In vivo identification of potential uranium protein targets in zebrafish ovaries after chronic waterborne exposure.

Ecotoxicological studies have indicated the reprotoxicity of uranium (U) in zebrafish, but its molecular mechanisms remain unclear. Due to the non-covalent nature of U-protein complexes, canonical proteomics approaches are often not relevant as they usually use denaturating reagents or solvents. In this study, non-denaturating (ND) methods were used to obtain insight into the nature of U potential targets in ovaries of reproduced and non-reproduced zebrafish after 20 days of exposure to an environmentally relevant U concentration (20 µg L-1). After the ND sample preparation, 1-dimensional (SEC) and 2-dimensional (OGE × SEC) separations followed by ICP-sector-field MS measurements (U, P, Fe, Cu, and Zn) enabled the determination of chemical characteristics (MW, pI) of the metal-protein complexes. Phosphorus and U coelution confirmed the affinity of U for P-containing proteins. In addition, 2D separation allowed the discrimination of Fe-metalloproteins as potential U targets. Finally, 20 protein candidates for U complexation were identified after tryptic digestion conditions by LC-ESI FT MS and a database search. Potential U targets were mainly involved in three biological processes: oxidative stress regulation (SOD, GST), cytoskeleton structure (actin) and embryo early development (vtg, initiation factor).

Assessment of committed effective dose due to the ingestion of (210)Po and (210)Pb in consumed Lebanese fish affected by a phosphate fertilizer plant.

Ingestion of radionuclides through seafood intake is a one of the sources contributing to the internal effective dose in the human organism. In order to evaluate the internal exposure and potential risks due to (210)Po and (210)Pb associated with fish consumption, these radionuclides were measured in commonly consumed fish species from a clean area and an area subjected to the impact of a Lebanese phosphate fertilizer plant. The highest concentration of (210)Pb was 98.7 Bq/kg fresh weight while (210)Po activity concentrations varied from 3.6 Bq/kg to 140 Bq/kg. A supplementary radiation exposure was detected; the highest committed effective dose due to (210)Po and (210)Pb was found to be 1110 µSv/y and 450 µSv/y, respectively. Moreover, the average mortality and morbidity risks due to the fish consuming were estimated.

Multiresidue analysis of 22 sulfonamides and their metabolites in animal tissues using quick, easy, cheap, effective, rugged, and safe extraction and high resolution mass spectrometry (hybrid linear ion trap-Orbitrap).

A new high performance liquid chromatography-high resolution mass spectrometry (HPLC-HRMS) method was developed for a simultaneous multi-residue analysis of 22 sulfonamides (SAs) and their metabolites in edible animal (pig, beef, sheep and chicken) tissues. Sample preparation was optimized on the basis of the "QuEChERS" protocol. The analytes were identified using their LC retention times and accurate mass; the identification was further confirmed by multi-stage high mass accuracy (<5ppm) mass spectrometry. The performance of the method was evaluated according to the EU guidelines for the validation of screening methods for the analysis of veterinary drugs residues. Acceptable values were obtained for: linearity (R(2)<0.99), limit of detection (LOD, 3-26µg/kg), limit of quantification (LOQ, 11-88µg/kg), accuracy (recovery 88-112%), intra- and inter-day precision 1-14 and 1-17%, respectively, decision limit (CCα) and detection capability (CCß) around the maximum residue limits (MRL) of SAs (100µg/kg). The method was validated by analysis of a reference material FAPAS-02188 "Pig kidney" with ǀ Z-scoreǀ<0.63. The method was applied to various matrices (kidney, liver, muscle) originated from pig, beef, sheep, and chicken) allowing the simultaneous quantification of target sulfonamides at concentration levels above the MRL/2 and the identification of untargeted compounds such as N(4)-acetyl metabolites using multi-stage high mass accuracy mass spectrometry.

Subcellular fractionation and chemical speciation of uranium to elucidate its fate in gills and hepatopancreas of crayfish Procambarus clarkii.

Knowledge of the organ and subcellular distribution of metals in organisms is fundamental for the understanding of their uptake, storage, elimination and toxicity. Detoxification via MTLP and MRG formation and chelation by some proteins are necessary to better assess the metal toxic fraction in aquatic organisms. This work focused on uranium, natural element mainly used in nuclear industry, and its subcellular fractionation and chemical speciation to elucidate its accumulation pattern in gills and hepatopancreas of crayfish Procambarus clarkii, key organs of uptake and detoxification, respectively. Crayfish waterborne exposure was performed during 4 and 10d at 0, 30, 600 and 4000 µg UL(-1). After tissue dissection, uranium subcellular fractionation was performed by successive ultracentrifugations. SEC-ICP MS was used to study uranium speciation in cytosolic fraction. The uranium subcellular partitioning patterns varied according to the target organ studied and its biological function in the organism. The cytosolic fraction accounted for 13-30% of the total uranium amount in gills and 35-75% in hepatopancreas. The uranium fraction coeluting with MTLPs in gills and hepatopancreas cytosols showed that roughly 55% of uranium remained non-detoxified and thus potentially toxic in the cytosol. Furthermore, the sum of uranium amount in organelle fractions and in the non-detoxified part of cytosol, possibly equivalent to available fraction, accounted for 20% (gills) and 57% (hepatopancreas) of the total uranium. Finally, the SEC-ICP MS analysis provided information on potential competition of U for biomolecules similar than the ones involved in endogenous essential metal (Fe, Cu) chelation.

Selenium metabolomics in yeast using complementary reversed-phase/hydrophilic ion interaction (HILIC) liquid chromatography-electrospray hybrid quadrupole trap/Orbitrap mass spectrometry.

A high efficiency chromatographic separation on a porous graphitic carbon stationary phase was developed for a large-scale separation of selenium metabolites in Se-rich yeast prior to their identification by electrospray hybrid quadrupole trap/Orbitrap mass spectrometry (Orbitrap MS(n)). The reversed-phase (RP) separation mode offered distinctly higher separation efficiency than the hydrophilic ion interaction (HILIC) mode. The latter was nevertheless complementary and useful to validate the detection of several compounds. The method allowed the detection of 64 metabolites including 30 SeSe or SeS conjugates (3 triple S/Se/S ones) and 14 selenoethers. 21 previously unreported metabolites were detected on the basis of the selenium isotopic pattern usually matched with the sub-ppm mass accuracy. 9 of these metabolites were subsequently identified using the multi-stage high mass accuracy (<5ppm) mass spectrometry. The identified metabolites (and their groups) were quantified on-line by ICP-MS fitted with a frequency-matching generator allowing a quasi-uniform response over the large (20-90%) acetonitrile mobile phase concentration range. The morphology of HPLC-ICP-MS chromatograms was remarkably similar to that of HPLC multi-ion extracted ESI-MS chromatograms. The detection limits obtained by ICP MS and ESI MS were 1 and 2ppb, respectively.

Identification of mercury and other metals complexes with metallothioneins in dolphin liver by hydrophilic interaction liquid chromatography with the parallel detection by ICP MS and electrospray hybrid linear/orbital trap MS/MS.

A novel analytical procedure for the identification of metal (Hg, Cd, Cu, Zn) complexes with individual metallothionein (MT) isoforms in biological tissues by electrospray MS/MS was developed. The sample preparation was reduced to three rapid steps: the two-fold dilution of the sample cytosol with acetonitrile, the recovery of the supernatant containing MT-complexes by centrifugation and its concentration under nitrogen flow. The replacement of reversed phase HPLC by hydrophilic interaction LC (HILIC) allowed the preservation of the unstable and low abundant metallothionein zinc-mercury mixed complexes (MT-Zn(6)Hg). The MT complexes eluted were detected by ICP MS and identified in terms of molecular mass by electrospray high resolution (100,000) MS. The identification was completed by on line demetallation and the determination of the molecular mass of the apoform, followed by amino acid sequencing in the top-down mode using high energy collision fragmentation (HCD). The method was applied to the identification of MT complexes in a white-sided dolphin (Lagenorhynchus acutus) liver homogenate. The Zn complex of the N-acetylated MT2 isoform was found to be predominant, the presence of mixed complexes with Cd, Cu and, for the first time ever, Hg, was demonstrated. The latter finding has the potential to shed new light on the mercury detoxification mechanism in marine organisms.

Direct sensitive simultaneous determination of fluorinated benzoic acids in oil reservoir waters by ultra high-performance liquid chromatography-tandem mass spectrometry.

A direct ultra-high performance reverse-phase HPLC (UHPLC)--electrospray MS/MS method was developed for the simultaneous determination of 16 fluorinated benzoic acids (FBAs) in oil reservoir waters. The separation was achieved within 5 min in a non-linear gradient mode using a 1-ml sample aliquot. The method detection limits were in the lower ng/ml range (between 0.05 and 50 ng/ml, depending on the compound) owing to the use of the travelling-wave collision cell technology. The method developed was more sensitive, faster (by avoiding sample preconcentration and purification steps) and more robust than the GC/MS methods currently used in oil industries. The accuracy of the method was verified by comparison with GC/MS results. It was applied to the determination of FBAs in water samples coming from reservoir tracing campaigns.

Imaging and speciation of trace elements in biological environment.

Mineral elements, often at the trace level, play a considerable role in physiology and pathology of biological systems. Metallogenomics, metalloproteomics, and metallomics are among the emerging disciplines which are critically dependent on spatially resolved concentration maps of trace elements in a cell or tissue, on information on chemical speciation, and on that on metal-binding coordination sites. The mini-review discusses recent progress in analytical techniques for element profiling on the genome scale, biological trace element imaging, and probing, identification and quantification of chemical species in the biological environment. Imaging of the element distribution in cells and tissue sections is becoming possible with sub-micrometer spatial resolution and picogram-level sensitivity owing to advances in laser ablation MS, ion beam and synchrotron radiation X-ray fluorescence microprobes. Progress in nanoflow chromatography and capillary electrophoresis coupled with element specific ICP MS and molecule-specific electrospray MS/MS and MALDI enables speciation of elements in microsamples in a complex biological environment. Laser ablation ICP MS, micro-SXRF, and micro-PIXE allow mapping of trace element distribution in 1D and 2D proteomics gels. The increasing sensitivity of EXAFS and XANES owing to the use of more intense synchrotron beams and efficient focusing optics provide information about oxidation state, fingerprint speciation of metal sites and metal-site structures.

An interdisciplinary approach to investigate the impact of cobalt in a human keratinocyte cell line.

Since in nuclear power plants, risks of skin contact contamination by radiocobalt are significant, we focused on the impact of cobalt on a human cutaneous cell line, i.e. HaCaT keratinocytes. The present paper reports an interdisciplinary approach aimed at clarifying the biochemical mechanisms of metabolism and toxicity of cobalt in HaCaT cells. Firstly, a brief overview of the used instrumental techniques is reported. The following parts present description and discussion of results concerning: (i) toxicological studies concerning cobalt impact towards HaCaT cells (ii) structural and speciation fundamental studies of cobalt-bioligand systems, through X-ray absorption spectroscopy (XAS), ab initio and thermodynamic modelling (iii) preliminary results regarding intracellular cobalt speciation in HaCaT cells using size exclusion chromatography/inductively coupled plasma-atomic emission spectroscopy (SEC/ICP-AES) and direct in situ analysis by ion beam micropobe analytical techniques.

Concentrations and bioavailability of cadmium and lead in cocoa powder and related products.

Concentrations and bioavailability of cadmium (Cd) and lead (Pb) were determined in cocoa powders and related products (beans, liquor, butter) of different geographical origins. Particular attention was paid to the fractionation of these metals, which was investigated by determining the metal fraction soluble in extractant solutions acting selectively with regard to the different classes of ligands. The targeted classes of Cd and Pb species included: water-soluble compounds, polypeptide and polysaccharide complexes, and compounds soluble in simulated gastrointestinal conditions. The bioavailability of Cd and Pb from cocoa powder, liquor and butter was evaluated using a sequential enzymolysis approach. The data obtained as a function of the geographical origin of the samples indicated strong differences not only in terms of the total Cd and Pb concentrations, but also with regard to the bioavailability of these metals. The Cd concentrations in the cocoa powders varied from 94 to 1833 microg kg(-1), of which 10-50% was potentially bioavailable. The bioavailability of Pb was generally below 10% and the concentrations measured in the cocoa powders were in the 11-769 microg kg(-1) range. Virtually all the Cd and most of Pb were found in the cocoa powder after the pressing of the liquor.

Development of a sequential enzymolysis approach for the evaluation of the bioaccessibility of Cd and Pb from cocoa.

An in vitro model simulating enzymatic activity in the gastrointestinal tract was developed for the assessment of the potential bioaccessibility of Cd and Pb in cocoa powder and liquor. The model was based on the sequential extraction with simulated gastric and intestinal juices; the residue after the latter extraction was further investigated by using, in parallel, solutions of phytase and cellulase. The solubility of Cd and Pb in the corresponding enzymatic extracts was measured by ICP MS. The bioaccessibility of Cd in cocoa varied from 10 to 50% in gastrointestinal conditions. An additional 20 or 30% of Cd could be recovered by phytase and cellulase, respectively. The bioaccessibility of Pb in gastrointestinal conditions did not exceed 5-10%. Only a few percent more of this metal could be recovered by extraction with phytase and cellulase.

Selenium from selenium-rich Spirulina is less bioavailable than selenium from sodium selenite and selenomethionine in selenium-deficient rats.

The bioavailabilty of selenium (Se) from selenium-rich Spirulina (SeSp) was assessed in Se-deficient rats by measuring tissue Se accumulation and glutathione peroxidase (GSH-Px) activity. For 42 d, rats were subjected to dietary Se depletion by consumption of a Torula yeast (TY)-based diet with no Se; controls were fed the same diet supplemented with 75 microg Se/kg diet as sodium selenite. Se-deficient rats were then repleted with Se (75 microg/kg) by the addition of sodium selenite, selenomethionine (SeMet) or SeSp to the TY basal diet. Selenium speciation in SeSp emphasized the quasi-absence of selenite (2% of total Se); organic Se comprised SeMet (approximately 18%), with the majority present in the form of two selenoproteins (20-30 kDa and 80 kDa). Gross absorption of Se from SeSp was significantly lower than from free SeMet and sodium selenite. SeMet was less effective than sodium selenite in restoring Se concentration in the liver but not in kidney. SeSp was always much less effective. Similarly, Se from SeSp was less effective than the other forms of Se in restoring GSH-Px activity, except in plasma and red blood cells where no differences were noted among the three sources. This was confirmed by measuring the bioavailability of Se by slope-ratio analysis using selenite as the reference form of Se. Although Se from SeSp did not replenish Se concentration and GSH-Px activity in most tissues to the same degree as the other forms of Se, we conclude that it is biologically useful and differently metabolized due to its chemical form.

Investigation of arsenic speciation in oyster test reference material by multidimensional HPLC-ICP-MS and electrospray tandem mass spectrometry (ES-MS-MS).

Multidimensional (size-exclusion-anion-exchange-cation-exchange) liquid chromatography with ICP-MS detection was developed to produce a map of water-soluble species in an oyster test reference material. The presence of arsenobetaine, trimethyl(2-carboxyethyl)arsonium inner salt, arsenocholine, dimethylarsonic acid, tetramethylammonium ion, As(v) and two arsenosugars was demonstrated by ES-MS-MS. A previously unreported compound was isolated and identified by ES-MS-MS as 5-dimethylarsinoyl-beta-ribofuranose. Anion-exchange chromatography was optimized to produce a chromatographically pure peak of arsenobetaine (accounting for ca. 64% of all water-soluble As present) that was used to quantify this compound.

Determination of phytochelatins by capillary zone electrophoresis with electrospray tandem mass spectrometry detection (CZE-ES MS/MS).

The coupling of capillary zone electrophoresis with electrospray mass spectrometry was optimized for the direct determination of phytochelatins (PCs) in extracts obtained from cells and plants that had been exposed to metal stress. Gluthathione and phytochelatins belonging to the different families (gamma Glu-Cys)nGly (n-PC), (gamma Glu-Cys)nSer, (gamma Glu-Cys)n beta Ala and (gamma Glu-Cys)n were separated in an uncoated capillary at pH 4 using a 5 mM ammonium acetate buffer, and detected by electrospray (ES) MS in the full scan mode (300-1100 u). The use of on-line tandem MS detection in the product ion scan mode of putative protonated molecules of PCs allowed the unambiguous confirmation of the identity of the compounds detected by ES MS. The operational conditions were optimized and the figures of merit were evaluated using n-PC2, n-PC3 and n-PC4 standards purified from a mixture obtained after the reaction of glutathione in the presence of Cd2+ and the enzyme PC-synthase. The method was applied to the characterization of bioinduced ligands in cell cultures of soybeans (Glycine max) and in rice (Oryza sativa) roots without the need for a preliminary sample cleanup by size-exclusion and/or reversed phase chromatography.

Dynamics of (Cd,Zn)-metallothioneins in gills, liver and kidney of common carp Cyprinus carpio during cadmium exposure.

Cadmium concentrations, (Cd,Zn)-metallothionein (MT) concentrations, MT synthesis and the relative amounts of cadmium bound to (Cd,Zn)-MTs were determined in gills, liver and kidney of common carp Cyprinus carpio exposed to 0, 0.5 microM (0.06 mg.l(-1)), 2.5 microM (0.28 mg.l(-1)) and 7 microM (0.79 mg.l(-1)) Cd for up to 29 days. Cadmium accumulation was in the order kidney > liver > gills. Control levels of hepatic (Cd,Zn)-MT were four times higher compared to those of gills and kidney. No increases in (Cd,Zn)-MT concentrations were observed in liver during the exposure period. In comparison with control carp, (Cd,Zn)-MT concentrations increased up to 4.5 times in kidney and two times in gills. In both these organs, (Cd,Zn)-MT concentrations were linearly related with cadmium tissue levels and with the de novo synthesis of MTs. Hepatic cadmium was almost completely bound to (Cd,Zn)-MT, while percentages of non-MT-bound cadmium were at least 40% in gills and 25% in kidney. This corresponded with a total saturation of (Cd,Zn)-MT by cadmium in kidney and a saturation of approximately 50 and 60% in gills and liver, respectively. The final order of non-MT-bound cadmium was kidney > gills > liver. Our results indicate that cadmium exposure causes toxic effects, which cannot be correlated with the accumulated levels of the metal in tissues. Although cadmium clearly leads to the de novo synthesis of MT and higher (Cd,Zn)-MT concentrations, the role of this protein in the detoxification process is clearly organ-specific and its synthesis does not keep track with cadmium accumulation.

Evaluation of the accuracy of the determination of lead isotope ratios in wine by ICP MS using quadrupole, multicollector magnetic sector and time-of-flight analyzers.

Different mass analysers [(quadrupole (Q), time-of-flight (TOF) and multicollector (MC) sector-field (SF)] of ions produced in an inductively coupled plasma were evaluated for the determination of lead isotope ratios in wine samples. A population of 20 wines of different origin including two reference wines from the EC Standards, Measurement and Testing Programme with concentrations varying between 7-140 mug Pb l(-1) was investigated. Wines were analyzed directly by Q ICP MS and MC ICP MS. The poor sensitivity of the TOF instrument, further aggravated by matrix signal suppression, did not allow the acquisition of data for wine samples that contained less than 50 mug l(-1) in the direct sample introduction mode. The separation and preconcentration of lead were therefore required. The precision obtained for the (206)Pb/(207)Pb and (208)Pb/(206)Pb were similar and equal to 0.14-2.7% for Q ICP MS, 0.04-0.17% for TOF ICP MS and 0.01-0.12% for MC ICP MS. The precision for (206)Pb/(204)Pb was 0.44-5.29, 0.15-1.7, 0.08-1.6%, respectively. On the level of accuracy, the data from TOF ICP MS and MC ICP MS were in good agreement. The accuracy of Q ICP MS data was judged satisfactory in comparison with the other techniques but their poor precision was a significant obstacle on the way of using these data for the determination of the geographic origin of wine.

Probing metal-complexes with metallothioneins by reversed phase microbore chromatography and capillary zone electrophoresis coupled with inductively coupled plasma and electrospray mass spectrometry.

Four different hyphenated techniques: microbore reversed phase (RP) HPLC-ICP MS, CZE-ICP MS, RP HPLC-ES MS and CZE-ES MS were investigated for the characterization of metallothionein-metal complexes under neutral pH conditions. Particular attention was given to the differentiation between metallothionein and artifact signals, identification of mixed-metal complexes, and the validity of the molecular mass as the identification parameter of the different MT iso- and sub-isoforms. Despite the similar morphology of chromatograms and electrophoregrams mass spectrometry revealed different origin of the apparently corresponding peaks. The performance of the four above mentioned techniques was characterized using the example of rabbit liver MT-1 preparation. Reversed-phase HPLC with post-column acidification prior to ES MS was judged to be the most versatile technique for the characterization of metal complexes with metallothioneins but other techniques offer valuable auxiliary information.

Species-selective analysis by microcolumn multicapillary gas chromatography with inductively coupled plasma mass spectrometric detection.

A glass rod (5-20 cm long, 2 mm o.d.) containing more than 1200 parallel microchannels (< 40 microns i.d.) was converted into a high-resolution (> 100 theoretical plates cm-1) GC column by coating the inside of each channel in a way that compensated for the dispersion of the channel inner diameter. The columns were evaluated for the separation of mixtures of several organometallic (Hg, Sn, Pb) compounds prior to on-line sensitive metalselective detection by ICPMS. Chromatographic separation conditions were optimized to enable a rapid (within a maximum 30 s) multielemental speciation analysis. Absolute detection limits were 0.1 pg for Hg, 0.05 pg for Sn, and 0.03 pg for Pb using the carrier gas flows of approximately 200 mL min-1. The microcolumn multicapillary GC/ICPMS developed was applied to the analysis of a number of environmental samples. The results were validated with certified reference materials for tin (BCR477, PACS-2) and mercury (DORM-1, TORT-1).

Detection of artefacts and peak identification in reversed-phase HPLC of metallothioneins by electrospray mass spectrometry.

The use of ion-spray mass spectrometry rendered it possible to characterize the signals obtained during studies of the polymorphism of metallothionein (MT) by reversed-phase (RP) HPLC in terms of the molecular mass. Artefact signals due to incomplete metallation, exchange of metals with the impurities of the column stationary phase and cross-contamination of the preparations purified by size-exclusion and anion-exchange chromatography may be present. On the other hand, some signals in RP HPLC with UV detection considered to belong to a single species were found to be composed of several complexes eluting precisely at the same time. On-line electrospray mass spectrometry was used to systematize the knowledge of the MT isoforms and subisoforms by attributing to each of the eluting peaks the molecular mass of the form involved and can be used to compare the results obtained for the different groups.