Detoxification Of Heavy Metal Ions Research Articles

Pxr- and Nrf2- mediated induction of ABC transporters by heavy metal ions in zebrafish embryos

Transcription factors including pregnane X receptor (Pxr) and nuclear factor-erythroid 2-related factor-2 (Nrf2) are important modulators of Adenosine triphosphate-binding cassette (ABC) transporters in mammalian cells. However, whether such modulation is conserved in zebrafish embryos remains largely unknown. In this manuscript, pxr- and nrf2-deficient models were constructed with CRISPR/Cas9 system, to evaluate the individual function of Pxr and Nrf2 in the regulation of ABC transporters and detoxification of heavy metal ions like Cd2+ and Ag+. As a result, both Cd2+ and Ag+ conferred extensive interactions with ABC transporters in wild type (WT) embryos: their accumulation and toxicity were affected by the activity of ABC transporters, and they significantly induced the mRNA expressions of ABC transporters. These induction effects were reduced by the mutation of pxr and nrf2, but elevations in the basal expression of ABC transporters compensated for the loss of their inducibility. This could be an explanation for remaining transporter function in both mutant models as well as the unaltered toxicity of metal ions in pxr-deficient embryos. However, mutation of nrf2 disrupted the production of glutathione (GSH), resulting in the enhanced toxicity of Cd2+/Ag+ in zebrafish embryos. In addition, elevated expressions of other transcription factors like aryl hydrocarbon receptor (ahr) 1b, peroxisome proliferator-activated receptor (ppar)-β, and nrf2 were found in pxr-deficient models without any treatment, while enhanced induction of ahr1b, ppar-β and pxr could only be seen in nrf2-deficient embryos after the treatment of metal ions, indicating different compensation phenomena for the absence of transcription factors. After all, pxr-deficient and nrf2-deficient zebrafish embryos are useful tools in the functional investigation of Pxr and Nrf2 in the early life stages of aquatic organisms. However, the compensatory mechanisms should be taken into consideration when interpreting the results and need in-depth investigations.

Functional Characterization of a Type 4 Metallothionein Gene (CsMT4) in Cucumber

The excess amounts of heavy metals are toxic to various physiological processes in plants. Plant metallothioneins (MTs) are low molecular weight, cysteine-rich metal-binding proteins that play important roles in the detoxification of heavy metal ions. In this study, we characterized CsMT4, a plant type 4 MT gene identified in a complementary DNA (cDNA) library prepared from young cucumber (Cucumis sativus) fruit. CsMT4 encodes a 90 amino acid protein with a predicted molecular mass of 9.028 kD. CsMT4 contains 17 cysteine residues in three highly conserved cysteine-rich domains. In contrast to the structures of other MTs, the highly conserved amino acid pattern CxCxxxCxCxxCxC is present in the middle of CsMT4. Furthermore, CsMT4 was markedly induced in various tissues by various concentrations of Cd2+ and Zn2+ (> 1.0 mmol•L−1). Similar to AtMT4b function, heterologous expression of CsMT4 in E. coli could also improved its tolerance to Cd2+ and led to increased uptake of Cd2+, and the rate of Cd2+ uptake was the highest in cells expressing a phytochelatin-like peptide. Our findings demonstrate that CsMT4 might be obviously induced by the metal stress in cucumber, and improves tolerance to Cd ions but not Zn ions when heterologously expressed in E. coli, and suggest that the composition and arrangement of N-terminal Cys-residues in MT4 are associated with their binding capacity and preference for different metal ions.

Complete genome sequence of Planococcus sp. PAMC21323 isolated from Antarctica and its metabolic potential to detoxify pollutants

The Planococcus sp. PAMC21323 is a yellow pigment-producing bacterium isolated from King George Island in Antarctica; it has a broad growth temperature range of 5–40 °C. Herein, we describe the complete genome sequence information of the genus Planococcus with its annotated sequence, genetic features for bioremediation, and oxidative stress capacity. The Planococcus sp. PAMC21323 possesses chromosomal DNA (3,196,500-bp) with plasmid DNA (3364-bp). The complete 3,199,864-bp of the genome consists of 3171 genes including 60 transfer RNAs and 24 ribosomal RNAs. Strain PAMC21323 encodes various genes associated with detoxification of heavy metal ions and aromatic hydrocarbons. Moreover, it is equipped with diverse stress response systems, which can be used to sense the internal and oxidative stresses caused by detoxification. This is the first report highlighting the genetic potential of Planococcus sp. PAMC21323 in bioremediation, suggesting application of this psychrotrophic strain in bioremediation in harsh environments.

The cadmium tolerance in Saccharomyces cerevisiae depends on inorganic polyphosphate.

The sensitivity to cadmium (Cd(II)), an important environmental pollutant, was studied in the cells of Saccharomyces cerevisiae strains with genetically altered polyphosphate metabolism. The strains overproducing polyphosphatases PPX1 or PPN1 were more sensitive to Cd(II) than the parent strain. The half maximal inhibitory concentrations were 0.02 and 0.05 mM for the transformants and the parent strain, respectively. Transformant strains cultivated in the presence of Cd(II) show a decrease in the content of short-chained cytosolic acid soluble polyphosphate. The role of this polyphosphate fraction in detoxification of heavy metal ions is discussed.

Characterization of a Type 1 Metallothionein Gene from the Stresses-Tolerant Plant Ziziphus jujuba.

Plant metallothioneins (MTs) are a family of low molecular weight, cysteine-rich, and metal-binding proteins, which play an important role in the detoxification of heavy metal ions, osmotic stresses, and hormone treatment. Sequence analysis revealed that the open-reading frame (ORF) of ZjMT was 225 bp, which encodes a protein composed of 75 amino acid residues with a calculated molecular mass of 7.376 kDa and a predicated isoelectric point (pI) of 4.83. ZjMT belongs to the type I MT, which consists of two highly conserved cysteine-rich terminal domains linked by a cysteine free region. Our studies showed that ZjMT was primarily localized in the cytoplasm and the nucleus of cells and ZjMT expression was up-regulated by NaCl, CdCl2 and polyethylene glycol (PEG) treatments. Constitutive expression of ZjMT in wild type Arabidopsis plants enhanced their tolerance to NaCl stress during the germination stage. Compared with the wild type, transgenic plants accumulate more Cd2+ in root, but less in leaf, suggesting that ZjMT may have a function in Cd2+ retension in roots and, therefore, decrease the toxicity of Cd2+.

Carbon nanotubes and graphene modified screen-printed carbon electrodes as sensitive sensors for the determination of phytochelatins in plants using liquid chromatography with amperometric detection

Nanomaterials are of great interest for the development of electrochemical sensors. Multi-walled carbon nanotubes and graphene were used to modify the working electrode surface of different screen-printed carbon electrodes (SPCE) with the aim of improving the sensitivity of the SPCE and comparing it with the conventional glassy carbon electrode. To assay the usability of these sensors, a HPLC methodology with amperometric detection was developed to analyze several phytochelatins in plants of Hordeum vulgare and Glycine max treated with Hg(II) or Cd(II) giving detection limits in the low μmolL−1 range.Phytochelatins are low molecular weight peptides with the general structure γ-(Glu-Cys)n-Gly (n=2–5) which are synthesized in plants in the presence of heavy metal ions. These compounds can chelate heavy metal ions by the formation of complexes which, are transported to the vacuoles, where the toxicity is not threatening. For this reason phytochelatins are essential in the detoxification of heavy metal ions in plants.The developed HPLC method uses a mobile phase of 1% of formic acid in water with KNO3 or NaCl (pH=2.00) and 1% of formic acid in acetonitrile. Electrochemical detection at different carbon-based electrodes was used. Among the sensors tested, the conventional glassy carbon electrode offers the best sensitivity although modification improves the sensitivity of the SPCE. Glutathione and several isoforms of phytochelatin two were found in plant extracts of both studied species.

Phytochelatin synthase: of a protease a peptide polymerase made

Of the mechanisms known to protect vascular plants and some algae, fungi and invertebrates from the toxic effects of non-essential heavy metals such as As, Cd or Hg, one of the most sophisticated is the enzyme-catalyzed synthesis of phytochelatins (PCs). PCs, (γ-Glu-Cys)(n) Gly polymers, which serve as high-affinity, thiol-rich cellular chelators and contribute to the detoxification of heavy metal ions, are derived from glutathione (GSH; γ-Glu-Cys-Gly) and related thiols in a reaction catalyzed by phytochelatin synthases (PC synthases, EC 2.3.2.15). Using the enzyme from Arabidopsis thaliana (AtPCS1) as a model, the reasoning and experiments behind the conclusion that PC synthases are novel papain-like Cys protease superfamily members are presented. The status of S-substituted GSH derivatives as generic PC synthase substrates and the sufficiency of the N-terminal domain of the enzyme from eukaryotic and its half-size equivalents from prokaryotic sources, for net PC synthesis and deglycylation of GSH and its derivatives, respectively, are emphasized. The question of the common need or needs met by PC synthases and their homologs is discussed. Of the schemes proposed to account for the combined protease and peptide polymerase capabilities of the eukaryotic enzymes vs the limited protease capabilities of the prokaryotic enzymes, two that will be considered are the storage and homeostasis of essential heavy metals in eukaryotes and the metabolism of S-substituted GSH derivatives in both eukaryotes and prokaryotes.

Functional comparison of metallothioneins MTT1 and MTT2 from Tetrahymena thermophila

Metallothioneins MTT1 and MTT2 from Tetrahymena thermophila have been characterized. The MTT1 contains mainly characteristic Cys-Cys-Cys and Cys-Cys clusters, but MTT2 contains mainly Cys-X-Cys cluster. Cd 16-MTT1 mainly consists of α-helix and β-turns, in contrast, Cd 11-MTT2 mainly consists of random coils. Reaction of Cd 16-MTT1 and Cd 11-MTT2 with nitric oxide leads to intramolecular disulfide bond formation, respectively. Binding stabilities of Cd 2+, Hg 2+ and Zn 2+ to MTT1 are stronger than those to MTT2. Cu 2+ can not replace Cd 2+ from Cd 16-MTT1 complex, but can replace Cd 2+ from Cd 11-MTT2 complex. The analysis of qRT-PCR revealed MTT2 mRNA levels were 31-fold higher than those of MTT1 under basal conditions. These results further suggest MTT1 possibly play a role in the detoxification of heavy metal ions, and MTT2 may be involved in the homeostasis of copper ions.

Structural and Metal Binding Characterization of the C-Terminal Metallochaperone Domain of Membrane Fusion Protein SilB from Cupriavidus metallidurans CH34

Detoxification of heavy metal ions in Proteobacteria is tightly controlled by various systems regulating their sequestration and transport. In Cupriavidus metallidurans CH34, a model organism for heavy metal resistance studies, the sil determinant is potentially involved in the efflux of silver and copper ions. Proteins SilA, SilB, and SilC form a resistance nodulation cell division (RND)-based transport system in which SilB is the periplasmic adaptor protein belonging to the membrane fusion protein (MFP) family. In addition to the four domains typical of known MFPs, SilB has a fifth additional C-terminal domain, called SilB(440-521), which is characterized here. Structure and backbone dynamics of SilB(440-521) have been investigated using nuclear magnetic resonance, and the residues of the metal site were identified from (15)N- and (13)C-edited HSQC spectra. The solution structure and additional metal binding experiments demonstrated that this C-terminal domain folds independently of the rest of the protein and has a conformation and a Ag(+) and Cu(+) binding specificity similar to those determined for CusF from Escherichia coli. The small protein CusF plays a role in metal trafficking in the periplasm. The similarity with CusF suggests a potential metallochaperone role for SilB(440-521) that is discussed in the context of simultaneous expression of different determinants involved in copper resistance in C. metallidurans CH34.

HbMT2, an ethephon-induced metallothionein gene from Hevea brasiliensis responds to H2O2 stress

Metallothioneins (MTs) are the cysteine-rich proteins with low molecular weight, which play important roles in maintaining intracellular ion homeostasis, detoxification of heavy metal ions and protecting against intracellular oxidative damages. In this study a novel ethephon-induced metallothionein gene, designated as HbMT2, was isolated and characterized from Hevea brasiliensis. The HbMT2 cDNA contained a 237 bp open reading frame encoding 78 amino acids and the deduced protein showed high similarity to the type 2 MTs from other plant species. Expression analysis revealed more significant accumulation of HbMT2 transcripts in leaves and latex than in roots and barks. The transcription of HbMT2 in latex was strongly induced by ethephon and hydrogen peroxide (H(2)O(2)) stress. Overproduction of recombinant HbMT2 protein gave the Escherichia coli cells more tolerance on Cu(2+) and Zn(2+), and the recombinant HbMT2 could scavenge the reactive oxidant species (ROS) in vitro. All these results indicated that HbMT2 could respond to ethephon stimulation and H(2)O(2) stress as a ROS scavenger in H. brasiliensis. It is also suggested that HbMT2 function in improving the tolerance of rubber trees to heavy metal ions, and repressing the ethephon-induced senilism and tapping panel dryness (TPD) development by ROS scavenge system in H. brasiliensis.

Plant MTs—long neglected members of the metallothionein superfamily

Occurrence of metallothioneins (MTs) was initially thought to be restricted to the animal kingdom, and the corresponding functions such as detoxification of heavy metal ions were assumed to be taken over in plants by the enzymatically synthesized phytochelatins. This perception was revised in the past years, and the existence of plant metallothioneins is generally accepted. Compared to the vertebrate forms, members of the plant MT family display a significantly larger sequence diversity, however, surprisingly little information is available concerning their possible functions, properties, and structures. Gene expression studies, and thus studies on the mRNA level, are the major source of data aiming at elucidating the function of plant MTs. However, so far it is not possible to unambiguously assign a specific function to a given metallothionein as proposed functions overlap, are complementary to each other, or even contradictory results are obtained. With respect to the structures and properties of plant metallothioneins even less scientific contributions are available illustrating the early stages, in which this research area resides. Existing data covers the metal ion content of the different plant metallothionein species and the pH stabilities of the resulting metal-thiolate clusters. Further, for a limited selection of proteins the number of clusters formed has been proposed and predictions towards the secondary structure of the protein backbone made. A recently determined three-dimensional structure of the larger domain of the wheat metallothionein E(c)-1 describes a metal ion coordination mode unprecedented for any metallothionein so far.

Enhancing the tolerance of zebrafish ( Danio rerio) to heavy metal toxicity by the expression of plant phytochelatin synthase

Phytochelatin synthase (PC synthase) catalyzes a biosynthesis of phytochelatins (PCs), which are small molecules and glutathione (GSH)-derived metal-binding peptides that are essential for the detoxification of heavy metal ions in plants, fungi and worms. In order to enhance tolerance to heavy metal cytotoxicity, mRNA coding for PC synthase from Arabidopsis thaliana ( AtPCS1) was introduced into the early embryos of zebrafish. As a result, the heterogeneous expression of PC synthase and the synthesis of PCs from GSH in embryos could be detected. The developing embryos expressing PC synthase (PC-embryos) became more tolerant to Cd toxicity (500 μM exposure). PC-embryos had significantly longer apparent lethal times for 50% of the population (LT50) of 8.17 ± 1.08 days, although control embryos had apparent LT50 of 5.43 ± 0.66 days. These data suggest that PC synthase can function in developmental zebrafish, and that PCs are highly effective in detoxifying Cd toxicity even in the whole body of a vertebrate species.

Phytochelatin synthase, papain's cousin, in stereo

Supraoptimal levels of essential heavy metals (e.g., Cu and Zn) and trace or higher levels of nonessential heavy metals (e.g., As, Cd, and Hg) pose a threat because they undergo aberrant capping reactions with the thiol groups of proteins, displace endogenous metal cofactors from their cellular binding sites, and promote the formation of active oxygen species (1). One of the most sophisticated mechanisms known to confer protection from metal toxicity is the enzyme-catalyzed synthesis of short-chain peptides, phytochelatins (PCs), which serve as high-affinity, thiol-rich cellular chelators and thereby contribute to the detoxification of heavy metal ions, most notably Cd2+, in vascular plants and some algae, diatoms, fungi, and invertebrates (2). Derived from glutathione (GSH) and related thiols in a γ-glutamylcysteinyl transpeptidation reaction catalyzed by phytochelatin synthases (PC synthases, EC 2.3.2.15) (1, 3, 4), PCs have the general structure (γ-Glu-Cys)n-Xaa, where n = 2–11 and Xaa is usually Gly (Fig. 1). Although studies of PC synthases have largely been concerned with the enzymes from eukaryotes, recent database searches have disclosed PC synthase-like sequences in the genomes of several prokaryotes (1, 5). In and of itself, this finding might not be of particular interest except that all of the prokaryotic PC synthase homologs identified are half the length of their cognates from eukaryotes (220–237 residues compared with 421–506 residues) because they lack the more sequence-variable C-terminal domain characteristic of the latter. Moreover, the one prokaryotic PC synthase homolog to have been assayed for activity, the alr0975 protein from the cyanobacterium Nostoc sp. PCC 7120 (NsPCS), catalyzes the deglycylation of GSH to γ-Glu-Cys at a high rate and the synthesis of PC2 at a relatively low rate (Fig. 1) (5, 6). It is as if the prokaryotic enzymes, despite an overall sequence identity of only 22–36% with the N-terminal domains of the eukaryotic enzymes, consist solely of the core catalytic domain of a canonical eukaryotic PC synthase (Fig. 1) (1, 7, 8). Against this background, the stereo structure of NsPCS in its native and γ-Glu-Cys-acylated state reported by Vivares et al. in a recent issue of PNAS (9) is seminal. The results presented not only represent the first crystal structure for a PC synthase but they also establish, as had been inferred tentatively from kinetic, protein chemical, and site mutagenic analyses of the prototypical eukaryotic PC synthase, Arabidopsis thaliana PC synthase 1 (AtPCS1) (Fig. 1) (1, 8, 10), that these enzymes belong to the papain superfamily and deploy a cysteine protease-like catalytic mechanism.

Identification of metaflothionein in Pleurodeles waltl.

The characterization of metallothionein in the Urodele amphibian species Pleurodeles waltl was achieved. A simple and rapid method for identification of metallothionein, based on its strong affinity for cadmium (109Cd), was used. We were able to show that metallothionein is constitutively synthesized in liver, ovary and brain. The property of metallothionein to strongly bind essential (Zn, Cu) as well as toxic (Cd, Hg) metals is consistent with a dual role in cellular metabolism, ie. homeostatis and detoxification of heavy metal ions.

Detection of root mucilage using an anti-fucose antibody.

Plant root mucilage is known to enhance soil quality by contributing towards the soil carbon pool, soil aggregation, detoxification of heavy metal ions and interactions with rhizospheric microflora. Mucilage consists of many monosaccharide units, including fucose which can be used as an indicator for plant root based polysaccharides. This is the first report of an immunological technique developed to use anti-fucose antibodies as markers for probing and localizing fucosyl residues in mucilage polysaccharide and, in turn, for localization of plant root mucilage. Fucose was complexed with bovine serum albumin to raise antibodies against fucose. A fucose-directed antibody was shown to cross-react with root cap mucilages from grasses. This antibody was used to localize root mucilage polysaccharide in maize and wheat root caps using immunogold electron microscopy. Abundant labelling could be localized on the cell wall, and in the intercellular matrix and vesicles of the peripheral root cap cells. Labelling was less intense in cells towards the centre of the root cap tissue. Control experiments confirmed that immunogold localization of fucose was specific and reliable.

Rapid method for detection and detoxification of heavy metal ions in water environments using phytochelation

Phytochelatins (PCs, (γGlu-Cys) n-Gly (n = 2–11)) are produced by higher plants, algae, and some fungi in response to heavy metal ion exposure. A rapid and convenient method for quantifying heavy metal ion concentrations in water environments was developed using a chemically synthesized PC as a mediator. The chelating ability of the PC and quantification of the thiol group were utilized to measure heavy metal ions at low concentrations. The method requires only ten minutes for measurement and only 1 ml of a liquid sample. A range of homogeneous PCs (n = 4–7) were chemically synthesized using a peptide synthesizer. These, especially PC 7, exhibited higher sensitivity and consistency of measurement than the native PC from Silene cucubalus, which produced a mixture of PC 2, PC 3, and PC 4. Detoxification of heavy metal ions in vitro by PC was also investigated. Using the paper disc method, the cell growth inhibition zone caused by cadmium ion against Salmonella typhimurium TA1538 was significantly decreased by addition of PC. Furthermore, at the minimum inhibitory concentration of cadmium ion (200 μM) in a nutrient broth culture of S. typhimurium, cell growth was almost completely recovered by addition of PC to the medium.

Sulfide stabilization of the cadmium-gamma-glutamyl peptide complex of Schizosaccharomyces pombe.

Addition of cadmium salts to the growth medium of Schizosaccharomyces pombe leads to synthesis of a Cd.gamma-Glu peptide complex and an enhanced generation of sulfide ions. The gamma-Glu peptide complex functions in the detoxification of heavy metal ions. Native Cd.gamma-Glu peptide complexes contain acid-labile sulfide in the metal-thiolate cluster. Two forms of the complex exist differing primarily in their sulfide content. Sulfide concentrations up to 0.2 and 1.2 mol/mol of peptide were observed in native isolates of forms I and II, respectively. Addition of sulfide to the low sulfide form I converted it to a complex similar to form II. Properties of the Cd.gamma-Glu peptide complex were altered by the incorporation of sulfide ions. Sulfide-dependent electronic transitions in the ultraviolet were evident, and the absorbance maximum of the transition was related to the sulfide content and the bound metal ion. High sulfide forms of the Cd and Zn complexes exhibited absorbance peaks at 318 nm and 255 nm, respectively. Incorporation of sulfide into the Cd.gamma-Glu peptide complex imparted greater thermodynamic stability to the complex, an increased Stokes radius, and an enhanced Cd(II) binding capacity. Sulfide generation may be a cellular response in part to enhance the effectiveness of the gamma-Glu peptide system for Cd(II) detoxification.