Plasmid-determined resistance to arsenic and antimony inPseudomonas aeruginosa

Resistance to arsenic salts in aPseudomonas aeruginosa clinical isolate was shown to be determined by a 100 kb transferable plasmid. The resistance pattern included arsenate, arsenite, and antimonate ions. Arsenate and arsenite resistances were inducible by previous exposure of cultures to subinhibitory amounts of either of the two ions. Phosphate ions protectedP. aeruginosa cells from the toxic effects of arsenate but did not alter arsenite toxicity.     

A novel arsenate reductase from the bacterium Thermus thermophilus HB27: Its role in arsenic detoxification

Microorganisms living in arsenic-rich geothermal environments act on arsenic with different biochemical strategies, but the molecular mechanisms responsible for the resistance to the harmful effects of the metalloid have only partially been examined. In this study, we investigated the mechanisms of arsenic resistance in the thermophilic bacterium Thermus thermophilus HB27. This strain, originally isolated from a Japanese hot spring, exhibited tolerance to concentrations of arsenate and arsenite up to 20 mM and 15 mM, respectively; it owns in its genome a putative chromosomal arsenate reductase (TtarsC) gene encoding a protein homologous to the one well characterized from the plasmid pI258 of the Gram + bacterium Staphylococcus aureus. Differently from the majority of microorganisms, TtarsC is part of an operon including genes not related to arsenic resistance; qRT-PCR showed that its expression was four-fold increased when arsenate was added to the growth medium. The gene cloning and expression in Escherichia coli, followed by purification of the recombinant protein, proved that TtArsC was indeed a thioredoxin-coupled arsenate reductase with a k_cat/K_M value of 1.2 × 10⁴ M^− 1 s^− 1. It also exhibited weak phosphatase activity with a k_cat/K_M value of 2.7 × 10^− 4 M^− 1 s^− 1. The catalytic role of the first cysteine (Cys7) was ascertained by site-directed mutagenesis. These results identify TtArsC as an important component in the arsenic resistance in T. thermophilus giving the first structural–functional characterization of a thermophilic arsenate reductase.     

Low-level arsenite activates the transcription of genes involved in adipose differentiation

In this study we analyzed gene expression in 3T3-F442A pre-adipocyte cells that differentiate in the presence of micro-molar arsenate concentration. Two concentrations of arsenite (As2O3, 0.25 micromol/L and 0.5 micromol/L) were applied for three days with and without insulin (170 nmol/L) and gene expressions were evaluated by quantitative RT-PCR. The genes included genes of oxidative-stress responses: heme-oxygenase-1 (HO1) and the hypoxia inducible factor 1a (HIF1alpha), genes of cell-cycle: c-jun and Kruppel like factor 5 (KLF5), and genes that play important roles in adipose determination: a peroxisome proliferator-activated receptor (PPARgamma) and a CCAAT/ enhancer binding protein (C/EBPalpha). Arsenite induced the expression of HO1, HIF1alpha, KLF5, PPARgamma and C/EBPalpha. These results suggest that under condition of oxidative stress arsenite induces genes that are required for adipose differentiation.     

Effects of protein deficient diets on the developmental toxicity of inorganic arsenic in mice

BACKGROUND: Inorganic arsenic, when given by injection to pregnant laboratory animals (mice, rats, hamsters), has been shown to induce malformations. Arsenic methylation may be a detoxification step, and diets deficient in protein are a poor source of methyl donors and may possibly result in impaired arsenic methylation. Human health effects from chronic arsenic exposure have been reported mainly in populations with low socioeconomic status. Individuals in such populations are likely to suffer from malnutrition, which can compromise embryonic/fetal development and diminish arsenic methylating capacity. We sought to determine if dietary protein deficiency affects the developmental toxicity of inorganic arsenic. METHODS: Mated females were randomly assigned to one of 12 treatment groups. Experimental groups received either AsIII or AsV i.p. on Gestation Day 8 (GD 8, plug=GD 0) and were maintained on a 5%, 10%, or 20% protein custom mixed diet from GD 1 until sacrifice. Controls received the custom diets alone, were given AsIII or AsV i.p. on GD 8 with Teklad LM-485 rodent diet, or were fed the LM-485 diet alone. Test females were sacrificed on GD 17, and their litters were examined for mortality and developmental defects. RESULTS: Arsenic plus dietary protein deficiency decreased maternal weight gain and increased the incidences of exencephaly, ablepharia, and skeletal defects, such as malformed vertebral centra, fused ribs, and abnormal sternebrae (bipartite, rudimentary, or unossified). CONCLUSIONS: These results demonstrate that dietary protein deficiency enhances the developmental toxicity of inorganic arsenic, possibly by impairment of arsenic methylation.     

Arsenite oxidase,an ancient bioenergetic enzyme

Operons coding for the enzyme arsenite oxidase have been detected in the genomes from Archaea and Bacteria by Blast searches using the amino acid sequences of the respective enzyme characterized in two different beta-proteobacteria as templates. Sequence analyses show that in all these species, arsenite oxidase is transported over the cytoplasmic membrane via the tat system and most probably remains membrane attached by an N-terminal transmembrane helix of the Rieske subunit. The biochemical and biophysical data obtained for arsenite oxidase in the green filamentous bacterium Chloroflexus aurantiacus allow a structural model of the enzyme's membrane association to be proposed. Phylogenies for the two constituent subunits (i.e., the molybdopterin-containing and the Rieske subunit) of the heterodimeric enzyme and their respective homologs in DMSO-reductase, formate dehydrogenase, nitrate reductase, and the Rieske/cytb complexes were calculated from multiple sequence alignments. The obtained phylogenetic trees indicate an early origin of arsenite oxidase before the divergence of Archaea and Bacteria. Evolutionary implications of these phylogenies are discussed.     

Pretreatment with periodate-oxidized adenosine enhances developmental toxicity of inorganic arsenic in mice

BACKGROUND: Inorganic arsenic, given by injection to pregnant laboratory animals, can induce malformations. Arsenic methylation can be inhibited by periodate‐oxidized adenosine (PAD). Severe human health effects from high chronic arsenic exposure have mainly been reported in populations with significant levels of malnutrition, which may enhance toxicity by diminishing arsenic methylating capacity. This study sought to determine the effect of inhibition of arsenic methylation on the developmental toxicity of arsenic in a mammalian model. METHODS: PAD (100 µM/kg, i.p.), was given to pregnant CD‐1 strain mice 30min before 7.5mg/kg sodium arsenite [As(III)], i.p., or 17.9mg/kg sodium arsenate [As(V)], i.p., on gestation day 8 (GD 8; copulation plug=GD 0). Control dams received As(III), As(V), or PAD alone or were untreated. Test dams were killed on GD 17, and their litters were examined for mortality and gross and skeletal defects. RESULTS: Pretreatment with PAD before either arsenical resulted in increased maternal toxicity and lower fetal weights. Pretreatment also caused higher prenatal mortality, with 8 of 21 and 5 of 17 litters totally resorbed in the PAD plus As(III) and PAD plus As(V) treatment groups, respectively. Significant increases in the incidences of exencephaly, ablepharia, and anomalies of the vertebral centra, sternebrae, and ribs were also associated with PAD pretreatment. Short tail (3 fetuses in 3 litters) was seen only following PAD plus As(III) treatment. CONCLUSIONS: These results demonstrate that the developmental toxicity of inorganic arsenic can be enhanced by PAD, due possibly to inhibited methylation of arsenic. Birth Defects Res B 68:335–343, 2003. © 2003 Wiley‐Liss, Inc.     

Multiple cysteine residues are necessary for sorting and transport activity of the arsenite permease Acr3p from Saccharomyces cerevisiae

The yeast transporter Acr3p is a low affinity As(III)/H⁺ and Sb(III)/H⁺ antiporter located in the plasma membrane. It has been shown for bacterial Acr3 proteins that just a single cysteine residue, which is located in the middle of the fourth transmembrane region and conserved in all members of the Acr3 family, is essential for As(III) transport activity. Here, we report a systematic mutational analysis of all nine cysteine residues present in the Saccharomyces cerevisiae Acr3p. We found that mutagenesis of highly conserved Cys151 resulted in a complete loss of metalloid transport function. In addition, lack of Cys90 and Cys169, which are conserved in eukaryotic members of Acr3 family, impaired Acr3p trafficking to the plasma membrane and greatly reduced As(III) efflux, respectively. Mutagenesis of five other cysteines in Acr3p resulted in moderate reduction of As(III) transport capacities and sorting perturbations. Our data suggest that interaction of As(III) with multiple thiol groups in the yeast Acr3p may facilitate As(III) translocation across the plasma membrane.