Discovery and characterization of UipA, a uranium- and iron-binding PepSY protein involved in uranium tolerance by soil bacteria.

Uranium is a naturally occurring radionuclide. Its redistribution, primarily due to human activities, can have adverse effects on human and non-human biota, which poses environmental concerns. The molecular mechanisms of uranium tolerance and the cellular response induced by uranium exposure in bacteria are not yet fully understood. Here, we carried out a comparative analysis of four actinobacterial strains isolated from metal and radionuclide-rich soils that display contrasted uranium tolerance phenotypes. Comparative proteogenomics showed that uranyl exposure affects 39-47% of the total proteins, with an impact on phosphate and iron metabolisms and membrane proteins. This approach highlighted a protein of unknown function, named UipA, that is specific to the uranium-tolerant strains and that had the highest positive fold-change upon uranium exposure. UipA is a single-pass transmembrane protein and its large C-terminal soluble domain displayed a specific, nanomolar binding affinity for UO22+ and Fe3+. ATR-FTIR and XAS-spectroscopy showed that mono and bidentate carboxylate groups of the protein coordinated both metals. The crystal structure of UipA, solved in its apo state and bound to uranium, revealed a tandem of PepSY domains in a swapped dimer, with a negatively charged face where uranium is bound through a set of conserved residues. This work reveals the importance of UipA and its PepSY domains in metal binding and radionuclide tolerance.

Fatty acid photodecarboxylase is an ancient photoenzyme that forms hydrocarbons in the thylakoids of algae.

Fatty acid photodecarboxylase (FAP) is one of the few enzymes that require light for their catalytic cycle (photoenzymes). FAP was first identified in the microalga Chlorella variabilis NC64A, and belongs to an algae-specific subgroup of the glucose-methanol-choline oxidoreductase family. While the FAP from C. variabilis and its Chlamydomonas reinhardtii homolog CrFAP have demonstrated in vitro activities, their activities and physiological functions have not been studied in vivo. Furthermore, the conservation of FAP activity beyond green microalgae remains hypothetical. Here, using a C. reinhardtii FAP knockout line (fap), we showed that CrFAP is responsible for the formation of 7-heptadecene, the only hydrocarbon of this alga. We further showed that CrFAP was predominantly membrane-associated and that >90% of 7-heptadecene was recovered in the thylakoid fraction. In the fap mutant, photosynthetic activity was not affected under standard growth conditions, but was reduced after cold acclimation when light intensity varied. A phylogenetic analysis that included sequences from Tara Ocean identified almost 200 putative FAPs and indicated that FAP was acquired early after primary endosymbiosis. Within Bikonta, FAP was retained in secondary photosynthetic endosymbiosis lineages but absent from those that lost the plastid. Characterization of recombinant FAPs from various algal genera (Nannochloropsis, Ectocarpus, Galdieria, Chondrus) provided experimental evidence that FAP photochemical activity was present in red and brown algae, and was not limited to unicellular species. These results thus indicate that FAP was conserved during the evolution of most algal lineages where photosynthesis was retained, and suggest that its function is linked to photosynthetic membranes.

Effects of gamma ionizing radiation exposure on Danio rerio embryo-larval stages - comparison with tritium exposure.

The objective was to investigate the effects of ionizing radiation induced in zebrafish early life stages by coupling responses obtained at the molecular (genotoxicity, ROS production, gene expression) and phenotypic (tissue alteration, embryo-larval development) levels. Here we present results obtained after exposure of 3 hpf larvae to 10 days of gamma irradiation at 3.3 × 101, 1.3 × 102 and 1.2 × 103 µGy/h, close to and higher than the benchmark for protection of ecosystems towards ionizing radiations of 101 µGy/h. Dose rates used in these studies were chosen to be in the 'derived consideration reference level' (DCRL) for gamma irradiation where deleterious effects can appear in freshwater fish. Also, these dose rates were similar to the ones already tested on tritium (beta ionizing radiation) in our previous work, in order to compare both types of ionizing radiation. Results showed that gamma irradiation did not induce any effect on survival and hatching. No effect was observed on DNA damages, but ROS production was increased. Muscle damages were observed for all tested dose rates, similarly to previous results obtained with tritium (beta ionizing radiation) at similar dose rates. Some molecular responses therefore appeared to be relevant for the study of gamma ionizing radiation effects in zebrafish.

Intracellular amorphous Ca-carbonate and magnetite biomineralization by a magnetotactic bacterium affiliated to the Alphaproteobacteria.

Bacteria synthesize a wide range of intracellular submicrometer-sized inorganic precipitates of diverse chemical compositions and structures, called biominerals. Their occurrences, functions and ultrastructures are not yet fully described despite great advances in our knowledge of microbial diversity. Here, we report bacteria inhabiting the sediments and water column of the permanently stratified ferruginous Lake Pavin, that have the peculiarity to biomineralize both intracellular magnetic particles and calcium carbonate granules. Based on an ultrastructural characterization using transmission electron microscopy (TEM) and synchrotron-based scanning transmission X-ray microscopy (STXM), we showed that the calcium carbonate granules are amorphous and contained within membrane-delimited vesicles. Single-cell sorting, correlative fluorescent in situ hybridization (FISH), scanning electron microscopy (SEM) and molecular typing of populations inhabiting sediments affiliated these bacteria to a new genus of the Alphaproteobacteria. The partially assembled genome sequence of a representative isolate revealed an atypical structure of the magnetosome gene cluster while geochemical analyses indicate that calcium carbonate production is an active process that costs energy to the cell to maintain an environment suitable for their formation. This discovery further expands the diversity of organisms capable of intracellular Ca-carbonate biomineralization. If the role of such biomineralization is still unclear, cell behaviour suggests that it may participate to cell motility in aquatic habitats as magnetite biomineralization does.

In vitro assessment of cobalt oxide particle dissolution in simulated lung fluids for identification of new decorporating agents.

Inhalation of 60Co3O4 particles may occur at the work place in nuclear industry. Their low solubility may result in chronic lung exposure to γ rays. Our strategy for an improved therapeutic approach is to enhance particle dissolution to facilitate cobalt excretion, as the dissolved fraction is rapidly eliminated, mainly in urine. In vitro dissolution of Co3O4 particles was assessed with two complementary assays in lung fluid surrogates to mimic a pulmonary contamination scenario. Twenty-one molecules and eleven combinations were selected through an extensive search in the literature, based on dissolution studies of other metal oxides (Fe, Mn, Cu) and tested for dissolution enhancement of cobalt particles after 1-28 days of incubation. DTPA, the recommended treatment following cobalt contamination did not enhance 60Co3O4 particles dissolution when used alone. However, by combining molecules with different properties, such as redox potential and chelating ability, we greatly improved the efficacy of each drug used alone, leading for the highest efficacy, to a 2.7 fold increased dissolution as compared to controls. These results suggest that destabilization of the particle surface is an important initiating event for a good efficacy of chelating drugs, and open new perspectives for the identification of new therapeutic strategies.

Tritiated Water Exposure in Zebrafish (Danio rerio): Effects on the Early-Life Stages.

Tritium, a radioactive isotope of hydrogen of natural and anthropogenic origin, is ubiquitously present in the environment. Effluents of nuclear centers of production are significant anthropogenic sources. With the upcoming project of thermonuclear fusion, tritium releases in the environment may increase. It is therefore important to characterize the ecological risk linked to tritium. The effects of tritiated water (HTO) were therefore studied in zebrafish larvae exposed for 10 d to different dose rates, 1.1 × 102 , 4.1 × 102 , and 3.8 × 103 µGy/h for larvae corresponding, respectively, to a water contamination of 104 , 105 , and 106 Bq/mL of HTO. Those dose rates were higher than 10 µGy/h, which is the threshold recommended to start monitoring ecosystems where radiological contaminants are present. Mortality, embryo-larval development, immune toxicity, genotoxicity, neurotoxicity, and alterations of tissues were investigated. The results showed that HTO exposure induced DNA damage and reactive oxygen species production and modulated the expression of genes involved in detoxification processes. Moreover, modifications of the muscular tissues (degradation of myofibrils at 4 d post fertilization and disorganization of mitochondria at later stages) were observed. The results differed with HTO dose rates and with developmental stages. These results will drive future research for the development of new HTO-sensitive biomarkers and will allow us to progress in the characterization of the modes of action of tritium in fish. Environ Toxicol Chem 2020;39:648-658. © 2019 SETAC.

Effects of tritiated water on locomotion of zebrafish larvae: a new insight in tritium toxic effects on a vertebrate model species.

Tritium (3H), a radioactive isotope of hydrogen, is ubiquitously present in the environment. In a previous study, we highlighted a mis-regulation of genes involved in muscle contraction, eye transparency and response to DNA damages after exposure of zebrafish embryo-larvae from 3 hpf to 96 hpf at 0.4 and 4 mGy/h of tritiated water (HTO). The present study aimed to link this gene mis-regulation to responses observed at higher biological levels. Analyses on spontaneous tail movement, locomotor activity and heart rate were performed. Histological sections of eyes were made to evaluate the impact of HTO on eye transparency and whole embryo immunostainings were realized to assess DNA double strand breaks repair using gamma-H2AX foci. We found a decrease of basal velocity as well as a decrease of response in 96 hpf larvae exposed at 0.4 mGy/h after a tactile stimulus as compared to controls. Histological sections of larvae eyes performed after the exposure to 4 mGy/h did not show obvious differences in lens transparency or retinal development between contaminated and control organisms. Gamma-H2AX foci detection revealed no differences in the number of foci between contaminated organisms and controls, for both dose rates. Overall, results highlighted more detrimental effects of HTO exposure on locomotor behavior in 96 hpf larvae exposed at the lowest dose rate. Those results could be linked to mis-regulation of genes involved in muscle contraction found in a previous study at the same dose rate. It appears that not all effects found at the molecular scale were confirmed using higher biological scales. These results could be due to a delay between gene expression modulation and the onset of physiological disruption or homeostatic mechanisms to deal with tritium effects. However, crossing data from different scales highlighted new pathways to explore, i.e. neurotoxic pathways, for better understanding HTO effects on organisms.

A systems biology approach reveals neuronal and muscle developmental defects after chronic exposure to ionising radiation in zebrafish.

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.

Adverse effects induced by chronic gamma irradiation in progeny of adult fish not affecting parental reproductive performance.

Multigenerational studies have become of great interest in ecotoxicology since the consequence of parental exposure to contaminants on offspring generations was established in situ or in laboratory conditions. The present study mainly examined the chronic effects of external Cs-137 gamma irradiation exposure at 4 dose rates (control, 0.5, 5, and 50 mGy h-1 ) on adult zebrafish (F0) exposed for 10 d and their progeny (F1) exposed or unexposed for 4 to 5 d. The main endpoints investigated included parental reproductive performance, embryo-larval survival, DNA alterations, and reactive oxygen species (ROS) production in F0 and F1. No effects on reproductive success, fecundity, or egg fertilization rate were observed. However, drastic effects were observed on F1 exposed to 50 mGy h-1 , resulting in a mortality rate of 100%. The drastic effects were also observed when the progeny was not irradiated. It was demonstrated that the sensitivity of the embryos was mainly attributable to parental irradiation. Moreover, these drastic effects induced by adult irradiation disappeared over time when 10 d-irradiated adults were placed in a nonirradiated condition. Alterations in larval DNA were observed for the 3 dose rates, and an increase of ROS production was also shown for the 2 lowest dose rates. The present study improves our understanding of the consequences of parental exposure conditions to the progeny. Furthermore, it provides an incentive to take transmitted generational effects into account in ecological risk assessments. Environ Toxicol Chem 2019;38:2556-2567. © 2019 SETAC.

Ectosymbiotic bacteria at the origin of magnetoreception in a marine protist.

Mutualistic symbioses are often a source of evolutionary innovation and drivers of biological diversification1. Widely distributed in the microbial world, particularly in anoxic settings2,3, they often rely on metabolic exchanges and syntrophy2,4. Here, we report a mutualistic symbiosis observed in marine anoxic sediments between excavate protists (Symbiontida, Euglenozoa)5 and ectosymbiotic Deltaproteobacteria biomineralizing ferrimagnetic nanoparticles. Light and electron microscopy observations as well as genomic data support a multi-layered mutualism based on collective magnetotactic motility with division of labour and interspecies hydrogen-transfer-based syntrophy6. The guided motility of the consortia along the geomagnetic field is allowed by the magnetic moment of the non-motile ectosymbiotic bacteria combined with the protist motor activity, which is a unique example of eukaryotic magnetoreception7 acquired by symbiosis. The nearly complete deltaproteobacterial genome assembled from a single consortium contains a full magnetosome gene set8, but shows signs of reduction, with the probable loss of flagellar genes. Based on the metabolic gene content, the ectosymbiotic bacteria are anaerobic sulfate-reducing chemolithoautotrophs that likely reduce sulfate with hydrogen produced by hydrogenosome-like organelles6 underlying the plasma membrane of the protist. In addition to being necessary hydrogen sinks, ectosymbionts may provide organics to the protist by diffusion and predation, as shown by magnetosome-containing digestive vacuoles. Phylogenetic analyses of 16S and 18S ribosomal RNA genes from magnetotactic consortia in marine sediments across the Northern and Southern hemispheres indicate a host-ectosymbiont specificity and co-evolution. This suggests a historical acquisition of magnetoreception by a euglenozoan ancestor from Deltaproteobacteria followed by subsequent diversification. It also supports the cosmopolitan nature of this type of symbiosis in marine anoxic sediments.

Tritiated water exposure disrupts myofibril structure and induces mis-regulation of eye opacity and DNA repair genes in zebrafish early life stages.

Tritium (3H) is a radioactive isotope of hydrogen. In the environment, the most common form of tritium is tritiated water (HTO). The present study aimed to identify early biomarkers of HTO contamination through the use of an aquatic model, the zebrafish (Danio rerio). We used the zebrafish embryo-larvae model to investigate the modes of action of HTO exposure at dose rates of 0.4 and 4 mGy/h, dose rates expected to induce deleterious effects on fish. Zebrafish were exposed to HTO from 3 hpf (hours post fertilization) to 96 hpf. The transcriptomic effects were investigated 24 h and 96 h after the beginning of the contamination, using mRNAseq. Results suggested an impact of HTO contamination, regardless of the dose rate, on genes involved in muscle contraction (tnnt2d, tnni2a.4, slc6a1a or atp2a1l) and eye opacity (crygm2d9, crygmxl1, mipb or lim2.3) after 24 h of contamination. Interestingly, an opposite differential expression was highlighted in genes playing a role in muscle contraction and eye opacity in 24 hpf embryos when comparing dose rates, suggesting an onset of DNA protective mechanisms. The expression of h2afx and ddb2 involved in DNA repair was enhanced in response to HTO exposure. The entrainment of circadian clock and the response to H2O2 signalling pathways were enriched at 96 hpf at 0.4 mGy/h and in both stages after 4 mGy/h. Genes involved in ROS scavenging were differentially expressed only after 24 h of exposure for the lowest dose rate, suggesting the onset of early protective mechanisms against oxidative stress. Effects highlighted on muscle at the molecular scale were confirmed at a higher biological scale, as electron microscopy observations revealed sarcomere impairments in 96 hpf larvae for both dose rates. Together with other studies, the present work provides useful data to better understand modes of action of tritium on zebrafish embryos-larvae.

Toxicokinetic and toxicodynamic of depleted uranium in the zebrafish, Danio rerio.

This study investigated the accumulation pattern and biological effects (genotoxicity and histopathology) to adult zebrafish (male and female) exposed to a nominal waterborne concentration of 20 µg L-1 of depleted uranium (DU) for 28 days followed by 27 days of depuration. Accumulation pattern showed that (i) DU accumulated in brain, (ii) levels in digestive tract were higher than those measured in gills and (iii) levels remained high in kidney, brain and ovary despite the 27 days of depuration period. Genotoxicity, assessed by comet assay, was significant not only during DU exposure, but also during depuration phase. Gonads, in particular the testes, were more sensitive than gills. The histology of gonads indicated severe biological damages in males. This study improved knowledge of ecotoxic profile of uranium, for which a large range of biological effects has already been demonstrated.

Zebrafish exposure to environmentally relevant concentration of depleted uranium impairs progeny development at the molecular and histological levels.

Uranium is an actinide naturally found in the environment. Anthropogenic activities lead to the release of increasing amounts of uranium and depleted uranium (DU) in the environment, posing potential risks to aquatic organisms due to radiological and chemical toxicity of this radionucleide. Although environmental contaminations with high levels of uranium have already been observed, chronic exposures of non-human species to levels close to the environmental quality standards remain scarcely characterized. The present study focused on the identification of the molecular pathways impacted by a chronic exposure of zebrafish to 20 µg/L of DU during 10 days. The transcriptomic effects were evaluated by the use of the mRNAseq analysis in three organs of adult zebrafish, the brain the testis and the ovaries, and two developmental stages of the adult fish progeny, two-cells embryo and four-days larvae. The results highlight generic effects on the cell adhesion process, but also specific transcriptomic responses depending on the organ or the developmental stage investigated. The analysis of the transgenerational effects of DU-exposure on the four-day zebrafish larvae demonstrate an induction of genes involved in oxidative response (cat, mpx, sod1 and sod2), a decrease of expression of the two hatching enzymes (he1a and he1b), the deregulation of the expression of gene coding for the ATPase complex and the induction of cellular stress. Electron microscopy analysis of skeletal muscles on the four-days larvae highlights significant histological impacts on the ultrastructure of both the mitochondria and the myofibres. In addition, the comparison with the transcriptomic data obtained for the acetylcholine esterase mutant reveals the induction of protein-chaperons in the skeletal muscles of the progeny of fish chronically exposed to DU, pointing towards long lasting effects of this chemical in the muscles. The results presented in this study support the hypothesis that a chronic parental exposure to an environmentally relevant concentration of DU could impair the progeny development with significant effects observed both at the molecular level and on the histological ultrastructure of organs. This study provides a comprehensive transcriptomic dataset useful for ecotoxicological studies on other fish species at the molecular level. It also provides a key DU responsive gene, egr1, which may be a candidate biomarker for monitoring aquatic pollution by heavy metals.

Epigenetic, histopathological and transcriptomic effects following exposure to depleted uranium in adult zebrafish and their progeny.

This study investigated the effects of adult zebrafish exposure to a nominal concentration of 20µgL-1 of depleted uranium (DU) for six days upon DNA methylation, gene expression and the appearance of histopathological damage in their progeny. In the embryos at the 2-8 cell stage, the parental exposure induced significant DU accumulation, with levels seven times higher than those measured in the control embryos, but in larvae 96h post-fertilisation (hpf), uranium concentration had already returned to a level identical to that of the control larvae. A significant two-fold increase in the global level of DNA methylation was observed in embryos as early as the prim5 (24 hpf) stage and was still maintained at the 96 hpf stage despite the fact that DU had already been depurated at the later stage. RNA sequencing analysis indicated an impact of parental exposure upon the total RNAs transmitted from the mother to eggs, and the up-regulated genes were those associated with post-traductional protein modification and trafficking and cellular signalling pathways, whereas the down-regulated genes concerned the translational process, cell cycle regulation and several cell signalling pathways. Alterations of photoreceptor cells and the axon-axon junctions between photoreceptors were observed in the eyes of adult fish exposed for 10days to DU. Actin and myosin filament disorganisation was observed in the skeletal muscles of 96 hpf larvae, at a stage when the maternally transmitted DU had already been excreted. These data reveal the extreme sensitivity of zebrafish embryos to DU transmitted through the oocyte by exposed females.

Acclimation capacity of the three-spined stickleback (Gasterosteus aculeatus, L.) to a sudden biological stress following a polymetallic exposure.

To get closer to the environmental reality, ecotoxicological studies should no longer consider the evaluation of a single pollutant, but rather combination of stress and their interaction. The aim of this study was to determine if responses of a fish to a sudden biological stress could be modified by a prior exposure to a chemical stress (a polymetallic contamination). For this purpose, in situ experiment was conducted in three ponds in the Haute-Vienne department (France). One pond was chosen for its high uranium concentration due to uranium mine tailings, and the two other ponds, which were not submitted to these tailings. Three-spined sticklebacks (Gasterosteus aculeatus) were caged in these ponds for 14 days. After this period, fish were submitted to a biological stress, exerted by lipopolysaccharides injection after anesthesia, and were sacrificed 4 days after these injections for multi-biomarkers analyses (leucocyte viability, phagocytic capacity and reactive oxygen species production, antioxidant peptide and enzymes, lipid peroxidation and DNA damage). The pond which received uranium mine tailings had higher metallic concentrations. Without biological stress, sticklebacks caged in this pond presented an oxidative stress, with increasing of reactive oxygen species levels, modification of some parts of the antioxidant system, and lipid peroxidation. Caging in the two most metal-contaminated ponds resulted in an increase of susceptibility of sticklebacks to the biological stress, preventing their phagocytic responses to lipopolysaccharides and modifying their glutathione contents and glutathione-S-transferase activity.

How toxic is the depleted uranium to crayfish Procambarus clarkii compared with cadmium?

Due to a lack of information on the assessment of uranium's (U) toxicity, our work aimed to compare the effects of U on the crayfish Procambarus clarkii with those of the well documented metal: cadmium (Cd). Accumulation and impacts at different levels of biological organization were assessed after acute (40 µM Cd or U; 4-10 days) and chronic (0.1 µM Cd or U; 30-60 days) exposures. The survival rates demonstrated the high tolerance of this species toward both metals and showed that Cd had a greater effect on the sustainability of crayfish. The concentration levels of Cd and U accumulated in gills and hepatopancreas were compared between both conditions. Distinctions in the adsorption capacities and the mobility of the contaminants were suspected. Differences in the detoxification mechanisms of both metals using transmission electron microscopy equiped with an energy dispersive X-ray were also pointed out. In contrast, comparison between the histological structures of contaminated hepatopancreas showed similar symptoms. Principal component analyses revealed different impacts of each metal on the oxidative balance and mitochondria using enzymatic activities and gene expression levels as endpoints. The observation that U seemed to generate more oxidative stress than Cd in our conditions of exposure is discussed.

Insights into the nature of uranium target proteins within zebrafish gills after chronic and acute waterborne exposures.

New data on the nature of the protein targets of uranium (U) within zebrafish gills were collected after waterborne exposure, with the aim of a better understanding of U toxicity mechanisms. Some common characteristics of the U protein target binding properties were found, such as their role in the regulation of other essential metals and their phosphorus content. In total, 21 potential protein targets, including hemoglobin, are identified and discussed in terms of the literature.

Biodistribution and Clearance of TiO2 Nanoparticles in Rats after Intravenous Injection.

Titanium dioxide (TiO2) nanoparticles are used in many applications. Due to their small size, easy body penetration and toxicological adverse effects have been suspected. Numerous studies have tried to characterize TiO2 translocation after oral, dermal or respiratory exposure. In this study, we focused on TiO2 nanoparticle biodistribution, clearance and toxicological effects after intravenous injection, considering TiO2 translocation in the blood occurs. Using ICP-OES, transmission electron microscopy, and histological methods, we found TiO2 accumulation in liver, lungs and spleen. We estimated TiO2 nanoparticles' half life in the body to about 10 days. Clinical biomarkers were also quantified for 56 days to identify potential toxicological impact on lungs, blood, liver, spleen and kidneys. Results showed absence of toxicological effects after TiO2 intravenous injection at concentrations of 7.7 to 9.4 mg/kg.

Effects of chronic exposure to environmentally relevant concentrations of waterborne depleted uranium on the digestive tract of zebrafish, Danio rerio.

Uranium is a naturally occurring element, but activities linked to the nuclear fuel cycle can increase background levels in the surrounding waters. For this reason it is important to understand how this affects organisms residing in the water column. The objective of this study was to assess histopathological effects of uranium on the gut wall of a widely used model organism: zebrafish, Danio rerio. To this end we exposed zebrafish to 84 and 420 nM depleted uranium for over a month and then examined the histology of intestines of exposed individuals compared to controls. The gut wall of individuals exposed to 84 and 420 nM of uranium had large regions of degraded mucosa. Using transmission electron microscopy (TEM) coupled to energy-dispersive X-ray spectroscopy microanalysis (EDX) we found that uranium induced a decrease in the amount of calcium containing mitochondrial matrix granules per mitochondria. This is suggestive of perturbations to cellular metabolism and more specifically to cellular calcium homeostasis. TEM-EDX of the gut wall tissue further showed that some uranium was internalized in the nucleus of epithelial cells in the 420 nM treatment. Fluorescent in situ hybridization using specific probes to detect all eubacteria was performed on frozen sections of 6 individual fish in the 84 nM and 420 nM treatments. Bacterial colonization of the gut of individuals in the 420 nM seemed to differ from that of the controls and 84 nM individuals. We suggest that host-microbiota interactions are potentially disturbed in response to uranium induced stress. The damage induced by waterborne uranium to the gut wall did not seem to depend on the concentration of uranium in the media. We measure whole body residues of uranium at the end of the experiment and compute the mean dose rate absorbed for each condition. We discuss why effects might be uncoupled from external concentration and highlight that it is not so much the external concentration but the dynamics of internalization which are important players in the game.