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).

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.

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.

Characterization of metallothionein isoforms by reversed-phase high-performance liquid chromatography with on-line post-column acidification and electrospray mass spectrometric detection.

Post-column acidification of the chromatographic effluent was developed to eliminate artefacts in investigations of the polymorphism of metallothionein (MT) by microbore reversed-phase HPLC with detection by pneumatically assisted electrospray mass spectrometry. Metallated species (Cd, Zn and mixed Cd-Zn complexes) were decomposed on-line to produce apo metallothioneins of which the molecular masses were determined by MS. Besides the simplification of the mass spectra taken at the apexes of the chromatographic peaks, the method resulted in a 10-fold improvement of the detection limit of metallothionein and allowed a more comprehensive and less ambiguous detection and identification of the iso- and subisoforms. The method was applied to the characterization of rabbit liver metallothioneins: rabbit liver MT (purified by size-exclusion chromatography only) and MT-1 and MT-2 isoform fractions purified additionally by anion-exchange chromatography.

Extraction-spectrophotometric determination of vanadium with 3,5-dinitrocatechol and Brilliant Green.

The formation and extraction of the ion-associates of the vanadium(V)-3,5-dinitrocatechol (DNC) anionic chelate complex with various basic dyes have been studied and a new sensitive extraction-spectrophotometric method for the determination of vanadium based on the system V(V)-DNC-Brilliant Green has been developed. Beer's law is obeyed up to a vanadium concentration of 0.3 mug/ml and the molar absorptivity is 1.7 x 1O(5) l.mole(-1). cm(-1) at 630 nm. The molar ratios of the components and the form of the vanadium(V) cation in the extracted compound have been determined, and the formula [VO(OH)(DNC)(2-)(2)][BG(+)](2) is proposed. Titanium, molybdenum, tungsten, EDTA and thiocyanate interfere seriously. The method becomes specific after a preliminary separation of vanadium by its extraction as the BPHA complex from H(2)SO(4)-HF medium, and is 40 times more sensitive than the spectrophotometric BPHA method. The proposed method has been applied to determination of traces of vanadium (about 10(-5)%) in alums.

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.

Analysis for metallothioneins using coupled techniques.

Analytical chemistry of metallothioneins based on the coupling of a high resolution separation technique with an element or species selective detection technique is discussed. The role of size-exclusion chromatography (SEC) with on-line atomic spectrometric detection for the quantification of metallothionein fraction in cell cytosols is evaluated. Particular attention is given to the conditions for the separation of metallated metallothionein isoforms (MT-1, MT-2, MT-3) and sub-isoforms within these classes by anion-exchange and reversed-phase HPLC. Techniques for interfacing chromatography with atomic absorption spectrometry (AAS), inductively coupled plasma atomic emission spectrometry (ICP AES) and ICP mass spectrometry (MS) are assessed. The potential of electrospray (tandem) mass spectrometry for the characterization of metallothionein isoforms with respect to molecular mass and aminoacid sequence is highlighted. Perspectives for capillary zone electrophoresis (CZE), microbore and capillary HPLC with ICP MS and electrospray MS(/MS) detection for the probing of metallothioneins are discussed. Applications of hyphenated techniques to the analysis of real-world samples are reviewed.

Validation of the determination of copper and zinc in blood plasma and urine by ICP MS with cross-flow and direct injection nebulization.

The simultaneous determination of copper and zinc in human plasma and urine by inductively coupled plasma mass spectrometry (ICP MS) is discussed. The performances of a cross-flow nebulizer and a direct-injection nebulizer (DIN) were compared. Flow-injection-DIN-ICP MS analysis of clinical samples using 1-2 mul samples was optimized. Isobaric interferences were discussed and were demonstrated to be eliminated for the (65)Cu and most of the Zn nuclides. The need for standard addition to compensate for signal suppression in the case of some serum samples was indicated. Results obtained by ICP MS using calibration with aqueous standard solutions were found to be in good agreement with those obtained by flame AAS for a batch of real blood plasma and urine samples. The methods developed were validated by analysis of several standard reference materials.

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.

Speciation analysis of organolead compounds in wine by capillary gas chromatography/microwave-induced-plasma atomic emission spectrometry.

A method was developed for the speciation analysis of ionic organolead compounds in wine. The analytes were extracted as diethyldithiocarbamate complexes into hexane and propylated with a Grignard reagent. The derivatized extract was analyzed by capillary gas chromatography/microwave-induced-plasma atomic emission spectrometry. The method of standard additions was used for calibration to correct for variable recoveries and signal enhancements. Red, rosé, and white wines from southern France were analyzed. Trimethyllead was the ubiquitous species. The wines made from grapes grown close to industrial zones showed elevated concentrations of ethyllead species. The concentrations of methyllead and ethyllead found in wines were compared with the concentrations of organolead found in rain water and plant sap collected in the viticultural regions. The ratio of methyllead to ethyllead in wines greatly exceeded the same ratio found in atmospheric deposits.

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.

Characterization of horse kidney metallothionein isoforms by electrospray MS and reversed-phase HPLC-electrospray MS.

Pneumatically assisted electrospray mass spectrometry (ESMS) in the direct mode and as a chromatographic detection technique was developed for the characterization of horse kidney metallothionein isoforms. Direct analysis in an acidic medium showed the presence of three major and five minor isoforms, the molecular masses of which were determined. The presence of the major isoforms (two of which matched the molecular masses calculated according to the published sequences) was confirmed by complexation with Cd at pH 4.0 and the determination of the stoichiometry of the complexes formed. Reversed-phase chromatography of Cd7-MT complexes (pH 6.0) gave two signals corresponding to the MT-1A and MT-1B isoforms. A post-column acidification procedure was developed to eliminate the possibility of artefacts associated with the formation of mixed-metal (Cd, Zn) complexes during chromatography in neutral media, and to improve the accuracy of the determination of the molecular mass of MT isoforms.

Polymorphism and identification of metallothionein isoforms by reversed-phase HPLC with on-line ion-spray mass spectrometric detection.

Microbore reversed-phase HPLC with on-line ion-spray mass spectrometric detection is proposed for a study of polymorphism of rabbit liver metallothionein (MTRL) and its major isoforms MT-1 and MT-2. Separation conditions had been optimized until each chromatographic peak could be attributed to a single metalloprotein species, of which the molecular mass could be determined by ionspray MS. At the optimized conditions (elution with the gradient 5-8% of acetonitrile within 50 min using a 5 mM acetate buffer at pH 6), the chromatogram of MTRL showed five peaks, whereas those of MT-1 and MT-2 showed nine and seven different peaks, respectively. The on-line determination of the molecular masses (+/- 1 Da) of the compounds eluted permits the unambiguous cataloguing and further referencing of putative and true MT subisoforms. The results obtained are compared with those obtained by direct ion-spray MS of the MT preparations at different pHs, with a goal to identify possible chromatographic artifacts. Metals (Cd, Cu) in the eluted complexes were studied by HPLC with on-line ICP MS detection.

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.

Purge-and-trap isothermal multicapillary gas chromatographic sample introduction accessory for speciation of mercury by microwave-induced plasma atomic emission spectrometry.

A compact device based on purge-and-trap multicapillary gas chromatography was developed for sensitive species-selective analysis of methylmercury and Hg2+ by atomic spectrometry. The operating mode includes in situ conversion of the analyte species to MeEtHg and HgEt2 and cryotrapping of the derivatives formed in a 0.53-mm-i.d. capillary, followed by their flash (< 30 s) isothermal low-temperature separation on a minimulticapillary (22 cm) column. The very low detection limits obtained (0.01 pg mL-1 of Hg for methylmercury) are due to the narrow injection band and reduced peak broadening in a bundle of 0.038-mm capillaries at high flow rates (> 60 mL min-1) compatible with an MIP AES detector (no dilution with a makeup gas is required). Developments regarding each of the steps of the analytical procedure and effects of operational variables (sample volume, purge flow, trap temperature, separation conditions) are discussed. The device allows speciation of MeHg+ and Hg2+ down to 5 pg g-1 in urine and, after a rapid microwave-assisted hydrolysis, down to 0.1 ng g-1 in solid biological samples with a throughput of 6 samples/h. The analytical protocols developed were validated by the analysis of DORM-1 (dogfish muscle), TORT-1 (lobster hepatopancreas), and Seronorm urine certified reference materials.