Dose rate dependent reduction in chromatin accessibility at transcriptional start sites long time after exposure to gamma radiation.

Ionizing radiation (IR) impact cellular and molecular processes that require chromatin remodelling relevant for cellular integrity. However, the cellular implications of ionizing radiation (IR) delivered per time unit (dose rate) are still debated. This study investigates whether the dose rate is relevant for inflicting changes to the epigenome, represented by chromatin accessibility, or whether it is the total dose that is decisive. CBA/CaOlaHsd mice were whole-body exposed to either chronic low dose rate (2.5 mGy/h for 54 d) or the higher dose rates (10 mGy/h for 14 d and 100 mGy/h for 30 h) of gamma radiation (60Co, total dose: 3 Gy). Chromatin accessibility was analysed in liver tissue samples using Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-Seq), both one day after and over three months post-radiation (>100 d). The results show that the dose rate contributes to radiation-induced epigenomic changes in the liver at both sampling timepoints. Interestingly, chronic low dose rate exposure to a high total dose (3 Gy) did not inflict long-term changes to the epigenome. In contrast to the acute high dose rate given to the same total dose, reduced accessibility at transcriptional start sites (TSS) was identified in genes relevant for the DNA damage response and transcriptional activity. Our findings link dose rate to essential biological mechanisms that could be relevant for understanding long-term changes after ionizing radiation exposure. However, future studies are needed to comprehend the biological consequence of these findings.

Calibration of the comet assay using ionising radiation.

Several trials have attempted to identify sources of inter-laboratory variability in comet assay results, aiming at achieving more equal responses. Ionising radiation induces a defined level of DNA single-strand breaks (per dose/base pairs) and is used as a reference when comparing comet results but relies on accurately determined radiation doses. In this ring test we studied the significance of dose calibrations and comet assay protocol differences, with the object of identifying causes of variability and how to deal with them. Eight participating laboratories, using either x-ray or gamma radiation units, measured dose rates using alanine pellet dosimeters that were subsequently sent to a specialised laboratory for analysis. We found substantial deviations between calibrated and nominal (uncalibrated) dose rates, with up to 46% difference comparing highest and lowest values. Three additional dosimetry systems were employed in some laboratories: thermoluminescence detectors and two aqueous chemical dosimeters. Fricke's and Benzoic Acid dosimetry solutions gave reliable quantitative dose estimations using local equipment. Mononuclear cells from fresh human blood or mammalian cell lines were irradiated locally with calibrated (alanine) radiation doses and analysed for DNA damage using a standardised comet assay protocol and a lab-specific protocol. The dose response of eight laboratories, calculated against calibrated radiation doses, was linear with slope variance CV= 29% with the lab-specific protocol, reduced to CV= 16% with the standard protocol. Variation between laboratories indicate post-irradiation repair differences. Intra-laboratory variation was very low judging from the dose response of 8 donors (CV=4%). Electrophoresis conditions were different in the lab-specific protocols explaining some dose response variations which were reduced by systematic corrections for electrophoresis conditions. The study shows that comet assay data obtained in different laboratories can be compared quantitatively using calibrated radiation doses and that systematic corrections for electrophoresis conditions are useful.

Assessing testicular germ cell DNA damage in the comet assay; introduction of a proof-of-concept.

The in vivo comet assay is widely used to measure genotoxicity; however, the current OECD test guideline (TG 489) does not recommend using the assay to assess testicular germ cells, due to the presence of testicular somatic cells. An adapted approach to specifically assess testicular germ cells within the comet assay is certainly warranted, considering regulatory needs for germ cell-specific genotoxicity data in relation to the increasing global production of and exposure to potentially hazardous chemicals. Here, we provide a proof-of-concept to selectively analyze round spermatids and primary spermatocytes, distinguishing them from other cells of the testicle. Utilizing the comet assay recordings of DNA content (total fluorescence intensity) and DNA damage (% tail intensity) of individual comets, we developed a framework to distinguish testicular cell populations based on differences in DNA content/ploidy and appearance. Haploid round spermatid comets are identified through (1) visual inspection of DNA content distributions, (2) setting DNA content thresholds, and (3) modeling DNA content distributions using a normal mixture distribution function. We also describe an approach to distinguish primary spermatocytes during comet scoring, based on their high DNA content and large physical size. Our concept allows both somatic and germ cells to be analyzed in the same animal, adding a versatile, sensitive, rapid, and resource-efficient assay to the limited genotoxicity assessment toolbox for germ cells. An adaptation of TG 489 facilitates accumulation of valuable information regarding distribution of substances to germ cells and their potential for inducing germ cell gene mutations and structural chromosomal aberrations.

Using prediction models to identify miRNA-based markers of low dose rate chronic stress.

Robust biomarkers of exposure to chronic low dose stressors such as ionizing radiation, particularly following chronic low doses and dose-rates, are urgently needed. MicroRNAs (miRNA) have emerged as promising markers of exposure to high dose and dose-rate. Here, we evaluated the feasibility of classifying γ-radiation exposure at different dose rates based on miRNA expression levels. Our objective was to identify miRNA-signatures discriminating between exposure to γ-radiation or not, including exposure to chronic low dose rates. We exposed male CBA/CaOlaHsd and C57BL/6NHsd wild-type mice to 0, 2.5, 10 and 100 mGy/h γ-irradiation (3 Gy total-dose). From an initial screening of 576 miRNAs, a set of 21 signature-miRNAs was identified based on differential expression (>± 2-fold or p < 0.05). This 21-signature miRNA panel was investigated in 39 samples from 4/5 livers/group/mouse strain. A set of significantly differentially expressed miRNAs was identified in all γ-irradiated samples. Most miRNAs were upregulated in all γ-irradiated groups compared to control, and functional analysis of these miRNAs revealed involvement in several cancer-related signaling pathways. To identify miRNAs that distinguished exposed mice from controls, nine prediction methods; i.e., six variants of generalized regression models, random-forest, boosted-tree and nearest-shrunken-centroid (PAM) were used. The generalized regression methods seem to outperform the other prediction methods for classification of irradiated and control samples. Using the 21-miRNA panel in the prediction models, we identified sets of candidate miRNA-markers that predict exposure to γ-radiation. Among the top10 miRNA predictors, contributing most in each of the three γ-irradiated groups, three miRNA predictors (miR-140-3p, miR-133a-5p and miR-145a-5p) were common. Three miRNAs, miR-188-3p/26a-5p/26b-5p, were specific for lower dose-rate γ-radiation. Similarly, exposure to the high dose-rates was also correctly predicted, including mice exposed to X-rays. Our approach identifying miRNA-based signature panels may be extended to classify exposure to environmental, nutritional and life-style-related stressors, including chronic low-stress scenarios.

Genotoxic effects of high dose rate X-ray and low dose rate gamma radiation in ApcMin/+ mice.

Risk estimates for radiation-induced cancer in humans are based on epidemiological data largely drawn from the Japanese atomic bomb survivor studies, which received an acute high dose rate (HDR) ionising radiation. Limited knowledge exists about the effects of chronic low dose rate (LDR) exposure, particularly with respect to the application of the dose and dose rate effectiveness factor. As part of a study to investigate the development of colon cancer following chronic LDR vs. acute HDR radiation, this study presents the results of genotoxic effects in blood of exposed mice. CBAB6 F1 Apc+/+ (wild type) and ApcMin/+ mice were chronically exposed to estimated whole body absorbed doses of 1.7 or 3.2 Gy 60 Co-γ-rays at a LDR (2.2 mGy h-1 ) or acutely exposed to 2.6 Gy HDR X-rays (1.3 Gy min-1 ). Genotoxic endpoints assessed in blood included chromosomal damage (flow cytometry based micronuclei (MN) assay), mutation analyses (Pig-a gene mutation assay), and levels of DNA lesions (Comet assay, single-strand breaks (ssb), alkali labile sites (als), oxidized DNA bases). Ionising radiation (ca. 3 Gy) induced genotoxic effects dependent on the dose rate. Chromosomal aberrations (MN assay) increased 3- and 10-fold after chronic LDR and acute HDR, respectively. Phenotypic mutation frequencies as well as DNA lesions (ssb/als) were modulated after acute HDR but not after chronic LDR. The ApcMin/+ genotype did not influence the outcome in any of the investigated endpoints. The results herein will add to the scant data available on genotoxic effects following chronic LDR of ionising radiation. Environ. Mol. Mutagen. 58:560-569, 2017. © 2017 The Authors Environmental and Molecular Mutagenesis published by Wiley Periodicals, Inc. on behalf of Environmental Mutagen Society.

No cancer predisposition or increased spontaneous mutation frequencies in NEIL DNA glycosylases-deficient mice.

Base excision repair (BER) is a major pathway for removal of DNA base lesions and maintenance of genomic stability, which is essential in cancer prevention. DNA glycosylases recognize and remove specific lesions in the first step of BER. The existence of a number of these enzymes with overlapping substrate specificities has been thought to be the reason why single knock-out models of individual DNA glycosylases are not cancer prone. In this work we have characterized DNA glycosylases NEIL1 and NEIL2 (Neil1 -/- /Neil2 -/-) double and NEIL1, NEIL2 and NEIL3 (Neil1 -/- /Neil2 -/- /Neil3 -/-) triple knock-out mouse models. Unexpectedly, our results show that these mice are not prone to cancer and have no elevated mutation frequencies under normal physiological conditions. Moreover, telomere length is not affected and there was no accumulation of oxidative DNA damage compared to wild-type mice. These results strengthen the hypothesis that the NEIL enzymes are not simply back-up enzymes for each other but enzymes that have distinct functions beyond canonical repair.

The Pig-a Gene Mutation Assay in Mice and Human Cells: A Review.

This MiniReview describes the principle of mutation assays based on the endogenous Pig-a gene and summarizes results for two species of toxicological interest, mice and human beings. The work summarized here largely avoids rat-based studies, as are summarized elsewhere. The Pig-a gene mutation assay has emerged as a valuable tool for quantifying in vivo and in vitro mutational events. The Pig-a locus is located at the X-chromosome, giving the advantage that one inactivated allele can give rise to a mutated phenotype, detectable by multicolour flow cytometry. For in vivo studies, only minute blood volumes are required, making it easily incorporated into ongoing studies or experiments with limited biological materials. Low blood volumes also allow individuals to serve as their own controls, providing temporal information of the mutagenic process, and/or outcome of intervention. These characteristics make it a promising exposure marker. To date, the Pig-a gene mutation assay has been most commonly performed in rats, while reports regarding its usefulness in other species are accumulating. Besides its applicability to in vivo studies, it holds promise for genotoxicity testing using cultured cells, as shown in recent studies. In addition to safety assessment roles, it is becoming a valuable tool in basic research to identify mutagenic effects of different interventions or to understand implications of various gene defects by investigating modified mouse models or cell systems. Human blood-based assays are also being developed that may be able to identify genotoxic environmental exposures, treatment- and lifestyle-related factors or endogenous host factors that contribute to mutagenesis.

Corrigendum: Reference cells and ploidy in the comet assay.

[This corrects the article on p. 61 in vol. 6, PMID: 25774164.].

Gamma radiation at a human relevant low dose rate is genotoxic in mice.

Even today, 70 years after Hiroshima and accidents like in Chernobyl and Fukushima, we still have limited knowledge about the health effects of low dose rate (LDR) radiation. Despite their human relevance after occupational and accidental exposure, only few animal studies on the genotoxic effects of chronic LDR radiation have been performed. Selenium (Se) is involved in oxidative stress defence, protecting DNA and other biomolecules from reactive oxygen species (ROS). It is hypothesised that Se deficiency, as it occurs in several parts of the world, may aggravate harmful effects of ROS-inducing stressors such as ionising radiation. We performed a study in the newly established LDR-facility Figaro on the combined effects of Se deprivation and LDR γ exposure in DNA repair knockout mice (Ogg1(-/-)) and control animals (Ogg1(+/-)). Genotoxic effects were seen after continuous radiation (1.4 mGy/h) for 45 days. Chromosomal damage (micronucleus), phenotypic mutations (Pig-a gene mutation of RBC(CD24-)) and DNA lesions (single strand breaks/alkali labile sites) were significantly increased in blood cells of irradiated animals, covering three types of genotoxic activity. This study demonstrates that chronic LDR γ radiation is genotoxic in an exposure scenario realistic for humans, supporting the hypothesis that even LDR γ radiation may induce cancer.

Reference cells and ploidy in the comet assay.

In the comet assay single cells are analyzed with respect to their level of DNA damage. Discrimination of the individual cell or cell type based on DNA content, with concomitant scoring of the DNA damage, is useful since this may allow analysis of mixtures of cells. Different cells can then be characterized based on their ploidy, cell cycle stage, or genome size. We here describe two applications of such a cell type-specific comet assay: (i) Testicular cell suspensions, analyzed on the basis of their ploidy during spermatogenesis; and (ii) reference cells in the form of fish erythrocytes which can be included as internal standards to correct for inter-assay variations. With standard fluorochromes used in the comet assay, the total staining signal from each cell - whether damaged or undamaged - was found to be associated with the cell's DNA content. Analysis of the fluorescence intensity of single cells is straightforward since these data are available in scoring systems based on image analysis. The analysis of testicular cell suspensions provides information on cell type specific composition, susceptibility to genotoxicants, and DNA repair. Internal reference cells, either untreated or carrying defined numbers of lesions induced by ionizing radiation, are useful for investigation of experimental factors that can cause variation in comet assay results, and for routine inclusion in experiments to facilitate standardization of methods, and comparison of comet assay data obtained in different experiments or in different laboratories. They can also be used - in combination with a reference curve - to quantify the DNA lesions induced by a certain treatment. Fish cells of a range of genome sizes, both greater and smaller than human, are suitable for this purpose, and they are inexpensive.

Genotoxic effects of two-generational selenium deficiency in mouse somatic and testicular cells.

Many studies have investigated genotoxic effects of high Se diets but very few have addressed the genotoxicity of Se deprivation and its consequences in germ cells and none in somatic cells. To address these data gaps, C57BL/6 male mice were subjected to Se deprivation starting in the parental generation, i.e. before conception. Mice were given a diet of either low (0.01mg Se/kg diet) or normal (0.23mg Se/kg diet) Se content. Ogg1-deficient (Ogg1 (-/-) ) mice were used as a sensitive model towards oxidative stress due to their reduced capacity to repair oxidised purines. Ogg1 (-/-) mice also mimic the repair characteristics of human post-meiotic male germ cells which have a reduced ability to repair such lesions. The genotoxicity of Se deficiency was addressed by measuring DNA lesions with the alkaline single cell gel electrophoresis (+ Fpg to detect oxidised DNA lesions) in somatic cells (nucleated blood cells and lung cells) and male germ cells (testicular cells). Total Se concentration in liver and GPx activity in plasma and testicular cells were measured. Gene mutation was evaluated by an erythrocyte-based Pig-a assay. We found that Se deprivation of F1 from their conception and until early adulthood led to the induction of DNA lesions in testicular and lung cells expressed as significantly increased levels of DNA lesions, irrespective of the mouse genotype. In blood cells, Se levels did not appear to affect DNA lesions or mutant cell frequencies. The results suggest that the testis was the most sensitive tissue. Thus, genotoxicity induced by the low Se diet in the spermatozoal genome has potential implications for the offspring.

Single cell gel electrophoresis (SCGE) and Pig-a mutation assay in vivo-tools for genotoxicity testing from a regulatory perspective: a study of benzo[a]pyrene in Ogg1(-/-) mice.

The OECD has developed test guidelines (TG) to identify agents with genotoxic effects. The in vivo alkaline single cell gel electrophoresis (SCGE) assay is currently being prepared to become such a TG. The performance of a combined SCGE/Pig-a gene mutation study was evaluated with the prototypical genotoxicant benzo[a]pyrene (BaP) at an exposure level known to induce germ cell mutation. We aimed to better understand (i) the strengths and weaknesses of the two methods applied in blood and their potential to predict germ cell mutagenicity, and (ii) the involvement of reactive oxygen species (ROS) following in vivo BaP-exposure. To explore the involvement of ROS on BaP genotoxicity, we utilised a mouse model deficient in a DNA glycosylase. Specifically, C57BL/6 mice (Ogg1(+/+) and Ogg1(-/-)) were treated for three consecutive days with 50 mg BaP/kg/day. DNA damage in nucleated blood cells was measured four hours after the last treatment with the SCGE assay, with and without formamidopyrimidine DNA glycosylase (Fpg). Pig-a mutant phenotype blood erythrocytes were analysed two and four weeks after treatment. BaP-induced DNA lesions were not significantly increased in either version of the SCGE assay. The phenotypic mutation frequencies for immature and mature erythrocytes were significantly increased after two weeks. These effects were not affected by genotype, suggesting oxidative damage may have a minor role in BaP genotoxicity, at least in the acute exposure situation studied here. While both assays are promising tools for risk assessment, these results highlight the necessity of understanding the limitations regarding each assay's ability to detect chemicals' genotoxic potential.

High-throughput comet assay using 96 minigels.

The single-cell gel electrophoresis--the comet assay--has proved to be a sensitive and relatively simple method that is much used in research for the analysis of specific types of DNA damage, and its use in genotoxicity testing is increasing. The efficiency of the comet assay, in terms of number of samples processed per experiment, has been rather poor, and both research and toxicological testing should profit from an increased throughput. We have designed and validated a format involving 96 agarose minigels supported by a hydrophilic polyester film. Using simple technology, hundreds of samples may be processed in one experiment by one person, with less time needed for processing, less use of chemicals and requiring fewer cells per sample. Controlled electrophoresis, including circulation of the electrophoresis solution, improves the homogeneity between replicate samples in the 96-minigel format. The high-throughput method described in this paper should greatly increase the overall capacity, versatility and robustness of the comet assay.