Carole Bresson

Low-solubility particles and a Trojan-horse type mechanism of toxicity: the case of cobalt oxide on human lung cells

BackgroundThe mechanisms of toxicity of metal oxide particles towards lung cells are far from being understood. In particular, the relative contribution of intracellular particulate versus solubilized fractions is rarely considered as it is very challenging to assess, especially for low-solubility particles such as cobalt oxide (Co3O4).MethodsThis study was possible owing to two highly sensitive, independent, analytical techniques, based on single-cell analysis, using ion beam microanalysis, and on bulk analysis of cell lysates, using mass spectrometry.ResultsOur study shows that cobalt oxide particles, of very low solubility in the culture medium, are readily incorporated by BEAS-2B human lung cells through endocytosis via the clathrin-dependent pathway. They are partially solubilized at low pH within lysosomes, leading to cobalt ions release. Solubilized cobalt was detected within the cytoplasm and the nucleus. As expected from these low-solubility particles, the intracellular solubilized cobalt content is small compared with the intracellular particulate cobalt content, in the parts-per-thousand range or below. However, we were able to demonstrate that this minute fraction of intracellular solubilized cobalt is responsible for the overall toxicity.ConclusionsCobalt oxide particles are readily internalized by pulmonary cells via the endo-lysosomal pathway and can lead, through a Trojan-horse mechanism, to intracellular release of toxic metal ions over long periods of time, involving specific toxicity.

Cobalt distribution in keratinocyte cells indicates nuclear and perinuclear accumulation and interaction with magnesium and zinc homeostasis.

Cobalt is known to be toxic at high concentration, to induce contact dermatosis, and occupational radiation skin damage because of its use in nuclear industry. We investigated the intracellular distribution of cobalt in HaCaT human keratinocytes as a model of skin cells, and its interaction with endogenous trace elements. Direct micro-chemical imaging based on ion beam techniques was applied to determine the quantitative distribution of cobalt in HaCaT cells. In addition, synchrotron radiation X-ray fluorescence microanalysis in tomography mode was performed, for the first time on a single cell, to determine the 3D intracellular distribution of cobalt. Results obtained with these micro-chemical techniques were compared to a more classical method based on cellular fractionation followed by inductively coupled plasma atomic emission spectrometry (ICP-AES) measurements. Cobalt was found to accumulate in the cell nucleus and in perinuclear structures indicating the possible direct interaction with genomic DNA, and nuclear proteins. The perinuclear accumulation in the cytosol suggests that cobalt could be stored in the endoplasmic reticulum or the Golgi apparatus. The multi-elemental analysis revealed that cobalt exposure significantly decreased magnesium and zinc content, with a likely competition of cobalt for magnesium and zinc binding sites in proteins. Overall, these data suggest a multiform toxicity of cobalt related to interactions with genomic DNA and nuclear proteins, and to the alteration of zinc and magnesium homeostasis.

Cobalt chloride speciation, mechanisms of cytotoxicity on human pulmonary cells, and synergistic toxicity with zinc

Cobalt is used in numerous industrial sectors, leading to occupational diseases, particularly by inhalation. Cobalt-associated mechanisms of toxicity are far from being understood and information that could improve knowledge in this area is required. We investigated the impact of a soluble cobalt compound, CoCl(2)·6H(2)O, on the BEAS-2B lung epithelial cell line, as well as its impact on metal homeostasis. Cobalt speciation in different culture media, in particular soluble and precipitated cobalt species, was investigated via theoretical and analytical approaches. The cytotoxic effects of cobalt on the cells were assessed. Upon exposure of BEAS-2B cells to cobalt, intracellular accumulation of cobalt and zinc was demonstrated using direct in situ microchemical analysis based on ion micro-beam techniques and analysis after cell lysis by inductively coupled plasma mass spectrometry (ICP-MS). Microchemical imaging revealed that cobalt was rather homogeneously distributed in the nucleus and in the cytoplasm whereas zinc was more abundant in the nucleus. The modulation of zinc homeostasis led to the evaluation of the effect of combined cobalt and zinc exposure. In this case, a clear synergistic increase in toxicity was observed as well as a substantial increase in zinc content within cells. Western blots performed under the same coexposure conditions revealed a decrease in ZnT1 expression, suggesting that cobalt could inhibit zinc release through the modulation of ZnT1. Overall, this study highlights the potential hazard to lung function, of combined exposure to cobalt and zinc.

Radionuclide speciation: A key point in the field of nuclear toxicology studies

The understanding of radionuclides (RN) action modes in the living organisms in case of contamination is a major aim in nuclear toxicology studies. The mechanisms of RN-involved toxicity at the molecular level are critically dependent on their speciation. In this framework, the calculating and experimental approaches of speciation determination are described in the first part of this paper. The selection of cobalt, uranium and plutonium as relevant examples of chemical and/or radiotoxic contaminants at diverse levels is further explained. The results regarding their speciation and interactions in various biological media, going from simple biorelevant systems to in vitro and in vivo systems are reported. An interdisciplinary approach, combining i) in silico studies, providing precise aspects of structural insights into protein-RN interactions, ii) in analytico studies, addressing fundamental speciation and structural studies and developments in biorelevant RN-ligands systems, iii) in vitro studies, concerning RN speciation and interactions in cellular systems and iv) in vivo studies describing the RN fate and behavior in part or whole organisms, has been undertaken to explore the results. In each case, the importance of the speciation knowledge is emphasized, in order to shed light on the understanding of RN impact and action modes at the molecular level.

X-ray absorption spectroscopy investigations on radioactive matter using MARS beamline at SOLEIL synchrotron

Abstract The MARS beamline at the SOLEIL synchrotron is dedicated to the characterization of radioactive material samples. One great advantage of the beamline is the possibility to characterize about 380 radionuclides by different X-ray techniques in the same place. This facility is unique in Europe. A wide energy range from around 3.5 keV to 36 keV K-edges from K to Cs, and L3 edges from Cd to Am and beyond can be used. The MARS beamline is optimized for X-ray absorption spectroscopy techniques (XANES/EXAFS), powder diffraction (XRD) but x-ray fluorescence (XRF) analysis, High Energy Resolution Fluorescence Detected -XAS (HERFD-XAS), X-ray Emission (XES) and μ-XAS/XRD are also possible. A description of the beamline as well as its performances are given in a first part. Then some scientific examples of XAS studies from users are presented which cover a wide variety of topics in radiochemistry and nuclear materials.

Comparison of the NMR Enantiodifferentiation of a Chiral Ruthenium(II) Complex of C2 Symmetry Using the TRISPHAT Anion and a Lanthanide Shift Reagent

The tris[tetrachlorobenzenediolato]phosphate(V) anion (TRISPHAT) is known to be an efficient NMR chiral shift agent for various chiral cationic species. Here we compare the efficiency of TRISPHAT and of a chiral lanthanide shift reagent for the determination of the enantiomeric purity of the chiral building block [Ru(phen)2py2]2+ which possesses C2 symmetry. We also discuss our results in terms of the geometry of interaction between the Ru(II) complex and the TRISPHAT anion.

XAS investigation of biorelevant cobalt complexes in aqueous media.

Cobalt is an essential element of biological cycles involved in numerous metallobiomolecules, but it becomes a toxic element at high concentration or a radiotoxic element because of its use in the nuclear industry. “Molecular speciation” in biological media is an essential prerequisite to evaluate its chemical behaviour as well as its toxic or beneficial effects. In this scheme, we have focused on the coordination properties of the thiol-containing amino acid cysteine (Cys) and the pseudo-peptide N-(2-mercaptopropionyl)glycine (MPG) towards the Co2+ cation in aqueous media. XAS at the Co K edge and traditional spectroscopic techniques have been coupled in order to structurally characterize the cobalt coordination sphere. Oxidation states and geometries of the bis- and tris-cysteinato Co(III) complexes are in agreement with the literature data. In addition, bond lengths between the metallic centre and the donor atoms have been determined. The structure of a new dimeric N-(2-mercaptopropionyl)glycinato Co(II) complex in solution is also reported. The coordination of MPG to Co(II) through the thiolate and carboxylate functions is ascertained. This work provides fundamental structural information about biorelevant complexes of cobalt, which will contribute to our understanding of the chemical behaviour and the biological role of this radionuclide.

Solvation of Co(III)-cysteinato complexes in water: a DFT-based molecular dynamics study.

Structural, dynamical, and vibrational properties of complexes made of metal cobalt(III) coordinated to different amounts of cysteine molecules were investigated with DFT-based Car-Parrinello molecular dynamics (CPMD) simulations in liquid water solution. The systems are composed of Co(III):3Cys and Co(III):2Cys immersed in liquid water which are modeled by about 110 explicit water molecules, thus one of the biggest molecular systems studied with ab initio molecular simulations so far. In such a way, we were able to investigate structural and dynamical properties of a model of a typical metal binding site used by several proteins. Cobalt, mainly a toxicological agent, can replace the natural binding metal and thus modify the biochemical activity. The structure of the surrounding solvent around the metal-ligands complexes is reported in detail, as well as the metal-ligands coordination bonds, using radial distribution functions and electronic analyses with Mayer bond orders. Structures of the Cocysteine complexes are found in very good agreement with EXAFS experimental data, stressing the importance of considering the surrounding solvent in the modeling. A vibrational analysis is also conducted and compared to experiment, which strengthens the reliability of the solvent interactions with the Cocysteine complexes from our molecular dynamics simulations, as well as the dynamics of the systems. From this preliminary analysis, we could suggest a vibrational fingerprint able to distinguish Co(III):2Cys from Co(III):3Cys. Our simulations also show the importance of considering a quantum explicit solvent, as solute-to-solvent proton transfer events have been observed.

A combined spectroscopic and theoretical approach to investigate structural properties of Co(II)/Co(III) tris-cysteinato complexes in aqueous medium.

Physiological and toxicological effects of metallic ions depend on their speciation and on the structure of their associated bioligand complexes. In the field of chemical and/or nuclear toxicological studies, we are investigating cobalt complexes with biorelevant ligands such as amino acids or peptides. The aqueous reaction of cobalt dichloride with an excess of cysteine (Cys, C3H5NSO22−) in a basic medium under an anaerobic atmosphere and subsequent oxidation by O2, afforded the mononuclear complexes Co(II):3Cys and Co(III):3Cys, respectively. A combination of X-ray absorption spectroscopy (XAS) measurements and Car-Parrinello molecular dynamics (CPMD) simulations allowed us to assess structural features of the already explored Co(III):3Cys complex. Inclusion of the temperature effects in the CPMD calculations gives an implicit access to disorder effects in the extended X-ray absorption fine structure (EXAFS) equation. The very good agreement between the measured and the simulated data showed the accuracy of these models provided by CPMD. The present investigation is completed by new UV-visible, X-ray absorption near edge structure (XANES) and electron paramagnetic resonance (EPR) data of Co(II):3Cys. These data are consistent with a Co(II) high-spin d7 complex in a distorted octahedral geometry. This work contributes to the knowledge of topics such as metal–bioligand interaction which is of major interest in the field of bioinorganic chemistry.

XAS examination of glutathione–cobalt complexes in solution

In the present work, we have investigated the coordination modes of cobalt with glutathione (γ-l-glutamyl-l-cysteinyl-glycine, GSH). A systematic study of cobalt-GSH complexes at basic and neutral pH has been undertaken with a multi-spectroscopic approach combined with quantum chemistry calculations. XAS (x-ray absorption spectroscopy) has been performed at the cobalt K edge in order to shed light into the cation coordination sphere and formal oxidation states. XANES (x-ray absorption near edge structure) enabled to show that in basic and neutral media, cobalt oxidation state is equal to +III and +II respectively. EXAFS (extended x-ray absorption fine structure) provided indications on the donor atoms involved in the coordination with cobalt as well as the bond lengths. DFT (density functional theory)-based calculations and NMR experiments have been performed to assess the most stable structure of the cobalt-GSH complex in basic conditions.