Radiation-induced transgenerational effects in animals.

According to the results of recent studies, parental exposure to ionizing radiation not only leads to mutation induction in the germline of irradiated animals but also affects their non-exposed offspring. These radiation-induced transgenerational effects belong to an epigenetic phenomenon that could not be defined as a transmission of altered phenotypes from the irradiated parents to their non-exposed offspring. In this review, we present the results of laboratory studies aimed to evaluate the transgenerational effects of parental irradiation on a number of traits in the offspring of exposed parents. The results of animal studies showing compromised viability, fertility and genome stability among the non-exposed offspring of irradiated parents are presented and discussed. So far, the epigenetic phenomenon of radiation-induced transgenerational effects has been established in laboratory studies. Future work should address the important issue of manifestation of radiation-induced transgenerational effects in populations inhabiting radioactive-contaminated areas, as well as the mechanisms of transgenerational effects.

From tangled banks to toxic bunnies; a reflection on the issues involved in developing an ecosystem approach for environmental radiation protection.

The objective of this paper is to present the results of discussions at a workshop held as part of the International Congress of Radiation Research (Environmental Health stream) in Manchester UK, 2019. The main objective of the workshop was to provide a platform for radioecologists to engage with radiobiologists to address major questions around developing an Ecosystem approach in radioecology and radiation protection of the environment. The aim was to establish a critical framework to guide research that would permit integration of a pan-ecosystem approach into radiation protection guidelines and regulation for the environment. The conclusions were that the interaction between radioecologists and radiobiologists is useful in particular in addressing field versus laboratory issues where there are issues and challenges in designing good field experiments and a need to cross validate field data against laboratory data and vice versa. Other main conclusions were that there is a need to appreciate wider issues in ecology to design good approaches for an ecosystems approach in radioecology and that with the capture of 'Big Data', novel tools such as machine learning can now be applied to help with the complex issues involved in developing an ecosystem approach.

The long-term effects of exposure to ionising radiation on gene expression in mice.

Despite great advancement in our understanding of the biological response to ionising radiation in mammals, a number of pertinent questions remain unanswered. For instance, the mechanisms underlying the long-term effects of acute radiation in vivo still eludes us. Here we report that acute exposure to X-rays in male mice significantly affects their transcriptome. Using microarrays and miRNA-sequencing, we profiled the gene expression pattern in the brain, the kidney, the liver and the sperm of irradiated and control from CBA/Ca and BALB/c in the timeline of 4 h, 24 h, 1 week and 10 weeks post-exposure. Acute exposure to 1 Gy of X-rays resulted in profound tissue- and strain-specific changes in gene expression pattern. There was profound change in the gene expression in the kidney of BALB/c irradiated mice over the period of 10 weeks after irradiation, whereas in the CBA/Ca strain the significant transcriptomic changes manifest over a shorter period of time up to 1 week post exposure. In the brain of irradiated CBA/Ca, significant changes in transcriptome were seen up to 10 weeks post-irradiation, while only short-term changes up to 4 h post-exposure was detected in the brain of irradiation BALB/c. Similarly, alteration in gene expression pattern was observed in the liver of irradiated BALB/c up to 10 weeks post-radiation, whereas only immediate but significant changes were observed in the CBA/Ca at 4 h post-irradiation. Furthermore, the analysis of miRNA in irradiated and control male mice also revealed highly tissue- and strain-specific changes in expression level, with no overlap between the differentially regulated miRNA genes across the three somatic tissues and the two inbred strains. We also analysed the pattern of miRNA expression in sperm of irradiated males, sacrificed at 24 h, 1 week and 10 weeks after irradiation. Only one miRNA (mmu-miR-217-5p) was significantly down-regulated in the CBA/Ca males. The results of our study may provide a plausible explanation for the delayed in vivo effects of irradiation.

The effects of DNA repair polymorphisms on chromosome aberrations in the population of Kazakhstan.

Purpose: To analyze the effects of DNA repair polymorphism and other factors on the frequency chromosome aberrations in an irradiated cohort of subjects living around the Semipalatinsk nuclear test site and non-exposed group of subjects from ecologically favorable zones of Kazakhstan.Materials and methods: Blood samples were collected in the rural areas of the East Kazakhstan district around the Semipalatinsk nuclear test site and ecologically favorable zones of Almaty region of Kazakhstan. Chromosome aberrations in the fresh and cryopreserved peripheral blood lymphocyte cultures were analyzed by Giemsa staining. Single nucleotide polymorphisms at eight DNA repair genes (XRCC1 rs1799782, XRCC1 rs25487, XRCC3 rs861539, ATM rs1801516, XPD rs1799793, XPD rs13181, APEX1 rs1130409, and hOGG1 rs1052133) were determined by PCR-RFLP method.Results: The age of donors and smoking significantly affected the frequency of chromosome aberrations among the irradiated and control subjects. In the irradiated and control cohorts, the frequency of chromosome aberrations was significantly increased in the heterozygous ATM rs1801516 (1853 Asp/Asn) individuals; for the rest of the loci no significant associations between polymorphism and the frequency of chromosome aberrations were detected.Conclusions: The age of donors, smoking, and the ATM rs1801516 polymorphism significantly affect the frequency of chromosome aberrations among individuals inhabiting contaminated area around the Semipalatinsk nuclear weapon test site, as well as among those inhabiting ecologically favorable zones of Kazakhstan.

Mutation Induction in Humans and Mice: Where Are We Now?

The analysis of mutation induction in human families exposed to mutagens provides the only source of reliable estimates of factors contributing to the genetic risk of human exposure to mutagens. In this paper, I briefly summarize the results of recent studies on the pattern of mutation induction in the human and mouse germline. The results of recent studies on the genome-wide effects of exposure to mutagens on mutation induction in the mammalian germline are presented and discussed. Lastly, this review also addresses the issue of transgenerational effects of parental exposure to mutagens on mutation rates in their non-exposed offspring, which are known as transgenerational instability. The possible contribution of transgenerational instability to the genetic risk of human exposure to mutagens is discussed.

The long-term effects of acute exposure to ionising radiation on survival and fertility in Daphnia magna.

The results of recent studies have provided strong evidence for the transgenerational effects of parental exposure to ionising radiation and chemical mutagens. However, the transgenerational effects of parental exposure on survival and fertility remain poorly understood. To establish whether parental irradiation can affect the survival and fertility of directly exposed organisms and their offspring, crustacean Daphnia magna were given 10, 100, 1000 and 10,000mGy of acute γ-rays. Exposure to 1000 and 10,000mGy significantly compromised the viability of irradiated Daphnia and their first-generation progeny, but did not affect the second-generation progeny. The fertility of F0 and F1Daphnia gradually declined with the dose of parental exposure and significantly decreased at dose of 100mGy and at higher doses. The effects of parental irradiation on the number of broods were only observed among the F0Daphnia exposed to 1000 and 10,000mGy, whereas the brood size was equally affected in the two consecutive generations. In contrast, the F2 total fertility was compromised only among progeny of parents that received the highest dose of 10,000mGy. We propose that the decreased fertility observed among the F2 progeny of parents exposed to 10,000mGy is attributed to transgenerational effects of parental irradiation. Our results also indicate a substantial recovery of the F2 progeny of irradiated F0Daphnia exposed to the lower doses of acute γ-rays.

Paternal irradiation perturbs the expression of circadian genes in offspring.

The circadian system represents a complex network which influences the timing of many biological processes. Recent studies have established that circadian alterations play an important role in the susceptibility to many human diseases, including cancer. Here we report that paternal irradiation in mice significantly affects the expression of genes involved in rhythmic processes in their first-generation offspring. Using microarrays, the patterns of gene expression were established for brain, kidney, liver and spleen samples from the non-exposed offspring of irradiated CBA/Ca and BALB/c male mice. The most over-represented categories among the genes differentially expressed in the offspring of control and irradiated males were those involved in rhythmic process, circadian rhythm and DNA-dependent regulation of transcription. The results of our study therefore provide a plausible explanation for the transgenerational effects of paternal irradiation, including increased transgenerational carcinogenesis described in other studies.

The genome-wide effects of ionizing radiation on mutation induction in the mammalian germline.

The ability to predict the genetic consequences of human exposure to ionizing radiation has been a long-standing goal of human genetics in the past 50 years. Here we present the results of an unbiased, comprehensive genome-wide survey of the range of germline mutations induced in laboratory mice after parental exposure to ionizing radiation and show irradiation markedly alters the frequency and spectrum of de novo mutations. Here we show that the frequency of de novo copy number variants (CNVs) and insertion/deletion events (indels) is significantly elevated in offspring of exposed fathers. We also show that the spectrum of induced de novo single-nucleotide variants (SNVs) is strikingly different; with clustered mutations being significantly over-represented in the offspring of irradiated males. Our study highlights the specific classes of radiation-induced DNA lesions that evade repair and result in germline mutation and paves the way for similarly comprehensive characterizations of other germline mutagens.

The effects of methyl-donor deficiency on the pattern of gene expression in mice.

SCOPE: Hepatocellular carcinoma is one of the most frequently occurring cancers in humans. Recent human and animal studies have provided strong evidence for the effects of dietary deficiency of methyl donors on the development of liver cancer. However, the mechanisms underlying the effects of methyl-group deficiency on cancer risk are not properly understood. METHODS AND RESULTS: Male BALB/c and CBA/Ca mice were maintained for 8 weeks on a synthetic diet lacking in choline and folic acid. Using microarrays, the pattern of gene expression was evaluated in their liver, kidney, and spleen. Methyl-donor deficient diet induced profound changes in gene expression in the liver of treated animals, whereas the effects of the methyl-deficient diet on the pattern of gene expression in the kidney and spleen were negligible. Methyl-donor dietary restriction induced strain-independent upregulation of genes involved in cellular proliferation in liver. CONCLUSION: The results of our study provide a plausible explanation of why diets lacking methyl donors can induce the development of liver cancers in rodents and humans.

Harnessing genomics to identify environmental determinants of heritable disease.

Next-generation sequencing technologies can now be used to directly measure heritable de novo DNA sequence mutations in humans. However, these techniques have not been used to examine environmental factors that induce such mutations and their associated diseases. To address this issue, a working group on environmentally induced germline mutation analysis (ENIGMA) met in October 2011 to propose the necessary foundational studies, which include sequencing of parent-offspring trios from highly exposed human populations, and controlled dose-response experiments in animals. These studies will establish background levels of variability in germline mutation rates and identify environmental agents that influence these rates and heritable disease. Guidance for the types of exposures to examine come from rodent studies that have identified agents such as cancer chemotherapeutic drugs, ionizing radiation, cigarette smoke, and air pollution as germ-cell mutagens. Research is urgently needed to establish the health consequences of parental exposures on subsequent generations.

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 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.

Exposure to anticancer drugs can result in transgenerational genomic instability in mice.

The genetic effects of human exposure to anticancer drugs remain poorly understood. To establish whether exposure to anticancer drugs can result not only in mutation induction in the germ line of treated animals, but also in altered mutation rates in their offspring, we evaluated mutation rates in the offspring of male mice treated with three commonly used chemotherapeutic agents: cyclophosphamide, mitomycin C, and procarbazine. The doses of paternal exposure were approximately equivalent to those used clinically. Using single-molecule PCR, the frequency of mutation at the mouse expanded simple tandem repeat locus Ms6-hm was established in DNA samples extracted from sperm and bone marrow of the offspring of treated males. After paternal exposure to any one of these three drugs, expanded simple tandem repeat mutation frequencies were significantly elevated in the germ line (sperm) and bone marrow of their offspring. This observed transgenerational instability was attributed to elevated mutation rates at the alleles derived from both the exposed fathers and from the nonexposed mothers, thus implying a genome-wide destabilization. Our results suggest that paternal exposure to a wide variety of mutagens can result in transgenerational instability manifesting in their offspring. Our data also raise important issues concerning delayed transgenerational effects in the children of survivors of anticancer therapy.

Age-related accumulation of mutations supports a replication-dependent mechanism of spontaneous mutation at tandem repeat DNA Loci in mice.

Expanded simple tandem repeat (ESTR) loci belong to the class of highly unstable loci in the mouse genome. The mechanisms underlying the very high spontaneous instability at these loci still remain poorly understood. Using single-molecule polymerase chain reaction, here we have compared the pattern of mutation accumulation in tissues with different proliferation capacities in male mice of age 12, 26, 48, and 96 weeks. In the nonproliferating brain, we did not observe any measurable age-related accumulation of ESTR mutations. In contrast, a highly elevated frequency of ESTR mutation was detected in the sperm samples taken from old mice; similar changes were also observed in the bone marrow tissue. The spectra of ESTR mutations accumulated in all tissues of young and old mice did not significantly differ. Taken together, these data clearly imply that spontaneous ESTR mutations occur almost exclusively in replication-proficient cells. To gain further insights into the mechanisms of ESTR mutation, we developed a stochastic model of age-related mutation accumulation. The observed spectra of ESTR mutants accumulated in the brain and sperm were fairly accurately approximated assuming the values of ESTR mutation rate, ranging from 0.01 to 0.04 per cell division. As these estimates dramatically exceed those for protein-coding genes and microsatellite loci, our data therefore suggest that ESTRs represent one of the most unstable loci in the mammalian genome. The results of our study also imply that ESTR loci can be regarded as a class of expanded microsatellites, with the mechanism of spontaneous mutation most probably attributed to replication slippage.

The effects of in utero irradiation on mutation induction and transgenerational instability in mice.

Epidemiological evidence suggests that the deleterious effects of prenatal irradiation can manifest during childhood, resulting in an increased risk of leukaemia and solid cancers after birth. However, the mechanisms underlying the long-term effects of foetal irradiation remain poorly understood. This study was designed to analyse the impact of in utero irradiation on mutation rates at expanded simple tandem repeat (ESTR) DNA loci in directly exposed mice and their first-generation (F(1)) offspring. ESTR mutation frequencies in the germline and somatic tissues of male and female mice irradiated at 12 days of gestation remained highly elevated during adulthood, which was mainly attributed to a significant increase in the frequency of singleton mutations. The prevalence of singleton mutations in directly exposed mice suggests that foetal irradiation results in genomic instability manifested both in utero and during adulthood. The frequency of ESTR mutation in the F(1) offspring of prenatally irradiated male mice was equally elevated across all tissues, which suggests that foetal exposure results in transgenerational genomic instability. In contrast, maternal in utero exposure did not affect the F(1) stability. Our data imply that the passive erasure of epigenetic marks in the maternal genome can diminish the transgenerational effects of foetal irradiation and therefore provide important clues to the still unknown mechanisms of radiation-induced genomic instability. The results of this study offer a plausible explanation for the effects of in utero irradiation on the risk of leukaemia and solid cancers after birth.

Single-molecule PCR analysis of germ line mutation induction by anticancer drugs in mice.

Understanding and estimating the genetic hazards of exposure to chemical mutagens and anticancer drugs in humans requires the development of efficient systems for monitoring germ line mutation. The suitability of a single-molecule PCR-based approach for monitoring mutation induction at the mouse expanded simple tandem repeat (ESTR) locus Ms6-hm by chemical mutagens and anticancer drugs has been validated. The frequency of ESTR mutation was evaluated in the germ line of male mice exposed to the well-characterized alkylating agent and mutagen, ethylnitrosourea, and four widely used anticancer drugs, bleomycin, cyclophosphamide, mitomycin C, and procarbazine. The dose-response of ethylnitrosourea-induced mutation was found to be very close to that previously established using a pedigree-based approach for ESTR mutation detection. Paternal exposure to the clinically relevant doses of bleomycin (15-30 mg/kg), cyclophosphamide (40-80 mg/kg), and mitomycin C (2.5-5 mg/kg) led to statistically significant, dose-dependent increases in ESTR mutation frequencies in the germ line of treated male mice. Exposure to procarbazine led to a maximal increase in mutation frequency at 50 mg/kg, with a plateau at the higher concentrations. The results of this study show that the single-molecule PCR technique provides a new and efficient experimental system for monitoring the genetic effects of anticancer drugs, capable of detecting increases in mutation rates at clinically relevant doses of exposure. In addition, this approach dramatically reduces the number of mice needed for the measurement of germ line mutation induction.

New methods for assessing male germ line mutations in humans and genetic risks in their offspring.

Germ line mutations resulting from chemical or radiation exposure are a particular problem in toxicology as they affect not only the exposed generation but also an infinite number of generations thereafter. Established methods to show that these mutations occur in an F1 or subsequent population require the use of a large number of progeny for statistical significance. Consequently, many thousands of animals have been used in the past. Such a use is no longer considered desirable and is also very expensive. Several new molecular techniques (including analysis of tandem repeats and randomly amplified polymorphic DNA) now provide alternative methods of assessment, which also allow the quantification of individual mutations in individual sperm cells. These can also be applied to human offspring, making extrapolation obsolete. The downside of these methods is that they effectively determine the mutation rate in certain regions of DNA and the relevance of these to diseases, particularly cancer, is not always apparent. Therefore, it must be assumed that an increase in mutation rates in these selected regions correlates with altered phenotype. However, disease types linked to changes in tandem repeat length indicate that these may act as relevant markers for the development of phenotypes. Further research and evaluation are required to more closely link changes in DNA with altered phenotype and validate the use of tandem repeats and randomly amplified polymorphic DNA in transgenerational genotoxicity testing. This paper introduces and compares recently developed methods to assess mutations in sperm due to stem cell damage.

The effects of Atm haploinsufficiency on mutation rate in the mouse germ line and somatic tissue.

Using single-molecule polymerase chain reaction, the frequency of spontaneous and radiation-induced mutation at an expanded simple tandem repeat (ESTR) locus was studied in DNA samples extracted from sperm and bone marrow of Atm knockout (Atm(+/-)) heterozygous male mice. The frequency of spontaneous mutation in sperm and bone marrow in Atm(+/-) males did not significantly differ from that in wild-type BALB/c mice. Acute exposure to 1 Gy of gamma-rays did not affect ESTR mutation frequency in bone marrow and resulted in similar increases in sperm samples taken from Atm(+/-) and BALB/c males. Taken together, these results suggest that the Atm haploinsufficiency analysed in our study does not affect spontaneous and radiation-induced ESTR mutation frequency in mice.

Evidence for alternative lengthening of telomeres in liposarcomas in the absence of ALT-associated PML bodies.

Immortalized and cancer cells maintain their telomeres by activation of a telomere maintenance mechanism (TMM). In approximately 85% of cancers telomerase is activated (TA) but in some tumours, in particular sarcomas, an alternative lengthening of telomeres (ALT) pathway is used. Liposarcomas are the most common soft-tissue sarcoma in adults and they activate ALT or telomerase with equal frequency, however no TMM has been identified in approximately 50% of liposarcomas. In our study, we have shown that instability at the minisatellite MS32, usually associated with ALT activation, aids the identification of liposarcomas that have recombination-like activity at telomeres in absence of ALT associated PML-bodies (APBs). Furthermore, using single molecule telomere analysis, we have detected complex telomere mutations directly in ALT positive liposarcomas and interestingly in some liposarcomas with an unknown TMM but high MS32 instability. We have shown by sequence analysis that some of these complex telomere mutations must arise by an inter-molecular recombination-like process rather than by deletion caused by t-loop excision or by unequal telomere-sister-chromatid-exchange (T-SCE), which is known to be elevated in ALT cell lines. Preliminary evidence also suggests that inter-molecular recombination events may be processed differently in liposarcomas with APBs compared to those without. In conclusion, we have shown for the first time, that some telomerase negative liposarcomas without APBs have other features associated with ALT, indicating that the incidence of ALT in these tumours has previously been under-estimated. This has major implications for the use of cancer treatments targeted at TMMs.

Paternal exposure to ethylnitrosourea results in transgenerational genomic instability in mice.

Recent data shows that the effects of ionizing radiation are not restricted to the directly exposed parental germ cells, but can also manifest in their nonexposed offspring, resulting in elevated mutation rates and cancer predisposition. The mechanisms underlying these transgenerational changes remain poorly understood. One of the most important steps in elucidating these mechanisms is to investigate the initial cellular events that trigger genomic instability. Here we have analyzed the effects of paternal treatment by ethylnitrosourea, an alkylating agent which is known to form specific types of DNA adducts, on the transgenerational effects in the first-generation (F1) offspring of exposed CBA/Ca and BALB/c male mice. Mutation rates at two expanded simple tandem repeat loci were significantly elevated in the F1 germline of both strains. Pre and postmeiotic exposures resulted in similar increases in mutation rate in the F1 germline. Within each strain mutation rates were equally elevated in the germline of male and female F1 offspring of the directly exposed males. The results of our study suggest that transgenerational instability is not attributed to a specific sub-set of DNA lesions, such as double strand breaks, and is most probably triggered by a stress-like response to a generalized DNA damage.