms to radiation-induced microsatellite instability was addressed. The spontaneous mutation frequency was significantly higher in msh2−/− males than in wild-type fish, whereas there was no difference in the frequency of radiation-induced mutations between msh2−/− and wild-type fish. By contrast, irradiated p53−/− fish exhibited markedly increased mutation frequencies, whereas their spontaneous mutation frequency was the same as that of wild-type fish. In the spermatogonia of the testis, radiation induced a high level of apoptosis both in wild-type and msh2−/− fish, but negligible levels in p53−/− fish. The results demonstrate that the msh2 and p53 genes protect genome integrity against spontaneous and radiation-induced mutation by two different pathways: direct removal of mismatches and elimination of damaged cells.

Increase of Radiation-induced Mutation Frequency in Regenerating Mouse Liver

Increase of Radiation-induced Mutation Frequency in Regenerating Mouse Liver

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.

Dietary· supplements of antioxidants reduce hprt mutant frequency in splenocytes of aging mice

The level of spontaneous and gamma-radiation-induced mutations in the hypoxanthine-guanine phosphoribosyl-transferase (hprt) locus as well as the decrease in frequency of these mutations in mice of various age pretreated with dietary supplements of an antioxidant mixture (vitamins C, E, beta-carotene, rutin, selenium, zinc) were studied in splenocytes of young (8-14-week-old) and aged (102-110-week-old) male C57BL/6 mice. The frequency of spontaneous mutations in splenocytes of 102-110-week-old mice was higher by 68-88% than that in mice aged 8-14 weeks. On gamma-irradiation (0.5-5.0 Gy) of mice, the frequency of radiation-induced mutations (Vf assay) in aged mice was 2.3 to 3.6 times (depending on dose) higher than in young ones. Daily supplements of an antioxidant mixture to the diet of mice prior to irradiation showed an antimutagenic effect. The values of mutant frequency reduction factor (MFRF) for 14-110-week-old mice fed with dietary antioxidants during 6 weeks prior to gamma-irradiation with doses of 2.0 and 5.0 Gy were 5.4 and 3.7, respectively. The frequency of radiation-induced mutations prevented or not prevented by antioxidants was much higher in aged mice than in young ones.

The effects of cycloheximide and WR-1065 on radiation-induced repair processes: a mechanism for chemoprevention.

The effects of cycloheximide (CHX) and 2-[(aminopropyl)-amino]ethanethiol (WR-1065), each alone or in combination, on radiation-induced mutation induction at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus and cell killing were investigated using a Chinese hamster ovary (CHO) AA8 cell system. Treatment with CHX, a potent inhibitor of protein synthesis, at a concentration of 10 micrograms/ml administered 30 min prior to irradiation with 7.5 Gy had no effect on cell survival but did reduce the radiation-induced mutation frequency (per 10(6) survivors) from 106.5 +/- 8.8 (SEM) to 36.2 +/- 5.6 (SEM). Exposure of cells to 4 mM WR-1065 reduced the mutation frequency to 44.8 +/- 4.2 (SEM), but the combination of agents afforded no additional protection, that is 41.1 +/- 3.3 (SEM). The mechanism of action attributed to CHX in reducing mutation frequency is its ability to prevent the induction of an error-prone repair system. Split-dose radiation experiments, that is 8 Gy versus 4 Gy + 4 Gy separated by 3 h, were performed to evaluate and contrast the relative abilities of CHX and WR-1065, each alone or in combination, in affecting cell survival. Cycloheximide administered to cells 30 min before the first radiation dose and present throughout the 3 h incubation time prior to the second dose inhibited split-dose repair as evidence by a reduction in surviving fraction by 60% as compared with the value obtained for non-CHX-treated cells that were exposed to two equal doses of 4 Gy. Cells exposed to 4 mM WR-1065 immediately following the first 4 Gy radiation dose and then washed free 2.5 h before exposure to a second Gy dose, which was also followed by a 30 min exposure to WR-1065, increased the surviving fraction by 80% over the value obtained for cells not exposed to WR-1065 during their split-dose radiation treatment. When CHX treatment was combined with WR-1065 was abolished, that is surviving cell fraction was again reduced by approximately 60% as compared with untreated control groups. These results indicate that protein synthesis is required for WR-1065 to affect split-dose related repair processes. Presumably, the inhibition of the induction of an error-phone repair system by CHX would account for its effects on both resultant decreases in mutation frequency and cell survival. In contrast, WR-1065 and/or its disulfide metabolite appear to facilitate the efficacy and fidelity of such a repair system once it is induced.(ABSTRACT TRUNCATED AT 400 WORDS)

Induction of error-free DNA repair inEscherichia coli by heat shock or nutritional stress

The thermal induction of resistance to UV inEscherichia coli JE1011 has now been shown to depend on an error-free DNA repair process. Heat shock causes a simultaneous reduction in the UV-induced frequency of mutation to prototrophy of two nutritional markers. In contrast, induction of SOS repair by thymine starvation causes the expected increase in mutation frequency upon UV irradiation. Thiamine deprivation produces an effect similar to heat shock, leading to increased UV resistance and a large decrease in the radiation-induced mutation frequency. Thus, two very different types of stress are able to induce error-free DNA repair in these cells.

The effect of genotype on the radiation-induced mutation frequencies of enzyme-activity alleles in spermatogonia of the mouse

The effect of genotype on the radiation-induced mutation frequencies of enzyme-activity alleles in spermatogonia of the mouse

Studies on mutagen-sensitive strains of Drosophila melanogaster: VI. The effect of DNA-repair deficiencies in spermatids, spermatocytes and spermatogonia irradiated in N 2 or O 2

This study was aimed at ascertaining the extent to which paternal repair processes possibly deficient in mei-9 a, mei-41 D5 and mus-101 D1 genotypes would affect the recovery of radiation-induced recessive lethals in early spermatids, spermatocytes and spermatogonia. These germ cell stages were sampled in two 2-day broods from freshly hatched males, that were irradiated as 24-h old pupae in O 2, or N 2 followed by N 2 or O 2 post-treatment. Spontaneous mutation frequencies were higher in mei-9 and mei-41 males, and thus appropriate corrections were applied to the radiation data. Only with mei-9 males a clear and consistent increase of the radiation-induced mutation frequency was observed. The effect is somewhat more pronounced in brood B, presumably representing spermatogonia, than in brood A and is observed after radiation in either O 2 or N 2. The paternal repair process thus differs from the maternal one in that it also responds to radiation damage induced in O 2. The finding that, following irradiation under anoxia, post-treatment with O 2 (versus that with N 2), also lowers the mutation frequency in mei-9 males, indicates that the repair defect in mei-9 does not interfere with oxygen-dependent post-radiation repair. Thus there are two different paternal repair processes in these early stages of spermatogenesis: that is, one controlled by mei-9 and one depending on oxygen. Mei-41 and mus-101 do not appear to interfere with the paternal repair process. The frequency of translocations recovered from these stages was likewise not affected by mus-101.

Cell Contact during Expression of Radiation Mutation in Chinese Hamster V79 Spheroids

The influence of cell to cell contact during expression of radiation mutation at the HGPRT locus was examined using Chinese hamster V79 spheroids. Spheroids left intact for up to 6 days following 7.5 Gy (and then dissociated into single cells for selection in 6-thioguanine) showed no significant decrease in radiation-induced mutation frequency compared to cells of spheroids dissociated immediately following irradiation and passaged in monolayers during the expression interval. These results suggest that the intimate cell contact which occurs between cells in spheroids does not inhibit mutant expression. However, the cell selection process did appear to reduce mutation frequency when spheroids were left intact for 8 days of expression, or when spheroids received 10 Gy.

Mutagen sensitivities and mutator effects of MMS-sensitive mutants in neurospora

7 mus (mutagen-sensitive) mutants of Neurospora crassa, which are more sensitive to the toxic effects of MMS (methyl methanesulfonate) than wild-type, were investigated for cross-sensitivities to other mutagens and inhibitors. These mutants have recently been mapped in 5 new genes, mus-7 to mus-11, and mutant alleles from each gene were checked for their effects on mutation frequencies. It was found that mutants in 3 of these 5 genes showed radiation-induced mutation frequencies similat to wild-type. These included 2 alleles of the gene mus-10, which were cross-sensitive only to UV and were the only mutants that produce some viable ascospores in homozygous crosses. The mutant of the second gene, mus-8, was especially sensitive to UV and mitomycin C and produced slightly reduced frequencies of spontaneous mutation. In contrast, the mutant of the third gene, mus-7, was not UV-sensitive but showed some cross-sensitivity to X-rays; mus-7 was highly sensitive to MMS and also to histidine, which inhibits various repair-defective mutants at concentrations well below those that reduce wild-type growth. None of these mus resemble mutants previously found in Neurospora, nor do they conform clearly to mutant types identified in E. coli or yeast. On the other hand mutants in 2 further genes, mus-11, and especially 2 alleles of mus-9, are very similar to uvs-3 of Neurospora and generally resemble mutants that are considered to be defective in “error-prone” repair. They were UV- as well as X-ray-sensitive, and showed strong spontaneous mutator effects but almost no increase in recessive lethal frequencies in heterokaryons after UV-treatments.

ACTINOMYCIN D EFFECTS ON THE FREQUENCY OF RADIATION-INDUCED MUTATIONS IN DROSOPHILA

ACTINOMYCIN D EFFECTS ON THE FREQUENCY OF RADIATION-INDUCED MUTATIONS IN DROSOPHILA

Two types of dose-rate dependence of radiation-induced mutation rates in spermatogonia and oogonia of the silkworm.

Dose-rate dependence of radiation-induced mutation rate was studied in silk-work spermatogonia and oogonia by the use of the specific locus method. Both sexes of a wild-type strain were exposed to x rays, Cs/sup 137/ gamma -rays of high dose rate, or to Co/sup 60/ gamma -rays of low dose rate at a definite stage or during a definite period in early larval stages. The dose-rate ratios between the acute and chronic irradiations were 2500: 1 to 7600: 1. The total doses given were 950 r or 1000 r. Two types of dose-rate dependence of radiationinduced mutation rate were found. In one type mutagenic effectiveness of chronic irradiation is lower than that of acute irradiation (Type I) and in the other mutagenic effectiveness is higher in chronic irradiation than acute irradiation (Type II) The former is observed only in the very prevalent in the testis, while the latter is found when later stages of spermatogonia or oogonia are irradiated. For Type I dose-rate dependence, repair of primary mutations seems to the responsible, whereas for Type II dose-rate dependence selective killing of a specific type of cells by intense exposure seems to play an important role. The relationship between our finding andmore » that of Russell is discussed. Radiation-induced mutation frequency is remarkably high at the very early stage of larva, i.e., around the time of hatching when the primordial germ cells are prevalent in the gonads. (auth)« less

Impairment of Fertility by Whole-body Irradiation of Female Mice

Summary(1) A proper appreciation of ovarian damage by radiation is possible only if enough time is given for the damage to develop. Simple fertility is a less adequate measure of damage than reproductive capacity–the total number of young produced in the reproductive life-span. In C57BL mice this is halved by a single dose of 25 r of x-rays and by about 80 r of gamma-rays given at 2·2 r daily.(2) The high radiosensitivity of the ovary and the variation of somatic ovarian damage with dose-rate need to be taken into account when interpreting experimental results obtained in the female mouse, especially in studies of the effect of dose-rate on the frequency of radiation-induced mutations. It is possible that more attention should be given to somatic ovarian sensitivity when considering radiation hazards.