Comparative cytotoxicity of fourteen trivalent and pentavalent arsenic species determined using real-time cell sensing

The occurrence of a large number of diverse arsenic species in the environment and in biological systems makes it important to compare their relative toxicity. The toxicity of arsenic species has been examined in various cell lines using different assays, making comparison difficult. We report real-time cell sensing of two human cell lines to examine the cytotoxicity of fourteen arsenic species： arsenite（As~Ⅲ）, monomethylarsonous acid（MMA~Ⅲ） originating from the oxide and iodide forms, dimethylarsinous acid（DMA~Ⅲ）, dimethylarsinic glutathione（DMAG~Ⅲ）, phenylarsine oxide（PAO~Ⅲ）, arsenate（AsV）, monomethylarsonic acid（MMA~Ⅴ）, dimethylarsinic acid（DMA~Ⅴ）,monomethyltrithioarsonate（MMTTA~Ⅴ）, dimethylmonothioarsinate（DMMTA~Ⅴ）,dimethyldithioarsinate（DMDTA~Ⅴ）, 3-nitro-4-hydroxyphenylarsonic acid（Roxarsone, Rox）,and 4-aminobenzenearsenic acid（p-arsanilic acid, p-ASA）. Cellular responses were measured in real time for 72 hr in human lung（A549） and bladder（T24） cells. IC50 values for the arsenicals were determined continuously over the exposure time, giving rise to IC50 histograms and unique cell response profiles. Arsenic accumulation and speciation were analyzed using inductively coupled plasma-mass spectrometry（ICP-MS）. On the basis of the 24-hr IC50 values, the relative cytotoxicity of the tested arsenicals was in the following decreasing order： PAO~Ⅲ？ MMA~Ⅲ≥ DMA~Ⅲ≥ DMAG~Ⅲ≈ DMMTA~Ⅴ≥ As~Ⅲ？ MMTTA~Ⅴ〉 AsV〉 DMDTA~Ⅴ〉DMA~Ⅴ〉 MMA~Ⅴ≥ Rox ≥ p-ASA. Stepwise shapes of cell response profiles for DMA~Ⅲ, DMAG~Ⅲ,and DMMTA~Ⅴcoincided with the conversion of these arsenicals to the less toxic pentavalent DMA~Ⅴ. Dynamic monitoring of real-time cellular responses to fourteen arsenicals provided useful information for comparison of their relative cytotoxicity.

Methylated and thiolated arsenic species for environmental and health research——A review on synthesis and characterization

Hundreds of millions of people around the world are exposed to elevated concentrations of inorganic and organic arsenic compounds, increasing the risk of a wide range of health effects. Studies of the environmental fate and human health effects of arsenic require authentic arsenic compounds. We summarize here the synthesis and characterization of more than a dozen methylated and thiolated arsenic compounds that are not commercially available. We discuss the methods of synthesis for the following14 trivalent（Ⅲ） and pentavalent（） arsenic compounds： monomethylarsonous acid（MMA~Ⅲ）, dicysteinylmethyldithioarsenite（MMA~Ⅲ（Cys）_2）, monomethylarsonic acid（MMA~Ⅴ）,monomethylmonothioarsonic acid（MMMTAⅤ） or monothio-MMA~Ⅴ, monomethyldithioarsonic acid（MMDTA~Ⅴ） or dithio-MMA~Ⅴ, monomethyltrithioarsonate（MMTTA~Ⅴ） or trithio-MMA~Ⅴ,dimethylarsinous acid（DMA~Ⅲ）, dimethylarsino-glutathione（DMA~Ⅲ（SG））, dimethylarsinic acid（DMA~Ⅴ）, dimethylmonothioarsinic acid（DMMTA~Ⅴ） or monothio-DMAⅤ, dimethyldithioarsinic acid（DMDTA~Ⅴ） or dithio-DMA~Ⅴ, trimethylarsine oxide（TMAO~Ⅴ）, arsenobetaine（AsB）, and an arsenicin-A model compound. We have reviewed and compared the available methods,synthesized the arsenic compounds in our laboratories, and provided characterization information. On the basis of reaction yield, ease of synthesis and purification of product, safety considerations, and our experience, we recommend a method for the synthesis of each of these arsenic compounds.

Supercritical fluid extraction and gas chromatography analysis of arsenic species from solid matrices

A method in combination with derivatization-supercritical fluid extraction（SFE） and gas chromatography（GC） for the speciation and quantitative determination of dimethylarsinate（DMA）, monomethylarsonate（MMA） and inorganic arsenic in solid matrices was investigated. Thioglycolic acid methyl ester（TGM） and thioglycolic acid ethyl ester（TGE） were evaluated as derivatization reagents. The effects of pressure, temperature, flow rate of supercritical CO_2, extraction time, modifier and microemulsion on the efficiency of extraction were systematically investigated. The procedure was applied to the analysis of real soil and sediment samples. Results showed that TGE was more effective for arsenic speciation as a derivatization reagent. Modifying supercritical CO_2 with methanol can greatly improve the extraction efficiency. Further, the addition of microemulsion containing surfactant Triton X-100 can further enhance recoveries of arsenic species. The optimum extraction conditions were 100 ℃, 30 MPa, 10 min static and 25 min dynamic extraction with 5%（v/v） methanol, and surfactant modified supercritical CO_2. Detection limits in solid matrices were 0.15, 0.3 and 1.2 mg/kg for DMA, MMA and inorganic arsenic,respectively. The method was validated by the recovery data. The resulting method was fast, easy to perform and selective in the extraction and detection of various arsenic species in solid matrices.

Multiple transformation pathways of p-arsanilic acid to inorganic arsenic species in water during UV disinfection

p-Arsanilic acid（p-ASA） is widely used in China as livestock and poultry feed additive for promoting animal growth.The use of organoarsenics poses a potential threat to the environment because it is mostly excreted by animals in its original form and can be transformed by UV–Vis light excitation.This work examined the initial rate and efficiency of p-ASA phototransformation under UV-C disinfection lamp.Several factors influencing p-ASA phototransformation,namely,p H,initial concentration,temperature,as well as the presence of Na Cl,NH4＋,and humic acid,were investigated.Quenching experiments and LC–MS were performed to investigate the mechanism of p-ASA phototransformation.Results show that p-ASA was decomposed to inorganic arsenic（including As（Ⅲ） and As（V））and aromatic products by UV-C light through direct photolysis and indirect oxidation.The oxidation efficency of p-ASA by direct photosis was about 32%,and those by HOU and1O2 were 19% and 49%,respectively.Cleavage of the arsenic–benzene bond through direct photolysis,HOU oxidation or1O2 oxidation results in simultaneous formation of inorganic As（Ⅲ）,As（IV）,and As（V）.Inorganic As（Ⅲ） is oxidized to As（IV） and then to As（V） by1O2 or HOU.As（IV） can undergo dismutation or simply react with oxygen to produce As（V） as well.Reactions of the organic moieties of p-ASA produce aniline,aminophenol and azobenzene derivatives as main products.The photoconvertible property of p-ASA implies that UV disinfection of wastewaters from poultry and swine farms containing p-ASA poses a potential threat to the ecosystem,especially agricultural environments.

The potential risk assessment for different arsenic species in the aquatic environment

The different toxicity characteristics of arsenic species result in discrepant ecological risk.The predicted no-effect concentrations（PNECs） 43.65, 250.18, and 2.00 × 10^3μg/L were calculated for As（III）, As（V）, and dimethylarsinic acid in aqueous phase, respectively. With these PNECs, the ecological risk from arsenic species in Pearl River Delta in China and Kwabrafo stream in Ghana was evaluated. It was found that the risk from As（III） and As（V）in the samples from Pearl River Delta was low, while much high in Kwabrafo stream. This study implies that ecological risk of arsenic should be evaluated basing on its species.

Evaluation of the ability of arsenic species to traverse cell membranes by simple diffusion using octanol-water and liposome-water partition coefficients

Arsenic metabolism in living organisms is dependent on the ability of different arsenic species to traverse biological membranes. Simple diffusion provides an alternative influx and efflux route to mediated transport mechanisms that can increase the amount of arsenic available for metabolism in cells. Using octanol-water and liposome-water partition coefficients, the ability of arsenous acid, arsenate, methylarsonate, dimethylarsinate, thio-methylarsonate, thiodimethylarsinic acid, arsenotriglutathione and monomethylarsonic diglutathione to diffuse through the lipid bilayer of cell membranes was investigated. Molecular modelling of arsenic species was used to explain the results. All arsenic species with the exception of arsenate, methylarsonate and thio-methylarsonate were able to diffuse through the lipid bilayer of liposomes, with liposome-water partition coefficients between 0.04 and 0.13. Trivalent arsenic species and thio-pentavalent arsenic species showed higher partition coefficients, suggesting that they can easily traverse cell membranes by passive simple diffusion. Given the higher toxicity of these species compared to oxo-pentavalent arsenic species, this study provides evidence supporting the risk associated with human exposure to trivalent and thio-arsenic species.

Determination of Inorganic Arsenic Species by Electrochemical Hydride Generation Atomic Absorption Spectrometry with Selective Electrochemical Reduction

A new direct procedure for the determination of inorganic arsenic species was developed by electrochemical hydride generation atomic absorption spectrometry （EcHG-AAS） with selective electrochemical reduction. The determination of inorganic arsenic species is based on the fact that As（Ⅲ） shows significantly higher absorbance at low electrolytic currents than As（Ⅴ） in 0.3 mol·L^-1 H2SO4. The electrolytic current used for the determination of As（Ⅲ） without considerable interferences of As（Ⅴ） was 0.4 A, whereas the current for the determination of As（Ⅲ） and As（Ⅴ） was 1.2 A. For equal concentrations of As（Ⅲ） and As（Ⅴ） in a sample, the interferences of As（Ⅴ） during the As（Ⅲ） determination were smaller than 5%. The absorbance for As（Ⅴ） could be calculated by subtracting that for As（Ⅲ） measured at 0.4 A from the total absorbance for As（Ⅲ） and As（Ⅴ） measured at 1.2 A, and then the concentration of As（Ⅴ） can be obtained by its calibration curve at 1.2 A. The methodology developed provided the detection limits of 0.3 and 0.6 ng·mL^-1 for As（Ⅲ） and As（Ⅴ）, respectively. The relative standard deviations were of 3.5% for 20 ng·mL^-1 As（Ⅲ） and 3.2% for 20 ng·mL^-1 As（Ⅴ）. The method was successfully applied to determination of soluble inorganic arsenic species in Chinese medicine.

Determination of arsenic speciation in secondary zinc oxide and arsenic leachability

The species of arsenic in secondary zinc oxide generated from fuming furnace were investigated. The results revealed that there are mainly three types of secondary zinc oxide based on three arsenic species. The main phase of As is As2O3 in type Ⅰ, zinc arsenite （Zn（AsO2）2） in type Ⅱ and lead arsenate （Pb（As206）, Pb4As2O9） in type Ⅲ, respectively. Selective leaching of zinc oxide of type Ⅱ was carried out. The leaching rate of As kept at 65%-70% with 30 g/L NaOH and L/S ratio of 3 at 20 ℃ for 1 h, while the losses of Pb and Zn were both below 1%.

Effects of soaking process on arsenic and other mineral elements in brown rice

One of the main food crops in the world,rice can accumulate high levels of arsenic from flooded paddy soils,which seriously threatens human health.Soaking,a common processing method for brown rice products,especially for brown rice noodles,was investigated in this study.Japonica rice(Dao Hua Xiang No.2)and Indica rice(Ye Xiang You No.3)were selected for studying the effects of soaking on arsenic concentrations,species,and distributions.Results revealed that soaking can efficiently remove arsenic in these two rice varieties,and the main part of removal is endosperm with the maximal rate of about 40%.Inorganic arsenic(I-As)(about 85%)is the main species of arsenic reduction.Meanwhile,the variations of four other elements(i.e.,Mg,Ca,Zn,and Fe)were analyzed.Collectively,the findings of this study indicate that soaking can efficiently remove arsenic in brown rice under controlled soaking conditions,which thereby reduces the arsenic intake for brown rice customers.

Application of iron and silicon fertilizers reduces arsenic accumulation by two Ipomoea aquatica varities

A 45 d pot experiment was conducted to examine the effects of silicon fertilizer or iron fertilizer on the growth of two typical Ipomoea aquatica cultivars（Daye and Liuye） and arsenic（As） accumuation of Daye and Liuye grown in As-contaminated soils at different As dosage levels. The results showed that the application of these two fertilizers generally enhanced the growth of the plants, which may be partly attributable to the reduction in As toxicity. The addition of these two fertilizers also significantly reduced the uptake of As by the plants though the iron fertilizer was more effective, as compared to the silicon fertilizer. The accumulation of As in shoot portion was weaker for Daye than for Liuye. The research findings obtained from this study have implications for developing cost-effective management strategies to minimize human health impacts from consumption of As-containing I. aquatica.

Speciation Analysis of Arsenic Compounds in Seafood by Ion Chromatography-Atomic Fluorescence Spectrometry

Ion chromatography-ultra violet-hydride generation-Atomic Florescence Spectrometry was applied to detect 5 arsenic species in seafoods. The arsenic species studied include arsenobetaine(As B), arsenite(As(III)), dimethylarsinic acid(DMA), monomethylarsonic acid(MMA), and arsenate(As(V)), which were extracted from samples using 2% formic acid. Gradient elution using 33 mmol L^(-1) CH_3COONH_4 and 15 mmol L^(-1) Na_2CO_3 with 10 mL CH_3CH_2OH at pH 8.4 allowed the chromatographic separation of all the species on a Hamilton PRP-X100 anion-exchange column in less than 8 min. In this study, an ultrasound extraction method was used to extract arsenic species from seafood. The extraction efficiency was good and the recoveries from spiked samples were in the range of 72.6%–109%; the precision between sample replicates was higher than 3.6% for all determinations. The detection limits were 3.543 μg L^(-1) for As B, 0.4261 μg L^(-1) for As(III), 0.216 μg L^(-1) for DMA, 0.211 μg L^(-1) for MMA, and 0.709 μg L^(-1) for As(V), and the linear coefficients were greater than 0.999. We also developed an application of this method for the determination of arsenic species in bonito, Euphausia superba, and Enteromorpha with satisfactory results. Therefore, it was confirmed that this method was appropriate for the detection of arsenic species in seafood.

Arsenic speciation in freshwater fish:challenges and research needs

Food and water are the main sources of human exposure to arsenic.It is important to determine arsenic species in food because the toxicities of arsenic vary greatly with its chemical speciation.Extensive research has focused on high concentrations of arsenic species in marine organisms.The concentrations of arsenic species in freshwater fish are much lower,and their determination presents analytical challenges.In this review,we summarize the current state of knowledge on arsenic speciation in freshwater fish and discuss challenges and research needs.Fish samples are typically homogenized,and arsenic species are extracted using water/methanol with the assistance of sonication and enzyme treatment.Arsenic species in the extracts are commonly separated using high-performance liquid chromatography(HPLC)and detected using inductively coupled plasma mass spectrometry(ICPMS).Electrospray ionization tandem mass spectrometry,used in combination with HPLC and ICPMS,provides complementary information for the identification and characterization of arsenic species.The methods and perspectives discussed in this review,covering sample preparation,chromatography separation,and mass spectrometry detection,are directed to arsenic speciation in freshwater fish and applicable to studies of other food items.Despite progress made in arsenic speciation analysis,a large fraction of the total arsenic in freshwater fish remains unidentified.It is challenging to identify and quantify arsenic species present in complex sample matrices at very low concentrations.Further research is needed to improve the extraction efficiency,chromatographic resolution,detection sensitivity,and characterization capability.

Photooxidation of arsenic(Ⅲ) to arsenic(Ⅴ) on the surface of kaolinite clay

As one of the most toxic heavy metals, the oxidation of inorganic arsenic has drawn great attention among environmental scientists. However, little has been reported on the solar photochemical behavior of arsenic species on top-soil. In the present work, the influencing factors（p H, relative humidity（RH）, humic acid（HA）, trisodium citrate, and additional iron ions） and the contributions of reactive oxygen species（ROS, mainly HO^- and HO2^-/O2^-） to photooxidation of As（Ⅲ） to As（Ⅴ） on kaolinite surfaces under UV irradiation（λ = 365 nm）were investigated. Results showed that lower p H facilitated photooxidation, and the photooxidation efficiency increased with the increase of RH and trisodium citrate.Promotion or inhibition of As（Ⅲ） photooxidation by HA was observed at low or high dosages, respectively. Additional iron ions greatly promoted the photooxidation, but excessive amounts of Fe^2＋competed with As（Ⅲ） for oxidation by ROS. Experiments on scavengers indicated that the HOUradical was the predominant oxidant in this system.Experiments on actual soil surfaces proved the occurrence of As（Ⅲ） photooxidation in real topsoil. This work demonstrates that the photooxidation process of As（Ⅲ） on the soil surface should be taken into account when studying the fate of arsenic in natural soil newly polluted with acidic wastewater containing As（Ⅲ）.

Impact of roxarsone on the UASB reactor performance and its degradation

Roxarsone （3-nitro-4-hydroxyphenylarsonic acid, ROX） has been widely used for decades as an organoarsenic feed additive to control intestinal parasites and improve feed efficiency in animal production. However, most of the ROX is excreted into the manure, causing arsenic contamination in wastewater. The arsenic compounds are toxic to microorganisms, but the influence of continuous ROX loading on upflow anaerobic sludge blanket （UASB） reactor is still unknown. In this study, the impact of ROX and its degradation products on the performance of the UASB reactor and the degradation and speciation of ROX in the reactor were investigated. The UASB reactor （hydraulic retention time： 1.75 d） was operated using synthetic wastewater supplemented with ROX for a period of 260 days. With continuous ROX addition at 25.0 mg.L-1, severe inhibition to methanogenic activity occurred after 87 days operation accompanied with an accumulation of volatile fatty acids （VFAs） and a decline in pH. The decrease of added ROX concentration to 13.2 mg.L-1 did not mediate the inhibition. As（III）, As （V）, MMA（V）, DMA（V）, HAPA and an unknown arsenic compound were detected in the reactor, and a possible biotransformation pathway of ROX was proposed. Mass balance analysis of arsenic indicated that 60%-70% of the arsenic was discharged into the effluent, and 30%-40% was precipitated in the reactor. The results from this study suggest that we need to pay attention to the stability in the UASB reactors treating organoarsenic-contaminated manure and wastewater, and the effluent and sludge from the reactor tO avoid diffusion of arsenic contamination.