Synchrotron-Based X-ray Fluorescence Imaging Elucidates Uranium Toxicokinetics in Daphnia magna.

A combination of synchrotron-based elemental analysis and acute toxicity tests was used to investigate the biodistribution and adverse effects in Daphnia magna exposed to uranium nanoparticle (UNP, 3-5 nm) suspensions or to uranium reference (Uref) solutions. Speciation analysis revealed similar size distributions between exposures, and toxicity tests showed comparable acute effects (UNP LC50: 402 µg L-1 [336-484], Uref LC50: 268 µg L-1 [229-315]). However, the uranium body burden was 3- to 5-fold greater in UNP-exposed daphnids, and analysis of survival as a function of body burden revealed a ∼5-fold higher specific toxicity from the Uref exposure. High-resolution X-ray fluorescence elemental maps of intact, whole daphnids from sublethal, acute exposures of both treatments revealed high uranium accumulation onto the gills (epipodites) as well as within the hepatic ceca and the intestinal lumen. Uranium uptake into the hemolymph circulatory system was inferred from signals observed in organs such as the heart and the maxillary gland. The substantial uptake in the maxillary gland and the associated nephridium suggests that these organs play a role in uranium removal from the hemolymph and subsequent excretion. Uranium was also observed associated with the embryos and the remnants of the chorion, suggesting uptake in the offspring. The identification of target organs and tissues is of major importance to the understanding of uranium and UNP toxicity and exposure characterization that should ultimately contribute to reducing uncertainties in related environmental impact and risk assessments.

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.

Multiomics Point of Departure (moPOD) Modeling Supports an Adverse Outcome Pathway Network for Ionizing Radiation.

While adverse biological effects of acute high-dose ionizing radiation have been extensively investigated, knowledge on chronic low-dose effects is scarce. The aims of the present study were to identify hazards of low-dose ionizing radiation to Daphnia magna using multiomics dose-response modeling and to demonstrate the use of omics data to support an adverse outcome pathway (AOP) network development for ionizing radiation. Neonatal D. magna were exposed to γ radiation for 8 days. Transcriptomic analysis was performed after 4 and 8 days of exposure, whereas metabolomics and confirmative bioassays to support the omics analyses were conducted after 8 days of exposure. Benchmark doses (BMDs, 10% benchmark response) as points of departure (PODs) were estimated for both dose-responsive genes/metabolites and the enriched KEGG pathways. Relevant pathways derived using the BMD modeling and additional functional end points measured by the bioassays were overlaid with a previously published AOP network. The results showed that several molecular pathways were highly relevant to the known modes of action of γ radiation, including oxidative stress, DNA damage, mitochondrial dysfunction, protein degradation, and apoptosis. The functional assays showed increased oxidative stress and decreased mitochondrial membrane potential and ATP pool. Ranking of PODs at the pathway and functional levels showed that oxidative damage related functions had relatively low PODs, followed by DNA damage, energy metabolism, and apoptosis. These were supportive of causal events in the proposed AOP network. This approach yielded promising results and can potentially provide additional empirical evidence to support further AOP development for ionizing radiation.

Synchrotron XRF and Histological Analyses Identify Damage to Digestive Tract of Uranium NP-Exposed Daphnia magna.

Micro- and nanoscopic X-ray techniques were used to investigate the relationship between uranium (U) tissue distributions and adverse effects to the digestive tract of aquatic model organism Daphnia magna following uranium nanoparticle (UNP) exposure. X-ray absorption computed tomography measurements of intact daphnids exposed to sublethal concentrations of UNPs or a U reference solution (URef) showed adverse morphological changes to the midgut and the hepatic ceca. Histological analyses of exposed organisms revealed a high proportion of abnormal and irregularly shaped intestinal epithelial cells. Disruption of the hepatic ceca and midgut epithelial tissues implied digestive functions and intestinal barriers were compromised. Synchrotron-based micro X-ray fluorescence (XRF) elemental mapping identified U co-localized with morphological changes, with substantial accumulation of U in the lumen as well as in the epithelial tissues. Utilizing high-resolution nano-XRF, 400-1000 nm sized U particulates could be identified throughout the midgut and within hepatic ceca cells, coinciding with tissue damages. The results highlight disruption of intestinal function as an important mode of action of acute U toxicity in D. magna and that midgut epithelial cells as well as the hepatic ceca are key target organs.

International expert group collaboration for developing an adverse outcome pathway for radiation induced leukemia.

PURPOSE: The concept of the adverse outcome pathway (AOP) has recently gained significant attention as to its potential for incorporation of mechanistic biological information into the assessment of adverse health outcomes following ionizing radiation (IR) exposure. This work is an account of the activities of an international expert group formed specifically to develop an AOP for IR-induced leukemia. Group discussions were held during dedicated sessions at the international AOP workshop jointly organized by the MELODI (Multidisciplinary European Low Dose Initiative) and the ALLIANCE (European Radioecology Alliance) associations to consolidate knowledge into a number of biological key events causally linked by key event relationships and connecting a molecular initiating event with the adverse outcome. Further knowledge review to generate a weight of evidence support for the Key Event Relationships (KERs) was undertaken using a systematic review approach. CONCLUSIONS: An AOP for IR-induced acute myeloid leukemia was proposed and submitted for review to the OECD-curated AOP-wiki (aopwiki.org). The systematic review identified over 500 studies that link IR, as a stressor, to leukemia, as an adverse outcome. Knowledge gap identification, although requiring a substantial effort via systematic review of literature, appears to be one of the major added values of the AOP concept. Further work, both within this leukemia AOP working group and other similar working groups, is warranted and is anticipated to produce highly demanded products for the radiation protection research community.

Adverse outcome pathways (AOPs) for radiation-induced reproductive effects in environmental species: state of science and identification of a consensus AOP network.

BACKGROUND: Reproductive effects of ionizing radiation in organisms have been observed under laboratory and field conditions. Such assessments often rely on associations between exposure and effects, and thus lacking a detailed mechanistic understanding of causality between effects occurring at different levels of biological organization. The Adverse Outcome Pathway (AOP), a conceptual knowledge framework to capture, organize, evaluate and visualize the scientific knowledge of relevant toxicological effects, has the potential to evaluate the causal relationships between molecular, cellular, individual, and population effects. This paper presents the first development of a set of consensus AOPs for reproductive effects of ionizing radiation in wildlife. This work was performed by a group of experts formed during a workshop organized jointly by the Multidisciplinary European Low Dose Initiative (MELODI) and the European Radioecology Alliance (ALLIANCE) associations to present the AOP approach and tools. The work presents a series of taxon-specific case studies that were used to identify relevant empirical evidence, identify common AOP components and propose a set of consensus AOPs that could be organized into an AOP network with broader taxonomic applicability. CONCLUSION: Expert consultation led to the identification of key biological events and description of causal linkages between ionizing radiation, reproductive impairment and reduction in population fitness. The study characterized the knowledge domain of taxon-specific AOPs, identified knowledge gaps pertinent to reproductive-relevant AOP development and reflected on how AOPs could assist applications in radiation (radioecological) research, environmental health assessment, and radiological protection. Future advancement and consolidation of the AOPs is planned to include structured weight of evidence considerations, formalized review and critical assessment of the empirical evidence prior to formal submission and review by the OECD sponsored AOP development program.

Ultraviolet B modulates gamma radiation-induced stress responses in Lemna minor at multiple levels of biological organisation.

Elevated levels of ionizing and non-ionizing radiation may co-occur and pose cumulative hazards to biota. However, the combined effects and underlying toxicity mechanisms of different types of radiation in aquatic plants remain poorly understood. The present study aims to demonstrate how different combined toxicity prediction approaches can collectively characterise how chronic (7 days) exposure to ultraviolet B (UVB) radiation (0.5 W m-2) modulates gamma (γ) radiation (14.9, 19.5, 43.6 mGy h-1) induced stress responses in the macrophyte Lemna minor. A suite of bioassays was applied to quantify stress responses at multiple levels of biological organisation. The combined effects (no-enhancement, additivity, synergism, antagonism) were determined by two-way analysis of variance (2 W-ANOVA) and a modified Independent Action (IA) model. The toxicological responses and the potential causality between stressors were further visualised by a network of toxicity pathways. The results showed that γ-radiation or UVB alone induced oxidative stress and programmed cell death (PCD) as well as impaired oxidative phosphorylation (OXPHOS) and photosystem II (PSII) activity in L. minor. γ-radiation also activated antioxidant responses, DNA damage repair and chlorophyll metabolism, and inhibited growth at higher dose rates (≥20 mGy h-1). When co-exposed, UVB predominantly caused non-interaction (no-enhancement or additive) effects on γ-radiation-induced antioxidant gene expression, energy quenching in PSII and growth for all dose rates, whereas antagonistic effects were observed for lipid peroxidation, OXPHOS, PCD, oxidative stress, chlorophyll metabolism and genes involved in DNA damage responses. Synergistic effects were observed for changes in photochemical quenching and non-photochemical quenching, and up-regulation of antioxidant enzyme genes (GST) at one or more dose rates, while synergistic reproductive inhibition occurred at all three γ-radiation dose rates. The present study provides mechanistic knowledge, quantitative understanding and novel analytical strategies to decipher combined effects across levels of biological organisation, which should facilitate future cumulative hazard assessments of multiple stressors.

Synchrotron XRF Analysis Identifies Cerium Accumulation Colocalized with Pharyngeal Deformities in CeO2 NP-Exposed Caenorhabditis elegans.

A combination of synchrotron radiation-based elemental imaging, in vivo redox status analysis, histology, and toxic responses was used to investigate the uptake, biodistribution, and adverse effects of Ce nanoparticles (CeO2 NP; 10 nm; 0.5-34.96 mg Ce L-1) or Ce(NO3)3 (2.3-26 mg Ce L-1) in Caenorhabditis elegans. Elemental mapping of the exposed nematodes revealed Ce uptake in the alimentary canal prior to depuration. Retention of CeO2 NPs was low compared to that of Ce(NO3)3 in depurated individuals. X-ray fluorescence (XRF) mapping showed that Ce translocation was confined to the pharyngeal valve and foregut. Ce(NO3)3 exposure significantly decreased growth, fertility, and reproduction, caused slightly reduced fecundity. XRF mapping and histological analysis revealed severe tissue deformities colocalized with retained Ce surrounding the pharyngeal valve. Both forms of Ce activated the sod-1 antioxidant defense, particularly in the pharynx, whereas no significant effects on the cellular redox balance were identified. The CeO2 NP-induced deformities did not appear to impair the pharyngeal function or feeding ability as growth effects were restricted to Ce(NO3)3 exposure. The results demonstrate the utility of integrated submicron-resolution SR-based XRF elemental mapping of tissue-specific distribution and adverse effect analysis to obtain robust toxicological evaluations of metal-containing contaminants.

A systems biology analysis of reproductive toxicity effects induced by multigenerational exposure to ionizing radiation in C. elegans.

Understanding the effects of chronic exposure to pollutants over generations is of primary importance for the protection of humans and the environment; however, to date, knowledge on the molecular mechanisms underlying multigenerational adverse effects is scarce. We employed a systems biology approach to analyze effects of chronic exposure to gamma radiation at molecular, tissue and individual levels in the nematode Caenorhabditis elegans. Our data show a decrease of 23% in the number of offspring on the first generation F0 and more than 40% in subsequent generations F1, F2 and F3. To unveil the impact on the germline, an in-depth analysis of reproductive processes involved in gametes formation was performed for all four generations. We measured a decrease in the number of mitotic germ cells accompanied by increased cell-cycle arrest in the distal part of the gonad. Further impact on the germline was manifested by decreased sperm quantity and quality. In order to obtain insight in the molecular mechanisms leading to decreased fecundity, gene expression was investigated via whole genome RNA sequencing. The transcriptomic analysis revealed modulation of transcription factors, as well as genes involved in stress response, unfolded protein response, lipid metabolism and reproduction. Furthermore, a drastic increase in the number of differentially expressed genes involved in defense response was measured in the last two generations, suggesting a cumulative stress effect of ionizing radiation exposure. Transcription factor binding site enrichment analysis and the use of transgenic strain identified daf-16/FOXO as a master regulator of genes differentially expressed in response to radiation. The presented data provide new knowledge with respect to the molecular mechanisms involved in reproductive toxic effects and accumulated stress resulting from multigenerational exposure to ionizing radiation.

Ionizing radiation, genotoxic stress, and mitochondrial DNA copy-number variation in Caenorhabditis elegans: droplet digital PCR analysis.

Mitochondria are vulnerable to the effects of ionizing radiation; damage to mitochondrial DNA (mtDNA) may be more extensive and persistent than damage to nuclear DNA (nDNA). Variation in mtDNA copy number has been proposed as a marker for mitochondrial dysfunction in response to ionizing radiation. We have developed a precise and sensitive duplex droplet digital PCR (ddPCR) method for quantitation of the mtDNA/nDNA ratio in the model organism Caenorhabditis elegans. The effect on this ratio was investigated over a wide range of doses (0.03-72 Gy) of chronic gamma irradiation. Five mitochondrial targets and two nuclear reference genes were amplified pairwise in duplex PCR format (one mitochondrial and one nuclear target per PCR) by both ddPCR and quantitative PCR (qPCR). The results showed that ddPCR but not qPCR enabled detection of a significant increase in mtDNA copy number (1.6 ± 0.1-fold) for nematodes exposed to high doses (≥24 Gy). Thus, ddPCR provided higher precision and greater sensitivity than qPCR for detection of mtDNA copy number variation. The variation followed a Hill-type dose response with threshold 10.3 ± 1 Gy. This strongly suggests that chronic genotoxic stress affects mtDNA replication. The duplex ddPCR method is a novel, high-precision, sensitive tool for determination of mitochondrial DNA copy number variation and function in C. elegans.

Nisin Z Production by Wild Strains of Lactococcus lactis Isolated from Brazilian (Italian Type) Fermented Sausage.

In this study, five bacteriocin-producing Lactococcus lactis strains were identified from different naturally fermented Brazilian sausages. Ion exchange and reversed-phase chromatographies were used to purify the bacteriocins from culture supernatant of the five strains. Mass spectrometry (MALDI-TOF/TOF) showed that the molecular masses of the bactericoins from L. lactis ID1.5, ID3.1, ID8.5, PD4.7, and PR3.1 were 3330.567 Da, 3330.514 Da, 3329.985 Da, 3329.561 Da, and 3329.591 Da, respectively. PCR product sequence analysis confirmed that the structural genes of bacteriocins produced by the five isolates are identical to the lantibiotic nisin Z. Optimal nisin Z production was achieved in tryptone and casein peptone, at pH 6.0 or 6.5. The most favorable temperatures for nisin Z production were 25°C and 30°C, and its production was better under aerobic than anaerobic condition. The type of carbon source appeared to be an important factor for nisin Z production. While sucrose was found to be the most efficient carbon source for nisin Z production by four L. lactis isolates, fructose was the best for one isolate. Lactose was also a good energy source for nisin Z production. Surprisingly, glucose was clearly the poorest carbon source for nisin Z production. The five isolates produced different amounts of the bacteriocin, L. lactis ID1.5 and ID8.5 isolates being the best nisin Z producers. DNA sequence analysis did not reveal any sequence differences in the nisZ and nisF promoter regions that could explain the differences in nisin Z production, suggesting that there should be other factors responsible for differential nisin Z production by the isolates.

In vivo assessment of reactive oxygen species production and oxidative stress effects induced by chronic exposure to gamma radiation in Caenorhabditis elegans.

In the current study, effects of chronic exposure to ionizing gamma radiation were assessed in the radioresistant nematode Caenorhabditis elegans in order to understand whether antioxidant defences (AODs) could ameliorate radical formation, or if increased ROS levels would cause oxidative damage. This analysis was accompanied by phenotypical as well as molecular investigations, via assessment of reproductive capacity, somatic growth and RNA-seq analysis. The use of a fluorescent reporter strain (sod1::gfp) and two ratiometric biosensors (HyPer and Grx1-roGFP2) demonstrated increased ROS production (H2O2) and activation of AODs (SOD1 and Grx) in vivo. The data showed that at dose-rates ≤10 mGy h-1 defence mechanisms were able to prevent the manifestation of oxidative stress. In contrast, at dose-rates ≥40 mGy h-1 the continuous formation of radicals caused a redox shift, which lead to oxidative stress transcriptomic responses, including changes in mitochondrial functions, protein degradation, lipid metabolism and collagen synthesis. Moreover, genotoxic effects were among the most over-represented functions affected by chronic gamma irradiation, as indicated by differential regulation of genes involved in DNA damage, DNA repair, cell-cycle checkpoints, chromosome segregation and chromatin remodelling. Ultimately, the exposure to gamma radiation caused reprotoxic effects, with >20% reduction in the number of offspring per adult hermaphrodite at dose-rates ≥40 mGy h-1, accompanied by the down-regulation of more than 300 genes related to reproductive system, apoptosis, meiotic functions and gamete development and fertilization.

Integrative assessment of low-dose gamma radiation effects on Daphnia magna reproduction: Toxicity pathway assembly and AOP development.

High energy gamma radiation is potentially hazardous to organisms, including aquatic invertebrates. Although extensively studied in a number of invertebrate species, knowledge on effects induced by gamma radiation is to a large extent limited to the induction of oxidative stress and DNA damage at the molecular/cellular level, or survival, growth and reproduction at the organismal level. As the knowledge of causal relationships between effects occurring at different levels of biological organization is scarce, the ability to provide mechanistic explanation for observed adverse effects is limited, and thus development of Adverse Outcome Pathways (AOPs) and larger scale implementation into next generation hazard and risk predictions is restricted. The present study was therefore conducted to assess the effects of high-energy gamma radiation from cobalt-60 across multiple levels of biological organization (i.e., molecular, cellular, tissue, organ and individual) and characterize the major toxicity pathways leading to impaired reproduction in the model freshwater crustacean Daphnia magna (water flea). Following gamma exposure, a number of bioassays were integrated to measure relevant toxicological endpoints such as gene expression, reactive oxygen species (ROS), lipid peroxidation (LPO), neutral lipid storage, adenosine triphosphate (ATP) content, apoptosis, ovary histology and reproduction. A non-monotonic pattern was consistently observed across the levels of biological organization, albeit with some variation at the lower end of the dose-rate scale, indicating a complex response to radiation doses. By integrating results from different bioassays, a novel pathway network describing the key toxicity pathways involved in the reproductive effects of gamma radiation were proposed, such as DNA damage-oocyte apoptosis pathway, LPO-ATP depletion pathway, calcium influx-endocrine disruption pathway and DNA hypermethylation pathway. Three novel AOPs were proposed for oxidative stressor-mediated excessive ROS formation leading to reproductive effect, and thus introducing the world's first AOPs for non-chemical stressors in aquatic invertebrates.

Gamma radiation induces life stage-dependent reprotoxicity in Caenorhabditis elegans via impairment of spermatogenesis.

The current study investigated life stage, tissue and cell dependent sensitivity to ionizing radiation of the nematode Caenorhabditis elegans. Results showed that irradiation of post mitotic L4 stage larvae induced no significant effects with respect to mortality, morbidity or reproduction at either acute dose ≤6 Gy (1500 mGy·h-1) or chronic exposure ≤15 Gy (≤100 mGy·h-1). In contrast, chronic exposure from the embryo to the L4-young adult stage caused a dose and dose-rate dependent reprotoxicity with 43% reduction in total brood size at 6.7 Gy (108 mGy·h-1). Systematic irradiation of the different developmental stages showed that the most sensitive life stage was L1 to young L4. Exposure during these stages was associated with dose-rate dependent genotoxic effects, resulting in a 1.8 to 2 fold increase in germ cell apoptosis in larvae subjected to 40 or 100 mGy·h-1, respectively. This was accompanied by a dose-rate dependent reduction in the number of spermatids, which was positively correlated to the reprotoxic effect (0.99, PCC). RNAseq analysis of nematodes irradiated from L1 to L4 stage revealed a significant enrichment of differentially expressed genes related to both male and hermaphrodite reproductive processes. Gene network analysis revealed effects related to down-regulation of genes required for spindle formation and sperm meiosis/maturation, including smz-1, smz-2 and htas-1. Furthermore, the expression of a subset of 28 set-17 regulated Major Sperm Proteins (MSP) required for spermatid production was correlated (R2 0.80) to the reduction in reproduction and the number of spermatids. Collectively these observations corroborate the impairment of spermatogenesis as the major cause of gamma radiation induced life-stage dependent reprotoxic effect. Furthermore, the progeny of irradiated nematodes showed significant embryonal DNA damage that was associated with persistent effect on somatic growth. Unexpectedly, these nematodes maintained much of their reproductive capacity in spite of the reduced growth.

Modes of action and adverse effects of gamma radiation in an aquatic macrophyte Lemna minor.

High dose rates of ionizing radiation have been reported to cause adverse effects such as reduction in reproduction and growth, and damage to protein and lipids in primary producers. However, the relevant effects of ionizing radiation are still poorly understood in aquatic plants. This study was intended to characterize the biological effects and modes of action (MoAs) of ionizing radiation using gamma radiation as the prototypical stressor and duckweed Lemna minor as a model organism. Lemna minor was exposed to 1, 14, 24, 46, 70 mGy/h gamma radiation dose rates from a cobalt-60 source for 7 days following the testing principles of the OECD test guideline 221. A suite of bioassays was applied to assess the biological effects of gamma radiation at multiple levels of biological organization, including detection of reactive oxygen species (ROS), oxidative stress responses (total glutathione, tGSH; lipid peroxidation, LPO), DNA damage, mitochondrial dysfunctions (mitochondrial membrane potential, MMP), photosynthetic parameters (chlorophyll a, chl a; chlorophyll b, chl b; carotenoids; Photosystem II (PSII) performance; CO2 uptake), intercellular signaling (Ca2+ release) and growth. Gamma radiation increased DNA damage, tGSH level and Ca2+ content together with reduction in chlorophyll content, maximal PSII efficiency and CO2 uptake at dose rates between 1 and 14 mGy/h, whereas increases in cellular ROS and LPO, inhibition of MMP and growth were observed at higher dose rates (≥24 mGy/h). A network of toxicity pathways was proposed to portray the causal relationships between gamma radiation-induced physiological responses and adverse outcomes to support the development of Adverse Outcome Pathways (AOPs) for ionizing radiation-mediated effects in primary producers.

Gamma radiation induces locus specific changes to histone modification enrichment in zebrafish and Atlantic salmon.

Ionizing radiation is a recognized genotoxic agent, however, little is known about the role of the functional form of DNA in these processes. Post translational modifications on histone proteins control the organization of chromatin and hence control transcriptional responses that ultimately affect the phenotype. The purpose of this study was to investigate effects on chromatin caused by ionizing radiation in fish. Direct exposure of zebrafish (Danio rerio) embryos to gamma radiation (10.9 mGy/h for 3h) induced hyper-enrichment of H3K4me3 at the genes hnf4a, gmnn and vegfab. A similar relative hyper-enrichment was seen at the hnf4a loci of irradiated Atlantic salmon (Salmo salar) embryos (30 mGy/h for 10 days). At the selected genes in ovaries of adult zebrafish irradiated during gametogenesis (8.7 and 53 mGy/h for 27 days), a reduced enrichment of H3K4me3 was observed, which was correlated with reduced levels of histone H3 was observed. F1 embryos of the exposed parents showed hyper-methylation of H3K4me3, H3K9me3 and H3K27me3 on the same three loci, while these differences were almost negligible in F2 embryos. Our results from three selected loci suggest that ionizing radiation can affect chromatin structure and organization, and that these changes can be detected in F1 offspring, but not in subsequent generations.

Ionizing radiation induces transgenerational effects of DNA methylation in zebrafish.

Ionizing radiation is known to cause DNA damage, yet the mechanisms underlying potential transgenerational effects of exposure have been scarcely studied. Previously, we observed effects in offspring of zebrafish exposed to gamma radiation during gametogenesis. Here, we hypothesize that these effects are accompanied by changes of DNA methylation possibly inherited by subsequent generations. We assessed DNA methylation in F1 embryos (5.5 hours post fertilization) with whole genome bisulfite sequencing following parental exposure to 8.7 mGy/h for 27 days and found 5658 differentially methylated regions (DMRs). DMRs were predominantly located at known regulatory regions, such as gene promoters and enhancers. Pathway analysis indicated the involvement of DMRs related to similar pathways found with gene expression analysis, such as development, apoptosis and cancers, which could be linked to previous observed developmental defects and genomic instability in the offspring. Follow up of 19 F1 DMRs in F2 and F3 embryos revealed persistent effects up to the F3 generation at 5 regions. These results indicate that ionizing radiation related effects in offspring can be linked to DNA methylation changes that partly can persist over generations. Monitoring DNA methylation could serve as a biomarker to provide an indication of ancestral exposures to ionizing radiation.

Parental exposure to gamma radiation causes progressively altered transcriptomes linked to adverse effects in zebrafish offspring.

Ionizing radiation causes a variety of effects, including DNA damage associated to cancers. However, the effects in progeny from irradiated parents is not well documented. Using zebrafish as a model, we previously found that parental exposure to ionizing radiation is associated with effects in offspring, such as increased hatching rates, deformities, increased DNA damage and reactive oxygen species. Here, we assessed short (one month) and long term effects (one year) on gene expression in embryonic offspring (5.5 h post fertilization) from zebrafish exposed during gametogenesis to gamma radiation (8.7 or 53 mGy/h for 27 days, total dose 5.2 or 31 Gy) using mRNA sequencing. One month after exposure, a global change in gene expression was observed in offspring from the 53 mGy/h group, followed by embryonic death at late gastrula, whereas offspring from the 8.7 mGy/h group was unaffected. Interestingly, one year after exposure newly derived embryos from the 8.7 mGy/h group exhibited 2390 (67.7% downregulated) differentially expressed genes. Overlaps in differentially expressed genes and enriched biological pathways were evident between the 53 mGy/h group one month and 8.7 mGy/h one year after exposure, but were oppositely regulated. Pathways could be linked to effects in adults and offspring, such as DNA damage (via Atm signaling) and reproduction (via Gnrh signaling). Comparison with gene expression analysis in directly exposed embryos indicate transferrin a and cytochrome P450 2x6 as possible biomarkers for radiation response in zebrafish. Our results indicate latent effects following ionizing radiation exposure from the lower dose in parents that can be transmitted to offspring and warrants monitoring effects over subsequent generations.

Combined Computed Nanotomography and Nanoscopic X-ray Fluorescence Imaging of Cobalt Nanoparticles in Caenorhabditis elegans.

Synchrotron radiation phase-contrast computed nanotomography (nano-CT) and two- and three-dimensional (2D and 3D) nanoscopic X-ray fluorescence (nano-XRF) were used to investigate the internal distribution of engineered cobalt nanoparticles (Co NPs) in exposed individuals of the nematode Caenorhabditis elegans. Whole nematodes and selected tissues and organs were 3D-rendered: anatomical 3D renderings with 50 nm voxel size enabled the visualization of spherical nanoparticle aggregates with size up to 200 nm within intact C. elegans. A 20 × 37 nm2 high-brilliance beam was employed to obtain XRF elemental distribution maps of entire nematodes or anatomical details such as embryos, which could be compared with the CT data. These maps showed Co NPs to be predominantly present within the intestine and the epithelium, and they were not colocalized with Zn granules found in the lysosome-containing vesicles or Fe agglomerates in the intestine. Iterated XRF scanning of a specimen at 0° and 90° angles suggested that NP aggregates were translocated into tissues outside of the intestinal lumen. Virtual slicing by means of 2D XRF tomography, combined with holotomography, indicated presumable presence of individual NP aggregates inside the uterus and within embryos.

Dose-dependent effects of gamma radiation on the early zebrafish development and gene expression.

Ionizing radiation from natural sources or anthropogenic activity has the potential to cause oxidative stress or genetic damage in living organisms, through the ionization and excitation of molecules and the subsequent production of free radicals and reactive oxygen species (ROS). The present work focuses on radiation-induced biological effects using the zebrafish (Danio rerio) vertebrate model. Changes in developmental traits and gene expression in zebrafish were assessed after continuous external gamma irradiation (0.4, 3.9, 15 and 38 mGy/h) with corresponding controls, starting at 2.5 hours post fertilization (hpf) and lasting through embryogenesis and the early larval stage. The lowest dose rate corresponded to recommended benchmarks at which adverse effects are not expected to occur in aquatic ecosystems (2-10 mGy/day). The survival observed at 96 hours post fertilization (hpf) in the 38 mGy/h group was significantly lower, while other groups showed no significant difference compared to controls. The total hatching was significantly lower from controls in the 15 mGy/h group and a delay in hatching onset in the 0.4 mGy/h group was observed. The deformity frequency was significantly increased by prolonged exposure duration at dose rates ≥ 0.4 mGy/h. Molecular responses analyzed by RNA-seq at gastrulation (5.5 hpf transcriptome) indicate that the radiation induced adverse effects occurred during the earliest stages of development. A dose-response relationship was found in the numbers of differentially regulated genes in exposure groups compared to controls at a total dose as low as 1.62 mGy. Ingenuity Pathway Analysis identified retinoic acid receptor activation, apoptosis, and glutathione mediated detoxification signaling as the most affected pathways in the lower dose rate (0.54 mGy/h), while eif2 and mTOR, i.e., involved in the modulation of angiogenesis, were most affected in higher dose rates (5.4 and 10.9 mGy/h). By comparing gene expression data, myc was found to be the most significant upstream regulator, followed by tp53, TNF, hnf4a, TGFb1 and cebpa, while crabp2b and vegfab were identified as most frequent downstream target genes. These genes are associated with various developmental processes. The present findings show that continuous gamma irradiation (≥ 0.54 mGy/h) during early gastrula causes gene expression changes that are linked to developmental defects in zebrafish embryos.