High arsenic tolerance in Brevundimonas aurantiaca PFAB1 from an arsenic-rich Indian hot spring

BACKGROUND Arsenic contamination in the ground water of rural India is a recurrent problem and decon tamination is mostly based on the chemical or physical treatments until now. Microbial bioremediation is eco-friendly, cheap, time-efficient and does not produce any toxic by-products. RESULT In the present study, a high arsenic tolerant bacteria Brevundimonas aurantiaca PFAB1 was iso lated from Panifala hot spring located in West Bengal, India. Previously Panifala was also reported to be an arsenic-rich hot spring. B. aurantiaca PFAB1 exhibited both positive arsenic reductase and arsenite oxidase activity. It was tolerant to arsenite up to 90 mM and arsenate up to 310 mM. Electron microscopy has proved significant changes in cellular micromorphology and stalk appearance under the presence of arsenic in growth medium. Bioaccumulation of arsenic in As (III) treated cells were 0.01% of the total cell weight, while 0.43% in case of As (V) treatment. CONCLUSIONS All experimental lines of evidence prove the uptake/accumulation of arsenic within the bac terial cell. All these features will help in the exploitation of B. aurantiaca PFAB1 as a potent biological weapon to fight arsenic toxicity in the near future

Determination of arsenic species in serum, liver, kidney, and spleen of realgar nanoparticles treated rats by HPLC-ICP-MS / 中草药

Objective Quantitative analysis of four arsenic species As (III), As (V), monomethyl arsenate (MMA), dimethyl arsenate (DMA) in rat serum, liver, kidney, and spleen was performed to compare their differences between realgar and realgar nanoparticles (NPs) groups. Methods SD rats were ig treated with blank solvent, realgar, and realgar NPs (800 mg/kg) respectively. After 28 d of continuous administration, serum and tissues were collected and four arsenic species were determined by high performance liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICP-MS). Results Four arsenic species were detected in serum and kidney of rats, three were detected in the liver and two in the spleen. The content of arsenic species in the realgar NPs group was significantly higher than that in the realgar group. Conclusion Nanotechnology enhanced the bioavailability of realgar, and more arsenic was absorbed into the body and underwent metabolic transformation, which might lead to increased toxicity of realgar NPs.

Combined effects of fluoride and arsenic exposure on proliferation, differentiation and beta-catenin expression in rat osteoblasts / 中华地方病学杂志

Objective To investigate the combined effects of fluoride (NaF) and arsenate (NaAsO2) exposure on proliferation,differentiation and bata-catenin expression in SD rat osteoblasts.Methods Osteoblasts were isolated from calvarias of twelve SD rats born in 1-3 days and cultured.The method was divided into 9 groups [F0.0As0.0 (control group),F0.5As0.0,F4.0As0.0,F0.0As0.1,F0.0As10.0,F0.5As0.1,F0.5As10.0,F4.0As0.1,F4.0As10.0] by factorial experiment design (3 factors and 2 levels).Osteoblasts were exposed to NaF (F-:0.0,0.5,4.0 mmol/L,F0.0,F0.5,F4.0),NaAsO2 (As3+:0.0,0.1,10.0 μmol/L,As0.0,As0.1,Asi10.0) and cultured for 72 hours.The proliferation and alkaline phosphatase (ALP) was determined by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl (MTT) and Enzyme-linked immunosorbent assay (ELISA).The expression of beta-catenin was analyzed by quantitative real-time PCR (qPCR) and Western blotting after 72 hours of experiment.Results ①There was significant difference in cell proliferation and the activity of ALP among groups after 72 hours (F =14.022,14.425,all P ＜ 0.05).Compared with control group,the proliferation and the activity of ALP were significantly induced in F0.5 treated osteoblasts groups (0.313 ±0.023 vs0.455 ± 0.152,4.46 ± 0.72 vs 6.09 ± 0.68,all P ＜ 0.05),and the proliferation was significantly suppressed in F4.0As0.0 group (0.029 ± 0.014,P ＜ 0.05),the activity of ALP was significantly induced in F0.0As10.0 group (0.156 ± 0.010,6.29 ± 0.67) and the proliferation was significantly suppressed in F0.0As0.1 group (0.370 ± 0.029,3.68 ± 0.45,all P ＜ 0.05).②There was statistical difference in beta-catenin mRNA and protein expressions among groups with F-(F =7.782,559.455,all P ＜ 0.05) at As0.0 condition.There was significant difference in the expression of betacatenin mRNA and beta-catenin protein in F0.5As0.0 group compared with control group (1.00 ± 0.32 vs 1.99 ± 0.14,3.56 ± 0.15 vs 5.11 ± 0.26,all P ＜ 0.05),the beta-catenin protein was significantly suppressed in F4.0As0.0 group (1.10 ± 0.02,P ＜ 0.05).There was significant difference in the expression of beta-catenin protein of all groups with As3+ (F =154.736,P ＜ 0.05) at F0.0 condition.③Factorial analysis showed that fluoride or arsenic alone could affect the proliferation and the expression level of beta-catenin mRNA and protein (F =82.081,11.991,514.741;19.302,8.753,523.698,all P ＜ 0.05),the effect of arsenic on ALP activity of osteoblasts was also the main effect (F =17.444,P ＜ 0.05);and there was an interaction between fluoride or arsenic to cell proliferation and the activity of ALP and the expression of beta-catenin mRNA and protein (F =13.085,18.157,4.936,426.036,all P ＜ 0.05).Conclusions A biphasic pattern of fluoride or arsenic on proliferation and differentiation has been induced in SD rat osteoblasts.Fluoride or arsenic can affect bone metabolic by beta-catenin.

A relationship between arsenite sodium, arsenate sodium metabolites and related metabolic enzymes in rat kidney / 中华地方病学杂志

Objective To investigate the relationship between metabolites of sodium arsenite and sodium dihydrogen arsenate with related metabolic enzymes in kidney of male rats.Methods According to body mass,thirty-five male Wistar rats(body mass 150-190 g) were divided into 7 groups by random number table.Control group drank deionized water; the contents of iAsⅢ in low,medium and high arsenite groups and the contents of iAsv in low,medium and high of sodium dihydrogen arsenate groups were 2.2,6.7 and 20.0 mg/kg,respectively.After 3 months,kidneys were collected and stored at-80 C; high performance liquid chromatography and hydride genesis atomic fluorescence spectroscopy (HPLC-HGAFS) was used to determine the level of arsenic metabolites in kidney,and enzyme-linked immunosorbent assay was used to detect and analyze the content or the activity of metabolic enzymes,meanwhile correlation studies between the level of metabolites and the activity of metabolic enzymes were carried out.Results The differences of total arsenic (TAs),dimethyl arsenic acid (DMA),monomethyl arsenic acid (MMA) and methyl transferase enzyme activity in kidneys of rats between groups were statistically significant (F =1874.672,H =33.513,31.002,F =79.607,all P ＜ 0.01).The TAs[(526.52 ± 25.56),(1 654.00 ± 101.55),(1 904.24 ± 104.76)μg,/kg] and DMA[(323.20 + 16.13),(1 444.40 ± 113.81),(1 765.40 ± 104.39)μg/kg] of sodium arsenite in low,medium and high dose groups were higher than those of the corresponding sodium dihydrogen arsenate groups [(235.70 ± 6.23),(471.05 ± 18.32),(1 677.40 ± 83.29)μg/kg,and(0.00 ± 0.00),(1.75 ± 0.16),(410.50 ± 19.76)μg/kg,P ＜ 0.0024 or ＜ 0.05] ; the MMA[(4.02 + 0.86),(4.20 ± 0.65),(4.04 ± 0.80)μg/kg] of sodium arsenite in low,medium and high dose groups were lower than those of the corresponding sodium dihydrogen arsenate groups[(98.90 ± 9.59),(376.50 ± 15.41),(1 131.90 ± 74.26) μg/kg,all P＜ 0.05]; the methyl transferase enzyme activities[(7.80 ± 0.93),(5.55 ± 0.49),(3.56 ± 0.26)U/g] of sodium arsenite in low,medium and high dose groups were lower than those of the corresponding sodium dihydrogen arsenate group[(11.59 ± 0.93),(8.93 ± 0.88),(6.52 ± 1.04)U/g,all P ＜ 0.0024].The DMA of sodium arsenite in low,medium and high dose groups,the MMA of sodium dihydrogen arsenate in medium and high dose groups were positively correlated with those of TAs in each group(r =0.970,0.984,0.997,0.947,0.961,all P ＜ 0.05).Conclusions Effects of sodium arsenite and sodium dihydrogen arsenate on arsenic metobdites and related metabolic enzymes in kidney of rats are different.The function of sodium dihydrogen arsenate in promoting methyl transferase activity is stronger than that of sodium arsenite,which affects the amount and distribution of arsenic methylation metabolites in kidney.

Controversy over the report on a bacterium that feeds on arsenic.

Arsenic (As), the metalloid, has been traditionally infamous for its toxicity. Because of its use by rulers to kill their rivals, it was once known as the poison of the kings or the king of poisons. It was also widely used as an agricultural insecticide. It kills humans quickly if consumed in large quantity. Also, regular small doses of arsenic cause various illnesses, such as patchy skin, digestive system disorders, peripheral neuritis, hepatic lesions and fatty degeneration of the heart, and other lifethreatening complications. Exposure to arsenic could be due to both natural and anthropogenic factors. In nature, arsenic occurs in four oxidation states: As(V), As(III), As(0) and As(−III). Occurrence of the highest oxidation states is more common and that of the two lowest oxidation states is rare. In aqueous aerobic environments, arsenate (AsO4 −3), the pentavalent form, predominates. It tends to be strongly adsorbed onto common minerals (e.g. alumina). Arsenite (AsO3 −3), the trivalent form, is more prevalent in anoxic environments and is substantially more toxic than arsenate. By virtue of its ability to bind sulphydryl groups, arsenic binds and inactivates enzymes. It inhibits the enzyme pyruvate dehydrogenase, essential for oxidation of pyruvate to acetyl-Co-A. In the process, it triggers cellular apoptosis. It also binds dithiols such as glutaredoxin and stimulates the production of hydrogen peroxide, thus leading to increase in oxidative stress. Inorganic arsenic trioxide, found in groundwater, affects voltage-gated potassium channels, thus creating neurological disturbances. This metabolic interference ultimately leads to death due to multiorgan failure. In the periodic table, arsenic is positioned just below phosphorus. Because of their similarity in electronegativity and radii, arsenate ion can replace phosphate in biological reactions. Thus, it can enter the early stages of metabolism, but fails to continue metabolism because of the rapid hydrolysis of the arsenate esters, compared with that of the phosphate esters. The hydrolysis of diesters is faster than that of triesters (Westheimer 1987). Arsenate esters are labile even if very low concentration of water is present. Presence of 0.5% water allows a half-life of less than 0.1 s at pH 9.0. Compared with phosphodiester-containing DNA, which has a half-life of 3×107 years, arsenodiestercontaining DNA spontaneously hydrolyses with an estimated half-life of 0.06 s at 25°C (Fekry et al. 2011). Analysis of arsenic-rich water and soil, sampled from different places, have so far revealed the presence of several types of bacteria belonging to the genera Acidithiobacillus, Bacillus, Deinococcus, Desulfitobacterium and Pseudomonas (Shivaji et al. 2005 and the references therein). These bacteria can tolerate arsenate in the presence of phosphate. The genes responsible for arsenic tolerance in these organisms are organized in an operon, called ars operon. Some bacteria can use arsenic as an electron donor; others can methylate inorganic arsenic or demethylate organic arsenic compounds. Bacillus macyae is an example of arsenate-respiring bacteria (Páez-Espino et al. 2009). However, until a couple of months ago, no bacterium was found to tolerate arsenic in absence of phosphorus. In fact, no form of life that could utilize arsenic was known to occur. In 2009, Felisa Lauren Wolfe-Simon, a NASA research fellow at the US Geological Survey, and her two colleagues postulated for the first time that arsenic might substitute phosphorus in ancient living systems (Wolfe-Simon et al. 2009). The postulation failed to evoke any response from the scientific community, the main reason being the instability of arsenate esters. Wolfe-Simon, however, stuck to her idea, and in order to pursue it, she collected mud samples from Mono Lake, a salty water body in California with high arsenic content. She started enriching it with bacterial growth medium containing carbon source, vitamins and trace metals with increasing concentrations of arsenate but no phosphorus. Several decimal-dilution transfers were performed to reduce any carryover from the phosphorus indigenous to the sample. Ultimately she reached a stage when the amount of phosphorus present in the medium (3.1 μM) was not sufficient to allow growth of any bacterium. Hence, she was highly surprised when she saw some organisms in the culture medium examined under a microscope. The isolate (named GFAJ-1) belonged to the Halomonadaceae family of γ-Proteobacteria, which could be maintained aerobically with 40 mM arsenate, 10 mM glucose and no added phosphorus. When grown in the presence of phosphorus, GFAJ-1 utilized 30-fold more phosphate compared with the same organism grown in the +As/−P condition. Inductively coupled plasma mass spectrometry (ICPMS) revealed very little intracellular phosphorus in the +As/-P condition. Hence, it appeared that arsenate did not replace the requirement for phosphate when phosphate was present, but it could do so in a phosphate-deprived environment. Distribution of arsenate in the cell, determined using radiolabelled arsenate (73AsO4 3−) in the medium, was consistent with the distribution of phosphate determined earlier. High-resolution secondary ion mass spectrometry (Nano-SIMS) of genomic DNA extracted from the organism showed higher concentration of arsenic in +As/−P DNA and higher phosphorus in −As/+P DNA. Evidence obtained from micro–X-ray absorption near-edge spectroscopy (μXANES) and micro–extended X-ray absorption fine-structure spectroscopy (μEXAFS) indicated that intracellular arsenic was in the +5 redox state. The bond lengths of arsenic with oxygen and carbon atoms did not match with models of small arsenical molecules, but matched with the crystal structure of DNA for the analogous structural position of phosphorus with respect to oxygen and carbon. Cellular ion ratios of 73As− :12C− and 31P− :12C−, determined by Nano- SIMS, confirmed the distribution of arsenic with carbon inside the cell. Besides replacing phosphorus in DNA, arsenic was also reported to be assimilated into proteins and other metabolites. The intracellular volume of the bacterium was found to increase 1.5-fold when it was grown on arsenic as compared with that observed in the phosphorus-containing medium. Large internal compartments were also observed under a transmission electron microscope. It was postulated that these vacuole-like regions were rich in poly-β-hydroxybutyrate, which stabilized the distribution of arsenate. The postulation was bolstered by the fact that in non-aqueous environments, hydrolysis of arsenate compounds is retarded. Despite being silent about the mechanism of incorporation of arsenic in the biomolecules, their’s is the first report of an organism that not only tolerates arsenic but also incorporates it into its cell. Unlike some other organisms that use arsenic compounds as terminal electron donor, GFAJ-1 could sustain and grow in the presence of arsenic and in the absence of phosphorus (Wolfe-Simon et al. 2010). This paper, which redefines the chemistry of life, has created a tumult. Appreciation as well disbelief has poured in through Twitter and journals. Some scientists such as Rosie Redfield of the University of British Columbia have written to Science, pointing out discrepancies in the work. Among several snags underscored by the scientific community, the purity of the genomic DNA has been called into question by many investigators. Wolfe-Simon and her team-mate Ronald Oremland have clarified that the isolated DNA passed thrice through phenol-chloroform and so it was expected to be pure. Dr Alex Bradley, a geochemist and microbiologist, has opined that the DNA could not contain arsenic as it passed through the aqueous phase during purification. He is also not convinced with Wolfe-Simon’s hypothesis on stabilization of arsenic-containing DNA as he believes that such a system could not function in the absence of proteins, which were removed during purification. In reply, Wolfe-Simon published her data on the kinetics of hydrolysis of arsenic compounds and demonstrated that arsenic compounds with longer carbon chains get hydrolysed slower than those with shorter ones. Bradley has also mentioned that bacteria survive in the Sargasso Sea, where the amount of phosphate present (10 nM) is far less than the contaminating phosphate present in the medium of GFAJ-1. It has been pointed out by Tawfik and Viola (2011) that arsenate is a good substrate for L-aspartate-β-semialdehyde dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase in terms of substrate affinity and catalytic efficiency, but the products are unstable. The kinetic instability in arsenic-containing DNA, as pointed out earlier, makes the concept of arsenic-dependent life unacceptable to many scientists as DNA is a biomolecule that maintains the vital genetic information through generations (Fekry et al. 2011). Besides Mono Lake, arsenic is known to occur in high concentration in some other environmental niches including terrestrial and deep-sea hydrothermal systems. Organoarsenicals (e.g, trimethyl- and dimethylarsinates), arsenosugars and arsenolipids have also been found in marine organisms. Therefore, evolutionarily speaking, one can envisage a scenario with the existence of primordial arsenate-based organisms in arsenate-rich environments under dysoxic conditions and at a neutral to slightly alkaline pH, in which arsenate and arsenite are thermodynamically stable. Primordial organisms with phosphate-based biology perhaps took over the ecosystems subsequently under aerobic conditions, restricting the arsenateutilizing organisms to only few environments, which have remained unexplored or have not been carefully examined so far. Regarding the future scope of this research, Jennifer Pett-Ridge, one of the co-authors of the disputed paper, said ‘The team hasn’t yet established how

Advance on Studies of Arsenic-resistant Microorganisms and Molecular Mechanisms / 微生物学通报

Arsenic is known as a toxic metalloid, which mainly exists in inorganic forms such as arsenite and arsenate in the natural environment. A number of microorganisms have evolved different resistant mechanisms for arsenic detoxification to cope with the widespread distribution of the poisonous arsenic. Four distinct microbial arsenic-resistant mechanisms have been described including As(III) oxidation, cytoplasmic As(V) reduction, respiratory As(V) reduction, and As(III) methylation. These mechanisms confer arsenic resistance in microorganisms that play important roles in the transformation and geological cycle of arsenic. This review mainly focuses on the researches on these molecular mechanisms and potential application for environmental arsenic bioremediation using microorganisms.

Inorganic Arsenics Have Different Actions in the Induction of DNA Strand Breaks in Primary Cultured Human Skin Fibroblasts / 环境与健康杂志

Objective To investigate the action of inorganic arsenic in the induction of DNA strand breaks in primary cultured human skin fibroblasts. Methods Sodium arsenate and sodium arsenite were used as test inorganic arsenics. DNA strand breaks were assessed by single cell gel electrophoresis assay (SCGE). Results Arsenate at 1～10 ?mol/L dose_dependently induced DNA strand breaks in cells. Arsenate at lower concentrations induced mainly degree I DNA strand breaks, while the proportion of cells with degree Ⅱ DNA strand breaks increased to 50% when treated with 10?mol/L arsenate,but no cells with degree ⅢDNA strand breaks were observed.Cells treated with 1?mol/L arsenite showed no significant increase in DNA strand breaks. At the concentration of 10?mol/L, however, arsenite induced DNA strand breaks with different degrees, and the apoptotic type DNA strand breaks were the major type. Conclusion Sodium arsenate mainly induced general type DNA strand breaks and sodium arsenite induced apoptotic type,beside general type of DNA strand breaks in primary cultured human skin fibroblasts. This could be explained by their different reaction modes with DNA, and further studies were needed.