Metal interactions in algal toxicology: conventional versus in vivo tests

Different studies on the toxicity of the same metals to algae have often shown divergent and sometimes contradictory results. The inconsistency of these findings was often attributed to environmental factors such as the degree of chelation, complexation and precipitation of metals. Conventionally, toxicity tests of metal mixtures were conducted by adding metals to algae growing in synthetic media. In our study, we examined the in vivo toxic effects of metals by pretreating the algal cells with one metal, resuspendeding them in fresh medium, then exposing them to a second metal. The algal response showed marked differences between the conventional and the new approach. The conventional approach shows that the toxicity depends upon the complexes formed externally, whereas the in vivo approach shows that the toxicity probably depends upon the molecular transformation of the metals internally.     

Comparative acute toxicity of gallium(III), antimony(III), indium(III), cadmium(II), and copper(II) on freshwater swamp shrimp (Macrobrachium nipponense)

Background

Acute toxicity testing were carried out the freshwater swamp shrimp, Macrobrachium nipponense, as the model animal for the semiconductor applied metals (gallium, antimony, indium, cadmium, and copper) to evaluate if the species is an suitable experimental animal of pollution in aquatic ecosystem.

Results

The static renewal test method of acute lethal concentrations determination was used, and water temperature was maintained at 24.0 ± 0.5°C. Data of individual metal obtained from acute toxicity tests were determined using probit analysis method. The median lethal concentration (96-h LC₅₀) of gallium, antimony, indium, cadmium, and copper for M. nipponense were estimated as 2.7742, 1.9626, 6.8938, 0.0539, and 0.0313 mg/L, respectively.

Conclusions

Comparing the toxicity tolerance of M. nipponense with other species which exposed to these metals, it is obviously that the M. nipponense is more sensitive than that of various other aquatic animals.     

Mixture toxicity of copper and zinc to barley at low level effects can be described by the Biotic Ligand Model

Background and aims

The biotic ligand model (BLM) is a bioavailability model for metals based on the concept that toxicity depends on the concentration of metal bound to a biological binding site; the biotic ligand. Here, we evaluated the BLM to interpret and explain mixture toxicity of metals (Cu and Zn).

Methods

The mixture toxicity of Cu and Zn to barley (Hordeum vulgare L.) was tested with a 4 days root elongation test in resin buffered nutrient solutions. Toxicity of one toxicant was tested in presence or absence of a low effect level of the other toxicant or in a ray design with constant toxicant ratios. All treatments ran at three different Ca concentrations (0.3, 2.2 and 10?mM) to reveal ion interaction effects.

Results

The 50 % effect level (EC50) of one metal, expressed as the free ion in solution, significantly (p?<?0.05) increased by adding a low level effect of the other metal at low Ca. Such antagonistic interactions were smaller or became insignificant at higher Ca levels. The Cu EC10 was unaffected by Zn whereas the Zn EC10 increased by Cu at low Ca. These effects obeyed the BLM combined with the independent action model for toxicants.

Conclusions

The BLM model explains the observed interactions by accounting for competition between both metals free ions and Ca²⁺ at the Cu and Zn biotic ligands. The implications of these findings for Cu/Zn interactions in soil are discussed.     

Effects of Soil Components and Liming Effect of CCA-Wood Ash upon Leaching of Cu,Cr, and As from CCA-Wood Ash in Ultisol Soil

The burning of wood that has been chemically treated with chromated copper arsenate (CCA) produces an ash containing high concentrations of copper, chromium, and arsenic (CCA-metals). The rainwater-leaching of these metals from burn sites can produce increased soil and water contamination. Soil systems have varying natural abilities to retard leaching and they also impact metals speciation and toxicity through sorption, conversion, and sedimentation-related mechanisms. Recent regulations restricting the use of CCA-treatment have resulted in increased quantities of CCA-treated lumber entering the waste stream, making studies of metals leaching from CCA-wood ash and soil/CCA-wood ash systems important areas of investigation.

Wood ash composition, soil composition, and CCA-metals speciation are all important factors determining the degree of the metal mobility in a soil system containing metals leached from CCA-wood ash. The CCA-metals composition of CCA-wood ash was determined by analytical methods. Both pH and batch leaching studies were used to postulate mobility mechanisms within the CCA-wood ash/soil system. The contrasting effects of untreated-wood ash and CCA-wood ash on soil components are presented in order to assess the potential for enhancement of immobilization mechanisms that increase the soil system retardance of CCA-metals mobility.

Results of this investigation show that the Ultisol test soil retards the mobility of As and Cr compared to CCA-wood ash alone, while Cu mobility is increased in the presence of the Ultisol test soil. Experimental results show that the alkalinity of a CCA-wood ash/Ultisol soil system is lower than that of an untreated-wood ash/Ultisol soil system. This indicates a difference in chemical composition and the potential consumption of hydroxyl ions during treatment by the retardance mechanisms affecting Cr and As mobility. Therefore, the study of these contaminated soil systems and the potential enhancement of immobilization mechanisms are important areas of investigation.     

Evaluation of the Relationship Between Height and Zinc,Copper, Iron,Calcium, and Magnesium Levels in Healthy Young Children in Beijing,China

Studies suggest a relationship between semen quality and the concentration of trace elements in serum or seminal plasma. However, trace elements may be linked to DNA and capable of altering the gene expression patterns. Thus, trace element interactions with DNA may contribute to the mechanisms for a trans-generational reproductive effect. We developed an analytical method to determine the amount of trace elements bound to the sperm DNA, and to estimate their affinity for the sperm DNA by the ratio: R = Log [metal concentration in the sperm DNA/metal concentration in seminal plasma]. We then analyzed the concentrations of 15 trace elements (Al, Cd, Cr, Cu, Hg, Mn, Mo, Ni, Pb, Ti, V, Zn, As, Sb, and Se) in the seminal plasma and the sperm DNA in 64 normal and 30 abnormal semen specimens with Inductively Coupled Plasma/Mass Spectrometry (ICP-MS). This study showed all trace elements were detected in the seminal plasma and only metals were detected in the sperm DNA. There was no correlation between the metals’ concentrations in the seminal plasma and the sperm DNA. Al had the highest affinity for DNA followed by Pb and Cd. This strong affinity is consistent with the known mutagenic effects of these metals. The lowest affinity was observed for Zn and Ti. We observed a significant increase of Al linked to the sperm DNA of patients with oligozoospermia and teratozoospermia. Al’s reproductive toxicity might be due to Al linked to DNA, by altering spermatogenesis and expression patterns of genes involved in the function of reproduction.     

Biological effects of contaminated bottom sediments of water bodies in Central and South Vietnam on aquatic organisms

The levels of metal content and toxicity have been determined in bottom sediments of water bodies in Central and South Vietnam. It is found that the concentration of metals (As, Cr, Cu, Hg, Ni, Pb, and Zn) exceed the value of the abundance of elements in the Earth’s crust, which may be associated with geochemical features of the province. Acute toxicity of water-extracted sediment elutriate for Ceriodaphnia and whole (native) sediments for chironomid larvae is not recorded in any investigated water bodies. An increase in the relative abundance of Chironomus riparius larvae with deformations of mouthpart structures compared to the control was observed for most of the water bodies. This index reaches the value in the Khe River (Cân Th? city). Antenna anomalies prevailed in pathomorphological disorders.     

Dinucleotide fold proteins. Interaction of arabinose binding protein with Cibacron Blue 3G-A.

Cibacron Blue 3G-A, the dye moiety of Blue dextran-Sepharose, has been shown to not specifically bind a protein with a dinucleotide fold-like supersecondary structure, L-arabinose binding protein from Escherichia coli. This shows that Cibacron Blue 3G-A is not suitable as a definitive probe for the dinucleotide fold as suggested earlier (Thompson et al., 1975; Stellwagen, 1977). An explanation for the large predominance of proteins containing this protein supersecondary structure that bind to this dye is presented.     

A field test of a sediment bioassay with the oligochaete worm Tubifex tubifex (Müller, 1774)

A sediment toxicity test using reproduction in the tubificid oligochaete Tubifex tubifex was tested in 11 geographic locations in the Laurentian Great Lakes where sediments have previously been documented as being contaminated. Samples were taken from both potentially contaminated and clean sites in each of the 11 areas. Using test endpoints such as survival and growth, results from 72 samples were classified using cluster and ordination techniques. Six response groups were identified and four groups of toxic sites determined. Toxic effects ranged from mortality of adult worms to reduced reproduction. Using correlation and discriminant analysis there was good evidence that high concentrations of metals such as copper and lead were responsible for the observed effects at some sites.     

Membrane transporters and protein traffic networks differentially affecting metal tolerance: a genomic phenotyping study in yeast

Background

The cellular mechanisms that underlie metal toxicity and detoxification are rather variegated and incompletely understood. Genomic phenotyping was used to assess the roles played by all nonessential Saccharomyces cerevisiae proteins in modulating cell viability after exposure to cadmium, nickel, and other metals.

Results

A number of novel genes and pathways that affect multimetal as well as metal-specific tolerance were discovered. Although the vacuole emerged as a major hot spot for metal detoxification, we also identified a number of pathways that play a more general, less direct role in promoting cell survival under stress conditions (for example, mRNA decay, nucleocytoplasmic transport, and iron acquisition) as well as proteins that are more proximally related to metal damage prevention or repair. Most prominent among the latter are various nutrient transporters previously not associated with metal toxicity. A strikingly differential effect was observed for a large set of deletions, the majority of which centered on the ESCRT (endosomal sorting complexes required for transport) and retromer complexes, which - by affecting transporter downregulation and intracellular protein traffic - cause cadmium sensitivity but nickel resistance.

Conclusion

The data show that a previously underestimated variety of pathways are involved in cadmium and nickel tolerance in eukaryotic cells. As revealed by comparison with five additional metals, there is a good correlation between the chemical properties and the cellular toxicity signatures of various metals. However, many conserved pathways centered on membrane transporters and protein traffic affect cell viability with a surprisingly high degree of metal specificity.     

Recovery of a stream macroinvertebrate community from mine drainage disturbance

Recovery of aquatic macroinvertebrates from the effects of mine drainage was documented using a weight-of-evidence approach which included measures of physical, chemical, and biological data. Taxa richness; number of taxa in the orders Ephemeroptera, Plecoptera, and Trichoptera; and shredder taxa richness all increased downstream of the point source after water treatment was initiated. Cluster analysis of aquatic macroinvertebrate community data along with abundance of a metals sensitive mayfly (Rhithrogena hageni) also suggested recovery from metals effects. Response to decreased metal inputs was rapid and biological measurements of impacted sites attained levels comparable to upstream reference sites in two years. Our results suggest that aquatic communities impacted by metals, in the absence of degraded habitat and with nearby colonist pools, will recover quickly if low instream concentrations of toxicants are achieved.     

Plant chitinase responses to different metal-type stresses reveal specificity

Key message

Chitinases in Glycine max roots specifically respond to different metal types and reveal a polymorphism that coincides with sensitivity to metal toxicity.

Abstract

Plants evolved various defense mechanisms to cope with metal toxicity. Chitinases (EC 3.2.1.14), belonging to so-called pathogenesis-related proteins, act as possible second line defense compounds in plants exposed to metals. In this work their activity was studied and compared in two selected soybean (Glycine max L.) cultivars, the metal-tolerant cv. Chernyatka and the sensitive cv. Kyivska 98. Roots were exposed to different metal(loid)s such as cadmium, arsenic and aluminum that are expected to cause toxicity in different ways. For comparison, a non-metal, NaCl, was applied as well. The results showed that the sensitivity of roots to different stressors coincides with the responsiveness of chitinases in total protein extracts. Moreover, detailed analyses of acidic and neutral proteins identified one polymorphic chitinase isoform that distinguishes between the two cultivars studied. This isoform was stress responsive and thus could reflect the evolutionary adaptation of soybean to environmental cues. Activities of the individual chitinases were dependent on metal type as well as the cultivar pointing to their more complex role in plant defense during this type of stress.     

Essential Metals in Cryptococcus neoformans: Acquisition and Regulation

Transition metals such as iron, zinc, and copper play key roles in numerous biological processes, including innate immunity in vertebrates. In fact, control of the availability of these essential metals is an important component of the immune response to limit the growth of pathogens during infection. In turn, pathogens must overcome the extreme limitation of essential metals and potential metal toxicity to successfully cause disease. Recent work illuminates the mechanisms by which the pathogenic fungus Cryptococcus neoformans manages iron, copper, and zinc availability in support of proliferation in vertebrate hosts. In particular, the fungus makes use of high-affinity metal transport systems and complex regulatory networks that play critical roles in the virulence of the fungus.     

A dialysis system with phytoplankton for monitoring chemical pollution in freshwater ecosystems by alkaline phosphatase assay

The effects of three groups of chemicals, heavy metals, pesticides and phenolics, on alkaline phosphatase activity of intact Chlorella vulgaris cells were investigated. There was a marked inhibitory effect of heavy metals and a slight one due to phenolics, but the pesticides tested showed no effect. In order to detect heavy metals in freshwater ecosystems, we propose a dialysis system, which can be placed in the field and then provide early warning signals of toxicity. This phosphatase inhibition test is recommended the first stage of an enzymatic screening system for chemical pollution in water.     

Noninvasive Evaluation of Heavy Metal Uptake and Storage in Micoralgae Using a Fluorescence Resonance Energy Transfer-Based Heavy Metal Biosensor

We have developed a fluorescence resonance energy transfer (FRET)-based heavy metal biosensor for the quantification of bioavailable free heavy metals in the cytoplasm of the microalga Chlamydomonas reinhardtii. The biosensor is composed of an end-to-end fusion of cyan fluorescent protein (CFP), chicken metallothionein II (MT-II), and yellow fluorescent protein (YFP). In vitro measurements of YFP/CFP fluorescence emission ratios indicated that the addition of metals to the purified biosensor enhanced FRET between CFP and YFP, consistent with heavy metal-induced folding of MT-II. A maximum YFP/CFP FRET ratio of 2.8 was observed in the presence of saturating concentrations of heavy metals. The sensitivity of the biosensor was greatest for Hg²⁺ followed by Cd²⁺ ≈ Pb²⁺ > Zn²⁺ > Cu²⁺. The heavy metal biosensor was unresponsive to metals that do not bind to MT-II (Na⁺ and Mg²⁺). When expressed in C. reinhardtii, we observed a differential metal-dependent response to saturating external concentrations (1.6 mm) of heavy metals (Pb²⁺ > Cd²⁺) that was unlike that observed for the isolated biosensor (in vitro). Significantly, analysis of metal uptake kinetics indicated that equilibration of the cytoplasm with externally applied heavy metals occurred within seconds. Our results also indicated that algae have substantial buffering capacity for free heavy metals in their cytosol, even at high external metal concentrations.Many proteins utilize metals to stabilize their structures or as cofactors to catalyze redox and other chemical reactions. Metals such as zinc, copper, iron, magnesium, cobalt, and manganese are required by most living organisms for their normal cellular functions. Essential metals are often present at low concentrations in the environment, however, and must be imported into cells, often at the expense of energy (Hanikenne et al., 2005; Merchant et al., 2006). In contrast to essential metals, toxic metals such as cadmium, lead, and mercury can disrupt cellular functions by competing with essential metals for their metal-binding sites and/or by altering the redox state of cells. Exposure of organisms to high concentrations of toxic metals can impair their cellular functions, growth, and reproduction. To prevent metal-induced cellular anomalies, organisms have evolved a variety of strategies to reduce the toxicity of heavy metals. One such strategy involves the selective binding of toxic metals in the cytoplasm by metal-binding proteins and other small molecules. As discussed below, both enzymatically and ribosomally synthesized Cys-rich peptides, including phytochelatins and metallothioneins (MTs), are utilized by a variety of organisms to sequester toxic heavy metals, including cadmium, mercury, lead, silver, and gold. The peptides may also serve as storage reserves for essential metals such as copper and zinc (Cobbett and Goldsbrough, 2002).Phytochelatins are enzymatically synthesized polypeptides containing repeating units of (γ-Glu-Cys)_n-Gly, where n = 2 to 11 (Rauser, 1990), whereas MTs are genetically encoded, ribosomally synthesized polypeptides (Cobbett and Goldsbrough, 2002). MTs have molecular mass values ranging from 6 to 7 kD and contain approximately 20 conserved Cys residues (Cobbett and Goldsbrough, 2002; Romero-Isart and Vasák, 2002). Metals are characteristically bound to MT via the thiolate sulfur ligands of Cys residues (Kägi, 1991). It is estimated that the metal-saturated MT contains about 10% thiolate sulfur and bound metals by mass (Romero-Isart and Vasák, 2002). Structural analyses of metal-free and metal-complexed MTs demonstrated that MTs undergo a structural transition from a metal-free random-coil structure to a metal-bound compact dumbbell-shaped structure having metal saturated α- and β-domains (Pearce et al., 2000; Romero-Isart and Vasak, 2002; Hong and Maret, 2003). The N-terminal β-domain binds three metal ion equivalents, and the C-terminal α-domain binds four metal ion equivalents (Romero-Isart and Vasák, 2002; Vasák, 2005). Furthermore, several decades of work on MTs have provided a great deal of information regarding their metal-binding affinity, specificity, and domain selectivity for select metals (Cobbett and Goldsbrough, 2002; Romero-Isart and Vasák, 2002; Vasák, 2005).Fluorescence resonance energy transfer (FRET) involves the nonradioactive transfer of energy between the excited state of a luminescent or fluorescent donor molecule and a nearby acceptor molecule that has overlapping excited state transitions. Proteins that are modified to have efficient energy donor and acceptor domains and that undergo structural changes upon binding a specific ligand are good candidates for FRET-based sensors. For ligand-specific FRET-based biosensors, the distance and/or the orientation between the energy donor and acceptor molecules is changed upon ligand binding in a concentration-dependent manner (Selvin, 1995; Weiss, 2000; Hong and Maret, 2003; Looger et al., 2005). Relevant to this discussion, a FRET-based biosensor with GFP variants fused to MT was previously shown to be an effective means to monitor metal release during nitric oxide-induced signaling in endothelial cells (Pearce et al., 2000).Unicellular algae such as Chlamydomonas species are often found in areas that might be contaminated by toxic heavy metals (Merchant et al., 2006). Chlamydomonas species have also been shown to sequester toxic metals (e.g. cadmium and mercury) and have potential use for bioremediation of these metals (Cai et al., 1999; Adhiya et al., 2002; Siripornadulsil et al., 2002; He et al., 2011; Priyadarshani et al., 2011). To determine the kinetics and selectivity of exogenous heavy metal uptake as well as free heavy metal concentration in the cytoplasm of Chlamydomonas species, we developed an MT, FRET-based metal-binding sensor and expressed this in the cytoplasm of the unicellular green alga Chlamydomonas reinhardtii. We demonstrate that heavy metal uptake is rapid in C. reinhardtii and that cytoplasmic free heavy metal concentrations are substantially lower than exogenous free heavy metal concentrations, implying that heavy metals are rapidly sequestered by various biological molecules in the cell.     

High genetic variation among closely related red oak (<Emphasis Type="Italic">Quercus rubra</Emphasis>) populations in an ecosystem under metal stress: analysis of gene regulation

Metal toxicity is a major abiotic stressor of plants. It has been established that changes in genetic variation occur very rapidly in plants in response to environmental stressors such as increased levels of metals. Quercus rubra (red oak) is a pioneer species in mining regions contaminated with metals in Northern Ontario (Canada). The objectives of the study were to (1) determine the level of genetic variation in Q. rubra populations from mining damaged ecosystems using RAPD marker system and (2) assess the level of gene expression of candidate genes for nickel resistance. Total gene diversity (H_T) and the mean gene diversity among populations (H_S) were 0.22 and 0.19, respectively. The percent of polymorphic loci within populations was high ranging from 61 % (Capreol) to 72 % (Daisy Lake) despite a high level of gene flow (2.4). The population differentiation (G_ST) value was low (0.17). No significant difference was found among the contaminated and reference sites for all the genetic parameters estimated. Hence, all the Q. rubra populations from the metal-contaminated and damaged ecosystems are genetically sustainable. Moreover, this study reveals that all populations were genetically closely related with genetic distance values varying from 0.17 to 0.35. A zinc finger protein of Arabidopsis thaliana (ZAT11) gene involved in nickel resistance was differentially expressed in samples analyzed. There was a 120 times higher of ZAT11 expression in samples from metal contaminated areas of Wahnapitae Dam compared to other metal contaminated and uncontaminated sites, but no association between soil metal levels and expression of ZAT11 was established.     

Accumulation of heavy metals by aquatic mosses. 3: Seasonal changes

The changes taking place at seven stream and river sites over a 14-month period were followed for the following:

1. % cover of Rhynchostegium riparioides;
2. relative abundance of Rhynchostegium as part of the whole plant community and, where present, also Amblystegium riparium and Fontinalis antipyretica;
3. 18 water chemistry variables;
4. concentrations of nine metals in 2-cm tips of Rhynchostegium at all sites and of Amblystegium and Fontinalis at one site each.

A decrease in the amounts of these mosses occurred in winter at most sites, the effect being most pronounced with Amblystegium. Nevertheless the majority of plants which remained had healthy shoots suitable for sampling for metal analysis at all times of year. There were marked temporal changes in the concentrations of metals in the 2-cm tips at each site, but few of these appeared to be related to seasonal events. The most plausible was for Pb in Rhynchostegium at a main river site, where increased accumulation in winter was probably an indirect effect of other chemical differences in the water during a period of high flows. Provided healthy shoots were available, there was no indication of seasonal differences in metal accumulation resulting from features of the mosses themselves.     

Bacterial resistance to arsenic protects against protist killing

Protists kill their bacterial prey using toxic metals such as copper. Here we hypothesize that the metalloid arsenic has a similar role. To test this hypothesis, we examined intracellular survival of Escherichia coli (E. coli) in the amoeba Dictyostelium discoideum (D. discoideum). Deletion of the E. coli ars operon led to significantly lower intracellular survival compared to wild type E. coli. This suggests that protists use arsenic to poison bacterial cells in the phagosome, similar to their use of copper. In response to copper and arsenic poisoning by protists, there is selection for acquisition of arsenic and copper resistance genes in the bacterial prey to avoid killing. In agreement with this hypothesis, both copper and arsenic resistance determinants are widespread in many bacterial taxa and environments, and they are often found together on plasmids. A role for heavy metals and arsenic in the ancient predator–prey relationship between protists and bacteria could explain the widespread presence of metal resistance determinants in pristine environments.     

Heavy-metal accumulation in crops grown on sewage sludge amended with metal salts

Poplar (Populus euramericana Robusta), oats (Avena sativa L. Leander), maize (Zea mays L. Ona 36), English ryegrass (Lolium perenne L.), butter head lettuce (Lactuca sativa L. Reskia), spinach (Spinacia oleracea L. Subito) and French beans (Phaseolus vulgaris Prelude), were grown in pots with pure sewage sludge (pH 6.7), amended with Cd, Cr, Cu, Ni, Pb and Zn acetates, either added singly or in combination, to study metal effects on plant growth and metal uptake. Phytotoxic metal doses varied with metal and plant species, increasing in the order Cd<Ni<Cu<Zn<Cr and Pb. The threshold dose of toxic metals applied in combination was generally lower than that of metals given singly. Addition of Cd, Ni and Zn was clearly reflected in the respective plant concentrations. This was much less so for Cu, whereas Cr and Pb concentrations were not affected in most plant species. Critical plant (leaf) metal concentrations were lower for metals applied in combination than for single metals. Because of such phenomena the use of critical levels as a diagnostic tool for determining potential multiple metal toxicity is limited.     

Alternative methods in toxicology tests: In vitro toxicity

Toxicity testing is required for new chemicals being introduced onto the market. The use of animals in evaluating chemical safety is costly and time consuming. Furthermore, there is the ethical need to develope alternative methods to reduce the required number of animals. The newinvitro assays offer numerous advantages such as speed, reproducibility and control of test conditions, and increased sensitivity. Although the dermal irritation assays might be substituted by theinvitro tests in the near future (Duffy, 1989), much work is required to evaluate organ toxicity withinvitro methods. We present data regarding the use of Balb/3T3 mice fibroblasts and primary rat hepatocytes as test systems forinvitro toxicity. The end-points we have analysed are total protein content, dye accumulation in lysosomes, reductase mytochondrial activity, intracellular content and leakage of enzymes into the medium.     

Genomic Regions Flanking E-Box Binding Sites Influence DNA Binding Specificity of bHLH Transcription Factors through DNA Shape

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Highlights? Cbf1 and Tye7 are paralogous TFs with virtually identical DNA binding-site motifs ? The two paralogous TFs bind different genomic target sites in vivo ? The genomic context of putative DNA binding sites affects TF binding specificity ? Structural analyses suggest that genomic context influences TF binding through DNA shape