Exploring uranium bioaccumulation in the brown alga Ascophyllum nodosum: insights from multi-scale spectroscopy and imaging.

Legacy radioactive waste can be defined as the radioactive waste produced during the infancy of the civil nuclear industry's development in the mid-20th Century, a time when, unfortunately, waste storage and treatment were not well planned. The marine environment is one of the environmental compartments worth studying in this regard because of legacy waste in specific locations of the seabed. Comprising nearly 70% of the earth's service, the oceans are the largest and indeed the final destination for contaminated fresh waters. For this reason, long-term studies of the accumulation biochemical mechanisms of metallic radionuclides in the marine ecosystem are required. In this context the brown algal compartment may be ecologically relevant because of forming large and dense algal beds in coastal areas and potential important biomass for contamination. This report presents the first step in the investigation of uranium (U, an element used in the nuclear cycle) bioaccumulation in the brown alga Ascophyllum nodosum using a multi-scale spectroscopic and imaging approach. Contamination of A. nodosum specimens in closed aquaria at 13 °C was performed with a defined quantity of U(VI) (10-5 M). The living algal uptake was quantified by ICP-MS and a localization study in the various algal compartments was carried out by combining electronic microscopy imaging (SEM), X-ray Absorption spectroscopy (XAS) and micro X-ray Florescence (µ-XRF). Data indicate that the brown alga is able to concentrate U(VI) by an active bioaccumulation mechanism, reaching an equilibrium state after 200 h of daily contamination. A comparison between living organisms and dry biomass confirms a stress-response process in the former, with an average bioaccumulation factor (BAF) of 10 ± 2 for living specimens (90% lower compared to dry biomass, 142 ± 5). Also, these results open new perspectives for a potential use of A. nodosum dry biomass as uranium biosorbent. The different partial BAFs (bioaccumulation factors) range from 3 (for thallus) to 49 (for receptacles) leading to a compartmentalization of uranium within the seaweed. This reveals a higher accumulation capacity in the receptacles, the algal reproductive parts. SEM images highlight the different tissue distributions among the compartments with a superficial absorption in the thallus and lateral branches and several hotspots in the oospheres of the female individuals. A preliminary speciation XAS analysis identified a distinct U speciation in the gametes-containing receptacles as a pseudo-autunite phosphate phase. Similarly, XAS measurements on the lateral branches (XANES) were not conclusive with regards to the occurrence of an alginate-U complex in these tissues. Nonetheless, the hypothesis that alginate may play a role in the speciation of U in the algal thallus tissues is still under consideration.

Uranium Speciation in a Chalky Soil.

In this article, the speciation and behavior of anthropogenic metallic uranium deposited on natural soil are approached by combining EXAFS (extended X-ray absorption fine structure) and TRLFS (time-resolved laser-induced fluorescence spectroscopy). First, uranium (uranyl) speciation was determined along the vertical profile of the soil and bedrock by linear combination fitting of the EXAFS spectra. It shows that uranium migration is strongly limited by the sorption reaction onto soil and rock constituents, mainly mineral carbonates and organic matter. Second, uranium sorption isotherms were established for calcite, chalk, and chalky soil materials along with EXAFS and TRLFS analysis. The presence of at least two adsorption complexes of uranyl onto carbonate materials (calcite) could be inferred from TRLFS. The first uranyl tricarbonate complex has a liebigite-type structure and is dominant for low loads on the carbonate surface (<10 mgU/kg(rock)). The second uranyl complex is incorporated into the calcite for intermediate (∼10 to 100 mgU/kg(rock)) to high (high: >100 mgU/kg(rock)) loads. Finally, the presence of a uranium-humic substance complex in subsurface soil materials was underlined in the EXAFS analysis by the occurrence of both monodentate and bidentate carboxylate (or/and carbonate) functions and confirmed by sorption isotherms in the presence of humic acid. This observation is of particular interest since humic substances may be mobilized from soil, potentially enhancing uranium migration under colloidal form.

Development of an Efficient FRET-Based Ratiometric Uranium Biosensor.

The dispersion of uranium in the environment can pose a problem for the health of humans and other living organisms. It is therefore important to monitor the bioavailable and hence toxic fraction of uranium in the environment, but no efficient measurement methods exist for this. Our study aims to fill this gap by developing a genetically encoded FRET-based ratiometric uranium biosensor. This biosensor was constructed by grafting two fluorescent proteins to both ends of calmodulin, a protein that binds four calcium ions. By modifying the metal-binding sites and the fluorescent proteins, several versions of the biosensor were generated and characterized in vitro. The best combination results in a biosensor that is affine and selective for uranium compared to metals such as calcium or other environmental compounds (sodium, magnesium, chlorine). It has a good dynamic range and should be robust to environmental conditions. In addition, its detection limit is below the uranium limit concentration in drinking water defined by the World Health Organization. This genetically encoded biosensor is a promising tool to develop a uranium whole-cell biosensor. This would make it possible to monitor the bioavailable fraction of uranium in the environment, even in calcium-rich waters.

Optimized Coordination of Uranyl in Engineered Calmodulin Site 1 Provides a Subnanomolar Affinity for Uranyl and a Strong Uranyl versus Calcium Selectivity.

As an alpha emitter and chemical toxicant, uranium toxicity in living organisms is driven by its molecular interactions. It is therefore essential to identify main determinants of uranium affinity for proteins. Others and we showed that introducing a phosphoryl group in the coordination sphere of uranyl confers a strong affinity of proteins for uranyl. In this work, using calmodulin site 1 as a template, we modulate the structural organization of a metal-binding loop comprising carboxylate and/or carbonyl ligands and reach affinities for uranyl comparable to that provided by introducing a strong phosphoryl ligand. Shortening the metal binding loop of calmodulin site 1 from 12 to 10 amino acids in CaMΔ increases the uranyl-binding affinity by about 2 orders of magnitude to log KpH7 = 9.55 ± 0.11 (KdpH7 = 280 ± 60 pM). Structural analysis by FTIR, XAS, and molecular dynamics simulations suggests an optimized coordination of the CaMΔ-uranyl complex involving bidentate and monodentate carboxylate groups in the uranyl equatorial plane. The main role of this coordination sphere in reaching subnanomolar dissociation constants for uranyl is supported by similar uranyl affinities obtained in a cyclic peptide reproducing CaMΔ binding loop. In addition, CaMΔ presents a uranyl/calcium selectivity of 107 that is even higher in the cyclic peptide.

Uranium Uptake in Paracentrotus lividus Sea Urchin, Accumulation and Speciation.

Uranium speciation and bioaccumulation were investigated in the sea urchin Paracentrotus lividus. Through accumulation experiments in a well-controlled aquarium followed by ICP-OES analysis, the quantification of uranium in the different compartments of the sea urchin was performed. Uranium is mainly distributed in the test (skeletal components), as it is the major constituent of the sea urchin, but in terms of quantity of uranium per gram of compartment, the following rating: intestinal tract > gonads ≫ test, was obtained. Combining both extended X-ray Absorption Spectroscopy and time-resolved laser-induced fluorescence spectroscopic analysis, it was possible to identify two different forms of uranium in the sea urchin, one in the test, as a carbonato-calcium complex, and the second one in the gonads and intestinal tract, as a protein complex. Toposome is a major calcium-binding transferrin-like protein contained within the sea urchin. EXAFS data fitting of both contaminated organs in vivo and the uranium-toposome complex from protein purified out of the gonads revealed that it is suspected to complex uranium in gonads and intestinal tract. This hypothesis is also supported by the results from two imaging techniques, i.e., Transmission Electron Microscopy and Scanning Transmission X-ray Microscopy. This thorough investigation of uranium uptake in sea urchin is one of the few attempts to assess the speciation in a living marine organism in vivo.

Is hydroxypyridonate 3,4,3-LI(1,2-HOPO) a good competitor of fetuin for uranyl metabolism?

Uranium is widespread in the environment, resulting both from natural occurrences and anthropogenic activities. Its toxicity is mainly chemical rather than radiological. In the blood it is transported as uranyl UO22+ cation and forms complexes with small ligands like carbonates and with some proteins. From there it reaches the skeleton, its main target organ for accumulation. Fetuin is a serum protein involved in biomineralization processes, and it was demonstrated to be the main UO22+-binder in vitro. Fetuin's life cycle ends in bone. It is thus suspected to be a key protagonist of U accumulation in this organ. Up to now, there has been no effective treatment for the removal of U from the body and studies devoted to the interactions involving chelating agents with both UO22+ and its protein targets are lacking. The present work aims at studying the potential role of 3,4,3-LI(1,2-HOPO) as a promising chelating agent in competition with fetuin. The apparent affinity constant of 3,4,3-LI(1,2-HOPO) was first determined, giving evidence for its very high affinity similar to that of fetuin. Chromatography experiments, aimed at identifying the complexes formed and quantifying their UO22+ content, and spectroscopic structural investigations (XAS) were carried out, demonstrating that 3,4,3-LI(1,2-HOPO) inhibits/limits the formation of fetuin-uranyl complexes under stoichiometric conditions. But surprisingly, possible ternary complexes stable enough to remain present after the chromatographic process were identified under sub-stoichiometric conditions of HOPO versus fetuin. These results contribute to the understanding of the mechanisms accounting for U residual accumulation despite chelation therapy after internal contamination.