Non-chromatographic Speciation Analysis of Tellurium by HG-ICP-MS/MS at Sub ng L-1 Concentration in Natural Waters Using TiIII as a Pre-Reducing Agent.

An automated and high-throughput (36 h-1) method for extremely sensitive determination of the two main tellurium species in the environment, namely, tellurite (TeIV) and tellurate (TeVI), was developed. Flow injection hydride generation was interfaced for the first time with inductively coupled plasma triple quadrupole mass spectrometry (ICP-MS/MS) detection to assure interference-free tellurium analysis. ICP-MS/MS conditions were studied in detail. Using a mixture of He + O2 gases in the reaction cell, the background signals significantly dropped and Xe isobaric interference was eliminated, allowing measurement with the most abundant Te isotopes, that is, 128Te and 130Te, and offering a huge increase in sensitivity. Volatile H2Te was selectively generated by a HCl/NaBH4 reaction from TeIV or from both TeIV and TeVI (TeIV+VI) after pre-reduction of TeVI by a TiCl3 solution. The optimum conditions for TiCl3 as a pre-reductant and the pre-reduction kinetics were also investigated. Different reduction rates were found depending on the sample stabilization media (HCl, HNO3, or EDTA). The same sensitivity was found for TeIV and TeVI, measured after pre-reduction, and no significant matrix effect was observed in both fresh and seawaters. Therefore, external calibration was used for quantification in real samples. Under optimal conditions, this method reached an unprecedented limit of detection of 0.07 ng L-1 for both TeIV and TeIV+VI and an intra-day repeatability of 5.2% at the 5 ng L-1 level. The methodology was successfully applied to the speciation analyses in commercially available certified reference materials of river water and seawater, and in bottled water and lake water samples.

Selected Ion Flow Tube Mass Spectrometry as a Tool to Understand Hydride Atomization and the Fate of Free Analyte Atoms in an Externally Heated Quartz Tube Atomizer.

Hydride atomization and the fate of free analyte atoms in an externally heated quartz tube atomizer (QTA) were investigated employing selected ion flow tube mass spectrometry (SIFT-MS). SIFT-MS proved to be ideally suited to study water concentration in gases leaving the atomizer. This made it possible to quantify the oxygen "contaminant" flow rate to QTA as 0.04-0.05 mL min-1. This is valid for typical conditions of hydride generation. Most significantly, studies of temperature influence on water concentration resulted in detailed insight into hydrogen radical-forming reactions between oxygen and hydrogen. Minimum QTA temperatures required to generate hydrogen radicals under a variety of different flow rates and compositions of the QTA atmosphere were found to be in the range between 585 and 800 °C. The ability of SIFT-MS to detect extremely low concentrations of arsane and selane was employed to quantify the fraction of As and Se removed from the QTA in the form of hydride in dependence on QTA temperature under typical conditions of hydride generation. It was found that free As atoms formed by atomization of arsane decay to different species than to arsane. In the case of selane under typical atomization conditions, the efficiency of the decay of free Se atoms to selane was between 50 and 100% in dependence on actual flow rates and compositions of the QTA atmosphere.

A mass spectrometric study of hydride generated arsenic species identified by direct analysis in real time (DART) following cryotrapping.

Hydride generation (HG) coupled to cryotrapping was employed to introduce, separately and with high selectivity, four gaseous arsanes into a direct analysis in real time source for high-resolution mass spectrometry (DART-HR-MS). The arsanes, i.e., arsane (AsH3), methylarsane (CH3AsH2), dimethylarsane ((CH3)2AsH), and trimethylarsane ((CH3)3As), were formed under HG conditions that were close to those typically used for analytical purposes. Arsenic containing ion species formed during ambient ionization in the DART were examined both in the positive and negative ion modes. It was clearly demonstrated that numerous arsenic ion species originated in the DART source that did not accurately reflect their origin. Pronounced oxidation, hydride abstraction, methyl group(s) loss, and formation of oligomer ions complicate the identification of the original species in both modes of detection, leading to potential misinterpretation. Suitability of the use of the DART source for identification of arsenic species in multiphase reaction systems comprising HG is discussed.

Arsenic accumulation and speciation in two cultivars of Pteris cretica L. and characterization of arsenate reductase PcACR2 and arsenite transporter PcACR3 genes in the hyperaccumulating cv. Albo-lineata.

Pollution and poisoning with carcinogenic arsenic (As) is of major concern globally. Interestingly, there are ferns that can naturally tolerate remarkably high As concentrations in soils while hyperaccumulating this metalloid in their fronds. Besides Pteris vittata in which As-related traits and molecular determinants have been studied in detail, the As hyperaccumulation status has been attributed also to Pteris cretica. We thus inspected two P. cretica cultivars, Parkerii and Albo-lineata, for As hyperaccumulation traits. The cultivars were grown in soils supplemented with 20, 100, and 250 mg kg-1 of inorganic arsenate (iAsV). Unlike Parkerii, Albo-lineata was confirmed to be As tolerant and hyperaccumulating, with up to 1.3 and 6.4 g As kg-1 dry weight in roots and fronds, respectively, from soils amended with 250 mg iAsV kg-1. As speciation analyses rejected that organoarsenical species and binding with phytochelatins and other proteinaceous ligands would play any significant role in the biology of As in either cultivar. While in Parkerii, the dominating As species, particularly in roots, occurred as iAsV, in Albo-lineata the majority of the root and frond As was apparently converted to iAsIII. Parkerii markedly accumulated iAsIII in its fronds when grown on As spiked soils. Considering the roles iAsV reductase ACR2 and iAsIII transporter ACR3 may have in the handling of iAs, we isolated Albo-lineata PcACR2 and PcACR3 genes closely related to P. vittata PvACR2 and PvACR3. The gene expression analysis in Albo-lineata fronds revealed that the transcription of PcACR2 and PcACR3 was clearly As responsive (up to 6.5- and 45-times increase in transcript levels compared to control soil conditions, respectively). The tolerance and uptake assays in yeasts showed that PcACRs can complement corresponding As-sensitive mutations, indicating that PcACR2 and PcACR3 encode functional proteins that can perform, respectively, iAsV reduction and membrane iAsIII transport tasks in As-hyperaccumulating Albo-lineata.

Inter-laboratory study for the certification of trace elements in seawater certified reference materials NASS-7 and CASS-6.

Certification of trace metals in seawater certified reference materials (CRMs) NASS-7 and CASS-6 is described. At the National Research Council Canada (NRC), column separation was performed to remove the seawater matrix prior to the determination of Cd, Cr, Cu, Fe, Pb, Mn, Mo, Ni, U, V, and Zn, whereas As was directly measured in 10-fold diluted seawater samples, and B was directly measured in 200-fold diluted seawater samples. High-resolution inductively coupled plasma mass spectrometry (HR-ICPMS) was used for elemental analyses, with double isotope dilution for the accurate determination of B, Cd, Cr, Cu, Fe, Pb, Mo, Ni, U, and Zn in seawater NASS-7 and CASS-6, and standard addition calibration for As, Co, Mn, and V. In addition, all analytes were measured using standard addition calibration with triple quadrupole (QQQ)-ICPMS to provide a second set of data at NRC. Expert laboratories worldwide were invited to contribute data to the certification of trace metals in NASS-7 and CASS-6. Various analytical methods were employed by participants including column separation, co-precipitation, and simple dilution coupled to ICPMS detection or flow injection analysis coupled to chemiluminescence detection, with use of double isotope dilution calibration, matrix matching external calibration, and standard addition calibration. Results presented in this study show that majority of laboratories have demonstrated their measurement capabilities for the accurate determination of trace metals in seawater. As a result of this comparison, certified/reference values and associated uncertainties were assigned for 14 elements in seawater CRMs NASS-7 and CASS-6, suitable for the validation of methods used for seawater analysis.

Direct Speciation Analysis of Arsenic in Whole Blood and Blood Plasma at Low Exposure Levels by Hydride Generation-Cryotrapping-Inductively Coupled Plasma Mass Spectrometry.

A method for analysis of toxicologically important arsenic species in blood plasma and whole blood by selective hydride generation with cryotrapping (HG-CT) coupled either to atomic absorption spectrometry (AAS) with a quartz multiatomizer or to inductively coupled plasma mass spectrometry (ICPMS) has been validated. Sample preparation, which involved only 5 times dilution with addition of Triton X-100, Antifoam B, and l-cysteine, suppressed excessive foaming in a hydride generator. Calibration slopes for whole blood and blood plasma spiked with arsenate, monomethylarsonate, and dimethylarsinate at 0.25-1 µg L-1 As and 0.025-0.1 µg L-1 As for AAS and ICPMS detection, respectively, did not differ from slopes in aqueous solutions. HG-CT-AAS was used to analyze samples with elevated levels of arsenic species-blood plasma from patients treated with arsenic trioxide for acute promyelocytic leukemia and whole blood from mice fed an arsenic-containing diet. A good agreement between results of the direct analysis and analysis after mild digestion in phosphoric acid proved the good efficiency of the direct HG-CT procedure for the arsenic species in these types of biological samples. In the next step, plasma and whole blood from healthy donors that were spiked with the plasma from leukemia patients at levels of 0.15-0.4 µg L-1 As were analyzed by direct HG-CT-ICPMS. Good recoveries for all species even at these low levels (88-104%) were obtained. Limits of detection in blood and plasma were 0.014 µg L-1 for inorganic arsenic and below 0.002 µg L-1 As for methylated arsenic species. Thus, the ultrasensitive direct HG-CT-ICPMS method is uniquely suited for analyses of blood plasma and whole blood from individuals at low exposure levels.

Speciation analysis of arsenic by selective hydride generation-cryotrapping-atomic fluorescence spectrometry with flame-in-gas-shield atomizer: achieving extremely low detection limits with inexpensive instrumentation.

This work describes the method of a selective hydride generation-cryotrapping (HG-CT) coupled to an extremely sensitive but simple in-house assembled and designed atomic fluorescence spectrometry (AFS) instrument for determination of toxicologically important As species. Here, an advanced flame-in-gas-shield atomizer (FIGS) was interfaced to HG-CT and its performance was compared to a standard miniature diffusion flame (MDF) atomizer. A significant improvement both in sensitivity and baseline noise was found that was reflected in improved (4 times) limits of detection (LODs). The yielded LODs with the FIGS atomizer were 0.44, 0.74, 0.15, 0.17 and 0.67 ng L(-1) for arsenite, total inorganic, mono-, dimethylated As and trimethylarsine oxide, respectively. Moreover, the sensitivities with FIGS and MDF were equal for all As species, allowing for the possibility of single species standardization with arsenate standard for accurate quantification of all other As species. The accuracy of HG-CT-AFS with FIGS was verified by speciation analysis in two samples of bottled drinking water and certified reference materials, NRC CASS-5 (nearshore seawater) and SLRS-5 (river water) that contain traces of methylated As species. As speciation was in agreement with results previously reported and sums of all quantified species corresponded with the certified total As. The feasibility of HG-CT-AFS with FIGS was also demonstrated by the speciation analysis in microsamples of exfoliated bladder epithelial cells isolated from human urine. The results for the sums of trivalent and pentavalent As species corresponded well with the reference results obtained by HG-CT-ICPMS (inductively coupled plasma mass spectrometry).

Quantification of potassium levels in cells treated with Bordetella adenylate cyclase toxin.

The aim of this study was to compare two methods for quantification of changes in intracellular potassium concentration (decrease from ∼140 to ∼20mM) due to the action of a pore-forming toxin, the adenylate cyclase toxin (CyaA) from the pathogenic bacterium Bordetella pertussis. CyaA was incubated with stably transfected K1 Chinese hamster ovary cells expressing the toxin receptor CD11b/CD18 and the decrease in potassium concentration in the cells was followed by inductively coupled plasma mass spectrometry (ICP-MS). It is shown that this method is superior in terms of sensitivity, accuracy, and temporal resolution over the method employing the potassium-binding benzofuran isophthalate-acetoxymethyl ester fluorescent indicator. The ICP-MS procedure was found to be a reliable and straightforward analytical approach enabling kinetic studies of CyaA action at physiologically relevant toxin concentrations (<1000ng/ml) in biological microsamples.

Methylated trivalent arsenicals are potent inhibitors of glucose stimulated insulin secretion by murine pancreatic islets.

Epidemiologic evidence has linked chronic exposure to inorganic arsenic (iAs) with an increased prevalence of diabetes mellitus. Laboratory studies have identified several mechanisms by which iAs can impair glucose homeostasis. We have previously shown that micromolar concentrations of arsenite (iAs(III)) or its methylated trivalent metabolites, methylarsonite (MAs(III)) and dimethylarsinite (DMAs(III)), inhibit the insulin-activated signal transduction pathway, resulting in insulin resistance in adipocytes. Our present study examined effects of the trivalent arsenicals on insulin secretion by intact pancreatic islets isolated from C57BL/6 mice. We found that 48-hour exposures to low subtoxic concentrations of iAs(III), MAs(III) or DMAs(III) inhibited glucose-stimulated insulin secretion (GSIS), but not basal insulin secretion. MAs(III) and DMAs(III) were more potent than iAs(III) as GSIS inhibitors with estimated IC(50)≤0.1 µM. The exposures had little or no effects on insulin content of the islets or on insulin expression, suggesting that trivalent arsenicals interfere with mechanisms regulating packaging of the insulin transport vesicles or with translocation of these vesicles to the plasma membrane. Notably, the inhibition of GSIS by iAs(III), MAs(III) or DMAs(III) could be reversed by a 24-hour incubation of the islets in arsenic-free medium. These results suggest that the insulin producing pancreatic ß-cells are among the targets for iAs exposure and that the inhibition of GSIS by low concentrations of the methylated metabolites of iAs may be the key mechanism of iAs-induced diabetes.

Comparative oxidation state specific analysis of arsenic species by high-performance liquid chromatography-inductively coupled plasma-mass spectrometry and hydride generation-cryotrapping-atomic absorption spectrometry.

The formation of methylarsonous acid (MAsIII) and dimethylarsinous acid (DMAsIII) in the course of inorganic arsenic (iAs) metabolism plays an important role in the adverse effects of chronic exposure to iAs. High-performance liquid chromatography-inductively coupled plasma-mass spectrometry (HPLC-ICP-MS) and hydride generation-cryotrapping-atomic absorption spectrometry (HG-CT-AAS) have been frequently used for the analysis of MAsIII and DMAsIII in biological samples. While HG-CT-AAS has consistently detected MAsIII and DMAsIII, HPLC-ICP-MS analyses have provided inconsistent and contradictory results. This study compares the capacities of both methods to detect and quantify MAsIII and DMAsIII in an in vitro methylation system consisting of recombinant human arsenic (+3 oxidation state) methyltransferase (AS3MT), S-adenosylmethionine as a methyl donor, a non-thiol reductant tris(2-carboxyethyl)phosphine, and arsenite (iAsIII) or MAsIII as substrate. The results show that reversed-phase HPLC-ICP-MS can identify and quantify MAsIII and DMAsIII in aqueous mixtures of biologically relevant arsenical standards. However, HPLC separation of the in vitro methylation mixture resulted in significant losses of MAsIII, and particularly DMAsIII with total arsenic recoveries below 25%. Further analyses showed that MAsIII and DMAsIII bind to AS3MT or interact with other components of the methylation mixture, forming complexes that do not elute from the column. Oxidation of the mixture with H2O2 which converted trivalent arsenicals to their pentavalent analogs prior to HPLC separation increased total arsenic recoveries to ~95%. In contrast, HG-CT-AAS analysis found large quantities of methylated trivalent arsenicals in mixtures incubated with either iAsIII or MAsIII and provided high (>72%) arsenic recoveries. These data suggest that an HPLC-based analysis of biological samples can underestimate MAsIII and DMAsIII concentrations and that controlling for arsenic species recovery is essential to avoid artifacts.

Selective hydride generation- cryotrapping- ICP-MS for arsenic speciation analysis at picogram levels: analysis of river and sea water reference materials and human bladder epithelial cells.

An ultra sensitive method for arsenic (As) speciation analysis based on selective hydride generation (HG) with preconcentration by cryotrapping (CT) and inductively coupled plasma- mass spectrometry (ICP-MS) detection is presented. Determination of valence of the As species is performed by selective HG without prereduction (trivalent species only) or with L-cysteine prereduction (sum of tri- and pentavalent species). Methylated species are resolved on the basis of thermal desorption of formed methyl substituted arsines after collection at -196°C. Limits of detection of 3.4, 0.04, 0.14 and 0.10 pg mL-1 (ppt) were achieved for inorganic As, mono-, di- and trimethylated species, respectively, from a 500 µL sample. Speciation analysis of river water (NRC SLRS-4 and SLRS-5) and sea water (NRC CASS-4, CASS-5 and NASS-5) reference materials certified to contain 0.4 to 1.3 ng mL-1 total As was performed. The concentrations of methylated As species in tens of pg mL-1 range obtained by HG-CT-ICP-MS systems in three laboratories were in excellent agreement and compared well with results of HG-CT-atomic absorption spectrometry and anion exchange liquid chromatography- ICP-MS; sums of detected species agreed well with the certified total As content. HG-CT-ICP-MS method was successfully used for analysis of microsamples of exfoliated bladder epithelial cells isolated from human urine. Here, samples of lysates of 25 to 550 thousand cells contained typically tens pg up to ng of iAs species and from single to hundreds pg of methylated species, well within detection power of the presented method. A significant portion of As in the cells was found in the form of the highly toxic trivalent species.

Arsenic in Lake Geneva (Switzerland, France): long term monitoring, and redox and methylation speciation in an As unpolluted, oligo-mesotrophic lake.

Arsenic speciation was followed monthly along the spring productivity period (January-June 2021) in the Petit Lac (76 m deep) and in April and June 2021 in the Grand Lac (309.7 m deep) of Lake Geneva (Switzerland/France). Lake Geneva is presently an oligo-mesotrophic lake, and As-unpolluted. The water column never becomes anoxic but the oxygen saturation at the bottom of the Grand Lac is now below 30% owing to lack of water column mixing since 2012. Thus, this lake offers excellent conditions to study As behaviour in an unpolluted, oxic freshwater body. The following 'dissolved' As species: iAs(III), iAs(III + V), MA(III), MA(III + V), DMA(III + V), and TMAO were analysed by HG-CT-ICP-MS/MS. Water column measurements were complemented with occasional sampling in the main rivers feeding the lake and in the interstitial waters of a sediment core. The presence of MA(III) and TMAO and the predominance of iAs(V) in lake and river samples has been confirmed as well as the key role of algae in the formation of organic species. While the total 'dissolved' As concentrations showed nearly vertical profiles in the Petit Lac, As concentrations steadily increase at deeper depths in the Grand Lac due to the lack of mixing and build up in bottom waters. The evaluation of 25 years of monthly data of 'dissolved' As concentrations showed no significant temporal trends between 1997 and 2021. The observed seasonal character of the 'dissolved' As along this period coincides with a lack of seasonality in As mass inventories, pointing to a seasonal internal cycling of As species in the water column with exchanges between the 'dissolved' and 'particulate' (i.e., algae) fractions.

Fate of arsenicals in mice carrying the human AS3MT gene exposed to environmentally relevant levels of arsenite in drinking water.

Although mice are widely used to study adverse effects of inorganic arsenic (iAs), higher rates of iAs methylation in mice than in humans may limit their utility as a model organism. A recently created 129S6 mouse strain in which the Borcs7/As3mt locus replaces the human BORCS7/AS3MT locus exhibits a human-like pattern of iAs metabolism. Here, we evaluate dosage dependency of iAs metabolism in humanized (Hs) mice. We determined tissue and urinary concentrations and proportions of iAs, methylarsenic (MAs), and dimethylarsenic (DMAs) in male and female Hs and wild-type (WT) mice that received 25- or 400-ppb iAs in drinking water. At both exposure levels, Hs mice excrete less total arsenic (tAs) in urine and retain more tAs in tissues than WT mice. Tissue tAs levels are higher in Hs females than in Hs males, particularly after exposure to 400-ppb iAs. Tissue and urinary fractions of tAs present as iAs and MAs are significantly greater in Hs mice than in WT mice. Notably, tissue tAs dosimetry in Hs mice resembles human tissue dosimetry predicted by a physiologically based pharmacokinetic model. These data provide additional support for use of Hs mice in laboratory studies examining effects of iAs exposure in target tissues or cells.

Impact of organic matter on As sulfidation in wetlands: An in situ experiment.

Arsenic incorporation into newly formed As sulfides has recently been identified as an important As sequestration pathway in both laboratory experiments and natural As-wetlands. Here, we used an in situ experimental technique with double nylon experimental bags (10-µm mesh) to study the effect of low-cost organic materials (sawdust, wood cubes and hemp shives) on As sulfidation in three naturally As-enriched wetland soils under water-saturated (~1 m depth) and neutral pH conditions. After 15 months of in situ incubation, all of the organic materials and their corresponding inner bags were covered by yellow-black mineral accumulations, dominantly composed of crystalline As4S4 polymorphs (realgar and bonazziite) and reactive Fe(II) sulfides (probably mackinawite); while the major fraction of As (~80%) was sequestered as AsS minerals. The amount of As accumulation in the experimental bags varied significantly (0.03-4.24 g As kg-1) and corresponded with different levels of As (0.23-9.4 mg As L-1) in the groundwater. Our findings suggest an authigenic formation of AsS minerals in strongly reducing conditions of experimental bags by a combination of reduced exchange of solutes through the pores of the bag and comparatively fast microbial production of dissolved sulfide. Arsenic sulfide formation, as an effective treatment mechanism for natural and human-constructed wetlands, appears to be favored for As(III)-rich waters with a low Fe(II)/As(III) molar ratio. These conditions prevent the consumption of dissolved As and sulfide by their preferential incorporation into natural organic matter, and newly-formed Fe(II) sulfides, respectively.

Atomization of As and Se volatile species in a dielectric barrier discharge atomizer after hydride generation: Fate of analyte studied by selected ion flow tube mass spectrometry.

Atomization of hydrides and their methylated analogues in a dielectric barrier discharge (DBD) plasma atomizer was investigated. Selected ion flow tube mass spectrometry (SIFT-MS) was chosen as a detector being capable of selective detection of non-atomized original volatile species allowing thus direct quantification of atomization efficiency. Selenium hydride (SeH2) and three volatile arsenic species, namely arsenic hydride (AsH3), monomethylarsane (CH3AsH2) and dimethylarsane ((CH3)2AsH), were selected as model analytes. The mechanistic study performed contributes to understanding of the atomization processes in atomic absorption spectrometry (AAS). The presented results are compatible with a complete atomization of arsenic hydride as well as its methylated analogues and with atomization efficiency of SeH2 below 80%. Using AsH3 as a model analyte and a combination of AAS and SIFT-MS detectors has revealed that the hydride is not atomized, but decomposed in the DBD atomizer in absence of hydrogen fraction in the carrier gas. Apart from investigation of analyte atomization, the SIFT-MS detector is capable of quantitative determination of water vapor content being either transported to, or produced in the atomizer. This information is crucial especially in the case of the low-power/temperature DBD atomizer since its performance is sensitive to the amount of water vapor introduced into the plasma.

Direct analysis of methylated trivalent arsenicals in mouse liver by hydride generation-cryotrapping-atomic absorption spectrometry.

Growing evidence suggest that the methylated trivalent metabolites of inorganic arsenic (iAs), methylarsonite (MAs(III)) and dimethylarsinite (DMAs(III)), contribute to adverse effects of iAs exposure. However, the lack of suitable methods has hindered the quantitative analysis of MAs(III) and DMAs(III) in complex biological matrices. Here, we show that hydride generation-cryotrapping-atomic absorption spectrometry can quantify both MAs(III) and DMAs(III) in livers of mice exposed to iAs. No sample extraction is required, thus limiting MAs(III) or DMAs(III) oxidation prior to analysis. The limits of detection are below 6 ng As/g of tissue, making this method suitable even for studies examining low exposures to iAs.

Speciation of germanium in environmental water reference materials by hydride generation and cryotrapping in combination with ICP-MS/MS.

A method for the speciation analysis of the three main species of germanium in environmental waters, namely inorganic germanium (iGe), monomethyl germanium (MMGe) and dimethyl germanium (DMGe), has been developed. Germanium species were volatilized by hydride generation (HG) prior to their preconcentration/separation in a semi-automated cryogenic trap (cryotrapping, CT) and detection by ICP-MS/MS. A procedure to minimize the iGe blanks from the chemicals and water is reported. One mL of water can be analyzed without any pretreatment. After application of this procedure, and the careful optimization of all experimental variables, limits of detection (LOD) of 0.015, 0.005 and 0.003 ng L-1 have been obtained for iGe, MMGe and DMGe, respectively. Standard addition experiments did not show any significant matrix effect, and, therefore, external calibration was used for sample analysis. In the Tris-HCl + L-Cysteine reaction media, additional experiments did not reveal any significant demethylation of MMGe to iGe in the process of HG-CT, which could affect the accuracy of the analysis in seawater. The method has been applied to the analysis of iGe, MMGe and DMGe in certified reference materials of unspiked natural waters: CASS-4, CASS-5 and CASS-6 (nearshore seawater); NASS-5 and NASS-7 (seawater); SLRS-4, SLRS-5 and SLRS-6 (river water).

A Unique Reverse Adaptation Mechanism Assists Bordetella pertussis in Resistance to Both Scarcity and Toxicity of Manganese.

The ability of bacterial pathogens to acquire essential micronutrients is critical for their survival in the host environment. Manganese plays a complex role in the virulence of a variety of pathogens due to its function as an antioxidant and enzymatic cofactor. Therefore, host cells deprive pathogens of manganese to prevent or attenuate infection. Here, we show that evolution of the human-restricted pathogen Bordetella pertussis has selected for an inhibitory duplication within a manganese exporter of the calcium:cation antiporter superfamily. Intriguingly, upon exposure to toxic levels of manganese, the nonfunctional exporter becomes operative in resister cells due to a unique reverse adaptation mechanism. However, compared with wild-type (wt) cells, the resisters carrying a functional copy of the exporter displayed strongly reduced intracellular levels of manganese and impaired growth under oxidative stress. Apparently, inactivation of the manganese exporter and the resulting accumulation of manganese in the cytosol benefited the pathogen by improving its survival under stress conditions. The inhibitory duplication within the exporter gene is highly conserved among B. pertussis strains, absent from all other Bordetella species and from a vast majority of organisms across all kingdoms of life. Therefore, we conclude that inactivation of the exporter gene represents an exceptional example of a flexible genome decay strategy employed by a human pathogen to adapt to its exclusive host. IMPORTANCE Bordetella pertussis, a respiratory pathogen restricted to humans, continuously adapts its genome to its exclusive host. We show that speciation of this reemerging pathogen was accompanied by loss of function of the manganese exporter. Intriguingly, the functionality of the exporter can be restored in the presence of toxic levels of manganese by a unique genetic modification. However, compared with the wt strain, the strain carrying the functional exporter failed to resist the oxidative stress in vitro. Thus, our data demonstrate that inactivation of the exporter resulting in manganese accumulation assists B. pertussis in adaptation to oxidative stress. We conclude that this sophisticated process of reverse adaptation enables B. pertussis to adjust to rapidly changing environments by facilitating its resistance to both manganese toxicity and manganese scarcity.

Blood Arsenic Levels as a Marker of Breast Cancer Risk among BRCA1 Carriers.

An important group of breast cancers is those associated with inherited susceptibility. In women, several predisposing mutations in genes involved in DNA repair have been discovered. Women with a germline pathogenic variant in BRCA1 have a lifetime cancer risk of 70%. As part of a larger prospective study on heavy metals, our aim was to investigate if blood arsenic levels are associated with breast cancer risk among women with inherited BRCA1 mutations. A total of 1084 participants with pathogenic variants in BRCA1 were enrolled in this study. Subjects were followed from 2011 to 2020 (mean follow-up time: 3.75 years). During that time, 90 cancers were diagnosed, including 67 breast and 10 ovarian cancers. The group was stratified into two categories (lower and higher blood As levels), divided at the median (<0.85 µg/L and ≥0.85 µg/L) As level among all unaffected participants. Cox proportional hazards models were used to model the association between As levels and cancer incidence. A high blood As level (≥0.85 µg/L) was associated with a significantly increased risk of developing breast cancer (HR = 2.05; 95%CI: 1.18-3.56; p = 0.01) and of any cancer (HR = 1.73; 95%CI: 1.09-2.74; p = 0.02). These findings suggest a possible role of environmental arsenic in the development of cancers among women with germline pathogenic variants in BRCA1.

Mercury volatile species generation from HCl and TRIS buffer media: Quantification of generation efficiency and characterization of severe changes in speciation information due to de-alkylation.

Severe changes in speciation information were observed during volatile species generation (VSG) due to de-alkylation of generated Hg species as proven by cryogenic trapping with inductively coupled plasma mass spectrometric detection (CT-ICP-MS). Methyl mercury hydride is de-methylated to Hg0 by 45% and 6%, respectively, in HCl and TRIS buffer media. Ethylmercury hydride is de-ethylated to Hg0 by 71% and 28%, respectively in HCl and TRIS buffer media. Only Hg0 as a volatile product was observed when generating from phenylmercury regardless of the reaction medium employed. These findings limit significantly the application of VSG to Hg speciation analysis, especially the possibility of generation of alkyl-substituted mercury hydrides for cryogenic trapping/separation or gas chromatography. On the contrary, post-column VSG of Hg species prior to spectrometric detection can be employed to enhance sensitivity without any negative effects on accuracy and precision of the results.