Immunotoxicity of Organic Arsenic Compounds in Marine Animals

In the present study, we demonstrated for the first time the immunotoxic effects of organic arsenic compounds in marine animals, namely arsenocholine [AsCho; trimethyl(2-hydroxyethyl)arsonium cation], arsenobetaine [AsBe; the trimethyl(carboxymethyl)arsonium zwitterion] and the tetramethylarsonium ion (TetMA), to murine principal immune effector cells (macrophages and lymphocytes), comparing them with the effects of inorganic arsenicals in vitro . Inorganic arsenicals (arsenite and arsenate) showed strong cytotoxicity to both macrophages and lymphocytes. The concentration of arsenite that reduced the number of surviving cells to 50% of that in untreated controls (IC₅₀) was 3–5 μmol dm⁻³, and the cytotoxicity of arsenate (IC₅₀=100 μ-1 m mol dm⁻³) was lower than that of arsenite. Compared with these findings, trimethylarsenic compounds in marine animals, AsCho and AsBe, were less toxic even at a concentration over 10 mmol dm⁻³ to both macrophages and lymphocytes; however, TetMA had weak, but significant, cytotoxicity to these cells (IC₅₀ was about 6 mmol dm⁻³).     

Influence of organic and inorganic arsenicals on glucose uptake in Madin-Darby canine kidney (MDCK) cells.

The effect of organic (oxophenylarsine; PhAsO) and inorganic (arsenite) arsenicals on the availability of glucose to Madin-Darby canine kidney (MDCK) cells was investigated. The MDCK cells revealed stereospecific D-glucose uptake which was inhibited by both arsenicals in a time- and concentration-dependent manner. After 10 min (37 degrees C), the effects on D-glucose and 2-deoxy-D-glucose accumulation were analogous, suggesting an impaired hexose uptake. With arsenite, 0.5-1 mmol dm-3 were required for half-maximum inhibition (IC50), whereas PhAsO inhibited glucose uptake in the micromolar range (IC50 5-30 mumol dm-3). Under these conditions neither cell morphology nor cellular viability was affected. After 60 min, however, the inhibition of glucose utilization was paralleled by the formation of blebs, detachment of the monolayer and a loss of cellular viability as confirmed by dye exclusion, lactate dehydrogenase and potassium release. It is concluded that inhibition of glucose uptake may contribute to the acute toxicity, especially of organic arsenicals, by further aggravating the depletion of intracellular carbohydrates.     

Conversion of arsenite and arsenate to methylarsenic and dimethylarsenic compounds by homogenates prepared from livers and kidneys of rats and mice

Pooled livers and pooled kidneys from rats or mice were homogenized and spiked with arsenite or arsenate in the concentration range 1.3–20 μmol dm^?3. Methylarsenic and dimethylarsenic compounds were determined by the hydride generation technique in the homogenates after a 90 min incubation at 37°C. The rat homogenates methylated arsenite and arsenate more efficiently than the mouse homogenates. Monomethylated arsenic was present in larger amounts than dimethylated arsenic in the rat homogenates. In the absence of reduced glutathione (GSH), no methylation occurred. Addition of GSH promoted monomethylation and dimethylation, whereas dithiothreitol and mercaptoethanol (10 mmol dm^?3) fostered only monomethylation. The amounts of monomethylated arsenic in the rat liver homogenates increased with increasing arsenite concentration (1.3–20 μmol dm^?3) however, the percentage of arsenic that had been methylated decreased. A similar trend, but with much less monomethylarsenic formed, was observed for arsenate-spiked homogenates. Rat kidney homogenates methylated arsenite and arsenate to a much smaller extent than rat liver homogenates. The K_m values for the monomethylation in rat liver homogenates were found to be 5.3 μmol dm^?3 for arsenite and 59 μmol dm^?3 for arsenate.     

Continuous flow hydride generation-atomic fluorescence spectrometric determination and speciation of arsenic in wine

Methods for the atomic fluorescence spectrometric (AFS) determination of total arsenic and arsenic species in wines based on continuous flow hydride generation (HG) with atomization in miniature diffusion flame (MDF) are described. For hydride-forming arsenic, l-cysteine is used as reagent for pre-reduction and complexation of arsenite, arsenate, monomethylarsonate and dimethylarsinate. Concentrations of hydrochloric acid and tetrahydroborate are optimized in order to minimize interference by ethanol. Procedure permits determination of the sum of these four species in 5–10-fold diluted samples with limit of detection (LOD) 0.3 and 0.6 μg l^− 1 As in white and red wines, respectively, with precision between 2% and 8% RSD at As levels within 0.5–10 μg l^− 1.Selective arsine generation from different reaction media is used for non-chromatographic determination of arsenic species in wines: citrate buffer at pH 5.1 for As(III); 0.2 mol l^− 1 acetic acid for arsenite + dimethylarsinate (DMA); 8 mol l^− 1 HCl for total inorganic arsenic [As(III) + As(V)]; and monomethylarsonate (MMA) calculated by difference. Calibration with aqueous and ethanol-matched standard solutions of As(III) is used for 10- and 5-fold diluted samples, respectively. The LODs are 0.4 μg l^− 1 for As(III) and 0.3 μg l^− 1 for the other three As species and precision is within 4–8% RSDs.Arsenic species in wine were also determined by coupling of ion chromatographic separation on an anion exchange column and HG-flame AFS detection. Methods were validated by means of recovery studies and comparative analyses by HG-AFS and electrothermal atomic absorption spectrometry after microwave digestion. The LODs were 0.12, 0.27, 0.15 and 0.13 μg l^− 1 (as As) and RSDs were 2–6%, 5–9%, 3–7% and 2–5% for As(III), As(V), MMA and DMA arsenic species, respectively. Bottled red and white wines from Bulgaria, Republic of Macedonia and Italy were analyzed by non-chromatographic and chromatographic procedures and the As(III), arsenite, has been confirmed as major arsenic species.     

Arsenic speciation in hair extracts

Ingested arsenic is known to be not only excreted by urine, but to be stored in sulphydryl-rich tissue like hair, nail or skin. We developed an extraction method for arsenic species from these tissues and studied the stability of different arsenic species during the extraction process. Inorganic and pentavalent methylated arsenic was found to be stable under the extraction conditions, whereas trivalent methylated arsenicals and the thio-analogue of DMA^V (DMAS) showed reduced stability. The absorption ability of hair for these different species was studied as well. Inorganic arsenic is better absorbed by hair than monomethyl- or dimethyl-arsenicals, whereby the trivalent forms are taken up better than the pentavalent forms. Independent of which methylated arsenical was used for the incubation, the pentavalent form was always the dominant form after extraction. Hair and nail samples from humans suffering from chronic arsenic intoxication contained dominantly inorganic arsenic with small and strongly varying amounts of DMA^V and MA^V present. DMAS was only found in some nail sample extracts containing unusually high amounts of DMA^V and is believed to be formed during the extraction process.     

Methylated Phenylarsenical Metabolites Discovered in Chicken Liver

We report the discovery of three toxicologically relevant methylated phenylarsenical metabolites in the liver of chickens fed 3‐nitro‐4‐hydroxyphenylarsonic acid (ROX), a feed additive in poultry production that is still in use in several countries. Methyl‐3‐nitro‐4‐hydroxyphenylarsonic acid (methyl‐ROX), methyl‐3‐amino‐4‐hydroxyphenylarsonic acid (methyl‐3‐AHPAA), and methyl‐3‐acetamido‐4‐hydroxyphenylarsonic acid (or methyl‐N ‐acetyl‐ROX, methyl‐N ‐AHPAA) were identified in such chicken livers, and the concentration of methyl‐ROX was as high as 90 μg kg⁻¹, even after a five‐day clearance period. The formation of these newly discovered methylated metabolites from reactions involving trivalent phenylarsonous acid substrates, S‐adenosylmethionine, and the arsenic (+3 oxidation state) methyltransferase enzyme As3MT suggests that these compounds are formed by addition of a methyl group to a trivalent phenylarsenical substrate in an enzymatic process. The IC₅₀ values of the trivalent phenylarsenical compounds were 300–30 000 times lower than those of the pentavalent phenylarsenicals.     

Arsenic Speciation in Marine Sediments: Effects of Redox Potential and Reducing conditions

Abstract

The speciation of arsenic in the environment is among others controlled by reduction, methylation and oxidation processes and therefore influenced by the prevailing redox conditions. In this study we have analyzed sediments taken from La Coruña estuary in the north west of Spain. Inorganic (trivalent and pentavalent) and the organic (MMA and DMA) arsenic speciation is related to Eh, Fe and Mn load. The various of the arsenic species concentration and other parameters was analyzed at different depths in some of the sampling points. Low arsenic concentrations (1–10 μg·g^–1) were found. In spite of oxidising conditions (Eh values between 31–96 mV), most of the samples showed a higher As(V) percentage than As (III). Principal component analysis was made to see a sample groups and the results showed that speciation depends on reducing conditions (Eh and Mn).     

Can Humans Metabolize Arsenic Compounds to Arsenobetaine?

Arsenic compounds were determined in 21 urine samples collected from a male volunteer. The volunteer was exposed to arsenic through either consumption of codfish or inhalation of small amounts of (CH₃)₃As present in the laboratory air. The arsenic compounds in the urine were separated and quantified with an HPLC–ICP–MS system equipped with a hydraulic high-pressure nebulizer. This method has a determination limit of 0.5 μg As dm⁻³ urine. To eliminate the influence of the density of the urine, creatinine was determined and all concentrations of arsenic compounds were expressed in μg As g⁻¹ creatinine. The concentrations of arsenite, arsenate and methylarsonic acid in the urine were not influenced by the consumption of seafood. Exposure to trimethylarsine doubled the concentration of arsenate and increased the concentration of methylarsonic acid drastically (0.5 to 5 μg As g⁻¹ creatinine). The concentration of dimethylarsinic acid was elevated after the first consumption of fish (2.8 to 4.3 μg As g⁻¹ creatinine), after the second consumption of fish (4.9 to 26.5 μg As g⁻¹ creatinine) and after exposure to trimethyl- arsine (2.9 to 9.6 μg As g⁻¹ creatinine). As expected, the concentration of arsenobetaine in the urine increased 30- to 50-fold after the first consumption of codfish. Surprisingly, the concentration of arsenobetaine also increased after exposure to trimethylarsine, from a background of approximately 1 μg As g⁻¹ creatinine up to 33.1 μg As g⁻¹ creatinine. Arsenobetaine was detected in all the urine samples investigated. The arsenobetaine in the urine not ascribable to consumed seafood could come from food items of terrestrial origin that—unknown to us—contain arsenobetaine. The possibility that the human body is capable of metabolizing trimethyl- arsine to arsenobetaine must be considered. © 1997 by John Wiley & Sons, Ltd.     

Study of interactions between arsenicals and thioredoxins (human and E. coli) using mass spectrometry

Thioredoxin (Trx) plays an important role in achieving redox balances in cells and protecting the cells from oxidative damage. However, little is known about how arsenic affects Trx chemically. It is conceivable that trivalent arsenicals may bind to Trx, which has a highly conserved -CysGlyProCys- sequence. The objective of this study is to characterize the binding of seven arsenic species with Trx from E. coli and humans, using two mass spectrometry techniques. The arsenic-Trx complexes and the free arsenicals were well separated by size-exclusion liquid chromatography (LC) and detected with inductively coupled plasma mass spectrometry (ICPMS). The LC/ICPMS analyses showed that the trivalent arsenic species were able to form complexes with both human and E. coli Trx. Determination of binding constants indicated that affinity to Trx was higher for monomethylarsonous acid (MMA(III)) and phenylarsine oxide (PhAs(III)) than inorganic arsenite (iAs(III)) and dimethylarsinous acid (DMA(III)), probably because MMA(III) and PhAs(III) were able to form stable complexes by binding to two vicinal cysteines in the -CysGlyProCys- region of the Trx. The complexes of arsenicals with both human and E. coli Trx were further characterized by nano-electrospray tandem mass spectrometry. Binding stoichiometries for different arsenic species were consistent with the available cysteine residues in the Trx. Mass spectral evidence also suggests that the pentavalent arsenicals could be reduced by Trx. This study provides the first detailed chemical characterization of the interactions between Trx and arsenic species.     

Arsenic Compounds in Terrestrial Organisms I: Collybia maculata,Collybia butyracea and Amanita muscaria from Arsenic Smelter Sites in Austria

Three mushroom species from two old arsenic smelter sites in Austria were analyzed for arsenic compounds. The total arsenic concentrations were determined by ICP–MS. Collybia maculata contained 30.0 mg, Collybia butyracea 10.9 mg and Amanita muscaria 21.9 mg As kg⁻¹ dry mass. The arsenic compounds extracted with methanol/water (9:1) from the dried mushroom powders were separated by HPLC on anion-exchange and reversed-phase columns and detected by ICP-MS using a hydraulic high-pressure nebulizer. In Collybia maculata almost all arsenic is present as arsenobetaine. Collybia butyracea contained mainly arsenobetaine (8.8 mg As kg⁻¹ dry mass) and dimethylarsinic acid (1.9 mg As kg⁻¹). Amanita muscaria contained arsenobetaine (15.1 mg As kg⁻¹), traces of arsenite, dimethylarsinic acid and arsenate, and surprisingly arsenocholine (2.6 mg As kg⁻¹) and a tetramethylarsonium salt (0.8 mg As kg⁻¹). © 1997 by John Wiley & Sons, Ltd.     

Enzymatic digestion and chromatographic analysis of arsenic species released from proteins

A method combining gel filtration chromatography (GFC), protease digestion, and ion pair chromatography with inductively coupled plasma mass spectrometry detection was developed for the determination of arsenic species bound to proteins. The method was first established by examining the interactions of two model proteins, metallothionein (MT) and hemoglobin, with three reactive trivalent arsenic species. It was then successfully applied to the speciation of arsenic in red blood cells of rats. Inorganic arsenite (iAs^III), monomethylarsonous acid (MMA^III), and dimethylarsinous acid (DMA^III) were efficiently released from the proteins by protease digestion at pH 8.0, with the recovery ranging from 93% to 106%. There was no oxidation of iAs^III or MMA^III during the protease digestion process. Up to 61% DMA^III (the least stable arsenic species) was unchanged, and the rest was oxidized to the pentavalent dimethylarsinic acid (DMA^V). The arsenic species in the red blood cells of control rats was present as DMA^III complex with hemoglobin. The method enabling the determination of the specific arsenic species that bind to cellular proteins is potentially useful for studying arsenic distribution, metabolism, and toxicity.     

The lon-chromatographic behavior of arsenite,arsenate, methylarsonic acid and dimethylarsinic acid on the hamilton PRP-X100 anion-exchange column

The HPLC separation of arsenite, arsenate, methylarsonic acid and dimethylarsinic acid has been studied in the past but not in a systematic manner. The dependence of the retention times of these arsenic compounds on the pH of the mobile phase, on the concentration and the chemical composition of buffer solutions (phosphate, acetate, potassium hydrogen phthalate) and on the presence of sodium sulfate or nickel sulfate in the mobile phase was investigated using a Hamilton PRP-X100 anion-exchange column. With a flame atomic absorption detector and arsenic concentrations of at least 10 mg dm^?3 all investigated mobile phases will separate the four arsenic compounds at appropriate pH values in the range 4–8. The shortest analysis time (?3 min) was achieved with a 0.006 mol dm^?3 potassium hydrogen phthalate mobile phase at pH 4, the longest (?10 min) with 0.006 mol dm^?3 sodium sulfate at pH 5.9 at a flow rate of 1.5 cm³ min^?1. With a graphite furnace atomic absorption detector at the required, much lower, flow rate of ?0.2 cm³ min^?1 acceptable separations were achievable only with the pH 6 phosphate buffer (0.03 mol dm^?3) and the nickel sulfate solution (0.005 mol dm^?3) as the mobile phase. To become detectable approximately 100 ng arsenic from each arsenic compound (100 μl injection) must be chromatographed with the phosphate buffer, and approximately 10 ng with the nickel sulfate solution.     

Fabrication of Co3O4 particles-modified multi-walled carbon nanotube and poly(phenosafranine) composite electrode for selective and sensitive rutin detection

The preparation and characterisation of a new composite electrode with Co₃O₄ particles-modified multi-walled carbon nanotube (MWCNT) and poly(phenosafranine), as well as its novel application for the voltammetric detection of rutin was described. The resulting composite electrode was characterised using scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS) and electrochemical impedance spectroscopy (EIS). In the optimised experimental conditions, the oxidation peak current (I_pa) of rutin showed a linear increase in concentration, between 0.008–0.6 and 0.80–6.0 μmol L⁻¹, with a detection limit of 0.00379 μmol L⁻¹. Due to its good selectivity and stability, the composite electrode was successfully applied in detecting rutin in pharmaceutical formulations.     

Methylation and Demethylation of Dimethylarsinic Acid in Rats Following Chronic Oral Exposure

Metabolites of dimethylarsinic acid (DMA) were studied in rats chronically exposed to DMA in drinking water. The urine was collected by forced urination at the end of 8, 20 and 30 weeks and the feces at the end of 30 weeks. The samples were analyzed for arsenic species by a combined system of ion chromatography and inductively coupled plasma mass spectrometry (IC–ICP–MS). Increases in arsenite, DMA, trimethylarsine oxide and a still-to-be-identified arsenic compound (which was eluted immediately after monomethylarsonic acid on the chromatogram) were detected in both urine and feces. At the 100 mg l⁻¹ dose, DMA was the main component in the urine; arsenite was a main component in the feces. The results indicate that, besides undergoing methylation, DMA can be demethylated to inorganic arsenic, and demethylation of DMA may be associated with intestinal bacteria     

Simultaneous Determination of Lipophilic Vitamin Esters Using Square-wave Voltammetry at the Glassy Carbon Electrode

Simple and rapid voltammetric method for simultaneous determination of all-trans-retinyl acetate (RAc) or all-trans-retinyl palmitate (RPa) and α-tocopheryl acetate (α-TOAc) has been proposed. The respective method was based on the anodic oxidation of the compounds of interest by square-wave voltammetry in acetone with 0.1 mol L⁻¹ LiClO₄ at the glassy carbon electrode. The procedure was also beneficial with respect to simple dissolution of sample directly in the supporting electrolyte. The all-trans-retinyl acetate could be quantified in two linear ranges (3.1–140 μmol L⁻¹ and 140–400 μmol L⁻¹) and α-tocopheryl acetate in linear range 5.3–400 μmol L⁻¹ with detection limits of 0.9 μmol L⁻¹ RAc (or 0.8 μmol L⁻¹ RPa) and of 1.6 μmol L⁻¹ α-TOAc. Selected commercial cosmetic products were analysed achieving satisfactory recoveries.     

HPLC-ICP-MS method development to monitor arsenic speciation changes by human gut microbiota

Inorganic arsenic (iAs) has been classified as a type 1 carcinogen and has also been linked to several noncancerous health effects. Prior to 1995, the As^V methylation pathway was generally considered to be a detoxification pathway, but cellular and animal studies involving MMA^III (mono metyl arsonous acid) and DMA^III (dimethyl arsinous acid) have indicated that their toxicities meet or exceed that of iAs, suggesting an activation process. In addition, thiolated arsenic metabolites were observed in urine after oral exposure of inorganic arsenic in some studies, for which the toxicological profile was not yet fully characterized in human cells. Studies have revealed that microorganisms from the gut environment are important contributors to arsenic speciation changes. This presystemic metabolism necessitates the development of protocols that enable the detection of not only inorganic arsenic species, but also pentavalent and trivalent methylated, thiolated arsenicals in a gastrointestinal environment. We aim to study the biotransformation of arsenic (As) using a Simulator of the Human Intestinal Microbial Ecosystem (SHIME). To be able to analyze the arsenicals resulting from biotransformation reactions occurring in this system, a method using liquid chromatography hyphenated to an inductively coupled plasma mass spectrometer (HPLC‐ICP‐MS) was developed. A Hamilton PRP‐X100 anion exchange column was used. The method allowed separation, identification and quantification of As^III(arsenite), As^V(arsenate), DMA^V(dimethylarsinicacid), MMA^V(monomethylarsonicacid) and MMMTA (monomethylmonothioarsenate). Attempts to optimize the same method for also separating MMA^III and DMA^III did not succeed. These compounds could be successfully separated using a method based on the use of a Zorbax C₁₈ column. The properties of the column, buffer strength, pH and polar nature of mobile phase were monitored and changed to optimize the developed methods. Linearity, sensitivity, precision, accuracy and resolution of both methods were checked. The combination of the two methods allowed successful quantification of arsenic species in suspensions sampled in vitro from the SHIME reactor or in vivo from the human colon and feces. Copyright © 2011 John Wiley & Sons, Ltd.     

Time Resolved Direct Determination of Arsenate in the Presence of Arsenite on Pencil Graphite Electrode Modified by Graphene Oxide and Zirconium

Trace amount of arsenate in the presence of arsenite was determined directly on pencil graphite electrode modified by graphene oxide and zirconium (Zr−G−PGE). The layer‐by‐layer modification of PGE was characterized by scanning electron microscopy (SEM), X‐ray photoelectron spectroscopy (XPS) and cyclic voltammetry (CV). Key point of the developed method was quick adsorption of arsenate than arsenite on the Zr−G−PGE. In optimal conditions, the Zr−G−PGE was applied for determination of arsenate using differential pulse voltammetry in a linear range 0.10–40.0 μg L⁻¹ with a limit of determination of 0.12±0.01 μg L⁻¹. The sensitivity of the electrode was 1.36±0.07 μA/μg L⁻¹. The modified electrode was used to measure the concentration of arsenate in the river water. A recovery test was performed by introducing 10 μg L⁻¹ arsenate into the rivers water in order and acceptable data of average recovery of 101.2 % was obtained. From the experimental results, the as‐prepared electrode can provide a satisfactory method for direct determination of arsenate in real samples.     

Speciation of arsenic in body fluids

Inorganic arsenic is metabolized by consecutive reduction and methylation reactions to dimethylated arsenic (DMA), and then excreted into the urine mostly in the form of DMA. Therefore, arsenic metabolites in the body fluids and organs/tissues are present in the form of inorganic (arsenite and arsenate) and methylated arsenics (MMA and DMA). Although pentavalent arsenics can be present mostly in the form of free ions, trivalent ones may be present more in the forms conjugated with thiol groups of glutathione (GSH) or proteins. Arsenic in the body fluids (plasma, bile and urine) is present in the soluble forms and can be speciated on ion exchange columns by HPLC with on-line detection by an inductively coupled argon plasma-mass spectrometer (ICP-MS). Free forms of arsenite, arsenate, and monomethylarsonous, monomethylarsonic, dimethylarsinous and dimethylarsinic acids in the body fluids have been demonstrated to be speciated simultaneously within 10 min or so on both anion and cation exchange columns together with arsenobetaine (AsB) and arsenocholine (AsC). Trivalent arsenics conjugated with GSH were eluted in intact forms on an anion exchange column but were liberated into free forms on a cation exchange column. Thus, free and GSH-conjugated arsenic metabolites in the bile and urine have been speciated simultaneously on ion exchange columns by HPLC-ICP-MS.     

Voltammetric Method for the Simultaneous Determination of Melatonin and Pyridoxine in Dietary Supplements Using a Cathodically Pretreated Boron‐doped Diamond Electrode

The simultaneous voltammetric determination of melatonin (MT) and pyridoxine (PY) has been carried out at a cathodically pretreated boron‐doped diamond electrode. By using cyclic voltammetry, a separation of the oxidation peak potentials of both compounds present in mixture was about 0.47 V in Britton‐Robinson buffer, pH 2. The results obtained by square‐wave voltammetry allowed a method to be developed for determination of MT and PY simultaneously in the ranges 1–100 μg mL⁻¹ (4.3×10⁻⁶–4.3×10⁻⁴ mol L⁻¹) and 10–175 μg mL⁻¹ (4.9×10⁻⁵–8.5×10⁻⁴ mol L⁻¹), with detection limits of 0.14 μg mL⁻¹ (6.0×10⁻⁷ mol L⁻¹) and 1.35 μg mL⁻¹ (6.6×10⁻⁶ mol L⁻¹), respectively. The proposed method was successfully to the dietary supplements samples containing these compounds for health‐caring purposes.     

The effect of arsenicals on cell suspension cultures of the Madagascar periwinkle (Catharanthus roseus)

Catharanthus roseus cells were grown in the presence of arsenite, arsenate, methylarsonate and dimethylarsinate. Cell growth and arsenical uptake were monitored. Reduction of arsenate, methylation of arsenic and demethylation of methylarsenic species are described. Alkaloid production by the cells is dramatically influenced by the presence of arsenicals. ¹H NMR studies of methylarsonate uptake by whole cells of C. roseus are reported.