THE ROLE OF REACTIVE OXYGEN SPECIES IN COPPER TOXICITY TO TWO FRESHWATER GREEN ALGAE1

The role of reactive oxygen species (ROS) in copper (Cu) toxicity to two freshwater green algal species, Pseudokirchneriella subcapitata (Korshikov) Hindák and Chlorella vulgaris Beij., was assessed to gain a better mechanistic understanding of this toxicity. Cu‐induced formation of ROS was investigated in the two algal species and linked to short‐term effects on photosynthetic activity and to long‐term effects on cell growth. A light‐ and time‐dependent increase in ROS concentrations was observed upon exposure to environmentally relevant Cu concentrations of 50 and 250 nM and was comparable in both algal species. However, effects of 250 nM Cu on photosynthesis were different, leading to a 12% reduction in photosynthetic activity in P. subcapitata, but not in C. vulgaris. These results indicate that differences in species‐specific sensitivities measured as photosynthetic activity were not caused by differences in the cellular ROS content of the algae, but probably by different species‐specific ROS defense systems. To investigate the role of ROS in Cu‐mediated inhibition of photosynthesis, the ROS scavenger N‐tert‐butyl‐α‐phenylnitrone (BPN) was used, resulting in a reduction of Cu‐induced ROS production up to control level and a complete restoration of photosynthetic activity of Cu‐exposed P. subcapitata. This finding implied that ROS play a primary role in Cu toxicity to algae. Furthermore, we observed a time‐dependent ROS release process across the plasma membrane. More than 90% of total ROS were determined to be extracellular in P. subcapitata, indicating an efficient method of cellular protection against oxidative stress.     

A note on copper bioaccumulation in Mozambique tilapia,Oreochromis mossambicus (Osteichthyes: Cichlidae)

Copper (Cu) is one of the most commonly reported metal pollutants in African water bodies, but there are few studies on African freshwater fish species of copper accumulation and copper toxicity. Adult O. mossambicus were exposed to 0mg l^?1 (control) and 0.75mg l^?1 Cu for 96h and 0 (control), 0.11, 0.29 and 0.47mg l^?1 copper for 64 days. Samples of liver and gills were collected after 96h, and after 1, 32 and 64 days, respectively. There were significant differences in the mean Cu accumulation values in the liver and gills between the control and the Cu-exposed fish after the 96-h exposure. In fish exposed to 0.11 and 0.29mg l^?1 Cu for 64 days there was an increase in copper level in the tissues. In fish exposed to 0.47mg l^?1 Cu the concentration in the gill and liver tissue did not increase between Day 1 and Day 32. At this time, Cu accumulation in the liver was higher than for fish exposed to 0.11 and 0.29mg l^?1 Cu for 64 days. Exposure to approximately 0.47mg l^?1 Cu for more than 32 days induced mortality.     

Antibacterial,antifungal and in vitro antileukaemia activity of metal complexes with thiosemicarbazones

1‐phenyl‐3‐methyl‐4‐benzoyl‐5‐pyrazolone 4‐ethyl‐thiosemicarbazone (HL) and its copper(II), vanadium(V) and nickel(II) complexes: [Cu(L)(Cl)]·C₂H₅OH·( 1 ), [Cu(L)₂]·H₂O ( 2 ), [Cu(L)(Br)]·H₂O·CH₃OH ( 3 ), [Cu(L)(NO₃)]·2C₂H₅OH ( 4 ), [VO₂(L)]·2H₂O ( 5 ), [Ni(L)₂]·H₂O ( 6 ), were synthesized and characterized. The ligand has been characterized by elemental analyses, IR, ¹H NMR and ¹³C NMR spectroscopy. The tridentate nature of the ligand is evident from the IR spectra. The copper(II), vanadium(V) and nickel(II) complexes have been characterized by different physico‐chemical techniques such as molar conductivity, magnetic susceptibility measurements and electronic, infrared and electron paramagnetic resonance spectral studies. The structures of the ligand and its copper(II) ( 2 , 4 ), and vanadium(V) ( 5 ) complexes have been determined by single‐crystal X‐ray diffraction. The composition of the coordination polyhedron of the central atom in 2 , 4 and 5 is different. The tetrahedral coordination geometry of Cu was found in complex 2 while in complex 4 , it is square planar, in complex 5 the coordination polyhedron of the central ion is distorted square pyramid. The in vitro antibacterial activity of the complexes against Escherichia coli, Salmonella abony, Staphylococcus aureus, Bacillus cereus and the antifungal activity against Candida albicans strains was higher for the metal complexes than for free ligand. The effect of the free ligand and its metal complexes on the proliferation of HL‐60 cells was tested.     

Copper toxicity response influences mesotrophic Synechococcus community structure

Picocyanobacteria from the genus Synechococcus are ubiquitous in ocean waters. Their phylogenetic and genomic diversity suggests ecological niche differentiation, but the selective forces influencing this are not well defined. Marine picocyanobacteria are sensitive to Cu toxicity, so adaptations to this stress could represent a selective force within, and between, ‘species’, also known as clades. Here, we compared Cu stress responses in cultures and natural populations of marine Synechococcus from two co‐occurring major mesotrophic clades (I and IV). Using custom microarrays and proteomics to characterize expression responses to Cu in the lab and field, we found evidence for a general stress regulon in marine Synechococcus. However, the two clades also exhibited distinct responses to copper. The Clade I representative induced expression of genomic island genes in cultures and Southern California Bight populations, while the Clade IV representative downregulated Fe‐limitation proteins. Copper incubation experiments suggest that Clade IV populations may harbour stress‐tolerant subgroups, and thus fitness tradeoffs may govern Cu‐tolerant strain distributions. This work demonstrates that Synechococcus has distinct adaptive strategies to deal with Cu toxicity at both the clade and subclade level, implying that metal toxicity and stress response adaptations represent an important selective force for influencing diversity within marine Synechococcus populations.     

Phosphate limitation influences the sensitivity to copper in periphytic algae

1. To assess the influence of nutrient limitation on copper toxicity, periphitic communities from an oligotrophic stream were exposed to copper for six to 12 days with and without the supply of nutrients (mainly P). 2. In contrast to the hypothesis that nutrient cycling in mature biofilms would protect them from Cu toxicity, low and high biomass biofilms did not differ in their physiological response to copper after 6 days of exposure. 3. A clear influence of P‐limitation on copper toxicity was observed. Periphytic communities that were previously fertilised for 18 days were three times more tolerant than control communities indicating that P‐limitation enhanced Cu toxicity and tolerance induction were probably related to the higher P‐availability. In addition, a compensation of Cu toxicity after P‐addition was observed in the long‐term (after 12 days). 4. We conclude that periphyton from oligotrophic streams is more sensitive to copper than periphyton from fertilised streams, and that therefore a higher effect of chronic copper exposure is expected to occur in oligotrophic P‐limited fluvial systems. 5. Extrapolation of our results to the area of study (Catalonia, north‐east of Spain) indicates that while the levels of Cu commonly found in the zone may negatively affect the periphyton from oligotrophic streams, because of the interaction between Cu and P, they are not able to control the growth of nuisance algae which is common under high nutrient conditions.     

INFLUENCE OF PHOSPHORUS NUTRITION ON COPPER TOXICITY TO THREE STRAINS OF SCENEDESMUS ACUTUS (CHLOROPHYCEAE)1

Three strains of Scenedesmus acutus f. alternans Hortobagyi with markedly different sensitivities to copper were examined to determine the relative importance of cellular polyphosphate content on acute copper toxicity to photosynthesis. By manipulating the phosphate concentration in semicontinuous cultures, the response of each strain was assessed at three cellular phosphorus states: P-loaded, P-sufficient, and P-deficient. The results demonstrated the importance of cellular polyphosphate content in reducing the toxic effect of copper on photosynthesis; the greater the cellular P content, the less inhibition of photosynthesis occurred during copper exposure. This relationship was evident for both Cu-tolerant strains (XCu and B-4) and a Cu-sensitive strain (X72). The ranges of response to 9.9 μM Cu (measured as the percentage of control rate of photosynthesis within strains) were, from P-loaded to P-deficient cells, X72, 78–54%; XCu, 95–77%; and B-4, 99–94%. The data suggest that polyphosphate plays a passive role in protecting cells from copper; however, with respect to the mechanism of Cu tolerance, polyphosphate appeared to be relatively unimportant because the sensitivity of the Cu-tolerant strains showed less dependence on cellular polyphosphate than did the Cu-sensitive strain.     

Toxicity of Tributyltin in Juvenile Common Carp (Cyprinus Carpio): Physiological Responses,Hepatic Gene Expression,and Stress Protein Profiling

In this study, the effects of tributyltin (TBT) on biochemical parameters (antioxidant responses and Na⁺‐K⁺‐ATPase) in different tissues were investigated by using juvenile common carp (Cyprinus Carpio) as well as growth and ion regulation–related genes expression and stress‐related proteins profiling in fish liver. Oxidative stress indices and Na⁺‐K⁺‐ATPase showed tissues‐specific responses in fish exposed to different TBT concentrations. All tested genes related to GH/IGF‐I axis and ion‐regulation were significantly induced in the TBT group with lower concentrations (except for the igfbp3 in 10 μg/L) and were inhibited in 20 μg/L. In addition, the profiling of two proteins Hsp 70 and MT were increasing in a dose‐dependent manner under TBT stress. In short, TBT‐induced biochemical and molecular responses in different tissues were reflected in the measured parameters in the test. On the basis of TBT residue levels in the natural environment, more long‐term experiments at lower concentrations will be necessary in the future.     

The role of the kinase OXI1 in cadmium‐ and copper‐induced molecular responses in Arabidopsis thaliana

The hypothesis that mitogen‐activated protein kinase (MAPK) signalling is important in plant defences against metal stress has become accepted in recent years. To test the role of oxidative signal‐inducible kinase (OXI1) in metal‐induced oxidative signalling, the responses of oxi1 knockout lines to environmentally realistic cadmium (Cd) and copper (Cu) concentrations were compared with those of wild‐type plants. A relationship between OXI1 and the activation of lipoxygenases and other initiators of oxylipin production was observed under these stress conditions, suggesting that lipoxygenase‐1 may be a downstream component of OXI1 signalling. Metal‐specific differences in OXI1 action were observed. For example, OXI1 was required for the up‐regulation of antioxidative defences such as catalase in leaves and Fe‐superoxide dismutase in roots, following exposure to Cu, processes that may involve the MEKK1‐MKK2‐WRKY25 cascade. Moreover, the induction of Cu/Zn superoxide dismutases in Cu‐exposed leaves was regulated by OXI1 in a manner that involves fluctuations in the expression of miRNA398. These observations contrast markedly with the responses to Cd exposure, which also involves OXI1‐independent pathways but rather involves changes in components mediating intracellular communication.     

Do Larvae and Ovipositing Chironomus riparius (Diptera: Chironomidae) Females Avoid Copper-Contaminated Environments?

Studies on the avoidance behavior of aquatic organisms to contaminants have confirmed that such behavior can be relevant in field situations. However, almost all toxicity tests involve the forced exposure of organisms to toxicants. In particular, despite the importance of Chironomus riparius Meigen larvae in sediment toxicity testing, only a few studies on avoidance behavior have been performed. This study investigated the ability of different life stages of C. riparius, including ovipositing females, first-, second-, and fourth-instar larvae, to avoid copper-contaminated environments. Ovipositing females were given a choice between a control and copper solution (1.3 mg Cu l^{? 1}). First-instar larvae were provided with a choice between a control and a copper (2.0 mg Cu l^{? 1})-spiked sediment. Both second- and fourth-instars were exposed to a copper gradient (0.38–3.4 mg Cu l^{? 1}) in a flow-through system. None of the life stages avoided copper, even though the highest concentrations caused lethal effects on midges. The avoidance behavior of C. riparius is not a sensitive endpoint to assess copper sublethal toxicity.     

Copper toxicity in Chinese cabbage is not influenced by plant sulphur status,but affects sulphur metabolism‐related gene expression and the suggested regulatory metabolites

The toxicity of high copper (Cu) concentrations in the root environment of Chinese cabbage (Brassica pekinensis) was little influenced by the sulphur nutritional status of the plant. However, Cu toxicity removed the correlation between sulphur metabolism‐related gene expression and the suggested regulatory metabolites. At high tissue Cu levels, there was no relation between sulphur metabolite levels viz. total sulphur, sulphate and water‐soluble non‐protein thiols, and the expression and activity of sulphate transporters and expression of APS reductase under sulphate‐sufficient or‐deprived conditions, in the presence or absence of H₂S. This indicated that the regulatory signal transduction pathway of sulphate transporters was overruled or by‐passed upon exposure to elevated Cu concentrations.     

EFFECT OF COPPER ON ALGAL COMMUNITIES FROM OLIGOTROPHIC CALCAREOUS STREAMS1

Two sets of experiments were done to quantify the effects of chronic copper exposure on natural peri‐ phyton in a nonpolluted calcareous river. The results of short‐term (up to 6 h exposure) experiments corroborated the significance of pH on copper toxicity. Copper toxicity increased when pH was reduced from 8.6 to 7.7, and this was related to the effect of pH on copper speciation (free copper concentration increased from 0.2% to 2.3% of total copper). Longer term experiments demonstrated that periphyton communities exposed to copper under pH variation (8.2–8.6) were already affected at 10 μg·L ^{? 1} (20–80 ng·L ^{? 1} Cu^{2 +} ) after 12 days of exposure. Copper exposure caused stronger effects on structural (algal biomass and community structure) than on functional (photosynthetic efficiency) parameters of peri‐ phyton. Changes in community composition included the enhancement of some taxa (Gomphonema gracile), the inhibition of others (Fragilaria capucina and Phormidium sp.), and the appearance of filament malformations (Mougeotia sp.). The results of our study demonstrated that several weeks of exposure to copper (10–20 μg·L ^{? 1}) were sufficient to cause chronic changes in the periphyton of oligotrophic calcareous rivers. This degree of copper pollution can be commonly found in the Mediterranean region as a result of agricultural practices and farming activities.     

Efficacy of multiple metals against copper-resistant bacterial strains

Aims: The antibacterial efficacy of zeolites containing copper (Cu) or silver (Ag) ions or a combination was assessed against several reported copper‐resistant (Cu^R) bacterial strains. Methods and Results: Comparison strains were obtained from the American Type Culture Collection that had no documented metal resistance. Reductions in bacterial populations were determined after exposure time intervals of 3, 6 and 24 h. All three Cu^R strains of Salmonella enterica exhibited resistance to Cu, Ag and Cu/Ag after three and 6 h of exposure. Both the Cu^R and comparison strain of Enterococcus faecium were resistant to both metals and the metal combination. Cu^RPseudomonas putida was significantly reduced by all zeolites within 3 h. The Cu^REscherichia coli strain was more sensitive to Cu, but more resistant to Ag than the comparison strain; however, significant reductions were achieved within 3 h with both Cu and Cu/Ag, and within 24 h with Ag. Conclusions: Some strains with reported resistance to Cu were also resistant to Ag, suggestive of a shared resistance mechanism such as an indiscriminate Cu efflux pump. Ent. faecium appears to have innate resistance to both metals. In general, Ent. faecium was the most resistant species to the individual metals and the combination of metals, Ps. putida the least resistant, and the Salmonella strains were more resistant than E. coli. Significance and Impact of the Study: Several of the comparison strains with no reported copper resistance were resistant to one or both metals. This may call into question the methods for determining bacterial metal resistance, which typically use nutrient‐rich media containing metals to assess the ability of the bacteria to grow in comparison with a wild‐type strain. Nevertheless, all the Cu^R strains evaluated in this study, with the exception of Ent. faecium, were reduced using the Cu and Ag zeolite combination.     

Enterobacter asburiae KE17 association regulates physiological changes and mitigates the toxic effects of heavy metals in soybean

This study aimed to elucidate the role played by Enterobacter asburiae KE17 in the growth and metabolism of soybeans during copper (100 μm Cu) and zinc (100 μm Zn) toxicity. When compared to controls, plants grown under Cu and Zn stress exhibited significantly lower growth rates, but inoculation with E. asburiae KE17 increased growth rates of stressed plants. The concentrations of plant hormones (abscisic acid and salicylic acid) and rates of lipid peroxidation were higher in plants under heavy metal stress, while total chlorophyll, carotenoid content and total polyphenol concentration were lower. While the bacterial treatment reduced the abscisic acid and salicylic acid content and lipid peroxidation rate of Cu‐stressed plants, it also increased the concentration of photosynthetic pigments and total polyphenol. Moreover, the heavy metals induced increased accumulation of free amino acids such as aspartic acid, threonine, serine, glycine, alanine, leucine, isoleucine, tyrosine, proline and gamma‐aminobutyric acid, while E. asburiae KE17 significantly reduced concentrations of free amino acids in metal‐affected plants. Co‐treatment with E. asburiae KE17 regulated nutrient uptake by enhancing nitrogen content and inhibiting Cu and Zn accumulation in soybean plants. The results of this study suggest that E. asburiae KE17 mitigates the effects of Cu and Zn stress by reprogramming plant metabolic processes.     

Crystal structure of dodecameric vanadium-dependent bromoperoxidase from the red algae Corallina officinalis

The three-dimensional structure of the vanadium bromoperoxidase protein from the marine red macroalgae Corallina officinalis has been determined by single isomorphous replacement at 2.3 A resolution. The enzyme subunit is made up of 595 amino acid residues folded into a single alpha+beta domain. There are 12 bromoperoxidase subunits, arranged with 23-point group symmetry. A cavity is formed by the N terminus of each subunit in the centre of the dodecamer. The subunit fold and dimer organisation of the Cor. officinalis vanadium bromoperoxidase are similar to those of the dimeric enzyme from the brown algae Ascophyllum nodosum, with which it shares 33 % sequence identity. The different oligomeric state of the two algal enzymes seems to reflect separate mechanisms of adaptation to harsh environmental conditions and/or to chemically active substrates and products. The residues involved in the vanadate binding are conserved between the two algal bromoperoxidases and the vanadium chloroperoxidase from the fungus Curvularia inaequalis. However, most of the other residues forming the active-site cavity are different in the three enzymes, which reflects differences in the substrate specificity and stereoselectivity of the reaction. A dimer of the Cor. officinalis enzyme partially superimposes with the two-domain monomer of the fungal enzyme.