Mercury speciation in selenium enriched wheat plants hydroponically exposed to mercury pollution.

Mercury (Hg) pollution in agricultural soils and its potential pathway to the human food chain can pose a serious health concern. Understanding the pathway of Hg in plants and how the speciation may change upon interaction with other elements used for biofortification can be critical to assess the real implications for the final plant-based product. In that respect, selenium (Se) biofortification of crops grown in Se-poor soil regions is becoming a common practice to overcome Se deficient diets. Therefore, it is important to assess the interplay between these two elements since Se may form complexes with Hg reducing its bioavailability and toxicity. In this work, the speciation of Hg in wheat plants grown hydroponically under the presence of Hg (HgCl2) and biofortified with Se (selenite, selenate, or a 1:1 mixture of both) has been investigated by X-ray absorption spectroscopy at the Hg L3-edge. The main Hg species found in wheat grains was the highly toxic methylmercury. It was found that the Se-biofortification of wheat did not prevent, in general, the Hg translocation to grains. Only the 1:1 mixture treatment seemed to have an effect in reducing the levels of Hg and the presence of methylmercury in grains.

First insight in element localisation in different body parts of the acanthocephalan Dentitruncus truttae using TEM and NanoSIMS.

Acanthocephalans, intestinal parasites of vertebrates, are characterised by orders of magnitude higher metal accumulation than free-living organisms, but the mechanism of such effective metal accumulation is still unknown. The aim of our study was to gain new insights into the high-resolution localization of elements in the bodies of acanthocephalans, thus taking an initial step towards elucidating metal uptake and accumulation in organisms under real environmental conditions. For the first time, nanoscale secondary ion mass spectrometry (NanoSIMS) was used for high-resolution mapping of 12 elements (C, Ca, Cu, Fe, N, Na, O, P, Pb, S, Se, and Tl) in three selected body parts (trunk spines, inner part of the proboscis receptacle and inner surface of the tegument) of Dentitruncus truttae, a parasite of brown trout (Salmo trutta) from the Krka River in Croatia. In addition, the same body parts were examined using transmission electron microscopy (TEM) and correlated with NanoSIMS images. Metal concentrations determined using HR ICP-MS confirmed higher accumulation in D. truttae than in the fish intestine. The chemical composition of the acanthocephalan body showed the highest density of C, Ca, N, Na, O, S, as important and constitutive elements in living cells in all studied structures, while Fe was predominant among trace elements. In general, higher element density was found in trunk spines and tegument, as body structures responsible for substance absorption in parasites. The results obtained with NanoSIMS and TEM-NanoSIMS correlative imaging represent pilot data for mapping of elements at nanoscale resolution in the ultrastructure of various body parts of acanthocephalans and generally provide a contribution for further application of this technique in all parasite species.

Direct and indirect selenium speciation in biofortified wheat: A tale of two techniques.

Wheat can be biofortified with different inorganic selenium (Se) forms, selenite or selenate. The choice of Se source influences the physiological response of the plant and the Se metabolites produced. We looked at selenium uptake, distribution and metabolization in wheat exposed to selenite, selenate and a 1:1 molar mixture of both to determine the impact of each treatment on the Se speciation in roots, shoots, and grains. To achieve a comprehensive quantification of the Se species, the complementarity of high-performance liquid chromatography coupled with inductively coupled plasma mass spectrometry and X-ray absorption spectroscopy was exploited. This approach allowed the identification of the six main selenium species: selenomethionine, selenocysteine, selenocystine, selenite, selenate, and elemental selenium. The three treatments resulted in similar total selenium concentration in grains, 90-150 mg Se kg-1 , but produced different effects in the plant. Selenite enhanced root accumulation (66% of selenium) and induced the maximum toxicity, whereas selenate favored shoot translocation (46%). With the 1:1 mixture, selenium was distributed along the plant generating lower toxicity. Although all conditions resulted in >92% of organic selenium in the grain, selenate produced mainly C-Se-C forms, such as selenomethionine, while selenite (alone or in the mixture) enhanced the production of C-Se-Se-C forms, such as selenocystine, modifying the selenoamino acid composition. These results provide a better understanding of the metabolization of selenium species which is key to minimize plant toxicity and any concomitant effect that may arise due to Se-biofortification.

High spatial resolution imaging of subcellular macro and trace element distribution during phagocytosis.

The bioavailability of trace elements in the course of evolution had an essential influence on the emergence of life itself. This is reflected in the co-evolution between eukaryotes and prokaryotes. In this study, the influence and cellular distribution of bioelements during phagocytosis at the host-pathogen interface were investigated using high-resolution nanoscale secondary ion mass spectrometry (NanoSIMS) and quantitative inductively coupled plasma mass spectrometry. In the eukaryotic murine macrophages (RAW 264.7 cell line), the cellular Fe/Zn ratio was found to be balanced, whereas the dominance of iron in the prokaryotic cells of the pathogen Salmonella enterica Serovar Enteritidis was ∼90% compared to zinc. This confirms the evolutionary increased zinc requirement of the eukaryotic animal cell. Using NanoSIMS, the Cs+ primary ion source allowed high spatial resolution mapping of cell morphology down to the subcellular level. At a comparable resolution, several low-abundant trace elements could be mapped during phagocytosis with a RF plasma O- primary ion source. An enrichment of copper and nickel could be detected in the prokaryotic cells. Surprisingly, an accumulation of cobalt in the area of the nuclear envelope was observed, indicating an interesting but still unknown distribution of this trace element in murine macrophages.

Comprehensive Speciation Analysis of Residual Gadolinium in Deep Cerebellar Nuclei in Rats Repeatedly Administered With Gadoterate Meglumine or Gadodiamide.

PURPOSE: Several preclinical studies have reported the presence of gadolinium (Gd) in different chemical forms in the brain, depending on the class (macrocyclic versus linear) of Gd-based contrast agent (GBCA) administered. The aim of this study was to identify, with a special focus on insoluble species, the speciation of Gd retained in the deep cerebellar nuclei (DCN) of rats administered repeatedly with gadoterate or gadodiamide 4 months after the last injection. METHODS: Three groups (N = 6/group) of healthy female Sprague-Dawley rats (SPF/OFA rats; Charles River, L'Arbresle, France) received a cumulated dose of 50 mmol/kg (4 daily intravenous administrations of 2.5 mmol/kg, for 5 weeks, corresponding to 80-fold the usual clinical dose if adjusted for man) of gadoterate meglumine (macrocyclic) or gadodiamide (linear) or isotonic saline for the control group (4 daily intravenous administrations of 5 mL/kg, for 5 weeks). The animals were sacrificed 4 months after the last injection. Deep cerebellar nuclei were dissected and stored at -80°C before sample preparation. To provide enough tissue for sample preparation and further analysis using multiple techniques, DCN from each group of 6 rats were pooled. Gadolinium species were extracted in 2 consecutive steps with water and urea solution. The total Gd concentrations were determined by inductively coupled plasma mass spectrometry (ICP-MS). Soluble Gd species were analyzed by size-exclusion chromatography coupled to ICP-MS. The insoluble Gd species were analyzed by single-particle (SP) ICP-MS, nanoscale secondary ion mass spectroscopy (NanoSIMS), and scanning transmission electron microscopy with energy-dispersive X-ray spectroscopy (STEM-EDX) for elemental detection. RESULTS: The Gd concentrations in pooled DCN from animals treated with gadoterate or gadodiamide were 0.25 and 24.3 nmol/g, respectively. For gadoterate, the highest amount of Gd was found in the water-soluble fractions. It was present exclusively as low-molecular-weight compounds, most likely as the intact GBCA form. In the case of gadodiamide, the water-soluble fraction of DCN was composed of high-molecular-weight Gd species of approximately 440 kDa and contained only a tiny amount (less than 1%) of intact gadodiamide. Furthermore, the column recovery calculated for this fraction was incomplete, which suggested presence of labile complexes of dissociated Gd3+ with endogenous molecules. The highest amount of Gd was detected in the insoluble residue, which was demonstrated, by SP-ICP-MS, to be a particulate form of Gd. Two imaging techniques (NanoSIMS and STEM-EDX) allowed further characterization of these insoluble Gd species. Amorphous, spheroid structures of approximately 100-200 nm of sea urchin-like shape were detected. Furthermore, Gd was consistently colocalized with calcium, oxygen, and phosphorous, strongly suggesting the presence of structures composed of mixed Gd/Ca phosphates. No or occasional colocalization with iron and sulfur was observed. CONCLUSION: A dedicated analytical workflow produced original data on the speciation of Gd in DCN of rats repeatedly injected with GBCAs. The addition, in comparison with previous studies of Gd speciation in brain, of SP element detection and imaging techniques allowed a comprehensive speciation analysis approach. Whereas for gadoterate the main fraction of retained Gd was present as intact GBCA form in the soluble fractions, for linear gadodiamide, less than 10% of Gd could be solubilized and characterized using size-exclusion chromatography coupled to ICP-MS. The main Gd species detected in the soluble fractions were macromolecules of 440 kDa. One of them was speculated to be a Gd complex with iron-binding protein (ferritin). However, the major fraction of residual Gd was present as insoluble particulate species, very likely composed of mixed Gd/Ca phosphates. This comprehensive Gd speciation study provided important evidence for the dechelation of linear GBCAs and offered a deeper insight into the mechanisms of Gd deposition in the brain.

Development of Mercury Analysis by NanoSIMS for the Localization of Mercury-Selenium Particles in Whale Liver.

Nanoscale secondary ion mass spectrometry (NanoSIMS) is a dynamic SIMS technique, which offers high spatial resolution allowing the mapping of chemical elements at the nanometer scale combined with high sensitivity. However, SIMS for mercury analysis is a challenging issue due to the low secondary ion yield and has never been done on NanoSIMS. The introduction of an rf plasma oxygen primary ion source on NanoSIMS enabled higher lateral resolution and higher sensitivity for electropositive elements such as most metals. In this paper, for the first time, mercury analysis by NanoSIMS was developed applying the new rf plasma O- ion source. All mercury isotopes could be detected as Hg+ secondary ions and the isotopic pattern corresponded to their natural isotopic abundances. Furthermore, Hg+ detection in HgSe nanocrystals has been investigated where polyatomic interferences from selenium clusters were identified and separated by high mass resolution (ΔM/M ≥ 3200). However, in the presence of selenium a strong matrix effect was observed, decreasing the Hg+ secondary ion yield. In addition, a detection of Se+ ions was possible, too. The newly developed method was successfully applied to nanoscale localization by chemical imaging of HgSe particles accumulated in the liver tissue of sperm whale (Physeter macrocephalus). This demonstrated the applicability of NanoSIMS not only for mercury detection in surface analysis but also for mercury mapping in biological samples.

A general covalent binding model between cytotoxic selenocompounds and albumin revealed by mass spectrometry and X-ray absorption spectroscopy.

Selenocompounds (SeCs) are promising therapeutic agents for a wide range of diseases including cancer. The treatment results are heterogeneous and dependent on both the chemical species and the concentration of SeCs. Moreover, the mechanisms of action are poorly revealed, which most probably is due to the detection methods where the quantification is based on the total selenium as an element. To understand the mechanisms underlying the heterogeneous cytotoxicity of SeCs and to determine their pharmacokinetics, we investigated selenium speciation of six SeCs representing different categories using liquid chromatography-mass spectrometry (LC-MS) and X-ray absorption spectroscopy (XAS) and the cytotoxicity using leukemic cells. SeCs cytotoxicity was correlated with albumin binding degree as revealed by LC-MS and XAS. Further analysis corroborated the covalent binding between selenol intermediates of SeCs and albumin thiols. On basis of the Se-S model, pharmacokinetic properties of four SeCs were for the first time profiled. In summary, we have shown that cytotoxic SeCs could spontaneously transform into selenol intermediates that immediately react with albumin thiols through Se-S bond. The heterogeneous albumin binding degree may predict the variability in cytotoxicity. The present knowledge will also guide further kinetic and mechanistic investigations in both experimental and clinical settings.

Rapid and Robust Quantification of p-Xyleneselenocyanate in Plasma via Derivatization.

p-Xyleneselenocyanate (p-XSC) is one of the most investigated selenium compounds in cancer-prevention and -therapy. Despite the potent anticancer property, there is still no proper method to perform the quantitative analysis of p-XSC in plasma. In this investigation, we aimed at developing a method based on liquid chromatography-mass spectrometry (LC-MS) for the measurement of p-XSC in plasma. Direct deproteinization was first used to extract parent p-XSC from plasma, but failed to achieve high recovery rate (<2%) due to formation of selenium-sulfur bond between p-XSC and plasma protein. To overcome this problem, we modified the extraction method to three steps: (1) break the selenium-sulfur bond by tris(2-carboxyethyl)phosphine; (2) stabilize the newly formed intermediate selenol by N-ethylmaleimide; (3) deproteinization. This three-step method efficiently recovered bound p-XSC by more than 75%. In in vivo study, p-XSC was injected intravenously into mice and plasma was collected for LC-MS analysis. Consistently, p-XSC was undetectable in its parent form, whereas the bound form was readily quantified, employing the modified extraction method. In summary, we describe a novel, robust, and sensitive method for quantification of p-XSC in plasma. The present method will enable pharmacokinetic and pharmacodynamic studies of p-XSC in both clinical and preclinical settings.