Article pubs.acs.org/IC
Actinide(IV) Deposits on Bone: Potential Role of the Osteopontin− Thorium Complex Gael̈ le Creff,*,† Samir Safi,‡ Jérôme Roques,‡ Hervé Michel,† Aurélie Jeanson,‡ Pier-Lorenzo Solari,§ Christian Basset,∥ Eric Simoni,‡ Claude Vidaud,∥ and Christophe Den Auwer† †
Institut de Chimie de Nice, UMR7272, Université Nice Sophia Antipolis, 28 Avenue Valrose, 06108 Nice, France Institut de Physique Nucléaire d’Orsay, UMR8608, Université Paris XI Orsay, 15 Rue Georges Clemenceau, 91405 Orsay, France § Synchrotron SOLEIL, L’Orme des Merisiers, BP 48, St Aubin, 91192 Gif sur Yvette, France ∥ CEA Marcoule, DSV, IBEB, LEPC, 30207 Bagnols sur Cèze, France ‡
S Supporting Information *
ABSTRACT: 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.
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INTRODUCTION Since the second half of the 20th Century, the large scale development of civil and military nuclear industry in countries of the northern hemisphere and, in some cases, southern hemisphere has raised some new issues in terms of defense, the fuel cycle for civil energy production, and environmental impact. From 2002 to 2008, for instance, the number of countries in discussion with IAEA for civil nuclear use has increased from 30 to 44. Although the actinide elements (An) are not the unique source of radioactive release in the case of a nuclear event, they are of particular concern because they are the only uncharacterized exogenous metals known to have no essential role in the normal biochemical processes occurring in living organisms (with the exception of some other heavy stable elements). Our understanding of biological pathways of this family of elements in the case of accidental contamination or chronic natural exposure (in the case of uranium rich soils, for instance) is therefore a crucial issue of public health and first and foremost of societal impact. However, human contamination can occur (inhalation, ingestion, or cutaneous); a © XXXX American Chemical Society
radioelement may be absorbed and then either directly excreted or transported by blood, linking with different biological ligands (amino acids, peptides, enzymes, and proteins), to target organs.1 Once absorbed by the organs, the radioelement might take the place of essential elements by chemical or steric analogy (calcium and iron, for example),2 leading to serious alterations of organ activity. Excretion rates and the nature of the target organs (mainly bone, kidney, and liver for actinides) depend on the radioelement itself, its oxidation state, and its speciation: for instance, U(VI) (under its uranyl {UO22+} form) is widely excreted through urination (65% after 1 day), while Np is more moderately eliminated [up to 40% for Np(V) and 15% for Np(IV) after 1 day]. Retention rates of Pu(IV) and “transplutonium” elements are very high: only 0.4% of Pu is excreted after 1 day, and the elimination rate of Am(III) and Cm(III) is 1.0. It is difficult in this context to assert that no mononuclear hydroxy species are present in solution, but they must be minor because no vibration modes
Figure 4. (a) k2-weighted Th LIII edge EXAFS spectra of the Th(IV)− H8V complex at pH 1 and 4 and the Th(IV)−OPNf notation complex at pH 4. (b) Corresponding Fourier transforms: black line for the experimental spectrum and dots for the corresponding fit.
Table 2. Fitted Values [R, ΔR, Ndegen (fixed during the procedure), σ2, Amp, and ΔE0] with Errors in Parentheses for R, ΔE, and Amp Determined for Each pH Condition for the H8V−Th(IV) Complex and the OPN−Th(IV) Complex path
N
fitted Ra (Å)
σ2 (×10−3 Å2)
Th−H8V
Th−O(P)
2
2.32(2)
2.4
pH 1
Th−O(wat) + O(Cbi) Th···Cbi Th···P Th−O(P)
6
2.46(4)
14.7
1 2 2
3.06(4) 3.86(2) 2.31(1)
2.3 2.5 3.4
Th−O(wat) + O(Cbi) Th···Cbi Th···P Th−O(P)
6
2.45(1)
14.8
1 2 2
3.06(3) 3.85(3) 2.34(3)
0.7 2.8 1.4
Th−O(wat) + O(Cbi) Th···Cbi Th···P
6
2.42(4)
16.0
1 2
3.06(2) 3.88(1)
0.9 0.5
(2.5;0.34)b Th−H8V pH 4 (2.5;0.32)b Th−OPNf pH 4 (3.1;0.81)b
ΔE0 (eV), Amp −3.61(4), 1.0(3)
−4.01(2), 1.0(3)
−3.57(4), 1.2(5)
a From the average R from the DFT model: Th−O(P), 2.29 Å; Th− O(wat) + O(Cbi), 2.52 Å; Th···Cbi, 2.99 Å; Th···P, 3.84 Å. bRefers to (r %; χi2/n), where r is the R factor of the fit in percent and χi2/n is the reduced χ2 of the fit, both being fit-quality factors.
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DOI: 10.1021/acs.inorgchem.5b02349 Inorg. Chem. XXXX, XXX, XXX−XXX
Article
Inorganic Chemistry
drastically, and this may be attributed to the large flexibility of the peptide (and protein) sequence.
corresponding to hydroxide can be observed on the infrared spectrum. However, the absence of a contribution at 3.90 Å represents a good indication that no polynuclear hydrolyzed species like dimers [Th2(OH)26+ and Th2(OH)35+], trimers [Th3(OH)57+], tetramers [Th4(OH)88+ and Th4(OH)124+], or even hexamers [Th6(OH)159+ and Th6(OH)1410+]28 are formed in solution. The Th−OP distances (2.31−2.34 Å) are also in good agreement with previous work of Th(IV) complexation by phosphate ligands in solution published by Sutton et al.29 or Den Auwer et al.30 These fitted data confirm the importance of the phosphate functions in the complexation ability of both H8V and OPNf, as suggested by the IR data. Last, we can assert that the Th(IV)−H8V complex can be transposed well with the OPNf fragment. Compared to our previous work concerning {UO22+} complexation with H8V,10 Th(IV) is complexed by a larger number of phosphate functions (both functions of the peptidic sequence are found in interaction with the cation; two P atoms are also found in the coordination sphere of the cation in the case of OPNf). With regard to the distances, U−O(P) binding was found to have distances (2.24−2.28 Å) shorter than those for Th−O(P) binding (2.31−2.34 Å), although the formal charge of the latter is larger. In that case, this may be attributed to steric effects because the constraints associated with the binding of two phosphate groups must be larger. Last, the affinity constant of H8V is slightly smaller for Th(IV) (log K = 4.2 ± 3) than for {U(VI)O22+} (log K = 5.2 ± 3), although in the same order.
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ASSOCIATED CONTENT
* Supporting Information S
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorgchem.5b02349. Entire infrared spectra of the free H8V peptide and the Th(IV)−H8V complex, xyz coordinates of the peptide− Th(IV) complex from DFT calculation, EXAFS spectra (experimental and fitted), and fitting parameters (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail: gaelle.creff@unice.fr. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors thank the ARLA Food Group for providing OPNenriched samples of cow’s milk. XAS data have been recorded on the MARS beamline of SOLEIL synchrotron, the French beamline dedicated to actinide materials. The authors thank B. Sitaud and S. Schultig for beamline assistance. Funding has been provided by the ToxNuc3 CEA program under the BIOMUROS project.
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CONCLUSION The work reported here aims to describe the mechanisms of uptake of actinides by the human bone system. We have restrained our study to Th(IV), taken here as a simple model for actinide +IV chemistry. However, the thorium case paves the way for plutonium mechanical studies and brings an essential point of comparison with respect to the uranyl case. The osteopontin (OPN) protein has been targeted as one important actor in the contamination process and is here at the center of our investigation. The combination of spectroscopic techniques allowed us to describe the structure of the Th(IV) coordination sphere with remarkable similarities between the biomimetic peptide H8V and OPNf itself. At first, ATR-FTIR revealed the functional interaction of phosphate and carboxylate moieties denoting bidentate bridging coordination to the carboxylate function. In addition, various structural models were refined using DFT calculations, and one of them was selected in accordance with the gathered spectroscopic data. The Th LIII edge EXAFS data confirmed the model assumptions with Th(IV) binding to two phosphoryl functional groups and one carboxyl functional group, with average distances of 3.06 and 3.86 Å between the thorium atom and the carbon and phosphorus atoms, respectively. These distances are indicative of an inner sphere complex with two oxygen atoms shared between Th(IV) and the phosphoryl group and two oxygens from a single carboxyl group (bidentate coordination mode). The coordination sphere is then fulfilled by four water molecules in this model. Remarkably, the structural data obtained with the peptide are independent of the pH, even at very low values (pH 1). The affinity constants determined via ITC for H8V and both Th(IV) and {U(VI)O22+} are very similar (4.2 ± 3 and 5.2 ± 3, respectively). This confirms the affinity of the structural motif discussed here for both cations, although their coordination spheres differ
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DOI: 10.1021/acs.inorgchem.5b02349 Inorg. Chem. XXXX, XXX, XXX−XXX
