Bifunctional protein ArsRM contributes to arsenite methylation and resistance in Brevundimonas sp. M20.

BACKGROUND: Arsenic (As) with various chemical forms, including inorganic arsenic and organic arsenic, is the most prevalent water and environmental toxin. This metalloid occurs worldwide and many of its forms, especially arsenite [As(III)], cause various diseases including cancer. Organification of arsenite is an effective way for organisms to cope with arsenic toxicity. Microbial communities are vital contributors to the global arsenic biocycle and represent a promising way to reduce arsenite toxicity. METHODS: Brevundimonas sp. M20 with arsenite and roxarsone resistance was isolated from aquaculture sewage. The arsHRNBC cluster and the metRFHH operon of M20 were identified by sequencing. The gene encoding ArsR/methyltransferase fusion protein, arsRM, was amplified and expressed in Escherichia coli BL21 (DE3), and this strain showed resistance to arsenic in the present of 0.25-6 mM As(III), aresenate, or pentavalent roxarsone. The methylation activity and regulatory action of ArsRM were analyzed using Discovery Studio 2.0, and its functions were confirmed by methyltransferase activity analysis and electrophoretic mobility shift assays. RESULTS: The minimum inhibitory concentration of the roxarsone resistant strain Brevundimonas sp. M20 to arsenite was 4.5 mM. A 3,011-bp arsenite resistance ars cluster arsHRNBC and a 5649-bp methionine biosynthesis met operon were found on the 3.315-Mb chromosome. Functional prediction analyses suggested that ArsRM is a difunctional protein with transcriptional regulation and methyltransferase activities. Expression of ArsRM in E. coli increased its arsenite resistance to 1.5 mM. The arsenite methylation activity of ArsRM and its ability to bind to its own gene promoter were confirmed. The As(III)-binding site (ABS) and S-adenosylmethionine-binding motif are responsible for the difunctional characteristic of ArsRM. CONCLUSIONS: We conclude that ArsRM promotes arsenite methylation and is able to bind to its own promoter region to regulate transcription. This difunctional characteristic directly connects methionine and arsenic metabolism. Our findings contribute important new knowledge about microbial arsenic resistance and detoxification. Future work should further explore how ArsRM regulates the met operon and the ars cluster.

Highly efficient persulfate catalyst prepared from modified electrolytic manganese residues coupled with biochar for the roxarsone removal.

The contamination of organoarsenic is becoming increasingly prominent while SR-AOPs were confirmed to be valid for their remediation. This study has found that the novel metal/carbon catalyst (Fe/C-Mn) prepared by solid waste with hierarchical pores could simultaneously degrade roxarsone (ROX) and remove As(V). A total of 95.6% of ROX (20 mg/L) could be removed at the concentration of 1.0 g/L of catalyst and 0.4 g/L of oxidant in the Fe/C-Mn/PMS system within 90 min. The scavenging experiment and electrochemical test revealed that both single-electron and two-electron pathways contributed to the ROX decomposition. Spectroscopic analysis suggested the ROX has been successfully mineralized while As(V) was fixed with the surface Fe and Mn. Density functional theory (DFT) calculation and chromatographic analysis indicated that the As7, N8, O9 and O10 sites of ROX molecule were vulnerable to being attacked by nucleophilic, electrophilic and radical, resulting in the formation of several intermediates such as phenolic compounds. Additionally, the low metal leaching concentration during recycling and high anti-interference ability in various water matrices manifested the practicability of Fe/C-Mn/PMS system.

The role of organic and inorganic substituents of roxarsone determines its binding behavior and mechanisms onto nano-ferrihydrite colloidal particles.

The retention and fate of Roxarsone (ROX) onto typical reactive soil minerals were crucial for evaluating its potential environmental risk. However, the behavior and molecular-level reaction mechanism of ROX and its substituents with iron (hydr)oxides remains unclear. Herein, the binding behavior of ROX on ferrihydrite (Fh) was investigated through batch experiments and in-situ ATR-FTIR techniques. Our results demonstrated that Fh is an effective geo-sorbent for the retention of ROX. The pseudo-second-order kinetic and the Langmuir model successfully described the sorption process. The driving force for the binding of ROX on Fh was ascribed to the chemical adsorption, and the rate-limiting step is simultaneously dominated by intraparticle and film diffusion. Isotherms results revealed that the sorption of ROX onto Fh appeared in uniformly distributed monolayer adsorption sites. The two-dimensional correlation spectroscopy and XPS results implied that the nitro, hydroxyl, and arsenate moiety of ROX molecules have participated in binding ROX onto Fh, signifying that the predominated mechanisms were attributed to the hydrogen bonding and surface complexation. Our results can help to better understand the ROX-mineral interactions at the molecular level and lay the foundation for exploring the degradation, transformation, and remediation technologies of ROX and structural analog pollutants in the environment.

Organoarsenical tolerance in Sphingobacterium wenxiniae, a bacterium isolated from activated sludge.

Organoarsenicals enter the environment from biogenic and anthropogenic sources. Trivalent inorganic arsenite (As(III)) is microbially methylated to more toxic methylarsenite (MAs(III)) and dimethylarsenite (DMAs(III)) that oxidize in air to MAs(V) and DMAs(V). Sources include the herbicide monosodium methylarsenate (MSMA or MAs(V)), which is microbially reduced to MAs(III), and the aromatic arsenical roxarsone (3-nitro-4-hydroxybenzenearsonic acid or Rox), an antimicrobial growth promoter for poultry and swine. Here we show that Sphingobacterium wenxiniae LQY-18T , isolated from activated sludge, is resistant to trivalent MAs(III) and Rox(III). Sphingobacterium wenxiniae detoxifies MAs(III) and Rox(III) by oxidation to MAs(V) and Rox(V). Sphingobacterium wenxiniae has a novel chromosomal gene, termed arsU1. Expressed in Escherichia coli arsU1 confers resistance to MAs(III) and Rox(III) but not As(III) or pentavalent organoarsenicals. Purified ArsU1 catalyses oxidation of trivalent methylarsenite and roxarsone. ArsU1 has six conserved cysteine residues. The DNA sequence for the three C-terminal cysteines was deleted, and the other three were mutated to serines. Only C45S and C122S lost activity, suggesting that Cys45 and Cys122 play a role in ArsU1 function. ArsU1 requires neither FMN nor FAD for activity. These results demonstrate that ArsU1 is a novel MAs(III) oxidase that contributes to S. wenxiniae tolerance to organoarsenicals.

Fabrication of Praseodymium Vanadate Nanoparticles on Disposable Strip for Rapid and Real-Time Amperometric Sensing of Arsenic Drug Roxarsone.

Nanomaterials have versatile properties owing to their high surface-to-volume ratio and can thus be used in a variety of applications. This work focused on applying a facile hydrothermal strategy to prepare praseodymium vanadate nanoparticles due to the importance of nanoparticles in today's society and the fact that their synthesis might be a challenging endeavor. The structural and morphological characterizations were carried out to confirm the influence of the optimizations on the reaction's outcomes, which revealed praseodymium vanadate (PrVO4) with a tetragonal crystal system. In this regard, the proposed development of electrochemical sensors based on the PrVO4 nanocatalyst for the real-time detection of arsenic drug roxarsone (RXS) is a primary concern. The detection was measured by amperometric (i-t) signals where PrVO4/SPCE, as a new electrochemical sensing medium for RXS detection, increased the sensitivity of the sensor to about ∼2.5 folds compared to the previously reported ones. In the concentration range of 0.001-551.78 µM, the suggested PrVO4/SPCE sensor has a high sensitivity for RXS, with a detection limit of 0.4 nM. Furthermore, the impact of several selected potential interferences, operational stability (2000 s), and reproducibility measurements have no discernible effect on RXS sensing, making it the ideal sensing device feasible for technical analysis. The real-time analysis reveals the excellent efficiency and reliability of the prosed sensor toward RXS detection with favorable recovery ranges between ±97.00-99.66% for chicken, egg, water, and urine samples.

Characterization of Two Highly Arsenic-Resistant Caulobacteraceae Strains of Brevundimonas nasdae: Discovery of a New Arsenic Resistance Determinant.

Arsenic (As), distributed widely in the natural environment, is a toxic substance which can severely impair the normal functions in living cells. Research on the genetic determinants conferring functions in arsenic resistance and metabolism is of great importance for remediating arsenic-contaminated environments. Many organisms, including bacteria, have developed various strategies to tolerate arsenic, by either detoxifying this harmful element or utilizing it for energy generation. More and more new arsenic resistance (ars) determinants have been identified to be conferring resistance to diverse arsenic compounds and encoded in ars operons. There is a hazard in mobilizing arsenic during gold-mining activities due to gold- and arsenic-bearing minerals coexisting. In this study, we isolated 8 gold enrichment strains from the Zijin gold and copper mine (Longyan, Fujian Province, China) wastewater treatment site soil, at an altitude of 192 m. We identified two Brevundimonas nasdae strains, Au-Bre29 and Au-Bre30, among these eight strains, having a high minimum inhibitory concentration (MIC) for As(III). These two strains contained the same ars operons but displayed differences regarding secretion of extra-polymeric substances (EPS) upon arsenite (As(III)) stress. B. nasdae Au-Bre29 contained one extra plasmid but without harboring any additional ars genes compared to B. nasdae Au-Bre30. We optimized the growth conditions for strains Au-Bre29 and Au-Bre30. Au-Bre30 was able to tolerate both a lower pH and slightly higher concentrations of NaCl. We also identified folE, a folate synthesis gene, in the ars operon of these two strains. In most organisms, folate synthesis begins with a FolE (GTP-Cyclohydrolase I)-type enzyme, and the corresponding gene is typically designated folE (in bacteria) or gch1 (in mammals). Heterologous expression of folE, cloned from B. nasdae Au-Bre30, in the arsenic-hypersensitive strain Escherichia coli AW3110, conferred resistance to As(III), arsenate (As(V)), trivalent roxarsone (Rox(III)), pentavalent roxarsone (Rox(V)), trivalent antimonite (Sb(III)), and pentavalent antimonate (Sb(V)), indicating that folate biosynthesis is a target of arsenite toxicity and increased production of folate confers increased resistance to oxyanions. Genes encoding Acr3 and ArsH were shown to confer resistance to As(III), Rox(III), Sb(III), and Sb(V), and ArsH also conferred resistance to As(V). Acr3 did not confer resistance to As(V) and Rox(V), while ArsH did not confer resistance to Rox(V).

Identification of a MarR Subfamily That Regulates Arsenic Resistance Genes.

In this study, comprehensive analyses were performed to determine the function of an atypical MarR homolog in Achromobacter sp. strain As-55. Genomic analyses of Achromobacter sp. As-55 showed that this marR is located adjacent to an arsV gene. ArsV is a flavin-dependent monooxygenase that confers resistance to the antibiotic methylarsenite [MAs(III)], the organoarsenic compound roxarsone(III) [Rox(III)], and the inorganic antimonite [Sb(III)]. Similar marR genes are widely distributed in arsenic-resistant bacteria. Phylogenetic analyses showed that these MarRs are found in operons predicted to be involved in resistance to inorganic and organic arsenic species, so the subfamily was named MarRars. MarRars orthologs have three conserved cysteine residues, which are Cys36, Cys37, and Cys157 in Achromobacter sp. As-55, mutation of which compromises the response to MAs(III)/Sb(III). GFP-fluorescent biosensor assays show that AdMarRars (MarR protein of Achromobacter deleyi As-55) responds to trivalent As(III) and Sb(III) but not to pentavalent As(V) or Sb(V). The results of RT-qPCR assays show that arsV is expressed constitutively in a marR deletion mutant, indicating that marR represses transcription of arsV. Moreover, electrophoretic mobility shift assays (EMSAs) demonstrate that AdMarRars binds to the promoters of both marR and arsV in the absence of ligands and that DNA binding is relieved upon binding of As(III) and Sb(III). Our results demonstrate that AdMarRars is a novel As(III)/Sb(III)-responsive transcriptional repressor that controls expression of arsV, which confers resistance to MAs(III), Rox(III), and Sb(III). AdMarRars and its orthologs form a subfamily of MarR proteins that regulate genes conferring resistance to arsenic-containing antibiotics. IMPORTANCE In this study, a MarR family member, AdMarRars was shown to regulate the arsV gene, which confers resistance to arsenic-containing antibiotics. It is a founding member of a distinct subfamily that we refer to as MarRars, regulating genes conferring resistance to arsenic and antimony antibiotic compounds. AdMarRars was shown to be a repressor containing conserved cysteine residues that are required to bind As(III) and Sb(III), leading to a conformational change and subsequent derepression. Here we show that members of the MarR family are involved in regulating arsenic-containing compounds.

Enhancing Roxarsone Degradation and In Situ Arsenic Immobilization Using a Sulfate-Mediated Bioelectrochemical System.

Roxarsone (ROX) is widely used in animal farms, thereby producing organoarsenic-bearing manure/wastewater. ROX cannot be completely degraded and nor can its arsenical metabolites be effectively immobilized during anaerobic digestion, potentially causing arsenic contamination upon discharge to the environment. Herein, we designed and tested a sulfate-mediated bioelectrochemical system (BES) to enhance ROX degradation and in situ immobilization of the released inorganic arsenic. Using our BES (0.5 V voltage and 350 µM sulfate), ROX and its metabolite, 4-hydroxy-3-amino-phenylarsonic acid (HAPA), were completely degraded within 13-22 days. In contrast, the degradation efficiency of ROX and HAPA was <85% during 32-day anaerobic digestion. In a sulfate-mediated BES, 75.0-83.2% of the total arsenic was immobilized in the sludge, significantly more compared to the anaerobic digestion (34.1-57.3%). Our results demonstrate that the combination of sulfate amendment and voltage application exerted a synergetic effect on enhancing HAPA degradation and sulfide-driven arsenic precipitation. This finding was further verified using real swine wastewater. A double-cell BES experiment indicated that As(V) and sulfate were transported from the anode to the cathode chamber and coprecipitated as crystalline alacranite in the cathode chamber. These findings suggest that the sulfate-mediated BES is a promising technique for enhanced arsenic decontamination of organoarsenic-bearing manure/wastewater.

NemA Catalyzes Trivalent Organoarsenical Oxidation and Is Regulated by the Trivalent Organoarsenical-Selective Transcriptional Repressor NemR.

Synthetic aromatic arsenicals such as roxarsone (Rox(V)) and nitarsone (Nit(V)) have been used as animal growth enhancers and herbicides. Microbes contribute to redox cycling between the relatively less toxic pentavalent and highly toxic trivalent arsenicals. In this study, we report the identification of nemRA operon from Enterobacter sp. Z1 and show that it is involved in trivalent organoarsenical oxidation. Expression of nemA is induced by chromate (Cr(VI)), Rox(III), and Nit(III). Heterologous expression of NemA in Escherichia coli confers resistance to Cr(VI), methylarsenite (MAs(III)), Rox(III), and Nit(III). Purified NemA catalyzes simultaneous Cr(VI) reduction and MAs(III)/Rox(III)/Nit(III) oxidation, and oxidation was enhanced in the presence of Cr(VI). The results of electrophoretic mobility shift assays and fluorescence assays demonstrate that the transcriptional repressor, NemR, binds to either Rox(III) or Nit(III). NemR has three conserved cysteine residues, Cys21, Cys106, and Cys116. Mutation of any of the three resulted in loss of response to Rox(III)/Nit(III), indicating that they form an Rox(III)/Nit(III) binding site. These results show that NemA is a novel trivalent organoarsenical oxidase that is regulated by the trivalent organoarsenical-selective repressor NemR. This discovery expands our knowledge of the molecular mechanisms of organoarsenical oxidation and provides a basis for studying the redox coupling of environmental toxic compounds.

Efficient Degradation of Organoarsenic by UV/Chlorine Treatment: Kinetics, Mechanism, Enhanced Arsenic Removal, and Cytotoxicity.

Roxarsone (ROX) has been widely used as an organoarsenic additive in animal feeding operations and poses a risk to the environment. Here, we first report the efficient degradation of ROX by UV/chlorine, where the kinetics, removal of total arsenic (As), and cytotoxicity were investigated. The kinetics study presented that reactive chlorine species (RCS) and HO• were the dominant species to react with ROX. Furthermore, the degradation rate of ROX can reach the maximum value at pH 7.5 due to the formation of more RCS. The degradation of ROX was affected by the amount of chlorine, pH, and water matrix. Through product analysis and Gauss theoretical calculation, two possible ROX degradation pathways were proposed. The free radicals attacked the As-C bond of ROX and resulted in releasing arsenate (As(V)). It was the reason that for an enhancement of the removal of total As by ferrous appeared after UV/chlorine, and over 98% of the total As was removed. In addition, cytotoxicity studies indicated that the cytotoxicity significantly enhanced during the degradation of ROX by UV/chlorine. However, by combination of UV/chlorine and adsorption, cytotoxicity can be greatly eliminated, probably due to the removal of As(V) and chlorinated products. These results further demonstrated that UV/chlorine treatment could be an effective method for the control of the potential environmental risks posed by organoarsenic.

Roxarsone transformation and its impacts on soil enzyme activity in paddy soils: A new insight into water flooding effects.

The aromatic arsenical roxarsone (ROX) has been used as feed additive for decades worldwide. The past or present application of animal manure containing ROX in paddy fields results in arsenic (As) accumulation in rice grain. However, the degradation and transformation mechanisms of ROX in paddy soil which determine As bioavailability and uptake by rice are still unclear. The current study investigated the variation of As speciation and soil enzyme activities in ROX-treated soils under flooded and non-flooded conditions for six months. Our results showed that 70.2% of ROX persisted in non-flooded paddy soils after 180 d while ROX degraded completely within 7 d in flooded soils. The rapid degradation of ROX under flooded conditions owed to the enhanced biotic transformation that was caused by the low Eh and the predominant presence of Clostridium spp. and Bacillus spp. ROX was not only transformed to As(III) and As(V) in non-flooded soils but also to 3-amino-4-hydroxyphenylarsonic acid and methyl arsenicals in flooded soils. The degradation products significantly inhibited soil enzyme activities for 7-30 d, but the inhibition effects disappeared after 90 d due to the sorption of transformed As products to amorphous Fe oxides. This study provides new insights into the flooding effect on ROX fate in paddy fields, which is important for the management of animal waste and risk control on polluted sites.

Role of ArsEFG in Roxarsone and Nitarsone Detoxification and Resistance.

Organoarsenical biotransformations are important components of the global cycling of arsenic. Roxarsone (3-nitro-4-hydroxybenzenearsenate or Rox(V)) and nitarsone (4-nitrobenzene arsenate or Nit(V)) are synthetic aromatic organoarsenicals used in the poultry industry as additives to prevent coccidiosis and improve feed efficiency. Here, we describe a novel pathway of resistance to roxarsone and nitarsone involving biotransformation of their trivalent forms (Rox(III)) and (Nit(III)) to the trivalent organoarsenicals HAPA(III) and pAsA(III), coupled to active extrusion of the aromatic aminobenezylarsenicals from the cells. The arsE, arsF, and arsG were cloned from the arsenic island in the chromosome of Shewanella putrefaciens 200. When expressed in Escherichia coli together, but not alone, arsEFG conferred resistance to Rox(III) and Nit(III) and decreased the accumulation of both. The cells transformed Rox(III) or Nit(III) to HAPA(III) or pAsA(III) by reducing the nitro group to an amine. Everted membrane vesicles from cells expressing arsG accumulated HAPA(III) or pAsA(III). Our data indicate that ArsE and ArsF together reduce Rox(III) or Nit(III) to HAPA(III) or pAsA(III), which are extruded from the cells by the efflux permease ArsG. Identification of the coupled pathway of ArsE, ArsF, and ArsG catalysis is a molecular description of a novel pathway for resistance to roxarsone and nitarsone.

Reduction of Organoarsenical Herbicides and Antimicrobial Growth Promoters by the Legume Symbiont Sinorhizobium meliloti.

Massive amounts of methyl [e.g., methylarsenate, MAs(V)] and aromatic arsenicals [e.g., roxarsone (4-hydroxy-3-nitrophenylarsonate, Rox(V)] have been utilized as herbicides for weed control and growth promotors for poultry and swine, respectively. The majority of these organoarsenicals degrade into more toxic inorganic species. Here, we demonstrate that the legume symbiont Sinorhizobium meliloti both reduces MAs(V) to MAs(III) and catalyzes sequential two-step reduction of nitro and arsenate groups in Rox(V), producing the highly toxic trivalent amino aromatic derivative 4-hydroxy-3-aminophenylarsenite (HAPA(III)). The existence of this process suggests that S. meliloti possesses the ability to transform pentavalent methyl and aromatic arsenicals into antibiotics to provide a competitive advantage over other microbes, which would be a critical process for the synthetic aromatic arsenicals to function as antimicrobial growth promoters. The activated trivalent aromatic arsenicals are degraded into less-toxic inorganic species by an MAs(III)-demethylating aerobe, suggesting that environmental aromatic arsenicals also undergo a multiple-step degradation pathway, in analogy with the previously reported demethylation pathway of the methylarsenate herbicide. We further show that an FAD-NADPH-dependent nitroreductase encoded by mdaB gene catalyzes nitroreduction of roxarsone both in vivo and in vitro. Our results demonstrate that environmental organoarsenicals trigger competition between members of microbial communities, resulting in gradual degradation of organoarsenicals and contamination by inorganic arsenic.

Effect of roxarsone metabolites in chicken manure on soil biological property.

Roxarsone (ROX), an organoarsenic feed additive, occurs as itself and its metabolites including As(V), As(III), monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA) in animal manure. Animal manure improves soil biological property, whereas As compounds impact microorganisms. The integral influence of animal manure bearing ROX metabolites on soil biological quality is not clear yet. Herein, the effect of four chicken manures excreted by chickens fed with four diets containing 0, 40, 80 and 120 mg ROX kg-1, on soil biological attributes. ROX addition in chicken diets increased total As and ROX metabolites in manures, but decreased manure total N, ammonium and nitrate. The elevated ROX metabolites in manures increased soil total As, As species and total N, and increased first and then decreased soil nitrate and nitrite, but did not affect soil ammonium in manure-applied soils. The promoting role of both soil As(III) and ammonium on soil microbial biomass carbon and nitrogen, respiration and saccharase activity, were exceeded or balanced by the inhibiting effect of soil nitrate. The suppression of soil catalase activity by soil As(V) was surpassed by the enhancement caused by soil nitrate and nitrite. Soil urease, acid phosphatase and polyphenol oxidase activities were not suitable bioindicators in the four manure-amended soils. Soil DMA did not affect soil biological properties, and MMA was not detectable in all manure-amended soils. The above highlights the complexity of joint influence of soil As and N on biological attributes. Totally, when ROX is used at allowable dose in chicken diet, soil biological quality would be suppressed in manure-amended soil.

Soil attribute regulates assimilation of roxarsone metabolites by rice (Oryza sativa L.).

Roxarsone (ROX), an organoarsenic feed additive, and its metabolites, can be present in animal manure used to fertilize rice. Rice is prone to absorb arsenic, and is subject to straighthead disorder, which reduces rice yield and is linked with organic arsenic compounds. This study aims to elucidate how soil property affect arsenic accumulation in rice plants fertilized with chicken manure containing ROX metabolites. Manures of chickens fed without or with ROX, designated as control manure and ROX treated manure (ROXCM), respectively, were applied in eight paddy soils of different origins, to investigate the assimilation of arsenic species in rice plants. The results show that inorganic arsenic (arsenate and arsenite), monomethylarsonic acid and dimethylarsinic acid (DMA) were detected in all brown rice and husk, trace tetramethylarsonium and trimethylarsine oxide were occasionally found in these both parts, whereas all these arsenic species were determined in straw, irrespective of manure type. ROXCM application specifically and significantly increased brown rice DMA (P = 0.002), which remarkably enhanced the risk of straighthead disease in rice. Although soil total As impacted grain biomass, soil free-iron oxides and pH dominated arsenic accumulation by rice plants. The significantly increased grain DMA suggests manure bearing ROX metabolites is not suitable to be used in soils with abundant free-iron oxides and/or high pH, if straighthead disorder is to be avoided in rice.

A screen-printed electrode modified with tungsten disulfide nanosheets for nanomolar detection of the arsenic drug roxarsone.

A sensitive electrochemical (voltammetric; DPV) sensor has been developed for the determination of coccidiostat drug (roxarsone) based on the use of an SPCE (screen-printed carbon electrode) modified with tungsten disulfide nanosheets (WS2 NSs). The electrochemical detection of roxarsone on the WS2-modified SPCE was examined by electrochemical strategies. XPS, XRD, Raman, SEM, TEM, EDS and EIS were used to characterize the nanosheets. The effects of scan rate, pH values (phosphate buffer) and buffer concentration were optimized. A selective roxarsone sensor was developed that works best at -0.64 V (vs. Ag/AgCl) and performs much better than the bare SPCE. Features include (a) a wider linear range (0.05 to 490 µM), (b) a nanomolar detection limit (0.03 µM) and (c) high sensitivity (29 µA·µM-1·cm-2). The modified SPCEs have been successfully applied to the determination of roxarsone in spiked meat samples where they gave high accuracy and good recoveries. Graphical abstract Synthesis of WS2 nanosheets and electrochemical detection of roxarsone.

Soil Behaviour of the Veterinary Drugs Lincomycin, Monensin, and Roxarsone and Their Toxicity on Environmental Organisms.

Lincomycin, monensin, and roxarsone are commonly used veterinary drugs. This study investigated their behaviours in different soils and their toxic effects on environmental organisms. Sorption and mobility analyses were performed to detect the migration capacity of drugs in soils. Toxic effects were evaluated by inhibition or acute toxicity tests on six organism species: algae, plants, daphnia, fish, earthworms and quails. The log Kd values (Freundlich model) of drugs were: lincomycin in laterite soil was 1.82; monensin in laterite soil was 2.76; and roxarsone in black soil was 1.29. The Rf value of lincomycin, roxarsone, monensin were 0.4995, 0.4493 and 0.8348 in laterite soil, and 0.5258, 0.5835 and 0.8033 in black soil, respectively. The EC50 for Scenedesmus obliquus, Arabidopsis thaliana, Daphnia magna and LC50/LD50 for Eisenia fetida, Danio rerio, and Coturnix coturnix were: 13.15 mg/L,32.18 mg/kg dry soil,292.6 mg/L,452.7 mg/L,5.74 g/kg dry soil and 103.9 mg/kg (roxarsone); 1.085 mg/L, 25 mg/kg dry soil, 21.1 mg/L, 4.76 mg/L, 0.346 g/kg dry soil and 672.8 mg/kg (monensin); 0.813 mg/L, 35.40 mg/kg dry soil, >400 mg/L, >2800 mg/L, >15 g/kg dry soil, >2000 mg/kg (lincomycin). These results showed that the environmental effects of veterinary drug residues should not be neglected, due to their mobility in environmental media and potential toxic effects on environmental organisms.

Functional organic material for roxarsone and its derivatives recognition via molecular imprinting.

Roxarsone, one of feed add drugs containing arsenic, has been most widely used in poultry and swine industry. Roxarsone discharged into the environment has caused serious pollution problem. Herein, a reusable functional material for selective recognition and adsorption of roxarsone and its derivatives were designed and synthesized. The interaction mechanism is based on acid-base interaction and surface molecular imprinting. Dual functionalized core-shell structure with silica gel as the core was prepared to use as carrier for surface molecularly imprinted polymers. Surface molecularly imprinted polymers for roxarsone was successfully designed and synthesized using 3-aminopropyltriethoxysilane and methyl acryloyloxypropyltriethoxy silane as functional monomers, Ethylene glycol dimethacrylate as crosslinker, Azobisisobutyronitrile as initiator, acetonitrile as solvent. Binding study showed that the recognition selectivity for roxarsone and its derivatives can be significantly improved (3.5-4 folds) with molecular imprinting. Moreover, the prepared functional material for selective recognition and adsorption of Roxarsone was reusable for multiple times without significant decreasing their adsorption capacities.

Efflux Transporter ArsK Is Responsible for Bacterial Resistance to Arsenite, Antimonite, Trivalent Roxarsone, and Methylarsenite.

Arsenic-resistant bacteria have evolved various efflux systems for arsenic resistance. Five arsenic efflux proteins, ArsB, Acr3, ArsP, ArsJ, and MSF1, have been reported. In this study, comprehensive analyses were performed to study the function of a putative major facilitator superfamily gene, arsK, and the regulation of arsK transcriptional expression in Agrobacterium tumefaciens GW4. We found that (i) arsK is located on an arsenic gene island in strain GW4. ArsK orthologs are widely distributed in arsenic-resistant bacteria and are phylogenetically divergent from the five reported arsenic efflux proteins, indicating that it may be a novel arsenic efflux transporter. (ii) Reporter gene assays showed that the expression of arsK was induced by arsenite [As(III)], antimonite [Sb(III)], trivalent roxarsone [Rox(III)], methylarsenite [MAs(III)], and arsenate [As(V)]. (iii) Heterologous expression of ArsK in an arsenic-hypersensitive Escherichia coli strain showed that ArsK was essential for resistance to As(III), Sb(III), Rox(III), and MAs(III) but not to As(V), dimethylarsenite [dimethyl-As(III)], or Cd(II). (iv) ArsK reduced the cellular accumulation of As(III), Sb(III), Rox(III), and MAs(III) but not to As(V) or dimethyl-As(III). (v) A putative arsenic regulator gene arsR2 was cotranscribed with arsK, and (vi) ArsR2 interacted with the arsR2-arsK promoter region without metalloids and was derepressed by As(III), Sb(III), Rox(III), and MAs(III), indicating the repression activity of ArsR2 for the transcription of arsK These results demonstrate that ArsK is a novel arsenic efflux protein for As(III), Sb(III), Rox(III), and MAs(III) and is regulated by ArsR2. Bacteria use the arsR2-arsK operon for resistance to several trivalent arsenicals or antimonials.IMPORTANCE The metalloid extrusion systems are very important bacterial resistance mechanisms. Each of the previously reported ArsB, Acr3, ArsP, ArsJ, and MSF1 transport proteins conferred only inorganic or organic arsenic/antimony resistance. In contrast, ArsK confers resistance to several inorganic and organic trivalent arsenicals and antimonials. The identification of the novel efflux transporter ArsK enriches our understanding of bacterial resistance to trivalent arsenite [As(III)], antimonite [Sb(III)], trivalent roxarsone [Rox(III)], and methylarsenite [MAs(III)].

Metabolism of a Phenylarsenical in Human Hepatic Cells and Identification of a New Arsenic Metabolite.

Environmental contamination and human consumption of chickens could result in potential exposure to Roxarsone (3-nitro-4-hydroxyphenylarsonic acid), an organic arsenical that has been used as a chicken feed additive in many countries. However, little is known about the metabolism of Roxarsone in humans. The objective of this research was to investigate the metabolism of Roxarsone in human liver cells and to identify new arsenic metabolites of toxicological significance. Human primary hepatocytes and hepatocellular carcinoma HepG2 cells were treated with 20 or 100 µM Roxarsone. Arsenic species were characterized using a strategy of complementary chromatography and mass spectrometry. The results showed that Roxarsone was metabolized to more than 10 arsenic species in human hepatic cells. A new metabolite was identified as a thiolated Roxarsone. The 24 h IC50 values of thiolated Roxarsone for A549 lung cancer cells and T24 bladder cancer cells were 380 ± 80 and 42 ± 10 µM, respectively, more toxic than Roxarsone, whose 24 h IC50 values for A549 and T24 were 9300 ± 1600 and 6800 ± 740 µM, respectively. The identification and toxicological studies of the new arsenic metabolite are useful for understanding the fate of arsenic species and assessing the potential impact of human exposure to Roxarsone.