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.

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.

Low-solubility particles and a Trojan-horse type mechanism of toxicity: the case of cobalt oxide on human lung cells.

BACKGROUND: The mechanisms of toxicity of metal oxide particles towards lung cells are far from being understood. In particular, the relative contribution of intracellular particulate versus solubilized fractions is rarely considered as it is very challenging to assess, especially for low-solubility particles such as cobalt oxide (Co3O4). METHODS: This study was possible owing to two highly sensitive, independent, analytical techniques, based on single-cell analysis, using ion beam microanalysis, and on bulk analysis of cell lysates, using mass spectrometry. RESULTS: Our study shows that cobalt oxide particles, of very low solubility in the culture medium, are readily incorporated by BEAS-2B human lung cells through endocytosis via the clathrin-dependent pathway. They are partially solubilized at low pH within lysosomes, leading to cobalt ions release. Solubilized cobalt was detected within the cytoplasm and the nucleus. As expected from these low-solubility particles, the intracellular solubilized cobalt content is small compared with the intracellular particulate cobalt content, in the parts-per-thousand range or below. However, we were able to demonstrate that this minute fraction of intracellular solubilized cobalt is responsible for the overall toxicity. CONCLUSIONS: Cobalt oxide particles are readily internalized by pulmonary cells via the endo-lysosomal pathway and can lead, through a Trojan-horse mechanism, to intracellular release of toxic metal ions over long periods of time, involving specific toxicity.

Mitochondrial gene expression, antioxidant responses, and histopathology after cadmium exposure.

The present study investigates cadmium effects on the transcription of mitochondrial genes of Procambarus clarkii after acute (0.05, 0.5, and 5 mg Cd/L; 4-10 days) and chronic exposures (10 µg Cd/L; 30-60 days). Transcriptional responses of cox1, atp6, and 12S using quantitative real-time RT-PCR were assessed in gills and hepatopancreas. Additionally, the expression levels of genes involved in detoxification and/or oxidative stress responses [mt, sod(Mn)] and enzymatic activities of antioxidants (SOD, CAT, GPX, and GST) were analyzed. The histopathological effects in hepatopancreas of crayfish were evaluated by light microscopy. Relationships between endpoints at different levels of biological organization and Cd bioaccumulation were also examined. Cd induced high levels of bioaccumulation, which was followed by mitochondrial dysfunction and histological alterations in both experiments. Moreover, perturbations in the defence mechanisms against oxidative stress tended to increase with time. Results also showed that molecular responses can vary depending on the intensity and duration of the chemical stress applied to the organisms and that the study of mt gene expression levels seemed to be the best tool to assess Cd intoxication.

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.

Biology-based modeling to analyze uranium toxicity data on Daphnia magna in a multigeneration study.

Recent studies have investigated chronic toxicity of waterborne depleted uranium on the life cycle and physiology of Daphnia magna. In particular, a reduction in food assimilation was observed. Our aims here were to examine whether this reduction could fully account for observed effects on both growth and reproduction, for three successive generations, and to investigate through microscope analyses whether this reduction resulted from direct damage to the intestinal epithelium. We analyzed data obtained by exposing Daphnia magna to uranium over three successive generations. We used energy-based models, which are both able to fit simultaneously growth and reproduction and are biologically relevant. Two possible modes of action were compared - decrease in food assimilation rate and increase in maintenance costs. In our models, effects were related either to internal concentration or to exposure concentration. The model that fitted the data best represented a decrease in food assimilation related to exposure concentration. Furthermore, observations of consequent histological damage to the intestinal epithelium, together with uranium precipitates in the epithelial cells, supported the assumption that uranium has direct effects on the digestive tract. We were able to model the data in all generations and showed that sensitivity increased from one generation to the next, in particular through a significant increase of the intensity of effect, once the threshold for appearance of effects was exceeded.

Effects of uranium uptake on transcriptional responses, histological structures and survival rate of the crayfish Procambarus clarkii.

This work aims to investigate the accumulation levels and effects (transcriptional responses, histopathology and survival rate) associated with a wide range of dissolved uranium (U) concentrations (0, 0.03, 0.6, 4 and 8 mg/L of U) on adult male crayfish Procambarus clarkii during 4 (T4) and 10 (T10) days of exposure. The follow-up of the crayfish mortality showed that P. clarkii was highly resistant to U. Increasing waterborne U concentrations led to increasing bioaccumulation in key crayfish organs and increasing histological damages. U distribution in tissues was also evaluated using transmission electron microscopy and showed the presence of a detoxified form of U in the gill's epithelium in the shape of flakes. Expression levels of mitochondrial genes (cox1, atp6 and 12S gene) and genes involved in oxidative stress (sod(Mn) and mt) were examined together with the housekeeping gene 18S. atp6 and mt genes of P. clarkii were cloned and sequenced before analysis. Significant correlations were observed between U bioaccumulation and the down-regulation of both cox1 and sod(Mn) genes. This work provides a first U toxicogenomic and histopathological pattern of P. clarkii, identify U biomarkers and associate gene expression endpoints to accumulation levels. It also provides new insights into the mechanisms involved in U stress.

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.

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.

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.

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.

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.

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.

Biodistribution and clearance of instilled carbon nanotubes in rat lung.

BACKGROUND: Constituted only by carbon atoms, CNT are hydrophobic and hardly detectable in biological tissues. These properties make biokinetics and toxicology studies more complex. METHODS: We propose here a method to investigate the biopersistence of CNT in organism, based on detection of nickel, a metal present in the MWCNT we investigated. RESULTS AND CONCLUSION: Our results in rats that received MWCNT by intratracheal instillation, reveal that MWCNT can be eliminated and do not significantly cross the pulmonary barrier but are still present in lungs 6 months after a unique instillation. MWCNT structure was also showed to be chemically modified and cleaved in the lung. These results provide the first data of CNT biopersistence and clearance at 6 months after respiratory administration.

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.

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.

Effect of selenate on growth and photosynthesis of Chlamydomonas reinhardtii.

Algal communities play a crucial role in aquatic food webs by facilitating the transfer of dissolved inorganic selenium (both an essential trace element and a toxic compound for a wide variety of organisms) to higher trophic levels. The dominant inorganic chemical species of selenium in freshwaters are selenite (SeO(3)(2-)) and selenate (SeO(4)(2-)). At environmental concentrations, selenite is not likely to have direct toxic effects on phytoplankton growth [Morlon, H., Fortin, C., Floriani, M., Adam, C., Garnier-Laplace, J., Boudou, A., 2005a. Toxicity of selenite in the unicellular green alga Chlamydomonas reinharditii: comparison between effects at the population and sub-cellular level. Aquat. Toxicol. 73(1), 65-78]. The effects of selenate, on the other hand, are poorly documented. We studied the effects of selenate on Chlamydomonas reinhardtii growth (a common parameter in phytotoxicity tests). Growth inhibition (96-h IC(50)) was observed at 4.5+/-0.2 microM selenate (p<0.001), an effective concentration which is low compared to environmental concentrations. Growth inhibition at high selenium concentrations may result from impaired photosynthesis. This is why we also studied the effects of selenate on the photosynthetic process (not previously assessed in this species to our knowledge) as well as selenate's effects on cell ultrastructure. The observed ultrastructural damage (chloroplast alterations, loss of appressed domains) confirmed that chloroplasts are important targets in the mechanism of selenium toxicity. Furthermore, the inhibition of photosynthetic electron transport evaluated by chlorophyll fluorescence induction confirmed this hypothesis and demonstrated that selenate disrupts the photosynthetic electron chain. Compared to the classical 'growth inhibition' parameter used in phytotoxicity tests, cell diameter and operational photosynthetic yield were more sensitive and may be convenient tools for selenate toxicity assessment in non-target plants.