Insights into the modes of action of tritium on the early-life stages of zebrafish, Danio rerio, using transcriptomic and proteomic analyses
ABSTRACT: In the environment, populations are exposed to different kinds of ionizing radiations. Little is known about their modes of actions on non-human species, and whether or not they are similar for alpha, beta and gamma radiations, considered as the reference. In this context, tritium effects (beta emitter) under the form of tritiated water (HTO) were investigated in zebrafish, a common model in toxicology and ecotoxicology with fully sequenced genome. Experiments were conducted on early life stages, considered to be highly sensitive to pollutants, by exposing eggs to 0.4 mGy/h of HTO until 10 days post fertilization. Tritium internalization was quantified and effects were investigated using a combined approach of transcriptomic and proteomic analyses. Results highlighted similarities in the biological pathways affected by HTO by both techniques, such as defense response, muscle integrity and contraction, and potential visual alterations. These results correlated well with previous data obtained on earlier developmental stages (1 and 4 dpf). Interestingly, HTO effects were partly overlapping those obtained after gamma irradiation, underlying potential common modes of action. This study therefore brought a body of evidence on the molecular effects of HTO on zebrafish larvae. Further studies could investigate if the effects persist in adult organisms. Overall design: RNAseq analysis of 7 and 10 days post-fertilization zebrafish larvae exposed to 0,4 mGy/h of tritiated water (HTO). Experiment was conducted with 3 biological replicates for both control and contaminated larvae for both stages.
Project description: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.
Project description:Contamination of the environment after the Chernobyl and Fukushima Daiichi nuclear power plant (NPP) disasters led to the exposure of a large number of humans and wild animals to radioactive substances. However, the sub-lethal consequences induced by these absorbed radiological doses remain understudied and the long-term biological impacts largely unknown. We assessed the biological effects of chronic exposure to ionizing radiation (IR) on embryonic development by exposing zebrafish embryo from fertilization and up to 120?hours post-fertilization (hpf) at dose rates of 0.5 mGy/h, 5 mGy/h and 50 mGy/h, thereby encompassing the field of low dose rates defined at 6 mGy/h. Chronic exposure to IR altered larval behaviour in a light-dark locomotor test and affected cardiac activity at a dose rate as low as 0.5 mGy/h. The multi-omics analysis of transcriptome, proteome and transcription factor binding sites in the promoters of the deregulated genes, collectively points towards perturbations of neurogenesis, muscle development, and retinoic acid (RA) signaling after chronic exposure to IR. Whole-mount RNA in situ hybridization confirmed the impaired expression of the transcription factors her4.4 in the central nervous system and myogenin in the developing muscles of exposed embryos. At the organ level, the assessment of muscle histology by transmission electron microscopy (TEM) demonstrated myofibers disruption and altered neuromuscular junctions in exposed larvae at 5 mGy/h and 50 mGy/h. The integration of these multi-level data demonstrates that chronic exposure to low dose rates of IR has an impact on neuronal and muscle progenitor cells, that could lead to motility defects in free swimming larvae at 120 hpf. The mechanistic understanding of these effects allows us to propose a model where deregulation of RA signaling by chronic exposure to IR has pleiotropic effects on neurogenesis and muscle development.
Project description:In zebrafish, parental exposure to ionizing radiation has been associated with effects in offspring, such as 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 hours 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). 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. One year after exposure, embryos from the 8.7 mGy/h group exhibited 2455(61.8% 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, which could be linked to effects in adults and offspring, such as DNA damage and lipid peroxidation. Interestingly, pathways between the two groups were oppositely regulated. Our results indicate latent effects following ionizing radiation exposure in parents that can be transmitted to offspring and warrants monitoring effects over subsequent generations. Overall design: One month after exposure, mRNA from F1 5.5 hpf embryos from parents exposed to 8.7 and 53 mGy/h gamma radiation during gametogenesis was sequenced on the Illumina 4000 platform with three replicas per treatment. One year after exposure, mRNA from F1 embryos from the same parents exposed to 8.7 mGy/h was sequenced with three biological replicates. In both cases, F1 embryos from non-exposed parents were used as control and mRNA sequenced in triplicates, taken at the same time points as the exposed samples.
Project description:The aim of this study was to carry out a comprehensive examination of potential genotoxic effects of low doses of tritium delivered chronically to mice and to compare these effects to the ones resulting from equivalent doses of gamma-irradiation. Mice were chronically exposed for one or eight months to either tritiated water (HTO) or organically bound tritium (OBT) in drinking water at concentrations of 10 kBq/L, 1 MBq/L or 20 MBq/L. Dose rates of internal β-particle resulting from such tritium treatments were calculated and matching external gamma-exposures were carried out. We measured cytogenetic damage in bone marrow and in peripheral blood lymphocytes (PBLs) and the cumulative tritium doses (0.009 - 181 mGy) were used to evaluate the dose-response of OBT in PBLs, as well as its relative biological effectiveness (RBE). Neither tritium, nor gamma exposures produced genotoxic effects in bone marrow. However, significant increases in chromosome damage rates in PBLs were found as a result of chronic OBT exposures at 1 and 20 M Bq/L, but not at 10 kBq/L. When compared to an external acute gamma-exposure <i>ex vivo</i>, the RBE of OBT for chromosome aberrations induction was evaluated to be significantly higher than 1 at cumulative tritium doses below 10 mGy. Although found non-existent at 10 kBq/L (the WHO limit), the genotoxic potential of low doses of tritium (>10 kBq/L), mainly OBT, may be higher than currently assumed.
Project description:In the current study a systematic investigation of life stage, tissue and cell dependent sensitivity to ionizing radiation in the nematode Caenorhabditis elegans was conducted. This revealed that individuals that have reached the post-mitotic L4 stage showed no significant effects with respect to mortality, morbidity or reproduction when subjected to either acute dose ≤6 Gy(1500 mGy/h) or chronic exposure ≤4 Gy( ≤ 100 mGy/h). In contrast, chronic exposure from embryo to young adult stage caused a dose and dose rate dependent reprotoxicitiy with 43% reduction in total brood size at 6.7Gy (107 mGy/h). Systematic targeted irradiation of developmental stages showed that exposure during L1 to young L4 was sufficient to induce reprotoxic effects. Exposure during these stages was associated with a dose rate dependent genotoxic effects on gonads with 1.7 to 3.2 fold increase in germ cell apoptosis in larvae subjected to 40-100 mGy/h, respectively. Importantly, exposure to gamma radiation significantly impaired spermatogenesis in a dose rate dependent manner. The observed reduction in the number of spermatids accounted for xx% of the reprotoxic effects, thus signifying spermatids as the most radiosensitive cell type in C. elegans. Molecular responses analyzed by RNAseq of nematodes irradiated from L1 to L4 stage revealed a significant enrichment of genes related to both male and hermaphrodite reproductive processes. Gene network analysis identified adverse genotoxic effects related to down-regulation of genes required for spindle formation and sperm meiosis/maturation, including smz-1, smz-2 and htas-1. The expression of a subset of 28 set-17 regulated Major Sperm Proteins (MSP) required for spermatids production was correlated to the reduction in reproduction and the number of spermatids, thus corroborating 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 did however maintain much of their reproductive capacity in spite of the reduced growth.
Project description:Zebrafish larvae (Danio rerio) are among the most used model species to test biological effects of different substances in biomedical research, neuroscience and ecotoxicology. Most tests are based on changes in swimming activity of zebrafish larvae by using commercially available high-throughput screening systems. These systems record and analyse behaviour patterns using visible (VIS) and near-infrared (NIR) light sources, to simulate day (VIS) and night (NIR) phases, which allow continuous recording of the behaviour using a NIR sensitive camera. So far, however, the sensitivity of zebrafish larvae to NIR has never been tested experimentally, although being a critical piece of information for interpreting their behaviour under experimental conditions. Here, we investigated the swimming activity of 96 hpf (hours post fertilization) and 120 hpf zebrafish larvae under light sources of NIR at 860 nm and at 960 nm wavelength and under VIS light. A thermal source was simultaneously presented opposite to one of the light sources as control. We found that zebrafish larvae of both larval stages showed a clear negative phototactic response towards 860 nm NIR light and to VIS light, but not to 960 nm NIR light. Our results demonstrated that zebrafish larvae are able to perceive NIR at 860 nm, which is almost identical to the most commonly used light source in commercial screening systems (NIR at 850 nm) to create a dark environment. These tests, however, are not performed in the dark from the zebrafish´s point of view. We recommend testing sensitivity of the used test organism before assuming no interaction with the applied light source of commonly used biosensor test systems. Previous studies on biological effects of substances to zebrafish larvae should be interpreted with caution.
Project description:Gamma radiation produces DNA instability and impaired phenotype. Previously, we observed negative effects on phenotype, DNA methylation, and gene expression profiles, in offspring of zebrafish exposed to gamma radiation during gametogenesis. We hypothesize that previously observed effects are accompanied with changes in the expression profile of non-coding RNAs, inherited by next generations. Non-coding RNA expression profile was analysed in F<sub>1</sub> offspring (5.5 h post-fertilization) by high-throughput sequencing 1 year after parental irradiation (8.7 mGy/h, 5.2 Gy total dose). Using our previous F<sub>1</sub>-? genome-wide gene expression data (GSE98539), hundreds of mRNAs were predicted as targets of differentially expressed (DE) miRNAs, involved in pathways such as insulin receptor, NFkB and PTEN signalling, linking to apoptosis and cancer. snRNAs belonging to the five major spliceosomal snRNAs were down-regulated in the F<sub>1</sub>-? group, Indicating transcriptional and post-transcriptional alterations. In addition, DEpiRNA clusters were associated to 9 transposable elements (TEs) (LTR, LINE, and TIR) (p?=?0.0024), probable as a response to the activation of these TEs. Moreover, the expression of the lincRNAs malat-1, and several others was altered in the offspring F<sub>1</sub>, in concordance with previously observed phenotypical alterations. In conclusion, our results demonstrate diverse gamma radiation-induced alterations in the ncRNA profiles of F<sub>1</sub> offspring observable 1 year after parental irradiation.
Project description:Zebrafish embryos/larvae are a choice model system for studying early stages of vertebrate development and how these stages can be perturbed by environmental stressors. Petrochemical combustion products elicit developmental toxicity that is associated with transcriptome changes in zebrafish embryos (ZFE). We used microarrays to characterize transcriptome changes in ZFE that had been exposed in the laboratory to oil emulsions collected from the GoM shoreline, ~9 weeks after the Deepwater Horizon blowout. ZFE were exposed to oil emulsions collected (July 1-4, 2010) at the GoM shoreline. ZFE were exposed to emulsions, consisting of varying amounts of alkanes, polynuclear aromatic hydrocarbons (PAHs), sand and seawater that had been M-bM-^@M-^\butteredM-bM-^@M-^] on the bottom of cups, in which ZFE were suspended in zebrafish medium. ZFE development was monitored by light microscopy. M-BM- Control ZFE were suspended in zebrafish medium without emulsions. Gross developmental abnormalities, including axial changes, altered swimming patterns and yolk sac and pericardial sac swelling were noted and photographed. M-BM- M-BM- ZFE/larvae were collected after exposures ended, and placed in clean medium until 192 hr post-fertilization, when larvae were collected for RNA extraction and hybridization on Affymetrix zebrafish microarrays.
Project description:Although radiation effects have been extensively studied, the biological effects of low-dose radiation (LDR) are controversial. This study investigates LDR-induced alterations in locomotive behavior and gene expression profiles of Drosophila melanogaster. We measured locomotive behavior using larval pupation height and rapid iterative negative geotaxis (RING) assay after exposure to 0.1 Gy gamma-radiation (dose rate of 16.7 mGy/h). We also observed chronic LDR effects on development (pupation and eclosion rates) and longevity (life span). To identify chronic LDR effects on gene expression, we performed whole-genome expression analysis using gene-expression microarrays, and confirmed the results using quantitative real-time PCR. Pupation height was significantly higher after LDR treatment at the first larval instar. Locomotive behavior of male flies was significantly greater approximately 3M-BM--5 weeks after LDR, but pupation and eclosion rates and life spans were not significantly different. Genome-wide expression analysis identified 344 genes that were differentially expressed in irradiated larvae compared with those of controls. We identified several genes belonging to larval behavior functional groups such as locomotive behavior and oxidation reduction, and genes involved in conventional functional groups modulated by irradiation such as defense response, sensory and perception. Four candidate genes were confirmed as differentially expressed genes in irradiated larvae using qRT-PCR. These data suggest that LDR stimulates locomotion-related genes, and these genes can be used as potential markers for LDR. Eggs were collected from 5-day-old female flies and cultivated for 24 h on standard medium. Then, twenty larvae were manually selected and seeded on fresh standard medium in a new vial. After transfer, the experimental group of first-instar larvae was immediately irradiated with chronic gamma-radiation at a dose rate of 16.7 mGy/h. After treatment, gamma-irradiated flies and non-irradiated control flies were maintained in the same incubator at 25degC.
Project description:For analysis of gene expression changes in the zebrafish larvae heart in response to TCDD exposure, three replicate samples of heart tissue were collected at 73, 74, 76 and 84 hours post fertilization from larvae exposed to 1 ng/ml TCDD or vehicle from 72 - 73 hours post fertilization. For analysis of gene expression changes in the extracardiac tissue in response to TCDD exposure, three replicate samples of zebrafish larvae bodies with the heart tissue removed were collected at 73, 74, 76 and 84 hours post fertilization from larvae exposed to 1 ng/ml TCDD or vehicle from 72 - 73 hours post fertilization.