To date, only few studies have addressed the effects of LDR radiation within a range which is of human relevance as it can be found after accidents like in Chernobyl and Fukushima. Even fewer have investigated the biological effects of continuous chronic LDR radiation despite their relevance in occupational and environmental contamination settings. This is likely due to the limited number of animal facilities allowing long term in vivo exposure to IR. Studies on genotoxic effects caused by Se depletion starting in utero are also scarce. Due to the central role of Se in the antioxidative defence system (Gpx), low Se levels may increase the susceptibility to radiation-induced ROS damaging the DNA or other macro-molecules. The current study is the first mouse study conducted in the newly established γ exposure facility Figaro where continuous and chronic low doses can be explored. To our knowledge this is also the first study to investigate genotoxic effects on the combined stressors LDR γ radiation and Se deficiency. In the following, the effects of the factors radiation, diet, and genotype are discussed separately prior to a discussion of their combined effects.

In the present study we demonstrate that exposure to a human relevant LDR γ radiation induces genotoxic effects in mouse blood cells assessed with three separate but complementary assays. These effects were expressed as increased levels of chromosomal damage (micronuclei), phenotypic mutations (RBCCD24−) and DNA lesions (ssb/als). The absolute measured changes were small, but significant. The formation of MN was observed in all irradiated groups independent of genotype or diet, and significant changes were seen in both immature and mature erythrocytes. This is an expected result given the chronic exposure and lack of splenic filtration of circulating MN-containing erythrocytes18.

Existing data on mutagenicity of LDR radiation is limited and the results in the literature are not consistent. Osipov and colleagues reported a genotoxic effect following very LDR exposure of mice with increased MN frequency in bone marrow cells and increased levels of ssb/als in spleen cells, applying a 20-fold lower dose rate (0.07 mGy/h) and 7-fold lower accumulated dose (70–200 mGy) compared to the present study design9. In contrast, Olipitz and colleagues did not observe increased levels of base lesions in spleen cells or micronucleated erythrocytes (applying a dose rate of 0.102 mGy/h and total dose of 105 mGy)5. Relative to the two above-mentioned reports which used microscopic analysis to detect MN, the current study benefitted from automated scoring using a flow cytometer. This facilitated objective analysis of 20,000 reticulocytes and hundreds of thousands of erythrocytes per mouse (compared to some thousand cells using the microscopic analysis), thereby providing greater statistical power to detect weak effects of the magnitude described herein. Wickliffe and colleagues exposed BigBlue® C57BL/6 hemizygous mice in the Red Forest area of Chernobyl, achieving the same LDR as in the present study (1.4 mGy/h)6. Even though the exposure time was twice as long as that of our study (90 days of irradiation), no increased levels of mutation frequencies were detected with the conventional lacI transgenic rodent mutation assay. However, the number of putative mutant clones assessed was low, reducing the possibility of identifying a genotoxic effect. In our study, we assessed mutations by the Pig-a gene mutation assay based on flow cytometry. There are only few studies exploring the mutagenic effect of ionising radiation by the Pig-a gene mutation assay19,20,21 applying acute exposure with x-rays, and not chronic exposure with LDR γ-rays as done in the present study. Significant increases of mutation frequencies were detected in RETCD24− and RBCCD24− after 1 Gy total dose (C57BL/6J mice)20,21 and in RBC CD24− after 2 Gy total dose (F344 rats)19 of acute x-ray exposure.

It has been estimated that one Gy of γ radiation as used herein causes 1000 ssb, 500 base damages, 40 double strand breaks (dsb) and 150 DNA protein cross-links in one mammalian cell (p. 351 in ref. 22). Based on this value and a simplistic linear extrapolation, our applied dose rate of 1.4 mGy/h would be expected to cause approximately 34 ssb per cell per day. This seems to be a negligible amount compared to the approximately 200,000 spontaneously induced ssb a mammalian cell has to cope with every day23. However, radiation-induced DNA lesions are more complex than endogenous DNA lesions24 and can therefore cause an overload of the natural repair capacity and potentially become deleterious over time25. Ionising radiation also produces free radicals that can cause clustered DNA lesions. These types of lesions occur when at least two radical hits occur within 20 base pairs26 and are assumed to contribute to the harmful effect of radiation. This is partly due to the compromised repair of such lesions and partly because IR causes a broad spectrum of DNA lesions27. The repair process depends strongly on the modified DNA components within the cluster. It has been suggested that dsb may be formed during the repair process of these clusters, contributing to mutagenesis28. In fact, in pro- and eukaryotic cells it has been shown that DNA clusters are prone to result in dsb29,30. Hence, this might explain the increased mutation frequencies that we have observed in some irradiated mice. However, studies on clustered DNA lesions have been using acute and high dose rates of ionising radiation. D. P. Hayes suggests that at a low dose/dose rate (as applied in this study) repair mechanisms might not be activated resulting in elimination of the cell by apoptotic or mitotic death31.

Interestingly, at day 90 (45 days after cessation of radiation) the levels of ssb/als in irradiated mice were approximately 50% lower than in non-irradiated mice (Fig. 5). A constant exposure to a stressor (e.g. LDR γ radiation) may not induce DNA repair when the total dose stays below a threshold level, as suggested in in vitro systems (eukaryotes)32 and in fish33. On the other hand, defence mechanisms (e.g. DNA repair mechanisms) might kick in when a certain total dose is approached34. Hence, we assume that the observed effect in our study could potentially reflect an induction of a protective response towards irradiation.

Se is an essential trace element which is, for example, incorporated in enzymes (e.g. GPx) as selenoprotein catalysing ROS-eliminating reactions (e.g. reducing H 2 O 2 and lipid hydroperoxides). An inadequate Se intake was therefore hypothesised to cause additional oxidative stress when combined with LDR γ radiation. Mice were fed a diet made out of locally grown wheat (resulting in Se deficient diet) or wheat fertilized with Se (normal Se diet) for two generations, thus assuring a complete Se depletion. The Se content in the diet had a noteworthy effect on the level of DNA lesions in blood measured at the end of γ exposure in mice (at day 45). The levels of ssb/als and oxidised DNA lesions (i.e. Fpg-sensitive sites, Fpg-ss) were marginal reduced in mice fed the Se deficient diet (p < 0.001 and p = 0.004, respectively). This observation was surprising since Se has a major role in the antioxidative defence system and a raised level of (oxidised) DNA lesions would have been expected. However, measurements of the GPx-activity in plasma (i.e. one part of the antioxidative defence system) were significantly reduced in the groups fed with low Se diet. It is possible that other antioxidant enzymes (e.g. superoxide dismutase and catalase which do not require secondary enzymes such as GPx) are up-regulated to back-up for the missing GPx activity. Even though the reduced levels of DNA lesions in the low Se group were significant, the difference is small (Figs 4 and 5) and should be weighed with care. In men with a low Se blood level elevated levels of DNA lesions (ssb/als) in leukocytes have been observed35. We have recently shown that the levels of DNA lesions in lung and testis of Se depleted mice were also elevated17. Furthermore, low Se blood levels have been associated with increased risk of cancers such as prostate cancer36, lung cancer37 and colorectal cancer38.

To study the implications of the lack of a DNA repair enzyme, namely DNA glycosylase OGG1, the Ogg1−/− genotype was included in this study. The Ogg1−/− accumulates oxidised DNA lesions due to its reduced capacity to excise 8-oxoG, a highly premutagenic oxidised base lesion (measurable in the SCGE in combination with the DNA repair enzyme Fpg). Interestingly, the lack of OGG1 had no impact on any studied endpoint in blood. Neither the chronic LDR of γ radiation nor the low Se diet led to additional levels of oxidised DNA lesions in Ogg1−/− mice detectable in the SCGE. It is possible that the low dose rate did not produce enough free radicals to induce a detectable level of oxidised DNA lesions. Another explanation might be that repair mechanisms for the induced oxidised DNA lesions may have been sufficiently active or up-regulated to compensate for radiation- or diet-induced DNA lesions. For instance, other DNA glycosylases besides OGG1 exist in mammalian cells in the base excision repair (BER) pathway (e.g., MYH, NEIL1, NEIL2, NEIL3 and NTH1). BER is the major DNA repair pathway for oxidised DNA lesions induced by ionising radiation39,40. Similar to OGG1, MYH prevents G to T mutations by removing misincorporated adenine opposite of 8-oxoG41. Thus, MYH might mask possible effects of LDR γ radiation.

The next issue was to identify potential interactions between the different stressors radiation, diet, and genotype. In general, there are few in vivo studies on the combined effects of Se and γ radiation. The few conducted studies have investigated the potential radio-protective character of Se supplementation when applying high doses and dose rates of irradiation (acute, not chronic)42,43. Studies on the consequences of Se deficiency in conjunction with realistic exposure to chronic LDR irradiation are missing, as well as studies on the genotoxic effect of these combinations. LDR γ radiation combined with low Se diet was hypothesised to give rise to additive or synergistic effects, and thus the opposite of the observed antagonistic effects in this study. Decreased levels of oxidised DNA lesions were measured in mice of the most stressed group, i.e. depleted of Se and subjected to γ radiation (Fig. 5), despite the fact that Se acts as co-factor for GPx. The Ogg1+/− mice showed a 40–50% reduced level of oxidised DNA lesions dependent on radiation and diet, with the lowest level in irradiated mice on low Se diet (at day 45) (Fig. 5a). Similar to observations of DNA breaks, an interaction between radiation and diet was also evident for phenotypic Pig-a gene mutation frequencies (Figs 1 and 3). The data proposes that defence mechanisms (such as antioxidant systems and repair of oxidised DNA lesions) might be triggered by the combined stressors (Se deficiency and γ radiation) to prevent the induction of harmful effects.

This study contributes with valuable data concerning genotoxic effects of chronic LDR γ radiation alone and in combination with another stressor, low Se diet. The observed changes are small, as expected for experiments using low doses and dose rates of exposure, but the applied doses are realistic in a human relevant context. Nevertheless, significant differences were identified supporting the high sensitivity for the assays, as shown previously for the SCGE assay44. We have found that chronic LDR γ radiation induces genotoxic effects in murine blood cells using three assays covering different endpoints (chromosomal fragmentations, phenotypic Pig-a gene mutations and DNA lesions). Data suggests that Se depletion partly influences the genotoxic effects of chronic LDR γ radiation with respect to levels of DNA lesions and phenotypic Pig-a gene mutation frequencies. A low Se diet seemed to moderate the levels of DNA lesions induced by γ radiation independently of the presence of DNA glycosylase OGG1. Furthermore, prolonged LDR γ radiation appeared to induce a protective response, with lower levels of ssb/als in irradiated mice compared with concurrent non-irradiated mice.

In summary, exposure to chronic LDR of ionising radiation is indeed genotoxic with potential implications for cancer development, and the response is modified by the availability of Se, an element involved in the antioxidative defence system.