Intracellular, time-resolved speciation and quantification of arsenic compounds in human urothelial and hepatoma cells

Hippler, Jörg; Zdrenka, Ricarda; Reichel, Robin; Weber, Daniel G.; Rozynek, P.; Johnen, Georg; Dopp, Elke; Hirner, Alfred V.

doi:10.1039/c1ja10150a

Cited by 13 publications

(18 citation statements)

References 37 publications

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“…Consistent with some of the previously published data, 37, 40 slopes of the calibration curves for individual As standards varied (Table S1, Supplementary Data). Specifically, the calibration slopes for MAs III and DMAs III in either solvent were lower than those for the other As standards, suggesting that these methylated trivalent arsenicals are partially retained on the column.…”

Section: Resultssupporting

confidence: 89%

“…38–40, 59 This method was tested in the present study using a 150 × 4.6 mm C 18 column heated to 30°C. A nearly base-line separation of six oxoarsenicals (iAs III , iAs V , MAs III , MAs V , DMAs III , and DMAs V ) and DMMTA was achieved with the mobile phase consisting of 4.7 mM TBAH, 2 mM malonic acid, and 4% methanol at pH 5.85 and with a flow rate of 1.5 mL/min (Figure 1B).…”

Section: Resultsmentioning

confidence: 99%

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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

Currier

Saunders

Lei

et al. 2013

J. Anal. At. Spectrom.

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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.

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Section: Resultssupporting

confidence: 89%

Section: Resultsmentioning

confidence: 99%

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

Currier

Saunders

Lei

et al. 2013

J. Anal. At. Spectrom.

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“…17 The difference in trivalent species portion of iAs and MAs compared to DMAs in BEC’s can be partially explained by low stability of the DMAs III documented in water and urine 30 . However, the MAs III a DMAs III species in cells are expected to be bound to cellular structures and proteins 31 and much more resistant to oxidation. 23 …”

Section: Resultsmentioning

confidence: 99%

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

Matoušek

Currier

Trojánková

et al. 2013

J. Anal. At. Spectrom.

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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.

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“…Despite the highly reducing environment of the cell, DMA V is detected in murine liver homogenates and human cell lines analyzed with oxidation state-specific hydride generationcryotrapping-atomic absorption spectroscopy (Currier et al, 2011). Furthermore, the highly reactive nature of DMA III combined with the lack of evidence for physiologic formation of dimethylarsenic glutathione [DMA(GS)] (Leslie, 2012) mean that DMA III is highly bound to protein and not available for transport (Hippler et al, 2011;Shen et al, 2013). MRP4 transport of DMA V was with high apparent affinity (K 0.5 , 0.22 mM) and would allow for the efficient efflux of DMA V at low cellular concentrations.…”

Section: Discussionmentioning

confidence: 99%

A Novel Pathway for Arsenic Elimination: Human Multidrug Resistance Protein 4 (MRP4/ABCC4) Mediates Cellular Export of Dimethylarsinic Acid (DMA^V) and the Diglutathione Conjugate of Monomethylarsonous Acid (MMA^III)

et al. 2014

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Hundreds of millions of people worldwide are exposed to unacceptable levels of arsenic in drinking water. This is a public health crisis because arsenic is a Group I (proven) human carcinogen. Human cells methylate arsenic to monomethylarsonous acid (MMA . Although the liver is the predominant site for arsenic methylation, elimination occurs mostly in urine. The protein(s) responsible for transport of arsenic from the liver (into blood), ultimately for urinary elimination, are unknown. Human multidrug resistance protein 1 (MRP1/ABCC1) and MRP2 (ABCC2) are established arsenic efflux pumps, but unlike the related MRP4 (ABCC4) are not present at the basolateral membrane of hepatocytes. MRP4 is also found at the apical membrane of renal proximal tubule cells, making it an ideal candidate for urinary arsenic elimination. V were the transported species. MMA(GS) 2 and DMA V transport was osmotically sensitive, allosteric (Hill coefficients of 1.4 6 0.2 and 2.9 6 1.2, respectively), and high affinity (K 0.5 of 0.70 6 0.16 and 0.22 6 0.15 mM, respectively). DMA V transport was pH-dependent, with highest affinity and capacity at pH 5.5. These results suggest that human MRP4 could be a major player in the elimination of arsenic.

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Intracellular, time-resolved speciation and quantification of arsenic compounds in human urothelial and hepatoma cells

Cited by 13 publications

References 37 publications

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

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

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

A Novel Pathway for Arsenic Elimination: Human Multidrug Resistance Protein 4 (MRP4/ABCC4) Mediates Cellular Export of Dimethylarsinic Acid (DMA^V) and the Diglutathione Conjugate of Monomethylarsonous Acid (MMA^III)

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Intracellular, time-resolved speciation and quantification of arsenic compounds in human urothelial and hepatoma cells

Cited by 13 publications

References 37 publications

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

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

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

A Novel Pathway for Arsenic Elimination: Human Multidrug Resistance Protein 4 (MRP4/ABCC4) Mediates Cellular Export of Dimethylarsinic Acid (DMAV) and the Diglutathione Conjugate of Monomethylarsonous Acid (MMAIII)

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A Novel Pathway for Arsenic Elimination: Human Multidrug Resistance Protein 4 (MRP4/ABCC4) Mediates Cellular Export of Dimethylarsinic Acid (DMA^V) and the Diglutathione Conjugate of Monomethylarsonous Acid (MMA^III)