Zijuan Liu

Arsenite transport by mammalian aquaglyceroporins AQP7 and AQP9

Much is known about the transport of arsenite and antimonite into microbes, but the identities of mammalian transport proteins are unknown. The Saccharomyces cerevisiae FPS1 gene encodes a membrane protein homologous to the bacterial aquaglyceroporin GlpF and to mammalian aquaglyceroporins AQP7 and AQP9. Fps1p mediates glycerol uptake and glycerol efflux in response to hypoosmotic shock. Fps1p has been shown to facilitate uptake of the metalloids arsenite and antimonite, and the Escherichia coli homolog, GlpF, facilitates the uptake and sensitivity to metalloid salts. In this study, the ability of mammalian aquaglyceroporins AQP7 and AQP9 to substitute for the yeast Fps1p was examined. The fps1Δ strain of S. cerevisiae exhibits increased tolerance to arsenite and antimonite compared to a wild-type strain. Introduction of a plasmid containing AQP9 reverses the metalloid tolerance of the deletion strain. AQP7 was not expressed in yeast. The fps1Δ cells exhibit reduced transport of 73As(III) or 125Sb(III), but uptake is enhanced by expression of AQP9. Xenopus laevis oocytes microinjected with either AQP7 or AQP9 cRNA exhibited increased transport of 73As(III). These results suggest that AQP9 and AQP7 may be a major routes of arsenite uptake into mammalian cells, an observation potentially of large importance for understanding the action of arsenite as a human toxin and carcinogen, as well as its efficacy as a chemotherapeutic agent for acute promyelocytic leukemia.

Transport pathways for arsenic and selenium : A minireview

Arsenic and selenium are metalloids found in the environment. Arsenic is considered to pose the most significant potential threat to human health based on frequency of occurrence, toxicity and human exposure. Selenium, on the other hand, ranks only 147th in toxicity but, in contrast to arsenic, is a required micronutrient. Whether a toxin or micronutrient, their metabolism requires that cells to accumulate these metalloids. In this review we discuss the membrane proteins that transport arsenic and selenium into cells, from bacteria to humans, as well as some of the efflux proteins involved in detoxification.

Methylarsonous acid transport by aquaglyceroporins

Many mammals methylate trivalent inorganic arsenic in liver to species that are released into the bloodstream and excreted in urine and feces. This study addresses how methylated arsenicals pass through cell membranes. We have previously shown that aquaglyceroporin channels, including Escherichia coli GlpF, Saccharomyces cerevisiae Fps1p, AQP7, and AQP9 from rat and human, conduct trivalent inorganic arsenic [As(III)] as arsenic trioxide, the protonated form of arsenite. One of the initial products of As(III) methylation is methylarsonous acid [MAs(III)], which is considerably more toxic than inorganic As(III). In this study, we investigated the ability of GlpF, Fps1p, and AQP9 to facilitate movement of MAs(III) and found that rat aquaglyceroporin conducted MAs(III) at a higher rate than the yeast homologue. In addition, rat AQP9 facilitates MAs(III) at a higher rate than As(III). These results demonstrate that aquaglyceroporins differ both in selectivity for and in transport rates of trivalent arsenicals. In this study, the requirement of AQP9 residues Phe-64 and Arg-219 for MAs(III) movement was examined. A hydrophobic residue at position 64 is not required for MAs(III) transport, whereas an arginine at residue 219 may be required. This is similar to that found for As(III), suggesting that As(III) and MAs(III) use the same translocation pathway in AQP9. Identification of MAs(III) as an AQP9 substrate is an important step in understanding physiologic responses to arsenic in mammals, including humans.

Arsenic transport by zebrafish aquaglyceroporins

BackgroundArsenic is one of the most ubiquitous toxins and endangers the health of tens of millions of humans worldwide. It is a mainly a water-borne contaminant. Inorganic trivalent arsenic (AsIII) is one of the major species that exists environmentally. The transport of AsIII has been studied in microbes, plants and mammals. Members of the aquaglyceroporin family have been shown to actively conduct AsIII and its organic metabolite, monomethylarsenite (MAsIII). However, the transport of AsIII and MAsIII in in any fish species has not been characterized.ResultsIn this study, five members of the aquaglyceroporin family from zebrafish (Danio rerio) were cloned, and their ability to transport water, glycerol, and trivalent arsenicals (AsIII and MAsIII) and antimonite (SbIII) was investigated. Genes for at least seven aquaglyceroporins have been annotated in the zebrafish genome project. Here, five genes which are close homologues to human AQP3, AQP9 and AQP10 were cloned from a zebrafish cDNA preparation. These genes were named aqp3, aqp3l, aqp9a, aqp9b and aqp10 according to their similarities to the corresponding human AQPs. Expression of aqp9a, aqp9b, aqp3, aqp3l and aqp10 in multiple zebrafish organs were examined by RT-PCR. Our results demonstrated that these aquaglyceroporins exhibited different tissue expression. They are all detected in more than one tissue. The ability of these five aquaglyceroporins to transport water, glycerol and the metalloids arsenic and antimony was examined following expression in oocytes from Xenopus leavis. Each of these channels showed substantial glycerol transport at equivalent rates. These aquaglyceroporins also facilitate uptake of inorganic AsIII, MAsIII and SbIII. Arsenic accumulation in fish larvae and in different tissues from adult zebrafish was studied following short-term arsenic exposure. The results showed that liver is the major organ of arsenic accumulation; other tissues such as gill, eye, heart, intestine muscle and skin also exhibited significant ability to accumulate arsenic. The zebrafish larvae also accumulate considerable amounts of arsenic.ConclusionThis is the first molecular identification of fish arsenite transport systems and we propose that the extensive expression of the fish aquaglyceroporins and their ability to transport metalloids suggests that aquaglyceroporins are the major pathways for arsenic accumulation in a variety of zebrafish tissues. Uptake is one important step of arsenic metabolism. Our results will contribute to a new understanding of aquatic arsenic metabolism and will support the use of zebrafish as a new model system to study arsenic associated human diseases.

Jen1p: a high affinity selenite transporter in yeast.

This report demonstrated selenite is transported through a monocarboxylate transporter Jen1p in Saccharomyces cerevisiae. Jen1p determined selenite sensitivity and uptake. Selenite had a similar affinity for Jen1p and a similar transport mechanism to the monocarboxylate lactate, which are both proton driven and exhibit reciprocal inhibition.

Pentavalent methylated arsenicals are substrates of human AQP9

Liver aquaglyceroporin AQP9 facilitates movement of trivalent inorganic arsenite (AsIII) and organic monomethylarsonous acid (MAsIII). However, the transport pathway for the two major pentavalent arsenic cellular metabolites, MAsV and DMAsV, remains unknown in mammals. These products of arsenic metabolism, in particular DMAsV, 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 MAsV and DMAsV 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 AsIII, MAsIII, MAsV and DMAsV via AQP9. However, phloretin, which inhibits water and glycerol permeation via AQP9, can only inhibit transport of pentavalent MAsV and DMAsV but not trivalent AsIII and MAsIII, 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 AsIII inside cells, AQP9 plays a dual role in the detoxification of arsenic metabolites by facilitating efflux from cells.

Identification of an S-adenosylmethionine (SAM) dependent arsenic methyltransferase in Danio rerio

Arsenic methylation is an important cellular metabolic process that modulates arsenic toxicity and carcinogenicity. Biomethylation of arsenic produces a series of mono-, di- and tri-methylated arsenic metabolites that can be detected in tissues and excretions. Here we report that zebrafish exposed to arsenite (As(III)) produces organic arsenicals, including MMA(III), MMA(V) and DMA(V) with characteristic tissue ratios, demonstrating that an arsenic methylation pathway exists in zebrafish. In mammals, cellular inorganic arsenic is methylated by a SAM-dependent arsenic methyltransferase, AS3MT. A zebrafish arsenic methyltransferase homolog, As3mt, was identified by sequence alignment. Western blotting analysis showed that As3mt was universally expressed in zebrafish tissues. Prominent expression in liver and intestine correlated with methylated arsenic metabolites detected in those tissues. As3mt was expressed in and purified from Escherichia coli for in vitro functional studies. Our results demonstrated that As3mt methylated As(III) to DMA(V) as an end product and produced MMA(III) and MMA(V) as intermediates. The activity of As3mt was inhibited by elevated concentrations of the substrate As(III) as well as the metalloid selenite, which is a well-known antagonistic micronutrient of arsenic toxicity. The activity As3mt was abolished by substitution of either Cys160 or Cys210, which corresponds to conserved cysteine residues in AS3MT homologs, suggesting that they are involved in catalysis. Expression in zebrafish of an enzyme that has a similar function to human and rodent orthologs in catalyzing intracellular arsenic biomethylation validates the applicability of zebrafish as a valuable vertebrate model for understanding arsenic-associated diseases in humans.

Inositol transporters AtINT2 and AtINT4 regulate arsenic accumulation in Arabidopsis seeds

Arsenic contamination of groundwater and soils threatens the health of tens of millions of people worldwide. Understanding the way in which arsenic is taken up by crops such as rice, which serve as a significant source of arsenic in the human diet, is therefore important. Membrane transport proteins that catalyse arsenic uptake by roots, and translocation through the xylem to shoots, have been characterized in a number of plants, including rice. The transporters responsible for loading arsenic from the xylem into the phloem and on into the seeds, however, are yet to be identified. Here, we show that transporters responsible for inositol uptake in the phloem in Arabidopsis also transport arsenic. Transformation of Saccharomyces cerevisiae with AtINT2 or AtINT4 led to increased arsenic accumulation and increased sensitivity to arsenite. Expression of AtINT2 in Xenopus laevis oocytes also induced arsenite import. Disruption of AtINT2 or AtINT4 in Arabidopsis thaliana led to a reduction in phloem, silique and seed arsenic concentrations in plants fed with arsenite through the roots, relative to wild-type plants. These plants also exhibited a large drop in silique and seed arsenic concentrations when fed with arsenite through the leaves. We conclude that in Arabidopsis, inositol transporters are responsible for arsenite loading into the phloem, the key source of arsenic in seeds.

Pentavalent arsenate transport by zebrafish phosphate transporter NaPi-IIb1.

Arsenate is a pentavalent form of arsenic that shares similar chemical properties to phosphate. It has been shown to be taken up by phosphate transporters in both eukaryotic and prokaryotic microbes such as yeast and Escherichia coli. Recently, the arsenate uptake in vertebrate cells was reported to be facilitated by mammalian type II sodium/phosphate transporter with different affinities. As arsenate is the most common form of arsenic exposure in aquatic system, identifying the uptake pathway of arsenate into aquatic animals is a crucial step in the elucidation of the entire metabolic pathway of arsenic. In this study, the ability of a zebrafish phosphate transporter, NaPi-IIb1 (SLC34a2a), to transport arsenate was examined. Our results demonstrate that a type II phosphate transporter in zebrafish, NaPi-IIb1, can transport arsenate in vitro when expressed in Xenopus laevis oocytes. NaPi-IIb1 mediates a high-affinity arsenate transport, with a K(m) of 0.22 mM. The natural substrate of NaPi-IIb1, dibasic phosphate, inhibits arsenate transport. Arsenate transport via NaPi-IIb1 is coupled with Na(+) and exhibits sigmoidal kinetics with a Hill coefficient of 3.24 ± 0.19. Consistent with these in vitro studies, significant arsenate accumulation is observed in all examined zebrafish tissues where NaPi-IIb1 is expressed, particularly intestine, kidney, and eye, indicating that zebrafish NaPi-IIb1 is likely the transport protein that is responsible for arsenic accumulation in vivo.

Zinc- and bicarbonate-dependent ZIP8 transporter mediates selenite uptake

Selenite (HSeO3−) is a monovalent anion of the essential trace element and micronutrient selenium (Se). In therapeutic concentrations, HSeO3− has been studied for treating certain cancers, serious inflammatory disorders, and septic shock. Little is known, however, about HSeO3− uptake into mammalian cells; until now, no mammalian HSeO3− uptake transporter has been identified. The ubiquitous mammalian ZIP8 divalent cation transporter (encoded by the SLC39A8 gene) is bicarbonate-dependent, moving endogenous substrates (Zn2+, Mn2+, Fe2+ or Co2+) and nonessential metals such as Cd2+ into the cell. Herein we studied HSeO3− uptake in: human and mouse cell cultures, shRNA-knockdown experiments, Xenopus oocytes, wild-type mice and two transgenic mouse lines having genetically altered ZIP8 expression, and mouse erythrocytes ex vivo. In mammalian cell culture, excess Zn2+ levels and/or ZIP8 over-expression can be associated with diminished viability in selenite-treated cells. Intraperitoneal HSeO3− causes the largest ZIP8-dependent increases in intracellular Se content in liver, followed by kidney, heart, lung and spleen. In every model system studied, HSeO3− uptake is tightly associated with ZIP8 protein levels and sufficient Zn2+ and HCO3− concentrations, suggesting that the ZIP8-mediated electroneutral complex transported contains three ions: Zn2+/(HCO3−)(HSeO3−). Transporters having three different ions in their transport complex are not without precedent. Although there might be other HSeO3− influx transporters as yet undiscovered, data herein suggest that mammalian ZIP8 plays a major role in HSeO3− uptake.