The study of the isotopic fractionation of endogen elements and toxic heavy metals in living organisms for biomedical applications, and for metabolic and toxicological studies, is a cutting-edge ...research topic. This paper shows that human neuroblastoma cells incorporated small amounts of uranium (U) after exposure to 10 μM natural U, with preferential uptake of the 235U isotope with regard to 238U. Efforts were made to develop and then validate a procedure for highly accurate n(238U)/n(235U) determinations in microsamples of cells. We found that intracellular U is enriched in 235U by 0.38 ± 0.13‰ (2σ, n = 7) relative to the exposure solutions. These in vitro experiments provide clues for the identification of biological processes responsible for uranium isotopic fractionation and link them to potential U incorporation pathways into neuronal cells. Suggested incorporation processes are a kinetically controlled process, such as facilitated transmembrane diffusion, and the uptake through a high-affinity uranium transport protein involving the modification of the uranyl (UO₂2+) coordination sphere. These findings open perspectives on the use of isotopic fractionation of metals in cellular models, offering a probe to track uptake/transport pathways and to help decipher associated cellular metabolic processes.
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(UO22+) and FETUA(UO22+)(CO32−) complexes, depending on the carbonate concentration.
Determining the affinity of proteins for uranyl is key to understand the toxicity of this cation and to further develop decorporation strategies. However, usual techniques to achieve that goal often ...require specific equipment and expertise. Here, we propose a simple, efficient, fluorescence‐based method to assess the affinity of proteins and peptides for uranyl, at equilibrium and in buffered solution. We first designed and characterized an original uranyl‐binding fluorescent probe. We then built a reference scale for uranyl affinity in solution, relying on signal quenching of our fluorescent probe in presence of high‐affinity uranyl‐binding peptides. We finally validated our approach by re‐evaluating the uranyl‐binding affinity of four native proteins. We envision that this tool will facilitate the reliable and reproducible assessment of affinities of peptides and proteins for uranyl.
A fluorescent uranyl probe was designed and validated for the reliable determination of uranyl–protein affinities, at equilibrium and in biologically relevant conditions.
The monitoring of isotopic fractionations in in vitro cultured human cell samples is a very promising and under-exploited tool to help identify the metabolic processes leading to disease-induced ...isotopic fractionations or decipher metabolic pathways of toxic metals in these samples. One of the limitations is that the analytes are often present at small amounts, ranging from tens to hundreds of ng, thus making challenging low-uncertainty isotope ratio determinations. Here we present a new procedure for U, Cu and Zn purification and isotope ratio determinations in cultured human neuron-like cells exposed to natural U. A thorough study of the influence of the limiting factors impacting the uncertainty of δ238U, δ66Zn and δ65Cu is also carried out. These factors include the signal intensity, which determines the within-day measurement reproducibility, the procedural blank correction and the matrix effects, which determine the accuracy of the mass bias correction models. Given the small Cu and U amounts in the cell samples, 15–30 and 20ng respectively, a highly efficient sample introduction system was employed in order to improve the analyte transport to the plasma and, hence, the signal intensity. With this device, the procedural blanks became the main uncertainty source of δ238U and δ65Cu values, accounting over 65% of the overall uncertainty. The matrix effects gave rise to inaccuracies in the mass bias correction models for samples finally dissolved in the minimal volumes required for the analysis, 100–150µL, leading to biases for U and Cu. We will show how these biases can be cancelled out by dissolving the samples in volumes of at least 300µL for Cu and 450µL for U. Using our procedure, expanded uncertainties (k = 2) of around 0.35‰ for δ238U and 0.15‰ for δ66Zn and δ65Cu could be obtained. The analytical approach presented in this work is also applicable to other biological microsamples and can be extended to other elements and applications.
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•High-precision isotope ratio of U, Cu and Zn have been determined at ng amounts.•A thorough and systematic study of the main sources of uncertainty was carried out.•Expanded uncertainty modelled with regard to the analyte mass in the sample.•Isotopic fractionation studies in cellular models are of major importance.•Our approach can be extended to microsamples containing very small analyte amounts.
The impact of natural uranium (U) on differentiated human neuron-like cells exposed to 1, 10, 125, and 250 µM of U for seven days was assessed. In particular, the effect of the U uptake on the ...homeostatic modulation of several endogenous elements (Mg, P, Mn, Fe, Zn, and Cu), the U isotopic fractionation upon its incorporation by the cells and the evolution of the intracellular Cu and Zn isotopic signatures were studied. The intracellular accumulation of U was accompanied by a preferential uptake of
U for cells exposed to 1 and 10 µM of U, whereas no significant isotopic fractionation was observed between the extra- and the intracellular media for higher exposure U concentrations. The U uptake was also found to modulate the homeostasis of Cu, Fe, and Mn for cells exposed to 125 and 250 µM of U, but the intracellular Cu isotopic signature was not modified. The intracellular Zn isotopic signature was not modified either. The activation of the non-specific U uptake pathway might be related to this homeostatic modulation. All together, these results show that isotopic and quantitative analyses of toxic and endogenous elements are powerful tools to help deciphering the toxicity mechanisms of heavy metals.
In case of a nuclear event, contamination (broad or limited) of the population or of specific workers might occur. In such a senario, the fate of actinide contaminants may be of first concern, in ...particular with regard to human target organs like the skeleton. To improve our understanding of the toxicological processes that might take place, a mechanistic approach is necessary. For instance, ∼50% of Pu(IV) is known from biokinetic data to accumulate in bone, but the underlining mechanisms are almost unknown. In this context, and to obtain a better description of the toxicological mechanisms associated with actinides(IV), we have undertaken the investigation, on a molecular scale, of the interaction of thorium(IV) with osteopontin (OPN) a hyperphosphorylated protein involved in bone turnover. Thorium is taken here as a simple model for actinide(IV) chemistry. In addition, we have selected a phosphorylated hexapeptide (His-pSer-Asp-Glu-pSer-Asp-Glu-Val) that is representative of the peptidic sequence involved in the bone interaction. For both the protein and the biomimetic peptide, we have determined the local environment of Th(IV) within the bioactinidic complex, combining isothermal titration calorimetry, attenuated total reflectance Fourier transform infrared spectroscopy, theoretical calculations with density functional theory, and extended X-ray absorption fine structure spectroscopy at the Th LIII edge. The results demonstrate a predominance of interaction of metal with the phosphate groups and confirmed the previous physiological studies that have highlighted a high affinity of Th(IV) for the bone matrix. Data are further compared with those of the uranyl case, representing the actinyl(V) and actinyl(VI) species. Last, our approach shows the importance of developing simplified systems Th(IV)–peptide that can serve as models for more biologically relevant systems.
Uranium (U) is the heaviest naturally occurring element ubiquitously present in the Earth’s crust. Human exposure to low levels of U is, therefore, unavoidable. Recently, several studies have clearly ...pointed out that the brain is a sensitive target for U, but the mechanisms leading to the observed neurological alterations are not fully known. To deepen our knowledge of the biochemical disturbances resulting from U(VI) toxicity in neuronal cells, two complementary strategies were set up to identify the proteins that selectively bind U(VI) in human dopaminergic SH-SY5Y cells. The first strategy relies on the selective capture of proteins capable of binding U(VI), using immobilized metal affinity chromatography, and starting from lysates of cells grown in a U(VI)-free medium. The second strategy is based on the separation of U-enriched protein fractions by size-exclusion chromatography, starting from lysates of U(VI)-exposed cells. High-resolution mass spectrometry helped us to highlight 269 common proteins identified as the urano-proteome. They were further analyzed to characterize their cellular localization and biological functions. Four canonical pathways, related to the protein ubiquitination system, gluconeogenesis, glycolysis, and the actin cytoskeleton proteins, were particularly emphasized due to their high content of U(VI)-bound proteins. A semi-quantification was performed to concentrate on the ten most abundant proteins, whose physico-chemical characteristics were studied in particular depth. The selective interaction of U(VI) with these proteins is an initial element of proof of the possible metabolic effects of U(VI) on neuronal cells at the molecular level.
The uranyl cation (UO(2) (2+)) can be suspected to interfere with the binding of essential metal cations to proteins, underlying some mechanisms of toxicity. A dedicated computational screen was used ...to identify UO(2) (2+) binding sites within a set of nonredundant protein structures. The list of potential targets was compared to data from a small molecules interaction database to pinpoint specific examples where UO(2) (2+) should be able to bind in the vicinity of an essential cation, and would be likely to affect the function of the corresponding protein. The C-reactive protein appeared as an interesting hit since its structure involves critical calcium ions in the binding of phosphorylcholine. Biochemical experiments confirmed the predicted binding site for UO(2) (2+) and it was demonstrated by surface plasmon resonance assays that UO(2) (2+) binding to CRP prevents the calcium-mediated binding of phosphorylcholine. Strikingly, the apparent affinity of UO(2) (2+) for native CRP was almost 100-fold higher than that of Ca(2+). This result exemplifies in the case of CRP the capability of our computational tool to predict effective binding sites for UO(2) (2+) in proteins and is a first evidence of calcium substitution by the uranyl cation in a native protein.
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.
During long-term exposure, uranium accumulates in bone. Since uranium in U(vi) complexes shares similar coordination properties to calcium, this toxicant is assumed to be exchanged with calcium ions ...at the surfaces of bone mineral crystals. Recently, two proteins involved in bone turnover, fetuin A and osteopontin, were shown to exhibit a high affinity for U(vi). A common biochemical feature of both fetuin A and osteopontin is their inhibiting role in calcium phosphate precipitation. Therefore it is conceivable that complexation of U(vi) with these proteins may alter their interaction with calcium and/or calcium phosphate. Quantitative analyses of calcium, phosphorus and uranium performed using inductively coupled plasma/mass spectrometry (ICP/MS) demonstrated the inhibition of the precipitation of calcium phosphate by fetuin A and osteopontin for 2 h. In addition, the presence of U(vi) did not seem to alter the duration of this process. However, dynamic light scattering studies revealed that the size of the colloidal particles formed with osteopontin was altered by the presence of U(vi) in a concentration-dependent manner. Finally, using hyphenated capillary electrophoresis-ICP/MS (CE-ICP/MS), we showed that in these systems, at a low concentration of U(vi) (protein : U(vi) 8 : 1), U(vi) might remain in solution by forming a complex with proteins and not by sequestration of a precipitate of either autunite or uranyl orthophosphate.
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