Arsenic retention in erythrocytes and excessive erythrophagocytosis is related to low selenium status by impaired redox homeostasis.

Arsenic (As) contamination in drinking water is a global public health problem. Epidemiological studies have shown that selenium (Se) deficiency is associated with an increasing risk of arsenism. However, the association between Se status and As retention in erythrocytes and mechanisms underlying this association have not been fully investigated. In the present study, a total of 165 eligible subjects were recruited and As was found to accumulate in blood mainly by retention in erythrocytes. Retention of As in erythrocytes was negatively correlated with Se status, antioxidant parameters related to Se and As methylation capacity, but positively correlated with the protein-binding capacity of As. Additionally, erythrocytes isolated from subjects with low Se status exhibited cellular damage along with lower protein levels of CD47, which could be aggravated by hydrogen peroxide treatment. Consistent with the human study, the erythrocytes from mice with sub-chronic As exposure exhibited similar cellular damage and shown to be phagocytosed by splenic macrophages, and these effects were mitigated by dietary Se supplementation. Furthermore, hydrogen peroxide treatment induced excessive phagocytosis of erythrocytes with As exposure by splenic macrophages, while co-treating erythrocytes with the reducing agent, N-Acetyl-l-cysteine, mitigated this excessive erythrophagocytosis. Hyperactivation of the NFκB pathway was also detected in splenic macrophages after excessive erythrophagocytosis. In conclusion, this study found that low Se status involving impaired redox homeostasis increased As retention in erythrocytes, which were subsequently phagocytosed by splenic macrophages and led to an increased inflammatory status of splenic macrophages. These findings provide insight into physiological features of arsenism related to Se status and redox homeostasis.

Pulsed Direct Current Electrospray: Enabling Systematic Analysis of Small Volume Sample by Boosting Sample Economy.

We had developed pulsed direct current electrospray ionization mass spectrometry (pulsed-dc-ESI-MS) for systematically profiling and determining components in small volume sample. Pulsed-dc-ESI utilized constant high voltage to induce the generation of single polarity pulsed electrospray remotely. This method had significantly boosted the sample economy, so as to obtain several minutes MS signal duration from merely picoliter volume sample. The elongated MS signal duration enable us to collect abundant MS(2) information on interested components in a small volume sample for systematical analysis. This method had been successfully applied for single cell metabolomics analysis. We had obtained 2-D profile of metabolites (including exact mass and MS(2) data) from single plant and mammalian cell, concerning 1034 components and 656 components for Allium cepa and HeLa cells, respectively. Further identification had found 162 compounds and 28 different modification groups of 141 saccharides in a single Allium cepa cell, indicating pulsed-dc-ESI a powerful tool for small volume sample systematical analysis.

Single cell analysis with probe ESI-mass spectrometry: detection of metabolites at cellular and subcellular levels.

Molecular analysis at cellular and subcellular levels, whether on selected molecules or at the metabolomics scale, is still a challenge now. Here we propose a method based on probe ESI mass spectrometry (PESI-MS) for single cell analysis. Detection of metabolites at cellular and subcellular levels was successfully achieved. In our work, tungsten probes with a tip diameter of about 1 µm were directly inserted into live cells to enrich metabolites. Then the enriched metabolites were directly desorbed/ionized from the tip of the probe for mass spectrometry (MS) detection. The direct desorption/ionization of the enriched metabolites from the tip of the probe greatly improved the sensitivity by a factor of about 30 fold compared to those methods that eluted the enriched analytes into a liquid phase for subsequent MS detection. We applied the PESI-MS to the detection of metabolites in single Allium cepa cells. Different kinds of metabolites, including 6 fructans, 4 lipids, and 8 flavone derivatives in single cells, have been successfully detected. Significant metabolite diversity was observed among different cells types of A. cepa bulb and different subcellular compartments of the same cell. We found that the inner epidermal cells had about 20 fold more fructans than the outer epidermal cells, while the outer epidermal cells had more lipids. We expected that PESI-MS might be a candidate in the future studies of single cell "omics".

Depth profiling of nanometer coatings by low temperature plasma probe combined with inductively coupled plasma mass spectrometry.

The development of material science increasingly calls for rapid characterization methods with low limits of detection and high spatial resolution. Here we report a depth profile analysis method for thin layer coatings by combining low temperature plasma (LTP) probe with inductively coupled plasma mass spectrometry (ICP-MS). The LTP probe with diameter of several tens of micrometers served as a tool for mass removal, which is generated by the discharge in a quartz capillary at ambient condition. The sample material is ablated by the LTP probe and converted into an aerosol, and transported by a carrier gas flow to the ICP-MS, where the decomposed and ionized aerosol particles are analyzed with high sensitivity. Scanning electron microscope (SEM) micrographs reveal that the trace after ablation by the LTP probe is a hole with a diameter less than 10 microm. A lateral resolution of approximately 200 microm has been achieved by analyzing an electron component with interval metal stripes. Depth profiling of a 100 nm single layer sample and a multiple layer sample (100 nm Al/250 nm SiO(2)/100 nm Au/50 nm Cr) on a silicon substrate have been successfully performed at ambient condition. The present method offers unique advantages in terms of high spatial resolution, fast analysis speed and ease of implementation. It might be considered a complementary technique to existing depth profiling methods such as GD-MS/OES, AES, and SIMS. In addition, the simple-to-fabricate LTP probe is easily coupled to various other elemental analysis tools for thin layer or direct solid sample analysis in micro area.

Inorganic arsenic modulates the expression of selenoproteins in mouse embryonic stem cell.

At least 25 selenoproteins in humans and 24 homologues in rodents have been identified. They play important roles in antioxidation, redox regulation and detoxification. The modulation of the expression of selenoproteins by inorganic arsenic (iAs) exposure may highlight the molecular mechanism for the arsenic toxicity. To investigate the effects of iAs exposure on the expression of selenoproteins, we determined how addition of iAs to culture medium affected all known selenoproteins in the mouse embryonic stem (ES) cells. Separated groups of ES cells were treated with arsenite (iAsIII) (0.25-0.5microM), arsenate (iAsV) (1.0-2.0microM) and co-treatment with sodium selenite (SeIV) (0.5microM). The mRNA levels of all selenoproteins were detected by real time quantitative PCR. The up-regulated selenoproteins were confirmed by immunoblotting analysis and enzymatic activity detection. Results showed that CGR8 cells treated with iAsIII (0.25-0.5microM) and iAsV (2.0microM) displayed significant increases of cellular reactive oxygen species (ROS) generation and nuclear accumulation of the transcription factor NF-E2-related factor 2 (Nrf2). Treatments of iAsIII (0.5microM) or iAsV (2.0microM) for 24h caused significant increases in the expression of the antioxidant selenoproteins (Gpx1, Gpx4, and Tr1), whereas led to significant decreases in the mRNA levels of selenoprotein H and some endoplasmic reticulum (ER) located selenoproteins (15-Sep, SelK, SelM, and SelS). Additionally, supplement of SeIV (0.5microM) could restore most of the down-regulated selenoproteins. These results suggested that iAs exposure modulated not only the antioxidant selenoproteins but also the ER stress associated selenoproteins. Further studies are required to clarify whether these modulated selenoproteins genes are targets for selenium supplement in the defense against the toxicity of iAs.

Effects of selenium on the structure and function of recombinant human S-adenosyl-L-methionine dependent arsenic (+3 oxidation state) methyltransferase in E. coli.

The effects of Se(IV) on the structure and function of recombinant human arsenic (+3 oxidation state) methyltransferase (AS3MT) purified from the cytoplasm of Escherichia coli were studied. The coding region of human AS3MT complementary DNA was amplified from total RNA extracted from HepG2 cell by reverse transcription PCR. Soluble and active human AS3MT was expressed in the E. coli with a Trx fusion tag under a lower induction temperature of 25 degrees C. Spectra (UV-vis, circular dichroism, and fluorescence) were first used to probe the interaction of Se(IV) and recombinant human AS3MT and the structure-function relationship of the enzyme. The recombinant human AS3MT had a secondary structure of 29.0% alpha-helix, 23.9% beta-pleated sheet, 17.9% beta-turn, and 29.2% random coil. When Se(IV) was added, the content of the alpha-helix did not change, but that of the beta-pleated sheet increased remarkably in the conformation of recombinant human AS3MT. Se(IV) inhibited the enzymatic methylation of inorganic As(III) in a concentration-dependent manner. The IC(50) value for Se(IV) was 2.38 muM. Double-reciprocal (1/V vs. 1/[inorganic As(III)]) plots showed Se(IV) to be a noncompetitive inhibitor of the methylation of inorganic As(III) by recombinant human AS3MT with a K (i) value of 2.61 muM. We hypothesized that Se(IV) interacts with the sulfhydryl group of cysteine(s) in the structural residues rather than the cysteines of the active site (Cys156 and Cys206). When Se(IV) was combined with cysteine(s) in the structural residues, the conformation of recombinant human AS3MT changed and the enzymatic activity decreased. Considering the quenching of tryptophan fluorescence, Cys72 and/or Cys226 are deduced to be primary targets for Se(IV).