Reactivity of U-associated osteopontin with lactoferrin: a one-to-many complex.

Uranium is the heaviest natural element, mainly found in aqueous medium as the hexavalent uranyl ion (UO22+). Bones are the main organs in which uranium accumulates, depending on as yet unknown molecular and cellular mechanisms. Recently, it has been revealed that osteopontin (OPN), a protein involved in bio-mineralization processes, and its main naturally occurring cleaved form (fOPN), have nanomolar affinities for UO22+. The binding of UO22+ is due to both the phosphorylation sites and acidic residues of these proteins and is accompanied by a slight gain in secondary structure. OPN is an Intrinsically Disordered Protein (IDP), a family of proteins which play a crucial role in several interaction networks, where phosphorylations are thought to be key elements. OPN has been shown to bind lactoferrin (LF) and the two proteins have antagonist functions in the modulation of the bio-mineralization process. However, to date, there has been no evidence that UO22+ and LF compete in their binding to OPN or not. Based on a series of convergent experimental data, this study first addressed in detail the LF/fOPN interaction and proposed a LF:fOPN 4/1 maximal stoichiometry. Moreover the phosphorylations were demonstrated to be necessary for the stability of such complexes. The interaction of preformed UO22+/fOPN complexes with LF was also investigated and the occurrence of several entities involving the three partners was demonstrated. These complexes did not reveal any significant conformational changes compared to those obtained in the absence of UO22+. The results have shown not only that LF and UO22+ do not compete, but also that these complexes are likely to be more stable than LF/fOPN complexes, as indicated by their melting temperature (Tm) values. The potential impact of those uranyl-stabilized ternary complexes on some biological pathways now remains to be assessed. Nonetheless, this work has contributed to shedding light on the formation of stable ternary complexes involving a large structured protein, an IDP and an exogenous metal.

Micro-distribution of uranium in bone after contamination: new insight into its mechanism of accumulation into bone tissue.

After internal contamination, uranium rapidly distributes in the body; up to 20 % of the initial dose is retained in the skeleton, where it remains for years. Several studies suggest that uranium has a deleterious effect on the bone cell system, but little is known regarding the mechanisms leading to accumulation of uranium in bone tissue. We have performed synchrotron radiation-based micro-X-ray fluorescence (SR µ-XRF) studies to assess the initial distribution of uranium within cortical and trabecular bones in contaminated rats' femurs at the micrometer scale. This sensitive technique with high spatial resolution is the only method available that can be successfully applied, given the small amount of uranium in bone tissue. Uranium was found preferentially located in calcifying zones in exposed rats and rapidly accumulates in the endosteal and periosteal area of femoral metaphyses, in calcifying cartilage and in recently formed bone tissue along trabecular bone. Furthermore, specific localized areas with high accumulation of uranium were observed in regions identified as micro-vessels and on bone trabeculae. These observations are of high importance in the study of the accumulation of uranium in bone tissue, as the generally proposed passive chemical sorption on the surface of the inorganic part (apatite) of bone tissue cannot account for these results. Our study opens original perspectives in the field of exogenous metal bio-mineralization.

Assessment of CE-ICP/MS hyphenation for the study of uranyl/protein interactions.

Identification of uranyl transport proteins is key to develop efficient detoxification approaches. Therefore, analytical approaches have to be developed to cope with the complexity of biological media and allow the analysis of metal speciation. CE-ICP/MS was used to combine the less-intrusive character and high separation efficiency of CE with the sensitive detection of ICP/MS. The method was based on the incubation of samples with uranyl prior to the separation. Electrophoretic buffers were compared to select a 10 mM Tris to 15 mM NaCl buffer, which enabled analyses at pH 7.4 and limited dissociation. This method was applied to the analysis of a serum. Two main fractions were observed. By comparison with synthetic mixtures of proteins, the first one was attributed to fetuin and in a lesser extent to HSA, and the second one to uranyl unbound to proteins. The analysis showed that fetuin was likely to be the main target of uranyl. CE-ICP/MS was also used to investigate the behavior of the fetuin-uranyl complex, in the presence of carbonate, an abundant complexing agent of uranyl in blood. This method enabled association constants determination, suggesting the occurrence of both FETUA(UO2(2+)) and FETUA(UO2(2+))(CO3(2-)) complexes, depending on the carbonate concentration.

Incorporation of uranium into a biomimetic apatite: physicochemical and biological aspects.

Bone is the main target organ for the storage of several toxic metals, including uranium. But the mode of action of uranium on bones remains poorly understood. To better assess the impact of uranium on bone cells, synthetic biomimetic apatites encompassing a controlled amount of uranium were prepared and analyzed. This study revealed the physicochemical impact of uranium on apatite mineralization: the presence of the metal induces a loss of crystallinity and a lower mineralization rate. The prepared samples were then used as substrates for bone cell culture. Osteoblasts were not sensitive to the presence of uranium in the support, whereas previous results showed a deleterious effect of uranium introduced into a cell culture solution. This work should therefore have some original prospects within the context of toxicological studies concerning the effect of metallic cations on bone cell systems.

Alternate dipping preparation of biomimetic apatite layers in the presence of carbonate ions.

The classical simulated body fluids method cannot be employed to prepare biomimetic apatites encompassing metallic ions that lead to very stable phosphates. This is the case for heavy metals such as uranium, whose presence in bone mineral after contamination deserves toxicological study. We have demonstrated that existing methods, based on alternate dipping into calcium and phosphate ions solutions, can be adapted to achieve this aim. We have also especially studied the impact of the presence of carbonate ions in the medium as these are necessary to avoid hydrolysis of the contaminating metallic cations. Both the apatite-collagen complex method and a standard chemical (STD) method employing only mineral solutions lead to biomimetic apatites when calcium and carbonate ions are introduced simultaneously. The obtained materials were fully characterized and we established that the STD method tolerates the presence of carbonate ions much better, and this leads to homogeneous samples. Emphasis was set on the repeatability of the method to ensure the relevancy of further work performed on series of samples. Finally, osteoblasts cultured on these samples also proved a similar yield and standard-deviation in their adenosine triphosphate content when compared to commercially available substrates designed to study of such cell cultures.