AP-PCR assay of DNA alterations in the progeny of male mice exposed to low-level gamma-radiation

By comparative analysis of fingerprints of arbitrarily primed polymerase chain reaction (AP-PCR) products, DNA alterations in somatic cells of the progeny (F1 generation) of male mice chronically exposed to low-doses of gamma-radiation was investigated. Male BALB/c mice exposed to 10-50 cGy were mated with unirradiated females 15 days after irradiation. DNA was isolated from biopsies taken from tail tips of 2-month-old progeny. Preliminary AP-PCRs were carried out with 17 primers representing core sequences of micro- and/or minisatellites or their flanking oligonucleotides. Best quantitatively reproduced AP-PCR fingerprints of genomic DNA were obtained with one of these primers, a 20-mer oligonucleotide flanking the micro-satellite locus Atplb2 on mouse chromosome 11. Comparative analysis of individual fingerprints of AP-PCR products obtained on DNA templates from the progeny of irradiated and intact males revealed an increased variability of micro-satellite-associated sequences and an increased frequency of "non-parental bands" in DNA-fingerprints from the progeny of males chronically exposed to gamma-radiation 15 days before mating (at the postmeiotic stage of spermatogenesis). The results show that increased micro-satellite instability can be initiated by irradiation of the male parent to subsequently arise or be transmitted to the soma of the F1 generations.     

The increase in the variability of microsatellite-associated repeats in the genome of gamma-irradiated male mice is tissue specific

The arbitrarily primed polymerase chain reaction (AP-PCR) was used to measure the level of polymorphism of microsatellite (MCS)-associated repeating sequences of spleen, lung, and brain DNA in the F1 progeny of male BALB/c mice exposed to acute gamma-radiation at doses of 50 cGy and 200 cGy 15 days before mating with unirradiated females. The variability of MCS-associated sequences in the genome of brain and lung cells was higher as compared to the spleen cells of the progeny of unirradiated males. In the progeny of irradiated males, a 20% increase in MCS polymorphism of spleen DNA was found as an increase in the frequency of "non-parent" bands in DNA-fingerprints as against to the progeny of unirradiated males. Significant changes in this parameter were revealed for brain tissue and not for lung tissue only in the progeny of males exposed to 200 cGy. The results suggest a tissue-specific character of transmission of radiation-induced alterations in the genome of germ cells of male parents to the somatic cells of the progeny.     

Study of genome instability using DNA fingerprinting of the offspring of male mice subjected to chronic low dose gamma irradiation

By a polymerase chain reaction with an arbitrary primer (AP-PCR), the possibility of transmission of genome instability to somatic cells of the offspring (F1 generation) from male parents of mice exposed to chronic low-level gamma-radiation was studied. Male BALB/c mice 15 days after exposure to 10-50 cGy were mated with unirradiated females. Biopsies were taken from tale tips of two month-old offspring mice and DNA was isolated. The primer in the AP-PCR was a 20-mer oligonucleotide flanking the microsatellite locus Atp1b2 on chromosome 11 of the mouse. A comparative analysis of individual fingerprints of AP-PCR products on DNA-templates from the offspring of irradiated and unirradiated male mice revealed an increased variability of microsatellite-associated sequences in the genome of the offspring of the males exposed to 25 and 50 cGy. The DNA-fingerprints of the offspring of male mice exposed to chronic irradiation with the doses 10 and 25 cGy 15 days before fertilization (at the post-meiotic stage of spermatogenesis) showed an increased frequency of "non-parent bands". The results of the study point to the possibility of transmission to the offspring somatic cells of changes increasing genome instability from male parents exposed to chronic low-level radiation prior to fertilization.     

Detection of genomic instability in offspring of male mice chronically exposed to gamma-radiation by the "adaptive response" test

The genomic instability (GI) in somatic cells of the progeny (F1 generation) of male mice chronically exposed to low-dose gamma-radiation was studied by comparative analysis of chromosome damage. BALB/C male mice exposed to 0.1 Gy (0.01 Gy/day) and 0.5 Gy (0.01 and 0.05 Gy/day) were mated with unirradiated females 15 days after irradiation. For comparison of radiosensitivity, two-month-old males, the descendants of irradiated and unirradiated animals, were subjected to irradiation with a dose of 1.5 Gy (0.47 Gy/min) from a 60Co source. GI was revealed by the standard scheme of adaptive response. The experiments indicated that, by using the test "adaptive response", it is possible to detect the transition of gamma-radiation-induced genomic instability in sex cells of male parent into somatic cells of mice (F1 generation) either from changes in radiosensitivity or by the absence of the adaptive response induced by a standard scheme.     

Transgenerational transmission of radiation induced genomic instability

Stability of genome is one of the evolutionary important trait of cells. Various mutations (gene, chromosomal, genomic) as well as artificial manipulations with genomes (inbreeding, DNA transfection, introduction of Br-DU in DNA) cause the genetic instability. Ionizing radiation is known as the factor which induced instability of genome in late mitotic descendants of cells after in vitro and in vivo exposure. Radiation induced genetic instability can be transmitted through germline cells. On the cell level both types of radiation induced genomic instability are manifested in elevated frequency of mutations, chromosome aberrations, micronuclei, increased radiosensitivity, disappearance of adaptive response, changes in gene expression. In studies of 1970-1980 years clear evidences on the different morphological and functional injuries in tissues of irradiated organisms as well as in tissues of the progeny of exposed parents were obtained. On the organism level the instability of mitotic and of meiotic progeny of irradiated cells is resulted in increased risk of cancer and of other somatic diseases. It seems to be useful to review the earlier radiobiology literature where delayed and transgenerational effects of ionizing radiation on tissues and on organisms level were clearly shown in animals. For the estimation of pathogenic role of radiation induced genomic instability in humans, particularly in children of exposed parents the parallel study of the same human cohorts using clinical parameters and various characteristic of genomic instability seems to be very important.     

The problem of induced genome instability as the basis of the increased morbidity in children exposed to low-intensity radiation at low doses

The main results of the complex examination of the genome instability are presented in children constantly living on territories contaminated with radionuclides as a result of the accident at the CNPP (Novozybkov district, Bryansk region, 16-18 Ci/km2, 137Cs) and in children exposed to low-intensity radiation at different stages of ontogenetic development: children exposed to postnatal irradiation in 1986 (born before the accident), children exposed to intrauterine irradiation during the accident in 1986, children of irradiated parents born after the accident in 1987-1992 and in 1994-2000. In all examined groups of irradiated children increased frequencies of certain radiation-induced chromosome aberrations were observed as well as a reduced activity of unscheduled synthesis of genomic DNA in lymphocytes and peculiarities in individual heterozygosity of genes encoding structural and enzymatic proteins of blood. An increased radiosensitivity of lymphocyte genomes to testing in vitro irradiation and peculiarities in the dynamics of the frequencies of chromosome aberrations and sister chromatid exchanges in 3 cell generations were revealed in children from the contaminated areas. The data obtained suggest a systemic character of dysgenomic effects, the reality of induction of genome instability in the growing organism of children exposed to low-intensity radiation at low doses the expression of which is determined by individual genotypic features of the organism. Biological significance of the phenomenon of the post-radiation genome instability, its relation to the state of health and the pathogenetic role in the development of somatic pathology are postulated.     

Elevated Micronucleus Frequency in Bone Marrow Erythrocytes in the Progeny of Male Mice Exposed to Chronic Low-Dose Gamma Irradiation

The micronucleus frequency in bone marrow erythrocytes from the F1 progeny of male mice exposed to chronic low-dose -irradiation was determined. Male BALB/c mice were irradiated with 10, 25 and 50 cGy at dose rates of 1, 5, and 15 cGy/day and mated with unirradiated females on day 15 after irradiation. The obtained offspring had an elevated micronucleus frequency in bone marrow erythrocytes at the age of 2 months. This suggests the transmission of genome instability from damaged germ-line cells of irradiated male parents to somatic cells of the progeny.     

Transmission of genome damage from irradiated male rats to their progeny

The effect of gamma-radiation (3Gy) on slowly proliferating liver tissue of male rats and their progeny was investigated with respect to induction and duration of latent damage. The irradiation caused latent cytogenetic damage in the liver in irradiated males of the F(0) generation, which manifested itself in different ways during proliferation of hepatocytes induced by partial hepatectomy: a reduced proliferating activity, a higher frequency of chromosomal aberrations and a higher proportion of cells with apoptotic DNA fragments were observed, compared with non-irradiated rats. In the progeny of irradiated males (F(1) and F(2) generation), the latent genome damage manifested itself during regeneration of the liver after partial hepatectomy by similar, but less pronounced changes compared with those seen in irradiated males of the parental generation. This finding gave evidence of the transfer of part of the radiation-induced genome damage from parents to their offspring. Irradiation of F(1) and F(2) progeny of irradiated males (their total radiation load being 3 + 3 and 3 + 0 + 3 Gy, respectively) caused less change as irradiation of progeny of non-irradiated control males (their total radiation load being 0 + 3 and 0 + 0 + 3 Gy, respectively).     

Germline genomic instability in PCNA mutants of Drosophila: DNA fingerprinting and microsatellite analysis

PCNA participates in multiple processes of DNA metabolism with an essential role in DNA replication and intervening in DNA repair. Temperature-sensitive PCNA mutants of Drosophila (mus209) are sensitive to mutagens, impair developmental processes and suppress positional-effect variegation. To investigate the role of proliferating cell nuclear antigen (PCNA) in germline genomic stability, independent mus209-defective and mus209-normal lines were established and maintained over six generations. A time course study was carried out and general genomic alterations were analyzed in the progeny by using arbitrarily primed PCR (AP-PCR) and microsatellite analysis. The AP-PCR analysis has shown that a dysfunctional PCNA leads to germline genomic instability, being the amount of genomic alterations transmitted to the progeny directly related to the number of mus209^B1 mutant alleles. In addition, we have found that the frequency of genomic alterations tends to increase over successive generations. Surprisingly, the highest microsatellite instability was found in the heterozygous mus209-defective lines, suggesting a greater mutation rate in these individuals, in comparison with the homozygous mus209-defective lines. In conclusion, our results clearly indicate that PCNA is an important factor to maintain genomic stability in germinal cells, both in the overall genome and in simple repeated sequences. The implication of PCNA mutations in transgenerational genomic instability and related to cancer susceptibility is also discussed.     

Genome instability in the F1-progeny of mice irradiated by ionizing radiation as determined by micronucleus assay

The F1-progeny of BALB/c male mice chronically exposed to low-dose gamma-radiation (0.1; 0.25 and 0.5 Gy; dose rate 0.01 Gy/day) as well as the F1-progeny of females exposed to acute X-radiation (0.5; 1.0 and 2.0 Gy; dose rate 0.1 Gy/min) shown the significant elevated micronuclei frequencies in bone marrow erythrocytes, as compared to the F1-progeny of unirradiated males and females. The increase in the micronuclei frequency in the F1-progeny was determined by the dose of irradiation of parents. The values of elevated micronuclei frequency in the F1-progeny of chronically irradiated males and acutely irradiated females for a dose of 0.5 Gy were comparable. The micronuclei frequencies in the F1-progeny of irradiated females and males for this dose were in 1.5 and in 1.6 times higher than ones in the F1-progeny of unirradiated mice correspondingly. The results suggest the possibility of transfer of genome instability from irradiated parents to the somatic cells of the F1-progeny via non-lethally damaged germ cells of parents.     

Transgenerational genomic instability in the first generation offspring of mice exposed to low-intensity red and near-infrared irradiation in vivo

Transgenerational genomic instability in the first generation offspring of mice exposed to lowintensity infrared laser (632.8 nm) and light-emitting-diode infrared irradiation (850 nm) was investigated in vivo. It was found that the level of spontaneous damage in bone marrow according to the micronucleus test, the level of reactive oxygen species in whole blood, and the mass index of lymphoid organs in all of the descendants of irradiated mice did not increase. After additional X-ray exposure of the progeny at a dose rate of 1.5 Gy, a decrease in the level of damage and the absence of an adaptive response were revealed upon testing according to “radiosensitivity” and the radiation-induced adaptive-response scheme (0.1 + 1.5 Gy), respectively, compared to the descendants of nonirradiated mice. The rate of tumor growth in the offspring of irradiated mice did not differ from that in the descendants of nonirradiated mice, although inhibition of the tumor growth rate was observed in their irradiated parents. The survival rate after irradiation at a dose rate of 6.5 Gy did not differ from both the parents and the control.

     

Dose-dependence, sex- and tissue-specificity, and persistence of radiation-induced genomic DNA methylation changes

Radiation is a well-known genotoxic agent and human carcinogen that gives rise to a variety of long-term effects. Its detrimental influence on cellular function is actively studied nowadays. One of the most analyzed, yet least understood long-term effects of ionizing radiation is transgenerational genomic instability. The inheritance of genomic instability suggests the possible involvement of epigenetic mechanisms, such as changes of the methylation of cytosine residues located within CpG dinucleotides. In the current study we evaluated the dose-dependence of the radiation-induced global genome DNA methylation changes. We also analyzed the effects of acute and chronic high dose (5Gy) exposure on DNA methylation in liver, spleen, and lung tissues of male and female mice and evaluated the possible persistence of the radiation-induced DNA methylation changes. Here we report that radiation-induced DNA methylation changes were sex- and tissue-specific, dose-dependent, and persistent. In parallel we have studied the levels of DNA damage in the exposed tissues. Based on the correlation between the levels of DNA methylation and DNA damage we propose that radiation-induced global genome DNA hypomethylation is DNA repair-related.     

Activation of the DNA repair system in tissues of mice subjected to chronic gamma irradiation

Mice exposed to gamma-quanta during 47 and 82 days at a dose-rate of 1.3 mGy/h and cumulative doses of 1.45 and 2.54 Gy, respectively, were subsequently subjected to a single acute irradiation with a dose of 20 Gy. Repair of DNA damages induced by the acute exposure was shown to proceed in the brain, pulmonary and splenic tissues of chronically exposed mice more readily than in the tissues of mice not subjected to chronic irradiation. The data obtained indicate that the induced adaptive response activates DNA repair in tissues of mice exposed to long-term low-level radiation.     

The influence of chronic and of acute irradiation on the polymorphism of RAPD-markers in offspring for irradiated mice

On mice lines BALB/c and CBA/lac was performed the study of molecular-genetics effects in mice progeny after the chronic (dose rate -0.0017 Gy/day, total dose -0.36 Gy) and acute (dose range 1-3 Gy) exposure of y-radiation on the parents. For variability analysis was used technique of amplification DNA with series of random primers (RAPD-assay). Random primers were used as single primer and in mixture of ones. In this work were held the comparative analysis of the genetic radiosensitivity for stem spermatogonia and spermatides. After the acute exposure the dose dependence for levels of polymorphism of RAPD-markers were obtained. After the chronic irradiation, significant differences from control group were obtained only by use primers mixture M1. Comparative analysis of the genetic radiosensitivity of different stages of mice spermatogenesis are display is similar sensitivity of stem spermatogonia and spermatides after doses of irradiation 1 Gy and 3 Gy. Indicated that after irradiation by dose 2 Gy, spermatogonia are more sensitivity than spermatides.     

Indicators of hippocampal neurogenesis are altered by 56Fe-particle irradiation in a dose-dependent manner

The health risks to astronauts exposed to high-LET radiation include possible cognitive deficits. The pathogenesis of radiation-induced cognitive injury is unknown but may involve loss of neural precursor cells from the subgranular zone (SGZ) of the hippocampal dentate gyrus. To address this hypothesis, adult female C57BL/6 mice received whole-body irradiation with a 1 GeV/nucleon iron-particle beam in a single fraction of 0, 1, 2 and 3 Gy. Two months later mice were given BrdU injections to label proliferating cells. Subsequently, hippocampal tissue was assessed using immunohistochemistry for detection of proliferating cells and immature neurons. Routine histopathological methods were used to qualitatively assess tissue/cell morphology in the hippocampal formation and adjacent areas. When compared to controls, irradiated mice showed progressively fewer BrdU-positive cells as a function of dose. This observation was confirmed by Ki-67 immunostaining in the SGZ showing reductions in a dose-dependent fashion. The progeny of the proliferating SGZ cells, i.e. immature neurons, were visualized by doublecortin staining and were significantly reduced by irradiation, with the decreases ranging from 34% after 1 Gy to 71% after 3 Gy. Histopathology showed that in addition to cell changes in the SGZ, (56)Fe particles induced a chronic and diffuse astrocytosis and changes in pyramidal neurons in and around the hippocampal formation. The present data provide the first evidence that high-LET radiation has deleterious effects on cells associated with hippocampal neurogenesis.     

Computerized video time-lapse analysis of apoptosis of REC:Myc cells X-irradiated in different phases of the cell cycle

Asynchronous rat embryo cells expressing Myc were followed in 50 fields by computerized video time lapse (CVTL) for three to four cycles before irradiation (4 Gy) and then for 6-7 days thereafter. Pedigrees were constructed for single cells that had been irradiated in different parts of the cycle, i.e. at different times after they were born. Over 95% of the cell death occurred by postmitotic apoptosis after the cells and their progeny had divided from one to six times. The duration of the process of apoptosis once it was initiated was independent of the phase in which the cell was irradiated. Cell death was defined as cessation of movement, typically 20-60 min after the cell rounded with membrane blebbing, but membrane rupture did not occur until 5 to 40 h later. The times to apoptosis and the number of divisions after irradiation were less for cells irradiated late in the cycle. Cells irradiated in G(1) phase divided one to six times and survived 40-120 h before undergoing apoptosis compared to only one to two times and 5-40 h for cells irradiated in G(2) phase. The only cells that died without dividing after irradiation were irradiated in mid to late S phase. Essentially the same results were observed for a dose of 9.5 Gy, although the progeny died sooner and after fewer divisions than after 4 Gy. Regardless of the phase in which they were irradiated, the cells underwent apoptosis from 2 to 150 h after their last division. Therefore, the postmitotic apoptosis did not occur in a predictable or programmed manner, although apoptosis was associated with lengthening of both the generation time and the duration of mitosis immediately prior to the death of the daughter cells. After the non-clonogenic cells divided and yielded progeny entering the first generation after irradiation with 4 Gy, 60% of the progeny either had micronuclei or were sisters of cells that had micronuclei, compared to none of the progeny of clonogenic cells having micronuclei in generation 1. However, another 20% of the non-clonogenic cells had progeny with micronuclei appearing first in generation 2 or 3. As a result, 80% of the non-clonogenic cells had progeny with micronuclei. Furthermore, cells with micronuclei were more likely to die during the generation in which the micronuclei were observed than cells not having micronuclei. Also, micronuclei were occasionally observed in the progeny from clonogenic cells in later generations at about the same time that lethal sectoring was observed. Thus cell death was associated with formation of micronuclei. Most importantly, cells irradiated in late S or G(2) phase were more radiosensitive than cells irradiated in G(1) phase for both loss of clonogenic survival and the time of death and number of divisions completed after irradiation. Finally, the cumulative percentage of apoptosis scored in whole populations of asynchronous or synchronous populations, without distinguishing between the progeny of individually irradiated cells, underestimates the true amount of apoptosis that occurs in cells that undergo postmitotic apoptosis after irradiation. Scoring cell death in whole populations of cells gives erroneous results since both clonogenic and non-clonogenic cells are dividing as non-clonogenic cells are undergoing apoptosis over a period of many days.     

Radiation induced chromosomal instability in human T-lymphocytes

Chromosomal instability in proliferating mammalian cells is characterized by a persistent increase of chromosomal aberrations and rearrangements occurring de novo during successive cell generations. Recent results from many laboratories using a variety of cells and cytogenetic end points show that this phenotype can be induced by low as well as high LET irradiation. A typical feature of chromosomal instability in primary human G₀-lymphocytes exposed to γ-irradiation at both high dose rate (45 Gy h⁻¹) and low dose rate (0.024 Gy h⁻¹) is the appearance of novel aberrations in the clonal progeny of the irradiated cell, many generations after the exposure. The same phenotype was observed in lymphocytes that were allowed to recover for 5 days in G₀ after the radiation exposure, as well as in hprt-mutant T cell clones. These results demonstrate that neither the acute genotoxic stress caused by high dose rate as compared to low dose rate irradiation, nor a hypothesized conflict between mitogen induced growth stimulation and growth arrest due to radiation damage, seem to be critical conditions for the development chromosomal instability in these cells. In contrast to observations in other cells, no evidence of a persistent decrease of cloning ability was observed in the progeny of radiation-exposed human lymphocytes, and no alteration was observed in their sensitivity to a second radiation exposure. Furthermore, the frequency of CA-repeat length variation at three loci was not increased in the progeny of X-irradiated T cells as compared to non-irradiated cells, which indicates that microsatellite instability is not part of the chromosomal instability phenotype in human T-lymphocytes.     

Protecting the heritable genome: DNA damage response mechanisms in spermatogonial stem cells

Spermatogonial stem cells (SSCs) must maintain the integrity of their genome to prevent reproduction failure and limit the hereditary risk associated with transmission to the progeny. SSCs must therefore have robust response mechanisms to counteract the potentially deleterious effects of DNA damage, with DNA double-strand breaks (DSBs) representing the greatest threat to genomic integrity. Through in vivo analysis of the DNA damage response of SSCs within their physiological tissue context, we aimed to gain insights into the mechanisms by which SSCs preserve genome integrity. After whole-body irradiation of repair-proficient and repair-deficient (DNA-PK- and ATM-deficient) mice, the formation and rejoining of DSBs was analyzed in SSCs of testis compared with somatic cells of other tissues by enumerating γH2AX-, MDC1-, and 53BP1-foci. Caspase-3 and PARP-1 were used as markers for apoptotic cell death. Our results show that DNA damage response mechanisms in SSCs characterized by unique chromatin compositions are markedly different from those of somatic cells. In SSCs lacking compact heterochromatin, histone-associated signaling components of the DNA repair machinery are completely absent and radiation-induced DSBs are rejoined predominantly by DNA-PK-independent pathways, suggesting the existence of alternative repair mechanisms. As a complimentary mechanism characterized by low thresholds for ATM-dependent checkpoint activation, the differentiating progeny, but not the SSCs themselves, promote apoptosis in response to low levels of DNA damage. By evaluating SSCs within their stem cell niche, we show that DNA repair, cell-cycle checkpoints, and apoptosis function together to maintain the integrity of the heritable genome.     

Radiation-induced apoptosis in developing mouse retina exhibits dose-dependent requirement for ATM phosphorylation of p53

Ionizing radiation (IR) induces DNA breakage to activate cell cycle checkpoints, DNA repair, premature senescence or cell death. A master regulator of cellular responses to IR is the ATM kinase, which phosphorylates a number of downstream effectors, including p53, to inhibit cell cycle progression or to induce apoptosis. ATM phosphorylates p53 directly at Ser15 (Ser18 of mouse p53) and indirectly through other kinases. In this study, we examined the role of ATM and p53 Ser18 phosphorylation in IR-induced retinal apoptosis of neonatal mice. Whole-body irradiation with 2 Gy IR induces apoptosis of postmitotic and proliferating cells in the neonatal retinas. This apoptotic response requires ATM, exhibits p53-haploid insufficiency and is defective in mice with the p53S18A allele. At a higher dose of 14 Gy, retinal apoptosis still requires ATM and p53 but can proceed without Ser18 phosphorylation. These results suggest that ATM activates the apoptotic function of p53 in vivo through alternative pathways depending on IR dose.     

Spontaneous and bleomycin-induced genomic alterations in the progeny of Drosophila treated males depends on the Msh2 status. DNA fingerprinting analysis

Deficiency in DNA mismatch repair (MMR) confers instability of simple repeated sequences and increases susceptibility to cancer. Some of the MMR genes are also implicated in other repair and cellular processes related to DNA damage response. Supposedly, lack of their function can lead to a global genomic instability, besides microsatellite instability (MSI). To study the spontaneous and induced genomic instability in germ cells, related to the Msh2 status, DNA alterations in the progeny of individual crosses of Drosophila deficient in one or two copies of the Msh2 gene, were analysed by the arbitrarily primed polymerase chain reaction (AP-PCR). The results indicate that the progeny of homozygous parents for the normal Msh2 allele (+/+) presents a significantly lower frequency of genomic alterations than those from heterozygous (+/-) or mutant homozygous (-/-) parents. In addition, the DNA damage transmitted to the progeny, after the adult parental males were exposed to bleomycin, indicates that whereas the induction of mutations related to MSI depends on the lack of the Msh2 function, the induction of other mutational events may require at least one functional Msh2 allele. Thus, the results obtained with heterozygous individuals may have special relevance for cancer development since they show that a disrupted Msh2 allele is enough to generate genomic instability in germ cells, increasing the genomic damage in the progeny of heterozygous individuals. This effect is enhanced by mutagenic stress, such as occurs after bleomycin exposure.