Uptake and toxicity of arsenite and arsenate in cultured brain astrocytes

Inorganic arsenicals are environmental toxins that have been connected with neuropathies and impaired cognitive functions. To investigate whether such substances accumulate in brain astrocytes and affect their viability and glutathione metabolism, we have exposed cultured primary astrocytes to arsenite or arsenate. Both arsenicals compromised the cell viability of astrocytes in a time- and concentration-dependent manner. However, the early onset of cell toxicity in arsenite-treated astrocytes revealed the higher toxic potential of arsenite compared with arsenate. The concentrations of arsenite and arsenate that caused within 24 h half-maximal release of the cytosolic enzyme lactate dehydrogenase were around 0.3 mM and 10 mM, respectively. The cellular arsenic contents of astrocytes increased rapidly upon exposure to arsenite or arsenate and reached after 4 h of incubation almost constant steady state levels. These levels were about 3-times higher in astrocytes that had been exposed to a given concentration of arsenite compared with the respective arsenate condition. Analysis of the intracellular arsenic species revealed that almost exclusively arsenite was present in viable astrocytes that had been exposed to either arsenate or arsenite. The emerging toxicity of arsenite 4 h after exposure was accompanied by a loss in cellular total glutathione and by an increase in the cellular glutathione disulfide content. These data suggest that the high arsenite content of astrocytes that had been exposed to inorganic arsenicals causes an increase in the ratio of glutathione disulfide to glutathione which contributes to the toxic potential of these substances.     

Uptake,Metabolic Effects and Toxicity of Arsenate and Arsenite in Astrocytes

The inorganic arsenic species arsenate and arsenite are common environmental toxins which contaminate the drinking water in many countries. Chronic intoxication with arsenicals has been connected with various diseases, but causes also neurological complications and impairs cognitive development, learning and memory. In brain, astrocytes have a pivotal role as partners of neurons in homeostatic and metabolic processes. In addition, astrocytes are the first parenchymal brain cell type which encounters substances which cross the blood–brain barrier and are considered as first line of defence against the toxic potential of xenobiotics. Therefore, astrocytes are likely to play a prominent role in the metabolism and potential detoxification of arsenicals in brain. This article summarizes the current knowledge on the uptake and toxicity of arsenate and arsenite in astrocytes and discusses the modulation of the astrocytic glucose and glutathione metabolism by arsenicals.     

Organic anion transporting polypeptide-C mediates arsenic uptake in HEK-293 cells

Summary Arsenic is an established human carcinogen. The role of aquaglyroporins (AQPs) in arsenic disposition was recently identified. In order to examine whether organic anion transporting polypeptide-C (OATP-C) also plays a role in arsenic transport, OATP-C cDNA was transfected into cells of a human embryonic kidney cell line (HEK-293). Transfection increased uptake of the model OATP-C substrate, estradiol-17β-D-glucuronide, by 10-fold. In addition, we measured uptake and cytotoxicity of arsenate, arsenite, monomethylarsonate(MMA^V), and dimethylarsinate (DMA^V). Transfection of OATP-C increased uptake and cytotoxicity of arsenate and arsenite, but not of MMA^V or DMA^V. Rifampin and taurocholic acid (a substrate of OATP-C) reversed the increased toxicity of arsenate and arsenite seen in OATP-C-transfected cells. The increase in uptake of inorganic arsenic was not as great as that of estradiol-17β-D-glucuronide. Our results suggest that OATP-C can transport inorganic arsenic in a (GSH)-dependent manner. However, this may not be the major pathway for arsenic transport.     

Mechanisms of arsenic-induced cell transformation

Arsenic is a well-established carcinogen in humans, but there is little evidence for its carcinogenicity in animals and it is inactive as an initiator or tumor promoter in two-stage models of carcinogenicity in mice. Studies with cells in culture have provided some possible mechanisms by which arsenic and arsenical compounds may exert a carcinogenic activity. Sodium arsenite and sodium arsenate were observed to induce morphological transformation of Syrian hamster embryo cells in a dose-dependent manner. The trivalent sodium arsenite was greater than tenfold more potent than the pentavalent sodium arsenate. The compounds also exhibited toxicity; however, transformation was observed at nontoxic as well as toxic doses. At low doses, enhanced colony forming efficiency of the cells was observed. To understand the mechanism of arsenic-induced transformation, the genetic effects of the two arsenicals were examined over the same doses that induced transformation. No arsenic-induced gene mutations were detected at two genetic loci. However, cell transformation and cytogenetic effects, including endoreduplication, chromosome aberrations, and sister chromatid exchanges, were induced by the arsenicals with similar dose responses. These results support a possible role for chromosomal changes in arsenic-induced transformation. The two arsenic salts also induced another form of mutation-gene amplification. Both sodium arsenite and sodium arsenate induced a high frequency of methotrexate-resistant 3T6 cells, which were shown to have amplified copies of the dihydrofolate reductase gene. The ability of arsenic to induce gene amplification may relate to its carcinogenic effects in humans since amplification of oncogenes is observed in many human tumors. Epidemiological studies suggest that arsenic acts late in the carcinogenic process in humans and oncogene amplification correlates with the progression of tumors. These observations lead us to propose the hypothesis that arsenic acts as a tumor progressor, rather than a tumor initiator or tumor promoter. Arsenic-induced chromosome aberrations or gene amplifications may play a role in tumor progression.     

Toxicity, transformation and accumulation of inorganic arsenic species in a microalga Scenedesmus sp. isolated from soil

Arsenic speciation and cycling in the natural environment are highly impacted via biological processes. Since arsenic is ubiquitous in the environment, microorganisms have developed resistance mechanisms and detoxification pathways to overcome the arsenic toxicity. This study has evaluated the toxicity, transformation and accumulation of arsenic in a soil microalga Scenedesmus sp. The alga showed high tolerance to arsenite. The 72-h 50 % growth inhibitory concentrations (IC₅₀ values) of the alga exposed to arsenite and arsenate in low-phosphate growth medium were 196.5 and 20.6 mg? L^?1, respectively. When treated with up to 7.5 mg? L^?1 arsenite, Scenedesmus sp. oxidised all arsenite to arsenate in solution. However, only 50 % of the total arsenic remained in the solution while the rest was accumulated in the cells. Thus, this alga has accumulated arsenic as much as 606 and 761 μg? g^?1 dry weight when exposed to 750 μg? L^?1 arsenite and arsenate, respectively, for 8 days. To our knowledge, this is the first report of biotransformation of arsenic by a soil alga. The ability of this alga to oxidise arsenite and accumulate arsenic could be used in bioremediation of arsenic from contaminated water and soil.     

Monomethylarsonate (MMAv) exerts stronger effects than arsenate on the structure and thermotropic properties of phospholipids bilayers

Methylation of inorganic arsenic has been regarded as a detoxification mechanism because its metabolites monomethylarsonic acid (MMA(v)) and dimethylarsinic acid (DMA(v)) are supposed to be less toxic than inorganic arsenite and arsenate. In recent years, however, this interpretation has been questioned. Additionally, there are insufficient reports concerning the effects of arsenic compounds on cell membrane structure and functions. With the aim to better understand the molecular mechanisms of the interaction of MMA(v) and arsenate with cell membranes, we have utilized molecular models consisting in bilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), representative of phospholipid classes located in the outer and inner monolayers of many cell membranes including that of the human erythrocyte. The capacity of MMA(v) and arsenate to perturb the bilayer structures of DMPC and DMPE was evaluated by X-ray diffraction; the modifications of their thermotropic behavior were followed by differential scanning calorimetry (DSC), while DMPC large unilamellar vesicles (LUV) were studied by fluorescence spectroscopy. It was found that MMA(v) and arsenate did not structurally perturb DMPC bilayers; however, DMPE bilayers did suffer structural perturbations by MMA(v). DSC measurements also revealed that DMPE's thermotropic properties were significantly affected by arsenicals, where MMA(v) was more effective than arsenate, whilst only slight modifications were observed in the case of DMPC-MMA(v) system.     

Diversity of inorganic arsenite biotransformation

Biotransformation of inorganic arsenic in mammals is catalyzed by three serial enzyme activities: arsenate reductase, arsenite methyltransferase, and monomethylarsonate methyltransferase. Our laboratory has purified and characterized these enzymes in order to understand the mechanisms and elucidate the variations of the responses to arsenate /arsenite challenge. Our results indicate a marked deficiency and diversity of these enzyme activities in various animal species.     

Biotransformation and volatilization of arsenic by three photosynthetic cyanobacteria

Arsenic (As) is a pervasive and ubiquitous environmental toxin that has created worldwide human health problems. However, there are few studies about how organisms detoxify As. Cyanobacteria are capable of both photolithotrophic growth in the light and heterotrophic growth in the dark and are ubiquitous in soils, aquatic systems, and wetlands. In this study, we investigated As biotransformation in three cyanobacterial species (Microcystis sp. PCC7806, Nostoc sp. PCC7120, and Synechocystis sp. PCC6803). Each accumulated large amounts of As, up to 0.39 g kg(-1) dry weight, 0.45 g kg(-1) dry weight, and 0.38 g kg(-1) dry weight when treated with 100 μM sodium arsenite for 14 d, respectively. Inorganic arsenate and arsenite were the predominant species, with arsenate making up >80% of total As; methylated arsenicals were detected following exposure to higher As concentrations. When treated with arsenate for 6 weeks, cells of each cyanobacterium produced volatile arsenicals. The genes encoding the As(III) S-adenosylmethionine methyltransferase (ArsM) were cloned from these three cyanobacteria. When expressed in an As-hypersensitive strain of Escherichia coli, each conferred resistance to arsenite. Two of the ArsM homologs (SsArsM from Synechocystis sp. PCC6803 and NsArsM from Nostoc sp. PCC7120) were purified and were shown to methylate arsenite in vitro with trimethylarsine as the end product. Given that ArsM homologs are widespread in cyanobacteria, we propose that they play an important role in As biogeochemistry.     

Relative toxicity of arsenite and arsenate on germination and early seedling growth of rice (Oryza sativa L.)

Elevated soil arsenic levels resulting from long-term use of arsenic contaminated ground for irrigation in Bangladesh may inhibit seed germination and seedling establishment of rice, the country's main food crop. A germination study on rice seeds and a short-term toxicity experiment with different concentrations of arsenite and arsenate on rice seedlings were conducted. Percent germination over control decreased significantly with increasing concentrations of arsenite and arsenate. Arsenite was found to be more toxic than arsenate for rice seed germination. There were varietal differences among the test varieties in response to arsenite and arsenate exposure. The performance of the dry season variety Purbachi was the best among the varieties. Germination of Purbachi was not inhibited at all up to 4 mg l^–1 arsenite and 8 mg l^–1 arsenate treatment. Root tolerance index (RTI) and relative shoot height (RSH) for rice seedlings decreased with increasing concentrations of arsenite and arsenate. Reduction of RTI caused by arsenate was higher than that of arsenite. In general, dry season varieties have more tolerance to arsenite or arsenate than the wet season varieties.     

Reduction kinetics of As (V) to As (III) by a dissimilatory arsenate-reducing bacterium, Bacillus sp. SF-1

This study proposes a kinetic model that accounts for the toxicity of both arsenate and arsenite and characterizes the arsenate reduction ability of a dissimilatory arsenate-reducing bacterium, Bacillus sp. SF-1 as a bioremediation agent. The model results correlated well with a series of batch reduction experiments conducted anaerobically in serum bottles with initial arsenate concentrations of 360, 735, and 1,500 mg-As/L. The reduction rate was expressed by the Haldane equation that describes the inhibitory effect of high concentrations of arsenate. The reduction rate constant k(r), half saturation constant K(S), and inhibition constant K(I) were estimated respectively as 1.2 x 10(9) mg-As/cells/h, 1.5 x 10(2) mg-As/L, and 4.2 x 10(2) mg-As/L. Lethal effects of arsenite that is accumulated as the end-product of arsenate reduction were expressed by the first-order term with a lethal constant of 2.7 x 10(-4) L/mg-As/h. The yield for the bacterial cells by arsenate respiration was estimated at 4.0 x 10(8) cells/mg-As.     

Arsenate, arsenite and dimethyl arsinic acid (DMA) uptake and tolerance in maize (Zea mays L.)

The influx of arsenate, arsenite and dimethyl arsinic acid (DMA) were studied in 7-day-old excised maize roots (Zea mays L.), and then related to arsenate, arsenite and DMA toxicity. Arsenate, arsenite and DMA influx was all found concentration dependent with significant genotypic differences for arsenite and DMA. Arsenate influx in phosphate starved plants best fitted the four-parameter Michaelis–Menten model corresponding to an additive high and low affinity uptake system, while the uptake of phosphate replete plants followed the two parameter model of Michaelis–Menten kinetics. Arsenite influx was well described by the two parameter model of ‘Michaelis–Menten’ kinetics. DMA influx was comprised of linear phase and a hyperbolic phase. DMA influx was much lower than that for arsenite and arsenate. Arsenate and DMA influx decreased when phosphate was given as a pre-treatment as opposed to phosphate starved plants. The +P treatment tended to decrease influx by 50% for arsenate while this figure was 90% for DMA. Arsenite influx increasing slightly at higher arsenite concentrations in P starved plants but at lower arsenite concentrations, there was little or no difference in arsenite uptake. Low toxicity was found for DMA on maize compared with arsenate and arsenite and the relative toxicity of arsenic species was As(V) > As(III) >> DMA.     

Microbial responses to environmental arsenic

Microorganisms have evolved dynamic mechanisms for facing the toxicity of arsenic in the environment. In this sense, arsenic speciation and mobility is also affected by the microbial metabolism that participates in the biogeochemical cycle of the element. The ars operon constitutes the most ubiquitous and important scheme of arsenic tolerance in bacteria. This system mediates the extrusion of arsenite out of the cells. There are also other microbial activities that alter the chemical characteristics of arsenic: some strains are able to oxidize arsenite or reduce arsenate as part of their respiratory processes. These type of microorganisms require membrane associated proteins that transfer electrons from or to arsenic (AoxAB and ArrAB, respectively). Other enzymatic transformations, such as methylation-demethylation reactions, exchange inorganic arsenic into organic forms contributing to its complex environmental turnover. This short review highlights recent studies in ecology, biochemistry and molecular biology of these processes in bacteria, and also provides some examples of genetic engineering for enhanced arsenic accumulation based on phytochelatins or metallothionein-like proteins.     

Arsenic hazards: strategies for tolerance and remediation by plants

Arsenic toxicity has become a global concern owing to the ever-increasing contamination of water, soil and crops in many regions of the world. To limit the detrimental impact of arsenic compounds, efficient strategies such as phytoremediation are required. Suitable plants include arsenic hyperaccumulating ferns and aquatic plants that are capable of completing their life cycle in the presence of high levels of arsenic through the concerted action of arsenate reduction to arsenite, arsenite complexation, and vacuolar compartmentalization of complexed or inorganic arsenic. Tolerance can also be conferred by lowering arsenic uptake by suppression of phosphate transport activity, a major pathway for arsenate entry. In many unicellular organisms, arsenic tolerance is based on the active removal of cytosolic arsenite while limiting the uptake of arsenate. Recent molecular studies have revealed many of the gene products involved in these processes, providing the tools to improve crop species and to optimize phytoremediation; however, so far only single genes have been manipulated, which has limited progress. We will discuss recent advances and their potential applications, particularly in the context of multigenic engineering approaches.     

Distribution and biotransformation of arsenic species in chicken cardiac and muscle tissues

This study evaluated the bioaccumulation and biotransformation of arsenic species in chicken heart and meat tissues. The experimental study was carried out using two sets of samples. In the first one, 10-d-old chickens were exposed to sodium arsenate, using spiked drinking water. These chickens grew normally and were killed after 50 d of arsenic exposure. The second set were edible chickens used as blanks for a parallel study. The total arsenic and arsenic species content in the exposed samples were at least twice those in the normal edible chicken. It has been demonstrated that sodium arsenate is biotransformed to arsenite and an unknown species and its distribution varies among the different cardiac and meat tissues. One important aspect is the capability of the auricle to preconcentrate the most toxic species, arsenite, in the exposed chicken. A nonidentified arsenic species from the edible chicken was detected. Arsenobetaine was also detected in several tissues. This article shows that chicken can be used as a representative animal when considering inorganic arsenic exposure in humans.     

Microbial oxidation of arsenite in a subarctic environment: diversity of arsenite oxidase genes and identification of a psychrotolerant arsenite oxidiser

Background  

Arsenic is toxic to most living cells. The two soluble inorganic forms of arsenic are arsenite (+3) and arsenate (+5), with arsenite the more toxic. Prokaryotic metabolism of arsenic has been reported in both thermal and moderate environments and has been shown to be involved in the redox cycling of arsenic. No arsenic metabolism (either dissimilatory arsenate reduction or arsenite oxidation) has ever been reported in cold environments (i.e. < 10°C).     

The effects of arsenate and arsenite on the growth and morphology of the marine unicellular algae Tetraselmis chui (Chlorophyta) and Hymenomonas carterae (Chrysophyta)

The marine phytoplanktonic algae, Tetraselmis chui Stein and Hymenomonas carterae (Braarud and Fagerland) Braarud, were grown in media containing various concentrations of arsenate or arsenite. The effects of arsenic on the algae varied with the oxidation state of the element, its concentration, and the degree of illumination. Arsenate affected mainly algal growth but also cell morphology, whereas arsenite caused only morphological changes. Studies on the incorporation of ⁷⁴As-arsenate into cells grown in artificial sea water indicated that arsenate was incorporated and later partially released by both T. chui and H. carterae. Both arsenate influx and efflux seemed to be energy-dependent phenomena, because they varied with the degree of illumination. Differences between the rates of uptake and release of arsenic suggested that arsenate undergoes chemical changes after having been transported into the algal cells.     

Response to Arsenate Treatment in Schizosaccharomyces pombe and the Role of Its Arsenate Reductase Activity

Arsenic toxicity has been studied for a long time due to its effects in humans. Although epidemiological studies have demonstrated multiple effects in human physiology, there are many open questions about the cellular targets and the mechanisms of response to arsenic. Using the fission yeast Schizosaccharomyces pombe as model system, we have been able to demonstrate a strong activation of the MAPK Spc1/Sty1 in response to arsenate. This activation is dependent on Wis1 activation and Pyp2 phosphatase inactivation. Using arsenic speciation analysis we have also demonstrated the previously unknown capacity of S. pombe cells to reduce As (V) to As (III). Genetic analysis of several fission yeast mutants point towards the cell cycle phosphatase Cdc25 as a possible candidate to carry out this arsenate reductase activity. We propose that arsenate reduction and intracellular accumulation of arsenite are the key mechanisms of arsenate tolerance in fission yeast.     

Toxicological properties of the thiolated inorganic arsenic and arsenosugar metabolite thio-dimethylarsinic acid in human bladder cells

Thio-dimethylarsinic acid (thio-DMA^V) has recently been identified as human metabolite after exposure toward both the human carcinogen inorganic arsenic and arsenosugars, which are the major arsenical constituents of marine algae. This study aims to get further insight in the toxic modes of action of thio-DMA^V in cultured human urothelial cells. Among others effects of thio-DMA^V on eight cell death related endpoints, cell cycle distribution, genotoxicity, cellular bioavailability as well as for the first time its impact on DNA damage induced poly(ADP-ribosyl)ation were investigated and compared to effects induced by arsenite. The data indicate that thio-DMA^V exerts its cellular toxicity in a similar or even lower concentration range, however most likely via different mechanisms, than arsenite. Most interestingly, thio-DMA^V decreased damage-induced cellular poly(ADP-ribosyl)ation by 35,000-fold lower concentrations than arsenite. The inhibition of this essential DNA-damage induced and DNA-repair related signaling reaction might contribute to inorganic arsenic induced toxicity, at least in the bladder. Therefore, and also because thio-DMA^V is to date by far the most toxic human metabolite identified after arsenosugar intake, thio-DMA^V should contemporary be fully (also in vivo) toxicologically characterized, to assess risks to human health related to inorganic arsenic but especially arsenosugar dietary intake.