[Show abstract][Hide abstract] ABSTRACT: Speciation analysis is essential when evaluating risks from arsenic (As) exposure. In an oral exposure scenario, the importance of presystemic metabolism by gut microorganisms has been evidenced with in vivo animal models and in vitro experiments with animal microbiota. However, it is unclear whether human microbiota display similar As metabolism, especially when present in a contaminated matrix.
We evaluated the metabolic potency of in vitro cultured human colon microbiota toward inorganic As (iAs) and As-contaminated soils.
A colon microbial community was cultured in a dynamic model of the human gut. These colon microbiota were incubated with iAs and with As-contaminated urban soils. We determined As speciation analysis using high-performance liquid chromatography coupled with inductively coupled plasma mass spectrometry.
We found a high degree of methylation for colon digests both of iAs (10 microg methylarsenical/g biomass/hr) and of As-contaminated soils (up to 28 microg/g biomass/hr). Besides the formation of monomethylarsonic acid (MMA(V)), we detected the highly toxic monomethylarsonous acid (MMA(III)). Moreover, this is the first description of microbial thiolation leading to monomethylmonothioarsonic acid (MMMTA(V)). MMMTA(V), the toxicokinetic properties of which are not well known, was in many cases a major metabolite.
Presystemic As metabolism is a significant process in the human body. Toxicokinetic studies aiming to completely elucidate the As metabolic pathway would therefore benefit from incorporating the metabolic potency of human gut microbiota. This will result in more accurate risk characterization associated with As exposures.
Environmental Health Perspectives 07/2010; 118(7):1004-9. · 7.26 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Developing a reliable and robust trace level method for organophosphates in the presence of relatively high inorganic phosphate is an area of interest for further method development. Since the advent of collision/reaction cell technology, inductively coupled plasma mass spectrometry (ICPMS) has been used with a variety of sample types for elemental phosphorus detection at m/z = 31. However, with many samples inorganic phosphate may be orders of magnitude higher than organophosphates, presenting a major interference to quantification. Therefore, removal of inorganic phosphate to levels low enough to minimize the interference would prove beneficial for more effective organophosphate analyses. In this study, applicable to most organophosphate containing samples, the illustration is to nerve agent degradation products as they might contaminate food and environmental systems after their initial formation. Calcium chloride was chosen as a coagulant mix and ammonium hydroxide was chosen to adjust the pH in order to remove inorganic phosphate at the sample preparation step. High performance liquid chromatography (HPLC) coupled with ICPMS was utilized for analysis. The results show that inorganic phosphate, at concentrations as high as 10000 µg mL−1, can be sufficiently removed by precipitation with calcium at pH > 9 while the organophosphorus chemical warfare agent degradation products, CWADPs, remain intact. Applications to apple juice and cola drink indicate that this method is suitable for more complex matrices containing relatively high levels of inorganic phosphate.
[Show abstract][Hide abstract] ABSTRACT: Rice generally contains higher levels of arsenic than most terrestrial-based foods. Studies related to dietary intake of arsenic from rice must take into account arsenic speciation due to toxicity differences in arsenic species. In this study, microwave-assisted extraction with trifluoroacetic acid was used to prepare rice samples for arsenic speciation analysis by high-performance liquid chromatography-inductively coupled plasma mass spectrometry. Fifty-three samples collected directly from the fields in four major rice-producing states in 1980 and 1981 were analysed for total and speciated arsenic and the results were compared with each other and with results for several more recently collected samples from local markets. The average content of total arsenic was 210 ± 190 ng As g(-1). This study demonstrates that US rice samples with higher levels of total arsenic have higher levels of dimethylarsinic acid; however, inorganic arsenic levels, regardless of the total arsenic content, rarely exceed 150 ng As g(-1) dry weight. These data are consistent with more recent findings, thus establishing trends that arsenic content in US-grown rice has been relatively constant throughout the last 30 years. To the authors' knowledge, the presented data are unique in that they provide a historical reference point for arsenic distribution in US-produced rice. These data would be invaluable for several applications including long-term arsenic exposure studies, environmental clean-up assessments, and to establish models for future trends in arsenic contribution in total diet studies.
Food Additives and Contaminants: Part B Surveillance 11/2009; 2(2):112-120.
[Show abstract][Hide abstract] ABSTRACT: The arsenic (+3 oxidation state) methyltransferase (As3mt) gene encodes a 43 kDa protein that catalyzes methylation of inorganic arsenic. Altered expression of AS3MT in cultured human cells controls arsenic methylation phenotypes, suggesting a critical role in arsenic metabolism. Because methylated arsenicals mediate some toxic or carcinogenic effects linked to inorganic arsenic exposure, studies of the fate and effects of arsenicals in mice which cannot methylate arsenic could be instructive. This study compared retention and distribution of arsenic in As3mt knockout mice and in wild-type C57BL/6 mice in which expression of the As3mt gene is normal. Male and female mice of either genotype received an oral dose of 0.5 mg of arsenic as arsenate per kg containing [(73)As]-arsenate. Mice were radioassayed for up to 96 h after dosing; tissues were collected at 2 and 24 h after dosing. At 2 and 24 h after dosing, livers of As3mt knockouts contained a greater proportion of inorganic and monomethylated arsenic than did livers of C57BL/6 mice. A similar predominance of inorganic and monomethylated arsenic was found in the urine of As3mt knockouts. At 24 h after dosing, As3mt knockouts retained significantly higher percentages of arsenic dose in liver, kidneys, urinary bladder, lungs, heart, and carcass than did C57BL/6 mice. Whole body clearance of [(73)As] in As3mt knockouts was substantially slower than in C57BL/6 mice. At 24 h after dosing, As3mt knockouts retained about 50% and C57BL/6 mice about 6% of the dose. After 96 h, As3mt knockouts retained about 20% and C57BL/6 mice retained less than 2% of the dose. These data confirm a central role for As3mt in the metabolism of inorganic arsenic and indicate that phenotypes for arsenic retention and distribution are markedly affected by the null genotype for arsenic methylation, indicating a close linkage between the metabolism and retention of arsenicals.
Chemical Research in Toxicology 09/2009; 22(10):1713-20. · 3.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Although metabolism of arsenicals to form methylated oxoarsenical species has been extensively studied, less is known about the formation of thiolated arsenical species that have recently been detected as urinary metabolites. Indeed, their presence suggests that the metabolism of ingested arsenic is more complex than previously thought. Recent reports have shown that thiolated arsenicals can be produced by the anaerobic microflora of the mouse cecum, suggesting that metabolism prior to systemic absorption may be a significant determinant of the pattern and extent of exposure to various arsenic-containing species. Here, we examined the metabolism of 34S labeled dimethylthioarsinic acid (34S-DMTA(V)) by the anaerobic microflora of the mouse cecum using HPLC-ICP-MS and HPLC-ESI-MS/MS to monitor for the presence of various oxo- and thioarsenicals. The use of isotopically enriched 34S-DMTA(V) made it possible to differentiate among potential metabolic pathways for production of the trimethylarsine sulfide (TMAS(V)). Upon in vitro incubation in an assay containing anaerobic microflora of mouse cecum, 34S-DMTA(V) underwent several transformations. Labile 34S was exchanged with more abundant 32S to produce 32S-DMTA(V), a thiol group was added to yield DMDTA(V), and a methyl group was added to yield 34S-TMAS(V). Because incubation of 34S-DMTA(V) resulted in the formation of 34S-TMAS(V), the pathway for its formation must preserve the arsenic-sulfur bond. The alternative metabolic pathway postulated for formation of TMAS(V) from dimethylarsinic acid (DMA(V)) would proceed via a dimethylarsinous acid (DMA(III)) intermediate and would necessitate the loss of 34S label. Structural confirmation of the metabolic product was achieved using HPLC-ESI-MS/MS. The data presented support the direct methylation of DMTA(V) to TMAS(V). Additionally, the detection of isotopically pure 34S-TMAS(V) raises questions about the sulfur exchange properties of TMAS(V) in the cecum material. Therefore, 34S-TMAS(V) was incubated and the exchange was monitored with respect to time. The data suggest that the As-S bond associated with TMAS(V) is less labile than the As-S bond associated with DMTA(V).
Toxicology and Applied Pharmacology 01/2009; 239(2):137-43. · 3.98 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The capacity of the anaerobic microflora from a mouse cecum to metabolize dimethylarsinic acid (DMAV) and trimethylarsine oxide (TMAO) was examined in an in vitro assay system containing cecal contents in modified VPIbuffer. Samples were incubated under anaerobic conditions at 37 °C for up to 24 hours and metabolic products were analyzed by HPLC-ICP-MS and HPLC-ESI-MS/MS. Under these conditions, DMAV was thiolated to dimethylthioarsinic acid (DMTAV) and dimethyldithioarsinic acid (DMDTA). The identities of DMTAV (m/z 154), DMDTA (m/z 170), and trimethylarsine sulfide (TMAS, m/z 152) were confirmed with HPLC-ESI-MS/MS. Three chromatographic separations were utilized to verify the lack of co-elution prior to quantification by ICP-MS. The predominant arsenical in reaction mixtures at 24 hours was DMDTA. The presence of TMAS in reaction mixtures after a six hour incubation implies a metabolism route from DMAV to TMAS possibly via reduction to dimethylarsinous acid, methylation to yield TMAO, and thiolation to TMAS. Addition of TMAO to in vitro assay systems containing cecal contents demonstrated that TMAO was almost quantitatively converted to TMAS within one hour. These combined results indicate that ingested arsenicals can undergo substantial metabolism mediated by the microflora of the gastrointestinal tract. Finally, metabolism of arsenicals occurring before absorption across the gastrointestinal barrier could be a modifier of exposure and dose.
[Show abstract][Hide abstract] ABSTRACT: With an increased awareness and concern for varying toxicities of the different chemical forms of environmental contaminants such as selenium and arsenic, effective methodologies for speciation are paramount. In general, chromatographic methodologies have been developed using a particular detection system and a unique matrix for single element speciation. In this study, a routine method to speciate selenium and arsenic in a variety of "real world" matrices with elemental and molecular mass spectrometric detection has been successfully accomplished. Specifically, four selenium species, selenite, selenate, selenomethionine and selenocystine, and four arsenic species, arsenite, arsenate, monomethlyarsonate and dimethylarsinate, were simultaneously separated using ion-pairing reversed phase chromatography coupled with inductively coupled plasma and electrospray ionization ion trap mass spectrometry. Using tetrabutylammonium hydroxide as the ion-pairing reagent on a C(18) column, the separation and re-equilibration time was attained within 18min. To illustrate the wide range of possible applications, the method was then successfully applied for the detection of selenium and arsenic species found naturally and spiked in river water, plant extract and urine matrices.
Journal of Chromatography A 09/2008; 1208(1-2):156-63. · 4.61 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The following work presents the exploration of three chromatographic separations in combination with inductively coupled plasma mass spectrometry (ICP-MS) for the analysis of chemical warfare agent degradation products (CWADPs). The robust ionization of ICP is virtually matrix independent thus enabling the examination of sample matrices generally considered too complicated for analysis by electrospray ionization (ESI) or atmospheric pressure chemical ionization MS with little to no sample preparation. The analysis was focused on detecting CWADPs in food matrices, as they present possible vehicles for terrorist contamination. Due to the specific detection of (31)P by ICP-MS, resolution of analytes of interest from other P-containing interferences (H(3)PO(4)) was a crucial part of each separation. Up to 10 CWADPs were separated in the presence of H(3)PO(4) with detection limits in the low part per billion levels using the methods described. Additionally, one method was tailored to be compatible with both ICP-MS and ESI-MS making structural verification possible.
Journal of Chromatography A 08/2008; 1202(2):124-31. · 4.61 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The simultaneous detection of arsenic and sulfur in thioarsenicals was achieved using xenon-based collision-cell inductively coupled plasma (ICP) mass spectrometry (MS) in combination with high-performance liquid chromatography. In an attempt to minimize the (16)O(16)O(+) interference at m/z 32, both sample introduction and collision-cell experimental parameters were optimized. Low flow rates (0.25 mL/min) and a high methanol concentration (8%) in the mobile phase produced a fourfold decrease in the m/z 32 background. A plasma sampling depth change from 3 to 7 mm produced a twofold decrease in background at m/z 32, with a corresponding fourfold increase in the signal associated with a high ionization surrogate for sulfur. The quadrupole bias and the octopole bias were used as a kinetic energy discriminator between background and analyte ions, but a variety of tuning conditions produced similar (less than twofold change) detection limits for sulfur ((32)S). A 34-fold improvement in the (32)S detection limit was achieved using xenon instead of helium as a collision gas. The optimized xenon-based collision cell ICP mass spectrometer was then used with electrospray ionization MS to provide elemental and molecular-based information for the analysis of a fortified sample of NIST freeze-dried urine. The 3sigma detection limits, based on peak height for dimethylthioarsinic acid (DMTA) and trimethylarsine sulfide (TMAS), were 15 and 12 ng/g, respectively. Finally, the peak area reproducibilities (percentage relative standard deviation) of a 5-ppm fortified sample of NIST freeze dried urine for DMTA and TMAS were 7.4 and 5.4%, respectively.
Analytical and Bioanalytical Chemistry 05/2008; 390(7):1731-7. · 3.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Determination of organophosphorus fire retardants and plasticizers at trace levels in wastewater is described. In this work, microwave assisted extraction (MAE) and solid-phase microextraction (SPME) are used for sample preparation to extract and preconcentrate the analytes, followed by analysis by gas chromatography coupled to inductively coupled plasma mass spectrometry (GC-ICP-MS) for phosphorus-specific detection. Gas chromatography coupled to time of flight mass spectrometry (GC-TOF-MS) was used to confirm the organphosphorus fire retardants in wastewater. The detection limits of organophosphorus fire retardants (OPFRs) were 29 ng L(-1) for tri-n-butyl phosphate (TnBP), 45 ng for L(-1) for tris(2-butoxyethyl)phosphate (TBEP), and 50 ng L(-1) for tris(2-ethylhexyl)phosphate (TEHP). Optimized extraction conditions were performed at 65 degrees C for 30 min and with 10% NaCl. Application of MAE during the sample preparation prior to the SPME allowed the detection of tris(2-ethylhexyl) phosphate, which has been difficult to determine in previous work. Application of the method to wastewater samples resulted in detecting 3.1 microg L(-1) P from TnBP, 5.0 microg L(-1) P from TBEP, and 4.0 microg L(-1) P from TEHP. The presence of these compounds were also confirmed by SPME-GC-TOF-MS.
Journal of Environmental Monitoring 01/2008; 9(12):1329-36. · 2.09 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In this study we demonstrate that the headspace selenium volatiles could be used as proxy to the metabolic pathways in the Se-accumulator plant Brassica juncea. The selenium metabolic pathways in wild type plants are compared to those of several genetically modified cultures. Complementary use of atomic and molecular mass spectrometric techniques also allowed for identification of yet unreported minor headspace Se-containing volatiles such as CH3SeSeSeCH3, CH3SeSSeCH3, and CH3SeCH2CH3. By combining the information resulting from this research with the previously known information about selenium metabolism in B. juncea, it is possible that a more efficacious phytoremediation tool can be constructed.
Environmental Science and Technology 04/2007; 41(6):1863-9. · 5.26 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The incorporation of Se to fungi has been studied, focusing on element distribution among different cellular compartments and, in particular, polysaccharide structures contained in cell walls. Se-enriched mycelia of Pleurotus ostreatus were obtained in submerged cultures. The incorporation of selenium from the growth medium to mycelia was observed with the relative distribution between cytosol plus cell membranes fraction (CCM) and cell walls fraction (CW) of about 44 and 56%, respectively. CCM fractions were analyzed by size exclusion chromatography with on-line UV (280 nm) and ICP-MS detection (80Se). The results obtained showed selenium binding to components of different molecular masses (about 24% of total selenium coeluted with the compounds of molecular mass > 10 kDa). A polysaccharide-containing fraction of mycelia was treated alternatively with Tris-HCl at pH 7.5 or with chitinase. Better solubility and increased contribution of low molecular mass compounds were observed in chitinase extracts (UV detection), confirming the degradation of polysacharides by the enzyme. The total area under the ICP-MS chromatogram of chitinase extract was 2 times higher with respect to the area for Tris-HCl extract. Furthermore, the relative contribution of selenium in the low molecular mass fraction (molecular mass < 1 kDa) in chitinase extract was 72% as compared to 45% in Tris-HCl extract (based on peak area measurements with respect to total area under the chromatogram). The results obtained suggest selenium binding to chitin-containing polysaccharide structures in fungi cell walls.
Journal of Agricultural and Food Chemistry 05/2006; 54(9):3440-4. · 2.91 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In this study, selenium species from Se containing proteins in mushrooms (Agaricus bisporus and Lentinula edodes) were investigated with size-exclusion liquid chromatography coupled to UV and inductively coupled plasma mass spectrometry (ICP-MS). Different protein extraction protocols were investigated. Variability of the fractionation patterns with three extraction media (0.1M NaOH, 30 mM Tris-HCl, and enzymatic digestions) was evaluated for both mushroom types. A 24 h Tris-HCl extraction followed by acetone addition was found to be optimal for protein precipitation. Presumably protein bound selenoamino acids were released using enzymes (proteinase K, protease XIV and trypsin). The selenium speciation of the proteolytic extract of the water soluble proteins fraction was carried out by using reversed-phase ion-pairing high performance liquid chromatography (RP-HPIPC) coupled on-line to ICP-MS for selenium specific detection. Selenocystine, selenomethionine, methylselenocysteine and inorganic selenium were established in both samples utilizing retention time standards and standard additions to the sample.
Journal of Chromatography A 02/2006; 1101(1-2):94-102. · 4.61 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Huitlacoche is the ethnic name of the young fruiting bodies of Ustilago maydis, a common parasite of maize. In Mexico and other Latin American countries, this fungus has been traditionally appreciated as a local delicacy. In this work a metallomics approach was used with the determination of eight elements in huitlacoche by electrothermal atomic absorption spectrometry as one facet of this approach. The results obtained indicated relatively lower concentrations of commonly analyzed metals, as referred to the data reported for other mushroom types. This effect was ascribed to different accessibilities of elements, depending on fungus substrate (lower from plant than from soil). Subcellular fractionation was accomplished by centrifugation of cell homogenates suspended in Tris-HCl buffer. Recoveries of the fractionation procedure were in the range of 71-103%. For six elements (Cr, Cu, Fe, Mn, Ni, and Pb), the mean relative contributions in cytosol, cell walls, and mixed membrane fraction were 50.7, 48.2, and 1.1% respectively. To attain the molecular weight distribution of compounds containing target elements as an additional aspect of the metallomics approach, the fungus extract (1% sodium dodecyl sulfate in Tris-HCl, 30 mmol L(-)(1), pH 7.0) was analyzed by size exclusion chromatography with UV and ICP-MS detection. With spectrophotometric detection (280 nm), the elution of high molecular weight compounds was observed in the form of one peak (MW > 10 kDa), and several lower peaks appeared at higher retention times (MW < 10 kDa). On ICP-MS chromatograms, a coelution of (59)Co, (63)Cu, (57)Fe, (202)Hg, (60)Ni, and (80)Se with the first peak on the UV chromatogram was clearly observed, indicating that a fraction of each element incorporated with high molecular weight compounds (12.7, 19.8, 33.7, 100, 19.4, and 45.8%, respectively, based on the peak area measurements). From a comparison of (80)Se and (33)S chromatograms (for sulfur analysis, the extract was obtained in the absence of SDS), both elements coeluted with the first UV peak, but their lower molecular weight compounds were apparently different. These findings may contribute to a better understanding of the accumulation of elements in mushrooms.
Journal of Agricultural and Food Chemistry 07/2005; 53(13):5138-43. · 2.91 Impact Factor