Preclinical Evaluation of Sodium Selenite in Mice: Toxicological and Tumor Regression Studies after Striatum Implantation of Human Glioblastoma Stem Cells.

Glioblastoma (GBM) is the most aggressive malignant glioma, with a very poor prognosis; as such, efforts to explore new treatments and GBM's etiology are a priority. We previously described human GBM cells (R2J-GS) as exhibiting the properties of cancer stem cells (growing in serum-free medium and proliferating into nude mice when orthotopically grafted). Sodium selenite (SS)-an in vitro attractive agent for cancer therapy against GBM-was evaluated in R2J-GS cells. To go further, we launched a preclinical study: SS was given orally, in an escalation-dose study (2.25 to 10.125 mg/kg/day, 5 days on, 2 days off, and 5 days on), to evaluate (1) the absorption of selenium in plasma and organs (brain, kidney, liver, and lung) and (2) the SS toxicity. A 6.75 mg/kg SS dose was chosen to perform a tumor regression assay, followed by MRI, in R2J-GS cells orthotopically implanted in nude mice, as this dose was nontoxic and increased brain selenium concentration. A group receiving TMZ (5 mg/kg) was led in parallel. Although not reaching statistical significance, the group of mice treated with SS showed a slower tumor growth vs. the control group (p = 0.08). No difference was observed between the TMZ and control groups. We provide new insights of the mechanisms of SS and its possible use in chemotherapy.

Optimization of elemental selenium (Se(0)) determination in yeasts by anion-exchange HPLC-ICP-MS.

An analytical method was developed for the speciation of elemental selenium (Se(0)) in selenized yeasts by anion-exchange HPLC-ICP-MS after its chemical transformation into SeSO32- by reaction with sodium sulfite. The presence of Se(0) in the yeasts was further confirmed by single-particle ICP-MS. Indeed, Se nanoparticles, if present, are expected to be, at least partly, Se(0). X-ray photoelectron spectroscopy, a well-recognized technique for chemical element speciation in the solid state, was also used with this objective. Both methods were able to confirm the presence of Se(0) in the selenized yeasts but failed to provide reliable quantitative results. Analytical performances of the HPLC-ICP-MS method were then evaluated for Se(0) determination. Quantification limits of 1 mg/kg were reached. The recovery levels from an added quantity comprised between 93 and 101%. Within-run and between-run precisions were both below 8%. The procedure developed was finally applied to quantify Se(0) content in a series of seven yeast batches from different suppliers. Se(0) was found to be present in all the studied yeasts and represented on average 10-15% of the total Se.

Potential of lead elemental and isotopic signatures for authenticity and geographical origin of Bordeaux wines.

Lead concentrations and lead isotope ratios of 43 authentic Bordeaux wines from prestigious châteaux and 14 suspicious Bordeaux origin were determined to evaluate their potential for authenticity and geographical origin assessment. Results have shown that the total Pb concentrations in Bordeaux wines drastically decreased over the previous 50 years with a clear shift of isotopic signatures towards geogenic values corresponding to an overall trend of European environmental lead monitoring. The Pb isotopic ratios determined in both series of samples clearly demonstrated that suspicious Bordeaux wines displayed Pb isotopic signatures statistically distinctive from those obtained for authentic wines. This observation was confirmed by the three-isotope mixing lines obtained between the geogenic and the anthropogenic Pb isotopes data that characterize European and Asian sources. The use these specific three-isotope plots allows a non-ambiguous discrimination between authentic Pauillac AOC and the counterfeited ones.

Strontium elemental and isotopic signatures of Bordeaux wines for authenticity and geographical origin assessment.

The 87Sr/86Sr ratio and Sr concentrations of 43 authentic Bordeaux wines from the world's most prestigious châteaux are presented in the context of their relation to the geographical origin of wine and authenticity. The results demonstrate relatively narrow spans of variabilities observed for 87Sr/86Sr ratio and Sr concentrations in authentic Bordeaux wines, which can be used with reasonable certainty as specific parameters for identifying regional wineries. For comparison, a set of imitated Bordeaux wines was studied for Sr isotopic and elemental compositions. A significant excess of both parameters in suspicious wines were found in reference to authentic values. Such natural and anthropogenically induced variations offer an enhanced discriminating potential of Sr. The unique Sr binary signature may detect imitated wines and trace genuine products from different regional wineries. The obtained results shown a promising perspective for wine authenticity control by means of Sr isotopic and elemental composition.

Total As and As speciation from worldwide collected red wine samples.

Total As and As speciation were measured in 147 red wines collected worldwide by ICP-MS and HPLC-ICP-MS, respectively. The samples included mid-priced to prestigious wines with vintages covering a period of almost 50 years. Total As concentration ranged from below 0.1 to 56 µg/L (average value: 4.0 ± 5.9 µg/L). None of the samples presented a concentration exceeding the limit set by the Office of Vine and Wine of 200 µg/L. Inorganic As was the most abundant form, representing from about half to all total As, mainly as As(III). Dimethylarsinic-acid (DMA) was detected in slightly less than half of the samples, accounting for a few to several dozens of percent. Monomethylarsonic-acid (MMA) was only detected in a few samples. In average, the DMA concentration seemed to be higher in the Bordeaux wines than in the other ones, irrespective of the total As concentration.

Total Selenium Quantification in Biological Samples by Inductively Coupled Plasma Mass Spectrometry (ICP-MS).

Selenium (Se) is an element readily absorbed during the intestinal tract, distributed in the body by means of blood and excreted mainly by urine or feces. Here, we describe the method allowing the determination of the total Se content in biological tissues and fluids by Inductively Coupled Plasma Mass Spectrometry (ICP MS).

Quantification of SeMet and SeCys in Biological Fluids and Tissues by Liquid Chromatography Coupled to Inductively Coupled Plasma Mass Spectrometry (HPLC-ICP MS).

Selenium (Se) is an element readily absorbed during the intestinal tract and distributed in the body. In biological fluids, tissues, and animal products, Se is known to be present mainly in the form of a selenoamino-acid (selenomethionine (SeMet) or selenocysteine (SeCys)). Both amino-acids have different biological activities which justifies their discrimination. Here, we describe the method allowing the simultaneous determination of SeMet and SeCys in blood/plasma, animal tissues, milk, and eggs by two-dimensional Liquid Chromatography coupled to Inductively Coupled Plasma Mass Spectrometry (2D HPLC-ICP MS).

Optimization and validation of the methods for the total mercury and methylmercury determination in breast milk.

The objective of the work was to develop and validate methods for the total Hg and methylmercury (MeHg) in breast milk that could be further used to obtain first data on chemical contamination of French breast milk. For total Hg determination, the potential of two techniques, namely Advanced Mercury Analyzer (AMA) and ICP MS, was compared. For MeHg determination, ICP MS detection associated to a quantification by isotopic dilution was used and the potential of a preliminary separation by gas or liquid chromatography was evaluated and discussed. The optimization studies have shown that AMA for total Hg determination and HPLC - ID - ICP MS, after a preconcentration step by freeze-drying, for MeHg quantification were the most relevant methods to use for epidemiologic studies. The figures of merit for both methods were evaluated by means of accuracy profiles in terms of limits of quantification (1.82 and 1.35µg Hg/kg dry weight, corresponding to 0.22 and 0.16µg Hg/kg wet basis for total Hg and MeHg, respectively), repeatability (2-11% and 3-8% for total Hg and MeHg respectively), intermediate precision reproducibility (4-12% and 4-8% for total Hg and MeHg respectively) and trueness bias (-0.1-9% and -4-0% for total Hg and MeHg respectively). The methods were then applied to 180 breast milk samples. Total Hg concentrations ranged from

Rapid ion-exchange matrix removal for a decrease of detection limits in the analysis of salt-rich reservoir waters for fluorobenzoic acids by liquid chromatography coupled with tandem mass spectrometry.

A matrix removal procedure with ion-exchange resin prior to analysis for 18 fluorinated benzoic acids (FBAs) tracers in saline (>25% salt) reservoir water was optimized. The elimination of >98% of salt and the simultaneous matrix sample cleanup allowed the direct analysis using the supernatant by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). This resulted in a gain in detection limits for most of the tracers in comparison with the reference method (direct analysis after minimum required dilution). The limits of detection (LODs) were in the range of 0.01-0.15 ng/ml and compared to other studies the developed method provided comparable limits of detection and advantage of simplified and shorter sample preparation. The presented method offers a considerable gain in simplicity and analysis time. Recoveries for all the tracers reached 80-100%, except for 2-FBA and 2,6-dFBA for which they were ca. 60%. The low recoveries were corrected by the use of five isotopically labeled internal standards. The method was validated by the analysis of spiked samples and by an independent comparison of the results with those obtained by solid-phase extraction LC-MS/MS method.

Beryllium in exhaled breath condensate as a biomarker of occupational exposure in a primary aluminum production plant.

OBJECTIVE: Low beryllium exposure can induce pulmonary granulomatosis, so called berylliosis. For occupational health monitoring, it is more relevant to assess the internal dose of Be received by the lungs than urinary or atmospheric Be. Exhaled breath condensate (EBC) is a matrix collected non-invasively that derives from the airway lining fluid. EBC beryllium (Be) levels were evaluated as a marker of occupational exposure in a primary aluminum production plant. METHODS: We collected urine and EBC from controls and workers recently exposed to beryllium in the pot room and the anode repair sectors, and calculated a cumulative beryllium exposure index (CBEI) summing the number of years of employment in each task and multiplying by the estimated average beryllium exposure for the task. Concentrations of beryllium and aluminum were measured in EBC (Be-EBC and Al-EBC) and in urine (Be-U and Al-U) by ICP-MS. RESULTS AND CONCLUSION: We have shown that it was possible to measure Be and Al in workers' EBC. Compared with controls and after adjustment for smoking status, levels of Be-EBC and Al-EBC were higher in pot room workers and exposed subjects, respectively. Due to its relationship with CBEI, but not with Be-U, it appears that Be-EBC could be a promising marker of occupational exposure and provide additional toxicokinetic information in occupational health studies.

Cr(VI) speciation in foods by HPLC-ICP-MS: investigation of Cr(VI)/food interactions by size exclusion and Cr(VI) determination and stability by ion-exchange on-line separations.

A method has been developed for the specific and sensitive determination of Cr(VI) in foods. First, the interactions between Cr(VI) and the matrices were investigated by size-exclusion HPLC-ICP-MS (SEC-ICP-MS). Evidence was found for the complexation of Cr(VI) potentially present with the ligands. For quantification of Cr(VI), the method was based on an alkaline extraction (NH4OH solution at pH 11.5) followed by Cr(VI) determination by anion-exchange HPLC-ICP-MS. Analytical performances of the method were satisfactory in terms of linearity, specificity, accuracy, repeatability, and intermediate precision. Detection limits ranged from 1 to 10 µg/kg, depending on the matrices investigated. The method was then applied for the determination of Cr(VI) in several products (dairy products, flour, chocolate, vegetables, fruits, meat, fish, eggs, and beverages) from different brands and origins. Cr(VI) was found in none of the samples investigated. To further investigate the reason for this absence, a stability study of spiked Cr(VI) was therefore conducted. A semi-skimmed cow milk was selected for this study. Cr(VI) was shown to be unstable in this matrix with a degradation rate increasing with the temperature.

Use of dried blood spots and inductively coupled plasma mass spectrometry for multi-element determination in blood.

The paper describes the development of an inductively coupled plasma mass spectrometry (ICP MS) method for multitrace element determination in dried blood spots (DBSs). The analytical conditions were optimized using Seronorm™ L-3 and L-1 Certified Reference Materials. The best results were obtained by sampling blood drops on a decontaminated PVDF filter membrane. After drying under metal-free conditions, the DBSs underwent acidic digestion and were analyzed with ICP MS. The method was then validated for As, Cd, Cu, Pb, Mo, Se and Zn. Using a matrix-matched calibration curve, the recovery levels ranged from 96% to 117%. The repeatability and reproducibility were generally below 15%. Limits of quantification ranging from 0.5 to 50 µg/L. In order to investigate the analytical procedure under real sampling conditions, the results obtained from DBSs and liquid blood aliquots (less subject to contamination) from two adult subjects were compared.

Determination of Zn-, Cu- and Mn-glycinate complexes in feed samples and in-vitro and in-vivo assays to assess their bioaccessibility in feed samples.

A method was developed for the quantification of Zn-, Cu- and Mn-glycinates in supplemented feed samples. The coupling of capillary electrophoresis (CE) with ICP MS detection after purification of the extract by ultrafiltration was shown to be efficient for the quantitative recovery of glycinates. The method developed was then applied to evaluate the bioaccessibility of glycinates using a sequential enzymolysis approach. The data obtained indicated a strong bioaccessibility of each element (79-94%). A new complex was also found to be formed during the digestion process. Bioavailability was then evaluated by analyzing plasma samples of horses supplemented with glycinates-rich feed. Intact glycinates could not be detected in plasma samples but a Cu-containing molecule was found more abundant after CuGly treatment.

Comparative study of a new organic selenium source v. seleno-yeast and mineral selenium sources on muscle selenium enrichment and selenium digestibility in broiler chickens.

Two experiments were conducted on broiler chickens to compare the effect of a new organic Se source, 2-hydroxy-4-methylselenobutanoic acid (HMSeBA; SO), with two practical Se additives, sodium selenite (SS) and Se yeast (SY). The relative bioavailability of the different Se sources was compared on muscle (pectoralis major) total Se, selenomethionine (SeMet) and selenocysteine (SeCys) concentrations and apparent digestibility of total Se (ADSe). In the first experiment, from day (d) 0 to d21, Se sources were tested at different supplied levels and compared with an unsupplemented diet (NC). No significant effects were observed on growth performance during the experimental period. However, the different Se sources and levels improved muscle Se concentration compared with the NC, with a significant source effect in the following order: SS < SY < SO (P<0·05). Seleno-amino acids speciation results for NC, SY and SO at 0·3 mg Se/kg feed indicated that muscle Se was only present as SeMet or SeCys, showing a full conversion of Se by the bird. The second experiment (d0-d24) compared SS, SY or SO at 0·3 mg Se/kg feed. The ADSe measurements carried out between d20 and d23 were 24, 46 and 49% for SS, SY and SO, respectively, with significant differences between the organic and mineral Se sources (P<0·05). These results confirmed the higher bioavailability of organic Se sources compared with the mineral source and demonstrated a significantly better efficiency of HMSeBA compared with SY for muscle Se enrichment.

Characterization of metal glycinate complexes by electrospray Q-TOF-MS/MS and their determination by capillary electrophoresis-ICP-MS: application to premix samples.

A method was developed for the determination of metal complexes with glycine (glycinates, [M(Gly)(x)(H(2)O)(y)(SO(4))(z)](n), where M denotes Zn, Cu, Mn and Fe) in premix samples used for the preparation of animal feeds enriched in essential trace elements. The method was based on the extraction of the glycinates with 10 mM ammonium acetate (pH 7.4) followed by their determination using capillary electrophoresis with ICP MS detection. The stability of the glycinates in solution was verified by electrospray TOF-MS. Each supplement was shown to be a mixture of complexes, with polymerization degrees ranging from n = 1 to n = 4 (depending on the metal), that were fully or partially dehydrated. The metal glycine complex moiety was found to be preserved during capillary electrophoresis. The detection limits, calculated as three times the standard deviation of the blank plus the blank, were between 0.05 and 0.2 microg mL(-1) (as the metal), and the calibration curves were linear, allowing the analysis of premix samples. Repeatability for glycinate standards was below 12%, and analytical precision was typically within 15%.

Simultaneous speciation of selenomethionine and 2-hydroxy-4-methylselenobutanoic acid by HPLC-ICP MS in biological samples.

An analytical method was developed for the simultaneous speciation of selenomethionine (SeMet) and 2-hydroxy-4-methylselenobutanoic acid (NutraSelen), a new SeMet precursor. The compounds could be baseline resolved by ion-pairing reversed-phase HPLC using ICP MS detection. Detection limits of 1 ng mL(-1) (Se content) could be reached. SELM-1 reference material was used to validate the SeMet measurement. Additionally, the quantification of NutraSelen was validated by standard addition together with checking the Se mass balance. The procedure developed was then applied to the monitoring of the conversion of NutraSelen into SeMet by yeast.

Determination of selenocysteine and selenomethionine in edible animal tissues by 2D size-exclusion reversed-phase HPLC-ICP MS following carbamidomethylation and proteolytic extraction.

A method was developed for the simultaneous determination of selenomethionine (SeMet) and selenocysteine (SeCys) in meat (chicken and lamb muscles) and different offal tissues (heart, liver, kidney). The analytical procedure was based on the protein extraction with urea under reducing conditions (dithiothreitol), derivatization of SeCys and SeMet by carbamidomethylation with iodoacetamide (IAM) followed by quantitative proteolysis. The mixture of the derivatized Se-amino acids was purified by size-exclusion liquid chromatography (LC) and analysed by ion-paring reversed-phase HPLC-inductively coupled plasma mass spectroscopy (ICP MS). The quantification of SeCys and SeMet was carried out by the method of standard additions. (77)SeMet was used to control the SeMet derivatization efficiency and recovery. The method was validated by the determination of the Se mass balance. The Se-amino acids accounted for 91 +/- 8% of the total selenium (mean of 95 samples of seven tissues analysed over a period of 18 months). The method was applied to the discrimination of the contribution of selenoproteins (containing SeCys) and other Se-containing proteins (containing SeMet) in tissues of animals during supplementation studies (dose-effect and tolerance).

Speciation of nickel in a hyperaccumulating plant by high-performance liquid chromatography-inductively coupled plasma mass spectrometry and electrospray MS/MS assisted by cloning using yeast complementation.

A novel analytical approach based on a combination of multidimensional hyphenated techniques and cloning of the Ni-resistance gene using yeast complementation screens was developed for the identification of nickel species in a Thlaspi caerulescens hyperaccumulating plant. The presence of an unknown strong Ni complex was demonstrated by size exclusion HPLC-capillary electrophoresis with ICPMS detection. The Ni-containing peak was characterized by electrospray MS (m/z 360) and shown by collision-induced dissociation MS to be a chelate with a tricarboxylic amino acid ligand. To identify the species and demonstrate its functional character, a cDNA library was constructed from T. caerulescens, expressed in the yeast, and screened on a toxic Ni2+ medium. The extract from the surviving transformant culture gave identical HPLC-ICPMS, CZE-ICPMS, and ES MS/MS data and contained a cDNA insert homologous to the nicotianamine synthase gene. This observation allowed the identification of nicotianamine as the nickel-binding ligand. The presence of the Ni-nicotianamine complex was ultimately demonstrated by comparing tandem mass spectra of the plant and yeast extracts with those of a synthetic standard.

Investigation of the response of wood-rotting fungi to copper stress by size-exclusion chromatography and capillary zone electrophoresis with ICP MS detection.

A method based on the coupling of size-exclusion chromatography (SEC) with inductively coupled plasma mass spectrometry (ICP MS) was developed for screening the changes in the bioligand composition of wood-rotting fungi as a function of their exposure to copper stress. Strains of four different species of wood-rotting fungi: Phanerochaete chrysosporium, Schizophyllum commune, Daedalea quercina and Pleurotus ostreatus were examined. Only one, namely Ph. chrysosporium, showed any significant difference in terms of the fingerprint of Cu-binding ligands between control and exposed cells which suggest trapping of Cu(II) by cell walls as the only resistance mechanisms. In the case of Ph. chrysosporium the bioinduction of a new Cu-binding ligand was demonstrated. The presence of a new compound in the SE chromatographic fraction of interest was confirmed by CZE-ICP MS. Attempts to identify the new compound by electrospray MS/MS failed because of insufficient sensitivity.