Pentavalent methylated arsenicals are substrates of human AQP9

Liver aquaglyceroporin AQP9 facilitates movement of trivalent inorganic arsenite (As^III) and organic monomethylarsonous acid (MAs^III). However, the transport pathway for the two major pentavalent arsenic cellular metabolites, MAs^V and DMAs^V, remains unknown in mammals. These products of arsenic metabolism, in particular DMAs^V, are the major arsenicals excreted in the urine of mammals. In this study, we examined the uptake of the two pentavalent organic arsenicals by human AQP9 in Xenopus laevis oocytes. Xenopus laevis oocytes microinjected with AQP9 cRNA exhibited uptake of both MAs^V and DMAs^V in a pH-dependent manner. The rate of transport was much higher at acidic pH (pH5.5) than at neutral pH. Hg(II), an aquaporin inhibitor, inhibited transport of As^III, MAs^III, MAs^V and DMAs^V via AQP9. However, phloretin, which inhibits water and glycerol permeation via AQP9, can only inhibit transport of pentavalent MAs^V and DMAs^V but not trivalent As^III and MAs^III, indicating the translocation mechanisms of these arsenic species are not exactly the same. Reagents such as FCCP, valinomycin and nigericin that dissipate transmembrane proton potential or change the transmemebrane pH gradient did not significantly inhibit all arsenic transport via AQP9, suggesting the transport of pentavalent arsenic is not proton coupled. The results suggest that in addition to the initial uptake of trivalent inorganic As^III inside cells, AQP9 plays a dual role in the detoxification of arsenic metabolites by facilitating efflux from cells.     

Role of AQP9 in transport of monomethyselenic acid and selenite

AQP9 is an aquaglyceroporin with a very broad substrate spectrum. In addition to its orthodox nutrient substrates, AQP9 also transports multiple neutral and ionic arsenic species including arsenic trioxide, monomethylarsenous acid (MAs^III) and dimethylarsenic acid (DMA^V). Here we discovered a new group of AQP9 substrates which includes two clinical relevant selenium species. We showed that AQP9 efficiently transports monomethylselenic acid (MSeA) with a preference for acidic pH, which has been demonstrated in Xenopus laevis oocyte following the overexpression of human AQP9. Specific inhibitors that dissipate transmembrane proton potential or change the transmembrane pH gradient, such as FCCP, valinomycin and nigericin did not significantly inhibit MSeA uptake, suggesting MSeA transport is not proton coupled. AQP9 was also found to transport ionic selenite and lactate, with much less efficiency compared with MSeA uptake. Selenite and lactate uptake via AQP9 is pH dependent and inhibited by FCCP and nigericin, but not valinomycin. The selenite and lactate uptake via AQP9 can be inhibited by different lactate analogs, indicating that their translocation share similar mechanisms. AQP9 transport of MSeA, selenite and lactate is all inhibited by a previously identified AQP9 inhibitor, phloretin, and the AQP9 substrate arsenite (As^III). These newly identified AQP9 selenium substrates imply that AQP9 play a significant role in MSeA uptake and possibly selenite uptake involved in cancer therapy under specific microenvironments.     

Flexible bacterial strains that oxidize arsenite in anoxic or aerobic conditions and utilize hydrogen or acetate as alternative electron donors

Arsenic is a carcinogenic compound widely distributed in the groundwater around the world. The fate of arsenic in groundwater depends on the activity of microorganisms either by oxidizing arsenite (As^III), or by reducing arsenate (As^V). Because of the higher toxicity and mobility of As^III compared to As^V, microbial-catalyzed oxidation of As^III to As^V can lower the environmental impact of arsenic. Although aerobic As^III-oxidizing bacteria are well known, anoxic oxidation of As^III with nitrate as electron acceptor has also been shown to occur. In this study, three As^III-oxidizing bacterial strains, Azoarcus sp. strain EC1-pb1, Azoarcus sp. strain EC3-pb1 and Diaphorobacter sp. strain MC-pb1, have been characterized. Each strain was tested for its ability to oxidize As^III with four different electron acceptors, nitrate, nitrite, chlorate and oxygen. Complete As^III oxidation was achieved with both nitrate and oxygen, demonstrating the novel ability of these bacterial strains to oxidize As^III in either anoxic or aerobic conditions. Nitrate was only reduced to nitrite. Different electron donors were used to study their suitability in supporting nitrate reduction. Hydrogen and acetate were readily utilized by all the cultures. The flexibility of these As^III-oxidizing bacteria to use oxygen and nitrate to oxidize As^III as well as organic and inorganic substrates as alternative electron donors explains their presence in non-arsenic-contaminated environments. The findings suggest that at least some As^III-oxidizing bacteria are flexible with respect to electron-acceptors and electron-donors and that they are potentially widespread in low arsenic concentration environments.     

A novel vanadium transporter of the Nramp family expressed at the vacuole of vanadium-accumulating cells of the ascidian Ascidia sydneiensis samea

Background

Vanadium is an essential transition metal in biological systems. Several key proteins related to vanadium accumulation and its physiological function have been isolated, but no vanadium ion transporter has yet been identified.

Methods

We identified and cloned a member of the Nramp/DCT family of membrane metal transporters (AsNramp) from the ascidian Ascidia sydneiensis samea, which can accumulate extremely high levels of vanadium in the vacuoles of a type of blood cell called signet ring cells (also called vanadocytes). We performed immunological and biochemical experiments to examine its expression and transport function.

Results

Western blotting analysis showed that AsNramp was localized at the vacuolar membrane of vanadocytes. Using the Xenopus oocyte expression system, we showed that AsNramp transported VO²⁺ into the oocyte as pH-dependent manner above pH 6, while no significant activity was observed below pH 6. Kinetic parameters (K_m and V_max) of AsNramp-mediated VO²⁺ transport at pH 8.5 were 90 nM and 9.1 pmol/oocyte/h, respectively. A rat homolog, DCT1, did not transport VO²⁺ under the same conditions. Excess Fe²⁺, Cu²⁺, Mn²⁺, or Zn²⁺ inhibited the transport of VO²⁺. AsNramp was revealed to be a novel VO²⁺/H⁺ antiporter, and we propose that AsNramp mediates vanadium accumulation coupled with the electrochemical gradient generated by vacuolar H⁺-ATPase in vanadocytes.

General Significance

This is the first report of identification and functional analysis on a membrane transporter for vanadium ions.     

Induction of DNA damage by free radicals generated either by organic or inorganic arsenic (AsIII, MMAIII, and DMAIII) in cultures of B and T lymphocytes

The aim of this work is based in the premise that inorganic arsenic (As^III) and trivalentmethylated metabolites monomethylarsonous (MMA^III) and dimethylarsinous (DMA^III) participate in DNA damage through the generation of reactive oxygen species (ROS). We have utilized two lymphoblastic lines, Raji (B cells) and Jurkat (T cells), which were treated with the trivalent arsenic species (dose: 0–100 μM) and analyzed by two assays (comet assay and flow cytometry) in the determination of DNA damage and ROS effects in vivo. The results showed that the damage to the DNA and the generation of ROS are different in both cellular lines with respect to the dose of organic arsenic, and the order of damage is MMA^III>DMA^III>As^III. This fact suggests that the DMA^III is not always the more cytotoxic intermediary xenobiotic, as has already been reported in another study.     

Arsenate and arsenite: the toxic effects on photosynthesis and growth of lettuce plants

Arsenate (As^V) and arsenite (As^III) contamination can promote several disturbances in plant metabolism, besides affecting directly human and animal health due to the insertion of this metalloid in the food chain. Therefore, the arsenic (As) uptake and accumulation, the changes in gas exchange and in chlorophyll a fluorescence parameters as well as the chloroplastic pigments content were measured. The As accumulation in leaves and roots increased with the increase of As^V and As^III concentration, except at the highest As^III concentration, probably because of As^III extrusion mechanism. Although the highest As concentration has been found in roots, significant amount was transported to the leaves, especially when plants were exposed to As^III. The As accumulation decreased the relative growth rate (RGR) of leaves and roots. However, at 6.6 μmol L^?1 As^V, an increase in leaves RGR was observed, possibly related to the changes in phosphate (P^V) nutrition caused by As. As^V and As^III interfered negatively in the photosynthetic process, except at 6.6 μmol L^?1 As^V. The observed reduction seemed to be associated to the interference in the photochemical and biochemical steps of photosynthesis; however, chlorophyll a fluorescence results indicate that the photosynthetic apparatus and chloroplastic pigments were not damaged. So, lettuce plants demonstrated to be able to accumulate As and also to protect the photosynthetic apparatus against the harmful effects of this metalloid, probably through the activation of tolerance mechanisms.     

Phosphate starvation response controls genes required to synthesize the phosphate analog arsenate

Environmental arsenic poisoning affects roughly 200 million people worldwide. The toxicity and mobility of arsenic in the environment is significantly influenced by microbial redox reactions, with arsenite (As^III) being more toxic than arsenate (As^V). Microbial oxidation of As^III to As^V is known to be regulated by the AioXSR signal transduction system and viewed to function for detoxification or energy generation. Here, we show that As^III oxidation is ultimately regulated by the phosphate starvation response (PSR), requiring the sensor kinase PhoR for expression of the As^III oxidase structural genes aioBA. The PhoRB and AioSR signal transduction systems are capable of transphosphorylation cross‐talk, closely integrating As^III oxidation with the PSR. Further, under PSR conditions, As^V significantly extends bacterial growth and accumulates in the lipid fraction to the apparent exclusion of phosphorus. This could spare phosphorus for nucleic acid synthesis or triphosphate metabolism wherein unstable arsenic esters are not tolerated, thereby enhancing cell survival potential. We conclude that As^III oxidation is logically part of the bacterial PSR, enabling the synthesis of the phosphate analog As^V to replace phosphorus in specific biomolecules or to synthesize other molecules capable of a similar function, although not for total replacement of cellular phosphate.     

Arsenic, cadmium and neuron specific enolase (ENO2, γ-enolase) expression in breast cancer

Background

Neuron specific enolase (ENO2, γ-enolase) has been used as a biomarker to help identify neuroendocrine differentiation in breast cancer. The goal of the present study was to determine if ENO2 expression in the breast epithelial cell is influenced by the environmental pollutants, arsenite and cadmium. Acute and chronic exposure of MCF-10A cells to As⁺³ and Cd⁺² sufficient to allow colony formation in soft agar, was used to determine if ENO2 expression was altered by these pollutants.

Results

It was shown that both As⁺³ and Cd⁺² exposure caused significant increases in ENO2 expression under conditions of both acute and chronic exposure. In contrast, ENO1, the major glycolytic enolase in non-muscle and neuronal cells, was largely unaffected by exposure to either As⁺³ or Cd⁺². Localization studies showed that ENO2 in the MCF-10A cells transformed by As⁺³ or Cd⁺² had both a cytoplasmic and nuclear localization. In contrast, ENO1 was localized to the cytoplasm. ENO2 localized to the cytoplasm was found to co-localized with ENO1.

Conclusion

The results are the first to show that ENO2 expression in breast epithelial cells is induced by acute and chronic exposure to As⁺³ or Cd⁺². The findings also suggest a possible link between As⁺³ and Cd⁺² exposure and neuroendocrine differentiation in tumors. Overall, the results suggest that ENO2 might be developed as a biomarker indicating acute and/or chronic environmental exposure of the breast epithelial cell to As⁺³ and Cd⁺².     

Identification and biochemical analysis of a homolog of a sulfate transporter from a vanadium-rich ascidian Ascidia sydneiensis samea

Background

Several species of ascidians accumulate extremely high levels of vanadium ions in the vacuoles of their blood cells (vanadocytes). The vacuoles of vanadocytes also contain many protons and sulfate ions. To maintain the concentration of sulfate ions, an active transporter must exist in the blood cells, but no such transporter has been reported in vanadium-accumulating ascidians.

Methods

We determined the concentration of vanadium and sulfate ions in the blood cells (except for the giant cells) of Ascidia sydneiensis samea. We cloned cDNA for an Slc13-type sulfate transporter, AsSUL1, expressed in the vanadocytes of A. sydneiensis samea. The synthetic mRNA of AsSUL1 was introduced into Xenopus oocytes, and its ability to transport sulfate ions was analyzed.

Results

The concentrations of vanadium and sulfate ions in the blood cells (except for the giant cells) were 38 mM and 86 mM, respectively. The concentration of sulfate ions in the blood plasma was 25 mM. The transport activity of AsSUL1 was dependent on sodium ions, and its maximum velocity and apparent affinity were 2500 pmol/oocyte/h and 1.75 mM, respectively.

General significance

This could account for active uptake of sulfate ions from blood plasma where sulfate concentration is 25 mM, as determined in this study.     

Speciation of Arsenic under Dynamic Conditions

In periodically flooded soils, reductive conditions can occur, which favor the dissolution of Fe (hydr)oxides. Fe (hydr)oxides such as goethite are important sorbents for arsenate (As^V), which is the dominant As species in soils under aerobic conditions. Hence, the dissolution of Fe (hydr)oxides under reductive conditions can result in the mobilization and reduction of As^V and, thus, in an increase in the bioavailability of arsenic. The temporal dynamics of these processes and possible re‐sorption or precipitation of arsenite (As^III) formed are poorly understood. Under controlled laboratory conditions, the temporal change in the redox potential and arsenic speciation with time after a simulated flooding event in a quartz‐goethite organic matter substrate, spiked with As^V, was examined. During a period of 6 weeks, substrate solutions were sampled weekly using micro‐suction cups and analyzed for pH, As^III and As^V, Fe, Mn and P concentrations. Redox potentials and matric potentials were determined in situ in the substrate‐bearing cylinders. The redox potential and the ratio between As^III and As^V concentrations remained unchanged during the experiment without organic matter application. With organic matter applied, the redox potential decreased and the As^III concentrations in the substrate solution increased while the total As concentrations in the substrate solution strongly decreased. An addition of goethite (1 g/kg) per se led to a decrease of the total As in the substrate solution (almost 50 %). In respect to the potential As availability for plants, and consequently, the transfer into the food chain, the results are difficult to evaluate. The lower the total As concentrations in the substrate solution, determined with decreasing redox potential, the least plant As uptake will occur. This effect may however be compensated by a shift of the molar P/As^V ratio in the solution in favor of As^V which is expected to increase the As uptake.     

Genomic survey,characterization and expression profile analysis of the peptide transporter family in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Background  

Peptide transporter (PTR) family whose member can transport di-/tripeptides and nitrate is important for plant growth and development. Although the rice (Oryza sativa L.) genome has been sequenced for a few years, a genomic survey, characterization and expression profile analysis of the PTR family in this species has not been reported.     

Fate of arsenate following arsenite oxidation in Agrobacterium tumefaciens GW4

The fate of arsenate (As^V) generated by microbial arsenite (As^III) oxidation is poorly understood. Agrobacterium tumefaciens wild‐type strain (GW4) was studied to determine how the cell copes with As^V generated in batch culture. GW4 grown heterotrophically with mannitol used As^III as a supplemental energy supply as reflected by enhanced growth and increased cellular levels of NADH and ATP. Under low phosphate (Pi) conditions and presence of As^III oxidation, up to ～ 50% of the resulting As^V was taken up and found associated with the periplasm, membrane or cytoplasm fractions of the cells. Arsenic was found associated with proteins and polar lipids, but not in nucleic acids or sugars. Thin‐layer chromatography and gas chromatography–mass spectrometry analysis suggested the presence of arsenolipids in membranes, presumably as part of the bilayer structure of the cell membrane and replacing Pi under Pi‐limiting conditions. The potential role of a Pi‐binding protein (PstS) for As^V uptake was assessed with the His‐tag purified protein. Intrinsic tryptophan fluorescence spectra analysis suggests that PstS can bind As^V, but with lower affinity as compared with Pi. In early stationary phase cells, the As^V : Pi ratio was approximately 4.3 and accompanied by an altered cell ultrastructure.     

SPARC Expression Is Selectively Suppressed in Tumor Initiating Urospheres Isolated from As+3- and Cd+2-Transformed Human Urothelial Cells (UROtsa) Stably Transfected with SPARC

Background

This laboratory previously analyzed the expression of SPARC in the parental UROtsa cells, their arsenite (As⁺³) and cadmium (Cd⁺²)-transformed cell lines, and tumor transplants generated from the transformed cells. It was demonstrated that SPARC expression was down-regulated to background levels in Cd⁺²-and As⁺³-transformed UROtsa cells and tumor transplants compared to parental cells. In the present study, the transformed cell lines were stably transfected with a SPARC expression vector to determine the effect of SPARC expression on the ability of the cells to form tumors in immune-compromised mice.

Methods

Real time PCR, western blotting, immunohistochemistry, and immunofluorescence were used to define the expression of SPARC in the As⁺³-and Cd⁺²-transformed cell lines, and urospheres isolated from these cell lines, following their stable transfection with an expression vector containing the SPARC open reading frame (ORF). Transplantation of the cultured cells into immune-compromised mice by subcutaneous injection was used to assess the effect of SPARC expression on tumors generated from the above cell lines and urospheres.

Results

It was shown that the As⁺³-and Cd⁺²-transformed UROtsa cells could undergo stable transfection with a SPARC expression vector and that the transfected cells expressed both SPARC mRNA and secreted protein. Tumors formed from these SPARC-transfected cells were shown to have no expression of SPARC. Urospheres isolated from cultures of the SPARC-transfected As⁺³-and Cd⁺²-transformed cell lines were shown to have only background expression of SPARC. Urospheres from both the non-transfected and SPARC-transfected cell lines were tumorigenic and thus fit the definition for a population of tumor initiating cells.

Conclusions

Tumor initiating cells isolated from SPARC-transfected As⁺³-and Cd⁺²-transformed cell lines have an inherent mechanism to suppress the expression of SPARC mRNA.     

Remodulation of central carbon metabolic pathway in response to arsenite exposure in Rhodococcus sp. strain NAU‐1

Arsenite‐tolerant bacteria were isolated from an organic farm of Navsari Agricultural University (NAU), Gujarat, India (Latitude: 20°55′39.04″N; Longitude: 72°54′6.34″E). One of the isolates, NAU‐1 (aerobic, Gram‐positive, non‐motile, coccobacilli), was hyper‐tolerant to arsenite (As^III, 23 mM) and arsenate (As^V, 180 mM). 16S rRNA gene of NAU‐1 was 99% similar to the 16S rRNA genes of Rhodococcus (Accession No. HQ659188). Assays confirmed the presence of membrane bound arsenite oxidase and cytoplasmic arsenate reductase in NAU‐1. Genes for arsenite transporters (arsB and ACR3(1)) and arsenite oxidase gene (aoxB) were confirmed by PCR. Arsenite oxidation and arsenite efflux genes help the bacteria to tolerate arsenite. Specific activities of antioxidant enzymes (catalase, ascorbate peroxidase, superoxide dismutase and glutathione S‐transferase) increased in dose‐dependent manner with arsenite, whereas glutathione reductase activity decreased with increase in As^III concentration. Metabolic studies revealed that Rhodococcus NAU‐1 produces excess of gluconic and succinic acids, and also activities of glucose dehydrogenase, phosphoenol pyruvate carboxylase and isocitrate lyase were increased, to cope with the inhibited activities of glucose‐6‐phosphate dehydrogenase, pyruvate dehydrogenase and α‐ketoglutarate dehydrogenase enzymes respectively, in the presence of As^III. Enzyme assays revealed the increase in direct oxidative and glyoxylate pathway in Rhodococcus NAU‐1 in the presence of As^III.     

Topographical analysis of As-induced folding of α-MT1a

Metallothionein binds multiple metals into two clustered domains. While the structure of the fully metalated protein is well known for the Cd- and Zn-containing protein, there is little known about the structures of the metal-free protein (apo-metallothionein) and even less about the partially metalated forms. However, the partially-metalated species are vitally important intermediates in the passage of the protein from translational synthesis to its homeostatic buffer or metal chaperone roles. Because multiple metals bind to metallothioneins, the partially-metalated species span a wide range depending on the metal bound. Up to 3 As³⁺ bind stepwise to the α-domain fragment in a manner that allows measurement of each of the 4 species simultaneously with the number of free cysteines diminishing by 3 for every As³⁺ bound: apo- (11 Cys), As₁- (8 Cys), As₂- (5 Cys) and As₃-α-MT (2 Cys). The cysteine modifier benzoquinone (Bq), was used to determine the relative accessibility of the free cysteines in the α-MT fragment as a function of the number of As³⁺ bound. The effect of each As³⁺ was to induce folding in the protein. The ESI-MS results show that the whole protein folds significantly even when just one of the three As³⁺ has bound. The profile of the Bq reacting with the unbound cysteines shows effects of steric hindrance in slowing down the reaction. By freezing the reaction midway to the endpoint, the mass spectral data show the ‘mid-flight’ concentrations of all the key species, 27 in all. Analysis of this mid-flight reaction profile gives insight into the topology of the partially metalated MT from the differential access to the unbound cysteinyl thiols by the Bq. Significantly, the metal-free, apo-α-MT also adopts a folded structure in the presence of the As³⁺ even though there is no As³⁺ bound. This can only happen if the apo-protein wraps around other metalated proteins in solution via protein–protein interactions.     

Evaluation of amino acid profile in contrasting arsenic accumulating rice genotypes under arsenic stress

Amino acids (AAs) play significant roles in metal binding, antioxidant defense, and signaling in plants during heavy metal stress. In the present study, the essential amino acids (EAAs), non-essential amino acids (NEAAs), as well as the enzymes of proline and cysteine biosynthetic pathways were studied in contrasting arsenic accumulating rice genotypes grown in hydroponic solutions with addition of arsenate (As^V) or arsenite (As^III). Under a mild As stress, the total AAs content significantly increased in both the rice genotypes with a greater increase in a low As accumulating rice genotype (LAARG; IET-19226) than in a high As accumulating rice genotype (HAARG; BRG-12). At the equimolar concentration (10 μM), As^III had a greater effect on EAAs than As^V. Conversely, As^V was more effective in inducing a proline accumulation than As^III. Among NEAAs, As significantly induced the accumulation of histidine, aspartic acid, and serine. In contrast, a higher As concentration (50 μM) reduced the content of most AAs, the effect being more prominent during As^III exposure. The inhibition of glutamate kinase activity was noticed in HAARG, conversely, serine acetyltransferase and cysteine synthase activities were increased which was positively correlated with the cysteine synthesis.     

Bacterial Community Structure and Functional arrA Gene Diversity Associated with Arsenic Reduction and Release in an Industrially Contaminated Soil

This study aimed at evaluating potential arsenic (As) mobility in an industrially contaminated soil (64 mg/kg of As) of the Meuse River basin, and at identifying key bacterial groups that drive soil As dynamics. Both speciation and release of As from this soil was followed under anaerobic conditions using a laboratory batch experiment. In the presence of exogenous carbon sources, As^V initially present in the soil matrix and/or adsorbed on synthetic hydrous ferric oxides were solubilized and mainly reduced to As^III by indigenous soil microflora. After a 1-month incubation period in these biotic conditions, As^III accounted for 80–85% of the total dissolved As and more than 60% of the solid-phase As. Bacterial community structure (i.e., 16S rDNA-based capillary electrophoresis single-strand conformation polymorphism profiles) changed with incubation time and As amendment. The detection of distantly related arsenate respiratory reductase genes (arrA), as functional markers of As^V respirers, indicates that novel dissimilatory As^V-reducing bacteria may be involved in As biotransformation and mobility in anoxic soils. Since As and iron were concomitantly released, a crucial role of indirect As-mobilizing bacteria on As behavior was also revealed. Our results show that the majority of As within the soil matrix was bioavailable and bioaccessible for heterotrophic As^V reduction to As^III, which may increase As toxicity and mobility in the contaminated soils.