Exposure to low-dose (56)Fe-ion radiation induces long-term epigenetic alterations in mouse bone marrow hematopoietic progenitor and stem cells.

There is an increasing need to better understand the long-term health effects of high-linear energy transfer (LET) radiation due to exposure during space missions, as well as its increasing use in clinical treatments. Previous studies have indicated that exposure to (56)Fe heavy ions increases the incidence of acute myeloid leukemia (AML) in mice but the underlying molecular mechanisms remain elusive. Epigenetic alterations play a role in radiation-induced genomic instability and the initiation and progression of AML. In this study, we assessed the effects of low-dose (56)Fe-ion irradiation on epigenetic alterations in bone marrow mononuclear cells (BM-MNCs) and hematopoietic progenitor and stem cells (HPSCs). Exposure to (56)Fe ions (600 MeV, 0.1, 0.2 and 0.4 Gy) resulted in significant epigenetic alterations involving methylation of DNA, the DNA methylation machinery and expression of repetitive elements. Four weeks after irradiation, these changes were primarily confined to HPSCs and were exhibited as dose-dependent hypermethylation of LINE1 and SINE B1 repetitive elements [4.2-fold increase in LINE1 (P < 0.001) and 7.6-fold increase in SINE B1 (P < 0.01) after exposure to 0.4 Gy; n = 5]. Epigenetic alterations were persistent and detectable for at least 22 weeks after exposure, when significant loss of global DNA hypomethylation (1.9-fold, P < 0.05), decreased expression of Dnmt1 (1.9-fold, P < 0.01), and increased expression of LINE1 and SINE B1 repetitive elements (2.8-fold, P < 0.001 for LINE1 and 1.9-fold, P < 0.05 for SINE B1; n = 5) were observed after exposure to 0.4 Gy. In contrast, exposure to (56)Fe ions did not result in accumulation of increased production of reactive oxygen species (ROS) and DNA damage, exhibited as DNA strand breaks. Furthermore, no significant alterations in cellular senescence and apoptosis were detected in HPSCs after exposure to (56)Fe-ion radiation. These findings suggest that epigenetic reprogramming is possibly involved in the development of radiation-induced genomic instability and thus, may have a causative role in the development of AML.

A single whole-body low dose X-irradiation does not affect L1, B1 and IAP repeat element DNA methylation longitudinally.

The low dose radioadaptive response has been shown to be protective against high doses of radiation as well as aging-induced genomic instability. We hypothesised that a single whole-body exposure of low dose radiation would induce a radioadaptive response thereby reducing or abrogating aging-related changes in repeat element DNA methylation in mice. Following sham or 10 mGy X-irradiation, serial peripheral blood sampling was performed and differences in Long Interspersed Nucleic Element 1 (L1), B1 and Intracisternal-A-Particle (IAP) repeat element methylation between samples were assessed using high resolution melt analysis of PCR amplicons. By 420 days post-irradiation, neither radiation- or aging-related changes in the methylation of peripheral blood, spleen or liver L1, B1 and IAP elements were observed. Analysis of the spleen and liver tissues of cohorts of untreated aging mice showed that the 17-19 month age group exhibited higher repeat element methylation than younger or older mice, with no overall decline in methylation detected with age. This is the first temporal analysis of the effect of low dose radiation on repeat element methylation in mouse peripheral blood and the first to examine the long term effect of this dose on repeat element methylation in a radiosensitive tissue (spleen) and a tissue fundamental to the aging process (liver). Our data indicate that the methylation of murine DNA repeat elements can fluctuate with age, but unlike human studies, do not demonstrate an overall aging-related decline. Furthermore, our results indicate that a low dose of ionising radiation does not induce detectable changes to murine repeat element DNA methylation in the tissues and at the time-points examined in this study. This radiation dose is relevant to human diagnostic radiation exposures and suggests that a dose of 10 mGy X-rays, unlike high dose radiation, does not cause significant short or long term changes to repeat element or global DNA methylation.

Characterization of non-CG genomic hypomethylation associated with gamma-ray-induced suppression of CMT3 transcription in Arabidopsis thaliana.

Ionizing radiation causes various epigenetic changes, as well as a variety of DNA lesions such as strand breaks, cross-links, oxidative damages, etc., in genomes. However, radiation-induced epigenetic changes have rarely been substantiated in plant genomes. The current study investigates whether DNA methylation of Arabidopsis thaliana genome is altered by gamma rays. We found that genomic DNA methylation decreased in wild-type plants with increasing doses of gamma rays (5, 50 and 200 Gy). Irradiation with 200 Gy significantly increased the expression of transcriptionally inactive centromeric 180-bp (CEN) and transcriptionally silent information (TSI) repeats. This increase suggested that there was a substantial release of transcriptional gene silencing by gamma rays, probably by induction of DNA hypomethylation. High expression of the DNA demethylase ROS1 and low expression of the DNA methyltransferase CMT3 supported this hypothesis. Moreover, Southern blot analysis following digestion of genomic DNA with methylation-sensitive enzymes revealed that the DNA hypomethylation occured preferentially at CHG or CHH sites rather than CG sites, depending on the radiation dose. Unlike CEN and TSI repeats, the number of Ta3, AtSN1 and FWA repeats decreased in transcription but increased in non-CG methylation. In addition, the cmt3-11 mutant showed neither DNA hypomethylation nor transcriptional activation of silenced repeats upon gamma irradiation. Furthermore, profiles of genome-wide transcriptomes in response to gamma rays differed between the wild-type and cmt3-11 mutant. These results suggest that gamma irradiation induced DNA hypomethylation preferentially at non-CG sites of transcriptionally inactive repeats in a locus-specific manner, which depends on CMT3 activity.

[The alterations in profiles of RAPD- and of ISSR-markers in progeny of the single gamma-irradiated mice of BALB/c strain].

Molecular-genetic effects in the offspring of BALB/c male mice exposed to single radiation doses of 1, 2 and 3 Gy were studied. Induced genetic variability was studied using such methods as assessment of variation RAPD- and ISSR-profiles. Comparative analysis of genetic radiosensitivity of stem spermatogonia and of spermatids is presented in the work. The frequency of changes in the patterns of the offsprings of irradiated mice was significantly different from the analogous parameters in the offsprings of the control group already at a dose of 1 Gy. Comparative analysis of genetic radiosensitivity at different stages of spermatogenesis revealed the similar sensitivity of spermatogonia and of spermatids at 1 and 3 Gy and a higer sensitivity of spematogonia at 2 Gy.

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.

Lack of spontaneous and radiation-induced chromosome breakage at interstitial telomeric sites in murine scid cells.

Interstitial telomeric sites (ITSs) in chromosomes from DNA repair-proficient mammalian cells are sensitive to both spontaneous and radiation-induced chromosome breakage. Exact mechanisms of this chromosome breakage sensitivity are not known. To investigate factors that predispose ITSs to chromosome breakage we used murine scid cells. These cells lack functional DNA-PKcs, an enzyme involved in the repair of DNA double-strand breaks. Interestingly, our results revealed lack of both spontaneous and radiation-induced chromosome breakage at ITSs found in scid chromosomes. Therefore, it is possible that increased sensitivity of ITSs to chromosome breakage is associated with the functional DNA double-strand break repair machinery. To investigate if this is the case we used scid cells in which DNA-PKcs deficiency was corrected. Our results revealed complete disappearance of ITSs in scid cells with functional DNA-PKcs, presumably through chromosome breakage at ITSs, but their unchanged frequency in positive and negative control cells. Therefore, our results indicate that the functional DNA double-strand break machinery is required for elevated sensitivity of ITSs to chromosome breakage. Interestingly, we observed significant differences in mitotic chromosome condensation between scid cells and their counterparts with restored DNA-PKcs activity suggesting that lack of functional DNA-PKcs may cause a defect in chromatin organization. Increased condensation of mitotic chromosomes in the scid background was also confirmed in vivo. Therefore, our results indicate a previously unanticipated role of DNA-PKcs in chromatin organisation, which could contribute to the lack of ITS sensitivity to chromosome breakage in murine scid cells.

Spontaneous and ultraviolet light-induced direct repeat recombination in mammalian cells frequently results in repeat deletion.

Recombination is enhanced by transcription and by DNA damage caused by ultraviolet light (UV). Recombination between direct repeats can occur by gene conversion without an associated crossover, which maintains the gross repeat structure. There are several possible mechanisms that delete one repeat and the intervening sequences (gene conversion associated with a crossover, unequal sister chromatid exchange, and single-strand annealing). We examined transcription-enhanced spontaneous recombination, and UV-induced recombination between neomycin (neo) direct repeats. One neo gene was driven by the inducible MMTV promoter. Multiple (silent) markers in the second neo gene were used to map conversion tracts. These markers are thought to inhibit spontaneous recombination, and our data suggest that this inhibition is partially overcome by high level transcription. Recombination was stimulated by transcription and by UV doses of 6-12J/m(2), but not by 18J/m(2). About 70% of spontaneous and UV-induced products were deletions. In contrast, only 3% of DSB-induced products were deletions. We propose that these product spectra differ because spontaneous and UV-induced recombination is replication-dependent, whereas DSB-induced recombination is replication-independent.

Radiation and germline mutation at repeat sequences: Are we in the middle of a paradigm shift?

Two assumptions are commonly made in the estimation of genetic risk: (1) that the seven specific loci in the mouse constitute a suitable basis for extrapolation to genetic disease in humans, and (2) that mutations are induced by radiation damage (energy-loss events leading to double-stranded damage) occurring within the gene and are induced linearly with dose, at least at low doses. Recent evidence on the mutability of repeat sequences is reviewed that suggests that neither of these assumptions is as well founded as we like to think. Repeat sequences are common in the human genome, and alterations in them may have health consequences. Many of them are unstable, both spontaneously and after irradiation. The fact that changes in DNA repeat sequences can clearly arise as a result of radiation damage outside the sequence concerned and the likely involvement of some sort of signal transduction process mean that the nature of the radiation dose response cannot be assumed. While the time has not come to abandon the current paradigms, it would seem sensible to invest more effort in exploring the induction of changes in repeat sequences after irradiation and the consequences of such changes for health.

Inactivation of 14-3-3sigma influences telomere behavior and ionizing radiation-induced chromosomal instability.

Telomeres are complexes of repetitive DNA sequences and proteins constituting the ends of linear eukaryotic chromosomes. While these structures are thought to be associated with the nuclear matrix, they appear to be released from this matrix at the time when the cells exit from G(2) and enter M phase. Checkpoints maintain the order and fidelity of the eukaryotic cell cycle, and defects in checkpoints contribute to genetic instability and cancer. The 14-3-3sigma gene has been reported to be a checkpoint control gene, since it promotes G(2) arrest following DNA damage. Here we demonstrate that inactivation of this gene influences genome integrity and cell survival. Analyses of chromosomes at metaphase showed frequent losses of telomeric repeat sequences, enhanced frequencies of chromosome end-to-end associations, and terminal nonreciprocal translocations in 14-3-3sigma(-/-) cells. These phenotypes correlated with a reduction in the amount of G-strand overhangs at the telomeres and an altered nuclear matrix association of telomeres in these cells. Since the p53-mediated G(1) checkpoint is operative in these cells, the chromosomal aberrations observed occurred preferentially in G(2) after irradiation with gamma rays, corroborating the role of the 14-3-3sigma protein in G(2)/M progression. The results also indicate that even in untreated cycling cells, occasional chromosomal breaks or telomere-telomere fusions trigger a G(2) checkpoint arrest followed by repair of these aberrant chromosome structures before entering M phase. Since 14-3-3sigma(-/-) cells are defective in maintaining G(2) arrest, they enter M phase without repair of the aberrant chromosome structures and undergo cell death during mitosis. Thus, our studies provide evidence for the correlation among a dysfunctional G(2)/M checkpoint control, genomic instability, and loss of telomeres in mammalian cells.

Induction of transcription from the long terminal repeat of the intracysternal particles type A (IAP) by X-irradiation.

Intracisternal A particles (IAPs) are retrovirus-like entities that are present in many embryonic and transformed cells of Mus musculus. They present long terminal repeats (LTRs) which control the promotion and regulation of their transcription. Using a construction expressing a reporter gene under the control of the entire long terminal repeat (LTR) of IAP in transfected murine fibroblast BALB/c 3T3 cells clone D152, we were able to show that the IAP-LTR is activated by X-irradiation in a time-dependent manner. The relative CAT activity increased with increasing X-irradiation doses, reaching a maximum at 75-150 cGy, followed by a drop in activation. In addition, X-induced D152 mouse cells produced extracellular factor(s), in response to X-irradiation, which activated the IAP-LTR in non-irradiated cells. This factor(s) was detected both when transfected cells were cocultured with inducing cells and when conditioned medium from irradiated cultures was added to the cell cultures. The use of suramin, a strong polyanonic molecule which has been reported to trap growth factors, induces a high reduction of the indirect activation.

X-irradiation activates the Drosophila 1731 retrotransposon LTR and stimulates secretion of an extracellular factor that induces the 1731-LTR transcription in nonirradiated cells.

Using constructs expressing the reporter gene under the control of the entire or deleted long terminal repeats (LTRs) of 1731, a Drosophila melanogaster retrotransposable element, we show that 1731-LTR is activated by X-irradiation in a dose- and time-dependent manner, and that a sequence located in the U3 region of these LTRs is required. The cis-acting element conferring X-responsiveness shows similarities to kappa B (kappa B)-like binding sequence. In response to X-irradiation, S2 Drosophila cells produced an extracellular factor which activates the 1731-LTR in nonirradiated cells. This factor was detected both when transfected cells were cocultured with inducing cells and when a conditioned medium taken from irradiated cultures was added.

Upregulation of the Drosophila 1731 retrotransposon long-terminal repeat by UV-B irradiation requires a short sequence in the U3 region.

A 1731 is a Drosophila melanogaster retrotransposon, the transcripts of which decrease in Drosophila cells after treatment by the 20-hydroxyecdysone (20-OH), the steroid-molting hormone of insects. In order to analyze the regulation of the long terminal repeat (LTR) directed transcription by UV-B, S2 Drosophila cells were transfected with various chimeric constructs carrying the LTR of 1731 linked to the bacterial chloramphenicol acetyltransferase (CAT) reporter gene and then subjected to UV-B irradiation. The results demonstrated that the 1731 LTR is activated by UV-B irradiation in a dose- and time-dependent manner. Using constructions expressing the reporter gene under the control of either the entire or deleted LTRs of 1731, we established that a sequence located in the U3 region was required for the retrotransposon to respond to UV-B. The cis-acting element is identical to the binding sequence of the dorsal transcription factors. In addition, the S2 Drosophila cell produced extracellular factor(s) in response to UV-B irradiation which activated the 1731 LTR in nonirradiated cells. This factor(s) was detected when responding cells were cocultured with inducing cells and when conditioned medium from irradiated cultures was added to the cell cultures.

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 induction of germline mutation at a hypervariable mouse minisatellite locus.

Paternal 60Co gamma-irradiation was tested for the induction of germline mutation at the mouse hypervariable minisatellite locus, Ms6hm. Male C3H/HeN mice were exposed to 3 Gy 60Co gamma-ray and mated with C57BL/6N females. Matings were made at 1-7, 15-21 and 71-77 days post-treatment to test spermatozoa, spermatids and spermatogonia stages. Reciprocal crosses were also made with irradiated C57BL/6N males. Southern analysis was carried out on DNA from parents and F1 mice. The paternal mutation frequencies per gamete of the Ms6hm locus were 8.3, 13, 28 and 15% for the C3H/HeN control, exposed spermatozoa, spermatids and spermatogonia stages, respectively. The paternal mutation frequencies per gamete were 7.7% for the C57BL/6N control and 13% for the C57BL/6N exposed spermatozoa stage. The increase in the paternal germline mutation frequency was statistically significant for C3H/HeN spermatids irradiation (p < 0.005). The induced mutation frequencies were of the order of 10(-1), and was too high to be accounted for by the direct action of radiation on the locus. These results suggest the presence of a previously unexpected mechanism of radiation induction of germline mutation. In addition, we demonstrate that the hypervariable minisatellite locus can serve as a sensitive monitor for genetic damages to germline cells.

Sequence specificity of cyclobutane pyrimidine dimers in DNA treated with solar (ultraviolet B) radiation.

Cyclobutane pyrimidine dimers were quantified at the sequence level after irradiation with solar ultraviolet (UVB) and nonsolar ultraviolet (UVC) light sources. The yield of photoproducts at specific sites was dependent on the nucleotide composition in and around the potential lesion as well as on the wavelength of ultraviolet light used to induce the damage. Induction was greater in the presence of 5' flanking pyrimidines than purines; 5' guanine inhibited induction more than adenine. UVB irradiation increased the induction of cyclobutane dimers containing cytosine relative to thymine homodimers. At the single UVC and UVB fluences used, the ratio of thymine homodimers (T mean value of T) to dimers containing cytosine (C mean value of T, T mean value of C, C mean value of C) was greater after UVC compared to UVB irradiation.

[Molecular mechanisms of interphase death of lymphoid cells. Distribution of nuclease attack sites in chromatin and kinetic complexity of DNA in PDN]. / Molekuliarnye mekhanizmy interfaznoi gibeli limfoidnykh kletok. Kharakter raspredeleniia mest nukleaznoi ataki v khromatine i kineticheskaia slozhnost' DNK v sostave PDN.

On the basis of a quantitative correlation between single--and double-helix fragments of different length within PDN, a study was made of the pattern of distribution of the sites, attacked by nuclease, in the chromatin. It was shown that under the effect of ionizing radiation products of enzymic digestion of the chromatin. It was shown that under the effect of ionizing radiation products of enzymic digestion of the chromatin accumulated in thymocytes due to internucleosome degradation were excised from randomly localized genome sites. The analysis of the reassociation curves did not reveal distinctions in the kinetic complexity of the fractions of PDN and total DNA.