Screening of potential uranium protein targets in fish ovaries after chronic waterborne exposure: Differences and similarities between roach and zebrafish.

Concentration of uranium (U), a naturally encountered radioactive element in earth's crust, can be enhanced in freshwater ecosystems (µg.L-1 - mg.L-1) due to various anthropogenic activities. The consequent aquatic organism exposure to U leads to its accumulation in all organs, particularly in the gonad, and in subcellular fractions (mainly the cytosol); then it is known to affect fish at several biological levels, and more particularly, at a reproduction endpoint, with a decrease in the total number of eggs, spawn events and larvae survival. The understanding of U reprotoxicity requires the fine knowledge of its speciation at molecular level, i.e., its interaction with cytosolic biomolecules. In this study, we focus on the U-protein interactions in gonads. A non-denaturating extraction protocol combined with size exclusion chromatography (SEC) allowed the separation of metal-protein complexes in ovaries of U-contaminated wild roaches before their elemental detection (ICP MS). This enables unprecedented information to be obtained about U distribution in ovaries of autochthonous fish, Rutilus rutilus, which is different in some points from that obtained in the model species, Danio rerio under controlled laboratory conditions at a similar concentration level. Finally, the ability to transpose results from model to autochthonous fish was briefly discussed.

Uranium transfer and accumulation in organs of Danio rerio after waterborne exposure alone or combined with diet-borne exposure.

Uranium (U) toxicity patterns for fish have been mainly determined under laboratory-controlled waterborne exposure conditions. Because fish can take up metals from water and diet under in situ exposure conditions, a waterborne U exposure experiment (20 µg L-1 , 20 d) was conducted in the laboratory to investigate transfer efficiency and target organ distribution in zebrafish Danio rerio compared with combined waterborne exposure (20 µg L-1 ) and diet-borne exposure (10.7 µg g-1 ). 233 Uranium was used as a specific U isotope tracer for diet-borne exposure. Bioaccumulation was examined in the gills, liver, kidneys, intestine, and gonads of D. rerio. Concentrations in the organs after waterborne exposure were approximately 500 ng g-1 fresh weight, except in the intestine (> 10 µg g-1 fresh wt) and the kidneys (200 ng g-1 fresh wt). No significant difference was observed between waterborne and diet-borne conditions. Trophic U transfer in organs was found but at a low level (< 10 ng g-1 fresh wt). Surprisingly, the intestine appeared to be the main target organ after both tested exposure modalities. The gonads (57% at 20 d) and the liver (41% at 20 d) showed the highest accumulated relative U burdens. Environ Toxicol Chem 2019;38:90-98. © 2018 SETAC.

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.

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.

Nested interactions in the combined toxicity of uranium and cadmium to the nematode Caenorhabditis elegans.

Uranium is a natural, ubiquitous radioactive element for which elevated concentrations can be found in the vicinity of some nuclear fuel cycle facilities or intensive farming areas, and most often in mixtures with other contaminants such as cadmium, due to co-occurrence in geological ores (e.g. U- or P-ore). The study of their combined effects on ecosystems is of interest to better characterize such multi-metallic polluted sites. In the present study, the toxicity of binary mixture of U and Cd on physiological parameters of the soil nematode Caenorhabditis elegans was assessed over time. Descriptive modeling using concentration and response addition reference models was applied to compare observed and expected combined effects and identify possible synergistic or antagonistic interactions. A strong antagonism between U and Cd was identified for length increase and brood size endpoints. The study revealed that the combined effects might be explained by two nested antagonistic interactions. We demonstrate that the first interaction occurred in the exposure medium. We also identified a significant second antagonistic interaction which occurred either during the toxicokinetic or toxicodynamic steps. These findings underline the complexity of interactions that may take place between chemicals and thus, highlight the importance of studying mixtures at various levels to fully understand underlying mechanisms.

Development of a non-denaturing 2D gel electrophoresis protocol for screening in vivo uranium-protein targets in Procambarus clarkii with laser ablation ICP MS followed by protein identification by HPLC-Orbitrap MS.

Limited knowledge about in vivo non-covalent uranium (U)-protein complexes is largely due to the lack of appropriate analytical methodology. Here, a method for screening and identifying the molecular targets of U was developed. The approach was based on non-denaturing 1D and 2D gel electrophoresis (ND-PAGE and ND-2D-PAGE (using ND-IEF as first dimension previously described)) in conjunction with laser ablation inductively coupled plasma mass spectrometry (LA-ICP MS) for the detection of U-containing proteins. The proteins were then identified by µbore HPLC-Orbitrap MS/MS. The method was applied to the analysis of cytosol of hepatopancreas (HP) of a model U-bioaccumulating organism (Procambarus clarkii). The imaging of uranium in 2D gels revealed the presence of 11 U-containing protein spots. Six protein candidates (i.e. ferritin, glyceraldehyde-3-phosphate dehydrogenase, triosephosphate isomerase, cytosolic manganese superoxide dismutase (Mn-SOD), glutathione S transferase D1 and H3 histone family protein) were then identified by matching with the data base of crustacea Decapoda species (e.g. crayfish). Among them, ferritin was the most important one. This strategy is expected to provide an insight into U toxicology and metabolism.

Different uranium distribution patterns in cytosolic protein pool of zebrafish gills after chronic and acute waterborne exposures.

The toxicity of uranium (U) to aquatic organisms depends notably on its compartmentalization in organs, tissues, cells as well as on its distribution among biomolecules. In order to contribute to the understanding of U accumulation and associated toxicity mechanisms in case of waterborne exposure, this study focused on U fate in the gills epithelia, uptake pathway, of the fish model Danio rerio (zebrafish). U distribution among cytosolic biomolecules was investigated after no addition (0µgL(-)(1) (c0) for 3 and 30d), chronic (20µgL(-)(1) (c20) for 30d) and acute (20µgL(-)(1) (c20) and 250µgL(-)(1) (c250) for 3d) exposures to depleted U. Cytosolic U accounted for an average of 24-32% of gills burden for c20 and c250, respectively. Size Exclusion Chromatography (SEC) coupled with Inductively Coupled Plasma-Sector Field Mass Spectrometry (ICP-SFMS) allowed identification of ecotoxicologically relevant U-containing fractions among cytosolic biomolecules as a function of exposure conditions. In c0 and c20 samples, most U (ca.80%) was found in the Low Molecular Weight fraction (LMW, <18kDa), often considered as a detoxifying fraction. In c250 exposed fish, U was equally distributed between LMW (40%) and High Molecular Weight (HMW, 150-670kDa; 40%) fractions, the latter including sensitive metalloproteins. Uranium-biomolecules were co-eluted with endogenous essential metal (Fe, Cu and Zn) species, however, no major influence on their cytosolic concentration and distribution pattern among cytosolic proteins was found.

Exploring ecotoxicological fish bioassay for the evaluation of uranium reprotoxicity.

Although reproduction in fish is known to be sensitive to metal exposure, few ecotoxicological studies have focused on the toxicological effects of metals. Because uranium (U) is naturally present in aquatic ecosystems (0.6-2 mg/L), freshwater organisms are subjected to chronic U exposure. Although new standardized assays are currently being developed to mimic realistic exposure conditions, they could be improved by taking into account the contamination that occurs throughout the life cycle of fish. The authors initially evaluated the effect of food (commercial flakes vs pure Spirulina) and ionic composition of the exposure medium on the reproductive performance of Danio rerio. The effects of U exposure on reproduction then were assessed 1) for the F0 adult stage at short exposure times (5 d, 20 d, and 40 d), and 2) for the F0 stage and the F1 generation after 200 d of exposure to control, low (20 µg U/L), and moderate (250 µg U/L) waterborne levels of U. Reproductive endpoints (reproductive success, fecundity, number of spawns, egg and larvae viability, and hatching) were measured mainly after the first spawn and after 10 d of cumulative spawns. The authors evaluated the plasticity of these endpoints and compared the effect of exposure conditions to identify the most relevant markers of the effect of U exposure on reproductive performance of D. rerio.

Development of non-denaturing off-gel isoelectric focusing for the separation of uranium-protein complexes in fish.

An off-gel non-denaturing isoelectric focusing (IEF) method was developed to separate uranium-biomolecule complexes from biological samples as a first step in a multidimensional metalloproteomic approach. Analysis of a synthetic uranium-bovine serum albumin complex demonstrated the focusing ability of the liquid-phase IEF method and the preservation of most of the uranium-protein interactions. The developed method was applied to gill cytosol prepared from zebrafish (Danio rerio) exposed to depleted uranium. The results were compared in terms of resolution, recovery, and protein identities with those obtained by in-gel IEF using an immobilized pH gradient gel strip.

Non-denaturating isoelectric focusing gel electrophoresis for uranium-protein complexes quantitative analysis with LA-ICP MS.

A non-denaturating isoelectric focusing (ND-IEF) gel electrophoresis protocol has been developed to study and identify uranium (U)-protein complexes with laser ablation-inductively coupled plasma mass spectrometry (LA-ICP MS) and electrospray ionization mass spectrometry (ESI-MS). The ND-IEF-LA-ICP MS methodology set-up was initiated using in vitro U-protein complex standards (i.e., U-bovine serum albumin and U-transferrin) allowing the assessment of U recovery to 64.4 ± 0.4 %. This methodology enabled the quantification of U-protein complexes at 9.03 ± 0.23, 15.27 ± 0.36, and 177.31 ± 25.51 nmol U L(-1) in digestive gland cytosols of the crayfish, Procambarus clarkii, exposed respectively to 0, 0.12, and 2.5 µmol of waterborne depleted U L(-1) during 10 days. ND-IEF-LA-ICP MS limit of detection was 19.3 pmol U L(-1). Elemental ICP MS signals obtained both in ND-IEF electropherograms and in size exclusion chromatograms of in vivo U-protein complexes revealed interactions between U- and Fe- and Cu-proteins. Moreover, three proteins (hemocyanin, pseudohemocyanin-2, and arginine kinase) out of 42 were identified as potential uranium targets in waterborne-exposed crayfish cytosols by microbore reversed phase chromatography coupled to molecular mass spectrometry (µRPC-ESI-MS/MS) after ND-IEF separation.

Use of diatom assemblages as biomonitor of the impact of treated uranium mining effluent discharge on a stream: case study of the Ritord watershed (Center-West France).

The rehabilitation of French former uranium mining sites has not prevented the contamination of the surrounding aquatic ecosystems with metal elements. This study assesses the impact of the discharge of treated uranium mining effluents on periphytic diatom communities to evaluate their potential of bioindication. A 7-month survey was conducted on the Ritord watercourse to measure the environmental conditions of microalgae, the non-taxonomic attributes of periphyton (photosynthesis and biomass) and to determine the specific composition of diatom assemblages grown on artificial substrates. The environmental conditions were altered by the mine waters, that contaminate the watercourse with uranium and with chemicals used in the pit-water treatment plants (BaCl2 and Al2(SO4)3). The biomass and photosynthetic activity of periphyton seemed not to respond to the stress induced by the treated mining effluents whereas the altered environmental conditions clearly impacted the composition of diatom communities. Downstream the discharges, the communities tended to be characterized by indicator species belonging to the genera Fragilaria, Eunotia and Brachysira and were highly similar to assemblages at acid mine drainage sites. The species Eunotia pectinalis var. undulata, Psammothidium rechtensis, Gomphonema lagenula and Pinnularia major were found to be sensitive to uranium effluents whereas Neidium alpinum and several species of Gomphonema tolerated this contamination. The relevance of diatoms as ecological indicator was illustrated through the changes in structure of communities induced by the discharge of uranium mining effluents and creates prospects for development of a bioindicator tool for this kind of impairment of water quality.

Relative importance of direct and trophic uranium exposures in the crayfish Orconectes limosus: Implication for predicting uranium bioaccumulation and its associated toxicity.

Pollutants that occur at sublethal concentrations in the environment may lead to chronic exposure in aquatic organisms. If these pollutants bioaccumulate, then organisms higher in the food chain may also be at risk. Increased attention has thus been focused on the relative importance of dietary uptake, but additional knowledge of the cellular distribution of metals after dietary exposure is required to assess the potential toxicity. The authors address concerns relating to increasing uranium (U) concentrations (from 12 µg/L to 2 mg/L) in the freshwater ecosystem caused by anthropogenic activities. The objective of the present study is to compare uranium bioaccumulation levels in tissues and in the subcellular environment. The authors focused on the cytosol fraction and its microlocalization (TEM-EDX) in the gills and the hepatopancreas (HP) of the crayfish Orconectes limosus after 10 d of direct exposure (at concentrations of 20, 100, and 500 µg/L) and five trophic exposure treatments (at concentrations from 1 to 20 µg/g). Results indicated that adsorption of uranium on the cuticle represents the main contribution of total uranium accumulation to the animal. Accumulation in the gills should be considered only as a marker of waterborne uranium exposure. Accumulation in the HP after trophic environmental exposure conditions was higher (18.9 ± 3.8 µg/g) than after direct exposure. Moreover, no significant difference in the subcellular distribution of uranium (50%) in HP was observed between animals that had been exposed to both types of treatment. A potential toxic effect after uranium accumulation could therefore exist after trophic exposure. This confirms the need to focus further studies on the metal (uranium) risk assessment.

Are antioxidant and transcriptional responses useful for discriminating between chemo- and radiotoxicity of uranium in the crayfish Procambarus clarkii?

The main objectives of this study were to evaluate uranium (U) toxicity in the crayfish Procambarus clarkii at a low dose of exposure and to discriminate between the chemotoxicity and radiotoxicity of U. We conducted two sets of experiments using either 30 µg L(-1) of depleted uranium (DU) or (233)U, which differ from each other only in their specific activity (DU=1.7×10(4)Bqg(-1), (233)U=3.57×10(8)Bqg(-1)). The endpoints were oxidative stress responses and mitochondrial functioning in the gills and hepatopancreas, which were measured in terms of enzyme activities and gene expression levels. U accumulation levels were measured in different organs (gills, hepatopancreas, stomach, intestine, green gland, muscles, and carapace), and internal dose rates in the hepatopancreas were compared after DU and (233)U exposures. Significant U accumulation occurred in the organs of P. clarkii, and mitochondrial damage and antioxidant responses were detected. Despite the huge difference (21,000×) in the specific activities of DU and (233)U, few significant differences in biological responses were detected in P. clarkii exposed to these two pollutants. This finding indicates that the radiotoxicity was low compared to the chemotoxicity under our exposure conditions. Finally, genes expression levels were more sensitive markers of U toxicity than enzyme activities.

Mitochondrial energetic metabolism perturbations in skeletal muscles and brain of zebrafish (Danio rerio) exposed to low concentrations of waterborne uranium.

Anthropogenic release of uranium (U), originating from the nuclear fuel cycle or military activities, may considerably increase U concentrations in terrestrial and aquatic ecosystems above the naturally occurring background levels found throughout the environment. With a projected increase in the world-wide use of nuclear power, it is important to improve our understanding of the possible effects of this metal on the aquatic fauna at concentrations commensurate with the provisional drinking water guideline value of the World Health Organization (15 µg U/L). The present study has examined the mitochondrial function in brain and skeletal muscles of the zebrafish, Danio rerio, exposed to 30 and 100 µg/L of waterborne U for 10 and 28 days. At the lower concentration, the basal mitochondrial respiration rate was increased in brain at day 10 and in muscles at day 28. This is due to an increase of the inner mitochondrial membrane permeability, resulting in a decrease of the respiratory control ratio. In addition, levels of cytochrome c oxidase subunit IV (COX-IV) increased in brain at day 10, and those of COX-I increased in muscles at day 28. Histological analyses performed by transmission electron microscopy revealed an alteration of myofibrils and a dilatation of endomysium in muscle cells. These effects were largest at the lowest concentration, following 28 days of exposure.

Determinations of uranium(VI) binding properties with some metalloproteins (transferrin, albumin, metallothionein and ferritin) by fluorescence quenching.

The interactions between uranium and four metalloproteins (Apo-HTf, HSA, MT and Apo-EqSF) were investigated using fluorescence quenching measurements. The combined use of a microplate spectrofluorometer and logarithmic additions of uranium into protein solutions allowed us to define the fluorescence quenching over a wide range of [U]/[Pi] ratios (from 0.05 to 1150) at physiologically relevant conditions of pH. Results showed that fluorescence from the four metalloproteins was quenched by UO(2)(2+). Stoichiometry reactions, fluorescence quenching mechanisms and complexing properties of metalloproteins, i.e. binding constants and binding sites densities, were determined using classic fluorescence quenching methods and curve-fitting software (PROSECE). It was demonstrated that in our test conditions, the metalloprotein complexation by uranium could be simulated by two specific sites (L(1) and L(2)). Results showed that the U(VI)-Apo-HTf complexation constant values (log K(1)=7.7, log K(2)=4.6) were slightly higher than those observed for U(VI)-HSA complex (log K(1)=6.1, log K(2)=4.8), U(VI)-MT complex (log K(1)=6.5, log K(2)=5.6) and U(VI)-Apo-EqsF complex (log K(1)=5.3, log K(2)=3.9). PROSECE fitting studies also showed that the complexing capacities of each protein were different: 550 moles of U(VI) are complexed by Apo-EqSF while only 28, 10 and 5 moles of U(VI) are complexed by Apo-HTf, HSA and MT, respectively.

Role of the olfactory receptor neurons in the direct transport of inhaled uranium to the rat brain.

Uranium presents numerous industrial and military uses and one of the most important risks of contamination is dust inhalation. In contrast to the other modes of contamination, the inhaled uranium has been proposed to enter the brain not only by the common route of all modes of exposure, the blood pathway, but also by a specific inhalation exposure route, the olfactory pathway. To test whether the inhaled uranium enter the brain directly from the nasal cavity, male Sprague-Dawley rats were exposed to both inhaled and intraperitoneally injected uranium using the (236)U and (233)U, respectively, as tracers. The results showed a specific frontal brain accumulation of the inhaled uranium which is not observed with the injected uranium. Furthermore, the inhaled uranium is higher than the injected uranium in the olfactory bulbs (OB) and tubercles, in the frontal cortex and in the hypothalamus. In contrast, the other cerebral areas (cortex, hippocampus, cerebellum and brain residue) did not show any preferential accumulation of inhaled or injected uranium. These results mean that inhaled uranium enters the brain via a direct transfer from the nasal turbinates to the OB in addition to the systemic pathway. The uranium transfer from the nasal turbinates to the OB is lower in animals showing a reduced level of olfactory receptor neurons (ORN) induced by an olfactory epithelium lesion prior to the uranium inhalation exposure. These results give prominence to a role of the ORN in the direct transfer of the uranium from the nasal cavity to the brain.

Alteration of mouse oocyte quality after a subchronic exposure to depleted Uranium.

Gametes and embryo tissues are known to represent a sensitive target to environmental toxicants exposure. Oocyte quality can impact subsequent developmental competence, pregnancy course and even adult health. The major health concern from depleted uranium (DU) is mainly centred on its chemotoxic properties as a heavy metal. Little attention was paid to the impact of uranium on female gamete quality. The aim of this research was to evaluate the effect of DU on mouse oocyte quality after 49 days of subchronic contamination in drinking water and to correlate the observed effects with the amount of DU accumulated in organs. Four different DU concentrations were investigated: 0 (control), 10 (DU10), 20 (DU20) and 40 mg L(-1) (DU40). DU did not influence the intensity of ovulation but affected oocyte quality. The proportion of healthy oocytes was reduced by half (P<0.001) from 20 mg L(-1) compared with control group (0.537; 0.497; 0.282 and 0.239 in control, DU10, DU20 and DU40 groups respectively) whereas no accumulation of DU was recorded in the ovaries whatever the dose tested. Abnormal perivitelline space (P<0.001) or absence of the 1st polar body (P<0.001) was identified as the main characteristic of DU impact. In the context of this study, the NOAEL for oocyte quality was determined at 10 mg L(-1) in drinking water (1.9 mg kg(-1)day(-1)). An increase in the dose of contamination over 20 mg L(-1) did not amplify the proportion of oocytes contracting a specific alteration but conducted to a diversification in oocytes abnormalities. Further investigations are necessary to correlate morphologic assessment of female gamete with its developmental competence.

Risk assessment after internal exposure to black sand from Camargue: uptake and prospective dose calculation.

Some beaches in the south of France present high levels of natural radioactivity mainly due to thorium (Th) and uranium (U) present in the sand. Risk assessment after internal exposure of members of the public by either inhalation or ingestion of black sand of Camargue was performed. This evaluation required some information on the human bioavailability of U and Th from this sand. In vitro assays to determine the solubility of U, Th and their progeny were performed either in simulated lung fluid, with the inhalable fraction of sand, or in both simulated gastric and intestinal fluids with a sample of the whole sand. The experimental data show that the bioavailability of these radionuclides from Camargue sand is low in the conditions of the study. Prospective dose assessment for both routes of intake show low risk after internal exposure to this sand.

Heterogeneous accumulation of uranium in the brain of rats.

Recent reports suggest that uranium can accumulate not only in known target organs, that is, kidneys or bones, but also in others such as central nervous system. In the present work, the accumulation of uranium in the brain of rats was studied after repeated exposure by inhalation, chronic exposure by ingestion and acute exposure by injection. For each route of administration, the amount of uranium entering the brain was low. The results showed different accumulation in the brain areas according to the route of intake. Injection gave a rather homogeneous distribution in the different brain areas, whereas both inhalation and ingestion yielded heterogeneous but specific accumulation. These differences in distribution suggest the operation of different mechanisms of delivery of uranium to the brain tissues.