Transient Glycolytic Complexation of Arsenate Enhances Resistance in the Enteropathogen Vibrio cholerae.

The ubiquitous presence of toxic arsenate (AsV) in the environment has raised mechanisms of resistance in all living organisms. Generally, bacterial detoxification of AsV relies on its reduction to arsenite (AsIII) by ArsC, followed by the export of AsIII by ArsB. However, how pathogenic species resist this metalloid remains largely unknown. Here, we found that Vibrio cholerae, the etiologic agent of the diarrheal disease cholera, outcompetes other enteropathogens when grown on millimolar concentrations of AsV. To do so, V. cholerae uses, instead of ArsCB, the AsV-inducible vc1068-1071 operon (renamed var for vibrio arsenate resistance), which encodes the arsenate repressor ArsR, an alternative glyceraldehyde-3-phosphate dehydrogenase, a putative phosphatase, and the AsV transporter ArsJ. In addition to Var, V. cholerae induces oxidative stress-related systems to counter reactive oxygen species (ROS) production caused by intracellular AsV. Characterization of the var mutants suggested that these proteins function independently from one another and play critical roles in preventing deleterious effects on the cell membrane potential and growth derived from the accumulation AsV. Mechanistically, we demonstrate that V. cholerae complexes AsV with the glycolytic intermediate 3-phosphoglycerate into 1-arseno-3-phosphoglycerate (1As3PG). We further show that 1As3PG is not transported outside the cell; instead, it is subsequently dissociated to enable extrusion of free AsV through ArsJ. Collectively, we propose the formation of 1As3PG as a transient metabolic storage of AsV to curb the noxious effect of free AsV. This study advances our understanding of AsV resistance in bacteria and underscores new points of vulnerability that might be an attractive target for antimicrobial interventions. IMPORTANCE Even though resistance to arsenate has been extensively investigated in environmental bacteria, how enteric pathogens tolerate this toxic compound remains unknown. Here, we found that the cholera pathogen V. cholerae exhibits increased resistance to arsenate compared to closely related enteric pathogens. Such resistance is promoted not by ArsC-dependent reduction of arsenate to arsenite but by an operon encoding an arsenate transporter (ArsJ), an alternative glyceraldehyde 3-phosphate dehydrogenase (VarG), and a putative, uncharacterized phosphatase (VarH). Mechanistically, we demonstrate that V. cholerae detoxifies arsenate by complexing it with the glycolytic intermediate 3-phosphoglycerate into 1-arseno-3-phosphoglycerate (1As3PG). 1As3PG is not transported outside the cell; instead, it is subsequently dissociated by VarH to enable extrusion of free arsenate through ArsJ. Collectively, this study proposes a novel mechanism for arsenate detoxification, entirely independent of arsenate reduction and arsenite extrusion, that enhances V. cholerae resistance to this metalloid compared to other enteric pathogens.

Biological characterization of Bacillus flexus strain SSAI1 transforming highly toxic arsenite to less toxic arsenate mediated by periplasmic arsenite oxidase enzyme encoded by aioAB genes.

Bacillus flexus strain SSAI1 isolated from agro-industry waste, Tuem, Goa, India displayed high arsenite resistance as minimal inhibitory concentration was 25 mM in mineral salts medium. This bacterial strain exposed to 10 mM arsenite demonstrated rapid arsenite oxidation and internalization of 7 mM arsenate within 24 h. The Fourier transformed infrared (FTIR) spectroscopy of cells exposed to arsenite revealed important functional groups on the cell surface interacting with arsenite. Furthermore, scanning electron microscopy combined with electron dispersive X-ray spectroscopy (SEM-EDAX) of cells exposed to arsenite revealed clumping of cells with no surface adsorption of arsenite. Transmission electron microscopy coupled with electron dispersive X-ray spectroscopic (TEM-EDAX) analysis of arsenite exposed cells clearly demonstrated ultra-structural changes and intracellular accumulation of arsenic. Whole-genome sequence analysis of this bacterial strain interestingly revealed the presence of large number of metal(loid) resistance genes, including aioAB genes encoding arsenite oxidase responsible for the oxidation of highly toxic arsenite to less toxic arsenate. Enzyme assay further confirmed that arsenite oxidase is a periplasmic enzyme. The genome of strain SSAI1 also carried glpF, aioS and aioE genes conferring resistance to arsenite. Therefore, multi-metal(loid) resistant arsenite oxidizing Bacillus flexus strain SSAI1 has potential to bioremediate arsenite contaminated environmental sites and is the first report of its kind.

Arsenite provides a selective signal that coordinates arsenate uptake and detoxification through the regulation of PHR1 stability in Arabidopsis.

In nature, plants acquire nutrients from soils to sustain growth, and at the same time, they need to avoid the uptake of toxic compounds and/or possess tolerance systems to cope with them. This is particularly challenging when the toxic compound and the nutrient are chemically similar, as in the case of phosphate and arsenate. In this study, we demonstrated that regulatory elements of the phosphate starvation response (PSR) coordinate the arsenate detoxification machinery in the cell. We showed that arsenate repression of the phosphate transporter PHT1;1 is associated with the degradation of the PSR master regulator PHR1. Once arsenic is sequestered into the vacuole, PHR1 stability is restored and PHT1;1 expression is recovered. Furthermore, we identified an arsenite responsive SKP1-like protein and a PHR1 interactor F-box (PHIF1) as constituents of the SCF complex responsible for PHR1 degradation.We found that arsenite, the form to which arsenate is reduced for compartmentalization in vacuoles, represses PHT1;1 expression, providing a highly selective signal versus phosphate to control PHT1;1 expression in response to arsenate. Collectively, our results provide molecular insights into a sensing mechanism that regulates arsenate/phosphate uptake depending on the plant's detoxification capacity.

Coordinated cytokinin signaling and auxin biosynthesis mediates arsenate-induced root growth inhibition.

Interactions between plant hormones and environmental signals are important for the maintenance of root growth plasticity under ever-changing environmental conditions. Here, we demonstrate that arsenate (AsV), the most prevalent form of arsenic (As) in nature, restrains elongation of the primary root through transcriptional regulation of local auxin biosynthesis genes in the root tips of Arabidopsis (Arabidopsis thaliana) plants. The ANTHRANILATE SYNTHASE ALPHA SUBUNIT 1 (ASA1) and BETA SUBUNIT 1 (ASB1) genes encode enzymes that catalyze the conversion of chorismate to anthranilate (ANT) via the tryptophan-dependent auxin biosynthesis pathway. Our results showed that AsV upregulates ASA1 and ASB1 expression in root tips, and ASA1- and ASB1-mediated auxin biosynthesis is involved in AsV-induced root growth inhibition. Further investigation confirmed that AsV activates cytokinin signaling by stabilizing the type-B ARABIDOPSIS RESPONSE REGULATOR1 (ARR1) protein, which directly promotes the transcription of ASA1 and ASB1 genes by binding to their promoters. Genetic analysis revealed that ASA1 and ASB1 are epistatic to ARR1 in the AsV-induced inhibition of primary root elongation. Overall, the results of this study illustrate a molecular framework that explains AsV-induced root growth inhibition via crosstalk between two major plant growth regulators, auxin and cytokinin.

Arsenate-Induced Changes in Bacterial Metabolite and Lipid Pools during Phosphate Stress.

Agrobacterium tumefaciens GW4 is a heterotrophic arsenite-oxidizing bacterium with a high resistance to arsenic toxicity. It is now a model organism for studying the processes of arsenic detoxification and utilization. Previously, we demonstrated that under low-phosphate conditions, arsenate [As(V)] could enhance bacterial growth and be incorporated into biomolecules, including lipids. While the basic microbial As(V) resistance mechanisms have been characterized, global metabolic responses under low phosphate remain largely unknown. In the present work, the impacts of As(V) and low phosphate on intracellular metabolite and lipid profiles of GW4 were quantified using liquid chromatography-mass spectroscopy (LC-MS) in combination with transcriptional assays and the analysis of intracellular ATP and NADH levels. Metabolite profiling revealed that oxidative stress response pathways were altered and suggested an increase in DNA repair. Changes in metabolite levels in the tricarboxylic acid (TCA) cycle along with increased ATP are consistent with As(V)-enhanced growth of A. tumefaciens GW4. Lipidomics analysis revealed that most glycerophospholipids decreased in abundance when As(V) was available. However, several glycerolipid classes increased, an outcome that is consistent with maximizing growth via a phosphate-sparing phenotype. Differentially regulated lipids included phosphotidylcholine and lysophospholipids, which have not been previously reported in A. tumefaciens The metabolites and lipids identified in this study deepen our understanding of the interplay between phosphate and arsenate on chemical and metabolic levels.IMPORTANCE Arsenic is widespread in the environment and is one of the most ubiquitous environmental pollutants. Parodoxically, the growth of certain bacteria is enhanced by arsenic when phosphate is limited. Arsenate and phosphate are chemically similar, and this behavior is believed to represent a phosphate-sparing phenotype in which arsenate is used in place of phosphate in certain biomolecules. The research presented here uses a global approach to track metabolic changes in an environmentally relevant bacterium during exposure to arsenate when phosphate is low. Our findings are relevant for understanding the environmental fate of arsenic as well as how human-associated microbiomes respond to this common toxin.

Flow-cytometry analysis reveals persister resuscitation characteristics.

BACKGROUND: Persisters and viable but non-culturable (VBNC) cells are two phenotypic variants known to be highly tolerant to antibiotics. Although both cell types are stained as live and often appear as nongrowing during antibiotic treatment, the only distinguishing feature is the ability of persisters to recolonize in standard culture media in the absence of antibiotics. Despite considerable progress in the characterization of persister formation mechanisms, their resuscitation mechanisms remain unclear due to technical limitations in detecting and isolating these cell types in culture environments that are highly heterogeneous. RESULTS: In this study, we used a methodology integrating flow cytometry, fluorescent protein expression systems and ampicillin-mediated cell lysing technique to monitor persister resuscitation at the single-cell level. With this method, we were able to investigate the effects of various culture conditions (e.g., antibiotic treatment time, the length of the stationary phase in overnight pre-cultures, or pretreatment of cells with a metabolic inhibitor) on persister resuscitation. Although we observed long-term pre-cultures have many more VBNC cells compared to short-term pre-cultures, only a small fraction of non-lysed cells was able to resuscitate in all conditions tested. Regardless of pre-culturing and ampicillin treatment times, these persister cells started to resuscitate within 1 hour, after they were transferred to fresh liquid media, with the same doubling time that normal cells have. Our analysis further showed that ampicillin was not able to lyse the cells in the presence of arsenate, a metabolic inhibitor commonly used to increase bacterial persistence. However, the removal of arsenate during antibiotic treatment resulted in cell lysis and a reduction in persister levels despite the significant decrease in ATP levels in the cells. CONCLUSIONS: The strategy presented in this study helps us monitor persister resuscitation at the single-cell level, and simultaneously quantify persister, VBNC and dead cell subpopulations in ampicillin-treated cultures. Our results indicate that the characterization of persister resuscitation with flow cytometry will enhance the current molecular-level understanding of persistence and its evolution.

Targeted cancer cell delivery of arsenate as a reductively activated prodrug.

Nanoformulations, prodrugs, and targeted therapies are among the most intensively investigated approaches to new cancer therapeutics. Human ferritin has been used extensively as a nanocarrier for the delivery of drugs and imaging agents to cancerous tumor cells both in vitro and in vivo. We report exploitation of the native properties of ferritin, which can be co-loaded with simple forms of iron (FeOOH) and arsenic (arsenate) in place of the native phosphate. The As(III) form arsenic trioxide has been successfully used to treat one blood cancer, but has so far proven too systemically toxic for use on solid tumors in the clinic. The As(V) form, arsenate, on the other hand, while much less systemically toxic upon bolus injection has also proven ineffective for cancer therapy. We extended the C-terminal ends of the human ferritin subunits with a tumor cell receptor targeting peptide and loaded this modified ferritin with ~ 800 arsenates and ~ 1100 irons. Our results demonstrate targeting and uptake of the iron, arsenate-loaded modified human ferritin by breast cancer cells. At the same arsenic levels, the cytotoxicity of the iron, arsenate-loaded human ferritin was equivalent to that of free arsenic trioxide and much greater than that of free arsenate. The iron-only loaded human ferritin was not cytotoxic at the highest achievable doses. The results are consistent with the receptor-targeted human ferritin delivering arsenate as a reductively activated 'prodrug'. This targeted delivery could be readily adapted to treat other types of solid tumor cancers.

Integrin αvß3 Receptor Overexpressing on Tumor-Targeted Positive MRI-Guided Chemotherapy.

Multifunctional nanomaterials with targeted imaging and chemotherapy have high demand with great challenge. Herein, we rationally aimed to design multifunctional drug delivery systems by RGD-modified chitosan (CH)-coated nanoneedles (NDs) of gadolinium arsenate (RGD-CH-Gd-AsNDs). These NDs have multifunctionality for imaging and targeted therapy. NDs on intravenous administration demonstrated significant accumulation of As ions/species in tumor tissues, which was monitored by the change in T1-weighted magnetic resonance (MR) imaging. Moreover, NDs were well opsonized in cells with high specificity, subsequently inducing apoptosis to the HepG2 cells. Consequent to this, the in vivo results demonstrated biosafety, enhanced tumor targeting, and tumor regression in a subcutaneously transplanted xenograft model in nude mice. These RGD-CH-Gd-AsNDs have great potential, and we anticipate that they could serve as a novel platform for real-time T1-weighted MR diagnosis and chemotherapy.

Ionomic responses of rice plants to the stresses of different arsenic species in hydroponics.

Different ionomic profiles of plants are associated with different external stresses to which they are exposed. Investigation of ionomic variation is necessary for understanding the migration and detoxification of toxic elements in plants. In the current study, rice plants were treated with arsenite, arsenate, monomethylarsonic acid and dimethylarsinic acid in hydroponics. The ionomic responses of the rice plants to different arsenic (As) species stresses were measured and analyzed. The multielement approach is more sensitive at detecting significant variations from external environmental stresses than the consideration of several individual elements. The pairs of significant correlations between elements varied based on the rice tissues and As species used in treatment, resulting in specific correlation networks. However, some pairs of correlations existed regardless of As species treatment used in this study. Positive correlations between P and Fe were observed in rice roots treated with any of the As species, implying that P and Fe share similar biological processes. The heatmap from hierarchical cluster analysis (HCA) agreed with the principal component analysis (PCA) results in ionomic differentiation between roots and shoots. Furthermore, ionomic differences between rice plants treated with different As species were identified through PCA. This study revealed that the ionomic profiles in rice plants are sufficient to detect responses to environmental perturbations. Association studies between ionomics and genomics are necessary to further understand the potential mechanisms that promote uptake or exclusion of elements in plants.

Characterization of arsenic resistant plant-growth promoting indigenous soil bacteria isolated from Center-East regions of India.

A total of 45 morphologically distinct arsenic (As)-resistant bacterial strains were isolated from the soils of different regions of Chhattisgarh, India. The minimum inhibitory concentration (MIC) values of these isolates varied widely in the range of 100-500 mM for arsenate [As(V)] and 15-30 mM for arsenite [As(III)]. Out of forty-five, three isolates viz; ARP3, ARRP3, and ADT5 also revealed plant growth-promoting properties, including phosphate solubilization and production of siderophores, indoleacetic acid, ammonia, and exopolysaccharide. Besides all these, the strains not only exhibited significant growth in the presence of As(V)/As(III) but also displayed higher efficiency (87%-94%) of As removal from the growth medium followed by intracellular accumulation (17-19 mg As/g). Hydride generation atomic absorption spectroscopic (HG-AAS) analysis revealed the intracellular accumulation of As, and the structural changes that took place in these isolates were further confirmed by microscopic studies. The 16 S rRNA and phylogenetic analyses unveiled that the isolates ARP3, ARRP3, and ADT5 belonged to genera Pseudomonas, Exiguobaterium, and Microbacterium, respectively. The conducted study suggested that such beneficial bacterial strains could be conveniently exploited at a commercial level for enhancing plant growth in As-contaminated agricultural fields, thereby improved productivity and enhanced bioremediation of soil having alarming strength of As.

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)].

Arsenate-mediated G2 cell cycle arrest in U-2OS cells involves phosphorylation of human polycomb protein 2 by p38 MAPK.

G2/M checkpoints ensure the proper timing of cell mitosis. We previously reported that p38 mitogen-activated protein kinase (MAPK) activation is essential for stress-induced G2 arrest in the U-2OS osteosarcoma cell line, but the molecular mechanism was obscure. Here, using the T7 phage display system, we find p38 directly binds to human polycomb protein 2 (HPC2), and arsenate-induced G2 arrest in U-2OS cell is p38- and phosphorylation of HPC2-dependent. Phosphorylation of HPC2 at threonine 495 is required for recruiting Ring1 and Rb family proteins to form the polycomb repressive complex (PRC), and PRC is required for arsenate-induced downregulation of CDC2 expression. Thus, p38 MAPK regulates cell cycle progression through phosphorylation of HPC2 to mediate transcriptional repression, providing a mechanistic link for arsenate-induced transcriptional silencing.

Transcriptomic comparison of two barley genotypes differing in arsenic tolerance exposed to arsenate and phosphate treatments.

Arsenic (As) is a ubiquitous metalloid and toxic to plants. Chemical similarity between arsenate and phosphate (P) indicates possible antagonism between them in uptake and transportation. However, there is little study to reveal the interaction of As and P at transcriptional level. In this study RNA-sequencing was conducted on the two barley genotypes differing in As tolerance. A total of 2942 differentially expressed genes (DEGs) were inclusively expressed in both genotypes under As (100 µM) and As (100 µM) + P (50 µM), and these DEGs included hormonal signaling, stress responsive, transport related and transcription factors. P addition in the culture solution inhibited the KEGG pathways related to ABC transporters, ether lipid metabolism, linolenic acid metabolism, endocytosis and RNA transport. ZDB160 had a higher expression of DEGs associated with hormone signaling, secondary metabolites and stress defense under P conditions compared to ZDB475, which might explain its tolerance mechanism to As under P condition. The abscisic acid, jasmonic acid and salicylic acid signaling pathways were also significantly regulated under As + P conditions, which may also account for genotypic differences. Finally we drew up a hypothetical model of high As + P stress tolerance mechanism in ZDB160. It may be concluded that ZDB160 achieves its tolerance to As under P by up-regulating P transporters, resulting in more P uptake and less As translocation. The identified candidate genes related to As + P tolerance may provide insights into understanding As tolerance under limited P conditions.

RiArsB and RiMT-11: Two novel genes induced by arsenate in arbuscular mycorrhiza.

Plants associated with arbuscular mycorrhizal fungi (AMF) increase their tolerance to arsenic-polluted soils. This study aims to investigate the genes involved in the AMF molecular response to arsenic pollution. Genes encoding proteins involved in arsenic metabolism were identified and their expression assessed by PCR or RT-qPCR. The As-inducible gene GiArsA (R. irregularis ABC ATPase component of the ArsAB arsenite efflux pump) and two new genes, an arsenate/arsenite permease component of ArsAB (RiArsB) and a methyltransferase type 11 (RiMT-11) were induced when arsenate was added to two-compartment in vitro monoxenic cultures of R. irregularis-transformed carrot roots. RiArsB and RiMT-11 expression in extraradical hyphae in response to arsenate displayed maximum induction 4-6 h after addition of 350 µM arsenate. Their expression was also detected in colonized root tissues grown in pots, or in the root-fungus compartment of two-compartment in vitro systems. We used a Medicago truncatula double mutant (mtpt4/mtpt8) to demonstrate that RiMT-11 and RiArsB transcripts accumulate in response to the addition of arsenate but not in response to phosphate. These results suggest that these genes respond to arsenate addition regardless of non-functional Pi symbiotic transport, and that RiMT-11 may be involved in arsenate detoxification by methylation in AMF-colonized tissues.

Arsenite exposure potentiates apoptosis-inducing effects of tumor necrosis factor-alpha- through reactive oxygen species.

Tumor necrosis factor-alpha (TNF-α) is a proinflammatory cytokine released by immune cells during inflammation process. Sodium arsenite (NaAsO2) is an environmental toxic metal. The effects of excess NaAsO2 on TNF-α response and its intracellular signaling are not well understood. We hypothesized that NaAsO2 exposure might affect cellular response to TNF-α. Using HeLa cell model, we found that the combination of NaAsO2 and TNF-α clearly decreased cell viability and mitochondrial membrane potential, but increased percentage of early and late apoptotic cells and cleaved-poly (ADP-ribose) polymerase (PARP). Moreover, the combination prolonged the phosphorylation of mitogen-activated protein kinase (MAPK) members, including c-Jun-N-terminal kinase (JNK), p38, and extracellular signal related kinases (ERK), and increased intracellular reactive oxygen species (ROS), in comparison to treatment of NaAsO2 or TNF-α alone. We further investigated the role of ROS and MAPK signaling on this event by inhibiting ROS production and MAPK. An antioxidant N-acetylcysteine pretreatment diminished the apoptosis-inducing effect of NaAsO2 and TNF-α combination and also inhibited MAPK signaling. Using specific inhibitor of p38 (SB203580) and siRNA-p38 surprisingly increased cell apoptosis and this effect was not observed by JNK and ERK inhibition. This study suggests that p38 may possibly be a survival mediator in response to environmental toxicant-related inflammation. In conclusion, NaAsO2 exposure might amplify inflammation-related tissue injury by potentiating the apoptosis-inducing effect of TNF-α through ROS-dependent mechanism.

Effects of Arsenic Compounds on Microminerals Content and Antioxidant Enzyme Activities in Rat Liver.

Interactions of arsenic with essential trace elements may result in disturbances on body homeostasis. In the present study, we aimed to investigate the effects of different arsenic compounds on micromineral content and antioxidant enzyme activities in rat liver. Male Wistar rats were randomly divided into five groups and exposed to sodium arsenite and sodium arsenate at 0.01 and 10 mg/L for 8 weeks in drinking water. The concentration of arsenic increased in the liver of all arsenic-exposed animals. The proportion of zinc and copper increased in animals exposed to 0.01 mg/L sodium arsenite. In addition, these animals presented a reduction in magnesium and sodium content. Superoxide dismutase activity decreased mainly in arsenite-exposed animals, whereas catalase activity decreased in animals exposed to 10 mg/L sodium arsenate. Further, exposure to sodium arsenate at 10 mg/L altered copper and magnesium content in the liver, and reduced total protein levels. Overall, both arsenic compounds altered the liver histology, with reduction in the proportion of cytoplasm and hepatocyte, and increased the percentage of sinusoidal capillaries and macrophages. In conclusion, our findings showed that oral exposure to arsenic compounds disturbs the trace elements balance in the liver, especially at low concentration, altering enzymatic and stereological parameters. We concluded that despite the increase in trace elements content, the antioxidant enzyme activities were downregulated and did not prevent morphological alterations in the liver of animals exposed to both arsenic compounds.

Evolution of copper arsenate resistance for enhanced enargite bioleaching using the extreme thermoacidophile Metallosphaera sedula.

Adaptive laboratory evolution (ALE) was employed to isolate arsenate and copper cross-resistant strains, from the copper-resistant M. sedula CuR1. The evolved strains, M. sedula ARS50-1 and M. sedula ARS50-2, contained 12 and 13 additional mutations, respectively, relative to M. sedula CuR1. Bioleaching capacity of a defined consortium (consisting of a naturally occurring strain and a genetically engineered copper sensitive strain) was increased by introduction of M. sedula ARS50-2, with 5.31 and 26.29% more copper recovered from enargite at a pulp density (PD) of 1 and 3% (w/v), respectively. M. sedula ARS50-2 arose as the predominant species and modulated the proportions of the other two strains after it had been introduced. Collectively, the higher Cu2+ resistance trait of M. sedula ARS50-2 resulted in a modulated microbial community structure, and consolidating enargite bioleaching especially at elevated PD.

Arsenic inorganic compounds cause oxidative stress mediated by the transcription factor PHO4 in Candida albicans.

Arsenic is a toxic metalloid widespread in nature. Recently, it has been demonstrated a main role of the transcription factor Pho4 in the acquisition of tolerance to arsenic-derived compounds, arsenite and arsenate in Candida albicans. Here, the effect of these compounds on this pathogenic yeast has been analyzed. In wild type cells, both arsenite and arsenate induced a marked increase in the endogenous production of Reactive Oxygen Species (ROS), together with the accumulation of intracellular trehalose and the activation of catalase, suggesting their role as generators of oxidative stress in this yeast. However, a pho4 null mutant showed a minor increase of intracellular ROS and a different kinetics of catalase activation upon exposure to arsenite and arsenate. Interestingly, the enzymatic activity of glutathione reductase and superoxide dismutase were exclusively triggered by arsenite but not by arsenate. pho4 mutant cells were also found to be sensitive to azide but significantly resistant to arsenate through a process dependent on an active electron transport chain and the alternative oxidase system. Therefore, arsenic-derived compounds induce a strong antioxidant response in C.albicans via different mechanisms.

Phosphorus deficiency modifies As translocation in the halophyte plant species Atriplex atacamensis.

Most arsenic in surface soil and water exists primarily in its oxidized form, as arsenate (As(V); AsO43-), which is an analog of phosphate (PO43-). Arsenate can be taken up by phosphate transporters. Atriplex atacamensis Phil. is native to northern Chile (Atacama Desert), and this species can cope with high As concentrations and low P availability in its natural environment. To determine the impact of P on As accumulation and tolerance in A. atacamensis, the plants were cultivated in a hydroponic system under four treatments: no As(V) addition with 323µM phosphate (control); 1000µM As(V) addition with 323µM phosphate; no As(V) and no phosphate; 1000µM As(V) addition and no phosphate. Phosphate starvation decreased shoot fresh weight, while As(V) addition reduced stem and root fresh weights. Arsenate addition decreased the P concentrations in both roots and leaves, but to a lesser extent than for P starvation. Phosphorus starvation increased the As concentrations in roots, but decreased it in shoots, which suggests that P deficiency reduced As translocation from roots to shoots. Arsenate addition increased total glutathione, but P deficiency decreased oxidized and reduced glutathione in As(V)-treated plants. Arsenate also induced an increase in S accumulation and nonprotein thiol and ethylene synthesis, and a decrease in K concentrations, effects that were similar for the P-supplied and P-starved plants. In contrast, in As(V)-treated plants, P starvation dramatically decreased total soluble protein content and increased lipid peroxidation, compared to plants supplied with P. Phosphorus nutrition thus appears to be an important component of A. atacamensis response to As toxicity.

Arsenic resistance genes of As-resistant purple nonsulfur bacteria isolated from As-contaminated sites for bioremediation application.

This study aimed to identify arsenic resistant mechanisms in As-resistant purple nonsulfur bacteria (PNSB) by screening them for presence of As-resistance genes and related enzymes. Resistance to As(III) and As(V) of four As-resistant PNSB determined in terms of median inhibition concentration (IC50 values) were in the order of strains Rhodopseudomonas palustris C1 > R. palustris AB3 > Rubrivivax benzoatilyticus C31 > R. palustris L28 which corresponded to the presence of As-resistance genes in these bacteria. The strain C1 showed all As-marker genes; arsC, arsM, aioA, and acr3, while aioA was not detected in strain AB3. Strains C31 and L28 had only Arsenite-transporter gene, acr3. Translation of all these detected gene sequences of strain C1 to amino acid sequences showed that these proteins have vicinal cysteine; Cys126, Cys105, and Cys178 of Acr3, ArsC, AioA, respectively. Tertiary structure of proteins Acr3, ArsC, AioA, and ArsM showed strain C1 exhibits the high activities of arsenite oxidase and arsenate reductase enzymes that are encoded by aioA and arsC genes, respectively. Moreover, strain C1 with arsM gene produced volatile-methylated As-compounds; monomethylarsonic acid (MMA), dimethylarsenic acid (DMA), and arsenobetaine (AsB) in the presence of either As(III) or As(V). In conclusion, the strain C1 has great potential for its application in bioremediation of As-contaminated sites.