Genomic instability in the offspring of irradiated parents: facts and interpretations

This review is devoted to genomic instability in the offspring of parents that were irradiated or treated with chemical mutagens. The evidence is presented, showing high frequency of cancer diseases and instability of the genome of somatic and germline cells in the offspring of radiation-exposed animals. Possible epigenetic mechanisms of these effects are considered, as well as their significance as components of genetic factors of radiation risk for humans.     

Trans-generational radiation-induced chromosomal instability in the female enhances the action of chemical mutagens

Genomic instability can be produced by ionising radiation, so-called radiation-induced genomic instability, and chemical mutagens. Radiation-induced genomic instability occurs in both germinal and somatic cells and also in the offspring of irradiated individuals, and it is characterised by genetic changes including chromosomal rearrangements. The majority of studies of trans-generational, radiation-induced genomic instability have been described in the male germ line, whereas the authors who have chosen the female as a model are scarce. The aim of this work is to find out the radiation-induced effects in the foetal offspring of X-ray-treated female rats and, at the same time, the possible impact of this radiation-induced genomic instability on the action of a chemical mutagen. In order to achieve both goals, the quantity and quality of chromosomal damage were analysed.

In order to detect trans-generational genomic instability, a total of 4806 metaphases from foetal tissues from the foetal offspring of X-irradiated female rats (5 Gy, acute dose) were analysed. The study's results showed that there is radiation-induced genomic instability: the number of aberrant metaphases and the breaks per total metaphases studied increased and were found to be statistically significant (p ≤ 0.05), with regard to the control group.

In order to identify how this trans-generational, radiation-induced chromosomal instability could influence the chromosomal behaviour of the offspring of irradiated rat females in front of a chemical agent (aphidicolin), a total of 2481 metaphases were studied. The observed results showed that there is an enhancement of the action of the chemical agent: chromosomal breaks per aberrant metaphases show significant differences (p ≤ 0.05) in the X-ray- and aphidicolin-treated group as regards the aphidicolin-treated group.

In conclusion, our findings indicate that there is trans-generational, radiation-induced chromosomal instability in the foetal cells from X-ray-treated female rats and that this RIGI enhances the chromosomal damage caused by the chemical agent aphidicolin.     

Chromosome analysis in childhood cancer survivors and their offspring--no evidence for radiotherapy-induced persistent genomic instability

Suggestions that the induction of genomic instability could play a role in radiation-induced carcinogenesis and heritable disease prompted the investigation of chromosome instability in relation to radiotherapy for childhood cancer. Chromosome analysis of peripheral blood lymphocytes at their first in vitro division was undertaken on 25 adult survivors of childhood cancer treated with radiation, 26 partners who acted as the non-irradiated control group and 43 offspring. A statistically significant increase in the frequency of dicentrics in the cancer survivor group compared with the partner control group was attributed to the residual effect of past radiation therapy. However, chromatid aberrations plus chromosome gaps, the aberrations most associated with persistent instability, were not increased. Therefore, there was no evidence that irradiation of the bone marrow had resulted in instability being transmitted to descendant cells. Frequencies of all aberration categories were significantly lower in the offspring group, compared to the partner group, apart from dicentrics for which the decrease did not reach statistical significance. The lower frequencies in the offspring provide no indication of transmissible instability being passed through the germline to the somatic cells of the offspring. Thus, in this study, genomic instability was not associated with radiotherapy in those who had received such treatment, nor was it found to be a transgenerational radiation effect.     

The dose and dose-rate effects of paternal irradiation on transgenerational instability in mice: a radiotherapy connection

The non-targeted effects of human exposure to ionising radiation, including transgenerational instability manifesting in the children of irradiated parents, remains poorly understood. Employing a mouse model, we have analysed whether low-dose acute or low-dose-rate chronic paternal γ-irradiation can destabilise the genomes of their first-generation offspring. Using single-molecule PCR, the frequency of mutation at the mouse expanded simple tandem repeat (ESTR) locus Ms6-hm was established in DNA samples extracted from sperm of directly exposed BALB/c male mice, as well as from sperm and the brain of their first-generation offspring. For acute γ-irradiation from 10-100 cGy a linear dose-response for ESTR mutation induction was found in the germ line of directly exposed mice, with a doubling dose of 57 cGy. The mutagenicity of acute exposure to 100 cGy was more pronounced than that for chronic low-dose-rate irradiation. The analysis of transgenerational effects of paternal irradiation revealed that ESTR mutation frequencies were equally elevated in the germ line (sperm) and brain of the offspring of fathers exposed to 50 and 100 cGy of acute γ-rays. In contrast, neither paternal acute irradiation at lower doses (10-25 cGy), nor low-dose-rate exposure to 100 cGy affected stability of their offspring. Our data imply that the manifestation of transgenerational instability is triggered by a threshold dose of acute paternal irradiation. The results of our study also suggest that most doses of human exposure to ionising radiation, including radiotherapy regimens, may be unlikely to result in transgenerational instability in the offspring children of irradiated fathers.     

The offspring of irradiated parents, are they stable?

Mutation induction in directly exposed cells is currently regarded as the main component of the genetic risk of ionising radiation for humans. However, recent studies showing that exposure to ionising radiation results in elevated mutation rates detectable in the non-irradiated progeny of exposed cells challenge the existing paradigm in radiation biology. This review describes some recent data on radiation-induced genomic instability in vitro and mainly focuses on the in vivo phenomenon of transgenerational instability, where elevated mutation rates are detected in the non-exposed offspring of irradiated parents. The possible mechanisms and implications of transgenerational instability are also discussed.     

Non-Mendelian transmission in dentatorubral-pallidoluysian atrophy and Machado-Joseph disease: the mutant allele is preferentially transmitted in male meiosis.

Autosomal dominant dentatorubral-pallidoluysian atrophy (DRPLA) and Machado-Joseph disease (MJD) are neurodegenerative disorders caused by CAG trinucleotide repeat expansions. An inverse correlation of age at onset with the length of the expanded CAG trinucleotide repeats has been demonstrated, and the intergenerational instability of the length of the CAG trinucleotide repeats, which is more prominent in paternal than in maternal transmissions, has been shown to underlie the basic mechanisms of anticipation in DRPLA and MJD. Our previous observations on DRPLA and MJD pedigrees, as well as a review of the literature, have suggested that the numbers of affected offspring exceed those of unaffected offspring, which is difficult to explain by the Mendelian principle of random segregation of alleles. In the present study, we analyzed the segregation patterns in 211 transmissions in 24 DRPLA pedigrees and 80 transmissions in 7 MJD pedigrees, with the diagnoses confirmed by molecular testing. Significant distortions in favor of transmission of the mutant alleles were found in male meiosis, where the mutant alleles were transmitted to 62% of all offspring in DRPLA (chi2 = 7.69; P<.01) and 73% in MJD (chi2 = 6.82; P<.01). The results were consistent with meiotic drive in DRPLA and MJD. Since more prominent meiotic instability of the length of the CAG trinucleotide repeats is observed in male meiosis than in female meiosis and meiotic drive is observed only in male meiosis, these results raise the possibility that a common molecular mechanism underlies the meiotic drive and the meiotic instability in male meiosis.     

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.     

Fluctuating asymmetry in farmed Atlantic salmon (Salmo salar) juveniles: also a maternal matter?

Developmental stability reflects the degree to which phenotypic expression is unaffected by random accidents or developmental noise. Developmental stability may be measured by phenodeviance or fluctuating asymmetry (FA), and estimation of developmental stability has attracted substantial interest because it appears to represent a relatively simple method to identify sub lethal stress exposure and to assess animal welfare. As a part of a long-term study, the work presented here primarily aimed to investigate impacts on developmental instability in farmed salmon offspring ten months post hatch attributable to maternal cortisol administration prior to spawning and mild hyperthermia exerted during incubation. Main results show that maternal cortisol enhancement increased the level of FA in pectoral and pelvic fins, but did not affect the frequency of malformations in offspring. Mild hyperthermia during incubation increased weight and fork length and also increased pelvic fin FA. Malformed fish were heavier and longer than the normal ones, and pelvic fin asymmetry was positively related to condition factor. These results illustrate plausible lasting impacts on offspring development due to the maternal endocrinological state at spawning and indicate that developmental instability in farmed salmon juveniles may mirror aspects of the broodstock’s housing conditions.     

Parental age influences developmental stability of the progeny in Drosophila

The stochastic nature of biochemical processes is a source of variability that influences developmental stability. Developmental instability (DI) is often estimated through fluctuating asymmetry (FA), a parameter that deals with within-individual variation in bilateral structures. A relevant goal is to shed light on how environment, physiology and genotype relate to DI, thus providing a more comprehensive view of organismal development. Using Drosophila melanogaster isogenic lines, we investigated the effect of parental age, parental diet and offspring heterozygosity on DI. In this work, we have uncovered a clear relationship between parental age and offspring asymmetry. We show that asymmetry of the progeny increases concomitantly with parental age. Moreover, we demonstrate that enriching the diet of parents mitigates the effect of age on offspring symmetry. We show as well that increasing the heterozygosity of the progeny eliminates the effect of parental age on offspring symmetry. Taken together, our results suggest that diet, genotype and age of the parents interact to determine offspring DI in wild populations. These findings provide us with an avenue to understand the mechanisms underlying DI.     

Ectoparasitism in mothers causes higher positional fluctuating asymmetry in their sons: implications for sexual selection

Random deviations from perfect bilateral symmetry, fluctuating asymmetry, arise from developmental instability. I tested experimentally whether parasitism in female Drosophila nigrospiracula increases fluctuating asymmetry in male offspring. I also developed a novel measure for estimating developmental instability in a meristic trait called positional fluctuating asymmetry, which is based on the difference in the position of thoracic bristles between the right and left sternopleuron. I expected this measure to be a more sensitive indicator of developmental instability than the traditional numerical fluctuating asymmetry, because the latter is based on the difference in the actual presence or absence of bristles. Female flies burdened with hematophagous mites, Macrocheles subbadius (Macrochelidae), produced sons with significantly higher positional fluctuating asymmetry than did females carrying no mites. This effect, which may have resulted from impaired provisioning of oocytes by infested females, was dose dependent and magnified in the progeny of younger (18-20 d) versus older (30-32 d) females. This apparent magnification resulted from a slight but not significant increase in asymmetry of offspring of the older and unparasitized females. In contrast, the same mite loads had no effect on offspring numerical fluctuating asymmetry. If low-positional fluctuating asymmetry males enjoy a mating advantage, then with appropriate genetic variability, sexual selection could drive the evolution of host resistance in host populations. However, variability in neither kind of asymmetry influenced male mating success in nature. Thus, although male positional fluctuating asymmetry is causally associated with parasitism via maternal effects, asymmetry-based sexual selection is unlikely to influence the evolution of mite resistance in D. nigrospiracula. The value of the sensitivity afforded by positional fluctuating asymmetry is discussed in the context of sexual selection and conservation biology.     

Manipulating developmental stress reveals sex-specific effects of egg size on offspring phenotype

The general lack of experimental evidence for strong, positive effects of egg size on offspring phenotype has led to suggestions that avian egg size is a neutral trait. To better understand the functional significance of intra-specific variation in egg size as a determinant of offspring fitness within a life-history (sex-specific life-history strategies) and an environmental (poor rearing conditions) context, we experimentally increased developmental stress (via maternal feather-clipping) in the sexually size-dimorphic European starling (Sturnus vulgaris) and measured phenotypic traits in offspring across multiple biological scales. As predicted by life-history theory, sons and daughters had different responses when faced with developmental stress and variation in egg size. In response to developmental stress, small egg size in normally faster-growing sons was associated with catch-up growth prior to attaining larger adult size, resulting in a reduction in developmental stability. Daughters apparently avoided this developmental instability by reducing growth rate and eventual adult body mass and size. Interestingly, large egg size provided offspring with greater developmental flexibility under poor growth conditions. Large-egg sons and daughters avoided the reduction in developmental stability, and daughters also showed enhanced escape performance during flight trials. Furthermore, large egg size resulted in elevated immune responses for both sexes under developmental stress. These findings show that there can be significant, but complex, context-specific effects of egg size on offspring phenotype at least up to fledging, but these can only be demonstrated by appreciating variation in the quality of the offspring environment and life histories. Results are therefore consistent with egg size playing a significant role in shaping the phenotypic outcome of offspring in species that show even greater intra-specific variation in egg size than starlings.     

Offspring size–number tradeoffs and food quality feedbacks impact population dynamics in a Daphnia–algae system

Population fluctuations can be affected by both extrinsic (e.g. weather patterns, food availability) and intrinsic (e.g. life‐history) factors. A key life‐history tradeoff is the production of offspring size versus number, ranging from many small offspring to few large offspring. Models show that this life‐history tradeoff in offspring size and number, through maturation time, can have significant impacts on population dynamics. However, few manipulative experiments have been conducted that can isolate life‐history effects from impacts of extrinsic factors in consumer–resource systems. We experimentally tested the effect of an offspring size–number tradeoff on population stability and food availability in a consumer–resource system. Using Daphnia pulex, we created a shift from many, small offspring being produced to fewer, larger offspring. Two sets of experiments were performed to examine the interaction of an extrinsic factor (light levels) and intrinsic population structure on dynamics, and we controlled for the ingestion pressure on algal prey at the time of the manipulation. We predicted that the tradeoff would impact internal consumer population characteristics, including biasing the stage structure towards adults, increasing adult size, and increasing average population‐level reproduction. This adult‐dominated stage structure was predicted to then lead to instability and a low quantity–high quality food state. Under all light levels, the manipulated populations became dominated by large adults. Contrary to predictions, the amplitudes of fluctuations in Daphnia biomass were lower in populations shifted to few–large offspring, representing higher stability in these populations. Furthermore, in high light conditions, a stable low Daphnia – high algae biomass (low food quality) state was observed in few–large offspring treatments but not in control (many–small offspring) treatments. Our results show a strong link between light levels as an extrinsic factor and the life‐history tradeoff of consumer offspring size versus number that impacts consumer–resource population dynamics through feedbacks with resource quality.     

Genetic mechanisms of formation of radiation-induced instability of the genome and its transgenerational effects in the descendants of chronically irradiated individuals of Drosophila melanogaster

The article is devoted to the study of the role of intracellular mechanisms in the formation of radiation-induced genetic instability and its transgenerational effect in cells of different tissues of the descendants of Drosophila melanogaster mutant strains whose parents were exposed to chronic radiation (0.42 and 3.5 mGy/h). The level of DNA damage (alkali-labile sites (ALS), single-strand (SSB) and double-strand (DSB) breaks) in cells of somatic (nerve ganglia, imaginal discs) and generative (testis) tissues from directly irradiated animals and their unirradiated offspring was evaluated. Confident transgenerational instability (on the level of ALSs and SSBs), observed only in somatic tissues and only at the higher dose rate, is characteristic for mus209 mutant strains defective in excision repair and, less often, for mus205 and mus210 mutant strains. The greatest manifestation of radiation-induced genetic instability was found in evaluating the DSBs. Dysfunction of the genes mus205, mus304, mei-9 and mei-41, which are responsible for postreplicative repair, excision repair, recombination and control of the cell cycle, affects transgenerational changes in the somatic tissues of the offspring of parents irradiated in both low and high dose rates. In germ cells, the key role in maintaining genetic stability under chronic irradiation is played by the non-recombination postreplication repair mus101 gene. We revealed the tissue specificity of the radiation-induced effects, transgenerational transmission and accumulation of DNA damage to descendants of chronically irradiated animals.     

Phenotype-based identification of mouse chromosome instability mutants

There is increasing evidence that defects in DNA double-strand-break (DSB) repair can cause chromosome instability, which may result in cancer. To identify novel DSB repair genes in mice, we performed a phenotype-driven mutagenesis screen for chromosome instability mutants using a flow cytometric peripheral blood micronucleus assay. Micronucleus levels were used as a quantitative indicator of chromosome damage in vivo. Among offspring derived from males mutagenized with the germline mutagen N-ethyl-N-nitrosourea (ENU), we identified a recessive mutation conferring elevated levels of spontaneous and radiation- or mitomycin C-induced micronuclei. This mutation, named chaos1 (chromosome aberration occurring spontaneously 1), was genetically mapped to a 1.3-Mb interval on chromosome 16 containing Polq, encoding DNA polymerase theta. We identified a nonconservative mutation in the ENU-derived allele, making it a strong candidate for chaos1. POLQ is homologous to Drosophila MUS308, which is essential for normal DNA interstrand crosslink repair and is unique in that it contains both a helicase and a DNA polymerase domain. While cancer susceptibility of chaos1 mutant mice is still under investigation, these data provide a practical paradigm for using a forward genetic approach to discover new potential cancer susceptibility genes using the surrogate biomarker of chromosome instability as a screen.     

Induction of stem cell cycling in mice increases their sensitivity to a chemical leukaemogen: implications for inherited genomic instability and the bystander effect

Preconception paternal irradiation (PPI) modifies haemopoietic and stromal tissues of offspring and increases risk of generating lympho-haemopopietic malignancy if those offspring are then exposed to a leukaemogen. We hypothesised that this increased risk was related to inherited damage which had caused increased stem cell proliferation rates. To test for this link, in vivo, rapid stem cell proliferation was established by giving sub-lethal irradiation (3Gy gamma-rays) and allowing 3 days recovery. At this stage, 60% of haemopoietic spleen colony-forming units (CFU-S) were in DNA-synthesis, compared to <10% in unirradiated controls. Two groups of mice, unirradiated controls and irradiated animals, were then injected with 50mg/kg methyl nitrosourea (MNU) and observed daily for onset of lympho-haemopoietic malignancy. In a further control group of 60 mice, irradiated but not injected with MNU, only one leukaemia developed. In unirradiated controls, 20% of the mice developed malignancies between 3 and 8 months later: in the irradiated, MNU-treated groups, 95% developed malignancies between 2 and 7 months later. Thus, at least one powerful potentiating mechanism for induction of lympho-haemopoietc malignancy following inherited damage can be related to haemopoietic stem cell proliferation. Genomic instability is exposed by cell proliferation and has been implicated in this type of damage. However, a regulatory stromal microenvironment plays a part in inducing that proliferation. Thus, the microenvironment is the effective "bystander" which is thought to promote and amplify genomic instability, and thereby influence the induction of malignancy both in PPI offspring and in mice with induced stem cell proliferation.     

Bet‐hedging as a complex interaction among developmental instability,environmental heterogeneity,dispersal, and life‐history strategy

One potential evolutionary response to environmental heterogeneity is the production of randomly variable offspring through developmental instability, a type of bet‐hedging. I used an individual‐based, genetically explicit model to examine the evolution of developmental instability. The model considered both temporal and spatial heterogeneity alone and in combination, the effect of migration pattern (stepping stone vs. island), and life‐history strategy. I confirmed that temporal heterogeneity alone requires a threshold amount of variation to select for a substantial amount of developmental instability. For spatial heterogeneity only, the response to selection on developmental instability depended on the life‐history strategy and the form and pattern of dispersal with the greatest response for island migration when selection occurred before dispersal. Both spatial and temporal variation alone select for similar amounts of instability, but in combination resulted in substantially more instability than either alone. Local adaptation traded off against bet‐hedging, but not in a simple linear fashion. I found higher‐order interactions between life‐history patterns, dispersal rates, dispersal patterns, and environmental heterogeneity that are not explainable by simple intuition. We need additional modeling efforts to understand these interactions and empirical tests that explicitly account for all of these factors.     

Low level chromosome instability in embryonic cells of primary aneuploid mice

Karyotypic analyses of Down syndrome patients have identified a low level of chromosome mosaicism, suggesting that the primary aneuploid status of the cells promotes further chromosomal segregation errors. Sycp3-null female mice produce aneuploid oocytes, which after fusion with normal haploid sperm, result in offspring with systemic whole chromosome, aneuploid embryo cells. Using the Sycp3-null female as a model, we observe an increase in the number of embryonic cells at E7.0 that exhibit abnormal chromosomal bridges at the anaphas estage of mitosis. This result suggests that global changes in gene expression patterns resulting from primary aneuploidy can affect mitotic chromosome segregation, resulting in a low level of chromosomal instability. The increased level of chromosomal instability could in the absence of mitotic checkpoints, lead to chromosomal mosaicism within the adult organism, as seen in Down syndrome patients.     

Instability of the sexual continuum

Maynard Smith and Szathmary have posed the problem of demonstrating the conjectured instability of a continuum of sexual types with finite interbreeding. Here, I propose a model in which one can analyse exactly when and how the existence of the instability can depend on an Allee effect, and how the growth rate and typical scale of the unstable perturbations depend on the strength and range of competition, mating preference, fecundity and offspring variance due to Mendelian segregation and mutation. Instabilities of various kinds are shown to occur in the majority of parameter regimes. In short, the continuum often breaks up into incipient species.     

The effects of methyl-donor deficiency on mutation induction and transgenerational instability in mice

The results of recent human and animal studies have provided strong evidence for the epigenetic effects of a dietary deficiency of methyl donors such as folate, choline and methionine on cancer risk and some other common diseases. However, the mechanisms underlying the links between epigenetic alterations and disease remain elusive. To establish whether a methyl-donor deficient diet can result in long-term changes in mutation rate in treated animals and their offspring, BALB/c male mice were maintained for 8 weeks, from 4 weeks of age, on a synthetic diet lacking in choline and folic acid. Using single-molecule PCR, the frequency of mutation at the mouse expanded simple tandem repeat (ESTR) locus Ms6-hm was established in sperm samples of treated males, as well as in sperm and brain of their first-generation offspring. ESTR mutation frequency in the germline of males sacrificed immediately after treatment or sampled 6 and 10 weeks after the end of dietary restriction did not significantly differ from that in age-matched control groups. The frequency of ESTR mutation in DNA samples extracted from sperm and brain of the first-generation offspring of treated mice was also similar to that in controls. The results of our study suggest that the effects of a methyl-donor deficient diet on mutation induction and transgenerational instability in mice are likely to be negligible.     

Involvement of early embryonic miR‐409‐3p in the establishment of anxiety levels in female mice

Small RNA molecules in early embryos, delivered from sperm to zygotes upon fertilization, are required for normal mouse embryonic development. Even modest changes in the levels of sperm‐derived miRNAs appear to influence early embryos and subsequent development. For example, stress‐associated behaviors develop in mice after injection into normal zygotes sets of sperm miRNAs elevated in stressed male mice. Here, we implicate early embryonic miR‐409‐3p in establishing anxiety levels in adult female, but not male mice. First, we found that exposure of male mice to chronic social instability stress, which leads to elevated anxiety in their female offspring across at least three generations through the paternal lineage, elevates sperm miR‐409‐3p levels not only in exposed males, but also in sperm of their F1 and F2 male offspring. Second, we observed that while injection of a mimic of miR‐409‐3p into zygotes from mating control males was incapable of mimicking this effect in offspring derived from them, injection of a specific inhibitor of this miRNA led to the opposite, anxiolytic effect in female, but not male, and offspring. These findings imply that baseline miR‐409‐3p activity in early female embryos is necessary for the expression of normal anxiety levels when they develop into adult females. In addition, elevated embryo miR‐409‐3p activity, possibly as a consequence of stress‐induced elevation of its expression in sperm, may participate in, but may not be sufficient for, the induction of enhanced anxiety.