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Extremely low-frequency magnetic fields (ELF-MF) are classified as possibly carcinogenic, despite inconsistent data and no plausible biological mechanism linking their universal exposure with childhood leukaemia and genotoxic effects. Given discrepancies in mutagenic data and widespread public concern over genotoxic effects, this study was designed to provide an in-depth analysis of potential molecular changes induced by ELF-MF exposure in vivo. Seven-week old, BALB/c x CBA/Ca hybrid F1 male mice were exposed to 50 Hz magnetic fields of 10, 100 and 300 μT for 2- or 15 hours. Blood and sperm DNA samples were collected 12 weeks post-exposure and mutation induction frequencies established at the Expanded Simple Tandem Repeat (ESTR) Ms6-hm loci using single-molecule PCR (SM-PCR). Likewise, Ms6-hm mutation induction frequencies were established in age-matched sham-treated hybrid males (control group) and those exposed to 1 Gy acute X-rays (positive controls). No significant increases in ESTR mutation frequencies were detected in either tissue at any ELF-MF exposure parameter compared to their sham-treated controls. Whilst a marginally significant increase was observed in the mutation induction frequency of pooled sperm data, these data should be regarded cautiously due to the lack of correlating dose-dependent responses. Conversely, germline and somatic ESTR mutation frequencies were significantly elevated in males exposed to acute 1 Gy X-rays. These data were validated in a high-throughput microarray pilot study, whereby no significant alterations in gene expression in kidney cells of hybrid males were detected following ELF-MF exposure. In contrast, five transcripts were significantly up-regulated in the irradiated males. Ultimately, these findings indicate that, within the analysed range of doses, the in vivo effects of ELF-MF exposure on mutation induction and gene expression are likely to be negligible. This study represents the first methodical attempt to determine mutation frequencies in vivo after continuous exposure to 50 Hz ELF-MFs up to 300 μT.

The main source of data used to assess genetic risks of radiation exposure for humans has been derived from experiments analysing the male germline, while the effects of maternal irradiation remain poorly understood. This project therefore aims to analyse the long term genetic effects of acute maternal irradiation. To investigate the effects of acute irradiation on genome stability in the germline of directly exposed females, adult BALB/c and CBA/Ca mice were exposed to 1 Gy of acute X-rays and mated with control males 2-5 days following exposure, enabling analyses of offspring that were conceived from irradiated dictyate oocytes in maturing follicles. The data revealed that frequency of mutation at expanded simple tandem repeat (ESTR) loci in the germline of directly exposed females did not differ from that in control families. To address the effect of parental irradiation on transgenerational instability, ESTR mutation frequency was also established in the germline and somatic tissues of first-generation offspring of exposed adult males and females using single-molecule PCR. The breeding scheme used implied that the offspring of irradiated males and females were derived from irradiated post-meiotic stages spermatozoa and meiotically arrested dictyate oocytes in maturing follicles, respectively. While the frequency ESTR mutation in the offspring of irradiated males was significantly elevated, maternal irradiation did not affect the F1 stability. The results of this project therefore show that, in sharp contrast to the paternal exposure to ionising radiation, the transgenerational effects of maternal high-dose acute irradiation are likely to be negligible. The analysis of transcription profiles of first-generation offspring of irradiated males and females reveals drastically different patterns of gene expression profiles in both groups. Specifically, a substantial number of genes significantly deregulated in the offspring of irradiated males belong to functional groups directly involved in maintaining the stability of the genome. In contrast, in the offspring of irradiated females none of the significantly deregulated genes can be implicated in the maintenance of genome stability. The work presented here therefore provide new evidence for striking differences in the manifestation of long-term effects of paternal and maternal acute exposure to ionising radiation in mice.

Transgenerational instability is characterized by genome-wide increases in mutation rates observed in the somatic and germline tissues of non-exposed offspring of irradiated parents. An increasing body of experimental evidence from animal studies suggests that the phenomenon of transgenerational instability is attributed to the yet unknown epigenetic changes induced in the germline of irradiated animals and manifested in their offspring. To establish whether paternal irradiation could result in the transgenerational changes in gene expression, CBA/Ca and BALB/c male mice were given 1 Gy of acute X-rays and mated 10 weeks after exposure to control females from the same strain. RNA samples were extracted from kidney, liver, spleen and brain of the first-generation offspring of irradiated and control parents. They were hybridised to the NimbleGen 12x135K multiplex expression array, containing 135,054 features on the expression profiles and covering 42,575 known mouse transcripts. Highly significant changes in gene expression at the FDR < 0.05 were detected in 39 transcripts, showing the same pattern in all four tissues. The GO analysis revealed three overlapping pathways showing highly significant enrichment (P < 10[superscript -6]) – rhythmic process, circadian rhythm and DNA-dependent regulation of transcription. Differentially expressed genes of interest were further validated by qPCR. The pattern of DNA methylation of a number of genes was established by single-molecule and conventional bisulfite sequencing. Paternal irradiation caused no differences in DNA methylation of the genes analysed. Methylation can be transmissible through many cell divisions, however, the transcripts and size of expression change varied between tissue-types suggesting a more permissive/modifiable epigenetic alteration including, perhaps, the histone code.

Data from various recent human and animal studies link epigenetic changes caused by dietary deficiency of methyl donors like folate with many common human diseases, including cancer (Esteller, 2007; Feinberg, 2007). However, the mechanisms underlying the observed links between epigenetic alterations and disease remain elusive (Ames & Wakimoto, 2002; Davis & Uthus, 2004). Since the development of cancer is a multistep process in which somatic cells acquire mutations in a specific clonal lineage (Loeb et al., 2003), it appears plausible that hypomethylation following dietary restriction may destabilise the genome of treated animals. To determine whether a methyl-donor deficient diet can cause longterm changes in the mutation rates of treated animals, BALB/c male mice were kept on a synthetic diet lacking in choline and folic acid for a duration of 8 weeks, directly after weaning. Sperm tissue was collected and analysed either at the end of treatment or 6 and 10 weeks after the end of dietary restriction. Single-molecule PCR technique was used to establish the frequency of mutation at the mouse expanded simple tandem repeat (ESTR) locus Ms6-hm in the treated males as well as their age-matched control groups. The results of this study showed that 8-weeks of dietary restriction did not affect ESTR mutation rate in the germline of treated male mice. Given that the epigenetic landscape of mammalian cell is not fixed and undergoes massive reprogramming during development (Reik, 2007), it can potentially be affected by a variety of environmental factors (Cropley et al., 2006; Waterland et al., 2006). To investigate any transgenerational effects of methyl-donor deficiency on genome stability, sperm and brain tissues from the first-generation offspring of treated animals were also analysed. The frequency of ESTR mutation in the offspring of treated males did not significantly differ from that in controls, therefore implying the lack of measurable transgenerational effects of paternal dietary restriction. Taken together, the results of this study suggest that the effects of methyl-donor deficient diet on mutation induction and transgenerational instability in mice are likely to be negligible (Voutounou et al., 2012).

Of the non-targeted, delayed consequences of exposure to ionising radiation, genomic instability is a particular area of concern, especially with regard to its manifestation in the non-exposed offspring of irradiated parents. However, further analysis of these effects and their implications is mainly limited by our understanding of the underlying mechanisms and the lack of reliable data for humans. As of yet, transgenerational instability has only been consistently demonstrated in animal models using high, acute doses of ionising radiation (> 1 Sv). To investigate the effects of low-dose acute and low dose-rate (chronic) irradiation and whether or not they are capable of destabilizing the genomes of the unexposed offspring, we exposed male BALB/c mice to a range of γ-ray doses (10- 100 cGy) and dose-rates (chronic and acute), and mated them to unexposed BALB/c females 10 weeks post-irradiation. The mutation frequency at the Ms6-hm locus was established in DNA samples extracted from the sperm of directly exposed mice, as well as from the sperm and brains of their F1, using the single-molecule PCR technique. A linear dose-response was observed for direct exposure across the range of acute doses, with a doubling dose of 57 cGy. Furthermore, 100 cGy of acute γ-rays was shown to be more mutagenic than chronic exposure to the same accumulated dose. However, acute exposure to 10-25 cGy failed to manifest genomic instability in the derived offspring. This was also true of low dose-rate exposure to 100 cGy. Only acute paternal exposure to 50 and 100 cGy resulted in transgenerational instability, to a similar extent for both doses. Analogous results were found for both tissues. Taken together, this would imply the presence of a stress-like response where a threshold of acute dose determines the onset of transgenerational instability. Our results also suggest that children whose fathers are subject to most forms of human exposure to ionizing radiation would be safe from the effect.

Despite its benefits in cancer treatment, ionising radiation (IR) can induce a series of adverse acute and/or long term effects. Studies on A-bomb survivors and radiotherapy patients have shown that acute whole-body exposure results in an increased risk for radiation-induced Acute Myeloid Leukaemia (r-AML), a bone marrow (BM) malignancy; whereas local-radiotherapy patients run the risk of developing acute and/or long term normal tissue reactions. Irradiated BM cells manifest persistent radiation-induced genomic instability. BM is one of the most susceptible tissues to radiation-induced cancer and one of the most radiosensitive tissues, which proposes a link between cancer susceptibility, genomic instability and radiosensitivity. The exact mechanism by which exposure of BM cells to IR leads to malignant transformation is still unclear, but the non-targeted nature of radiation-induced damage and genomic instability could suggest that an epigenetic mechanism is also involved; and DNA methylation is a good candidate. This thesis investigated genetic and epigenetic factors that may be involved in biological responses to ionising radiation exposure. Stem cell frequency is genetically determined in mouse haemopoiesis and is possibly part of the equation that defines cancer risk. Global DNA methylation levels were assessed in control haemopoietic tissues and radiation-induced leukaemias. The methylation status of haemopoietic malignancies reflects that of their untransformed tissue. Additionally, DNA methylation levels of untransformed haemopoietic tissues were found to correlate with their relative radiosensitivity in vivo. In vivo responses to ionising radiation were found to be under the influence of both genetic and epigenetic factors, highlighting the complexity of such biological reactions.